Vitamin D Receptor Gene Expression Research Articles

Age and Vitamin D Receptor (VDR) Expression and Functionality in Peripheral Blood Mononuclear Cells (PBMC)

ObjectiveCharacterize PBMC VDR expression and functionality by age and serum 25(OH) D.Methods40 adults ≥ 50 yrs provided pre‐influenza vaccination blood for PBMC isolation in fall 2009. Serum 25(OH) D was measured by liquid chromatography/mass spectrometry. Gene expression of VDR, 1α‐hydroxylase (1αOHase), cathelicidin, triggering receptor expressed on myeloid cells 1 (TREM1), retinoic acid‐inducible gene 1 (RIG1), and interferon β (IFNβ) was measured by quantitative reverse transcription polymerase chain reaction in unstimulated and lipopolysaccharide (LPS) or vitamin D‐treated PBMC.ResultsMean ± SD serum 25(OH) D was 30 ± 4 ng/mL and not associated with age. Unstimulated mRNA expression of VDR, 1αOHase, TREM1, and RIG1 did not differ by age. IFNβ mRNA expression was higher in subjects ≥ 80 vs. <70 yrs (p<0.05). LPS stimulation upregulated expression of VDR (p<0.01) and 1αOHase (p<0.05). 25(OH) D and 1, 25(OH)2 D added in vitro upregulated TREM1 and cathelicidin. Serum 25(OH) D was inversely associated with unstimulated (r = −0.31) and LPS‐stimulated (r = −0.41) TREM1 expression, and LPS‐stimulated VDR expression (r = −0.33), adjusting for age, self‐rated health, and functional status.ConclusionAge had no consistent association with expression or functionality of the VDR or 1αOHase genes. 25(OH) D levels were inversely associated with TREM1 and LPS‐stimulated VDR expression. This project has been funded by CDC grants 5 U18 IP000183–02 and 1 U18 IP000184–01.

Vitamin D inquiry in hippocampal neurons: consequences of vitamin D-VDR pathway disruption on calcium channel and the vitamin D requirement

Vitamin D receptor (VDR) and the enzymes involved in bioactivation of vitamin D, shown to be expressed in the central nervous system, particularly in areas affected by neurodegenerative disorders, especially in hippocampus. We showed that amyloid beta (Aβ) pathology includes VDR protein depletion and vitamin D-VDR pathway disruption either induced by Aβ or by VDR siRNA have very similar effects on cortical neurons. The goal of this study is to show the presence of 25 hydroxy vitamin D3-24 hydroxylase (24OHase) which is essential for vitamin D catabolism in hippocampal and cortical neurons. Additional goal is to compare the expression pattern of VDR and 24OHase both in hippocampal and in cortical neurons and to investigate the effects of VDR suppression in hippocampal neurons in order to see whether similar mechanisms work in hippocampus and cerebral cortex. Primary neuronal cultures were prepared from Sprague-dawley rat embryos. qRT-PCR was performed to determine VDR, 24OHase, and LVSCC-A1C mRNA expression levels. Cytotoxicity levels were determined by ELISA. Our findings illustrate that 24OHase mRNA was present both in hippocampal and in cortical neurons. VDR and 24OHase mRNA were higher in hippocampal neurons than the cortical ones. LVSCC-A1C mRNA levels increased in hippocampal neurons when VDR is down-regulated. Our results indicate that hippocampal neurons response to VDR suppression similar as cortical neurons, regarding calcium channel regulation. Higher gene expression of 24OHase and VDR might indicate "higher requirement of vitamin D" in hippocampus and potential consequences of vitamin D deficiency in cognitive decline, neurodegeneration, and Alzheimer's disease.

