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The active form of vitamin D, 1α,25-(OH) 2 D 3 , not only promotes intestinal calcium absorption, but also regulates the formation of osteoclasts (OCs) and their capacity for bone mineral dissolution. Gal-3 is a newly discovered bone metabolic regulator involved in the proliferation, differentiation, and apoptosis of various cells. However, the role of galectin-3 (gal-3) in OC formation and the regulatory effects of 1α,25-(OH) 2 D 3 have yet to be explored. To confirm whether gal-3 contributes to the regulatory effects of 1α,25-(OH) 2 D 3 on osteoclastogenesis, osteoclast precursors (OCPs) were induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). TRAP staining and bone resorption analyses were used to verify the formation and activation of OCs. qPCR, Western blotting, co-immunoprecipitation, and immunofluorescence assays were used to detect gene and protein expression. The regulatory effects of gal-3 in OC formation after treatment with 1α,25-(OH) 2 D 3 were evaluated using gal-3 siRNA. The results showed that 1α,25-(OH) 2 D 3 significantly increased gal-3 expression and inhibited OC formation and bone resorption. Expression levels of OC-related genes and proteins, matrix metalloproteinase 9 (MMP-9), nuclear factor of activated T cells 1 (NFATc1), and cathepsin K (Ctsk) were also inhibited by 1α,25-(OH) 2 D 3 . Gal-3 knockdown attenuated the inhibitory effects of 1α,25-(OH) 2 D 3 on OC formation, activation, and gene and protein expression. In addition, gal-3 was co-localized with the vitamin D receptor (VDR). These data suggest that gal-3 contributes to the osteoclastogenesis inhibitory effect of lα,25-(OH) 2 D 3 , which is involved in bone and calcium homeostasis.

Vitamin D 3 is an essential micronutrient mediating pleiotropic effects in multiple tissues and cell types via its metabolite 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), which activates the transcription factor vitamin D receptor. In this study, we used peripheral blood mononuclear cells (PBMCs) obtained from five healthy adults and investigated transcriptome-wide, whether the precursor of 1,25(OH) 2 D 3 , 25-hydroxyvitamin D 3 (25(OH)D 3 ), has gene regulatory potential on its own. Applying thresholds of >2 in fold change of gene expression and <0.05 as a false discovery rate, in this ex vivo approach the maximal physiological concentration of 25(OH)D 3 (250 nM (nmol/L)) none of the study participants had a significant effect on their PBMC transcriptome. In contrast, 1000 and 10,000 nM 25(OH)D 3 regulated 398 and 477 genes, respectively, which is comparable to the 625 genes responding to 10 nM 1,25(OH) 2 D 3 . The majority of these genes displayed specificity to the tested individuals, but not to the vitamin D metabolite. Interestingly, the genes MYLIP (myosin regulatory light chain interacting protein) and ABCG1 (ATP binding cassette subfamily G member 1) showed to be specific targets of 10,000 nM 25(OH)D 3 . In conclusion, 100- and 1000-fold higher 25(OH)D 3 concentrations than the reference 10 nM 1,25(OH) 2 D 3 are able to affect the transcriptome of PBMCs with a profile comparable to that of 1,25(OH) 2 D 3 .

Although vitamin D is critical for the function of the intestine, most studies have focused on the duodenum. We show that transgenic expression of the vitamin D receptor (VDR) only in the distal intestine of VDR null mice (KO/TG mice) results in the normalization of serum calcium and rescue of rickets. Although it had been suggested that calcium transport in the distal intestine involves a paracellular process, we found that the 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]-activated genes in the proximal intestine associated with active calcium transport ( Trpv6 , S100g , and Atp2b1 ) are also induced by 1,25(OH) 2 D 3 in the distal intestine of KO/TG mice. In addition, Slc30a10 , encoding a manganese efflux transporter, was one of the genes most induced by 1,25(OH) 2 D 3 in both proximal and distal intestine. Both villus and crypt were found to express Vdr and VDR target genes. RNA sequence (RNA-seq) analysis of human enteroids indicated that the effects of 1,25(OH) 2 D 3 observed in mice are conserved in humans. Using Slc30a10 -/- mice, a loss of cortical bone and a marked decrease in S100g and Trpv6 in the intestine was observed. Our findings suggest an interrelationship between vitamin D and intestinal Mn efflux and indicate the importance of distal intestinal segments to vitamin D action., (Copyright © 2020 American Society for Microbiology.)

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)

We have reported that 25-hydroxyvitamin D 3 [25(OH)D 3 ] binds to vitamin D receptor and exhibits several biological functions directly in vitro. To evaluate the direct effect of 25(OH)D 3 in vivo, we used Cyp27b1 knockout (KO) mice, which had no detectable plasma 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] when fed a diet containing normal Ca and vitamin D. Daily treatment with 25(OH)D 3 at 250 μg kg -1  day -1 rescued rachitic phenotypes in the Cyp27b1 KO mice. Bone mineral density, female sexual cycles, and plasma levels of Ca, P, and PTH were all normalized following 25(OH)D 3 administration. An elevated Cyp24a1 mRNA expression was observed in the kidneys, and plasma concentrations of Cyp24a1-dependent metabolites of 25(OH)D 3 were increased. To our surprise, 1,25(OH) 2 D 3 was detected at a normal level in the plasma of Cyp27b1 KO mice. The F1 to F4 generations of Cyp27b1 KO mice fed 25(OH)D 3 showed normal growth, normal plasma levels of Ca, P, and parathyroid hormone, and normal bone mineral density. The curative effect of 25(OH)D 3 was considered to depend on the de novo synthesis of 1,25(OH) 2 D 3 in the Cyp27b1 KO mice. This suggests that another enzyme than Cyp27b1 is present for the 1,25(OH) 2 D 3 synthesis. Interestingly, the liver mitochondrial fraction prepared from Cyp27b1 KO mice converted 25(OH)D 3 to 1,25(OH) 2 D 3 . The most probable candidate is Cyp27a1. Our findings suggest that 25(OH)D 3 may be useful for the treatment and prevention of osteoporosis for patients with chronic kidney disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

Genetic forms of vitamin D-dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR; however, using whole exome sequencing analysis, we identified a recurrent de novo missense mutation, c.902T>C (p.I301T), in CYP3A4 in both subjects that alters the conformation of substrate recognition site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than WT CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide insight into vitamin D metabolism and demonstrate that accelerated inactivation of vitamin D metabolites represents a mechanism for vitamin D deficiency.

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 endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D 3 to its hormonal form, 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1 , are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH) 2 D 3 -mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH) 2 D 3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1 We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH) 2 D 3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

Although fibroblast growth factor (FGF) 23 was recently identified as a phosphatonin that influences vitamin D metabolism, the underlying signaling mechanisms remain unclear. FGF23 elevates the renal levels of membrane-associated klotho as well as soluble klotho. Klotho is expressed on distal tubules. Upon enzymatic cleavage, soluble klotho is released into the renal interstitial space and then into the systemic circulation. The expression of 25-hydroxyvitamin D3 1α-hydroxylase (1-OH) on proximal tubular cells is controlled by parathyroid hormone (PTH). Klotho binds to various membrane proteins to alter their function. Here, the interaction between the PTH receptor and klotho was studied using various approaches, including immunoprecipitation, in vitro cell culture, and in vivo animal experiments. Immunoprecipitation studies demonstrate, for the first time, that recombinant human klotho protein interacts with human PTH receptors to inhibit the binding of human PTH. Furthermore, when applied to human proximal tubular cells, recombinant human klotho suppresses PTH-stimulated generation of inositol trisphosphate in vitro. Moreover, PTH-induced increase of cyclic AMP secretion and 1α,25-dihydroxyvitamin D3 (1,25VD) was attenuated by recombinant human klotho in vivo. In addition, recombinant human klotho inhibits the expression of 1-OH by PTH both in vitro and in vivo. These results suggest that free klotho mediates the FGF23-induced inhibition of 1,25VD synthesis., (© 2015 American Society for Bone and Mineral Research.)

Fibroblast growth factor-23 (Fgf23) is a bone-derived hormone, suppressing phosphate reabsorption and vitamin D hormone (1,25(OH)2 D3 ) production in the kidney. It has long been an enigma why lack of Fgf23 or of Klotho, the coreceptor for Fgf23, leads to severe impairment in bone mineralization despite the presence of hypercalcemia and hyperphosphatemia. Using Fgf23(-/-) or Klotho(-/-) mice together with compound mutant mice lacking both Fgf23 or Klotho and a functioning vitamin D receptor, we show that in Klotho(-/-) mice the mineralization defect is solely driven by 1,25(OH)2 D3 -induced upregulation of the mineralization-inhibiting molecules osteopontin and pyrophosphate in bone. In Fgf23(-/-) mice, the mineralization defect has two components, a 1,25(OH)2 D3 -driven component similar to Klotho(-/-) mice and a component driven by lack of Fgf23, causing additional accumulation of osteopontin. We found that FGF23 regulates osteopontin secretion indirectly by suppressing alkaline phosphatase transcription and phosphate production in osteoblastic cells, acting through FGF receptor-3 in a Klotho-independent manner. Hence, FGF23 secreted from osteocytes may form an autocrine/paracrine feedback loop for the local fine-tuning of bone mineralization., (© 2015 American Society for Bone and Mineral Research.)

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.

Purpose: Vitamin D pathway single nucleotide polymorphisms (SNPs) are potentially useful proxies for investigating whether circulating vitamin D metabolites [total 25-hydroxyvitamin-D, 25(OH)D; 1,25-dihydroxyvitamin, 1,25(OH)2D] are causally related to prostate cancer. We investigated associations of sixteen SNPs across seven genes with prostate-specific antigen-detected prostate cancer., Methods: In a nested case-control study (within the ProtecT trial), we estimated odds ratios and 95 % confidence intervals (CIs) quantifying associations between SNPs and prostate cancer. Subgroup analyses investigated whether associations were stronger in men who had high/low sun exposure [a proxy for 25(OH)D]. We quantified associations of SNPs with stage (T1-T2/T3-T4) and grade (<7/≥7). Multiple variant scores included SNPs encoding proteins involved in 25(OH)D synthesis and metabolism., Results: We included 1,275 prostate cancer cases (141 locally advanced, 385 high grades) and 2,062 healthy controls. Vitamin D-binding protein SNPs were associated with prostate cancer (rs4588-A: OR 1.20, CI 1.01, 1.41, p = 0.04; rs7041-T: OR 1.19, CI 1.02, 1.38, p = 0.03). Low 25(OH)D metabolism score was associated with high (vs low) grade (OR 0.76, CI 0.63, 0.93, p = 0.01); there was a similar association of its component variants: rs6013897-A in CYP24A1 (OR 0.78, CI 0.60, 1.01, p = 0.06) and rs10877012-T in CYP27B1 (OR 0.80, CI 0.63, 1.02, p = 0.07). There was no evidence that associations differed by level of sun exposure., Conclusion: We found some evidence that vitamin D pathway SNPs were associated with prostate cancer risk and grade, but not stage. There was no evidence of an association in men with deficient vitamin D (measured by having low sun exposure).

Throughout evolution, vertebrates including humans have depended on the sunshine vitamin D for their calcified skeletons. As our hunter gatherer forefathers ventured from the equator, their skin tone became much lighter in order to permit an adequate amount of ultraviolet B radiation to enter the skin to produce the vitally important vitamin D. Although sensible sun exposure does not significantly increase risk of skin cancer, it has remained a mystery as to why. Jiang and Bikle in their viewpoint provide a novel insight as to how Mother Nature was able to balance the need for receiving adequate sun exposure to produce vitamin D while limiting damage caused by the DNA absorbing the ultraviolet B radiation. Long non-coding RNAs which are plentiful in cells have a dual personality. Some enhance malignancy, while others act as tumor suppressors. Jiang and Bikle provide compelling evidence that these long non-coding RNAs in skin cells are responsive to 1,25-dihydroxyvitamin D3 by decreasing their carcinogenic activity while enhancing their tumor suppression function presumably as a strategy for reducing ultraviolet-induced non-melanoma skin cancer. Mother Nature got it right. Sensible sun exposure is important for maintaining an adequate vitamin D status. Once formed in the skin, vitamin D can exit into the circulation to carry out its physiologic functions on calcium and bone metabolism. Some vitamin D however remains in the skin and is activated to interact with its vitamin D receptor to control cell proliferation using a variety of strategies including interacting with long non-coding RNAs to reduce risk of photocarcinogenesis., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Sunlight, vitamin D and skin cancer form a controversial brew. While too much sunlight exposure causes skin cancer, it is the major source of vitamin D from skin. We propose that these processes can be balanced. Vitamin D signalling (VDS) protects against skin cancer as demonstrated by the susceptibility of the skin to tumor formation in VDR null mice and protection from UVB-induced mutations when VDR agonists are administered. The question is how is protection afforded. Previously, we have focused on the Wnt/β-catenin/hedgehog and DNA damage repair (DDR) pathways. As VDR regulates hundreds of genes with thousands of VDR response elements (VDRE) throughout the genome, and many VDREs are in non-coding regions, we decided to explore long non-coding RNAs (lncRNA). LncRNAs are mRNA-like transcripts ranging from 200 bases ~100 kb lacking significant open reading frames. They are aberrantly expressed in human cancers and involved in a spectrum of tumorigenic/metastatic processes (cell proliferation/apoptosis/angiogenesis). We discovered that VDS regulated the expression of certain lncRNAs in a manner consistent with VDS protection against skin cancer. Given the huge variation in genes actively regulated by 1,25(OH)2 D from different cell types, it is conceivable that our results could apply to personalized medicine based on the distinctive lncRNA profiles. These lncRNAs could also serve as skin cancer biomarkers secreted into the blood or urine via exosomes as demonstrated in other cancer types (breast, prostate). Modulation of lncRNA profile by VDS may also provide insight into regulating pathways such as Wnt/ß-catenin and hedgehog., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Fibroblast growth factor 23 (FGF23) gain of function mutations can lead to autosomal dominant hypophosphatemic rickets (ADHR) disease onset at birth, or delayed onset following puberty or pregnancy. We previously demonstrated that the combination of iron deficiency and a knock-in R176Q FGF23 mutation in mature mice induced FGF23 expression and hypophosphatemia that paralleled the late-onset ADHR phenotype. Because anemia in pregnancy and in premature infants is common, the goal of this study was to test whether iron deficiency alters phosphate handling in neonatal life. Wild-type (WT) and ADHR female breeder mice were provided control or iron-deficient diets during pregnancy and nursing. Iron-deficient breeders were also made iron replete. Iron-deficient WT and ADHR pups were hypophosphatemic, with ADHR pups having significantly lower serum phosphate (p < 0.01) and widened growth plates. Both genotypes increased bone FGF23 mRNA (>50 fold; p < 0.01). WT and ADHR pups receiving low iron had elevated intact serum FGF23; ADHR mice were affected to a greater degree (p < 0.01). Iron-deficient mice also showed increased Cyp24a1 and reduced Cyp27b1, and low serum 1,25-dihydroxyvitamin D (1,25D). Iron repletion normalized most abnormalities. Because iron deficiency can induce tissue hypoxia, oxygen deprivation was tested as a regulator of FGF23, and was shown to stimulate FGF23 mRNA in vitro and serum C-terminal FGF23 in normal rats in vivo. These studies demonstrate that FGF23 is modulated by iron status in young WT and ADHR mice and that hypoxia independently controls FGF23 expression in situations of normal iron. Therefore, disturbed iron and oxygen metabolism in neonatal life may have important effects on skeletal function and structure through FGF23 activity on phosphate regulation., Competing Interests: The other authors state that they have no conflicts of interest., (© 2014 American Society for Bone and Mineral Research.)

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.)

Protein 4.1R was first identified in the erythrocyte membrane skeleton. It is now known that the protein is expressed in a variety of epithelial cell lines and in the epithelia of many tissues, including the small intestine. However, the physiological function of 4.1R in the epithelial cells of the small intestine has not so far been explored. Here, we show that 4.1R knock-out mice exhibited a significantly impaired small intestinal calcium absorption that resulted in secondary hyperparathyroidism as evidenced by increased serum 1,25-(OH)2-vitamin D3 and parathyroid hormone levels, decreased serum calcium levels, hyperplasia of the parathyroid, and demineralization of the bones. 4.1R is located on the basolateral membrane of enterocytes, where it co-localizes with PMCA1b (plasma membrane calcium ATPase 1b). Expression of PMCA1b in enterocytes was decreased in 4.1(-/-) mice. 4.1R directly associated with PMCA1b, and the association involved the membrane-binding domain of 4.1R and the second intracellular loop and C terminus of PMCA1b. Our findings have enabled us to define a functional role for 4.1R in small intestinal calcium absorption through regulation of membrane expression of PMCA1b.

Vitamin D is a secosteroid hormone that has expanding importance for a healthy lifestyle and disease prevention. A multitude of studies have highlighted that vitamin D acts not only in bone and calcium homeostasis but is critically important for human immunity. The discovery that the storage form of vitamin D (25-hydroxyvitamin D3) can be locally converted to the active form (1,25-hydroxyvitamin D3) in immune cells, epithelial cells and numerous other non-renal tissues highlights the importance of maintaining sufficient stores. When responding to a specific external stimulus, like bacterial invasion, intracrine synthesis of active vitamin D has the ability to regulate gene expression providing a specific response and directing cellular actions. These responses include the generation of antimicrobial peptides with production of these peptides dependent on vitamin D status. Vitamin D deficiency is associated with an increased rate of infection. This paper highlights the antibiotic like actions of vitamin D and importance of vitamin D sufficiency.

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.

In addition to its traditional role in the regulation of calcium homeostasis and bone metabolism, vitamin D also exhibits immunomodulatory, anti-proliferative and cancer preventive activities. Molecular mechanisms that confer the chemo-preventive properties to vitamin D are poorly understood. We previously reported that constitutive phosphorylation of histone H2AX on Ser139 (γH2AX) and activation of ATM (Ser1981 phosphorylation), seen in untreated normal or tumor cells predominantly in S phase of the cell cycle, is to a large extent indicative of DNA replication stress occurring as a result of persistent DNA damage caused by endogenous oxidants, by-products of oxidative metabolism. In the present study we observed that exposure of mitogenically stimulated human lymphocytes, pulmonary carcinoma A549 and lymphoblastoid TK6 cells to 1,25-dihydroxyvitamin D3 (1,25-VD) reduced the level of constitutive expression of γH2AX and ATM-S1981P. We also observed that the H2O2-induced rise in the level of γH2AX in lymphocytes was attenuated by 1,25-VD. Whereas in lymphocytes 1,25-VD reduced by 50-70% the level of endogenous oxidants as determined by their ability to oxidize 2,7-dichlorodihydrofluorescein (DCFH) in A549 and TK6 cells the attenuation of DNA damage signaling by 1,25-VD was seen in the absence of detectable reduction in DCFH oxidation. These findings suggest that while the anti-oxidant activity of 1,25-VD may contribute to a reduction in the intensity of DNA replication stress in lymphocytes, other factors play a role in the 1,25-VD effects seen in A549 and TK6 cells. The data are consistent with the recent report on the interaction between DNA damage signaling (ATM activation) and 1,25D receptor (VDR) phosphorylation that lead to enhancement of DNA repair efficiency, and provide further support for the chemo-preventive and anti-aging properties of this vitamin/hormone.

