From the Departments of Pathology and Laboratory
Medicine and
Pediatric Oncology and Hematology, University
Hospital Groningen, 9713 G2 Groningen, The Netherlands and the
¶ United States Department of Commerce, National Ocean Service,
Charleston, South Carolina 29412 and the § Department of
Biochemistry and Molecular Biology, Medical University of South
Carolina, Charleston, South Carolina 29425
Received for publication, October 18, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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B-cells, triggered via their surface B-cell
receptor (BcR), start an apoptotic program known as activation-induced
cell death (AICD), and it is widely believed that this phenomenon plays
a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD,
the contribution of either and the exact molecular events through which
AICD commences are still unknown. Here we show that in Ramos B-cells,
BcR-triggered cell death is associated with an early rise of C16
ceramide that derives from activation of the de novo
pathway, as demonstrated using a specific inhibitor of ceramide
synthase, fumonisin B1 (FB1), and using pulse labeling with the
metabolic sphingolipid precursor, palmitate. There was no evidence for
activation of sphingomyelinases or hydrolysis of sphingomyelin.
Importantly, FB1 inhibited several specific apoptotic hallmarks such as
poly(A)DP-ribose polymerase cleavage and DNA fragmentation.
Electron microscopy revealed morphological evidence of mitochondrial
damage, suggesting the involvement of mitochondria in BcR-triggered
apoptosis, and this was inhibited by FB1. Moreover, a loss of
mitochondrial membrane potential was observed in Ramos cells after BcR
cross-linking, which was inhibited by the addition of FB1.
Interestingly, benzyloxycarbonyl-Val-Ala-DL-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de
novo generated C16 ceramide in the execution of AICD, and they
further suggest an ordered and more specific sequence of biochemical
events in which de novo generated C16 ceramide is involved
in mitochondrial damage resulting in a downstream activation of
caspases and apoptosis.
Apoptosis has been shown to be an important means by which
organisms maintain homeostasis in proliferating tissues and systems such as the immune system (1-3). The term apoptosis is commonly used
to denote the appearance of one, or a combination of, cellular events
characterized by nuclear condensation, membrane blebbing, chromatin
fragmentation, and loss of membrane integrity resulting in
phosphatidylserine exposure and trypan blue uptake (4, 5). Biochemical
mechanisms by which each of these cellular characteristics are
regulated remain largely unknown. However, it has become evident that
the activation of a family of cysteine proteases known as caspases
plays an important role in the progression of the apoptotic process (6,
7). Within this caspase family, initiator caspases are activated
through an apoptotic stimulus and subsequently activate downstream
effector caspases. These effector caspases in turn have a multitude of
intracellular substrates, among which are components that are
critically needed for cellular homeostasis. Cleavage of one or more of
these substrates disregulates cell function and promotes specific
morphological characteristics of the apoptotic program (4, 6, 7).
Ceramide accumulation has been widely described to be associated with a
number of apoptotic hallmarks such as
PARP1 cleavage, DNA
fragmentation, phosphatidylserine exposure, and trypan blue uptake
(8-11). Exogenously added ceramides are generally able to mimic
stress-induced apoptosis in a stereospecific manner, and inhibition of
the formation of ceramide has been shown, at least in some cases, to
inhibit progression of apoptosis (12-15). Ceramide can be generated
through different metabolic routes in the cell (16). The stress-induced
metabolic conversion of sphingomyelin (SM) into ceramide by the enzyme
SMase has been described extensively in response to various treatments
of cells such as tumor necrosis factor- AICD of B-cells triggered via their BcR provides a physiologically
relevant model to study molecular and biochemical events leading to
induction of apoptosis. Cross-linking of surface-expressed Ig in
B-cells sets off an apoptotic program leading to cell death that is
characterized by a number of the above-mentioned apoptotic features
including PARP cleavage and DNA fragmentation (13). However,
BcR-induced cell death has been sown to commence via an initial
caspase-independent step (20). In addition to activation of caspases,
ceramide formation has been shown to occur in response to BcR
triggering (12, 21). However, the exact metabolic pathway underlying
this elevated ceramide, as well as the role of ceramide in the
progression of apoptosis, has not been determined.
In this study we have investigated how BcR-induced generation of
ceramide is involved in the induction of apoptosis. We first provide
evidence that this ceramide appears to arise exclusively from de
novo synthesis. We also show that early changes in de novo derived C16 ceramide are linked to a loss of function of mitochondria and subsequent activation of the apoptotic program. We
postulate a role for de novo generated C16 ceramide in
alterations in the mitochondrial membrane leading to cytochrome
c release and subsequent activation of a cascade of
downstream effectors involved in the further execution of apoptosis.
Cell Culture--
The Epstein-Barr virus negative
Burkitt's lymphoma Ramos cells were purchased from ATCC
(Manassas, VA) and grown in RPMI 1640 supplemented with 25 mM Hepes, 10% fetal calf serum, 2 mM
L-glutamine (Life Technologies, Inc.), 1 mN
sodium pyruvate (Life Technologies, Inc.), 100 units/ml
penicillin (Life Technologies, Inc.), and 100 g/ml streptomycin (Life
Technologies, Inc.) under standard incubator conditions (humidified
atmosphere: 95% air, 5% CO2; 37 °C).
