Glucocorticoid Signaling From Molecules To Mice To Man

Advances in Experimental Medicine and Biology 872
Jen-Chywan Wang
Charles Harris Editors
Glucocorticoid
Signaling
From Molecules to Mice to Man
Advances in Experimental Medicine and Biology
Editorial Board:
IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel

ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, Italy
More information about this series at http://www.springer.com/series/5584

Jen-Chywan Wang • Charles Harris
Editors
Glucocorticoid Signaling
From Molecules to Mice to Man
Editors
Jen-Chywan Wang, PhD Charles Harris, MD, PhD
Department of Nutritional Division of Endocrinology
Science & Toxicology Metabolism & Lipid Research
University of California Berkeley Department of Internal Medicine
Berkeley, CA, USA Washington University School of Medicine
St. Louis, MO, USA
ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-1-4939-2894-1 ISBN 978-1-4939-2895-8 (eBook)
DOI 10.1007/978-1-4939-2895-8
Library of Congress Control Number: 2015945236
Springer New York Heidelberg Dordrecht London

© Springer Science+Business Media New York 2015
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material contained herein or for any errors
or omissions that may have been made.
Printed on acid-free paper
Springer Science+Business Media LLC New York is part of Springer Science+Business Media
(www.springer.com)
Acknowledgements
JCW would like to thank his parents and family for support. He would also like to
thank his graduate and postdoctoral mentors, Daryl Granner and Keith Yamamoto,
his former and current lab members, and many collaborators and friends for their
immense help during his research career.
CH would like to thank his parents, Stewart and Helen; his wife, Audrey; and his
son, Benjamin for their support. He would like to thank countless mentors who
helped form his perspective as a physician scientist. Specifically, he would like to
thank basic science mentors, Diane Lipscombe, Eugene M Johnson Jr., and Robert
V. Farese Jr., and clinical mentor, James Blake Tyrrell, who introduced him to the
clinical care of patients with the fascinating Cushing syndrome.
Contents
Part I Introductory Materials
1 Regulatory Actions of Glucocorticoid Hormones:

From Organisms to Mechanisms ........................................................... 3
Daryl K. Granner, Jen-Chywan Wang, and Keith R. Yamamoto
2 Molecular Biology of Glucocorticoid Signaling ................................... 33
Margarita Arango-Lievano, W. Marcus Lambert,
and Freddy Jeanneteau
3 Mechanisms of Glucocorticoid-Regulated
Gene Transcription ................................................................................. 59
Sebastiaan H. Meijsing
4 Clinical Perspective: What Do Addison and Cushing
Tell Us About Glucocorticoid Action? .................................................. 83
Charles Harris
Part II Effects of Glucocorticoids on Metabolism
5 Regulation of Glucose Homeostasis by Glucocorticoids...................... 99

Taiyi Kuo, Allison McQueen, Tzu-Chieh Chen,
and Jen-Chywan Wang
6 How Do Glucocorticoids Regulate Lipid Metabolism? ....................... 127
Roldan M. de Guia and Stephan Herzig
7 Glucocorticoids and Skeletal Muscle .................................................... 145
Sue C. Bodine and J. David Furlow
vii
viii Contents
Part III Specific Effects of Glucocorticoids on Tissues
8 Glucocorticoid-Induced Osteoporosis ................................................... 179

Baruch Frenkel, Wendy White, and Jan Tuckermann
9 Effects of Glucocorticoids in the Immune System ............................... 217
Emmanuel Oppong and Andrew C.B. Cato
10 Glucocorticoids and the Brain: Neural Mechanisms
Regulating the Stress Response ............................................................. 235
Shawn N. Shirazi, Aaron R. Friedman, Daniela Kaufer,
and Samuel A. Sakhai
11 Glucocorticoid Regulation of Reproduction ......................................... 253
Anna C. Geraghty and Daniela Kaufer
12 Glucocorticoids and the Lung ................................................................ 279
Anthony N. Gerber
13 Glucocorticoids and the Cardiovascular System ................................. 299
Julie E. Goodwin
14 Glucocorticoids and Cancer ................................................................... 315
Miles A. Pufall
Part IV Miscellaneous Topics
15 Animal Models of Altered Glucocorticoid Signaling ........................... 337

Charles Harris
16 The Dehydrogenase Hypothesis ............................................................. 353
Conor Woods and Jeremy W. Tomlinson
17 Conclusions and Future Directions ....................................................... 381
Jen-Chywan Wang and Charles Harris
Index ................................................................................................................. 383

Contributors
Margarita Arango-Lievano, Ph.D. Inserm U1191, CNRS UMR5203, Institute

for Functional Genomics, Montpellier, France
Sue C. Bodine, Ph.D. Department of Neurobiology, Physiology and Behavior,
University of California, Davis, Davis, CA, USA
Department of Physiology and Membrane Biology, University of California, Davis,
Davis, CA, USA
Andrew C.B. Cato, Ph.D. Institute of Toxicology and Genetics, Karlsruhe Institute
of Technology, Karlsruhe, Baden Württemberg, Germany
Tzu-Chieh Chen, M.S. Department of Nutritional Sciences and Toxicology,
University of California Berkeley, Berkeley, CA, USA
Baruch Frenkel, D.M.D., Ph.D. Department of Orthopaedic Surgery, Keck
School of Medicine, Institute for Genetic Medicine, University of Southern
California, Los Angeles, CA, USA
Department of Biochemistry and Molecular Biology, Keck School of Medicine,
Institute for Genetic Medicine, University of Southern California, Los Angeles,
CA, USA
Aaron R. Friedman, B.A., Ph.D. Department of Integrative Biology, University
of California, Berkeley, Berkeley, CA, USA
J. David Furlow, Ph.D. Department of Neurobiology, Physiology and Behavior,
University of California, Davis, Davis, CA, USA
Anna C. Geraghty, B.A. Department of Integrative Biology, University of
California, Berkeley, CA, USA
Anthony N. Gerber, M.D., Ph.D. Department of Medicine, National Jewish
Health, University of Colorado, Denver, Denver, CO, USA
ix
x Contributors
Julie E. Goodwin, M.D. Department of Pediatrics, Yale University School of

Medicine, New Haven, CT, USA
Daryl K. Granner, M.D. Department of Molecular Physiology and Biophysics,
University of Iowa Carver College of Medicine, Iowa City, IA, USA
Department Internal Medicine, Fraternal Order of Eagles Diabetes Research Center,
University of Iowa Carver College of Medicine, Iowa City, IA, USA
Roldan M. de Guia, Ph.D. Department of Molecular Metabolic Control, German
Cancer Research Center, Center for Molecular Biology and University Hospital
Heidelberg, Heidelberg, Germany
Charles Harris, M.D., Ph.D. Division of Endocrinology, Metabolism and Lipid
Research, Department of Internal Medicine, Washington University School of
Medicine, St. Louis, MO, USA
Stephan Herzig, Ph.D. Department of Molecular Metabolic Control, German
Cancer Research Center, Center for Molecular Biology and University Hospital
Heidelberg, Heidelberg, Germany
Freddy Jeanneteau, Ph.D. Inserm U1191, CNRS UMR5203, Institute for
Functional Genomics, Montpellier, France
Daniela Kaufer, Ph.D. Department of Integrative Biology, University of California,
Berkeley, CA, USA
Taiyi Kuo, Ph.D. Department of Nutritional Sciences and Toxicology, University
of California Berkeley, Berkeley, CA, USA
W. Marcus Lambert, Ph.D. Department of Microbiology, New York University
School of Medicine, New York, NY, USA
Allison McQueen, B.S. Department of Nutritional Sciences and Toxicology,
Sebastiaan H. Meijsing, Ph.D. Department of Computational Molecular Biology,
Max Planck Institute for Molecular Biology, Berlin, Germany
Emmanuel Oppong, Ph.D. Institute of Toxicology and Genetics, Karlsruhe
Institute of Technology, Karlsruhe, Baden Württemberg, Germany
Miles A. Pufall, M.S., Ph.D. Department of Biochemistry, Carver College of
Medicine, Holden Comprehensive Cancer Center, Iowa City, IA, USA
Samuel A. Sakhai, Ph.D. Department of Psychology, University of California,
Berkeley, CA, USA
Shawn N. Shirazi, B.A. Department of Integrative Biology, University of California,
Berkeley, CA, USA
Contributors xi
Jeremy W. Tomlinson, FRCP, Ph.D. Oxford Centre for Diabetes,

Endocrinology & Metabolism, University of Oxford, Radcliffe Department of
Medicine, Oxford, UK
Jan Tuckermann, Ph.D. Institute for Comparative Molecular Endocrinology,
University of Ulm, Ulm, Germany
Jen-Chywan Wang, Ph.D. Department of Nutritional Sciences and Toxicology,
Wendy White, M.D. Department of Diabetes and Endocrinology, Eisenhower
Medical Center, Rancho Mirage, CA, USA
Conor Woods, M.B. B.Ch., M.R.C.P. Department of Diabetes and Endocrinology,
St. Vincent’s University Hospital, Dublin, Leisnter, Ireland
Keith R. Yamamoto, Ph.D. Cellular & Molecular Pharmacology, University of
California, San Francisco, San Francisco, CA, USA
Part I
Introductory Materials
Chapter 1
Regulatory Actions of Glucocorticoid
Hormones: From Organisms to Mechanisms
Daryl K. Granner, Jen-Chywan Wang, and Keith R. Yamamoto
Abstract The history of glucocorticoid hormone research is an excellent example of

“bedside to bench” investigation. It started with two very insightful clinical observa-
tions. Thomas Addison described the syndrome of what came to be known as adrenal
hormone insufficiency and Harvey Cushing the syndrome of glucocorticoid hormone
excess. These dramatic and life-threatening conditions spawned 150 years of active
research that has involved many disciplines; indeed some of the fundamental obser-
vations of molecular biology are the result of this work. We have a fundamental
knowledge of how glucocorticoids regulate gene transcription, their major effect.
The challenge facing current and future investigators is to discern how to use this
information to make these powerful therapeutic agents safer and more effective.
Dedication We dedicate this chapter to Gordon M. Tomkins. Gordon was a visionary who, after
direct exposure to the Paris bacterial genetics group in the early 1960s, quite clearly foresaw the
field of mammalian gene regulation. He was one of the founders of the discipline now known as
Molecular Endocrinology. Most importantly, as regards the topic of this book, his scientific passion
was glucocorticoid action. A generation of young scientists was fortunate to spend time in his
laboratory; many others were influenced by his writings, entertaining lectures and the informal
talks he gave during his many visits to universities and research institutes. Gordon was a direct
mentor to two of us, D.K.G. and K.R.Y., and a second generation mentor to J.-C.W.
D.K. Granner, M.D. (*)
Department of Molecular Physiology and Biophysics, University of Iowa Carver
College of Medicine, 169 Newton Road, 4312 PBDB, Iowa City, IA 52242, USA
Department Internal Medicine, Fraternal Order of Eagles Diabetes Research Center,
University of Iowa Carver College of Medicine, 169 Newton Road, 4312 PBDB, Iowa City,
IA 52242, USA
Vanderbilt University School of Medicine, Nashville, TN, USA
e-mail: [email protected]
J.-C. Wang, Ph.D.
Department of Nutritional Sciences and Toxicology, University of California Berkeley,
119 Morgan Hall, Berkeley, CA 94720-3104, USA
K.R. Yamamoto, Ph.D.
Cellular & Molecular Pharmacology, University of California, San Francisco, 600 16th Street,
Genentech Hall S572D, Campus Box 2280, San Francisco, CA 94143-2280, USA
© Springer Science+Business Media New York 2015 3

J.-C. Wang, C. Harris (eds.), Glucocorticoid Signaling, Advances in Experimental
Medicine and Biology 872, DOI 10.1007/978-1-4939-2895-8_1
4 D.K. Granner et al.
Keywords Glucocorticoids • Gene expression • Nuclear receptors • Transcription

factors • DNA elements
The survival of multicellular organisms requires rapid and efficient adaptation to an

ever-changing external environment. Many mechanisms have evolved to ensure the
effective coupling of external cues to internally generated signals that affect
Table 1.1 The pleiotropic effects of glucocorticoid hormones

Effects on intermediary metabolism
1. Increase glucose production by: (a) increasing the delivery of amino acids and glycerol
(the gluconeogenic substrates) from peripheral tissues; (b) increasing the rate of
gluconeogenesis by increasing the amount and activity of several key enzymes; and (c)
“permitting” other metabolic reactions to operate at maximal rates
2. Inhibiting the uptake of glucose by tissues excepting the nervous system, heart and red
blood cells
3. Increase hepatic glycogen deposition by promoting the activation of glycogen synthase
4. Promote lipolysis, but can cause lipogenesis in some sites (face and trunk) especially at
higher than physiologic levels
5. Promote protein metabolism. This is an anabolic effect, particularly in liver, at physiologic
levels. Can be catabolic in certain conditions as a means of supplying amino acids for
gluconeogenesis
Effects on host mechanisms
1. Suppress the immune response. These hormones cause a species- and cell type-specific lysis
of lymphocytes
2. Suppress the inflammatory response by: (a) decreasing the number of circulating leukocytes
and the migration of tissue leukocytes; (b) inhibiting fibroblast proliferation; (c) inducing
lipocortins, which by inhibiting phospholipase A2, blunt the production of the potent
anti-inflammatory prostaglandins and leukotrienes; and (d) inhibit the action of NF-kB by
increasing synthesis of the inhibitor IkB; GR tethering to p65 subunit of NF-kB, competing
with Pol II CTD kinase P-TEFb association with p65
Effects on development/differentiation
1. Development of the lung, including the production of surfactants and an inducible sodium
channel
2. Development of neural crest-derived chromaffin cells (catecholamine production) in the adrenal
medulla. The delivery of high concentrations of glucocorticoids to the medulla through the
intra-adrenal portal system allows for the induction of phenylethanolamine-N-methyltransferase,
which catalyzes the conversion of norepinephrine to epinephrine
Other effects
1. Necessary with catecholamines for maintenance of normal blood pressure and cardiac output
2. Required for maintenance of normal water and electrolyte balance, perhaps by restraining
ADH release (H2O) and by increasing angiotensinogen (Na+). These effects contribute to the
effect on blood pressure
3. Necessary, with the hormones of the adrenal medulla, allowing the organism to respond to
stress
1 Regulatory Actions of Glucocorticoid Hormones: From Organisms to Mechanisms 5
complex processes such as the response to food deprivation, exercise, stress, trauma,
and infection. A constant supply of energy is of central importance in all of these
functions. Glucocorticoid hormones are named for the central role they play in glu-
cose homeostasis, which is an important source of energy for all tissues; in particu-
lar, the brain depends almost entirely on glucose metabolism.
Glucocorticoids are also of interest because of the direct and indirect effects they
have on a large number of apparently diverse physiologic and biochemical pro-
cesses (Table 1.1). And, they play an important therapeutic role as life-saving
replacement treatment in adrenal insufficiency (Addison’s disease; Chap. 4), as a
key component in the therapy of certain malignancies (Chap. 14), as an immuno-
suppressant in transplantation and autoimmune diseases and as an anti-inflammatory
agent (Chap. 9). It is amazing that this class of hormones, which are small (cortisol
is 362 Da), relatively simple derivatives of cholesterol, can accomplish so much.
The current understanding of how these hormones work started first with a descrip-
tion of the adrenal glands, then with a remarkable clinical observation that led to
more than 150 years of research that has employed, and also helped formulate,
many of the basic principles of physiology, biochemistry and molecular biology.
This introductory chapter will trace the discoveries that have led to our current
understanding of glucocorticoid action, and will attempt to set the stage for the suc-
ceeding chapters that delve more deeply into the different processes affected by
these interesting hormones.
The Importance of the Adrenal Glands Is Established
The story begins in 1563 when Eustachius described two small organs, located near
and just above (ad-) the kidneys (renal) in humans. Adrenal glands, as they were
subsequently named, are found across vertebrates, but their role in biology remained
unknown until the mid-1800s when Kolliker placed the adrenals among the group
of ductless glands that communicate only with the blood system. Adrenals were
shown to consist of two discrete areas: a firm outer layer, or cortex, and a soft,
spongy inner layer, the medulla. The function of each of these areas was unknown,
and the concept of hormones, molecules synthesized in one organ and transported
through the vascular system to one or more distant target organs, was not formulated
until the studies of the control of secretin secretion were reported by Bayliss and
Starling in 1901–1902 [1, 2].
Thomas Addison made a brilliant clinical-pathologic observation in 1855 that
really launched this field of research. He described a syndrome that included
intense skin pigmentation, weakness, feeble pulse, and general debility with a fatal
outcome in a group of 11 patients, all of whom had small, diseased or absent adre-
nal glands. The title of his monograph, published posthumously, was “On the
Constitutional and Local Effects of Disease of the Suprarenal Capsule” [3].
Curiously, also in 1855, Claude Bernard first described his studies on the glyco-
genic function of the liver whereby this organ “prepares sugar at the expense of the
elements of the blood passing through it” [4], a concept of central importance once
the full manifestations of adrenal insufficiency were known. Bernard also first
wrote about the importance of maintaining a “stable internal environment”, which
led to the concept of “homeostasis”, a term first used by Cannon in 1926 [5].
Although presented in the same year, the observations by Addison and Bernard
were not connected for about 75 years.
Brown-Séquard showed that adrenalectomy resulted in the death of experimental
animals [6], as was observed in humans affected with what had become known as
Addison’s disease. Attempts to treat persons (or experimental animals subjected to
adrenalectomy) commenced in the latter part of the nineteenth century, even though
the active agent(s) in the adrenals was unknown. Aqueous extracts of the entire
adrenal often had effects on heart rate and blood pressure, but did not resolve the
life-threatening symptoms of Addison’s disease. With clarification of the medullary
source of adrenaline (epinephrine) and its subsequent purification and synthesis, the
separate role of this glandular structure, and its role in the sympathetic nervous
system, became apparent. The search for the critical adrenal cortical factor “cortin”
became the focal point of interest during this time and continued during the early
part of the twentieth century. Cortin was suspected of being a hormone, a concept
which Starling had by then defined [7], but the structure of cortin was a complete
mystery.
The Active Adrenal Cortical Hormones Are Identified

and Synthesized
The identification of the active hormone proved to be an arduous task. A major

breakthrough came when organic solvents were used in place of water to make
adrenal cortical extracts; the subsequent demonstration of the lipophilic nature of
steroids explained this important discovery. These extracts led to the survival of
adrenalectomized animals and in the improvement of patients with Addison’s dis-
ease [8, 9]. The race to discovery continued, and in the 1920s and 1930s many
groups, most notably those of Kendall and Reichstein, developed techniques for the
crystallization of adrenal corticosteroids and the subsequent synthesis of many of
these molecules [10, 11]. The significant difficulties encountered in this work were
understood much later when it was realized that there are dozens of steroids in the
adrenal cortex, most of which are intermediates in the synthesis of the active hor-
mones from cholesterol. The problem was made even more difficult by the fact that
many of these molecules co-purified and co-crystallized. To complicate matters
even further, very small molecular changes had large effects on activity, thus the
chemical synthesis had to be very precise [10, 11]. These obstacles were eventually
overcome. Cortisone, synthesized by Sarett in 1947 [12], was the first glucocorticoid
to be extensively used clinically.
The Metabolic and Therapeutic Effects of Glucocorticoids

Are First Explored
By the early part of the twentieth century, when various clinical parameters could
be reliably quantitated, the syndrome of Addison’s disease was further expanded
to include metabolic and renal components. The inability to maintain glucose
homeostasis, coupled with extreme insulin sensitivity (hypoglycemia), was a seri-
ous problem in patients with Addison’s disease. In time this was attributed to a
reduced ability of the liver to convert amino acids or glycerol into glucose (impaired
gluconeogenesis) or to convert glycogen into glucose, a validation of the early ideas
formulated by Claude Bernard. These observations also led to studies of the hor-
monal regulation of these processes, as is discussed in detail below. The renal
manifestations include excessive retention of potassium and diuresis associated
with excessive loss of sodium in the urine, which contributes to the severe hypoten-
sion noted in these patients. The eventual availability of molecules of known struc-
ture led to the categorization of adrenal corticosteroids into glucocorticoids and
mineralocorticoids, according to their predominant, but not exclusive, action.
Hydrocortisone (cortisol) and corticosterone are the major glucocorticoids in
humans and rodents, respectively; aldosterone is the major mineralocorticoid.
Persons with primary adrenal insufficiency are now usually treated with both a glu-
cocorticoid and a mineralocorticoid.
The production of adrenal androgens (mostly androstenedione) was defined
later, based in part on the serendipitous synthesis of steroids with androgenic activ-
ity in the course of efforts to make glucocorticoids, and on subsequent clinical
observations, which helped explain why some persons with adrenal hyperplasia
develop masculinization. The structures of the primary adrenal hormones are shown
in Fig. 1.1.
A second clinical observation played a major role in advancing this research field.
Harvey Cushing, in 1912, described a syndrome in which an adenoma of the ante-
rior pituitary gland caused hypertrophy of the adrenal glands and a characteristic set
of clinical signs and symptoms [13]. The manifestations of Cushing’s disease were,
in many ways, the opposite of those seen in Addison’s disease: hypertension, fluid
retention, weight gain, obesity with ectopic fat deposition, hyperglycemia with
insulin resistance, masculinization, and thin friable skin with bruising, among oth-
ers. Now known to be due to excessive, unsuppressed release of adrenocorticotropic
hormone (ACTH) from a tumor of the basophilic cells in the anterior pituitary
(or corticotrophin releasing hormone (CRH) from the hypothalamus), the condition
is mimicked when excessive amounts of exogenous glucocorticoids are adminis-
tered therapeutically, or when primary adrenal tumors overproduce the hormones, so-
called Cushing’s syndrome. Philip Hench, a colleague of Kendall at the Mayo
Clinic, first used a glucocorticoid (cortisone) to treat persons with rheumatoid
arthritis [14]. This treatment had remarkable beneficial effects and led to its subse-
quent use as an anti-inflammatory/immunosuppressant agent. Unfortunately, clinical
Fig. 1.1 Basic structures and trivial names of major hormones of the adrenal cortex
remission requires long-term use at high doses, and this often leads to the serious
complication of Cushing’s syndrome with its attendant, devastating complications.
These studies, in collection, are an excellent example of how early endocrine
research evolved. The general sequence of discovery was: (1) ablate a gland of inter-
est; (2) observe and quantitate the physiologic and biochemical events that ensue;
(3) isolate, purify and synthesize the putative hormone; and (4) prove the role of the
latter by replacing the pure hormone and restoring normal homeostasis. The next
challenge was to elucidate how glucocorticoids accomplish all these physiologic
and pathophysiologic events.
Once the physiologic effects of glucocorticoid deficiency or excess were defined,
and pure molecules were readily available, the question became “what exactly do
these hormones do and how do they do it?” Emphasis was placed early on the regu-
lation of glucose metabolism because of the notable effects glucocorticoids appeared
to have on this process and because of a considerable body of relevant knowledge
which had been developing contemporaneously. Important concepts such as:
(a) enzymes have a unique structure, (b) precise metabolic pathways exist and they
are coordinated, (c) proteins turnover independent of cell replication, and (d)
enzyme adaptation (induction and repression) were all applied to the study of glu-
cocorticoid hormone action.
Enzymes Are Defined and the Metabolic Pathways Are

Elucidated
The concept that the conversion of foodstuffs into cellular constituents, or into
energy, involved an orderly progression of discrete biochemical reactions, each
catalyzed by an enzyme, first began attracting attention as early as 1752 when de
Réaumur showed that gastric secretions could digest meat [15]. Others demon-
strated that saliva could convert starch to sugar, and in 1833 diastase (amylases)
was described [16]. This work is associated with the subsequent convention of
adding “-ase” to the name of an enzyme, although the word “enzyme” was appar-
ently not used until 1877 [17]. The nature and function of enzymes was unknown.
The prevailing theory, based on the fermentation of sugar into alcohol, was that
the process required a living cell. When Buchner showed, in 1907, that yeast
extracts accomplish the same purpose, the cell-free action of enzymes was estab-
lished [18].
Investigators had shown that enzymatic activity was associated with proteins,
but had not proven that a protein, per se, was capable of this action. In 1926 Sumner
purified and crystallized the protein urease; he repeated this with catalase in 1937
[19]. Northrup and Stanley, who studied pepsin, trypsin and chymotrypsin, among
several other proteins, presented further proof that enzymes are proteins [20, 21].
And importantly, as Northrop stated, “the enzymatic activity is a property of the
protein itself and not due to a non-protein impurity”.
Studies by hundreds of investigators, which started even before the exact nature of
enzymes was established, led to the construction of the “metabolic chart”. The chart
presents a picture (although details are still being added) of the complex, interacting
metabolic events that occur within a cell. This is truly one of the great scientific
accomplishments of the twentieth century, especially when one considers that a great
many of the enzymes on this chart were purified using virtually none of the overex-
pression, chromatographic and affinity techniques available today; very tedious,
nonspecific techniques (e.g., salt fractionation, alcohol and acetone fractionation)
were among those commonly employed.
Knowledge of the metabolic pathways made it possible to finally understand that
the conversion of foodstuffs into cellular constituents or energy involves an orderly
progression of discrete biochemical reactions, each catalyzed by an enzyme. This
begged the question of whether these processes are regulated, particularly in view
of observations such as those that suggested glucocorticoids might play a role in one
or more of these processes and thereby account for some of the manifestations of
Addison’s disease. Subsequent experiments in this area focused on the coordination
and regulation of these complex pathways. A number of investigators formulated
the hypothesis that hormones might provide the means of metabolic coordination
[22]. But another important concept had to be developed before this hypothesis
could be tested.
The Concept of Differential Turnover of Cellular Constituents

Is Established
Until the late 1930s cells and cellular constituents were thought to turn over at the
same rate. The stable components of a cell somehow replicated themselves and were
equally distributed into the two daughter cells with cell division. This was a conserva-
tive mechanism, but it would not allow for adaptation based on changing the amount
of a cellular constituent (protein) independent of cell replication. The pioneering
work by Schoenheimer, Shemin, Rittenberg, and others, who were among the first
to use isotopes to address biologic questions, showed convincingly that various
lipids and proteins have turnover rates different from that of the cell itself, and have
different turnover rates within a given cell [23–25]. For example, hepatocytes were
found to have a small component of proteins (~3 %) with a t1⁄2 of ~140 days (about
the t1⁄2 of the cell) and a much larger component consisting of two subclasses with
t1⁄2 values of 4.5 and 12 days [24].
The concept that turnover occurs, and is dynamic, was a major advance, as subtle
adjustments of an active synthesis/degradation process could allow for flexible,
rapid and accurate adaptive responses to the challenges of an acutely changing
external and internal environment. This observation offered the possibility that the
enzymatic reactions that govern a certain metabolic pathway could be regulated by
changes of the amount and/or activity of one or more enzymes. These changes could
be facilitated by intercellular signals (e.g., glucocorticoid hormones) that would
allow a cell to respond to various metabolic and environmental challenges.
Enzymes Show Adaptive Changes and Glucocorticoids

Regulate Gene Expression
The observation that cellular components turnover led directly to the concept that
organisms could show adaptive responses to their environment. Remarkable
changes in the amount of enzymes in microorganisms had been demonstrated in the
1940s, generally in response to alterations of substrate concentration [26–28]. This
phenomenon was demonstrated in mammalian cells when tryptophan was shown to
induce a six to eightfold increase in tryptophan oxygenase (TO) [29]. This effect,
which appeared to be an example of substrate induction similar to that observed in
bacteria, was rapid and self-limited. However, subsequent studies showed that other
amino acids and compounds, which were not substrates of TO, also increase activity
of the enzyme. All the substances tested appeared to stress the animals, and the
response only occurred in those with an intact pituitary-adrenal axis [30]. Selye had
proposed that this axis was involved in the stress response [31], which led to the
hypothesis that the adrenal cortical hormones were responsible for the induction of
Table 1.2 The timetable of Enzyme

glucocorticoid induction of
Function TAT TO PEPCK
hepatic enzymes
Increased enzyme activity 1957 1954 1963
Increased enzyme amount 1962 1962 x
Increased enzyme synthesis 1962 1965 1975
Increased mRNA activity 1976 1973 1977
Increased mRNA amount 1983 1982 1983
Increased transcription 1987 1983 1983
TO. This concept was validated shortly thereafter when a purified glucocorticoid
(see above) administered to adrenalectomized rats resulted in the induction of TO
[32]. By 1956, many examples of changes of enzyme activity in response to adre-
nalectomy, thyroidectomy, hypophysectomy, and diabetes were known [33]. When
glucocorticoids were also found to induce tyrosine aminotransferase (TAT) [34], the
era of research on the hormonal regulation of enzyme induction by these hormones
was well underway. The timetable of progress is shown in Table 1.2.
Knox and Mahler observed that the changes in TO activity could result from a
change in the amount of enzyme rather than to a change in the catalytic activity of
the protein—“the production of a potential increase in metabolism by increasing the
amount of enzyme, but without affecting the catalytic activity of a given amount of
enzyme may therefore be a general means of metabolic regulation” [29]. This state-
ment seems obvious today but it was presented when much of the effort to deter-
mine the mechanism of action of steroid hormones was confined to cell free systems,
since a prevailing idea was that they acted to alter catalytic activity by serving as
energy transducers, enzyme cofactors or allosteric regulators.
Proof that increased activity of an enzyme was due to an increased amount of the
protein required a purified protein, which was used to produce a specific antibody
that could then be used to selectively immunoprecipitate the radioactively labeled
protein. Such evidence was obtained for TAT [35], and for TO [36]. The concept of
turnover implied that an increased amount of protein could result from an increased
rate of synthesis, from a decreased rate of degradation, or from some combination
of these processes. The theoretical basis for such experiments was defined by
Schimke [37] who then showed that tryptophan slowed hepatic TO degradation
while hydrocortisone enhanced TO synthesis [38]. By contrast, rat liver TAT syn-
thesis was enhanced by hydrocortisone without an effect on degradation [39].
Tryptophan and tyrosine are not significant gluconeogenic substrates, so the
regulation of TO and TAT served mostly as model systems for studies of enzyme
regulation. By contrast, phosphoenolpyruvate carboxykinase (PEPCK), which cata-
lyzes the conversion of oxaloacetate (from pyruvate) to phosphoenolpyruvate, is a
major gluconeogenic enzyme. Thus, the demonstration of the induction of PEPCK
by glucocorticoids was especially significant [40].
Glucocorticoids Regulate the Transcription of Specific Genes
An enormous conceptual advance occurred as a result of the studies of the regulation

of the E. coli lac operon by Jacob and Monod [41]. Two major concepts arose from
these, and subsequent, experiments. The first was that genes consist of structural
and regulatory components. The second was the “information flow” hypothesis,
which states that genes direct the synthesis of a messenger RNA (mRNA), which
then directs the synthesis of the corresponding protein. The studies of the lac operon
gave immediate direction to studies of regulation of gene expression in eukaryotic
cells, even though it would take many years to develop the techniques necessary for
performing these investigations.
Gordon Tomkins was one of the first persons to propose that the approach Jacob
and Monod used to study gene regulation in prokaryotes might be applied to the
analysis of the hormonal regulation of enzyme synthesis in cultured mammalian cells.
This idea was not enthusiastically accepted at first, to say the least, but the demonstra-
tion that glucocorticoids induce TAT in cultured H4IIE and HTC hepatoma cells was
a game-changer [42, 43]. A subsequent study showed that the basic observations of
the induction of TAT in liver were replicated in HTC cells [44], and it soon became
clear that the ability to precisely control the hormonal environment, select for
mutants, synchronize cells, adapt them to growth as single cells in suspension, etc.
offered a system amenable to the molecular biology studies that were to follow.
The conceptual framework used to analyze how glucocorticoids affect enzyme syn-
thesis was applied to the analysis of the role of mRNA in this process. Changes of
the rate of synthesis of a specific enzyme could result from changes of the transla-
tional activity of a fixed amount of mRNA, or a changed amount of mRNA from
either an alteration of mRNA stability or of its rate of synthesis (transcription).
Unfortunately, many of the glucocorticoid-regulated enzymes exist in very small
amount; TO, TAT and PEPCK are each present at ≤1 % of cytosolic protein. Later
studies showed, as expected, that the corresponding basal levels of the mRNAs for
these enzymes comprise less than 0.1 % of total poly A+ RNA in hepatocytes [45].
The basal rate of PEPCK gene transcription is 0.01 % of the total [46]. Because
procedures had to be developed to account for this lack of abundance (there were no
commercially available reagents or kits, DNA had to be sequenced by manual pro-
cedures, none of the genes had been isolated or characterized, etc.), it took many
years before specific assays of mRNA activity, amount, or transcription, measured
by various cell-free translation systems or by hybridization to specific cDNA probes,
were established. In the meantime, results obtained from experiments using various
inhibitors of RNA synthesis were used to make inferences about the mediating role
of mRNA. Since many of these compounds, most notably actinomycin D, inhibited
glucocorticoid induction of TAT [43, 47], TO [47], and PEPCK [40], it was assumed
that ongoing mRNA synthesis was necessary for the response [45].
The assumption that hormones regulate mRNA synthesis was directly tested in more
than a decade of research starting in the early 1970s. mRNA activity was assessed by
translating total nuclear poly A+ RNA in a wheat germ or reticulocyte lysate translation
system. The amount of radiolabel incorporated into specific protein (again detected by
immunoprecipitation) was compared to that in the total protein synthesized. The amount
of mRNA was assessed by hybridization to specific cDNA probes once those were avail-
able. Transcription assays, much easier to perform in cultured cells, used longer cDNA
probes to detect the amount of radioisotope incorporated into a specific mRNA. The
progression illustrated in Table 1.2 shows that the glucocorticoid-induced increase of
enzyme activity is accomplished through an enhanced rate of transcription of the TO
[48], TAT [49] and PEPCK [46] genes, measured using an elongation assay. It is note-
worthy that 20 years elapsed between first concept and the final accomplishment, which
underscores the difficulties encountered in performing these experiments.
Glucocorticoids often do not act in isolation on important metabolic processes. An
example is hepatic gluconeogenesis. This process is increased by glucocorticoids
and glucagon (cAMP) and decreased by insulin. It thus is of interest to note that
each of these hormones affect PEPCK activity in parallel with their effect on gluco-
neogenesis. The changes in PEPCK activity caused by the hormones are due to
changes of specific mRNA amount, which are, in turn, directly proportional to the
rate of transcription of the PEPCK gene [46, 50]. The basal rate of transcription of
the PEPCK gene is ~100 ppm of total RNA synthesized; glucocorticoids and cAMP
each increase this rate several fold and their effects are additive. Insulin inhibits
basal and induced transcription, and the insulin effect is dominant [46]. All of these
actions could be studied in the H4IIE rat hepatoma cell line, thus, as is discussed
below, a system existed for analyzing how several hormones interact at the level of
a single gene to regulate an important metabolic function.
A Specific Receptor Mediates the Action of Glucocorticoids
While studies of the effect of glucocorticoids on gene expression were progressing,

several investigators were establishing the physiologic and biochemical parameters
of what became known as the glucocorticoid hormone signal transduction pathway.
The following is a brief summary of these important observations. Cortisol, synthe-
sized in the fasciculata and reticularis zones of the adrenal cortex, is secreted directly
into plasma. Cortisol exists in two forms in plasma: (1) bound to transcortin
(corticosteroid-binding globulin; CBG) and (2) as free, unbound, cortisol. The latter,
which is a small percentage of the total circulating hormone (<10 %), is the biologically
active form. Free cortisol readily crosses the plasma membrane, where it initiates
action by binding, with high affinity, to a specific glucocorticoid receptor (GR).
The concept of specific receptors for steroid hormones began with the work of
Talwar et al., who showed that estradiol binds with high affinity to a uterine
cytosolic substance [51], later shown to be a protein. Definitive evidence for a GR
was presented a few years later, as summarized in comprehensive reviews [52, 53].
Early biochemical studies, performed before the purified GR was available, revealed
several key points: (1) GR is located in the cytosol in the absence of ligand. (2) The
ligand · GR interaction is of high affinity and is rapidly reversible. (3) The binding
of ligands to GR correlates well with the biologic activity of the ligand, and the
biologic effect disappears quickly following removal of the ligand. Cortisol, corti-
costerone and aldosterone all bind to the GR with high affinity, but in humans the
dominant glucocorticoid is cortisol because of its much greater plasma concentra-
tion. (4) The absence of GR in a cell results in a loss of biologic activity of the
ligand. For example, lymphocytes that lack GR are resistant to the cell-killing
effects of glucocorticoids. (5) An activation process results in the transfer of GR into
the nucleus of target cells. GR is associated with one or more chaperones (i.e.,
hsp90) in the absence of ligand. This large, multimeric complex dissociates upon
ligand binding, and the ligand·GR complex can then translocate into the nucleus.
(6) The DNA component of chromatin binds GR. This observation is based on
direct binding studies using DNA, and on the observation that DNase treatment of
chromatin reduces GR binding. Actually, GR binding to DNA exceeds that to chro-
matin, which was early evidence that chromatin can occlude transcription factor
binding to DNA. (7) All tissues known to respond to glucocorticoids exhibit this
pattern of GR behavior. Extension of these studies depended on the availability of
purified GR, and the subsequent isolation of a cDNA specific for the GR.
The Glucocorticoid Receptor Is a Transcriptional

Regulatory Factor
The GR is not an abundant protein, is relatively unstable, and forms complexes with
several other proteins, so its purification is difficult. In the late 1970s Gustafsson
and colleagues reported substantial success in purifying the GR [54, 55]. A single
polypeptide of ~90 kDa, with hormone binding properties virtually identical to
those of crude cytosol preparations, was obtained. The sequence of GR, deduced
from the open reading frames of cDNAs from human [56], rat [57] and mouse [58],
show that this molecule has been highly conserved across evolution.
Evidence accumulated in the 1970s showed that glucocorticoid receptors bind to
DNA [52, 59, 60], but selective binding, in a region likely to affect gene transcrip-
tion, was not possible until the early 1980s when purified GR became available.
Several studies suggested that the possible association of binding and function
might be established by analyzing the glucocorticoid-enhanced production of mam-
mary tumor virus (MTV) [61], an effect due to enhanced transcription of
chromosomally-integrated MTV proviral DNA [62, 63]. The virus contains all the
information required for glucocorticoid action in the MTV long-terminal repeat
(LTR) segment [64–66], but the regions of the LTR involved, and the specifics of
how the hormone accomplishes this induction, had not been established.
The specific binding of purified glucocorticoid receptor to DNA was demonstrated
by Payvar and colleagues in 1981 [67]. A number of GR binding sequences (GBSs)
in MTV DNA were identified in this and subsequent investigations [67–71]. The
identification of specific receptor-DNA interactions in vitro allowed investigators to
next test whether they were sufficient to regulate transcription and whether they
were independent from the DNA region in the LTR known to be required for tran-
scription initiation.
The strategy employed to demonstrate that ligand-receptor binding to specific
DNA segments could affect the transcription of a specific gene involved the con-
struction of a reporter gene, based in this case on the thymidine kinase (tk) gene,
which has a promoter that is not responsive to glucocorticoids. A 340 bp sequence
of MTV LTR DNA previously shown to contain several sites that bind the ligand-
GR complex [67–71], and known to be lacking in transcription initiation sequences,
was inserted into the reporter gene in various positions and orientations upstream
from the tk promoter. The addition of dexamethasone (a potent, synthetic glucocor-
ticoid) to these fusion genes led to robust induction of tk transcription from the
endogenous tk promoter [72]. This study led to several important conclusions:
(1) the MTV LTR contains a “glucocorticoid response element”, or GRE, the first
response element (i.e., a genomic segment that confers a particular transcriptional
regulatory effect in vivo) and the prototype for all hormone response elements
(HREs); (2) the GR is a transcriptional regulatory factor, the first such factor identi-
fied that is encoded in a eukaryotic genome; (3) the GR combines the two functions
of a receptor, ligand binding and signal transduction, in a single molecule; (4) the
location and orientation of the GRE relative to the transcription initiation site is
quite flexible, revealing a general functional explanation for the phenomenon of
transcriptional “enhancement”, which had been described in the SV40 tumor virus;
and (5) the transcription enhancing function afforded by the GR-GRE interaction is
separable from the process of transcription initiation [72, 73].
The Glucocorticoid Receptor Is Modular, Containing Discrete

Functional Domains
The successful isolation of a cDNA molecule for GR was a landmark achievement

[74]. Biochemical, immunologic and genetic studies had suggested that the GR has
at least three functional domains: DNA-binding (DBD), ligand binding (LBD) and
an N-terminal modulator (NTD) (Fig. 1.2 and [55, 59, 75, 76]). The availability of
a GR cDNA allowed investigators to express and test different portions of the mol-
ecule for functional activity, to perform domain swap experiments within the same
molecule, and to ligate various regions of GR to completely unrelated molecules to
test for transference of function. Selected studies, summarized below, led to the
conclusion that GR, the founding member of the nuclear receptor superfamily, is
indeed composed of several functional domains, or modules (Fig. 1.2) and these
modules are independent from one another.
The N-terminal region of the GR varies in length and amino acid sequence from
other members of the nuclear receptor superfamily [77]. It contains antigenic sites
and a transcription activation domain (AF1), deletion of which results in a partial
reduction of activation by GR. AF1 is highly acidic, and it contains many phos-
phorylation sites which can affect the basic functions of the receptor i.e., ligand,
Fig. 1.2 Schematic diagram of the structure of the human glucocorticoid receptor. The amino- and
carboxy-termini are illustrated as positions 1 and 777, respectively. The major domain structures:
amino-terminal (NTD), DNA-binding (DBD) and ligand binding (LBD) are demarcated by brack-
ets at the top. AF1, T2 and AF2 represent the transactivation domains. Regions corresponding to
specialized functions are shown as horizontal lines at the bottom
DNA and co-regulator binding [78–80]. This domain is particularly important for
assembly of the co-regulators involved in the assembly of an active transcription
apparatus. Additional domains functional in transcription activation are located in
the LBD (AF2 and tau 2) and in the dimerization region of the DNA binding domain.
Like AF1, AF2 and tau 2 are highly acidic, but they appear to be structurally unrelated
to AF1. The three domains function from different positions relative to promoters,
and when fused to heterologous DBDs [79, 81].
The DNA-binding domain (DBD) is a highly conserved, cysteine-rich, ~75 amino
acid segment located in the middle of the molecule (Fig. 1.2). Analysis of the
cDNA-deduced structure of the DBD revealed an arrangement of eight cysteine resi-
dues (perfectly conserved in all members of the receptor superfamily) that form two
zinc finger structures, each of which coordinates the association of a zinc atom to four
cysteine residues [82]. The first zinc finger is primarily involved in DNA binding,
whereas the second finger stabilizes this binding. GBS recognition is a function of
the first, or N-terminal, zinc finger domain. A three amino acid segment subdomain,
the P-box, is critical to binding specificity for all nuclear receptors. In GR this sequence
is Gly-Ser-Val, whereas in the estrogen receptor it is Glu-Gly-Ala [83].
Modularity of receptor function was demonstrated in domain swap experiments.
When the DBD of the GR was placed into the corresponding region of the estrogen
receptor, this chimeric receptor converted a gene that was normally glucocorticoid-
responsive into an estrogen responsive one [84]. In another experiment, the GR
DBD was replaced by the DBD of the bacterial transcription repressor Lex A, a
helix-turn-helix transcription factor. This chimeric molecule activated transcription
from a Lex promoter-operator construct in response to dexamethasone [85].
The GR binds to GREs as a dimer. The DBD of each receptor monomer contains a
five amino acid segment, the D box, which is located between the two most
N-terminal cysteine residues at the base of the second finger. The D box is involved
in mediating the dimerization and cooperative binding of two receptor molecules to

a GRE. Mutations of the D-box compromise the cooperativity of occupying the two
half-sites of the GBS in vitro, and reduce GR activity in vivo. NMR and X-ray
crystallographic studies show that receptors interact with the GRE as a dimer in a
head-to-head arrangement where each arm of the palindromic DNA element contacts
a single receptor molecule [82, 86].
The C-terminal ligand-binding domain (LBD) (Fig. 1.2) is a ~260 amino acid segment
that displays greater sequence divergence that the DBD among nuclear receptors.
Several widely separated and discontinuous amino acids form the hormone-binding
surfaces of this domain [81, 87]. The LBD has other functions, including hormone-
dependent nuclear localization (the NL1 and NL2 subdomains), hsp90 binding and
transcription activation (AF2 and tau2). Starting with the binding of hormone to the
LBD, a sequence of events links these functions. First, hsp90 appears to dissociate
from the LBD, resulting in a conformational change of the receptor. This has two con-
sequences. The nuclear localization signals are functional, so GR translocates stably to
the nucleus where it can now function as a transcriptional regulatory factor [88].
Interestingly, inactivation of functional domains by the hsp90-associated unli-
ganded LBD is maintained when the LBD is repositioned to the N-terminus of GR,
and even when the LBD is fused to unrelated proteins, such as the viral E1a protein
[88]. The mechanism of this global inactivation is unknown. As expected, however,
GR lacking the LBD, and the inhibitory action of the bound hsp90, activates tran-
scription constitutively [79, 85].
These representative experiments provide direct evidence of the modular nature
of the GR, and show that each of the domains can function independently. Importantly,
however, they do not imply that the domains do not interact functionally in the intact
GR. Indeed, as described below, there is abundant evidence of extensive allosteric
signaling between domains.
The GBS Is Sufficient for GR-Regulated Transcription

Activation in a Reporter Context
The availability of purified receptors and the promoter regions of several hormone
responsive genes led to tests of the relationship between in vitro glucocorticoid
binding sequence (GBS) activity and in vivo GR-mediated transcriptional regula-
tory activity. In these experiments, purified GR was bound to cloned target gene
promoter regions, and the bound sequences determined by DNase I footprinting
[68, 89]. To discover segments of DNA sufficient to confer hormonal regulation,
various promoter-proximal fragments were ligated into expression vectors bearing
basal promoter elements (e.g., from the thymidine kinase (tk) gene) driving a
reporter gene (e.g. chloramphenicol acetyltransferase (CAT) or luciferase). The
chimeric construct was transfected into a recipient cell, hormone was added and
expression of the reporter gene quantitated. Such experiments demonstrated that the
canonical idealized GBS represents a large family of 15 base pair elements related
to the sequence GGTACAnnnTGTTCT [77], and inferred that the GBS is sufficient
for regulatory activity [83, 89, 90]. Importantly, however, as described below,
GBSs are not essential for GR regulation, and bona fide GREs associated with
chromosomal GR responsive genes are substantially more complex than GBSs.
Context-Specific Glucocorticoid Regulation Is Specified

by Complex Arrays of DNA Elements and Binding Proteins
The notion that natural genes, be they chromosomal or viral, would be regulated by
glucocorticoids solely by GR binding to GBSs (termed “simple GREs” in early
work) was soon challenged by experimental observations: two new classes of GREs
were described [91]. Composite GREs are ~0.5–2 kb compound elements [92] com-
posed of one or more GBSs together with binding sites for one, or more typically,
multiple nonreceptor transcriptional regulatory factors, producing functional cross-
talk between the different classes of factors that affects the regulatory outcome.
Tethering GREs are also compound elements encompassing binding sequences for
nonreceptor regulators, but lack GBSs; GR associates at tethering GREs through
protein · protein interactions with one or more of the bound nonreceptor factor
instead of directly with DNA.
The first composite element was described at the proliferin gene, wherein a GBS
and an AP-1 binding site are contiguous (Fig. 1.3). The proteins c-fos and c-jun, two
prominent members of the phorbol ester-activated AP-1 transcription factor family,
activate proliferin expression via either c-jun homodimers or c-fos·c-jun heterodi-
mers. GR regulates proliferin expression through its GBS, but only in the presence
of AP-1, further activating the homodimeric species and repressing the heterodimer
[93]. The ratio of c-jun to c-fos in the cell is therefore the determinant of whether
GR mediates a positive or negative transcription response.
GR represses transcription at tethering GREs proximal to the collagenase gene,
where GR associates through protein · protein interactions with a specifically bound
c-Jun subunit of AP-1 [94, 95], and to the IL-8 gene, where GR binds to the p65
subunit of a NFκB factor bound at a κB binding sequence [96]. Hence, tethering
elements can be viewed as a subset of composite elements in which both GR and
nonreceptor regulators occupy a specific genomic site, but that GRE activity in
these tethering contexts proceeds in the absence of GBS elements.
Involvement of nonreceptor factors in GR-mediated regulation was also suggested
for MTV, where binding sites for nuclear factor-1 (NF-1) and octamer transcription
factor-1 (Oct-1), appear to increase glucocorticoid-induced transcription [97].
The binding sites for these factors lie between the proximal GRE region and the
TATA-box (Fig. 1.3). Binding of NF-1 in vivo occurs only in the presence of
hormone, but there is no evidence of a direct GR·NF-1 interaction. In this case, a
GR-induced alteration in chromatin structure, resulting in loss of a nucleosome near
Fig. 1.3 Presumptive glucocorticoid binding sequences (GBSs) proximal to several glucocorticoid-
regulated genes. The mammary tumor virus (MTV), tyrosine aminotransferase (TAT), tryptophan
oxygenase (TO), proliferin, and angiotensinogen (angioten) promoter-proximal regions are illus-
trated. Glucocorticoid receptor binding sequences (GBSs) each of which binds a GR dimer in vitro,
are shown as shaded ovals or circles. Binding sequences for various nonreceptor transcriptional
regulatory factors are shown as open forms. The numbers indicate the approximate positions of the
elements in relation to the transcription initiation site (arrow)
the transcription initiation site may “open” the chromatin structure, facilitating
binding of general transcription factors, which are required for transcription initia-
tion [98, 99]. Mutation of the NF-1 binding site reduced modestly the glucocorti-
coid response, whereas mutation of the two Oct-1 binding sites located between the
NF-1 site and the TATA box (Fig. 1.3) resulted in a markedly reduced response of
the reporter gene to glucocorticoids [100]; studies suggesting cooperative DNA
binding between GR and Oct-1 on the MTV promoter imply a direct protein · pro-
tein interaction between these factors.
As more candidate GREs close to target genes have been analyzed, it became
apparent that all contain binding sequences for various non-receptor regulatory
factors, either together with or in the absence of GBSs. As illustrated in Figs. 1.3
and 1.4, this includes candidate GREs at the TAT, TO and PEPCK genes, the first
proteins known to be induced by glucocorticoids, as well as other GC-regulated
genes such as proliferin and angiotensinogen. Deletion or mutation of these accessory
DNA elements, with the associated loss of binding of the cognate transcription factor,
blunts or abolishes the response of these promoters to glucocorticoids in reporter
assays (for review see [101]). Also important were the precise sequences of the
GBSs, as were their spacing, multiplicity and location. It may be significant in this
Fig. 1.4 Promoter-proximal region of the PEPCK gene. Binding sequences for various regulatory
and general transcription factors are shown across the top line. The numbers above these represent
the approximate center of each element with respect to the transcription initiation site (demarcated
by the arrow). The five boxes below represent hormone-specific response units, and the regulatory
factors thought to be bound, for glucocorticoids (GRU), cyclic AMP (CRU), retinoic acid (RARU),
thyroid hormone (TRU) and insulin (IRU) that mediate each hormone’s regulation of PEPCK
transcription. Insofar as the CRU, RARU, TRU and IRU impact the actions of the GRU under
physiological conditions, all of these response units interact functionally, cooperating or compet-
ing, to comprise the PEPCK gene hormone response domain. Details are described in the text
regard that the receptors for glucocorticoids, mineralocorticoids, progestins and

androgens have very similar DBDs, and all can regulate transcription through an
idealized GBS, despite their very different physiologic actions in vivo (for review
see [101]; and see below).
It is now apparent that GREs (and by extension, all genomic transcriptional
response elements) are nucleation centers for the dynamic assembly and disassem-
bly of multifactor regulatory complexes containing context-specific combinations
of >100 different genome-associated regulatory and coregulatory proteins associ-
ated through protein·DNA and protein · protein interactions [102]. It is the combina-
torial assembly of these complexes, displaying both precision in a given context and
plasticity to shift readily to different assembly instructions in a different context,
that enables GCs and GR to regulate arrays of distinct gene transcription networks
with exquisite gene, cell and physiologic specificity. These three crucial contexts
are conveyed to GR by a combination of cellular signals (GR interactions with
hormone or ligand, DNA sequence, and other regulatory proteins), which are
received, interpreted and integrated as allosteric alterations in GR conformation
[86, 90]. In turn, these conformations define functional GR surfaces that serve as
“assembly instructions” for multi-component transcriptional regulatory complexes

comprised of unique combinations of regulatory factors and co-regulators [103].
The PEPCK GRE Provides Finely Tuned, Versatile Regulation

of Transcription
A key element of regulatory crosstalk is the integration of information from multiple

signaling pathways, which occurs at response elements and provides precise control
of remarkably complex biological processes involving gene networks in multiple
organs and tissues. For example, metabolic processes such as gluconeogenesis
involve interacting regulatory mechanisms that provide additive, synergistic, positive,
negative and dominant control. To illustrate this, we focus here on the integration of
multiple hormone signals in particular cultured cell contexts, at the level of gene
transcription, that govern expression of hepatic phosphoenolpyruvate carboxyki-
nase (PEPCK), which catalyzes a critical, first step in gluconeogenesis. The amount
and activity of the cytosolic form this enzyme (PEPCK-C) is determined by several
hormones through their effects on transcription of the PEPCK gene (Pck-1) [46].
Glucagon (acting through cyclic AMP), retinoic acid,1 thyroid hormone and gluco-
corticoids stimulate transcription, whereas insulin and glucose exert dominant,
inhibitory effects [46, 50].
In the contexts examined, several DNA elements with associated regulatory
factors have been implicated in the glucocorticoid response of the PEPCK gene.
A promoter-proximal segment, denoted here as a glucocorticoid response unit
(GRU), contains two adjacent GBSs (GBS1 and GBS2) located between positions
−349 to −395 relative to the transcription initiation site (Fig. 1.4). GBS1 and GBS2
can each bind GR dimers in vitro, albeit with 30-fold lower affinity than that of the
idealized GBS, probably because they resemble the latter at only 7/12 and 6/12
positions, respectively [105]. Unlike the TAT GRE, neither of the PEPCK GBSs, by
themselves, confers a glucocorticoid response in transient reporter assays [105].
Rather, glucocorticoid regulation in this context requires flanking DNA bearing
binding sequences for non-receptor regulators collectively termed glucocorticoid
accessory factor elements (gAF)2: gAF1 binds HNF-4 α and COUP-TF [106], both
members of the nuclear receptor family; gAF2 binds FoxA2 (HNF-3 β) [107,
108]; and gAF3 binds COUP-TF (Fig. 1.4) [109]. Deletion of either GBS1 or GBS2
compromises the glucocorticoid response; the effect of GBS1 is greater than that of
GBS2, but both are required for maximal activity in the contexts tested [105].
1
This effect helps explain an observation made more than 50 years ago. Wolf et al. showed that,
in vitamin A deficient rats, hepatic cholesterol and fatty acid synthesis, the citric acid cycle,
glycogen metabolism and glycolysis were all normal. Gluconeogenesis, however, was markedly
impaired [104].
2
The GC accessory factor elements in the PEPCK gene promoter were originally referred to as
AF1-3. As the designation of the transactivation domains in nuclear receptors became known as
AF1 and AF2, the DNA elements in the PEPCK gene were designated gAF1-3.
Deletion of either gAF1/gAF3 (gAF1 > gAF3) or the gAF2 element reduces the
glucocorticoid response by ~50 %; deletion of both abolishes the response.
Chromatin immunoprecipitation (ChIP) assays revealed that HNF-4 α, COUP-TF
and FoxA2 occupy the GRU in the absence of dexamethasone; as expected GR does
not [110]. Those gAF proteins increase the affinity of GR for GBS1 and GBS2, as
demonstrated by fluorescence anisotropy [111], thereby nucleating assembly of a
transcriptional regulatory complex that includes co-regulators SRC-1, CBP/p300,
PGC-1, FoxO1, FoxO3, which appear to assemble through protein · protein inter-
actions [112–114]. ChIP assays confirmed recruitment of those factors, as well as
polymerase II, to the PEPCK GRU following addition of dexamethasone [110].
Interestingly, gAF1 and gAF3 also contain binding sequences for heterodimers
of retinoic acid receptor (RAR) and retinoid X receptor (RXR), and in the absence
of dexamethasone but in the presence of retinoic acid, activate PEPCK gene
transcription (Fig. 1.4) [115]. Thus, gAF1 and gAF3, in one context GRU compo-
nents, serve in another as a retinoic acid response unit (RARU).
Similarly, the GRU/RARU, in the absence of their cognate signals, serves as a
cyclic AMP response unit (CRU) to enhance PEPCK transcription upon glucagon
stimulation (Fig. 1.4); in that context, the CRU includes some DNA elements and
corresponding regulatory factors that overlap with those in the GRU and RARU, as
well as others that are distinct [116, 117].
Finally, the dominant effect of insulin is mediated, in part, through the multifunc-
tional gAF2 element [118]; an epigenetic effect involving insulin-induced demeth-
ylation of arginine-17 on histone H3 may also be operative [110]. FoxO1 is clearly
involved in this insulin effect, as it rapidly leaves the IRU (see below) and exits the
nucleus in response to insulin [119]. The ChIP assay was used to demonstrate how
quickly this dominant inhibition happens. H4IIE cells3 were treated with dexameth-
asone and the maximally active transcription complex was allowed to assemble.
Within 3 min following the addition of insulin, p300, FoxO1and FoxO3 are removed
from the IRU and by 10 min most components of the assembly are at, or below, the
basal level, including polymerase II [110].
This complex system, comprised of overlapping but distinct composite response
units and associated regulatory factors, underscores the importance of finely tuned
homeostatic regulation of gluconeogenesis. A mutation of one DNA element or
accessory factor blunts, but does not abolish, the effect of a given stimulatory hor-
mone. Indeed, the complete loss of the response to one of these hormones (or the
absence of the hormone itself) blunts, but does not abolish, the positive regulation
of gluconeogenesis. By contrast, insulin stands alone as the hormonal inhibitor
of gluconeogenesis. Under physiological conditions, the actions of the five
hormones (and other cellular signals) are integrated in a highly context-dependent
manner, so with respect to glucocorticoid-mediated regulation, all of the response
units and their regulatory factors operate in aggregate, cooperating or competing, as
the PEPCK GRE.
3
All the experiments described in this section were performed using this cell line, which was
derived from a rat hepatoma [42].
Multiple Context Effects Determine GRE Activities

and Mechanisms
Molecular analyses showing that glucocorticoid-responsive genes are governed by

composite GREs, bolstered by the dissection of multi-hormonal regulation of
PEPCK expression, demonstrate that the remarkable specificity of GR-mediated
transcriptional regulation emerges from the integration of multiple context effects
that converge on GR in a given setting.
The consequences of this context dependence are striking: some specific GR
binding regions (GBRs; genomic segments occupied by GR in certain contexts in
vivo) are functional GREs in some cell types but not in others; a given GRE-bound
regulatory complex may activate transcription in one setting and repress in another;
a GC ligand may be a strong agonist in one cell or gene context, a weak agonist in
another, and an antagonist in a third. It is this extreme context dependence that
enables GR to orchestrate different transcription networks in every cell and tissue
type, transducing a simple molecular signal into an array of distinct physiological
outcomes (Table 1.1).
The remarkable effects of context, imposed as noted above by the integrated
actions of multiple classes of signaling inputs and mediated by allosteric transitions in
GR structure, result in the assembly of regulatory complexes with different composi-
tions, structures and mechanistic actions on transcription. The production and accu-
mulation of mRNA is itself an exceedingly complicated process, comprised of coupled
ordered reactions—initiation, elongation, splicing, cleavage, polyadenylation, termi-
nation, nuclear export, degradation—that are themselves complex and each a potential
point of positive or negative regulation. Quantitative methods for distinguishing these
processes have been devised [120, 121] but typically only mRNA accumulation,
the endpoint of all of these steps, is monitored by methods such as qPCR or RNA-seq.
To date, GR has been implicated in regulating initiation [122], elongation [96] and
stabilization [123], but other steps have not been ruled out.
Application of novel survey methods together with a better understanding of the
mechanistic effects of coregulatory factors resident in GR-containing regulatory
complexes [124, 125] will provide the full picture of the varied strategies by which
GR modulates gene transcription. In framework, it is apparent that the coregulators
can catalyze chromatin remodeling, chemically modify histones and other factors,
and recruit or occlude general transcription factors; each of these activities could
potentially alter one or more steps in mRNA production or accumulation.
The powerful and varied actions of GR on specific gene transcription, together
with the knowledge that eukaryotic transcriptional regulatory complexes, especially
in higher metazoans such as Drosophila or mammals, can operate from very long
range, raise the challenging question of the determinants of which GRE(s) will con-
fer regulation on which target gene. Chromatin immunoprecipitation (ChIP) meth-
ods for determination of the genomic sites of occupancy by GR in vivo, assessed
initially on selected genomic regions (ChIP-qPCR) and subsequently across the
entire genome (ChIP-seq) have large numbers of GR binding regions (GBRs), many
of which are specific to particular contexts, such as cell type or developmental stage.
As the sensitivity, range and discrimination of the methods have increased, the
number of reported GBRs has increased, from hundreds to thousands to tens of
thousands [126–132].
Which of these GBRs are functional GREs, and which GC-responsive target
gene is controlled by which GRE? It is thought that not every GBR is a functional
GRE, at least in the restricted contexts examined, as some can be deleted without
apparent effect, and many are located many megabases from GC-responsive genes.
Lacking unequivocal methods for assessing activity in native chromosomal environ-
ments (rather than reporters or transgenes), researchers have resorted to proximity,
“assigning” GRE activity to one or more GBRs that neighbor GC-responsive genes.
Even with this proviso, most assigned GREs are >10 kb from their presumptive
target promoters [127]. Whether GBRs that appear to lack function in one context
may function in another context is an intriguing untested possibility.
To date, only a single GBR, which resides some 25 kb downstream of the tran-
scription start site for the Per2 circadian rhythm regulatory gene, has been unequiv-
ocally assigned to its target gene [133]. This was discovered only because a deletion
constructed for another purpose fortuitously removed the GBS and the mouse that
ensued had lost GR regulation of Per2 expression [133]. Fortunately, powerful and
facile gene editing methods such as CRISPR [134, 135] now make possible targeted
changes in genome sequence that will enable GRE activities on specific target genes
to be determined with certainty.
The ability to analyze GC regulated genes across the entire genome, in all organs
and tissues, enables rather direct access to many interesting and important questions.
For example, can relationships be discerned between regulatory complex structure
and mechanism and the physiologic processes that they control, e.g., complexes
involved in GC-mediated anti-inflammation or immunosuppression. If this were the
case, it might then be possible to design small molecules that target those genes with-
out affecting the different assemblies involved in metabolism, growth and develop-
ment, and thus avoid or minimize the devastating side effects that complicate, and
limit, current glucocorticoid therapy. This might not have to be an “all or none”
phenomenon. As informed by the regulation of the PEPCK gene, where a complex
array of hormone signals, accessory proteins and co-regulators appears to allow for
a linear degree of gene expression from 0 to 100 %, subtle modifications of the
expression of one class of genes, with full expression of another, may be sufficient
to control unwanted side effects.
Epilogue
Here we have provided an historical perspective on the progression of research on

the actions of glucocorticoids, and how early attempts to treat the consequences of
adrenal insufficiency eventually evolved to become key drivers of the conceptual
and experimental understanding of metazoan transcriptional regulation. The
outstanding contributors to this volume build on our overview to describe in detail

many of the physiologic and pathophysiologic effects of these hormones, and the
molecular mechanisms involved in these processes.
Future work promises to reveal still more regulatory processes, perhaps demon-
strating roles for intranuclear position, chromosome topology, disassembly of regu-
latory complexes, or hormone transport across plasma membranes. Other studies
will illuminate the relationships between such regulatory mechanisms and the phys-
iologic outcomes they specify, relationships currently shrouded in the complexity of
combinatorial processes yet to be deeply understood. And with those advances
could come the capacity for the prediction and design of mechanisms, and of
hormone-like ligands that trigger or inhibit them, This, in turn, will lead to a more
selective, effective and safer class of therapeutics.
Acknowledgements The authors wish to thank Ms. Stacie Vik and Allison McQueen for their
assistance in preparing the manuscript and Michael Stallcup for his incisive comments and encour-
agement during its preparation. We thank the many people who, over the years, performed experi-
ments in our laboratories. All of their contributions could not be discussed, but all are a part of this
story. We also are grateful to the legion of colleagues who, over years of their own work, and in
formal and informal discussions, made our participation in this field so interesting.
References
1. Bayliss WM, Starling FH. The movements and innervation of the small intestine. J Physiol.
1901;26:125–38.
2. Bayliss WM, Starling FH. The mechanism of pancreatic secretion. J Physiol. 1902;28:
325–53.
3. Addison T. On the constitutional and local effects of disease of the suprarenal capsule. In: A
collection of the published writings of the late Thomas Addison MD. London: New Sydenham
Society; 1868.
4. Bernard C. Remarques sur le sécrétion du sucre dans la foie, faites à l’ occasion de la com-
munication de M Lehman. CR Acad Sci Paris. 1855;40:589–92.
5. Cannon WB. The wisdom of the body. New York: WW Norton; 1932.
6. Henderson J. Ernest starling and “hormones”: an historical commentary. J Endocrinol.
2005;184:5–10.
7. Starling FH. Croonian lecture: On the chemical correlation of the functions of the body.
Lancet. 1905;2:339–41.
8. Swingle WW, Pfiffner JJ. The revival of comatose adrendectomized cats with an extract of the
suprarenal cortex. Science. 1930;72:75–6.
9. Hartman FA, Brownell KA. The hormone of the adrenal cortex. Science. 1930;72:76.
10. Kendall EC. The development of cortisone as a therapeutic agent. In: Nobel lectures in
physiology or medicine: 1942–1962. Amsterdam: Elsevier; 1964. p. 270–288.
11. Reichstein T. Chemistry of the adrenal cortex hormones. In: Nobel lectures in physiology or
medicine: 1942–1962. Amsterdam: Elsevier; 1964. p. 291–308.
12. Sarett LH. A new method for the preparation of 17α-hydroxy-20-ketopregnanes. J Am Chem
Soc. 1948;70:1454–8.
13. Cushing H. The basophil adenomas of the pituitary body and their clinical manifestations
(pituitary basophilism). Bull Johns Hopkins Hosp. 1932;50(4):137–95.
14. Hench PS. The reversibility of certain rheumatic and non-rheumatic conditions by the use of
the cortisone or of the pituitary adrenocorticotropic hormone. In: Nobel lectures in physiology
or medicine 1942–1962. Amsterdam: Elsevier; 1964. p. 311–43.
15. de Réaumur RAF. Observations sur la digestion des oiseaux. Hist Acad Roy Sci. 1752;266:461.
16. Payen A, Persoz J-F. Memoire sur la diastase, les principaux produits de ses réactions, et leurs
applications aux arts industriels. Ann Chim Phys. 1833;53:73–92.
17. Kühne W. On the behavior of various organized and so-called unformed ferments. Verb. d.
Nat. Med. Ver. Heidelberg. 1877;1:190–8.
18. Buchner E. Cell-free fermentation. In: Nobel lectures in chemistry 1901–1921. Amsterdam:
Elsevier; 1966. p. 103–20.
19. Sumner JB. The chemical nature of enzymes. In: Nobel lectures in chemistry 1901–1921.
Amsterdam: Elsevier; 1964. p. 114–21.
20. Northrop JH. The chemical nature of enzymes. In: Nobel lectures in chemistry 1942–1962.
21. Stanley WM. The chemical nature of enzymes. In: Nobel lectures in chemistry 1942–1962.
22. Levine R. Insulin action: 1948–80. Diabetes Care. 1981;4(1):38–44.
23. Schoenheimer R. The dynamic state of body constituents. Cambridge: Harvard University
Press; 1942.
24. Shemin D, Rittenberg D. Life span of human red blood cell. J Biol Chem. 1946;
166:627–36.
25. Ballou JE, Thompson RC. Studies of metabolic turnover with tritium as a tracer: V. The
predominantly non-dynamic state of body constituents in the rat. J Biol Chem. 1956;
223:795–809.
26. Dubos RJ. The adaptive production of enzymes by bacteria. Bacteriol Rev. 1940;4(1):1–16.
27. Monod J. The phenomenon of enzymatic adaptation and its bearings on problems of genetics
and cellular differentiation. Growth. 1947;11:223–89.
28. Spiegelman S. Nuclear and cytoplasmic factors controlling enzymatic constitution. Cold
Spring Harbor Symp Quant Biol. 1946;11:256–77.
29. Knox WE, Mehler AH. Adaptive increase of tryptophan peroxidase-oxidase system of liver.
Science. 1951;113:237–8.
30. Knox WE. Two mechanisms which increase in vivo liver tryptophan peroxidase activity:
specific enzyme adaptation and stimulation of the pituitary-adrenal system. Br J Exp Pathol.
1951;32(5):462–9.
31. Selye H. The general adaptation syndrome and the diseases of adaptation. J Clin Endocrinol
Metab. 1946;6:117–230.
32. Thompson JF, Mikuta ET. Effect of total body x-irradiation on tryptophan peroxidase activity
of rat liver. Proc Soc Exp Biol Med. 1954;85(1):29–32.
33. Knox WE, Auerbach VH, Lin ECC. Enzymatic and metabolic adaptation in animals. Physiol
Rev. 1956;36:164–254.
34. Lin ECC, Knox WE. Adaption of the rat liver-tyrosine-alpha-ketoglutarate transaminase.
Biochim Biophys Acta. 1957;26:85–8.
35. Kenney FT. Induction of tyrosine-α-ketoglutarate transaminase in rat liver. II: enzyme purifi-
cation and preparation of antitransaminase. J Biol Chem. 1962;237(5):1605–9.
36. Feigelson P, Greengard O. Immunochemical evidence for increased titers of liver tryptophan
pyrrolase during substrate and hormonal enzyme induction. J Biol Chem. 1962;237:3714–7.
37. Schimke RT. Control of enzyme levels in mammalian issues. Adv Enzymol Relat Areas Mol
Biol. 1973;37:135–87.
38. Schimke RT, Sweeney EW, Berlin CM. The roles of synthesis and degradation in the control
of rat liver tryptophan pyrrolase. J Biol Chem. 1965;240:322–31.
39. Kenney FT. Induction of tyrosine-α-ketoglutarate transaminase in rat liver: IV. Evidence for
an increase in the rate of enzyme synthesis. J Biol Chem. 1962;237:3495–8.
40. Shrago E, Lardy HA, Nordlie RC, Foster DO. Metabolic and hormonal control of phospho-
enolpyruvate carboxykinase and malic enzyme in rat liver. J Biol Chem. 1963;238:3188–92.
41. Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of protein. J Mol Biol.
1961;3:318–56.
42. Pitot H, Peraino C, Morse P, Potter V. Hepatomas in tissue culture compared with adapting
liver in vivo. Natl Cancer Inst Monogr. 1964;13:229–45.
43. Thompson EB, Tomkins GM, Curran JF. Induction of tyrosine alpha-ketoglutarate transami-
nase by steroid hormones in a newly established tissue culture cell line. Proc Natl Acad Sci
U S A. 1966;56:296–303.
44. Granner DK, Hayashi S-I, Thompson EB, Tomkins GM. Stimulation of tyrosine amino-
transferase synthesis by dexamethasone phosphate in cell culture. J Mol Biol. 1968;
35:291–301.
45. Granner DK, Beale EG. Regulation of the synthesis of tyrosine aminotransferase and phos-
phoenolpyruvate carboxykinase by glucocorticoid hormones. In: Biochemical actions of hor-
mones, vol. 12. New York: Academic; 1985. p. 89–138.
46. Sasaki K, Cripe T, Koch S, et al. Multihormonal regulation of PEPCK gene transcription: the
dominant role of insulin. J Biol Chem. 1984;259:15242–51.
47. Greengard O, Acs G. The effect of actinomycin on the substrate and hormonal induction of
liver enzymes. Biochim Biophys Acta. 1962;61:652–3.
48. Danesch U, Hashimoto S, Renkawitz R, Schütz G. Transcriptional regulation of the trypto-
phan oxygenase gene in rat liver by glucocorticoids. J Biol Chem. 1983;258(8):4750–3.
49. Schmid E, Schmid W, Jantzen M, Mayer D, Jastorff B, Schütz G. Transcription activation of
the tyrosine aminotransferase gene by glucocorticoids and cAMP in primary hepatocytes.
Eur J Biochem. 1987;165(3):499–506.
50. Lucas PC, O’Brien RM, Mitchell JA, et al. A retinoic acid response element is part of a pleio-
tropic domain in the phosphoenolpyruvate carboxykinase gene. Proc Natl Acad Sci U S A.
1991;88:2184–8.
51. Talwar GP, Segal SJ, Evans A, Davison OW. The binding of estradiol in the uterus: a mecha-
nism for depression of RNA synthesis. Proc Natl Acad Sci U S A. 1964;52:1059–66.
52. Rousseau GG, Baxter JD. Glucocorticoid receptors. In: Glucocorticoid hormone action.
Heidelberg: Springer; 1979. p. 49–77.
53. Baxter JD. Glucocorticoid hormone action. Pharmacol Ther B. 1976;2(3):605–59.
54. Wrange O, Carlstedt-Duke J, Gustafsson JǺ. Purification of the glucocorticoid receptor from
rat liver cytosol. J Biol Chem. 1979;254:9284–90.
55. Wrange O, Okret S, Radojćić M, Carlstedt-Duke J, Gustafsson J. Characterization of the
purified activated glucocorticoid receptor from rat liver cytosol. J Biol Chem. 1984;
259:4534–41.
56. Hollenberg SM, Moldenhauer C, Ong ES, et al. Primary structure and expression of a func-
tional human glucocorticoid receptor cDNA. Nature. 1985;318:635–41.
57. Miesfeld R, Rusconi S, Godowski PJ, et al. Genetic complementation of a glucocorticoid
receptor deficiency by expression of cloned receptor cDNA. Cell. 1986;46(3):389–99.
58. Danielson M, Northrop JP, Ringold GM. The mouse glucocorticoid receptor: mapping of
functional domains by cloning, sequencing and expression of wild-type and mutant receptor
proteins. EMBO J. 1986;5(10):2513–22.
59. Yamamoto KR, Gehring U, Stampfer MR, Sibley CH. Genetic approaches to steroid hormone
action. Recent Prog Horm Res. 1976;32:3–32.
60. Yamamoto KR. Steriod hormone receptors: interaction with deoxyribonucleic acid and
transcription factors. Annu Rev Genet. 1985;19:209–52.
61. Ringold GM, Yamamoto KR, Tomkins GM, Bishop M, Varmus HE. Dexamethasone-
mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorti-
coid action. Cell. 1975;6(3):299–305.
62. Ringold GM, Yamamoto KR, Bishop JM, Varmus HE. Glucocorticoid-stimulated accumulation
of mouse mammary tumor virus RNA: increased rate of synthesis of viral RNA. Proc Natl
Acad Sci U S A. 1977;74(7):2879–83.
63. Ucker DS, Ross SR, Yamamoto KR. Mammary tumor virus DNA contains sequences
required for its hormone-regulated transcription. Cell. 1981;27:257–66.
64. Huang AL, Ostrowski MC, Berard D, Hager GL. Glucocorticoid regulation of the Ha-Mu SV
p21 gene conferred by sequences from mouse mammary tumor virus. Cell. 1981;27:245–55.
65. Yamamoto KR, Chandler VL, Ross SR, Ucker DS, Ring JC, Feinstein SC. Integration and
activity of mammary tumor virus genes: regulation by hormone receptors and chromosomal
position. Cold Spring Harb Symp Quant Biol. 1981;45:687–97.
66. Groner B, Kennedy N, Rahmsdorf U, Herrlich P, Van Ooyen A, Hynes NE. Introduction of a
proviral MMTV gene and a chimeric MMTV-tk gene into L cells results in their glucorticoid
responsive expression. In: Hormones and cell regulation. Amsterdam: Elsevier Biomedical
Press; 1982. p. 217–28.
67. Payvar R, Wrange O, Carlstedt-Duke J, Okret S, Gustafsson JǺ, Yamamoto KR. Purified
glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment whose transcrip-
tion is regulated by glucocorticoids in vivo. Proc Natl Acad Sci U S A. 1981;78:6628–32.
68. Payvar F, Firestone GL, Ross S, et al. Multiple specific binding sites for purified glucocorticoid
receptors on mammary tumor virus DNA. J Cell Biochem. 1982;19:241–7.
69. Govindan MV, Spiess E, Majors J. Purified glucocorticoid receptor-hormone complex from
rat liver cytosol binds specifically to cloned mouse mammary tumor virus long terminal
repeats in vitro. Proc Natl Acad Sci U S A. 1982;79(17):5157–61.
70. Geisse S, Scheidereit C, Westphal HM, Hynes NE, Groner B, Beato M. Glucocorticoid
receptors recognize DNA sequences in and around murine mammary tumour virus DNA.
EMBO J. 1982;1(12):1613–9.
71. Pfahl M. Specific binding of the glucocorticoid-receptor complex to the mouse mammary
tumor proviral promoter region. Cell. 1982;31:475–82.
72. Chandler VL, Maler BA, Yamamoto KR. DNA sequences bound specifically by glucocorticoid
receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell.
1983;33(2):489–99.
73. Yamamoto KR. Transcriptional enhancement by specific regulatory protein: DNA com-
plexes. In: Ginsberg HS, Vogel HJ, editors. Transfer and expression of eukaryotic genes.
New York: Academic; 1983. p. 79–92.
74. Miesfeld R, Okret S, Wikstrom AC, Wrange O, Gustafsson JA, Yamamoto KR. Characterization
of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature.
1984;312(5996):779–81.
75. Carlstedt-Duke J, Okret S, Wrange O, Gustafsson JǺ. Immunochemical analysis of the glu-
cocorticoid receptor: identification of a third domain separate from the steroid-binding and
DNA-binding domains. Proc Natl Acad Sci U S A. 1982;79:4260–4.
76. Westphal HM, Moldenhauer G, Beato M. Monoclonal antibodies to the rat liver glucocorti-
coid receptor. EMBO J. 1982;1:1467–71.
77. Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988;240:889–95.
78. Ismaili N, Garabedian MJ. Modulation of glucocorticoid receptor function via phosphorylation.
Ann N Y Acad Sci. 2004;1024:86–101.
79. Hollenberg SM, Evans RM. Multiple and cooperative trans-activation domains of the human
glucocorticoid receptor. Cell. 1988;55:899–906.
80. Galliher-Beckley AJ, Cidlowski JA. Emerging roles of glucocorticoid receptor phosphoryla-
tion in modulating glucocorticoid hormone action in health and disease. IUBMB Life.
2009;61(10):979–86.
81. Carson-Jurica MA, Schrader WT, O’Malley BW. Steroid receptor family: structure and functions.
Endocr Rev. 1990;11(2):201–20.
82. Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler PB. Crystallographic
analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991;
352:497–505.
83. Mader S, Kumar V, de Verneuil H, Chambon P. Three amino acids of the oestrogen receptor
are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive
element. Nature. 1989;339:271–4.
84. Green S, Chambon P. Oestradiol induction of a glucocorticoid-responsive gene by a chimae-
ric receptor. Nature. 1987;325(6099):75–8.
85. Godowski PJ, Picard D, Yamamoto KR. Signal transduction and transcriptional regulation by
glucocorticoid receptor-LexA fusion proteins. Science. 1988;241(4867):812–6.
86. Watson LC, Kuchenbecker KM, Schiller BJ, Gross JD, Pufall MA, Yamamoto KR. The glu-
cocorticoid receptor dimer interface allosterically transmits sequence-specific DNA signals.
Nat Struct Mol Biol. 2013;20(7):876–83.
87. Danielson M. Structure and function of the glucocorticoid receptor. In: Parker MG, editor.
Nuclear hormone receptors: molecular mechanisms, cellular functions, clinical abnormali-
ties. London: Academic; 1991. p. 39–78.
88. Picard D, Salser SJ, Yamamoto KR. A movable and regulable inactivation function within the
steroid binding domain of the glucocorticoid receptor. Cell. 1988;54(7):1073–80.
89. LaBaer J, Yamamoto KR. Analysis of the DNA-binding affinity, sequence specificity and
context dependence of the glucocorticoid receptor zinc finger region. J Mol Biol.
1994;239:664–88.
90. Meijsing SH, Pufall MA, So AY, Bates DL, Chen L, Yamamoto KR. DNA binding site
sequence directs glucocorticoid receptor structure and activity. Science. 2009;
324(5925):407–10.
91. Miner JN, Yamamoto KR. Regulatory crosstalk at composite response elements. Trends
Biochem Sci. 1991;16:423–6.
92. So AY, Cooper SB, Feldman BJ, Manuchehri M, Yamamoto KR. Conservation analysis pre-
dicts in vivo occupancy of glucocorticoid receptor-binding sequences at glucocorticoid-
induced genes. Proc Natl Acad Sci U S A. 2008;105(15):5745–9.
93. Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR. Transcription factor interactions:
selectors of positive or negative regulation from a single DNA element. Science.
1990;249(4974):1266–72.
94. Jonat C, Rahmsdorf HJ, Park KK, et al. Antitumor promotion and antiinflammation: down-
modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell. 1990;62(6):
1189–204.
95. Miner JN, Diamond MI, Yamamoto KR. Joints in the regulatory lattice: composite regulation
by steroid receptor-AP1 complexes. Cell Growth Differ. 1991;2(10):525–30.
96. Luecke HF, Yamamoto KR. The glucocorticoid receptor blocks P-TEFb recruitment by
NFkappaB to effect promoter-specific transcriptional repression. Genes Dev. 2005;
19(9):1116–27.
97. Truss M, Beato M. Steroid hormone receptors: interaction with deoxyribonucleic acid and
transcription factors. Endocr Rev. 1993;14(4):459–79.
98. Zaret KS, Yamamoto KR. Reversible and persistent changes in chromatin structure accom-
pany activation of a glucocorticoid-dependent enhancer element. Cell. 1984;38(1):29–38.
99. Richard-Foy H, Hager GL. Sequence-specific positioning of nucleosomes over the steroid-
inducible MMTV promoter. EMBO J. 1987;6(8):2321–8.
100. Brüggemeier U, Kalff M, Franke S, Scheidereit C, Beato M. Ubiquitous transcription factor
OTF-1 mediates induction of the MMTV promoter through synergistic interaction with hor-
mone receptors. Cell. 1991;64(3):565–72.
101. Lucas PC, Granner DK. Hormone response domains in gene transcription. Annu Rev
Biochem. 1992;61:1131–73.
102. Yamamoto KR, Darimont BD, Wagner RL, Iniguez-Liuhi JA. Building transcriptional
regulatory complexes: signals and surfaces. Cold Spring Harb Symp Quant Biol. 1998;63:
587–98.
103. Chodankar R, Wu DY, Schiller BJ, Yamamoto KR, Stallcup MR. Hic-5 is a transcription
coregulator that acts before and/or after glucocorticoid receptor genome occupancy in a gene-
selective manner. Proc Natl Acad Sci U S A. 2014;111(11):4007–12.
104. Wolf G, Lane MD, Johnson BC. Studies on the function of vitamin A in metabolism. J Biol
Chem. 1957;225:995–1008.
105. Imai E, Stromstedt PE, Quinn PG, Carlstedt-Duke J, Gustafsson JǺ, Granner DK.
Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate
carboxykinase gene. Mol Cell Biol. 1990;10(9):4712–9.
106. Hall RK, Sladek FM, Granner DK. The orphan receptors COUP-TF and HNF-4 serve as
accessory factors required for induction of phosphoenolpyruvate carboxykinase gene tran-
scription by glucocorticoids. Proc Natl Acad Sci U S A. 1995;92(2):412–6.
107. Wang JC, Stromstedt PE, O’Brien PM, Granner DK. Hepatic nuclear factor 3 is an accessory
factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription
by glucocorticoids. Mol Endocrinol. 1996;10:794–800.
108. Wang JC, Stafford JM, Scott DK, Sutherland C, Granner DK. The molecular physiology of
hepatic nuclear factor 3 (HNF3) in the regulation of gluconeogenesis. J Biol Chem. 2000;
275:14717–21.
109. Scott DK, Mitchell JA, Granner DK. The orphan receptor COUP-TF binds to a third gluco-
corticoid accessory factor element within the phosphoenolpyruvate carboxykinase gene
promoter. J Biol Chem. 1996;271:31909–14.
110. Hall RK, Wang XL, George L, Koch SR, Granner DK. Insulin represses phosphoenolpyruvate
carboxykinase gene transcription by causing the rapid disruption of an active transcription
complex: a potential epigenetic effect. Mol Endocrinol. 2007;21(2):550–63.
111. Stafford JM, Wilkinson JC, Beechem JM, Granner DK. Accessory factors facilitate the
binding of glucocorticoid receptor to the PEPCK gene promoter. J Biol Chem. 2001;
276(43):39885–91.
112. Wang JC, Stafford JM, Granner DK. SRC-1 and GRIP1 coactivate transcription with hepato-
cyte nuclear factor 4. J Biol Chem. 1998;273:30847–50.
113. Wang JC, Stromstedt PE, Sugiyama T, Granner DK. The phosphoenolpyruvate carboxyki-
nase gene glucocorticoid response unit: identification of the functional domains of accessory
factors HNF3β and HNF4 and the necessity of proper alignment of their cognate binding
sites. Mol Endocrinol. 1999;13:604–18.
114. Yoon JC, Puigserver P, Chen G, et al. Control of hepatic gluconeogenesis through the tran-
scriptional coactivator PGC-1. Nature. 2001;413:131–8.
115. Lucas PC, Forman BM, Samuels HH, Granner DK. Specificity of a retinoic acid response
element in the phosphoenolpyruvate carboxykinase gene promotor: consequences of both
retinoic acid and thyroid hormone receptor binding. Mol Cell Biol. 1991;11(10):5164–70.
116. Short JM, Wynshaw-Boris A, Short HP, Hanson RW. Characterization of the phosphoenol-
pyruvate carboxykinase (GTP) promoter-regulatory region. J Biol Chem. 1986;261:9721–6.
117. Bokar JA, Roesler WJ, Vandenbark GR, Kaetzel DM, Hanson RW, Nilson JH. Characterization
of the cAMP responsive elements from the genes for the alpha-subunit of glycoprotein
hormones and phosphoenolpyruvate carboxykinase (GTP). Conserved features of nuclear
protein binding between tissues and species. J Biol Chem. 1988;263(36):19740–7.
118. O’Brien RM, Bonovich MT, Forest CD, Granner DK. Signal transduction convergence: phor-
bol esters and insulin inhibit phosphoenolpyruvate carboxykinase gene transcription through
the same 10-base-pair sequence. Proc Natl Acad Sci U S A. 1991;88(15):6580–4.
119. Matsumoto M, Pocai A, Rossetti L, DePinho RA, Accili D. Impaired regulation of hepatic
glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell
Metab. 2007;6:208–16.
120. Churchman LS, Weissman JS. Nascent transcript sequencing visualizes transcription at
nucleotide resolution. Nature. 2011;469(7330):368–73.
121. Kwak H, Fuda NJ, Core LJ, Lis JT. Precise maps of RNA polymerase reveal how promoters
direct initiation and pausing. Science. 2013;339(6122):950–3.
122. Ucker DS, Yamamoto KR. Early events in the stimulation of mammary tumor virus RNA
synthesis by glucocorticoids. Novel assays of transcription rates. J Biol Chem. 1984;
259(12):7416–20.
123. Peterson DD, Koch SR, Granner DK. The 3′ noncoding region of phosphoenolpyruvate
carboxykinase mRNA contains a glucocorticoid-responsive mRNA-stabilizing element. Proc
Natl Acad Sci U S A. 1989;86:7800–4.
124. Bittencourt D, Wu D-Y, Jeong KW, et al. G9a functions as a molecular scaffold for assembly
of transcriptional coactivators on a subset of glucocorticoid receptor target genes. Proc Natl
Acad Sci U S A. 2012;109(48):19673–8.
125. Engel KB, Yamamoto KR. The glucocorticoid receptor and the coregulator Brm selectively
modulate each other’s occupancy and activity in a gene-specific manner. Mol Cell Biol.
2011;31(16):3267–76.
126. Yu C-Y, Mayba O, Lee JV, et al. Genome-wide analysis of glucocorticoid receptor binding
regions in adipocytes reveal gene network involved in triglyceride homeostasis. PLoS One.
2010;5:1–13.
127. So AY-L, Chaivorapol C, Bolton E, et al. Determinants of cell-and gene-specific transcrip-
tional regulation by the glucocorticold receptor. PLoS Genet. 2007;94:1–16.
128. Kuo T, Lew M, Mayba O, et al. Genome-wide analysis of glucocorticoid receptor-binding
sites in myotubes identifies gene networks modulating insulin signaling. Proc Natl Acad Sci
U S A. 2012;109(28):11160–5.
129. Grontved L, John S, Baek S, Liu Y, et al. C/EBP maintains chromatin accessibility in liver
and facilitates glucocorticoid receptor recruitment to steroid response elements. EMBO
J. 2013;32(11):1568–83.
130. John S, Sabo PJ, Thurman RE, et al. Chromatin accessibility pre-determines glucocorticoid
receptor binding patterns. Nat Genet. 2011;43(3):264–8.
131. Polman JA, Welten JE, Bosch DS, et al. A genome-wide signature of glucocorticoid receptor
binding in neuronal PC12 cells. BMC Neurosci. 2012;13:118.
132. Schiller BJ, Chodankar R, Watson LC, Stallcup MR, Yamamoto KR. Glucocorticoid receptor
binds half sites as a monomer and regulates specific target genes. Genome Biol. 2014;15(7):418.
133. So AY, Bernal TU, Pillsbury ML, Yamamoto KR, Feldman BJ. Glucocorticoid regulation of
the circadian clock modulates glucose homeostasis. Proc Natl Acad Sci U S A. 2009;
106(41):17582–7.
134. Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA. Sequence- and structure-specific
RNA processing by a CRISPR endonuclease. Science. 2010;329(5997):1355–8.
135. Hochstrasser ML, Doudna JA. Cutting it close: CRISPR-associated endoribonuclease structure
and function. Trends Biochem Sci. 2015;40(1):58–66.
Chapter 2
Molecular Biology of Glucocorticoid Signaling
Margarita Arango-Lievano, W. Marcus Lambert, and Freddy Jeanneteau
Abstract Well-defined as signaling hormones for the programming of cell type-

specific and context-dependent gene expression signatures, glucocorticoids control
experience-driven allostasis. One unifying model is that glucocorticoids help main-
taining the integrity and plasticity of cellular networks in changing environments
through the mobilization of cellular energy stores, profiling of gene expression, and
changes in the electrical and morphological properties of cells. The nucleus is their
primary site of action, yet recent discoveries point to additional gene transcription-
independent functions at the plasma membrane of neuronal synapses. Glucocorticoids
are secreted factors that reflect intrinsically the changes coming from the external
world, temporally and regionally, during development and adulthood. In this review,
we will enumerate the properties and signaling attributes of glucocorticoids and
their receptors that characterize them as allostatic modulators. The molecular mech-
anisms used to support their role at the synapse will be highlighted.
Keywords Allostasis • Transcription • MAPK • Phosphorylation • Context-

dependent signaling
Introduction
Glucocorticoid hormones are chemical messengers that signal via alterations of

gene expression in cells expressing cognate receptors. Nuclear receptors are an evo-
lutionarily conserved class of transcription factors that regulate gene expression in
a cell type-specific and context-dependent manner. Importantly, ligand-activated
M. Arango-Lievano, Ph.D. • F. Jeanneteau, Ph.D. (*)

Inserm U1191, CNRS UMR5203, Institute for Functional Genomics,
141 rue de la Cardonille, Montpellier Cedex 05 34094, France
e-mail: [email protected]; [email protected]
W.M. Lambert, Ph.D.
Department of Microbiology, New York University School of Medicine,
550 First Avenue, New York, NY 10016, USA

34 M. Arango-Lievano et al.
nuclear receptors are able to integrate multiple signals giving rise to distinct patterns of
gene expression, and rapid non-genomic signaling outcomes yet to be discovered.
Converging signaling pathways act in part by altering receptor phosphorylation that
fosters the recruitment of interacting co-regulatory molecules in the mitochondria,
cytoplasm, synapse and nucleus. This chapter explores how glucocorticoid-mediated
allostasis employs rapid non-genomic and slow genomic mechanisms together with
ongoing neuronal activity, thus providing a molecular framework for understanding
normal and pathological glucocorticoid functions. In particular, the mechanisms
underlying the slow appearance of glucocorticoid resistance in numerous human
disease-states are not understood although several molecular correlates have been
identified. One hypothesis that shows great promise is that the survival/ growth
MAPK pathway modulates glucocorticoid-mediated allostasis, in part, through
the phosphorylation of the glucocorticoid receptors. This chapter synthesizes our
current understanding of glucocorticoid signaling with an emphasis on cellular
networks of the brain.
Ligands
Sources
Glucocorticoids are cholesterol-derived steroid hormones that prepare the organism

to adapt to changing environments during development and adulthood. Metabolism,
immunity, cognition, circadian learning and allostatic response (defined as the physi-
ological processes engaged to return to homeostasis [1]) to physical or psychological
threats are well-characterized physiological responses that engage glucocorticoid
hormone signaling. Cortisol, the major endogenous glucocorticoid in humans (corti-
costerone in rodents), is secreted by the adrenal cortex where biosynthetic enzymes
are produced [2]. The classical view is that cortisol reaching the brain comes from
the adrenal gland. Yet, the expression of some of these enzymes like 11βhydroxysteroid
dehydrogenase (11βHSD1) exists in other tissues like the brain, which supports the
possibility of local production sites for glucocorticoids and related hormones called
neurosteroids [3]. Therefore, adrenal glands are the major source of glucocorticoids
but the presence of biosynthetic or degradation enzymes modulate the availability of
these hormones at target organs. Future investigations shall clarify the impact of the
presence of 11βHSD1 in the brain. Interestingly, it is the brain that processes sensory
information and activates the hypothalamic-pituitary-adrenal (HPA) axis that trig-
gers the secretion of cortisol. Hypothalamic paraventricular neurons secrete corti-
cotropin releasing hormone (CRH) and arginine-vasopressin (AVP) into the portal
vessels to reach the anterior lobe of the pituitary gland and stimulate adrenocorti-
cotropic hormone (ACTH) secretion. Circulating ACTH binds to cognate receptor
in the cortex of the adrenal gland, which stimulates the (i) biosynthesis and
(ii) release of cortisol in the bloodstream. Through the vasculature, crossing the
blood–brain-barrier, cortisol can readily access every organ to prepare a coordinated
cellular allostatic response.
2 Molecular Biology of Glucocorticoid Signaling 35
Fig. 2.1 Typical superimposed ultradian (fine line) and circadian (thick line) circulating corticos-
terone plasma level. Due to a very high affinity for corticosterone, MR are constantly activated
contrary to GR only activated at secretion peaks (adapted from [6])
Thanks to robust anti-inflammatory and immunosuppressive effects, glucocorticoids

are a mainstay of treatment for numerous inflammatory and immune diseases, and
against trauma despite a wide range of side effects in multiple organs (brain, bone,
liver, lung, eye, muscle). Side effects of glucocorticoid therapy that involve the brain
include manifestations of emotional liability, psychosis, gain or loss of appetite,
insomnia, and memory impairments.
Secretion Modes
Pulsatile
Glucocorticoids are secreted in synchrony with circadian rhythms and in response

to stress. At rest, the HPA axis displays a circadian pattern of activity, which pro-
duces a cortisol secretion peak during the active period of the day and a trough dur-
ing the inactive phase (Fig. 2.1). A recent study demonstrates that glucocorticoid
peaks and troughs are critical determinants of circadian learning [4]. That is, mice
learning at glucocorticoid peaks (evening) acquire a learned motor coordination
task better than mice trained at glucocorticoid trough (morning). If learning is sensi-
tive to glucocorticoid circadian rhythms, recall of the learned motor task is not
sensitive to glucocorticoid peaks and troughs. This study illustrates how naturally
occurring glucocorticoid oscillations impacts distinct phases required for behav-
ioral adaptation to novelty. Circadian activation of the HPA axis is controlled by the
suprachiasmatic nucleus, which functions as a light–dark oscillator with direct out-
put projections to the hypothalamic PVN. Thus, neurons of the suprachiasmatic
nucleus order CRH-producing neurons to activate the HPA axis, at the time period
that precedes the awakening and active phase [5–7].
Superimposed to the circadian rhythms are the ultradian oscillations. The fre-
quency of ultradian pulsatory release of glucocorticoid is much more rapid than that
of circadian rhythms. An ultradian oscillation usually resolves within a 2 h period

[8–10]. The coincidence of ultradian and circadian peaks and troughs produces the
highest amplitudes of glucocorticoid oscillations [6] (Fig. 2.1). Counter-intuitively,
glucocorticoid secretion at ultradian trough during the circadian peak may be lower
than that resulting from ultradian peak at circadian trough. One question that arises
from this notion is whether learning at the ascending ultradian peak is more effec-
tive than learning at the ultradian trough. According to proposed mathematical mod-
els, this ultradian oscillation is the consequence of the slightly delayed feedforward
control of pituitary ACTH on glucocorticoid release and the feedback control of
adrenal glucocorticoids on ACTH release that depends on an underlying CRH drive
but does not necessitate CRH pulsatility [11]. This model contrast with the best
described endocrine system controlled by ultradian oscillations in which the oscil-
lating secretion of gonadotropin-releasing hormone from the hypothalamus directly
controls pulses of LH and FSH in the pituitary [12, 13]. Disruption of circadian
glucocorticoid rhythms in animal models is sufficient to impair learning and mem-
ory and to produce symptoms of depression [4, 14]. Consistently, perturbation of
glucocorticoid oscillations have been associated with numerous diseases of the cen-
tral nervous system [6].
In Response to Stress
Circulating levels of glucocorticoids not only vary as a result of intrinsic rhythms,

they are also secreted in response to stress. Polysynaptic limbic circuits converge on
the hypothalamic paraventricular nucleus (PVN) to activate CRH-producing neurons
[15]. These input circuits involve multiple brain regions including the hippocampus,
prefrontal cortex, amygdala, bed nucleus of the stria terminalis (BNST), and locus
coeruleus (LC). The intricacy of these circuits allow for a diversity of stressor spe-
cific responses like physical stress or emotionally arousing experiences, leading to
the activation of the HPA axis. Deactivation of the HPA axis is critical to prevent
escalation of glucocorticoid levels beyond physiological range. A remarkable feature
of this pathway is the ability of glucocorticoid to exert feedback inhibition on CRH,
AVP, ACTH and glucocorticoid levels, resulting in the deactivation of the HPA axis.
The dexamethasone-suppression test allows for the determination of the strength
of the negative feedback in humans and animal models. About 50 % of depressed
individuals in multiple cohorts present with diminished negative feedback [16]. Such
signs of glucocorticoid resistance gave rise to the glucocorticoid hypothesis of
depression [17]. Therefore, maintenance of the HPA axis involves homeostatic
equilibrium between the activation pathway and inhibitory feedback.
Availability
Bioavailability of glucocorticoids to target tissues is not only regulated by its secretion

patterns but also by carrier proteins in the blood. Corticosteroid binding globulin
(CBG) a high affinity plasma protein and albumin bind up to 95 % of plasma
glucocorticoids. Only the remaining unbound fraction is free to diffuse across

plasma membranes and bind receptors, be degraded by the liver or processed by
converting enzymes [18]. CBG is saturated at glucocorticoid concentrations corre-
sponding to the peak concentration of each pulse (400–500 nmol cortisol for
humans), permitting an increase of free cortisol only at these time points [6, 19].
CBG-glucocorticoid coupling regulation, allows for spatial and temporal modula-
tion of glucocorticoid availability. Minute temperature rise leads to a reversible
decrease in CBG affinity for glucocorticoids, permitting in case of fever or inflam-
mation an increment of glucocorticoid availability [19]. Inflammation sites are rich in
proteinases, like elastase that cleaves CBG such that steroid binding is irreversibly
lost [20, 21]. Glucocorticoid availability in target tissues is also regulated by locally
expressed 11βHSD enzymes (see Chap. 16 for more discussion on this topic). These
exist in two isoforms that convert corticosteroid precursors into cortisol and vice-
versa [22]. Type 2 (11βHSD-2) catalyzes inactivation by converting cortisol in inac-
tive cortisone. Type 1 (11βHSD-1) catalyzes activation by converting inactive
keto-forms like cortisone in active cortisol, locally within the cells. 11βHSD-1 is
abundant in the liver, adipose tissue and brain, where it plays a role in ageing related
cognitive decline, as demonstrated by stable learning ability of 11βHSD-1 KO mice
[23, 24]. Finally, to access target tissues like the brain, glucocorticoids need to cross
the blood–brain barrier, a specialized layer of endothelial cells. Despite their lipo-
philic nature permitting diffusion through plasma membranes, transporters pump
glucocorticoids out of the intracellular space. Among these transporters, multidrug
resistance-p-glycoproteins 1a and b (Mdr1) are expressed by endothelial cells of the
blood–brain barrier and cortical and hippocampal neurons [25]. Interestingly, Mdr1a
expression in neurons is upregulated following seizures [25, 26].
Receptors
Early postnatal development is characterized by a glucocorticoid hypo-responsive

period during which the organism is highly vulnerable to extreme environmental
changes. Extensive research using early life stress paradigms indicates that gluco-
corticoid signaling is not fully mature during embryogenesis and the first weeks of
life. In consequence, pups are unable to cope with extreme stress and easily develop
defects that persist during adulthood. For example, early life stress (or glucocorti-
coid treatment during the same period) impairs neuronal growth and differentiation
that could result in neural circuit wiring defects responsible for the development of
cognitive and neuropsychiatric illnesses during adulthood [27]. It is believed that such
critical period of glucocorticoid responsiveness depends on receptor expression
patterning that is temporally and spatially regulated.
To date, glucocorticoids have been shown to signal trough two nuclear receptors,
the mineralocorticoid receptor (MR), and the glucocorticoid receptor (GR), denom-
inations that correspond to their peripheral activity, as MR regulates electrolyte
balance, and GR is involved in gluconeogenesis. Both of these receptors belong to
the nuclear hormone receptor family. Nuclear hormone receptors are comprised of
Fig. 2.2 A schematic diagram of the functional domains and best characterized phosphorylation
sites of human GR. Sites in red are BDNF-dependent sites. NTD amino terminal domain, AF-1
activation function-1, DBD DNA-binding domain, HR hinge region, LBD ligand-binding domain,
AF-2 activation function-2, S serine, and P proline
multiple, independent functional domains [28]. These include (i) a variable amino-
terminal domain (NTD or A/B region), containing an activation function (AF)-1
with ligand-independent transactivation activity; (ii) a conserved DNA binding
domain (DBD or C region) involved in the recognition of specific DNA ligand
sequences; (iii) a variable hinge region (D) connecting the DBD to the ligand bind-
ing domain (LBD); (iv) and a conserved LBD (E) containing an additional region
for transactivation activity named AF-2. Some receptors contain an additional
highly variable carboxyl-terminal region of unknown function (Fig. 2.2). Of these
functional domains, the NTD is the most variable among nuclear receptors in terms
of length and sequence similarities and is the major target for ligand-dependent
phosphorylation at multiple serine residues [29, 30].
The Cloned Receptor Subtypes
Mineralocorticoid Receptor
MR is encoded by the NR3C2 gene. The human gene is composed of ten exons and
eight introns. The first two exons, 1β and 1α undergo alternative splicing, giving rise
to two different mRNA forms [31]. This alternative transcription is under control of
two distinct promoters whose activities are tissue specific and regulated during
development. The alternative spliced exons encode the 5′ untranslated region of the
transcript and translation starts at exon 2. Both transcript variants result in the same
protein of 984 amino acids [31, 32]. In the adult brain, MR is mostly expressed in
the dorsolateral septum and hippocampus. The highest levels of MR receptors are
detected in the pyramidal layer of the CA1 and CA2 region of the hippocampus and
the granular layer of the dentate gyrus, with lower expression on the pyramidal layer
of the CA3 region of the hippocampus [33, 34]. Outside the brain, the main ligand
for MR is aldosterone, a corticoid hormone also secreted by the adrenal gland at a
concentration 100-fold less than glucocorticoids. Having a similar affinity for glu-
cocorticoids and mineralocorticoids, the coexpression of MR with 11βHSD-2 in
peripheral organs like the kidney provides deterministic signaling properties.
Indeed, the degradation of intra-kidney cortisol by 11βHSD-2 allows MR to bind to
the less abundant aldosterone [22]. Because 11βHSD-2 is mostly absent from the
adult brain, the preferred MR ligand within the brain is cortisol in humans and
corticosterone in rodents. Corticosterone binding affinity to MR Kd of 0.1–0.3 nM
is very high compared to that of GR Kd of 2–5 nM [35, 36]. One consequence of this
very high affinity is the relatively high occupation of MR with endogenous corticos-
terone that can reach 80 % at a low corticosterone circadian trough level. This trans-
lates into a constant activation of MR signaling regardless of circadian and ultradian
rhythms (Fig. 2.1). What could be the function of a hippocampal glucocorticoid
receptor that would be constantly activated? The hippocampus plays an important
role in the HPA axis regulation as demonstrated by lesion studies [37]. Using differ-
ent pharmacological antagonists, it was demonstrated that blocking MR receptor
activates the HPA axis, suggesting a role of MR on the tonic inhibition of the HPA
axis [36, 38, 39]. Additionally, dentate granular neurons death resulting from adre-
nalectomy can be rescued by hormonal replacement with aldosterone, a specific
MR receptor agonist [40]. Aldosterone treatment also enhances the proliferation
and survival of newly-born granule cells of naïve mice [41]. Given that GR activa-
tion inhibits granule cell proliferation in the hippocampus, it is believed that a
balance of GR/MR activation along the glucocorticoid circadian and ultradian
oscillations controls the electrical activity and number of newly-born neurons in
the adult hippocampus.
Glucocorticoid Receptor
GR is encoded by the NR3C1 gene, comprising 9 exons and 11 introns, and the
protein is coded from exons 2 to 9. Exons 1 and 9 undergo alternative splicing.
Alternative splicing of exon 9 gives rise to two isoforms: the most prevalent GRα
and, and GRβ form that has a shorter C terminus transactivation region [42, 43].
GRα is the majoritarian form and classically shuttles between the cytoplasm and
nucleus depending on signaling. GRβ resides permanently in the nucleus and acts as
a dominant negative inhibitor of the GRα isoform, but can also directly regulate
genes that are not regulated by the α isoform [43, 44]. Additional isoforms of GR
are generated by alternative translation via different initiation sites giving rise to
eight variants with truncated N termini. The resulting isoforms all bind glucocorti-
coids with similar affinities but have different transcriptional activity depending on
the presence or not of the AF1 domain [43]. The expression of GR is ubiquitous and
several isoforms can be co-expressed in many tissues adding to the complexity of GR
signaling. Consequently, GR signaling depends on tissue-specific isoforms, expres-
sion level and glucocorticoid availability (time at exposure, duration and dose).
Given that mice lacking NR3C1 die of lung maturation defects [45, 46], further
tissue specific gene inactivation studies provided valuable information regarding the
role of limbic circuits in the control of the HPA axis and allostatic responses to drugs
of abuse and fear [47–49].
In stark contrast with the MR, it is unlikely that GR is activated at circadian and
ultradian glucocorticoid troughs due to its low affinity for endogenous corticoids.
This is one important feature of glucocorticoid signaling that alternates between
cycles of MR followed by a MR/GR co-activation both of which are critical for cellular
networks of the limbic system known to co-express both receptor subtypes (Fig. 2.1).
For instance, glucocorticoid circadian peaks are as important as glucocorticoid
troughs for learning and memory. If training at glucocorticoid peaks facilitates the
acquisition of a motor coordination task, the glucocorticoid trough is paramount
for its retention. In vivo imaging studies indicate that glucocorticoid peaks via GR
enhance the formation of dendritic spines, the post-synaptic entity of excitatory
synapses whereas the following troughs via MR permits the elimination of pre-
existing old spines [4]. Overall, the coincidence of glucocorticoid naturally occur-
ring circadian oscillations with procedural learning may favor the patterning of
dendritic spines needed to adjust neural circuit wiring with behavioral demands.
Sub-cellular Distribution
The classic view is that both MR and GR reside in the cytoplasm of cells and shifts
to their nucleus upon ligand binding for transcriptional regulation. Yet, unliganded
GR can be found in the nucleus unbound to chromatin and yet, readily accessible to
cortisol [50–52]. Sub-cellular distribution is actually controlled by the rates of
import and export through the nuclear pores, the directionality being determined by
glucocorticoid binding and recruitment of specific co-factors like importins, HSP90,
FKBP52 and FKBP51. For instance, cytoplasmic location is favored in the absence
of ligand, when GR is bound to a HSP90-FKBP51-based chaperone complex. This
chaperone complex stabilizes the receptor, represses its regulatory activities, and
favors a conformation that facilitates ligand binding [53]. Upon glucocorticoid
binding, the receptor undergoes a conformational change and is released from the
cytoplasmic chaperone-complex. Structural rearrangements elicited upon ligand
binding notably expose nuclear localization signal sequences facilitating the bind-
ing to import proteins and active transport through the nuclear pores. Once in the
nucleus, the interaction of MR and GR with DNA ligands and transcription factors
specify the transcriptional targets involved in the glucocorticoid genomic signaling.
The nucleus is not the only organelle where DNA ligands for the GR have been
characterized [54]. Such discovery opens new avenue for understanding cell auton-
omous metabolic effects of glucocorticoids through the mitochondria [55] (Fig. 2.3).
Since then, mitochondrial GR has been purified in brain extracts from mice sub-
jected to chronic stress [56]. The functional role of mitochondrial GR is still in its
infancy but experiments that forced GR localization to the mitochondria suggests it
is highly toxic [57]. Beyond the dogmatic genomic effects, which are slow at onset
(minutes to hours from the transcription to the bioactivity of regulated genes) exists
rapid non-genomic effects that could rely upon the subcellular distribution of the
receptors. One example is illustrated by the effects of glucocorticoids on neuronal
excitability that cannot be accounted for genomic effects [58, 59], because they are
not only faster than expected from a transcriptional response, but also resistant to
protein synthesis inhibitors [58, 60].
Fig. 2.3 Subcellular distribution of GR associates with distinct signaling outcomes. Neuronal GR
is abundant in the nucleus, somatic cytoplasm and less abundant but detectable in the mitochondria
and synaptic terminals like post-synaptic dendritic spines. (1) Ligand activated nuclear GR binds
to DNA to induce or repress gene transcription, which target Y may also function as transcription
factor to trigger a second wave of glucocorticoid response involving the gene Z. GR phosphoryla-
tion is tightly control by kinases and phosphatases that regulates the binding of biased signaling
co-factors. (2) Ligand activated GR can translocate to the mitochondria to regulate the production
of ATP and the release of cytochrome C known to endanger the survival prognosis of targeted cells.
(3) Ligand-activated GR signaling from a membrane origin rapidly activates the LIMK1-Cofilin
pathway that impinges on the turnover of the actin cytoskeleton. As a result, glucocorticoids rap-
idly enhance the formation of post-synaptic dendritic spines. (4) The postsynaptic membrane
bound GR first facilitates neurotransmission by enhancing the transport of AMPA receptor sub-
units to the active zone of the synapse, and diminish neurotransmission through a pre-synaptic
mechanism that requires the release of a retrograde messenger (endocannabinoids) that employs a
G-protein coupled receptor (CB1) to suppress the release of the excitatory neurotransmitter
glutamate
Other Receptors
Rapid glucocorticoid effects on neuronal excitability can be reproduced with a

functional synthetic glucocorticoid conjugated with BSA that cannot penetrate the
plasma membrane nor access the cytoplasmic and nuclear receptors. Such gluco-
corticoid signaling of membrane-bound origin has been replicated in numerous
paradigms and frequently associated with a MR type of pharmacology because it
can be abolished by a MR antagonist and does not occur in mice lacking MR [61].
Yet, in some tissues, the membrane-bound glucocorticoid receptor is also sensitive

to GR antagonists and GR knockdown at least on dendritic spine formation [4].
Localization of MR and GR in close proximity to the plasma membrane notably at
the synapse was evidenced by biochemical purification and electron microscopy
[60, 62–65]. The existence of membrane-bound MR and GR has been functionally
proved in other models [66, 67] (Fig. 2.3). Despite the evidence, the pharmacology
of membrane-bound cortisol receptors supports the possibility that several trans-
membrane proteins coupled to small heterotrimeric G proteins and sensitive to MR
and or GR could signal rapidly.
Associated Protein Complexes
Ligand-Independent Partners
Chaperone Complex
The unliganded MR and GR are permanently scanning the cellular environment

thanks to a large complex of chaperone proteins that control the architecture of the
glucocorticoid ligand binding domain. Numerous GR and MR binding partners
have been characterized. Here, we present only a few examples. Invariably, HSP90
serves as scaffold for the ‘foldosome’ complex (HSP70, HSP40 and Hop) that
molds the ligand-binding pocket of GR, in an ATP-dependent manner [53, 68, 69].
Binding of p23 by this heterocomplex, further stabilizes the conformational change
[69]. The release of Hop from the complex leads to the dynamic and competitive
addition of other co-chaperones like the immunophilins FKBP51, FKBP52 and cyp
40 and the immunophilin-like phosphatase PP5 that mediate changes in receptor
mobility, ligand affinity and signaling capacity [70, 71]. For example, FKBP52
facilitates GR signaling activity by bringing together GR and motor proteins onto
the microtubules for active nuclear transport [70, 72]. In contrast, binding of
FKBP51 to the chaperone supercomplex attenuates glucocorticoid signaling by
facilitating the nuclear export of GR. The robust induction of FKBP51 expression
by glucocorticoid, unlike FKBP52, suggests that glucocorticoid signaling feedback
can also be cell autonomous [73].
Ligand Dependent Partners
Numerous proteins have been functionally associated with the liganded MR and
GR. By no means is it the goal of this chapter to enumerate them. Only a small
selection of relevant partners will be emphasized, as it is dependent on post-
translational modifications and subcellular distribution.
Post-translational Modifications
With low basal phosphorylation at rest, MR and GR get hyperphosphorylated upon

ligand binding offering binding sites for the 14-3-3 family of proteins. Despite
multiple phosphorylated serine or threonine residues identified in the N-terminal
domain of GR [74], only a select number of sites have been functionally character-
ized. For instance, glucocorticoid-dependent phosphorylation at conserved serines
203 (S203), 211 (S211), 226 (S226), and 404 (S404) in the human GR numbering
scheme impacts on transcriptional capabilities at specific target gene. Three of
these sites demonstrate some interdependency upon one another [75]. All four sites
exhibit low basal phosphorylation in the absence of cortisol as long as the protein
phosphatase PP5 remains associated with GR and the HSP90 chaperone complex
[76]. S203 phosphorylation, however, is characteristically higher without hormone
compared to S211 and S226 sites. Upon glucocorticoid binding, the S203 phos-
phorylated form of the receptor appears perinuclear, suggesting that GR phosphor-
ylated at S203 does not participate in DNA-bound GR transcriptional regulation
[75]. Consistent with this idea, S203 phosphorylated GR does not occupy select
GREs whereas S211 and S226 phospho-isoforms can bind to DNA ligands in the
genome [77]. Classically, phosphorylation of GR at S211 serves as surrogate
marker of ligand-activated GR because basal phosphorylation is null in absence of
glucocorticoids and ligand-induced S211 phosphorylation correlates with the mag-
nitude of GR transcriptional activity [77]. In mitotic cells, S211 phosphorylation
depends on CDK2 whereas CDK5 or ERK may phosphorylate GR in postmitotic
neurons [78, 79]. In contrast, phosphorylation at S226 by JNK serve as docking
site for specific cofactors that increases nuclear export and reduces GR transcrip-
tional activation [77, 80, 81]. S404, a substrate of GSK3β reduces GR transactiva-
tion by increasing the turnover of the liganded GR and by hindering GR-mediated
NF-κB repression [82].
Other post-translational modifications are dependent on the phosphorylation
code of GR. For example, JNK-dependent phosphorylation of S246 enhances
sumoylation at K293 and K277, which are implicated in the control of GR trans-
activation at multiple GREs [83, 84]. Lastly, ligand-mediated GR degradation by
the proteasome requires ubiquitination of K419, which is also dependent upon
phosphorylation, as a phospho-defective GR mutant is resistant to ligand-induced
degradation [84, 85]. Functionally, GR phosphorylation at rat serine 232 (analo-
gous to S211 in humans), responds to neurotoxic insults to induce specifically
the ectopic expression of hdac2 that correlates with the occurrence of cognitive
defects [86]. GR phosphorylation at serine 232 also increases in the rodent brain
after exposure to glucocorticoids or stress [87, 88]. The MR is also phosphory-
lated at rest and upon ligand binding, yet the functional characterization of indi-
vidual sites is far less advanced [89]. Although the kinases have more or less
been characterized for most sites, it is unclear how glucocorticoid signaling can
activate kinases.
Nuclear Trafficking
It was long believed that nuclear translocation of GR necessitated the release from
the chaperone complex [90]. The emerging picture is that the entire complex moves
along the microtubule cytoskeleton depending on the recruitment of subtype specific
immunophilins, importins and exportins [91, 92]. GR has two nuclear localization
signals, NL1 and NL2. NL1 supports rapid and hormone independent translocation
[93], whereas NL2 facilitates slower hormone dependent nuclear import [84, 94].
Several importins interact with GR NL regions and mediate its nuclear translocation
across the nuclear pore complex. Importins 7 and 8 bind NL1 and NL2, whereas
importin α/β bind exclusively to NL1 [94]. Evidence of importins and nuclear pore
proteins binding with components of the chaperone complex shed light into the
molecular mechanism of nuclear import. Importin β and nuclear pore glycoprotein
Nup62 bind GR and HSP90, p23, and FKBP52 to accelerate nuclear import rate
[92, 95]. Nuclear export is dependent on exportin/CRM1 as pharmacological block-
ade abolishes GR translocation to the cytosol [52]. Nuclear calcium levels also
impacts upon GR nuclear residency via a direct interaction between the NES
sequences and the calcium-sensing protein calreticulin and other related co-factors
such as SRC-1 and 14-3-3σ [79, 84, 96–98].
DNA Binding
Despite a high degree of homology between MR and GR, common protein complexes,
and similar post-translational modifications such as ubiquitination, sumoylation and
phosphorylation, MR and GR affect distinct gene targets [99]. Yet, GR and MR can
form heterodimers [100, 101], which may modulate transcription of a few target
genes in a unique way that differ from either GR or MR homodimers. One described
example is the serotonin receptor 5HT1A gene which transcription is repressed by GR
and MR alone but GR/MR heterodimers exert an even stronger inhibition [102].
Zinc fingers of the DNA binding domain mediate of GR-DNA interaction that
usually requires the receptor to dimerize with itself or other transcription factors.
Yet, the GRdim mutant that cannot homodimerize is still capable of robust tran-
scriptional regulation at multiple but not all targets-genes [43]. The DNA sequence
also acts as an allosteric ligand that influences GR structure and activity [99].
Classically, GREs have been grouped into three classes: (i) simple GREs;(ii) com-
posite GREs; (iii) and tethering elements [103]. Each class requires GR binding in
different conformations and orientations as follows. Simple GREs are most often
inverted, repeat hexameric sequences separated by three nucleotides, supporting the
homodimerization model of GR. A few negative GREs (nGREs) that mediate
GR-dependent repression have been well characterized [104]. The inverted repeat
IR nGREs found in numerous glucocorticoid-repressed genes [105, 106] support a
model whereby direct binding of GR as monomer is not always required but may
instead involve the recruitment of specific co-factors like nuclear receptor corepressors
1 and 2 (N-CoR) and (SMRT) [105, 107]. Composite GREs contain non-GR binding
sequences surrounding a core GR-binding site important for gene-specific synergistic

or antagonistic regulation. GR may interact physically and/or functionally with the
transcription factors associated with those motifs. An increasing number of mecha-
nistic and genome-wide studies suggest that most functional transactivating GREs
are composite elements, composed of binding sites for GR as well as additional
DNA-binding regulatory factors that act in synergy with GR in the particular
context of each GRE [108, 109]. Such composite elements might explain some of
the cell-type specific regulation by GR and is discussed in additional detail in the
following Chapter.
Transcription Cofactors
The typical inverted U-shape responses to increasing dose of glucocorticoids could

be in part explained by “squelching” or titration of a limiting co-factor required for
full response by the activated GR [110–112]. At high concentrations of activated GR,
a factor becomes limiting for full GR activity and as such, transcriptional activation
is reduced. Thus, the squelching model emphasizes the availability of transcription
co-factors. In the case of tethering GREs, it is the DNA bound co-factor of MR and
GR that specifies the glucocorticoid-targets genes. Such factors are AP-1, CREB,
NF-κB, or STAT5, C/EBP, SP-1, Egr-1 and others less characterized. Most tethering
GREs do not contain canonical sequence motifs for GR, but rather contain motifs
for the interacting transcription factor that may also mediate induction of target
genes in the absence of glucocorticoids. For example, tethering of GR to AP-1 or
NF-κB alters the assembly of coactivator complexes and recruits the corepressor
glucocorticoid receptor interacting protein 1 (GRIP1) [113 , 114]. In the case
of direct GR-DNA binding, the transcriptional output depends on recruitment of a
distinct class of co-factors that include P300/CBP, HDAC2, p160, MED1,14,
and SWI/SNF complex. These factors are thought to affect chromatin structure or
the stability of transcriptional machinery at the transcription start site. Coactivator
complexes are assembled through interactions with GR’s AF-1 or more commonly
the AF-2 domain [115]. The p160 family of coactivators include SRC-1, SRC-2/
GRIP1/transcriptional intermediary factor 2 (TIF2), and SRC-3 (also known as
pCIP/ACTR/AIB1/RAC3). P160 proteins often increase GR transcriptional activity,
but GRIP1 was shown to act both as a coactivator and corepressor via intrinsic acti-
vation and repression domains [113, 116]. GRIP1 specifically utilizes its repression
domain at AP-1 and NF-κB tethering GREs, whereas SRC-1 and SRC-2 lack this
domain and fail to assist repression at AP-1 [82]. Many p160 proteins facilitate
transcriptional activation by recruiting p300/CBP. P300/CBP contains potent acet-
yltransferase activity that target histones and other proteins. GR has also been
shown to interact with a number of histone deacetylases (HDACs) including: HDAC
1 and 6 as coactivators [117–119] and HDAC2 as both a coactivator and corepres-
sor, and its recruitment depend on S211 phosphorylation [82, 86, 119, 120]. Through
its AF1 and AF2 domains GR interacts with MED14 and MED1 two components of
the mediator complex [121]. The mediator complex is a large multiunit complex
that bridges transcription factors and RNA pol II, affecting transcription initiation
[122] and elongation [123]. The assembly of select mediator subunits alters
promoter responsiveness to various transcription factors, so distinct subunits and
confirmations are important for GR transcriptional activity. In cell-based reporter
assays, overexpression of MED14 enhances GR transcriptional activity, while
MED1 only enhanced GR activity in the context of MED14 [124].
Signaling
A large number of well-documented rapid and slow cellular responses to glucocorti-

coids contribute to glucocorticoid-mediated allostasis. It is remarkable that slow effects
can either oppose or reinforce the rapid effects. In this section, we will describe a few
examples illustrating how pertinent the spatial and temporal resolution of glucocorti-
coid signaling is to adjust neuronal networks to changing environments.
Minutes
Rapid glucocorticoid signaling has been particularly studied in the context of neu-
ronal excitability within the range of seconds to minutes. For instance, the excit-
ability of dorsal hippocampal CA1 neurons is sensitive to glucocorticoids within
minutes of exposure as demonstrated by an increase in the frequency of spontane-
ous excitatory neurotransmission (Fig. 2.3). Pharmacological and genetic proof of
concept studies revealed the contribution of the membrane-bound MR [125, 126].
Pharmacological characterization of downstream signaling revealed the requirement
of the ERK1/2 pathway [126]. An increase in frequency of spontaneous excitatory
neurotransmission usually results from an increased quantal release of the neu-
rotransmitter glutamate, and extracellular glutamate levels are augmented following
acute glucocorticoid treatment. As much as glucocorticoids affect presynaptic
neurotransmitters release, rapid glucocorticoid effects on the post-synaptic neuro-
nal terminal were also characterized. Indeed, glucocorticoids through the mem-
brane-bound MR rapidly (few minutes) increase the mobility and dwell time at the
post-synaptic density of glutamate receptors GluR2-AMPAR [127] (Fig. 2.3). Such
effects are usually interpreted as enhancing neurotransmission. Similar observa-
tions were reported in the basoloateral amygdala (BLA) that yet, expresses much
lower MR levels than the hippocampus [66]. Interestingly, the rapid increase of
neuronal excitability by the membrane-bound MR is context dependent as it varied
with a history of glucocorticoid exposure or stress [66]. That is, a second pulse
stimulation of brain slice preparations with corticosterone after washout of a first
pulse decreased the excitability of BLA neurons that a single pulse could increase
[21, 66]. This suggests that time at exposure with glucocorticoids also determines
the signaling outcome. This picture is not the rule because glucocorticoids cause a
rapid (few minutes) suppression of excitatory synaptic inputs in hypothalamic
CRH- and AVP-releasing neurons of the paraventricular nucleus (PVN) [58, 128].
Physiologically, this effect contributes to one of the rapid components of the nega-
tive feedback that deactivates the HPA axis. In this paradigm, a membrane-bound
GR pathway was highlighted using a synthetic membrane impermeant GR agonist
that curiously cannot be abolished by a specific GR antagonist, suggesting the
involvement of a membrane bound GR with non-canonical pharmacological fea-
tures [58]. Suppression of PVN neuronal excitation employs endocannabinoids
released from the post-synaptic membrane and signaling through the CB1 receptor
at the pre-synaptic terminals to suppress glutamate release [58, 129, 130]. Given
this signaling pathway involves small heterotrimeric G protein, this finding recon-
ciles previous data indicating that glucocorticoid signaling of membrane origin is
sensitive to antagonists of MR, GR and G proteins [58, 129]. Finally, glucocorti-
coids enhance dendritic spine formation in the living cortex within minutes of expo-
sure via GR-mediated activation of a LIMK-cofilin pathway that impinges on the
dynamics of the post-synaptic actin cytoskeleton [4] (Fig. 2.3).
Hours
While the rapid glucocorticoid signaling serves immediate purposes like the deac-
tivation of the HPA axis, slow glucocorticoid signaling is classically viewed as an
adaptive response of cellular networks to changing environments like the suppres-
sion of CRH transcription to maintain HPA axis homeostasis [131]. Both the rapid
and slow signaling components often studied separately shall be considered as
integrated response overtime. For instance, it is intriguing that only slow effects of
GR and MR phosphorylation on transcriptional activity have been studied given
that phosphorylation occurs within minutes of stimulation with cortisol. In the hip-
pocampus, GR mediated genomic effects (transcription and RNA decay) follow
the rapid increase of hippocampal excitability mediated by MR in order to reduce
neuronal excitability by changing the expression levels of signaling molecules and
ion channels like the L type calcium channel [21, 132]. Such a short-term increase
of hippocampal neurons excitability is viewed as means to consolidate stress-
related memory whereas the following decrease in excitability is viewed as means
to protect hippocampal cell networks from noise information [21]. This picture is
not the rule because glucocorticoids increase excitability in the BLA that persists
after drug washout and is sensitive to protein synthesis inhibitors, GR inhibitors,
and absent in GR knockout mice, pointing towards a genomic GR-dependent
mechanism [66, 133].
Cell-type specific GR-dependent genomic effects are well-illustrated by the
transcription of crh, the major molecular trigger of the HPA axis that is increased in
the amygdala but decreased in the hypothalamic PVN as a function of elevated lev-
els of glucocorticoids [134]. In the cortex, learning-dependent weaving of neural
networks relies on the formation and elimination of synaptic connectivity as a func-
tion of glucocorticoid levels. At glucocorticoid circadian trough when only MR is
activated, elimination of dendritic spines compensate for the increased GR-dependent
spine formation that occurred during the glucocorticoid circadian peak [4]. This result
is interpreted as if learning associated new spines are offset by the elimination of
pre-existing old spines within distinct temporal domains to shape neural circuits as
a function of novelty.
Context-Dependent Glucocorticoid Signaling
Glucocorticoid-mediated allostasis employs genomic and non-genomic mechanisms

together with ongoing, experience-driven neural activity mediated by excitatory
amino acids neurotransmitters, neurotrophic factors such as BDNF, neuropeptides
such as CRH and cell adhesion molecules (NCAM) [135, 136]. Therefore, it is par-
ticularly interesting to discuss how glucocorticoid activity superimposes with other
signaling pathways. For instance, the enhancement of emotionally arousing memo-
ries by glucocorticoids requires co-incident norepinephrine signaling in the amyg-
dala [137]. Norepinephrine is capable of potentiating ligand-induced GR
transcriptional capabilities and DNA binding in a PI3K-dependent manner [138].
Adrenergic receptor agonists, generally used in the treatment of asthma, synergisti-
cally enhance GR-dependent transcription in a cAMP/PKA-dependent manner.
Thus, maximal GR transcriptional capabilities are achieved approximately at ten-
fold lower glucocorticoid concentrations in the presence of cAMP-elevating drugs
[139]. Another set of factors are proinflammatory cytokines, such as interleukin
IL1, IL-2, IL-6, TNF-a, and interferon (IFN)-a, signaling pathways that alter
GR signaling and neuroendocrine function [140, 141]. Co-incident growth factor
signaling may also intersect with GR actions. For instance, bFGF and IGF1 can
enhance ligand-induced GR transcriptional capabilities in a PI3K-dependent man-
ner [138]. The neurotrophin BDNF through its receptor TrkB can also specify the
transcription of select GR-mediated genes [142] that may be relevant for glucocor-
ticoid-induced memory consolidation of fear and inhibitory avoidance [143].
Mechanistically, BDNF signaling results in phosphorylation of rat GR at serines
134 and 267, which fosters cofactor recruitment to promote a novel gene expression
signature. Thus, BDNF utilizes GR as a transcription factor to alter glucocorticoid-
regulated transcription in neurons [142] (Fig. 2.4). Similarly, GR phosphorylation at
rat serine 232 responds to neurotoxic insults to induce specifically the ectopic
expression of hdac2 that correlates with the occurrence of cognitive defects [86].
Pharmacological blockade of HDAC2 activity resolved cognitive impairments pro-
duced by the prolonged activation of GR in a model of Alzheimer’s disease featur-
ing chronic high glucocorticoid levels known to worsen neuropathological features
[86, 144, 145]. In contrast, the expression of HDAC2 is downregulated in glucocorti-
coid-resistant cases of severe asthma and pharmacological activation of HDAC2 with
theophiline and antioxidants present therapeutic value [146, 147]. Glucocorticoid
activity may also diverge as a function of estrogen signaling, which increases the
expression of the GR phosphatase-PP5 [148]. The opposing effect of cortisol on
crh expression in the hypothalamus and the amygdala is another good example of
Fig. 2.4 Glucocorticoid transcriptional effects diverge as a function of BDNF signaling. On top of
glucocorticoid-mediated GR phosphorylation exists a parallel converging pathway that allows
for site-specific GR phosphorylation by the neurotrophic factor BDNF and its receptor TrkB.
The resulting hyperphosphorylated GR can activate or repress new select target genes enriched
with the indicated transcription factors binding sites, as well as potentiate the expression of
GR-sensitive genes. Thus, BDNF-induced GR phosphorylation rewrites the GR transcriptome
toward a cellular network signature by fostering the recruitment of phospho-specific cofactors
context-dependent glucocorticoid signaling [134]. It is the CREB co-activator

CRTC2 that determines the ability of neurotrophins and glucocorticoids to activate
or suppress hypothalamic CRH expression, respectively [112, 149] (Fig. 2.5).
Conclusions
One paramount feature of the body’s allostasis resides in the flexibility of the naturally
occurring glucocorticoid rhythms and signaling to changing environments. For
instance, glucocorticoid-mediated allostasis is critical for behavioral adaptation to
novelty, stress coping, learning and memory. Disruption of glucocorticoid circadian
and ultradian rhythms is a hallmark of numerous diseases notably neuropsychiatric. In
contrast, administration of glucocorticoids, which are mainstay of treatment for
Fig. 2.5 Glucocorticoids employ GR-dependent mechanisms to enhance the formation and the
elimination of post-synaptic dendritic spines within distinct temporal domains. Rapid transcription-
independent GR signaling facilitates the formation of dendritic spines associated with learning
capabilities whereas the slow genomic GR/MR signaling accounts for the stabilization as well as
the elimination of pre-existing old spines, a process that is critical for memory retention. The pat-
terning of dendritic spines through the processes of formation and elimination is critical to adjust
neural connectivity networks in changing environments
numerous disorders, can produce side effects related to brain functions, like psycho-
sis, depression and memory loss. One hypothesis to account for these effects is that
glucocorticoid resistance whether innate or acquired increases the vulnerability to
neurotoxic insults that slowly contribute to the development of numerous disorders of
the nervous and immune systems. Two examples: glucocorticoids do not cause but
worsen the neuropathological feature of Alzheimer’s disease and glucocorticoid resis-
tance that interferes with inflammation, increases the sensitivity to develop a common
cold. Several studies suggest that the growth/survival MAPK pathway regulates
glucocorticoid signaling because diseases featuring glucocorticoid resistance also
exhibit disrupted MAPK activity like asthma and depression. Evolution may have
selected the MAPK pathway to cope with the allostatic overload of stress and form
new memories. One putative mechanism that shows great promises is that MAPKs
modulate glucocorticoid activities in part through the phosphorylation of the gluco-
corticoid receptors. Indeed, MAPK-mediated modulation of GR function appears to
be a central player in the development of glucocorticoid resistance. Future functional
characterization of the GR and MR phosphorylation codes could help comprehend
how glucocorticoid actions can be changed from harmful to protective.
Acknowledgement We are thankful to Michael Garabedian (New York University) for support
and Inserm’s AVENIR funding program.
References
1. McEwen BS, Wingfield JC. The concept of allostasis in biology and biomedicine. Horm
Behav. 2003;43(1):2–15.
2. de Kloet ER, Vreugdenhil E, Oitzl MS, Joëls M. Brain corticosteroid receptor balance in
health and disease. Endocr Rev. 1998;19(3):269–301.
3. Compagnone NA, Mellon SH. Neurosteroids: biosynthesis and function of these novel
neuromodulators. Front Neuroendocrinol. 2000;21(1):1–56.
4. Liston C, Cichon JM, Jeanneteau F, Jia Z, Chao MV, Gan WB. Circadian glucocorticoid
oscillations promote learning-dependent synapse formation and maintenance. Nat Neurosci.
2013;16(6):698–705.
5. Keller-Wood ME, Dallman MF. Corticosteroid inhibition of ACTH secretion. Endocr Rev.
1984;5(1):1–24.
6. Lightman SL, Conway-Campbell BL. The crucial role of pulsatile activity of the HPA axis
for continuous dynamic equilibration. Nat Rev Neurosci. 2010;11(10):710–8.
7. Kalsbeek A, Buijs RM. Output pathways of the mammalian suprachiasmatic nucleus: coding
circadian time by transmitter selection and specific targeting. Cell Tissue Res.
2002;309(1):109–18.
8. de Kloet ER, Sarabdjitsingh RA. Everything has rhythm: focus on glucocorticoid pulsatility.
Endocrinology. 2008;149(7):3241–3.
9. Cook CJ. Measuring of extracellular cortisol and corticotropin-releasing hormone in the
amygdala using immunosensor coupled microdialysis. J Neurosci Methods. 2001;110(1–2):
95–101.
10. Jasper MS, Engeland WC. Synchronous ultradian rhythms in adrenocortical secretion
detected by microdialysis in awake rats. Am J Physiol. 1991;261(5 Pt 2):R1257–68.
11. Walker JJ, Terry JR, Lightman SL. Origin of ultradian pulsatility in the hypothalamic-
pituitary-adrenal axis. Proc Biol Sci. 2010;277(1688):1627–33.
12. Papavasiliou SS, Zmeili S, Khoury S, Landefeld TD, Chin WW, Marshall JC. Gonadotropin-
releasing hormone differentially regulates expression of the genes for luteinizing hormone
alpha and beta subunits in male rats. Proc Natl Acad Sci U S A. 1986;83(11):4026–9.
13. Wildt L, Hausler A, Marshall G, et al. Frequency and amplitude of gonadotropin-releasing
hormone stimulation and gonadotropin secretion in the rhesus monkey. Endocrinology.
1981;109(2):376–85.
14. Gregus A, Wintink AJ, Davis AC, Kalynchuk LE. Effect of repeated corticosterone injections
and restraint stress on anxiety and depression-like behavior in male rats. Behav Brain Res.
2005;156(1):105–14.
15. Ulrich-Lai YM, Herman JP. Neural regulation of endocrine and autonomic stress responses.
Nat Rev Neurosci. 2009;10(6):397–409.
16. Holsboer F, Ising M. Stress hormone regulation: biological role and translation into therapy.
Annu Rev Psychol. 2010;61:81–109.
17. Pariante CM, Miller AH. Glucocorticoid receptors in major depression: relevance to patho-
physiology and treatment. Biol Psychiatry. 2001;49(5):391–404.
18. Rosner W. The functions of corticosteroid-binding globulin and sex hormone-binding globulin:
recent advances. Endocr Rev. 1990;11(1):80–91.
19. Cameron A, Henley D, Carrell R, Zhou A, Clarke A, Lightman S. Temperature-responsive
release of cortisol from its binding globulin: a protein thermocouple. J Clin Endocrinol
Metab. 2010;95(10):4689–95.
20. Klieber MA, Underhill C, Hammond GL, Muller YA. Corticosteroid-binding globulin, a
structural basis for steroid transport and proteinase-triggered release. J Biol Chem.
2007;282(40):29594–603.
21. Joels M, Sarabdjitsingh RA, Karst H. Unraveling the time domains of corticosteroid hormone
influences on brain activity: rapid, slow, and chronic modes. Pharmacol Rev. 2012;
64(4):901–38.
22. Wyrwoll CS, Holmes MC, Seckl JR. 11beta-hydroxysteroid dehydrogenases and the brain:
from zero to hero, a decade of progress. Front Neuroendocrinol. 2011;32(3):265–86.
23. Yau JL, Noble J, Kenyon CJ, et al. Lack of tissue glucocorticoid reactivation in 11beta-
hydroxysteroid dehydrogenase type 1 knockout mice ameliorates age-related learning
impairments. Proc Natl Acad Sci U S A. 2001;98(8):4716–21.
24. Seckl JR, Walker BR. 11beta-hydroxysteroid dehydrogenase type 1 as a modulator of gluco-
corticoid action: from metabolism to memory. Trends Endocrinol Metab. 2004;15(9):
418–24.
25. Pariante CM. The role of multi-drug resistance p-glycoprotein in glucocorticoid function:
studies in animals and relevance in humans. Eur J Pharmacol. 2008;583(2–3):263–71.
26. Volk HA, Burkhardt K, Potschka H, Chen J, Becker A, Loscher W. Neuronal expression of
the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures.
Neuroscience. 2004;123(3):751–9.
27. Akers KG, Nakazawa M, Romeo RD, Connor JA, McEwen BS, Tang AC. Early life modula-
tors and predictors of adult synaptic plasticity. Eur J Neurosci. 2006;24(2):547–54.
28. Kumar R, Thompson EB. The structure of the nuclear hormone receptors. Steroids.
1999;64(5):310–9.
29. Bodwell JE, Webster JC, Jewell CM, Cidlowski JA, Hu JM, Munck A. Glucocorticoid recep-
tor phosphorylation: overview, function and cell cycle-dependence. J Steroid Biochem Mol
Biol. 1998;65(1–6):91–9.
30. Ismaili N, Garabedian MJ. Modulation of glucocorticoid receptor function via phosphoryla-
tion. Ann N Y Acad Sci. 2004;1024:86–101.
31. Zennaro MC, Keightley MC, Kotelevtsev Y, Conway GS, Soubrier F, Fuller PJ. Human min-
eralocorticoid receptor genomic structure and identification of expressed isoforms. J Biol
Chem. 1995;270(36):21016–20.
32. Martinerie L, Munier M, Le Menuet D, Meduri G, Viengchareun S, Lombes M. The miner-
alocorticoid signaling pathway throughout development: expression, regulation and patho-
physiological implications. Biochimie. 2013;95(2):148–57.
33. Van Eekelen JA, Jiang W, De Kloet ER, Bohn MC. Distribution of the mineralocorticoid
and the glucocorticoid receptor mRNAs in the rat hippocampus. J Neurosci Res. 1988;
21(1):88–94.
34. Gesing A, Bilang-Bleuel A, Droste SK, Linthorst AC, Holsboer F, Reul JM. Psychological
stress increases hippocampal mineralocorticoid receptor levels: involvement of corticotropin-
releasing hormone. J Neurosci. 2001;21(13):4822–9.
35. Reul JM, de Kloet ER. Two receptor systems for corticosterone in rat brain: microdistribution
and differential occupation. Endocrinology. 1985;117(6):2505–11.
36. Reul JM, Gesing A, Droste S, et al. The brain mineralocorticoid receptor: greedy for ligand,
mysterious in function. Eur J Pharmacol. 2000;405(1-3):235–49.
37. Jacobson L, Sapolsky R. The role of the hippocampus in feedback regulation of the
hypothalamic-pituitary-adrenocortical axis. Endocr Rev. 1991;12(2):118–34.
38. Ratka A, Sutanto W, Bloemers M, de Kloet ER. On the role of brain mineralocorticoid (type
I) and glucocorticoid (type II) receptors in neuroendocrine regulation. Neuroendocrinology.
1989;50(2):117–23.
39. Cole MA, Kalman BA, Pace TW, Topczewski F, Lowrey MJ, Spencer RL. Selective blockade
of the mineralocorticoid receptor impairs hypothalamic-pituitary-adrenal axis expression of
habituation. J Neuroendocrinol. 2000;12(10):1034–42.
40. Woolley CS, Gould E, Sakai RR, Spencer RL, McEwen BS. Effects of aldosterone or
RU28362 treatment on adrenalectomy-induced cell death in the dentate gyrus of the adult rat.
Brain Res. 1991;554(1–2):312–5.
41. Fischer AK, von Rosenstiel P, Fuchs E, Goula D, Almeida OF, Czeh B. The prototypic min-
eralocorticoid receptor agonist aldosterone influences neurogenesis in the dentate gyrus of
the adrenalectomized rat. Brain Res. 2002;947(2):290–3.
42. Duma D, Jewell CM, Cidlowski JA. Multiple glucocorticoid receptor isoforms and mechanisms
of post-translational modification. J Steroid Biochem Mol Biol. 2006;102(1–5):11–21.
43. Kadmiel M, Cidlowski JA. Glucocorticoid receptor signaling in health and disease. Trends
Pharmacol Sci. 2013;34(9):518–30.
44. Lewis-Tuffin LJ, Jewell CM, Bienstock RJ, Collins JB, Cidlowski JA. Human glucocorticoid
receptor beta binds RU-486 and is transcriptionally active. Mol Cell Biol. 2007;27(6):
2266–82.
45. Kellendonk C, Eiden S, Kretz O, et al. Inactivation of the GR in the nervous system affects
energy accumulation. Endocrinology. 2002;143(6):2333–40.
46. Tronche F, Kellendonk C, Kretz O, et al. Disruption of the glucocorticoid receptor gene in the
nervous system results in reduced anxiety. Nat Genet. 1999;23(1):99–103.
47. Howell MP, Muglia LJ. Effects of genetically altered brain glucocorticoid receptor action on
behavior and adrenal axis regulation in mice. Front Neuroendocrinol. 2006;27(3):275–84.
48. Kolber BJ, Roberts MS, Howell MP, Wozniak DF, Sands MS, Muglia LJ. Central amygdala
glucocorticoid receptor action promotes fear-associated CRH activation and conditioning.
Proc Natl Acad Sci U S A. 2008;105(33):12004–9.
49. Schmidt MV, Sterlemann V, Wagner K, et al. Postnatal glucocorticoid excess due to pituitary
glucocorticoid receptor deficiency: differential short- and long-term consequences.
Endocrinology. 2009;150(6):2709–16.
50. Nishi M, Ogawa H, Ito T, Matsuda KI, Kawata M. Dynamic changes in subcellular localiza-
tion of mineralocorticoid receptor in living cells: in comparison with glucocorticoid receptor
using dual-color labeling with green fluorescent protein spectral variants. Mol Endocrinol.
2001;15(7):1077–92.
51. Piwien Pilipuk G, Vinson GP, Sanchez CG, Galigniana MD. Evidence for NL1-independent
nuclear translocation of the mineralocorticoid receptor. Biochemistry. 2007;46(5):
1389–97.
52. Hache RJ, Tse R, Reich T, Savory JG, Lefebvre YA. Nucleocytoplasmic trafficking of steroid-
free glucocorticoid receptor. J Biol Chem. 1999;274(3):1432–9.
53. Dittmar KD, Banach M, Galigniana MD, Pratt WB. The role of DnaJ-like proteins in gluco-
corticoid receptor.hsp90 heterocomplex assembly by the reconstituted hsp90.p60.hsp70
foldosome complex. J Biol Chem. 1998;273(13):7358–66.
54. Lee SR, Kim HK, Song IS, et al. Glucocorticoids and their receptors: insights into specific
roles in mitochondria. Prog Biophys Mol Biol. 2013;112(1–2):44–54.
55. Du J, Wang Y, Hunter R, et al. Dynamic regulation of mitochondrial function by glucocorti-
coids. Proc Natl Acad Sci U S A. 2009;106(9):3543–8.
56. Adzic M, Lukic I, Mitic M, et al. Brain region- and sex-specific modulation of mitochondrial
glucocorticoid receptor phosphorylation in fluoxetine treated stressed rats: effects on energy
metabolism. Psychoneuroendocrinology. 2013;38(12):2914–24.
57. Sionov RV, Cohen O, Kfir S, Zilberman Y, Yefenof E. Role of mitochondrial glucocorticoid
receptor in glucocorticoid-induced apoptosis. J Exp Med. 2006;203(1):189–201.
58. Di S, Malcher-Lopes R, Halmos KC, Tasker JG. Nongenomic glucocorticoid inhibition via
endocannabinoid release in the hypothalamus: a fast feedback mechanism. J Neurosci.
2003;23(12):4850–7.
59. Di S, Maxson MM, Franco A, Tasker JG. Glucocorticoids regulate glutamate and GABA
synapse-specific retrograde transmission via divergent nongenomic signaling pathways.
J Neurosci. 2009;29(2):393–401.
60. Groeneweg FL, Karst H, de Kloet ER, Joels M. Mineralocorticoid and glucocorticoid receptors
at the neuronal membrane, regulators of nongenomic corticosteroid signalling. Mol Cell
Endocrinol. 2012;350(2):299–309.
61. Karst H, Berger S, Turiault M, Tronche F, Schutz G, Joels M. Mineralocorticoid receptors
are indispensable for nongenomic modulation of hippocampal glutamate transmission by
corticosterone. Proc Natl Acad Sci U S A. 2005;102(52):19204–7.
62. Qiu S, Champagne DL, Peters M, et al. Loss of limbic system-associated membrane protein
leads to reduced hippocampal mineralocorticoid receptor expression, impaired synaptic
plasticity, and spatial memory deficit. Biol Psychiatry. 2010;68(2):197–204.
63. Prager EM, Brielmaier J, Bergstrom HC, McGuire J, Johnson LR. Localization of mineralo-
corticoid receptors at mammalian synapses. PLoS One. 2010;5(12), e14344.
64. Wang CC, Wang SJ. Modulation of presynaptic glucocorticoid receptors on glutamate release
from rat hippocampal nerve terminals. Synapse. 2009;63(9):745–51.
65. Johnson LR, Farb C, Morrison JH, McEwen BS, LeDoux JE. Localization of glucocorticoid
receptors at postsynaptic membranes in the lateral amygdala. Neuroscience. 2005;136(1):
289–99.
66. Karst H, Berger S, Erdmann G, Schutz G, Joels M. Metaplasticity of amygdalar responses
to the stress hormone corticosterone. Proc Natl Acad Sci U S A. 2010;107(32):
14449–54.
67. Evanson NK, Tasker JG, Hill MN, Hillard CJ, Herman JP. Fast feedback inhibition of the
HPA axis by glucocorticoids is mediated by endocannabinoid signaling. Endocrinology.
2010;151(10):4811–9.
68. Dittmar KD, Demady DR, Stancato LF, Krishna P, Pratt WB. Folding of the glucocorticoid
receptor by the heat shock protein (hsp) 90-based chaperone machinery. J Biol Chem.
1997;272(34):21213–20.
69. Stancato LF, Silverstein AM, Gitler C, Groner B, Pratt WB. Use of the thiol-specific deriva-
tizing agent N-iodoacetyl-3-[125I]iodotyrosine to demonstrate conformational differences
between the unbound and hsp90-bound glucocorticoid receptor hormone binding domain.
J Biol Chem. 1996;271(15):8831–6.
70. Pratt WB, Silverstein AM, Galigniana MD. A model for the cytoplasmic trafficking of
signalling proteins involving the hsp90-binding immunophilins and p50cdc37. Cell Signal.
1999;11(12):839–51.
71. Chen S, Smith DF. Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90
chaperone machinery. J Biol Chem. 1998;273(52):35194–200.
72. Pratt WB, Toft DO. Steroid receptor interactions with heat shock protein and immunophilin
chaperones. Endocr Rev. 1997;18(3):306–60.
73. Jaaskelainen T, Makkonen H, Palvimo JJ. Steroid up-regulation of FKBP51 and its role in
hormone signaling. Curr Opin Pharmacol. 2011;11(4):326–31.
74. Miller ML, Jensen LJ, Diella F, et al. Linear motif atlas for phosphorylation-dependent
signaling. Sci Signal. 2008;1(35):ra2.
75. Wang Z, Frederick J, Garabedian MJ. Deciphering the phosphorylation “code” of the gluco-
corticoid receptor in vivo. J Biol Chem. 2002;277(29):26573–80.
76. Wang Z, Chen W, Kono E, Dang T, Garabedian MJ. Modulation of glucocorticoid receptor
phosphorylation and transcriptional activity by a C-terminal-associated protein phosphatase.
Mol Endocrinol. 2007;21(3):625–34.
77. Chen W, Dang T, Blind RD, et al. Glucocorticoid receptor phosphorylation differentially
affects target gene expression. Mol Endocrinol. 2008;22(8):1754–66.
78. Galliher-Beckley AJ, Cidlowski JA. Emerging roles of glucocorticoid receptor phosphoryla-
tion in modulating glucocorticoid hormone action in health and disease. IUBMB Life.
2009;61(10):979–86.
79. Kino T, Ichijo T, Amin ND, et al. Cyclin-dependent kinase 5 differentially regulates the
transcriptional activity of the glucocorticoid receptor through phosphorylation: clinical
implications for the nervous system response to glucocorticoids and stress. Mol Endocrinol.
2007;21(7):1552–68.
80. Itoh M, Adachi M, Yasui H, Takekawa M, Tanaka H, Imai K. Nuclear export of glucocorti-
coid receptor is enhanced by c-Jun N-terminal kinase-mediated phosphorylation. Mol
Endocrinol. 2002;16(10):2382–92.
81. Avenant C, Kotitschke A, Hapgood JP. Glucocorticoid receptor phosphorylation modulates
transcription efficacy through GRIP-1 recruitment. Biochemistry. 2010;49(5):972–85.
82. Galliher-Beckley AJ, Williams JG, Collins JB, Cidlowski JA. Glycogen synthase kinase
3β-mediated serine phosphorylation of the human glucocorticoid receptor redirects gene
expression profiles. Mol Cell Biol. 2008;28(24):7309–22.
83. Davies L, Karthikeyan N, Lynch JT, et al. Cross talk of signaling pathways in the regulation
of the glucocorticoid receptor function. Mol Endocrinol. 2008;22(6):1331–44.
84. Beck IM, De Bosscher K, Haegeman G. Glucocorticoid receptor mutants: man-made tools
for functional research. Trends Endocrinol Metab. 2011;22(8):295–310.
85. Webster JC, Jewell CM, Bodwell JE, Munck A, Sar M, Cidlowski JA. Mouse glucocorticoid
receptor phosphorylation status influences multiple functions of the receptor protein. J Biol
Chem. 1997;272(14):9287–93.
86. Graff J, Rei D, Guan JS, et al. An epigenetic blockade of cognitive functions in the neurode-
generating brain. Nature. 2012;483(7388):222–6.
87. Adzic M, Djordjevic J, Djordjevic A, et al. Acute or chronic stress induce cell compartment-
specific phosphorylation of glucocorticoid receptor and alter its transcriptional activity in
Wistar rat brain. J Endocrinol. 2009;202(1):87–97.
88. Jeanneteau F, Garabedian MJ, Chao MV. Activation of Trk neurotrophin receptors by glucocor-
ticoids provides a neuroprotective effect. Proc Natl Acad Sci U S A. 2008;105(12):4862–7.
89. Faresse N, Vitagliano JJ, Staub O. Differential ubiquitylation of the mineralocorticoid receptor
is regulated by phosphorylation. FASEB J. 2012;26(10):4373–82.
90. Denis M, Poellinger L, Wikstom AC, Gustafsson JA. Requirement of hormone for thermal
conversion of the glucocorticoid receptor to a DNA-binding state. Nature. 1988;
333(6174):686–8.
91. Davies TH, Ning YM, Sanchez ER. A new first step in activation of steroid receptors:
hormone-induced switching of FKBP51 and FKBP52 immunophilins. J Biol Chem.
2002;277(7):4597–600.
92. Vandevyver S, Dejager L, Libert C. On the trail of the glucocorticoid receptor: into the
nucleus and back. Traffic. 2012;13(3):364–74.
93. Savory JG, Hsu B, Laquian IR, et al. Discrimination between NL1- and NL2-mediated
nuclear localization of the glucocorticoid receptor. Mol Cell Biol. 1999;19(2):1025–37.
94. Freedman ND, Yamamoto KR. Importin 7 and importin alpha/importin beta are nuclear
import receptors for the glucocorticoid receptor. Mol Biol Cell. 2004;15(5):2276–86.
95. Echeverria PC, Mazaira G, Erlejman A, Gomez-Sanchez C, Piwien Pilipuk G, Galigniana MD.
Nuclear import of the glucocorticoid receptor-hsp90 complex through the nuclear pore
complex is mediated by its interaction with Nup62 and importin beta. Mol Cell Biol.
2009;29(17):4788–97.
96. Olkku A, Mahonen A. Calreticulin mediated glucocorticoid receptor export is involved in
beta-catenin translocation and Wnt signalling inhibition in human osteoblastic cells. Bone.
2009;44(4):555–65.
97. Holaska JM, Black BE, Rastinejad F, Paschal BM. Ca2+-dependent nuclear export mediated
by calreticulin. Mol Cell Biol. 2002;22(17):6286–97.
98. Amazit L, Alj Y, Tyagi RK, et al. Subcellular localization and mechanisms of nucleocytoplas-
mic trafficking of steroid receptor coactivator-1. J Biol Chem. 2003;278(34):32195–203.
99. Datson NA, van der Perk J, de Kloet ER, Vreugdenhil E. Identification of corticosteroid-
responsive genes in rat hippocampus using serial analysis of gene expression. Eur J Neurosci.
2001;14(4):675–89.
100. Liu W, Wang J, Sauter NK, Pearce D. Steroid receptor heterodimerization demonstrated
in vitro and in vivo. Proc Natl Acad Sci U S A. 1995;92(26):12480–4.
101. Nishi M, Tanaka M, Matsuda K, Sunaguchi M, Kawata M. Visualization of glucocorticoid
receptor and mineralocorticoid receptor interactions in living cells with GFP-based fluores-
cence resonance energy transfer. J Neurosci. 2004;24(21):4918–27.
102. Ou XM, Storring JM, Kushwaha N, Albert PR. Heterodimerization of mineralocorticoid and
glucocorticoid receptors at a novel negative response element of the 5-HT1A receptor gene.
J Biol Chem. 2001;276(17):14299–307.
103. Newton R, Holden NS. Separating transrepression and transactivation: a distressing divorce
for the glucocorticoid receptor? Mol Pharmacol. 2007;72(4):799–809.
104. Sakai DD, Helms S, Carlstedt-Duke J, Gustafsson JA, Rottman FM, Yamamoto KR. Hormone-
mediated repression: a negative glucocorticoid response element from the bovine prolactin
gene. Genes Dev. 1988;2(9):1144–54.
105. Surjit M, Ganti Krishna P, Mukherji A, et al. Widespread negative response elements mediate
direct repression by agonist-liganded glucocorticoid receptor. Cell. 2011;145(2):224–41.
106. Reddy TE, Pauli F, Sprouse RO, et al. Genomic determination of the glucocorticoid response
reveals unexpected mechanisms of gene regulation. Genome Res. 2009;19(12):2163–71.
107. Hudson WH, Youn C, Ortlund EA. The structural basis of direct glucocorticoid-mediated
transrepression. Nat Struct Mol Biol. 2013;20(1):53–8.
108. So AY-L, Chaivorapol C, Bolton EC, Li H, Yamamoto KR. Determinants of cell- and gene-
specific transcriptional regulation by the glucocorticoid receptor. PLoS Genet. 2007;3(6), e94.
109. Reddy TE, Gertz J, Crawford GE, Garabedian MJ, Myers RM. The hypersensitive glucocor-
ticoid response specifically regulates period 1 and expression of circadian genes. Mol Cell
Biol. 2012;32(18):3756–67.
110. Wright AP, Gustafsson JA. Mechanism of synergistic transcriptional transactivation by the
human glucocorticoid receptor. Proc Natl Acad Sci U S A. 1991;88(19):8283–7.
111. Lefstin JA, Thomas JR, Yamamoto KR. Influence of a steroid receptor DNA-binding domain
on transcriptional regulatory functions. Genes Dev. 1994;8(23):2842–56.
112. Jeanneteau FD, Lambert WM, Ismaili N, et al. BDNF and glucocorticoids regulate
corticotrophin-releasing hormone (CRH) homeostasis in the hypothalamus. Proc Natl Acad
Sci U S A. 2012;109(4):1305–10.
113. Rogatsky I, Luecke HF, Leitman DC, Yamamoto KR. Alternate surfaces of transcriptional
coregulator GRIP1 function in different glucocorticoid receptor activation and repression
contexts. Proc Natl Acad Sci. 2002;99(26):16701–6.
114. Glass CK, Saijo K. Nuclear receptor transrepression pathways that regulate inflammation in
macrophages and T cells. Nat Rev Immunol. 2010;10(5):365–76.
115. McInerney EM, Rose DW, Flynn SE, et al. Determinants of coactivator LXXLL motif speci-
ficity in nuclear receptor transcriptional activation. Genes Dev. 1998;12(21):3357–68.
116. Chinenov Y, Gupte R, Dobrovolna J, et al. Role of transcriptional coregulator GRIP1 in the
anti-inflammatory actions of glucocorticoids. Proc Natl Acad Sci. 2012;109(29):11776–81.
117. Espallergues J, Teegarden SL, Veerakumar A, et al. HDAC6 regulates glucocorticoid receptor
signaling in serotonin pathways with critical impact on stress resilience. J Neurosci.
2012;32(13):4400–16.
118. Qiu Y, Zhao Y, Becker M, et al. HDAC1 acetylation is linked to progressive modulation of
steroid receptor-induced gene transcription. Mol Cell. 2006;22(5):669–79.
119. Govindan MV. Recruitment of cAMP-response element-binding protein and histone deacety-
lase has opposite effects on glucocorticoid receptor gene transcription. J Biol Chem.
2010;285(7):4489–510.
120. L-b L, Leung DYM, Martin RJ, Goleva E. Inhibition of histone deacetylase 2 expression by
elevated glucocorticoid receptor β in steroid-resistant asthma. Am J Respir Crit Care Med.
2010;182(7):877–83.
121. Hittelman AB, Burakov D, Iniguez-Lluhi JA, Freedman LP, Garabedian MJ. Differential
regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins.
EMBO J. 1999;18(19):5380–8.
122. Malik S, Roeder RG. The metazoan Mediator co-activator complex as an integrative hub for
transcriptional regulation. Nat Rev Genet. 2010;11(11):761–72.
123. Takahashi H, Parmely TJ, Sato S, et al. Human mediator subunit MED26 functions as a docking
site for transcription elongation factors. Cell. 2011;146(1):92–104.
124. Chen W, Rogatsky I, Garabedian MJ. MED14 and MED1 differentially regulate target-specific
gene activation by the glucocorticoid Receptor. Mol Endocrinol. 2006;20(3):560–72.
125. Karst H, Joels M. Corticosterone slowly enhances miniature excitatory postsynaptic current
amplitude in mice CA1 hippocampal cells. J Neurophysiol. 2005;94(5):3479–86.
126. Olijslagers JE, de Kloet ER, Elgersma Y, van Woerden GM, Joels M, Karst H. Rapid changes
in hippocampal CA1 pyramidal cell function via pre- as well as postsynaptic membrane
mineralocorticoid receptors. Eur J Neurosci. 2008;27(10):2542–50.
127. Groc L, Choquet D, Chaouloff F. The stress hormone corticosterone conditions AMPAR
surface trafficking and synaptic potentiation. Nat Neurosci. 2008;11(8):868–70.
128. Tasker JG, Herman JP. Mechanisms of rapid glucocorticoid feedback inhibition of the
hypothalamic-pituitary-adrenal axis. Stress. 2011;14(4):398–406.
129. Di S, Malcher-Lopes R, Marcheselli VL, Bazan NG, Tasker JG. Rapid glucocorticoid-mediated
endocannabinoid release and opposing regulation of glutamate and gamma-aminobutyric acid
inputs to hypothalamic magnocellular neurons. Endocrinology. 2005;146(10):4292–301.
130. Malcher-Lopes R, Di S, Marcheselli VS, et al. Opposing crosstalk between leptin and gluco-
corticoids rapidly modulates synaptic excitation via endocannabinoid release. J Neurosci.
2006;26(24):6643–50.
131. Watts AG. Glucocorticoid regulation of peptide genes in neuroendocrine CRH neurons: a
complexity beyond negative feedback. Front Neuroendocrinol. 2005;26(3–4):109–30.
132. Karst H, Karten YJ, Reichardt HM, de Kloet ER, Schutz G, Joels M. Corticosteroid actions
in hippocampus require DNA binding of glucocorticoid receptor homodimers. Nat Neurosci.
2000;3(10):977–8.
133. Duvarci S, Pare D. Glucocorticoids enhance the excitability of principal basolateral amyg-
dala neurons. J Neurosci. 2007;27(16):4482–91.
134. Makino S, Gold PW, Schulkin J. Corticosterone effects on corticotropin-releasing hormone
mRNA in the central nucleus of the amygdala and the parvocellular region of the paraven-
tricular nucleus of the hypothalamus. Brain Res. 1994;640(1–2):105–12.
135. McEwen BS. The ever-changing brain: cellular and molecular mechanisms for the effects of
stressful experiences. Dev Neurobiol. 2011;72(6):878–90.
136. Prager EM, Johnson LR. Stress at the synapse: signal transduction mechanisms of adrenal
steroids at neuronal membranes. Sci Signal. 2009;2(86):5.
137. Roozendaal B, Okuda S, Van der Zee EA, McGaugh JL. Glucocorticoid enhancement of
memory requires arousal-induced noradrenergic activation in the basolateral amygdala. Proc
Natl Acad Sci U S A. 2006;103(17):6741–6.
138. Schmidt P, Holsboer F, Spengler D. Beta(2)-adrenergic receptors potentiate glucocorticoid
receptor transactivation via G protein beta gamma-subunits and the phosphoinositide 3-kinase
pathway. Mol Endocrinol. 2001;15(4):553–64.
139. Kaur M, Chivers JE, Giembycz MA, Newton R. Long-acting beta2-adrenoceptor agonists
synergistically enhance glucocorticoid-dependent transcription in human airway epithelial
and smooth muscle cells. Mol Pharmacol. 2008;73(1):203–14.
140. Miller AH, Pariante CM, Pearce BD. Effects of cytokines on glucocorticoid receptor expres-
sion and function. Glucocorticoid resistance and relevance to depression. Adv Exp Med Biol.
1999;461:107–16.
141. Raison CL, Capuron L, Miller AH. Cytokines sing the blues: inflammation and the pathogenesis
of depression. Trends Immunol. 2006;27(1):24–31.
142. Lambert WM, Xu CF, Neubert TA, Chao MV, Garabedian MJ, Jeanneteau FD. Brain-derived
neurotrophic factor signaling rewrites the glucocorticoid transcriptome via glucocorticoid
receptor phosphorylation. Mol Cell Biol. 2013;33(18):3700–14.
143. Chen DY, Bambah-Mukku D, Pollonini G, Alberini CM. Glucocorticoid receptors recruit the
CaMKIIalpha-BDNF-CREB pathways to mediate memory consolidation. Nat Neurosci.
2012;15(12):1707–14.
144. de Quervain DJ, Poirier R, Wollmer MA, et al. Glucocorticoid-related genetic susceptibility
for Alzheimer’s disease. Hum Mol Genet. 2004;13(1):47–52.
145. Green KN, Billings LM, Roozendaal B, McGaugh JL, LaFerla FM. Glucocorticoids increase
amyloid-beta and tau pathology in a mouse model of Alzheimer’s disease. J Neurosci.
2006;26(35):9047–56.
146. Ito K, Ito M, Elliott WM, et al. Decreased histone deacetylase activity in chronic obstructive
pulmonary disease. N Engl J Med. 2005;352(19):1967–76.
147. Barnes PJ, Adcock IM. Glucocorticoid resistance in inflammatory diseases. Lancet.
2009;373(9678):1905–17.
148. Zhang Y, Leung DY, Nordeen SK, Goleva E. Estrogen inhibits glucocorticoid action via pro-
tein phosphatase 5 (PP5)-mediated glucocorticoid receptor dephosphorylation. J Biol Chem.
2009;284(36):24542–52.
149. Altarejos JY, Montminy M. CREB and the CRTC co-activators: sensors for hormonal and
metabolic signals. Nat Rev Mol Cell Biol. 2011;12(3):141–51.
Chapter 3
Mechanisms of Glucocorticoid-Regulated
Gene Transcription
Sebastiaan H. Meijsing
Abstract One fascinating aspect of glucocorticoid signaling is their broad range of

physiological and pharmacological effects. These effects are at least in part a conse-
quence of transcriptional regulation by the glucocorticoid receptor (GR). Activation
of GR by glucocorticoids results in tissue-specific changes in gene expression levels
with some genes being activated whereas others are repressed. This raises two ques-
tions: First, how does GR regulate different subsets of target genes in different tis-
sues? And second, how can GR both activate and repress the expression of genes?
To answer these questions, this chapter will describe the function of the various
“components” and how they cooperate to mediate the transcriptional responses to
glucocorticoids. The first “component” is GR itself. The second “component” is the
chromatin and its role in specifying where in the genome GR binds. Binding to the
genome however is just the first step in regulating the expression of genes and tran-
scriptional regulation by GR depends on the recruitment of coregulator proteins that
either directly or indirectly influence the recruitment and or activity of RNA poly-
merase II. Ultimately, the integration of inputs including GR isoform, DNA
sequence, chromatin and cooperation with coregulators determines which genes are
regulated and the direction of their regulation.
Keywords Transcription • Coregulators • Chromatin • Cis-regulatory elements

• Glucocorticoid receptor
Structure of the Glucocorticoid Receptor
Although glucocorticoids have been used clinically from the 1940s [1], it wasn’t
until 1984 when the coding sequence for its receptor was initially isolated from rat [2]
and soon after its human homolog was cloned [3]. The human gene coding for GR
S.H. Meijsing, Ph.D. (*)

Department of Computational Molecular Biology, Max Planck Institute
for Molecular Biology, Ihnestrasse 63-73, Berlin 14195, Germany

60 S.H. Meijsing
Fig. 3.1 Domain structure of GR and sites of post-translational modifications. Shown are the
functional domains of GR: The activation function 1 and -2 (AF1, AF2), the DNA-binding domain
(DBD), hinge region (H) and ligand-binding domain (LBD). Also shown are post-translational
modifications (phosphorylation (P); acetylation (A); ubiquitination (U); sumoylation (S)) of either
Serine (S) or Lysine (K) residues. Amino acid numbering refers to human GR
consists of nine exons and can produce a variety of different gene products through
alternative splicing, alternative translational initiation and by post-translational
modifications. Here we describe the functional domains of GR and how alternative
splicing, translational initiation and post-translational modifications generate recep-
tor isoforms with distinct expression profiles and target genes.
Functional Domains of the Glucocorticoid Receptor
A combination of biochemical (limited proteolysis, [4]) and molecular biological

(mutagenesis and domain fusions) approaches have uncovered that the glucocorti-
coid receptor is a modular protein with several functional domains (Fig. 3.1).
The N-terminal domain of the glucocorticoid receptor contains the activation
function 1 domain (AF1, amino acids 77–262, throughout amino acid numbering
refers to human GR), which is involved in transcriptional regulation [5]. In contrast
to the AF2 domain (see below), the AF1 domain is constitutively active meaning
that its activity does not rely on the presence of hormone [5]. GR-dependent tran-
scriptional regulation critically depends on its interaction with several coregulator
proteins that either directly or indirectly recruit or influence the activity of RNA
polymerase II (the role of coregulators in transcription is described in section
“Transcriptional Regulation by GR”). For the AF1 domain these interaction part-
ners include p160, TIF2, DRIP/TRAP and TBP [6]. How these proteins interact
with the AF1 domain is largely unknown. No clear interaction domains have been
identified in either GR or in the proteins interacting with AF1 and computational
predictions and experimental approaches indicate that large parts of the AF1 domain
are intrinsically disordered [7]. This may allow the AF1 to adopt different confor-
mations to create interaction surfaces for a variety of coregulators.
DNA binding by GR is mediated by the DNA binding domain (DBD, amino
acids 420–480), which is conserved across steroid hormone receptor proteins. GR can
bind as a homodimer to DNA sequences consisting of inverted repeats of a loosely
defined recognition sequence separated by a three base pair spacer (Fig. 3.2) [8].
The three-dimensional structure shows that the DBD contains several alpha-helices.
3 Mechanisms of Glucocorticoid-Regulated Gene Transcription 61
Fig. 3.2 Classical GR signaling pathway. Upon ligand binding, cytoplasmic GR dissociates from
chaperone proteins and translocates to the nucleus where it interacts with specific DNA sequences
to control the expression of associated target genes
One helix mediates base-specific DNA contacts whereas an alpha-helix at the

C-terminus of the DBD makes several non-specific phosphate backbone and minor
groove contacts [9, 10]. Two zinc-fingers ascertain proper folding of the DBD to
coordinate DNA recognition and dimerization [9, 11, 12]. Like other domains, the
DBD interacts with several coregulators including JDP1, JDP2, HMG1, HMG2,
GT198 and SET/TAFI-β(beta) [13–16]. In addition to direct DNA binding, GR can
also be tethered to the DNA for example via its interaction with activator protein 1
(AP1), NFκ(kappa)B or STAT3 (Fig. 3.3) [17–22]. Interestingly, also here the DBD
appears responsible for tethered DNA interactions by directly interacting with the
c-Jun/c-Fos or p65 subunits of AP1 and NFκ(kappa)B respectively [17, 21, 23].
62 S.H. Meijsing
Fig. 3.3 DNA binding by GR. Glucocorticoid-activated GR can interact with DNA either directly
(top), indirectly via tethering (middle) or can bind to composite elements where it engages in
cross-talk with neighboring DNA-bound transcriptional regulatory factors (bottom)
Arguing for an important role of the DBD in tethered DNA binding, mutations in
this domain interfere with GR’s function as a transcriptional repressor at sites where
it is tethered to the DNA by either AP1 or NFκ(kappa)B [17, 23].
C-terminal to the DBD the hinge region connects the DBD to the ligand binding
domain (LBD). The LBD consists of 12 alpha helixes [24] and ligand binding is
facilitated by several alpha helixes that together form a hydrophobic pocket [24].
Furthermore, the LBD harbors a second dimerization domain, sequences involved
in nuclear translocation upon hormone binding and the activation function 2 (AF2
domain), which mediates the interaction with several coregulators (reviewed in [25]).
In the absence of ligand, GR is predominantly cytoplasmic where the interaction of
the LBD with chaperone proteins such as hsp90 and p23 keep GR in a hormone-
binding competent state (Fig. 3.2) (reviewed in [26]). Ligand binding results in
Hsp90 dissociation, nuclear translocation and conformational changes in helix 12
(Fig. 3.2) [26]. These conformational changes facilitate the interaction of the AF2
domain with a variety of coregulators containing LXXLL motifs [24, 26] including
p160 coactivator family members SRC1 and GRIP1 [27, 28].
Although the domains of GR can function in isolation, recent studies indicate
that the domains of nuclear hormone receptors are both functionally and structurally
connected [29–32]. These domain-connections can be rewired depending on the
context in which GR is active and accordingly, different combinations of GR
domains are required to regulate the expression of individual genes [33].
Creating Functional Diversity: Glucocorticoid Receptor

Isoforms and Post-translational Modifications
Although a single gene (NR3C1) codes for the glucocorticoid receptor protein,
this gene can give rise to several isoforms with unique expression profiles [34, 35].
In addition, post-translational modifications of these isoforms further expand the
diversity of responses to glucocorticoids. Besides the predominant GR isoform
GRα(alpha), alternative splicing of GR can generate at least four additional iso-
forms: GRβ(beta), GRγ(gamma), GR-A and GR-P [25]. GRβ(beta) differs from
GRα(alpha) in its LBD and is unable to bind hormone [36]. The GRβ(beta) isoform
appears to be transcriptionally inactive and can antagonize the activity of GRα(alpha)
[37]. Accordingly, increased GRβ(beta) levels have been linked to glucocorticoid
resistance in a variety of diseases including asthma, rheumatoid arthritis and acute
lymphoblastic leukemia [37]. Use of an alternative splice-donor site generates the
GRγ(gamma) isoform, which differs from GRα(alpha) in having a single additional
Arginine inserted in the DBD [38]. The Arginine insertion results in gene-specific
effects with most genes being unaffected, whereas some genes are regulated more
strongly and others more weakly [30]. Consistent with a reduced activity towards
certain target genes, GRγ(gamma) has been linked to glucocorticoid resistance in
childhood acute lymphoblastic leukemia and small cell lung carcinoma cells [39, 40].
The GR-A and GR-P isoforms lack exons encoding the LBD and consequently lack
the ability to bind ligand [41]. Similar to GRβ(beta) and GRγ(gamma), GR-P can
antagonize the transcriptional activity of GRα(alpha) and has been linked to gluco-
corticoid-resistance [42].
Additional GR isoforms are produced as a consequence of alternative transla-
tional initiation, which generates GR proteins with shorter N-terminal domains
[43]. These translational isoforms differ in their tissue-specific expression and the
transcriptional programs they initiate [44]. Another mechanism that can generate
functional diversity are post-translational modifications. Such modifications can
alter the function of GR and include phosphorylation, acetylation, sumoylation and
ubiquitination (Fig. 3.1) [25]. One example of a post-translational modification that
influences GR activity is the phosphorylation of Serine residues in the N-terminus
of GR. The phosphorylation modulates GR’s interaction with coregulators and
64 S.H. Meijsing
differentially affects its activity towards individual target genes [45]. Another exam-
ple is acetylation of GR by CLOCK, a histone acetyltransferase (HAT) involved in
circadian rhythm. The CLOCK-dependent acetylation of multiple Lysines in the
hinge region of GR interferes with DNA binding resulting in changes in the expression
level of a subset of GR target genes [46, 47].
Together, alternative splicing, translational initiation and post-translational
modifications generate a variety of GR variants with different target genes.
Consequently, differences between cell types and tissues in the expression level of
these isoforms and of the enzymes responsible for post-translational modifications
likely contribute to the highly tissue-specific effects of glucocorticoids.
Chromosomal Binding of GR
Hormone binding by GR results in nuclear translocation and allows the receptor to

bind to specific genomic sequences (Fig. 3.2). The binding of GR to glucocorticoid
response elements (GREs) constitutes an essential first step in the regulation of the
expression of associated target genes. Here we discuss the contributions of DNA
sequence elements and the chromatin landscape in guiding GR to its appropriate
genomic destination.
Binding to the Genome: “Classical” GR Binding Sequences
The first described “classical” mode of DNA binding is for liganded GR to associate
as a dimer to GR binding sequences (GBSs) [8]. GBSs are typically imperfect pal-
indromic hexameric half-sites separated by a 3bp spacer (Fig. 3.2). Historically,
mostly for practical reasons, studies to identify regulatory sequences exploited by
GR to regulate target genes were focused on promoter regions and have uncovered
numerous promoter-proximal GBSs [48–50]. In support of a role of these GBSs in
the regulation of associated target genes, genomic regions that harbor a GBS as well
as simply the 15bp GBS are sufficient to facilitate GR-dependent transcriptional
activation when localized upstream of heterologous promoters [10, 49]. However,
up until recently it was unclear whether promoter-proximal binding by GR is the
exception or the rule that governs genomic binding and the control of target gene
expression. Technological advances that combine chromatin immunoprecipitation
(ChIP) with next generation sequencing (ChIP-seq) allow the unbiased genome-
wide identification of GR binding sites [51, 52]. Several ChIP-seq studies have
revealed that promoter-proximal binding by GR appears to be the exception and that
the majority of GR binding is at promoter-distal locations [53–56]. One representa-
tive study showed that for genes that are up regulated in response to glucocorticoid
treatment (likely GR target genes), 50 % of the binding sites were located at a dis-
tance greater than 10 kb from the transcriptional start-site (TSS) [54]. Even more
striking, for down regulated genes the median distance to the TSS was >100 kb [54].
The finding that only the minority of GR binds promoter-proximal is not specific
for GR but is also seen for related hormone receptors including ER, PPAR and AR
[55, 57, 58]. This suggests that long-range regulation by GR and other hormone
receptors might be responsible for the regulation of a large fraction of target genes.
In support of this idea, a study using chromatin conformation capture showed that
the promoter of the GR-regulated gene Ciz1 was contacted by a GR binding region
located nearly 30 kb away [59].
Bioinformatical analysis of genomic regions bound by GR shows that the canon-
ical 15 bp GBS is highly enriched at such binding sites with one study reporting that
58 % of the bound regions contains a GBS [56]. This underscores the important role
of the canonical GBS in guiding GR to specific genomic locations. It does however
also hint at the existence of alternative sequences that facilitate GR binding at the
remaining 42 % of GR-bound regions.
Binding to the Genome: Other Sequences
Several ChIP-seq studies made the striking observation that only a fraction of all GR
binding regions appears to contain the canonical 15 bp GBS [54, 56]. This indicates
that GR may be able to bind to very degenerate sequences with the assistance of
another transcriptional regulatory factor. Moreover, sequences other than the canoni-
cal GBS might be able to recruit GR to specific genomic loci (Fig. 3.3). Such sequences
could either bind proteins that tether GR to the DNA or alternatively GR might be able
to interact directly with a broader spectrum of DNA sequences. In support of this,
studies with the hormone-repressed gene POMC uncovered GR-bound sequences that
resemble the canonical GBSs somewhat but lack similarity to the consensus motif at
key positions [60]. Interestingly, whereas regulation from canonical GBSs is typically
associated with activation of transcription, the promoter region of the POMC gene
mediated transcriptional repression when fused to a luciferase reporter gene and was
therefore called negative glucocorticoid response element (nGRE). This repression
was lost when the GBS-like sequence was changed to resemble a canonical GBS [60].
In isolation however, this sequence failed to confer repression arguing that its function
relies on other functional elements present at the POMC promoter [60]. Another class
of sequences that has been proposed to directly interact with GR are inverted repeats
of CTCC that have a spacing of either 0, 1 or 2 base pairs [61]. These sequences are
associated with genes that are repressed by GR. Notably, binding of GR to canoni-
cal GBSs strictly requires a 3 bp spacer to position two GR molecules such that they
can effectively dimerize [9]. The variable spacing for these nGREs suggests that
dimerization might not be required at these nGREs and accordingly structural studies
suggest monomeric GR-binding to the half sites (Fig. 3.3) [62].
Together these studies suggest that GR is able to interact directly with a variety
of sequence motifs to control the expression of associated target genes.
66 S.H. Meijsing
Binding to the Genome: Tethered Binding
The absence of canonical GBSs in ChIP-seq peaks can also be explained by tethered
DNA binding by GR (Fig. 3.3). Tethered GR binding has predominantly been linked
to transcriptional repression and has been proposed for several transcriptional regu-
latory factors including NFκ(kappa)B [21], AP1 [18–20], STAT3 [22, 63] and
NGFI-B [64]. For NFκ(kappa)B, the p65 (RelA) subunit physically interacts with
GR [21] and recruits GR to NFκ(kappa)B response elements [65]. The ability of GR
to repress from NFκ(kappa)B sites can be recapitulated using reporters plasmids
simply harboring NFκ(kappa)B sites driving the expression of a luciferase reporter
gene arguing that tethered binding to NFκ(kappa)B mediates the repressive effects
of GR [66]. Genome-wide studies using ChIP-seq, showed that co-treatment of
cells with dexamethasone, a synthetic GR ligand, and with TNFα(alpha) to activate
NFκ(kappa)B resulted in GR binding to approximately a thousand additional
genomic regions when compared to the binding profile when cells were treated with
dexamethasone alone [67]. These additional binding regions are enriched for
NFκ(kappa)B binding sites suggesting that tethered binding might occur quite fre-
quently [67]. However, it could also be that part or all of the gained binding is a
simple consequence of NFκ(kappa)B-induced changes in chromatin accessibility
that makes previously inaccessible GR binding regions available.
AP1 is another factor that physically interacts with GR [18] and has been impli-
cated in tethering GR to DNA [68]. Similar to the observation for NFκ(kappa)B,
tethered binding by AP1 is linked to transcriptional repression. This repression can
be recapitulated using a luciferase reporter that contains a single copy of the AP1
consensus sequence driving expression of a luciferase reporter gene [69]. Other
proteins implicated in tethering GR to the DNA are members of the signal trans-
ducer and activator of transcription (STAT) family. GR physically interacts with
several STAT proteins including STAT1 [70], STAT3 [63] and STAT5 [71]. Genome-
wide profiling of STAT3 and GR binding suggests that GR may be tethered to the
DNA by STAT3 at about 300 genomic binding sites and that such binding events are
almost exclusively associated with transcriptional repression by GR [22].
Combinatorial Binding by GR and Regulation
Binding sites for GR in the genome are not present in isolation but are surrounded
by sequence motifs that can be occupied by other transcriptional regulatory factors
(Fig. 3.3). Accordingly, analysis of GR ChIP-seq peaks shows a cell-type specific
overrepresentation of various sequence motifs [56]. Recent studies underscore the
important role of combinatorial binding in transcriptional regulation by GR and for
transcriptional regulatory factors in general [72, 73]. The study by Siersbaek and
coworkers analyzed five transcriptional regulatory factors involved in adipogenesis
including GR (out of the more than a thousand transcriptional regulatory factors
encoded in the human genome) [72]. ChIP-seq of these factors showed combinatorial
binding of GR with at least one other factor for >93 % and simultaneous binding of all
5 factors for 25 % of all GR binding events [72]. These “hotspots” of transcriptional
regulatory factor binding were also found by the encode consortium that looked at
>100 transcriptional regulatory factors [73, 74]. The co-occurrence of a GR binding
site with recognition sequences for “partner” transcriptional regulatory factors can
give rise to a broad spectrum of signaling cross-talk. A commonly observed type of
cross-talk is a synergetic interaction between GR and other transcription factors.
For example, knockdown of C/EBPβ(beta) results in a reduction of GR binding at
co-occupied sites whereas binding at control sites that are not co-occupied are not
affected [72]. The knockdown of factors co-occupying “hotspots” revealed a highly
cooperative nature of transcriptional regulatory factor binding at these sites [72].
Synergetic interactions likely reflect at least in part effects of chromatin (chromatin
accessibility) where several transcriptional regulatory factors cooperate to keep
genomic sites accessible. This might explain the many synergetic interactions with
other transcriptional regulatory factors that have been described for GR which
include SP1, NF1, STAT3, COUP-TFII and AP1 [75–78].
The cross-talk between GR and other transcriptional regulatory factors at combina-
torial binding sites can also be antagonistic. For example, at the osteocalcin promoter,
the GR binding site overlaps the TATA box and GR binding thereby antagonizes TFIID
binding to the TATA box and transcriptional initiation [79]. Another example of an
antagonistic interaction between GR due to overlapping binding sites is found at the
prolactin gene where the GR binding sites overlaps site for Oct1 and Pbx1 [80]. GR can
also antagonize the activity of other factors via non-overlapping binding sites as was
shown for the glutathione S-transferase A2 gene [81]. Here binding of GR to a GBS-
like sequence results in the recruitment of the transcriptional co-repressor SMRT to
repress C/EBP- and NRF2-mediated activation [81]. For the mouse proliferin gene,
depending on the composition of the proteins that bind to the dimeric AP1 binding site,
GR can either act antagonistically or synergistically [82].
The complex nature of interactions between GR and other transcriptional regu-
latory proteins illustrates the complexity of signaling cross-talk occurring at com-
posite elements. This complexity can potentiate the ability of GR to regulate genes
in a cell type specific manner and to tailor its activity towards individual genes.
Gene-specific effects can for instance be a consequence of differences in the local
sequence of the GR binding site. Similarly, the cell-type specific expression and
binding of transcriptional regulatory factors that engage in synergistic interactions
with GR can explain tissue-specific effects.
DNA Binding: Influence of Chromatin Structure

on GR Binding
Another fascinating fact that the genome-wide analysis uncovered is that the
genomic binding pattern of GR shows little overlap (<5 % [53]) between cell-types
[56], and personal unpublished results). The highly tissue-specific binding by GR
68 S.H. Meijsing
Fig. 3.4 Chromatin features influence DNA binding by GR. Chromatin features either negatively
or positively correlating with genomic DNA binding by GR. Negative: closed chromatin (top).
Positive: closed chromatin, nucleosome free DNA, enrichment for the H2A.Z histone variant, pres-
ence of other transcriptional regulatory factors and of histone modifications H3K4me1 and
H3K27ac (bottom)
indicates that the sequence of GR binding sites alone is insufficient to explain where
in the genome GR binds. Thus, DNA sequence specifies where in the genome GR
could bind and other inputs including the chromatin landscape are needed to specify
where GR actually does bind (Fig. 3.4). One aspect of the chromatin landscape that
appears to be a major contributor is chromatin-accessibility as assayed by DNase-I
accessibility assays [53, 78]. These studies showed that the majority of GR binding
occurs in genomic regions that are DNase-I accessible (“open”) prior to hormone
treatment [53, 78]. Interestingly, which regions of the genome are actually “open”
appears to be highly cell-type specific [53, 73]. So an emerging picture is that
cell-type specific “chromatin-accessibility factors”, other than GR, specify which

regions of the genome are “open” and thereby where in the genome GR can bind
and which genes it can regulate. One such “chromatin-accessibility factor” is AP1,
whose binding, according to one study, overlaps with >50 % of GR binding sites
[78]. Consistent with a role in facilitating GR binding, dominant negative AP1 and
depletion of AP1 levels by siRNAs resulted in reduced chromatin accessibility and
a loss of GR binding at co-occupied sites [78]. Another factor linking chromatin
accessibility and steroid receptor binding is the forkhead box A1 (FoxA1) protein.
FoxA1 induces DNase-I hypersensitivity [83, 84] indicative of open chromatin and
facilitates GR binding [83]. Conversely, depletion of FoxA1 results in a redistribu-
tion of genomic estrogen receptor (ER), androgen receptor (AR) and GR binding
[85, 86]. However, for ER depletion of FoxA1 results in an almost complete loss of
ER binding [85] whereas for AR and GR FoxA1 depletion results in a redistribution
of binding [86] indicating that the role of FoxA1 for GR and AR is more complex
than simply facilitating access to the genome.
The observation that transcriptional regulatory factors typically bind together to
“hotspots” and mutually stimulate genomic binding [72] suggests that they might
cooperatively keep certain regulatory regions open. This can also explain the tissue-
specific binding patterns observed for GR due to cell-type-specific expression of
these cooperation partners. Notably, not all GR binding occurs at open regions and
for a subset of binding events GR appears to act as a “chromatin-accessibility
factor” [53, 78]. Analysis of sequence motifs for closed chromatin GR binding sites
showed that binding to these sites is mediated by GBSs with high motif scores [87]
suggesting that high-affinity binding might be a prerequisite for GR binding at
closed chromatin. Interestingly, whereas GR binding sites in “open” chromatin
show little overlap between cell types, binding at “closed” sites is often shared
between cell types [87] indicating that for these sites GR might not rely on other
factors for binding.
Other chromatin features linked to GR binding are the presence of nucleosomes,
the post-translational modification state of nucleosomal histones and the presence
of histone variants. GR binding sites are enriched for several chromatin features
linked to enhancers including monomethylation of histone H3 Lysine 4, acetylation
of histone H3 Lysine 27 and enrichment of the histone variant H2A.Z [88, 89] and
unpublished data from my group). The enrichment profile of these histone modifi-
cations shows a bimodal peak flanking the site of GR-binding, which indicates that
GR typically binds to DNA located between two nucleosomes (Fig. 3.4) [88].
However, despite the significant correlation between histone modifications and GR
binding, future studies are needed to determine if and how these are causatively
connected.
In conclusion, the integration of DNA sequence information, cooperation with
other transcriptional regulatory factors and chromatin features appears to determine
where in the genome GR binds and ultimately which genes it regulates in a particular
cell type.
70 S.H. Meijsing
Transcriptional Regulation by GR
The transcriptional process begins with the recruitment of RNA polymerases to the
transcriptional start site (TSS) by the pre-initiation complex (PIC). After recruitment,
the RNA polymerases proceed through distinct steps of the transcription cycle:
initiation, elongation and termination. RNA polymerases are multi-protein com-
plexes, which change their composition and/or carry different modifications depen-
dent on the step in the transcription cycle. For example, RNA polymerase II is
differentially phosphorylated in the C-terminal tail domain (CTD) of its largest sub-
unit dependent on whether it is initiating, elongating or terminating (reviewed in
[90]). Gene regulation depends on the action of transcriptional regulatory factors,
like GR. GR can exploit a broad spectrum of mechanisms to influence the expres-
sion level of genes. Such mechanisms include influencing RNA stability [91–93],
sequestering or influencing the activity state of other transcriptional regulatory factors
by protein:protein interactions [94, 95] that thus does not require direct interactions
of GR with DNA or with the RNA polymerase machinery. Here however, we will
focus on transcriptional effects in response to glucocorticoids that involve DNA
binding and RNA polymerase II. GR may affect the state of RNA Polymerase II
directly (e.g., the phosphorylation state of the CTD or the assembly of the PIC).
Alternatively, GR can modulate RNA polymerase II’s regulatory role indirectly by
recruiting coregulators such as histone modifying enzymes, chromatin remodelers
or the mediator complex that bridges the interaction with RNA polymerase II
(Fig. 3.5). GR can either increase the transcription rate (hence acting as an activator)
or can reduce—or even eliminate—transcription (acting as a repressor). This para-
graph presents an overview of different classes of coregulators and their role in
mediating the transcriptional effects of GR.
Interaction with Coregulators: Interactions with the Basal

Transcriptional Machinery
Perhaps the most straightforward way for GR to influence transcription is by inter-

acting directly or indirectly with components of the basal transcriptional machinery
(Fig. 3.5). A direct interaction of the GR’s AF1 domain with TBP, which is part of
the TFIID component of the pre-initiation complex, suggests that GR can promote
transcriptional initiation by recruiting TFIID to promoters of target genes [96].
Furthermore, GR interacts with p300/CBP, which in turn interacts with TFIIB,
another component of the pre-initiation complex, and thereby indirectly linking GR
to the basal transcriptional machinery [97–99]. The glucocorticoid receptor can also
recruit RNA polymerase II via its interaction both physically and functionally with
components (MED1 and MED14) of the mediator complex, which interacts with
the CTD of RNA polymerase II [100, 101].
Fig. 3.5 Coregulators and their role in GR-dependent regulation of promoter activity. Overview
of interacting coregulators (proteins and RNA) of GR that can either directly or indirectly influ-
ence the recruitment or activity of RNA polymerase II and thereby the transcriptional output.
Abbreviations: HATS histone acetyltransferases, HDACs histone deacetylases, NELF negative
elongation factor, PIC pre-initiation complex, TBP TATA-binding protein, eRNA enhancer RNA
Transcriptional control by GR is also exerted at the level of transcriptional

elongation, the step in the transcription cycle downstream of transcriptional initiation.
For example, GR can interact with proteins that stimulate elongation (elongation
factor RNA polymerase (ELL)), resulting in increased levels of transcript [102].
Conversely, at the IL8 gene GR displaces p-TEFb, a factor that stimulates elongation
[65]. The displacement prevents the p-TEFb-dependent Serine 2 phosphorylation of
the CTD of RNA polymerase II and consequently reduces transcriptional elongation
at the IL8 gene [65, 103]. Furthermore, GR interacts with suppressors of elongation
(negative elongation factor (NELF), [104]). An elegant study in macrophages shows
that GR can either repress the expression of genes at the level of RNA polymerase II
recruitment or by recruiting NELF, which results in a pausing of RNA polymerase II
[104]. In agreement with a role for NELF in mediating the effects of GR, repression
was specifically lost for the elongation-controlled genes in NELF-deficient macro-
phages [104]. Together these studies indicate that GR directly or indirectly contacts
components of the basal RNA polymerase II machinery and thereby can influence
gene expression by affecting different stages of the transcription cycle.
72 S.H. Meijsing
Coregulators That Influence Chromatin Structure

and Histone Modification States
In eukaryotes DNA accessibility and chromatin structure play an important role in

specifying the expression level of genes. Eukaryotic genomes are packaged into
chromatin, whose basic repeating unit is the nucleosome [105, 106]. Nucleosomes
form by wrapping 147 base pairs of DNA around an octamer of the four core his-
tones (H2A, H2B, H3 and H4) and can be found approximately every 200 base pairs
throughout the genome [107]. Their presence affects all DNA-dependent processes,
including DNA-repair, DNA replication and transcription. For instance, in vitro
a chromatinized DNA template prevents RNA polymerase II from initiating tran-
scription [108]. Even before the identification of the coregulators responsible, stud-
ies of promoters of hormone-activated genes showed that transcriptional regulation
by GR is tightly coupled to chromatin remodeling [109, 110]. Especially studies
with the mouse mammary tumor virus (MMTV) have been instrumental in dissect-
ing the steps needed for transcriptional activation (reviewed in [110]). These steps
include the recruitment of chromatin modifying enzymes, nucleosome reposition-
ing and changes in sensitivity to nucleases, ultimately resulting in the recruitment of
RNA polymerase II. GR-dependent chromatin remodeling is mediated by its inter-
action with a variety of coregulators that modify chromatin structure and thereby
indirectly the recruitment of RNA polymerase II. The first class of GR-interacting
chromatin modifiers are members of ATP-dependent chromatin remodelers that can
move and remove nucleosomes. Specifically, GR interacts with the ATP-dependent
chromatin remodeling complex SWI/SNF [98, 111]. This interaction is mediated by
BAF proteins that are part of the SWI/SNF complex [112]. The SWI/SNF complex
contains one of two possible core ATP-ase subunits [110] Brm or BRG1 and a
physical and functional connection between both ATPase subunits and GR activity
has been shown [89, 113, 114]. The interaction with the SWI/SNF complex is
essential for GR-dependent transcriptional activation of the MMTV promoter [98].
Here, the SWI/SNF complex repositions nucleosomes to allow other transcriptional
regulatory factors and TBP to bind and thereby facilitates the assembly of the pre-
initiation complex at the promoter (reviewed in [110]). For endogenous genes, the
effects of disrupting Brm or BRG1 activity, by either dominant negative versions of
these proteins or by knocking down their expression using siRNA, results in gene-
specific effects with some genes being affected whereas others are not [89, 114].
The mechanisms responsible for the facultative requirement for Brm and BRG1 are
unknown, but might reflect the fact that for certain genes alternative mechanisms
ensure appropriate nucleosome positioning and transcriptional initiation.
The second class of chromatin modifying enzymes that interact with GR are
enzymes that post-translationally modify histones. These histone modifications
can act as recognition signals for proteins [115]. For example, trimethylated
Lysine 4 of histone H3 is recognized by TFIID providing a direct link between
histone modifications and the basal transcriptional machinery [116]. Additionally,
histone modifications might influence transcription by loosening the chromatin.

This occurs when Lysines are acetylated which removes its positive charge thereby
reducing the affinity between DNA and histones [117]. One coregulator that acts as
a coactivator of GR is the histone acetyltransferase p300 [118]. Conversely,
enzymes that remove acetylation marks, histone deacetylases (HDACs), can act as
corepressors of GR [119]. Examples of HDACs or complexes containing HDACs
that interact with GR are NcoR, SMRT and HDAC2 [61, 119]. Although the activi-
ties of HATs and HDACs might be a consequence of “loosening” the chromatin,
their role is likely to be more complex. One added level of complexity is that in
addition to histones, these enzymes can also modify transcriptional regulatory fac-
tors, chaperones like hsp90 and coregulators [47, 120]. For example, acetylation of
GR by either CLOCK or GCN5 interferes with GR’s ability to interact with DNA
[47, 121]. Further illustrating the complexity of the interaction, the GR-interacting
coregulator GRIP1 acts as a coactivator for some GR target genes whereas it acts
as a corepressor at others [122]. In addition to coregulators that modify the acetyla-
tion state of Lysines, GR also interacts with histone modifying enzymes CARM1
and G9a that methylate respectively Arginine or Lysine residues of histones and
other proteins [123, 124].
In conclusion, genomic binding by GR coordinates the recruitment of a large
variety of coregulator proteins. These coregulators specify the activity as well as the
direction of the transcriptional responses to glucocorticoids at individual target
genes. However, the underlying mechanisms responsible for the context-specific
requirement of coregulators remain largely unknown. One possible explanation
could be that the combinatorial binding of GR and another factor creates an inter-
action surface for coregulators that is not present when these factors bind in isola-
tion. Furthermore, the DNA binding site responsible for GR recruitment appears to
play an important role as tethering sites and non-canonical GBSs are typically asso-
ciated with repression and preferentially recruit corepressors whereas canonical
GBSs direct the assembly of regulatory complexes that usually activate transcription.
The role of the DNA sequence might in part be explained by the fact that DNA
induces sequence-specific conformational changes in the DBD of GR [10, 125].
These conformational changes could be propagated to domains of GR engaged in
protein:protein interactions thereby explaining the context specific signaling cross-talk
between GR and coregulators.
Concluding Remarks and Future Perspectives
The past decades have generated a wealth of mechanistic insight into how GR
orchestrates the transcriptional response of cells and tissues to glucocorticoid
hormones. It is becoming increasingly clear that these responses are highly context-
specific and that chromatin plays a key role in dictating which transcriptional
program is initiation in a particular cell type. In addition to the cell-type-specific
74 S.H. Meijsing
effects, GR also appears to have highly gene-specific effects within a cell. This might
complicate research as there are perhaps few universally applicable operating prin-
ciples for GR in transcriptional regulation. It also provides an opportunity to try to
activate GR in a targeted way that may selectively affect the expression of a subset
of genes and thereby might result in therapeutic usage of glucocorticoids with fewer
side effects. One approach in this regard has been to develop synthetic GR ligands
with selective activities [126, 127]. Several such ligands do indeed regulate subsets
of GR target genes [126, 127]. However, if and to what extend ligands can be identi-
fied that display such selectivity towards the therapeutically relevant target genes
remains to be seen.
There are still lots of open-ended questions related to transcriptional responses to
glucocorticoids that will keep researchers busy for decades to come. For example,
ChIP-seq experiments have uncovered thousands of GR binding sites and although
regulated genes tend to have more GR binding sites in their vicinity, there are plenty
of genes that are not regulated despite having a GR binding site nearby. This raises
the question: What distinguishes a productive GR binding site (resulting in the regu-
lation of associated genes) from ones where nearby genes are not regulated?
A major complication in answering this question is that binding sites are assigned to
a gene based on proximity along the linear DNA chain and not based on established
functional connections between binding sites and genes. Some of these binding
sites are located 100 s of kb away from the TSS and therefore could just as well be
connected to other genes that are perhaps closer when the three dimensional organi-
zation of the nucleus is taken into account. Recently developed techniques to edit
the genome like zinc finger nucleases (ZFNs), transcription activator-like effector
nucleases (TALENs) and the CRISPR/Cas9 RNA-guided system provide the oppor-
tunity to disrupt genomic binding sites of GR and thereby to determine functional
connections between binding sites and regulated genes [128–130]. Another
approach to link binding sites to genes is to systematically determine the physical
contacts between GR binding sites and TSSs of genes. Such long-range looping
interactions can be identified with the use of chromatin conformation capture
(3C)-based techniques and have shown a clear correlation between long-range con-
tacts and transcriptional regulation by transcriptional regulatory factors [131, 132].
A final challenge is to understand how the integration of various inputs warrants that
the right genes are expressed at the correct level in response to glucocorticoids.
Many of these inputs that modulate the transcriptional responses have been identi-
fied including receptor isoform, post-translational modification state, ligand and
interaction with other biological macromolecules including proteins and
DNA. Likely however, additional inputs exist. For example, the role of the non-
coding RNA universe is still largely unexplored and studies with ER have shown
that so called enhancer RNAs (eRNAs, see Fig. 3.5) produced at ER binding sites
are required for long-range looping and the transcriptional regulation of ER target
genes [133]. Ultimately, a detailed knowledge of the signaling inputs and how they
are integrated at individual genes will yield a greater understanding of the heteroge-
neity in GR signaling in health and disease and may one day improve the therapeu-
tic use of glucocorticoids in the clinic.
References
1. Benedek TG. History of the development of corticosteroid therapy. Clin Exp Rheumatol.
2011;29(5 Suppl 68):5–12.
2. Miesfeld R, Okret S, Wikstrom AC, Wrange O, Gustafsson JA, Yamamoto KR. Characterization
of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature.
1984;312(5996):779–81.
3. Hollenberg SM, Weinberger C, Ong ES, et al. Primary structure and expression of a func-
tional human glucocorticoid receptor cDNA. Nature. 1985;318(6047):635–41.
4. Carlstedt-Duke J, Stromstedt PE, Wrange O, Bergman T, Gustafsson JA, Jornvall H. Domain
structure of the glucocorticoid receptor protein. Proc Natl Acad Sci U S A. 1987;
84(13):4437–40.
5. Kumar R, Thompson EB. Gene regulation by the glucocorticoid receptor: structure:function
relationship. J Steroid Biochem Mol Biol. 2005;94(5):383–94.
6. Kumar R, Thompson EB. Transactivation functions of the N-terminal domains of nuclear
hormone receptors: protein folding and coactivator interactions. Mol Endocrinol.
2003;17(1):1–10.
7. Kumar R, Thompson EB. Folding of the glucocorticoid receptor N-terminal transactivation
function: dynamics and regulation. Mol Cell Endocrinol. 2012;348(2):450–6.
8. Strahle U, Klock G, Schutz G. A DNA sequence of 15 base pairs is sufficient to mediate both
glucocorticoid and progesterone induction of gene expression. Proc Natl Acad Sci U S A.
1987;84(22):7871–5.
9. Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler PB. Crystallographic
analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991;
352(6335):497–505.
10. Meijsing SH, Pufall MA, So AY, Bates DL, Chen L, Yamamoto KR. DNA binding site sequence
directs glucocorticoid receptor structure and activity. Science. 2009;324(5925):407–10.
11. Freedman LP, Luisi BF, Korszun ZR, Basavappa R, Sigler PB, Yamamoto KR. The function
and structure of the metal coordination sites within the glucocorticoid receptor DNA binding
domain. Nature. 1988;334(6182):543–6.
12. Severne Y, Wieland S, Schaffner W, Rusconi S. Metal binding 'finger' structures in the gluco-
corticoid receptor defined by site-directed mutagenesis. EMBO J. 1988;7(8):2503–8.
13. Hill KK, Roemer SC, Jones DN, Churchill ME, Edwards DP. A progesterone receptor co-
activator (JDP2) mediates activity through interaction with residues in the carboxyl-terminal
extension of the DNA binding domain. J Biol Chem. 2009;284(36):24415–24.
14. Boonyaratanakornkit V, Melvin V, Prendergast P, et al. High-mobility group chromatin pro-
teins 1 and 2 functionally interact with steroid hormone receptors to enhance their DNA
binding in vitro and transcriptional activity in mammalian cells. Mol Cell Biol.
1998;18(8):4471–87.
15. Ichijo T, Chrousos GP, Kino T. Activated glucocorticoid receptor interacts with the INHAT
component Set/TAF-Ibeta and releases it from a glucocorticoid-responsive gene promoter,
relieving repression: implications for the pathogenesis of glucocorticoid resistance in acute
undifferentiated leukemia with Set-Can translocation. Mol Cell Endocrinol. 2008;
283(1-2):19–31.
16. Ko L, Cardona GR, Henrion-Caude A, Chin WW. Identification and characterization of a
tissue-specific coactivator, GT198, that interacts with the DNA-binding domains of nuclear
receptors. Mol Cell Biol. 2002;22(1):357–69.
17. Tao Y, Williams-Skipp C, Scheinman RI. Mapping of glucocorticoid receptor DNA binding
domain surfaces contributing to transrepression of NF-kappa B and induction of apoptosis.
J Biol Chem. 2001;276(4):2329–32.
18. Jonat C, Rahmsdorf HJ, Park KK, et al. Antitumor promotion and antiinflammation: down-
modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell. 1990;62(6):
1189–204.
76 S.H. Meijsing
19. Yang-Yen HF, Chambard JC, Sun YL, et al. Transcriptional interference between c-Jun and
the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein
interaction. Cell. 1990;62(6):1205–15.
20. Schule R, Rangarajan P, Kliewer S, et al. Functional antagonism between oncoprotein c-Jun
and the glucocorticoid receptor. Cell. 1990;62(6):1217–26.
21. Ray A, Prefontaine KE. Physical association and functional antagonism between the p65
subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad
Sci U S A. 1994;91(2):752–6.
22. Langlais D, Couture C, Balsalobre A, Drouin J. The Stat3/GR interaction code: predictive
value of direct/indirect DNA recruitment for transcription outcome. Mol Cell. 2012;
47(1):38–49.
23. Heck S, Kullmann M, Gast A, et al. A distinct modulating domain in glucocorticoid recep-
tor monomers in the repression of activity of the transcription factor AP-1. EMBO J.
1994;13(17):4087–95.
24. Bledsoe RK, Montana VG, Stanley TB, et al. Crystal structure of the glucocorticoid receptor
ligand binding domain reveals a novel mode of receptor dimerization and coactivator recog-
nition. Cell. 2002;110(1):93–105.
25. Oakley RH, Cidlowski JA. The biology of the glucocorticoid receptor: new signaling mecha-
nisms in health and disease. J Allergy Clin Immunol. 2013;132(5):1033–44.
26. Pratt WB, Toft DO. Steroid receptor interactions with heat shock protein and immunophilin
chaperones. Endocr Rev. 1997;18(3):306–60.
27. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR. GRIP1, a novel mouse protein that
serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci U S A. 1996;93(10):4948–52.
28. Kucera T, Waltner-Law M, Scott DK, Prasad R, Granner DK. A point mutation of the AF2
transactivation domain of the glucocorticoid receptor disrupts its interaction with steroid
receptor coactivator 1. J Biol Chem. 2002;277(29):26098–102.
29. Hall JM, McDonnell DP, Korach KS. Allosteric regulation of estrogen receptor structure,
function, and coactivator recruitment by different estrogen response elements. Mol
Endocrinol. 2002;16(3):469–86.
30. Thomas-Chollier M, Watson LC, Cooper SB, et al. A naturally occurring insertion of a single
amino acid rewires transcriptional regulation by glucocorticoid receptor isoforms. Proc Natl
Acad Sci U S A. 2013;110(44):17826–31.
31. Chandra V, Huang P, Hamuro Y, et al. Structure of the intact PPAR-gamma-RXR-nuclear
receptor complex on DNA. Nature. 2008;456(7220):350–6.
32. Zhang J, Chalmers MJ, Stayrook KR, et al. DNA binding alters coactivator interaction sur-
faces of the intact VDR-RXR complex. Nat Struct Mol Biol. 2011;18(5):556–63.
33. Rogatsky I, Wang JC, Derynck MK, et al. Target-specific utilization of transcriptional regula-
tory surfaces by the glucocorticoid receptor. Proc Natl Acad Sci U S A. 2003;100(24):
13845–50.
34. Nehme A, Lobenhofer EK, Stamer WD, Edelman JL. Glucocorticoids with different chemi-
cal structures but similar glucocorticoid receptor potency regulate subsets of common and
unique genes in human trabecular meshwork cells. BMC Med Genomics. 2009;2:58.
35. Cao Y, Bender IK, Konstantinidis AK, et al. Glucocorticoid receptor translational isoforms
underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and
humans. Blood. 2013;121(9):1553–62.
36. Kino T, Su YA, Chrousos GP. Human glucocorticoid receptor isoform beta: recent under-
standing of its potential implications in physiology and pathophysiology. Cell Mol Life Sci.
2009;66(21):3435–48.
37. Lewis-Tuffin LJ, Cidlowski JA. The physiology of human glucocorticoid receptor beta
(hGRbeta) and glucocorticoid resistance. Ann N Y Acad Sci. 2006;1069:1–9.
38. Rivers C, Levy A, Hancock J, Lightman S, Norman M. Insertion of an amino acid in the
DNA-binding domain of the glucocorticoid receptor as a result of alternative splicing. J Clin
Endocrinol Metab. 1999;84(11):4283–6.
39. Haarman EG, Kaspers GJ, Pieters R, Rottier MM, Veerman AJ. Glucocorticoid receptor
alpha, beta and gamma expression vs in vitro glucocorticod resistance in childhood leukemia.
Leukemia. 2004;18(3):530–7.
40. Ray DW, Davis JR, White A, Clark AJ. Glucocorticoid receptor structure and function in
glucocorticoid-resistant small cell lung carcinoma cells. Cancer Res. 1996;56(14):3276–80.
41. Sanchez-Vega B, Krett N, Rosen ST, Gandhi V. Glucocorticoid receptor transcriptional iso-
forms and resistance in multiple myeloma cells. Mol Cancer Ther. 2006;5(12):3062–70.
42. de Lange P, Segeren CM, Koper JW, et al. Expression in hematological malignancies of a
glucocorticoid receptor splice variant that augments glucocorticoid receptor-mediated effects
in transfected cells. Cancer Res. 2001;61(10):3937–41.
43. Oakley RH, Cidlowski JA. Cellular processing of the glucocorticoid receptor gene and pro-
tein: new mechanisms for generating tissue-specific actions of glucocorticoids. J Biol Chem.
2011;286(5):3177–84.
44. Lu NZ, Cidlowski JA. Translational regulatory mechanisms generate N-terminal glucocorti-
coid receptor isoforms with unique transcriptional target genes. Mol Cell. 2005;
18(3):331–42.
45. Chen W, Dang T, Blind RD, et al. Glucocorticoid receptor phosphorylation differentially
affects target gene expression. Mol Endocrinol. 2008;22(8):1754–66.
46. Charmandari E, Chrousos GP, Lambrou GI, et al. Peripheral CLOCK regulates target-tissue
glucocorticoid receptor transcriptional activity in a circadian fashion in man. PLoS One.
2011;6(9), e25612.
47. Nader N, Chrousos GP, Kino T. Circadian rhythm transcription factor CLOCK regulates the
transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine
cluster: potential physiological implications. FASEB J. 2009;23(5):1572–83.
48. Karin M, Haslinger A, Holtgreve H, et al. Characterization of DNA sequences through which
cadmium and glucocorticoid hormones induce human metallothionein-IIA gene. Nature.
1984;308(5959):513–9.
49. Jantzen HM, Strahle U, Gloss B, et al. Cooperativity of glucocorticoid response elements
located far upstream of the tyrosine aminotransferase gene. Cell. 1987;49(1):29–38.
50. Chandler VL, Maler BA, Yamamoto KR. DNA sequences bound specifically by glucocorti-
coid receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell.
1983;33(2):489–99.
51. Solomon MJ, Larsen PL, Varshavsky A. Mapping protein-DNA interactions in vivo with
formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell.
1988;53(6):937–47.
52. Robertson G, Hirst M, Bainbridge M, et al. Genome-wide profiles of STAT1 DNA associa-
tion using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods.
2007;4(8):651–7.
53. John S, Sabo PJ, Thurman RE, et al. Chromatin accessibility pre-determines glucocorticoid
receptor binding patterns. Nat Genet. 2011;43(3):264–8.
55. Yu CY, Mayba O, Lee JV, et al. Genome-wide analysis of glucocorticoid receptor binding
2010;5(12), e15188.
56. Polman JA, Welten JE, Bosch DS, et al. A genome-wide signature of glucocorticoid receptor
binding in neuronal PC12 cells. BMC Neurosci. 2012;13:118.
57. Nielsen R, Pedersen TA, Hagenbeek D, et al. Genome-wide profiling of PPARgamma:RXR
and RNA polymerase II occupancy reveals temporal activation of distinct metabolic path-
ways and changes in RXR dimer composition during adipogenesis. Genes Dev.
2008;22(21):2953–67.
58. Welboren WJ, van Driel MA, Janssen-Megens EM, et al. ChIP-Seq of ERalpha and RNA
polymerase II defines genes differentially responding to ligands. EMBO J. 2009;28(10):
1418–28.
78 S.H. Meijsing
59. Hakim O, John S, Ling JQ, Biddie SC, Hoffman AR, Hager GL. Glucocorticoid receptor
activation of the Ciz1-Lcn2 locus by long range interactions. J Biol Chem. 2009;284(10):
6048–52.
60. Drouin J, Sun YL, Chamberland M, et al. Novel glucocorticoid receptor complex with DNA
element of the hormone-repressed POMC gene. EMBO J. 1993;12(1):145–56.
61. Surjit M, Ganti KP, Mukherji A, et al. Widespread negative response elements mediate direct
repression by agonist-liganded glucocorticoid receptor. Cell. 2011;145(2):224–41.
63. Zhang Z, Jones S, Hagood JS, Fuentes NL, Fuller GM. STAT3 acts as a co-activator of glu-
cocorticoid receptor signaling. J Biol Chem. 1997;272(49):30607–10.
64. Philips A, Maira M, Mullick A, et al. Antagonism between Nur77 and glucocorticoid recep-
tor for control of transcription. Mol Cell Biol. 1997;17(10):5952–9.
65. Luecke HF, Yamamoto KR. The glucocorticoid receptor blocks P-TEFb recruitment by
NFkappaB to effect promoter-specific transcriptional repression. Genes Dev. 2005;
19(9):1116–27.
66. Bladh LG, Liden J, Dahlman-Wright K, Reimers M, Nilsson S, Okret S. Identification of
endogenous glucocorticoid repressed genes differentially regulated by a glucocorticoid
receptor mutant able to separate between nuclear factor-kappaB and activator protein-1
repression. Mol Pharmacol. 2005;67(3):815–26.
67. Rao NA, McCalman MT, Moulos P, et al. Coactivation of GR and NFKB alters the repertoire
of their binding sites and target genes. Genome Res. 2011;21(9):1404–16.
68. Rogatsky I, Zarember KA, Yamamoto KR. Factor recruitment and TIF2/GRIP1 corepressor
activity at a collagenase-3 response element that mediates regulation by phorbol esters and
hormones. EMBO J. 2001;20(21):6071–83.
69. Rogatsky I, Waase CL, Garabedian MJ. Phosphorylation and inhibition of rat glucocorticoid
receptor transcriptional activation by glycogen synthase kinase-3 (GSK-3). Species-specific
differences between human and rat glucocorticoid receptor signaling as revealed through
GSK-3 phosphorylation. J Biol Chem. 1998;273(23):14315–21.
70. Aittomaki S, Pesu M, Groner B, Janne OA, Palvimo JJ, Silvennoinen O. Cooperation among
Stat1, glucocorticoid receptor, and PU.1 in transcriptional activation of the high-affinity Fc
gamma receptor I in monocytes. J Immunol. 2000;164(11):5689–97.
71. Cella N, Groner B, Hynes NE. Characterization of Stat5a and Stat5b homodimers and het-
erodimers and their association with the glucocortiocoid receptor in mammary cells. Mol
Cell Biol. 1998;18(4):1783–92.
72. Siersbaek R, Nielsen R, John S, et al. Extensive chromatin remodelling and establishment of
transcription factor 'hotspots' during early adipogenesis. EMBO J. 2011;30(8):1459–72.
73. Thurman RE, Rynes E, Humbert R, et al. The accessible chromatin landscape of the human
genome. Nature. 2012;489(7414):75–82.
74. Wang J, Zhuang J, Iyer S, et al. Sequence features and chromatin structure around the genomic
regions bound by 119 human transcription factors. Genome Res. 2012;22(9):1798–812.
75. Strahle U, Schmid W, Schutz G. Synergistic action of the glucocorticoid receptor with tran-
scription factors. EMBO J. 1988;7(11):3389–95.
76. Lerner L, Henriksen MA, Zhang X, Darnell Jr JE. STAT3-dependent enhanceosome assem-
bly and disassembly: synergy with GR for full transcriptional increase of the alpha
2-macroglobulin gene. Genes Dev. 2003;17(20):2564–77.
77. De Martino MU, Alesci S, Chrousos GP, Kino T. Interaction of the glucocorticoid receptor
and the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII): implica-
tions for the actions of glucocorticoids on glucose, lipoprotein, and xenobiotic metabolism.
Ann N Y Acad Sci. 2004;1024:72–85.
78. Biddie SC, John S, Sabo PJ, et al. Transcription factor AP1 potentiates chromatin accessibil-
ity and glucocorticoid receptor binding. Mol Cell. 2011;43(1):145–55.
79. Stromstedt PE, Poellinger L, Gustafsson JA, Carlstedt-Duke J. The glucocorticoid receptor
binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential
mechanism for negative regulation. Mol Cell Biol. 1991;11(6):3379–83.
80. Subramaniam N, Cairns W, Okret S. Glucocorticoids repress transcription from a negative

glucocorticoid response element recognized by two homeodomain-containing proteins, Pbx
and Oct-1. J Biol Chem. 1998;273(36):23567–74.
81. Ki SH, Cho IJ, Choi DW, Kim SG. Glucocorticoid receptor (GR)-associated SMRT binding
to C/EBPbeta TAD and Nrf2 Neh4/5: role of SMRT recruited to GR in GSTA2 gene repres-
sion. Mol Cell Biol. 2005;25(10):4150–65.
82. Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR. Transcription factor interactions:
selectors of positive or negative regulation from a single DNA element. Science.
1990;249(4974):1266–72.
83. Belikov S, Astrand C, Wrange O. FoxA1 binding directs chromatin structure and the func-
tional response of a glucocorticoid receptor-regulated promoter. Mol Cell Biol. 2009;
29(20):5413–25.
84. Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS. Opening of compacted chro-
matin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell.
2002;9(2):279–89.
85. Hurtado A, Holmes KA, Ross-Innes CS, Schmidt D, Carroll JS. FOXA1 is a key determinant
of estrogen receptor function and endocrine response. Nat Genet. 2011;43(1):27–33.
86. Sahu B, Laakso M, Ovaska K, et al. Dual role of FoxA1 in androgen receptor binding to
chromatin, androgen signalling and prostate cancer. EMBO J. 2011;30(19):3962–76.
87. Gertz J, Savic D, Varley KE, et al. Distinct properties of cell-type-specific and shared tran-
scription factor binding sites. Mol Cell. 2013;52(1):25–36.
88. Grontved L, John S, Baek S, et al. C/EBP maintains chromatin accessibility in liver and
facilitates glucocorticoid receptor recruitment to steroid response elements. EMBO
J. 2013;32(11):1568–83.
89. John S, Sabo PJ, Johnson TA, et al. Interaction of the glucocorticoid receptor with the chro-
matin landscape. Mol Cell. 2008;29(5):611–24.
90. Buratowski S. Progression through the RNA polymerase II CTD cycle. Mol Cell.
2009;36(4):541–6.
91. Murasawa S, Matsubara H, Kizima K, Maruyama K, Mori Y, Inada M. Glucocorticoids regu-
late V1a vasopressin receptor expression by increasing mRNA stability in vascular smooth
muscle cells. Hypertension. 1995;26(4):665–9.
92. Lasa M, Brook M, Saklatvala J, Clark AR. Dexamethasone destabilizes cyclooxygenase 2
mRNA by inhibiting mitogen-activated protein kinase p38. Mol Cell Biol. 2001;21(3):
771–80.
93. Ing NH. Steroid hormones regulate gene expression posttranscriptionally by altering the sta-
bilities of messenger RNAs. Biol Reprod. 2005;72(6):1290–6.
94. Beermann F, Schmid E, Ganss R, Schutz G, Ruppert S. Molecular characterization of the
mouse tyrosinase gene: pigment cell-specific expression in transgenic mice. Pigment Cell
Res. 1992;5(5 Pt 2):295–9.
95. Ratman D, Vanden Berghe W, Dejager L, et al. How glucocorticoid receptors modulate the
activity of other transcription factors: a scope beyond tethering. Mol Cell Endocrinol.
2013;380(1–2):41–54.
96. Ford J, McEwan IJ, Wright AP, Gustafsson JA. Involvement of the transcription factor IID
protein complex in gene activation by the N-terminal transactivation domain of the glucocor-
ticoid receptor in vitro. Mol Endocrinol. 1997;11(10):1467–75.
97. Shiama N. The p300/CBP family: integrating signals with transcription factors and chroma-
tin. Trends Cell Biol. 1997;7(6):230–6.
98. Fryer CJ, Archer TK. Chromatin remodelling by the glucocorticoid receptor requires the
BRG1 complex. Nature. 1998;393(6680):88–91.
99. Almlof T, Wallberg AE, Gustafsson JA, Wright AP. Role of important hydrophobic amino
acids in the interaction between the glucocorticoid receptor tau 1-core activation domain and
target factors. Biochemistry. 1998;37(26):9586–94.
100. Hittelman AB, Burakov D, Iniguez-Lluhi JA, Freedman LP, Garabedian MJ. Differential
regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins.
EMBO J. 1999;18(19):5380–8.
80 S.H. Meijsing
101. Chen W, Roeder RG. The Mediator subunit MED1/TRAP220 is required for optimal gluco-
corticoid receptor-mediated transcription activation. Nucleic Acids Res. 2007;35(18):
6161–9.
102. Pascual-Le Tallec L, Simone F, Viengchareun S, Meduri G, Thirman MJ, Lombes M.
The elongation factor ELL (eleven-nineteen lysine-rich leukemia) is a selective coregulator
for steroid receptor functions. Mol Endocrinol. 2005;19(5):1158–69.
103. Conaway JW, Shilatifard A, Dvir A, Conaway RC. Control of elongation by RNA
polymerase II. Trends Biochem Sci. 2000;25(8):375–80.
104. Gupte R, Muse GW, Chinenov Y, Adelman K, Rogatsky I. Glucocorticoid receptor represses
proinflammatory genes at distinct steps of the transcription cycle. Proc Natl Acad Sci U S A.
2013;110(36):14616–21.
105. Germond JE, Hirt B, Oudet P, Gross-Bellark M, Chambon P. Folding of the DNA double
helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A.
1975;72(5):1843–7.
106. Kornberg RD. Chromatin structure: a repeating unit of histones and DNA. Science.
1974;184(4139):868–71.
107. Richmond TJ, Davey CA. The structure of DNA in the nucleosome core. Nature. 2003;
423(6936):145–50.
108. Workman JL, Roeder RG. Binding of transcription factor TFIID to the major late promoter
during in vitro nucleosome assembly potentiates subsequent initiation by RNA polymerase
II. Cell. 1987;51(4):613–22.
109. Hager GL, Archer TK, Fragoso G, et al. Influence of chromatin structure on the binding of
transcription factors to DNA. Cold Spring Harb Symp Quant Biol. 1993;58:63–71.
110. King HA, Trotter KW, Archer TK. Chromatin remodeling during glucocorticoid receptor
regulated transactivation. Biochim Biophys Acta. 2012;1819(7):716–26.
111. Yoshinaga SK, Peterson CL, Herskowitz I, Yamamoto KR. Roles of SWI1, SWI2, and SWI3
proteins for transcriptional enhancement by steroid receptors. Science. 1992;258(5088):
1598–604.
112. Hsiao PW, Fryer CJ, Trotter KW, Wang W, Archer TK. BAF60a mediates critical interactions
between nuclear receptors and the BRG1 chromatin-remodeling complex for transactivation.
Mol Cell Biol. 2003;23(17):6210–20.
113. Johnson TA, Elbi C, Parekh BS, Hager GL, John S. Chromatin remodeling complexes
interact dynamically with a glucocorticoid receptor-regulated promoter. Mol Biol Cell.
2008;19(8):3308–22.
114. Engel KB, Yamamoto KR. The glucocorticoid receptor and the coregulator Brm selectively
modulate each other's occupancy and activity in a gene-specific manner. Mol Cell Biol.
2011;31(16):3267–76.
115. Nightingale KP, O'Neill LP, Turner BM. Histone modifications: signalling receptors and
potential elements of a heritable epigenetic code. Curr Opin Genet Dev. 2006;16(2):125–36.
116. Vermeulen M, Mulder KW, Denissov S, et al. Selective anchoring of TFIID to nucleosomes
by trimethylation of histone H3 lysine 4. Cell. 2007;131(1):58–69.
117. Bode J, Henco K, Wingender E. Modulation of the nucleosome structure by histone acetylation.
Eur J Biochem. 1980;110(1):143–52.
118. Glass CK, Rose DW, Rosenfeld MG. Nuclear receptor coactivators. Curr Opin Cell Biol.
1997;9(2):222–32.
119. Ito K, Barnes PJ, Adcock IM. Glucocorticoid receptor recruitment of histone deacetylase 2
inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12. Mol Cell Biol.
2000;20(18):6891–903.
120. Kovacs JJ, Murphy PJ, Gaillard S, et al. HDAC6 regulates Hsp90 acetylation and chaperone-
dependent activation of glucocorticoid receptor. Mol Cell. 2005;18(5):601–7.
121. Zelin E, Zhang Y, Toogun OA, Zhong S, Freeman BC. The p23 molecular chaperone and
GCN5 acetylase jointly modulate protein-DNA dynamics and open chromatin status. Mol
Cell. 2012;48(3):459–70.
contexts. Proc Natl Acad Sci U S A. 2002;99(26):16701–6.
123. Chen D, Huang SM, Stallcup MR. Synergistic, p160 coactivator-dependent enhancement of
estrogen receptor function by CARM1 and p300. J Biol Chem. 2000;275(52):40810–6.
124. Lee DY, Northrop JP, Kuo MH, Stallcup MR. Histone H3 lysine 9 methyltransferase G9a is
a transcriptional coactivator for nuclear receptors. J Biol Chem. 2006;281(13):8476–85.
125. Watson LC, Kuchenbecker KM, Schiller BJ, Gross JD, Pufall MA, Yamamoto KR. The glu-
cocorticoid receptor dimer interface allosterically transmits sequence-specific DNA signals.
Nat Struct Mol Biol. 2013;20(7):876–83.
126. Wang JC, Shah N, Pantoja C, et al. Novel arylpyrazole compounds selectively modulate
glucocorticoid receptor regulatory activity. Genes Dev. 2006;20(6):689–99.
127. De Bosscher K, Vanden Berghe W, Beck IM, et al. A fully dissociated compound of plant
origin for inflammatory gene repression. Proc Natl Acad Sci U S A. 2005;102(44):
15827–32.
128. Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat
Methods. 2013;10(10):957–63.
129. Campbell JM, Hartjes KA, Nelson TJ, Xu X, Ekker SC. New and TALENted genome engi-
neering toolbox. Circ Res. 2013;113(5):571–87.
130. Carroll D. Genome engineering with zinc-finger nucleases. Genetics. 2011;188(4):773–82.
131. Dixon JR, Selvaraj S, Yue F, et al. Topological domains in mammalian genomes identified by
analysis of chromatin interactions. Nature. 2012;485(7398):376–80.
132. Jin F, Li Y, Dixon JR, et al. A high-resolution map of the three-dimensional chromatin
interactome in human cells. Nature. 2013;503(7475):290–4.
133. Li W, Notani D, Ma Q, et al. Functional roles of enhancer RNAs for oestrogen-dependent
transcriptional activation. Nature. 2013;498(7455):516–20.
Chapter 4
Clinical Perspective: What Do Addison
and Cushing Tell Us About Glucocorticoid
Action?
Charles Harris
Abstract This chapter is distinct from the others in its clinical subject matter. I will
attempt to outline the major points of interest in glucocorticoids clinically. To aid
the illustration in the evaluation of a patient with Cushing disease I have created a
case study.
Keywords Glucocorticoids • Cushing syndrome • Cushing disease • Addison

• Hypothalamus • Hypothalamic pituitary adrenal axis (HPA axis) • CRH • ACTH
• Adrenal gland • Cortisol
The Hypothalamic Pituitary Adrenal Axis
The HPA axis is a classic endocrine feedback system. The hypothalamus produces
CRH in response to circadian cues as well as in response to stress from other
regions of the brain. CRH binds to CRH receptors found in the pituitary and
stimulates ACTH release. ACTH binds to the ACTH receptors (MC2R) in the
adrenal gland and stimulates the synthesis and release of cortisol. Cortisol in turn
via the glucocorticoid receptor inhibits release of CRH and ACTH. Glucocorticoid
excess can be broken down into endogenous excess and iatrogenic due to exoge-
nous glucocorticoid administration for the treatment of any of a number of
diseases. Endogenous glucocorticoid excess could theoretically arise from hyper-
activity at each of the levels of the HPA axis. Although there are case reports of
excess CRH secretion causing Cushing’s syndrome, it is a very rare event. The
most common cause of endogenous hypercortisolemia is due to pituitary tumors
of the ACTH secreting corticotroph lineage. Excess glucocorticoid exposure
C. Harris, M.D., Ph.D. (*)

Division of Endocrinology, Metabolism and Lipid Research, Department of Internal
Medicine, Washington University School of Medicine, 660 S Euclid Avenue,
Campus Box 8127, St. Louis, MO 63110, USA

84 C. Harris
results in Cushing syndrome and when this is due to an ACTH producing pituitary
tumor it is known as Cushing disease. Less commonly, a tumor of the adrenal
gland can become autonomous of regulation by ACTH and secrete excess gluco-
corticoids. The least common form of endogenous hypercortisolemia is due to
ectopic ACTH syndrome, when a non-pituitary tumor secretes ACTH which can
stimulate the adrenal gland. Therefore, endogenous glucocorticoid excess can be
broken into ACTH-dependent disease (pituitary tumor and ectopic ACTH) and
ACTH-independent disease (adrenal tumor). So the overall evaluation of a patient
with signs of glucocorticoid excess is to determine if it is exogenous or endoge-
nous, confirm the diagnosis of endogenous hypercortisolism and determine
the source.
Approach to the Patient with Potential Glucocorticoid Excess
It has been greater than 100 years since Harvey Cushing published a case report of
23-year old Minnie G who presented with central obesity, elevated systolic blood
pressure and laevulose intolerance. Today, the most common cause of glucocorticoid
excess unfortunately is iatrogenic use of glucocorticoid medications for the treat-
ment of inflammatory diseases. Endogenous glucocorticoid excess is a rare disease,
but must never be forgotten as a cause of Cushing syndrome. The clinician’s job has
become even more difficult given the increasing prevalence of metabolic syndrome
which is a subset of Cushing syndrome. However, patients with metabolic syn-
drome have normal serum cortisol levels. The approach to diagnose Cushing’s syn-
drome varies by nation, medical center and practitioner, but I will put forth a general
approach. First, the diagnosis of endogenous glucocorticoid excess must be made.
This can be done by several complementing approaches. A 24 h urine cortisol is
helpful. A midnight serum cortisol would be very helpful because the normal circa-
dian pattern of cortisol secretion is lost in endogenous glucocorticoid excess.
Therefore, cortisol is very low in normals and does not suppress at night in patients
with endogenous hypercortisolism. Because of the practical difficulties in obtaining
such testing (patient laboratories are generally not open at that hour, and it is not
practical for patients to go to the ER for a blood draw) salivary cortisol levels have
been used to obtain similar information. Salivary tests have the advantage of being
less invasive, which not only do patients prefer, but also are less likely to induce a
stress response as phlebotomy might. The patient collects a saliva sample at midnight
in the convenience of their home and returns the sample to the clinical lab for analy-
sis. In addition to a 24 h urine cortisol, a dexamethasone suppression test is helpful
in establishing a diagnosis of endogenous glucocorticoid excess. The patient takes
a 1 mg dose of the potent glucocorticoid dexamethasone at 11 p.m. at home and then
serum cortisol is measured at 8 a.m. the next day. In normal patients the dexametha-
sone will suppress CRH and ACTH secretion and therefore cortisol secretion to less
than 2. If the patient does not perform their portion of the test properly, i.e., they do
4 Clinical Perspective: What Do Addison and Cushing Tell Us About Glucocorticoid… 85
not take it at the correct time or do not take it at all, the “normal” AM cortisol will
appear non-suppressed and the patient will have a false positive result. In these
cases, and in a few cases of altered dexamethasone metabolism [1], one can obtain
a serum dexamethasone level to determine if the patient has adequate dexametha-
sone levels to suppress cortisol production, but this is rarely necessary in practice.
Once the diagnosis of endogenous glucocorticoid excess is established, one needs
to determine the source. An AM measurement of ACTH indicates whether the
patient has ACTH dependent disease (ACTH is normal or high) or ACTH indepen-
dent disease (ACTH is suppressed). If the ACTH is in the low normal range, the
clinician will need to consider both ACTH dependent or independent disease.
ACTH independent disease is most likely due to adrenal adenomas and a CT scan
is indicated to evaluate for this. If there is a single adenoma, unilateral resection
should be curative. If there is bilateral disease one must decide whether to resect the
dominant (larger) nodule containing adrenal, pursue definitive curative bilateral
adrenalectomy, or medical therapy. If removal of the adrenal containing the domi-
nant nodule is not curative, a second surgery to remove the remaining cortical ade-
noma may be necessary. This is particularly true in cases of macronodular adrenal
hyperplasia. Some centers can perform cortical- sparing resection of such tumors,
or depending on the severity of Cushing’s syndrome, patient may opt for medical
therapy. Cortical sparing adrenalectomy has usually been performed in the setting
of medullary disease, such as pheochromocytoma, but has been reported for
Cushings due to adrenal tumors as well [2].
The advantage of bilateral adrenalectomy is it is a definitive cure, not just for
adrenal Cushing’s, but all forms of endogenous hypercortisolism. The disadvantage
of this procedure is it renders the patient permanently adrenally insufficient and this
has been correlated with decreased quality of life [3]. If bilateral adrenalectomy is
pursued for Cushing’s disease it leaves open the possibility of accelerated growth of
a pituitary tumor. This is because the pituitary tumor has been partially suppressed
by cortisol and if this is removed, unopposed growth of the tumor, so called Nelson’s
syndrome can occur [4]. Furthermore, bilateral adrenalectomy in ectopic ACTH
does not address the underlying problem of the ectopic tumor. For ACTH dependent
disease the clinician must determine if the source of the ACTH is from the pituitary
or an ectopic source. Some centers rely on a second dexamethasone suppression
test, using higher amounts of dexamethasone administered over 2 days and the
percent dexamethasone suppression as an indicator of pituitary vs. ectopic disease.
This “hi-dose dexamethasone suppression test” takes advantage of the fact that pitu-
itary tumors can be partially suppressed, while suppression is completely lacking in
ectopic ACTH. However, given that the pre-test probability of pituitary disease in
ACTH dependent disease is ~90 % and is comparable to the sensitivity of this
high-dose dexamethasone suppression test, I do not use it in my practice. The next
step is an MRI of the pituitary gland. Because most pituitary adenomas causing
Cushing’s disease are small, a negative MRI does not exclude the possibility of
Cushing’s disease. The sensitivity of MRI is ~3–4 mm and remarkably pituitary
tumors smaller than this can cause full-blown Cushing’s disease. Generally, if there
86 C. Harris
is an obvious pituitary microadenoma greater or equal to 6 mm on the pituitary

MRI, transphenoidal surgery is indicated. Because of the possibility of smaller find-
ings being incidental, if no obvious adenoma is seen on MRI, it is recommended to
undergo petrosal venous sampling. The principle of this test is that the pituitary is
the point source of ACTH secretion. The closer you get to the pituitary, the higher
the concentration of ACTH in the blood. During this study a trained interventional
radiologist will sample blood from the periphery as well as near the petrosal sinus.
For patients with pituitary secretion of ACTH, such a point source will result in a
gradient of ACTH between the petrosal sinus and peripheral blood of two or more.
A patient with an ectopic source of ACTH should not have higher ACTH levels in
the petrosal sinus because the HPA axis would be suppressed due to the ectopic
ACTH mediated secretion of cortisol. The sensitivity can be increased by adminis-
tering agents that enhance ACTH secretion. The most commonly used agent is
CRH. Generally a gradient of 2 before stimulation is considered a positive gradient
and a gradient of 3 after stimulation is considered positive. Patients with confirmed
pituitary disease should undergo transphenoidal surgery by a neurosurgeon with
experience in such cases. It should be noted the importance in correctly diagnosing
patient with glucocorticoid excess because a normal patient without glucocorticoid
excess would also be expected to have a pituitary gradient of ACTH. If the petrosal
sampling points to a non-pituitary source (no gradient means ectopic ACTH) the
source of ACTH must be determined. Since the most common source is from bron-
chial carcinoids, a CT scan of the chest is usually the next test performed. If this
does not reveal a single obvious suspicious lesion additional imaging modalities can
be used to identify the lesion. In addition, there is case report of pulmonary vein
sampling in a patient with ectopic ACTH and bilateral pulmonary nodules to deter-
mine the culpable lesion [5].
All patients with endogenous glucocorticoid excess will be rendered adrenally
insufficient if they are surgically cured regardless of the tumor type. To assess for
surgical cure, cortisol measurements in the blood are made in the immediate post-
operative period. If the cortisol level is not very low, surgical cure probably has not
occurred and the patient may still have glucocorticoid excess or is at high risk for
relapse in the near future. In the case of adrenal disease, chronic excess cortisol
suppresses the HPA axis. HPA axis suppression also occurs in ectopic ACTH syn-
drome. HPA axis suppression occurs in Cushing’s disease as well as the normal
corticotropes are suppressed. Therefore, all patients will require replacement dosing
of glucocorticoids. This period of adrenal insufficiency can last upwards of 12
months. Patients should undergo ACTH stimulation testing prior to weaning off
replacement glucocorticoids. Patients with Cushing’s disease require long-term
post-operative follow-up as recurrence can occur, even years after resection.
Periodic urine cortisol and pituitary MRI testing is appropriate. In addition, patients
should be educated about their symptoms of glucocorticoid excess and instructed to
seek care if these symptoms return.
Medical therapy for endogenous Cushing’s syndrome is an unmet need. Currently,
two medications are approved for Cushing’s syndrome. Korlym (mifepristone) is
FDA approved for hyperglycemia associated with Cushing’s syndrome. Mifepristone

is a glucocorticoid antagonist. Because of its unique mechanism of action, serum
and urine cortisol levels increase but the net effect is decreased signaling through
the glucocorticoid receptor. Serum and urine cortisol increase because the GR that
mediates peripheral effects of glucocorticoids is the same molecule that mediates
negative feedback inhibition in the pituitary. Patients with pituitary disease still
have some degree of feedback inhibition by glucocorticoids, i.e., secretion is not
entirely autonomous, hence the increase in serum and urine cortisol. Increased
circulating cortisol can overcome blockade by the renal 11-β-HSD2 enzyme and
lead to apparent mineralocorticoid excess. In fact, many patients on mifepristone
experience increased blood pressure and hypokalemia. This can be addressed by
combining mifepristone with a mineralocorticoid antagonist such as eplerenone.
In addition, prolonged blockade of the progesterone receptor chronically has
unknown health effects, particularly in women. Currently, mifepristone is approved
by the FDA for the treatment of hyperglycemia associated with endogenous gluco-
corticoid excess, but it likely has beneficial effects on other organs adversely
affected by glucocorticoid excess. The other medication approved for endogenous
hypercortisolism is pasireotide, but because of this drug’s mode of action, it is only
effective against Cushing’s disease (pituitary disease). Pasireotide is an octreotide
analog with high affinity for the somatostatin receptor 5. It only has efficacy in
~25 % of patients at the highest dose and has adverse effects including hyperglyce-
mia [6]. The anti-fungal ketoconazole has been used off-label to treat endogenous
hypercortisolism. Since it is an inhibitor of glucocorticoid biosynthesis, it is effec-
tive in all subtypes of endogenous hypercortisolism. Ketoconazole is becoming
increasingly difficult to procure because it has fallen out of favor as an anti-fungal,
it’s only approved indication. Ketoconazole has rare adverse effect of hepatitis and
is also known to prolong QT interval (increasing the risk of ventricular tachycardia,
a potentially life threatening arrhythmia of the heart), especially when combined
with other medications. For these reasons, a specific enantiomer is being developed
for the treatment of endogenous hypercortisolism (http://clinicaltrials.gov/show/
NCT01838551). Other molecules in development are more specific GR antagonists,
other inhibitors of glucocorticoid synthesis including LCI-699, an inhibitor of
11-β-hydroxylase [7].
Case Study
A 45 year old female presents with worsening weight gain, diabetes and hypertension
over the last 3 years. During that time she has gradually gained 30 lbs and gone
from having a normal blood pressure to elevated blood pressure that is poorly con-
trolled on three blood pressure medications. In addition, she stopped menstruating 2
years ago, and notices some disturbing growth of facial hair. She developed excess
urination 1 year ago and was diagnosed with diabetes at that time. On physical
88 C. Harris
exam she is afebrile, hypertensive (BP 180/100) with a pulse of 90, she has a round
“moon” facies, dorsorcervical fat (buffalo hump), supraclavicular fat, her heart is
beating with regular rate and rhythm, and her breath sounds are normal. Her abdo-
men shows central obesity and her extremities are thin. Her skin is thin when rubbed
between the examiners fingers and she has purple stretch marks (striae) on the sides
of her abdomen. She is tearful, when describing her recent problems and adds that
her moods have been much more labile over the last 2 years. She is alert and ori-
ented to person, place and time. She has mildly decreased strength and has difficulty
rising from a chair without using her hands.
Her current medications include: Lisinopril, hydrochlorothiazide, atenolol, met-
formin, glipizide. She denies taking any glucocorticoid medications.
She has had some blood work done by her primary care provider
HgbA1C 9.0 (normal <6.0)
Cortisol 22 mcg/dL (done at 9 a.m., normal 9–24 mcg/dL)
ACTH 65 pg/mL (done at 9 a.m., normal 6–55 pg/mL)
You tell her, you suspect she has Cushing’s syndrome due to endogenous gluco-
corticoid excess, but that you will need to run some additional tests. You order a test
for 24-h urine cortisol. In addition, you order a blood serum cortisol to be done
8–10 a.m. the morning after she takes a 1 mg dose of dexamethasone that you write
a prescription for. Importantly, you tell her to do the dexamethasone suppression
test after she has completed the 24-h urine collection.
She returns to the clinic 1 week later with the following results
24-h urine cortisol is 302 mcg (normal <50 mcg), creatinine is 900 mg in a volume
of 2.1 L.
Cortisol in the AM 10.0 (after having taken 1 mg of dexamethasone the night prior
at 11 p.m.).
The case is informative as to the effects of glucocorticoids on the body. One of
the most important elements of this patient’s history is that she did not take gluco-
corticoids. This is important because glucocorticoids are given as medications for
several common conditions including asthma, emphysema (COPD), inflammatory
bowel disease and rheumatoid arthritis. As a practical point, since patients may
not be familiar with the term “glucocorticoid” it is useful to list the names of the
commonly used glucocorticoids such as prednisone, hydrocortisone, cortef, dexa-
methasone, etc. Usually Cushing’s syndrome is only seen with systemic glucocorti-
coid exposure, but it is possible to receive enough systemic exposure from injections
into the joint, inhaled preparations, or topical preparations [8–10]. There were a
large number of cases of Cushing’s syndrome in HIV patients taking the inhaled
glucocorticoid fluticasone because the commonly used anti-viral medication ritona-
vir inhibited the body’s system of breakdown for the inhaled glucocorticoid, thereby
raising levels of the inhaled glucocorticoid in the blood [11]. Another relevant point is
that one cannot rule in or rule out Cushing’s disease with the serum cortisol levels alone.
Cortisol is secreted in a circadian manner with peaks in the early morning and nadirs
in the evening and night. Patients with endogenous hypercortisolemia often have
normal peak cortisol levels but have failure to reduce levels in the evening. This is
why either a 24-h urine collection or a midnight salivary cortisol test is needed.
Glucocorticoids are known to increase adiposity, but the exact mechanisms under-
lying this are unknown. Glucocorticoids are known to increase appetite, and in fact
are used off-label for this purpose for such conditions such as cancer cachexia [12].
Megestrol is a commonly used glucocorticoid appetite stimulant in patients with
cachexia due to cancer or AIDS. Glucocorticoids are also known to stimulate adipo-
genesis, the formation of fat cells from fat cell precursors such as adipocytes. In fact
much of what we know about adipocyte biology has been learned using a cell line
3T3-L1 that is a fibroblast, but can be differentiated into adipocytes with a chemical
cocktail including glucocorticoids. This patient has evidence of lipodystrophy with
the buffalo hump, supraclavicular fat, central obesity, and loss of fat from her arms
and legs. Although there is a net gain of fat mass the redistribution of fat in Cushing’s
syndrome is more prominent. This redistribution is due to increased breakdown of
fat in some depots and enhanced synthesis in other. All depots undergo futile cycling
of fat continuously breaking it down and resynthesizing it, so it is not difficult to see
if the net synthesis/breakdown of the various depots was altered, one could get
fat redistribution. In fact, it is remarkable that we are able to keep our fat depots
in a constant distribution. Similarly, it is known that glucocorticoids raise blood
pressure. This is likely due to effects on the endothelium, the kidney, and the heart
(see Chap. 13). In addition, some of the effects of glucocorticoids to raise blood
pressure are mediated by the mineralocorticoid receptor and some are mediated
through the glucocorticoid receptor. Glucocorticoids can bind to the mineralocorti-
coid receptor, but when glucocorticoids are present at normal levels, they are inac-
tivated locally in the kidney via the 11-b-HSD2 enzyme. However, when endogenous
glucocorticoids are found at very high levels, they can overcome this inhibition and
are free to bind to the mineralocorticoid receptor which increases blood pressure
and lowers serum potassium. However, it is also clear that glucocorticoids raise
blood pressure through the glucocorticoid receptor as well because synthetic gluco-
corticoids that do not bind the mineralocorticoid receptor also raise blood pressure.
Usually, the increase in blood pressure is more severe than that seen in the general
population (“essential” hypertension). The fact that this patient’s blood pressure is
very high while on three blood pressure medications is an indicator of the severity
of the problem. The loss of menses can also be directly attributed to glucocorticoids
as glucocorticoids suppress the hypothalamic-pituitary gonadal axis. Of course, loss
of menses could be due to menopause. This patient had loss of menses at age 43,
which is several standard deviations away from the population mean of age 51. If she
did not have an obvious cause such as her Cushing’s syndrome, other hormone testing
would be helpful. It is possible she is having early menopause. In that case her FSH
and LH would be elevated due to loss of negative feedback from estrogen that is no
longer being produced by her ovaries. If her lack of periods (amenorrhea) is due to
Cushing’s syndrome, we expect her serum FSH and LH to be low. Ockham’s razor,
that the simplest explanation is usually the correct one, would dictate her amenorrhea
90 C. Harris
is due to Cushing’s. Other common hormonal abnormalities that could cause loss
of menses include hypothyroidism, hyperthyroidism, and elevated prolactin levels.
These scenarios would involve a distinct set of associated signs and symptoms.
The growth of facial hair is common in endogenous glucocorticoid excess. In both
ACTH-dependent and independent disease the adrenal gland is stimulated. The
adrenal gland makes both glucocorticoids and androgens and the latter class of
compounds will cause the increased facial hair in women. The excess urination
(polyuria) is a direct consequence of diabetes and the diabetes could also be due to
glucocorticoid excess. Although the exact mechanisms for this are not known,
glucocorticoids are known to stimulate gluconeogenesis by the liver. Increased
production of glucose by the liver can raise the serum glucose causing diabetes.
In addition, glucocorticoids cause insulin resistance in peripheral tissues such as
muscle and adipose tissue. Normally insulin stimulates these tissues to take up
glucose from the blood, but in a state of insulin resistance glucose uptake is reduced,
also raising serum glucose. Her skin is thin, because glucocorticoids suppress the
synthesis of collagens. It is difficult to appreciate skin thickness visually so it is
necessary to pick up the skin, usually on the back of the patients hand, between the
examiner’s thumb and forefinger. Of course, skin thickness decreases with age in
the general population, so this must be taken into account. The glucocorticoids also
cause the pigmented stretch marks, known as striae. Striae are virtually pathogno-
monic for Cushing’s syndrome; i.e., when they occur, the patient usually has
Cushing’s syndrome. Her neurological exam was notable for muscle weakness as
evidenced by her difficulty rising from a chair without using her arms. Glucocorticoids
cause myopathy or muscle wasting. The pattern shows a proximal myopathy, with
the larger muscles towards the center of the body being preferentially affected.
Because, these are the muscles required to rise from a seat (without using one’s
hands), this is a good clinical test for proximal myopathy. Again, there are multiple
causes of proximal myopathy, but in the setting of this patient it is likely due to
glucocorticoid excess. Glucocorticoids induce muscle wasting by transcriptionally
activating genes encoding proteases that breakdown muscle protein into amino
acids. These amino acids are sent to the liver where they are converted to glucose.
Her neurological history and exam are notable for mood changes and labile moods.
Glucocorticoids cause pronounced cognitive changes including depression, psychoses
and impairments in learning and memory. The patient’s further laboratory testing
confirms endogenous glucocorticoid excess as evidenced by increased urine corti-
sol and failure to suppress cortisol secretion in the dexamethasone suppression test.
Ingesting 1 mg of dexamethasone at 11 p.m. should suppress serum cortisol levels
to less than 2 mcg/dL the next morning (reference range in the morning in the
absence of dexamethasone is 9–24 mcg/dL). Since, a diagnosis of endogenous
hypercortisolism has been confirmed we can use the serum ACTH to distinguish
whether she has ACTH-dependent or -independent disease. Since her ACTH is
above 20, she has ACTH dependent disease. We tell her she needs an MRI of her
pituitary gland, which is obtained and shows a 4 mm tumor in her pituitary. Because
the tumor is quite small and could be an incidental finding in any person “off the
street”, we tell her we need to confirm that this small tumor is the source of ACTH
that is causing her glucocorticoid excess. In fact the incidence of pituitary tumors in
autopsy series and MRI or CT scans of normal people ranges from 5 to 20 % and have
been given the humorous name pituitary incidentaloma [13, 14]. If her pituitary
tumor had been larger (>6 mm), such petrosal sampling would be deemed unneces-
sary by some and she could be referred directly to a neurosurgeon. She undergoes
petrosal sampling and has a petrosal-peripheral gradient of ACTH of 4 before CRH
and 6 after CRH. This confirms that the pituitary is the source of excess ACTH driv-
ing her adrenal to produce excess cortisol. You tell her she stands a good chance of
being cured following surgical resection of the tumor. You send her to a neurosur-
geon that has experience operating on these tumors using the transphenoidal
approach. The pituitary is in the center of your head, underneath the region of the
brain known as the hypothalamus. A craniotomy (cutting through the skull) can be
avoided if the surgeon goes through the sphenoid sinus by cutting through the back
of the patient’s nose. The patient goes for neurosurgery and the surgeon finds the
tumor, excises it and sends it to the pathology lab. The next morning the patient’s
cortisol level is 0.6 and she is started on glucocorticoids: hydrocortisone 20 mg in
the AM and 10 mg at 3 p.m. It does seem counter-intuitive to give glucocorticoids
to the patient after their body has been plagued by too much glucocorticoids, but it
is necessary as the patient will have adrenal insufficiency because the HPA axis has
been suppressed. If the patient is not given glucocorticoids, they could have adrenal
crisis and die. Patients can be started on a replacement dose of glucocorticoids.
However, patients on a replacement dose often have symptoms of glucocorticoid
deficiency, so-called “glucocorticoid withdrawal syndrome”, because they are
accustomed to much higher doses of glucocorticoids. There is likely a reset rheostat
of glucocorticoid “tone”. The clinician must weigh the need to wean patients to
replacement doses of glucocorticoids to minimize excess glucocorticoid exposure
with patient discomfort at physiological levels of glucocorticoids. Patients should
be able to be weaned to replacement dose of glucocorticoids within a few months of
surgery. At this point, one can assess patients periodically with AM cortisol to deter-
mine when their HPA axis will become non-suppressed. I typically check an AM
cortisol every 3 months, until the AM cortisol is greater or equal than 7 at which
point I perform an ACTH stimulation test. If they pass the ACTH stimulation test,
I will slowly withdraw the replacement glucocorticoids. Again here one is balanc-
ing minimizing the need for glucocorticoids with the inconvenience and cost of
performing too many ACTH stimulation tests. Two-weeks after her surgery our
patient develops right calf pain and swelling. She undergoes ultrasound of the veins
in her leg which show a deep vein thrombosis. She is started on anticoagulant ther-
apy. Our patient was told to hold her dose of hydrocortisone the morning of her
blood test and obtain an AM cortisol 3 month post-operatively. It is 4 (low) so she
is told to obtain another test 3 months later (6 months post-operatively) and it is now
9 (lower limit of normal). She returns for an ACTH stimulation test and her cortisol
is now 21 45 min after receiving an IM injection of 250 μg of cosyntropin (ACTH).
Her dose of hydrocortisone is weaned by 2.5 mg every week (alternating reducing the
AM and PM dose) so that after another 6 weeks she is off all medication. Her anti-
coagulant therapy is discontinued. On her next visit she states her menses have
92 C. Harris
returned and a repeat 24-h urine cortisol is in the normal change. She obtains an
MRI 6 months post-operatively to obtain a baseline image of the post-surgical
changes. Given all these obvious signs and symptoms, one may ask whether there
are any negative health consequences of Cushing’s syndrome that are less obvious.
In fact, one of the gravest health concerns in these patients is silent: osteoporosis.
The rates of fracture in patients with Cushing’s syndrome are quite high and higher
than one would predict based on patients’ bone mineral density [15]. This is likely
due to poor bone quality, but also other non-bone related factors that contribute to
fractures. Glucocorticoids impact bone on many levels. Glucocorticoids cause osteo-
blast apoptosis, osteoclast activation. In addition glucocorticoids cause hypogonad-
ism in males and females which has a negative impact on bone health. In addition,
glucocorticoids cause GH deficiency further impacting bone. In addition, glucocor-
ticoids act as functional antagonists of vitamin D decreasing absorption of calcium.
In addition, the glucocorticoid induced muscle weakness contributes to falls, which
increases the likelihood of fractures. Bisphosphonates and recombinant PTH
(Forteo, Teriparatide) have been shown to treat glucocorticoid induced osteoporo-
sis. Although it is prudent to treat with either an anabolic or anti-resorptive in a
patient with active endogenous hypercortisolism to prevent active bone loss, the
case for anti-resorptive after a surgical cure is less clear. This is due to the fact that
there is spontaneous recovery of bone mass following cure [16], which theoretically
could be blunted by an anti-resorptive.
Approach to the Patient with Adrenal Insufficiency
Many of the same principles discussed above pertain to the converse situation of
adrenal insufficiency. Adrenal insufficiency can be primary or secondary. In pri-
mary adrenal insufficiency, the problem lies in the adrenal gland. In this case there
is loss of negative feedback at the level of pituitary ACTH and the circulating level
of this hormone is very high. Since ACTH is derived from a larger protein pro-
opiomelanocortin (POMC) which also yields melanin stimulating peptides the
result is the dark pigmentation associated with primary adrenal insufficiency. In
primary adrenal insufficiency both glucocorticoid and mineralocorticoid secretion
is diminished. Patients with primary adrenal insufficiency will need replacement of
glucocorticoid and mineralocorticoids, whereas patients with secondary adrenal
insufficiency only require glucocorticoids. This is an important distinction as
inappropriate use of mineralocorticoid can have negative effects including hyper-
tension, hypokalemia, and heart failure. Conversely, if one were to omit mineralo-
corticoids in a patient with primary adrenal insufficiency they would develop
hyperkalemia. This is due to the major effects of mineralocorticoids to promote
sodium absorption and potassium excretion in the kidney. The distinction between
primary and secondary adrenal insufficiency is usually obvious based on patient
history, physical exam, and laboratory testing.
Because of the circadian fluctuation in cortisol secretion, sampling is best

performed in the morning since reference ranges are available. However, the gold
standard for diagnosing adrenal insufficiency is the ACTH stimulation test during
which ACTH is injected (either intramuscularly or intravenously) and a cortisol
level is obtained 30 and or 60 min after ACTH administration. Although the exact
diagnostic cut off is arguable most use a stimulated level of greater than 18, but
some use up to 20 as a cut off. Several points are worth considering. A controversial
issue of glucocorticoid biology in medicine is the diagnosis of adrenal insufficiency
in critically ill patients, e.g., patients in intensive care units. An early clinical trial
found the incidence of adrenal insufficiency in such patients to be quite high, but
was flawed in that a large number of these patients had received etomidate as an
anesthetic prior to intubation [17]. Etomidate is a known inhibitor of glucocorticoid
synthesis so the adrenal insufficiency in these patients may have been due to etomi-
date and not critical illness. In addition, serum cortisol levels in critically ill patients
may appear low due to low levels of serum proteins that carry glucocorticoids
(again due to critical illness) whereas the more important levels of free cortisol are
normal [18]. A final misconception of adrenal insufficiency is the requirement that
the serum cortisol increase a preset amount (usually set at 7–9) following adminis-
tration of ACTH in addition to achieving a set level (of 18–20). In my opinion, this
is an illogical requirement. If serum cortisol levels are very high, this should be
sufficient evidence for adrenal sufficiency. One may not be able to respond to ACTH
if endogenous ACTH is at a maximal effect. More recent studies have not demon-
strated a benefit of glucocorticoid treatment for critically ill patients with septic
shock [19]. Because adrenal insufficiency is often considered in hospitalized patient,
glucocorticoids may be given before such a diagnosis is certain. These patients can
still undergo an ACTH stimulation test with glucocorticoids “on board” i.e., one can
still stimulate through the potential suppression from glucocorticoid therapy. In this
case, it may be helpful to switch the patient to a synthetic glucocorticoid such as
dexamethasone that does not interfere with the cortisol assay. Another point to con-
sider is that the loss of the adrenal response to ACTH does not occur acutely in
secondary adrenal insufficiency, but rather takes approximately 6 weeks to develop.
The clinical implication of this is that a patient with secondary adrenal insufficiency
may have a falsely normal ACTH stimulation test because the adrenals have not yet
lost the ability to respond. Therefore, the clinical scenario is key to diagnosis and
clinicians should not hesitate to treat and then perform repeat ACTH stimulation
testing when indicated. Another less commonly used test of adrenal function is the
metyrapone test. Metyrapone is an inhibitor of the 11-β-hydroxylase, which cata-
lyzes the last step of cortisol synthesis. In a metyrapone test 2–3 g (the exact dose
depending on patient weight) of metyrapone are ingested at midnight, and then
cortisol and deoxycortisol are measured 8 h later at 8 a.m. when glucocorticoid syn-
thesis is at its peak circadian phase. With inhibition of 11-β-hydroxylase, cortisol
levels fall and in an individual with normal adrenal function there should be build-
up of the substrate for 11-β-hydroxylase, 11-deoxycortisol. However, if there is low
steroid synthesis in the setting of adrenal insufficiency, the 11-deoxycortisol level
94 C. Harris
will not be elevated. One advantage of this test is that it has an “internal control”.
If the cortisol level is not low, it means the patient did not take the metyrapone
properly. So, in a metyrapone test a normal patient has low cortisol levels and ele-
vated deoxycortisol levels and a patient with AI has low cortisol and deoxycortisol
after ingesting metyrapone. Specifically, adequate metyrapone exposure is evident
with a cortisol <5 mcg/dL and normal adrenal function is defined as deoxycortisol
levels >7 mcg/dL. Of note, in the absence of metyrapone, the normal circulating
levels of deoxycortisol are <0.1 mcg/dL. Of course deoxycortisol can be elevated
not only with metyrapone, but also with genetic mutations in 11-β-hydroxylase,
which is a cause of congenital adrenal hyperplasia, beyond the scope of this chapter.
It is essential to determine if the patient has primary or secondary adrenal insuffi-
ciency. These distinct entities have different causes, treatments and implications for
the patient. Patients with primary adrenal insufficiency have a problem at the level
of the adrenal glands themselves. This can be caused by an autoimmune process,
hemorrhage, infection. Tuberculosis used to be a common cause, but is rare today.
The patient with primary adrenal insufficiency will have an elevated ACTH (due to
loss of negative feedback of cortisol on POMC transcription in the pituitary).
Because POMC pro-protein is cleaved to melanocyte stimulating hormone peptides
in addition to ACTH, the patient with primary adrenal insufficiency will be hyper-
pigmented. This is evident on the skin as well as on mucous membranes. The patient
with primary adrenal insufficiency will require replacement of both glucocorticoids
and mineralocorticoids. It is not feasible to replace catecholamines produced by the
adrenal medulla, and they are also made by the sympathetic nervous system. The
adrenal cortex makes androgens, but in males, the adrenal production is much
smaller than that produced in the testes. Androgen replacement in women with pri-
mary adrenal insufficiency is a controversial issue [20]. Some have postulated low
dose androgens should be replaced to ensure normal sexual function and libido in
women. However, no androgen formulation exists that can be safely administered
to women without the risk of virilization. Some have also proposed patients with
primary adrenal insufficiency would benefit from replacement of DHEA-S, but
there are not strong data to support this [21]. DHEA has not been shown to have
function other than as a precursor to adrenal hormones. Therefore, if the end-prod-
uct hormone is being replaced, there should be no need to replace DHEAS. Similarly,
in the absence of normal adrenal function, the DHEAS will unlikely be metabolized
into end product hormones. Therefore, the standard regimen includes a mineralo-
corticoid and a glucocorticoid. Although cortisol has inherent mineralocorticoid
activity, a high dose would be required to overcome HSD2 in the kidney and would
result in iatrogenic Cushing syndrome. The dose of mineralocorticoid should be
titrated to the patients’ blood pressure and serum potassium. There are a large num-
ber of glucocorticoid agonists available for replacement. In my practice, I usually
use hydrocortisone with a total daily dose of 15 mg that is broken up into 10 mg in
the morning and 5 p.m. mid-afternoon. This is done to try to mimic the circadian
fluctuation in serum cortisol levels. Other commonly used oral GC agonists include
prednisone. A replacement dose of prednisone is 4–5 mg per day as it is ~4 times
more potent that hydrocortisone. Prednisone has MR agonist activity. The synthetic
GC dexamethasone is 30 times more potent than hydrocortisone so a replacement
dose is usually 0.25 mg and dexamethasone has no mineralocorticoid agonism.
Dexamethasone is rarely used for adrenal insufficiency because of its higher
potency, difficulty in finding an appropriate replacement dose without inducing
iatrogenic Cushing’s. It does have a role in the non-compliant patient given its very
long biological half-life. Patients with adrenal insufficiency should be monitored
for adequate replacement as well as iatrogenic Cushing syndrome. Patients with
primary adrenal insufficiency usually require 100–200 mcg of a synthetic mineralo-
corticoid such as fludrocortisone. Interestingly, the most common cause of gluco-
corticoid deficiency today is glucocorticoids themselves. When glucocorticoids are
taken for a length of time, there is suppression of the hypothalamic-pituitary adrenal
axis. There are no clear cut rules as to the amount and duration of exposure needed
to suppress the HPA axis. Certainly patients taking greater than 15 mg of prednisone
for longer than 3 months are at risk and should undergo ACTH stimulation testing
prior to discontinuing therapy If patients are taking prednisone, it is best to slowly
lower their dose to 5 mg per day, the equivalent to what the body makes on its own.
At that point, I find it helpful to switch to hydrocortisone. Given its short half-life
and twice a day dosing to more closely mimic the circadian variation seen with
cortisol secretion and may contribute to more paid recovery of the HPA axis.
Because of the different potency the replacement dose of hydrocortisone is given as
10 mg first thing in the morning and 5 mg in the afternoon. A common error is to
prescribe it merely as “bid” which means twice a day. Patients should be instructed
to take the medication first thing in the morning and at 3 p.m., not bedtime, to more
closely approximate endogenous rhythms. Once the patient is on replacement glu-
cocorticoids one can perform ACTH stimulation testing every 6 months for 2 years
or until they pass. Once they pass, and only once they pass, should their dose of
glucocorticoid be lowered. Theoretically, once they pass an ACTH stimulation test
they could stop glucocorticoids cold turkey, but I usually do this over a 6-week
period having the patient lower their dose of hydrocortisone by 2.5 mg per week. If
patients do not pass an ACTH stimulation test after 2 years of being on a replace-
ment dose, it is unlikely they will recover HPA function and will likely require life-
long glucocorticoid treatment. I will end this chapter with another case presentation.
A 70 year-old male with severe emphysema presents to the emergency room with
nausea and vomiting. He has required oral glucocorticoids to control his emphy-
sema and has been on 20 mg of prednisone daily for 3 years. He ran out of predni-
sone 1 week ago and has had nausea and vomiting for the past 5 days. On physical
exam, he appears Cushingoid with moon facies, central obesity, a buffalo hump and
pigmented striae. When he comes to the emergency room he has some laboratory
testing of blood which shows a serum cortisol level of 1 mcg/dL (taken at 8 a.m.)
and his ACTH is undetectable (<5 pg/mL). This patient has both Cushing’s syn-
drome and adrenal insufficiency! He has iatrogenic exogenous Cushing’s syndrome
from the prednisone, but adrenal insufficiency from suppression of his hypotha-
lamic-pituitary-adrenal axis from said prednisone.
96 C. Harris
References
1. Cassidy F, Ritchie JC, Verghese K, Carroll BJ. Dexamethasone metabolism in dexamethasone

suppression test suppressors and nonsuppressors. Biol Psychiatry. 2000;47:677–80.
2. He HC, et al. Retroperitoneal adrenal-sparing surgery for the treatment of Cushing’s syndrome
caused by adrenocortical adenoma: 8-year experience with 87 patients. World J Surg.
2012;36:1182–8. doi:10.1007/s00268-012-1509-0.
3. Hawn MT, Cook D, Deveney C, Sheppard BC. Quality of life after laparoscopic bilateral adre-
nalectomy for Cushing’s disease. Surgery 2002;132:1064–8; discussion 1068–9. doi:10.1067/
msy.2002.128482.
4. Nelson DH, Meakin JW, Thorn GW. ACTH-producing pituitary tumors following adrenalectomy
for Cushing’s syndrome. Ann Intern Med. 1960;52:560–9.
5. Vu L, Theodore PR. Localization of a corticotropin-secreting tumor by thoracoscopic pulmonary
venous sampling. N Engl J Med. 2005;353:851–2. doi:10.1056/NEJMc050486.
6. Colao A, et al. A 12-month phase 3 study of pasireotide in Cushing’s disease. N Engl J Med.
2012;366:914–24. doi:10.1056/NEJMoa1105743.
7. Bertagna X, et al. LCI699, a potent 11beta-hydroxylase inhibitor, normalizes urinary cortisol
in patients with Cushing’s disease: results from a multicenter, proof-of-concept study. J Clin
Endocrinol Metab. 2014;99:1375–83. doi:10.1210/jc.2013-2117.
8. Yombi JC, Maiter D, Belkhir L, Nzeusseu A, Vandercam B. Iatrogenic Cushing’s syndrome
and secondary adrenal insufficiency after a single intra-articular administration of triamcino-
lone acetonide in HIV-infected patients treated with ritonavir. Clin Rheumatol. 2008;27 Suppl
2:S79–82. doi:10.1007/s10067-008-1022-x.
9. Kumar S, Singh RJ, Reed AM, Lteif AN. Cushing’s syndrome after intra-articular and intra-
dermal administration of triamcinolone acetonide in three pediatric patients. Pediatrics.
2004;113:1820–4.
10. O’Sullivan MM, Rumfeld WR, Jones MK, Williams BD. Cushing’s syndrome with suppression
of the hypothalamic-pituitary-adrenal axis after intra-articular steroid injections. Ann Rheum
Dis. 1985;44:561–3.
11. Bernecker C, West TB, Mansmann W, Scherbaum WA, Willenberg HS. Hypercortisolism
caused by ritonavir associated inhibition of CYP 3A4 under inhalative glucocorticoid therapy.
2 case reports and a review of the literature. Exp Clin Endocrinol Diabetes. 2012;120:125–7.
doi:10.1055/s-0031-1297993.
12. Yeh SS, Schuster MW. Megestrol acetate in cachexia and anorexia. Int J Nanomedicine.
2006;1:411–6.
13. Freda PU, et al. Pituitary incidentaloma: an endocrine society clinical practice guideline. J Clin
14. Molitch ME, Russell EJ. The pituitary “incidentaloma”. Ann Intern Med. 1990;112:925–31.
15. Van Staa TP, et al. Bone density threshold and other predictors of vertebral fracture in patients
receiving oral glucocorticoid therapy. Arthritis Rheum. 2003;48:3224–9. doi:10.1002/art.11283.
16. Randazzo ME, Grossrubatscher E, Dalino Ciaramella P, Vanzulli A, Loli P. Spontaneous
recovery of bone mass after cure of endogenous hypercortisolism. Pituitary. 2012;15:193–201.
doi:10.1007/s11102-011-0306-3.
17. Annane D, et al. Effect of treatment with low doses of hydrocortisone and fludrocortisone on
mortality in patients with septic shock. JAMA. 2002;288:862–71.
18. Hamrahian AH, Oseni TS, Arafah BM. Measurements of serum free cortisol in critically ill
patients. N Engl J Med. 2004;350:1629–38. doi:10.1056/NEJMoa020266.
19. Sprung CL, et al. Hydrocortisone therapy for patients with septic shock. N Engl J Med.
2008;358:111–24. doi:10.1056/NEJMoa071366.
20. Arlt W, et al. Dehydroepiandrosterone replacement in women with adrenal insufficiency. N Engl
J Med. 1999;341:1013–20. doi:10.1056/NEJM199909303411401.
21. Alkatib AA, et al. A systematic review and meta-analysis of randomized placebo-controlled
trials of DHEA treatment effects on quality of life in women with adrenal insufficiency. J Clin
Part II
Effects of Glucocorticoids on Metabolism
Chapter 5
Regulation of Glucose Homeostasis
by Glucocorticoids
Taiyi Kuo, Allison McQueen, Tzu-Chieh Chen, and Jen-Chywan Wang
Abstract Glucocorticoids are steroid hormones that regulate multiple aspects of

glucose homeostasis. Glucocorticoids promote gluconeogenesis in liver, whereas in
skeletal muscle and white adipose tissue they decrease glucose uptake and utiliza-
tion by antagonizing insulin response. Therefore, excess glucocorticoid exposure
causes hyperglycemia and insulin resistance. Glucocorticoids also regulate glyco-
gen metabolism. In liver, glucocorticoids increase glycogen storage, whereas in
skeletal muscle they play a permissive role for catecholamine-induced glycogenoly-
sis and/or inhibit insulin-stimulated glycogen synthesis. Moreover, glucocorticoids
modulate the function of pancreatic α and β cells to regulate the secretion of gluca-
gon and insulin, two hormones that play a pivotal role in the regulation of blood
glucose levels. Overall, the major glucocorticoid effect on glucose homeostasis is to
preserve plasma glucose for brain during stress, as transiently raising blood glucose
is important to promote maximal brain function. In this chapter we will discuss the
current understanding of the mechanisms underlying different aspects of
glucocorticoid-regulated mammalian glucose homeostasis.
Keywords Glucocorticoids • Glucocorticoid receptor • Gluconeogenesis • Insulin

• Glucose utilization • Glycogen • Pancreas • Glucose metabolism
Introduction
Glucocorticoids (GC) are stress hormones that play a key role in the regulation of
mammalian glucose homeostasis. The name “glucocorticoids” originates from their
profound effects on plasma glucose levels. GC regulate multiple aspects of glucose
T. Kuo, Ph.D. • A. McQueen, B.S. • T.-C. Chen, M.S.