Vitamin D from the diet or synthesized in the skin upon UV-B irradiation is converted in the organism into the active metabolite 1α,25- dihydroxyvitamin D 3 [1,25(OH) 2D 3, calcitriol], a pleiotropic hormone with wide regulatory actions. The classical model of 1,25(OH)2D3 action implies the activation of the vitamin D receptor, which binds specific DNA sequences in its target genes and modulates their transcription rate. We have recently shown that 1,25(OH) 2D 3 induces the expression of the JMJD3 gene coding for a histone demethylase that is involved in epigenetic regulation. JMJD3 mediates the effects of 1,25(OH) 2D 3 on a subset of target genes and affects the expression of ZEB1, ZEB2 and SNAI1, inducers of epithelial-mesenchymal transition. Novel data indicate that 1,25(OH) 2D 3 has an unanticipated wide regulatory action on the expression of genes coding for histone demethylases of the Jumonji C (JmjC) domain and lysine-specific demethylase (LSD) families. Moreover, JMJD3 knockdown decreases the expression of miR‑200b and miR‑200c, two microRNAs targeting ZEB1 RNA. This may explain the upregulation of this transcription factor found in JMJD3-depleted cells. Thus, 1,25(OH) 2D 3 exerts an ample regulatory effect on the expression of histone-modifying enzymes involved in epigenetic regulation that may mediate its actions on gene transcription and cell phenotype.

In this study, we employed 1α-hydroxylase knockout (1α-(OH)ase(-/-) ) mice to investigate the influence of 1,25-dihydroxy vitamin D(3) (1,25-(OH)(2) D(3) ) deficiency on the adult neurogenesis in the hippocampal dentate gyrus (DG). The numbers of both 24-hr-old BrdU(+) cells and proliferating cell nuclear antigen positive cells in 8-week-old 1α-(OH)ase(-/-) mice increased approximately twofold compared with wild-type littermates. In contrast, the numbers of 7- and 28-day-old BrdU(+) cells in 1α-(OH)ase(-/-) mice decreased by 50% compared with wild-type mice, while the proportion of BrdU(+) /NeuN(+) cells in BrdU(+) population showed no difference between 1α-(OH)ase(-/-) and wild-type mice. Apoptotic cells in the subgranular zone (SGZ) of DG markedly increased in 1α-(OH)ase(-/-) mice. Replenishment of 1,25-(OH)(2) D(3) , but not correction of serum calcium and phosphorus levels, completely prevented changes in the neurogenesis in 1α-(OH)ase(-/-) mice. The absence of 1,25-(OH)(2) D(3) led to an increase in the expression of L-type voltage-gated calcium channel (L-VGCC) and a decrease in the nerve growth factor (NGF) mRNA level. Treatment with the L-VGCC inhibitor nifedipine blocked the increased cell proliferations by 1,25-(OH)(2) D(3) deficiency. Administration of NGF significantly attenuated the loss of newborn neurons in 1α-(OH)ase(-/-) mice., (Copyright © 2010 Wiley Periodicals, Inc.)

Background: We previously demonstrated that 25-hydroxyvitamin D(3) concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D(3) can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells., Methodology/principal Findings: Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D(3), human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D(3) that resulted in the production of 1α,25-dihydroxyvitamin D(3). Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D(3) and 1α,25-dihydroxyvitamin D(3) production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D(3) synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells., Conclusions: The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.

The protein ERp57/GRP58 is a stress-responsive protein and a component of the protein disulfide isomerase family. Its functions in the endoplasmic reticulum are well known, concerning mainly the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class 1. However, ERp57 is present in many other subcellular locations, where it is involved in a variety of functions, primarily suggested by its participation in complexes with other proteins and even with DNA. While in some instances these roles need to be confirmed by further studies, a great number of observations support the participation of ERp57 in signal transduction from the cell surface, in regulatory processes taking place in the nucleus, and in multimeric protein complexes involved in DNA repair.

Background: Genetic Hypophosphatemic Rickets (HR) is a group of diseases characterized by renal phosphate wasting with inappropriately low or normal 1,25-dihydroxyvitamin D3 (1,25(OH)2D) serum levels. The most common form of HR is X-linked dominant HR (XLHR) which is caused by inactivating mutations in the PHEX gene. The purpose of this study was to perform genetic diagnosis in a cohort of patients with clinical diagnosis of HR, to perform genotype-phenotype correlations of those patients and to compare our data with other HR cohort studies., Methods: Forty three affected individuals from 36 non related families were analyzed. For the genetic analysis, the PHEX gene was sequenced in all of the patients and in 13 cases the study was complemented by mRNA sequencing and Multiple Ligation Probe Assay. For the genotype-phenotype correlation study, the clinical and biochemical phenotype of the patients was compared with the type of mutation, which was grouped into clearly deleterious or likely causative, using the Mann-Whitney and Fisher's exact test., Results: Mutations in the PHEX gene were identified in all the patients thus confirming an XLHR. Thirty four different mutations were found distributed throughout the gene with higher density at the 3' end. The majority of the mutations were novel (69.4%), most of them resulted in a truncated PHEX protein (83.3%) and were family specific (88.9%). Tubular reabsorption of phosphate (TRP) and 1,25(OH)2D serum levels were significantly lower in patients carrying clearly deleterious mutations than in patients carrying likely causative ones (61.39 ± 19.76 vs. 80.14 ± 8.80%, p = 0.028 and 40.93 ± 30.73 vs. 78.46 ± 36.27 pg/ml, p = 0.013)., Conclusions: PHEX gene mutations were found in all the HR cases analyzed, which was in contrast with other cohort studies. Patients with clearly deleterious PHEX mutations had lower TRP and 1,25(OH)2D levels suggesting that the PHEX type of mutation might predict the XLHR phenotype severity.

RANKL is a stromal cell-derived tumor necrosis factor (TNF)-like factor that plays a primary role in osteoclast formation and function. Recent studies suggest that 1,25(OH)(2) D(3) induces Rankl expression via vitamin D receptor (VDR) interaction at several enhancers located up to 76  kb upstream of the gene's transcriptional start site (TSS). In the current studies, we explored these interactions further using ChIP-chip and RNA analysis. We confirm VDR and RXR binding to the five enhancers described previously and identify two additional sites, one located within the Rankl coding region. We also show that RNA polymerase II is recruited to these enhancers, most likely through transcription factors TBP, TFIIB, and TAF(II) 250. Interestingly, the recruitment of these factors leads to the production of RNA transcripts, although their role at present is unknown. We also discovered that histone H4 acetylation (H4ac) marks many upstream Rankl enhancers under basal conditions and that H4ac is increased upon 1,25(OH)(2) D(3) treatment. Surprisingly, the hormone also induces C/EBPβ binding across the Rankl locus. C/EBPβ binding correlates directly with increased H4ac activity following 1,25(OH)(2) D(3) treatment. Finally, elevated H4ac is restricted to an extended region located between two potential insulator sites occupied by CTCF and Rad21. These data suggest a mechanism whereby 1,25(OH)(2) D(3) functions via the VDR and C/EBPβ to upregulate Rankl expression., (Copyright © 2011 Wiley-Liss, Inc.)

Vitamin D(3) hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D(3) (VD(3)) to 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)VD(3)) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD(3), i.e. the conversion of VD(3) to 25-hydroxyvitamin D(3) (25(OH)VD(3)) and then of 25(OH)VD(3) to 1α,25(OH)(2)VD(3), a hormonal form of VD(3). Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD(3)-bound, and 25(OH)VD(3)-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substrate-bound structure of Vdh-K1 accommodates both VD(3) and 25(OH)VD(3) but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD(3) hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD(3) 25-hydroxylase and 25(OH)VD(3) 1α-hydroxylase activities.

Parathyroid hormone (PTH), the active form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D], and PTH-related peptide (PTHrP), the mediator of hypercalcemia of malignancy, are all osteolytic hormones. Recent studies have demonstrated that endogenous PTH and PTHrP also exert bone anabolic activity and that PTHrP is a crucial modulator of growth plate development. At least part of these PTHrP functions can be mediated by intracrine effects, involving a unique interplay of cell surface membrane and intracellular signaling. 1,25(OH)2D also exerts bone anabolic effects and, as with PTHrP, acts on multiple extraskeletal tissues. The skeletal functions of these hormones now extend beyond modulating bone resorption, and important extraskeletal activities have been discovered which involve unique local modes of action., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background: Megalin and cubilin bind and internalize the 25(OH)D3-DBP complex in the kidney. Once the complex is internalized, 25(OH)D3 is released and activated to 1,25(OH)2D3 the ligand for the vitamin D receptor (VDR). Supporting the important role of cubilin in this process recent findings showed that cubilin deficiency results in decrease of 25(OH)D3 and 1,25(OH)2D3 plasma levels., Methods: Two hundred T1D patients and healthy controls (n=200) were genotyped for five polymorphisms (rs3740168, rs3740165, rs1801233, rs1801229 and rs2796835) within the cubilin gene. The polymorphisms were analyzed by RFLP or real time PCR. Statistic analyses were performed by using allele-wise and genotype-wise chi2 testing by using BiAS software. A p-value<0.05 was considered as significant., Results: We found that the genotype "AA" of the rs3740165 was more frequent in T1D patients compared to healthy controls (26.7% vs. 5.1%, p=4x10(-7)). Nevertheless no association between the rs3740165 polymorphism and the 25(OH)D3 or 1,25(OH)2D3 plasma levels was found. No association with the other studied polymorphisms was observed., Conclusion: Thus our findings reveal a novel association of the cubilin rs3740165 polymorphism with type 1 diabetes. Nevertheless how exactly this polymorphism could increase the risk to develop type 1 diabetes is subject for further investigations., (Copyright (c) 2010. Published by Elsevier Ltd.)

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.)

