Molecular basis of the selective activity of vitamin D analogues

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures.     

Ligand-based pharmacophore modeling and docking studies on vitamin D receptor inhibitors

In recent years, pharmacophore modeling and molecular docking approaches have been extensively used to characterize the structural requirements and explore the conformational space of a ligand in the binding pocket of the selected target protein. Herein, we report a pharmacophore modeling and molecular docking of 45 compounds comprising of the indole scaffold as vitamin D receptor (VDR) inhibitors. Based on the selected best hypothesis (DRRRR.61), an atom-based three-dimensional quantitative structure-activity relationships model was developed to rationalize the structural requirement of biological activity modulating components. The developed model predicted the binding affinity for the training set and test set with R²_(training) = 0.8869 and R²_(test) = 0.8139, respectively. Furthermore, molecular docking and dynamics simulation were performed to understand the underpinning of binding interaction and stability of selected VDR inhibitors in the binding pocket. In conclusion, the results presented here, in the form of functional and structural data, agreed well with the proposed pharmacophores and provide further insights into the development of novel VDR inhibitors with better activity.     

Novel regulators of vitamin D action and metabolism: Lessons learned at the Los Angeles zoo

We undertook an investigation of an outbreak of rachitic bone disease in the Emperor Tamarin New World primate colony at the Los Angeles Zoo in the mid-1980s. The disease phenotype resembled that observed in humans with an inactivating mutation of the vitamin D receptor (VDR), hypocalcemia, high 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) levels, and rickets in rapidly growing adolescent primates. In contrast to the human disease, the New World primate VDR was functionally normal in all respects. The proximate cause of vitamin D hormone resistance in New World primates was determined to be the constitutive overexpression of a heterogeneous nuclear ribonucleoprotein in the A family which we coined the vitamin D response element binding protein (VDRE-BP). VDRE-BP competed in trans with the VDR-retinoid X receptor (RXR) for binding to the vitamin D response element. VDRE-BP-legislated resistance to 1,25-(OH)(2)D was antagonized (i.e., compensated) by another set of constitutively overexpressed proteins, the hsp-70-related intracellular vitamin D binding proteins (IDBPs). IDBPs, present but expressed at much lower levels in Old World primates including man, exhibited a high capacity for 25-hydroxylated vitamin D metabolites and functioned to traffic vitamin Ds to specific intracellular destinations to promote their action and metabolism.     

Expression patterns of vitamin D receptor in human prostate

UV exposure and serum levels of vitamin D have been linked in several studies with prostate cancer risk. At the cellular level, the principal action of vitamin D is mediated though vitamin D receptors (VDR). Since prostate cancer is a disease strongly associated with age, we examined the presence of VDR in normal prostate from donors of various ages to determine if the VDR expression pattern changed with age. We also compared the VDR expression in the peripheral and central zones of the prostate to determine if the expression pattern varied by location. Immunohistochemical studies were performed on paraffin-embedded tissue from cases selected by the following age decades; 10-19, 20-29, 30-39, 40-49, 50-59, and 60-69. Both the central and peripheral zones were examined for VDR expression. The intensity of VDR expression in prostate was compared with expression in different types of human tissues. Mean VDR expression was lowest in the 10-19 years of age group. The intensity of the nuclear VDR was higher though the fifth decade, and then declined in cases of ages 60-70. When multiple sections of the same donor prostate were compared, VDR expression was greater in the peripheral zone compared to the central zone.     

Effects of 1,25-dihydroxyvitamin D3 on the distribution of androgen and vitamin D receptors in human prostate neonatal epithelial cells

Although many studies have examined the mechanisms of 1,25-dihydroxyvitamin D(3) (calcitriol or 1,25 D) action in different prostate cancer cell lines, little is known regarding the influence of this steroid on the normal prostate. The presence of both VDR and AR in normal prostatic tissues raises the distinct possibility of an important role for this hormone in the normal gland. In order to ascertain the possible role of 1,25 D on both AR and VDR in the normal prostate, the effects of calcitriol and dihydrotestosterone (DHT) on the normal human neonatal prostatic epithelial cell line, 267B-1, were examined. These studies were approached by focusing on how 1,25 D in the presence or absence of DHT affects the distribution of AR and VDR in the cytoplasmic and nuclear compartments of the cells in terms of their protein levels and DNA binding activities. Immunoblot analyses show that 1,25 D increases the AR protein level in both the cytoplasmic and nuclear fractions but not the VDR protein level. On the other hand, the gel shift assays demonstrate that 1,25 D increases both the AR- and VDR-DNA binding activities in the nuclear fraction, whereas there is no increase in DNA binding activities in the cytoplasmic fraction. Addition of DHT along with 1,25 D does not affect the DNA binding activities of both AR and VDR. Overall, these studies suggest that 1,25 D actions on the normal prostate cells may be mediated independently through AR and VDR, respectively.     

