Vitamin D Receptor Gene Promoter Research Articles

Growth, Dietary Intake, and Vitamin D Receptor (VDR) Promoter Genotype in Indonesian School-Age Children.

Nutrition has been known as a predominant factor associated with stunting. However, some studies have discovered a genetic contribution in calcium absorption that will affect growth, known as the VDR gene. The aim of this study was to assess the association between VDR gene polymorphism and dietary intake towards height-for-age z-score (HAZ) of elementary school children in Malang District, East Java. This study analyzed the baseline of a randomized trial in East Java, Indonesia. School children aged 8–10 years old (n = 142) were included in this study. Energy, protein, calcium, and vitamin D intakes were obtained using 4-day 24-h dietary recalls. Two SNPs located in the promoter region of VDR gene were selected (rs11568820 and rs4516035) and analyzed using Real-Time PCR. The result showed a significant correlation between energy and protein intake with HAZ of the children (p = 0.030 and p = 0.016, respectively). The association between VDR gene and HAZ was not found (p > 0.05). Adjusted by other factors, protein intake was significantly correlated with HAZ (β = 0.034, 95% CI 0.015–0.052, p < 0.001, adj. R2 = 0.089). The children in our study had a favorable VDR gene genotype, however the effect of VDR gene promoter activity might not be revealed due to very low vitamin D and calcium intake to stimulate intestinal calcium absorption which in turn affects HAZ.

Study of vitamin D receptor gene methylation and the risk of gestational diabetes mellitus in Iranian pregnant women

Vitamin D acting through vitamin D receptor (VDR) plays a crucial role in regulating multiple pathways, including immunomodulatory properties, insulin secretion, and calcium homeostasis. Gestational diabetes mellitus (GDM) is a metabolic disorder with glucose intolerance that occurs in pregnancy. It has been lately suggested that epigenetic mechanisms as well as genetic factors have a significant role in GDM pathogenesis. Our study's intention was to investigate the methylation pattern of CpG islands of the VDR gene promoter in GDM and non-GDM population and predisposition to GDM in Iranian pregnant women. A total of 240 of pregnant Iranian women including, 120 women with GDM and 120 non-GDM were recruited in this study. Diagnosis of GDM was confirmed via a 75 g oral glucose tolerance test (OGTT) at 24–28 weeks of pregnancy. Peripheral blood DNA was isolated and underwent chemical modification using sodium bisulfite. The status of VDR promoter methylation of was determined by methylation-specific PCR (MSP). Analysis of MSP data uncovered the unmethylated promoter of VDR gene both in GDM and non-GDM pregnant women. Indeed, we found no differences in the DNA methylation patterns of the VDR promoter between GDM and non-GDM groups. Our study was the first report of VDR promoter methylation in GDM. In conclusion, we found no significant association between methylation status of VDR promoter and susceptibility to GDM in our studied population.

Methylation Status of VDR Gene and its Association with Vitamin D Status and VDR Gene Expression in Pediatric Tuberculosis Disease

ABSTRACT Deficiency in circulatory vitamin D level and vitamin D receptor DNA methylation could be associated with weakened innate immune response and increased susceptibility to tuberculosis (TB) disease in children. Therefore, we aimed to study the effect of vitamin D receptor (VDR) gene methylation on plasma vitamin D level and the expression of the VDR gene in children with active-TB disease. A cross-sectional comparative study was conducted in 43 children with active-TB and 33 healthy control children (HC). The vitamin D level was measured in plasma, while the levels of VDR gene promoter methylation and VDR gene expression were measured in peripheral blood. Children with active-TB showed a significantly lower median vitamin D level than HC [Cases 17.18 ng/mL (IQR, 8.3–18.6 ng/mL); HC 41.34 ng/mL (IQR, 40.2–43.49 ng/mL) (p<0.0001)] and decreased mRNA expression level of VDR gene [Cases 0.51 (IQR, 0.40–0.70); HC 1.06 (IQR, 0.8–1.2) (p<0.0001)] and increased VDR DNA methylation [Cases 75% (IQR, 50–75%); HC 10% (IQR, 10–25%) (p<0.0001)]. The VDR hypermethylation is significantly associated with reduced vitamin D level and decreased expression level of VDR gene. Therefore this inverse association could be involved in the impairment in the VDR mediated cytolytic and antimicrobial effector cell response in pediatric TB disease.

Vitamin D receptor gene methylation in hepatocellular carcinoma

Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p < 0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcumin-adequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.

THE INVOLVEMENT OF VDR PROMOTER METHYLATION, CDX-2 VDR POLYMORPHISM AND VITAMIN D LEVELS IN MALE INFERTILITY.

