Vitamin D Receptor Ligand Research Articles

Pharmacologic or genetic interference with atrogene signaling protects against glucocorticoid-induced musculoskeletal and cardiac disease.

Despite their beneficial actions as immunosuppressants, glucocorticoids (GC) have devastating effects on the musculoskeletal and cardiac systems, as long-term treated patients exhibit high incidence of falls, bone fractures, and cardiovascular events. Herein, we show that GC upregulate simultaneously in bone, skeletal muscle, and the heart the expression of E3 ubiquitin ligases (atrogenes), known to stimulate the proteasomal degradation of proteins. Activation of vitamin D receptor (VDR) signaling with the VDR ligands calcitriol or eldecalcitol prevented GC-induced atrogene upregulation in vivo and ex vivo in bone/muscle organ cultures and preserved tissue structure/mass and function of the 3 tissues in vivo. Direct pharmacologic inhibition of the proteasome with carfilzomib also conferred musculoskeletal protection. Genetic loss of the atrogene MuRF1-mediated protein ubiquitination in ΔRING mice afforded temporary or sustained protection from GC excess in bone or skeletal and heart muscle. We concluded that the atrogene pathway downstream of MuRF1 underlies GC action in bone, muscle, and the heart, and it can be pharmacologically or genetically targeted to confer protection against the damaging actions of GC simultaneously in the 3 tissues.

Vitamin D in Melanoma: Potential Role of Cytochrome P450 Enzymes.

Vitamin D is a promising anticancer agent for the prevention and treatment of several cancers, including melanoma. Low 25-hydroxyvitamin D levels, a routinely used marker for vitamin D, have been suggested as one of the factors in the development and progression of melanoma. The parent vitamin D needs activation by cytochrome P450 (CYP) enzymes to exert its actions via the vitamin D receptor (VDR). This review discusses the role of vitamin D in melanoma and how CYP-mediated metabolism can potentially affect the actions of vitamin D. Through interacting with the retinoid X receptor, VDR signaling leads to anti-inflammatory, antioxidative, and anticancer actions. Calcitriol, the dihydroxylated form of vitamin D3, is the most active and potent ligand of VDR. CYP27A1, CYP27B1, and CYP2R1 are involved in the activation of vitamin D, whereas CYP24A1 and CYP3A4 are responsible for the degradation of the active vitamin D. CYP24A1, the primary catabolic enzyme of calcitriol, is overexpressed in melanoma tissues and cells. Several drug classes and natural health products can modulate vitamin D-related CYP enzymes and eventually cause lower levels of vitamin D and its active metabolites in tissues. Although the role of vitamin D in the development of melanoma is yet to be fully elucidated, it has been proposed that melanoma prevention may be significantly aided by increased vitamin D signaling. Furthermore, selective targeting of the catabolic enzymes responsible for vitamin D degradation could be a plausible strategy in melanoma therapy. Vitamin D signaling can be improved by utilizing dietary supplements or by modulating CYP metabolism. A positive association exists between the intake of vitamin D supplements and improved prognosis for melanoma patients. Further investigation is required to determine the function of vitamin D supplementation and specific enzyme targeting in the prevention of melanoma.

Vitamin D and the epigenome: basic definitions, mechanisms and clinical effects

Epigenetic mechanisms play a crucial role in the regulation of gene expression. The underlying mechanisms include DNA methylation and covalent modification of histones by methylation, acetylation, phosphorylation, or ubiquitination. The complex interplay of various epigenetic mechanisms is mediated by enzymes operating in the cell nucleus. Modifications in DNA methylation are carried out primarily by DNA methyltransferases (DNMTs) and ten-eleven translocation proteins (TETs), while a variety of enzymes such as histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), and histone demethylases (HDMs)) regulate histone covalent modifications. In many pathological conditions such as cancer, autoimmune, microbial inflammatory, metabolic, allergic diseases and/or low vitamin D availability, the epigenetic regulatory system is often disrupted. Vitamin D interacts with the epigenome at several levels. First, critical genes in the vitamin D signaling system that encode for the vitamin D receptor (VDR) and the enzymes 25-hydroxylase (CYP2R1), 1α-hydroxylase (CYP27B1), and 24-hydroxylase (CYP24A1) have large CpG islands in their promoters. areas and therefore can be suppressed by DNA methylation. Second, the VDR protein physically interacts with coactivator and corepressor proteins, which in turn are in contact with chromatin modifiers such as HATs, HDACs, HMTS, and chromatin remodelers. Third, a number of genes encoding chromatin modifiers and remodelers, such as HDM from the Jumonji C (JmjC) domain containing proteins and lysine-specific demethylase (LSD) families, are primary targets for VDR and its ligands. Finally, there is evidence that certain VDR ligands have DNA demethylating effects. In this review, the authors discuss the regulation of the vitamin D system by epigenetic modifications and how vitamin D contributes to the maintenance of the epigenome and assess its impact on health and disease.

