Role Of RAGE And Krt7 In Hyperpermeability Of Alveolar Epithelial Cells In Acute Lung Injury

Research background:Sepsis is the systemic inflammatory response syndrome(SIRS)induced by infection.Lung is the most susceptible target organ for sepsis,and acute lung injury(ALI)is the earliest event in sepsis.A large number of inflammatory mediators might injure alveolar epithelial cells and vascular endothelial cells and increase their permeability,resulting in pulmonary edema in which proteins and liquid of blood leak into the lung tissue.As one of the major inflammatory mediators,high mobility group box-1(HMGB1)binds to receptor for advanced glycation end products(RAGE)to activate downstream signaling pathways involved in inflammatory responses.The FH1 segment of mammalian diaphanous 1(mDia1)could combine with cytoplasmic segment of RAGE,and mDia1 knockout cells inhibit RAGE induced downstream signaling pathways.Early studies shown that HMGB1 binding to RAGE regulate cytoskeletal movement and cell migration through cell division cycle protein(Cdc42)and Rac1.We presume that RAGE,mDia1 and Cdc42 are involved in HMGB1-induced hyperpermeability of lung cancer cells(A549 cells).Previously,our laboratory has confirmed that keratin 7(Krt 7)express different in the lung tissue of septic shock mice.Krt 7 is an epithelial cytoskeletal protein that is closely related to epithelial cell barrier dysfunction.Accordingly,we hypothesized that Krt7 were involved in the hyperpermeability of alveolar epithelial cells in acute lung injury.p38 MAPK is known to involve in the regulation of keratin phosphorylation and directly induce serine phosphorylation of Krt8 at position 73.Since both Krt7 and Krt8 belong to the keratin family,we assumed that p38 MAPK is involved in the regulation of Krt7 phosphorylation.Studies have shown that type II keratin 5 interacts with desmosomes the epithelial cells connection protein to form keratin-desmosomes complexes,whose stability plays a major role in wound healing and epidermal barrier function.Krt7 is type Ⅱ keratin,which is highly expressed in lung epithelial cells,we suggesting that Krt7 and desmosomes are involved in regulating epithelial cell barrier function.Objective:This study aims to elucidate whether RAGE and Krt7 are involved in hyperpermeability of alveolar epithelial cells induced by HMGB1 and explore the underlying mechanismsMethods:A series of experiments were accomplished in cells by using cells culture,primary isolated mouse alveolar epithelial cells and lung cancer cells.Western blot,immunofluorescence,siRNA interference,transfected plasmids,adenovirus,epithelial monolayer permeability assay and so on,to observe the role of RAGE and Krt7 involved in hyperpermeability of alveolar epithelial cells.Results:1.RAGE gene deletion has a protective effect on CLP model mice induced acute lung injury.Compared with the WT-Sham group,lung water content,total protein concentration of BALF,the Neutrophil count in the BALF,the content of HMGB1 in serum and BALF significantly increased in WT-CLP group.Multiple sizes of alveolar cavities,interstitial hyperemia,edema,a large number of inflammatory cells and red blood cell infiltration were observed through pathological observation of lung tissue under microscopy.RAGE(-/-)-Sham group compared with RAGE(-/-)-CLP group had no significant changes.2.mDia1 was involved in the process of hyperpermeability evoked by HMGB1 via binding to RAGE.Compared with control group,HMGB1 significantly decreased TER value and increased FITC-dextran leakage.Comparaed with HMGB1 group,cells transfected with RAGE siRNA,mDia1 siRNA,RAGE mutant plasmid,infecting interference adenovirus of mDia1,displayed significantly increased TER and decreased FITC-dextran leakage induced by HMGB1.While cells transfected with RAGE wild-type plasmid,showed significantly decreased TER and increased FITC-dextran leakage,with NC siRNA,control empty virus and control empty vector plasmid had no effect.3.Krt7 was associated with hyperpermeability of A549 cells induced by HMGB1.A549 cells Transfected with Krt7 siRNA significantly increased TER and decreased FITC-dextran leakage induced by HMGB1,while NC siRNA had no effect.4.HMGB1-induced phosphorylation of p38 MAPK,Cdc42-p38 MAPK was associated with hyperpermeability of A549 cells induced by HMGB1.Pretreatment with Cdc42 inhibitor ML141,cells significantly increased TER and decreased FITC-dextran leakage.Cells were incubated with 100ng/mL of HMGB1 for diferent duration and phosphorylation of p3 8 MAPK was determined by western blot.Results showed that phosphorylation of p38 MAPK was rapidly enhanced,reached a peak at 30min.Cells Transfected with p38 MAPK siRNA significantly increased TER and decreased FITC-dextran leakage,with NC siRNA had no effect.Cells pretreated with p38 MAPK inhibitor SB203580 significantly increased TER and decreased FITC-dextran leakage.5.p38 MAPK is involved in the regulation of Krt7 phosphorylation.HMGB1 induced p38 MAPK/Krt7 association in A549 cells.A549 cells were treated with 100ng/mL HMGB1 for 30min and lysed,and co-immunoprecipitation(CO-IP)for p38 MAPK was performed.Increased expression of Krt7 was detected by western blot.A549 cells were treated with 100ng/mL HMGB1 for 30min and lysed,and CO-IP for Krt7 was performed.Increased expression of p38 MAPK was detected by western blot.GST-p38 MAPK and Krt7 wild-type plasmid was performed.Increased expression of Krt7 was detected by western blot.Pretreatment with p38 MAPK upstream kinase MKK6(b)E,increased expression of phosphorylation of serine(55kDa)was detected by western blot.Using the bioinformatics analysis software GPS 3.0 online analysis tool,the phosphorylation of Krt7 protein sequence was predicted and p38 MAPK was found to be involved in the regulation of phosphorylation of Krt7.6.The morphological changes of Krt7、Desmoplakin、Desmoglein3 induced by HMGB1.Conclusion:1.RAGE was involved in the process of acute lung injury induced by CLP model mice.HMGB1 induced A549 cells hyperpermeability through HMGB1-RAGE binding.2.mDia1-RAGE binding was contributed to HMGB1-induced hyperpermeability of A549 cells.3.HMGB1 induced phosphorylation of p38 MAPK,HMGB1-induced hyperpermeability of A549 cells via Cdc42-p38 MAPK.4.Krt7 was involved in HMGB1-induced hyperpermeability of A549 cells,preliminarily verified p38 MAPK is involved in the regulation of Krt7 phosphorylation.5.The morphological changes of Krt7、Desmoplakin、Desmoglein3 induced by HMGB1.