Autophagy, a process that helps maintain homeostatic balance between the synthesis, degradation, and recycling of organelles and proteins to meet metabolic demands, plays an important regulatory role in cellular senescence and differentiation. Here we examine the regulatory role of autophagy in idiopathic pulmonary fibrosis (IPF) pathogenesis. We test the hypothesis that epithelial cell senescence and myofibroblast differentiation are consequences of insufficient autophagy. Using biochemical evaluation of in vitro models, we find that autophagy inhibition is sufficient to induce acceleration of epithelial cell senescence and myofibroblast differentiation in lung fibroblasts. Immunohistochemical evaluation of human IPF biospecimens reveals that epithelial cells show increased cellular senescence, and both overlaying epithelial cells and fibroblasts in fibroblastic foci (FF) express both ubiquitinated proteins and p62. These findings suggest that insufficient autophagy is an underlying mechanism of both accelerated cellular senescence and myofibroblast differentiation in a celltype-specific manner and is a promising clue for understanding the pathogenesis of IPF. myofibroblast; p62; senescence

CUB domain-containing protein 1 (CDCP1) is a membrane protein that is highly expressed in several solid cancers. We reported previously that CDCP1 regulates anoikis resistance as well as cancer cell migration and invasion, although the underlying mechanisms have not been elucidated. In this study, we found that expression of CDCP1 in pancreatic cancer tissue was significantly correlated with overall survival and that CDCP1 expression in pancreatic cancer cell lines was relatively high among solid tumor cell lines. Reduction of CDCP1 expression in these cells suppressed extracellular matrix (ECM) degradation by inhibiting matrix metalloproteinase-9 secretion. Using the Y734F mutant of CDCP1, which lacks the tyrosine phosphorylation site, we showed that CDCP1 regulates cell migration, invasion, and ECM degradation in a tyrosine phosphorylation-dependent manner and that these CDCP1-associated characteristics were inhibited by blocking the association of CDCP1 and protein kinase Cdelta (PKCdelta). CDCP1 modulates the enzymatic activity of PKCdelta through the tyrosine phosphorylation of PKCdelta by recruiting PKCdelta to Src family kinases. Cortactin, which was detected as a CDCP1-dependent binding partner of PKCdelta, played a significant role in migration and invasion but not in ECM degradation of pancreatic cells. These results suggest that CDCP1 expression might play a crucial role in poor outcome of pancreatic cancer through promotion of invasion and metastasis and that molecules blocking the expression, phosphorylation, or the PKCdelta-binding site of CDCP1 are potential therapeutic candidates.

(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.

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.

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.

Involvement of Ras in cancer initiation is known, but recent evidence indicates a role in cancer progression, including metastasis and invasion; however, the mechanism is still unknown. In this study, it was determined that human lung cancer cells with Ras mutations, among other popular mutations, showed significantly higher expression of CUB domain-containing protein 1 (CDCP1) than those without. Furthermore, activated Ras clearly induced CDCP1, whereas CDCP1 knockdown or inhibition of CDCP1 phosphorylation by Src-directed therapy abrogated anoikis resistance, migration, and invasion induced by activated-Ras. Activation of MMP2 and secretion of MMP9, in a model of Ras-induced invasion, was found to be regulated through induction of phosphorylated CDCP1. Thus, CDCP1 is required for the functional link between Ras and Src signaling during the multistage development of human malignant tumors, highlighting CDCP1 as a potent target for treatment in the broad spectrum of human cancers associated with these oncogenes.

Cigarette smoke induces damage to proteins and organelles by oxidative stress, resulting in accelerated epithelial cell senescence in the lung, which is implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Although the detailed molecular mechanisms are not fully understood, cellular energy status is one of the most crucial determinants for cell senescence. Creatine kinase (CK) is a constitutive enzyme, playing regulatory roles in energy homeostasis of cells. Among two isozymes, brain-type CK (CKB) is the predominant CK in lung tissue. In this study, we investigated the role of CKB in cigarette smoke extract (CSE)-induced cellular senescence in human bronchial epithelial cells (HBECs). Primary HBECs and Beas2B cells were used. Protein carbonylation was evaluated as a marker of oxidative protein damage. Cellular senescence was evaluated by senescence-associated β-galactosidase staining. CKB inhibition was examined by small interfering RNA and cyclocreatine. Secretion of IL-8, a hallmark of senescence-associated secretary phenotype, was measured by ELISA. CKB expression levels were reduced in HBECs from patients with COPD compared with that of HBECs from nonsmokers. CSE induced carbonylation of CKB and subsequently decreased CKB protein levels, which was reversed by a proteasome inhibitor. CKB inhibition alone induced cell senescence, and further enhanced CSE-induced cell senescence and IL-8 secretion. CSE-induced oxidation of CKB is a trigger for proteasomal degradation. Concomitant loss of enzymatic activity regulating energy homeostasis may lead to the acceleration of bronchial epithelial cell senescence, which is implicated in the pathogenesis of COPD.

BackgroundPostoperative pulmonary complications (PPC) in patients with pulmonary diseases remain to be resolved clinical issue. However, most evidence regarding PPC has been established more than 10 years ago. Therefore, it is necessary to evaluate perioperative management using new inhalant drugs in patients with obstructive pulmonary diseases.MethodsApril 2014 through March 2015, 346 adult patients with pulmonary diseases (257 asthma, 89 chronic obstructive pulmonary disease (COPD)) underwent non-pulmonary surgery except cataract surgery in our university hospital. To analyze the risk factors for PPC, we retrospectively evaluated physiological backgrounds, surgical factors and perioperative specific treatment for asthma and COPD.ResultsFinally, 29 patients with pulmonary diseases (22 asthma, 7 COPD) had PPC. In patients with asthma, smoking index (≥ 20 pack-years), peripheral blood eosinophil count (≥ 200/mm3) and severity (Global INitiative for Asthma(GINA) STEP ≥ 3) were significantly associated with PPC in the multivariate logistic regression analysis [odds ratio (95% confidence interval) = 5.4(1.4–20.8), 0.31 (0.11–0.84) and 3.2 (1.04–9.9), respectively]. In patients with COPD, age, introducing treatment for COPD, upper abdominal surgery and operation time (≥ 5 h) were significantly associated with PPC [1.18 (1.00–1.40), 0.09 (0.01–0.81), 21.2 (1.3–349) and 9.5 (1.2–77.4), respectively].ConclusionsHistory of smoking or severe asthma is a risk factor of PPC in patients with asthma, and age, upper abdominal surgery, or long operation time is a risk factor of PPC in patients with COPD. Adequate inhaled corticosteroids treatment in patients with eosinophilic asthma and introducing treatment for COPD in patients with COPD could reduce PPCs.