Differential gene regulation by a synthetic vitamin D receptor ligand and active vitamin D in human cells

Vitamin D (VD) exerts a wide variety of biological functions including calcemic activity. VD nutritional status is closely associated with the onset and development of chronic diseases. To develop a VD analog with the desired VD activity but without calcemic activity, we screened synthetic VDR antagonists. We identified 1α,25-dihydroxyvitamin D3-26-23-lactams (DLAM)-2a-d (DLAM-2s) as nuclear vitamin D receptor (VDR) ligands in a competitive VDR binding assay for 1α,25(OH)2 vitamin D3 (1α,25(OH)2D3), and DLAM-2s showed an antagonistic effect on 1α,25(OH)2 D3-induced cell differentiation in HL60 cells. In a luciferase reporter assay in which human VDR was exogenously expressed in cultured COS-1 cells, DLAM-2s acted as transcriptional antagonists. Consistently, DLAM-2s had an antagonistic effect on the 1α,25(OH)2D3-induced expression of a known VD target gene [Cytochrome P450 24A1 (CYP24A1)], and VDR bound DLAM-2s was recruited to an endogenous VD response element in chromatin in human keratinocytes (HaCaT cells) endogenously expressing VDR. In an ATAC-seq assay, the effects of 1α,25(OH)2 D3 and DLAM-2b on chromatin reorganization were undetectable in HaCaT cells, while the effect of an androgen receptor (AR) antagonist (bicalutamide) was confirmed in prostate cancer cells (LNCaP) expressing endogenous AR. However, whole genome analysis using RNA-seq and ATAC (Assay for Transposase Accessible Chromatin)-seq revealed differential gene expression profiles regulated by DLAM-2b versus 1α,25(OH)2D3. The upregulated and downregulated genes only partially overlapped between cells treated with 1α,25(OH)2D3 and those treated with DLAM-2b. Thus, the present findings illustrate a novel VDR ligand with gene regulatory activity differing from that of 1α,25(OH)2D3.

Advances in the Administration of Vitamin D Analogues to Support Bone Health and Treat Chronic Diseases.

Vitamin D (VD) exerts a wide variety of biological actions in addition to its well-known roles in calcium homeostasis. Nutritional VD deficiency induces rachitic abnormalities in growing children and osteomalacia in adults, and it has been proposed to underlie the onset and development of multiple non-communicable chronic diseases. Therefore, the administration of VD or synthetic VD analogues represents a promising therapeutic strategy; indeed, VD and a VD agonist have shown clinical promise in mitigating osteoporosis and symptoms of insufficient calcium intake. However, even though high doses of VD analogues have shown pre-clinical efficacy against several diseases, including cancers, they have not yet had wide-spread clinical success. This difference may be due to limitation of clinical doses in light of the inherent calcemic action of VD. An approach to overcome this problem involves the development of VD analogues with lower calcemic activity, which could be administered in high doses to attenuate the onset and progress of disease. In a similar strategy, selective estrogen receptor modulators have had success as anti-osteoporosis drugs, and they have shown benefit for other estrogen target organs by serving as partial antagonists or agonists of estrogen receptor α. It is thus conceivable to generate synthetic partial antagonists or agonists for the VD receptor (VDR) that would exert beneficial effects on bone and other VD target organs. In this review, we discuss the molecular basis of the development of such synthetic VDR ligands from the viewpoint of roles of VDR in gene regulation.

Syntheses of 25-Adamantyl-25-alkyl-2-methylidene-1α,25-dihydroxyvitamin D3 Derivatives with Structure-Function Studies of Antagonistic and Agonistic Active Vitamin D Analogs.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a major regulator of calcium homeostasis through activation of the vitamin D receptor (VDR). We have previously synthesized vitamin D derivatives with large adamantane (AD) rings at position 24, 25, or 26 of the side chain to study VDR agonist and/or antagonist properties. One of them-ADTK1, with an AD ring and 23,24-triple bond-shows a high VDR affinity and cell-selective VDR activity. In this study, we synthesized novel vitamin D derivatives (ADKM1-6) with an alkyl group substituted at position 25 of ADTK1 to develop more cell-selective VDR ligands. ADKM2, ADKM4, and ADKM6 had VDR transcriptional activity comparable to 1,25(OH)2D3 and ADTK1, although their VDR affinities were weaker. Interestingly, ADKM2 has selective VDR activity in kidney- and skin-derived cells-a unique phenotype that differs from ADTK1. Furthermore, ADKM2, ADKM4, and ADKM6 induced osteoblast differentiation in human dedifferentiated fat cells more effectively than ADTK1. The development of vitamin D derivatives with bulky modifications such as AD at position 24, 25, or 26 of the side chain is useful for increased stability and tissue selectivity in VDR-targeting therapy.

Rapid non-genomic actions of 25(OH)D3: state of the art

Over recent years, the secosteroid hormone calcitriol (1α,25(OH)2D3) has been attracting growing attention due to its essential role in calcium absorption and bone mineralization. This hormone elicits these functions through genomic and non-genomic mechanisms. In the former case, the interaction of 1α,25(OH)2D3 with vitamin D receptor (VDR) results in the transcription of genes involved in the regulation of calcium homeostasis. Compared with their genomic counterparts, non-transcriptional effects, on the other hand, occur rapidly and are not subject to the effects of transcription and protein synthesis inhibitors; they have also been shown to be responsible for the multiple actions of vitamin D. The direct precursor metabolite of 1α,25(OH)2D3, calcifediol (25(OH)D3), which also exhibits anti-proliferative and gene regulatory properties, was recently described as an agonistic ligand of VDR, albeit with lower affinity than 1α,25(OH)2D3. This mini-review attempts to offer an overview of the non-genomic actions of calcifediol and the possible mechanisms underlying the generation of these rapid responses. Insights into the rapid non-genomic mechanisms of 25(OH)D3 could help to increase knowledge of the vitamin D endocrine system, and thus result in the identification of novel therapeutic strategies able to regulate non-genomic actions, which could prove crucial in 25(OH)D3 deficiency-related disorders.

Vitamin D Receptor Mediates Attenuating Effect of Lithocholic Acid on Dextran Sulfate Sodium Induced Colitis in Mice.

Bile acids are major components of bile; they emulsify dietary lipids for efficient digestion and absorption and act as signaling molecules that activate nuclear and membrane receptors. The vitamin D receptor (VDR) is a receptor for the active form of vitamin D and lithocholic acid (LCA), a secondary bile acid produced by the intestinal microflora. Unlike other bile acids that enter the enterohepatic circulation, LCA is poorly absorbed in the intestine. Although vitamin D signaling regulates various physiological functions, including calcium metabolism and inflammation/immunity, LCA signaling remains largely unknown. In this study, we investigated the effect of the oral administration of LCA on colitis in a mouse model using dextran sulfate sodium (DSS). Oral LCA decreased the disease activity of colitis in the early phase, which is a phenotype associated with the suppression of histological injury, such as inflammatory cell infiltration and goblet cell loss. These protective effects of LCA were abolished in VDR-deleted mice. LCA decreased the expression of inflammatory cytokine genes, but this effect was at least partly observed in VDR-deleted mice. The pharmacological effect of LCA on colitis was not associated with hypercalcemia, an adverse effect induced by vitamin D compounds. Therefore, LCA suppresses DSS-induced intestinal injury in its action as a VDR ligand.

Involvement of CYP3A4 and MDR1 in altered metabolism and transport of indinavir in 1,25(OH)2D3-treated Caco-2 cells

Altered drug concentrations may induce unexpected toxicity or treatment failure; thus, understanding the factors that alter the pharmacokinetic profiles of drugs is crucial for optimal disease treatment. Vitamin D receptor (VDR), a nuclear receptor, regulates the expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1), which are crucial determinants of drug pharmacokinetics. In this study, we investigated the effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], a VDR ligand, on the metabolism, transport, and pharmacokinetics of indinavir, a dual substrate of CYP3A4 and MDR1. 1,25(OH)2D3 treatment for three days upregulated the expression levels of CYP3A4 and MDR1 in Caco-2 cells and consequently led to an increase in the level of a metabolite formed via CYP3A4 (indinavir M6) and the efflux ratio of indinavir in transport study. The increase in the metabolic reaction was also confirmed through a metabolism assay performed using the lysate of 1,25(OH)2D3-treated Caco-2 cells. In the Ussing chamber study conducted with the rat intestine, 1,25(OH)2D3 treatment did not alter the transport of indinavir into the basolateral side but increased indinavir M6 formation. Similarly, plasma levels of the metabolite increased in 1,25(OH)2D3-treated rats; however, systemic exposure to indinavir led to insignificant alterations. Considering the overlapping substrate specificities for CYP3A4 and MDR1 and their significant roles in drug pharmacokinetics, VDR may play an important role in drug interactions of CYP3A4 and MDR1 substrates for accessing more effective and safe disease treatments.

Deciphering the importance of MD descriptors in designing Vitamin D Receptor agonists and antagonists using machine learning

The Vitamin D Receptor (VDR) ligand-binding domain undergoes conformation change upon the binding of VDR agonists/antagonists. Helix 12 ((H)12) is one of the important helices at VDR ligand binding and its conformational changes are controlled by the binding of agonists and antagonists molecules. Various molecular modeling studies are available to explain the agonistic and antagonistic activity of vitamin D analogs. In this work, for the first time, we attempted to generate a machine learning model with fingerprints, 2D, 3D and MD descriptors that are specific to Vitamin D analogs and VDR. Initially, 2D and 3D descriptors and fingerprints of 1003 vitamin D analogs were calculated using CDK and RDKit. The machine learning model was generated using descriptors and fingerprints. Further, 80 Vitamin D analogs (40 VDR agonists + 40 VDR antagonists) were docked in the VDR active site. 50ns MD simulation was performed for each protein-ligand complex. Different MD descriptors such as Solvent Accessible Surface Area (SASA), radius of gyration, PC1 and PC2 were calculated and considered along with CDK and RDKit descriptors as features for machine learning calculations. A few other descriptors that are related to VDR conformational changes such as conformation of the (H)12, the angle at kink were considered for machine learning model generation. It was observed that the descriptors calculated from VDR conformational changes i) were able to distinguish between agonists and antagonists ii) provide key and comprehensive information about the unique binding characteristics of agonists and antagonists iii) provide a strong basis for the machine learning model generation. Overall, this study attempts the utilization of descriptors that are specific to a protein conformation will be helpful for the generation of an efficient machine learning model.

Vitamin D Analogs Bearing C-20 Modifications Stabilize the Agonistic Conformation of Non-Responsive Vitamin D Receptor Variants

The Vitamin D receptor (VDR) plays a key role in calcium homeostasis, as well as in cell proliferation and differentiation. Among the large number of VDR ligands that have been developed, we have previously shown that BXL-62 and Gemini-72, two C-20-modified vitamin D analogs are highly potent VDR agonists. In this study, we show that both VDR ligands restore the transcriptional activities of VDR variants unresponsive to the natural ligand and identified in patients with rickets. The elucidated mechanisms of action underlying the activities of these C-20-modified analogs emphasize the mutual adaptation of the ligand and the VDR ligand-binding pocket.

