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Background: Retinoid X receptors (RXRs) heterodimerize with other nuclear hormone receptors and control ligand mediated transcription. To address how RXRs function as heterodimers, we investigated activities of truncated RXRα and RXRβ that lack ≈ 20 conserved C-terminal amino acids. Results: The truncated RXRs formed heterodimers and bound to respective DNA elements in vitro. By transient reporter assays we found that these RXRs act as dominant negative receptors and inhibit ligand dependent transcription by the retinoic acid receptor (RAR) and vitamin D receptor. P19 embryonal carcinoma cells stably expressing the truncated RXRβ (termed ΔC2) were deficient in activating the endogenous RARβ gene and an RA responsive reporter. To study the dominant negative activity of ΔC2 further, genomic footprinting analysis was performed for the RARβ2 promoter. In control P19 clones, the RA responsive element (RARE) and other elements in the promoter were protected after RA treatment. However, in ΔC2 clones RA-induced protection was markedly inhibited at all elements. Conclusions: These results indicate that the C-terminal region of RXR is required for full RARE occupancy in vivo, a RA dependent process that leads to the recruitment of other factors to the promoter and the subsequent transcriptional activation. Thus, RXRs play an integral role in ligand dependent transcription.

Genomic actions of the calciotropic hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)2D3 to 1 alpha, 25-dihydroxyvitamin D3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-3H]vitamin D3-3-deoxy-3 beta-bromoacetate (1,25(OH)2[3H]D3-BE) and 1 alpha,25-dihydroxyvitaminD3-3 beta-[1-14C]bromoacetate(1,25(OH)2D3-[14C]BE) binding-site directed analogs of 1,25(OH)2D3, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)2[3H]D3-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1,25(OH)2D3-[14C]BE). In each case, specificity of labeling was demonstrated by the drastic reduction in labeling when the incubation was carried out in the presence of an excess of nonradioactive 1 alpha,25(OH)2D3. These results strongly suggested that 1,25(OH)2[3H]D3-BE and 1,25(OH)2D3-[14C]BE covalently modified the 1,25(OH)2D3-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.

Residues located between amino acids 244 and 263 in the human vitamin D receptor (hVDR) show extensive homology with other members of the steroid/thyroid/retinoid hormone receptor superfamily. The corresponding region of the glucocorticoid receptor has been shown to interact with the 90-kilodalton heat shock protein (hsp90), yet hVDR does not appear to bind to hsp90. Herein we report a study of hVDR in which the functional role of five conserved residues was tested by replacing Phe-244, Lys-246, Leu-254, Gln-259, and Leu-262 with glycines by site-directed mutagenesis. Initial screening of these mutants indicated that all were significantly impaired in their ability to activate transcription from a vitamin D-responsive reporter construct when expressed in transfected VDR-deficient COS-7 cells. Further characterization revealed two classes of mutants: the predominant class binds the 1,25-dihydroxyvitamin D3 ligand normally but is defective in its ability to form a heterodimeric complex with the retinoid X receptor (RXR) on a vitamin D responsive element (VDRE). A second unique class, represented by a single mutant at Lys-246, is normal both with respect to ligand binding and complex formation but still very impaired in transactivation ability. The distinction between these two classes was confirmed by the demonstration that a member of the first class, with a mutation at Gln-259, could be restored to near wild type transactivation ability by supplying excess RXR, while the Lys-246 mutant could not be so rescued. We therefore conclude that the primary function of this conserved domain in hVDR is the mediation of heterodimerization with RXR, leading to VDRE binding and transactivation. The possibility also exists that the Lys-246 mutant may be impaired in a step of transactivation that is distal to complex formation with RXR on the VDRE, perhaps in interactions with the transcriptional machinery itself.

Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D(3) ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (-31 to -46 kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D(3) are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.

The conformationally flexible secosteroid, 1α,25(OH)₂vitamin D₃ (1α,25(OH)₂D₃) initiates biological responses via binding to the vitamin D receptor (VDR). The VDR contains two overlapping ligand binding sites, a genomic pocket (VDR-GP) and an alternative pocket (VDR-AP), that respectively bind a bowl-like ligand configuration (gene transcription) or a planar-like ligand shape (rapid responses). When occupied by 1α,25(OH)₂D₃, the VDR-GP interacts with the retinoid X receptor to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1α,25(OH)₂D₃. By recruiting complexes of either coactivators or corepressors, activated VDR modulates the transcription of genes encoding proteins that promulgate the traditional genomic functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. 1α,25(OH)₂D₃/VDR control of gene expression and rapid responses also delays chronic diseases of aging such as osteoporosis, cancer, type-1 and -2 diabetes, arteriosclerosis, vascular disease, and infection.

Publisher Summary Small Angle X-ray Scattering and Fluorescence Resonance Energy Transfer techniques have determined the structure of the hypocalcemic vitamin-D-resistant rickets (hVDR) DNA-binding domain (DBD) and ligand-binding domain (LBD) together in the full-length receptor, heterodimerized with full-length RXR α, docked on a VDRE, and occupied with 1,25(OH)2D3 plus a single coactivator, allowing one to begin to understand the interactions between the DBD and the ligand-binding/heterodimerization domains of both VDR and its RXR heteropartner. A second major realm of accomplishment has been the identification of numerous additional VDR-regulated genes, including those mediating calcium and particularly phosphate homeostasis, bone metabolism, detoxification, cell proliferation, differentiation, migration and death, immunity and antimicrobial action, as well as carbohydrate, lipid, and amino acid metabolism. Equally exciting has been the discovery of additional natural VDR ligands, especially in the more-recently identified and studied VDR target tissues such as the hair follicle/skin and immune systems. The availability of this information will facilitate molecular investigations of transcriptional control by VDR in target-cell specific environments in the presence of novel ligands and the context of a myriad of promoters/genes. Such experiments are likely to extend the understanding of the variety of conformations and coregulator associations that the VDR-RXR heterodimer is capable of achieving while performing its multitude of extraosseous effects to lower the risk of the chronic diseases of aging.

We report here that highly purified human vitamin D receptor (hVDR) derived from E. coli or baculovirus expression systems does not exhibit saturable, high affinity 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand binding when these preparations alone are analyzed. Inclusion of rat liver nuclear extract, which does not itself contain detectable 1,25(OH)2D3 binding activity, is required to endow hVDR isolated from bacterial or insect cells with the property of high affinity hormone binding (Kd 0.13-0.22 nM). This observation should facilitate the valid assay of 1,25(OH)2D3 binding activity and kinetics in samples of overexpressed hVDR. Moreover, since rat liver nuclear extract contains retinoid X receptors and possibly other auxiliary factors capable of forming heterodimers with hVDR that in turn associate with vitamin D responsive elements, we hypothesize that like DNA binding, 1,25(OH)2D3 binding to hVDR requires the cooperation of a co-receptor or some uncharacterized receptor activating/stabilizing factor.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10(-8) M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P]orthophosphate results in a rapid (< or = 30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25(OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-beta (PKC-beta), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P.W., Galligan, M.A., Terpening, C.M., Haussler, C.A., Samuels, D.S., Shimizu, Y., Shimizu, N., and Haussler, M.R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-beta, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-beta substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-beta, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineral-ocorticoid, and androgen receptors, eliminated PKC-beta phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-beta elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.

We report that the human 1,25-dihydroxyvitamin D3 receptor is an efficient substrate for cAMP-dependent protein kinase, in vitro. This phosphorylation reaction is rapid and neither dependent upon nor significantly affected by the presence of the 1,25-dihydroxyvitamin D3 ligand. Preliminary mapping experiments utilizing C-terminal truncation mutants reveal that the primary site(s) of phosphorylation, in vitro, is localized between amino acids 133 and 201. Cotransfection of the catalytic subunit of murine cAMP-dependent protein kinase and the human 1,25-dihydroxyvitamin D3 receptor into monkey kidney (COS-7) cells not only results in a dramatic kinase-dependent increase in receptor phosphorylation but also elicits an attenuation in 1,25-dihydroxyvitamin D3-dependent transcriptional activation of a reporter gene. These observations suggest a potential role for cAMP-dependent protein kinase in the modulation of 1,25-dihydroxyvitamin D3 receptor-mediated gene regulation.

The human 1,25-dihydroxyvitamin D3 receptor (hVDR), like other members of the steroid/thyroid receptor superfamily, has been observed to undergo rapid phosphorylation. We report here that the hVDR is a substrate for casein kinase II (CK-II), a regulatory enzyme of significance in the function of nuclear proteins. Intact hVDRs produced by in vitro transcription/translation or in a baculovirus overexpression system served as efficient substrates for purified bovine CK-II, and the magnitude of this phosphorylation was not affected by the addition of 1,25-dihydroxyvitamin D3. CK-II-catalyzed phosphorylation of truncated hVDRs suggested that phosphorylated residues may occur between Arg121 and Asp232, including the region of hVDR which we have previously demonstrated to contain a major site(s) of phosphorylation in intact cells (Jones, B.B., Jurutka, P.W., Haussler, C.A., Haussler, M.R., and Whitfield, G.K. (1991) Mol. Endocrinol. 5, 1137-1146). Site-directed mutagenesis of serine/threonine residues in this region now reveals a site of phosphorylation at Ser208 contained within the sequence -S208 (P)EEDSDD-, a classic CK-II consensus recognition site. Mutation of this serine to a glycine drastically reduces phosphorylation of hVDR by CK-II, in vitro. The Ser208 mutant receptor also shows a dramatic decrease in [32P]orthophosphate incorporation when transfected into COS-7 cells. We therefore propose that phosphorylation of hVDR at Ser208 in target cells is mediated by casein kinase II or a similar enzyme, and that this quantitatively significant post-translational modification is a potential mechanism for the modulation of the activity of hVDR in controlling gene transcription.

A primary response of the avian intestine to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is increased synthesis of a 28-kD calcium-binding protein, calbindin-D28k (CaBP). This study examined whether 1,25-(OH)2D3 regulates CaBP gene transcription by an interaction of the vitamin D receptor (VDR) with a vitamin D-responsive element (VDRE) in the CaBP promoter. A genomic clone of CaBP containing about 1 kb of 5'-flanking DNA and 13 kb of the structural gene was isolated. 5'-Flanking DNA from -320 to -306 had considerable sequence similarity to VDREs identified in other genes. Indeed, a portion of the CaBP gene containing this region (-743 to +47) linked to a growth hormone reporter construct elicited a 1,25-(OH)2D3-dependent, VDR-dependent increase in reporter expression in transiently transfected chicken embryo fibroblasts. However, deletion analysis demonstrated that the sequences responsible for this induction reside 3' to -133 and the putative VDRE at -320 to -306 was not involved in the response. Furthermore, transfection of heterologous promoter constructs consisting of a Ban I fragment (-354 to -252) linked to the Herpes simplex thymidine kinase promoter revealed no effect of this region on reporter expression. Gel mobility shift analysis confirmed that this putative VDRE in the CaBP promoter was not a high-affinity binding site for VDR. Consequently, functional significance with respect to the primary induction of CaBP by 1,25-(OH)2D3 cannot be ascribed to this region of the CaBP promoter.

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 (LCA), 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 (VDRE) 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 VDR-expressing 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) 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 alpha, beta, and gamma isozymes of PKC, and VDR proved to be an effective substrate for PKC-beta, 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-beta, 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, whereas that of the Ser-51 mutant hVDR was unaffected. Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo. To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element. Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation. Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.