Reagents--
Polyclonal rabbit anti-PARP antibody and
peroxidase-conjugated anti-rabbit antibody were obtained from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA). Fumonisin B1 and ribonuclease
A were purchased from Sigma. C6-ceramide was obtained from Matreya Inc.
(Pleasant Gap, PA). Propidium Iodide and DiOC6 were
purchased from Molecular Probes (Eugene, OR).
[3H]Palmitate,
[methyl-3H]choline, and
[ Ceramide Measurements (Diacylglycerol Kinase
Assay)--
Cell pellets (2.0 × 106) were lysed in
chloroform:methanol (1:2), and lipids were extracted by the method of
Bligh and Dyer. Aliquots were dried down and used for ceramide and
phosphate measurements. Ceramide levels were measured using the
Escherichia coli diacylglycerol kinase assay (22, 23).
Briefly, lipids were incubated at room temperature for 30 min in the
presence of Ceramide Measurements (Tandem Mass Spectrometry)--
Cell
pellets (2.0 × 106) were lysed in chloroform:methanol
(1:2), and lipids were extracted by the method of Bligh and Dyer. Aliquots were dried down and used for ceramide and phosphate
measurements. For mass spectrometric analysis we utilized normal phase
high performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization. Briefly, separations were conducted using
Iatrobead (Iatron Laboratories, Tokyo, Japan) beaded silica columns
utilized in a normal phase of operation. Elutions were completed on an
Agilent (Palo Alto, CA) model 1100 HPLC system equipped with a binary
pumping system of iso-octane and ethyl acetate. All mass spectrometry
analyses were conducted on a Finnigan (Foster City, CA) LCQ ion
trap mass spectrometer. For atmospheric pressure chemical ionization
mass spectrometry, the entire flow from the HPLC column was directed to
the atmospheric pressure chemical ionization source. For all
experiments, source ion optics were adjusted to accomplish desolvation
of ions while minimizing fragmentation of analyte ions in the inlet
region of the mass spectrometer. Ceramide and dihydroceramide
subspecies were identified by a combination of mass
(m/z), intensity ratios in the mass
spectrometry fragmentation pattern, and column retention. Analysis was
then standardized by programming the LC-Quant software followed by manual confirmation (the full details of this method are in
preparation).2
[3H]Palmitate Labeling of Cells--
Ramos cells
(2.0 × 106/4 ml of RPMI, 10% fetal calf serum) were
labeled with 4 µCi of [3H]palmitate. Anti-IgM and
inhibitors (fumonisin B1) were added together with the
radionucleotides. After 6 h, cells were washed once with PBS, and
subsequently the lipids were extracted by the method of Bligh and Dyer.
Lipids were separated using TLC analysis in ethyl
acetate:iso-octane:acetic acid (9:5:2). A ceramide standard, visualized
by iodine vapor, was used as a reference. TLC plates were sprayed by
En3Hance spray, and radioactivity was visualized by
autoradiography for 48 h in Analysis of SMase Activity--
The activities of both neutral
and acid SMase were determined using radiolabeled substrate in a mixed
micelle assay as described (24). For analysis of neutral SMase
activity, cells were lysed in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1% Triton X-100, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin and leupeptin. A cell lysate (50 µl) containing 50 µg of protein was added to a 50-µl reaction mixture containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
0.05% Triton X-100, 5 mM dithiothreitol, 5 nmol of
[14C]sphingomyelin (100,000 dpm), and 10 nmol of cold SM
per reaction. For analysis of acid SMase activity, 50 µl of cell
lysate containing 50 µg of protein was added to a 50-µl
reaction mixture containing 100 mM sodium acetate, pH 5.0, 5 nmol of [14C]sphingomyelin (100,000 dpm), and 10 nmol
of cold SM per reaction. Exogenously added SM was solubilized in the
reaction mixture by sonication prior to the addition of enzyme.
Reactions were allowed to proceed for 45 min at 37 °C and stopped by
the addition of 1.5 ml of cloroform: methanol (2:1) and 0.2 ml of
H2O. Separate phases were clarified by centrifugation for 5 min at 3000 rpm, and 0.4 ml of the aqueous upper phase was counted by
liquid scintillation counting. Total hydrolyzed SM never exceeded 5%
of the total amount of radiolabeled SM added to the assay.
Analysis of Cellular SM Mass--
Cells were labeled for 48 h with [methyl-3H]choline chloride (final
specific activity, 0.5 µCi/ml). After cell treatment as described,
cells were washed with PBS, and lipids were extracted from the cell
pellets by the method of Bligh and Dyer. Part of the extracted lipids
were used for phosphate measurement. Remaining lipids were subjected to
mild base hydrolysis (25), and [3H]sphingomyelin was
determined by TLC analysis in chloroform: methanol: acetic acid:
H2O (50:30:8:5), followed by scraping and counting
radioactivity by liquid scintilation.
