From the Dipartimento di Medicina Sperimentale e Patologia,
Università "La Sapienza," Roma, viale Regina Elena 324, 00161 Rome, Italy, the Department of Ultrastructures,
Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy, and the § Dipartimento di Medicina
Sperimentale, Università di L'Aquila, Via Vetoio Coppito
2, 67100 L'Aquila, Italy
Received for publication, July 29, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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In this investigation we show that the
death-inducing signaling complex (DISC) associates with
glycosphingolipid-enriched microdomains (GEM) upon CD95/Fas engagement.
We primarily analyzed the ganglioside pattern and composition of GEM
after triggering through CD95/Fas and observed that GM3 is the main
ganglioside constituent of GEM. Stimulation with anti-CD95/Fas did not
cause translocation of gangliosides within or from the GEM fraction. Scanning confocal microscopy showed that triggering through CD95/Fas induced a significant GM3-caspase-8 association, as revealed by nearly
complete colocalization areas. Coimmunoprecipitation experiments demonstrated that GM3 and GM1 were immunoprecipitated by anti-caspase-8 only after triggering through CD95/Fas. This association was supported by the recruitment of caspase-8, as well as of CD95/Fas, to GEM upon
CD95/Fas engagement, as revealed by the analysis of linear sucrose
gradient fractions. It indicates that the DISC associates with GEM; no
changes were observed in the distribution of caspase-9. The disruption
of GEM by methyl--cyclodextrin prevented DNA fragmentation, as well
as CD95/Fas clustering on the cell surface, demonstrating a role for
GEM in initiating of Fas signaling.
These findings strongly suggest a role for gangliosides as structural
components of the membrane multimolecular signaling complex involved in
CD95/Fas receptor-mediated apoptotic pathway.
Gangliosides, sialic acid-containing glycosphingolipids, are
ubiquitous constituents of cell plasma membranes (1). However, each
cell type shows a peculiar ganglioside expression pattern. In human T
lymphocytes monosialoganglioside
GM31 represents the main
ganglioside constituent of cell plasma membrane (2), where it is
concentrated in glycosphingolipid-enriched microdomains (GEM) (3, 4).
The presence of tyrosine kinase receptors, mono-(Ras, Rap) and
heterotrimeric G proteins, Src-like tyrosine kinases (Lck, Lyn,
Fyn), PKC isozymes, glycosylphosphatidylinositol-anchored proteins (5, 6) and, after T cell activation, the Syk family kinase
Zap-70 (7) prompts these portions of the plasma membrane to be
considered as "glycosignaling domains" (8, 9). Recently, we studied
the glycosphingolipid composition of these specialized portions of
plasma membrane in human peripheral blood lymphocytes (7),
demonstrating by high performance thin layer chromatography, gas
chromatographic and mass spectrometric analysis that GM3 represents one
of the main markers of microdomains in these cells. Minor components
are sialosyl paragloboside (NeuAc-nLac4Cer), migrating between GM3 and GM1, sialosyl lactohexaosyl ceramide, and
disialoganglioside GD3 (2, 10, 11). Gangliosides may be involved in
modulating signal transduction, mainly by interaction with specific
signal transducer molecules detected in these domains (8), such as p56lck (12) or Zap-70 (7). In addition, we demonstrated an
association of GM3 and GD3 gangliosides with the cytoskeletal protein
ezrin, induced by CD95/Fas-mediated apoptosis in lymphoblastoid T
cells (13).
Apoptosis is a type of cell death characterized by chromatin
condensation, DNA fragmentation, and membrane blebbing (14). Triggering
of cell apoptosis is strictly regulated by ligand-receptor systems,
including Fas ligand-CD95/Fas (15, 16). The binding of CD95/Fas by its
ligand results in trimerization of the receptor, recruitment of
Fas-associated death domain (FADD) protein to the death domain of CD95,
and binding of caspase-8 to the death-effector domain of FADD (17).
This process induces the formation of the DISC. Binding and activation
of caspase-8 results in transmission of the activation signal to other
caspases, in particular caspase-3, involvement of mitochondria with
release of cytochrome c and membrane depolarization, and
release of apoptosis-inducing factor (18). Two types of cells have been
classified on the basis of the CD95/Fas signaling (19). Type-1 cells
show a rapid recruitment of the DISC to the receptor resulting in the
activation of caspase-8, whereas type-2 cells, including lymphoblastoid
T cells (i.e. CEM) (20), have slow apoptotic kinetics
depeding on mitochondrial activation. Evidence suggests that many
receptors aggregate in distinct plasma membrane microdomains or rafts
(21). This notion is supported by the finding that disruption of
microdomains prevents clustering of many receptors, including TNF-R
(22) and CD95/Fas (23).
On the basis of these considerations and following the observation that
the main gangliosides present in GEM associate with ezrin-CD95 complex
after the receptor triggering (13), we decided to analyze whether GEM
were involved in the initiation of Fas/CD95 signaling.
Thus, in this investigation we analyzed the ganglioside pattern of GEM
after CD95/Fas engagement and then we focused on the interaction of the
death-inducing signaling complex with these domains.
Cells--
Human lymphoblastoid CEM cells (24) were maintained
in RPMI 1640 (Invitrogen Italia srl, Milan, Italy), containing
10% fetal calf serum plus 100 units/ml penicillin, 100 µg/ml
streptomicin, at 37 °C in a humidified 5% CO2 atmosphere.
Cells were stimulated with anti-CD95/Fas (Cl CH11, 250 ng/ml, Upstate
Biotechnology, Lake Placid, NY) antibodies for the indicated incubation
times at 37 °C.
Ganglioside Extraction and Analysis by High Performance Thin
Layer Chromatography (HPTLC)--
Ganglioside extraction was performed
according to the method of Svennerholm and Fredman (25), with minor
modifications. Briefly, glycosphingolipids were extracted twice in
chloroform:methanol:water (4:8:3) (v:v:v) and subjected to Folch
partition by the addition of water resulting in a final
chloroform:methanol:water ratio of 1:2:1.4. The upper phase, containing
polar glycosphingolipids, was purified of salts and low molecular
weight contaminants using Bond Elut-C18 columns, 3 ml (Superchrom,
Harbor City, CA), according to the method of Williams and McCluer (26).
The eluted glycosphingolipids were dried down and separated by HPTLC,
using silica gel 60 HPTLC plates (Merck, Darmstadt, Germany).
Chromatography was performed in chloroform:methanol:0.25% aqueous KCl
(5:4:1) (v:v:v). Plates were then air-dried and gangliosides visualized
with resorcinol (27).
Alternatively, the ganglioside extract was run on HPTLC aluminum-backed
silica gel 60 (20 × 20) plates (Merck). Plates were soaked in a
0.2% solution of polyisobutylmethacrylate in hexane for 90 s,
air-dried, and incubated in blocking solution consisting of 3% albumin
(Sigma) in phosphate-buffered saline (PBS) pH 7.4, for 1 h
at room temperature. The blocking solution was removed and replaced by
washing buffer (PBS). The plates were then incubated for 1 h
at room temperature with HRP-conjugated cholera toxin, B subunit (CTxB,
Sigma). Immunoreactivity was assessed by chemiluminescence reaction
using the ECL Western blocking detection system (Amersham Biosciences, Buckinghamshire, UK).
Isolation and Analysis of GEM Fraction--
GEM fraction from
lymphoblastoid CEM cells, either untreated or treated with
anti-CD95/Fas (250 ng/ml for 1 or 2 h at 37 °C), was isolated
as described previously (28). Briefly, 2 × 108 cells
were suspended in 1 ml of lysis buffer containing 1%
Triton-X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM
NaVO4, and 75 units of aprotinin and allowed to stand for
20 min. The cell suspension was mechanically disrupted by Dounce
homogenization (10 strokes). The lysate was centrifuged for 5 min at
1300 × g to remove nuclei and large cellular debris.
The supernatant fraction (postnuclear fraction) was subjected to
sucrose density gradient centrifugation, i.e. the fraction
was mixed with an equal volume of 85% sucrose (w/v) in lysis buffer
(10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA). The resulting diluent was placed at the bottom of
a linear sucrose gradient (5-30%) in the same buffer and centrifuged
at 200,000 × g for 16-18 h at 4 °C in a SW41 rotor (Beckman Instruments). After centrifugation, the gradient was fractionated, and 11 fractions were collected starting from the top of
the tube. All steps were done at 0-4 °C. The amount of protein in
each fraction was first quantified by Bio-Rad protein assay (Bio-Rad
GmbH, Munchen, Germany).
Finally, the GEM fraction from untreated and CD95/Fas-stimulated cells
was subjected to ganglioside extraction, as reported above.
Analysis of GM3-Caspase-8 Colocalization by Scanning Confocal
Microscopy--
CEM cells, either untreated or treated with
anti-CD95/Fas (250 ng/ml for 1 h at 37 °C), were fixed with 4%
formaldehyde in PBS for 30 min at 4 °C and labeled with
anti-caspase-8 MoAb (Upstate Biotechnology) for 1 h at 4 °C,
followed by addition (30 min at 4 °C) of Texas Red-conjugated
anti-mouse IgG (Calbiochem). After three washes in PBS, cells were
incubated with GMR6 anti-GM3 MoAb (Seikagaku Corp., Chuo-ku, Tokyo,
Japan) (29) for 1 h at 4 °C, followed by three washes in PBS
and addition (30 min at 4 °C) of fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgM (Sigma). In parallel experiments,
cells were stained with anti-GM3 MoAb before fixing the
cells. Cells were finally washed three times in PBS and
resuspended in glycerol/Tris-HCl (pH 9.2). The images were acquired
through a confocal laser scanning microscope Leica, equipped with an
argon ion laser. Simultaneously, the green (FITC) and the red (Texas
Red) fluorophores were excited at 488 and 518 nm. Acquisition of single
FITC-stained samples in dual fluorescence scanning configuration did
not show contribution of green signal in red. Images were collected at
512 × 512 pixels.
