Extracellular Heat Shock Protein90? Mediates The Mechanism Of Hdm-induced Bronchial Epithelial Barrier Dysfunction

Background:Bronchial asthma(referred to as asthma)is a persistent chronic airway inflammatory disease which multiple kind of cells and many cellular components involved in.The destruction of airway epithelial barrier and hyperpermeability is essential to the initiation and development of asthma.Previous studies have shown that house dust mite(HDM)can increase the permeability of the airway epithelial barrier,participated in the initiation and development of asthma.However,the specific mechanism is unclear.Heat shock protein(Hsp)90? exerts its cell motility through secreting into the extracellular microenvironment.The correlative researches mainly focused on wound healing and tumor metastasis.RhoA signaling plays a key role in many biological processes.It reported that RhoA signaling was involved in the intergrity defect of vascular endothelial and intestinal epithelial barrier.Hsp90 inhibition prevents and restores endotoxin(LPS)-induced lung endothelial barrier dysfunction via suppressing Src-mediated RhoA activity and signaling.Therefore,extracellular Hsp90a(eHsp90a)may play a key role in HDM-induced airway epithelial barrier dysfunction.Objective:1,To explore specific mechanisms that eHsp90a mediate HDM-induced airway epithelial barrier dysfunction process and the role of activation of RhoA/myosin light chain(MLC)induced by eHsp90a mediate HDM-induced destruction of the bronchial epithelial barrier.2.To investigate the effect of house dust mite(HDM)on bronchial epithelial actin stress fibers(F-actin)reorganization and the involvement of epidermal growth factor receptor(EGFR)signaling.Method:The airway epthelial barrier function induced by HDM was assessed by measuring transepithelial electrical resistance(TEER)and FITC-dextran flux(FITC-DX)in human bronchial airway epithelial 16HBE14o-(16HBE)cells.Western blotting and immunofluorescence(Cofocal)were used to evaluate the expression and delocalization of E-cadherin and ?-catenin.The secretion of eHsp90a by airway epithelial cells16HBE was detected by concentration and purification condition medium.The activity of RhoA was determined by the Rho G-LISA(?)RhoA activation assay kit TM biochem kit,and the MLC phosphorylation level was determined by Western blotting.Meanwhile,we detected the levels of EGFR,phospho-EGFR,and F-actin by Western Blot method.The location of F-actin in bronchial epithelia was measured by Immunofluorescence technique.Results:Compared with the control group,HDM stimulated 16HBE cells to induce hyperpermeability of monolayer of epithelial cells,which were showed by decreasing in TEER values and rising of FITC-DX values,especially in 400 U/ml of HDM concentration.Meanwhile,HDM can also induce delocalization of adhesion junctional proteins E-cadherin and ?-catenin,showing the diffusion from membrane to cytoplasm.In addition,the protein level of eHsp90a was significantly increased in HDM stimulated 16HBE cells,especially in 400 U/ml of HDM concentration.Furthermore,inhibition of Hsp90a by down-regulated Hsp90a or pretreatment with eHsp90a monoclonal antibody(1G6-D7)prevented HDM-induced airway epithelial barrier dysfunction,which were showed by improving TEER values and FITC-DX values or restoring the delocalization of E-cadherin and p-catenin.Moreover,400 U/ml of HDM could increase the activity of RhoA at 6 h,and the same concentration of HDM could induce the increase of MLC phosphorylation at 6 h and peak at 24 h.However,inhibition of Hsp90a by down-regulated Hsp90a or pretreatment with 1G6-D7 suppressed HDM-induced RhoA activity and MLC phosphorylation levels.Furthermore,pretreatment with inhibitors of Rho kinase,GSK429286A and Y27632 2HC1,could protect HDM-induced loss of bronchial epithelium barrier integrity in 16HBE cells monolayer,which were showed by improving TEER values and FITC-DX values or restoring the delocalization of E-cadherin and ?-catenin.In addition,human recombinant(hr)Hsp90a,but not hrHsp90p,stimulated 16HBE cells to induce hyperpermeability of the bronchial epithelial barrier and activation of RhoA/MLC signaling.Besides,cells was stimulated by HDM and pretreated by AG-1478,the EGFR inhibitor.There are four groups:Control group,AG-1478 group,HDM group,AG-1478+HDM group.Compared with Control group,the levels of phospho-EGFR in HDM group were significantly increased.Cells pretreated by EGFR inhibitor exhibited an apparent improvement of the delocalization of E-cadherin and ?-catenin and increased TEER value and hyperpermeability of FITC-DX induced by HDM.F-actin expression was also increased and the reorganization of F-actin was observedin 16HBE cells after HDM stimulation,which could be inhibited by AG-1478,an EGFR inhibiter.Conclusions:1.Our study show that eHsp90a? mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling,suggesting that the eHsp90a? is a potential therapeutic target for treatment of asthma.2.EGFR signaling mediates F-actin reorganization and contributes to house dust mite-induced epithelial barrier dysfunction.