Human gonadotropin-releasing hormone receptor gene transcription: up-regulation by 3',5'-cyclic adenosine monophosphate/protein kinase A pathway.
Transient transfection of mouse gonadotrope-derived (alphaT3-1) cells with a 2297 bp human GnRHR promoter-luciferase construct (p2300-LucF) showed a dose- and time-dependent increase in the human gonodotropin-releasing hormone receptor (GnRHR) promoter activity after forskolin treatment. An average of 4.8-fold increase in promoter activity was observed after 12 h of 10 microM forskolin treatment. This effect was mimicked by administration of cholera toxin, cAMP analog or pituitary adenylate cyclase activating polypeptide 38 (PACAP). A specific adenylate cyclase (AC) inhibitor (ACI) or protein kinase A (PKA) inhibitor (PKAI) pretreatment reversed the forskolin- and PACAP-induced increase in the human GnRHR promoter activity. These results not only confirm the stimulatory effect of Cyclic adenosine monophosphate (cAMP) in human GnRHR promoter activation, but also suggest that hormones or neurotransmitters that activate adenylate cyclase in pituitary gonadotropes may increase the expression of human GnRHR gene in transcriptional level. 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 have identified two putative AP-1/CREB binding sites, namely hGR-AP/CRE-1 and hGR-AP/CRE-2 that participated in mediating the cAMP-stimulatory effect. Mutation of the putative hGR-AP/CRE-1 and hGR-CRE-2 resulted in a 38 and 32% decrease in the forskolin-induced stimulation. However, mutation of both binding sites did not completely abolish the cAMP-stimulatory effect, suggesting that multiple transcription factor binding sites were involved in full response in cAMP stimulation. The binding of CREB to these motifs was confirmed by gel mobility shift assay and antibody supershift assay.
Cheng
K W
KW
Department of Obstetrics and Gynaecology, The University of British Columbia, B.C. Women's Hospital, 2H30-4490 Oak Street, BC, V6H 3V5, Vancouver, Canada.
Leung
P C
PC
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009479:Neuropeptides / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011966:Receptors, LHRH / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion; D015398:Signal Transduction; D014158:Transcription, Genetic / Q000235:genetics*; D015854:Up-Regulation / Q000187:drug effects
C495401:ADCYAP1 protein, human; C495402:Adcyap1 protein, mouse; D009479:Neuropeptides; D009687:Nuclear Proteins; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D011966:Receptors, LHRH; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA; D002772:Cholera Toxin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases