Extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and c-Src are involved in gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone follicle-stimulating hormone β-subunit promoter
Extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and c-Src are involved in gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone follicle-stimulating hormone β-subunit promoter
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DOI：
10.1210/en.2003-1418
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发表时间：
2004-05-01
期刊：
影响因子：
4.8
通讯作者：
Naor, Z
中科院分区：
文献类型：
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作者：
Bonfil, D;Chuderland, D;Naor, Z
在 LbetaT-2 促性腺激素细胞系中检查了 ERK、Jun N 末端激酶 (JNK)、p38 和 c-Src 在 GnRH 刺激的 FSHbeta 亚基启动子活性中的作用。将细胞与 GnRH 激动剂一起孵育会导致 ERK、JNK、p38 和 c-Src 的激活。 ERK 激活峰值出现在 5 分钟，而 JNK、p38 和 c-Src 激活峰值出现在 30 分钟，此后逐渐下降。 GnRH 引起的 ERK 激活依赖于蛋白激酶 C (PKC)，这一点可从 12-O-十四烷酰佛波醇-13-乙酸盐敏感性 PKC 亚种的激活、抑制和耗竭中看出。 ERK 激活需要 Ca2+ 流入，但不需要 Ca2+ 动员。 GnRH 向 ERK 发出的信号部分由动力蛋白和蛋白酪氨酸激酶（显然是 c-Src）介导。 LbetaT-2 细胞中 GnRH 激活 ERK 不涉及表皮生长因子受体的反式激活或通过 Gbetagamma 或 β-arrestin 的介导。一旦被 GnRH 激活，ERK 就会转移到细胞核。我们检查了 ERK、JNK、p38 和 c-Src 在 GnRH 刺激的绵羊 FSHbeta 启动子中的作用，该启动子与荧光素酶报告基因 (-4741oFSHbeta-LUC) 连接。 PKC 激活剂 12-O-十四烷酰佛波醇-13-乙酸酯（而非 Ca2+ 离子载体离子霉素）刺激 FSHbeta-荧光素酶 (LUC) 活性。此外，下调 PKC，但不去除 Ca2+，会抑制 GnRH 反应。 FSHbeta-LUC 以及 Raf-1 和 MEK 的组成型活性形式的共转染刺激了 FSHbeta-LUC 活性，而 Ras、Raf-1 和 MEK 以及选择性 MEK 抑制剂 PD98059 的显性失活则消除了 GnRH 诱导的 FSHbeta-LUC 活性。 CDC42 和 JNK 的显性失活分别使 GnRH 反应降低 36% 和 49%。将细胞与 p38 或 c-Src 抑制剂 SB203580 和 PP1 一起孵育也降低了 GnRH 反应。令人惊讶的是，两个近端激活蛋白 1 位点对 GnRH 反应的贡献很小。因此，PKC、ERK、JNK、p38 和 c-Src（但不包括 Ca2+）参与绵羊 FSHbeta 基因的 GnRH 诱导。
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. 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 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 Ca2+ ionophore ionomycin, stimulated FSHbeta-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca2+, 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 Ca2+, are involved in GnRH induction of the ovine FSHbeta gene.