Context: Vitamin D deficiency is associated with many adverse health outcomes, yet little is known about the genetic epidemiology of vitamin D or its metabolites., Objective: Our objective was to examine the relationship among three vitamin D-related genes and levels of 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] in Hispanics (HAs) and African Americans (AAs)., Design and Setting: The cross-sectional Insulin Resistance Atherosclerosis Family Study recruited and examined subjects in: Los Angeles, California (AAs; 513 individuals from 42 families); San Luis Valley (SLV), Colorado (HAs; 513 individuals from 30 families); and San Antonio (SA), Texas (HAs; 504 individuals from 58 families)., Main Outcome Measures: Plasma levels of 25(OH)D and 1,25(OH)2D were measured., Results: Levels of 25(OH)D were highest in SLV-HAs [18.3 +/- 7.7 ng/ml (45.7 +/- 19.2 nmol/liter)], lower in SA-HAs [14.6 +/- 6.4 ng/ml (36.4 +/- 16.0 nmol/liter)], and lowest in AAs [11.0 +/- 5.4 ng/ml (27.5 +/- 13.5 nmol/liter)]. Levels of 1,25(OH)2D were similar in AAs [43.5 +/- 13.9 pg/ml (113.1 +/- 36.1 pmol/liter)] and SLV-HAs [43.2 +/- 13.3 pg/ml (112.3 +/- 34.6 pmol/liter)], but higher in SA-HAs [48.6 +/- 17.0 pg/ml (126.4 +/- 44.2 pmol/liter)]. After adjusting for gender and age within the site, two single nucleotide polymorphisms (SNPs) in the vitamin D binding protein gene (DBP), rs4588 and rs7041, were associated with 25(OH)D, and one SNP in the DBP, rs4588, was associated with 1,25(OH)2D at all three study centers., Conclusions: SNPs in the DBP are associated with levels of 25(OH)D and 1,25(OH)2D in HA and AA participants in the Insulin Resistance Atherosclerosis Family Study.

Recent advances in bone and calcium (Ca) metabolism have relied upon genetically modified mice. However, although human studies have identified gender as an important modulator of Ca metabolism, its effect on Ca metabolism has not been examined in mice. Here we examined basal and vitamin D-regulated Ca absorption (in situ ligated loops) and mRNA levels for the apical membrane calcium channel, TRPV6, and the calcium binding protein, calbindin D(9k) (CaBP) mRNA levels (real-time PCR) in duodenum of female and male mice. At 2 mo of age, females fed a 5 g Ca/kg diet had higher Ca absorption (62.3 +/- 4.8 vs. 47 +/- 3.6%) and TRPV6 mRNA levels than males even though plasma 1,25 dihydroxyvitamin D [1,25(OH)(2) D] was not different. In mice fed high (20 g/kg), normal (5 g/kg), or low (0.2 g/kg) Ca diets for 7 d to alter plasma 1,25(OH)(2) D (91 +/- 12, 322 +/- 25, and 587 +/- 43 pmol/L, respectively), the relation between Ca absorption (slope = 0.116 vs. 0.084, P = 0.021) or duodenum TRPV6 mRNA (slope = 0.042 vs. 0.025, P = 0.034) and circulating 1,25(OH)(2) D was steeper in females. After a single 1,25(OH)(2) D injection (200 ng/100 g body weight), peak induction of TRPV6 mRNA was 2-fold greater (at 6 h) and CaBP mRNA was 20% higher in females (at 16 h). Duodenal vitamin D receptor mRNA levels did not differ between genders. Our data indicate that female mice are more sensitive to changes in serum 1,25(OH)(2) D levels than males and that this must be considered when using mice to study calcium and bone biology.

Objective: The biologically active 1,25(OH)2 vitamin D3 and its analogs have been shown to have antiproliferative and differentiating effects in a variety of malignant and non-malignant cells. For squamous carcinoma cell lines of the head and neck (SCCHN) we could show that this antiproliferative activity of 1,25(OH)2 vitamin D3 is due to induced expression of the cell-cycle inhibitory proteins p21 and p27, causing an arrest in the G0/G1 cell-cycle phase., Material and Methods: In this work we investigated the effects of three vitamin D3 analogs, EB1089, MC1288 and CB1093, on proliferation behavior and cell-cycle status in a laryngeal carcinoma cell line (JPPA) as well as in control human immortalized keratinocytes (HaCaT). To study the molecular mechanism the functional activity of the promoter region of p21, a potential target gene of vitamin D3 transcriptional regulation, was investigated. For this reason a 2.7-kb fragment of the p21 promoter was isolated by polymerase chain reaction from HaCaT, JPPA and SCC9 (tongue carcinoma) cells and directionally cloned into an enhanced green fluorescence protein (EGFP) reporter gene vector system. A construct was used to stably transfect HaCaT cells and to monitor the expression of the EGFP gene by confocal microscopy., Results: Analysis of proliferation and cell-cycle status revealed decreased growth rates and G0/G1I cell-cycle arrest in cells treated with 1,25(OH)2 vitamin D3 and its analogs The EGFP reporter gene-transfected cells showed distinct fluorescence under the influence of 1,25(OH)2 vitamin D3 and its analogs compared to control cells., Conclusion: These results demonstrate that the cell-cycle inhibitor protein p21 is a direct target gene of biologically active 1,25(OH)2 vitamin D3, inducing G0/G1 cell-cycle arrest. The ability of vitamin D analogs to act via the same molecular mechanism as the natural hormone but with less hypercalcemic activity may have therapeutic implications for patients with SCCHN malignancy.

Vitamin D is emerging as an important dietary factor that affects the incidence and progression of many malignancies including prostate cancer. The active form of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)], inhibits the growth and stimulates the differentiation of prostate cancer cells. We have studied primary cultures of normal and cancer-derived prostatic epithelial cells as well as established human prostate cancer cell lines to elucidate the molecular pathways of 1,25(OH)(2)D(3) actions. These pathways are varied and appear to be cell specific. In LNCaP cells, 1,25(OH)(2)D(3) mainly causes growth arrest through the induction of insulin-like growth factor binding protein-3 and also stimulates apoptosis to a much smaller extent. We have used cDNA-microarray analyses to identify additional genes that are regulated by 1,25(OH)(2)D(3) and to raise novel therapeutic targets for use in the chemoprevention or treatment of prostate cancer. Less calcemic analogs of 1,25(OH)(2)D(3) that have more antiproliferative activity are being developed that will be more useful clinically. In target cells, 1,25(OH)(2)D(3) induces 24-hydroxylase, the enzyme that catalyzes its self inactivation. Cotreatment with 24-hydroxylase inhibitors enhances the antiproliferative activity of 1,25(OH)(2)D(3). The combination of other anticancer agents such as retinoids with vitamin D offers another promising therapeutic approach. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate-specific antigen increase in prostate cancer patients, which demonstrates proof of the concept that vitamin D or its analogs are clinically effective. Our research is directed at understanding the mechanisms of vitamin D action in prostate cells with the goal of developing chemoprevention and treatment strategies to improve prostate cancer therapy.

The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17beta-estradiol (17beta-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17beta-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17beta-E2. Treatment with 17beta-E2 upregulated mRNA levels of duodenal TRPV6 in these 1alpha-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 +/- 0.10 to 2.03 +/- 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1alpha-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17beta-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only.

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a secosteroid whose genomic mechanism of action is similar to that of other steroid hormones and is mediated by stereospecific interaction of 1,25(OH)(2)D(3) with the vitamin D receptor (VDR) which heterodimerizes with the retinoid X receptor (RXR). After interaction with the vitamin D response element (VDRE) in the promoter of target genes, transcription proceeds through the interaction of VDR with coactivators and with the transcription machinery. The identification of the steps involved in this process has been a major focus of recent research in the field. However, the functional significance of target proteins as well as the functional significance of proteins involved in the transport and metabolism of vitamin D is also of major importance. Within the past few years much new information has been obtained from studies using knockout and transgenic mice. New insight has been obtained using this technology related to the physiological significance of the vitamin D binding protein (DBP), used to transport vitamin D metabolites, as well as the physiological significance of target proteins including 25-hydroxyvitamin D(3) 24-hydroxylase (24(OH)ase), 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-(OH)ase), VDR, and osteopontin. The crystal structure of the DBP and the ligand binding domain of the VDR have recently been reported, explaining, in part, the unique properties of these proteins. In addition novel 1,25(OH)(2)D(3) target genes have been identified including the epithelial calcium channel, present in the proximal intestine and in the distal nephron. Thus in recent years a number of exciting discoveries have been made that have enhanced our understanding of mechanisms involved in the pleiotropic actions of 1,25(OH)(2)D(3)., (Copyright 2002 Wiley-Liss, Inc.)

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.)

Our aim was to determine the molecular targets involved in the antiproliferative effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in a normal murine mammary epithelial cell line, HC11. Among the early response genes analyzed, c-myc, junB, junD, c-jun, c-fos, fosB, fra, as well as max, mad1-4, sin3, only c-jun and fra-2 mRNAs were up-regulated after 1,25(OH)(2)D(3) exposure. Cyclin C was reduced and cyclin A2 and E were slightly enhanced; however, cyclins D1, D3, B1, B2, F, G1, G2, I and H, as well as TGF beta 1, TGF beta 3, T beta RI and T beta RII transcripts were not modulated by 1,25(OH)(2)D(3). Although p27(KIP1) protein content was unchanged, enhancement of p21(WAF1/CIP1) low basal levels in cell extracts and IGFBP-3 abundance on the culture medium was detected after 1,25(OH)(2)D(3) induction. Using differential display analysis, we identified eight down-modulated clones in exposed cells: 26S proteasome non-ATPase subunit Pad1, ubiquitin-conjugating enzyme Ube2i, extracellular proteinase inhibitor Expi or Wdnm1, cytochrome-c oxidase Cox7c, microtubule-associated protein-1 light chain-3 (Map1lc3), nascent-associated complex alpha Naca, transforming acidic coiled-coil Tacc3, stearoyl-CoA desaturase (Scd), keratin 6 alpha, and 1 up-regulated, fork head transcription factor Hfh-1L. Hence, the antiproliferative effect of 1,25(OH)(2)D(3) seems associated to enhancement of c-jun, Fra-2, IGFBP3 and p21(WAF1/CIP1). Decreased Pad1 and Ube2i might account for increased stability of cell cycle inhibitory proteins while reduced Wdnm1, Tacc3 and Scd might be secondary to accumulation of cells in G0/G1 phase.

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.