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J. Biol. Chem., Vol. 278, Issue 31, 29184-29191, August 1, 2003
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Necrotic Cell Death in Response to Oxidant Stress Involves the Activation of the Apoptogenic Caspase-8/Bid Pathway*
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From the
Division of Pulmonary, Allergy, and
Critical Care Medicine, Department of Medicine, the
Department of Pathology, and the
¶Center for Biologic Imaging, Department of Cell
Biology and Physiology, School of Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania 15213
Received for publication, February 14, 2003 , and in revised form, May 14, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Human epithelial (A549) cells exposed to hyperoxia die by cellular necrosis. In the current study, we demonstrated the involvement of apoptogenic factors in epithelial cell necrosis in response to hyperoxia, including the formation of the Fas-related death-inducing signaling complex and initiation of mitochondria-dependent apoptotic pathways. We showed increased activation of both Bid and Bax in A549 cells subjected to hyperoxia. Bax activation involved a Bid-assisted conformational change. We discovered that the response to hyperoxia in vivo predominantly involved the activation of the Bid/caspase-8 pathway without apparent increases in Bax expression. Disruption of the Bid pathway by gene deletion protected against cell death in vivo and in vitro. Likewise, inhibition of caspase-8 by Flip also protected against cell death. Taken together, we have demonstrated the involvement of apoptogenic factors in epithelial cell responses to hyperoxia, despite a final outcome of cellular necrosis. We have, for the first time, identified a predominant role for the caspase-8/Bid pathway in signaling associated with hyperoxic lung injury and cell death in vivo and in vitro.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The clinical treatment of respiratory failure often requires supplemental oxygen therapy. Unfortunately, exposure to an elevated oxygen tension (hyperoxia) may cause acute and chronic lung injury. Prolonged hyperoxia triggers an extensive inflammatory response in the lung that degrades the alveolar-capillary barrier, leading to impaired gas exchange and pulmonary edema (1). The pathological changes in hyperoxia-injured lungs coincide with the injury or death of pulmonary capillary endothelial cells and alveolar epithelial cells, which maintain the integrity of the alveolar-capillary barrier and serve as a first-line defense against oxidative injury. Compromised epithelial cell function may permit fluid and macromolecules to leak into the airspace, resulting in clinical respiratory failure and death (2). Epithelial cell homeostasis requires stringent control over proliferative and apoptotic pathways.
Hyperoxia-induced cell death may involve apoptosis and necrosis, two distinct mechanisms of cell death with different biochemical, morphological, and functional characteristics (3). In necrosis, an extensive cell lysis results from acute, accidental, or non-physiological injury (4). In contrast, apoptosis represents a regulated form of cell death that participates in tissue development and homeostasis, requiring the action of proteases and nucleases within an intact plasma membrane (5). The delineation of apoptosis and necrosis as mutually exclusive processes, however, has not been convincingly demonstrated in vivo or in vitro.
Previous studies describe apoptosis as a major histological feature of hyperoxia-induced lung injury in vivo (6–8). Petrache et al. (9) demonstrated induction of apoptosis in murine macrophage cell lines in response to in vitro hyperoxia. In contrast, hyperoxia primarily caused necrosis in A549 and in Type-I epithelial and murine lung bronchial cells (10–12). Furthermore, pre-treatment with hyperoxia inhibited oxidant-induced apoptosis in A549 cells (13). Thus, the mechanisms underlying hyperoxic lung injury and cell death may vary in a tissue-specific manner and may involve apoptosis, necrosis, or a mixed cell-death phenotype.
Members of the death receptor superfamily, including Fas
(14) and tumor necrosis
factor-
(TNF-)1
(15), as well as Bcl family
members (i.e. Bax and Bcl-XL) have been implicated in
hyperoxic lung injury. Adult mice exposed to hyperoxia (>95% O2)
for 72 h displayed increased whole-lung Bax and Bcl-XL mRNA levels;
unaltered Bak, Bad, or Bcl-2 mRNA levels; and decreased Bcl-w and Bfl-1 mRNA
levels (14,
16). Increases in
Bcl-XL protein, but not Bax protein, have been reported in response
to hyperoxia in the mouse lung
(16,
17). Hyperoxia may induce
other cell death-related molecules, such as p53 and
p21Cip1/WAF1/Sdi1 (p21)
(14,
16,
18–20).
