Li J, Wu N, Shen Y
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS, PUMC, Beijing 100005, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2000 Aug;22(4):327-31.
To study the effect of mitogen-activated protein kinase (MAPK) pathways on hsp90 genes expression in Jurkat cells.
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 hsp90 alpha and hsp90 beta 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.
In the Jurkat cells treated at 45 degrees C 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 hsp90 alpha gene which reduced to 68% of that of control. After transfected with anti-p38, the constitutive expression of hsp90 alpha gene and the heat-induced expression of hsp90 beta gene increased by 26% and 22% over the control. The constitutive expression of hsp90 alpha and hsp90 beta 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.
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.
研究丝裂原活化蛋白激酶（MAPK）信号通路对Jurkat细胞中hsp90基因表达的影响。
首先，采用蛋白质免疫印迹增强化学发光（ECL）系统检测Jurkat细胞中细胞外信号调节蛋白激酶（ERK）、p38激酶和c-Jun的磷酸化水平。其次，分别用c-Jun氨基末端激酶（JNK）显性负性质粒DN-JNK1、p38 cDNA反义质粒anti-p38瞬时转染Jurkat细胞，或在ERK激酶特异性抑制剂PD98059存在的情况下孵育细胞。然后，通过竞争性逆转录聚合酶链反应（RT-PCR）定量测定这些细胞中hsp90α和hsp90β的组成型和热诱导型mRNA水平。将hsp90的相对mRNA水平分别与转染空载体（pCDNA3）的细胞或未处理细胞中的水平进行比较。
在45℃处理15分钟的Jurkat细胞中，c-Jun和p38激酶的磷酸化明显增强，而与对照组相比，ERK的磷酸化有一定程度的增加。转染DN-JNK1的Jurkat细胞中hsp90基因的组成型和热诱导型表达均降低，尤其是hsp90α基因的热诱导型表达降低至对照组的68%。转染anti-p38后，hsp90α基因的组成型表达和hsp90β基因的热诱导型表达分别比对照组增加了26%和22%。用PD98059处理的Jurkat细胞中hsp90α和hsp90β基因的组成型表达分别增加至对照组的46%和16%，而hsp90基因的热诱导型表达没有变化。
有证据表明ERK信号通路抑制Jurkat细胞中hsp90基因的组成型表达。JNK信号通路促进hsp90基因的组成型和热诱导型表达，而p38信号通路可能在hsp90基因表达中不起重要作用。
