Vitamin D Nuclear Receptor Research Articles

Ginsenoside AD-2 Ameliorating Lipopolysaccharide-Induced Activation in HSC-T6 Cells and Carbon Tetrachloride-Induced Hepatic Fibrosis in Mice via Regulation of VD-VDR Axis.

Ginsenoside 25-hydroxy protopanaxadiol (AD-2) isolated from ginseng was proved to have anti-hepatic fibrosis (HF) effect in our previous study. But the mechanism is unknown. The present study investigated the anti-HF effects and mechanisms of AD-2 on the lipopolysaccharide (LPS)-induced activation in HSC-T6 cells and carbon tetrachloride (CCl4)-induced hepatic fibrosis (HF) in mice. Results showed that AD-2 significantly inhibited the LPS-induced activated HSC-T6 cells in vitro and markedly reduced the serum transaminase and hydroxyproline levels, pathological changes, and hepatic body ratio in CCl4-induced HF mice, indicating AD-2 ameliorated liver injury and reversed HF notably. Moreover, AD-2 decreased the expression of TGF-β1, α-SMA, and MMP2, and maintained TIMP1/MMP9 in balance with the level of vitamin D (VD) and the expression of VD nuclear receptor (VDR) and Sirt3 increased. In conclusion, the anti-HF mechanism of AD-2 is related to the inhibition of HSC activation, promotion of collagen degradation, and regulation of the VD/VDR axis.

Upregulation of reduced folate carrier by vitamin D enhances brain folate uptake in mice lacking folate receptor alpha

Folates are critical for central nervous system function. Folate transport is mediated by 3 major pathways, reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor alpha (FRα/Folr1), known to be regulated by ligand-activated nuclear receptors. Cerebral folate delivery primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems can result in very low folate levels in the cerebrospinal fluid causing childhood neurodegenerative disorders. These disorders have devastating effects in young children, and current therapeutic approaches are not sufficiently effective. Our group has previously reported in vitro that functional expression of RFC at the blood-brain barrier (BBB) and its upregulation by the vitamin D nuclear receptor (VDR) could provide an alternative route for brain folate uptake. In this study, we further demonstrated in vivo, using Folr1 knockout (KO) mice, that loss of FRα led to a substantial decrease of folate delivery to the brain and that pretreatment of Folr1 KO mice with the VDR activating ligand, calcitriol (1,25-dihydroxyvitamin D3), resulted in over a 6-fold increase in [13C5]-5-formyltetrahydrofolate ([13C5]-5-formylTHF) concentration in brain tissues, with levels comparable to wild-type animals. Brain-to-plasma concentration ratio of [13C5]-5-formylTHF was also significantly higher in calcitriol-treated Folr1 KO mice (15-fold), indicating a remarkable enhancement in brain folate delivery. These findings demonstrate that augmenting RFC functional expression at the BBB could effectively compensate for the loss of Folr1-mediated folate uptake at the choroid plexus, providing a therapeutic approach for neurometabolic disorders caused by defective brain folate transport.

Structure-activity relationship study of vitamin D analogs with oxolane group in their side chain

Synthetic analogs of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) have been developed with the goal of improving the biological profile of the natural hormone for therapeutic applications. Derivatives of 1,25(OH)2D3 with the oxolane moiety branched in the side chain at carbon C20, act as Vitamin D nuclear Receptor (VDR) superagonists being several orders of magnitude more active than the natural ligand. Here, we describe the synthesis and biological evaluation of three diastereoisomers of (1S, 3R)-Dihydroxy-(20S)-[(2″-hydroxy-2″-propyl)-tetrahydrofuryl]-22,23,24,25,26,27-hexanor-1α-hydroxyvitamin D3, with different stereochemistry at positions C2 and C5 of the oxolane ring branched at carbon C22 (1, C2RC5S; 2, C2SC5R; 3, C2SC5S). These compounds act as weak VDR agonist in transcriptional assays with compound 3 being the most active. X-ray crystallographic analysis of the VDR ligand-binding domain accommodating the three compounds indicates that the oxolane group branched at carbon C22 is not constrained as in case of compound with oxolane group branched at C20 leading to the loss of interactions of the triene group and increased flexibility of the C/D-rings and of the side chain.

