缺氧时丝裂原激活蛋白激酶类对人肾小管上皮细胞富含半胱氨酸蛋白61基因转录活性的调控  

Role of mitogen-activated protein kinases in the trans-activation of Cyr61 induced by hypoxia in HKC cells

作　　者：徐岩[1] 梅长林[1] 沈学飞[1] 吉程程[1] 刘亚伟[1] 

机构地区：[1]第二军医大学附属长征医院肾内科,上海200003

出　　处：《中华肾脏病杂志》2007年第3期178-183,共6页Chinese Journal of Nephrology

摘　　要：目的 探讨丝裂原激活蛋白激酶类（MAPKs）对缺氧条件下人近端肾小管上皮细胞（HKC）中富含半胱氨酸蛋白61（Cyr61）基因转录活性的调控机制。方法 缺氧培养HKC，Northern印迹检测Cyr61mRNA表达；Western印迹检测Cyr61、p38、细胞外信号调节激酶（ERK1／2）、c—Jun—N末端蛋白激酶（JNK）以及缺氧诱导因子1c（HIF-1α）的表达。构建含有人Cyr61基因启动子的报告基因Cyr61-luc质粒，将其单独或者分别与表达活性MAPKs的质粒Ca—MEK1和Ca—MKK6共同瞬时转染HKC。通过荧光素酶活性检测观察缺氧、MAPKs抑制剂和MAPKs活性酶对Cyr61基因转录活性的调控。结果 缺氧时HKC表达cyr61、HIF-1α增高，ERK1／2、JNK、p38总量不变，而其各自的磷酸化形式均明显增加。HKC转染Cyr—luc后，p38通路抑制剂SB203580和ERK通路抑制剂PD98059显著抑制缺氧时Cyr61的转录活性，两者协同作用时抑制作用显著增强。Ca—MEK1与Cyr—luc共转染HKC后，Cyr61转录活性无改变；而Ca—MKK6与Cyr—luc共转染后，Cyr61转录活性显著增高。对缺氧培养的HKC，PD98059处理使HIF-1α和Cyr61蛋白表达显著降低；SB203580处理可显著降低Cyr61蛋白表达，但对HIF-1α无影响。结论 在HKC中，缺氧可通过p38通路直接上调Cyr61基因启动子活性，也可通过ERK1／2途径促进HIF-1α表达，间接调节Cyr61基因启动子活性。Objective To investigate the regulatory mechanism of mitogen-activated protein kinase （MAPK） in Cyr61 trans-activation induced by hypoxia in HKC cells. Methods The expression levels of Cyr61, p38, ERK1/2, JNK, and HIF-1α in HKC cells induced by hypoxia were analyzed by Nothern and Western blot. Full length of Cyr61 promoter region was amplified by PCR and recombinant plasmid of Cyr-luc were constructed. After transfection alone or coexpression with vector overexpressing constitutively active forms of either MEK1 （Ca-MEK1）, or MKK6 （Ca-MKK6） , the trans-activation of Cyr61 in HKC cells induced by hypoxia, MAPK pathway inhibitors or MAPK kinase were assayed by means of luciferase reporter gene assay system. Results Cyr61 and HIF-1α were expressed at an obviously high level in HKC cells cultured under hypoxia, The phosphorylation content of ERK, JNK,p38 in the HKC cells increased along with the time of hypoxia, while there were no significant differences in total ERK, JNK and p38 expression. The luciferase activity was inhibited separately by PD98059 or SB203580 in HKC cells transfected with Cyr-luc plasmid,and it was obviously enhanced by the cotreated with PD98059 and SB203580. The trans-activation of Cyr61 in HKC ceils transfected with Cyr-luc was not affected after cotransfected with Ca-MEKl,but was significantly increased with Ca-MKK6. The protein expression of Cyr61 and HIF-1α was inhibited by PD98059 in HKC cells under hypoxia, and the same effect of SB203580 on Cyr61 was seen as well. The inhibition of Cyr61 protein expression was significantly improved when cotreated by PD98059 and SB203580. Conclusion In HKC cells, hypoxia stimulates the trans-activation of Cyr61 through p38 MAPK pathway directly , and also adjusts HIF-1α expression through ERK1/2 pathway,which could stimulates the trans-activation of Cyr61 indirectly.

关 键 词：缺氧 丝裂原激活蛋白激酶类 基因 半胱氨酸蛋白61 

分 类 号：R686[医药卫生—骨科学]