title: Human chorionic gonadotropin-activated cAMP pathway regulates human placental GnRH receptor gene transcription in choriocarcinoma JEG-3 cells.
abstract: 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.
authors.affiliation: Department of Obstetrics and Gynaecology, The University of British Columbia, 2H30-4490 Oak Street, British Columbia Women's Hospital, Vancouver, BC, Canada V6H 3V5.
authors.forename: Kwai Wa
authors.initials: KW
authors.lastname: Cheng
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Leung
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000502:physiology; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006801:Humans; D010920:Placenta / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D011966:Receptors, LHRH / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000502:physiology
chemical_list: D006063:Chorionic Gonadotropin; D017362:Cyclic AMP Response Element-Binding Protein; D011966:Receptors, LHRH; D018808:Transcription Factor AP-1; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases