The process of macroautophagy can extend cell survival under conditions of starvation or nutrient depletion by recycling metabolic precursors from damaged organelles or denatured proteins (21
). Furthermore, autophagy is now known to play regulatory roles in innate and adaptive immunity, and may directly assist in the clearance of pathogens (33
). The function of autophagy in stress is not always clear, and has been linked to both pro- and antiapoptotic mechanisms. For example, in cigarette smoke exposure, autophagy may promote, rather than inhibit, the progression of apoptosis (27
). In this study, we show for the first time, that autophagy, a cellular homeostatic program, can be regulated by CO exposure in epithelial cell culture. We show that CO induces biochemical and morphological markers of autophagy, including the enhanced expression of autophagic proteins, and the formation of autophagosomes. Given that CO can provide pleiotropic cyto- and tissue-protective effects at low concentration, these observations add the modulation of autophagy and/or autophagic proteins as additional candidate mechanism(s) by which CO can protect cellular function and preserve homeostasis under stress.
A role for autophagic regulator proteins in CO-dependent cytoprotection is indicated by studies showing that genetic interference of LC3B abolishes the cytoprotective effects of CO in hyperoxic lung cell death in epithelial cells. LC3B is a major regulator of autophagosome formation, and is the sole atg
protein retained by mature autophagosomes. We cannot exclude the possibility that nonautophagic functions of LC3B may contribute to the apparent cytoprotection, including possible effects on mRNA translation, microtubule organization, or interaction with signaling mechanisms (34
). Recently, we have shown that LC3B can modulate the apoptotic program in epithelial cells challenged with cigarette smoke extract (27
The mechanism(s) by which CO can modulate the autophagic program remain incompletely clear. The current study is the first to show that autophagy can be modulated in epithelial cells by CO, through an ROS-dependent signaling component. Several other recent studies have implicated intracellular ROS and/or redox signaling mechanisms in the regulation of the autophagy. For example, starvation, a well known activator of autophagy, was associated with increased intracellular ROS production in CHO and HeLa cells (36
). Treatment of CHO cells with antioxidant (i.e., NAC) or catalase reduced starvation–induced autophagosome formation. The authors identified autophagic protein, Atg4, as a potential target for ROS, and proposed a redox regulation mechanism for the activity of this protein (36
Currently, intracellular ROS are regarded not only as a potential source of toxicity under pathophysiological states, but also as integral components of signaling pathways at physiological levels. In hyperoxia, the excess production of ROS leads not only to the activation of autophagic markers, as shown in this study, but also to epithelial cell killing. In contrast, nontoxic levels of CO (250 ppm) generate sufficient levels of ROS, as shown by MitoSOX staining, to activate autophagic process without cell killing. Consistently, antioxidant compounds can both reduce overt toxicity by hyperoxia, as shown by LDH assays, as well as inhibit LC3B expression induced by exposure to CO alone.
Intracellular ROS generation can occur at several sites, including reduced NAD phosphate (NADPH) oxidase, xanthine oxidase, and mitochondrial respiratory chain complexes I and III (37
). CO exposure has previously been reported to modulate intracellular ROS generation (18
) at membrane and/or mitochondrial sources. The down-regulation of plasma membrane–dependent ROS production by CO has been implicated in antiapoptotic (18
), antiproliferative (29
), and anti-inflammatory effects (28
) of CO through inhibition of NADPH oxidase activity and consequent effects on cell signaling. For example, down-regulation of NADPH oxidase–dependent ROS production by CO inhibited Toll-like receptor–dependent trafficking to the lipid raft after LPS stimulation (28
). Furthermore, the up-regulation of mitochondrial ROS by CO, through deregulated electron flow through the respiratory chain, has also been implicated in antiproliferative (29
) and anti-inflammatory (30
) effects of CO. For example, up-regulation of peroxisome proliferator–activated receptor–γ by CO-dependent ROS production led to down-regulation of Egr-1 and downstream inflammatory responses in macrophages (30
In this study, we show that scavenging of mitochondrial ROS with Mito-TEMPO, a triphenylphosphonium-conjugated antioxidant that localizes to the mitochondria (38
), inhibits the activation of LC3B by CO, indicating a role for mitochondrial-derived ROS in the activation of autophagy by CO. Recent literature has identified the preferential mitochondrial localization and ROS scavenging effect of Mito-TEMPO (38
). Although mitochondria-derived ROS are thought to arise primarily from respiratory activity, we cannot completely exclude the potential contribution of ROS from other cytosolic sources, such as nonphagocytic NADPH oxidase isoforms, in the signaling effects of CO.
Although both hyperoxia and CO alone can increase cellular ROS, the simultaneous application of CO results in a net protective effect, associated with the stabilization of LC3B. Interestingly, the stimulatory effects of hyperoxia treatment as a known activator of mitochondrial ROS production were diminished by the simultaneous inclusion of CO, suggesting that CO antagonizes ROS production under high oxygen tension. Thus, although both treatments appear to stimulate LC3B conversion, hyperoxia leads to a faster degradation of LC3B, probably due to a higher degree of oxidative stress than that induced by CO alone.
Recent studies also implicate ROS-dependent signaling in the stimulation of mitochondrial biogenesis by CO through redox-dependent activation of nuclear respiratory factor 1, which, together with nuclear respiratory factor 2 and peroxisome proliferator–activated receptor γ coactivator–1α, stimulate the transcription of mitochondrial transcription factor A, a key regulator of mitochondrial DNA replication (40
). These studies, taken together with our identification of autophagy as an inducible response to CO exposure, indicate that CO can potentially accelerate the turnover of mitochondria by stimulating both mitochondrial degradation and biosynthesis pathways. However, we cannot exclude the possibility that the role of LC3B in CO-dependent cytoprotection involves signaling effects of LC3B independent of autophagic process.
These studies lend further support to the potential therapeutic application of CO, which has been demonstrated in numerous in vitro
and in vivo
models of acute lung injury (6
). Although the clinical application of CO in humans for diseases of the lung remains unrealized, the feasibility of low-dose CO application in humans and nonhuman primates has now been tested (42
). A recent study shows potential therapeutic effects of CO in human subjects with chronic obstructive pulmonary disease (44
). The further examination of therapeutic efficacy of CO in human acute lung injury/acute respiratory distress syndrome awaits approved clinical trials.