LE Yan-Qing, Dong Hang-Ming, Wang Yan-Hong, Zhao Hai-Jin, Cai Shao-Xi
Laboratory of Chronic Airway Diseases, Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2017 Jun 20;37(6):737-743. doi: 10.3969/j.issn.1673-4254.2017.06.04.
To investigate the role of epidermal growth factor receptor (EGFR) signaling pathway in bronchial epithelial actin stress fiber (F-actin) rearrangement induced by house dust mite (HDM).
Normal human bronchial epithelial cells (16HBE) were stimulated with HDM with or without pretreatment with AG-1478, an EGFR inhibitor. The levels of phospho(p)-EGFR, F-actin, E-cadherin and β-catenin in the cell cultures were detected with Western blotting. The localizations of F-actin, E-cadherin and β-catenin in the bronchial epithelial cells were determined with immunofluorescence assay, and the transmembrane electrical resistance (TER) and FITC-dextran flux (FITC-DX) in the cells were measured to assess the barrier function of the bronchial epithelia.
HDM stimulation of the cells for 10 min resulted in significantly increased p-EGFR expression (P<0.05) without causing obvious changes in the expression of E-cadherin (P>0.05) or β-catenin (P>0.05). Immunofluorescence assay revealed delocalization of E-cadherin and β-catenin in HDM-treated 16HBE cells, shown by their diffusion from the cell membrane to the cytoplasm. In HDM-treated cells, the TER was significantly decreased to (70.00∓4.33)% and the FITC-DX was significantly increased to (115.98∓4.34)%; Inhibition of EGFR reversed the delocalization of E-cadherin and β-catenin, improved the TER to (90.00∓3.75)% and lowered the FITC-DX to (101.10∓2.10)%. HDM induced increased expression and rearrangement of F-actin, which was obviously inhibited by pretreatment of the cells with AG-1478 (P<0.05).
EGFR signaling pathway mediates HDM-induced F-actin rearrangement in human bronchial epithelial cells to contribute to epithelial barrier dysfunction.
探讨表皮生长因子受体（EGFR）信号通路在屋尘螨（HDM）诱导的支气管上皮肌动蛋白应力纤维（F-肌动蛋白）重排中的作用。
用或不用EGFR抑制剂AG-1478预处理正常人支气管上皮细胞（16HBE），然后用HDM刺激。采用蛋白质免疫印迹法检测细胞培养物中磷酸化（p）-EGFR、F-肌动蛋白、E-钙黏蛋白和β-连环蛋白的水平。采用免疫荧光法测定支气管上皮细胞中F-肌动蛋白、E-钙黏蛋白和β-连环蛋白的定位，并测量细胞的跨膜电阻（TER）和异硫氰酸荧光素-葡聚糖通量（FITC-DX），以评估支气管上皮的屏障功能。
HDM刺激细胞10分钟导致p-EGFR表达显著增加（P<0.05），而E-钙黏蛋白（P>0.05）或β-连环蛋白（P>0.05）的表达无明显变化。免疫荧光法显示，HDM处理的16HBE细胞中E-钙黏蛋白和β-连环蛋白发生错位，表现为它们从细胞膜扩散到细胞质中。在HDM处理的细胞中，TER显著降低至（70.00±4.33）%，FITC-DX显著增加至（115.98±4.34）%；抑制EGFR可逆转E-钙黏蛋白和β-连环蛋白的错位，使TER提高至（90.00±3.75）%，并使FITC-DX降低至（101.10±2.10）%。HDM诱导F-肌动蛋白表达增加和重排，而用AG-1478预处理细胞可明显抑制这种现象（P<0.05）。
EGFR信号通路介导HDM诱导的人支气管上皮细胞F-肌动蛋白重排，导致上皮屏障功能障碍。
