Molecular Characterization and Role of Bovine Upstream Stimulatory Factor 1 and 2 in the Regulation of the Prostaglandin G/H Synthase-2 Promoter in Granulosa Cells

Abstract The transcriptional activation of the prostaglandin G/H synthase-2 (PGHS-2) gene in granulosa cells is required for ovulation. To directly study the ability of upstream stimulatory factor 1 (USF1) and USF2 to trans-activate the bovine PGHS-2 promoter in granulosa cells, USF1 or USF2 expression vectors were cotransfected with the PGHS-2/luciferase (LUC) chimeric construct, -149/-2PGHS-2.LUC. Results revealed that overexpression of USF1 or USF2 caused a marked and significant increase in basal and forskolin-inducible promoter activities (p < 0.05), and these effects were dependent on the presence of a consensus E-box cis-element within the promoter fragment. Co-transfections with different N- and C-terminal truncated USF mutants led to significant reductions in promoter activation, as compared with full-length constructs (p < 0.05), thus allowing identification of putative bovine USF functional domains. Overexpression of a USF2 truncated mutant lacking the first 220 residues (U2Δ1-220) acted as a dominant negative mutant and blocked endogenous and USF-stimulated PGHS-2 promoter activation. Interestingly, transfections with U2Δ1-220 blocked the forskolin-dependent induction of PGHS-2 mRNA in granulosa cells, whereas transfections with full-length USF2 increased PGHS-2 transcript levels. Immunoblot analyses confirmed overexpression of full-length and truncated USF proteins, and electrophoretic mobility shift assays (EMSAs) and supershift EMSAs established that the observed effects were dependent on specific interactions between USF proteins and the consensus E-box cis-element. Stimulation of cells with forskolin increased, whereas treatment of extracts with phosphatase decreased USF binding activities to the E-box. Thus, this study presents for the first time direct evidence for a role of USF proteins in the regulation of the PGHS-2 promoter in preovulatory granulosa cells.