Idiopathic pulmonary fibrosis (IPF), characterized by progressive decline in lung function, is associated with histological appearance of usual interstitial pneumonia (UIP). To define pathogenesis of lung fibrosis, several mouse models have been developed. Bleomycin is the most frequently used agent to drive lung fibrosis in mice. Apart from bleomycin model, AECII injury and apoptosis were reported as important triggers in other mouse models like Hermansky-Pudlak syndrome (HPS) associated lung fibrosis and amiodarone induced lung fibrosis. HPS is an autosomal recessive disorder characterized by bleeding diathesis, occuculocutaneous albinism and pulmonary fibrosis in patients. Aim of this study is to analyze regulation of autophagy in HPSIP and bleomycin model of lung fibrosis. Autophagy is a lysosome dependent quality control mechanism of cell to maintain cellular homeostasis and promote cell survival under stressful conditions.In our study, key autophagy proteins including LC3BII and p62 were found to be increased in HPS1/2 double mutant mice lungs. Electron microscopy revealed preferable LC3B binding to the lumen of lamellar bodies in HPS1/2 while it was found in both the lumen as well as on the limiting membrane of lamellar bodies in wildtype mice. After HPS1 knockdown in A549 cells, p62, an autophagy substrate protein accumulated indicating defective autophagy pathway. Exogenous LC3B normalized HPS1 knockdown induced p62 protein levels. A549 cells post HPS1 knockdown also showed a decrease in the formation of autophagolysosomes. We overexpressed and immunoprecipitated HPS1 protein from A549 cells and identified specific autophagy related proteins as HPS1 interacting proteins via mass spectrometric analysis. Bleomycin treated mice lungs showed increase in autophagy proteins. MLE12 cells, upon bleomycin treatment revealed decrease in autophagy flux but showed increased levels of cleaved caspase-3 indicating induction of apoptosis post bleomycin treatment. We performed co-imunoprecipitation followed by mass spectrometric analysis to identify interaction partners for autophagy marker protein, LC3B within the alveolar epithelial cells. Our analysis revealed novel interacting partners for LC3B.This study concludes that defective autophagy pathway observed both, in response to HPS1 knockdown and bleomycin challenge might play a critical role in the development and progression of pulmonary fibrosis. Understanding functions of autophagy by analyzing novel interaction partners of its marker proteins within the alveolar epithelial cells may help to further understand the mechanisms behind the contribution of defective autophagy towards the development of lung fibrosis.
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