Specific inhibition of protein kinase A in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter.
Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5'-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 microM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was cotransfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either gonadotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.
Young
S L
SL
Division of Reproductive Biology, Oregon Regional Primate Research Center, Beaverton 97006.
Searles
R P
RP
Kaynard
A H
AH
Melner
M H
MH
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006062:Gonadotropins / Q000502:physiology*; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors; D051381:Rats; D016385:TATA Box; D014162:Transfection
D006062:Gonadotropins; D047428:Protein Kinase Inhibitors; D011333:Pro-Opiomelanocortin; D005640:Follicle Stimulating Hormone; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D007545:Isoproterenol