212 Morgan Hall, Berkeley, CA 94720, USA
e-mail: [email protected]; [email protected]; [email protected]
J.-C. Wang, Ph.D. (*)
119 Morgan Hall, Berkeley, CA 94720-3104, USA

100 T. Kuo et al.
1. Promote gluconeogenesis
Liver 2. Increase glycogen storage
1. Inhibit glucose uptake and oxidation

Skeletal 2. Reduce glycogen storage
Cortisol 3. Increase protein degradation
Muscle
to provide amino acids as precursors
for gluconeogenesis
1. Inhibit glucose uptake and oxidation

WAT 2. Increase lipolysis
to provide glycerol as precursors
for gluconeogenesis
1. Inhibit insulin secretion in cells

Pancreas
2. Increase glucagon secretion in cells
3. Induce cell hyperplasia
Fig. 5.1 Glucocorticoid effects on glucose homeostasis. The effects of cortisol on glucose homeo-
stasis in peripheral tissues
homeostasis (Fig. 5.1). First, GC promote hepatic gluconeogenesis [1, 2] and reduce
glucose uptake and utilization in skeletal muscle and white adipose tissue (WAT) [3,
4]. These effects are critical for metabolic adaptation during stress, such as fasting/
starvation, when plasma glucose needs to be preserved because it is the brains’ pri-
mary energy source, and transiently raising blood glucose is important to promoting
maximal brain functions [5]. Insulin, a hormone secreted from pancreatic β cells,
exerts opposite effects on these physiological processes by inhibiting hepatic gluco-
neogenesis and promoting glucose utilization in skeletal muscle and WAT. Thus, to
exert their responses, GC need to antagonize insulin actions. These effects are criti-
cal during stress, which in short term does not affect or even enhances glucose toler-
ance. However, chronic GC exposure results in hyperglycemia and insulin resistance
[3, 4, 6]. Second, GC exert tissue-specific effects on glycogen metabolism. In liver,
GC increase glycogen storage, whereas in skeletal muscle GC play a permissive
role for catecholamine-induced glycogenolysis or inhibit insulin-stimulated glyco-
gen synthesis [7–9]. Third, GC modulate insulin and glucagon secretion from pan-
creas. GC treatment increases plasma glucagon levels [10, 11], whereas the effects
of GC on insulin secretion are complex [12–16]. GC induce pancreas islet hyperpla-
sia in vivo that leads to hyperinsulinemia [12, 17–19] and have been shown to exert
cytotoxic effects on β cells [20, 21]. Overall, in this chapter we will discuss the
current understanding of the mechanisms underlying these distinct aspects of
GC-regulated glucose homeostasis.
5 Regulation of Glucose Homeostasis by Glucocorticoids 101
Gluconeogenesis
The gluconeogenic pathway generates glucose from non-carbohydrate substrates

(Fig. 5.2) [22, 23]. Gluconeogenesis mainly occurs in liver, though kidney and
intestine are also contributors. The major gluconeogenic precursors are lactate,
glycerol and gluconeogenic amino acids, such as alanine. Both lactate and alanine
can be converted to pyruvate, which is then carboxylated to oxaloacetate (OAA) by
pyruvate carboxylase (PC) [24, 25], a step that occurs in mitochondria. OAA needs
to be converted to malate to be shuttled to the cytoplasm where it is converted back
to OAA. Cytosolic phosphoenolpyruvate carboxykinase (PCK1) [26, 27] then cata-
lyzes OAA to phosphoenolpyruvate (PEP), which then enters the gluconeogenic
pathway. There is also a mitochondrial form of PCK1 (m-PCK1) that can directly
convert OAA to PEP in mitochondria, which is then transported to cytoplasm and
participates in gluconeogenesis. Recent studies suggest that m-PCK1 could also
play a role in gluconeogenesis [28–31].
Conversion of PEP to fructose-1,6-phosphate (F1,6BP) requires five enzymatic
steps that are essentially the reverse of glycolysis (Fig. 5.2). The “bifunctional”
enzyme, phosphofructokinase 2 (PFK2)/fructose bisphosphatase 2 (FBPase2)
(a.k.a. PFKFB1) [32, 33], plays a critical role in the switch between gluconeogen-
esis and glycolysis. PFKFB1 regulates the production of fructose 2,6 bisphosphate
(F2,6BP), which is an allosteric activator of phosphofructokinase 1 (PFK1), an
enzyme in the glycolytic pathway. When circulating glucose levels are low, such as
during fasting and starvation, glucagon inactivates PFK2, which allows FBPase2
activity to be favored. This results in decreased production of F2,6BP, and reduced
glycolysis and enhanced gluconeogenesis. Fructose 1,6-bisphosphatase (FBP1)
converts F1,6BP to fructose-6-phosphate (F6P) [34], which is then converted to
glucose-6-phosphate (G6P). G6P enters the endoplasmic reticulum (ER), where the
enzyme glucose-6-phosphatase (G6PC) [35–37] converts G6P to glucose (Fig. 5.2).
Notably, distinct gluconeogenic amino acids can be converted to specific intermedi-
ates in the tricarboxylic acid (TCA) cycle. These TCA cycle intermediates are con-
verted to OAA to enter the gluconeogenic cycle. Glycerol enters gluconeogenesis
through conversion to dihydroxyacetone phosphate (DHAP), which can then be
metabolized to glycerol-3-phosphate (G3P) or directly to F1,6BP (Fig. 5.2) thus
entering the gluconeogenic cycle above PCK1.
The positive effect of GC on hepatic gluconeogenesis is well established and has
been under extensive study for several decades. Injecting GC into humans [38, 39]
and rodents increases hepatic gluconeogenesis. In addition, GC are important for
other hormones to activate gluconeogenesis. Although glucagon is regarded as a
major hormone that activates gluconeogenesis during fasting, fasting-induced
gluconeogenesis is reduced in adrenalectomized mice, an effect that is restored by
treating mice with GC [40–42]. In fact, glucagon-, epinephrine-, or cyclic AMP
(cAMP)-induced gluconeogenesis are all attenuated in adrenalectomized mice.
Giving GC to adrenalectomized mice restores the ability of these hormones to induce
gluconeogenesis. Thus, GC play a “permissive” role promoting the optimal ability of
102 T. Kuo et al.
endoplasmic reticulum
G6P G6P glucose

G6PC
PFKFB1
F2,6BP F6P
PFK1
alanine
FBP1
lactate
F1,6BP
pyruvate
glycerol DHAP G3P
pyruvate 1,3-BPG
PC malate malate
3-PG
OAA
malate-aspartate shuttle OAA
2-PG
m-PCK1
aspartate aspartate PCK1
PEP PEP cytosol
mitochondria
Fig. 5.2 Gluconeogenic pathway in hepatocytes. Lactate and alanine are converted to pyruvate,
which enters the mitochondria and is then converted to OAA by enzyme PC. Through malate-
aspartate shuttle, OAA exits the mitochondria to form PEP. OAA can also be converted to PEP
directly within the mitochondria. PEP then feeds into the gluconeogenic pathway. In addition,
glycerol is metabolized to DHAP, which is then converted directly or indirectly through G3P to
F1,6BP. The final product, glucose, is produced in the ER by enzyme G6PC. The key enzymes are
boxed, with GR primary targets shown in yellow. Abbreviation: OAA oxaloacetate, PEP phospho-
enolpyruvate, DHAP dihydroxyacetone phosphate, G3P glyceraldehyde-3-phosphate, F1,6BP
fructose-1,6-bisphosphate, 2-PG 2-phosphoglycerate, 3-PG 3-phosphoglycerate, 1,3-BPG
1,3-bisphosphoglycerate, G3P glyceraldehyde-3-phosphate, F1,6BP fructose-1,6-bisphosphate,
F2,6BP fructose-2,6-bisphosphate, F6P fructose-6-phosphate, and G6P glucose-6-phosphate.
Enzyme abbreviation: PC pyruvate carboxylase, m-PCK1 mitochondrial phosphoenolpyruvate
carboxykinase, PCK1 cytosolic phosphoenolpyruvate carboxykinase, FBP1 fructose-1,6-
bisphosphatase 1, PFK1 phosphofructokinase 1, PFKFB1 phosphofructokinase 2/fructose bispho-
sphatase 2, G6PC glucose-6-phosphatase catalytic subunit
these hormones in gluconeogenesis. GC also provide gluconeogenic precursors by

promoting protein degradation in skeletal muscle to generate gluconeogenic amino
acids [1, 4]. They also enhance lipolysis in WAT. This releases glycerol and fatty
acids. Glycerol is used as a gluconeogenic precursor, whereas fatty acids provide
energy to drive the gluconeogenic pathway [8, 43, 44].
GC promote gluconeogenesis mainly through activation of the transcription of

genes encoding enzymes in the gluconeogenic pathway. The transcription of PC,
PCK1, FBP1, PFKFB1, G6PC and G6P transporter (SLC37A4) are all stimulated
by GC (Fig. 5.2). GC convey their signals mainly through an intracellular receptor,
the glucocorticoid receptor (GR). Before binding to GC, GR resides in cytoplasm
and is associated with Hsp90 chaperone complex. Upon binding to GC, GR dissoci-
ates from Hsp90 chaperone complex and enters the nucleus, where the GR is
recruited to specific genomic sequences, called glucocorticoid response elements
(GREs). The GR:GRE association could be a direct GR:DNA interaction or an indi-
rect association through other DNA-binding transcription factors. In any case, once
GR associates with a specific GRE, it recruits a number of transcriptional coregula-
tors. While they do not bind to DNA directly, these coregulators are able to assist the
GR in modulation of the transcriptional rate of nearby genes through distinct mech-
anisms: altering chromatin structure, inducing histone modifications; recruiting
RNA polymerase II containing basal transcription machinery; and modulating tran-
scriptional elongation. Notably, most genomic GREs are “composite” GREs (also
called glucocorticoid response units, GRUs) that consist of multiple cis-acting ele-
ments, which include binding sites for GR and other transcription factors (called
accessory elements and accessory factors, respectively), to mediate a complete GC
response. Because different DNA binding transcription factors other than GR are
involved in the regulation of distinct GR primary target genes, the multi-protein
transcriptional regulatory complex assembled on each GRE is likely distinct.
Composite GREs could allow GC to differentially regulate distinct target genes in
different cell types depending on the presence of cell type specific accessory factors
and transcriptional coregulators. Another advantage of employing composite GREs
is to allow specific cross-talk between GC and other signaling pathways at the
accessory elements and their respective accessory factors. Thus, cross-talk does not
need to occur directly through GR.
The GREs of Pck1, Pfkfb1, and G6pc genes have been identified. In particular,
the mechanism of GR-regulated Pck1 gene transcription has been extensively stud-
ied. By contrast, the mechanisms governing GC-activated Pc and Fbp1 gene are
unclear. Below we will discuss the mechanisms of GR-stimulated Pck1, Pfkfb1, and
G6pc gene transcription.
PCK1
Rat Pck1 gene contains two GREs, GRE1 and GRE2 (Fig. 5.3) [45]. GRE1 is
located between −388 and −374 (relative to transcription start site, TSS), whereas
GRE2 is located between and −367 and −354 in the Pck1 gene promoter. When
GRE1 or GRE2 is placed in front of TATA box in a synthetic reporter gene, neither
mediates a GC response [46]. The combination of GRE1 and GRE2 also fails to
confer GC-induced transcription [46]. In fact, both GRE1 and GRE2 bind GR very
weakly in vitro [46]. However, in cooperation with other accessory elements on the
104 T. Kuo et al.
-445 -410 -380 -360 -325 -90 -27

gAF1 gAF3
gAF2 GRE1 GRE2 CRE TATA
RARE1 RARE2
HNF4 FoxA2 GRU

GR GR COUP-TF C/EBP-
COUP-TF FoxO1?
COUP-TF COUP-TF CREB CRU
RAR/RXR RAR/RXR CREB RARU
FoxO1 IRU
Fig. 5.3 Hormone response units in the PEPCK gene. Binding sites for various regulatory and
transcription factors are shown in the top row, with the number indicating the center nucleotide
position of each element with respect to the transcription start site. Four hormone-specific response
units are drawn: proximal glucocorticoid response unit (GRU), cyclic AMP response unit (CRU),
retinoic acid response unite (RARU), and insulin response unit (IRU). In the absence of the other
hormones, the components of each response unit are depicted. These units interact functionally,
cooperating or competing, to comprise the PEPCK promoter. Except for gAF2, DNA elements
involved in IRU are not yet identified
Pck1 promoter, they confer a robust GC response. These accessory elements include
gAF1 [45], gAF2 [45], gAF3 [47] and the cAMP response element (CRE) [48].
Both gAF1 (between −451 and −434 of rat PCK1 promoter) and gAF2 (−416 and
−407) are located 5′ from GRE1 and GRE2, whereas gAF3 and CRE are located in
3′ of these GREs (Fig. 5.3). Hepatic nuclear factor 4 (HNF4, NR2A1) and chicken
ovalbumin upstream transcription factor (COUP-TF, NR2F2) bind to gAF1 and
serve as accessory factors for a complete GC response [49]. The gAF1 element also
serves as a retinoic acid response element (RARE). An all-trans retinoic acid recep-
tor (RAR) and 9-cis retinoic acid receptor (RXR) heterodimer binds to gAF1 and
confers retinoic acid (RA)-activated Pck1 gene transcription [50]. RA has been
shown to synergize with GC to stimulate Pck1 gene transcription [51].
The gAF2 element binds to members of the forkhead box transcription factor fam-
ily that include FoxA1 (also called hepatic nuclear factor 3 α, HNF3α), FoxA2
(hepatic nuclear factor 3 β, HNF3β), FoxO1 (FKHR) and FoxO3A (FKHRL1).
FoxA2 have been shown to act as accessory factors for GR-regulated Pck1 gene tran-
scription in vitro [52]. Liver specific deletion of FoxO1 but not FoxO3A significantly
reduces fasting-induced Pck1 gene expression [53]. Because GC play an important
role in fasting-induced Pck1 gene transcription, these results suggest that FoxO1 may
serve as an accessory factor for GR in vivo. The gAF2 element also serves as an insu-
lin response sequence (IRS) that confers at least part of repressive effect of insulin on
Pck1 gene transcription [54, 55]. The ability of insulin to suppress Pck1 gene expres-
sion is compromised in liver specific FoxO1 knockout mice [53] or mice overex-
pressed dominant negative FoxO1 [56]. Notably, the ability of insulin to reduce Pck1
gene expression is not affected in FoxO3A deletion mice [53]. These results support
the key role of FoxO1 in the regulation of Pck1 gene expression in vivo. The potential
role of FoxA2 inhibiting insulin effects has been reported [57], although more studies
are needed to confirm the importance of FoxA2 in insulin-suppressed Pck1 gene
expression.
Streptozotocin is a relatively specific pancreas β cell cytotoxin. Treatment of
mice with streptozotocin induces a state that mimics type 1 diabetes. Circulating
GC levels are increased in such animals and Pck1 gene expression is augmented. In
mice bearing a reporter gene containing a construct containing −2 kb rat Pck1 gene
promoter with a mutation at gAF2, streptozotocin-induced reporter gene expression
is markedly reduced [58]. These results confirm the importance of the gAF2 ele-
ment in GC-activated Pck1 gene in vivo.
The gAF3 element (−337 and −321) binds to COUP-TF [47] and, like gAF1, also
serves as a RARE that binds to RAR/RXR heterodimer [59]. The cAMP response
element (CRE, between −90 and −82) is also required for a complete GC-stimulated
Pck1 gene transcription [48]. CCAAT enhancer binding protein β (C/EBPβ) binds
to the CRE to mediate the accessory activity for the GC response [60].
Chromatin immunoprecipitation (ChIP) was used to monitor the recruitment of
GR and various accessory factors to their respective binding sites in rat H4IIE hepa-
toma cells. GC treatment increases the recruitment of GR, FoxO1, FoxO3A and RNA
polymerase II (Pol II) to the Pck1 promoter [61]. FoxA2, C/EBPβ, HNF4 and
COUP-TF occupy the Pck1 promoter before GC treatment and their occupancy is not
altered after GC treatment [61]. The recruitment of transcriptional coregulators, SRC-
1, p300 and CREB binding protein (CBP), to the Pck1 GRU is markedly increased by
GC treatment [61]. This suggests that GC treatment initiates the assembly of multi-
protein transcriptional regulatory complex on the Pck1 promoter. Insulin treatment
for just 3 min markedly decreases the GC-induced recruitment of GR, FoxO1,
FoxO3A, FoxA2, SRC-1, p300, CBP, and Pol II, and only the occupancy of C/EBPβ,
HNF-4 and COUP-TF remains unchanged [61]. Thus, insulin treatment rapidly dis-
rupts the assembly of GC-induced transcriptional complex on the Pck1 GRU. Analyzing
epigenetic marks showed that the most significant change is the methylation at his-
tone H3 arginine residue 17, which is significantly increased upon GC treatment and
is abolished by insulin [61]. CARM1/PRMT4, is the histone methyltransferase that
methylates histone H3 tail arginine 17 residue (H3R17) [62, 63]. CARM1 has been
shown to serve as a transcriptional coactivator for GR. However, the occupancy of
CARM1 on the Pck1 promoter is not significantly changed upon GC or insulin treat-
ment. One way to explain these results is that CARM1 is present on the Pck1 GRU
before GC treatment and the activity of CARM1 is modulated by insulin treatment.
The activity of CARM1 is regulated by post-translational modifications [64].
Alternatively, it is possible that an unknown histone methyltransferase is involved in
the elevation of H3R17 methylation on the Pck1 GRU.
An analysis of the rat Pck1 promoter in human hepatoma HepG2 cells identified
two additional accessory elements (dAF1 and dAF2) that are involved in
GC-stimulated Pck1 gene transcription. The dAF1 element is located at −993 and
has sequence similarity to the gAF1 element, whereas the dAF2 element, located at
−1365, resembles more proximal gAF2 [65]. HNF4 and peroxisome proliferator
106 T. Kuo et al.
activated receptor α (PPARα) and RXR heterodimer (PPARα/RXR) bind to dAF1,

whereas FoxA1, FoxA2 and FoxO1 bind to dAF2 [65]. ChIP experiments showed
that GC treatment increases FoxO1 and PPARα recruitment to the gAF2 and the
dAF2, and HNF4 to the dAF1 in mouse liver [65]. Overexpressing PPARα or HNF4
synergizes with GR to activate a reporter gene harboring 2 kb of rat Pck1 promoter
[65]. A role for PPARα in GC-induced Pck1 expression in vivo is consistent with the
observation that GC-induced Pck1 gene expression is markedly reduced in Pparα
null mice [66]. Notably, streptozotocin-induced Pck1 gene expression and blood
glucose levels are reduced in transgenic mice that harbor a targeted ablation of the
dAF1 in the Pck1 gene promoter. These results support the importance of the
dAF1 in GC-activated Pck1 gene expression in vivo [65].
Overall, GC activate Pck1 gene transcription through a complex GRU. Intriguingly,
all accessory elements in the Pck1 GRU are involved in the responses of other hor-
mones that provide potential cross-talk between GC and other hormones, including
glucagon, retinoic acid and insulin (Fig. 5.3). The complexity of GRU also allows
various signaling pathways to fine tune the transcription levels of Pck1 gene.
Interestingly, GC repress the transcription of Pck1 gene in adipocytes [67, 68] where
the major metabolic role of Pck1 is glyceroneogenesis [69, 70]. It is not entirely
clear why Pck1 GRU is not functional in adipocytes, although it is proposed that GR
inhibits Pck1 gene transcription through antagonizing C/EBP family of transcrip-
tion factors in adipocytes [67, 68]. Nonetheless, the requirement of accessory fac-
tors to act with GR on “weak” GREs provides the flexibility for GC to regulate Pck1
gene transcription in a tissue specific manner. While it is unknown how accessory
factors participate in GR-regulated Pck1 gene transcription, there are two potential
mechanisms. First, accessory factors may aid in recruitment of transcriptional
coregulators to the GRU to stimulate the transcription. Previous studies have shown
that the transactivation domain of HNF4 and FoxA2 are required for their accessory
activities [71]. When gAF1, gAF2 or gAF3 is replaced by the binding site of a yeast
transcription factor Gal4, a fusion protein that consists of GAL4 DNA binding
domain and a transcriptional coregulator, SRC1, is able to provide accessory activ-
ity [72]. Moreover, another transcriptional coregulator, peroxisome proliferator
activated receptor γ coactivator-1α (PGC1α), has been shown to interact with HNF4
to participate in GC-activated Pck1 gene transcription. PGC1α also interacts with
and coactivates FoxO1 [73] that binds to the gAF2 element. But the role of PGC1α-
FoxO1 interaction in GC-stimulated Pck1 gene transcription has not been exam-
ined. Second, accessory factors can potentiate GR:GRE association. Using
quantitative, real time equilibrium and stopped-flow fluorescence anisotropy mea-
surements of nuclear protein-DNA interactions it was shown that GR binds to the
Pck1 GREs poorly. However, the presence of the gAF1 and the gAF2 elements
markedly enhanced the association between GR and the Pck1 GREs [74]. It is pos-
sible that the assembly of a multi-protein complex that includes GR, accessory fac-
tors and transcriptional coregulators enhances the association between GR and the
two Pck1 GREs.
G6PC
G6PC gene transcription is induced by GC, whereas insulin suppresses both basal
and GC-activated G6PC gene transcription. Mouse G6pc also contains a complex
GRU in the proximal promoter region. This GRU consists of three GREs, a CRE, a
HNF4 binding site, a hepatic nuclear factor 1 (HNF1) binding site, and multiple
FoxO/FoxA binding sites (FREs) [75]. GC significantly activate the expression of a
reporter gene that contains the G6pc GRU. Mutation at any of these accessory ele-
ments in the G6pc GRU reporter gene reduces the GC response [75]. Mutations at
FREs that bind FoxO1 and FoxO3A also reduce both the inhibitory effect of insulin
and basal expression of G6pc gene [76]. In liver specific FoxO1 deletion mice the
expression of G6pc in 18 h fasted mice is markedly lower than that of 18 h fasted
wild type mice [53]. Moreover, the ability of insulin to suppress the expression of
G6pc is abolished in liver specific FoxO1 deletion mice [53] or mice overexpressed
dominant negative FoxO1 [56]. In contrast, liver specific FoxO3A deletion does not
affect basal expression of G6pc and the ability of insulin to inhibit G6pc remains
intact [53]. These results are reminiscent of the regulation of Pck1 gene and sug-
gests that FoxO1 plays a key role in the regulation of gluconeogenic genes in vivo.
Also, similar to the regulation of Pck1 gene, transcription coregulator PGC1α posi-
tively regulates basal G6pc gene transcription and enhances GC-stimulated G6pc
gene transcription through interaction with HNF4 [77, 78]. FoxO1 and PGC1α
appear to synergistically activate G6pc gene transcription [73, 79].
GC also activate the transcription of the G6P transporter (SLC37A4) gene, which
encodes a protein that is responsible for shuttling G6P from the cytoplasm to the ER
lumen. The mouse Slc37a4 gene promoter contains a GRE [80, 81] and a FoxO1
binding site is identified nearby the GRE [81]. GC increase the activity of a luciferase
reporter gene under the control of the Slc37a4 gene promoter in 293 cells, whereas the
mutation at the FoxO1 binding site reduces the ability of GC to potentiate this reporter
gene activity [81]. Overexpression of FoxO1 in 293 cells potentiates the ability of GC
to activate the reporter gene activity [81]. These results suggest that GC stimulate the
Slc37a4 gene through a GRU that contains at least a GRE and a FoxO1 binding site.
PFKFB1
Hepatic rat Pfkfb1 gene transcription is stimulated by GC and a GRU has been
identified in the intronic region of this gene. In addition to the GRE, this GRU con-
sists of binding sites for FoxA2, hepatic nuclear factor 6 (HNF6, a.k.a. Onecut1), C/
EBP and Nuclear factor 1 (NF1) [82]. Insulin antagonizes the stimulatory effect of
GC on Pfkfb1 gene [82, 83]. While insulin acts through PI3K and Akt to inhibit
Pck1 and G6pc gene expression [84, 85], this pathway is apparently not involved in
the suppressive effect of insulin on GC-induced Pfkfb1 gene. Instead, insulin acti-
vates the Jun N-terminal Kinase (JNK) pathway to inhibit GC-induced Pfkfb1 gene
expression [86].
108 T. Kuo et al.
Factors Regulating GC-Stimulated Hepatic Gluconeogenesis
Many other factors regulate gluconeogenesis by modulating GC signaling. Liver X

receptor β (LXRβ) is involved in GC-activated Pck1 gene expression. In Lxrβ null
mice (Lxrβ−/−) GC-induced Pck1 gene expression and glucose production are
reduced, and GC-induced recruitment of GR to the Pck1 GRE is impaired [87]. In
contrast, in Lxrα null mice (Lxra−/−) GC-regulated Pck1 gene expression is not
affected. Moreover, GC-stimulated expression of tyrosine aminotransferase (Tat)
gene, which encodes the enzyme that converts tyrosine to 4-hydroxyphenolpyruvate,
is not affected in Lxrb−/− mice. Thus, the role of LXRβ in GC action is relatively
specific to the Pck1 gene. The mechanisms governing LXRβ action in GC-activated
Pck1 gene transcription are unclear, especially in view of another study, which
showed that treating hepatoma cells with LXR ligands suppresses GC-stimulated
Pck1 and G6pc gene expression [88]. Microarray analyses showed that LXR ligands
affect only a subset of GC-regulated genes. Both gel shift and ChIP experiments sug-
gest that an LXRα/RXRα heterodimer competes with GR for binding at rat G6pc
GRE [88]. In agreement with its effects on GC-induced gluconeogenic gene expres-
sion is the observation that treating rats with an LXR ligand attenuates GC-augmented
plasma glucose levels [88]. In summary, unliganded LXRβ is required for maximal
GC-induced Pck1 gene transcription and is necessary for GC-induced recruitment of
GR to the Pck1 GRE. By contrast, LXR ligands suppress GC-activated gluconeo-
genic gene transcription by inhibiting the recruitment of GR to the GREs of these
genes.
The expression of transcription factor ying yang 1 (YY1) is increased upon fast-
ing and in insulin resistant state [89]. Overexpression of YY1 in mouse liver increases
gluconeogenesis [89]. In contrast, the deletion of YY1 in mouse liver results in
hypoglycemia. YY1 potentiates gluconeogenesis through the increase of hepatic GR
expression, which in turn augments the expression of gluconeogenic genes [89].
The expression of farnesoid x receptor (FXR, NR1H4), a bile acid receptor, is also
increased during fasting [90]. Fxr null mice become hypoglycemic during fasting and
have a reduced glucose production after a pyruvate challenge and a decreased expres-
sion of Pck1 and G6pc [90]. The treatment of fasted mice with an FXR ligand,
6α-ethylchenodeoxycholic acid (6E-CDCA), increases hepatic glucose production
and Pck1 and G6pc gene expression. These effects are not observed in Fxr null mice,
nor are they seen in fed mice [90]. 6E-CDCA elevates the expression of GR. By con-
trast, the expression of GR is decreased in Fxr null mice. Reducing GR expression in
liver abolishes 6E-CDCA-induced glucose production and the expression of Pck1
and G6pc [90]. Thus, FXR activation elevates GR expression, which in turn results in
enhanced gluconeogenesis.
Ubiquitin-specific protease 2 (USP2) expression is induced by fasting by both
GC and glucagon, and by PGC1α overexpression [91]. Overexpression of Usp2 in
mouse liver increases glucose production and exacerbates high fat diet-induced glu-
cose intolerance, whereas the reduction of Usp2 expression in mouse liver improves
systemic glycemic control [91]. Usp2 induces the expression of 11β-hydroxysteroid
dehydrogenase type 1 (11β-HSD1), the enzyme that converts the inactive GC,
11-DHC (rodents) or cortisone (humans), to active corticosterone (rodents) or cor-

tisol (humans) in liver. Thus, Usp2 increases hepatic gluconeogenesis by increasing
the active GC levels in hepatocytes. Usp2 is a ubiquitin specific protease. How Usp2
increases the expression of 11β-HSD1 is unclear.
Transforming growth factor β (TGFβ) decreases hepatic gluconeogenesis.
TGFβ increases the expression of SMAD6, which directly associates with the
N-terminus of GR [92]. When SMAD6 associates with GRE-bound GR, it recruits
histone deacetylase 3 (HDAC3) which antagonizes the acetylation of histone H3
and H4 on genomic regions near the GREs [92]. This results in an inhibition of the
transactivation activity of GR. Overexpression of SMAD6 in liver reduces
GC-induced Pck1 transcription and blood glucose levels, which mimics TGFβ
effects [92].
GR associates with the Hsp90-containing chaperone complex in the cytoplasm,
an interaction that induces a GR conformation favorable for binding GC. The
Hsp90-containing chaperone complex also plays a role in the translocation of GR
into the nucleus. Acetylation of the lysine 294 residue of Hsp90 reduces its interac-
tion with GR [93]. Therefore, keeping lysine 294 in a deacetylated state is critical
for the GR-Hsp90 interaction. Histone deacetylase 6 (HDAC6) deacetylates lysine
294 of Hsp90. Ablation of Hdac6 results in a decreased GR response due to defec-
tive GR translocation into the nucleus [93]. In Hdac6 knockout mice, GC-induced
hepatic Pck1 and G6pc gene expression and glucose production are reduced [93].
GC-induced glucose production in primary hepatocytes isolated from Hdac6 knock-
out mice is markedly lower than in primary hepatocytes isolated from wild type
mice [93]. Notably, Hdac6 ablation generally reduces GR signaling. Not surpris-
ingly, other GR-regulated processes, including GC-induced insulin resistance and
lipolysis in adipocytes, are also affected when Hdac6 is deleted.
Glucose Utilization
GC inhibit glucose utilization by reducing both glucose uptake and glucose oxida-
tion in skeletal muscle and WAT, two major tissues involved in insulin-responsive
glucose utilization [94, 95]. These GC effects counteract those of insulin, which
promote glucose uptake, glycolysis and glucose oxidation. These result in a tran-
sient increase of circulating glucose, which is considered beneficial during stress
[5]. In both mouse and human myotubes GC reduce insulin-stimulated glucose
uptake [96–98] by attenuating insulin-induced GLUT4 translocation to the cell
membrane. By contrast, reduced GC signaling improves insulin sensitivity and glu-
cose utilization in mouse and human skeletal muscle. Circulating GC levels are
increased in genetically obese ob/ob, db/db and lipotrophic A-ZIP/F-1 [99] mice as
compared to wild type. These mice are insulin resistant, but adrenalectomy improves
insulin-stimulated muscle glucose disposal [100, 101]. In high fat diet-induced
obese mice, adrenalectomy or treatment with the GR antagonist, RU-486, improves
insulin sensitivity and increases glucose utilization in skeletal muscle. Moreover,
110 T. Kuo et al.
GC
Insulin
Myotube Liver
IR Palmitoyl-CoA
pIR Tyr
Sptlc2
pIRS-1 Ser307 IRS-1
3-keto-dihydro sphingosine
pIRS-1 Tyr608 Pik3r1
p110
dihydroceramide
pAkt Ser473 Des1

Akt
pAkt Thr308
Ceramide
mTOR Cers1
Ceramidase
Cers6
pS6K Thr389 S6K
sphingosine
Fig. 5.4 Models of glucocorticoid-regulated insulin action. Mechanisms of glucocorticoid-

induced insulin resistance are depicted. In myotubes, glucocorticoids (GC) decrease the tyrosine
phosphorylation of insulin receptor (IR) and the expression of IRS1. They increase the serine 307
phosphorylation while decrease the tyrosine 608 phosphorylation of IRS1. GC also increase the
expression of Pik3r1, which results in decreased activity of Akt and p70 S6 kinase (S6K). In the
liver, GC increase the gene expression of enzymes involved in ceramide synthesis, including
Sptlc2, Cers1 and Cers6, which results in increased levels of ceramides. These ceramides then
interfere with insulin signaling. The GC-regulated genes are shown in yellow
11β-HSD1-specific inhibitors, which reduce corticosterone levels in various tissues,

improve insulin sensitivity and skeletal muscle glucose utilization in animal models
of diabetes or insulin resistance [96].
The ability of GC to inhibit glucose uptake and glucose oxidation in skeletal mus-
cle is due to, at least in part, a direct effect of GCs on myotubes. Treating cultured
myotubes with GC inhibits insulin-stimulated glucose utilization [96, 102]. One
mechanism by which GC reduce glucose utilization is the inhibition of insulin sig-
naling [96, 103, 104]. Insulin binds to and activates the cell-surface insulin receptor
(IR) tyrosine kinase, which in turn phosphorylates the members of insulin receptor
substrate (IRS) protein family [105] (Fig. 5.4). Tyrosine-phosphorylated IRS proteins
associate with the IR and initiate downstream signaling events [105] (Fig. 5.4). Mice
treated with GCs have reduced levels of tyrosine-phosphorylated IR and total IRS-1
proteins in skeletal muscle [96], and the activities of phosphoinositide-3-kinase
(PI3K) and Akt, two signaling molecules downstream of IRS-1, are decreased [96,
106, 107] (Fig. 5.4).
The ability of GC to inhibit glucose utilization in myotubes requires protein

synthesis. A list of potential GR primary target genes that can suppress insulin action
has been identified in mouse C2C12 myotubes [108]. Among these genes, the role of
Pik3r1 (a.k.a. p85α) in the GC response was examined in vitro. Pik3r1 encodes the
regulatory subunit of PI3K, which binds to activate IRS1 (Fig. 5.4) through its SRC
homology 2 (SH2) domain to bring the catalytic subunit of PI3K, Pik3ca (a.k.a.
p110), to the plasma membrane [109, 110]. Pik3ca then catalyzes the conversion of
phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-bispho-
sphate (PIP3) [109, 110]. PIP3 anchors the protein kinase Akt protein kinase family
to the plasma membrane and thus initiates downstream signaling events [110]. Pik3r1
is a key component in the insulin signaling pathway. But monomeric Pik3r1 is thought
to compete with the Pik3r1/Pik3ca heterodimer to interact with IRS-1 to suppress
insulin action [111, 112]. In addition, Pik3r1 is required for the maximal activity of
phosphatase and tensin homolog (PTEN) [113], which antagonizes PI3K activity. In
C2C12 mouse myotubes, GC treatment reduces the activity of several components of
the insulin signaling pathway. These GC effects are markedly decreased in C2C12
myotubes that have reduced Pik3r1 expression [108]. Thus, Pik3r1 is a potential GR
primary target gene in the mediation of the suppressive effect of GC on glucose utili-
zation in skeletal muscle, though this notion needs to be confirmed in vivo.
Interestingly, global heterozygous deletion of Pik3r1 gene in mice has improved
whole body insulin sensitivity [114, 115]. Elevated expression of PIK3R1 is found in
patients with insulin resistance [116]. Notably, Pik3r1 is likely not the only GR target
gene that mediating suppressive effects of GC in insulin response, and additional GR
primary target genes could also participate in this process.
GC also modulate insulin sensitivity and glucose utilization through the genera-
tion of specific lipid mediators. GC treatment increases ceramide levels in mouse
liver and portal circulation [117]. These ceramides cause hepatic insulin resistance
[118]. They are also delivered to skeletal muscle so one might expect systemic
effects. In fact, hyperinsulinemic-euglycemic clamp studies in mice show that GC
decrease the glucose infusion rate required to maintain euglycemia, prevent insulin-
inhibited hepatic glucose output, and inhibit 2-deoxyglucose uptake into skeletal
muscle, all evidence of reduced insulin sensitivity, whereas mice pretreat with
myriocin, an inhibitor of serine palmitoyaltransferase (Sptlc1 and Sptlc2), an enzyme
in the ceramide synthetic pathway, have reduced GC-induced insulin resistance in
skeletal muscle and hepatic glucose output (Fig. 5.4) [117]. Notably, GC augment
the expression of enzymes in the ceramide synthetic and metabolic pathway in liver,
such as Sptlc2, ceramide synthase 1 (Cers1, a.k.a. Lass1), and ceramide synthase 6
(Cers6, a.k.a. Lass6) (Fig. 5.4) [117]. However, it is not yet clear whether the genes
encoding these enzymes are primary targets of GR signaling.
In addition to increased ceramide levels in liver, GC-promoted lipolysis in WAT
could impair whole body glucose homeostasis. Acipimox, an inhibitor of lipolysis
in adipocytes, improves whole body glucose homeostasis in human subjects treated
with GC [119]. Although it is not clear how GC-induced lipolysis affects glucose
homeostasis, it is likely that the fatty acids generated from lipolysis are mobilized
to skeletal muscle and liver and converted to lipid mediators, such as diacylglycerol
(DAG) and ceramides, that cause insulin resistance.
112 T. Kuo et al.
Notably, Brennan-Speranza et. al. also reported that GC inhibit the expression of
osteocalcin, a secreted protein from bone that reduce adiposity and hepatic steato-
sis, to decrease insulin sensitivity.
GC also inhibit glucose oxidation by stimulating the expression of several mem-
bers of the pyruvate dehydrogenase kinase family (PDK). PDK regulates glucose
oxidation by inhibiting the pyruvate dehydrogenase complex that converts pyruvate
to acetyl-CoA [120]. Among the PDK family, PDK4 is a GR primary target gene;
GREs have been identified in human PDK4 and rat Pdk4 genes [121, 122]. FoxO1
binding sites have been identified near the human PDK4 gene GRE and they are
required for the maximal induction of PDK4 gene transcription by GC. These FREs
also mediate the inhibitory response of insulin on PDK4 gene transcription [122].
For the rat Pdk4 gene, an FRE located approximately 6 kb away from the GREs is
thought to participate in both the insulin and GC responses [121]. Thus, the mecha-
nisms governing the transcriptional regulation of the PDK4 gene by GC appear to
be similar to GC-activated Pck1 and G6pc gene transcription discussed above.
In addition to skeletal muscle, GC reduce glucose uptake and glucose oxidation in
many other tissues. GC inhibit insulin-stimulated glucose uptake in both mouse 3T3-
L1 and primary adipocytes. The mechanisms governing these GC effects are mostly
unknown. Overexpression of dual specificity protein phosphatase 1 (Dusp1, a.k.a.
MAP kinase phosphatase 1, Mkp1), a primary GR target gene, inhibits insulin-
stimulated glucose uptake in 3T3-L1 adipocytes [123]. However, the exact role of
Dusp1 in GC-induced insulin resistance in adipocytes has not been established. Most
reports indicate that GC inhibit glucose uptake by antagonizing the insulin response,
though the direct inhibition of glucose transporter 4 (Glut4) trafficking process by
GC in 3T3-L1 adipocytes has also been reported [124]. Pik3r1 expression is also
increased by GC in adipocytes [125]. Thus, Pik3r1 may also participate in the
GC-inhibited insulin response in adipocytes. Studies of human adipocytes found that
GC inhibit insulin-stimulated glucose uptake and signaling in omental but not subcu-
taneous adipocytes [126]. In fact, studies in human primary subcutaneous adipocytes
show that GC pre-treatment potentiates insulin-stimulated glucose uptake [102, 127].
This suggests that GC affect insulin signaling in a depot-specific manner in humans.
The ability of GC to inhibit glucose oxidation has been linked to GC-induced
apoptosis in leukemia cells. GC inhibit the expression of glucose transporter 1
(GLUT1) that results in a decreased glucose uptake into leukemia cells [128]. GC
also suppress glucose uptake and oxidation in certain regions of brain, such as the
hypothalamus and hippocampus [129–131]. The exact mechanisms of these GC
effects on these cell types are mostly unclear.
Glycogen Metabolism
GC regulate glycogen metabolism in a tissue-specific manner. In liver the adminis-

tration of GC to fasted mice increases liver glycogen content [40, 132]. GC induce
the activity of glycogen synthase [7, 133, 134]. Glycogen synthase activity is
regulated by post-translational modification. Protein kinase A (PKA) and glycogen

synthase kinase 3 (GSK3) phosphorylates and inactivates glycogen synthase [135–
137]. In contrast, protein phosphatase 1 (PP1) dephosphorylates glycogen synthase,
which potentiates its activity [138, 139]. The active form of glycogen phosphory-
lase, glycogen phosphorylase a, breaks down glycogen to glucose units. Glycogen
phosphorylase a also inhibits the dephosphorylation of glycogen synthase by PP1.
PKA can also inhibit PP1 activity. Data suggest that the activation of glycogen syn-
thase phosphatase, PP1, by GC [140, 141], though the exact the mechanism of this
activation is unclear.
Epinephrine plays a key role in skeletal muscle glycogenolysis. This effect is
blunted in adrenalectomized rats [142]. The stimulatory effects of epinephrine on
muscle glycogen phosphorylase and phosphorylase kinase are all attenuated in the
skeletal muscle of adrenalectomized rats. PP1 dephosphorylates glycogen phos-
phorylase and phosphorylase kinase and suppresses their activities. PP1 activity is
increased in skeletal muscle of adrenalectomized rats and the ability of epinephrine
to inhibit PP1 is reduced in the skeletal muscle of adrenalectomized rats. Cortisol
treatment in adrenalectomized rats restores normal epinephrine effects through the
activation of phosphorylase kinase and glycogen phosphorylase and the inhibition
of PP1. The induction of PDK4 gene transcription by GR may contribute to
GC-regulated glycogen metabolism in myotubes. One report shows that a reduction
of the expression of PDK4 in human primary myotubes diminishes GC-repressed
glycogen synthesis [143]. Notably, GC also inhibits insulin-stimulated glycogen
synthesis and the activity of glycogen synthase [9]. These GC effects are mainly due
to their ability to reduce insulin signaling in skeletal muscle. Interestingly, one
report shows that glycogen storage is actually increased in soleus muscle by dexa-
methasone treatment despite a decrease of glycogen synthesis [9]. The mechanisms
underlying these phenotypes are not clear.
In cardiac muscle, adrenalectomy also blocks epinephrine-induced glycogenoly-
sis [144]. GC treatment, however, has been shown to facilitate glycogen storage in
cardiac muscle [145]. GC increase AMP-activated protein kinase (AMPK) activity
that leads to the elevation of glucose uptake, the induction of glycogen synthase,
and the reduction of glycogen phosphorylase. These contribute to an augmentation
of glycogen content in cardiac muscle. Brain stores certain amounts of glycogen,
though the levels are lower than in skeletal muscle or liver. Astrocytes store most
of the brain glycogen, whose levels are increased by norepinephrine. In primary
astrocyte culture norepinephrine induces glycogen synthesis, an effect which is
suppressed by GC [146].
GC Effects on Pancreas
As an endocrine organ, the pancreas secretes several hormones that include insulin
(from β cells in the islets of Langerhans), glucagon (from α cells), and somatostatin
(from δ cells). Insulin secreted during the fed state promotes glucose uptake and
114 T. Kuo et al.
utilization, and inhibits gluconeogenesis. In contrast, glucagon secreted during fast-

ing stimulates gluconeogenesis and glycogenolysis. Somatostatin suppresses both
insulin and glucagon secretion.
The effect of GC on insulin secretion is an area of vigorous research; however, the
direct mechanistic relationship between GC and β cell function in vivo remains elu-
sive. This is largely due to the difficulty of separating the indirect effects of
GC-induced peripheral insulin resistance from the direct actions of GC on pancreatic
β cells. Hypercortisolism induces insulin resistance in tissues such as liver, adipose
and skeletal muscle, as discussed above. In murine models and human studies, this
GC-induced insulin resistance can lead to compensatory β cell hyperplasia and
hyperinsulinemia, and consequent normoglycemia. However, long-term GC treat-
ment that exceeds the β cell compensatory capacity leads to insufficient insulin secre-
tion, hyperglycemia, and eventually Type 2 diabetes [15, 17, 147–150]. Overall, the
effects of GC on β cell function are dependent on the length and dosage of the treat-
ment, the experimental animal model under investigation, as well as the susceptibil-
ity of the strain exposed [151]. It is important to note that short-term effects of GC on
β cells are likely reversible [152]. To begin to dissect the direct in vivo effects of GC
on β cells, a mouse model of insulin promoter-driven GR overexpression was gener-
ated [14]. These mice show glucose intolerance due to blunted insulin secretion.
Insulin secretion is mainly triggered by a postprandial increase in the concentra-
tion of plasma glucose, which enters pancreatic β cells through GLUT2 transport-
ers. Glucokinase phosphorylates glucose to produce glucose-6-phosphate, which
enters the glycolytic cycle. Through mitochondrial β-oxidation, the ratio of ATP to
ADP increases, which leads to the closure of ATP-sensitive potassium channels and
plasma membrane depolarization. The increase in cellular electrical conductivity
drives the opening of voltage-sensitive calcium ion channels. The consequent influx
of calcium ions eventually leads to exocytosis of insulin-containing granules.
Compared to islets isolated from GC-treated animals, isolated islets directly
treated with GC present opposite results. First, synthetic GC dexamethasone pro-
motes posttranslational degradation of GLUT2 and decreases glucose-stimulated
insulin secretion (GSIS) in isolated rat pancreatic islets [153]. Second, dexametha-
sone inhibits glucokinase mRNA expression in RIN cells, a rat pancreatic islet
tumor cell line [154]. GC have also been reported to suppress the gene expression
of insulin [155] and pancreatic and duodenal homeobox 1 (Pdx1) in β cells [156].
Pdx1 is a transcription factor essential for pancreatic development and β cell matu-
ration. Third, in ob/ob mice, GC enhance the activity of glucose-6-phosphatase,
which dephosphorylates glucose-6-phosphate to produce glucose, resulting in a
futile cycle [157]. Fourth, in the INS-1 rat pancreatic β cell line, the transcription of
voltage-gated potassium channel Kv1.5 is upregulated by dexamethasone treatment
[158]. This results in an increase in the repolarizing outward current and reduces the
influx of calcium ions, which compromises GSIS. On the contrary, islets purified
from GC-treated rats display enhanced insulin secretion, likely due to compensa-
tory hyperinsulinemia [159–161], and have improved glucose sensitivity and oxida-
tive metabolism [162]. Furthermore, transmission electron microscopy shows an
increased amount of docked secretory granules in GC-treated β cells [148].
GC also regulates the expression of genes that modulate insulin secretion. The
blunting of GSIS by dexamethasone is significantly restored in islets isolated from
serum and glucocorticoid-induced kinase (Sgk1) knockout mice compared to wild
type [158]. Sgk1 is a well-established GR primary target gene. Moreover, GC treat-
ment of MIN6 mouse insulinoma cells and mouse islets augments the expression of
corticotropin-releasing factor receptor type 2α (CRFR2α) and inhibits the expression
of CRFR type 1 (CRFR1) [163]. CRFR1 potentiates both glucose-induced insulin
secretion in vitro and in vivo and the proliferation of neonatal rat β cells [164].
GC can stimulate β cell proliferation in vivo; however, GC exposure could also
have cytotoxic effects on mouse isolated pancreatic islets of Langerhans and MIN6
cells [165]. Interestingly, increased intracellular cAMP levels have previously been
shown to attenuate dexamethasone-induced β cell death using exendin-4 [20] and
forskolin [166]. Co-treatment with RU486, an antagonist of GC, abolished these
GC effects. Mitogen-activated protein kinases (MAPK) such as p38 MAPK and
JNK regulate apoptosis. In MIN6 cells, inhibition of p38 MAPK reduces
glucocorticoid-induced apoptosis [167]. This may be due to decreased phosphoryla-
tion of mouse GR at serine residue 220 by p38 MAPK. Phosphorylation of GR at
serine 220 is positively associated with GR transcriptional activity [168, 169].
Moreover, in isolated islets, inhibition of p38 MAPK decreases glucocorticoid-
induced formation of cleaved caspase 3, which plays a key role in the execution
phase of apoptosis [167]. Thus, p38 MAPK is required for glucocorticoid-induced
cytotoxicity. In contrast, JNK signaling dampens glucocorticoid-induced cytotoxic-
ity, as inhibition of JNK potentiates the cytotoxic effect of GC in MIN6 cells [167].
A plausible explanation is that JNK is responsible for phosphorylating GR at serine
234, which is negatively associated with the transcriptional activity of GR [170].
Reactive oxygen species (ROS) may also mediate glucocorticoid-induced cyto-
toxicity [171]. Thioredoxin-interacting protein (Txnip) could exert its pro-apoptotic
effect by blocking the activity of thioredoxin, which is involved in a major pro-
survival thiol-reducing pathway in cells. In db/db mice, overexpression of thiore-
doxin suppresses the progression of hyperglycemia, likely through the prevention of
the reduction of Pdx1 and V-maf musculoaponeurotic fibrosarcoma oncogene
homologue A (MafA) transcription factors [172]. GC induces Txnip expression in
human and mouse islets, and in MIN6 cells [165]. Overexpressing Txnip mimicked
pro-apoptotic effect of GC, while knocking down Txnip partially rescued this phe-
notype in MIN6 cells. Furthermore, thioredoxin overexpression protected MIN6
cells from GC-induced cytotoxicity [165]. Interestingly, GC-induced Txnip is
dependent on the presence of p38 MAPK. Studies in leukemia and other cell types
have shown that Txnip is a primary GR target gene [173].
In addition to insulin secretion, GC has been shown to modulate glucagon secre-
tion. GC treatment causes both hyperinsulinemia and hyperglucagonemia. Rodents
treated with GC have unaltered insulin/glucagon ratio from the fasted state to the
fed state. They tend to have increased α cell mass, and show impaired high glucose-
suppressed glucagon secretion. This hyperglucagonemia leads to hyperglycemia
through the activation of hepatic glycogenolysis and gluconeogenesis [174].
Notably, co-treatment of GC and a glucagon receptor antagonist on rats resulted in
116 T. Kuo et al.
normoglycemia [11]. The mechanisms underlying the effects of GC on glucagon

secretion, however, are unclear.
Conclusion and Future Directions
The physiological responses of GC that modulate glucose homeostasis have been

well documented. Significant progress has been made to understand these GC
actions in the last three decades. However, the precise manner by which GC regu-
lates glucose homeostasis is still unclear. The key to understanding the mechanisms
of GC-regulated glucose homeostasis is the identification of GR primary target
genes that mediate GC actions, and understanding how these primary target genes
are transcriptionally regulated by GR in various processes, including gluconeogen-
esis, glucose uptake and utilization, glycogen metabolism, and pancreatic endocrine
secretions. For transcriptional regulation, GC-activated Pck1 gene transcription has
received the most attention. Nevertheless, further studies are needed to understand
how different accessory factors coordinate with GR and transcriptional coregulators
to activate Pck1 gene transcription. Analyzing the transcriptional regulatory mecha-
nisms of these GC-regulated gluconeogenic genes should allow the identification of
common molecular features in GR-stimulated gluconeogenesis. In vivo, GC actions
are affected by other hormones and environmental cues. Understanding the cross-
talk and integration of these signals should be the focus of future work.
The emergence of advanced techniques and technologies during the last decade
have facilitated the characterization of molecular features of GC-regulated glucose
homeostasis. Using high throughput DNA sequencing technology, genomic GR
binding regions, transcriptional coregulator occupancy sites, and global chromatin
structure changes were identified. The discovery of GC-regulated non-coding RNA
and enhancer RNA, using genomic run-on sequencing (GRO-seq) and RNA
sequencing (RNA-seq), unveiled another layer of glucose homeostasis regulation
[175–178]. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
genome editing technology can now be used to efficiently ablate a GR primary tar-
get gene, and modify a particular residue of GRE to study its role in the natural
chromosomal context [179–183]. These new approaches in combination with
refined genetic, physiological and biochemical tools will play a critical role in
improving our understanding of GC-regulated biology in metabolic tissues.
Ultimately, the goal of the studying the basic mechanisms of GC-regulated glu-
cose homeostasis is to develop a pharmacotherapy targeting GC signaling for treat-
ing metabolic diseases. Reducing GC signaling in vivo improves insulin sensitivity
and decreases plasma glucose levels, which would largely benefit type 2 diabetes
patients. However, reducing GC actions globally is not ideal. For example, the
body’s inflammatory status would be elevated with reduced GC signaling. In fact,
as inflammation promotes insulin resistance, the anti-inflammatory activity of GR
could also improve insulin sensitivity. Overall, identifying approaches to dissociate
metabolic and anti-inflammatory actions of GR could provide novel ways to treat
metabolic disease.
Acknowledgment We thank Drs. Daryl Granner and Richard O’Brien for their insightful
comments and suggestions for this chapter. The Wang laboratory is supported by NIH DK83591.
References
1. Exton JH. Regulation of gluconeogenesis by glucocorticoids. Monogr Endocrinol. 1979;12:

535–46.
2. Kraus-Friedmann N. Hormonal regulation of hepatic gluconeogenesis. Physiol Rev.
1984;64:170–259.
3. Di Dalmazi G, Pagotto U, Pasquali R, Vicennati V. Glucocorticoids and type 2 diabetes: from
physiology to pathology. J Nutr Metab. 2012;2012:525093. doi:10.1155/2012/525093.
4. Kuo T, Harris CA, Wang JC. Metabolic functions of glucocorticoid receptor in skeletal mus-
cle. Mol Cell Endocrinol. 2013;380:79–88. doi:10.1016/j.mce.2013.03.003.
5. Charmandari E, Tsigos C, Chrousos G. Endocrinology of the stress response. Annu Rev
Physiol. 2005;67:259–84. doi:10.1146/annurev.physiol.67.040403.120816.
6. Andrews RC, Walker BR. Glucocorticoids and insulin resistance: old hormones, new targets.
Clin Sci (Lond). 1999;96:513–23.
7. Stalmans W, Laloux M. Glucocorticoids and hepatic glycogen metabolism. Monogr
Endocrinol. 1979;12:517–33.
8. Exton JH, Friedmann N, Wong EH, Brineaux JP, Corbin JD, Park CR. Interaction of gluco-
corticoids with glucagon and epinephrine in the control of gluconeogenesis and glycogenoly-
sis in liver and of lipolysis in adipose tissue. J Biol Chem. 1972;247:3579–88.
9. Ruzzin J, Wagman AS, Jensen J. Glucocorticoid-induced insulin resistance in skeletal mus-
cles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3
inhibitor. Diabetologia. 2005;48:2119–30. doi:10.1007/s00125-005-1886-0.
10. Wise JK, Hendler R, Felig P. Influence of glucocorticoids on glucagon secretion and plasma
amino acid concentrations in man. J Clin Invest. 1973;52:2774–82. doi:10.1172/JCI107473.
11. Rafacho A, Goncalves-Neto LM, Santos-Silva JC, Alonso-Magdalena P, Merino B, Taboga
SR, Carneiro EM, Boschero AC, Nadal A, Quesada I. Pancreatic alpha-cell dysfunction con-
tributes to the disruption of glucose homeostasis and compensatory insulin hypersecretion in
glucocorticoid-treated rats. PLoS One. 2014;9:e93531. doi:10.1371/journal.pone.0093531.
12. Beaudry JL, Riddell MC. Effects of glucocorticoids and exercise on pancreatic beta-cell
function and diabetes development. Diabetes Metab Res Rev. 2012;28:560–73. doi:10.1002/
dmrr.2310.
13. Longano CA, Fletcher HP. Insulin release after acute hydrocortisone treatment in mice.
Metabolism. 1983;32:603–8.
14. Delaunay F, Khan A, Cintra A, Davani B, Ling ZC, Andersson A, Ostenson CG, Gustafsson
J, Efendic S, Okret S. Pancreatic beta cells are important targets for the diabetogenic effects
of glucocorticoids. J Clin Invest. 1997;100:2094–8. doi:10.1172/JCI119743.
15. Ogawa A, Johnson JH, Ohneda M, McAllister CT, Inman L, Alam T, Unger RH. Roles of
insulin resistance and beta-cell dysfunction in dexamethasone-induced diabetes. J Clin
Invest. 1992;90:497–504. doi:10.1172/JCI115886.
16. Dinneen S, Alzaid A, Miles J, Rizza R. Metabolic effects of the nocturnal rise in cortisol on
carbohydrate metabolism in normal humans. J Clin Invest. 1993;92:2283–90.
17. Rafacho A, Cestari TM, Taboga SR, Boschero AC, Bosqueiro JR. High doses of dexametha-
sone induce increased beta-cell proliferation in pancreatic rat islets. Am J Physiol Endocrinol
Metab. 2009;296:E681–9. doi:10.1152/ajpendo.90931.2008.
18. Morisset J, Jolicoeur L. Effect of hydrocortisone on pancreatic growth in rats. Am J Physiol.
1980;239:G95–8.
19. Like AA, Chick WL. Pancreatic beta cell replication induced by glucocorticoids in subhuman
primates. Am J Pathol. 1974;75:329–48.
118 T. Kuo et al.
20. Ranta F, Avram D, Berchtold S, Dufer M, Drews G, Lang F, Ullrich S. Dexamethasone

induces cell death in insulin-secreting cells, an effect reversed by exendin-4. Diabetes.
2006;55:1380–90.
21. Weinhaus AJ, Bhagroo NV, Brelje TC, Sorenson RL. Dexamethasone counteracts the effect
of prolactin on islet function: implications for islet regulation in late pregnancy. Endocrinology.
2000;141:1384–93. doi:10.1210/endo.141.4.7409.
22. Pilkis SJ, el-Maghrabi MR, Claus TH. Hormonal regulation of hepatic gluconeogenesis and
glycolysis. Annu Rev Biochem. 1988;57:755–83. doi:10.1146/annurev.bi.57.070188.003543.
23. Pilkis SJ, Granner DK. Molecular physiology of the regulation of hepatic gluconeogenesis and
glycolysis. Annu Rev Physiol. 1992;54:885–909. doi:10.1146/annurev.ph.54.030192.004321.
24. Jitrapakdee S, Wallace JC. Structure, function and regulation of pyruvate carboxylase.
Biochem J. 1999;340(Pt 1):1–16.
25. Menefee AL, Zeczycki TN. Nearly 50 years in the making: defining the catalytic mechanism
of the multifunctional enzyme, pyruvate carboxylase. FEBS J. 2014;281:1333–54.
doi:10.1111/febs.12713.
26. Hanson RW, Garber AJ. Phosphoenolpyruvate carboxykinase. I. Its role in gluconeogenesis.
Am J Clin Nutr. 1972;25:1010–21.
27. Hanson RW, Patel YM. Phosphoenolpyruvate carboxykinase (GTP): the gene and the
enzyme. Adv Enzymol Relat Areas Mol Biol. 1994;69:203–81.
28. Mendez-Lucas A, Duarte JA, Sunny NE, Satapati S, He T, Fu X, Bermudez J, Burgess SC,
Perales JC. PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C medi-
ated gluconeogenesis. J Hepatol. 2013;59:105–13. doi:10.1016/j.jhep.2013.02.020.
29. Stark R, Guebre-Egziabher F, Zhao X, Feriod C, Dong J, Alves TC, Ioja S, Pongratz RL,
Bhanot S, Roden M, Cline GW, Shulman GI, Kibbey RG. A role for mitochondrial phospho-
enolpyruvate carboxykinase (PEPCK-M) in the regulation of hepatic gluconeogenesis. J Biol
Chem. 2014;289:7257–63. doi:10.1074/jbc.C113.544759.
30. Arinze IJ, Garber AJ, Hanson RW. The regulation of gluconeogenesis in mammalian liver. The
role of mitochondrial phosphoenolpyruvate carboxykinase. J Biol Chem. 1973;248:2266–74.
31. Cheng SC, Cheng RH. A mitochondrial phosphoenolpyruvate carboxykinase from rat brain.
Arch Biochem Biophys. 1972;151:501–11.
32. Rider MH, Bertrand L, Vertommen D, Michels PA, Rousseau GG, Hue L. 6-phosphofructo-
2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls
glycolysis. Biochem J. 2004;381:561–79. doi:10.1042/BJ20040752.
33. Rousseau GG, Hue L. Mammalian 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a
bifunctional enzyme that controls glycolysis. Prog Nucleic Acid Res Mol Biol. 1993;45:
99–127.
34. Marcus F, Rittenhouse J, Gontero B, Harrsch PB. Function, structure and evolution of
fructose-1,6-bisphosphatase. Arch Biol Med Exp. 1987;20:371–8.
35. Burchell A, Waddell ID. The molecular basis of the hepatic microsomal glucose-6-
phosphatase system. Biochim Biophys Acta. 1991;1092:129–37.
36. Hutton JC, O’Brien RM. Glucose-6-phosphatase catalytic subunit gene family. J Biol Chem.
2009;284:29241–5. doi:10.1074/jbc.R109.025544.
37. van Schaftingen E, Gerin I. The glucose-6-phosphatase system. Biochem J. 2002;362:513–32.
38. Schneiter P, Tappy L. Kinetics of dexamethasone-induced alterations of glucose metabolism
in healthy humans. Am J Physiol. 1998;275:E806–13.
39. Tounian P, Schneiter P, Henry S, Delarue J, Tappy L. Effects of dexamethasone on hepatic
glucose production and fructose metabolism in healthy humans. Am J Physiol.
1997;273:E315–20.
40. Exton JH, Miller TB, Harper SC, Park CR. Carbohydrate metabolism in perfused livers of
adrenalectomized and steroid-replaced rats. Am J Physiol. 1976;230:163–70.
41. Exton JH, Park CR. Control of gluconeogenesis in the perfused liver of normal and adrenal-
ectomized rats. J Biol Chem. 1965;240:955–7.
42. Sistare FD, Haynes Jr RC. Acute stimulation by glucocorticoids of gluconeogenesis from
lactate/pyruvate in isolated hepatocytes from normal and adrenalectomized rats. J Biol Chem.
1985;260:12754–60.
43. Exton JH, Mallette LE, Jefferson LS, Wong EH, Friedmann N, Miller Jr TB, Park CR. The
hormonal control of hepatic gluconeogenesis. Recent Prog Horm Res. 1970;26:411–61.
44. Menon RK, Sperling MA. Carbohydrate metabolism. Semin Perinatol. 1988;12:157–62.
45. Imai E, Stromstedt PE, Quinn PG, Carlstedt-Duke J, Gustafsson JA, Granner
DK. Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate
carboxykinase gene. Mol Cell Biol. 1990;10:4712–9.
46. Scott DK, Stromstedt PE, Wang JC, Granner DK. Further characterization of the glucocorti-
coid response unit in the phosphoenolpyruvate carboxykinase gene. The role of the glucocor-
ticoid receptor-binding sites. Mol Endocrinol. 1998;12:482–91.
47. Scott DK, Mitchell JA, Granner DK. The orphan receptor COUP-TF binds to a third gluco-
corticoid accessory factor element within the phosphoenolpyruvate carboxykinase gene pro-
moter. J Biol Chem. 1996;271:31909–14.
48. Imai E, Miner JN, Mitchell JA, Yamamoto KR, Granner DK. Glucocorticoid receptor-cAMP
response element-binding protein interaction and the response of the phosphoenolpyruvate
carboxykinase gene to glucocorticoids. J Biol Chem. 1993;268:5353–6.
49. Hall RK, Sladek FM, Granner DK. The orphan receptors COUP-TF and HNF-4 serve as
accessory factors required for induction of phosphoenolpyruvate carboxykinase gene tran-
scription by glucocorticoids. Proc Natl Acad Sci U S A. 1995;92:412–6.
50. Hall RK, Scott DK, Noisin EL, Lucas PC, Granner DK. Activation of the phosphoenolpyru-
vate carboxykinase gene retinoic acid response element is dependent on a retinoic acid recep-
tor/coregulator complex. Mol Cell Biol. 1992;12:5527–35.
51. Wang XL, Herzog B, Waltner-Law M, Hall RK, Shiota M, Granner DK. The synergistic
effect of dexamethasone and all-trans-retinoic acid on hepatic phosphoenolpyruvate car-
boxykinase gene expression involves the coactivator p300. J Biol Chem. 2004;279:34191–
200. doi:10.1074/jbc.M403455200.
52. Wang JC, Stromstedt PE, O’Brien RM, Granner DK. Hepatic nuclear factor 3 is an accessory
factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription
by glucocorticoids. Mol Endocrinol. 1996;10:794–800.
53. Zhang K, Li L, Qi Y, Zhu X, Gan B, DePinho RA, Averitt T, Guo S. Hepatic suppression of
Foxo1 and Foxo3 causes hypoglycemia and hyperlipidemia in mice. Endocrinology.
2012;153:631–46.
54. O’Brien RM, Lucas PC, Forest CD, Magnuson MA, Granner DK. Identification of a sequence
in the PEPCK gene that mediates a negative effect of insulin on transcription. Science.
1990;249:533–7.
55. Forest CD, O’Brien RM, Lucas PC, Magnuson MA, Granner DK. Regulation of phospho-
enolpyruvate carboxykinase gene expression by insulin. Use of the stable transfection
approach to locate an insulin responsive sequence. Mol Endocrinol. 1990;4:1302–10.
doi:10.1210/mend-4-9-1302.
56. Nakae J, Kitamura T, Silver DL, Accili D. The forkhead transcription factor Foxo1 (Fkhr)
confers insulin sensitivity onto glucose-6-phosphatase expression. J Clin Invest. 2001;108:
1359–67.
57. Wolfrum C, Asilmaz E, Luca E, Friedman JM, Stoffel M. Foxa2 regulates lipid metabolism
and ketogenesis in the liver during fasting and in diabetes. Nature. 2004;432:1027–32.
doi:10.1038/nature03047.
58. Lechner PS, Croniger CM, Hakimi P, Millward C, Fekter C, Yun JS, Hanson RW. The use of
transgenic mice to analyze the role of accessory factor two in the regulation of
phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes. J Biol Chem.
2001;276:22675–9. doi:10.1074/jbc.M102422200.
59. Scott DK, Mitchell JA, Granner DK. Identification and characterization of the second retinoic
acid response element in the phosphoenolpyruvate carboxykinase gene promoter. J Biol
Chem. 1996;271:6260–4.
60. Yamada K, Duong DT, Scott DK, Wang JC, Granner DK. CCAAT/enhancer-binding protein
beta is an accessory factor for the glucocorticoid response from the cAMP response element in
the rat phosphoenolpyruvate carboxykinase gene promoter. J Biol Chem. 1999;274:5880–7.
120 T. Kuo et al.
61. Hall RK, Wang XL, George L, Koch SR, Granner DK. Insulin represses phosphoenolpyru-
vate carboxykinase gene transcription by causing the rapid disruption of an active transcrip-
tion complex: a potential epigenetic effect. Mol Endocrinol. 2007;21:550–63. doi:10.1210/
me.2006-0307.
62. Schurter BT, Koh SS, Chen D, Bunick GJ, Harp JM, Hanson BL, Henschen-Edman A,
Mackay DR, Stallcup MR, Aswad DW. Methylation of histone H3 by coactivator-associated
arginine methyltransferase 1. Biochemistry. 2001;40:5747–56.
63. Lee DY, Teyssier C, Strahl BD, Stallcup MR. Role of protein methylation in regulation of
transcription. Endocr Rev. 2005;26:147–70.
64. Feng Q, He B, Jung SY, Song Y, Qin J, Tsai SY, Tsai MJ, O’Malley BW. Biochemical control
of CARM1 enzymatic activity by phosphorylation. J Biol Chem. 2009;284:36167–74.
doi:10.1074/jbc.M109.065524.
65. Cassuto H, Kochan K, Chakravarty K, Cohen H, Blum B, Olswang Y, Hakimi P, Xu C,
Massillon D, Hanson RW, Reshef L. Glucocorticoids regulate transcription of the gene for
phosphoenolpyruvate carboxykinase in the liver via an extended glucocorticoid regulatory
unit. J Biol Chem. 2005;280:33873–84. doi:10.1074/jbc.M504119200.
66. Bernal-Mizrachi C, Weng S, Feng C, Finck BN, Knutsen RH, Leone TC, Coleman T, Mecham
RP, Kelly DP, Semenkovich CF. Dexamethasone induction of hypertension and diabetes is
PPAR-alpha dependent in LDL receptor-null mice. Nat Med. 2003;9:1069–75.
67. Beale EG, Forest C, Hammer RE. Regulation of cytosolic phosphoenolpyruvate carboxyki-
nase gene expression in adipocytes. Biochimie. 2003;85:1207–11.
68. Olswang Y, Blum B, Cassuto H, Cohen H, Biberman Y, Hanson RW, Reshef L. Glucocorticoids
repress transcription of phosphoenolpyruvate carboxykinase (GTP) gene in adipocytes by
inhibiting its C/EBP-mediated activation. J Biol Chem. 2003;278:12929–36. doi:10.1074/
jbc.M300263200.
69. Hanson RW, Reshef L. Glyceroneogenesis revisited. Biochimie. 2003;85:1199–205.
70. Cadoudal T, Leroyer S, Reis AF, Tordjman J, Durant S, Fouque F, Collinet M, Quette J,
Chauvet G, Beale E, Velho G, Antoine B, Benelli C, Forest C. Proposed involvement of adi-
pocyte glyceroneogenesis and phosphoenolpyruvate carboxykinase in the metabolic syn-
drome. Biochimie. 2005;87:27–32. doi:10.1016/j.biochi.2004.12.005.
71. Wang JC, Stromstedt PE, Sugiyama T, Granner DK. The phosphoenolpyruvate carboxyki-
nase gene glucocorticoid response unit: identification of the functional domains of accessory
factors HNF3 beta (hepatic nuclear factor-3 beta) and HNF4 and the necessity of proper
alignment of their cognate binding sites. Mol Endocrinol. 1999;13:604–18.
72. Stafford JM, Waltner-Law M, Granner DK. Role of accessory factors and steroid receptor
coactivator 1 in the regulation of phosphoenolpyruvate carboxykinase gene transcription by
glucocorticoids. J Biol Chem. 2001;276:3811–9. doi:10.1074/jbc.M009389200.
73. Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, Kitamura Y, Altomonte J,
Dong H, Accili D, Spiegelman BM. Insulin-regulated hepatic gluconeogenesis through
FOXO1-PGC-1alpha interaction. Nature. 2003;423:550–5. doi:10.1038/nature01667.
74. Stafford JM, Wilkinson JC, Beechem JM, Granner DK. Accessory factors facilitate the
binding of glucocorticoid receptor to the phosphoenolpyruvate carboxykinase gene promoter.
J Biol Chem. 2001;276:39885–91.
75. Vander Kooi BT, Onuma H, Oeser JK, Svitek CA, Allen SR, Vander Kooi CW, Chazin WJ,
O’Brien RM. The glucose-6-phosphatase catalytic subunit gene promoter contains both posi-
tive and negative glucocorticoid response elements. Mol Endocrinol. 2005;19:3001–22.
76. Onuma H, Vander Kooi BT, Boustead JN, Oeser JK, O’Brien RM. Correlation between
FOXO1a (FKHR) and FOXO3a (FKHRL1) binding and the inhibition of basal glucose-6-
phosphatase catalytic subunit gene transcription by insulin. Mol Endocrinol. 2006;20:2831–
47. doi:10.1210/me.2006-0085.
77. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR,
Granner DK, Newgard CB, Spiegelman BM. Control of hepatic gluconeogenesis through the
transcriptional coactivator PGC-1. Nature. 2001;413:131–8.
78. Boustead JN, Stadelmaier BT, Eeds AM, Wiebe PO, Svitek CA, Oeser JK, O’Brien
RM. Hepatocyte nuclear factor-4 alpha mediates the stimulatory effect of peroxisome
proliferator-activated receptor gamma co-activator-1 alpha (PGC-1 alpha) on glucose-6-
phosphatase catalytic subunit gene transcription in H4IIE cells. Biochem J. 2003;369:17–22.
doi:10.1042/BJ20021382.
79. Matsumoto M, Pocai A, Rossetti L, Depinho RA, Accili D. Impaired regulation of hepatic
glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell
Metab. 2007;6:208–16.
80. Hiraiwa H, Chou JY. Glucocorticoids activate transcription of the gene for the glucose-6-
phosphate transporter, deficient in glycogen storage disease type 1b. DNA Cell Biol.
2001;20:447–53. doi:10.1089/104454901316976073.
81. Kallwellis-Opara A, Zaho X, Zimmermann U, Unterman TG, Walther R, Schmoll
D. Characterization of cis-elements mediating the stimulation of glucose-6-phosphate trans-
porter promoter activity by glucocorticoids. Gene. 2003;320:59–66.
82. Pierreux CE, Urso B, De Meyts P, Rousseau GG, Lemaigre FP. Inhibition by insulin of
glucocorticoid-induced gene transcription: involvement of the ligand-binding domain of the
glucocorticoid receptor and independence from the phosphatidylinositol 3-kinase and
mitogen-activated protein kinase pathways. Mol Endocrinol. 1998;12:1343–54. doi:10.1210/
mend.12.9.0172.
83. Lemaigre FP, Lause P, Rousseau GG. Insulin inhibits glucocorticoid-induced stimulation of
liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene transcription. FEBS Lett.
1994;340:221–5.
84. Sutherland C, O’Brien RM, Granner DK. Phosphatidylinositol 3-kinase, but not p70/p85 ribo-
somal S6 protein kinase, is required for the regulation of phosphoenolpyruvate carboxykinase
(PEPCK) gene expression by insulin. Dissociation of signaling pathways for insulin and phor-
bol ester regulation of PEPCK gene expression. J Biol Chem. 1995;270:15501–6.
85. Liao J, Barthel A, Nakatani K, Roth RA. Activation of protein kinase B/Akt is sufficient to
repress the glucocorticoid and cAMP induction of phosphoenolpyruvate carboxykinase gene.
J Biol Chem. 1998;273:27320–4.
86. De Los Pinos E, Fernandez De Mattos S, Joaquin M, Tauler A. Insulin inhibits glucocorticoid-
stimulated L-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression by
activation of the c-Jun N-terminal kinase pathway. Biochem J. 2001;353:267–73.
87. Patel R, Patel M, Tsai R, Lin V, Bookout AL, Zhang Y, Magomedova L, Li T, Chan JF, Budd
C, Mangelsdorf DJ, Cummins CL. LXRbeta is required for glucocorticoid-induced hypergly-
cemia and hepatosteatosis in mice. J Clin Invest. 2010;121:431–41.
88. Nader N, Ng SS, Wang Y, Abel BS, Chrousos GP, Kino T. Liver X receptors regulate the
transcriptional activity of the glucocorticoid receptor: implications for the carbohydrate
metabolism. PLoS One. 2012;7:e26751.
89. Lu Y, Xiong X, Wang X, Zhang Z, Li J, Shi G, Yang J, Zhang H, Ning G, Li X. Yin Yang 1
promotes hepatic gluconeogenesis through upregulation of glucocorticoid receptor. Diabetes.
2013;62:1064–73. doi:10.2337/db12-0744.
90. Renga B, Mencarelli A, D’Amore C, Cipriani S, Baldelli F, Zampella A, Distrutti E, Fiorucci
S. Glucocorticoid receptor mediates the gluconeogenic activity of the farnesoid X receptor in
the fasting condition. FASEB J. 2012;26:3021–31. doi:10.1096/fj.11-195701.
91. Molusky MM, Li S, Ma D, Yu L, Lin JD. Ubiquitin-specific protease 2 regulates hepatic
gluconeogenesis and diurnal glucose metabolism through 11beta-hydroxysteroid dehydroge-
nase 1. Diabetes. 2012;61:1025–35. doi:10.2337/db11-0970.
92. Ichijo T, Voutetakis A, Cotrim AP, Bhattachryya N, Fujii M, Chrousos GP, Kino T. The
Smad6-histone deacetylase 3 complex silences the transcriptional activity of the glucocorti-
coid receptor: potential clinical implications. J Biol Chem. 2005;280:42067–77. doi:10.1074/
jbc.M509338200.
93. Winkler R, Benz V, Clemenz M, Bloch M, Foryst-Ludwig A, Wardat S, Witte N, Trappiel M,
Namsolleck P, Mai K, Spranger J, Matthias G, Roloff T, Truee O, Kappert K, Schupp M,
Matthias P, Kintscher U. Histone deacetylase 6 (HDAC6) is an essential modifier of
122 T. Kuo et al.
glucocorticoid-induced hepatic gluconeogenesis. Diabetes. 2012;61:513–23. doi:10.2337/

db11-0313.
94. Ferrannini E, Simonson DC, Katz LD, Reichard Jr G, Bevilacqua S, Barrett EJ, Olsson M,
DeFronzo RA. The disposal of an oral glucose load in patients with non-insulin-dependent
diabetes. Metabolism. 1988;37:79–85.
95. DeFronzo RA, Tripathy D. Skeletal muscle insulin resistance is the primary defect in type 2
diabetes. Diabetes Care. 2009;32 Suppl 2:S157–63.
96. Morgan SA, Sherlock M, Gathercole LL, Lavery GG, Lenaghan C, Bujalska IJ, Laber D, Yu
A, Convey G, Mayers R, Hegyi K, Sethi JK, Stewart PM, Smith DM, Tomlinson JW. 11beta-
hydroxysteroid dehydrogenase type 1 regulates glucocorticoid-induced insulin resistance in
skeletal muscle. Diabetes. 2009;58:2506–15.
97. Dimitriadis G, Leighton B, Parry-Billings M, Sasson S, Young M, Krause U, Bevan S, Piva T,
Wegener G, Newsholme EA. Effects of glucocorticoid excess on the sensitivity of glucose
transport and metabolism to insulin in rat skeletal muscle. Biochem J. 1997;321(Pt 3):707–12.
98. Weinstein SP, Wilson CM, Pritsker A, Cushman SW. Dexamethasone inhibits insulin-
stimulated recruitment of GLUT4 to the cell surface in rat skeletal muscle. Metabolism.
1998;47:3–6.
99. Moitra J, Mason MM, Olive M, Krylov D, Gavrilova O, Marcus-Samuels B, Feigenbaum L,
Lee E, Aoyama T, Eckhaus M, Reitman ML, Vinson C. Life without white fat: a transgenic
mouse. Genes Dev. 1998;12:3168–81.
100. Ohshima K, Shargill NS, Chan TM, Bray GA. Effects of dexamethasone on glucose transport
by skeletal muscles of obese (ob/ob) mice. Int J Obes. 1989;13:155–63.
101. Haluzik M, Dietz KR, Kim JK, Marcus-Samuels B, Shulman GI, Gavrilova O, Reitman
ML. Adrenalectomy improves diabetes in A-ZIP/F-1 lipoatrophic mice by increasing both
liver and muscle insulin sensitivity. Diabetes. 2002;51:2113–8.
102. Gathercole LL, Bujalska IJ, Stewart PM, Tomlinson JW. Glucocorticoid modulation of insulin
signaling in human subcutaneous adipose tissue. J Clin Endocrinol Metab. 2007;92:4332–9.
103. Pivonello R, De Leo M, Vitale P, Cozzolino A, Simeoli C, De Martino MC, Lombardi G,
Colao A. Pathophysiology of diabetes mellitus in Cushing’s syndrome. Neuroendocrinology.
2010;92 Suppl 1:77–81.
104. Schakman O, Gilson H, Thissen JP. Mechanisms of glucocorticoid-induced myopathy.
J Endocrinol. 2008;197:1–10.
105. Lee YH, White MF. Insulin receptor substrate proteins and diabetes. Arch Pharm Res.
2004;27:361–70.
106. Saad MJ, Folli F, Kahn JA, Kahn CR. Modulation of insulin receptor, insulin receptor sub-
strate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats.
J Clin Invest. 1993;92:2065–72.
107. Giorgino F, Almahfouz A, Goodyear LJ, Smith RJ. Glucocorticoid regulation of insulin
receptor and substrate IRS-1 tyrosine phosphorylation in rat skeletal muscle in vivo. J Clin
Invest. 1993;91:2020–30.
108. Kuo T, Lew MJ, Mayba O, Harris CA, Speed TP, Wang JC. Genome-wide analysis of
glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insu-
lin signaling. Proc Natl Acad Sci U S A. 2012;109:11160–5. doi:10.1073/pnas.1111334109.
109. Backer JM. The regulation of class IA PI 3-kinases by inter-subunit interactions. Curr Top
Microbiol Immunol. 2010;346:87–114. doi:10.1007/82_2010_52.
110. Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002;296:1655–7. doi:10.1126/
science.296.5573.1655.
111. Barbour LA, Shao J, Qiao L, Leitner W, Anderson M, Friedman JE, Draznin B. Human placental
growth hormone increases expression of the p85 regulatory unit of phosphatidylinositol 3-kinase
and triggers severe insulin resistance in skeletal muscle. Endocrinology. 2004;145:1144–50.
112. Draznin B. Molecular mechanisms of insulin resistance: serine phosphorylation of insulin
receptor substrate-1 and increased expression of p85alpha: the two sides of a coin. Diabetes.
2006;55:2392–7.
113. Chagpar RB, Links PH, Pastor MC, Furber LA, Hawrysh AD, Chamberlain MD, Anderson
DH. Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase.
Proc Natl Acad Sci U S A. 2010;107:5471–6. doi:10.1073/pnas.0908899107.
114. Terauchi Y, Tsuji Y, Satoh S, Minoura H, Murakami K, Okuno A, Inukai K, Asano T, Kaburagi
Y, Ueki K, Nakajima H, Hanafusa T, Matsuzawa Y, Sekihara H, Yin Y, Barrett JC, Oda H,
Ishikawa T, Akanuma Y, Komuro I, Suzuki M, Yamamura K, Kodama T, Suzuki H, Yamamura
K, Kodama T, Suzuki H, Koyasu S, Aizawa S, Tobe K, Fukui Y, Yazaki Y, Kadowaki
T. Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of
phosphoinositide 3-kinase. Nat Genet. 1999;21:230–5. doi:10.1038/6023.
115. Mauvais-Jarvis F, Ueki K, Fruman DA, Hirshman MF, Sakamoto K, Goodyear LJ, Iannacone
M, Accili D, Cantley LC, Kahn CR. Reduced expression of the murine p85alpha subunit of
phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. J Clin Invest.
2002;109:141–9.
116. Bandyopadhyay GK, Yu JG, Ofrecio J, Olefsky JM. Increased p85/55/50 expression and
decreased phosphotidylinositol 3-kinase activity in insulin-resistant human skeletal muscle.
Diabetes. 2005;54:2351–9.
117. Holland WL, Brozinick JT, Wang LP, Hawkins ED, Sargent KM, Liu Y, Narra K, Hoehn KL,
Knotts TA, Siesky A, Nelson DH, Karathanasis SK, Fontenot GK, Birnbaum MJ, Summers
SA. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-
induced insulin resistance. Cell Metab. 2007;5:167–79.
118. Chavez JA, Summers SA. A ceramide-centric view of insulin resistance. Cell Metab.
2012;15:585–94. doi:10.1016/j.cmet.2012.04.002.
119. Tappy L, Randin D, Vollenweider P, Vollenweider L, Paquot N, Scherrer U, Schneiter P,
Nicod P, Jequier E. Mechanisms of dexamethasone-induced insulin resistance in healthy
humans. J Clin Endocrinol Metab. 1994;79:1063–9.
120. Sugden MC, Holness MJ. Recent advances in mechanisms regulating glucose oxidation at
the level of the pyruvate dehydrogenase complex by PDKs. Am J Physiol Endocrinol Metab.
2003;284:E855–62. doi:10.1152/ajpendo.00526.2002.
121. Connaughton S, Chowdhury F, Attia RR, Song S, Zhang Y, Elam MB, Cook GA, Park EA.
Regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) gene expression by glucocor-
ticoids and insulin. Mol Cell Endocrinol. 2010;315:159–67. doi:10.1016/j.mce.2009.08.011.
122. Kwon HS, Huang B, Unterman TG, Harris RA. Protein kinase B-alpha inhibits human pyru-
vate dehydrogenase kinase-4 gene induction by dexamethasone through inactivation of
FOXO transcription factors. Diabetes. 2004;53:899–910.
123. Bazuine M, Carlotti F, Tafrechi RS, Hoeben RC, Maassen JA. Mitogen-activated protein
kinase (MAPK) phosphatase-1 and -4 attenuate p38 MAPK during dexamethasone-induced
insulin resistance in 3T3-L1 adipocytes. Mol Endocrinol. 2004;18:1697–707.
124. Sakoda H, Ogihara T, Anai M, Funaki M, Inukai K, Katagiri H, Fukushima Y, Onishi Y, Ono
H, Fujishiro M, Kikuchi M, Oka Y, Asano T. Dexamethasone-induced insulin resistance in
3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal trans-
duction. Diabetes. 2000;49:1700–8.
125. Yu CY, Mayba O, Lee JV, Tran J, Harris C, Speed TP, Wang JC. Genome-wide analysis of
glucocorticoid receptor binding regions in adipocytes reveal gene network involved in tri-
glyceride homeostasis. PLoS One. 2010;5:e15188.
126. Hazlehurst JM, Gathercole LL, Nasiri M, Armstrong MJ, Borrows S, Yu J, Wagenmakers AJ,
Stewart PM, Tomlinson JW. Glucocorticoids fail to cause insulin resistance in human subcu-
taneous adipose tissue in vivo. J Clin Endocrinol Metab. 2013;98:1631–40. doi:10.1210/
jc.2012-3523.
127. Gathercole LL, Morgan SA, Bujalska IJ, Stewart PM, Tomlinson JW. Short- and long-term
glucocorticoid treatment enhances insulin signalling in human subcutaneous adipose tissue.
Nutr Diabetes. 2011;1:e3. doi:10.1038/nutd.2010.3.
128. Buentke E, Nordstrom A, Lin H, Bjorklund AC, Laane E, Harada M, Lu L, Tegnebratt T,
Stone-Elander S, Heyman M, Soderhall S, Porwit A, Ostenson CG, Shoshan M, Tamm KP,
Grander D. Glucocorticoid-induced cell death is mediated through reduced glucose metabo-
lism in lymphoid leukemia cells. Blood Cancer J. 2011;1:e31. doi:10.1038/bcj.2011.27.
124 T. Kuo et al.
129. de Leon MJ, McRae T, Rusinek H, Convit A, De Santi S, Tarshish C, Golomb J, Volkow N,
Daisley K, Orentreich N, McEwen B. Cortisol reduces hippocampal glucose metabolism in
normal elderly, but not in Alzheimer’s disease. J Clin Endocrinol Metab. 1997;82:3251–9.
doi:10.1210/jcem.82.10.4305.
130. Horner HC, Packan DR, Sapolsky RM. Glucocorticoids inhibit glucose transport in cultured
hippocampal neurons and glia. Neuroendocrinology. 1990;52:57–64.
131. Cherian AK, Briski KP. Effects of adrenalectomy on neuronal substrate fuel transporter and
energy transducer gene expression in hypothalamic and hindbrain metabolic monitoring
sites. Neuroendocrinology. 2010;91:56–63. doi:10.1159/000264919.
132. Mersmann HJ, Segal HL. Glucocorticoid control of the liver glycogen synthetase-activating
system. J Biol Chem. 1969;244:1701–4.
133. Ray PD, Foster DO, Lardy HA. Mode of action of glucocorticoids. I. Stimulation of gluconeo-
genesis independent of synthesis de novo of enzymes. J Biol Chem. 1964;239:3396–400.
134. De Wulf H, Hers HG. The stimulation of glycogen synthesis and of glycogen synthetase in
the liver by glucocorticoids. Eur J Biochem. 1967;2:57–60.
135. Huang TS, Krebs EG. Amino acid sequence of a phosphorylation site in skeletal muscle
glycogen synthetase. Biochem Biophys Res Commun. 1977;75:643–50.
136. Rylatt DB, Aitken A, Bilham T, Condon GD, Embi N, Cohen P. Glycogen synthase from rab-
bit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase
kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by
phosphorylase kinase. Eur J Biochem. 1980;107:529–37.
137. Pugazhenthi S, Khandelwal RL. Regulation of glycogen synthase activation in isolated hepa-
tocytes. Mol Cell Biochem. 1995;149–150:95–101.
138. Brady MJ, Nairn AC, Saltiel AR. The regulation of glycogen synthase by protein phosphatase
1 in 3T3-L1 adipocytes. Evidence for a potential role for DARPP-32 in insulin action.
J Biol Chem. 1997;272:29698–703.
139. Printen JA, Brady MJ, Saltiel AR. PTG, a protein phosphatase 1-binding protein with a role
in glycogen metabolism. Science. 1997;275:1475–8.
140. Laloux M, Stalmans W, Hers HG. On the mechanism by which glucocorticoids cause the
activation of glycogen synthase in mouse and rat livers. Eur J Biochem. 1983;136:175–81.
141. Vanstapel F, Dopere F, Stalmans W. The role of glycogen synthase phosphatase in the
glucocorticoid-induced deposition of glycogen in foetal rat liver. Biochem J. 1980;192:
607–12.
142. Green GA, Chenoweth M, Dunn A. Adrenal glucocorticoid permissive regulation of muscle
glycogenolysis: action on protein phosphatase(s) and its inhibitor(s). Proc Natl Acad Sci U S
A. 1980;77:5711–5.
143. Salehzadeh F, Al-Khalili L, Kulkarni SS, Wang M, Lonnqvist F, Krook A. Glucocorticoid-
mediated effects on metabolism are reversed by targeting 11 beta hydroxysteroid dehydroge-
nase type 1 in human skeletal muscle. Diabetes Metab Res Rev. 2009;25:250–8. doi:10.1002/
dmrr.944.
144. Miller TB, Exton JH, Park CR. A block in epinephrine-induced glycogenolysis in hearts from
adrenalectomized rats. J Biol Chem. 1971;246:3672–8.
145. Puthanveetil P, Rodrigues B. Glucocorticoid excess induces accumulation of cardiac glyco-
gen and triglyceride: suggested role for AMPK. Curr Pharm Des. 2013;19:4818–30.
146. Baltrons MA, Agullo L, Garcia A. Dexamethasone up-regulates a constitutive nitric oxide
synthase in cerebellar astrocytes but not in granule cells in culture. J Neurochem. 1995;64:
447–50.
147. Binnert C, Ruchat S, Nicod N, Tappy L. Dexamethasone-induced insulin resistance shows no
gender difference in healthy humans. Diabetes Metab. 2004;30:321–6.
148. Rafacho A, Marroqui L, Taboga SR, Abrantes JL, Silveira LR, Boschero AC, Carneiro EM,
Bosqueiro JR, Nadal A, Quesada I. Glucocorticoids in vivo induce both insulin hypersecre-
tion and enhanced glucose sensitivity of stimulus-secretion coupling in isolated rat islets.
Endocrinology. 2010;151:85–95. doi:10.1210/en.2009-0704.
149. Besse C, Nicod N, Tappy L. Changes in insulin secretion and glucose metabolism induced by
dexamethasone in lean and obese females. Obes Res. 2005;13:306–11. doi:10.1038/
oby.2005.41.
150. Fransson L, Franzen S, Rosengren V, Wolbert P, Sjoholm A, Ortsater H. beta-Cell adaptation
in a mouse model of glucocorticoid-induced metabolic syndrome. J Endocrinol. 2013;219:
231–41. doi:10.1530/JOE-13-0189.
151. Jeong IK, Oh SH, Kim BJ, Chung JH, Min YK, Lee MS, Lee MK, Kim KW. The effects of
dexamethasone on insulin release and biosynthesis are dependent on the dose and duration of
treatment. Diabetes Res Clin Pract. 2001;51:163–71.
152. van Raalte DH, Nofrate V, Bunck MC, van Iersel T, Elassaiss Schaap J, Nassander UK, Heine
RJ, Mari A, Dokter WH, Diamant M. Acute and 2-week exposure to prednisolone impair
different aspects of beta-cell function in healthy men. Eur J Endocrinol. 2010;162:729–35.
doi:10.1530/EJE-09-1034.
153. Gremlich S, Roduit R, Thorens B. Dexamethasone induces posttranslational degradation of
GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with
the effects of fatty acids. J Biol Chem. 1997;272:3216–22.
154. Borboni P, Porzio O, Magnaterra R, Fusco A, Sesti G, Lauro R, Marlier LN. Quantitative
analysis of pancreatic glucokinase gene expression in cultured beta cells by competitive poly-
merase chain reaction. Mol Cell Endocrinol. 1996;117:175–81.
155. Goodman PA, Medina-Martinez O, Fernandez-Mejia C. Identification of the human insulin
negative regulatory element as a negative glucocorticoid response element. Mol Cell
Endocrinol. 1996;120:139–46.
156. Sharma S, Jhala US, Johnson T, Ferreri K, Leonard J, Montminy M. Hormonal regulation of
an islet-specific enhancer in the pancreatic homeobox gene STF-1. Mol Cell Biol. 1997;17:
2598–604.
157. Khan A, Ostenson CG, Berggren PO, Efendic S. Glucocorticoid increases glucose cycling and
inhibits insulin release in pancreatic islets of ob/ob mice. Am J Physiol. 1992;263:E663–6.
158. Ullrich S, Berchtold S, Ranta F, Seebohm G, Henke G, Lupescu A, Mack AF, Chao CM, Su
J, Nitschke R, Alexander D, Friedrich B, Wulff P, Kuhl D, Lang F. Serum- and
glucocorticoid-inducible kinase 1 (SGK1) mediates glucocorticoid-induced inhibition of
insulin secretion. Diabetes. 2005;54:1090–9.
159. Negrato CA, Jovanovic L, Rafacho A, Tambascia MA, Geloneze B, Dias A, Rudge
MV. Association between different levels of dysglycemia and metabolic syndrome in preg-
nancy. Diabetol Metab Syndr. 2009;1:3. doi:10.1186/1758-5996-1-3.
160. Rafacho A, Quallio S, Ribeiro DL, Taboga SR, Paula FM, Boschero AC, Bosqueiro JR. The adap-
tive compensations in endocrine pancreas from glucocorticoid-treated rats are reversible after the
interruption of treatment.Acta Physiol. 2010;200:223–35. doi:10.1111/j.1748-1716.2010.02146.x.
161. Rafacho A, Abrantes JL, Ribeiro DL, Paula FM, Pinto ME, Boschero AC, Bosqueiro
JR. Morphofunctional alterations in endocrine pancreas of short- and long-term
dexamethasone-treated rats. Horm Metab Res. 2011;43:275–81. doi:10.1055/s-0030-1269896.
162. Rafacho A, Giozzet VA, Boschero AC, Bosqueiro JR. Functional alterations in endocrine
pancreas of rats with different degrees of dexamethasone-induced insulin resistance.
Pancreas. 2008;36:284–93. doi:10.1097/MPA.0b013e31815ba826.
163. Huising MO, Pilbrow AP, Matsumoto M, van der Meulen T, Park H, Vaughan JM, Lee S, Vale
WW. Glucocorticoids differentially regulate the expression of CRFR1 and CRFR2alpha in
MIN6 insulinoma cells and rodent islets. Endocrinology. 2011;152:138–50. doi:10.1210/
en.2010-0791.
164. Huising MO, van der Meulen T, Vaughan JM, Matsumoto M, Donaldson CJ, Park H,
Billestrup N, Vale WW. CRFR1 is expressed on pancreatic beta cells, promotes beta cell
proliferation, and potentiates insulin secretion in a glucose-dependent manner. Proc Natl
Acad Sci U S A. 2010;107:912–7. doi:10.1073/pnas.0913610107.
165. Reich E, Tamary A, Sionov RV, Melloul D. Involvement of thioredoxin-interacting protein
(TXNIP) in glucocorticoid-mediated beta cell death. Diabetologia. 2012;55:1048–57.
doi:10.1007/s00125-011-2422-z.
126 T. Kuo et al.
166. Avram D, Ranta F, Hennige AM, Berchtold S, Hopp S, Haring HU, Lang F, Ullrich S. IGF-1
protects against dexamethasone-induced cell death in insulin secreting INS-1 cells indepen-
dent of AKT/PKB phosphorylation. Cell Physiol Biochem. 2008;21:455–62.
doi:10.1159/000129638.
167. Fransson L, Rosengren V, Saha TK, Grankvist N, Islam T, Honkanen RE, Sjoholm A, Ortsater
H. Mitogen-activated protein kinases and protein phosphatase 5 mediate glucocorticoid-
induced cytotoxicity in pancreatic islets and beta-cells. Mol Cell Endocrinol. 2014;383:126–
36. doi:10.1016/j.mce.2013.12.010.
168. Bodwell JE, Webster JC, Jewell CM, Cidlowski JA, Hu JM, Munck A. Glucocorticoid
receptor phosphorylation: overview, function and cell cycle-dependence. J Steroid Biochem
Mol Biol. 1998;65:91–9.
169. Ismaili N, Garabedian MJ. Modulation of glucocorticoid receptor function via phosphoryla-
tion. Ann N Y Acad Sci. 2004;1024:86–101. doi:10.1196/annals.1321.007.
170. Rogatsky I, Logan SK, Garabedian MJ. Antagonism of glucocorticoid receptor transcrip-
tional activation by the c-Jun N-terminal kinase. Proc Natl Acad Sci U S A. 1998;95:
2050–5.
171. Roma LP, Oliveira CA, Carneiro EM, Albuquerque GG, Boschero AC, Souza
KL. N-acetylcysteine protects pancreatic islet against glucocorticoid toxicity. Redox Rep.
2011;16:173–80. doi:10.1179/1351000211Y.0000000006.
172. Yamamoto M, Yamato E, Toyoda S, Tashiro F, Ikegami H, Yodoi J, Miyazaki J. Transgenic
expression of antioxidant protein thioredoxin in pancreatic beta cells prevents progression of
type 2 diabetes mellitus. Antioxid Redox Signal. 2008;10:43–9. doi:10.1089/ars.2007.1586.
173. Wang Z, Rong YP, Malone MH, Davis MC, Zhong F, Distelhorst CW. Thioredoxin-interacting
protein (txnip) is a glucocorticoid-regulated primary response gene involved in mediating
glucocorticoid-induced apoptosis. Oncogene. 2006;25:1903–13. doi:10.1038/sj.onc.1209218.
174. Quesada I, Tuduri E, Ripoll C, Nadal A. Physiology of the pancreatic alpha-cell and glucagon
secretion: role in glucose homeostasis and diabetes. J Endocrinol. 2008;199:5–19.
doi:10.1677/JOE-08-0290.
175. Everett LJ, Lazar MA. Cell-specific integration of nuclear receptor function at the genome.
Wiley Interdiscip Rev Syst Biol Med. 2013;5:615–29. doi:10.1002/wsbm.1231.
176. Hah N, Danko CG, Core L, Waterfall JJ, Siepel A, Lis JT, Kraus WL. A rapid, extensive, and
transient transcriptional response to estrogen signaling in breast cancer cells. Cell.
2011;145:622–34. doi:10.1016/j.cell.2011.03.042.
177. Hah N, Kraus WL. Hormone-regulated transcriptomes: lessons learned from estrogen signal-
ing pathways in breast cancer cells. Mol Cell Endocrinol. 2014;382:652–64. doi:10.1016/j.
mce.2013.06.021.
178. Madsen JG, Schmidt SF, Larsen BD, Loft A, Nielsen R, Mandrup S. iRNA-seq: computa-
tional method for genome-wide assessment of acute transcriptional regulation from total
RNA-seq data. Nucleic Acids Res. 2015;43(6):e40. doi:10.1093/nar/gku1365.
179. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in
human cells. eLife. 2013;2:e00471. doi:10.7554/eLife.00471.
180. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-
guided human genome engineering via Cas9. Science. 2013;339:823–6. doi:10.1126/
science.1232033.
181. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA,
Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:
819–23. doi:10.1126/science.1231143.
182. Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the
Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31:230–2. doi:10.1038/nbt.2507.
183. Yang H, Wang H, Jaenisch R. Generating genetically modified mice using CRISPR/
Cas-mediated genome engineering. Nat Protoc. 2014;9:1956–68. doi:10.1038/nprot.2014.134.
Chapter 6
How Do Glucocorticoids Regulate
Lipid Metabolism?
Roldan M. de Guia and Stephan Herzig
Abstract Glucocorticoids (GCs) and their cognate, intracellular receptor, the

glucocorticoid receptor (GR) have been characterized as critical checkpoints in the
hormonal control of energy homeostasis in mammals. Whereas physiological levels
of GCs are required for proper metabolic control, aberrant GC action has been linked
to a variety of severe metabolic diseases, including type 2 diabetes and obesity.
As a member of the nuclear receptor superfamily of transcription factors, the GR
translocates into the cell nucleus upon GC binding where it serves as a transcrip-
tional regulator of distinct GC-responsive target genes that are in many cases associ-
ated with lipid regulatory pathways and thereby intricately control both physiological
and pathophysiological systemic lipid homeostasis. Thus, this chapter focuses on
the current knowledge of GC/GR function in lipid handling and its implications for
systemic metabolic dysfunction.
Keywords Glucocorticoid receptor • Glucocorticoid hormones • Liver • Adipose

tissue • Lipid metabolism • Lipoprotein metabolism • Cholesterol metabolism
Lipid Metabolism at a Glance
The maintenance of metabolic homeostasis depends on the highly regulated interac-

tion of nutritional, neural, and endocrine stimuli. The continuous cycle of nutrient
acquisition from the ingested food and the depletion of nutrients involves the coor-
dination of several biomolecular processes at all levels of the animal’s biology. Each
of the major metabolic organs of the body—the liver, muscles, adipose tissues, intes-
tines, pancreas, and the brain—depends on the major food groups of carbohydrates,
proteins, and fats to support various physiologic processes of the organism [1, 2].
R.M. de Guia, Ph.D. • S. Herzig, Ph.D. (*)

Department of Molecular Metabolic Control, German Cancer Research Center,
Center for Molecular Biology and University Hospital Heidelberg,
Im Neuenheimer Feld 280, Heidelberg 69120, Germany

128 R.M. de Guia and S. Herzig
Compared to carbohydrates and proteins, lipids, due to their amphipathic or

non-polar nature, are digested, absorbed, and processed in the body differently.
Lipids in food require emulsification by liver-derived bile salts in the digestive tract
to facilitate pancreas-derived lipase action and intestinal absorption. Cholesterol
esters and re-formed triglycerides in the intestinal enterocytes are packaged into
lipoproteins, the chylomicrons (Fig. 6.1), which drain via the lymphatics before
emptying into the blood vessels [1, 2]. Triglycerides in lipoproteins are acted upon
by lipoprotein lipases (LPL) which are tethered to the walls of capillaries by proteo-
glycans and GPIHBP1 [3, 4]. Fatty acid then move into the cells with the help of
transport proteins (fatty acid transport proteins, FATP; fatty acid translocase, FAT/
CD36; caveolin-1 and fatty acid-binding protein, FABP) [5]. Cholesterol esters
inside lipoproteins are transported into the cell via receptor-mediated processes
involving the low-density lipoprotein receptor (LDLR), lipolysis-stimulated lipo-
protein receptor (LSR), LDLR-related protein (LRP), and the scavenger receptor
(SRB1). The majority of the fatty acid transporters are located on the surface of
adipocytes, hepatocytes, and muscle cells, while most lipoprotein receptors are
expressed by hepatocytes [6].
Apart from chylomicrons, four other types of lipoprotein particles are recognized
to be involved in lipid transport (Fig. 6.1). They differ in size and density which
Fig. 6.1 The lipoprotein pathway. ABCA1 ATP-binding cassette transporter A1, CM chylomi-
crons, HDL high-density lipoproteins, IDL intermediate density lipoproteins, LDLR LDL receptor,
LPL lipoprotein lipase, LDL low-density lipoproteins, LRP LDL receptor-related protein, LSR
lipolysis-stimulated lipoprotein receptor, SRB1 Scavenger receptor class B member 1, TRL
triglyceride-rich lipoproteins, VLDL very-low-density-lipoproteins
6 How Do Glucocorticoids Regulate Lipid Metabolism? 129
depend on the varying lipid and apolipoprotein contents of each particle [6].
Triglycerides and cholesterol esters comprise the core of the particle while amphipa-
thic phospholipids, unesterified cholesterol, and proteins are found in the outer
shell. Apolipoproteins function by activating or inhibiting enzymes important in the
transport process and by serving as ligands for cell surface receptors [6]. ApoB and
apoE, for example, can be recognized by the LDLR and LSR receptors while
ApoCII activates lipoprotein lipase that hydrolyzes triglycerides [7]. Dietary (exog-
enous) lipids are transported through chylomicrons while endogenous lipids from
the liver are transported by very-low density lipoproteins (VLDL) which can give
rise to intermediate density (IDL), low-density (LDL), and remnant lipoprotein par-
ticles. High density lipoproteins (HDL) are involved primarily in the transport of
cholesterol from the peripheral tissues to the liver [2, 6].
The triglycerides and fatty acids being transported from the liver come from dif-
ferent sources: (1) from the chylomicron remnants, (2) de novo lipogenic pathway
(DNL), and (3) fatty acids from lipolysis in adipocytes [1, 8]. Cholesterol, on the
other hand, is provided from (1) the diet via chylomicron remnants, (2) de novo
synthesis of cholesterol, and (3) from other lipoproteins internalized by the liver [9].
In the cell, fatty acids can be converted into triglycerides or used as source of
energy during starvation through mitochondrial β-oxidation. Triglycerides can be
packaged into lipoprotein particles (liver) or stored in depots (adipose tissues). In
β-oxidation, long-chain fatty acids (LCFAs) are channeled across the mitochondrial
membrane, with the help of carnitine palmitoyltransferase-I (CPT-I), and acetyl-
CoAs are generated and extracted for energy via the Kreb’s cycle and electron
transport chain. Fatty acids can also be used for the synthesis of eicosanoids (pros-
taglandins, thromboxanes, leukotrienes), glycerophospholipids (plasmalogens and
phosphatidates), and sphingolipids (sphingomyelin, cerebrosides, gangliosides) [10].
Cholesterol is then used as component of biomembranes and for the synthesis of
bile acids (cholic, glycocholic, taurocholic, lithocholic acids), steroid hormones
(androgens, progestins, estrogens, glucocorticoids, and mineralocorticoids), and
secosteroids (vitamin D) [11].
Insulin and counter-regulatory hormones, which include the glucocorticoids
(GCs), play major roles in keeping the balance of metabolites in the cell by activat-
ing or deactivating key enzymes involved in different anabolic and catabolic path-
ways [12]. Furthermore, inter-organ nutrient cross-talk depends primarily on the
hormonal signals perceived by the cells. These are true for glucose, amino acid, and
lipid metabolism. Below, we present collective information on how GCs regulate
lipid metabolism in adipocytes, the liver, and other tissues. Most of the enzymes that
are regulated by GCs in lipid pathways are in fact the same ones that are dysregu-
lated in chronic conditions of GC excess i.e. Metabolic and Cushing’s syndromes.
GCs and Adipose Tissue
The adipose tissues are the major fat depot of the body. Excess triglycerides that can-
not be used by the body as well as excess glucose can be stored as fats in white adi-
pose tissues [2]. Here, triglycerides remain until needed in times of nutrient deprivation.
Abdominal and visceral adiposity are now regarded as an important criteria contributing
to the collective dysfunctional phenotypes of the Metabolic Syndrome [13].
Interestingly, this abnormal deposition of fats in the body is likewise observed in
Cushings syndrome where the individual suffers from hyperactive HPA axis and
hypercortisolemia [14, 15]. However, one key distinction between metabolic syn-
drome and Cushing syndrome is that patients with Metabolic Syndrome have normal
or increased amounts of peripheral adipose and patients with Cushings syndrome
typically have decreased peripheral adipose. This partially overlapping phenotype
between Metabolic and Cushings syndrome demonstrates potential important role
of GCs in adipose tissue physiology.
Effects of GCs on Adipocyte Differentiation
GCs have been reported to possess pro-adipogenic function. This was realized in
transgenic mice over-expressing 11beta (β)-HSD1 in adipocytes thus increasing
active local GC levels. In these mice, a significant increase in abdominal fat was
observed whereas peripheral fats were less affected [16]. Furthermore, the specific
elevation of GCs in adipose tissues resulted in the manifestation of major pheno-
types of the metabolic syndrome such as abdominal obesity, glucose intolerance,
and hypertriglyceridemia. The adipocyte hypertrophic phenotype observed in these
animals also results in decreased levels of adiponectin, an insulin-sensitizing adipo-
kine, and increased local and systemic levels of the cytokine tumor necrosis factor-
alpha (TNF-α), a marker of insulin resistance [16–19].
Apart from the adipocyte hypertrophic phenotype of mice over-expressing 11β
(beta)-HSD1, GCs have been shown to promote differentiation in vitro [20]. Cortisol
and dexamethasone, a synthetic glucocorticoid, have been widely used as a compo-
nent of the adipogenic induction cocktail for adipose stromal cells, primary and
3T3-L1 pre-adipocytes [21, 22]. This function of GCs can be attributed to both
transcriptional and non-transcriptional action of the GR. The GR has been reported
to act on specific histone deacetylase 1 complex for degradation by the 26S protea-
some, thus promoting the expression of transcription factors (i.e. C/EBP-alpha (α)
and STAT5) necessary for the differentiation process [23, 24]. In primary adipo-
cytes isolated from 11β (beta)-HSD1-over-expressing mice, peroxisome-proliferator
activated receptor—gamma (PPARγ), an important regulator of lipogenesis and
adipogenesis, has been reported to be up-regulated [25–27]. In other recent reports
where the GR function was rendered deficient pharmacologically or genetically [28],
adipogenesis was shown to be inhibited in vitro which may depend on GR-induced
expression of genes such as Krüppel-like factor 15 (KLF15) [29]. Since elevated
11β (beta)-HSD1 activity has been reported in both human and mouse obesity, these
are therefore, indicative of the possible contribution of GC/GR signaling in the
development of the Metabolic Syndrome by regulating the expression of transcrip-
tional regulators and complexes [30–32].
Effects of GCs on Adipose Tissue Lipolysis
During fasting and starvation, fatty acids and glycerol are release from their storage
form as triglycerides in adipose tissue fat depots. Lipases are involved in catalyzing
the hydrolysis of the ester bonds between fatty acids and glycerol. Adipose triglyc-
eride lipase (ATGL/desnutrin) hydrolyzes triglyceride into diacylglyceride (DAG)
and fatty acid. DAG is in turn hydrolyzed by hormone-sensitive lipase (HSL) into
monoacylglyceride (MAG) and another molecule of fatty acid. The final hydrolysis
step that completely liberates fatty acids and glycerol is catalyzed by monoglyceride
lipase (MGL). Under normal, basal (fasted) physiologic state, GCs promote lipolysis
in adipose tissues by inducing activity of all three lipases and reducing LPL activity
[33–37]. This lipolytic function of GCs is more pronounce in peripheral adipose
than in abdominal depots where adipogenic and lipogenic programs are favored
[38, 39]. GCs are also known to induce expression of FoxO transcription factors
which are known regulators of the lipases [40, 41].
In addition to the possible direct GR regulation [34] or indirect effects via FoxO,
GCs can also affect lipolysis by acting on upstream regulators. Cyclic adenosine
monophosphate (cAMP)–protein kinase A (PKA) pathway which is stimulated by
catecholamines via G-protein coupled, β (beta)-adrenergic receptor activates HSL
and the lipid droplet surface protein perilipin which facilitates fatty acid release by
promoting ATGL and HSL function [42, 43]. This process is actually inhibited by
insulin via phosphoinositide-3 kinase (PI3K)-protein kinase B (PKB/Akt) signaling
which activates the cAMP-hydrolyzing enzyme, phosphodiesterase 3B (PDE3B)
[42, 43]. The lipolytic action of catecholamines via beta (β)-adrenergic stimulation
occurs rapidly compared to glucocorticoids which takes several hours [44]. It has
been postulated that GCs increase intracellular cAMP levels by (1) increasing PKA
activity, (2) inhibiting PDE3B expression and/or (3) by inducing expression of
angiopoietin-like 4 (ANGPTL4) [44–46].
ANGPTL4 is a hypoxia-induced, secreted glycoprotein that can interact with
proteoglycans of the extracellular matrix [47, 48]. It has been reported that GCs
promote secretion of ANGPTL4 from the liver and white adipose tissues [49].
ANGPTL4 in turn activates adipose tissue lipolysis and inhibits LPL activity result-
ing in elevation of free fatty acids in the circulation [50]. Furthermore, ANGPTL4
expression is found to be highly induced during fasting, when the levels of GCs are
high, and is inhibited by treatment with RU486, a GR antagonist. [51] Mice lacking
ANGPTL4 have reduced fasting-induced WAT lipolysis further demonstrating
the possible role of the GC/GR signaling in the process [51]. In line with this, a
conserved GRE is present in the rat, mouse, and human ANGPTL4 gene locus [45].
The exact mechanism by which GC-dependent, ANGPTL4-induced lipolysis works
remains to be elucidated, but the main hypothesis points to cAMP activation which
was observed to be deficient in WAT of ANGPTL4 knockout mice [45]. The impor-
tance of this protein in adipose tissue biology, nevertheless, is justifiable as increased
ANGPTL4 expression is demonstrated to be associated with elevated fatty acids in
both Type 1 and Type 2 diabetes mouse models [52]. This process is also implicated
in fatty acid redistribution from adipose tissue to the liver which in turn increases
triglyceride synthesis in hepatocytes [33].
Studies on the lipolytic action of GCs in humans have been largely inconsistent
across different steroidal preparations, length of GC administration, distinct fat
depots, and characteristics of the study population itself. To mention a few, in
Cushing’s patients [53, 54] and individuals who received chronic GC treatments
[55–57], the rate of non-esterified fatty acid (NEFA) turnover is unaffected. This
potential anti-lipolytic activity of GCs is also observed in vitro in 3T3-L1 cells
treated with high concentration of corticosterone (>1 μM). The effect though, is
reversed upon removal of the GC from the medium favoring once more lipolysis
[35]. In other studies in humans involving infusion of fatty acid stable-isotope and
acute physiological hypercortisolemia, an increase in blood NEFA levels is observed
[58, 59]. This is probably due to an increased lipolysis from subcutaneous fat depots
when insulin levels were normal [58].
With regards to fatty acid oxidation, there has been no concrete report on how
GCs could possibly affect this pathway. Furthermore, no study has examined the
role of adipose tissue GR using genetic gain- or loss-of-function models which
could further provide insight on how GCs regulate lipid metabolism.
Effects of GCs on de novo Lipogenesis and (β)-Oxidation
Fatty acids can be synthesized from non-lipid materials, like glucose, via the de
novo lipogenic pathway (DNL). DNL takes place both in the liver and the adipose
tissues with synthesized fatty acids stored as triglycerides as cytosolic lipid drop-
lets [60]. In adipocytes, TG is made by successive acylation and dephosphorylation
of a glycerol-3-phosphate backbone. Specifically, glycerol-3-phosphate is acylated
by GPAT resulting in lysophosphatidic acid which is acylated by AGPAT to form
phosphatidic acid. Phosphatidic acid is dephosphosphorylated by phosphatidic acid
phosphatase, otherwise known as lipins to yield diacylglycerol. In the final step
DAG is acylated by DGAT to form triglyceride. There are several isoforms of each
enzyme class. Interestingly, some, but not all isoforms are induced by glucocorti-
coids in mice [34].
In contrast to lipolysis, lipogenesis is activated by insulin in times of nutrient
excess. One might expect that in this case, GCs might be inhibitory but experimen-
tal evidence said otherwise. Despite the low levels in the fed state, GCs have been
shown to potentiate the lipogenic function of insulin by regulating the expression of
some genes in the pathway [61]. This synergistic response in lipogenesis is shown
to be important also in adrenalectomised rats [62] and human adipocytes [63].
The synthesis of fatty acids from glucose starts with acetyl-CoA, the product of the
action of pyruvate dehydrogenase on the final metabolite of the glycolytic pathway.
Acetyl-CoA carboxylase (ACC) then converts acetyl-CoA to malonyl-CoA which
is in turn the substrate for fatty acid synthase (FASN), a rate-determining enzyme
of lipogenesis. GCs have been reported, both in vivo and in vitro, to regulate the
expression of ACC and FASN [44, 61, 64, 65]. GR-binding regions in or nearby
the genes coding for these enzymes have been identified in both mouse and human
genome [34, 63, 66]. In one study, mRNA levels of ACC and FASN in a human
adipocyte cell line are shown to be induced by GC treatment [64]. The lipogenic
program, however, is decreased and could only be increased by addition of insulin
which supports previous reports on the GC-insulin synergism [62, 63].
GCs are known to affect the oxidation of fatty acids in both adipose tissues and
the liver. In the former, one proposed mechanism is via the induction of expression
of Tribbles-homologue 3 (TRB3) by GCs. TRB3 expression is induced in adipose
tissues during fasting and over-expression of the protein resulted in increased fatty
acid oxidation and protection of mice against diet-induced obesity [67]. In contrast
to pro-lipogenic effect of GCs, TRB3 has been shown to promote ubiquitin-
mediated degradation of ACC explaining how the oxidative pathway could have
been favored [67]. In a similar report but in chronic lymphocytic leukemia (CLL)
cells where resistance has been observed with GC treatments [68], dexamethasone
is found to induce the expression of PPAR-α (alpha) and δ (delta) [69]. PPARα and
δ are known to regulate fatty acid oxidation [70]. Whether the same mechanism
works in adipose tissues and the liver remains to be investigated. Nevertheless, all
these reports prove that GCs play an important role in the regulation of lipid homeo-
stasis in adipose tissues.
The Hepatic Functions of GCs
The liver is a central metabolic organ involved in the control of mammalian glucose
and lipid homeostasis [71]. Genome-wide analysis of GC-regulated target gene net-
works has shown that the GR controls many aspects of hepatic energy metabolism.
More than 50 genes seem to be direct, regulatory targets of GC action. In many cases,
the GR functionally interacts with other transcription factors to control specific
genetic networks in the liver [72], among which only few have been characterized.
Control of Hepatic Triglyceride Metabolism

and VLDL by GCs
Besides ACC and FASN, other enzymes involved in lipogenesis and TG synthesis
have been reported to be regulated by GCs in the liver. Stearoyl-CoA desaturase
(SCD1/2), glycerol-3-phosphate acyltransferase (GPAT3/4), 1-acylglycerol-3-
phosphate acyltransferase (AGPAT2), lipin 1 (LPIN1), and diacylglyceride acyl-
transferase (DGAT1/2) are all implicated to be regulated directly or indirectly by
the GR [73–75]. As in adipose tissues, GC/GR pro-lipogenic activity in the liver
seems to require insulin signaling [74, 76]. This process is possibly important in
the development of hepatic steatosis prior to the onset of insulin resistance in

Metabolic Syndrome [8]. Hepatic steatosis is, in fact, a prominent negative side-effect
of long-term GCs treatment [39]. In accordance to this, liver specific GR loss-of-
function reduces TG levels in normal [77] as well as diabetic [78] mice and reduces
accumulation of TG in liver during liver regeneration [28].
Several mechanisms have been proposed on how GCs promote TG synthesis and
hepatic steatosis one being the inhibition of the hairy and enhancer of split 1 (HES1)
repressor by GCs [78]. GR loss-of-function manipulations in the liver and hepato-
cytes resulted in up-regulation of HES1 that increases expression of pancreatic
lipase (PNL) and pancreatic lipase-related protein (PLRP2), proteins involved in the
“lipase arm” of GR-dependent TG metabolism. Both PNL and PLRP2 are repressed
by GC-mediated HES1 inhibition thereby explaining hepatic triglyceride accumula-
tion. Furthermore, liver HES1 expression is repressed in mouse models of hepatic
steatosis and overexpressing HES1 in these models partially reverses the steatotic
phenotype [78]. Another possible mechanism by which GCs control triglyceride
turnover in the liver is via carboxyesterase 1d (CES1D/triacylglycerol hydrolase,
TGH). TGH is believed to be involved in triglyceride lipolysis of fats droplets in
hepatocytes [75]. Just like PNL and PLRP2, TGH mRNA levels are repressed upon
dexamethasone treatment which could be explained by GC-induced destabilization
of the mRNA at the 3′-UTR [75].
The composite function of the GR in concert with other transcription factors
which could be a repressor or activator adds additional layer to the role of GCs in
controlling hepatic lipid metabolism. The liver X receptor (LXR), for example, is
known to interact with the GR [79] and it has been reported that mice lacking
LXR-β (beta) has reduced GC-induced triglyceride accumulation [80]. The same
phenotype is observed in mice where MED1 is specifically inhibited in the liver
(Med1Δliver) [81]. MED1 is a co-activator subunit of the Mediator complex which in
turn participates in RNA Pol II-dependent transcription [82]. In dexamethasone-
treated Med1Δliver mice, levels of fatty acid oxidation genes—short-chain and
medium-chain acyl-CoA dehydrogenase (SCAD & MCAD)—are normalized com-
pared to wild type mice where the genes are repressed by GC treatment [81]. The
mechanism could also involve PPARγ which is known to be induced by GCs [27].
In a follow-up study in the same Med1Δliver mice where PPARγ is inhibited with an
adenovirus, mice are protected from high-fat diet induced hepatic steatosis with
impaired induction of lipogenic genes, adiponectin, and lipid droplet-associated
genes [83].
Apart from potential direct effects on the enzymes of DNL and TG synthesis,
GCs can regulate expression of secreted factors that can likewise affect lipid load in
hepatocytes. As mentioned above, GCs induce secretion of ANGPTL4 from the
liver and adipose tissues. ANGPTL4, with its pro-lipolytic activity, increases NEFA
being released in the circulation which can serve as substrate for TG synthesis in
the liver. Indeed, ANGPTL4 null mice are protected from GC-induced hepatic ste-
atosis and hyperlipidemia [45]. Another intriguing recent study has shown that
osteoblast specific GR knockout reduces the negative metabolic side effects of
chronic GC treatments probably involving the bone secretion of osteocalcin or the

bone γ-carboxyglutamate protein (BGALP) which has been shown to inhibit adi-
posity and hepatic steatosis [84].
The liver, aside from being at the receiving end for NEFA, can transport lipids to
peripheral tissues via VLDL. The assembly and secretion of VLDL is a complicated
process that depends not only on substrate availability but also on co-translational
regulation of ApoB and the microsomal triglyceride transfer protein (MTTP); re-
uptake of under-lipidated, immature particles; and the status of the intracellular
secretory pathway [85]. Both in vitro and in vivo studies done on primary hepato-
cytes and dexamethasone-treated mice, respectively, showed increased VLDL tri-
glycerides and plasma concentration of HDL particles [86–88]. Promotion of VLDL
secretion by GCs is possibly due to increased production and stabilization of ApoB
together with increased triglyceride synthesis [89]. Similarly, increased VLDL-TG
production resulting in hypertriglyceridemia is observed in rats treated with dexa-
methasone for 2 weeks [90]. The rats also had impaired adipose tissue LPL activity
which contributes to hypertriglyceridemia. In human cases of Cushing’s syndrome,
the rate of VLDL production, and consequently VLDL and LDL levels, is found to
be elevated and peripheral clearance is unaffected [91]. Besides VLDL secretion,
the receptor-mediated clearance of the remnants of triglyceride-rich lipoproteins
(TRLs: chylomicrons, VLDL, IDL, and LDL) is also possibly regulated by GCs.
It has been reported that the levels of these remnant particles are elevated in condi-
tions of metabolic syndrome where hypertriglyceridemia and HPA hyperactivity are
likewise observed [7, 92]. The major remnant receptors include the lipolysis-
stimulated lipoprotein receptor (LSR), LDL receptor (LDLR), LDLR-related pro-
tein (LRP), and heparan sulfate proteoglycans (HSPGs) [7, 93]. With the exception
of LDLR which has been shown to be down-regulated by GC treatment [94], no
other studies have been done on how GC or the GR can affect receptor-mediated
clearance of TRL remnant particles that can then impact on systemic triglyceride
levels.
Effects of GCs on Cholesterol and Bile Acid Homeostasis
Cholesterol is an important lipid metabolite serving as a component of biomem-

branes and as precursor for the synthesis of steroids. Cholesterol transport in the
body is largely dependent on LDL and HDL particles. Both interact with its respec-
tive receptors enabling endocytosis of the entire particle. Besides VLDL and TRL
remnants, GCs are also shown to elevate HDL in the plasma of rats which can be
attributed to the inhibition of hepatic lipase (HL) and activation of lecithin-
cholesterol acyltransferase (LCAT) activity [95]. Similar results are reported in
dexamethasone-treated livers of adrenalectomized rats [88], in overweight and
obese individuals [96], and persons who received prednisone for 1 month as part of
rheumatic disease management [97]. Furthermore, GCs are shown to increase

transcriptional activity of apolipoprotein A-I (ApoA1), the major protein compo-
nent of HDL particles which is important for the transport of cholesterol from the
peripheral tissues to the liver [98]. A dose-dependent effect of dexamethasone is
also observed in the hepatocyte binding capacity of HDL in dexamethasone-treated
adrenalectomized rats [99]. The HDL scavenger receptor (SCRAB1/SRB1) pro-
motes glucocorticoid production by the adrenal gland [100] particularly in response
to inflammation and bacterial endotoxins [101]. Furthermore, adrenal-specific
SRB1 deficiency resulted into glucocorticoid insufficiency and lower VLDL and
LDL levels suggesting potential cross-talk between the liver and adrenal glands
[102]. In case of LDL, reports on the effects of GCs on its metabolism have been
elusive. The negative effects of GCs on hepatic LDLR levels [94] enables one to
speculate the pathophysiologic consequences of elevated GC levels but the limited,
often contradicting, reports warrant further studies.
In the field of cholesterol handling by the liver, hepatocyte-specific GR loss-of-
function reduced the serum hypercholesterolemia in obese mice [78]. This is also
accompanied by higher hepatic expression of the sterol regulatory element binding
protein 2 (SREBP2) and increased liver cholesterol levels suggesting functional
role of the GR in hepatic cholesterol homeostasis. In another study, as well as
Addison’s patients, serum bile acids were elevated in the fasted state and systemic
bile acid homeostasis was disrupted upon the fasted-fed transition [103]. This
abnormality in bile acid homeostasis can be attributed to lower expression of the
major basolateral hepatocyte bile acid transporter, Na+-taurocholate transport pro-
tein (NTCP/SLC10A1) resulting in faulty trans-hepatic bile acid recycling [103].
The gene coding for NTCP is shown to be a direct GR target based on avidin-biotin
DNA-binding (ABCD) and chromatin immunoprecipitation (ChIP) assays and
studies in mice with mutant GR where DNA-binding and dimerization function
was lost (GRdim) [103, 104]. The impaired bile acid uptake likewise exacerbated
development of gallstones in mice with hepatocyte-specific GR knockdown [103].
Furthermore, the mice did not gain as much weight as the control owing to reduced
fat mass which could be due to lower feed efficiency brought by ineffective lipid
digestion and absorption [103]. The findings though, are contradictory to what is
observed in Cushing’s patients who have elevated serum bile acid and in a separate
study where liver-specific GR knockdown results in lower serum bile acid due to
inhibition of FXR activity which then blunts bile acid synthesis [105]. Further stud-
ies are therefore required to clarify these discrepancies.
GC-Dependent Lipid Processes in Muscles and Macrophages
With the exception of macrophages and smooth muscles, little attention has been
given to how glucocorticoids affect lipid metabolism in other tissues. In muscles,
most studies have focused on the effects of GCs on protein metabolism. With regards
to lipids, dexamethasone has been shown to promote intramuscular fat accumulation

in chicken by AMPK inhibition and mTOR activation [106]. The process is aggra-
vated by saturated fats and alleviated by unsaturated fatty acids. This is contradic-
tory to what has been reported in C2C12 myotubes where dexamethasone treatment
resulted into dose-dependent reduction of expression of lipogenic genes (FASN,
ACC, and GPAT) which can be reversed by the GR antagonist RU486 [107]. It has
also been reported that GCs can induce excessive NEFA oxidation that can lead into
muscle insulin resistance [108] and perturbation of equilibrium between fats and
glucose as energy sources [109]. In rats, GC-induced insulin resistance has been
shown to increase serum insulin and NEFA levels and decrease oxidative phosphor-
ylation by producing ATP from phosphocreatine [110]. How GC/GR signaling
exactly orchestrates these entire events in skeletal muscles, with the possibility of
inter-organ cross-talk, remains to be studied. In smooth muscles, dexamethasone has
been demonstrated to increase the rate of cholesteryl ester formation in cultured
human smooth muscle cells [111] and in vivo [112] which has implications on the
atherogenic effect of GCs. But whether the mechanism of GC-induced lipid oxida-
tion in muscle is similar as in fats and the liver and how GCs promote cholesteryl
ester accumulation in smooth muscles remain to be investigated. In macrophages,
increased GC-dependent, esterification of cholesterol is likewise observed [112, 113],
which provide a unifying theme by which GCs can adversely affect atherogenesis.
Furthermore, it has been demonstrated also in macrophages that dexamethasone
can decrease both mRNA and protein levels of the cholesterol transporter ATP-
binding cassette transporter-A1 (ABCA1) and that apoA1-mediated cholesterol
efflux is impeded [113] which would further support the hypothesis of GC-associated
cardiovascular risk.
Concluding Remarks and Outlook
Apart from the peripheral functions of the GC/GR axis in metabolic control
(Fig. 6.2), recent studies have established further ties between GC/GR signaling and
regulatory functions of the central nervous system, osteoblasts as well as intestinal
cells in energy homeostasis and lipid handling.
Together, these studies have substantially broadened the spectrum of GC target
organs in energy homeostasis and revealed unexpected communication routes
between peripheral and/or central organ compartments. GC/GR-dependent endo-
crine control beyond the classical GC-mediated pathways therefore represents a
largely unexplored area of research that holds the promise for exciting discoveries
in endocrine circuitry in the future. It can be anticipated that recent advances in –
OMICS technologies and systems biology will provide substantial support for our
further understanding of endocrine GC/GR signaling and its impact on metabolic
health and disease.
138
R.M. de Guia and S. Herzig
Fig. 6.2 GC/GR targets in lipid metabolism. Overview of lipid metabolic processes regulated by the GC/GR signaling in adipose tissues, the liver,
bone, muscles, macrophages, and adrenal glands. Genes regulated by GCs in each metabolic process are listed in square-round boxes: genes in black
texts are up-regulated; genes in red texts are inhibited by GCs
References
1. Nguyen P, Leray V, Diez M, et al. Liver lipid metabolism. J Anim Physiol Anim Nutr (Berl).
2008;92(3):272–83.
2. Werner A, Kuipers F, Verkade HJ. Fat absorption and lipid metabolism in cholestasis. Landes
Biosci; 2000-2013.
3. Mead JR, Irvine SA, Ramji DP. Lipoprotein lipase: structure, function, regulation, and role in
disease. J Mol Med (Berl). 2002;80(12):753–69.
4. Young SG, Davies BS, Voss CV, Gin P, Weinstein MM, Tontonoz P, Reue K, Bensadoun A,
Fong LG, Beigneux AP. GPIHBP1, an endothelial cell transporter for lipoprotein lipase.
J Lipid Res. 2011;52(11):1869–84.
5. Williams KJ, Fisher EA. Globular warming: how fat gets to the furnace. Nat Med.
2011;17(2):157–9.
6. Ginsberg HN. Lipoprotein physiology in nondiabetic and diabetic states. Relationship to ath-
erogenesis. Diabetes Care. 1991;14(9):839–55.
7. Narvekar P, Berriel Diaz M, Krones-Herzig A, et al. Liver-specific loss of lipolysis-stimulated
lipoprotein receptor triggers systemic hyperlipidemia in mice. Diabetes. 2009;58(5):
1040–9.
8. Choi SH, Ginsberg HN. Increased very low density lipoprotein (VLDL) secretion, hepatic
steatosis, and insulin resistance. Trends Endocrinol Metab. 2011;22(9):353–63.
9. Huff MW. Dietary cholesterol, cholesterol absorption, postprandial lipemia and atherosclero-
sis. Can J Clin Pharmacol. 2003;10 Suppl A:26A–32A.
10. Mathews CK, van Holde KE, Ahern KG. Biochemistry. 3rd ed. Upper Saddle River, NJ:
Prentice Hall; 1999.
11. Russell DW. Cholesterol biosynthesis and metabolism. Cardiovasc Drugs Ther. 1992;6(2):
103–10.
12. Desvergne B, Michalik L, Wahli W. Transcriptional regulation of metabolism. Physiol Rev.
2006;86(2):465–514.
13. Grundy SM, Cleeman JI, Daniels SR, et al. Diagnosis and management of the metabolic
syndrome: an American Heart Association/National Heart, Lung, and Blood Institute scien-
tific statement. Curr Opin Cardiol. 2006;21(1):1–6.
14. Arnaldi G, Mancini T, Tirabassi G, Trementino L, Boscaro M. Advances in the epidemiology,
pathogenesis, and management of Cushing’s syndrome complications. J Endocrinol Invest.
2012;35(4):434–48.
15. Anagnostis P, Athyros VG, Tziomalos K, Karagiannis A, Mikhailidis DP. Clinical review: the
pathogenetic role of cortisol in the metabolic syndrome: a hypothesis. J Clin Endocrinol
Metab. 2009;94(8):2692–701.
16. Masuzaki H, Paterson J, Shinyama H, et al. A transgenic model of visceral obesity and the
metabolic syndrome. Science. 2001;294(5549):2166–70.
17. Viengchareun S, Zennaro MC, Pascual-Le Tallec L, Lombes M. Brown adipocytes are novel
sites of expression and regulation of adiponectin and resistin. FEBS Lett. 2002;532(3):
345–50.
18. Fasshauer M, Klein J, Neumann S, Eszlinger M, Paschke R. Hormonal regulation of adipo-
nectin gene expression in 3T3-L1 adipocytes. Biochem Biophys Res Commun.
2002;290(3):1084–9.
19. Masuzaki H, Yamamoto H, Kenyon CJ, et al. Transgenic amplification of glucocorticoid
action in adipose tissue causes high blood pressure in mice. J Clin Invest. 2003;112(1):
83–90.
20. Gregoire FM, Smas CM, Sul HS. Understanding adipocyte differentiation. Physiol Rev.
1998;78(3):783–809.
21. Pantoja C, Huff JT, Yamamoto KR. Glucocorticoid signaling defines a novel commitment
state during adipogenesis in vitro. Mol Biol Cell. 2008;19(10):4032–41.
22. Hauner H, Schmid P, Pfeiffer EF. Glucocorticoids and insulin promote the differentiation of
human adipocyte precursor cells into fat cells. J Clin Endocrinol Metab. 1987;64(4):832–5.
23. Wiper-Bergeron NWD, Pope L, Schild-Poulter C, Hache RJ. Stimulation of preadipocyte
differentiation by steroid through targeting of an HDAC1 complex. EMBO J. 2003;22(9):
2135–45.
24. Floyd ZE, Stephens JM. STAT5A promotes adipogenesis in nonprecursor cells and associates
with the glucocorticoid receptor during adipocyte differentiation. Diabetes. 2003;52(2):
308–14.
25. Liu Y, Sun WL, Sun Y, Hu G, Ding GX. Role of 11-beta-hydroxysteroid dehydrogenase type
1 in differentiation of 3T3-L1 cells and in rats with diet-induced obesity. Acta Pharmacol Sin.
2006;27(5):588–96.
26. Liu Y, Yan C, Wang Y, et al. Liver X receptor agonist T0901317 inhibition of glucocorticoid
receptor expression in hepatocytes may contribute to the amelioration of diabetic syndrome
in db/db mice. Endocrinology. 2006;147(11):5061–8.
27. Wu Z, Bucher NL, Farmer SR. Induction of peroxisome proliferator-activated receptor
gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta,
C/EBPdelta, and glucocorticoids. Mol Cell Biol. 1996;16(8):4128–36.
28. Shteyer E, Liao Y, Muglia LJ, Hruz PW, Rudnick DA. Disruption of hepatic adipogenesis is
associated with impaired liver regeneration in mice. Hepatology. 2004;40(6):1322–32.
29. Asada M, Rauch A, Shimizu H, et al. DNA binding-dependent glucocorticoid receptor activity
promotes adipogenesis via Kruppel-like factor 15 gene expression. Lab Invest. 2011;91(2):
203–15.
30. Rask E, Olsson T, Soderberg S, et al. Tissue-specific dysregulation of cortisol metabolism in
human obesity. J Clin Endocrinol Metab. 2001;86(3):1418–21.
31. Livingstone DE, Jones GC, Smith K, et al. Understanding the role of glucocorticoids in obesity:
tissue-specific alterations of corticosterone metabolism in obese Zucker rats. Endocrinology.
2000;141(2):560–3.
32. Baudrand R, Carvajal CA, Riquelme A, et al. Overexpression of 11beta-hydroxysteroid
dehydrogenase type 1 in hepatic and visceral adipose tissue is associated with metabolic
disorders in morbidly obese patients. Obes Surg. 2010;20(1):77–83.
33. Peckett AJ, Wright DC, Riddell MC. The effects of glucocorticoids on adipose tissue lipid
metabolism. Metabolism. 2011;60(11):1500–10.
34. Yu CY, Mayba O, Lee JV, et al. Genome-wide analysis of glucocorticoid receptor binding
2010;5(12):e15188.
35. Campbell JE, Peckett AJ, D‘Souza AM, Hawke TJ, Riddell MC. Adipogenic and lipolytic
effects of chronic glucocorticoid exposure. Am J Physiol Cell Physiol. 2011;300(1):
C198–209.
36. Slavin BG, Ong JM, Kern PA. Hormonal regulation of hormone-sensitive lipase activity and
mRNA levels in isolated rat adipocytes. J Lipid Res. 1994;35(9):1535–41.
37. Villena JA, Roy S, Sarkadi-Nagy E, Kim KH, Sul HS. Desnutrin, an adipocyte gene encoding
a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic
expression of desnutrin increases triglyceride hydrolysis. J Biol Chem. 2004;279(45):
47066–75.
38. Ebbert JO, Jensen MD. Fat depots, free fatty acids, and dyslipidemia. Nutrients. 2013;5(2):
498–508.
39. Vegiopoulos A, Herzig S. Glucocorticoids, metabolism and metabolic diseases. Mol Cell
Endocrinol. 2007;275(1–2):43–61.
40. Wang JC, Gray NE, Kuo T, Harris CA. Regulation of triglyceride metabolism by glucocorti-
coid receptor. Cell Biosci. 2012;2(1):19.
41. Macfarlane DP, Forbes S, Walker BR. Glucocorticoids and fatty acid metabolism in humans:
fuelling fat redistribution in the metabolic syndrome. J Endocrinol. 2008;197(2):189–204.
42. Carmen GY, Victor SM. Signalling mechanisms regulating lipolysis. Cell Signal. 2006;18(4):
401–8.
43. Holm C. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem
Soc Trans. 2003;31(Pt 6):1120–4.
44. Xu C, He J, Jiang H, et al. Direct effect of glucocorticoids on lipolysis in adipocytes. Mol
Endocrinol. 2009;23(8):1161–70.
45. Koliwad SK, Kuo T, Shipp LE, et al. Angiopoietin-like 4 (ANGPTL4, fasting-induced adi-
pose factor) is a direct glucocorticoid receptor target and participates in glucocorticoid-
regulated triglyceride metabolism. J Biol Chem. 2009;284(38):25593–601.
46. van Raalte DH, Brands M, Serlie MJ, et al. Angiopoietin-like protein 4 is differentially regu-
lated by glucocorticoids and insulin in vitro and in vivo in healthy humans. Exp Clin
Endocrinol Diabetes. 2012;120(10):598–603.
47. Le Jan S, Amy C, Cazes A, et al. Angiopoietin-like 4 is a proangiogenic factor produced dur-
ing ischemia and in conventional renal cell carcinoma. Am J Pathol. 2003;162(5):1521–8.
48. Cazes A, Galaup A, Chomel C, et al. Extracellular matrix-bound angiopoietin-like 4 inhibits
endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton. Circ Res.
2006;99(11):1207–15.
49. Mandard S, Zandbergen F, van Straten E, et al. The fasting-induced adipose factor/
angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma
lipid levels and adiposity. J Biol Chem. 2006;281(2):934–44.
50. Lichtenstein L, Berbee JF, van Dijk SJ, et al. Angptl4 upregulates cholesterol synthesis in
liver via inhibition of LPL- and HL-dependent hepatic cholesterol uptake. Arterioscler
Thromb Vasc Biol. 2007;27(11):2420–7.
51. Gray NE, Lam LN, Yang K, Zhou AY, Koliwad S, Wang JC. Angiopoietin-like 4 (Angptl4)
protein is a physiological mediator of intracellular lipolysis in murine adipocytes. J Biol
Chem. 2012;287(11):8444–56.
52. Mizutani N, Ozaki N, Seino Y, et al. Reduction of insulin signaling upregulates angiopoietin-
like protein 4 through elevated free fatty acids in diabetic mice. Exp Clin Endocrinol Diabetes.
2012;120(3):139–44.
53. Birkenhager JC, Timmermans HA, Lamberts SW. Depressed plasma FFA turnover rate in
Cushing’s syndrome. J Clin Endocrinol Metab. 1976;42(1):28–32.
54. Saunders J, Hall SE, Sonksen PH. Glucose and free fatty acid turnover in Cushing’s syn-
drome. J Endocrinol Invest. 1980;3(3):309–11.
55. Miyoshi H, Shulman GI, Peters EJ, Wolfe MH, Elahi D, Wolfe RR. Hormonal control of
substrate cycling in humans. J Clin Invest. 1988;81(5):1545–55.
56. Gravholt CH, Dall R, Christiansen JS, Moller N, Schmitz O. Preferential stimulation of
abdominal subcutaneous lipolysis after prednisolone exposure in humans. Obes Res. 2002;
10(8):774–81.
57. Johnston DG, Gill A, Orskov H, Batstone GF, Alberti KG. Metabolic effects of cortisol in
man—studies with somatostatin. Metabolism. 1982;31(4):312–7.
58. Djurhuus CB, Gravholt CH, Nielsen S, et al. Effects of cortisol on lipolysis and regional
interstitial glycerol levels in humans. Am J Physiol Endocrinol Metab. 2002;283(1):E172–7.
59. Samra JS, Clark ML, Humphreys SM, MacDonald IA, Bannister PA, Frayn KN. Effects of
physiological hypercortisolemia on the regulation of lipolysis in subcutaneous adipose tissue.
J Clin Endocrinol Metab. 1998;83(2):626–31.
60. Ferramosca A, Zara V. Modulation of hepatic steatosis by dietary fatty acids. World J
Gastroenterol. 2014;20(7):1746–55.
61. Hillgartner FB, Salati LM, Goodridge AG. Physiological and molecular mechanisms involved
in nutritional regulation of fatty acid synthesis. Physiol Rev. 1995;75(1):47–76.
62. Williams BH, Berdanier CD. Effects of diet composition and adrenalectomy on the lipogenic
responses of rats to starvation-refeeding. J Nutr. 1982;112(3):534–41.
63. Wang Y, Jones Voy B, Urs S, et al. The human fatty acid synthase gene and de novo lipogen-
esis are coordinately regulated in human adipose tissue. J Nutr. 2004;134(5):1032–8.
64. Gathercole LL, Morgan SA, Bujalska IJ, Hauton D, Stewart PM, Tomlinson JW. Regulation
of lipogenesis by glucocorticoids and insulin in human adipose tissue. PLoS One. 2011;
6(10):e26223.
65. Sul HS, Wang D. Nutritional and hormonal regulation of enzymes in fat synthesis: studies of
fatty acid synthase and mitochondrial glycerol-3-phosphate acyltransferase gene transcrip-
tion. Annu Rev Nutr. 1998;18:331–51.
66. Lu Z, Gu Y, Rooney SA. Transcriptional regulation of the lung fatty acid synthase gene by
glucocorticoid, thyroid hormone and transforming growth factor-beta 1. Biochim Biophys
Acta. 2001;1532(3):213–22.
67. Qi L, Heredia JE, Altarejos JY, et al. TRB3 links the E3 ubiquitin ligase COP1 to lipid metab-
olism. Science. 2006;312(5781):1763–6.
68. Hala M, Hartmann BL, Bock G, Geley S, Kofler R. Glucocorticoid-receptor-gene defects and
resistance to glucocorticoid-induced apoptosis in human leukemic cell lines. Int J Cancer.
1996;68(5):663–8.
69. Tung S, Shi Y, Wong K, et al. PPARalpha and fatty acid oxidation mediate glucocorticoid
resistance in chronic lymphocytic leukemia. Blood. 2013;122(6):969–80.
70. Grygiel-Gorniak B. Peroxisome proliferator-activated receptors and their ligands: nutritional
and clinical implications—a review. Nutr J. 2014;13:17.
71. van den Berghe G. The role of the liver in metabolic homeostasis: implications for inborn
errors of metabolism. J Inherit Metab Dis. 1991;14(4):407–20.
72. Le Phuc P, Friedman JR, Schug J, et al. Glucocorticoid receptor-dependent gene regulatory
networks. PLoS Genet. 2005;1(2):e16.
73. Legrand P, Catheline D, Hannetel JM, Lemarchal P. Stearoyl-CoA desaturase activity in pri-
mary culture of chicken hepatocytes. Influence of insulin, glucocorticoid, fatty acids and
cordycepin. Int J Biochem. 1994;26(6):777–85.
74. Dich J, Bro B, Grunnet N, Jensen F, Kondrup J. Accumulation of triacylglycerol in cultured
rat hepatocytes is increased by ethanol and by insulin and dexamethasone. Biochem
J. 1983;212(3):617–23.
75. Dolinsky VW, Douglas DN, Lehner R, Vance DE. Regulation of the enzymes of hepatic
microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexametha-
sone. Biochem J. 2004;378(Pt 3):967–74.
76. Mangiapane EH, Brindley DN. Effects of dexamethasone and insulin on the synthesis of tria-
cylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and
lysophosphatidylcholine by monolayer cultures of rat hepatocytes. Biochem J. 1986;
233(1):151–60.
77. Opherk C, Tronche F, Kellendonk C, et al. Inactivation of the glucocorticoid receptor in
hepatocytes leads to fasting hypoglycemia and ameliorates hyperglycemia in streptozotocin-
induced diabetes mellitus. Mol Endocrinol. 2004;18(6):1346–53.
78. Lemke U, Krones-Herzig A, Berriel Diaz M, et al. The glucocorticoid receptor controls
hepatic dyslipidemia through Hes1. Cell Metab. 2008;8(3):212–23.
79. Nader N, Ng SS, Wang Y, Abel BS, Chrousos GP, Kino T. Liver x receptors regulate the tran-
scriptional activity of the glucocorticoid receptor: implications for the carbohydrate metabo-
lism. PLoS One. 2012;7(3):e26751.
80. Patel R, Patel M, Tsai R, et al. LXRbeta is required for glucocorticoid-induced hyperglyce-
mia and hepatosteatosis in mice. J Clin Invest. 2011;121(1):431–41.
81. Jia Y, Viswakarma N, Fu T, et al. Conditional ablation of mediator subunit MED1 (MED1/
PPARBP) gene in mouse liver attenuates glucocorticoid receptor agonist dexamethasone-
induced hepatic steatosis. Gene Expr. 2009;14(5):291–306.
82. Zhang Y, Xiaoli, Zhao X, Yang Y. The mediator complex and lipid metabolism. J Biochem
Pharmacol Res. 2013;1(1):51–5.
83. Bai L, Jia Y, Viswakarma N, et al. Transcription coactivator mediator subunit MED1 is
required for the development of fatty liver in the mouse. Hepatology. 2011;53(4):1164–74.
84. Brennan-Speranza TC, Henneicke H, Gasparini SJ, et al. Osteoblasts mediate the adverse
effects of glucocorticoids on fuel metabolism. J Clin Invest. 2012;122(11):4172–89.
85. Sundaram M, Yao Z. Recent progress in understanding protein and lipid factors affecting
hepatic VLDL assembly and secretion. Nutr Metab (Lond). 2010;7:35.
86. Martin-Sanz P, Vance JE, Brindley DN. Stimulation of apolipoprotein secretion in very-low-
density and high-density lipoproteins from cultured rat hepatocytes by dexamethasone.
Biochem J. 1990;271(3):575–83.
87. Duerden JM, Bartlett SM, Gibbons GF. Long-term maintenance of high rates of very-low-
density-lipoprotein secretion in hepatocyte cultures. A model for studying the direct effects
of insulin and insulin deficiency in vitro. Biochem J. 1989;263(3):937–43.
88. Cole TG, Wilcox HG, Heimberg M. Effects of adrenalectomy and dexamethasone on hepatic
lipid metabolism. J Lipid Res. 1982;23(1):81–91.
89. Wang CN, Hobman TC, Brindley DN. Degradation of apolipoprotein B in cultured rat hepa-
tocytes occurs in a post-endoplasmic reticulum compartment. J Biol Chem. 1995;270(42):
24924–31.
90. Bagdade JD, Yee E, Albers J, Pykalisto OJ. Glucocorticoids and triglyceride transport: effects
on triglyceride secretion rates, lipoprotein lipase, and plasma lipoproteins in the rat.
Metabolism. 1976;25(5):533–42.
91. Taskinen MR, Nikkila EA, Pelkonen R, Sane T. Plasma lipoproteins, lipolytic enzymes, and
very low density lipoprotein triglyceride turnover in Cushing’s syndrome. J Clin Endocrinol
Metab. 1983;57(3):619–26.
92. Chan DC, Watts GF, Barrett PH, Mamo JC, Redgrave TG. Markers of triglyceride-rich lipo-
protein remnant metabolism in visceral obesity. Clin Chem. 2002;48(2):278–83.
93. Foley EM, Gordts PL, Stanford KI, et al. Hepatic remnant lipoprotein clearance by heparan
sulfate proteoglycans and low-density lipoprotein receptors depend on dietary conditions in
mice. Arterioscler Thromb Vasc Biol. 2013;33(9):2065–74.
94. Hazra A, Pyszczynski NA, DuBois DC, Almon RR, Jusko WJ. Modeling of corticosteroid
effects on hepatic low-density lipoprotein receptors and plasma lipid dynamics in rats. Pharm
Res. 2008;25(4):769–80.
95. Jansen H, van Tol A, Auwerx J, Skretting G, Staels B. Opposite regulation of hepatic lipase
and lecithin: cholesterol acyltransferase by glucocorticoids in rats. Biochim Biophys Acta.
1992;1128(2–3):181–5.
96. Wang X, Magkos F, Patterson BW, Reeds DN, Kampelman J, Mittendorfer B. Low-dose
dexamethasone administration for 3 weeks favorably affects plasma HDL concentration and
composition but does not affect very low-density lipoprotein kinetics. Eur J Endocrinol.
2012;167(2):217–23.
97. Ettinger WH, Klinefelter HF, Kwiterovitch PO. Effect of short-term, low-dose corticosteroids
on plasma lipoprotein lipids. Atherosclerosis. 1987;63(2–3):167–72.
98. Taylor AH, Raymond J, Dionne JM, et al. Glucocorticoid increases rat apolipoprotein A-I
promoter activity. J Lipid Res. 1996;37(10):2232–43.
99. Bocharov AV, Huang W, Vishniakova TG, et al. Glucocorticoids upregulate high-affinity,
high-density lipoprotein binding sites in rat hepatocytes. Metabolism. 1995;44(6):730–8.
100. Temel RE, Trigatti B, DeMattos RB, Azhar S, Krieger M, Williams DL. Scavenger receptor
class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein choles-
terol to the steroidogenic pathway in cultured mouse adrenocortical cells. Proc Natl Acad Sci
U S A. 1997;94(25):13600–5.
101. Cai L, Ji A, de Beer FC, Tannock LR, van der Westhuyzen DR. SR-BI protects against endo-
toxemia in mice through its roles in glucocorticoid production and hepatic clearance. J Clin
Invest. 2008;118(1):364–75.
102. Hoekstra M, van der Sluis RJ, Van Eck M, Van Berkel TJ. Adrenal-specific scavenger recep-
tor BI deficiency induces glucocorticoid insufficiency and lowers plasma very-low-density
and low-density lipoprotein levels in mice. Arterioscler Thromb Vasc Biol. 2013;33(2):
e39–46.
103. Rose AJ, Berriel Diaz M, Reimann A, et al. Molecular control of systemic bile acid homeo-
stasis by the liver glucocorticoid receptor. Cell Metab. 2011;14(1):123–30.
104. Eloranta JJ, Jung D, Kullak-Ublick GA. The human Na+-taurocholate cotransporting poly-
peptide gene is activated by glucocorticoid receptor and peroxisome proliferator-activated
receptor-gamma coactivator-1alpha, and suppressed by bile acids via a small heterodimer

partner-dependent mechanism. Mol Endocrinol. 2006;20(1):65–79.
105. Lu Y, Zhang Z, Xiong X, et al. Glucocorticoids promote hepatic cholestasis in mice by inhib-
iting the transcriptional activity of the farnesoid X receptor. Gastroenterology. 2012;143(6):
1630–40. e1638.
106. Wang X, Wei D, Song Z, Jiao H, Lin H. Effects of fatty acid treatments on the dexamethasone-
induced intramuscular lipid accumulation in chickens. PLoS One. 2012;7(5):e36663.
107. Morgan S, Gathercole L, Stewart S, Smith D, Tomlinson J. Impact of glucocorticoids upon
lipogenesis and β-oxidation in skeletal muscle. Paper presented at Society for Endocrinology
BES 20102010; Manchester, UK.
108. Guillaume-Gentil C, Assimacopoulos-Jeannet F, Jeanrenaud B. Involvement of non-esterified
fatty acid oxidation in glucocorticoid-induced peripheral insulin resistance in vivo in rats.
Diabetologia. 1993;36(10):899–906.
109. Venkatesan N, Davidson MB, Hutchinson A. Possible role for the glucose-fatty acid cycle in
dexamethasone-induced insulin antagonism in rats. Metabolism. 1987;36(9):883–91.
110. Dumas JF, Bielicki G, Renou JP, et al. Dexamethasone impairs muscle energetics, studied by
(31)P NMR, in rats. Diabetologia. 2005;48(2):328–35.
111. Petrichenko IE, Daret D, Kolpakova GV, Shakhov YA, Larrue J. Glucocorticoids stimulate
cholesteryl ester formation in human smooth muscle cells. Arterioscler Thromb Vasc Biol.
1997;17(6):1143–51.
112. Stein O, Dabach Y, Hollander G, Ben-Naim M, Halperin G, Stein Y. Dexamethasone impairs
cholesterol egress from a localized lipoprotein depot in vivo. Atherosclerosis. 1998;137(2):
303–10.
113. Ayaori M, Sawada S, Yonemura A, et al. Glucocorticoid receptor regulates ATP-binding
cassette transporter-A1 expression and apolipoprotein-mediated cholesterol efflux from
macrophages. Arterioscler Thromb Vasc Biol. 2006;26(1):163–8.
Chapter 7
Glucocorticoids and Skeletal Muscle
Sue C. Bodine and J. David Furlow
Abstract Glucocorticoids are known to regulate protein metabolism in skeletal

muscle, producing a catabolic effect that is opposite that of insulin. In many catabolic
diseases, such as sepsis, starvation, and cancer cachexia, endogenous glucocorticoids
are elevated contributing to the loss of muscle mass and function. Further, exogenous
glucocorticoids are often given acutely and chronically to treat inflammatory condi-
tions such as asthma, chronic obstructive pulmonary disease, and rheumatoid arthri-
tis, resulting in muscle atrophy. This chapter will detail the nature of
glucocorticoid-induced muscle atrophy and discuss the mechanisms thought to be
responsible for the catabolic effects of glucocorticoids on muscle.
Keywords Muscle atrophy • Protein synthesis • Proteolysis • Ubiquitin proteasome

pathway • Gene transcription
Introduction
Skeletal muscle comprises ~40 % of body mass and is a major target of glucocor-
ticoids (GC), whose actions primarily alter protein and glucose metabolism.
Glucocorticoids are released endogenously in response to a variety of physically
and psychologically stressful conditions including fasting, illness and exercise.
The effect of elevated levels of circulating GCs on skeletal muscle is a decrease in
S.C. Bodine, Ph.D. (*)

Department of Neurobiology, Physiology and Behavior, University of California, Davis,
One Shields Avenue, Davis, CA 95616, USA
Department of Physiology and Membrane Biology, University of California, Davis,
J.D. Furlow, Ph.D.
Department of Neurobiology, Physiology and Behavior, University of California, Davis,

