Browsing by Author "Fernandes, S. M. (Sandra Maria)"
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- ItemTranscriptional regulation of the gonadotropin-releasing hormone receptor (GnRHR) gene by glucocorticoids(Stellenbosch : Stellenbosch University, 2007-03) Fernandes, S. M. (Sandra Maria); Hapgood, J. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor is a G-protein-coupled receptor in the pituitary gonadotropes and is an important control point for reproduction. GnRH binds to the GnRH receptor (GnRHR) resulting in the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of the pituitary to GnRH can be directly correlated with GnRHR levels. The mouse GnRHR promoter contains three cis elements containing binding sites for steroidogenic factor-1 (SF-1), namely site 1 (-15/-7), site 2 (-244/- 236) and site 3 (-304/-296) as well as an activator protein-1 (AP-1)-like consensus sequence (TGAGTCA) at position –336/-330. While sites 1 and 2 and the AP-1 site have been previously shown to be involved in regulation of transcription of the mouse GnRHR (mGnRHR) promoter in some cell lines, the role of site 3 has not been previously investigated. This study investigated whether transcription of the mGnRHR gene is regulated by GnRH and glucocorticoids in the LβT2 gonadotrope pituitary cell line, and the role therein of site 3 and the AP-1 site and their cognate proteins, using a combination of in vitro protein- DNA binding studies and promoter-reporter assays. The role played by site 3 and the AP-1 site in basal transcription of the mGnRHR gene in LβT2 cells was the first area of investigation during this study. Luciferase reporter plasmids containing 600 bp of the mGnRHR promoter were used where the site 3 and AP-1 sites were either wild-type or mutated. Two constructs were prepared from the wild-type construct, i.e. wild type (LG), site 3 mutant (m3) and AP-1 mutant (mAP-1). Transfection of LG, m3 and mAP-1 plasmids into LβT2 cells was carried out to determine the effect of these mutations on the basal expression of the mGnRHR gene. Mutation of site 3 resulted in a 1.5 fold increase in the transcriptional activity of the mGnRHR promoter. This suggests that site 3 plays a role in the inhibition of basal transcriptional levels of the mGnRHR promoter in LβT2 cells. Mutation of the AP-1 site resulted in a 50% decrease in basal transcriptional levels of the mGnRHR promoter in LβT2 cells. This suggests that the AP-1 site is involved in positively mediating the basal transcriptional response of the GnRHR promoter in LβT2 cells. Experiments towards the understanding of the mechanism of the cis elements (site 3 and AP-1 site) on the mGnRHR promoter were carried out along with the role of protein kinase A (PKA) pathways, proteins involved and the effect of varying doses for varying times of GnRH, as well as the overexpression of PKA and the SF-1 protein. It was found that site 3 and the AP-1 site are not involved in the GnRH response. Results suggest that site 3 is partially involved in the PKA response in LβT2 cells. Site 3 can bind SF-1 protein as shown via competitive electrophoretic mobility shift assays (EMSA). When EMSA’s were performed on the AP-1 site the findings were that the c-Fos protein was not involved in the activation of the AP-1 site. A factor was found to bind to the AP-1 site, which did not require the intact AP-1 site, suggesting that it could be the c-Jun protein that binds to the AP-1 site under basal conditions. Another area that was investigated was whether the mGnRHR promoter can be regulated by dexamethasone (dex) either via the AP-1 site or site 3. A dose and time-dependent increase in promoter activity was observed with dex. This effect appears to require site 3 and the AP-1 site, as shown by the complete loss of response when these sites were individually mutated, consistent with a functional interaction between site 3 and the AP-1 site in LβT2 cells.