The hormonal form of vitamin D, 1␣,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], transiently stimul... more The hormonal form of vitamin D, 1␣,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], transiently stimulates the transcription of the c-fos proto-oncogene in osteoblastic cells. We have identified and characterized a vitamin D response element (VDRE) in the promoter of c-fos. The 1,25-(OH) 2 D 3 -responsive region was delineated between residues ؊178 and ؊144 upstream of the c-fos transcription start site. A mutation that inhibited binding to the sequence concomitantly abolished 1,25-(OH) 2 D 3 -induced transcriptional responsiveness; similarly, cloning of the site upstream of a heterologous promoter conferred copy-number-dependent vitamin D responsiveness to a reporter gene, demonstrating that we have identified a functional response element. The structure of the c-fos VDRE was found to be unusual. Mutational analysis revealed that the c-fos VDRE does not conform to the direct repeat configuration in which hexameric core-binding sites are spaced by a few nucleotide residues. In contrast, the entire 36-bp sequence was essential for binding. We identified the vitamin D receptor and the retinoid X receptor ␣ as components of the complex that bound the c-fos VDRE. However, our results also show that a putative CCAAT-binding transcription factor/nuclear factor 1 (CTF/NF-1) family member bound the response element in conjunction with the nuclear hormone receptors. The expression of this CTF/NF-1 family member appeared restricted to bone cells. These data hint at new molecular mechanisms of action for vitamin D.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 12, 2015
To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive... more To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive elements (VDREs) at -7/-10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)-retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25-dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate-limiting enzyme in serotonin synthesis, was induced 2.2-fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8-fold in rat serotonergic RN46A-B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1-fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis-regulatory module; CRM) at -28 kb that 1,25D-dependently docked VDR, RXR, C/EBP...
In a closed endocrine loop, 1,25-dihydroxyvitamin D3 (1,25D) induces the expression of fibroblast... more In a closed endocrine loop, 1,25-dihydroxyvitamin D3 (1,25D) induces the expression of fibroblast growth factor 23 (FGF23) in bone, with the phosphaturic peptide in turn acting at kidney to feedback repress CYP27B1 and induce CYP24A1 to limit the levels of 1,25D. In 3T3-L1 differentiated adipocytes, 1,25D represses FGF23 and leptin expression and induces C/EBPβ, but does not affect leptin receptor transcription. Conversely, in UMR-106 osteoblast-like cells, FGF23 mRNA concentrations are upregulated by 1,25D, an effect that is blunted by lysophosphatidic acid, a cell-surface acting ligand. Progressive truncation of the mouse FGF23 proximal promoter linked in luciferase reporter constructs reveals a 1,25D-responsive region between -400 and -200 bp. A 0.6 kb fragment of the mouse FGF23 promoter, linked in a reporter construct, responds to 1,25D with a fourfold enhancement of transcription in transfected K562 cells. Mutation of either an ETS1 site at -346 bp, or an adjacent candidate vitamin D receptor (VDR)/Nurr1-element, in the 0.6 kb reporter construct reduces the transcriptional activity elicited by 1,25D to a level that is not significantly different from a minimal promoter. This composite ETS1-VDR/Nurr1 cis-element may function as a switch between induction (osteocytes) and repression (adipocytes) of FGF23, depending on the cellular setting of transcription factors. Moreover, experiments demonstrate that a 1 kb mouse FGF23 promoter-reporter construct, transfected into MC3T3-E1 osteoblast-like cells, responds to a high calcium challenge with a statistically significant 1.7- to 2.0-fold enhancement of transcription. Thus, the FGF23 proximal promoter harbors cis elements that drive responsiveness to 1,25D and calcium, agents that induce FGF23 to curtail the pathologic consequences of their excess.
The Journal of Steroid Biochemistry and Molecular Biology, 2010
The nuclear vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D 3 (1,25D), its high affinity r... more The nuclear vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D 3 (1,25D), its high affinity renal endocrine ligand, to signal intestinal calcium and phosphate absorption plus bone remodeling, generating a mineralized skeleton free of rickets/osteomalacia with a reduced risk of osteoporotic fractures. 1,25D/VDR signaling regulates the expression of TRPV6, BGP, SPP1, LRP5, RANKL and OPG, while achieving feedback control of mineral ions to prevent age-related ectopic calcification by governing CYP24A1, PTH, FGF23, PHEX, and klotho transcription. Vitamin D also elicits numerous intracrine actions when circulating 25-hydroxyvitamin D 3 , the metabolite reflecting vitamin D status, is converted to 1,25D locally by extrarenal CYP27B1, and binds VDR to promote immunoregulation, antimicrobial defense, xenobiotic detoxification, antiinflammatory/anticancer actions and cardiovascular benefits. VDR also affects Wnt signaling through direct interaction with β-catenin, ligand-dependently blunting β-catenin mediated transcription in colon cancer cells to attenuate growth, while potentiating β-catenin signaling via VDR ligand-independent mechanisms in osteoblasts and keratinocytes to function osteogenically and as a pro-hair cycling receptor, respectively. Finally, VDR also drives the mammalian hair cycle in conjunction with the hairless corepressor by repressing SOSTDC1, S100A8/S100A9, and PTHrP. Hair provides a shield against UV-induced skin damage and cancer in terrestrial mammals, illuminating another function of VDR that facilitates healthful aging.
Proceedings of the National Academy of Sciences, 1991
The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino ... more The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC}-namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the et, (, and y isozymes of PKC, and VDR proved to be an effective substrate for PKC-,B, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-fi, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced,
The human vitamin D receptor (hVDR) is a ligandregulated transcription factor that mediates the a... more The human vitamin D receptor (hVDR) is a ligandregulated transcription factor that mediates the actions of the 1,25-dihydroxyvitamin D 3 hormone to effect bone mineral homeostasis. Employing mutational analysis, we characterized Arg-18/Arg-22, hVDR residues immediately N-terminal of the first DNA binding zinc finger, as vital for contact with human basal transcription factor IIB (TFIIB). Alteration of either of these basic amino acids to alanine also compromised hVDR transcriptional activity. In contrast, an artificial hVDR truncation devoid of the first 12 residues displayed both enhanced interaction with TFIIB and transactivation. Similarly, a natural polymorphic variant of hVDR, termed F/M4 (missing a FokI restriction site), which lacks only the first three amino acids (including Glu-2), interacted more efficiently with TFIIB and also possessed elevated transcriptional activity compared with the full-length (f/M1) receptor. It is concluded that the functioning of positively charged Arg-18/Arg-22 as part of an hVDR docking site for TFIIB is influenced by the composition of the adjacent polymorphic N terminus. Increased transactivation by the F/M4 neomorphic hVDR is hypothesized to result from its demonstrated enhanced association with TFIIB. This proposal is supported by the observed conversion of f/M1 hVDR activity to that of F/M4 hVDR, either by overexpression of TFIIB or neutralization of the acidic Glu-2 by replacement with alanine in f/M1 hVDR. Because the f VDR genotype has been associated with lower bone mineral density in diverse populations, one factor contributing to a genetic predisposition to osteoporosis may be the F/f polymor-phism that dictates VDR isoforms with differential TFIIB interaction. (Molecular Endocrinology 14: 401-420, 2000)
The activity of β-catenin, commonly dysregulated in human colon cancers, is inhibited by the vita... more The activity of β-catenin, commonly dysregulated in human colon cancers, is inhibited by the vitamin D receptor (VDR), and this mechanism is postulated to explain the putative anti-cancer activity of vitamin D metabolites in the colon. We investigated the effect of a common FokI restriction site polymorphism (F/f) in the human VDR gene as well as the effect of antitumorigenic 1,25-dihydroxyvitamin D 3 (1,25D) and pro-tumorigenic lithocholic acid (LCA) VDR ligands on β-catenin transcriptional activity. Furthermore, the influence of a major regulatory protein of β-catenin, the APC tumor suppressor gene, on VDR-dependent inhibition of β-catenin activity was examined. We report herein that β-catenin-mediated transcription is most effectively suppressed by the VDR FokI variant F/M4 when 1,25D is limiting. Using Caco-2 colorectal cancer cells, it was observed that VDR ligands, 1,25D and LCA, both suppress β-catenin transcriptional activity, though 1,25D exhibited significantly greater inhibition. Moreover, 1,25D, but not LCA, suppressed endogenous expression of the β-catenin target gene DKK-4 independent of VDR DNA-binding activity. These results support β-catenin sequestration away from endogenous gene targets by 1,25D-VDR. This activity is most efficiently mediated by the FokI gene variant F/M4, a VDR allele previously associated with protection against colorectal cancer. Interestingly, we found the inhibition of β-catenin activity by 1,25D-VDR was significantly enhanced by wildtype APC. These results reveal a previously unrecognized role for 1,25D-VDR in APC/β-catenin cross-talk. Collectively, these findings strengthen evidence favoring a direct effect on the Wnt-signaling molecule β-catenin as one anti-cancer target of 1,25D-VDR action in the colorectum.
The functional significance of two unlinked human vitamin D receptor (hVDR) gene polymorphisms wa... more The functional significance of two unlinked human vitamin D receptor (hVDR) gene polymorphisms was evaluated in twenty human fibroblast cell lines. Genotypes at both a Fok I restriction site (F/f ) in exon II and a singlet (A) repeat in exon IX (L/S) were determined, and relative transcription activities of endogenous hVDR proteins were measured using a transfected, 1,25-dihydroxyvitamin D 3 -responsive reporter gene. Observed activities ranged from 2 -100-fold induction by hormone, with higher activity being displayed by the F and the L biallelic forms. Only when genotypes at both sites were considered simultaneously did statistically significant differences emerge. Moreover, the correlation between hVDR activity and genotype segregated further into clearly defined high and low activity groups with similar genotypic distributions. These results not only demonstrate functional relevance for both the F/f and L/S common polymorphisms in hVDR, but also provide novel evidence for a third genetic variable impacting receptor potency.
The nuclear vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D 3 (1,25D) to... more The nuclear vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D 3 (1,25D) to regulate gene transcription. Recently, the secondary bile acid, lithocholate, was recognized as a novel VDR ligand. Using reporter gene and mammalian two-hybrid systems, immunoblotting, competitive ligand displacement, and quantitative real time PCR, we identified curcumin (CM), a turmeric-derived bioactive polyphenol, as a likely additional novel ligand for VDR. CM (10 −5 M) activated transcription of a luciferase plasmid containing the distal vitamin D responsive element from the human CYP3A4 gene at levels comparable to 1,25D (10 −8 M) in transfected human colon cancer cells (Caco-2). While CM also activated transcription via a retinoid X receptor (RXR) responsive element, activation of the glucocorticoid receptor (GR) by CM was negligible. Competition binding assays with radiolabeled 1,25D confirmed that CM binds directly to VDR. In mammalian two hybrid assays employing transfected Caco-2 cells, CM (10 −5 M) increased the ability of VDR to recruit its heterodimeric partner, RXR, and steroid receptor coactivator-1 (SRC-1). Real time PCR studies revealed that CM-bound VDR can activate VDR target genes CYP3A4, CYP24, p21, and TRPV6 in Caco-2 cells. Numerous studies have shown chemoprotection by CM against intestinal cancers via a variety of mechanisms. Small intestine and colon are important VDRexpressing tissues where 1,25D has known anticancer properties that may, in part, be elicited by activation of CYP-mediated xenobiotic detoxification and/or up-regulation of the tumor suppressor p21. Our results suggest the novel hypothesis that nutritionally-derived CM facilitates chemoprevention via direct binding to, and activation of, VDR.
The vitamin D receptor (VDR), but not its hormonal ligand, 1,25-dihydroxyvitamin D3 (1,25D), is r... more The vitamin D receptor (VDR), but not its hormonal ligand, 1,25-dihydroxyvitamin D3 (1,25D), is required for the progression of the mammalian hair cycle. We studied three genes relevant to hair cycle signaling, DKKL1 (Soggy), SOSTDC1 (Wise), and HR (Hairless), to determine whether their expression is regulated by VDR and/or its 1,25D ligand. DKKL1 mRNA was repressed 49-72% by 1,25D in primary human and CCD-1106 KERTr keratinocytes; a functional vitamin D responsive element (VDRE) was identified at -9590 bp in murine Soggy. Similarly, SOSTDC1 mRNA was repressed 41-59% by 1,25D in KERTr and primary human keratinocytes; a functional VDRE was located at -6215 bp in human Wise. In contrast, HR mRNA was upregulated 1.56- to 2.77-fold by 1,25D in primary human and KERTr keratinocytes; a VDRE (TGGTGAgtgAGGACA) consisting of an imperfect direct repeat separated by three nucleotides (DR3) was identified at -7269 bp in the human Hairless gene that mediated dramatic induction, even in the absence of 1,25D ligand. In parallel, a DR4 thyroid hormone responsive element, TGGTGAggccAGGACA, was identified at +1304 bp in the human HR gene that conferred tri-iodothyronine (T3)-independent transcriptional activation. Because the thyroid hormone receptor controls HR expression in the CNS, whereas VDR functions in concert with the HR corepressor specifically in skin, a model is proposed wherein unliganded VDR upregulates the expression of HR, the gene product of which acts as a downstream comodulator to feedback-repress DKKL1 and SOSTDC1, resulting in integration of bone morphogenic protein and Wnt signaling to drive the mammalian hair cycle and/or influencing epidermal function.
Six patients with primary hyperparathyroidism (PHPT) and one with squamous cell carcinoma of the ... more Six patients with primary hyperparathyroidism (PHPT) and one with squamous cell carcinoma of the esophagus with parathyroid hormone excess received disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) at a daily dose of 20 mg/kg orally. During treatment, the decrease in urinary calcium, total urinary hydroxyproline, and fasting urinary calcium suggested an inhibition of bone resorption. Serum calcium intestinal absorption of calcium and urinary cyclic adenosine monophosphate (cAMP) did not change significantly. This preliminary study indicates a possible role of diphosphonates in the management of inoperable cases of primary hyperparathyroidism or pseudohyperparathyroidism.
Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) action are the poss... more Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) action are the possible signi®cance of VDR homodimers and the functional role of receptor phosphorylation. To address these issues, milligram quantities of baculovirus-expressed hVDR were puri®ed to 97% homogeneity, and then tested for binding to the rat osteocalcin vitamin D responsive element (VDRE) via electrophoretic mobility shift and half-site competition assays in the presence or absence of a CV-1 nuclear extract containing retinoid X receptor (RXR). Methylation interference analysis revealed that both the hVDR homodimer and the VDR±RXR heterodimer display similar patterns of VDRE G-base protection. However, in competition studies, the relative dissociation of the homodimeric hVDR complex from the VDRE was extremely rapid (t 1/2 < 30 s) compared to the dissociation of the heteromeric complex (t 1/2 > 5 min), thus illustrating the relative instability and low af®nity of homodimeric VDR binding to DNA. These results indicate that VDR±RXR heterodimers are the preferred VDRE binding species. Further, two dimensional gel electrophoresis of hVDR demonstrated several isoelectric forms of the receptor, suggesting that it is subject to multiple phosphorylation events. In vitro kinase assays con®rmed that puri®ed hVDR is an ef®cient substrate for protein kinases A and Cb, as well as casein kinase II. In vivo studies of the expressed receptor in intact cells, namely baculovirus vector infected Sf9 insect cells and transfected mammalian COS-7 cells, demonstrated that hVDR was phosphorylated in a hormone-enhanced fashion. Functional consequences of hVDR phosphorylation were suggested by the observations that: (i) potato acid phosphatase (PAP)-treated hVDR no longer interacted with the VDRE as either a homodimer or a heteromeric complex with RXR, and (ii) treatment of transfected COS-7 cells with a phosphatase inhibitor (okadaic acid) along with 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) resulted in a synergistic enhancement of both hVDR phosphorylation and transactivation of a VDRElinked reporter gene, compared to the effect of treatment with either agent alone. These studies point to a signi®cant role for phosphorylation of VDR in regulating high-af®nity VDR±RXR interactions with VDREs, and also in modulating 1,25(OH) 2 D 3 -elicited transcriptional activation in target cells.
The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned... more The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA-binding zinc fingers that is similar to well-characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D-responsive element in DNA, in vitro, and hVDR-mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR-specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS-7 cells. Although wild-type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3 may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg-49 and Lys-55 contains a novel constitutive nuclear localization signal, RRSMKRK.
The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25-dihydroxyvitamin D(3) ... more The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) hormone to alter gene transcription. A newly recognized VDR ligand is the carcinogenic bile acid, lithocholic acid (LCA). We demonstrate that, in HT-29 colon cancer cells, both LCA and 1,25(OH)(2)D(3) induce expression of cytochrome P450 3A4 (CYP3A4), an enzyme involved in cellular detoxification. We also show that LCA-VDR stimulates transcription of gene reporter constructs containing DR3 and ER6 vitamin D responsive elements (VDREs) from the human CYP3A4 gene. Utilizing gel mobility shift, pulldown, and mammalian two-hybrid assays, we observe that: (i) 1,25(OH)(2)D(3) enhances retinoid X receptor (RXR) heterodimerization with VDR more effectively than LCA, (ii) the 1,25(OH)(2)D(3)-liganded VDR-RXR heterodimer recruits full-length SRC-1 coactivator, whereas this interaction is minimal with LCA unless LXXLL-containing fragments of SRC-1 are employed, and (iii) both 1,25(OH)(2)D(3) and LCA enhance the binding of VDR to DRIP205/mediator, but unlike 1,25(OH)(2)D(3)-VDR, LCA-VDR does not interact detectably with NCoA-62 or TRIP1/SUG1, suggesting a different pattern of LCA-VDR comodulator association. Finally, residues in the human VDR (hVDR) ligand binding domain (LBD) were altered to create mutants unresponsive to 1,25(OH)(2)D(3)- and/or LCA-stimulated transactivation, identifying S237 and S225/S278 as critical for 1,25(OH)(2)D(3) and LCA action, respectively. Therefore, these two VDR ligands contact distinct residues in the binding pocket, perhaps generating unique receptor conformations that determine the degree of RXR and comodulator binding. We propose that VDR is a bifunctional regulator, with the 1,25(OH)(2)D(3)-liganded conformation facilitating high affinity endocrine actions, and the LCA-liganded configuration mediating local, lower affinity cellular detoxification by upregulation of CYP3A4 in the colon.
The characterization of the superfamily of nuclear receptors, in particular the steroid/retinoid/... more The characterization of the superfamily of nuclear receptors, in particular the steroid/retinoid/thyroid hormone receptors, has resulted in a more complete understanding of how a repertoire of hormonally and nutritionally derived lipophilic ligands controls cell functions to effect development and homeostasis. As transducers of hormonal signaling in the nucleus, this superfamily of DNA-binding proteins appears to represent a crucial link in the emergence of multicellular organisms. Because nuclear receptors bind and are conformationally activated by a chemically diverse array of ligands, yet are closely related in general structure, they present an intriguing example of paralogous evolution. It is hypothesized that an ancient prototype receptor evolved into an intricate set of dimerizing isoforms, capable of recognizing an ensemble of hormone-responsive element motifs in DNA, and exerting ligand-directed combinatorial control of gene expression. The effector domains of nuclear receptors mediate transcriptional activation by recruiting coregulatory multisubunit complexes that remodel chromatin, target the initiation site, and stabilize the RNA polymerase II machinery for repeated rounds of transcription of the regulated gene. Because some nuclear receptors also function in gene repression, while others are constitutive activators, this superfamily of proteins provides a number of avenues for investigating hormonal regulation of gene expression. This review surveys briefly the latest findings in the nuclear receptor field and identifies particular areas where future studies should be fruitful. J. Cell. Biochem. Suppls. 32/33:110-122, 1999.
The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with... more The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schräder et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and triiodothyronine (T(3)). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR-TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR-mediated signaling were demonstrated: a T(3)-independent action, presumably via direct TR-RXR competition for DNA binding, and a T(3)-dependent repression, likely by diversion of limiting RXR from VDR-RXR toward the formation of TR-RXR heterodimers. The relative importance of these two mechanisms differed in a response element-specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis.
The active metabolite of vitamin D, 1α,25(OH) 2 D 3 , regulates calcium and phosphate metabolism ... more The active metabolite of vitamin D, 1α,25(OH) 2 D 3 , regulates calcium and phosphate metabolism by effects on intestine, bone, kidney, and parathyroid gland tissues. (1,2) The virtually ubiquitous presence of vitamin D receptors (VDRs), however, suggests a more universal function of ...
