Csk/Src/EGFR Signaling Regulates Migration Of Myofibroblasts And Alveolarization

Objects: Bronchopulmonary dysplasia(BPD)is a major chronic pulmonary complication in preterm infants.It is characterized by premature alveolar developmental arrest with enlarged alveolar spaces,decreased terminal space numbers and reduced secondary septa counts.Antenatal exposure to bacterial infections and inflammation has been an important risk factor for BPD occurence.During lung development,alveolar myofibroblasts play an important role in extracellular collagen and elastic fiber deposition,leading to the growth of secondary septa,saccule subdivision and respiratory space enlargement.Our previous studies observed abnormal location of alveolar myofibroblasts in neonatal rats with lipopolysaccharide(LPS)-induced lung injury,a model for BPD.Next,we try to explore whether LPS disrupts the directional migration of myofibroblasts,resulting in their abnormal location and alveolar developmental arrest.We also investigate the role of Csk/Src/EGFR signaling in the directional migration of myofibroblasts.Methods: Part 1: LPS was injected into the amniotic sac of pregnant rats at 16.5 days post coitum(E16.5)and primary lung myofibroblast was isolated from newborn rats at postnatal day 7(P7).Golgi polarization assays and transwell migration assays were carried out to observe the directional migration of myofibroblasts.The effect of LPS on cell migration was also observed in cultured human lung myofibroblasts.Part 2: Protein expressions in primary myofibroblast were assessed by western blot analysis.Csk activity was detected by a spectrophotometric-based kit while EGFR and Src activation were evaluated by measuring the ratio of phosphorylated to total protein.The effect of LPS on activities of protein was also observed in cultured myofibroblasts.Part 3: Exogenous rhTGF-α or the EGFR-specific inhibitor AG1478 was applied to test the role of EGFR in cell migration.Silencing of Csk by shRNA,inhibition of Src with PP1 or transfection of plasmid encoding dominant negative Src(DN-Src)was administrated to examine whether Csk/Src acts upstream of the EGFR in regulating cell migration.Part 4: LPS with or without EGFR inhibitor erlotinib were injected into the amniotic sac of pregnant rat.Lung sections stained with hematoxylin and eosin(H&E)or immunostained with α–SMA for myofibroblast identification were analyzed to determine the effect of erlotinib on LPS-induced lung injury.Results: Part 1: Primary myofibroblasts isolated from newborn rat lungs with intra-amniotic LPS exposure exhibited significantly reduced directional persistence.Compared with the saline group,cells in the LPS group exhibited significantly reduced Golgi reorientation toward the wound(74.39±0.6848% vs 56.95±1.457%,p<0.01)and the number of migrated cells was decreased(46.27±5.347 vs 20.07±4.631,p<0.05).Similar trends were found in cultured myofibroblasts treated with LPS in vitro.LPS disrupted the directional migration of myofibroblasts(60.12±0.7442% vs 41.65±0.7777% for Golgi polarization assay,37.6±1.208 vs 19.6±0.8778 for transwell migration assay,p<0.01).Part 2: No significant changes in the expression of protein were observed in myofibroblasts from LPS-exposed rats.However,Csk activity was decreased in myofibroblasts from LPS-exposed rats(73.6%).In contrast,phosphorylation levels of Src and EGFR were significantly elevated by LPS treatment(2.6 fold and 1.96 fold respectively).Similarly,Csk activity was decreased in cultured myofibroblasts treated with LPS(70.3%)while Src and EGFR activation was increased(2.2 fold and 1.5 fold respectively).Part 3: With additional application of rhTGF-α,myofibroblasts also exhibited significantly reduced directional persistence and the EGFR-specific inhibitor AG1478 can rescue the myofibroblast migration deficiency induced by LPS or rhTGF-α treatment.Src-blockade with PP1 attenuated EGFR activation and impaired directional migration caused by LPS treatment.Furthermore,expression of DN-Src in myofibroblasts also abrogated LPS-induced EGFR activation and migration impairment.In addition,Silencing of Csk by shRNA significantly up-regulated EGFR activation and reduced directional persistence of myofibroblast migration.Part 4: Co-treatment with the EGFR inhibitor erlotinib and LPS increased terminal airspace counts,secondary septa counts and the ratio of myofibroblasts in secondary septa compared to LPS treatment alone,suggesting erlotinib effectively alleviated the lung injury and abnormal localization of myofibroblasts induced by LPS.Conclusion: Our study demonstrated that LPS may induce EGFR activation through Csk/Src signaling pathway,disrupt the directional migration of myofibroblast,preventing correct localization of these cells and thus alveolar septation and maturation.Furthermore,EGFR inhibition partially restored alveolar development impaired by LPS,indicating that this signaling pathway serves as a key pathophysiological mechanism of BPD.