Structural Studies of Vitamin D Nuclear Receptor Ligand-Binding Properties.

The vitamin D nuclear receptor (VDR) and its natural ligand, 1α,25-dihydroxyvitamin D3 hormone (1,25(OH)2D3, or calcitriol), classically regulate mineral homeostasis and metabolism but also much broader range of biological functions, such as cell growth, differentiation, antiproliferation, apoptosis, adaptive/innate immune responses. Being widely expressed in various tissues, VDR represents an important therapeutic target in the treatment of diverse disorders. Since ligand binding is a key step in VDR-mediated signaling, numerous 1,25(OH)2D3 analogs have been synthesized in order to selectively modulate the receptor activity. Most of the synthetic analogs have been developed by modification of a parental compound and some of them mimic 1,25(OH)2D3 scaffold without being structurally related to it. The ability of ligands that have different size and conformation to bind to VDR and to demonstrate biological effects is intriguing, and therefore, ligand-binding properties of the receptor have been extensively investigated using a variety of biochemical, biophysical, and computational methods. In this chapter, we describe different aspects of the structure-function relationship of VDR in complex with natural and synthetic ligands coming from structural analysis. With the emphasis on the binding modes of the most promising compounds, such as secosteroidal agonists and 1,25(OH)2D3 mimics, we also highlight the action of VDR antagonists and the evidence for the existence of an alternative ligand-binding site within the receptor. Additionally, we describe the crystal structures of VDR mutants associated with hereditary vitamin D-resistant rickets that display impaired ligand-binding function.

Current Topics on Vitamin D. Evolution of animals and vitamin D

Vitamin D3 is already found in the early evolution of life, but essentially as inactive products of the photochemical reaction of 7-dehydrocholesterol. The full vitamin D endocrine system characterized by the specific vitamin D transport protein (DBP) , specific vitamin D-metabolizing CYP P450 enzymes, active vitamin D metabolites, 1α,25 (OH) 2D3, specific vitamin D nuclear receptor (VDR), and fibroblast growth factor 23 (FGF23) became essential for maintaining calcium and bone homeostasis in terrestrial animals cope with the challenging of higher gravity and calcium-poor environment. The present review describes the story about the evolution of animals and vitamin D.

Modulators of vitamin D nuclear receptor: recent advances from structural studies.

The vitamin D nuclear receptor (VDR) and its ligand, 1α, 25-dihydroxyvitamin D3 (1,25(OH)2D3, or calcitriol) regulate numerous biological functions. Therefore, VDR represents an important therapeutic target in the treatment of various diseases such as cancers, psoriasis, rickets, renal osteodystrophy, and autoimmune dysfunctions. Despite the number of newly synthesized 1,25(OH)2D3 analogues, the need for highly potential modulators of VDR with precise cell-, gene- or coregulator-selectivity still exists. The information coming from the analysis of crystal structures of VDR-ligand complexes remains one of the most powerful tools to explain and validate the properties of the compounds and, furthermore, to gain new rationales for their modification. The number of reports on VDR-ligand recognition is constantly rising, and herein we review the recently published structural data. With the emphasis on the most promising compounds, such as secosteroidal compounds and 1,25(OH)2D3 mimics, we also highlight other natural ligands for VDR, evidence for the existence of an alternative ligand binding site within LBP, and identification of novel VDR modulators.

