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Originally published In Press as doi:10.1074/jbc.M607610200 on November 29, 2006

J. Biol. Chem., Vol. 282, Issue 3, 1718-1726, January 19, 2007
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Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation*

Xue Wang, Yong Wang, Hong Pyo Kim, Kiichi Nakahira, Stefan W. Ryter, and Augustine M. K. Choi1

From the Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213

Received for publication, August 9, 2006 , and in revised form, November 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47phox subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved in both intrinsic and extrinsic apoptotic signaling. Carbon monoxide inhibited the activation of Bid and the expression and mitochondrial translocation of Bax, whereas promoted Bcl-XL/Bax interaction and increased Bad phosphorylation. We also show that carbon monoxide promoted an interaction of heme oxygenase-1 with Bax. These results define novel mechanisms underlying the antiapoptotic effects of carbon monoxide during hyperoxic stress.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The clinical treatment of respiratory failure often requires supplemental oxygen therapy. Prolonged exposure to an elevated oxygen tension (hyperoxia) in animal models causes acute and chronic lung injury that resembles acute respiratory distress syndrome. In rodent models, hyperoxia triggers an extensive inflammatory response in the lung that degrades the alveolar-capillary barrier, leading to impaired gas exchange and pulmonary edema (1, 2). Lung tissue damage results from the direct action of increased intracellular reactive oxygen species (ROS)2 or as a secondary consequence of inflammatory responses of the host (3, 4). The source(s) of intracellular ROS during oxygen exposure remain unclear but may involve increased mitochondrial generation and/or activation of NADPH oxidases (5, 6). The pathological changes in hyperoxia-injured lungs coincide with the injury or death of pulmonary capillary endothelial cells and alveolar epithelial cells (2, 4–7). Epithelial cells maintain the integrity of the alveolar-capillary barrier and defend against oxidative injury. Compromised epithelial cell function may permit fluid and macromolecules to leak into the airspace, resulting in clinical respiratory failure and death (3, 8, 9).

The mechanisms underlying hyperoxic lung injury and cell death vary in a tissue-specific manner and can involve apoptosis, necrosis, or mixed cell death phenotypes. Apoptosis signaling pathways play an important role in hyperoxia-induced lung cell death, regardless of whether the final phenotypic outcome resembles apoptosis or necrosis (10).

Two apoptotic pathways have been identified by which cells can initiate and execute the cell death process: an intrinsic (mitochondria-dependent) pathway and an extrinsic (receptor-dependent) pathway. Mitochondria play key roles in directing the fate of cells by maintaining the cellular levels of ATP and by releasing apoptogenic factors upon cellular stimulation with pro-apoptotic signals. Members of the Bcl-2 family of proteins act as critical regulators of the intrinsic apoptotic pathway. Antiapoptotic members of this family, such as Bcl-XL, localize to the outer membrane of the mitochondria, whereas pro-apoptotic Bcl-2 family members such as Bax and Bid translocate to the mitochondria upon cellular stimulation with diverse agents. Bax forms channels upon assimilating into the mitochondria, thus increasing outer mitochondrial membrane permeability and thereby facilitating the release of cytochrome c and other proapoptotic molecules from the mitochondrial intermembrane space (11–13). Alternatively, hyperoxia may directly cause irreversible mitochondrial damage, also leading to increases in mitochondrial outer membrane permeability (14). Once released to the cytosol, cytochrome c forms an apoptosome complex with Apaf-1, which activates caspase-9 and, in turn, its downstream caspase-3, resulting in the morphological features of apoptosis (15). On the other hand, the extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand (FasL), interacts with its cell surface receptor (i.e. Fas), forming a death-inducing signal complex (DISC) (16). Activation of Fas triggers its oligomerization and the rapid recruitment of FADD (Fas-associated death domain protein) and caspase-8. Activated caspase-8 subsequently cleaves Bid into truncated Bid (tBid), which translocates from the cytosol to the mitochondrial membrane, where it assists in Bax activation (16).

CO can provide protection against hyperoxic injury in mice (17) and rats (18), but the underlying mechanisms remain incompletely understood. CO occurs in nature as a product of the combustion of organic materials. CO also arises endogenously in cells and tissues as a product of heme oxygenase (HO) activity, which degrades heme to biliverdin-IX{alpha} and ferrous iron (19). HO-1, the inducible form of HO, responds to transcriptional up-regulation by multiple forms of cellular stress. HO-1 confers cytoprotection against oxidative stress in vitro and in vivo (20). HO-derived CO acts as a vasorelaxant and inhibits other vascular functions such as platelet aggregation and smooth muscle proliferation (21). When applied at low concentration, CO exerts potent cytoprotective effects mimicking those of HO-1 induction, which include anti-inflammatory effects in macrophages (22, 23), as well as anti-apoptotic effects in vascular cells (24, 25). HO-1 and/or CO provide tissue protection in a number of in vivo models, including vascular injury and ischemia/reperfusion injury (26, 27). The mechanisms of CO action potentially involve modulation of intracellular signaling pathways, including activation of p38 mitogen-activated protein kinase and soluble guanylyl cyclase (22, 27–29).

In the current study, we demonstrate that CO prevented hyperoxia-induced apoptosis by inhibition of both extrinsic and intrinsic apoptotic pathways by blocking Bax and Bid activation and cytochrome c release from the mitochondria. Additionally, CO inhibited plasma membrane DISC formation by attenuating ROS production and preventing DISC trafficking from the Golgi apparatus. We describe for the first time the occurrence of an interaction between HO-1 with Bax that is enhanced by CO treatment and that suggests a novel antiapoptotic mechanism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—Antibodies anti-Bax, anti-Bid, anti-caspase-8, anti-caspase-9, anti-cytochrome c, anti-Fas, and protein A-agarose were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Bax 6A7 antibody was purchased from BD PharMingen (San Diego, CA). Anti-p110 was from Calbiochem (San Diego, CA). Anti-heme oxygenase-1 was from Stressgen (Vancouver, Canada). The lactate dehydrogenase (LDH) assay kit was from Roche Applied Science. Apocyanin and diphenyleneiodoniun were from Calbiochem. Digitonin and all other chemicals were from Sigma.

Animals—The mice were acclimated for 1 week with rodent chow and water ad libitum. The animals were housed according to guidelines from the American Association for Laboratory Animal Care and Research Protocols and were approved by the Animal Care and Use Committee (University of Pittsburgh School of Medicine). C57BL/6 wild type mice and p47phox–/– mice (C57BL/6 background) were purchased from Jackson Laboratory and used for preparation of endothelial cells as described below.

Isolation and Culture of Murine Lung Endothelial Cells—The isolation of mouse lung endothelial cells (MLEC) by an immunobead protocol has been reported elsewhere (30). Briefly, mouse lungs were digested in collagenase and filtered through 100-µm cell strainers, centrifuged, and washed twice with medium. Cell suspensions were incubated with a monoclonal antibody (rat anti-mouse) against platelet endothelial cell adhesion molecule-1 for 30 min at 4 °C. The cells were washed twice to remove unbound antibody and resuspended in a binding buffer containing washed magnetic beads coated with sheep anti-rat immunoglobulin G. Attached cells were washed four to five times in culture medium and then digested with trypsin/EDTA to detach the beads. Bead-free cells were centrifuged and resuspended for culture. After two passages, the cells were incubated with fluorescent-labeled diacetylated low density lipoprotein, which is only absorbed by endothelial cells and macrophages, and sorted to homogeneity by fluorescence-activated cell sorting.

Cell Culture and Treatments—The MLEC were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 6.32 g/liter Hepes, and 3.3 ml of endothelial cell growth supplements in humidified incubators at 37 °C. For adenoviral infections, the cells were grown to 30% confluence and changed to serum-free medium containing 106 plaque-forming units/ml of an adenoviral vector inserted with lacZ, or ho-1. Infected cells were incubated for 3 h and then restored to Dulbecco's modified Eagle's medium containing 10% fetal calf serum for an additional 2 days of incubation. For hyperoxia treatment, the MLEC were grown to 95% confluence, changed to fresh medium, and transferred to a COY anaerobic chamber (COY Laboratory Products, Inc., Ann Arbor, MI) containing a hyperoxic atmosphere (95% O2 and 5% CO2) in the absence or presence of CO (250 ppm). Control cells were cultured in standard tissue culture conditions (95% room air, 5% CO2).

LDH Release Assay—LDH release was measured using a commercially available assay (cytotoxicity detection kit; Roche Applied Science). After gentle agitation, 200 µl of medium was removed at various times to be used for the assay. The samples were incubated (30 min) with buffer containing NAD+, lactate, and tetrazolium. LDH converts lactate to pyruvate generating NADH. The NADH then reduces tetrazolium (yellow) to formazan (red), which was detected by absorbance at 490 nm. Additionally, the percentage of cell death was determined by exclusion of trypan blue.

ROS Measurement by DCFDA Fluorescence—The formation of ROS in MLEC was determined by the DCFDA fluorescence method (6). MLEC were loaded with 10 µM DCFDA for 30 min in Dulbecco's modified Eagle's medium at 37 °C in a 95% air, 5% CO2 environment. At the end of the incubation, the medium containing DCFDA was aspirated, and the cells were washed once in media. After the addition of 5 ml of growth media, the cultures were exposed to hyperoxia (95% O2, 5% CO2) for 3 h in the absence or presence of CO (250 ppm). At the end of exposure to hyperoxia, the cultures were scraped on ice, and the resulting cell suspensions were centrifuged at 8,000 x g for 10 min at 4 °C. The medium was aspirated, and the cell pellet was washed twice with ice-cold phosphate-buffered saline. The cells were then sonicated on ice with a probe sonicator at a setting of 5 for 15 s in 500 µl of ice-cold phosphate-buffered saline to prepare cell lysates. The fluorescence of oxidized DCFDA in cell lysates, an index of formation of ROS, was measured on an Aminco Bowman series-2 spectrofluorometer at 490/530-nm excitation/emission, using appropriate blanks. The extent of ROS formation was expressed in arbitrary fluorescence units.


Figure 1
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  		FIGURE 1.
HO-1/CO protected against hyperoxia-induced cell death. MLEC at 30% confluence were cultured in serum-free medium in the presence of 106 CPU/ml of adeno-HO-1 or adeno-lacZ for 3 h and then restored to normal medium. Two days later, the cells were exposed to hyperoxia (95% O2, 5% CO2) for the indicated times. Viability was determined by trypan blue exclusion (A). LDH assay was performed on cell culture supernatants (B). Cytosolic or mitochondrial fractions were prepared from total lysates as described under "Experimental Procedures." The fractions were subjected to immunoblotting (IB) to detect cytochrome c, with total cytochrome c as the standard (C). Western results are representative of three experiments. beta-Actin served as the loading standard. The data represent the means ± S.E. of three independent experiments. *, p < 0.05.

 
Cell Fractionation—Cellular fractionation was performed essentially as previously described (31). For mitochondrial isolation, at various times after exposure to hypoxia, MLEC were harvested in 0.05% of digitonin in extraction buffer containing 50 mM Hepes, pH 7.5, 50 mM KCl, 5 mM EGTA, and 2 mM MgCl2, 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin, 1 mM sodium orthovanadate. The cell extracts were spun at 700 x g for 10 min, and the supernatants were moved to new tubes and then centrifuged again at 14,000 x g at 4 °C for 20 min. The supernatants (cytosol) and the pellets were retained for Western blotting.

For plasma membrane isolation, the homogenates of the cells in MBS (25 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5, 0.15 M NaCl) containing 1% Triton X-100 were adjusted to 40% sucrose by the addition of 2 ml of 80% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube. A 5–30% discontinuous sucrose gradient was formed above (4 ml of 5% sucrose, 4 ml of 30% sucrose, both in MBS lacking detergent) and centrifuged at 39,000 rpm for 18 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA). A band at the interface of 5 and 30% sucrose was collected and used for immunoprecipitation and Western blotting.

The Golgi complex was isolated using sucrose density gradient centrifugation, as described elsewhere (31). After washing with phosphate-buffered saline, the cells were harvested in G buffer (10 mM Tris-HCl, 0.25 M sucrose, 2 mM MgCl2, pH 7.4) containing 10 mM CaCl2 and protease inhibitors. The cells were disrupted with 20 strokes in a Potter-type homogenizer. The homogenate was centrifuged at 2,500 x g for 10 min, and the pellet was discarded. The resulting post nuclear supernatant was harvested, and the sucrose concentration was adjusted to a final concentration of 1.4 M. This suspension was loaded onto the bottom of an ultracentrifuge tube and overlaid in succession with 1.2, 1.0, and 0.8 M sucrose in G buffer. The samples were then centrifuged at 95,000 x g for 2.5 h. Two bands from the top, representing 0.8/1.0 and 1.0/1.2 M interfaces were carefully removed, diluted with G buffer without sucrose, collected by centrifugation at 80,000 x g for 30 min, and used for the experiments. Golgi fraction purity was assessed by enzymatic activity and immunoblotting methods as previously described (31).

Western Blot Analysis and Immunoprecipitation—The proteins were isolated from MLEC cultures in radioimmunoprecipitation assay buffer (1x phosphate-buffered saline, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin, and 1 mM sodium orthovanadate). For immunoprecipitation, 1 µg of antibody (anti-Fas, anti-6A7, anti-Bax, or anti-phosphoserine) was added to 500 µg of total protein in 500 µl, rotated for 2 h at 4°C, and then incubated with 20 µl of beads (protein A-sucrose; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h, spun down at 500 x g, and washed three times with radioimmunoprecipitation buffer. Then 20 µl of loading buffer (100 mM Tris-HCl, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, and 20% glycerol) was added. For SDS-PAGE, protein samples at the indicated and equal amounts were boiled in the loading buffer and separated on SDS-PAGE, followed by transfer to polyvinylidene difluoride membranes. The membrane was blocked with 5% nonfat milk and stained with the primary antibodies for 2 h at the optimal concentrations. After five washes in phosphate-buffered saline with 0.2% Tween 20, the horseradish peroxidase-conjugated secondary antibody was applied, and the blot was developed with enhanced chemiluminescence reagents (Amersham Biosciences).


Figure 2
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  		FIGURE 2.
CO protected against hyperoxia-induced cell death. MLEC were cultured under 95% air/5% CO2 (normoxia), or 95% O2, 5% CO2 (hyperoxia) in the absence or presence of CO (250 ppm) for the times indicated. Viability was determined by trypan blue exclusion (A). Supernatants were analyzed by LDH release assay (B). Cytosolic or mitochondrial fractions were prepared from total lysates as described under "Experimental Procedures." The indicated fractions were subjected to immunoblotting (IB) to detect cytochrome c (C) or caspase-9 (D). The lysates were also assayed for caspase-3 activity (E). Western results are representative of three experiments. beta-Actin served as the loading standard. The data represent the means ± S.E. of three independent experiments. *, p < 0.05.

 
Caspase Activity Assays—Caspase-3 fluorogenic substrate, Ac-DEVD-AFC and caspase-8 fluorogenic substrate, and Ac-IETD-AFC were from BD Biosciences (Franklin Lakes, NJ). Caspase activity in cell lysates was determined according to the manufacturer's instructions, using an Aminco Bowman series-2 spectrofluorometer (440/500-nm excitation/emission), and expressed as arbitrary fluorescence units.

Statistical Analysis—All of the values are expressed as the means ± S.E. Statistical significance was determined by Student's t test, and a value of p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HO-1 and CO Inhibit Hyperoxic MLEC Death—MLEC were exposed to hyperoxia (95% O2, 5% CO2) in vitro and then monitored for hyperoxia-induced toxicity by assaying LDH in the supernatants. Hyperoxia caused a time-dependent cell death in MLEC as determined by viability and LDH release assays, evident at 24 h, which increased until 72 h of exposure (Fig. 1, A and B). Hyperoxia caused the time-dependent release of cytochrome c from the mitochondria and corresponding increase in cytosolic cytochrome c levels in MLEC (Fig. 1C).

Overexpression of HO-1 by infection with ho-1 containing adenovirus inhibited hyperoxia-induced cell death, as assessed by cell viability and LDH release assays (Fig. 1, A and B), relative to LacZ-infected cells. Furthermore, ho-1 infection dramatically decreased cytochrome c release from the mitochondria and corresponding accumulation in the cytosol (Fig. 1C). Because many of the effects of HO-1 expression can be duplicated by the exogenous application of CO and vice versa, we also tested the effect of CO on hyperoxia-induced cell death.

The presence of CO at a concentration of 250 ppm in the hyperoxic environment significantly inhibited hyperoxia-induced cell death and LDH release (Fig. 2, A and B). The presence of CO (250 ppm) inhibited hyperoxia-dependent cytochrome c release from the mitochondria and its corresponding accumulation in the cytosol (Fig. 2C). We also examined the effect of CO on the hyperoxia-dependent execution of executioner caspases. CO inhibited hyperoxia-dependent caspase-9 activation (Fig. 2D) in MLEC. Finally, CO inhibited the time-dependent activation of caspase-3 during hyperoxic treatment of MLEC (Fig. 2E).

CO Inhibits the Extrinsic Apoptotic Pathway—We tested the hypothesis that the antiapoptotic effects of CO during hyperoxic stress involve the inhibition of the extrinsic apoptotic pathway. MLEC were exposed to hyperoxia for various periods of time in the absence or presence of CO (250 ppm). Cell lysates were immunoprecipitated with Fas followed by immunoblotting with caspase-8 to detect DISC formation. Exposure of MLEC to hyperoxia induced time-dependent DISC formation associated with the recruitment of pro-caspase-8 to Fas, the cleavage of pro-caspase-8, and the appearance of caspase-8 activity (Fig. 3A). The presence of CO during the hyperoxia treatment decreased the DISC formation and delayed the cleavage of caspase-8 (Fig. 3A). We also examined cellular signaling events upstream of DISC formation in response to hyperoxia. Previously, several investigations have demonstrated interactions between Fas and the epidermal growth factor receptor (EGFR) and demonstrated tyrosine phosphorylation of Fas by EGFR as a prerequisite for Fas plasma membrane trafficking and DISC formation (32, 33).


Figure 3
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  		FIGURE 3.
CO inhibits extrinsic apoptotic pathways induced by hyperoxia. MLEC were cultured under 95% room air, 5% CO2 (normoxia) or 95% O2, 5% CO2 (hyperoxia) in the absence or presence of CO (250 ppm) for the times indicated (A and B). Their lysates were then subjected to immunoprecipitation with anti-Fas followed by Western blot analysis to detect caspase-8 (A, upper panel), The total lysates were also subjected to Western blot analysis to detect caspase-8 (A, middle panel). MLEC were cultured under 95% O2, 5% CO2 (hyperoxia) in the absence or presence of 250 ppm of CO (B) or NADPH oxidase inhibitors, apocynin (300 µM), or diphenyleneiodoniun (DPI, 10 µM) (C), for the times indicated. Their lysates were then subjected to immunoprecipitation with anti-Fas followed by Western blot analysis as indicated to detect EGFR (B and C, upper panels), or caspase-8 (C, lower panel). The lysates were also subjected to Western blot analysis to detect EGFR phosphorylation (B). Western blots are representative of three independent experiments. Total EGFR served as the standard (B). MLEC were loaded with DCFDA (10 µM) and then exposed to hyperoxia (95% O2, 5% CO2) for 3 h. ROS generation was determined by the fluorimetric measurement of DCFDA oxidation (D) as described under "Experimental Procedures." The measurements represent the means ± S.E. of triplicate determinations. **, p < 0.01 (D). MLEC isolated from wild type or p47–/– mice were cultured under 95% room air, 5% CO2 (normoxia) or 95% O2, 5% CO2 (hyperoxia) for the indicated times. Their lysates were subjected to immunoprecipitation with anti-Fas followed by Western blot analysis to detect caspase-8 (E). IB, immunoblot; IP, immunoprecipitation; WT, wild type.

 


Figure 4
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  		FIGURE 4.
CO inhibits the DISC trafficking from the Golgi complex to the plasma membrane. MLEC were cultured under 95% O2, 5% CO2 (hyperoxia) in the absence or presence of CO (250 ppm) for the times indicated. Proteins from the Golgi complex or plasma membrane were separated from the cell lysates and subjected to immunoprecipitation with anti-Fas followed by Western blot analysis as indicated to detect caspase-8. Western blots are representative of three independent experiments. IB, immunoblot; IP, immunoprecipitation.

 
Hyperoxia increased EGFR phosphorylation and promoted its association with Fas (Fig. 3B). The presence of CO during the hyperoxia treatment significantly inhibited EGFR phosphorylation and decreased the association of Fas with EGFR (Fig. 3B). We hypothesized that hyperoxia-induced ROS generation regulates early events in extrinsic apoptosis. MLEC were treated with a NADPH oxidase inhibitor, dipheyleneiodonium or apocynin, prior to their exposure to hyperoxia. Interestingly, the inhibition of NADPH oxidase with such compounds dramatically decreased the DISC formation, as represented by the associations between caspase-8 and/or EGFR with Fas (Fig. 3C). CO, which attenuated DISC formation, also inhibited hyperoxia-induced ROS production (Fig. 3D). To confirm these observations with a genetic strategy, MLEC were then isolated from mice deficient in the p47phox subunit of NADPH oxidase. Previous studies show that p47phox is essential for ROS production in endothelial cells in response to stimuli such as phorbol esters and tumor necrosis factor-{alpha} (34). Hyperoxia induced the association of caspase-8 and Fas (DISC formation) in wild type MLEC. However, hyperoxia did not induce DISC formation in p47–/– MLEC (Fig. 3E). These results suggest that NADPH oxidase-derived ROS play a critical role in the activation of the extrinsic apoptotic pathway in response to hyperoxia and provide a mechanism by which CO inhibits the activation of this pathway.

CO Inhibits DISC Trafficking—Recent evidence suggests that DISC formation at the plasma membrane is preceded by translocation from the Golgi apparatus. To test the effect of hyperoxia on translocation of the DISC, cell lysates from MLEC exposed to hyperoxia were separated into Golgi complex and plasma membrane fractions. DISC formation was assessed by immunoprecipitation with Fas followed by immunoblotting with caspase-8. Hyperoxia induced DISC formation in the Golgi complex and in the plasma membrane (Fig. 4). Inclusion of CO during the hyperoxic exposure resulted in the retention of the DISC in the Golgi complex and decreased DISC formation in the plasma membrane relative to exposure to hyperoxia alone. These results suggested that the DISC is preformed in the Golgi complex and translocates to the plasma membrane during hyperoxia exposure, whereas the presence of CO inhibits the DISC trafficking.

CO Inhibits Bax Activation and Translocation—We tested the hypothesis that CO inhibits hyperoxia-induced cell death by inhibiting the intrinsic apoptotic pathway. Bax is a major regulator of intrinsic apoptosis but also amplifies extrinsic apoptosis through interaction with Bid, which facilitates its activation. Hyperoxia induced the total expression level of Bax in MLEC in a time-dependent fashion with an apparent maximum at 48 h of exposure, relative to normoxic cultures (Fig. 5A). The presence of CO (250 ppm) during the hyperoxia delayed the time-dependent expression of Bax, resulting in diminished Bax expression at 48 h (Fig. 5A). Furthermore, hyperoxia induced the time-dependent cleavage of Bid, whereas the presence of CO significantly decreased hyperoxia-inducible Bid activation (Fig. 5B).


Figure 5
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  		FIGURE 5.
CO inhibits Bax activation. MLEC were cultured under 95% room air, 5% CO2 (normoxia) or 95% O2, 5% CO2 (hyperoxia) in the absence or presence of CO (250 ppm) for the times indicated (A and B). Their lysates were then subjected to Western blot analysis to detect Bax (A) or tBid (B). Lysates corresponding to 48 exposure were also subjected to immunoprecipitation with anti-6A7 followed by immunoblotting with Bax to detect activated Bax (C) or separated into cytosolic (C, middle panel) and mitochondrial fractions (C, lower panel) and subjected to immunoblotting to detect Bax. The lysates (48 h) were segregated into total or mitochondrial fractions and subjected to immunoprecipitation with anti-6A7 and Western blot analysis to detect Bid (D). p110 mitochondrial protein served as the standard for mitochondrial fractions (C and D). IB, immunoblot; IP, immunoprecipitation.

 
We also tested the hypothesis that CO may inhibit Bax activation and translocation from cytosol to mitochondria. Cell lysates were immunoprecipitated with the anti-Bax monoclonal antibody (6A7) that specifically recognizes a conformational change in Bax protein associated with its activation (10). The presence of CO inhibited the activation of Bax after 48 h of exposure to hyperoxia, relative to high levels of activated Bax observed in cells exposed to hyperoxia alone (Fig. 5C). Next we isolated cytosolic and mitochondrial fractions from MLEC after 48 h of exposure to hyperoxia. At this time point, Bax was detected primarily in the mitochondrial fraction relative to the cytosolic fraction (Fig. 5C). In the presence of CO (250 ppm), Bax was detected primarily in the cytosolic fraction relative to the mitochondrial fraction (Fig. 5C). These results indicate that CO inhibited Bax trafficking from the cytosol to the mitochondria during hyperoxia exposure. We have previously shown that Bid binds to and assists in the conformational change of Bax required for its activation (10). To test the potential association of Bid and Bax under these conditions, MLEC were subjected to hyperoxia (48 h) in the absence or presence of CO (250 ppm), and total cell lysates were immunoprecipitated with 6A7 antibody followed by immunoblotting with Bid. In cells exposed to hyperoxia, an association of Bid with the activated form of Bax was detected in the total lysates and in the mitochondrial fractions. The presence of CO inhibited the association of Bid with activated Bax in the mitochondrial fraction of MLEC exposed to hyperoxia, relative to that observed in MLEC treated with hyperoxia alone (Fig. 5D). We also hypothesized that CO may modulate the interaction of Bax with anti-apoptotic proteins. The presence of CO during hyperoxia promoted the interaction of Bcl-XL with Bax and increased the phosphorylation of Bad relative to hyperoxia treatment alone (Fig. 6A). Interestingly, CO treatment promoted an association of HO-1 with Bax, specifically in mitochondrial fractions (Fig. 6B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies from this laboratory and others describe apoptosis as a major histological feature of hyperoxia-induced lung injury in vivo (4, 7–9, 35, 36) and murine macrophage cell lines in vitro (37). In contrast, hyperoxia primarily causes necrosis in A549 alveolar type II epithelial cells. Similarly, necrosis, but not apoptosis, was observed in type II cells isolated from rats exposed to hyperoxia (10, 38). Recent in vitro studies indicate that the signaling pathways used to initiate cell death do not predict the final outcome of cell necrosis or apoptosis. For example, A549 cells exposed to hyperoxia, although presenting caspase-8 and caspase-9 activation, did not undergo apoptotic cell death but presented morphological features of necrosis (10, 39). However, deletion of Bid or inhibition of caspase-8 by c-FLIP protected against cell death in this model (10). Integrin-dependent interactions with the extracellular matrix may determine whether type II cells repair oxygen-induced DNA strand breaks or activate the apoptotic machinery (40). The differential ability to affect repair or apoptosis potentially explains why SV40-transformed MLE12 mouse type II cells died by apoptosis after hyperoxia exposure, whereas MLE15 cells, another SV40-transformed line of mouse type II cells, died primarily by necrosis (41, 42). In pulmonary endothelial cells, we observed both apoptosis and necrosis after hyperoxia exposure by annexin-V/propidium iodide staining (data not shown).

Previous work from this laboratory demonstrated that exogenous CO prevented hyperoxia-induced lung injury in rats, even when endogenous HO enzyme activity was inhibited with tin protoporphyrin, a competitive inhibitor of HO activity. Rats exposed to CO also exhibited a marked attenuation of hyperoxia-induced neutrophil infiltration into the airways and total lung apoptotic index (18). However, the mechanisms of CO protection against hyperoxic lung cell death have not been well defined. In the current study, we demonstrated that CO inhibited cytochrome c and caspase-9 activation in hyperoxic MLEC by blocking both extrinsic and intrinsic apoptotic pathways. CO diminished DISC formation and associated activation of caspase-8 and Bid. CO exposure inhibited Bax activation and blocked its translocation into mitochondria, thus preventing cytochrome c release and subsequent caspase-9 activation (Fig. 5C). We also showed that CO blocked the association of Bid with the activated form of Bax (Fig. 5D). We previously reported that Bid assists in the conformational change associated with Bax activation (10), and the current data suggest that CO inhibits this processing. tBid assists Bax not only through protein-protein interactions but also by protein-lipid interactions to form lipid pore-type structures in the outer mitochondrial membrane through which intermembrane pro-death molecules exit the mitochondria during apoptosis (43).


Figure 6
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  		FIGURE 6.
CO promotes Bax complex formation with Bcl-XL and HO-1. MLEC were cultured under 95% room air, 5% CO2 (normoxia) or 95% O2, 5% CO2 (hyperoxia) in the absence or presence of CO (250 ppm). Total lysates (0–24 h of exposure) were subjected to immunoprecipitation with anti-Bcl-XL and Western blot analysis to detect Bax (A, upper panel). The total lysates were also subjected to Western blot analysis to detect phosphorylated-Bad (A, middle panel). Total Bax served as the standard (A, lower panel). Proteins from mitochondria and cytosol were separated from the cell lysates and were immunoprecipitated (IP) with anti-HO-1 followed by Western blot analysis (IB) to detect Bax (B). Western blots are representative of three experiments. beta-Actin served as the standard. p110 mitochondrial protein served as the standard for mitochondrial fractions (B, lower panel). IB, immunoblot; IP, immunoprecipitation.

 
Bcl-XL is a pro-survival member of the Bcl-2 family that inhibits the activities of the pro-apoptotic members of the family, such as Bax and Bad. Bad occurs in the cytosol of normal cells, where it is phosphorylated at multiple serine residues (11, 14). An apoptotic signal triggering Bad dephosphorylation results in the binding of Bad to Bcl-XL and the inactivation of the pro-survival function of Bcl-XL. Uncomplexed Bcl-XL can counteract the pro-apoptotic activity of Bax by inhibiting Bax mitochondrial translocation and oligomerization, thus preventing cytochrome c release (44). In the current study, we found that CO promoted the interaction of Bcl-XL with Bax and enhanced Bad phosphorylation (Fig. 6A). These data suggest that CO inhibits intrinsic apoptotic signaling pathways through increased Bad phosphorylation and by promoting the binding of Bax with Bcl-XL, which blocks the oligomerization of Bax necessary for cytochrome c release.

In death receptor (Fas)-mediated apoptosis, the current model suggests that the DISC, consisting of Fas/FADD/caspase-8, forms in the plasma membrane upon ligand (FasL) stimulation. Autoproteolytic generation of caspase-8 from procaspase-8 occurs within the DISC. However, the precise mechanisms underlying DISC formation and the translocations of Fas, FADD, and caspase-8 are not entirely known. In the hypoxia/reoxygenation model, we previously reported that the DISC forms initially in the Golgi complex and then translocates to the plasma membrane (31, 45). DISC formation in the Golgi complex is important for initiating apoptotic signaling (31), whereas its formation in the plasma membrane is critical for caspase-8 activation.

Recent studies in hepatocytes show that FasL-induced apoptosis involved ROS generation and, consequently, the c-Jun NH2-terminal kinase-dependent tyrosine phosphorylation and activation of EGFR and its subsequent association with Fas (33). In this model, EGFR-mediated tyrosine phosphorylation of Fas was required for the trafficking of Fas to the membrane, for DISC formation, and for the initiation of the extrinsic apoptotic pathway (33). Consistent with this model, we observed that hyperoxia treatment promoted EGFR phosphorylation and its association with Fas in MLEC, in parallel with DISC activation (Fig. 3B). Fas/caspase-8 association and Fas/EGFR association depended on ROS generation, because these processes were inhibitable by NADPH oxidase inhibitors (Fig. 3C), absent in p47–/– MLEC (Fig. 3E) and were also inhibited by CO (Fig. 3B), which down-regulated ROS production (Fig. 3D). Recently, we have also demonstrated that CO inhibited ROS-dependent trafficking of Toll-like receptor-4 to lipid rafts by down-regulating NADPH oxidase activity (47). The dependence of ROS in DISC activation during hyperoxia and the inhibition of these processes by CO provide a distinct mechanism by which CO inhibits extrinsic apoptosis.

CO has previously been shown to confer cellular or tissue protection in multiple models of stress or injury. In rodent models of hyperoxia-induced lung injury, CO exerts potent anti-inflammatory effects with reduced inflammatory cell influx into the lungs and marked attenuation in the expression of pro-inflammatory cytokines (17). CO suppressed arteriosclerotic lesion formation associated with chronic graft rejection and with balloon injury (27) and inhibited apoptosis during ischemia-reperfusion injury (26, 48, 49). These effects were associated with the CO-dependent activation of the p38 mitogen-activated protein kinase pathway.

HO-1, a stress-inducible protein, responds to transcriptional up-regulation by hyperoxic conditions. Exogenous administration of HO-1 by adenoviral gene transfer increased rat survival and attenuated lung neutrophil influx and lung cell apoptosis during hyperoxia (50). Similarly, administration of low dose CO may prevent lung oxidative stress through secondary increases in HO-1 (21, 46). However, the exact pathway by which HO-1 increases cell survival has not yet been elucidated. Consistent with previously described antiapoptotic roles, we observed that adenoviral-mediated HO-1 overexpression prevented hyperoxia-induced cytochrome c release from the mitochondria. HO-1 also inhibited hyperoxia-induced membrane damage, as assessed by LDH release assays, which also suggests general protection against necrotic cell death. Here, we described for the first time that exogenous CO treatment promotes a previously undescribed binding interaction of HO-1 with Bax in the mitochondria (Fig. 6B). The functional significance of this interaction is currently unknown, although we speculate that this association blocks the oligomerization of Bax, thus precluding its ability to destabilize mitochondrial membrane function and initiate cytochrome c release (Fig. 7).


Figure 7
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  		FIGURE 7.
CO inhibits extrinsic and intrinsic apoptotic pathways during hyperoxia. The diagram depicts the mechanisms by which CO provides protection against cell death. Hyperoxia triggers both mitochondrial (intrinsic) and death receptor-dependent (extrinsic) apoptotic pathways in MLEC. CO inhibited hyperoxic cell death by decreasing ROS production, EGFR phosphorylation, as well as the interaction between EGFR and Fas. CO reduced the formation of the DISC in the plasma membrane, by retaining the DISC in the Golgi apparatus. CO increased Bad phosphorylation and promoted an association of Bcl-XL and/or HO-1 with Bax.

 
In summary, we have examined cell death pathways in cultured MLEC exposed to hyperoxia and found that hyperoxia caused significant apoptosis, involving both extrinsic (Fas/caspase-8) and intrinsic (Bax/mitochondria)-dependent apoptotic pathways (Fig. 7). CO inhibited hyperoxic cell death by inhibiting cellular ROS production and antagonizing ROS-dependent DISC formation in the plasma membrane, thereby inhibiting caspase-8 activation and Bid cleavage. CO also blocked Bax mitochondrial translocation by increasing Bad phosphorylation and promoting a novel interaction of HO-1 with Bax. Strategies to attenuate hyperoxia-induced apoptotic pathways may expand the currently limited therapeutic options.


   FOOTNOTES
 
* This work was supported by American Heart Association Grants 0335035N (to S. W. R.) and 0525552U (to H. P. K.) and National Institutes of Health Grants R01-HL60234, R01-HL55330, R01-HL079904, and P01-HL70807 (to A. M. K. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Division of Pulmonary, Allergy and Critical Care Medicine, Dept. of Medicine, University of Pittsburgh Medical Center, 3459 Fifth Ave., MUH 628NW, Pittsburgh, PA 15213. Tel.: 412-692-2210; Fax: 412-692-2260; E-mail: choiam{at}upmc.edu.

2 The abbreviations used are: ROS, reactive oxygen species; DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; DISC, Death-inducing signal complex; EGFR, epidermal growth factor receptor; HO, heme oxygenase; MLEC, mouse lung endothelial cells; LDH, lactate dehydrogenase. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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