Vitamin D (VD) levels were correlated with different health conditions, including reproductive disorders in males. Vitamin D action is mediated through vitamin D receptor (VDR), which acts as a transcription factor. VDR gene promoter is embedded in a GC-rich island. The VDR gene has been shown to have several polymorphisms that affect the receptor function. To examine the relationship between Cdx-2 polymorphism (rs17883968), the methylation status of VDR's promoter and serum levels of 25-hydroxyvitamin D in male infertility. A total of 69 infertile men and 37 age-matched controls were enrolled in this study. Vitamin D level assessments were detected using the electrochemiluminescent method. Cdx-2 VDR polymorphism identification was performed by PCR on DNA samples from blood, followed by restriction. Methylation of VDR gene promoter was assessed by qMS-PCR using bisulfite-treated DNA from fresh sperm. Vitamin D levels was found to be significantly decreased in infertile groups compared the controls (p=0.0279). The GG genotype was found in a higher percentage in controls and the AA genotype was higher in infertile group (p=0.0056). Infertile homozygote (GG) and heterozygote (GA) individuals had significantly higher vitamin D levels than AA homozygote. Methylation is higher in individuals with lower vitamin D levels and AA genotype is characterized by higher methylation values. The results provide new insights of Cdx-2 polymorphism is involved in vitamin D deficiency, highlighting the important role of epigenetic modification of vitamin D receptor and male infertility along with the genetic context.

Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors.

We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5′ regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas) were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesis.

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.

Calcium intake and prostate cancer among African Americans: Effect modification by vitamin D receptor calcium absorption genotype

High dietary intake of calcium has been classified as a probable cause of prostate cancer, although the mechanism underlying the association between dietary calcium and prostate cancer risk is unclear. The vitamin D receptor (VDR) is a key regulator of calcium absorption. In the small intestine, VDR expression is regulated by the CDX-2 transcription factor, which binds a polymorphic site in the VDR gene promoter. We examined VDR Cdx2 genotype and calcium intake, assessed by a food frequency questionnaire, in 533 African-American prostate cancer cases (256 with advanced stage at diagnosis, 277 with localized stage) and 250 African-American controls who participated in the California Collaborative Prostate Cancer Study. We examined the effects of genotype, calcium intake, and diet-gene interactions by conditional logistic regression. Compared with men in the lowest quartile of calcium intake, men in the highest quartile had an approximately twofold increased risk of localized and advanced prostate cancer (odds ratio [OR] = 2.20, 95% confidence interval [CI] = 1.40, 3.46), with a significant dose-response. Poor absorbers of calcium (VDR Cdx2 GG genotype) had a significantly lower risk of advanced prostate cancer (OR = 0.41, 95% CI = 0.19, 0.90). The gene-calcium interaction was statistically significant (p = 0.03). Among men with calcium intake below the median (680 mg/day), carriers of the G allele had an approximately 50% decreased risk compared with men with the AA genotype. These findings suggest a link between prostate cancer risk and high intestinal absorption of calcium.

The transcription factors Snail1 and Snail2 repress vitamin D receptor during colon cancer progression

Vitamin D receptor (VDR) mediates the antitumoral action of the active vitamin D metabolite 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). However, VDR expression is lost during colon cancer progression, possibly causing unresponsiveness to 1,25(OH) 2D 3. Although several mechanisms responsible for resistance to 1,25(OH) 2D 3 action in different types of cancer had been reported, none explained the loss of VDR expression. We have found that the transcription factors Snail1 and Snail2, known as inducers of epithelial-to-mesenchymal transition (EMT), inhibit VDR expression and block 1,25(OH) 2D 3 action in colon cancer cells. Snail1 and Snail2 have an additive repressing effect on VDR gene promoter. These effects are specific to the Snail family, as other transcription factors that function as EMT inducers do not inhibit VDR expression in colon cancer cells. Moreover, we also found that the RNA expression of SNAI1 and SNAI2 is upregulated in human colorectal tumors and inversely correlates with that of VDR. Our results suggest that high levels of SNAIL1 and SNAIL2 are a probable cause of VDR downregulation and 1,25(OH) 2D 3 unresponsiveness in colon cancer. In addition, they may contribute to the improvement of protocols for the clinical use of vitamin D compounds, as they indicate that advanced colon cancer patients overexpressing SNAIL1 and SNAIL2 are not suitable candidates for this therapy.

Higher Milk Requirements for Bone Mineral Accrual in Adolescent Girls Bearing Specific Caucasian Genotypes in the VDR Promoter

Low milk intakes hamper bone mineral acquisition during adolescence, especially in European girls. We hypothesized that ethnic-specific polymorphisms of the vitamin D receptor gene promoter (VDRp) influence this milk/bone association. We evaluated lumbar spine BMC and BMD, milk/dairy products and calcium intakes, markers of P-Ca metabolism, and VDRp polymorphisms at the Cdx-2 binding (rs11568820) and -1012 (rs4516035) loci in 117 healthy European peri- and postmenarcheal girls (14.9 +/- 1.6 yr) during a 4-yr follow-up. Calcium intakes from milk, nonmilk dairy products, and nondairy products averaged 199, 243, and 443 mg/d at the initiation of the study. Results show no association between milk intakes and bone mass accrual in girls bearing an A/A genotype at the -1012 VDRp locus (30% of the cohort). In contrast, A/G or G/G girls had lower spine BMC (-13%, p = 0.031), BMD (-10%, p = 0.004), and BMD Z-score (-0.84 SD, p = 0.0003) when their milk intakes were <260 ml/d compared with genotype-matched girls with higher milk intakes and with girls with an A/A genotype. The negative impact of low milk intake persisted up to 19.0 +/- 1.7 yr. These findings suggest that European girls bearing a -1012 A/G or G/G VDRp genotype should have higher milk/calcium intakes for optimal vertebral mass accrual during adolescence than girls bearing an A/A genotype, a genotype found in 30% of European and 98% of Asian and Sub-Saharan African populations. VDRp genotype diversity may contribute to the ethnic differences observed in milk requirements for bone health during adolescence.

