原癌基因c-jun对毒胡萝卜素内酯处理的小鼠胚胎成纤维细胞生存率的影响

c-jun affects thapsigargin-induced mouse fibroblasts survival

目的观察原癌基因c-jun对经毒胡萝卜素内酯(TG)处理的小鼠胚胎成纤维细胞生存率的影响,并探讨其机制。方法应用内质网应激诱导剂TG、钙调神经磷酸酶(Ca N)的结构活性形式Cn A(p CMV-Cn A)和附加血凝素表位的c-jun表达载体(p SRα-HA-c-jun)处理基因敲除型(c-jun-/-)和基因重组型(c-jun Re)两组小鼠胚胎成纤维细胞后观察细胞生存率;蛋白免疫印迹技术分析蛋白c-jun、p-c-jun、JNK、p-JNK、Adapt78和α-tubulin的表达。结果经TG处理引起p-JNK与p-c-jun在c-jun Re细胞中的强烈激活,而Ca N活性在c-jun-/-细胞中显著增加(P<0.05);Ca N通过p CMV-Cn A的外源性表达导致Adapt78表达在TG处理的c-jun Re细胞中有增强;在c-jun-/-细胞中由TG与p CM V-Cn A同时处理后其细胞生存率低于同样处理后的c-jun Re细胞(P<0.05);c-jun外显基因在c-jun-/-细胞的过表达,同时引起了c-jun在靶细胞的磷酸化及诱导产生的Adapt78表达上调,c-jun基因的外部表达抑制了TG诱导的细胞死亡。结论 c-jun基因表达水平的改变影响TG诱导的小鼠胚胎成纤维细胞生存率的变化,其变化与细胞内Ca N和Adapt78蛋白表达的改变有关,c-jun基因表达增强导致Adapt78蛋白表达增强,从而减弱Ca N活性,并产生出抵抗TG诱导细胞死亡的保护机制。

Objective To examine the impact of c-jun on thapsigargin-induced mouse fibroblasts survival and its mechanism. Methods Thapsigargin,p CMV-Cn A and p SRα-HA-c-jun were used to treat c-jun-/- and c-jun Re mouse fibroblasts. Cell viability was evaluated with trypan blue dye exclusion. Calcineurin assays were performed according to the manufacturers instructions. c-jun,p-c-jun,JNK,p-JNK,Adapt78 and α-tubulin were analyzed with immunoblotting. Results TG treatment led to strong JNK activation and phosphorylation of c-jun in c-jun Re cells. We observed a higher level of calcineurin activity in c-jun-/-cells compared with c-jun Re cells（ P〈0. 05）. TG treatment resulted in significantly increased calcineurin activity in c-jun-/-cells but not in c-jun Re cells. Exogenous expression of calcineurin via p CMV-Cn A caused increased Adapt78 expression in both c-jun-/- and c-jun Re cells,although TG treatment induced Adapt78 expression only in c-jun Re cells. Adapt78 expression induced by p CMV-Cn A in c-jun-/- cells was associated with protection against TG-induced cell death. Overexpression of exogenous c-jun in c-jun-/- cells caused both an autophosphorylation of c-jun in the target cells and exogenous expression of c-jun recovered thapsigargin-induced Adapt78 up-regulation and inhibited TG-induced cell death. Conclusion The change of c-jun gene expression level can affect TG-induced mouse cell survival rate. The changes of calcineurin and Adapt78 protein expression are related to thealteration of c-jun gene expression. The increased c-jun gene expression results in enhanced expression of Adapt78 protein,thus weakens the calcineurin activity and produces protective mechanism against TG-induced cell death.