Thesis (PhD (Biochemistry))--University of Stellenbosch, 2006. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1495 |
| Date | 03 1900 |
| Creators | Sadie, Hanél |
| Contributors | Hapgood, Janet P., University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2747790 bytes, application/pdf |
| Rights | University of Stellenbosch |
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