PARENT SESSION
GENE REGULATION AND FUNCTION - A

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(385) ROLE OF UPSTREAM STIMULATORY FACTOR 1 AND 2 PHOSPHORYLATION IN THE TRANSCRIPTIONAL REGULATION OF THE BOVINE PROSTAGLANDIN G/H SYNTHASE-2 PROMOTER IN GRANULOSA CELLS.

Sayasith, Khampoune¹, Lussier, Jacques G.¹, Sirois, Jean¹, ¹ CRRA, Faculté de Médecine Vétérinaire, Saint-Hyacinthe, (Qué), Canada

ABSTRACT- Ovulation requires the transcriptional activation of the prostaglandin G/H synthase-2 (PGHS-2) gene in granulosa cells, and binding of upstream stimulatory factor 1 (USF1) and USF2 to an E-box cis-element plays a central role in the gonadotropin-dependent regulation of the PGHS-2 promoter. The objective of this study was 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 PGHS-2 promoter/luciferase (LUC) chimeric construct -149/-2PGHS-2.LUC in the absence or presence of forskolin (FSK), the protein kinase A (PKA) inhibitor H89 or expression vectors encoding USF1, USF2, the catalytic subunit of PKA (cPKA) or a PKA inhibitor protein (PKI). Electrophoretic mobility shift assays (EMSAs) were performed to study protein/DNA interactions using granulosa cell nuclear extracts and a ³²P-labelled proximal PGHS-2 promoter fragment containing the E-box element. Results showed that FSK stimulation and cPKA overexpression caused a marked and significant increase of USF-dependent DNA binding and PGHS-2 promoter activities (P < 0.05). Both activities were decreased by H89 treatment or PKI overexpression. RT-PCR analyses revealed that these treatments had similar effects (induction by FSK and cPKA, and inhibition by H89 and PKI) on the levels of PGHS-2 mRNA in granulosa cells. Cotransfection studies with a USF2 mutant lacking N-terminal activation domains (U21-220) strongly inhibited USF-dependent PGHS-2 promoter activities, while chromatin immunoprecipitation assays revealed that binding of wildtype (wt) USF2 and U21-220 to the endogenous PGHS-2 promoter was stimulated by FSK. EMSAs showed that binding of wtUSFs with the promoter fragment was competed by the presence of U21-220. Immunoprecipitation/Western blot analyses revealed an increase in levels of serine- and threonine-phosphorylated USF1 and USF2 after FSK treatment. Collectively, results from this study identify for the first time phosphorylation as a putative post-translational modification involved in USF action, which ultimately leads to an increase in USF binding to the E-box cis-element and a marked rise in PGHS-2 promoter activation in granulosa cells.

KEY WORDS: USF phosphorylation, Ovulation, PGHS-2, Bovine granulosa cells