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BACKGROUND Obesity has become a global public health problem. Obesity increases the risk of several lethal diseases. This study aimed to assess whether the obesity susceptibility was associated with genetic variation in vitamin D receptor (VDR) gene by conducting a meta-analysis. MATERIAL AND METHODS PubMed, EMBASE and Cochrane Library databases were screened for all relevant articles published up to October 2018. The pooled odds ratios (OR) were calculated using STATA 13.0 software for 4 polymorphisms in the VDR gene (ApaI, BsmI, FokI and TaqI). RESULTS Seven case-control studies, including 1188 obese patients and 1657 healthy controls, were recruited. The pooled findings showed that there were no associations between obesity risk and the VDR polymorphisms in ApaI, BsmI and TaqI loci overall. However, VDR TaqI polymorphism was associated with the risk of obesity in Asian under homozygous [TT versus tt: odds ratio (OR)=0.26, 95% confidence interval (CI)=0.14-0.49; P<0.001], heterozygous (Tt versus tt: OR=0.34, 95% CI=0.18-0.64; P=0.001), and dominant (TT+Tt versus tt: OR=0.30, 95% CI=0.17-0.52; P<0.001) models; FokI variant was related with increased risk of obesity only under dominant model (FF+Ff versus ff: OR=1.54, 95% CI=1.15-2.06; P=0.004). CONCLUSIONS Our meta-analysis results suggest that the T allele of TaqI may have a protective effect, while the F allele of FokI is proposed as a risk factor related to obesity.

Vitamin D receptor (VDR) null fetuses have normal serum minerals, parathyroid hormone (PTH), skeletal morphology, and mineralization but increased serum calcitriol, placental calcium transport, and placental expression of Pthrp, Trpv6, and (as reported in this study) Pdia3. We examined Cyp27b1 null fetal mice, which do not make calcitriol, to determine if loss of calcitriol has the same consequences as loss of VDR. Cyp27b1 null and wild-type (WT) females were mated to Cyp27b1 +/- males, which generated Cyp27b1 null and Cyp27b1 +/- fetuses from Cyp27b1 null mothers, and Cyp27b1 +/- and WT fetuses from WT mothers. Cyp27b1 null fetuses had undetectable calcitriol but normal serum calcium and phosphorus, PTH, fibroblast growth factor 23 (FGF23), skeletal mineral content, tibial lengths and morphology, placental calcium transport, and expression of Trpv6 and Pthrp; conversely, placental Pdia3 was downregulated. However, although Cyp27b1 +/- and null fetuses of Cyp27b1 null mothers were indistinguishable, they had higher serum and amniotic fluid calcium, lower amniotic fluid phosphorus, lower FGF23, and higher 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D than in WT and Cyp27b1 +/- fetuses of WT mothers. In summary, loss of fetal calcitriol did not alter mineral or bone homeostasis, but Cyp27b1 null mothers altered mineral homeostasis in their fetuses independent of fetal genotype. Cyp27b1 null fetuses differ from Vdr null fetuses, possibly through high levels of calcitriol acting on Pdia3 in Vdr nulls to upregulate placental calcium transport and expression of Trpv6 and Pthrp. In conclusion, maternal calcitriol influences fetal mineral metabolism, whereas loss of fetal calcitriol does not. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

A super-enhancer (SE) is a cluster of enhancers with a relatively high density of particular chromatin features. SEs typically regulate key genes that can determine cell identity and differentiation. Identifying SEs and their effects may be critical in predicting key regulatory genes, such as master transcription factor genes or oncogenes. Signal inducible SEs are dense stretches of signal terminal transcription factor (TF) binding regions, and may modulate the interaction between environmental factors (e.g., Vitamin D) and genetic factors (i.e., risk variants) in complex diseases such as multiple sclerosis (MS). As a complex autoimmune disease, the etiology and progression of MS, including the interaction between Vitamin D and MS risk variants, is still unclear and can be explored from the aspect of signal SEs. Vitamin D [with its active form: 1,25(OH) 2 D 3 ], is an environmental risk factor for MS. It binds the Vitamin D receptor (VDR) and regulates gene expression. This study explores the association between VDR super-enhancers (VSEs) and MS risk variants. Firstly, we reanalyse public ChIP-seq and RNA-seq data to classify VSEs into three categories according to their combinations of persistent and secondary VDR binding. Secondly, we indicate the genes with VSE regions that are near MS risk variants. Furthermore, we find that MS risk variants are enriched in VSE regions, and we indicate some genes with a VSE overlapping MS risk variant for further exploration. We also find two clusters of genes from the set of genes showing correlation of expression patterns with the MS risk gene ZMIZ1 that appear to be regulated by VSEs in THP-1 cells. It is the first time that VSEs have been analyzed, and we directly connect the genetic risk factors for MS risk with Vitamin D based on VSEs.

