Supplemental Material can be found at:

http://www.jlr.org/cgi/content/full/M400294-JLR200/DC1

Selective activation of vitamin D receptor by lithocholic

acid acetate, a bile acid derivative
Ryutaro Adachi,1,* Yoshio Honma,2,† Hiroyuki Masuno,§ Katsuyoshi Kawana,*,**
Iichiro Shimomura,*,†† Sachiko Yamada,§ and Makoto Makishima3,*,**,††
Graduate School of Medicine* and Graduate School of Frontier Biosciences,†† Osaka University, Suita, Osaka
565-0871, Japan; Department of Chemotherapy,† Saitama Cancer Center Research Institute, Ina-machi,
Saitama 362-0806, Japan; Institute of Biomaterials and Bioengineering and School of Biomedical Science,§
Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan; and Department of
Biochemistry,** Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan

Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010

Abstract The vitamin D receptor (VDR), a member of the of malignant cells, including breast, prostate, colon, skin,
nuclear receptor superfamily, mediates the biological ac- and brain cancer cells, as well as myeloid leukemia cells in
tions of the active form of vitamin D, 1!,25-dihydroxyvita- vitro (2). The administration of 1,25(OH)2D3 or its ana-
min D3. It regulates calcium homeostasis, immunity, cellular logs has therapeutic effects in mouse models of malignan-
differentiation, and other physiological processes. Recently,
cies such as leukemia and colon cancer (3, 4). The biologi-
VDR was found to respond to bile acids as well as other nu-
clear receptors, farnesoid X receptor (FXR) and pregnane cal activities of 1,25(OH)2D3 are mediated through
X receptor (PXR). The toxic bile acid lithocholic acid binding to its intracellular receptor, the vitamin D recep-
(LCA) induces its metabolism through VDR interaction. To tor (VDR; NR1I1), a member of the nuclear receptor su-
elucidate the structure-function relationship between VDR perfamily. Additional nongenomic mechanisms of action
and bile acids, we examined the effect of several LCA deriv- may be mediated by a poorly characterized membrane re-
atives on VDR activation and identified compounds with ceptor (5). Structure-function relationship studies of the
more potent activity than LCA. LCA acetate is the most po- interaction of vitamin D analogs with VDR reveal different
tent of these VDR agonists. It binds directly to VDR and ac- binding modes in the ligand-binding pocket of VDR (6).
tivates the receptor with 30 times the potency of LCA and
has no or minimal activity on FXR and PXR. LCA acetate ef- These differences in ligand-receptor interaction may con-
fectively induced the expression of VDR target genes in in- tribute to the differential recruitment of coactivators to
testinal cells. Unlike LCA, LCA acetate inhibited the prolif- VDR and selective biological actions (7).
eration of human monoblastic leukemia cells and induced Nuclear receptors are ligand-inducible transcription
their monocytic differentiation. We propose a docking factors that are involved in many biological processes, in-
model for LCA acetate binding to VDR. The development cluding cell growth and differentiation, embryonic devel-
of VDR agonists derived from bile acids should be useful to opment, and metabolic homeostasis (8). Recently, nuclear
elucidate ligand-selective VDR functions.—Adachi, R., Y. receptors belonging to the NR1H and NR1I subfamilies,
Honma, H. Masuno, K. Kawana, I. Shimomura, S. Yamada,
including VDR, have been shown to control cholesterol
and M. Makishima. Selective activation of vitamin D recep-
tor by lithocholic acid acetate, a bile acid derivative. J. Lipid
Res. 2005. 46: 46–57.
Abbreviations: BSEP, bile salt export pump; CAR, constitutive an-
drostane receptor; ER, estrogen receptor; FXR, farnesoid X receptor;
Supplementary key words nuclear receptor • structure-function rela-
IBABP, ileal bile acid binding protein; 3-keto-LCA, 3-keto-cholanic
tionship • colon cancer • intestine • leukemia
acid; LCA, lithocholic acid; LXR, liver X receptor; NBT, nitroblue tetra-
zolium; N-CoR, nuclear receptor corepressor; 1,25(OH)2D3, 1!,25-dihy-
droxyvitamin D3; PPAR, peroxisome proliferator-activated receptor; PXR,
The active form of vitamin D, 1!,25-dihydroxyvitamin D3 pregnane X receptor; RAR, retinoic acid receptor; RXR, retinoid X re-
ceptor; SRC-1, steroid receptor coativator-1; TR, thyroid hormone re-
[1,25(OH)2D3], regulates calcium homeostasis, immunity, ceptor; VDR, vitamin D receptor.
and cellular growth and differentiation (1). 1,25(OH)2D3 1 Present address of R. Adachi: Pharmaceutical Discovery Center,

has been demonstrated to be able to inhibit the prolifera- Pharmaceutical Research Division, Takeda Chemical Industries, 2-17-
85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan.
tion and/or to induce the differentiation of various types 2 Present address of Y. Honma: Department of Life Science, Shi-

mane University School of Medicine, 89-1 Enya-cho, Izumo, Shimane

693-8501, Japan.
Manuscript received 2 August 2004 and in revised form 17 September 2004 3 To whom correspondence should be addressed.
and in re-revised form 30 September 2004. e-mail: [email protected]
Published, JLR Papers in Press, October 16, 2004. The online version of this article (available at http://www.jlr.org)
DOI 10.1194/jlr.M400294-JLR200 contains an additional figure.

Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc.
46 Journal of Lipid Research Volume 46, 2005 This article is available online at http://www.jlr.org
and bile acid metabolism (9). Liver X receptor ! (LXR!; inoid X receptor (RXR!; accession number NM_002957), and
NR1H3) and LXR" (NR1H2) function as oxysterol recep- mouse peroxisome proliferator-activated receptor ! (PPAR!;
tors and regulate cholesterol metabolism in liver, intes- accession number NM_011144), PPAR# (accession number
NM_011145), and PPAR$ (accession number NM_011146) were
tine, adipose tissue, and macrophages. Bile acids, which
inserted into the pCMX-GAL4 vector to make pCMX-GAL4-VDR,
are major metabolites of cholesterol in the body, bind to pCMX-GAL4-FXR, pCMX-GAL4-TR!, pCMX-GAL4-RAR!, pCMX-
farnesoid X receptor (FXR; NR1H4) and induce negative GAL4-LXR!, pCMX-GAL4-CAR, pCMX-GAL4-ER!, pCMX-GAL4-
feedback regulation of their synthesis from cholesterol. RXR!, pCMX-GAL4-PPAR!, pCMX-GAL4-PPAR#, and pCMX-
Primary bile acids, produced in the liver, are excreted in GAL4-PPAR$, respectively. A full-length fragment of human VDR
the bile after conjugation with taurine and glycine and are was inserted into the pCMX-VP16 vector to make pCMX-VP16-
subsequently reabsorbed in the intestine. Bile acids that VDR. Nuclear hormone receptor-interacting domains of steroid
escape reabsorption are converted to secondary bile acids receptor coactivator-1 (SRC-1) (amino acids 595–771; GenBank
by the intestinal microflora. Pregnane X receptor (PXR; accession number U90661) and nuclear receptor corepressor
(N-CoR) were inserted into the pCMX-GAL4 vector to make
NR1I2), which acts as a receptor for various xenobiotics,
pCMX-GAL4-SRC-1 and pCMX-GAL4-N-CoR (14). Mutations were
senses the levels of secondary bile acids and induces their introduced into pCMX-GAL4-VDR using the QuikChange Site-
metabolism in the liver (10, 11). VDR was also found to Directed Mutagenesis Kit (Stratagene, La Jolla, CA). hCYP3A4-
function as a receptor for secondary bile acids such as ER-6x3-tk-LUC, IR-1x3-tk-LUC, and GAL4-responsive MH100
lithocholic acid (LCA) and to be involved in bile acid me- (UAS)x4-tk-LUC reporters were used to evaluate the activities of
tabolism by inducing a LCA detoxification mechanism in VDR and PXR, FXR, and GAL4-chimera receptors, respectively.
the liver and intestine (12). All plasmids were sequenced before use to verify DNA sequence
Previously, we analyzed the structure-function relation- fidelity.

Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010

ships of the endocrine [1,25(OH)2D3] and xenobiotic Cell lines and cell culture
(LCA) ligands with VDR and revealed that 1,25(OH)2D3 HEK293 cells were cultured in Dulbecco’s modified Eagle’s
and LCA interact with a different set of amino acids in the medium containing 5% fetal bovine serum and antibiotic-anti-
ligand binding pocket of VDR (13, 14). These results sug- mycotic (Nacalai) at 37%C in a humidified atmosphere of 5%
gest the possibility that VDR adopts distinct conforma- CO2 in air. Human hepatoblastoma HepG2 cells and colon can-
tions in response to 1,25(OH)2D3 and LCA binding and cer SW480 cells were cultured in Dulbecco’s modified Eagle’s
provides a possible mechanism for the compounds’ differ- medium containing 10% fetal bovine serum and antibiotic-anti-
ent biological actions. The docking models of LCA and mycotic (Nacalai) at 37%C in a humidified atmosphere of 5%
CO2 in air. Human myeloid leukemia THP-1 cells were cultured
3-keto-cholanic acid (3-keto-LCA), which is a metabolite of
in suspension in RPMI 1640 medium containing 10% fetal bo-
LCA, reveal that these compounds are accommodated vine serum and 80 &g/ml gentamicin at 37%C in a humidified at-
in the VDR ligand binding pocket more weakly than mosphere of 5% CO2 in air (16).
1,25(OH)2D3 (13, 14), suggesting that modification of
these bile acids can increase the VDR transactivation activ- Cotransfection assay
ity. In this study, we examined the ability of several LCA Transfections were performed by the calcium phosphate co-
analogs to activate VDR and found that modification of precipitation assay as described previously (14). Eight hours after
the 3 position of LCA increased VDR transactivation by transfection, test compounds were added. Cells were harvested
16–24 h later, and luciferase and "-galactosidase activities were
more than 30-fold. Furthermore, the LCA acetate analog
assayed using a luminometer and a microplate reader (Molecu-
can induce differentiation of myeloid leukemia cells. lar Devices, Sunnyvale, CA). Cotransfection experiments used 50
ng of reporter plasmid, 20 ng of pCMX-"-galactosidase, 15 ng of
each receptor and/or cofactor expression plasmid, and pGEM
MATERIALS AND METHODS carrier DNA to give a total of 150 ng of DNA per well of a 96-well
plate. Luciferase data were normalized to the internal "-galac-
Chemical compounds tosidase control and represent means ' SD of triplicate assays.
LCA formate and LCA isobutyrate were synthesized in our lab-
oratory (H. Masuno and S. Yamada, unpublished results), and Competitive ligand binding assay
other bile acids and derivatives were purchased from Sigma- Human VDR protein was generated using the TNT Quick
Aldrich (St. Louis, MO), Wako (Osaka, Japan), Nacalai (Kyoto, Coupled Transcription/Translation System (Promega, Madison,
Japan), or Steraloids (Newport, RI). 1,25(OH)2D3 was obtained WI). The protein was diluted 5-fold with ice-cold TEGWD buffer
from Calbiochem (San Diego, CA). (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 20
mM sodium tungstate, and 10% glycerol). The diluted lysate was
Plasmids incubated with 1 nM of [26,27-methyl-3H]1,25(OH)2D3 for 16 h
Fragments of human VDR (GenBank accession number at 4%C in the presence or absence of nonradioactive compet-
NM_000376), FXR (accession number NM_005123), and PXR ing compounds (14). Bound and free compounds were sepa-
(accession number NM_022002) were inserted into the pCMX rated by the dextran-charcoal method (17). Bound and labeled
vector to make pCMX-VDR, pCMX-FXR, and pCMX-PXR, re- 1,25(OH)2D3 was quantitated using scintillation counting.
spectively (12, 14, 15). The ligand binding domains of human
VDR, FXR, thyroid hormone !1 (TR!1) (accession number Graphic manipulation and docking
NM_199334), retinoic acid receptor ! (RAR!) (accession num- Graphic manipulations were performed using SYBYL 6.8 (Tri-
ber NM_000964), LXR! (accession number NM_005693), consti- pos, St. Louis, MO) (13, 14). The atomic coordinates of the hu-
tutive androstane receptor (CAR) (accession number NM_005122), man VDR ligand binding domain ((165–215) crystal structure
estrogen receptor ! (ER!) (accession number NM_000125), ret- were retrieved from the Protein Data Bank (PDB #1DB1). LCA

Adachi et al. A lithocholic acid-derived agonist for vitamin D receptor 47

acetate was docked into VDR using the docking software FlexX activities were compared (Fig. 1B). Transcriptional activa-
(version 1.10; Tripos) (18). tion by 1,25(OH)2D3 and LCA was similar to previous re-
Animal studies ports (12). Esterification of the side chain carboxyl group
C57BL/6J mice were obtained from Japan SLC (Hamamatsu,
of LCA with methyl, ethyl, and benzyl groups drastically
Japan) and were maintained under controlled temperature (23 ' decreased the activity on VDR (Fig. 1B). Next, we examined
1%C) and humidity (45–65%) with free access to water and chow the effects of LCA derivatives modified at the 3!-hydroxyl
(Oriental Yeast, Tokyo, Japan). Experiments were conducted with group (Fig. 1A). LCA formate and LCA acetate were able
male mice between 8 and 9 weeks of age. Mice were treated orally to activate VDR as efficiently as LCA at the concentration
with LCA or LCA acetate in a polyethylene glycol-Tween 80 (4:1) of 10 &M. LCA isobutyrate activated VDR moderately,
formulation or with vehicle alone (19). Mice were analyzed 12 h whereas LCA hemisuccinate was not an effective VDR ago-
after treatment under fasting conditions. The experimental pro- nist. The data indicate that addition of a large acyl group
tocol was approved by the Ethics Review Committee for Animal
at the 3!-hydroxyl group of LCA abolishes VDR activa-
Experimentation of Osaka University.
tion. The stereochemistry, as well as the substituent of the
Quantitative real-time RT-PCR analysis 3-hydroxyl group, is also important for LCA activity. Iso-
Total RNAs from samples were prepared with an RNA STAT-60 LCA with a 3"-hydroxyl group and ursocholanic acid with
kit (Tel-Test, Friendswood, TX). The cDNA was synthesized using no hydroxyl group at C-3 (Fig. 1A) have little activity on
the ThermoScript RT-PCR System (Invitrogen, Carlsbad, CA). VDR (Fig. 1B). Interestingly, although the effect of LCA
Real-time PCR was performed on a LightCycler using the Fast- methyl ester on VDR activation was weak, LCA acetate
Start DNA Master SYBR Green I (Roche Diagnostics, Tokyo, Ja-
methyl ester was able to induce VDR activation effectively.
pan) according to the instructions provided by the manufacturer
3-Keto-LCA, a metabolite of LCA, is another potent bile

Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010

(19). Primers were as follows: human VDR, 5)-GCTGACCTGGT-
CAGTTACAGCA-3) and 5)-CACGTCACTGACGCGGTACTT-3); acid for VDR (12). The esterification on the side chain of
RXR!, 5)-AAGATGCGGGACATGCAGAT-3) and 5)-CAGGCGGA- 3-keto-LCA modestly decreased its activity on VDR (Fig.
GCAAGAGCTTAG-3); cyclophilin, 5)-CCCACCGTGTTCTTCGA- 1B). 6-Keto-LCA is a very weak VDR agonist, and 7-keto-
CAT-3) and 5)-CCAGTGCTCAGAGCACGAAA-3); CYP24A1, 5)-TGA- LCA and 12-keto-LCA were not able to activate VDR (12).
ACGTTGGCTTCAGGAGAA-3) and 5)-AGGGTGCCTGAGTGT- Transactivation of VDR by 3,6-diketo-LCA, 3,7-diketo-LCA,
AGCATCT-3); CYP3A4, 5)-AGTGTGGGGCTTTTATGATG-3) and and 3,12-keto-LCA was almost absent (Fig. 1B). These data
5)-ATACTGGGCAATGATAGGGA-3); CaT1, 5)-AGCCTACATGA-
indicate that addition of a ketone moiety at position 6, 7, or
CCCCTAAGGACG-3 ) and 5) -GTAGAAGTGGCCTAGCTCCT-
CGG-3); E-cadherin, 5)-GAAGGTGACAGAGCCTCTGGATAG-3)
12 to LCA or 3-keto-LCA disturbs the interaction with VDR.
and 5)-CTGGAAGAGCACCTTCCATGA-3); bile salt export pump
(BSEP), 5)-TCTTTACTGGATTCGTGTGG-3) and 5)-TGACACT- LCA acetate is a potent agonist for VDR
GAGGAAAATCTGG-3); mouse cyclophilin, 5)-CAGACGCCACT- We compared VDR dose-response curves for LCA, LCA
GTCGCTTT-3) and 5)-TGTCTTTGGAACTTTGTCTGCAA-3); formate, LCA acetate, LCA acetate methyl ester, and 3-keto-
Cyp24a1, 5)-CCCATTACTCAGGGAAGCAC-3) and 5)-CCACTCA- LCA. LCA acetate activated VDR with an EC50 of 0.40 &M,
GACAATGAAGCCA-3); Cyp3a11, 5)-CCAACAAGGCACCTCCC- followed in rank order by LCA formate (EC50 * 4.0 &M),
ACG-3) and 5)-TGGAATTCTTCAGGCTCTGA-3); ileal bile acid 3-keto-LCA (6.8 &M), and LCA (12.1 &M) (Fig. 2A). Nota-
binding protein (IBABP), 5)-GGTACCACCATGGCCTTCAGTG-
bly, the potency of VDR activation by LCA acetate on VDR
GCAAATAT-3) and 5)-GCTAGCTCAAGCCAGCCTCTTGCTTAC-
3). The RNA values were normalized to the amount of cyclophi- was 30-fold greater than that of LCA.
lin mRNA and are represented in arbitrary units. Upon ligand binding, nuclear receptors undergo a con-
formational change that induces recruitment of coactiva-
Growth and differentiation of myeloid leukemia cells tors, such as SRC-1, and dissociation of corepressors, such
Cell suspensions were cultured with or without test com- as N-CoR (21). To assay ligand-dependent interactions of
pound. The cells were counted in a model ZM Coulter Counter VDR with cofactors, the receptor-interacting domains of
(Coulter Electronics, Luton, UK). Cell morphology was exam-
SRC-1 and N-CoR were fused to the GAL4 DNA binding do-
ined in cell smears stained with May-Gruenwald-Giemsa. !-Naph-
thyl acetate esterase was determined cytochemically, nitroblue
main (14). Cotransfection of GAL4-cofactors with VDR
tetrazolium (NBT) reduction was assayed colorimetrically, and fused to the transactivation domain of herpesvirus VP16
expression of monocytic antigens CD11b and CD14 on the cell protein allowed for the detection of ligand-dependent co-
surface was determined using indirect immunofluorescent stain- factor interaction (15). Although there was no association
ing and a flow cytometer (Epics XL; Coulter Electronics) (16, 20). between control GAL4 protein and VP16-VDR, LCA acetate
at 10 &M and 1,25(OH)2D3 at 100 nM strongly induced
the association of VDR with SRC-1 (Fig. 2B). The effects of
RESULTS LCA formate and LCA acetate methyl ester on this interac-
tion were modest, and activation by LCA and 3-keto-LCA
Transactivation of VDR by LCA derivatives were weak at 10 &M concentration. LCA acetate, LCA for-
To elucidate the structure-activity relationship of LCA mate, LCA acetate methyl ester, and 3-keto-LCA dissociated
derivatives (Fig. 1A) on VDR function, we transiently trans- N-CoR from VDR as effectively as 1,25 (OH)2D3 (Fig. 2B).
fected HEK293 cells with a VDR expression vector and a The effects of these LCA derivatives on N-CoR dissociation
luciferase reporter containing a VDR-responsive everted were stronger than that of LCA. Thus, LCA acetate is a po-
repeat-6 element from the CYP3A4 promoter (12). Cells tent regulator of VDR-cofactor interaction.
were treated with test compounds and the induced luciferase Next, we assessed the ability of LCA derivatives to bind

48 Journal of Lipid Research Volume 46, 2005

 Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010
Fig. 1. Lithocholic acid (LCA) derivatives activate vitamin D receptor (VDR). A: Structures of LCA, its derivatives, and 1!,25-dihydroxyvi-
tamin D3 [1,25(OH)2D3] are shown. B: Activation of VDR by LCA derivatives. HEK293 cells were cotransfected with CMX-VDR and
CYP3A4-ER-6x3-tk-LUC and then treated with vehicle control (ethanol), 1,25(OH)2D3 (100 nM), or bile acid derivatives (10 &M) for 24 h.
Luciferase activity of the reporter is expressed as fold induction with compound treatment relative to vehicle control. The values represent
means ' SD.

directly to VDR in vitro using the competitive binding as- transcription factor GAL4 to examine the effect of LCA
say. Isotopically labeled 1,25(OH)2D3 was incubated with acetate on these receptors. The GAL4-chimera receptors
in vitro translated VDR protein in the absence or presence were cotransfected with a GAL4-responsive luciferase re-
of test compounds. The binding of labeled 1,25(OH)2D3 porter into HEK293 cells (15). Because this reporter is ac-
to VDR was competed by the addition of unlabeled tivated only by GAL4-chimera receptors, the potentially
1,25(OH)2D3 (Fig. 2C). LCA acetate and LCA formate confounding effects of endogenous receptors are elimi-
also inhibited the binding of labeled 1,25(OH)2D3 to nated. LCA acetate at 30 &M induced the activation of
VDR, indicating that these LCA derivatives directly bind to GAL4-VDR (Fig. 3A). It induced weak activation of FXR
VDR. Competition with 3-keto-LCA and LCA was weaker but was not effective on TR!, RAR!, PPAR!, PPAR#,
than that of LCA acetate and LCA formate. Interestingly, PPAR$, LXR!, CAR, RXR!, or ER!. FXR has been previ-
although LCA acetate methyl ester showed enhanced acti- ously shown to respond to various bile acids, such as cheno-
vation of VDR compared with 3-keto-LCA in the luciferase deoxycholic acid and deoxycholic acid (12, 15). Next, we
reporter assay, as shown Fig. 2A, its direct interaction with determined FXR dose-response curves for LCA derivatives
VDR protein was weaker than those of LCA and 3-keto- modified at position 3. As reported previously (15), cheno-
LCA (Fig. 2C). LCA acetate did not inhibit the binding deoxycholic acid was a potent FXR agonist (Fig. 3B). In-
of labeled estradiol to ER! (data not shown). Taken to- terestingly, ursocholanic acid and iso-LCA, which were not
gether, these data indicate that LCA acetate activates VDR effective on VDR (Fig. 1B), strongly induced the activa-
by direct binding. tion of FXR (Fig. 3B). LCA formate and LCA acetate, as
well as LCA, were weak FXR agonists. PXR was reported to
LCA acetate is not a potent agonist for other bile respond to high concentrations of LCA (10, 11). To exam-
acid receptors ine the effects of LCA derivatives on PXR, we transfected
The ligand binding domains of various nuclear recep- VDR or PXR expression vectors with a reporter contain-
tors were fused to the DNA binding domain of the yeast ing a CYP3A4 element, which can be activated by both re-

