Transcriptional regulation of the mouse gonadotropin-releasing hormone receptor gene in pituitary gonadotrope cell lines

Sadie, Hanel (Stellenbosch : University of Stellenbosch, 2006-03)

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2006.

Thesis

Gonadotropin-releasing hormone (GnRH), acting via its cognate receptor (GnRHR) is the primary regulator of mammalian reproductive function. Pituitary sensitivity to GnRH can be directly correlated with GnRHR levels on the surface of the pituitary gonadotrope cells, which can be regulated at transcriptional, post-transcriptional and post-translational levels. This study investigated mechanisms of transcriptional regulation of mouse GnRHR expression in two mouse gonadotrope cell lines, αT3-1 and LβT2, using a combination of endogenous mRNA expression studies, promoter-reporter studies, a two-hybrid protein-protein interaction assay, Western blotting, and in vitro protein-DNA binding studies. In the first part of the study, the role of two GnRHR promoter nuclear receptor binding sites (NRSs) and their cognate transcription factors in basal and Protein Kinase A (PKA)-stimulated regulation of GnRHR promoter activity was investigated in αT3-1 cells. The distal NRS was found to be crucial for basal promoter activity in these cells. While the NRSs were not required for the PKA response in these cells, results indicate a modulatory role for the transcription factors Steroidogenic Factor-1 (SF-1) and Nur77 via these promoter elements. The second part of the study focused on elucidating the mechanism of homologous regulation of GnRHR transcription in LβT2 cells, with a view to defining the respective roles of PKA and Protein Kinase C (PKC) in the transcriptional response to GnRH. In addition, the respective roles of the NRSs, the cyclic AMP response element (CRE) and the Activator Protein-1 (AP-1) promoter cis elements, together with their cognate transcription factors, in basal and GnRH-stimulated GnRHR promoter activity, were investigated. Homologous upregulation of transcription of the endogenous gene was confirmed, and was quantified by means of real-time RTPCR. The GnRH response of the endogenous gene and of the transfected promoter-reporter construct required PKA and PKC activity, and the GnRH response of the promoter-reporter construct was found to be dependent on a functional AP-1 site. Furthermore, GnRH treatment resulted in increased binding of phosphorylated cAMP-response element binding protein (phospho-CREB) and decreased expression and binding of SF-1 to their cognate cis elements in vitro, and stimulated a direct interaction between SF-1 and CREB, suggesting that these events are also required for the full transcriptional response to GnRH. This study is the first providing detail regarding the mechanism of transcriptional regulation of GnRHR expression in LβT2 cells by GnRH. Based on results from this study, a model has been proposed which outlines for the first time the kinase pathways, the promoter cis elements and the cognate transcription factors involved in homologous regulation of GnRHR transcription in the LβT2 cell line. As certain aspects of this model have been confirmed for the endogenous GnRHR gene, the model is likely to be physiologically relevant, and provides new ideas and hypotheses to be tested in future studies.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/1495
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