Detection of GM3 and GM1 in the Immunoprecipitates of Caspase-8
by HPTLC Immunostaining--
Briefly, CEM cells, either untreated or
treated with anti-CD95/Fas (250 ng/ml for 1 h at 37 °C) were
lysed in lysis buffer (20 mM HEPES (pH 7.2), 1% Nonidet
P-40, 10% glycerol, 50 mM NaF, 1 mM
phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml). Cell-free lysates were mixed with protein G-acrylic beads and stirred by a rotary
shaker for 2h at 4 °C to preclear nonspecific binding. After
centrifugation (500 × g for 1 min), the supernatant
was immunoprecipitated with anti-caspase-8 MoAb (Medical & Biological Laboratory, Naka-ku Nagoya, Japan) plus protein G-acrylic beads. A
mouse IgG isotypic control (Sigma) was employed.
The immunoprecipitates were subjected to ganglioside extraction as
reported above. The ganglioside extract was split into two aliquots. A
portion was separated by HPTLC, using silica gel 60 HPTLC plates
(Merck) and stained with resorcinol (27). Another portion was run on
HPTLC aluminum-backed silica gel 60 (20 × 20) plates (Merck), as
reported above. The plates were immunostained for 1 h at room
temperature with HRP-conjugated CTxB (Sigma) or, alternatively, with
GMR6 anti-GM3 MoAb (Seikagaku Corp.) and then HRP-conjugated anti-mouse
IgM (Sigma). Immunoreactivity was assessed by chemiluminescence
reaction using the ECL Western blocking detection system (Amersham Biosciences).
Detergent Solubilization--
Lymphoblastoid CEM cells, either
untreated or treated with anti-CD95/Fas (250 ng/ml for 5 min, 30 min or
1 h at 37 °C), were detergent solubilized according to Skibbens
et al. (30). Briefly, cells were lysed on ice with 1 ml of
extraction buffer (25 mM HEPES (pH 7.5), 0.15 NaCl, 1%
Triton X-100, and 100 kallikrein units/ml aprotinin) for 20 min on ice.
Lysates were collected and centrifuged for 2 min in a Brinkmann
microfuge at 12,000 rpm at 4 °C. Sovranatants, containing Triton
X-100-soluble material, were collected; pellets were undertaken to a
second centrifugation (30 s) to remove the remaining soluble material.
The pellets were then solubilized in 100 µl of buffer containing 50 mM Tris-HCl (pH 8.8), 5 mM EDTA, and 1% SDS.
DNA was sheared by passage through a 22-gauge needle. Both Triton
X-100-soluble and -insoluble material were analyzed by Western blot as
described above.
Immunoblotting Analysis of GEM Fraction--
All the
fractions obtained as reported above were subjected to 10% sodium
dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE). The
proteins were electrophoretically transferred to nitrocellulose
membrane (Bio-Rad) and then, after blocking with PBS, containing 1%
albumin, probed with anti-caspase-8 MoAb (Upstate Biotechnology),
anti-caspase-9 polyclonal Ab (Upstate Biotechnology), or anti-CD95/Fas
MoAb (Medical & Biological Laboratory). Bound antibodies were
visualized with HRP-conjugated anti-mouse or anti-rabbit IgG (Sigma)
and immunoreactivity assessed by chemiluminescence reaction using the
ECL Western blocking detection system (Amersham Biosciences). As a
control for nonspecific reactivity, parallel blots were performed as
above, using an anti-mouse IgG (Sigma).
Densitometric scanning analysis was performed by Mac OS 9.0 (Apple
Computer International) using NIH Image 1.62 software. The density of
each band in the same gel was analyzed, values were totaled, and then
the percent distribution across the gel was detected.
Immunofluorescence and Intensified Video
Microscopy--
CD95/Fas distribution was analyzed by
immunofluorescence in cells treated with anti-CD95/Fas (250 ng/ml for
2 h at 37 °C) or in cells preincubated with 5 mM
methyl- Evaluation of DNA Fragmentation--
DNA fragmentation was
studied by propidium iodide staining followed by flow cytometric
analysis (EPICS Profile, Coulter Electronics, Hialeah, FL) (31). It was
evaluated in control cells, in cells treated with anti-CD95/Fas (250 ng/ml for 2 h at 37 °C), or in cells preincubated with 5 mM M Ganglioside Pattern of Lymphoblastoid CEM Cells--
We primarily
analyzed the ganglioside pattern of lymphoblastoid CEM cells.
