Study Of EGFR-TNFR1 Signaling In Inflamed Endothelium During Acute Lung Injury

Background and objectiveAcute lung injury(ALI)is a relatively common acute respiratory failure syndrome caused by intrapulmonary or extrapulmonary factors,which can be fatal or disabling.The most severe stage is acute respiratory distress syndrome characterized by progressive hypoxemia,which lacks specific drugs and has a very high mortality rate.Endothelial dysfunction is the core of ALI,and its pathogenesis is varied,among which TNFa is closely related to endothelial dysfunction.TNFa acts primarily on TNFR1 receptors and regulates cellular immunity,inflammation,and cell death,but the exact mechanism is still not fully understood.Epidermal growth factor receptor(EGFR),an important member of the ErbB family,has been widely reported in cancer,cell proliferation and inflammatory injury,and has become a therapeutic target.Since EGFR inhibitors inhibit the abnormal expression of EGFR and TNF-α in inflammatory states,EGFR may be a key molecule in the TNFR1 signaling pathway.The purpose of this study was to investigate the regulatory effect of EGFR-TNFR1 signal on pulmonary vascular endothelial injury in ALI and its potential mechanism,so as to provide an effective drug therapeutic target for the treatment of ALI.MethodsIn vivo:ALI model was established by intratracheal TNFα instillation on wild type mice.EGFR activation was inhibited by erlotinib,EGFR tyrosine kinase inhibitor,gavage pretreatment.Lung tissues were collected for HE staining,damage scoring,quantitative PCR,and western blot detection.Bronchoalveolar lavage fluid was collected for protein concentration and flow cytometry detection.In vitro:HUVEC cells were cultured and stimulated by TNFα after pretreatment of PD or EGFR silencing.The cells were collected for immunocoprecipitation,immunofluorescence,quantitative polymerase chain reaction and western blotting.3T3 cells were co-transfected with His-TNFR1 and Flag-EGFR plasmid,and collected for co-immunoprecipitation and western blot detection.Results1.EGFR physically interacted with TNFR1,increased with TNFα stimulation.(Co-localization existed in immunoprecipitation and immunofluorescence.)2.Early endocytosis of TNFR1 occurred after TNF-α stimulated HUVEC cells for 20min,and EGFR could promote the endocytosis of TNFR1(increased colocalization of TNFR1 and EEA1).3.EGFR promoted the assembly of TNFR1 complex Ⅰ(increased binding of TRAF2 to TNFR1),and EGFR inhibitors can reduce NF-κB/MAPK-mediated inflammatory responses(reduced expression of inflammatory genes)by regulating the nuclear localization of AP-1.4.EGFR did not affect endothelial cell apoptosis regulated by TNFR1 complexⅡa.EGFR inhibitors reduced cleaved caspase3 protein expression,but did not affect cell annexin V fluorescence staining.5.EGFR affected necroptosis of endothelial cells mediated by TNFR1 complexⅡb.EGFR inhibitors reduced the binding and respective phosphorylation of RIP1 and RIP3.6.Inhibition of EGFR tyrosine kinase resulted in reduced lung injury in TNFa induced ALI(decreased lung injury score,lung permeability,and neutrophil recruitment)and inhibited activation of NF-κB/MAPK signaling pathways in lung tissue(decreased signaling proteins,inflammatory factors).ConclusionsEGFR can regulate TNFR1-mediated NF-κB/MAPK inflammatory response and RIP3-dependent necroptosis through interacting with TNFR1,resulting in enhanced pulmonary vascular endothelial injury,increased neutrophil infiltration,and activation of inflammatory response.Targeting EGFR-TNFR1 signaling could be the therapeutic method for ALI.