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J. Biol. Chem., Vol. 280, Issue 32, 28885-28893, August 12, 2005

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Role of Upstream Stimulatory Factor Phosphorylation in the Regulation of the Prostaglandin G/H Synthase-2 Promoter in Granulosa Cells^*

Khampoune Sayasith, 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, Saint-Hyacinthe, Québec J2S 7C6, Canada

To investigate the role of USF phosphorylation in the regulation of the PGHS-2 promoter in granulosa cells, promoter activity assays were performed in primary cultures of bovine granulosa cells transfected with the chimeric PGHS-2 promoter/luciferase (LUC) construct –149/–2PGHS-2.LUC. Transfections were done in the absence or presence of forskolin; the protein kinase A (PKA) inhibitor H-89; or an expression vector encoding USF1, USF2, the catalytic subunit of PKA (cPKA), or a PKA inhibitor protein (PKI). Electrophoretic mobility shift assays were performed to study USF/DNA interactions using granulosa cell nuclear extracts and a ³²P-labeled proximal PGHS-2 promoter fragment containing the E-box element. The results show that forskolin stimulation and cPKA overexpression caused a marked and significant increase in USF-dependent DNA binding and PGHS-2 promoter activities (p < 0.05). In contrast, both activities were decreased by H-89 treatment or PKI overexpression. Reverse transcription-PCR analyses revealed that these treatments had similar effects on endogenous PGHS-2 mRNA levels in granulosa cells. Cotransfection studies with a USF2 mutant lacking N-terminal activation domains (U21–220) repressed forskolin-, cPKA-, and USF-dependent PGHS-2 promoter activities. Electrophoretic mobility shift assays showed that U21–220 was able to compete with full-length USF proteins and to saturate the E-box element. Immunoprecipitation/Western blot analyses revealed an increase in the levels of phosphorylated USF1 and USF2 after forskolin treatment, whereas chromatin immunoprecipitation assays showed that binding of USF proteins to the endogenous PGHS-2 promoter was stimulated by forskolin. Site-directed mutagenesis of a consensus PKA phosphorylation site within USF proteins abolished their transactivating capacity. Collectively, these results characterize the role of USF phosphorylation in PGHS-2 expression and identify the phosphorylation-dependent increase in USF binding to the E-box as a putative molecular basis for the increase in PGHS-2 promoter transactivation in granulosa cells.

Received for publication, November 29, 2004 , and in revised form, May 26, 2005.

^* This work was supported in part by Canadian Institutes of Health Research 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 Canadian Institutes of Health Research investigator award. To whom correspondence should be addressed: CRRA, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte, 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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