146 S.C. Bodine and J.D. Furlow
protein synthesis and an increase in proteolysis in order to generate an increase in

free amino acids that can serve as substrates for gluconeogenesis in the liver and
help to maintain blood glucose levels. Thus, acute increases in glucocorticoids are
beneficial in that they maintain circulating glucose levels to support brain function
under stressful conditions. However, when circulating glucocorticoid levels are
elevated chronically due to a pathological condition or as a course of medical treat-
ment the effects on skeletal muscle are negative causing loss of mass and weak-
ness, as well as insulin resistance. In this chapter, our discussion will concentrate
on the effects of elevated glucocorticoids on skeletal muscle mass and function, as
the effects on skeletal muscle metabolism are discussed in detail in other chapters
of this book.
Glucocorticoid Induced Muscle Atrophy
Glucocorticoids are known to have catabolic effects on skeletal muscle, either as an

endocrine hormone released in response to stress or as a drug given to treat inflam-
mation. Elevated endogenous glucocorticoids are a major component of Cushing’s
syndrome, as well as other diseases such as sepsis and cancer cachexia [1]. In addi-
tion, millions of adults are treated with glucocorticoid therapy for a variety of acute
and chronic inflammatory conditions such as asthma, chronic obstructive pulmo-
nary disease, rheumatoid arthritis, and neurological conditions such as spinal cord
injury, brain tumors, and myasthenia gravis [2], and in an apparent paradox, mus-
cular dystrophy (see below). In many cases, treatment with glucocorticoids can
exceed 5 years leading to a variety of side effects including muscle wasting and
weakness.
To demonstrate the catabolic effects of glucocorticoids on muscle, many studies
have utilized healthy juvenile and adult rodents, and delivered a variety of synthetic
glucocorticoids (e.g. dexamethasone, methylprednisolone, prednisolone, and triam-
cinolone acetonide) at relatively high doses on a daily basis for days to weeks. The
effect of excess glucocorticoids on muscle mass is well characterized, and in gen-
eral, is dependent on the fiber type composition of the muscle. Muscle fibers are
classified into types based on their myosin heavy chain composition (I, IIa, IIx, or
IIb) and metabolic properties (SO, slow oxidative; FOG, fast oxidative glycolytic;
and FG, fast glycolytic) [3] (Fig. 7.1). Chronic, excess levels of GCs cause a loss of
muscle mass that is greatest in muscles composed predominantly of type II, fast
twitch fibers [4, 5]. At the individual fiber level, the loss of fiber cross-sectional area
is greatest in the fast-twitch glycolytic fibers (FG, type IIb or IIx) and least in the
slow-twitch oxidative fibers (SO, type I) [6–8]. In rat muscles of mixed fiber type
composition, such as the medial gastrocnemius, atrophy usually follows the order
(from highest to lowest) of FG > FOG > SO [6, 9]. Moreover, there can be regional
differences in the degree of atrophy of the fast glycolytic fibers, in that the FG fibers
in the predominantly “white” region of the medial gastrocnemius atrophy more than
the FG fibers in the predominantly “red” region [7].
7 Glucocorticoids and Skeletal Muscle 147
Fig. 7.1 Fiber types and glucocorticoid-induced atrophy. (a) Skeletal muscles are composed of
fibers that express different myosin heavy chain isoforms (MHC) and have varying metabolic
properties. There are four MHCs expressed in rodent skeletal muscles (I, IIa, IIx and IIb), while
there are only three MHCs expressed in human skeletal muscle (I, IIa, and IIx). The metabolic
profiles of fibers vary with respect to their capacity for oxidative metabolism and glycolysis. Fibers
can be classified into three types based on their myosin ATPase activity, oxidative capacity and,
glycolytic capacity: SO slow oxidative, FOG fast oxidative glycolytic, and FG fast glycolytic. The
cross-sectional area of muscle fibers vary with the largest fibers being the FG fibers and the small-
est being the SO fibers. (b) Data taken from Gardiner et al. [6] demonstrating fiber type specific
atrophy following 6 weeks of glucocorticoid treatment (1 mg/kg triamcinoloneacetonide-21 phos-
phate) in male rats. Gastrocnemius muscle weights and mean areas of each fiber type in GC-treated
and pair-fed rats are expressed as a percentage of control values. The control means ± SE are given
to the right of the bars. MGR and MGW refer to regions of the medial gastrocnemius muscle con-
taining high percentages of “Red” (i.e. oxidative) and “White” (i.e. non-oxidative) fibers. Gardiner,
PF Montanaro, G Simpson, DR Edgerton, VR Effects of glucocorticoid treatment and food restric-
tion on rat hindlimb muscles. The American journal of physiology.1980; 238(2): E124–30
The extent to which oxidative fibers are affected by exogenous glucocorticoids

depends in part on the dose and type of synthetic glucocorticoid used, and also on the
specific muscle. For example, the fluorinated synthetic glucocorticoids (dexametha-
sone and triamcinolone acetonide) are more potent atrophy-inducing agents than the
nonfluorinated preparations (prednisolone and predisone) [10, 11]. Additional fac-
tors to be taken into consideration when studying the effects of glucocorticoids on
muscle mass are the sex of the animal and the animal species. In general, rats are
more sensitive to the atrophy-inducing effects of glucocorticoids than mice. Whereas
a dose of <0.5 mg/kg of dexamethasone will induce significant muscle atrophy in the
rat over a 7-day period, doses in the range of 1–3 mg/kg are required to induce atro-
phy in the mouse. Furthermore, male and female rodents show different sensitivities
to glucocorticoids, with male rodents requiring higher doses than female rodents to
achieve similar amounts of atrophy [12]. The mechanisms responsible for the sex
differences are unclear, but may relate to the ability of testosterone to protect against
the catabolic effects of glucocorticoids [13, 14, 15].
Food restriction and starvation, which cause an increase in circulating glucocor-
ticoids, also induce fiber-type specific atrophy [6, 8]. Supraphysiologic doses of
synthetic glucocorticoids can cause a reduction in food intake, especially in rats,
and thus the atrophy inducing effects of glucocorticoids are often compared to
pair-fed animals (i.e. food intake matched to the glucocorticoid-treated animals).
In general, the degree of body weight loss, or muscle atrophy, observed in response
to pair-feeding is significantly less than that observed in response to glucocorticoid
treatment [6, 7]. Thus, the mechanisms regulating muscle atrophy under
glucocorticoid-treatment and food deprivation may overlap, but likely are not
identical. Furthermore, glucocorticoids are thought to increase food intake in
humans and still cause muscle atrophy. One indication that this may be the case is
the observation that deletion of the E3 ubiquitin ligase, MuRF1, can spare muscle
fibers from atrophy induced by glucocorticoid-treatment, but not nutritional depri-
vation [12].
Synthetic glucocorticoids are used to treat a variety of inflammatory diseases in
humans and can induce muscle atrophy in both the acute and chronic phase of
treatment. Glucocorticoid-induced myopathies are most often associated with
fluorinated glucocorticoids, but can occur with all formulations depending on
dose, length of treatment, and underlying diseases [11]. The risk of acquiring a
glucocorticoid-induced myopathy is higher in the elderly and in patients with can-
cer, respiratory distress syndrome, and those who are physically inactive and in
negative nitrogen balance at the onset of glucocorticoid treatment [1, 16]. Acute
muscle atrophy associated with glucocorticoid treatment often occurs in the inten-
sive care setting where high doses of glucocorticoids are given to patients who are
immobilized, often are mechanically ventilated, given paralyzing agents, and have
additional complications such as nutritional deficiencies and sepsis.
Acute muscle wasting associated with high doses of glucocorticoids can affect
both respiratory and limb muscles [1, 17]. In humans, chronic glucocorticoid-
induced weakness and wasting is generally associated with proximal muscles,
especially the pelvic girdle muscles, but can also affect distal muscles [11].
Glucocorticoid-induced atrophy in humans is fiber type specific, as observed in
animal models, being greatest in the type IIx, glycolytic, non-oxidative fibers [18,
19]. It should be noted that in human limb muscles the fast glycolytic fibers express
the type IIx myosin heavy chain and not IIb, which is not found in human limb
muscles (Fig. 7.1). Type II specific atrophy is also observed in diseases with ele-
vated endogenous glucocorticoids, such as Cushing’s syndrome and sepsis [20].
Glucocorticoids and Muscle Function
Excess glucocorticoids also cause a decrease in muscle strength that parallels the
loss of fiber cross-sectional area. For example, patients with Cushing’s syndrome
and patients receiving glucocorticoid treatment have decreases in muscle strength
[20–22]. On physical exam, a patient with chronic glucocorticoid excess (Cushing
syndrome) will have difficulty rising from a chair without use of the hands, a test of
proximal muscle function. The use of glucocorticoids as treatment of an inflamma-
tory disease can often exacerbate the deleterious effects of the disease on muscle
strength. For example, measurement of isometric and isokinetic muscle strength in
rheumatoid arthritis patients found significantly greater decreases in strength in those
patients treated with prednisone compared to those patients who received no treat-
ment [23]. Furthermore, glucocorticoid-mediated muscle atrophy in patients with
chronic obstructive pulmonary disease can negatively affect their pulmonary func-
tion due to decrease muscle (bellows) function.
Decreases in muscle force output are also found in animal disease models associ-
ated with increases in endogenous glucocorticoids, such as sepsis and critical care
illness [24–26]. In some animals, exogenous glucocorticoid treatment produces
comparable decreases in both muscle contractile force and fiber cross-sectional [9, 27].
Interestingly, while treatment of rodents with no underlying disease with a synthetic
glucocorticoid, such as dexamethasone, causes a loss of muscle mass, especially in
predominantly fast-twitch muscle, there is often no measurable decrease in maxi-
mum muscle force output leading to an increase in specific force output (i.e., force
per cross-sectional area) [12, 28, 29]. This increase in specific force is apparent in
limb muscles, especially fast-twitch muscles, but not in the diaphragm of juvenile
rodents given synthetic glucocorticoids [8, 30]. In rodents, the mechanism respon-
sible for the increase in specific force is unclear, but does not appear to be related to
a change in sarcolemmal excitability or excitation-contraction coupling [29].
Glucocorticoids and Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is the most prevalent muscular dystrophy

occurring in 1 out of 3500 live male births, and caused by mutations in the dystro-
phin gene [31]. DMD is a severe progressive neuromuscular disease that presents
with gait disturbances between the ages of 3 and 5 years and loss of ambulation by
12. There are no cures for DMD, but the use of glucocorticoid therapy (prednisone,
prednisolone and deflazacort) has been shown to improve muscle strength during
the first 6 months of usage followed by stabilization of the course of the disease for
up to 2 years, after which there is a decline in function. There are many potential
adverse effects of glucocorticoid therapy including osteoporosis, excessive weight
gain, and behavioral abnormalities [32]. While there appear to be short-term benefits
of glucocorticoid therapy in DMD patients, the long-term functional benefits are
unclear [31]. A study performed on mdx mice showed that daily administration of
prednisolone resulted in early (initial 50 days) improvements in muscle strength and
motor coordination, but these benefits were lost after 100 days of continuous treat-
ment [33]. Further, there was a deterioration of cardiac function and increased fibro-
sis of the heart with prolonged glucocorticoid treatment [34].
The mechanism by which glucocorticoids improve muscle strength in DMD
patients is unknown, and thought to occur through a suppression of inflammation.
DMD patients often show pseudohypertrophy of muscles, especially in the calf,
which is thought to be due to inflammation. However, other immunosuppressive
drugs that reduce inflammatory infiltrates in the muscles of DMD patients do not
improve strength, as seen with prednisone. In mdx mice, treatment with predniso-
lone increases specific force in muscle while having no affect on muscle fiber size
[35, 36]. The increase in specific force output is similar to what is seen in normal
mice and rats given glucocorticoids. The mechanism by which glucocorticoids
improve force output in DMD and mdx is still unclear and may be distinct from the
anti-inflammatory actions of the glucocorticoids.
Glucocorticoids and Protein Turnover
It is well established that the overall effect of supraphysiologic levels of glucocorti-

coids on skeletal muscle is catabolic resulting in a loss of muscle mass, fiber cross-
sectional, and force output. Loss of muscle protein occurs as the result of an
inbalance between protein synthesis and breakdown, with net breakdown exceeding
synthesis. The response of skeletal muscle to catabolic doses of exogenous gluco-
corticoids is both a decrease in protein synthesis and an increase in protein degrada-
tion. The extent to which protein synthesis and degradation are altered by
exogenously delivered glucocorticoids is dependent on both the type of steroid used
and the dose given.
Alteration in Protein Synthesis: The earliest changes in muscle protein metabolism
following glucocorticoid administration appear to be related primarily to a decrease
in the rate of protein synthesis, rather than an increase in protein breakdown. Protein
synthesis is the process by which mRNA is translated on ribosomes into amino acids
and can be divided into three distinct phases: initiation, elongation and termination
[37]. Translation initiation is a key rate limiting process that is controlled at two key
steps: (1) modulation of the binding of the initiator methionyl-tRNA (met-tRNAi) to
the 40S ribosomal subunit to form the 43S preinitiation complex, and (2) the bind-
ing of the mRNA to the 43S preinitiation complex to form the 48S preinitiation
complex. Regulation of these translation initiation steps is controlled through the
phosphorylation of initiation factors and protein kinases that are controlled by sig-
naling pathways downstream of phosphatidylinositol 3-kinase (PI3K) [38] (Fig. 7.2).
In mammalian muscle, muscle mass is critically regulated by activation of the Ser/
Thr protein kinase, the mammalian (or mechanical) target of rapamycin (mTOR),
Fig. 7.2 Signaling pathways downstream of insulin and insulin-like growth factor 1 (IGF-1) that
are affected by glucocorticoids. The mammalian target of rapamycin complex 1 (mTORC1) is
activated by growth factors such as insulin and IGF-1 and branch chain amino acids such as leucine,
leading to increases in mRNA translation and protein synthesis. Glucocorticoids potentially inhibit
protein synthesis through multiple mechanisms including (1) a decrease in amino acid uptake, (2)
a decrease in insulin and IGF1 levels (3) a decrease in the activity of phosphatidylinositol 3-kinase
(PI3K) leading to a decrease in Protein Kinase B/Akt activity, (4) a decrease in mTORC1 activity
leading to a decrease in the phosphorylation and activation of S6K1 and 4E-BP1, and (5) an increase
in GSK3β activity leading to a decrease in β-catenin and possibly inhibition of eIF2B
and its downstream targets the ribosomal protein S6 kinase (S6K1/p70s6k) and the
translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1).
In muscle, mTOR can be found in at least two distinct complexes: the rapamycin-
sensitive mTOR complex 1 (mTORC1) and the rapamycin-insensitive mTOR
complex 2 (mTORC2) [39]. Activation of mTORC1 is critical for the regulation of
muscle mass and is regulated by many pathways including growth factor signaling
through PI3K/Protein Kinase B (PKB/Akt), amino acids, and mechanical loading
[40, 41].
In response to moderate to high doses of glucocorticoids the synthesis of muscle
proteins is significantly reduced in both humans and rodents [10, 42, 43].
Glucocorticoids delivered acutely at low doses do not produce significant decreases
in basal protein synthesis, but do attenuate the ability of branched chain amino acids
and insulin to activate protein translation [44, 45]. Chronic levels of low doses of
glucocorticoids could induce muscle loss through anabolic resistance to elevated
levels of insulin and amino acids as would occur following a meal.
The decrease in protein synthesis that occurs in response to glucocorticoids could

be related to a number of mechanisms. One possible mechanism is through a
decrease in the ability of muscle fibers to uptake amino acids from extracellular
fluids [46, 47]. Further, decreases in ribosome content, as well as, decreases in the
efficiency of protein translation have been measured following glucocorticoid treat-
ment and reflect a decrease in the capacity for protein synthesis. Initial studies
revealed both a decrease in the number of polyribosomes, and a reduced ability to
incorporate amino acids into proteins in fast-twitch muscle following glucocorticoid-
treatment [48, 49]. Later studies by Rannels et al. revealed that the 55 % decrease in
protein synthesis of rat fast-twitch muscles measured over 5 days of glucocorticoid
treatment was due primarily to disaggregation of actively engaged polysomes
through inhibition of peptide-chain initiation and partly to a ~15 % decrease in RNA
content [43, 50]. Subsequent studies demonstrated that acute delivery (4 h) of glu-
cocorticoids in rats induced rapid inhibition of protein synthesis (59 % decrease)
independent of changes in total RNA, but through a mechanism that involved
dephosphorylation of 4E-BP1 and S6K1, and stabilization of the inhibitory complex
4E-BP · eIF4E, leading to inhibition of the assembly of the cap-recognition complex,
eIF4F (which is composed of the scaffolding protein eIF4G, the mRNA cap-recog-
nition protein eIF4E and the ATP-dependent RNA helicase eIF4A) [51, 52]. Acute
glucocorticoid treatment in vivo was not associated with a decrease in the rate of
eIF2B-catalyzed guanine nucleotide exchange, which is a rate-limiting step in for-
mation of the ternary complex (eIF2 · GTP, Met-tRNAi, 40S ribosomal subunit,
eIF1A and eIF3). The reduced phosphorylation of both 4E-BP1 and S6K1 suggested
that glucocorticoids inhibit protein synthesis through inhibition of mTORC1.
Alterations to Protein Degradation: The inhibitory effect of glucocorticoids on pro-
tein synthesis were the first to be established, while the ability of glucocorticoids to
increase protein degradation was debated for some time due to conflicting findings.
Many early studies were able to measure a decrease in protein synthesis following
hours to days of glucocorticoid treatment, but failed to measure a significant increase
in proteolysis. Some of the conflicting proteolysis data was contributed to differ-
ences in the type and dose of glucocorticoid used, the muscles studied, and the
nutritional status of the animals.
Goldberg (1969) was one of the first to measure an increase in proteolysis, as
well as a decrease in protein synthesis, in the rat plantaris muscle (but not the
soleus) following a high dose of cortisone acetate (10 mg/100 g body weight) that
induced a 30 % and 50 % decrease in muscle mass 7 and 12 days following the
initiation of GC treatment, respectively [4]. In 1979, Tomas et al. examined the
effect of a range of corticosterone doses on rat skeletal muscle proteolysis as mea-
sured by 3-methyhistidine (N-tau-methylhistidine, 3MeH) excretion [53]. The
excretion of 3-methylhistidine is often used to measure the breakdown of muscle
since this amino acid is formed by the methylation of certain histidine residues in
the myofibrillar proteins (actin and myosin) during proteolysis, and since it unable
to be reincorporated into other proteins it is excreted in the urine [54]. Skeletal
muscle is the major source of excreted 3MeH, with skin and intestine contributing
a small amount [55]. Tomas et al. found that skeletal muscle proteolysis increased
only when plasma glucocorticoid levels were significantly greater than resting lev-
els, which occurred only at the highest doses of corticosterone treatment (5 and
10 mg/100 g body weight/day) and not at lower doses (0.2, 0.5 or 1.0 mg/100 g
BW/day) [53]. The peak plasma corticosterone concentrations (40–80 μg/100 mL
of plasma) that caused muscle proteolysis were in the range of corticosterone levels
measured following burn injury and prolonged psychological/physical stress (50–
100 μg/100 mL plasma).
These data revealed that in a fed state, glucocorticoids can increase muscle pro-
teolysis but only if given in exceptionally high doses. In a fed state, low levels of
glucocorticoids do not induce muscle breakdown due to the presence of insulin.
However, in a fasted state, elevated levels of glucocorticoids and low insulin pro-
duce increases in muscle proteolysis [56, 57]. Glucocorticoids given directly to iso-
lated muscles in a perfused bath or to myotubes in culture increase proteolysis,
however, the effect is suppressed in the presence of insulin [58].
The importance of glucocorticoids in promoting muscle proteolysis under condi-
tions of fasting and acidosis was demonstrated using adrenalectomized rats. Wing
and Goldberg showed that the rate of protein degradation increased ~50 % in the
extensor digitorum longus muscle of rats fasted for 24 h. In contrast, fasting of
adrenalectomized rats induced a significantly smaller increase in muscle degrada-
tion (2.5-fold difference between fasted and fasted adrenalectomized rats).
Administration of dexamethasone to fasted, adrenalectomized rats elevated the rate
of protein breakdown to that observed in normal fasted rats thus demonstrating the
role of glucocorticoids in regulating muscle proteolysis under conditions of fasting.
Elevated levels of plasma glucocorticoids have also been shown to be required for
the increase in muscle protein breakdown observed in response to metabolic acido-
sis [59, 60]. Glucocorticoids together with elevated cytokines also contribute to an
increase in muscle proteolysis in various catabolic diseases such as sepsis, cancer
and burns [57].
The Ubiquitin Proteasome Pathway and Glucocorticoids
Accelerated protein breakdown in skeletal muscle in response to glucocorticoids

has been shown to be largely due to activation of the Ubiquitin Proteasome Pathway
(UPP) [61, 62]. The UPP is an ATP-dependent proteolytic pathway made up of 3
classes of proteins involved in tagging proteins with the 76 amino acid protein ubiq-
uitin, and the 26S proteasome, which recognizes polyubiquitinated proteins and
degrades them into small peptides [63] (Fig. 7.3). Proteins can be monoubiquiti-
nated, diubiquitinated, or polyubiquitinated via the actions of an E1 ubiquitin-activating
enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase enzyme.
To form a polyubiquitin chain, ubiquitin proteins are linked at one of their seven
lysine residues [64]. Proteins containing a chain of at least four lysine-48 linked
ubiquitin molecules are recognized and degraded by the proteasome [65].
Fig. 7.3 The ubiquitin proteasome pathway (UPP). Excess glucocorticoids can lead to an increase
in protein degradation through an increase in components of the UPP and an increase in the number
of ubiquitin-tagged proteins. The process of ubiquitination is controlled by the ubiquitin-activating
enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3). MuRF1 and
MAFbx are both E3 ubiquitin ligases that control the ubiquitination of specific substrates. Ubiquitin
can be added to a substrate as a chain of ubiquitins of variable length on a single lysine (polyubiq-
uitination). Polyubiquitination of a substrate with an ubiquitin chain using K48 linkages generally
results in proteasomal degradation. Deubiquitinating enzymes (DUB) associated with the protea-
some or in the cytoplasm are responsible for disassembly of the polyubiquitin chains and mainte-
nance of the pool of free ubiquitin
The 26S proteasome is a large (2-MDa) multi-subunit complex consisting of a

20S core particle and two 19S regulatory particles found at each end of the 20S
core, and is responsible for the breakdown of the majority (60–70 %) of muscle
proteins, especially under catabolic conditions. The 19S particle is involved in the
recognition of polyubiquitinated proteins and functions to unfold these proteins and
guide them into the 20S catalytic core [66]. The hydrolysis of ATP drives the unfold-
ing of large globular proteins and translocation of the proteins into the 20S core.
The 20S core proteasome is a cylindrical structure made up of two outer α rings and
two inner β rings. Each alpha ring contains seven α subunits and each beta ring
contain seven β subunits, with the proteolytic sites located on the inner surface of
the β1, β2, and β5 subunits [67]. The β1 subunit possesses caspase-like proteolytic
activity, the β2 subunit has trypsin-like proteolytic activity, and the β5 subunit has
chymotrypsin-like proteolytic activity [67]. The substrate protein that enters the
20S core particle exits as small peptides ranging in size from 3 to 25 residues in length
[68]. These peptides are quickly digested into amino acids that can be reused to
synthesize new proteins or metabolized to provide energy. Deubiquitinating
enzymes (DUBs), associated with the 26S proteasome and also found in the cyto-
plasm, are responsible for disassembly of the polyubiquitin chain and release of free
ubiquitin that can be reused to tag additional substrates.
The selection of proteins to be ubiquitinated and subsequently degraded by the
proteasome is dependent on the specific E2 and E3 ligases present in the cell.
Specificity of the process is determined largely by the E3 ligase, which binds the
substrate and interacts with the E2 ligase to direct the transfer of ubiquitin to the
substrates [69]. The specific E2-E3 pairings dictate the type of ubiquitin linkage and
the length of the ubiquitin chains. In mammals, ~40 E2 ligases and over 600 E3
ligases have been identified. The E3 ligases can be classified into two major groups:
the HECT (Homologous to the E6AP Carboxyl Terminus) E3 ligases and the RING
(Really Interesting New Gene) E3 ubiquitin ligases [70]. The RING E3 ligases con-
stitute the majority of E3s found in mammals and function as a scaffold that medi-
ates the transfer of ubiquitin directly from the E2 ~ Ub to substrate.
Under catabolic conditions in which glucocorticoids are elevated, genes encod-
ing components of the ubiquitin proteasome pathway and proteasome subunits have
been shown to increase in skeletal muscle. An increase in ATP-dependent protea-
some activity has been measured in both L6 and C2C12 myotubes exposed to dexa-
methasone in vitro and in hindlimb muscles of rats treated with dexamethasone [71,
72]. Interestingly, no increase in 20S or 26S proteasome activity was measured in
hindlimb muscles of C57BL6 mice treated with dexamethasone for up to 14 days
[12, 73]. In these mice, significant muscle atrophy occurred and was associated with
a decrease in protein synthesis, as opposed to elevated proteolysis. The difference in
the reported proteolytic response of rats versus mice to dexamethasone may be
related to the nutritional status of the animals and the levels of plasma glucocorti-
coids achieved. In general, glucocorticoid-treatment in rats, is accompanied with a
decrease in food consumption and rapid loss of muscle mass (especially when fluo-
rinated glucocorticoids are used). In contrast, in mice no changes in food consump-
tion are observed, at least at doses of 3–5 mg/kg of dexamethasone. Increases in
proteasome-mediated degradation have been noted in catabolic disease states such
as starvation, metabolic acidosis, sepsis, diabetes and cancer [74–80]. Associated
with increases in proteasome activity are increases in the amount of ubiquitin-
conjugates isolated from skeletal muscle. In addition, increases in the mRNA
expression of ubiquitin, proteasome subunits and several E2 ligases (E214k, E217k)
have been measured.
The E3 Ubiquitin Ligases: MuRF1 and MAFbx
A number of E3 ubiquitin ligases have been identified in skeletal muscle, but two
muscle-specific E3s, Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-Box
(MAFbx)/atrogin-1, have been shown to be transcriptionally increased under a
variety of atrophy conditions including glucocorticoid treatment, as well as other

catabolic conditions such as starvation, renal failure, cancer cachexia and diabetes
[81]. Muscle RING Finger 1 (gene name:Trim63) is a simple RING E3 that belongs
to a family of genes (MuRF1, 2, and 3) which represent a subgroup of the TRIM
superfamily of multidomain ubiquitin E3 ligases characterized by the presence of the
N-terminal tripartite motif: RING domain, zinc-finger B-box domain, and leucine-
rich coiled-coil domain [82]. Muscle Atrophy F-Box (gene name:FBX032), also
known as atrogin-1, is 1 of ~70F-box proteins and belongs to the cullin–RING E3
ligase (CRL) superfamily. MAFbx functions in a multi-subunit complex consisting
of: SKP1 and a F-box protein which serve as the substrate adaptor module, a cullin
protein (CUL1) that acts as a scaffold, and a RING finger protein (RBX1 or RBX2)
that associates with CUL1 and recruits the ubiquitin charged E2s [83]. In resting
skeletal muscle, MuRF1 and MAFbx are expressed at relatively low levels, but
become transcriptionally upregulated in response to a variety of stressors, including
glucocorticoids and cytokines [84].
The analysis of mice with null alleles in genes encoding either MuRF1 or MAFbx
has provided evidence that both genes are key regulators of muscle atrophy. Baehr
et al. examined the response of both male and female MuRF1 and MAFbx null mice
to 14 days of dexamethasone-treatment and to 3 days of nutritional deprivation
(80 % deficit) [12]. Glucocorticoid-induced muscle atrophy was significantly atten-
uated in both male and female MuRF1 KO mice, but not MAFbx KO mice. Muscle
sparing was evident as measured by the maintenance of muscle mass, muscle fiber
cross-sectional area, and muscle contractile force. In contrast, the deletion of
MuRF1 or MAFbx did not result in muscle sparing in response to nutritional depri-
vation, even though glucocorticoids are elevated under conditions of starvation.
These results and others [85] suggest that starvation activates additional glucocorti-
coid independent pathways that contribute to muscle atrophy.
Anti-anabolic Effects of Glucocorticoids
Suppression of protein synthesis in skeletal muscle by glucocorticoids can occur

through a number of mechanisms. As mentioned previously, one mechanism by
which glucocorticoids could decrease protein synthesis is related to its ability to
inhibit the transport of amino acids into the muscle. Glucocorticoids can also inhibit
protein synthesis by altering the activation level of specific signaling pathways
(such as the PI3K/Akt/mTOR pathway), which mediate the anabolic actions of
amino acids (primarily leucine), growth factors (such as insulin-like growth factor-
1, IGF-1), and insulin.
As a key example, IGF-1 has been shown to have both an anabolic and an anti-
catabolic role in skeletal muscle with the anabolic effects being mediated through its
ability to increase protein synthesis through activation of the PI3K/Akt/mTOR path-
ways [86]. Under catabolic conditions and in response to excess glucocorticoids,
circulating and muscle IGF-1 levels are often reduced [87, 88]. In young growing
rats, a catabolic dose of dexamethasone has been shown to decrease muscle IGF-1
levels, as well as, decrease the phosphorylation levels (and thus activation) of both
PKB/Akt and S6K1 [89, 90]. Evidence to support a role for decreased IGF-1 levels
and reduced activation of PKB/Akt in glucocorticoid-induced muscle atrophy has
come primarily from over-expression studies. Using in vitro myotube culture sys-
tems, IGF-1 has been shown to prevent glucocorticoid-induced loss of myotube
diameter through the activation of PI3K/Akt and blockage of the nuclear transloca-
tion of the FOXO transcription factors and downregulation of the E3 ubiquitin
ligases, MuRF1 and MAFbx/atrogin-1 [91, 92]. Moreover, in vivo systemic admin-
istration [93–95] and direct overexpression of IGF-1 into skeletal muscle [90] has
been shown to attenuate glucocorticoid-induced muscle atrophy.
The attenuation of muscle atrophy in response to overexpression of either IGF-1
or constitutively active PKB/Akt is associated with an increase in mTOR signaling,
a decrease in the nuclear translocation of FOXO1/3a, decreased expression of
MuRF1 and MAFbx/atrogin-1 and an increase in β-catenin expression [96]. The
responses to IGF1 and PKB/Akt-1 activation suggest that the attenuated atrophy is
related to both an anabolic effect (maintenance of protein synthesis through activa-
tion of mTOR and its downstream targets) and anti-catabolic effect (reduced activa-
tion of the FOXO transcription factors and reduced expression of genes associated
with elevated proteolysis, such as MuRF1, MAFbx and cathepsin-L). The increase
in β-catenin expression in response to IGF-1 and PKB/Akt activation was interest-
ing given that the transcription factor has been reported to play a role in skeletal
muscle hypertrophy [97].
The amount of β-catenin in the cell is regulated by its phosphorylation and deg-
radation by the proteasome. Phosphorylation of β-catenin is controlled by glycogen
synthase kinase 3β (GSK-3β) whose activity in turn is inhibited by phosphorylation
by PKB/Akt. In response to dexamethasone-treatment, a significant reduction in
β-catenin protein levels were also reported, which is consistent with the hypothesis
that GSK3β is activated in response to glucocorticoids leading to an increase in the
phosphorylation and degradation of β-catenin [90]. An increase in β-catenin in
response to IGF-1 or Akt activation is consistent with inactivation of GSK3β. Over-
expression of a stable β-catenin protein, which is resistant to proteasomal degrada-
tion, in skeletal muscle is capable of blocking GC-induced muscle atrophy, thus
providing some evidence that β-catenin may be an important regulator of the nega-
tive effects of glucocorticoids [90].
Independent of changes in growth factor availability, glucocorticoids can also
decrease the activation of PI3K through mechanisms that modify the amount of
insulin receptor substrate-1 (IRS-1) and the relative levels of the regulatory subunit
of PI3K, i.e., p85α, and not the catalytic subunit, p110. Glucocorticoid-treatment
consistently leads to a decrease in the amount of IRS-1 protein and inactivation of
IRS-1 by serine-phosphorylation (ser307) in vitro in C2C12 myotubes [98–100].
Acceleration of the degradation of IRS-1 could come about through multiple mech-
anisms involving either an increase in the amount of the tyrosine phosphatase,
C1-Ten [101] or the E3 ubiquitin ligase, Cblb [98].
Another mechanism that can account for a decrease in PI3K activity following
glucocorticoid treatment is the selective increase in the regulatory subunit of PI3K,
p85α (a.k.a. Pik3r1). An increase in the regulatory subunit of PI3K (p85α), without
a concomitant increase in the catalytic subunit (p110) occurs in response to gluco-
corticoids, and could contribute to the inhibition of PI3K activity [102, 103].
Overexpression of p85α in vitro results in a decrease in the diameter of C2C12 myo-
tubes [102]. In contrast, inhibition of p85α in C2C12 myotubes, with small interfer-
ing RNA (siRNA), reduced the ability of glucocorticoids to suppress PI3K activity,
inhibit protein synthesis and induce muscle atrophy. These data suggest that
increased expression of p85α, the regulatory subunit of PI3K, is a major regulator
of glucocorticoid-induced atrophy through the suppression of PI3K activity and
protein synthesis.
Downstream of PI3K/Akt is the serine-threonine kinase, mTOR, which is a
major regulator of skeletal muscle size [40]. As previously noted, mTORC1 activ-
ity, as measured by the phosphorylation of S6K1 and 4E-BP1, is suppressed in
response to glucocorticoid treatment. Suppression of mTOR activity may be related
to the inhibition of PI3K/Akt activation, but could also be a consequence of other
mechanisms. Recently, it has been shown that suppression of mTORC1 in response
to glucocorticoids is related to the up-regulation of genes such as REDD1 [104,
105]. The upregulation of REDD1 has been directly associated with a decrease in
phosphorylation of the mTOR targets S6K1, 4E-BP1, and ULK1, as well as a
decrease in protein synthesis. More recently it was demonstrated that mice with a
deletion of REDD1 were resistant to the effects of dexamethasone [73]. The lack of
REDD1 expression in skeletal muscle prevented dexamethasone-induced muscle
atrophy and prevented the suppression of mTOR activity and protein synthesis. The
mechanism underlying glucocorticoid-mediated mTOR inhibition has been unclear,
however, recent studies suggest that inhibition is dependent on Akt/PRAS40 signal-
ing [73]. It appears that overexpression of REDD1 leads to a decrease in Akt thr308
phosphorylation, which lessens the inhibition on PRAS40, allowing it to inhibit
mTOR. An alternative hypothesis is that the REDD1 induced dephosphorylation of
Akt on Thr308 leads to TSC1-TSC2 complex assembly and attenuation of Rheb-
GTP and mTORC1 activation [106].
Molecular Mode of Action of Glucocorticoids

in Skeletal Muscle
The Glucocorticoid Receptor and the “Classical” Genomic

Actions of Glucocorticoids
As discussed in more detail in other chapters, the major means by which natural and
synthetic GCs exert their actions in skeletal muscle as in other tissues is through the
intracellular glucocorticoid receptor (GR), a member of the steroid receptor gene
superfamily [107]. Although widely expressed in mammalian tissues, GR is
relatively highly expressed in skeletal muscle and at higher levels than in more well-
studied tissues such as the liver or adipose tissue. The GR is most closely related to
the mineralocorticoid receptor (MR) that is best known for its role in sodium and
potassium balance via its cognate hormone, aldosterone, in many vertebrates.
However, the MR is expressed at much lower levels in skeletal muscle than other
cell types. There is only one GR gene in mammals (GRα), although humans express
a GRβ splicing isoform that appears to be expressed at low levels in skeletal muscle
[108]. Internal ribosome entry may generate further diversity in the major GRα
isoform (GR A-D), which alters target gene selectivity. Additional GR encoding
genes and various alternately spliced isoforms have been reported in other verte-
brates, most notably in fish [109]. In this chapter, for simplicity sake, we will refer
to the skeletal muscle GRα generically as the GR.
The GR is expressed at much higher levels in fast-twitch glycolytic fibers versus
slow-twitch oxidative fibers, which is consistent with the much greater sensitivity
of fast twitch glycolytic fibers to the atrophy-inducing effects of glucocorticoids
[110]. GR expression in muscle is not static, but is commonly down-regulated by its
own cognate ligand [111] and also varies in a circadian manner with peak levels in
the morning in rodents when endogenous glucocorticoids are low [112]. GR expres-
sion levels have also been reported to vary as a consequence of aging and obesity,
although the significance of these expression changes has not been clearly estab-
lished [113]. Skeletal muscle GR expression is also regulated by other nuclear
receptors such as retinoic acid (RARs) [114], and estrogen receptor-related (ERR)
receptors [115]; in the latter case, this has been linked to metabolic reprogramming
by this orphan member of the gene superfamily.
The GR is a modular protein with interacting but separable domains that allow
the versatile receptor to bind ligand, as well as, interact with specific DNA sequences,
additional DNA-bound transcription factors, and transcriptional coregulatory pro-
teins. As a result, activation of the GR by synthetic or natural ligands leads to a
change in the expression of dozens to hundreds of genes in skeletal muscle, with the
number and magnitude reported dependent on expression platform, dose and dura-
tion of exposure, and whether cultured myotubes or intact animals were used. The
identified regulated genes in skeletal muscle include: (1) known, classical GR target
genes that have been identified in many other tissues and (2) genes involved in sub-
strate metabolism, insulin signaling, inflammation, the extracellular matrix, and
angiogenesis as the most common functional clusters [102, 103, 116–118]. A subset
of these genes, including their presumed mode of regulation by glucocorticoids, are
highlighted in Table 7.1. In addition to protein encoding genes, potential roles for
GR mediated transcriptional upregulation of microRNAs such as miR1 have been
described [119].
In the unliganded state, the GR is retained in the cytoplasm as a complex with
Hsp90 and other chaperones, and upon binding ligand releases these chaperones
and exhibits nuclear translocation [107]. Recent chromatin immunoprecipitation
experiments in C2C12 myotubes identified multiple direct genomic targets of the
GR in muscle [102]. A DNA sequence was commonly observed in the vicinity of
both up and down-regulated primary response genes that was similar to a consensus
Table 7.1 Selected glucocorticoid regulated genes in skeletal muscle related to atrophy
Gene Mode of regulation Roles References
MuRF1/TRIM63/RNF GRE/FOXO1/KLF15 E3 ubiquitin ligase [84, 110,
(directa) 134]
MAFbx/Atrogin-1/Fbxo32 FOXOs (indirect) E3 ubiquitin ligase [83, 84, 167]
PI3kr1 GRE (direct) PI3 kinase subunit [102]
REDD1/Ddit4 GRE (direct) mTOR inhibitor [73, 102,
104]
KLF15 GRE (direct) Transcription factor [110]
FOXOs GRE (direct) (FOXO1b) Transcription [128, 168]
factors
Myostatin/GDF8 GRE (direct) Muscle growth [169, 170]
inhibitor
a
Both direct (via GRE) and indirect (via induced FOXO, KLF15 and other transcription factors) possible
b
Only FOXO1 regulation by glucocorticoids has been investigated to date
glucocorticoid response element (GRE) recognized by ligand bound GR homodimers

(5′ AGAACAnnnTGTTCT-3′, where n is any nucleotide in the three base pair
spacer between the hexameric inverted repeat half sites), However, only a minority
of these binding sites are found in the proximal promoter region of target genes
(i.e. <5 kb upstream of the promoter); most GR binding sites are found more than
25 kb up or downstream of target gene transcription units or within introns.
Glucocorticoids are also capable of direct transcriptional suppression of genes in
many cell types, including skeletal muscle. A motif similar (but not identical) to the
GRE described in up-regulated genes is enriched in skeletal muscle down-regu-
lated genes [102], although protein–protein interactions with other transcription fac-
tors has traditionally been ascribed as the major mode of gene expression inhibition
by glucocorticoids. For example, the GR is able to bind to NF-κB and AP-1 sub-
units as a major means of suppressing gene expression in fibroblasts (i.e. structural
genes like collagen) and immune cells (i.e. pro-inflammatory cytokine genes)
[120], but most of the work in skeletal muscle to date has focused on up-regulated
genes.
To accomplish transcriptional regulation of its target genes, the GR must recruit
additional transcriptional cofactors that serve to post-translationally modify chro-
matin or other transcription factors (primarily via acetylation and methylation), or
otherwise collaborate to influence RNA polymerase II activity. For example, the GR
both transcriptionally induces and physically associates with the histone acetyl-
transferase p300 and its paralog CBP [121]as well as the p300/CBP interacting pro-
tein Cited2 in skeletal muscle cells [122], in both sepsis and glucocorticoid excess
models. Inhibition of global acetylation reduces muscle atrophy in cultured myo-
tubes [123, 124], although the relative contribution of p300/CBP versus other GR
interacting and non-interacting acetyltransferases/deacetylases is unknown at pres-
ent. In addition to acetylation, the methyltransferase SMYD3 is also involved in
glucocorticoid-induced atrophy in vitro [125], and interestingly is specifically
induced by glucocorticoids in C2C12 cells, whereas other SMYD family members
are not. SMYD3 recruits the bromodomain containing, chromatin remodeling protein,
BRD4, to specific muscle promoters activated during atrophy. A specific role in
skeletal muscle for these transcriptional cofactors is proposed, however, they may
simply be generally supporting GR transcriptional activity in myotubes given that
HATs like p300/CBP are recruited to GR regulated promoters in many other cell
types and target gene promoters. Indeed, SMYD3 also interacts with other steroid
receptors as a mediator of steroid hormone action in several contexts [126].
While glucocorticoids directly up-regulate a number of genes implicated in
skeletal muscle protein turnover and lipid/carbohydrate metabolism, multiple tran-
scription factors are also induced by glucocorticoids in this tissue, serving to
expand and integrate the hormone regulated transcriptional network in these cells.
One important class of transcription factors discussed in previous sections is the
forkhead domain containing class O transcription factors (FOXOs). FOXO1, 3 and
4 are all induced by exogenous glucocorticoids in skeletal muscle and are also con-
sistently induced under a variety of conditions that elevate endogenous glucocorti-
coids [117, 127, 128]. Functional GREs may be present in at least one FOXO gene
promoter [129]. FOXO transcription factors are important mediators of insulin
action in multiple tissues, and are also involved in the transcriptional regulation of
metabolic enzymes, response to oxidative stress, and autophagy [130, 131]. FOXO
activity is inhibited via activation of Akt and phosphorylation at specific serine resi-
dues that inhibit FOXO nuclear localization. FOXO binding sites have been reported
in multiple atrophy-associated and metabolic enzyme genes in skeletal muscle and
inhibition of FOXO activity by growth factor addition or genetic means strongly
suppresses muscle atrophy and associated gene expression [92, 132–135], suggest-
ing that FOXOs may serve as key mediators of glucocorticoid-induced atrophy.
FOXOs are also subject to acetylation by p300 in addition to phosphorylation, but
in an isoform specific manner [136]. For example, nuclear translocation of FOXO1,
the family member particularly linked to GR actions, is activated by p300-mediated
acetylation, whereas FOXO3 transcriptional activity and localization are inhibited.
Class I histone deacetylase activity supports muscle atrophy in disuse models pre-
sumably by targeting FOXO3a (at least in part) [137]; however, isoform specific
FOXO activity in the context of glucocorticoid treatment models, where histone
acetyltransferases are actively recruited by the GR to target gene promoters will
need to be clarified.
In addition to FOXOs, several other glucocorticoid regulated transcription factors
have been identified that may also play a key role in mediating the steroid’s effects
on muscle mass and function. For example, members of the C/EBP family, in particu-
lar C/EBPβ and δ, are up-regulated in cultured myotubes and in vivo [138] in
response to glucocorticoids, and C/EBPβ may be necessary but not sufficient to
support atrophy and associated gene expression in cultured myotubes [139]. Another
transcription factor of note, Kruppel-like factor 15 (KLF15), is a strongly induced,
direct GR target in multiple cell types, including skeletal muscle. This factor partici-
pates in regulation of previously described atrophy associated genes such as MuRF1
and MAFbx, on one hand, as well as a key enzyme involved in branch chained
amino acid metabolism, BCAT2, on the other, resulting in compromised mTOR
function and suppressed protein synthesis [110]. Lastly, overexpression of GILZ, a

widely induced GR target gene that is strongly up-regulated in primary and C2C12
myoblasts, inhibits myotube differentiation, thus providing a distinct pathway by
which glucocorticoids can affect skeletal muscle mass beyond its effects in mature
muscle fibers [140]. The function of GILZ is not fully understood since it may not be
a transcription factor as originally believed based on its leucine zipper motif, but
rather may act via interactions with several cytoplasmic signal transduction proteins
[141].
Several other transcription factor genes have been identified as being glucocorti-
coid up- and down-regulated by microarray and candidate gene analysis. While
these genes are less well characterized to date in terms of regulating muscle mass
and function, they may be important in executing or fine-tuning the integrative
response to glucocorticoid exposure in these cells in concert with the factors we
have focused on so far. Despite significant progress in identifying key downstream
transcription factors, the particular code that produces a skeletal muscle specific
array of glucocorticoid regulated, functionally relevant genes has not been estab-
lished. Presumably, this specificity depends on developmental programming of
access to specific GREs that allows expression of the muscle specific glucocorticoid
response, as has been demonstrated with specific “pioneering” factors such as FoxA
isoforms in liver development [142]. While glucocorticoids regulate FOXO, C/
EBP, and KLF15 transcription factors genes in many cell types, for example, the
expression of a key muscle atrophy gene, MuRF1, is exclusive to heart and skeletal
muscle. Even in this context, MuRF1 is induced to a greater degree and from a
much lower baseline in skeletal muscle versus the heart, suggesting differential set
points in MuRF1 gene activity in its two major sites of expression and action that
are not well understood.
GR Interactions with Cytoplasmic Signal Transduction Proteins
While the previous section focused on the “classical” actions of the GR in skeletal
muscle, there is growing evidence that the GR can also mediate the effects of glu-
cocorticoids via specific protein-protein interactions with signal transduction
machinery in the cytoplasm. For example, the GR can directly interact with and
inhibit the activity of mitogen activated kinases (MAPK), IkB kinase, and PI3
kinase [143]. While most of these interactions have been studied in the context of
immune system modulation, the best studied with respect to skeletal muscle mass is
the direct physical interaction between GR and the p85 regulatory subunit of PI3
kinase. GR/p85 binding inhibits the p110 catalytic subunit interaction with IRS-1,
thus contributing to subsequent downstream Akt inhibition, FOXO activation, and
transcriptional regulation of atrophy-associated genes, among other consequences
[144]. Elevated growth factor exposure, including insulin and IGF-1, can override
the inhibitory PI3K/GR interaction; thus, atrophy stimulated by physiological glu-
cocorticoid levels must be coupled with abrogated insulin signaling or decreased
insulin levels as is observed in diabetes mellitus or starvation [144]. The importance

of this aspect of glucocorticoid signaling in muscle will be discussed in more detail
below.
In turn, the GR itself is phosphorylated by a variety of kinases, some of which
have been implicated in the regulation of muscle mass and metabolism. Interestingly,
while on one hand activated GSK3β has been implicated as an important mediator
of glucocorticoid induced muscle atrophy [145], this kinase also directly phosphory-
lates the GR [146]. This results in increased activation of downstream targets such
as GILZ; however, the effect of GSK3β phosphorylation of the GR appears to be
highly target gene specific. Since these studies were conducted in nonmuscle cell
lines, it will be interesting to examine GSK3β/GR interactions in the context of skel-
etal muscle atrophy in that it could serve as a selective feed forward mechanism to
amply GR actions in the face of decreased insulin or other growth factor
stimulation.
Potential Non-GR Mediated Effects of Glucocorticoids

in Skeletal Muscle?
Despite considerable evidence for the bona fide GR in control of skeletal muscle
mass and metabolism, nongenomic, non-GR mediated effects of glucocorticoids
have been reported, particularly in the brain [147]. Rapid effects of the steroid
appear in these cases to be mediated by a membrane bound receptor distinct from
the GR itself [148]. While this response is still understudied relative to the classical,
nuclear receptor pathway, a few reports of very rapid effects of glucocorticoids have
appeared that support an alternative mode of glucocorticoid action in both cardiac
and skeletal muscle [149–151]. Since the pharmacological profile of putative mem-
brane receptors maybe distinct from the GR, it is noteworthy that particularly in
cultured myotubes, very high doses of synthetic glucocorticoids are often used in
order to observe the most robust effects on atrophy and associated gene expression
[152, 153]. The GR equilibrium dissociation constant (Kd) for dexamethasone is
very low (low nanomolar to subnanomolar range) yet concentrations of 100 μM are
often reported as being required to observe maximal responses in these models. The
necessity for very high doses of synthetic glucocorticoids in cultured myotubes has
not adequately been explained to date.
Prereceptor Control of Glucocorticoid Actions
While the GR is critical for the actions of glucocorticoids in skeletal muscle, prere-
ceptor control of access to high affinity ligands is another important regulatory
point. Cells that express 11β hydroxysteroid dehydrogenase type I (11β HSD I) are
able to convert the inactive cortisone and deoxycortisone to respectively to the

active forms cortisol and corticosterone (the major endogenous glucocorticoid in
man and rodent respectively), thereby increasing local glucocorticoid activity [154].
Interestingly, increased 11β-HSD type I activity in skeletal muscle has been linked
to development of insulin resistance in skeletal muscle and as well as decreases in
muscle mass and increases in atrophy associated gene expression [99]. Interestingly,
11β-HSD type I activity increases with age and obesity in multiple cell types,
including skeletal muscle.
Non Cell Autonomous Actions of GCs on Skeletal

Muscle Structure and Function
Most of the discussion thus far has focused on the direct actions of glucocorticoids
in myofibers and myoblasts or satellite cells. However, systemic glucocorticoid
exposure has important effects in several other cell types within skeletal muscle, in
particular connective tissue and blood vessels. Glucocorticoids such as methylpred-
nisolone acutely decrease blood flow to skeletal muscle, which correlates with
decreased glucose up-take and systemic hypertension observed with prolonged glu-
cocorticoid treatment [44, 155]. Transient up-regulation of transcripts related to
vasoconstriction have been observed (e.g. endothelin-1) [156], presumably originating
in associated blood vessel endothelial cells rather than in myofibers themselves.
Likewise, in several gene expression profiling experiments, collagen gene expres-
sion is suppressed in intact muscle consistent with prior biochemical studies,
presumably in fibroblasts supporting the muscle extracellular matrix.
Lastly, elevation of systemic glucocorticoids alters the levels of other circulating
hormones and various metabolites that may indirectly impact muscle mass and
function, in addition to metabolic adaptation and development of insulin resistance.
In addition to effects on IGF1 described above, elevated glucocorticoids affect the
levels of growth hormone, catecholamines, inflammatory cytokines, and other cir-
culating factors which all may affect skeletal muscle structure and function indi-
rectly [157–159]. In addition, central effects of glucocorticoids on food intake are
well known [160], but this response varies among species. Furthermore, glucocor-
ticoid excess causes hypogonadism, which could also indirectly affect muscle.
Therefore, to examine the direct actions of glucocorticoids in skeletal muscle fibers
themselves, it was important to develop experimental models with skeletal muscle
specific reduction of or absent glucocorticoid signaling.
Genetic Approaches for GR Functional Analysis

in Skeletal Muscle
As discussed in previous sections, the classical approach to establishing a role for

glucocorticoids has been to challenge adrenalectomized animals or animals treated
with the GR (and progesterone receptor) antagonist RU486. Since (a) the adrenal
ablation or systemic pharmacological approaches are not specific enough to assess
a direct role of the GR in muscle itself, and (b) germline knockout of the GR in
mice is lethal around birth, a series of tissue specific knockout models have been
developed in the intact organism (Table 7.2). These models allow for an analysis of
cell autonomy in glucocorticoid induced atrophy as well as unequivocal identifica-
tion of the GR as the bona fide target of excess glucocorticoids or elevated endog-
enous glucocorticoids in a range of atrophy models.
Three groups separately developed and published findings on muscle specific
GR knockout mice (MGRKO), and challenged these mice with mostly different
experimental models of atrophy. The first publication describing a MGRKO was
used to investigate muscle atrophy caused by streptozotocin induced diabetes
mellitus [144]. Mice with loxp sites flanking GR exon 2 were crossed with mice
bearing a transgene with the muscle specific creatine kinase promoter driving cre
recombinase expression in skeletal muscle (MCK-cre), although the promoter is
also active at some level in the heart as well. Diabetes induced muscle atrophy and
associated decreases in IRS-1 association with PI3 kinase was inhibited in the
MGRKO mice. Interestingly, expression of a mutant GR that lacks the ability to
translocate to the nucleus upon ligand binding in the MGRKO background at least
partially rescues the atrophy and MAFbx/atrogin-1 induction in the diabetes
model. Their data support a two step model for glucocorticoid induced atrophy in
response to induced diabetes and starvation [144]: namely, insulin signaling must
decrease or be otherwise impaired in order for the ligand activated GR to effec-
tively further reduce PI3 kinase activity via a cytoplasmic interaction and activate
Table 7.2 Genetic approaches to studying glucocorticoid receptor activity in skeletal muscle
atrophy
GR mutant Atrophy model References
GR dimerization Dexamethasone [134]
mutant mice
GR floxed exon Diabetes/starvation [144]
2 × MCK-cre mice
GR floxed exon Dexamethasone/starvation/denervation [161]
3 × MCK-cre mice
GR floxed exon Cachexia/lipopolysaccharide/chemotherapy [162, 163]
2 × MCK-cre mice
atrophy associated gene expression via reduced Akt activity and increased FOXO
activity in the nucleus.
In a separate set of studies, Watson et al. [161] created a MGRKO mouse model
(MGRe3KO) by targeting a floxed exon 3 allele of the GR, again by crossing those
mice with the MCK-cre transgene. These authors demonstrated that lack of the GR
in skeletal muscle essentially prevents excess synthetic glucocorticoid induced atro-
phy and up-regulation of several atrophy associated genes including MuRF1 and
MAFbx/atrogin-1. Up-regulation of atrophy-associated genes was blunted, but not
completely prevented, in a nutritional deprivation model in the MGRe3KO mice.
Atrophy induced in a very different model, denervation of the sciatic nerve, was not
different between wild type or MGRe3KO mice nor was atrophy induced gene
expression altered, demonstrating that the GR is not uniformly required for all
conditions resulting in atrophy and it may be restricted to catabolic states [161].
These studies followed from prior work with a dimerization mutant GR expressing
mouse line that surprisingly did not prevent excess glucocorticoid induced muscle
atrophy, despite the presence of a near perfect palindromic GRE in the MuRF1
promoter [134]. MuRF1 induction was inhibited to a degree in the treated mice, but
not enough to inhibit atrophy. Interestingly, dimerization mutant GRs alone fail to
activate the palindromic GRE in the proximal MuRF1 promoter in transfection
assays, but co-expression of FOXO1 re-establishes the strong synergistic activation
of the promoter that is greater than observed upon expression of either the GR or
FOXO1 on their own [134].
In a third mouse model, muscle specific disruption of the GR gene with the
same strategy as in Hu et al. [144], and muscle atrophy was induced in a model of
inflammation using systemic lipopolysaccharide (LPS) injection as well as a can-
cer cachexia model via implantation of Lewis lung carcinoma cells [162]. Muscle
atrophy and induction of several associated muscle atrophy genes including
MuRF1 and MAFbx/atrogin-1 were strongly abrogated in both challenges in exon
2 deleted MGRKO mice. The study also clarified the role of cytokines in inflam-
mation and cancer induced atrophy with strong evidence that rather than acting
directly on muscle itself, instead, increase circulating glucocorticoids that then in
turn induce gene expression and subsequent atrophy via activation of the skeletal
muscle GR [162]. Recently, this group also used the exon 2 deleted MGRKO
model to demonstrate the requirement for skeletal muscle GR in atrophy induced
by cytotoxic chemotherapeutic agents, again likely via increasing endogenous glu-
cocorticoids that in turn induce muscle atrophy via the GR [163]. Taken together,
the above studies clearly establish the critical central role of the skeletal muscle
GR in regulating atrophy in rodent models of diabetes, excess glucocorticoid treat-
ment, starvation, inflammation (as modeled by LPS exposure), cancer cachexia,
and chemotherapy.
In addition to genetically modified mouse models, other approaches to investi-
gate the role of the GR in skeletal muscle are emerging. Small interfering RNA
(siRNA) directed against the GR inhibited glucocorticoid regulated atrophy and
gene expression in cultured myotubes [164]. In an alternative intact vertebrate
model, morpholinos directed against the zebrafish GR significantly affected proper

muscle development [165], but atrophy has not been investigated in this system to
date. The recent emergence of rapid genome editing technologies using TAL
directed endonucleases (TALENs) or CRISPR/Cas9 [166], provides an even greater
opportunity to specifically alter the GR coding sequence and/or its downstream
effectors in a variety of cell based or in vivo models, including human stem cells or
rats for example.
Summary of Glucocorticoid Regulated Processes

in Skeletal Muscle Atrophy
The preponderance of evidence published to date is consistent with a central role for
the GR, when bound by natural or synthetic glucocorticoids, in (a) interaction with
cytoplasmic growth factor signal transduction machinery and (b) direct activation of
genes that result in an interacting gene expression network leading to imbalances in
protein synthesis and degradation pathways and ultimately muscle atrophy. A simplified
model for the mechanisms underlying acute and chronic effects of glucocorticoids in
initiating muscle atrophy is shown in Fig. 7.4. Under normal circumstances,
Fig. 7.4 Schematic for the mechanism underlying glucocorticoid induced skeletal muscle atro-
phy. More details are found in the text. Red, steps or proteins whose expression and/or activity are
inhibited by glucocorticoids; Blue, steps or proteins whose expression or activity are induced by
glucocorticoids. GR glucocorticoid receptor, GRE glucocorticoid response element, FOXO fork-
head class O transcription factor, FBE forkhead transcription factors, PIK3r1 phosphatidylinositol
3-phosphate kinase regulatory subunit 1 (PI3K p85), p110 PI3K catalytic subunit
adequate insulin and growth factor activity keeps the pro-catabolic actions of nor-
mally fluctuating endogenous glucocorticoids in check via PI3 kinase, Akt, and
mTOR activities. However, in cases of compromised insulin/IGF-1 levels or signal
transduction (diabetes, starvation, inflammation), elevated endogenous glucocorti-
coids can acutely and directly interact with and inhibit PI3 kinase activity, decreas-
ing downstream Akt activity and activating FOXO transcription factors that enter the
nucleus and activate their pro-atrophy associated target genes. Excess synthetic glu-
cocorticoid exposure, as the result of anti-inflammatory steroid use, may also over-
come the brakes normally in place via insulin or IGF-1. The endogenous or synthetic
ligand activated GR also translocates to the nucleus and directly activates atrophy
associated genes via GREs; some of these targets are regulated in close collaboration
with FOXO1 like MuRF1, and others (perhaps) more independently such as the
regulatory subunit of PI3 kinase (p85), FOXOs themselves, and transcription fac-
tors or other effector genes like KLF15 and REDD1. Increased PI3 kinase p85 and
FOXO expression with continued ligand occupation of the GR may then “feed-
forward” to further inhibit Akt and downstream targets (including preventing inhibi-
tion of accumulating FOXOs), leading to an expanding network of downstream
events and accelerated muscle loss seen in chronic glucocorticoid exposure.
References
1. Pereira RM, Freire de Carvalho J. Glucocorticoid-induced myopathy. Joint Bone Spine.

2011;78:41–4.
2. Overman RA, Yeh JY, Deal CL. Prevalence of oral glucocorticoid usage in the United States:
a general population perspective. Arthritis Care Res (Hoboken). 2013;65:294–8.
3. Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiol Rev.
2011;91:1447–531.
4. Goldberg AL. Protein turnover in skeletal muscle. II. Effects of denervation and cortisone on
protein catabolism in skeletal muscle. J Biol Chem. 1969;244:3223–9.
5. Kelly FJ, Goldspink DF. The differing responses of four muscle types to dexamethasone
treatment in the rat. Biochem J. 1982;208:147–51.
6. Gardiner PF, Montanaro G, Simpson DR, Edgerton VR. Effects of glucocorticoid treatment
and food restriction on rat hindlimb muscles. Am J Physiol. 1980;238:E124–30.
7. Roy RR, Gardiner PF, Simpson DR, Edgerton VR. Glucocorticoid-induced atrophy in differ-
ent fibre types of selected rat jaw and hind-limb muscles. Arch Oral Biol. 1983;28:639–43.
8. Dekhuijzen PN, Gayan-Ramirez G, Bisschop A, De Bock V, Dom R, Decramer
M. Corticosteroid treatment and nutritional deprivation cause a different pattern of atrophy in
rat diaphragm. J Appl Physiol. 1995;78:629–37.
9. Gardiner PF, Botterman BR, Eldred E, Simpson DR, Edgerton VR. Metabolic and contractile
changes in fast and slow muscles of the cat after glucocorticoid-induced atrophy. Exp Neurol.
1978;62:241–55.
10. Bullock GR, Carter EE, Elliott P, Peters RF, Simpson P, White AM. Relative changes in the
function of muscle ribosomes and mitochondria during the early phase of steroid-induced
catabolism. Biochem J. 1972;127:881–92.
11. Anagnos A, Ruff RL, Kaminski HJ. Endocrine neuromyopathies. Neurol Clin. 1997;15:
673–96.
12. Baehr LM, Furlow JD, Bodine SC. Muscle sparing in muscle RING finger 1 null mice:
response to synthetic glucocorticoids. J Physiol. 2011;589:4759–76.
13. Zhao W, Pan J, Zhao Z, Wu Y, Bauman WA, Cardozo CP. Testosterone protects against
dexamethasone-induced muscle atrophy, protein degradation and MAFbx upregulation. J
Steroid Biochem Mol Biol. 2008;110:125–9.
14. Ragnarsson O, Burt MG, Ho KK, Johannsson G. Effect of short-term GH and testosterone
administration on body composition and glucose homoeostasis in men receiving chronic glu-
cocorticoid therapy. Eur J Endocrinol. 2013;168:243–51.
15. Capaccio JA, Kurowski TT, Czerwinski SM, Chatterton Jr RT, Hickson RC. Testosterone
fails to prevent skeletal muscle atrophy from glucocorticoids. J Appl Physiol. 1987;63:
328–34.
16. Batchelor TT, Taylor LP, Thaler HT, Posner JB, DeAngelis LM. Steroid myopathy in cancer
patients. Neurology. 1997;48:1234–8.
17. Levin OS, Polunina AG, Demyanova MA, Isaev FV. Steroid myopathy in patients with
chronic respiratory diseases. J Neurol Sci. 2014;338:96–101.
18. Decramer M, de Bock V, Dom R. Functional and histologic picture of steroid-induced myop-
athy in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1996;153:
1958–64.
19. Hatakenaka M, Soeda H, Okafuji T, et al. Steroid myopathy: evaluation of fiber atrophy with
T2 relaxation time—rabbit and human study. Radiology. 2006;238:650–7.
20. Khaleeli AA, Edwards RH, Gohil K, et al. Corticosteroid myopathy: a clinical and pathologi-
cal study. Clin Endocrinol (Oxf). 1983;18:155–66.
21. Horber FF, Scheidegger JR, Grunig BE, Frey FJ. Thigh muscle mass and function in patients
treated with glucocorticoids. Eur J Clin Invest. 1985;15:302–7.
22. Decramer M, Stas KJ. Corticosteroid-induced myopathy involving respiratory muscles in
patients with chronic obstructive pulmonary disease or asthma. Am Rev Respir Dis.
1992;146:800–2.
23. Danneskiold-Samsoe B, Grimby G. Isokinetic and isometric muscle strength in patients with
rheumatoid arthritis. The relationship to clinical parameters and the influence of corticoste-
roid. Clin Rheumatol. 1986;5:459–67.
24. Rossignol B, Gueret G, Pennec JP, et al. Effects of chronic sepsis on contractile properties of
fast twitch muscle in an experimental model of critical illness neuromyopathy in the rat. Crit
Care Med. 2008;36:1855–63.
25. Alamdari N, Toraldo G, Aversa Z, et al. Loss of muscle strength during sepsis is in part regu-
lated by glucocorticoids and is associated with reduced muscle fiber stiffness. Am J Physiol
Regul Integr Comp Physiol. 2012;303:R1090–9.
26. Supinski GS, Wang L, Song XH, Moylan JS, Callahan LA. Muscle-specific calpastatin over-
expression prevents diaphragm weakness in cecal ligation puncture-induced sepsis. J Appl
Physiol. 2014;117:921–9.
27. Robinson AJ, Clamann HP. Effects of glucocorticoids on motor units in cat hindlimb mus-
cles. Muscle Nerve. 1988;11:703–13.
28. Gardiner PF, Edgerton VR. Contractile responses of rat fast-twitch and slow-twitch muscles
to glucocorticoid treatment. Muscle Nerve. 1979;2:274–81.
29. Ruff RL, Martyn D, Gordon AM. Glucocorticoid-induced atrophy is not due to impaired
excitability of rat muscle. Am J Physiol. 1982;243:E512–21.
30. Van Balkom RH, Zhan WZ, Prakash YS, Dekhuijzen PN, Sieck GC. Corticosteroid effects on
isotonic contractile properties of rat diaphragm muscle. J Appl Physiol. 1997;83:1062–7.
31. Manzur AY, Kuntzer T, Pike M, Swan A. Glucocorticoid corticosteroids for Duchenne mus-
cular dystrophy. Cochrane Database Syst Rev 2008;CD003725.
32. Hanaoka BY, Peterson CA, Horbinski C, Crofford LJ. Implications of glucocorticoid therapy
in idiopathic inflammatory myopathies. Nat Rev Rheumatol. 2012;8:448–57.
33. Sali A, Guerron AD, Gordish-Dressman H, et al. Glucocorticoid-treated mice are an inap-
propriate positive control for long-term preclinical studies in the mdx mouse. PLoS One.
2012;7:e34204.
34. Janssen PM, Murray JD, Schill KE, et al. Prednisolone attenuates improvement of cardiac
and skeletal contractile function and histopathology by lisinopril and spironolactone in the
mdx mouse model of Duchenne muscular dystrophy. PLoS One. 2014;9:e88360.
35. Baltgalvis KA, Call JA, Nikas JB, Lowe DA. Effects of prednisolone on skeletal muscle
contractility in mdx mice. Muscle Nerve. 2009;40:443–54.
36. Fisher I, Abraham D, Bouri K, Hoffman EP, Muntoni F, Morgan J. Prednisolone-induced
changes in dystrophic skeletal muscle. FASEB J. 2005;19:834–6.
37. Hershey JW, Sonenberg N, Mathews MB. Principles of translational control: an overview.
Cold Spring Harb Perspect Biol. 2012;4. pii: a011528.
38. Gordon BS, Kelleher AR, Kimball SR. Regulation of muscle protein synthesis and the effects
of catabolic states. Int J Biochem Cell Biol. 2013;45:2147–57.
39. Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translational control. Nat Rev
Mol Cell Biol. 2009;10:307–18.
40. Marcotte GR, West DW, Baar K. The molecular basis for load-induced skeletal muscle
hypertrophy. Calcif Tissue Int. 2015;96(3):196–210.
41. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes
and ageing. Nat Rev Mol Cell Biol. 2011;12:21–35.
42. Millward DJ, Garlick PJ, Nnanyelugo DO, Waterlow JC. The relative importance of muscle
protein synthesis and breakdown in the regulation of muscle mass. Biochem J. 1976;156:
185–8.
43. Rannels SR, Rannels DE, Pegg AE, Jefferson LS. Glucocorticoid effects on peptide-chain
initiation in skeletal muscle and heart. Am J Physiol. 1978;235:E134–9.
44. Short KR, Nygren J, Bigelow ML, Nair KS. Effect of short-term prednisone use on blood
flow, muscle protein metabolism, and function. J Clin Endocrinol Metab. 2004;89:
6198–207.
45. Liu Z, Li G, Kimball SR, Jahn LA, Barrett EJ. Glucocorticoids modulate amino acid-induced
translation initiation in human skeletal muscle. Am J Physiol Endocrinol Metab. 2004;287:
E275–81.
46. Mayer M, Shafrir E, Kaiser N, Milholland RJ, Rosen F. Interaction of glucocorticoid hor-
mones with rat skeletal muscle: catabolic effects and hormone binding. Metabolism. 1976;
25:157–67.
47. Kostyo JL, Redmond AF. Role of protein synthesis in the inhibitory action of adrenal steroid
hormones on amino acid transport by muscle. Endocrinology. 1966;79:531–40.
48. Young VR, Chen SC, Macdonald J. The sedimentation of rat skeletal-muscle ribosomes.
Effect of hydrocortisone, insulin and diet. Biochem J. 1968;106:913–9.
49. Peters RF, Richardson MC, Small M, White AM. Some biochemical effects of triamcinolone
acetonide on rat liver and muscle. Biochem J. 1970;116:349–55.
50. Rannels DE, Rannels SR, Li JB, Pegg AE, Morgan HE, Jefferson LS. Effects of glucocorti-
coids on peptide chain initiation in heart and skeletal muscle. Adv Myocardiol. 1980;1:
493–501.
51. Shah OJ, Kimball SR, Jefferson LS. Acute attenuation of translation initiation and protein
synthesis by glucocorticoids in skeletal muscle. Am J Physiol Endocrinol Metab. 2000;278:
E76–82.
52. Shah OJ, Kimball SR, Jefferson LS. Glucocorticoids abate p70(S6k) and eIF4E function in
L6 skeletal myoblasts. Am J Physiol Endocrinol Metab. 2000;279:E74–82.
53. Tomas FM, Munro HN, Young VR. Effect of glucocorticoid administration on the rate of
muscle protein breakdown in vivo in rats, as measured by urinary excretion of N tau-
methylhistidine. Biochem J. 1979;178:139–46.
54. Nagasawa T, Funabiki R. Quantitative determination of urinary N-tau-methylhistidine output
as an index of myofibrillar protein degradation. J Biochem. 1981;89:1155–61.
55. Millward DJ, Bates PC. 3-Methylhistidine turnover in the whole body, and the contribution
of skeletal muscle and intestine to urinary 3-methylhistidine excretion in the adult rat.
Biochem J. 1983;214:607–15.
56. Mitch WE, Clark AS, May RC. Relationships between protein degradation and glucose
metabolism in skeletal muscle. Prog Clin Biol Res. 1985;180:623–5.
57. Mitch WE, Goldberg AL. Mechanisms of muscle wasting. The role of the ubiquitin-
proteasome pathway. N Engl J Med. 1996;335:1897–905.
58. Wing SS, Goldberg AL. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic
system in skeletal muscle during fasting. Am J Physiol. 1993;264:E668–76.
59. Mitch WE, Medina R, Grieber S, et al. Metabolic acidosis stimulates muscle protein degrada-
tion by activating the adenosine triphosphate-dependent pathway involving ubiquitin and
proteasomes. J Clin Invest. 1994;93:2127–33.
60. Price SR, Bailey JL, England BK. Necessary but not sufficient: the role of glucocorticoids in
the acidosis-induced increase in levels of mRNAs encoding proteins of the ATP-dependent
proteolytic pathway in rat muscle. Miner Electrolyte Metab. 1996;22:72–5.
61. Price SR, Mitch WE. Mechanisms stimulating protein degradation to cause muscle atrophy.
Curr Opin Clin Nutr Metab Care. 1998;1:79–83.
62. Medina R, Wing SS, Haas A, Goldberg AL. Activation of the ubiquitin-ATP-dependent pro-
teolytic system in skeletal muscle during fasting and denervation atrophy. Biomed Biochim
Acta. 1991;50:347–56.
63. Lecker SH, Solomon V, Mitch WE, Goldberg AL. Muscle protein breakdown and the critical
role of the ubiquitin-proteasome pathway in normal and disease states. J Nutr. 1999;129:
227S–37S.
64. Chau V, Tobias JW, Bachmair A, et al. A multiubiquitin chain is confined to specific lysine in
a targeted short-lived protein. Science. 1989;243:1576–83.
65. D’Azzo A, Bongiovanni A, Nastasi T. E3 ubiquitin ligases as regulators of membrane protein
trafficking and degradation. Traffic. 2005;6:429–41.
66. Tisdale MJ. The ubiquitin-proteasome pathway as a therapeutic target for muscle wasting.
J Support Oncol. 2005;3:209–17.
67. Xie Y. Structure, assembly and homeostatic regulation of the 26S proteasome. J Mol Cell
Biol. 2010;2:308–17.
68. Lecker SH, Goldberg AL, Mitch WE. Protein degradation by the ubiquitin-proteasome path-
way in normal and disease states. J Am Soc Nephrol. 2006;17:1807–19.
69. Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem. 2009;78:
399–434.
70. Metzger MB, Hristova VA, Weissman AM. HECT and RING finger families of E3 ubiquitin
ligases at a glance. J Cell Sci. 2012;125:531–7.
71. Auclair D, Garrel DR, Chaouki Zerouala A, Ferland LH. Activation of the ubiquitin pathway
in rat skeletal muscle by catabolic doses of glucocorticoids. Am J Physiol. 1997;272:
C1007–16.
72. Combaret L, Taillandier D, Dardevet D, et al. Glucocorticoids regulate mRNA levels for
subunits of the 19S regulatory complex of the 26S proteasome in fast-twitch skeletal mus-
cles. Biochem J. 2004;378:239–46.
73. Britto FA, Begue G, Rossano B, et al. REDD1 deletion prevents dexamethasone-induced
skeletal muscle atrophy. Am J Physiol Endocrinol Metab. 2014;307:E983–93.
74. Price SR, Bailey JL, Wang X, et al. Muscle wasting in insulinopenic rats results from activa-
tion of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism
including gene transcription. J Clin Invest. 1996;98:1703–8.
75. Tawa Jr NE, Odessey R, Goldberg AL. Inhibitors of the proteasome reduce the accelerated
proteolysis in atrophying rat skeletal muscles. J Clin Invest. 1997;100:197–203.
76. Wing SS, Haas AL, Goldberg AL. Increase in ubiquitin-protein conjugates concomitant with
the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation.
Biochem J. 1995;307(Pt 3):639–45.
77. Tiao G, Hobler S, Wang JJ, et al. Sepsis is associated with increased mRNAs of the ubiquitin-
proteasome proteolytic pathway in human skeletal muscle. J Clin Invest. 1997;99:163–8.
78. Voisin L, Breuille D, Combaret L, et al. Muscle wasting in a rat model of long-lasting sepsis
results from the activation of lysosomal, Ca2+-activated, and ubiquitin-proteasome proteo-
lytic pathways. J Clin Invest. 1996;97:1610–7.
79. Tiao G, Fagan JM, Samuels N, et al. Sepsis stimulates nonlysosomal, energy-dependent pro-
teolysis and increases ubiquitin mRNA levels in rat skeletal muscle. J Clin Invest. 1994;94:
2255–64.
80. Baracos VE, DeVivo C, Hoyle DH, Goldberg AL. Activation of the ATP-ubiquitin-
proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma. Am J Physiol.
1 9 9 5 ; 2 6 8 :
E996–1006.
81. Bodine SC, Baehr LM. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and
MAFbx/atrogin-1. Am J Physiol Endocrinol Metab. 2014;307:E469–84.
82. Centner T, Yano J, Kimura E, et al. Identification of muscle specific ring finger proteins as
potential regulators of the titin kinase domain. J Mol Biol. 2001;306:717–26.
83. Gomes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL. Atrogin-1, a muscle-specific
F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci U S A. 2001;
98:14440–5.
84. Bodine SC, Latres E, Baumhueter S, et al. Identification of ubiquitin ligases required for
skeletal muscle atrophy. Science. 2001;294:1704–8.
85. Files DC, D’Alessio FR, Johnston LF, et al. A critical role for muscle ring finger-1 in acute
lung injury-associated skeletal muscle wasting. Am J Respir Crit Care Med. 2012;185:
825–34.
86. Frost RA, Lang CH. Multifaceted role of insulin-like growth factors and mammalian target
of rapamycin in skeletal muscle. Endocrinol Metab Clin North Am. 2012;41:297–322.
87. Gayan-Ramirez G, Vanderhoydonc F, Verhoeven G, Decramer M. Acute treatment with cor-
ticosteroids decreases IGF-1 and IGF-2 expression in the rat diaphragm and gastrocnemius.
Am J Respir Crit Care Med. 1999;159:283–9.
88. Inder WJ, Jang C, Obeyesekere VR, Alford FP. Dexamethasone administration inhibits skel-
etal muscle expression of the androgen receptor and IGF-1–implications for steroid-induced
myopathy. Clin Endocrinol (Oxf). 2010;73:126–32.
89. Schakman O, Gilson H, de Coninck V, et al. Insulin-like growth factor-I gene transfer by
electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats. Endocrinology.
2005;146:1789–97.
90. Schakman O, Kalista S, Bertrand L, et al. Role of Akt/GSK-3beta/beta-catenin transduction
pathway in the muscle anti-atrophy action of insulin-like growth factor-I in glucocorticoid-
treated rats. Endocrinology. 2008;149:3900–8.
91. Sacheck JM, Ohtsuka A, McLary SC, Goldberg AL. IGF-1 stimulates muscle growth by sup-
pressing protein breakdown and expression of atrophy-related ubiquitin-ligases, atrogin-1
and MuRF1. Am J Physiol Endocrinol Metab. 2004;287(4):E591–601.
92. Stitt TN, Drujan D, Clarke BA, et al. The IGF-1/PI3K/Akt pathway prevents expression of
muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol
Cell. 2004;14:395–403.
93. Fournier M, Huang ZS, Li H, Da X, Cercek B, Lewis MI. Insulin-like growth factor I pre-
vents corticosteroid-induced diaphragm muscle atrophy in emphysematous hamsters. Am J
Physiol Regul Integr Comp Physiol. 2003;285:R34–43.
94. Chrysis D, Underwood LE. Regulation of components of the ubiquitin system by insulin-like
growth factor I and growth hormone in skeletal muscle of rats made catabolic with dexa-
methasone. Endocrinology. 1999;140:5635–41.
95. Kanda F, Takatani K, Okuda S, Matsushita T, Chihara K. Preventive effects of insulin like
growth factor-I on steroid-induced muscle atrophy. Muscle Nerve. 1999;22:213–7.
96. Schakman O, Kalista S, Barbe C, Loumaye A, Thissen JP. Glucocorticoid-induced skeletal
muscle atrophy. Int J Biochem Cell Biol. 2013;45:2163–72.
97. Armstrong DD, Esser KA. Wnt/beta-catenin signaling activates growth-control genes during
overload-induced skeletal muscle hypertrophy. Am J Physiol Cell Physiol. 2005;289:
C853–9.
98. Nakao R, Hirasaka K, Goto J, et al. Ubiquitin ligase Cbl-b is a negative regulator for insulin-
like growth factor 1 signaling during muscle atrophy caused by unloading. Mol Cell Biol.
2009;29:4798–811.
99. Morgan SA, Sherlock M, Gathercole LL, et al. 11beta-hydroxysteroid dehydrogenase type 1
regulates glucocorticoid-induced insulin resistance in skeletal muscle. Diabetes. 2009;58:
2506–15.
100. Zheng B, Ohkawa S, Li H, Roberts-Wilson TK, Price SR. FOXO3a mediates signaling cross-
talk that coordinates ubiquitin and atrogin-1/MAFbx expression during glucocorticoid-
induced skeletal muscle atrophy. FASEB J. 2010;24:2660–9.
101. Koh A, Lee MN, Yang YR, et al. C1-Ten is a protein tyrosine phosphatase of insulin receptor
substrate 1 (IRS-1), regulating IRS-1 stability and muscle atrophy. Mol Cell Biol. 2013;33:
1608–20.
102. Kuo T, Lew MJ, Mayba O, Harris CA, Speed TP, Wang JC. Genome-wide analysis of gluco-
corticoid receptor-binding sites in myotubes identifies gene networks modulating insulin sig-
naling. Proc Natl Acad Sci U S A. 2012;109:11160–5.
103. Furlow JD, Watson ML, Waddell DS, et al. Altered gene expression patterns in muscle ring
finger 1 null mice during denervation- and dexamethasone-induced muscle atrophy. Physiol
Genomics. 2013;45:1168–85.
104. Wang H, Kubica N, Ellisen LW, Jefferson LS, Kimball SR. Dexamethasone represses signal-
ing through the mammalian target of rapamycin in muscle cells by enhancing expression of
REDD1. J Biol Chem. 2006;281:39128–34.
105. Kumari R, Willing LB, Jefferson LS, Simpson IA, Kimball SR. REDD1 (regulated in devel-
opment and DNA damage response 1) expression in skeletal muscle as a surrogate biomarker
of the efficiency of glucocorticoid receptor blockade. Biochem Biophys Res Commun.
2011;412:644–7.
106. Dennis MD, Coleman CS, Berg A, Jefferson LS, Kimball SR. REDD1 enhances protein
phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling. Sci
Signal. 2014;7:ra68.
107. Kadmiel M, Cidlowski JA. Glucocorticoid receptor signaling in health and disease. Trends
Pharmacol Sci. 2013;34:518–30.
108. Duma D, Jewell CM, Cidlowski JA. Multiple glucocorticoid receptor isoforms and mecha-
nisms of post-translational modification. J Steroid Biochem Mol Biol. 2006;102:11–21.
109. Schaaf MJ, Champagne D, van Laanen IH, et al. Discovery of a functional glucocorticoid
receptor beta-isoform in zebrafish. Endocrinology. 2008;149:1591–9.
110. Shimizu N, Yoshikawa N, Ito N, et al. Crosstalk between glucocorticoid receptor and nutri-
tional sensor mTOR in skeletal muscle. Cell Metab. 2011;13:170–82.
111. Oakley RH, Cidlowski JA. Homologous down regulation of the glucocorticoid receptor: the
molecular machinery. Crit Rev Eukaryot Gene Expr. 1993;3:63–88.
112. Yao Z, DuBois DC, Almon RR, Jusko WJ. Modeling circadian rhythms of glucocorticoid
receptor and glutamine synthetase expression in rat skeletal muscle. Pharm Res. 2006;23:
670–9.
113. Witchel SF, DeFranco DB. Mechanisms of disease: regulation of glucocorticoid and receptor
levels–impact on the metabolic syndrome. Nat Clin Pract Endocrinol Metab. 2006;2:
621–31.
114. Aubry EM, Odermatt A. Retinoic acid reduces glucocorticoid sensitivity in C2C12 myotubes
by decreasing 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor
activities. Endocrinology. 2009;150:2700–8.
115. Wang SC, Myers S, Dooms C, Capon R, Muscat GE. An ERRbeta/gamma agonist modulates
GRalpha expression, and glucocorticoid responsive gene expression in skeletal muscle cells.
Mol Cell Endocrinol. 2010;315:146–52.
116. Almon RR, DuBois DC, Yao Z, Hoffman EP, Ghimbovschi S, Jusko WJ. Microarray analysis
of the temporal response of skeletal muscle to methylprednisolone: comparative analysis of
two dosing regimens. Physiol Genomics. 2007;30:282–99.
117. Lecker SH, Jagoe RT, Gilbert A, et al. Multiple types of skeletal muscle atrophy involve a
common program of changes in gene expression. FASEB J. 2004;18:39–51.
118. Carraro L, Ferraresso S, Cardazzo B, et al. Expression profiling of skeletal muscle in young
bulls treated with steroidal growth promoters. Physiol Genomics. 2009;38:138–48.
119. Kukreti H, Amuthavalli K, Harikumar A, et al. Muscle-specific microRNA1 (miR1) targets
heat shock protein 70 (HSP70) during dexamethasone-mediated atrophy. J Biol Chem.
2013;288:6663–78.
120. Busillo JM, Cidlowski JA. The five Rs of glucocorticoid action during inflammation: ready,
reinforce, repress, resolve, and restore. Trends Endocrinol Metab. 2013;24:109–19.
121. Yang H, Menconi MJ, Wei W, Petkova V, Hasselgren PO. Dexamethasone upregulates the
expression of the nuclear cofactor p300 and its interaction with C/EBPbeta in cultured myo-
tubes. J Cell Biochem. 2005;94:1058–67.
122. Tobimatsu K, Noguchi T, Hosooka T, et al. Overexpression of the transcriptional coregulator
Cited2 protects against glucocorticoid-induced atrophy of C2C12 myotubes. Biochem
Biophys Res Commun. 2009;378:399–403.
123. Yang H, Wei W, Menconi M, Hasselgren PO. Dexamethasone-induced protein degradation in
cultured myotubes is p300/HAT dependent. Am J Physiol Regul Integr Comp Physiol.
2007;292:R337-4.
124. Alamdari N, Aversa Z, Castillero E, Hasselgren PO. Acetylation and deacetylation—novel
factors in muscle wasting. Metabolism. 2013;62:1–11.
125. Proserpio V, Fittipaldi R, Ryall JG, Sartorelli V, Caretti G. The methyltransferase SMYD3
mediates the recruitment of transcriptional cofactors at the myostatin and c-Met genes and
regulates skeletal muscle atrophy. Genes Dev. 2013;27:1299–312.
126. Kim H, Heo K, Kim JH, Kim K, Choi J, An W. Requirement of histone methyltransferase
SMYD3 for estrogen receptor-mediated transcription. J Biol Chem. 2009;284:19867–77.
127. Furuyama T, Kitayama K, Yamashita H, Mori N. Forkhead transcription factor FOXO1
(FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy
deprivation. Biochem J. 2003;375:365–71.
128. Cho JE, Fournier M, Da X, Lewis MI. Time course expression of Foxo transcription factors
in skeletal muscle following corticosteroid administration. J Appl Physiol. 2010;108:
137–45.
129. Sun H, Gong Y, Qiu J, Chen Y, Ding F, Zhao Q. TRAF6 inhibition rescues dexamethasone-
induced muscle atrophy. Int J Mol Sci. 2014;15:11126–41.
130. Webb AE, Brunet A. FOXO transcription factors: key regulators of cellular quality control.
Trends Biochem Sci. 2014;39:159–69.
131. Gross DN, Wan M, Birnbaum MJ. The role of FOXO in the regulation of metabolism. Curr
Diab Rep. 2009;9:208–14.
132. Zhao J, Brault JJ, Schild A, et al. FoxO3 coordinately activates protein degradation by the
autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab.
2007;6:472–83.
133. Mammucari C, Milan G, Romanello V, et al. FoxO3 controls autophagy in skeletal muscle
in vivo. Cell Metab. 2007;6:458–71.
134. Waddell DS, Baehr LM, van den Brandt J, et al. The glucocorticoid receptor and FOXO1
synergistically activate the skeletal muscle atrophy-associated MuRF1 gene. Am J Physiol
Endocrinol Metab. 2008;295:E785–97.
135. Reed SA, Sandesara PB, Senf SM, Judge AR. Inhibition of FoxO transcriptional activity
prevents muscle fiber atrophy during cachexia and induces hypertrophy. FASEB J. 2012;26:
987–1000.
136. Senf SM, Sandesara PB, Reed SA, Judge AR. p300 Acetyltransferase activity differentially
regulates the localization and activity of the FOXO homologues in skeletal muscle. Am J
Physiol Cell Physiol. 2011;300:C1490–501.
137. Beharry AW, Sandesara PB, Roberts BM, Ferreira LF, Senf SM, Judge AR. HDAC1 activates
FoxO and is both sufficient and required for skeletal muscle atrophy. J Cell Sci. 2014;127:
1441–53.
138. Yang H, Mammen J, Wei W, et al. Expression and activity of C/EBPbeta and delta are upreg-
ulated by dexamethasone in skeletal muscle. J Cell Physiol. 2005;204:219–26.
139. Gonnella P, Alamdari N, Tizio S, Aversa Z, Petkova V, Hasselgren PO. C/EBPbeta regulates
dexamethasone-induced muscle cell atrophy and expression of atrogin-1 and MuRF1. J Cell
Biochem. 2011;112:1737–48.
140. Bruscoli S, Donato V, Velardi E, et al. Glucocorticoid-induced leucine zipper (GILZ) and
long GILZ inhibit myogenic differentiation and mediate anti-myogenic effects of glucocorti-
coids. J Biol Chem. 2010;285:10385–96.
141. Ayroldi E, Riccardi C. Glucocorticoid-induced leucine zipper (GILZ): a new important medi-
ator of glucocorticoid action. FASEB J. 2009;23:3649–58.
142. Kaestner KH. The FoxA factors in organogenesis and differentiation. Curr Opin Genet Dev.
2010;20:527–32.
143. Revollo JR, Cidlowski JA. Mechanisms generating diversity in glucocorticoid receptor sig-
naling. Ann N Y Acad Sci. 2009;1179:167–78.
144. Hu Z, Wang H, Lee IH, Du J, Mitch WE. Endogenous glucocorticoids and impaired insulin
signaling are both required to stimulate muscle wasting under pathophysiological conditions
in mice. J Clin Invest. 2009;119:3059–69.
145. Fang CH, Li BG, James JH, et al. Protein breakdown in muscle from burned rats is blocked
by insulin-like growth factor i and glycogen synthase kinase-3beta inhibitors. Endocrinology.
2005;146:3141–9.
146. Rubio-Patino C, Palmeri CM, Perez-Perarnau A, et al. Glycogen synthase kinase-3beta is
involved in ligand-dependent activation of transcription and cellular localization of the glu-
cocorticoid receptor. Mol Endocrinol. 2012;26:1508–20.
147. Sarabdjitsingh RA, Joels M, de Kloet ER. Glucocorticoid pulsatility and rapid corticosteroid
actions in the central stress response. Physiol Behav. 2012;106:73–80.
148. Tasker JG, Di S, Malcher-Lopes R. Minireview: rapid glucocorticoid signaling via membrane-
associated receptors. Endocrinology. 2006;147:5549–56.
149. Perez MH, Cormack J, Mallinson D, Mutungi G. A membrane glucocorticoid receptor medi-
ates the rapid/non-genomic actions of glucocorticoids in mammalian skeletal muscle fibres.
J Physiol. 2013;591:5171–85.
150. Kewalramani G, Puthanveetil P, Kim MS, et al. Acute dexamethasone-induced increase in
cardiac lipoprotein lipase requires activation of both Akt and stress kinases. Am J Physiol
151. Lee SR, Kim HK, Youm JB, et al. Non-genomic effect of glucocorticoids on cardiovascular
system. Pflugers Arch. 2012;464:549–59.
152. Stitt TN, Drujan D, Clarke BA, et al. The IGF-1/PI3K/Akt pathway prevents expression of
muscle atrophy-induced ubiquitin liganses by inhibiting FOXO transcription factors. Mol
Cell. 2004;14:1–14.
153. Latres E, Amini AR, Amini AA, et al. Insulin-like growth factor-1 (IGF-1) inversely regu-
lates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of
rapamycin (PI3K/Akt/mTOR) pathway. J Biol Chem. 2005;280:2737–44.
154. Chapman K, Holmes M, Seckl J. 11beta-hydroxysteroid dehydrogenases: intracellular gate-
keepers of tissue glucocorticoid action. Physiol Rev. 2013;93:1139–206.
155. Short KR, Bigelow ML, Nair KS. Short-term prednisone use antagonizes insulin's anabolic
effect on muscle protein and glucose metabolism in young healthy people. Am J Physiol
156. Almon RR, Dubois DC, Jin JY, Jusko WJ. Temporal profiling of the transcriptional basis for
the development of corticosteroid-induced insulin resistance in rat muscle. J Endocrinol.
2005;184:219–32.
157. Patel R, Bookout AL, Magomedova L, et al. Glucocorticoids regulate the metabolic hormone
FGF21 in a feed-forward loop. Mol Endocrinol. 2015;29(2):213–23.
158. Mazziotti G, Giustina A. Glucocorticoids and the regulation of growth hormone secretion.
Nat Rev Endocrinol. 2013;9:265–76.
159. Wurtman RJ. Stress and the adrenocortical control of epinephrine synthesis. Metabolism.
2002;51:11–4.
160. la Fleur SE. The effects of glucocorticoids on feeding behavior in rats. Physiol Behav.
2006;89:110–4.
161. Watson ML, Baehr LM, Reichardt HM, Tuckermann JP, Bodine SC, Furlow JD. A cell-
autonomous role for the glucocorticoid receptor in skeletal muscle atrophy induced by sys-
temic glucocorticoid exposure. Am J Physiol Endocrinol Metab. 2012;302:E1210–20.
162. Braun TP, Grossberg AJ, Krasnow SM, et al. Cancer- and endotoxin-induced cachexia require
intact glucocorticoid signaling in skeletal muscle. FASEB J. 2013;27:3572–82.
163. Braun TP, Szumowski M, Levasseur PR, et al. Muscle atrophy in response to cytotoxic che-
motherapy is dependent on intact glucocorticoid signaling in skeletal muscle. PLoS One.
2014;9:e106489.
164. Zhao W, Qin W, Pan J, Wu Y, Bauman WA, Cardozo C. Dependence of dexamethasone-
induced Akt/FOXO1 signaling, upregulation of MAFbx, and protein catabolism upon the
glucocorticoid receptor. Biochem Biophys Res Commun. 2009;378:668–72.
165. Nesan D, Kamkar M, Burrows J, Scott IC, Marsden M, Vijayan MM. Glucocorticoid receptor
signaling is essential for mesoderm formation and muscle development in zebrafish.
Endocrinology. 2012;153:1288–300.
166. Segal DJ, Meckler JF. Genome engineering at the dawn of the golden age. Annu Rev
Genomics Hum Genet. 2013;14:135–58.
167. Sandri M, Sandri C, Gilbert A, et al. Foxo transcription factors induce the atrophy-related
ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell. 2004;117:399–412.
168. Qin W, Pan J, Qin Y, Lee DN, Bauman WA, Cardozo C. Identification of functional glucocor-
ticoid response elements in the mouse FoxO1 promoter. Biochem Biophys Res Commun.
2014;450:979–83.
169. Ma K, Mallidis C, Bhasin S, et al. Glucocorticoid-induced skeletal muscle atrophy is associ-
ated with upregulation of myostatin gene expression. Am J Physiol Endocrinol Metab.
2003;285:E363–71.
170. Qin J, Du R, Yang YQ, et al. Dexamethasone-induced skeletal muscle atrophy was associated
with upregulation of myostatin promoter activity. Res Vet Sci. 2013;94:84–9.
Part III
Specific Effects of Glucocorticoids
on Tissues
Chapter 8
Glucocorticoid-Induced Osteoporosis
Baruch Frenkel, Wendy White, and Jan Tuckermann
Abstract Osteoporosis is among the most devastating side effects of glucocorticoid

(GC) therapy for the management of inflammatory and auto-immune diseases.
Evidence from both humans and mice indicate deleterious skeletal effects within
weeks of pharmacological GC administration, both related and unrelated to a
decrease in bone mineral density (BMD). Osteoclast numbers and bone resorption
are also rapidly increased, and together with osteoblast inactivation and decreased
bone formation, these changes lead the fastest loss in BMD during the initial disease
phase. Bone resorption then decreases to sub-physiological levels, but persistent
and severe inhibition of bone formation leads to further bone loss and progressively
increased fracture risk, up to an order of magnitude higher than that observed in
untreated individuals. Bone forming osteoblasts are thus considered the main cul-
prits in GC-induced osteoporosis (GIO). Accordingly, we focus this review primar-
ily on deleterious effects on osteoblasts: inhibition of cell replication and function
and acceleration of apoptosis. Mediating these adverse effects, GCs target pivotal
regulatory mechanisms that govern osteoblast growth, differentiation and survival.
Specifically, GCs inhibit growth factor pathways, including Insulin Growth Factors,
Growth Hormone, Hepatocyte Growth/Scatter Factor and IL6-type cytokines. They
also inhibit downstream kinases, including PI3-kinase and the MAP kinase ERK,
the latter attributable in part to direct transcriptional stimulation of MAP kinase
phosphatase 1. Most importantly, however, GCs inhibit the Wnt signaling pathway,
B. Frenkel, D.M.D., Ph.D. (*)

Department of Orthopaedic Surgery, Keck School of Medicine,
Institute for Genetic Medicine, University of Southern California,
2250 Alcazar Street, CSC-240, Los Angeles, CA 90033, USA
Department of Biochemistry and Molecular Biology, Keck School of Medicine,
Institute for Genetic Medicine, University of Southern California,
2250 Alcazar Street, CSC-240, Los Angeles, CA 90033, USA
W. White, M.D.
Department of Diabetes and Endocrinology, Eisenhower Medical Center,
39000 Bob Hope Drive, Rancho Mirage, CA 92270, USA
J. Tuckermann
Institute for Comparative Molecular Endocrinology, University of Ulm, Helmholtzstrasse 8/1,
Ulm, D-89081, Germany

180 B. Frenkel et al.
which plays a pivotal role in osteoblast replication, function and survival. They
transcriptionally stimulate expression of Wnt inhibitors of both the Dkk and Sfrp
families, and they induce reactive oxygen species (ROS), which result in loss of
ß-catenin to ROS-activated FoxO transcription factors. Identification of dissociated
GCs, which would suppress the immune system without causing osteoporosis, is
proving more challenging than initially thought, and GIO is currently managed by
co-treatment with bisphosphonates or PTH. These drugs, however, are not ideally
suited for GIO. Future therapeutic approaches may aim at GC targets such as those
mentioned above, or newly identified targets including the Notch pathway, the
AP-1/Il11 axis and the osteoblast master regulator RUNX2.
Keywords GIO • Proliferation • Osteoblast • Wnt • Notch • ERK • Akt • MKP-1

• FoxO • RUNX2
Glucocorticoid-Induced Osteoporosis (GIO)
The Clinical Problem
Since 1950’s Nobel laureates P. Hench, E. Kendall and T. Reichstein introduced

glucocorticoids (GCs) for the treatment of rheumatoid arthritis, these drugs have
been widely used in the management of a myriad autoimmune and inflammatory
diseases. GCs mainly act through the NR3C1-encoded glucocorticoid receptor
(GR). Once bound, the activated GR translocates to the nucleus and regulates tran-
scription of target genes either as a homodimer or a monomer that can interact with
other transcription factors. Physicians frequently prescribe synthetic GCs to treat
patients with advanced rheumatoid arthritis, asthma, multiple sclerosis, systemic
lupus erythematosus, inflammatory bowel disease, and, in combination with other
drugs, patients with hematologic and other malignancies and after organ transplan-
tation. The high efficacy of synthetic GCs as immune suppressants is blemished,
however, by deleterious side effects, in particular rapid bone loss leading to
GC-induced osteoporosis (GIO). The GR is ubiquitously expressed, including in the
various cell types present in bone, such as bone forming osteoblasts, matrix embed-
ded osteocytes, and bone resorbing osteoclasts. GIO results from effects of GCs on
all of these, as well as additional cell types, of which the effects on osteoblasts are
generally considered the most important in mediating the progressive bone loss in
patients chronically treated with GCs.
Following bone loss in post-menopausal women and in aging individuals of both
sexes, GIO is the third most-common etiology of pathological bone loss, approach-
ing by some estimates 20 % of all patients with osteoporosis [1]. Several large
population-based and cohort studies have evaluated the relationship between oral
GC and bone mineral density (BMD) as well as fracture risk, and the largest of
these, the General Practice Research Database (GPRD) in the UK, has shown that
8 Glucocorticoid-Induced Osteoporosis 181
even daily doses as low as 2.5 mg of prednisone are associated with increased risk
for vertebral and hip fracture [2, 3]. BMD decreases as a function of cumulative
exposure [4] and fracture risk depends additionally on the maximum daily dose [4].
The highest risk is observed in the spine, where the incidence of fracture compared
to controls increases by more than fivefold with daily doses equivalent to or greater
than 7.5 mg prednisolone [3]. Within the first year after treatment initiation, patients
on oral GCs show an average of 54 % increased fracture risk, which far outpaces the
decrease seen in BMD [3]. The apparent BMD-independent fracture risk may reflect
effects of GCs on neurons and muscle cells, as well as microanatomical changes to
the bone microarchitecture and/or material properties not captured by conventional
imaging techniques [5]. Nonetheless, a decrease in BMD is readily detectable by
the 12th week of GC administration and rapid bone loss continues through the 24th
week of treatment. Thereafter, the BMD loss rate appears to slow, although fracture
risk continues to accumulate [4, 6, 7]. Bone is lost from both the cortical and the
trabecular compartments, and even though the latter is affected more severely, the
trabecular thinning is usually not associated with perforations [4, 8, 9].
In addition to oral GC administration, many patients with severe obstructive
respiratory diseases such as asthma and COPD are treated with high-dose inhaled
corticosteroids. While there has been some controversy over whether or not inhaled
GCs lead to an increased risk of fracture, or whether increased fracture rates were
secondary to the underlying disease, the EOLO (Evaluation of Obstructive Lung
Disease and Osteoporosis) Study group has recently shown that inhaled GCs at the
highest doses, >1500 μg/day, have a 1.4-fold increased risk of fracture compared
with controls and patients on lower doses of inhaled steroids. Additionally, univari-
ate and multivariate analyses did not show any independent association between
obstructive lung disease and fracture risk [10, 11].
Several recent reviews provide excellent coverage of clinical manifestations and
management of GIO [12, 13]. When applicable, local GC administration should be
preferred over systemic treatment, and chronic treatment with oral GCs should be
considered a last resort. Once prescribed, oral GCs should be accompanied with
prophylactic therapy to minimize deleterious side effects. This includes cases where
high dose GCs are prescribed for short periods of time to control “flares” (exacerba-
tions) of common inflammatory diseases because, as mentioned above, GC increase
fracture risk within a few weeks of administration. Specialists treating autoimmune
and inflammatory diseases often fail to take the necessary measures to prevent
osteoporosis, and GIO-related fractures are frequently the basis for successful liti-
gation [12]. The American College of Rheumatology advocates management of
patients initiating ≥3-month treatment with a daily dose of ≥7.5 mg of prednisone
equivalents with anti-resorptive therapy (e.g., bisphosphonates) to ameliorate GIO
[14]. Recently, however, the bone anabolic peptide PTH(1-34) has been shown to
counteract deleterious effects of GCs on osteoblasts in vitro and in mice [15], and a
clinical trial suggested that it was in fact superior to anti-resorptive therapy for GIO
[16]. The results of this clinical trial are consistent with the central role of osteo-
blasts in GIO (section “Cellular Mechanisms of GIO: Osteoblasts at the Center
Stage”), and provide the prospect that further improvement of patient care may be
achieved through better understanding of the underlying cellular and molecular

disease mechanisms. Most of the published work on cellular and molecular mecha-
nisms underlying GIO has focused on osteoblasts and their precursors, which is
reviewed in detail in this chapter. We open, however, with a brief review of mouse
models, which also highlight the less well-investigated roles for GCs in osteocytes
and osteoclasts.
The Mouse as a Model
The adult mammalian skeleton undergoes continuous remodeling throughout life.

Bone resorbing osteoclasts, bone forming osteoblasts and matrix-embedded osteo-
cytes that derive from osteoblasts are the major cell types responsible for this
process. Early studies with several animal models resulted in paradoxical observa-
tions, which impeded progress with in vivo investigation of GIO, but recent work
shows that principal GIO mechanisms can be usefully modeled in mice of certain
strains, including Swiss-Webster [17], Balb/c [18] and FVB/N [19]. Similar to the
human disease, the chronic phase of decreased osteoblastogenesis and bone forma-
tion in these mouse models is preceded by an early phase dominated by exaggerated
osteoclast-mediated bone resorption, resulting in the highest rates of bone loss early
on after commencement of GC administration [17, 20].
Mechanistic investigation of GIO in vivo, including in mice, is limited because
results of molecular analyses, for example gene expression data, are typically
obtained at the tissue level and not from individual cell types at specific differentia-
tion stages. Mouse genetics, however, has proven invaluable for functional assess-
ment, in vivo, of the significance of various molecular aspects of GR signaling in
particular cell types. For example, a direct role in GIO has been unequivocally
assigned to osteoblasts using two mouse models where GC signaling was abrogated
specifically in this cell type. In one model, GC signaling was ablated by knocking
out the GR gene in cells that express Cre recombinase under the control of Runx2
regulatory sequences [19]. In an earlier study, GC signaling in osteoblasts was
abrogated by over-expression of the GC inactivating enzyme 11ß-HSD2 under the
control of the Osteocalcin Gene 2 (OG2) promoter [21]. In both mouse models,
administration of prednisolone resulted in less severe GIO compared to that
observed in control mice, indicating that GC signaling in osteoblasts plays a critical
role in GIO [19, 21] (section “Cellular Mechanisms of GIO: Osteoblasts at the
Center Stage” below). Interestingly, the OG2-HSD2 transgenic mice had no bone
phenotype at the basal state [21]. In contrast, some skeletal deficiencies at the basal
state were observed in transgenic mice expressing 11ß-HSD2 in osteoblasts under
the control of the Collagen α1(I) promoter [22, 23] and in mice lacking the GR in
the osteoblast lineage [19], indicating that endogenous GC signaling plays a bone
anabolic role during early stages of osteoblast differentiation.
Another mouse model was used to assess the contribution of GR homodimer-
ization to GIO. Classically, transcriptional stimulation in response to GCs occurs
through binding of GR homodimers to palindromic GC response elements (GREs)

at target gene enhancers, and for many years this was considered the predominant
mechanism underlying GIO. A paradigm shift, however, was offered by administra-
tion of prednisolone to so-called GRdim mice, harboring a GR mutant with an
impaired dimerization interface [24, 25]. After 2 weeks of GC treatment, these mice
had reduced osteoblast colony forming units (CFU-OBs) in the bone marrow,
reduced osteoblast numbers on the bone surface, lower bone formation rates, and
decreased bone mass, all similar to GC-treated wild type mice [19]. Thus,
dimerization-independent mechanisms, such as binding of GR monomers to non-
palindromic DNA response elements [26, 27], appear to be critical for the develop-
ment of GIO. The significance of this paradigm shift to the future of GIO research
is discussed in section “Glucocorticoids Without Osteoporosis?”.
Finally, although this chapter addresses the contribution of osteoclastogenesis to
GIO only briefly, such contribution appears quite sizable, especially at the early
phase of GIO. Indeed, GCs promote osteoclast survival and function in vivo [20, 28]
and the GC-induced bone loss (albeit without loss of bone strength) in the afore-
mentioned OG2-11ß-HSD2 mice that lack GC signaling in osteoblasts [21] could
result from persistent activation of osteoclasts in the presence of GCs. GC-stimulated
bone resorption likely occurs through their receptors in cells of the osteoblast
lineage (see section “Involvement of Cells Other than Osteoblasts in GIO”),
although involvement of osteoclast GR in increased resorption has been suggested
based on evidence from mice with conditional GR inactivation in the monocytic
lineage [29, 30].
Cellular Mechanisms of GIO: Osteoblasts at the Center Stage
The multifaceted and complex mechanisms underlying GIO have been extensively
reviewed [12, 13, 31–33]. Early anecdotal evidence suggested indirect effects of
GCs on bone through their actions in the gonads and in calcium-regulating organs
(kidney, intestine). However, more recent clinical observations and in vivo investi-
gation of mouse models argue against such indirect effects as primary pathogenic
mechanisms in GIO [12, 13, 44]. Instead, it is now widely accepted that GIO is
caused primarily through direct effects of GCs in bone cells.
Bone loss in the chronic state of GIO is mostly attributable to decreased bone
formation by osteoblasts [13], secondary to impaired osteoblast cell replication
(section “Glucocorticoids Inhibit Osteoblast Cell Cycle” below), diminished osteo-
blast differentiation and function (section “Glucocorticoids Inhibit Osteoblast
Differentiation and Function” below), and accelerated osteoblast and osteocyte
apoptosis (section “Glucocorticoids Promote Osteoblast Apoptosis” below).
Additional considerations will be briefly reviewed in the section “Involvement of
Cells Other than Osteoblasts in GIO”.
Glucocorticoids Inhibit Osteoblast Cell Cycle
Reports on GC-mediated inhibition of osteoblast proliferation in vitro date back to

the 1970s [35]. Definitive in vivo evidence for inhibition of osteoblastic cell prolif-
eration was demonstrated in GC-treated mice, where a dramatic decrease was
observed in the number of bone marrow-derived CFU-Ob representing mesenchy-
mal progenitors capable of bone formation [17, 19].
While acting as anti-mitogens in a variety of cell types, including fibroblasts,
lymphocytes, hepatocytes, and lung alveolar cells, GCs engage different cell cycle
regulatory mechanisms in a context-dependent manner. Even among osteoblast
models, effects of GCs on cell cycle progression and the underlying molecular
mechanisms vary as a function of the particular culture system and the differentia-
tion stage. Treatment of mouse calvaria-derived osteoblasts with dexamethasone
(dex) resulted in up to ~50 % reduction in the proportion of cells traversing through
the active cell cycle phases (S/G2/M), but this inhibition occurred only at and after,
not before, a well-defined developmental stage marked by a commitment to termi-
nal differentiation [36, 37]. This differentiation stage-related anti-mitogenic effect
of GCs was demonstrable in both the MC3T3-E1 immortalized cell line [36] and
primary osteoblast cultures derived from newborn mouse calvariae [37], and in
both cases inhibition of cell cycle progression was most strongly associated with
suppression of cyclin A expression [36, 37]. In MC3T3-E1 cells, inhibition of cell
cycle progression (as well as promotion of apoptosis) was also associated with
activation of p53 [38]. In primary human osteoblast culture models, dex decreased
thymidine incorporation into newly synthesized DNA and the proportion of cells
traversing through the S/G2/M phases of the cell cycle [39]. In this system, as well
as in human osteosarcoma cell lines, the anti-mitogenic effect of GCs is associated
with either down-regulation of cell cycle stimulators such as CDK2, 4 and 6, cyclin
D, c-Myc, and E2F-1, or upregulation of the cyclin-dependent inhibitors p21 and
p27 [39, 40].
GC-mediated inhibition of thymidine incorporation was also demonstrated in the
murine MBA-15.4 and the human MG-63 cell lines [41]. Co-treatment with sodium
orthovanadate, a protein tyrosine phosphatase inhibitor, reversed the anti-mitogenic
effect of dex, implicating tyrosine phosphatase(s) in the anti-mitogenic activity of
GCs in these cells (see section “Additional Signaling Pathways”). Additional
GC-regulated mechanisms involved in their anti-mitogenic effect in osteoblasts
include the Wnt signaling pathway, Akt and MAPK signaling, as well as transcrip-
tion factors such as FoxO3 (see section “Molecular Targets of Glucocorticoids in
Osteoblasts”). Although the relative contribution of the aforementioned mecha-
nisms to the inhibition of osteoblast replication remains to be determined, they all
likely require the GC receptor (GR), as indicated by their sensitivity to GR antago-
nism by RU486 [36, 41].
Glucocorticoids Inhibit Osteoblast Differentiation and Function
Mesenchymal progenitors that reside in the bone marrow give rise to osteoblasts for
the life-long process of filling eroded surfaces with new bone matrix. The early
progenitors, commonly referred to as bone marrow stromal pluripotent cells, or
mesenchymal stem cells, can select one of several developmental fates with
appreciable plasticity, including a well documented reciprocal relationship between
osteoblastogenesis and adipogenesis [42–47]. Many in vitro studies suggest that
GCs influence this competitive cell fate choice, promoting the differentiation of
bone marrow stromal cells into adipocytes at the expense of osteoblasts. Treatment
of ST2 bone marrow-derived pluripotent cells with cortisol strongly stimulated the
adipocytic genes PPARγ, C/EBPα, C/EBPδ and Adipsin [43] and inhibited RUNX2
(See also section “RUNX2”). Stimulation of C/EBPα and C/EBPδ was also observed
in a microarray-based global gene expression study of dex-treated MC3T3-E1 pre-
osteoblasts [48]. Similar to the commitment stage described in primary osteoblast
cultures [37] and in MC3T3-E1 cells [49], GCs drive ST2 cells towards the adipo-
cyte lineage and away from the osteoblastic cell fate only when administered early;
once cells undergo commitment to the osteoblast lineage, the bone phenotype con-
tinues to develop normally even in the presence of GCs [50]. The idea that GIO is
mediated in part by favoring differentiation of bone marrow pluripotent mesenchy-
mal cells into adipocytes at the expense of osteoblasts is circumstantially supported
by the increased marrow adiposity observed in GC-treated mice and humans [44],
although direct in vivo evidence is lacking. Recent advances in the identification of
skeletal stem cells may facilitate lineage tracing experiments to overcome this
shortcoming [51, 52].
GCs at pharmacological concentrations suppress fundamental osteoblast func-
tions, which can be partially traced to their influence, as described above, on cell
fate at the osteoblast/adipocyte decision fork. Most significantly, GCs inhibit the
biosynthesis of type I collagen, the predominant organic component of the bone
matrix, in vitro [53] and in vivo [54]. Contributing to this inhibition, GCs first inhibit
Procollagen α1(I) transcription; this was demonstrated in primary rat calvarial osteo-
blast cultures using nuclear run-off assays, where strong inhibition was evident
within as little as two hours of treatment with 1 μM cortisol [55]. Secondly, GCs
destabilize the Procollagen α1(I) transcript [55]. Thirdly, GCs inhibit collagen accu-
mulation in a manner independent on Procollagen α1 mRNA; this was demon-
strated, for example, by Sirius red-based assay of collagen accumulation in
BMP2-treated MC3T3-E1 cultures. Co-treatment of these cultures with 0.1 or 1 μM
dex resulted in a ~4-fold decline in the collagen accumulation rate even though
Procollagen α1(I) mRNA levels did not decrease, but in fact increased [56]. The
mRNA-independent decrease in collagen accumulation is attributable to inhibition
of collagen translation, secretion, assembly, and/or accelerated collagen breakdown
by GC-inducted collagenases [57].
Beside collagen accumulation, investigators in the GIO field often rely on assays
of alkaline phosphatase (ALP) activity and mineral deposition to assess GC-mediated
inhibition of osteoblast differentiation and function in culture models. Indeed, GCs

at pharmacological concentrations inhibit both of these outcome parameters [19, 36,
53, 58–61]. The clinical relevance of ALP and mineralization assays to GIO, how-
ever, is not trivial because unlike inhibition of osteoblast proliferation and collagen
synthesis, the results of ALP and mineralization assays can be species-dependent. In
rodent osteoblast cultures, GCs at pharmacological levels usually inhibit ALP
activity and mineralization, whereas physiological GC concentrations usually have
stimulatory effects in these models [53, 62]. In human osteoblast cultures, on the
other hand, GCs, often at low concentration, but sometimes even at pharmacologi-
cal concentrations have been shown to stimulate ALP and mineral deposition [63,
64], and such stimulatory effects were also observed in murine cultures under
specific experimental conditions [48, 65]. These observations initially led to a con-
cept that GC might drive precocious osteoblast differentiation and thus exhaustion
of a mesenchymal stem cell pool that otherwise continues to supply osteoblasts for
bone formation throughout life. However, the paradoxical stimulation of osteo-
blast differentiation in vitro is typically observed at physiological rather than
pharmacological GC concentrations [65], and most investigators no longer consider
this a significant in vivo mechanism contributing to GIO. Among considerations
arguing against this paradigm, regain of bone mass is observed shortly following
GC withdrawal [6].
It is generally assumed that GC-mediated inhibition of ALP activity and miner-
alization, which are most reproducible in murine osteoblast cultures, capture a
GC-sensitive phase of osteoblast differentiation. An alternative interpretation, how-
ever, is that some of the inhibitory effects of GCs in murine osteoblast cultures
(e.g., on mineralization) are not as relevant to the human disease, and that other
inhibitory effects (e.g., on cell replication and collagen synthesis) better model GIO
in humans. Another cautionary note for interpreting results of ALP and mineraliza-
tion assays in the context of GIO is that the two are not always coupled, and there-
fore inhibition of any one or even both of them by GCs may ultimately prove
irrelevant to GIO. This is suggested, for example, by the good correlation between
mineralization and the activity SMAD-BMPs, but not between mineralization and
ALP activity in MC3T3-E1 cultures treated with dex along with recombinant
BMP-2 or BMP-4 [67]. In addition ALP and mineralization assays can provide data
that does not parallel collagen accumulation [68]. That cell proliferation and colla-
gen accumulation assays are likely more relevant to GIO than mineralization assays
is suggested by the lack of evidence for poorly mineralized osteoid in GIO in vivo.
In section “Molecular Targets of Glucocorticoids in Osteoblasts”, we review several
signaling pathways adversely affected by exposure of osteoblast cultures to GCs;
some of these pathways may ultimately provide the basis for assays most relevant to
molecular mechanisms underlying GIO and to developing bone-sparing GC-based
anti-inflammatory therapies. Some of these assays may capture mechanisms that
reflect control of both osteoblast replication and maturation, two processes that clas-
sically represent opposite aspects of cell growth and differentiation, but may in fact
operate hand-in-hand during development of the osteoblast phenotype [36, 69–71].
Glucocorticoids Promote Osteoblast Apoptosis
Many studies demonstrated GC-driven apoptosis of osteoblasts and osteocytes, both

in vivo and in vitro [13, 33, 41, 72]. A milestone study invigorated this research
avenue in 1998, showing by TUNEL staining increased apoptosis of osteoblasts and
osteocytes in bone specimens from GC-treated mice and humans [17]. GC-mediated
promotion of osteoblast apoptosis is a cell autonomous effect because apoptosis is
not observed in transgenic mice with osteoblast-specific overexpression of the GC
inactivating enzyme 11β-HSD2 [21].
Modeling osteoblast apoptosis in GIO, dex induced apoptosis in both the
MC3T3-E1 and UMR-106 osteoblastic cell lines, and in both culture systems this
was associated with activation of Caspase 3, a common downstream effector of
multiple apoptotic signaling pathways [38, 73]. Apoptosis in the MC3T3-E1 culture
model was also linked to activation of p53 [38]. GCs dose-dependently induced
apoptosis in cultures of primary human osteoblasts derived from surgical bone
chips, and this was attributable to increased mRNA and protein expression of Bak,
as well as decreased mRNA and protein expression of Bcl-XL [39]. Annotations of
genes differentially expressed in GC-treated versus control primary osteoblast cul-
tures were highly enriched for apoptosis-related functions [74]. The same gene-set
was also enriched for oxidative stress-related genes, hinting to one mechanism
underlying GC-induced osteoblast apoptosis [74]. Related mechanisms of
GC-induced apoptosis include inhibition of major survival pathways, such as Wnt,
PI3K and ERK (section “Molecular Targets of Glucocorticoids in Osteoblasts”).
Inhibition of these pathways by GCs leaves unopposed pro-apoptotic pathways that
are activated by Fas, TNF, TRAIL [75] and Reactive Oxygen Species [ROS; sec-
tions “Pyk2, JNK and p66shc” and “FoxO Proteins”].
Several open questions related to the role of osteoblast and osteocyte apoptosis
in GIO should be noted. In the aforementioned milestone study [17], less than 1 %
of murine osteoblasts in vivo were TUNEL-positive, and GCs increased this value
threefold. The significance of these observations to the debilitation of bone forma-
tion, by up to ~80 %, in GIO remains a matter of debate. Under-estimation of the
magnitude of basal and/or GC-induced apoptosis could result from technical diffi-
culties with demonstration and quantitation of osteoblast apoptosis in vivo.
Alternative techniques and/or approaches may shed light on this controversy.
Furthermore, GC did not appear to induce apoptosis in osteoblasts during certain
developmental stages [75] and in mice of certain strains [19]. Additionally, preven-
tion of GC-induced osteoblast apoptosis did not rescue the low bone formation rates
or the loss of spinal BMD in the aforementioned OG2-11ß-HSD2 mice (although
the decrease in vertebral compression strength was prevented; see section
“Involvement of Cells Other than Osteoblasts in GIO” below) [21]. Despite the
uncertainty as to the relative contributions of effects of GCs on pre-osteoblast pro-
liferation, impaired osteoblast function and increased apoptosis, the combined
effect on all of these aspects together appears to create a “perfect storm” that leaves
osteoblasts incapable of balancing bone resorption in GIO.
Involvement of Cells Other than Osteoblasts in GIO
Osteocytes. Not all osteoblasts undergo apoptosis after depositing new bone mate-
rial at sites that have just been resorbed. Some give rise to flat lining cells that
remain on the bone surface and many others incorporate into the newly formed
matrix, where they continue to live for lengthy periods of time, contributing to the
largest subpopulation of bone cells—the osteocytes. Through neuronal-like pro-
cesses embedded in a highly interconnected canalicular system, osteocytes serve as
biological relays, which stimulate osteoclasts, osteoblasts and their precursors in
response to microdamage and mechanical loading. In fact, osteocytes are a major
source of RANKL, a quintessential osteoclastogenic factor [77, 78], and their role
in bone homeostasis is increasingly appreciated [79]. Thus, skeletal effects of GCs
through osteocytes are both direct (as discussed immediately below) and indirect
via osteoclastogenesis (see ‘osteoclasts’ thereafter).
There is significant evidence that high-dose GCs increase fracture risk not only
by decreasing bone mass, but also by compromising bone material quality [13]. One
of several explanations for this phenomenon entails GC-induced osteocyte apopto-
sis [17]. Contrasting osteocyte autophagy induced by GCs at physiological concen-
trations, which may protect these cells against stress [80], osteocyte apoptosis in
response to high-dose GCs may deprive osteoclasts and osteoblasts the input, based
on which they would otherwise respond to biomechanical needs. GC-induced osteo-
cyte apoptosis is attributable to inhibition of survival mechanisms including Wnt
signaling, Akt, and Pyk2 (section “Molecular Targets of Glucocorticoids in
Osteoblasts” below). Additionally, recent evidence suggests that osteocytes directly
modify the bone matrix in which they are embedded and that GCs interfere with a
post-osteoblast mineralization process, whereby osteocytes regulate their immedi-
ate microenvironment [5]. This novel effect of GCs, hypomineralization of peri-
osteocytic bone material, was demonstrated using a nanoindentation technique
assisted by atomic force microscopy, and cannot be detected by conventional
imaging or histmorphometric methods [5].
Osteoclasts. The early and most destructive phase of GIO is driven not only by the
inhibition of osteoblastic bone formation as described above, but also by simultane-
ous stimulation of osteoclastic bone resorption. After prolonged treatment, how-
ever, bone resorption is suppressed to sub-physiological levels [28], contributing to
the overall low bone turnover rates typical of GIO.
Stimulation of osteoclastogenesis by GCs at the early disease phase is attribut-
able to both cell autonomous and paracrine mechanisms. Because osteoclastogene-
sis is a process that usually requires a few days for completion, the fast increase in
osteoclast number observed immediately after GC administration has been attrib-
utable to extended life span of pre-existing osteoclasts. GC-mediated extension of
the osteoclast life-span appears to be cell-autonomous because it occurred in iso-
lated osteoclasts in vitro [28], and because osteoclast number was lower in wild
type GC-treated mice compared to GC-treated transgenic mice over-expressing the

GC-inactivating enzyme 11β-HSD2 is osteoclasts [30]. Additionally, in vitro data
suggest enhancement of osteoblast-driven osteoclastogenesis by GCs, potentially
contributing to the increased bone resorption observed in the early phase of GIO
[29]. In particular, GCs dramatically suppress OPG mRNA and protein expression
in human osteoblast cultures [18]. It remains to be clarified whether GC administra-
tion also stimulates bone resorption through osteocyte-borne RANKL [77, 78] and/
or other osteoclastogenenic factors, and whether GC-induced osteocyte apoptosis
facilitates the release of such factors to promote osteoclastogenesis [81].
The decreased osteoclastic bone resorption observed after prolonged GC treat-
ment periods is generally considered secondary to attenuation of cell number and
function in the osteoblast lineage as described above. There is intriguing evidence
in vivo, however, suggesting that the GR in myeloid progenitor cells decreases the
resorptive activity of osteoclasts, and that this is mediated by their failure to form
cytoskeletal structures necessary for attachment to and resorption of the bone
matrix [29].
Non-bone cells. In addition to mineralized tissue, the bone as an organ consists of
soft tissues, such as cartilage, bone marrow, vessels and nerves. Potential contribu-
tions of these tissues to GIO are largely unknown. Furthermore, GCs may have
indirect negative effects on bone through their actions in the neuroendocrine system,
the gonads, the intestine, and the kidney (reviewed in [13]). Finally, adverse effects
of GCs on motor function and muscle strength may contribute to fracture risk by
increasing fall rates.
Molecular Targets of Glucocorticoids in Osteoblasts
The Wnt Signaling Pathway

Glucocorticoids Inhibit Wnt Signaling in Osteoblasts
As with several other cell types, Wnt signaling in osteoblasts increases cell prolif-
eration by promoting cell cycle progression and by inhibiting apoptosis [82].
Because Wnt ligands are known to promote asymmetric stem cell division [83],
they can be expected to allow early pre-osteoblast pool expansion to supply cells for
the formative arm of bone remodeling, while preserving a mesenchymal stem cell
pool in the bone marrow microenvironment. The central role of Wnt signaling in
bone biology was initially indicated by the inactivating mutations in LRP5, coding
a Wnt co-receptor, in patients with the familial bone disease osteoporosis-pseudo-
glioma [84]. The opposite, high bone mass (HBM), is observed in patients carrying
LRP5 activating mutations [85, 86]. Alterations to many additional Wnt-related
genes have been linked to bone mass control, and experiments manipulating such
genes in mice solidified the notion that Wnt signaling plays a pivotal role in bone
metabolism and bone mass control (reviewed in [82]). Accordingly, experimental
stimulation of Wnt signaling in osteoblasts results in increased proliferation,
increased expression of differentiation markers, decreased apoptosis, as well as
attenuated osteoblast-driven osteoclastogenesis [82].
Bioinformatic analysis of global gene expression in bone tissue of GC-treated
versus control mice linked the differentially expresed genes to the Wnt pathway,
with indications for reduced signaling due, in part, to low expression of Wnt ligands
[5]. GCs also decrease Wnt signaling in osteoblast cultures as indicated by decreased
expression of Wnt targets, both endogenous genes and reporter constructs [37, 60,
87–90]. Mechanisms underlying inhibition of Wnt signaling in GIO are reviewed
below and schematically summarized in Fig. 8.1.
Role of Dkks in GIO
DKK1, encoding dickkopf-1 (DKK1), a secreted antagonist of canonical WNT sig-

naling, is one of few examples available to date for classical direct transcriptional
stimulation by GCs in osteoblasts. Treatment of human osteoblasts with ≥10 nM
dex resulted in robust stimulation of DKK1 mRNA expression, attributable to a
glucocorticoid response element (GRE) located 0.8 kb upstream of the DKK1 tran-
scription start site [91]. In a microarray study of primary human osteoblast cultures,
DKK1 was among the genes most strongly upregulated by GCs [74]. GCs also stim-
ulated Dkk1 expression in primary rat calvarial osteoblast cultures [92]. A role for
GC-induced Dkk1 in inhibiting Wnt signaling in osteoblasts is indicated by reversal
of the suppressive effect of dex on Wnt signaling upon addition of anti-Dkk1 anti-
body to the osteoblast culture medium [88].
Members of the Dkk family inhibit Wnt signaling through interaction with
LRP5/LRP6 and with Kremen1/Kremen2 in the Wnt receptor complex [93, 94].
The critical role of DKKs in regulating Wnt signaling in osteoblasts and conse-
quently controlling bone mass is demonstrated by the fact that the G171V mutation
in LRP5 that leads to a HBM phenotype [85] is associated with abrogation of its
binding to Dkk-1 [95, 96]. The HBM phenotype observed in patients with the
G171V LRP5 mutation is therefore indicative of high sensitivity of the Wnt pathway
and bone mass to reduced DKK1 activity upon LRP5 in osteoblasts. This is also
demonstrated by the strong anabolic response and increased bone mass in DKK1
hypomorphic or heterozygous mice [97, 98]. The opposite effect, decreased bone
mass, can therefore be expected to follow increased activity of DKK1 on osteo-
blasts’ LRP5 in GIO. Indeed, GC-mediated suppression of Wnt/β-catenin signaling
and osteoblast differentiation [86] was attenuated when stimulation of Dkk1 was
prevented by siRNA-mediated Dkk1 knockdown [60]. In addition to the stimulation
of Dkk1 in human and rat cells, GCs have also been shown to stimulate expression
of the closely related Dkk2 in primary mouse calvarial osteoblast cultures [37],
potentially resulting in the observed inhibition of Wnt signaling in osteoblasts
Fig. 8.1 GCs stimulate GSK3ß and inhibit Wnt signaling. Filled (colored) shapes represent com-
ponents of the canonical Wnt pathway and empty (white) shapes represent signaling molecules
that intersect with the Wnt pathway. GSK3ß is positioned at an interesection between the Wnt
pathway and protein tyrosine kinase signaling. Lightning bolts indicate phosphorylation. Inhibitory
and stimulatory effects of GCs are depicted by red and green circled G’s, respectively. GCs inhibit
growth factors (GF) and the downstream PI3K/Akt pathway. Consequently, the inhibitory phos-
phoryltion of GSK3ß on Ser9 (octagon) is attenuated and GSK3ß is thus activated. Outside the
canonical Wnt pathway, active GSK3ß phophorylates c-Myc on Thr58, resulting in c-Myc degrada-
tion. Within the canonical Wnt pathway, GSK3ß phosphorylates ß-catenin, resulting in its degrada-
tion. This adds to increased rates of ß-catenin degradation due to stimulation of Wnt inhibitors of
the SFRP and DKK families, as well as inhibition of Wnt ligands. Inactivation of the Wnt receptor
complex stabilizes the ß-catenin destruction complex, where ß-catenin is phosphorylated, tagging
it for degradation. Accumulation of ß-catenin is thus inhibited. Because ß-catenin is a critical co-
activator for LEF/TCF transcription factors, their target genes (i.e., Wnt target genes) are sup-
pressed. Additionally, GCs attenuate expression of some LEF/TCF transcription factors and
stimulate that of HDAC proteins, which inhibit both ß-catenin and LEF/TCF. Abbreviations: APC
adenomatous polyposis coli, CK-Iα casein kinase-Iα, DVL disheveled, GSK glycogen synthase
kinase, HDAC histone deacetylase, LRP5 low density lipoprotein-related protein 5, SFRP secreted
freezled-related protein
through a similar mechanism, again leading to decreased osteoblast function and

loss of bone mass.
Consistent with a potential role for DKK1 in GIO, its levels were elevated in the
sera of children with 21-hydroxylase deficiency and chronically treated with high-
dose glucocorticoids (10–25 mg/m2 of hydrocortisone). The patients’ sera inhib-
ited osteoblast differentiation in vitro (although the inhibition—of ALP
activity—was small), and the effect was ameliorated after administration of anti-
DKK1 antibodies [99]. Additional data obtained from these patients suggested that
the excess DKK1 could be partly biosynthesized by leukocytes, and that it played
a role in regulating RANKL levels in these patients [99]. Dkk1 mRNA was also
elevated in mouse bones in vivo after 56 days of prednisolone treatment [20]; this
study, however, raises the question of whether GC-mediated stimulation of Dkk1 is
a primary event in the mouse because the early time point in vivo (7-days) indi-
cated decreased, not increased DKK1 mRNA levels [20]. Further questioning the
role of DKK1 in GIO, its levels decreased, not increased in a prospective study
with patients initiating GC therapy [100]. Additional work is therefore needed, for
example using conditional knockout mice, to rigorously test the potential role of
DKK1 in GIO.
Role of Secreted Frizzled-Related Proteins (SFRPs) in GIO
The first step in activating Wnt signaling is the binding of Wnt ligands to frizzled
family receptors. A group of decoy receptors, the secreted frizzled-related proteins
(SFRPs) compete with membrane bound frizzled receptors for Wnt binding (Fig. 8.1),
thus attenuating both canonical and non-canonical Wnt signaling [93, 101]. Sfrp1
knockout mice have increased trabecular bone mineral density [102] and injection of
rats with recombinant SFRP1 decreased bone mineral density [62]. Both canonical
and non-canonical Wnt signaling in osteoblasts have been implicated in the regulation
of osteoblast proliferation, differentiation and apoptosis by SFRP1 [102].
Dex at concentrations ≥0.1 μM dramatically stimulated Sfrp1 mRNA expression
in primary rat bone marrow stromal cell cultures, and this was independent of new
protein synthesis [62]. Dex also stimulated Sfrp1 expression in mouse primary cal-
varial osteoblast cultures [92]. Furthermore, siRNA knock down of SFRP1 led to
increased β-catenin accumulation, enhanced Runx2 activity and high levels of ALP
and osteocalcin expression, culminating in robust nodule formation even at high dex
concentrations [62]. Hence, SFRP could serve as a therapeutic target, inhibition of
which may ameliorate GIO.
Additional Wnt-Related GC Targets
Wnt ligands. GCs regulate the expression of some Wnt ligands, potentially contrib-
uting to inhibition Wnt signaling in osteoblasts. For example, corticosterone at 100
nM inhibited by ~50 % expression of Wnt 7b and Wnt 10b in mature green fluores-
cent protein (GFP)-expressing osteoblasts of Col2.3-GFP mice. Interestingly, 10
nM corticosterone had the opposite effect, potentially accounting for paradoxical
anabolic effects often observed with low GC doses [92]. Loss of the autocrine/para-
crine activity of Wnt ligands at high GC concentrations may amplify the aforemen-
tioned anti-Wnt effects of Dkk1, which were confirmed in the Col2.3-GFP-expressing
cells [92].
GSK3ß. GC-treated osteoblasts from both human and mouse origin display a
decrease in the inhibitory phosphorylation of GSK3β on its Ser9 residue, resulting in
increased enzyme activity [46, 49]. The role of GSK3β in the anti-mitogenic effect
of GCs was demonstrated by the rescue of cell cycle progression in GC-arrested
MC3T3-E1 osteoblasts co-treated with lithium chloride, a GSK3β inhibitor [49].
GC-stimulated GSK3β attenuates cell cycle progression both by inhibiting ß-catenin/
LEF-mediated transcription [87] and by phosphorylation of c-MYC on Thr58, which
marks the protein for proteasomal degradation [49]. The phosphorylation of GSK3β
represents an important point of intersection between growth factor signaling and
the canonical Wnt pathway (Fig. 8.1). Specifically, following the activation of PI3K
by receptor tyrosine kinases [section “Akt”], Akt phosphorylates GSK3β’s serine9
residue, which results in loss of GSK3β activity upon its targets, such as β-catenin
and c-Myc. Accordingly, pharmacological and molecular inhibition of PI3K/Akt in
GC-treated MC3T3-E1 osteoblasts is associated with decreased phosphorylation of
GSK3β’s Ser9 as well as c-MYC’s Thr58 [49]. Thus, GC-mediated stimulation of the
inhibitory kinase GSK3β results in (i) attenuation of β-catenin/LEF-driven tran-
scription, adding to other inhibitory effects of GCs within the canonical Wnt path-
way; and (ii) abrogation of GSK3β functions outside the canonical Wnt pathway
(Fig. 8.1).
ß-catenin. Ligand-bound GR has been shown to physically interact with β-catenin
itself in U2OS/GR cells [103]. This could contribute to inhibition of LEF/TCF-
mediated cyclin D transcription and to GIO in vivo, even though GCs did not inhibit
cell cycle progression in the U2OS/GR cell culture model [103]. Additionally, GCs
may inhibit Wnt signaling by translocating β-catenin from the cell nucleus to the
cytoplasmic membrane, which is mediated though interactions of GR with calre-
ticulin. Indeed, silencing of calreticulin abolished dex-mediated inhibition of cyclin
D1 expression [104]. Finally, as will be described in section “FoxO Proteins”, GCs
interfere with canonical Wnt signaling at the level of β-catenin by generating
reactive oxygen species, resulting in activation of FoxO transcription factors, which
interact with β-catenin at the expense of LEF/TCF transcription factors.
Recent work suggests that GC-mediated suppression of Wnt/β-catenin signaling
is mediated in part through inhibition of mir-29a [105]. In murine calvarial osteo-
blasts, both primary and MC3T3-E1 cells, mir-29a promotes bone phenotypic prop-
erties by suppressing expression of HDAC4, a β-catenin deacetylase [105].
GC-mediated downregulation of mir-29a, and the subsequent deacetylation and
inactivation of ß-catenin by HDAC4 appear critical for suppression of the bone
phenotype because anti-sense-mediated silencing of HDAC4 rendered the cultures
resistant to GCs. Consistent with these findings, GC-mediated inhibition of cell
cycle progression in MC3T3-E1 cultures was partially negated in the presence of
the HDAC inhibitor trichostatin A [87].
LEF/TCF. Signals elicited by binding of Wnt ligands to their cell surface receptors
ultimately lead to changes in gene expression by the binding of activated β-catenin to
transcription factors of the LEF/TCF family (Fig. 8.1). In newborn mouse calvarial
osteoblast cultures, 1 μM dex decreased the expression of Lef1, Tcf1 and Tcf4 (but not
Tcf3) mRNA [37]. Interestingly, the effect of dex on Lef1 and Tcf1 expression depended
on the developmental stage with respect to a commitment stage defined based on
resistance that these cultures develop on day 6–7 to GC-mediated attenuation of

mineral deposition. Specifically, dex inhibited Lef1 only before the commitment stage,
whereas the inhibition of Tcf1 was most robust after that stage [37].
Axin2. As discussed in section “Glucocorticoids Inhibit Osteoblast Differentiation
and Function”, GCs drive osteoblast precursors towards adipogenesis at the expense
of osteogenesis [46, 90, 106]. In murine MC3T3-E1 pre-osteoblasts and ROB-C26
rat mesenchymal progenitor cells, this was attributable in part to a dex-mediated
3-fold increase in Axin2 mRNA expression [90, 107]. Indeed, dex also abrogated
ß-catenin activation and this was no longer obvious after depletion of Axin2 in
ROB-C26 cells [90]. Consistently, knockdown of Axin2 antagonized dex-mediated
adipogenesis, although inhibition of ALP by dex persisted in Axin2-depleted ROB-
C26 cultures [90].
Additional Signaling Pathways
In addition to the well documented role of the Wnt signaling pathway in bone
pathophysiology in general, and GIO in particular, GCs affect several other path-
ways in osteoblasts, any of which may ultimately prove an effective target for thera-
peutic intervention. We briefly review here evidence for the involvement of Notch
and BMP signaling, as well as several growth factor pathways, in GIO.
Notch Signaling
Glucocorticoids strongly stimulate transcription of Notch1 and Notch2 in osteo-

blasts, resulting in several-fold increased mRNA expression within hours of treat-
ment [108]. The activated Notch Intracellular Domain (NICD) is known to inhibit
osteoblast differentiation by targeting RUNX2 both directly and indirectly [109,
110]. Although manipulation of Notch signaling in vivo results in a complex skel-
etal phenotype that depends on age, sex and bone tissue type [110–111], GC-mediated
stimulation of Notch signaling likely plays an important role in GIO, which may be
mediated in part by inhibition of RUNX2 [section “RUNX2”].
BMP Signaling
Comprehensive gene expression analysis in GC-arrested MC3T3-E1 osteoblast cul-

tures indicated a threefold increase in the expression of Follistatin and Dan mRNAs,
encoding inhibitors of BMP signaling [49]. In the same culture model, GCs also
strongly inhibited Bmp2 gene expression, and recombinant BMP2 reversed the
inhibitory effects of GCs on mineral deposition, ALP activity, osteocalcin expres-
sion, as well as (transiently) cell cycle progression [56, 68]. These, however, remain
indirect lines of evidence for a role that BMP signaling may play in GIO. In fact,
dex did not inhibit the activity of a SMAD-BMP reporter in cultures of MC3T3-E1
cells [67], and some investigators even demonstrated stimulation of BMP signaling
by GCs in osteoblasts [32]. Paradoxically, stimulation of BMP signaling by GCs
may contribute to GIO through inhibition of Wnt signaling [112], although this
conjuncture remains to be tested. Another interesting speculation is that GCs con-
comitantly stimulate and inhibit BMP signaling in a target gene-dependent manner.
Be that as it may, global inhibition of BMP-SMAD signaling does not appear to
occur in GIO, or at least not in the MC3T3-E1 culture model; high expression levels
of other BMPs, in particular BMP-4, could have sustained activity of the BMP-
SMAD reporter in the presence of GCs [67]. Still, decreased BMP2 levels may
contribute to GIO when alternative osteogenic BMP genes are not expressed. Under
such circumstances (and not in MC3T3-E1 cells), inhibition of Bmp2 transcription,
through regulatory sequences located >50-kb downstream of the transcription start
site [67], might reduce BMP-SMAD signaling below a threshold necessary for
development of the osteoblast phenotype.
Growth Factors
GCs inhibit the biosynthesis of hepatocyte growth factor (HGF, a.k.a. Scatter Factor)
in human osteoblasts [113, 114], which could interrupt an autocrine mechanism
whereby HGF stimulates osteoblast proliferation by binding to its c-Met receptor on
the osteoblast membrane [115]. Indeed, inhibition of HGF signaling mimicked the
anti-mitogenic effect of GCs in human osteoblast-like cultures, and GCs no longer
inhibited cell proliferation in the presence of added recombinant HGF [114]. These
results suggest that HGF may play an important role in GIO, although direct evi-
dence to this effect is lacking.
Growth hormone (GH) and insulin-like growth factors (IGFs) influence bone
metabolism through both endocrine and paracrine/autocrine mechanisms, of which
the latter are more likely to play a role in GIO [116]. Indeed, osteoblast proliferation
and collagen synthesis, probably the two most important functions inhibited in GIO,
are stimulated by IGF-I and IGF-II, and GCs have been shown to inhibit IGF signal-
ing at several levels. First, GCs inhibit IGF-I transcription and secretion in primary
rat calvarial osteoblast cultures [117, 118], which again may be related to the pro-
motion of the alternative, adipocyte cell fate decision. Indeed, the GC-induced adip-
ogenic factors C/EBPα and C/EBPδ appear to bind Igf-1 regulatory sequences and
block transcriptional initiation or elongation [119]. Second, GCs inhibit expression
of IGF-binding protein-3 (IGFBP-3) and IGFBP-5 in human osteoblast cultures
[120]. The strong inhibition of Igfbp5, which occurs at the transcriptional level in
rat calvarial osteoblast cultures [121], is of particular interest because IGFBP5 has
an additional, autonomous effect on human osteoblast proliferation [122] and a
selective bone anabolic effect in mice in vivo [123–125].
Downstream Kinases and Phosphatases
ERK
In addition to kinase activity directly associated with the receptors reviewed above,
downstream kinases with roles in osteoblast growth and differentiation have been
implicated in GIO. Chief among them, ERK is a central hub downstream of a vari-
ety of osteogenic stimuli elicited by interaction of growth factors and extracellular
matrix proteins with their respective receptor tyrosine kinases and integrin receptors
[126–129]. Activated ERK executes much of its function in the osteoblast nucleus,
where it associates with specific DNA elements to activate key regulators of cell
growth and differentiation, including the osteoblast master regulator RUNX2 [130,
131]. Accordingly, stimulation and inhibition of ERK results in enhancement and
impediment, respectively, of osteoblast differentiation and bone formation in vivo
and in vitro [41, 132, 133].
High dose GCs inhibit ERK signaling and its downstream effectors, and these
inhibitory activities are similar to those observed after treatment of osteoblasts with
the MEK/ERK inhibitor U0126 [41]. Both dex and U0126 decreased thymidine
incorporation into newly synthesized DNA in serum- and TPA-stimulated MBA-
15.4 and MG-63 osteoblastic cells [41]. Attenuated ERK activity in GC-treated
osteoblasts is likely related to many aspects of GIO, including inhibition of osteo-
blast proliferation, differentiation and survival [75, 129]. Protection of ERK from
GCs has the potential of partially reversing GIO [134].
Akt
Activation of the PI3K/Akt pathway by hormones and growth factors, including

PTH and IGFs, is required for the differentiation of mesenchymal pluripotent cells
into osteoblasts, as well as survival of the committed cells [135–137]. Mechanisms
of action of Akt in regulating canonical signaling pathways such as Wnt (through
GSK3β) and mTOR are well established [138, 139]. Additionally in osteoblasts,
Akt promotes RUNX2 activity [140], in part through post-translational regulation
of Smurf2 [141], and may also activate transcription factors directly within the cell
nucleus [139]. GCs attenuate the activity of Akt in osteoblasts by decreasing growth
factor availability [see section “Growth Factors”], and by compromising the cellular
response to such growth factors [87], which is partially attributable to oxidative
stress [89, 143].
Pyk2, JNK and p66shc
In MLO-Y4 osteocyte-like cells, the two highly homologous kinases, FAK and
Pyk2, play opposing roles with regard to interaction with the extracellular matrix
(ECM). Whereas FAK promotes ECM attachment and cell survival, Pyk2 activation
results in loss of cellular processes and anoikis, i.e., detachment-mediated pro-

grammed cell death [144]. GCs activates Pyk2 in a manner independent of either
RNA or protein synthesis; the underlying mechanism of action, likely employing
membrane-associated GR, involves stimulation of JNK [144]. Indeed, GCs no lon-
ger induce anoikis in Pyk2-depleted cells or in cells that express either an inactive
Pyk2 mutant or a dominant negative JNK [143, 144]. GC-mediated detachment of
cells from the ECM through the Pyk2/JNK axis may play a role in the apoptosis of
both osteocytes and osteoblasts in vivo [17]. JNK also plays a role in GC-mediated
ROS-driven apoptosis through activation of FoxO transcription factors [see section
“FoxO Proteins”]. Indeed, GC-mediated FoxO-driven transcription and apoptosis
was associated with activation of JNK, and both were severely compromised in
fibroblasts derived from JNK1/2 double-knockout mouse embryos [89].
GCs also activated the p66shc kinase in bone in vivo and in cultured osteoblasts in
vitro, leading to accumulation of reactive oxygen species (ROS) [89]. Indeed, GCs
no longer stimulate ROS accumulation in osteoblast cultures in which p66shc is
absent, or in which PKCß, the kinase that phosphorylates p66shc, is pharmacologi-
cally inhibited [89]. ROS have many deleterious effects in osteoblasts. As described
above they activate JNK, which results in apoptosis through FoxO-dependent and
independent mechanisms, and they inhibit Wnt signaling through activation of
FoxO transcription factors [section “FoxO Proteins” below] and inhibition of Akt
[section “Akt” above].
MKP-1/DUSP
Protein tyrosine phosphatases (PTPs) have been strongly implicated in GC-mediated

inhibition of ERK signaling and osteoblast function, as they are the dominant active
phosphatase class in the lineage [41]. Inhibition of PTP with sodium orthovanadate
restored ERK activity and osteoblast proliferation in dex-inhibited osteoblast cul-
tures [134] and in methylprednisolone-treated Sprague–Dawley rats [145].
The main phosphatase implicated in GC-mediated inhibition of ERK is MAPK
phosphatase-1 (MKP-1), also named dual specificity phosphatase-1 (DUSP1),
which colocalizes with ERK and limits its effects on target gene transcription [146–
149]. Although JNK and p38 can also be inactivated by MKP-1 [150] and even
though MKP-3 is most efficient in inactivating ERK1/2 [148], studies in osteoblasts
clearly indicate that GCs stimulate MKP-1 [41, 151, 152] while inhibiting MKP-3
[41], and that MKP-1 inactivates ERK [152]. In addition, knockdown of MKP-1
with siRNA in human MG-63 osteosarcoma cells prevented dex-mediated ERK
dephosphorylation [152].
Stimulation of MKP-1 expression by GCs is rapid and robust. MKP-1 mRNA
levels increased by >10-fold within 30 min of treatment of mouse MBA-15.4 and
human MG-63 pre-osteoblasts with dex, an effect that lasted >24 h [41, 152]. This
was associated with a similar >10-fold induction of MKP-1 protein, which precisely
correlated with inhibition of ERK phosphorylation [41, 152]. GC-induced expres-
sion of MKP-1 was confirmed in a global microarray analysis of dex-treated
MC3T3-E1 osteoblast-like cells [48], as well as in fibroblast-like synoviocytes

