From the Department of Pathology and Laboratory
Medicine, Medical Biology branch, University Hospital Groningen,
Hanzeplein 1, 9713 GZ Groningen, The Netherlands, ¶ Department of
Biochemistry and Molecular Biology, Medical University of South
Carolina, Charleston, South Carolina 29425,
Department of
Pathology, University Hospital Groningen, Hanzeplein 1, 9713 GZ
Groningen, The Netherlands, and ** Department of Cell
Biology, University of Groningen, Antonius Deusinglaan 1, 9713AV
Groningen, The Netherlands
Received for publication, October 21, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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In this study, we describe an ordered
formation of long- and very long-chain ceramide species in relation to
the progression of B-cell receptor (BcR) triggering induced apoptosis.
An early and caspase-independent increase in long-chain ceramide
species, in which C16- ceramide predominated, was
observed 6 h after BcR triggering. In contrast, very long-chain
ceramide species were generated later, 12-24 h after BcR triggering.
The formation of these very long-chain ceramide species, in which
C24-ceramide predominated, required the activation of
effector caspases. BcR-induced formation of long-chain ceramide species
resulted in proteasomal activation and degradation of XIAP and
subsequent activation of effector caspases, demonstrating an important
cell-biological mechanism through which long-chain ceramides may be
involved in the progression of BcR triggering induced apoptosis and
subsequent formation of very long-chain ceramide species. BcR-induced
activation of the proteasome was blocked with ISP-1/myriocin, a
potent and selective inhibitor of serine palmitoyl transferase that
catalyzes the first and rate-limiting step in the de novo
formation of ceramide. Both ISP-1 and clasto-lactacystin
Activation of B-cells via their surface B-cell receptor
(BcR)1 initiates an apoptotic
program known as activation-induced cell death (AICD). AICD serves to
protect the body from the outgrowth of autoreactive immune cells and,
as such, plays a key role in the maintenance of immune homeostasis.
BcR-induced AICD is initiated as part of a complex signaling cascade in
which multiple molecular components participate upon recognition of
specific antigens. These signaling moieties comprise both positive and
negative regulators of B-cell mitogenesis and survival, but in the
absence of adequate rescue signals, they will generally skew the cells
into the activation of a cell death program (1-4). BcR-induced
apoptosis has been shown to involve the formation of ceramide, a
bioactive lipid involved in coordinating the cellular response to
various stress signals (5-11).
Ceramide is generated de novo from the condensation of
L-serine and palmitoyl-CoA followed by an acylation
reaction of the resulting sphinganine and subsequent reduction of the
product dihydroceramide to ceramide. The term ceramide is often used as a generic term for the many different cellular ceramide species present. These are distinguished by the degree of saturation, fatty
acyl chain length, and ( To date, very little is known on the differential regulation of
specific ceramide species and how this contributes to the regulation of
specific cell-biological events. In Ramos B-cells, we recently
described that ceramide accumulation after BcR cross-linking is
biphasic with an early rise in de novo derived
C16-ceramide followed by the formation of both
C16- and C24-ceramide after prolonged
stimulation (5). Although mitochondria were found to be involved in the
BcR-induced ceramide formation, molecular targets of the ceramides
generated upon BcR cross-linking remain unknown. One recently described
mechanism by which ceramide may regulate cellular responses is through
the activation of a ubiquitin-dependent protein degradation
pathway (13, 14).
Cellular protein degradation by the proteasome is a strictly regulated
process in which tagging of proteins with ubiquitin is the committing
step for their degradation by this 26 S multicatalytic protease (15,
16). The proteasome plays a crucial role in cellular homeostasis,
regulating the cellular life span of proteins involved in phenomena
such as cell activation, cell cycle progression, and apoptosis (17,
18). Proteasome activation linked to the degradation of anti-apoptotic
proteins such as Bcl-2 and XIAP has been described as an early event in
various forms of programmed cell death (18-23). In addition, the
disruption of sphingolipid formation through the de novo
pathway of sphingolipid biosynthesis has been shown to prevent
ubiquitin-dependent proteolysis during heat stress in
Saccharomyces cerevisiae (13). Thus, de novo derived sphingolipids may be regulating the life span of specific proteins that are prone to degradation by the 26 S proteasome. Moreover, because many of the proteins involved in cellular homeostasis have been described to be subject to degradation by the proteasome, sphingolipid-mediated regulation of proteasomal activity may provide cells with an attractive means to regulate various aspects of cellular
homeostasis including cell activation, cell cycle progression, and apoptosis.
In this study, we demonstrate that ceramide formation in response to
BcR cross-linking is a highly ordered process in that specific
long-chain ceramide species are generated early and independent from
effector caspase activation, whereas very long-chain ceramide species
are generated relatively late and downstream from effector caspase
activation. In addition, it is shown that the early formation of
long-chain ceramide species is linked to proteasomal degradation of
XIAP and subsequent activation of effector caspases, which demonstrates
an important cell-biological mechanism through which long-chain
ceramide species may be involved in the activation of effector caspases
and subsequent formation of very long-chain ceramide species.
Cell Culture
The Epstein-Barr virus-negative Burkitt's lymphoma Ramos
cells were purchased from ATCC (Manassas, VA) and grown in RPMI
1640 medium supplemented with 25 mM Hepes, 10% fetal calf
serum, 2 mM L-glutamine (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 100 units/ml penicillin
(Invitrogen), and 100 g/ml streptomycin (Invitrogen) 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). Anti-XIAP was purchased from
R&D Systems Inc. (Minneapolis, MN); anti-caspase-3 was purchased from
BD Biosciences; and anti-actin was purchased from ICN Biomedicals
(Zoetermeer, The Netherlands). ISP-1 and ribonuclease A were purchased
from Sigma. Suc-Leu-Leu-Val-Tyr-7-AMC was obtained from Bachem AG
(Bubendorf, Switzerland). Propidium iodide and DiOC6
were purchased from Molecular Probes (Eugene, OR).
[ Ceramide Measurements
Diacylglycerol Kinase (DGK) Assay--
Cell pellets (2.0 × 106) were lysed in chloroform:methanol (1:2), and lipids
were extracted by the method of Bligh and Dyer (51). Aliquots
were dried down and used for ceramide and phosphate measurements.
Ceramide levels were measured using the Escherichia coli
diacylglycerol kinase assay (24, 25). Lipids were incubated at room
temperature for 30 min in the presence of
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 coupled to atmospheric pressure chemical
ionization (APCI) as described previously (5). Separations were
conducted using Iatrobead (Iatron Laboratories, Tokyo, Japan) beaded
silica columns utilized in a normal phase of operation. Elutions were
completed on a Agilent (Palo Alto, CA) model 1100 high performance
liquid chromatography system equipped with a binary pumping system of
iso-octane and ethyl acetate. All MS analyses were conducted on a
Finnigan (Foster City, CA) LCQ ion trap mass spectrometer. For
APCI-MS, the entire flow from the high performance liquid
chromatography column was directed to the APCI source. For all of the
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 MS fragmentation pattern and column retention. The analysis was then standardized by
programming the LC-Quant software followed by manual
confirmation. Quantitative analysis was
made possible by the use of C17-ceramide as an internal
standard with comparison to external calibration standards. The full
details of this method have been submitted.2
Mitochondrial Transmembrane Potential ( To evaluate changes in DNA Fragmentation Analysis
DNA fragmentation was essentially performed as described
previously (26). 5 × 106 cells were labeled with 1 µCi/ml [3H]thymidine at 37 °C, 5% CO2
overnight for 18 h. Cells were then washed three times with
culture medium to remove excess free label. 1 × 104
cells were plated in a round bottom 96-well plate (Corning Life Sciences, Schiphol-Rijk, The Netherlands) in 100 µl of culture medium
and treated as described (26) at 37 °C, 5% CO2
for 24 h. Samples were then harvested onto fiberglass filters
retaining non-fragmented DNA. Radioactivity was measured by standard
liquid scintillation counting.
Gel Electrophoresis
1 × 106 cells were treated as described and
lysed in 90 µl of lysis buffer containing 0.5% N-lauroyl
sarcosine (Sigma), 0.5 mg/ml RNase (Sigma), 1 mg/ml proteinase K
(Invitrogen) in 50 mM Tris-HCl, pH 8, and incubated at
50 °C for 2 h. Samples were then sheared by 10 strokes through
a 25-gauge needle, and the concentration of DNA per sample was
determined by UV at
A260/A280 nm to allow equal sample loading. DNA laddering was visualized by UV after separation at 100 V on a 1.5% agarose gel.
Cloning and Expression of XIAP Mutants in Ramos
Wild type XIAP (XIAPwt) and RING-less XIAP
(XIAP 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
aprotein, 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 by SDS-polyacrylamide gel electrophoresis. Proteins were
transferred to nitrocellulose membranes (Bio-Rad), which were
subsequently blocked by 5% nonfat dry milk in phosphate-buffered
saline-Tween 20 (0.1%, v/v). Blots were incubated with anti-PARP
(1:1000 dilution, 1 h), anti-XIAP (1:200 dilution, 4 h),
anti-active caspase-3 (1:1000 dilution, 1 h), or anti-actin
(1:4000 dilution, 2 h) in 5% nonfat dry milk. After subsequent
incubation with a 1:2000 dilution of horseradish peroxidase anti-rabbit
IgG (for PARP, caspase-3, and XIAP) or 1:2500 dilution of horseradish
peroxidase anti-mouse IgG (for actin) for 1 h, proteins were
visualized by ECL.
Analysis of Proteasome Activity
1.5 × 106 cells were treated in a
volume of 3 ml as noted. After treatment, cells were washed once
in phosphate-buffered saline, and 1 × 106 cells were
suspended in 200 µl of phosphate-buffered saline in a white flat
bottom 96-well plate (Corning Life Sciences). Directly thereafter, 20 µl of Suc-LLVY-7AMC (0.1 mM) was added and the plate was
placed at 37 °C until measurement. Substrate conversion was measured
with a dual-scanning spectrofluorometer (Spectramax Gemini XS,
Molecular Devices Corp., Sunnyvale, CA) using an excitation wavelength
of 355 nm, measuring fluorescence at an emission wavelength of 440 with
a cutoff set at 435 nm.
BcR Cross-linking Induces the Formation of Ceramide through the de
Novo Pathway of Sphingolipid Biosynthesis--
BcR cross-linking of
Ramos B-cells is associated with the formation of ceramide. This was
shown using both the DGK method for quantitation of ceramide (Fig.
1, A and B) and
using normal phase high performance liquid chromatography coupled to
atmospheric pressure chemical ionization (tandem mass spectrometry)
(Fig. 1, C and D). In Ramos cells, the
C16- and C24-ceramide species are the
predominant species (5), and the changes in their levels in response to
BcR cross-linking therefore are shown specifically in Fig. 1,
C and D. An analysis of ceramide by DGK yields
two ceramide spots upon separation by TLC. The upper spot corresponds to very long-chain ceramide species
(C22-C26-ceramide + dihydroceramides), whereas
the lower spot corresponds to long-chain ceramide species (C14-C20) (5). The early increase at 6 h
after BcR cross-linking of C16-ceramide is seen both by DGK
analysis as an increase in the lower ceramide-1-P spot upon separation
by TLC (Fig. 1B) and by tandem MS analysis (Fig.
1D). The increase in very long-chain ceramide species
becomes apparent after 12-24 h of BcR cross-linking as an increase in
the upper ceramide-1-P spot upon separation by TLC (Fig. 1A)
and as C24-ceramide by tandem MS analysis (Fig. 1C). We have previously shown that the increase in
ceramide after BcR cross-linking results from activation of the
de novo pathway of sphingolipid biosynthesis (5). Indeed,
ISP-1, a potent inhibitor of serine palmitoyltransferase, which
catalyzes the first and rate-limiting step in the de novo
pathway of sphingolipid biosynthesis, blocked ceramide formation.
BcR Cross-linking Activates the 26 S Proteasome via de Novo Derived
Sphingolipids--
Recently, it was shown that sphingolipids derived
via the de novo pathway of sphingolipid biosynthesis are
required to induce ubiquitin-dependent proteolysis in
S. cerevisiae upon heat stress (13). In light of these
results, we examined the effects of BcR cross-linking and ceramide
formation in Ramos cells on proteasome activation. The treatment of
Ramos cells with anti-IgM resulted in significant up-regulation of
proteasomal activity as shown by enhanced substrate processing 6 h
after start of the treatment (Fig. 2).
Anti-IgM-induced activation of the proteasome could be detected up to
24 h after BcR cross-linking. The induced activity of the
proteasome could be blocked using clasto-lactacystin
As shown in Fig. 1, co-incubation with ISP-1 inhibits the early
accumulation of ceramide upon BcR cross-linking. Moreover, as shown in
Fig. 2B, ISP-1 was found to inhibit BcR
cross-linking-induced proteasome activation. Thus, the formation of
sphingolipids via the de novo pathway of sphingolipid
biosynthesis is a required step in the activation of the proteasome
upon BcR cross-linking.
XIAP Is Degraded as a Result of Sphingolipid-dependent
Proteasome Activation--
To further delineate the significance of
the observed BcR-induced activation of the proteasome, we investigated
the fate of XIAP, an anti-apoptotic protein known to be degraded in a
ubiquitin proteasome-dependent manner. As shown in Fig.
3, BcR cross-linking caused a reduction
in the levels of XIAP. This was first seen 6 h after BcR
cross-linking and more pronounced in cells treated for 24 h (Fig.
3A). CLC shown to specifically inhibit proteasome activation
(see also Fig. 2) prevented BcR cross-linking-induced XIAP degradation
(Fig. 3A), thus supporting a role for the proteosome in
degradation of XIAP.
Ramos cells were treated next with anti-IgM in the presence of ISP-1
(Fig. 3B) or the caspase inhibitors, zVAD and IETD,
(Fig. 3, C and D) to investigate the effect of
inhibition of sphingolipid formation and effector caspases on XIAP
stability and apoptosis. As shown in Fig. 3B, ISP-1
dose-dependently prevented BcR-induced XIAP degradation.
These results are in line with the observation that ISP-1 prevented BcR
cross-linking-induced activation of the proteasome (Fig.
2B). Thus, de novo generated sphingolipids
mediate the effects of BcR cross-linking on degradation of XIAP. In
addition and in agreement with previous studies (28), XIAP degradation could not be prevented using zVAD, demonstrating that XIAP degradation was independent from effector caspase activation (Fig. 3C).
Furthermore, ISP-1 prevented BcR-induced PARP cleavage when used in
combination with the initiation caspase inhibitor IETD (Fig.
3D), which has been shown previously not to be involved
directly in BcR-induced mitochondrial damage (Fig. 3D) (4,
29).
Overexpression of a Degradation-resistant XIAP Confers Resistance
to Specific Aspects of BcR-induced Apoptosis--
The role of XIAP in
BcR cross-linking-induced apoptosis and its relation to
sphingolipid-mediated degradation through the proteasome was further
investigated. To this end, we constructed a XIAP lacking the RING
domain, XIAP
XIAPwt- or XIAP
The significance of XIAP as an anti-apoptotic protein in B-cell
homeostasis was investigated next. To this end, Ramos,
Ramos-XIAPwt, and Ramos-XIAP
Because XIAP-mediated protection against BcR cross-linking-induced
apoptosis occurs at the level of caspases, we also determined mitochondrial damage in response to BcR cross-linking, which is proximal to caspase activation in BcR cross-linking-induced apoptosis. As shown in Fig. 5C, the overexpression of neither
XIAPwt nor XIAP Formation of C16- and C24-Ceramide Species
Is Differentially Regulated Upstream and Downstream of Effector
Caspases--
We previously described that early following BcR
cross-linking, before signs of apoptosis associated with
caspase activation are observed, long-chain ceramides with
C16-ceramide being the predominant species are formed. At
later time points (i.e. 24 h after the start of the
treatment), very long-chain ceramides with C24-ceramide
being the predominant species are formed (see also Fig. 1).
In light of the distinct mitochondrial and
caspase-dependent phases of apoptosis that had become
apparent using overexpression of XIAP, we next investigated the effects
of caspase inhibition by XIAP on the formation of ceramide after BcR
cross-linking. To this end, we used the DGK assay in which long chain
ceramide species (C14-C18) migrate in the
lower ceramide-1-P spot, whereas very long-chain ceramide species
(C20-C26) migrate in the upper ceramide-1-P
spot upon separation by TLC (5). Because C16 and C24 are by far the predominant ceramide species present in
Ramos cells (5), separation and quantification of DGK-phosphorylated ceramide species by TLC in the upper and lower spot provide an accurate
measurement of specific changes in C16- and
C24-ceramide after treatment. As shown in Fig.
6, caspase inhibition using XIAPwt, XIAP We previously described the involvement of de novo
derived ceramide in BcR-induced apoptosis (5). This study was initiated to investigate the mechanisms by which specific ceramide species are
generated and how these ceramide species may be involved in BcR-induced
apoptosis in Ramos cells. After 6 h of stimulation prior to the
detection of classical apoptosis-related phenomena such as
PARP-cleavage and DNA fragmentation, BcR cross-linking is characterized
by a rise in long-chain ceramide species in which C16-ceramide predominates. In contrast, very long-chain
ceramide species in which C24-ceramide predominates are
formed after prolonged BcR stimulation and become detectable after
24 h of stimulation. Predominant accumulation of
C16-ceramide during apoptosis has been described previously
(33). To date, no reports are available on the regulation of this
differentiated ceramide formation. In addition, it remains unclear
exactly how ceramide may be involved in the progression of BcR-induced apoptosis.
Recent reports have linked sphingolipid synthesis with the activation
of the ubiquitin proteasome pathway (13, 14). Inactivation of the
ubiquitin-proteasomal pathway was shown in a mutant strain of
S. cerevisiae, lcb1-100, a temperature-sensitive
mutant of serine palmitoyltransferase that catalyzes the first and
rate-limiting step in the de novo formation of ceramide
(13). In addition, the activation of the proteasome has been described
as an integral part of the initiation phase of apoptosis (19, 22, 23). Likewise, we found increased proteasomal activity early after BcR
cross-linking. In addition, blocking ceramide formation with ISP-1, a
potent inhibitor of serine palmitoyltransferase (34), inhibited the
activation of the proteasome, linking the formation of ceramide through
the de novo pathway of sphingolipid biosynthesis with the
increased proteasomal activity. This is in line with studies showing
sphingolipid-dependent modulation of the proteasome complex
via phosphorylation of the C8 and C9 subunits of the proteasome complex
(35) and activation of transcription of genes encoding subunits of the
proteasome (36, 37). Proteasome activation does not seem to be a direct
consequence of mitochondrial damage because anti-CD20-induced
apoptosis, which involves mitochondrial damage without formation of
ceramide,3 does not involve
proteasome activation. Activation of the ubiquitin proteasome pathway
by sphingolipids is not likely to be via direct interaction because
direct modulation of proteasomal activity using synthetic short-chain
and long-chain ceramides in vitro was not observed (data not
shown). Proteasome activation in relation to apoptosis has been well
documented (18). In addition, pharmacological blockers of proteasome
function may induce apoptosis too, showing the importance of this
protease complex in maintaining cellular homeostasis (38, 39). In
general, it may be postulated that the ubiquitin proteasome pathway for
protein degradation is especially well suited to regulate cellular
levels of short-lived proteins and to control unidirectional cellular
processes such as the execution of a cell death program. The latter
typically involves degradation of protective proteins such as that
recently described for the anti-apoptotic protein XIAP (19, 28).
Indeed, we observed significant degradation of XIAP in Ramos cells upon
BcR cross-linking. In agreement with previously described studies, XIAP
degradation was completely blocked using CLC, an irreversible and
specific inhibitor of the proteasome (19, 40). Similar results were
obtained using a recently described (41) potent and selective
proteasome inhibitor, AdaAhxL3VS (data not shown). Moreover, the
pan-caspase inhibitor zVAD could not prevent BcR-induced XIAP
degradation, which is in agreement with the results described by others
(19, 28), and shows that BcR cross-linking-induced XIAP degradation is
caspase-independent. In addition, BcR-induced proteasome activation
appears to occur upstream of caspase activation. Indeed, BcR-induced
proteasome activation was not blocked by zVAD (data not shown). The
anti-apoptotic properties of XIAP reside in the BIR domains, which
interact with the substrate recognition sequences of caspases-3, -7, and -9, and mask these for their cellular targets. XIAP degradation by
the proteasome has been shown to involve the ubiquitin-ligase activity
of the C-terminal RING finger domain (19, 31, 42). Mutation analysis
has shown that the removal of this domain results in a protein that is
as competent as its full-length counterpart in inhibiting apoptosis, whereas the protein itself becomes resistant to proteasomal
degradation. Therefore, we overexpressed a RING-less XIAP construct in
Ramos cells first to verify its stability upon BcR cross-linking and second to investigate downstream effects of stable
degradation-resistant XIAP overexpression during BcR cross-linking.
We show that the RING-less XIAP, XIAP Overexpression of wild type XIAP and, even more so, overexpression of
the degradation-resistant XIAP Parallel with the differentiated effects of XIAP overexpression on the
protection of mitochondrial damage, we found that overexpression of
XIAP had profound effects on the generation of specific ceramide species after BcR cross-linking. To date, no reports exist on the
differentiated regulation of specific ceramide species during apoptosis. Here we provide evidence for such a differentiated regulation upstream and downstream of effector caspases. XIAP overexpression did not abrogate the early formation of ceramide retained in the lower ceramide-1-P TLC spot, which consists of long-chain ceramide species. In contrast, XIAP overexpression completely blocked the formation of ceramide species retained in the
upper ceramide-1-P TLC spot, which is normally increased at later time
points (i.e. 24 h) after the start of BcR
cross-linking. Ceramide species that migrate in this upper spot
identified as very long-chain ceramide species (5) apparently are
formed not only later in the apoptotic process but also as a result of caspase activation. Interestingly, the differentially regulated formation of long- and very long-chain ceramide species coincides with
the effects of XIAP seen on mitochondrial damage and therefore appears
to be linked. The source of specific ceramide species generated 24 h after BcR cross-linking remains to be investigated. Whereas both
ISP-1 and fumonisin B1 (FB1) abrogate the formation of these
ceramide species at 24 h, suggesting the de novo
pathway of sphingolipid biosynthesis to be the main source, we cannot rule out activation of SMase enzymes or other more complex
conversion reactions leading to the observed increase of ceramide
24 h after start of the treatment. SM levels do not drop
significantly upon BcR stimulation (5), making the involvement of any
SMase-driven form of ceramide formation unlikely. Other pools of
sphingolipids that may serve as a source for ceramide include
sphingosine, glycosphingolipids, and ceramide-1-phosphate. Future
experiments need to clarify how effector caspase activation may lead to
the activation of enzymes involved in the generation of ceramide from
any of these sources.
As summarized in Fig. 7, our results
suggest the involvement of the early formation of long-chain ceramide
species in the initial mitochondrial phase of apoptosis. In contrast,
the very long-chain ceramide species appear to result from activated
caspases and might play a role in the downstream effector phase of
apoptosis. In this respect, it may be interesting to note that in the
presence of ISP-1, ceramide levels initially dropped to below control
values. However, at the later time points after BcR cross-linking,
ISP-1 lost its ability to completely block the formation of ceramide, especially the very long-chain ceramide. This finding suggests that the
total ceramide response after BcR cross-linking may be regulated in the
early mitochondrial phase through the de novo pathway,
although the secondarily formed very long-chain ceramide species may be
differently regulated, e.g. through caspase-activated SMases
or turnover of other complex sphingolipids. A combined response
involving both SMase activity and de novo derived ceramide in MCF7 cells upon tumor necrosis factor--lactone, an irreversible inhibitor of the proteasome, prevented BcR
cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the
ubiquitin-ligating ring finger motif was completely resistant to
proteasome-mediated degradation, and Ramos cells overexpressing XIAP
became highly resistant to BcR cross-linking-induced activation of
caspases. The formation of C16-ceramide in response to BcR
cross-linking was found unaltered in XIAP overexpressing Ramos cells,
whereas C24-ceramide formation was completely abolished.
These results demonstrate how de novo generated long-chain
ceramide species may be involved in the activation of downstream
effector caspases and subsequent formation of very long-chain ceramide
species. As such, these results provide novel and important insights
into the significance of specific ceramide species in defined stages of apoptosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)-hydroxylation of the
N-linked fatty acids. In addition, specific ceramide
species may reside preferentially at topologically distinct
cellular locations, which may relate to the intracellular location of
specific enzymes involved in the metabolism of ceramide (10, 12).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was obtained from PerkinElmer Life Sciences
products. zVAD-fmk (Z-Val-Ala-DL-Asp-fluoromethylketone) was from
Calbiochem.
-octylglucoside/dioleoylphosphatidylglycerol micelles, 2 mM dithiothreitol, 5 µg of proteins of diacylglycerol kinase membranes, and 2 mM ATP (mixed with
[
-32-P]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.
)
, cells (5 × 105/ml) were treated as as noted earlier. After
washing and resuspension in medium without fetal bovine serum, cells
were labeled with DiOC6 (0.1 µM) for 25 min at 37 °C.
After incubation, cells were washed and analyzed directly on a flow
cytometer using excitation and emission wavelengths of 495 and 525 nm, respectively.
