Intracellular mechanisms of gonadotropin-stimulated gene expression in granulosa cells.
Previous studies have shown that the gonadotropins follicle-stimulating hormone and luteinizing hormone stimulate proopiomelanocortin (POMC) promoter activity and mRNA levels in ovarian granulosa cells. The objective of these studies was to determine the role of cAMP-dependent protein kinases (pKA) in gonadotropin-stimulated gene expression. Primary cultures of rat granulosa cells were transfected with a gene construct consisting of the POMC promoter (-150 to +63; designated pOMC-CAT) fused to the chloramphenicol acetyltransferase (CAT) reporter gene either alone or cotransfected with an expression plasmid (designated mutant RI), which overexpresses a mutant form of the murine RI subunit incapable of binding cAMP and serving as an irreversible inhibitor of the catalytic subunit of pKA. Follicle-stimulating hormone or isoproterenol caused a significant stimulation of pOMC-CAT activity in transfected cells. Cotransfection of pOMC-CAT with mutant RI caused a significant inhibition of basal pOMC-CAT activity and abolished the gonadotropin stimulation. As a control, transfection of the SV-40 viral enhancer-promoter fused to CAT (pSV2-CAT) was unresponsive to follicle-stimulating hormone stimulation and cotransfection with mutant RI had no significant effect on pSV2-CAT activity. These studies suggest that gonadotropin regulation of the POMC promoter is mediated by pKA and that promoter activity is stringently controlled by pKA.
Melner
M H
MH
Division of Reproductive Biology, Oregon Regional Primate Research Center, Beaverton 97006.
Young
S L
SL
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006064:Gonadotropins, Equine / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology*; D051381:Rats; D014162:Transfection / Q000235:genetics
D006064:Gonadotropins, Equine; D047428:Protein Kinase Inhibitors; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases