Anoxia-induced apoptosis occurs through a mitochondria-dependent pathway in lung epithelial cells

Matthew T. Santore, David S. McClintock, Vivian Y. Lee, G. R. Scott Budinger, and Navdeep S. Chandel

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60601


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The intracellular signaling pathways that control O2 deprivation (anoxia)-induced apoptosis have not been fully defined in lung epithelial cells. We show here that the lung epithelial cell line A549 releases cytochrome c and activates caspase-9 followed by DNA fragmentation and plasma membrane breakage in response to anoxia. The antiapoptotic protein Bcl-XL prevented the anoxia-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. A549 cells devoid of mitochondrial DNA (rho °-cells) and lacking a functional electron transport chain were resistant to anoxia-induced apoptosis. A549 cells preconditioned with either hypoxia (1.5% O2) or tumor necrosis factor-alpha , which activated the transcription factors hypoxia-inducible factor-1 or nuclear factor-kappa B, respectively, did not provide protection from anoxia-induced cell death. These results indicate that A549 cells require a functional electron transport chain and the release of cytochrome c for anoxia-induced apoptosis.

Bcl-XL; hypoxia; hypoxia inducible factor-1; tumor necrosis factor-alpha ; nuclear factor-kappa B


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

APOPTOSIS IS A GENETICALLY ENCODED, regulated multistep process of cell death (32). Presently, there are two discrete mechanisms that can initiate apoptosis (6, 29). The first mechanism, commonly associated with growth factor withdrawal and radiation, involves the activation of proapoptotic Bcl-2 family members such as Bax or Bak (18, 34). These proteins initiate the loss of outer mitochondrial membrane integrity, resulting in the release of cytochrome c and other proapoptotic proteins that normally reside in the mitochondrial intermembrane space (8, 12). Cytochrome c released into the cytoplasm initiates the formation of a complex known as the "apoptosome" by binding to the adaptor molecule apoptotic protease-activating factor (Apaf)-1 (17, 19). This triggers the oligomerization of Apaf-1 followed by the recruitment and activation of procaspase-9. Activated caspase-9 can activate downstream effector caspases-3 and -7, resulting in morphological features of apoptosis. Cytochrome c release and subsequent caspase-9 activation are considered early events in apoptosis (6). Bcl-XL or Bcl-2 inhibits apoptosis in response to a variety of death stimuli by preserving mitochondrial integrity and preventing cytochrome c release (11, 31, 35). The second apoptotic mechanism involves the ligation of death receptors such as Fas/CD95, which recruit and activate caspase-8 (22, 23). In a variety of cells, caspase-8 is sufficient to activate downstream caspases, such as caspase-3 or -7, that execute the morphological features associated with apoptosis. By contrast, some cells display a weak caspase-8 activation and thereby require caspase-8 to cleave Bid, a proapoptotic factor. Cleavage of Bid induces the loss of mitochondrial integrity leading to cytochrome c release and caspase-9 activation (16, 20).

Clinically, O2 deprivation-induced cell death is an important phenomenon (31). The mechanisms responsible for O2 deprivation-induced cell death in lung epithelial cells have relevance to clinical lung injury after severe shock or lung transplantation. Cells that become exposed to PO2 <1 Torr (anoxia) for prolonged periods of time begin to undergo cell death. Previous studies have suggested that a variety of nonlung cells die through apoptosis in response to anoxia (9, 10, 25, 28). These studies have also shown that the antiapoptotic Bcl-2 family members such as Bcl-XL or caspase inhibitors prevent anoxia-induced cell death. However, to date studies have not examined the mechanisms underlying anoxia-induced apoptosis in lung epithelial cells. In the present study, we examined whether mitochondrial electron transport and the release of mitochondrial protein cytochrome c are involved in initiating anoxia-induced apoptosis in the human lung epithelial A549 cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. A549 cells were cultured at 30-50% confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with HEPES (10 mM), pyruvate (1 mM), penicillin (100 units/ml), streptomycin (100 µg/ml) and 10% heat-inactivated fetal bovine serum (GIBCO). The Bcl-XL and the control neomycin vector (Neo) were gifts of Dr. Craig Thompson (30). A549 cells transfected with Bcl-XL or Neo were cultured in DMEM with 1 mg/ml of G418, and stable clones were isolated. Wild-type A549 cells were incubated in DMEM containing ethidium bromide (100 ng/ml), sodium pyruvate (1 mM), and uridine (100 µg/ml) to generate rho °-A549 cells. Anoxic conditions (0% O2, 85% N2, 10% H2, and 5% CO2) were achieved in an anaerobic workstation (BugBox, Ruskinn Technologies). Hypoxic conditions (1.5% O2, 93.5% N2, and 5% CO2) were achieved in a variable anaerobic workstation (INVIVO O2; Ruskinn Technologies).

Measurement of cell death. We assayed cell death by measuring lactate dehydrogenase (LDH) activity in culture supernatants using the cytotoxicity detection kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. We detected apoptosis either as a percentage of cells that had condensed/fragmented nuclei by staining with Hoechst no. 33258 (1 µg/ml; Sigma) as previously described (4) or by measuring cytoplasmic histone-associated DNA fragments after cell death using the Cell Death Detection ElizaPlus(Roche Molecular Biochemicals) according to the manufacturer's protocol.

Cytochrome c immunostaining. A549 cells were plated on 60-mm culture dishes at 20-30% confluence and exposed to experimental conditions. Both adherent and nonadherent cells were collected and centrifuged for 5 min at 200 g. The resulting pellet was then washed with PBS and centrifuged again for 5 min at 200 g. The cells were subsequently resuspended in 100 µl of PBS and centrifuged to microscope glass plates at 200 g for 5 min (Cytospin 3 Cytocentrifuge; Shandon). The cells were then incubated at 20°C for 5 min in methanol-acetone (1:1) and blocked for 45 min in PBS containing 0.1% bovine serum albumin (BSA; Sigma). After being blocked, the cells were removed, air-dried, and incubated for 2 h with a 1:500 dilution of the 7H8.2C12 anti-cytochrome c monoclonal antibody (PharMingen) at 37°C in a humidified environment. The cells were then washed for 4 × 15 min in PBS containing 0.1% BSA, air-dried, and incubated for 1 h with a 1:50 dilution of a rhodamine-conjugated secondary antibody (Chemicon International). Subsequently, the cells were washed as before, air-dried, and stained with 4,6-diamidino-2-phenylindole and 1,4-diazabicyclo-(2.2.2)octane. The cells were then visualized via microscopy.

Measurement of caspase activity. Caspase-9 enzymatic activity was measured with a fluorometric assay kit specific to the caspase (R&D Systems). Cell were plated onto 100-mm culture dishes at 40-60% confluence, and caspase activity was measured according to the manufacturer's protocol with a fluorescent microplate reader. Data were normalized using total protein concentration as determined by the Bio-Rad protein assay (Bio-Rad). The caspase inhibitor z-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk; Enzyme Systems Products) was administered at a concentration of 100 µM 1 h before incubation of the cells under O2 deprivation.

Immunoblotting analysis of hypoxia-inducible factor-1alpha . A549 cells were plated on 100-mm plates at 60-80% confluence and assayed for hypoxia-inducible factor (HIF)-1alpha protein levels from nuclear extracts by Western analysis as previously described (5).

Transfection and reporter gene assays. A549 cells were plated on 35-mm culture dishes at 40-60% confluence and transfected with 0.5 µg plasmid using LipofectAMINE reagent (Life Technologies) according to the manufacturer's protocol. Transfections were performed using either a HIF-1-responsive luciferase reporter plasmid containing a trimer of hypoxia response elements (HRE) from the murine Epo 3' enhancer or a nuclear factor (NF)-kappa B-responsive luciferase reporter plasmid containing two canonical kappa B sites (1, 5). Cells were exposed to various conditions 24 h after transfection. Cells were lysed and assayed for luciferase reporter activity using the Luciferase Assay System (Promega).

Measurement of mitochondrial membrane potential. To assess the change in mitochondrial membrane potential (Delta psi ) in cells exposed to anoxia, cells were plated onto 60-mm culture dishes at 40-60% confluence and incubated 1 h before the time point in the presence of two fluorescent probes, tetramethylrhodamine ethyl ester (TMRE, excitation = 550 nm and emission = 580 nm; 200 nM) and Mitotracker green (MITO, excitation = 490 nm and emission = 515 nm; 1 µM) (Molecular Probes). Cells were lysed with 1% (vol/vol) Triton X-100, and fluorescence was measured on a SpectraMax Gemini microplate reader (Molecular Devices). TMRE localizes within mitochondria, and its fluorescence increases in proportion to the psi . MITO fluorescence localizes to mitochondria independently of Delta psi and reflects the number of mitochondria within a given cell. The ratio between TMRE fluorescence and MITO fluorescence reflects psi  normalized to the number of mitochondria. As a control for each condition, cells were incubated with both TMRE and MITO in the presence of the protonophore carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP, 20 µM; Sigma), which dissipates the psi . For each condition, the ratio of TMRE and MITO was subtracted from the TMRE-to-MITO ratio in the presence of FCCP [(TMRE/MITO) - (TMRE/MITO)FCCP].

Statistical analysis. Data are presented as mean values ± SE. The experimental n values reported reflect the number of independent experiments. Data were analyzed using one-way analysis of variance (ANOVA). When the ANOVA indicated that a significant difference was present, we explored individual differences with the Student's t-test, using the Bonferroni correction for multiple comparisons. Statistical significance was determined at the 0.05 level. Experimental samples were compared with control cells exposed to 21% O2.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bcl-XL protects A549 cells from anoxia-induced apoptosis. A549 cells stably transfected with Neo or Bcl-XL were exposed to 21 or 0% O2 for 24 and 48 h. Cellular death was detected by assaying the samples for LDH release. Neo control cells and Bcl-XL cells did not display a significant increase in cell death after 24-h exposure to 0% O2. After 48 h of anoxia, there was a significant increase in cell death in the Neo A549 cells compared with cells transfected with Bcl-XL (Fig. 1A). To determine if the cellular death due to anoxia was apoptotic, Neo and Bcl-XL A549 cells were examined for DNA fragmentation after 48 h of 0% O2. Neo A549 cells revealed a 4.3 ± 0.23-fold increase in DNA fragmentation over basal levels, whereas Bcl-XL A549 cells did not show a significant increase in fragmentation (Fig. 1B). These observations were further confirmed by examining condensed/fragmented nuclei with Hoechst staining in fixed Neo and Bcl-XL A549 cells exposed to 0% O2 for 48 h. Neo control cells under 0% O2 alone displayed apoptotic nuclei, whereas Bcl-XL cells under 0% O2 did not display apoptotic nuclei (Fig. 1C). These results illustrate that Bcl-XL protects cells from anoxia-induced apoptosis.


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Fig. 1.   Bcl-XL protects cells from anoxia-induced apoptosis. A: A549 cells stably transfected with a control vector [neomycin (Neo)] or Bcl-XL were exposed to 21% O2 or 0% O2 for 24 and 48 h. The samples were assayed for lactate dehydrogenase (LDH) release in culture media, a marker of cell death. B: A549 cells were exposed to 21 or 0% O2 for 48 h and assayed for cytoplasmic histone-associated DNA fragments indicative of apoptosis. C: percentage of apoptotic cells scored by Hoechst staining of Neo control and Bcl-XL A549 cells exposed to 21 or 0% O2 for 48 h. *P < 0.05; n = 4. Values are means ± SE.

Bcl-XL prevents anoxia-induced release of cytochrome c from mitochondria. The release of mitochondrial cytochrome c is an early event in a variety of cells undergoing apoptosis (11, 31, 35). To examine whether cells release cytochrome c during anoxia, we exposed Neo and Bcl-XL A549 cells to 24 or 36 h of 21 or 0% O2. Neo and Bcl-XL A549 cells exposed to 21% O2 with an anticytochrome c antibody gave a punctate immunofluorescent staining pattern characteristic of mitochondrial localization (Fig. 2, A and C). Neo and Bcl-XL A549 cells exposed to 24 h of 0% O2 also displayed a punctate staining indicative of cytochrome c localization of mitochondria (data not shown). By contrast, Neo A549 cells after 36 h of 0% O2 displayed a diffuse staining pattern characteristic of a loss of mitochondrial cytochrome c staining (Fig. 2B). Bcl-XL A549 cells after 36 h of 0% O2 continued to exhibit a punctate staining pattern indicative that cytochrome c was localized to mitochondria (Fig. 2D). The irreversible broad-range caspase inhibitor zVAD-fmk (100 µM) did not prevent the release of cytochrome c (data not shown), indicating that anoxia-induced release of cytochrome c was not dependent on caspase activation. Collectively, these results suggest that Bcl-XL inhibits anoxia-induced apoptosis by preventing the release of cytochrome c.


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Fig. 2.   Bcl-XL prevents the release of cytochrome c in response to anoxia. Neo and Bcl-XL cells were exposed to 21 or 0% O2 for 36 h, the release of cytochrome c was detected via immunofluorescence imaging, and nuclear staining was done with 4,6-diamidino-2-phenylindole (DAPI). Neo control cells display a diffuse staining pattern for cytochrome c under 0% O2 (B) compared with the punctate staining pattern observed under 21% O2 (A). Bcl-XL cells display a punctate staining pattern of cytochrome c under 21% O2 (C) or 0% O2 (D).

Bcl-XL prevents anoxia-induced caspase-9 activation. Cytochrome c released from mitochondria has been shown to bind to Apaf-1, which then undergoes a conformational change that allows the cleavage and activation of caspase-9 (17, 19). The activated form of caspase-9 subsequently triggers a caspase cascade that results in the death of the cell. To test whether the release of cytochrome c during anoxia led to the activation of caspase-9, Neo- and Bcl-XL-transfected A549 cells were exposed to 0% O2 for 24 and 36 h, and caspase-9 activity was measured (Fig. 3A). Neo control cells exhibit a significant increase in caspase-9 activity after 36 h of anoxia compared with cells transfected with Bcl-XL. To further understand the role of caspases during anoxia induced cell death, we treated A549 cells with zVAD-fmk (100 µM) before exposure to 0% O2 conditions for 48 h. Cells exposed to 0% O2 displayed a significant increase in cell death compared with cells exposed to 21% O2 or cells exposed to 0% O2 in the presence of zVAD-fmk (Fig. 3B). These results suggest that caspases are important executioners of the cell death pathway in A549 cells after exposure to anoxia.


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Fig. 3.   Caspases are involved in anoxia-induced cell death. A: Neo and Bcl-XL A549 cells were exposed to 0% O2 for 24 and 36 h and assayed for caspase-9 activity. B: the caspase inhibitor z-Val-Ala-asp-fluoromethyl-ketone (zVAD-fmk, 100 µM) protects against anoxia-induced cell death. *P < 0.05; n = 4. Values are means ± SE.

Bcl-XL maintains a partial psi  during anoxia. Previous studies have shown that loss of cytochrome c is accompanied by a loss of psi  (11, 31, 35). We examined changes in psi  in Neo control and Bcl-XL transfected A549 cells exposed to 0% O2 for 12, 24, 36, and 48 h. psi  was measured using the ratio of TMRE and MITO. TMRE fluorescence is dependent on psi , whereas MITO fluorescence is independent of Delta psi and reflects the number of mitochondria within a given cell. Neo A549 cells had a significant drop in potential at the point of cytochrome c release as demonstrated by a 78.3 ± 4.8% and 84.8 ± 3.2% decrease in potential after 36 and 48 h of 0% O2, respectively (Fig. 4A). Bcl-XL maintained a potential of 49.7 ± 5.8% and 54.5 ± 3.0% after 36 and 48 h of 0% O2 after 48 h. Thus Bcl-XL prevents the loss of mitochondrial integrity during anoxia as indicated by the maintenance of a psi  and the prevention of cytochrome c release in A549 cells overexpressing Bcl-XL.


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Fig. 4.   Bcl-XL prevents the depolarization of the mitochondrial membrane potential in response to anoxia. A: control (Neo) and Bcl-XL A549 cells were exposed to 0% O2 for 12, 24, 36, and 48 h, and tetramethylrhodamine ethyl ester (TMRE) fluorescence was measured. B: the permeability transition pore inhibitor cyclosporin A does not prevent anoxia-induced cell death. Wild-type A549 cells were exposed 21 and 0% O2 in the presence of 0.5 or 5 µM cyclosporin A for 48 h and assayed for LDH release. *P < 0.05; n = 4. Values are means ± SE.

The permeability transition pore (PTP) has been implicated as a possible mechanism of mitochondrial release of cytochrome c and loss of mitochondrial potential during anoxia (15). Furthermore, Bcl-XL has been shown to prevent the formation of the PTP. To test whether the involvement of the PTP was required during anoxia-induced cell death, A549 cells were exposed to 0% O2 for 48 h in the presence of the PTP inhibitor cyclosporin A and assayed for LDH release. Cyclosporin A at 0.5 or 5 µM did not prevent anoxia-induced cell death (Fig. 4B). These results indicate that the formation of the PTP is not involved in the execution of anoxia-induced death in A549 cells.

Anoxia-induced cell death requires a functional mitochondrial electron transport chain. To determine whether electron transport chain was required for anoxia-induced death in A549 cells, wild-type and rho °-A549 cells were exposed to 21 or 0% O2 for 24 or 48 h and assayed for cell death. rho °-cells have been depleted of mitochondrial DNA, which encodes for 13 genes, including critical subunits of the electron transport chain (3). Previous studies have demonstrated rho °-cells were able to die in response to growth factor withdrawal (7). However, our current results indicate that rho °-A549 cells did not undergo death after exposure to anoxia. Wild-type A549 cells exposed to 0% O2 exhibited 40.2 ± 4.8% cell death compared with 13.4 ± 1.9% cell death in rho °-A549 cells (Fig. 5). Thus these results indicate that a functional electron transport is required for anoxia-induced cell death.


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Fig. 5.   Cells lacking functional mitochondrial electron transport chain (rho °-cells) are resistant against anoxia-induced cell death. Wild-type and rho °-A549 cells were exposed to 21 and 0% O2 for 24 and 48 h and assayed for LDH release. *P < 0.05; n = 4. Values are means ± SE.

Hypoxia activates HIF-1 but does not prevent anoxia-induced cell death in A549 cells. HIF-1 is a basic helix-loop-helix/PAS protein consisting of 120-kDa HIF-1alpha and 91- to 94-kDa HIF-1beta subunits. HIF-1alpha protein is present only in hypoxic cells, whereas HIF-1beta protein levels are constitutively expressed and not significantly affected by O2. HIF-1-targeted genes include the glycolytic enzymes and glucose transporters (27). We tested whether the activation of HIF-1 would prevent anoxia-induced cell death by exposing wild-type A549 cells to 1.5% O2 for 24 h to activate HIF-1, followed by a 48-h exposure to anoxia. Figure 6A demonstrates that an HIF-1-dependent luciferase reporter construct increased 130 ± 21-fold after 24 h of 1.5% O2 compared with 21% O2. Furthermore, a Western blot analysis to detect HIF1-alpha showed a drastic increase at 1.5% O2 (Fig. 6A). However, wild-type A549 cells exposed to 1.5% O2 for 24 h to activate HIF-1 and subsequently exposed to 0% O2 for 48 h displayed similar level of cell death as cells that were exposed to 48 h of anoxia without the hypoxic preconditioning (Fig. 6B). A549 cells exposed to 1.5% O2 for 48 h did not display an increase cell death compared with cells under 21% O2. Collectively, these results indicate that hypoxic preconditioning activates HIF-1 but does not affect anoxia-induced cell death.


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Fig. 6.   Hypoxia activates hypoxia-inducible factor (HIF)-1 in A549 cells but does not prevent anoxia-induced cell death. A: wild-type A549 cells were exposed to 1.5% O2 for 24 h. HIF-1 activity was measured with a hypoxia response element-dependent luciferase reporter construct, and HIF-1alpha stabilization was measured by Western blot analysis. B: A549 cells were preconditioned at 1.5% O2 for 24 h and subsequently exposed to 0% O2 for 48 h. The cells were assayed for LDH release to determine cell death.

Tumor necrosis factor-alpha activates NF-kappa B but does not prevent anoxia-induced cell death in A549 cells. Tumor necrosis factor (TNF)-alpha can activate either the intracellular death machinery or a signaling pathway, resulting in stimulation of the transcription factor NF-kappa B (2). Hypoxia can stimulate NF-kappa B activation, which itself can then activate a variety of antiapoptotic genes. To examine whether activation of NF-kappa B by TNF-alpha would prevent anoxia-induced cell death, wild-type A549 cells were exposed to TNF-alpha for 24 h followed by exposure to 0% O2 for 48 h. TNF-alpha activation of NF-kappa B was measured with a luciferase reporter construct containing two NF-kappa B sites. A 9.0 ± 2.3-fold increase was seen in NF-kappa B activity after 24 h of exposure to TNF-alpha , whereas a smaller increase of 4.6 ± 1.6-fold was found after exposure to 1.5% O2 for 24 h (Fig. 7A). Exposure of A549 cells to TNF-alpha (10 ng/ml) for 48 h at 21% O2 did not trigger cell death. A549 cells exposed to TNF-alpha (10 ng/ml) for 24 h to activate NF-kappa B and subsequently exposed to 0% O2 for 48 h displayed a similar level of cell death as cells that were exposed to 48 h of anoxia without the TNF-alpha preconditioning (Fig. 7B). Taken together, these results indicate that TNF-alpha preconditioning resulting in NF-kappa B activation does not prevent anoxia-induced cell death.


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Fig. 7.   Tumor necrosis factor (TNF)-alpha activates nuclear factor (NF)-kappa B but does not prevent anoxia-induced cell death. A: wild-type A549 cells were exposed to 21% O2, 1.5% O2, and TNF-alpha for 24 h, and NF-kappa B activity was measured via a luciferase assay. B: A549 cells were preconditioned with TNF-alpha for 24 h at 21% O2 and subsequently exposed to 48 h of 21 and 0% O2 in the presence of TNF-alpha (10 ng/ml). The cells were assayed for LDH release to determine cell death.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An early feature of apoptosis in response to a variety of death stimuli is the disruption of outer mitochondrial membrane integrity resulting in the release of the cytochrome c and other proapoptotic molecules from the mitochondria to the cytosol (6). In the present study we examined whether a loss of mitochondrial integrity was an early event in apoptosis induced by anoxia in A549 cells. Cytochrome c release, followed by caspase-9 activation, was observed before plasma membrane breakage and DNA fragmentation in A549 cells exposed to anoxia. The loss of cytochrome c was preceded by a depolarization in psi , whereas an almost complete dissipation of psi  was observed after cytochrome c release. The broad-based caspase inhibitor zVAD-fmk did not prevent the release of cytochrome c during anoxia, which suggests that caspases are not involved in the initiation of cytochrome c release. However, zVAD-fmk was able to significantly prevent anoxia-induced cell death, indicating that caspases play an important role in cell death after cytochrome c release. By contrast, Bcl-XL, an antiapoptotic protein that localizes to the outer mitochondrial membrane, was able to prevent the dissipation of psi , release of cytochrome c, caspase-9 activation, and cell death during anoxia. These results indicate that the loss of mitochondrial integrity is an important early regulatory step in the apoptotic pathway induced by anoxia.

A possible mechanism proposed, in a variety of cells, for cytochrome c release and mitochondrial depolarization is the opening of the PTP (13). This pore is a large-conductance channel that spans both mitochondrial membranes. Opening of the pore is characterized by an abrupt increase in the permeability of the inner mitochondrial membrane to molecules as large as 1,500 Da. Primary evidence for a PTP model comes from experiments showing that cyclosporin A, an inhibitor of PTP formation, prevents cell death. However, our present data indicate that cyclosporin A does not prevent cell death in response to anoxia in A549 cells. These data suggest that PTP may not be a major regulator of death in these cells. Other possible mechanisms for cytochrome c release could be specific cytochrome c-conducting channels in the outer membrane or the nonspecific disruption of the outer membrane as a result of proapoptotic Bcl-2 family members such as Bax or Bak (21). In this case, antiapoptotic proteins such as Bcl-XL would bind to Bax or Bak to prevent cytochrome c release.

A fundamental question in understanding anoxia-induced apoptosis is determining the initiating mechanism for cell death. Under normal physiological conditions, the oxidation of NADH is coupled to the reduction of O2 through the respiratory chain (24). O2 is reduced to water by cytochrome c oxidase. Electron transfer through the respiratory chain is coupled to the directional movement of protons across the inner mitochondrial membrane. This movement across the membrane establishes an electrochemical potential that provides the thermodynamic driving force for the F1F0-ATP synthase to generate ATP in the matrix. Thus a potential mechanism to initiate anoxia-induced apoptosis is that a lack of O2 inhibits cytochrome c oxidase within the mitochondrial electron transport chain, resulting in the activation of programmed cell death. Experimental evidence in the present study indicates that rho °-A549 cells that lack mitochondrial DNA do not undergo anoxia-induced cell death. Mitochondrial DNA encodes the three catalytic subunits of cytochrome c oxidase, whereas nuclear DNA encodes the proapoptotic protein cytochrome c. Previous studies have shown that rho °-cells are able to undergo cell death in response to a variety of apoptotic stimuli, including growth factor withdrawal, ligation of death receptors, and staurosporine (7). Therefore our current results indicate that the initiating mechanism of apoptosis during anoxia is different from other classic apoptotic stimuli.

A potential therapeutic mechanism to prevent anoxia-induced apoptosis could be to activate genes encoded by the transcription factors HIF-1 and NF-kappa B. HIF-1 is able to induce the expression of glycolytic genes and the angiogenic factor vascular endothelial growth factor (VEGF) (26). HIF-1 is necessary for the normal embryonic and cardiovascular system development and is also implicated in cancer progression and apoptosis. Activation of HIF-1 in vivo could prevent cells from being exposed to anoxic conditions by stimulating VEGF production, thereby promoting angiogenesis. By contrast, the activation of HIF-1 in vitro could activate glycolytic enzymes that could provide ATP that is normally generated by electron transport during anoxia to prevent cell death. Our current data suggest that activation of HIF-1 is not sufficient to prevent cell death during anoxia in vitro. A549 cells preconditioned with 24 h of 1.5% O2 to activate HIF-1 target genes did not prevent subsequent anoxia-induced cell death. Hence, glycolysis upregulation via HIF-1 is not likely to be an important regulator of anoxia-induced cell death.

The NF-kappa B pathway is also a key mediator of genes involved in the control of apoptosis (14). Antiapoptotic genes that are directly activated by NF-kappa B include the cellular inhibitors of apoptosis (c-IAP1, c-IAP2, and IXAP), the TNF-alpha receptor-associated factors (TRAF1 and TRAF2), and IEX-1L. TNF-alpha is one of the classical inducers of NF-kappa B and can induce cells to undergo apoptosis (33). The sensitivity of different cell types to TNF-alpha -induced apoptosis can vary dramatically, but most cells become very sensitive upon simultaneous treatment with inhibitors of protein synthesis. It has been suggested therefore that a gene, or set of genes, is induced upon TNF-alpha receptor activation that downregulates the apoptosis signal. Recent results have shown that NF-kappa B activated by TNF-alpha is at least partly responsible for this effect. Indeed, our current results show that A549 cells do not undergo cell death with TNF-alpha treatment alone. TNF-alpha is sufficient to activate NF-kappa B in these cells. However, A549 cells that were preconditioned for 24 h with TNF-alpha to activate NF-kappa B could not protect against subsequent anoxia-induced cell death. These results suggest that NF-kappa B target genes are not likely to regulate anoxia-induced apoptosis.

In summary, our findings provide evidence that anoxia-induced apoptosis requires the inhibition of a functional electron transport chain and the release of cytochrome c in lung epithelial cells. The antiapoptotic Bcl-XL protein can prevent the release of cytochrome c and subsequent cell death during anoxia, whereas preconditioning with hypoxia or TNF-alpha does not prevent cell death during anoxia. Collectively, these results indicate that the main regulatory step of anoxia-induced apoptosis is at the level of the mitochondria.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of General Medicine Sciences Grant GM-60472-02 (to N. S. Chandel) and the Crane Asthma Center.


    FOOTNOTES

Address for reprint requests and other correspondence: N. S. Chandel, Div. of Pulmonary & Critical Care, Tarry Bldg. 14-707, 300 E. Superior St., Chicago, IL 60611-3010 (E-mail: nav{at}northwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00281.2001

Received 24 July 2001; accepted in final form 6 November 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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