1,25-Dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D.

To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive elements (VDREs) at -7/-10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)-retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25-dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate-limiting enzyme in serotonin synthesis, was induced 2.2-fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8-fold in rat serotonergic RN46A-B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1-fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis-regulatory module; CRM) at -28 kb that 1,25D-dependently docked VDR, RXR, C/EBPß, and RUNX2. This CRM harbored 3 VDREs and single C/EBPß and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at -7/-10 kb and -28 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.

Resveratrol potentiates vitamin D and nuclear receptor signaling.

The 1,25-dihydroxyvitamin D3 (1,25D) hormone is derived from vitamin D generated in skin or obtained from the diet, and binds to and activates the vitamin D receptor (VDR) in target tissues including kidney, colon/small intestine, and bone/muscle. We tested resveratrol for its ability to modulate VDR signaling, using vitamin D responsive element (VDRE) and mammalian 2-hybrid (M2H) transcriptional system technology. Via VDRE-based assays in kidney, colon and myoblast cells, VDR-mediated transcription was activated by resveratrol, and a cooperative effect on transactivation was observed with resveratrol plus 1,25D. The M2H assay revealed a modest, resveratrol-induced dimerization of VDR with its retinoid X receptor (RXR) heteropartner. Cells treated with both resveratrol and 1,25D displayed synergistic stimulation of VDR-RXR heterodimerization, while resveratrol antagonized rexinoid-mediated RXR-RXR homodimerization. Increased transactivation in response to resveratrol was also observed with a subset of other nuclear receptors and their respective cognate responsive elements. Evaluation of wild-type versus a ligand-binding domain mutant VDR revealed that hormone-responsiveness to 1,25D was severely depressed, while the response to resveratrol was only moderately attenuated. Moreover, radiolabeled 1,25D-displacement assays demonstrated an increase in VDR-bound 1,25D in the presence of resveratrol. Thus, resveratrol may affect VDR and other nuclear receptors indirectly, likely via the ability of resveratrol to: (1) potentiate 1,25D binding to VDR; (2) activate RXR; and/or (3) stimulate SIRT1, an enzyme known to deacetylate nuclear receptors. The results of this study elucidate a possible pathway for crosstalk between two nutritionally derived lipids, vitamin D and resveratrol, both of which converge on VDR signaling.

Regulation of late cornified envelope genes relevant to psoriasis risk by plant-derived cyanidin.

The PSORS4 genetic risk factor for psoriasis is a deletion of two late cornified envelope (LCE) genes (LCE3C_LCE3Bdel) in a cluster of five LCE3 genes with a proposed role in skin repair. We previously showed that 1,25-dihydroxyvitamin D3 (1,25D) modestly upregulates transcripts from all five LCE3 genes as monitored by real time PCR in primary human keratinocytes. Herein we report that cyanidin, a plant-derived compound with anti-inflammatory/anti-oxidant properties, upregulates expression of all five LCE3 genes in cultures of differentiating primary human keratinocytes to a greater extent that does 1,25D. This action of cyanidin is dependent on the differentiation state of the keratinocytes, with a stronger effect after the cells have been incubated with 1.2mM calcium for 24h. Competition displacement assays using radiolabeled 1,25D revealed that cyanidin directly competes as a ligand for vitamin D receptor (VDR) binding with an estimated IC50 of 500µM. However, 20µM cyanidin is sufficient to upregulate LCE3 genes. The 25-fold discrepancy between the cyanidin concentration required for upregulating LCE3 genes in intact keratinocytes vs. that required for direct binding to VDR in vitro suggests that cyanidin may be: (a) metabolized to a more active VDR ligand in keratinocytes and/or (b) functioning via a non-VDR mediated mechanism. The fact that cyanidin is the most potent upregulator of global LCE3 gene expression reported to date suggests that this or related compounds may have potential in psoriasis therapy.

1,25-dihydroxyvitamin D(3) regulation of fibroblast growth factor-23 expression in bone cells: evidence for primary and secondary mechanisms modulated by leptin and interleukin-6.

Fibroblast growth factor-23 (FGF23) is a circulating hormone that acts to correct hyperphosphatemic states by inhibiting renal phosphate reabsorption and to prevent hypervitaminosis D by feedback repressing 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) biosynthesis. FGF23 gene expression in the osteoblast/osteocyte is induced by the nuclear vitamin D receptor (VDR) bound to 1,25(OH)2D3, but cycloheximide sensitivity of this induction suggests that it may occur largely via secondary mechanisms requiring cooperating transcription factors. We therefore sought to identify 1,25(OH)2D3-regulated transcription factors that might impact FGF23 expression. Although neither leptin nor interleukin-6 (IL-6) alone affects FGF23 expression, leptin treatment was found to potentiate 1,25(OH)2D3 upregulation of FGF23 in UMR-106 cells, whereas IL-6 treatment blunted this upregulation. Genomic analyses revealed conserved binding sites for STATs (signal transduction mediators of leptin and IL-6 action) along with transcription factor ETS1 in human and other mammalian FGF23 genes. Further, STAT3, STAT1, ETS1, and VDR mRNAs were induced in a dose-dependent manner by 1,25(OH)2D3 in UMR-106 cells. Bioinformatic analysis identified nine potential VDREs in a genomic interval containing human FGF23. Six of the putative VDREs were capable of mediating direct transcriptional activation of a heterologous reporter gene when bound by a 1,25(OH)2D3-liganded VDR complex. A model is proposed wherein 1,25(OH)2D3 upregulates FGF23 production directly via multiple VDREs and indirectly via induction of STAT3, ETS1, and VDR transcription factors that are then activated via cell surface and intracellular signaling to cooperate in the induction of FGF23 through DNA looping and generation of euchromatin architecture.

Molecular mechanisms of vitamin D action.

The hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D), initiates biological responses via binding to the vitamin D receptor (VDR). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either coactivators or corepressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. Thus, vitamin D action in a particular cell depends upon the metabolic production or delivery of sufficient concentrations of the 1,25D ligand, expression of adequate VDR and RXR coreceptor proteins, and cell-specific programming of transcriptional responses to regulate select genes that encode proteins that function in mediating the effects of vitamin D. For example, 1,25D induces RANKL, SPP1 (osteopontin), and BGP (osteocalcin) to govern bone mineral remodeling; TRPV6, CaBP(9k), and claudin 2 to promote intestinal calcium absorption; and TRPV5, klotho, and Npt2c to regulate renal calcium and phosphate reabsorption. VDR appears to function unliganded by 1,25D in keratinocytes to drive mammalian hair cycling via regulation of genes such as CASP14, S100A8, SOSTDC1, and others affecting Wnt signaling. Finally, alternative, low-affinity, non-vitamin D VDR ligands, e.g., lithocholic acid, docosahexaenoic acid, and curcumin, have been reported. Combined alternative VDR ligand(s) and 1,25D/VDR control of gene expression may delay chronic disorders of aging such as osteoporosis, type 2 diabetes, cardiovascular disease, and cancer.

Genomically anchored vitamin D receptor mediates an abundance of bioprotective actions elicited by its 1,25-dihydroxyvitamin D hormonal ligand.

The nuclear vitamin D receptor (VDR) mediates the actions of its physiologic 1,25-dihydroxyvitamin D3 (1,25D) ligand produced in kidney and at extrarenal sites during times of physiologic and cellular stress. The ligand-receptor complex transcriptionally controls genes encoding factors that regulate calcium and phosphate sensing/transport, bone remodeling, immune function, and nervous system maintenance. With the aid of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23), 1,25D/VDR primarily participates in an intricate network of feedback controls that govern extracellular calcium and phosphate concentrations, mainly influencing bone formation and mineralization, ectopic calcification, and indirectly supporting many fundamental roles of calcium. Beyond endocrine and intracrine effects, 1,25D/VDR signaling impacts multiple biochemical phenomena that potentially affect human health and disease, including autophagy, carcinogenesis, cell growth/differentiation, detoxification, metabolic homeostasis, and oxidative stress mitigation. Several health advantages conferred by 1,25D/VDR appear to be promulgated by induction of klotho, an anti-aging renal peptide hormone which functions as a co-receptor for FGF23 and, like 1,25D, regulates nrf2, foxo, mTOR and other cellular protective pathways. Among hundreds of genes for which expression is modulated by 1,25D/VDR either primarily or secondarily in a cell-specific manner, the resulting gene products (in addition to those expressed in the classic skeletal mineral regulatory tissues kidney, intestine, and bone), fall into multiple biochemical categories including apoptosis, cholesterol homeostasis, glycolysis, hypoxia, inflammation, p53 signaling, unfolded protein response and xenobiotic metabolism. Thus, 1,25D/VDR is a bone mineral control instrument that also signals the maintenance of multiple cellular processes in the face of environmental and genetic challenges.

The role of vitamin D in the FGF23, klotho, and phosphate bone-kidney endocrine axis.

1,25-dihydroxyvitamin D (1,25D), through association with the nuclear vitamin D receptor (VDR), exerts control over a novel endocrine axis consisting of the bone-derived hormone FGF23, and the kidney-expressed klotho, CYP27B1, and CYP24A1 genes, which together prevent hyperphosphatemia/ectopic calcification and govern the levels of 1,25D to maintain bone mineral integrity while promoting optimal function of other vital tissues. When occupied by 1,25D, VDR interacts with RXR to form a heterodimer that binds to VDREs in the region of genes directly controlled by 1,25D (e.g., FGF23, klotho, Npt2c, CYP27B1 and CYP24A1). By recruiting complexes of comodulators, activated VDR initiates a series of events that induces or represses the transcription of genes encoding proteins such as: the osteocyte-derived hormone, FGF23; the renal anti-senescence factor and protein co-receptor for FGF23, klotho; other mediators of phosphate transport including Npt2a/c; and vitamin D hormone metabolic enzymes, CYP27B1 and CYP24A1. The mechanism whereby osteocytes are triggered to release FGF23 is yet to be fully defined, but 1,25D, phosphate, and leptin appear to play major roles. The kidney responds to FGF23 to elicit CYP24A1-catalyzed detoxification of the 1,25D hormone while also repressing both Npt2a/c to mediate phosphate elimination and CYP27B1 to limit de novo 1,25D synthesis. Comprehension of these skeletal and renal actions of 1,25D should facilitate the development of novel mimetics to prevent ectopic calcification, chronic renal and vascular disease, and promote healthful aging.

Vitamin D receptor controls expression of the anti-aging klotho gene in mouse and human renal cells.

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

Vitamin D Receptor Mediates a Myriad of Biological Actions Dependent on Its 1,25-Dihydroxyvitamin D Ligand: Distinct Regulatory Themes Revealed by Induction of Klotho and Fibroblast Growth Factor-23.

The hormonal vitamin D metabolite, 1,25-dihydroxyvitamin D [1,25(OH)2D], produced in kidney, acts in numerous end organs via the nuclear vitamin D receptor (VDR) to trigger molecular events that orchestrate bone mineral homeostasis. VDR is a ligand-controlled transcription factor that obligatorily heterodimerizes with retinoid X receptor (RXR) to target vitamin D responsive elements (VDREs) in the vicinity of vitamin D-regulated genes. Circulating 1,25(OH)2D concentrations are governed by PTH, an inducer of renal D-hormone biosynthesis catalyzed by CYP27B1 that functions as the key player in a calcemic endocrine circuit, and by fibroblast growth factor-23 (FGF23), a repressor of the CYP27B1 renal enzyme, creating a hypophosphatemic endocrine loop. 1,25(OH)2D/VDR-RXR acts in kidney to induce Klotho (a phosphaturic coreceptor for FGF23) to correct hyperphosphatemia, NPT2a/c to correct hypophosphatemia, and TRPV5 and CaBP28k to enhance calcium reabsorption. 1,25(OH)2D-liganded VDR-RXR functions in osteoblasts/osteocytes by augmenting RANK-ligand expression to paracrine signal osteoclastic bone resorption, while simultaneously inducing FGF23, SPP1, BGLP, LRP5, ANK1, ENPP1, and TNAP, and conversely repressing RUNX2 and PHEX expression, effecting localized control of mineralization to sculpt the skeleton. Herein, we document the history of 1,25(OH)2D/VDR and summarize recent advances in characterizing their physiology, biochemistry, and mechanism of action by highlighting two examples of 1,25(OH)2D/VDR molecular function. The first is VDR-mediated primary induction of Klotho mRNA by 1,25(OH)2D in kidney via a mechanism initiated by the docking of liganded VDR-RXR on a VDRE at -35 kb in the mouse Klotho gene. In contrast, the secondary induction of FGF23 by 1,25(OH)2D in bone is proposed to involve rapid nongenomic action of 1,25(OH)2D/VDR to acutely activate PI3K, in turn signaling the induction of MZF1, a transcription factor that, in cooperation with c-ets1-P, binds to an enhancer element centered at -263 bp in the promoter-proximal region of the mouse fgf23 gene. Chronically, 1,25(OH)2D-induced osteopontin apparently potentiates MZF1. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Analysis of hairless corepressor mutants to characterize molecular cooperation with the vitamin D receptor in promoting the mammalian hair cycle.

The mammalian hair cycle requires both the vitamin D receptor (VDR) and the hairless (Hr) corepressor, each of which is expressed in the hair follicle. Hr interacts directly with VDR to repress VDR-targeted transcription. Herein, we further map the VDR-interaction domain to regions in the C-terminal half of Hr that contain two LXXLL-like pairs of motifs known to mediate contact of Hr with the RAR-related orphan receptor alpha and with the thyroid hormone receptor, respectively. Site-directed mutagenesis indicates that all four hydrophobic motifs are required for VDR transrepression by Hr. Point mutation of rat Hr at conserved residues corresponding to natural mutants causing alopecia in mice (G985W and a C-terminal deletion DeltaAK) and in humans (P95S, C422Y, E611G, R640Q, C642G, N988S, D1030N, A1040T, V1074M, and V1154D), as well as alteration of residues in the C-terminal Jumonji C domain implicated in histone demethylation activity (C1025G/E1027G and H1143G) revealed that all Hr mutants retained VDR association, and that transrepressor activity was selectively abrogated in C642G, G985W, N988S, D1030N, V1074M, H1143G, and V1154D. Four of these latter Hr mutants (C642G, N988S, D1030N, and V1154D) were found to associate normally with histone deacetylase-3. Finally, we identified three regions of human VDR necessary for association with Hr, namely residues 109-111, 134-201, and 202-303. It is concluded that Hr and VDR interact via multiple protein-protein interfaces, with Hr recruiting histone deacetylases and possibly itself catalyzing histone demethylation to effect chromatin remodeling and repress the transcription of VDR target genes that control the hair cycle.

Vitamin D receptor ligands, adenomatous polyposis coli, and the vitamin D receptor FokI polymorphism collectively modulate beta-catenin activity in colon cancer cells.

The activity of beta-catenin, commonly dysregulated in human colon cancers, is inhibited by the vitamin D receptor (VDR), and this mechanism is postulated to explain the putative anti-cancer activity of vitamin D metabolites in the colon. We investigated the effect of a common FokI restriction site polymorphism (F/f) in the human VDR gene as well as the effect of anti-tumorigenic 1,25-dihydroxyvitamin D(3) (1,25D) and pro-tumorigenic lithocholic acid (LCA) VDR ligands on beta-catenin transcriptional activity. Furthermore, the influence of a major regulatory protein of beta-catenin, the APC tumor suppressor gene, on VDR-dependent inhibition of beta-catenin activity was examined. We report herein that beta-catenin-mediated transcription is most effectively suppressed by the VDR FokI variant F/M4 when 1,25D is limiting. Using Caco-2 colorectal cancer (CRC) cells, it was observed that VDR ligands, 1,25D and LCA, both suppress beta-catenin transcriptional activity, though 1,25D exhibited significantly greater inhibition. Moreover, 1,25D, but not LCA, suppressed endogenous expression of the beta-catenin target gene DKK-4 independent of VDR DNA-binding activity. These results support beta-catenin sequestration away from endogenous gene targets by 1,25D-VDR. This activity is most efficiently mediated by the FokI gene variant F/M4, a VDR allele previously associated with protection against CRC. Interestingly, we found the inhibition of beta-catenin activity by 1,25D-VDR was significantly enhanced by wild-type APC. These results reveal a previously unrecognized role for 1,25D-VDR in APC/beta-catenin cross talk. Collectively, these findings strengthen evidence favoring a direct effect on the Wnt-signaling molecule beta-catenin as one anti-cancer target of 1,25D-VDR action in the colorectum.

Pomegranate derivative urolithin A enhances vitamin D receptor signaling to amplify serotonin-related gene induction by 1,25-dihydroxyvitamin D.

Mediated by the nuclear vitamin D receptor (VDR), the hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25D), is known to regulate expression of genes impacting calcium and phosphorus metabolism, the immune system, and behavior. Urolithin A, a nutrient metabolite derived from pomegranate, possibly acting through AMP kinase (AMPK) signaling, supports respiratory muscle health in rodents and longevity in C. elegans by inducing oxidative damage-reversing genes and mitophagy. We show herein that urolithin A enhances transcriptional actions of 1,25D driven by co-transfected vitamin D responsive elements (VDREs), and dissection of this genomic effect in cell culture reveals: 1) urolithin A concentration-dependency, 2) occurrence with isolated natural VDREs, 3) nuclear receptor selectivity for VDR over ER, LXR and RXR, and 4) significant 3- to 13-fold urolithin A-augmentation of 1,25D-dependent mRNA encoding the widely expressed 1,25D-detoxification enzyme, CYP24A1, a benchmark vitamin D target gene. Relevant to potential behavioral effects of vitamin D, urolithin A elicits enhancement of 1,25D-dependent mRNA encoding tryptophan hydroxylase-2 (TPH2), the serotonergic neuron-expressed initial enzyme in tryptophan metabolism to serotonin. Employing quantitative real time-PCR, we demonstrate that TPH2 mRNA is induced 1.9-fold by 10 nM 1,25D treatment in culture of differentiated rat serotonergic raphe (RN46A-B14) cells, an effect magnified 2.5-fold via supplementation with 10 µM urolithin A. This potentiation of 1,25D-induced TPH2 mRNA by urolithin A is followed by a 3.1- to 3.7-fold increase in serotonin concentration in culture medium from the pertinent neuronal cell line, RN46A-B14. These results are consistent with the concept that two natural nutrient metabolites, urolithin A from pomegranate and 1,25D from sunlight/vitamin D, likely acting via AMPK and VDR, respectively, cooperate mechanistically to effect VDRE-mediated regulation of gene expression in neuroendocrine cells. Finally, gedunin, a neuroprotective natural product from Indian neem tree that impacts the brain derived neurotropic factor pathway, similarly potentiates 1,25D/VDR-action.

Bioactive Dietary VDR Ligands Regulate Genes Encoding Biomarkers of Skin Repair That Are Associated with Risk for Psoriasis.

Treatment with 1,25-dihydroxyvitamin D3 (1,25D) improves psoriasis symptoms, possibly by inducing the expression of late cornified envelope (LCE)3 genes involved in skin repair. In psoriasis patients, the majority of whom harbor genomic deletion of LCE3B and LCE3C (LCE3C_LCE3B-del), we propose that certain dietary analogues of 1,25D activate the expression of residual LCE3A/LCE3D/LCE3E genes to compensate for the loss of LCE3B/LCE3C in the deletant genotype. Herein, human keratinocytes (HEKn) homozygous for LCE3C_LCE3B-del were treated with docosahexaenoic acid (DHA) and curcumin, two low-affinity, nutrient ligands for the vitamin D receptor (VDR). DHA and curcumin induce the expression of LCE3A/LCE3D/LCE3E mRNAs at concentrations corresponding to their affinity for VDR. Moreover, immunohistochemical quantitation revealed that the treatment of keratinocytes with DHA or curcumin stimulates LCE3 protein expression, while simultaneously opposing the tumor necrosis factor-alpha (TNFα)-signaled phosphorylation of mitogen activated protein (MAP) kinases, p38 and Jun amino-terminal kinase (JNK), thereby overcoming inflammation biomarkers elicited by TNFα challenge. Finally, DHA and curcumin modulate two transcription factors relevant to psoriatic inflammation, the activator protein-1 factor Jun B and the nuclear receptor NR4A2/NURR1, that is implicated as a mediator of VDR ligand-triggered gene control. These findings provide insights into the mechanism(s) whereby dietary VDR ligands alter inflammatory and barrier functions relevant to skin repair, and may provide a molecular basis for improved treatments for mild/moderate psoriasis.

Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (SERT) and degradation (MAO-A) gene expression in cultured rat serotonergic neuronal cell lines.

BACKGROUND: Diminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D3 (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D. RESULTS: Employing quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51-59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system. CONCLUSIONS: These results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.

1,25-Dihydroxyvitamin D3/VDR-mediated induction of FGF23 as well as transcriptional control of other bone anabolic and catabolic genes that orchestrate the regulation of phosphate and calcium mineral metabolism.

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

SIRT1 enzymatically potentiates 1,25-dihydroxyvitamin D3 signaling via vitamin D receptor deacetylation.

The hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), binds to the vitamin D receptor (VDR) and promotes heterodimerization of VDR with a retinoid-X-receptor (RXR) to genomically regulate diverse cellular processes. Herein, it is revealed for the first time that VDR is post-translationally acetylated, and that VDR immunoprecipitated from human embryonic kidney (HEK293) cells displays a dramatic decrease in acetylated receptor in the presence of 1,25D-ligand, sirtuin-1 (SIRT1) deacetylase, or the resveratrol activator of SIRT1. To elucidate the functional significance of VDR deacetylation, vitamin-d-responsive-element (VDRE)-based transcriptional assays were performed to determine if deacetylase overexpression affects VDR/VDRE-driven transcription. In HEK293 kidney and TE85 bone cells, co-transfection of low amounts (1-5ng) of a SIRT1-expression vector elicits a reproducible and statistically significant enhancement (1.3- to 2.6-fold) in transcription mediated by VDREs from the CYP3A4 and cyp24a1 genes, where the magnitude of response to 1,25D-ligand is 6- to 30-fold. Inhibition of SIRT1 via EX-527, or utilization of a SIRT1 loss-of-function mutant (H363Y), resulted in abrogation of SIRT1-mediated VDR potentiation. Studies with a novel, non-acetylatable VDR mutant (K413R) showed that the mutant VDR possesses enhanced responsiveness to 1,25D, in conjunction with reduced, but still significant, sensitivity to exogenous SIRT1, indicating that acetylation of lysine 413 is relevant, but that other acetylated residues in VDR contribute to modulation of its activity. We conclude that the acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This regulatory loop is reversed by SIRT1-catalyzed deacetylation of VDR to amplify VDR signaling and 1,25D actions.

1,25-Dihydroxyvitamin D and Klotho: A Tale of Two Renal Hormones Coming of Age.

1,25-Dihydroxyvitamin D3 (1,25D) is the renal metabolite of vitamin D that signals through binding to the nuclear vitamin D receptor (VDR). The ligand-receptor complex transcriptionally regulates genes encoding factors stimulating calcium and phosphate absorption plus bone remodeling, maintaining a skeleton with reduced risk of age-related osteoporotic fractures. 1,25D/VDR signaling exerts feedback control of Ca/PO4 via regulation of FGF23, klotho, and CYP24A1 to prevent age-related, ectopic calcification, fibrosis, and associated pathologies. Vitamin D also elicits xenobiotic detoxification, oxidative stress reduction, neuroprotective functions, antimicrobial defense, immunoregulation, anti-inflammatory/anticancer actions, and cardiovascular benefits. Many of the healthspan advantages conferred by 1,25D are promulgated by its induction of klotho, a renal hormone that is an anti-aging enzyme/coreceptor that protects against skin atrophy, osteopenia, hyperphosphatemia, endothelial dysfunction, cognitive defects, neurodegenerative disorders, and impaired hearing. In addition to the high-affinity 1,25D hormone, low-affinity nutritional VDR ligands including curcumin, polyunsaturated fatty acids, and anthocyanidins initiate VDR signaling, whereas the longevity principles resveratrol and SIRT1 potentiate VDR signaling. 1,25D exerts actions against neural excitotoxicity and induces serotonin mood elevation to support cognitive function and prosocial behavior. Together, 1,25D and klotho maintain the molecular signaling systems that promote growth (p21), development (Wnt), antioxidation (Nrf2/FOXO), and homeostasis (FGF23) in tissues crucial for normal physiology, while simultaneously guarding against malignancy and degeneration. Therefore, liganded-VDR modulates the expression of a "fountain of youth" array of genes, with the klotho target emerging as a major player in the facilitation of health span by delaying the chronic diseases of aging.

Association between Circulating Vitamin D Metabolites and Fecal Bile Acid Concentrations.

Although hydrophobic bile acids have been demonstrated to exhibit cytotoxic and carcinogenic effects in the colorectum, ursodeoxycholic acid (UDCA) has been investigated as a potential chemopreventive agent. Vitamin D has been shown to play a role in both bile acid metabolism and in the development of colorectal neoplasia. Using a cross-sectional design, we sought to determine whether baseline circulating concentrations of the vitamin D metabolites 25(OH)D and 1,25(OH)2D were associated with baseline fecal bile acid concentrations in a trial of UDCA for the prevention of colorectal adenoma recurrence. We also prospectively evaluated whether vitamin D metabolite concentrations modified the effect of UDCA on adenoma recurrence. After adjustment for age, sex, BMI, physical activity, and calcium intake, adequate concentrations of 25(OH)D (≥30 ng/mL) were statistically significantly associated with reduced odds for high levels of total [OR, 0.61; 95% confidence interval (CI), 0.38-0.97], and primary (OR, 0.61; 95% CI, 0.38-0.96) bile acids, as well as individually with chenodeoxycholic acid (OR, 0.39; 95% CI, 0.24-0.63) and cholic acid (OR, 0.56; 95% CI, 0.36-0.90). No significant associations were observed for 1,25(OH)2D and high versus low fecal bile acid concentrations. In addition, neither 25(OH)D nor 1,25(OH)2D modified the effect of UDCA on colorectal adenoma recurrence. In conclusion, this is the first study to demonstrate an inverse relationship between circulating levels of 25(OH)D and primary fecal bile acid concentrations. These results support prior data demonstrating that vitamin D plays a key role in bile acid metabolism, and suggest a potential mechanism of action for 25(OH)D in colorectal cancer prevention. Cancer Prev Res; 9(7); 589-97. ©2016 AACR.

