摘要
目的:了解激活蛋白-1(AP-1)参与调控香烟烟雾诱导的气道黏蛋白(MUC)5AC表达的作用机制,探讨此过程中AP-1活化的可能信号途径。方法:体外培养人气道上皮细胞BEAS-2B,给予香烟烟雾提取物(CSE)刺激,干预实验用c-Jun的显性负性突变体TAM67转染细胞阻断AP-1的DNA结合活性;用SP600125和PD98059预处理分别阻断c-Jun氨基端激酶(JNK)和细胞外信号调节激酶(ERK)的活性。以ELISA法检测MUC5AC蛋白含量,Western印迹检测磷酸化JNK(p-JNK)、磷酸化ERK(p-ERK)和磷酸化P38(p-P38)含量,RT-PCR检测MUC5ACmRNA表达水平,EMSA测定AP-1的DNA结合活性。结果:CSE(1g/L)刺激后MUC5ACmRNA表达水平和蛋白含量明显高于对照组(均P<0.01),AP-1的DNA结合活性也明显强于对照组(P<0.01),伴随p-ERK和p-JNK蛋白含量显著增加,与对照组相比,差异有统计学意义(均P<0.01),而P38含量无明显变化(P>0.05);与CSE组相比,TAM67转染细胞后MUC5ACmRNA表达水平和蛋白含量均显著降低(P<0.01);与CSE组相比,用SP600125或PD98059处理细胞后可明显抑制AP-1的DNA结合活性(均P<0.05),并下调MUC5AC蛋白含量和mRNA的表达(均P<0.05)。结论:AP-1参与调控CSE诱导的气道MUC5AC基因表达的过程,此过程是由JNK和ERK信号转导通路激活AP-1,再由AP-1与MUC5AC启动子上AP-1DNA反应元件结合后在转录水平上调MUC5AC的表达而实现的。
Objective To investigate the mechanism of activator protein-1 ( AP-1 ) on cigarette smoke-induced airway mucous hypersecretion and to explore the possible signal transduction pathway that activates AP-1. Methods The airway epithelial cell line ( BEAS-2B) was cultured in vivo and treated with cigarette smoke extract ( CSE) . The DNA binding activity of AP-1 was blocked by the transfection of c-Jun dominant negative mutant TAM67 into the cells. SP600125 and PD98059 were used to block the activation of c-Jun terminal kinase ( JNK) and extracellular signal-regulated kinase ( ERK) respectively. MUC5AC protein was detected by enzyme-linked immunosorbent assay,MUC5AC mRNA level was analyzed by RT-PCR,while the protein contents of p-JNK, p-ERK and p-P38 were detected by Western blot,and the DNA binding activity of AP-1 was determined by electrophoretic mobility shift assay. Results The MUC5AC protein production and mRNA expression in the CSE group were significantly higher than those in the control group,and the DNA binding activity of AP-1 was also higher than that in the control group ( P 0. 01 ) . The protein contents of p-ERK and p-JNK in the CSE group were higher than those in the control group ( P 0. 01 ) ,but the p-P38 level was not significantly different from that in the control group ( P 0. 05 ) . After the transfection of TAM67 into the cells,the expression levels of MUC5AC protein and mRNA and the binding activity of AP-1 decreased significantly ( P 0. 01) . The DNA binding activity of AP-1 and the expression levels of MUC5AC protein and mRNA were lower in the SP600125 group and in the PD98059 group than those in the CSE group ( P 0. 05 ) . Conclusion After being activated by JNK and ERK which are phosphorylated by cigarette smoke,AP-1 binds to its DNA binding elements on the promoter of MUC5AC gene and up-regulates the MUC5AC expression at the transcriptional level.
出处
《中南大学学报(医学版)》
CAS
CSCD
北大核心
2010年第11期1150-1155,共6页
Journal of Central South University :Medical Science
基金
国家自然科学基金(30770951)~~