Suppression of PTH by the vitamin D analog eldecalcitol is modulated by its high affinity for the serum vitamin D-binding protein and resistance to metabolism

Eldecalcitol [1α,25‐dihydroxy‐2β‐(3‐hydroxypropyloxy)vitamin D₃], a vitamin D analog with enhanced efficacy for treatment of osteoporosis, has been found to be less potent than 1,25‐dihydroxyvitamin D₃ (calcitriol) in suppressing PTH in vivo. To define the mechanism for the latter observation, we compared the effects of eldecalcitol and calcitriol on PTH secretion by bovine parathyroid cells. While the two compounds showed similar potency when the cells were cultured in medium containing 15% newborn calf serum, eldecalcitol was 100 times more potent than calcitriol in the absence of serum. Eldecalcitol has a higher affinity for the serum vitamin D‐binding protein (DBP), and therefore binding to DBP, and possibly other serum components, appears to limit the uptake and activity of eldecalcitol in parathyroid cells, providing an explanation for the lower PTH suppressing activity in vivo (100% serum). However, the 100‐fold higher activity of eldecalcitol in the absence of serum was unexpected since the VDR affinity for eldecalcitol is eightfold lower than for calcitriol. The enhanced activity was not due to preferential uptake, but to a resistance to metabolism. While 1 nM [³H]calcitriol was completely degraded within 24 h, [³H]eldecalcitol was not metabolized, despite the induction of the vitamin D catabolic enzyme, 24‐hydroxylase (CYP24A). The resistance to metabolism is the likely explanation for the higher potency of eldecalcitol in suppressing PTH in cell culture lacking serum. Thus, the unique properties of eldecalcitol in vivo can be attributed, at least in part, to its high‐DBP affinity which increases the half‐life, but limits the uptake of eldecalcitol, and to its reduced metabolism, which prolongs the activity of this analog in target tissues. J. Cell. Biochem. 112: 1348–1352, 2011. © 2011 Wiley‐Liss, Inc.     

Protective effects of activated vitamin D receptor on radiation-induced intestinal injury

Radiation-induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) -induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti-injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation-induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation-induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation-induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.     

Association of vitamin D receptor BsmI gene polymorphism with the risk of type 2 diabetes mellitus

Abstract

Relationship between vitamin D receptor (VDR) BsmI (rs1544410) gene polymorphism and the type 2 diabetes mellitus (T2DM) susceptibility is still conflicting at present. This meta-analysis was conducted to assess the association between VDR BsmI gene polymorphism and the risk of T2DM. The association studies were identified from PubMed, and Cochrane Library on 1 January 2014, and eligible investigations were included and synthesized using meta-analysis method. Eleven reports were recruited into this meta-analysis for the association of VDR BsmI gene polymorphism with T2DM susceptibility. In overall populations, B allele, BB genotype and bb genotype were not associated with T2DM risk. VDR BsmI gene polymorphism was also not associated with the T2DM risk in Asians and Caucasians. In conclusion, VDR BsmI gene polymorphism was also not associated with T2DM risk in overall populations, Asians and Caucasians. However, more studies should be conducted to confirm it.     

The importance of nuclear import in protection of the vitamin D receptor from polyubiquitination and proteasome‐mediated degradation

Others and we previously showed that the vitamin D receptor (VDR) is subject to degradation by the 26S proteasome and that treatment with 1,25‐dihydroxyvitamin D₃ (1,25D₃) inhibited this degradation. In the present study, we found that in osteoblasts, but not in intestinal epithelial cells, the VDR was susceptible to degradation by the 26S proteasome. The subcellular site for degradation of the VDR in osteoblasts is the cytoplasm and the site for ligand‐dependent protection of the VDR from the 26S proteasome is the chromatin. These direct relationships between nuclear localization and protection of the VDR from 26S proteasome degradation led us to hypothesize that the unoccupied cytoplasmic VDR is a substrate for polyubiquitination, which targets VDR for degradation by the 26S proteasome, and that nuclear localization has the ability to protect the VDR from polyubiquitination and degradation. To test these hypotheses, we used Cos‐1 cells transfected with human VDR and histidine‐tagged ubiquitin expression vectors. We found that unoccupied VDR was polyubiquitinated and that 1,25D₃ inhibited this modification. Mutations in the nuclear localization signal of VDR (R49W/R50G and K53Q/R54G/K55E) or in the dimerization interface of VDR with retinoid X receptor (M383G/Q385A) abolished the ability of 1,25D₃ to protect the VDR from polyubiquitination, although these mutations had no effect on the ligand‐binding activity of VDR. Therefore, we concluded that in some cellular environments unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation and that ligand‐dependent heterodimerization and nuclear localization protect the VDR from these modifications. J. Cell. Biochem. 110: 926–934, 2010. © 2010 Wiley‐Liss, Inc.     

Osteoblasts lacking the vitamin D receptor display enhanced osteogenic potential in vitro

1,25-dihydroxyvitamin D plays an important role in the regulation of osteoblast gene expression, regulating the expression of bone matrix proteins as well as that of Runx2, a key regulator of osteoblast differentiation. Studies in mice lacking the vitamin D receptor (VDR) have revealed that the actions of the VDR on the skeleton are not required in the setting of normal mineral ion homeostasis. Since paracrine and endocrine factors can compensate for gene defects in vivo, studies were performed to determine whether ablation of the VDR alters the program of osteoblast differentiation in vitro. Studies in primary calvarial cultures revealed that ablation of the VDR enhanced osteoblast differentiation. The cells from the VDR null mice exhibited an earlier onset and increased magnitude of alkaline phosphatase activity, as well as an earlier and sustained increase in mineralized matrix formation, demonstrating that this enhancement persists throughout the program of osteoblast differentiation. The expression of bone sialoprotein, which enhances mineralization, was also increased in the VDR null cultures. To determine whether the increase in osteoblast differentiation was associated with an increase in the number of osteogenic progenitors, the number of osteoblastic colony forming units (CFU-OB) was evaluated. There was a twofold increase in the number of CFU-OB in the cultures isolated from the VDR null mice. Furthermore, the VDR null CFU-OB demonstrated an earlier onset and higher magnitude of expression of alkaline phosphatase activity when compared to the CFU-OB from their wild-type control littermates. These studies demonstrate that the VDR attenuates osteoblast differentiation in vitro and suggest that other endocrine and paracrine factors modulate the effect of the VDR on osteoblast differentiation in vivo.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

A meta-analysis of association of vitamin D receptor BsmI gene polymorphism with the risk of type 1 diabetes mellitus

Abstract

Association between vitamin D receptor (VDR) BsmI (rs1544410) gene polymorphism and the risk of type 1 diabetes mellitus (T1DM) from the published reports are still conflicting. This study was conducted to evaluate the relationship between VDR BsmI gene polymorphism and the risk of T1DM using meta-analysis method. The association studies were identified from PubMed, and Cochrane Library on 1 December 2013, and eligible investigations were included and synthesized using meta-analysis method. Twenty-three reports were recruited into this meta-analysis for the association of VDR BsmI gene polymorphism with T1DM susceptibility. In overall populations, bb genotype was associated with T1DM, but the B allele and BB genotype were not. In Asians and Latino population, B allele and bb genotype were associated with TIDM risk, but BB genotype was not. In Caucasians, VDR BsmI gene polymorphism was not associated with the T1DM risk. In Africans, B allele and BB genotype were associated with T1DM risk, but the bb genotype was not. However, the sample size for Latino population and Africans was small. In conclusion, VDR BsmI B allele, bb genotype was associated with T1DM risk in Asians, and bb genotype was associated with T1DM risk in overall populations. However, more studies should be conducted to confirm it.     

Glycosylation status of vitamin D binding protein in cancer patients

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase‐derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O‐linked trisaccharide glycosylation of serum‐derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization‐based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.