Adachi et al. A lithocholic acid-derived agonist for vitamin D receptor 49

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Fig. 2. LCA acetate is a potent VDR agonist. A: Concentration-dependent activation of VDR by LCA ace-
tate and its related compounds. HEK293 cells were cotransfected with CMX-VDR and CYP3A4-ER-6x3-tk-
LUC reporter and treated with several concentrations of LCA, LCA formate, LCA acetate, LCA acetate me-
thyl ester (LCA acetate ME), and 3-keto-cholanic acid (3-keto-LCA) for 16 h. B: Interactions of VDR with ste-
roid receptor coativator-1 (SRC-1) and nuclear receptor corepressor (N-CoR) induced by LCA acetate and
its related compounds. HEK293 cells were cotransfected with GAL4 control vector or GAL4-chimera vectors
for SRC-1 or N-CoR, in combination with VP16-VDR and MH100(UAS)x4-tk-LUC reporter, and were treated
with ethanol (EtOH) control, 10 &M LCA acetate, or related bile acids. C: Direct binding of LCA acetate to
VDR. In vitro translated VDR proteins were incubated with 1 nM [ 3H]1,25(OH)2D3 in the presence or ab-
sence of nonradioactive 10 nM 1,25(OH)2D3 or 50 &M or 200 &M bile acid derivatives. The values represent
means ' SD.

50 Journal of Lipid Research Volume 46, 2005

 the LCA derivatives (Fig. 3C). This effect may be derived
from endogenous receptors such as VDR. LCA acetate
and LCA formate strongly induced the activity of trans-
fected VDR, indicating that these LCA derivatives activate
VDR in HepG2 cells. The PXR agonist rifampicin did not
activate VDR. When HepG2 cells were cotransfected with
PXR, rifampicin and 3-keto-LCA increased the reporter
activity, but LCA acetate and LCA formate were not effec-
tive PXR ligands (Fig. 3C). These findings indicate that
LCA acetate is selective for VDR activation.

Effect of VDR mutation on LCA acetate response

To elucidate the structure-activity relationship of LCA
acetate and VDR, we examined the effects of LCA acetate
on the activation of several VDR mutants. Wild-type GAL4-
VDR and several alanine mutants, Y143A, S237A, S275A,
S278A, W286A, and H305A, were introduced into HEK293
cells and activation by LCA, LCA acetate, and 1,25(OH)2D3
were compared (Fig. 4A). According to the crystal struc-
ture of the VDR-1,25(OH)2D3 complex (22), Y143 and S278

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interact with the 3"-hydroxyl group of 1,25(OH)2D3,
S237 hydrogen bonds with the 1!-hydroxyl group, H305
coordinates the 25-hydroxyl group, and S275 and W286
mediate hydrophobic interaction with 1,25(OH)2D3. The
Y143A and W286A mutations inhibited activation by LCA
acetate, LCA, and 1,25(OH)2D3. The effect of S237A was
modest on LCA, LCA acetate, and 1,25(OH)2D3 activity.
Whereas S275A and S278A almost abolished the activity of
LCA, LCA acetate and 1,25(OH)2D3 still activated these
mutants. Interestingly, although H305A had significant ef-
fects on the activity of LCA and 1,25(OH)2D3, this muta-
tion had little effect on the activity of LCA acetate. Thus,
LCA acetate is similar to 3-keto-LCA in its ability to acti-
vate VDR-H305A (13). In a previous study, we found that
the VDR-S278V mutant is activated by 1,25(OH)2D3 but
not by LCA, whereas VDR-S237M can respond to LCA but
not to 1,25(OH)2D3 (14). We next examined the effects
of LCA acetate on these mutants (Fig. 4B). The S237M
mutation weakly affected the activity of LCA acetate as
Fig. 3. LCA acetate is a selective agonist for VDR. A: Receptor-
well as that of LCA. S278V drastically decreased LCA ace-
specific activation by LCA acetate. GAL4-chimera receptors for vari-
ous nuclear receptors were expressed with MH100(UAS)x4-tk-LUC tate activity. Based on these findings, we modeled LCA
reporter in HEK293 cells and assayed for activation by 30 &M LCA acetate in the VDR ligand binding domain ((165–215)
acetate. Luciferase activity of the reporter is expressed as fold in- (PDB #1DB1) using FlexX software (13). In contrast to
duction with compound treatment relative to vehicle control. the LCA docking model, the side chain of LCA acetate di-
PPAR, peroxisome proliferator-activated receptor. B: Concentra- rects to the "-turn site (Fig. 4C, left panel) (13). The oxy-
tion-dependent activation of farnesoid X receptor (FXR) by LCA
acetate and its related compounds. HEK293 cells were cotrans-
gen of the side chain carboxyl group and the carbonyl ox-
fected with CMX-FXR and IR-1x3-tk-LUC reporter and treated with ygen of the 3-O-acetyl acetate group nearly overlap with
several concentrations of LCA, LCA formate, LCA acetate, iso-LCA, the 3"-hydroxyl oxygen and 25-hydroxyl oxygen, respec-
ursocholanic acid, or chenodeoxycholic acid (CDCA). C: Compara- tively, of 1,25(OH)2D3 in the crystal structure of VDR-
tive response of VDR and pregnane X receptor (PXR) to LCA ace- 1,25(OH)2D3 (Fig. 4C, right panel). The proximity of
tate in liver HepG2 cells. HepG2 cells were transfected with CMX
these amino acid residues to hydrogen bond acceptors
control vector (+), CMX-VDR, or CMX-PXR with CYP3A4-ER-6x3-
tk-LUC and treated with vehicle control [ethanol (EtOH)], 30 &M within 1,25(OH)2D3 may be responsible for the strong ac-
LCA, LCA formate, LCA acetate, 3-keto-LCA, or rifampicin. The tivity of LCA acetate on VDR.
values represent means ' SD.
Induction of VDR target genes by LCA acetate in
ceptors (12). Liver-derived HepG2 cells were used for this intestinal cells
experiment, because PXR activation is cell type depen- VDR is highly expressed in intestinal mucosa cells and
dent (data not shown). In the absence of transfected re- regulates the expression of genes involved in calcium ho-
ceptors, the luciferase activity was increased by addition of meostasis and bile acid metabolism (1, 12, 23). We investi-

