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.
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 (U2Delta1-220) acted as a dominant negative mutant and blocked endogenous and USF-stimulated PGHS-2 promoter activation. Interestingly, transfections with U2Delta1-220 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.
Sayasith
Khampoune
K
Centre de Recherche en Reproduction Animale and the Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, CP 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada.
Bouchard
Nadine
N
Sawadogo
Michèle
M
Lussier
Jacques G
JG
Sirois
Jean
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D051546:Cyclooxygenase 2; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D005260:Female; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006107:Granulosa Cells / Q000378:metabolism*; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D051720:Upstream Stimulatory Factors
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases