Vitamin D Receptor Protein Research Articles

Differential Effects of Vitamin D Receptor Agonists on Gene Expression in Neonatal Rat Cardiomyocytes

Vitamin D receptor (VDR) activation is associated with cardiovascular benefits in chronic kidney disease patients, but whether VDR's hormone and prehormone exhibit similar effects requires more studies. Neonatal rat cardiomyocytes were treated with VDR agonists (calcitriol and/or paricalcitol) and the prehormone calcidiol in the presence of aldo (1μM). The expression of VDR target genes were determined by real-time PCR and Western blotting. The expression and activity of CYP27B1 (the enzyme responsible for converting calcidiol to calcitriol) was measured. Treating cells with aldo (1μM) for 24h significantly reduced the VDR mRNA (29%) and protein levels (>90%). Calcitriol and calcidiol induced VDR expression in the presence of aldo with EC(50) at 0.3 and 7,952nM, respectively. Calcitriol, paricalcitol and calcidiol stimulated CYP24A1 (EC(50) at 6.4, 4.5 and 992nM, respectively) and suppressed NPPB expression (IC(50) at 1.9, 0.1 and 210nM, respectively) in the presence of 1μM aldo. Neonatal rat cardiomyocytes expressed CYP27B1 and converted calcidiol to calcitriol at a low rate (~10% in 24h). VDR hormone calcitriol and its analog paricalcitol exhibit more potent effects than the prehormone calcidiol in cardiomyocytes.

The Effects of Vitamin D Receptor Silencing on the Expression of LVSCC-A1C and LVSCC-A1D and the Release of NGF in Cortical Neurons

BackgroundRecent studies have suggested that vitamin D can act on cells in the nervous system. Associations between polymorphisms in the vitamin D receptor (VDR), age- dependent cognitive decline, and insufficient serum 25 hydroxyvitamin D3 levels in Alzheimer's patients and elderly people with cognitive decline have been reported. We have previously shown that amyloid β (Aβ) treatment eliminates VDR protein in cortical neurons. These results suggest a potential role for vitamin D and vitamin D-mediated mechanisms in Alzheimer's disease (AD) and neurodegeneration. Vitamin D has been shown to down-regulate the L-type voltage-sensitive calcium channels, LVSCC-A1C and LVSCC-A1D, and up-regulate nerve growth factor (NGF). However, expression of these proteins when VDR is repressed is unknown. The aim of this study is to investigate LVSCC-A1C, LVSCC-A1D expression levels and NGF release in VDR-silenced primary cortical neurons prepared from Sprague-Dawley rat embryos.Methodology/Principal FindingsqRT-PCR and western blots were performed to determine VDR, LVSCC-A1C and -A1D expression levels. NGF and cytotoxicity levels were determined by ELISA. Apoptosis was determined by TUNEL. Our findings illustrate that LVSCC-A1C mRNA and protein levels increased rapidly in cortical neurons when VDR is down-regulated, whereas, LVSCC-A1D mRNA and protein levels did not change and NGF release decreased in response to VDR down-regulation. Although vitamin D regulates LVSCC-A1C through VDR, it may not regulate LVSCC-A1D through VDR.Conclusions/SignificanceOur results indicate that suppression of VDR disrupts LVSCC-A1C and NGF production. In addition, when VDR is suppressed, neurons could be vulnerable to aging and neurodegeneration, and when combined with Aβ toxicity, it is possible to explain some of the events that occur during neurodegeneration.

Functional Significance of Vitamin D Receptor FokI Polymorphism in Human Breast Cancer Cells

BackgroundThe FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)2D3, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)2D3 downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.Methods/FindingsTo elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)2D3 treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.Conclusions/SignificanceThese results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.

Vitamin D and Susceptibility of Chronic Lung Diseases: Role of Epigenetics

Vitamin D deficiency is linked to accelerated decline in lung function, increased inflammation, and reduced immunity in chronic lung diseases. Epidemiological studies have suggested that vitamin D insufficiency is associated with low lung function in susceptible subjects who are exposed to higher levels of environmental agents (airborne particulates). Recent studies have highlighted the role of vitamin D and vitamin D receptor (VDR) in regulation of several genes that are involved in inflammation, immunity, cellular proliferation, differentiation, and apoptosis. Vitamin D has also been implicated in reversal of steroid resistance and airway remodeling, which are the hallmarks of chronic obstructive pulmonary disease (COPD) and severe asthma. VDR protein level is decreased in lungs of patients with COPD. VDR deficient mice develop an abnormal lung phenotype with characteristics of COPD, such as airspace enlargement and decline in lung function associated with increased lung inflammatory cellular influx, and immune-lymphoid aggregates formation. Dietary vitamin D may regulate epigenetic events, in particular on genes which are responsible for COPD susceptibility. Active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 plays an essential role in cellular metabolism and differentiation via its nuclear receptor (VDR) that cooperates with several other chromatin modification enzymes (histone acetyltransferases and histone deacetylases), thereby mediating complex epigenetic events in vitamin D signaling and metabolism. This review provides an update on the current knowledge and understanding on vitamin D, and susceptibility of chronic lung diseases in relation to the possible role of epigenetics in its molecular action. Understanding the molecular epigenetic mechanism of vitamin D/VDR would provide rationale for dietary vitamin D-mediated intervention in prevention and management of chronic lung diseases linked with vitamin D deficiency.

Role for vitamin D receptor in the neuronal control of the hematopoietic stem cell niche

AbstractHematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow to the circulation by the cytokine, granulocyte colony-stimulating factor, via sympathetic nervous system (SNS)–mediated osteoblast suppression. Because the orientation of HSPCs in their osteoblastic niche is reported to be guided by [Ca2+], we speculated on a cooperation between the calcium-regulating hormones and SNS in the regulation of HSPC trafficking. Here, we present the severe impairment of granulocyte colony-stimulating factor–induced osteoblast suppression and subsequent HSPC mobilization in vitamin D receptor (VDR)–deficient mice. In osteoblasts, functional VDR possessing, at least in part, a transcriptional activity, was specifically induced by β2-adrenergic receptor (AR) agonists. While β2-AR agonists transiently increased mRNA expression of Vdr and its downstream gene, Rankl, 1α,25-dihydroxyvitamin-D3 sustained the β2-AR–induced Rankl expression at high level by stabilizing VDR protein. These data suggest that VDR is essential for durable β2-AR signaling in the stem cell niche. Our study demonstrates not only a novel function of VDR as a critical modulator of HSPC trafficking, but also the presence of a SNS-mediated, bone-remodeling mechanism through VDR. VDR contributes to brain-bone-blood integration in an unanticipated way distinct from other classical calcium-regulating hormones.

PTPH1 cooperates with vitamin D receptor to stimulate breast cancer growth through their mutual stabilization

Tyrosine phosphorylation is tightly regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), and plays a critical role in malignant transformation and progression. While PTKs have a well-established role in regulating breast cancer growth, contribution of PTPs remains mostly unknown. Here, we report that the tyrosine phosphatase PTPH1 stimulates breast cancer growth through regulating vitamin D receptor (VDR) expression. PTPH1 was shown to be over-expressed in 49% of primary breast cancer and levels of its protein expression positively correlate with the clinic metastasis, suggesting its oncogenic activity. Indeed, PTPH1 promotes breast cancer growth by a mechanism independent of its phosphatase activity but dependent of its stimulatory effect on the nuclear receptor VDR protein expression and depletion of induced VDR abolishes the PTPH1 oncogenic activity. Additional analyses showed that PTPH1 binds VDR and increases its cytoplasmic accumulation leading to their mutual stabilization and stable expression of a nuclear localization deficient VDR abolishes the growth-inhibitory activity of the receptor independent of 1, 25-dihydroxyvitamin D3 (vitamin D3). These results reveal a new paradigm in which a protein tyrosine phosphatase may stimulate breast cancer growth through increasing cytoplasmic translocation of a nuclear receptor leading to their mutual stabilization.

