热激活Rac-MEKK-JNK信号通路与hsp90β基因表达

Heat Shock Activated Rac-MEKK-JNK Pathway and hsp90βGene Expression

目的探讨Rac-MEKK-JNK(Rac-丝裂原活化的蛋白激酶的激酶的激酶-C-junN端蛋白激酶)信号通路对热诱导hsp90β基因表达的调控作用以及Hsp90蛋白对Rac信号通路传递的调节过程。方法将载体PcDNA3、显性负突变Rac(DN-Rac),显性负突变MEKK(DN-MEKK)和显性负突变JNK(DN-JNK)表达质粒分别与hsp90β基因转录调控片段介导的氯霉素乙酰基转移酶(CAT)报告基因质粒β3.1共转染Jurkat和LETPa-2细胞,采用以竞争性RT-PCR为基础的系统检测CAT报告基因转录水平。用Western印迹分析检测转染了DN-Rac、DN-MEKK和DN-JNK的细胞在热刺激前后c-Jun的表达水平和磷酸化状态。用体外激酶活性分析法检测geldanamycin(GA)对热诱导的JNK活性的影响。结果转染DN-Rac、DN-MEKK及DN-JNK均能抑制42℃1h诱导的β3.1报告基因的表达,Jurkat细胞中CAT的热诱导表达分别为对照组的(72.8±5)%、(60±13.2)%及(47.7±12.1)%;LETPa-2细胞中CAT的热诱导表达分别为对照组的(16.17±5.1)%、(50.2±8.7)%及(47.5±10)%。转录因子c-Jun的表达和磷酸化水平也在一定程度上被抑制。GA处理细胞可明显抑制热诱导激活的JNK激酶活性和c-Jun的磷酸化水平,但不影响JNK和c-Jun的蛋白表达水平。结论Rac-MEKK-JNK通路参与hsp90β基因的热诱导表达,hsp90可能对热激活化?

Objective To study the effect of Rac-MEKK-JNK(Rac-mitogen activated protein kinase kinase kinase-C-jun N-terminal protein kinase)signal pathway on heat shock-induced hsp90βgene expression and the impact of Hsp90on the regulation of the pathway.Methods DN-Rac,DN-MEKK or DN-JNK were cotransfected with hsp90βCAT reporter plasmidβ3.1into Jurkat or LETPa-2cells individually,the CAT mRNA expression was then determined quantitatively by competitive RT-PCR based system.Western blot was carried out to detect the expression level and phosphorylation of c-Jun in Jurkat and LETPa-2cells that were transfected with DN-Rac,DN-MEKK or DN-JNK.By in vitro kinase activity assay and Western blot,the effect of geldnamycin(GA)on heat induced JNK activity were evaluated.Results In Jurkat cell transfected with DN-Rac,DN-MEKK or DN-JNK,heat shock induced relative CAT mRNA expression level was decreased to(72.8±5)%,(60±13.2)%and(47.7±12.1)%of the control respectively;while in LETPa-2cell hsp90β3.1reporter gene expression was accordingly suppressed to(16.17±5.1)%,(50.2±8.7)%and(47.5±10)%of control.C-Jun expression and phosphorylation were inhibited by the transfection of either one of DN-Rac,DN-MEKK or DN-JNK.With GA treatment,heat shock induced JNK activity was repressed,while the expression level of JNK or c-Jun was not obviously changed.Conclusions Rac-MEKK-JNK pathway promotes heat shock induced hsp90βgene expression and hsp90may participate in the regulation of heat shock activated Rac-MEKK-JNK signal pathway in both Jurkat and LETPa-2cells.