Abstract
Introduction: Cigarette smoke-induced mitochondrial damage accompanying enhanced ROS production has been implicated in COPD pathogenesis in terms of acceleration of cell senescence. Mitophagy may play a pivotal role for removal of CSE-induced damaged mitochondria and phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)-Parkin pathway has been proposed as a part of the mechanism for mitophagic degradation.
Methods: Using HBEC, senescence associated beta-galactosidase (SA-β-gal) staining and western blotting of p21 were performed to evaluate senescence. Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3, using confocal microscopy to measure colocalization between TOM20 stained mitochondria and EGFP-LC3 dots as a representation of autophagosome formation. Mitophagy was also evaluated using electron microscopy in HBEC. To elucidate the involvement of Parkin and PINK1 in mitophagy, knockdown (KD) by transfection of siRNAs was performed.
Results: CSE markedly induced EGFP-LC3 dot formation with concomitant colocalization with TOM20 in the presence of Bafilomycin A, suggesting that CSE treatment induces mitophagy, which was subsequently reduced by Parkin KD. CSE-induced mitophagy was further confirmed by electron microscopic detection of autophagosomes with mitochondria inside. Inhibition of mitophagy via PINK1 and Parkin KD increased mitochondrial ROS production and enhanced cell senescence in HBEC.
Conclusion: These findings suggest PINK1-Parkin pathway-mediated mitophagy plays a key regulatory role in CSE-induced oxidative stress and cell senescence through the degradation of damaged mitochondria.
- © 2014 ERS