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J. Biol. Chem., Vol. 276, Issue 23, 20589-20596, June 8, 2001
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From the Department of Immunology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105, the § Kekulé
Institute for Organic Chemistry and Biochemistry, University of Bonn,
Gerhard-Domagk-Str.1, 53121 Bonn, the
Received for publication, February 7, 2001, and in revised form, March 8, 2001
Clustering seems to be employed by many receptors
for transmembrane signaling. Here, we show that acid sphingomyelinase
(ASM)-released ceramide is essential for clustering of CD95.
In vitro and in vivo, extracellularly
orientated ceramide, released upon CD95-triggered translocation of ASM
to the plasma membrane outer surface, enabled clustering of CD95 in
sphingolipid-rich membrane rafts and apoptosis induction. Whereas
ASM deficiency, destruction of rafts, or neutralization of surface
ceramide prevented CD95 clustering and apoptosis, natural ceramide only
rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.
Stimulation of a variety of surface receptors including the T-cell
receptor/CD3 complex (1, 2), B-cell receptor (3), tumor necrosis factor
receptor (TNF-R)1 (4), CD2,
CD44, L-selectin, or integrins (5) results in clustering of these
receptors, which appears to be required for rapid and efficient
receptor-mediated signaling. Recent studies on peptide antigen-induced
signaling via the T-cell receptor/CD3 complex indicated that receptor
aggregation rather than conformational changes of the intracellular
part of the T-cell receptor/CD3 complex upon ligand binding is the
predominant mechanism mediating signal transmission (1). Evidence
suggests that many receptors aggregate in distinct cholesterol- and
sphingolipid-rich membrane microdomains or rafts (6-8). This notion is
supported by the finding that disruption of rafts prevents clustering
of many receptors including the B-cell receptor (9), CD48 (10), Fc In the present studies, we have investigated whether a hydrolysis of
sphingomyelin to ceramide and, thus, a change in the composition of
rafts contributes to clustering of receptor molecules. Ceramide is
released by the activity of at least three forms of sphingomyelinases
with an acidic, neutral, or basic pH optimum (14). Of these enzymes
only the acid sphingomyelinase (ASM) is rapidly, i.e. within
seconds, activated upon stimulation via e.g. CD95 or TNF and
therefore, could release ceramide within the time frame of receptor
clustering (15-17). Further, ASM is activated by diverse receptors
including CD95 (15, 16), TNF-R (17), CD28 (18), CD5 (19), or ICAM (20)
suggesting a general role of the ASM in receptor signaling. Finally, at
least for the p75NGF- or IL-1 receptor ceramide generation
predominantly localizes to sphingolipid-rich rafts (21, 22).
Here, we suggest a novel mechanism for receptor clustering and show
that ASM translocates from an intracellular compartment to the
extracellular surface of the cell membrane upon stimulation via CD95.
Translocated ASM localizes to sphingolipid-rich rafts and releases
extracellularly orientated ceramide that mediates selective clustering
of CD95 and constitutes an essential prerequisite for signaling.
Cells and Stimulation--
Human ASM- or acid ceramidase
(AC)-deficient lymphocytes or fibroblasts, respectively, were obtained
from patients with Niemann Pick disease type A (NPDA) or Farber
disease. JY and Jurkat were from ATCC. All lymphocytes were grown in
phenol red-free RPMI 1640 supplemented with 10% fetal calf serum, 10 mM HEPES, pH 7.4, 2 mM L-glutamine,
1 mM sodium pyruvate, 100 µM non-essential
amino acids, 100 units/ml penicillin, 100 µg/ml streptomycin (all
Life Technologies, Inc.) and 50 µM
Reconstitution of NPDA or Farber B cells for ASM or AC, respectively,
was by electroporation using ASM and AC constructs subcloned into the
pJK or pEF vectors (pJK-asm, pEF-asm, which
regulate gene expression under control of an elongation factor
promoter). In addition, JY B cells were transfected with wild-type ASM
or ASM fused with tag sequences of VSV and Myc (pJK-vsv-asm,
pEF-myc-asm). Empty vectors (pJK, pEF) were used as
controls. The pJK vector also encodes a single chain antibody fused
with a Myc tag permitting isolation of transfected cells by panning
with anti-Myc-9E10 (Roche Molecular Biochemicals) as described (23).
Expression of ASM constructs was confirmed by FACS, and measurement of
ASM activity revealed a 3-5-fold overexpression.
Stimulation with anti-CD95 CH11 was performed at 30 ng/ml if not
otherwise indicated. CD95 ligand was added to cells at 20 ng/ml for 10 min at 4 °C, cells were washed, and cellular activation was
initiated by addition of 100 ng/ml anti-FLAG F(ab)2
antibodies at 37 °C. Cells were stimulated for 2 min for capping
experiments and 4 h for apoptosis studies, unless otherwise indicated.
Nystatin was used at 10 µg/ml, filipin at 0.5 µg/ml, Microscopy--
For fluorescence microscopy, lymphocytes were
immobilized on glass coverslips coated with 1% (v/v)
poly-L-lysine for 15 min, stimulated or left unstimulated,
fixed for 15 min in 1% paraformaldehyde (PFA) (w/v) in PBS (PFA/PBS),
and washed in 0.05% Tween 20/PBS and 0.1% bovine serum albumin-C/PBS
(w/v, Aurion EM Reagents). Where indicated cells were permeabilized by
a 10-min incubation in 0.1% Triton X-100/PBS. Cells were stained for
45 min each with a polyclonal goat or a monoclonal mouse anti-ASM (23)
followed by incubation with 0.5 µg/ml Texas Red (TR)-conjugated
F(ab)2-fragments of anti-goat or mouse antibodies
(PharMingen), respectively. Cells were washed and stained for 45 min
with 200 ng/ml FITC-labeled anti-CD95 CH11. Control stainings were
performed with irrelevant monoclonal mouse antibodies or pre-bled goat
antiserum. Fc receptors on B-cells were blocked with a 45-min
incubation with 20 µg/ml of an irrelevant rabbit antibody. All other
antibodies did not bind to human Fc receptors. Staining was viewed
using a conventional Zeiss fluorescence microscope or a Leica TCS NT
scanning confocal microscope.
Cells for scanning electron microscopy were immobilized as above on
plastic coverslips, stimulated, fixed in 4% PFA/PBS, blocked with a
15-min incubation with 0.1% BSA-C/PBS, and incubated for 60 min each
with mouse anti-ASM and a Nanogold-coupled goat anti-mouse antibody
(Nanoprobes, NY). Samples were postfixed in 4% PFA/PBS, dehydrated,
critical-point-dried from CO2, sputter-coated with 1 nm Cr
and examined at 10 kV accelerating voltage in a Hitachi S-800 field
emission scanning electron microscope equipped with a detector for
backscattered electrons of the YAG type. Magnification was × 30,000.
FACS Studies--
FACS studies were performed with control and
ASM-retransfected NPDA lymphocytes or fibroblasts, respectively. Intact
cells were stained with 500 ng/ml of the indicated antibody diluted in
PBS, 1% FCS, and 0.1% NaN3.
Video Imaging--
Video imaging was performed on a Zeiss
Axiovert 135 microscope with a 100 × Zeiss Fluar oil immersion
objective connected to an intensified CCD video camera (Proxitronic)
and the appropriate filters to observe FITC fluorescence. The images
were processed and digitized using Axon Imaging Systems software.
Clustering was defined in all assays as one or several intense spots on
the cell surface, whereas on resting cells, fluorescence was
distributed homogenously throughout the membrane. In each experiment,
100-200 cells were scored for clustering by a blinded observer and
confirmed by a second independent observer.
Lipid Studies--
Binding of ceramide to intact cells was
determined with a 2-min incubation with
[14C16]ceramide (1 µCi/ml) dissolved in
0.01% octyl-glucopyranoside. Cells were then extracted with
CHCl3/CH3OH/H20/pyridine
(60:160:6:1) and lipids were separated by silica G60 TLC with
CHCl3/CH3OH/CaCl2 (60:35:8).
Alternatively, the cells were dounced in 2 ml of 25 mM
Tris/HCl, pH 7.4, 150 mM NaCl, and 1% Triton X-100 and 10 µM each aprotinin/leupeptin (A/L), adjusted to 40%
sucrose, overlaid with 4 ml each of 38 and 5% sucrose, centrifuged at
31,000 rpm in a Beckman 70.1 rotor for 18 h, and 15 fractions were
collected to determine radioactivity and ASM activity.
Apoptosis--
Apoptosis of lymphocytes was measured as
previously described (16) by binding of Fluos-labeled annexin V (Roche
Molecular Biochemicals). Apoptosis was confirmed by DNA fragmentation
using 10 µCi/ml [3H]thymidine (8.3 Ci/mmol, PerkinElmer
Life Sciences) labeling of the mammalian cells. To this end, cells were
disrupted by one cycle of freezing and thawing, and unfragmented DNA
was collected by filtration through glass fiber filters (Amersham
Pharmacia Biotech) and counted by liquid scintillation spectrometry.
Finally an aliquot of all samples was trypan-blue stained to
investigate morphological changes typical of apoptosis.
Infections--
Normal or ASM-deficient fibroblasts were
infected at a cell/bacteria ratio of 1:1000 with the well defined
laboratory strain ATCC 27853 or the clinical isolate 762 (24). Cells
were incubated with precross-linked or monomeric anti-CD95 ZB4 (200 ng/ml) during the infection. Cross-linking was achieved by addition of
1 µg/ml anti-mouse Ig. All cells used for the infection experiments
were grown in medium free of antibiotics.
ASM and Ceramide Mediate CD95 Clustering in Sphingolipid-rich
Rafts--
Initially, we investigated whether stimulation via CD95
results in receptor clustering. To mimick the physiological interaction of membrane-bound CD95 ligand with CD95, Jurkat T or JY B lymphocytes were co-incubated with CD95 ligand-positive LN229 glioma cells. Stimulation via CD95 rapidly triggered CD95 clustering in the lymphocytes (Fig. 1A).
Neutralization of tumor-associated CD95 ligand with a soluble
recombinant Fc-CD95 protein completely abrogated clustering providing
evidence for specificity of the receptor-ligand interaction. CD95
clustering was quantitatively comparable with that after stimulation
with 30 ng/ml of anti-CD95 CH11 or 20 ng/ml cross-linked recombinant
CD95 ligand (Fig. 1A). These doses were used in all
subsequent experiments. Fig. 1B shows that clustering occurred within seconds after stimulation with anti-CD95 CH11 (Fig.
1B). Further, clustering was preceded by the primary
formation of several small CD95 patches, which appeared to fuse to a
large cluster/cap.
To gain insight into the molecular mechanism of CD95 clustering, we
investigated whether ASM and ceramide might be involved. Whereas
stimulation of ASM-retransfected lymphocytes with the stimulatory
anti-CD95 CH11 or CD95 ligand resulted in rapid CD95 clustering (Fig.
1C), ASM deficiency abrogated CD95 clustering. Deficiency of
ASM did not result in a general defect of receptor clustering, as
capping of L-selectin or
To examine the role of ASM/ceramide in clustering of surface receptors,
we determined the subcellular localization of ASM prior or after CD95
stimulation. Confocal microscopy on permeabilized (Fig.
2A) and intact cells (Fig.
2B), scanning electron microscopy (Fig. 2C), and
FACS studies (Fig. 2D) revealed that ASM translocates onto
the outer leaflet of the cell membrane and co-localizes with clustered
CD95 in cholesterol- and sphingolipid-rich rafts upon CD95 stimulation.
A small portion of ASM was located outside the cluster, a finding
consistent with the primary formation of small microdomains that then
fuse to a large cluster, as depicted in Fig. 1B. Consistent
with an extracellular orientation of ASM, we detected binding of a
monoclonal anti-ceramide antibody (15B4) to intact cells by FACS or
fluorescence microscopy upon stimulation with anti-CD95 CH11 (Fig.
2E), whereas resting cells were essentially unreactive.
These studies further revealed that ceramide was concentrated in
distinct membrane domains of activated cells reminiscent of CD95- and
ASM-containing clusters (Fig. 2E). There is precedent for
extracellular phospholipase action as PLA2 also
translocates to the outer plasma membrane to release arachidonic acid
from surface phospholipid (26, 27).
The role of sphingolipid rafts in CD95 clustering was further
investigated by disruption of cellular cholesterol metabolism using
filipin, nystatin, or
Finally, addition of C16-ceramide (1 µM) to
ASM-deficient B lymphocytes restored CD95 clustering whereas addition
of C16-ceramide to normal cells was without effect (Fig.
3C). Incubation of cells with anti-ceramide antibody 15B4
abrogated the effect of C16-ceramide on CD95 clustering.
C16-ceramide was incorporated into the same distinct
membrane domains targeted by ASM upon stimulation as evidenced by
co-localization of [14C16]ceramide and ASM
activity after subcellular fractionation across a sucrose density
gradient (Fig. 3D). These studies suggest that ceramide
within rafts is required for capping.
Ceramide-mediated Clustering Is Important for CD95-induced
Apoptosis--
Our studies showing that extracellularly orientated
ceramide triggers CD95 clustering in rafts, raise the question of the significance of clustering for CD95-induced apoptosis. We therefore determined apoptosis via CD95 under conditions that block receptor clustering. We observed that genetic deficiency of ASM prevented anti-CD95 CH11-triggered apoptosis, an event restored by addition of
natural C16-ceramide (Fig.
