Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD
Kenji Mizumura, Suzanne M. Cloonan, Kiichi Nakahira, Abhiram R. Bhashyam, Morgan Cervo, Tohru Kitada, Kimberly Glass, Caroline A. Owen, Ashfaq Mahmood, George R. Washko, Shu Hashimoto, Stefan W. Ryter, Augustine M.K. Choi
Kenji Mizumura, Suzanne M. Cloonan, Kiichi Nakahira, Abhiram R. Bhashyam, Morgan Cervo, Tohru Kitada, Kimberly Glass, Caroline A. Owen, Ashfaq Mahmood, George R. Washko, Shu Hashimoto, Stefan W. Ryter, Augustine M.K. Choi
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Research Article Pulmonology

Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

Abstract

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1–/– mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.

Authors

Kenji Mizumura, Suzanne M. Cloonan, Kiichi Nakahira, Abhiram R. Bhashyam, Morgan Cervo, Tohru Kitada, Kimberly Glass, Caroline A. Owen, Ashfaq Mahmood, George R. Washko, Shu Hashimoto, Stefan W. Ryter, Augustine M.K. Choi

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Figure 5

CSE-induced mitophagy decreases ΔΨm, leading to cell death in pulmonary epithelial cells.

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CSE-induced mitophagy decreases ΔΨm, leading to cell death in pulmonary ...
(A) Flow cytometry of alveolar epithelial cells obtained from Pink1–/– and Pink1+/+ mice. Alveolar epithelial cells were treated with 20% CSE for 4 hours and labeled with TMRE. The x axis shows the fluorescent signal intensity of TMRE, and the y axis shows the cell number with corresponding fluorescence intensity. Numbers above bracketed lines indicate the percentage of cells with loss of mitochondrial membrane potential. Data are representative of 2 experiments. (B) Beas-2B cells were incubated for 1 hour with CsA (10 μM) or vehicle (DMSO) and treated with 20% CSE for 15 hours. Cell death was determined by calcein AM/EthD-1 flow cytometry. The x axes show calcein AM staining, and the y axes show EthD-1 staining (B and D). Data are representative of 3 independent experiments. (C) Alveolar epithelial cells were obtained from Pink1–/– and Pink+/+ mice. Alveolar epithelial cells were treated with 10% or 20% CSE for 15 hours. Cytotoxicity was measured by LDH release. Data are representative of 2 experiments. (D) Beas-2B cells were incubated for 3 hours with Mdivi-1 (50 μM) or vehicle (DMSO) and treated with 20% CSE for 15 hours. Cell death was determined by calcein AM/EthD-1 flow cytometry. Data are representative of 3 experiments. Data represent the mean ± SEM (B–D). **P < 0.01 by unpaired, 2-tailed Student’s t test (B–D).