Cis-regulatory elements conferring cyclic 3',5'-adenosine monophosphate responsiveness of the progesterone receptor gene in transfected rat granulosa cells.
We have previously shown that both pituitary gonadotropins and forskolin induce progesterone receptor (PR) messenger RNA expression at the level of transcription in granulosa cells of the rat ovary. To determine the DNA regulatory elements that are important for cAMP-induced transcription of the PR gene in the ovary, we examined the cAMP-induced activity of promoter sequences in rat granulosa cells transfected with various fusion constructs containing PRB promoter sequences linked to the luciferase reporter gene. When cells were transfected with a luciferase fusion construct containing the 1375-base pair 5'-flanking region of the rat PRB gene, forskolin treatment substantially increased luciferase activity. Analysis of a series of 5'-deletion mutants indicated that a minimal PRB promoter containing 116 base pairs of upstream sequence (-116/3) was sufficient to increase luciferase activity in response to forskolin in transfected rat granulosa cells. This promoter contains a consensus CCAAT site in reverse orientation (5'-ATTGG-3') and a consensus GC box (5'-GGGGCGGGCC-3'), but no known cAMP-responsive element. Site-specific mutation of the GC box notably decreased both basal and cAMP-induced activity of this minimal PRB promoter. In addition, site-specific mutation of the CCAAT binding site within this proximal promoter of the PRB gene substantially decreased cAMP-induced activity, but did not significantly affect the basal activity of this promoter. Either mutation alone failed to abolish cAMP inducibility. In contrast, double mutation of both the GC box and the CCAAT box completely abolished cAMP inducibility, suggesting that the GC box and the CCAAT box act together to mediate cAMP-induced transcription of the PRB gene. Gel shift analysis shows that the minimal PRB promoter sequences form multiple complexes with nuclear proteins of granulosa cells, all of which are specifically competed by oligonucleotides containing the GC box and the CCAAT box. Taken together, our results suggest a functional role for transcription factors binding the GC box and the CCAAT box in mediating cAMP-induced transcription of the rat PRB promoter in rat granulosa cells.
Park-Sarge
O K
OK
Department of Physiology, University of Kentucky, Lexington 40536-0084, USA.
Sarge
K D
KD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011980:Receptors, Progesterone / Q000235:genetics*; D014162:Transfection
D011980:Receptors, Progesterone; D000242:Cyclic AMP