Kenji Mizumura

Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

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.

The Impact of Autophagy on Cell Death Modalities

Autophagy represents a homeostatic cellular mechanism for the turnover of organelles and proteins, through a lysosome-dependent degradation pathway. During starvation, autophagy facilitates cell survival through the recycling of metabolic precursors. Additionally, autophagy can modulate other vital processes such as programmed cell death (e.g., apoptosis), inflammation, and adaptive immune mechanisms and thereby influence disease pathogenesis. Selective pathways can target distinct cargoes (e.g., mitochondria and proteins) for autophagic degradation. At present, the causal relationship between autophagy and various forms of regulated or nonregulated cell death remains unclear. Autophagy can occur in association with necrosis-like cell death triggered by caspase inhibition. Autophagy and apoptosis have been shown to be coincident or antagonistic, depending on experimental context, and share cross-talk between signal transduction elements. Autophagy may modulate the outcome of other regulated forms of cell death such as necroptosis. Recent advances suggest that autophagy can dampen inflammatory responses, including inflammasome-dependent caspase-1 activation and maturation of proinflammatory cytokines. Autophagy may also act as regulator of caspase-1 dependent cell death (pyroptosis). Strategies aimed at modulating autophagy may lead to therapeutic interventions for diseases in which apoptosis or other forms of regulated cell death may play a cardinal role.

Histone deacetylase 6–mediated selective autophagy regulates COPD-associated cilia dysfunction

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) that are associated with epithelial cell dysfunction, cilia shortening, and mucociliary clearance disruption. Exposure to CS reduced cilia length and induced autophagy in vivo and in differentiated mouse tracheal epithelial cells (MTECs). Autophagy-impaired (Becn1+/- or Map1lc3B-/-) mice and MTECs resisted CS-induced cilia shortening. Furthermore, CS increased the autophagic turnover of ciliary proteins, indicating that autophagy may regulate cilia homeostasis. We identified cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure. Mice bearing an X chromosome deletion of Hdac6 (Hdac6-/Y) and MTECs from these mice had reduced autophagy and were protected from CS-induced cilia shortening. Autophagy-impaired Becn1-/-, Map1lc3B-/-, and Hdac6-/Y mice or mice injected with an HDAC6 inhibitor were protected from CS-induced mucociliary clearance (MCC) disruption. MCC was preserved in mice given the chemical chaperone 4-phenylbutyric acid, but was disrupted in mice lacking the transcription factor NRF2, suggesting that oxidative stress and altered proteostasis contribute to the disruption of MCC. Analysis of human COPD specimens revealed epigenetic deregulation of HDAC6 by hypomethylation and increased protein expression in the airways. We conclude that an autophagy-dependent pathway regulates cilia length during CS exposure and has potential as a therapeutic target for COPD.

ASK1 regulates influenza virus infection-induced apoptotic cell death.

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

Background Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined.

Mitochondrial iron chelation ameliorates cigarette smoke–induced bronchitis and emphysema in mice

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element–binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

Emerging role of selective autophagy in human diseases

Autophagy was originally described as a highly conserved system for the degradation of cytosol through a lysosome-dependent pathway. In response to starvation, autophagy degrades organelles and proteins to provide metabolites and energy for its pro-survival effects. Autophagy is recognized as playing a role in the pathogenesis of disease either directly or indirectly, through the regulation of vital processes such as programmed cell death, inflammation, and adaptive immune mechanisms. Recent studies have demonstrated that autophagy is not only a simple metabolite recycling system, but also has the ability to degrade specific cellular targets, such as mitochondria, cilia, and invading bacteria. In addition, selective autophagy has also been implicated in vesicle trafficking pathways, with potential roles in secretion and other intracellular transport processes. Selective autophagy has drawn the attention of researchers because of its potential importance in clinical diseases. Therapeutic strategies to target selective autophagy rather than general autophagy may maximize clinical benefit by enhancing selectivity. In this review, we outline the principle components of selective autophagy processes and their emerging importance in human disease, with an emphasis on pulmonary diseases.

Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells

ObjectiveReactive oxygen species initiate pulmonary vascular endothelial cell damage leading to an increase in endothelial permeability resulting in the production of pulmonary edema. Apoptosis signal-regulating kinase (ASK)-1 is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the MKK3/MKK6-p38 MAPK and the SEK1-c-Jun N-terminal kinase (JNK) signaling cascade. ASK1 has been implicated in cytokine- and stress-induced apoptosis. However, little is known about the role of ASK1 in apoptosis in hydrogen peroxide (H2O2)-stimulated pulmonary vascular endothelial cells and how ASK1-mediated apoptosis is executed. To clarify this issue, we examined the role of ASK1-p38 MAPK/JNK cascade in apoptosis and caspase-3 activation in H2O2-stimulated pulmonary vascular endothelial cells. DesignExperimental laboratory study. SettingUniversity laboratory. SubjectsNormal human pulmonary artery endothelial cells. InterventionsWestern blot analysis and quantification of apoptosis in cells. Measurements and Main ResultsThe results showed that H2O2 induced ASK1 phosphorylation and concomitantly p38 MAPK and JNK phosphorylation as well as induced caspase-3 activation in pulmonary vascular endothelial cells. To further characterize the role of ASK1 cascade in H2O2-induced apoptosis of pulmonary vascular endothelial cells, the dominant negative form of ASK1-stably transfected porcine artery endothelial cells was used. p38 MAPK and JNK phosphorylation, caspase-3 activation, and apoptosis in the dominant negative form of ASK1-stably transfected porcine artery endothelial cells were depressed compared with those in the parental porcine artery endothelial cells. ConclusionASK1-p38 MAPK/JNK cascade regulates apoptosis of H2O2-stimulated human pulmonary vascular endothelial cells.

Heme Oxygenase-1 Promotes Survival of Renal Cancer Cells through Modulation of Apoptosis- and Autophagy-regulating Molecules

Background: The cytoprotective enzyme HO-1 promotes tumor growth. Results: HO-1 down-regulated apoptosis- and autophagy-regulating proteins, and attenuated Rapamycin- and Sorafenib-induced apoptosis and autophagy in renal cancer cells. Conclusion: HO-1 protects cancer cells from chemotherapeutic drug-induced death by down-regulating apoptosis and autophagy. Significance: Inhibition of HO-1 augments efficiency of chemotherapeutic agents to kill cancer cells by promoting both apoptosis and autophagy. The cytoprotective enzyme heme oxygenase-1 (HO-1) is often overexpressed in different types of cancers and promotes cancer progression. We have recently shown that the Ras-Raf-ERK pathway induces HO-1 to promote survival of renal cancer cells. Here, we examined the possible mechanisms underlying HO-1-mediated cell survival. Considering the growing evidence about the significance of apoptosis and autophagy in cancer, we tried to investigate how HO-1 controls these events to regulate survival of cancer cells. Rapamycin (RAPA) and sorafenib, two commonly used drugs for renal cancer treatment, were found to induce HO-1 expression in renal cancer cells Caki-1 and 786-O; and the apoptotic effect of these drugs was markedly enhanced upon HO-1 knockdown. Overexpression of HO-1 protected the cells from RAPA- and sorafenib-induced apoptosis and also averted drug-mediated inhibition of cell proliferation. HO-1 induced the expression of anti-apoptotic Bcl-xL and decreased the expression of autophagic proteins Beclin-1 and LC3B-II; while knockdown of HO-1 down-regulated Bcl-xL and markedly increased LC3B-II. Moreover, HO-1 promoted the association of Beclin-1 with Bcl-xL and Rubicon, a novel negative regulator of autophagy. Drug-induced dissociation of Beclin-1 from Rubicon and the induction of autophagy were also inhibited by HO-1. Together, our data signify that HO-1 is up-regulated in renal cancer cells as a survival strategy against chemotherapeutic drugs and promotes growth of tumor cells by inhibiting both apoptosis and autophagy. Thus, application of chemotherapeutic drugs along with HO-1 inhibitor may elevate therapeutic efficiency by reducing the cytoprotective effects of HO-1 and by simultaneous induction of both apoptosis and autophagy.

Autophagy: A Crucial Moderator of Redox Balance, Inflammation, and Apoptosis in Lung Disease

SIGNIFICANCE Autophagy is a fundamental cellular process that functions in the turnover of subcellular organelles and protein. Activation of autophagy may represent a cellular defense against oxidative stress, or related conditions that cause accumulation of damaged proteins or organelles. Selective forms of autophagy can maintain organelle populations or remove aggregated proteins. Autophagy can increase survival during nutrient deficiency and play a multifunctional role in host defense, by promoting pathogen clearance and modulating innate and adaptive immune responses. RECENT ADVANCES Autophagy has been described as an inducible response to oxidative stress. Once believed to represent a random process, recent studies have defined selective mechanisms for cargo assimilation into autophagosomes. Such mechanisms may provide for protein aggregate detoxification and mitochondrial homeostasis during oxidative stress. Although long studied as a cellular phenomenon, recent advances implicate autophagy as a component of human diseases. Altered autophagy phenotypes have been observed in various human diseases, including lung diseases such as chronic obstructive lung disease, cystic fibrosis, pulmonary hypertension, and idiopathic pulmonary fibrosis. CRITICAL ISSUES Although autophagy can represent a pro-survival process, in particular, during nutrient starvation, its role in disease pathogenesis may be multifunctional and complex. The relationship of autophagy to programmed cell death pathways is incompletely defined and varies with model system. FUTURE DIRECTIONS Activation or inhibition of autophagy may be used to alter the progression of human diseases. Further resolution of the mechanisms by which autophagy impacts the initiation and progression of diseases may lead to the development of therapeutics specifically targeting this pathway.