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) 90 alpha is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90 alpha (eHsp90 alpha) on the bronchial epithelial barrier disruption induced by HDM has never been reported. Methods: To investigate the involvement of eHsp90 alpha 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) Hsp90a and hrHsp90 alpha respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90 alpha monoclonal antibody (mAb). Hsp90a-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 beta-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90 alpha was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA (R) RhoA activation assay kit (TM) 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 beta-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90a secretion. All of which were rescued in Hsp90 alpha-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 hrHsp90 alpha instead of hrHsp90 alpha that promoted barrier dysfunction and activated RhoA/ MLC signaling in 16HBE cells. Conclusions: The eHsp90 alpha mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/ MLC signaling, suggesting that eHsp90 alpha is a potential therapeutic target for treatment of asthma.