Ferroptosis is a necrotic form of regulated cell death (RCD) mediated by phospholipid peroxidation in association with free iron-mediated Fenton reactions. Disrupted iron homeostasis resulting in excessive oxidative stress has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we demonstrate the involvement of ferroptosis in COPD pathogenesis. Our in vivo and in vitro models show labile iron accumulation and enhanced lipid peroxidation with concomitant non-apoptotic cell death during cigarette smoke (CS) exposure, which are negatively regulated by GPx4 activity. Treatment with deferoxamine and ferrostatin-1, in addition to GPx4 knockdown, illuminate the role of ferroptosis in CS-treated lung epithelial cells. NCOA4-mediated ferritin selective autophagy (ferritinophagy) is initiated during ferritin degradation in response to CS treatment. CS exposure models, using both GPx4-deficient and overexpressing mice, clarify the pivotal role of GPx4-regulated cell death during COPD. These findings support a role for cigarette smoke-induced ferroptosis in the pathogenesis of COPD.

(2015) PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis , Autophagy, 11:3, 547-559, DOI: 10.1080/15548627.2015 Abbreviations: Baf A1, bafilomycin A 1 ; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; CSE, cigarette smoke extract; EM, electron microscopy; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; HBEC, human bronchial epithelial cell; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; NAC, N-acetylcysteine; PCD, programmed cell death; PINK1, PTEN-induced putative kinase 1; ROS, reactive oxygen species; SA-b-Gal, senescence-associated b-galactosidase;TLR, toll-like receptor; WB, western blotting.Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by PINK1 and PARK2 knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.

Reepithelialization of remodeled air spaces with bronchial epithelial cells is a prominent pathological finding in idiopathic pulmonary fibrosis (IPF) and is implicated in IPF pathogenesis. Recent studies suggest that epithelial senescence is a risk factor for development of IPF, indicating such reepithelialization may be influenced by the acceleration of cellular senescence. Among the sirtuin (SIRT) family, SIRT6, a class III histone deacetylase, has been demonstrated to antagonize senescence. We evaluated the senescence of bronchiolization in association with SIRT6 expression in IPF lung. Senescence-associated β-galactosidase staining and immunohistochemical detection of p21 were performed to evaluate cellular senescence. As a model for transforming growth factor (TGF)-β-induced senescence of abnormal reepithelialization, we used primary human bronchial epithelial cells (HBEC). The changes of SIRT6, p21, and interleukin (IL)-1β expression levels in HBEC, as well as type I collagen expression levels in fibroblasts, were evaluated. In IPF lung samples, an increase in markers of senescence and SIRT6 expression was found in the bronchial epithelial cells lining cystically remodeled air spaces. We found that TGF-β induced senescence in primary HBEC by increasing p21 expression, and, whereas TGF-β also induced SIRT6, it was not sufficient to inhibit cellular senescence. However, overexpression of SIRT6 efficiently inhibited TGF-β-induced senescence via proteasomal degradation of p21. TGF-β-induced senescent HBEC secreted increased amounts of IL-1β, which was sufficient to induce myofibroblast differentiation in fibroblasts. These findings suggest that accelerated epithelial senescence plays a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions, which can be antagonized by SIRT6.

Tobacco smoke-induced accelerated cell senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cell senescence is accompanied by the accumulation of damaged cellular components suggesting that in COPD, inhibition of autophagy may contribute to cell senescence. Here we look at whether autophagy contributes to cigarette smoke extract (CSE) - induced cell senescence of primary human bronchial epithelial cells (HBEC), and further evaluate p62 and ubiquitinated protein levels in lung homogenates from COPD patients. We demonstrate that CSE transiently induces activation of autophagy in HBEC, followed by accelerated cell senescence and concomitant accumulation of p62 and ubiquitinated proteins. Autophagy inhibition further enhanced accumulations of p62 and ubiquitinated proteins, resulting in increased senescence and senescence-associated secretory phenotype (SASP) with interleukin (IL)-8 secretion. Conversely, autophagy activation by Torin1, a mammalian target of rapamycin (mTOR inhibitor), suppressed accumulations of p62 and ubiquitinated proteins and inhibits cell senescence. Despite increased baseline activity, autophagy induction in response to CSE was significantly decreased in HBEC from COPD patients. Increased accumulations of p62 and ubiquitinated proteins were detected in lung homogenates from COPD patients. Insufficient autophagic clearance of damaged proteins, including ubiquitinated proteins, is involved in accelerated cell senescence in COPD, suggesting a novel protective role for autophagy in the tobacco smoke-induced senescence-associated lung disease, COPD.

Mitochondria are dynamic organelles that continuously change their shape through fission and fusion. Disruption of mitochondrial dynamics is involved in disease pathology through excessive reactive oxygen species (ROS) production. Accelerated cellular senescence resulting from cigarette smoke exposure with excessive ROS production has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Hence, we investigated the involvement of mitochondrial dynamics and ROS production in terms of cigarette smoke extract (CSE)-induced cellular senescence in human bronchial epithelial cells (HBEC). Mitochondrial morphology was examined by electron microscopy and fluorescence microscopy. Senescence-associated β-galactosidase staining and p21 Western blotting of primary HBEC were performed to evaluate cellular senescence. Mitochondrial-specific superoxide production was measured by MitoSOX staining. Mitochondrial fragmentation was induced by knockdown of mitochondrial fusion proteins (OPA1 or Mitofusins) by small-interfering RNA transfection. N-acetylcysteine and Mito-TEMPO were used as antioxidants. Mitochondria in bronchial epithelial cells were prone to be more fragmented in COPD lung tissues. CSE induced mitochondrial fragmentation and mitochondrial ROS production, which were responsible for acceleration of cellular senescence in HBEC. Mitochondrial fragmentation induced by knockdown of fusion proteins also increased mitochondrial ROS production and percentages of senescent cells. HBEC senescence and mitochondria fragmentation in response to CSE treatment were inhibited in the presence of antioxidants. CSE-induced mitochondrial fragmentation is involved in cellular senescence through the mechanism of mitochondrial ROS production. Hence, disruption of mitochondrial dynamics may be a part of the pathogenic sequence of COPD development.

BackgroundAccumulation of profibrotic myofibroblasts in fibroblastic foci (FF) is a crucial process for development of fibrosis during idiopathic pulmonary fibrosis (IPF) pathogenesis, and transforming growth factor (TGF)-β plays a key regulatory role in myofibroblast differentiation. Reactive oxygen species (ROS) has been proposed to be involved in the mechanism for TGF-β-induced myofibroblast differentiation. Metformin is a biguanide antidiabetic medication and its pharmacological action is mediated through the activation of AMP-activated protein kinase (AMPK), which regulates not only energy homeostasis but also stress responses, including ROS. Therefore, we sought to investigate the inhibitory role of metformin in lung fibrosis development via modulating TGF-β signaling.MethodsTGF-β-induced myofibroblast differentiation in lung fibroblasts (LF) was used for in vitro models. The anti-fibrotic role of metfromin was examined in a bleomycin (BLM)-induced lung fibrosis model.ResultsWe found that TGF-β-induced myofibroblast differentiation was clearly inhibited by metformin treatment in LF. Metformin-mediated activation of AMPK was responsible for inhibiting TGF-β-induced NOX4 expression. NOX4 knockdown and N-acetylcysteine (NAC) treatment illustrated that NOX4-derived ROS generation was critical for TGF-β-induced SMAD phosphorylation and myofibroblast differentiation. BLM treatment induced development of lung fibrosis with concomitantly enhanced NOX4 expression and SMAD phosphorylation, which was efficiently inhibited by metformin. Increased NOX4 expression levels were also observed in FF of IPF lungs and LF isolated from IPF patients.ConclusionsThese findings suggest that metformin can be a promising anti-fibrotic modality of treatment for IPF affected by TGF-β.

Cigarette smoke (CS)–induced cellular senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease, and SIRT6, a histone deacetylase, antagonizes this senescence, presumably through the attenuation of insulin-like growth factor (IGF)-Akt signaling. Autophagy controls cellular senescence by eliminating damaged cellular components and is negatively regulated by IGF-Akt signaling through the mammalian target of rapamycin (mTOR). SIRT1, a representative sirtuin family, has been demonstrated to activate autophagy, but a role for SIRT6 in autophagy activation has not been shown. Therefore, we sought to investigate the regulatory role for SIRT6 in autophagy activation during CS-induced cellular senescence. SIRT6 expression levels were modulated by cDNA and small interfering RNA transfection in human bronchial epithelial cells (HBECs). Senescence-associated β-galactosidase staining and Western blotting of p21 were performed to evaluate senescence. We demonstrated that SIRT6 expression levels were decreased in lung homogenates from chronic obstructive pulmonary disease patients, and SIRT6 expression levels correlated significantly with the percentage of forced expiratory volume in 1 s/forced vital capacity. CS extract (CSE) suppressed SIRT6 expression in HBECs. CSE-induced HBEC senescence was inhibited by SIRT6 overexpression, whereas SIRT6 knockdown and mutant SIRT6 (H133Y) without histone deacetylase activity enhanced HBEC senescence. SIRT6 overexpression induced autophagy via attenuation of IGF-Akt-mTOR signaling. Conversely, SIRT6 knockdown and overexpression of a mutant SIRT6 (H133Y) inhibited autophagy. Autophagy inhibition by knockdown of ATG5 and LC3B attenuated the antisenescent effect of SIRT6 overexpression. These results suggest that SIRT6 is involved in CSE-induced HBEC senescence via autophagy regulation, which can be attributed to attenuation of IGF-Akt-mTOR signaling.

Fibroblastic foci, known to be the leading edge of fibrosis development in idiopathic pulmonary fibrosis (IPF), are composed of fibrogenic myofibroblasts. Autophagy has been implicated in the regulation of myofibroblast differentiation. Insufficient mitophagy, the mitochondria-selective autophagy, results in increased reactive oxygen species, which may modulate cell signaling pathways for myofibroblast differentiation. Therefore, we sought to investigate the regulatory role of mitophagy in myofibroblast differentiation as a part of IPF pathogenesis. Lung fibroblasts were used in in vitro experiments. Immunohistochemical evaluation in IPF lung tissues was performed. PARK2 was examined as a target molecule for mitophagy regulation, and a PARK2 knockout mouse was employed in a bleomycin-induced lung fibrosis model. We demonstrated that PARK2 knockdown-mediated mitophagy inhibition was involved in the mechanism for activation of the platelet-derived growth factor receptor (PDGFR)/PI3K/AKT signaling pathway accompanied by enhanced myofibroblast differentiation and proliferation, which were clearly inhibited by treatment with both antioxidants and AG1296, a PDGFR inhibitor. Mitophagy inhibition–mediated activation of PDGFR signaling was responsible for further autophagy suppression, suggesting the existence of a self-amplifying loop of mitophagy inhibition and PDGFR activation. IPF lung demonstrated reduced PARK2 with concomitantly increased PDGFR phosphorylation. Furthermore, bleomycin-induced lung fibrosis was enhanced in PARK2 knockout mice and subsequently inhibited by AG1296. These findings suggest that insufficient mitophagy-mediated PDGFR/PI3K/AKT activation, which is mainly attributed to reduced PARK2 expression, is a potent underlying mechanism for myofibroblast differentiation and proliferation in fibroblastic foci formation during IPF pathogenesis.