Gangliosides were extracted in chloroform:methanol:water and separated
in HPTLC as reported above. Five main resorcinol-positive bands were
detected: a main GM3 comigrating double band, a band migrating between
GM3 and GM1, a GM1 comigrating band, one comigrating with GD3, and one
with GD1a (Fig. 1a). The GM3
double band is due to the heterogeneity of fatty acid composition, as
described previously (7, 33).
The identity of the band comigrating with GM1 was verified by HPTLC
immunostaining using the CTxB. The analysis revealed that in these
cells the band was immunostained by the CTxB (Fig. 1b), indicating that it is GM1.
Ganglioside Pattern of GEM after Triggering through
CD95/Fas--
We investigated the ganglioside composition
of sucrose gradient fractions obtained from lymphoblastoid CEM cells in
the absence or in the presence of triggering through CD95/Fas. The
resorcinol-positive bands were present mainly in the fraction 5 that,
under our experimental condition, corresponds to GEM and, to a less
extent, in the fractions 4, 6, and 7. About 90% of ganglioside content
present in the total cell extract was recovered in the fractions 4-7.
No bands were detectable in the Triton X-100-soluble fractions 10 and 11.
The comparative analysis of the ganglioside pattern of sucrose gradient
fractions revealed that triggering through CD95/Fas did not modify the
distribution pattern of gangliosides (Fig. 2B).
Analysis of the Association of GM3 with Caspase-8 after Triggering
through CD95/Fas--
To study the possible association of
caspase-8 with GEM after triggering through CD95/Fas, we analyzed, by
scanning confocal microscopy, the distribution of caspase-8 and GM3
(Fig. 3). As expected, in untreated cells
caspase-8 staining was mostly diffuse in the cytoplasm (A,
panel 1), whereas after stimulation, it appeared uneven and
punctate near the plasma membrane (A, panel 2),
indicating that the protein translocates mostly in correspondence of
specific membrane domains. This change of localization pattern is
generally associated with protein activation (17). The results revealed that most of the cells showed an uneven signal distribution of ganglioside molecules over the cell surface, either costitutively (B, panel 1) or after treatment with
anti-CD95/Fas (250 ng/ml) for 2 h at 37 °C (B,
panel 2). To determine the possible association between
caspase-8 and GM3, we superimposed the double immunostaining of
anti-caspase 8 and anti-GM3 in the absence or in the presence of
triggering through CD95/Fas. In the absence of cell stimulation, GM3
and caspase-8 showed weak colocalization (C, panel
1). This finding suggests that GM3 and caspase-8 are not
associated in untreated CEM. After cell stimulation through
CD95/Fas, the merged image of anti-CD95/Fas and anti-GM3 staining
revealed brown yellow areas, resulting from overlap of green
and red fluorescence, which correspond to nearly complete
colocalization areas (C, panel 2). Therefore,
cell triggering through CD95/Fas preferentially promotes translocation
of caspase-8 in selective membrane microdomains in which GM3 is highly
enriched.
Coimmunoprecipitation of Gangliosides and Caspase-8 after
Triggering through CD95/Fas--
To verify whether
caspase-8 may interact with gangliosides, cell-free lysates from
anti-CD95/Fas-treated and untreated cells were immunoprecipitated with
the anti-caspase-8 MoAb, followed by protein G-acrylic beads. Acidic
glycosphingolipids were extracted from the caspase-8 immunoprecipitates
and analyzed by HPTLC analysis followed by either resorcinol staining
or immunostaining. The analysis revealed a main GM3 comigrating double
band and a GM1 comigrating band after triggering through CD95/Fas (Fig.
4, lane A). No
resorcinol-positive bands were detected in the immunoprecipitates from
untreated cells (Fig. 4, lane B). TLC immunostaining
revealed that the extract from the immunoprecipitates from
anti-CD95/Fas-treated cells were immunostained by both anti-GM3 MoAb
(Fig. 4, lane D) and B subunit cholera toxin (Fig. 4,
lane F). On the contrary, no bands were detected in the
immunoprecipitates from untreated cells (Fig. 4, lanes E and
G), as well as in control samples immunoprecipitated with a
mouse IgG with irrelevant specificity, under the same condition (Fig.
4, lane H).
Recruitment of Caspase-8 to GEM after Triggering through
CD95/Fas--
To analyze the distribution of caspase-8, we
investigated the presence of this protein in both Triton
X-100-insoluble and -soluble fractions after different incubation times
with anti-CD95/Fas. As shown in Fig.
5A, caspase-8 was almost
enterely soluble in Triton X-100. However, the protein became
progressively less soluble in the detergent after triggering through
CD95/Fas. By 1 h, about 90% was insoluble. To better
clarify the distribution of the protein, GEM fractions of
CEM cells, obtained by a 5-30% linear sucrose gradient, in the
absence or in the presence of anti-CD95/Fas (250 ng/ml for 1 h at
37 °C), were analyzed. The results revealed that in nonstimulated
cells caspase-8 was present in fractions 6-10, but was almost
completely absent in the fraction 5, corresponding to GEM (Fig.
5B). After triggering through CD95/Fas, caspase-8 was
detected in the detergent-insoluble fraction 5 and in fractions 4, 6, 7, and 8, but not in fractions 9 and 10 (Fig. 5C),
indicating that caspase-8 recruited to GEM upon CD95/Fas cross-linking.
On the contrary, caspase-9 was detected in fractions 6-11 in both anti-CD95/Fas-treated (Fig. 5E) and untreated (Fig.
5D) cells.
Distribution of CD95/Fas in Sucrose Density Gradient
Fractions--
To analyze the distribution of CD95/Fas, we primarily
investigated the presence of this protein in both Triton
X-100-insoluble and -soluble fractions after different incubation times
with anti-CD95/Fas. As shown in Fig.
6A, the receptor was mainly
soluble in Triton X-100 and became progressively less soluble in the
detergent after triggering through CD95/Fas. To better clarify the
distribution of the protein, we also investigated the presence of
CD95/Fas in the GEM fractions of CEM cells in the absence or in the
presence of cross-linking with the antibody (250 ng/ml for 1 h at
37 °C). The results revealed that in control cells only a small
amount of CD95/Fas was detectable in the fraction 5, corresponding to GEM (5% of the total content, as revealed by densitometric analysis) (Fig. 6B). In cells treated with the antibody, CD95/Fas
content in fraction 5 increased to 12% of the total content (Fig.
6C). It indicated that the antibody triggering induced
CD95/Fas enrichment in GEM fraction, suggesting that GEM represent the
plasma membrane sites from which CD95/Fas initiates signaling cascade
upon binding to its ligand.
Effect of M
In all samples the percentage of necrotic cells was less than 2%, as a
result of trypan blue exclusion test.
In this investigation we provide evidence that in CEM
lymphoblastoid T cells the death-inducing signaling complex associates with GEM upon CD95/Fas engagement. This finding extends our preliminary observation that in the same cells gangliosides GM3 and GD3
coimmunoprecipitated with the cytoskeletal protein ezrin (13), which,
in turn, was complexed with CD95/Fas (34) after triggering through
anti-Fas Ab, but not after treatment with staurosporine (13). Here, we primarily analyzed whether triggering through CD95/Fas modified the
ganglioside pattern and composition of GEM. In agreement with our
previous reports on human peripheral blood lymphocytes (3, 7), we
observed that GM3 is the main ganglioside constituent of GEM and can be
considered a marker of these specialized portions of plasma membrane in
CEM lymphoblastoid T cells. Interestingly, we revealed by CTxB staining
that also GM1 is present in these cells, although as a minor
constituent. Moreover, stimulation with anti-CD95/Fas did not cause
translocation of gangliosides within or from the GEM fraction.
Following our observation that T cell activation induced Zap-70-GM3
interaction within GEM (7) and the recent studies that pointed out the
role of GEM in initiating of CD95/Fas triggered cell death in mouse
thymocytes (35) or in lymphoblastoid T cells (13, 23), we analyzed the
possible association of DISC components with GM3 during
CD95/Fas-triggered apoptosis. Scanning confocal microscopic
observations showed the presence of GM3 clusters, revealing the
existence on the cell surface of GEM with GM3 molecule concentration
either in the absence or in the presence of triggering through
CD95/Fas. These observations are consistent with previously reported
thermodynamic results (36) and with our immunoelectron microscopic (3)
and immunofluorescence (7) data in human peripheral blood lymphocytes.
We now demonstrate by scanning confocal microscopy the association of
caspase-8 with GEM, as revealed by nearly complete colocalization areas
between caspase-8 and GM3 after triggering through CD95/Fas.
Interestingly, T cell stimulation does not modify the ganglioside
distribution, revealing that CD95/Fas engagement does not promote a
redistribution of GEM, but induces a preferential translocation of DISC
to discrete microdomains of cell plasma membrane in which it associates
with GM3. This association was supported by coimmunoprecipitation
experiments which demonstrated that not only GM3 but also GM1 were
immunoprecipitated by anti-caspase 8 after triggering through CD95/Fas.
In addition, the analysis of linear sucrose gradient fractions further
clarified the recruitment of caspase-8, as well as of Fas, to GEM upon
CD95/Fas engagement. Thus, we provide evidence that CD95/Fas triggering induces the lateral organization of rafts, bringing the CD95/Fas receptor together with GEM and demonstrating that the DISC associates with GEM. This finding strongly suggests a role for GEM in triggering of T cell apoptosis, since binding and activation of caspase-8 results in transmission of the activation signal to other caspases and
involvement of mitochondria. Indeed, the binding of CD95/Fas by its
ligand results in trimerization of the receptor, recruitment of FADD to
the death domain of CD95 and binding of procaspase-8 to the
death-effector domain of FADD (17). As a control, we show that
caspase-9 did not modify its distribution pattern after triggering
through CD95/Fas, which is consistent with the cytoplasmic localization
of this caspase.
