TNF-α通过激活蛋白磷酸酶4调节HepG2细胞中JNK磷酸化

TNF-α Promotes Phosphorylation of JNK by Activation of PP4 in Human Hepatocarcinoma Cell HepG2

目的 探讨蛋白磷酸酶4（protein phosphatase 4,PP4）在肿瘤坏死因子（tumor necrosis factorα,TNF-α）诱导JNK磷酸化中的作用.方法 TNF-α处理人肝癌细胞HepG2,免疫印迹检测JNK磷酸化水平和PP4表达水平,免疫沉淀结合磷酸酶活性测定法分析PP4活性变化.构建PP4磷酸酶活性缺失突变体 PP4-RL,转染HepG2细胞,经过G418筛选获得稳定表达FLAG-PP4-RL的克隆,进一步观察PP4对TNF-α诱导的JNK磷酸化的影响.结果 TNF-α处理后迅速引起HepG2细胞JNK的磷酸化,在20 min时达到峰值.PP4的表达在TNF-α处理后60 min内没有显著变化,但活性迅速增强,在20 min时达到峰值.在稳定表达FLAG-PP4-RL的HepG2细胞中,TNF-α诱导的JNK磷酸化被PP4-RL阻断.结论 TNF-α通过激活蛋白磷酸酶4调节HepG2细胞中JNK磷酸化.

Objective The aim of this study was to investigate the role of PP4 in TNF-α-in- duced JNK phosphorylation. Methods Human hepatocarcinoma cells HepG2 were treated with TNF-α. Western blot was performed to measure the levels of JNK phosphorylation and PP4 expression. PP4 activity was analyzed by immunoprecipitation combined with phosphatase assays. PP4-RL （ dominant negative mutant of PP4） plasmid was constructed. HepG2 cell clones expressing PP4-RL were established. Results Treatment of HepG2 cells with TNF-α rapidly resulted in JNK phosphorylation and increase of PP4 activity with a maximal level at 20 min, while PP4 expression level remained unchanged within 60 rain. In the TNF-α treated HepG2 cells, however, TNF-α induced phosphorylation of JNK induced was blocked by the dominant negative mutant PP4-RL. Conclusion TNF-α promotes phosphorylation of JNK by activating PP4 in HepG2 cells.