Mitochondrial Transmembrane Potential ( DNA Fragmentation Analysis--
Cells (0.5 × 106/2 ml) were treated as described. After treatment, cells
were pelleted and resuspended in 2 ml of ice-cold 70% ethanol and
incubated for 1 h at 4 °C. After incubation, cells were washed
twice with PBS and finally resuspended in PBS containing RNase (final
concentration, 0.5 mg/ml) and incubated for 30 min at 37 °C.
Finally, propidium iodide was added at a final concentration of 50 µg/ml and incubated overnight in the dark at 4 °C. The cell cycle
was analyzed by flow cytometry.
Western Blot Analysis--
Cells were lysed for 15 min on ice in
lysis buffer (20 mM Tris-HCl, 5 mM EDTA, 2 mM EGTA, 100 mM NaCl, 0.05% SDS, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin). Lysates were centrifuged
at 10,000 rpm for 10 min and mixed with reducing SDS sample buffer.
Proteins (10 µg, as determined by Bio-Rad protein assay) were
separated on 7.5% SDS-polyacrylamide minigels. Proteins were
transferred to nitrocellulose membranes, which were subsequently
blocked by 5% nonfat dry milk in PBS, Tween 20 (0.1%, v/v).
Blots were incubated with anti-PARP at a 1:1000 dilution with 5%
nonfat dry milk. After subsequent incubation with a 1:2000 dilution of
horseradish peroxidase anti-rabbit IgG, proteins were visualized by ECL
(Amersham Pharmacia Biotech).
BcR Cross-linking Induces Ceramide Generation and Apoptotic
Features--
BcR cross-linking using anti-IgM antiserum has been
shown to induce apoptosis over a time period of ~48 h. As shown in
Fig. 1A, PARP cleavage was
observed starting at 12 h after BcR cross-linking and gradually
increased over the next 36 h. In accordance with this, a gradual
increase in DNA fragmentation up to 31% after 48 h was observed
in similarly treated cells (Fig. 1B). Similar to BcR
cross-linking, direct addition of exogenous ceramide to cells has been
shown to induce apoptosis in various cell lines. As shown in Fig.
2, addition of up to 10 µM
C6-ceramide to Ramos cells for 24 h induced PARP cleavage in a
dose-dependent manner. We next determined ceramide levels
in Ramos cells upon BcR cross-linking. As shown in Fig.
3A, ceramide levels increased
gradually, reaching a 2.5-fold increase at 48 h after the start of
the treatment. These changes are consistent with a previous study in
WEHI B-cells (12). The changes in ceramide were further analyzed by
TLC. It is important to note that our TLC system resolves ceramide (after derivatization into ceramide phosphate) into two spots (see Fig.
3D). Interestingly, ceramide in the lower spot started to
increase as soon as 6 h after BcR cross-linking, whereas an increase in the upper ceramide spot could not be detected until 24 h after treatment (Fig. 3, B and C). We find that
the derivatized ceramide phosphate comigrates with the lower spot,
whereas dihydroceramide phosphate migrates with the upper spot (26). In
addition, longer chain ceramides migrate with the upper spot as
compared with C16-ceramides, which migrate in the lower
spot.3 No changes in ceramide
were detected at earlier time points (5 min to 3 h) after BcR
cross-linking (data not shown).
Specific Generation of C16-Ceramide--
To clarify the molecular
nature of these two spots, tandem mass spectrometric analysis was
employed. Such analysis has been able to routinely detect 14 ceramide
and dihydroceramide subspecies in Ramos cells ranging from C14 to C26
in fatty acid length. Of note was the presence of a double bond on the
fatty acid of ceramides longer than C24. C24:0 and C26:0 were also
present, but absolute levels of C24:1 to C24:0 were difficult to
resolve because, in contrast to the dihydroceramides, there is no
difference in column retention with this double bond (ceramides have a
double bond in resonance association with a hydroxyl group), and
isotope ratios make a m/z of 2 units difficult to
quantitate. We speculate that the ratio is nearly 1:1, with this ratio
remaining constant during ceramide changes studied thus far.
Analysis by this method under anti-IgM-induced cross-linking of BcR
revealed that early ceramide changes were specific to long chain
ceramides (C14, C16, and C18 fatty acid lengths) and their respective
dihydroceramide counterparts. The composition of the lower spot was
overwhelmingly a C16 ceramide, with a 1.5-fold change at 6 h
corresponding well to the increase in ceramide seen in the lower
spot by diacylglycerol kinase at 6 h (Fig.
4A). Similarly, the
composition at 24 h of treatment revealed a nearly 3-fold increase
in C16 ceramide, with similar, approximately 2-fold changes in C14,
C18, and the dihydroceramides matching the 2.5-fold increase in the
lower spot at this time. However, at 24 h the upper spot began to
change significantly, as could be seen by a 1.5-fold increase by
diacylglycerol kinase. Mass spectrometry revealed a nearly
2-fold increase in the very long chain C24-ceramide, with possible
slight increases in C22 and C26 ceramides and the corresponding
dihydroceramides (Fig. 4B). Therefore, mass spectrometry was
able to resolve distinct temporal changes in ceramide species; the
C16-ceramide was the early detected species and corresponded to the
lower spot seen by diacylglycerol kinase, and C24-ceramides came up
late and corresponded to the upper spot by diacylglycerol kinase.
BcR-induced Ceramide Is Generated de Novo--
Ceramide can be
generated from diverse metabolic routes, e.g. from SM
through activation of SMase or from activation of the de
novo pathway. Thus, the activity of divergent enzymes involved in
the generation of ceramide may be regulated differently. Moreover, ceramide derived from different pathways may be involved differently in
various cellular processes such as proliferation, differentiation, and
apoptosis. To determine the nature of the BcR-induced ceramide formation, we measured SMase activity and determined SM levels in Ramos
cells after BcR cross-linking. As shown in Fig.
5, A and B,
induction of neither neutral- nor acid-type SMase activity was observed
after BcR cross-linking over the time period where ceramide levels
increased. In addition, no decrease in SM levels was detected after IgM
cross-linking, which would have been indicative of any type of SMase
activity. Because SMase activity did not seem to be involved in the
formation of ceramide after BcR cross-linking, we next investigated the
possibility that the observed ceramide was formed de novo.
To this end we made use of fumonisin B1 (FB1), a selective inhibitor of
ceramide synthase that catalyzes the acylation step in the de
novo pathway of ceramide formation. As shown in Fig.
6 for the lower ceramide spot, treatment
of Ramos cells with 50 µM FB1 completely prevented
ceramide accumulation in response to anti-IgM-induced BcR
cross-linking. This strongly suggested the involvement of ceramide
synthase in the formation of ceramide upon BcR ligation. In addition,
the increase in ceramide migrating with the upper ceramide phosphate
spot was completely inhibited, whereas SM levels remained unchanged
(data not shown).
To further substantiate that BcR-induced ceramide
accumulation was generated de novo, we performed
pulse-labeling experiments using radioactive palmitate, which serves as
a substrate for both serine palmitoyl transferase and ceramide synthase
in the de novo pathway. As shown in Fig.
7, palmitate added as a pulse label after
BcR ligation was incorporated into ceramide, resulting in a 175%
increase over control-treated cells 6 h after the start of the
treatment. The increase in ceramide from palmitate was nearly totally
blocked by adding FB1.
Generation of Ceramide Is Directly Linked to
Caspase-dependent Apoptotic Features and Involves
Mitochondria--
Because ceramide has been proposed to be involved
directly in multiple aspects of apoptosis, we determined whether FB1
could inhibit Ramos cells from entering the apoptotic program. As shown in Fig. 8, incubation of Ramos cells with
FB1 during BcR cross-linking inhibited both induction of PARP cleavage
(Fig. 8A) and DNA fragmentation (Fig. 8B). FB1 by
itself did not significantly affect PARP cleavage (Fig. 8A)
or DNA fragmentation (data not shown). These results suggest a
necessary role for the de novo generated ceramide in activating these downstream effector functions of apoptosis that are
mediated by caspases (see below).
It is now generally accepted that mitochondria play key roles in the
regulation of apoptosis. To investigate the involvement of mitochondria
in BcR cross-linking-induced ceramide formation and apoptosis, we
examined the mitochondrial morphology and function of Ramos cells by
electron microscopy and flow cytometry, respectively, after anti-IgM
treatment in the presence or absence of fumonisin or z-VAD, a broad
spectrum caspase inhibitor. BcR cross-linking resulted in distinct
morphological changes of mitochondria. Control cells showed a multitude
of mostly small intact mitochondria. In contrast, anti-IgM treatment
resulted in significant mitochondrial swelling along with extensive
disruption of the mitochondrial membranes (Fig.
9). A protective effect on mitochondrial
morphology was observed when Ramos cells were treated with anti-IgM in
the presence of FB1 (Fig. 9), suggesting the involvement of de
novo derived ceramide in the observed mitochondrial damage.
Interestingly, no such protective effect on anti-IgM-induced
mitochondrial damage was observed using z-VAD, suggesting that
mitochondrial effects of BcR cross-linking are independent of
activation of caspases 3, 4, 7, and possibly 9.
Mitochondrial damage can be assessed biochemically by measuring the
mitochondrial transmembrane potential (
In contrast to the inability of z-VAD to protect mitochondrial
integrity, anti-IgM-induced PARP cleavage (data not shown), as well as
DNA fragmentation (Fig. 10), was almost completely abrogated in the
presence of z-VAD, which demonstrates the critical involvement of
caspases in the execution phase of BcR-mediated apoptosis.
Immune homeostasis is maintained by a strictly coordinated
regulation of cell proliferation, differentiation, and apoptosis. AICD
has been described as serving immune homeostasis, and it occurs in both
T- and B-cells to avoid autoimmunity and to self-limit the immune
response (1-3). AICD in B-cells occurs after antigenic triggering in
the absence of survival signals such as those provided by interaction
of CD40 with CD40L, which is expressed by activated Th-cells
(28, 29). Although a number of key players involved in BcR-induced cell
death have been described, the molecular/biochemical route through
which AICD in B-cells proceeds has still not been resolved.
Recently, BcR-induced apoptosis has been shown to involve mitochondrial
loss of function, resulting in the activation of the caspase cascade
(20, 30). In addition, BcR cross-linking has been postulated to involve
the generation of ceramide (13, 21) and reactive oxygen species (31).
Earlier it was shown that ceramide directly results in the production
of reactive oxygen species by mitochondria in a cell-free system
(32), and exogenous ceramide-induced mitochondrial hydrogen peroxide
has been suggested to be involved in apoptosis (33, 34).
We therefore investigated the role of ceramide, resulting from BcR
cross-linking, in anti-IgM-induced apoptosis. Our results show that BcR
cross-linking on Ramos B-cells results in apoptosis, which is preceded
by the generation of ceramide. We show that this ceramide response
arises from activation of the de novo pathway of
sphingolipid biosynthesis. Mass spectrometric data support this by
showing significant and specific generation of the long chain ceramide
subspecies (mainly C16-ceramide) in early data points prior to
apoptotic features. The fact that changes in dihydroceramide subspecies
occur is also significant, because it supports the hypothesis of
de novo synthesis, which would generate dihydroceramide prior to desaturation to ceramide in that pathway. In addition, the
studies described here show that BcR-induced apoptosis proceeds via
mitochondrial loss of function, which in turn is dependent on the
accumulation of ceramide, thus linking BcR-induced ceramide formation
with mitochondrial damage and progression of apoptosis.
Importantly, our results also provide evidence that BcR-induced
apoptosis is dependent on the generation of ceramide derived from the
de novo pathway of sphingolipid biosynthesis. In accordance with this was the observation that cotreatment with FB1, a selective ceramide synthase inhibitor, not only blocked ceramide formation but
also inhibited PARP cleavage, DNA fragmentation, and changes in
mitochondrial There is now a growing body of evidence to implicate ceramide
accumulation in induction of apoptosis. Addition of cell-permeable synthetic short chain ceramides closely mimics apoptosis-inducing stimuli, and elevation of endogenous ceramide has been described as a
hallmark of the apoptotic death program (10, 12, 35). There are several
studies documenting necessary roles for ceramide in regulating various
aspects of apoptosis. Mechanistically, ceramide has been implicated as
a pleiotropic bioactive molecule giving rise to effector-caspase
activation as well as other stress-related biochemical events such as
production of reactive oxygen species in mitochondria through as yet
unidentified mechanisms (32, 35-37).
Mitochondrial damage has been widely accepted as an evolutionary
conserved initiating event in the onset of apoptosis (38). Loss of
mitochondrial function results in the activation of caspase 9 through
the release of cytochrome c, which, together with the ubiquitous cellular Apaf-1, has been described to result in activation of pro-caspase 9 (27, 38). The release of cytochrome
c can be inhibited by proapoptotic members of the Bcl-2
protein family and can be inhibited by the anti-apoptotic members.
Indeed, it has been described that overexpression of Bcl-XL
can significantly inhibit BcR-induced apoptosis.
Ceramide generation was completely inhibitable by FB1 and thus seemed
to be formed exclusively through the de novo synthesis pathway. This is in contrast to the results reported by Wiesner et al. (12), who described ceramide formation after BcR
cross-linking due to increased neutral SMase activity, which was
monitored 24 h after BcR cross-linking. No data on cellular SM
levels after BcR cross-linking were provided in that study. As shown in
the present study, we failed to detect increased neutral or acid SMase activity at any of the assayed time points. In addition, we did not
detect even a small decrease in SM levels after BcR cross-linking, which is necessary to result in ceramide formation and which might have
been suggestive of additional unassayed SMase activities that we may
have failed to detect. In this respect Ramos cells seem to respond
differently to BcR cross-linking than WEHI-231 cells, which might
reflect differential phases of B-cell maturation. Whereas Ramos cells
represent mature germinal B-cells (39), WEHI-231 cells have been
reported to represent an immature stage in B-cell development (40).
Our results are the first to implicate de novo ceramide in
BcR-induced apoptosis and the first to implicate de novo
ceramide in regulation of the mitochondrial response to AICD. Very
recently, Kawatani et al. (41) also showed the involvement
of protein kinase C-regulated ceramide induction in inostamycin-induced
apoptosis. Using FB1, a decrease in the release of cytochrome
c was noted in that study, suggesting activation of the
de novo pathway in that process (but this was not directly
demonstrated). The fact that, in our hands, FB1 did not completely
inhibit apoptosis, whereas it did completely inhibit ceramide
accumulation, could be indicative of other signaling mechanisms
independent of the de novo pathway of sphingolipid
biosynthesis. It should be noted, however, that such alternative,
sphingolipid-independent pathways seem to be independent of caspase
activation, because z-VAD did not totally inhibit apoptosis, and the
combination of FB1 and z-VAD did not further decrease the small amount
of apoptosis that was still observed using z-VAD alone. In addition, we
cannot rule out the possibility that topological redistribution of
ceramide or metabolites thereof accounts (at least in part) for the
apoptotic response observed in our model.
Our studies place ceramide formation upstream of activation of caspases
in BcR-induced apoptosis, which is in line with earlier studies
demonstrating that z-VAD does not abrogate ceramide formation in
response to BcR cross-linking (13, 20). This is also consistent with
previous results showing that, with tumor necrosis factor, ceramide
formation is dependent on the upstream, but not downstream/effector, caspases, whereas the action of exogenous ceramide leads to activation of the downstream, but not upstream, caspases (42, 43). In a recent
study, Grullich et al. (44) showed that increased ceramide generation occurs early in the apoptotic response to CD95 triggering, prior to the commitment step to the execution phase of apoptosis. Although the increase in ceramide levels as determined by the diacylglycerol kinase assay excellently fitted the elevation of ceramide quantified by mass spectrometry, no specific ceramide species
were reported to increase in that study (44). By contrast, we were able
to detect early changes of C16 ceramide specifically, followed by later
increments of both C16 and C24 ceramides. Earlier changes in ceramide
levels (range of minutes to 3 h), measured by the diacylglycerol
kinase assay, were not detected. We did not measure SM levels or SMase
activity at very early time points after BcR cross-linking. Therefore,
we cannot rule out activation of SMase activity at these early time
points. However, the lack of detectable levels of ceramide at these
time points suggests that early activation of SMase is unlikely.
The nature of ceramide generated in response to a stimulus may hold
important clues as to the divergent cell biological effects observed
and described to be related to ceramide. In this respect it was
important to note the differential kinetics of ceramide generated in
the upper versus lower TLC spots. Molecular properties that
have been described as determining the separation of ceramide into two
spots are the degree of saturation, fatty acyl chain length, and
( This raises another significant question that deserves further
investigation, namely, whether de novo derived and
SMase-derived ceramide have fundamentally different roles in cell
functioning. It is well established that the generation of ceramide
through the de novo and SMase synthesis routes occurs at
different cellular sites. Whereas enzymes with SMase-type activity have
been shown to be associated with a number of cellular compartments
including the lysosomes (acid SMase) and possibly the cell membrane
(neutral SMase), the enzymes involved in the de novo
synthesis of ceramide are all found in the endoplasmic reticulum
(although mitochondrial localization of at least a subpopulation has
not been ruled out). This raises another important issue, which relates
to the site of generation of sphingolipids and the site of action. To
date, no models exist on the specific mitochondrial delivery of
naturally formed ceramide or sphingolipids in general. Transport from
the endoplasmic reticulum to the trans Golgi network of de
novo generated ceramide has been reported as not being
vesicle-mediated (45) and as not being dependent on protein transport.
Whether a similar transport mechanism applies for de novo
derived ceramide from endoplasmic reticulum to mitochondria is not
known. Theoretically, it may be that a more intricate metabolic network
occurs in which de novo formed ceramide is first converted
to sphingosine and sphingosine-1-phosphate, which may move more freely
compared with ceramide. Alternatively, ceramide might even be generated
in situ in mitochondria. In rodent and bovine brain, a
mitochondrial localization of a ceramide synthase has been described
(46, 47). A very recent study has identified a novel mitochondrial
ceramidase with reciprocal (ceramide synthase) activity indicative of
the existence of a topologically distinct sphingolipid pathway with a
direct possible impact on mitochondria (48-50). (49) Whether or not human lymphocytes contain this mitochondrial ceramide synthase activity
is unknown. Interestingly, an enzyme, carnitine palmitoyltransferase I,
that facilitates passage of long chain fatty acids into mitochondria has been shown to become up-regulated in response to BcR cross-linking. Carnitine palmitoyl transferase I catalyzes the rate-limiting step in
the mitochondrial transmembrane transport of fatty acids used for
In conclusion, our results demonstrate activation of de novo
ceramide synthesis following BcR cross-linking in B-cells. We were able
to distinguish a subset of ceramide species centered around
C16-ceramide that preceded features of apoptosis. Moreover, this
de novo ceramide is important in activating the
mitochondrial pathway of apoptosis in response to BcR
cross-linking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, anti-Fas, serum withdrawal,
and other agents. However, ceramide can also be generated through the
de novo synthesis pathway, in which activation of serine
palmitoyl transferase and/or ceramide synthase may play a pivotal role
(15, 17). However, how ceramide generated from each pathway is involved
in the apoptotic program is as yet largely unknown. In previous
studies, using model systems in which apoptosis was induced with tumor
necrosis factor or Fas activation via the caspase
8-dependent pathway, it was shown that ceramide formation
occurs in between initiator caspases and effector caspases (18,
19).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP were obtained from PerkinElmer Life
Sciences. z-VAD (Z-Val-Ala-DL-Asp)-fluoromethylketone was
from Calbiochem (La Jolla, CA).
-octylglucoside/dioleoyl-phosphatidyl glycerol micelles,
2 mM dithiothreitol, 5 µg of proteins of diacylglycerol kinase membranes, and 2 mM ATP (mixed with
[
-32P]ATP) in a final volume of 100 µl. Lipids were
again extracted according to Bligh and Dyer. 32P-Ceramide
was determined by TLC analysis in chloroform:acetone:methanol:acetic acid:water (50:20:15:10:5), followed by scraping the ceramide doublet
and counting the radioactivity by liquid scintillation. Ceramide levels
were normalized to total lipid phosphate.
80 °C. The ceramide spot was
scraped, and radioactivity was quantitated by liquid scintillation
counting. Ceramide levels were normalized to total lipid phosphate.
)--
To evaluate
changes in
, cells (5 × 105/ml) were treated as
described. After washing and resuspension in medium without fetal calf
serum, cells were labeled with DiOC6 (40 nM)
for 15 min at 37 °C. After incubation, cells were washed and
analyzed directly on a flow cytometer using extinction and emission
wavelengths of 495 and 525 nm, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
Anti-IgM-induced BcR cross-linking
triggers PARP cleavage (A) and DNA fragmentation
(B) in Ramos B-cells. A, Ramos B-cells
(5 × 105/ml) were cultured for the indicated time
periods in the presence of anti-IgM (10 µg/ml) and lysed, followed by
SDS-polyacrylamide gel electrophoresis and Western blotting as
described. A gradual increase in cleaved PARP over time was apparent.
B, in parallel, a gradual increase of fragmented cellular
DNA was observed. Ramos B-cells (5 × 105/ml) were
cultured for the indicated time periods in the presence of anti-IgM (10 µg/ml) and prepared for flow cytometric analysis of DNA content as
described using propidium iodide staining. The hypodiploid peak,
indicative of apoptotic cells, was fitted using Modfit-2 software and
plotted as the percentage of total cells. Representative results from
four independently performed experiments are shown.
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Fig. 2.
Treatment of Ramos cells with cell permeable
C6-ceramide mimics anti-IgM-induced PARP cleavage. Ramos cells
(5 × 105/ml) were treated with anti-IgM (10 µg/ml)
or C6-ceramide in the concentrations shown. After 24 h of
treatment, cells were lysed, followed by SDS-polyacrylamide gel
electrophoresis, and Western blotting was performed as described. Both
treatments, with either anti-IgM or ceramide, induced PARP
cleavage. Representative results from three independently performed
experiments are shown.
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Fig. 3.
B-cell receptor cross-linking is associated
with elevation of endogenous ceramide levels. Ramos cells (5 × 105/ml) were treated with anti-IgM (10 µg/ml) for the
indicated time periods and prepared for measurement of cellular
ceramide (cer.) as described. Ceramide mass (closed
bars), normalized to lipid phosphate, is plotted as the total
ceramide measured (A), the upper ceramide phosphate spot
(B), and the lower ceramide phosphate spot (C),
as visible on TLC (D). Time-matched controls (open
bars) obtained from untreated Ramos cells are included.
Representative results from five independently performed experiments
are shown.
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Fig. 4.
Ceramide subspecies analysis by tandem mass
spectrometry (MS) at (A) 6 h and
(B) 24 h after BcR cross-linking. Data
represent semiquantitative analysis expressed as signal peak
integration normalized to lipid phosphate. On the x axis,
the different ceramide subspecies analyzed are shown. The number
before the colon designates the fatty acid length, the
number after the colon designates the number of double bonds
on this fatty acid, and dh denotes a dihydroceramide. Data
shown are the average of two experiments. Similar results were obtained
in a third experiment.
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Fig. 5.
B-cell receptor cross-linking does not result
in increased SMase-type enzymatic activity (A and
B) or significant changes in the level of cellular SM
(C). Ramos cells (5 × 105/ml)
were treated with anti-IgM (10 µg/ml) for the indicated time periods
and prepared for measurement of neutral (A) or acid
(B) SMase enzyme activity (closed bars) as
described. Time-matched controls (open bars) obtained from
untreated Ramos cells are included. Data are presented as the amount of
[14C]choline recovered from the upper aqueous phase after
phase separation. No increase in either neutral- or acid-type SMase
enzyme activity was apparent over any of the treated time periods.
C, possible hydrolysis of SM to ceramide in vivo
was determined by measurement of total SM mass after steady-state
choline labeling as described. No significant decrease in total levels
of SM, normalized to total lipid phosphate, was observed after
treatment of Ramos cells with anti-IgM (closed bars).
Time-matched controls (open bars) obtained from untreated
Ramos cells are also included. Representative results from five
independently performed experiments are shown.
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Fig. 6.
FB1 inhibits anti-IgM-induced accumulation of
ceramide. Ramos cells (5 × 105/ml) were
treated with anti-IgM (10 µg/ml) for the indicated time periods in
the presence or absence of FB1 and prepared for measurement of cellular
ceramide (cer.) as described. Anti-IgM-induced ceramide
accumulation (closed bars) was prevented in the presence of
FB1 (50 µM) (hatched bars). Time-matched
controls obtained from untreated (open bars) and FB1-treated
(cross hatched bars) Ramos cells are included.
Representative results from three independently performed experiments
are shown.
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Fig. 7.
BcR cross-linking results in a significant
incorporation of palmitate into ceramide. Ramos cells (5 × 105/ml) were treated with or without anti-IgM (10 µg/ml)
in the presence or absence of FB1 (50 µM) as indicated.
Treatment in all cases was performed in the presence of 4 µCi of
[3H]palmitate. After the treatment, cells were harvested,
and lipids were extracted and analyzed by TLC as described. The amount
of radioactivity incorporated into ceramide was determined and
normalized to total phospholipid mass. Representative results from
three independently performed experiments are shown.
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Fig. 8.
Pretreatment of Ramos cells with FB1
significantly inhibits PARP cleavage (A) and DNA
fragmentation (B). Ramos cells (5 × 105/ml) were treated 24 h with or without anti-IgM (10 µg/ml) in the presence or absence of FB1 (50 µM) as
indicated. A, after the treatment, cells were lysed,
followed by SDS-polyacrylamide gel electrophoresis and Western blotting
to detect PARP as described. PARP cleavage was quantified by measuring
the optical density and plotted in arbitrary units. B, Ramos
cells treated as shown were analyzed by flow cytometry for DNA
fragmentation as described. The hypodiploid peak, indicative of
apoptotic cells, was fitted using Modfit-2 software and plotted as the
percentage of total cells analyzed. Representative results from four
independently performed experiments are shown.
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Fig. 9.
Electron microscopy analysis of
anti-IgM-induced changes on mitochondrial morphology in Ramos cells and
the effects of FB1 and z-VAD. Ramos cells (5 × 105/ml) were treated for 24 h with (B-D)
or without (A) anti-IgM (10 µg/ml) in the presence of
(C) FB1 (50 µM) or (D) z-VAD (50 µM). After treatment, cells were harvested and prepared
for electron microscopic analysis as described. Treatment with anti-IgM
resulted in significant swelling and damage of mitochondria, which was
partly reversed by pretreatment with FB1 but not with z-VAD.
), which has been shown to
correlate with the release of cytochrome c, giving rise to
caspase 9 activation that can then activate caspase 3 (27).
DiOC6 is a strong cationic dye that can be used to assay the
. Damaged mitochondria lose membrane integrity and as a consequence are no longer able to maintain their transmembrane potential, resulting in a decreased binding of DiOC6.
Indeed, a clear decrease in the binding of DiOC6 was
observed in anti-IgM-treated Ramos cells (Fig.
10). Moreover, as shown in Fig. 10, the
drop in DiOC6 binding was inhibited by FB1 but not by
z-VAD, which, along with the results shown in Fig. 8, suggests a direct
caspase-independent involvement of anti-IgM-induced ceramide in the
loss of function of mitochondria.
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Fig. 10.
Left panel, BcR cross-linking-mediated
changes in mitochondrial can be reversed by FB1 but not by
z-VAD. Right panel, both FB1 and z-VAD effectively prevent
anti-IgM-induced DNA fragmentation. Ramos cells (5 × 105/ml) were treated for 24 h with (B-D
and F-H) or without (A and E)
anti-IgM (10 µg/ml) in the presence of FB1 (50 µM)
(C and G) or z-VAD (50 µM)
(D and H). After the treatment, cells were
harvested and prepared for flow cytometric analysis of mitochondrial
(A-D) using staining with DiOC6 (40 nM) or for DNA fragmentation (E-H) as
described. Representative results from four independently performed
experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. By contrast, the decrease in mitochondrial
, observed after BcR cross-linking, is not blocked by z-VAD. Taken together, these results place ceramide generation and subsequent loss in mitochondrial
upstream of caspase activation in
BcR-induced apoptosis. We therefore postulate a model in which BcR
cross-linking induces increased de novo ceramide generation,
which is involved directly or indirectly in mitochondrial damage.
)hydroxylation of the N-linked fatty acids (8, 26).
Indeed, this differential response was critical in convincing us of the
changes in ceramide preceding any sign of apoptosis by several hours.
Confirmation by mass spectrometric analysis, therefore, is significant
in several regards. First, it allowed us to identify the ceramide
subspecies involved in the early stages of the signal, highlighting the
significant and specific way in which these changes occurred. Second,
it added further support to the hypothesized role of de novo
synthesis by showing similarly specific changes in the precursor
dihydroceramide subspecies. Changes in longer chain (C18-C24)
ceramides observed at later time points after BcR cross-linking might
be due to metabolic conversion of the initially formed C16-ceramide or
other complex sphingolipids.
-oxidation. Palmitoyl-CoA, an important precursor for de
novo sphingolipid synthesis, is a substrate for this enzyme, and
BcR cross-linking-induced up-regulation of carnitine palmitoyl transferase I mRNA might thus allow enhanced mitochondrial
transmembrane transport of fatty acids that could be used as precursors
for ceramide synthesis.
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ACKNOWLEDGEMENTS |
---|
We acknowledge C. Enockson for assistance with flow cytometric analysis and Hazel Martin for assistance with electron microscopic analysis. We also acknowledge Peter Moeller at the United States Department of Commerce, National Ocean Service, Charleston laboratory, for access to the Tandem Mass Spectrometry Facility.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM43825 and NIGMS GM08716.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M009517200
2 B. Pettus, M. Bussmann, and Y. Hannun, manuscript in preparation.
3 A. Bielawska and Y. Hannun, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: PARP, poly(A)DP-ribose polymerase; SM, sphingomyelin; SMase, sphingomyelinase; AICD, activation-induced cell death; BcR, B-cell receptor; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; z-VAD, Z-Val-Ala-DL-Asp; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; FB1, fumonisin B1.
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