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GRP75经由Raf/Mek/Erk1/2信号转导通路抑制缺糖诱导的细胞凋亡

李洪岩, 杨玲^*, 刘雯, 左伋

复旦大学上海医学院细胞与遗传医学系，上海 200032

摘要

本文旨在探讨促存活信号通路Raf/Mek/Erk1/2是否参与了葡萄糖调节蛋白75 (glucose-regulated protein 75, GRP75)对缺糖诱导的细胞凋亡的抑制作用。GRP75过表达的PC12细胞给予Raf/Mek/Erk1/2通路抑制剂U0126预处理之后，无糖培养6、12和24 h，同时以DMSO预处理的GRP75过表达PC12细胞组为对照。Western blot检测Erk1/2的磷酸化和表达水平，MTT实验检测细胞存活率，Hoechst 33258染色观察凋亡细胞核的形态学改变，流式细胞仪检测细胞亚二倍体峰，免疫荧光检测细胞色素c (cytochrome c, Cyt c)向胞浆的弥散情况。结果显示：U0126在没有影响Erk1/2表达水平的前提下，阻断了GRP75对Erk1/2磷酸化水平的维持；U0126处理组的凋亡率明显高于对照组；U0126处理组Cyt c从线粒体向胞浆释放的时间明显早于对照组，同时Cyt c向胞浆的弥散程度大于对照组。以上结果提示，U0126通过抑制Erk1/2磷酸化，阻断了缺糖状态下GRP75对Cyt c释放和细胞凋亡的抑制作用，这表明GRP75是通过Raf/Mek/Erk1/2级联反应抑制缺糖诱导的线粒体凋亡途径的进程。

关键词： 葡萄糖调节蛋白75; Raf/Mek/Erk1/2; 凋亡

分类号：R3

[GRP75 overexpression inhibits apoptosis induced by glucose deprivation via Raf/Mek/Erk1/2 signaling pathway.] [Article in Chinese]

LI Hong-Yan, YANG Ling^*, LIU Wen, ZUO Ji

Department of Cellular and Genetic Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China

Abstract

The purpose of the present study is to investigate whether glucose-regulated protein 75 (GRP75) overexpression inhibits apoptosis induced by glucose deprivation through Raf/Mek/Erk1/2 signaling pathway. After pretreatment with Mek-specific inhibitor U0126, GRP75 overexpressing PC12 cells were incubated in glucose-free DMEM medium for indicated time (6, 12 and 24 h). And DMSO-treated GRP75 overexpressing PC12 cells were applied as control. Western blot was used to determine the expression and phosphorylation level of Erk1/2. MTT assay was used to measure cell viability. Hoechst 33258 staining and flow cytometry using propidium iodide (PI) staining was used to analysis apoptosis. Immunofluorescence with antibody against cytochrome c (Cyt c) was used to detect Cyt c release from mitochondrion. The results showed U0126 prevented the activation of Erk1/2 maintained by GRP75, but the total Erk1/2 expression was not affected. U0126-treated group showed lower cell viability and higher apoptotic rate compared with control group. Immunofluorescence indicated the delay in release of Cyt c was blocked by U0126. These results suggest U0126 prevents protective effect of GRP75 on PC12 cells by inhibiting Erk1/2 phosphorylation, which certifies that GRP75 can inhibit the mitochondria-dependent apoptotic pathway through Raf/Mek/Erk1/2 signaling cascade.

Key words: glucose-regulated protein 75; Raf/Mek/Erk1/2; apoptosis

收稿日期：2010-11-08　　录用日期：2010-12-14

通讯作者：杨玲　　E-mail: yangling@fudan.edu.cn

引用本文：

李洪岩, 杨玲, 刘雯, 左伋. GRP75经由Raf/Mek/Erk1/2信号转导通路抑制缺糖诱导的细胞凋亡[J]. 生理学报 2011; 63 (1): 69-74.

LI Hong-Yan, YANG Ling, LIU Wen, ZUO Ji. [GRP75 overexpression inhibits apoptosis induced by glucose deprivation via Raf/Mek/Erk1/2 signaling pathway.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (1): 69-74 (in Chinese with English abstract).