Endocrine Abstracts (2003) 6 OC24


RC Fowkes1,2, M Desclozeaux2, HA Ingraham2 & JM Burrin1

1Endocrinology, Barts & the London School of Medicine & Dentistry, London, UK; 2Physiology, University of California, San Francisco, USA.

The orphan nuclear receptor, steroidogenic factor-1 (SF-1) regulates gonadotroph development and gene expression. In the absence of an identified ligand, SF-1 has been shown to be constitutively active and recent studies reveal that post-translational modification may regulate its transcriptional activity. The Ser 203 residue, proximally located to helix 1 of the ligand binding domain (LBD) of SF-1, resides in a consensus MAPK phosphorylation site, and is required for full transcriptional activity. We have investigated the post-translational modification of SF-1 in alphaT3-1 gonadotrophs, and examined the influence upon hormone-stimulated gene transcription in vitro. alphaT3-1 cells were co-transfected with increasing concentrations of wild-type or S203A mutant SF-1 and a human glycoprotein hormone alpha-subunit promoter-luciferase construct (-517alphaLUC), before stimulation with 0 or 100 nM PACAP for 8 h. Basal -517alphaLUC activity was enhanced following wild-type SF-1 over-expression (by 7.4+1.5-fold, P<0.01), but not in the presence of the S203A mutant. Furthermore, the S203A mutant reduced the PACAP-stimulated activation of -517alphaLUC to 51.1+11% (P<0.05). Using a modified mammalian 2-hybrid assay, alphaT3-1 cells were co-transfected with Gal4-hinge-helix-1-SF-1 or Gal4-S203A-hinge-helix-1-SF-1 and the activation domain of VP16-helix-2-12-SF-1, and the Gal4RE-LUC reporter. PACAP stimulation (100 nM, 8h) enhanced basal LBD activity by up to 10-fold (P<0.01), but the S203A mutation abolished this effect. Co-transfection of MAP kinase phosphatase-1 or treatment with MAPK kinase (MEK) inhibitor (UO126, 1 mM) also inhibited the PACAP effect (to 64.2+4.3% and 53.5+9% of control, respectively P<0.01). Finally, Western blotting of PACAP-stimulated alphaT3-1 cells revealed enhanced phosphorylation of endogenous SF-1 at Ser 203 within 5 min, an effect which was inhibited by blockade of MEK (UO126). Collectively, these data are the first to describe post-translational modification of SF-1 in gonadotrophs and suggest that MAPK-dependent re-modelling of the SF-1 LBD contributes to PACAP-stimulated gene transcription.

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