SHP-2通过ERK和JNK通路调节PC12细胞应对NGF的细胞生存和凋亡

SHP-2 regulates PC12 cell survival and apoptosis in response to NGF via activation of ERK and JNK

目的:探讨蛋白酪氨酸磷酸酶SHP-2在神经生长因子(NGF)作用下大鼠嗜铬细胞瘤PC12细胞生存及NGF撤除后细胞凋亡的过程中的作用及机制。方法:SHP-2抑制剂NSC87877作用于PC12细胞,MTT测定PC12细胞存活率;流式细胞仪检测细胞凋亡率。将pIRES-GFP空载体、pIRES-GFP-SHP-2野生型和pIRES-GFP-SHP-2C459S突变体通过脂质体方法转染PC12细胞;加入NGF作用1h和撤除NGF5h后分别用Westernblotting方法检测细胞外信号调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)、c-Jun氨基末端激酶(JNK)及磷酸化JNK(p-JNK)表达变化。结果:MTT和流式细胞仪检测表明SHP-2可以促进PC12细胞生存,抑制细胞凋亡。Western blotting结果显示无SHP-2抑制剂组和转染pIRES-SHP-2野生型组p-ERK表达在加入NGF的过程中升高;撤除NGF后,各组p-ERK表达均降低,pIRES-GFP-SHP-2C459S突变体组和pIRES-GFP组p-ERK表达明显低于pIRES-GFP-SHP-2野生型组,NGF去除+SHP-2抑制剂组的表达水平明显低于NGF去除对照组;NGF去除+SHP-2抑制剂组p-JNK表达高于NGF去除对照组;pIRES-GFP-SHP-2C459S突变体组高于pIRES-GFP空载体组,pIRES-GFP-SHP-2野生型组低于pIRES-GFP空载体组。结论:SHP-2可能通过对ERK的正向激活,抑制JNK的激活,增强NGF作用下PC12细胞生存及抑制NGF撤除后所致细胞凋亡,从而在NGF的信号转导中起到一个中心环节的作用。

AIM： To investigate the central role of the protein tyrosine phosphatase SHP- 2 in the survival of PC12 cells upon NGF treatment and apoptosis after NGF withdrawal. METHODS： PC12 cells were treated with SHP- 2 inhibitor （NSC87877）. Cell survival rate was detected by MTF method and apoptosis was determined by flow cytometry. Moreover, plRES - GFP （ vector alone）, plRES - GFP - SHP - 2 （ wild type） and pIRES - GFP - SHP - 2^c459s （ dominant negative mutant form） were transfected into PC12 cells. ERK, p-ERK, JNK and p-JNK were immunoblotted at 1 h in the presence of NGF and 5 h after NGF withdrawal. RESULTS： SHP -2 potentially enhanced survival and attenuated apoptosis of PC12 cells. The activation of ERK was significantly enhanced with NGF treatment either in the group without SHP - 2 inhibitor or the SHP - 2 wild type group. On the other hand, phosphorylation level of JNK was significantly increased after NGF withdrawal when PC12 cells were treated with SHP -2 inhibitor or transfected with SHP -2 mutant. CONCLUSION： SHP -2 may play a central role in mediating the survival and apoptosis of PC12 cells upon NGF exposure and with- drawal. The underlying mechanisms may be through the activation of ERK and JNK.