A baculovirus expression vector system (BEVS) was used to overproduce the full-length human vitamin D receptor (hVDR) in Spodoptera frugiperda ovarian cells. hVDR was expressed to a level of 0.5% of the total soluble protein in this system. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties equivalent to those of hVDR expressed in mammalian cells. The BEVS-derived receptor displayed specificity and high affinity (apparent Kd = 0.7 nM) for the 1,25(OH)2D3 ligand. Recombinant hVDR generated a specific protein-DNA complex with a duplex oligomer containing a vitamin D-responsive element (VDRE) in gel mobility shift assays. The intensity of the VDR.VDRE complex was not affected by 1,25(OH)2D3. However, the complex exhibited increased mobility in the presence of hormone, possibly the result of a 1,25(OH)2D3-dependent conformational change. A nuclear extract obtained from CV-1 cells markedly enhanced the intensity of this VDR.VDRE complex and produced an additional distinct VDR-dependent complex, thus implicating a role for nuclear auxiliary factors in multiple high affinity VDR.VDRE interactions. Finally, methylation interference studies defined the guanine residues contacted when the putative VDR-auxiliary factor complex associates with the rat osteocalcin VDRE; specifically, all of the GC base pairs in the sequence GGGTGAATGAGGACA. Therefore, these results show that the BEV system elicits high level expression of hVDR with critical functional characteristics being preserved.

Glucocorticoid treatment of certain lymphoma cell lines and thymocytes activates a self-destructive pathway of programmed cell death referred to as apoptosis. Calcium and calmodulin (CaM) may be important signals in the apoptotic cascade because an early event is a sustained elevation in cytosolic Ca2+ and CaM inhibitors interfere with the death pathway. In the present study, expression of the CaM gene was examined during glucocorticoid-induced apoptosis in WEHI7.2 lymphocytes. Steady state levels of CaM mRNA were increased up to 10-fold following a 4-6-h exposure of WEHI7.2 cells to 10(-6) M dexamethasone. This increase was mediated through the glucocorticoid receptor since the response was not observed in WEHI7.418, a variant line which does not express active glucocorticoid receptor. Induction of CaM mRNA was dose-dependent and highly specific for glucocorticoids, as other steroids were unable to elicit the response. A stringent cell specificity was also observed. Pretreatment of WEHI7.2 lymphocytes with cycloheximide did not interfere with dexamethasone-dependent increases in CaM mRNA levels, and studies with actinomycin D demonstrated that the stability of the transcript was not altered by hormone, Finally, a calmodulin inhibitor elicited a protective effect on WEHI7.2 cells following glucocorticoid exposure. These results indicate that CaM mRNA levels were hormonally controlled in WEHI7.2 lymphocytes and support the putative involvement of CaM in glucocorticoid-induced apoptosis.

The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormone-binding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for casein kinase-II or a related protein kinase.

Increasing evidence suggests an immunoregulatory function of the potent steroid hormone 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) which has been successfully applied for treatment of psoriasis. The skin is both a site of production and a target of 1,25(OH) 2 D 3 . In vitro, 1,25(OH) 2 D 3 inhibits proliferation and stimulates differentiation of keratinocytes. We investigated the in situ expression of vitamin D-receptors (VDR) in normal and psoriatic skin by immunochemical methods. The VDR were visualized using the monoclonal antibody (MoAb) 9A7g to the VDR and the labeled avidinbiotin technique. Immunoreactivity was consistently confined to nuclei in all skin biopsies. In normal skin specimens (n = 10) VDR antigens were expressed in keratinocytes of all epidermal layers (except those of the stratum corneum) and in cells of the epidermal appendages. Double labeling experiments with MoAb to cluster-defined antigens indicated that melanocytes and approximately 75% of Langerhans cells exhibit 1,25(OH) 2 D 3 receptors in normal skin biopsies (n = 5). Depending on their localization in skin compartments 42–62% of CD11b + positive macrophages and 45–75% of CD3 + T lymphocytes expressed VDR. Non-lesional psoriatic skin specimens (n = 8) revealed nearly identical staining patterns. Lesional psoriatic skin specimens (n = 8) exhibited a significant increase of VDR expression both in basal and suprabasal epidermal layers as measured by computer-assisted morphometry and showed a remarkable change of the immune cell pattern: the densitity and proportion of VDR positive T lymphocytes and macrophages were higher in the epidermal and the perivascular papillary loop compartment. These in vivo findings strongly support the hypothesis that 1,25(OH) 2 D 3 modulates immune response and cell proliferation/differentiation in human skin.

The human vitamin D receptor (VDR) is a key nuclear receptor that binds nutritionally derived ligands and exerts bioeffects that contribute to bone mineral homeostasis, detoxification of exogenous and endogenous compounds, cancer prevention, and mammalian hair cycling. Liganded VDR modulates gene expression via heterodimerization with the retinoid X receptor and recruitment of coactivators or corepressors. VDR interacts with the corepressor hairless (Hr) to control hair cycling, an action independent of the endocrine VDR ligand, 1,25-dihydroxyvitamin D(3). We report novel dietary ligands for VDR including curcumin, gamma-tocotrienol, and essential fatty acid derivatives that likely play a role in the bioactions of VDR.

The vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds with high affinity to the nuclear vitamin D receptor (VDR), which recruits its retinoid X receptor (RXR) heterodimeric partner to recognize vitamin D responsive elements (VDREs) in target genes. 1,25(OH)(2)D(3) is known primarily as a regulator of calcium, but it also controls phosphate (re)absorption at the intestine and kidney. Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced in osteoblasts that, like PTH, lowers serum phosphate by inhibiting renal reabsorption through Npt2a/Npt2c. Real-time PCR and reporter gene transfection assays were used to probe VDR-mediated transcriptional control by 1,25(OH)(2)D(3). Reporter gene and mammalian two-hybrid transfections, plus competitive receptor binding assays, were used to discover novel VDR ligands. 1,25(OH)(2)D(3) induces FGF23 78-fold in osteoblasts, and because FGF23 in turn represses 1,25(OH)(2)D(3) synthesis, a reciprocal relationship is established, with FGF23 indirectly curtailing 1,25(OH)(2)D(3)-mediated intestinal absorption and counterbalancing renal reabsorption of phosphate, thereby reversing hyperphosphatemia and preventing ectopic calcification. Therefore, a 1,25(OH)(2)D(3)-FGF23 axis regulating phosphate is comparable in importance to the 1,25(OH)(2)D(3)-PTH axis that regulates calcium. 1,25(OH)(2)D(3) also elicits regulation of LRP5, Runx2, PHEX, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic. Regulation of mouse RANKL by 1,25(OH)(2)D(3) supports a cloverleaf model, whereby VDR-RXR heterodimers bound to multiple VDREs are juxtapositioned through chromatin looping to form a supercomplex, potentially allowing simultaneous interactions with multiple co-modulators and chromatin remodeling enzymes. VDR also selectively binds certain omega3/omega6 polyunsaturated fatty acids (PUFAs) with low affinity, leading to transcriptionally active VDR-RXR complexes. Moreover, the turmeric-derived polyphenol, curcumin, activates transcription of a VDRE reporter construct in human colon cancer cells. Activation of VDR by PUFAs and curcumin may elicit unique, 1,25(OH)(2)D(3)-independent signaling pathways to orchestrate the bioeffects of these lipids in intestine, bone, skin/hair follicle, and other VDR-containing tissues.

A fragment of the complementary deoxyribonucleic acid to the human 1,25-dihydroxyvitamin D 3 receptor protein containing essentially the entire open reading frame was transcribed and translated in vitro . The resulting protein was then demonstrated to exhibit the physical and functional features, i.e. molecular weight, immunoreactivity, 1,25-dihydroxyvitamin D 3 binding, and DNA-cellulose binding, of the native human receptor from the T47D cell line. This validates the authenticity of the cDNA in a cell free system and provides a biochemical means of generating this rare and labile macromolecule to use in heretofore difficult structure/function studies.

1,25-Dihydroxyvitamin D(3) (1,25D) is known primarily as a regulator of calcium, but 1,25D also promotes phosphate absorption from intestine, reabsorption from kidney, and bone mineral resorption. FGF23 is a newly discovered phosphaturic hormone that, like PTH, lowers serum phosphate by inhibiting renal reabsorption via Npt2a. We show that 1,25D strongly upregulates FGF23 in bone. FGF23 then represses 1alpha-OHase activity in kidney, thus preventing spiraling induction of FGF23 by 1,25D. We also report that LRP5, Runx2, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic, are transcriptionally regulated by 1,25D. This coordinated regulation together with that of FGF23 and PTH allows 1,25D to play a central role in maintaining calcium and phosphate homeostasis and bone metabolism. In the cases of LRP5, Runx2, TRPV6, and Npt2c we show that transcriptional regulation results at least in part from direct binding of VDR near the relevant gene promoter. Finally, because 1,25D induces FGF23, and FGF23 in turn represses 1,25D synthesis, a reciprocal relationship is established with FGF23 indirectly curtailing 1,25D-mediated intestinal absorption and counterbalancing renal reabsorption of phosphate. This newly revealed FGF23/1,25D/Pi axis is comparable in significance to phosphate and bone metabolism as the PTH/1,25D/Ca axis is to calcium homeostasis.

The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [γ- 32 P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [ 32 P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50% of the transactivation capacity in response to 1,25(OH) 2 D 3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH) 2 D 3 target genes.

The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D (1,25(OH)2D3), the, active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)2D3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)2D3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)2D3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)2D3 response and is involved in basal transcription. South-western blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)2D3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)2D3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.

Both the vitamin D receptor (VDR) and hairless (hr) genes play a role in the mammalian hair cycle, as inactivating mutations in either result in total alopecia. VDR is a nuclear receptor that functions as a ligand-activated transcription factor, whereas the hairless gene product (Hr) acts as a corepressor of both the thyroid hormone receptor (TR) and the orphan nuclear receptor, RORalpha. In the present study, we show that VDR-mediated transactivation is strikingly inhibited by coexpression of rat Hr. The repressive effect of Hr is observed on both synthetic and naturally occurring VDR-responsive promoters and also when VDR-mediated transactivation is augmented by overexpression of its heterodimeric partner, retinoid X receptor. Utilizing in vitro pull down methods, we find that Hr binds directly to VDR but insignificantly to nuclear receptors that are not functionally repressed by Hr. Coimmunoprecipitation data demonstrate that Hr and VDR associate in a cellular milieu, suggesting in vivo interaction. The Hr contact site in human VDR is localized to the central portion of the ligand binding domain, a known corepressor docking region in other nuclear receptors separate from the activation function-2 domain. Coimmunoprecipitation and functional studies of Hr deletants reveal that VDR contacts a C-terminal region of Hr that includes motifs required for TR and RORalpha binding. Finally, in situ hybridization analysis of hr and VDR mRNAs in mouse skin demonstrates colocalization in cells of the hair follicle, consistent with a hypothesized intracellular interaction between these proteins to repress VDR target gene expression, in vivo.

The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.

The vitamin D receptor (VDR) mediates the effects of the calcemic hormone 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. We show that VDR also functions as a receptor for the secondary bile acid lithocholic acid (LCA), which is hepatotoxic and a potential enteric carcinogen. VDR is an order of magnitude more sensitive to LCA and its metabolites than are other nuclear receptors. Activation of VDR by LCA or vitamin D induced expression in vivo of CYP3A, a cytochrome P450 enzyme that detoxifies LCA in the liver and intestine. These studies offer a mechanism that may explain the proposed protective effects of vitamin D and its receptor against colon cancer.

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.

The human vitamin D receptor (hVDR) is a ligand-regulated transcription factor that mediates the actions of the 1,25-dihydroxyvitamin D3 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 polymorphism that dictates VDR isoforms with differential TFIIB interaction.

Nonsteroidal analogues of 1alpha,25(OH)2D3, lacking either the full five-membered D ring (C-ring analogues) or the full six-membered C ring (D-ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1alpha,25(OH)2D3. Maximal superagonistic activity was seen for the C-ring analogue with a 24(R)-hydroxyl group in the side chain [30- to 60-fold the activity of 1alpha,25(OH)2D3]. The 19-nor-16-ene-26,27-bishomo C-ring analogue showed the best ratio of antiproliferative to calcemic effects (1275-fold better than 1alpha,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D-dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR-ligand complex more resistant against protease digestion than is 1alpha,25(OH)2D3. In contrast to 20-epimer steroidal vitamin D analogues, 20-epimer C-ring analogues were less potent than analogues with a natural C-20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1alpha,25(OH)2D3 despite lower receptor affinity and, for the C-ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published.

The vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D(3) and mediates its actions on gene transcription by heterodimerizing with retinoid X receptors (RXRs) on direct repeat (DR+3) vitamin D responsive elements (VDREs) located in target genes. The VDRE binding function of VDR has been primarily ascribed to the zinc finger region (residues 24-87). To define the minimal VDRE binding domain for human VDR (hVDR), a series of C-terminally truncated hVDR mutants (Delta134, Delta113, Delta102, Delta90, Delta84, Delta80, and Delta60) was generated and expressed in bacteria. Only the Delta134 and Delta113 mutants bound the VDRE (predominantly as monomers), suggesting that, in addition to the conserved zinc finger region of hVDR, as many as 25 amino acids in a C-terminal extension (CTE) participate in DNA binding. Site-directed mutagenesis of conserved charged residues in full-length hVDR was then performed to dissect the functional significance of the CTE (residues 88-112) in the context of the complete hVDR-RXR-VDRE interaction. Functional assays revealed that E98K/E99K, R102A/K103A/R104A, and K109A/R110A/K111A mutant hVDRs possessed dramatically reduced DNA binding and transcriptional activities, whereas distinct point mutants, such as K103A, bound to DNA normally but lacked transcriptional activity. Therefore, the boundary for the minimal DNA-binding domain in hVDR extends C-terminal of the zinc fingers to Lys-111, with clusters of highly conserved charged amino acids playing a crucial role in binding to the DR+3 element. Further, individual residues in this region (e.g., Lys-103) may lie on the opposing face of a DNA-binding alpha-helix, where they could contact transcriptional coactivators or basal transcription factors.

IN THE DECADE SINCE THE VITAMIN D RECEPTOR (VDR) was cloned and recognized as a member of the superfamily of nuclear receptors that regulate gene expression in a ligand-dependent manner, the central role of VDR in the biology of vitamin D action has been illuminated and is being defined at the molecular level. Following renal production as the hormonal metabolite of vitamin D, 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) functions as the ligand for VDR, with the hormone–receptor complex inducing calcemic and phosphatemic effects that result in normal bone mineralization and remodeling. VDR not only mediates the action of 1,25(OH)2D3 in calcium/phosphate translocating tissues, primarily intestine, but also elicits a myriad of apparent bioactivities in other major cell systems in the organism, including immune, neural, epithelial, and endocrine. The scope of this review will be limited to highlighting the actions of 1,25(OH)2D3 mediated by nuclear VDR and discussing new developments in the structure/function analysis of the receptor, including the phenotype of VDR knockout mice and the biochemical classification of patients with point mutations in the receptor. These new advances, along with other recent research, will be interpreted to update our understanding of the molecular role of VDR, ranging from characterization of its natural gene and clinically significant polymorphisms, through its DNA contact sites and protein partners, to novel ligand analogs that hold the promise of influencing VDR conformation in a therapeutically beneficial fashion. VDR BIOLOGY

To investigate a potential ligand-dependent transcriptional activation domain (AF-2) in the C-terminal region of the human vitamin D receptor (hVDR), two conserved residues, Leu-417 and Glu-420, were replaced with alanines by site-directed mutagenesis (L417A and E420A). Transcriptional activation in response to 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) was virtually eliminated when either point mutant was transfected into several mammalian cell lines. Furthermore, both mutants exhibited a dominant negative phenotype when expressed in COS-7 cells. Scatchard analysis at 4 degrees C and a ligand-dependent DNA binding assay at 25 degrees C revealed essentially normal 1,25-(OH)2D3 binding for the mutant hVDRs, which were also equivalent to native receptor in associating with the rat osteocalcin vitamin D responsive element as a presumed heterodimer with retinoid X receptor. Glutathione S-transferase-human transcription factor IIB (TFIIB) fusion protein linked to Sepharose equally coprecipitated the wild-type hVDR and the AF-2 mutants. These data implicate amino acids Leu-417 and Glu-420, residing in a putative alpha-helical region at the extreme C terminus of hVDR, as critical in the mechanism of 1, 25-(OH)2D3-stimulated transcription, likely mediating an interaction with a coactivator(s) or a component of the basal transcriptional machinery distinct from TFIIB.

