1 Renal Section, Department of Medicine, Boston Medical Center, Boston University, Boston 02118-2518; 2 Department of Pathology, Tufts University and New England Medical Center, Boston, Massachusetts 02111-1533; and 3 Department of Nephrology, First Affiliated Hospital, Zhongshan University, GuangZhou, China 510080
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ABSTRACT |
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The events that precipitate cell death and the stress proteins responsible for cytoprotection during ATP depletion remain elusive. We hypothesize that exposure to metabolic inhibitors damages mitochondria, allowing proapoptotic proteins to leak into the cytosol, and suggest that heat stress-induced hsp72 accumulation prevents mitochondrial membrane injury. To test these hypotheses, renal epithelial cells were transiently ATP depleted with sodium cyanide and 2-deoxy-D-glucose in the absence of medium dextrose. Recovery from ATP depletion was associated with the release into the cytosol of cytochrome c and apoptosis-inducing factor (AIF), proapoptotic proteins that localize to the intermitochondrial membrane space. Concomitant with mitochondrial cytochrome c leak, a seven- to eightfold increase in caspase 3 activity was observed. In controls, state III mitochondrial respiration was reduced by 30% after transient exposure to metabolic inhibitors. Prior heat stress preserved mitochondrial ATP production and significantly reduced both cytochrome c release and caspase 3 activation. Despite less cytochrome c release, prior heat stress increased binding between cytochrome c and hsp72. The present study demonstrates that mitochondrial injury accompanies exposure to metabolic inhibitors. By reducing outer mitochondrial membrane injury and by complexing with cytochrome c, hsp72 could inhibit caspase activation and subsequent apoptosis.
hsp72; cytochrome c; caspase 3; apoptosis-inducing factor; mitochondrial membrane potential
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INTRODUCTION |
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APOPTOSIS HAS EMERGED as an important cause of cell death in renal epithelial cells subjected to stress. Observations in both in vivo and in vitro models demonstrate that ischemia (38, 47), oxidant stress (16), hypoxia (45), ultraviolet (UV) irradiation (25), urinary tract obstruction (11), glomerulonephritis (58), and kidney transplantation (12) cause apoptosis. Exposure to metabolic inhibitors, an in vitro model of ischemia, also induces apoptosis in renal epithelial cells (29, 45, 53). Prior heat stress sufficient to induce hsp72, a cytoprotectant protein, reduces apoptosis in ATP-depleted renal cells (53). However, the biochemical pathways that mediate ischemia-induced apoptosis and the mechanism(s) of cytoprotection by heat stress proteins (HSPs) have not been clarified (46).
The mitochondrion has recently been implicated as the primary regulator
of apoptosis after diverse forms of cell injury (21, 45,
50, 51, 59). Disruption of the inner mitochondrial membrane
dissipates the 220 mV proton gradient responsible for the
mitochondrial membrane voltage potential (
m,)
(13, 43, 48, 57). In contrast, changes in the permeability
of the outer membrane release proteins that are normally restricted to
the intermitochondrial membrane space into the cytosol that can cause apoptosis (2, 24, 49). These two events may be
independent of one another (5). Proapoptotic
mitochondrial proteins in the intermembranous space include (but are
not limited to) cytochrome c, several caspases, and
apoptosis-inducing factor (AIF) (14, 24, 46, 50).
On release of mitochondrial cytochrome c into the cytosol,
the "apoptosome" is formed. This multimeric complex consists of
procaspase 3, caspase 9, apoptosis-activating factor (APAF)-1,
and cytochrome c (1, 4). The apoptosome
activates caspase 9, initiating the self-amplifying apoptotic
enzyme cascade (1). In contrast to cytochrome
c, mitochondrial AIF migrates to the nucleus, causing DNA
fragmentation and partial chromatin condensation (14, 49).
In the present study, we tested the hypothesis that exposure to cyanide and 2-deoxy-D-glucose in the absence of exogenous dextrose induces apoptosis in renal epithelial cells by injuring the outer mitochondrial membrane. Furthermore, we evaluated the possibility that the induction of HSPs, including hsp72, decreases apoptosis by ameliorating outer mitochondrial membrane injury and inhibiting subsequent caspase activation. The present studies show that inhibition of electron transport causes a rapid loss of mitochondrial membrane potential associated with the redistribution of both mitochondrial cytochrome c and AIF to the cytosolic compartment. Loss of mitochondrial cytochrome c and AIF is accompanied by a marked fall in maximal mitochondrial ATP production and activation of caspase 3. In contrast, the accumulation of hsp72 is associated with preserved mitochondrial function, a decrement in cytochrome c release after exposure to metabolic inhibitors, and significantly less activation of caspase 3. We describe for the first time an interaction between cytochrome c and hsp72 and suggest two mechanisms by which hsp72 might inhibit apoptosis.
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METHODS |
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Materials. All reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise indicated.
Cell culture. Renal epithelial cells from the opossum kidney (OK) were obtained from the American Type Culture Collection (ATCC CRL-1840) and were grown in DMEM (GIBCO BRL, Grand Island, NY) supplemented with 10% FCS. Cells were used within 72 h of achieving confluence.
ATP depletion and induction of hsp72. To induce ATP depletion, cells were incubated for 1 h at 37°C in glucose-free medium (DMEM; GIBCO BRL no. 23800-014) that contained sodium cyanide and 2-deoxy-D-glucose (5 mM each) as previously described (55). Fresh DMEM containing 10 mM glucose (without metabolic inhibitors) was used to initiate recovery. Parallel medium changes were made in controls using glucose-containing DMEM. To induce hsp72, OK cells were heated to 42.5 ± 0.5°C for 45 min in a temperature-regulated incubator followed by incubation at 37°C for 16-18 h (55).
m.
m was assessed in viable OK cells with the use of
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine
iodide (JC-1; Molecular Probes, Eugene, OR), a lipophilic, cationic
dye, according to the manufacturer's protocol. Cells were plated on coverslips coated with rat tail collagen in 12-well plates in DMEM
supplemented with 10% FCS. Subconfluent monolayers of cells were
washed with PBS three times and then incubated with DMEM containing
cyanide (5 mM) and 2-deoxy-D-glucose (5 mM) in glucose-free DMEM at 37°C. JC-1 (10 µg/ml) was added during the final 10 min of
exposure to the metabolic inhibitors (for 30, 60, and 90 min in the
absence of recovery). To intentionally disrupt
m, 1 µM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone
(FCCP) was added to cells for 10 min before addition of JC-1. The cells
were rinsed twice, and then coverslips were mounted on glass slides. Dual-emission images were simultaneously obtained by using an inverted
laser scanning confocal microscope (Zeiss LSM 510, Thornwood, NY) at
475 nm (excitation) and 525 and 590 nm (emission) in
1-µm-thick sections.
ATP production rate. State III mitochondrial respiration, the maximal capacity for mitochondria to generate ATP, was estimated by use of an assay developed in our laboratory (6, 55). In this assay, cells are selectively permeabilized with digitonin (0.15 mg/ml) in the presence of metabolic substrates (5 mM glutamate, 1 mM butyrate, 5 mM malate, and 600 nmol ADP) for 10 min at 37°C. ATP production was measured with luciferase (Analytical Luminescence Laboratory, San Diego, CA) in a luminometer (Turner Designs, Sunnyvale, CA). The results are expressed in nanomoles of ATP per milligram of protein.
Immunoblot analysis and coimmunoprecipitation.
Harvested cells were resuspended in cell lysis buffer (containing 150 mM NaCl, 10 mM Tris · HCl, 5 mM EDTA, 1 mM EGTA, and 1% Triton
X-100) and protease inhibitors (5 µM AEBSF-HCl, 10 nM leupeptin, 1.5 nM aprotinin, 10 nM E-64, and 5 µM EDTA; pH 7.40; Calbiochem-Novabiochem, San Diego, CA). The cells were sonicated and
then centrifuged at 10,000 g for 10 min at 4°C. Antigens
in the supernatant were detected by immunoblot with the use of
commercially available monoclonal antibodies directed against
cytochrome c (Research Diagnostics, Flanders, NJ; catalog
no. RDI-cytoC12-abm), hsp72 (Amersham, Arlington Heights,
IL), and AIF (Santa Cruz Biotechnology, Santa Cruz, CA). Specific
protein bands were detected with an anti-IgG antibody coupled to a
horseradish peroxidase-based enzyme-linked chemiluminescence system
(Lumigolow; Kirkegaard and Perry, Gaithersberg, MD). After digitization
of the image of each immunoblot (Hewlett-Packard, Desk Scan II), band
densities were quantified by use of NIH Image Quant software. Cytosolic
protein fractions were obtained by incubation of cells with 0.15 mg/ml
digitonin for 10 min at 4°C before immunoprecipitation (IP). Samples
were then incubated overnight at 4°C with a polyclonal rabbit
antibody directed against cytochrome c (1-2
µg · mg protein1 · ml
buffer
1; Santa Cruz Biotechnology) or hsp72
(1-2 µg · mg protein
1 · ml
buffer
1; Stress Gen). The IP buffer contained 150 mM
NaCl, 10 mM Tris · HCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100,
0.5% NP-40, and protease inhibitors at pH 7.4. To prevent the
potential release of proteins attached to hsp72 during
isolation, apyrase (10 U/ml), a compound that causes ATP hydrolysis,
was added. The absence of ATP prevents the dissociation of HSP70 from
its bound ligands during sample preparation (53),
permitting coimmunoprecipitation studies to be performed
(54). After protein separation by 12% SDS gel
electrophoresis, the immunoblots were probed with one or more specific antibodies.
Cellular distribution of cytochrome c and AIF. To visualize mitochondria, subconfluent, live cells grown on glass coverslips were incubated with Mitotracker Green-FM (600 nM; Molecular Probes), a mitochondrial specific marker, for 30 min at 37°C. Cells were then fixed in methanol (4°C for 20 min), and routine immunohistochemistry was performed with an anti-cytochrome c antibody (cytoC12-abm; Research Diagnostics) that was detected with a Cy3-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Confocal microscopy was used to localize cytochrome c and Mitotracker Green.
To quantify "cytosolic" and "mitochondrial" pools of cytochrome c and AIF, immunoblot analysis was performed in samples of cells exposed to either digitonin or SDS, respectively. Exposure to digitonin, a selective detergent, permeabilizes the plasma membrane without disrupting mitochondria (45) or their ability to generate ATP (6). In contrast, SDS solubilizes virtually all cell and organelle membranes, permitting cytochrome c (and AIF) within mitochondria to be detected (45). After removal of the medium, cell monolayers were incubated for 10 min at 4°C in 100-mm2 dishes with 2 ml of an intracellular-like buffer containing 120 mM KCl, 5 mM KH2PO4, 10 mM HEPES, 2 mM EGTA, 1% bovine serum albumin, 0.15 mg/ml digitonin, and a mixture of protease inhibitors (described above) and then harvested with a rubber policeman as previously described (55). Samples were centrifuged at 14,000 g for 5 min at 4°C. The supernatant was designated as the "digitonin-soluble" protein fraction. The pellet was incubated in 2% SDS at 4°C for 5 min, sonicated, and then centrifuged at 14,000 g for 5 min. This supernatant was designated as the "SDS-soluble" protein fraction. The content of cytochrome c and AIF was examined in both fractions by immunoblot analysis.Caspase 3 activity. Caspase 3 enzyme activity was measured with the use of a fluorometric assay according to the manufacturer's protocol (ApoAlert caspase 3 fluorescence assay; Clontech Laboratories, Palo Alto, CA). OK cells grown on 60-mm2 dishes were lysed with 200 µl of chilled cell lysis buffer (Clontech Laboratories) on ice for 10 min. After centrifugation (12,000 rpm × 3 min, 4°C), 50 µl of supernatant, 50 µl of 2× reaction buffer containing 1 mM 1,4-dithiothreitol, and 5 µl of caspase substrate (DEVD-AFC; 50 µM) were added to each well of a 96-well plate. After incubation at 37°C for 1 h, fluorescence was determined with the use of a plate reader with a 400-nm excitation filter and 505-nm emission filter (Spectra Max Gemini, Molecular Devices, Sunnyvale, CA). To confirm assay specificity, parallel reactions were performed either without substrate or in the presence of a specific caspase 3 inhibitor (1 µl DEVD-CHO; Clontech Labs). The role of hsp72 per se in caspase 3 activation was evaluated by adding 1 µg of purified human hsp72 (Stress Gen, SPP-755, Victoria, BC, Canada) to the assay mixture and then repeating measurements of caspase 3 activity. This concentration of hsp72 exceeds the concentration of this protein detected in cells subjected to heat stress (44).
Protein assay. Protein concentrations were determined with a colorometric dye-binding assay [bicinchoninic acid (BCA) assay; Pierce, Rockford, IL]. Results are expressed in milligrams of protein per milliliter.
Statistical analysis. Data are expressed as means ± SE. Comparison of two groups was performed using a two-tailed Student's t-test. Results involving more than one group were compared using analysis of variance (ANOVA) and were then analyzed with the Fishers post hoc test. A result was considered significant if P value < 0.05.
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RESULTS |
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To determine the effect of exposure to cyanide and
2-deoxy-D-glucose in the absence of exogenous dextrose on
m, live cells were incubated with JC-1 (Fig.
1). In control cells (Fig.
1A), mitochondria demonstrated a heterogeneous
pattern of fluorescence. Many mitochondria exhibited red fluorescence,
demonstrating JC-1 aggregation in response to a normal
m. Some mitochondria exhibited green fluorescence,
demonstrating the presence of JC-1 monomers, a finding consistent with
a low
m. In other control cells, a white pseudocolor
(representing colocalization of JC-1 monomers and aggregates) was
observed. Higher magnification confirmed the heterogeneous pattern of
m within a single cell (Fig. 1A,
inset). After 30-min exposure to metabolic inhibitors
(without recovery), most JC-1 was present in the monomeric (green) form
(Fig. 1B). At higher magnification, most mitochondria were
depolarized (Fig. 1B, inset). Green JC-1 monomers
also predominated after 60 and 90 min of exposure to metabolic
inhibitors without recovery (data not shown). Incubation with FCCP, a
mitochondrial uncoupling agent (data not shown), produced similar
increases in the monomeric form of JC-1 as did exposure to metabolic
inhibitors. Prior heat stress (Fig. 1C) did not have an
independent effect on
m, since the pattern of JC-1
staining was similar to that observed in control cells. During exposure
to metabolic inhibitors, most mitochondria in previously heated cells
were depolarized (Fig. 1D).
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If the mitochondria were a target for injury by metabolic inhibitors,
then organelle dysfunction would be expected. To assess mitochondrial
function, the rate of ATP production was examined in selectively
permeabilized OK cells under conditions that estimate maximal (state
III) mitochondrial respiration. Compared with control, the maximal rate
of ATP production fell by ~50% after 60 min of exposure to metabolic
inhibitors (Fig. 2). In this experiment, cyanide and 2-deoxy-D-glucose were removed during the
measurements of mitochondrial respiration. Prior heat stress prevented
the mitochondrial dysfunction associated with exposure to metabolic inhibitors (P < 0.05; ATP deplete vs. heat stress + ATP deplete; n = 6). The observation that previously
heated cells exposed to metabolic inhibitors exhibited similar rates of
respiration as did controls suggests that these agents had little if
any residual effect on mitochondrial metabolism. Prior heat stress
alone (43°C followed by 16 h of recovery at 37°C) did not
significantly alter mitochondrial ATP production compared with controls
(P > 0.05).
