Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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It has been proposed that ceramide acts as a cellular messenger
to mediate tumor necrosis factor- (TNF-
)-induced apoptosis. Based
on this hypothesis, it was postulated that resistance of some cells to
TNF-
cytotoxicity was due to an insufficient production of ceramide
on stimulation by TNF-
. The present study was initiated to
investigate whether this was the case in mesangial cells, which normally are insensitive to TNF-
-induced apoptosis. Our results indicate that although C2 ceramide
was toxic to mesangial cells, the cell death it induced differed both
morphologically and biochemically from that induced by TNF-
in the
presence of cycloheximide (CHX). The most apparent effect of
C2 ceramide was to cause cells to swell, followed by disruption of the cell membrane. It is evident that
C2 ceramide caused cell death by
necrosis, whereas TNF-
in the presence of CHX killed the cells by
apoptosis. C2 ceramide did not
mimic the effects of TNF-
on the activation of c-Jun NH2-terminal protein kinase and
nuclear factor-
B transcription factor. Although mitogen-activated
protein kinase [extracellular signal-related kinase (ERK)]
was activated by both C2 ceramide and TNF-
, such activation appeared to be mediated by different mechanisms as judged from the kinetics of ERK activation. Furthermore, the cleavage of cytosolic phospholipase
A2 during cell death induced by
C2 ceramide and by TNF-
in the
presence of CHX showed distinctive patterns. The present study provides
evidence that apoptosis and necrosis use distinctive signaling
machinery to cause cell death.
apoptosis; mitogen-activated protein kinase; c-Jun
NH2-terminal protein kinase; nuclear factor-B
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INTRODUCTION |
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CELL DEATH CAN OCCUR through two very different ways, termed apoptosis (or programmed cell death) and necrosis. Apoptosis is characterized by such morphological changes as cell shrinkage, cytoplasmic blebbing, chromatin condensation, and DNA fragmentation, whereas necrosis is defined by cell swelling, destruction of organelles, and cell lysis resulting from disruption of the cell membrane (3, 34). Apoptosis occurs generally in a highly regulated manner and is thought to be a physiological form of cell death for the purpose of removing unwanted or damaged cells. It is important for such processes as embryogenesis and the immune response. On the other hand, necrosis is considered to represent a degenerative cell death induced by direct toxic, chemical, or physical injuries. It is usually involved in processes such as inflammatory reactions (26, 34).
Tumor necrosis factor- (TNF-
) is a cytokine produced by many cell
types. It was originally identified on the basis of its cytotoxic
effect on certain cells. It has been found to elicit a wide range of
biological responses including inflammatory reaction, cell
proliferation, and differentiation in addition to its ability to induce
cell death. Which response is elicited appears to depend on the cell
type and its state of differentiation (3). Many tumor cells are
sensitive to TNF-
-induced apoptosis, but normal cells are usually
resistant. Some cells lose their viability only when they are treated
with TNF-
in the presence of other agents or when the cells are
damaged (27, 37). Although cell death by apoptosis and necrosis have
been well characterized by morphological criteria, the initial events
in the signal transduction pathways responsible for the later phases of
cell death are poorly understood. Among the several signaling pathways
stimulated by TNF-
, production of ceramide through the sphingomyelin
cycle is thought to be critical for the initiation of apoptosis (18,
24). This hypothesis was advanced because exogenous ceramide
derivatives can mimic the effect of TNF-
in activating the signaling
pathways leading to apoptosis in certain cells (32, 33, 41). It is
supported by the findings that the resistance of some cells to TNF-
cytotoxicity is due to defects in the sphingomyelin cycle because
restoring this pathway resulted in the cells becoming susceptible to
TNF-
-induced apoptosis (4, 7, 30). Furthermore, sphingomyelinase
knockout mice failed to hydrolyze sphingomyelin and generate ceramide
and also showed defective apoptotic responses (24). Although these findings strongly support the role of ceramide as a cellular messenger for TNF-
-induced apoptosis in certain cells, there are a number of
recent reports that indicate that this is not a universal response. For
instance, in human vascular endothelial cells and neutrophils, ceramide
did not mediate TNF-
-induced gene expression (36, 43). In leukemia
ML-1 cells, it was found that ceramide is necessary but not sufficient
for TNF-
-induced apoptosis (21). In other cells, although both
TNF-
and ceramide were cytotoxic, they seemed to cause cell death
through different mechanisms (1, 12, 36). It is evident that the role
of ceramide in cellular processes is likely to be cell-type dependent.
A general role for ceramide as a second messenger for TNF-
-induced
apoptosis is clearly debatable.
Mesangial cells are a prominent cell type of the kidney glomerulus.
They are smooth muscle-like contractile cells that take part in the
regulation of the glomerular microcirculation, ultrafiltration, and
immune response. Like many primary cells, mesangial cells are normally
resistant to TNF- cytotoxicity, but they rapidly undergo apoptosis
when treated with TNF-
in the presence of the protein synthesis
inhibitor cycloheximide (CHX) (14). These data support the hypothesis
that a TNF-
-inducible factor is responsible for the protective
effect against further TNF-
cytotoxicity in resistant cell types
(40). Previous studies by Guo et al. (15) and others (10)
indicated that mitogen-activated protein (MAP) kinase phosphatase-1 may
act as such a protective factor by repressing a sustained c-Jun
NH2-terminal protein kinase (JNK)
activity. Although our hypothesis provided a plausible explanation for
the resistance to TNF-
-induced cell death in mesangial cells, we cannot rule out the possibility that this resistance could also be due
to an insufficient production of ceramide stimulated by TNF-
as
demonstrated in some cell types (7, 30). Here, we present evidence that
C2 ceramide, a cell-permeable
ceramide analog, did not mimic the effects of TNF-
on the activation
of MAP kinases and nuclear factor-
B (NF-
B)
transcription factor. Although C2 ceramide is toxic to mesangial cells, it causes cell death through a
mechanism distinct from TNF-
plus CHX-induced apoptosis.
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METHODS |
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Materials. Recombinant
TNF- was obtained from Chemicon International (Temecula, CA).
Anti-cytosolic phospholipase A2
(cPLA2), anti-I
B
, and
anti-NF-
B antibodies were purchased from Santa Crutz Biotechnology
(Santa Cruz, CA). Phosphorylated anti-extracellular signal-related
kinase (ERK) was from Promega (Madison, WI).
C2 ceramide
(N-acetyl-D-erythro-sphingosine)
and C2 dihydroceramide (dihydro-N-acetyl-D-erythro-sphingosine)
were obtained from Calbiochem (San Diego, CA). Hoechst 33258, propidium
iodide, and fluorescein-labeled anti-rabbit IgG were from Molecular
Probes (Eugene, OR).
Cell culture and cell viability assays. Rat mesangial cells were isolated from male Sprague-Dawley rats under sterile conditions with the sieving technique as previously described (25). The cells were maintained in RPMI 1640 medium containing 20% FCS and 0.6 U/ml of insulin at 37°C in a humidified incubator (5% CO2-95% air). Cells from 5 to 20 passages were used. After the cells were grown to 80-90% confluence, they were made quiescent by incubation for 16-18 h in insulin-free RPMI 1640 medium containing 2% FCS.
For cell viability assays, mesangial cells were grown in 12-well plates. The quiescent cells were treated with reagents for the indicated times. Uptake of neutral red dye was used as a measurement of cell viability (14). At the end of the incubations, the medium was removed, and the cells were incubated in DMEM with 2% FCS and 0.001% neutral red for 90 min at 37°C. Uptake of the dye by viable cells was terminated by removing the medium, washing the cells briefly with 1 ml of 4% paraformaldehyde in PBS (pH 7.4), and solubilizing the internalized dye with 1 ml of a solution containing 50% ethanol and 1% glacial acetic acid. The absorbencies, which correlate with the amount of live cells, were determined at 540 nm.
Morphological analysis of apoptosis and necrosis. Cells were grown on 25-mm glass coverslips in six-well plates to 80% confluence. After treatment with various reagents as indicated, the cells were fixed with 4% paraformaldehyde in PBS (pH 7.4). The morphological features of the cells were examined under a light microscope at different time intervals during incubation. Apoptosis and necrosis were analyzed according to the protocols described by Yoshimura et al. (44) with some modifications. Briefly, the fixed cells were stained with 10 µM Hoechst 33258 and 10 µM propidium iodide for 30 min and analyzed under a fluorescence microscope, with excitation at 340 nm. Hoechst 33258 stains nuclei of viable and apoptotic cells with a blue color, whereas propidium iodide stains nuclei of cells with a disrupted cell membrane with a red color. Therefore, nuclei of viable, apoptotic, and necrotic cells were identified as blue intact nuclei, fragmented nuclei, and intact red nuclei, respectively (44).
Immunodetection of
NF-B. Cells grown on 25-mm glass
coverslips in six-well plates were fixed with 4% paraformaldehyde in PBS (pH 7.4) after treatment with various reagents as indicated. The
coverslips were incubated with anti-NF-
B (65-kDa subunit) antibodies
[1:200 in Tris-buffered saline and 0.1% Triton X-100 (TBS-T)
containing 5% BSA] for 2 h at room temperature, washed with
TBS-T three times, and incubated with fluorescein-conjugated anti-rabbit IgG for 1 h at room temperature. After three washes with
PBS, the coverslips were mounted on slides. The NF-
B was localized
with fluorescence microscopy.
Effect of TNF- on sphingomyelin
metabolism. Mesangial cells were grown to 60%
confluence in 60-mm dishes. To label sphingomyelin in vivo,
[3H]serine (20 µCi/ml) was added to the culture medium. After 48 h, the medium was
replaced with insulin-free RPMI 1640 medium containing 2% FCS, and the
cells were incubated overnight. The cells were stimulated with 10 ng/ml
of TNF-
. Lipids were extracted from the cells according to the
method described by Bligh and Dyer (5) and were separated on silica gel
thin-layer plates. Thin-layer chromatography was carried out by the
method of Gomez-Munoz et al. (13). Sphingomyelin and ceramide were
identified based on the migration of authentic standards. The matrix on
the appropriate spots was scraped and transferred to scintillation
vials. Radioactivity was determined by scintillation counting.
Cell lysate preparation. The quiescent cells were treated with reagents for the indicated times, washed twice with ice-cold PBS, and scraped into cell lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM Na3VO4, 50 mM pyrophosphate, 100 mM NaF, 1 mM EGTA, 1.5 mM MgCl2, 1% Triton X-100, 10% glycerol, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The cells were incubated for 10 min on ice, lysed by sonication (25 pulses, output control 3) in a sonicator (Sonifier 450, Branson), and centrifuged at 15,000 g for 15 min. The supernatant was designated as whole cell lysate. Protein concentration was determined with the method of Bradford (6), with BSA as standard.
Protein kinase assays. JNK activity
was measured with a solid-phase kinase assay method. Glutathione
S-transferase (GST)-c-Jun(179) (GST-Jun) fusion protein was isolated from bacterial cells expressing pGEX-c-Jun plasmid. JNK activity was determined with GST-Jun as substrate as previously described (14). Briefly, 50 µg of cell lysate
were incubated with 2 µg of GST-Jun agarose beads at 4°C for 2 h
with rotation and centrifuged at 10,000 g for 1 min. The beads were washed
three times with washing buffer [25 mM HEPES, pH 7.5, 50 mM NaCl,
0.1 mM EDTA, 2.5 mM MgCl2, 0.05%
(vol/vol) Triton X-100, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin,
1 mM phenylmethylsulfonyl fluoride, 20 mM
-glycerophosphate, and 10 mM NaF]. The beads were then resuspended in 10 µl of kinase
buffer containing (final concentrations) 20 mM HEPES, pH 7.5, 10 mM
MgCl2, 1 mM
Na3VO4,
20 mM
-glycerophosphate, 5 mM NaF, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, 40 µM ATP, and 1 µCi of
[
-32P]ATP. After
incubation at room temperature for 20 min, the reaction was terminated
by adding SDS sample buffer followed by heating at 100°C for 3 min.
The proteins were separated by SDS-PAGE, and the phosphorylated
proteins were detected by autoradiography. ERK activation was
determined by Western blot analysis with anti-ERK antibodies that only
recognize the phosphorylated forms of ERK1 and ERK2 (15).
Western blot analysis. The protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in TBS-T and incubated with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's instructions. The immunnoblots were visualized with an enhanced chemiluminescence kit obtained from Amersham Pharmacia Biotech.
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RESULTS |
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TNF- does not induce production of
ceramide in mesangial cells. The cells were labeled in
vivo with [3H]serine,
a commonly used method for the study of sphingolipid metabolism. In one
experiment, 15 × 103
counts/min were incorporated into ceramide and 34 × 103 counts/min were incorporated
into sphingomyelin after the cells were labeled for 48 h. During the
first 45 min after TNF-
stimulation, ceramide and sphingomyelin
contents remained at levels indistinguishable from baseline values. A
second experiment gave essentially the same results.
Effect of C2 ceramide on the viability of
mesangial cells in presence and absence of TNF-.
Treatment of mesangial cells with TNF-
at concentrations from 10 to
50 ng/ml, which effectively induced apoptosis in susceptible cells (37,
39), caused neither detectable cell death (14) nor measurable
production of ceramide in mesangial cells. These results indicate that
resistance to TNF-
-induced apoptosis exhibited by normal mesangial
cells could be due to insufficient generation of ceramide. To test this
possibility, the cells were treated with
C2 ceramide (a synthetic
cell-permeable ceramide analog) alone and in combination with TNF-
.
As shown in Fig. 1, treatment of the cells
with 12 µM C2 ceramide alone,
which is sufficient to induce apoptosis in susceptible cells, or in
combination with 10 ng/ml of TNF-
did not affect their viability
within a 24-h period of incubation (Fig.
1A). However, >50% of the cells
lost their viability within 4 h when they were incubated with 50 µM C2 ceramide. This toxicity was not
significantly potentiated by TNF-
or CHX. The same concentration of
C2 dihydroceramide showed no
apparent effect on cell viability (Fig.
1B). A dose response of the cells to
ceramide toxicity is shown in Fig. 2. Cell
death was prominent when C2
ceramide concentrations were >18 µM during 24 h of incubation.
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Morphological features of cell death induced by
C2 ceramide and TNF- in
presence of CHX.
For comparison, time courses of cell death caused by
C2 ceramide and TNF-
in the
presence of CHX are shown in Fig. 3. Both treatments caused an ~80% loss in cell viability after 4-5 h. These conditions provided us with a convenient time frame in which to
study the morphological changes and their correlation with pertinent
signaling pathways during cell death. Normal mesangial cells exhibited
typical stellate morphology, with large round nuclei when they were
subconfluent (Fig. 4,
top,
A). The cells began to increase in
cell volume and lose their fiberlike structures after 30 min of
incubation with 50 µM C2
ceramide. Thereafter, the cell membrane was progressively disrupted
while the nuclei remained intact, exhibiting typical features of
necrosis (Fig, 4, top,
D). Incubation with
C2 dihydroceramide showed no
apparent effect on cell morphology (Fig, 4, top,
B). On the other hand, when cells
were treated with TNF-
plus CHX, they underwent apoptosis, exhibiting characteristic nuclear condensation and cytoplasm blebbing (Fig, 4, top,
C). The nuclei of cells under these
conditions were analyzed further by double staining with Hoechst 33258 and propidium iodide. Hoechst 33258 stains nuclei of viable and
apoptotic cells with a blue color, whereas propidium iodide stains
nuclei of cells having a disrupted cell membrane with a red color. As
shown in Fig. 4, bottom, the nuclei of
cells treated with C2
dihydroceramide were stained blue
(B), similar to the nuclei of
control cells (A). Nuclei of cells
treated with TNF-
plus CHX were disrupted and fragmented, exhibiting
typical features of apoptosis (Fig. 4,
bottom,
C). In these cells, the fragmented
nuclei were still confined in the membrane vesicles. Although propidium
iodide could enter the vesicles, its red color was overshadowed by the
blue color of a larger amount of Hoechst 33258; therefore, the
fragmented nuclei appear as a bright blue color. On the other hand,
after treatment with C2 ceramide,
the nuclei were stained red (Fig. 4,
bottom,
D), indicating that although these
cells had intact nuclei, their membrane systems were disrupted. The
characteristics of nuclei staining correlated well with the observed
cell morphological changes for apoptosis and necrosis as defined for
other cells (44). DNA, isolated from the cells treated as described in
Fig. 4, was analyzed by agarose gel electrophoresis. Only the
cells that were incubated with TNF-
plus CHX showed DNA
fragmentation (data not shown). These results clearly demonstrate that
cell death induced by TNF-
plus CHX and by
C2 ceramide was through different
mechanisms, i.e., apoptosis and necrosis, respectively.
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Effects of C2 ceramide on ERK and JNK
activities.
A selective activation of JNK compared with ERK has been considered as
major evidence for a role of ceramide in mediating apoptosis induced by
TNF-, ultraviolet light, and Fas ligand (39, 41). However, further
studies have shown that the selectivity of activation of
MAP kinase family members by ceramide seems to vary depending on the
cell type (12). In our case, C2
ceramide did not activate JNK to a detectable level (Fig.
5A) but
strongly activated ERK (Fig. 5B).
TNF-
plus CHX strongly activated both JNK and ERK as described
previously by Guo et al. (15). It was used as positive control in this
experiment (Fig. 5). It should be pointed out that both TNF-
and
ceramide activated ERK, but the activation patterns were different in
the two cases; TNF-
only transiently activated ERK at 15 min and the
ERK activity declined to the basal level after 30 min (14), whereas
C2 ceramide activated ERK in a
sustained manner, with the activation of ERK being apparent at 60 min
and lasting for at least 3 h (Fig.
5B).
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Effect of C2 ceramide on activation of
NF-B.
NF-
B is a transcription factor present as a heterodimer complexed
with I
B in the cytoplasm of unstimulated cells. On cell stimulation,
I
B is degraded, resulting in the release of NF-
B, which
translocates to the nucleus where it initiates transcription activity
(2). TNF-
is known to induce NF-
B activation in many cells,
whereas the ability of ceramide to activate NF-
B varies greatly from
cell to cell; in some cells, it mimics the effect of TNF-
(22, 29),
but in other cells, it had no effect (12, 36). Degradation of I
B
has been used as an indirect indication of NF-
B activation (17). In
mesangial cells, TNF-
induced an initial decrease in I
B
,
evident after 15 min, before it returned to initial levels after 2 h
(Fig.
6A). CHX
did not affect the degradation of I
B
induced by TNF-
but
prevented the de novo synthesis of I
B
as expected (Fig.
6B). In comparison, treatment of the
cells with C2 ceramide did not
cause degradation of I
B
within 3 h. However, the level of
I
B
decreased after 5 h of incubation (Fig.
6C), at the time when >70% of the
cells had lost their viability (Fig. 3).
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Cleavage of cPLA2 during apoptosis and
necrosis.
cPLA2 hydrolyzes membrane
phospholipids with the release of arachidonic acid. TNF--induced
apoptosis has been found to be associated with increased
cPLA2 activity (9, 20). Recently, Wissing et al. (42) found that TNF-
-induced apoptosis was correlated with the proteolytic cleavage of
cPLA2 by caspase-3 and an increase in phospholipase activity. They proposed that a 70-kDa fragment of
cPLA2 was responsible for the
increased phospholipase activity and was critical for the induction of
subsequent apoptosis. As illustrated in Fig.
8B,
the cleavage of cPLA2 and
generation of a 70-kDa fragment was also observed in mesangial cells at
the onset of TNF-
plus CHX-induced apoptosis (~2 h, as shown in
Fig. 3). Degradation of cPLA2 was
also associated with C2
ceramide-induced cell death, but a 70-kDa cleavage product of
cPLA2 was never detected (Fig.
8C). In addition, degradation of
cPLA2 was only observed at the
terminal stage of necrosis (~4 h, as shown in Fig. 3). These results
indicate that distinct cleavage of signaling molecules was involved in
the two modes of cell death.
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DISCUSSION |
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Many tumor cells are sensitive to TNF--induced apoptosis, but normal
cells are usually resistant. At least three hypotheses have been
proposed to explain how some cells achieve resistance to this
cytokine-induced apoptosis. First, TNF-
may activate an
antiapoptotic signaling pathway that counteracts the cytotoxicity of
the apoptotic pathway (8, 11). Second, TNF-
may elicit the synthesis
of a protective factor, with the result that the cells become
insensitive to TNF-
cytotoxicity (2, 38). Third, the apoptotic
pathway inducible by TNF-
is not activated or is not sufficiently
activated to initiate the apoptotic process (21). Each hypothesis has
gained some support in certain cell types, but none of them has been
fully established to date. Given the complexity of TNF-
signaling
pathways, it is not surprising that different cells may use different mechanisms.
TNF- activates sphingomyelinase, with a concurrent generation of
ceramide and a decrease in sphingomyelin in some cells, which usually
take place within 5-20 min of stimulation (36, 39). The addition
of exogenous C2 ceramide also
mimics the effect of TNF-
on the induction of apoptosis in these
cells, supporting the hypothesis that ceramide acts as a second
messenger for TNF-
to induce apoptosis. However, under similar
conditions, TNF-
failed to cause a measurable production of ceramide
in mesangial cells. It has been proposed that resistance of some cells
to TNF-
cytotoxicity may be due to insufficient production of
ceramide resulting from defects in the sphingomyelin cycle (7, 30). However, data from the present study are not in favor of this possibility in mesangial cells. Exogenous
C2 ceramide at a concentration of
12 µM, which was sufficient to mimic the effect of TNF-
for the
induction of apoptosis in susceptible cells, did not cause the death of
mesangial cells either by itself or in combination with TNF-
. These
results were not due to an inability of the cells to respond to
C2 ceramide because they exhibited
an apparent morphological change (i.e., cell volume increase) on
stimulation by C2 ceramide even at
6 µM. Furthermore, C2 ceramide
from the same source effectively inhibited thrombin-induced
proliferation of CCL-39 cells at concentrations as low as 5 µM (16).
None of these effects was observed when cells were treated with the biologically inactive analog of C2
ceramide, C2 dihydroceramide, or
with TNF-
. Higher concentrations of
C2 ceramide caused cell death by
necrosis rather than by apoptosis as confirmed by various morphological
and biochemical criteria.
The signaling pathways activated by
C2 ceramide and TNF- also
showed distinctive patterns in mesangial cells. Neither JNK nor
NF-
B, which was strongly stimulated by TNF-
(14), was activated
by C2 ceramide. Although ERK was
activated by both C2 ceramide and
TNF-
, such activation appeared to be mediated by different
mechanisms as judged from the kinetics of ERK activation. Activation of
caspases, which takes place in a highly controlled manner, is a
hallmark of apoptosis induced by various stimuli (31). The cleavage of
specific proteins by caspases results in the irreversible commitment to
cell death. The partial cleavage of
cPLA2 during TNF-
-induced
apoptosis represents an excellent example of this event. It was
proposed that a 70-kDa fragment of
cPLA2 generated by caspase-3 was
responsible for the increased phospholipase activity, which was
critical for TNF-
-induced apoptosis in MCF-7S1 cells (42). A very
similar cleavage pattern of cPLA2 during apoptosis was observed in mesangial cells. On the other hand,
C2 ceramide-induced degradation of
cPLA2 occurred only at the late
stage of cell death, and a 70-kDa fragment of
cPLA2 seen during apoptosis was
never detected. These results indicate that C2 ceramide- and TNF-
plus
CHX-induced degradation of cPLA2
was brought about by different proteolytic mechanisms and may have fundamentally different implications for the two types of cell death.
Unlike the well-controlled cleavage of
cPLA2 and I
B
during apoptosis, the degradation of these molecules in
C2 ceramide-induced necrosis is
more likely a result of cell lysis rather than of signaling steps that
lead to cell death. These results support the notion that, unlike
apoptosis, necrosis seems to be a less-controlled degenerative process
characterized by a widespread lysis of cell structures and membrane
systems without systematic signaling events (28, 34).
Essentially none of the responses of mesangial cells to
C2 ceramide that were investigated
resembled those elicited by TNF-. A similar disparity between the
signaling pathways induced by TNF-
and ceramide has previously been
reported in endothelial cells and SW480 cells (23, 36). The effect of
CHX to sensitize the cell to TNF-
toxicity is attributed to its
effect on inhibiting protein synthesis (2, 38). However, the
possibility that ceramide could be produced during TNF-
plus
Chx-induced apoptosis and, therefore, may influence the process
directly or indirectly cannot be ruled out. Because of complex
nonselective effects of CHX on cellular metabolism, it is difficult to
ascertain whether generation of ceramide under such conditions is a
consequence of cell injury or a cause of cell death. Another study (35) indicated that ceramide did not mediate Fas-induced apoptosis and
suggested that the observed generation of ceramide was more likely to
be a consequence rather than the cause of apoptosis. It is worth noting
that due to the membrane impermeability of natural ceramide, most
studies have used synthetic ceramides, mainly
C2 and
C6 ceramides. In most cases, the
synthetic ceramides mimicked the effect of natural ones as confirmed by
various experimental approaches. One criterion of specificity between
the compounds has been that dihydoceramide should have no effect in
comparison with ceramide. We saw this in our experiments, which
therefore agree with the bulk of data in the literature. However, a
recent report (19) indicated that the effects of the synthetic
ceramides may be also related to the
N-acyl chain length. Together with the
potential of synthetic ceramides to cause damage to the membrane, this
finding gives cause for concern and caution in interpreting results.
For the past several years, considerable effort has been made to clarify the mechanisms leading to apoptosis. However, the signaling pathways leading to necrosis have not attracted appreciable attention although cell death from necrosis is prominent in many physiological or pathological processes such as inflammation and hypoxic conditions (44). In fact, ceramide-caused necrosis was recently noted in synovial fibroblasts (12) and hepatocytes (1). Thus, in addition to its recognized role as a cellular messenger to mediate apoptosis in some cells, it is clear that ceramide can cause cell death by necrosis in other cell types. The present study provides important evidence that apoptosis and necrosis use distinctive mechanisms to cause cell death. It will be of interest to find out the nature of ceramide targets and how they may mediate the necrotic effect of ceramide. In this respect, activation of the ERK pathway by C2 ceramide deserves further investigation.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. K. Westwick for providing the pGEX-c-Jun plasmid and Dr. K. Baysal for performing ceramide assays.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-15120 and DK-48493.
Y.-L. Guo is a recipient of National Institute of Diabetes and Digestive and Kidney Diseases Training Grant DK-07314.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. R. Williamson, Dept. of Biochemistry and Biophysics, Univ. of Pennsylvania, 601 Goddard Labs, 37th and Hamilton Walk, Philadelphia, PA 19104 (E-mail: johnrwil{at}mail.med.upenn.edu).
Received 19 August 1998; accepted in final form 2 December 1998.
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