R) were cloned from Ramos cells by reverse
transcriptase-PCR using the following primers (Biolegio, Malden, The
Netherlands). Forward primer for both XIAPwt and
XIAP
R was 5'-ATCTCGAGATGACTTTTAACAGTTTTGAA-3'. Reverse
primer XIAPwt was 5'-ATGCGGCCGCACATGCCTACTATAGAGTTAG-3'. Reverse primer for XIAP
R was
5'-ATGCGGCCGCACACTCCTCAAGTGAATGAGT-3'. PCR products were
subcloned in the pCR4Blunt-TOPO vector (Invitrogen) and then subcloned
into the retroviral LZRS-pBMN-IRES-EGFP (kindly provided by H. Spits, NKI, The Netherlands), which was constructed from the
LZRS-LacZ(A) originally described by Kinsella and Nolan (27) by
replacing the LacZ by the IRES-EGFP sequence, allowing an easy
selection of transduced cells. LZRS-pBMN-IRES-EGFP-XIAPwt and LZRS-pBMN-IRES-EGFP-XIAP
R plasmid DNA were used to
transduce the retroviral packaging cell line FNX. Ramos cells
were transduced by co-culture of Ramos cells with
FNX-XIAPwt or FNX-XIAP
R, after which
transduced Ramos cells were selected flow cytometrically by gating for
EGFP using excitation and emission wavelengths of 495 and 525 nm,
respectively. Cellular expression of XIAPwt or XIAP
R was confirmed by Western blotting.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BcR cross-linking-induced long-chain and
short-chain ceramides are generated with different kinetics.
Ramos cells (5 × 105/ml) were treated with
anti-IgM (10 µg/ml) in the presence or absence of ISP-1 in 5%
CO2 at 37 °C. After the indicated time periods, cells
were prepared for measurement of cellular ceramide by DGK analysis
(A, upper ceramide-phosphate spot; B, lower
ceramide-phosphate spot) or tandem mass spectrometry (C,
C24-ceramide; D, C16-ceramide) as
described under "Materials and Methods." Ceramide mass normalized
to lipid phosphate is plotted. Data (mean ± S.E.) are from one of
three experiments performed in duplicate. Significant increase compared
with control (p < 0.05, Student's t test)
is noted by asterisks.
-lactone (CLC), a specific inhibitor of the proteasome, affirming
the specificity of the observed substrate processing by the proteasome
(Fig. 2A).
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Fig. 2.
BcR cross-linking is associated with
sphingolipid-dependent activation of the proteasome.
Ramos cells (5 × 105/ml) were treated with
anti-IgM (10 µg/ml) for 6 or 24 h in the presence or absence of
the proteasome inhibitor CLC (5 µM) (A) or
ISP-1 (10 nM) (B) in 5% CO2 at
37 °C. Proteasome activity was analyzed in live cells (1 × 106) 2 h after addition of the proteasome substrate
Suc-LLVY-7-AMC (0.1 mM) as described under "Materials and
Methods." Inset, kinetics of proteasomal substrate
conversion after the addition (T = 0 h) of substrate following
treatment for 6 h with anti-IgM. Data (mean ± S.E.) are from
one of three experiments performed in duplicate.
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Fig. 3.
BcR-induced proteasome activation is
associated with the degradation of XIAP. Ramos cells
(5 × 105/ml) were treated with anti-IgM (10 µg/ml)
for the indicated time (A) or 24 h (B-D) in
5% CO2 at 37 °C. Cells were co-incubated with or
without CLC (5 µM), ISP-1 (10 nM) as
indicated, zVAD (50 µM), or IETD (20 µM) as
shown. Cells were prepared for analysis of XIAP degradation by Western
blotting as described under "Materials and Methods." Equal
protein loading (10 µg/lane) was verified using actin as a reference
protein. Representative results of four independent experiments are
shown.
R, by reverse transcriptase-PCR from Ramos
cells as described by Yang et al. (19) (Fig.
4A). The C-terminal RING
domain is a required element of XIAP for ubiquitination and
proteasome-mediated degradation. In contrast, the anti-apoptotic properties of XIAP reside in the three BIR domains with the
BIR2 domain being sufficient to inhibit caspases-3 and -7 and the BIR3 domain being sufficient to block caspase-9 activation (30, 31).
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Fig. 4.
Expression and stability of mutant XIAP in
Ramos. Ramos cells were transduced with XIAPwt and
XIAP R by retroviral infection as described under
"Materials and Methods." A, schematic of
XIAPwt and XIAP
R. N and C termini as well as
the location of the anti-apoptotic BIR and the RING domains are shown.
B, Ramos cells (5 × 105/ml) were treated
with anti-IgM (10 µg/ml) for 24 h in 5% CO2 at
37 °C. Cells were prepared for analysis of XIAP degradation by
Western blotting as described under "Materials and Methods." The
anti-XIAP antibody recognizes an epitope on the linker between BIR2 and
BIR3 (amino acids 244-263) present in both XIAPwt and
XIAP
R. Representative results of five independent
experiments are shown.
R-expressing sublines of
Ramos were established as described under "Materials and Methods."
Western blot analysis of Ramos-XIAP
R and Ramos
XIAPwt revealed overexpression of the wild type 57-kDa XIAP
in Ramos-XIAPwt cells, whereas Ramos-XIAP
R showed overexpression of a 40-kDa protein, corresponding to the estimated molecular mass of the RING-less XIAP protein (Fig.
4B). In addition, whereas both endogenous XIAP and the
XIAPwt were subject to BcR cross-linking-induced
degradation, XIAP
R was found to be totally resistant
toward degradation after BcR cross-linking. Because the RING domain has
been described to mediate in the ubiquitination of XIAP, thus tagging
it for degradation by the proteasome, these results confirm the
involvement of the ubiquitin proteasome pathway in XIAP degradation
upon BcR cross-linking.
R cells were
treated with anti-IgM and various characteristic aspects of apoptosis
were measured. As shown in Fig.
5A, BcR cross-linking-induced
PARP-cleavage was significantly reduced in Ramos-XIAPwt
cells and even more so in Ramos-XIAP
R cells. Also, in
Fig. 5A, it is shown that the activation of caspase-3 was
significantly reduced in Ramos-XIAPwt cells and virtually undetectable in Ramos-XIAP
R cells. In addition to
cleavage of PARP, caspase-3 directly activates endonucleases resulting
in the fragmentation of nuclear DNA. The JAM assay provides a
convenient way to quantify fragmentation of cellular DNA during
apoptosis. Quantification of DNA fragmentation by this method has been
shown to correlate well with the percentage of apoptotic cells (26) and
various other markers of apoptosis (32). As shown in Fig. 5B, both XIAPwt and XIAP
R
conferred resistance toward BcR cross-linking-induced DNA fragmentation
with the degradation-resistant XIAP
R being slightly more
effective compared with XIAPwt. Similar clear results were
obtained by direct visualization of DNA laddering using agarose gel
electrophoresis (data not shown). Taken together, these results demonstrate that indeed 1) XIAP is actively involved in protecting against BcR cross-linking-induced apoptosis and 2) degradation of XIAP
is required for the activation of the caspase-mediated apoptotic
program in response to BcR cross-linking.
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Fig. 5.
XIAP overexpression protects against BcR
cross-linking-induced caspase-dependent apoptotic
events. Ramos, Ramos-XIAPwt, and
Ramos-XIAP R cells (5 × 105/ml) were
treated with anti-IgM (10 µg/ml) or the indicated concentration of
anti-IgM (B) for 24 h or the indicated time period
(C-E) in 5% CO2 at 37 °C. A,
cells were prepared for analysis of PARP cleavage or presence of
activated caspase-3 as described under "Materials and Methods."
Equal protein loading (10 µg/lane) was verified using actin as a
reference protein. Equal presence of total caspase-3 in all cell lines
was verified using polyclonal antibodies directed against amino acids
1-277, representing the full-length precursor form of caspase-3.
B, [3H]thymidine prelabeled cells were used
and analyzed for the induction of DNA fragmentation after treatment
with the indicated concentrations of anti-IgM using the JAM assay as
described under "Materials and Methods." C-E, after 6 and 24 h of treatment, cells were prepared for flow cytometrical
analysis of mitochondrial damage by staining with DiOC6 (0.1 µM) as described under "Materials and Methods." All
results shown are representative of at least three independent
experiments.
R protected against early
mitochondrial damage. After the first 6 h of treatment, an
approximately 2-fold induction of mitochondrial damage was observed in
Ramos, Ramos-XIAPwt, and Ramos-XIAP
R cells (Fig. 5, C and E). In contrast, 24 h after
treatment, mitochondrial damage in Ramos cells further increased
8-fold, whereas XIAPwt and XIAP
R
significantly protected against further mitochondrial damage. This is
consistent with a caspase-mediated feedback loop, enhancing
mitochondrial damage and the effector-phase of BcR
cross-linking-induced apoptosis.
R, or the pan-caspase inhibitor
zVAD had no effect on the early BcR cross-linking-induced formation of
long-chain ceramides (compare Fig. 6B in the absence of zVAD
with Fig. 6D in the presence of zVAD). In contrast,
the formation of very long-chain ceramide species in response to BcR
cross-linking was completely blocked by XIAP overexpression or
treatment with zVAD (compare Fig. 6A in the absence of zVAD
with Fig. 6C in the presence of zVAD). Thus, although
long-chain ceramide species, specifically C16-ceramide, are
generated upstream of caspase activation, the formation of very
long-chain ceramide species, specifically C24-ceramide, seem to be dependent on the activation of effector caspases.
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Fig. 6.
Short-chain ceramides but not long-chain
ceramides are generated upstream of BcR-induced caspase
activation. Ramos and Ramos-XIAP R cells (5 × 105/ml) were treated with anti-IgM (10 µg/ml) without
(A and B) or with (C and D)
zVAD (50 µM) for 24 h in 5% CO2 at
37 °C. Cells were prepared for analysis of cellular ceramide by DGK
analysis as described under "Materials and Methods." Ceramide mass
normalized to lipid phosphate retained in the upper ceramide-phosphate
spot (A and C) and lower ceramide-phosphate spot
(B and D) after separation by TLC is plotted. The
mean (± S.E.) from three independently performed experiments is
shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
R, remains stably
expressed after BcR cross-linking. Incubation over prolonged time periods with anti-IgM did not result in a noticeable diminution of
XIAP
R. In contrast, endogenous XIAP in these same cells
as well as overexpressed wild type XIAP remained subject to
degradation, which places the ubiquitin proteasome pathway as an
important regulator upstream of caspase activation in BcR-induced apoptosis.
R profoundly affected the
execution of BcR cross-linking-induced apoptosis. Besides ceramide
formation, BcR-induced apoptosis involves mitochondrial dysfunction and
caspase activation followed by cleavage of caspase-sensitive target
proteins, such as PARP, and ultimately DNA fragmentation. Caspase-3
activation as well as PARP cleavage and DNA fragmentation were
significantly reduced in Ramos-XIAPwt cells and virtually undetectable in Ramos-XIAP
R cells. This confirms the
retention of the anti-apoptotic properties of XIAP
R and
signifies the roles of effector caspases such as caspases-3, -7, and -9 in the execution phase of BcR-induced apoptosis. The effects of XIAP
overexpression on the loss of BcR cross-linking-induced mitochondrial
function displayed a more differentiated pattern. XIAP did not offer
protection against early mitochondrial damage measured after treatment
with anti-IgM for 6 h. However, XIAP significantly protected
against BcR cross-linking-induced mitochondrial damage measured at
later time points after the start of the treatment. This finding is consistent with the proposed apoptotic feedback loop, inflicting effector caspase-dependent mitochondrial damage (43).
Together with previous results showing that zVAD did not protect
against initial mitochondrial damage (5), these results further
underline an initiating role for mitochondria in BcR
cross-linking-induced apoptosis.
treatment has recently been described (44) and may thus represent a common mode by which
cellular ceramide may accumulate and exert specific effects at
distinct intracellular locations.
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Fig. 7.
Schematic representation of the sequence of
events following BcR triggering in Ramos cells. Anti-IgM-mediated
BcR triggering induces the formation of long-chain ceramide species
(preferentially C16-ceramide) prior to the formation of
very long-chain ceramide species (preferentially
C24-ceramide). Whereas the formation of
C16-ceramide occurs relatively early (at 6 h) and
independently from caspase activation, C24-ceramide occurs
at a late stage of apoptosis (at 24 h) and is dependent on the
prior activation of effector caspases. Effector caspase activation
involves sphingolipid-dependent down-regulation of XIAP via
the ubiquitin proteasomal-dependent protein degradation
pathway. Dotted lines represent possible routes of
interaction of the very long-chain ceramide species.
This complexity of ceramide formation may explain many of the confusing results in the literature that attempt to place (total) ceramides at various points upstream or downstream in the apoptotic program. Our results clearly demonstrate that different ceramide species are formed with distinct kinetics by different mechanisms and participate in distinct mechanisms and phases of the apoptotic program.
The interrelation of sphingolipids and mitochondrial function has been
the subject of recent interest. Studies have identified mitochondrial
presence of enzymes and metabolites thereof involved in a mitochondrial
network of sphingolipid metabolism (45-47). In addition, mitochondrial
targeting of bacterial SMase was the only intracellular location where
transfection-induced ceramide formation positively correlated with the
induction of apoptosis (47, 48). Furthermore, BcR cross-linking has
been shown to up-regulate the enzyme CPT-I, which catalyzes import of
fatty acids used for -oxidation into mitochondria (49). It has been shown that the import of palmitoyl CoA, which is a prerequisite precursor for C16-ceramide, into mitochondria by CPT-I
correlated with apoptosis (49, 50). These studies suggest an interplay between mitochondrial ceramide and other cellular components, such
as the ubiquitin proteasome pathway, in the regulation of apoptosis. In
addition, they establish a strong basis for future studies to further
dissect molecular mechanism underlying the generation of specific
ceramide species during apoptosis and their role in the progression of apoptosis.
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FOOTNOTES |
---|
* 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. Pathology and Laboratory Medicine/Medical Biology Branch, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Tel.: 31-50-3614291; Fax: 31-50-3619911; E-mail: b.j.kroesen@thorax.azg.nl.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M210756200
2 B. Pettus, M. Busman, P. Moeller, B. Kroesen, Z, Szulc, A. Bielawska, and Y. Hannun, manuscript submitted.
3 B.-J. Kroesen, S. Jacobs, B. J. Pettus, H. Sietsma, J. W. Kok, Y. A. Hannun, and L. F. M. H. de Leij, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
BcR, B-cell
receptor;
AICD, activation-induced cell death;
DGK, diacylglycerol
kinase;
zVAD-fmk, Z-Val-Ala-DL-Asp-fluoromethylketone;
IETD, 2-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F;
APCI, atmospheric
pressure chemical ionization;
MS, mass spectrometry;
XIAPwt, wild type XIAP;
XIAPR, RING-less
XIAP;
EGFP, enhanced green fluorescent protein;
CLC, clasto-lactacystin
-lactone;
XIAP, X-linked inhibitor of
apoptosis;
PARP, poly (ADP-ribose)polymerase;
IRES, internal ribosome
entry site;
BIR, Baculovirus IAP repeat;
sn, sphingomyelin.
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