From the Laboratory of Signal Transduction, the
Departments of
Radiation Oncology and ¶ Surgery, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021 and the
§ Department of Immunology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105
Received for publication, September 25, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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The role of the second messenger ceramide
in Fas-mediated death requires clarification. To address this issue, we
generated hepatocytes from paired acid sphingomyelinase
(ASMase; asmase)+/+ and
asmase Engagement of Fas/CD95/APO-1 receptors by Fas ligand or anti-Fas
antibody results in formation of a death-inducing signaling complex
comprised of the adaptor molecules FADD/MORT-1 and caspase 8, resulting
in release of active caspase 8 to initiate the apoptotic process (1).
Apoptosis, however, proceeds only after an ensuing commitment step,
which allows effector caspase activation. Recently, Peter and
co-workers (2) provided evidence that the quality and quantity of the
activated caspase 8 signal are regulated in a cell type-specific
fashion and suggested that Fas initiates apoptosis via two different
mechanisms. In type I cells, apoptosis occurs after high caspase 8 activation, which signals exclusively via a hierarchical caspase
cascade, independent of mitochondrial dysfunction. In contrast,
apoptosis in type II cells proceeds after minimal caspase 8 activation
through an amplification cascade involving mitochondrial dysfunction,
release of mitochondrial cytochrome c, and activation of
Apaf-1 and caspase 9. Although many of the upstream elements in type II
apoptosis are unknown, this event is inhibited by Bcl-2 and can be
mimicked by exogenous addition of short chain ceramides. In both
mechanisms, apoptosis eventually ensues after effector caspases, such
as 3 and 7, are activated.
There are numerous reports of early ceramide elevation upon Fas
activation (3, 4). Ceramide is a second messenger in an evolutionarily
conserved stress response pathway that, in different cells, signals
events as diverse as differentiation, proliferation, and apoptosis (3).
Ceramide is generated from sphingomyelin by the action of a neutral or
acid sphingomyelinase
(NSMase1 or ASMase) or by
de novo synthesis coordinated through the enzyme ceramide
synthase (5). In response to Fas ligation, ceramide elevation is often
biphasic, a rapid elevation with return to baseline is followed by a
more prolonged elevation that is progressive. Evidence has been
provided that both ASMase and NSMase might contribute to Fas-mediated
ceramide elevation (6, 7), with ASMase involvement in the acute event.
Recent investigations titrated down the amount of FADD or procaspase 8 transfected into HeLa and 293T cells to the point where they no longer
induced apoptosis (8). Under these conditions, FADD and procaspase 8 nonetheless induced maximal ceramide generation, which was inhibitable
by the initiator caspase inhibitor CrmA. These experiments provided
evidence that ceramide generation is initiator
caspase-dependent and occurs prior to commitment to the
effector phase of the apoptotic process.
Whether the ceramide generated in response to Fas ligation is involved
in the apoptotic process or is an epiphenomenon is a matter of ongoing
debate. Testi and co-workers (9) claimed that Epstein-Barr
virus-transformed lymphoblasts from Niemann-Pick disease (NPD)
patients, which have an inherited deficiency of ASMase activity,
displayed deficits in ceramide generation and apoptosis, whereas Borst
and co-workers (10) found no differences. Recently, Green and
co-workers (7) reported partial resistance to Jo2 anti-Fas-induced
liver failure and death in
asmase To address directly the issue of the involvement of ceramide in
Fas-mediated death, we examined the ability of primary cultures of
hepatocytes derived from asmase+/+ and
asmase Animals--
ASMase knockout mice, maintained in a sv129 × C57BL/6 background, are propagated using heterozygous breeding pairs
and genotyped as described (16). Experimental mice were 8-12 weeks old
and sacrificed by carbon dioxide asphyxiation.