Adachi et al. A lithocholic acid-derived agonist for vitamin D receptor 51

 Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010
Fig. 4. Structure-function analysis of LCA acetate and VDR. A: Activation of VDR or its mutants by LCA ac-
etate. GAL4-VDR and alanine mutants (Y143A, S237A, S275A, S278A, W286A, and H305A) were cotrans-
fected with MH100(UAS)x4-tk-LUC reporter in HEK293 cells and treated with vehicle control [ethanol
(EtOH)] or the indicated concentrations of test compounds. WT, wild type. B: Dose response of VDR S237M
and S278V mutants for LCA acetate. HEK293 cells were cotransfected with GAL4-VDR, GAL4-VDR-S237M,
or GAL4-VDR-S278V with MH100(UAS)x4-tk-LUC reporter in HEK293 cells. The values represent means '
SD. C: Docking model of VDR interaction with LCA acetate. Left panel: The side chain carboxyl group is di-
rected to the "-turn site interacting with S278. The Connolly channel surface of the VDR ligand binding
pocket is shown in translucent gray. Right panel: Overlay of LCA acetate (yellow) and 1,25(OH)2D3 (gray)
accommodated in the VDR ligand binding pocket.

gated the ability of LCA acetate to activate endogenous the expression of VDR target genes, including CYP24A1,
VDR target genes in intestinal cells. Colon cancer-derived CYP3A4, CaT1, and E-cadherin, was examined. CYP24A1
SW480 cells were incubated with LCA, LCA acetate, and CaT1 are involved in calcium homeostasis and CYP3A4
1,25(OH)2D3, chenodeoxycholic acid, or rifampicin, and metabolizes LCA (1, 24). E-cadherin was reported to be

52 Journal of Lipid Research Volume 46, 2005

 Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010
Fig. 5. Induction of VDR target genes by LCA acetate in intestinal cells. A: LCA acetate induced VDR tar-
get genes more effectively than LCA in colon cancer-derived SW480 cells. Cells were treated with vehicle con-
trol [ethanol (EtOH)], 10 or 100 &M LCA or LCA acetate, 100 nM 1,25(OH)2D3, 100 &M chenodeoxy-
cholic acid (CDCA), or 30 &M rifampicin for 24 h. Quantitative real-time PCR from mRNA for CYP24A1,
CYP3A4, CaT1, E-cadherin, VDR, and retinoid X receptor ! (RXR!) was performed. B: LCA acetate in-
creased VDR target genes in mouse intestine more effectively than LCA. Mice were orally administrated with
200 mg/kg LCA or LCA acetate. Twelve hours after administration, total RNA was extracted from intestinal
mucosa and quantitative real-time PCR from mRNA for CYP24a1, Cyp3a11, and ileal bile acid binding pro-
tein (IBABP) was performed. The values represent means ' SD.

associated with cell growth inhibition induced by 1,25 acts as a potent VDR agonist in colon cancer cells. There
(OH)2D3 (25). As shown in Fig. 5A, 1,25(OH)2D3 in- were no changes in the expression of VDR and RXR!,
duced the expression of CYP24A1, CYP3A4, CaT1, and which forms a heterodimer with VDR, after treatment
E-cadherin. LCA acetate induced these VDR target genes with the VDR ligands. The FXR agonist chenodeoxycholic
more effectively than LCA, indicating that LCA acetate acid and the PXR agonist rifampicin were not able to in-

Adachi et al. A lithocholic acid-derived agonist for vitamin D receptor 53

duce the expression of these genes, although PXR was re- rivatives modified at position 3, such as LCA formate and
ported to be involved in CYP3A4 gene regulation (11). LCA acetate, have stronger activity than LCA (Figs. 1, 2).
The inability of PXR agonist to increase gene expression The docking model shown in Fig. 4 indicates that LCA ac-
is likely attributable to the fact that PXR is not expressed etate is accommodated in the VDR ligand binding pocket
in SW480 cells (data not shown). Next, we examined the in the same manner as 3-keto-LCA and that LCA acetate
expression of VDR target genes in vivo. Mice were orally can form hydrogen bonds with the same amino acid resi-
administered LCA or LCA acetate, and the expression of dues that coordinate 1,25(OH)2D3 binding. LCA acetate
intestinal Cyp24a1 and Cyp3a11 genes was evaluated. methyl ester has much stronger activity than LCA methyl
Both LCA and LCA acetate increased the mRNA expres- ester. This may be attributable to different docking modes
sion of Cyp24a1 and Cyp3a11 significantly, LCA acetate of these two LCA esters. LCA acetate and LCA can activate
being more effective than LCA (Fig. 5B). LCA and LCA VDR-S237M (Fig. 4B), which does not respond to 1,25
acetate did not induce the FXR target IBABP gene expres- (OH)2D3 (14). S237 is located in H3 and may mediate al-
sion (Fig. 5B). These data indicate that LCA acetate is a losteric communication with the cofactor interaction sur-
potent agonist for endogenous VDR in intestinal cells. face. These findings suggest the possibility that LCA ace-
tate induces an alternative conformation in VDR, which
LCA acetate induces the differentiation of monoblastic results in differential cofactor recruitment and selective
leukemia cells physiological function. Further study is required to eluci-
1,25(OH)2D3 is known as an inducer of myeloid leuke- date the structure-function relationship of VDR and LCA
mia differentiation (1). We examined the effects of LCA derivatives such as LCA acetate.
acetate on the growth and differentiation of human FXR is activated by both primary bile acids (chenode-

Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010

monoblastic leukemia THP-1 cells. 1,25(OH)2D3 inhibited oxycholic acid and cholic acid) and secondary bile acids
the proliferation of THP-1 cells and enhanced NBT-reduc- (LCA and deoxycholic acid) (15, 27, 28). In contrast, VDR
ing activity, a differentiation marker of myeloid leukemia responds to only LCA and its derivatives (12). In the previ-
cells, as reported previously (26). LCA acetate inhibited ous study, 6-keto-LCA was identified as a selective ligand
cell proliferation more effectively than LCA and 3-keto- for VDR, but its activity was very weak (12). The potent
LCA (Fig. 6A), and it induced NBT-reducing activity in VDR agonist LCA acetate activated FXR to low levels, simi-
the cells. In contrast, LCA and 3-keto-LCA were not able lar to the weak FXR agonist LCA, and much more weakly
to induce this activity even at concentrations that com- than chenodeoxycholic acid (Fig. 3B). In HepG2 cells,
pletely inhibit cell proliferation (Fig. 6B). Untreated THP-1 chenodeoxycholic acid induced the expression of the
cells have large nuclei with visible nucleoli and basophilic BSEP gene, which is an FXR target (9), but LCA and LCA
cytoplasmic staining. LCA acetate induced a concentra- acetate were not effective in its induction, although LCA
tion-dependent increase in the percentage of differenti- acetate increased the VDR target CYP24A1 expression
ated cells (Fig. 6C). In cells treated with LCA acetate, the (see supplementary data online). Although LCA and 3-keto-
nuclei were condensed, nucleoli were no longer apparent, LCA were agonists for PXR at high concentrations, LCA
and the cytoplasm appeared gray, indicating monocytic dif- acetate did not activate PXR (Fig. 3C). These data indi-
ferentiation (Fig. 6C). Esterase activity, a functional marker cate that LCA acetate is a selective agonist for VDR. Inter-
of monocytic differentiation, was also induced by LCA ace- estingly, although iso-LCA and ursocholanic acid were not
tate (Fig. 6D). LCA and 3-keto-LCA did not induce mor- able to activate VDR, they were more potent FXR agonists
phological and functional differentiation of THP-1 cells. than LCA. Recently, crystal structures of FXR and PXR
LCA acetate increased the expression of surface makers, have been reported (29–31). Mutational analysis of FXR
such as CD11b and CD14, as effectively as 1,25(OH)2D3 and PXR should be useful in elucidating the structure-
(Fig. 6E). Therefore, the VDR agonist LCA acetate is a po- function relationship of these LCA derivatives and in the
tent inducer of monocytic differentiation in THP-1 leuke- development of selective ligands for the bile acid recep-
mia cells. tors VDR, FXR, and PXR.
Vitamin D has been identified as a protective agent
against the development of colorectal cancer (32). Epide-
DISCUSSION miological analysis revealed that solar exposure, which re-
sults in vitamin D production in the skin, or vitamin D up-
In this study, we found that the modification of the 3!- take reduces the incidence of colorectal cancer (32).
hydroxyl group of LCA increases the transactivation activ- Protective effects of vitamin D in colon carcinogenesis are
ity and selectivity on VDR. Structure-function relationship mediated through its receptor VDR. VDR activation in-
analysis of the VDR-LCA interaction using several VDR duces the expression of genes involved in growth inhibi-
mutants shows that the side chain of LCA faces H12 of the tion, differentiation, and apoptosis (1, 33). In contrast to
receptor and 3-keto-LCA is directed toward the "-turn site vitamin D, the secondary bile acid LCA is considered to be
(13). As shown in Fig. 1, esterification of the side chain a promoter of colon carcinogenesis (34). LCA induces
carboxyl group of LCA abolished VDR activation. How- DNA strand breaks, forms DNA adducts, inhibits DNA re-
ever, in 3-keto-LCA, the corresponding esterifications had pair enzymes, and can promote colon cancer in rodent
only moderate effects. This may be ascribed to the oppos- models (35). CYP3A was reported to detoxify LCA to a
ing docking modes of LCA and 3-keto-LCA. The LCA de- nontoxic hyodeoxycholic acid and is a VDR target gene

