Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis*

(Received for publication, August 15, 1996, and in revised form, April 15, 1997)

Anne Quillet-Mary Dagger §, Jean-Pierre Jaffrézou Dagger , Véronique Mansat Dagger , Christine Bordier Dagger , Javier Naval and Guy Laurent Dagger par

From the Dagger  CJF INSERM 9503, Centre Claudius Régaud, Toulouse Cedex, France, the  Department of Biochemistry and Molecular Biology, University of Zaragoza, 50009 Zaragoza, Spain, and the par  CHU Purpan, Service d'Hématologie, Toulouse, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
Note Added in proof
REFERENCES


ABSTRACT

The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 µM C6-ceramide induced significant H2O2 production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect. Furthermore, no H2O2 production was observed in C6-ceramide-treated U937 rho ° cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-kappa B and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H2O2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor). H2O2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramide-induced oxidative stress. Finally, C6-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis.


INTRODUCTION

Ceramide has emerged as a potentially important mediator of a number of natural or pharmacological agents that affect cell growth, viability, and differentiation (1, 2). This lipid second messenger is the breakdown product of sphingomyelin (SPM)1 generated by the activation of a neutral and/or an acidic sphingomyelinase. Agonists of the SPM-ceramide pathway include: cytokines or growth factors such as tumor necrosis factor-alpha (TNF-alpha ) (3), interleukin-1beta (4), gamma -interferon (5), nerve growth factor (6); antibodies directed against functional molecules such as Fas/APO-1 (7) or CD28 (8) proteins; as well as stress-inducing agents such as UV (9) and ionizing radiation (10, 11); antileukemic agents (12, 13); and H2O2 (9). The observation that cell-permeant ceramides or natural ceramide (generated by treating cells with bacterial sphingomyelinase) could mimic the biological effects of most SPM-ceramide cycle agonists has provided significant weight as to the role of ceramide in signal transduction. Finally, ceramide has been shown to exert a wide range of biological effects, depending on the cellular model, including cell activation, mitogenic signaling, survival promoting effect, growth inhibition, and apoptosis. As an example, ceramide has been described to induce growth inhibition in Molt-4 cells (14), proliferation in fibroblasts (15), and apoptosis in many other cellular models such as U937 (3) and lymphoblastoid (16) and endothelial (10) cells.

The multiplicity of biological activities of ceramide suggests that it possesses several downstream targets, which, in turn, mediate distinct intracellular pathways. Among these targets there may be a serine-threonine phosphatase termed ceramide-activated protein phosphatase (17) and/or a proline-directed protein kinase termed ceramide-activated protein kinase (18), which, in turn, could activate the mitogen-activated protein kinase cascade in U937 cells (19). Ceramide has also been described to activate stress-activated protein kinases (SAPK/c-Jun kinase), which may be more closely related to ceramide-induced apoptosis in U937 and in endothelial cells (9). In addition, ceramide has been shown to activate transcription factors such as NF-kappa B (20) and AP-1, which could also play an important role since, for example, inhibitors of AP-1 activation or antisense oligonucleotides for c-jun prevented ceramide-induced apoptosis of HL-60 cells (21). Nevertheless, to what extent these various signaling proteins are directly or indirectly involved within the SPM-ceramide pathway remains elusive.

Despite the characterization of various biochemical changes associated with the SPM-ceramide signaling, such as apoptosis, a consensus on the sequence of cellular events has not been reached. However, there is mounting evidence that radical oxygen species (ROS) may be central (22). Indeed, ROS are involved in apoptosis induced by agents such as TNF-alpha (23), UV light (24), ionizing radiation (25), and anthracyclines (26), which have been all documented to activate the sphingomyelin cycle. Furthermore, ROS themselves, such as low doses of H2O2, or prooxidant conditions, such as UV or gamma -irradiation, activate signaling pathways and transcription factors that have been involved in ceramide-induced apoptosis such as mitogen-activated protein kinase (27) or stress-activated protein kinases cascades (28) as well as AP-1 and NF-kappa B activation (for review, see Ref. 29). Moreover, oxidant production has been shown to be accompanied by cell death triggered by a cell permeant ceramide (C2-ceramide) in lymphoid B cells (30). Altogether, these observations suggest ROS as a common mediator in ceramide-induced apoptosis.

Here we show that C6-ceramide induces intracellular H2O2 production followed by DNA fragmentation in U937 cells whereas the inactive analogue (dihydro-C6 ceramide) was ineffective. Ceramide-induced apoptosis was inhibited by ROS scavengers such as dithiocarbamates (PDTC) and N-acetylcysteine, a thiol antioxidant and a glutathione (GSH) precursor. In this study, we provide evidence that H2O2 is produced at the ubiquinone site of the mitochondrial respiratory chain with mitochondrial Ca2+ homeostasis alterations followed by disregulation of mitochondrial membrane potential (Delta Psi m). Finally, we show that C6-ceramide could activate the transcriptional factor NF-kappa B and AP-1 via mitochondrial ROS production.


EXPERIMENTAL PROCEDURES

Drugs and Reagents

The following reagents were purchased from Sigma: N-hexanoyl-D-sphingosine (C6-ceramide), carbonyl cyanide m-chlorophenyl hydrazone, N-acetylcystein, PDTC, rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, ruthenium red (RR). Dihydro-C6-ceramide was a generous gift from Dr. Salem Chouaib (CJF INSERM 9411, Villejuif, France). C2938 (6-carboxy-2',7' dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester)) and DiOC6 (3,3'-dihexyloxacarbocyanine iodide) were purchased from Molecular Probes (Interchim, France). Stock solutions of the reagents were routinely prepared in phosphate-buffered saline (PBS), dimethyl sulfoxide or ethanol as appropriate. [methyl-3H]Thymidine (79 Ci/mmol) was purchased from Amersham (Les Ulis, France). TNF-alpha was purchased from PeproTech-Tebu (Le Perray en Yvelines, France), and daunorubicin (Cerubidine®) was from Laboratoire Roger Bellon (Neuilly-sur -Seine, France).