4A). Likewise, neutralization
of surface ceramide using the anti-ceramide antibody 15B4 or
destruction of lipid rafts by
To confirm the significance of CD95 clustering for apoptosis in an
in vivo situation, we employed the induction of apoptosis in
mammalian cells upon infection with Pseudomonas aeruginosa (24); infection with P. aeruginosa induces apoptosis by
up-regulation of endogenous CD95 and CD95 ligand on the cell surface
resulting in activation of CD95 by its ligand (24). Genetic studies
confirmed the strict requirement of the CD95/CD95 ligand system for
P. aeruginosa-triggered cell death (24). Thus, this system
enabled us to study the function of ASM and ceramide for CD95-triggered
death without exogenous manipulation of the receptor/ligand system. The
results show that ASM-deficient NPDA fibroblasts were resistant to
P. aeruginosa-triggered apoptosis, whereas ASM-retransfected
NPDA fibroblasts readily died (Fig. 4C). Up-regulation of
CD95 and CD95 ligand on the cells was not affected by expression of ASM
(data not shown). The resistance of ASM-deficient cells indicates a
role of the ASM for CD95-triggered apoptosis.
To define the role of CD95 clustering itself for apoptosis, we tested
whether artificial cross-linking of CD95 restores apoptosis in
asm Receptor clustering appears to be critical for transmembrane
signaling through many receptors. Our results provide insight into
molecular mechanisms of the clustering process and indicate a novel
mechanism for receptor clustering. The data identify ASM, translocated
onto the surface of sphingolipid-rich rafts, as a key player in CD95
clustering and signaling. The experiments using P. aeruginosa-induced up-regulation of CD95 and CD95 ligand indicate the significance of ASM/ceramide-mediated clustering of CD95 for apoptosis even under (patho)physiological conditions. Our data suggest
the following function of ASM/ceramide for CD95 clustering: Interaction
of membrane-bound CD95 ligand with CD95 results in a transient and weak
activation of a limited number of CD95 trimers or oligomers
insufficient to trigger apoptosis but sufficient for ASM translocation
to the outer membrane leaflet. The notion of a primary weak activation
of CD95 is supported by the findings that mere trimerization of CD95 by
its ligand is insufficient to induce apoptosis (28) and by our
preliminary data showing a 50-100-fold reduction of caspase 8 activation in cells lacking ASM upon CD95 stimulation, an event
restored by ASM transfection. Translocated ASM, now in the proximity of
sphingomyelin that resides in the outer surface of mammalian membranes,
will release ceramide. In model membrane systems, ceramide has the
unique capacity to spontaneously self-aggregate into microdomains
(29-31). If this event occurred in a preformed microdomain such as
caveolae or caveolar-like domains, re-ordering of this domain into a
signaling structure might ensue. In fact, a number of groups have now
published data demonstrating that ASM functions in
sphingolipid-enriched microdomains in various cell types (21, 22). In
addition, in model membranes ceramide-rich microdomains tend to fuse
(32), and thus, ceramide might further trigger the fusion of rafts to larger platforms. This notion is supported by unpublished findings from
our group that CD95 stimulation of NPDA cells expressing an inactive
mutant of ASM induces translocation of this mutant protein onto
multiple small spots on the cell surface, which fail to fuse into a
large cluster. These platforms may finally interact with activated CD95
at the synapse between CD95 ligand and CD95, greatly increasing the
stability of their interaction by (a) trapping and locking
CD95 molecules within rafts, thus increasing the time of interaction
with the ligand, (b) recruiting intracellular signaling molecules to CD95, (c) excluding inhibitory pathways, and/or
(d) directly altering the affinity/avidity of CD95 for its
ligand. As a result of receptor clustering, intracellular effector
molecules mediating CD95 action are brought into close proximity,
enabling their transactivation. Thus, e.g. CD95 clustering
may concentrate caspase 8 molecules within a confined area amplifying
local proteolysis, even if primarily only a few dispersed caspase 8 molecules were active. In accordance with that model, high
overexpression of the TNF-R results in its spontaneous multimerization
and apoptosis of the transfected cells (33).
In the present study, we employed two anti-ASM antibodies, a
polyclonal goat and a monoclonal mouse antibody. Both antibodies were
raised against the full-length human ASM protein. To assure that ASM
and not a cross-reacting protein was being recognized at the cell
surface upon CD95 ligation, we repeated our studies using cells
transfected with a Myc- or VSV-tagged ASM construct (see Fig.
2D). The pattern of detection of transfected ASM with either
anti-Myc 9E10 or anti-VSV P5D4 antibodies was identical to that
detected using the anti-ASM antibodies. Further, lymphocytes derived
from ASM knockout mice stained with either the goat or mouse anti-ASM
antibodies failed to show a signal, supporting the specificity of these
antibodies for ASM.2 Finally,
CD95 stimulation of NPDA cells expressing a mutated inactive ASM showed
translocation of this protein to the cell surface but no clustering.