Vitamin D nuclear receptor deficiency promotes cholestatic liver injury by disruption of biliary epithelial cell junctions in mice

Alterations in apical junctional complexes (AJCs) have been reported in genetic or acquired biliary diseases. The vitamin D nuclear receptor (VDR), predominantly expressed in biliary epithelial cells in the liver, has been shown to regulate AJCs. The aim of our study was thus to investigate the role of VDR in the maintenance of bile duct integrity in mice challenged with biliary-type liver injury. Vdr−/− mice subjected to bile duct ligation (BDL) displayed increased liver damage compared to wildtype BDL mice. Adaptation to cholestasis, ascertained by expression of genes involved in bile acid metabolism and tissue repair, was limited in Vdr−/− BDL mice. Furthermore, evaluation of Vdr−/− BDL mouse liver tissue sections indicated altered E-cadherin staining associated with increased bile duct rupture. Total liver protein analysis revealed that a truncated form of E-cadherin was present in higher amounts in Vdr−/− mice subjected to BDL compared to wildtype BDL mice. Truncated E-cadherin was also associated with loss of cell adhesion in biliary epithelial cells silenced for VDR. In these cells, E-cadherin cleavage occurred together with calpain 1 activation and was prevented by the silencing of calpain 1. Furthermore, VDR deficiency led to the activation of the epidermal growth factor receptor (EGFR) pathway, while EGFR activation by EGF induced both calpain 1 activation and E-cadherin cleavage in these cells. Finally, truncation of E-cadherin was blunted when EGFR signaling was inhibited in VDR-silenced cells. Conclusion: Biliary-type liver injury is exacerbated in Vdr−/− mice by limited adaptive response and increased bile duct rupture. These results indicate that loss of VDR restricts the adaptation to cholestasis and diminishes bile duct integrity in the setting of biliary-type liver injury. (Hepatology 2013;58:1401–1412)

Study of vitamin D metabolism and signaling in human hepatocellular carcinoma and surrounding liver tissues

Aim: 1,25-DihydroxyvitaminD3 (1,25(OH)2D3), the active form of vitamin D, mediates antitumor effects mostly by the vitamin D nuclear receptor (VDR) in various cancers. We aimed to examine mRNA and protein expression differences of 1,25(OH)2D3-inactivating CYP24A1 and mRNA expression of the activating CYP27B1 enzymes, as well as that of VDR between human non-tumorous liver and hepatocellular carcinoma (HCC) tissue samples, and to study the correlation with clinicopathological characteristics.

Structural Basis for the Accommodation of Bis- and Tris-Aromatic Derivatives in Vitamin D Nuclear Receptor

Actual use of the active form of vitamin D (calcitriol or 1α,25-dihydroxyvitamin D(3)) to treat hyperproliferative disorders is hampered by calcemic effects, hence the continuous development of chemically modified analogues with dissociated profiles. Structurally distinct nonsecosteroidal analogues have been developed to mimic calcitriol activity profiles with low calcium serum levels. Here, we report the crystallographic study of vitamin D nuclear receptor (VDR) ligand binding domain in complexes with six nonsecosteroidal analogues harboring two or three phenyl rings. These compounds induce a stimulated transcription in the nanomolar range, similar to calcitriol. Examination of the protein-ligand interactions reveals the mode of binding of these nonsecosteroidal compounds and highlights the role of the various chemical modifications of the ligands to VDR binding and activity, notably (de)solvation effects. The structures with the tris-aromatic ligands exhibit a rearrangement of a novel region of the VDR ligand binding pocket, helix H6.

Innate immune gene polymorphisms in tuberculosis.

Tuberculosis (TB) is a leading cause worldwide of human mortality attributable to a single infectious agent. Recent studies targeting candidate genes and "case-control" association have revealed numerous polymorphisms implicated in host susceptibility to TB. Here, we review current progress in the understanding of causative polymorphisms in host innate immune genes associated with TB pathogenesis. We discuss genes encoding several types of proteins: macrophage receptors, such as the mannose receptor (MR, CD206), dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209), Dectin-1, Toll-like receptors (TLRs), complement receptor 3 (CR3, CD11b/CD18), nucleotide oligomerization domain 1 (NOD1) and NOD2, CD14, P2X7, and the vitamin D nuclear receptor (VDR); soluble C-type lectins, such as surfactant protein-A (SP-A), SP-D, and mannose-binding lectin (MBL); phagocyte cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), IL-6, IL-10, IL-12, and IL-18; chemokines, such as IL-8, monocyte chemoattractant protein 1 (MCP-1), RANTES, and CXCL10; and other important innate immune molecules, such as inducible nitric oxide synthase (iNOS) and solute carrier protein 11A1 (SLC11A1). Polymorphisms in these genes have been variably associated with susceptibility to TB among different populations. This apparent variability is probably accounted for by evolutionary selection pressure as a result of long-term host-pathogen interactions in certain regions or populations and, in part, by lack of proper study design and limited knowledge of molecular and functional effects of the implicated genetic variants. Finally, we discuss genomic technologies that hold promise for resolving questions regarding the evolutionary paths of the human genome, functional effects of polymorphisms, and corollary impacts of adaptation on human health, ultimately leading to novel approaches to controlling TB.

Genetic variants in vitamin D metabolism-related genes and body mass index: analysis of genome-wide scan data of approximately 7000 Chinese women

Vitamin D deficiency has been consistently associated with obesity. However, it is unclear whether vitamin D deficiency is the cause or consequence of obesity. We investigated this question by evaluating the association between genetic variants in vitamin D metabolism pathway genes and obesity-related traits. Using directly genotyped and imputed data from a genome-wide association study of 6922 women aged 25-70 years, we examined the association of 198 single-nucleotide polymorphisms (SNPs) in vitamin D pathway genes (CYP27A1, CYP27B1, CYP24A1, CYP2R1, group-specific component (GC) and vitamin D nuclear receptor (VDR)) with body mass index (BMI) and body weight. Per allele beta (β) estimates were calculated for this association using linear regression models, controlling for age, square of age, menopausal status and sample sets. Overall, only two SNPs (rs2248359 in CYP24A1 and rs10832313 in CYP2R1) had a nominally significant association with BMI and weight (P<0.05 for all), with no variation observed by menopausal status, physical activity or dietary energy intake. None of the SNPs examined in the VDR gene were associated with BMI or weight. Our findings suggest that common genetic variants in vitamin D pathway genes do not have a major role in obesity among Chinese women. This comprehensive evaluation of genetic polymorphisms in vitamin D metabolism-related genes and obesity-related traits did not provide strong evidence to support low vitamin D levels as a cause of obesity.

1α,25(OH)2-3-Epi-Vitamin D3, a Natural Physiological Metabolite of Vitamin D3: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor

BackgroundThe 1α,25-dihydroxy-3-epi-vitamin-D3 (1α,25(OH)2-3-epi-D3), a natural metabolite of the seco-steroid vitamin D3, exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. In vivo action of 1α,25(OH)2-3-epi-D3 is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1α,25(OH)2D3. To further unveil the structural mechanism and structure-activity relationships of 1α,25(OH)2-3-epi-D3 and its receptor complex, we characterized some of its in vitro biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD).Methodology/Principal FindingsIn the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1α,25(OH)2D3. We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1α,25(OH)2-3-epi-D3 in primary human keratinocytes and biochemical properties are comparable to 1α,25(OH)2D3.Conclusions/SignificanceThe physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)2D3 lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)2D3.

Vitamin D and the vitamin D receptor in liver pathophysiology

Vitamin D through the vitamin D nuclear receptor (VDR) plays a key role in mineral ion homeostasis. The liver is central in vitamin D synthesis, however the direct involvement of the vitamin D-VDR axis on the liver remains to be evaluated. In this review, we will describe vitamin D metabolism and the mechanisms of homeostatic control. We will also address the associations between the vitamin D-VDR axis and pathological liver entities, such as non-alcoholic fatty liver disease, autoimmune liver disease, viral hepatitis and liver cancer. The link between liver diseases and the vitamin D-VDR axis will be discussed in light of evidences arising from in vitro and in vivo studies. Finally, we will consider the therapeutic potential of the vitamin D-VDR axis in liver diseases.

Dysregulation of the Vitamin D Nuclear Receptor May Contribute to the Higher Prevalence of Some Autoimmune Diseases in Women

Researchers have noted that the incidence of autoimmune diseases, such as Hashimoto's thyroiditis, is markedly higher in women than in men, but to date the reason for this disparity has been unclear. The vitamin D nuclear receptor (VDR) is expressed in the human cycling endometrium. Because the VDR controls expression of the cathelicidin and beta-defensin antimicrobial peptides (AmPs), dysregulation of the receptor greatly compromises the innate immune response. Increasing evidence indicates the presence of a chronic, intraphagocytic, metagenomic microbiota in patients with autoimmune disease that may survive by dysregulating the VDR. VDR dysregulation, in turn, prevents the breakdown of the active vitamin D metabolite 1,25-hydroxyvitamin D (1,25-D) by CYP24. In silico data suggest that when 1,25-D rises above its normal range, it binds the alpha/beta thyroid receptors, the glucocorticoid receptor (GCR), and the androgen receptor (AR), displacing their native ligands and causing an array of hormonal imbalances. If T3 is displaced from alpha-thyroid, thyroiditis may result. Because the VDR, GCR, and AR also express multiple families of AmPs, expression of these natural antibiotics further wanes in response to dysregulation by 1,25-D. The end result is a system-wide drop in AmP expression that may allow pathogens to spread with greater ease. Because women have an extra site of VDR expression in the endometrium, the drop in AmP expression associated with nuclear receptor dysregulation may disproportionately affect them. This would cause women to accumulate higher bacterial loads than their male counterparts, particularly during early pregnancy when 1,25-D levels rise by 40%.

Structure-Based Design of a Superagonist Ligand for the Vitamin D Nuclear Receptor

Vitamin D nuclear receptor (VDR), a ligand-dependent transcriptional regulator, is an important target for multiple clinical applications, such as osteoporosis and cancer. Since exacerbated increase of calcium serum level is currently associated with VDR ligands action, superagonists with low calcium serum levels have been developed. Based on the crystal structures of human VDR (hVDR) bound to 1alpha,25-dihydroxyvitamin D(3) and superagonists-notably, KH1060-we designed a superagonist ligand. In order to optimize the aliphatic side chain conformation with a subsequent entropy benefit, we incorporated an oxolane ring and generated two stereo diasteromers, AMCR277A and AMCR277B. Only AMCR277A exhibits superagonist activity in vitro, but is as calcemic in vivo as the natural ligand. The crystal structures of the complexes between the ligand binding domain of hVDR and these ligands provide a rational approach to the design of more potent superagonist ligands for potential clinical application.

Crystal structure of the vitamin D nuclear receptor ligand binding domain in complex with a locked side chain analog of calcitriol

The crystal structures of vitamin D nuclear receptor (VDR) have revealed that all compounds are anchored by the same residues to the ligand binding pocket (LBP). Based on this observation, a synthetic analog with a locked side chain (21-nor-calcitriol-20(22),23-diyne) has been synthesized in order to gain in entropy energy with a predefined active side chain conformation. The crystal structure of VDR LBD bound to this locked side chain analogue while confirming the docking provides a structural basis for the activity of this compound.

Novel 1α,25-Dihydroxyvitamin D3Analogues with the Side Chain at C12

The plethora of actions of 1alpha,25(OH)2D3 in various systems suggested wide clinical applications of vitamin D nuclear receptor (VDR) ligands in treatments of inflammation, dermatological indication, osteoporosis, cancers, and autoimmune diseases. More than 3000 vitamin D analogues have been synthesized in order to reduce the calcemic side effects while maintaining the transactivation potency of the natural ligand. In light of the crystal structures of the vitamin D nuclear receptor (VDR), novel analogues of the hormone 1alpha,25(OH)2D3 with side chains attached to C-12 were synthesized via the convergent Wittig-Horner approach. Among the compounds studied, the analogue 2b showed the highest binding affinity for VDR and was the most potent at inducing VDR transcriptional activity in a transient transfection assay (20% of the transactivation activity of the natural ligand).