Snail2 cooperates with Snail1 in the repression of vitamin D receptor in colon cancer

Vitamin D receptor (VDR) mediates the antitumoral action of the active vitamin D metabolite 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). VDR expression is lost during colon cancer progression causing unresponsiveness to 1,25(OH)(2)D(3) and its analogs. Previously, Snail1, an inducer of epithelial-to-mesenchymal transition (EMT), was reported to inhibit VDR expression. Here, we show that Snail2/Slug, but not other EMT inducers such as Zeb1, Zeb2, E47 or Twist1, represses VDR gene promoter. Moreover, Snail2 and Snail1 show additive repressing effect on VDR promoter. Snail2 inhibits VDR RNA and protein and blocks the induction of E-cadherin and an adhesive phenotype by 1,25(OH)(2)D(3). Snail2 reduces the ligand-induced VDR transcriptional activation of a consensus response element and of the CYP24 promoter. Concordantly, Snail2 inhibits the induction of CYP24 RNA and p21(CIP1), filamin A and vinculin proteins and the repression of c-MYC by 1,25(OH)(2)D(3). Additionally, Snail2 abrogates beta-catenin nuclear export and the antagonism of the transcriptional activity of beta-catenin-T-cell factor complexes by 1,25(OH)(2)D(3). SNAI2 expression is upregulated in 58% of colorectal tumors and correlates inversely with that of VDR. However, VDR downregulation is higher in tumors coexpressing SNAI2 and SNAI1 than in those expressing only one of these genes. Together, these data indicate that Snail2 and Snail1 cooperate for VDR repression in colon cancer.

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.

The polymorphism in the caudal-related homeodomain protein Cdx-2 binding element in the human vitamin D receptor gene.

The major physiological activity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the regulation of calcium absorption in the small intestine, and the level of vitamin D receptor (VDR) is an important factor in this regulation. In a previous study, we indicated-that the caudal-related homeodomain Cdx-2 played an important role in the intestine-specific transcription of the human VDR gene. In this study, the polymorphism was identified in the core sequence 5'-ATAAAAACTTAT-3' in the Cdx-2 binding site in the VDR gene promoter. In 261 Japanese women with genotyped VDR polymorphisms, 48 were genotype Cdx-A (adenine at -3731 nucleotides [nt] relative to the transcription start site of human VDR gene 5-ATAAAAACTTAT), 82 were genotype Cdx-G (guanine at -3731 nt, 5'-GTAAAAACTTAT-3'), and 131 were genotype Cdx-A/G (heterozygote). In postmenopausal Japanese women, the bone mineral density (BMD) in the lumbar spine (L2-L4) with the Cdx-G homozygote was 12% lower than that with the Cdx-A homozygote (p < 0.05). In electrophoretic gel mobility shift assay (EMSA), the oligonucleotide with Cdx-G allele markedly decreased the binding to Cdx-2 compared with that in the Cdx-A allele. The transcriptional activity of the VDR promoter with Cdx-G allele was decreased to 70% of the Cdx-A allele. In addition, in the herpes simplex virus thymidine kinase promoter, the Cdx-2 binding element with the G allele showed significantly lower transcriptional activity than that of the A allele. Thus, the polymorphism in the Cdx-2 binding site of the VDR gene (Cdx-polymorphism) would affect the expression of VDR in the small intestine. In addition, this polymorphism may modulate BMD in postmenopausal Japanese women.

The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets.

I. The Syndrome of Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets (HVDRR) A. Historical B. Clinical features of HVDRR C. Pathophysiology D. Alopecia E. 1,25-Dihydroxyvitamin D [1,25-(OH)2D] action and HVDRR II. Vitamin D Physiology A. Metabolism B. 1α-Hydroxylase deficiency III. 1,25-Dihydroxyvitamin D Action Mediated by the Vitamin D receptor (VDR) A. Historical aspects of VDR structure and function B. The domain structure of the VDR C. The regulation of gene expression by the VDR IV. Cellular Basis of HVDRR A. Studies in cultured skin fibroblasts B. Studies in other cells V. The VDR Gene and the Molecular Basis of HVDRR A. The VDR chromosomal gene B. The VDR gene promoter C. Polymorphisms of the VDR gene VI. HVDRR Mutations Causing the Ligand-Binding Positive Phenotype A. Initial description of DNA-binding domain (DBD) mutations B. Characterization of additional DBD mutations C. Structural analysis of DBD mutations VII. HVDRR Mutations Causing the Ligand-Binding Negative Phenotype A. Initial descr...