[147]. Whereas GR occupancy at the MKP-1 locus in osteoblasts has not been
mapped systematically, it has been shown to associate, either directly or through a
tethering mechanism, with a GRE-C/EBP composite element located ~1.3-kb
upstream of the DUSP transcription start site in A549 human lung adenocarcinoma
cells [153, 154]. GCs have also been shown to increase MKP-1 stability in mast
cells and fibroblasts [151], although this mechanism is less likely relevant to bone
cells because GCs no longer increase MKP-1 levels in cyclohexamide-treated
MG-63 cells [152].
Does inhibition of MKP-1 offer a realistic approach for the management of GIO?
Despite the pivotal roles of ERK in osteoblast growth and differentiation, side
effects of global MKP-1 inhibition are more than likely to occur, which would
necessitate specific targeting to bone. Additionally, the efficacy of prospective anti-
MKP-1 approaches for GIO has been questioned by observations in MKP-1 knock-
out mice treated for 28 days with methylprednisolone [155]. The absence of MKP-1
did not negate the GC-mediated decrease in bone formation, suggesting that inhibi-
tion of MKP-1 alone is insufficient for protection against GIO [155].
Transcription Factors
FoxO Proteins
The FoxO (forkhead box O) family, consisting of FoxO1, FoxO3a, FoxO4, and
FoxO6 [156], play important roles in cellular responses to ROS as well as regulation
of cell cycle progression and apoptosis [157]. As in other cells, FoxO family mem-
bers defend osteoblasts against ROS [89, 158]. The balance between protective and
deleterious effects of ROS and FoxO transcription factors is therefore key to for
bone health [143]. GCs severely impair this balance by super-activating FoxO tran-
scription factors. This results in the concomitant inhibition of Wnt signaling [143,
159, 160], thus compromising osteoblast proliferation and differentiation (see sec-
tions “Glucocorticoids Inhibit Osteoblast Cell Cycle”–“Glucocorticoids Promote
Osteoblast Apoptosis”).
Similar to the transcriptional and post-transcriptional regulation of FoxO3 in
non-bone cells [161], GCs stimulate FoxO transcription factors in osteoblasts
through several independent mechanisms. First, FoxO mRNA levels are upregu-
lated by GCs. Indeed, FoxO3a and FoxO1a were two of the mRNAs most strongly
upregulated in a microarray study of GC-treated primary human osteoblasts [74].
Second, GC-induced ROS stimulate the PKCβ/p66shc axis, resulting in activation of
JNK and the subsequent phosphorylation of FoxO [143] [see section “Pyk2, JNK
and p66shc”]. Third, GCs inhibit Akt [see section “Akt”], which results in the activa-
tion of FoxO proteins at the expense of LEF/TCF transcription factors [89].
When treated with pharmacologic doses of GCs, activated FoxO proteins bind
and compete for a limited supply of ß-catenin [143, 162, 163]. Direct interaction
between ß-catenin and FoxO3a was demonstrated by co-immunoprecipitation of

assays in C2C12 cells [143]. Serving as a co-activator, ß-catenin stimulates expres-
sion of FoxO target genes at the expense of Wnt/TCF target genes [163]. Indeed,
mimicry of GC treatment by over-expression of FoxO3a in Wnt3a-treated C2C12
cultures abrogated development of the osteoblast phenotype, illustrated by a
decrease in ALP activity [143]. Furthermore, overexpression of ß-catenin partially
overcame FoxO-mediated suppression of TCF/LEF-driven transcription, again sug-
gesting that a limited pool of ß-catenin is shared for the activation of LEF/TCF and
FoxO target genes [143]. Furthermore, unlike osteoblasts isolated from WT mice,
Wnt3a-driven LEF/TCF activity was not inhibited by GCs in osteoblasts isolated
from mice lacking FoxO1, FoxO2 and FoxO3, illustrating the critical role of FoxO
proteins in GC-mediated inhibition of Wnt signaling [89].
AP-1
Much of the anti-inflammatory activity of GCs is attributable to both direct and

indirect interactions between the GR and other transcription factors. Direct interac-
tions occur both at cis-acting regulatory DNA elements and away from DNA. Indirect
interactions involve, for example, regulation of phosphorylation and competition
for common co-activators [164]. Perhaps the most important GR-interacting pro-
teins in the context of immune suppression are AP1 (FOS/JUN) and NF-κB tran-
scription factors. Of these, interaction with NF-kB does not appear to play a role in
GIO because GCs suppress ALP activity in primary osteoblast cultures even when
the cells are impaired for NF-κB activation [19]. In contrast, interactions of the GR
with AP-1 family members, which are well documented in contexts other than
osteoblasts [164–169], appear to play a role in GIO. This notion is consistent with
the pivotal roles that FOS and JUN family members play in osteoblast growth and
differentiation [170–172]. Indeed, GRdim mice, in which classical transcriptional
stimulation by GR dimers is impaired but inhibition of AP-1 is preserved, devel-
oped GIO [19]. Furthermore, abrogation of AP-1-depedent expression of IL-11
[174], a cytokine critical for bone formation in vivo [175], has been implicated in
GIO, and administration of IL-11 restored ALP activity, mineralization, and RUNX2
expression in GC-inhibited murine primary calvarial osteoblast cultures [19].
Intriguingly, PTH administration overcame GC-mediated suppression of IL11
expression by stimulating Smad1 and the FOS family member delta FosB [176], a
mechanism that could be exploited to combat GIO.
RUNX2
RUNX2 is the most powerful osteoblast lineage specifying factor known to date.
Runx2 ablation in the mouse resulted in absence of osteoblasts and a general failure
to form any mineralized tissue [177, 178]. Accordingly, manipulation of RUNX2 in
cell culture models led to corresponding gain or loss of osteoblast phenotypic
properties [179–181]. Because functional osteoblasts are needed not only for
embryonic bone development, but also to balance the resorptive activity of osteo-
clasts at bone multicellular units (BMUs) throughout life, suppression of RUNX2 in
adults can be expected to result in bone loss [182] [183]. Therefore, the inhibition
of Runx2 expression by GCs, observed in several osteoblast culture systems and
in vivo, may play an important role in GIO [19, 43, 184, 185].
Because RUNX2 activity is strongly regulated post-translationally by covalent
modifications and protein-protein interactions [186, 187], data on its mRNA and
even protein expression levels can be misleading. It is therefore important to note
that, in addition to inhibition RUNX2 expression, GCs have been reported to sup-
press the expression of RUNX2 targets, both endogenous genes such as osteocalcin
and artificial constructs designed to specifically report on RUNX activity [61, 184].
Furthermore, a recent study demonstrated that GCs inhibit the activity of RUNX2
even when constitutively expressed from an exogenous lentiviral vector [61]. The
implied post-translational inhibition of RUNX2, without a decrease in its mRNA or
protein levels [61], is likely the primary effect leading secondarily to changes in the
expression levels of endogenous RUNX2 because endogenous Runx2 is subjected to
auto-regulation [188]. Indeed, exceptional cases where endogenous Runx2 expres-
sion is not inhibited by GCs [185] may represent culture models, in which RUNX2
autoregulatory loops are not operative. Alternatively, inhibition of RUNX2 activity
by GCs may be specific for differentiation stages represented by some and not other
tissue culture models [56, 185].
Like several other steroid hormone receptors [189–191], the GR physically inter-
acts with RUNX2 [61, 192], possibly inhibiting its DNA-binding and/or transcrip-
tional activation activity. Such inhibition may vary depending on the target gene,
including the topographic relationships between local sites occupied by RUNX2,
GR and possibly other transcription factors [61, 193]. Additionally, GCs may inhibit
RUNX2 indirectly, by suppressing Wnt [section “The Wnt Signaling Pathway”],
ERK [section “ERK”] and Akt signaling [section “Akt”], all of which have been
implicated in stimulating RUNX2 [130, 131, 140, 141, 194], or by stimulating
Notch signaling, which inhibits RUNX2 [109, 110] [section “Notch Signaling”]. A
comprehensive understanding of GC-mediated regulation of RUNX2 activity may
ultimately lead to the development of novel therapeutic approaches for the manage-
ment of GIO.
Additional Transcription Factors
The suppression of RUNX2 discussed in the previous section may constitute a criti-
cal mechanism underlying GC-mediated inhibition of osteocalcin, both a clinical
marker of bone formation and a classical model for osteoblast-specific gene expres-
sion. The inhibition of osteocalcin expression by GCs, reproducibly observed both
in vitro and in vivo, both in humans and mice, has been investigated for decades,
with initial reports focusing on GR binding to osteocalcin proximal promoter ele-
ments [20, 195–201]. The inhibition of RUNX2 itself, however, is likely much more
relevant to GIO than the inhibition of Osteocalcin, because Osteocalcin does not
play any critical role in bone formation [202]. Still, an additional mechanism of
osteocalcin transcriptional repression has been discovered using the MC3T3-E1 cell
line, in which GCs do not inhibit Runx2 [56, 185]. In these cells, GCs inhibit osteo-
calcin transcription by strongly repressing expression of Krox20 [48, 203], which
has been implicated in embryonal bone development in vivo [204]. Recent studies,
however, have raised a doubt regarding the role of Krox20 in osteoblast suppression
in GIO because its main function in the adult mouse skeleton in vivo appears to be
inhibition of osteoclastogenesis and bone resorption [205, 206].
Microarray-assisted profiling of gene expression in GC-arrested MC3T3-E1
osteoblast cultures [48] confirmed the GC-mediated stimulation of the adipogenic
regulators C/EBPß and C/EBPδ and the inhibition of Krox20 (see section
“Glucocorticoids Inhibit Osteoblast Differentiation and Function” and previous
paragraph, respectively). Together with Krox20, another zinc finger transcription
factor gene, the Kruppel-like factor 10 gene (Klf10; a.k.a TGFß-inducible growth
response, or Tieg), displayed the strongest suppression (6-fold) in the GC-arrested
as compared to control cultures [48]. The relevance of these repressed transcription
factor genes to GIO, as well as that of GC-stimulated transcription factors including
Klf 13, Period circadian clock 1 (Per1) [48] and Glucocorticoid-Inducible Leucine
Zipper (Gilz) [207], is less certain. Unexpectedly, some of the GC-upregulated
genes play positive roles in osteoblast differentiation [207] and may explain para-
doxical anabolic effects of GCs. Alternatively, these genes may play a role in GIO
by abrogating a finely tuned circadian rhythm of gene expression [208, 209], and
thus mediate the impact of GCs on proliferation and differentiation of osteoblasts.
Glucocorticoids Without Osteoporosis?
The current standard of care for GIO management is administration of bisphospho-

nates, which suppresses osteoclast activity. In contrast to high turnover osteoporosis
(e.g., after estrogen loss), the use of bisphosphonates for patients undergoing long-
term GC therapy is questionable, because it does not address osteoblast suppression
and abrogation of bone formation, the hallmark of GIO. In fact, the outcome of
bisphosphonate therapy for GIO is a further decrease of the bone turnover rate that
is already low due to GC administration [15]. In this sense, intermittent treatment
with recombinant PTH appears better suited for the management of GIO because it
increases bone mass through stimulation of osteoblast function, directly counteract-
ing adverse effects of GCs in osteoblasts [15, 210]. However, PTH therapy is costly,
limited to 18–24 months, and requires daily subcutaneous injections, which together
warrant efforts for the development of solutions more suitable for the prevention
and treatment of GIO.
At least two further strategies can be envisioned for the development of bone-
sparing GC-based therapy. One option is to develop GR ligands, which promote its
anti-inflammatory properties without eliciting adverse effects in osteoblasts. This

could be accomplished as a selective GR modulator monotherapy, or by taking advan-
tage of bone targeting strategies [211]. Alternatively, rather than targeting GR itself,
one can envision the development of combined therapies, whereby conventional GCs
are administered along with an anabolic compound, preferably one that restores
mechanisms that are compromised in osteoblasts and osteocytes exposed to GCs.
Dissociated Glucocorticoids
Transactivation versus Transrepression: A Model Too Simplistic for the

Development of Osteoblast-Forgiving, Bone-Sparing GCs
A strategy used by pharmaceutical companies to reduce side effects of steroid therapy

such as osteoporosis has been the design of ligands that avoid GR dimerization-depen-
dent transactivation, but still transrepress inflammatory genes [212]. Limitations of
this strategy, however, have quickly come to attention as compounds that specifically
promote transrepression were not necessarily successful in avoiding bone loss [213].
Furthermore, such compounds demonstrated limited efficacy, and only a few have
been tested in clinical trials [214]. Part of the problem with this approach was that first
generation steroidal SGRMs were identified in in vitro high throughput screens, but
appeared to be metabolized in vivo into non-dissociated compounds. It remains to be
seen if second generation non-steroidal SGRMs will avoid such shortcomings.
The limited clinical success in identifying bone-sparing GR ligands also reflects
under-appreciation of the complexity of GR-mediated transcriptional control.
Basically, it was assumed that GR employs fundamentally different mechanisms in
mediating immune suppression versus side effects. Specifically, an over-simplistic
paradigm predominated, which associated side effects with GR homodimers at palin-
dromic GREs, while ascribing the repression of pro-inflammatory genes to a tether-
ing mechanism whereby the GR is recruited to pro-inflammatory genes by transcription
factors such as NF-κB, AP-1 and IRF3 [215]. Recent progress, however, revealed a
more complex picture. Specifically, chromatin immunoprecipitation (ChIP)-based
techniques are currently allowing investigators to map transcription factor locations,
as well as capture large-scale chromatin landscapes genome wide. The results high-
light cell type-dependent GR-mediated mechanisms that are specific for activation
states of cells and individual genes. Based on the emerging paradigm, the limited
success of prior efforts is not surprising because both the immune suppression prop-
erty and the side effects of GCs employ similar fundamental mechanisms of action.
According to the new paradigm, cell type-specific effects of GCs are based on the
principle that GR binding is dependent to a large extent on chromatin accessibility,
which itself is tissue-specific [216]. Furthermore, direct binding of GR monomers to
DNA seems to be more common than initially anticipated and treatment with pharma-
cological GCs favors binding of GR dimers to classical palindromic DNA elements
[26, 27]. Tissue-specific accessibility, in turn, depends on histone modifications and
nucleosome positioning within gene regulatory regions [217]. Transcription factors
such as C/EBP in liver and fat cells [218, 219], Stat3 in pituitary cells, and AP-1 in
mammary epithelial cells [220] often determine accessibility. They are often lineage-
specific master regulators, and serve as pioneering transcription factors. Thus, the
view of interactions between GR and other transcription factors is changing: early
studies of individual genes were mostly interpreted as tethering of the GR by other
transcription factors via direct protein-protein interaction. The more recent, genome
wide analyses, however, suggest a more complex picture. For example the combinato-
rial activation of GR and NF-κB leads to the creation of novel binding sites in addition
to those occupied after activation of either GR alone or NF-κB alone [221]. Moreover
NF-κB target genes sometimes bear GR DNA binding sites close to the NF-κB sites,
leading to gene activation or repression dependent on the inflammatory state of innate
immune cells [222]. These complex regulatory mechanisms explain, in retrospect,
why early attempts to develop bone-sparing dissociating GR ligands were unsuccess-
ful; they relied on transactivation/transrepression of a small number of genes and
reporter constructs, which miss the big picture, where GR-mediated transcriptional
control is heavily context-dependent. It greatly varies as a function of the individual
target gene and it strongly depends on the cellular milieu, which itself depends on the
cell type, on whether it is cycling or quiescent, and on the stage of differentiation.
New Requirements for Selective GR Agonists
New principles are sought after towards the identification of bone-sparing glucocor-
ticoids, which do not depend on dissociating dimerization-dependent transactivation
from dimerization-independent transrepression. Candidate dissociating ligands will
have to be assessed for their bone-sparing property in primary cell systems. That
such efforts may be fruitful is demonstrated by the discovery of the plant-derived GR
ligand compound A (CpdA). CpdA does not compromise osteoblast differentiation
[223]. In contrast to the classical ligand dex, CpdA does not antagonize AP-1-
dependent IL-11 expression [223], a pivotal mechanism leading to inhibition of
osteoblast differentiation [19]. Consequently, the anti-inflammatory activity of CpdA
in arthritis [224, 225], experimental autoimmune encephalomyelitis (EAE) [226,
227] and asthma [225], which is attributable to inhibition of NF-κB [225] and thus
decreased levels of cytokines such as IL-6 [223], is not associated with a decrease in
bone mass [229]. Future cell-based screens may result in the discovery of additional
GR ligands that spare osteoblasts, and some of these ligands may serve as lead com-
pounds for the development of novel bone-sparing anti-inflammatory GC drugs.
Aiming at GR Targets
In addition to employing novel strategies for identification and optimization of

novel GR ligands, parallel efforts are warranted towards the development of
improved combination therapies for GIO. An optimal combination therapy would
replace bisphosphonates or PTH with a treatment modality best suited to counteract

pathogenic mechanisms of GIO. Several examples are provided below.
IL11 (interleukin-11), LIF1 (leukemia inhibitory factor 1). As described in sec-
tion “AP-1”, GCs inhibit AP-1-mediated stimulation of Il11 in osteoblasts in
vitro and in vivo. The GC-mediated decrease in IL11, as well as LIF1 expression,
appears to significantly contribute to the inhibition of osteoblast differentiation [19,
176]. Supplementation of LIF and IL11 in animal models counteracts GIO at least
partially (unpublished observations), consistent with data from transgenic mouse
models indicating an important role for IL-11 in bone formation and bone mass
control in vivo [230]. However, the safety profile of IL-11, as well as LIF, can be
problematic; due to their pleiotropic effects, they might induce hematologic and
other complications [231].
DKK1, sclerostin (SOST). We reviewed in section “Glucocorticoids Inhibit Wnt
Signaling in Osteoblasts” evidence for the critical role of the Wnt pathway in osteo-
blast replication, differentiation and survival, and cited many lines of evidence
implicating inhibition of Wnt signaling in GIO. One of the most appealing lines of
evidence is the direct stimulation of Dkk1, a Wnt inhibitor, by GCs (section “Role
of Dkks in GIO”). Regardless of whether or not GCs stimulate Dkk-1 expression
in vivo as they do in vitro (see section “Role of Dkks in GIO”), restoration of Wnt
signaling is an attractive option for the management of GIO. This can be achieved,
for example, by neutralizing DKK1 using anti-DKK1 antibodies. Another potential
approach to restore Wnt signaling in GC-treated patients is co-administration of
antibodies against sclerostin, another Wnt antagonist. An advantage of the latter is
that the sclerostin-coding gene Sost is primarily expressed in osteocytes [232], and
its neutralization is therefore less likely to have undesirable extraskeletal effects.
Encouraging results from a recent preclinical study, in which mice were co-treated
with GCs and anti-sclerostin antibodies, warrant further efforts towards the devel-
opment of Wnt-targeting strategies for the management of GIO [233].
microRNAs. Restoration of Wnt signaling in the presence of GCs may also be
achieved by manipulation of microRNAs, an emerging therapeutic modality that
remains to be exploited in the context of GIO. We reviewed in section “Additional
Wnt-Related GC Targets” the suppression of HDAC4 by mir29-a and the conse-
quential stimulation of β-catenin and the osteoblast phenotype. Because GCs inhibit
mir29-a expression, its pharmacological stimulation appears an attractive avenue
towards shielding osteoblasts from adverse effects of GCs.
Unbiased screens. With the advent of high throughput screening technologies, iden-
tification of lead compounds, steroidal or otherwise, for the management of GIO
may be achieved in an agnostic fashion. Synthetic steroids or large chemical librar-
ies can be screened in the presence of dex, for example, using cell-based assays that
report on cellular features such as proliferation, differentiation and/or apoptosis
[234]. Alternatively, the assay, typically fluorescent, may report on a distinct aspect
of osteoblast differentiation. For example, the readout may reflect the activity of
ALP, RUNX2, or a molecular pathway such as Notch or Wnt signaling [235].
Establishing such assays in primary mesenchymal stem cells, pre-osteoblastic cells

or osteocytes may be challenging, but worth the effort. With recent advancements in
the understanding of cellular and molecular mechanisms underlying GIO, the iden-
tification of target genes and compounds for combating this devastating iatrogenic
disease is increasingly within reach.
Acknowledgements We thank Gillian H. Little, Michael R. Stallcup (University of Southern

California) and Tamas Röszer (University of Ulm) for insightful comments. BF, who holds the
J. Harold and Edna L. LaBriola Chair in Genetic Orthopaedic Research, acknowledges support
from the National Institutes of Health (RO1 DK071122). JT acknowledges support from Deutsche
Forschungsgemeinschaft Immunbone (SPP 1468 Tu220/6-2 INST 40/492 SFB 1149, Collaborative
Research Centre 1149) and from KaroBioScience Foundation.
References
1. Soen S, Tanaka Y. Glucocorticoid-induced osteoporosis: skeletal manifestations of glucocor-

ticoid use and 2004 Japanese Society for Bone and Mineral Research-proposed guidelines for
its management. Mod Rheumatol. 2005;15:163–8.
2. van Staa TP, et al. A simple score for estimating the long-term risk of fracture in patients
using oral glucocorticoids. QJM. 2005;98:191–8.
3. Van Staa TP, Leufkens HG, Abenhaim L, Zhang B, Cooper C. Use of oral corticosteroids and
risk of fractures. J Bone Miner Res. 2000;15:993–1000.
4. van Staa TP, Leufkens HG, Cooper C. The epidemiology of corticosteroid-induced osteopo-
rosis: a meta-analysis. Osteoporos Int. 2002;13:777–87.
5. Lane NE, et al. Glucocorticoid-treated mice have localized changes in trabecular bone mate-
rial properties and osteocyte lacunar size that are not observed in placebo-treated or estrogen-
deficient mice. J Bone Miner Res. 2006;21:466–76.
6. Laan RF, et al. Low-dose prednisone induces rapid reversible axial bone loss in patients with
rheumatoid arthritis. A randomized, controlled study. Ann Intern Med. 1993;119:963–8.
7. McKenzie R, et al. Decreased bone mineral density during low dose glucocorticoid adminis-
tration in a randomized, placebo controlled trial. J Rheumatol. 2000;27:2222–6.
8. Aaron JE, Francis RM, Peacock M, Makins NB. Contrasting microanatomy of idiopathic and
corticosteroid-induced osteoporosis. Clin Orthop Relat Res. 1989;243:294–305.
9. Laan RF, et al. Differential effects of glucocorticoids on cortical appendicular and cortical
vertebral bone mineral content. Calcif Tissue Int. 1993;52:5–9.
10. Maggi S, et al. Osteoporosis risk in patients with chronic obstructive pulmonary disease: the
EOLO study. J Clin Densitom. 2009;12:345–52.
11. Gonnelli S, et al. Effect of inhaled glucocorticoids and beta(2) agonists on vertebral fracture
risk in COPD patients: the EOLO study. Calcif Tissue Int. 2010;87:137–43.
12. Weinstein RS. Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinol Metab
Clin North Am. 2012;41:595–611.
13. Canalis E, Mazziotti G, Giustina A, Bilezikian JP. Glucocorticoid-induced osteoporosis:
pathophysiology and therapy. Osteoporos Int. 2007;18:1319–28.
14. Grossman JM, et al. American College of Rheumatology 2010 recommendations for the pre-
vention and treatment of glucocorticoid-induced osteoporosis. Arthritis Care Res.
2010;62:1515–26.
15. Weinstein RS, Jilka RL, Almeida M, Roberson PK, Manolagas SC. Intermittent parathyroid
hormone administration counteracts the adverse effects of glucocorticoids on osteoblast and
osteocyte viability, bone formation, and strength in mice. Endocrinology. 2010;151:2641–9.
16. Gluer CC, et al. Comparative effects of teriparatide and risedronate in glucocorticoid-induced
osteoporosis in men: 18-month results of the EuroGIOPs trial. J Bone Miner Res. 2013;28:
1355–68.
17. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC. Inhibition of osteoblastogenesis and
promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mecha-
nisms of their deleterious effects on bone. J Clin Invest. 1998;102:274–82.
18. Hofbauer LC, et al. Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin
production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mecha-
nisms of glucocorticoid-induced osteoporosis. Endocrinology. 1999;140:4382–9.
19. Rauch A, et al. Glucocorticoids suppress bone formation by attenuating osteoblast differen-
tiation via the monomeric glucocorticoid receptor. Cell Metab. 2010;11:517–31.
20. Yao W, et al. Glucocorticoid excess in mice results in early activation of osteoclastogenesis
and adipogenesis and prolonged suppression of osteogenesis: a longitudinal study of gene
expression in bone tissue from glucocorticoid-treated mice. Arthritis Rheum. 2008;58:
1674–86.
21. O’Brien CA, et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their
apoptosis and reduce bone formation and strength. Endocrinology. 2004;145:1835–41.
22. Sher LB, et al. Transgenic expression of 11beta-hydroxysteroid dehydrogenase type 2 in
osteoblasts reveals an anabolic role for endogenous glucocorticoids in bone. Endocrinology.
2004;145:922–9.
23. Yang M, et al. Col3.6-HSD2 transgenic mice: a glucocorticoid loss-of-function model span-
ning early and late osteoblast differentiation. Bone. 2010;47:573–82.
24. Reichardt HM, et al. Repression of inflammatory responses in the absence of DNA binding
by the glucocorticoid receptor. EMBO J. 2001;20:7168–73.
25. Reichardt HM, et al. DNA binding of the glucocorticoid receptor is not essential for survival.
Cell. 1998;93:531–41.
26. Lim HW et al. Genomic redistribution of GR monomers and dimers mediates transcriptional
response to exogenous glucocorticoids in vivo. Genome Research 2015 May 8 [Epub ahead
of print].
binds half sites as a monomer and regulates specific target genes. Genome Biol. 2014;15:3181.
28. Weinstein RS, et al. Promotion of osteoclast survival and antagonism of bisphosphonate-
induced osteoclast apoptosis by glucocorticoids. J Clin Invest. 2002;109:1041–8.
29. Kim HJ, et al. Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest.
2006;116:2152–60.
30. Jia D, O’Brien CA, Stewart SA, Manolagas SC, Weinstein RS. Glucocorticoids act directly
on osteoclasts to increase their life span and reduce bone density. Endocrinology. 2006;
147:5592–9.
31. Alesci S, De Martino MU, Ilias I, Gold PW, Chrousos GP. Glucocorticoid-induced osteopo-
rosis: from basic mechanisms to clinical aspects. Neuroimmunomodulation. 2005;12:1–19.
32. Moutsatsou P, Kassi E, Papavassiliou AG. Glucocorticoid receptor signaling in bone cells.
Trends Mol Med. 2012;18:348–59.
33. Baschant U, Lane NE, Tuckermann J. The multiple facets of glucocorticoid action in rheuma-
toid arthritis. Nat Rev Rheumatol. 2012;8:645–55.
34. Weinstein RS, et al. The skeletal effects of glucocorticoid excess override those of orchidec-
tomy in mice. Endocrinology. 2004;145:1980–7.
35. Chen TL, Aronow L, Feldman D. Glucocorticoid receptors and inhibition of bone cell growth
in primary culture. Endocrinology. 1977;100:619–28.
36. Smith E, et al. Glucocorticoids inhibit developmental stage-specific osteoblast cell cycle.
Dissociation of cyclin A-cyclin-dependent kinase 2 from E2F4-p130 complexes. J Biol
Chem. 2000;275:19992–20001.
37. Gabet Y, Noh T, Lee C, Frenkel B. Developmentally regulated inhibition of cell cycle pro-
gression by glucocorticoids through repression of cyclin A transcription in primary osteo-
blast cultures. J Cell Physiol. 2011;226:991–8.
38. Li H, et al. Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates
apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells. PLoS One. 2012;7:e37030.
39. Chang JK, et al. Anti-inflammatory drugs suppress proliferation and induce apoptosis through
altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human
osteoblasts. Toxicology. 2009;258:148–56.
40. Rogatsky I, Trowbridge JM, Garabedian MJ. Glucocorticoid receptor-mediated cell cycle
arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms. Mol
Cell Biol. 1997;17:3181–93.
41. Engelbrecht Y, et al. Glucocorticoids induce rapid up-regulation of mitogen-activated protein
kinase phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase and
impair proliferation in human and mouse osteoblast cell lines. Endocrinology. 2003;144:
412–22.
42. Lecka-Czernik B, et al. Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differen-
tiation by PPARgamma2. J Cell Biochem. 1999;74:357–71.
43. Pereira RC, Delany AM, Canalis E. Effects of cortisol and bone morphogenetic protein-2 on
stromal cell differentiation: correlation with CCAAT-enhancer binding protein expression.
Bone. 2002;30:685–91.
44. Rosen CJ, Bouxsein ML. Mechanisms of disease: is osteoporosis the obesity of bone? Nat
Clin Pract Rheumatol. 2006;2:35–43.
45. Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrow-derived
mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci.
2009;66:236–53.
46. Naito M, Omoteyama K, Mikami Y, Takahashi T, Takagi M. Inhibition of Wnt/beta-catenin
signaling by dexamethasone promotes adipocyte differentiation in mesenchymal progenitor
cells, ROB-C26. Histochem Cell Biol. 2012;138:833–45.
47. Berendsen AD, Olsen BR. Osteoblast-adipocyte lineage plasticity in tissue development,
maintenance and pathology. Cell Mol Life Sci. 2014;71:493–7.
48. Leclerc N, et al. Gene expression profiling of glucocorticoid-inhibited osteoblasts. J Mol
Endocrinol. 2004;33:175–93.
49. Smith E, Coetzee GA, Frenkel B. Glucocorticoids inhibit cell cycle progression in differenti-
ating osteoblasts via glycogen synthase kinase-3beta. J Biol Chem. 2002;277:18191–7.
50. Mikami Y, Lee M, Irie S, Honda MJ. Dexamethasone modulates osteogenesis and adipogen-
esis with regulation of osterix expression in rat calvaria-derived cells. J Cell Physiol.
2011;226:739–48.
51. Worthley et al. Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular
Stromal Potential Cell. 2015;160:269–284.
52. Chan et al. Identification and Specification of the Mouse Skeletal Stem Cell. Cell. 2015;
160:285–298.
53. Canalis E. Effect of glucocorticoids on type I collagen synthesis, alkaline phosphatase activ-
ity, and deoxyribonucleic acid content in cultured rat calvariae. Endocrinology. 1983;112:
931–9.
54. Harris C, et al. Large increases in adipose triacylglycerol flux in Cushingoid CRH-Tg mice
are explained by futile cycling. Am J Physiol Endocrinol Metab. 2013;304:E282–93.
55. Delany AM, Gabbitas BY, Canalis E. Cortisol downregulates osteoblast alpha 1 (I) procolla-
gen mRNA by transcriptional and posttranscriptional mechanisms. J Cell Biochem.
1995;57:488–94.
56. Luppen CA, et al. Brief bone morphogenetic protein 2 treatment of glucocorticoid-inhibited
MC3T3-E1 osteoblasts rescues commitment-associated cell cycle and mineralization without
alteration of Runx2. J Biol Chem. 2003;278:44995–5003.
57. Delany AM, Jeffrey JJ, Rydziel S, Canalis E. Cortisol increases interstitial collagenase
expression in osteoblasts by post-transcriptional mechanisms. J Biol Chem. 1995;270:
26607–12.
58. Lian JB, et al. Species-specific glucocorticoid and 1,25-dihydroxyvitamin D responsiveness

in mouse MC3T3-E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vita-
min D down-regulates osteocalcin gene expression. Endocrinology. 1997;138:2117–27.
59. Chen TL, Fry D. Hormonal regulation of the osteoblastic phenotype expression in neonatal
murine calvarial cells. Calcif Tissue Int. 1999;64:304–9.
60. Butler JS, et al. Silencing Dkk1 expression rescues dexamethasone-induced suppression of
primary human osteoblast differentiation. BMC Musculoskelet Disord. 2010;11:210.
61. Koromila T, et al. Glucocorticoids antagonize RUNX2 during osteoblast differentiation in
cultures of ST2 pluripotent mesenchymal cells. J Cell Biochem. 2014;115:27–33.
62. Wang FS, et al. Secreted frizzled-related protein 1 modulates glucocorticoid attenuation of
osteogenic activities and bone mass. Endocrinology. 2005;146:2415–23.
63. Subramaniam M, et al. Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and
proto-oncogenes in normal human osteoblast-like cells. J Cell Biochem. 1992;50:411–24.
64. Cheng SL, Yang JW, Rifas L, Zhang SF, Avioli LV. Differentiation of human bone marrow
osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone.
Endocrinology. 1994;134:277–86.
65. Shalhoub V, et al. Glucocorticoids promote development of the osteoblast phenotype by
selectively modulating expression of cell growth and differentiation associated genes. J Cell
Biochem. 1992;50:425–40.
66. Ishida Y, Heersche JN. Glucocorticoid-induced osteoporosis: both in vivo and in vitro con-
centrations of glucocorticoids higher than physiological levels attenuate osteoblast differen-
tiation. J Bone Miner Res. 1998;13:1822–6.
67. Luppen CA, Chandler RL, Noh T, Mortlock DP, Frenkel B. BMP-2 vs. BMP-4 expression
and activity in glucocorticoid-arrested MC3T3-E1 osteoblasts: Smad signaling, not alkaline
phosphatase activity, predicts rescue of mineralization. Growth Factors. 2008;26:226–37.
68. Luppen CA, Smith E, Spevak L, Boskey AL, Frenkel B. Bone morphogenetic protein-2
restores mineralization in glucocorticoid-inhibited MC3T3-E1 osteoblast cultures. J Bone
Miner Res. 2003;18:1186–97.
69. Smith E, et al. Expression of cell cycle regulatory factors in differentiating osteoblasts: post-
proliferative up-regulation of cyclins B and E. Cancer Res. 1995;55:5019–24.
70. Smith E, et al. Post-proliferative cyclin E-associated kinase activity in differentiated osteo-
blasts: inhibition by proliferating osteoblasts and osteosarcoma cells. J Cell Biochem.
1997;66:141–52.
71. Carcamo-Orive I, et al. Regulation of human bone marrow stromal cell proliferation and dif-
ferentiation capacity by glucocorticoid receptor and AP-1 crosstalk. J Bone Miner Res. 2010;
25:2115–25.
72. Gu G, Hentunen TA, Nars M, Harkonen PL, Vaananen HK. Estrogen protects primary osteo-
cytes against glucocorticoid-induced apoptosis. Apoptosis. 2005;10:583–95.
73. Liu Y, et al. Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by
calbindin-D28k. J Bone Miner Res. 2004;19:479–90.
74. Hurson CJ, et al. Gene expression analysis in human osteoblasts exposed to dexamethasone
identifies altered developmental pathways as putative drivers of osteoporosis. BMC
Musculoskelet Disord. 2007;8:12.
75. Conradie MM, et al. Vanadate prevents glucocorticoid-induced apoptosis of osteoblasts
in vitro and osteocytes in vivo. J Endocrinol. 2007;195:229–40.
76. Zalavras C, Shah S, Birnbaum MJ, Frenkel B. Role of apoptosis in glucocorticoid-induced
osteoporosis and osteonecrosis. Crit Rev Eukaryot Gene Expr. 2003;13:221–35.
77. Nakashima T, et al. Evidence for osteocyte regulation of bone homeostasis through RANKL
expression. Nat Med. 2011;17:1231–4.
78. Xiong J, et al. Matrix-embedded cells control osteoclast formation. Nat Med. 2011;17:
1235–41.
79. Dallas SL, Prideaux M, Bonewald LF. The osteocyte: an endocrine cell … and more. Endocr
Rev. 2013;34:658–90.
80. Jia J, et al. Glucocorticoid dose determines osteocyte cell fate. FASEB J. 2011;25:3366–76.
81. Gu G, Mulari M, Peng Z, Hentunen TA, Vaananen HK. Death of osteocytes turns off the
inhibition of osteoclasts and triggers local bone resorption. Biochem Biophys Res Commun.
2005;335:1095–101.
82. Monroe DG, McGee-Lawrence ME, Oursler MJ, Westendorf JJ. Update on Wnt signaling in
bone cell biology and bone disease. Gene. 2012;492:1–18.
83. Habib SJ, et al. A localized Wnt signal orients asymmetric stem cell division in vitro. Science.
2013;339:1445–8.
84. Gong Y, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye develop-
ment. Cell. 2001;107:513–23.
85. Little RD, et al. A mutation in the LDL receptor-related protein 5 gene results in the autoso-
mal dominant high-bone-mass trait. Am J Hum Genet. 2002;70:11–9.
86. Boyden LM, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N
Engl J Med. 2002;346:1513–21.
87. Smith E, Frenkel B. Glucocorticoids inhibit the transcriptional activity of LEF/TCF in
differentiating osteoblasts in a glycogen synthase kinase-3beta-dependent and -independent
manner. J Biol Chem. 2005;280:2388–94.
88. Ohnaka K, Tanabe M, Kawate H, Nawata H, Takayanagi R. Glucocorticoid suppresses the
canonical Wnt signal in cultured human osteoblasts. Biochem Biophys Res Commun.
2005;329:177–81.
89. Almeida M, Han L, Ambrogini E, Weinstein RS, Manolagas SC. Glucocorticoids and tumor
necrosis factor alpha increase oxidative stress and suppress Wnt protein signaling in osteo-
blasts. J Biol Chem. 2011;286:44326–35.
90. Naito M, Mikami Y, Takagi M, Takahashi T. Up-regulation of Axin2 by dexamethasone
promotes adipocyte differentiation in ROB-C26 mesenchymal progenitor cells. Cell Tissue
Res. 2013;354:761–70.
91. Ohnaka K, Taniguchi H, Kawate H, Nawata H, Takayanagi R. Glucocorticoid enhances the
expression of dickkopf-1 in human osteoblasts: novel mechanism of glucocorticoid-induced
osteoporosis. Biochem Biophys Res Commun. 2004;318:259–64.
92. Mak W, Shao X, Dunstan CR, Seibel MJ, Zhou H. Biphasic glucocorticoid-dependent regulation
of Wnt expression and its inhibitors in mature osteoblastic cells. Calcif Tissue Int.
2009;85:538–45.
93. Cruciat CM, Niehrs C. Secreted and transmembrane wnt inhibitors and activators. Cold
Spring Harb Perspect Biol. 2013;5:a015081.
94. Bafico A, Liu G, Yaniv A, Gazit A, Aaronson SA. Novel mechanism of Wnt signalling inhibi-
tion mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat Cell Biol. 2001;3:683–6.
95. Patel MS, Karsenty G. Regulation of bone formation and vision by LRP5. N Engl J Med.
2002;346:1572–4.
96. Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature.
2003;423:349–55.
97. Morvan F, et al. Deletion of a single allele of the Dkk1 gene leads to an increase in bone
formation and bone mass. J Bone Miner Res. 2006;21:934–45.
98. MacDonald BT, et al. Bone mass is inversely proportional to Dkk1 levels in mice. Bone.
2007;41:331–9.
99. Brunetti G, et al. High dickkopf-1 levels in sera and leukocytes from children with
21-hydroxylase deficiency on chronic glucocorticoid treatment. Am J Physiol Endocrinol
Metab. 2013;304:E546–54.
100. Gifre L, et al. Effect of glucocorticoid treatment on Wnt signalling antagonists (sclerostin and
Dkk-1) and their relationship with bone turnover. Bone. 2013;57:272–6.
101. Jones SE, Jomary C. Secreted frizzled-related proteins: searching for relationships and pat-
terns. Bioessays. 2002;24:811–20.
102. Bodine PV, et al. The Wnt antagonist secreted frizzled-related protein-1 is a negative regula-
tor of trabecular bone formation in adult mice. Mol Endocrinol. 2004;18:1222–37.
103. Takayama S, Rogatsky I, Schwarcz LE, Darimont BD. The glucocorticoid receptor represses
cyclin D1 by targeting the Tcf-beta-catenin complex. J Biol Chem. 2006;281:17856–63.
104. Olkku A, Mahonen A. Calreticulin mediated glucocorticoid receptor export is involved in

beta-catenin translocation and Wnt signalling inhibition in human osteoblastic cells. Bone.
2009;44:555–65.
105. Ko J-Y, et al. MicroRNA-29a ameliorates glucocorticoid-induced suppression of osteoblast
differentiation by regulating β-catenin acetylation. Bone. 2013;57:468–75.
106. Nuttall ME, Patton AJ, Olivera DL, Nadeau DP, Gowen M. Human trabecular bone cells are
able to express both osteoblastic and adipocytic phenotype: implications for osteopenic dis-
orders. J Bone Miner Res. 1998;13:371–82.
107. Hayashi K, et al. BMP/Wnt antagonists are upregulated by dexamethasone in osteoblasts and
reversed by alendronate and PTH: potential therapeutic targets for glucocorticoid-induced
osteoporosis. Biochem Biophys Res Commun. 2009;379:261–6.
108. Pereira RM, Delany AM, Durant D, Canalis E. Cortisol regulates the expression of Notch in
osteoblasts. J Cell Biochem. 2002;85:252–8.
109. Engin F, et al. Dimorphic effects of Notch signaling in bone homeostasis. Nat Med.
2008;14:299–305.
110. Hilton MJ, et al. Notch signaling maintains bone marrow mesenchymal progenitors by
suppressing osteoblast differentiation. Nat Med. 2008;14:306–14.
111. Zanotti S, Canalis E. Notch1 and Notch2 expression in osteoblast precursors regulates femo-
ral microarchitecture. Bone. 2014;62:22–8.
112. Kamiya N, et al. BMP signaling negatively regulates bone mass through sclerostin by inhibit-
ing the canonical Wnt pathway. Development. 2008;135:3801–11.
113. Skrtic S, Ohlsson C. Cortisol decreases hepatocyte growth factor levels in human osteoblast-
like cells. Calcif Tissue Int. 2000;66:108–12.
114. Tsunashima Y, Kondo A, Matsuda T, Togari A. Hydrocortisone inhibits cellular proliferation
by downregulating hepatocyte growth factor synthesis in human osteoblasts. Biol Pharm
Bull. 2011;34:700–3.
115. Grano M, et al. Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts
in vitro. Proc Natl Acad Sci U S A. 1996;93:7644–8.
116. Giustina A, Mazziotti G, Canalis E. Growth hormone, insulin-like growth factors, and the
skeleton. Endocr Rev. 2008;29:535–59.
117. McCarthy TL, Centrella M, Canalis E. Cortisol inhibits the synthesis of insulin-like growth
factor-I in skeletal cells. Endocrinology. 1990;126:1569–75.
118. Delany AM, Canalis E. Transcriptional repression of insulin-like growth factor I by glucocor-
ticoids in rat bone cells. Endocrinology. 1995;136:4776–81.
119. Delany AM, Durant D, Canalis E. Glucocorticoid suppression of IGF I transcription in osteo-
blasts. Mol Endocrinol. 2001;15:1781–9.
120. Chevalley T, Strong DD, Mohan S, Baylink D, Linkhart TA. Evidence for a role for insulin-
like growth factor binding proteins in glucocorticoid inhibition of normal human osteoblast-
like cell proliferation. Eur J Endocrinol. 1996;134:591–601.
121. Gabbitas B, Pash JM, Delany AM, Canalis E. Cortisol inhibits the synthesis of insulin-like
growth factor-binding protein-5 in bone cell cultures by transcriptional mechanisms. J Biol
Chem. 1996;271:9033–8.
122. Andress DL, Birnbaum RS. Human osteoblast-derived insulin-like growth factor (IGF) bind-
ing protein-5 stimulates osteoblast mitogenesis and potentiates IGF action. J Biol Chem.
1992;267:22467–72.
123. Richman C, Baylink DJ, Lang K, Dony C, Mohan S. Recombinant human insulin-like growth
factor-binding protein-5 stimulates bone formation parameters in vitro and in vivo.
Endocrinology. 1999;140:4699–705.
124. Miyakoshi N, et al. Evidence that IGF-binding protein-5 functions as a growth factor. J Clin
Invest. 2001;107:73–81.
125. Salih DA, et al. Insulin-like growth factor-binding protein-5 induces a gender-related decrease
in bone mineral density in transgenic mice. Endocrinology. 2005;146:931–40.
126. Chaudhary LR, Avioli LV. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1
and ERK2) by FGF-2 and PDGF-BB in normal human osteoblastic and bone marrow stromal
cells: differences in mobility and in-gel renaturation of ERK1 in human, rat, and mouse
osteoblastic cells. Biochem Biophys Res Commun. 1997;238:134–9.
127. Xiao G, et al. Bone morphogenetic proteins, extracellular matrix, and mitogen-activated pro-
tein kinase signaling pathways are required for osteoblast-specific gene expression and dif-
ferentiation in MC3T3-E1 cells. J Bone Miner Res. 2002;17:101–10.
128. Xiao G, Jiang D, Gopalakrishnan R, Franceschi RT. Fibroblast growth factor 2 induction of
the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcrip-
tion factor, Cbfa1/Runx2. J Biol Chem. 2002;277:36181–7.
129. Greenblatt MB, Shim JH, Glimcher LH. Mitogen-activated protein kinase pathways in osteo-
blasts. Annu Rev Cell Dev Biol. 2013;29:63–79.
130. Xiao G, et al. MAPK pathways activate and phosphorylate the osteoblast-specific transcription
factor, Cbfa1. J Biol Chem. 2000;275:4453–9.
131. Li Y, Ge C, Franceschi RT. Differentiation-dependent association of phosphorylated extracel-
lular signal-regulated kinase with the chromatin of osteoblast-related genes. J Bone Miner
Res. 2010;25:154–63.
132. Lai CF, et al. Erk is essential for growth, differentiation, integrin expression, and cell function
in human osteoblastic cells. J Biol Chem. 2001;276:14443–50.
133. Ge C, Xiao G, Jiang D, Franceschi RT. Critical role of the extracellular signal-regulated
kinase-MAPK pathway in osteoblast differentiation and skeletal development. J Cell Biol.
2007;176:709–18.
134. Hulley PA, Gordon F, Hough FS. Inhibition of mitogen-activated protein kinase activity and
proliferation of an early osteoblast cell line (MBA 15.4) by dexamethasone: role of protein
phosphatases. Endocrinology. 1998;139:2423–31.
135. Xian L, et al. Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal
stem cells. Nat Med. 2012;18:1095–101.
136. Yamamoto T, et al. Parathyroid hormone activates phosphoinositide 3-kinase-Akt-Bad
cascade in osteoblast-like cells. Bone. 2007;40:354–9.
137. Mukherjee A, Rotwein P. Akt promotes BMP2-mediated osteoblast differentiation and bone
development. J Cell Sci. 2009;122:716–26.
138. Liang J, Slingerland JM. Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progres-
sion. Cell Cycle. 2003;2:339–45.
139. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the PI3K/AKT pathway for
cancer drug discovery. Nat Rev Drug Discov. 2005;4:988–1004.
140. Fujita T, et al. Runx2 induces osteoblast and chondrocyte differentiation and enhances their
migration by coupling with PI3K-Akt signaling. J Cell Biol. 2004;166:85–95.
141. Choi YH, et al. Akt enhances Runx2 protein stability by regulating Smurf2 function during
osteoblast differentiation. FEBS J. 2014;281(16):3656–66.
142. Borgatti P, et al. Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells
exposed to proliferative growth factors. FEBS Lett. 2000;477:27–32.
143. Almeida M, Han L, Martin-Millan M, O’Brien CA, Manolagas SC. Oxidative stress antago-
nizes Wnt signaling in osteoblast precursors by diverting beta-catenin from T cell factor- to
forkhead box O-mediated transcription. J Biol Chem. 2007;282:27298–305.
144. Plotkin LI, Manolagas SC, Bellido T. Glucocorticoids induce osteocyte apoptosis by block-
ing focal adhesion kinase-mediated survival. Evidence for inside-out signaling leading to
anoikis. J Biol Chem. 2007;282:24120–30.
145. Hulley PA, Conradie MM, Langeveldt CR, Hough FS. Glucocorticoid-induced osteoporosis
in the rat is prevented by the tyrosine phosphatase inhibitor, sodium orthovanadate. Bone.
2002;31:220–9.
146. Clark AR, Lasa M. Crosstalk between glucocorticoids and mitogen-activated protein kinase
signalling pathways. Curr Opin Pharmacol. 2003;3:404–11.
147. Toh ML, Yang Y, Leech M, Santos L, Morand EF. Expression of mitogen-activated protein
kinase phosphatase 1, a negative regulator of the mitogen-activated protein kinases, in rheu-
matoid arthritis: up-regulation by interleukin-1beta and glucocorticoids. Arthritis Rheum.
2004;50:3118–28.
148. Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases: a gene family for control
of MAP kinase function. FASEB J. 2000;14:6–16.
149. Slack DN, Seternes OM, Gabrielsen M, Keyse SM. Distinct binding determinants for ERK2/
p38alpha and JNK map kinases mediate catalytic activation and substrate selectivity of map
kinase phosphatase-1. J Biol Chem. 2001;276:16491–500.
150. Franklin CC, Kraft AS. Conditional expression of the mitogen-activated protein kinase
(MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated protein
kinase in U937 cells. J Biol Chem. 1997;272:16917–23.
151. Kassel O, et al. Glucocorticoids inhibit MAP kinase via increased expression and decreased
degradation of MKP-1. EMBO J. 2001;20:7108–16.
152. Horsch K, et al. Mitogen-activated protein kinase phosphatase 1/dual specificity phosphatase
1 mediates glucocorticoid inhibition of osteoblast proliferation. Mol Endocrinol. 2007;
21:2929–40.
153. Johansson-Haque K, Palanichamy E, Okret S. Stimulation of MAPK-phosphatase 1 gene
expression by glucocorticoids occurs through a tethering mechanism involving C/EBP. J Mol
Endocrinol. 2008;41:239–49.
154. Shipp LE, et al. Transcriptional regulation of human dual specificity protein phosphatase 1
(DUSP1) gene by glucocorticoids. PLoS One. 2010;5:e13754.
155. Conradie MM, et al. MKP-1 knockout does not prevent glucocorticoid-induced bone disease
in mice. Calcif Tissue Int. 2011;89:221–7.
156. Katoh M, Katoh M. Human FOX gene family (Review). Int J Oncol. 2004;25:1495–500.
157. Kenyon C. The plasticity of aging: insights from long-lived mutants. Cell. 2005;120:
449–60.
158. Houstis N, Rosen ED, Lander ES. Reactive oxygen species have a causal role in multiple
forms of insulin resistance. Nature. 2006;440:944–8.
159. Essers MA, et al. FOXO transcription factor activation by oxidative stress mediated by the
small GTPase Ral and JNK. EMBO J. 2004;23:4802–12.
160. Iyer S, et al. FOXOs attenuate bone formation by suppressing Wnt signaling. J Clin Invest.
2013;123:3409–19.
161. Lutzner N, Kalbacher H, Krones-Herzig A, Rosl F. FOXO3 is a glucocorticoid receptor target
and regulates LKB1 and its own expression based on cellular AMP levels via a positive auto-
regulatory loop. PLoS One. 2012;7:e42166.
162. Essers MA, et al. Functional interaction between beta-catenin and FOXO in oxidative stress
signaling. Science. 2005;308:1181–4.
163. Hoogeboom D, et al. Interaction of FOXO with beta-catenin inhibits beta-catenin/T cell fac-
tor activity. J Biol Chem. 2008;283:9224–30.
164. De Bosscher K, Vanden Berghe W, Haegeman G. The interplay between the glucocorticoid
receptor and nuclear factor-kappaB or activator protein-1: molecular mechanisms for gene
repression. Endocr Rev. 2003;24:488–522.
165. Jonat C, et al. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/
Jun) activity by glucocorticoid hormone. Cell. 1990;62:1189–204.
166. Yang-Yen HF, et al. Transcriptional interference between c-Jun and the glucocorticoid recep-
tor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell.
1990;62:1205–15.
167. Subramaniam N, Cairns W, Okret S. Studies on the mechanism of glucocorticoid-mediated
repression from a negative glucocorticoid response element from the bovine prolactin gene.
DNA Cell Biol. 1997;16:153–63.
168. Miner JN, Yamamoto KR. The basic region of AP-1 specifies glucocorticoid receptor activity
at a composite response element. Genes Dev. 1992;6:2491–501.
169. Tuckermann JP, et al. The DNA binding-independent function of the glucocorticoid receptor
mediates repression of AP-1-dependent genes in skin. J Cell Biol. 1999;147:1365–70.
170. McCabe LR, et al. Developmental expression and activities of specific fos and jun proteins
are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentia-
tion. Endocrinology. 1996;137:4398–408.
171. Sabatakos G, et al. Overexpression of DeltaFosB transcription factor(s) increases bone for-
mation and inhibits adipogenesis. Nat Med. 2000;6:985–90.
172. Wagner EF. Functions of AP1 (Fos/Jun) in bone development. Ann Rheum Dis. 2002;61
Suppl 2:ii40–2.
173. Roohk DJ, et al. Dexamethasone-mediated changes in adipose triacylglycerol metabolism are
exaggerated, not diminished, in the absence of a functional GR dimerization domain.
Endocrinology. 2013;154:1528–39.
174. Matsumoto T, Kuriwaka-Kido R, Kondo T, Endo I, Kido S. Regulation of osteoblast differ-
entiation by interleukin-11 via AP-1 and Smad signaling. Endocr J. 2012;59:91–101.
175. Sims NA, et al. Interleukin-11 receptor signaling is required for normal bone remodeling. J
Bone Miner Res. 2005;20:1093–102.
176. Kuriwaka-Kido R, et al. Parathyroid hormone (1-34) counteracts the suppression of interleu-
kin-11 expression by glucocorticoid in murine osteoblasts: a possible mechanism for stimu-
lating osteoblast differentiation against glucocorticoid excess. Endocrinology. 2013;154:
1156–67.
177. Komori T, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation
owing to maturational arrest of osteoblasts [see comments]. Cell. 1997;89:755–64.
178. Otto F, et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for
osteoblast differentiation and bone development [see comments]. Cell. 1997;89:765–71.
179. Banerjee C, et al. Runt homology domain proteins in osteoblast differentiation: AML3/
CBFA1 is a major component of a bone-specific complex. J Cell Biochem. 1997;66:1–8.
180. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator
of osteoblast differentiation [see comments]. Cell. 1997;89:747–54.
181. Komori T. Signaling networks in RUNX2-dependent bone development. J Cell Biochem.
2011;112:750–5.
182. Ducy P, et al. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic
development. Genes Dev. 1999;13:1025–36.
183. Estrada K, et al. Genome-wide meta-analysis identifies 56 bone mineral density loci and
reveals 14 loci associated with risk of fracture. Nat Genet. 2012;44:491–501.
184. Chang DJ, et al. Reduction in transforming growth factor beta receptor I expression and
transcription factor CBFa1 on bone cells by glucocorticoid. J Biol Chem. 1998;273:4892–6.
185. Prince M, et al. Expression and regulation of Runx2/Cbfa1 and osteoblast phenotypic mark-
ers during the growth and differentiation of human osteoblasts. J Cell Biochem.
2001;80:424–40.
186. Bae SC, Lee YH. Phosphorylation, acetylation and ubiquitination: the molecular basis of
RUNX regulation. Gene. 2006;366:58–66.
187. Li X, Decker M, Westendorf JJ. TEThered to Runx: novel binding partners for runx factors.
Blood Cells Mol Dis. 2010;45:82–5.
188. Drissi H, et al. Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene. J Cell
Physiol. 2000;184:341–50.
189. Paredes R, et al. Bone-specific transcription factor Runx2 interacts with the 1alpha,25-
dihydroxyvitamin D3 receptor to up-regulate rat osteocalcin gene expression in osteoblastic
cells. Mol Cell Biol. 2004;24:8847–61.
190. Khalid O, et al. Modulation of Runx2 activity by estrogen receptor-alpha: implications for
osteoporosis and breast cancer. Endocrinology. 2008;149:5984–95.
191. Baniwal SK, et al. Repression of Runx2 by androgen receptor (AR) in osteoblasts and pros-
tate cancer cells: AR binds Runx2 and abrogates its recruitment to DNA. Mol Endocrinol.
2009;23:1203–14.
192. Ning YM, Robins DM. AML3/CBFalpha1 is required for androgen-specific activation of the
enhancer of the mouse sex-limited protein (Slp) gene. J Biol Chem. 1999;274:30624–30.
193. Little GH, et al. Differential effects of RUNX2 on the androgen receptor in prostate cancer:
synergistic stimulation of a gene set exemplified by SNAI2 and subsequent invasiveness.
Cancer Res. 2014;74:2857–68.
194. Gaur T, et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2
gene expression. J Biol Chem. 2005;280:33132–40.
195. Ekenstam E, Stalenheim G, Hallgren R. The acute effect of high dose corticosteroid treat-
ment on serum osteocalcin. Metabolism. 1988;37:141–4.
196. Heinrichs AA, et al. Identification of multiple glucocorticoid receptor binding sites in the rat
osteocalcin gene promoter. Biochemistry. 1993;32:11436–44.
197. Cosman F, Nieves J, Herbert J, Shen V, Lindsay R. High-dose glucocorticoids in multiple
sclerosis patients exert direct effects on the kidney and skeleton. J Bone Miner Res. 1994;9:
1097–105.
198. Morrison N, Eisman J. Role of the negative glucocorticoid regulatory element in glucocorticoid
repression of the human osteocalcin promoter. J Bone Miner Res. 1993;8:969–75.
199. Morrison NA, et al. 1,25-dihydroxyvitamin D-responsive element and glucocorticoid repres-
sion in the osteocalcin gene. Science. 1989;246:1158–61.
200. Stromstedt PE, Poellinger L, Gustafsson JA, Carlstedt-Duke J. The glucocorticoid receptor
binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential
mechanism for negative regulation. Mol Cell Biol. 1991;11:3379–83.
201. Meyer T, Gustafsson JA, Carlstedt-Duke J. Glucocorticoid-dependent transcriptional repres-
sion of the osteocalcin gene by competitive binding at the TATA box. DNA Cell Biol.
1997;16:919–27.
202. Ducy P, et al. Increased bone formation in osteocalcin-deficient mice. Nature. 1996;382:
448–52.
203. Leclerc N, Noh T, Khokhar A, Smith E, Frenkel B. Glucocorticoids inhibit osteocalcin tran-
scription in osteoblasts by suppressing Egr2/Krox20-binding enhancer. Arthritis Rheum.
2005;52:929–39.
204. Levi G, et al. Defective bone formation in Krox-20 mutant mice. Development. 1996;122:
113–20.
205. Gabet Y, et al. Gender-specific control of peak bone mass by the Wnt pathway: androgen
signaling protects against lef1 haploinsufficiency-induced bone loss. Bone. 2008;42:S50
(meeting abstract).
206. Kim HJ, et al. Early growth response 2 negatively modulates osteoclast differentiation
through upregulation of Id helix-loop-helix proteins. Bone. 2012;51:643–50.
207. Shi X, et al. A glucocorticoid-induced leucine-zipper protein, GILZ, inhibits adipogenesis of
mesenchymal cells. EMBO Rep. 2003;4:374–80.
208. Fu L, Patel MS, Karsenty G. The circadian modulation of leptin-controlled bone formation.
Prog Brain Res. 2006;153:177–88.
209. Wu X, et al. Circadian mechanisms in murine and human bone marrow mesenchymal stem
cells following dexamethasone exposure. Bone. 2008;42:861–70.
210. Jilka RL, et al. Increased bone formation by prevention of osteoblast apoptosis with parathy-
roid hormone. J Clin Invest. 1999;104:439–46.
211. Whyte MP, et al. Enzyme-replacement therapy in life-threatening hypophosphatasia. N Engl
J Med. 2012;366:904–13.
212. Schäcke H, Berger M, Rehwinkel H, Asadullah K. Selective glucocorticoid receptor agonists
(SEGRAs): novel ligands with an improved therapeutic index. Mol Cell Endocrinol.
2007;275:109–17.
213. Belvisi MG, et al. Therapeutic benefit of a dissociated glucocorticoid and the relevance of
in vitro separation of transrepression from transactivation activity. J Immunol. 2001;166:
1975–82.
214. Schäcke H, et al. Characterization of ZK 245186, a novel, selective glucocorticoid receptor
agonist for the topical treatment of inflammatory skin diseases. Br J Pharmacol. 2009;158:
1088–103.
215. Kassel O, Herrlich P. Crosstalk between the glucocorticoid receptor and other transcription
factors: molecular aspects. Mol Cell Endocrinol. 2007;275:13–29.
216. John S, et al. Chromatin accessibility pre-determines glucocorticoid receptor binding pat-
terns. Nat Genet. 2011;43:264–8.
217. Miranda TB, Morris SA, Hager GL. Complex genomic interactions in the dynamic regulation
of transcription by the glucocorticoid receptor. Mol Cell Endocrinol. 2013;380:16–24.
218. Grøntved L, et al. C/EBP maintains chromatin accessibility in liver and facilitates glucocor-
ticoid receptor recruitment to steroid response elements. EMBO J. 2013;32:1568–83.
219. Siersbæk R, et al. Extensive chromatin remodelling and establishment of transcription factor
‘hotspots’ during early adipogenesis. EMBO J. 2011;30:1459–72.
220. Biddie SC, et al. Transcription factor AP1 potentiates chromatin accessibility and glucocorti-
coid receptor binding. Mol Cell. 2011;43:145–55.
221. Rao NAS, et al. Coactivation of GR and NFKB alters the repertoire of their binding sites and
target genes. Genome Res. 2011;21:1404–16.
222. Uhlenhaut NH, et al. Insights into negative regulation by the glucocorticoid receptor from
genome-wide profiling of inflammatory cistromes. Mol Cell. 2013;49(1):158–71.
223. Rauch A, et al. An anti-inflammatory selective glucocorticoid receptor modulator preserves
osteoblast differentiation. FASEB J. 2011;25:1323–32.
224. Rauner M, et al. Effects of the selective glucocorticoid receptor modulator compound A on
bone metabolism and inflammation in male mice with collagen-induced arthritis.
Endocrinology. 2013;154(10):3719–28.
225. De Bosscher K, et al. Selective modulation of the glucocorticoid receptor can distinguish
between transrepression of NF-κB and AP-1. Cell Mol Life Sci. 2013. doi:10.1007/
s00018-013-1367-4.
226. Wüst S, et al. Peripheral T cells are the therapeutic targets of glucocorticoids in experimental
autoimmune encephalomyelitis. J Immunol. 2008;180:8434–43.
227. Van Loo G, et al. Antiinflammatory properties of a plant-derived nonsteroidal, dissociated
glucocorticoid receptor modulator in experimental autoimmune encephalomyelitis. Mol
Endocrinol. 2010;24(2):310–22.
228. Reber LL, et al. A dissociated glucocorticoid receptor modulator reduces airway hyperre-
sponsiveness and inflammation in a mouse model of asthma. J Immunol. 2012;188(7):
3478–87.
229. Thiele S, et al. Selective glucocorticoid receptor modulation maintains bone mineral density
in mice. J Bone Miner Res. 2012;27:2242–50.
230. Sims NA, Walsh NC. GP130 cytokines and bone remodelling in health and disease. BMB
Rep. 2010;43:513–23.
231. Metcalf D, Nicola NA, Gearing DP. Effects of injected leukemia inhibitory factor on hema-
topoietic and other tissues in mice. Blood. 1990;76:50–6.
232. van Bezooijen RL, ten Dijke P, Papapoulos SE, Lowik CW. SOST/sclerostin, an osteocyte-
derived negative regulator of bone formation. Cytokine Growth Factor Rev.
2005;16:319–27.
233. Marenzana M, et al. Sclerostin antibody treatment enhances bone strength but does not pre-
vent growth retardation in young mice treated with dexamethasone. Arthritis Rheum.
2011;63:2385–95.
234. Bickle M. The beautiful cell: high-content screening in drug discovery. Anal Bioanal Chem.
2010;398:219–26.
235. Borchert KM, et al. High-content screening assay for activators of the Wnt/Fzd pathway in
primary human cells. Assay Drug Develop Technol. 2005;3:133–41.
Chapter 9
Effects of Glucocorticoids in the Immune
System
Emmanuel Oppong and Andrew C.B. Cato
Abstract Glucocorticoids (GCs) are steroid hormones with widespread effects.

They control intermediate metabolism by stimulating gluconeogenesis in the liver,
mobilize amino acids from extra hepatic tissues, inhibit glucose uptake in muscle
and adipose tissue, and stimulate fat breakdown in adipose tissue. They also medi-
ate stress response. They exert potent immune-suppressive and anti-inflammatory
effects particularly when administered pharmacologically. Understanding these
diverse effects of glucocorticoids requires a detailed knowledge of their mode of
action. Research over the years has uncovered several details on the molecular
action of this hormone, especially in immune cells. In this chapter, we have
summarized the latest findings on the action of glucocorticoids in immune cells
with a view of identifying important control points that may be relevant in gluco-
corticoid therapy.
Keywords Glucocorticoid receptor • Inflammation • Signaling pathways • Immune cells
Introduction
The human body is constantly exposed to a host of pathogens that disturbs its proper
function and survival. These pathogens, which are mainly microorganisms, gain
access to the body through infections or through tissue injuries. However, the body
is able to defend itself by making use of cells, tissues and organs which function to
protect it from harm. Collectively, the network of these interacting cells, tissues and
organs and the mechanisms for protection, form the immune system.
Without a proper functioning immune system, exposure of the human body to
simple infections can be detrimental. The first line of defense by the immune system
E. Oppong, Ph.D. (*) • A.C.B. Cato

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology,
Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen, Karlsruhe,
Baden Württemberg 76344, Germany

218 E. Oppong and A.C.B. Cato
is the innate immune system [1]. It is a nonspecific response and it acts rapidly.
Components of this system are the physical, chemical and cellular barriers that offer
protection against an invading pathogen [2]. These include the skin, the mucous
lining of the digestive tract, the respiratory and the urogenital systems. Certain flu-
ids found in the body such as tears, sweat, saliva, urine and gastric juice contain
components that are not conducive for microbial growth and these form chemical
barriers to infection [2]. The cellular aspect of the innate immune system is made up
of cells that are programmed to recognize and destroy microorganisms. They act by
engulfing or releasing toxic contents to destroy the invading organisms. In addition
to the rapid innate immune system, there is a second line of defense, termed adap-
tive immune system, which has a slow onset of action and is very specific in the type
of immune response it initiates [2, 3]. With regards to this type of immune protec-
tion, the primary response to an exposure of antigen elicits the production of
immunoglobulins. These immunoglobulins subsequently initiate a robust response
when the same antigen is later encountered.
In its quest to protect the body from injurious agents, the immune system triggers
reactions that may damage tissues and organs in the body. This response, which
alters the normal physiological function of various tissues and organs, occurs
through the release of mediators such as cytokines and chemokines as well as
prostaglandins and is termed inflammation [4, 5]. Inflammation seeks to establish
tissue homeostasis, but it is also associated with deleterious effects such as pain,
swelling, itching, redness and heat that do more harm than good. The inflammatory
response involves a complex interplay of cells of the immune system. While some
cells function to detect and alert the immune system of an impending danger, others
are involved in terminating the response [5, 6].
As inflammation is a “harmful” process, there are efforts to attenuate or control
it by steroids. Glucocorticoids are steroidal compounds that influence both arms of
the immune system at multiple levels [7, 8]. Their effective action in terminating
inflammatory responses has made them a gold standard in anti-inflammatory ther-
apy. Dexamethasone, prednisolone methylprednisolone, triamcinolone and beta-
methasone are some of the synthetic glucocorticoid analogs that have been
successfully used to manage various inflammatory diseases, ever since Hench and
his colleagues first reported the anti-inflammatory effect of the natural hormone,
cortisol, in patients with rheumatoid arthritis in the early 1950s [9–11].
The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR),
a ligand inducible transcription factor (TF). In target cells, the hormone activated
receptor diminishes inflammation by either inhibiting the activity of pro-
inflammatory TFs, terminating signaling pathways or upregulating the expression
of anti-inflammatory proteins. Over the years, studies carried out in different
immune cell types have revealed novel insights into the mechanism of action of
glucocorticoids in the immune system. Thus, the objective of this chapter is to give
an overview of various mechanisms used by glucocorticoids to modulate the behav-
ior and function of the different immune cells.
9 Effects of Glucocorticoids in the Immune System 219
Cells of the Immune System
The white blood cells, also referred to as leucocytes, form the class of cells involved
in the defense against pathogens. They are derived from specialized progenitor cells
in the bone marrow and they differentiate and mature into other cell types under the
influence of different cytokines and transcription factors [12]. Based on the pres-
ence or absence of granules in their cytoplasm, leucocytes can be classified as either
granulocytes or agranulocytes respectively [13]. Granulocytes have multi-lobed
nuclei and are made up of neutrophils, basophils, eosinophils and mast cells. They
can be distinguished from each other microscopically by the appearance of charac-
teristic granules upon staining with hematoxylin and eosin as well as by the expres-
sion of cell type specific genes. Each cell type plays a specific role during an injury,
infection or inflammation. For example, at inflammatory sites, local signals gener-
ated in response to chemokines and cytokines act as chemoattractants for neutro-
phils. These cells are recruited to the inflammatory sites where they exhibit very
potent phagocytic and microbicidal activity which is characterized by the activation
of myeloperoxidase that contributes to clearance and resolution of the inflammatory
response [14–16]. Eosinophils are involved in protection against parasites and hel-
minth infections [17, 18] and mast cells mediate allergic responses. These latter
cells are characterized by the presence of cytoplasmic granules which consist of
histamine, heparin and the proteases tryptase and chymase. When activated by aller-
gens, they degranulate and release the contents of their cytoplasmic granules and
they also increase the expression of cytokine genes [19, 20]. These events contribute
to the symptoms of allergic and inflammatory reactions that are characteristic of
mast cells. Basophils form a class of granulocytes with a rather unknown physiolog-
ical function although they are thought to have a role in host defense just like other
immune cells [21].
The second major class of immune cells is made up of agranulocytes. They are
single nucleated cells that consist of monocytes and lymphocytes. Monocytes
migrate from the blood and differentiate into either macrophages or dendritic cells.
Macrophages are large, irregularly shaped cells which contain numerous vacuoles
that are used for phagocytosis of cellular debris from tissue necrosis and invading
microorganisms [22]. In contrast, dendritic cells are star shaped cells whose main
functions are to capture, process and present antigens to B and T lymphocytes to
prime and boost immunity [23, 24]. B lymphocytes are involved in antigen-specific
antibody production and T lymphocytes are involved in cell-mediated immunity
and cytokine production. Nearly all these immune cells are sensitive to the immune
suppressive and anti-inflammatory actions of glucocorticoids and a summary of
their response to the hormone is presented in Table 9.1.
Table 9.1 Summary of the effects of glucocorticoids on immune cells

Immune cell Glucocorticoid action References
Neutrophil Increased survival and accumulation of cells [92–94]
at inflammatory sites
Basophil Induction of apoptosis, rapid non-genomic [110, 111]
inhibition of cell activation
Eosinophil No reported action
Dendritic cells Inhibition of cell differentiation, reduced T cell [63, 64, 112–115]
stimulatory capacity, increased endocytic
activity, induction of apoptosis, inhibition of
pro-inflammatory cytokines, inhibition of cell
migration
Macrophages Activation of JNK and p38 MAPK are inhibited, [57, 61, 62]
expression of pro inflammatory genes are
attenuated, contact hypersensitivity is suppressed
Mast cells Activation of FcεRI dependent signaling is (see Oppong
attenuated, histamine and calcium release are et al., 2013 for
downregulated, Phosphatases such as DUSP1 review) [37]
are upregulated
T lymphocytes Apoptosis is induced, release of [71, 72, 78–80,
pro-inflammatory cytokines are inhibited, 83–86]
Non genomic inhibition of LCK and Fyn,
rapid phosphorylation of ZAP70
B lymphocytes Induction of apoptosis, production of [116–118]
immunoglobulins is induced, suppression of cell
activation, proliferation and differentiation
Mechanism of Glucocorticoid Action
The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a

cytoplasmic modular protein that consist of a central DNA binding domain, a
N-terminal domain that contains transactivation function and a C-terminal ligand
binding domain that allows for recruitment of coregulators [25]. Upon hormone
binding, a conformational change is induced in the receptor that allows it to translo-
cate to the nucleus where it binds chromatin and induces or represses the expression
of target genes [26]. To facilitate binding of the GR to chromatin, an “open” chro-
matin conformation is established by the transcription factor, activation protein-1
(AP-1) that allows the receptor to recognize its target sequences [27]. The hormone
activated receptor then homodimerizes and binds in a head-to-head manner to glu-
cocorticoid response elements (GREs), that are made up of two hexameric half
motifs separated by three base pairs [28, 29]. Subsequently, the GR recruits coacti-
vator proteins (e.g., steroid receptor) coactivator 1, 2, 3 (SRC1, SRC2 and SRC3)
with histone acetyltransferase activity or chromatin remodeling complexes to the
promoters of the target genes to enhance gene activation [30, 31]. The GR also
downregulates gene expression. In this case, the liganded receptor is tethered as a
monomer to DNA bound TFs such as AP-1 or nuclear factor kappa-light-chain-

enhancer of activated B cells (NF-κB) to inhibit their action [32]. The GR uses
another mode of action to bind as monomers to negative glucocorticoid response
elements (nGRE) in a head to tail conformation to inhibit gene expression [33, 34].
In this case, the consensus sequence for the nGREs consists of two inverted repeats
separated by one base pair. Herein, the receptor assembles a repression complex via
its association with corepressor proteins such as nuclear receptor co-repressor
(NCoR) and histone deacetylases (HDACs) to inhibit gene expression [33, 34]. At
certain DNA binding sites, termed “composite GRE”, the binding motifs for the GR
and other TFs are side by side such that the two proteins influence each other’s
activity to synergistically activate or repress gene expression [35]. All these mecha-
nisms are classical actions of the GR, also referred to as its genomic mechanisms
that are used in regulating immune cell function.
In addition, the hormone also elicits rapid non-genomic actions which are
thought to arise from the plasma cell membrane. These actions of glucocorticoids
involve modulation of signaling pathways, and they occur within seconds to min-
utes and are insensitive to transcriptional and translation inhibitors [36].
Mechanistically, the rapid actions of glucocorticoids are not clearly understood and
are therefore described by three theories. First, they are thought to emanate from a
membrane localized GR (mGR) that is distinct from the classical receptor. Second
they are described as merely coming from receptor independent effects originating
from hormone-mediated changes in membrane fluidity and third they are thought to
arise from the classical intracellular GR that is recruited to the plasma membrane
(for reviews see Stahl and Buttegreit 2010 and Oppong et al., 2013) [37, 38]. Over
the years, different methods have been used to demonstrate the localization and
action of the mGR. One of the early approaches was the use of a monoclonal anti-
body conjugated to fluorescein isothiocyanate to show the presence of mGR on
S-49 lymphoma cells [39]. From these and later studies, it was postulated that they
may be different from the classical receptor [40–44]. However, the identity of this
receptor still remains obscure, as it has not been cloned. The second theory on
glucocorticoid-mediated changes in membrane fluidity could only be achieved with
high concentrations of the hormone which are different from the reported rapid
effects with physiological concentrations of the hormone [45]. Thus, it is more
likely that the classical GR is recruited to the plasma membrane to mediate the rapid
action of the hormone. However, the membrane-localized classical GR represents a
very small population of the intracellular receptor and this has made its detection
challenging. Recently, antibody-conjugated magnetofluorescent liposomes were
used to demonstrate membrane bound GR in peripheral monocytes [46]. This tech-
nique has the advantage of amplifying fluorescence signal up to 1000-fold to detect
membrane bound GR [47–49]. Another technique that has been used to demonstrate
membrane-bound GR in both monocytes and T cells is termed Fluorescence
Amplification by Sequential Employment of Reagents (FASER) [50]. With this
method, the fluorescence signal from cells labeled with antibody conjugated to a
fluorochrome is amplified by the sequential addition of two reagents: a fluoro-
chrome specific activator and an enhancer. In yet another technique using mast
cells, allergens were patterned on a glass surface, to immobilize and activate the
mast cells [51]. This approach was used to visualize in real time the recruitment of
a GR tagged with GFP to the site of interaction of the allergen with the cell [52].
Further analyses using photobleaching techniques revealed slower dynamics of the
receptor at the membrane of the activated mast cells compared to non-activated
cells, indicating possible interaction of the GR with membrane bound components
in the activated cells [52]. With these brief descriptions of the pathways and meth-
ods used for studying glucocorticoid action in immune cells, we will now take a
look at the effect of the hormone on individual immune cells.
Glucocorticoid Modulation of Immune Cell Action
Macrophages
Macrophages act as scavenger cells of the immune system since they clear cel-
lular debris and invading microorganisms by phagocytosis [22]. They are acti-
vated by bacteria components such as lipopolysaccharide (LPS) or endotoxin that
bind to toll like receptors (TLRs) located on their surfaces. This triggers intracel-
lular signaling cascades such as the MAP kinases (p38 MAPK, ERK and JNK)
resulting in enhanced transcription of pro-inflammatory cytokine genes.
Glucocorticoids interfere with these signaling pathways to dampen the inflamma-
tory responses. For example, pretreatment of bone marrow derived macrophages
with the synthetic glucocorticoid, dexamethasone, following TLR activation by
LPS, resulted in the suppression of phosphorylation of JNK and p38 MAP kinase.
However, phosphorylation of ERK was unaffected [53], indicating a differential
action of glucocorticoids on the MAPK pathway in these cells. Additionally, pro-
inflammatory cytokines and chemokines such as interferon gamma (IFNγ), inter-
leukin 1α and interleukin 1β (IL-1α and IL-1β) were inhibited at both the mRNA
and protein levels [53]. The anti-inflammatory action of glucocorticoids in mac-
rophages was also reported to occur through an upregulation of the expression of
dual specificity phosphatase one, DUSP1, a negative regulator of the MAPKs.
Ablation of DUSP1 impaired the anti-inflammatory action of glucocorticoids in
these cells [53, 54].
The ligand activated GR also negatively regulates pro-inflammatory gene expres-
sion through crosstalk with TFs such as AP-1 and NF-κB at the promoter of target
genes [35]. Downregulation of the activities of these TFs by the GR was reported to
involve the coregulator protein GRIP1, that was previously identified as a coacti-
vator of the GR [55, 56]. GRIP1, possesses a unique domain that confers repression
to target genes when it is bound by the GR and it is through this region that the
negative regulation by the GR is achieved [57]. Conventional GRIP1 knock-out
mice show defects in normal growth of the adrenal glands and glucocorticoid
secretion as well as impaired metabolic and reproductive functions [58–60].

However to further clarify the contribution of GRIP1 to the anti-inflammatory action
of glucocorticoids, macrophage-specific GRIP1 knockout mice were generated.
Genome wide transcriptome analyses of bone marrow derived macrophages iso-
lated from these mice, showed that the expression of approximately forty percent of
the LPS-induced inflammatory genes repressed by glucocorticoids in the wide-type
were not repressed by glucocorticoids in the macrophage-specific knockout mice
[61]. Some of the pro-inflammatory cytokine genes that showed impaired inhibition
by glucocorticoids in the knockout cells are IL-1α, IL-1β, TNFα and CCL4 [61].
Together these results confirm that GRIP1 is indeed an essential mediator of the
negative action of GR in macrophages.
The role of macrophages as targets for the anti-inflammatory action of gluco-
corticoids has also been investigated in animal models of immune diseases. For
example, contact hypersensitivity (CHS) induced by the allergens, 2, 4-dinitrofluo-
robenezene or oxazolone, which are characterized by swollen ears, was not sup-
pressed by glucocorticoids in mice with a macrophage specific GR knockout
although control mice showed glucocorticoid inhibition [62]. Furthermore, in the
CHS model, mice expressing a dimerization defective GR were also unresponsive
to glucocorticoids [62]. These mice exhibited increased accumulation of macro-
phages in the swollen ear [62], indicating that macrophages are important mediators
of the anti-inflammatory action of glucocorticoids and that dimerization of the GR
is necessary for the anti-inflammatory effect of the hormone.
Dendritic Cells
Dendritic cells (DCs) are antigen presenting cells that differentiate from precursor
monocytes [23]. They exist either as freshly differentiated immature cells or termi-
nally differentiated mature cells in resident tissues such as the spleen and lymph
node. In response to tissue damage or injury, immature DCs capture invading patho-
gen, process them and present them to T cells for initiation of an immune response
[23]. In general, immature DCs have increased antigen capturing and processing
capability but low T cell stimulatory potential [23]. On the contrary, mature DCs are
characterized by low antigen uptake capacity, increased ability of antigen presenta-
tion and are effective T-cell stimulators [24]. Dexamethasone interferes with the
lifecycle of DCs. In vitro maturation of DCs by TNFα or CD40 ligand to express
costimulatory molecules such as MHC I, MHC II and CD80 is impaired by dexa-
methasone and as such glucocorticoid treated DCs exhibit reduced T-cell stimula-
tory capacity and production of cytokines [63, 64]. Thus, it is likely that by keeping
DCs in an immature state, glucocorticoids may enhance the clearance of invading
pathogens and microorganisms. Other effects of glucocorticoids on DCs are
presented in Table 9.1.
T Lymphocytes
T Lymphocytes, also referred to as T cells are immune cells that arise from lym-
phoid progenitors in the bone marrow and mature in the thymus [65]. They are clas-
sified as naïve, effector or memory cells based on their function during the immune
response. Naïve T cells are nonactivated, short-lived cells that die when no antigen is
encountered. However, upon activation, naïve T cells differentiate into distinct effec-
tor T cell types (Th1, Th2, Th17 and Tregs cells) based on their pattern of cytokine
production [66, 67]. For example, IL-12, IFN-α and IFN-γ induce differentiation of
Th1 phenotype to secrete IL-2, TNF-α and IFN-γ whereas IL-4 drives the Th2 to
produce IL-4, IL-10 and IL-13 [68, 69]. Generally T effector cells disappear after
removal of the antigenic agent. However, a subpopulation remains to form a third
class of cells, termed memory T cells [70]. Upon reexposure to the same antigen,
memory T lymphocytes are activated to mount a rapid and potent immune response.
Nearly all the subtypes of T lymphocytes are sensitive to the actions of glucocorti-
coids, which result in changes in their survival, differentiation or function.
Glucocorticoids also influence the pattern of cytokines that drive the differentiation
of T cells into the distinct subtypes. These effects of the hormone have been USE
extensively instead of intensively reviewed by Elenkov 2004 and Flammer et al. 2011
[71, 72] and readers are referred to these articles for further information on this topic.
One of the mechanisms by which the immune system elicits its protective role is
by inducing programmed cell death, also termed apoptosis, of the different immune
cells [73]. The Bcl-2 family of proteins which contain up to four Bcl-2 homology
domains (BH1-BH4) mediate programmed cell death [74]. While some members of
this class of proteins protect against cell death, other members initiate cellular apop-
tosis. In T lymphocytes, apoptosis is induced either by the intrinsic pathway which
is usually activated by cellular stress or by the extrinsic pathway induced by aggre-
gation of surface receptors such as Fas [73, 75]. The intrinsic pathway is initiated by
a subset of proapoptotic proteins that contains only the BH3 domain such as Bid,
Bad and Blk. These proteins trigger cell death by either activating other proapop-
totic bcl-2 family members (Bax and Bak) or by inhibiting the antiapoptotic pro-
teins Mcl, Bcl-2 and Bcl-x [73]. Consequently, cytochrome c is released from the
mitochondria and an apoptosome consisting of cytochrome c, protease activating
factor 1 (Apaf-1) and caspase 9 activate effector caspases that lead to cell death [76,
77]. In the extrinsic pathway, activation of cell surface “death” receptors recruits
caspase 8 which in turn activates downstream effectors such as caspase 3 to trigger
apoptosis of the cells [73]. Alternatively, caspase 8 can also cleave the BH3-only
propaptotic member, Bid, resulting in the release of cytochrome c and initiation of
apoptosis as described above [73, 74]. Glucocorticoids potently induce T cell death
via both mechanisms. Studies documenting this action of the hormone have been
reviewed by Distelhorst 2002, Ashwell et al. 2000 and Herold et al. 2006 [78–80].
The effects of glucocorticoids in T cells described above are not the only
action of the hormone in these cells. There are reports that glucocorticoids also
exert non-genomic actions in these cells by modulating the activities of kinases.
Several kinases are activated when the T cell receptor (TCR) expressed on
T lymphocytes are cross-linked. Some of these kinases include the Src-like

kinases, LCK and FYN [81, 82]. In an analysis of the kinome of human CD4+ T
lymphocytes using a peptide array that consists of about 1176 kinase substrates,
Lowenberg et al. 2005 showed that glucocorticoids differentially altered the
phosphorylation of these substrates with about 116 kinases showing a rapid
increase or decrease in phosphorylation following glucocorticoid treatment [83].
Importantly, within minutes of hormone administration, the activities of LCK
and FYN were downregulated causing them to dissociate from the TCR [83, 84].
In another study, tyrosine phosphorylation of ZAP-70, an adapter molecule in the
TCR pathway was rapidly enhanced by glucocorticoid treatment [85, 86]. These
rapid actions of the hormone in T cells were all shown to require membrane
localization of the GR.
Neutrophils
Neutrophils are immune cells that are rapidly recruited to inflammatory sites for
phagocytosis and destruction of invading organisms [87]. After their phagocytic
action, they undergo apoptosis and are subsequently cleared from the inflamed tis-
sue by macrophages. The removal of neutrophils is valuable to the resolution of
inflammation and return to tissue homeostasis [88]. However, when neutrophils per-
sist for long periods, they accumulate and release cytotoxic serine proteases such as
neutrophil elastase and metalloproteinases which further aggravate the inflamma-
tory process [89–91]. Long-lived neutrophils at inflammatory sites are therefore
undesirable. Intriguingly, glucocorticoids have been shown to increase the accumu-
lation and survival of neutrophils [92, 93] with the half-lives of glucocorticoid
treated neutrophils doubling compared to non-treated controls [92]. This
glucocorticoid-mediated prolongation of neutrophil survival involves induced
expression of anti-apoptotic proteins such as members of the Bcl-2 family of pro-
teins and/or suppression of apoptotic signaling pathways [94]. Thus the action of
glucocorticoids on neutrophils maybe detrimental since the hormone enhances the
ability of neutrophils to survive and to secrete pro-inflammatory mediators [95].
This notion is supported by the finding that glucocorticoids are ineffective in neu-
trophilic inflammation [94]. This pro-inflammatory effect of neutrophils is therefore
thought to contribute to the clinical disorder of glucocorticoid resistance [94, 96].
Besides, glucocorticoids also increase the concentration of neutrophils in the blood
by causing demargination of these cells from the walls of the blood vessels [97].
Mast Cells
Mast cells are effector cells of allergic and inflammatory reactions. They express on
their surface IgE receptor (FcεRI), TLRs, mast/stem cell growth factor receptor
(SCFR), (also referred to as c-kit receptor) and IgG receptor that are involved in the
activation of the cells. However, most studies exploring the behavior and function of
these cells have concentrated on the FcεRI pathway since this is the main signaling
pathway of mast cells. The FcεRI consists of α, β and γ subunits that play distinct
roles during activation of mast cells (Fig. 9.1). The α subunit contains an extracel-
lular domain that is important for IgE binding while the β and γ domains contain
immunoreceptor tyrosine-based activation motifs (ITAMs) for signal amplification
and propagation respectively [98, 99]. Allergen-mediated aggregation of FcεRI trig-
gers a chain of signaling events where Lyn, a proximal Src-like kinase, is recruited
to phosphorylate the ITAMs of the β and γ subunits. This leads to the recruitment of
spleen tyrosine kinase (Syk) which further transduces the signal to downstream
targets [100, 101] (Fig. 9.1). Signal transduction in mast cells can also occur through
an alternative pathway involving Fyn kinase, Grb2- associated binder 2 (Gab2) and
phosphatidylinositol 3-kinase (PI3K) [102] (Fig. 9.1). All these signaling events
result in three main downstream effects: degranulation, cytokine/chemokine gene
expression and arachidonic acid and eicosanoid production (Fig. 9.1). Glucocorticoids
inhibit mast cell activation at different levels in this signaling cascade as recently
described (Oppong et al [37] and Fig. 9.1). For example, following long-term treat-
ment (16–24 h), glucocorticoids downregulate the activity of Erk1/2 [103], which
could have an impact on their effect on arachidonic release and cytokine gene
expression. Activation of PI3K and the release of calcium from intracellular store as
well as degranulation are other examples of markers of mast cell activation that are
inhibited by long-term glucocorticoid treatment of mast cells [37, 104].
Glucocorticoids also use rapid non-genomic actions to modulate mast cell action
[105, 106]. Here, it has been shown that following allergen-mediated crosslinking
of FcεRI of mast cells, the GR is rapidly recruited to the plasma membrane (Fig. 9.1)
and this process is further transiently enhanced by glucocorticoid treatment [52]. It
therefore appears that the membrane-localized GR may serve as an early sensor for
the hormone to amplify its main genomic action [52]. In kinetic studies of hormone-
dependent nuclear translocation of the GR, a transient delay in the mobility of the
GR was observed in allergen-activated compared to non-activated mast cells [52].
This lag phase in nuclear translocation of the GR was suggested to have arisen from
the time the receptor spent at the plasma membrane. The function of the membrane
localized-GR was therefore postulated to modulate cell signaling pathways that
could eventually be relevant to the genomic action of the receptor [52], a definitive
proof for this mechanism is however lacking.
One of the downstream targets for this rapid action of glucocorticoids is the
rapid- and transient- increase in FcεRI-dependent phosphorylation of Erk1 and 2
[52]. Along with the long-term action on phosphorylation of Erk-1/2, glucocorticoids
therefore exert a dual effect on Erk phosphorylation in mast cells. The significance
of these two regulatory pathways is not fully understood. However it is likely that
the rapid upregulation of phosphorylation of the Erk-1/2 may prepare the cell for the
later events that lead to the downregulation of the kinase activity. The latter negative
action of the glucocorticoid is however thought to be the basis for the anti-allergic
and anti-inflammatory actions of glucocorticoids since Erk1/2 regulates several of
the pro-inflammatory pathways in mast cells that are downregulated by
glucocorticoids.
ALLERGEN
β α FcεRI
L L
A GR Lyn A
Fyn
T γ T PLCγ
2 Gab2 Syk
GR PI3K SLP PIP2
76
BTK
Vav1
Non Genomic PLCγ
Pathway IP3 DAG
Ras-Raf-MEK
IP3
GC H8090
GR Ca2+
Ca2+
Genomic MAPK
Pathway (p38/ERK/JNK)
Degranulation
GR Cyokine gene
+ expression
DUSP1
G GR
Lipid mediators
production
Fig. 9.1 Signal transduction in mast cells following crosslinking of FcεRI and activation of the
glucocorticoid receptor (GR) in mast cells. Signaling pathways through the Lyn-Syk-LAT pathway
following FcεRI crosslinking are indicated by red arrows. An alternative pathway involving Fyn-
Gab2-PI3K initiated after aggregation of the FcεRI is also indicated by red arrows. Activation of
the mast cell leads to three main downstream events: degranulation, cytokine gene expression and
production of lipid mediators. The GR signaling pathway in mast cells is shown by black arrows.
In the genomic effect of the GR, the receptor dissociates from heat shock proteins upon hormone
binding and translocates to the nucleus to modulate gene expression such as the induction of
expression of the DUSP1 gene, whose product inhibits MAPK activation. In the non-genomic
action of glucocorticoids, allergen mediated activation of mast cells recruit the GR to the FcεRI
complex. Components of the mast cell signaling pathway reported to be inhibited by glucocorti-
coids are indicated by the symbol
Conclusion
In the management of inflammatory diseases, both experimental and clinical data

underscore the importance of glucocorticoids as the drugs of choice [107]. The
potent anti-inflammatory effect of this hormone is due in part to its ability to modulate
the behavior and function of immune cells. Over the years, much progress has been
made in elucidating the effects of glucocorticoids in immune cells. However, the
molecular details involved in the action of the hormone are still not completely
understood since glucocorticoids exert diverse effects in these cells. Some effects of
the hormone are apparently beneficial as they negatively regulate signaling pathways
and downregulate pro-inflammatory cytokine gene expression. On the other hand,
other actions such as hormone-increased survival and accumulation of neutrophils
or enhanced Erk1/2 phosphorylation in mast cells are still not understood. However,
positive regulatory effects of glucocorticoids may contribute to the anti-inflammatory
action of glucocorticoids. In T-cells, glucocorticoids positively regulate the expres-
sion of IL-10, a cytokine that possesses anti-inflammatory effects and that has a
valuable role in diseases such as asthma [108, 109]. These complexities in the action
of glucocorticoids emphasize the need for further research to be carried out to
clarify their effects in the immune system.
References
1. Grasso P, Gangolli S, Gaunt I. Essentials of pathology for toxicologist. Boca Raton: CRC;
2002. ISBN 978-0-415-25795-4.
2. Mayer G. Immunology: Innate (Non-Specific) Immunity (Chapter 1). In: Microbiology and
Immunology On-line. University of South Carolina. 2009.
3. Janeway CPT, Walport M, Shlomchik M. Immunobiology. New York and London: Garland
Science; 2001.
4. Medzhitov R. Origin and physiological roles of inflammation. Nature. 2008;454(7203):
428–35.
5. Okin D, Medzhitov R. Evolution of inflammatory diseases. Curr Biol. 2012;22(17):R733–40.
6. Medzhitov R. Inflammation 2010: new adventures of an old flame. Cell. 2010;140(6):771–6.
7. Baschant U, Tuckermann J. The role of the glucocorticoid receptor in inflammation and
immunity. J Steroid Biochem Mol Biol. 2010;120(2–3):69–75.
8. Chinenov Y, Rogatsky I. Glucocorticoids and the innate immune system: crosstalk with the
toll-like receptor signaling network. Mol Cell Endocrinol. 2007;275(1–2):30–42.
9. Hench P. Effects of cortisone in the rheumatic diseases. Lancet. 1950;2(6634):483–4.
10. Hench PS, Kendall EC, Slocumb CH, Polley HF. The antirheumatic effects of cortisone and
pituitary ACTH. Trans Stud Coll Physicians Phila. 1950;18(3):95–102.
11. Hench PS, Kendall EC, Slocumb CH, Polley HF. Cortisone, its effects on rheumatoid arthri-
tis, rheumatic fever, and certain other conditions. Merck Rep. 1950;59(4):9–14.
12. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of the cell.
4th ed. New York: Garland Science; 2002.
13. Gartner LP, Hiatt JL. Color textbook of histology. 3rd ed. Philadelphia: Elsevier; 2007.
14. Borregaard N. Neutrophils, from marrow to microbes. Immunity. 2010;33(5):657–70.
15. Nathan C. Points of control in inflammation. Nature. 2002;420(6917):846–52.
16. Kobayashi SD, Voyich JM, Burlak C, DeLeo FR. Neutrophils in the innate immune response.
Arch Immunol Ther Exp (Warsz). 2005;53(6):505–17.
17. Hogan SP, Foster PS, Rothenberg ME. Experimental analysis of eosinophil-associated gas-
trointestinal diseases. Curr Opin Allergy Clin Immunol. 2002;2(3):239–48.
18. Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol. 2006;24:147–74.
19. Turner H, Kinet JP. Signalling through the high-affinity IgE receptor Fc epsilonRI. Nature.
1999;402(6760 Suppl):B24–30.
20. Gilfillan AM, Rivera J. The tyrosine kinase network regulating mast cell activation. Immunol
Rev. 2009;228(1):149–69.
21. Prussin C, Metcalfe DD. 4. IgE, mast cells, basophils, and eosinophils. J Allergy Clin
Immunol. 2003;111(2 Suppl):S486–94.
22. Mills CD. M1 and M2 macrophages: oracles of health and disease. Crit Rev Immunol.
2012;32(6):463–88.
23. Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol.
2000;18:767–811.
24. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature.
1998;392(6673):245–52.
25. Kumar R, Thompson EB. Gene regulation by the glucocorticoid receptor: structure:function
relationship. J Steroid Biochem Mol Biol. 2005;94(5):383–94.
26. Grad I, Picard D. The glucocorticoid responses are shaped by molecular chaperones. Mol
Cell Endocrinol. 2007;275(1–2):2–12.
27. Biddie SC, John S, Sabo PJ, et al. Transcription factor AP1 potentiates chromatin accessibil-
ity and glucocorticoid receptor binding. Mol Cell. 2011;43(1):145–55.
28. Revollo JR, Cidlowski JA. Mechanisms generating diversity in glucocorticoid receptor sig-
naling. Ann N Y Acad Sci. 2009;1179:167–78.
29. Meijsing SH, Pufall MA, So AY, Bates DL, Chen L, Yamamoto KR. DNA binding site
sequence directs glucocorticoid receptor structure and activity. Science. 2009;324(5925):
407–10.
30. Lonard DM, Kumar R, O’Malley BW. Minireview: the SRC family of coactivators: an entree
to understanding a subset of polygenic diseases? Mol Endocrinol. 2010;24(2):279–85.
31. Lonard DM, O’Malley BW. Nuclear receptor coregulators: judges, juries, and executioners of
cellular regulation. Mol Cell. 2007;27(5):691–700.
32. Heck S, Kullmann M, Gast A, et al. A distinct modulating domain in glucocorticoid receptor
monomers in the repression of activity of the transcription factor AP-1. EMBO J. 1994;
13(17):4087–95.
33. Surjit M, Ganti KP, Mukherji A, et al. Widespread negative response elements mediate direct
repression by agonist-liganded glucocorticoid receptor. Cell. 2011;145(2):224–41.
35. Kassel O, Herrlich P. Crosstalk between the glucocorticoid receptor and other transcription
factors: molecular aspects. Mol Cell Endocrinol. 2007;275(1–2):13–29.
36. Croxtall JD, Choudhury Q, Flower RJ. Glucocorticoids act within minutes to inhibit recruit-
ment of signalling factors to activated EGF receptors through a receptor-dependent,
transcription-independent mechanism. Br J Pharmacol. 2000;130(2):289–98.
37. Oppong E, Flink N, Cato AC. Molecular mechanisms of glucocorticoid action in mast cells.
Mol Cell Endocrinol. 2013;380(1–2):119–26.
38. Stahn C, Buttgereit F. Genomic and nongenomic effects of glucocorticoids. Nat Clin Pract
Rheumatol. 2008;4(10):525–33.
39. Gametchu B. Glucocorticoid receptor-like antigen in lymphoma cell membranes: correlation
to cell lysis. Science. 1987;236(4800):456–61.
40. Gametchu B, Watson CS, Pasko D. Size and steroid-binding characterization of membrane-
associated glucocorticoid receptor in S-49 lymphoma cells. Steroids. 1991;56(8):402–10.
41. Gametchu B, Watson CS, Shih CC, Dashew B. Studies on the arrangement of glucocorticoid
receptors in the plasma membrane of S-49 lymphoma cells. Steroids. 1991;56(8):411–9.
42. Gametchu B, Watson CS, Wu S. Use of receptor antibodies to demonstrate membrane gluco-
corticoid receptor in cells from human leukemic patients. FASEB J. 1993;7(13):1283–92.
43. Powell CE, Watson CS, Gametchu B. Immunoaffinity isolation of native membrane gluco-
corticoid receptor from S-49++ lymphoma cells: biochemical characterization and interac-
tion with Hsp 70 and Hsp 90. Endocrine. 1999;10(3):271–80.
44. Gametchu B, Chen F, Sackey F, Powell C, Watson CS. Plasma membrane-resident glucocor-
ticoid receptors in rodent lymphoma and human leukemia models. Steroids. 1999;64(1–2):
107–19.
45. Buttgereit F, Scheffold A. Rapid glucocorticoid effects on immune cells. Steroids. 2002;67(6):
529–34.
46. Scheffold A, Assenmacher M, Reiners-Schramm L, Lauster R, Radbruch A. High-sensitivity

immunofluorescence for detection of the pro- and anti-inflammatory cytokines gamma inter-
feron and interleukin-10 on the surface of cytokine-secreting cells. Nat Med. 2000;6(1):
107–10.
47. Bartholome B, Spies CM, Gaber T, et al. Membrane glucocorticoid receptors (mGCR) are
expressed in normal human peripheral blood mononuclear cells and up-regulated after
in vitro stimulation and in patients with rheumatoid arthritis. FASEB J. 2004;18(1):70–80.
48. Spies CM, Bartholome B, Berki T, et al. Membrane glucocorticoid receptors (mGCR) on
monocytes are up-regulated after vaccination. Rheumatology (Oxford). 2007;46(2):364–5.
49. Tryc AB, Spies CM, Schneider U, et al. Membrane glucocorticoid receptor expression on
peripheral blood mononuclear cells in patients with ankylosing spondylitis. J Rheumatol.
2006;33(11):2249–53.
50. Strehl C, Gaber T, Jakstadt M, et al. High-sensitivity immunofluorescence staining: a com-
parison of the liposome procedure and the FASER technique on mGR detection. J Fluoresc.
2013;23(3):509–18.
51. Sekula-Neuner S, Maier J, Oppong E, Cato AC, Hirtz M, Fuchs H. Allergen arrays for anti-
body screening and immune cell activation profiling generated by parallel lipid dip-pen nano-
lithography. Small. 2012;8(4):585–91.
52. Oppong E, Hedde PN, Sekula-Neuner S, et al. Localization and dynamics of glucocorticoid
receptor at the plasma membrane of activated mast cells. Small. 2014;10(10):1991–8.
53. Abraham SM, Lawrence T, Kleiman A, et al. Antiinflammatory effects of dexamethasone are
partly dependent on induction of dual specificity phosphatase 1. J Exp Med. 2006;
203(8):1883–9.
54. Abraham SM, Clark AR. Dual-specificity phosphatase 1: a critical regulator of innate immune
responses. Biochem Soc Trans. 2006;34(Pt 6):1018–23.
55. Nissen RM, Yamamoto KR. The glucocorticoid receptor inhibits NFkappaB by interfering
with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes
Dev. 2000;14(18):2314–29.
56. Rogatsky I, Zarember KA, Yamamoto KR. Factor recruitment and TIF2/GRIP1 corepressor
activity at a collagenase-3 response element that mediates regulation by phorbol esters and
hormones. EMBO J. 2001;20(21):6071–83.
contexts. Proc Natl Acad Sci U S A. 2002;99(26):16701–6.
58. Chopra AR, Louet JF, Saha P, et al. Absence of the SRC-2 coactivator results in a glyco-
genopathy resembling Von Gierke’s disease. Science. 2008;322(5906):1395–9.
59. Gehin M, Mark M, Dennefeld C, Dierich A, Gronemeyer H, Chambon P. The function of
TIF2/GRIP1 in mouse reproduction is distinct from those of SRC-1 and p/CIP. Mol Cell Biol.
2002;22(16):5923–37.
60. Patchev AV, Fischer D, Wolf SS, et al. Insidious adrenocortical insufficiency underlies neuro-
endocrine dysregulation in TIF-2 deficient mice. FASEB J. 2007;21(1):231–8.
61. Chinenov Y, Gupte R, Dobrovolna J, et al. Role of transcriptional coregulator GRIP1 in the
anti-inflammatory actions of glucocorticoids. Proc Natl Acad Sci U S A. 2012;109(29):
11776–81.
62. Tuckermann JP, Kleiman A, Moriggl R, et al. Macrophages and neutrophils are the targets for
immune suppression by glucocorticoids in contact allergy. J Clin Invest. 2007;117(5):
1381–90.
63. Matasic R, Dietz AB, Vuk-Pavlovic S. Dexamethasone inhibits dendritic cell maturation by
redirecting differentiation of a subset of cells. J Leukoc Biol. 1999;66(6):909–14.
64. Woltman AM, de Fijter JW, Kamerling SW, Paul LC, Daha MR, van Kooten C. The effect of
calcineurin inhibitors and corticosteroids on the differentiation of human dendritic cells. Eur
J Immunol. 2000;30(7):1807–12.
65. Schwarz BA, Bhandoola A. Trafficking from the bone marrow to the thymus: a prerequisite
for thymopoiesis. Immunol Rev. 2006;209:47–57.
66. Broere F, Apasov SG, Sitkovsky MV, van Eden W. T cell subsets and T cell-mediated
immunity. 3rd ed. New York: Springer; 2011.
67. Stemberger C, Neuenhahn M, Buchholz VR, Busch DH. Origin of CD8+ effector and mem-
ory T cell subsets. Cell Mol Immunol. 2007;4(6):399–405.
68. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O’Garra A, Murphy KM. Development of TH1
CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science.
1993;260(5107):547–9.
69. Rogge L, D’Ambrosio D, Biffi M, et al. The role of Stat4 in species-specific regulation of Th
cell development by type I IFNs. J Immunol. 1998;161(12):6567–74.
70. Ahmed R, Gray D. Immunological memory and protective immunity: understanding their
relation. Science. 1996;272(5258):54–60.
71. Flammer JR, Rogatsky I. Minireview: glucocorticoids in autoimmunity: unexpected targets
and mechanisms. Mol Endocrinol. 2011;25(7):1075–86.
72. Elenkov IJ. Glucocorticoids and the Th1/Th2 balance. Ann N Y Acad Sci. 2004;1024:
138–46.
73. Opferman JT, Korsmeyer SJ. Apoptosis in the development and maintenance of the immune
system. Nat Immunol. 2003;4(5):410–5.
74. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in
apoptosis. Genes Dev. 1999;13(15):1899–911.
75. Budd RC. Activation-induced cell death. Curr Opin Immunol. 2001;13(3):356–62.
76. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from
mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997;275(5303):
1132–6.
77. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from
mitochondria blocked. Science. 1997;275(5303):1129–32.
78. Ashwell JD, Lu FW, Vacchio MS. Glucocorticoids in T cell development and function*.
Annu Rev Immunol. 2000;18:309–45.
79. Herold MJ, McPherson KG, Reichardt HM. Glucocorticoids in T cell apoptosis and function.
Cell Mol Life Sci. 2006;63(1):60–72.
80. Distelhorst CW. Recent insights into the mechanism of glucocorticosteroid-induced apopto-
sis. Cell Death Differ. 2002;9(1):6–19.
81. Zamoyska R, Basson A, Filby A, Legname G, Lovatt M, Seddon B. The influence of the
src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation. Immunol
Rev. 2003;191:107–18.
82. Palacios EH, Weiss A. Function of the Src-family kinases, Lck and Fyn, in T-cell develop-
ment and activation. Oncogene. 2004;23(48):7990–8000.
83. Lowenberg M, Tuynman J, Bilderbeek J, et al. Rapid immunosuppressive effects of glucocor-
ticoids mediated through Lck and Fyn. Blood. 2005;106(5):1703–10.
84. Lowenberg M, Verhaar AP, Bilderbeek J, et al. Glucocorticoids cause rapid dissociation of a
T-cell-receptor-associated protein complex containing LCK and FYN. EMBO Rep. 2006;
7(10):1023–9.
85. Bartis D, Boldizsar F, Szabo M, Palinkas L, Nemeth P, Berki T. Dexamethasone induces rapid
tyrosine-phosphorylation of ZAP-70 in Jurkat cells. J Steroid Biochem Mol Biol.
2006;98(2–3):147–54.
86. Boldizsar F, Szabo M, Kvell K, et al. ZAP-70 tyrosines 315 and 492 transmit non-genomic
glucocorticoid (GC) effects in T cells. Mol Immunol. 2013;53(1–2):111–7.
87. Fox S, Leitch AE, Duffin R, Haslett C, Rossi AG. Neutrophil apoptosis: relevance to the
innate immune response and inflammatory disease. J Innate Immun. 2010;2(3):216–27.
88. Hallett JM, Leitch AE, Riley NA, Duffin R, Haslett C, Rossi AG. Novel pharmacological
strategies for driving inflammatory cell apoptosis and enhancing the resolution of inflamma-
tion. Trends Pharmacol Sci. 2008;29(5):250–7.
89. Savill J. Apoptosis in resolution of inflammation. J Leukoc Biol. 1997;61(4):375–80.
90. Filep JG, El Kebir D. Neutrophil apoptosis: a target for enhancing the resolution of inflam-
mation. J Cell Biochem. 2009;108(5):1039–46.
91. Lacy P. Mechanisms of degranulation in neutrophils. Allergy Asthma Clin Immunol. 2006;
2(3):98–108.
92. Cox G. Glucocorticoid treatment inhibits apoptosis in human neutrophils. Separation of
survival and activation outcomes. J Immunol. 1995;154(9):4719–25.
93. Aoki K, Ishida Y, Kikuta N, Kawai H, Kuroiwa M, Sato H. Role of CXC chemokines in the
enhancement of LPS-induced neutrophil accumulation in the lung of mice by dexametha-
sone. Biochem Biophys Res Commun. 2002;294(5):1101–8.
94. Saffar AS, Ashdown H, Gounni AS. The molecular mechanisms of glucocorticoids-mediated
neutrophil survival. Curr Drug Targets. 2011;12(4):556–62.
95. Heasman SJ, Giles KM, Ward C, Rossi AG, Haslett C, Dransfield I. Glucocorticoid-mediated
regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implica-
tions for the resolution of inflammation. J Endocrinol. 2003;178(1):29–36.
96. Barnes PJ. Inhaled corticosteroids are not beneficial in chronic obstructive pulmonary dis-
ease. Am J Respir Crit Care Med. 2000;161(2 Pt 1):342–4. discussion 344.
97. Nakagawa M, Terashima T, D’Yachkova Y, Bondy GP, Hogg JC, van Eeden SF. Glucocorticoid-
induced granulocytosis: contribution of marrow release and demargination of intravascular
granulocytes. Circulation. 1998;98(21):2307–13.
98. Kraft S, Kinet JP. New developments in FcepsilonRI regulation, function and inhibition. Nat
Rev Immunol. 2007;7(5):365–78.
99. Rivera J, Gilfillan AM. Molecular regulation of mast cell activation. J Allergy Clin Immunol.
2006;117(6):1214–25. quiz 1226.
100. Scharenberg AM, Lin S, Cuenod B, Yamamura H, Kinet JP. Reconstitution of interactions
between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor
clustering. EMBO J. 1995;14(14):3385–94.
101. Scharenberg AM, Kinet JP. Early events in mast cell signal transduction. Chem Immunol.
1995;61:72–87.
102. Metcalfe DD, Peavy RD, Gilfillan AM. Mechanisms of mast cell signaling in anaphylaxis. J
Allergy Clin Immunol. 2009;124(4):639–46. quiz 647-638.
103. Kassel O, Sancono A, Kratzschmar J, Kreft B, Stassen M, Cato AC. Glucocorticoids inhibit
MAP kinase via increased expression and decreased degradation of MKP-1. EMBO
J. 2001;20(24):7108–16.
104. Andrade MV, Hiragun T, Beaven MA. Dexamethasone suppresses antigen-induced activation
of phosphatidylinositol 3-kinase and downstream responses in mast cells. J Immunol.
2004;172(12):7254–62.
105. Zhou J, Liu D, Liu C, Kang ZM, Shen XH, Chen YZ, Xu T, Jiang CL. Glucocorticoids inhibit
degranulation of mast cells in allergic asthma via nongenomic mechanism. Allergy.
2008;63:1177–85.
106. Liu C, Zhou J, Zhang LD, Wang YX, Kang ZM, Chen YZ, Jiang CL. Rapid inhibitory effect
of corticosterone on histamine release from rat peritoneal mast cells. Horm Metab Res.
2007;39:273–7.
107. Barnes PJ. Anti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin Sci
(Lond). 1998;94(6):557–72.
108. Kui Wu YB. Kun Sun and Changzheng Wang IL-10-producing type 1 regulatory T cells and
allergy. Cell Mol Immunol. 2007;4(4):269–75.
109. Hawrylowicz CM, O’Garra A. Potential role of interleukin-10-secreting regulatory T cells in
allergy and asthma. Nat Rev Immunol. 2005;5(4):271–83.
110. Yamagata S, Tomita K, Sano H, et al. Non-genomic inhibitory effect of glucocorticoids on
activated peripheral blood basophils through suppression of lipid raft formation. Clin Exp
Immunol. 2012;170(1):86–93.
111. Yoshimura C, Miyamasu M, Nagase H, et al. Glucocorticoids induce basophil apoptosis. J
Allergy Clin Immunol. 2001;108(2):215–20.
112. Moser M, De Smedt T, Sornasse T, et al. Glucocorticoids down-regulate dendritic cell func-
tion in vitro and in vivo. Eur J Immunol. 1995;25(10):2818–24.
113. Piemonti L, Monti P, Allavena P, et al. Glucocorticoids affect human dendritic cell differen-
tiation and maturation. J Immunol. 1999;162(11):6473–81.
114. Piemonti L, Monti P, Allavena P, Leone BE, Caputo A, Di Carlo V. Glucocorticoid increase
the endocytic activity of human dendritic cells. Int Immunol. 1999;11(9):1519–26.
115. Vizzardelli C, Pavelka N, Luchini A, et al. Effects of dexamethazone on LPS-induced activa-
tion and migration of mouse dendritic cells revealed by a genome-wide transcriptional analy-
sis. Eur J Immunol. 2006;36(6):1504–15.
116. Gruver-Yates AL, Quinn MA, Cidlowski JA. Analysis of glucocorticoid receptors and their
apoptotic response to dexamethasone in male murine B cells during development.
Endocrinology. 2014;155(2):463–74.
117. Cupps TR, Edgar LC, Thomas CA, Fauci AS. Multiple mechanisms of B cell immunoregula-
tion in man after administration of in vivo corticosteroids. J Immunol. 1984;132(1):170–5.
118. Cupps TR, Gerrard TL, Falkoff RJ, Whalen G, Fauci AS. Effects of in vitro corticosteroids on
B cell activation, proliferation, and differentiation. J Clin Invest. 1985;75(2):754–61.
Chapter 10
Glucocorticoids and the Brain: Neural
Mechanisms Regulating the Stress Response
Shawn N. Shirazi, Aaron R. Friedman, Daniela Kaufer,

and Samuel A. Sakhai
Abstract In this chapter, we describe the central role of the brain in the glucocor-
ticoid mediated stress response. We describe the mechanisms by which the brain
gauges the severity of stress, mechanisms of hypothalamic-pituitary-adrenal axis
(HPA) regulation, and how various sub-systems of the brain respond to glucocor-
ticoid (GC) signaling to regulate stress behavior. In particular, we focus on the
hippocampus, pre-frontal cortex, and amygdala, where GCs can induce a series
of changes. Finally, we briefly discuss an apparent paradox in GC signaling:
while exposure to glucocorticoids promotes the survival of an organism during
acute stress, these same hormones in chronic excess can also cause damage and
promote illness.
Keywords Stress • HPA axis • Negative feedback • Glucocorticoids • Behavior

• Amygdala • Prefrontal cortex • Hippocampus
Introduction
Organisms face a wide variety of environmental conditions that can perturb

homeostasis. To effectively respond to these “stressors,” the organism must initiate
a coordinated response across a variety of physiological systems. For example, the
S.N. Shirazi • A.R. Friedman

Department of Integrative Biology, University of California,
430 Li Ka Shing Center #3370, Berkeley, CA 94720, USA
D. Kaufer, Ph.D. (*)
Department of Integrative Biology, Kaufer Lab, University of California,
400 Li Ka Shing Center, Berkeley, CA 94720, USA
S.A. Sakhai, Ph.D. (*)
Department of Psychology, University of California,
3210 Tolman Hall, Berkeley, CA 94720, USA

236 S.N. Shirazi et al.
organism must perceive the stressor and select appropriate behavioral strategies
(brain) and optimize energy resources towards a “fight, flight or freeze” response
(cardiac, respiratory, skeletal) in part by shutting down systems that are not imme-
diately essential (digestive, reproductive, growth).1 In the vertebrate stress response,
the activation of these various systems is initiated by the release of glucocorticoid
(GC) stress hormones from the adrenal glands, and also by catecholamine signaling.
Importantly, the stressful situations that an organism encounters are diverse.
Stressors may be acute and severe (e.g., predation), chronic and severe (e.g.,
drought), or mild (e.g., social interactions) and each type of stressor requires a
unique adaptive response. On the other hand, some types of challenges are predict-
able, and in these cases GC secretion can allow the organism to prime its physiolog-
ical response in anticipation of the pending challenge. For example, in diurnal
animals GCs are secreted in a daily circadian cycle, with high GC secretion induc-
ing arousal during the early morning and a GC trough promoting rest during the
evening. To respond to these wide variety of environmental challenges, ranging
from mild to severe and predictable to unpredictable, vertebrates have evolved a
complex regulatory system, the hypothalamic-pituitary-adrenal (HPA) axis, to
perceive the severity of environmental challenge and release an appropriate amount
of GCs for a measured, homeostatic behavioral response.
In this chapter, we describe the central role of the brain in the GC-mediated stress
response. We describe the mechanisms by which the brain gauges the severity of
stress and initiates an appropriate systemic response—in other words, regulation via
the HPA axis. Secondly, we describe how various sub-systems of the brain respond
to GC signaling to regulate stress behavior. In particular, we focus on the hippocam-
pus, pre-frontal cortex, and amygdala, where GCs can induce a series of changes
(Fig. 10.1). These include alterations that underpin behavioral responses such as
alertness and cognitive function, appetitive versus aversive thresholds to various
threatening stimuli and rewards (i.e., motivation vs. avoidance), fear, and memory
formation. On a cellular and molecular level, this entails modulations of neurotrans-
mitter levels, alterations in dendritic morphology, receptor density, and changes in
signal transduction. Thirdly, we briefly discuss an apparent paradox in GC signal-
ing: while exposure to glucocorticoids promotes the survival of an organism during
acute stress, these same hormones in chronic excess can also cause damage and
promote illness. Chronic stress is a risk factor for multiple diseases, including
diseases of central and peripheral nervous systems such as stroke, mental illness,
and multiple sclerosis [122–127]. Within the CNS, chronic glucocorticoid exposure
can suppress neurogenesis, bias cell fates of neural precursor cells, contribute to
dendritic atrophy, and alter neuronal excitability in key regions of the brain involved
in anxiety and depression [reference]. Therefore, an organism’s best option is to
mount as efficient a stress response as possible, limiting its exposure to high levels
of catabolic and metabolically demanding glucocorticoids. Fine-tuning of the stress
1
Hans Selye, the father of modern stress research defined stress as the “non-specific response of
the body to any demand made upon it” [96].
10 Glucocorticoids and the Brain: Neural Mechanisms… 237
Fig. 10.1 Selected limbic structures involved with HPA axis regulation
response can have a dramatic influence on health. Importantly, the calibration and
reactivity of the stress response is partly dependent upon early life environmental
contexts and developmental programming, which help prepare organisms for
future and current environmental challenges.
The Hypothalamic-Pituitary Adrenal Axis
As a first step in activating the HPA axis, the brain integrates external and internal
sensory information pertaining to the immediate challenge, and this information is
transduced into endocrine responses within the paraventricular nucleus of the hypo-
thalamus (PVN) [45]. The hypophysiotropic neurons within the PVN secrete
corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) into the
hypophyseal portal system, a system of blood vessels that link the hypothalamus
with the pituitary gland. Upon reaching the anterior pituitary, CRH stimulates the
release of adrenocorticotropic hormone (ACTH) into circulation. Elevated ACTH
levels, in turn, stimulate the synthesis and release of glucocorticoids via binding to
melancortin-2 receptors within the cortex of the adrenal glands [1]. HPA activation
results in a maximal rise in circulating GCs after 15–30 min, and returns to baseline
levels at roughly one hour after the termination of a stressor [93]. The crucial ability
to terminate the stress response, or inhibit the secretion of CRH and ACTH, is via
glucocorticoid negative feedback on key neural regions, such as the PVN, anterior
pituitary, medial prefrontal cortex (mPFC), and hippocampus.
The stress response, as a whole, does not solely depend on GCs to alter physiol-
ogy and behavior—it also requires the concerted actions of several other neuropep-
tides. These include: urocortins, which interact with CRH; vasopressin, which is
implicated in stress-related social memory and emotionality; and orexins, which are
involved with stress-related energy and circadian homeostasis. Furthermore, CRH
acts in many other brain regions outside of the PVN of the hypothalamus. For example,
CRH is released in the bed nucleus of the stria terminalis (BnST) where it plays a
role in stress-related anxiety. In the nucleus accumbens, CRH acts to suppress dopa-
mine release in response to rewards, and shift appetitive and aversive behaviors [61,
113] while in the amygdala and in the hippocampus it is involved in stress-related
emotional memories, anxiety, and learning processes [89, 92]. A review of the
actions of GCs on the brain is incomplete without considering the coordinated influ-
ence of the aforementioned peptide mediators, however, its discussion exists outside
the scope of this chapter. For an excellent review of CRH, see [53].
HPA Negative Feedback
The ability of the HPA axis to respond dynamically to stress or to tonic secretion of
glucocorticoids via circadian rhythm is determined, in part, by the ability of
glucocorticoids to adjust ACTH secretion. This negative feedback occurs when GCs
penetrate the blood–brain barrier and exert rapid (non-genomic) and slower
(genomic) effects on the various neural regions that regulate ACTH release [24, 38,
101]. Two classes of brain steroid receptors mediate negative feedback: the miner-
alocorticoid receptor (MR), and glucocorticoid receptor (GR). Both MR and GR
belong to the nuclear receptor superfamily and function as transcription factors
regulating gene expression [85].
MRs have a relatively limited distribution, exhibiting the highest expression
within the subiculum/CA1 field and dentate gyrus of the hippocampus [85]
(Fig. 10.2a). GRs are expressed nearly ubiquitously (Fig. 10.2b). There are, how-
ever, areas of greater GR density within the hippocampus, amygdala, cerebellum,
hypothalamus (most notably the PVN), neurons of the ascending aminergic path-
ways of the brainstem, and to a lesser extent, the caudate nucleus and putamen
[34, 78]. While both receptor subtypes bind corticosterone, MRs have a roughly
tenfold greater affinity for GCs relative to GRs (Kd of ~0.5 nM for MR vs. Kd
~2.0–5.0 nM for GR) [85]. Consequently, MRs preferentially bind GCs over GRs
and reach near-saturation levels during troughs of the circadian cycle (i.e., low basal
levels), and are fully saturated during circadian peaks and stress. GRs are activated
only when glucocorticoid levels reach a high concentration beyond the level that
saturates MRs, such as during an acute stressor or during the zenith of the circadian
rhythm. It is hypothesized that MRs are a critical component of the circadian regu-
lation of baseline HPA tone (via fast-feedback, non-genomic actions), while GRs,
occupied at higher corticosteroid concentrations, mediate feedback actions follow-
ing stress [7]. Thus, the balance of MR and GR receptor types, their occupancy, and
Fig. 10.2 (a) MR distribution in the mouse brain. (b) GR distribution in the mouse brain
their associated mechanism of action are intricately involved in HPA regulation.

Might be worth citing the papers where they make tissue specific KO of GR in
hypothalamus or hypothalamus + GR and get Cushings syndrome.
Major Brain Structures Involved in HPA Regulation
Four brain regions are strongly implicated as sites for HPA regulation and synthesis
of CRH and AVP. These include the PVN of the hypothalamus, frontal cortex,
amygdala and hippocampus.
Paraventricular Nucleus of the Hypothalamus
The PVN is the main gateway for initiating the hormonal stress response, and thus
a primary target for regulating HPA negative feedback. It contains one of the densest
populations of CRH neurons, which express GRs [17, 110]. Exogenous application
of GCs in the PVN results in a rapid decrease in CRH mRNA expression [56] lead-
ing to a corollary decrease in HPA activation. Conversely, lesioning PVN afferents
serves to increase expression of CRH and AVP mRNA demonstrating that neuronal
inhibitory pathways are also necessary for the maintenance of HPA tone [44, 45].
Non-genomic, fast feedback inhibition of the HPA axis within the PVN is depen-
dent on both endocannabinoid and GABAergic mechanisms [106]. GCs stimulate
the synthesis and release of endocannabinoids within the PVN by binding to
membrane-bound MRs. These endocannabinoids then bind to presynaptic CB1
receptors to suppress glutamatergic transmission, thus inhibiting the activation of
PVN neurons and reducing secretion of CRH [24, 29, 48]. GCs also bind to receptors
on inhibitory magnocellular neurons of the PVN to stimulate fast, G-protein-dependent
release of GABA to inhibit downstream CRH secretion [106]. In this fashion, MRs
act in a rapid, non-genomic pathway for negative feedback within the PVN of the
hypothalamus.
Medial Prefrontal Cortex
In rodents, the medial prefrontal cortex (mPFC) is comprised of infra-limbic (IL),

pre-limbic (PL), and anterior cingulate cortices (AC) (based on structural connec-
tivity and function, these areas are thought to be homologous to human Brodman
areas 25, 32 and 24b respectively) [111, 112]. The mPFC is a region of the brain that
is involved in cognitive and executive functioning, including working memory, the
ability to shift attention across perceptual dimensions, and rule-guided action to
plan and guide behavioral sequences. Receiving diverse afferent inputs from the
amygdala and ventral hippocampus, as well as providing direct efferent connections
to hypothalamic and monoamine brain nuclei, the mPFC is well situated to regulate
cognitive, emotional and physiological responses to stress [13].
The mPFC is highly involved in autonomic control and HPA inhibition [2, 6, 75].
Evidence for HPA suppression arises from lesion studies in which mPFC lesions
lead to significantly increased plasma levels of ACTH and corticosterone following
restraint stress and increased c-Fos activation in the PVN and medial amygdala [10,
27, 31, 102]. Furthermore, local injections of corticosterone into the mPFC are
capable of dampening plasma levels of these same hormones [3]. However, expo-
sure to chronic stressors, such as 4 weeks of daily restraint, leads to a down regula-
tion of GR mRNA and protein levels in the PFC, resulting in attenuated PFC-mediated
HPA negative feedback [74]. Of note, the primate brain expresses significantly
different GR and MR distributions compared to rodents. In primates, GR levels are
in greater abundance in the mPFC than the hippocampus, where there is a relative
paucity in expression [91]. This suggests that the primate PFC may play a larger
role in GR mediated feedback than the hippocampus.
The mPFC influence over the HPA axis is both intra-region specific and exhibits
hemispheric functional lateralization [84, 102]. Pre-limbic and infra-limbic cortices
exert opposing control over HPA tone. The pre-limbic cortex can be thought of as
the ‘brakes’ whereas the infra-limbic cortex can be considered the ‘gas pedal’ of
HPA regulation. Electrical stimulation of pre-limbic cortex activates parasympa-
thetic systems, whereas infra-limbic stimulation results in robust HPA activation.
More recent work has confirmed that this dual control of HPA regulation is GR
dependent. GR knockdown in pre-limbic or infra-limbic cortices via short-hairpin
RNA leads to differential regulation of HPA secretion, such that infra-limbic disrup-
tion leads to HPA hyper-reactivity, while pre-limbic GR knockdown contributes to
stress hypo-reactivity in response to an acute psychogenic stressor [70].
HPA control also exhibits hemispheric lateralization. HPA activation is markedly
lower after right mPFC lesions, but not left [102]. HPA axis down-regulation after
right mPFC lesion was found to be greater in response to chronic stress than to acute
stress, suggesting that the mPFC is associated with regulating HPA activity during
highly stressful conditions [13].
Intra-region specificity of the mPFC (brake vs. gas) is stressor specific. It modu-
lates its responses based on the nature of the environmental challenges presented,
such as psychological stress vs. physical stress. The pre-limbic cortex (brake) is of
particular note in its role in inhibiting the HPA axis, especially with respect to
psychogenic stressors. This is evidenced by inhibition of CRH and AVP expression
after GC infusion into mPFC during restraint stress, a psychological stressor, but
not the anesthetic ether, a physical stressor. Conversely, the infra-limbic cortex can
initiate HPA activity. It responds robustly to both physical and psychogenic stress
[70, 84, 103]. For instance, repeated social stress, but not noise stress, significantly
increases ∆FosB expression, an immediate early gene used as a marker for neuronal
activation, within the infra-limbic mPFC [50]. This suggests that the mPFC plays a
role in the ability to discriminate between psychogenic and physical stressors,
thereby increasing the efficiency and specificity of HPA axis regulation [27, 31, 84].
Similarly to the PVN, mPFC-mediated HPA inhibition is dependent in part on
GR-mediated endocannabinoid signaling. CB1 receptor antagonism within the
mPFC up-regulates HPA activity and results in prolonged GC secretions.
Furthermore, GC exposure results in endocannabinoid release, indicating homeo-
static negative feedback. Mechanistically, increases in endocannabinoids lead to a
decrease in GABA release. This results in a net gain in excitation on pre-limbic
(brake) neurons [49]. The pre-limbic cortex has direct afferents onto GABAergic
neurons within the BnST, which in turn send projections to the neurosecretory cells
of the PVN. In this fashion, GC-induced CB1 activation of the pre-limbic mPFC
results in an activation of inhibitory BnST-PVN circuitry. The net result is the sup-
pression of HPA activity.
Responding to psychogenic and physiological stressors, the mPFC is a key
component in the top-down regulation of the HPA axis. Part of this regulation is
mediated by serotonergic mPFC-amygdala connectivity [32]. For example, decou-
pling serotonergic mPFC-amygdala circuitry leads to alterations in stress related
behaviors [5, 116]. However, the amygdala also regulates the mPFC, thus is another
critical component in the emotional guidance of behavior [26].
Amygdala
Receiving direct and indirect connections from limbic structures, including mPFC
and hippocampus, the amygdala is thought to be a major integrating center for emo-
tional and arousing stimuli. The amygdala is highly involved in the systemic stress
response and is sensitive to both glucocorticoids and catecholamines. Direct stereo-
tactic infusions of GC to the amygdala greatly increase CRH mRNA expression
within the PVN during psychogenic stress, illustrating the amygdala’s capacity to
alter HPA activity during elevated GC exposure [97].
Like the mPFC, the amygdala’s influence over HPA systems is region specific.
The amygdala is a complex of many sub-nuclei, often segregated into three regions:
corticomedial (MeA), central (CeA), and basolateral (BLA) nuclei groups [100].
The CeA is further divided into a lateral component (CeL), and a medial (CeM)
component. The balance of excitation and inhibition in each of these sub-regions
modulates HPA reactivity [12, 68, 98, 100]. The amygdala has both “anxiogenic”
and “anxiolytic” pathways. Stimulation of the BLA itself has been shown to increase
HPA activity (anxiogenic), while direct stimulation of the CeM, results in anxiolytic
effects. Since both the CeA and the BLA sends projections to the PVN via the
BNST, the net effect of amygdala activation on the HPA axis is contingent upon
the circuitry that is invoked [80, 109].
Hippocampus
Involved in cognition and memory formation, the hippocampus is a critical locus in