Nongenotropic, Anti-Apoptotic Signaling of 1α,25(OH)2-Vitamin D3 and Analogs through the Ligand Binding Domain of the Vitamin D Receptor in Osteoblasts and Osteocytes: MEDIATION BY Src, PHOSPHATIDYLINOSITOL 3-, AND JNK KINASES

Because sex steroids regulate the life span of bone cells by modulating cytoplasmic kinase activity via a nongenotropic action of their classical receptors, we have explored the possibility that the vitamin D nuclear receptor (VDR) might exhibit similar nongenotropic actions. We report that the conformationally flexible full VDR agonist, 1alpha,25(OH)2-vitamin D3 (1alpha,25(OH)2D3), and the 6-s-cis-locked 1alpha,25(OH)2-lumisterol3 (JN) analog, also acting through the VDR but with poor transcriptional activity, protected murine osteoblastic or osteocytic cells from apoptosis. This effect was reproduced in HeLa cells transiently transfected with either wild type VDR or a mutant consisting of only the VDR ligand binding domain. The VDR ligand binding domain bound [3H]1alpha,25(OH)2D3 as effectively as wild type VDR but did not induce vitamin D response element-mediated transcription. The anti-apoptotic effects of 1alpha,25(OH)2D3 and the 6-s-cis-locked 1alpha,25(OH)2-lumisterol3 analog in calvaria cells were blocked by three cytoplasmic kinase inhibitors: Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), phosphatidylinositol 3 kinase inhibitor Wortmannin, and the JNK kinase inhibitor SP600125. However, inhibition of p38 with SB203580 or ERK with either U0126 or a transfected dominant negative MEK did not interfere with these anti-apoptotic actions. Further, 1alpha,25(OH)2D3 induced rapid (5 min) association of VDR with Src kinase in OB-6 cells. Finally, actinomycin D or cycloheximide prevented the anti-apoptotic effect of 1alpha,25(OH)2D3, indicating that transcriptional events are also required. These findings suggest that nongenotropic modulation of kinase activity is also a general property of the VDR and that ligands that activate nongenotropic signals, but lack transcriptional activity, display different biological profiles from the steroid hormone 1alpha,25(OH)2D3.

Synthesis of 1α,25‐Dihydroxyvitamin D3‐26,23‐Lactams (DLAMs), a Novel Series of 1α,25‐Dihydroxyvitamin D3 Antagonist.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Role of residues 143 and 278 of the human nuclear Vitamin D receptor in the full-length and Δ165-215 deletion mutant

Most of the actions of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] are mediated by binding to the Vitamin D nuclear receptor (VDR). The crystal structure of a deletion mutant (Δ165-215) of the VDR ligand-binding domain (LBD) bound to 1,25(OH) 2D 3 indicates that amino acid residues tyrosine-143 and serine-278 form hydrogen bonding interactions with the 3-hydroxyl group of 1,25(OH) 2D 3. Studies of VDR and three mutants (Y143F, S278A, and Y143F/S278A) did not indicate any differences in the binding affinity between the variant receptors and the wild-type receptor. This might indicate that the 3-hydroxyl group binds differently to the full-length VDR than the to deletion mutant. To further investigate, four deletion VDR mutants were constructed: VDR Δ165-215, VDR Δ165-215 (Y143F), VDR Δ165-215 (S278A), VDR Δ165-215 (Y143F/S278A). There were no significant differences in binding affinity between the wild-type receptor and the deletion mutants except for VDR Δ165-215 (Y143F/S278A). In gene activation assays, VDR constructs with the single mutation Y143F and the double mutation Y143F/S278A, but not the single mutation S278A required higher doses of 1,25(OH) 2D 3 for half-maximal response. This suggests that there are some minor structural and functional differences between the wild-type VDR and the Δ165-215 deletion mutant and that Y143 residue is more important for receptor function than residue S278.