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.

Mediated by the nuclear vitamin D receptor (VDR), the hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D 3 (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.

Bexarotene, a drug approved for the treatment of cutaneous T-cell lymphoma (CTCL), is classified as a rexinoid due to its ability to act as a retinoid X receptor (RXR) agonist with high specificity. Rexinoids are capable of inducing RXR homodimerization leading to the induction of apoptosis and inhibition of proliferation in human cancers. Numerous studies have shown that bexarotene is effective in reducing viability and proliferation in CTCL cell lines. However, many treated patients present with cutaneous toxicity, hypothyroidism, and hyperlipidemia due to crossover activity with retinoic acid receptor (RAR), thyroid hormone receptor (TR), and liver X receptor (LXR) signaling, respectively. In this study, fourteen novel analogs were evaluated for their potential to bind RXR through modeling and then assayed in an RXR-RXR mammalian-2-hybrid (M2H) system and in RXRE-mediated luciferase reporter assays. In addition, these analogs were tested for their effectiveness to inhibit proliferation in CTCL cells relative to bexarotene. Furthermore, the most effective analogs were analyzed via qPCR to determine efficacy in modulating expression of two critical tumor suppressor genes, ATF3 and EGR3. Our results suggest that these new compounds possess similar or enhanced therapeutic potential as several of our novel rexinoids display more selective RXR activation with equivalent or greater reduction in CTCL cell proliferation, as well as the ability to induce ATF3 and EGR3. This work broadens our understanding of RXR-ligand relationships and permits development of more highly efficacious pharmaceutical drugs. Modifications of RXR agonists can yield agents with enhanced biological selectivity and potency when compared to the parent compound, potentially leading to improved patient outcomes. NCUIRE Undergraduate Research Program (ASU) & National Institutes of Health (NIH) CA140285 (to PWJ, CEW and PAM) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vitamin D is an essential nutrient, commonly acquired via dietary intake and/or from endogenous cutaneous synthesis in response to ultraviolet radiation. The biologically active form of vitamin D, 1,25‐dihydroxyvitamin D (1,25D), binds to the vitamin D receptor (VDR) and stimulates formation of an active hetero‐complex with the retinoid X receptor (RXR). This VDR‐RXR heterodimer controls vitamin D‐regulated genes in such target tissues as kidney and colon, modulates immune defenses, and controls cellular proliferation. VDR may also play a significant role in preventing oxidative damage, potentially delaying the aging process, and serving as an anti‐carcinogenic mediator. We hypothesize that VDR may target genes encoding antioxidant enzymes which contain antioxidant‐responsive elements (AREs) that act as binding sites for transcriptional regulators such as nuclear factor (erythroid‐derived 2)‐like 2 (Nrf‐2). This study aims to investigate the influence of vitamin D‐VDR signaling on Nrf‐2 activity. In order to probe a possible molecular mechanism, an ARE‐luciferase reporter plasmid was employed to measure Nrf‐2 activity in human embryonic kidney cells (HEK‐293) in the presence of 1,25D/VDR. Results indicate cells transfected with both Nrf‐2 and VDR displayed Nrf‐2 activity that was modulated in a 1,25D‐ and VDR‐dependent manner; with low 1,25D enhancing Nrf‐2 activity while higher concentrations inhibited Nrf‐2. When treating cells with 1,25D and/or urolithin‐A (UA), a nutraceutical hypothesized to cooperate with vitamin D, Nrf‐2 activity was instead consistently upregulated. Moreover, in qPCR studies with Nrf‐2 target genes GCLC and HMOX1, UA and 1,25D treatment resulted in similar enhancement and/or suppression of Nrf‐2, consistent with the luciferase‐based assays. Collectively, these results imply that VDR likely targets Nrf‐2 genes indirectly perhaps by influencing the activity of Nrf‐2 transfactors and/or by post‐translational modification of Nrf‐2 to either activate or suppress Nrf‐2‐directed gene regulation. The modulation of Nrf‐2 activity by a VDR‐mediated pathway identifies a possible regulatory role for vitamin D in anti‐oxidation and establishes the significance of vitamin D to human senescence and aging.

The active vitamin D hormone, 1,25‐dihydroxyvitamin D (1,25D), mediates its biological effects by binding to the nuclear vitamin D receptor (VDR) and promoting heterodimerization with retinoid X receptors (RXRs). Various nutraceuticals, including resveratrol and curcumin, have been postulated to interact with the vitamin D signaling pathway. More recently, health benefits attributed to pomegranate have been associated with its high content of polyphenols, specifically ellagitannins, which are metabolized by the gut microbiota to produce Urolithin A (UA). In this study, we investigated the ability of UA to modulate 1,25D signaling via transcription and qPCR assays. We hypothesized that 1,25D in combination with urolithin will stimulate VDR activity more than 1,25D alone, increasing transcription of 1,25D target genes. The increased activation of anti‐oxidation genes by 1,25D and UA could help combat reactive oxygen species and attenuate cellular “aging”. Our results indicate that there is a significant increase in VDR transcriptional activity when HEK‐293 kidney cells are treated with UA in conjunction with 1,25D at several 1,25D andUA concentrations. A similar effect was also observed using three structurally‐distinct 1,25D response elements. The potentiation of VDR activity via UA retains receptor‐selectivity since UA did not promote further activation of other receptor‐ligand complexes including estrogen‐ER, rexinoid‐RXR and TO‐LXR. Finally, amplification of serotonin production in brain cells was greatest with a combined cocktail of 1,25D/UA/resveratrol via qPCR (TPH2 gene) and ELISA (5‐HT). In order to probe the molecular mechanism of UA amplification in VDR activity, we tested VDR‐RXR heterodimerization and observed no effect of UA. However, UA activates AMP‐kinase, and we found potential AMP kinase phosphorylation consensus sites in the VDR protein sequence. Collectively, our novel results position gut microbiota‐derived UA as a putative VDR modulator, suggesting that the influences of 1,25D and UA converging on VDR may potentially mediate anti‐aging and promote longevity. Support or Funding Information Financial support/grant sponsor: National Institutes of Health; grant numbers: DK033351 and CA140285