Cells were plated at a density of 2.5 × 10^5 cells/mL on BioFlex plates pre-coated with collagen, and incubated for 24 h. siRNA was subsequently diluted with DMEM/F12 to remove FBS. INTERFERin and siRNA were diluted and subsequently incubated at room temperature for 10 min prior to further incubation for 48 h.

BACKGROUND AND OBJECTIVE: Ventilator-induced lung injury (VILI) is commonly associated with respiratory barrier dysfunction; however, the mechanisms have not been fully elucidated. This study aimed to determine the order and components of the signalling pathway that mediates the degradation of adherin junction of p120-catenin in VILI. METHODS: For the in vivo study, C57BL/6 mice were pre-treated with inhibitors for 60 min prior to 4 h of mechanical ventilation. For the in vitro study, mouse lung epithelial 12 (MLE-12) cells were pre-treated with inhibitors for 60 min or small interfering RNA (siRNA) for 48 h prior to cyclic stretch at 20% for 4 h. The protein levels of protein kinase Calpha (PKCalpha), activated c-Src and p120-catenin were determined via western blot analysis. Lung injury was determined via HE staining, immunofluorescence, wet/dry ratio and lung injury scores. RESULTS: High tidal volume mechanical ventilation and 20% cyclic stretch resulted in the degradation of p120-catenin. Inhibitors of PKCalpha blocked c-Src kinase activation and p120-catenin degradation in VILI. Inhibitors of c-Src kinase or PP2 or siRNA blocked p120-catenin degradation but not PKCalpha activation. CONCLUSION: The current findings demonstrate that PKCalpha and c-Src kinase participate in VILI. PKCalpha activation phosphorylates c-Src kinase and further decreases p120-catenin in VILI.