HPA regulation. It exhibits tremendous plasticity to stress and glucocorticoids.
Within the rodent brain, the hippocampus expresses the highest level of GR and
MR, hence, it is of little surprise that it serves as an important negative feedback
center and regulator of the stress response [46]. Evidence for HPA negative feed-
back arises from early studies in which lesioning or blocking hippocampal GC
receptors results in an up-regulation of CRH and AVP mRNA within the PVN. The
consequence of this is hypersecretion of glucocorticoids [52, 93]. Conversely, acti-
vation of hippocampal GC receptors results in HPA axis inhibition [93].
However, the relationship between hippocampus and HPA regulation are specific
to particular hippocampal sub-regions. Structurally and functionally heterogeneous,
the hippocampus can be segregated across a septotemporal axis [8, 30]. In rodents,
the dorsal hippocampus appears to be more involved in learning and memory, while
the ventral hippocampus is implicated in the modulation of anxiety-like behavior
and HPA regulation [8]. Lesions to dorsal hippocampus result in spatial memory
deficits, whereas ventral hippocampal lesions result in anxiety-like behaviors alter-
ations [8, 30, 79]. These differences in function can be explained in part by the
connectivity of each region. For instance, regions of the ventral hippocampus proj-
ect to areas involved in emotional regulation, most notably, the mPFC, amygdala,
BnST, and the PVN [30].
The hippocampus is also one of two brain regions in which resident populations
of neural stem cells (NSCs) produce new neurons in adult animals [33, 35]. These
new neurons are thought to contribute to the plasticity of hippocampal networks and
have roles in the classical hippocampal functions of learning and memory. However,
several lines of recent evidence also suggest that NSCs play a role in HPA regula-
tion. First, chronic, but not acute, activation of hippocampal GRs is associated with
a general increase in HPA reactivity [86]. This failure in HPA axis negative feed-
back may be due, in part, to a reduction of the neurogenic pool resulting from
chronic GC exposure. More direct evidence comes from ablation of hippocampal
neurogenesis, through the use of techniques such as irradiation or by transgenic

animal models, which results in impaired HPA negative feedback and elevated GC
levels following recovery from restraint stress [99]. Animals with impaired neuro-
genesis also exhibit a depressed phenotype at baseline that can be reversed by
antidepressant treatment [99]. Lastly, hippocampal neurogenesis appears to be
required for antidepressants to restore HPA axis inhibition following chronic stress
[104]. Taken as a whole, these and other findings have implicated hippocampal
neurogenesis as a component of HPA axis regulation.
Effects of GCs on Brain and Behavior
As described above, many brain regions that integrate sensory information, such as
the mPFC, amygdala, and hippocampus, can exert control of the PVN to fine tune
the stress response according to the immediate experiences of the animal. In turn,
once the stress response is initiated, the animal also has to enact appropriate behav-
ioral strategies to cope with the stressor. Thus, beyond acting as a negative feedback
signal, GCs also modulate brain function in these same regions to coordinate appro-
priate stress-response behaviors.
Medial Prefrontal Cortex
Stress, both mild and severe, can lead to functional and structural changes in the
prefrontal cortex [6, 39, 51]. This includes alterations in dendritic arborization and
spine density in all regions of the mPFC (IL, PL, and AC) and neighboring orbito-
frontal cortex, which is driven in part, by GR signaling [11, 15, 62, 63, 82, 121]. For
instance, 3 weeks of corticosterone administration [115] or daily restraint stress
[20], is capable of reducing dendritic arborization and spine density in the mPFC
and dorsomedial striatum [21, 25]. Functionally, both glucocorticoid administration
and stress leads to deficits in working memory [11, 15, 76, 90], mPFC dependent
set-shifting [63], as well as reversal learning [14, 15, 60].
Paradoxically, under some conditions, chronic stress can facilitate reversal
learning [40, 41]. One hypothesis by Dias-Ferreira and colleagues as well as
Schwab and Wolf, posits that stress leads to a disinhibition of PFC functions and
towards striatal mediated learning [25]. The effect is bias in an organism’s behavior
towards habit formation [25, 41, 94, 95]. Indeed, severe, repeated stressors result in
an increase in apical dendrite arborization in both the dorsolateral striatum and
orbitofrontal cortex, regions involved in habitual strategies, reward valuation, and
reversal learning [25, 63]. These studies suggest that the effects of stress and gluco-
corticoids may be beneficial to shift behaviors toward optimal behavioral adaption
to environmental stress.
Amygdala
Its activation by GCs can lead to alterations in learning and memory [88]. However,
it is important to note that amygdala contributions to autonomic functioning are
with respect to emotional arousal, not circadian or homeostatic HPA regulation.
Both acute and chronic stress results in the remodeling of synapses and dendritic
branching within the amygdala [72, 73]. Stress induced synaptic plasticity is modu-
lated by GABAergic inputs [23]. These changes are correlated with an increase in
anxiety-like behaviors and enhanced fear conditioning [19, 73, 118]. High levels of
corticosterone reduce GABA transmission, which results in an increase in the firing
rate of excitatory neurons in the basolateral amygdala [28]. This suggests that high
levels of glucocorticoids can change the balance between excitation and inhibition,
resulting in modifications in synaptic connectivity. These changes can influence
neuronal plasticity even in distal brain regions. Recent findings demonstrate that the
BLA can alter synaptic plasticity and long-term potentiation in the striatum and
hippocampus. Therefore, it is becoming increasingly clear that glucocorticoids
within the amygdala can be far-reaching and impactful [4, 81].
Finally, the amygdala also plays a central role in enhancing memory consolida-
tion following emotionally arousing events. High levels of circulating GCs can
improve the recall of a stressful event [9, 77, 89]. However, GCs effects may be
mediated by ß-adrenergic activation; blockade of ß-ardrenergic receptors within the
BLA prevents memory enhancements following GR activation. Furthermore, acti-
vation of BLA via emotional arousal is critical in GC-mediated memory enhance-
ments [83]. Enhanced memory performance following a stressful event can be
advantageous, as future encounters to similarly arousing stimuli would result in a
feed-forward HPA activation to prime physiological systems in anticipation of a
stressor. However, more investigations are needed to fully determine how stress,
NE, and GCs influence different phases of fear learning and its expression in
memory.
Hippocampus
The hippocampus responds dynamically to changes in GCs levels by modulating

neuronal structure and function. GCs directly influence hippocampal function by
acting as neuromodulators to influence neural excitability and signaling [16, 54, 55,
59, 64]. More broadly, GCs also affect the structural connectivity of the hippocam-
pus by affecting dendritic arborization and formation of synapses [114, 119, 120].
Together, a model has emerged from these studies in which mild or acute stress
increases hippocampal dendritic branching and long-term potentiation to boost
hippocampal learning and memory, while chronic or high GC concentrations have
opposite effects [19, 47, 58, 92]. However, the distribution of MR and GR receptors
differs in dorsal versus ventral hippocampus, with ventral hippocampus having a
much higher relative concentration of MR [87]. This suggests that the effect of
stress on hippocampal function may be more nuanced and region-specific, such that
high levels of GCs do not simply suppress hippocampal memory function in
general, but rather specifically suppress the contextual memory functions of dorsal
hippocampus while promoting the emotional cognitive functions of the ventral hip-
pocampus [65, 66]. This model fits well with the overall paradigm of the stress
response as an adaptive mechanism that manifests stress-specific behavioral strate-
gies suited to overcoming stressful challenges [65, 66].
Stress effects on hippocampal-mediated behaviors may also be regulated
through the contributions of hippocampal NSCs. NSCs express functional GRs (but
not MRs) [18, 36], and their rate of proliferation and differentiation, as well as
the survival of the new neurons that they produce, are altered by GCs [58, 117,
128–130]. The effects can be via direct activation of GR in the NSC [36] or
indirectly, through activation of GR-dependent mechanisms in other cells in the
hippocampal niche. For instance, acute corticosterone exposure elicits release of
fibroblast growth factor-2 from astrocytes in the dorsal hippocampus, leading to
increased proliferation of neural stem cells in the area [58].
The effects of stress on adult neurogenesis can be divided into the effects of acute
stress and repeated, chronic stress. Chronic, repeated stressors inhibit NSC survival,
proliferation, and neuronal differentiation within the dentate gyrus [57, 71, 117].
However, the effects of acute stress display a more mixed picture, ranging from a
decrease, increase, or no change in NSC proliferation [22, 43, 107, 108]. One expla-
nation for discrepancies in the literature may be that, like cognitive performance in
response to stress, adult hippocampal neurogenesis follows an inverted U function—
increasing in response to acute stressors and decreasing in response to high, chronic
GC exposure. For example, high levels of transient GCs can inhibit NSC prolifera-
tion in the SGZ, and this effect can be blocked through adrenalectomy [105].
Beyond proliferation, high levels of GCs may also reduce the total number of
new neurons by decreasing the survival of immature neurons as they begin to incor-
porate into the network [117]. Furthermore, GCs cause a shift in the cell fate of
differentiation NSCs, causing them to more frequently differentiate into oligoden-
drocytes at the expense of neurogenesis [18]. Given that new neurons ultimately
confer additional plasticity onto hippocampal networks, by forming new synaptic
connections and showing enhanced capacity for LTP [37, 42, 67], the reductions in
neurogenesis in response to elevated GCs may be one of the mechanisms underly-
ing reduced memory capacity in stressed animals.
Conclusion
Glucocorticoids regulate the brain and behavior in multiple domains. They help
adjust basal and peak HPA axis reactivity [93], as well as alter limbic structures
(PVN, mPFC, amygdala, hippocampus), both structurally and functionally. This
includes alterations in synaptic plasticity, long-term potentiation, and neurogenesis,
which result in changes in appetitive and avoidant behaviors, and modifications in

learning and memory. Additionally, the limbic system is responsible for the top-
down and bottom-up regulation of the HPA axis through its complex micro-
circuitry. In this sense, HPA regulation can be a recursive process since
glucocorticoids modulate both initiators and terminators of the stress response.
Ultimately, stress is necessary for the optimization of behavior to environmental
pressures. With respect to humans, it is HPA axis dysregulation that is implicated in
the pathogenesis of many disease phenotypes such as anxiety and depression [69].
It is of paramount importance to efficiently initiate a stress response, as it promotes
survival, while equally important to terminate the stress response, as glucocorti-
coids are metabolically demanding and can lead to disease.
References
1. Abdel-Malek ZA. Melanocortin receptors: their functions and regulation by physiological

agonists and antagonists. Cell Mol Life Sci. 2001;58:434–41.
2. Ahima RS, Harlan RE. Charting of type II glucocorticoid receptor-like immunoreactivity in
the rat central nervous system. Neuroscience. 1990;39:579–604.
3. Akana SF, Chu A, Soriano L, Dallman MF. Corticosterone exerts site-specific and state-
dependent effects in prefrontal cortex and amygdala on regulation of adrenocorticotropic
hormone, insulin and fat depots. J Neuroendocrinol. 2001;13:625–37.
4. Akirav I, Richter-Levin G. Mechanisms of amygdala modulation of hippocampal plasticity. J
Neurosci. 2002;22:9912–21.
5. Andolina D, Maran D, Valzania A, Conversi D, Puglisi-Allegra S. Prefrontal/amygdalar
system determines stress coping behavior through 5-HT/GABA connection. Neuropsycho-
pharmacology. 2013;38:2057–67.
6. Arnsten AFT. Stress signalling pathways that impair prefrontal cortex structure and function.
Nat Rev Neurosci. 2009;10:410–22.
7. Atkinson HC, Wood SA, Castrique ES, Kershaw YM, Wiles CC, Lightman SL. Corticosteroids
mediate fast feedback of the rat hypothalamic-pituitary-adrenal axis via the mineralocorti-
coid receptor. Am J Physiol. 2008;294:E1011–22.
8. Bannerman DM, Rawlins JN, McHugh SB, Deacon RM, Yee BK, Bast T, Zhang WN,
Pothuizen HH, Feldon J. Regional dissociations within the hippocampus–memory and anxi-
ety. Neurosci Biobehav Rev. 2004;28:273–83.
9. Bohannon 3rd JN. Flashbulb memories for the space shuttle disaster: a tale of two theories.
Cognition. 1988;29:179–96.
10. Brake WG, Flores G, Francis D, Meaney MJ, Srivastava LK, Gratton A. Enhanced nucleus
accumbens dopamine and plasma corticosterone stress responses in adult rats with neonatal
excitotoxic lesions to the medial prefrontal cortex. Neuroscience. 2000;96:687–95.
11. Butts KA, Weinberg J, Young AH, Phillips AG. Glucocorticoid receptors in the prefrontal
cortex regulate stress-evoked dopamine efflux and aspects of executive function. Proc Natl
Acad Sci. 2011;108:18459–64.
12. Carlsen J. Immunocytochemical localization of glutamate decarboxylase in the rat basolat-
eral amygdaloid nucleus, with special reference to GABAergic innervation of amygdalostria-
tal projection neurons. J Comp Neurol. 1988;273:513–26.
13. Cerqueira JJ, Almeida OF, Sousa N. The stressed prefrontal cortex. Left? Right! Brain Behav
Immun. 2008;22:630–8.
14. Cerqueira JJ, Mailliet F, Almeida OF, Jay TM, Sousa N. The prefrontal cortex as a key target
of the maladaptive response to stress. J Neurosci. 2007;27:2781–7.
15. Cerqueira JJ, Pêgo JM, Taipa R, Bessa JM, Almeida OF, Sousa N. Morphological correlates
of corticosteroid-induced changes in prefrontal cortex-dependent behaviors. J Neurosci.
2005;25:7792–800.
16. Chameau P, Qin Y, Spijker S, Smit G, Joëls M. Glucocorticoids specifically enhance L-type
calcium current amplitude and affect calcium channel subunit expression in the mouse
hippocampus. J Neurophysiol. 2007;97:5–14.
17. Chen J, Gomez-Sanchez CE, Penman A, May PJ, Gomez-Sanchez E. Expression of miner-
alocorticoid and glucocorticoid receptors in preautonomic neurons of the rat paraventricular
nucleus. Am J Physiol. 2014;306:R328–40.
18. Chetty S, Friedman AR, Taravosh-Lahn K, Kirby ED, Mirescu C, Guo F, Krupik D, Nicholas
A, Geraghty AC, Krishnamurthy A, Tsai MK, Covarrubias D, Wong AT, Francis DD,
Sapolsky RM, Palmer TD, Pleasure D, Kaufer D. Stress and glucocorticoids promote oligo-
dendrogenesis in the adult hippocampus. Mol Psychiatry. 2014;19(12):1275–83.
19. Conrad CD, LeDoux JE, Magarinos AM, McEwen BS. Repeated restraint stress facilitates
fear conditioning independently of causing hippocampal CA3 dendritic atrophy. Behav
Neurosci. 1999;113:902–13.
20. Cook SC, Wellman CL. Chronic stress alters dendritic morphology in rat medial prefrontal
cortex. J Neurobiol. 2004;60:236–48.
21. Czeh B, Muller-Keuker JI, Rygula R, Abumaria N, Hiemke C, Domenici E, Fuchs E. Chronic
social stress inhibits cell proliferation in the adult medial prefrontal cortex: hemispheric
asymmetry and reversal by fluoxetine treatment. Neuropsychopharmacology. 2007;32:
1490–503.
22. Dagytė G, Van der Zee EA, Postema F, Luiten PGM, Den Boer JA, Trentani A, Meerlo
P. Chronic but not acute foot-shock stress leads to temporary suppression of cell proliferation
in rat hippocampus. Neuroscience. 2009;162:904–13.
23. Davis M, Rainnie D, Cassell M. Neurotransmission in the rat amygdala related to fear and
anxiety. Trends Neurosci. 1994;17:208–14.
24. Di S, Malcher-Lopes R, Halmos KC, Tasker JG. Nongenomic glucocorticoid inhibition via
endocannabinoid release in the hypothalamus: a fast feedback mechanism. J Neurosci.
2003;23:4850–7.
25. Dias-Ferreira E, Sousa JC, Melo I, Morgado P, Mesquita AR, Cerqueira JJ, Costa RM, Sousa
N. Chronic stress causes frontostriatal reorganization and affects decision-making. Science.
2009;325:621–5.
26. Dilgen J, Tejeda HA, O’Donnell P. Amygdala inputs drive feedforward inhibition in the
medial prefrontal cortex. J Neurophysiol. 2013;110:221–9.
27. Diorio D, Viau V, Meaney MJ. The role of the medial prefrontal cortex (cingulate gyrus) in
the regulation of hypothalamic-pituitary-adrenal responses to stress. J Neurosci.
1993;13:3839–47.
28. Duvarci S, Pare D. Glucocorticoids enhance the excitability of principal basolateral amyg-
dala neurons. J Neurosci. 2007;27:4482–91.
29. Evanson NK, Tasker JG, Hill MN, Hillard CJ, Herman JP. Fast feedback inhibition of the
HPA axis by glucocorticoids is mediated by endocannabinoid signaling. Endocrinology.
2010;151:4811–9.
30. Fanselow MS, Dong HW. Are the dorsal and ventral hippocampus functionally distinct struc-
tures? Neuron. 2010;65:7–19.
31. Figueiredo HF, Bruestle A, Bodie B, Dolgas CM, Herman JP. The medial prefrontal cortex
differentially regulates stress-induced c-fos expression in the forebrain depending on type of
stressor. Eur J Neurosci. 2003;18:2357–64.
32. Fisher PM, Meltzer CC, Price JC, Coleman RL, Ziolko SK, Becker C, Moses-Kolko EL,
Berga SL, Hariri AR. Medial prefrontal cortex 5-HT2A density is correlated with amygdala
reactivity, response habituation, and functional coupling. Cereb Cortex. 2009;19:2499–507.
33. Friedman AR, Kaufer D. Emerging roles for hippocampal adult neural stem cells in memory.
Princeton: Biota Publishing; 2013.
34. Fuxe K, Härfstrand A, Agnati LF, Yu ZY, Cintra A, Wikström AC, Okret S, Cantoni E,
Gustafsson JÅ. Immunocytochemical studies on the localization of glucocorticoid receptor
immunoreactive nerve cells in the lower brain stem and spinal cord of the male rat using a
monoclonal antibody against rat liver glucocorticoid receptor. Neurosci Lett. 1985;60:1–6.
35. Gage FH. Mammalian neural stem cells. Science. 2000;287:1433–8.
36. Garcia A, Steiner B, Kronenberg G, Bick-Sander A, Kempermann G. Age-dependent expres-
sion of glucocorticoid- and mineralocorticoid receptors on neural precursor cell populations
in the adult murine hippocampus. Aging Cell. 2004;3:363–71.
37. Ge S, Yang C-H, Hsu K-S, Ming G-L, Song H. A critical period for enhanced synaptic plas-
ticity in newly generated neurons of the adult brain. Neuron. 2007;54:559–66.
38. Giguère V, Hollenberg SM, Rosenfeld MG, Evans RM. Functional domains of the human
glucocorticoid receptor. Cell. 1986;46:645–52.
39. Goldwater DS, Pavlides C, Hunter RG, Bloss EB, Hof PR, McEwen BS, Morrison
JH. Structural and functional alterations to rat medial prefrontal cortex following chronic
restraint stress and recovery. Neuroscience. 2009;164:798–808.
40. Graybeal C, Feyder M, Schulman E, Saksida LM, Bussey TJ, Brigman JL, Holmes
A. Paradoxical reversal learning enhancement by stress or prefrontal cortical damage: rescue
with BDNF. Nat Neurosci. 2011;14:1507–9.
41. Graybeal C, Kiselycznyk C, Holmes A. Stress-induced impairments in prefrontal-mediated
behaviors and the role of the N-methyl-D-aspartate receptor. Neuroscience. 2012;211:
28–38.
42. Gu Y, Arruda-Carvalho M, Wang J, Janoschka SR, Josselyn SA, Frankland PW, Ge S. Optical
controlling reveals time-dependent roles for adult-born dentate granule cells. Nat Neurosci.
2012;15:1700–6.
43. Hanson ND, Owens MJ, Boss-Williams KA, Weiss JM, Nemeroff CB. Several stressors fail
to reduce adult hippocampal neurogenesis. Psychoneuroendocrinology. 2011;36:1520–9.
44. Herman JP. In situ hybridization analysis of vasopressin gene transcription in the paraven-
tricular and supraoptic nuclei of the rat: regulation by stress and glucocorticoids. J Comp
Neurol. 1995;363:15–27.
45. Herman JP, Cullinan WE. Neurocircuitry of stress: central control of the hypothalamo-
pituitary-adrenocortical axis. Trends Neurosci. 1997;20:78–84.
46. Herman JP, Cullinan WE, Young EA, Akil H, Watson SJ. Selective forebrain fiber tract
lesions implicate ventral hippocampal structures in tonic regulation of paraventricular
nucleus corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA
expression. Brain Res. 1992;592:228–38.
47. Herman JP, Spencer R. Regulation of hippocampal glucocorticoid receptor gene transcription
and protein expression in vivo. J Neurosci. 1998;18:7462–73.
48. Hill MN, McEwen BS. Involvement of the endocannabinoid system in the neurobehavioural
effects of stress and glucocorticoids. Prog Neuropsychopharmacol Biol Psychiatry. 2010;
34:791–7.
49. Hill MN, McLaughlin RJ, Pan B, Fitzgerald ML, Roberts CJ, Lee TT, Karatsoreos IN,
Mackie K, Viau V, Pickel VM, McEwen BS, Liu QS, Gorzalka BB, Hillard CJ. Recruitment
of prefrontal cortical endocannabinoid signaling by glucocorticoids contributes to termina-
tion of the stress response. J Neurosci. 2011;31:10506–15.
50. Hinwood M, Tynan RJ, Day TA, Walker FR. Repeated social defeat selectively increases
deltaFosB expression and histone H3 acetylation in the infralimbic medial prefrontal cortex.
Cereb Cortex. 2011;21:262–71.
51. Holmes A, Wellman CL. Stress-induced prefrontal reorganization and executive dysfunction
in rodents. Neurosci Biobehav Rev. 2009;33:773–83.
52. Jacobson L, Sapolsky R. The role of the hippocampus in feedback regulation of the
hypothalamic-pituitary-adrenocortical axis. Endocr Rev. 1991;12:118–34.
53. Joels M, Baram TZ. The neuro-symphony of stress. Nat Rev Neurosci. 2009;10:459–66.
54. Karst H, Joëls M. Corticosterone slowly enhances miniature excitatory postsynaptic current
amplitude in mice CA1 hippocampal cells. J Neurophysiol. 2005;94:3479–86.
55. Karst H, Karten Y, Reichardt H, De Kloet E, Schütz G, Joels M. Corticosteroid actions in
hippocampus require DNA binding of glucocorticoid receptor homodimers. Nat Neurosci.
2000;3:977–8.
56. Keller-Wood ME, Dallman MF. Corticosteroid inhibition of ACTH secretion. Endocr Rev.
1984;5:1–24.
57. Kirby ED, Kaufer D. Stress and adult neurogenesis in the mammalian central nervous sys-
tem. In: Soreq H, Friedman A, Kaufer D, editors. Stress—from molecules to behavior: a
comprehensive analysis of the neurobiology of stress responses. Darmstadt: Wiley-VCH;
2009. p. 71–91.
58. Kirby ED, Muroy SE, Sun WG, Covarrubias D, Leong MJ, Barchas LA, Kaufer D. Acute
stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via
secreted astrocytic FGF2. eLife. 2013;2:e00362.
59. Krugers HJ, Alfarez DN, Karst H, Parashkouhi K, van Gemert N, Joëls M. Corticosterone
shifts different forms of synaptic potentiation in opposite directions. Hippocampus. 2005;
15:697–703.
60. Lapiz-Bluhm MD, Soto-Pina AE, Hensler JG, Morilak DA. Chronic intermittent cold stress
and serotonin depletion induce deficits of reversal learning in an attentional set-shifting test
in rats. Psychopharmacology (Berl). 2009;202:329–41.
61. Lemos JC, Wanat MJ, Smith JS, Reyes BAS, Hollon NG, Van Bockstaele EJ, Chavkin C,
Phillips PEM. Severe stress switches CRF action in the nucleus accumbens from appetitive
to aversive. Nature. 2012;490:402–6.
62. Liston C, Gan W-B. Glucocorticoids are critical regulators of dendritic spine development
and plasticity in vivo. Proc Natl Acad Sci. 2011;108:16074–9.
63. Liston C, Miller MM, Goldwater DS, Radley JJ, Rocher AB, Hof PR, Morrison JH, McEwen
BS. Stress-induced alterations in prefrontal cortical dendritic morphology predict selective
impairments in perceptual attentional set-shifting. J Neurosci. 2006;26:7870–4.
64. Maggio N, Segal M. Differential modulation of long-term depression by acute stress in the
rat dorsal and ventral hippocampus. J Neurosci. 2009;29:8633–8.
65. Maggio N, Segal M. Corticosteroid regulation of synaptic plasticity in the hippocampus.
Scientific World J. 2010;10:462–9.
66. Maggio N, Segal M. Steroid modulation of hippocampal plasticity: switching between cogni-
tive and emotional memories. Front Cell Neurosci. 2012;6:12.
67. Marín-Burgin A, Mongiat LA, Pardi MB, Schinder AF. Unique processing during a period of
high excitation/inhibition balance in adult-born neurons. Science. 2012;335:1238–42.
68. McDonald AJ. Cytoarchitecture of the central amygdaloid nucleus of the rat. J Comp Neurol.
1982;208:401–18.
69. McEwen BS. Stress, adaptation, and disease: allostasis and allostatic load. Ann N Y Acad Sci.
1998;840:33–44.
70. McKlveen JM, Myers B, Flak JN, Bundzikova J, Solomon MB, Seroogy KB, Herman
JP. Role of prefrontal cortex glucocorticoid receptors in stress and emotion. Biol Psychiatry.
2013;74:672–9.
71. Mirescu C, Gould E. Stress and adult neurogenesis. Hippocampus. 2006;16:233–8.
72. Mitra R, Jadhav S, McEwen BS, Vyas A, Chattarji S. Stress duration modulates the spatio-
temporal patterns of spine formation in the basolateral amygdala. Proc Natl Acad Sci U S A.
2005;102:9371–6.
73. Mitra R, Sapolsky RM. Acute corticosterone treatment is sufficient to induce anxiety and
amygdaloid dendritic hypertrophy. Proc Natl Acad Sci U S A. 2008;105:5573–8.
74. Mizoguchi K, Ishige A, Aburada M, Tabira T. Chronic stress attenuates glucocorticoid nega-
tive feedback: involvement of the prefrontal cortex and hippocampus. Neuroscience.
2003;119:887–97.
75. Mizoguchi K, Ishige A, Takeda S, Aburada M, Tabira T. Endogenous glucocorticoids are

essential for maintaining prefrontal cortical cognitive function. J Neurosci. 2004;24:5492–9.
76. Mizoguchi K, Yuzurihara M, Ishige A, Sasaki H, Chui D-H, Tabira T. Chronic stress induces
impairment of spatial working memory because of prefrontal dopaminergic dysfunction. J
Neurosci. 2000;20:1568–74.
77. Neisser U, Winograd E, Bergman ET, Schreiber CA, Palmer SE, Weldon MS. Remembering
the earthquake: direct experience vs. hearing the news. Memory. 1996;4:337–57.
78. Patel PD, Lopez JF, Lyons DM, Burke S, Wallace M, Schatzberg AF. Glucocorticoid and
mineralocorticoid receptor mRNA expression in squirrel monkey brain. J Psychiatr Res.
2000;34:383–92.
79. Pentkowski NS, Blanchard DC, Lever C, Litvin Y, Blanchard RJ. Effects of lesions to the
dorsal and ventral hippocampus on defensive behaviors in rats. Eur J Neurosci. 2006;23:
2185–96.
80. Pitts MW, Takahashi LK. The central amygdala nucleus via corticotropin-releasing factor is
necessary for time-limited consolidation processing but not storage of contextual fear mem-
ory. Neurobiol Learn Mem. 2011;95:86–91.
81. Popescu AT, Saghyan AA, Pare D. NMDA-dependent facilitation of corticostriatal plasticity
by the amygdala. Proc Natl Acad Sci U S A. 2007;104:341–6.
82. Popoli M, Yan Z, McEwen BS, Sanacora G. The stressed synapse: the impact of stress and
glucocorticoids on glutamate transmission. Nat Rev Neurosci. 2011;13:22–37.
83. Quirarte GL, Roozendaal B, McGaugh JL. Glucocorticoid enhancement of memory storage
involves noradrenergic activation in the basolateral amygdala. Proc Natl Acad Sci U S A.
1997;94:14048–53.
84. Radley JJ, Arias CM, Sawchenko PE. Regional differentiation of the medial prefrontal cortex
in regulating adaptive responses to acute emotional stress. J Neurosci. 2006;26:12967–76.
85. Reul JM, de Kloet ER. Two receptor systems for corticosterone in rat brain: microdistribution
and differential occupation. Endocrinology. 1985;117:2505–11.
86. Ridder S, Chourbaji S, Hellweg R, Urani A, Zacher C, Schmid W, Zink M, Hortnagl H, Flor
H, Henn FA, Schutz G, Gass P. Mice with genetically altered glucocorticoid receptor expres-
sion show altered sensitivity for stress-induced depressive reactions. J Neurosci. 2005;25:
6243–50.
87. Robertson DA, Beattie J, Reid I, Balfour D. Regulation of corticosteroid receptors in the rat
brain: the role of serotonin and stress. Eur J Neurosci. 2005;21:1511–20.
88. Roozendaal B. Glucocorticoids and the regulation of memory consolidation. Psychoneuro-
endocrinology. 2000;25:213–38.
89. Roozendaal B, McEwen BS, Chattarji S. Stress, memory and the amygdala. Nat Rev
Neurosci. 2009;10:423–33.
90. Roozendaal B, McReynolds JR, McGaugh JL. The basolateral amygdala interacts with the
medial prefrontal cortex in regulating glucocorticoid effects on working memory impairment.
J Neurosci. 2004;24:1385–92.
91. Sanchez MM, Young LJ, Plotsky PM, Insel TR. Distribution of coricosteriod receptors in the
rhesus brain: relative absence of gluococorticoid receptors in the hippocampal formation. J
Neurosci. 2000;20:4657–68.
92. Sapolsky R. Stress and plasticity in the limbic system. Neurochem Res. 2003;28:1735–42.
93. Sapolsky RM, Krey LC, McEwen BS. Glucocorticoid-sensitive hippocampal neurons are
involved in terminating the adrenocortical stress response. Proc Natl Acad Sci. 1984;
81:6174–7.
94. Schwabe L, Wolf OT. Stress prompts habit behavior in humans. J Neurosci. 2009;29:
7191–8.
95. Schwabe L, Wolf OT. Stress-induced modulation of instrumental behavior: from goal-
directed to habitual control of action. Behav Brain Res. 2011;219:321–8.
96. Selye H. Stress in health and disease. Boston: Butterworths; 1976.
97. Shepard JD, Barron KW, Myers DA. Stereotaxic localization of corticosterone to the amyg-
dala enhances hypothalamo-pituitary–adrenal responses to behavioral stress. Brain Res.
2003;963:203–13.
98. Smith Y, Pare D. Intra-amygdaloid projections of the lateral nucleus in the cat: PHA-L
anterograde labeling combined with postembedding GABA and glutamate immunocyto-
chemistry. J Comp Neurol. 1994;342:232–48.
99. Snyder JS, Soumier A, Brewer M, Pickel J, Cameron HA. Adult hippocampal neurogenesis
buffers stress responses and depressive behaviour. Nature. 2011;476:438–62.
100. Solano-Castiella E, Anwander A, Lohmann G, Weiss M, Docherty C, Geyer S, Reimer E,
Friederici AD, Turner R. Diffusion tensor imaging segments the human amygdala in vivo.
Neuroimage. 2010;49:2958–65.
101. Stahn C, Buttgereit F. Genomic and nongenomic effects of glucocorticoids. Nature clinical
practice. Rheumatology. 2008;4:525–33.
102. Sullivan RM, Gratton A. Lateralized effects of medial prefrontal cortex lesions on neuroen-
docrine and autonomic stress responses in rats. J Neurosci. 1999;19:2834–40.
103. Sullivan RM, Gratton A. Behavioral effects of excitotoxic lesions of ventral medial prefrontal
cortex in the rat are hemisphere-dependent. Brain Res. 2002;927:69–79.
104. Surget A, Tanti A, Leonardo ED, Laugeray A, Rainer Q, Touma C, Palme R, Griebel G,
Ibarguen-Vargas Y, Hen R, Belzung C. Antidepressants recruit new neurons to improve stress
response regulation. Mol Psychiatry. 2011;16:1177–88.
105. Tanapat P, Hastings NB, Rydel TA, Galea LA, Gould E. Exposure to fox odor inhibits cell
proliferation in the hippocampus of adult rats via an adrenal hormone-dependent mechanism.
J Comp Neurol. 2001;437:496–504.
106. Tasker JG, Di S, Malcher-Lopes R. Minireview: rapid glucocorticoid signaling via membrane-
associated receptors. Endocrinology. 2006;147:5549–56.
107. Thomas RM, Hotsenpiller G, Peterson DA. Acute psychosocial stress reduces cell survival in
adult hippocampal neurogenesis without altering proliferation. J Neurosci. 2007;27:
2734–43.
108. Thomas RM, Urban JH, Peterson DA. Acute exposure to predator odor elicits a robust
increase in corticosterone and a decrease in activity without altering proliferation in the adult
rat hippocampus. Exp Neurol. 2006;201:308–15.
109. Ulrich-Lai YM, Herman JP. Neural regulation of endocrine and autonomic stress responses.
Nat Rev Neurosci. 2009;10:397–409.
110. Uth RM, McKelvy JF, Harrison RW, Bohn MC. Demonstration of glucocorticoid receptor-
like immunoreactivity in glucocorticoid-sensitive vasopressin and corticotropin-releasing
factor neurons in the hypothalamic paraventricular nucleus. J Neurosci Res. 1988;19:
405–11.
111. Uylings HBM, Groenewegen HJ, Kolb B. Do rats have a prefrontal cortex? Behav Brain Res.
2003;146:3–17.
112. Wallis JD. Cross-species studies of orbitofrontal cortex and value-based decision-making.
Nat Neurosci. 2012;15:13–9.
113. Wanat MJ, Bonci A, Phillips PEM. CRF acts in the midbrain to attenuate accumbens dopa-
mine release to rewards but not their predictors. Nat Neurosci. 2013;16:383–5.
114. Watanabe Y, Gould E, Daniels DC, Cameron H, McEwen BS. Tianeptine attenuates stress-
induced morphological changes in the hippocampus. Eur J Pharmacol. 1992;222:157–62.
115. Wellman CL. Dendritic reorganization in pyramidal neurons in medial prefrontal cortex after
chronic corticosterone administration. J Neurobiol. 2001;49:245–53.
116. Wellman CL, Izquierdo A, Garrett JE, Martin KP, Carroll J, Millstein R, Lesch K-P, Murphy
DL, Holmes A. Impaired stress-coping and fear extinction and abnormal corticolimbic mor-
phology in serotonin transporter knock-out mice. J Neurosci. 2007;27:684–91.
117. Wong EY, Herbert J. The corticoid environment: a determining factor for neural progenitors’
survival in the adult hippocampus. Eur J Neurosci. 2004;20:2491–8.
118. Wood GE, Norris EH, Waters E, Stoldt JT, McEwen BS. Chronic immobilization stress alters
aspects of emotionality and associative learning in the rat. Behav Neurosci. 2008;122:
282–92.
119. Woolley CS, Gould E, McEwen BS. Exposure to excess glucocorticoids alters dendritic mor-
phology of adult hippocampal pyramidal neurons. Brain Res. 1990;531:225–31.
120. Yoshiya M, Komatsuzaki Y, Hojo Y, Ikeda M, Mukai H, Hatanaka Y, Murakami G, Kawata
M, Kimoto T, Kawato S. Corticosterone rapidly increases thorns of CA3 neurons via synap-
tic/extranuclear glucocorticoid receptor in rat hippocampus. Front Neural Circuits.
2013;7:191.
121. Yuen EY, Liu W, Karatsoreos IN, Feng J, McEwen BS, Yan Z. Acute stress enhances gluta-
matergic transmission in prefrontal cortex and facilitates working memory. Proc Natl Acad
Sci U S A. 2009;106:14075–9.
122. Bebbington P, Der G, MacCarthy B, Wykes T, Brugha T, Sturt P, Potter J. Stress incubation
and the onset of affective disorders. Br J Psychiatry. 1993;162(3):358–62.
123. Brindley DN, Rolland Y. Possible connections between stress, diabetes, obesity, hypertension
and altered lipoprotein metabolism that may result in atherosclerosis. Hypertension.
1989;23:33–351.
124. Cohen S, Herbert TB. Health psychology: psychological factors and physical disease from
the perspective of human psychoneuroimmunology. Annu Rev Psychol. 1996;47(1):113–42.
125. Johnson EO, Kamilaris TC, Chrousos GP, Gold PW. Mechanisms of stress: a dynamic over-
view of hormonal and behavioral homeostasis. Neurosci Biobehav Rev. 1990;16:115–30.
126. Lupien SJ, McEwen BS, Gunnar MR, Heim C. Effects of stress throughout the lifespan on
the brain, behavior and cognition. Nat Rev Neurosci. 2009;10:434–45.
127. Sternberg EM, Chrousos GP, Wilder RL, Gold PW. The stress response and the regulation of
inflammatory disease. Ann Intern Med. 1992;117(10):854–66.
128. Dranovsky A, Hen R. Hippocampal neurogenesis: regulation by stress and antidepressants. Biol
Psychiatry. 2006;59(12):1136–43. doi:10.1016/j.biopsych.2006.03.082.
129. Wong EYH, Herbert J. The corticoid environment: a determining factor for neural progenitors’
survival in the adult hippocampus. Eur J Neurosci. 2006;20(10):2491–8.
doi:10.1111/j.1460-9568.2004.03717.x.
130. Gould TD, Dao DT, Kovacsics CE. Mood and anxiety related phenotypes in mice, vol. 42.
New York: Humana Press; 2009. p. 1–20.
Chapter 11
Glucocorticoid Regulation of Reproduction
Anna C. Geraghty and Daniela Kaufer
Abstract It is well accepted that stress, measured by increased glucocorticoid

secretion, leads to profound reproductive dysfunction. In times of stress, glucocorti-
coids activate many parts of the fight or flight response, mobilizing energy and
enhancing survival, while inhibiting metabolic processes that are not necessary for
survival in the moment. This includes reproduction, an energetically costly procedure
that is very finely regulated. In the short term, this is meant to be beneficial, so that
the organism does not waste precious energy needed for survival. However, long-
term inhibition can lead to persistent reproductive dysfunction, even if no longer
stressed. This response is mediated by the increased levels of circulating glucocor-
ticoids, which orchestrate complex inhibition of the entire reproductive axis. Stress
and glucocorticoids exhibits both central and peripheral inhibition of the reproduc-
tive hormonal axis. While this has long been recognized as an issue, understanding
the complex signaling mechanism behind this inhibition remains somewhat of a
mystery. What makes this especially difficult is attempting to differentiate the many
parts of both of these hormonal axes, and new neuropeptide discoveries in the last
decade in the reproductive field have added even more complexity to an already
complicated system. Glucocorticoids (GCs) and other hormones within the hypo-
thalamic-pituitary-adrenal (HPA) axis (as well as contributors in the sympathetic
system) can modulate the hypothalamic-pituitary-gonadal (HPG) axis at all lev-
els—GCs can inhibit release of GnRH from the hypothalamus, inhibit gonadotropin
synthesis and release in the pituitary, and inhibit testosterone synthesis and release
from the gonads, while also influencing gametogenesis and sexual behavior. This
chapter is not an exhaustive review of all the known literature, however is aimed at
giving a brief look at both the central and peripheral effects of glucocorticoids on
the reproductive function.
Keywords Reproduction • HPG axis • HPA axis • Glucocorticoids • Hypothalamus
A.C. Geraghty, B.A. • D. Kaufer, Ph.D. (*)

Department of Integrative Biology, University of California, Berkeley, CA, USA

254 A.C. Geraghty and D. Kaufer
Introduction
It is well accepted that stress, measured by increased glucocorticoid secretion, leads to

profound reproductive dysfunction. In times of stress, glucocorticoids activate many
parts of the fight or flight response, mobilizing energy and enhancing survival, while
inhibiting metabolic processes that are not necessary for survival in the moment. This
includes reproduction, an energetically costly procedure that is very finely regulated.
In the short term, this is meant to be beneficial, so that the organism does not waste
precious energy needed for survival. However, long-term inhibition can lead to persis-
tent reproductive dysfunction, even after the stressor has been removed. This response
is mediated by the increased levels of circulating glucocorticoids, which orchestrate
complex inhibition of the entire reproductive axis. The reproductive and stress sys-
tems share a common architecture with central (hypothalamic stimulation of pituitary
in both) and peripheral (gonads and adrenal respectively) components. Stress and glu-
cocorticoids exhibits both central and peripheral inhibition of the reproductive hor-
monal axis. While this has long been recognized as an issue, understanding the
complex signaling mechanism behind this inhibition remains somewhat of a mystery.
In fact, the presenting complaint of Harvey Cushing’s first patient with the syndrome
that bears his name was secondary amenorrhea. She had normal menarche at age 14,
but menses ceased two years later. What makes this especially difficult is attempting
to differentiate the many parts of both of these hormonal axes, and new neuropeptide
discoveries in the last decade in the reproductive field have added even more complex-
ity to an already complicated system. Glucocorticoids (GCs) and other hormones
within the hypothalamic-pituitary-adrenal (HPA) axis (as well as contributors in the
sympathetic system) can modulate the hypothalamic-pituitary-gonadal (HPG) axis at
all levels—GCs can inhibit release of GnRH from the hypothalamus, inhibit gonado-
tropin synthesis and release in the pituitary, and inhibit testosterone synthesis and
release from the gonads, while also influencing gametogenesis and sexual behavior.
This chapter is not an exhaustive review of all the known literature however is aimed
at giving a brief look at both the central and peripheral effects of glucocorticoids on the
reproductive function.
Reproductive Physiology: A Primer
Reproduction in mammals is a complex and precisely regulated hormonal process

that requires the coordination of both the central nervous system and the peripheral
reproductive organs for successful procreation. Negative and positive feedback signals
tightly regulate the reproductive hormonal axis, also known as the hypothalamo-
pituitary-gonadal (HPG) axis to maintain homeostasis. Perturbations in the axis,
such as those caused by stress, can therefore have profound effects on reproductive
ability, as even small changes can have large effects downstream, or even stop the
axis in its tracks. Much of this chapter is focused on research conducted in rodents,
however there are many species and sex differences in reproductive research indi-
cating that stress can exert a myriad of effects to inhibit reproduction.
11 Glucocorticoid Regulation of Reproduction 255
Studying the reproductive system of males offers a simpler “model” of HPG axis
to begin examining reproductive physiology, due to its relative consistency over the
lifetime post-puberty, as opposed to the cycling of female reproductive hormones
during the post-pubertal and pre-menopausal years. Gonadotropin-releasing hor-
mone (GnRH) is secreted from the hypothalamus in a pulsatile manner, crossing the
hypophyseal portal system into the anterior pituitary. In the pituitary, GnRH stimu-
lates the synthesis and release of luteinizing hormone (LH) and follicle-stimulating
hormone (FSH). LH and FSH circulate systemically to trigger testosterone release
from the testes and gametogenesis. In turn, testosterone (T), as well and LH and
FSH, negatively feedback on the axis to maintain homeostasis [1–6] (Fig. 11.1).
Females, however, present a much more complicated picture. Unlike males,
females experience hormonal surges to trigger ovulation, a phenomenon where
estrogen switches from exerting negative feedback on the axis to positive feedback
[7–12]. In rats, ovulation occurs once every 4 days. Similarly to males, GnRH is
released from the hypothalamus, and LH and FSH circulate to the ovaries to trigger
the release of estradiol (E2) and progesterone (P) as well as development of the
ovum [6, 12–14]. Estradiol from the ovaries tightly regulates the HPG axis, exerting
negative feedback onto the hypothalamus to inhibit GnRH release. However, when
the developing ovum is near completion, and ovulation is due to occur, there is a
Fig. 11.1 General schematic

of hypothalamic-pituitary-
gonadal axis in the rodent
model
switch in the hormonal system. Increasing estradiol secretion from the developing
follicle triggers a change from negative to positive feedback of estradiol on the
hypothalamus, resulting in an increase of GnRH secretion and leading to a surge of
LH secretion from the pituitary to trigger ovulation [15, 16]. However, until recently
it was unknown what triggered the switch between negative and positive feedback.
Research in the last decade has shed more light on that switch during ovulation
while also revealing that the axis is not nearly as simple as it appeared. There are
currently two different hypotheses on what triggers the switch between negative and
positive feedback in the HPG axis: The first one focuses on progesterone as the
culprit, whereas the second one points to allopregnanolone. Supporting the first
hypothesis, earlier research found that the estrogen surge initiates synthesis of pro-
gesterone receptors (PRs) in the hypothalamus, indicating progesterone is just as
critical for ovulation as the estrogen surge [17–19]. However, studies on ovariecto-
mized (OVX) and andrenalectomized (ADX) rats indicate that this progesterone
does not come from the ovaries or adrenals, areas typical for steroid synthesis and
systemic release. OVX rats given a normal dose of E2 can still exhibit LH surges
[20]. Additionally, this surge can be blocked by administering trilostane, which
inhibits the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), an enzyme criti-
cal for synthesis of progesterone [20]. This data suggests that not only is pre-LH
surge progesterone necessary for a successful LH surge, it is progesterone synthe-
sized outside of the ovary and adrenals that regulates the surge. Hypothalamic pro-
gesterone synthesis, also known as neuroprogesterone, is a likely location,
particularly hypothalamic astrocytes, which have been found to be the main source
of neuroprogesterone in the hypothalamus, as they have all the steroidogenic
enzyme machinery necessary for synthesis, regulate releasing factors and possess
both types of estradiol receptors—Erα and ERβ receptors, which enable them to
respond to the estradiol peak that precedes the LH surge [20–25].
While GnRH is the main coordinator of the HPG axis, secreted from the hypo-
thalamus through the hypophyseal portal system to the anterior pituitary to stimu-
late release of LH, there are many factors upstream of GnRH that can impact its
release, preventing successful ovulation. Two in particular, directly in the HPG
axis, have been recently discovered: kisspeptin (KISS1) and gonadotropin-inhibi-
tory hormone (GnIH) [26–34]. These two neuropeptides have opposing effects—
KISS1 stimulates GnRH release from the hypothalamus [35–40], while GnIH
inhibits it [30, 41–45]. A proposed mechanism of KISS1 action in the neuroproges-
terone model of the LH surge states that the peak of estradiol also triggers an
increase of progesterone receptors (PRs) on KISS1 neurons in the hypothalamus,
which respond to the neuroprogesterone secreted from the astrocytes (activated
also by estradiol to trigger the neuroprogesterone synthesis, likely through mem-
brane estrogen receptors, specifically mERα). Neuroprogesterone, binding to the
PRs on the KISS neurons, trigger the release of KISS, which then activates the
GnRH neurons, leading to increased secretion of GnRH to trigger the LH surge
from the pituitary [24, 46–48].
An alternative hypothesis for the switch between negative and positive feedback
of E2 in the hypothalamus argues that progesterone is not the driving factor of ovu-
lation, but it is a derivative of progesterone, allopregnanolone, that modulates GnRH
release from the hypothalamus via increasing glutamate release. This in turn could
act on NMDA receptors on the GnRH neurons, stimulating GnRH release [49–51].
While it is still not clear exactly what mechanism is truly responsible for positive
feedback of E2 during ovulation, the many regulators of GnRH and GnRH itself are
all impacted by glucocorticoids, and stress can lead to a disruption of homeostasis
that results in both short-term reproductive dysfunction and long-term infertility.
Stress, whether psychological or physical, via the activation of the HPA axis, and
subsequent increase in serum concentration of glucocorticoids, can inhibit the
reproductive axis at every level, from the hypothalamus down to the ovaries or testes.
Due to how closely regulated the HPG axis is, even small interferences in the
hormonal milieu responsible for successful breeding can cause major dysfunction
in the system (Fig. 11.2). The female ovulatory system discussed above, as well as
Fig. 11.2 Summary schematic of glucocorticoid and HPA axis interaction with the HPG axis and
reproductive function
GnRH afferents such as GnIH and KISS, exhibit many points at which stress can
disrupt the axis and cause fertility issues. While acute stress inhibiting the repro-
ductive axis is found to be adaptive, preventing animals from breeding when times
are not optimal for raising young, chronic stress and long-term shutdown of the
reproductive axis can lead to prolonged dysfunction and infertility. In addition to
causing infertility, this hypogonadism can contribute to other medical conditions
such as osteoporosis. We will examine how stress impacts each part of the reproduc-
tive axis individually, and how this can add up to detrimental fertility issues.
Glucocorticoid Effects on the Hypothalamus
Stress causes the activation of the hypothalamic-pituitary-adrenal axis by a cascade

of hormonal release: neural signals onto the hypothalamus cause the release of
corticotropin-releasing hormone (CRH, previously known as CRF) to the hypoph-
yseal portal system, via the median eminence. CRH in turn, activates cells in the
pituitary to release adrenocorticotrophic hormone (ACTH) to the general circula-
tion. ACTH stimulates cells in the adrenal cortex to synthesize and release the third
hormone in this cascade of the glucocorticoid family, which in rodents is corticos-
terone (CORT). Stress and high glucocorticoids have a profound negative impact on
the reproductive system, and within the hypothalamus both CRH and CORT affect
GnRH and its afferents to inhibit reproductive success, as detailed in the next two
sections. As discussed above, the timing of ovulation in female rodents is highly
regulated by multiple factors controlling GnRH levels. Stress has been shown to
impact each one of these factors, combining to effectively shut down the reproduc-
tive axis from the top. A difficulty in studying the effects of stress on reproduction
is to accurately differentiate between the effects of CRH and the effects of CORT,
both elevated during stress exposure, as well as determining specifically which
level of the HPG axis is being directly affected. While it is well accepted that there
are central effects of CRH directly on GnRH, completely separating those effects
from downstream peripheral effects of CORT on GnRH is complex. Many manipu-
lations of either CORT or CRH influence the other by feedback regulation, making
the dissection of mechanisms even more difficult. In this section, we will try to dif-
ferentiate the two to better understand how stress influences reproductive success.
Direct Regulation of GnRH by Glucocorticoids
All central influences on reproduction driven by stress converge on GnRH, and an

inhibition of the GnRH signal from the hypothalamus can occur both directly on the
GnRH neuron itself and indirectly via influence on GnRH afferents. While many
peptides, steroids and neurotransmitters regulate the GnRH surge, stress and gluco-
corticoids can directly inhibit GnRH release from the hypothalamus. To regulate
LH secretion from the pituitary, GnRH is released from the hypothalamus in pulses,
and modulation of the pulsatile release of GnRH affects downstream gonadotropin

release from the pituitary. Many types of stress have been found to affect the pulse
generator of GnRH, and both CRH and CORT have been implicated in this mecha-
nism of action [52–54]. GnRH crosses the hypophyseal portal system, a small cir-
culatory system between the hypothalamus and the pituitary, to stimulate release of
LH and FSH from the pituitary. Inhibiting the pulse generator of GnRH leads to a
decrease in GnRH secretion from the hypothalamus. However, measuring GnRH
release from the rodent hypothalamus is incredibly difficult, as the hypophyseal
portal system is small and hard to sample from. To determine whether GC’s are
inhibiting GnRH release from the hypothalamus, researchers use downstream
release of LH from the pituitary as a proxy for GnRH. If stress leads to a decrease
in LH release, it is assumed that this is due to a decrease in GnRH release from the
hypothalamus. Intracerebroventricular (ICV) administration of CRH has been
found to inhibit LH pulses in rats [55] and this response can be blocked or reversed
by administration of CRH antagonists [55, 56]. It appears that this suppression is
mediated in part by both CRH receptor subtypes, CRH-R1 and CRH-R2, however
which receptor is predominant depends on the type of stressor utilized [57–59].
CRH axon terminals directly interact with GnRH dendrites, and in vitro studies
have shown that CRH can inhibit GnRH release. Other studies have also shown that
infusing CRH into the medial preoptic area of females rats lead to a 60 % decrease
in GnRH release from the hypothalamus [60, 61]. Though many studies have used
in vitro systems to determine how GnRH can be inhibited by high CRH and CORT,
recent studies using sheep, however, which have a much larger hypophyseal portal
system than rodents, have allowed researchers to show in vivo that glucocorticoids
inhibit GnRH release in the hypothalamus. Prolonged corticosterone administration
IV caused a drop in GnRH release in the portal system of ewes [62] supporting the
long-standing theory that glucocorticoids’ central actions function to inhibit GnRH
release from the hypothalamus. Release has also been inhibited in hypothalamic
explants incubated with CORT and the glucocorticoid receptor agonist dexa-
methasone (DEX) in a dose-dependent manner [63].
Populations of GnRH neurons have been found to express both CRH receptors
(CRH-R1, R2) [64] and glucocorticoid receptors (GRs) directly in both mice and
rats [65, 66], indicating a method for which glucocorticoids (GCs) can act to directly
inhibit GnRH synthesis and release. Glucocorticoids have also been found to inhibit
GnRH transcription [67–69] and identified glucocorticoid response elements near
the GnRH gene that regulate GnRH transcription. The GT1 cell line derived from
GnRH-neurons, which synthesize and release GnRH [70, 71] express glucocorti-
coid receptors [72]. Further studies show that GCs can represses GnRH gene
expression and release from these cells [73]. Studies in vivo have shown that chronic
CORT treatment can inhibit GnRH expression, leading to a decrease in serum LH
levels [74]. This however, had no effect on FSH levels, nor did it affect gonadotro-
pin mRNA.
The effect of peripheral circulating glucocorticoids on GnRH and consequently
the LH surge is highly variable and likely dependent on the severity and length of
the stressor. In different acute stress studies glucocorticoids are shown to exert a full
spectrum of effects on the LH surge that range from complete inhibition, to little or
no effect to a positive activator of LH release. It emphasizes the importance of
comparing stress paradigms and understanding how different stressors trigger the
HPA axis. Acute stress tends to provide variable results on many different
outcomes—life history, age and gender can influence this response greatly. In con-
trast, chronic stress has been consistently shown to inhibit the LH surge and ovula-
tion in the literature, indicating that while short-term stressors may exert variable
effects on reproduction, likely due to the type of stressor, long-term stress reliably
causes reproductive dysfunction.
Indirect Regulation of GnRH by Glucocorticoids
Glucocorticoids may also exert an inhibitory influence on reproduction and GnRH

output by influencing hormones upstream of GnRH, adding another level of control
over reproduction. Two hormones in particular have been recently identified
upstream of GnRH that respond directly to stress, affecting GnRH synthesis and
secretion from the hypothalamus. Kisspeptin (KISS1) and gonadotropin-inhibitory
hormone (GnIH), mentioned earlier, have opposing effects on GnRH. KISS1 is an
activator of GnRH and plays a critical role in the maintenance of the GnRH pulse
generator in the hypothalamus as well as the estrogen surge responsible for ovu-
lation. Many different types of stressors, including exposure to the endotoxin lipo-
polysaccharide, acute hypoglycemia, immobilization (restraint) stress and social
isolation, lead to downregulation of KISS1 and it’s receptor KISS1R in the popula-
tion of kisspeptin neurons in the median pre-optic area (mPOA) of the hypothala-
mus, as well as decreases in KISS1 expression in the arcuate nucleus, leading to
downstream decreases in LH secretion [75–77]. Kisspeptin neurons have been
shown to express both CRH-R and GR, implying that they could potentially respond
directly to increase in either hormones following a stress stimulus [78]. These
changes can transmit downstream into inhibition of the GnRH pulse generator,
which has been shown to rely on kisspeptin input, and ultimately translate to down-
stream inhibition of LH secretion from the pituitary.
A subset of neurons in the hypothalamus expressing kisspeptin project directly to
GnRH neurons [79–82], and has been implicated in modulation of the GnRH pulse
generator. These neurons, known as the KNDy neurons, express two other neuro-
peptides, neurokinin B and dynorphin, and strongly respond to CORT. This sub-
group offers yet another pathway in which stress via glucocorticoids can
indirectly inhibit the reproductive axis. Dynorphin, an endogenous opioid in the
brain signals through the kappa-subtype of the opioid receptor (KOR) in the
hypothalamus, and administering dynorphin to female rats has been shown to
inhibit LH pulses from the pituitary [83]. Stress has been shown to increase dynor-
phin release [84] which could lead to downstream inhibition of LH [85].
Gonadotropin-inhibitory hormone (GnIH) is another hormone upstream of

GnRH that is regulated by stress to inhibit reproduction. GnIH was originally
discovered in birds [30] and a mammalian orthologue, Rfamide-related peptide-3
(RFRP3) has since been identified in many species including rats, mice, hamsters
and humans [26, 27, 86]. RFRP3 inhibits the GnRH pulse generator, decreasing
GnRH release from the hypothalamus and leading to decreased gonadotropin
secretion from the pituitary [41, 45, 87]. Both acute and chronic immobilization
stress in male rats were found to increase RFRP3 mRNA and peptide levels in the
hypothalamus and adrenalectomy blocked this effect, revealing that this is due to
circulating glucocorticoid levels [88]. This increase in RFRP3 by glucocorticoids
led to downstream inhibition of LH release from the pituitary. In vitro studies utiliz-
ing a cell line derived from RFRP3-ergic neurons has shown that RFRP3 neurons
express the glucocorticoid receptor (GR) and also possess two glucocorticoid
response elements (GREs) at the RFRP promoter region, further evidence pointing
to a direct regulation of RFRP3 neurons by glucocorticoids [89, 90]. RFRP3 neu-
rons in mammals can directly inhibit GnRH pulses from the hypothalamus, a
response enhanced by high glucocorticoids after stress. In female rats, research has
shown that high glucocorticoids after chronic stress can lead to an increase in
RFRP3 levels in all stages of the estrous cycle and that this increase is sustained for
at least one more estrous cycle after the stress has ceased. This increase leads to
downstream reproductive dysfunction with fewer copulatory events in females
exposed to chronic stress, lower pregnancy rates and decreased litter sizes [91]. This
new research into the effect of glucocorticoids on RFRP3 levels and long-term
reproductive dysfunction in female rodents could shed light on a more detailed
mechanism of chronic inhibition of the HPG axis and reproductive success by
glucocorticoids.
These are only a small sample of the possible indirect mechanisms of glucocor-
ticoids on GnRH secretion. Much is still not well understood about the mechanism
of action of glucocorticoids on GnRH levels, especially since there have been a slew
of novel discoveries of peptides upstream of GnRH that increases the complexity of
the regulation of reproductive neural systems by glucocorticoids. The difficulty in
measuring GnRH levels from rodents has also made it difficult to come to generate
conclusions about the mechanism of action of GCs on GnRH.
Glucocorticoid Effects in the Pituitary
While many of the stress effects on GnRH can lead to downstream pituitary dys-
function, glucocorticoids also directly influence reproduction at the level of the
pituitary. This inhibition can happen via many different mechanisms, including
modulating the sensitivity of the pituitary to changes in GnRH secretion, a decrease
in synthesis of the gonadotropes LH and FSH, as well as decreasing the secretion of
LH and FSH from the pituitary. However, the effects of glucocorticoids directly on
the pituitary in secretion and synthesis are highly variable, indicating that the type
of stressor and duration is very important when discussing this. This variability may
stem from only looking at a part of the inhibition, such as focusing on just LH
secretion as the output, rather than examining the mechanisms of synthesis and
responsiveness of the pituitary itself.
Synthesis of LH and FSH is a highly regulated process involving not just GnRH
levels, but steroid gonadal hormones as well [92, 93]. LH and FSH are glycoprotein
heterodimers that consist of a common glycoprotein alpha-subunit (aGSU) and a
specific β-subunit for either FSH or LH (LHβ and FSHβ) [94]. As rat and mice
gonadotrope cells express GRs [95], it is likely that GR transcriptional regulation
influences the synthesis of these subunits, both the specific β-subunits as well as the
common alpha subunit to also regulate expression levels. Breen et.al. found that
both daily immobilization stress and CORT administration led to reduced LH-β
mRNA levels, as well as decreased LH release from the pituitary in vivo. They also
showed in vitro, using a gonadotope cell line, Lβ T2 that CORT decreases GnRH-
induced increase in LHβ mRNA levels as well as identified the LHβ promoter
regions that are CORT and DEX responsive [96].
Similar to the effects seen in the hypothalamus, acute stress leads to variable
results. Several studies utilizing multiple types of acute stressors on gonadotropin
synthesis and release found that acute stress stimulated the HPG axis, leading to
increased levels of LH, prolactin (PRL) and FSH in the plasma [97, 98]. Others have
found though that acute administration of glucocorticoids can reduce the LH peak
in females [99, 100] and injection of CRH peripherally has been shown to inhibit
ovulation and block the LH surge completely [54]. Baldwin and Sawyer found that
an acute injection of DEX early in the estrous cycle of rats can delay the onset of
ovulation. Administration of LH though, in addition to DEX, can recover the ovula-
tory event, indicating that this delay of the estrous cycle is due to DEX inhibiting the
LH surge necessary for ovulation, rather than a problem within the ovary [101].
There is some debate over timing of sample collection—some studies see an initial
stimulation of LH but an inhibitory response later [102]. Collu et.al. found that after
the first 15 min of an acute stressor, female rats exhibited an increase in LH plasma
levels, however after 6 h of immobilization stress LH plasma levels in the females
had dropped below control levels, supporting the theory that the acute stimulation
of the HPG axis is only transitory [102]. Differences in the stressors, duration,
sample collection and timing of the experiment may explain the differences found
in LH release from the pituitary after acute stress and so it is hard to make concrete
conclusions about acute stress and pituitary function in reproduction.
Chronic immobilization stress, on the other hand, has been shown to reliably
reduce LH levels in both males and females [102–110]. One such study found that
chronic immobilization stress reduced plasma levels of LH, and prolactin (PRL)
with no change in FSH. However, pituitary levels of LH and FSH protein increased,
showing that synthesis and secretion are not always matched up-it appears in this
study, chronic stress did not influence synthesis, but inhibited release in some way
[109]. This difference is critical when comparing studies—most studies do not look
at both synthesis and secretion simultaneously, which may explain differences in
findings.
Glucocorticoids can also change the responsiveness of the pituitary to GnRH

secretion. This may occur via regulation of the GnRH receptor in the pituitary, how-
ever other studies have shown that this can also occur independently of changes in
the GnRH receptor [111]. Baldwin (1979) found that stress affects estrogen feed-
back onto the pituitary, making it less responsive to GnRH secretion [112]. GnRH
binding to its receptor on the gonadotropes is necessary to induce synthesis of
aGSU, LHβ and FSHβ subunits, as well as stimulate the dimerization of the sub-
units for successful synthesis and release from the gonadotrope cells. GnRH recep-
tors appear to be transcriptionally regulated by GR [113, 114].
This section focused predominately on the influence CORT and GR has on LH
synthesis and secretion. CORT has been shown to also decrease FSH mRNA levels
as well in some studies [110, 115], however, there is extreme variability in the FSH
response in regards to stress. FSH secretion levels are rarely affected by stress, espe-
cially acute stress, showing little to no change in most studies looking at it [110,
116]. This likely is due to other regulatory signals on FSH beyond corticosterone.
Many studies, in fact, show increases in FSHβ (beta) mRNA post-stress, both after
immobilization stress and corticosterone or cortisol administration in many species
in both acute and chronic studies, sometimes accompanied by decreases in LHβ or
with no change in LH at all [96, 111, 117–119]. This also happens with little to no
change in actual FSH secretion, so it is hard to draw conclusions on how stress regu-
lates FSH levels.
In summary, based on current literature it seems impossible to draw clear answers
about glucocorticoid inhibition of reproduction at the level of the pituitary. The
range of studies utilizing different species, different sexes, a variety of stressors as
well as different lengths of time of the stressors themselves is likely part of the
cause of the confusion. Some studies show that acute stress increases the pituitary
gonadotropins while some find decreases in gonadotropins. Some studies show
changes in the synthesis of the gonadotropins, but no change in release and others
find that release is altered with no change in synthesis. Much research is still to be
done in this area to fully elucidate how glucocorticoids affect the pituitary directly
in terms of reproduction.
Glucocorticoids and the Gonads
The final component of the HPG axis, the gonads, is yet another level in which GCs
regulate the HPG axis. In the gonads, GCs can act to inhibit many critical steps to
complete the reproductive process. Corticosterone can inhibit steroidogenesis,
inhibiting the synthesis of testosterone (T), estrogen (E2) and progesterone (P), as
well as directly inhibiting the release of these steroids from the gonads. GCs modu-
late the expression of the LH-receptor (LHR) on the gonads, changing how the
gonads respond to LH and leading to downstream effects on steroids. GCs can also
regulate gametogenesis, the development of mature sperm and ovum, to inhibit
reproduction at the levels of the gamete. These effects can all be completed in the
absence of influences from the hypothalamus and pituitary, emphasizing how pro-
foundly stress can influence reproduction.
Glucocorticoid Effects in the Testes
Research has shown that GR is localized in several different cell populations within
the testes, including importantly the Leydig cells, which is where steroidogenesis
occurs within the testes, as well as in the Sertoli cells, primary spermatocytes and
the epididymis [121–123]. This indicates that GRs can regulate not only steroido-
genesis and the release of T, but spermatogenesis as well, either through affecting
the primary population of cells or affecting the last steps of maturation in the epi-
didymis. Both acute and chronic stress experiments have shown that high GCs
inhibits testosterone secretion, spermatogenesis and libido [124–128] as expected.
This effect is due specifically to circulating GC levels in the blood and action via
GR because ACTH treatment in adrenalectomized animals fails to replicate these
findings [129]. Some studies show that this decreased testosterone release can occur
either via downregulation of the LH receptor in Leydig cells [130] or through inhi-
bition of the enzymes necessary for testosterone biosynthesis [125, 126, 128, 129,
131–133]. Overall, these changes result in decreased testosterone synthesis and
release from the gonads.
Glucocorticoids may also impact spermatogenesis, as GRs are present on the
primary spermatocytes as well as within the epididymis. High glucocorticoids have
been found to induce testicular germ cell apoptosis [134, 135] as well as Leydig
cell apoptosis [136], which has a profound inhibitory influence on male reproduc-
tive abilities. Chronic stress has been shown to also decrease the number of sper-
matids within the testis [137], and in humans it has been shown that chronic stress
leads to decreased sperm numbers, likely through a combination of the above
responses [138]. Expression of GR in all these spermatogenic area indicates that
glucocorticoids can act directly on the testes to regulate sperm production. Stress
and high levels of GCs likely inhibit reproduction both indirectly and directly at the
level of the gonads, with decreased secretion of LH from the pituitary decreasing
testosterone release, and direct inhibition of testosterone synthesis and sperm
production by GCs.
Glucocorticoid Effects in the Ovaries
The role of glucocorticoids within the ovary is somewhat more complicated than it
is within the testes. Rather than a straight inhibitory role of GCs on ovarian func-
tion, some GC effects are actually beneficial to the ovaries and are necessary for
maintenance of the follicular development pathway. During each cycle, many
follicles are activated for development within the ovaries, however not all fully
develop to maturity and it appears GCs are an active part in that selection process.
This is necessary for normal ovarian function, but likely is very finely controlled,
and high stress may tip the balance between a “good” level of GCs and a “maladap-
tive” level that leads to ovarian dysfunction.
A way in which the ovaries control levels of GCs through follicular development
during the female estrous or menstrual cycle is via expression of the enzyme
11β-hydroxysteroid dehydrogenase (11β-HSD). 11β-HSD is a family of enzymes
responsible for catalyzing the conversion of inactive cortisone to cortisol or vice
versa to regulate glucocorticoid exposure. 11β-HSD1 activates cortisol predomi-
nately, however the reaction is bidirectional, and 11β-HSD1 can also inactivate cor-
tisol. 11β-HSD2 on the other hand unidirectionally inactivates cortisol, converting
it back to cortisone [139, 140]. Researchers have identified that many of the cells
within the ovaries, including the follicles and corpus luteum, express 11β-HSD1,
11β-HSD2 and GR [141–145], indicating that there are possibly many regulatory
effects of glucocorticoids on follicular development and ovarian function.
Interestingly, the ovaries differentially regulate 11β-HSD1 and HSD2 throughout
the cycle. 11β-HSD2, which inactivates GCs, is highly expressed in developing fol-
licles in the ovary, while 11β-HDS1, which activates GCs, is highly expressed in
follicles that have been luteinized, meaning they have been activated by an LH
surge and ready for an ovulatory event [141, 146, 147]. This indicates that the ova-
ries upregulate 11β-HSD2 while developing in order to inactivate GCs present in
the ovary while the follicles are maturing in order to enhance development, but
choose to activate circulating GCs once the follicle is released for ovulation. These
activated and functional GCs may act as an anti-inflammatory response triggered by
the rupturing of the ovarian surface epithelium during ovulation [148, 149]. These
two examples show how GCs are likely necessary for normal function of the ova-
ries, however their levels are tightly regulated via variability in expression of the
11β-HSD1 and 2. These enzymes are actually manipulated via gonadotropin signals
from the pituitary, with LH controlling expression of 11β-HSD1 expression (thus
activating GCs during ovulation). This regulation via gonadotropins provides a
mechanism through which excess GCs could influence enzymatic regulation of
GCs. As these two enzymes are so narrowly regulated during the ovarian cycle,
stress and high GC secretion from the adrenals can easily dysregulate these signals
and cause profound fertility problems in both ovarian function during ovulation and
uterine function during fertilization, implantation and pregnancy.
In the ovaries, high amounts of GCs, surpassing the amount that is typically
inactivated by 11β-HSD2, can suppress LH function and inhibit estrogen release
and synthesis [131, 146, 150]. Studies both in vivo and in vitro have shown that GCs
can influence not only LH response, but also inhibit transcription of the enzymes
necessary for steroid biosynthesis, critically inhibiting p450 aromatase, necessary
for conversion of testosterone to estrogen. In rat granulosa cell cultures, FSH trig-
gers the increase of aromatase activity, promoting estrogen synthesis for ovulation.
Administration of both CORT and DEX inhibited this FSH-induced increase, how-
ever stimulated progesterone synthesis and did not inhibit pre-existing aromatase
function. This indicates that GCs act to inhibit induction of aromatase activity spe-
cifically, not necessarily affecting granulosa cell function as a whole [151].
Glucocorticoid treatment was also found to decrease LH receptor in cultured granu-
losa cells [152], indicating that GCs can act directly on the ovarian cells to decrease
FSH-stimulated functions, including aromatase activity and LH receptor binding.
Interestingly, GC effects on oocyte maturation appear to be species dependent.
Studies in humans and pig have shown that GCs can inhibit meiotic development in
the oocytes [153, 154], however studies in sheep and mice have shown no effect of
GCs on final oocyte maturation [155, 156]. However a recent study in mice showed
that high levels of CRH in the cytoplasm of the ovaries due to restraint stress induced
ovarian apoptosis, decreasing follicular development independent of GR. This
increase of CRH was acting on thecal cells in the ovary, decreasing testosterone and
estrogen levels and increasing progesterone, creating a hormonal imbalance between
estrogen and progesterone that led to decreased oocyte success [157]. These differ-
ences are likely due to problems intrinsic to in vitro models that utilize only the
ovarian granulosa cells. In addition, another in vivo study in mice utilizing preda-
tory stress found that while high GCs did not affect oocyte maturation, blastocyst
formation was significantly decreased in these mice, showing that GCs may have a
stronger effect on embryo development or the oocyte potential for fertilization,
rather than maturation of the oocytes in general [158]. The next section will explore
GCs effects on pregnancy and fertilization more closely.
The role of glucocorticoid function in the ovary is incredibly complex and nar-
rowly regulated. The actions of GCs are regulated via differential transcription of
the two 11β-HSD enzymes, transcription of which is controlled through gonadotro-
pin release form the pituitary. Glucocorticoids are critical for maintenance of ovar-
ian function, involved in functional apoptosis of follicles to maintain normal
follicular development, as well as its anti-inflammatory role necessary for ovulation
to occur. However, high stress can tip the scales from functional to dysfunctional,
overwhelming the ability of 11β-HSD to regulate GC levels and causing ovarian
problems ranging from a decreased responsiveness to LH levels, decreased synthe-
sis of estrogen due to inhibition of aromatase release, and potentially inhibiting the
final step of oocyte maturation.
Glucocorticoids, Implantation and Pregnancy Success
Even if an ovum can be successfully developed in times of stress, and the HPG axis
still functional enough to trigger ovulation, GCs can still act to influence the uterus
to prevent successful implantation and completion of pregnancy. Glucocorticoids
typically act in opposition to estrogenic actions, and this becomes increasingly criti-
cal in implantation. For successful implantation of a blastocyst, progesterone and
estrogen regulate uterine cell proliferation, and are necessary for the changes in
both the blastocyst and uterine epithelium for successful adhesion. Glucocorticoids
inhibit estradiol-stimulated uterine growth and decreases estrogen receptor concen-

trations in the uterus [159–162].
Pregnancy itself requires a delicate immune balance and regulation of the mater-
nal immune cells in order for survival of both the fetus and the mother. It is sug-
gested that the high levels of progesterone (P) released from the corpus luteum of
the ovary after ovulation and sustained by the placenta throughout pregnancy help
regulate the mothers immune system. There is some research indicating that
membrane-bound progesterone receptors act to inhibit maternal T-cell during preg-
nancy [163, 164]. This combines with a series of other downstream immune events
that allows the maternal immune system to accept the foreign fetus and expression
of progesterone and related progesterone factors such as progesterone-induced
blocking factor (PIBF) continues to increase through pregnancy. High levels of Th1
cytokines in mice have been shown to be abortogenic, and progesterone during
pregnancy binding to progesterone receptors have been shown to release PIBF,
which in turn decreases natural killer (NK) cells in the uterus and induces Th2 cyto-
kine development, changing the balance towards an anti-abortive immune response
[163–167]. However studies using restraint stress in rodents has found that stress
early in pregnancy leads to decreased embryo success, showing higher abortion
rates in the mice and smaller litter sizes [168–170]. Wiebold et.al. found that this
was due to decreases in corpora lutea, lower levels of serum progesterone and fewer
implantation sites [168]. In humans, circadian cortisol levels are suppressed in early
pregnancy, and women who have been found to have high morning cortisol levels
in the first weeks of pregnancy were more likely to experience spontaneous abor-
tions [130]. This however appears to be specific to the peri-implantation time, as
studies have not found that circadian levels of cortisol are indicative of likelihood of
a miscarriage later in the first trimester [131]. Human data on the subject is incon-
clusive, but the immunosuppressive effects of high cortisol, as well GC’s influence
on decreasing progesterone and PIBF release support the idea that stress can have a
profound influence on early miscarriage rates.
Glucocorticoids play a significant role in pregnancy maintenance, opposing
estrogen’s ability to ready the uterus for implantation and inhibiting progesterone’s
anti-abortive immune response. While much of research into this focuses on stress
during the pregnancy itself, there could be long-term effects of stress prior to the
pregnancy that could affect pregnancy success as well, maybe via long-term inhibi-
tion of progesterone.
Conclusion: Stress and Its Many Effects

on Reproductive Ability
Physiologically, glucocorticoids exert many effects on surrounding cells and are

necessary for life. Within normal ranges, GCs regulate homeostasis and are critical
for our stress response. In times of stress, high levels of GCs shut down physiological
processes not relevant for survival in that time, including reproduction. GCs and the
HPA axis can act upon every level of the HPG axis, both directly and indirectly
inhibiting gonadotropin release from the pituitary and exerting direct effects on the
gonads. Stress and high GCs decrease the release of GnRH from the hypothalamus,
either by directly inhibiting GnRH pulses or inhibiting upstream regulators of
GnRH release. This can lead to downstream decreases in LH release form the pitu-
itary, however GCs can also directly inhibit the synthesis and release of gonadotro-
pins from the anterior pituitary. The decrease of LH and sometimes FSH from the
pituitary can decrease steroid release form the gonads, and circulating GCs can also
act directly on the gonads to inhibit the transcription of enzymes necessary for
gonadal steroid biosynthesis. There are sex and species differences in all these
responses. It is a complex and confusing field, however new techniques utilizing
cell-specific knockdowns of GR and/or other peptides involved in this response can
help clarify the more specific roles of GCs and reproductive dysfunction. This
becomes increasingly important as we find that infertility rates continue to increase
in humans, likely due to high stress exposure in day-to-day lives. Understanding the
molecular mechanisms behind how stress impairs fecundity and reproductive suc-
cess, especially in females, is critical to helping improve fertility rates.
References
1. Handa RJ, Weiser MJ. Gonadal steroid hormones and the hypothalamo-pituitary-adrenal
axis. Front Neuroendocrinol. 2014;35(2):197–220. doi:10.1016/j.yfrne.2013.11.001.
2. Jennes L, Conn PM. Gonadotropin-releasing hormone and its receptors in rat brain. Front
Neuroendocrinol. 1994;15(1):51–77. doi:10.1006/frne.1994.1003.
3. King JC, Tobet SA, Snavely FL, Arimura AA. LHRH immunopositive cells and their projec-
tions to the median eminence and organum vasculosum of the lamina terminalis. J Comp
Neurol. 1982;209(3):287–300. doi:10.1002/cne.902090307.
4. Levine JE, Bauer-Dantoin AC. Neuroendocrine regulation of the luteinizing hormone-
releasing hormone pulse generator in the rat. Recent Prog Horm Res. 1991;47:97–151.
5. Moenter SM, Anthony DeFazio R, Pitts GR, Nunemaker CS. Mechanisms underlying epi-
sodic gonadotropin-releasing hormone secretion. Front Neuroendocrinol. 2003;24(2):79–93.
doi:10.1016/S0091-3022(03)00013-X.
6. Haisenleder DJ, Dalkin AC, Ortolano GA, Marshall JC, Shupnik MA. A pulsatile
gonadotropin-releasing hormone stimulus is required to increase transcription of the gonado-
tropin subunit genes: evidence for differential regulation of transcription by pulse frequency
in vivo. Endocrinology. 1991;128(1):509–17. doi:10.1210/endo-128-1-509.
7. Sarkar DK, Chiappa SA, Fink G, Sherwood NM. Gonadotropin-releasing hormone surge in
pro-oestrous rats. Nature. 1976;264(5585):461–3. doi:10.1038/264461a0.
8. Park O-K, Ramirez VD. Spontaneous changes in LHRH release during the rat estrous cycle,
as measured with repetitive push-pull perfusions of the pituitary gland in the same female
rats. Neuroendocrinology. 1989;50(1):66–72. doi:10.1159/10.1159/000125203.
9. Marshall JC, Griffin ML. The role of changing pulse frequency in the regulation of ovulation.
Hum Reprod. 1993;8 Suppl 2:57–61. http://www.ncbi.nlm.nih.gov/pubmed/8276970.
Accessed 27 May 2014.
10. Marshall JC, Dalkin AC, Haisenleder DJ, Griffin ML, Kelch RP. GnRH pulses—the regula-
tors of human reproduction. Trans Am Clin Climatol Assoc. 1993;104:31–46. http://www.
pubmedcentral.nih.gov/articlerender.fcgi?artid=2376610&tool=pmcentrez&rendertype=abs
tract. Accessed 27 May 2014.
11. Herbison AE. Estrogen positive feedback to gonadotropin-releasing hormone (GnRH) neu-
rons in the rodent: the case for the rostral periventricular area of the third ventricle (RP3V).
Brain Res Rev. 2008;57(2):277–87. doi:10.1016/j.brainresrev.2007.05.006.
12. Chazal G, Faudon M, Gogan F, Laplante E. Negative and positive effects of oestradiol upon
luteinizing hormone secretion in the female rat. J Endocrinol. 1974;61(3):511–2. doi:10.1677/
joe.0.0610511.
13. Shupnik MA. Gonadotropin gene modulation by steroids and gonadotropin-releasing hor-
mone. Biol Reprod. 1996;54(2):279–86. doi:10.1095/biolreprod54.2.279.
14. Knobil E. The neuroendocrine control of the menstrual cycle. Recent Prog Horm Res.
1980;36:53–88.
15. Baird DT, McNeilly AS. Gonadotrophic control of follicular development and function dur-
ing the oestrous cycle of the ewe. J Reprod Fertil Suppl. 1981;30:119–33. http://europepmc.
org/abstract/MED/6300383. Accessed 2 Sept 2014.
16. Legan SJ, Karsch FJ. A daily signal for the LH surge in the rat. Endocrinology. 1975;96(1):57–
62. doi:10.1210/endo-96-1-57.
17. Ferin M, Tempone A, Zimmering PE, Van de Wiele RL. Effect of antibodies to 17beta-
estradiol and progesterone on the estrous cycle of the rat. Endocrinology. 1969;85(6):1070–8.
doi:10.1210/endo-85-6-1070.
18. Labhsetwar AP. Role of estrogens in ovulation: a study using the estrogen-antagonist, I.C.I.
46,474. Endocrinology. 1970;87(3):542–51. doi:10.1210/endo-87-3-542.
19. Chappell PE, Levine JE. Stimulation of gonadotropin-releasing hormone surges by estrogen.
I. Role of hypothalamic progesterone receptors. Endocrinology. 2000;141(4):1477–85.
doi:10.1210/endo.141.4.7428.
20. Micevych P, Sinchak K, Mills RH, Tao L, LaPolt P, Lu JKH. The luteinizing hormone surge
is preceded by an estrogen-induced increase of hypothalamic progesterone in ovariectomized
and adrenalectomized rats. Neuroendocrinology. 2003;78(1):29–35. doi:10.1159/000071703.
21. Kuo J, Hamid N, Bondar G, Prossnitz ER, Micevych P. Membrane estrogen receptors stimu-
late intracellular calcium release and progesterone synthesis in hypothalamic astrocytes. J
Neurosci. 2010;30(39):12950–7. doi:10.1523/JNEUROSCI.1158-10.2010.
22. Micevych P, Soma KK, Sinchak K. Neuroprogesterone: key to estrogen positive feedback?
Brain Res Rev. 2008;57(2):470–80. doi:10.1016/j.brainresrev.2007.06.009.
23. Micevych PE, Chaban V, Ogi J, Dewing P, Lu JKH, Sinchak K. Estradiol stimulates proges-
terone synthesis in hypothalamic astrocyte cultures. Endocrinology. 2007;148(2):782–9.
doi:10.1210/en.2006-0774.
24. Micevych P, Sinchak K. The neurosteroid progesterone underlies estrogen positive feedback
of the LH surge. Front Endocrinol (Lausanne). 2011;2:90. doi:10.3389/fendo.2011.00090.
25. Chaban VV, Lakhter AJ, Micevych P. A membrane estrogen receptor mediates intracellular
calcium release in astrocytes. Endocrinology. 2004;145(8):3788–95. doi:10.1210/
en.2004-0149.
26. Ubuka T, Inoue K, Fukuda Y, et al. Identification, expression, and physiological functions of
Siberian hamster gonadotropin-inhibitory hormone. Endocrinology. 2012;153(1):373–85.
papers://cf7c60b8-94a1-4c79-88e4-6c57345fd583/Paper/p1434.
27. Ubuka T, Morgan K, Pawson A, et al. Identification of human GnIH homologs, RFRP-1 and
RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis. PLoS
One. 2009;4(12):1334–9. papers://cf7c60b8-94a1-4c79-88e4-6c57345fd583/Paper/p1233.
28. Ubuka T, Lai H, Kitani M, et al. Gonadotropin-inhibitory hormone identification, cDNA
cloning, and distribution in rhesus macaque brain. J Comp Neurol. 2009;517:841–55.
doi:10.1002/cne.22191.
29. Ukena K, Iwakoshi E, Minakata H, Tsutsui K. A novel rat hypothalamic RFamide-related
peptide identified by immunoaffinity chromatography and mass spectrometry. FEBS Lett.
2002;512(1–3):255–8. papers://cf7c60b8-94a1-4c79-88e4-6c57345fd583/Paper/p1299.
30. Tsutsui K, Saigoh E, Ukena K, et al. A novel avian hypothalamic peptide inhibiting gonado-
tropin release. Biochem Biophys Res Commun. 2000;275(2):661–7. doi:10.1006/
bbrc.2000.3350.
31. De Roux N, Genin E, Carel J-C, Matsuda F, Chaussain J-L, Milgrom E. Hypogonadotropic
hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc
Natl Acad Sci U S A. 2003;100(19):10972–6. doi:10.1073/pnas.1834399100.
32. Thompson EL, Patterson M, Murphy KG, et al. Central and peripheral administration of
kisspeptin-10 stimulates the hypothalamic-pituitary-gonadal axis. J Neuroendocrinol.
2004;16(10):850–8. doi:10.1111/j.1365-2826.2004.01240.x.
33. Gottsch ML, Cunningham MJ, Smith JT, et al. A role for kisspeptins in the regulation of
gonadotropin secretion in the mouse. Endocrinology. 2004;145(9):4073–7. doi:10.1210/
en.2004-0431.
34. Navarro VM, Castellano JM, Fernández-Fernández R, et al. Effects of KiSS-1 peptide, the
natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology.
2005;146(4):1689–97. doi:10.1210/en.2004-1353.
35. Dhillo WS, Chaudhri OB, Patterson M, et al. Kisspeptin-54 stimulates the hypothalamic-
pituitary gonadal axis in human males. J Clin Endocrinol Metab. 2005;90(12):6609–15.
doi:10.1210/jc.2005-1468.
36. Li X-F, Kinsey-Jones JS, Cheng Y, et al. Kisspeptin signalling in the hypothalamic arcuate
nucleus regulates GnRH pulse generator frequency in the rat. Tena-Sempere M, ed. PLoS
One. 2009;4(12):e8334. doi:10.1371/journal.pone.0008334.
37. Maeda K-I, Ohkura S, Uenoyama Y, et al. Neurobiological mechanisms underlying GnRH
pulse generation by the hypothalamus. Brain Res. 2010;1364:103–15. doi:10.1016/j.
brainres.2010.10.026.
38. Roseweir AK, Kauffman AS, Smith JT, et al. Discovery of potent kisspeptin antagonists
delineate physiological mechanisms of gonadotropin regulation. J Neurosci.
2009;29(12):3920–9. doi:10.1523/JNEUROSCI.5740-08.2009.
39. Messager S, Chatzidaki EE, Ma D, et al. Kisspeptin directly stimulates gonadotropin-
releasing hormone release via G protein-coupled receptor 54. Proc Natl Acad Sci U S A.
2005;102(5):1761–6. papers://cf7c60b8-94a1-4c79-88e4-6c57345fd583/Paper/p1232.
40. Millar RP, Roseweir AK, Tello JA, et al. Kisspeptin antagonists: unraveling the role of
kisspeptin in reproductive physiology. Brain Res. 2010;1364:81–9. doi:10.1016/j.
brainres.2010.09.044.
41. Pineda R, Garcia-Galiano D, Sanchez-Garrido MA, et al. Characterization of the inhibitory
roles of RFRP3, the mammalian ortholog of GnIH, in the control of gonadotropin secretion
in the rat: in vivo and in vitro studies. Am J Physiol Endocrinol Metab. 2010;299(1):E39–46.
42. Khan AR, Kauffman AS. The role of kisspeptin and RFamide-related peptide-3 neurones in
the circadian-timed preovulatory luteinising hormone surge. J Neuroendocrinol.
43. Clarke I, Smith J, Henry B, et al. Gonadotropin-inhibitory hormone is a hypothalamic peptide
that provides a molecular switch between reproduction and feeding. Neuroendocrinology.
44. Wu M, Dumalska I, Morozova E, van den Pol AN, Alreja M. Gonadotropin inhibitory hor-
mone inhibits basal forebrain vGluT2-gonadotropin-releasing hormone neurons via a direct
postsynaptic mechanism. J Physiol. 2009;587(7):1401. papers://cf7c60b8-94a1-4c79-88e4-
6c57345fd583/Paper/p1234.
45. Kriegsfeld LJ, Gibson EM, Williams WP, et al. The roles of RFamide-related peptide-3 in
mammalian reproductive function and behaviour. J Neuroendocrinol. 2010;22(7):692–700.
doi:10.1111/j.1365-2826.2010.02031.x.
46. Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH, Herbison AE. Kisspeptin-
GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron acti-
vation and the luteinizing hormone surge. J Neurosci. 2008;28(35):8691–7. doi:10.1523/
JNEUROSCI.1775-08.2008.
47. Sinchak K, Wagner EJ. Estradiol signaling in the regulation of reproduction and energy bal-
ance. Front Neuroendocrinol. 2012;33(4):342–63. doi:10.1016/j.yfrne.2012.08.004.
48. Christensen A, Bentley GE, Cabrera R, et al. Hormonal regulation of female reproduction.
Horm Metab Res. 2012;44(8):587–91. doi:10.1055/s-0032-1306301.
49. Giuliani FA, Yunes R, Mohn CE, Laconi M, Rettori V, Cabrera R. Allopregnanolone induces
LHRH and glutamate release through NMDA receptor modulation. Endocrine. 2011;40(1):21–
6. doi:10.1007/s12020-011-9451-8.
50. Sim JA, Skynner MJ, Herbison AE. Direct regulation of postnatal GnRH neurons by the
progesterone derivative allopregnanolone in the mouse. Endocrinology. 2001;142(10):4448–
53. doi:10.1210/endo.142.10.8451.
51. el-Etr M, Akwa Y, Fiddes RJ, Robel P, Baulieu EE. A progesterone metabolite stimulates the
release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the
gamma-aminobutyric acid type A receptor. Proc Natl Acad Sci U S A. 1995;92(9):3769–73.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=42043&tool=pmcentrez&render
type=abstract. Accessed 27 May 2014.
52. Breen KM, Karsch FJ. New insights regarding glucocorticoids, stress and gonadotropin sup-
pression. Front Neuroendocrinol. 2006;27(2):233–45. doi:10.1016/j.yfrne.2006.03.335.
53. Li XF, Knox AMI, O’Byrne KT. Corticotrophin-releasing factor and stress-induced inhibi-
tion of the gonadotrophin-releasing hormone pulse generator in the female. Brain Res.
2010;1364:153–63. doi:10.1016/j.brainres.2010.08.036.
54. Rivier C, Vale W. Influence of corticotropin-releasing factor on reproductive functions in the
rat. Endocrinology. 1984;114(3):914–21. doi:10.1210/endo-114-3-914.
55. Cates PS, Li XF, O’Byrne KT. The influence of 17beta-oestradiol on corticotrophin-releasing
hormone induced suppression of luteinising hormone pulses and the role of CRH in hypogly-
caemic stress-induced suppression of pulsatile LH secretion in the female rat. Stress.
2004;7(2):113–8. doi:10.1080/1025389042000218988.
56. Bowe JE, Li XF, Kinsey-Jones JS, Brain SD, Lightman SL, O’Byrne KT. The role of
corticotrophin-releasing hormone receptors in the calcitonin gene-related peptide-induced
suppression of pulsatile luteinising hormone secretion in the female rat. Stress.
2008;11(4):312–9. doi:10.1080/10253890701801448.
57. Li XF, Bowe JE, Kinsey-Jones JS, Brain SD, Lightman SL, O’Byrne KT. Differential role of
corticotrophin-releasing factor receptor types 1 and 2 in stress-induced suppression of pulsa-
tile luteinising hormone secretion in the female rat. J Neuroendocrinol. 2006;18(8):602–10.
58. Li XF, Bowe JE, Lightman SL, O’Byrne KT. Role of corticotropin-releasing factor receptor-
2 in stress-induced suppression of pulsatile luteinizing hormone secretion in the rat.
Endocrinology. 2005;146(1):318–22. papers://cf7c60b8-94a1-4c79-88e4-6c57345fd583/
Paper/p710.
59. Kinsey‐Jones J, Li X, Knox A, et al. Corticotrophin‐releasing factor alters the timing of
puberty in the female rat. J Neuroendocrinol. 2010;22(2):102–109. file:///Users/annager-
aghty/Documents/Papers/2010/Kinsey‐Jones/Journal of Neuroendocrinology 2010 Kinsey‐
Jones.pdf.
60. Rivier C, Rivest S. Effect of stress on the activity of the hypothalamic-pituitary-gonadal axis:
peripheral and central mechanisms. Biol Reprod. 1991;45(4):523–32. http://www.ncbi.nlm.
nih.gov/pubmed/1661182. Accessed 9 Sept 2013.
61. Rivest S, Rivier C. Central mechanisms and sites of action involved in the inhibitory effects
of CRF and cytokines on LHRH neuronal activity. Ann N Y Acad Sci. 1993;697
(1 Corticotropin):117–41. doi:10.1111/j.1749-6632.1993.tb49928.x.
62. Oakley AE, Breen KM, Clarke IJ, Karsch FJ, Wagenmaker ER, Tilbrook AJ. Cortisol reduces
gonadotropin-releasing hormone pulse frequency in follicular phase ewes: influence of ovar-
ian steroids. Endocrinology. 2009;150(1):341–9. doi:10.1210/en.2008-0587.
63. Calogero AE, Burrello N, Bosboom AM, Garofalo MR, Weber RF, D’Agata R. Glucocorticoids
inhibit gonadotropin-releasing hormone by acting directly at the hypothalamic level. J
Endocrinol Invest. 1999;22(9):666–70. http://www.ncbi.nlm.nih.gov/pubmed/10595829.
64. Jasoni CL, Todman MG, Han S-K, Herbison AE. Expression of mRNAs encoding receptors
that mediate stress signals in gonadotropin-releasing hormone neurons of the mouse.
Neuroendocrinology. 2005;82(5–6):320–8. doi:10.1159/000093155.
65. Ahima RS, Harlan RE. Glucocorticoid receptors in LHRH neurons. Neuroendocrinology.
1992;56(6):845–50. http://www.ncbi.nlm.nih.gov/pubmed/1369593. Accessed 27 May 2014.
66. Dondi D, Piccolella M, Messi E, et al. Expression and differential effects of the activation of
glucocorticoid receptors in mouse gonadotropin-releasing hormone neurons.
Neuroendocrinology. 2005;82(3–4):151–63. doi:10.1159/000091693.
67. DeFranco DB, Attardi B, Chandran UR. Glucocorticoid receptor-mediated repression of
GnRH gene expression in a hypothalamic GnRH-secreting neuronal cell line. Ann N Y Acad
Sci. 1994;746:473–5. http://www.ncbi.nlm.nih.gov/pubmed/7825918. Accessed 28 May 2014.
68. Tellam DJ, Perone MJ, Dunn IC, et al. Direct regulation of GnRH transcription by CRF-like
peptides in an immortalized neuronal cell line. Neuroreport. 1998;9(14):3135–40.
doi:10.1097/00001756-199810050-00003.
69. Tellam DJ, Mohammad YN, Lovejoy DA. Molecular integration of hypothalamo-pituitary-
adrenal axis-related neurohormones on the GnRH neuron. 2011. http://www.nrcresearch-
press.com/doi/abs/10.1139/o00-060#.U4Z5rlhdX-Y. Accessed 29 May 2014.
70. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI. Immortalization of
hypothalamic GnRH by genetically targeted tumorigenesis. Neuron. 1990;5(1):1–10.
doi:10.1016/0896-6273(90)90028-E.
71. Wetsel WC, Mellon PL, Weiner RI, Negro-Vilar A. Metabolism of pro-luteinizing hormone-
releasing hormone in immortalized hypothalamic neurons. Endocrinology. 1991;129(3):1584–
95. doi:10.1210/endo-129-3-1584.
72. Chandran UR, Attardi B, Friedman R, Dong KW, Roberts JL, DeFranco DB. Glucocorticoid
receptor-mediated repression of gonadotropin-releasing hormone promoter activity in GT1
hypothalamic cell lines. Endocrinology. 1994;134(3):1467–74. doi:10.1210/endo.134.3.8119188.
73. Attardi B, Tsujii T, Friedman R, et al. Glucocorticoid repression of gonadotropin-releasing
hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7
cells. Mol Cell Endocrinol. 1997;131(2):241–55. http://www.ncbi.nlm.nih.gov/pubmed/
9296383. Accessed 28 May 2014.
74. Gore A, Attardi B, DeFranco D. Glucocorticoid repression of the reproductive axis: effects on
GnRH and gonadotropin subunit mRNA levels. Mol Cell Endocrinol. 2006;256(1-2):40–8.
Papers
75. Kinsey‐Jones J, Li X, Knox A, et al. Down‐regulation of hypothalamic kisspeptin and its
receptor, Kiss1r, mRNA expression is associated with stress‐induced suppression of luteinis-
ing hormone secretion in the female rat. J Neuroendocrinol. 2009;21(1):20–29. file:///Users/
annageraghty/Documents/Papers/2009/Kinsey‐Jones/Journal of Neuroendocrinology 2009
Kinsey‐Jones-1.pdf.
76. Iwasa T, Matsuzaki T, Murakami M, et al. Decreased expression of kisspeptin mediates acute
immune/inflammatory stress-induced suppression of gonadotropin secretion in female rat.
J Endocrinol Invest. 2008;31(7):656–9. http://europepmc.org/abstract/MED/18787387.
77. Grachev P, Li XF, O’Byrne K. Stress regulation of kisspeptin in the modulation of reproduc-
tive function. Adv Exp Med Biol. 2013;784:431–54. doi:10.1007/978-1-4614-6199-9_20.
78. Takumi K, Iijima N, Higo S, Ozawa H. Immunohistochemical analysis of the colocalization
of corticotropin-releasing hormone receptor and glucocorticoid receptor in kisspeptin neu-
rons in the hypothalamus of female rats. Neurosci Lett. 2012;531(1):40–5. doi:10.1016/j.
neulet.2012.10.010.
79. Grachev P, Li XF, Hu MH, et al. Neurokinin B signaling in the female rat: a novel link
between stress and reproduction. Endocrinology. 2014;155(7):2589–601. doi:10.1210/
en.2013-2038.
80. Goodman RL, Hileman SM, Nestor CC, et al. Kisspeptin, neurokinin B, and dynorphin act in
the arcuate nucleus to control activity of the GnRH pulse generator in ewes. Endocrinology.
2013;154(11):4259–69. doi:10.1210/en.2013-1331.
81. Okamura H, Tsukamura H, Ohkura S, Uenoyama Y, Wakabayashi Y, Maeda K. Kisspeptin

and GnRH pulse generation. Adv Exp Med Biol. 2013;784:297–323. doi:10.1007/978-1-
4614-6199-9_14.
82. Wakabayashi Y, Yamamura T, Sakamoto K, Mori Y, Okamura H. Electrophysiological and
morphological evidence for synchronized GnRH pulse generator activity among Kisspeptin/
neurokinin B/dynorphin A (KNDy) neurons in goats. J Reprod Dev. 2013;59(1):40–8. http://
www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3943231&tool=pmcentrez&rendertyp
e=abstract. Accessed 28 May 2014.
83. Bowe JE, Li XF, Kinsey-Jones JS, et al. Calcitonin gene-related peptide-induced suppression
of luteinizing hormone pulses in the rat: the role of endogenous opioid peptides. J Physiol.
2005;566(Pt 3):921–8. doi:10.1113/jphysiol.2005.085662.
84. Nabeshima T, Katoh A, Wada M, Kameyama T. Stress-induced changes in brain Met-
enkephalin, Leu-enkephalin and dynorphin concentrations. Life Sci. 1992;51(3):211–7.
http://www.ncbi.nlm.nih.gov/pubmed/1352028. Accessed 28 May 2014.
85. Petraglia F, Vale W, Rivier C. Opioids act centrally to modulate stress-induced decrease in
luteinizing hormone in the rat. Endocrinology. 1986;119(6):2445–50. doi:10.1210/
endo-119-6-2445.
86. Ukena K, Iwakoshi E, Minakata H, Tsutsui K. A novel rat hypothalamic RFamide-related
peptide identified by immunoaffinity chromatography and mass spectrometry. FEBS Lett.
2002;512(1–3):255–8. http://www.ncbi.nlm.nih.gov/pubmed/11852091. Accessed 9 Sept
2013.
87. Sari IP, Rao A, Smith JT, Tilbrook AJ, Clarke IJ. Effect of RF-amide-related peptide-3 on
luteinizing hormone and follicle-stimulating hormone synthesis and secretion in ovine pitu-
itary gonadotropes. Endocrinology. 2009;150(12):5549–56. papers://cf7c60b8-94a1-4c79-
88e4-6c57345fd583/Paper/p1243.
88. Kirby ED, Geraghty AC, Ubuka T, Bentley GE, Kaufer D. Stress increases putative gonado-
tropin inhibitory hormone and decreases luteinizing hormone in male rats. Proc Natl Acad
Sci U S A. 2009;106(27):11324–9. papers://cf7c60b8-94a1-4c79-88e4-6c57345fd583/Paper/
p1273.
89. Lee Son Y, Ubuka T, Narihiro M, et al. Molecular basis for the activation of gonadotropin-
inhibitory hormone gene transcription by corticosterone. Endocrinology. 2014;155(5):1817–
26. doi:10.1210/en.2013-2076.
90. Gojska NM, Belsham DD. Glucocorticoid receptor-mediated regulation of Rfrp (GnIH) and
Gpr147 (GnIH-R) synthesis in immortalized hypothalamic neurons. Mol Cell Endocrinol.
2014;384(1–2):23–31. doi:10.1016/j.mce.2013.12.015.
91. Geraghty A, Muroy S, Zhao S, Bentley G, Kriegsfeld L, Kaufer D. Chronic stress causes an
increase in RFRP expression and leads to reproductive dysfunction in the adult female rat.
[abstract]. In: 2013 Neuroscience Meet Plan, Society for Neuroscience; 2013.
92. Kaiser UB, Jakubowiak A, Steinberger A, Chin WW. Differential effects of gonadotropin-
releasing hormone (GnRH) pulse frequency on gonadotropin subunit and GnRH receptor
messenger ribonucleic acid levels in vitro. Endocrinology. 1997;138(3):1224–31.
doi:10.1210/endo.138.3.4968.
93. Vale W, Rivier C, Brown M. Regulatory peptides of the hypothalamus. Annu Rev Physiol.
1977;39:473–527. doi:10.1146/annurev.ph.39.030177.002353.
94. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem.
1981;50:465–95. doi:10.1146/annurev.bi.50.070181.002341.
95. Kononen J, Honkaniemi J, Gustafsson JA, Pelto-Huikko M. Glucocorticoid receptor colocal-
ization with pituitary hormones in the rat pituitary gland. Mol Cell Endocrinol. 1993;93(1):97–
103. http://www.ncbi.nlm.nih.gov/pubmed/8319836. Accessed 28 May 2014.
96. Breen KM, Thackray VG, Hsu T, Mak-McCully RA, Coss D, Mellon PL. Stress levels of
glucocorticoids inhibit LHβ-subunit gene expression in gonadotrope cells. Mol Endocrinol.
2012;26(10):1716–31. doi:10.1210/me.2011-1327.
97. Armario A, Lopez-Calderon A, Jolin T, Balasch J. Response of anterior pituitary hormones to

chronic stress. The specificity of adaptation. Neurosci Biobehav Rev. 1986;10(3):245–50.
doi:10.1016/0149-7634(86)90011-4.
98. Lopez-Calderon A, Gonzalez-Quijano MI, Tresguerres JAF, Ariznavarreta C. Role of LHRH
in the gonadotrophin response to restraint stress in intact male rats. J Endocrinol. 1990;
124(2):241–6. doi:10.1677/joe.0.1240241.
99. Blake CA. Effects of “stress” on pulsatile luteinizing hormone release in ovariectomized rats.
Proc Soc Exp Biol Med. 1975;148(3):813–5. http://www.ncbi.nlm.nih.gov/pubmed/165534.
100. Kamel F, Kubajak CL. Modulation of gonadotropin secretion by corticosterone: interaction
with gonadal steroids and mechanism of action. Endocrinology. 1987;121(2):561–8.
doi:10.1210/endo-121-2-561.
101. Baldwin DM, Sawyer CH. Effects of dexamethasone on LH release and ovulation in the
cyclic rat. Endocrinology. 1974;94(5):1397–403. doi:10.1210/endo-94-5-1397.
102. Collu R, Taché Y, Ducharme J. Hormonal modifications induced by chronic stress in rats. J
Steroid Biochem. 1979;11(1):989–1000. doi:10.1016/0022-4731(79)90042-6.
103. Vreeburg JT, de Greef WJ, Ooms MP, van Wouw P, Weber RF. Effects of adrenocorticotropin
and corticosterone on the negative feedback action of testosterone in the adult male rat.
Endocrinology. 1984;115(3):977–83. doi:10.1210/endo-115-3-977.
104. Taché Y, Ducharme JR, Charpenet G, Haour F, Saez J, Collu R. Effect of chronic intermittent
immobilization stress on hypophyso-gonadal function of rats. Acta Endocrinol (Copenh).
105. Briski KP, Sylvester PW. Differential impact of naltrexone on luteinizing hormone release
during single versus repetitive exposure to restraint stress. Psychoneuroendocrinology.
1992;17(2–3):125–33. http://www.ncbi.nlm.nih.gov/pubmed/1332097. Accessed 28 May
2014.
106. Li XF, Edward J, Mitchell JC, et al. Differential effects of repeated restraint stress on pulsatile
lutenizing hormone secretion in female Fischer, Lewis and Wistar rats. J Neuroendocrinol.
2004;16(7):620–7. doi:10.1111/j.1365-2826.2004.01209.x.
107. Briski KP, Sylvester PW. Effects of repetitive daily acute stress on pituitary LH and prolactin
release during exposure to the same stressor or a second novel stress. Psychoneuroendocrinology.
108. Sakakura M, Takebe K, Nakagawa S. Inhibition of luteinizing hormone secretion induced by
synthetic LRH by long-term treatment with glucocorticoids in human subjects. J Clin
Endocrinol Metab. 1975;40(5):774–9. doi:10.1210/jcem-40-5-774.
109. Du Ruisseau P, Taché Y, Brazeau P, Collu R. Effects of chronic immobilization stress on
pituitary hormone secretion, on hypothalamic factor levels, and on pituitary responsiveness
to LHRH and TRH in female rats. Neuroendocrinology. 1979;29(2):90–9. http://www.ncbi.
nlm.nih.gov/pubmed/116141. Accessed 9 Sept 2013.
110. Rivier C, Vale W. Effect of the long-term administration of corticotropin-releasing factor
on the pituitary-adrenal and pituitary-gonadal axis in the male rat. J Clin Invest.
1985;75(2):689–94. doi:10.1172/JCI111748.
111. Suter DE, Schwartz NB, Ringstrom SJ. Dual role of glucocorticoids in regulation of pituitary
content and secretion of gonadotropins. Am J Physiol. 1988;254(5 Pt 1):E595–600. http://
ajpendo.physiology.org/content/254/5/E595.abstract. Accessed 20 May 2014.
112. Baldwin DM. The effect of glucocorticoids on estrogen-dependent luteinizing hormone
release in the ovariectomized rat and on gonadotropin secretin in the intact female rat.
113. Maya-Núñez G, Conn PM. Transcriptional regulation of the GnRH receptor gene by glucocor-
ticoids. Mol Cell Endocrinol. 2003;200(1–2):89–98. doi:10.1016/S0303-7207(02)00419-7.
114. Kotitschke A, Sadie-Van Gijsen H, Avenant C, Fernandes S, Hapgood JP. Genomic and non-
genomic cross talk between the gonadotropin-releasing hormone receptor and glucocorticoid
receptor signaling pathways. Mol Endocrinol. 2009;23(11):1726–45. doi:10.1210/
me.2008-0462.
115. Bronson FH. Establishment of social rank among grouped male mice: relative effects on
circulating FSH, LH, and corticosterone. Physiol Behav. 1973;10(5):947–51. doi:10.1016/0031-
9384(73)90065-6.
116. Du Ruisseau P, Taché Y, Brazeau P, Colin R. Effects of chronic immobilization stress on
pituitary hormone secretion, on hypothalamic factor levels, and on pituitary responsiveness
to LHRH and TRH in female rats. Neuroendocrinology. 1979;29(2):90–9. doi:10.1159/
000122910.
117. Ringstrom SJ, Schwartz NB. Differential effect of glucocorticoids on synthesis and secretion
of luteinizing hormone (LH) and follicle stimulating hormone (FSH). J Steroid Biochem.
1987;27(1–3):625–30. doi:10.1016/0022-4731(87)90362-1.
118. Ringstrom SJ, McAndrews JM, Rahal JO, Schwartz NB. Cortisol in vivo increases FSH beta
mRNA selectively in pituitaries of male rats. Endocrinology. 1991;129(5):2793–5.
doi:10.1210/endo-129-5-2793.
119. Thackray VG, McGillivray SM, Mellon PL. Androgens, progestins, and glucocorticoids
induce follicle-stimulating hormone beta-subunit gene expression at the level of the gonado-
trope. Mol Endocrinol. 2006;20(9):2062–79. doi:10.1210/me.2005-0316.
120. Smals AG, Kloppenborg PW, Benraad TJ. Plasma testosterone profiles in Cushing’s
syndrome. J Clin Endocrinol Metab. 1977;45(2):240–5. doi:10.1210/jcem-45-2-240.
121. Whirledge S, Cidlowski JA. Glucocorticoids, stress, and fertility. Minerva Endocrinol.
2010;35(2):109–25. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3547681&t
ool=pmcentrez&rendertype=abstract. Accessed 20 May 2014.
122. Schultz R, Isola J, Parvinen M, et al. Localization of the glucocorticoid receptor in testis and
accessory sexual organs of male rat. Mol Cell Endocrinol. 1993;95(1–2):115–20.
doi:10.1016/0303-7207(93)90036-J.
123. Silva EJR, Queiróz DBC, Honda L, Avellar MCW. Glucocorticoid receptor in the rat epididy-
mis: expression, cellular distribution and regulation by steroid hormones. Mol Cell
Endocrinol. 2010;325(1–2):64–77. doi:10.1016/j.mce.2010.05.013.
124. Bernier M, Gibb W, Collu R, Ducharme JR. Effect of glucocorticoids on testosterone
production by porcine Leydig cells in primary culture. Can J Physiol Pharmacol.
1984;62(9):1166–9. doi:10.1139/y84-195.
125. Orr T. Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell
LH/HCG receptors, and in vitro testicular steroidogenesis in adult rats. Horm Behav.
1990;24(3):324–41. doi:10.1016/0018-506X(90)90013-N.
126. Orr T. Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis
in adult male rats. Horm Behav. 1992;26(3):350–63. doi:10.1016/0018-506X(92)90005-G.
127. Cumming DC, Quigley ME, Yen SS. Acute suppression of circulating testosterone levels by
cortisol in men. J Clin Endocrinol Metab. 1983;57(3):671–3. doi:10.1210/jcem-57-3-671.
128. Marić D, Kostić T, Kovačević R. Effects of acute and chronic immobilization stress on rat
Leydig cell steroidogenesis. J Steroid Biochem Mol Biol. 1996;58(3):351–5.
129. Saez JM, Morera AM, Haour F, Evain D. Effects of in vivo administration of dexamethasone,
corticotropin and human chorionic gonadotropin on steroidogenesis and protein and DNA
synthesis of testicular interstitial cells in prepuberal rats. Endocrinology. 1977;101(4):1256–63.
doi:10.1210/endo-101-4-1256.
130. Bambino TH, Hsueh AJ. Direct inhibitory effect of glucocorticoids upon testicular luteiniz-
ing hormone receptor and steroidogenesis in vivo and in vitro. Endocrinology. 1981;108(6):
2142–8. doi:10.1210/endo-108-6-2142.
131. Hales DB, Payne AH. Glucocorticoid-mediated repression of P450scc mRNA and de novo
synthesis in cultured Leydig cells. Endocrinology. 1989;124(5):2099–104. doi:10.1210/
endo-124-5-2099.
132. Payne AH, Sha LL. Multiple mechanisms for regulation of 3 beta-hydroxysteroid dehydroge-
nase/delta 5––delta 4-isomerase, 17 alpha-hydroxylase/C17-20 lyase cytochrome P450, and
cholesterol side-chain cleavage cytochrome P450 messenger ribonucleic acid levels in pri-
mary cultures of mouse Leydig cells. Endocrinology. 1991;129(3):1429–35. doi:10.1210/
endo-129-3-1429.
133. Martin LJ, Tremblay JJ. Glucocorticoids antagonize cAMP-induced Star transcription in
Leydig cells through the orphan nuclear receptor NR4A1. J Mol Endocrinol. 2008;41(3):
165–75. doi:10.1677/JME-07-0145.
134. Sasagawa I, Yazawa H, Suzuki Y, Nakada T. Stress and testicular germ cell apoptosis. 2009.
http://informahealthcare.com/doi/abs/10.1080/014850101753145924. Accessed 20 May
2014.
135. Yazawa H. Apoptosis of testicular germ cells induced by exogenous glucocorticoid in rats.
Hum Reprod. 2000;15(9):1917–20. doi:10.1093/humrep/15.9.1917.
136. Gao H-B, Tong M-H, Hu Y-Q, Guo Q-S, Ge R, Hardy MP. Glucocorticoid induces apoptosis
in rat Leydig cells. 2013. http://press.endocrine.org/doi/abs/10.1210/endo.143.1.8604?url_
ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed. Accessed 20 May
2014.
137. Almeida SA, Petenusci SO, Anselmo-Franci JA, Rosa-e-Silva AAM, Lamano-Carvalho
TL. Decreased spermatogenic and androgenic testicular functions in adult rats submitted to
immobilization-induced stress from prepuberty. Braz J Med Biol Res. 1998;31(11):1443–8.
doi:10.1590/S0100-879X1998001100013.
138. Zorn B, Auger J, Velikonja V, Kolbezen M, Meden-Vrtovec H. Psychological factors in male
partners of infertile couples: relationship with semen quality and early miscarriage. Int J
Androl. 2008;31(6):557–64. doi:10.1111/j.1365-2605.2007.00806.x.
139. Seckl JR. 11Beta-hydroxysteroid dehydrogenase in the brain: a novel regulator of glucocor-
ticoid action? Front Neuroendocrinol. 1997;18(1):49–99. doi:10.1006/frne.1996.0143.
140. Seckl JR, Walker BR. Minireview: 11beta-hydroxysteroid dehydrogenase type 1—a tissue-
specific amplifier of glucocorticoid action. Endocrinology. 2001;142(4):1371–6. doi:10.1210/
endo.142.4.8114.
141. Tetsuka M. Expression of 11 beta-hydroxysteroid dehydrogenase, glucocorticoid receptor,
and mineralocorticoid receptor genes in rat ovary. Biol Reprod. 1999;60(2):330–5.
doi:10.1095/biolreprod60.2.330.
142. Benediktsson R, Yau JLW, Brett LP, Cooke BE, Edwards CRW, Seckl JR. 11β-Hydroxysteroid
dehydrogenase in the rat ovary: high expression in the oocyte. J Endocrinol. 1992;135(1):53–58.
http://www.scopus.com/inward/record.url?eid=2-s2.0-0026757114&partnerID=tZOtx3y1.
143. McDonald SE, Henderson TA, Gomez-Sanchez CE, Critchley HOD, Mason JI. 11Beta-
hydroxysteroid dehydrogenases in human endometrium. Mol Cell Endocrinol. 2006;248
(1–2):72–8. doi:10.1016/j.mce.2005.12.010.
144. Michael AE, Evagelatou M, Norgate DP, et al. Isoforms of 11β-hydroxysteroid dehydroge-
nase in human granulosa-lutein cells. Mol Cell Endocrinol. 1997;132(1–2):43–52.
doi:10.1016/S0303-7207(97)00118-4.
145. Schreiber JR, Nakamura K, Erickson GF. Rat ovary glucocorticoid receptor: identification
and characterization. Steroids. 1982;39(5):569–84. doi:10.1016/0039-128X(82)90057-5.
146. Michael A, Cooke B. A working hypothesis for the regulation of steroidogenesis and germ
cell development in the gonads by glucocorticoids and 11β-hydroxysteroid dehydrogenase
(11βHSD). Mol Cell Endocrinol. 1994;100(1–2):55–63. doi:10.1016/0303-7207(94)
90279-8.
147. Albiston AL, Smith RE, Krozowski ZS. Changes in the levels of 11β-hydroxysteroid dehy-
drogenase mRNA over the oestrous cycle in the rat. J Steroid Biochem Mol Biol.
1995;52(1):45–8. doi:10.1016/0960-0760(94)00154-E.
148. Hillier SG. Molecular biology of the female reproductive system. Amsterdam: Elsevier;
1994. p. 1–37. doi:10.1016/B978-0-08-091819-8.50005-9.
149. Hillier S, Tetsuka M. An anti-inflammatory role for glucocorticoids in the ovaries? J Reprod
Immunol. 1998;39(1–2):21–7. doi:10.1016/S0165-0378(98)00011-4.
150. Michael AE, Pester LA, Curtis P, Shaw RW, Edwards CR, Cooke BA. Direct inhibition of
ovarian steroidogenesis by cortisol and the modulatory role of 11 beta-hydroxysteroid dehy-
drogenase. Clin Endocrinol (Oxf). 1993;38(6):641–4. http://www.ncbi.nlm.nih.gov/
pubmed/8334750. Accessed 29 May 2014.
151. Hsueh A. Glucocorticoid inhibition of FSH-induced estrogen production in cultured rat gran-
ulosa cells. Steroids. 1978;32(5):639–48. doi:10.1016/0039-128X(78)90074-0.
152. Schoonmaker JN, Erickson GF. Glucocorticoid modulation of follicle-stimulating hormone-
mediated granulosa cell differentiation. Endocrinology. 1983;113(4):1356–63. doi:10.1210/
endo-113-4-1356.
153. Yang J-G. Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertil-
izing capacity in vitro. Biol Reprod. 1999;60(4):929–36. doi:10.1095/biolreprod60.4.929.
154. Jimena P, Castilla JA, Peran F, et al. Adrenal hormones in human follicular fluid. Eur J
Endocrinol. 1992;127(5):403–6. doi:10.1530/acta.0.1270403.
155. González R, Ruiz-León Y, Gomendio M, Roldan ERS. The effect of glucocorticoids on ERK-
1/2 phosphorylation during maturation of lamb oocytes and their subsequent fertilization and
cleavage ability in vitro. Reprod Toxicol. 2010;29(2):198–205. doi:10.1016/j.
reprotox.2009.10.009.
156. Andersen CY. Effect of glucocorticoids on spontaneous and follicle-stimulating hormone
induced oocyte maturation in mouse oocytes during culture. J Steroid Biochem Mol Biol.
2003;85(2–5):423–7. doi:10.1016/S0960-0760(03)00190-0.
157. Liang B, Wei D-L, Cheng Y-N, et al. Restraint stress impairs oocyte developmental potential
in mice: role of CRH-induced apoptosis of ovarian cells. Biol Reprod. 2013;89(3):64.
doi:10.1095/biolreprod.113.110619.
158. Liu Y-X, Cheng Y-N, Miao Y-L, et al. Psychological stress on female mice diminishes the
developmental potential of oocytes: a study using the predatory stress model. PLoS One.
2012;7(10), e48083. doi:10.1371/journal.pone.0048083.
159. Rabin DS. Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the
uterine estradiol receptor. Biol Reprod. 1990;42(1):74–80. doi:10.1095/biolreprod42.1.74.
160. Bever AT, Hisaw FL, Velardo JT. Inhibitory action of desoxycorticosterone acetate, cortisone
acetate, and testosterone on uterine growth induced by estradiol-17beta. Endocrinology.
1956;59(2):165–9. doi:10.1210/endo-59-2-165.
161. Johnson DC, Dey SK. Role of Histamine in implantation: dexamethasone inhibits estradiol-
induced implantation in the rat. Biol Reprod. 1980;22(5):1136–1141. http://www.biolreprod.
org/content/22/5/1136.abstract. Accessed 26 May 2014.
162. Zhao L-H, Cui X-Z, Yuan H-J, et al. Restraint stress inhibits mouse implantation: temporal
window and the involvement of HB-EGF, estrogen and progesterone. PLoS One. 2013;8(11),
e80472. doi:10.1371/journal.pone.0080472.
163. Chien EJ, Liao C-F, Chang C-P, et al. The non-genomic effects on Na+/H+-exchange 1 by
progesterone and 20alpha-hydroxyprogesterone in human T cells. J Cell Physiol.
2007;211(2):544–50. doi:10.1002/jcp.20962.
164. Szekeres-Bartho J, Barakonyi A, Miko E, Polgar B, Palkovics T. The role of gamma/delta T
cells in the feto-maternal relationship. Semin Immunol. 2001;13(4):229–33. doi:10.1006/
smim.2000.0318.
165. Arck P, Hansen PJ, Mulac Jericevic B, Piccinni MP, Szekeres-Bartho J. Progesterone during
pregnancy: endocrine-immune cross talk in mammalian species and the role of stress. Am J
Reprod Immunol (New York, NY 1989). 2007;58(3):268–279. papers://cf7c60b8-94a1-4c79-
88e4-6c57345fd583/Paper/p1437.
166. Szekeres-Bartho J, Wegmann TG. A progesterone-dependent immunomodulatory protein
alters the Th1/Th2 balance. J Reprod Immunol. 1996;31(1–2):81–95. http://www.ncbi.nlm.
nih.gov/pubmed/8887124. Accessed 29 May 2014.
167. Polgar B, Kispal G, Lachmann M, et al. Molecular cloning and immunologic characterization
of a novel cDNA coding for progesterone-induced blocking factor. J Immunol.
2003;171(11):5956–63. http://www.ncbi.nlm.nih.gov/pubmed/14634107. Accessed 29 May
2014.
168. Wiebold JL, Stanfield PH, Becker WC, Hillers JK. The effect of restraint stress in early preg-
nancy in mice. Reproduction. 1986;78(1):185–92. doi:10.1530/jrf.0.0780185.
169. Joachim R, Zenclussen AC, Polgar B, et al. The progesterone derivative dydrogesterone abro-
gates murine stress-triggered abortion by inducing a Th2 biased local immune response.
Steroids. 2003;68(10–13):931–40. doi:10.1016/j.steroids.2003.08.010.
170. Blois SM, Joachim R, Kandil J, et al. Depletion of CD8+ cells abolishes the pregnancy
protective effect of progesterone substitution with dydrogesterone in mice by altering the
Th1/Th2 cytokine profile. J Immunol. 2004;172(10):5893–9. doi:10.4049/jimmunol.172.10.
5893.
171. Kalinka J, Szekeres-Bartho J. The impact of dydrogesterone supplementation on hormonal
profile and progesterone-induced blocking factor concentrations in women with threatened
abortion. Am J Reprod Immunol. 2005;53(4):166–71. doi:10.1111/j.1600-0897.2005.00261.x.
172. Raghupathy R, Al Mutawa E, Makhseed M, Azizieh F, Szekeres-Bartho J. Modulation of
cytokine production by dydrogesterone in lymphocytes from women with recurrent miscar-
riage. BJOG. 2005;112(8):1096–101. doi:10.1111/j.1471-0528.2005.00633.x.
Chapter 12
Glucocorticoids and the Lung
Anthony N. Gerber
Abstract The lung is a major clinical target of glucocorticoid-based therapeutics,

and GR signaling has broad effects on respiratory physiology and inflammation.
During lung development, expression of GR in the mesenchyme is required for
normal terminal alveolar epithelial differentiation. Prenatal administration of exog-
enous glucocorticoids (GCs) to prevent neonatal respiratory distress syndrome,
however, promotes alveolar maturation and accelerates surfactant expression in a
manner consistent with direct effects on the developing alveolar epithelium.
Likewise, cell autonomous effects of GCs in regulating gene expression and pheno-
type of the airway epithelium and airway smooth muscle have been demonstrated to
control important therapeutic effects of GCs in treating asthma and chronic obstruc-
tive pulmonary disease. Here, mechanisms and consequences of GR signaling in the
developing lung and in treating obstructive lung disease are reviewed, with a focus
on direct effects of GR signaling on alveolar differentiation, surfactant expression,
and airway epithelial and smooth muscle pathophysiology.
Keywords Glucocorticoid receptor • Surfactant • Asthma • Airway smooth muscle

• Airway epithelium • Feed-forward loop
Introduction: Glucocorticoids and the Lung
Glucocorticoids are used to treat a wide variety of lung disease, ranging from mater-
nal administration to prevent newborn respiratory distress syndrome of prematurity
[1], to their dominant role in treating inflammatory lung diseases, such as asthma, in
both children and adults [2]. A great deal of research has therefore focused on the
effects of exogenous glucocorticoids on various aspects of lung biology, and our
overall understanding of glucocorticoid signaling in the lung is largely informed by
in vitro and in vivo studies on the effects of supplemental, generally synthetic, GR
agonists on a subset of lung cell and tissue types, and pulmonary developmental
A.N. Gerber, M.D., Ph.D. (*)

Department of Medicine, National Jewish Health, University of Colorado, Denver,
1400 Jackson Street, Room K621b, Denver, CO 80206, USA

280 A.N. Gerber
processes. Reflecting clinical use, particular areas of focus have been aimed at
understanding how glucocorticoids regulate lung maturation and surfactant expres-
sion, and also how glucocorticoids exert therapeutic effects on airway inflamma-
tion, notably in airway epithelial and smooth muscle cells. In this chapter, I will
review the literature that has established a key role for supplemental glucocorticoids
in promoting airway maturation and surfactant expression, and I will provide an
overview of our understanding of the molecular mechanisms underpinning these
effects. I will also summarize recent loss of function studies that have indicated a
seemingly contradictory role for endogenous GR signaling in the developing lung.
In addition, I will review the use of glucocorticoids in the treatment of obstructive
lung disease, and summarize the large and growing body of literature exploring the
mechanisms responsible for therapeutic effects of glucocorticoids in airway inflam-
mation. Important areas for future research will also be discussed.
Glucocorticoids and Neonatal Respiratory Distress Syndrome
In 1969, Dr. Graham Liggins published a landmark paper entitled Premature

Delivery of Foetal Lambs Infused With Glucocorticoid [3]. In this study, Liggins
found that injection of pre-term pregnant ewes with dexamethasone resulted in the
delivery of viable premature lambs. Lungs from these animals were inflated after
birth, in contrast to a complete lack of aeration in control animals. This observation
led Liggins to speculate that glucocorticoid (GC) signaling promoted surfactant
expression, thus allowing postnatal maintenance of alveolar integrity. Based on this
hypothesis, Liggins and his colleague, Dr. Ross Howie, conducted a remarkable
randomized clinical trial in which betamethasone or control was infused antepartum
to 282 women with threatened premature labor or planned early delivery for medi-
cal reasons (e.g. pre-eclampsia). The primary endpoint of the trial was the occur-
rence of neonatal respiratory distress syndrome (RDS), which is a major complication
of preterm birth that has high associated morbidity and mortality. In infants born to
the subset of mothers with premature labor who were treated with betamethasone,
the rate of RDS was 9 %, while the rate of RDS in babies born to mothers with
premature labor that were in the control arm was 25.8 % (P < 0.03) [4]. The benefi-
cial effect was confined to babies of less than 32 weeks gestational age. This extraor-
dinary finding, which has been replicated in numerous subsequent studies [5], led to
the prevention of hundreds of thousand of potentially fatal cases of neonatal respira-
tory distress syndrome and transformed the standard of care for pre-term labor.
Glucocorticoid Signaling, Surfactogenesis, and Lung

Maturation
The potent clinical effect of maternal GC administration in reducing the incidence

of newborn RDS following premature birth has spawned substantial research into
the mechanisms responsible for the effects of GCs on lung maturation and function.
12 Glucocorticoids and the Lung 281
GR is highly expressed in the developing lung. Administration of exogenous GCs to

pregnant rats, rabbits and monkeys, among other animals, leads to accelerated tran-
sition through the pseudoglandular phase of lung development, in association with
premature terminal differentiation [6–8], precocious secretion of surfactant, and the
initiation of structural changes required for gas exchange, most notably alveolar
thinning [9–11]. These effects of GCs on terminal lung maturation in vivo are
reflected in vitro in both explants from developing lungs and in cultured cell models.
In human lung explants, glucocorticoids induce expression of surfactant B (SFTPB)
and surfactant C (SFTPB) mRNA, and have a complex biphasic effect on surfactant
A (SFTPA) expression [12–14]. In cultured Type II alveolar cells, exogenous GCs
induce the expression of numerous markers terminal differentiation of the respira-
tory epithelium [15], including TTF-1 (also known as NKX2-1), a transcription fac-
tor that is implicated in directly regulating surfactant C expression [16], and the
expression of other genes associated with mature alveolar epithelium. The effects of
GCs on isolated alveolar epithelial cells thus appear to recapitulate many of the
important effects of exogenous GCs in vivo, supporting an epithelial cell autono-
mous model for the effects of GCs on surfactogenesis in which ligand-activated GR
in the developing epithelium drives terminal differentiation.
Glucocorticoids and Surfactant Expression: Molecular

Mechanisms
A number of studies have addressed putative epithelial-autonomous mechanisms

through which GR signaling induces surfactant gene expression and promotes dif-
ferentiation. Similar to explants, which include both epithelial and mesenchymal
components, exposure of isolated cultured fetal alveolar type II cells to a differen-
tiation cocktail containing dexamethasone led to SFTPB and SFTPC mRNA levels
increasing over time [17]. In contrast, induction of SFTPA expression by treatment
with cAMP and IL1B was inhibited by GCs such as dexamethasone [18]; these
dichotomous effects are concordant with complex biphasic changes in SFTPA
mRNA levels that have been noted in association with GC treatment in explants [13,
19]. Repression of SFTPA by GCs occurs both through down-regulation of SFTPA
transcription and through post-transcriptional destabilization of SFTPA mRNA [18,
20, 21]. Work from Mendelson and colleagues, encompassed in several publica-
tions, indicates that direct repression of SFTPA transcription by GCs results from
agonist-bound GR interacting with a composite TTF-1/NF-kB regulatory element
upstream of the SFTPA start site [20, 22, 23]. This TTF binding element has been
implicated in the induction of SFTPA by both TTF-1 and NF-kB in association with
histone modifications indicative of active transcription. Activation of GR signaling
modified the histone acetylation pattern at the SFTPA locus to marks reflective of
transcriptional repression [18]. This suggests that antagonism of TTF-1 and NF-kB
activity at the SFTPA promoter by GR occurs through canonical trans-repression
mechanisms in which GR tethering to a TTF-1/NF-kB complex results in altered
co-regulator recruitment and assembly of a repressive complex [24, 25].
282 A.N. Gerber
Fig. 12.1 The structures of the Type 1 coherent and incoherent feed-forward loop (FFL) as indi-
cated. (a) In a coherent FFL, factor A induces factor B, and both factors exert the same effects on
expression of a third downstream gene, referred to here as target gene Z. (b) In an incoherent FFL,
the two factors exert opposing effects on target gene Z expression
The transrepression model for the effects of GR on SFTPA expression is, however,
complicated by data indicating that treatment of Type II cells with GCs results in
increased TTF-1 expression [26]. Thus, GR appears to exert an inductive effect on
SFTPA expression through promoting TTF-1 expression, while also directly antago-
nizing TTF-1 function at the SFTPA promoter. These opposing effects provide a
possible mechanistic basis for the complex biphasic expression response of SFTPA
to GCs. The pattern of seemingly antagonistic regulation of SFTPA expression by
GR is consistent with GR and TTF-1 comprising an incoherent feed-forward loop
that controls SFTPA expression. The general circuit design of coherent and incoher-
ent feed-forward loops are depicted in Fig. 12.1. Feed-forward loops are widely
utilized in bacteria and yeast to confer specific properties to downstream gene
expression, including delayed induction and biphasic expression patterns [27, 28].
Two recent studies have indicated that feed-forward logic may result in similar
expression patterns in response to GCs [29, 30], with incoherent feed-forward regu-
latory logic implicated as the mechanism underlying biphasic expression responses
of several genes to GCs in U2OS and A549 cells. A theoretical GR:TTF-1 incoher-
ent feed-forward loop controlling SFTPA expression is shown in Fig. 12.2. Whether
GR directly induces TTF-1, thus fulfilling the formal requirements for GR:TTF1
feed-forward control of SFTPA, remains to be determined.
Similar to SFTPA, SFTPB and SFTPC are both transcriptional targets of TTF-1
[31–33]. This suggests that the inductive effect of GCs on TTF-1 expression is at
least one mechanism through which GR signaling promotes SFTPB and SFTPC
expression in a cell-autonomous manner during alveolar epithelial differentiation. It
is likely, however, that GCs exert additional effects on SFTPB and SFTPC expres-
sion beyond a simple linear cascade in which GR induces TTF-1, with TTF-1 in turn
regulating SFTPB and SFTPC. In elegant work by Kolla et al, the effects of viral-
driven TTF-1 over-expression on various markers of differentiated alveolar epithe-
lium were assessed [17]. In this study, TTF-1 over-expression increased SFTPB and
SFTPC expression. However, expression of both SFTPB and SFTPC was further
increased when a dexamethasone containing cocktail was added to the culture
medium. The mechanisms underlying the additional induction of SFTPB and
SFTPC expression have yet to be established. One possibility is that GR, in addition
Fig. 12.2 Putative feed-forward regulation of SFTPA and SFTPB by GR and TTF-1. (a) In this
model, GR induces TTF-1 expression, which induces SFTPA. However, GR also negatively
regulates SFTPA expression, thus creating a feed-forward loop that may be responsible for biphasic
expression patterns of SFTPA. (b) In this case, GR induces TTF-1 and both GR and TTF-1 induce
SFTPB expression
to inducing TTF-1, also enhances the expression of a second transcriptional driver

of surfactant expression. An alternative, but not mutually exclusive possibility, is
that GR itself directly regulates SFTPB and SFTPC expression, possibly in coopera-
tion with TTF-1. ChIP-seq data from Reddy et al. suggest that this may be the case
[34]. In this study, A549 cells, an alveolar adenocarcinoma-derived cell line, were
treated with dexamethasone, and genome wide GR binding events were assayed
using chromatin immunoprecipitation (ChIP) followed by deep sequencing.
Identified binding sites were subsequently annotated in the public domain through
ENCODE and can be visualized on the UCSC genome browser [35]. Scanning of
the SFTPB and SFTPC loci in the UCSC genome browser revealed GR binding in
regions upstream of both the SFTPB and SFTPC promoters. These putative binding
sites have not been validated by ChIP-PCR, however, the original publication
showed that a high percentage of randomly selected GR binding sites identified by
sequencing could also be detected through standard PCR-based analysis [34]. A
second limitation of this study in the context of understanding surfactant regulation
is that the interactions between GR and the SFTPB and SFTPC loci were detected
in A549 cells, and thus may not reflect events in the immature respiratory epithe-
lium. In addition, GR binding to these areas has not been functionally implicated in
al regulation of SFTPB and SFTPC. Nevertheless, despite these caveats, the A549-
based GR binding data support a model in which GR and TTF1 regulate SFTPB and
SFTPC expression though a coherent feed-forward loop system (see Figs. 12.1 and
12.2b). Establishing whether GR:TTF1 coherent feed-forward loops regulate
SFTPB and SFTPC expression could be accomplished through temporal analysis of
genome wide binding of TTF1 and GR during alveolar epithelial cell differentia-
tion, along with functional characterization of any binding sites identified within the
SFTPB and SFTPC loci.
284 A.N. Gerber
Function of Endogenous GR in Lung Development
The data reviewed above strongly suggest that exogenous administration of gluco-
corticoids promotes surfactant expression and epithelial maturation through epithe-
lial cell-autonomous mechanisms. In further support of this notion, deletion of GR
in the lung epithelium during murine development reduced, but did not eliminate
surfactant expression [36], and also reduced viability. However, additional experi-
ments comparing systemic and mesenchymal restricted knockout of GR in mice
indicate that the dominant physiological effects of endogenous GR signaling during
lung development likely occur through mesenchymally expressed GR. Systemic
knockout of the murine GR resulted in highly penetrant perinatal lethality second-
ary to defective lung development. In particular, there was a marked reduction in
alveolar Type I cell number and surfactant expression, indicating a defect in termi-
nal alveolar differentiation [37]. In an initially surprising finding, given the impor-
tant effects of exogenous GCs on epithelial maturation, the pulmonary phenotype of
GR−/− mice was largely recapitulated with tissue-specific removal of GR from the
mesenchyme [38, 39]. For example, in one recent study, germline deletion of GR
was compared with conditional removal of GR in the endothelium, mesenchyme
and epithelium. Mice lacking GR in the mesenchyme were found to display lethal
effects similar to germline GR knockout, while the epithelial and endothelial KO
strains did not exhibit perinatal respiratory failure [40]. Although there was reduced
viability in a third publication that reported on epithelial deletion of GR [36], and
hyper-cellularity has been observed consistently with epithelial GR deletion, in
aggregate, the data indicate that normal lung development and surfactogenesis has
a greater requirement for GR activity in the mesenchyme than within the developing
airway epithelium. Thus, the effects of exogenous GCs, which can drive cell auton-
omous maturation of the epithelium, and the physiologic role of endogenous GR
signaling in murine lung development, which appears to predominantly depend on
GR activity in the mesenchyme, are somewhat divergent.
Efforts to explain this non-cell autonomous role of GR signaling on epithelial
development have focused primarily on well-known, obligate signaling between the
epithelial and mesenchyme layers that mediates branching morphogenesis and later
stages of lung development [41]. One model that is consistent with mesenchymal
GR regulating epithelial proliferation and differentiation is that GR induces a
secreted mesenchymal factor (or factors) that is required for normal alveolar
differentiation [42]. In support of this general notion, Sweezey and colleagues have
shown that LGL1 (also known as CRIPSDL2) is a mesenchymal target of GR that
signals to the epithelium [42]. Specifically, in rat lung, immunohistochemistry
revealed mesenchymal LGL1 expression and co-localization of LGL1 with markers
of the Golgi and ER, consistent with it functioning as a secreted protein. Moreover
epithelial cells were shown to import extracellular LGL1. Antisense inhibition of
LGL1 in developing rats resulted in decreased branching morphogenesis [43]. Thus,
LGL1, although unlikely to be the primary mesenchymal factor that drives the
effects of GR on the epithelium, exemplifies the notion that secreted GR-regulated
proteins in the mesenchyme can control epithelial development. Intriguingly, gluco-
corticoids induce the expression of LGL1 in Beas-2B airway epithelial cells [44].
This suggests that high doses of exogenous GCs may promote ectopic expression of
mesenchymal factors such as LGL1, thus driving cell-autonomous effects of GR
signaling on epithelial cells, as is observed in cultured systems. In that regard, it
would be interesting to determine whether prenatal administration of exogenous
GCs to fetal mice would reverse, at least partially, the severe basal phenotype asso-
ciated with mesenchymal GR deletion.
In summary, GR signaling is required for normal lung development and
treatment of prenatal mammals with exogenous GCs accelerates alveolar matura-
tion and surfactant expression. The latter effect is the basis for widespread and
effective use of GCs during threatened pre-term labor to prevent neonatal respira-
tory distress. However, the mechanisms underpinning the discordance between the
effects of exogenous GCs on the epithelium, and the requirement for mesenchymal
GR in mediating normal lung development, have yet to be fully elucidated. Future
mechanistic studies of GR action in the pulmonary mesenchyme, epithelium,
and representative cultured cells, using both loss of function and exogenous GC
administration, will undoubtedly further enhance our understanding of the molecu-
lar basis of the clinical effects of GCs on alveolar maturation and surfactogenesis.
Glucocorticoids and Obstructive Lung Disease
In addition to the use of GCs to prevent RDS of prematurity, GCs are used to treat a
spectrum of lung disease in children and adults, including autoimmune associated
interstitial lung disease [45], hypersensitivity pneumonitis [46], cryptogenic orga-
nizing pneumonia [47], eosinsophilic pneumonia [48], asthma [49], and chronic
obstructive pulmonary disease (COPD) [50]. Of these, the most extensive literature
involves the use of GCs in two of the most prevalent chronic diseases of the lung—
asthma and COPD. Moreover, therapeutic responses in these diseases are at least in
part due to direct effects of GCs on pulmonary structural cells, notably the airway
epithelium and airway smooth muscle, which are the primary tissue types found in
the conducting airways (see Fig. 12.3). Although tissue resident immune cells cer-
tainly contribute to airway pathology and corticosteroid effects [51], the role of GCs
in modulating immune cell function is covered elsewhere in this book. In the
remainder of this chapter, therefore, I will predominantly focus on mechanisms and
consequences of GR signaling in the airway epithelium and smooth muscle.
Introduction to Asthma and COPD: Similarities

and Differences
Asthma and COPD both fall within the broad category of obstructive lung disease
and there is considerable overlap in the symptoms associated with both diseases
[52], and to a lesser extent, their associated physiologic perturbations and
286 A.N. Gerber
Fig. 12.3 A schematic of airway structure and a partial list of therapeutically relevant targets of
glucocorticoid signaling in the airway epithelium and in airway smooth muscle
molecular pathology [53, 54]. Obstructive lung disease is defined as an abnormal-

ity in pulmonary air movement that limits exhalation; the primary symptom associ-
ated with obstructive lung disease is shortness of breath [55]. In asthma, airway
obstruction and symptoms are generally intermittent and are caused, in part, by
exuberant contraction of the smooth muscle layer that surrounds conducting air-
ways [56]. Alterations in the epithelium also occur in asthma, including excessive
production of mucus, which can also serve to limit airflow and to precipitate cough-
ing. Additionally, allergic inflammation is frequently, but not always, present in
asthmatic airways. In partial contrast, while airway obstruction in COPD often has
a reversible component in which the airway smooth muscle response to beta ago-
nists results in improved lung function, COPD also consistently features a compo-
nent of “fixed” airway obstruction that is essentially irreversible and is associated
with persistent shortness of breath. Fixed airway obstruction in COPD can result
from several distinct pathologic processes including airway thickening, excess
mucus production, and emphysematous destruction of the integrity of distal bron-
chioles and alveoli [53]. Although as we will review below, both diseases are
treated with inhaled GCs, the distinct pathophysiologic features of COPD and
asthma indicate that therapeutic responses to GCs likely involve both overlapping
and non-overlapping molecular actions of GR on the airway epithelium and airway
smooth muscle.
GCs in Asthma and COPD: Clinical Perspective
In the late 1940s and 1950s it was recognized that administration of cortisone, and
various derivative formulations, provides substantial clinical benefit to asthma
patients [57, 58]. In 1951, a paper in the New England Journal of Medicine from
Gelfand reported on the use of an aerosolized cortisone preparation to treat asthma
[59]; this intervention improved symptoms in a small cohort of patients and fore-
shadowed the eventual predominant role of inhaled corticosteroids (ICS) in asthma
control. By the early 1970s, with the development of more modern ICS formula-
tions, the promise of the pilot data from the Gelfand study was realized and expanded
on by numerous other investigators [60, 61]. For example, Lal et al. reported on an
inhaled preparation of beclomethasone that exhibited similar clinical efficacy to
systemic prednisolone [62]. Increasingly potent ICS formulations have led to
dramatically improved control of asthma symptoms, with the localized dosing
afforded through inhalation resulting in substantially fewer side effects and inci-
dence of adrenal suppression in comparison to systemic GCs [24, 63]. Consequently,
ICSs are now the standard of care for treating the majority of asthma patients [2].
Similarly, secondary to reducing the frequency of disease exacerbations, ICSs are
also frequently prescribed in COPD [50], whereas systemic use of GCs has been
associated with a number of debilitating morbidities.
In addition to these important clinical benefits of ICSs over systemic GCs, the
efficacy of ICS formulations in limiting asthma symptoms suggests that airway
structural cells, which receive the majority of GCs dosed through inhaled prepa-
rations, are likely crucial in mediating therapeutic effects of GCs. The primary
constituents of the airway are the airway epithelium and smooth muscle. A vari-
ety of studies indicate that both tissues harbor an active GR signaling apparatus
and that both tissues respond to inhaled steroids in human subjects at the tran-
scriptional level. Mechanisms and consequences of GCs on both tissue types are
reviewed below.
Effects of GCs on Airway Epithelial Cells
The airway contains an internal epithelial layer that serves as the conducting surface
through which air passes to and from the alveoli during the respiratory cycle
(Fig. 12.3). Although this surface contains a number of different cell types, includ-
ing goblet and club cells, few studies have discriminated between epithelial cell
subtype and GC effect, nor shall distinct effects of GCs on cell types within the
airway epithelium be addressed here. With that general caveat, beginning in the
early 1990s, a large number of studies have employed various airway epithelial
cell models to examine the effects of GCs on the expression of individual cytokines
and other factors implicated in asthma and/or COPD pathogenesis. For example, in
a representative publication, van de Stolpe and colleagues showed that ICAM
288 A.N. Gerber
expression was repressed by GCs in H292 cells [64], a cancer cell model of mucus
producing airway epithelium. Similarly a study by Levine and colleagues in 1993
established that corticosteroids could block the secretion of IL-6, IL-8 and G-CSF
from Beas2B cells [65], a standard model of cultured human airway epithelial cells.
Another study showed that TSLP, whose misregulation is implicated as a genetic
risk factor in asthma [66], is induced by dsRNA in normal human bronchial epithe-
lial cells; this induction is repressed by GCs. Several groups have also shown that
GCs reduce mucin gene expression, notably MUC5AC, both in cultured human air-
way epithelial cells and in ex vivo murine airway epithelial progenitor cells [67]. As
increased mucus is strongly implicated as driving symptoms in asthma and in
COPD, down-regulation of MUC5AC by GCs in the airway epithelium is thus
implicated as at least part of the underlying therapeutic effects of GCs in both dis-
eases. Taken together, these and other in vitro studies, as reviewed in greater detail
by Stellato [68], clearly establish that GCs modulate transcriptional programming
in a cell autonomous manner in airway epithelial cells, including exerting repressive
effects on cytokine expression and other pathways implicated in asthma and COPD
pathogenesis.
In Vivo Activity of GCs in the Airway Epithelium
These in vitro studies have been mirrored by various clinical studies in which
inhaled corticosteroids have been shown to reduce the expression of pro-
inflammatory cytokines [69] and other aspects of airway pathology in airway epi-
thelial cells in patients. For example in a study by Sousa et al. [69], inhaled
beclomethasone led to a significant decrease in the expression of GM-CSF and
IL-8 in the airway epithelium. Likewise RANTES expression was reduced in patients
after treatment with corticosteroids [70]. Thus, the clinical efficacy of GCs in treat-
ing asthma is associated with both in vivo and in vitro effects on airway epithelial
cells, establishing the airway epithelium as a steroid responsive tissue that mediates
important therapeutic effects of GCs in the lung.
More recent investigations have expanded on these studies through applying
genomics technology to study in vivo effects of glucocorticoids on airway epithelial
gene expression. One particularly well–designed study assessed airway gene
expression on a genome-wide basis in normal volunteers, smokers and asthmatics,
both before and after treatment with an ICS for 2 weeks [71]. Microarray-based
expression profiling of epithelial cells from airway brushings identified a set of
genes whose expression changed significantly after 1 week of inhaled fluticasone.
The investigators also found a set of genes whose expression levels differed in the
airway epithelium between asthmatics and healthy controls. The overlap of these
sets was comprised of three genes, periostin, Clca1, and serpinB2. Each of these
three genes exhibited elevated expression in asthmatic epithelium in comparison to
control, and expression of each gene was reduced after treatment with fluticasone.
Moreover, the decrease in serpinB2 expression caused by fluticasone was correlated
with treatment-associated improvements in lung function. The authors went on to

use cultured normal human bronchial epithelial cells to show that IL-13, a major
contributor to the Th2-dominant asthma phenotype, increased the expression of
periostin, Clca1, and serpinB2. Conversely, expression of these three genes was
repressed by dexamethasone. Taken together, this study, along with several other
studies, support a model in which Th2-dominant asthma responds to GC treatment,
at least in part through direct repression of specific IL-4/IL-13 targets in the airway
epithelium.
A recent study also applied microarray technology to examine the effects of fluti-
casone delivered in combination with a long acting beta agonist, salmeterol, on
airway gene expression in COPD [72]. They found a set of genes whose expression
is increased COPD that tended to return to normal expression levels with treatment;
conversely, a second set of genes exhibited decreased expression in COPD that nor-
malized after fluticasone/salmeterol therapy. Gene set enrichment analysis indicated
that epithelial barrier function genes were overrepresented amongst genes with
COPD specific expression patterns that normalized with fluticasone/salmeterol
treatment, suggesting that beneficial effects of GCs in COPD may occur through
non-inflammatory mechanisms. Moreover, these data, similar to data from the
asthma study of Woodruff et al. described previously above, support an epithelial
cell-autonomous mode of GC action contributing to therapeutic effects in COPD.
Molecular Mechanisms of GR Signaling

in the Airway Epithelium
The wide range of studies, which indicate that GCs exert beneficial effects in
obstructive airway disease through induction of GR signaling in the epithelium, has
led to a growing body of research focused on the molecular mechanisms underpin-
ning GR-mediated gene regulation in the airway epithelium. Although such work is
complicated by intricacies inherent to airway disease, including multiple pheno-
types, and environmental and infectious contributions to airway pathology, a num-
ber of underlying mechanistic principals governing GR activity in airway epithelial
cells have emerged. First, and similar to results from a range of non-airway cell
types and disease models, repression of inflammatory transcription factor activity,
exemplified by NF-kB, appears to be an important mechanism through which GCs
repress cytokine expression in the airway epithelium. This appears to occur through
a combination of direct effects of GR on NF-kB (or on other inflammatory tran-
scription factors, e.g. AP-1) activity, and through two-step processes in which GR
induces an inhibitor of inflammatory gene expression, such as DUSP1 [73].
Evidence for the first mode of repression (generally referred to as transrepression)
occurring specifically in normal airway epithelial cells (i.e. not tumor-derived cell)
is somewhat limited, with support garnered from studies that applied protein syn-
thesis and or transcriptional inhibitors to infer both direct and indirect repression of
cytokine transcription by ligand-activated GR [73, 74]. Corroborating these
290 A.N. Gerber
findings, repression of NF-kB activity in airway epithelial cells has been associated
with changes in histone acetylation to marks consistent with gene repression at
several loci [75]. For example, work from Rose and colleagues encompassed in
several publications established that GR directly represses MUC5AC expression
through recruiting HDAC2 [76]. Moreover, stimulation of MUC5AC expression by
IL-1b, which occurs through induction of NF-kB activity, is antagonized by dexa-
methasone [77], implicating GR-mediated recruitment of HDAC2 as a mechanism
through which NF-kB activity is repressed by GCs in the airway epithelium.
Despite these insights into GR-mediated repression of MUC5AC, and several
other studies of the molecular events involved in repression of NF-kB activity at
specific loci in airway epithelial cells [78], genome-wide analysis of GR cross-talk
with inflammatory transcriptional regulators implicated in airway disease, such as
NF-kB, in relationship to RNA Polymerase II occupancy and histone modification
has yet to be reported on in airway epithelial models. Such studies would dramati-
cally enhance our understanding of mechanisms through which GR represses cyto-
kine expression in the airway and would also serve as a foundation for studying the
mechanisms through which specific airway disease phenotypes and environmental
factors reduce the efficacy of GCs, which is a major clinical problem in asthma and
COPD [79]. In that regard, although a number of mechanisms through which GR
activity is potentially reduced in airway disease have been proposed [80, 81], with-
out a more detailed understanding of GR activity in specific disease-relevant mod-
els, e.g. mechanisms whereby GR reduces symptoms in IL-4/IL-13 dominant
asthma, it will be difficult to fully characterize resistance mechanisms, and to ratio-
nally improve GC-based therapies.
In addition to the direct effects of GR on NF-kB and other inflammatory tran-
scription factors, it is also clear the GR induces the expression of various factors that
negatively regulate inflammatory gene expression. For example, the induction of
DUSP1 by GR, which occurs through GR binding to several canonical GC response
elements within the DUSP1 locus [82], is strongly implicated as contributing to
repression of cytokine expression in airway epithelial cells by GCs [73]. Likewise,
we have recently shown that TNFAIP3, a potent negative feedback regulator of
NF-kB, is induced by ligand-activated GR [83]. Intriguingly, induction of TNFAIP3
by GR involves cooperation with NF-kB, indicating that crosstalk between GR and
inflammatory transcription factors is likely to be combinatorially complex and
result in divergent regulatory outcomes depending on the specific target gene. Yet a
third GR-induced gene that mediates inflammatory repression is ZFP36, which
decreases the stability of various cytokine mRNAs, thus implicating post-
transcriptional regulation as another important element of GR-mediated inflamma-
tory repression [84]. Thus, genes that are induced by GCs are implicated in
mediating a variety of effects on airway epithelial inflammatory gene expression,
and likely contribute to therapeutic effects.
In summary, agonist-induced GR directly regulates gene expression in the air-
way epithelium, which contributes to the effectiveness of GC-based therapies in
treating airway disease. A number of specific genes and mechanisms have been
identified as important in this process, with notable findings including profound
repressive effects of GCs on cytokine expression, a reduction in the expression of

specific IL-13 targets in TH2 predominant asthma, and an impact of GCs on airway
epithelial barrier function in COPD. Both the targets and mechanisms of GR action
in the airway remain under active investigation, with an expectation that increas-
ingly sophisticated genomics technology will greatly expand our understanding of
GR signaling in the airway epithelium.
GCs and Airway Smooth Muscle: Another GR-Responsive