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Because exposure to metabolic inhibitors induced mitochondrial
depolarization and impaired oxidative phosphorylation, release of
cytochrome c was examined by two complementary techniques. First, cytochrome c (detected with a primary antibody to
cytochrome c and a secondary antibody coupled to Cy3) and
Mitotracker Green (a fluorescent probe that is highly concentrated
within mitochondria) were localized in renal epithelial cells by use of
immunohistochemistry. In control cells, cytochrome c and
Mitotracker colocalized, represented as yellow-orange fluorescence
(Fig. 3A). After 60 min of
exposure to metabolic inhibitors, cytochrome c and
Mitotracker no longer colocalized in many cells (Fig. 3B).
By immunofluorescence, cytochrome c (red) was not observed
in the cytosol of cells exposed to metabolic inhibitors. This may be
due to the translocation of a relatively small amount of cytochrome
c into a large compartment (i.e., the cytosol). To quantify
cytochrome c release, immunoblot analysis was performed on
two cell fractions: a cytosolic fraction (normally containing no
immunoreactive cytochrome c) obtained after exposure of
intact cells to digitonin, a plasma membrane-selective detergent, and a
whole cell protein fraction (including mitochondria) solubilized with
SDS, a nonselective detergent. In control cells, leak of cytochrome
c (14 kDa) into the cytosol could be observed after 60 min
of exposure to cyanide and 2-deoxy-D-glucose in the absence of dextrose (Fig. 4A).
Cytosolic cytochrome c increased 30 min after removal of the
metabolic inhibitors (ATP repletion). In contrast, prior heat stress
decreased the translocation of cytochrome c. In both control
and previously heated cells, exposure to metabolic inhibitors or
recovery caused minimal changes in the amount of residual mitochondrial
cytochrome c detected in the whole cell fraction (SDS). To
quantify the leak of cytochrome c during and after exposure
to metabolic inhibitors, data from several studies were analyzed by
densitometry (Fig. 4B). In these studies, prior heat stress
significantly reduced the leak of cytochrome c associated with exposure to metabolic inhibitors (P < 0.05, n = 3).
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To confirm injury to the mitochondrial membrane, the translocation of
AIF (57 kDa) was also examined after exposure to metabolic inhibitors.
In control cells, virtually no AIF was detected in the protein fraction
obtained by exposing cells to digitonin (Fig. 5). In contrast to normal cells at
baseline, progressively larger amounts of AIF were detected in the
cytosolic protein fraction after the removal of metabolic inhibitors.
The major immunoreactive band present in the protein fraction obtained
after exposure to SDS confirmed the localization of AIF at the expected
molecular mass. A single minor band (~40-45 kDa) was also
detected in the SDS protein fraction. Although not characterized, this
minor band has been previously detected by others (15).
Similar to cytochrome c, most AIF remained in the
mitochondrial compartment even in injured cells.
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The release of intermitochondrial membrane proteins into the cytosol
could result from nonspecific mitochondrial swelling/rupture to
selective opening of a channel in the outer membrane (5, 51) or to both. To distinguish between these possibilities, cells were preincubated with bongkrekic acid (50 µM × 2 h), an inhibitor of the membrane permeability transition pore (MPT;
Refs. 5 and 10). Exposure to bongkrekic acid alone did not
cause AIF release (data not shown). This MPT inhibitor significantly reduced AIF leak (Fig. 6) during ATP
depletion as well as during both time points of recovery
(P < 0.05 vs. control).
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To determine whether the leak of cytochrome c in
ATP-depleted cells activated the apoptotic pathway, caspase 3 activity was measured. This assay did not require digitonin. This is
important in that detergent exposure could increase the release of
cytochrome c and/or AIF from mitochondria injured by
exposure to metabolic inhibitors. Compared with control cells, exposure
to metabolic inhibitors resulted in a seven- to eightfold increase in
caspase 3 activity (P < 0.5, n = 3;
Fig. 7). Prior heat stress significantly ameliorated the activation of caspase 3 caused by exposure to metabolic
inhibitors (P < 0.05, n = 3). Purified
hsp72 (1 µg) added to the caspase 3 reaction mixture (in
vitro) did not alter the activity of caspase 3 in cell lysates obtained
from cells exposed to metabolic inhibitors (data not shown). In
contrast, the addition of a specific inhibitor of caspase 3 completely
abolished caspase activity, confirming the specificity of this assay
(data not shown).
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Potent suppression of caspase 3 despite measurable cytochrome
c release in heat-stressed cells suggested that an
additional mechanism might inhibit apoptosis. To explore the
possibility that hsp72 might prevent caspase activation by
binding cytosolic cytochrome c, coimmunoprecipitation of the
two proteins was investigated. In control cells, interaction between
hsp72 and cytochrome c was observed immediately
after 60 min of exposure to metabolic inhibitors and to a lesser degree
during recovery (Fig. 8). Despite the
reduction in cytosolic cytochrome c in previously heated
cells (Fig. 8B), the interaction between these two proteins
after exposure to metabolic inhibitors was markedly increased (Fig.
8A). Immunoblot analysis confirmed the adequacy of heat
stress in increasing the content of hsp72 (Fig.
8C). Persistent nonspecific bands precluded interpretation of studies in which hsp72 was immunoprecipitated and the
blots were probed for cytochrome c (data not shown).
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DISCUSSION |
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Historically, the mitochondrion has been overlooked as an early mediator of apoptosis (30, 51). This is largely due to the fact that major changes in mitochondrial membrane integrity and organelle function occur well before the classic morphological signs of cell death are evident (30, 51). Subsequent investigations have shown that mitochondrial products are rate limiting for activating caspase and endonuclease in cell free systems, that stabilization of mitochondrial membranes prevents apoptosis, and that selective overexpression of mitochondrial protective proteins (e.g., Bcl family members) ameliorates apoptosis (reviewed in Ref 51). These observations emphasize the role of mitochondria in regulating apoptosis.
In the present study, mitochondrial injury accompanied exposure to
metabolic inhibitors in vitro. m decreased within 30 min of exposure to metabolic inhibitors and remained low up to 90 min.
This observation is similar to that reported by Weinberg et al.
(57) in renal epithelial cells subjected to
hypoxia/reoxygenation. The observed decrement in
m is
not surprising, since ATP depletion included exposure to cyanide, a
potent inhibitor of mitochondrial respiration. The loss of
m has been attributed to dissipation of the proton
gradient responsible for the voltage across the inner mitochondrial
membrane that in some circumstances is associated with the opening of
the MPT (51). The MPT is a regulated megachannel composed
of the adenine nucleotide translocator (ANT), the voltage-dependent anion channel (VDAC), cyclophilin D, hexokinase, and other
yet-unidentified proteins (43, 51) that are susceptible to
stress (9, 23, 31).
In addition to disrupting m, exposure to metabolic
inhibitors caused a 50% reduction in state III mitochondrial
respiration (Fig. 2). This decrease in mitochondrial energy production
is multifactoral. First, the protein gradient across the mitochondrial membrane is directly coupled to ATP production. A decrease in
m during ATP depletion would reduce ATP production by
oxidative phosphorylation. Second, loss of cytochrome c
during the period of ATP depletion and early recovery would directly
compromise electron transport. Third, loss of AIF, a flavoprotein
(35), could adversely affect metabolic function by
mitochondria. Last, Weinberg et al. (57) have recently
demonstrated that hypoxia/reoxygentation of rabbit tubules in
suspension damages complex I enzymes in the oxidative phosphorylation
pathway proximal to cytochrome c (57). A
decrement in the ability to generate ATP ultimately leads to a fall in
ATP content. Both the absolute level of ATP and the duration of ATP
depletion are critical determinants of whether the renal epithelial
cell dies by apoptosis or necrosis (18, 29).
In the present study, it is important to note that the release of cytochrome c and AIF (Figs. 4 and 5) precedes morphological evidence of apoptosis (e.g., the formation of apoptotic bodies, chromatin condensation, and endonucleosomal DNA fragmentation) previously observed in these same cells after ATP depletion (53). In addition, substantial evidence suggests that the release of either cytochrome c or AIF is sufficient to cause apoptosis (15, 43, 49). In forming the apoptosome, cytochrome c activates caspase 9, the first step in the enzyme cascade that ultimately causes apoptosis (7, 8). In contrast, AIF induces nuclear changes that are characteristic of apoptosis by a "caspase-independent" mechanism that directly activates endonucleases and causes chromatin condensation (15, 17, 24, 49). In the present study, release of both cytochrome c and AIF into the cytosol implies that outer mitochondrial membrane permeability has been altered during ATP depletion and recovery. Loss of mitochondrial membrane integrity could result from nonspecific membrane injury or more selective opening of the MPT. To distinguish between these possibilities, cells were preincubated with bongkrekic acid, an MPT inhibitor that binds to the ANT (10). Because this agent is reportedly more specific and effective for inhibiting MPT than cyclosporine A (51), the later agent was not used in the present study. Bongkrekic acid significantly inhibited AIF release during and after exposure to metabolic inhibitors (Fig. 6). Incomplete protection of AIF release by bongkrekic acid could be due to a suboptimal drug concentration or inadequate exposure to the agent before injury. Alternatively, AIF leak could also occur by an MPT-independent mechanism precipitated by mitochondrial swelling or rupture (41).
What factors disrupt the outer mitochondrial membrane? Recent investigations have focused on the role of two proteins, BAX and Bcl2, as mediators of mitochondrial permeability transition (PT) and apoptosis (9, 23, 31, 32, 34, 45). Insults that increase the ratio of BAX to Bcl2 promote apoptosis by allowing BAX to form homodimers within the mitochondrial membrane (26, 34). The mechanism by which BAX causes membrane injury remains unclear. Some investigators suggest that BAX cooperates with the ANT, promoting MPT (9). Others suggest that BAX does not require association with components of the MPT to create an outer membrane channel (34). In contrast to BAX, Bcl2 prevents MPT opening by interfering with BAX (9, 26, 34). In renal epithelial cells, hypoxia and ATP depletion are associated with marked changes in both BAX and Bcl2. After induction of ATP depletion with a mitochondrial uncoupling agent, BAX translocates to the mitochondrial membrane (45). Translocation of BAX is associated with the release of cytochrome c into the cytosol and subsequent apoptosis (45). In a similar in vitro model, as described in the present study, marked proapoptotic changes in the ratio of Bcl2 to BAX were observed in OK cells exposed to metabolic inhibitors (53). These studies do not preclude the possibility that mitochondrial "toxins" such as reactive oxygen species contribute to membrane injury and promote the leak of proapoptotic proteins (31).
The accumulation of hsp72, either selectively or induced by
heat stress, ameliorates apoptosis in a variety of experimental models (4, 19, 39, 42, 44, 53). In the present study, the
accumulation of hsp72 was associated with preservation of the outer mitochondrial membrane (Fig. 4) and improved state III mitochondrial respiration (Fig. 2). This protective effect occurred despite similar reductions in m (Fig. 1) and ATP
content during exposure to metabolic inhibitors (55).
These observations suggest that both heat-stress and control cells are
equally sensitive to the effects of cyanide, a potent inhibitor of
electron transport that causes mitochondrial depolarization, a function
of the inner mitochondrial membrane (5). In contrast, heat
stress prevents the loss of proapoptotic proteins from the
intermembranous mitochondrial space, suggesting that disruption of the
outer membrane does not necessarily accompany mitochondrial
depolarization. Preservation of state III respiration in heat-stressed
cells occurred despite some loss of cytochrome c (Fig.
4B). This is likely due to the fact that renal epithelial
cells have a large reserve for oxidative ATP production
(52). Preservation of respiration could also be explained
by an excess of available cytochrome c and incomplete release from mitochondria. The presence of oxidative reserve is supported by the observation that resting cells exhibit heterogeneity in
m (Fig. 1) and is consistent with prior reports in
nonrenal cells in which
m was measured by lipophilic,
cationic probes (27, 37).
Recent studies in our laboratory confirm that the selective overexpression of hsp72 (in the absence of heat stress) is sufficient to inhibit caspase 3 activation and improve cell survival in renal epithelial cells exposed to metabolic inhibitors (56). By protecting the mitochondrial membrane, hsp72 could inhibit apoptosis and improve cell survival. Mosser et al. (36) have shown that HSP70 prevents cytochrome c release and caspase activation after thermal injury. Although the mechanism by which hsp72 protects the mitochondrial membrane has not been characterized, Bcl2 is a likely candidate for protecting mitochondria in our model. Selective overexpression of Bcl2 ameliorates mitochondrial membrane damage in hypoxic renal epithelial cells (45). Furthermore, hsp72 immunoprecipitates with Bcl2 but not Bax in renal cells exposed to metabolic inhibitors (53). Interaction between hsp72 and Bcl2 could afford cytoprotection by restoring Bcl2 function, a role compatible with the chaperone function of this stress protein (36, 40). Regardless of the mechanism by which hsp72 protects the outer mitochondrial membrane during exposure to metabolic inhibitors, reduction of cytochrome c release would prevent apoptosome assembly and caspase activation, central events in the apoptosis pathway (3).
In this study, a previously unreported interaction between hsp72 and cytochrome c was observed. The interaction was greatest in ATP-depleted cells subjected to prior heat stress (Fig. 7), despite the marked decrement in cytochrome c released by these cells (Fig. 4). In both control and heated cells, this interaction was most apparent immediately after the period of ATP depletion. The observation is consistent with function of HSP70 members as chaperones that require ATP to release bound ligands (40). It is conceivable that by binding cytosolic cytochrome c, hsp72 interferes with the assembly of the apoptosome complex, thereby reducing caspase activation. Interestingly, HSP70 has been reported to inhibit apoptosome assembly, although the proposed mechanism did not include interaction with cytochrome c (4). Binding to hsp72 could also interfere with the immunodetection of cytochrome c in the cytosol of cells exposed to metabolic inhibitors (Fig. 3B). In addition to preventing cytochrome c-dependent caspase activation, HSP70 can inhibit apoptosis by antagonizing AIF, a proapoptotic protein responsible for DNA injury (44). This finding may be important in our model, since prior heat stress significantly reduced DNA fragmentation in OK cells exposed to metabolic inhibitors (53). Other investigators have shown that HSP70 prevents the activation of proapoptotic stress kinases such as c-Jun NH2-terminal kinase (JNK) (20) and inhibits "executioner" caspases both proximal (28) and distal (22) to caspase 3. The failure of hsp72 to inhibit caspase 3 activity in vitro suggests that this cytoprotectant protein acts at an earlier step in the apoptotic cascade in our model. Given the existing data, it is likely that hsp72 modulates apoptosis at a variety of checkpoints that may differ by cell type and the nature of the stress.
In some studies, the morphological and biochemical manifestations of apoptosis have been inhibited without improving long-term cell viability (33, 45). Although the present studies were short-term (i.e., limited to 1 h post-ATP depletion), hsp72 accumulation significantly increased renal epithelial cell survival for at least 6 days after exposure to metabolic inhibitors (55). The improvement in cell survival associated with the induction of hsp72 contrasts with biochemical maneuvers that prevented caspase activation downstream of mitochondrial injury but failed to enhance viability (33, 45) and emphasizes the role of mitochondria in determining the fate of a cell. The present study demonstrates that the outer mitochondrial membrane is an early target of injury in cells exposed to metabolic inhibitors and that upregulation of HSPs including hsp72 may ameliorate apoptosis by preventing the release and possibly by inhibiting the action of proapoptotic proteins.
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ACKNOWLEDGEMENTS |
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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-53387 (S. C. Borkan) and DK-5298 (J. H. Schwartz) and a supplemental award from the American Society of Nephrology (S. C. Borkan).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. C. Borkan, Evans Biomedical Research Center, Renal Section, Rm. 547, 650 Albany St., Boston, MA 02118-2518 (E-mail: sborkan{at}bu.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.
May 15, 2002;10.1152/ajpcell.00517.2001
Received 26 October 2001; accepted in final form 2 May 2002.
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