The key role of GEM in initiating of Fas signaling gained further
support from the demonstration that disruption of GEM prevents DNA
fragmentation as well as CD95/Fas clustering on the cell surface. These
conclusions are fully in agreement with the observations in mouse
thymocytes (35), or in human CD4+ cells (37), and are supported by the
demonstration that CD95/Fas clustering and signaling were mediated by
ceramide-rich membrane rafts (23, 38). Here, we show that apoptosis was
prevented not only by neutralization of surface ceramide or inhibition
of ceramide release (38), but also by cholesterol depletion on cell
plasma membrane. These observations reveal an additional difference in
the apoptotic pathways of type-1 and type-2 cells, since CD95/Fas
signaling is GEM-independent in type-1 cells (20).
These findings strongly suggest a role for gangliosides as structural
components of the membrane multimolecular signaling complex involved in
cell apoptosis pathways. In our previous work (7) we demonstrated their
involvement in T cell activation; the present one deals with cell death
proneness. Hence, to understand the regulation pathways supervising
both cell activation (and proliferation) and, on the opposite side,
cell death by apoptosis could provide useful information on the
subcellular mechanisms influencing T cell fate. The present work,
according to literature (39), allows us to hypothesize that
microdomains might represent a sort of "closed chamber," where
specific key reactions can take place, including the phosphorylation of
tyrosine kinases (8, 9) and of the membrane/cytoskeletal linker ezrin
(13, 34), hijacking T cells toward their survival or death. This could
also be of relevance in the elucidation of the regulatory mechanisms underlying cell death process in terms of both resistance and susceptibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin (M
CD) (20 min at 37 °C) in 10 mM HEPES, either treated or untreated with anti-CD95/Fas.
Control and treated cells were collected by centrifugation, attached to
glass coverslips precoated with polylysine, and fixed with 4%
paraformaldehyde in PBS for 30 min at room temperature. After washing
in the same buffer, cells were permeabilized with 0.5% Triton X-100 in
PBS for 5 min at room temperature. Samples were incubated at 37 °C
for 30 min with polyclonal antibodies to CD95 (Santa Cruz
Biotechnology, Santa Cruz, CA). Cells were then incubated with
FITC-conjugated anti-rabbit IgG (Sigma). After washing, all samples
were mounted with glycerol/PBS (2:1) and observed with a Nikon
Microphot fluorescence microscope. Images were captured by a color
chilled 3CCD camera (Hamamatsu Photonics, Hamamatsu City,
Japan). Normalization and background subtraction were performed
for each image. Figures were obtained by the OPTILAB (Graftek)
software for image analysis.
CD (Sigma) in 10 mM HEPES, either treated or untreated with anti-CD95/Fas. Cells were fixed with cold
70% ethanol in PBS for 1 h at 4 °C. After centrifugation at
200 × g for 10 min at 4 °C, cells were washed once
in PBS. The pellet was resuspended in 0.5 ml PBS, 50 µl of RNase
(Type I-A, Sigma, 10 mg/ml in PBS) was added, followed by 50 µg/ml
propidium iodide (Sigma) in PBS. The cells were incubated in the dark
at room temperature for 15 min and kept at 4 °C until measured. In parallel samples the amount of cholesterol was evaluated as described previously (32). Free cholesterol was quantitated from TLC plates by
densitometric scanning and comparison with standard. The density of the
bands used to quantitate cholesterol concentration fell within the
linear range of compound concentration versus absorbance. A
trypan blue exclusion test was performed to evaluate the viability of
the cultures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
a, HPTLC analysis of the ganglioside
pattern of CEM cells. Gangliosides were extracted in
chloroform/methanol/water. The plate was stained with resorcinol
(ganglioside-specific stain). Lane A, gangliosides obtained
from total CEM cells; lane B, standard gangliosides GM3,
GM1, GD3, GD1a, GD1b, and GT1b. b, TLC immunostaining of the
ganglioside extract with HRP-conjugated CTxB. Gangliosides were
separated using HPTLC aluminium-backed silica gel plates.
Immunoreactivity was assessed by chemiluminescence reaction using the
ECL Western blocking detection system. Lane A, gangliosides
obtained from CEM cells; lane B, standard GM1.
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Fig. 2.
Ganglioside distribution in CEM cells sucrose
gradient fractions. Lymphoblastoid CEM cells were lysed in lysis
buffer, and the supernatant fraction (postnuclear fraction) was
subjected to sucrose density gradient. After centrifugation the
gradient was fractionated, and each gradient fraction was recovered.
