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(The FASEB Journal. 2000;14:847-858.)
© 2000 FASEB

Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation

CARMEN GARCÍA-RUIZ1, ANNA COLELL1, RAQUEL PARÍS and J. C. FERNÁNDEZ-CHECA2

Liver Unit, Department of Medicine, Hospital Clinic i Provincial and Instituto de Investigaciones Biomedicas August Pi Suñer, Consejo Superior de Investigaciones Científicas, Barcelona, 08036, Spain

2Correspondence: Liver Unit, Hospital Clinic i Provincial Instituto Investigaciones Biomédicas, Consejo Superior Investigaciones Científicas Villarroel, 170, 08036-Barcelona, Spain. E-mail: checa{at}medicina.ub.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycosphingolipids, including gangliosides, are emerging as signaling intermediates of extracellular stimuli. Because mitochondria play a key role in the orchestration of death signals, we assessed the interaction of GD3 ganglioside (GD3) with mitochondria and the subsequent cascade of events that culminate in cell death. In vitro studies with isolated mitochondria from rat liver demonstrate that GD3 elicited a burst of peroxide production within 15–30 min, which preceded the opening of the mitochondrial permeability transition, followed by cytochrome c (cyt c) release. These effects were mimicked by lactosylceramide and N-acetyl-sphingosine but not by sphinganine or sphingosine and were prevented by cyclosporin A and butylated hydroxytoluene (BHT). Reconstitution of mitochondria pre-exposed to GD3 with cytosol from rat liver in a cell-free system resulted in the proteolytic processing of procaspase 3 and subsequent caspase 3 activation. Intact hepatocytes or U937 cells selectively depleted of glutathione in mitochondria by 3-hydroxyl-4-pentenoate (HP) with the sparing of cytosol reduced glutathione (GSH) were sensitized to GD3, manifested as an apoptotic death. Inhibition of caspase 3 prevented the apoptotic phenotype of HP-treated cells caused by GD3 without affecting cell survival; in contrast, BHT fully protected HP-treated cells to GD3 treatment. Treatment of cells with tumor necrosis factor increased the level of GD3, whereas blockers of mitochondrial respiration at complex I and II protected sensitized cells to GD3 treatment. Thus, the effect of GD3 as a lipid death effector is determined by its interaction with mitochondria leading to oxidant-dependent caspase activation. Mitochondrial glutathione plays a key role in controlling cell survival through modulation of the oxidative stress induced by glycosphingolipids.—García-Ruiz, C., Colell, A., París, R., Fernández-Checa, J. C. Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation.


Key Words: oxidative stress • apoptosis • necrosis • glutathione


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INCREASING EVIDENCE HAS identified mitochondria as a strategic center that determines cell survival. Indeed, mitochondria control the commitment to cell death triggered by certain stimuli through the release of vital components that assist in the presence of cytosolic factors to the assembly of the apoptosome, the molecular machinery responsible for the programmed cell death (1 2 3 4) . In this context, it has been shown that cytochrome c (cyt c) (1) , which is released from mitochondria in cells undergoing apoptosis, interacts with specific cytosolic components, resulting in the activation of downstream caspases that account for the organized dissasembly of the cell (5 , 6) . Furthermore, recent evidence has demonstrated that procaspases are sequestered within the mitochondrial intermembrane space and undergo a redistribution into the cytosol where they become fully active in response to cell death triggers (7 8 9) . Moreover, an apoptotic-inducing factor, which is released from mitochondria into the cytosol, initiates the apoptotic demise of cells without the need for cooperative factors (10) . Consistent with the key role of mitochondria in the control of cell death, survival or apoptotic factors (e.g., Bcl-2 or Bax) act on mitochondria where they prevent or facilitate, respectively, the release of apoptogenic factors, e.g., cyt c (11 12 13) .

The mitochondrial permeability transition (PT), a phenomenon characterized by a sudden increase in the permeability of the inner mitochondrial membrane, plays a major role in the onset of cell death and has been shown to be key in apoptosis by facilitating the release of apoptogenic factors. Indeed, agents that induce or prevent PT modulate cell survival (1 2 3 , 14) . Furthermore, PT collapses ion gradients across the inner mitochondrial membrane, leading to mitochondrial depolarization, loss of oxidative phosphorylation, and reactive oxygen species (ROS) overgeneration that result in the necrotic death of cells. Thus, PT functions as a common mechanism that is key for necrosis and apoptosis (14) . On the other hand, PT and oxidative stress, subsequent to ROS overgeneration, are two reciprocally regulated processes through the redox state of critical sulfhydryls, which control the open/close state of PT (15) .

Sphingolipids and glycosphingolipids are constituents of eukaryotic membranes, where they play a critical structural role (16) . However, growing evidence points to these structural lipids as signaling intermediates of inflammatory cytokines and death factors (17 18 19 20) . In this regard, ceramide, a sphingolipid whose levels increase rapidly in cells in response to a variety of inducing stimuli, mediates the biological effects of inflammatory cytokines and growth factors (18 , 19) . Thus, previous studies have demonstrated that cell-permeable ceramide analogs interact with the mitochondrial respiratory chain, resulting in ROS overproduction and subsequent oxidative stress, mimicking the oxidative stress induced by inflammatory cytokines (21 22 23 24) . On the other hand, ceramide is a precursor for the biosynthesis of complex glycosphingolipids within the Golgi, and, hence, the translocation of ceramide to the Golgi complex may regulate glycosphingolipid metabolism including gangliosides.

Recent studies have provided evidence that GD3 ganglioside (GD3) mediates the Fas- and ceramide-induced apoptosis of lymphoid and myeloid tumor cells (20) . However, the mechanism involved was not examined in detail, although it was demonstrated that GD3 induced mitochondrial depolarization in cells undergoing apoptosis. Furthermore, whether mitochondrial depolarization caused by GD3 was cause or consequence of apoptosis was not addressed in these studies (20) . Hence, our study was undertaken to examine whether GD3 and related analogs directly interact with mitochondria and to determine the sequence of events leading to the activation of dowstream caspases and apoptosis. We provide evidence that GD3 elicits a burst of ROS overproduction from the complex III of the mitochondrial electron transport chain, which triggers the opening of the mitochondrial PT, leading to the cyt c-dependent caspase 3 activation. Furthermore, selective depletion of GSH in mitochondria sensitizes intact hepatocytes to GD3-induced apoptosis by enhancing the oxidative stress caused by GD3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Lactosylceramide (LactC), GD3, GM1, GD1a, sphinganine, sphingosine, C2-ceramide, antimycin A, atractyloside, dATP, ATP, cyclosporin A (CsA), rotenone, leupeptin, pepstatin A, and Hoeschst 33258 were purchased from Sigma (St. Louis, Mo.). Myxothiazol and DTT were from Boehringer Mannheim (Mannheim, Germany). 2'-7'-Dichlorofluorescein diacetate (DCFDA), dihydrorhodamine 123 (DHR), and tetramethylrhodamine methyl ester (TMRM) were from Molecular Probes (Eugene, Ore.). Caspase 3 inhibitor and substrate, Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AMC), respectively, were from Bachem (Bubendorf, Switzerland). (R, S)-3-Hydroxyl-4-pentenoate (HP) was a generous gift from Dr. W. Anders (University of Rochester, N.Y.).

Cell culture
Rat hepatocytes were isolated by collagenase digestion, plated on rat tail collagen, and cultured routinely in DMEM/F12 medium as described previously (25) . Alternatively, freshly isolated hepatocytes were incubated for up to 4 h in Fisher’s medium at 37°C under 95% CO2 and 5% CO2. The human leukemia cell line U937 was obtained from the ATCC (Rockville, Md.) and grown in RPMI 1640, and culture medium was supplemented with 10% heat-inactivated feat calf serum, 2 mM L-glutamine, and antibiotics, streptomycin (100 ìg/ml), and penicillin (200 units/ml).

Mitochondria preparation
Rat liver mitochondria were isolated from liver homogenates by differential centrifugation. Alternatively, highly purified mitochondria were prepared by a rapid centrifugation through Percoll density gradient as described in detail previously (26 , 27) . Enrichment and recovery of mitochondria were ascertained by the specific activity of succinic dehydrogenase (SDH). Mitochondrial purity was confirmed by estimating the contamination with other subcellular organelles such as sinusoidal and canalicular plasma membrane, microsomes, and lysosomes assessed by the activity of Na+-K+ ATPase, Mg2+ ATPase, G-6-P-phosphatase, and acid phosphatase, respectively, (27) . Mitochondrial integrity was determined by the acceptor control ratio as the ADP-stimulated oxygen consumption over its absence as described previously (27) .

Determining ROS and mitochondrial membrane potential by flow cytometry
Hydrogen peroxide and membrane potential were determined using DCFDA and TMRM. Mitochondria were incubated with the corresponding fluorescent probe, analyzing the fluorescence and side light scatter (SSC, 90 angle) in 10,000 events/test using a FACStar flow cytometer (Becton Dickinson, San Jose, Calif.) (22) . Data on mitochondrial fluorescence and light scatter were obtained using a 5-W argon ion laser tuned at 488 nm and 250 mW. DCF fluorescence from oxidation of DCFDA was measured through a 530-nm bandpass filter placed in front of the green photomultiplier tube using a four-decade log amplifier. The mean intensity of the green fluorescence in the presence or absence of GD3 (1 µM) was determined using the Cell Quest software program and expressed as fluorescence channels (scale from 0 to 10,000 arbitrary units). Graphics were plotted using the Cell Quest software program (Becton Dickinson). Alternatively, hydrogen peroxide quantitation was performed spectrofluorimetrically as described previously (28) .

Stock solutions of glycosphingolipids were made up in ethanol and stored at –80°C under nitrogen. When added to aqueous reaction mixtures containing mitochondria, the final concentration of the carrier solvent did not exceed 0.1%. Control incubations contained only the carrier solvent, which did not affect any of the parameter studied.

Measuring mitochondrial membrane permeability transition
Large amplitude swelling was measured spectrophotometrically by recording absorbance at 540 nm. An increase in mitochondrial swelling results in a decrease in optical density. Isolated rat liver mitochondria were suspended in a buffer consisting of 200 mM sucrose, 10 mM Tris-MOPS, 5 mM succinate, 1 mM potassium phosphate, 2 µM rotenone, 1 µg/ml oligomycin, 10 ìM EGTA, pH 7.4, at 25°C. Glycosphingolipids (1 µM) were added to mitochondrial suspension, and absorbance at 540 nm was determined at 25°C over time (22) . Opening of the pore was induced by the adenine nucleotide translocator ligand atractyloside (2 mM) and prevented by preincubation with cyclosporin A (5 µM).

Cyt c release assay
Mitochondria were incubated with glycosphingolipids with or without the presence of atractyloside or CsA for up to 2 h at 25°C. Mitochondria were pelleted by centrifugation at 10,000 g for 5 min, and the resulting supernatant was resolved by SDS/PAGE immunoblotting in 15% gels with high Tris (75 mM). Proteins were transferred to nitrocellulose, and the blots were incubated with mAb anticyt c (clone 7H8.2C12, PharMingen, San Diego, Calif.) followed by ECL-based detection. Parallel aliquots were analyzed by immunoblotting for the release of cytochrome oxidase using mAb anticytochrome oxidase subunit II (Molecular Probes) to confirm the specificity of cyt c release.

Cell-free assay of cyt c-induced caspase activation
Cytosolic extracts from rat liver were prepared by centrifugation liver homogenates at 100,000 g for 1 h. Supernatants from GD3-treated mitochondria were incubated with cytosol extracts at 30°C for 1 h in the presence of dATP or ATP and an ATP-regenerating system. Caspase 3 activation was detected by immunoblotting using a polyclonal mAb anticaspase 3 (Santa Cruz Biotechnology, Santa Cruz, Calif.) following ECL detection, as in the case of cyt c. Caspase activity was performed by release of 7-amino-4-trifluoromethyl coumarin (AMC) from Ac-DEVD-AMC, and fluorescence was continuously recorded with emission at 460 nm and excitation at 355 nm.

Selective mitochondrial reduced glutathione (GSH) depletion of hepatocytes and apoptotic cell death
Hepatocytes were incubated with HP (1 mM for 5 min), followed by washing of cells to remove excess HP. Subsequently, cells were fractionated into cytosol and mitochondria as described (26 , 27) to confirm selective depletion of mitochondrial GSH, which was determined by HPLC as described previously (29) . In some instances, cells were preincubated with Ac-DEVD-CHO (100 µM) or butylated hydroxytoluene [(BHT), 50 µM in dimethyl sulfoxide (DMSO)] for 30 min and then incubated with GD3 (1 µM) for various periods of time. At every time point, an aliquot of cells was removed, and viability was determined by the release of glutathione S-transferase (GST) into the medium with comparable results obtained by trypan blue exclusion. The morphological changes in nuclear chromatin as an index of apoptosis were assessed by staining of cells with the DNA-binding fluorochrome Hoeschst 33258. Incidence of apoptotic chromatin changes includes condensation of chromatin, its margination along the periphery of the nuclei, and segmentation of the nuclei into more than three fragments. Alternatively, internucleosomal DNA fragmentation was determined as described previously (30) .

Determination of GD3
GD3 levels in hepatocytes (6–10x107 cells) or mitochondrial fraction were determined by high-performance thin layer chromatrography (HPTLC) as described previously (31) . Lipid extracts were washed and dissolved in C6H14O/CH3(CH2)3OH (3:2, v/v) with the addition of NaCl (0.85 mM final concentration) and lyophilized. Samples were dissolved in CH3OH/H2O (1:1, v/v), sonicated, and loaded in a Sep-Pak C18 cartridge, prewashed with CH3OH, CH3OH/CHCl3 (1:1, v/v), CH3OH, and CH3OH/H2O (1:1, v/v). Gangliosides were eluted by washing cartridges with CH3OH and CH3OH/CHCl3 (1:1, v/v) and were dried under N2. The eluate was applied to HPTLC plates (Merck, Rahway, N.J.) and developed in CHCl3/CH3OH/H2O (50:42:11, v/v) containing 0.2% (w/v) CaCl2. After drying, plates were sprayed with C2H5OH/37% (w/v) HCl (4:1, v/v) containing 300 mg dimethylaminobenzaldehyde (Ehrlich’s spray) and heated to 180°C for 15 min. Samples were run with gangliosides standards including GD3, GM1. The level of GD3 was calculated by densitometric analyses of HPTLC plates and compared with a standard curve generated using known amounts of GD3.

Statistical analyses
Statistical analyses for comparison of mean values for multiple comparisons between mitochondrial or cellular preparations were made by one-way analysis of variance (ANOVA) followed by Fisher’s test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oxidative stress in isolated mitochondria by glycosphingolipids precedes mitochondrial depolarization
The mitochondrial PT caused by the opening of a transmembrane pore is known to cause cell damage, although its requirement for apoptosis has remained controversial (2 3 4 , 32 , 33) . Because of the reciprocal regulation between the mitochondrial PT and ROS overproduction and subsequent oxidative stress, we determined in a time-dependent fashion the relationship between the production of hydrogen peroxide and the mitochondrial depolarization in isolated rat liver mitochondria exposed to GD3. As shown in Fig. 1 , isolated mitochondria were labeled with DCFDA monitoring DCF fluorescence in a time-dependent fashion based on the treatment of isolated mitochondria to GD3. In parallel incubations, the TMRM fluorescence, which reflects the mitochondrial membrane potential, was followed over time after GD3 exposure. An increase in DCF fluorescence was detected after 30 min of incubation with GD3 preceding the mitochondrial depolarization observed at 60 min (Fig. 1B ). A greater magnitude of ROS generation and subsequent mitochondrial depolarization were observed with increasing concentrations of GD3 (1–20 µM, not shown).



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  		Figure 1. Time-dependent changes in hydrogen peroxide and membrane potential in rat liver mitochondria incubated with GD3. Isolated mitochondria from rat liver (1 mg/ml) were incubated with succinate to drive electron flow at complex II. Mitochondria were then labeled with DCFDA or TMRM, washed to remove excess probes, and then incubated with GD3 (1 µM) at 25°C for 1 h. At the indicated periods of time, an aliquot of mitochondrial suspension was analyzed for DCF (A) and TMRM (B) fluorescence by flow cytometry as described in Materials and Methods. A representative flow cytometric profile of at least four repeats is shown.

Furthermore, we determined the specificity of the ability of GD3 to overproduce ROS by assessing the effect of related analogs in the induction of hydrogen peroxide in isolated mitochondria. Glucosylceramide, LactC, and GM1 (Table 1 ) mimicked the effect evoked by GD3; in addition, the synthetic ceramide analog C2 led to a significant increase in the generation of hydrogen peroxide, confirming previous results (22 , 24) that suggest that the presence of sugar residues in the structure backbone of gangliosides is dispensable for the stimulation of ROS production. Furthermore, the sugar backbone of sphingolipids, sphingosine, or its precursor sphinganine failed to generate hydrogen peroxide (Table 1) , indicating that the N-fatty acyl-sphingosine moiety of gangliosides is required to stimulate ROS production. Blocking electron flow at complexes I and II with thenoyltrifluoroacetone (TTFA) and rotenone prevented the hydrogen peroxide increase caused by GD3. However, interference of electron flow at center i and center o of complex III of the respiratory chain by antimycin A or myxothiazol (34) potentiated and prevented, respectively, the inducing effect of GD3 in hydrogen peroxide generation (not shown).


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  		Table 1. Structural specificity of sphingolipids in generating hydrogen peroxide in isolated rat liver mitochondriaa

Antioxidants prevent GD3-induced hydrogen peroxide generation and mitochondrial swelling
Mitochondrial swelling is another functional consequence of the mitochondrial PT. Hence, we followed the change in absorbance at 540 nm of mitochondria incubated with GD3 and examined the effect of antioxidants compared with known inhibitors of the permeability transition. As shown, the time-dependent change in A540 nm caused by GD3 mirrored that of mitochondrial membrane potential, because the loss of A540 nm was undetected within the first 30 min, observed at 60 min incubation with GD3 (Fig. 2). Consistent with the burst of hydrogen peroxide caused by LactC, this analog mimicked the effect of GD3 on mitochondrial swelling (Fig. 2) . The magnitude of mitochondrial swelling caused by GD3 was lower than that elicited by atractyloside, a positive trigger of PT, which induced mitochondrial swelling as quickly as 15 min of incubation. Mitochondrial incubation with CsA, a known inhibitor of mitochondrial PT, prevented GD3-induced mitochondrial swelling caused by GD3 (Fig. 2) . However, this agent did not affect the early generation of hydrogen peroxide caused by GD3 (Fig. 2) .



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  		Figure 2. Effect of BHT and CsA on hydrogen peroxide generation and mitochondrial swelling induced by GD3. Isolated rat liver mitochondria (1 mg/ml) were incubated in the presence of GD3, LactC, or C2-ceramide (1 µM each), and the DCF (A) fluorescence was determined spectrofluorimetrically at the indicated times. Parallel aliquots were examined for change in absorbance at 540 nm (B) as described in Materials and Methods. As indicated, CsA, BHT, or atractyloside were added at 5 µM, 50 ìM, and 2 mM, respectively. Results are the mean ± SD of three independent experiments with different mitochondrial preparations.

To test the effect of antioxidants on the induction of mitochondrial PT, we determined the generation of hydrogen peroxide and change in A540 nm in mitochondria incubated in the presence of BHT and GD3. As shown, BHT efficiently blocked the overproduction of hydrogen peroxide and the loss of A540 nm caused by GD3 (Fig. 2) . Thus, these findings indicate that the overgeneration of hydrogen peroxide in isolated mitochondria induced by GD3 precedes the mitochondrial PT and that antioxidants control the opening state of the mitochondrial transition pore by regulating the level of ROS production.

GD3 releases cyt c from isolated mitochondria
The release of cyt c from mitochondria has been identified as a key step in the apoptotic cascade (1 2 3) . Therefore, having determined that GD3 elicits a burst of ROS production followed by PT opening, we next assessed whether GD3 elicits the release of cyt c. Isolated mitochondria were incubated with GD3 for various time periods, analyzing by immunoblotting the presence of cyt c in the supernatant. The levels of cytochrome oxidase in the supernatants were used to confirm the specificity of cyt c release. As shown in Fig. 3 , GD3 caused a progressive cyt c release in the medium in the absence of detectable cytochrome oxidase. GD3 released a discrete amount of cyt c from mitochondria compared with the level of cyt c detected in supernatants from intact mitochondria permeabilized with Triton X-100. The time pattern of cyt c release in the medium caused by LactC was similar to that caused by GD3 (Fig. 3) .



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  		Figure 3. GD3 induces the release of cytochrome c from isolated mitochondria. Mitochondria were incubated with GD3 or LactC in the absence (A) or presence (B) of BHT, atractyloside (Atract.), and CsA for the indicated periods of time. Mitochondria were pelleted, and the resulting supernatant was analyzed for the presence of cyt c and cytochrome oxidase (Cyt ox.) by immunoblotting as described in Materials and Methods. Parallel aliquots of mitochondria were treated with Triton X-100, and release of cyt c and oxidase was examined in the supernatant. Mitochondrial pellets were analyzed for cytochrome oxidase by immunoblotting.

To analyze the effect of mitochondrial PT regulators on GD3-induced cyt c release, we monitored the effect of CsA on cyt c release caused by GD3. CsA prevented the release of cyt c induced by GD3, highlighting the critical role of the mitochondrial PT in the release of apoptogenic factors. The cyt c level released from intact mitochondria when exposed to atractiloside was similar to that caused by solubilization of mitochondria with Triton X-100 (Fig. 3A, B ). Incubation of mitochondria with BHT blocked the GD3-induced release of cyt c, mimicking the inhibitory effect of CsA (Fig. 3) .

In vitro caspase 3 activation by treatment of mitochondria with GD3
Because the release of cyt c from mitochondria plays a key role in the activation of downstream caspases (1 2 3) , we monitored the role of GD3 on caspase 3 activation in a cell-free system. Mitochondria were incubated with GD3, and the resulting supernatant obtained after centrifugation was added to a cytosol fraction prepared from normal rat liver. Caspase activation was followed by the proteolytic processing of procaspase 3, resulting in the appearance of a lower 17-kd fragment. As seen (Fig. 4 ), this fragment was detectable by treatment of mitochondria with GD3. Other analogs, LactC and permeable C2-ceramide, mimicked the effect of GD3 on procaspase 3 activation (not shown). Furthermore, to document the correlation between the appearance of the active caspase 3 fragment and the onset of activity, we determined the fluorescence of AMC released from the synthetic fluorescent peptide DEVD-AMC, which mimics the target sequence of the downstream caspases. As seen, GD3 increased the fluorescence from DEVD-AMC peptide (Fig. 4B ). The presence of a caspase inhibitor, Ac-DEVD-CHO, did not affect the proteolytic processing of procaspase 3 in the cell-free system, although it prevented the release of the fluorescent peptide Ac-DEVD-AMC. Omission of either cytosol or mitochondria from the cell-free system failed to activate caspase 3. A similar pattern of caspase 3 activation was observed in the presence of inhibitors of lysosomal proteases (leupeptin and pepstatin A, 10 µg/ml), which discard a contributory role for contaminating lyosomes to caspase 3 activation (35) . Consistent with the abolishing effect of BHT on the release of cyt c induced by GD3 in isolated mitochondria, this antioxidant prevented the proteolytic processing of procaspase leading to caspase 3 activation (Fig. 4) .



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  		Figure 4. Proteolytic processing and caspase 3 activation induced by GD3 in a cell-free system. Mitochondria were incubated with GD3 (1 µM) for 1 h, and the resulting supernatant was added to rat liver cytosol and incubated for 1h. The proteolytic processing (A) of procaspase 3 was monitored by immunoblotting as described in Materials and Methods. Following incubation of the mitochondrial supernatant with cytosol, DEVD-AMC peptide was added to the mixture, and the fluorescence of AMC released was monitored over time (B). In some cases, mitochondrial GSH levels were depleted with HP to 60–70% of control values (5.5 ± 0.7 nmol/mg protein) before exposure to GD3 in the presence or absence of BHT (50 µM). The mitochondrial supernatant was incubated with cytosol in the presence or absence of Ac-DEVD-CHO (100 µM), and the proteolytic processing (A) and caspase 3 activity (B) were determined as described in Materials and Methods. Results are the mean of n=4–5 individual experiments.

