蛋白激酶Cσ介导GHRP-6诱导的大鼠生长激素腺瘤GH3细胞CREB磷酸化

Protein Kinase C σ mediates GHRP-6-induced phosphorylation of cAMP response element binding protein in GH3 cells of rats growth hormone-secreting adenomas

目的探讨生长激素释放肽-6(GHRP-6)对大鼠生长激素腺瘤细胞GH3细胞cAMP反应元件结合蛋白(CREB)磷酸化水平的影响及其细胞内信号传导机制。方法用GHRP-6处理大鼠生长激素腺瘤GH3细胞并检测其对GH3细胞CREB磷酸化水平的影响;初步筛选参与GHRP-6激活的细胞内信号通路的蛋白激酶C(PKC)亚型;然后应用RNA干扰技术确认发挥中介作用的PKC亚型;Western免疫印迹法检测蛋白质磷酸化水平。结果 GHRP-6诱导的CREB磷酸化呈时间依赖性;PKCσ特殊抑制剂楸毒素可以明显减弱GHRP-6的这种效应;PKCσ表达沉默后GHRP-6诱导的CREB磷酸化水平明显降低;并且GHRP-6可以诱导PKCσ磷酸化。结论 PKC,特别是PKCσ,介导了GHRP-6诱导的CREB磷酸化。此结果为了解垂体生长激素腺瘤细胞内信号通路间"交叉通讯"提供了新的依据。

Objective To explore the effect of growth hormone releasing peptide-6 （GHRP-6） on the phosphorylation level of cAMP response element binding protein（CREB） in GH3 cells of rat growth hormone（GH）-seereting adenoma and intracellular signal transduction mechanism of GHRP-6-induced CREB phosphorylation. Methods The level of CREB phosphorylation in the cultured GH3 cells of rats GH-secreling adenomas was determined after GHRP-6 treatment. Protein kinase C（PKC） subtype involved in GHRP-6-induced CREB phosphorylation was examined by RNA interference. CREB phosphorylafion and the PKC subtype activation were determined by Western blot. Results GHRP-6-induced CREB phosphorylation was time-dependent. The level of GHRP-6-induced CREB phosphorylation was significantly reduced by rottlerin, a special PKCσ inhibitor （P〈0.01）. A dominant negative mutant of PKCσ also significantly reduced the level of GHRP-6-induced CREB phosphorylafion （P〈0.01）. PKCσ activation was induced by GHRP-6 in time-dependent manner. Conclusion PKCσ may mediate CREB phosphorylation induced by GHRP-6 in GH3 cells. This result provides new basis for understanding the ＂cross talk＂ between signal paths in GH-seereting adenoma cells.