Abstract 3084: Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription by suppressing vitamin D receptor gene expression in mouse colon epithelial cells

Abstract The active metabolite of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), binds to the vitamin D receptor (VDR) and stimulates anti-cancer transcriptional events in many cell lines including colon cancer cells. However, we found that tumors which form in the distal colon of mice with an APC allele deletion had significantly lower VDR mRNA levels compared to RNA isolated from the distal colon of wild-type mice (-92%, p≤0.05). These data suggest that colon carcinogenesis may impair 1,25(OH)2D-regulated chemoprevention in colon epithelial cells. APC inactivation and KRAS activation are early events in the development of distal colon that may suppress VDR gene expression. Compared to immortalized colonic epithelial cells derived from the “Immorto” mouse (YAMC), cells from a cross between APCmin and “Immorto” mice (IMCE), and YAMC cells overexpressing the v-Ha-RAS gene (YAMC-RAS) have significantly lower VDR mRNA (-80%; p≤0.05) expression. Previous research shows that constitutively active (ca) RAS signaling suppresses 1,25(OH)2D action in many cell lines so we conducted additional studies on the impact of this mutation. YAMC-RAS cells had a significant reduction in 1,25(OH)2D-induced CYP24 mRNA expression (-40%; p≤0.05). Consistent with lower VDR mRNA levels, YAMC-RAS cells also had lower VDR protein levels than YAMC cells and the suppression of 1,25(OH)2D-induced CYP24 expression in YAMC-RAS was overcome with VDR over-expression. The mechanism for RAS-mediated suppression of VDR mRNA levels was further explored. First we hypothesized that caRAS signaling expression reduced VDR mRNA stability, however, YAMC and YAMC-RAS cells treated with 4 µg/ml of the transcription inhibitor actinomycin D for up to 12 h had no significant difference in the VDR mRNA half-life (about 8 h). Next we examined whether caRAS signaling reduced accessibility within chromosomal regions in the VDR gene previously identified as necessary for VDR gene expression. Using a DNAse I hypersensitivity assay we found that in YAMC-RAS cells there was a significant reduction (p≤0.05) in chromosome accessibility on the proximal promoter (-23%, 300 bp upstream of exon 1), an enhancer region located 6 kb upstream of the transcriptional start site (-56%), and an enhancer region located in exon 2 (-100%) compared to the YAMC cells. These data show that caRAS signaling suppresses 1,25(OH)2D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription. Supported by NIDDK Award DK54111and AICR Award 09A098 to JCF Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3084. doi:1538-7445.AM2012-3084

Hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D together stimulate human bone marrow-derived stem cells toward the osteogenic phenotype by HGF-induced up-regulation of VDR

Bone formation and remodeling require generation of osteoprogenitors from bone marrow stem cells (MSC), which are regulated by growth factors and hormones, with putative roles in mesenchymal cell differentiation. Hepatocyte growth factor (HGF) is a pleiotropic growth factor, and together with its high affinity receptor cMet are widely expressed in normal tissues. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D; produced mainly in the kidney, but also by osteoblasts. We previously reported that HGF and 1,25OHD act together to increase osteogenic differentiation of human MSC (hMSC) potentially through increasing p53. Although p53 does not induce the vitamin D receptor (VDR), p63, a member of the p53 family of transcription factors has been reported to up-regulate VDR expression in some tumor cell lines, and thus might play a part in HGF-regulated VDR expression. Our hypothesis is that the combination of HGF and 1,25OHD can induce hMSC differentiation by up-regulation of 1,25OHD and/or VDR expression to increase cell response(s) to 1,25OHD. Using real-time RT-qPCR, Western blots, luciferase reporter assays, and siRNAs, as well as antibodies to specific signaling molecules we showed that HGF induced VDR gene expression, as well as up-regulated p63 gene expression. p63 gene knockdown by siRNA eliminated the effects of HGF on VDR gene expression as measured by RT-qPCR, Western blots and luciferase reporter assay, and downstream on osteogenic differentiation markers, including alkaline phosphatase staining. Differentiation is a coordinated process of cell cycle exit and tissue-specific gene expression. These results suggest HGF might be a good candidate to coordinate the regulation of these two processes during hMSC osteogenic differentiation. p63 could be a key connecting molecule on the pathway of HGF-induced VDR expression. Understanding the role of these factors and their actions could have important clinical implications for the use of hMSC in the development of novel stem cell therapies.

Curcumins Promote Monocytic Gene Expression Related to β-Amyloid and Superoxide Dismutase Clearance

Neurodegenerative diseases are associated with accumulation of modified proteins or peptides including amyloid-β (Aβ) in Alzheimer’s disease (AD), and misfolded superoxide dismutase-1 (SOD-1) in amyotrophic lateral sclerosis (ALS). Clearance of Aβ or SOD-1 by the innate immune system may be important for controlling or preventing disease onset. Curcumins restore Aβ phagocytosis by peripheral blood mononuclear cells (PBMCs) from AD patients and Aβ clearance with upregulation of key genes including MGAT3, vitamin D receptor (VDR) and Toll-like receptors (TLRs). Certain curcumins inhibit inflammatory processes of PBMCs from ALS patients. We developed an in vitro system using human monocytes from patients and monocytic cell lines (i.e. U-937, THP-1) for evaluating curcuminoid potency of innate immune cell stimulation. Bisdemethoxycurcumin and certain analogs potentiated MGAT3,VDR and TLR gene expression 3- to 300-fold in U-937 cells. The effect of curcumins on inflammation in monocytes from patients with ALS was examined. Recursive medicinal chemistry was applied to identify compounds that stimulate the innate immune system for use in the clearance of Aβ in AD and the reversal of neuroinflammation and defective SOD-1 accumulation in ALS.

Osteoblast-Specific Transcription Factor Osterix Increases Vitamin D Receptor Gene Expression in Osteoblasts

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

Abstract B36: Development of calcitrol-resistance in triple-negative breast cancer cells through SLUG-mediated coordinate repression of CYP2R1, CYP27B1, and VDR gene promoters

Abstract Metastatic breast and other cancers are often found to be refractory to vitamin D therapy. Vitamin D is synthesized from 7-dehydrocholesterol, an intermediate metaboltie in cholesterol synthesis. Ultraviolet irradiation in sunlight-exposed skin induces a photochemical reaction of 7–25-position by vitamin D 25-hydroxylase (CYP2R1), to yield 25-hydroxyvitamin D3 (cholecalciferol). Vitamin D3 is hydroxylated at the 25-position by vitamin D 25-hydroxylase (CYP2R1), to yield 25-hydroxyvitamin D3 (25(OH)D3; 25-hydroxycholecalciferol), the major form of vitamin D in the circulation. 25(OH)D3 is further hydroxylated at the 1 -position by the 25-hydroxyvitamin D 1 -hydroxylase (CYP27B1) to produce calcitrol (VD3). VD3 exhibits physiological and pharmacological effects by binding to the vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Although photoactivated cholecalciferol is mainly hydroxylated in the liver and kidney, many other cells including breast cells have significant expressions of CYP2R1, CYP27B1 and VDR. Breast cells thus should be able to activate and utilize cholecalciferol if these proteins are not suppressed. We report here that the metastasis modulator protein SLUG, which is often overexpressed in metastatic triple negative breast cancer cells, coordinately repressse the levels of CYP2R1, CYP27B1 and VDR proteins in the breast cancer cells to induce vitamin D-resistance in these cells. SLUG inhibited CYP2R1, CYP27B1 and VDR gene promoter activities in these cells. ChIP assays revealed that SLUG is recruited at the CYP2R1, CYP27B1 and VDR gene promoters. Knockdown of SLUG in highly invasive MDA-MB-231 and BT549 cells increased their CYP2R1, CYP27B1 and VDR gene expression and decreased their resistance their resistance to VD3 in vitro. Our data established that SLUG regulates vitamin D metabolism and contributes to the induction of VD3-resistance in human breast cancer cells through the inhibition of CYP2R1, CYP27B1 and VDR gene promoters epigenetically through chromatin remodeling. Supported in parts by the DOD-CDMRP grants BC050641 and BC103645. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B36.

Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice

ObjectivesThe presence of vitamin D receptor (VDR) and the identification of localized vitamin D3 synthesis in placenta and decidua implicate the importance of vitamin D3 in reproductive function. There is, however, no data on the expression profile of VDR in the mouse placenta and endometrium throughout the pregnancy period. Study designIn the present work expression of VDR in reproductive tissues of pregnant mice at different gestational phases has been addressed. Expression of VDR was determined by semi-quantitative RT-PCR, Western blotting and immunohistochemistry. ResultsThe results showed that VDR mRNA and protein were expressed in decidua, placenta and ovary throughout the pregnancy. VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy. Additionally, VDR expression level in decidua rose significantly as pregnancy progressed from early to mid stages. VDR expression in decidua of pregnant mice was higher in comparison to endometrium of non-pregnant mice. Immunohistochemical analysis revealed that VDR protein is consistently expressed by luminal and glandular epithelial cells of decidua, giant cells, glycogen rich cells and labyrinth cells of placenta and by almost all follicular cell types of ovary. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. ConclusionIt seems that expression of VDR in reproductive organs is finely tuned during pregnancy indicating its eminent role in reproductive biology.

Alveolar bone level is not associated with vitamin D receptor gene polymorphism and bone density in mandible

The objective of this study was to determine, using digital panoramic radiographs, whether the bone level at the alveolar crest is related to the mandibular bone density and/or to vitamin D receptor (VDR) gene polymorphisms. We analyzed 319 digital panoramic radiographs from the same number of patients. Alveolar bone level was expressed as percentage of root length. The mandibular cortical width index was calculated as a measure of mandibular bone density, and, in 72 randomly selected cases, the haplotype of the VDR gene (BsmL) was determined by polymerase chain reaction. Alveolar bone level was not related to the mandibular cortical width index (p = 0.568) or VDR gene expression (p = 0.575). Bone loss was greater in smokers than in non-smokers (p = 0.036), and the mandibular cortical width index was higher in males (p = 0.04), the older age group (p = 0.032), and in those with more teeth (p = 0.01). Multivariate analysis confirmed the association between these variables and alveolar bone loss. Alveolar bone loss showed no significant relationship with the mandibular bone density evaluated on digital panoramic radiographs or with VDR genotype (BsmL) in Caucasian females and males aged under 47years.

Decreased Anti-Inflammatory Responses to Vitamin D in Neonatal Neutrophils

Neutrophil activity is prolonged in newborns, suggesting decreased exposure and/or responses to immunosuppressive modulators, such as 1,25-hydroxyvitamin D3 (1,25-vit D3). We hypothesized that 1,25-vit D3 suppresses neutrophil activation and that this response is impaired in newborns. Consistent with this, 1,25-vit D3 decreased LPS-induced expression of macrophage inflammatory protein-1β and VEGF in adult, but not neonatal, neutrophils. Expression of vitamin D receptor (VDR) and 25-hydroxyvitamin D3-1α-hydroxylase was reduced in neonatal, relative to adult neutrophils. Moreover, 1,25-vit D3 induced VDR gene expression in activated adult, but not neonatal, neutrophils. 1,25-vit D3 also suppressed expression of cyclooxygenase-2 and induced expression of 5-lipoxygenase in LPS-exposed adult neutrophils, while neonatal cells were not affected. 1,25-vit D3 had no effect on respiratory burst in either adult or neonatal cells. Anti-inflammatory activity of vitamin D is impaired in neonatal neutrophils, and this may be due to decreased expression of VDR and 1α-hydroxylase. Insensitivity to 1,25-vit D3 may contribute to chronic inflammation in neonates.

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.

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.

Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines

Vitamin D receptor (VDR) regulates the expression of many genes involved in mineral metabolism, cellular proliferation, differentiation and drug biotransformation. We studied the expression and activity of VDR and its heterodimerization partner retinoid X receptor-alpha (RXRalpha) in choriocarcinoma trophoblast cell lines BeWo and JEG-3, in comparison with human isolated placental cytotrophoblasts and human full term placenta. We found that VDR and RXRalpha are localised in the human term placenta trophoblast and expressed in isolated cytotrophoblasts. However, we found low expression and no transcriptional activity of VDR in used choriocarcinoma cell lines. The inhibitor of DNA methylation, 5-deoxy-3'-azacytidine, and histone deacetylase inhibitor sodium butyrate partially restored the expression of VDR, suggesting an epigenetic suppression of the gene in choriocarcinoma cells. Differentiation of BeWo cells resulted in up-regulation of VDR mRNA. Finally, we observed a non-genomic effect of 1,25(OH)(2)D(3) in the activation of the extracellular signal-regulated kinase (ERK) signalling pathway in JEG-3 cells. In conclusion, our results suggest an epigenetic repression of VDR gene expression and activity in choriocarcinoma cell lines, and a non-genomic effect of 1,25(OH)(2)D(3) in JEG-3 cells.

Epidemiological Studies of Vitamin D and Breast Cancer

Abundant experimental evidence provides a rationale for examining the role of vitamin D in relation to breast cancer risk. Specifically, the main actions of the biologically active form of vitamin D, 1,25(OH)2D, are mediated via the vitamin D receptor. The vitamin D receptor (VDR) is present in normal breast tissue. 1,25(OH)2D has antiproliferative effects on and promotes the differentiation of breast cancer cells. In MCF-7 cells, vitamin D and vitamin D analogs have been shown to induce cell cycle arrest and apoptosis, to downregulate estrogen receptor expression, to limit responsiveness to the mitogenic effects of 17beta-estradiol, and to limit induction of the progesterone receptor. Epidemiologic evidence bearing on the relationship between vitamin D and breast cancer risk has come from several sources: ecologic studies, studies of vitamin D in relation to breast density, studies of VDR polymorphisms and breast cancer risk, studies of circulating vitamin D levels and risk, and studies of dietary and supplemental vitamin D intake and risk. There have been several ecologic studies of the association between sunlight or solar radiation exposure and breast cancer incidence or mortality. These are of interest because synthesis in the skin resulting from exposure to sunlight (in particular, exposure to UV-B radiation) is the major source of endogenous vitamin D production in humans, given that few food sources contain vitamin D. For example, one study showed a strong inverse association between per capita average annual total solar energy exposure (calories/cm/d) and breast cancer incidence rates in republics of the former Soviet Union. However, as is well known, ecologic studies suffer from a number of limitations, including the fact that because measurements are averaged over individuals, associations observed at the population level may not reflect associations at the individual level and generally do not account for potential confounding. Therefore, this type of information is suggestive (hypothesis-generating) at best. Mammographic (breast) density reflects the epithelial and stromal components of the breast; fat appears dark and epithelium and stroma appear light or white. Women with very dense breast tissue, as seen on mammography, are at increased risk of subsequent breast cancer. Mammographic density can be modified, and such changes represent a potential biological marker for assessing the effects of dietary/supplemental factors on breast cancer risk. Four studies have examined the association between vitamin D and breast density. Of these, two showed an inverse association between vitamin D intake and breast density in premenopausal women only (one focused on vitamin D from foods only and the other on total vitamin D intake from foods and supplements), one study showed an inverse association in both premenopausal and postmenopausal women (dietary vitamin D only), and one study showed no association. However, all of these studies were cross-sectional, which limits the inferences that can be drawn. Interestingly, in one study, the association was independent of sunlight exposure but weakened after adjustment for calcium intake, highlighting the need to include assessment of calcium intake in studies of vitamin D. A recent cross-sectional study showed no association between serum 25-hydroxyvitamin D levels and mammographic density. The vitamin D receptor is a nuclear transcription regulating factor that is the crucial mediator of the cellular growth and differentiation effects of vitamin D. It is expressed in normal and malignant breast cells. Genetic polymorphisms in the VDR might influence breast cancer risk due to their effects on VDR gene expression and protein function. Of the polymorphisms that have been identified in the VDR gene (largely through their restriction endonuclease cleavage sites), Fok1, Bsm1, Apa1, Taq1, and Poly(A) have been studied most frequently. Whereas the Fok1 polymorphism (a T-to-C transition in exon 2) has functional consequences, the functional significance of the Bsm1, Apa1, Taq1, and Poly(A) polymorphisms, located at the 3 end of the Dr. Rohan is with the Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York, USA. Please address all correspondence to: Dr. Tom Rohan, Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA; Phone: 718-4303355; Fax: 718-430-8653; E-mail: rohan@aecom.yu.edu. doi: 10.1301/nr.2007.aug.S80–S83

Simultaneous changes in the calcium-sensing receptor and the vitamin D receptor under the influence of calcium and calcitriol

The regulatory mechanisms of parathyroid hormone (PTH) synthesis are complex, involving calcium, calcitriol, the calcium-sensing receptor (CaR) and the vitamin D receptor (VDR). In this study, the effects of calcium and calcitriol on the simultaneous expression of CaR and VDR mRNA and protein levels were assessed in parathyroid glands cultured in vitro. Parathyroid glands (N = 424) were removed and cultured for 24 h to study the effect of calcium on the CaR, VDR and PTH. In addition, the effect of calcitriol at low calcium concentrations (0.6 mM) on CaR and VDR levels was studied after 48 h of incubation. CaR, VDR and PTH mRNAs were measured by quantitative real-time PCR (qRT-PCR), and CaR and VDR protein levels were measured by immunohistochemistry. PTH gene expression was reduced by high calcium concentration. No differences were found in the CaR mRNA levels among the different calcium concentrations tested (0.6 mM calcium: 100%; 1.2 mM calcium: 120%; 2.0 mM calcium: 112%; median values), but VDR gene expression rose when calcium increased (0.6 mM calcium: 100%; 1.2 mM calcium: 164%; 2.0 mM calcium: 195%; median values). Calcitriol increased both CaR (control: 100%; 10(-8) M calcitriol: 196%; median values) and VDR genes expression (control: 100%; 10(-8) M calcitriol: 176%; median values). The same findings were corroborated at protein levels for both CaR and VDR. In parathyroid glands cultured in vitro, calcium up-regulates VDR but not CaR. Conversely, calcitriol up-regulates both VDR and CaR mRNAs and protein levels, even at low calcium concentrations.

In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells

The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628 bp fragment (−613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling.

Enhancers Located within Two Introns of the Vitamin D Receptor Gene Mediate Transcriptional Autoregulation by 1,25-Dihydroxyvitamin D3

The biological actions of 1,25-(OH)2D3 are mediated by the vitamin D receptor (VDR), a protein that binds to target genes and alters their expression. 1,25-(OH)2D3 is also capable of inducing transcription of the VDR gene itself. In the present study, we explored both the capacity of 1,25-(OH)2D3 to induce VDR gene expression in bone cells and the mechanism instrumental to this up-regulation. After establishing the ability of 1,25-(OH)2D3 to stimulate VDR mRNA up-regulation both in bone in vivo and in osteoblastic cells, we screened the mouse VDR gene locus from 20 kb upstream of the gene's transcriptional start site (TSS) to 10 kb downstream of the final exon to identify VDR binding sites using chromatin immunoprecipitation-DNA microarray (ChIP-chip) analysis. Three conserved regions were identified 20, 27, and 29 kb downstream of the TSS. VDR binding to these sites in response to 1,25-(OH)2D3 was confirmed by ChIP analysis and was accompanied by differential localization of retinoid X receptor, histone acetylation, and RNA polymerase II recruitment. One of these regions was able to confer 1,25-(OH)2D3 regulation to downstream promoters, thereby permitting identification and characterization of the regulatory element located within. Importantly, a highly conserved region within the human VDR gene analogous to that discovered in the mouse was also capable of mediating 1,25-(OH)2D3 response. Our results demonstrate that 1,25-(OH)2D3 and its receptor autoregulate the expression of the VDR gene. The location of these regulatory regions and their apparent distances from the TSS are consistent with new findings suggesting the emerging relevance of distant enhancers.

Effects of vitamin D analogue EB1089 on proliferation and apoptosis of hepatic carcinoma cells

This study aimed at investigating the effects of vitamin D analogue EB1089 on the proliferation and apoptosis of hepatic carcinoma cells. Hepatic carcinoma cell strain G(2) (Hep-G(2)) in which prominent vitamin D receptor (VDR) mRNA could be expressed and the cell strain T (HCC-T) negative in VDR gene expression were incubated in culture media with 100 nmol/L, 10 nmol/L and 1 nmol/L EB1089 for 2 d, 4 d and 6 d, respectively. Survival and proliferation of the cells were detected by blue tetrazolium colorimetric test and plate clone-forming test, the VDR mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and apoptosis of the cells was detected by flow cytometry (FCM) and electron microscopy. EB1089 could inhibit the proliferation of hepatocellular cell line Hep-G(2) that expressed prominent vitamin D receptor mRNA, the inhibitory rate is 17.5% approximately 72.1%. On the other hand, EB1089 had no anti-proliferative effect on hepatocellular cell line HCC-T in which the gene expression of vitamin D receptors was negative. The electron microscope results showed that EB1089 could induce apoptosis of hepatocarcinoma cells and the percentages of apoptotic cells measured by flow cytometer was 21.4%. Cell cycle progression was blocked at G(1) phase with EB1089. EB1089 could inhibit proliferation of human Hep-G(2), probably through VDR, and induce apoptosis of the cells.

Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer

By using array comparative genomic hybridization (CGH), the increased copy number of CYP24 (which encodes vitamin D 24-hydroxylase) at 20q13.2 was previously reported, leading to the identification of CYP24 as a candidate oncogene in breast cancer. CYP24 leads to abrogate growth control mediated by vitamin D. We examined CYP24 expression as well as VDR (vitamin D receptor) gene expression in 42 esophageal cancer cases using semi-quantitative RT-PCR assay. We induced CYP24 in seven esophageal cancer cell lines using 25-hydroxyvitamin D3 [25(OH)D3] and compared cell growth rate, measured using the 3-(4, 5-dimethylthiazol-2-y)-2, 5-diphenyltetrazolium bromide (MTT) assay system. The overall survival rate was significantly higher in 25 cases of lower CYP24 expression than 17 cases of higher CYP24 expression (P <0.05); on the other hand, 23 cases of low VDR expression had a poorer prognosis than 19 cases of high VDR expression. Moreover, we disclosed that the inverse correlation between CYP24 and VDR expression is significant in esophageal cancer cases (P <0.05). Furthermore, the cell growth evaluated by MTT assay was greatly increased in CYP24-induced and VDR-diminished cells than non-responding cells by 25(OH)D3 activity (P <0.01). Overexpression of the candidate oncogene CYP24 is inversely correlated to vitamin D receptor expression, and may play an important role in determination of the malignant potential of esophageal cancer.

Exclusive underexpression of vitamin D receptor exon 1f transcripts in tumors of primary hyperparathyroidism.

Primary hyperparathyroidism (pHPT) is characterized by excessive production of parathyroid hormone (PTH) due to parathyroid adenomas while uremic secondary HPT (sHPT) is caused by parathyroid hyperplasia in response to renal failure. Active vitamin D, 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), with the vitamin D receptor (VDR) is involved in regulation of the calcium homeostasis together with PTH. In a feedback loop, 1,25-(OH)(2)D(3) has a direct action on the parathyroid gland to regulate PTH transcription, PTH secretion and cell proliferation. We have previously demonstrated reduced VDR mRNA expression in parathyroid adenomas and hyperplasia of sHPT using a probe detecting all 14 variant VDR transcripts expressed in parathyroid cells. Here we have assessed which of the 5'-terminal exon 1a, 1d and 1f variant VDR transcripts are reduced in pathological parathyroid glands. The relative VDR/glyceraldehyde-3-phosphate dehydrogenase mRNA levels for each VDR exon were determined by real-time quantitative RT-PCR in five normal parathyroid glands, seventeen parathyroid adenomas and ten hyperplastic glands of sHPT. The results demonstrated exclusive underexpression of VDR exon 1f transcripts in parathyroid adenoma, while all measured VDR transcripts were reduced in secondary hyperplasia. We suggest that exclusive underexpression of VDR exon 1f transcripts in adenomas of pHPT, which derive from a distal promoter active in tIssues involved in calcium regulation by 1,25-(OH)(2)D(3), may either reflect a defective cell type-specific transcription factor or other physiologically important pathway(s) for tIssue-specific VDR gene expression.