Mitochondrial GSH controls the survival of hepatocytes to GD3 treatment
Having shown that GD3 targets mitochondria, eliciting a burst of oxidative stress, we next evaluted the role of mitochondrial GSH in controlling the oxidative stress and its consequences on cell survival using a sublethal dose of GD3 (1 µM). Freshly isolated rat hepatocytes were selectively depleted of GSH in mitochondria on exposure to HP, examining the viability of hepatocytes after GD3 incubation. As seen, the mitochondrial pool of GSH was depleted by HP incubation to 50–60% of control levels with the sparing of cytosol GSH (Fig. 5 ). Whereas, control hepatocytes were resistant to GD3 treatment, HP sensitized hepatocytes to GD3 as cell survival decreased gradually over time. Similar sensitization by mitochondrial GSH depletion to GD3 treatment was observed in U937 cells (not shown). Similar findings were observed with increasing GD3 concentrations up to 10 µM; however, higher GD3 doses (>10 µM) were cytotoxic to control hepatocytes in the absence of HP pretreatment (not shown). Because necrosis and apoptosis share common mechanisms (14) , we next examined the type of death induced by GD3 treatment. As shown, sensitized hepatocytes by HP displayed chromatin disruption and DNA degradation, indicating that GD3 killed HP-treated hepatocytes by apoptosis (Fig. 5) . Consistent with the role of caspase 3 in this type of cell death, HP potentiated the GD3-induced caspase 3 activation (Fig. 4) .



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  		Figure 5. Mitochondrial GSH controls the survival of rat hepatocytes to GD3 exposure. Hepatocytes were selectively depleted of mitochondrial GSH by HP preincubation (1 mM for 5 min) and fractionated into cytosol and mitochondria for GSH analysis shown in the inset. Cells were then incubated with GD3 (1 µM), and viability (A) was determined by the release of cytosolic enzyme GST in the medium. Parallel cell aliquots were taken to analyze the morphology of chromatin and DNA fragmentation, to monitor apoptotic cell death (B). Results are the mean ± SD of n=4 individual cell preparation. *P<0.05 vs. control, GD3 or HP.

Furthermore, we next evaluated the role of caspase 3 inhibitors on survival of HP-treated hepatocytes to GD3 treatment. Ac-DEVD-CHO, which blocked the activation of caspase 3 (Fig. 4) , prevented the phenotype of apoptosis of HP-hepatocytes caused by GD3, as shown by the morphological chromatin appearance and DNA integrity (Fig. 6 ). However, Ac-DEVD-CHO, which did not prevent PT induction (not shown), failed to protect HP-treated cells to GD3 treatment, as shown by the loss of viability of HP-treated hepatocytes (Fig. 6) . In contrast, BHT fully protected HP-treated hepatocytes to GD3, because the survival of these cells was similar to control hepatocytes (Fig. 6) . Thus, these findings reveal a critical role of mitochondrial GSH in the control of cell death through modulation of oxidant-dependent caspase activation. Blocking downstream caspases modulates the phenotype of cell death, whereas prevention of mitochondrial-induced oxidative stress prolongs cell survival.



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  		Figure 6. Effect of caspase 3 inhibitor and BHT on survival of mitochondrial GSH-depleted hepatocytes. Hepatocytes were treated with HP as in Fig. 5 to deplete mitochondrial GSH levels. Hepatocytes were then incubated with GD3 in the absence or presence of Ac-DEVD-CHO (100 µM) or BHT (50 µM), and the survival (A) and apoptotic phenotype (B) were determined as in Fig. 5 . Mrk=size markers of DNA fragments. Results are the mean ± SD of n=4 individual cell preparation. *P<0.05 vs. HP and HP+BHT+GD3.

GD3 levels in hepatocytes treated with TNF-{alpha}
To determine the biological significance of the apoptotic ability of GD3, we determined the levels of GD3 in intact hepatocytes and in mitochondrial fraction after treatment with TNF-{alpha}, which at 10,000 U/ml led to a progressive increase in the levels of GD3 in lipid extracts obtained from cellular homogenate and mitochondrial fraction. These were significant 2 h after incubation of hepatocytes with TNF-{alpha} (Fig. 7 ). The levels of GD3 in response to TNF-{alpha} were unaffected by the pretreatment of hepatocytes with HP, although the survival of HP-treated hepatocytes incubated with TNF-{alpha} decreased over time (not shown). Thus, these findings are consistent with a putative intermediate role of GD3 in TNF-{alpha} signaling.



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  		Figure 7. GD3 levels in hepatocytes incubated with TNF-á. Hepatocytes were treated with or without TNF-{alpha} (10,000 U/ml) for 2 h, and then lipid extracts from cellular homogenate (open bar) or mitochondrial fraction (closed bar) were extracted and dissolved in chloroform:methanol (3:2, v/v). Lipid extracts were applied to HPTLC plates, and gangliosides were resolved and quantitated as described in Materials and Methods. Results are expressed as the mean ± SD of n=3 individual experiments.

To further evaluate the relevance of the interaction of GD3 with mitochondrial components in the induction of apoptosis, the survival of HP-treated hepatocytes in response to GD3 treatment was assessed in the presence of inhibitors of the mitochondrial electron flow at complexes I and II. As shown, rotenone and TTFA prevented the cytotoxicity of GD3 in HP-treated hepatocytes (Fig. 8 ), suggesting that the apoptotic capability of GD3 is mediated by its interaction with mitochondrial respiratory complex, similar to what has been previously demonstrated for ceramides (22 23 24) .



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  		Figure 8. Effect of rotenone and TTFA on the survival of HP-treated hepatocytes to GD3 treatment. Hepatocytes were pretreated with HP as described and then incubated with GD3 (1 µM) in the presence or absence of rotenone (Rot, 20 µM) and TTFA (15 µM). Survival of hepatocytes after the end of 3 h was determined by release of cytosolic GST in the medium. Results are the mean ± SD of n=4 individual experiments. *P<0.05 vs. control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Glycosphingolipids are characteristic components of plasma membranes, which have been considered mainly as structural lipids. However, glycosphingolipids are increasingly recognized as signaling intermediates involved in the regulation of multiple cellular functions—cell proliferation and differentiation; cell-cell interaction; as receptors for microbes and their toxins; and as modulators of the sensitivity of cancer cells to anticancer drugs (36 37 38 39) . The present study examined the role of gangliosides (e.g., GD3) as death effectors, and the mechanism and sequence of events involved in this function. Our findings uncover the ability of GD3 ganglioside and structurally related analogs to elicit a burst of ROS in purified mitochondria from rat liver, which precedes the PT opening leading to mitochondrial depolarization and swelling. Evidence in support for this sequence of events is provided by the temporal relationship between hydrogen peroxide generation and mitochondrial depolarization and swelling caused by GD3 treatment of mitochondria, and by the fact that antioxidants prevent mitochondrial swelling through attenuation of ROS generation. A similar time-dependent pattern between hydrogen peroxide generation and mitochondrial swelling has been observed previously for cell-permeable ceramide analogs (e.g., C2-ceramide) in isolated mitochondrial (22) . This suggests that ceramides and gangliosides share a common structural requirement, namely the N-fatty acyl-sphingosine, which is essential for the interaction of gangliosides with the complex III of the mitochondrial electron transport chain causing ROS overgeneration. Indeed, the retention of this structural feature of gangliosides is consistent with the known role of ceramide as the precursor for glycosphingolipids (16) . In line with this, recent data from our lab have established a functional link between ceramide generated from the acidic sphingomyelinase and ganglioside synthesis as the triggering of ROS overgeneration induced by acidic sphingomylinase. Further, subsequent apoptosis of rat hepatocytes were prevented by pharmacological inhibition of glucosyltransferase, which abolished GD3 ganglioside formation (García-Ruiz et al., unpublished results) (40) .

Because PT is a critical event in the assembly of the apoptotic machinery, the interaction of GD3 with mitochondria may lead to the activation of caspases through release of apoptogenic factors (1 2 3) . Our data provide compelling evidence that GD3 induces the release of cyt c from intact mitochondria, which, in cooperation with cytosolic factors, results in the proteolytic processing and activation of dowstream caspases. Whether PT is required in apoptosis is not well established yet (1 2 3 , 32 , 33) . However, our work demonstrates that this process is needed for cyt c release and subsequent caspase activation induced by GD3, because CsA, which blocks PT (14) , prevents cyt c release and caspase 3 activation. Furthermore, our data show that the mitochondrial-derived oxidative stress induced by GD3 controls PT. This is indicated because the attenuation of the GD3-induced ROS generation by antioxidants (e.g., BHT) abolished cyt c release and caspase activation. Recent observations have shown that C2-ceramide induced the release of cyt c from isolated mitochondria through modulation of the mitochondrial redox state (41) . Thus, these findings and the results from the present study reveal that ceramides and gangliosides (e.g., GD3) share a common function in the activation of the apoptotic network by interaction with mitochondria.

Consistent with the significance of the burst of ROS overproduction caused by GD3 in the assembly and activation of caspase 3, our work assessed the role of mitochondrial GSH, a critical line of defense to control the mitochondrial oxidative stress (42 , 43) in the survival of hepatocytes to GD3 treatment. Selective mitochondrial GSH depletion in hepatocytes was accomplished by HP. Through its predominant metabolism within mitochondria by the (R)-3-hydroxybutanoate NAD+ oxidoreductase (44) , HP generates a Michael acceptor that reacts with GSH, resulting in its depletion with the sparing of the cytosolic pool of GSH. Using this approach, our data show that HP treatment sensitizes hepatocytes to GD3 exposure, reflected by the gradual loss of cell viability induced by GD3, an effect that is accompanied by chromatin disruption and DNA fragmentation. Thus, these data demonstrate that the interaction of GD3 with mitochondria can be regulated by the modulation of specific mitochondrial antioxidant status. This highlights the critical importance of the mitochondrial pool of GSH in controlling the fate of cells in response to stimuli that cause mitochondrial-induced oxidative stress. In line with this, we have recently described the sensitization of hepatocytes to TNF-{alpha} by selective mitochondrial GSH depletion (26) .

To estimate the significance of the relative contribution of oxidative stress and caspase activation in the sensitization of hepatocytes by HP treatment, we examined the effect of Ac-DEVD-CHO, a caspase 3 inhibitor, and BHT on the survival of HP-treated hepatocytes to GD3 exposure. Treatment of sensitized hepatocytes with caspase 3 inhibitor did not block cell death, whereas it was effective in preventing apoptotic features—e.g., DNA fragmentation and chromatin disruption. However, other consequences of GD3 exposure, such as ROS overgeneration and subsequent PT, were unaffected by the caspase 3 inhibitor. Consequently, although this treatment avoids the development of the apoptotic phenotype, it fails to protect sensitized hepatocytes against the necrosis induced by GD3—consistent with the key role of PT in cell death (14) . In contrast, the ability of BHT to prevent the triggering of PT induced by GD3 confers complete protection of sensitized hepatocytes to GD3. Thus, our findings confirm the key role of PT in determining cell survival, regardless of the phenotype of cell death. Further, PT induction by GD3 is upstream of caspase 3 activation, as demonstrated recently with other stimuli (32 , 33) .

Our work extends recent observations in lymphoid and myeloid tumor cells in which GD3 plays a key role in the Fas- and ceramide-induced apoptosis (20 , 45) . Although the mechanism involved was not addressed in these studies, an intriguing finding was the fact that the apoptosis of hematopoietic cells elicited by GD3 was independent of caspases, because apoptosis still occurred in the presence of a wide range of caspase inhibitors (20) . However, according to the present results, a direct effect of GD3 with mitochondria leading to PT, subsequent to the induction of oxidative stress, emerges as a mechanism of action of GD3. This is consistent with recent observations (46 , 47) , demonstrating that PT is a key step in the apoptosis induced by GD3 and related analogs. Furthermore, our work has assessed the biological significance of GD3’s ability to propagate apoptotic signals as its levels increased 2- and 3-fold in hepatocytes following TNF-{alpha} treatment or exposure to acidic sphingomyelinase (40) . In line with this outcome, it has been demonstrated recently in HuT78 cells that GD3 levels increased to a similar range (5-fold) after CD95 crosslinking (20) . Furthermore, unlike these findings, our data reveal an increase in GD3 levels in the mitochondrial fraction of TNF-{alpha} -treated hepatocytes, similar to that observed in cellular homogenate. Whether a sudden rise in mitochondrial GD3 levels is sufficient to initiate the cascade of events leading to cell death may depend not only on the magnitude of GD3 increase but also on the available mitochondrial GSH, as demonstrated recently in the sensitization of hepatocytes from chronic ethanol-fed rats to TNF-{alpha} (26) . Moreover, the hepatotoxicity of GD3 is abolished by inhibitors of mitochondrial respiratory complexes I and II, highlighting the in vivo significance of the targeting by GD3 to mitochondria. Thus, our findings in hepatocytes and in human endothelial cells (17) suggest that gangliosides function as mediators of inflammatory cytokines (e.g., TNF-{alpha}). Hence, it is conceivable that the interaction of GD3 with mitochondria, leading to ROS overgeneration and subsequent apoptosis, may account for the cytotoxicity of TNF-{alpha}. The mechanism whereby GD3, which is synthesized in the Golgi complex, targets mitochondria is presently unknown. Previous studies, however, have indicated that newly synthesized gangliosides are shuttled to mitochondria and other subcellular sites by an as yet uncharacterized mechanism (48) .

An interesting aspect of our work is the inability of caspase inhibitors, as opposed to antioxidants, to rescue hepatocytes from the cytotoxicity induced by GD3. Evidence has been shown for the potential use of caspase inhibitors as a therapy directed specifically at reducing apoptosis associated with brain damage in acute bacterial meningitis or lethal normothermic liver ischemia (49 , 50) . In light of our findings, however, the use of antioxidants, along with caspase inhibitors, may be a more efficient therapeutical approach to guarantee protection against necrosis and apoptosis through modulation of the mitochondrial-derived oxidative stress.

In summary, our findings reveal the potential of gangliosides as lipid death effectors through the targeting to mitochondria where they cause a ROS overproduction initiating a chain of processes that activate the apoptotic machinery. Because of the key role of mitochondria in the control of cell death/survival pathways, our study highlights the consequences of the synergism between disease-promoting factors (e.g., gangliosides as intermediates of inflammatory cytokines causing increased oxidative stress within mitochondria as a result of ROS generation) and the decreased GSH defense in the mitochondrial compartment. At least in the alcohol-induced liver injury, the impaired transport of GSH from cytosol into the mitochondrial matrix induced by ethanol (26 , 27 , 43) will favor conditions that lead to the escalating cell damage. Induced by death effectors generated from the signaling of inflammatory cytokines, this cell damage initiates a chain of processes contributing to the progressive dysfunction of the liver.


   ACKNOWLEDGMENTS
 
The work presented was supported by grants from the U.S. National Institute of Alcohol Abuse and Alcoholism Grant AA 09526 and Alcohol Center Grant P50 AA11999; Dirección General Política Científica y Técnica PM 95–0185; Plan Nacional de I+D Grants SAF 97–0087-001 and SAF99–0138; and Europharma. C.G.-R. is an SNS investigator of the Fondo Investigaciones Sanitarias.


   FOOTNOTES
 
1 These authors contributed equally to the work.

Received for publication August 17, 1999. Revised for publication December 6, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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