Vitamin D is an anti-inflammatory molecule and has a role in prevention of arthritis development. Biologically active form 1, 25(OH) 2 D3 of vitamin D can only exert its action after binding its definite vitamin D receptor encoded by VDR gene. VDR gene polymorphism leads to dysfunctioning of 1, 25(OH) 2 D 3 ultimately disease onset. The purpose of current study was to evaluate the effect of vitamin D level and VDR gene polymorphism on rheumatoid arthritis and osteoarthritis. Blood samples were collected from case and control after taking written consent. Serum was separated and vitamin D level as determined from each sample by ELISA. DNA was extracted from each blood sample and amplified by using gene specific primers. Genotyping was performed by Sangers sequencing and PCR-RFLP technique. It was found that vitamin D level was not significantly different among patients and controls. The rs10735810, rs1544410, rs7975232, and rs731236 were associated with the onset of arthritis at both allelic and genotypic level (p < 0.01). Nucleotide change on rs10735810 site leads to change of tryptophan with arginine. The frequencies of haplotype CGAT, CGGA, CGGT, CTAA, CTAT, TGAA, TGAT, TGGA, and TTGA were higher in patients and act as risk factors of RA onset, whereas haplotypes CGAT, CGAT, CGGT, CTGA, TGAT, TGGA, TTAA, and TTGA were associated with OA onset. In conclusion, serum vitamin D level may be normal among arthritis patients but polymorphism on VDR gene restricts vitamin D to perform its anti-inflammatory function by altering the 1, 25(OH)2 D3 binding sites.

Breast cancer is the leading cause of death among women worldwide. It is a multi-factorial disease caused by genetic and environmental factors. Vitamin D has been hypothesized to lower the risk of breast cancer via the nuclear vitamin D receptor (VDR). Genetic variants of these vitamin D metabolizing genes may alter the bioavailability of vitamin D, and hence modulate the risk of breast cancer. Materials and Methods: The distribution of Fok1 VDR gene (rs2228570) polymorphism and its association with breast cancer was analysed in a case–control study based on 125 breast cancer patients and 125 healthy females from North Indian population, using PCR-RFLP. An In silico exploration of the probable mechanism of increased risk of breast cancer was performed to investigate the role of single nucleotide polymorphisms (SNPs) in cancer susceptibility. Results: The Fok1 ff genotype was significantly associated with an increased risk of breast cancer (p=0.001; χ2=13.09; OR=16.909; %95 CI=2.20 - 130.11). In silico analysis indicated that SNPs may lead to a loss in affinity of VDR to calcitriol, and may also cause the impairment of normal interaction of liganded VDR with its heterodimeric partner, the retinoid X receptor (RXR), at protein level, thereby affecting target gene transcription. Conclusion: Breast cancer risk and pathogenesis in females can be influenced by SNPs. SNPs in VDR may cause alterations in the major molecular actions of VDR, namely ligand binding, heterodimerization and transactivation. VDRE binding and co-activator recruitment by VDR appear to be functionally inseparable events that affect vitamin D-elicited gene transcription. This indicates that breast cancer risk and pathogenesis in females may be influenced by SNPs., (Creative Commons Attribution License)

Resistance to vitamin D has been known for decades as vitamin D resistant rickets, caused by mutations of the gene encoding for vitamin D receptor (VDR). Findings of extra-skeletal effects of vitamin D and learning of the molecular mechanisms used by its biologically active metabolite calcitriol revealed other ways leading to its impaired sensitivity. Calcitriol takes advantage of both genomic and non-genomic mechanisms through its binding to vitamin D receptor, located not only in the cell nuclei but also in a perinuclear space. On the genomic level the complex of calcitriol bound to VDR binds to the DNA responsive elements of the controlled gene in concert with another nuclear receptor, retinoid X receptor, and expression of the VDR itself is controlled by its own ligand. These elements were found not only in the promotor region, but are scattered over the gene DNA. The gene expression includes a number of nuclear transcription factors which interact with the responsive elements and with each other and learning how they operate would further contribute to revealing causes of the impaired vitamin D sensitivity. Finally, the examples of major disorders are provided, associated with impairment of the vitamin D function and its receptor.

Vitamin D, besides its classical role in bone metabolism, plays a distinct role in multiple pathways of the feto-maternal unit. Calcitriol is the major active ligand of the nuclear vitamin D receptor (VDR). The vitamin D receptor (VDR) is expressed in different uteroplacental parts and exerts a variety of functions in physiologic pregnancy. It regulates decidualisation and implantation, influences hormone secretion and placental immune modulations. This review highlights the role of the vitamin D receptor in physiologic and disturbed pregnancy, as preeclampsia, fetal growth restriction, gestational diabetes and preterm birth. We discuss the existing literature regarding common VDR polymorphisms in these pregnancy disorders., Competing Interests: The authors declare no conflict of interest.

The vitamin D receptor (VDR) is the single known regulatory mediator of hormonal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in higher vertebrates. It acts in the nucleus of vitamin D target cells to regulate the expression of genes whose products control diverse, cell type-specific biological functions that include mineral homeostasis. In this Review we describe progress that has been made in defining new cellular sites of action of this receptor, the mechanisms through which this mediator controls the expression of genes, the biology that ensues, and the translational impact of this receptor on human health and disease. We conclude with a brief discussion of what comes next in understanding vitamin D biology and the mechanisms that underlie its actions.

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), whose expression in bone cells is regulated positively by 1,25(OH)2D3, retinoic acid, and parathyroid hormone through both intergenic and intronic enhancers. In this report, we used ChIP-sequencing analysis to confirm the presence of these Vdr gene enhancers in mesenchyme-derived bone cells and to describe the epigenetic histone landscape that spans the Vdr locus. Using bacterial artificial chromosome-minigene stable cell lines, CRISPR/Cas9 enhancer-deleted daughter cell lines, transient transfection/mutagenesis analyses, and transgenic mice, we confirmed the functionality of these bone cell enhancers in vivo as well as in vitro. We also identified VDR-binding sites across the Vdr gene locus in kidney and intestine using ChIP-sequencing analysis, revealing that only one of the bone cell-type enhancers bound VDR in kidney tissue, and none were occupied by the VDR in the intestine, consistent with weak or absent regulation by the 1,25(OH)2D3 hormone in these tissues, respectively. However, a number of additional sites of VDR binding unique to either kidney or intestine were present further upstream of the Vdr gene, suggesting the potential for alternative regulatory loci. Importantly, virtually all of these regions retained histone signatures consistent with those of enhancers and exhibited unique DNase I hypersensitivity profiles that reflected the potential for chromatin access. These studies define mechanisms associated with hormonal regulation of the Vdr and hint at the differential nature of VDR binding activity at the Vdr gene in different primary target tissues in vivo., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Signaling by hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D) has attracted increasing interest because of its non-classical actions, particularly its putative anticancer properties and its role in controlling immune system function. Notably, the hormone-bound vitamin D receptor (VDR) suppresses signaling by pro-inflammatory NF-κB transcription factors, although the underlying mechanisms have remained elusive. Recently, the VDR was shown to enhance the turnover of the oncogenic transcription factor cMYC mediated by the E3 ligase and tumor suppressor FBW7. As FBW7 also controls the turnover of the p100 (NF-κB2) subunit of the family, we determined whether the 1,25D enhanced FBW7-dependent turnover of NF-κB subunits p100, p105 (NF-κB1) and p65 (RELA). Protein levels of all three subunits declined markedly in the presence of 1,25D in multiple cell lines in the absence of substantial changes in mRNA expression. The VDR coimmunoprecipitated with all three subunits, and 1,25D treatment accelerated subunit turnover in cycloheximide-treated cells. Importantly, we observed an association of FBW7 with p105 and p65, as well as p100, and knockdown of FBW7 eliminated 1,25D-dependent subunit turnover. Moreover, expression of NF-κB target genes was elevated in FBW7-depleted cells. These results reveal that 1,25D signaling suppresses NF-κB function by enhancing FBW7-dependent subunit turnover.

The current study aimed to examine the gene specific mechanisms by which the actions of the vitamin D receptor (VDR) are distorted in prostate cancer. Transcriptional responses toward the VDR ligand, 1α,25(OH)2D3, were examined in non-malignant prostate epithelial cells (RWPE-1) and compared to the 1α,25(OH)2D3-recalcitrant prostate cancer cells (PC-3). Time resolved transcriptional studies for two VDR target genes revealed selective attenuation and repression of VDR transcriptional responses in PC-3 cells. For example, responses in PC-3 cells revealed suppressed responsiveness of IGFBP3 and G0S2. Furthermore, Chromatin Immunoprecipitation (ChIP) assays revealed that suppressed transcriptional responses in PC-3 cells of IGFBP3 and G0S2 were associated with selective VDR-induced NCOR1 enrichment at VDR-binding regions on target-gene promoter regions. We propose that VDR inappropriately recruits co-repressors in prostate cancer cells. Subsequent direct and indirect mechanisms may induce local DNA methylation and stable transcriptional silencing. Thus a transient epigenetic process mediated by co-repressor binding, namely, the control of H3K9 acetylation, is distorted to favor a more stable epigenetic event, namely DNA methylation. This article is part of a Special Issue entitled 'Vitamin D Workshop'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of genes involved in xenobiotic metabolism, including CYP1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I subfamily of the nuclear receptor superfamily, which also regulates expression of xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR signaling pathways and found that 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a potent physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human monocytic U937 cells and THP-1 cells, breast cancer cells, and kidney epithelium-derived cells. 1,25(OH)(2)D(3) alone did not induce CYP1A1, and 1,25(OH)(2)D(3) plus BaP did not increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. The combination of 1,25(OH)(2)D(3) and BaP increased CYP1A1 protein levels, BaP hydroxylation activity, and BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP alone. The combined effect of 1,25(OH)(2)D(3) and BaP on CYP1A1 mRNA expression in U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat (ER) 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP-mediated toxicity and the physiological function of this enzyme.

Compelling evidence suggests that vitamin D3 insufficiency may contribute causally to multiple sclerosis (MS) risk. Experimental autoimmune encephalomyelitis (EAE) research firmly supports this hypothesis. Vitamin D3 supports 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) synthesis in the CNS, initiating biological processes that reduce pathogenic CD4+ T cell longevity. MS is prevalent in Sardinia despite high ambient UV irradiation, challenging the vitamin D-MS hypothesis. Sardinian MS patients frequently carry a low Ifng expresser allele, suggesting that inadequate IFN-γ may undermine vitamin D3-mediated inhibition of demyelinating disease. Testing this hypothesis, we found vitamin D3 failed to inhibit EAE in female Ifng knockout (GKO) mice, unlike wild-type mice. The two strains did not differ in Cyp27b1 and Cyp24a1 gene expression, implying equivalent vitamin D3 metabolism in the CNS. The 1,25-(OH)2D3 inhibited EAE in both strains, but 2-fold more 1,25-(OH)2D3 was needed in GKO mice, causing hypercalcemic toxicity. Unexpectedly, GKO mice had very low Vdr gene expression in the CNS. Injecting IFN-γ intracranially into adult mice did not increase Vdr gene expression. Correlating with low Vdr expression, GKO mice had more numerous pathogenic Th1 and Th17 cells in the CNS, and 1,25-(OH)2D3 reduced these cells in GKO and wild-type mice without altering Foxp3+ regulatory T cells. Thus, the Ifng gene was needed for CNS Vdr gene expression and vitamin D3-dependent mechanisms that inhibit EAE. Individuals with inadequate Ifng expression may have increased MS risk despite high ambient UV irradiation because of low Vdr gene expression and a high encephalitogenic T cell burden in the CNS.

Two rare genetic diseases can cause rickets in children. The critical enzyme to synthesize calcitriol from 25-hydroxyvitamin D, the circulating hormone precursor, is 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). When this enzyme is defective and calcitriol can no longer be synthesized, the disease 1alpha-hydroxylase deficiency develops. The disease is also known as vitamin D-dependent rickets type 1 or pseudovitamin D deficiency rickets. When the VDR is defective, the disease hereditary vitamin D-resistant rickets, also known as vitamin D-dependent rickets type 2, develops. Both diseases are rare autosomal recessive disorders characterized by hypocalcemia, secondary hyperparathyroidism, and early onset severe rickets. In this article, these 2 genetic childhood diseases, which present similarly with hypocalcemia and rickets in infancy, are discussed and compared., (Copyright 2010 Elsevier Inc. All rights reserved.)

The actions of the vitamin D hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mediated by the vitamin D receptor (VDR), a ligand-activated transcription factor that functions to control gene expression. After ligand activation, the VDR binds directly to specific sequences located near promoters and recruits a variety of coregulatory complexes that perform the additional functions required to modify transcriptional output. Recent advances in transcriptional regulation, which permit the unbiased identification of the regulatory regions of genes, are providing new insight into how genes are regulated. Surprisingly, gene regulation requires the orchestrated efforts of multiple modular enhancers often located many kilobases upstream, downstream, or within the transcription units themselves. These studies are transforming our understanding of how 1,25(OH)(2)D(3) regulates gene transcription., (Copyright 2010 Elsevier Inc. All rights reserved.)

The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) through its ability to regulate cellular programs of gene expression. We identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides using a mammalian two-hybrid system and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. Peptides were identified that were reactive to RXR alone as well as to both VDR and RXR. Peptide fusion proteins were then examined in MC3T3 E1 cells for their ability to block induction of the osteocalcin promoter by 1,25(OH)(2)D(3) or stimulation of an RARE-TK reporter by 9-cis RA. Peptides that interacted with both VDR and RXR blocked 1,25(OH)(2)D(3)-dependent transcription by up to 75%. Peptides that interacted with RXR blocked 9-cis RA induced transcription. Two RXR-interacting peptides, however, were also found to block 1,25(OH)(2)D(3) response effectively. These studies support the idea that comodulator recruitment is essential for VDR- and RXR-mediated gene expression and that RXR is required for 1,25(OH)(2)D(3)-induced osteocalcin gene transcription. This approach may represent a novel means of assessing the contribution of RXR in various endogenous biological responses to 1,25(OH)(2)D(3)., (Copyright 2002 Wiley-Liss, Inc.)

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures., (Copyright 2002 Wiley-Liss, Inc.)

Background: The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) acts through the transcription factor vitamin D receptor (1alpha,25(OH)(2)D(3) receptor, VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins and specific DNA binding sites (1alpha,25(OH)(2)D(3) response elements, VDREs). Ligand-mediated conformational changes of the VDR are the basis of the molecular mechanisms of nuclear 1alpha,25(OH)(2)D(3) signaling. Cell-specific VDR antagonists would allow to dissect and fine regulate the pleiotropic 1alpha,25(OH)(2)D(3) endocrine system affecting the regulation of calcium homeostasis, bone mineralization and other cellular functions., Results: Two carboxylic ester analogues of 1alpha,25(OH)(2)D(3), ZK159222 and ZK168281, which have additional cyclopropyl rings and allylic alcohol substructures in their side chain, were characterized in different 1alpha, 25(OH)(2)D(3) target tissues as functional antagonists of 1alpha, 25(OH)(2)D(3) signaling. In all tested systems, ZK168281 showed lower residual agonistic activity and higher antagonistic effects than ZK159222, but the strength of these effects was cell-specific. Both antagonists were shown to act via the same mechanisms: they selectively stabilize an antagonistic conformation of the ligand-binding domain of the VDR within VDR-RXR-VDRE complexes, which then inhibits the interaction of the VDR with coactivator proteins and an induction of transactivation. Interestingly, cells that have been treated with antagonists were found to contain VDR-RXR heterodimers in a different conformation than cells that were stimulated with an agonist. Moreover, the strength of the functional antagonism of ZK159222 and ZK168281 appears to depend on the VDR/RXR expression ratio and high RXR levels were found to reduce the antagonistic effect of both compounds. In support of this observation, the overexpression of an transactivation function 2 (AF-2) deletion mutant of RXR resulted for both ZK159222 and ZK168281 in a reduced agonistic activity and an increased antagonistic effect., Conclusions: A novel, more potent VDR antagonist, ZK168281, was identified, which stabilizes VDR-RXR heterodimers in living cells in a different conformation than agonists. In addition, the VDR/RXR ratio was found as the major discriminating factor for understanding cell-specific effects of VDR antagonists.

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.

Vitamin D is an important regulator of calcium homeostasis in the body and it plays an essential role in bone metabolism. 1,25(OH)2D (calcitriol), the active metabolite of vitamin D stimulates intestinal calcium and phosphate absorption, thereby maintaining sufficient concentrations of these ions in the extracellular fluids, necessary for normal mineralization of bone matrix. Furthermore, calcitriol exerts a number of important functions in regulating bone metabolism. Similar to other classical steroid hormones, calcitriol regulates the transcription of a large number of target genes. A large number of recent studies of the mechanisms of genomic actions of calcitriol have been published. The aim of this review is to give a summary of these recent findings. The interaction between calcitriol and the vitamin D receptor as well as receptor structure and function in relation to transcriptional regulation are discussed.

The active metabolite of vitamin D, 1,25 (OH)2D3, exerts its cell cycle regulating effects via binding to VDR (Vitamin D Receptor). This complex forms a heterodimer with RXR (Retinoic X Receptor). The VDR-RXR heterodimer binds to promoter regions of cell cycle regulating genes through a vitamin D response element (VDRE). The tumour suppressor gene cyclin kinase inhibitor (Cki) p21, one of the well known cell cycle regulating genes, is one of the genes regulated in this manner. Its tumour suppressive action is through inhibition of cell division. These molecular biological mechanisms and large epidemiological investigations give strong support for the benefits of vitamin D in preventing colon cancer and prostate cancer.

We showed previously that liganded vitamin D receptor (VDR) effects a suppression of human atrial natriuretic peptide (hANP) gene-promoter activity in cultured neonatal rat atrial myocytes. In the present study, we have attempted to identify the structural domains of the VDR that are involved in mediating this suppression. We examined the effects of a series of VDR mutants on a cotransfected hANP promoter-driven chloramphenicol acetyltransferase (CAT) reporter. Neither the native VDR nor any of the mutants tested displayed inhibitory activity in the absence of the 1,25-dihydroxyvitamin D3 (VD3) ligand. Delta134, a deletant harboring solely the DNA binding region of the VDR, and L254G, a mutant shown to be defective in retinoid X receptor (RXR) heterodimer formation in other systems, were as effective as the native VDR in reducing promoter activity. HBD, a deletant containing only the hormone-binding domain of the VDR, and K246G, a point mutant that is defective in the activation function of the receptor, did not attenuate reporter activity. A similar activity profile was displayed when a positively regulated promoter containing a direct-repeat vitamin D responsive element (DR3-CAT) was examined in these cells. Liganded VDR, the delta134 mutant, and liganded L254G effected increases in DR3-CAT activity of 2.5-, 2-, and 4-fold, respectively. Two nonhypercalcemic analogues of VD3 (RO 23-7553 and RO 25-6760) displayed the same inhibitory activity as VD3. These studies suggest that the inhibition of hANP promoter activity requires both the DNA binding and activation functions of the receptor but does not appear to require formation of a classic RXR alpha-VDR heterodimer.

Analogs of 1,25-dihydroxyvitamin D3 (1,25D3) can be used to elucidate details of vitamin D receptor (VDR) activation. The A ring-modified analog, (TN-2) has 15-fold less affinity for VDR, but its transcriptional activity is diminished 1000-fold. Likewise, the ability of TN-2 to induce a protease-resistant conformation in VDR is 1/1000 that of 1,25D3. The stability of the VDR-TN-2 complexes is also significantly lower than VDR-1,25D3 complexes. Mapping the VDR-binding site of TN-2 showed that it had a significantly greater requirement for transcription activation function 2 (AF-2) residues than 1,25D3 did. These results suggest that the increased requirement for AF-2 residues that was induced by the A ring modifications is associated with diminished receptor activation. To determine whether restoring the potency of TN-2 by additional structural modifications would change the requirements for AF-2 residues, we synthesized hybrid analogs with 1beta-hydroxymethyl-3-epi groups and with dimethyl groups at positions 26 and 27 of the side chain, without or with a double bond between CD ring positions 16 and 17. We found that the side chain modification enhanced transcriptional activity 150-fold, increased the ability of the receptor to form a protease-resistant conformation 100-fold, and stabilized the VDR-analog complexes. The addition of the 16-ene group further reduced the analog's dissociation rate and increased its potency in the protease assays. These functional changes in the hybrid analogs were associated with a significant reduction in interaction with AF-2 residues. We conclude that there is an inverse relationship between analogs' potencies and their interaction with AF-2 residues of VDR.

Recent studies have identified a heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) as the active complex for mediating positive transcriptional effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active hormonal form of vitamin D. The VDR-RXR heterodimer has been shown to bind to direct repeat vitamin D-responsive elements (VDREs) upstream of positively controlled genes in the target tissues for vitamin D, including bone (osteocalcin, osteopontin, and beta 3 integrin), kidney (24-hydroxylase) and intestine (calbindin). Residues that participate in heterodimer formation have been identified in the C-terminal hormone-binding domain by analysis of VDR mutants. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is not entirely clear, but studies of two natural VDR mutants suggest that the binding of both hormone and RXR are required to induce a receptor conformation that is competent to activate transcription. A final level of complexity is added by recent observations that VDR is modified by phosphorylation. Thus, the VDR-mediated action of 1,25(OH)2D3 is now known to involve multiple factors that may provide a conceptual basis for future understanding of the tissue-specific genomic effects of 1,25(OH)2D3.

Objective: Hereditary vitamin D resistant rickets (HVDRR) has been shown to be due to mutations in the gene encoding the vitamin D receptor (VDR). In two patients with the characteristic phenotype we have investigated the functional defect and sequenced the VDR cDNA. We report two new mutations in the DNA binding domain of the VDR gene and we have used the crystallographic structure of the glucocorticoid and oestrogen receptors (GR and ER respectively) as models to explain the stereochemical consequences of these mutations., Design: Patient and control cell lines prepared from skin fibroblasts were used to measure binding of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and functional responses to this hormone. These cells were also used to isolate VDR mRNA from which cDNA was prepared and sequenced. VDR cDNA from affected and control patients was also transfected into receptor defective cells to analyse further functional responses to 1,25(OH)2D3. Computer analysis of mutations in the VDR gene was carried out using the glucocorticoid and oestrogen receptors as model systems., Patients: Two patients with HVDRR from unrelated families., Measurements: Cytosolic binding and nuclear association of 1,25(OH)2D3 were determined in control and affected patients, and functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamin D-24-hydroxylase activity (24-hydroxylase). VDR cDNA was sequenced and transfected into VDR-deficient CV-1 cells for further analysis of functional response to 1,25(OH)2D3 following cotransfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid., Results: Cells from HVDRR patients I and II showed detectable numbers of VDR with normal hormone binding. However, unlike controls, the HVDRR cells did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)2D3. Sequencing of cDNA revealed single mutations, in patient I (Phe44-->IIe) and in patient II (Lys42-->Glu). Both these residues are conserved in the steroid/thyroid hormone receptor superfamily and stereochemical analysis has been used to deduce the importance of these amino acids and the deleterious effect of these and other mutations in the DNA-binding domain of the VDR., Conclusions: Two new mutations in the vitamin D receptor which cause hereditary vitamin D resistant rickets have been described and using molecular modelling we have been able to analyse the genesis of this inherited disease at the level of stereochemistry.