Abstract
BackgroundThe 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.MethodsTo 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 hrHsp90β 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 β-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.ResultsThe epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and β-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 hrHsp90β that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells.ConclusionsThe 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.
Highlights
The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma
House dust mite (HDM) induced bronchial epithelial hyperpermeability To clarify the effect of HDM on loss of bronchial epithelial barrier integrity, 16HBE cells were stimulated with different concentrations of HDM (200, 400, 800 U/ml) for 24 h, which were used for measurement of Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX)
Results indicated that HDM (400 U/ml or 800 U/ml) induced the fall of TEER values (Fig. 1a, P < 0.05) and the raise of FITC-DX (Fig. 1b, P < 0.05) compared to con HDM induced delocalization of E-cadherin and β-catenin E-cadherin/β-catenin complex plays an important role in maintaining epithelial integrity [6]
Summary
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. Presence of dysfunctional epithelial barrier in the asthmatic airway included hyperpermeability to allergens and disrupted expression of the junction molecule E-cadherin at sites of epithelial detachment [2, 3]. The decrease of epithelial E-cadherin protein expression in patients with atopic asthma contributed to defective airway epithelial barrier [4, 5]. E-cadherin/β-catenin complex, the AJs complex (AJC), plays an important role in maintaining epithelial integrity and disrupting this complex affect the adhesive repertoire of a cell, and the Wnt-signaling pathway [6], leading to the initiation and chronicity of asthma. Dysregulation of epithelial repair in asthmatic airways, including epithelial-mesenchymal transition, contributed to asthmatic airway remodeling [7]
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