Tissue Implicated in Mediating Therapeutic Effects
The other major tissue type that is targeted by glucocorticoid-based therapies in the
treatment of obstructive airway diseases is airway smooth muscle (ASM), which
forms the outside layer of the conducting airways (Fig. 12.3). ASM dysfunction,
including hyperresponsiveness to methacholine, a specific constrictor of ASM, is
one of the major pathophysiologic hallmarks of asthma [85], and ASM also exhibits
significant pathology in COPD [86]. In addition to hyperresponsiveness and exuber-
ant contractility, ASM can also show both hypertrophy and hyperplasia in asthma
[87, 88], which is believed to contribute to chronic airway thickening and fixed
airway obstruction [89]. Given these central roles for ASM pathology in obstructive
airway disease, a growing body of research has been focused on the potential that
GC signaling in ASM may result in therapeutic benefit.
Investigations of GC action in ASM have been enabled by the development of
techniques that allow for culturing ASM from explanted and post-mortem lungs
[90], including following fatal asthma attacks, and also from material obtained
with airway biopsies [91]. Culture cells derived from these sources have been
shown to have many characteristics that are associated with airway smooth in vivo.
These include response to various asthma associated mitogenic factors, expression
of smooth muscle contractile markers, and hypertrophy in response to stimuli such
as TGF beta, which is also implicated in hypertrophy in vivo [92–94]. With physi-
ological relevance of the cultured ASM cell model established, investigators have
gone on to study specific effects of GCs on ASM that may be responsible for limit-
ing inflammation and ASM dysfunction. Similar to analogous work on cultured
airway epithelial cells described above, in the pre-genomics era, a number of stud-
ies analyzed the effects of glucocorticoids on gene and protein expression to
establish that glucocorticoids suppress ASM cytokine expression [95]. This led to
the notion that ASM, in addition to serving a physiologic role in asthma pathol-
ogy, likely also serves an immunomodulatory role in which cytokines released
from ASM promote epithelial dysfunction [96]. Again, similar to epithelial cells,
mechanisms underlying the effects of GCs on ASM include trans-repression of
inflammatory transcription factor function by GR, with co-regulators such as
GRIP1 and IRF1 implicated in this process [97]. Induction of negative regulators
by GR also appears to be important for cytokine repression, with DUSP1 well
characterized as a mediator of IL6 repression by GCs in ASM; GC-mediated
292 A.N. Gerber
repression of CD38, which is implicated in regulating aberrant ASM contraction

in asthma and is the target of several inflammatory pathways [98, 99], also appears
to involve DUSP1 [100, 101].
The genomics era ushered in a number of genome-wide studies on the effects of
GCs on ASM that highlighted both a role for GR signaling in suppressing ASM-
mediated cytokine secretion, and also in modulating other aspects of ASM gene
expression, potentially including pathways associated with hypertrophy and hyper-
plasia. For example, studies by Hakonarson et al, Misior et al, and Masuno et al
showed robust changes in ASM gene expression caused by GC treatment [102–
104]; these effects were modulated by co-treatment with cytokines, growth factors,
PKA activation, and also depended on treatment time. A single study has reported
on in vivo effects of GCs on human ASM [105]; this work defined two genes
SYNPO2 and FAM129A, whose expression changed in ASM after systemic GC
treatment and that correlated with lung function. Additional studies are needed to
validate these findings and also to test whether genes such as RGS2, implicated in
animal and cell culture models as mediating therapeutic effects of GCs via modulat-
ing calcium signaling, are also regulated in patients by GCs.
Another area of intense investigation in ASM and the glucocorticoid response in
asthma has been cross-talk between GR and beta agonists [106]. Indeed these two
drugs are frequently packaged in combination, with beta agonists primarily impli-
cated as promoting ASM relaxation. However a variety of studies have suggested
that GCs and beta-agonists have synergistic effects on gene regulation in ASM
[107]. For example, RGS2 appears to be co-regulated by both agents [108].
Paradoxically, there is evidence that GCs may promote ASM proliferation in a beta-
agonist dependent fashion [103], potentially indicating that long term GC/beta-ago-
nist treatment may have deleterious effects in a subset of patients. The underlying
molecular control of transcriptional interactions between GC and beta-agonist sig-
naling remains incompletely understood and requires further investigation.
In closing, in addition to effects of GR on the developing lung and the airway
epithelium, GR signaling is also active in ASM where it appears to mediate thera-
peutic effects on ASM pathology in asthma and COPD through a range of mecha-
nisms. These include down-regulation of cytokine expression, and induction of
genes such as RGS2, which promote ASM relaxation. GR also cooperates with beta-
agonists at the transcriptional level to modulate ASM gene expression and pheno-
type. Additional in vivo determination of ASM targets of GR may aid in developing
better therapies to treat ASM pathology in obstructive airway disease.
References
1. Hallman M, Peltoniemi O, Kari MA. Enhancing functional maturity before preterm birth.
Neonatology. 2010;97(4):373–8.
2. Stoloff SW, Kelly HW. Updates on the use of inhaled corticosteroids in asthma. Curr Opin
Allergy Clin Immunol. 2011;11(4):337–44.
3. Liggins GC. Premature delivery of foetal lambs infused with glucocorticoids. J Endocrinol.
1969;45(4):515–23.
4. Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for preven-
tion of the respiratory distress syndrome in premature infants. Pediatrics. 1972;50(4):
515–25.
5. Brownfoot FC, Gagliardi DI, Bain E, Middleton P, Crowther CA. Different corticosteroids
and regimens for accelerating fetal lung maturation for women at risk of preterm birth.
Cochrane Database Syst Rev. 2013;(8):CD006764.
6. Oshika E, Liu S, Ung LP, et al. Glucocorticoid-induced effects on pattern formation and
epithelial cell differentiation in early embryonic rat lungs. Pediatr Res. 1998;43(3):305–14.
7. Mendelson CR, Gao E, Young PP, Michael LF, Alcorn JL. Transcriptional regulation of the
surfactant protein-A gene in fetal lung. Chest. 1997;111(6 Suppl):96S–104.
8. Vyas J, Kotecha S. Effects of antenatal and postnatal corticosteroids on the preterm lung.
Arch Dis Child Fetal Neonatal Ed. 1997;77(2):F147–50.
9. Mendelson CR. Role of transcription factors in fetal lung development and surfactant protein
gene expression. Annu Rev Physiol. 2000;62:875–915.
10. Blanco LN, Massaro GD, Massaro D. Alveolar dimensions and number: developmental and
hormonal regulation. Am J Physiol. 1989;257(4 Pt 1):L240–7.
11. Whitsett JA, Matsuzaki Y. Transcriptional regulation of perinatal lung maturation. Pediatr
Clin North Am. 2006;53(5):873–87, viii.
12. Venkatesh VC, Iannuzzi DM, Ertsey R, Ballard PL. Differential glucocorticoid regulation of
the pulmonary hydrophobic surfactant proteins SP-B and SP-C. Am J Respir Cell Mol Biol.
1993;8(2):222–8.
13. Boggaram V, Smith ME, Mendelson CR. Regulation of expression of the gene encoding the
major surfactant protein (SP-A) in human fetal lung in vitro. Disparate effects of glucocorti-
coids on transcription and on mRNA stability. J Biol Chem. 1989;264(19):11421–7.
14. Odom MJ, Snyder JM, Boggaram V, Mendelson CR. Glucocorticoid regulation of the major
surfactant associated protein (SP-A) and its messenger ribonucleic acid and of morphological
development of human fetal lung in vitro. Endocrinology. 1988;123(4):1712–20.
15. Gonzales LW, Guttentag SH, Wade KC, Postle AD, Ballard PL. Differentiation of human
pulmonary type II cells in vitro by glucocorticoid plus cAMP. Am J Physiol Lung Cell Mol
Physiol. 2002;283(5):L940–51.
16. Maeda Y, Hunter TC, Loudy DE, Dave V, Schreiber V, Whitsett JA. PARP-2 interacts with
TTF-1 and regulates expression of surfactant protein-B. J Biol Chem. 2006;281(14):
9600–6.
17. Kolla V, Gonzales LW, Gonzales J, et al. Thyroid transcription factor in differentiating type
II cells: regulation, isoforms, and target genes. Am J Respir Cell Mol Biol. 2007;36(2):
213–25.
18. Islam KN, Mendelson CR. Glucocorticoid/glucocorticoid receptor inhibition of surfactant
protein-A (SP-A) gene expression in lung type II cells is mediated by repressive changes in
histone modification at the SP-A promoter. Mol Endocrinol. 2008;22(3):585–96.
19. Boggaram V, Smith ME, Mendelson CR. Posttranscriptional regulation of surfactant protein-
A messenger RNA in human fetal lung in vitro by glucocorticoids. Mol Endocrinol.
1991;5(3):414–23.
20. Alcorn JL, Islam KN, Young PP, Mendelson CR. Glucocorticoid inhibition of SP-A gene
expression in lung type II cells is mediated via the TTF-1-binding element. Am J Physiol
Lung Cell Mol Physiol. 2004;286(4):L767–76.
21. Huang HW, Bi W, Jenkins GN, Alcorn JL. Glucocorticoid regulation of human pulmonary
surfactant protein-B mRNA stability involves the 3′-untranslated region. Am J Respir Cell
Mol Biol. 2008;38(4):473–82.
22. Liu D, Yi M, Smith M, Mendelson CR. TTF-1 response element is critical for temporal and
spatial regulation and necessary for hormonal regulation of human surfactant protein-A2 pro-
moter activity. Am J Physiol Lung Cell Mol Physiol. 2008;295(2):L264–71.
294 A.N. Gerber
23. Islam KN, Mendelson CR. Potential role of nuclear factor kappaB and reactive oxygen spe-
cies in cAMP and cytokine regulation of surfactant protein-A gene expression in lung type II
cells. Mol Endocrinol. 2002;16(6):1428–40.
24. Barnes PJ. Glucocorticosteroids: current and future directions. Br J Pharmacol. 2011;163(1):
29–43.
25. De Bosscher K, Van Craenenbroeck K, Meijer OC, Haegeman G. Selective transrepression
versus transactivation mechanisms by glucocorticoid receptor modulators in stress and
immune systems. Eur J Pharmacol. 2008;583(2–3):290–302.
26. Wade KC, Guttentag SH, Gonzales LW, et al. Gene induction during differentiation of human
pulmonary type II cells in vitro. Am J Respir Cell Mol Biol. 2006;34(6):727–37.
27. Kim D, Kwon YK, Cho KH. The biphasic behavior of incoherent feed-forward loops in bio-
molecular regulatory networks. Bioessays. 2008;30(11–12):1204–11.
28. Eichenberger P, Fujita M, Jensen ST, et al. The program of gene transcription for a single
differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol. 2004;2(10):e328.
29. Sasse SK, Mailloux CM, Barczak AJ, et al. The glucocorticoid receptor and KLF15 regulate
gene expression dynamics and integrate signals through feed-forward circuitry. Mol Cell
Biol. 2013;33(11):2104–15.
30. Chen SH, Masuno K, Cooper SB, Yamamoto KR. Incoherent feed-forward regulatory logic
underpinning glucocorticoid receptor action. Proc Natl Acad Sci U S A. 2013;110(5):
1964–9.
31. Yang MC, Guo Y, Liu CC, Weissler JC, Yang YS. The TTF-1/TAP26 complex differentially
modulates surfactant protein-B (SP-B) and -C (SP-C) promoters in lung cells. Biochem
Biophys Res Commun. 2006;344(2):484–90.
32. Park KS, Whitsett JA, Di Palma T, Hong JH, Yaffe MB, Zannini M. TAZ interacts with
TTF-1 and regulates expression of surfactant protein-C. J Biol Chem. 2004;279(17):
17384–90.
33. Margana RK, Boggaram V. Functional analysis of surfactant protein B (SP-B) promoter. Sp1,
Sp3, TTF-1, and HNF-3alpha transcription factors are necessary for lung cell-specific activa-
tion of SP-B gene transcription. J Biol Chem. 1997;272(5):3083–90.
35. ENCODE Project Consortium. A user’s guide to the encyclopedia of DNA elements
(ENCODE). PLoS Biol. 2011;9(4):e1001046.
36. Manwani N, Gagnon S, Post M, et al. Reduced viability of mice with lung epithelial-specific
knockout of glucocorticoid receptor. Am J Respir Cell Mol Biol. 2010;43(5):599–606.
37. Cole TJ, Solomon NM, Van Driel R, et al. Altered epithelial cell proportions in the fetal lung
of glucocorticoid receptor null mice. Am J Respir Cell Mol Biol. 2004;30(5):613–9.
38. Habermehl D, Parkitna JR, Kaden S, et al. Glucocorticoid activity during lung maturation is
essential in mesenchymal and less in alveolar epithelial cells. Mol Endocrinol. 2011;
25(8):1280–8.
39. Li A, Hardy R, Stoner S, Tuckermann J, Seibel M, Zhou H. Deletion of mesenchymal gluco-
corticoid receptor attenuates embryonic lung development and abdominal wall closure. PLoS
One. 2013;8(5):e63578.
40. Bird AD, Choo YL, Hooper SB, McDougall AR, Cole TJ. Mesenchymal glucocorticoid
receptor regulates the development of multiple cell layers of the mouse lung. Am J Respir
Cell Mol Biol. 2014;50(2):419–28.
41. Morrisey EE, Hogan BL. Preparing for the first breath: genetic and cellular mechanisms in
lung development. Dev Cell. 2010;18(1):8–23.
42. Oyewumi L, Kaplan F, Sweezey NB. Lgl1, a mesenchymal modulator of early lung branch-
ing morphogenesis, is a secreted glycoprotein imported by late gestation lung epithelial cells.
Biochem J. 2003;376(Pt 1):61–9.
43. Oyewumi L, Kaplan F, Gagnon S, Sweezey NB. Antisense oligodeoxynucleotides decrease
LGL1 mRNA and protein levels and inhibit branching morphogenesis in fetal rat lung. Am J
Respir Cell Mol Biol. 2003;28(2):232–40.
44. Greer S, Page CW, Joshi T, Yan D, Newton R, Giembycz MA. Concurrent agonism of ade-
nosine A2B and glucocorticoid receptors in human airway epithelial cells cooperatively
induces genes with anti-inflammatory potential: a novel approach to treat chronic obstructive
pulmonary disease. J Pharmacol Exp Ther. 2013;346(3):473–85.
45. Kim R, Meyer KC. Therapies for interstitial lung disease: past, present and future. Ther Adv
Respir Dis. 2008;2(5):319–38.
46. Selman M, Buendia-Roldan I. Immunopathology, diagnosis, and management of hypersensi-
tivity pneumonitis. Semin Respir Crit Care Med. 2012;33(5):543–54.
47. Epler GR. Bronchiolitis obliterans organizing pneumonia, 25 years: a variety of causes, but
what are the treatment options? Expert Rev Respir Med. 2011;5(3):353–61.
48. Rhee CK, Min KH, Yim NY, et al. Clinical characteristics and corticosteroid treatment of
acute eosinophilic pneumonia. Eur Respir J. 2013;41(2):402–9.
49. Calhoun WJ, Ameredes BT, King TS, et al. Comparison of physician-, biomarker-, and
symptom-based strategies for adjustment of inhaled corticosteroid therapy in adults with
asthma: the BASALT randomized controlled trial. JAMA. 2012;308(10):987–97.
50. Calverley PM, Anderson JA, Celli B, et al. Salmeterol and fluticasone propionate and sur-
vival in chronic obstructive pulmonary disease. N Engl J Med. 2007;356(8):775–89.
51. Wang JH, Trigg CJ, Devalia JL, Jordan S, Davies RJ. Effect of inhaled beclomethasone dipro-
pionate on expression of proinflammatory cytokines and activated eosinophils in the bron-
chial epithelium of patients with mild asthma. J Allergy Clin Immunol. 1994;94(6 Pt
1):1025–34.
52. Mosenifar Z. Differentiating COPD from asthma in clinical practice. Postgrad Med.
2009;121(3):105–12.
53. Carolan BJ, Sutherland ER. Clinical phenotypes of chronic obstructive pulmonary disease
and asthma: recent advances. J Allergy Clin Immunol. 2013;131(3):627–34, quiz 635.
54. Postma DS, Reddel HK, ten Hacken NH, van den Berge M. Asthma and chronic obstructive
pulmonary disease: similarities and differences. Clin Chest Med. 2014;35(1):143–56.
55. Scano G, Stendardi L. Dyspnea and asthma. Curr Opin Pulm Med. 2006;12(1):18–22.
56. Hershenson MB, Brown M, Camoretti-Mercado B, Solway J. Airway smooth muscle in
asthma. Annu Rev Pathol. 2008;3:523–55.
57. Randolph TG, Rollins JP. The effect of cortisone on bronchial asthma. J Allergy.
1950;21(4):288–95.
58. Friedlaender S, Friedlaender AS. Effect of cortisone administered orally in bronchial asthma.
J Am Med Assoc. 1951;146(15):1381–2.
59. Gelfand ML. Administration of cortisone by the aerosol method in the treatment of bronchial
asthma. N Engl J Med. 1951;245(8):293–4.
60. Clark TJ. Effect of beclomethasone dipropionate delivered by aerosol in patients with asthma.
Lancet. 1972;1(7765):1361–4.
61. Brown HM, Storey G, George WH. Beclomethasone dipropionate: a new steroid aerosol for
the treatment of allergic asthma. Br Med J. 1972;1(5800):585–90.
62. Lal S, Harris DM, Bhalla KK, Singhal SN, Butler AG. Comparison of beclomethasone dipro-
pionate aerosol and prednisolone in reversible airways obstruction. Br Med
J. 1972;3(5822):314–7.
63. Inhaled corticosteroids compared with oral prednisone in patients starting long-term cortico-
steroid therapy for asthma. A controlled trial by the British Thoracic and Tuberculosis
Association. Lancet. 1975;2(7933):469–73.
64. van de Stolpe A, Caldenhoven E, Raaijmakers JA, van der Saag PT, Koenderman
L. Glucocorticoid-mediated repression of intercellular adhesion molecule-1 expression in
human monocytic and bronchial epithelial cell lines. Am J Respir Cell Mol Biol.
1993;8(3):340–7.
65. Levine SJ, Larivee P, Logun C, Angus CW, Shelhamer JH. Corticosteroids differentially
regulate secretion of IL-6, IL-8, and G-CSF by a human bronchial epithelial cell line. Am J
Physiol. 1993;265(4 Pt 1):L360–8.
296 A.N. Gerber
66. Kato A, Favoreto Jr S, Avila PC, Schleimer RP. TLR3- and Th2 cytokine-dependent produc-
tion of thymic stromal lymphopoietin in human airway epithelial cells. J Immunol.
2007;179(2):1080–7.
67. Chen H, Sun X, Chi R, et al. Glucocorticoid dexamethasone regulates the differentiation of
mouse conducting airway epithelial progenitor cells. Steroids. 2014;80:44–50.
68. Stellato C. Glucocorticoid actions on airway epithelial responses in immunity: functional
outcomes and molecular targets. J Allergy Clin Immunol. 2007;120(6):1247–63. quiz
1264–5.
69. Sousa AR, Poston RN, Lane SJ, Nakhosteen JA, Lee TH. Detection of GM-CSF in asthmatic
bronchial epithelium and decrease by inhaled corticosteroids. Am Rev Respir Dis. 1993;147(6
Pt 1):1557–61.
70. Wang JH, Devalia JL, Xia C, Sapsford RJ, Davies RJ. Expression of RANTES by human
bronchial epithelial cells in vitro and in vivo and the effect of corticosteroids. Am J Respir
Cell Mol Biol. 1996;14(1):27–35.
71. Woodruff PG, Boushey HA, Dolganov GM, et al. Genome-wide profiling identifies epithelial
cell genes associated with asthma and with treatment response to corticosteroids. Proc Natl
Acad Sci U S A. 2007;104(40):15858–63.
72. van den Berge M, Steiling K, Timens W, et al. Airway gene expression in COPD is dynamic
with inhaled corticosteroid treatment and reflects biological pathways associated with disease
activity. Thorax. 2014;69(1):14–23.
73. King EM, Holden NS, Gong W, Rider CF, Newton R. Inhibition of NF-kappaB-dependent
transcription by MKP-1: transcriptional repression by glucocorticoids occurring via p38
MAPK. J Biol Chem. 2009;284(39):26803–15.
74. Newton R, King EM, Gong W, et al. Glucocorticoids inhibit IL-1beta-induced GM-CSF
expression at multiple levels: roles for the ERK pathway and repression by MKP-1. Biochem
J. 2010;427(1):113–24.
75. Kim SH, Kim DH, Lavender P, et al. Repression of TNF-alpha-induced IL-8 expression by
the glucocorticoid receptor-beta involves inhibition of histone H4 acetylation. Exp Mol Med.
2009;41(5):297–306.
76. Chen Y, Watson AM, Williamson CD, et al. Glucocorticoid receptor and histone deacetylase-
2 mediate dexamethasone-induced repression of MUC5AC gene expression. Am J Respir
Cell Mol Biol. 2012;47(5):637–44.
77. Chen Y, Garvin LM, Nickola TJ, Watson AM, Colberg-Poley AM, Rose MC. IL-1beta induc-
tion of MUC5AC gene expression is mediated by CREB and NF-kappaB and repressed by
dexamethasone. Am J Physiol Lung Cell Mol Physiol. 2014;306(8):L797–807.
78. Matsukura S, Kokubu F, Kurokawa M, et al. Molecular mechanisms of repression of eotaxin
expression with fluticasone propionate in airway epithelial cells. Int Arch Allergy Immunol.
2004;134 Suppl 1:12–20.
79. Barnes PJ. Corticosteroid resistance in patients with asthma and chronic obstructive pulmo-
nary disease. J Allergy Clin Immunol. 2013;131(3):636–45.
80. Papi A, Contoli M, Adcock IM, et al. Rhinovirus infection causes steroid resistance in airway
epithelium through nuclear factor kappaB and c-Jun N-terminal kinase activation. J Allergy
Clin Immunol. 2013;132(5):1075–85.e6.
81. Tliba O, Cidlowski JA, Amrani Y. CD38 expression is insensitive to steroid action in cells
treated with tumor necrosis factor-alpha and interferon-gamma by a mechanism involving the
up-regulation of the glucocorticoid receptor beta isoform. Mol Pharmacol. 2006;69(2):
588–96.
82. Shipp LE, Lee JV, Yu CY, et al. Transcriptional regulation of human dual specificity protein
phosphatase 1 (DUSP1) gene by glucocorticoids. PLoS One. 2010;5(10):e13754.
83. Altonsy MO, Sasse SK, Phang TL, Gerber AN. Context-dependent cooperation between
nuclear factor kappaB (NF-kappaB) and the glucocorticoid receptor at a TNFAIP3 intronic
enhancer: a mechanism to maintain negative feedback control of inflammation. J Biol Chem.
2014;289(12):8231–9.
84. Fan J, Ishmael FT, Fang X, et al. Chemokine transcripts as targets of the RNA-binding protein
HuR in human airway epithelium. J Immunol. 2011;186(4):2482–94.
85. Prakash YS. Airway smooth muscle in airway reactivity and remodeling: what have we
learned? Am J Physiol Lung Cell Mol Physiol. 2013;305(12):L912–33.
86. Mauad T, Dolhnikoff M. Pathologic similarities and differences between asthma and chronic
obstructive pulmonary disease. Curr Opin Pulm Med. 2008;14(1):31–8.
87. Woodruff PG, Fahy JV. Airway remodeling in asthma. Semin Respir Crit Care Med.
2002;23(4):361–7.
88. Woodruff PG, Dolganov GM, Ferrando RE, et al. Hyperplasia of smooth muscle in mild to
moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med.
2004;169(9):1001–6.
89. Tliba O, Panettieri Jr RA. Noncontractile functions of airway smooth muscle cells in asthma.
Annu Rev Physiol. 2009;71:509–35.
90. Panettieri RA, Murray RK, DePalo LR, Yadvish PA, Kotlikoff MI. A human airway smooth
muscle cell line that retains physiological responsiveness. Am J Physiol. 1989;256(2 Pt
1):C329–35.
91. Chang PJ, Bhavsar PK, Michaeloudes C, Khorasani N, Chung KF. Corticosteroid insensitiv-
ity of chemokine expression in airway smooth muscle of patients with severe asthma. J
Allergy Clin Immunol. 2012;130(4):877–85.e5.
92. Zhou L, Hershenson MB. Mitogenic signaling pathways in airway smooth muscle. Respir
Physiol Neurobiol. 2003;137(2-3):295–308.
93. Bentley JK, Hershenson MB. Airway smooth muscle growth in asthma: proliferation, hyper-
trophy, and migration. Proc Am Thorac Soc. 2008;5(1):89–96.
94. Ma L, Brown M, Kogut P, et al. Akt activation induces hypertrophy without contractile phe-
notypic maturation in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2011;
300(5):L701–9.
95. Ammit AJ, Lazaar AL, Irani C, et al. Tumor necrosis factor-alpha-induced secretion of
RANTES and interleukin-6 from human airway smooth muscle cells: modulation by gluco-
corticoids and beta-agonists. Am J Respir Cell Mol Biol. 2002;26(4):465–74.
96. Damera G, Tliba O, Panettieri Jr RA. Airway smooth muscle as an immunomodulatory cell.
Pulm Pharmacol Ther. 2009;22(5):353–9.
97. Bhandare R, Damera G, Banerjee A, et al. Glucocorticoid receptor interacting protein-1
restores glucocorticoid responsiveness in steroid-resistant airway structural cells. Am J
Respir Cell Mol Biol. 2010;42(1):9–15.
98. Tirumurugaan KG, Kang BN, Panettieri RA, Foster DN, Walseth TF, Kannan MS. Regulation
of the cd38 promoter in human airway smooth muscle cells by TNF-alpha and dexametha-
sone. Respir Res. 2008;9:26.
99. Abcejo AJ, Sathish V, Smelter DF, et al. Brain-derived neurotrophic factor enhances calcium
regulatory mechanisms in human airway smooth muscle. PLoS One. 2012;7(8):e44343.
100. Kang BN, Jude JA, Panettieri Jr RA, Walseth TF, Kannan MS. Glucocorticoid regulation of
CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase
1. Am J Physiol Lung Cell Mol Physiol. 2008;295(1):L186–93.
101. Jude JA, Panettieri Jr RA, Walseth TF, Kannan MS. TNF-alpha regulation of CD38 expres-
sion in human airway smooth muscle: role of MAP kinases and NF-kappaB. Adv Exp Med
Biol. 2011;691:449–59.
102. Hakonarson H, Halapi E, Whelan R, Gulcher J, Stefansson K, Grunstein MM. Association
between IL-1beta/TNF-alpha-induced glucocorticoid-sensitive changes in multiple gene
expression and altered responsiveness in airway smooth muscle. Am J Respir Cell Mol Biol.
2001;25(6):761–71.
103. Misior AM, Deshpande DA, Loza MJ, Pascual RM, Hipp JD, Penn RB. Glucocorticoid- and
protein kinase A-dependent transcriptome regulation in airway smooth muscle. Am J Respir
Cell Mol Biol. 2009;41(1):24–39.
104. Masuno K, Haldar SM, Jeyaraj D, et al. Expression profiling identifies Klf15 as a glucocorti-
coid target that regulates airway hyperresponsiveness. Am J Respir Cell Mol Biol.
2011;45(3):642–9.
298 A.N. Gerber
105. Yick CY, Zwinderman AH, Kunst PW, et al. Glucocorticoid-induced changes in gene expres-
sion of airway smooth muscle in patients with asthma. Am J Respir Crit Care Med.
2013;187(10):1076–84.
106. Kaur M, Chivers JE, Giembycz MA, Newton R. Long-acting beta2-adrenoceptor agonists
synergistically enhance glucocorticoid-dependent transcription in human airway epithelial
and smooth muscle cells. Mol Pharmacol. 2008;73(1):203–14.
107. Manetsch M, Rahman MM, Patel BS, et al. Long-acting beta2-agonists increase fluticasone
propionate-induced mitogen-activated protein kinase phosphatase 1 (MKP-1) in airway
smooth muscle cells. PLoS One. 2013;8(3):e59635.
108. Holden NS, Bell MJ, Rider CF, et al. Beta2-adrenoceptor agonist-induced RGS2 expression
is a genomic mechanism of bronchoprotection that is enhanced by glucocorticoids. Proc Natl
Acad Sci U S A. 2011;108(49):19713–8.
Chapter 13
Glucocorticoids and the Cardiovascular
System
Julie E. Goodwin
Abstract Glucocorticoids affect the developing and mature cardiovascular system

in profound and, at times, contradictory ways. The glucocorticoid receptor is ubiq-
uitous in most cell types and conserved across species, highlighting its importance
in development and homeostasis. Despite the fact that the glucocorticoid receptor is
widely expressed, tissue-specific effects of glucocorticoids may have pronounced
effects on whole organism phenotypes. Here we will review the interactions between
glucocorticoids and the cardiovascular system.
Keywords Glucocorticoids • Hypertension • Vascular smooth muscle • Endothelium

• Dexamethasone • Nitric oxide • Cardiomyocyte • Nephron • Stent
Introduction
Glucocorticoids affect the developing and mature cardiovascular system in profound

and, at times, contradictory ways. The glucocorticoid receptor is ubiquitous in most
cell types and conserved across species, highlighting its importance in development
and homeostasis. Despite the fact that the glucocorticoid receptor is widely
expressed, tissue-specific effects of glucocorticoids may have pronounced effects
on whole organism phenotypes. Here we will review the interactions between
glucocorticoids and the cardiovascular system.
J.E. Goodwin, M.D. (*)

Department of Pediatrics, Yale University School of Medicine,
333 Cedar Street, PO Box 208064, New Haven, CT 06520-8064, USA

300 J.E. Goodwin
Blood Pressure Regulation
Excess glucocorticoid, whether endogenous as in Cushing’s syndrome, or exoge-

nous, via pharmacologic administration of glucocorticoids, induces hypertension.
Approximately 80 % of patients with Cushing’s syndrome have hypertension,
while 20 % of patients receiving chronic pharmacologic treatment with glucocor-
ticoids have hypertension [1]. In the general population, hypertension increases
the risk for cardiovascular disease with a doubling in mortality from ischemic
heart disease and stroke for every 20 mmHg systolic or 10 mmHg diastolic
increase in blood pressure [2]. This likely contributes to increased mortality;
patients with Cushing’s disease have a fivefold increased standard mortality ratio
for vascular disease [3].
Given the widespread usage of glucocorticoids in the treatment of a variety of
diseases the significance of glucocorticoid-induced hypertension is clear. Despite
this, however, the mechanisms underlying this association are unclear. This form
of hypertension has traditionally been believed to be mediated through promiscu-
ous activation of the mineralocorticoid receptor by excess glucocorticoid [2, 4].
Cortisol is able to bind the mineralocorticoid receptor in the kidney, but is nor-
mally prevented by local inactivation of glucocorticoids in the kidney by the
enzyme 11-b-HSD2. In cases of endogenous hypercortisolism, the levels of cortisol
can overcome inhibition by 11-b-HSD to activate the mineralocorticoid receptor
resulting in hypertension and hypokalemia. However, increasing evidence argues
against this being the only, or perhaps even the most significant, factor. The hyper-
tensive response to glucocorticoids in patients suffering from Addisonian shock
occurs much too rapidly to be explained by renal mechanisms alone and suggests
a crucial role of vascular tissues in acute blood pressure responses [5]. More signifi-
cantly, however, many lines of evidence indicate that glucocorticoids are capable of
inducing sustained elevations of blood pressure in both humans and in animal mod-
els via effects entirely independent of the mineralocorticoid receptor. For example,
budesonide, a potent glucocorticoid with essentially no activity at the mineralocor-
ticoid receptor, produces sustained hypertension in humans [6], while RU486, an
anti-glucocorticoid agent without anti-mineralocorticoid effects, reduces blood
pressure in both humans with Cushing’s syndrome [7–9] as well as in rat models of
glucocorticoid-induced hypertension [10, 11]. Similarly, spironolactone does not
inhibit glucocorticoid-mediated hypertension in humans [12] or in rat models [13, 14]
despite blocking salt and water retention.
In fact, in vivo studies consistently demonstrate that glucocorticoids acutely
increase renal sodium excretion in adrenalectomized rats [15–18], although
sustained effects of glucocorticoids on renal sodium handling are more challenging
to assess.
13 Glucocorticoids and the Cardiovascular System 301
Glucocorticoids and the Kidney
The glucocorticoid receptor is widely expressed in the kidney, and yet, its role there
is poorly understood. While glucocorticoid receptor mRNA has been identified in
most cells of the (rat and rabbit) nephron, it is most abundant in the glomerulus,
proximal tubule, and thick ascending limb segments [19]. Conversely, immunoreactive
(human) glucocorticoid receptor has been demonstrated by light microscopy pri-
marily in the distal convoluted tubules and collecting ducts, with moderate detection
in the glomerulus, and a faint presence in the proximal tubule. Within the glomeru-
lus, light microscopy and electron microscopy identified the glucocorticoid receptor
in all cell types, including visceral epithelial cells, parietal epithelial cells, mesan-
gial cells, and endothelial cells [20].
Despite its wide expression within the kidney, the role of glucocorticoids within
any cell of the nephron is not clear, as data on functional effects of glucocorticoids
in the kidney have been contradictory [19]. Glucocorticoids have been ascribed a
number of roles within the kidney via in vitro studies, including a role in increasing
glomerular filtration rate [21], regulation of renal ammoniagenesis [22], gluconeo-
genesis [23], sodium-hydrogen ion exchange [24, 25], and sodium-phosphate
cotransport [24, 26]. Micropuncture studies suggest that dexamethasone increases
the rate of sodium transport in the loop of Henle [27], as well as Na-K-ATPase
activity in the medullary thick ascending limb [28], but in situ microperfusion
showed no effect of glucocorticoid hormones on loop sodium transport [29]. In the
distal nephron, there is increasing evidence that glucocorticoids serve to regulate
the sodium reabsorption machinery. Dexamethasone increases mRNA abundance of
a number of genes central to renal sodium reabsorption, including SGK1, α-ENaC,
and GILZ [17]. RU28362, a pure glucocorticoid agonist devoid of mineralocorti-
coid effects, induces mineralocorticoid like effects in cultured collecting duct cells;
this activity is blocked by glucocorticoid but not mineralocorticoid antagonists [30].
Finally, dexamethasone induces a kidney-specific WNK1 isoform in cultured cells
[31] while decreasing expression of WNK4 as well [32]; elevated expression of
WNK1 and decreased activity of WNK4 have both been associated with a heredi-
tary form of human hypertension and hyperkalemia via up regulation of thiazide
sensitive cotransporter activity [33–35]. It should be noted, of course, that this
potential activation of the distal nephron glucocorticoid receptor by glucocorticoids,
as with activation of the mineralocorticoid receptor, would require glucocorticoids
to evade 11β-HSD2 machinery metabolizing glucocorticoids, at least in aldosterone-
sensitive epithelia.
Although the sum of these in vitro observations would seem to point to an anti-
natriuretic role of glucocorticoids in the distal nephron, studies performed in live
animals have consistently demonstrated that glucocorticoids induce natriuresis in
conjunction with a more prominent kaliuresis [16, 17]. As such, the contribution of
302 J.E. Goodwin
glucocorticoids to renal sodium handling has remained difficult to comprehend.

We believe glucocorticoid infusion acutely increases blood pressure in these rats via
vascular mechanisms, as noted above, thus increasing renal perfusion and triggering
a pressure natriuresis independent of any renal effects of glucocorticoids.
Investigation of the role of the glucocorticoid receptor in the distal nephron has
been accomplished via a mouse model yielding a tissue-specific deletion of the
glucocorticoid receptor, using the Ksp-cadherin Cre model. Ksp-Cre mice express
Cre under the control of the cadherin 16 promoter which is active in the renal tubules
and collecting ducts. The only extrarenal site is the Mullerian and Wolffian ducts.
Though the initial prediction was that the mice lacking the glucocorticoid receptor
in the distal nephron would become acutely hypertensive but would be unable to
sustain this elevated blood pressure chronically, in vivo studies have shown that
these mutant mice develop the same degree of acute and chronic hypertension as
controls when treated with dexamethasone [36]. These mice also demonstrated sim-
ilar nephron number and aldosterone levels as controls though they had a small but
significant elevation in mean blood pressure (7 mmHg) at baseline. Since the exci-
sion of the glucocorticoid receptor using the Cre-lox system is not 100 % efficient
the authors could not exclude the possibility that only a small percentage of existing
glucocorticoid receptor need be present to perpetuate steroid-mediated hyperten-
sion, nor could they rule out the possibility that proximal up regulation of the glu-
cocorticoid receptor was playing a role.
Glucocorticoids and Vascular Smooth Muscle
The rapidity with which glucocorticoids induce a rise in blood pressure is inconsis-
tent with glucocorticoids inducing hypertension via augmentation of renal sodium
reabsorption and suggests, perhaps, a profound effect of glucocorticoids on vascular
tone. Perhaps the most concentrated efforts to date have been focused on trying to
understand the vascular effects of glucocorticoids and how they may contribute to
clinically observed hypertension. There is evidence that the glucocorticoid receptor
is present both in vascular smooth muscle [37–39] and in the vascular endothelium
[38, 40, 41]. The majority of studies that have investigated glucocorticoid activity at
either of these sites have employed in vitro models.
Studies in smooth muscle cell culture have observed an up regulation in angio-
tensin II type I (AT I) receptors induced by glucocorticoids, which are hypothesized,
to result in alterations in blood pressure [42, 43]. Other in vitro studies using vascu-
lar smooth muscle cells have indicated that the glucocorticoid receptor as well as
the mineralocorticoid receptor may mediate the effect of glucocorticoids on the
influx of Na+ and/or Ca2+ into cells [38, 44]. Molnar et al. previously demonstrated
that corticosterone-induced signaling pathways in rat aortic vascular smooth muscle
cells involved phosphorylation of the mitogen-activated protein kinases, Src and Akt
which is believed to be mediated through the classical mineralocorticoid receptor. In the
same study, the authors showed that corticosterone enhanced phenylephrine-induced
vasoconstriction in isolated aortic rings in a dose-dependent manner, and that this

effect was blocked by pre-treatment of the rings with L-NAME or indomethacin,
suggesting that the effect of corticosterone is endothelial-dependent. Pre-incubation
with RU486, a glucocorticoid receptor blocker lessened the observed corticoste-
rone-dependent effects, while pre-incubation with spironolactone, a mineralocorti-
coid blocker, did not. Ultimately, the authors suggest that glucocorticoids may
activate signaling of the mineralocorticoid receptor in non-epithelial tissues result-
ing in the formation of a protein complex that may also include the glucocorticoid
receptor, which then transmits the signal [45].
A recent study in rats by Ong et al. is one of the only attempts in vivo to try to
dissect the underlying mechanism of hypertension by examining regional blood
flow [46]. In this study, central and regional blow flows of dexamethasone-treated
rats were compared to those of rats treated with minoxidil and dexamethasone,
saline alone and minoxidil alone. The authors were able to measure cardiac output
and regional blood flow directly by placing a perivascular probe around the aorta,
renal artery, superior mesenteric artery and common iliac artery of anesthetized
animals. Dexamethasone increased blood pressure in association with an increase in
total peripheral resistance. Interestingly, there was no detectable increase in the
peripheral resistance in any of the regional vascular beds assessed. The authors
speculated that this discrepancy could be explained by the possibility that the
increase in total peripheral resistance might be a reflection of a cumulative increase
in many different peripheral vascular beds, only a few of which were measured in
this study. Furthermore, they acknowledged that their technique may not be sensi-
tive enough to measure small changes in resistance in individual vascular beds. In
the group treated with both minoxidil and dexamethasone, the authors show that
these rats have significantly lower total peripheral resistance and significantly higher
cardiac output than the rats treated with dexamethasone alone, though there is no
significant decrease in mean arterial pressure. Since the increase in total peripheral
resistance was prevented while the increase in blood pressure was not, the authors
suggest that increased total peripheral resistance is not necessary for the develop-
ment of glucocorticoid-induced hypertension. They speculate that changes in car-
diac output and specifically stroke volume and resulting plasma expansion may
have offset the vasodilator effects of minoxidil though there were no observed dif-
ferences in hematocrit between the dexamethasone only treated rats and the minoxi-
dil and dexamethasone treated rats in this study [46].
A second study has used advances in mouse genetics to enable a more targeted
in vivo approach. In this study, a mouse model with a tissue-specific deletion of the
glucocorticoid receptor in the vascular smooth muscle tissues via the use of SM22-
Cre was characterized. While knockout and control mice had similar baseline blood
pressures with normal diurnal variation, knockout animals demonstrated a statistically
significant attenuation of the acute hypertensive response to exogenous dexametha-
sone [47]. This persisted through 1 week of dexamethasone administration, but the
difference in blood pressure was not statistically significant after 2 weeks.
Interestingly, in this study, the acute natriuretic response to dexamethasone was pre-
served in the knockout animals even though the acute hypertensive response was not.
304 J.E. Goodwin
This finding raises the possibility that the preserved “pressure natriuresis” is respon-
sible for the absent hypertensive response and that the glucocorticoid receptor’s pres-
ence in the vascular smooth muscle serves as an obstacle to pressure natriuresis.
This proposal is speculative, but it seems clear that the preserved “pressure natriure-
sis” in the absence of elevated blood pressure suggests a complex relationship
between glucocorticoids and the systemic and renal vasculature, which requires
further investigation.
Glucocorticoids and the Endothelium
There is evidence to confirm the presence of the glucocorticoid in the vascular

endothelium [48–50], but little is known about its role here. In vitro experiments
with endothelial cell culture models have suggested that glucocorticoids regulate
vascular reactivity via suppression of the production of vasodilators such as prosta-
cyclin and nitric oxide, but conflicting evidence exists in this regard. Provencher
et al. demonstrated an increase in angiotensin II receptor levels, yet a decrease in
endothelin-1 levels in response to synthetic glucocorticoids in a cell culture model
of vascular smooth muscle cells [37]. Endothelin-1, secreted only by the endothe-
lium, is the strongest vasoconstrictor known. These contradictory results led the
authors to speculate that glucocorticoids function as modulators of vascular inflam-
mation and not solely as vasoconstrictive agents.
Experiments performed using vascular endothelial models have generally
employed vessels in which the endothelium has been mechanically stripped, and
hence is devoid of all endothelial function. Wallerath et al. suggest that it is the
ability of glucocorticoids to destabilize endothelial nitric oxide synthase (eNOS)
mRNA and reduce eNOS protein expression that is responsible for the ensuing
hypertension observed in rats treated with dexamethasone [51]. More recent studies
in isolated rat aortas showed that, through the glucocorticoid receptor, glucocorti-
coids could decrease expression of guanosine triphosphate cyclohydrolase 1
(GTPCH1) mRNA, the rate-limiting enzyme in the production of tetrahydrobiop-
terin (BH4), a cofactor for nitric oxide synthase [52]. However, in this series, eNOS
mRNA levels were not significantly different in control vessels than in those treated
with dexamethasone.
Using a mouse model used to investigate tissue-specific contributions of the
glucocorticoid receptor, this receptor was removed from the vascular endothelium
by using Tie-1 Cre. Interestingly, these knockout animals had a small but statisti-
cally significant increase in their baseline blood pressure, the source of which could
not be entirely explained. In addition, the knockout animals were almost completely
resistant to glucocorticoid-induced hypertension and conspicuously lacked the pressure
natriuresis observed in the vascular smooth muscle glucocorticoid receptor knock-
out model. Intravital microscopy studies done in real-time on resistance vessels
from these knockout animals revealed a statistically significant decrease in vessel
contractility to the glucocorticoid receptor-specific ligand dexamethasone when
compared to wild-type animals. The contractility to phenylephrine was similar

between the two groups suggesting that loss of the endothelial glucocorticoid
receptor confers a specific contractile defect in these animals, presumably prevent-
ing them from mounting a hypertensive response to systemic dexamethasone. In
this study whole blood nitric oxide levels were not different between the two groups
though the possibility of differences in nitric oxide in local vascular beds could not
be ruled out [53].
Other laboratories have examined nitric oxide derangements via ex vivo investi-
gations in rats. In 2009, Aras-Lopez et al. evaluated electrical-field stimulation
induced neuronal nitric oxide release in mesenteric arteries of Wistar-Kyoto (WKY)
rats and spontaneously hypertensive rats (SHRs) and the role of protein kinase C
(PKC) in these responses [54]. Through a series of manipulations involving various
PKC inhibitors, the authors demonstrated that dexamethasone was able to reduce
neuronal nitric oxide release in arteries from SHRs but not WKY rats, and that this
effect was mediated though activation of glucocorticoid receptors. This is the first
study to address the possibility that glucocorticoids exert their hypertensive effects,
at least in part, by altering the neuronal nitric oxide release of the perivascular inner-
vation of these tissues [54]. This study raises important questions with regard to
differential effects of glucocorticoids on the various nitric oxide isoforms, which
appear to be affected by the overall milieu.
To date, there have been few reliable ways to assess the role of glucocorticoids
in the vascular endothelium in vivo. In addition, there is increasing focus on the
purported non-genomic effects of glucocorticoids which would allow very rapid
signaling responses as opposed to the traditional notion of nuclear translocation and
transcription which theoretically take much longer. The mystery as to how a recep-
tor with only one known gene can effect such a vast array of physiologic and phar-
macologic consequences suggests that there must be multiple promoters, mRNAs,
translational isoforms and/or post-translational modifications to account for such
diversity. The discovery of profound species diversity in glucocorticoid sensitivity
also suggests that there may be specific environmental or epigenetic influences that
impact cellular responses to glucocorticoids. Given all of these variables, it is con-
ceivable that a dozen or more GR isoforms may be able to be generated in a single
cell [55]. Therefore, it is not particularly surprising that there is much that is poorly
understood about vascular responses to glucocorticoids.
Glucocorticoids and Adipose Tissue
Transgenic expression of 11-b-HSD1 in adipose tissue increases local glucocorti-

coid levels in adipose tissue and causes a metabolic syndrome-like picture in mice.
This is in further support of the dehydrogenase hypothesis that states that the meta-
bolic syndrome is caused in part by Cushings disease of fat: normal circulating
cortisol levels, but increased adipose tissue glucocorticoid levels [56]. Interestingly
in this study, glucocorticoid levels were found to be elevated in the portal
306 J.E. Goodwin
circulation, suggesting downstream effects of glucocorticoids, particularly in the

liver, could be playing a role in the some of the observed metabolic derangements.
At least one other study has suggested a link between features of the metabolic
syndrome, specifically insulin resistance, glucocorticoids and the liver. Bernal-
Mizrachi et al. demonstrated that activation of PPAR-alpha in the setting of dexa-
methasone treatment, both in vivo in a mouse model and in vitro in human
hepatocytes, resulted in hyperinsulinemia, hypertension and induction of gluconeo-
genic gene expression. These studies would in turn explain the coupling of increased
blood pressure with insulin resistance. Adipose tissue could directly regulate blood
pressure by secretion of adipokines such as angiotensinogen. An indirect effect
mediated via downstream portal flow of newly generated glucocorticoids might also
be a possibility. For a more complete discussion, please see the Chapter dedicated
to this subject.
Glucocorticoids and Development and Cardiovascular Risk
Glucocorticoids are essential for fetal health and development and are tightly regu-
lated during the embryonic period. Fetal glucocorticoid levels rise abruptly toward
the end of gestation in mammals in preparation for post-natal life [57]. It has been
shown that pre-natal glucocorticoid treatment can elicit growth-inhibitory [58] or
stimulatory [59] effects in cardiomyocytes, though direct in vivo effects on the heart
are unknown. Recent work using a mouse model with global absence of glucocorti-
coid signaling has shown that fetal heart function is severely impaired under these
conditions [60]. The authors of this study proceed to show that deficiency of gluco-
corticoid signaling in the cardiomyocytes and vascular smooth muscle only produce
nearly the identical phenotype demonstrating that many aspects of heart maturation
are highly dependent on glucocorticoid signaling within these tissues. In addition
there are robust data showing that post-natal vessel reactivity and even onset of
hypertension may be determined by the pre-natal steroid milieu. Roghair et al.
have nicely demonstrated that administration of dexamethasone to pregnant ewes
results in increased coronary artery vasoconstriction to acetylcholine and attenu-
ated vasodilatation to adenosine in newborn lambs [61]. In addition lambs from
steroid-treated mothers had decreased endothelial nitric oxide synthesis compared
to controls. In a follow-up study, coronary arteries from dexamethasone-exposed
lambs demonstrated greater vascular smooth muscle cell proliferation and greater
production of reactive oxygen species compared to controls and suggest a link
between abnormal intrauterine environment and future risk for coronary artery
disease [62].
While excess prenatal glucocorticoid has been linked to adverse cardiovascular
outcomes in adulthood, prenatal glucocorticoid deficiency is no less worrisome.
Preterm birth increases the risk of cardiovascular disease [63], occurring before the
surge of cortisol in late gestation. Cardiovascular complications in pre-term infants
include persistence of the foramen ovale and the ductus arteriosus, delay in the
decrease of pulmonary vascular resistance and delay in the increase in cardiac out-
put necessary to support post-natal life [64]. Many of these complications can be
improved by exogenous steroids. While it is difficult to dissect the long-term effects
of prematurity vs. the effects of glucocorticoid in humans, animal models are useful
in this regard to evaluate the effect of ‘programming’ [65, 66].
Non-genomic Effects of Glucocorticoids in the Cardiovascular

System
It has long been known that glucocorticoids exert many of their effects on the
cardiovascular system via genomic effects, i.e. rapid (within hours) nuclear translo-
cation of cytoplasmic glucocorticoid receptor and binding to glucocorticoid
response elements (GREs) in the promoter region of target genes. However, genomic
effects cannot explain all the effects of glucocorticoids on the cardiovascular system.
Non-genomic effects, that is responses that occur on the order of several minutes, in the
absence of transcription may also be playing a role. The importance of non-genomic
effects in the cardiovascular system is further obscured by the fact that endogenous
cortisol rises with stress, which is itself a risk factor for cardiovascular disease.
In general, non-genomic effects may be separated into (i) those with GR and
another cytoplasmic signaling molecule and (ii) protein-protein interactions. The
rationale for these protein interactions lies in purported alteration in biochemical
properties in adjacent membranes via insertion of glucocorticoid in plasma mem-
branes or secondary to altered lipophilicity or polarity [67]. Components of the GR
complex released during GR-ligand binding, including heat shock proteins (HSPs),
have been shown to influence cell signaling. For example, Src, which is released
from this complex has been shown to initiate a signaling cascade in non-
cardiovascular systems [68]. GR has also been reported to activate the PI3K-Akt
pathway [69] which in turn can activate eNOS and has been shown to reduce myo-
cardial infarct size in a mouse model [70]. Additional non-genomic effects mediated
via stimulation of cytoplasmic signaling include dexamethasone-induced increase
in coronary lipoprotein lipase by an AMP-activated protein kinase and p38 activated
protein kinase in cardiomyocytes resulting in decreased cardiac glucose oxidation
[71, 72], stimulation of the phosphoinositide system in vascular smooth muscle
cells by cortisol [73], and increased inotropic effect by possible direct potentiation
of calcium channels by hydrocortisone in cardiomyocytes [74].
The non-genomic effects of protein-protein interactions are most notably illus-
trated by the anti-inflammatory effects of dexamethasone in endothelial cells by
direct NF-κB-GR interaction [75]. There is also some evidence that corticosteroid-
binding globulins (CBG), responsible for transporting glucocorticoids in the blood,
may exert a non-genomic role in the cardiovascular system as it has been shown that
elevated CBG levels could result in increased blood pressure [76]. It has also been
shown that bovine serum albumin-bound glucocorticoids can cause non-genomic
effects at the cell surface [77] begging the question of whether glucocorticoid bound
308 J.E. Goodwin
CBG influences functioning of glucocorticoids in many cell types. Currently work

is ongoing to identify synthetic glucocorticoids with nongenomic mechanisms of
action and fewer systemic side effects [78, 79].
Glucocorticoids as Therapy for Cardiovascular Disease
While the developmental milieu vis-a-vis glucocorticoids is often underappreciated

or overlooked, use of these agents as a therapy for adult-onset cardiovascular
disease is the subject of a great deal of investigation, both in animal models and in
clinical trials in humans. There remains ambiguity as to whether glucocorticoids,
and at what doses, are helpful or harmful in cardiovascular disease.
Rodent models have generally been employed to investigate serious morbidities
such as stroke or myocardial infarction. It is well known that elevated glucocorti-
coid levels are a risk factor for cardiovascular disease and, in particular, stroke [80].
Balkaya et al. demonstrated in mouse model that chronic stress, induced by various
restraint manipulations resulted in increased blood pressure, reduced endothelial
nitric oxide synthase levels and impaired endothelium dependent vasorelaxation in
aortic rings and substantial increases in ischemic lesion size when subjected to mid-
dle cerebral artery occlusion [81]. Interestingly all of these detrimental effects,
which are also risk factors for stroke, were completely reversed by treatment with
the glucocorticoid antagonist RU486. Similarly, in an isolated perfused rat heart
model that is used to mimic heart failure, cortisol, as well as aldosterone, resulted in
increased infarct area and apoptosis [82]. Interestingly perfusion with the synthetic
steroid dexamethasone as well as the antagonist RU486 also resulted in worsened
outcomes, though there is some evidence for RU486 to be a partial glucocorticoid
agonist [83]. The phenotype was improved by treatment with spironolactone which
physically occupies the mineralocorticoid receptor as well as by a free-radical scav-
enger, suggesting that oxidative stress plays a role in the cardiac damage in this
model. At least one other rat MI model has shown adverse outcomes with adminis-
tration of exogenous steroid in the form of methylprednisolone, particularly when
given at the late (>2 days post MI) time point, as assayed by matrix metalloprotein-
ase activity, inflammation and caliber of scar formation [84].
In contrast, there are several studies in rodents which demonstrate a beneficial
effect of glucocorticoid administration on cardiovascular disease outcomes. For
example, Xu et al. have shown that provision of dexamethasone prior to left coro-
nary artery occlusion surgery can induce myocardial transcription of the gene Bcl-xl
which protects the heart from ischemia reperfusion injury by preventing mitochondrial
release of cytochrome C [85]. These authors also showed that, in vitro, dexamethasone
induced Bcl-xL expression in cultured cardiomyocytes. All therapeutic effects of
dexamethasone were blocked by the glucocorticoid antagonist, RU486. Interestingly,
the dose of dexamethasone that was used in vivo was quite high at 20 mg/kg.
Steroids have also been investigated in combination with hyperoxia, which has been
shown to provide some benefit in ischemia-reperfusion injury, to determine if the
preconditioning effects of hyperoxia and steroids are additive. Kaljusto et al. did
indeed show that combined pretreatment of male rats with hyperoxia (95 % O2) and
a modest dose of dexamethasone (3 mg/kg) before 30 min of ischemia followed by
120 min of reperfusion did result in more attenuation of infarct size and enhanced
endothelium-independent vessel relaxation compared to groups without dexameth-
asone pretreatment [86].
As opposed to existing in such black and white terms, there is probably a set of
conditions under which steroids will be helpful and another set under which they
will be harmful for any given disease. Nakamura et al. explored this using a mouse
model of viral myocarditis [87]. In a series of experiments the authors showed that
modest dose dexamethasone (0.75 mg/kg) administration before or directly follow-
ing cocksackie virus infection resulted in improved survival and attenuated fibrosis
and systolic dysfunction. However, if the dexamethasone was given at a late time
point (>7 days after induction of myocarditis) then these beneficial effects were lost
and it resulted in decreased mouse survival and increased cardiac tumor necrosis
factor-α. As the beneficial effects were shown to be completely reversed by a selec-
tive COX-2 inhibitor, the authors speculated that it was the action of dexamethasone
on cardiomyocytes directly and not its anti-inflammatory effects which conferred
the beneficial effects at the earlier time point.
While rodent models seem to indicate a somewhat more negative view of using
steroids in the treatment of cardiovascular disease, the opposite is generally true for
human trials. One cardiovascular intervention which lends itself with relative ease
to outcomes testing with regard to steroid effect is drug-eluting stent placement
after myocardial infarction. Coronary stent implantation has superior outcomes
compared to fibrinolysis or balloon angioplasty [88–90], but is limited by re-stenosis
with repeat intervention rates of about 10 % at 6 months post-procedure [89].
Coronary restenosis after stenting is a highly inflammatory process driven by the
secretion of cytokines and adhesion molecules in response to endothelial damage
[91]. Dexamethasone-eluting stents have been used with good effect and in several
trials have been associated with low rates of target lesion revascularization, reinfarc-
tion and late stent thrombosis [91, 92].
Oral steroid therapy in combination with bare metal stents has also been studied
in patients who had received percutaneous coronary interventions after myocardial
infarction. Interestingly there appears to be a prednisone dose response effect in this
setting. Known as the IMPRESS (Immunosuppressive Therapy for the Prevention
of Restenosis After Coronary Stent Implantation) trials, there were both high dose
(HD) and low dose (LD) protocols tested [93, 94]. HD treatment consisted of a total
of 45 days of oral prednisone after myocardial infarction with a dose of 1 mg/kg/day
prescribed in the first 10 days, 0.5 mg/kg/day for days 11–30 and 0.25 mg/kg/day
for days 31–45. LD therapy was considered to be prednisone 1 mg/kg/day for 5 days,
0.5 mg/kg/day for days 5–15 and 0.25 mg/kg/day for days 16–30 [95]. In these studies
the LD prednisone regimen was ineffective in preventing major adverse cardiac
events and had a restenosis rate of ~5× greater than that observed in the HD group.
The authors speculated that the LD protocol, with its abbreviated duration and lower
total steroid dose was insufficient to enact the systemic suppression of inflammation
310 J.E. Goodwin
that was observed in the HD group. These results argue that the systemic effect of
steroids may, in some cases, be necessary for beneficial cardiovascular outcomes and
perhaps superior to tissue-specific steroid effects under some conditions.
Conclusions
In summary, despite an enormous body of basic science research as well as a robust

clinical experience, the role of glucocorticoids in the regulation of the cardiovascular
system remains poorly understood. In vitro studies and work in animal models have
generally highlighted the effects or responses of glucocorticoids in a single cell
type. In reality, clinical studies in humans are, by necessity much more heteroge-
neous and cannot as reliably examine cell-specific effects. Though pre-clinical and
bench research has and will continue to direct and inform human clinical trials,
there is much that still needs to be clarified regarding the role of the glucocorticoids
in the development and function of the cardiovascular system.
References
1. Mantero F, Boscaro M. Glucocorticoid-dependent hypertension. J Steroid Biochem Mol Biol.

1992;43:409–13.
2. Baid S, Nieman LK. Glucocorticoid excess and hypertension. Curr Hypertens Rep. 2004;
6:493–9.
3. Etxabe J, Vazquez JA. Morbidity and mortality in Cushing’s disease: an epidemiological
approach. Clin Endocrinol (Oxf). 1994;40:479–84.
4. Whitworth JA, Mangos GJ, Kelly JJ. Cushing, cortisol, and cardiovascular disease.
Hypertension. 2000;36:912–6.
5. Sabharwal P, Fishel RS, Breslow MJ. Adrenal insufficiency—an unusual cause of shock in
postoperative patients. Endocr Pract. 1998;4:387–90.
6. De Wachter E, Vanbesien J, De Schutter I, Malfroot A, De Schepper J. Rapidly developing
Cushing syndrome in a 4-year-old patient during combined treatment with itraconazole and
inhaled budesonide. Eur J Pediatr. 2003;162:488–9.
7. Bertagna X, Bertagna C, Laudat MH, Husson JM, Girard F, et al. Pituitary-adrenal response to
the antiglucocorticoid action of RU 486 in Cushing’s syndrome. J Clin Endocrinol Metab.
1986;63:639–43.
8. Nieman LK, Chrousos GP, Kellner C, Spitz IM, Nisula BC, et al. Successful treatment of
Cushing’s syndrome with the glucocorticoid antagonist RU 486. J Clin Endocrinol Metab.
1985;61:536–40.
9. Sartor O, Cutler Jr GB. Mifepristone: treatment of Cushing’s syndrome. Clin Obstet Gynecol.
1996;39:506–10.
10. Kalimi M. Role of antiglucocorticoid RU 486 on dexamethasone-induced hypertension in rats.
Am J Physiol. 1989;256:E682–5.
11. Grunfeld JP, Eloy L, Moura AM, Ganeval D, Ramos-Frendo B, et al. Effects of antiglucocor-
ticoids on glucocorticoid hypertension in the rat. Hypertension. 1985;7:292–9.
12. Mangos GJ, Whitworth JA, Williamson PM, Kelly JJ. Glucocorticoids and the kidney.
Nephrology (Carlton). 2003;8:267–73.
13. Williamson PM, Kelly JJ, Whitworth JA. Dose-response relationships and mineralocorticoid
activity in cortisol-induced hypertension in humans. J Hypertens Suppl. 1996;14:S37–41.
14. Montrella-Waybill M, Clore JN, Schoolwerth AC, Watlington CO. Evidence that high dose
cortisol-induced Na+ retention in man is not mediated by the mineralocorticoid receptor. J Clin
Endocrinol Metab. 1991;72:1060–6.
15. Campen TJ, Vaughn DA, Fanestil DD. Mineralo- and glucocorticoid effects on renal excretion
of electrolytes. Pflugers Arch. 1983;399:93–101.
16. Funder JW, Pearce PT, Myles K, Roy LP. Apparent mineralocorticoid excess, pseudohypoal-
dosteronism, and urinary electrolyte excretion: toward a redefinition of mineralocorticoid
action. FASEB J. 1990;4:3234–8.
17. Muller OG, Parnova RG, Centeno G, Rossier BC, Firsov D, et al. Mineralocorticoid effects in
the kidney: correlation between alphaENaC, GILZ, and Sgk-1 mRNA expression and urinary
excretion of Na+ and K+. J Am Soc Nephrol. 2003;14:1107–15.
18. Stewart PM, Corrie JE, Shackleton CH, Edwards CR. Syndrome of apparent mineralocorticoid
excess. A defect in the cortisol-cortisone shuttle. J Clin Invest. 1988;82:340–9.
19. Todd-Turla KM, Schnermann J, Fejes-Toth G, Naray-Fejes-Toth A, Smart A, et al. Distribution
of mineralocorticoid and glucocorticoid receptor mRNA along the nephron. Am J Physiol.
1993;264:F781–91.
20. Yan K, Kudo A, Hirano H, Watanabe T, Tasaka T, et al. Subcellular localization of glucocorti-
coid receptor protein in the human kidney glomerulus. Kidney Int. 1999;56:65–73.
21. Baylis C, Handa RK, Sorkin M. Glucocorticoids and control of glomerular filtration rate.
Semin Nephrol. 1990;10:320–9.
22. Welbourne TC. Glucocorticoid control of ammoniagenesis in the proximal tubule. Semin
Nephrol. 1990;10:339–49.
23. Rodriguez HJ, Sinha SK, Starling J, Klahr S. Regulation of renal Na+-K+-ATPase in the rat by
adrenal steroids. Am J Physiol. 1981;241:F186–95.
24. Freiberg JM, Kinsella J, Sacktor B. Glucocorticoids increase the Na+-H+ exchange and
decrease the Na+ gradient-dependent phosphate-uptake systems in renal brush border mem-
brane vesicles. Proc Natl Acad Sci U S A. 1982;79:4932–6.
25. Kinsella J, Cujdik T, Sacktor B. Na+-H+ exchange activity in renal brush border membrane
vesicles in response to metabolic acidosis: the role of glucocorticoids. Proc Natl Acad Sci U S
A. 1984;81:630–4.
26. Frick A, Durasin I. Proximal tubular reabsorption of inorganic phosphate in adrenalectomized
rats. Pflugers Arch. 1980;385:189–92.
27. Welch WJ, Ott CE, Guthrie Jr GP, Kotchen TA. Renin secretion and loop of Henle chloride
reabsorption in the adrenalectomized rat. Am J Physiol. 1985;249:F596–602.
28. Doucet A, Hus-Citharel A, Morel F. In vitro stimulation of Na-K-ATPase in rat thick ascending
limb by dexamethasone. Am J Physiol. 1986;251:F851–7.
29. Stanton BA. Regulation by adrenal corticosteroids of sodium and potassium transport in loop
of Henle and distal tubule of rat kidney. J Clin Invest. 1986;78:1612–20.
30. Naray-Fejes-Toth A, Fejes-Toth G. Glucocorticoid receptors mediate mineralocorticoid-like
effects in cultured collecting duct cells. Am J Physiol. 1990;259:F672–8.
31. Naray-Fejes-Toth A, Snyder PM, Fejes-Toth G. The kidney-specific WNK1 isoform is induced
by aldosterone and stimulates epithelial sodium channel-mediated Na+ transport. Proc Natl
Acad Sci U S A. 2004;101:17434–9.
32. Li C, Li Y, Liu H, Sun Z, Lu J, et al. Glucocorticoid repression of human with-no-lysine (K)
kinase-4 gene expression is mediated by the negative response elements in the promoter. J Mol
Endocrinol. 2008;40:3–12.
33. Yang CL, Angell J, Mitchell R, Ellison DH. WNK kinases regulate thiazide-sensitive Na-Cl
cotransport. J Clin Invest. 2003;111:1039–45.
34. Wilson FH, Disse-Nicodeme S, Choate KA, Ishikawa K, Nelson-Williams C, et al. Human
hypertension caused by mutations in WNK kinases. Science. 2001;293:1107–12.
35. Kahle KT, Wilson FH, Leng Q, Lalioti MD, O’Connell AD, et al. WNK4 regulates the balance
between renal NaCl reabsorption and K+ secretion. Nat Genet. 2003;35:372–6.
36. Goodwin JE, Zhang J, Velazquez H, Geller DS. The glucocorticoid receptor in the distal nephron
is not necessary for the development or maintenance of dexamethasone-induced hypertension.
Biochem Biophys Res Commun. 2010;394:266–71.
312 J.E. Goodwin
37. Provencher PH, Saltis J, Funder JW. Glucocorticoids but not mineralocorticoids modulate
endothelin-1 and angiotensin II binding in SHR vascular smooth muscle cells. J Steroid
Biochem Mol Biol. 1995;52:219–25.
38. Kornel L, Nelson WA, Manisundaram B, Chigurupati R, Hayashi T. Mechanism of the effects
of glucocorticoids and mineralocorticoids on vascular smooth muscle contractility. Steroids.
1993;58:580–7.
39. Tsugita M, Iwasaki Y, Nishiyama M, Taguchi T, Shinahara M, et al. Differential regulation of
11beta-hydroxysteroid dehydrogenase type-1 and -2 gene transcription by proinflammatory
cytokines in vascular smooth muscle cells. Life Sci. 2008;83:426–32.
40. Wallerath T, Witte K, Schafer SC, Schwarz PM, Prellwitz W, et al. Down-regulation of the
expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated
hypertension. Proc Natl Acad Sci U S A. 1999;96:13357–62.
41. Ray KP, Searle N. Glucocorticoid inhibition of cytokine-induced E-selectin promoter activation.
Biochem Soc Trans. 1997;25:189S.
42. Yang S, Zhang L. Glucocorticoids and vascular reactivity. Curr Vasc Pharmacol. 2004;2:1–12.
43. Sato A, Suzuki H, Nakazato Y, Shibata H, Inagami T, et al. Increased expression of vascular
angiotensin II type 1A receptor gene in glucocorticoid-induced hypertension. J Hypertens.
1994;12:511–6.
44. Kornel L, Prancan AV, Kanamarlapudi N, Hynes J, Kuzianik E. Study on the mechanisms of
glucocorticoid-induced hypertension: glucocorticoids increase transmembrane Ca2+ influx in
vascular smooth muscle in vivo. Endocr Res. 1995;21:203–10.
45. Molnar GA, Lindschau C, Dubrovska G, Mertens PR, Kirsch T, et al. Glucocorticoid-related
signaling effects in vascular smooth muscle cells. Hypertension. 2008;51:1372–8.
46. Ong SL, Zhang Y, Sutton M, Whitworth JA. Hemodynamics of dexamethasone-induced
hypertension in the rat. Hypertens Res. 2009;32:889–94.
47. Goodwin JE, Zhang J, Geller DS. A critical role for vascular smooth muscle in acute
glucocorticoid-induced hypertension. J Am Soc Nephrol. 2008;19:1291–9.
48. Imai Y, Abe K, Sasaki S, Minami N, Munakata M, et al. Exogenous glucocorticoid eliminates
or reverses circadian blood pressure variations. J Hypertens. 1989;7:113–20.
49. Piovesan A, Panarelli M, Terzolo M, Osella G, Matrella C, et al. 24-hour profiles of blood
pressure and heart rate in Cushing’s syndrome: relationship between cortisol and cardiovascu-
lar rhythmicities. Chronobiol Int. 1990;7:263–5.
50. Fallo F, Fanelli G, Cipolla A, Betterle C, Boscaro M, et al. 24-hour blood pressure profile in
Addison’s disease. Am J Hypertens. 1994;7:1105–9.
51. Wallerath T, Godecke A, Molojavyi A, Li H, Schrader J, et al. Dexamethasone lacks effect on
blood pressure in mice with a disrupted endothelial NO synthase gene. Nitric Oxide.
2004;10:36–41.
52. Mitchell BM, Dorrance AM, Mack EA, Webb RC. Glucocorticoids decrease GTP cyclohy-
drolase and tetrahydrobiopterin-dependent vasorelaxation through glucocorticoid receptors.
J Cardiovasc Pharmacol. 2004;43:8–13.
53. Goodwin JE, Zhang J, Gonzalez D, Albinsson S, Geller DS. Knockout of the vascular endo-
thelial glucocorticoid receptor abrogates dexamethasone-induced hypertension. J Hypertens.
2011;29(7):1347–56.
54. Aras-Lopez R, Xavier FE, Ferrer M, Balfagon G. Dexamethasone decreases neuronal nitric
oxide release in mesenteric arteries from hypertensive rats through decreased protein kinase C
activation. Clin Sci (Lond). 2009;117:305–12.
55. Yudt MR, Cidlowski JA. The glucocorticoid receptor: coding a diversity of proteins and
responses through a single gene. Mol Endocrinol. 2002;16:1719–26.
56. Masuzaki H, Paterson J, Shinyama H, Morton NM, Mullins JJ, et al. A transgenic model of
visceral obesity and the metabolic syndrome. Science. 2001;294:2166–70.
57. Fowden AL, Li J, Forhead AJ. Glucocorticoids and the preparation for life after birth: are there
long-term consequences of the life insurance? Proc Nutr Soc. 1998;57:113–22.
58. Slotkin TA, Seidler FJ, Kavlock RJ, Gray JA. Fetal dexamethasone exposure accelerates devel-
opment of renal function: relationship to dose, cell differentiation and growth inhibition. J Dev
Physiol. 1992;17:55–61.
59. Torres A, Belser 3rd WW, Umeda PK, Tucker D. Indicators of delayed maturation of rat heart
treated prenatally with dexamethasone. Pediatr Res. 1997;42:139–44.
60. Rog-Zielinska EA, Thomson A, Kenyon CJ, Brownstein DG, Moran CM, et al. Glucocorticoid
receptor is required for foetal heart maturation. Hum Mol Genet. 2013;22:3269–82.
61. Roghair RD, Segar JL, Sharma RV, Zimmerman MC, Jagadeesha DK, et al. Newborn lamb
coronary artery reactivity is programmed by early gestation dexamethasone before the
onset of systemic hypertension. Am J Physiol Regul Integr Comp Physiol. 2005;289:
R1169–76.
62. Volk KA, Roghair RD, Jung F, Scholz TD, Lamb FS, et al. Coronary endothelial function and
vascular smooth muscle proliferation are programmed by early-gestation dexamethasone
exposure in sheep. Am J Physiol Regul Integr Comp Physiol. 2010;298:R1607–14.
63. Crump C, Sundquist K, Sundquist J, Winkleby MA. Gestational age at birth and mortality in
young adulthood. JAMA. 2011;306:1233–40.
64. Huhta JC. Fetal congestive heart failure. Semin Fetal Neonatal Med. 2005;10:542–52.
65. Rog-Zielinska EA, Richardson RV, Denvir MA, Chapman KE. Glucocorticoids and foetal
heart maturation; implications for prematurity and foetal programming. J Mol Endocrinol.
2014;52:R125–35.
66. Vuguin PM. Animal models for small for gestational age and fetal programming of adult disease.
Horm Res. 2007;68:113–23.
67. Lee SR, Kim HK, Youm JB, Dizon LA, Song IS, et al. Non-genomic effect of glucocorticoids
on cardiovascular system. Pflugers Arch. 2012;464:549–59.
68. Losel R, Wehling M. Nongenomic actions of steroid hormones. Nat Rev Mol Cell Biol.
2003;4:46–56.
69. Kfir-Erenfeld S, Sionov RV, Spokoini R, Cohen O, Yefenof E. Protein kinase networks regulat-
ing glucocorticoid-induced apoptosis of hematopoietic cancer cells: fundamental aspects and
practical considerations. Leuk Lymphoma. 2010;51:1968–2005.
70. Hafezi-Moghadam A, Simoncini T, Yang Z, Limbourg FP, Plumier JC, et al. Acute cardiovas-
cular protective effects of corticosteroids are mediated by non-transcriptional activation of
endothelial nitric oxide synthase. Nat Med. 2002;8:473–9.
71. Kewalramani G, Puthanveetil P, Kim MS, Wang F, Lee V, et al. Acute dexamethasone-induced
increase in cardiac lipoprotein lipase requires activation of both Akt and stress kinases. Am J
Physiol Endocrinol Metab. 2008;295:E137–47.
72. Puthanveetil P, Wang Y, Wang F, Kim MS, Abrahani A, et al. The increase in cardiac pyruvate
dehydrogenase kinase-4 after short-term dexamethasone is controlled by an Akt-p38-forkhead
box other factor-1 signaling axis. Endocrinology. 2010;151:2306–18.
73. Steiner A, Locher R, Sachinidis A, Vetter W. Cortisol-stimulated phosphoinositide metabolism
in vascular smooth muscle cells: a role for glucocorticoids in blood pressure control?
J Hypertens Suppl. 1989;7:S140–1.
74. Yano K, Tsuda Y, Kaji Y, Kanaya S, Fujino T, et al. Effects of hydrocortisone on transmem-
brane currents in guinea pig ventricular myocytes—possible evidence for positive inotropism.
Jpn Circ J. 1994;58:836–43.
75. Brostjan C, Anrather J, Csizmadia V, Stroka D, Soares M, et al. Glucocorticoid-mediated
repression of NFkappaB activity in endothelial cells does not involve induction of IkappaBalpha
synthesis. J Biol Chem. 1996;271:19612–6.
76. Whitworth JA, Kelly JJ, Brown MA, Williamson PM, Lawson JA. Glucocorticoids and
hypertension in man. Clin Exp Hypertens. 1997;19:871–84.
77. Hua SY, Chen YZ. Membrane receptor-mediated electrophysiological effects of glucocorticoid
on mammalian neurons. Endocrinology. 1989;124:687–91.
78. Schmidt BM, Gerdes D, Feuring M, Falkenstein E, Christ M, et al. Rapid, nongenomic steroid
actions: a new age? Front Neuroendocrinol. 2000;21:57–94.
79. Schoneveld JL, Fritsch-Stork RD, Bijlsma JW. Nongenomic glucocorticoid signaling: new
targets for immunosuppressive therapy? Arthritis Rheum. 2011;63:3665–7.
80. DeVries AC, Joh HD, Bernard O, Hattori K, Hurn PD, et al. Social stress exacerbates stroke
outcome by suppressing Bcl-2 expression. Proc Natl Acad Sci U S A. 2001;98:11824–8.
314 J.E. Goodwin
81. Balkaya M, Prinz V, Custodis F, Gertz K, Kronenberg G, et al. Stress worsens endothelial
function and ischemic stroke via glucocorticoids. Stroke. 2011;42:3258–64.
82. Mihailidou AS, Le Loan TY, Mardini M, Funder JW. Glucocorticoids activate cardiac miner-
alocorticoid receptors during experimental myocardial infarction. Hypertension. 2009;54:
1306–12.
83. Schulz M, Eggert M, Baniahmad A, Dostert A, Heinzel T, et al. RU486-induced glucocorticoid
receptor agonism is controlled by the receptor N terminus and by corepressor binding. J Biol
Chem. 2002;277:26238–43.
84. Garcia RA, Go KV, Villarreal FJ. Effects of timed administration of doxycycline or methyl-
prednisolone on post-myocardial infarction inflammation and left ventricular remodeling in
the rat heart. Mol Cell Biochem. 2007;300:159–69.
85. Xu B, Strom J, Chen QM. Dexamethasone induces transcriptional activation of Bcl-xL gene
and inhibits cardiac injury by myocardial ischemia. Eur J Pharmacol. 2011;668:194–200.
86. Kaljusto ML, Stenslokken KO, Mori T, Panchenko A, Frantzen ML, et al. Preconditioning
effects of steroids and hyperoxia on cardiac ischemia-reperfusion injury and vascular reactivity.
Eur J Cardiothorac Surg. 2008;33:355–63.
87. Nakamura H, Kunitsugu I, Fukuda K, Matsuzaki M, Sano M. Diverse stage-dependent effects
of glucocorticoids in a murine model of viral myocarditis. J Cardiol. 2013;61:237–42.
88. Keeley EC, Boura JA, Grines CL. Primary angioplasty versus intravenous thrombolytic
therapy for acute myocardial infarction: a quantitative review of 23 randomised trials. Lancet.
2003;361:13–20.
89. Stone GW, Grines CL, Cox DA, Garcia E, Tcheng JE, et al. Comparison of angioplasty with
stenting, with or without abciximab, in acute myocardial infarction. N Engl J Med.
2002;346:957–66.
90. Nordmann AJ, Hengstler P, Harr T, Young J, Bucher HC. Clinical outcomes of primary stenting
versus balloon angioplasty in patients with myocardial infarction: a meta-analysis of random-
ized controlled trials. Am J Med. 2004;116:253–62.
91. Jimenez-Valero S, Santos B, Pajin F, Canton T, Lazaro E, et al. Clinical outcomes of
dexamethasone-eluting stent implantation in ST-elevation acute myocardial infarction.
Catheter Cardiovasc Interv. 2007;70:492–7.
92. Liu X, Huang Y, Hanet C, Vandormael M, Legrand V, et al. Study of antirestenosis with the
BiodivYsio dexamethasone-eluting stent (STRIDE): a first-in-human multicenter pilot trial.
Catheter Cardiovasc Interv. 2003;60:172–8; discussion 179.
93. Ribichini F, Tomai F, Ferrero V, Versaci F, Boccuzzi G, et al. Immunosuppressive oral predni-
sone after percutaneous interventions in patients with multi-vessel coronary artery disease.
The IMPRESS-2/MVD study. EuroIntervention. 2005;1:173–80.
94. Versaci F, Gaspardone A, Tomai F, Ribichini F, Russo P, et al. Immunosuppressive therapy for
the prevention of restenosis after coronary artery stent implantation (IMPRESS Study). J Am
Coll Cardiol. 2002;40:1935–42.
95. Ferrero V, Ribichini F, Rognoni A, Marino P, Brunelleschi S, et al. Comparison of efficacy and
safety of lower-dose to higher-dose oral prednisone after percutaneous coronary interventions
(the IMPRESS-LD study). Am J Cardiol. 2007;99:1082–6.
Chapter 14
Glucocorticoids and Cancer
Miles A. Pufall
Abstract Unlike other steroid hormone receptors, the glucocorticoid receptor (GR)
is not considered an oncogene. In breast cancer, the estrogen receptor (ER) drives
cell growth, proliferation, and metastasis, and the androgen receptor (AR) plays a
similar role in prostate cancer. Accordingly, treatment of these diseases has focused
on blocking steroid hormone receptor function. In contrast, glucocorticoids (GCs)
work through GR to arrest growth and induce apoptosis in lymphoid tissue.
Glucocorticoids are amazingly effective in this role, and have been deployed as the
cornerstone of lymphoid cancer treatment for decades. Unfortunately, not all patients
respond to GCs and dosage is restricted by immediate and long term side effects. In
this chapter we review the treatment protocols that employ glucocorticoids as a cura-
tive agent, elaborate on what is known about their mechanism of action in these
cancers, and also summarize the palliative uses of glucocorticoids for other cancers.
Keywords Leukemia • Apoptosis • Lymphoma • Chemotherapy • Palliative
Glucocorticoids in Cancer
Unlike other highly related nuclear hormone receptors, the glucocorticoid receptor
(GR) is not considered an oncogene. In breast cancer, the estrogen receptor (ER)
drives cell growth, proliferation and metastasis. The androgen receptor (AR) plays
a similar role in prostate cancer. Accordingly, treatment of these diseases has
focused on blocking estrogen or testosterone production, or directly blocking
steroid binding to their respective receptors. In contrast, glucocorticoids (GCs)
work through GR to perform a variety of functions, including arresting growth or
inducing apoptosis in lymphocytes. Glucocorticoids are so effective in this role that
they are the cornerstone of treatment for all lymphatic cancers, though often
M.A. Pufall, M.S., Ph.D. (*)

Department of Biochemistry, Carver College of Medicine, Holden Comprehensive Cancer
Center, 51 Newton Road, Bowen Science Building, Room 4-430, Iowa City, IA 52242, USA

316 M.A. Pufall
hampered by a panoply of off-target consequences. In this chapter, we will review

the treatment protocols that employ glucocorticoids as a curative agent, elaborate on
what is known about the mechanism of how they function in those cancers, and also
summarize the palliative uses of glucocorticoids in treatment of a variety of can-
cers, and the implications of that use.
Lymphoid Cancers
Glucocorticoids in Childhood Leukemia
Much of what is known about glucocorticoids as a chemotherapeutic, and indeed

many other cytotoxic agents, was learned from treatment of childhood leukemia. The
predominant role of glucocorticoids in cancer is in the treatment of lymphoid malig-
nancies. Building on an observation that there is an inverse relationship between the
size of the adrenal cortex and thymus [1], Dougherty and White demonstrated that
administration of ACTH reduced the size of most lymphoid tissue (excluding the
spleen) [2]. Around the same time, cells from a tumor of unknown origin, later
described to be “lymphosarcoma,” were injected into mice and were found to form
tumors. These tumors did not spontaneously regress, but instead shrunk when exposed
to Compound E, otherwise known as cortisone [3]. A similar effect was observed in
rats [4]. The increased catabolism observed at the time was thought to be responsible
for the consumption of the tumor. These findings were rapidly moved to the clinic
where is was reported in 1949 that either ACTH or cortisone acetate dramatically
reduced the size of lymphoid tumors or leukemias, but not other carcinomas [5].
Emboldened by these findings, much larger studies were undertaken to explore the
effect of cortisone and ACTH on hematological disorders. Patients were recruited
with lymphoid malignancies, including CLL, lymphoma, Hodgkin’s, acute lympho-
blastic leukemia, and multiple myeloma (plasma cell myeloma), as well as the myeloid
derived AML and CML. Beneficial effects were observed specifically in lymphoid,
but not myeloid disease, ranging from symptomatic relief (multiple myeloma) to
complete, but temporary remission in childhood ALL [6]. Despite the effectiveness of
GCs in reducing lymphoid disease, as single agents they did not produce durable remis-
sions, much less a cures.
Combination Therapy: Glucocorticoids Have a Central Role
Arround the same time, in the early 1950s, other agents were being tested for their
anti-tumorigenic properties. Folic acid had been shown to increase the growth of
lymphoid tumors, likely by upregulating amino acid and purine biosynthetic path-
ways. Much as with GCs blocking these pathways with anti-folates, such as the
early aminopterin and later methotrexate, produced, a dramatic, but temporary
remission in lymphoid malignancies. A dark, but serendipitous, observation brought
14 Glucocorticoids and Cancer 317
another agent to the fore. Exposure to mustard gases in World War I was shown to
deplete bone marrow and lymph nodes. The alkylating properties of these gasses
were developed into drugs, such as cyclophosphamide, that proved to be potent
anti-tumor agents in rodent models and later in human trials. Inhibition of nucleo-
tide synthesis was effective as well, with 6-mercaptopurine showing promise both
in acute leukemias and other cancers [7]. Interestingly, although each of these
classes of drugs was shown to reduce or even clear the cancerous disease, none of
the remissions proved durable.
Three treatment breakthroughs came in the early 1960s. First, vinca alkaloids
(later vincristine), isolated from plants, were found to disrupt microtubules and have
potent anti-tumorogenic properties [7]. Next, it was discovered that leukemic blasts
are unable to make their own asparagine, and thus needed to absorb it from their
medium to survive. To exploit this, a component of guinea pig serum, l-asparaginase,
was isolated that converts free asparagine to aspartic acid, effectively starving the
cells [8]. Perhaps most importantly, in the early 1960s intrepid cancer physicians
took the radical step of administering agents in combination. The first successful
protocol, called VAMP (vincristine, amethopterin, 6-mercaptopurine, and predni-
sone), improved. The 5 year survival of children with leukemia from 25 % with
single agents to over 60 % [7].
For childhood leukemia, these basic components have evolved over the years to
today’s treatment, which is administered in three phases: remission induction, inten-
sification (consolidation), and maintenance. Glucocorticoids can be administered
during all three phases, but are used most intensely during remission induction, with
the goal of eliminating greater than 99 % of the disease tissue (minimum residual
disease, MRD) [9]. During this phase a glucocorticoids are administered with vin-
cristine and asparaginase and are 98 % effective in inducing remission in childhood
B-cell leukemias. In intensification, mercaptopurine is combined with polyethylene
glycol conjugated asparaginase (pegasparaginase), and methotrexate. Intensification
may also include cyclophosphamide or cytarabine (araC) or cycles of reinduction,
using the same agents described above. GCs may again be administered during
maintenance therapy, though at a lower dose, and less frequently [9]. These advances
in treatment have led to cure rates approaching 90 % in children with both B and
T-cell acute lymphoblastic leukemia, making it one of the most treatable cancers.
In addition, the success of these trials have informed the treatment of other lymphoid
cancers, discussed below.
GC Response Predicts Treatment Response
Though only effective as a curative agent in combination, GCs are central to the
effectiveness of treatment. This has been best elucidated in European studies from
the Berlin-Frankfurt-Muenster (BFM) group, who have developed treatment proto-
cols in parallel with those in the US. This consortium showed that the initial response
of infants and children with leukemia to prednisone alone was the best predictor of
eventual outcome to full treatment [10, 11]. Importantly, other groups showed that
318 M.A. Pufall
patient response to prednisone, and overall response, could also be predicted by

treatment of leukemic blasts ex vivo with GCs [12, 13]. This suggested that the func-
tion of GCs in inducing leukemic blast cell death was not necessarily dependent on
the environment, but the cell autonomous program initiated by the drug.
The importance of GCs in treatment of leukemias is perhaps best highlighted by
comparisons of patient response to dexamethasone and prednisone. Dex and pred are
both derivatives of cortisol, with dex differing by addition of a fluorine at the 9α and a
methyl at C16. These two differences make dex more specific for GR, with little to no
MR activity, and about 10–16× more potent according to established indices [14].
In clinical trials, substituting dex for pred in high risk ALL patients improves outcome
by over 10 % (81–94 % overall survival) (COG AALL0232), despite each inducing
indistinguishable MRD after induction. This indicates that response to GCs in not
only predictive of eventual outcome, but is a major determinant of outcome.
Side and Late Effects
Unfortunately, despite the clear benefits of using high-dose potent GCs in disease
treatment, the side effects and potential late effects limit what can be administered.
Although dex is much more effective than pred in children, it is only well-tolerated
in children under ten. For children over ten, and adults, dex is significantly more
likely to cause avascular necrosis (AVN), psychiatric issues, muscle wasting, and
mortality [14, 15]. The late effects, or effects that arise years after cessation of treat-
ment, are also a concern. The muscle wasting, osteoporosis and metabolic effects of
GCs can persist after treatment ends, and the eventual neuro-psychiatric effects are
of concern. More recently it has been shown that pulsing dex during maintenance
can produce the same outcomes with more acceptable side effects. Nonetheless,
physicians are currently challenged with the choice of which GC to use, what dose,
and for how long. A more complete understanding of how GCs function in leukemic
blasts and other tissues will help inform these choices.
Mechanism of Action
The biological function of GCs in hematopoietic cells and why they induce cell
death of lymphoid cells is not clear. However, seminal work performed by John
Ashwell at the NIH indicates that glucocorticoids serve as a negative signal in lym-
phoid development. By knocking out GR in developing thymocytes he showed that
the mice were immunocompromised due to a reduction in T cell repertoire. He
further showed that intact glucocorticoid signaling was important for proper T cell
selection, perhaps pointing to a role for endogenous GCs in developing lympho-
cytes [16]. More recently, experiments in mice indicate that GCs may have a similar
role in B cells [17].
Although non-genomic effects of GR have been proposed, cell death appears to
require GC-induced gene regulation. General blocks of transcription or translation
by actinomycin D and cycloheximide, respectively, block GC-induced cell death
[18]. In addition, a mutation that weakens DNA-induced GR dimerization and

blunts activation also impairs GC-induced cell death. Further, the effect of GCs
appears to proceed in two steps; an initial growth arrest that lasts about 24 h, and
subsequent cell death. Continuous administration of GCs is required through arrest
to induce cell death, which takes 2–3 days more [19]. The most frequently reported
mechanism of cell death is apoptosis, though cases of necrosis [20] and necroptosis
[21] have also been reported.
The GR-regulated genes that are required to induce apoptosis have not been well
defined, but studies on both patient samples and cell lines have lent some insight.
The clearest role is in driving apoptosis genes. Activation of GR has been shown to
tip the balance of the BH3 domain containing Bcl2 family of apoptotic factors
towards apoptosis through activation of pro-apoptotic BIM (BCL2L11), and down
regulation of the anti-apoptotic BCL2 [22, 23]. Less directly, GCs consistently
upregulate thioredoxin interacting protein (TXNIP), which results in accumulation
of reactive oxygen species and apoptosis [24]. Part of the initial growth arrest likely
involves regulation of cell-survival genes, such as repression of pro-growth c-MYC
and Hexokinase II, and cell cycle genes [19]. Although blocking these pathways
does not necessarily block GC induced apoptosis, they may contribute eventually to
cell death [25]. There is some evidence that by inhibiting NFκB and growth signal-
ing pathways (e.g. ERK, MEK) GR can arrest cells and induce cell death, though a
clear direct mechanism has not been elaborated [24, 26]. In addition, there is now
also evidence that GR suppresses miRNA expression, and that repression of miR-
17-92 correlates with apoptosis. Thus, although GCs can alter apoptotic and cell
survival pathways it is not clear how prevalent either mechanism is in inducing cell
death, or whether these pathways are directly regulated by GR.
Glucocorticoid Resistance
Misregulation of apoptotic factors have been implicated in resistance. For exam-

ple, resistant ALL patients exhibit higher expression levels of the anti-apoptotic
genes BCL2 and MCL1 [25, 27, 28], which likely blunt the apoptotic signal affected
by GCs. Overexpression of MCL1 has been linked to activation of mTor signaling,
and can be alleviated by inhibition of this pathway with rapamycin. On the other
side of the coin, activation of Akt stimulates the mTor pathway (as well as perhaps
inhibiting GR directly, see below) and makes cells resistant [28]. It has also been
observed that a failure of GCs to activate the pro-apoptotic BIM contributes to
resistance [29]. Thus, in cells where the balance of Bcl2 family members cannot be
sufficiently biased toward pro-apoptosis, GC-induced cell death is likely to be
impaired.
In addition to apoptosis, pathway analysis of resistant samples compared to
sensitive ones has implicated repression of cell cycle genes [30], and increased
carbohydrate consumption through overexpression of associated genes, including
carbonic anhydrase 4 (CA4), glucose transporter 3 (GLUT3/SLC2A3), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [27, 31, 32].
320 M.A. Pufall
More general changes in transcription have been also been linked to GC resistance.
For example, ALLs that have translocations in the MLL gene are more likely to not
respond to treatment [33]. MLL encodes a histone methyl transferase that methyl-
ates lysine 4 of histone H3 and is a mark of active enhancers including response
elements and active genes [34, 35]. Translocations that impair the methyltransferase
activity of MLL show widespread changes in the chromatin state that are thought to
reprogram, and generally downregulate gene expression [36]. In addition, muta-
tional analysis of diagnostic and relapsed patients shows a significant enrichment
for transcription factor mutations over other gene sets, again implicating transcrip-
tional defects [37].
More recently, alterations in GR regulation have been implicated in resistance.
Phosphorylation of S134 of GR was shown in to be associated with GC resistance in a
T-ALL line (CEM). In a heterologous cell system, AKT phosphorylation of GR at this
site appeared to impair translocation, providing a mechanism of resistance [38]. This
finding, however, is at odds with another study showing that phosphorylation of S134
is able to translocate to the nucleus, but alters gene expression [39]. In other studies, a
failure to increase GR levels through a positive feedback loop has been shown to impair
GC induced apoptosis, though how this feedback is disrupted has not been established
[40, 41]. Lastly, not all GR isoforms have the ability to induce apoptosis, suggesting
that mechanisms that regulate isoform selection may play a role [42].
Adult Acute Lymphoblastic Leukemia
The treatment for adult ALL is modeled on the treatments that are so successful in
children. Unfortunately, response rates in adults are significantly worse, with an
80–90 % initial response rate, but only 25–50 % disease free survival after 5 years.
Why the response of adults is so much worse is not clear, but has been attributed to
two factors. The first is that significantly more mutations accumulate in adult ALL
blasts, though few have been directly attributable to treatment response. The second
is that adults are not able to tolerate the treatment regimens as well as children. For
example, with similar dosing of vincristine, daunorubicin, and dexamethasone dur-
ing induction, treatment related death is almost 10 % in adults compared to ~1 % in
children. To avoid some of these side effects, dex is given in pulses, rather than
continuously during induction [43]. Because response rates are worse, bone marrow
transplants are often the best route to a durable remission.
Intensification (consolidation) and maintenance are also similar to the children’s
protocol. Typical consolidation includes methotrexate, cytarabine, cyclophos-
phamide, and asparaginase. Clinical trials using hyper CVAD (cyclophosphamide,
vincristine, doxorubicin, and dex) have shown somewhat better responses [44].
Maintenance therapy consists of 6-mecaptopurine and methotrexate with monthly
pulses of vincristine and prednisone.
Treatment of T-ALLs are similar [43], except for cases with mutation in the
NOTCH pathway. Notch is a cell surface receptor, whose intracellular domain is
liberated by cleavage with γ-secretase upon ligand binding. This domain translo-
cates to the nucleus and acts as a coactivator of transcription. Activating mutations
in NOTCH are found in ~50 % of T cell ALLs and correlate with GC resistance.
Administration of γ-secretase inhibitors reverses this resistance, but causes gut tox-
icity. Fortunately, GCs protect against gut toxicity [45], allowing inhibitors to be
used in clinical trials (NCT01088763).
Multiple Myeloma
Multiple Myeloma is the clonal expansion of plasma cells, the mature B cells that
emerge from germinal centers. The resulting cells both overpopulate the bone marrow
and secrete excessive immunoglobulin, resulting in impaired immune function,
renal disease, and bone lesions. At this point, multiple myeloma is not curable, but
can be managed with chemotherapy. Like ALL, chemotherapeutic regimens have
dramatically improved prognosis, from months in 1950s to 7 years or more for stan-
dard risk patients today [46].
The treatment for multiple myeloma has, until recently, involved alkylating
agents and glucocorticoids almost exclusively. Like ALL, mustard gases and their
derivatives, such as malphalan, were initially used for treatment of the disease.
They also induced remission, but at a lower rate (about 1/3 response), and also
quickly relapsed. Prednisone also exhibited initial effectiveness, with an average
complete response rate of 44 %. In 1969 these two agents were combined to pro-
duce a much more robust response rate of 60 %. Unlike ALL, however, these treat-
ments did not fully cure the disease, but improved survival. In the early 1970s,
multiple alkylating agents (carmustine, cyclophosphamide, and melphalan) were
combined with GCs and vincristine to increase initial response rates, though sur-
vival was not improved. Subsequently, dex was combined with doxorubicin and
vincristine under the VAD protocol, which was used for years as the main treat-
ment [46]. While these formulations were being used in the clinic, researchers
were exploring the use of thalidomide, the teratogenic treatment for nausea and
morning sickness used in the early 1960s. In 1990s, thalidomide was shown to
have anti-angiogenic properties, including specific action on multiple myeloma.
Combination of thalidomide and its derivatives with GCs and cyclophosphamide
proved effective in treatment of relapsed multiple myeloma and was installed as
the main treatment for most patients [47].
Proteasome inhibitors have also emerged as having activity in myeloma and syn-
ergy with GCs [48]. Combination therapy of bortezomib with GCs began in 2003,
with other proteasome inhibitors being developed since then that have proven effec-
tive both in initial and relapse treatment. These include carfilzomib, which target
different proteolytic activities within the proteasome.
322 M.A. Pufall
Mechanism
Although less is known about how GCs induce cell death in multiple myeloma, there
are some clear parallels with their mechanism of action in ALL. First, they modulate
the expression of Bcl2 family members, tipping the balance to apoptotis. Second,
GCs also inhibit proliferation by suppressing c-MYC. Lastly, it has been shown that
GCs also affect the redox balance of multiple myeloma cells, which makes them
more susceptible to cell death. GC activation of the transcription factor GilZ has also
been implicated in apoptosis. GilZ is regulated by GR in all known tissues, but, in
contrast to other tissues, induces apoptosis in multiple myeloma [49].
Resistance to GCs show similarities, but also some differences. Like ALL, acti-
vation of Akt attenuates the cytotoxic effects of GCs. However, in multiple myeloma
the disease microenvironment shows a clear effect. Secretion of IL6 by either the
disease itself or the supporting tissue severely impairs the response of multiple
myeloma to GCs and treatment in general, and appears to work though NFκB [50].
Like ALL, unfortunately, not enough is known about the GC-induced program of
cell death to account for how resistance arises in most cases.
Hodgkin’s Disease
Hodgkin’s disease occurs within lymph nodes as the clonal expansion of mature
B cells. The disease is characterized by starting in one node then spreading systemi-
cally. First described in 1832, it was initially treated with radiation. Response was
poor, and the treatment was stopped in 1920s. Many of the agents that worked in
ALL were tried for Hodgkin’s disease, including alkylating agents such as chloram-
bucil and cyclophosphamide, vincristine, and glucocorticoids. ACTH and cortisone
used as single agents were found to induce remarkable, but temporary remission. It
was not until 1967 that an effective combination therapy was formulated. The
MOPP protocol included a mustard alkylator, vincristine (oncovin), procarbazine
(another alkylator), and prednisone and achieved a 50 % cure rate. When combined
with involved field radiation, MOPP produced even better results, with response
rates as high as 70 % [43]. The MOPP protocol was unfortunately associated with a
number of late effects, including nerve damage, infertility, and secondary malignan-
cies such as acute leukemia. A better tolerated alternative called ABVD (Adriamycin,
bleomycin, vinblastine, and dacarbazine) was developed in 1970s, and by 1990s
had supplanted MOPP as more effective an better tolerated. Over the last few years,
a new protocol, once again involving GCs, was developed called BEACOPP (bleo-
mycin, etoposide, Adriamycin, cyclophosphamide, vincristine, procarbazine, and
prednisone), and has demonstrated better initial response. This response comes at a
cost. First, the BEACOPP treatment has a higher mortality rate, a higher secondary
malignancy rate, and sterility risk. Second, the rate of recovery for those who fail to
respond or relapse, called the “salvage rate” is lower. When taken into account, the
rate of initial response plus the salvage rate for ABVD and BEACOPP are not signifi-
cantly different [51]. The relative benefits of BEACOPP vs. ABVD are the subject
of debate as of the writing of this chapter, though it is clear that ABVD is better
tolerated by most patients.
Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia differs from ALL in that it is a disease of later stage
B cells that accumulates over time, rather than as a result of hyperproliferation. CLL
cells are refractory to apoptosis, and accumulate in the blood stream, lymph nodes,
and bone marrow. The disease becomes pathological when CLL cells crowd out the
production of other blood cells. In the early 1950s, the effect of ACTH or GCs on
CLL was tested along with several other lymphoid malignancies [52]. When tested
as a monotherapy, only ~11 % of patients had even a partial response [53]. Later,
when used as part of combination therapies such as CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone), GCs were found to have no effect on
eventual outcome while still causing side effects, and were not included in treatment
regimens. When high dose prednisone was tested in patients in 1990s, a better initial
response was observed, but the response was not durable [53]. The most severe side
effects in these studies were opportunistic infections, and have limited the useful-
ness of GCs in CLL.
More recently monoclonal antibodies (mAbs) have become an essential agent
in treatment of CLL. The most common therapy for CLL is FCR which consists of:
an alkylating agent, such as cyclophosphamide; flutarabine, a nucleoside analog;
and rituximab, a mAb directed against the B cell specific CD20 cell surface marker.
This combination therapy has a very high initial response rate (some reports as high
as 90 %) with an overall response rate over 50 % [43]. Based on the success of
rituximab, other mAbs have been developed, including Ofatumumab (also against
CD20), and alemtuzumab (against CD52). Recently, high dose GCs have been com-
bined with mAbs in clinical trials for CLLs refractory to standard therapy. Although
overall response rates have been over 50 %, the median progression free survival is
less than a year [53].
Use of GCs in CLL is still being considered because of their role not in cell
death, but in lymphocyte redistribution. In the late 1940s and early 1950s, patients
with CLL treated with ACTH or cortisone experienced a reduction in nodal or
splenic tumor masses. Surprisingly, this was accompanied by an increase in the
circulating leukocytes, called leukocytosis. It was thought that, in addition to prob-
ably modest cell death, GCs induce a redistribution of leukocytes to the blood
stream that is reversed upon removal of GCs. This behavior mirrors the normal,
circadian redistribution of B cells. Under non-pathological conditions, when GCs
are low, B and T cell circulation is high, but when cortisol spikes in the morning,
cells home to tissue locations. Why CLL cells would leave tumors is not clear,
but GC induced expression of CXCR4 causes B and T cells to enter the bloodstream,
and eventually migrate to environments, such as the bone marrow or lymph nodes,
that express CXCR12 [54]. This window of time when lymphocytes are circulating
provides an opportunity for other agents to attack.
324 M.A. Pufall
Non-Hodgkin’s Lymphomas
Non-Hodgkin’s Lymphoma (NHL) is the most common hematological malignancy

diagnosed in the US. It represents a collection of over 30 subtypes of lymphoid
malignancies that are distinct, for the most part, from leukemias in that they are not
circulating. The subtypes are distinguished by their lineage, developmental stage, or
location. Despite this heterogeneity, most NHLs are treated with a similar protocol
that involves GCs [55]. Follicular, Mantle cell, diffuse large B cell lymphoma
(DLBCL), and T cell lymphomas are treated with the CHOP protocol, which com-
prises cyclophosphamide, doxorubicin (hydroxyduanomycin), vincristine
(Oncovin), and prednisone. Outcome can be improved in most B cell lymphomas
with the addition of the mAb rituximab targeted against the B-cell specific antigen
CD20 (R-CHOP). For more advanced case of DLBCL, ACVBP (doxycycline,
cyclophosphamide, vincristine, bleomycin, and prednisone) or R-CHOP with eto-
poside can be used. Higher grade, or more aggressive NHLs are can be treated with
hyperCVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone),
which is the same combination as CHOP, but with the more potent dexamethasone
[43]. As observed in childhood ALL, the upgrade to Dex is clearly more effective,
but harbors the risk of more side effects and late effects.
Solid Tumors
Prostate
Prostate cancer afflicts about one in five men in the US, making it their second
most common cancer. The growth and proliferation of prostate cancer is driven by
androgens, which work through the androgen receptor exclusively. Accordingly, an
important part of treatment for prostate cancer is to block production of androgens,
typically through castration. Though this is effective in blocking production of
testosterone and inducing cancer regression, the cancer often returns in 2–3 years
[56]. The relapsed tumor is able to grow in the apparent absence of testosterone,
and is termed either castration resistant prostate cancer (CRPC) or Hormone
Refractory Prostate Cancer (HRPC). Small molecule inhibitors that block testos-
terone binding to the androgen receptor have been developed, and can once again
induce regression [57]. However, once resistance to these anti-androgens develops,
the treatment options for CRPC are significantly less effective. Glucocorticoids are
not used in initial therapy, but the glucocorticoid receptor has two opposing func-
tions in anti-androgen refractory CRPC.
Prostate cancer is diagnosed and staged based on biopsies of the prostate gland
and invasion to nearby tissues. Concomitant measurement of Prostate Specific
Antigen (PSA), a gene driven specifically by AR in the prostate, serves as a
marker for activity of AR in the prostate and a potential indicator of PC growth.
Although the accuracy of PSA as an indicator of prostate cancer progression or

aggressiveness is controversial, it is nonetheless a widely accepted metric of AR
activity in the prostate [43]. After successful initial therapy, when the prostate is
either removed or regressed by androgen deprivation therapy followed by radia-
tion therapy, PSA levels drop precipitously (normal, though it can vary widely, is
~4 ng/mL in the blood) [58]. During subsequent monitoring, a PSA level doubling
time of 15 months or more is not associated with poor outcome, whereas a dou-
bling time of <3 months is an indicator of recurrent disease. Radiographic evi-
dence is more definitive. In these recurrent patients, AR activity is still observed
despite low levels (<50 ng/dL) of circulating testosterone. This behavior is
indicative of androgen independent activity of AR, hypersensitivity of AR to even
low levels of testosterone, or an AR independent mechanism [58].
The former of these two possibilities, androgen independence and hypersensitivity
to androgens, often result from overexpression of AR and are difficult to distinguish.
One common therapy for such patients is glucocorticoids either alone or combined
with other chemotherapeutics, such as paclitaxel or mitoxantrone. The effectiveness
of this therapy varies widely, with 20–79 % of patients exhibiting suppressed PSA
levels [56].
How patients derive any benefit from glucocorticoids is not clear. The adminis-
tration of GCs provides a negative feedback on the pituitary gland, suppressing
production of adrenal testosterone [59]. Though the adrenal gland is thought to be a
minor source of androgens, a decrease may nonetheless provide relief if the patient
is androgen hypersensitive. Quite separate from this mechanism, GR may act as a
tumor suppressor in PC itself. GR is highly expressed in normal prostate, but often
suppressed in PC. Studies in cell line models of PC show that GCs can suppress
pro-growth or tumorogenic pathways such as IL-6, NFκB, and MAP and ERK
kinases, as well as induce growth arrest through upregulation of TGFβ, p21 and p27
[60, 61]. However, for as many examples of GR regulated genes that potentially
block PC proliferation, there are examples of the opposite, such as downregulation
of p53 or upregulation of anti-apoptotic S100P. A definitive mechanism for how
GCs prevent PC growth and progression awaits further study in primary tissue.
For locally advanced PC, the androgen deprivation therapy in combination with
competitive AR blockers and radiation therapy are often used. Goserelin, a
gonadotropin releasing hormone (GnRH) agonist, blocks androgen production by
interrupting the endogenous feedback loop in the pituitary gland [57]. Androgen
analogs, such as bicalutamide and flutamide, then inhibit AR function by blocking
the testosterone-binding domain without activating the receptor. Despite the success
of this therapy, resistance can emerge, and for those patients second-generation
AR competitive antagonists have been developed, including enzaludamide [58].
Two modes of resistance to these second generation inhibitors have been described:
mutation of AR and surprisingly upregulation of GR. High GR levels have been
observed in PC bone metastases of enzaludamide resistant patients. In a preclinical
model of PC in which GR is overexpressed, it was shown that GR could substitute
for AR by regulating some of the same genes, including SGK1, STK39, and the
PSA gene (KLK3). In all, about 80 % of GR regulated genes overlapped with those
326 M.A. Pufall
regulated by AR. Further, it was shown that GCs could induce growth of these cells
in the presence of AR antagonists, and that effect could be blocked by GR antago-
nists [62]. This recent work suggests that in cells conditioned with anti-androgens,
the highly homologous AR and GR can complement each other. The cellular factors
that allow this complementation have not been identified, but this dependence on
GR function for PC growth suggests that combination therapy with anti-glucocorti-
coids, such as RU-486, may be useful in resistant PC.
These two examples highlight the potential dangers of using GC therapy in hor-
mone dependent cancers in which the mechanism is not well understood. GCs have
a clear though modest, effect on CRPC, with a >20 % response rate. However, GCs
can also be AR-like in hormone resistant PCs. Since the cellular determinants of GC
action in these two relapsed PCs have not been determined, administering GCs may
carry substantial risk.
Kaposi Sarcoma
Kaposi’s Sarcoma is a virally induced cancer that is best known for being activated in
patients with HIV/AIDS. Although mostly disfiguring, KS can have cause serious
problems, including lymphedema, gastrointestinal blockage, and in rare cases, death.
First-line treatment includes ABV (doxorubicin, bleomycin, and vincristine) among
several formulations. These treatments manage the disease, but are not a cure.
A retrospective study concluded that the Hodgkin’s formulation of EVAD (eto-
poside, vincristine, doxorubicin and dexamethasone) (EVAD) was an effective
treatment for advanced or relapsed Kaposi’s Sarcoma, though no mechanism was
proposed [63].
Cancers Where Glucocorticoids Are Not Used

as a Curative Agent
Glucocorticoids are often administered to help patients tolerate treatment, rather

than as a chemotherapeutic that targets the cancer itself. Reflective of the biology of
glucocorticoids described elsewhere in this book, their palliative effects are diverse.
In some chemotherapeutic regimens, for example those that include cisplatin, GCs
are first-line antiemetics (see below). For others, such as folate inhibitors, they are
used to blunt hypersensitivity, which can result in severe skin rashes (see Table 1.1).
Glucocorticoids are used for their anti-inflammatory properties to relieve bone pain
other discomfort that may arise from metastatic disease and CNS compression due
to metastatic disease. Though effective for these purposes, the use of glucocorti-
coids in patients with cancer caries some risk of protecting the tumor against che-
motherapeutic agents, or even increasing proliferation rates.
Table 1.1 Palliative uses for glucocorticoids

Tumor type Chemotherapy regimen Glucocorticoid Purpose Reference
Metastatic Pemetrexed (Altima), folic Dexamethasone Prevent skin [64]
bladder canceracid, vitamin B12 (4 mg) rashes
(vitamins)
Metastatic Pemetrexed (Altima) and Dexamethasone Prevent skin [65–67]
lung cancer cisplatin or bevacizumab (4 mg) rashes
(Avastin) and vitamins
Metastatic Paclitaxel, carboplatin Dexamethsone Blunt [68]
lung cancer premedicate with: (20, 8 mg if no hypersensitivity
diphenhydramine hypersensitivity)
cimetidine, Ranitidine or
Zantac
Mesolthelioma Cisplatin or carboplatin and Dexamethasone Prevent skin [69, 70]
pemetrexed (4 mg) rashes
Kaposi’s Paclitaxel premedicate with: Dexamethsone Blunt [71]
sarcoma diphenhydramine, (20, 8 mg if no hypersensitivity
cimetidine hypersensitivity)
Breast cancer Ixabepilone premedicate Dexamethasone Allergic [72–74]
with: diphenhydramine, (20 mg) reaction
Ranitidine
Use of GCs as Antiemetics
Chemotherapeutics, radiation, and surgery, all of which are important tools in the
fight against cancer, are often poorly tolerated by patients. In addition to their side
effects, they can cause severe nausea and vomiting that result in weakness, dehydra-
tion, and an unwillingness of patients to continue with therapy. The relative emetic
risk of chemotherapeutic agents have been categorized and published by American
Society of Clinical Oncology [75]. The categories range from high likelihood
(>90 %), with cisplatin being at the top of this range, to minimal likelihood (<10 %),
which includes agents such as vincristine and rituximab. The virtual universal reac-
tion of patients to cisplatin, which began being used in 1978, prompted the search
for effective anti-emetic agents [76, 77].
There are a number of agents that are used to ameliorate these effects, including:
dopaminergic blockers (e.g. metoclopramide); serotonin type 3 (5-HT3) receptor
inhibitors antagonists; NK1 receptor inhibitors (aprepitant); and low dose glucocor-
ticoids such as dexamethasone and methylprednisolone. The ASCO guidelines rec-
ommend how to administer these classes of drugs. For moderately emetogenic
treatments, a combination of dex and 5-HT is recommended. For highly emetogenic
agents, such as cisplatin, combinations of GCs, 5-HT, and NK1 inhibitors are rec-
ommended. Although the mechanism for dopaminergic blockers, 5-HT, and NK1
inhibitors are well established, how GCs work is not well understood [78].
Some mechanisms for how GCs work have been hypothesized. First, physiological
levels of GCs appear to be required for general well-being. Low levels of GC in and
328 M.A. Pufall
of themselves have been linked to nausea and vomiting [79]. Second, the anti-
inflammatory actions may be sufficient on some cases. Cyclooxegenase inhibitors
or ibuprofen, both of which suppress inflammation, can ameliorate the effects of
both radiation and some chemotherapeutic agents [80]. Third, GCs have been shown
to reduce 5-HT production, perhaps effectively blocking serotonin receptors in the
vagal nerve complexes that transmit the vomiting response [81]. Lastly, GCs inhibit
production of prostaglandins and substance P, both of which have been implicated
in the vomiting response [80]. Other mechanisms have been suggested as well, such
as reducing pain and direct effects on brain centers, but further research needs to be
done to uncover which GC effects are most beneficial. In addition GCs are used as
appetite stimulants in patients with cancer cachexia [82].
The Future of GCs in Cancer Treatment
GCs are still a critical component of chemotherapy for hematopoietic malignancies.

As described above, they are very effective in treatment of lymphoid malignancies,
including leukemia, lymphomas, and multiple myeloma, and much work is being
done to enhance their effect and overcome resistance. However, the use of GCs as
chemotherapeutic agents is considerably limited by their side effects, most promi-
nently osteonecrosis [14]. A good deal of effort has been invested in the develop-
ment of selective GR modulators (SGRMs, also sometime referred to as
SEGRMs)—compounds that work through GR to enhance the beneficial effect and
minimize or eliminate the side effects [83]. The prevailing model has been that GCs
exert their beneficial effects by repression of genes with side effects resulting from
gene activation [84]. Accordingly, the search for SGRMs has been focused on
development of what are known as dissociating compounds, or those that selec-
tively only allow GR to repress, but not activate genes. As more has been learned
about GR gene regulation in a variety of tissues and conditions, this model has
proven too simplistic (though it should be noted that the general trend holds). BIM
and BCL2, positive and negative regulators of apoptosis, respectively, are good
examples of how this model fails. To induce efficient apoptosis in leukemic blasts,
GCs activate BIM, but repress BCL2 [22, 85]. Thus in this tissue, both activation
and repression are beneficial. Further, both BIM and BCL2 appear to be similarly
regulated in bone, contributing to osteonecrosis [86]. Therefor, for leukemia, a
SGRM that represses but doesn’t activate would be a less effective chemotherapeutic
because BIM would not be activated. Further, even if a dissociating SGRM was
developed to preserve regulation of BIM and BCL2, it would still have severe osteo-
necrotic side effects.
The future use of SGRMs for treatment of hematopoietic malignancies thus
requires a model of GC function with tissue and gene-specific resolution. One
method is to first identify genes that are regulated by GR specifically in lymphoid
cells that contribute to cell death, but do not perform similar function in bone.
Recently, KLF13 was identified as a GR regulated gene that helps coordinate B and
T cell development and GC-induced cell death [85]. As this gene does not yet appear
to have a function in bone, development of compounds that separate allow KLF13
regulation, but not BIM or BCL2, might allow GC-induced apoptosis in leukemic
blasts but not bone. There are other strategies currently under investigation to develop
activity and tissue specific GC function. One is to develop a deeper of understanding
of not just which genes are regulated, but how they are regulated in different tissues
to identify alternative targets that enhance specific GR functions. One key may be in
understanding which GR cofactors are used in each tissue. For examples, the GR
cofactors NCOA1, 2, and 3, are differentially expressed in tissues [87]. If one, such
as NCOA2, is the primary GR cofactor in bone, but not B cells, then it could be
targeted to block bone-cell death but still allow GC-induced apoptosis in leukemic
blasts. Lastly, it may be possible to develop GCs whose chemical makeup or deliv-
ery method partition uptake specifically to lymphoid cells over bone. In this way,
the drug would provide selective modulation of GR function at the tissue level, but
not at the level of gene regulation. Each of these strategies are the subject of current
research efforts.
References
1. Kendall EC. Hormones. Annu Rev Biochem. 1941;10:285–336.

2. Dougherty TF, White A. Effect of pituitary adrenotropic hormone on lymphoid tissue. Exp
Biol Med. 1943;53(2):132–3. doi:10.3181/00379727-53-14219P.
3. Heilman FR, Kendall EC. The influence of 11-dehydr0-17-hydroxycorticosterone (compound
E) on the growth of a malignant tumor in the Mouse1. Endocrinology. 1944;34(6):416–20.
4. Murphy JB, Sturm E. The effect of adrenal cortical and pituitary adrenotropic hormones on
transplanted leukemia in rats. Science. 1944;99(2572):303. doi:10.1126/science.99.2572.303.
5. Pearson OH, Eliel LP, Rawson RW, Dobriner K, Rhoads CP. Acth‐ and cortisone‐induced
regression of lymphoid tumors in man. A preliminary report. Cancer. 1949;2(6):943–5.
doi:10.1002/1097-0142(194911)2:6<943::AID-CNCR2820020602>3.0.CO;2-P.
6. Pearson OH, Eliel LP. Use of pituitary adrenocorticotropic hormone (ACTH) and cortisone in
lymphomas and leukemias. J Am Med Assoc. 1950;144(16):1349–53.
7. DeVita VT, Chu E. A history of cancer chemotherapy. Cancer Res. 2008;68(21):8643–53.
doi:10.1158/0008-5472.CAN-07-6611.
8. Broome JD. Evidence that the L-asparaginase of guinea pig serum is responsible for its
antilymphoma effects. J Exp Med. 1963;118:121–48.
9. Pui C-H, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med.
2006;354(2):166–78. doi:10.1056/NEJMra052603.
10. Dördelmann M, Reiter A, Borkhardt A, et al. Prednisone response is the strongest predictor of
treatment outcome in infant acute lymphoblastic leukemia. Blood. 1999;94(4):1209–17.
11. Lonnerholm G, Thorn I, Sundstrom C, et al. In vitro cellular drug sensitivity at diagnosis is
correlated to minimal residual disease at end of induction therapy in childhood acute lympho-
blastic leukemia. Leuk Res. 2009;33(1):46–53. doi:10.1016/j.leukres.2008.06.012.
12. Hongo T, Yajima S, Sakurai M, Horikoshi Y, Hanada R. In vitro drug sensitivity testing can
predict induction failure and early relapse of childhood acute lymphoblastic leukemia. Blood.
1997;89(8):2959–65.
13. Kaspers GJ, Veerman AJ, Pieters R, et al. In vitro cellular drug resistance and prognosis in
newly diagnosed childhood acute lymphoblastic leukemia. Blood. 1997;90(7):2723–9.
330 M.A. Pufall
14. Inaba H, Pui C-H. Glucocorticoid use in acute lymphoblastic leukaemia. Lancet Oncol.
2010;11(11):1096–106. doi:10.1016/S1470-2045(10)70114-5.
15. Teuffel O, Kuster SP, Hunger SP, et al. Dexamethasone versus prednisone for induction ther-
apy in childhood acute lymphoblastic leukemia: a systematic review and meta-analysis.
Leukemia. 2011;25(8):1232–8. doi:10.1038/leu.2011.84.
16. Mittelstadt PR, Monteiro JP, Ashwell JD. Thymocyte responsiveness to endogenous glucocor-
ticoids is required for immunological fitness. J Clin Invest. 2012;122(7):2384–94. doi:10.1172/
JCI63067.
17. Gruver-Yates AL, Quinn MA, Cidlowski JA. Analysis of glucocorticoid receptors and their
apoptotic response to dexamethasone in male murine B cells during development.
Endocrinology. 2014;155(2):463–74. doi:10.1210/en.2013-1473.
18. Smith LK, Cidlowski JA. Glucocorticoid-induced apoptosis of healthy and malignant lympho-
cytes. Prog Brain Res. 2010;182:1–30. doi:10.1016/S0079-6123(10)82001-1.
19. Ploner C, Schmidt S, Presul E, et al. Glucocorticoid-induced apoptosis and glucocorticoid
resistance in acute lymphoblastic leukemia. J Steroid Biochem Mol Biol. 2005;93(2–5):153–60.
doi:10.1016/j.jsbmb.2004.12.017.
20. Lambrou GI, Vlahopoulos S, Papathanasiou C, et al. Prednisolone exerts late mitogenic and
biphasic effects on resistant acute lymphoblastic leukemia cells: relation to early gene expres-
sion. Leuk Res. 2009;33(12):1684–95. doi:10.1016/j.leukres.2009.04.018.
21. Bonapace L, Bornhauser BC, Schmitz M, et al. Induction of autophagy-dependent necroptosis
is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resis-
tance. J Clin Invest. 2010;120(4):1310–23. doi:10.1172/JCI39987DS1.
22. Ploner C, Rainer J, Niederegger H, et al. The BCL2 rheostat in glucocorticoid-induced apoptosis
of acute lymphoblastic leukemia. Leukemia. 2007;22(2):370–7. doi:10.1038/sj.leu.2405039.
23. Schmidt S, Rainer J, Ploner C, Presul E, Riml S, Kofler R. Glucocorticoid-induced apoptosis
and glucocorticoid resistance: molecular mechanisms and clinical relevance. Cell Death
Differ. 2004;11:S45–55. doi:10.1038/sj.cdd.4401456.
24. Tissing WJE, den Boer ML, Meijerink JPP, et al. Genomewide identification of prednisolone-
responsive genes in acute lymphoblastic leukemia cells. Blood. 2007;109(9):3929–35.
doi:10.1182/blood-2006-11-056366.
25. Sionov RV, Spokoini R, Kfir-Erenfeld S, Cohen O, Yefenof E. Chapter 6 mechanisms regulat-
ing the susceptibility of hematopoietic malignancies to glucocorticoid‐induced apoptosis.
Adv Cancer Res. 2008;101:127–248. doi:10.1016/S0065-230X(08)00406-5.
26. Schmidt S. Identification of glucocorticoid-response genes in children with acute lymphoblas-
tic leukemia. Blood. 2006;107(5):2061–9. doi:10.1182/blood-2005-07-2853.
27. Holleman A, Cheok MH, den Boer ML, et al. Gene-expression patterns in drug-resistant acute
lymphoblastic leukemia cells and response to treatment. N Engl J Med. 2004;351(6):533–42.
doi:10.1056/NEJMoa033513.
28. Wei G, Twomey D, Lamb J, et al. Gene expression-based chemical genomics identifies
rapamycin as a modulator of MCL1 and glucocorticoid resistance. Cancer Cell. 2006;
10(4):331–42. doi:10.1016/j.ccr.2006.09.006.
29. Bachmann PS. Dexamethasone resistance in B-cell precursor childhood acute lymphoblastic
leukemia occurs downstream of ligand-induced nuclear translocation of the glucocorticoid
receptor. Blood. 2005;105(6):2519–26. doi:10.1182/blood-2004-05-2023.
30. Cario G, Fetz A, Bretscher C, et al. Initial leukemic gene expression profiles of patients with
poor in vivo prednisone response are similar to those of blasts persisting under prednisone
treatment in childhood acute lymphoblastic leukemia. Ann Hematol. 2008;87(9):709–16.
doi:10.1007/s00277-008-0504-x.
31. Hulleman E, Kazemier KM, Holleman A, et al. Inhibition of glycolysis modulates predniso-
lone resistance in acute lymphoblastic leukemia cells. Blood. 2009;113(9):2014–21.
doi:10.1182/blood-2008-05-157842.
32. Buentke E, Nordström A, Lin H, et al. Glucocorticoid-induced cell death is mediated through
reduced glucose metabolism in lymphoid leukemia cells. Blood Cancer J. 2011;1(7):e31–9.
doi:10.1038/bcj.2011.27.
33. Chen CS, Sorensen PH, Domer PH, et al. Molecular rearrangements on chromosome 11q23
predominate in infant acute lymphoblastic leukemia and are associated with specific biologic
variables and poor outcome. Blood. 1993;81(9):2386–93.
34. Briggs SD, Bryk M, Strahl BD, et al. Histone H3 lysine 4 methylation is mediated by Set1 and
required for cell growth and rDNA silencing in Saccharomyces cerevisiae. Genes Dev.
2001;15(24):3286–95. doi:10.1101/gad.940201.
35. Milne TA, Briggs SD, Brock HW, et al. MLL targets SET domain methyltransferase activity to
Hox gene promoters. Mol Cell. 2002;10(5):1107–17.
36. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-
cell development. Nat Rev Cancer. 2007;7(11):823–33. doi:10.1038/nrc2253.
37. Mullighan CG, Phillips LA, Su X, et al. Genomic analysis of the clonal origins of relapsed acute
lymphoblastic leukemia. Science. 2008;322(5906):1377–80. doi:10.1126/science.1164266.
38. Piovan E, Yu J, Tosello V, et al. Direct reversal of glucocorticoid resistance by AKT inhibition in
acute lymphoblastic leukemia. Cancer Cell. 2013;24(6):766–76. doi:10.1016/j.ccr.2013.10.022.
39. Galliher-Beckley AJ, Williams JG, Cidlowski JA. Ligand-independent phosphorylation of the
glucocorticoid receptor integrates cellular stress pathways with nuclear receptor signaling.
Mol Cell Biol. 2011;31(23):4663–75. doi:10.1128/MCB.05866-11.
40. Gruber G, Carlet M, Türtscher E, et al. Levels of glucocorticoid receptor and its ligand deter-
mine sensitivity and kinetics of glucocorticoid-induced leukemia apoptosis. Leukemia. 2009;
23(4):820–3. doi:10.1038/leu.2008.360.
41. Schmidt S, Irving JAE, Minto L, et al. Glucocorticoid resistance in two key models of acute
lymphoblastic leukemia occurs at the level of the glucocorticoid receptor. FASEB
J. 2006;20(14):2600–2. doi:10.1096/fj.06-6214fje.
42. Wu I, Shin SC, Cao Y, et al. Selective glucocorticoid receptor translational isoforms reveal
glucocorticoid-induced apoptotic transcriptomes. Cell Death Dis. 2013;4, e453. doi:10.1038/
cddis.2012.193.
43. DeVita VT, Lawrence TS, Rosenberg SA. DeVita, Hellman, and Rosenberg’s cancer: princi-
ples & practice of oncology, vol. 2. Philadelphia, PA: Lippincott Williams & Wilkins; 2008.
44. Thomas DA. Outcome with the hyper-CVAD regimens in lymphoblastic lymphoma. Blood.
2004;104(6):1624–30. doi:10.1182/blood-2003-12-4428.
45. Real PJ, Tosello V, Palomero T, et al. γ-secretase inhibitors reverse glucocorticoid resistance
in T cell acute lymphoblastic leukemia. Nat Med. 2008;15(1):50–8. doi:10.1038/nm.1900.
46. Rajkumar SV. Multiple myeloma. Curr Probl Cancer. 2009;33(1):7–64. doi:10.1016/j.
currproblcancer.2009.01.001.
47. Kyle RA, Rajkumar SV. Multiple myeloma. Blood. 2008;111(6):2962–72. doi:10.1182/
blood-2007-10-078022.
48. Munshi NC, Anderson KC. New strategies in the treatment of multiple myeloma. Clin Cancer
Res. 2013;19(13):3337–44. doi:10.1158/1078-0432.CCR-12-1881.
49. Grugan KD, Ma C, Singhal S, Krett NL, Rosen ST. Dual regulation of glucocorticoid-induced
leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in
multiple myeloma. J Steroid Biochem Mol Biol. 2008;110(3–5):244–54. doi:10.1016/j.
jsbmb.2007.11.003.
50. Raab MS, Podar K, Breitkreutz I, Richardson PG, Anderson KC. Multiple myeloma. Lancet.
2009;374(9686):324–39. doi:10.1016/S0140-6736(09)60221-X.
51. Viviani S, Zinzani PL, Rambaldi A. ABVD versus BEACOPP for Hodgkin’s lymphoma
when high-dose salvage is planned. N Engl J Med. 2011;365(3):203–12. doi:10.1056/
NEJMoa1100340.
52. Rosenthal MC, Saunders RH, Schwartz LI, Zannos L, Perez Santiago E, Dameshek W. The use
of adrenocorticotropic hormone and cortisone in the treatment of leukemia and leukosarcoma.
Blood. 1951;6(9):804–23.
53. Smolej L. The role of high-dose corticosteroids in the treatment of chronic lymphocytic leuke-
mia. Expert Opin Investig Drugs. 2012;21(7):1009–17. doi:10.1517/13543784.2012.690393.
54. Burger JA, Montserrat E. Coming full circle: 70 years of chronic lymphocytic leukemia cell
redistribution, from glucocorticoids to inhibitors of B-cell receptor signaling. Blood.
2013;121(9):1501–9. doi:10.1182/blood-2012-08-452607.
332 M.A. Pufall
55. Connors JM. Non-Hodgkin lymphoma: the clinician’s perspective—a view from the receiving
end. Mod Pathol. 2013;26(s1):S111–8. doi:10.1038/modpathol.2012.184.
56. Kassi E, Moutsatsou P. Glucocorticoid receptor signaling and prostate cancer. Cancer Lett.
2011;302(1):1–10. doi:10.1016/j.canlet.2010.10.020.
57. Agarwal N, Di Lorenzo G, Sonpavde G, Bellmunt J. New agents for prostate cancer. Ann
Oncol. 2014;25(9):1700–9. doi:10.1093/annonc/mdu038.
58. Cookson MS, Roth BJ, Dahm P, et al. Castration-resistant prostate cancer: AUA guideline.
J Urol. 2013;190(2):429–38. doi:10.1016/j.juro.2013.05.005.
59. Montgomery B, Mostaghel E, Cheng H, Drechsler J. Glucocorticoids and prostate cancer
treatment: friend or foe? Asian J Androl. 2014;16(3):354–8. doi:10.4103/1008-682X.125392.
60. Yemelyanov A, Czwornog J, Chebotaev D, et al. Tumor suppressor activity of glucocorticoid
receptor in the prostate. Oncogene. 2006;26(13):1885–96. doi:10.1038/sj.onc.1209991.
61. Cavarretta IT, Neuwirt H, Untergasser G, et al. The antiapoptotic effect of IL-6 autocrine loop
in a cellular model of advanced prostate cancer is mediated by Mcl-1. Oncogene.
2006;26(20):2822–32. doi:10.1038/sj.onc.1210097.
62. Arora VK, Schenkein E, Murali R, et al. Glucocorticoid receptor confers resistance to antian-
drogens by bypassing androgen receptor blockade. Cell. 2013;155(6):1309–22. doi:10.1016/j.
cell.2013.11.012.
63. Zhong DT, Shi CM, Chen Q, Huang JZ, Liang JG, Lin D. Etoposide, vincristine, doxorubicin
and dexamethasone (EVAD) combination chemotherapy as second-line treatment for advanced
AIDS-related Kaposi’s sarcoma. J Cancer Res Clin Oncol. 2011;138(3):425–30. doi:10.1007/
s00432-011-1109-7.
64. Sweeney CJ. Phase II, study of pemetrexed for second-line treatment of transitional cell cancer
of the urothelium. J Clin Oncol. 2006;24(21):3451–7. doi:10.1200/JCO.2005.03.6699.
65. Scagliotti GV, Parikh P, von Pawel J, et al. Phase III study comparing cisplatin plus gem-
citabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage
non-small-cell lung cancer. J Clin Oncol. 2008;26(21):3543–51. doi:10.1200/
JCO.2007.15.0375.
66. Hanna N. Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-
cell lung cancer previously treated with chemotherapy. J Clin Oncol. 2004;22(9):1589–97.
doi:10.1200/JCO.2004.08.163.
67. Herbst RS, O’Neill VJ, Fehrenbacher L, et al. Phase II study of efficacy and safety of bevaci-
zumab in combination with chemotherapy or erlotinib compared with chemotherapy alone for
treatment of recurrent or refractory non small-cell lung cancer. J Clin Oncol. 2007;25(30):4743–50.
doi:10.1200/JCO.2007.12.3026.
68. Belani CP, Ramalingam S, Perry MC, et al. Randomized, phase III study of weekly paclitaxel
in combination with carboplatin versus standard every-3-weeks administration of carboplatin
and paclitaxel for patients with previously untreated advanced non-small-cell lung cancer.
J Clin Oncol. 2008;26(3):468–73. doi:10.1200/JCO.2007.13.1912.
69. Vogelzang NJ. Phase III, study of pemetrexed in combination with cisplatin versus cisplatin
alone in patients with malignant pleural mesothelioma. J Clin Oncol. 2003;21(14):2636–44.
doi:10.1200/JCO.2003.11.136.
70. Ceresoli GL, Phase II. Study of pemetrexed plus carboplatin in malignant pleural mesothelioma.
J Clin Oncol. 2006;24(9):1443–8. doi:10.1200/JCO.2005.04.3190.
71. Gill PS, Tulpule A, Espina BM, et al. Paclitaxel is safe and effective in the treatment of
advanced AIDS-related Kaposi’s sarcoma. J Clin Oncol. 1999;17(6):1876–83.
72. Perez EA, Lerzo G, Pivot X, et al. Efficacy and safety of ixabepilone (BMS-247550) in a phase
II study of patients with advanced breast cancer resistant to an anthracycline, a taxane, and
capecitabine. J Clin Oncol. 2007;25(23):3407–14. doi:10.1200/JCO.2006.09.3849.
73. Roche H, Yelle L, Cognetti F, et al. Phase II clinical trial of ixabepilone (BMS-247550), an
epothilone B analog, as first-line therapy in patients with metastatic breast cancer previously
treated with anthracycline chemotherapy. J Clin Oncol. 2007;25(23):3415–20. doi:10.1200/
JCO.2006.09.7535.
74. Thomas E, Tabernero J, Fornier M, et al. Phase II clinical trial of ixabepilone (BMS-247550),
an epothilone B analog, in patients with taxane-resistant metastatic breast cancer. J Clin Oncol.
2007;25(23):3399–406. doi:10.1200/JCO.2006.08.9102.
75. American Society of Clinical Oncology, Kris MG, Hesketh PJ, et al. American Society of
Clinical Oncology guideline for antiemetics in oncology: update 2006. J Clin Oncol.
2006;24(18):2932–47. doi:10.1200/JCO.2006.06.9591.
76. Aapro MS, Alberts DS. Dexamethasone as an antiemetic in patients treated with cisplatin.
N Engl J Med. 1981;305(9):520.
77. Rich WM, Abdulhayoglu G, DiSaia PJ. Methylprednisolone as an antiemetic during cancer
chemotherapy—a pilot study. Gynecol Oncol. 1980;9(2):193–8.
78. Chu C-C, Hsing C-H, Shieh J-P, Chien C-C, Ho C-M, Wang J-J. The cellular mechanisms of
the antiemetic action of dexamethasone and related glucocorticoids against vomiting. Eur J
Pharmacol. 2014;722(C):48–54. doi:10.1016/j.ejphar.2013.10.008.
79. Hursti TJ, Fredrikson M, Steineck G, Börjeson S, Fürst CJ, Peterson C. Endogenous cortisol
exerts antiemetic effect similar to that of exogenous corticosteroids. Br J Cancer.
1993;68(1):112–4.
80. Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids—new mechanisms for old
drugs. N Engl J Med. 2005;353(16):1711–23. doi:10.1056/NEJMra050541.
81. Mantovani G, Maccio A, Massa E, Lai P, Esu S. Cisplatin induces serotonin release from
human peripheral blood mononuclear cells of cancer patients and methylprednisolone inhibits
this effect. Oncol Rep. 1997;4(5):1051–3.
82. Inui A. Cancer anorexia-cachexia syndrome: current issues in research and management. CA
Cancer J Clin. 2002;52(2):72–91.
83. Quax RAM, Peeters RP, Feelders RA. Selective glucocorticoid receptor modulators: future
of glucocorticoid immunosuppressive therapy? Endocrinology. 2011;152(8):2927–9.
doi:10.1210/en.2011-1258.
84. Clark AR, Belvisi MG. Maps and legends: the quest for dissociated ligands of the glucocorticoid
receptor. Pharmacol Ther. 2012;134(1):54–67. doi:10.1016/j.pharmthera.2011.12.004.
85. Jing D, Bhadri VA, Beck D, et al. Opposing regulation of BIM and BCL2 controls
glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells. Blood.
2014;125(2):273–83. doi:10.1182/blood-2014-05-576470.
86. Moutsatsou P, Kassi E, Papavassiliou AG. Glucocorticoid receptor signaling in bone cells.
Trends Mol Med. 2012;18(6):348–59. doi:10.1016/j.molmed.2012.04.005.
87. McKenna NJ, O’Malley BW. Combinatorial control of gene expression by nuclear receptors
and coregulators. Cell. 2002;108(4):465–74.
Part IV
Miscellaneous Topics
Chapter 15
Animal Models of Altered Glucocorticoid
Signaling
Charles Harris
Abstract In this chapter we will review genetically engineered mice with alterations
in glucocorticoid signaling. Most of the mice involve direct alterations to the gluco-
corticoid receptor locus, but we will touch briefly on other relevant models includ-
ing 11-β-HSD transgenics which alter tissue levels of ligand as well as mice with
glucocorticoid excess. Of course, the number of mice with mutations in genes such
as GR targets and transcriptional coregulators is beyond the scope of this chapter.
Keywords Altered glucocorticoid signaling • Animal models • 11-β-HSD transgen-

ics • Hypomorphs • GR-null mice • Tissue specific KO • Tissue-specific transgenics
Version 1.0: Hypomorphs
The scientific community was quite eager to know the function of the glucocorticoid
receptor in vivo. Transgenic mice were introduced in 1981 [1], but gene targeting via
homologous recombination was not introduced until 1990 [2] and did not become
commonplace until the mid-1990s. GR was cloned in 1985 [3–5]. Therefore, in order
to probe the function of GR in vivo in the early 1990s, multiple groups created trans-
genic animals with transgenes encoding antisense for GR coding sequence, thereby
creating in essence a “tissue specific knockdown” governed by the promoter driving
the anti-sense construct. The first paper expressed a rat antisense GR sequence under
the control of a human neurofilament gene promoter. These mice were obese, had
decreased but detectable glucocorticoid binding and GR mRNA in the brain, mildly
increased serum corticosterone and very increased ACTH levels [6]. Another group
used a similar approach to knockdown GR with a transgene encoding 3′ UTR rat GR
antisense driven by lck, an early T-cell specific promoter. These mice had small
C. Harris (*)
Campus Box 8127, St. Louis, MO 63110, USA