The T-box mediates binding of retinoid X receptor (RXR) homodimers to DNA while the P- and D-box in the zinc fingers of steroid hormone receptors play roles in DNA-binding specificity and homodimerization, respectively. We investigated the function of these elements in the human vitamin D receptor (hVDR) by mutating a Lys-Glu pair of amino acids in the T-box, and by altering the P- and D-boxes to the corresponding residues of the glucocorticoid receptor (GR). The T-box mutant hVDR displayed attenuated vitamin D responsive element (VDRE) binding in the presence of RXR and was severely compromised in transcriptional activation. In contrast, GR P/D-box mutant hVDRs bound to the rat osteocalcin VDRE and elicited near normal transcriptional activation. The T-box mutant uniquely exhibited dominant negative properties, highlighting the significance of this region of hVDR for heterodimeric transcriptional activation.

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]hormone with high affinity and elicits its actions to regulate gene expression in target cells by binding to vitamin D-responsive elements (VDREs). VDREs in positively controlled genes such as osteocalcin, osteopontin, beta 3-integrin, and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The VDR associates with these VDREs with the greatest affinity as a heterodimer with one of the family of retinoid X receptors (RXRs). VDR consists of an N-terminal zinc finger domain that determines DNA binding, a "hinge" segment and a C-terminal hormone binding domain which also contains two conserved regions that engage in heterodimerization with an RXR on the VDRE. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is to alter the conformation of the hormone-binding domain of VDR to facilitate strong dimerization with RXR, which results in ligand-enhanced association with the VDRE. Thus RXR is recruited into a heterocomplex by liganded VDR. The natural ligand for the RXR coreceptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3-stimulated transcription, indicating that 9-cis retinoic acid diverts RXR away from being the silent partner of VDR to instead form RXR homodimers. Recent data reveal that after binding RXR, a subsequent target for VDR in the vitamin D signal transduction cascade is basal transcription factor IIB (TFIIB).(ABSTRACT TRUNCATED AT 250 WORDS)

Organ culture of 19-day-old chick embryo duodena was utilized to evaluate the mechanism of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent calbindin-D28k (CaBP) expression. Duodenal CaBP and 1,25(OH)2D3 receptor (VDR) expression were assessed by Western blot analysis, while CaBP and VDR mRNA levels were determined by Northern blot analysis. In untreated duodena, both VDR protein and mRNA were present, while CaBP protein and mRNA were undetectable. Treatment of cultured duodena with 25 nM 1,25(OH)2D3 resulted in detectable CaBP mRNA after 4 h which continued to increase during a 24 h time period. Under these conditions, localization of [3H-1 beta]1 alpha,25(OH)2D3 in duodenal chromatin is rapid (< or = 30 min). Thus, the delayed accumulation of detectable CaBP mRNA cannot be explained by slow nuclear binding of 1,25(OH)2D3. The inclusion of 1.6 microM actinomycin D in the organ culture partially inhibited the 1,25(OH)2D3-regulated increase in CaBP mRNA, which implies that there is a transcriptional component involved in the increased CaBP mRNA levels. Similarly, quantitative polymerase chain reaction studies allowed the detection of CaBP pre-mRNA and mRNA sequences 1 h after hormone treatment, suggesting that CaBP gene transcription is initiated rapidly. Treatment of cultures with 36 microM cycloheximide 1 h prior to 1,25(OH)2D3 addition resulted in superinduction of VDR mRNA levels but sharply reduced CaBP steady-state mRNA levels. This dramatic reduction in CaBP mRNA reveals that 1,25(OH)2D3-mediated CaBP expression is dependent on ongoing protein synthesis. Thus, we propose that a labile auxiliary protein or other cofactor, which may or may not be 1,25(OH)2D3-dependent, is necessary for 1,25(OH)2D3-mediated CaBP gene transcription in chick duodena.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates gene transcription through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily. Sequence-specific transcription factors, including nuclear hormone receptors, are thought to interact with the basal transcription complex to regulate transcription. In glutathione S-transferase fusion-based protein-protein binding assays we found that VDR specifically binds to TFIIB, a component of the basal complex, and that the interaction requires select domains of each protein. To assess the functional significance of this interaction, transfection assays were performed with a 1,25(OH)2D3-responsive reporter construct. In P19 embryonal carcinoma cells cotransfection of VDR and TFIIB cooperatively activated reporter transcription, while each factor alone gave very low to no activation. This activation was dependent on 1,25(OH)2D3 and the dose of TFIIB and VDR transfected, demonstrating that a nuclear hormone receptor functionally interacts with TFIIB in vivo. In contrast, transfection of NIH 3T3 cells generated strong reporter activation by 1,25(OH)2D3 in the presence of VDR alone, and cotransfection of TFIIB led to specific dose-dependent repression of reporter activity. Taken together, these results indicate that TFIIB-nuclear hormone receptor interaction plays a critical role in ligand-dependent transcription, which is apparently modulated by a cell-type-specific accessory factor.

The human vitamin D receptor (hVDR) requires another nuclear protein(s), designated receptor auxiliary factor (RAF), for optimal binding to the vitamin D-responsive element (VDRE). To determine the region in hVDR required to form a heterodimer with RAF on the VDRE, mutant hVDR cDNAs were constructed by site-directed mutagenesis and transfected into COS-7 cells. A truncated hVDR, lacking 25 C-terminal amino acids (delta 403-427), showed complex production in combination with endogenous RAF in COS-7 cells. Complex development was markedly enhanced by adding a rat liver nuclear fraction, which contains RAF activity, or either the alpha or beta form of the retinoid-X receptor, which has been reported to be closely related or identical to RAF. In contrast, either a C-terminal truncation of 46 amino acids (delta 382-427) or single point mutations at lysine-382, methionine-383, glutamine-385, or leucine-390 dramatically reduced the ability of hVDR to heterodimerize with RAF. Binding of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] hormone was undetectable in delta 382-427 truncated hVDR, whereas the delta 403-427 mutant hVDR exhibited significant 1,25-(OH)2D3 ligand binding, although the dissociation constant was approximately 10-fold higher than that of the wild-type receptor. Surprisingly, the delta 403-427 mutant hVDR did not mediate measurable transcriptional activation in cotransfection experiments with a VDRE-GH reporter gene construct. These results indicate that hVDR residues between cysteine-403 and serine-427 are required for very high affinity 1,25-(OH)2D3 ligand binding and transcriptional activation, but are not involved in heterodimerization. The region of hVDR between lysine-382 and arginine-402, probably the domain containing heptad 9, plays an essential role in the heterodimerization of hVDR with RAF. However, based upon additional point mutagenesis experiments, it is likely that other regions of the hormone-binding domain, such as that including heptad 4 (leucine-325 to leucine-332), also contribute to the protein-protein interactions required for the high affinity, specific binding of hVDR to the VDRE.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Summary The effect of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) administration on duodenal vitamin D receptor (VDR) mRNA levels in hypophosphatemic ( Hyp ) mice, a murine homologue of human X-linked hypophosphatemic rickets, was examined. Basal levels of VDR mRNA in Hyp mice were similar to those of normal littermates and, in normal mice, VDR mRNA levels were up-regulated 1.8–2.7-fold after injection of 1 μg/kg 1,25(OH) 2 D 3 . In contrast, no significant change in VDR mRNA was observed in Hyp mice treated with 1,25(OH) 2 D 3 . To determine the effect of phosphate repletion on VDR mRNA levels, high-phosphate diet was fed to Hyp mice. Although plasma phosphorus concentration was restored to normal, up-regulation of VDR mRNA was not recovered with phosphate supplementation. These results indicate that the vitamin D-resistance in Hyp mice is not caused by hypophosphatemia, per se, and may result from a fundamental molecular defect in vitamin D action at the intestine which could be related to ineffective up-regulation of VDR mRNA by 1,25(OH) 2 D 3 .

The WEHI7.2 thymoma cell line undergoes apoptotic cell death when exposed to glucocorticoids and agents that increase intracellular cAMP. Several lines of evidence indicate that calcium may play an important role in events culminating in lymphocyte apoptosis. In these studies, calbindin-D28K was stably overexpressed in WEHI7.2 cells to determine if increasing the Ca(2+)-binding capacity of the cell interferes with the apoptotic pathway. Indeed, stable expression of calbindin-D28K decreased the apoptotic effects of dexamethasone and forskolin, and the level of resistance to these agents correlated with the relative amount of calbindin expressed in each line. Overexpression of calbindin also increased cell survival in the presence of the calcium ionophore A23187. The stably expressed calcium-binding protein appeared to exert its protective effect subsequent to transcriptional activation, since glucocorticoid- and cAMP-induced gene expression were not affected. These data support the proposal that calcium fluxes are involved in apoptosis and suggest that high level expression of proteins that buffer calcium fluxes can effectively suppress death in apoptosis-susceptible cells.

Clinical observations have demonstrated a positive effect of estrogens and androgens on the maintenance of structural bone integrity. This study examines the direct effects of androgenic hormones on the osteoblast-like human osteosarcoma cell line, HOS TE85. Employing radiolabeled dihydrotestosterone (DHT), 2800 saturable, high-affinity (dissociation constant = 0.66 nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific in that DHT and testosterone (T) displayed significantly greater competition than the progestins, progesterone and medroxyprogesterone. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. A human androgen receptor complementary DNA probe revealed a 9.5 kilobase transcript which corresponds to the predominant human androgen receptor transcript detected in human male reproductive tissues. Androgens were also found to elicit biological responses in HOS TE85 cells. Physiological concentrations of DHT and T decreased HOS TE85 cell proliferation as assessed by cell count. This finding suggests that DHT may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with T (24 h, 10 nM) enhanced the abundance of both alpha 1(I)-procollagen messenger RNA (mRNA) (5-fold) and transforming growth factor-beta mRNA (2.2 fold). The nonaromatizable androgen DHT (24 h, 10 nM) elicited an increase in the steady state concentration of alpha 1(I)-procollagen mRNA similar to the increase observed with T treatment. Thus, in addition to the recent discovery of estradiol receptors and estrogenic regulation of HOS TE85 cells, it is now evident that these osteoblast-like osteosarcoma cells also express high affinity androgen binding sites and can respond biologically to androgens.

A finite human cell line was established from trabecular bone explants obtained from a 48-year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using the following independent criteria: (1) the presence of histochemically detectable alkaline phosphatase (AP) activity; (2) response to the calciotropic hormone 1,25-(OH)2D3 as assessed by increased AP activity; (3) synthesis and secretion of the osteoblast-specific marker bone gla protein; and (4) expression of alpha 1(I)-procollagen and alpha 1(III)-procollagen mRNAs in a pattern similar to that of other osteoblast-like cell lines. In addition to these classic osteoblast markers, BG688 cells also possess approximately 2400 high-affinity (Kd = 0.45 nM) 17 beta-estradiol (E2) binding sites per cell. The binding of E2 to these sites is specific, and of the steroid hormone agonists tested, E2 and diethylstilbestrol elicited the greatest amount of competition with radiolabeled E2. BG688 cells were also shown to respond to a physiologic concentration (10 nM) of E2. In vitro translation products of poly(A)+ RNA obtained from control and hormone-treated cells revealed a pleiotropic influence of E2 on the relative abundance of several mRNAs as assessed by two-dimensional gel electrophoretic analysis of their corresponding peptides. E2 also elicits a twofold increase in the steady-state concentration of alpha 1(I)-procollagen mRNA as demonstrated by northern blot hybridization. Thus, we here extend our previous data obtained in osteoblast-like osteosarcoma cells to indicate that a normal osteoblastic cell line is a target for the action of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene for rat bone gla protein (BGP) was isolated and 1250 basepairs (bp), including 1100 bp of 5' flanking DNA, were placed up-stream of the human GH reporter gene. After transient transfection into the osteoblast-like rat osteosarcoma cell line ROS 17/2.8, the BGP promoter demonstrated a low level of basal activity that was increased approximately 10-fold by the addition of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. A single 250-bp fragment (-523 to -274) was sufficient to confer hormone inducibility upon both heterologous and homologous promoters. Deletion studies, complemented by evaluation with synthetic oligomers, enabled localization of the 1,25-(OH)2D3 response element to within 19 bp (-456 to -438), containing an element with an imperfect direct repeat [GGTGA(N4)GGACA] and homology to other steroid-responsive elements. Gel retardation assays demonstrated that partially purified chick intestinal 1,25-(OH)2D3 receptor bound specifically and with high affinity to a DNA fragment containing the putative 1,25-(OH)2D3 response element, and this binding was perturbed by monoclonal antibodies to the 1,25-(OH)2D3 receptor. Surprisingly, the 250-bp fragment, when linked in an antisense orientation with respect to the BGP promoter, blocked basal and hormone-dependent gene expression. However, a 246-bp fragment 5' to the 250-bp element (-1100 to -855) restored 20-fold inducibility when linked to the first fragment in the same orientation, suggesting cooperativity between at least two elements to achieve the hormonal regulation observed in this gene.