Expression of the p53 protein responds to DNA damage and, in turn, regulates
genes involved in growth control, DNA repair, and apoptosis. By increasing the
expression of pro-apoptotic Bcl-2 family members such as Bax, p53 may promote
cell death. A major regulatory target of p53, the cyclin-dependent kinase
inhibitor p21, may protect against oxidative lung injury by inhibiting cell
proliferation and DNA replication, and promoting DNA repair
(11).
Mice with different genetic backgrounds differ in oxygen sensitivity. In vivo studies using genetically modified mouse strains have revealed the relative roles of apoptosis-related proteins in oxygen sensitivity of the lung. Fas–/– (lpr) mice (14) and TNFRI/II–/– mice (21) did not display resistance to hyperoxia. Murine embryonic fibroblasts derived from Bax–/– and Bak–/– mice resisted hyperoxia-induced cytotoxicity (22). In contrast, p21–/– mice were more sensitive than wild-type mice, whereas p53–/– mice did not display any modulation of oxygen sensitivity (11, 14, 16). Hyperoxia induced the expression of p21 and of growth arrest and DNA damage-inducible genes in p53–/– mice (11, 23). These observations suggest that downstream regulatory targets of p53 such as p21 and Bax modulate cellular sensitivity to hyperoxia, although p53 itself is apparently not essential.
Despite these observations, the one or more signal transduction pathways involved in hyperoxic lung injury and cell death remain incompletely understood. In the current study, we demonstrate the involvement of apoptogenic factors in epithelial cell responses to hyperoxia, despite a final outcome of cellular necrosis. We have, for the first time, identified a predominant role for the caspase-8/Bid pathway in signaling associated with hyperoxic lung injury and cell death in vivo and in vitro. A second pathway involving Bax gains importance under conditions that inhibit or abolish the caspase-8/Bid pathway. We therefore provide an explanation for the lack of Bax modulation in the lungs of wild-type mice subjected to a lethal dose of hyperoxia.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Chemicals and Reagents—Digitonin, disuccinimidyl suberate, etoposide, and other chemicals were from Sigma Chemical Co. (St. Louis, MO). Rabbit or goat polyclonal antibodies against Bax, Bcl-XL, Bid, caspase-8, caspase-9, cytochrome c, Fas, TNFR-1, and protein A-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA) and were used for Western blotting. Anti-Bax 6A7 antibody was purchased from BD Pharmingen (San Diego, CA) and used for immunoprecipitation experiments. The Flip, Bcl-XL, and LacZ inserted in an adenovirus expression system were from the Molecular Medicine Institute Programs of Excellence in Gene Therapy Vector Core Facility, University of Pittsburgh.
Hyperoxia Exposure and Animal Experimentations—Normal, lpr (Fas null, Fas–/–), and gld (Fas ligand null, FasL–/–) adult (7 weeks) pathogen-free male C3H/HeJ mice and appropriate controls were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL Bid wild-type (Bid+/+) and Bid-null (Bid–/–) adult (7 weeks) mice were bred at the University of Pittsburgh. Mice were kept in room air (control) or exposed to >98% oxygen by placing the cages inside a plexiglass chamber. Animals were injected with 1 x 108 of adenovirus (inserted with appropriate gene) per mouse through the trachea into the lung 2 days prior to oxygen exposure. Food and water were provided normally. Animals were observed closely for overall mortality, and the time of death was recorded. Immediately after death, mice were necropsied, and their lungs were harvested for histochemical and biochemical analyses.
Cell Culture and Treatments—Human lung adenocarcinoma A549 cells (ATCC CCL185) were grown in F12K medium (Invitrogen) supplemented with 10% fetal bovine serum. Cells were maintained at 37 °C in 95% room air, 5% CO2 in a humidified chamber. Subconfluent cultures were used in all experiments, with the cells adhered 24 h prior to the experimental treatment. Cells were cultured in sealed chambers flushed with 95% O2 and 5% CO2. Control cells were cultured in 95% room air, 5% CO2. For cells expressing the appropriate gene, 1 x 105 of adenovirus per milliliter was added to cells grown to about 30% confluence into the medium 2 days before exposure to O2. Increased glucose consumption has been observed in a number of cell types exposed to hyperoxia in culture. In 10 ml of medium, glucose was depleted and cellular ATP decreased after 36 h (24). We refreshed the medium and gases daily to control for the equilibrium between apoptosis and necrosis that is influenced by maintaining the ATP levels (5). At each time point, cell viability was determined by Trypan blue dye exclusion analysis (Invitrogen). Apoptosis was determined by morphological changes and TUNEL assays.
Preparation of Primary Fibroblast Cultures from Mouse Lung—Lung tissue was excised from either wild-type or Bid–/– mice. The lung tissue was soaked in a 1% antibiotic-antimycotic PBS (Invitrogen, Carlsbad, CA) twice for 20 min. The tissue was minced into 1-mm pieces, which were plated onto p100 dishes (15–20 pieces per plate). Fetal bovine serum was added dropwise over each tissue piece, and then the plates were incubated for 4 h at 37 °C. Then 2 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics was added to each plate. The plates were restored to the incubator and monitored every day until the fibroblasts reached confluence.
Cross-linking of Fas—After treatment of hyperoxia, A549 cells were grown to 90% confluence and were then washed three times with cold PBS. Freshly prepared disuccinimyl suberate in Me2SO was then added to a final concentration of 0.25 mM to the cells in 1 ml of PBS. The dishes were incubated for 30 min on a rocker platform. After cross-linking, each dish was washed two times with PBS. The cells were then harvested for Western blotting analysis.
Annexin V/Propidium Iodide Staining—Using the annexin V-FITC kit from BD Pharmingen we followed the manufacturer's protocol. Briefly, after hyperoxia treatment for 72 h, A549 cells were washed with cold PBS and resuspended with binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) before transferring 1 x 105 cells to a 5-ml tube. Then 5 µl of annexin V and 5 µl of propidium iodide were added, and the cells were incubated for 15 min in the dark. Binding buffer (400 µl) was then added to each tube and analyzed by flow cytometry.
DNA Ladder—A549 cells treated with hyperoxia were trypsinized and washed with PBS, and then the cells were spun down and resuspended in lysis buffer containing 50 mM Tris-HCl, pH 7.8, 10 mM EDTA-2Na+, and 0.5% SDS. RNase A was then added at a concentration of 0.5 mg/ml and incubated at 37 °C for 60 min. Next, the protein was degraded using 0.5 mg/ml proteinase K at 50 °C for 60 min. DNA fragmentation was visualized with ethidium bromide staining after electrophoresis on a 1.2% agarose gel.
Electron Microscopy Assays—Cell cultures were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at 4 °C. The cells were then postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in LX112 (Ladd Corp., Burlington, VT). Thin sections (60 nm) were cut, stained with uranyl acetate and lead citrate, and examined on a Zeiss EM 10 transmission electron microscope.
Cytosol Isolation—At different times after exposure to hyperoxia, A549 cells 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 with protease inhibitors. The cell extracts were spun at 14,000 x g for 20 min, and the supernatants were removed and used for Western blotting.
Lactate Dehydrogenase Release Assay—LDH release was measured using a commercially available assay (Cytotoxicity Detection Kit, Roche Applied Science, Indianapolis, IN). Lung fibroblasts derived from the wild-type or Bid–/– strains were treated with hyperoxia. After gentle agitation, 200 µl of medium was removed at different time points to be used for the assay. The samples were incubated (30 min) with buffer containing NAD+, lactate, and tetrazolium. LDH converts lactate to pyruvate, thus generating NADH. The NADH then reduces tetrazolium (yellow) to formazan (red), which was detected by absorbance (490 nm). Statistical Analysis—Fisher's exact test was used for analysis of the survival rate. The unpaired Student's t test was used for other data analyses as indicated, and a value of p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Hyperoxia Induced Necrosis in A549 Epithelial Cells Involves the Release of Apoptogenic Factors from the Mitochondria— Morphological signs of injury appear in alveolar endothelial and epithelial cells within 48–72 h of exposure to lethal levels of oxygen (2). Continued exposure for more than 72 h can result in epithelial cell death, and consequently, loss of alveolar integrity, airway fluid accumulation, and mortality. Hyperoxia primarily caused necrosis in A549 alveolar epithelial cells, as determined by fluorescence DNA labeling, in situ end labeling of DNA, and electron microscopy (10).
We exposed human epithelial cells (A549) to in vitro hyperoxia and then monitored the cells for survival. Flow cytometry with PI/Annexin-V double staining and DNA-laddering analyses demonstrated that hyperoxia caused A549 cell death primarily by necrosis (Fig. 1, B and C). Etoposide-induced apoptosis of Jurkat cells was used as positive controls (Fig. 1A). Quantitative data from our flow cytometry analyses (PI/Annexin-V double staining) demonstrate that >96% of the dead cells were necrotic, whereas 3% were apoptotic after 72 h of hyperoxia exposure. Furthermore, we also performed electron microscopy analysis of A549 cells at 72 h of hyperoxia, confirming the classic feature of necrosis as shown in Fig. 2. The epithelial cell growth medium was refreshed daily to maintain high levels of intracellular ATP (24). Hyperoxia induced the necrotic death of A549 cells even in the presence of a high glucose concentration in the medium. Under these experimental conditions, <50% of these cells survived after 72 h of hyperoxia, by trypan blue analysis (Fig. 3A).
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We hypothesized that apoptogenic factors may participate in
hyperoxia-induced A549 cell necrosis. In many cell types, apoptosis may
proceed through receptor-dependent pathways initiated by Fas ligand (FasL) or
tumor necrosis factor- (TNF-). FasL-mediated apoptosis begins
with the formation of a death-inducing signal complex (DISC), that involves
the death receptor Fas, Fas-associated death domain, and caspase-8. The
consumption and recruitment of Fas leads to apparent decreases in Fas protein
levels. Activated caspase-8 may in turn either activate effector caspases
(i.e. caspase-3) or cleave Bid. The activated form of Bid triggers
apoptosis by mitochondrial translocation, which stimulates cytochrome
c release (25). On
the other hand, receptor-independent apoptosis may involve the
mitochondrial-translocation of pro-apoptotic Bcl-2 family members such as Bax.
In the mitochondria, Bax oligomers form membrane channels by opening the
permeability transition pore, leading to cytochrome c release and
caspase activation (26). Once
released in the cytosol, cytochrome c binds to Apaf-1, stimulating
its ATP-dependent oligomerization, which in turn leads to the recruitment of
procaspase-9 and its activation to caspase-9.
The role of apoptotic pathways in hyperoxia-induced A549 epithelial cell necrosis was evaluated in vitro, by monitoring the expression of death receptors (Fas and TNFR-I) and Bcl-2 family proteins. Following treatment of A549 cells with hyperoxia (24–72 h), Fas protein levels decreased, whereas TNFR-I protein levels remained unchanged (Fig. 3B). Anti-Fas immunoprecipitation revealed an interaction of caspase-8 with Fas (Fig. 3B), demonstrating DISC formation, the initiation of the Fas-induced apoptotic pathway. We show here for the first time, that hyperoxia up-regulated the activation of both Bax and Bid in A549 cells (Fig. 3C) with release of cytochrome c into the cytosol (Fig. 3D). After 24 h exposure to hyperoxia, active caspase-9 appeared in A549 cells (Fig. 3D).
We next examined the expression of the anti-apoptotic Bcl-2 family member Bcl-XL as a function of hyperoxic exposure in A549 cells. Bcl-XL inhibits mitochondrial cytochrome c release and subsequent caspase activation (27–29). After 48 h in hyperoxia, the expression of Bcl-XL increased in A549 cells (Fig. 3E), whereas Bcl-2 expression did not change (Fig. 3E). The high expression of Bcl-XL apparently did not protect A549 cells from cell death.
Overexpression of FLIP Increased Survival under Hyperoxia in Vitro and in Vivo—Because hyperoxia treatment led to caspase-8 and Bid activation, and increased Bcl-XL expression in A549 cells, we hypothesized that modulation of these apoptosis-related proteins may influence oxygen sensitivity in vitro. A549 cells were infected with adenoviral constructs inserted with Bcl-XL, Flip, or LacZ (Fig. 4A), exposed to in vitro hyperoxia for 72 h, and monitored for necrotic cell death. Overexpression of the caspase-8 inhibitor, FLIP (but not Bcl-XL), significantly protected the cells from death during hyperoxia, relative to the LacZ control (p < 0.05) (Fig. 4B).
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We hypothesized that inhibition of caspase-8 would also modulate oxygen sensitivity in vivo. We infected C3H/HeJ mice with adenoviral constructs inserted with Flip, Bcl-XL, or LacZ. After 48 h, the mice were exposed to >95% O2 and monitored for overall survival. Interestingly, the FLIP group was significantly more resistant to oxygen toxicity than the LacZ-infected group (Fig. 4C). On the other hand, there was no significant difference in hyperoxia resistance between the Bcl-XL-expressing group and the LacZ control group (Fig. 4C). These results emphasize an important role for the caspase-8/Bid pathway in sensitivity to hyperoxia. These results also suggest that increases in Bcl-XL protein expression do not protect against hyperoxia-induced lung injury or cell death.
Overexpression of FLIP in A549 Epithelial Cells Inhibited Bid Expression, and Bax Conformational Change Induced by Hyperoxia—Bid assists Bax in a conformational change that unmasks the Bax NH2-terminal domain and coincides with mitochondrial cytochrome c release (30). Thus, the Bax mitochondrial membrane insertion triggered by Bid may represent a key step in the pathways leading to apoptosis (31). Therefore we investigated the hypothesis that Bax conformational change may respond to the levels of active Bid in A549 cells subjected to hyperoxia. A549 cells were infected with adenoviral constructs containing Bcl-XL, Flip, or LacZ insertions and then grown under hyperoxia (72 h). Cell lysates were immunoprecipitated with the anti-Bax monoclonal antibody (6A7) that specifically recognizes the conformational change in Bax protein (32). FLIP overexpression decreased Bid protein levels and inhibited the conformational change of Bax, compared with LacZ controls (Fig. 5). In contrast, Bcl-XL overexpression only slightly decreased the expression of Bid protein and Bax conformational change relative to LacZ controls (Fig. 5). These results support the hypothesis that Bid assists in a Bax conformational change that increases the efficiency of Bax in promoting cell death.
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lpr (Fas–/–) and gld (FasL–/–) Mice Did Not Display Increased Resistance to Hyperoxia but Displayed Increased Bax Protein Expression Levels—We hypothesized that a Fas-induced apoptosis-like process may precede necrotic cell death in vivo. lpr mice (Fas–/–), however, did not display increased resistance to hyperoxia (14). We challenged lpr (Fas–/–) and gld (FasL–/–) mice with hyperoxia and monitored survival. As shown in Fig. 6A, both Fas–/– and FasL–/– mice exhibited similar survival curves in response to hyperoxia relative to the C3H/HeJ wild-type mice, without significant statistical difference (p > 0.05) (Fig. 6A). We therefore investigated whether a Fas-independent pathway, involving the expression of the Bcl-2 family member Bax, might participate in the response to hyperoxia. Under ambient conditions, Bax protein was elevated in the lungs of the Fas–/– mice relative to the wild-type, with higher expression in Fas–/– mice than in FasL–/– mice (Fig. 6B). Further increases in Bax protein were detected in the lungs of Fas–/– and FasL–/– mice at the time of death by hyperoxia, relative to hyperoxia-treated wild-type mice. Conversely, the activated form of Bid (p15) appeared only in the lungs of wild-type mice subjected to hyperoxia (Fig. 6B). We also found equivalent Bcl-XL expression and caspase-9 activation in the lungs of all three mice strains after hyperoxia (Fig. 6, B and C).
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These results suggest that the activation of Bid appears to represent the dominant pathway leading to cell death in pulmonary cells during the hyperoxia response in vivo. Bid may assist in the conformational change of pre-existing Bax-protein, which is not directly increased by hyperoxia in vivo. On the other hand, the deletion of the death receptor Fas promotes increased Bax expression during in vivo hyperoxia, which likely represents a compensatory activation of the Bax-induced pathway.
