(Received for publication, August 15, 1996, and in revised form, April 15, 1997)
From the CJF INSERM 9503, Centre Claudius
Régaud, Toulouse Cedex, France, the ¶ Department of
Biochemistry and Molecular Biology, University of Zaragoza, 50009 Zaragoza, Spain, and the
CHU Purpan, Service d'Hématologie,
Toulouse, France
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 ° cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-
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.
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- (TNF-
) (3), interleukin-1
(4),
-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-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- (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
-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-
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 (m). Finally,
we show that C6-ceramide could activate the transcriptional factor
NF-
B and AP-1 via mitochondrial ROS production.
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-
was purchased from
PeproTech-Tebu (Le Perray en Yvelines, France), and daunorubicin
(Cerubidine®) was from Laboratoire Roger Bellon
(Neuilly-sur -Seine, France).
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 ° 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.
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 StainingExponentially 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).
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-1ICE 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 ofTo evaluate 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
m, at 50 µM for 15 min at 37 °C.
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 AssaysLabeling of NF-B
(5
-AGTTGAGGGGACTTTCCCAGGC-3
) and AP-1
(5
-CGCTTGATGAGTCAGCCGGAA-3
) consensus oligonucleotides
(binding sites are underlined) was performed using T4 polynucleotide
kinase and [
-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-
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-
B or AP-1 consensus oligonucleotide, as well as supershift assays
(data not shown) for NF-
B using p65-, p55-, and c-Rel-specific
antibodies generously provided by Dr. J. Imbert (INSERM U119,
Marseille).
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-. Treatment of U937 cells by TNF-
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.
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).
Furthermore, we tested H2O2 production in U937
° 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
° 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 ROSWe 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).
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).
Alteration of
It has been described
that a reduction in m precedes apoptosis and may represent an
early signaling event (34). Using the DiOC6 fluorescent probe, we
analyzed the
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
m) and DiOC6 showed a drastic reduction
of
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
m reduction at 6 h. However, after 20 h of incubation with C6-ceramide, 20% of cells showed a drastic
reduction of the
m (Fig. 6),
suggesting that reduction of mitochondrial transmembrane potential is a
distal event in DNA fragmentation triggered by C6-ceramide.
Activation of Transcriptional Factors NF-
In light of the fact
that ceramide and many other conditions which induce an oxidative
stress result in increased activation of NF-B and AP-1 (21) (for
review, see Ref. 44), we tested the effect of the different respiratory
chain inhibitors or antioxidants on NF-
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-
B and AP-1 within 1 h. The activation of both NF-
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-
B (1.5-2-fold) and AP-1
(2-3-fold) activation compared with C6-ceramide alone.
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 (O2),
H2O2 which is a catalytically derived
intermediate in the conversion of O
2 to H2O and
O2, and the hydroxyl radical (OH·) which is a
by-product of both O
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 O2 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
° 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 m followed by a rotenone- and RR-sensitive ROS
production in U937 cells treated with TNF-
(34). In our study we
found that ceramide-induced H2O2 production (1 h) largely preceded reduction of
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
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-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-
B activation as assessed by gel
shift assays and that depletion of mitochondrial GSH with
diethylmaleate further increased antimycin-induced NF-
B activation
(55). These important studies strongly suggest that oxidative stress
within mitochondria can promote extramitochondrial activation of
NF-
B (55). In our study, we found that ceramide-induced activation
of NF-
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-
B and AP-1 activation. These observations suggest a redox regulation of ceramide-induced NF-
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.
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.
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.