Transcriptional activation of the ovine follicle-stimulating hormone β-subunit gene by gonadotropin-releasing hormone:: Involvement of two activating protein-1-binding sites and protein kinase C

FSH is an alpha/beta heterodimeric glycoprotein, the formation of which is regulated primarily by expression of its beta-subunit. Recent studies on transcriptional regulation of the ovine FSH beta-subunit gene (oFSH beta) have defined two functional activating protein-1 (AP-1) enhancers in the proximal promoter (located at -120 and -83 bp) that are probably physiologically important for FSH beta expression. As GnRH is a major regulator of FSH beta expression and is also known to stimulate the synthesis of Jun and Fos family members (AP-1), we investigated the possibility that oFSH beta transcription may be regulated by GnRH through AP-1. Here we report the use of an in vitro cell system involving transient transfection of GnRH receptors (GnRHR) into HeLa cells to define regulatory elements involved in GnRHmediated induction of oFSH beta. This system was used to show that expression of luciferase constructs containing either the - 4741/+ 759 region of the oFSH beta gene ( -4741oFSH beta-Luc) or the -846/+44 region of the human or gene (alpha-Luc; a positive control) was stimulated 3.1 +/- 0.3- and 7.7 +/- 1.9-fold, respectively, by 100 nM GnRH. Another luciferase expression plasmid containing the Rous sarcoma virus promoter (a negative control) showed no response to GnRH. Similar results with these constructs were obtained in COS-7 cells. Studies with progressive 5'-deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on each AP-1 site in the proximal promoter of oFSH beta. Gel shift assays demonstrated the ability of GnRHR in HeLa cells to increase AP-1 binding activity. Responses in the HeLa cell system were dependent on GnRH (ED50 = 0.5 nM) and GnRHR, which was identified by photoaffinity labeling. In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium. Finally, as protein kinase C (PKC) is a known target of GnRH action in gonadotropes, the role of PKC in transcriptional regulation of oFSH beta and alpha-subunit genes by GnRH in HeLa cells was investigated. Although 12-O-tetradecanoyl 13-acetate induction of alpha-Luc and -215oFSH beta-Luc could be completely blocked in a dose-dependent manner by the specific PKC inhibitor bisindolylmaleimide I, only 57-65% of the GnRHmediated stimulation of these promoters was blocked, demonstrating the involvement of PKC as well as other signaling systems in GnRH induction. These data define a molecular action of GnRH on oFSH beta gene transcription that involves two proximal AP-1 enhancer elements and PKC activation. Furthermore, these studies establish the usefulness of HeLa and COS-7 cells to investigate specific aspects of GnRH action on gonadotropin subunit gene expression, as similar signaling pathways and transcription factors that are activated by GnRH in gonadotropes (such as PKC, mitogen-activated protein kinase, Ca2+, and AP-1) exist in these cells.