Bid–/– Mice Resisted Hyperoxia-induced Pulmonary Cell Death—We then hypothesized that deletion of apoptosis-related factors downstream of Fas (i.e. Bid) would modulate oxygen sensitivity in vivo. Bid knock-out (Bid–/–) mice and corresponding wild-type (Bid+/+) C57BL mice were exposed to >95% O2 and monitored for overall survival and pulmonary cell death. Interestingly, the Bid–/– group was significantly more resistant to oxygen toxicity than the corresponding wild-type littermates (Fig. 7A). We also observed increased resistance to hyperoxia in cells isolated from Bid–/– null mice when compared with cells from negative littermate controls (Fig. 7B). These results again emphasize an important role for the caspase-8/Bid pathway in sensitivity to hyperoxia.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The therapeutic application of supplemental oxygen to elevate arterial pO2 and reduce tissue hypoxia can be offset by its cytotoxic properties, which increase the risk of morbidity and mortality. Over the last 30 years, morphological studies in animal models have described hyperoxia-induced lung injury. The first signs of damage include focal cytoplasm swelling of microvascular endothelial cells, interstitial edema, and endothelial cell fragmentation. After 72 h of exposure, type-I pulmonary epithelial cells begin to die by necrosis (33–35). Many studies have also demonstrated that lung cells exposed to hyperoxia exhibit features of apoptosis, including chromatin condensation, DNA fragmentation, changes in the expression of Bcl-2 family genes, and increases in TUNEL-positive cells (12, 14, 16, 19, 20). We have also observed increases in TUNEL-positive staining in mouse lungs after hyperoxia treatment relative to air-treated controls but without apparent differences in the response between the various genetically modified strains or during overexpression of FLIP or Bcl-XL, as used in this study (data not shown). Intravenous infusion of the caspase inhibitor N-benzylcarbonyl-Val-Ala-Asp-fluoromethyl-ketone into mice, however, did not protect pulmonary cells from death, nor affect lung weights and DNA laddering (14). These features of hyperoxic lung injury suggest that apoptosis and necrosis may occur exclusively in the different lung cell types, and in some cases, may occur concomitantly in the same cell type.
The intracellular ATP concentration may represent a downstream signal capable of directing cells toward either type of cell death, according to the hypothesis that high energy levels are required for the execution of the apoptotic program, whereas they are dissipated during necrosis (36). A number of additional cellular factors, independent of caspase activation, may influence the decision between apoptosis and necrosis, including the duration of mitochondrial membrane pore opening, oxidative stress, and Bcl-2 expression.
Caspases play an indispensable role in regulating many of the morphological and biochemical features of apoptosis. Two main pathways of procaspase activation have been proposed: (i) extrinsic activation of initiator pro-caspases, triggered by a death receptor (i.e. Fas and TNF-R1)-initiated receptosome complex and (ii) intrinsic activation of initiator procaspases initiated by formation of an apoptosome complex, triggered by environmental insults, senescence, and developmental programs, that involves the release of cytochrome c from the mitochondrial intermembrane space to the cytosol (5). The Bcl-2 family of proteins controls mitochondrial integrity, whereby the balance of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2 and Bcl-XL) proteins determines the relative sensitivity of cells to apoptotic stimuli (37). Bcl-XL protects the mitochondria by inhibiting the release of cytochrome c and subsequent procaspase activation, and therefore modulates both extrinsic and intrinsic apoptotic cell death. The existence of necrotic cell death pathways regulated by an intrinsic death program distinct from that of apoptosis has also been proposed (38).
Previous studies have suggested that human A549 epithelial cells subjected to hyperoxia died primarily by necrosis (10). Our results (Fig. 1) support that hyperoxia induces necrotic cell death in A549 cells. The outcome of necrosis did not strictly depend on depletion of cellular ATP levels, because we refreshed the medium daily to maintain high levels of glucose and intracellular ATP (24). The induction of necrotic cell death in A549 cells activated an apoptotic process that involved the formation of the Fas-related DISC. We demonstrated the increased expression, activation, and mitochondrial translocation of both Bid and Bax in A549 cells subjected to hyperoxia. Bid activation by caspase-8 involved cleavage to the p15 form, whereas Bax activation involved a Bid-assisted conformational change. Activation of both Bax and Bid stimulated the release of mitochondrial cytochrome c and cleavage of caspase-9 (Fig. 8). The inhibition of caspase-8 protected epithelial cells from hyperoxia-induced necrotic death. We conclude that the early events in hyperoxia-induced epithelial cell death involve the initiation of both receptor-mediated and mitochondria-dependent apoptotic pathways, despite a final outcome of cellular necrosis.
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In mouse models, previous studies show that hyperoxia induces high levels of mRNA transcription of Bcl-XL and Bax, but not Bax protein, in mouse lungs. Here, we confirm that Bax protein has not changed in normal mouse lung in response to hyperoxia (Fig. 6B). We demonstrated for the first time that Bid activation occurs in normal mouse lung in response to hyperoxia. We therefore propose that a Bid-dependent pathway dominates over a Bax-dependent pathway in wild-type mice subjected to hyperoxia, with Bid assisting in the conformational change of pre-existing Bax protein (Fig. 8). This model is consistent with the lack of apparent modulation of Bax protein in vivo during hyperoxia (Refs. 16 and 17 and Fig. 6B).
In our studies we also found that the Bid–/– genotype significantly conferred resistance to hyperoxia-induced mortality in vivo and in vitro. Likewise, inhibition of caspase-8 by overexpression of Flip also resulted in a marked hyperoxia-resistant phenotype. Although caspase-8 activates Bid with the highest efficiency, other activating species may include granzyme-B, and caspase-3 (39–41). The existence of alternative routes to Bid activation may explain the apparent lack of a hyperoxia resistance phenotype in Fas–/– mice.
In the present study, we also found that the anti-apoptotic molecule, Bcl-XL, could not protect against hyperoxic lung injury and cell death in vivo and in vitro (Fig. 4, B and C). Indeed it has been reported that Bcl-XL does not prevent Bax-induced mitochondrial damage (42). Bax induces cell death via apoptosis in cells with a low level of Bcl-XL, whereas induces necrosis in cells with high expression of Bcl-XL (42). Meilhac et al. (43) described a similar balance between apoptosis and necrosis responding to Bcl-2 expression levels.
This evidence suggests that the intracellular events leading to apoptosis and necrosis can occur sequentially. In the initial step, there is a collapse of mitochondrial membrane potential and loss of cellular glutathione. At this stage, pharmacologic intervention in the form of caspase inhibitors can prevent cell death. In the second phase, a disruption of the plasma membrane leads to necrosis (44). Indeed, the term "programmed cell death" now refers to any kind of cell death mediated by an intracellular death program, irrespective of the trigger, where the morphological outcome can resemble apoptosis, necrosis, or a mixed phenotype (45). Based on our observations, we reason that, in some cell types or in some conditions, the process of necrosis shares a similar regulatory mechanism with apoptosis, which may include the activation of caspases. The final manifestation of death is distinct from apoptosis, in that it escapes inhibition by Bcl-XL.
In summary, hyperoxia induces lung injury and leads to necrotic cell death in lung epithelial cells, mainly through a two stage process: (i) the initiation of apoptosis-related molecules leading to loss of normal mitochondrial function, which involves both death receptor-initiated (Bid-dependent) and mitochondria-dependent pathways leading to release of cytochrome c and (ii) a necrotic cell death. The activation of Bid appears to represent the dominant pathway in pulmonary cells during the hyperoxia response in vivo. Bid assists Bax to change its conformation into the active form (Fig. 8). These data, taken together, suggest that cell death in the mouse lung and in cultured human A549 epithelial cells involves a cellular death pathway sharing features of both apoptosis and necrosis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01-HL60234, R01-AI42365, and R01-HL55330 (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.
|| To whom correspondence should be addressed: Dept. of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, 3459 Fifth Ave., Montefiore University Hospital, 628 NW, Pittsburgh, PA 15213. Tel.: 412-692-2210; Fax: 412-692-2260; E-mail: Choiam{at}msx.upmc.edu.
1 The abbreviations used are: TNF, tumor necrosis factor; TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling; PBS,
phosphate-buffered saline; LDH, lactate dehydrogenase; FasL, Fas ligand; DISC,
death-inducing signal complex; TNFR-I, TNF receptor type I.
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