54 Journal of Lipid Research Volume 46, 2005

 Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010
Fig. 6. Effects of LCA acetate on growth and differentiation of human myeloid leukemia THP-1 cells. A:
Growth inhibition of THP-1 cells by 1,25(OH)2D3 and LCA acetate. B: Induction of nitroblue tetrazolium
(NBT)-reducing activity in THP-1 cells by 1,25(OH)2D3 and LCA acetate. THP-1 cells were treated with
1,25(OH)2D3, LCA acetate, LCA, or 3-keto-LCA for 4 days. C: LCA acetate induces the morphological differ-
entiation of THP-1 cells. Cells were treated with LCA acetate, LCA, or 3-keto-LCA for 6 days, and differenti-
ated cells shown in the left panel were counted. D: LCA acetate induces monocyte-specific esterase activity.
Cells were treated with LCA acetate, LCA, or 3-keto-LCA for 6 days. E: LCA acetate increases the expression
of CD11b and CD14 surface antigens. Cells were treated with LCA or 1,25(OH)2D3 for 4 days and CD11b
and CD14 expression was examined using monoclonal antibodies and flow cytometry. EtOH, ethanol. The
values represent means ' SD.

(11, 36). By binding to VDR, 1,25(OH)2D3 and LCA in- successful because of their hypercalcemic activities (38).
duce CYP3A expression in the intestine. VDR may serve as Structure-function analysis of vitamin D analogs suggests
a sensor for LCA and function to protect intestinal mu- that 1,25(OH)2D3 and its analogs also induce nonge-
cosa from its harmful effects. Recently, a significant corre- nomic VDR actions and that adverse effects are at least
lation between a VDR polymorphism and colorectal can- partly attributable to nongenomic mechanisms (5, 39).
cer risk was reported in a Singapore Chinese population Ligand-dependent dissociation of nongenomic from ge-
(37). These findings suggest that VDR functions as an an- nomic activity was reported for the estrogen receptor
ticancer factor and indicate that it is a promising molecu- (40). An estrogen receptor ligand, pyrazole, induced the
lar target for chemoprevention against colorectal cancer. transactivation of an estrogen receptor target gene but
Clinical trials of vitamin D and its analogs have been un- had weak nongenomic activity, whereas another ligand, es-

Adachi et al. A lithocholic acid-derived agonist for vitamin D receptor 55

tren, induced strong nongenomic action of the estrogen anisms of LCA acetate- and 1,25(OH)2D3-induced leuke-
receptor without altering gene expression. There has mia cell differentiation.
been no reported physiological correlation between bile
acids and intestinal calcium absorption, suggesting that The authors thank E. Kaneko of Osaka University for assistance
LCA or its derivatives may relatively induce genomic ac- in animal experiments, Dr. David J. Mangelsdorf of the Howard
tions in the intestine, such as bile acid metabolism and Hughes Medical Institute, University of Texas Southwestern
cell growth control, without inducing hypercalcemia. LCA Medical Center (Dallas, TX), for providing plasmids, Dr.
acetate induced VDR target genes via genomic action, in- Shigeyuki Uno of Nihon University School of Medicine for
cluding the LCA-detoxifying enzyme CYP3A, in colon can- technical assistance, and members of the Makishima, Shimo-
cer cells and mouse intestines more effectively than LCA mura, Honma, and Yamada laboratories for helpful comments.
(Fig. 4). Nongenomic action of bile acids and derivatives K.K. is a Research Fellow of the Japan Society for the Promo-
should be further investigated. The development of more tion of Science. This work was supported in part by grants from
potent LCA derivatives that are nontoxic and less hyper- the Ministry of Education, Culture, Sports, Science, and Tech-
calcemic should be useful for chemoprevention against nology, Japan (M.M.), the Ministry of Health, Labor, and Wel-
colon carcinogenesis. fare, Japan (M.M.), the Japan Research Foundation for Clinical
1,25(OH)2D3 was found to induce the differentiation Pharmacology (M.M.), and the Yasuda Medical Research Foun-
dation (M.M.) and was partly funded by Kyowa Hakko Co.,
of mouse myeloid leukemia M1 cells more than 20 years
Ltd., Japan.
ago (41). Treatment with 1,25(OH)2D3 or 1!-hydroxyvi-
tamin D3, which is rapidly metabolized to 1,25(OH)2D3,
was reported to prolong survival in mice inoculated with

Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010

REFERENCES
M1 leukemia cells (3). The differentiation-inducing ef-
fects of 1,25(OH)2D3 were also demonstrated in human 1. Haussler, M. R., G. K. Whitfield, C. A. Haussler, J. C. Hsieh, P. D.
leukemia cells (42, 43). However, the molecular mecha- Thompson, S. H. Selznick, C. E. Dominguez, and P. W. Jurutka.
nisms of differentiation induced by 1,25(OH)2D3 have 1998. The nuclear vitamin D receptor: biological and molecular
regulatory properties revealed. J. Bone Miner. Res. 13: 325–349.
not been elucidated. We found that the potent VDR ago- 2. Brown, A. J., A. Dusso, and E. Slatopolsky. 1999. Vitamin D. Am. J.
nist LCA acetate was able to induce the differentiation of Physiol. Renal Physiol. 277: F157–F175.
human monoblastic leukemia THP-1 cells at concentra- 3. Honma, Y., M. Hozumi, E. Abe, K. Konno, M. Fukushima, S. Hata,
Y. Nishii, H. F. DeLuca, and T. Suda. 1983. 1!,25-Dihydroxyvitamin
tions that induce VDR activation (Fig. 6). LCA and 3-keto-
D3 and 1!-hydroxyvitamin D3 prolong survival time of mice inoc-
LCA inhibited proliferation but did not induce differenti- ulated with myeloid leukemia cells. Proc. Natl. Acad. Sci. USA. 80:
ation. The growth-inhibiting activity of these bile acids 201–204.
may be attributable to their cytotoxic effects. Zimber et al. 4. Kumagai, T., J. O’Kelly, J. W. Said, and H. P. Koeffler. 2003. Vita-
min D2 analog 19-nor-1,25-dihydroxyvitamin D2: antitumor activ-
(44) reported that bile acids, including deoxycholic acid, ity against leukemia, myeloma, and colon cancer cells. J. Natl. Can-
chenodeoxycholic acid, and LCA, induced the differentia- cer Inst. 95: 896–905.
tion of human promyelocytic leukemia HL-60 cells. We 5. Huhtakangas, J. A., C. J. Olivera, J. E. Bishop, L. P. Zanello, and
A. W. Norman. 2004. The vitamin D receptor is present in caveo-
did not observe differentiation-inducing activity of these lae-enriched plasma membranes and binds 1!,25(OH)2-vitamin
bile acids in HL-60 cells (data not shown). This is proba- D3 in vivo and in vitro. Mol. Endocrinol. 18: 2660–2671.
bly because of differences between subclones of leukemia 6. Yamada, S., M. Shimizu, and K. Yamamoto. 2003. Structure-func-
tion relationships of vitamin D including ligand recognition by the
cell lines, which could affect sensitivity to the compounds. vitamin D receptor. Med. Res. Rev. 23: 89–115.
Regardless, LCA acetate did induce differentiation mark- 7. Takeyama, K., Y. Masuhiro, H. Fuse, H. Endoh, A. Murayama, S. Ki-
ers in HL-60 cells (data not shown). These findings indi- tanaka, M. Suzawa, J. Yanagisawa, and S. Kato. 1999. Selective in-
teraction of vitamin D receptor with transcriptional coactivators by
cate that LCA acetate is a more effective inducer of leuke- a vitamin D analog. Mol. Cell. Biol. 19: 1049–1055.
mia differentiation than bile acids such as LCA and 8. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schutz,
chenodeoxycholic acid. Zimber et al. (45) reported that K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, and
LCA alone did not induce the differentiation of THP-1 R. M. Evans. 1995. The nuclear receptor superfamily: the second
decade. Cell. 83: 835–839.
cells but that it enhanced the response to all-trans-retinoic 9. Lu, T. T., J. J. Repa, and D. J. Mangelsdorf. 2001. Orphan nuclear
acid, which is a potent differentiation inducer of myeloid receptors as eLiXiRs and FiXeRs of sterol metabolism. J. Biol.
leukemia cells. The combinational effects of LCA acetate Chem. 276: 37735–37738.
10. Staudinger, J. L., B. Goodwin, S. A. Jones, D. Hawkins-Brown, K. I.
and other differentiation inducers are now under investi- MacKenzie, A. LaTour, Y. Liu, C. D. Klaassen, K. K. Brown, J. Rein-
gation. The protein kinase C inhibitor sphingosine de- hard, T. M. Willson, B. H. Koller, and S. A. Kliewer. 2001. The nu-
creased the NBT-reducing activity induced by deoxycholic clear receptor PXR is a lithocholic acid sensor that protects
against liver toxicity. Proc. Natl. Acad. Sci. USA. 98: 3369–3374.
acid and chenodeoxycholic acid in HL-60 cells but did not 11. Xie, W., A. Radominska-Pandya, Y. Shi, C. M. Simon, M. C. Nelson,
alter the response to LCA (44), suggesting that the effect E. S. Ong, D. J. Waxman, and R. M. Evans. 2001. An essential role
of LCA is mediated by mechanisms distinct from those for nuclear receptors SXR/PXR in detoxification of cholestatic
bile acids. Proc. Natl. Acad. Sci. USA. 98: 3375–3380.
used by deoxycholic acid and chenodeoxycholic acid. Ex- 12. Makishima, M., T. T. Lu, W. Xie, G. K. Whitfield, H. Domoto, R. M.
pression of some VDR target genes was increased in THP-1 Evans, M. R. Haussler, and D. J. Mangelsdorf. 2002. Vitamin D re-
cells after treatment with LCA acetate (data not shown). ceptor as an intestinal bile acid sensor. Science. 296: 1313–1316.
The data indicate that LCA acetate functions as a VDR ag- 13. Choi, M., K. Yamamoto, T. Itoh, M. Makishima, D. J. Mangelsdorf,
D. Moras, H. F. DeLuca, and S. Yamada. 2003. Interaction between
onist in leukemia cells and induces cell differentiation. vitamin D receptor and vitamin D ligands: two-dimensional ala-
Further studies are required to elucidate the precise mech- nine scanning mutational analysis. Chem. Biol. 10: 261–270.

56 Journal of Lipid Research Volume 46, 2005

14. Adachi, R., A. I. Shulman, K. Yamamoto, I. Shimomura, S. Yamada, tural analysis of the nuclear bile acid receptor FXR. Mol. Cell. 11:
D. J. Mangelsdorf, and M. Makishima. 2004. Structural determi- 1079–1092.
nants for vitamin D receptor response to endocrine and xenobi- 30. Mi, L. Z., S. Devarakonda, J. M. Harp, Q. Han, R. Pellicciari, T. M.
otic signals. Mol. Endocrinol. 18: 43–52. Willson, S. Khorasanizadeh, and F. Rastinejad. 2003. Structural ba-
15. Makishima, M., A. Y. Okamoto, J. J. Repa, H. Tu, R. M. Learned, A. sis for bile acid binding and activation of the nuclear receptor
Luk, M. V. Hull, K. D. Lustig, D. J. Mangelsdorf, and B. Shan. 1999. FXR. Mol. Cell. 11: 1093–1100.
Identification of a nuclear receptor for bile acids. Science. 284: 31. Watkins, R. E., G. B. Wisely, L. B. Moore, J. L. Collins, M. H. Lam-
1362–1365. bert, S. P. Williams, T. M. Willson, S. A. Kliewer, and M. R. Redinbo.
16. Makishima, M., J. Okabe-Kado, and Y. Honma. 1998. Growth inhibi- 2001. The human nuclear xenobiotic receptor PXR: structural de-
tion and differentiation induction in human monoblastic leu- terminants of directed promiscuity. Science. 292: 2329–2333.
kaemia cells by 1!-hydroxyvitamin D derivatives and their enhance- 32. Garland, C. F., F. C. Garland, and E. D. Gorham. 1999. Calcium
ment by combination with hydroxyurea. Br. J. Cancer. 77: 33–39. and vitamin D. Their potential roles in colon and breast cancer
17. Yamamoto, K., H. Masuno, M. Choi, K. Nakashima, T. Taga, H. Oo- prevention. Ann. N.Y. Acad. Sci. 889: 107–119.
izumi, K. Umesono, W. Sicinska, J. VanHooke, H. F. DeLuca, and 33. Palmer, H. G., M. Sanchez-Carbayo, P. Ordonez-Moran, M. J. Lar-
S. Yamada. 2000. Three-dimensional modeling of and ligand dock- riba, C. Cordon-Cardo, and A. Munoz. 2003. Genetic signatures of
ing to vitamin D receptor ligand binding domain. Proc. Natl. Acad. differentiation induced by 1!,25-dihydroxyvitamin D3 in human
Sci. USA. 97: 1467–1472. colon cancer cells. Cancer Res. 63: 7799–7806.
18. Rarey, M., B. Kramer, T. Lengauer, and G. Klebe. 1996. A fast flexi- 34. Nagengast, F. M., M. J. Grubben, and I. P. van Munster. 1995. Role
ble docking method using an incremental construction algorithm. of bile acids in colorectal carcinogenesis. Eur. J. Cancer. 31A: 1067–
J. Mol. Biol. 261: 470–489. 1070.
19. Kaneko, E., M. Matsuda, Y. Yamada, Y. Tachibana, I. Shimomura, 35. Narisawa, T., N. E. Magadia, J. H. Weisburger, and E. L. Wynder.
and M. Makishima. 2003. Induction of intestinal ATP-binding cas- 1974. Promoting effect of bile acids on colon carcinogenesis after
sette transporters by a phytosterol-derived liver X receptor agonist. intrarectal instillation of N-methyl-N)-nitrosoguanidine in rats. J.
J. Biol. Chem. 278: 36091–36098. Natl. Cancer Inst. 53: 1093–1097.
20. Makishima, M., K. Shudo, and Y. Honma. 1999. Greater synergism 36. Thummel, K. E., C. Brimer, K. Yasuda, J. Thottassery, T. Senn, Y.
of retinoic acid receptor (RAR) agonists with vitamin D3 than that Lin, H. Ishizuka, E. Kharasch, J. Schuetz, and E. Schuetz. 2001.