The hormonal form of vitamin D, 1␣,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], transiently stimul... more The hormonal form of vitamin D, 1␣,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], transiently stimulates the transcription of the c-fos proto-oncogene in osteoblastic cells. We have identified and characterized a vitamin D response element (VDRE) in the promoter of c-fos. The 1,25-(OH) 2 D 3 -responsive region was delineated between residues ؊178 and ؊144 upstream of the c-fos transcription start site. A mutation that inhibited binding to the sequence concomitantly abolished 1,25-(OH) 2 D 3 -induced transcriptional responsiveness; similarly, cloning of the site upstream of a heterologous promoter conferred copy-number-dependent vitamin D responsiveness to a reporter gene, demonstrating that we have identified a functional response element. The structure of the c-fos VDRE was found to be unusual. Mutational analysis revealed that the c-fos VDRE does not conform to the direct repeat configuration in which hexameric core-binding sites are spaced by a few nucleotide residues. In contrast, the entire 36-bp sequence was essential for binding. We identified the vitamin D receptor and the retinoid X receptor ␣ as components of the complex that bound the c-fos VDRE. However, our results also show that a putative CCAAT-binding transcription factor/nuclear factor 1 (CTF/NF-1) family member bound the response element in conjunction with the nuclear hormone receptors. The expression of this CTF/NF-1 family member appeared restricted to bone cells. These data hint at new molecular mechanisms of action for vitamin D.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 12, 2015
To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive... more To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive elements (VDREs) at -7/-10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)-retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25-dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate-limiting enzyme in serotonin synthesis, was induced 2.2-fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8-fold in rat serotonergic RN46A-B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1-fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis-regulatory module; CRM) at -28 kb that 1,25D-dependently docked VDR, RXR, C/EBP...
In a closed endocrine loop, 1,25-dihydroxyvitamin D3 (1,25D) induces the expression of fibroblast... more In a closed endocrine loop, 1,25-dihydroxyvitamin D3 (1,25D) induces the expression of fibroblast growth factor 23 (FGF23) in bone, with the phosphaturic peptide in turn acting at kidney to feedback repress CYP27B1 and induce CYP24A1 to limit the levels of 1,25D. In 3T3-L1 differentiated adipocytes, 1,25D represses FGF23 and leptin expression and induces C/EBPβ, but does not affect leptin receptor transcription. Conversely, in UMR-106 osteoblast-like cells, FGF23 mRNA concentrations are upregulated by 1,25D, an effect that is blunted by lysophosphatidic acid, a cell-surface acting ligand. Progressive truncation of the mouse FGF23 proximal promoter linked in luciferase reporter constructs reveals a 1,25D-responsive region between -400 and -200 bp. A 0.6 kb fragment of the mouse FGF23 promoter, linked in a reporter construct, responds to 1,25D with a fourfold enhancement of transcription in transfected K562 cells. Mutation of either an ETS1 site at -346 bp, or an adjacent candidate vitamin D receptor (VDR)/Nurr1-element, in the 0.6 kb reporter construct reduces the transcriptional activity elicited by 1,25D to a level that is not significantly different from a minimal promoter. This composite ETS1-VDR/Nurr1 cis-element may function as a switch between induction (osteocytes) and repression (adipocytes) of FGF23, depending on the cellular setting of transcription factors. Moreover, experiments demonstrate that a 1 kb mouse FGF23 promoter-reporter construct, transfected into MC3T3-E1 osteoblast-like cells, responds to a high calcium challenge with a statistically significant 1.7- to 2.0-fold enhancement of transcription. Thus, the FGF23 proximal promoter harbors cis elements that drive responsiveness to 1,25D and calcium, agents that induce FGF23 to curtail the pathologic consequences of their excess.
The Journal of Steroid Biochemistry and Molecular Biology, 2010
The nuclear vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D 3 (1,25D), its high affinity r... more The nuclear vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D 3 (1,25D), its high affinity renal endocrine ligand, to signal intestinal calcium and phosphate absorption plus bone remodeling, generating a mineralized skeleton free of rickets/osteomalacia with a reduced risk of osteoporotic fractures. 1,25D/VDR signaling regulates the expression of TRPV6, BGP, SPP1, LRP5, RANKL and OPG, while achieving feedback control of mineral ions to prevent age-related ectopic calcification by governing CYP24A1, PTH, FGF23, PHEX, and klotho transcription. Vitamin D also elicits numerous intracrine actions when circulating 25-hydroxyvitamin D 3 , the metabolite reflecting vitamin D status, is converted to 1,25D locally by extrarenal CYP27B1, and binds VDR to promote immunoregulation, antimicrobial defense, xenobiotic detoxification, antiinflammatory/anticancer actions and cardiovascular benefits. VDR also affects Wnt signaling through direct interaction with β-catenin, ligand-dependently blunting β-catenin mediated transcription in colon cancer cells to attenuate growth, while potentiating β-catenin signaling via VDR ligand-independent mechanisms in osteoblasts and keratinocytes to function osteogenically and as a pro-hair cycling receptor, respectively. Finally, VDR also drives the mammalian hair cycle in conjunction with the hairless corepressor by repressing SOSTDC1, S100A8/S100A9, and PTHrP. Hair provides a shield against UV-induced skin damage and cancer in terrestrial mammals, illuminating another function of VDR that facilitates healthful aging.