Cell Culture

The human leukemia cell line U937 (monocytic) was obtained from the ATCC (Rockville, MD) and grown in RPMI 1640 at 37 °C in 5% CO2. Culture medium was supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and antibiotics, streptomycin (100 µg/ml) and penicillin (200 units/ml). All experiments with C6-ceramide were done in RPMI 1640 supplemented with 1% fetal calf serum, L-glutamine, and antibiotics. Cell stocks were screened routinely for Mycoplasma by the polymerase chain reaction method (Stratagene Mycoplasma PCR kit, La Jolla, CA).

The U937 rho ° subline was maintained in the same medium as U937 cells but supplemented with glucose (4.5 mg/ml), pyruvate (0.1 mg/ml), and uridine (50 µg/ml) (31). For some experiments U937 cells were grown in RPMI 1640 without glucose supplemented with 10% heat-inactivated fetal calf serum dialyzed against PBS.

Determination of ROS

Production of ROS was detected with C2938 fluorescent probe. C2938 is an uncharged cell-permeant molecule. Inside the cells, this probe is cleaved by nonspecific esterases, formed carboxydichlorofluroscein which is oxidized in the presence of H2O2. Exponentially growing cells (5 × 105 cells/ml) were labeled with 0.5 µM C2938 for 1 h and then incubated in the absence or presence of C6-ceramide at 37 °C for various periods of time. The cells were washed in PBS, and cell fluorescence was determined using flow cytometry on a FACScan (Becton Dickinson). The presence of ethanol (final concentration 0.25%) in the culture medium did not affect the fluorescence of C2938. Additional experiments were performed by preincubating the cells with nontoxic concentrations of specific mitochondrial inhibitors (rotenone, TTFA, antimycin A, RR) or by coincubation with antioxidant (PDTC). The optimal effective nontoxic concentrations for 3 h continuous incubation of these inhibitors were determined by dose-effect studies with rotenone 1-5 µM, TTFA 10-50 µM, and antimycin A 5-10 µM (data not shown).

Cytochemical Staining

Exponentially growing cells (5 × 105 cells/ml) were preincubated with or without various inhibitors before the addition of C6-ceramide. Cells were harvested by centrifugation onto glass slides and stained with May-Grünwald-Giemsa stain. Changes in cell morphology were examined by light microscopy using a Zeiss microscope. Apoptotic cells were scored and expressed as the number of cells exhibiting morphology typical of apoptosis (chromatin condensation and fragmentation, and cytoplasmic volume reduction) per 200 cells counted (5-10 fields).

For photography, cells were fixed in 4% paraformaldehyde, washed in PBS. Cells were then stained with 0.1 µg/ml 4',6'-diamidino-2-phenylindol for 1 h, washed, and mounted for fluorescence microscopy (Leica model Diaplan).

Quantification of DNA Fragmentation

DNA fragmentation was quantified as described previously (26). Exponentially growing cells (5 × 105 cells/ml) were labeled with 0.5 µCi/106 cells of [methyl-3H]thymidine for 24 h and washed three times with nonradioactive fresh medium. Labeled cells were exposed to C6-ceramide for 6- or 24-h continuous exposure. In additional experiments, cells were preincubated with various inhibitors before the addition of C6-ceramide. Cells were harvested by centrifugation, and the pellets were suspended in lysis buffer containing Tris 15 mM, EDTA 20 mM, Triton X-100 0.5%, pH 8.0. After 30 min on ice, samples were centrifugated at 20,000 × g for 30 min, and the pellets were resuspended in lysis buffer. The radioactivity present in the supernatant (detergent-soluble low molecular weight DNA) and in the pellet (intact chromatin DNA or large chromatin fragments, >50 kilobase pairs) was determined by liquid scintillation counting. In control cells DNA fragmentation with [3H]thymidine alone was below 2-3%.

Interleukin-1beta -converting Enzyme (ICE) Substrate Cleavage Assay

ICE activation was measured as described previously (32). Briefly, after treatment with C6-ceramide, cells were made permeable with 20 µM digitonin for 5 min. Cells were washed in PBS and resuspended in 100 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol, and 0.1 mg/ml ovalbumin at 25 °C and incubated 30 min with 20 µM of the fluorogenic ICE substrate DABC-YL-YVADAP-EDANS (BACHEM, Voisins le Bretonneux, France). The fluorescence was analyzed by spectrofluorometry using an excitation wavelength of 340 nm and an emission wavelength of 490 nm (33).

Assessment of Delta Psi m

To evaluate Delta Psi m, exponentially growing cells (5 × 105 cells/ml) were incubated with C6-ceramide for various periods of time. 15 min before the end of incubation, cells were labeled with DiOC6 (40 nM in PBS) at 37 °C (34). After washing, cells were analyzed by flow cytometry. Control experiments were performed in the presence of carbonyl cyanide m-chlorophenyl hydrazone, an uncoupling agent that abolishes the Delta Psi m, at 50 µM for 15 min at 37 °C.

Nuclear Extract Preparation

Extracts were prepared as described previously (19). Cells (5 × 106) were incubated with or without C6-ceramide in the presence or absence of different inhibitors. Cells were then washed twice with ice-cold PBS and resuspended in 10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µM pepstatin A, 0.6 µM leupeptin, 1 µg/ml aprotinin, and 0.6% Nonidet P-40. After 15 min on ice, the nuclear pellet was recovered after centrifugation at 1200 × g and resuspended in 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA. Aliquots were then incubated at 4 °C for 30 min and centrifuged at 21,000 × g, and supernatants containing nuclear proteins were collected. Protein concentrations were determined according to Smith et al. (35) using bicinchoninic acid (Sigma).

Electrophoretic Mobility Shift Assays

Labeling of NF-kappa B (5'-AGTTGAGGGGACTTTCCCAGGC-3') and AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3') consensus oligonucleotides (binding sites are underlined) was performed using T4 polynucleotide kinase and [gamma -32P]ATP (specific activity, 5000 Ci/mmol, Amersham, Les Ulis, France). Binding reactions were carried out in 2 mM HEPES (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 2% glycerol, 0.5 mM dithiothreitol, 1 µg poly(dI-dC), and 2 µg of bovine serum albumin. Typical reactions contained 50,000 cpm of end-labeled NF-kappa B or AP-1 consensus oligonucleotide (Promega, Madison, WI) with 2-6 µg of nuclear extract. After 20 min of incubation, the mixture was electrophoresed through a low ionic strength 4% polyacrylamide gel (acrylamide:bisacrylamide ratio 80:1) containing 6.7 mM Tris-HCl (pH 7.9), 3.3 mM sodium acetate, 2 mM EDTA. The gel was preelectrophoresed for 90 min at 10 V/cm. Electrophoresis was carried out at the same voltage for 3 h at room temperature with buffer recirculation. The gel was then dried and autoradiographed with intensifying screens at -70 °C. Quantification of bands was performed by densitometry and by radioactivity counting of excised bands. Band specificity was determined by competition experiments using 100-fold excess unlabeled NF-kappa B or AP-1 consensus oligonucleotide, as well as supershift assays (data not shown) for NF-kappa B using p65-, p55-, and c-Rel-specific antibodies generously provided by Dr. J. Imbert (INSERM U119, Marseille).


RESULTS

H2O2 Production by C6-Ceramide

The fluorescence distribution of the C2938 dye, revealing the presence of hydrogen peroxide, was measured by flow cytometry in the viable cell population. Fig. 1A shows the increase in the mean C2938 fluorescence in 25 µM C6-ceramide-treated cells relative to untreated cells. The mean fluorescence increased as a function of time, reflecting H2O2 generation in U937 cells induced by C6-ceramide. Within 60 min, significant H2O2 was detected, and this level increased up to 180 min. At low doses (5 µM and 10 µM) we observed only a low level of H2O2 generation at 180 min (Fig. 1A). In comparison, Fig. 1B presents the H2O2 produced, after 60 min, by SPM-ceramide agonists daunorubicin and TNF-alpha . Treatment of U937 cells by TNF-alpha generated comparable H2O2 production, whereas daunorubicin induced significantly higher H2O2 generation. The mean fluorescence emitted by C2938 in cells treated with exogenous H2O2 is presented, as well as dihydro-C6-ceramide, an inactive ceramide analogue, which had no significant effect. In addition, glucose deprivation and addition of deoxyglucose (2.5 mM) did not change H2O2 production in U937 cells treated with C6-ceramide (data not shown). These results suggest that, in this model, H2O2 production is independent of the pentose cycle.


Fig. 1. Flow cytometry analysis of ROS production. A, kinetics of ROS production induced by C6-ceramide in U937 cells; (triangle ) 5 µM, (square ) 10 µM, (bullet ) 25 µM C6-ceramide. Results represent the mean (± S.D.) of four separate experiments. B, comparison of ROS production induced by different agents at 1 h: C6-ceramide (25 µM), dihydro-C6-ceramide (25 µM), daunorubicin (DNR) (1 µM), TNF-alpha (20 ng/ml), H2O2 (15 µM). Results represent the mean of four separate experiments. Bars, S.D. *p < 0.05 as compared with C6-ceramide alone and evaluated by Student's t test. a.u., arbitrary units.
[View Larger Version of this Image (16K GIF file)]

Role of Mitochondria in H2O2 Production Induced by C6-Ceramide

To further investigate the role of mitochondria in the generation of H2O2 induced by C6-ceramide, we tested several classic specific mitochondrial respiratory chain inhibitors (36-39). Two sites of the mitochondrial respiratory chain have been identified as sources of ROS. One depends on the autooxidation of complex I, whereas the other is dependent on the autooxidation of complex III (ubiquinone site) (40). When U937 cells were pretreated with rotenone (a specific inhibitor of complex I that interferes with the electron flow from NADH-linked substrates and NADH dehydrogenase to the ubiquinone pool), or TTFA (a specific inhibitor of complex II that interferes with the electron transport flow from succinate dehydrogenase to the ubiquinone pool) followed by treatment with C6-ceramide, we observed an almost complete inhibition of H2O2 generation (Fig. 2). On the other hand, pretreatment by antimycin A (a specific inhibitor of complex III, which inhibits the electron flow from ubiquinone to complex IV), increased by approximately 2-fold H2O2 production induced by C6-ceramide (Fig. 2).


Fig. 2. Effects of mitochondrial inhibitors on ROS production induced by C6-ceramide. U937 cells were pretreated for 30 min with rotenone (5 µM), TTFA (50 µM), or antimycin A (AA) (10 µM), or 1 h with RR (25 µM), followed by C6-ceramide treatment (25 µM) during 1 h continuous exposure. Results represent the mean (± S.D.) of four separate experiments. *p < 0.01 as compared with C6-ceramide alone and evaluated by Student's t test. Inset, ROS production in 25 µM C6-ceramide treated U937 rho ° cells. a.u., arbitrary units
[View Larger Version of this Image (28K GIF file)]

Furthermore, we tested H2O2 production in U937 rho ° cells. These cells have been selected in the presence of ethidium bromide and present mitochondrial DNA depletion and a reduced complex III activity in the mitochondrial respiratory chain (31). We did not observe significant H2O2 production in U937 rho ° cells treated for 1 h with 25 µM C6-ceramide (Fig. 2, inset). These results strongly support the role of complex III in C6-ceramide-induced H2O2 production.

In addition, since calcium has been shown to play a role in apoptosis in certain experimental systems (41), we tested the contribution of calcium to the production of H2O2 induced by C6-ceramide. U937 cells were pretreated with ruthenium red (an inhibitor of the mitochondrial calcium uptake) (42) and then analyzed for the production of H2O2 generated by C6-ceramide. As shown in Fig. 2, ruthenium red inhibited C6-ceramide induced H2O2 generation by 60-70%.

C6-Ceramide-induced Apoptosis, Implication of ROS

We tested the relationship between H2O2 production and apoptosis induced by C6-ceramide in U937 cells. U937 cells were treated in kinetics experiments with different doses of C6-ceramide and analyzed for typical morphological features of apoptosis (Fig. 3A) as well as DNA fragmentation using the [3H]thymidine release assay. At 6 h, approximately 10% of U937 cells treated by 25 µM C6-ceramide presented typical apoptotic features. This population grew to approximately 30% at 24 h (Fig. 3B). Analysis of DNA fragmentation also reflected a similar effect (8% at 6 h; 37% at 24 h) and we observed that C6-ceramide dose-dependent DNA fragmentation correlated closely with H2O2 production (data not shown). In addition, cleavage of ICE-substrate (32) was observed as early as 4 h after 25 µM C6-ceramide treatment (Fig. 3C).


Fig. 3. Kinetics of C6-ceramide-induced apoptosis and DNA fragmentation. Cells were treated with 25 µM C6-ceramide during 6 or 24 h continuous exposure. A, morphological 4',6'-diamidino-2-phenylindol staining of U937 untreated cells (a), treated during 6 h (b), or 24 h (c) C6-ceramide continuous exposure (magnification of × 50). B, apoptotic cells were analyzed by cytochemical staining. C, ICE activation. U937 cells were treated (square ) or not (black-square) with 25 µM C6-ceramide during 1 or 4 h. ICE substrate cleavage was measured as described under "Experimental Procedures." Results represent the mean (±S.D.) of three separate experiments. a.u., arbitrary units
[View Larger Version of this Image (68K GIF file)]

We tested the influence of different ROS scavengers on apoptosis and DNA fragmentation induced by C6-ceramide. U937 cells were pretreated with different concentrations of N-acetylcysteine, the thiol antioxidant and GSH precursor (22), or PDTC, the oxygen radical scavenger and iron chelator (43), in combination with 6 h (data not shown) or 24 h of continuous exposure to 25 µM C6-ceramide. C6-Ceramide induced-apoptosis (data not shown) and DNA fragmentation were inhibited by both N-acetylcysteine (Fig. 4A) and PDTC (Fig. 4B) in a dose-dependent manner. While confirming that C6-ceramide is able to induce apoptosis in U937 cells, these results implicated ROS within this process, since PDTC was also able to inhibit ROS production induced by C6-ceramide (Fig. 4B, inset). To further substantiate the role of ROS in ceramide-mediated apoptosis, we evaluated the impact of both rotenone and TTFA on this cell death process. Both mitochondrial respiratory chain inhibitors significantly inhibited C6-ceramide-triggered apoptosis (not shown) and DNA fragmentation (Fig. 5). In addition, pretreatment of U937 cells with antimycin A greatly increased the apoptotic population induced by C6-ceramide (not shown) as well as DNA fragmentation (Fig. 5). The effective nontoxic concentrations for 6 h of continuous exposure of these inhibitors were determined by dose-effect studies (data not shown).


Fig. 4. Inhibition of C6-ceramide-induced DNA fragmentation by antioxidants. A, after 2 h of preincubation with different concentrations of N-acetylcysteine (N-ac), cells were incubated with C6-ceramide (25 µM) for 24 h continuous exposure. B, cells were cotreated with different concentrations of PDTC and C6-ceramide during 24 h of continuous exposure. Inset, inhibition of ROS production by PDTC (5 µM) at 1 h. For all experiments specific DNA fragmentation was quantified as described under "Experimental Procedures." Results represent the mean (±S.D.) of three separate experiments. *p < 0.05 as evaluated by Student's t test in comparison with C6-ceramide alone. a.u., arbitrary units
[View Larger Version of this Image (23K GIF file)]


Fig. 5. Differential effects of mitochondrial respiratory chain inhibitors on C6-ceramide-induced DNA fragmentation. Cells were pretreated 30 min with rotenone (1 µM), TTFA (10 µM), or antimycin A (5 µM) before treatment with C6-ceramide during 6 h of continuous exposure. DNA fragmentation was analyzed as described under "Experimental Procedures." Results represent the mean (±S.D.) of three separate experiments. *p < 0.05 as compared with C6-ceramide alone evaluated by Student's t test.
[View Larger Version of this Image (30K GIF file)]

Alteration of Delta Psi m by C6-Ceramide

It has been described that a reduction in Delta Psi m precedes apoptosis and may represent an early signaling event (34). Using the DiOC6 fluorescent probe, we analyzed the Delta Psi m of U937 treated with C6-ceramide. Cells were exposed to DiOC6 probe at 40 nM, 15 min before the end of incubation with C6-ceramide. As described previously, under these conditions, only mitochondria are labeled as confirmed by confocal microscopy experiments (data not shown). In addition, treatment with carbonyl cyanide m-chlorophenyl hydrazone (an uncoupling agent that abolishes the Delta Psi m) and DiOC6 showed a drastic reduction of Delta Psi m, whereas incubation in the presence of high concentration of KCl (120 mM) showed no difference, confirming that, under these conditions, only mitochondrial membrane potential is measured (data not shown). In our hands, C6-ceramide had no significant influence on mitochondrial membrane potential at 2 h, and only 5% of cells showed a Delta Psi m reduction at 6 h. However, after 20 h of incubation with C6-ceramide, 20% of cells showed a drastic reduction of the Delta Psi m (Fig. 6), suggesting that reduction of mitochondrial transmembrane potential is a distal event in DNA fragmentation triggered by C6-ceramide.


Fig. 6. Assessment of Delta Psi m. U937 cells were treated in continuous exposure with (black line) EtOH; (blue line) C6-ceramide (25 µM) 2 h; (red line) C6-ceramide (25 µM) 6 h; (green line) C6-ceramide (25 µM) 20 h. After treatment, cells were stained with DiOC6 and analyzed by flow cytometry.
[View Larger Version of this Image (16K GIF file)]

Activation of Transcriptional Factors NF-kappa B and AP-1 by C6-Ceramide, Implication of Mitochondria

In light of the fact that ceramide and many other conditions which induce an oxidative stress result in increased activation of NF-kappa B and AP-1 (21) (for review, see Ref. 44), we tested the effect of the different respiratory chain inhibitors or antioxidants on NF-kappa B and AP-1 activation induced by C6-ceramide. As shown in Fig. 7, A and B, C6-ceramide induced a 2-3-fold activation of both NF-kappa B and AP-1 within 1 h. The activation of both NF-kappa B and AP-1 induced by C6-ceramide was inhibited by a 30-min preincubation with rotenone (>60 and >25%, respectively), TTFA (>60 and >25%, respectively), a 2-h preincubation with N-acetylcysteine (>65 and >40%, respectively), and a coincubation with PDTC (>70 and >30%, respectively). In contrast, cells treated with antimycin A, which by itself is a potent activator of the transcription factors, presented slightly higher levels of NF-kappa B (1.5-2-fold) and AP-1 (2-3-fold) activation compared with C6-ceramide alone.


Fig. 7. Effects of mitochondrial inhibitors and antioxidants on NF-kappa B and AP-1 activation induced by C6-ceramide. U937 cells were pretreated with mitochondrial inhibitors or antioxidants, followed by 1-h exposure to C6 ceramide (25 µM). Nuclear extracts were isolated and gel shift assays were performed as described under "Experimental Procedures." A, NF-kappa B activation; B, AP-1 activation. For both transcriptional activation, nuclear extracts were from untreated cells (lanes 1, 3, 5, 7, 9, and 11), C6-ceramide treated cells (lanes 2, 4, 6, 8, 10, and 12); rotenone (5 µM) (lanes 3 and 4); TTFA (50 µM) (lanes 5 and 6); antimycin A (10 µM) (lanes 7 and 8); PDTC (10 µM) (lanes 9 and 10); N-acetylcysteine (25 mM) (lanes 11 and 12). ns, nonspecific binding.
[View Larger Version of this Image (56K GIF file)]


DISCUSSION

ROS are produced in all mammalian cells, partly as a result of normal cellular metabolism and in response to various stimuli. ROS comprise essentially the superoxide anion radical (Obardot 2), H2O2 which is a catalytically derived intermediate in the conversion of Obardot 2 to H2O and O2, and the hydroxyl radical (OH·) which is a by-product of both Obardot 2 and H2O2 in iron-dependent radical reactions (45). In our study, we demonstrate that a cell-permeant ceramide (C6-ceramide) induced intracellular H2O2 accumulation in U937 cells. This H2O2 generation was significant at 1 h, and preceded ICE activation by 3 h. C6-Ceramide-mediated ROS appeared specific since dihydro-C6-ceramide was essentially ineffective in generating H2O2.

Since ceramide does not possess a prooxidant chemical structure, we hypothesized that ceramide acted by stimulating ROS production within cells or cell organelles. We observed that glucose deprivation and addition of deoxyglucose did not change ceramide-induced H2O2 production. These data rule out a role for the cytosolic pentose cycle in C6-ceramide-induced H2O2 production in U937 cells. Furthermore, we cannot exclude that C6-ceramide could inhibit catalase, leading to H2O2 overproduction. In our experiments (not shown), we observed no differences in catalase activity, measuring by continuous recording of the absorbance at 240 nm of a 10 mM solution of H2O2, in extracts from U937 cells treated or not with C6-ceramide. Several lines of evidence suggested that mitochondria could be involved in ceramide-induced H2O2 production. Indeed, ROS are produced at a high rate as a by-product of aerobic metabolism in mitochondria, which, in fact, constitute the greatest source of ROS as the mitochondrial electron transport system consumes 85-90% of the oxygen utilized by the cell (46). Intrinsic to this process is the generation of ROS derived from specific segments of the electron transport chain, mainly at the ubiquinone site in complex III. This site catalyzes the conversion of O2 to Obardot 2 and, in turn, can lead to the formation of other potent oxygen-derived free radicals (47). In fact, our study shows that inhibition of the electron transport at complex I by rotenone resulted in a reduction in ceramide-induced H2O2 accumulation. This suggests that ceramide-induced H2O2 production was distal to complex I. Inhibition at complex II by TTFA also decreased ceramide-induced H2O2 production, suggesting that ceramide did not interfere with complex II function. However, when electron flow was inhibited distal to the ubiquinone pool, by the addition of antimycin A, a marked potentiation of both H2O2 production and DNA fragmentation was observed. Moreover, the observation that C6-ceramide did not lead to H2O2 generation in U937 rho ° cells strongly argues for a mitochondrial electron transport origin for H2O2. Taken together, these results imply that ceramide-induced H2O2 production is centered at the ubiquinone site of the mitochondrial respiratory chain.

Ubiquinone (also known as coenzyme Q) is the only non-protein component of the electron transport chain that can capture one or two electrons, thereby forming ubiquinol (hydroquinone). Since the ubiquinone molecule is not tightly bound to proteins, it can play a strategic role as a mobile carrier of electrons. The function of ubiquinone as a H2O2-producing site is not surprising, since it has previously been shown that the mitochondrial respiratory chain generates superoxide anion secondary to the interruption of electron flow between the rotenone- and antimycin-sensitive sites through one-electron reduction (36-39).

Another respiratory linked function of the mitochondrion is Ca2+ accumulation that has been described to be mediated by a RR-sensitive uniporter (48). In the last few years a correlation between transmembrane mitochondrial Ca2+ exchange and oxidative stress has been discovered; most reports suggest that oxidative stress may induce the release of mitochondrial Ca2+ via a Na+-independent pathway (41). Conversely, other studies have suggested that alterations in Ca2+ homeostasis may initiate oxidative stress. Thus, it has been reported that an increase in intracellular Ca2+ concentration in isolated hepatocytes results in the depletion of mitochondrial GSH, probably as a result of mitochondrial Ca2+ cycling. More recently, RR attenuated doxorubicin-induced ROS in isolated heart mitochondria, suggesting that alteration in mitochondrial Ca2+ transport may be involved in the release of ROS (49). Our study shows that RR inhibited H2O2 production in ceramide-treated cells, suggesting that ceramide induced mitochondrial Ca2+ homeostasis alterations which, in turn, could be involved in ceramide-induced oxidative stress. Our experiments using flow cytometry and the calcium-specific INDO-1 AM fluorescent probe (Molecular Probes, Eugene, OR) (50), in C6-ceramide-treated U937 cells (not shown) and two earlier reports in fibroblasts and T cells have shown that permeant ceramides had no effect on whole cellular Ca2+ content (51, 52).

In our study, we attempted to determine if ROS were important in ceramide signal transduction. We observed that N-acetylcysteine or PDTC, two potent antioxidants, significantly decreased in parallel both H2O2 production and ceramide-induced apoptosis and DNA fragmentation. Moreover, rotenone decreased, whereas antimycin A increased ceramide-induced apoptosis and DNA fragmentation. Therefore it appears that mitochondrial ROS production is involved in these processes. However, it should be noted that N-acetylcysteine, PDTC, and rotenone significantly decreased but did not abrogate ceramide-induced DNA fragmentation. This observation can be related to the inability of antioxidants to totally block ceramide-induced H2O2 production. Alternatively, it is conceivable that ROS mediated some but not all the multiple downstream signaling cascades activated by ceramide and that other mediators also play an important role in ceramide-induced apoptosis. Further studies are needed to evaluate the effects of ROS produced by ceramide on ceramide-activated protein kinase and ceramide-activated protein phosphatase activities as well as their role in the activation of downstream signaling events that have been involved in ceramide-induced apoptosis such as mitogen-activated protein kinase/stress-activated protein kinases cascade activation.

By altering the generation of ROS through inhibition of the electron transport chain and Ca2+ homeostasis, our study demonstrates a potentially significant role of ROS in triggering downstream signaling cascades leading to apoptosis in U937 cells. It has recently been reported that apoptosis is preceded by a sequential disregulation of mitochondrial functions, characterized by an initial reduction of Delta Psi m followed by a rotenone- and RR-sensitive ROS production in U937 cells treated with TNF-alpha (34). In our study we found that ceramide-induced H2O2 production (1 h) largely preceded reduction of Delta Psi mq, which in fact occurred mainly at 20 h. This suggests that the initial ROS generation produced a ripple effect consisting in the loss of Delta Psi m of mitochondria, which, in turn, may accelerate the ongoing cell death process through self-amplification of ROS production and Ca2+ homeostasis disturbances. Altogether our study suggests that mitochondrial ROS production may represent a crucial event for ceramide-induced signaling and apoptosis.

ROS have largely been described as potent activators of transcription factors, such as NF-kappa B and AP-1 (21, 44, 53, 54), two transcription factors activated in ceramide-induced apoptosis (20, 21). It has also recently been reported that inhibition of mitochondrial complex III by antimycin A led to a 2-3-fold NF-kappa B activation as assessed by gel shift assays and that depletion of mitochondrial GSH with diethylmaleate further increased antimycin-induced NF-kappa B activation (55). These important studies strongly suggest that oxidative stress within mitochondria can promote extramitochondrial activation of NF-kappa B (55). In our study, we found that ceramide-induced activation of NF-kappa B and AP-1 was blocked not only by N-acetylcysteine or PDTC but also, and at a similar level, by rotenone and TTFA. Conversely, pretreatment with antimycin A led to further enhancement of ceramide-mediated NF-kappa B and AP-1 activation. These observations suggest a redox regulation of ceramide-induced NF-kappa B and AP-1 activation, which appeared to be essentially mediated by mitochondrial ROS production.

In conclusion, our study shows that C6-ceramide could induce mitochondrial disturbances such as H2O2 production, alteration of calcium homeostasis followed by alteration of mitochondrial transmembrane potential. In light of these findings, it is conceivable that increased antioxidative defenses through detoxifying enzymes, such as glutathione peroxydase, or radical scavengers can decrease the apoptotic effect of ceramide in U937 cells, and that of anti-tumor agents which are SPM-ceramide agonists. In addition, the role of mitochondrial ROS production in ceramide-induced apoptosis could explain the protective effect of Bcl-2 (56-58) and Bcl-xL (30, 58) against ceramide-induced apoptosis since this protein, which has been described as an antioxidant, is preferentially located in the mitochondrial inner membrane (59). Finally, two very recent studies have proposed a central role for mitochondrial cytochrome c release within the apoptotic signaling pathway (60, 61). Therefore, it would be of great interest to determine whether ceramide could directly trigger cytochrome c release and if this process is closely related to the electron-transport chain.


FOOTNOTES

*   This work was supported in part by la Fédération Nationale des Centres de Lutte Contre le Cancer (A. Q. M., J. P. J.), le Conseil Régional Midi-Pyrénées (J. P. J.), l'Association pour la Recherche sur le Cancer Grants 6749 (G. L.) and 2069 (J. P. J.), and by La Ligue Nationale Contre le Cancer (G. L.).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.
§   To whom correspondence should be addressed: CJF INSERM 9503, Centre Claudius Régaud, 20-24 rue du Pont St Pierre, 31052 Toulouse Cedex, France. Tel.: 33 05 61 42 41 73; Fax: 33 05 61 42 46 06.

ACKNOWLEDGEMENTS

We thank Drs. D Lautier, T. Levade and A. Bettaïeb for stimulating discussions, Dr. H. Conjeaud for calcium experiments, and A. Rousse for technical assistance.


Note Added in proof

While this manuscript was being reviewed, another paper also demonstrated the mitochondrial complexe III origin of hydrogen peroxide generated by ceramide (62). In concert, both studies underline the novel role of ceramide as an inducer of oxidative stress or perhaps more precisely of a reactive oxygen species-mediated signaling pathway.


REFERENCES

  1. Kolesnick, R. N. (1991) Prog. Lipid Res. 30, 1-38 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hannun, Y. A. (1996) Science 274, 1855-1859 [Abstract/Free Full Text]
  3. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771 [Medline] [Order article via Infotrieve]
  4. Andrieu, N., Salvayre, R., and Levade, T. (1994) Biochem. J. 270, 24518-24524
  5. Kim, M.-Y., Linardic, C., Obeid, L., and Hannun, Y. (1991) J. Biol. Chem. 266, 484-489 [Abstract/Free Full Text]
  6. Dobrowsky, R. T., Werner, M. H., Castellino, A. M., Chao, M. V., and Hannun, Y. A. (1994) Science 265, 1596-1599 [Medline] [Order article via Infotrieve]
  7. Cifone, M. C., De Maria, R., Roncaioli, P., Rippo, M. R., Azuma, L. L. M., Lanier, L. L., Santoni, A., and Testi, R. (1994) J. Exp. Med. 177, 1547-1552
  8. Boucher, L. M., Wiegmann, K., Füttere, A., Pfeffer, K., Machleidt, T., Schütze, S., Mak, T. W., and Krönke, M. (1995) J. Exp. Med. 181, 2059-2068 [Abstract]
  9. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Nature 380, 75-79 [CrossRef][Medline] [Order article via Infotrieve]
  10. Haimovitz-Friedman, A., Kan, C. C, Ehleiter, D., Persaud, R. S., McLoughlin, M., Fuks, Z., and Kolesnick, R. N. (1994) J. Exp. Med. 180, 525-535 [Abstract]
  11. Santana, P., Pena, I. A., Haimovitz-Friedmann, A., Martin, S., Green, D., Mc Loughlin, M., Cordon-Cardo, C., Schuchman, E. H., Fuks, Z., and Kolesnick, R. (1996) Cell 86, 189-199 [Medline] [Order article via Infotrieve]
  12. Strum, J. C., Small, G. W., Pauig, S. B., and Daniel, L. W. (1994) J. Biol. Chem. 269, 15493-15497 [Abstract/Free Full Text]
  13. Jaffrézou, J. P., Levade, T., Bettaïeb, A., Andrieu, N., Bezombes, C., Maestre, N., Vermeersch, S., Rousse, A., and Laurent, G. (1996) EMBO J. 15, 2417-2424 [Abstract]
  14. Jayadev, S., Liu, B., Bielawaska, A. E., Lee, J. Y., Nazaire, F., Pushkareva, M. Y., Obeid, L. M., and Hannun, Y. A. (1995) J. Biol. Chem. 270, 2047-2052 [Abstract/Free Full Text]
  15. Olivera, A., Buckley, N. E., and Spiegel, S. (1992) J. Biol. Chem. 267, 26121-26127 [Abstract/Free Full Text]
  16. Tepper, C. G., Jayadev, S., Liu, B., Bielawska, A., Wolff, R., Yonehara, S., Hannun, Y. A., and Seldin, M. F. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8443-8447 [Abstract]
  17. Dobrowsky, R. T., and Hannun, Y. A. (1992) J. Biol. Chem. 267, 5048-5051 [Abstract/Free Full Text]
  18. Mathias, S., Dressler, K. A., and Kolesnick, R. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10009-10013 [Abstract]
  19. Yao, B., Zhang, Y., Delikat, S., Mathias, S., Basu, S., and Kolesnick, R. N. (1995) Nature 378, 307-310 [CrossRef][Medline] [Order article via Infotrieve]
  20. Schütze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K., and Krönke, M. (1992) Cell 71, 765-776 [Medline] [Order article via Infotrieve]
  21. Sawai, H., Okazaki, T., Yamamoto, H., Okano, H., Takeda, Y., Tashima, M., Sawada, H., Okuma, M., Ishikura, H., Umehara, H., and Domae, N. (1995) J. Biol. Chem. 270, 27326-27331 [Abstract/Free Full Text]
  22. Buttke, T. M., and Sandstrom, P. A. (1994) Immunol. Today 15, 7-10 [CrossRef][Medline] [Order article via Infotrieve]
  23. Larrick, J. W., and Wright, S. C. (1990) FASEB J. 4, 3215-3223 [Abstract]
  24. Devary, Y., Rosette, C., DiDonato, J. A., and Karin, M. (1993) Science 261, 1442-1445 [Medline] [Order article via Infotrieve]
  25. Manome, Y., Datta, R., Taneja, N., Shafman, T., Bump, E., Hass, R., Weichselbaum, R., and Kufe, D. (1993) Biochemistry 32, 10607-10613 [Medline] [Order article via Infotrieve]
  26. Quillet-Mary, A., Mansat, V., Duchayne, E., Come, M. G., Allouche, M., Bailly, J. D., Bordier, C., and Laurent, G. (1996) Leukemia 10, 417-425 [Medline] [Order article via Infotrieve]
  27. Stevenson, M. A., Pollock, S. S., Coleman, N., and Calderwood, S. K. (1994) Cancer Res. 54, 12-15 [Abstract]
  28. Shafman, T. D., Saleem, A., Kyriakis, J., Weichselbaum, R., Kharbanda, S., and Kufe, D. W. (1995) Cancer Res. 55, 3242-3245 [Abstract]
  29. Weichselbaum, R. R., Hallahan, D., Fuks, Z., and Kufe, D. (1994) Int. J. Radiat. Oncol. Biol. Phys. 30, 229-234 [Medline] [Order article via Infotrieve]
  30. Fang, W., Rivard, J. J., Ganser, J. A., LeBien, T. W., Nath, K. A., Mueller, D. L., and Behrens, T. W. (1995) J. Immunol. 155, 66-75 [Abstract]
  31. Gamen, S., Anel, A., Montoya, J., Marzo, I., Pineiro, A., and Naval, J. (1995) FEBS Lett. 376, 15-18 [CrossRef][Medline] [Order article via Infotrieve]
  32. Los, M., Van de Caren, M., Penning, L. C., Schenk, H., Westendorp, M., Bauerle, P. A., Dröge, M., Krammer, P., Fiers, W., and Schulze-Osthoff, K. (1995) Nature 375, 81-83 [CrossRef][Medline] [Order article via Infotrieve]
  33. Pennington, M. W., and Thornberry, N. A. (1994) Pept. Res. 7, 72-76 [Medline] [Order article via Infotrieve]
  34. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsh, T., Susin, S. A., Petit, P. X., Mignotte, B., and Kroemer, G. (1995) J. Exp. Med. 182, 367-377 [Abstract]
  35. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85 [Medline] [Order article via Infotrieve]
  36. Boveris, A., Cadenas, E., and Stroppani, A. O. M. (1976) Biochem. J. 156, 435-445 [Medline] [Order article via Infotrieve]
  37. Cadenas, E., and Boveris, A. (1980) Biochem. J. 188, 31-37 [Medline] [Order article via Infotrieve]
  38. Konstantinov, A. A., Peskin, A. V., Popova, E. Y., Khomutov, G. B., and Ruuge, E. K. (1987) Biochim. Biophys. Acta 894, 1-10 [Medline] [Order article via Infotrieve]
  39. Cino, M., and Del Maestro, R. F. (1989) Arch. Biochem. Biophys. 269, 623-638 [Medline] [Order article via Infotrieve]
  40. Turrens, J. F., Alexandre, A., and Lehninger, A. L. (1985) Arch. Biochem. Biophys. 237, 408-414 [Medline] [Order article via Infotrieve]
  41. Richter, C., and Kass, G. E. N. (1991) Chem.-Biol. Interact. 77, 1-23 [CrossRef][Medline] [Order article via Infotrieve]
  42. Hennet, T., Richter, C., and Peterhans, E. (1993) Biochem. J. 289, 587-592 [Medline] [Order article via Infotrieve]
  43. Schreck, R., Meier, B., Männel, D., Dröge, W., and Bauerle, P. A. (1992) J. Exp. Med. 175, 1181-1194 [Abstract]
  44. Schulze-Osthoff, K., Los, M., and Baeuerle, P. A. (1995) Biochem. Pharmacol. 50, 735-741 [CrossRef][Medline] [Order article via Infotrieve]
  45. Fridovich, I. (1978) Science 201, 875-880 [Medline] [Order article via Infotrieve]
  46. Shigenaga, M. K. T., Hagen, T. M., and Ames, B. N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10771-10778 [Abstract/Free Full Text]
  47. Chance, B., Sies, H., and Boveris, A. (1979) Physiol. Rev. 59, 527-605 [Free Full Text]
  48. Nicholls, D. G., and Akerman, K. (1982) Biochim. Biophys. Acta 683, 57-88 [Medline] [Order article via Infotrieve]
  49. Chacon, E., and Acosta, D. (1991) Toxicol. Appl. Pharmacol. 107, 117-128 [Medline] [Order article via Infotrieve]
  50. Gil, M. L., Vita, N., Lebel-Binay, S., Miloux, B., Chalon, P., Kaghad, M., Marchiol-Fournigault, C., Conjeaud, H., Caput, D., Ferrara, P., Fradelizi, D., and Quillet-Mary, A. (1992) J. Immunol. 148, 2826-2833 [Abstract/Free Full Text]
  51. Chao, C. P., Laulerderkind, J. F., and Ballou, L. R. (1994) J. Biol. Chem. 269, 5849-5856 [Abstract/Free Full Text]
  52. Breittmayer, J.-P., Bernard, A., and Aussel, C. (1994) J. Biol. Chem. 269, 5054-5058 [Abstract/Free Full Text]
  53. Schreck, R., Rieber, P., and Bauerle, P. A. (1991) EMBO J. 10, 2247-2258 [Abstract]
  54. Schulze-Osthoff, K., Beyaert, R., Vandevoorde, V., Haegemen, G., and Fiers, W. (1993) EMBO J. 12, 3095-3104 [Abstract]
  55. Garcia-Ruiz, C., Colell, A., Morales, A., Kaplowitz, N., and Fernandez-Checa, J. C. (1995) Mol. Pharmacol. 48, 825-834 [Abstract]
  56. Chen, M., Quintans, J., Fuks, Z., Thompson, C., Kufe, D. W., and Weichselbaum, R. R. (1995) Cancer Res. 55, 991-994 [Abstract]
  57. Martin, S. J., Takayama, S., McGahon, A. J., Miyashita, T., Corbeil, J., Kolesnick, R. N., Reed, J. C., and Green, D. R. (1995) Cell Death Differ. 26, 253-257
  58. Decaudin, D., Geley, S., Hirsch, T., Castedo, M., Marchetti, P., Macho, A., Kofler, R., and Kroemer, G. (1997) Cancer Res. 57, 62-67 [Abstract]
  59. Korsmeyer, S. J. (1992) Blood 80, 879-886 [Medline] [Order article via Infotrieve]
  60. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., and Wang, X. (1997) Science 275, 1129-1132 [Abstract/Free Full Text]
  61. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997) Science 275, 1132-1136 [Abstract/Free Full Text]
  62. García-Ruiz, C., Colell, A., Marí, M., Morales, A., and Fernández-Checa, J. C. (1997) J. Biol. Chem. 272, 11369-11377 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.