Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-kB pathways in A549 cells.
Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-kB pathways in A549 cells.
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DOI：
10.3904/kjim.2005.20.4.275
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发表时间：
2005-12
期刊：
影响因子：
--
通讯作者：
Park SH
中科院分区：
文献类型：
--
作者：
Song JS;Cho KS;Yoon HK;Moon HS;Park SH
研究发现中性粒细胞弹性蛋白酶（NE）可以增加呼吸道粘蛋白基因MUC 5AC，尽管该过程的分子机制仍不清楚。我们试图确定NE诱导支气管上皮细胞MUC 5AC基因表达的信号转导途径。将已克隆到pGL 3-Basic荧光素酶载体中的1.3Kb MUC 5AC启动子片段转染到A549细胞中。通过测量荧光素酶活性，我们能够评估MUC 5AC启动子在A549细胞中的活性。蛋白质印迹法证实了丝裂原活化蛋白激酶（MAPK）的参与。为了证实核因子κ B（NF-kB）的参与，我们使用定点突变和电泳迁移率变动分析（EMSA）放射自显影。通过RT-PCR证实MUC 5AC mRNA的表达。NE可增加A549细胞中MUC 5AC启动子的转录活性。发现NE增加MUC 5AC启动子的转录活性与增加的NF-κ B活性相关。定点突变显示，转染来自PGL 3-MUC 5AC-3752启动子荧光素酶报告质粒的突变的NF-κ B结合位点降低NE刺激后的荧光素酶活性。在MAPKs中，只有细胞外信号调节激酶（ERK）参与NE诱导的MUC 5AC粘蛋白表达。RT-PCR结果显示NE可增加MUC 5AC mRNA的表达。EMSA放射自显影显示NE诱导NF-κ B：DNA结合。这些结果表明，在A549细胞中，人NE通过表皮生长因子受体（EGF-R）、ERK和NF-kB途径诱导MUC 5AC粘蛋白。
Neutrophil elastase (NE) was found to increase the respiratory mucin gene, MUC5AC, although the molecular mechanisms of this process remain unknown. We attempted to determine the signal transduction pathway through which NE induces MUC5AC gene expression in bronchial epithelial cells. A fragment of 1.3 Kb MUC5AC promoter which had been cloned into the pGL3-Basic luciferase vector was transfected to the A549 cells. By measuring the luciferase activity, we were able to evaluate the MUC5AC promoter activity in A549 cells. The involvement of mitogen-activated protein kinases (MAPK) was confirmed by Western blotting. To confirm the involvement of nuclear factorkappaB (NF-kB), we used site-directed mutagenesis and electrophoretic mobility shift assay (EMSA) autoradiogram. The MUC5AC mRNA expression was confirmed by RT-PCR. NE increased the transcriptional activity of the MUC5AC promoter in A549 cells. The increased transcriptional activity of the MUC5AC promoter by NE was found to be associated with increased NF-kB activity. Site-directed mutagenesis showed that the transfection of the mutated NF-kB binding sites from the PGL3-MUC5AC-3752 promoter luciferase reporter plasmid decreased the luciferase activity after NE stimulation. Among the MAPKs, only extracellular signal-regulated kinases (ERK) were involved in this NE-induced MUC5AC mucin expression. RT-PCR also showed that NE increased MUC5AC mRNA. An EMSA autoradiogram revealed that NE induced NF-kB:DNA binding. These results indicate that human NE induces MUC5AC mucin through the epidermal growth factor receptor (EGF-R), ERK, and NF-kB pathways in A549 cells.