Interaction of SF-1 and Nur77 proteins from a gonadotrope cell line with the promoter of the GnRH receptor gene : implications for gene regulation

[摘要] ENGLISH ABSTRACT:The regulation of gonadotropin releasing hormone (GnRH) receptor numbers in the pituitary is acrucial control point in reproduction. Pituitary sensitivity to GnRH can be directly correlated withGnRH receptor levels, which can be regulated at transcriptional and post-transcriptional level. Theproximal promoter of the mouse GnRH receptor gene contains two cis elements bearing theconsensus sequence for a Steroidogenic Factor-l (SF -1) binding site. The distal site has previouslybeen shown to be involved in basal and tissue-specific transcriptional regulation, whereas thefunction of the proximal site was not established. SF-I, a member of the nuclear receptorsuperfamily of transcription factors, is involved in the transcriptional regulation of a large numberof genes involved in steroidogenesis and reproduction. The consensus SF-I binding site can serveas a binding site for several members of the nuclear receptor superfamily. The aim of this study wasto investigate the binding of SF-I protein from the aT3-1 gonadotrope cell line to the two putativeSF-I binding sites in the mouse GnRH receptor promoter in vitro, in order to provide supportingevidence for their functional roles in GnRH receptor gene regulation. It was shown by Westernblotting that SF-I and Nur77, another nuclear receptor transcription factor, are both expressed inaT3-1 cells, in a manner that is influenced by cell culture conditions. Gel mobility shift assaysusing specific antibodies showed that both SF-I and Nur77 protein in aT3-1 nuclear extracts bindto both sites in a mutually exclusive fashion. As shown by competition assays using mutatedversions of the two sites, Nur77 protein had different base pair requirements than that of SF-Iprotein for binding to the sites. Additionally, SF-I mRNA was shown by Northern blotting to beincreased in aT3-1 cells in response to stimulation of the Protein Kinase A (PKA) pathway byforskolin. These results highlight unexpected degeneracy in so-called consensus nuclear receptorbinding sites. Furthermore, since Nur77 protein is involved in the stress response of thehypothalamic-pituitary-adrenal (HPA) axis, the unexpected presence of Nur77 protein in agonadotrope cell line has potentially important implications for cross-talk between the HPA andhypothalamic-pituitary-gonadal (HPG) axes.

[发布机构] Stellenbosch University