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

[摘要] 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 thesynthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). Thesensitivity of pituitary gonadotropes to GnRH depends on the number of GnRH receptors presenton the gonadotrope cell surface. GnRH-R is regulated at a transcriptional, post-transcriptional andpost-translational level. Hormones such as GnRH and glucocorticoids (GCs) regulate GnRH-Rs ina time- and dose-dependent manner. Previous studies have shown that the GnRH-R promoterconfers glucocorticoid-dependent activation via the activating protein 1 (AP-1) site in the nongonadotropeGGH3 cell line. The mechanism by which GCs regulate the GnRH-R promoter is notprecisely known as the literature is contradictory. Therefore this study investigates the mechanismof transcriptional regulation of the mouse GnRH-R promoter in the mouse gonadotrope cell lineLβT2, treated with the synthetic GC dexamethasone (dex). Assays used include promoter-reporterstudies, Western blotting, endogenous mRNA expression studies, electrophoretic mobility shiftassay (EMSA) as well as the in vivo chromatin immunoprecipitation (ChIP) assay. A transfectedpromoter-reporter plasmid containing 600 bp of the mouse GnRH-R promoter was used toinvestigate the effect of dex on transcriptional regulation. Previously it was determined in ourlaboratory that the GnRH-R promoter is activated via an AP-1 binding site in the LβT2 cell line, andis regulated in a time- and dose-dependent manner by dex. In the present study in the LβT2 cellline a small induction was indeed seen upon dex treatment. Cotransfecting a expression vector forrat GR succeeded in inducing a 2 fold positive dex response. Western blot analysis revealed thatGR levels remain consistent even after 8 hours dex induction. The effect of dex on the endogenousGnRH-R gene was investigated by means of real-time RT-PCR. Dex did indeed upregulate thegene in a time-dependant manner. Maximal induction (7.4 fold) was obtained after at least 12 hoursof dex treatment. Untreated LβT2 nuclear extracts were investigated using EMSA, for proteinbinding 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 tetheringmechanism to mediate the positive dex response. The results of an in vivo ChIP assay wereconsistent 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 GRtethering to AP-1 has not been shown before in the LβT2 cell line.

[发布机构] Stellenbosch University