Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.

BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.

[Role of epidermal growth factor receptor in house dust mite-induced airway epithelial barrier dysfunction].

OBJECTIVE: 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). METHODS: 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 ß-catenin in the cell cultures were detected with Western blotting. The localizations of F-actin, E-cadherin and ß-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. RESULTS: 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 ß-catenin (P>0.05). Immunofluorescence assay revealed delocalization of E-cadherin and ß-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 ß-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). CONCLUSION: EGFR signaling pathway mediates HDM-induced F-actin rearrangement in human bronchial epithelial cells to contribute to epithelial barrier dysfunction.