FGF23 gene regulation by 1,25-dihydroxyvitamin D: opposing effects in adipocytes and osteocytes.

In a closed endocrine loop, 1,25-dihydroxyvitamin D3 (1,25D) induces the expression of fibroblast growth factor 23 (FGF23) in bone, with the phosphaturic peptide in turn acting at kidney to feedback repress CYP27B1 and induce CYP24A1 to limit the levels of 1,25D. In 3T3-L1 differentiated adipocytes, 1,25D represses FGF23 and leptin expression and induces C/EBPß, but does not affect leptin receptor transcription. Conversely, in UMR-106 osteoblast-like cells, FGF23 mRNA concentrations are upregulated by 1,25D, an effect that is blunted by lysophosphatidic acid, a cell-surface acting ligand. Progressive truncation of the mouse FGF23 proximal promoter linked in luciferase reporter constructs reveals a 1,25D-responsive region between -400 and -200  bp. A 0.6  kb fragment of the mouse FGF23 promoter, linked in a reporter construct, responds to 1,25D with a fourfold enhancement of transcription in transfected K562 cells. Mutation of either an ETS1 site at -346  bp, or an adjacent candidate vitamin D receptor (VDR)/Nurr1-element, in the 0.6  kb reporter construct reduces the transcriptional activity elicited by 1,25D to a level that is not significantly different from a minimal promoter. This composite ETS1-VDR/Nurr1 cis-element may function as a switch between induction (osteocytes) and repression (adipocytes) of FGF23, depending on the cellular setting of transcription factors. Moreover, experiments demonstrate that a 1 kb mouse FGF23 promoter-reporter construct, transfected into MC3T3-E1 osteoblast-like cells, responds to a high calcium challenge with a statistically significant 1.7- to 2.0-fold enhancement of transcription. Thus, the FGF23 proximal promoter harbors cis elements that drive responsiveness to 1,25D and calcium, agents that induce FGF23 to curtail the pathologic consequences of their excess.

Two basic amino acids C-terminal of the proximal box specify functional binding of the vitamin D receptor to its rat osteocalcin deoxyribonucleic acid-responsive element.

Nuclear hormone receptor-responsive element binding specificity has been reported to reside predominantly in the proximal box (P-box), three amino acids located in a DNA-recognition alpha-helix situated on the C-terminal side of the first zinc finger. To further define the residues in the vitamin D receptor (VDR) DNA binding domain (DBD) that mediate its interaction as a retinoid X receptor (RXR) heterodimer with the rat osteocalcin vitamin D-responsive element (VDRE), chimeric receptors were created in which the core DBD of VDR was replaced with that of the homodimerizing glucocorticoid receptor (GR). Systematic alteration of GR DBD amino acids in these chimeras to VDR DBD residues identified arg-49 and lys-53, just C-terminal of the P-box within the base recognition alpha-helix of human VDR (hVDR), as the only two amino acids among 36 differences required to convert the GR core zinc finger domain to that of the VDR. Gel mobility shift and 1,25-dihydroxyvitamin D3-stimulated transcription assays verified that an hVDR-GR DBD chimera is functional on the rat osteocalcin VDRE with only the conservative change of lys-49 to arg, and of the negatively charged glu-53 to a basic amino acid (lys or arg). Thus, for RXR heterodimerizing receptors like VDR, the P-box requires redefinition and expansion to include a DNA specificity element corresponding to arg-49 and lys-53 of hVDR. Examination of DNA specificity element amino acids in other nuclear receptors in terms of conservation and base contact in cocrystal structures supports the conclusion that these residues are crucial for selective DNA recognition.