By
From the * Department of Pediatrics, Department of Medicine, and ¶ Department of Cell Biology,
Duke University Medical Center, Durham, North Carolina 27710; and the
Laboratory of Molecular
Endocrinology, Laval University, Sainte-Foy, Quebec, G1V-4G2, Canada
Proteases are now firmly established as major regulators of the "execution" phase of apoptosis.
Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor- (TNF-
)-induced pathway of cell death. Ceramide
induced activation of prICE, the protease that cleaves the death substrate poly(ADP-ribose)
polymerase. Bcl-2 inhibited ceramide-induced death, but not ceramide generation. In contrast,
Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1
converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-
-induced death.
Exogenous ceramide could overcome the CrmA block to cell death, but not the Bcl-2 block.
CrmA, however, did not inhibit the activation of nuclear factor (NF)-
B by TNF-
, demonstrating that other signaling functions of TNF-
remain intact and that ceramide does not play a role in the activation of NF-
B. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.
Apoptosis is an essential, highly conserved, and tightly
regulated cellular process of cell death that is important
for development, host defense, and suppression of oncogenesis (1). The intracellular mediators of apoptosis are
poorly defined. Recently, proteases belonging to the newly
described IL-1 Another important group of cell death regulators are
members of the Bcl-2 family that act as either inhibitors or
promoters of apoptosis (17). Bcl-2 can inhibit apoptosis
induced by a wide variety of stimuli (17, 20). However, the
mechanism of action of Bcl-2 remains unknown.
Inhibition of apoptosis is used by some DNA viruses to
attenuate host defense and increase viral progeny (21). The
cowpox virus cytokine response modifier A (CrmA) protein
that exhibits cross-class inhibition of both serine and cysteine proteases (22) is a potent inhibitor of apoptosis induced by serum withdrawal (23), activation of the Fas or
TNF- Based on recent studies, the sphingolipid ceramide is
emerging as a possible regulator of apoptosis. Ceramide
can potently induce apoptosis in a number of different systems (27). TNF- Here we investigate whether the antiapoptotic molecules, CrmA and Bcl-2, interact with the ceramide pathway. We show that CrmA targets this pathway upstream of
ceramide generation in response to TNF- Cell Lines and Cultivation.
The previously described cell lines
expressing CrmA or mutant CrmA and those overexpressing Bcl-2
were derived from a TNF- Ceramide Measurement.
Cells were seeded at 2 × 105 cells/ml
in a 10 ml volume, rested overnight, and then treated with 1.2 nM TNF- Measurement of Ceramide Uptake.
Cells were seeded at 2.5 × 105/well of a 6-well plate in a 2-ml volume of RPMI with 2%
FBS and treated with 14C6ceramide at 20 µM. Cells were harvested by scraping at the indicated time points, washed twice
with PBS, and the radioactivity retained in the pellet was counted
in a scintillation counter.
Preparation of Cell Lysates.
For experiments analyzing PARP
proteolysis, cells were seeded and treated as described for the ceramide measurement experiments. At the indicated time points,
cells were harvested by scraping in media followed by centrifugation at 4°C and washed once with ice-cold PBS. The cell pellet
was then resuspended in 50 µl PBS and lysed with sample buffer
(30 mM Tris-HCL [pH 6.8], 10% glycerol, 6% Western Blotting.
Equal amounts of protein, usually 100 µg,
were resolved by SDS-PAGE and transferred to a nitrocellulose
membrane. PARP and its cleaved fragment were detected using a
rabbit polyclonal antiserum at a dilution of 1:2,000, and a goat
anti-rabbit secondary antibody at a dilution of 1:5,000. The signal
was visualized by enhanced chemiluminescence (Amersham Intl.,
Buckinghamshire, UK).
Nuclear Factor- We treated MCF-7 breast carcinoma cells with
TNF-
The ability of CrmA
to inhibit, with variable efficiency, the ICE family of cysteine proteases (6, 7, 22, 26) was used to examine the relationship of these proteases to ceramide along the death
pathway. MCF-7 cells stably expressing CrmA were treated
with 2 nM TNF-
Since endogenous ceramide elevation may drive the
cell to die, we next examined whether CrmA interferes
with ceramide generation. We measured endogenous ceramide levels in response to TNF- As ceramide generation occurred in cells destined to
die due to treatment with TNF-
To determine the relationship between TNF-
Next, it became important to determine the effect of
CrmA or Bcl-2 expression on ceramide-induced PARP
cleavage in MCF-7 cells. Although both Bcl-2 and CrmA inhibited TNF- To further dissociate the site of action of
CrmA from that of Bcl-2, we employed the alkylating agent
mitomycin C. Mitomycin C did not cause any significant
change in endogenous ceramide levels (data not shown).