Ex Vivo Hepatocyte Culture Prepared by Mechanical
Disruption--
Harvested livers were washed three times with
phosphate-buffered saline at 37 °C, cut into small pieces, and
separated into small clumps with a spatula. Individual hepatocytes were
dispersed mechanically by passage through an 18-gauge needle, filtered
through a 100-µM cell strainer, washed once with RPMI
1640 complete medium, and resuspended into the same medium containing
10% fetal bovine serum, as described (11). Viability was always
greater than 95% as defined by trypan blue exclusion analysis. After
1 h, cells (5 × 106/ml) were placed in 24-well
plates pretreated with 10 mg/ml bovine serum albumin fraction V (Sigma)
in an incubator at 37 °C with 5% CO2 atmosphere. Cells
were treated with anti-Fas Jo2 antibody (Pharmingen) or staurosporine
(Sigma) for the indicated times while under constant agitation using a
nutator (Becton Dickinson).
Ex Vivo Hepatocyte Culture Prepared by Collagenase
Perfusion--
Hepatocytes were isolated by cannulation of the portal
vein and retrograde in situ collagenase perfusion according
the method described previously by Picardo et al. (17).
Briefly, livers were perfused under sterile conditions at 40 °C
consecutively with Leffert's buffer (100 mM HEPES, pH 7.4, 30 mM KCl, 1.3 M NaCl, 10 mM
NaH2PO4, 100 mM
D-glucose) with 0.5 mM EGTA for 4 min, without
EGTA for 2 min, and then with 33% (w/v) collagenase type V (Roche) and
5 mM CaCl2 for 12 min using an Easy Load
perfusion pump (Masterflex, Cole Parmer; flow rate of 16 ml/min). The
liver was then minced in cold Leffert's buffer containing 5 mM CaCl2, and isolated hepatocytes were
filtered through a 100-µm cell strainer. Isolated hepatocytes were
washed twice at 50 × g and resuspended into RPMI 1640 complete medium containing 10% fetal bovine serum. Viability was
always greater than 95% using this method.
Sphingolipid Treatment--
Cells (5 × 106/ml)
were pretreated for 10 min with C16-ceramide, sphingosine,
sphingomyelin, sphinganine, sphingosine 1-phosphate (Biomol), or
C16-dihydroceramide (Toronto Research Chemicals, Inc.) in dodecane:ethanol (2:98, v/v; 0.05% final concentration) or
diluent alone prior to the addition of Jo2 antibody.
Fluorescence-activated Cell Sorting (FACS)--
A single cell
suspension of hepatocytes was washed twice and resuspended into 100 µl of H/S (132 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, 0.8 mM MgSO4)
containing 2% fetal calf serum and 0.2% NaN3 supplemented
with 1 µg/ml anti-Fas Jo2 antibody or an isotype-specific control
immunoglobulin. After 45 min on ice, cells were washed with the same
buffer and stained with a fluorescein isothiocyanate-coupled
anti-hamster antibody (Pharmingen 12084D). Cell surface Fas was
determined in labeled cells by flow cytometry, as described (11).
Diacylglycerol Kinase Assay--
Hepatocytes, stimulated for the
indicated times with Jo2, were lysed in 25 mM HEPES, pH
7.4, 0.5% SDS, 1% Triton X-100, 10 mM EDTA, 10 mM each sodium pyrophosphate and sodium fluoride, 10 µg/ml each aprotinin and leupeptin, 125 mM NaCl. Lipids
were extracted, and ceramide was quantified by the diacylglycerol
kinase reaction as described previously (18).
Terminal Nucleotidyl Transferase Assay--
Hepatocytes
were trypsinized after treatment with anti-Fas antibody or
staurosporine and then permeabilized with a solution of 0.1% Triton
X-100 and 0.1% sodium citrate at 4 °C for 5 min. Apoptosis was
assessed by terminal nucleotidyl transferase according to the
manufacturer's instructions (Roche, Indianapolis, IN). At least 200 cells were counted for each point.
Ethidium Bromide/Acridine Orange Staining--
Cells were
stained with 4 µg/ml each ethidium bromide and acridine orange for 10 min and analyzed by fluorescence microscopy as described (19).
