Lipopolysaccharide Disrupts Polarization And Directional Migration Of Alveolar Myofibroblasts

Objects: Bronchopulmonary dysplasia(BPD)is characterized by alveolarization arrest with fewer,larger alveoli.It is clear that alveolarization involves a process known as the outgrowth of secondary septation triggered by myofibroblasts.Our previous studies had demonstrated that lipopolysaccharide(LPS)exposure induced activation of EGFR and overexpression of its ligand,TGF-? in myofibroblasts,then leading to affected directional migration and final abnormal location of myofibroblasts,which may contribute to the arrested alveolar development in BPD.Nevertheless,it is still not clear what are the specific intracellular signal transduction mechanisms in response to LPS to disturb directional migration of myofibroblasts.Methods: Part 1: In general,the initial response of cell migration is to polarize,so we speculate LPS may affect the directional migration of myofibroblasts by disturbing the polarization of myofibroblasts.We assessed the role of LPS and TGF-? on the polarization of myofibroblast using scrape wounding assays combined with Golgi tracking.Then we further verified the role of LPS and TGF-? on the directional migration of myofibroblast using transwell migration assay.To identify the mechanism of LPS-induced polarization and migration abnormality,pretreatment of cells with AG1478,a selective inhibitor of EGFR.Part 2: Pull-down assays were performed to isolate the active GTP-bound form using the RhoA activation assay kits following the manufacturer's protocol.Part 3: To determine whether 14-3-3? is involved in the regulation of RhoA activity,we obtained siRNA for 14-3-3?.Results: Part 1: Compared with control groups,LPS and TGF-? led to a significantly decrease of polarization(orientation score: 60.03 ± 0.22 in LPS vs 41.90± 0.87 in saline,p<0.01;59.37 ± 0.8762 in rhTGF-? vs 42.73 ± 0.4410 in saline,p<0.01).Then we further verified the role of LPS and TGF-? on the directional migration of myofibroblast using transwell migration assay.We found that LPS and TGF-? significantly suppressed a motile phenotype,as detected by a decrease in the number of migrated cells/field compared with control groups(37.80 ± 1.66 in LPS vs 20.80±1.11 in saline,p<0.01;37.80 ± 1.65 in rhTGF-? vs 25.20 ± 0.9 in saline,p<0.01).Pretreatment of cells with AG1478 increased polarization and directional migration on LPS/TGF-?-induced myofibroblasts.Part 2: Western blot showed LPS or TGF-? was able to activate RhoA;pretreated with AG1478 for 30 min,LPS or TGF-? couldn't activate RhoA.In the presence of Y27632,a ROCK-specific inhibitor,LPS and TGF-? couldn't disturb the polarization and directional migration of myofibroblasts.Part 3: Treatment with LPS and TGF-? result in a marked increase of RhoA activation and disrupt polarization and directional migration of myofibroblasts,an effect that was eliminated by depletion of 14-3-3? by siRNA.Conclusion: In summary,our results establish a new molecular mechanism to explain how TGF-? affects directional migration of myofibroblasts.We speculate that LPS/TGF-? / EGFR signaling induces up-regulation of RhoA activity,and in this process 14-3-3? is absolutely necessary as an adaptor of GAPs.Then up-regulation of RhoA activity contributes to increase of stress fibers and focal adhesions,and decrease of cell polarization,leading to decreased cell directional migration of myofibroblasts and final abnormal location of myofibroblasts,which is important to secondary septa outgrowth and may contribute to the arrested alveolar development in BPD.