We next compared the effects of TNF-
Activation of the transcription
factor NF-
The results from this study show that ceramide accumulation observed after treatment of MCF-7 cells with TNF- The antiapoptotic activity of CrmA has been thought to
be due to its ability to inhibit members of the ICE family of
cysteine proteases that are homologues of the C. elegans cell
death protease CED-3 (2, 4, 25). However, the potency by
which CrmA inhibits the various members of this family
varies significantly. Whereas picomolar concentrations of
CrmA can potently inhibit ICE, concentrations that are
over 1,000-fold higher are required to achieve comparable inhibition of Yama/CPP32/apopain, a putative PARP cleaving protease (7). Similarly, CrmA is a poor inhibitor of
ICH-1 (23) and CED-3 (3, 55), indicating that it preferentially targets only a subset of ICE family members. This
may explain why CrmA is not a universal inhibitor of apoptosis. Based on our findings, the more physiologically relevant target of CrmA appears to be a protease that is activated by TNF-
Our findings define a novel site of CrmA-induced protection from apoptosis that is clearly distinct from that of
Bcl-2. Whereas CrmA interferes with ceramide accumulation, Bcl-2 does not modulate this event and appears to
work downstream by inhibiting the execution machinery
of apoptosis (Fig. 8). This is additionally supported by the
ability of both CrmA and Bcl-2 to protect from cell death when induced with TNF- These studies also define distinct pathways that lead to
NF- Taken together, these findings are consistent with an important role for ceramide in TNF- In conclusion, ceramide accumulation is emerging as an
important component in a TNF- converting enzyme (ICE)1 family emerged
as central constituents of the apoptotic machinery. The evidence implicating these proteases in the induction of apoptosis stems from the following observations: (a) homology
to the ced-3 gene product of the nematode Caenorhabditis
elegans that is required for cell death during development
(2), (b) induction of apoptosis when these proteases are overexpressed in their active form (3), (c) inhibition of apoptosis
when these proteases are specifically inhibited (4), and
(d) identification of a number of cellular protein substrates
that are proteolytically cleaved through the specific action of
these proteases during apoptosis including poly(ADP-ribose)
polymerase (PARP) (9, 10), the 70-kD protein component
of the U1 small nuclear ribonucleoprotein (11), and others
(12). Current evidence implicates certain members of the
ICE family, particularly CPP32/Yama/apopain and ICELAP3, in the execution phase of apoptosis (6, 7, 13, 14).
Also, MACH/FLICE has been recently identified as a protease in close association with the Fas receptor (15, 16).
receptors (4, 24), or withdrawal of nerve growth
factor in primary chicken neuronal cultures (25). Although
CrmA has been shown to have preferential activity against
ICE (26), it is also able to inhibit the proteolytic activity of
other members of the ICE family implicated in apoptosis, although less potently.
and other inducers of apoptosis (Fas
ligation, serum withdrawal, some chemotherapeutic agents,
-irradiation; 29, 31-34) have been shown to elevate cellular levels of ceramide. In turn, the addition of cell-permeable ceramide induces apoptosis in several cell types, raising
the possibility that this may be a final pathway common to
a variety of inducers (28, 32). More recently, the death signal from both Fas and TNF-
-receptor 1 was shown to
converge proximally on FADD, a "death domain" containing protein that belongs to a new family of signaling molecules that associate with members of the TNF-
receptor
family (35). Expression of a dominant negative mutant
of FADD inhibited ceramide accumulation, ICE-related protease activation, and apoptosis after treatment with Fas
antibody by blocking the proximal signal. Exogenous ceramide was able to bypass this block and produce apoptosis
(38). Additionally, the Drososphila melanogaster protein
REAPER, which is critical for the normal development of
the Drosophila embryo, was recently found to activate apoptosis by the activation of an ICE-like protease associated
with ceramide generation and apoptosis (39). These effects
were blocked by the use of a specific peptide inhibitor of
ICE-like proteases. Based on these observations, it has been suggested that ceramide may play a central role in the regulation of apoptosis (40).
. In contrast, we
show that Bcl-2 protects from both TNF-
and ceramide-
induced cell death without interfering with ceramide generation, suggesting that it functions further downstream along the ceramide pathway. These studies identify a novel target
for inhibition of apoptosis, namely, the inhibition of ceramide generation, and clarify the order by which CrmA,
ceramide, and Bcl-2 interact.
-sensitive MCF-7 parental line (4, 6,
41) (gift of Muneesh Tewari and Dr. Vishva Dixit, University of
Michigan, Ann Arbor). Cells were grown in RPMI 1640 medium
supplemented with 10% fetal bovine serum (FBS) and 0.2% sodium bicarbonate. 500 µg/ml G418 was added to the CrmA cell
line and its vector while 150 µg/ml hygromycin was added to the
Bcl-2 cell line and its vector. Experiments were done in the absence of G418 or hygromycin. Cell viability was determined by
the ability to exclude trypan blue.
. Lipids were collected according to the method of
Bligh and Dyer (42). In brief, cells were pelleted, washed once
with PBS, and then extracted with 3 ml chloroform/methanol (1:2,
vol/vol) in 13 × 100 mm screw-top glass tubes. The monophase
was mixed, 0.7 ml water was added, and the samples were rested
for 10 min. The organic and aqueous phases were subsequently
separated by addition of 1 ml chloroform and 1 ml water followed by vigorous shaking and centrifugation at 1,000 rpm. The
organic phase was carefully removed and transferred to a new
tube, and the samples were dried under N2. Lipids were then resuspended in 1 ml of chloroform. Ceramide levels were measured using a modified diacylglycerol kinase assay (43, 44) using external standards. In brief, 80% of the lipid sample was dried under
N2. The dried lipid was solubilized in 20 µl of an octyl-
-D-glucoside/dioleoyl phophatidylglycerol micellar solution (7.5% octyl
-D-glucoside, 25 mM dioleoyl phosphatidylglycerol) by 2 cycles
of sonication in a bath sonicator for 60 s followed by resting at room
temperature for 15-20 min. The reaction buffer was prepared as a
2 × solution containing 100 mM imidazole HCL, pH 6.6, 100 mM LiCl, 25 mM MgCl2, 2 mM EGTA. To the lipid micelles, 50 µl of the 2 × reaction buffer were added, 0.2 µl of 1 M dithiothreitol, 5 µg of diglycerol kinase membranes, and dilution buffer (10 mM imidazole, pH 6.6, 1 mM diethylenetriaminepentaacetic acid,
pH 7) to a final volume of 90 µl. The reaction was started by adding
10 µl 2.5 mM [
-32P]ATP solution (specific activity of 75,000-
200,000 cpm/nmol). The reaction was allowed to proceed at
25°C for 30 min. Lipids were extracted as described above and a
1.5-ml aliquot of the organic phase was dried under N2. Lipids
were then resuspended in a volume of 100 µl methanol/chloroform (1:20, vol/vol) and 20 µl was spotted on a 20-cm silica gel
thin layer chromatography plate. Plates were developed with chloroform/acetone/methanol/acetic acid/H2O (50:20:15:10:5), air
dried, and then subjected to autoradiography. The radioactive spots
corresponding to phosphatidic acid and ceramide phosphate, and
the phosphorylated products of diacylglycerol, and ceramide, respectively, were scraped into a scintillation vial containing 4 ml
scintillation fluid and counted on a scintillation counter. Linear
curves of phosphorylation were produced over a concentration
range of 0-640 pM of external standards (dioleoyl glycerol and CIII
ceramide; Sigma Chemical Co., St. Louis, MO). Diacyglycerol and
ceramide levels were always normalized to lipid phosphate which
was measured according to the method of Rouser et al. (45). In
brief, 20% of the lipid sample was dried down under N2 and oxidized with 150 µl of 70% perchloric acid on a heating block at
160°C for 45 min. The tubes were allowed to cool, and then 830 µl
of H20 was added, followed by 170 µl of 2.5% ammonium molybdate, and 170 µl of 10% ascorbic acid with vortexing after each addition. The tubes were then incubated at 50°C for 15 min, allowed to cool, and absorbance read at 820 nm and compared to standard.
-mercaptoethanol, 4% SDS) and boiled for 10 min. Protein concentrations were
determined using the Bio-Rad assay.
B Assay.
