Transcription profiling of human osteosarcom) cell lines expressing wild type hGR or N363SA to establish a link between the N363S glucocorticoid teceptor polymorphism and altered gene expression related to human disease

ABSTRACT: 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 manufacturer’s 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).

ORGANISM(S): Homo sapiens

SUBMITTER: NIEHS Microarray Group 

PROVIDER: E-GEOD-5796 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress