Regulation of expression of the CYP11A (P450scc) gene in bovine ovarian luteal cells by forskolin and phorbol esters.
This study examines the transcriptional regulation of the bovine CYP11A (P450scc) gene by activators of protein kinase A and protein kinase C in bovine ovarian luteal cells. Cells were transfected with reporter gene constructs containing deletion mutations of the 5'-flanking region of the bovine CYP11A gene linked to the minimal beta-globin gene. A construct containing -118/-101 base pairs of CYP11A sequence retains the same degree of stimulation by forskolin and inhibition by co-treatment with phorbol 12-myristate 13-acetate as larger constructs. This sequence contains two putative binding sites for nuclear proteins, an AP1-like sequence and an overlapping GA box element. Gel shift analysis using nuclear extracts of bovine ovarian luteal cells demonstrated that both the wild-type -118/-101-base pair sequence and a consensus GC box bound Sp1 or Sp1-like proteins. Mutation of the GA box element completely suppressed stimulation by forskolin. Absence of binding using the same mutated sequence correlated with the reporter gene transcription results. Mutation of the AP1-like site had little effect on forskolin induction of phorbol 12-myristate 13-acetate inhibition. These results indicate that both stimulation by forskolin and inhibition by phorbol esters are mediated by the same GA box element, which binds Sp1 or an Sp1-like protein.
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235.
M
M
Begeot
U
U
Shetty
M
M
Kilgore
M
M
Waterman
E
E
Simpson
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002417:Cattle; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology*; D003338:Corpus Luteum / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D016329:Sp1 Transcription Factor; D005576:Colforsin; D004247:DNA; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D013755:Tetradecanoylphorbol Acetate