Nuclear extracts were prepared as
described previously (46, 47). In brief, 2 × 106 cells were washed
once in PBS. The cell pellet was rapidly frozen in dry ice and
isopropanol, and then thawed by adding 100 µl of ice-cold buffer
A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM
dithiothreitol) resulting in 100% cell lysis. The nuclei were pelleted by microcentrifugation at 3,500 rpm for 10 min at 4°C. The
supernatant was discarded and the nuclei suspended in 15 µl of
buffer C (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1.5 mM MgCl2, 25% vol/vol glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). The suspension was mixed
gently for 20 min at 4°C, and then microcentrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was diluted with 50-70 µl
of buffer D (20 mM Hepes, pH 7.9, 50 mM KCl, 20% vol/vol
glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and aliquots stored at
80°C. Protein
concentrations were determined using the Bio-Rad assay. Electrophoretic mobility-shift assay reactions were performed in 20 µl
volume, using 8-10 µg of nuclear extract in the presence of 1 µg
of poly(d[I-C]) 1 µg of pd(N)6, and 10 µg of BSA. Incubations
were in the presence of HDKE buffer with the following final
concentrations: 20 mM Hepes, pH 7.9, 50 mM KCl, 1 mM
EDTA, and 5 mM dithiothreitol. Reactions were started by the
addition of 10,000-50,000 cpm radiolabeled nuclear factor (NF)-
B oligonucleotide probe (Promega Corp., Madison, WI). After
incubation for 10 min, the reactions were terminated by adding 6 µl of 15% ficoll solution containing indicator dyes. Equal
amounts of the reactions were loaded on 5% nondenaturing polyacrylamide gels in 1 × Tris/Borate/EDTA and run at 200 volts. Specificity of the NF-
B bands was determined by competition
with excess unlabeled probe and lack of competition with 100fold excess of a probe containing a one basepair mutation in the
NF-
B binding sequence.
Ceramide Accumulation Is Delayed, but Precedes Early Apoptotic Signs.
and measured ceramide levels and cell death concomitantly at several time points (Fig. 1 A). Ceramide levels did not change appreciably in the first 5 h (data not
shown) but were significantly increased between 7 and 9 h
and continued to increase with time, up to 400% by 20 h.
This accumulation was not dependent on new protein synthesis since the addition of cycloheximide to the cells before treatment with TNF-
enhanced the accumulation of
ceramide (Fig. 1 B). Although these are delayed and persistent changes in ceramide, it is becoming increasingly apparent that this is the pattern most closely related to the apoptotic responses. Although this raises the concern that ceramide
accumulation is a consequence of death, studies with Bcl-2
(see below) negate this possibility. Cell death, as measured by the inability to exclude trypan blue, was not seen until
20 h, occurring several hours after the increase in ceramide
levels, indicating that ceramide accumulation occurs long
before loss of membrane integrity. To verify that cell death
was occurring through induction of apoptosis, we assayed
for cleavage of the 116-kD PARP polypeptide to a specific
85-kD apoptotic fragment (9, 10). This proteolytic cleavage has been shown to occur in apoptosis and to be mediated by Yama/CPP32/apopain or related proteases. As
shown previously (6), treatment of MCF-7 cells with
TNF-
resulted in specific cleavage of PARP to the 85-kD fragment (Fig. 2). Significant PARP cleavage did not occur
until 12 h after treatment with TNF-
and was maximal by
25 h. These results indicate that ceramide accumulation
precedes one of the early signs of apoptosis, i.e., PARP
cleavage, by at least 3-4 h. Moreover, loss of cell membrane integrity, as determined by trypan blue uptake, is unlikely to contribute to ceramide accumulation since it occurs several hours after the dramatic increase in endogenous ceramide levels.
Fig. 1.
(A) Effects of TNF-
on cell death and ceramide levels
in MCF-7 cells. MCF-7 breast
carcinoma cells were treated
with TNF-
at 1.2 nM. At the
indicated time points, adherent
and floating cells were harvested
and the number of dead cells was
determined by their inability to
exclude trypan blue (open circles).
Concomitantly, lipids were collected and ceramide levels were
measured (filled circles) and compared to time-matched controls.
Levels of ceramide in control
cells ranged between 4-6 pmole/
nmole of lipid phosphate. Results are averages of three experiments. Standard deviation for all
points is indicated. (B) Effect of cycloheximide on ceramide accumulation after TNF-
. MCF-7 cells were treated with TNF-
at 1.2 nM as in A in the
presence of increasing concentrations of cycloheximide as indicated. Cells were harvested at 18 h after TNF-
treatment, and ceramide levels were measured in the lipid extracts.
[View Larger Versions of these Images (16 + 17K GIF file)]
Fig. 2.
Kinetics of PARP cleavage after TNF- treatment. MCF-7
cells were seeded at 2 × 106 cells/10-cm plate, rested overnight, and then
treated as in Fig. 1. At the indicated time points, cells were harvested by
scraping in media to insure inclusion of the floating cells at later time points. Cell lysis and Western blotting were performed as described in
Materials and Methods. Densitometric analysis of the two resulting bands
was performed. The cleaved PARP fragment is represented as a percent
of the total of both fragments. A representative experiment is shown (out
of three).
