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.
Cheng
Kwai Wa
KW
Department of Obstetrics and Gynaecology, The University of British Columbia, 2H30-4490 Oak Street, British Columbia Women's Hospital, Vancouver, BC, Canada V6H 3V5.
Leung
Peter C K
PC
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
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