Download Induction of Apoptosis through B-cell Receptor Cross-linking

THE JOURNAL
OF
BIOLOGICAL CHEMISTRY
Vol. 276, No. 17, Issue of April 27, pp. 13606 –13614, 2001
Printed in U.S.A.
Induction of Apoptosis through B-cell Receptor Cross-linking
Occurs via de Novo Generated C16-Ceramide and Involves
Mitochondria*
Received for publication, October 18, 2000, and in revised form, January 16, 2001
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M009517200
Bart-Jan Kroesen‡, Benjamin Pettus§, Chiara Luberto§, Mark Busman¶, Hannie Sietsma储,
Lou de Leij‡, and Yusuf A. Hannun§**
From the Departments of ‡Pathology and Laboratory Medicine and 储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
B-cells, triggered via their surface B-cell receptor
(BcR), start an apoptotic program known as activationinduced 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
* 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. This
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.
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-␣,
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).
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 sur1
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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This paper is available on line at http://www.jbc.org
Role of Ceramide and Mitochondria in BcR-induced Apoptosis
face-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.
MATERIALS AND METHODS
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 peroxidaseconjugated 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 [␥-32P]ATP were obtained from PerkinElmer Life Sciences.
z-VAD (Z-Val-Ala-DL-Asp)-fluoromethylketone was from Calbiochem
(La Jolla, CA).
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 ␤-octylglucoside/dioleoyl-phosphatidyl glycerol micelles, 2 mM dithiothreitol, 5 ␮g of proteins of diacylglycerol kinase
membranes, and 2 mM ATP (mixed with [␥-32P]ATP) in a final volume
of 100 ␮l. Lipids were again extracted according to Bligh and Dyer.
32
P-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.
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
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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 ␮Ci 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 ⫺80 °C. The
ceramide spot was scraped, and radioactivity was quantitated by liquid
scintillation counting. Ceramide levels were normalized to total lipid
phosphate.
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 ␮g/ml
aprotinin and leupeptin. A cell lysate (50 ␮l) containing 50 ␮g of protein
was added to a 50-␮l 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 ␮l of cell lysate containing 50 ␮g
of protein was added to a 50-␮l 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 ␮Ci/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 (⌬⌿)—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.
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 ␮g/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 ␮g/ml
aprotinin, and 10 ␮g/ml leupeptin). Lysates were centrifuged at 10,000
rpm for 10 min and mixed with reducing SDS sample buffer. Proteins
(10 ␮g, 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 antirabbit IgG, proteins were visualized by ECL (Amersham Pharmacia
Biotech).
RESULTS
BcR Cross-linking Induces Ceramide Generation and Apoptotic Features—BcR cross-linking using anti-IgM antiserum
2
B. Pettus, M. Bussmann, and Y. Hannun, manuscript in
preparation.
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Role of Ceramide and Mitochondria in BcR-induced Apoptosis
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 ␮g/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 ␮g/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.
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 ␮M 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 crosslinking. 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
3
A. Bielawska and Y. Hannun, unpublished observations.
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 ␮g/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.
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 signif-
Role of Ceramide and Mitochondria in BcR-induced Apoptosis
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 ␮g/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.
icantly, 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
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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 ␮M 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,
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Role of Ceramide and Mitochondria in BcR-induced Apoptosis
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.
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 (⌬⌿),
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 caspaseindependent involvement of anti-IgM-induced ceramide in the
loss of function of mitochondria.
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 BcRmediated apoptosis.
DISCUSSION
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 C16ceramide) 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
Role of Ceramide and Mitochondria in BcR-induced Apoptosis
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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 ␮g/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 ␮M) (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.
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 ␮g/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.
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 BcRinduced 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
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 ␮g/ml) in the presence or absence of FB1
(50 ␮M) as indicated. Treatment in all cases was performed in the
presence of 4 ␮Ci 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.
blocked ceramide formation but also inhibited PARP cleavage,
DNA fragmentation, and changes in mitochondrial ⌬␺. 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 crosslinking induces increased de novo ceramide generation, which
is involved directly or indirectly in mitochondrial damage.
There is now a growing body of evidence to implicate ceramide accumulation in induction of apoptosis. Addition of cellpermeable 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
13612
Role of Ceramide and Mitochondria in BcR-induced Apoptosis
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 ␮g/ml)
in the presence or absence of FB1 (50 ␮M)
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.
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 ␮g/ml) in the presence of (C)
FB1 (50 ␮M) or (D) z-VAD (50 ␮M). 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.
(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 zVAD 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
Role of Ceramide and Mitochondria in BcR-induced Apoptosis
13613
FIG. 10. Left panel, BcR cross-linkingmediated 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 ␮g/ml)
in the presence of FB1 (50 ␮M) (C and G)
or z-VAD (50 ␮M) (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.
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 (␣)hydroxylation of the Nlinked 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.
13614
Role of Ceramide and Mitochondria in BcR-induced Apoptosis
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.
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-1phosphate, 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 ␤-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.
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.
Acknowledgments—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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