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.

There is considerable interest in identifying effective and safe drugs for neurodegenerative disorders. Cell culture and animal model work have demonstrated that modulating gene expression through RXR-mediated pathways may mitigate or reverse cognitive decline. However, because RXR is a dimeric partner for several transcription factors, activating off-target transcription is a concern with RXR ligands (rexinoids). This off-target gene modulation leads to unwanted side effects that can include low thyroid function and significant hyperlipidemia. There is a need to develop rexinoids that have binding specificity for subsets of RXR heterodimers, to drive desired gene modulation, but that do not induce spurious effects. Herein, we describe experiments in which we analyze a series of novel and previously reported rexinoids for their ability to modulate specific gene pathways implicated in neurodegenerative disorders employing a U87 cell culture model. We demonstrate that, compared to the FDA-approved rexinoid bexarotene (1), several of these compounds are equally or more effective at stimulating gene expression via LXREs or Nurr1/NBREs and are superior at inducing ApoE and/or tyrosine hydroxylase (TH) gene and protein expression, including analogs 8, 9, 13, 14, 20, 23, and 24, suggesting a possible therapeutic role for these compounds in Alzheimer's or Parkinson's disease (PD). A subset of these potent RXR agonists can synergize with a presumed Nurr1 ligand and antimalarial drug (amodiaquine) to further enhance Nurr1/NBREs-directed transcription. This novel discovery has potential clinical implications for treatment of PD since it suggests that the combination of an RXR agonist and a Nurr1 ligand can significantly enhance RXR-Nurr1 heterodimer activity and drive enhanced therapeutic expression of the TH gene to increase endogenous synthesis of dopamine. These data indicate that is it possible and prudent to develop novel rexinoids for testing of gene expression and side effect profiles for use in potential treatment of neurodegenerative disorders, as individual rexinoids can have markedly different gene expression profiles but similar structures.

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.

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.

Agonists are chemical compounds that bind to and activate their cognate receptors allowing for the propagation of a downstream signal. Receptor agonists exhibit varying pharmacodynamic properties, including binding affinity and receptor specificity. Bexarotene, a drug approved by the FDA in 1999 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. RXR is known to associate with other receptors in the nuclear receptor superfamily as either permissive or nonpermissive heterodimers that then modulate gene expression of numerous target genes. In a similar fashion, RXR‐selective rexinoids, such as bexarotene, also act as RXR agonists and influence RXR homodimerization leading to the induction of apoptosis and inhibition of cell proliferation in human cancers. In fact, several studies have shown that bexarotene is effective in reducing viability and proliferation in CTCL cell lines. However, the side effects of bexarotene therapy are significant and can include cutaneous toxicity, hypothyroidism, and hyperlipidemia due to crossover activity with retinoic acid receptor, thyroid hormone receptor, and liver X receptor signaling, respectively. Thus, the discovery of compounds with greater RXR specificity is an important goal in cancer therapeutics and drug development. In the current study, we have evaluated 14 novel analogs for their potential to bind RXR through modeling and via an RXR‐RXR mammalian‐2‐hybrid system. In addition, RXR response element‐mediated luciferase reporter assays were employed to determine the degree of transcriptional activation induced by each analog. Moreover, using a cell proliferation assay, these analogs were tested for their effectiveness to inhibit proliferation in CTCL cells relative to bexarotene. Finally, the most effective analogs were analyzed via qPCR to determine efficacy in modulating expression of two critical tumor suppressor genes, ATF3 and EGR3. Current results suggest that these new compounds possess similar or even 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 the development of more highly efficacious pharmaceutical drugs. Our results reveal that modifications of RXR agonists can yield agents with enhanced biological selectivity and potency when compared to the parent bexarotene compound, potentially leading to improved patient outcomes.