DEVDase Assay--
The fluorogenic substrate Ac-DEVD-AFC
was used to measure caspase 3-like activity according to the
manufacturer's instructions (Kamiya, Seattle, WA).
Statistical Analysis--
Statistical analysis was performed by
Student's t test and t test for correlation coefficient.
Characterization of asmase asmase
Studies were also performed to determine the time course of ceramide
elevation after Jo2 treatment using a 500-ng/ml dose (Fig.
2B). In asmase+/+ hepatocytes,
ceramide levels increased to 1.6-fold of control by 30 s
(p < 0.01), peaked at 5 min, and remained elevated for at least 20 min. asmase asmase
Because the hepatocytes used in these studies were obtained by
mechanical dispersion, we compared the effect of Jo2 to induce death
using asmase+/+ and
asmase
The apoptotic response was also assessed using a biochemical assay for
caspase 3 activity after a dose of 1,000 ng/ml Jo2 (see Fig.
5B). Consistent with the results shown in Fig. 3A and 3B,
asmase+/+ hepatocytes showed a 4.1 ± 0.1-fold increase in caspase activity at 8 h of Jo2 treatment
while asmase asmase Natural Ceramide Can Rescue the Apoptotic Response of
asmase Natural C16-ceramide but Not Other Sphingolipids
Rescues ASMase-deficient Hepatocytes--
To address further the
specific role of ceramide in Fas-mediated apoptosis, we investigated
the capacity of other sphingolipids to restore Jo2-mediated death to
asmase The present studies show that primary cultures of hepatocytes from
asmase Prior studies reported that ASMase-deficient mice were partially
resistant to animal death upon intravenous injection of Jo2. In
contrast to the 1-log defect in apoptosis observed in hepatocytes ex vivo, ASMase-deficient mice displayed only a moderate
reduction in sensitivity to the lethal effect of Jo2. The reason for
the difference in requirement for ASMase for these events is presently uncertain. However, animals expressing a Bcl-2 transgene only in their
livers were defective in Fas-induced hepatitis yet displayed little
resistance to death (34). Thus, it is not clear that rapid hepatic
failure is causative in animal death after anti-Fas. Because the dose
dependence for hepatocyte death in vivo has not been
determined in animals treated with anti-Fas, a comparison of the
effects of Jo2 in vivo and in vitro is not
presently possible.
The present studies add to emerging data indicating that young
ASMase-deficient animals display normal sphingomyelin levels and hence
do not manifest evidence of NPD (35). NPD is caused by the progressive
accumulation of sphingomyelin (35). It is therefore not surprising that
hepatocytes, murine embryonic fibroblasts, and thymocytes from
8-10-week-old asmase The present studies extend the literature documenting rapid
sphingolipid metabolism in cells destined to undergo apoptosis. In our
survey of the literature we found 50 publications of stimulated sphingomyelinase activation, sphingomyelin hydrolysis, and/or ceramide
elevation within the first 5-15 min of exposure to proapoptotic stimuli, well prior to the effector stage of the apoptotic process. The
distribution of these reports is as follows: 14 for Fas (6-9, 38-47),
6 for tumor necrosis factor- In summary, the present investigation provides evidence that ceramide
is required for optimal Fas signaling of the death response in
hepatocytes. Preliminary data suggest that ceramide is required for
capping of Fas, an event necessary for transmembrane signal transmission for other cell surface
receptors.2 Overcoming
apoptosis resistance by natural ceramide is evidence that it is the
lack of ceramide, and not ASMase, which determines the Fas-resistant
phenotype in hepatocytes.