338 C. Harris
thymi and had decreased number of thymocytes due to decreased differentiation of

mature thymocytes as well as increased apoptosis [7].
Version 2.0: Whole Body Knockouts
GR-null mice have been generated separately from several different groups [8–10].
GR-null mice are born at the expected Mendelian frequency, but die within hours of
birth due to respiratory difficulties and defects in lung maturation. This finding is
concordant with clinical observations showing infants born prematurely suffer from
similar defects in lung maturity if mothers are not given glucocorticoids shortly
before birth. In addition, in GR-null mice, circulating ACTH and corticosterone
levels are high due to loss of negative feedback. The elevated corticosterone does
not activate GR in these animals (there is none), but would be predicted to activate
MR and cause a syndrome of apparent mineralocorticoid excess. This phenomenon
has been described in persons with resistance to glucocorticoid hormone [11].
GR-null mice also have cortical adrenal hyperplasia, presumably from increased
ACTH levels stimulating intact ACTH receptors (MC2R) in the adrenal cortex.
The role of GR in the adrenal chromaffin lineage has been subject to reinterpretation
over the years. GR-null mice were originally reported to be missing adrenal medullae
[8], but more careful studies showed the existence of adrenal chromaffin cells in
normal numbers with the more specific phenotype of conversion of adrenergic to
noradrenergic cells due to loss of GR-mediated activation of the enzyme PNMT
(Phenylethanolamine N-methyltransferase), that converts noradrenaline to adrena-
line). More recently, a study of tissue-specific loss of GR in noradrenergic cells
(using the cre-lox system and a dopamine beta hydroxylase driven cre line) revealed
loss of chromaffin cells as adults [12]. The synthesis of these findings is that GR is
not required for the formation of adrenal chromaffin cells but does help maintain
these cells into adulthood. An attempt to rescue neonatal mortality due to the lung
phenotype by crossing GR-null mice to transgenic mice expressing GR under the
SP-C promoter was unsuccessful [13]. GR-null mice have a defect in epidermal dif-
ferentiation and skin barrier function [14]. Because neonatal mortality has pre-
cluded analysis of adult GR-null animals, several investigators have alternatively
studied GR-null cells in vitro, in transplant experiments, GR heterozygous mice
in vivo [10] and later tissue specific null mice. GR-null erythroid progenitors have
decreased erythropoiesis in vitro [15]. In an elegant set of experiments it was dem-
onstrated that E18.5 GR-null mice have a disorganized endocrine pancreas. GR-null
pancreas appeared normal at E15.5 and the disorganization that occurred between
E15 and E18.5 could be rescued by transplanting the pancreas under the kidney
capsule of a GR expressing mouse. The conclusion of these experiments was the
disorganization of the GR-null pancreas was an indirect effect [16].
15 Animal Models of Altered Glucocorticoid Signaling 339
Version 3.0: Tissue Specific KO

and Tissue-Specific Transgenics
The advent of cre lox technology allows specific deletion of gene products from
tissues or cell types of interest. A thorough review of the subject can be found here
[17]. Briefly, a critical region of the gene of interest is modified to be flanked by 34
base pair loxP sites, which are introduced by homologous recombination in such a
manner so as not to disrupt gene function. So called “floxed” (flanking loxP mice)
are then crossed to transgenic mice expressing the bacteriophage cre recombinase
driven by the promoter of interest. The cre recombinase is expressed only in the tis-
sue of interest where it excises the DNA between the two loxP sites, resulting in a
null allele.
The pleiotropic actions of glucocorticoids have made it difficult to draw direct
links between specific gene activations with physiological observations. For exam-
ple, glucocorticoids are known to cause osteoporosis, but this could be due to direct
actions on bone, both osteoblasts and osteoclasts, but also indirectly through reduc-
tions in sex hormones, reductions in Growth Hormone, along with actions on the
intestinal absorption of calcium. Another example is the known sarcopenic effects of
glucocorticoids which could be explained by direct actions on muscle, but indirectly
through suppression of the GH-IGF1 axis as well. Tissue specific deletion of GR
has allowed a dissection of these pleiotropic actions to determine the contribution of
cell-autonomous effects on respective phenotypes. Tissue-specific deletion of GR
has confirmed effects on bone (osteoblast) and muscle are largely cell autonomous
(see below). This is of clinical importance as novel therapeutics with tissue-specific
delivery of GR modulators becomes a possible avenue in the future [18–20].
Glucocorticoid Receptor has been deleted in many different tissues. We do not have
sufficient room to include all of these mice, but rather describe a handful and direct
the reader to MGI for a complete listing. With respect to GR several different floxed
mice have been generated with loxP sites flanking different exons of GR genomic
locus [21–25]. Recently a comparison between exon 2 floxed mice and exon 3
floxed mice was used using the Sim1-cre mice with expression in the hypothalamus.
Exon 3 mice gave an interesting expected phenotype of Cushing’s syndrome (due to
loss of GR-mediated negative feedback on CRH in the hypothalamus, but exon 2
flanked mice crossed to the same cre transgenic mouse did not display the pheno-
type. This may be due to inefficient excision of exon 2 in exon 2 floxed mice bearing
certain cre transgenes. Alternatively, there may be truncated GR after exon 2 exci-
sion that can retain some transcriptional activity [26]. These observations do not
derail previous results obtained with exon 2 floxed mice, as some cre lines do result
in complete excision and loss of function when exon 2 is excised. However, given
the potential for residual function with exon 2, we anticipate greater use of exon 3
floxed mice for future studies.
340 C. Harris
Lung
Since GR-null mice died due to defects in lung maturation, it was of great interest
to know which cell type was responsible for the phenotype. Unfortunately there has
not been consensus among investigators in this area. One group found that mesen-
chymal expression of GR governed the phenotype since mesenchymal-specific
deletion of GR using col1-cre or dermo1-cre mice recapitulated the neonatal mor-
tality phenotype [27, 28] but deletion using epithelial SPC-cre did not have a
phenotype whereas another group observed decreased viability with an epithelial
specific GR KO mouse that was generated by crossing GR floxed mice to mice
carrying SPC-rtTA and tetO-Cre transgenes [29]. So, there is disagreement as to
the necessity of GR in these cell types. As mentioned above, attempts to rescue
the GR-null mouse by crossing to a transgenic expressing GR under the control of
surfactant protein C regulatory elements were unsuccessful. It would be interest-
ing to attempt a similar rescue with a mesenchymal targeted GR transgene.
However, it is not clear whether a GR-null mouse rescued from the lung pheno-
type would survive given defects in other cell types or gain of function phenotype
from excessive action on the mineralocorticoid receptor (syndrome of apparent
mineralocorticoid excess).
Skin
The skin phenotype of global GR-null mice was described above. This phenotype is
recapitulated in keratinocyte specific GR KO mice generated using cre lox technology
and keratin14-cre mice [30]. These epidermal GR KO mice also have increased
susceptibility to chemical induced skin tumors [31]. Overexpression of GR in trans-
genic mice driven by the keratin5 promoter results in abnormal skin including hypo-
plasia and impaired hyperplastic response to topical TPA [32].
Cardiovascular System
GR action in the distal nephron is not critical for glucocorticoid mediated hyperten-
sion [33] as mice with GR deleted in the distal nephron (Ksp-cre) had a hypertensive
response to dexamethasone similar to WT mice. In contrast, mice with endothelial
specific GR KO mice were created by using tie2-cre mice. Endothelial GR KO mice
are resistant to dexamethasone mediated hypertension and have increased sensitivity
to hemodynamic shock associated with sepsis as mimicked by systemic LPS injec-
tions [34]. This effect is thought to be mediated by loss of negative regulation of
iNOS in maintaining vascular tone. A conditional induction of a GR transgene in the
heart resulted in arrhythmia [35]. Deletion of GR in the heart (α-MHC-cre) results in
death at age 6 months from heart failure, indicating a previously unknown role for
GR signaling in the heart [22]. These findings have potential clinical implications for
the development of GR antagonists in metabolic disease. In addition global GR-null
embryos were found to have decreased cardiac function [36].
Muscle
Muscle specific deletion of GR was accomplished using muscle creatine kinase-cre

mice. Muscle specific GR KO mice were completely protected from dexamethasone
mediated muscle atrophy [37] indicating this is a cell autonomous process. Induction
of the atrogins MURF1 and MAFbx1 was abrogated in muscle specific GR KO mice
treated with glucocorticoid. This was in contrast to the lack of effect seen in muscle-
specific GRKO mice on muscle atrophy and atrogin gene activation seen with sciatic
nerve transection. There was a partial effect on both atrogin gene induction and
muscle atrophy seen with nutritional deprivation suggesting that glucocorticoids
combine with other signals in that paradigm. A separate group has shown muscle GR
is required for muscle atrophy following either chemotherapy or endotoxin [38, 39].
These reports did not indicate the contribution of muscle GR to whole body changes
in glucose homeostasis, but may be the subject of future studies.
Gastrointestinal System
GR is dispensable for the development of the intestine as GR-null intestines are

morphologically normal [40]. However, the perinatal death of GR-null mice pre-
cluded examination of GR in adult intestine. Enterocyte specific GR KO mice were
created using villin-cre. Intestine-specific GR KO mice had loss of dexamethasone-
stimulated intestinal glucose absorption that was thought to be mediated by transcrip-
tional activation of genes encoding glucose transporters [41]. Recently, it was shown
dexamethasone caused gastroparesis in wild type mice. This effect was not mediated
by enterocytes as it was preserved in enterocyte specific GR KO animals (villin-cre).
Rather, the authors believed it is due to loss of NO signaling in the stomach [42].
Immune System
Because GR-null animals die at birth one must use either tissue-specific knockouts
or alternatively one can isolate fetal liver from GR-null animals and perform bone
marrow transplants or perform bone marrow transplants from a tissue-specific KO to
narrow down the cell type. Such bone marrow transplants were performed to show
that macrophage GR was dispensable for atherogenesis in LDLR-null mice, but
342 C. Harris
macrophage GR contributed to vascular calcification [43]. Glucocorticoids are effec-

tive in multiple sclerosis and appear to act on T-cells in mouse models of autoimmune
encephalitis [44] as evidenced by T-cell specific (lck) and macrophage specific KO
(LysM-cre) mice. In a contact hypersensitivity model myeloid cells seemed to be the
predominant mediator of glucocorticoid effects as myeloid specific GR KO mice
(LysM-cre) were resistant to the anti-inflammatory effects of dexamethasone whereas
T-cell (lck-Cre) and keratinocyte (K14cre-ER following tamoxifen injection) were
unaffected [45]. T-cells were identified as the critical site of action for the therapeutic
effects of glucocorticoid in an antigen-induced model of rheumatoid arthritis [46].
Bone
One group found osteoblast specific deletion of GR (Runx2-cre) rendered mice resis-
tant to glucocorticoid-induced osteoporosis whereas deletion of GR in osteoclasts
(LysM-cre) had no effect [47]. This same group did not see any defects in bone
development as assessed by GR-null mice analyzed at E18.5 and also reported com-
plete sensitivity of GR dim mice (see below) to glucocorticoid induced osteoporosis,
implicating GR monomers in this process. Another group has implicated an anabolic
function of glucocorticoids in bone development from the phenotype of transgenic
mice expressing the glucocorticoid inactivating 11-β-HSD2 in osteoblasts [48].
Another group showed that action of GR in osteoclasts was the dominant determinant
on the bone phenotype using a similar LysM-cre breeding strategy to create osteo-
clast deficiency of GR [49]. From these studies it is clear GR action on the osteoblast
is critical for glucocorticoid induced osteoporosis, but there may be a role in the
osteoclast as well. The actions of GR mediating osteoporosis do not require func-
tional dimers. This has implications for the possible development of selective GR
modulators as treatments for osteoporosis or to be used in conjunction with another
glucocorticoid to attenuate glucocorticoid induced osteoporosis.
Tissues Involved in Metabolism
Deletion of GR in hepatocytes using albumin-cre resulted in neonatal mortality

with incomplete penetrance [50]. These mice displayed fasting hypoglycemia and
amelioration of streptozotocin induced diabetes [51]. The growth impairment in
these mice phenocopied liver-specific deletion of Stat5 using the same cre line. The
authors went on to show via microarrays that GR and Stat5 regulate similar sets of
genes in the liver [52]. Deletion of GR in the liver with a different albumin-cre dis-
played no such viability defects. In addition, the latter mice had normal response to
hepatectomy mediated hepatic regeneration [53]. The discrepant data with respect
to viability is likely due to the timing of the two liver-specific cre activations, with
early deletion being responsible for mortality and runting. The albumin-cre lines
used in these studies are similar, but made by different investigators. The early
expressing albumin-cre that resulted in mortality and a growth phenotype contained

additional alpha fetoprotein enhancer elements which may explain its earlier expres-
sion. Alternatively the timing of expression could have been affected by the genomic
integration site. Liver-specific GR KO mice have also demonstrated a role for GR
in bile acid homeostasis [54]. Specific deletion of GR in pancreatic beta cells using
RIP-cre had no effect on alpha or beta cell mass whereas deletion of GR in the pre-
cursor, using Pdx1-cre resulted in a doubling of beta cell mass [55]. Furthermore, as
noted above GR-null embryos have disorganized pancreata.
CNS and HPA Axis
Glucocorticoids are known to cause negative feedback on hypothalamic CRH and

pituitary ACTH secretion and the phenotypes of tissue-specific GR-null mice are
informative as to the relative contribution of these sites. Mice with neuron specific
deletion of GR (nestin-Cre) displayed Cushing’s syndrome due to loss of negative
feedback at the hypothalamic CRH producing neurons. These mice also had reduced
anxiety [25]. The phenotype of this mouse is similar to the CRH-Tg mouse (see
below) as well as the mice with deletion of GR in the hypothalamus using Sim1-cre
and to the phenotype of mice with the (-120) mutation in the CRH promoter (see
below), all resulting in loss of negative feedback at the level of the hypothalamus.
Mice with loss of GR in the ACTH producing cells of the pituitary (POMC-cre)
have elevated corticosterone levels in the first week of life, but normal peak and
nadir corticosterone levels as adults [56]. Pituitary GR KO mice did have impaired
suppression of corticosterone in a dexamethasone suppression test. In contrast dele-
tion of GR in both the pituitary and hypothalamus (calcium/calmodulin-dependent
protein kinase II alpha-cre) results in death in the first week of life from an advanced
form of Cushing’s syndrome [57]. Therefore, loss of pituitary negative feedback has
modest effects, loss of the hypothalamic site results in Cushing’s syndrome but is
compatible with life, but complete loss of negative feedback is not. Given the role
of stress on psychiatric conditions a number of GR cell type specific KO mice have
been analyzed in behavioral paradigms. GR in dopamine responding neurons (dopa-
mine receptor 1-cre) was found to be a critical mediator of social aggression whereas
GR in dopaminergic neurons (dopamine active transporter, DAT-cre) was dispens-
able. Mice subject to social aggression show characteristic behaviors which were
abolished in mice lacking GR in dopaminoceptive neurons [58]. Mice with deletion
of GR in dopaminoceptive neurons (D1R-cre) self-administered less cocaine than
wildtype mice, but no effect was seen when GR was deleted from dopaminergic
neurons (DAT-cre) [59]. GR has been found to be a mediator of MPTP-induced
Parkinsonism in microglia. Microglial GR KO mice (LysM-cre) had more death of
dopaminergic neurons following MPTP-insult, whereas the loss was not effected by
deletion of GR from dopaminergic neurons (DAT-cre) [60]. Microglia from microg-
lia GR KO mice also had increased activation, consistent with a role for glucocorti-
coids in regulating microglial activation status as an anti-inflammatory.
344 C. Harris
Other Tissues
Deletion of GR in the mammary epithelium (whey acidic protein-cre) had no effect on

lactation, but there was a modest defect in glandular proliferation during pregnancy
[61]. Deletion of GR in sertoli cells (using anti-Mullerian hormone-cre) resulted in
decreased sertoli cell number, circulating FSH and secondarily decreased LH and
abnormal Leydig cell morphology, but these mice were fertile [62]. As mentioned
above, deletion of GR in noradrenergic cells (using dopamine beta hydroxylase cre)
resulted in decreased numbers of adrenal chromaffin cells in adults.
Version 4.0: Qualitative Differences (Point Mutants)
The most well studied GR knockin mouse is the dim mouse (MGI:1931329). Site
directed mutagenesis studies identified this mutation in vitro as inhibiting transacti-
vation while leaving repression intact [63]. Alanine to threonine mutation was gen-
erated by homologous recombination. Unlike GR-null animals, GR dim mice are
viable [64]. However, we have observed most GRdim/dim mice on a C57/BL6
background die at birth [65]. Interestingly, viability is normal on a mixed (BALBC
and C57) background (C.H., unpublished observation). This could be due to genetic
modifiers affecting GR signaling. In addition, although the dim allele was originally
described as being a loss of function allele for all transactivation targets [64], more
detailed analysis in the age of genomics has uncovered that some targets are still
activated by the dim allele and in addition the dim allele has some gain of function
properties [66, 67]. In other words, a subset of genes is activated in a ligand depen-
dent manner by the dim allele that is not normally activated by wildtype GR. These
so called “rogue” genes have been identified in vitro [66, 68] as well as in vivo [69
and our unpublished results]. One potential explanation for gain of function proper-
ties of the dim allele is gene activation by an increased concentration of GR mono-
mers in the absence of dimer formation. Therefore, though the dim mouse is a
powerful tool to dissect whether biological effects are mediated by dimers experi-
ments using them must be interpreted with this in mind.
GR dim mice had blunted response to anemia by phenylhydrazine and also did
not increase red blood cell counts in response to hypoxia [15]. As opposed to wild-
type mice, GR dim mice did not potentiate hippocampal calcium currents or enhance
serotonin response in the hippocampus when treated with glucocorticoids [70].
Water maze experiments demonstrated that GR dim mice have impairments in spa-
tial memory [71]. GR dimerization is required for inhibition of contact hypersensitiv-
ity by glucocorticoids [45]. The defects in skin observed in GR-null mice were not
seen in GR dim mice suggesting that GR dimers are dispensable for skin development
[14]. GR dim mice undergo a muscle atrophy similar to WT mice when treated with
glucocorticoids despite attenuated induction of MuRF1, indicating that muscle atro-
phy is a dimer independent process [72]. Glucocorticoids induce osteoporosis in GR
dim mice indicating this is a dimer independent process [47, 65]. GR dim mice were
resistant to the therapeutic effects of glucocorticoid in an antigen induced arthritis
model of rheumatoid arthritis [46] indicating GR dimers are essential for this pro-
cess. GR dim mice did not increase intestinal glucose uptake in response to glucocor-
ticoids indicating this process is dependent on GR dimers [41]. GR dim mice were
not immune to glucocorticoid-mediated changes in triglyceride metabolism in adi-
pose, indicating this is a GR dimer independent process [65].
Another knockin mouse (MGI strain 3842978), of interest is a mutation that
made GR more sensitive to ligand by mutation M610L in the LBD. These mice
did not have a phenotype of glucocorticoid excess because their HPA axis was
reset such that the circulating levels of glucocorticoids were lower than WT [73].
Muglia generated a knockin for a GFP-GR fusion protein allowing visualization of
GR in vivo (MGI:3577992) [74].
Version 5.0: The Future of Mice with Altered GR Signaling
The advent of CRISPRs has made gene targeting more accessible given reduced
cost, time and labor involved. First generation CRISPRs experiments were able to
introduce double-strand breaks repaired in a stochastic manner involving non-
homologous end-joining resulting in deletions and insertions [75]. Newer approaches
allowed coinjection of a large single stranded donor oligonucleotide or double
stranded DNA donor plasmid to induce targeted mutations via homologous recom-
bination. Further advances have been made rapidly allowing the introduction of
loxP sites to create floxed mice for conditional null alleles [76]. We predict this
technology will lead to the creation of even more mice genetically modified in GR
signaling. What mice will we see in the near future? Additional knock-in mice with
point mutations leading to altered AF1 and AF2 function [77] would be insightful.
In addition, point mutations for GR residues shown to be post-translationally modi-
fied would be informative as to the role of these post-translational modifications
in vivo. In addition, there are a number of additional polymorphisms in GR that
have been linked to human disease states including metabolic syndrome and depres-
sion [78] which could be informative in the function of GR in vivo. Finally, the ease
of using CRISPRs to generate targeted germ line mutations could be the solution to
a problem that has existed in the field of transcriptional regulation. We have access
to whole genome transcriptional data and chromatin-immunoprecipitation data for
regulation by glucocorticoids. It has been difficult to demonstrate that a given bind-
ing site is directly responsible for a given gene regulation event. Often this correla-
tion is made by way of parsimony: Ockham’s razor argues that the closest GR
binding region to a gene regulated by GR is responsible for that gene’s regulation.
However, it is clear that there are a number of transcriptional events regulated from
distal DNA elements. Therefore, gene targeting of a GRE would enable one to
determine what genes are governed by the GRE. While it is not feasible to do this
on a wide scale, and it is more feasible to do this in cell lines, we would not be sur-
prised if we saw some GRE targeted CRISPRs in the near future. A handful of such
mice might reveal rules to the logic of transcriptional regulation previously undeci-
pherable. Candidates for such approaches would be GR primary target genes that
are critical in a glucocorticoid-mediated phenotype with a well-defined GRE.
346 C. Harris
Non-GR Mutants Relevant to Glucocorticoid Signaling
We will not have space to describe all of the genetically altered mice related to
glucocorticoid action, but a few notable mutations are described here. Given the
regulation of glucocorticoid synthesis by the hypothalamic–pituitary–adrenal axis,
it was of great interest to know the phenotype of CRH-null mice. CRH-null mice
have reduced but detectable corticosterone levels. CRH-null animals born to hetero-
zygous mothers survive, but CRH-null mice born to CRH-null mothers die at birth
due to defects in lung maturation, similar to GR-null animals. If CRH-null mothers
are given corticosterone in the drinking water, their CRH-null pups will survive.
The peptide ACTH is encoded by the POMC gene which gives rise to several other
peptides. Therefore, the phenotype of the POMC-null mice can not be attributed
only to defects in ACTH. Mice lacking 7B2, a cofactor for proconvertase 2 (a pro-
tease that processes several endocrine protein hormones) display Cushing’s disease,
due to hypersecretion of ACTH [79]. Recently, a Cushingoid mouse was identified
as part of an ENU mutagenesis screen [80]. The mutation was mapped to the CRH
promoter (120 basepairs upstream of the transcriptional start site) in a caudal-type
homeobox response element (CDXARE). The mutation leads to increased CRH
expression and is recapitulated with a luciferase reporter. The phenotype of this
mouse is similar to the CRH-Tg mouse as well as the mice with deletion of GR in
tissues governing negative feedback in the hypothalamus including Sim1-cre and
Nestin-Cre. Vale’s group created a Cushingoid mouse by placing the CRH gene
under a constitutive metallothionein promoter [81]. Interestingly, the investigators
found that the transgene was only expressed in the hypothalamus and CRH levels
could not be detected in the circulation. Yet, these mice had increased ACTH levels.
The investigators postulated that a cryptic regulatory site within the coding region
was responsible for this tissue specific transgene expression. While their peak corti-
costerone levels are not significantly different from WTs, their nadir levels are much
higher, abrogating the circadian rhythmicity of corticosterone production similar to
patients with endogenous glucocorticoid excess. CRH-Tg mice have increased adi-
posity, decreased bone density, decreased skin thickness and skin collagen produc-
tion. Interestingly, CRH-Tg mice have large increases in adipose triglyceride futile
cycling with increased rate of both lipolysis and reesterification [82]. CRH-Tg mice
display age-dependent malabsorption that is due to a “transdifferentiation” of exo-
crine pancreas to express hepatocyte markers [83].
Dehydrogenase Mutants
To test the hypothesis that increased glucocorticoid signaling in adipose tissue plays
a role in the etiology of metabolic syndrome Flier’s group created an aP2-11-β-
HSD1 transgenic mouse. Selective expression of this GC activating enzyme in adi-
pose should raise tissue corticosterone levels in adipose. These mice displayed
features of metabolic syndrome while on a chow diet [84]. Similarly, the converse
was shown, i.e. transgenic expression of 11-β-HSD2, the GC deactivating enzyme,

protected mice from metabolic abnormalities when fed a high-fat diet [85]. A similar
approach was used to modulate glucocorticoid levels in the bone [48].
GR Targets
A full description of genetic modifications to primary GR causative target genes is

beyond the scope of this chapter, but we would like to highlight a few key observa-
tions. Given that glucocorticoid receptor alters transcription of hundreds to thou-
sands of genes in most cell types it is amazing that elimination of a single GR target
gene can affect the phenotype associated with glucocorticoid exposure. Furthermore,
many glucocorticoid targets have functions distinct from their regulation by gluco-
corticoids. Glucocorticoid-mediated muscle atrophy occurs via transcriptional acti-
vation of atrogins such as MuRF1 and MAFbx (atrogin1). Deletion of MURF1
attenuates glucocorticoid-mediated muscle atrophy [86]. Similarly, deletion of the
GR target Hes1 attenuates metabolic effects of glucocorticoids in the liver [87].
Summary and Looking Ahead
The last 25 years have seen a proliferation of our knowledge about glucocorticoid
signaling. Essential to this has been the ability to probe the role of GR in vivo in mice
using first anti-sense transgenics, the traditional knockouts, and then tissue specific
knockouts. The tissue-specific GR KO mice have allowed a better understanding of
which effects of glucocorticoids are cell autonomous and which are indirect. For
example, muscle atrophy which could have been due to effects directly on muscle as
well as indirectly through effects on liver (IGF1 secretion), the HPA axis (reduced sex
steroids) appears to be largely cell autonomous. Similarly, a priori, effects on bone
could be direct or indirect through reductions in other pituitary axes including
GH-IGF1, hypothalamic–pituitary–gonadal, intestinal absorption of calcium, etc.
Here, too effects seem to be direct on bone, and while the osteoblast appears to be
critical, the osteoclast may play a role. Furthermore, some phenotypes are agreed
upon by multiple investigators, such as the muscle specific KO mouse which has
been made by three different investigators. Areas of discrepant data include the
critical cell type in the lung responsible for the lethality of GR-null mice. The appar-
ent discrepancy in phenotype of liver specific GR-null mice can be explained by the
timing of activation of the cre lines used by the different investigators. Given the
recent breakthrough in gene editing with CRISPRs, it is likely many more GR mutant
mice will be generated and contribute further to our understanding of how GR signals.
In addition CRISPRs may enable investigators that use other experimental models
such as rat to also generate additional animals with gene modifications.
After detailing the multiple effects of the glucocorticoid receptor in the sections
above, it is quite remarkable that the GR-null mouse makes it to birth and is not
348 C. Harris
embryonic lethal. In other words, although GR has been implicated in cell fate deci-
sions, GR-null mice do not have an obvious loss of an essential cell type. The excep-
tions are the maintenance of adrenal chromaffin cells as adults and maintenance of
some pancreatic cell types as mentioned above. Similarly, glucocorticoid excess
alters some cell fate decisions with the best evidence in vivo being hepatic conver-
sion of exocrine pancreas in CRH-Tg mice. Although the focus regarding neonatal
mortality of GR-null mice has been on the lung, it is possible than even if this were
rescued, GR-null mice might die due to effects on other tissues, such as skin or the
heart. We predict the answers to these questions will come in the years ahead.
Additional Resources
In addition to this chapter, we suggest the reader use the following websites to
assemble the most up to date information. Mouse genome informatics (http://www.
informatics.jax.org/). This is a comprehensive listing of mouse genes with links to
individual mice (i.e. separate listings for different strains created by different labs
including knockout projects). There is a separate listing for the GR gene (NR3C1)
as well as a listing for each mutant mouse. Each mutant mouse line is assigned a
unique MGI number which is occasionally referenced in this chapter. There are
links to references, as well as to many other data bases, including IMSR (interna-
tional mouse strain resource, http://www.findmice.org/), a database listing where
various mouse strains are held internationally.
Nuclear Receptor Signaling Atlas (NURSA, http://www.nursa.org/) contains
valuable information on nuclear receptors and coregulators including links to MGI
pages.
References
1. Brinster RL, et al. Somatic expression of herpes thymidine kinase in mice following injection
of a fusion gene into eggs. Cell. 1981;27:223–31.
2. Thomas KR, Capecchi MR. Targeted disruption of the murine int-1 proto-oncogene resulting
in severe abnormalities in midbrain and cerebellar development. Nature. 1990;346:847–50.
doi:10.1038/346847a0.
3. Miesfeld R, et al. Characterization of a steroid hormone receptor gene and mRNA in wild-type
and mutant cells. Nature. 1984;312:779–81.
4. Weinberger C, et al. Identification of human glucocorticoid receptor complementary DNA
clones by epitope selection. Science. 1985;228:740–2.
5. Govindan MV, Devic M, Green S, Gronemeyer H, Chambon P. Cloning of the human gluco-
corticoid receptor cDNA. Nucleic Acids Res. 1985;13:8293–304.
6. Pepin MC, Pothier F, Barden N. Impaired type II glucocorticoid-receptor function in mice
bearing antisense RNA transgene. Nature. 1992;355:725–8. doi:10.1038/355725a0.
7. King LB, et al. A targeted glucocorticoid receptor antisense transgene increases thymocyte
apoptosis and alters thymocyte development. Immunity. 1995;3:647–56.
8. Cole TJ, et al. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chro-
maffin cell development and severely retards lung maturation. Genes Dev. 1995;9:1608–21.
9. Brewer JA, Kanagawa O, Sleckman BP, Muglia LJ. Thymocyte apoptosis induced by T cell
activation is mediated by glucocorticoids in vivo. J Immunol. 2002;169:1837–43.
10. Michailidou Z, et al. Glucocorticoid receptor haploinsufficiency causes hypertension and
attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet.
FASEB J. 2008;22:3896–907. doi:10.1096/fj.08-111914.
11. Chrousos GP, et al. Primary cortisol resistance in man. A glucocorticoid receptor-mediated
disease. J Clin Invest. 1982;69:1261–9.
12. Parlato R, et al. Conditional inactivation of glucocorticoid receptor gene in dopamine-beta-
hydroxylase cells impairs chromaffin cell survival. Endocrinology. 2009;150:1775–81.
doi:10.1210/en.2008-1107.
13. Gagnon S, et al. Transgenic glucocorticoid receptor expression driven by the SP-C promoter
reduces neonatal lung cellularity and midkine expression in GRhypo mice. Biol Neonate.
2006;90:46–57. doi:10.1159/000091844.
14. Bayo P, et al. Glucocorticoid receptor is required for skin barrier competence. Endocrinology.
2008;149:1377–88. doi:10.1210/en.2007-0814.
15. Bauer A, et al. The glucocorticoid receptor is required for stress erythropoiesis. Genes Dev.
1999;13:2996–3002.
16. Gesina E, et al. Glucocorticoid signalling affects pancreatic development through both direct
and indirect effects. Diabetologia. 2006;49:2939–47. doi:10.1007/s00125-006-0449-3.
17. Bouabe H, Okkenhaug K. Gene targeting in mice: a review. Methods Mol Biol. 2013;1064:315–
36. doi:10.1007/978-1-62703-601-6_23.
18. Shah K, et al. Discovery of liver selective non-steroidal glucocorticoid receptor antagonist as
novel antidiabetic agents. Bioorg Med Chem Lett. 2012;22:5857–62. doi:10.1016/j.
bmcl.2012.07.078.
19. Zinker B, et al. Liver-selective glucocorticoid receptor antagonism decreases glucose production
and increases glucose disposal, ameliorating insulin resistance. Metabolism. 2007;56:380–7.
doi:10.1016/j.metabol.2006.10.021.
20. von Geldern TW, et al. Liver-selective glucocorticoid antagonists: a novel treatment for type 2
diabetes. J Med Chem. 2004;47:4213–30. doi:10.1021/jm0400045.
21. Jeanneteau FD, et al. BDNF and glucocorticoids regulate corticotrophin-releasing hormone
(CRH) homeostasis in the hypothalamus. Proc Natl Acad Sci U S A. 2012;109:1305–10.
doi:10.1073/pnas.1114122109.
22. Oakley RH, et al. Essential role of stress hormone signaling in cardiomyocytes for the preven-
tion of heart disease. Proc Natl Acad Sci U S A. 2013;110:17035–40. doi:10.1073/
pnas.1302546110.
23. Mittelstadt PR, Monteiro JP, Ashwell JD. Thymocyte responsiveness to endogenous glucocor-
ticoids is required for immunological fitness. J Clin Invest. 2012;122:2384–94. doi:10.1172/
JCI63067.
24. Brewer JA, et al. T-cell glucocorticoid receptor is required to suppress COX-2-mediated lethal
immune activation. Nat Med. 2003;9:1318–22. doi:10.1038/nm895.
25. Tronche F, et al. Disruption of the glucocorticoid receptor gene in the nervous system results
in reduced anxiety. Nat Genet. 1999;23:99–103. doi:10.1038/12703.
26. Mittelstadt PR, Ashwell JD. Disruption of glucocorticoid receptor exon 2 yields a ligand-
responsive C-terminal fragment that regulates gene expression. Mol Endocrinol. 2003;17:1534–42.
doi:10.1210/me.2002-0429.
27. Habermehl D, et al. Glucocorticoid activity during lung maturation is essential in mesenchy-
mal and less in alveolar epithelial cells. Mol Endocrinol. 2011;25:1280–8. doi:10.1210/
me.2009-0380.
28. Li A, et al. Deletion of mesenchymal glucocorticoid receptor attenuates embryonic lung devel-
opment and abdominal wall closure. PLoS One. 2013;8:e63578. doi:10.1371/journal.
pone.0063578.
350 C. Harris
29. Manwani N, et al. Reduced viability of mice with lung epithelial-specific knockout of gluco-
corticoid receptor. Am J Respir Cell Mol Biol. 2010;43:599–606. doi:10.1165/
rcmb.2009-0263OC.
30. Sevilla LM, Latorre V, Sanchis A, Perez P. Epidermal inactivation of the glucocorticoid recep-
tor triggers skin barrier defects and cutaneous inflammation. J Invest Dermatol. 2013;133:361–
70. doi:10.1038/jid.2012.281.
31. Latorre V, Sevilla LM, Sanchis A, Perez P. Selective ablation of glucocorticoid receptor in
mouse keratinocytes increases susceptibility to skin tumorigenesis. J Invest Dermatol.
2013;133:2771–9. doi:10.1038/jid.2013.255.
32. Perez P, et al. Altered skin development and impaired proliferative and inflammatory responses
in transgenic mice overexpressing the glucocorticoid receptor. FASEB J. 2001;15:2030–2.
doi:10.1096/fj.00-0772fje.
33. Goodwin JE, Zhang J, Velazquez H, Geller DS. The glucocorticoid receptor in the distal neph-
ron is not necessary for the development or maintenance of dexamethasone-induced hyperten-
sion. Biochem Biophys Res Commun. 2010;394:266–71. doi:10.1016/j.bbrc.2010.02.123.
34. Goodwin JE, Zhang J, Gonzalez D, Albinsson S, Geller DS. Knockout of the vascular endo-
thelial glucocorticoid receptor abrogates dexamethasone-induced hypertension. J Hypertens.
2011;29:1347–56. doi:10.1097/HJH.0b013e328347da54.
35. Sainte-Marie Y, et al. Conditional glucocorticoid receptor expression in the heart induces atrio-
ventricular block. FASEB J. 2007;21:3133–41. doi:10.1096/fj.07-8357com.
36. Rog-Zielinska EA, et al. Glucocorticoid receptor is required for foetal heart maturation. Hum
Mol Genet. 2013;22:3269–82. doi:10.1093/hmg/ddt182.
37. Watson ML, et al. A cell-autonomous role for the glucocorticoid receptor in skeletal muscle
atrophy induced by systemic glucocorticoid exposure. Am J Physiol Endocrinol Metab.
2012;302:E1210–20. doi:10.1152/ajpendo.00512.2011.
38. Braun TP, et al. Muscle atrophy in response to cytotoxic chemotherapy is dependent on intact
glucocorticoid signaling in skeletal muscle. PLoS One. 2014;9:e106489. doi:10.1371/journal.
pone.0106489.
39. Braun TP, et al. Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling
in skeletal muscle. FASEB J. 2013;27:3572–82. doi:10.1096/fj.13-230375.
40. Gartner H, Graul MC, Oesterreicher TJ, Finegold MJ, Henning SJ. Development of the fetal
intestine in mice lacking the glucocorticoid receptor (GR). J Cell Physiol. 2003;194:80–7.
doi:10.1002/jcp.10189.
41. Reichardt SD, et al. Glucocorticoids enhance intestinal glucose uptake via the dimerized glu-
cocorticoid receptor in enterocytes. Endocrinology. 2012;153:1783–94. doi:10.1210/
en.2011-1747.
42. Reichardt SD, et al. Glucocorticoids induce gastroparesis in mice through depletion of
l-arginine. Endocrinology. 2014;155:3899–908. doi:10.1210/en.2014-1246.
43. Preusch MR, et al. Critical role of macrophages in glucocorticoid driven vascular calcification
in a mouse-model of atherosclerosis. Arterioscler Thromb Vasc Biol. 2008;28:2158–64.
doi:10.1161/ATVBAHA.108.174128.
44. Wust S, et al. Peripheral T cells are the therapeutic targets of glucocorticoids in experimental
autoimmune encephalomyelitis. J Immunol. 2008;180:8434–43.
45. Tuckermann JP, et al. Macrophages and neutrophils are the targets for immune suppression by
glucocorticoids in contact allergy. J Clin Invest. 2007;117:1381–90. doi:10.1172/JCI28034.
46. Baschant U, et al. Glucocorticoid therapy of antigen-induced arthritis depends on the dimer-
ized glucocorticoid receptor in T cells. Proc Natl Acad Sci U S A. 2011;108:19317–22.
doi:10.1073/pnas.1105857108.
47. Rauch A, et al. Glucocorticoids suppress bone formation by attenuating osteoblast differentia-
tion via the monomeric glucocorticoid receptor. Cell Metab. 2010;11:517–31. doi:10.1016/j.
cmet.2010.05.005.
48. Sher LB, et al. Transgenic expression of 11beta-hydroxysteroid dehydrogenase type 2 in
osteoblasts reveals an anabolic role for endogenous glucocorticoids in bone. Endocrinology.
2004;145:922–9. doi:10.1210/en.2003-0655.
49. Kim HJ, et al. Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest.
2006;116:2152–60. doi:10.1172/JCI28084.
50. Tronche F, et al. Glucocorticoid receptor function in hepatocytes is essential to promote post-
natal body growth. Genes Dev. 2004;18:492–7. doi:10.1101/gad.284704.
51. Opherk C, et al. Inactivation of the glucocorticoid receptor in hepatocytes leads to fasting
hypoglycemia and ameliorates hyperglycemia in streptozotocin-induced diabetes mellitus.
Mol Endocrinol. 2004;18:1346–53. doi:10.1210/me.2003-0283.
52. Engblom D, et al. Direct glucocorticoid receptor-Stat5 interaction in hepatocytes controls
body size and maturation-related gene expression. Genes Dev. 2007;21:1157–62. doi:10.1101/
gad.426007.
53. Shteyer E, Liao Y, Muglia LJ, Hruz PW, Rudnick DA. Disruption of hepatic adipogenesis is
associated with impaired liver regeneration in mice. Hepatology. 2004;40:1322–32.
doi:10.1002/hep.20462.
54. Rose AJ, et al. Molecular control of systemic bile acid homeostasis by the liver glucocorticoid
receptor. Cell Metab. 2011;14:123–30. doi:10.1016/j.cmet.2011.04.010.
55. Gesina E, et al. Dissecting the role of glucocorticoids on pancreas development. Diabetes.
2004;53:2322–9.
56. Schmidt MV, et al. Postnatal glucocorticoid excess due to pituitary glucocorticoid receptor
deficiency: differential short- and long-term consequences. Endocrinology. 2009;150:2709–16.
doi:10.1210/en.2008-1211.
57. Erdmann G, Schutz G, Berger S. Loss of glucocorticoid receptor function in the pituitary results
in early postnatal lethality. Endocrinology. 2008;149:3446–51. doi:10.1210/en.2007-1786.
58. Barik J, et al. Chronic stress triggers social aversion via glucocorticoid receptor in dopamino-
ceptive neurons. Science. 2013;339:332–5. doi:10.1126/science.1226767.
59. Ambroggi F, et al. Stress and addiction: glucocorticoid receptor in dopaminoceptive neurons
facilitates cocaine seeking. Nat Neurosci. 2009;12:247–9. doi:10.1038/nn.2282.
60. Ros-Bernal F, et al. Microglial glucocorticoid receptors play a pivotal role in regulating dopa-
minergic neurodegeneration in parkinsonism. Proc Natl Acad Sci U S A. 2011;108:6632–7.
doi:10.1073/pnas.1017820108.
61. Wintermantel TM, Bock D, Fleig V, Greiner EF, Schutz G. The epithelial glucocorticoid receptor
is required for the normal timing of cell proliferation during mammary lobuloalveolar develop-
ment but is dispensable for milk production. Mol Endocrinol. 2005;19:340–9. doi:10.1210/
me.2004-0068.
62. Hazra R, et al. In vivo actions of the Sertoli cell glucocorticoid receptor. Endocrinology.
2014;155:1120–30. doi:10.1210/en.2013-1940.
63. Heck S, et al. A distinct modulating domain in glucocorticoid receptor monomers in the repres-
sion of activity of the transcription factor AP-1. EMBO J. 1994;13:4087–95.
64. Reichardt HM, et al. DNA binding of the glucocorticoid receptor is not essential for survival.
Cell. 1998;93:531–41.
65. Roohk DJ, et al. Dexamethasone-mediated changes in adipose triacylglycerol metabolism are
exaggerated, not diminished, in the absence of a functional GR dimerization domain.
Endocrinology. 2013;154:1528–39. doi:10.1210/en.2011-1047.
66. Rogatsky I, et al. Target-specific utilization of transcriptional regulatory surfaces by the glucocor-
ticoid receptor. Proc Natl Acad Sci U S A. 2003;100:13845–50. doi:10.1073/pnas.2336092100.
binds half sites as a monomer and regulates specific target genes. Genome Biol. 2014;15:418.
doi:10.1186/s13059-014-0418-y.
68. Jewell CM, Scoltock AB, Hamel BL, Yudt MR, Cidlowski JA. Complex human glucocorticoid
receptor dim mutations define glucocorticoid induced apoptotic resistance in bone cells. Mol
Endocrinol. 2012;26:244–56. doi:10.1210/me.2011-1116.
69. Frijters R, et al. Prednisolone-induced differential gene expression in mouse liver carrying
wild type or a dimerization-defective glucocorticoid receptor. BMC Genomics. 2010;11:359.
doi:10.1186/1471-2164-11-359.
352 C. Harris
70. Karst H, et al. Corticosteroid actions in hippocampus require DNA binding of glucocorticoid
receptor homodimers. Nat Neurosci. 2000;3:977–8. doi:10.1038/79910.
71. Oitzl MS, Reichardt HM, Joels M, de Kloet ER. Point mutation in the mouse glucocorticoid
receptor preventing DNA binding impairs spatial memory. Proc Natl Acad Sci U S A.
2001;98:12790–5. doi:10.1073/pnas.231313998.
72. Waddell DS, et al. The glucocorticoid receptor and FOXO1 synergistically activate the skeletal
muscle atrophy-associated MuRF1 gene. Am J Physiol Endocrinol Metab. 2008;295:E785–97.
doi:10.1152/ajpendo.00646.2007.
73. Zhang J, et al. Characterization of a novel gain of function glucocorticoid receptor knock-in
mouse. J Biol Chem. 2009;284:6249–59. doi:10.1074/jbc.M807997200.
74. Brewer JA, Sleckman BP, Swat W, Muglia LJ. Green fluorescent protein-glucocorticoid
receptor knockin mice reveal dynamic receptor modulation during thymocyte development.
J Immunol. 2002;169:1309–18.
75. Wang H, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/
Cas-mediated genome engineering. Cell. 2013;153:910–8. doi:10.1016/j.cell.2013.04.025.
76. Yang H, et al. One-step generation of mice carrying reporter and conditional alleles by
CRISPR/Cas-mediated genome engineering. Cell. 2013;154:1370–9. doi:10.1016/j.
cell.2013.08.022.
77. Iniguez-Lluhi JA, Lou DY, Yamamoto KR. Three amino acid substitutions selectively disrupt
the activation but not the repression function of the glucocorticoid receptor N terminus. J Biol
Chem. 1997;272:4149–56.
78. Koper JW, van Rossum EF, van den Akker EL. Glucocorticoid receptor polymorphisms and
haplotypes and their expression in health and disease. Steroids. 2014;92:62–73. doi:10.1016/j.
steroids.2014.07.015.
79. Sarac MS, Zieske AW, Lindberg I. The lethal form of Cushing’s in 7B2 null mice is caused by
multiple metabolic and hormonal abnormalities. Endocrinology. 2002;143:2324–32.
doi:10.1210/endo.143.6.8808.
80. Bentley L, et al. An N-ethyl-N-nitrosourea induced corticotropin-releasing hormone promoter
mutation provides a mouse model for endogenous glucocorticoid excess. Endocrinology.
2014;155:908–22. doi:10.1210/en.2013-1247.
81. Stenzel-Poore MP, Cameron VA, Vaughan J, Sawchenko PE, Vale W. Development of
Cushing’s syndrome in corticotropin-releasing factor transgenic mice. Endocrinology.
1992;130:3378–86. doi:10.1210/endo.130.6.1597149.
82. Harris C, et al. Large increases in adipose triacylglycerol flux in Cushingoid CRH-Tg mice are
explained by futile cycling. Am J Physiol Endocrinol Metab. 2013;304:E282–93. doi:10.1152/
ajpendo.00154.2012.
83. Wallace K, Flecknell PA, Burt AD, Wright MC. Disrupted pancreatic exocrine differentiation
and malabsorption in response to chronic elevated systemic glucocorticoid. Am J Pathol.
2010;177:1225–32. doi:10.2353/ajpath.2010.100107.
84. Masuzaki H, et al. A transgenic model of visceral obesity and the metabolic syndrome.
Science. 2001;294:2166–70. doi:10.1126/science.1066285.
85. Kershaw EE, et al. Adipocyte-specific glucocorticoid inactivation protects against diet-induced
obesity. Diabetes. 2005;54:1023–31.
86. Baehr LM, Furlow JD, Bodine SC. Muscle sparing in muscle RING finger 1 null mice: response
to synthetic glucocorticoids. J Physiol. 2011;589:4759–76. doi:10.1113/jphysiol.2011.212845.
87. Lemke U, et al. The glucocorticoid receptor controls hepatic dyslipidemia through Hes1. Cell
Metab. 2008;8:212–23. doi:10.1016/j.cmet.2008.08.001.
Chapter 16
The Dehydrogenase Hypothesis
Conor Woods and Jeremy W. Tomlinson
Abstract Circulating glucocorticoid (GC) levels are controlled by the

Hypothalamo–Pituitary–Adrenal (HPA) axis, but within tissues, GC availability is
controlled by the isoforms of 11β (Beta)-Hydroxysteroid Dehydrogenase 11β
(Beta)-HSD that interconvert inactive cortisone and active cortisol. Two isoforms
have been identified; in key metabolic target tissues (including liver and adipose),
expression of 11β (Beta)-HSD1 predominates that in vivo converts cortisone to cor-
tisol and thus amplifies local GC action. In contrast, in mineralocorticoid target
tissues 11β (Beta)-HSD2 is the isoform that is most abundantly expressed. This
inactivates cortisol to cortisone and offers protection for the mineralocorticoid
receptor form occupation and activation by cortisol. Dysregulated 11β (Beta)-HSD1
activity has been implicated in many metabolic diseases such as obesity and diabe-
tes and inhibition of 11β (Beta)-HSD1 represents a promising therapeutic target.
Mutations within the gene encoding 11β (Beta)-HSD2 cause the Syndrome of
Apparent Mineralocorticoid Excess and decreases in activity are linked to hyperten-
sion as well as impairment in placental function and neonatal growth. We will dis-
cuss the molecular biology and enzymology of 11β (Beta)-HSD and its role in
normal physiology and discuss altered 11β (Beta)-HSD activity in pathological
states and the potential for therapeutic targeting.
Keywords 11β (Beta)-HSD • Endoplasmic reticulum (ER) • Cortisol • Cortisone

• H6PDH • NADPH
C. Woods, M.B.B.Ch., M.R.C.P.

Department of Diabetes and Endocrinology, St Vincent’s University Hospital,
Dublin, Leisnter, Ireland
J.W. Tomlinson (*)
Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford,
Radcliffe Department of Medicine, Churchill Hospital, Oxford, UK

354 C. Woods and J.W. Tomlinson
Introduction
Glucocorticoids (GC) (cortisol in man and corticosterone in rodents) are crucial in

regulating many important physiological functions including glucose and amino
acid metabolism, inflammation, immunity and general health and well-being [1].
GCs bind to the glucocorticoid receptor (GR), a member of the nuclear receptor
family and alter gene transcription up-regulating anti-inflammatory protein synthe-
sis and reducing expression of pro-inflammatory cytokines.
Since their discovery in the 1940s by Kendall and Hench, GCs have become a main-
stay of therapy in many clinical conditions including rheumatoid arthritis, asthma and as
a cornerstone in anti-rejection medication regimes in organ transplant recipients.
Whereas circulating GC levels are regulated by the Hypothalamo–pituitary–
adrenal (HPA) axis, tissue cortisol metabolism is under the control of the enzymes
of the 11β (Beta)-Hydroxysteroid Dehydrogenase (11β (Beta)-HSD) system. Two
isozymes exist, namely 11β (Beta)-HSD1 and 11β (Beta)-HSD2. In recent decades,
attention has focussed on tissue cortisol metabolism and the pre-receptor regulation
of GCs within tissues such as fat, muscle and liver [2–6]. 11β (Beta)-HSD1 is a bi-
directional enzyme, which in vivo acts primarily as an oxo-reductase, converting
inactive cortisone (and inactive 11-dehydrocorticosterone in rodents) to active cor-
tisol. 11β (Beta)-HSD1 is located in a number of key metabolic tissues including
liver, skeletal muscle, gonads and adipose tissue [6, 7] and has key metabolic influ-
ence acting as an amplification step in tissue cortisol exposure. Tissue exposure to
GC therefore is not only reliant on circulating levels of cortisol but can also be
altered by pre receptor regulation via 11β (Beta)-HSD1. Dysregulated 11β (Beta)-
HSD1 activity has recently been implicated in many metabolic and inflammatory
diseases and is a promising target to alter tissue glucocorticoid exposure. Selective
11β (Beta)-HSD1 inhibitors exist and have been trialled in early phase 2 studies
primarily in type 2 diabetes [8].
11β (Beta)-HSD2 is expressed in aldosterone selective tissues, mainly in the distal
nephrons, colonic epithelium, salivary and sweat glands (mineralocorticoid receptor
(MR) expressing tissues) and in the foetus and placenta during gestation. It acts
uniquely as a dehydrogenase deactivating cortisol to inactive cortisone, protecting
tissues from excess glucocorticoid exposure [6, 9]. 11β (Beta)-HSD2 deficiency or
inhibition causes hypertension secondary to apparent mineralocorticoid excess
whereby cortisol activates the MR for which it shares the same affinity as aldoste-
rone. A similar syndrome manifests in people ingesting large amounts of European
licorice which contains an inhibitor of this enzyme. 11β (Beta)-HSD2 is important
in gestation with significant activity in both placenta and fetal brain development by
preventing early exposure to GCs.
Hypothalamic Pituitary Adrenal Axis and Cortisol Production
GCs, synthesized from cholesterol precursors, are produced primarily by the adrenal
cortex (zona fasiculata) under the influence of the HPA axis in a classical circadian
rhythm, regulated by negative feedback.
16 The Dehydrogenase Hypothesis 355
Healthy adults secrete 10–15 mg s cortisol/day [10]. The majority of cortisol is

protein bound to cortisol binding globulin (CBG) although some is albumin bound.
Estimates suggest that only 5 % of circulating cortisol is “free” and biologically
active [11, 12]. The half-life of free cortisol is only a few minutes whereas protein
bound cortisol has a much longer half life of between 70 and 120 min [11, 13, 14].
Cortisone has similar unbound concentrations to that of cortisol but has a lower
affinity for both CBG and GR [12, 15]. Importantly the biological availability of
GCs represents a balance between synthesis/secretion and metabolism/clearance.
Cortisol Metabolism and Clearance
There are multiple pathways involved in cortisol and cortisone metabolism. These
include A-ring reduction to from tetrahydrocortisol and it’s 5α (alpha)-isomer allo-
tetrahydrocortisol, hydroxylation to 6β (Beta) hydroxycortisol and reduction of the
20-oxo group giving cortols. Among the various distinct pathways, perhaps the
most critical of these is the inter-conversion of cortisol and cortisone mediated by
the 11β (Beta)-HSD isozymes. 11β (Beta)-HSD interconverts cortisol and cortisone
by altering the hydroxyl group at C11 (see Fig. 16.1).
Tissue Cortisol Metabolism: 11β (Beta) HSD1
11β (Beta) HSD1: Discovery
Glucocorticoids were discovered in the 1940s and 1950s and were heralded as a
potentially curative treatment for many ailments [16]. Kendall et al. published the
discovery of what they believed to be a treatment that could reverse rheumatoid arthri-
tis in the 1950s [17]. What they referred to as Compound E; was actually cortisone, a
Fig. 16.1 Isoforms of 11β (Beta)-Hydroxysteroid dehydrogenase as pre-receptor regulators of

glucocorticoid availability
precursor of cortisol (Compound F). We now know that the cortisone administered,
was converted to cortisol by 11β (Beta)-HSD1. The hydroxyl group at C11 is crucially
important for cortisol to be effective. 11β (Beta)-HSD1 reduces inactive cortisone to
active cortisol by adding a hydroxyl group at C11. Thus, unbeknownst to the authors
they had actually made two discoveries—the first that the glucocorticoid cortisol has
dramatic therapeutic effect on arthritis and second that the enzyme 11β (Beta)-HSD1
has the ability to convert inactive cortisone to active steroid.
Subsequently to Kendall’s paper, cortisol, and not cortisone, was established as
the active steroid. It also became apparent that inter-conversion between cortisone
and cortisol is possible [18, 19]. This conversion is made possible by the enzyme
11β (Beta)-HSD1, a member of the dehydrogenase/reductase superfamily. Short-
chain Dehydrogenase/Reducatse (SDR) enzymes are NADP(H) dependent enzymes,
that are involved primarily in breaking down hormones and chemical messengers
[20]. There are more than 3,000 members in the SDR family [7].
Early studies identified 11β (Beta)-HSD activity in different tissues such as pla-
centa, kidney and liver. However, 11β (Beta)-HSD did not have the same activity
between different tissues. The “set point” of the enzyme varied from tissue to tissue
with dehydrogenase activity (cortisol conversion to cortisone) predominating in the
placenta and kidney and reductive activity (cortisone conversion to cortisol) predomi-
nating in liver [7]. The answer lay in the fact that there are two distinct isozymes of
11β (Beta)-HSD namely type 1 being predominantly reductive and type 2 acting
solely as a dehydrogenase. In addition 11β (Beta)-HSD type 1 is a bi-directional
capable of acting in either direction but dependent on co-factors such as NADPH.
In vivo in intact tissues the 11β (Beta)-HSD1 enzyme acts as an oxo-reductase
converting cortisone to cortisol. Purified 11β (Beta)-HSD1 enzyme behaves princi-
pally as a dehydrogenase, oxidizing cortisol to cortisone.
Human 11β (Beta)-HSD1
11β (Beta)-HSD1 was initially purified and cloned from rodents in the 1980s by
Carl Monder’s group [21, 22]. In humans the gene for 11β (Beta)-HSD1 (HSD11B1)
is located on chromosome 1, is 30 kb in length and has six exons and five introns.
It comprises of 292 amino acids and shares 77 % homology with rat amino acid
sequence [23]. Human 11β (Beta)-HSD1, cloned in 2002 [24], exits as a dimer [25]
and is bound to the endoplasmic reticulum (ER) with its catalytic domain within the
ER lumen [26] (see Fig. 16.2). The catalytic directionality of the enzyme is based
on the position of 11β (Beta)-HSD1 within the ER lumen where it co-localises with
Hexose 6 Phosphate Dehydrogenase (H6PD). H6PD generates the reduced co-
substrate NADPH. Thus the ratio of NADPH/NADP confers directionality to 11β
(Beta)-HSD1 [27, 28]. Purified 11β (Beta)-HSD1, in the absence of H6PD, behaves
principally as a dehydrogenase, oxidizing cortisol to cortisone. The enzyme 11β
(Beta)-HSD1, has Michaelis Menten (Km) constants of 1.83 ± 0.06 μm for corticos-
terone and 17.3 ± 2.2406 μm for cortisol [7].
Fig. 16.2 Localization and co-factor dependency of 11β (Beta)-HSD1 within the endoplasmic
reticulum
11β (Beta)-HSD1 Ontogeny
The ontogeny of 11β (Beta)-HSD1 has been studied predominantly in rodents and
sheep animal models. The expression of mammalian 11β (Beta)-HSD1 is predomi-
nantly post-natal. In general 11β (Beta)-HSD1 is detectable in many tissues but
there is a lack of activity in early gestation with reductase activity only becoming
apparent after delivery and rising steadily throughout infancy [7, 29]. The ontogeny
of 11β (Beta)-HSD1 in humans is not well documented with few published studies.
Cortisone therapy is not useful for treating congenital adrenal hyperplasia in early
infancy most likely due to absent or significantly reduced liver 11β (Beta)-HSD1
activity [30]. Both reductase and dehydrogenase activity has been demonstrated in
fetal lung tissue [31, 32]. 11β (Beta)-HSD1 activity remains unchanged throughout
childhood in both boys and girls [33]. At puberty there is a reduction in 11β (Beta)-
HSD1 activity in women [33] and this appears to remain. In adults, there is a well
described dimorphism in cortisol metabolism between men and women with an
apparent reduction in 11β (Beta)-HSD1 activity in women [34, 35]. However, not
all studies have shown this dimorphism [36].
Regulation of 11β (Beta)-HSD1 Expression
11β (Beta)-HSD1 expression increases steadily until 1 year of age at which time
peak levels are reached. 11β (Beta)-HSD1 is expressed in many tissues including
liver, adipose tissue, gonads, GI tract, kidney, eye, anterior pituitary, leukocytes and
bone [7]. 11β (Beta)-HSD1 expression is highest in liver, brain gonads and adipose
tissues [37]. Many factors contribute to alterations in tissue expression of 11β
(Beta)-HSD1. In general glucocorticoids, pro-inflammatory cytokines (TNFα,
IL-1β) peroxisome proliferator-activated receptor γ agonists and CEBPs increase
11β (Beta)-HSD1 expression whereas Growth Hormone (GH) and liver X receptor
agonists inhibit 11β (Beta)-HSD1 expression [7]. Recently salicylates have been
shown to down regulate 11β (Beta)-HSD1 expression [38] in adipose tissue and
improve insulin sensitivity. The effects of sex steroids, insulin and other hormones
are variable across tissues and between species. 11β (Beta)-HSD1 has been shown
to be expressed more in adipose tissue in lean women compared to lean men [39].
High dose oestradiol has been shown to repress 11β (Beta)-HSD1 expression in rat
liver and kidney but testosterone did not alter expression [40].
Tissue-Specific Role of 11β (Beta)-HSD1
Adipose Tissue
11β (Beta)-HSD1 is highly expressed in human adipose tissue [3, 41]. Broadly
speaking, studies have shown similar expression levels of 11β (Beta)-HSD1 between
visceral and subcutaneous compartments [39, 42, 43]. However, H6PD and GR
have been shown to have different levels of expression between adipose tissue
depots [43].
As in other tissues, 11β (Beta)-HSD1 acts primarily as a reductase generating
active GCs. Its expression and activity are induced by GCs and inflammatory cyto-
kines such as interleukin 1 and TNFα [7, 44–47]. 11β (Beta)-HSD1 expression
and activity increases upon adipocyte differentiation [48] and inhibition of 11β
(Beta)-HSD1 blocks cortisone-induced adipocyte differentiation [49]. 11β (Beta)-HSD1
activity within adipose tissue depots is controversial with some studies demonstrating
increased activity in omental tissue compared to subcutaneous adipose tissue.
Dysregulation of 11β (Beta)-HSD1 activity has been postulated to be critical in
the pathogenesis of metabolic complications associated with obesity. In rodent
models increased 11β (Beta)-HSD1 activity has been demonstrated in visceral
adipose tissue of obese rodents compared to wild type [50, 51] although expression
in adipose tissue was reduced following high fat feeding in mice [52]. Short term
high fat diet reduced the activity of 11β (Beta)-HSD1 in both subcutaneous and
visceral adipose tissue in Wistar rats [53]. Genetically modified rodent models have
contributed significantly to our understanding of the role of 11β (Beta)-HSD1 in
adipose tissue. 11β (Beta)-HSD1 knockout mice are resistant to the metabolic side
effects of a high fat diet compared to wild type [54]. Conversely over expression of
11β (Beta)-HSD1 in adipose tissue leads to weight gain and metabolic complications
compared to wild type [55] even on a chow diet.
Clinical studies have yielded varied and sometimes conflicting results. In human
adipose tissue, the majority of studies have examined subcutaneous adipose tissue.
11β (Beta)-HSD1 activity in subcutaneous adipose tissue has been shown to be
increased in obese patients compared to non-obese controls [56]. Both 11β (Beta)-
HSD1 activity and expression were shown to correlate positively with BMI by Lindsay
et al. in 2003 [57]. Whilst the majority of studies show a positive relationship of 11β
(Beta)-HSD1 expression and activity with BMI, this is not the case for all studies.
Data with regards to the omental depot are more sparse, but some studies have identi-
fied increased expression in women in association with increased omental adiposity
[58]. The impact of diet induced obesity on 11β (Beta)-HSD1 activity and expression
is not clear. Obese Zucker rats have increased visceral adipose tissue 11β (Beta)-
HSD1 expression compared to lean rats [51] however Wistar rats, fed a short term
high fat diet show reduced 11β (Beta)-HSD1 activity [53]. Diet induced obese mice
also have reduced expression of 11β (Beta)-HSD1 [59]. In these diet induced obese
mice 11β (Beta)-HSD1 expression is increased by NFκ (Kappa) B and reduced by
HIF-1. H6PD is also important in the directionality of 11β (Beta)-HSD1 and some
studies suggest a role in visceral adipose tissue [60]. Pro inflammatory cytokines and
glucocorticoids (increased in obesity) are also known to induce 11β (Beta)-HSD1
expression in adipose tissue [7]. In vitro studies highlight the role of transcription fac-
tors (CEBPs) for controlling 11β (Beta)-HSD1 expression in adipocytes [61, 62].
Liver
GCs have key metabolic effects on the liver, augmenting insulin stimulated lipogenesis
[63] and reducing lipolysis in hepatic tissue [64]. Indirectly, they increase hepatic
lipid accumulation by inducing surrounding adipose tissue lipolysis thus increasing
the delivery of free fatty acids to the liver via the portal circulation [65].
11β (Beta)-HSD1 expression and activity has been extensively studied in hepatic
tissue. In animal studies global 11β (Beta)-HSD1 knock out mice are protected from
diet induced hepatic steatosis when fed a high fat diet [54, 55]. Transgenic mice
with hepatic 11β (Beta)-HSD1 overexpression, develop hypertension, hepatic ste-
atosis and dyslipidemia but interestingly do not develop steatohepatitis and minimal
insulin resistance [66]. Other factors including HPA axis activation and adipose
tissue 11β (Beta)-HSD1 activity may play a role in the development of hepatosteati-
tis. Liver specific HSD1-null mice have minimal phenotype (ref) suggesting vis-
ceral adipose HSD1 is the key site of action and excess adipose-generated GCs act
on the liver via visceral portal drainage.
11β (Beta)-HSD1 is highly expressed in human liver [23], predominantly in a
centripetal pattern histologically with maximum expression around the central vein
[67]. Hepatic 11β (Beta)-HSD1 appears to act exclusively as an oxo-reductase gen-
erating active GC [68]. In animal rodent models over expression of 11β (Beta)-
HSD1 in visceral adipose tissue was associated with Non Alcoholic Fatty Liver
Disease (NAFLD) [69]. In humans, some studies have demonstrated increased
expression of 11β (Beta)-HSD1 in liver tissue in obese persons with metabolic syn-
drome [70] however other studies have not confirmed these findings [71, 72]. Liver
11β (Beta)-HSD1 activity, as measured by urinary steroid metabolite analysis is
reduced in obesity compared to non-obese controls [73]. Liver 11β (Beta)-HSD1
activity (using serum cortisol generation form oral cortisone) is also reduced in
obesity compared to non-obese controls [73]. These reductions in both liver 11β
(Beta)-HSD1 activity and expression contrast with increased activity and expres-
sion in adipose tissue in obesity. Ahmed et al. have described a switch in the expres-
sion of 11β (Beta)-HSD1 across the spectrum of liver disease from lower expression
of 11β (Beta)-HSD1 in steatosis to higher levels of 11β (Beta)-HSD1 expression
associated with steatohepatitis [74]. The authors concluded these changes might
reflect a response to a more inflammatory phenotype.
Skeletal Muscle
GCs play an important role in protein metabolism. In Cushing’s syndrome the

effect of excess GCs is clearly seen with myopathy and muscle atrophy. 11β
(Beta)-HSD1 is expressed in skeletal muscle [75, 76], whereas 11β (Beta)-HSD2
is not. 11β (Beta)-HSD1 activity has been demonstrated in skeletal muscle [77]
and it may have a role in insulin sensitivity and metabolic disease. Increased
expression [78] and activity [79] have been demonstrated in rats and humans with
type 2 DM.
The exact role of 11β (Beta)-HSD1 in sarcopaenia remains unexplained. It is
thought that increased 11β (Beta)-HSD1 activity may play a role in allowing
increased muscle cortisol exposure. Increased 11β (Beta)-HSD1 expression in skel-
etal muscle is associated with reduced muscle strength in older adults compared to
younger controls [80].
Skin
Data on tissue cortisol metabolism within skin is only recently becoming an area
of interest and investigation. Skin has been shown to be an active site of cortisol
production and metabolism [81, 82]. Excess skin exposure to GC’s cause skin
changes similar to the natural aging process. These include reduced elasticity,
reduced collagen and fibroblast numbers, thinning of dermis and epidermis and a
general reduction in the repair capacity of skin [83]. Increased exposure to GC’s has
been postulated as a factor in age related changes, inflammatory and auto-immunity
changes seen in skin [84]. It has been postulated that skin changes seen over time
are in part as a result of 11β (Beta)-HSD1 activity [82].
Both 11β (Beta)-HSD1 and 2 are expressed in skin [81, 82, 85]. 11β (Beta)-HSD2
is expressed in association with the mineralocorticoid receptor on sweat glands how-
ever its role (if any) within the dermis and epidermis is debated. In wound healing,
11β (Beta)-HSD2 expression has been shown to be induced 48 h after tissue injury
with subsequent return to basal levels at 96 h [81]. This has been postulated to be a
mechanism to reduce local cortisol excess following inflammation.
11β (Beta)-HSD1 is widely expressed in human and mouse dermis and epider-
mis [82, 85, 86]. Upon differentiation of keratinocytes 11β (Beta)-HSD1 expression
increases [85], somewhat akin to the changes seen with pre adipocyte differentiation
[87]. Interestingly despite reducing levels of expression of 11β (Beta)-HSD1 in
elderly subjects, a paradoxical rise in 11β (Beta)-HSD1 activity is seen with increasing
age in both humans and mice [82]. This gives credence to the concept of age related
skin atrophic changes being in part due to increased cortisol exposure secondary to
increased 11β (Beta)-HSD1 activity.
11β (Beta)-HSD1 has been shown to have a pivotal role in skin repair following
injury [88] and tissue remodeling [85]. In mice 11β (Beta)-HSD1 is contributory to
impaired wound healing. Blocking 11β (Beta)-HSD1 improved wound healing in
mice and prevented age induced skin changes [89]. These data suggest that 11β
(Beta)-HSD1 generated local cortisol is critically important in wound healing and
in aging skin changes. Inhibitors of 11β (Beta)-HSD1 inhibitors (topical or oral)
may therefore have therapeutic potential.
Both isozymes are expressed in blood vessel walls [90] and heart [91], however oxo
reductase directionality (11β (Beta)-HSD1) predominates in vascular smooth
muscle [88]. There is evidence linking 11β (Beta)-HSD1 activity with atheroscle-
rosis. Mediastinal adipose tissue 11β (Beta)-HSD1 has been linked with coronary
atherosclerosis [92]. The same authors demonstrated increased 11β (Beta)-HSD1
expression in aortas of obese patients with the metabolic syndrome [93]. 11β (Beta)-
HSD1 inhibition in apoE knockout mice achieved significant reduction atheroscle-
rotic load suggesting a role in plaque formation [94]. Carbenoxolone treatment has
been shown to reduce atherosclerosis in mice [95]. 11β (Beta)-HSD1 knock out mice
show improved angiogenesis to infarcted regions possibly through reduced GC
regeneration locally [96].
Central Nervous System (CNS)
GCs are required for normal brain development and normal brain function. Excess
GCs are associated with alterations in mood, memory and brain function [97, 98].
Both 11β (Beta)-HSD1 and 2 are expressed in the brain [99–102], but 11β (Beta)-
HSD2 is expressed at lower levels. 11β (Beta)-HSD1 acts principally as an oxo-
reductase in brain tissue [103] but interestingly H6PD does not always co-localise
with 11β (Beta)-HSD1 in the CNS [101]. This lack of universal co-localisation sug-
gests other enzymes providing the necessary co factors for 11β (Beta)-HSD1.
Elevated 11β (Beta)-HSD1 is seen with ageing and is associated with cognitive
decline. Altered 11β (Beta)-HSD1 activity in the CNS is associated with changes in
appetite, affective behavior and circadian rhythm [104].
A putative role for 11β (Beta)-HSD1 in cognitive impairment is postulated
along with investigations looking at the link between 11β (Beta)-HSD1 and human
eye disease including thyroid eye disease [105] and glaucoma. 11β (Beta)-HSD1
inhibitors have been shown to improve cognitive function in elderly persons with
T2 DM [106].
Inflammation and Immunity
Glucocorticoids in pharmacological doses are immunosuppressive and produce

powerful anti-inflammatory effects [107]. They achieve this by altering gene tran-
scription and altering pro and anti-inflammatory mediators including cytokines and
signaling pathways. 11β (Beta)–HSD1 is believed to play a key role in local inflam-
mation and immune response to stimuli and allergens [108].
11β (Beta)-HSD1 is expressed on numerous immune cell types in humans includ-
ing mast cells, mononuclear cells and macrophages, lymphocytes, B cells and den-
dritic cells [109–112]. 11β (Beta)-HSD1 is not expressed in non-stimulated
monocytes but 11β (Beta)-HSD1 expression is increased upon differentiation of
monocytes cells into macrophages with interleukin 4 and 13 [110]. A further increase
in 11β (Beta)–HSD1 expression is seen when monocytes differentiate into dendritic
cells under the influence of interleukin-4 and granulocyte-macrophage colony-stim-
ulating factor. Other pro-inflammatory cytokines do not increase 11β (Beta)-HSD1.
In macrophages, 11β (Beta)-HSD1 activity increases with exposure to lipopolysac-
charide [110]. 11β (Beta)-HSD1 expression has also been seen in murine lymphocytes
and B cells with increases seen when CD4-positive lymphocytes polarize into Th1 or
Th2 subsets [111].
Separate to expression, the functional role of 11β (Beta)-HSD1 in immunity
remains debated as it is difficult to separate out the specific subsets of immune cells
and to be confident of the role (if any) of 11β (Beta)-HSD1. Our understanding of
11β (Beta)-HSD1 expression and activity on human immune cell actions and
inflammation is incomplete and warrants further investigation.
There is evidence both in animal models and in human studies that demonstrate
that 11β (Beta)-HSD1 is implicated in immune response to infection and inflamma-
tion. In 11β (Beta)-HSD1 knock out mice defective macrophage phagocytosis of
apoptotic neutrophils is seen in peritonitis [113]. KO mice have increased suscepti-
bility to endotoxaemia compared to wild type [114]. Of note, a study in mice demon-
strated that in various models of joint and lung inflammation 11β (Beta)-HSD1
knock out mice developed inflammation earlier, substantially more inflammation and
slower resolution of inflammation compared to wild type controls [115]. This study
suggests that 11β (Beta)-HSD1 plays a key role as an anti-inflammatory regulator
and raises concerns for drug therapy inhibiting 11β (Beta)-HSD1.
In humans (and rats) 11β (Beta)-HSD1 expression is elevated in colitis [116, 117]
with a concomitant reduction in 11β (Beta)-HSD2 expression. Acute exacerbations of
inflammatory bowel disease are associated with elevated 11β (Beta)-HSD1 expres-
sion [118] but interestingly 11β (Beta)-HSD1 activity was elevated in patients who
were in remission, suggesting that high local glucocorticoid levels are important in
limiting inflammation. In a rat model, chemically induced inflammation increased
11β (Beta)-HSD1 expression both in colonic tissue and lymphoid tissue [119].
Recently, 11β (Beta)-HSD1 has been shown to provide tonic inhibition of mast
cell deactivation. 11β (Beta)-HSD1 knock out mice had increased mast cell num-
bers and a lower threshold for deactivation suggesting that reduced 11β (Beta)-
HSD1 activity increases allergy and anaphylaxis [109].
Bone and Joint
11β (Beta)-HSD1 is expressed in human bone [120], predominantly in osteo-

blasts [121].
In rheumatoid arthritis, both 11β (Beta)-HSD1 and 11β (Beta)-HSD2 isozymes
are expressed in synovial tissue with conflicting reports as to which is the dominant
isoform. Global 11β (Beta)-HSD1 activity is increased in rheumatoid arthritis
(as measured by urinary corticosteroid metabolites) compared to non-arthritic controls
[122]. Enzyme activity correlates with synovial inflammation severity [123]. In ani-
mal models 11β (Beta)-HSD1 knock out mice develop more severe arthritis and
earlier compared to wild type controls [115]and mice treated with 11β (Beta)-HSD1
inhibitors (Carbenoxolone) developed worse arthritis [124].
Cortisone Reductase Deficiency (CRD) and Apparent Cortisone Reductase

Deficiency (ACRD)
Genetic defects in both HSDB1 and H6PD encoding genes demonstrate the effect
of alterations in tissue 11β (Beta)-HSD1 on clinical phenotype. Both Cortisone
Reductase Deficiency (CRD) from HSDB1 gene defects and apparent Cortisone
Reductase Deficiency (ACRD) from H6PD gene defects show the impact of
reduction in tissue 11β (Beta)-HSD1 activity with low urine cortisol, significantly
elevated cortisone with subsequent compensatory increased HPA activity leading to
hyperandrogenism, early adrenarche and PCOS in women [7, 125].
CRD was first described in the 1980s. The majority of cases are female and
present with clinical and biochemical hyperandrogenism, with males presenting with
precocious puberty [7, 37]. CRD has been described as the “human 11β (Beta)-HSD1
knockout” [7]. The condition is ameliorable to dexamethasone therapy. It shares
some clinical and biochemical features and should not be confused with non-classical
congenital adrenal hyperplasia.
11β (Beta)-HSD1 Inhibition in Clinical Studies
Due to evidence that has implicated 11β (Beta)-HSD1 in the pathogenesis of disease
states including obesity, diabetes and the metabolic syndrome, it represents an
exciting therapeutic target to limit local GC availability [2, 126]. Many inhibitors
of the 11β (Beta)-HSD enzymes have been described. These include naturally
occurring inhibitor compounds such as liquorice derived glycyrrhetinic acid [127],
flavanone/hydroxyl flavanones [128], bile acids [129], progesterone metabolites
[130] and even coffee [131]. Most of these naturally occurring compounds inhibit
both 11β (Beta)-HSD1 and 11β (Beta)-HSD2, with subsequent hypertension and
hypokalaemia limiting their possible benefits.
Carbenoxolone, a non-selective inhibitor derived from glycyrrhetinic acid, was
the first drug to show benefit in human studies [4, 132]. In healthy volunteers it
improved insulin sensitivity and reduced glucose production rates via a reduction in
glycogenolysis but not gluconeogenesis in patients with type 2 diabetes [132].
Carbenoxolone has also been shown to reduce local cortisol availability in subcuta-
neous adipose tissue and inhibits glucocorticoid induced lipolysis [4]. These early
small “proof of principal” studies demonstrated that 11β (Beta)-HSD1 in metabolic
disease could be targeted for drug manipulation and importantly that tissue specific
effect could be demonstrated, despite a relative lack of specificity of carbenexolone
on 11β (Beta)-HSD1. Several pharmaceutical companies have developed potent
selective 11β (Beta)-HSD1 inhibitors. Indeed 11β (Beta)-HSD inhibition has
become a significant area of investment for many companies [133] and an extensive
array of chemical compounds have been patented and reviewed extensively else-
where [134]. In general, they are highly selective for 11β (Beta)-HSD1 over 11β
(Beta)-HSD2 and have high potency for inhibition.
A small number of clinical trials have been published with relatively short dura-
tions of intervention (all no more than 12 weeks) and have demonstrated improve-
ments in biomarkers including cholesterol profiles, weight, glycaemic control and
blood pressure [8, 135, 136]. The first outcome study to be published investigated
the addition of an 11β (Beta)-HSD1 inhibitor (INCN13739) to metformin in
patients with type 2 Diabetes. The drug was well tolerated and the 12 week study
demonstrated improvements in weight, glycaemic control and lipid profiles in
those people that received the inhibitor [135]. Of note, in this trial there was a
compensatory increase in the HPA axis with a dose dependent increase in ACTH
and subsequent increase in certain androgens. In women there was a small and
significant increase in testosterone levels but biologically active testosterone was
felt to be unchanged as sex hormone binding globulin also increased. Whilst all
blood results remained in the normal reference range these alterations and small
elevations in androgens from HPA activation will remain a clinical concern in the
long term, especially for women.
In a further study using a different compound (MK0916) again in the setting of
type 2 diabetes and metabolic syndrome, modest improvements in glycaemic con-
trol with no reduction in fasting glucose levels were seen [8]. Again however there
was mild activation of HPA with elevations in adrenal androgen secretion. Overall,
the long-term side effects of 11β (Beta)-HSD1 inhibition remain unknown and
while the benefits may outweigh any side effects the possible disruption of HPA
axis will remain a concern alongside the magnitude of the clinical response.
Tissue Cortisone Metabolism: 11β (Beta)-HSD2
11β (Beta)-HSD2 Discovery
In 1993, Seckl et al. isolated an enzyme with exclusive 11β (Beta)-HSD dehydroge-
nase activity from both human placenta and rat kidney [137]. In 1994, Krozoski et al.
isolated human 11β (Beta)-HSD from human kidney [138], identical to the dehydro-
genase enzyme found in placenta. This second enzyme was found to be distinct from
11β (Beta)-HSD1 and was called 11β (Beta)-HSD2 and is also a member of the SDR
family [139]. Both 11β (Beta)-HSD1 and 11β (Beta)-HSD2 isozymes are members of
the Short-Chain Dehydrogenase/Reductase (SDR) superfamily of enzymes, however
each isozyme has a distinct gene sequence with exons found on different chromo-
somes. There is little similarity or overlap in sequence between isozymes (18 % iden-
tity), except for similar co-factor binding regions at the NH2-terminal [6].
The human 11β (Beta)-HSD2 gene is located on chromosome 16, has 5 exons
and is only 6 kbs in length [140]. Human 11β (Beta)-HSD2 measures 405 amino
acids in length and has a molecular mass of 44 kDa [138]. It is also anchored to the
ER and loses its dehydrogenase activity once removed from tissue membranes
[141]. 11β (Beta)-HSD2 universally acts as a dehydrogenase across species [6] and
has a Km for cortisol of 50–60 nM and 10–13 nM for cortisone [6, 142]. Mutations
in 11β (Beta)-HSD2 leading to apparent mineralocorticoid excess (AME) and
hypertension have been extensively reported [143–145].
11β (Beta)-HSD2 Ontogeny
11β (Beta)-HSD2 has an important role in fetal development with intra uterine
“programming” affecting subsequent adult physiology. 11β (Beta)-HSD2 plays an
important role in gestation in humans and mammals protecting tissues against GC
exposure prematurely. 11β (Beta)-HSD2 is expressed and active in placenta [36, 51]
and steadily rises throughout gestation and declines 2 weeks prior to labour [29,
137, 146]. Placenta 11β (Beta)-HSD2 is localized to the syncytiotrophoblast where
it has been described as a barrier to maternal corticosteroid which is considerably
more concentrated [6]. GCs play a critical role in the development of fetal organs,
in particular, towards the end of pregnancy, in lung tissue. Excess GC exposure in
utero is associated with physiological and metabolic complications [147–149].
There is mounting evidence that 11β (Beta)-HSD2 plays a key protective role in
normal development of the fetus and in particular brain development [148]. Altered
or disrupted 11β (Beta)-HSD2 activity with subsequent excess intra-uterine expo-
sure to glucocorticoid has a “programming” effect on the fetus leading to low birth
weight and lifelong physiological consequences such as increased cardiovascular,
metabolic and psychiatric complications [150]. This role in the development of the
fetus and its role in subsequent lifelong physiology has led some to consider the
degree of prenatal GC exposure as a potential prognostic biomarker [6].
Regulation 11β (Beta)-HSD2 Expression
Unlike 11β (Beta)-HSD1, there is considerable data published on the epigenetic

influence on 11β (Beta)-HSD2 activity in humans and in rodent models.
The 11β (Beta)-HSD2 gene is susceptible to epigenetic influence, with methyla-
tion of the promoter region of particular interest. Increased methylation of this
region has been inversely associated with 11β (Beta)-HSD2 expression and influences
the development of hypertension, intrauterine growth, birth weight and neurobehav-
ioural movement [151, 152] in rats. Intrauterine growth retardation has been associ-
ated with increased methylation of 11β (Beta)-HSD2 gene promoter with subsequent
repression of 11β (Beta)-HSD2 in adult kidneys [153].
Factors that increase 11β (Beta)-HSD1 expression tend to reduce 11β (Beta)-HSD2
such as TNFα [154]. Oestrogen increases 11β (Beta)-HSD2 expression [40, 155].
Vasopressin has been shown to stimulate 11β (Beta)-HSD2 [156]. Glucocorticoids
down-regulate 11β (Beta)-HSD2 in foetal placenta but not foetal kidney [157].
Dexamethasone up-regulates 11β (Beta)-HSD2 in lung cells [158]. Hypoxia has been
shown to reduce 11β (Beta)-HSD2 expression [159]. In colonic epithelium, aldoste-
rone increases 11β (Beta)-HSD2 expression [160].
Tissue-Specific Role of 11β (Beta)-HSD2
CNS
In the adult brain, 11β (Beta)-HSD2 is expressed in a select few regions [161].
Before birth, 11β (Beta)-HSD2 is expressed in several additional brain regions,
including the thalamus and cerebellum, where this enzyme protects proliferating
granule cells from the growth-limiting effects of glucocorticosteroids [162, 163].
In adults, however, mRNA and protein are no longer detectable in these regions.
Instead, 11β (Beta)-HSD2 expression is found in just a few small sites in adult mice
and rats [161, 164] most prominently in a group of neurons found inside the nucleus
of the solitary tract (NTS). NTS neurons with 11β (Beta)-HSD2 expression are the
only cells in the brain shown to express both this enzyme and the mineralocorticoid
receptor (MR); these “HSD2 neurons” are activated along with salt appetite after
dietary sodium deprivation or volume depletion, and may trigger salt appetite. The
only other brain sites in which 11β (Beta)-HSD2 expression (mRNA and protein)
was identified consistently in adult animals are the subcommissural organ (a cir-
cumventricular organ comprised of modified ependymal cells which do not express
MR) and a small subdivision of the ventromedial hypothalamic nucleus (neurons
that lack MR immunolabeling). Information regarding 11β (Beta)-HSD2 expression
remains incomplete for non-neuronal tissues such as the meninges, choroid plexus,
ventricular ependyma, and cerebral vasculature.
11β (Beta)-HSD2 is expressed in vascular endothelium [90]. 11β (Beta)-HSD2

knockout mice develop endothelial dysfunction [165]. Lack of 11β (Beta)-HSD2
and MR activation is implicated in generation of severe atherosclerosis in mouse
models [166].
Kidney
11β (Beta)-HSD2 is perhaps best known for its role in the kidney where it protects
the mineralocorticoid receptor from excess exposure to GC. 11β (Beta)-HSD2 is
widely expressed in distal nephrons [141]. Although the inherent enzyme ability
of 11β (Beta)-HSD2 to clear cortisol (converting it to cortisone) should not be
enough, given concentrations and binding affinities, in reality it protects the miner-
alocorticoid receptor from GC exposure [6, 167]. Lack of 11β (Beta)-HSD2 in
kidney (AME discussed below), leads to hypertension and other sequelae. 11β
(Beta)-HSD2 activity, measured by urinary metabolite ratios, reduces with age
suggesting a role in age related hypertension [168]. Some studies show a role of 11β
(Beta)-HSD2 in hypertension although not all studies are in agreement [169].
In kidney disease reduced 11β (Beta)-HSD2 activity has been shown in persons
with hypertension [170, 171].
Colon
11β (Beta)-HSD2 is expressed in colonic epithelium [172]. Expression is increased

by aldosterone in rats [160]. In Inflammatory bowel disease 11β (Beta)-HSD2
expression is down regulated in both humans and rats [116]. This is accompanied by
an increase in 11β (Beta)-HSD1 expression and so is presumed to be an attempt to
locally control GC exposure to inflamed tissue. Zhang et al. showed that inhibiting
11β (Beta)-HSD2 reduces colon carcinogenesis by inhibiting COX 2 pathways. The
reduction in 11β (Beta)-HSD2 blocked colorectal adenocarcinoma angiogenesis
and metastasis [173].
Salivary Gland and Skin
As mineralocorticoid target tissues, both skin and salivary glands express 11β
(Beta)-HSD2. In the skin expression is mainly restricted to sweat glands [174] 11β
(Beta)-HSD2 is expressed in both parotid and sub-mandibular glands [174, 175].
Measuring salivary cortisone has been postulated a potential biomarker of serum
free cortisol [176]. In addition, reduced activity of 11β (Beta)-HSD2 in sweat glands
has also been linked with essential hypertension [177].
Pituitary
In normal anterior pituitary tissue 11β (Beta)-HSD2 mRNA expression is seen, but
immunofluorescence reveals absent 11β (Beta)-HSD2 isozyme. Interestingly,
ACTH secreting tumours induce 11β (Beta)-HSD2 expression and may in part
explain the re-setting of GC feedback control seen in Cushing’s disease [99].
Apparent Mineralocorticoid Excess (AME)
AME is a rare clinical disease that presents in early childhood with hypertension,
sodium retention, potassium loss, suppressed renin activity and a metabolic alkalo-
sis [178]. This condition was hallmarked by an increased ratio of urinary cortisol to
cortisone metabolites as a result of a lack of conversion of cortisol to cortisone.
There was also a low level of circulating of mineralocorticoid despite having evi-
dence of apparent excess with metabolic derangement. Usually fatal in childhood
adults with AME were discovered and successfully treated with dexamethasone.
Physiological replacement with cortisol caused a return of symptoms and signs [179].
Stewart et al. in 1996 demonstrated that AME was caused by a defect in the 11β
(Beta)-HSD2 gene [180] (similar to the effect seen with liquorice ingestion [181]).
These and other observations led to the understanding that 11β (Beta)-HSD2 in the
kidney protected the mineralocorticoid receptor from glucocorticoid binding. Under
normal circumstances 11β (Beta)-HSD2 deactivates cortisol to cortisone and thus
protects the MR receptor. In AME, with loss of 11β (Beta)-HSD2 activity unop-
posed cortisol binds to MR with subsequent clinical mineralocorticoid excess.
The Future of the Dehydrogenase Hypothesis
There is strong evidence that dysregulated 11β (Beta)-HSD1 activity is involved in

many pathological processes including obesity and type 2 diabetes. We have also
begun to see early clinical trials that demonstrate clinical benefit in 11β (Beta)-
HSD1 inhibition. However, whether or not these compounds eventually enter the
market with a licence to treat diabetes and metabolic disease remains to be seen.
However, they may have utility in other conditions including glaucoma, idiopathic
intracranial hypertension and low bone mineral density and clinical trials in these
areas are ongoing. In addition, recent work based on observations in a patient with
Cushing’s disease [182] has suggested that 11β (Beta)-HSD1 may have a role in
regulating the phenotype of circulating active GC excess [183]. This not only raises
the possibility for the use of 11β (Beta)-HSD1 inhibitors in the treatment of
Cushing’s syndrome, but also that basal activity may predict the susceptibility to the
adverse effects of exogenous GCs and that 11β (Beta)-HSD1 inhibitors could ame-
liorate the adverse effects, without compromise to the desired actions, of therapeuti-
cally indicated GCs.
11β (Beta)-HSD2 has an established role in the regulation of blood pressure and
this is highlighted in patients with AME. However, evidence points to an additional
role in placental development and function that may have implications for neonatal
growth, either directly or through programming. Inhibiting 11β (Beta)-HSD2
action has been shown to stop colorectal adenocarcinoma spread and development
and this warrants further investigation [173]. Importantly though, whilst much
attention in recent years has focussed on 11β (Beta)-HSD1 activity and therapeutic
inhibition, we must not forget that these two enzymes are tightly associated not
least of all because the activity of 11β (Beta)-HSD1 is entirely dependent upon
substrate availability (cortisone) that is generated by 11β (Beta)-HSD2. It is entirely
plausible that cortisone availability may represent a rate-limiting step regulating
11β (Beta)-HSD1 activity through substrate availability and this needs to be further
explored [184].
Inhibition of 11β (Beta)-HSD1 and specifically within tissues is a promising
target as a potential disease prevention/modifying pathway. There is strong evidence
to date that dysregulated 11β (Beta)-HSD1 activity is involved in many disease
states including obesity and diabetes among many. We have also begun to see early
human clinical trials that demonstrate clinical benefit with 11β (Beta)-HSD1 inhibition.
However, as outlined above both the small number, and the short nature of studies
to date have not yielded strong robust evidence to use 11β (Beta)-HSD1 inhibitors.
Limited benefits and possible side effects including HPA activation will likely
impede and slow the process of these inhibitor compounds from entering phase 3
trials and into clinical use.
One area of possible clinical use, which has not been looked at yet, is to help
antagonise the effects of excess glucocorticoid side effects. Published data demon-
strate a key role of 11β (Beta)-HSD1 in contributing to GC side effects in Cushing’s
[182] and in bone metabolism [184]. 11β (Beta)-HSD1 global knock out mice treated
with excess GC are protected against GC side effects when compared to wild type
controls [183]. This suggests that in conditions with excess circulating GC’s, it is the
re-activation of cortisone to cortisol within tissues by 11β (Beta)-HSD1, rather than
simple cortisol delivery to tissue from the circulation that is the crucial step deter-
mining Cushingoid side effects. Data on the role of 11β (Beta)-HSD1 activity in
humans with excess GC’s is lacking. GC’s are widely prescribed with estimates of
1–2 % [185] of the population taking prescribed steroids for various inflammatory
conditions. Despite their efficacy, up to 70 % of patients experience an adverse
systemic side-effect profile [186]. Inhibiting 11β (Beta)-HSD1 activity may play a
beneficial role in preventing glucocorticoid side effects therefore making 11β (Beta)-
HSD1 an exciting therapeutic target for patients with Cushing’s syndrome.
Lastly, as mentioned previously there is evidence in the literature that 11β (Beta)-
HSD1 activity increases with age and is associated with tissue damage and dysfunc-
tion. Therefore using tissue specific inhibitors or 11β (Beta)-HSD1 may be a target
in conditions such as sarcopaenia and osteoporosis.
Conclusions
Tissue-specific GC metabolism is under the control of the 11β (Beta)-HSD

dehydrogenase/reductase enzymes. 11β (Beta)-HSD2 exists to primarily protect
mineralocorticoid receptor from excess cortisol exposure in tissues such as kidney.
11β (Beta)-HSD1 exists in many tissues and amplifies tissue exposure to cortisol by
activating cortisol from inert cortisone. Both isoforms have a potent ability to
manipulate clinical phenotype entirely independent of circulating GC levels.
The complexity of this system and the intricate and finely tuned control that is able
to exert at a tissue-specific level to govern ligand access to corticosteroid receptors
has highlighted a fundamental shift in our approach. It adds weight to the argument that
simple measurement of circulating steroid hormone levels provides an over-simplistic
and perhaps misleading view of GC action.
References
1. Munck A, Náray-Fejes-Tóth A. The ups and downs of glucocorticoid physiology. Permissive

and suppressive effects revisited. Mol Cell Endocrinol. 1992;90(1):C1–4. http://www.ncbi.
nlm.nih.gov/pubmed/1301388. Accessed 31 Dec 2013.
2. Anagnostis P, Katsiki N, Adamidou F, et al. 11beta-Hydroxysteroid dehydrogenase type 1
inhibitors: novel agents for the treatment of metabolic syndrome and obesity-related disorders?
Metabolism. 2013;62(1):21–33. doi:10.1016/j.metabol.2012.05.002.
3. Bujalska IL, Kumar SSP. Does central obesity reflect “Cushing’s disease of the omentum”?
Lancet. 1997;349(9060):1210–3.
4. Tomlinson JW, Sherlock M, Hughes B, et al. Inhibition of 11beta-hydroxysteroid dehydroge-
nase type 1 activity in vivo limits glucocorticoid exposure to human adipose tissue and decreases
lipolysis. J Clin Endocrinol Metab. 2007;92(3):857–64. doi:10.1210/jc.2006-2325.
5. Tomlinson JW, Moore JS, Clark PMS, Holder G, Shakespeare L, Stewart PM. Weight loss
increases 11beta-hydroxysteroid dehydrogenase type 1 expression in human adipose tissue.
J Clin Endocrinol Metab. 2004;89(6):2711–6. doi:10.1210/jc.2003-031376.
6. Chapman K, Holmes M, Seckl J. 11β-Hydroxysteroid dehydrogenases: intracellular gate-
keepers of tissue glucocorticoid action. Physiol Rev. 2013;93(3):1139–206. doi:10.1152/
physrev.00020.2012.
7. Tomlinson JW, Walker EA, Bujalska IJ, et al. 11Beta-hydroxysteroid dehydrogenase type 1:
a tissue-specific regulator of glucocorticoid response. Endocr Rev. 2004;25(5):831–66.
doi:10.1210/er.2003-0031.
8. Feig PU, Shah S, Hermanowski-Vosatka A, et al. Effects of an 11β-hydroxysteroid dehydroge-
nase type 1 inhibitor, MK-0916, in patients with type 2 diabetes mellitus and metabolic syn-
drome. Diabetes Obes Metab. 2011;13(6):498–504. doi:10.1111/j.1463-1326.2011.01375.x.
9. Edwards CR, Stewart PM, Burt D, et al. Localisation of 11 beta-hydroxysteroid dehydroge-
nase—tissue specific protector of the mineralocorticoid receptor. Lancet. 1988;2(8618):986–
9. http://www.ncbi.nlm.nih.gov/pubmed/2902493. Accessed 23 Feb 2014.
10. Esteban NV, Loughlin T, Yergey AL, et al. Daily cortisol production rate in man determined
by stable isotope dilution/mass spectrometry. J Clin Endocrinol Metab. 1991;72(1):39–45.
doi:10.1210/jcem-72-1-39.
11. Keenan DM, Roelfsema F, Veldhuis JD. Endogenous ACTH concentration-dependent drive
of pulsatile cortisol secretion in the human. Am J Physiol Endocrinol Metab.
2004;287(4):E652–61. doi:10.1152/ajpendo.00167.2004.
12. Siiteri PK, Murai JT, Hammond GL, Nisker JA, Raymoure WJ, Kuhn RW. The serum trans-
port of steroid hormones. Recent Prog Horm Res. 1982;38:457–510. http://www.ncbi.nlm.
nih.gov/pubmed/6750727. Accessed 30 Dec 2013.
13. Dorin RI, Qiao Z, Qualls CR, Urban FK. Estimation of maximal cortisol secretion rate in
healthy humans. J Clin Endocrinol Metab. 2012;97(4):1285–93. doi:10.1210/jc.2011-2227.
14. Toothaker RD, Welling PG. Effect of dose size on the pharmacokinetics of intravenous
hydrocortisone during endogenous hydrocortisone suppression. J Pharmacokinet Biopharm.
1982;10(2):147–56. http://www.ncbi.nlm.nih.gov/pubmed/7120045. Accessed 9 Feb 2014.
15. Meulenberg PM, Hofman JA. Differences between concentrations of salivary cortisol and
cortisone and of free cortisol and cortisone in plasma during pregnancy and postpartum.
Clin Chem. 1990;36(1):70–5. http://www.ncbi.nlm.nih.gov/pubmed/2297937. Accessed 7
Mar 2014.
16. Kendall EC. Cortisone: memoirs of a hormone hunter. New York: Charles Scribner’s Sons;
1971.
17. Hench PS, Kendall EC. The effect of a hormone of the adrenal cortex (17-hydroxy-11-
dehydrocorticosterone; compound E) and of pituitary adrenocorticotropic hormone on rheu-
matoid arthritis. Proc Staff Meet Mayo Clin. 1949;24(8):181–97. http://www.ncbi.nlm.nih.
gov/pubmed/18118071.
18. Burton RB, Keutmann EH, Waterhouse C, Schuler EA. The conversion of cortisone acetate
to other alphaketolic steroids. J Clin Endocrinol Metab. 1953;13(1):48–63. doi:10.1210/
jcem-13-1-48.
19. Amelung D, Hubener HJ, Roka L, Meyerheim G. Conversion of cortisone to compound F.
J Clin Endocrinol Metab. 1953;13(9):1125–6. doi:10.1210/jcem-13-9-1125.
20. Kavanagh KL, Jörnvall H, Persson B, Oppermann U. Medium- and short-chain dehydroge-
nase/reductase gene and protein families: the SDR superfamily: functional and structural
diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci.
2008;65(24):3895–906. doi:10.1007/s00018-008-8588-y.
21. Agarwal AK, Monder C, Eckstein B, White PC. Cloning and expression of rat cDNA encod-
ing corticosteroid 11 beta-dehydrogenase. J Biol Chem. 1989;264(32):18939–43. http://
www.ncbi.nlm.nih.gov/pubmed/2808402.
22. Lakshmi V, Monder C. Purification and characterization of the corticosteroid 11 beta-
dehydrogenase component of the rat liver 11 beta-hydroxysteroid dehydrogenase complex.
23. Tannin GM, Agarwal AK, Monder C, New MI, White PC. The human gene for 11 beta-
hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal local-
ization. J Biol Chem. 1991;266(25):16653–8.
24. Nobel CSI, Dunås F, Abrahmsén LB. Purification of full-length recombinant human and rat
type 1 11beta-hydroxysteroid dehydrogenases with retained oxidoreductase activities.
Protein Expr Purif. 2002;26(3):349–56. http://www.ncbi.nlm.nih.gov/pubmed/12460758.
Accessed 5 Jan 2014.
25. Maser E, Völker B, Friebertshäuser J. 11 Beta-hydroxysteroid dehydrogenase type 1 from
human liver: dimerization and enzyme cooperativity support its postulated role as glucocor-
ticoid reductase. Biochemistry. 2002;41(7):2459–65. http://www.ncbi.nlm.nih.gov/
pubmed/11841241. Accessed 31 Dec 2013.
26. Odermatt A. The N-terminal anchor sequences of 11beta-hydroxysteroid dehydrogenases
determine their orientation in the endoplasmic reticulum membrane. J Biol Chem.
1999;274(40):28762–70. doi:10.1074/jbc.274.40.28762.
27. Bujalska IJ, Draper N, Michailidou Z, et al. Hexose-6-phosphate dehydrogenase confers oxo-
reductase activity upon 11 beta-hydroxysteroid dehydrogenase type 1. J Mol Endocrinol.
2005;34(3):675–84. doi:10.1677/jme.1.01718.
28. Draper N, Walker EA, Bujalska IJ, et al. Mutations in the genes encoding 11beta-
hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to
cause cortisone reductase deficiency. Nat Genet. 2003;34(4):434–9. doi:10.1038/ng1214.
29. Murphy VE, Clifton VL. Alterations in human placental 11beta-hydroxysteroid dehydroge-
nase type 1 and 2 with gestational age and labour. Placenta. 2003;24(7):739–44. http://www.
ncbi.nlm.nih.gov/pubmed/12852864. Accessed 12 Feb 2014.
30. Jinno K, Sakura N, Nomura S, Fujitaka M, Ueda K, Kihara M. Failure of cortisone acetate
therapy in 21-hydroxylase deficiency in early infancy. Pediatr Int. 2001;43(5):478–82. http://
www.ncbi.nlm.nih.gov/pubmed/11737708. Accessed 22 Jan 2014.
31. Murphy BE. Cortisol production and inactivation by the human lung during gestation and
infancy. J Clin Endocrinol Metab. 1978;47(2):243–8. doi:10.1210/jcem-47-2-243.
32. Abramovitz M, Branchaud CL, Murphy BE. Cortisol-cortisone interconversion in human
fetal lung: contrasting results using explant and monolayer cultures suggest that 11
beta-hydroxysteroid dehydrogenase (EC 1.1.1.146) comprises two enzymes. J Clin

Endocrinol Metab. 1982;54(3):563–8. doi:10.1210/jcem-54-3-563.
33. Dimitriou T, Maser-Gluth C, Remer T. Adrenocortical activity in healthy children is associ-
ated with fat mass. Am J Clin Nutr. 2003;77(3):731–6. http://www.ncbi.nlm.nih.gov/
pubmed/12600869.
34. Toogood AA, Taylor NF, Shalet SM, Monson JP. Sexual dimorphism of cortisol metabolism
is maintained in elderly subjects and is not oestrogen dependent. Clin Endocrinol (Oxf).
2000;52(1):61–6. http://www.ncbi.nlm.nih.gov/pubmed/10651754. Accessed 22 Jan 2014.
35. Vierhapper H, Heinze G, Nowotny P. Sex-specific difference in the interconversion of corti-
sol and cortisone in men and women. Obesity (Silver Spring). 2007;15(4):820–4. doi:10.1038/
oby.2007.592.
36. Finken MJ, Andrews RC, Andrew R, Walker BR. Cortisol metabolism in healthy young
adults: sexual dimorphism in activities of A-ring reductases, but not 11beta-hydroxysteroid
dehydrogenases. J Clin Endocrinol Metab. 1999;84(9):3316–21. doi:10.1210/jcem.84.9.6009.
37. Gathercole LL, Lavery GG, Morgan SA, et al. 11β-hydroxysteroid dehydrogenase 1: transla-
tional and therapeutic aspects. Endocr Rev. 2013;34(4):525–55. doi:10.1210/er.2012-1050.
38. Nixon M, Wake DJ, Livingstone DE, et al. Salicylate downregulates 11β-HSD1 expression in
adipose tissue in obese mice and in humans, mediating insulin sensitization. Diabetes.
2012;61(4):790–6. doi:10.2337/db11-0931.
39. Paulsen SK, Pedersen SB, Fisker S, Richelsen B. 11Beta-HSD type 1 expression in human
adipose tissue: impact of gender, obesity, and fat localization. Obesity (Silver Spring).
2007;15(8):1954–60. doi:10.1038/oby.2007.233.
40. Gomez-Sanchez EP, Ganjam V, Chen YJ, et al. Regulation of 11 beta-hydroxysteroid dehy-
drogenase enzymes in the rat kidney by estradiol. Am J Physiol Endocrinol Metab.
2003;285(2):E272–9. doi:10.1152/ajpendo.00409.2002.
41. Quirk SJ, Slattery JA, Funder JW. Epithelial and adipose cells isolated from mammary
glands of pregnant and lactating rats differ in 11 beta-hydroxysteroid dehydrogenase activity.
J Steroid Biochem Mol Biol. 1990;37(4):529–34. http://www.ncbi.nlm.nih.gov/
pubmed/2278836. Accessed 31 Dec 2013.
42. Goedecke JH, Wake DJ, Levitt NS, et al. Glucocorticoid metabolism within superficial sub-
cutaneous rather than visceral adipose tissue is associated with features of the metabolic
syndrome in South African women. Clin Endocrinol (Oxf). 2006;65(1):81–7.
doi:10.1111/j.1365-2265.2006.02552.x.
43. Veilleux A, Laberge PY, Morency J, Noël S, Luu-The V, Tchernof A. Expression of genes
related to glucocorticoid action in human subcutaneous and omental adipose tissue. J Steroid
Biochem Mol Biol. 2010;122(1–3):28–34. doi:10.1016/j.jsbmb.2010.02.024.
44. Tomlinson JW, Moore J, Cooper MS, et al. Regulation of expression of 11β-hydroxysteroid
dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines. Endocrinology.
2001;142(5):1982–9.
45. Friedberg M, Zoumakis E, Hiroi N, Bader T, Chrousos GP, Hochberg Z. Modulation of 11
beta-hydroxysteroid dehydrogenase type 1 in mature human subcutaneous adipocytes by
hypothalamic messengers. J Clin Endocrinol Metab. 2003;88(1):385–93. doi:10.1210/
jc.2002-020510.
46. Handoko K, Yang K, Strutt B, Khalil W, Killinger D. Insulin attenuates the stimulatory
effects of tumor necrosis factor alpha on 11beta-hydroxysteroid dehydrogenase 1 in human
adipose stromal cells. J Steroid Biochem Mol Biol. 2000;72(3–4):163–8. http://www.ncbi.
nlm.nih.gov/pubmed/10775808. Accessed 31 Dec 2013.
47. Esteves CL, Kelly V, Breton A, et al. Proinflammatory cytokine induction of 11β-hydroxysteroid
dehydrogenase type 1 (11β-HSD1) in human adipocytes is mediated by MEK, C/EBPβ, and
NF-κB/RelA. J Clin Endocrinol Metab. 2014;99(1):E160–8. doi:10.1210/jc.2013-1708.
48. Bujalska IJ, Walker EA, Hewison M, Stewart PM. A switch in dehydrogenase to reductase
activity of 11 beta-hydroxysteroid dehydrogenase type 1 upon differentiation of human
omental adipose stromal cells. J Clin Endocrinol Metab. 2002;87(3):1205–10. doi:10.1210/
jcem.87.3.8301.
49. Bujalska IJ, Gathercole LL, Tomlinson JW, et al. A novel selective 11beta-hydroxysteroid
dehydrogenase type 1 inhibitor prevents human adipogenesis. J Endocrinol. 2008;
197(2):297–307. doi:10.1677/JOE-08-0050.
50. Livingstone DE, Kenyon CJ, Walker BR. Mechanisms of dysregulation of 11 beta-
hydroxysteroid dehydrogenase type 1 in obese Zucker rats. J Endocrinol. 2000;167(3):533–9.
http://www.ncbi.nlm.nih.gov/pubmed/11115781. Accessed 7 Mar 2014.
51. Livingstone DE, Jones GC, Smith K, et al. Understanding the role of glucocorticoids in
obesity: tissue-specific alterations of corticosterone metabolism in obese Zucker rats.
Endocrinology. 2000;141(2):560–3. doi:10.1210/endo.141.2.7297.
52. Morton NM, Ramage L, Seckl JR. Down-regulation of adipose 11beta-hydroxysteroid dehy-
drogenase type 1 by high-fat feeding in mice: a potential adaptive mechanism counteracting
metabolic disease. Endocrinology. 2004;145(6):2707–12. doi:10.1210/en.2003-1674.
53. Drake AJ, Livingstone DEW, Andrew R, Seckl JR, Morton NM, Walker BR. Reduced
adipose glucocorticoid reactivation and increased hepatic glucocorticoid clearance as an
early adaptation to high-fat feeding in Wistar rats. Endocrinology. 2005;146(2):913–9.
doi:10.1210/en.2004-1063.
54. Morton NM, Holmes MC, Fiévet C, et al. Improved lipid and lipoprotein profile, hepatic
insulin sensitivity, and glucose tolerance in 11beta-hydroxysteroid dehydrogenase type 1 null
mice. J Biol Chem. 2001;276(44):41293–300. doi:10.1074/jbc.M103676200.
55. Masuzaki H, Paterson J, Shinyama H, et al. A transgenic model of visceral obesity and the
metabolic syndrome. Science. 2001;294(5549):2166–70. doi:10.1126/science.1066285.
56. Rask E. Tissue-specific dysregulation of cortisol metabolism in human obesity. J Clin
Endocrinol Metab. 2001;86(3):1418–21. doi:10.1210/jc.86.3.1418.
57. Lindsay RS. Subcutaneous adipose 11-hydroxysteroid dehydrogenase type 1 activity and
messenger ribonucleic acid levels are associated with adiposity and insulinemia in Pima
Indians and Caucasians. J Clin Endocrinol Metab. 2003;88(6):2738–44. doi:10.1210/
jc.2002-030017.
58. Veilleux A, Rhéaume C, Daris M, Luu-The V, Tchernof A. Omental adipose tissue type 1 11
beta-hydroxysteroid dehydrogenase oxoreductase activity, body fat distribution, and meta-
bolic alterations in women. J Clin Endocrinol Metab. 2009;94(9):3550–7. doi:10.1210/
jc.2008-2011.
59. Lee JH, Gao Z, Ye J. Regulation of 11β-HSD1 expression during adipose tissue expansion by
hypoxia through different activities of NF-κB and HIF-1α. Am J Physiol Endocrinol Metab.
2013;304(10):E1035–41. doi:10.1152/ajpendo.00029.2013.
60. McCormick KL, Wang X, Mick GJ. Evidence that the 11 beta-hydroxysteroid dehydrogenase
(11 beta-HSD1) is regulated by pentose pathway flux. Studies in rat adipocytes and micro-
somes. J Biol Chem. 2006;281(1):341–7. doi:10.1074/jbc.M506026200.
61. Gout J, Tirard J, Thévenon C, Riou J-P, Bégeot M, Naville D. CCAAT/enhancer-binding
proteins (C/EBPs) regulate the basal and cAMP-induced transcription of the human 11beta-
hydroxysteroid dehydrogenase encoding gene in adipose cells. Biochimie. 2006;88(9):1115–24.
doi:10.1016/j.biochi.2006.05.020.
62. Esteves CL, Kelly V, Bégay V, et al. Regulation of adipocyte 11β-hydroxysteroid dehydroge-
nase type 1 (11β-HSD1) by CCAAT/enhancer-binding protein (C/EBP) β isoforms LIP and
LAP. PLoS One. 2012;7(5):e37953. doi:10.1371/journal.pone.0037953.
63. Amatruda JM, Danahy SA, Chang CL. The effects of glucocorticoids on insulin-stimulated
lipogenesis in primary cultures of rat hepatocytes. Biochem J. 1983;212(1):135–41. http://
www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1152020&tool=pmcentrez&rendertyp
e=abstract.
64. Dolinsky VW, Douglas DN, Lehner R, Vance DE. Regulation of the enzymes of hepatic
microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexametha-
sone. Biochem J. 2004;378(Pt 3):967–74. doi:10.1042/BJ20031320.
65. Baxter JD, Forsham PH. Tissue effects of glucocorticoids. Am J Med. 1972;53(5):573–89.
http://www.ncbi.nlm.nih.gov/pubmed/4342884. Accessed 22 Jan 2014.
66. Paterson JM, Morton NM, Fievet C, et al. Metabolic syndrome without obesity: hepatic
overexpression of 11beta-hydroxysteroid dehydrogenase type 1 in transgenic mice. Proc
Natl Acad Sci U S A. 2004;101(18):7088–93. doi:10.1073/pnas.0305524101.
67. Ricketts ML, Verhaeg JM, Bujalska I, Howie AJ, Rainey WE, Stewart PM.
Immunohistochemical localization of type 1 11beta-hydroxysteroid dehydrogenase in human
tissues. J Clin Endocrinol Metab. 1998;83(4):1325–35. doi:10.1210/jcem.83.4.4706.
68. Jamieson PM, Walker BR, Chapman KE, Andrew R, Rossiter S, Seckl JR. 11 beta-
hydroxysteroid dehydrogenase type 1 is a predominant 11 beta-reductase in the intact perfused
rat liver. J Endocrinol. 2000;165(3):685–92. http://www.ncbi.nlm.nih.gov/pubmed/10828853.
Accessed 7 Mar 2014.
69. Candia R, Riquelme A, Baudrand R, et al. Overexpression of 11β-hydroxysteroid dehydroge-
nase type 1 in visceral adipose tissue and portal hypercortisolism in non-alcoholic fatty liver
disease. Liver Int. 2012;32(3):392–9. doi:10.1111/j.1478-3231.2011.02685.x.
70. Baudrand R, Carvajal CA, Riquelme A, et al. Overexpression of 11beta-hydroxysteroid
dehydrogenase type 1 in hepatic and visceral adipose tissue is associated with metabolic
disorders in morbidly obese patients. Obes Surg. 2010;20(1):77–83. doi:10.1007/
s11695-009-9937-0.
71. Konopelska S, Kienitz T, Hughes B, et al. Hepatic 11beta-HSD1 mRNA expression in fatty
liver and nonalcoholic steatohepatitis. Clin Endocrinol (Oxf). 2009;70(4):554–60.
doi:10.1111/j.1365-2265.2008.03358.x.
72. Torrecilla E, Fernández-Vázquez G, Vicent D, et al. Liver upregulation of genes involved in
cortisol production and action is associated with metabolic syndrome in morbidly obese
patients. Obes Surg. 2012;22(3):478–86. doi:10.1007/s11695-011-0524-9.
73. Valsamakis G, Anwar A, Tomlinson JW, et al. 11Beta-hydroxysteroid dehydrogenase type 1
activity in lean and obese males with type 2 diabetes mellitus. J Clin Endocrinol Metab.
2004;89(9):4755–61. doi:10.1210/jc.2003-032240.
74. Ahmed A, Rabbitt E, Brady T, et al. A switch in hepatic cortisol metabolism across the spec-
trum of non alcoholic fatty liver disease. PLoS One. 2012;7(2):e29531. doi:10.1371/journal.
pone.0029531.
75. Whorwood CB, Donovan SJ, Flanagan D, Phillips DIW, Byrne CD. Increased glucocorticoid
receptor expression in human skeletal muscle cells may contribute to the pathogenesis of the meta-
bolic syndrome. Diabetes. 2002;51(4):1066–75. http://www.ncbi.nlm.nih.gov/pubmed/11916927.
76. Whorwood CB, Donovan SJ, Wood PJ, Phillips DI. Regulation of glucocorticoid receptor
alpha and beta isoforms and type I 11β-hydroxysteroid dehydrogenase expression in human
skeletal muscle cells: a key role in the pathogenesis of insulin resistance? J Clin Endocrinol
Metab. 2001;86(5):2296–308.
77. Morgan SA, Sherlock M, Gathercole LL, et al. 11beta-hydroxysteroid dehydrogenase type 1
regulates glucocorticoid-induced insulin resistance in skeletal muscle. Diabetes.
2009;58(11):2506–15. doi:10.2337/db09-0525.
78. Abdallah BM, Beck-Nielsen H, Gaster M. Increased expression of 11beta-hydroxysteroid
dehydrogenase type 1 in type 2 diabetic myotubes. Eur J Clin Invest. 2005;35(10):627–34.
doi:10.1111/j.1365-2362.2005.01552.x.
79. Zhang M, Lv X-Y, Li J, Xu Z-G, Chen L. Alteration of 11beta-hydroxysteroid dehydrogenase
type 1 in skeletal muscle in a rat model of type 2 diabetes. Mol Cell Biochem. 2009;324(1–
2):147–55. doi:10.1007/s11010-008-9993-0.
80. Kilgour AHM, Gallagher IJ, Maclullich AMJ, et al. Increased skeletal muscle 11βHSD1
mRNA is associated with lower muscle strength in ageing. PLoS One. 2013;8(12):e84057.
doi:10.1371/journal.pone.0084057.
81. Vukelic S, Stojadinovic O, Pastar I, et al. Cortisol synthesis in epidermis is induced by IL-1
and tissue injury. J Biol Chem. 2011;286(12):10265–75. doi:10.1074/jbc.M110.188268.
82. Tiganescu A, Walker EA, Hardy RS, Mayes AE, Stewart PM. Localization, age- and site-
dependent expression, and regulation of 11β-hydroxysteroid dehydrogenase type 1 in skin.
J Invest Dermatol. 2011;131(1):30–6. doi:10.1038/jid.2010.257.
83. Fisher GJ, Varani J, Voorhees JJ. Looking older: fibroblast collapse and therapeutic implications.
Arch Dermatol. 2010;144(5):666–72. doi:10.1001/archderm.144.5.666.Looking.
84. Slominski A, Zbytek B, Nikolakis G, et al. Steroidogenesis in the skin: implications for
local immune functions. J Steroid Biochem Mol Biol. 2013;137:107–23. doi:10.1016/j.
jsbmb.2013.02.006.
85. Terao M, Murota H, Kimura A, et al. 11β-Hydroxysteroid dehydrogenase-1 is a novel regula-
tor of skin homeostasis and a candidate target for promoting tissue repair. PLoS One.
2011;6(9):e25039. doi:10.1371/journal.pone.0025039.
86. Cirillo N, Prime SS. Keratinocytes synthesize and activate cortisol. J Cell Biochem.
2011;112(6):1499–505. doi:10.1002/jcb.23081.
87. Napolitano A, Voice MW, Edwards CR, Seckl JR, Chapman KE. 11Beta-hydroxysteroid
dehydrogenase 1 in adipocytes: expression is differentiation-dependent and hormonally regu-
lated. J Steroid Biochem Mol Biol. 1998;64(5–6):251–60. http://www.ncbi.nlm.nih.gov/
pubmed/9618026. Accessed 3 Feb 2014.
88. Tiganescu A, Hupe M, Uchida Y, Mauro T, Elias PM, Holleran W. Increased glucocorticoid
activation during mouse skin wound healing. J Endocrinol. 2014;221(1):51–61. doi:10.1530/
JOE-13-0420.
89. Tiganescu A, Tahrani AA, Morgan SA, et al. 11β-Hydroxysteroid dehydrogenase blockade
prevents age-induced skin structure and function defects. J Clin Invest. 2013;123(7):3051–60.
doi:10.1172/JCI64162.
90. Brem AS, Bina RB, King TC, Morris DJ. Localization of 2 11beta-OH steroid dehydrogenase
isoforms in aortic endothelial cells. Hypertension. 1998;31(1 Pt 2):459–62. http://www.ncbi.
nlm.nih.gov/pubmed/9453345. Accessed 14 Feb 2014.
91. Walker BR, Yau JL, Brett LP, et al. 11 beta-hydroxysteroid dehydrogenase in vascular smooth
muscle and heart: implications for cardiovascular responses to glucocorticoids. Endocrinology.
1991;129(6):3305–12. doi:10.1210/endo-129-6-3305.
92. Atalar F, Gormez S, Caynak B, et al. The role of mediastinal adipose tissue 11β-hydroxysteroid
dehydrogenase type 1 and glucocorticoid expression in the development of coronary athero-
sclerosis in obese patients with ischemic heart disease. Cardiovasc Diabetol. 2012;11:115.
doi:10.1186/1475-2840-11-115.
93. Atalar F, Vural B, Ciftci C, et al. 11β-hydroxysteroid dehydrogenase type 1 gene expression
is increased in ascending aorta tissue of metabolic syndrome patients with coronary artery
disease. Genet Mol Res. 2012;11(3):3122–32. doi:10.4238/2012.August.31.10.
94. Hermanowski-Vosatka A, Balkovec JM, Cheng K, et al. 11beta-HSD1 inhibition ameliorates
metabolic syndrome and prevents progression of atherosclerosis in mice. J Exp Med.
2005;202(4):517–27. doi:10.1084/jem.20050119.
95. Nuotio-Antar AM, Hachey DL, Hasty AH. Carbenoxolone treatment attenuates symptoms of
metabolic syndrome and atherogenesis in obese, hyperlipidemic mice. Am J Physiol
Endocrinol Metab. 2007;293(6):E1517–28. doi:10.1152/ajpendo.00522.2007.
96. Small GR, Hadoke PWF, Sharif I, et al. Preventing local regeneration of glucocorticoids by
11beta-hydroxysteroid dehydrogenase type 1 enhances angiogenesis. Proc Natl Acad Sci U
S A. 2005;102(34):12165–70. doi:10.1073/pnas.0500641102.
97. Brown ES. Effects of glucocorticoids on mood, memory, and the hippocampus. Treatment and
preventive therapy.Ann NYAcad Sci. 2009;1179:41–55. doi:10.1111/j.1749-6632.2009.04981.x.
98. Swaab DF, Bao A-M, Lucassen PJ. The stress system in the human brain in depression and
neurodegeneration. Ageing Res Rev. 2005;4(2):141–94. doi:10.1016/j.arr.2005.03.003.
99. Korbonits M, Bujalska I, Shimojo M, et al. Expression of 11 beta-hydroxysteroid dehydroge-
nase isoenzymes in the human pituitary: induction of the type 2 enzyme in corticotropinomas
and other pituitary tumors. J Clin Endocrinol Metab. 2001;86(6):2728–33. doi:10.1210/
jcem.86.6.7563.
100. Moisan MP, Seckl JR, Edwards CR. 11 beta-hydroxysteroid dehydrogenase bioactivity and
messenger RNA expression in rat forebrain: localization in hypothalamus, hippocampus, and
cortex. Endocrinology. 1990;127(3):1450–5. doi:10.1210/endo-127-3-1450.
101. Gomez-Sanchez EP, Romero DG, de Rodriguez AF, Warden MP, Krozowski Z, Gomez-Sanchez
CE. Hexose-6-phosphate dehydrogenase and 11beta-hydroxysteroid dehydrogenase-1 tissue
distribution in the rat. Endocrinology. 2008;149(2):525–33. doi:10.1210/en.2007-0328.
102. Lakshmi V, Sakai RR, McEwen BS, Monder C. Regional distribution of 11 beta-
hydroxysteroid dehydrogenase in rat brain. Endocrinology. 1991;128(4):1741–8. doi:10.1210/
endo-128-4-1741.
103. Rajan V, Edwards RW, Seckl JR. 11 beta-hydroxysteroid dehydrogenase in cultured cells reac-
tivates inert 11-dehydrocorticosterone, potentiating neurotoxicity hippocampal. J Neurosci.
1996;76(1):65–70.
104. Wyrwoll CS, Holmes MC, Seckl JR. 11β-hydroxysteroid dehydrogenases and the brain: from
zero to hero, a decade of progress. Front Neuroendocrinol. 2011;32(3):265–86. doi:10.1016/j.
yfrne.2010.12.001.
105. Tomlinson JW, Durrani OM, Bujalska IJ, et al. The role of 11beta-hydroxysteroid dehydro-
genase 1 in adipogenesis in thyroid-associated ophthalmopathy. J Clin Endocrinol Metab.
2010;95(1):398–406. doi:10.1210/jc.2009-0873.
106. Sandeep TC, Yau JLW, MacLullich AMJ, et al. 11Beta-hydroxysteroid dehydrogenase inhibition
improves cognitive function in healthy elderly men and type 2 diabetics. Proc Natl Acad Sci
U S A. 2004;101(17):6734–9. doi:10.1073/pnas.0306996101.
107. Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids—new mechanisms for
old drugs. N Engl J Med. 2005;353(16):1711–23. doi:10.1056/NEJMra050541.
108. Chapman KE, Coutinho AE, Gray M, Gilmour JS, Savill JS, Seckl JR. The role and regula-
tion of 11beta-hydroxysteroid dehydrogenase type 1 in the inflammatory response. Mol Cell
Endocrinol. 2009;301(1-2):123–31. doi:10.1016/j.mce.2008.09.031.
109. Coutinho AE, Brown JK, Yang F, et al. Mast cells express 11β-hydroxysteroid dehydrogenase
type 1: a role in restraining mast cell degranulation. PLoS One. 2013;8(1):e54640.
doi:10.1371/journal.pone.0054640.
110. Thieringer R, Le Grand CB, Carbin L, et al. 11 Beta-hydroxysteroid dehydrogenase type 1 is
induced in human monocytes upon differentiation to macrophages. J Immunol.
2001;167(1):30–5. http://www.ncbi.nlm.nih.gov/pubmed/11418628.
111. Zhang TY, Ding X, Daynes RA, Alerts E. Regulation of glucocorticoid activities 1. J Immunol.
2005;174(2):879–89.
112. Freeman L, Hewison M, Hughes SV, et al. Expression of 11β-hydroxysteroid dehydrogenase
type 1 permits regulation of glucocorticoid bioavailability by human dendritic cells. Blood.
2005;106(6):2042–9. doi:10.1182/blood-2005-01-0186.
113. Gilmour JS, Coutinho AE, Cailhier J, et al. Local amplification of glucocorticoids by
11β-hydroxysteroid dehydrogenase type 1 promotes macrophage phagocytosis of apoptotic
leukocytes. J Immunol. 2006;176(12):7605–11.
114. Zhang TY, Daynes RA. Macrophages from 11beta-hydroxysteroid dehydrogenase type
1-deficient mice exhibit an increased sensitivity to lipopolysaccharide stimulation due to
TGF-beta-mediated up-regulation of SHIP1 expression. J Immunol. 2007;179(9):
6325–35.
115. Coutinho AE, Gray M, Brownstein DG, et al. 11β-Hydroxysteroid dehydrogenase type 1, but
not type 2, deficiency worsens acute inflammation and experimental arthritis in mice.
Endocrinology. 2012;153(1):234–40. doi:10.1210/en.2011-1398.
116. Zbánková S, Bryndová J, Leden P, Kment M, Svec A, Pácha J. 11beta-hydroxysteroid dehy-
drogenase 1 and 2 expression in colon from patients with ulcerative colitis. J Gastroenterol
Hepatol. 2007;22(7):1019–23. doi:10.1111/j.1440-1746.2006.04529.x.
117. Stegk JP, Ebert B, Martin H-J, Maser E. Expression profiles of human 11beta-hydroxysteroid
dehydrogenases type 1 and type 2 in inflammatory bowel diseases. Mol Cell Endocrinol.
2009;301(1–2):104–8. doi:10.1016/j.mce.2008.10.030.
118. Cooper MS, Kriel H, Sayers A, et al. Can 11β-hydroxysteroid dehydrogenase activity predict
the sensitivity of bone to therapeutic glucocorticoids in inflammatory bowel disease? Calcif
Tissue Int. 2011;89(3):246–51. doi:10.1007/s00223-011-9512-2.
119. Ergang P, Vytáčková K, Svec J, Bryndová J, Mikšík I, Pácha J. Upregulation of

11β-hydroxysteroid dehydrogenase 1 in lymphoid organs during inflammation in the rat.
J Steroid Biochem Mol Biol. 2011;126(1–2):19–25. doi:10.1016/j.jsbmb.2011.04.002.
120. Cooper MS, Walker EA, Bland R, Fraser WD, Hewison M, Stewart PM. Expression and
functional consequences of 11beta-hydroxysteroid dehydrogenase activity in human bone. Bone.
121. Bellows CG, Ciaccia A, Heersche JN. Osteoprogenitor cells in cell populations derived from
mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone. Bone.
122. Hardy R, Rabbitt EH, Filer A, et al. Local and systemic glucocorticoid metabolism in inflam-
matory arthritis. Ann Rheum Dis. 2008;67(9):1204–10. doi:10.1136/ard.2008.090662.
123. Schmidt M, Weidler C, Naumann H, Anders S, Schölmerich J, Straub RH. Reduced capacity
for the reactivation of glucocorticoids in rheumatoid arthritis synovial cells: possible role
of the sympathetic nervous system? Arthritis Rheum. 2005;52(6):1711–20. doi:10.1002/
art.21091.
124. Ergang P, Leden P, Vagnerová K, et al. Local metabolism of glucocorticoids and its role in rat
adjuvant arthritis. Mol Cell Endocrinol. 2010;323(2):155–60. doi:10.1016/j.mce.2010.03.003.
125. Lavery GG, Walker EA, Tiganescu A, et al. Steroid biomarkers and genetic studies reveal
inactivating mutations in hexose-6-phosphate dehydrogenase in patients with cortisone
reductase deficiency. J Clin Endocrinol Metab. 2008;93(10):3827–32. doi:10.1210/
jc.2008-0743.
126. Morgan SA, Tomlinson JW. 11beta-hydroxysteroid dehydrogenase type 1 inhibitors for the
treatment of type 2 diabetes. Expert Opin Investig Drugs. 2010;19(9):1067–76. doi:10.1517/
13543784.2010.504713.
127. Monder C, Lakshmi V. Evidence for kinetically distinct forms of corticosteroid 11 beta-
dehydrogenase in rat liver microsomes. J Steroid Biochem. 1989;32(1A):77–83. http://www.
ncbi.nlm.nih.gov/pubmed/2913404. Accessed 23 Jan 2014.
128. Schweizer RAS, Atanasov AG, Frey BM, Odermatt A. A rapid screening assay for inhibitors
of 11beta-hydroxysteroid dehydrogenases (11beta-HSD): flavanone selectively inhibits
11beta-HSD1 reductase activity. Mol Cell Endocrinol. 2003;212(1–2):41–9. http://www.
ncbi.nlm.nih.gov/pubmed/14654249. Accessed 23 Jan 2014.
129. Diederich S, Grossmann C, Hanke B, et al. In the search for specific inhibitors of human
11beta-hydroxysteroid-dehydrogenases (11beta-HSDs): chenodeoxycholic acid selectively
inhibits 11beta-HSD-I. Eur J Endocrinol. 2000;142(2):200–7. http://www.ncbi.nlm.nih.gov/
pubmed/10664531.
130. Latif SA, Pardo HA, Hardy MP, Morris DJ. Endogenous selective inhibitors of 11beta-
hydroxysteroid dehydrogenase isoforms 1 and 2 of adrenal origin. Mol Cell Endocrinol.
2005;243(1–2):43–50. doi:10.1016/j.mce.2005.08.006.
131. Atanasov AG, Dzyakanchuk AA, Schweizer RAS, Nashev LG, Maurer EM, Odermatt
A. Coffee inhibits the reactivation of glucocorticoids by 11beta-hydroxysteroid dehydroge-
nase type 1: a glucocorticoid connection in the anti-diabetic action of coffee? FEBS Lett.
2006;580(17):4081–5. doi:10.1016/j.febslet.2006.06.046.
132. Andrews RC, Rooyackers O, Walker BR. Effects of the 11 beta-hydroxysteroid dehydro-
genase inhibitor carbenoxolone on insulin sensitivity in men with type 2 diabetes. J Clin
Endocrinol Metab. 2003;88(1):285–91. doi:10.1210/jc.2002-021194.
133. Reuters T. The changing role of chemistry in drug discovery. 2011. http://thomsonreuters.
com/content/dam/openweb/documents/pdf/pharma-life-sciences/report/international-year-
of-chemistry-report-drug-discovery.pdf.
134. Boyle CD, Kowalski TJ. 11beta-hydroxysteroid dehydrogenase type 1 inhibitors: a review of
recent patents. Expert Opin Ther Pat. 2009;19(6):801–25. doi: 10.1517/13543770902967658.
135. Rosenstock J, Banarer S, Fonseca VA, et al. The 11-beta-hydroxysteroid dehydrogenase type
1 inhibitor INCB13739 improves hyperglycemia in patients with type 2 diabetes inadequately
controlled by metformin monotherapy. Diabetes Care. 2010;33(7):1516–22. doi:10.2337/

dc09-2315.
136. Shah S, Hermanowski-Vosatka A, Gibson K, et al. Efficacy and safety of the selective
11β-HSD-1 inhibitors MK-0736 and MK-0916 in overweight and obese patients with hyper-
tension. J Am Soc Hypertens. 2011;5(3):166–76. doi:10.1016/j.jash.2011.01.009.
137. Brown RW, Chapman KE, Edwards CR, Seckl JR. Human placental 11 beta-hydroxysteroid
dehydrogenase: evidence for and partial purification of a distinct NAD-dependent isoform.
Endocrinology. 1993;132(6):2614–21. doi:10.1210/endo.132.6.8504762.
138. Albiston AL, Obeyesekere VR, Smith RE, Krozowski ZS. Cloning and tissue distribution of
the human 11 beta-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol.
1994;105(2):R11–7. http://www.ncbi.nlm.nih.gov/pubmed/7859916. Accessed 5 Feb 2014.
139. Persson B, Kallberg Y, Bray JE, et al. The SDR (short-chain dehydrogenase/reductase and
related enzymes) nomenclature initiative. Chem Biol Interact. 2009;178(1–3):94–8.
doi:10.1016/j.cbi.2008.10.040.
140. Agarwal AK, Rogerson FM, Mune T, White PC. Gene structure and chromosomal localiza-
tion of the human HSD11K gene encoding the kidney (type 2) isozyme of 11 beta-
hydroxysteroid dehydrogenase. Genomics. 1995;29(1):195–9. doi:10.1006/geno.1995.1231.
141. Brown RW, Chapman KE, Kotelevtsev Y, et al. Cloning and production of antisera to human
placental 11β-hydroxysteroid dehydrogenase type 2. Biochem J. 1996;313:1007–17.
142. Stewart PM, Murry BA, Mason JI. Human kidney 11 beta-hydroxysteroid dehydrogenase is
a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the
cloned type I isoform. J Clin Endocrinol Metab. 1994;79(2):480–4. doi:10.1210/jcem.
79.2.8045966.
143. Dave-Sharma S, Wilson RC, Harbison MD, et al. Examination of genotype and phenotype
relationships in 14 patients with apparent mineralocorticoid excess. J Clin Endocrinol Metab.
1998;83(7):2244–54. doi:10.1210/jcem.83.7.4986.
144. Wilson RC, Krozowski ZS, Li K, et al. A mutation in the HSD11B2 gene in a family with
apparent mineralocorticoid excess. J Clin Endocrinol Metab. 1995;80(7):2263–6.
doi:10.1210/jcem.80.7.7608290.
145. Mune T, Rogerson FM, Nikkilä H, Agarwal AK, White PC. Human hypertension caused by
mutations in the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase. Nat Genet.
1995;10(4):394–9. doi:10.1038/ng0895-394.
146. Osinski PA. Steroid 11beta-ol dehydrogenase in human placenta. Nature. 1960;187:777.
http://www.ncbi.nlm.nih.gov/pubmed/14429221. Accessed 31 Dec 2013.
147. Waffarn F, Davis EP. Effects of antenatal corticosteroids on the hypothalamic-pituitary-
adrenocortical axis of the fetus and newborn: experimental findings and clinical consider-
ations. Am J Obstet Gynecol. 2012;207(6):446–54. doi:10.1016/j.ajog.2012.06.012.
148. Wyrwoll CS, Holmes MC. Prenatal excess glucocorticoid exposure and adult affective disor-
ders: a role for serotonergic and catecholamine pathways. Neuroendocrinology.
2012;95(1):47–55. doi:10.1159/000331345.
149. Huang WL, Beazley LD, Quinlivan JA, Evans SF, Newnham JP, Dunlop SA. Effect of corti-
costeroids on brain growth in fetal sheep. Obstet Gynecol. 1999;94(2):213–8. http://www.
ncbi.nlm.nih.gov/pubmed/10432130. Accessed 14 Feb 2014.
150. Seckl JR, Holmes MC. Mechanisms of disease: glucocorticoids, their placental metabolism
and fetal “programming” of adult pathophysiology. Nat Clin Pract Endocrinol Metab.
2007;3(6):479–88. doi:10.1038/ncpendmet0515.
151. Alikhani-Koopaei R, Fouladkou F, Frey FJ, Frey BM. Epigenetic regulation of 11 beta-
hydroxysteroid dehydrogenase type 2 expression. J Clin Invest. 2004;114(8):1146–57.
doi:10.1172/JCI21647.
152. Marsit CJ, Maccani MA, Padbury JF, Lester BM. Placental 11-beta hydroxysteroid dehydro-
genase methylation is associated with newborn growth and a measure of neurobehavioral
outcome. PLoS One. 2012;7(3):e33794. doi:10.1371/journal.pone.0033794.
153. Baserga M, Kaur R, Hale MA, et al. Fetal growth restriction alters transcription factor bind-
ing and epigenetic mechanisms of renal 11β-hydroxysteroid dehydrogenase type 2 in a sex-
specific manner. Am J Physiol Regul Integr Comp Physiol. 2010;299(1):334–42. doi:10.1152/

ajpregu.00122.2010.
154. Kostadinova RM, Nawrocki AR, Frey FJ, Frey BM. Tumor necrosis factor alpha and phorbol
12-myristate-13-acetate down-regulate human 11beta-hydroxysteroid dehydrogenase type 2
through p50/p50 NF-kappaB homodimers and Egr-1. FASEB J. 2005;19(6):650–2.
doi:10.1096/fj.04-2820fje.
155. Low SC, Assaad SN, Rajan V, Chapman KE, Edwards CR, Seckl JR. Regulation of 11
beta-hydroxysteroid dehydrogenase by sex steroids in vivo: further evidence for the existence
of a second dehydrogenase in rat kidney. J Endocrinol. 1993;139(1):27–35. http://www.ncbi.
nlm.nih.gov/pubmed/8254291. Accessed 13 Feb 2014.
156. Rubis B, Krozowski Z, Trzeciak WH. Arginine vasopressin stimulates 11beta-hydroxysteroid
dehydrogenase type 2 expression in the mineralocorticosteroid target cells. Mol Cell
Endocrinol. 2006;256(1–2):17–22. doi:10.1016/j.mce.2006.04.032.
157. Clarke KA, Ward JW, Forhead AJ, Giussani DA, Fowden AL. Regulation of 11 beta-
hydroxysteroid dehydrogenase type 2 activity in ovine placenta by fetal cortisol. J Endocrinol.
158. Suzuki S, Koyama K, Darnel A, et al. Dexamethasone upregulates 11beta-hydroxysteroid
dehydrogenase type 2 in BEAS-2B cells. Am J Respir Crit Care Med. 2003;167(9):1244–9.
doi:10.1164/rccm.200210-1139OC.
159. Heiniger CD, Kostadinova RM, Rochat MK, et al. Hypoxia causes down-regulation of 11
beta-hydroxysteroid dehydrogenase type 2 by induction of Egr-1. FASEB J. 2003;17(8):917–
9. doi:10.1096/fj.02-0582fje.
160. Fukushima K, Funayama Y, Yonezawa H, et al. Aldosterone enhances 11beta-hydroxysteroid
dehydrogenase type 2 expression in colonic epithelial cells in vivo. Scand J Gastroenterol.
2005;40(7):850–7. doi:10.1080/00365520510015700.
161. Geerling JC, Loewy AD. Aldosterone in the brain. Am J Physiol Renal Physiol.
2009;297(3):F559–76. doi:10.1152/ajprenal.90399.2008.
162. Holmes MC, Sangra M, French KL, et al. 11beta-hydroxysteroid dehydrogenase type 2 pro-
tects the neonatal cerebellum from deleterious effects of glucocorticoids. Neuroscience.
2006;137(3):865–73. doi:10.1016/j.neuroscience.2005.09.037.
163. Noguchi KK, Lau K, Smith DJ, Swiney BS, Farber NB. Glucocorticoid receptor stimulation
and the regulation of neonatal cerebellar neural progenitor cell apoptosis. Neurobiol Dis.
2011;43(2):356–63. doi:10.1016/j.nbd.2011.04.004.
164. Roland BL, Li KX, Funder JW. Hybridization histochemical localization of 11 beta-
hydroxysteroid dehydrogenase type 2 in rat brain. Endocrinology. 1995;136(10):4697–700.
doi:10.1210/endo.136.10.7664691.
165. Christy C, Hadoke PWF, Paterson JM, Mullins JJ, Seckl JR, Walker BR. 11beta-hydroxysteroid
dehydrogenase type 2 in mouse aorta: localization and influence on response to glucocorticoids.
Hypertension. 2003;42(4):580–7. doi:10.1161/01.HYP.0000088855.06598.5B.
166. Deuchar GA, McLean D, Hadoke PWF, et al. 11β-hydroxysteroid dehydrogenase type 2
deficiency accelerates atherogenesis and causes proinflammatory changes in the endothelium
in apoe-/- mice. Endocrinology. 2011;152(1):236–46. doi:10.1210/en.2010-0925.
167. Leckie C, Chapman KE, Edwards CR, Seckl JR. LLC-PK1 cells model 11 beta-hydroxysteroid
dehydrogenase type 2 regulation of glucocorticoid access to renal mineralocorticoid recep-
tors. Endocrinology. 1995;136(12):5561–9. doi:10.1210/endo.136.12.7588309.
168. Henschkowski J, Stuck AE, Frey BM, et al. Age-dependent decrease in 11beta-hydroxysteroid
dehydrogenase type 2 (11beta-HSD2) activity in hypertensive patients. Am J Hypertens.
2008;21(6):644–9. doi:10.1038/ajh.2008.152.
169. Ferrari P. The role of 11β-hydroxysteroid dehydrogenase type 2 in human hypertension.
Biochim Biophys Acta. 2010;1802(12):1178–87. doi:10.1016/j.bbadis.2009.10.017.
170. Mongia A, Vecker R, George M, et al. Role of 11βHSD type 2 enzyme activity in essential
hypertension and children with chronic kidney disease (CKD). J Clin Endocrinol Metab.
2012;97(10):3622–9. doi:10.1210/jc.2012-1411.
171. Watson B, Bergman SM, Myracle A, Callen DF, Acton RT, Warnock DG. Genetic association
of 11 beta-hydroxysteroid dehydrogenase type 2 (HSD11B2) flanking microsatellites with
essential hypertension in blacks. Hypertension. 1996;28(3):478–82. http://www.ncbi.nlm.

nih.gov/pubmed/8794836. Accessed 2 Mar 2014.
172. Whorwood CB, Ricketts ML, Stewart PM. Epithelial cell localization of type 2 11 beta-
hydroxysteroid dehydrogenase in rat and human colon. Endocrinology. 1994;135(6):2533–41.
doi:10.1210/endo.135.6.7988441.
173. Zhang M-Z, Xu J, Yao B, et al. Inhibition of 11beta-hydroxysteroid dehydrogenase type II
selectively blocks the tumor COX-2 pathway and suppresses colon carcinogenesis in mice
and humans. J Clin Invest. 2009;119(4):876–85. doi:10.1172/JCI37398.
174. Smith RE, Maguire JA, Stein-Oakley AN, et al. Localization of 11 beta-hydroxysteroid dehy-
drogenase type II in human epithelial tissues. J Clin Endocrinol Metab. 1996;81(9):3244–8.
doi:10.1210/jcem.81.9.8784076.
175. Shimojo M, Ricketts ML, Petrelli MD, et al. Immunodetection of 11 beta-hydroxysteroid
dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear local-
ization. Endocrinology. 1997;138(3):1305–11. doi:10.1210/endo.138.3.4994.
176. Perogamvros I, Keevil BG, Ray DW, Trainer PJ. Salivary cortisone is a potential biomarker
for serum free cortisol. J Clin Endocrinol Metab. 2010;95(11):4951–8. doi:10.1210/
jc.2010-1215.
177. Bocchi B, Kenouch S, Lamarre-Cliche M, et al. Impaired 11-beta hydroxysteroid dehydroge-
nase type 2 activity in sweat gland ducts in human essential hypertension. Hypertension.
2004;43(4):803–8. doi:10.1161/01.HYP.0000121362.64182.ad.
178. Ulick S, Levine LS, Gunczler P, et al. A syndrome of apparent mineralocorticoid excess
associated with defects in the peripheral metabolism of cortisol. J Clin Endocrinol Metab.
1979;49(5):757–64. doi:10.1210/jcem-49-5-757.
179. Stewart PM, Corrie JE, Shackleton CH, Edwards CR. Syndrome of apparent mineralocorti-
coid excess. A defect in the cortisol-cortisone shuttle. J Clin Invest. 1988;82(1):340–9.
doi:10.1172/JCI113592.
180. Stewart PM, Krozowski ZS, Gupta A, et al. Hypertension in the syndrome of apparent min-
eralocorticoid excess due to mutation of the 11 beta-hydroxysteroid dehydrogenase type 2
gene. Lancet. 1996;347(8994):88–91. http://www.ncbi.nlm.nih.gov/pubmed/8538347.
Accessed 12 Feb 2014.
181. Stewart PM, Wallace AM, Valentino R, Burt D, Shackleton CH, Edwards CR.
Mineralocorticoid activity of liquorice: 11-beta-hydroxysteroid dehydrogenase defi-
ciency comes of age. Lancet. 1987;2(8563):821–4. http://www.ncbi.nlm.nih.gov/
pubmed/2889032. Accessed 12 Feb 2014.
182. Tomlinson JW, Draper N, Mackie J, et al. Absence of Cushingoid phenotype in a patient with
Cushing’s disease due to defective cortisone to cortisol conversion. J Clin Endocrinol Metab.
2002;87(1):57–62. doi:10.1210/jcem.87.1.8189.
183. Morgan SA, McCabe EL, Gathercole LL, Hassan-Smith ZK, Larner DP, Bujalska IJ, Stewart
PM, Tomlinson JW, Lavery GG. 11β-HSD1 is the major regulator of the tissue-specific
effects of circulating glucocorticoid excess. Proc Natl Acad Sci USA. 2014;111(24):E2482-
91. doi: 10..1073/pnas.1323681111
184. Cooper MS, Syddall HE, Fall CHD, et al. Circulating cortisone levels are associated with
biochemical markers of bone formation and lumbar spine BMD: the Hertfordshire Cohort
Study. Clin Endocrinol (Oxf). 2005;62(6):692–7. doi:10.1111/j.1365-2265.2005.02281.x.
185. Overman RA, Yeh J-Y, Deal CL. Prevalence of oral glucocorticoid usage in the United States:
a general population perspective. Arthritis Care Res (Hoboken). 2013;65(2):294–8.
doi:10.1002/acr.21796.
186. Fardet L, Flahault A, Kettaneh A, et al. Corticosteroid-induced clinical adverse events: fre-
quency, risk factors and patient’s opinion. Br J Dermatol. 2007;157(1):142–8.
doi:10.1111/j.1365-2133.2007.07950.x.
Chapter 17
Conclusions and Future Directions
Jen-Chywan Wang and Charles Harris
We hope this book has provided a robust introduction to glucocorticoid action.

We have provided a historical perspective on discoveries related to glucocorticoid
action: from the discovery of the hormones, their use as therapeutics for inflamma-
tory disease, biochemical identification of receptors and finally the cloning of the
glucocorticoid receptor. This has led to identification of not only the primary amino
acid sequence, but structural information gleaned from both X-ray crystallography as
well as nuclear magnetic resonance approaches. We hope the reader comes away
with a better understanding of the many actions of glucocorticoids on various tissues
and cell types. Indeed, one of the unanswered questions in our field is how one
hormone and receptor can have such varied transcriptional responses in different cell
types. Surely, some of this will be explained by epigenetic modifications that occur
in different cell types. Recent work has also made it clear that GR does not act alone
to modulate transcription as numerous transcriptional coregulators have been
described to modulate GR’s transcriptional program. Additional combinatorial com-
plexity comes about from composite response elements between GR and other tran-
scription factors. Subtle changes in the glucocorticoid response element as well as
levels of GR (both the major alpha isoform as well as alternatively spliced and beta
and gamma isoforms) could also modulate the transcriptional program. Analysis of
mice with altered glucocorticoid signaling has reinforced the critical importance
of this pathway. Mice lacking GR die at birth due to defects in lung maturation and
J.-C. Wang (*)

119 Morgan Hall, Berkeley, CA, USA
C. Harris (*)
Campus Box 8127, St. Louis, MO, USA

382 J.-C. Wang and C. Harris
it is a well described phenomenon that premature infants can have their lung maturation
accelerated by administering glucocorticoids when pregnant mothers are threatening
preterm labor. The treatment of these women with glucocorticoids has greatly
reduced infant mortality due to prematurity. Thus it is clear, that understanding glu-
cocorticoid action has great clinical benefits. In addition to treating patient with adre-
nal insufficiency (Addison’s disease) glucocorticoids are used to treat a wide variety
of common inflammatory diseases such as asthma, inflammatory bowel disease and
rheumatoid arthritis. However, treatment of inflammatory disease with glucocorti-
coids has powerful adverse effects including osteoporosis, insulin, resistance, hyper-
tension and behavioral problems. Therefore, the identification of selective GR
modulators that retain anti-inflammatory properties with less adverse effects has
great promise for safer steroids in the twenty-first century. Hope from this model
comes from prior successes developing selective modulators of the estrogen receptor
for the treatment of osteoporosis. Key to the development of selective GR modula-
tors will be a better understanding of how these effects are mediated. It is clear that
early models linking GR transactivation to adverse effects and repression to immu-
nosuppression are overly simplistic. We are hopeful, that new models will replace
them so that such selective molecules will be possible in the future. One barrier to a
more complete understanding of glucocorticoid action is linking GR binding sites
identified in whole genome approaches (such as Chip-seq) to the genes that are
regulated by these binding sites. Early hopes that a binding site would regulate the
gene nearest to it do not appear to always be the case. Techniques such as HiC and
FaireSeq have shown that binding sites can regulate genes far away due to looping
of chromatin. Therefore, understanding which genes are regulated by which binding
sites is currently a hurdle. New techniques such as CRISPRs have facilitated genetic
modification so that in the future it might be possible to mutate binding sites indi-
vidually to determine the exact transcriptional consequences. We are confident the
coming years will be an exciting time for research on glucocorticoid action.
Index
A B
ACTH. See Adrenocorticotropic hormone Behavior, 14, 35, 40, 49, 149, 218, 225, 227,
(ACTH) 236, 238, 240–246, 254, 323, 325,
Addison, T., 5–7, 9, 83–95, 136, 382 343, 361, 382
Adipose tissue, 37, 90, 127, 129–135, 138, 11-β-HSD transgenics, 337
159, 305–310, 346, 354, 357–361,
364
Adrenal gland, 5–7, 34, 38, 83, 84, 90, 92, 94, C
136, 138, 222, 236, 237, 325 Cardiomyocyte, 306–309
Adrenocorticotropic hormone (ACTH), 7, 34, Chemotherapy, 166, 316, 321, 325–328, 341
36, 83–86, 88, 90–95, 237, 238, Cholesterol metabolism, 127
240, 258, 264, 316, 322, 323, 337, Chromatin, 14, 18, 19, 23, 40, 45, 64–70, 72–74,
338, 343, 346, 364, 367 103, 116, 160, 161, 202, 220, 320, 382
Airway epithelium, 280, 284–292 Cis-regulatory elements, 59
Airway smooth muscle (ASM), 280, 285–287, Context-dependent signaling, 48–49
291–292 Coregulators, 23, 60–63, 70–73, 103, 105,
Akt, 107, 110, 111, 131, 151, 156–158, 106, 116, 220, 348, 381
161, 162, 166, 168, 184, 188, Corticotrophin releasing hormone (CRH), 7,
191, 193, 196–198, 200, 302, 34–36, 47–49, 83, 84, 86, 91,
319, 320, 322 237–242, 258–260, 262, 266, 339,
Allostasis, 34, 39, 46, 48–50 343, 346, 348
Altered glucocorticoid signaling, 48, 337–348, Cortisol, 5, 7, 13, 14, 34, 35, 37–40, 43, 47,
381 83–95, 100, 109, 113, 130, 164,
Amygdala, 36, 47, 48, 236, 238–245 185, 218, 263, 265, 267, 300,
Animal models, 36, 110, 114, 148, 149, 182, 305–308, 318, 323, 354–357,
204, 223, 243, 300, 307, 308, 310, 359–361, 363–365, 368–370
337–348, 357, 359, 362, 363 Cortisone, 6, 7, 37, 109, 152, 164, 265, 287,
Apoptosis, 92, 112, 115, 183, 184, 187–190, 316, 322, 323, 354–359, 363–365,
192, 197, 198, 204, 224, 225, 264, 367–370
266, 308, 315, 319, 320, 322, 323, CRH. See Corticotrophin releasing hormone
328, 329, 338 (CRH)
Asthma, 48, 50, 63, 88, 146, 180, 181, 203, Cushing disease, 84
228, 279, 285–292, 354, 382 Cushing syndrome, 84, 94, 95, 130, 149

Medicine and Biology 872, DOI 10.1007/978-1-4939-2895-8
384 Index
D Glucose metabolism, 5, 8, 145

Dexamethasone, 15, 36, 66, 84, 113, 130, Glucose utilization, 100, 109–112
146, 184, 218, 259, 280, 301, 318, Glycogen, 4, 5, 7, 100, 112–113, 115, 116,
340, 363 157, 191, 364
DNA elements, 17–22, 104, 196, 199, 202, 345 GR-null mice, 338, 340–344, 347, 348
E H
Endoplasmic reticulum (ER), 101, 356, 357 Hippocampus, 36, 38, 39, 46, 47, 112,
Endothelium, 89, 284, 302, 304–305, 308, 236–245, 344
309, 366 HPA axis. See Hypothalamic pituitary adrenal
ERK, 43, 187, 196–198, 200, 222, 319, 325 axis (HPA axis)
H6PDH, 353
HPG axis. See Hypothalamic-pituitary-
F gonadal (HPG) axis
Feed-forward loop (FFL), 282, 283 Hypertension, 7, 87, 89, 92, 164, 300–304,
FFL. See Feed-forward loop (FFL) 306, 340, 354, 359, 363, 365–368,
FoxO, 107, 131, 157, 160–162, 166–168, 187, 382
193, 197–199 Hypomorphs, 337
Hypothalamic pituitary adrenal axis (HPA
axis), 34–36, 39, 47, 83–84, 86, 91,
G 95, 130, 135, 236–246, 254, 257,
Gene expression, 10–13, 24, 33, 34, 48, 64, 258, 260, 343, 345–347, 354–355,
71, 104–110, 114, 160, 161, 163, 359, 363, 364, 369
164, 166, 167, 182, 185, 190, 193, Hypothalamic-pituitary-gonadal (HPG)
194, 200, 220–222, 226–228, 259, axis, 89, 254–258, 261–263,
281, 288–290, 306, 320, 346 266, 268, 347
Gene transcription, 12, 14, 20–23, 41, 59–74, Hypothalamus, 7, 36, 48, 83, 91, 112,
103–108, 112, 113, 116, 160, 197, 237–240, 254–262, 264, 268, 339,
354, 362 343, 346
GIO. See Glucocorticoid-Induced
Osteoporosis (GIO)
Glucocorticoid hormones, 3–25, 33, 34, 73, I
301, 338 Immune cells, 160, 203, 218–227, 267, 285,
Glucocorticoid-Induced Osteoporosis (GIO), 362
92, 179–205, 342 Inflammation, 37, 50, 116, 136, 146, 150, 159,
Glucocorticoid receptor (GR), 13, 34, 59, 83, 166, 168, 218, 219, 225, 280, 286,
103, 158, 180, 218, 238, 259, 299, 291, 304, 308, 309, 328, 354, 360,
315, 337, 354, 381 362, 363
Glucocorticoids (GC), 3–25, 33–50, 59–74, Insulin, 7, 90, 100, 129, 146, 195, 306,
83–95, 99–116, 127–138, 145–168, 358, 382
179–205, 217–228, 235–246,
253–268, 279–292, 299–310,
315–329, 337–348, 354–356, 358, L
359, 362, 364–366, 368, 369, 381, 382 Leukemia, 63, 112, 115, 133, 204, 316–318,
Gluconeogenesis, 4, 7, 13, 21, 22, 37, 90, 320–324, 328
100–103, 108–109, 113, 115, 116, Lipid metabolism, 127–138
146, 301, 364 Lipoprotein metabolism, 127
Index 385
Liver, 4, 35, 90, 100, 127, 146, 202, 306, 245, 266, 284, 292, 306, 315,
341, 354 322–326, 344, 347
Lymphoma, 221, 316, 324, 328 Protein synthesis, 13, 40, 47, 111, 146, 150–152,
155–158, 162, 167, 192, 197, 289, 354
Proteolysis, 60, 146, 152, 153, 155, 157
M
MAPK. See Mitogen-activated protein kinases
(MAPK) R
Mitogen-activated protein kinases (MAPK), Reproduction, 253–268
34, 50, 115, 162, 184, 197, 220, RUNX2, 182, 185, 192, 194, 196, 199–201, 204
222, 227, 302
MKP-1, 197–198
Muscle atrophy, 146–149, 155–158, 160–163, S
165–167, 341, 344, 347, 360 Stent, 309
Stress, 4, 35, 83, 99, 145, 187, 224, 235, 254,
307, 343
N Surfactant, 4, 280–285, 340
NADPH, 356
Negative feedback, 36, 47, 87, 89, 92, 94,
237–243, 255, 290, 325, 338, 339, T
343, 346, 354 Tissue specific KO, 239, 339–344
Nephron, 301, 302, 340, 354, 367 Tissue-specific transgenics, 339–344, 346
Nitric oxide, 304–306, 308 Transcription, 12, 33, 59, 90, 103, 130, 157,
Notch, 194, 200, 204, 321 180, 221, 238, 259, 281, 305, 318,
Nuclear receptors, 15–17, 21, 33, 34, 37, 339, 354, 381
38, 41, 44, 159, 163, 221, 238, Transcription factors, 14, 16, 18–20, 33, 40,
354 41, 44–46, 49, 104, 106, 114, 130,
133, 157, 160–162, 167, 180, 191,
193, 198–203, 218–220, 281,
O 289–291, 320, 322, 359, 381
Osteoblast, 92, 134, 180, 339, 363,
U
P Ubiquitin proteasome pathway, 153–155
Palliative, 316, 326, 327
Pancreas, 100, 105, 113–115, 127, 128, 338,
346, 348 V
Phosphorylation, 15, 34, 38, 41, 43–45, 47–50, Vascular smooth muscle, 302–304, 306, 307, 361
60, 63, 70, 71, 110, 113, 115, 132,
137, 150–152, 157, 158, 161, 163,
191–193, 197–199, 220, 222, 225, W
226, 228, 302, 320 Wnt signaling pathway, 21, 34, 47, 48, 61,
Prefrontal cortex, 36, 237, 240–241, 243 103, 106, 111, 150, 151, 156, 184,
Proliferation, 4, 39, 115, 184, 186, 187, 189, 186–200, 204, 218, 221, 222,
190, 192, 195–198, 201, 204, 220, 225–228, 302, 319, 362

Glucocorticoid Signaling From Molecules To Mice To Man

Uploaded by

Copyright:

Available Formats

Glucocorticoid Signaling From Molecules To Mice To Man

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Glucocorticoid Signaling From Molecules To Mice To Man

Uploaded by

Copyright:

Available Formats

Advances in Experimental Medicine and Biology 872

IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel

More information about this series at http://www.springer.com/series/5584

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Library of Congress Control Number: 2015945236

Springer New York Heidelberg Dordrecht London

Printed on acid-free paper

Part I Introductory Materials

1 Regulatory Actions of Glucocorticoid Hormones:

Part II Effects of Glucocorticoids on Metabolism

5 Regulation of Glucose Homeostasis by Glucocorticoids...................... 99

Part III Specific Effects of Glucocorticoids on Tissues

8 Glucocorticoid-Induced Osteoporosis ................................................... 179

Part IV Miscellaneous Topics

15 Animal Models of Altered Glucocorticoid Signaling ........................... 337

Index ................................................................................................................. 383

Margarita Arango-Lievano, Ph.D. Inserm U1191, CNRS UMR5203, Institute

Julie E. Goodwin, M.D. Department of Pediatrics, Yale University School of

Jeremy W. Tomlinson, FRCP, Ph.D. Oxford Centre for Diabetes,

Daryl K. Granner, Jen-Chywan Wang, and Keith R. Yamamoto

Abstract The history of glucocorticoid hormone research is an excellent example of

© Springer Science+Business Media New York 2015 3

Keywords Glucocorticoids • Gene expression • Nuclear receptors • Transcription

The survival of multicellular organisms requires rapid and efficient adaptation to an

Table 1.1 The pleiotropic effects of glucocorticoid hormones

The Importance of the Adrenal Glands Is Established

The Active Adrenal Cortical Hormones Are Identified

The identification of the active hormone proved to be an arduous task. A major

The Metabolic and Therapeutic Effects of Glucocorticoids

Enzymes Are Defined and the Metabolic Pathways Are

The Concept of Differential Turnover of Cellular Constituents

Enzymes Show Adaptive Changes and Glucocorticoids

Table 1.2 The timetable of Enzyme

Glucocorticoids Regulate the Transcription of Specific Genes

An enormous conceptual advance occurred as a result of the studies of the regulation

A Specific Receptor Mediates the Action of Glucocorticoids

While studies of the effect of glucocorticoids on gene expression were progressing,

The Glucocorticoid Receptor Is a Transcriptional

The Glucocorticoid Receptor Is Modular, Containing Discrete

The successful isolation of a cDNA molecule for GR was a landmark achievement

in mediating the dimerization and cooperative binding of two receptor molecules to

The GBS Is Sufficient for GR-Regulated Transcription

Context-Specific Glucocorticoid Regulation Is Specified

regard that the receptors for glucocorticoids, mineralocorticoids, progestins and

“assembly instructions” for multi-component transcriptional regulatory complexes

The PEPCK GRE Provides Finely Tuned, Versatile Regulation

A key element of regulatory crosstalk is the integration of information from multiple

Multiple Context Effects Determine GRE Activities

Molecular analyses showing that glucocorticoid-responsive genes are governed by

Here we have provided an historical perspective on the progression of research on

outstanding contributors to this volume build on our overview to describe in detail

Margarita Arango-Lievano, W. Marcus Lambert, and Freddy Jeanneteau

Abstract Well-defined as signaling hormones for the programming of cell type-

Keywords Allostasis • Transcription • MAPK • Phosphorylation • Context-

Glucocorticoid hormones are chemical messengers that signal via alterations of

M. Arango-Lievano, Ph.D. • F. Jeanneteau, Ph.D. (*)

© Springer Science+Business Media New York 2015 33

Glucocorticoids are cholesterol-derived steroid hormones that prepare the organism

Thanks to robust anti-inflammatory and immunosuppressive effects, glucocorticoids