CIS-REGULATORY ELEMENTS CONFERRING CYCLIC 3',5'-ADENOSINE-MONOPHOSPHATE RESPONSIVENESS OF THE PROGESTERONE-RECEPTOR GENE IN TRANSFECTED RATGRANULOSA-CELLS

We have previously shown that both pituitary gonadotropins and forskol in induce progesterone receptor (PR) messenger RNA expression at the l evel of transcription in granulosa cells of the rat ovary. To determin e the DNA regulatory elements that are important for cAMP-induced tran scription of the PR gene in the ovary, we examined the cAMP-induced ac tivity of promoter sequences in rat granulosa cells transfected with v arious fusion constructs containing PR(B) promoter sequences linked to the luciferase reporter gene. When cells were transfected with a luci ferase fusion construct containing the 1375-base pair 5'-flanking regi on of the rat PR(B) gene, forskolin treatment substantially increased luciferase activity. Analysis of a series of 5'-deletion mutants indic ated that a minimal PR(B) promoter containing 116 base pairs of upstre am sequence (-116/3) was sufficient to increase luciferase activity in response to forskolin in transfected rat granulosa cells. This promot er contains a consensus CCAAT site in reverse orientation (5'-ATTGG-3' ) and a consensus GC box (5'GGGGCGGGCC-3'), but no known cAMP-responsi ve element. Site-specific mutation of the GC box notably decreased bot h basal and cAMP-induced activity of this minimal PR(B) promoter. In a ddition, site-specific mutation of the CCAAT binding site within this proximal promoter of the PR(B) gene substantially decreased cAMP-induc ed activity, but did not significantly affect the basal activity of th is promoter. Either mutation alone failed to abolish cAMP inducibility . In contrast, double mutation of both the GC box and the CCAAT box co mpletely abolished cAMP inducibility, suggesting that the GC box and t he CCAAT box act together to mediate cAMP-induced transcription of the PR(B) gene. Gel shift analysis shows that the minimal PR(B) promoter sequences form multiple complexes with nuclear proteins of granulosa c ells, all of which are specifically competed by oligonucleotides conta ining the GC box and the CCAAT box. Taken together, our results sugges t a functional role for transcription factors binding the GC box and t he CCAAT box in mediating cAMP-induced transcription of the rat PR(B) promoter in rat granulosa cells.