Dana Chuderlandת David Shahbazian, Ilan Friedberg , Zvi Naor - The George S. Wise Faculty of Life Sciences, Tel Aviv University

Sarah Kraus, Rony Seger - Department of Biological Regulation (S.K., R.S.), The Weizmann Institute of Science

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The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHβ-subunit promoter activity was examined in the LβT-2 gonadotroph cell line. Incubation of the cells with a GnRH agonist resulted in activation of ERK, JNK, p38, and c-Src. The peak of ERK activation was observed at 5 min, whereas that of JNK, p38, and c-Src at 30 min, declining thereafter. ERK activation by GnRH is dependent on protein kinase C (PKC), as evident by activation, inhibition, and depletion of 12-O-tetradecanoylphorbol-13-acetate-sensitive PKC subspecies. Ca²⁺ influx, but not Ca²⁺ mobilization, is required for ERK activation. GnRH signaling to ERK is partially mediated by dynamin and a protein tyrosine kinase, apparently c-Src. ERK activation by GnRH in LβT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gβγ or β-arrestin. Once activated by GnRH, ERK translocates to the nucleus. We examined the role of ERK, JNK, p38, and c-Src in GnRH-stimulated ovine FSHβ promoter, linked to a luciferase reporter gene (−4741oFSHβ-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca²⁺ ionophore ionomycin, stimulated FSHβ-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca²⁺, inhibited the GnRH response. Cotransfection of FSHβ-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHβ-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHβ-LUC activity. The dominant negatives of CDC42 and JNK reduced the GnRH response by 36 and 49%, respectively. Incubation of the cells with the p38 or the c-Src inhibitors SB203580 and PP1 also reduced the GnRH response. Surprisingly, two proximal activator protein-1 sites contribute very little to the GnRH response. Thus, PKC, ERK, JNK, p38, and c-Src, but not Ca²⁺, are involved in GnRH induction of the ovine FSHβ gene.