Downloaded from www.jlr.org at Kuopio University Library on July 15, 2010

of retinoid X receptor (RXR) agonists with regard to growth inhi- Transcriptional control of intestinal cytochrome P-4503A by 1!,25-
bition and differentiation induction in monoblastic leukemia cells. dihydroxy vitamin D3. Mol. Pharmacol. 60: 1399–1406.
Biochem. Pharmacol. 57: 521–529. 37. Wong, H-L., A. Seow, K. Arakawa, H-P. Lee, M. C. Yu, and S. A. In-
21. Glass, C. K., and M. G. Rosenfeld. 2000. The coregulator exchange gles. 2003. Vitamin D receptor start codon polymorphism and co-
in transcriptional functions of nuclear receptors. Genes Dev. 14: lorectal cancer risk: effect modification by dietary calcium and fat
121–141. in Singapore Chinese. Carcinogenesis. 24: 1091–1095.
22. Rochel, N., J. M. Wurtz, A. Mitschler, B. Klaholz, and D. Moras. 38. Guyton, K. Z., T. W. Kensler, and G. H. Posner. 2003. Vitamin D
2000. The crystal structure of the nuclear receptor for vitamin D and vitamin D analogs as cancer chemopreventive agents. Nutr.
bound to its natural ligand. Mol. Cell. 5: 173–179. Rev. 61: 227–238.
23. Berger, U., P. Wilson, R. A. McClelland, K. Colston, M. R. Haussler, 39. Zanello, L. P., and A. W. Norman. 2004. Rapid modulation of os-
J. W. Pike, and R. C. Coombes. 1988. Immunocytochemical detec- teoblast ion channel responses by 1!,25(OH)2-vitamin D3 re-
tion of 1,25-dihydroxyvitamin D receptors in normal human tis- quires the presence of a functional vitamin D nuclear receptor.
sues. J. Clin. Endocrinol. Metab. 67: 607–613. Proc. Natl. Acad. Sci. USA. 101: 1589–1594.
24. Van Cromphaut, S. J., M. Dewerchin, J. G. J. Hoenderop, I. 40. Kousteni, S., T. Bellido, L. I. Plotkin, C. A. O’Brien, D. L. Boden-
Stockmans, E. Van Herck, S. Kato, R. J. M. Bindels, D. Collen, P. ner, L. Han, K. Han, G. B. DiGregorio, J. A. Katzenellenbogen,
Carmeliet, R. Bouillon, and G. Carmeliet. 2001. Duodenal cal- B. S. Katzenellenbogen, P. K. Roberson, R. S. Weinstein, R. L. Jilka,
cium absorption in vitamin D receptor-knockout mice: func- and S. C. Manolagas. 2001. Nongenotropic, sex-nonspecific signal-
tional and molecular aspects. Proc. Natl. Acad. Sci. USA. 98: 13324– ing through the estrogen or androgen receptors: dissociation from
13329. transcriptional activity. Cell. 104: 719–730.
25. Palmer, H. G., J. M. Gonzalez-Sancho, J. Espada, M. T. Berciano, 41. Abe, E., C. Miyaura, H. Sakagami, M. Takeda, K. Konno, T. Yamazaki,
I. Puig, J. Baulida, M. Quintanilla, A. Cano, A. G. de Herreros, M. S. Yoshiki, and T. Suda. 1981. Differentiation of mouse myeloid
Lafarga, and A. Munoz. 2001. Vitamin D3 promotes the differen- leukemia cells induced by 1!,25-dihydroxyvitamin D3. Proc. Natl.
tiation of colon carcinoma cells by the induction of E-cadherin Acad. Sci. USA. 78: 4990–4994.
and the inhibition of "-catenin signaling. J. Cell Biol. 154: 369– 42. Miyaura, C., E. Abe, T. Kuribayashi, H. Tanaka, K. Konno, Y. Nishii,
388. and T. Suda. 1981. 1!,25-Dihydroxyvitamin D3 induces differentia-
26. Makishima, M., Y. Kanatani, Y. Yamamoto-Yamaguchi, and Y. tion of human myeloid leukemia cells. Biochem. Biophys. Res. Com-
Honma. 1996. Enhancement of activity of 1!,25-dihydroxyvitamin mun. 102: 937–943.
D3 for growth inhibition and differentiation induction of human 43. Mangelsdorf, D. J., H. P. Koeffler, C. A. Donaldson, J. W. Pike, and
myelomonocytic leukemia cells by tretinoin tocoferil, an alpha- M. R. Haussler. 1984. 1,25-Dihydroxyvitamin D3-induced differen-
tocopherol ester of all-trans retinoic acid. Blood. 87: 3384–3394. tiation in a human promyelocytic leukemia cell line (HL-60): re-
27. Parks, D. J., S. G. Blanchard, R. K. Bledsoe, G. Chandra, T. G. Con- ceptor-mediated maturation to macrophage-like cells. J. Cell Biol.
sler, S. A. Kliewer, J. B. Stimmel, T. M. Willson, A. M. Zavacki, D. D. 98: 391–398.
Moore, and J. M. Lehmann. 1999. Bile acids: natural ligands for an 44. Zimber, A., A. Chedeville, C. Gespach, and J-P. Abita. 1994. Inhibi-
orphan nuclear receptor. Science. 284: 1365–1368. tion of proliferation and induction of monocytic differentiation in
28. Wang, H., J. Chen, K. Hollister, L. C. Sowers, and B. M. Forman. HL60 human promyelocytic leukemia cells treated with bile acids
1999. Endogenous bile acids are ligands for the nuclear receptor in vitro. Int. J. Cancer. 59: 71–77.
FXR/BAR. Mol. Cell. 3: 543–553. 45. Zimber, A., A. Chedeville, J-P. Abita, V. Barbu, and C. Gespach.
29. Downes, M., M. A. Verdecia, A. J. Roecker, R. Hughes, J. B. Hoge- 2000. Functional interactions between bile acids, all-trans retinoic
nesch, H. R. Kast-Woelbern, M. E. Bowman, J. L. Ferrer, A. M. An- acid, and 1,25-dihydroxy-vitamin D3 on monocytic differentiation
isfeld, P. A. Edwards, J. M. Rosenfeld, J. G. Alvarez, J. P. Noel, K. C. and myeloblastin gene down-regulation in HL-60 and THP-1 hu-
Nicolaou, and R. M. Evans. 2003. A chemical, genetic, and struc- man leukemia cells. Cancer Res. 60: 672–678.

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