Investigating the mechanism of transcriptional regulation of the gonadotropin-releasing hormone receptor (GnRHR) gene by dexamethasone

Von Boetticher, S. (2008-12)

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008.

Thesis

Gonadotropin-releasing hormone (GnRH) acting through the cognate GnRH receptor (GnRH-R) plays an important role in the regulation of mammalian reproductive function by regulating the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of pituitary gonadotropes to GnRH depends on the number of GnRH receptors present on the gonadotrope cell surface. GnRH-R is regulated at a transcriptional, post-transcriptional and post-translational level. Hormones such as GnRH and glucocorticoids (GCs) regulate GnRH-Rs in a time- and dose-dependent manner. Previous studies have shown that the GnRH-R promoter confers glucocorticoid-dependent activation via the activating protein 1 (AP-1) site in the nongonadotrope GGH3 cell line. The mechanism by which GCs regulate the GnRH-R promoter is not precisely known as the literature is contradictory. Therefore this study investigates the mechanism of transcriptional regulation of the mouse GnRH-R promoter in the mouse gonadotrope cell line LβT2, treated with the synthetic GC dexamethasone (dex). Assays used include promoter-reporter studies, Western blotting, endogenous mRNA expression studies, electrophoretic mobility shift assay (EMSA) as well as the in vivo chromatin immunoprecipitation (ChIP) assay. A transfected promoter-reporter plasmid containing 600 bp of the mouse GnRH-R promoter was used to investigate the effect of dex on transcriptional regulation. Previously it was determined in our laboratory that the GnRH-R promoter is activated via an AP-1 binding site in the LβT2 cell line, and is regulated in a time- and dose-dependent manner by dex. In the present study in the LβT2 cell line a small induction was indeed seen upon dex treatment. Cotransfecting a expression vector for rat GR succeeded in inducing a 2 fold positive dex response. Western blot analysis revealed that GR levels remain consistent even after 8 hours dex induction. The effect of dex on the endogenous GnRH-R gene was investigated by means of real-time RT-PCR. Dex did indeed upregulate the gene in a time-dependant manner. Maximal induction (7.4 fold) was obtained after at least 12 hours of dex treatment. Untreated LβT2 nuclear extracts were investigated using EMSA, for protein binding to the mouse GnRH-R promoter AP-1 binding site, and these proteins were identified as c- Fos and GR. This suggests that the GR interacts with the AP-1 transcription factor via a tethering mechanism to mediate the positive dex response. The results of an in vivo ChIP assay were consistent with this hypothesis, showing that the GR interacted with a genomic fragment containingthe AP-1 site, in response to dex. The transactivation of the GnRH-R promoter by means of the GR tethering to AP-1 has not been shown before in the LβT2 cell line.

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