Gangliosides were extracted in chloroform-methanol-water. The plate was
stained with resorcinol (ganglioside-specific stain). A,
untreated CEM cells; B, anti-CD95/Fas-treated (250 ng/ml for
2 h at 37 °C) CEM cells.
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Fig. 3.
Scanning confocal microscopic analysis of
GM3-caspase-8 association on CEM cell plasma membrane.
Cells were stimulated with anti-CD95/Fas (250 ng/ml for 1 h
at 37 °C). Cells were labeled with anti-caspase-8 MoAb, followed by
the addition of Texas Red-conjugated anti-mouse IgG. After washing with
PBS cells were incubated with anti-GM3 (GMR6), followed by the addition
of FITC-conjugated anti-mouse IgM. A, panel 1,
untreated cell stained with anti-caspase-8; A, panel
2, anti-CD95/Fas-treated cell stained with anti-caspase-8;
B, panel 1, untreated cell stained with anti-GM3;
B, panel 2, anti-CD95/Fas-treated cell stained
with anti-GM3; C, panel 1, dual immunolabeling of
anti-GM3 (green) and anti-caspase-8 (red) in
untreated cell; C, panel 2, dual immunolabeling
of anti-GM3 (green) and anti-caspase-8 (red) in
anti-CD95/Fas-treated cell. Colocalization areas are stained in
brown yellow. Bar, 2 µm.
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Fig. 4.
Coimmunoprecipitation of gangliosides and
caspase-8 upon CD95/Fas triggering. CEM cells were treated with
anti-CD95/Fas (250 ng/ml for 1 h at 37 °C) and
immunoprecipitated with anti-caspase-8. The immunoprecipitates were
analyzed for the presence of ganglioside molecules by HPTLC analysis
followed by either resorcinol staining or immunostaining. Gangliosides
were extracted in chloroform/methanol/water. Lane A,
resorcinol staining of extract from caspase-8 immunoprecipitate of
anti-Fas treated cells; lane B, resorcinol staining of
extract from the caspase-8 immunoprecipitate of control cells;
lane C, standard gangliosides GM3, GM1, GD3, GD1a, GD1b, and
GT1b stained with resorcinol; lane D, anti-GM3 reactivity of
extract from caspase-8 immunoprecipitate of anti-Fas treated cells;
lane E, anti-GM3 reactivity of extract from caspase-8
immunoprecipitate of control cells; lane F, CTxB reactivity
of extract from caspase-8 immunoprecipitate of anti-Fas treated cells;
lane G, CTxB reactivity of extract from caspase-8
immunoprecipitate of control cells; lane H, anti-GM3
reactivity of extract from immunoprecipitate with IgG with irrelevant
specificity of anti-Fas-treated cells.
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Fig. 5.
A, lymphoblastoid CEM cells, either
untreated or treated with anti-CD95/Fas (250 ng/ml for 5 min, 30 min,
or 1 h at 37 °C), were detergent-solubilized as described under
"Experimental Procedures." Both Triton X-100-soluble and -insoluble
materials were analyzed by Western blot and probed with anti-caspase-8
MoAb followed by incubation with HRP-conjugated anti-mouse IgG.
Immunoreactivity was assessed by chemiluminescence reaction.
B, caspase-8 distribution in control CEM sucrose gradient
fractions. CEM cells were lysed in lysis buffer, and the supernatant
fraction (postnuclear fraction) was subjected to sucrose density
gradient. After centrifugation the gradient was fractionated, and each
gradient fraction was recovered and analyzed by Western blotting with
anti-caspase-8 MoAb. C, caspase-8 distribution in stimulated
CEM (anti-CD95/Fas, 250 ng/ml for 1 h at 37 °C) sucrose
gradient fractions. CEM cells were lysed in lysis buffer, and the
supernatant fraction was subjected to sucrose density gradient. After
centrifugation the gradient was fractionated, and each gradient
fraction was recovered and analyzed by Western blotting with
anti-caspase-8 MoAb. D, caspase-9 distribution in control
CEM sucrose gradient fractions. CEM cells were lysed in lysis buffer,
and the supernatant fraction was subjected to sucrose density gradient.
After centrifugation the gradient was fractionated, and each gradient
fraction was recovered and analyzed by Western blotting with
anti-caspase-9 polyclonal Ab. E, caspase-9 distribution in
stimulated CEM (anti-CD95/Fas, 250 ng/ml for 1 h at 37 °C)
sucrose gradient fractions. CEM cells were lysed in lysis buffer, and
the supernatant fraction was subjected to sucrose density gradient.
After centrifugation the gradient was fractionated, and each gradient
fraction was recovered and analyzed by Western blotting with
anti-caspase-9 polyclonal Ab.
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Fig. 6.
A, lymphoblastoid CEM cells, either
untreated or treated with anti-CD95/Fas (250 ng/ml for 5 min, 30 min,
or 1 h at 37 °C), were detergent-solubilized as described under
"Experimental Procedures." Both Triton X-100-soluble and -insoluble
material were analyzed by Western blot and probed with anti-CD95/Fas
MoAb followed by incubation with HRP-conjugated anti-mouse IgG.