Ethanol extract of Fructus Ligustri Lucidi increases circulating 1,25-dihydroxyvitamin D3 by inducing renal 25-hydroxyvitamin D-1α hydroxylase activity

The present study was designed to determine whether Fructus Ligustri Lucidi (FLL) ethanol extract can directly regulate vitamin D metabolism both in vivo and in vitro. Eleven-month-old, aged Sprague-Dawley sham-operated and ovariectomized (OVX) female rats were fed a normal-calcium (Ca) diet (0.6% Ca, 0.65% phosphorus) and received either FLL (700 mg/kg) or vehicle daily for 12 weeks. The in vitro effects of FLL on vitamin D metabolism were studied using primary cultures of the rat renal proximal tubules. mRNA and protein expressions of 25-hydroxyvitamin D-1α hydroxylase (1-OHase) and vitamin D receptor (VDR) in the kidney and proximal tubule were measured using real-time polymerase chain reaction and Western blotting, respectively. The concentrations of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) synthesized by renal 1-OHase were measured by a competitive enzyme immunoassay. FLL treatment significantly increased serum 1,25(OH)2D3 levels in both sham (P < 0.01) and OVX (P < 0.05) rats. FLL increased renal 1-OHase and VDR protein and mRNA expressions in sham rats. Protein expression of renal 1-OHase, but not VDR, was also up-regulated in OVX rats during FLL treatment. 1-OHase mRNA and 1-OHase activity were increased by FLL treatment in primary cultures of renal proximal tubule cells. FLL could increase the circulating levels of 1,25(OH)2D3 in vivo in aged female rats by directly stimulating 1-OHase activity. Thus, it might be an ideal oral agent that can help to improve the ability to induce 1,25(OH)2D3 synthesis and Ca balance in postmenopausal women who are of high risk of developing osteoporosis.

Thyroid Cancer Resistance to Vitamin D Receptor Activation Is Associated with 24-Hydroxylase Levels But Not theffFokI Polymorphism

The vitamin D receptor (VDR) has been studied as a novel target for cancer therapy in many tissue types as VDR ligands decrease cell proliferation in vitro and decrease tumor growth in vivo in sensitive cells. The objective of this study was to analyze the response to VDR agonist therapy in a panel of validated thyroid cancer cells and assess genetic markers predicting sensitivity and resistance to calcitriol and the noncalcemic analog DP006. Thyroid cancer cell lines were analyzed for VDR and RXR protein by Western blot. Cell growth after VDR agonist treatment (calcitriol or DP006) was assessed after 6 days of treatment by viable cell assay. To investigate calcitriol/DP006 resistance in VDR-expressing cells, the VDR was sequenced and 1-α and 24-hydroxylase mRNA expression was assessed. VDR protein was variably expressed in the thyroid cancer cell lines and its presence was not sufficient for decreased viable cell count in response to calcitriol or DP006. The most sensitive cells (TPC1) have an ff FokI VDR polymorphism and the most resistant cells (HTh7 and 8505C) have an FF FokI VDR. This is a unique finding given that the balance of the literature of VDR polymorphisms describes an association of the ff FokI polymorphism with cancer risk and/or decreased VDR transactivation. 1-α and 24-hydroxylase mRNA expression before and after VDR agonist therapy was examined. 1-α-Hydroxylase levels did not change after calcitriol treatment. However, we found that higher baseline 24-hydroxylase levels and/or lower stimulation of 24-hydroxylase levels after calcitriol treatment were associated with relative resistance to calcitriol/DP006. The VDR represents a novel therapeutic target in poorly differentiated thyroid cancer; however, the efficacy of VDR agonist therapy to decrease viable thyroid cancer cell count cannot be predicted solely on the presence of the VDR. The FF FokI VDR genotype and high baseline 24-hydroxylase levels were associated with relative resistance to calcitriol and DP006. Therefore, identifiable markers of sensitivity or resistance to VDR agonist therapy may allow for a personalized use of these agents in poorly differentiated thyroid cancer.