[View Larger Versions of these Images (11 + 18K GIF file)]
or increasing concentrations of C6-ceramide, a cell-permeable ceramide analogue, and compared
with control (vector) cells. As shown previously (4), CrmA
offered almost complete protection from TNF-
-induced
cytotoxicity (Fig. 3 A). However, CrmA offered no protection from the cytotoxic effects of ceramide so that cells
died equally in the presence or absence of CrmA (Fig. 3 A).
Comparison of another pair of vector and CrmA-expressing MCF-7 clones described previously (4) yielded similar results (data not shown). Therefore, CrmA appeared not to
interfere with the downstream effects of ceramide.
Fig. 3.
Effects of CrmA on the TNF--activated ceramide pathway.
(A) CrmA protects from TNF-
-induced, but not ceramide-induced, cell death. TNF-
-sensitive MCF-7 cells transfected with either pcDNA3 vector (open bars) or pcDNA3/crmA (filled bars) were seeded in 24-well plates
at 5 × 104 cells/well in 2% FBS, rested overnight, and then treated with the indicated concentrations of C6-ceramide or 2 nM TNF-
. Cell death,
determined by the inability to exclude trypan blue, was evaluated at 48 h.
(B) CrmA inhibits ceramide generation after TNF-
stimulation. The
CrmA-expressing MCF-7 cells (filled circles) and their vector control cells
(open circles) were treated with TNF-
as in Fig. 1. Ceramide levels were
measured at the indicated time points as in Fig. 1. Results are the average
of three experiments with the standard deviation indicated. (C) Antiproteolytic activity of CrmA is important in inhibiting ceramide accumulation. Cells expressing CrmA, point-mutant CrmA, or wild-type MCF-7
cells pretreated 1 h with 50 µm Ac-YVAD-CHO (Bachem, King of
Prussia, PA) were treated, along with their respective controls, with 1.2 nM TNF-
for 18 h. Ceramide was then measured as in Fig. 1. Percent
inhibition was calculated by comparison with the results from TNF-
-
treated controls. The average of three experiments is shown.
[View Larger Versions of these Images (21 + 18 + 51K GIF file)]
in control and in CrmAexpressing MCF-7 cells. Ceramide levels increased dramatically in vector cells at 18 and 24 h (Fig. 3 B). However, CrmA-expressing cells treated similarly with TNF-
showed
no increase in ceramide levels. To determine whether the
inhibition of ceramide accumulation was dependent on the
ability of CrmA to inhibit proteases, we used two approaches. First, we used a point-mutant of CrmA that has
no antiproteolytic activity due to the substitution of Arg for
Thr at amino acid 291 in the reactive site loop (6). In
MCF-7 cells expressing this mutant, there was no significant inhibition of ceramide accumulation (Fig. 3 C) and no
protection from TNF-
-induced apoptosis (6). Second,
we used the synthetic peptide Ac-YVAD-CHO that, like
CrmA, is a potent competitive inhibitor of ICE (7). Pretreatment of cells with this peptide, resulted in significant
inhibition of ceramide accumulation after TNF-
treatment (Fig. 3 C). In contrast, other protease inhibitors not known to inhibit the ICE family of proteases, including
N-tosyl-L-phenylalanine chloromethyl ketone and leupeptin, were unable to inhibit ceramide accumulation after
TNF-
treatment (data not shown). Therefore, CrmA inhibits TNF-
-induced apoptosis by acting at a point upstream of the generation of ceramide, and this inhibition is
dependent on its antiproteolytic activity.
and not in the protected,
CrmA-expressing cells, it became important to verify that
this increase in ceramide levels, as well as the suppression of
this increase by CrmA, were specific events correlating
with cell death or survival, respectively. We used Bcl-2,
another antiapoptotic molecule that has recently been
shown to protect from ceramide-induced apoptosis (48,
49). MCF-7 cells expressing Bcl-2 or vector controls were
treated with TNF-
or increasing concentrations of C6-ceramide. Control cells died in response to both treatments,
but Bcl-2-expressing cells displayed resistance to TNF
-induced cell death as seen in cells expressing CrmA (Fig.
4 A). However, unlike CrmA-expressing cells, Bcl-2-expressing cells were resistant to ceramide-induced cell death. Additionally, generation of endogenous ceramide was nearly equal
in both vector and Bcl-2 cells in response to TNF-
, indicating that Bcl-2 does not interfere with ceramide generation (Fig. 4 B). Therefore, Bcl-2 functions at a point
downstream of ceramide to inhibit apoptosis. More importantly, this delayed accumulation of ceramide is not a consequence of cell death since it is still observed in the viable
Bcl-2-expressing cells. Thus, these data demonstrate that the
elevation in ceramide is proximal to the biochemical and
morphological changes of cell death.
Fig. 4.
Effects of Bcl-2 overexpression on the TNF--activated ceramide pathway. (A) Bcl-2
prevents both TNF-
- and ceramide-induced cell death. TNF-
-
and Fas-sensitive MCF-7 cells
overexpressing pEBS7 (open bars)
or pEBS7/Bcl-2 (filled bars) were
treated as in Fig. 3 A and evaluated for cell death by trypan blue
at 48 h. (B) Bcl-2 does not prevent the TNF-
-induced elevation in ceramide levels. The Bcl2-overexpressing MCF-7 cells
(filled diamonds) and control vector cells (open circles) were treated
as in Fig. 3 B, and ceramide levels were measured at the indicated time points.
[View Larger Versions of these Images (18 + 19K GIF file)]
,
ceramide, and subsequent PARP cleavage and apoptosis, it
became imperative to study the effects and kinetics of ceramide on PARP cleavage. Consistent with results in Molt
4 cells (50), treatment of MCF-7 cells with ceramide resulted in cleavage of PARP (Fig. 5 A), and PARP cleavage
after 4 h of ceramide treatment was equivalent to that caused
by 12-16 h of TNF-
treatment (compare with Fig. 2).
By using 14C-labeled ceramide and evaluating the kinetics
of its uptake by MCF-7 cells at several time points, we
found that ceramide was taken up slowly by these cells
with maximal uptake reached at only 4 h (Fig. 5 B). Therefore, the delay in PARP cleavage after ceramide treatment
can be attributed to the delay in uptake of exogenous ceramide. Hence, these experiments support the hypothesis that
ceramide accumulation following TNF-
treatment may
represent a trigger for PARP cleavage and apoptosis.
Fig. 5.
(A) Kinetics of PARP cleavage after ceramide treatment.
MCF-7 cells were seeded and treated with ceramide, and then harvested at the indicated time points. PARP cleavage was assayed as in Fig. 2. Intact PARP (116 kD) and its cleaved product (85 kD) are indicated. (B)
Kinetics of exogenous ceramide uptake. MCF-7 cells were seeded and
treated with 14C6-ceramide (specific activity of 1.5 × 1013 cpm/mole) at a
similar concentration. At the indicated time points, cells were harvested,
washed twice with PBS, and the radioactivity retained in the pellet was
counted and presented as a percent of total radioactivity delivered. (C) Effects of CrmA and Bcl-2 on ceramide-induced PARP cleavage. Vector,
CrmA-expressing, or Bcl-2-overexpressing cells were seeded at 2.5 × 105
cells/well of a 6-well plate. The cells were rested overnight then treated
with vehicle (V) or ceramide (C) for 8 h or TNF- (T) for 16 h. The final
concentration of ceramide was 0.32 pmole/cell, and 1.2 nM for TNF-
.
Cells from a total of six wells for each treatment were then harvested,
combined, and PARP cleavage was assayed as in Fig. 2.
[View Larger Versions of these Images (33 + 37 + 14K GIF file)]
-induced PARP cleavage, only Bcl-2 expression provided protection from ceramide-induced cleavage.
PARP was cleaved after exogenous ceramide treatment despite high levels of expression of CrmA, indicating that
ceramide bypasses the CrmA target and activates a downstream protease capable of cleaving PARP (i.e., prICE)
(Fig. 5 C). Densitometric quantitation of PARP cleavage
showed 17.7 and 20.4% cleavage in the ceramide- or
TNF-
-treated vector cells, respectively, whereas CrmAexpressing cells treated with ceramide or TNF-
had 16.6 and 0% cleavage, respectively. Therefore, in MCF-7 cells,
CrmA expression does not significantly interfere with activation of PARP cleavage by ceramide.
, C6-ceramide, and
mitomycin C on the survival of CrmA- or Bcl-2-expressing MCF-7 cells. Whereas the CrmA-expressing cells were
protected from TNF-
-induced apoptosis, they were equally
susceptible to mitomycin C (Fig. 6 A) as were vector cells.
However, cells expressing Bcl-2 were protected from the
cytotoxic effects of mitomycin C as well as TNF-
and ceramide. Biochemically, expression of CrmA prevented PARP
cleavage after activation of the ceramide-dependent TNF-
pathway (Fig. 6 B). However, PARP cleavage was almost
complete, despite expression of CrmA, after treatment with
mitomycin C that activates a ceramide-independent apoptotic pathway. In contrast, Bcl-2 overexpression prevented
PARP cleavage induced by both the TNF-
(ceramidedependent) and mitomycin C (ceramide-independent) pathways. These results provide additional evidence that
CrmA functions distinctly from Bcl-2. Specifically, CrmA
appears to inhibit an event proximal to ceramide accumulation in ceramide-dependent pathways whereas Bcl-2 works
by inhibiting a more distal target that is common to both
apoptotic pathways.
Fig. 6.
Differential protection from cell death by CrmA and Bcl-2.
(A) Vector, CrmA-expressing, and Bcl-2-overexpressing MCF-7 cells were
treated as in Fig. 3 A with C6-ceramide (10 µM), TNF- (2 nM), or mitomycin C (2.5 µg/ml). Cell death was evaluated at 48 h as in Fig. 2 A. Results are the average of three experiments. (B) Vector, CrmA-expressing, and Bcl-2-overexpressing MCF-7 cells were seeded as in Fig. 2 and
treated with PBS vehicle (lane 1), 1.2 nM TNF-
(lane 2), or 10 µg/ml
mitomycin C (lane 3). Cells were harvested after 20 h of treatment and
PARP cleavage was assayed as in Fig. 2. The bands representing intact or
cleaved (apoptotic) PARP are shown. One out of three different experiments is shown.
[View Larger Versions of these Images (27 + 42K GIF file)]
B Activation.
B is one of the major functions of TNF-
. For
activation to occur, NF-
B has to dissociate from its cytoplasmic inhibitory protein I-
B so that it can translocate to
the nucleus (51). This is accomplished through site-specific
serine phosphorylation followed by proteolytic degradation
of I-
B (52, 53). The mediators of this activation have not
been clarified, although the "death domain"-containing protein TRADD, but not FADD, has been shown to be
important for its activation (35, 36). Recently, ceramide
generated through the action of an acidic sphingomyelinase
was implicated in the activation of NF-
B (54). The antiproteolytic activity of CrmA, as well as its ability to inhibit
ceramide accumulation after TNF-
treatment led us to
examine the effect of CrmA expression on NF-
B activation. We used electrophoretic mobility shift assays which
rely on the ability of activated NF-
B to bind to its specific DNA sequence resulting in retardation of the protein-
DNA complex on nondenaturing polyacrylamide gels. Treatment of MCF-7 cells expressing CrmA, Bcl-2, or their
corresponding vector with 2 nM TNF-
resulted in equal
activation of NF-
B (Fig. 7, A and B). Therefore, CrmA
does not interfere with the signaling pathway leading to activation of NF-
B, despite its demonstrated ability to completely inhibit ceramide accumulation. This suggests that a
physiologic role for ceramide in NF-
B activation is unlikely.
Fig. 7.
Activation of NF-B is not inhibited by CrmA (A) or Bcl-2
(B). Electrophoretic mobility shift assay (EMSA) for the transcription factor NF-
B in MCF-7 cells expressing CrmA, Bcl-2, or vector is shown.
Cells were seeded at 2 × 106 cells/10-cm dish in RPMI media containing
10% FBS and rested overnight. Treatment with 2 nM TNF-
proceeded
for 30 min after which the cells were trypsinized, nuclear extracts prepared,
and EMSA performed using 10 µg of nuclear protein and a 32P-labeled
NF-
B oligonucleotide probe as described in Materials and Methods. Bands
representing the specific NF-
B-DNA complex, a nonspecific band (n.s.),
and the free probe are indicated.
[View Larger Versions of these Images (42 + 33K GIF file)]
is completely inhibited by CrmA (Fig. 3 B). Exogenous ceramide can bypass this block and produce apoptosis by activation of downstream proteases (Fig. 3 A and 5 A). Similar
results were obtained with BJAB cells after ligation of the
Fas receptor (data not shown). These data suggest that
CrmA targets a protease acting upstream of ceramide generation. Since CrmA did not inhibit activation of prICE by ceramide or mitomycin C, these results raise the possibility
that CrmA may target proteases that act upstream of the
PARP protease.
and is involved in the activation phase,
rather than the execution phase, of apoptosis. The recently described Fas- and TNF-
receptor-associated ICE protease FLICE/MACH, is a likely candidate since it was
demonstrated that apoptosis induced by its overexpression
can be inhibited by CrmA (16). Other apoptotic stimuli
such as mitomycin or sodium azide (data not shown) that
use a ceramide-independent pathway to activate the death
proteases are not inhibited by CrmA (Fig. 8).
Fig. 8.
Schematic presentation of the proposed sites of inhibition of
the ceramide pathway by CrmA and Bcl-2. Activation of sphingomyelinases requires several stages of proteolytic processing (63). The inhibition
of ceramide accumulation by CrmA is hypothesized to be due to its ability to inhibit cysteine or serine proteases probably involved in the processing of sphingomyelinase(s). Bcl-2 functions further downstream by inhibiting effector molecules involved in the execution of the death order
without interfering with the generation of ceramide. The specific target
of Bcl-2 is not yet known. The diagram illustrates the possibility that either proteases or regulatory molecules are targeted by Bcl-2.
[View Larger Version of this Image (17K GIF file)]
while only Bcl-2 is capable of
protecting cells treated with C6-ceramide or mitomycin C. These findings implicate Bcl-2 as a more generalized inhibitor of apoptosis acting on a target common to different apoptotic pathways whereas CrmA may specifically inhibit pathways that lead to a sustained ceramide signal such as that
stimulated by TNF-
or Fas.
B activation and apoptosis. The activation of the ceramide pathway has been implicated by some groups to be a
necessary step for activation of NF-
B (54, 56, 57). Others
have been unable to reproduce these results (47, 58).
The ability of CrmA to inhibit the generation of ceramide
provided a new tool to help resolve these contradictions.
Our finding that CrmA does not inhibit NF-
B activation
by TNF-
despite a complete shutdown of ceramide generation makes a role for ceramide in NF-
B activation highly questionable. Rather, ceramide accumulation appears to be
more intimately related to apoptosis and growth suppression. These results are consistent with published studies
showing that CrmA does not interfere with NF-
B activation stimulated by TRADD overexpression, but blocks
TRADD-driven apoptosis (36), and that the dominant negative FADD mutant can potently suppress FADD-dependent apoptosis without interfering with NF-
B activation
(38). Additionally, in cells undergoing Fas-induced apoptosis which is accompanied by massive accumulation of ceramide, NF-
B activation is strikingly absent (Gamard, C.,
G. Dbaibo, L. Obeid, and Y. Hannun, manuscript submitted). These studies clearly support the previous findings that
ceramide is neither necessary nor sufficient for NF-
B activation.
-induced apoptosis.
The sustained elevations in endogenous ceramide levels
observed over several hours that are inhibited by CrmA are
more closely related to apoptosis than the transient, but
more modest, increases in ceramide observed within minutes in other cell lines and implicated in mediating early
signaling events (27). Indeed, these sustained levels of ceramide are more compatible with cellular levels achieved with 1-20 µM of exogenous ceramide. Accumulation of
ceramide with delayed kinetics has been implicated as a
stress response that drives the cell to either undergo apoptosis (29, 34) or cell cycle arrest (29, 62). Indeed, these delayed
kinetics of ceramide accumulation are more commensurate
with the observed delay in the onset of apoptotic morphology and subsequent cell death after TNF-
. Our findings
that this accumulation occurs before PARP cleavage and
the ability of Bcl-2 to prevent ceramide-induced death support this emerging role for ceramide. The results obtained using MCF-7 cells suggest that, in the apoptotic response of
these cells, ceramide functions much more as a downstream
sensor and integrator rather than as an upstream acute signaling switch.
-induceable pathway of
apoptosis. This pathway can be inhibited by CrmA that
blocks ceramide generation or Bcl-2 that interferes with
the function of effector molecules downstream of ceramide
(Fig. 8). Although both Bcl-2 and CrmA appear to inhibit
activation of proteases, these results show that they interfere with the death pathway at two different points. These
results will need to be evaluated in other cell lines and further exploration of this pathway will be essential for the understanding of this complex aspect of cell regulation and
may identify more specific targets for inhibition of apoptosis.
Address correspondence to Yusuf A. Hannun, Duke University Medical Center, Box 3355, Durham, NC 27710.
Received for publication 25 September 1996
This work was supported by a National Institute of Child Health and Human Development grant 5P30HD28828-03 to G. Dbaibo, National Institutes of Health grant GM-43825 to Y. Hannun, and Department of the Army Medical Division grant 17-94-J4301 to Y. Hannun.We thank Muneesh Tewari and Dr. Vishva M. Dixit for providing us with different MCF-7 cell lines and for helpful discussions and advice, Alicja Bielawska for C6-ceramide and 14C6-ceramide synthesis, Milton Campbell and William E. Kraus for help with densitometry, and Marsha Mangum for expert secretarial assistance.
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