Immunoreactivity was assessed by chemiluminescence reaction.
B, CD95/Fas distribution in control CEM sucrose gradient
fractions. CEM cells were lysed in lysis buffer, and the supernatant
fraction was subjected to sucrose density gradient. After
centrifugation the gradient was fractionated, and each gradient
fraction was recovered and analyzed by Western blotting with
anti-CD95/Fas MoAb. The numbers indicate the percent distribution
across the gel of fractions 5 (GEM) and 10 and 11 (Triton-soluble), as
detected by densitometric scanning analysis. C, CD95/Fas
distribution in stimulated CEM (anti-CD95/Fas, 250 ng/ml for 1 h
at 37 °C) sucrose gradient fractions. CEM cells were lysed in lysis
buffer, and the supernatant fraction was subjected to sucrose density
gradient. After centrifugation the gradient was fractionated, and each
gradient fraction was recovered and analyzed by Western blotting with
anti-CD95/Fas MoAb. The numbers indicate the percent distribution
across the gel of fractions 5 (GEM) and 10 and 11 (Triton-soluble), as
detected by densitometric scanning analysis.
CD on CD95/Fas Distribution and
CD95/Fas-induced Apoptosis--
To analyze the contribution
of GEM in CD95/Fas-induced apoptosis, we analyzed Fas distribution and
DNA fragmentation after triggering through CD95/Fas of cells pretreated
with 5 mM M
CD (20 min at 37 °C), which is known to
induce cholesterol efflux from plasma membrane and, consequently, GEM
desruption (9). The total cellular cholesterol concentration was about
60% that found in normal cells. Consistent with previous papers (13, 34), immunoflorescence analysis showed that CD95/Fas might be detected
at one pole of control CEM cells (Fig.
7a), and it was markedly
polarized in anti-CD95 MoAb-treated cells (Fig. 7c). The
disruption of lipid rafts by M
CD led to the loss of cell polarity
with redistribution of CD95/Fas molecule all over the CEM cells (Fig.
7b) and prevented the receptor polarization observed in
anti-CD95 MoAb-treated cells (Fig. 7d). As expected, DNA
staining with propidium iodide of anti-Fas treated cells followed by
cytofluorimetric analysis showed a subdiploid peak of fluorescence,
consistent with apoptosis. Fig. 7e shows that M
CD greatly
prevented the CD95/Fas-triggered apoptosis, suggesting that GEM play a
key role in CD95/Fas signaling cascade, leading to programmed cell
death.
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Fig. 7.
Effect of M CD on
CD95/Fas distribution and DNA fragmentation. a,
IVM analysis of CD95/Fas distribution in control untreated cells;
b, IVM analysis of CD95/Fas distribution in cells treated
with 5mM M
CD; c, IVM analysis of CD95/Fas distribution in
stimulated CEM (anti-CD95/Fas, 250 ng/ml for 2 h at 37 °C);
d, IVM analysis of CD95/Fas distribution in CEM cells
treated with 5mM M
CD and then with anti-CD95/Fas, 250 ng/ml for
2 h at 37 °C; e, DNA fragmentation studied by
propidium iodide staining followed by flow cytometric analysis. It was
evaluated in control cells, in cells treated with anti-CD95/Fas (250 ng/ml for 2 h at 37 °C), or in cells preincubated with 5 mM M
CD, either treated or untreated with
anti-CD95/Fas.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR) and Ateneo 2001.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 all correspondence should be addressed: Dip. Medicina Sperimentale e Patologia, Università "La Sapienza," Roma, viale Regina Elena 324, Roma 00161, Italy. Tel.: 39-6-49972675; Fax: 39-6-4454820; E-mail: maurizio.sorice@uniroma1.it.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M207618200
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ABBREVIATIONS |
---|
The abbreviations used are:
GM3, II3NeuAc-LacCer;
GM1, II3NeuAc-GgOse4Cer;
GD1a, IV3NeuAc,II3NeuAc-GgOse4Cer;
GD1b, II3(NeuAc)2-GgOse4Cer;
GD3, II3(NeuAc)2-LacCer;
GT1b, IV3Neu
NAc II(NeuNAc)2-GgOse4Cer;
DISC, death-inducing
signaling complex;
GEM, glycosphingolipid-enriched microdomains;
FADD, Fas-associated death domain;
HPTLC, high performance thin
layer chromatography;
PBS, phosphate-buffered saline;
CTxB, cholera
toxin, B subunit;
MoAb, monoclonal antibody;
FITC, fluorescein
isothiocyanate;
HRP, horseradish peroxidase;
MCD, methyl-
-cyclodextrin;
IVM, intensified video
microscopy.
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REFERENCES |
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