丝裂原活化蛋白激酶信号途径在热休克蛋白90基因表达中的作用

The Role of MAPK Pathways in hsp90 Genes Expression

目的 研究丝裂原活化蛋白激酶 - (MAPK)信号传导途径对 Jurkat细胞中热休克蛋白 90基因(hsp90 )表达的影响。方法 用 Western免疫印迹 -增强型化学发光系统 (ECL)检测热休克前后 Jurkat细胞胞外信号调节激酶 (ERK)、p38激酶及 c- Jun N-末端蛋白激酶 (JNK)的底物 c- Jun的磷酸化程度 ;分别用 JNK1的显性负突变质粒 DN- JNK1、p38c DNA反义表达质粒 anti- p38及 ERK激酶的特异性抑制剂 PD980 5 9瞬时转染或处理Jurkat细胞 ,再行 RT- PCR检测 hsp90 α和 hsp90 β基因的表达水平。分别以转染空载体 (p CDNA3)及未经任何处理的 Jurkat细胞作为相应对照组。结果 45℃热休克 15 min后 ,Jurkat细胞中 JNK1和 p38激酶的磷酸化程度与热休克前相比明显增强 ,ERK的磷酸化较热休克前有所增强。转染 DN- JNK1的细胞中 hsp90 α和 hsp90 β基因的组成性和热诱导表达均较转染空载体的对照组降低 ,尤其对 hsp90 α基因的热诱导表达的影响更为明显 (仅为对照组的6 8% ) ;转染 anti- p38后 ,hsp90 α基因的组成性表达和 hsp90 β基因热诱导表达分别比转染空载体的对照组增加2 6 %和 2 2 % ;经 PD980 5 9处理的 Jurkat细胞中 ,hsp90 α基因的组成性表达比未处理的细胞增加 46 % ,但 PD980 5 9处理对 hsp90 α和

Objective To study the effect of mitogen activated protein kinase (MAPK) pathways on hsp90 genes expression in Jurkat cells. Methods Firstly,the phosphorylation of extracellular signal regulated protein kinase (ERK), p38 kinase and c Jun in Jurkat cells were detected by using Western blotting enhanced chemically lightening (ECL) system.Secondly,Jurkat cells were transfected transiently with c Jun N terminal kinase (JNK) dominant negative plasmid, DN JNK1 , p38 cDNA antisense plasmid,anti p38,respectively or incubated in the present of PD98059,specific inhibitor of ERK kinase.Then the constitutive and heat induced mRNA levels of hsp90α and hsp90β in these cells were determined quantitatively by competitive RT PCR .The relative mRNA level of hsp90 was compared with those in the cells transfected with mock vector (pCDNA3) or untreated cells respectively. Results In the Jurkat cells treated at 45℃ for 15 min,the phosphorylation of c Jun and p38 kinase enhanced obviously, whereas the phosphorylation of ERK increased at certain degree as compared with control.Both constitutive and heat induced expression of hsp90 genes decreased in the Jurkat cells transfected with DN JNK1,especially for the heat induced expression of hsp90α gene which reduced to 68% of that of control.After transfected with anti p38,the constitutive expression of hsp90α gene and the heat induced expression of hsp90β gene increased by 26% and 22% over the control.The constitutive expression of hsp90α and hsp90β genes in the Jurkat cells treated with PD98059,increased to 46% and 16% of the control respectively,whereas the heat induced expression of hsp90 genes did not change. Conclusions Evidences showed that ERK signal pathway inhibited the constitutive expression of hsp90 genes in Jurkat cell.JNK pathway promoted both constitutive and heat induced expression of hsp90 genes, whereas p38 pathway may not play an important role in the hsp90 genes expression.