/
mice.
asmase
/
hepatocytes, derived
from 8-week-old mice, manifested normal sphingomyelin content and
normal morphological, biochemical, and biologic features. Nonetheless,
ASMase-deficient hepatocytes did not display rapid ceramide elevation
or apoptosis in response to Jo2 anti-Fas antibody.
asmase
/
hepatocytes were not
inherently resistant to apoptosis because staurosporine, which did not
induce early ceramide elevation, stimulated a normal apoptotic
response. The addition of low nanomolar quantities of natural
C16-ceramide, which by itself did not induce apoptosis,
completely restored the apoptotic response to anti-Fas in
asmase
/
hepatocytes. Other
sphingolipids did not replace natural ceramide and restore Fas
sensitivity. Overcoming resistance to Fas in
asmase
/
hepatocytes by natural
ceramide is evidence that it is the lack of ceramide and not ASMase
which determines the apoptotic phenotype. The ability of natural
ceramide to rescue the phenotype without reversing the genotype
provides evidence that ceramide is obligate for Fas induction of
apoptosis in hepatocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice in vivo.
Furthermore, the establishment of primary cultures of several cell
types derived from asmase
/
mice
showed that hepatocytes (11) but not thymocytes (7) displayed
resistance to Fas-mediated death. Although these genetic models tended
to support a possible cell type-specific role for ASMase, and
indirectly for ceramide, in Fas-induced apoptosis, other
interpretations are possible. For instance, the NPD lymphoblasts described above were transformed with Epstein-Barr virus and the transformation process itself, as well as known Epstein-Barr virus strain-specific phenotypic differences (12-14), might have affected the outcome. Further, it remains possible that the resistance of cells
from asmase
/
mice to Fas-induced
apoptotic death might result from subtle alterations in membrane
structure/function independent of ceramide itself.
/
mice to undergo
apoptosis in response to anti-Fas antibody. Like embryonic fibroblasts
and thymocytes from asmase
/
mice
(15), asmase
/
hepatocytes
displayed normal sphingomyelin content and were indistinguishable from
asmase+/+ hepatocytes by numerous morphological,
biochemical, and biologic criteria. In contrast to wild type
hepatocytes, however, ASMase-deficient hepatocytes failed to generate
ceramide upon activation of Fas and were markedly resistant to
Fas-mediated apoptotic death. Further, the addition of low nanomolar
quantities of natural C16-ceramide, which by itself does
not effect apoptosis, restored Fas-mediated apoptosis in
asmase
/
hepatocytes. Overcoming
apoptosis resistance by provision of natural ceramide is evidence that
it is the lack of ceramide and not ASMase that determines the Fas
sensitivity. The ability of natural ceramide to rescue the phenotype
without reversing the genotype provides evidence that ceramide is
obligate for efficient Fas induction of apoptosis in hepatocytes.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
Hepatocytes--
We reported recently that 8-10 week old
asmase
/
mice derived from our
colony display normal sphingomyelin content in multiple tissues (15,
20). Consequently, these animals are physiologically normal and do not
manifest NPD, as NPD requires sphingomyelin accumulation (21). In the
present studies, we generated primary cultures of hepatocytes from
livers of asmase+/+ and
asmase
/
mice. Livers from
8-week-old ASMase-deficient mice displayed normal architecture, and
isolated hepatocytes manifested physiologic morphology. No measurable
ASMase activity was detected in the asmase
/
hepatocytes (Table
I); NSMase activity was unchanged.
Further, asmase
/
hepatocytes
manifested normal sphingomyelin and ceramide content (Table I),
consistent with the observation that these animals develop normally
into adulthood (15, 22, 23). The distribution of
glycosphingolipid-enriched microdomains, a compartment putatively involved in ceramide signaling (24) and their content was similar in
asmase+/+ and
asmase
/
hepatocytes (Table I),
as was the expression of Fas on the cell surface, measured by FACS
using the Jo2 anti-Fas antibody (Fig. 1).
Similarly, the levels of factors known to participate in Fas-mediated apoptosis in hepatocytes including FADD/MORT-1, Bid, Bcl-2, Apaf-1, and
caspases 3, 8, and 9 were identical in asmase+/+
and asmase
/
hepatocytes (Table
I). Moreover, the activation patterns of p38, c-Jun terminal, and
extracellular signal-regulated kinases in response epidermal growth
factor, tumor necrosis factor-
, heat shock, and hyperosmolarity were
also similar in asmase+/+ and
asmase
/
hepatocytes (Table I).
Thus, asmase+/+ and
asmase
/
hepatocytes appeared
similar as assessed by numerous criteria.
Comparison of asmase+/+ and asmase/
hepatocytes
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Fig. 1.
asmase+/+ and
asmase /
hepatocytes express comparable levels of cell surface Fas.
The level of Fas on the surface of hepatocytes was determined by flow
cytometry as described (11). To detect Fas,
asmase+/+ and
asmase
/
hepatocytes were stained
with Jo2 and a fluorescein isothiocyanate-coupled anti-hamster
secondary antibody (green and blue lines,
respectively). For controls, asmase+/+ and
asmase
/
hepatocytes were stained
with an isotype-specific control antibody followed by fluorescein
isothiocyanate-coupled secondary antibody (orange and
red lines, respectively).
/
Hepatocytes Are
Defective in Fas-induced Ceramide Generation--
To investigate
whether ASMase-deficient cells displayed defects in Fas-mediated
signaling through the sphingomyelin pathway, we treated primary
cultures of asmase+/+ and
asmase
/
hepatocytes with
increasing concentrations of Jo2 anti-Fas antibody. Hepatocytes were
treated in suspension, as Kass and co-workers (25) showed that plating
hepatocytes as monolayers conferred Fas resistance, i.e. the
development of a requirement for cycloheximide for death induction
(25). In contrast, hepatocytes in suspension retained sensitivity to
Fas-mediated death. Fig. 2A
shows that asmase+/+ hepatocytes displayed
dose-dependent elevation in ceramide content when measured
at 5 min of treatment. As little as 50 ng/ml Jo2 increased ceramide
content from a baseline of 170 ± 20 pmol/106 cells to
210 ± 20 pmol/106 cells, and a maximal increase to
515 ± 15 pmol/106 cells (p < 0.01)
was achieved with 1,000 ng/ml. In contrast, asmase
/
hepatocytes failed to
manifest increased ceramide levels at any Jo2 dose up to 8,000 ng/ml.
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Fig. 2.
asmase /
hepatocytes are deficient in ceramide generation after Jo2
treatment. Panel A, ceramide levels in
asmase+/+ and
asmase
/
hepatocytes were
determined by diacylglycerol kinase assay after treatment with the
indicated concentrations of Jo2 monoclonal antibody. The data
(mean ± S.D.) are compiled from two studies performed in
duplicate. Panel B, time course of ceramide generation in
asmase+/+ and
asmase
/
hepatocytes after 500 ng/ml Jo2 monoclonal antibody determined as in panel A. The
data (mean ± S.D.) are compiled from two studies performed in
duplicate.
/
hepatocytes, however, did not demonstrate elevation of ceramide levels
at any time up to 20 min. Thus,
asmase
/
hepatocytes display a
complete defect in the early phase of ceramide generation upon Jo2 treatment.
/
Hepatocytes Are
Resistant to Anti-Fas-mediated Apoptosis--
To evaluate whether
asmase
/
hepatocytes also
displayed defects in Fas-mediated apoptosis, hepatocytes were rested
for 1 h in RPMI 1640 complete medium containing 10% fetal bovine
serum prior to treatment with Jo2 anti-Fas antibody. Prior
investigations showed that Jo2 antibody induced time- and
dose-dependent apoptosis in wild type hepatocytes in
vivo and ex vivo (7, 11, 26-30). Initial studies
determined the time course of induction of apoptosis in
asmase+/+ hepatocytes as measured by terminal
nucleotidyl transferase using 1,000 ng/ml Jo2 antibody, a maximally
effective dose. Apoptosis was detected by 2 h and was maximal by
8 h (not shown). Subsequent investigations measured the dose
dependence of Jo2-induced apoptosis in asmase+/+
hepatocytes after 8 h of treatment. As shown in Fig.
3A, apoptosis occurred over
2-3 logs of Jo2 doses; as little as 100 ng/ml Jo2 was effective; a
maximal apoptotic effect was achieved using 2,000 ng/ml. Thus, the dose
dependence for Jo2-induced ceramide generation is slightly lower than
that for apoptosis. asmase
/
hepatocytes, which contain normal numbers of cell surface Fas receptors
(Fig. 1), were nonetheless resistant to treatment with anti-Fas
antibody. In contrast to the total resistance to ceramide generation,
apoptosis was inducible by increasing the dose of Jo2 antibody about 1 log. Thus, apoptosis was elicited with a 2,000-ng/ml dose of Jo2 in
asmase
/
hepatocytes and
increased to a maximum at 8,000 ng/ml Jo2. Similar results were
obtained using ethidium bromide and acridine orange staining to detect
morphological changes of apoptosis (Fig. 3B). These studies
indicate that asmase
/
hepatocytes can undergo ASMase-dependent and -independent
apoptosis after Jo2 treatment.
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Fig. 3.
asmase /
hepatocytes are relatively resistant to anti-Fas Jo2 antibody.
Panel A, hepatocytes were isolated from
asmase+/+ and
asmase
/
littermates, placed in
24-well plates precoated with bovine serum albumin, and treated with
the indicated concentrations of anti-Fas monoclonal antibody. Cells
were harvested after 8 h, and apoptosis assessed by terminal
nucleotidyl transferase assay. 200 cells were evaluated per point. The
data (mean ± S.E.) are compiled from three studies performed in
duplicate. Panel B, apoptosis was also assessed in
hepatocytes from asmase+/+ and
asmase
/
littermates 8 h
after the indicated doses of Jo2 by ethidium bromide/acridine orange
staining as described (19). The data (mean ± S.E.) are compiled
from six studies performed in triplicate.
/
hepatocytes obtained by
an alternative technique, in vivo collagenase treatment. For
these studies, collagenase was delivered to intact livers after
cannulation of the inferior hepatic vein and isolated hepatocytes
derived as described (17). Using this approach, we again observed a
1-log deficit in apoptotic death in
asmase
/
hepatocytes in
suspension culture (not shown). Thus, by two separate techniques for
hepatocyte isolation asmase
/
hepatocytes are defective in Fas-mediated apoptotic death. Unless otherwise indicated, subsequent studies used mechanical dispersion for
hepatocyte isolation.
/
hepatocytes did
not exhibit any increase in caspase 3 activity. Hence, multiple assays
confirm that lack of ASMase renders cells resistant to Jo2-induced apoptosis.
/
Hepatocytes Are
Sensitive to Staurosporine-induced Apoptosis--
To evaluate whether
the mutation leading to the asmase
/
phenotype resulted in a general inactivation of the apoptotic
machinery, hepatocytes were exposed to a different proapoptotic
stimulus. Staurosporine is a potent protein kinase inhibitor capable of inducing apoptosis by a mechanism that does not involve ASMase-mediated ceramide generation (20). As in other cell types, staurosporine failed
to generate ceramide within the first 30 min in either asmase+/+ or
asmase
/
hepatocytes (not shown).
Nonetheless, staurosporine induced apoptosis in
asmase+/+ hepatocytes within 6-8 h (Fig.
4), which peaked by 12 h (not shown). A dose as low as 0.1 µM staurosporine induced
apoptosis in 30% of the population at 12 h, whereas 75%
apoptosis was observed with 5-10 µM staurosporine (Fig.
4). In contrast to the Fas experiments, the
asmase
/
hepatocytes showed no
resistance to staurosporine-induced cell death, and the level of
apoptosis was essentially identical to that of the
asmase+/+ hepatocytes (Fig. 4). These studies
indicate that asmase
/
hepatocytes are fully competent in generating an apoptotic response and
suggest that the lack of apoptosis to anti-Fas may be specifically associated with ASMase deficiency.
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Fig. 4.
Staurosporine induces apoptosis in
asmase /
hepatocytes. The dose-response of staurosporine-induced apoptosis
after 12 h was measured by terminal nucleotidyl transferase assay
as in Fig. 3A. The data (mean ± S.D.) are compiled
from two studies performed in duplicate.
