Regulation of CYP11A gene expression in bovine ovarian granulosa cells in primary culture by cAMP and phorbol esters is conferred by a common cis-acting element.
Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechanisms by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc (CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5' flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-globin-derived reporter gene were transfected into granulosa cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Lauber
M E
ME
Cecil H. and Ida Green Center for Reproductive Biology Sciences, UT Southwestern Medical Center, Dallas 75235-9051.
Picton
H M
HM
Begeot
M
M
Momoi
K
K
Waterman
M R
MR
Simpson
E R
ER
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000032:analysis / Q000235:genetics*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000032:analysis / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics* / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics*; D006107:Granulosa Cells / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology*; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015290:Second Messenger Systems / Q000502:physiology; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D010703:Phorbol Esters; D012333:RNA, Messenger; D005576:Colforsin; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; C022433:pregnenolone 17-alpha-hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate