Human Chorionic Gonadotropin-Activated cAMP Pathway Regulates Human Placental GnRH Receptor Gene Transcription in Choriocarcinoma JEG-3 Cells

A dose- and time-dependent increase in the human GnRH receptor (GnRHR) promoter activity after forskolin treatment was observed after transient transfection of human placental choriocarcinoma JEG-3 cells with a 2297-bp human GnRHR promoter-luciferase construct (p2300-LucF). This stimulatory effect was mimicked by administrating of cholera toxin, cAMP analog, or human chorionic gonadotropin. A specific adenylate cyclase inhibitor or protein kinase A inhibitor pretreatment reversed the forskolin- and human chorionic gonadotropin-induced increase in the human GnRHR promoter activity. Progressive 5' deletion assays identified a 412-bp fragment (-577 to -167) in the human GnRHR 5'-flanking region that is essential in maintaining the basal responsiveness to cAMP. Mutagenesis, coupled with functional studies, has identified two putative activating protein-1 (AP-1)/cAMP-responsive element (CRE) binding protein binding sites, namely human GnRHR (hGR)-AP/CRE-1 and hGR-AP/CRE-2, mediating the cAMP-stimulatory effect. Mutation of the putative hGR-AP/CRE-1 and hGR-AP/CRE-2 resulted in 32% and 35% decreases in the forskolin-induced stimulation, respectively. The binding of CRE binding protein to these motifs was confirmed by gel mobility shift assay and antibody supershift assay.