Proceedings of the National Academy of Sciences, 1991
The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino ... more The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC}-namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the et, (, and y isozymes of PKC, and VDR proved to be an effective substrate for PKC-,B, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-fi, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced,
The human vitamin D receptor (hVDR) is a ligandregulated transcription factor that mediates the a... more The human vitamin D receptor (hVDR) is a ligandregulated transcription factor that mediates the actions of the 1,25-dihydroxyvitamin D 3 hormone to effect bone mineral homeostasis. Employing mutational analysis, we characterized Arg-18/Arg-22, hVDR residues immediately N-terminal of the first DNA binding zinc finger, as vital for contact with human basal transcription factor IIB (TFIIB). Alteration of either of these basic amino acids to alanine also compromised hVDR transcriptional activity. In contrast, an artificial hVDR truncation devoid of the first 12 residues displayed both enhanced interaction with TFIIB and transactivation. Similarly, a natural polymorphic variant of hVDR, termed F/M4 (missing a FokI restriction site), which lacks only the first three amino acids (including Glu-2), interacted more efficiently with TFIIB and also possessed elevated transcriptional activity compared with the full-length (f/M1) receptor. It is concluded that the functioning of positively charged Arg-18/Arg-22 as part of an hVDR docking site for TFIIB is influenced by the composition of the adjacent polymorphic N terminus. Increased transactivation by the F/M4 neomorphic hVDR is hypothesized to result from its demonstrated enhanced association with TFIIB. This proposal is supported by the observed conversion of f/M1 hVDR activity to that of F/M4 hVDR, either by overexpression of TFIIB or neutralization of the acidic Glu-2 by replacement with alanine in f/M1 hVDR. Because the f VDR genotype has been associated with lower bone mineral density in diverse populations, one factor contributing to a genetic predisposition to osteoporosis may be the F/f polymor-phism that dictates VDR isoforms with differential TFIIB interaction. (Molecular Endocrinology 14: 401-420, 2000)
The activity of β-catenin, commonly dysregulated in human colon cancers, is inhibited by the vita... more The activity of β-catenin, commonly dysregulated in human colon cancers, is inhibited by the vitamin D receptor (VDR), and this mechanism is postulated to explain the putative anti-cancer activity of vitamin D metabolites in the colon. We investigated the effect of a common FokI restriction site polymorphism (F/f) in the human VDR gene as well as the effect of antitumorigenic 1,25-dihydroxyvitamin D 3 (1,25D) and pro-tumorigenic lithocholic acid (LCA) VDR ligands on β-catenin transcriptional activity. Furthermore, the influence of a major regulatory protein of β-catenin, the APC tumor suppressor gene, on VDR-dependent inhibition of β-catenin activity was examined. We report herein that β-catenin-mediated transcription is most effectively suppressed by the VDR FokI variant F/M4 when 1,25D is limiting. Using Caco-2 colorectal cancer cells, it was observed that VDR ligands, 1,25D and LCA, both suppress β-catenin transcriptional activity, though 1,25D exhibited significantly greater inhibition. Moreover, 1,25D, but not LCA, suppressed endogenous expression of the β-catenin target gene DKK-4 independent of VDR DNA-binding activity. These results support β-catenin sequestration away from endogenous gene targets by 1,25D-VDR. This activity is most efficiently mediated by the FokI gene variant F/M4, a VDR allele previously associated with protection against colorectal cancer. Interestingly, we found the inhibition of β-catenin activity by 1,25D-VDR was significantly enhanced by wildtype APC. These results reveal a previously unrecognized role for 1,25D-VDR in APC/β-catenin cross-talk. Collectively, these findings strengthen evidence favoring a direct effect on the Wnt-signaling molecule β-catenin as one anti-cancer target of 1,25D-VDR action in the colorectum.
The functional significance of two unlinked human vitamin D receptor (hVDR) gene polymorphisms wa... more The functional significance of two unlinked human vitamin D receptor (hVDR) gene polymorphisms was evaluated in twenty human fibroblast cell lines. Genotypes at both a Fok I restriction site (F/f ) in exon II and a singlet (A) repeat in exon IX (L/S) were determined, and relative transcription activities of endogenous hVDR proteins were measured using a transfected, 1,25-dihydroxyvitamin D 3 -responsive reporter gene. Observed activities ranged from 2 -100-fold induction by hormone, with higher activity being displayed by the F and the L biallelic forms. Only when genotypes at both sites were considered simultaneously did statistically significant differences emerge. Moreover, the correlation between hVDR activity and genotype segregated further into clearly defined high and low activity groups with similar genotypic distributions. These results not only demonstrate functional relevance for both the F/f and L/S common polymorphisms in hVDR, but also provide novel evidence for a third genetic variable impacting receptor potency.
The nuclear vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D 3 (1,25D) to... more The nuclear vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D 3 (1,25D) to regulate gene transcription. Recently, the secondary bile acid, lithocholate, was recognized as a novel VDR ligand. Using reporter gene and mammalian two-hybrid systems, immunoblotting, competitive ligand displacement, and quantitative real time PCR, we identified curcumin (CM), a turmeric-derived bioactive polyphenol, as a likely additional novel ligand for VDR. CM (10 −5 M) activated transcription of a luciferase plasmid containing the distal vitamin D responsive element from the human CYP3A4 gene at levels comparable to 1,25D (10 −8 M) in transfected human colon cancer cells (Caco-2). While CM also activated transcription via a retinoid X receptor (RXR) responsive element, activation of the glucocorticoid receptor (GR) by CM was negligible. Competition binding assays with radiolabeled 1,25D confirmed that CM binds directly to VDR. In mammalian two hybrid assays employing transfected Caco-2 cells, CM (10 −5 M) increased the ability of VDR to recruit its heterodimeric partner, RXR, and steroid receptor coactivator-1 (SRC-1). Real time PCR studies revealed that CM-bound VDR can activate VDR target genes CYP3A4, CYP24, p21, and TRPV6 in Caco-2 cells. Numerous studies have shown chemoprotection by CM against intestinal cancers via a variety of mechanisms. Small intestine and colon are important VDRexpressing tissues where 1,25D has known anticancer properties that may, in part, be elicited by activation of CYP-mediated xenobiotic detoxification and/or up-regulation of the tumor suppressor p21. Our results suggest the novel hypothesis that nutritionally-derived CM facilitates chemoprevention via direct binding to, and activation of, VDR.
The vitamin D receptor (VDR), but not its hormonal ligand, 1,25-dihydroxyvitamin D3 (1,25D), is r... more The vitamin D receptor (VDR), but not its hormonal ligand, 1,25-dihydroxyvitamin D3 (1,25D), is required for the progression of the mammalian hair cycle. We studied three genes relevant to hair cycle signaling, DKKL1 (Soggy), SOSTDC1 (Wise), and HR (Hairless), to determine whether their expression is regulated by VDR and/or its 1,25D ligand. DKKL1 mRNA was repressed 49-72% by 1,25D in primary human and CCD-1106 KERTr keratinocytes; a functional vitamin D responsive element (VDRE) was identified at -9590 bp in murine Soggy. Similarly, SOSTDC1 mRNA was repressed 41-59% by 1,25D in KERTr and primary human keratinocytes; a functional VDRE was located at -6215 bp in human Wise. In contrast, HR mRNA was upregulated 1.56- to 2.77-fold by 1,25D in primary human and KERTr keratinocytes; a VDRE (TGGTGAgtgAGGACA) consisting of an imperfect direct repeat separated by three nucleotides (DR3) was identified at -7269 bp in the human Hairless gene that mediated dramatic induction, even in the absence of 1,25D ligand. In parallel, a DR4 thyroid hormone responsive element, TGGTGAggccAGGACA, was identified at +1304 bp in the human HR gene that conferred tri-iodothyronine (T3)-independent transcriptional activation. Because the thyroid hormone receptor controls HR expression in the CNS, whereas VDR functions in concert with the HR corepressor specifically in skin, a model is proposed wherein unliganded VDR upregulates the expression of HR, the gene product of which acts as a downstream comodulator to feedback-repress DKKL1 and SOSTDC1, resulting in integration of bone morphogenic protein and Wnt signaling to drive the mammalian hair cycle and/or influencing epidermal function.
Six patients with primary hyperparathyroidism (PHPT) and one with squamous cell carcinoma of the ... more Six patients with primary hyperparathyroidism (PHPT) and one with squamous cell carcinoma of the esophagus with parathyroid hormone excess received disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) at a daily dose of 20 mg/kg orally. During treatment, the decrease in urinary calcium, total urinary hydroxyproline, and fasting urinary calcium suggested an inhibition of bone resorption. Serum calcium intestinal absorption of calcium and urinary cyclic adenosine monophosphate (cAMP) did not change significantly. This preliminary study indicates a possible role of diphosphonates in the management of inoperable cases of primary hyperparathyroidism or pseudohyperparathyroidism.
Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) action are the poss... more Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) action are the possible signi®cance of VDR homodimers and the functional role of receptor phosphorylation. To address these issues, milligram quantities of baculovirus-expressed hVDR were puri®ed to 97% homogeneity, and then tested for binding to the rat osteocalcin vitamin D responsive element (VDRE) via electrophoretic mobility shift and half-site competition assays in the presence or absence of a CV-1 nuclear extract containing retinoid X receptor (RXR). Methylation interference analysis revealed that both the hVDR homodimer and the VDR±RXR heterodimer display similar patterns of VDRE G-base protection. However, in competition studies, the relative dissociation of the homodimeric hVDR complex from the VDRE was extremely rapid (t 1/2 < 30 s) compared to the dissociation of the heteromeric complex (t 1/2 > 5 min), thus illustrating the relative instability and low af®nity of homodimeric VDR binding to DNA. These results indicate that VDR±RXR heterodimers are the preferred VDRE binding species. Further, two dimensional gel electrophoresis of hVDR demonstrated several isoelectric forms of the receptor, suggesting that it is subject to multiple phosphorylation events. In vitro kinase assays con®rmed that puri®ed hVDR is an ef®cient substrate for protein kinases A and Cb, as well as casein kinase II. In vivo studies of the expressed receptor in intact cells, namely baculovirus vector infected Sf9 insect cells and transfected mammalian COS-7 cells, demonstrated that hVDR was phosphorylated in a hormone-enhanced fashion. Functional consequences of hVDR phosphorylation were suggested by the observations that: (i) potato acid phosphatase (PAP)-treated hVDR no longer interacted with the VDRE as either a homodimer or a heteromeric complex with RXR, and (ii) treatment of transfected COS-7 cells with a phosphatase inhibitor (okadaic acid) along with 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) resulted in a synergistic enhancement of both hVDR phosphorylation and transactivation of a VDRElinked reporter gene, compared to the effect of treatment with either agent alone. These studies point to a signi®cant role for phosphorylation of VDR in regulating high-af®nity VDR±RXR interactions with VDREs, and also in modulating 1,25(OH) 2 D 3 -elicited transcriptional activation in target cells.
The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned... more The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA-binding zinc fingers that is similar to well-characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D-responsive element in DNA, in vitro, and hVDR-mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR-specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS-7 cells. Although wild-type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3 may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg-49 and Lys-55 contains a novel constitutive nuclear localization signal, RRSMKRK.
The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25-dihydroxyvitamin D(3) ... more The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) hormone to alter gene transcription. A newly recognized VDR ligand is the carcinogenic bile acid, lithocholic acid (LCA). We demonstrate that, in HT-29 colon cancer cells, both LCA and 1,25(OH)(2)D(3) induce expression of cytochrome P450 3A4 (CYP3A4), an enzyme involved in cellular detoxification. We also show that LCA-VDR stimulates transcription of gene reporter constructs containing DR3 and ER6 vitamin D responsive elements (VDREs) from the human CYP3A4 gene. Utilizing gel mobility shift, pulldown, and mammalian two-hybrid assays, we observe that: (i) 1,25(OH)(2)D(3) enhances retinoid X receptor (RXR) heterodimerization with VDR more effectively than LCA, (ii) the 1,25(OH)(2)D(3)-liganded VDR-RXR heterodimer recruits full-length SRC-1 coactivator, whereas this interaction is minimal with LCA unless LXXLL-containing fragments of SRC-1 are employed, and (iii) both 1,25(OH)(2)D(3) and LCA enhance the binding of VDR to DRIP205/mediator, but unlike 1,25(OH)(2)D(3)-VDR, LCA-VDR does not interact detectably with NCoA-62 or TRIP1/SUG1, suggesting a different pattern of LCA-VDR comodulator association. Finally, residues in the human VDR (hVDR) ligand binding domain (LBD) were altered to create mutants unresponsive to 1,25(OH)(2)D(3)- and/or LCA-stimulated transactivation, identifying S237 and S225/S278 as critical for 1,25(OH)(2)D(3) and LCA action, respectively. Therefore, these two VDR ligands contact distinct residues in the binding pocket, perhaps generating unique receptor conformations that determine the degree of RXR and comodulator binding. We propose that VDR is a bifunctional regulator, with the 1,25(OH)(2)D(3)-liganded conformation facilitating high affinity endocrine actions, and the LCA-liganded configuration mediating local, lower affinity cellular detoxification by upregulation of CYP3A4 in the colon.
The characterization of the superfamily of nuclear receptors, in particular the steroid/retinoid/... more The characterization of the superfamily of nuclear receptors, in particular the steroid/retinoid/thyroid hormone receptors, has resulted in a more complete understanding of how a repertoire of hormonally and nutritionally derived lipophilic ligands controls cell functions to effect development and homeostasis. As transducers of hormonal signaling in the nucleus, this superfamily of DNA-binding proteins appears to represent a crucial link in the emergence of multicellular organisms. Because nuclear receptors bind and are conformationally activated by a chemically diverse array of ligands, yet are closely related in general structure, they present an intriguing example of paralogous evolution. It is hypothesized that an ancient prototype receptor evolved into an intricate set of dimerizing isoforms, capable of recognizing an ensemble of hormone-responsive element motifs in DNA, and exerting ligand-directed combinatorial control of gene expression. The effector domains of nuclear receptors mediate transcriptional activation by recruiting coregulatory multisubunit complexes that remodel chromatin, target the initiation site, and stabilize the RNA polymerase II machinery for repeated rounds of transcription of the regulated gene. Because some nuclear receptors also function in gene repression, while others are constitutive activators, this superfamily of proteins provides a number of avenues for investigating hormonal regulation of gene expression. This review surveys briefly the latest findings in the nuclear receptor field and identifies particular areas where future studies should be fruitful. J. Cell. Biochem. Suppls. 32/33:110-122, 1999.
The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with... more The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schräder et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and triiodothyronine (T(3)). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR-TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR-mediated signaling were demonstrated: a T(3)-independent action, presumably via direct TR-RXR competition for DNA binding, and a T(3)-dependent repression, likely by diversion of limiting RXR from VDR-RXR toward the formation of TR-RXR heterodimers. The relative importance of these two mechanisms differed in a response element-specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis.
The active metabolite of vitamin D, 1α,25(OH) 2 D 3 , regulates calcium and phosphate metabolism ... more The active metabolite of vitamin D, 1α,25(OH) 2 D 3 , regulates calcium and phosphate metabolism by effects on intestine, bone, kidney, and parathyroid gland tissues. (1,2) The virtually ubiquitous presence of vitamin D receptors (VDRs), however, suggests a more universal function of ...
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Papers by Mark Haussler