Investigating the mechanism of transcriptional regulation of the gonadotropin-releasing hormone receptor (GnRHR) gene by dexamethasone
Date
2008-12
Authors
Von Boetticher, S.
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
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.
Description
Thesis (MSc (Biochemistry))--Stellenbosch University, 2008.
Keywords
Dexamethasone, Gonadotropin, Hormone receptors, Genetic regulation, Dissertations -- Biochemistry, Theses -- Biochemistry, Luteinizing hormone releasing hormone, Adrenocortical hormones