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J. Biol. Chem., Vol. 279, Issue 8, 6327-6336, February 20, 2004

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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^*

Khampoune Sayasith, Nadine Bouchard, Michèle Sawadogo¶, Jacques G. Lussier, and Jean Sirois||

From the Centre de Recherche en Reproduction Animale and the Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada and the ^¶Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

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 (U21-220) acted as a dominant negative mutant and blocked endogenous and USF-stimulated PGHS-2 promoter activation. Interestingly, transfections with U21-220 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.

Received for publication, October 13, 2003 , and in revised form, November 30, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY241931 and AY239291.

^* This work was supported by Canadian Institutes of Health Research (CIHR) Grant MT-13190 (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a CIHR Investigator Award.

^|| To whom correspondence should be addressed: CRRA, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada. Tel.: 450-773-8521 (ext. 8542); Fax: 450-778-8103; E-mail: jean.sirois{at}umontreal.ca.

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