FGF23 is a newly recognized regulator of serum phosphate, acting reciprocally to 1,25-dihydroxyvitamin D3 (1,25D), the hormonally active form of vitamin D. The hormones also act to regulate each other, with FGF23 down-regulating the production of 1,25D, and 1,25D acting via its nuclear receptor to up-regulate FGF23 production. Purpose To elucidate the mechanism whereby FGF23 gene transcription is up-regulated by the vitamin D receptor bound to 1,25D. Methods An initial approach to address this question was to construct a luciferase reporter construct containing the FGF23 proximal promoter. A second approach was to search mammalian genomic sequences for conserved vitamin D response elements (VDREs) and also conserved binding sites for other transcription factors in the vicinity of the FGF23 gene. Finally, chromatin immunoprecipitation (ChIP) experiments were performed using antibodies directed against modified histones to investigate the role of chromatin remodeling in 1,25D-stimulated FGF23 expression. A collaborator used real-time PCR (rt-PCR) to test whether 1,25D regulates two transcription factors for which conserved binding sites were found. Results The FGF23 reporter construct yielded a strong level of expression but very weak up-regulation (1.5X) by 1,25D. These data led us to believe that either our promoter segment did not contain the relevant control elements or that in vivo regulatory events (eg, chromatin remodeling) could not be replicated in our plasmid reporter construct. Other factors led us to conclude that 1,25D regulation of FGF23 might be a secondary effect: (a) genomic searches failed to yield conserved VDREs and (b) cyclohexamide experiments implied that protein synthesis was required for the 1,25D effect. We identified conserved binding sites for c-Ets-1 and GATA-3 in the FGF23 proximal promoter; however, we were unable to show 1,25D regulation by either factor using real-time PCR. Finally, ChIP experiments using various antisera to modified histones showed that histone H4 acetylation is increased in the FGF23 promoter in response to 1,25D. Conclusions Our results, so far, are consistent with FGF23 up-regulation occurring via a secondary mechanism. Unfortunately, a transcription factor that fits the criteria of a primary 1,25D target has not yet been identified. We have nevertheless been able to demonstrate that histone modification is part of the mechanism for FGF23 activation and are hopeful that further ChIP and bioinformatics approaches will allow us to focus on a specific region of the FGF23 gene where key regulatory elements may reside.

Fibroblast Growth Factor-23 (FGF23) is a systemic phosphaturic hormone that inhibits osteoblastic mineralization, blocks renal 25-OH-vitamin D3 bioactivation to 1,25(OH)2D3 (D3) and elicits hypophosphatemia by repressing the renal type IIa sodium-phosphate cotransporter. It has been shown that FGF23 causes a reduction in serum phosphate and D3 concentrations that appear to be caused by a preceding decrease and increase in the expression of 1αOHase and 24OHase genes, respectively. Also, serum FGF23 increases after treatment with D3, suggesting that D3 negatively feedback controls its circulating concentrations by inducing FGF23. We thus undertook this investigation to determine the molecular mechanism by which D3 increases the circulating level of FGF23. To this end we examined by Real-Time PCR the effect of D3 on FGF23 mRNA expression in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. The results reveal a dose- and time-dependent stimulation of endogenous FGF23 mRNA level in UMR osteoblasts in response to D3. The maximal increase was 117-fold at 10-6M D3, although for further experiments we chose a 10-7 M concentration of D3, to represent a more physiological concentration of the hormone. The increase of FGF23 mRNA was 55.8-fold after 24 hrs treatment, but statistically significant differences were observed as early as after 4-8 hrs. In addition, using cotreatment with actinomycin D or cyclohexamide, we observed that D3 regulates FGF23 expression at the transcriptional level, likely via the synthesis of an intermediary transfactor. To evaluate the biological significance of vitamin D regulation of FGF23 mRNA expression in vivo, we administered D3 (6μg/kg B.W.) to 4-5 week old C57BL/6 mice by IP injection. Calvaria were collected at 48 hrs after single injection and FGF23 mRNA levels were measured. The expression of FGF23 mRNA in this bone tissue was significantly up-regulated by D3. These results suggest that FGF23 and D3 are reciprocal phosphate regulatory hormones, with D3 initially acting to enhance blood phosphate levels via intestinal absorption and renal reabsorption, followed by a negative feedback effect of D3 to reduce phosphate by inducing FGF23 both diminishing D3 through 1;gaOHase repression and eliciting phosphaturia.