钙/钙调素依赖性蛋白激酶Ⅱ对热休克小鼠胚胎成纤维细胞HSP70表达的影响

Calcium /Calmodulin-dependent Protein Kinase Ⅱ Contributes to HSP70 Expression in Mouse Embryonic Fibroblasts

为证实热休克小鼠胎儿成纤维细胞中钙/钙调素依赖性蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)对热休克蛋白70(Heat shock protein70,HSP70)基因表达的影响及其作用机理,将体外培养的小鼠胎儿成纤维细胞(Mouse Embryonic Fibroblasts,MEF)随机分为39℃和41℃热处理组(分别处理0.5,1,1.5,2 h)和常温对照组(37℃),测定各组细胞CaMKⅡ和HSF1 mRNA的量。此外,将培养的细胞分为37℃和39℃CaMKⅡ特异性抑制剂(myr-AIP)处理组和相应的空白对照组,分别测定各组细胞HSP70、HSF1及其mRNA的量。结果表明,39℃热休克1 h后HSF1和CaMKⅡ的mRNA表达量极显著高于常温对照组,经过myr-AIP处理的39℃组细胞HSP70及其mR-NA含量显著低于39℃空白对照组,而37℃myr-AIP处理组与37℃对照组没有显著差异;myr-AIP对37℃组和39℃组的HSF1 mRNA和蛋白含量影响均不显著,却使p-HSF1的含量显著降低。热休克能增加CaMKⅡ和HSF1 mRNA的转录,且CaMKⅡ通过磷酸化HSF1促进HSP70基因表达。

The objectives of the study were to verify if the CaMK Ⅱ （Ca^2＋/calmodulin-dependent protein kinase Ⅱ ） influences the expression of HSP70 gene in the mouse embryonic fibroblasts （MEF） with heat shock and to clarify its mechanism. The fibroblasts were heat-treated at 37℃ ,39℃ and 41℃ individually before the expressions of both CaMK Ⅱ and HSF1 were detected by RT-PCR at 0.5,1,1.5 and 2 h of heat shock, respectivelly. Moreover, the fibroblasts were divided into control group and myr-AIP group randomly, and cultured at 37 ℃ and 39℃ ,respec- tively. The expressions of both HSP70 and HSF1 were detected. The expressions of HSF1 and CaMK Ⅱ mRNA in the MEF treated at 39℃ for 1 h were both extremely significantly higher （P 〈0. 01 ） than that of the control group. There was a significantly （P 〈 0.05） decrease of HSP70 expression in the MEF cultured at 39℃ treated with myr- AlP compared with its blank control. However,there was no difference in HSP70 exoression between the MEF cul- tured at 37℃ with or without myr-AIP. The expressions of HSF1 in the MEF treated with myr-AIP were not signifi- cantly influenced. However,the p-HSF1 expression in the MEF treated with myr-AIP was significantly （P 〈 0. 05） decreased. Moderate heat shock increases both expressions of CaMK Ⅱ and HSF1 in mouse embryonic fibroblasts. CaMK Ⅱ participates in both heat shock response and HSP70 gene expression by p-HSF1.