The Delayed Activation of the Prostaglandin G/H Synthase-2 Promoter in Bovine Granulosa Cells Is Associated with Down-regulation of Truncated Upstream Stimulatory Factor-2

To elucidate the molecular mechanisms involved in the delayed induction of PGHS-2 in species with a long ovulatory process, a 1. 6-kilobase fragment of the bovine PGHS-2 promoter was isolated, and its activity was characterized in primary cultures of bovine granulosa cells. Promoter activity assays performed with a series of deletion mutants revealed that the promoter region from -149 to -2 (+1 = transcription start site) confers full-length promoter activity in response to forskolin (10 microM). Four consensus cis-elements were identified within this region, including an E-box, ATF/CRE, C/EBP, and AP2 site. Site-directed mutagenesis showed that the E-box was required for PGHS-2 promoter activity, that disruption of the C/EBP element decreased forskolin inducible activity by 29%, whereas point mutation within the ATF/CRE and AP2 element had no inhibitory effect. Electrophoretic mobility shift assays (EMSAs) performed with the -149/-2 fragment and granulosa cell nuclear extracts obtained before (0 h) and after (18 and 20 h) human chorionic gonadotropin (hCG) revealed the regulation of multiple DNA-protein complexes. The 0-h extract generated four complexes at the E-box, whereas only one complex was produced at this site with the 18-h extract. Supershift EMSAs identified that upstream stimulatory factor-1 and -2 (USF-1 and -2) were part of these complexes. Interestingly, the presence of the amino-terminal truncated USF-2, which lacks the transcription activation domain, was detected in the 0-h extract, but not in extracts prepared post-hCG. Supershift EMSAs also indicated high levels of C/EBPbeta binding to its cis-element in the 0-h extract, which contrasts with results previously reported in rats. Thus, high levels of amino-terminal truncated USF-2 and C/EBPbeta in bovine granulosa cells prior to hCG treatment could repress gene expression, and be involved in the delayed induction of PGHS-2 in species with a long ovulatory process.