Robust osteogenic efficacy of 2α-heteroarylalkyl vitamin D analogue AH-1 in VDR (R270L) hereditary vitamin D-dependent rickets model rats

Active vitamin D form 1α,25-dihydroxtvitamin D3 (1,25(OH)2D3) plays pivotal roles in calcium homeostasis and osteogenesis via its transcription regulation effect via binding to vitamin D receptor (VDR). Mutated VDR often causes hereditary vitamin D-dependent rickets (VDDR) type II, and patients with VDDR-II are hardly responsive to physiological doses of 1,25(OH)D3. Current therapeutic approaches, including high doses of oral calcium and supraphysiologic doses of 1,25(OH)2D3, have limited success and fail to improve the quality of life of affected patients. Thus, various vitamin D analogues have been developed as therapeutic options. In our previous study, we generated genetically modified rats with mutated Vdr(R270L), an ortholog of human VDR(R274L) isolated from the patients with VDDR-II. The significant reduced affinity toward 1,25(OH)2D3 of rat Vdr(R270L) enabled us to evaluate biological activities of exogenous VDR ligand without 1α-hydroxy group such as 25(OH)D3. In this study, 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exerted much higher affinity for Vdr(R270L) in in vitro ligand binding assay than both 25(OH)D3 and 1,25(OH)2D3. A robust osteogenic activity of AH-1 was observed in Vdr(R270L) rats. Only a 40-fold lower dose of AH-1 than that of 25(OH)D3 was effective in ameliorating rickets symptoms in Vdr(R270L) rats. Therefore, AH-1 may be promising for the therapy of VDDR-II with VDR(R274L).

Calcipotriol abrogates cancer-associated fibroblast-derived IL-8-mediated oxaliplatin resistance in gastric cancer cells via blocking PI3K/Akt signaling.

Activation of vitamin D receptor (VDR) in cancer-associated fibroblasts (CAFs) has been implicated in hesitating tumor progression and chemoresistance of several human malignancies. Yet, the role of VDR in CAF-induced chemotherapy resistance of gastric cancer (GC) cells remains elusive. In this study we first conducted immunohistochemistry analysis on tissue microarrays including 88 pairs of GC and normal mucosa samples, and provided clinical evidence that VDR was mainly expressed in gastric mucous cells but almost invisible in CAFs, and VDR expression was negatively correlated with malignant clinical phenotype and advanced stages, low VDR expression confers to poor overall survival rate of patients with GC. In a co-culture system of primary CAFs and cancer cells, we showed that treatment of HGC-27 and AGS GC cells with VDR ligand calcipotriol (Cal, 500 nM) significantly inhibited CAF-induced oxaliplatin resistance. By using RNA-sequencing and Human Cytokine Antibody Array, we demonstrated that IL-8 secretion from CAFs induced oxaliplatin resistance via activating the PI3K/AKT pathway in GC, whereas Cal treatment greatly attenuated the tumor-supportive effect of CAF-derived IL-8 on GC cells. Taken together, this study verifies the specific localization of VDR in GC tissues and demonstrates that activation of VDR abrogates CAF-derived IL-8-mediated oxaliplatin resistance in GC via blocking PI3K/Akt signaling, suggesting vitamin D supplementation as a potential strategy of enhancing the anti-tumor effect of chemotherapy in GC.

Significance of the Vitamin D Receptor on Crosstalk with Nuclear Receptors and Regulation of Enzymes and Transporters.

The vitamin D receptor (VDR), in addition to other nuclear receptors, the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), is involved in the regulation of enzymes, transporters and receptors, and therefore intimately affects drug disposition, tissue health, and the handling of endogenous and exogenous compounds. This review examines the role of 1α,25-dihydroxyvitamin D3 or calcitriol, the natural VDR ligand, on activation of the VDR and its crosstalk with other nuclear receptors towards the regulation of enzymes and transporters, notably many of the cytochrome P450s including CYP3A4 and sulfotransferase 2A1 (SULT2A1) as well as cholesterol 7α-hydroxylase (CYP7A1). Moreover, the VDR upregulates the intestinal channel, TRPV6, for calcium absorption, LDL receptor-related protein 1 (LRP1) and receptor for advanced glycation end products (RAGE) in brain for β-amyloid peptide efflux and influx, the sodium phosphate transporters (NaPi), theapical sodium-dependent bile acid transporter (ASBT) and organic solute transporters (OSTα-OSTβ) for bile acid absorption and efflux, respectively, the renal organic anion transporter 3 (OAT3) and several of the ATP-binding cassette protein transporters-the multidrug resistance protein 1 (MDR1) and the multidrug resistance-associated proteins (MRPs). Hence, the role of the VDR is increasingly being recognized for its therapeutic potential and pharmacologic activity, giving rise to drug-drug interactions (DDI). Therapeutically, ligand-activated VDR shows anti-inflammatory effects towards the suppression of inflammatory mediators, improves cognition by upregulating amyloid-beta (Aβ) peptide clearance in brain, and maintains phosphate, calcium, and parathyroid hormone (PTH) balance and kidney function and bone health, demonstrating the crucial roles of the VDR in disease progression and treatment of diseases.

Plasma Levels of Bioactive Vitamin D and A5 Ligands Positively Correlate with Clinical Atopic Dermatitis Markers

Background: Over the past decade, several controversial studies described a relationship between vitamin D and atopic diseases. Low plasma vitamin D levels or even vitamin D deficiency was associated with an increased incidence of atopic disease, postulating that a higher dietary intake of vitamin D may be a beneficial strategy against atopic diseases such as atopic dermatitis (AD). Objective: Our aim was to determine the relationship between plasma 25-hydroxyvitamin D3 (25(OH)D₃) levels, the levels of the ligand of the vitamin D receptor (VDR) heterodimerization partner as well as the retinoid X receptor (RXR) and the active vitamin A5 derivative 9-cis-13,14-dihydroretinoic acid (9CDHRA) and AD severity. Methods/Results: Samples from AD patients (n = 20) and healthy volunteers (n = 20) were assessed. In our study, the frequently measured VDR ligand precursor 25(OH)D₃ in addition to the VDR-ligand 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) and 9CDHRA displayed no different levels when compared with the plasma of AD patients and healthy volunteers. When performing further correlation studies focusing on AD patients, plasma 25(OH)D₃ levels showed a negative correlation with eosinophils in blood (EOS) and SCORing Atopic Dermatitis (SCORAD) values, while 1,25(OH)₂D₃ and 9CDHRA levels correlated positively with plasma IgE, EOS, and SCORAD values. Conclusion: In consequence, the metabolic activation of vitamin D from 25(OH)D₃ towards 1,25(OH)₂D₃ as well as the co-liganding of the RXR by 9CDHRA may be an important signalling mechanism, an important marker for AD development and severity as well as the basis for novel nutritional and pharmaceutical AD treatment options.

Synergistic autophagy blockade and VDR signaling activation enhance stellate cell reprogramming in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic tumor microenvironment (TME) consisting of abundant activated pancreatic stellate cells (PSCs). PSCs play a key role in the refractory responses of PDAC to immunotherapy and chemotherapy and deactivating PSCs into quiescence through vitamin D receptor (VDR) signaling activation is a promising strategy for PDAC treatment. We observed p62 loss in PSCs hindered the deactivation efficacy of VDR ligands, and hypothesized that reversing p62 levels by inhibiting autophagy processing, which is responsible for p62 loss, could sensitize PSCs toward VDR ligands. Herein, we constructed a PSC deactivator with dual functions of VDR activation and autophagy inhibition, utilizing a pH-buffering micelle (LBM) with an inherent ability to block autophagic flux to encapsulate calcipotriol (Cal), a VDR ligand. This Cal-loaded LBM (C-LBM) could efficiently reprogram PSCs, modulate the fibrotic TME, and alter immunosuppression. In combination with PD-1 antagonists and chemotherapy, C-LBM showed superior antitumor efficacy and significantly prolonged the survival of PDAC mice. These findings suggest that synergistic autophagy blockade and VDR signaling activation are promising therapeutic approaches to reprogram PSCs and improve the PDAC response to immunotherapy.

In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells.

Several recent studies have demonstrated that the direct precursor of vitamin D3, the calcifediol [25(OH)D3], through the binding to the nuclear vitamin D receptor (VDR), is able to regulate the expression of many genes involved in several cellular processes. Considering that itself may function as a VDR ligand, although with a lower affinity, respect than the active form of vitamin D, we have assumed that 25(OH)D3 by binding the VDR could have a vitamin’s D3 activity such as activating non-genomic pathways, and in particular we selected mesenchymal stem cells derived from human adipose tissue (hADMSCs) for the in vitro assessment of the intracellular Ca2+ mobilization in response to 25(OH)D3. Our result reveals the ability of 25(OH)D3 to activate rapid, non-genomic pathways, such as an increase of intracellular Ca2+ levels, similar to what observed with the biologically active form of vitamin D3. hADMSCs loaded with Fluo-4 AM exhibited a rapid and sustained increase in intracellular Ca2+ concentration as a result of exposure to 10−5 M of 25(OH)D3. In this work, we show for the first time the in vitro ability of 25(OH)D3 to induce a rapid increase of intracellular Ca2+ levels in hADMSCs. These findings represent an important step to better understand the non-genomic effects of vitamin D3 and its role in endocrine system.

Ligand-Independent Actions of the Vitamin D Receptor: More Questions Than Answers.

ABSTRACTOur predominant understanding of the actions of vitamin D involve binding of its ligand, 1,25(OH)D, to the vitamin D receptor (VDR), which for its genomic actions binds to discrete regions of its target genes called vitamin D response elements. However, chromatin immunoprecipitation‐sequencing (ChIP‐seq) studies have observed that the VDR can bind to many sites in the genome without its ligand. The number of such sites and how much they coincide with sites that also bind the liganded VDR vary from cell to cell, with the keratinocyte from the skin having the greatest overlap and the intestinal epithelial cell having the least. What is the purpose of the unliganded VDR? In this review, I will focus on two clear examples in which the unliganded VDR plays a role. The best example is that of hair follicle cycling. Hair follicle cycling does not need 1,25(OH)2D, and Vdr lacking the ability to bind 1,25(OH)2D can restore hair follicle cycling in mice otherwise lacking Vdr. This is not true for other functions of VDR such as intestinal calcium transport. Tumor formation in the skin after UVB radiation or the application of chemical carcinogens also appears to be at least partially independent of 1,25(OH)2D in that Vdr null mice develop such tumors after these challenges, but mice lacking Cyp27b1, the enzyme producing 1,25(OH)2D, do not. Examples in other tissues emerge when studies comparing Vdr null and Cyp27b1 null mice are compared, demonstrating a more severe phenotype with respect to bone mineral homeostasis in the Cyp27b1 null mouse, suggesting a repressor function for VDR. This review will examine potential mechanisms for these ligand‐independent actions of VDR, but as the title indicates, there are more questions than answers with respect to this role of VDR. © 2021 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The Vitamin D Receptor and RXR Heteropartner Cooperate via Synergistic Activation to Control Target Gene Expression

Vitamin D is a fat-soluble vitamin that is essential for maintaining cellular growth and development. The biologically active form of vitamin D, 1,25-dihydroxycholecalciferol (1,25D), binds to the vitamin D receptor (VDR), a nuclear protein that mediates 1,25D actions. 1,25D binding to VDR also triggers heterodimerization with the retinoid X receptor (RXR) and the VDR-RXR heterocomplex then binds to vitamin D response elements (VDREs) to modulate target gene expression. In the present study, we probed RXR-specific ligands (rexinoids) for their potential role in activating VDR-RXR by employing luciferase reporter plasmids that contain distinct VDREs in human embryonic kidney cells (HEK-293). When cells were transfected with or without VDR/RXR expression plasmids and a direct repeat VDRE (XDR3), treatment with either 1,25D alone, or with RXR-specific ligands such as bexarotene (Bex) or novel Bex analogs alone, showed modest transcriptional activity of the luciferase reporter gene. However, VDR-RXR-mediated activity was significantly increased in the presence of both RXR and VDR ligands. Similar results were obtained using an everted repeat VDRE (PER6). These results suggest that rexinoids alone either: 1) bind and partially activates VDR and/or 2) that when rexinoids binds to RXR, the liganded RXR can partially activates unliganded VDR. Thus, when both ligands are present, the significantly elevated heterodimer activity implicates a synergistic effect via a dual ligand activation process. VDR-RXR is thought to be a nonpermissive heterodimer, which can be activated only by the “primary” (VDR) ligand, but our novel results suggest that VDR may in fact possess permissive behavior and be activated by either RXR or VDR ligands. In summary, this study illuminates a coordinated interaction between VDR and RXR in transcriptional regulation of vitamin D target genes.

Lithocholic acid-based design of noncalcemic vitamin D receptor agonists

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands.