Bonfil David, Chuderland Dana, Kraus Sarah, Shahbazian David, Friedberg Ilan, Seger Rony, Naor Zvi
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel.
Endocrinology. 2004 May;145(5):2228-44. doi: 10.1210/en.2003-1418. Epub 2004 Jan 21.
The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHbeta-subunit promoter activity was examined in the LbetaT-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. Ca(2+) influx, but not Ca(2+) 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 LbetaT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gbetagamma or beta-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 FSHbeta promoter, linked to a luciferase reporter gene (-4741oFSHbeta-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca(2+) ionophore ionomycin, stimulated FSHbeta-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca(2+), inhibited the GnRH response. Cotransfection of FSHbeta-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHbeta-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHbeta-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 Ca(2+), are involved in GnRH induction of the ovine FSHbeta gene.
在LbetaT - 2促性腺激素细胞系中研究了细胞外信号调节激酶（ERK）、c - Jun氨基末端激酶（JNK）、p38和c - Src在促性腺激素释放激素（GnRH）刺激的促卵泡激素β亚基（FSHbeta）启动子活性中的作用。用GnRH激动剂孵育细胞导致ERK、JNK、p38和c - Src激活。ERK激活峰值在5分钟时观察到，而JNK、p38和c - Src的激活峰值在30分钟时出现，此后下降。GnRH对ERK的激活依赖于蛋白激酶C（PKC），12 - O - 十四酰佛波醇 - 13 - 乙酸酯敏感的PKC亚型的激活、抑制和消耗证明了这一点。ERK激活需要Ca(2+)内流，但不需要Ca(2+)动员。GnRH向ERK的信号传导部分由发动蛋白和一种蛋白酪氨酸激酶介导，显然是c - Src。GnRH在LbetaT - 2细胞中对ERK的激活不涉及表皮生长因子受体的反式激活或通过Gbetagamma或β - 抑制蛋白的介导。一旦被GnRH激活，ERK就会转位到细胞核。我们研究了ERK、JNK、p38和c - Src在GnRH刺激的与荧光素酶报告基因（-4741oFSHbeta - LUC）相连的绵羊FSHbeta启动子中的作用。PKC激活剂12 - O - 十四酰佛波醇 - 13 - 乙酸酯刺激了FSHbeta - 荧光素酶（LUC）活性，但Ca(2+)离子载体离子霉素没有。此外，PKC的下调而非Ca(2+)的去除抑制了GnRH反应。FSHbeta - LUC与组成型活性形式的Raf - 1和MEK共转染刺激了FSHbeta - LUC活性，而Ras、Raf - 1和MEK的显性负性突变体以及选择性MEK抑制剂PD98059消除了GnRH诱导的FSHbeta - LUC活性。CDC42和JNK的显性负性突变体分别使GnRH反应降低了36%和49%。用p38或c - Src抑制剂SB203580和PP1孵育细胞也降低了GnRH反应。令人惊讶的是，两个近端激活蛋白 - 1位点对GnRH反应贡献很小。因此，PKC、ERK、JNK、p38和c - Src而非Ca(2+)参与了GnRH对绵羊FSHbeta基因的诱导。