/
Hepatocytes to
Anti-Fas--
To provide definitive evidence that the lack of ceramide
is the cause of the resistance of
asmase
/
cells to anti-Fas
treatment, we pretreated asmase
/
hepatocytes with low doses of natural C16-ceramide for 10 min prior to the treatment with 1,000 ng/ml Jo2. This dose was selected because it provides the greatest discrimination in apoptotic responses between asmase+/+ and
asmase
/
hepatocytes. Fig.
5A shows that 5-25
nM C16-ceramide was without direct effect on
apoptosis, nor did it alter the apoptotic response to 1,000 ng/ml Jo2
in asmase+/+ hepatocytes. However, 5-25
nM C16-ceramide restored the sensitivity of
asmase
/
hepatocytes to Jo2
antibody. In fact, at 25 nM C16-ceramide the apoptotic response of asmase
/
hepatocytes was indistinguishable from that of
asmase+/+ hepatocytes. Identical results were
obtained using hepatocytes isolated by collagenase digestion (not
shown). The capacity of natural ceramide to rescue the ASMase-deficient
apoptotic phenotype was also evaluated using the caspase 3 biochemical
assay. As shown in Fig. 5B, natural ceramide alone at 25 nM also conferred Jo2-induced caspase activation onto
asmase
/
hepatocytes. Thus, the
addition of natural ceramide to the
asmase
/
hepatocytes rescues the
apoptotic phenotype without reverting the genotype, providing proof
that the resistance of ASMase-deficient hepatocytes to anti-Fas
antibody is mediated by the lack of ceramide generation and not by the
lack of ASMase.
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Fig. 5.
Natural C16-ceramide but not
other sphingolipids restores the sensitivity of
asmase /
hepatocytes to anti-Fas Jo2 antibody. Panel A,
hepatocytes were handled as in Fig. 3A and for some
incubations pretreated for 10 min with the indicated concentration of
C16-ceramide in dodecane:ethanol (2:98, v/v; 0.05% final
concentration) or diluent alone prior to the addition of 1,000 ng/ml
Jo2 antibody. The data (mean ± S.E.) are compiled from three
studies performed in duplicate. Panel B, caspase 3-like
activity measured by cleavage of the fluorogenic substrate Ac-DEVD-AFC.
Hepatocytes were handled as in Fig. 3A, then cell lysates
were prepared in RIPA buffer, and DEVDase activity was measured as
described under "Materials and Methods." The data (mean ± S.D.) are compiled from two studies performed in duplicate. Panel
C, hepatocytes were handled as in Fig. 4A except some
incubations received 50 nM C16-dihydroceramide
(C16 dh ceramide), sphingomyelin, sphingosine,
sphinganine, or sphingosine 1-phosphate (S 1-P), as
indicated. The data (mean ± S.E.) are compiled from five studies
performed in duplicate.
/
hepatocytes. Fig.
5C shows that although 25 nM
C16-ceramide rescued the apoptotic phenotype of
asmase
/
hepatocytes, as much as
a 50 nM concentration of other sphingolipids including
C16-dihydroceramide, sphingomyelin, sphingosine
1-phosphate, sphingosine, or sphinganine failed to rescue Jo2-induced
apoptosis. It should be noted that these other sphingolipids also did
not induce apoptosis in untreated asmase+/+ or
asmase
/
hepatocytes (not shown).
