Carbon Monoxide Activates Autophagy via Mitochondrial Reactive Oxygen Species Formation

Abstract

Autophagy, an autodigestive process that degrades cellular organelles and protein, plays an important role in maintaining cellular homeostasis during environmental stress. Carbon monoxide (CO), a toxic gas and candidate therapeutic molecule, confers cytoprotection in animal models of acute lung injury. The mechanisms underlying CO-dependent lung cell protection and the role of autophagy in this process remain unclear. Here, we demonstrate that CO exposure time-dependently increased the expression and activation of the autophagic protein, microtubule-associated protein-1 light chain-3B (LC3B) in mouse lung, and in cultured human alveolar (A549) or human bronchial epithelial cells. Furthermore, CO increased autophagosome formation in epithelial cells by electron microscopy and green fluorescent protein (GFP)-LC3 puncta assays. Recent studies indicate that reactive oxygen species (ROS) play an important role in the activation of autophagy. CO up-regulated mitochondria-dependent generation of ROS in epithelial cells, as assayed by MitoSOX fluorescence. Furthermore, CO-dependent induction of LC3B expression was inhibited by N-acetyl-L-cysteine and the mitochondria-targeting antioxidant, Mito-TEMPO. These data suggest that CO promotes the autophagic process through mitochondrial ROS generation. We investigated the relationships between autophagic proteins and CO-dependent cytoprotection using a model of hyperoxic stress. CO protected against hyperoxia-induced cell death, and inhibited hyperoxia-associated ROS production. The ability of CO to protect against hyperoxia-induced cell death and caspase-3 activation was compromised in epithelial cells infected with LC3B-small interfering (si)RNA, indicating a role for autophagic proteins. These studies uncover a new mechanism for the protective action of CO, in support of potential therapeutic application of this gas.Keywords: apoptosis; autophagy; carbon monoxide; epithelial cells; hyperoxia Carbon monoxide (CO), a low-molecular weight diatomic gas, can induce clinical toxicity and even death at high ambient concentrations (1). The adverse effects of CO in humans are associated with hypoxemia as the result of competitive binding of CO with the heme iron centers of the oxygen carrier protein hemoglobin (2, 3). The cellular toxicity of CO may involve complex formation and inhibition of respiratory chain enzymes (i.e., cytochrome c: oxidase) (4, 5). In contrast to these toxic mechanisms, recent studies over the last decade have revealed cyto-and tissue-protective effects of CO when applied at low ambient concentrations in several models of lung or vascular injury, including acute lung injury (i.e., endotoxin shock, hyperoxia, mechanical ventilation), and ischemia-reperfusion injury (6-12). The protective effects of CO in these models depend on anti-inflammatory, antiapoptotic, and/or antiproliferative activities of this gas (13). Several signaling pathways have been implicated in CO-dependent cytoprotection, including the p38 mitogen activated protein kinase (MAPK) and NF-kB ...

The Emerging Importance of Autophagy in Pulmonary Diseases

“…10 CO increased the expression of LC3B in mouse lung and in cultured human alveolar or human bronchial epithelial cells as well as increased autopha gosome formation in epithelial cells as seen by electron microscopy and green fluorescent protein-LC3 puncta assays. This increase in autophagy activity was inhibited by N-acetyl-l -cysteine and the mitochondriatargeting antioxidant Mito-TEMPO, suggesting that CO promotes the autophagic process through mitochondrial reactive oxygen species (ROS) generation.…”

“…This increase in autophagy activity was inhibited by N-acetyl-l -cysteine and the mitochondriatargeting antioxidant Mito-TEMPO, suggesting that CO promotes the autophagic process through mitochondrial reactive oxygen species (ROS) generation. 10 The protection afforded by CO against hyperoxiainduced cell death was also compromised on treatment with siRNA against LC3B. These fi ndings suggest that LC3B plays an important role in the cytoprotection afforded by CO through the production of mitochondrial ROS.…”

Autophagy Promotes Intracellular Degradation of Type I Collagen Induced by Transforming Growth Factor (TGF)-β1

“…CO is an endogenous product of heme oxygenase activity that has been shown to exert potent cytoprotection against tissue injury including kidney fibrosis (34), and recently CO has been shown to induce autophagy (48). Lowdose CO (250 ppm) increased the expression and activation of the autophagic protein LC3 in the mouse lung, and in cultured human alveolar (A549) or bronchial epithelial cells in a timedependent manner (48). Our present study also demonstrated that CO induced Beclin 1 and LC3 expression in the mouse kidneys.…”

Inhibition of Regulatory-Associated Protein of Mechanistic Target of Rapamycin Prevents Hyperoxia-Induced Lung Injury by Enhancing Autophagy and Reducing Apoptosis in Neonatal Mice

“…Autophagy has recently been implicated in the regulation of hyperoxia-induced epithelial cell death (13). The hallmark of autophagy lies in the conversion of LC3-I activated by ATG7, transferred to ATG3, and conjugated to phosphatidylethanolamine at the outer and inner autophagosomal membrane to form LC3-II (13).…”

“…The hallmark of autophagy lies in the conversion of LC3-I activated by ATG7, transferred to ATG3, and conjugated to phosphatidylethanolamine at the outer and inner autophagosomal membrane to form LC3-II (13). Although LC3-II increase is considered a standard marker of autophagy activation, it is important to assess the multiple components of the signaling pathway and the autophagic flux (14).…”