Fluorescence microscopy studies on those NPDA cells similarly showed a
dispersed surface pattern of translocated ASM suggesting that ASM
detection by these antibodies is not restricted to aggregated ASM (data
not shown).
Cell surface ceramide was immunodetected using the anti-ceramide
antibody 15B4 raised against C16-ceramide coupled to bovine serum albumin. Several lines of evidence attest to the specific binding
of this antibody to ceramide. ELISA revealed that 15B4 binding to
C16-ceramide is
dose-dependent.3
Further, loading intact cells with C16-ceramide conferred
strong antibody binding to the cell surface. In addition, the antibody does not significantly bind to unstimulated cells in FACS or
fluorescence microscopy excluding a substantive reaction with
cholesterol, sphingomyelin, or other phospholipids (see Fig. 2,
E and F). ASM deficiency also abrogated
antibody binding upon CD95 ligation suggesting that only lipids
generated after ASM activation are detected. Finally, thin layer
chromatography immunostaining of lipid extracts from CD95 stimulated JY
cells showed that the antibody binds to a single band co-migrating with
C16-ceramide, effectively excluding significant interaction
with other lipids. Consistent with this observation, the antibody did
not bind C16-dihydroceramide or sphingomyelin in immune
thin layer chromatography.2
In summary, our data provide evidence for a previously unreported
biological function of ASM-released ceramide, i.e. the
generation of receptor clusters, which constitute an important
prerequisite for receptor signaling. Thus, the data may shed some light
on modifications of rafts that trigger receptor clustering.
Ceramide-mediated receptor clustering might be operational in many
receptor systems explaining the diverse functions of ceramide ranging
from induction of apoptosis to invasion of bacterial pathogens.
Preliminary results from our group show that ASM and ceramide not only
trigger clustering of CD95 and CD40, but may play a similar role in
T-cell receptor/CD3, B-cell receptor or CD28 signaling. In
addition, some pathogenic bacteria may employ ASM-mediated receptor
clustering for invasion into mammalian cells. Thus, our data suggest
sphingolipid-mediated clustering as a generic mechanism for
transmembrane signal transmission.
We thank Dr. M. Weller for valuable reagents
and Dr. Y. Stierhof for excellent technical help.
*
The study was supported by Deutsche Forschungsgemeinschaft
Grants Gu 335/2-2 and 335/9-4, American Lebanese Syrian
Associated Charities (to E. G.), and National Institutes of Health
Grant CA21765.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.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M101207200
2
E. Gulbins, unpublished observations.
3
R. Kolesnick, unpublished observations.
The abbreviations used are:
TNF-R, tumor
necrosis factor receptor;
ASM, acid sphingomyelinase;
AC, acid
ceramidase;
SM, sphingomyelin;
NPDA, Niemann-Pick disease Type A;
FACS, fluorescence-activated cell sorter;
PFA, paraformaldehyde;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
IL, interleukin.
CD95 Signaling via Ceramide-rich Membrane Rafts*
,,
Max-Planck-Institute for Developmental Biology,
Spemannstrasse 35, 72076 Tuebingen, and the ¶ Laboratory of
Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York,
New York 10021
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(11), or the TNF-R (12). Rafts seem to exist as preformed entities in
the membrane of resting cells (13); whether receptor stimulation
induces a biologically relevant modification of these rafts is unknown. Likewise, mechanisms mediating the trapping of activated receptor molecules within rafts require definition.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol.
Fibroblasts and LN229 glioma cells (kindly provided by Dr. M. Weller)
were cultured in phenol red-free DMEM supplemented as above. Anti-human
CD95 antibodies were from UBI, anti-human CD95 ligand NOK-1 from
PharMingen, anti-ceramide 15B4, recombinant CD95 ligand, and anti-FLAG
antibodies from Alexis. Anti-CD40 5C3, anti-L-selectin DREG 56 and
anti-integrin 6.7 were from PharMingen, anti-VSV P5D4 from Roche
Molecular Biochemicals.
and
-cyclodextrins at 1 mM each. They were added 30 or 15 min prior to infection or anti-CD95 stimulation, respectively. The
anti-ceramide antibody 15B4 was used at 1 µg/ml.
C16-ceramide, C20:4 arachidonic acid, sphingomyelin,
dihydro-C2-ceramide, and dihydro-C16-ceramide were used at 1 µM each in octyl-glucopyranoside.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CD95 clusters upon stimulation.
A, co-incubation (10 min) of Jurkat T or JY B lymphocytes
with LN229 glioma cells or stimulation for 2 min via CD95 using
anti-CD95 CH11 or cross-linked CD95 ligand rapidly results in CD95
clustering. The specificity of the clustering process is evident as
stimulation via CD95 does not result in clustering of other receptors
including CD40, the B-cell receptor, L-selectin, or
2-integrin. Shown is the percentage of cells displaying
CD95 clusters. The panel displays the mean ± S.D. of
30 experiments with analysis of a total of 3000 cells. B, a
time course of clustering reveals very rapid clustering of CD95 with
the primary formation of small rafts, which then seem to fuse to a
large cap. Shown is a typical video image. The appearance of the red
color in the video image reflects the increased intensity of the signal
because of a higher concentration of CD95. The discrimination between
red (capping) and green (not capping) enables an
exact quantification of clustering. Shown is a typical result from 30 experiments. C, ASM deficiency prevents CD95 clustering upon
stimulation. D, vice versa AC deficiency enhances
CD95 clustering (n = 5 each; mean ± S.D.).
Clustering of L-selectin or 2-integrin after specific
receptor stimulation was not affected in ASM-deficient NPDA cells
excluding a general clustering defect in these cells.
2-integrin, which do not activate ASM (25),
was unaltered in ASM-deficient cells (Fig. 1C). Genetic
deficiency of AC strongly enhanced CD95 clustering (Fig.
1D). Because AC metabolizes ceramide and should function as
a negative regulator of ceramide-mediated clustering by reducing cellular ceramide, these data support a pivotal role of ceramide for clustering.
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Fig. 2.
CD95 induces translocation of ASM onto the
cell surface and release of extracellularly orientated ceramide.
Confocal (A and B) and scanning electron
microscopy (C) of JY cells revealed a translocation of the
ASM onto the surface of the cell membrane and a co-localization of ASM
with clustered CD95 in rafts. Rafts are indicated by FITC-labeled
cholera toxin, which binds GM1 gangliosides enriched in rafts (34). In
A, the cells were permeabilized; in B, the cells
were left intact and thus do not show significant staining of
(intracellular) ASM in the unstimulated sample. Because the GM1-binding
-subunit of cholera toxin forms a pore (35), some intracellular
staining was observed for the toxin. In the scanning electron
microscopy samples, the ASM was visualized by gold-coupled anti-ASM
appearing on the cell surface as white dots. Shown is a representative
result of 10 (A and B) or 3 (C)
similar independent experiments. In A and B, a
total of ~500 cells each were analyzed. D, transfection of
Myc- or VSV-tagged ASM constructs confirms the translocation of the ASM
onto the cell surface upon CD95 stimulation. JY B cells were stably
transfected with c-Myc- or VSV-tagged ASM (pJK/myc-asm or
pJK/vsv-asm) or the control vector (pJK) stimulated with
anti-CD95 CH11 for 2 min, stained with FITC-labeled anti-Myc 9E10,
anti-VSV P5D4, or goat anti-ASM and subjected to FACS analysis. The
wild-type ASM- or pJK-transfected JY B cells served as controls.
E, ceramide is exposed on the cell surface and localizes
into distinct domains on the extracellular leaflet of the cell membrane
upon stimulation via CD95. Shown is a representative FACS analysis and
a typical video fluorescence image of five independent experiments
each. Cells were stained with FITC-labeled anti-ceramide 15B4. The
video imaging experiments analyzed a total of 500 cells. Please note
that the sensitivity of the camera for unstimulated cells was 100 times
higher than for the stimulated cells in order to detect a signal in
resting cells.
-cyclodextrin. Loss of cholesterol destroys
sphingolipid-enriched rafts. All three drugs, but not the inactive
stereoisomer -cyclodextrin, abrogated CD95 clustering (Fig.
3A). Further, neutralization
of surface ceramide by anti-ceramide 15B4, but not an irrelevant IgM,
also abrogated CD95 clustering (Fig. 3B).
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Fig. 3.
Surface ASM and ceramide mediate CD95
clustering. A and B, destruction of
cholesterol- and sphingolipid-rich rafts (A) or
neutralization of surface ceramide by binding of anti-ceramide 15B4
specifically prevented CD95 clustering (B). Clustering was
analyzed by video-fluorescence microscopy. Shown is the mean ± S.D. of three independent experiments each. C, addition of
natural C16-ceramide (1 µM) restores CD95
clustering in ASM-deficient cells upon stimulation with anti-CD95 CH11.
C16-ceramide was added immediately prior to application of
anti-CD95 CH11. Neither the solvent 0.01% octyl-glucopyranoside nor
arachidonic acid (AA), sphingomyelin (SM),
dihydro-C2-ceramide, or dihydro-C16-ceramide
restored CD95 clustering in anti-CD95 CH11-treated NPDA cells. None of
the reagents including C16-ceramide triggered any CD95
clustering per se. D, C16-ceramide is
incorporated into the same distinct membrane domains targeted by ASM
upon stimulation. This is evidenced in experiments determining
[14C16]ceramide incorporation and ASM
activity after subcellular fractionation of CD95-stimulated cells
across a sucrose density gradient. Squares represent ASM
activity; circles, radioactive ceramide.
