Ezrin介导中性粒细胞弹性蛋白酶诱导的人气道上皮细胞株分泌黏蛋白增加

Ezrin mediates neutrophil elastase-induced MUC5AC secretion in human airway epithelial cells

目的探讨膜连接蛋白Ezrin在中性粒细胞弹性蛋白酶(NE)诱导气道黏液高分泌的作用及相关调节机制。方法以中性粒细胞弹性蛋白酶刺激培养的人气道上皮HBE16细胞,ELISA法、real-time PCR法测定黏蛋白(MUC)5AC的蛋白及mRNA水平。免疫荧光法检测Ezrin蛋白含量。结果 NE刺激30 min后MUC5AC mRNA表达增强,细胞中及培养上清中黏蛋白MUC5AC的含量均显著高于对照组(P<0.01);转染Ezrin永久磷酸化载体pEGFPN1-Ezrin-T567D的细胞经NE刺激后,胞质内磷酸化Ezrin表达明显增强,且胞膜分布增多。同时,细胞培养上清液中的MUC5AC蛋白含量亦显著高于单纯NE组(P<0.01)。遏制Ezrin磷酸化pEGFP-N1-Ezrin-T567A载体转染组在NE刺激后,Ezrin蛋白与pEGFP-N1-Ezrin-T567D组相比有明显减少,且未出现向胞膜聚集的趋势,同时分泌至培养上清的MUC5AC蛋白也有明显减少(P<0.01),同时伴随胞质内MUC5AC蛋白有所增加(P<0.05)。结论 Ezrin蛋白参与了NE诱导的MUC5AC蛋白分泌,是气道上皮细胞MUC5AC分泌的重要调控分子。

Objective To explore the role of Ezrin in neutrophil elastase（NE）-induced mucin（MUC）5AC pro-duction in human HBE16 airway epithelial cells. Methods HBE16 airway epithelial cells were transfected with pEGFP-N1-Ezrin-T567D and pEGFP-N1-Ezrin-T567A,respectively,then each group was treated with 0. 5 μmol / L NE. The level of MUC5AC protein in culture medium,MUC5AC protein in cytoplasm,MUC5AC mRNA,Ezrin pro-tein in culture cells were detected with ELISA,real-time PCR,and immunofluorescence,respectively. Results There was an obvious increase of MUC5AC protein production and mRNA expression in cells exposed to NE,with elevation of Ezrin protein,all showed significant differences when compared with normal control group. Cell trans-fected with pEGFP-N1-Ezrin-T567D,MUC5AC secretion was increased in culture medium（P 〈0. 01） as compared with single NE-stimulated cells. Ezrin protein was increased,and manily concentrated in cytoplasmic membranes（P 0. 05） as ompared with single NE-stimulated cells. pEGFP-N1-Ezrin-T567A restrained Ezrin expression in cells and decreased MUC5AC protein in culture medium. Conclusions Ezrin is involved in NE-mediated MUC5AC protein secretion but not MUC5AC mRAN expression in HBE16 cells. The phosphrylation of Thr567 site in Ezrin may play a critical role.