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Molecular evidence for a link between the N363S glucocorticoid receptor polymorphism and altered gene expression.

ABSTRACT: CONTEXT:A single-nucleotide polymorphism (SNP) in the human glucocorticoid receptor (hGR) N363S (rs6195) has been the focus of several clinical studies, and some epidemiological data link this SNP to increased glucocorticoid sensitivity, coronary artery disease, and increased body mass index. However, molecular studies in vitro using reporter gene expression systems have failed, for the most part, to define a link between this polymorphism and altered glucocorticoid receptor function. OBJECTIVE:The objective of this study was to address the biological relevancy of N363S SNP in GR function by establishing stable U-2 OS (human osteosarcoma) cell lines expressing wild-type hGR or N363S and examining these receptors under a variety of conditions that probe for GR activity including human gene microarray analysis. DESIGN:Functional assays with reporter gene systems, Western blotting, and human microarray analysis were used to evaluate the activity of wild-type and N363S GR in both transiently and stably expressing cells. In addition, quantitative RT-PCR was used to confirm the microarray analysis. RESULTS:Functional assays with reporter gene systems and homologous down-regulation revealed only minor differences between the wild-type hGR and N363S receptors in both transiently and stably expressing cell lines. However, examination of the two receptors by human gene microarray analysis revealed a unique gene expression profile for N363S. CONCLUSIONS:These studies demonstrate that the N363S SNP regulates a novel set of genes with several of the regulated genes supporting a potential role for this GR polymorphism in human diseases.

SUBMITTER: Jewell CM

PROVIDER: S-EPMC2692663 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Molecular evidence for a link between the N363S glucocorticoid receptor polymorphism and altered gene expression.

Jewell Christine M CM Cidlowski John A JA

The Journal of clinical endocrinology and metabolism 20070529 8

<h4>Context</h4>A single-nucleotide polymorphism (SNP) in the human glucocorticoid receptor (hGR) N363S (rs6195) has been the focus of several clinical studies, and some epidemiological data link this SNP to increased glucocorticoid sensitivity, coronary artery disease, and increased body mass index. However, molecular studies in vitro using reporter gene expression systems have failed, for the most part, to define a link between this polymorphism and altered glucocorticoid receptor function.<h4 ...[more]

PMID: 17535992

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Project description:A single nucleotide polymorphism (SNP) in the human glucocorticoid receptor (GR), N363S, has been the focus of several clinical studies, and some epidemiological data link this SNP to increased glucocorticoid sensitivity, coronary artery disease and increased body mass index (BMI). However, molecular studies in vitro using reporter gene expression systems have failed to define a link between this polymorphism and altered glucocorticoid receptor function. To address the biological relevancy of N363S in glucocorticoid receptor mechanisms and function, we established stable U-2 OS (human osteosarcoma) cell lines expressing wild type hGR or N363S using a tetracycline-regulated expression system. Functional assays with reporter gene systems revealed only minor differences between the wild type hGR and N363S receptors under a variety of conditions that probe for GR function. However, examination of this polymorphism by human gene microarray analysis showed, for the first time, that there are significant differences between wild type hGR and the N363S SNP in their ability to selectively regulate gene expression. Several of these genes may define the link between the N363S SNP and human disease. Experiment Overall Design: U-2 OS cells were transfected with the BD Clontech pTET-OFF regulatory plasmid to establish the U-OFF parental cell line. MluI and EcoRV ends were generated onto the coding region of hGRa using PCR amplification of the pCMVhGRa plasmid. The pTRE2hyg vector was digested with MluI and EcoRV and the two DNAs were ligated to form the pTRE2hGRa plasmid (Lu and Cidlowski). Site-directed mutagenesis was then performed to make pTRE2N363S. The wild type hGR and the N363S mutant were individually transfected into the U-OFF cells and clones were selected which stably expressed either hGRa or N363S using 200 mg/ml of geneticin and 500 mg/ml of hygromycin. Several clones were obtained for each receptor, and the receptor levels were compared using western blot analyses. In these cell lines, the expression of hGR can be repressed by the addition of tetracycline or the derivative doxycycline to the media. U-2 OS (human osteosarcoma) cells were maintained in DMEM/F-12 supplemented with 10% FCS:CS, 2 mM glutamine and pen-strep and selected clones were maintained in the same media with the addition of 200 mg/ml Geneticin and 200 mg/ml hygromycin. All cells were maintained in a humidified, 5% CO2 atmosphere. For the Microarray: U-2 OS cells stably expressing either wild type hGR or N363S were treated for six hours with 10 nM dexamethasone or vehicle, and total RNA was isolated using the Qiagen RNeasy midi kit (Qiagen, Valencia, CA). Experiment Overall Design: Gene expression analysis was conducted using Agilent Human1Av2 arrays (Agilent Technologies, Palo Alto, CA). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturerâs protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters. For each comparison, two biological replicates were used, each with a dye reversal (totaling four arrays per comparison).

Project description:BACKGROUND: Since both excess glucocorticoid secretion and central obesity are clinical features of some obese patients, it is worthwhile to study a possible association of glucocorticoid receptor gene (GRL) variants with obesity. Previous studies have linked the N363S variant of the GRL gene to increased glucocorticoid effects such as higher body fat, a lower lean-body mass and a larger insulin response to dexamethasone. However, contradictory findings have been also reported about the association between this variant and obesity phenotypes. Individual studies may lack statistical power which may result in disparate results. This limitation can be overcome using meta-analytic techniques. METHODS: We conducted a meta-analysis to assess the association between the N363S polymorphism of the GRL gene and obesity risk. In addition to published research, we included also our own unpublished data -three novel case-control studies- in the meta-analysis The new case-control studies were conducted in German and Spanish children, adolescents and adults (total number of subjects: 1,117). Genotype was assessed by PCR-RFLP (Tsp509I). The final formal meta-analysis included a total number of 5,909 individuals. RESULTS: The meta-analysis revealed a higher body mass index (BMI) with an overall estimation of +0.18 kg/m2 (95% CI: +0.004 to +0.35) for homo-/heterozygous carriers of the 363S allele of the GRL gene in comparison to non-carriers. Moreover, differences in pooled BMI were statistically significant and positive when considering one-group studies from the literature in which participants had a BMI below 27 kg/m2 (+ 0.41 kg/m2 [95% CI +0.17 to +0.66]), but the differences in BMI were negative when only our novel data from younger (aged under 45) and normal weight subjects were pooled together (-0.50 kg/m2 [95% CI -0.84 to -0.17]). The overall risk for obesity for homo-/heterozygous carriers of the 363S allele was not statistically significant in the meta-analysis (pooled OR = 1.02; 95% CI: 0.56-1.87). CONCLUSION: Although certain genotypic effects could be population-specific, we conclude that there is no compelling evidence that the N363S polymorphism of the GRL gene is associated with either average BMI or obesity risk.

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Molecular evidence for a link between the N363S glucocorticoid receptor polymorphism and altered gene expression.

Publications

Molecular evidence for a link between the N363S glucocorticoid receptor polymorphism and altered gene expression.

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