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.

Figures

Figure 1.

Autophagic markers are increased in lung tissues of carbon monoxide (CO)–exposed C57BL/6 mice. C57BL/6 mice were exposed to 250 ppm CO for 0 (control), 24, and 72 hours. Lung tissues were harvested at the end of each experiment. (A) Western blot analysis of microtubule-associated protein–1 light chain 3B (LC3B)–I/–II expression in lung tissue from C57BL/6 mice. Total LC3B expression was normalized to β-actin. (B) The quantification of LC3B protein levels from each group (n = 6) was measured using ImageJ Software. β-actin was used as the standard. Data represent mean (±SD). *P < 0.05 compared with room air.

Figure 2.

CO increases autophagy in cultured human epithelial cells in vitro. (A) A549 human alveolar epithelial cells were exposed to 250 ppm CO for 0–24 hours and evaluated for time-dependent expression of LC3B-I and -II by Western blot analysis; β-actin served as the standard. (B) A549 transfected with green fluorescent protein (GFP)-LC3 were exposed to 250 ppm CO or room air for 24 hours. Cells were visualized by confocal laser scanning microscopy. The percentage of cells exhibiting punctuated GFP-LC3 fluorescence was calculated relative to all GFP-positive cells. Data are presented as average (±SE) of three independent experiments. *P < 0.05, #P < 0.01. (C–E) Human bronchial epithelial (HBE) cells were exposed to (C) CO alone (250 ppm) for 0–24 hours, (D) hyperoxia alone (>95% O2) for 0–24 hours, or (E) combination treatments (CO and/or hyperoxia) for 0–72 hours and evaluated for time-dependent expression of LC3B-I and -II by Western blot analysis; β-actin served as the standard.

Figure 3.

CO increases autophagosome formation in human epithelial cells in vitro. (A) Electron microscopic (EM) analysis of A549 cells after 24 hours of 250 ppm CO exposure. We indicate the presence of immature autophagic vacuoles (AVis) or degradative AVs (AVds). (B) Quantification of autophagosomes (AVi + AVd) per cell in A549 cells exposed to CO or room air. Data represent mean (±SD) (n = 15 EM images per condition); #P < 0.01.

Figure 4.

CO modulates mitochondrial reactive oxygen species (ROS) generation. A549 cells (A and B) or HBE cells (C–H) were exposed to 250 ppm CO or room air (Control), in the absence or presence of antioxidants, N-acetyl-L-cysteine (NAC) or Mito-TEMPO (D), or in the presence of high oxygen tension (>95% O2) (E) (stained with MitoSOX Red and analyzed by flow cytometry). Rotenone was used as a positive control for mitochondrial ROS production (C and F). Cells left unstained served as an additional negative control (NC). Representative histograms of flow cytometry experiments demonstrate increase in fluorescent intensity of MitoSOX Red (5 μM) after CO treatment for 2 hours (A and C), which is reversible by antioxidant treatment (D). Hyperoxia increases MitoSOX Red signal, which is diminished in the presence of 250 ppm CO (E). Quantification data (B and F–H) are presented as average (±SE) of three independent experiments; *P < 0.05.

Figure 5.

CO-induced LC3B is dependent on ROS production in A549 cells. (A) A549 cells were pretreated with 5 mM NAC for 30 minutes followed by CO treatment (250 ppm) for 24 hours. LC3B protein levels were determined by Western blotting. (B) A549 cells were treated with Mito-TEMPO (100 μM) for 24 hours in the presence of CO (250 ppm) or room air. (C) HBE cells were pretreated with 5 mM NAC for 30 minutes followed by CO treatment (250 ppm) for 24 hours, or were treated with Mito-TEMPO (100 μM) for 24 hours in the presence of 250 ppm CO or room air; β-actin was used as the standard (A–C). (D) HBE cells were treated with 5 mM NAC or Mito-TEMPO (100 μM) for 24 or 48 hours. Lactate dehydrogenase (LDH) levels were detected in cell culture supernatants by colorimetric assay (see Materials and Methods). Data are presented as average (±SE) of three independent experiments. *P < 0.05, #P < 0.01 (A–B and D).

Figure 6.

CO protection against hyperoxia-induced cell death in human epithelial cells requires autophagic protein, LC3B. A549 cells (A and B) or HBE cells (C and D) were transfected with small interfering (si)RNA of LC3B for 12 hours and recovered in fresh medium containing 10% FBS. After 24 hours, cells were cultured in serum-containing medium and further incubated with hyperoxia (95% O2) in the presence (250 ppm) or absence of CO. (A and C) After 72 hours, cell viability was measured by the crystal violet staining method. (B and D) After 72 hours, the protein levels of cleaved caspase-3, LC3B, and β-actin were determined by Western blot analysis. Data shown in (A) represent the mean (±SD) (n ≥ 3); *P < 0.05 versus hyperoxia alone.