-cyclodextrin, filipin, or nystatin
inhibited CD95-induced apoptosis (Fig. 4B).
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Fig. 4.
CD95 clustering is required for
CD95-triggered apoptosis. A, genetic deficiency of
ASM in NPDA B lymphocytes prevented apoptosis by anti-CD95 CH11, which
is restored by simultaneous application of C16-ceramide (1 µM). None of the indicated controls had any effect. Shown
is the mean ± S.D. of five independent experiments.
AA, arachidonic acid; SM, sphingomyelin.
B, neutralization of surface ceramide by treatment with
anti-ceramide 15B4 or disruption of rafts using the indicated drugs
inhibited CD95-triggered apoptosis. Shown is the mean ± S.D. of
five independent experiments. C, deficiency of the ASM
abrogated apoptosis in fibroblasts infected for 30 min with the
P. aeruginosa strains ATCC 27853 or 762, whereas ASM
positive fibroblasts died by apoptosis. Artificial cross-linking of
CD95 by addition of multimeric anti-CD95 ZB4 restored apoptosis in
ASM-deficient cells upon infection. Monomeric anti-CD95 ZB4 was without
effect. Shown is the mean ± S.D. of four independent
experiments.
/ cells after infection with P. aeruginosa. If artificial cross-linking of CD95 overcomes
apoptosis resistance in asm/ cells,
clustering can be considered pivotal for CD95 signaling. To this end,
ASM-deficient NPDA fibroblasts were treated with aggregates of the
non-stimulatory anti-CD95 antibody ZB4 during infection with P. aeruginosa. Anti-CD95 ZB4 binds but ordinarily does not activate
CD95 or induce apoptosis. This antibody does not interfere with binding
of CD95 ligand but blocks binding of anti-CD95 CH11. Therefore, it can
be used as a tool to study the biological relevance of CD95 clustering;
cross-linking of CD95 by addition of aggregated anti-CD95 ZB4 restored
apoptosis in ASM-deficient NPDA cells to a level comparable with that
in ASM-retransfected cells (Fig. 4C). Addition of
(non-cross-linking) ZB4 monomers was not sufficient to overcome
apoptosis resistance in ASM-deficient cells. Uninfected cells were not
altered by anti-CD95 ZB4. Fluorescence microscopy studies confirmed the
(artificial) formation of CD95 clusters by addition of the cross-linked
anti-CD95 ZB4 (not shown). Thus, artificial CD95 cross-linking is
sufficient to restore apoptosis sensitivity to ASM-deficient cells
upon P. aeruginosa infection, providing evidence that
ASM-mediated clustering of CD95 is required for apoptosis induction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Immunology, St. Jude Children's Research Hospital, 332 North
Lauderdale, Memphis, TN 38105. Tel.: 901-495-3085; Fax: 901-495-3107;
E-mail: erich.gulbins@stjude.org.
ABBREVIATIONS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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 Y.-Y. Fan, D. N. McMurray, L. H. Ly, and R. S. Chapkin Dietary (n-3) Polyunsaturated Fatty Acids Remodel Mouse T-Cell Lipid Rafts J. Nutr., June 1, 2003; 133(6): 1913 - 1920. [Abstract] [Full Text] [PDF]
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 R. Barsacchi, C. Perrotta, S. Bulotta, S. Moncada, N. Borgese, and E. Clementi Activation of Endothelial Nitric-Oxide Synthase by Tumor Necrosis Factor-alpha : A Novel Pathway Involving Sequential Activation of Neutral Sphingomyelinase, Phosphatidylinositol-3' kinase, and Akt Mol. Pharmacol., April 1, 2003; 63(4): 886 - 895. [Abstract] [Full Text] [PDF]
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M. Tani, H. Iida, and M. Ito O-Glycosylation of Mucin-like Domain Retains the Neutral Ceramidase on the Plasma Membranes as a Type II Integral Membrane Protein J. Biol. Chem., March 14, 2003; 278(12): 10523 - 10530. [Abstract] [Full Text] [PDF]
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T. Garofalo, R. Misasi, V. Mattei, A. M. Giammarioli, W. Malorni, G. M. Pontieri, A. Pavan, and M. Sorice Association of the Death-inducing Signaling Complex with Microdomains after Triggering through CD95/Fas. EVIDENCE FOR CASPASE-8-GANGLIOSIDE INTERACTION IN T CELLS J. Biol. Chem., February 28, 2003; 278(10): 8309 - 8315. [Abstract] [Full Text] [PDF]
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K. C. Switzer, D. N. McMurray, J. S. Morris, and R. S. Chapkin (n-3) Polyunsaturated Fatty Acids Promote Activation-Induced Cell Death in Murine T Lymphocytes J. Nutr., February 1, 2003; 133(2): 496 - 503. [Abstract] [Full Text] [PDF]
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P. Ducoroy, O. Micheau, S. Perruche, L. Dubrez-Daloz, D. de Fornel, P. Dutartre, P. Saas, and E. Solary LF 15-0195 immunosuppressive agent enhances activation-induced T-cell death by facilitating caspase-8 and caspase-10 activation at the DISC level Blood, January 1, 2003; 101(1): 194 - 201. [Abstract] [Full Text] [PDF]
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G. Jarad, B. Wang, S. Khan, J. DeVore, H. Miao, K. Wu, S. L. Nishimura, B. A. Wible, M. Konieczkowski, J. R. Sedor, et al. Fas Activation Induces Renal Tubular Epithelial Cell beta 8 Integrin Expression and Function in the Absence of Apoptosis J. Biol. Chem., November 27, 2002; 277(49): 47826 - 47833. [Abstract] [Full Text] [PDF]
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A. Krippner-Heidenreich, F. Tubing, S. Bryde, S. Willi, G. Zimmermann, and P. Scheurich Control of Receptor-induced Signaling Complex Formation by the Kinetics of Ligand/Receptor Interaction J. Biol. Chem., November 8, 2002; 277(46): 44155 - 44163. [Abstract] [Full Text] [PDF]
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 J. K. Pru, I. R. Hendry, J. S. Davis, and B. R. Rueda Soluble Fas Ligand Activates the Sphingomyelin Pathway and Induces Apoptosis in Luteal Steroidogenic Cells Independently of Stress-Activated p38MAPK Endocrinology, November 1, 2002; 143(11): 4350 - 4357. [Abstract] [Full Text] [PDF]
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 J. H. Li, M. S. Kluger, L. A. Madge, L. Zheng, A. L. M. Bothwell, and J. S. Pober Interferon-{gamma} Augments CD95(APO-1/Fas) and Pro-Caspase-8 Expression and Sensitizes Human Vascular Endothelial Cells to CD95-Mediated Apoptosis Am. J. Pathol., October 1, 2002; 161(4): 1485 - 1495. [Abstract] [Full Text] [PDF]
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H. Grassme, J. Bock, J. Kun, and E. Gulbins Clustering of CD40 Ligand Is Required to Form a Functional Contact with CD40 J. Biol. Chem., August 9, 2002; 277(33): 30289 - 30299. [Abstract] [Full Text] [PDF]
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V. Ayllon, A. Fleischer, X. Cayla, A. Garcia, and A. Rebollo Segregation of Bad from Lipid Rafts Is Implicated in the Induction of Apoptosis J. Immunol., April 1, 2002; 168(7): 3387 - 3393. [Abstract] [Full Text] [PDF]
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 A. Algeciras-Schimnich, L. Shen, B. C. Barnhart, A. E. Murmann, J. K. Burkhardt, and M. E. Peter Molecular Ordering of the Initial Signaling Events of CD95 Mol. Cell. Biol., January 1, 2002; 22(1): 207 - 220. [Abstract] [Full Text] [PDF]
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H. Grassme, V. Jendrossek, J. Bock, A. Riehle, and E. Gulbins Ceramide-Rich Membrane Rafts Mediate CD40 Clustering J. Immunol., January 1, 2002; 168(1): 298 - 307. [Abstract] [Full Text] [PDF]
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A. Cremesti, F. Paris, H. Grassme, N. Holler, J. Tschopp, Z. Fuks, E. Gulbins, and R. Kolesnick Ceramide Enables Fas to Cap and Kill J. Biol. Chem., June 22, 2001; 276(26): 23954 - 23961. [Abstract] [Full Text] [PDF]
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X. Xu, R. Bittman, G. Duportail, D. Heissler, C. Vilcheze, and E. London Effect of the Structure of Natural Sterols and Sphingolipids on the Formation of Ordered Sphingolipid/Sterol Domains (Rafts). COMPARISON OF CHOLESTEROL TO PLANT, FUNGAL, AND DISEASE-ASSOCIATED STEROLS AND COMPARISON OF SPHINGOMYELIN, CEREBROSIDES, AND CERAMIDE J. Biol. Chem., August 31, 2001; 276(36): 33540 - 33546. [Abstract] [Full Text] [PDF]
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