Preventive effects of 1,25-(OH)2VD3 against ConA-induced mouse hepatitis through promoting vitamin D receptor gene expression

To investigate the immunosuppressive effects of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)VD(3)) on concanavalin A (ConA)-induced hepatitis and elucidate the action mechanism. Female BALB/C mice were intravenously administered ConA (20 mg/kg) to induce acute immunological liver injury. Liver damage was evaluated in respect to serum alanine transaminase (ALT) level and liver histological changes. The proliferation of splenocytes was measured by using [(3)H]-thymidine incorporation. The cytokine level in the cultured splenocyte supernatant was determined by using enzyme-linked immunosorbent assays (ELISAs). The percentage of different splenic T cell subtypes was analyzed by using flow cytometry. The expression of splenic vitamin D receptor (VDR) mRNA and protein was detected by using real-time qRT-PCR and Western blot, respectively. 1,25-(OH)(2)VD(3) (2.5 microg/kg, ip) significantly decreased the serum ALT levels and markedly attenuated the histological liver damage. The beneficial effect of 1,25-(OH)(2)VD(3) was associated with: (i) inhibition of CD4(+) T cell activation; (ii) reduction of interferon-gamma (IFN-gamma) and elevation of both IL-4 and IL-5 in supernatants of cultured splenocytes; and (iii) elimination of activated T cells by increasing VDR mRNA and protein expression in the spleen. 1,25-(OH)(2)VD(3) had a significant protective effect against ConA-induced hepatitis, and its mechanism of action was associated with down-regulation of T cell-mediated immunity and up-regulation of VDR gene expression.

Abstract 5718: Convergence of vitamin D and PPARγ signaling in human breast cancer cell lines

Abstract Previous studies have demonstrated a positive interaction between vitamin D and peroxisome proliferator- activated receptor γ (PPARγ) mediated signaling while other studies have shown an inverse relationship between these two pathways. Therefore, we sought to determine the functional interaction between the vitamin D receptor (VDR) and PPARγ in human breast cancer cells. We report the physical interaction and nuclear colocalization of VDR and PPARγ in the T47D human breast cancer cell line as determined by co-immunoprecipitation and immunofluorescence respectively. Furthermore, 1alpha,25-dihydroxyvitamin D3 (1,25D3) treatment of MCF-7 and T47D cells significantly upregulated PPARγ protein expression in a dose and time dependent manner. Conversely, knockdown of unliganded VDR also upregulated PPARγ protein expression, suggesting that it is a potential VDR target gene. Thus, VDR itself in the absence of ligand, can suppress PPARγ basal expression. This paradoxical effect may be due to the differential binding of VDR to its coactivators or corepressors during the liganded and unliganded state. Consistent with this, the basal expression of PPARγ mRNA was inversely correlated with VDR mRNA expression in several human breast cancer cell lines. Reciprocally, a PPARγ agonist attenuated vitamin D mediated upregulation of vitamin D target genes CYP24 and p21. Similarly, the activity of VDRE, a vitamin D responsive reporter was decreased by 2 fold in the presence of the PPARγ agonist. Together, these observations infer that VDR and PPARγ protein interactions negatively influence their respective pathways. (This work was supported by NCI predoctoral fellowship 1F31CA132619 (F.A.) and NCI R03 CA121365 (X.P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5718.

Elevated vitamin D receptor levels in genetic hypercalciuric stone-forming rats are associated with downregulation of Snail.

Patients with idiopathic hypercalciuria (IH) and genetic hypercalciuric stone-forming (GHS) rats, an animal model of IH, are both characterized by normal serum Ca, hypercalciuria, Ca nephrolithiasis, reduced renal Ca reabsorption, and increased bone resorption. Serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels are elevated or normal in IH and are normal in GHS rats. In GHS rats, vitamin D receptor (VDR) protein levels are elevated in intestinal, kidney, and bone cells, and in IH, peripheral blood monocyte VDR levels are high. The high VDR is thought to amplify the target-tissue actions of normal circulating 1,25(OH)2D levels to increase Ca transport. The aim of this study was to elucidate the molecular mechanisms whereby Snail may contribute to the high VDR levels in GHS rats. In the study, Snail gene expression and protein levels were lower in GHS rat tissues and inversely correlated with VDR gene expression and protein levels in intestine and kidney cells. In human kidney and colon cell lines, ChIP assays revealed endogenous Snail binding close to specific E-box sequences within the human VDR promoter region, whereas only one E-box specifically bound Snail in the rat promoter. Snail binding to rat VDR promoter E-box regions was reduced in GHS compared with normal control intestine and was accompanied by hyperacetylation of histone H3. These results provide evidence that elevated VDR in GHS rats likely occurs because of derepression resulting from reduced Snail binding to the VDR promoter and hyperacetylation of histone H3. © 2010 American Society for Bone and Mineral Research.

Heat shock protein 90 interacts with vitamin D receptor in human leukemia cells

The active form of vitamin D, 1α,25-dihydroxyvitamin D 3 (1,25D), has a broad range of effects which are mediated by nuclear vitamin D receptor (VDR). Many experiments that investigate the role of VDR can be done in human acute myeloid leukemia (AML) cells, since these cells are responsive to 1,25D and express VDR in a 1,25D-regulated manner. In this paper we show that in HL60 and in THP-1 cells VDR protein interacts with heat shock protein 90 (Hsp90) and that Hsp90 is important for differentiation of AML cells. Geldanamycin (GA), an Hsp90 inhibitor, is able to suppress 1,25-induced differentiation of HL60 cells.

Mitochondrial localization of vitamin D receptor in human platelets and differentiated megakaryocytes.

BackgroundLike other steroid hormones, vitamin D elicits both transcriptional events and rapid non genomic effects. Vitamin D receptor (VDR) localization and mechanisms of VDR-triggered non genomic responses are still controversial. Although anticoagulant effects of vitamin D have been reported and VDR signalling has been characterized in monocytes and vascular cells, nothing is known about VDR expression and functions in human platelets, anucleated fragments of megakaryocytes which are known targets of other steroids.Methodology/Principal FindingsIn this study we characterized the expression and cellular localization of VDR in human platelets and in a megakaryocyte lineage. Human platelets and their TPA-differentiated precursors expressed a classical 50 kDa VDR protein, which increased with megakaryocytes maturation. By biochemical fractionation studies we demonstrated the presence of the receptor in the soluble and mitochondrial compartment of human platelets, and the observation was confirmed by immunoelectron microscopy analysis. Similar localization was found in mature megakaryocytes, where besides its classical nuclear localization the receptor was evident as soluble and mitochondria resident protein.ConclusionsThe results reported here suggest that megakaryocytopoiesis and platelet activation, which are calcium-dependent events, might be modulated by a mitochondrial non genomic activity of VDR. These data open challenging future studies on VDR physiological role in platelets and more generally in mitochondria.

Abstract PR-01: Vitamin D receptor expression is inversely associated with prostate cancer progression

Abstract Background: Vitamin D is inversely associated with risk of several malignancies. Circulating vitamin D interacts with the vitamin D receptor (VDR) at the cellular level to inhibit proliferation, increase apoptosis and decrease angiogenesis. Thus, although vitamin D levels appear to be unrelated to total prostate cancer incidence, VDR levels in tumor tissue may influence prostate cancer prognosis. Methods: We examined the immunohistochemical expression of VDR on archival tumor tissue from 841 prostate cancer cases diagnosed between 1982 and 2004 within two ongoing, prospective cohorts: the Physicians' Health Study and Health Professionals Follow-up Study. VDR expression was measured quantitatively using the CRi Vectra™ system. We used Cox proportional hazards regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association of VDR expression with lethal prostate cancer (N=73) through 2008. On a subset of cases, we also examined correlation of tumor VDR expression with circulating 25(OH)D3 and 1alpha,25(OH)2D3 measured at baseline (N=84) in 1982 and two SNPs in VDR: Fok1 and bsm1 (N=140). Results: Men with high tumor VDR expression had significantly lower Gleason score, lower prostate specific antigen (PSA) levels at diagnosis, and were less likely to have advanced tumor stage (p=0.008, p=0.012, p=0.001, respectively). Compared to the lowest quartile, men in the highest quartile of VDR expression were at significantly lower risk of developing lethal prostate cancer (age-adjusted HRs across quartiles Q2=0.95, 95% CI: 0.51–1.79; Q3=0.93, 95% CI: 0.49–1.76; Q4 = 0.23, 95% CI:0.09–0.55). This association was attenuated (HRQ4vsQ1 = 0.42, 95% CI: 0.16–1.09) after further adjustment for pathological tumor stage, Gleason grade, and PSA level at diagnosis. Tumors expressing high levels of VDR had modest downregulation of cell proliferation as measured by Ki67 (r=-0.11). Moreover, expression of estrogen receptor alpha (r=0.40, p&amp;lt;0.001) and androgen receptor (r=0.41, p&amp;lt;0.001) were positively correlated with VDR expression. Neither prediagnostic plasma vitamin D levels nor gene variants in VDR were associated with VDR protein expression in tumors. Conclusion: In this large prospective study, men with tumors that demonstrated upregulation of VDR had significantly improved clinical features and reduced risk of lethal prostate cancer. In line with experimental studies, the positive correlation between VDR expression, and androgen receptor and estrogen receptor provides evidence that that VDR acts in an androgen- and/or estrogen-dependent manner. These data highlight the potential role of the vitamin D system in preventing prostate cancer progression. Citation Information: Cancer Prev Res 2010;3(1 Suppl):PR-01.

Evolution of the interaction between Runx2 and VDR, two transcription factors involved in osteoblastogenesis

BackgroundThe mineralized skeleton is a major evolutionary novelty that has contributed to the impressive morphological diversifications of the vertebrates. Essential to bone biology is the solidified extracellular matrix secreted by highly specialized cells, the osteoblasts. We now have a rather complete view of the events underlying osteogenesis, from a cellular, molecular, genetic, and epigenetic perspective. Because this knowledge is still largely restricted to mammals, it is difficult, if not impossible, to deduce the evolutionary history of the regulatory network involved in osteoblasts specification and differentiation. In this study, we focused on the transcriptional regulators Runx2 and VDR (the Vitamin D Receptor) that, in mammals, directly interact together and stabilize complexes of co-activators and chromatin remodellers, thereby allowing the transcriptional activation of target genes involved in extracellular matrix mineralization. Using a combination of functional, biochemical, and histological approaches, we have asked if the interaction observed between Runx2 and VDR represents a recent mammalian innovation, or if it results from more ancient changes that have occurred deep in the vertebrate lineage.ResultsUsing immunohistochemistry and in situ hybridization in developing embryos of chick, frog and teleost fishes, we have revealed that the co-expression of Runx2 and VDR in skeletal elements has been particularly strengthened in the lineage leading to amniotes. We show that the teleost Runx2 orthologue as well as the three mammalian Runx1, Runx2 and Runx3 paralogues are able to co-immunoprecipitate with the VDR protein present in nuclear extracts of rat osteoblasts stimulated with 1α,25-dihydroxyvitamin D3. In addition, the teleost Runx2 can activate the transcription of the mammalian osteocalcin promoter in transfection experiments, and this response can be further enhanced by 1α,25-dihydroxyvitamin D3. Finally, using pull-down experiments between recombinant proteins, we show that the VDR homologue from teleosts, but not from ascidians, is able to directly interact with the mammalian Runx2 homologue.ConclusionsWe propose an evolutionary scenario for the assembly of the molecular machinery involving Runx2 and VDR in vertebrates. In the last common ancestor of actinopterygians and sacropterygians, the three Runx paralogues possessed the potential to physically and functionally interact with the VDR protein. Therefore, 1α,25-dihydroxyvitamin D3 might have been able to modulate the transcriptional activity of Runx1, Runx2 or Runx3 in the tissues expressing VDR. After the split from amphibians, in the lineage leading to amniotes, Runx2 and VDR became robustly co-expressed in developing skeletal elements, and their regulatory interaction was incorporated in the genetic program involved in the specification and differentiation of osteoblasts.

Two New Unrelated Cases of Hereditary 1,25-Dihydroxyvitamin D-resistant Rickets with Alopecia resulting from the same Novel Nonsense Mutation in the Vitamin D Receptor Gene

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) an important regulator of bone homeostasis, mediates its actions by binding to the vitamin D receptor (VDR), a nuclear transcription factor. Mutations in the VDR cause the rare genetic disease hereditary vitamin D resistant rickets (HVDRR). In this study, we examined two unrelated young female patients who exhibited severe early onset rickets, hypocalcemia, and hypophosphatemia. Both patients had partial alopecia but with different unusual patterns of scant hair. Sequencing of the VDR gene showed that both patients harbored the same unique nonsense mutation that resulted in a premature stop codon (R50X). Skin fibroblasts from patient #1 were devoid of VDR protein and 1,25(OH)2D3 treatment of these cells failed to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 action. In conclusion, we identified a novel nonsense mutation in the VDR gene in two patients with HVDRR and alopecia. The mutation truncates the VDR protein and causes 1,25(OH)2D3 resistance.

The Antiproliferative Effect of Calcitriol on Breast Cancer Cell Lines Depends on Vitamin D Receptor Status and the Expression of Prostaglandin Metabolizing Enzymes.

Abstract Background: Both first studies in prostate cancer and our results in breast cancer suggest a link between vitamin D and prostaglandin metabolism. There is a growing body of evidence that COX-2 expression is a fundamental step in breast cancer pathogenesis acting through prostaglandin-dependent and independent mechanisms. The antiproliferative effects of calcitriol (1,25(OH)2D3) mediated via the vitamin D receptor (VDR), render the biologically active form of vitamin D a promising target in breast cancer therapy.Materials and methods: The expression of VDR, COX-2 and 15 PGDH was determined by Western blot in MCF-10F cells, a human benign epithelial cell line, in MCF-7 cells, a moderate invasive and in MDA-MB-231 cells, a highly invasive breast cancer cell line. Measurement of basal PGE2 secretion was performed with ELISA. The influence of calcitriol on cell proliferation was determined by 5-bromo-2-deoxyuridine (BrDU) incorporation (BrDU ELISA). In addition we examined the effect of calcitriol on COX-2, 15 PGDH and VDR protein levels compared to untreated cells.Results: After 72 hours the proliferation of MCF-10F cells was dose-dependent inhibited to 0.83± 0.1 by 1 nM calcitriol. Concurrently, we observed a reduction of COX-2 expression to 0.63± 0.05 as compared to the untreated control group, but we also found a reduction of 15 PGDH to 0.73± 0.16. These effects were associated with a higher expression of VDR to 1.64± 0.16. Interestingly, MCF-7 cell growth was reduced to 0.52± 0.04 by 10 nM calcitriol and to 0.7± 0.06 by 1 nM. These effects do not seem to be associated with COX-2 and 15 PGDH and are independently of VDR. VDR was detected neither in stimulated nor in unstimulated cells. A PGE2 secretion could not be detected by ELISA. In contrast, we found no effect on cell growth, COX-2 and 15-PGDH expression of the MDA-MB-231 cells when treated by calcitriol (10 nM and 1 nM). Although PGE2 was synthesized 2.3-fold more by MDA-MB-231 cells than MCF-10F cells; the COX-2 expression was consistent with the MCF-10F cells, but 15-PDGH was expressed about 50 % less. As we have shown in MCF-7 cells, an expression of the VDR could not be detected.Conclusions: The growth inhibiting effect of calcitriol in MCF10F cells is associated with the increasing expression of VDR. and a remaining question is whether the loss of VDR goes along with the invasiveness of the breast cancer cell lines. Additionally, we suggest that the invasiveness of the breast cells is abetted by the increased COX-2 expression (MCF-7) or by the decreased expression of 15 PGDH (MDA-MB-231). These results suggest even more a link between vitamin D and prostaglandin metabolism and therefore a possible synergism between COX-2 inhibition and calcitriol in breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5155.

Effects of 1α,25‐dihydroxyvitamin D3 on transporters and enzymes of the rat intestine and kidney in vivo

1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR-rich target organ in which VDR regulation on xenobiotic transporters and enzymes is ill-defined. Hence, changes in protein and mRNA expression of nuclear receptors, transporters and enzymes of the rat intestine and kidney in response to 1,25(OH)2D3 treatment (0 to 2.56 nmol/kg/day intraperitoneally in corn oil for 4 days) were studied. In the intestine, protein and not mRNA levels of Mrp2, Mrp3, Mrp4 and PepT1 in the duodenum and proximal jejunum were induced, whereas Oat1 and Oat3 mRNA were decreased in the ileum after 1,25(OH)2D3 treatment. In the kidney, VDR, Cyp24, Asbt and Mdr1a mRNA and protein expression increased significantly (2- to 20-fold) in 1,25(OH)2D3-treated rats, and a 28-fold increase of Cyp3a9 mRNA but not of total Cy3a protein nor Cyp3a1 and Cyp3a2 mRNA was observed, implicating that VDR played a significant, renal-specific role in Cyp3a9 induction. Additionally, renal mRNA levels of PepT1, Oat1, Oat3, Ostalpha, and Mrp4, and protein levels of PepT1 and Oat1 were decreased in a dose-dependent manner, and the approximately 50% concomitant reduction in FXR, SHP, HNF-1alpha and HNF-4alpha mRNA expression suggests the possibility of cross-talk among the nuclear receptors. It is concluded that the effects of 1,25(OH)2D3 changes are tissue-specific, differing between the intestine and kidney which are VDR-rich organs.

Abstract A52: Mechanisms of 1,25-dihdyroxyvitamin D-mediated growth arrest and apoptosis in transformed mammary cells

Abstract 1,25-Dihdyroxyvitamin D (1,25D), the biologically active form of vitamin D, exerts pro-differentiating and anti-proliferative actions on epithelial cells, but the exact role of the vitamin D receptor (VDR) in mediating these effects remains to be defined. The goal of this project is to link specific functional domains of the VDR with regulation of cancer cell proliferation, invasion, and apoptosis. Epithelial cell lines derived from DMBA induced mammary tumors generated in VDR knock-out (KO) mice and their wild-type (WT) littermates were characterized for growth arrest and apoptosis following 1,25D treatment. Cells derived from WT tumors expressed VDR and exhibited growth arrest and apoptosis in response to 1,25D. In VDR positive cells, 1,25D mediated apoptosis was associated with activation of multiple caspases and calpain, resulting in PARP cleavage. In contrast, cells derived from KO tumors lacked VDR and failed to undergo growth arrest and apoptosis upon 1,25D treatment. To recreate VDR signaling in KO cells, human VDR was stably expressed in the KO240 cell line, generating the KOhVDR cell line. KOhVDR cells expressed transcriptionally active VDR protein at levels comparable to that of WT cells. Re-introduction of VDR did not affect doubling time of KOhVDR cells in the absence of ligand, but did sensitize cells to growth arrest and apoptosis in response to VDR agonists. PCR screening arrays and independent qPCR validation were used to identify VDR target genes in this model system. In WT cells, 6-24h treatment with 100nM 1,25D induced PDGF-B (up to 10 fold) and VEGF-A (up to 5 fold). No changes in PDGF-B or VEGF-A expression were observed in KO cells treated with 1,25D, but both genes were inducible by 1,25D in KOhVDR cells stably expressing hVDR. Thus, 1,25D can activate either human or murine VDR to induce endogenous PDGF-B and VEGF-A gene expression in these murine cells. In summary, these data indicate that VDR does not exert ligand-independent effects on mammary cell growth, but is essential to trigger gene expression, growth arrest and apoptosis in response to 1,25D. Supported by NIH CA69700. Citation Information: Cancer Res 2009;69(23 Suppl):A52.

Presence of a Deletion Mutation (c.716delA) in the Ligand Binding Domain of the Vitamin D Receptor in an Indian Patient with Vitamin D-Dependent Rickets Type II

Vitamin D-dependent rickets type II (VDDR-type II) is a rare disorder caused by mutations in the vitamin D receptor (VDR) gene. Here, we describe a patient with VDDR-type II with severe alopecia and rickets. She had hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and elevated serum alkaline phosphatase and 1,25-dihydroxyvitamin D(3). Sequence analysis of the lymphocyte VDR cDNA revealed deletion mutation c.716delA. Sequence analysis of her genomic DNA fragment amplified from exon 6 of the VDR gene incorporating this mutation confirmed the presence of the mutation in homozygous form. This frameshift mutation in the ligand binding domain (LBD) resulted in premature termination (p.Lys240Argfs) of the VDR protein. The mutant protein contained 246 amino acids, with 239 normal amino acids at the N terminus, followed by seven changed amino acids resulting in complete loss of its LBD. The mutant VDR protein showed evidence of 50% reduced binding with VDR response elements on electrophoretic mobility assay in comparison to the wild-type VDR protein. She was treated with high-dose calcium infusion and oral phosphate. After 18 months of treatment, she gained 6 cm of height, serum calcium and phosphorus improved, alkaline phosphatase levels decreased, and intact PTH normalized. Radiologically, there were signs of healing of rickets. Her parents and one of her siblings had the same c.716delA mutation in heterozygous form. Despite the complete absence of LBD, the rickets showed signs of healing with intravenous calcium.