Thus, natural ceramide specifically restores Jo2-induced apoptosis to
asmase
/
hepatocytes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice display a large
deficit in anti-Fas induced death ex vivo compared with
hepatocytes from asmase+/+ littermates. In
contrast to asmase+/+ hepatocytes,
asmase
/
hepatocytes failed to
demonstrate an early ceramide elevation despite containing normal
levels of sphingomyelin. Further, the apoptosis resistance of
asmase
/
hepatocytes was overcome
by natural ceramide. This provides proof that it is the lack of
ceramide, and not ASMase, which determines the
asmase
/
phenotype. Consistent
with this notion, only natural ceramide and not other sphingolipids
could restore Fas sensitivity. Apoptosis resistance could also be
overcome by very high doses of Jo2 anti-Fas. The mechanism for this
ceramide-independent apoptosis induction by Jo2 may involve
superaggregation of trimerized Fas (31-33). However, it is not certain
that this ceramide-independent apoptotic mechanism is operative under
physiologic conditions. In this regard, two independent in
vivo events, phytohemagglutinin-induced hepatitis, and
anti-CD4-induced deletion of CD3+ and CD4+
cells, which are mediated by Fas ligand up-regulation on peripheral blood lymphocytes, are completely defective in our
asmase
/
mice despite normal Fas
ligand up-regulation (11).
/
mice
appear normal as assessed by a battery of biochemical, histological, and biological tests (7, 15, 20, 22, 23). Consistent with this
observation, the asmase
/
mice
develop normally into adulthood. In our colony, the earliest clinical
manifestation of NPD, a resting tremor, begins at 12-16 weeks of age.
Routinely, our asmase
/
mice
survive to 9.6 ± 0.4 (mean ± S.D.) months of age.
Nevertheless, asmase
/
mice and
primary cultures of cells from young
asmase
/
mice display specific
defects in apoptosis. asmase
/
hepatocytes display resistance to Fas-induced death ex vivo
and, as mentioned above, to phytohemagglutinin-induced hepatitis
in vivo (11). Similarly, murine embryonic fibroblasts
from asmase
/
mice display
complete resistance to ionizing radiation-induced apoptosis and
partial resistance to tumor necrosis factor-
/actinomycin D- and
serum withdrawal-induced apoptosis (20). In both hepatocytes and murine
embryonic fibroblasts, however, sensitivity to staurosporine is
maintained, indicating that the use of the sphingomyelin pathway within
any cell may be stress type-specific.
asmase
/
mice also display
defects in apoptosis in vivo. Endothelium in the lung and
throughout the central nervous system of these mice is almost
completely resistant to induction of apoptosis with doses as high as
100 Gray, whereas asmase+/+ littermates display
dose-dependent apoptosis beginning with as little as 5 Gray
(22, 23). ASMase-deficient mice also manifest a marked defect in the
ovarian developmental program (36). A failure to delete oocytes
normally in asmase mutant females during embryogenesis
results in ovarian hyperplasia at birth (37). Further, oocytes isolated
from adult ASMase-deficient females are markedly resistant to
doxorubicin-induced apoptotic death (36). In contrast, thymic cells
from asmase
/
mice display no
resistance to apoptosis in vivo or ex vivo by any
stimulus tested so far (7, 23). These data show the use of the
sphingomyelin pathway for induction of apoptosis is stress type- and
cell type-specific.
(48-53), 8 for ionizing radiation (23,
54-60), 11 for chemical agents (61-71), and 11 for other stresses
(72-82). Thus we cannot explain the inability of Borst and co-workers
(83) to detect early sphingolipid changes after many of the same
proapoptotic stimuli. However, we do not dispute that ceramide might be
generated in some instances, as they have suggested, at a later stage,
prior to or possibly even during the effector stage of apoptosis.
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ACKNOWLEDGEMENTS |
---|
We thank Desiree Ehleiter, Brian DeRubertis, and Niraj Gusani for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA42385 (to R. K.), CA52462 (to Z. F.), CA72632 (to Y. F.), and CA61524 (to Y. F.) and by Deutsche Forschungsgemeinschaft Grant Gu 335/2-3 (to E. G.).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: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7558; Fax: 212-639-2767; E-mail: r-kolesnick@ski.mskcc.org.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008732200
2 E. Gulbins and R. Kolesnick, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NSMase, neutral sphingomyelinase; ASMase, acid sphingomyelinase; NPD, Niemann-Pick disease; FACS, fluorescence-activated cell sorting; AFC, 7- amino-4-trifluro-coumarin.
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