Yu Hongmei, Zhou Xiangdong
Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2010 Nov;35(11):1150-5. doi: 10.3969/j.issn.1672-7347.2010.11.006.
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.
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.
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).
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.
探讨活化蛋白-1（AP-1）在香烟烟雾诱导气道黏液高分泌中的作用机制，并探索激活AP-1的可能信号转导途径。
体外培养气道上皮细胞系（BEAS-2B），用香烟烟雾提取物（CSE）处理。通过将c-Jun显性负性突变体TAM67转染入细胞来阻断AP-1的DNA结合活性。分别用SP600125和PD98059阻断c-Jun氨基末端激酶（JNK）和细胞外信号调节激酶（ERK）的激活。采用酶联免疫吸附测定法检测MUC5AC蛋白，用逆转录聚合酶链反应分析MUC5AC mRNA水平，用蛋白质免疫印迹法检测p-JNK、p-ERK和p-P38的蛋白含量，用电泳迁移率变动分析测定AP-1的DNA结合活性。
CSE组MUC5AC蛋白产量和mRNA表达均显著高于对照组，AP-1的DNA结合活性也高于对照组（P<0.01）。CSE组p-ERK和p-JNK的蛋白含量高于对照组（P<0.01），但p-P38水平与对照组相比差异无统计学意义（P>0.05）。将TAM67转染入细胞后，MUC5AC蛋白和mRNA的表达水平以及AP-1的结合活性均显著降低（P<0.01）。SP600125组和PD98059组AP-1的DNA结合活性以及MUC5AC蛋白和mRNA的表达水平均低于CSE组（P<0.05）。
香烟烟雾使JNK和ERK磷酸化而激活后，AP-1与其在MUC5AC基因启动子上的DNA结合元件结合，在转录水平上调MUC5AC表达。
