From the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, District of Columbia 20007
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Physiologic cell death occurs through an evolutionary conserved
suicide process, termed apoptosis, which plays a considerable role in
early development and homeostasis of adult tissues (1). Ceramide has
recently emerged as a critical component of apoptosis (2, 3). A variety
of stress stimuli, such as tumor necrosis factor (TNF
),1 Fas ligand, growth
factor withdrawal, anticancer drugs, oxidative stress, heat shock,
ionizing radiation, and ultraviolet light increase cellular ceramide
which, in turn, is capable of inducing apoptosis (3, 4). This apoptotic
effect is blocked by addition of the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) or
diacylglycerol, both activators of protein kinase C (PKC) (2, 5),
suggesting that PKC activation counteracts ceramide-mediated apoptosis.
Activation of PKC in diverse cell types stimulates sphingosine kinase
activity resulting in intracellular accumulation of sphingosine
1-phosphate (SPP) (6, 7). Recently, we showed that SPP prevented the
hallmarks of apoptosis resulting from elevated levels of ceramide
induced by TNF
, anti-Fas antibody, sphingomyelinase, or
cell-permeable ceramide (7). Furthermore, inhibition of PKC, as well as
inhibition of sphingosine kinase, induces apoptosis, which can be
overcome by the addition of SPP (7). These results indicate that PKC
may inhibit ceramide-induced apoptosis by activating sphingosine
kinase.
Recently, attention has been focused on the role of a novel family of
aspartate-specific cysteine proteases, called caspases, which are
intimately associated with apoptosis (reviewed in Ref. 8). Genetic
studies of the nematode Caenorhabditis elegans have led to
the identification of two genes, ced-3 and ced-4, that are required for apoptotic cell death (9). The ced-3
gene encodes a caspase similar to the prototype mammalian interleukin 1-converting enzyme (ICE) (9). Ten other homologs of ICE/CED-3 have
been identified and phylogenetically classified into three subfamilies:
(i) the CED-3 subfamily, consisting of caspase-3 (CPP32/Yama/apopain),
caspase-6 (Mch2), caspase-7 (Mch3/ICE-LAP3/CMH-1), caspase-8
(FLICE/MACH/Mch5), caspase-9 (ICE-LAP6/Mch6), and caspase-10 (Mch4);
(ii) the ICE subfamily, which includes caspase-1 (ICE), caspase-4
(TX/ICH2/ICE rel-II), caspase-5 (TY/ICE rel-III), and caspase-11
(ICH3); and (iii) the Nedd-2 subfamily, which is represented by
caspase-2 (ICH1/murine Nedd-2).
Previous studies have identified a number of substrates for the caspases, particularly the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) (10) and the nuclear lamins (11). Proteolysis of these substrates may account for many of the biochemical and morphological nuclear changes associated with apoptosis. The early cleavage of PARP can be catalyzed by several caspases belonging to the CED-3 subfamily (10, 12-14). Of these, caspase-3/CPP32 is thought to be the most efficient PARP protease (10, 12), followed by caspase-7/Mch3 (13). The degradation of nuclear lamins is required for packaging of the condensed chromatin into apoptotic bodies, a typical morphology observed during the late stages of apoptosis (11). Caspase-6/Mch2 is the only known caspase capable of cleaving lamins (14, 15).
Although the relative intracellular levels of ceramide and SPP have
been proposed to be a critical gauge of cell fate (7), the molecular
mechanism of actions of these sphingolipid metabolites are not well
understood. Recently, ceramide has been implicated in PARP
cleavage (16, 17) and caspase-3/CPP32 activation (18). In addition, it
appears that ceramide can be generated via a CrmA-inhibitable caspase,
most likely caspase-8/FLICE, since overexpression of CrmA blocks both
cell death and ceramide accumulation induced by TNF, while exogenous
ceramide can bypass this sensitive step and induce apoptosis (16).
In this study, the relationship between sphingolipid metabolites and the caspases was examined. We demonstrated that both SPP and TPA decrease PARP cleavage by inhibiting activation of caspase-3/CPP32 and caspase-7/Mch3 induced by Fas ligation or cell-permeable ceramide. Furthermore, we also report that SPP and TPA inhibit activation of caspase-6/Mch2 and subsequent lamin B cleavage. Finally, ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- Anti-Fas monoclonal antibody (clone CH-11) was from Upstate Biotechnology (Lake Placid, NY). C2-ceramide and SPP were from Biomol (Plymouth Meeting, PA). Ac-DEVD-AMC was from Bachem (King of Prussia, PA). [methyl-3H]Thymidine (70-90 Ci/mmol) was from NEN Life Science Products. Peroxidase-conjugated anti-goat IgG (Mch2) was from Boehringer Mannheim. Anti-mouse (lamin B1) and anti-rabbit (CPP32, Mch3 and FLICE) IgG conjugated to peroxidase were from Bio-Rad.
Cell Culture-- Jurkat T cells (clone E6-1, ATCC, Rockville, MD) were grown in RPMI 1640 supplemented with 10% fetal bovine serum. On the day of the experiment, cells were washed twice in RPMI 1640 containing 5 µg/ml transferrin and 5 µg/ml insulin in place of serum and resuspended in this serum-free medium (0.75-1 × 106 cells/ml).
DNA Fragmentation Assay and Staining of Apoptotic Nuclei-- DNA fragmentation was measured in cells prelabeled with [methyl-3H]thymidine (0.5 µCi/ml) for 16 h (19). Cells were washed twice, resuspended in serum-free medium, plated in 12-well plates (3 × 106 cells/well), and then exposed to the indicated agents. After 3 h, cells were lysed in TTE (10 mM Tris (pH 7.4), 10 mM EDTA, 0.2% (v/v) Triton X-100) and incubated for 15 min on ice. Fragmented DNA and intact chromatin were separated by centrifugation at 12,000 × g for 10 min. Pellets were resuspended in TTE, and trichloroacetic acid was added to a final concentration of 12.5% (v/v). DNA fragmentation was calculated as follows: percent DNA fragmentation = (fragmented/(fragmented + intact chromatin)) × 100. All results were determined in triplicate and expressed as means ± standard deviations.
Apoptosis was also assessed by staining cells with bisbenzimide trihydrochloride (8 µg/ml in 30% glycerol/PBS; Hoechst 33258, Calbiochem, San Diego, CA) for 10 min. Cells were then examined with a Zeiss Photoscope II fluorescent microscope (Petersburg, VA).Cleavage of in Vitro Translated Poly(ADP)-ribose
Polymerase--
A full-length cDNA clone for PARP (gift of Dr.
Donald Nicholson) was used to drive the synthesis of PARP labeled with
[35S]methionine (NEN Life Science Products) by coupled T7
transcription/translation in a reticulocyte lysate system (Promega,
Madison, WI). [35S]PARP was purified by gel filtration
chromatography on a Superdex-75 FPLC column (Pharmacia Biotech Inc.)
using 10 mM HEPES-KOH (pH 7.4) buffer containing 2 mM EDTA, 0.1% (w/v) CHAPS, and 5 mM
dithiothreitol. Cytosolic extracts were prepared by homogenizing
PBS-washed cell pellets in 10 mM HEPES-KOH (pH 7.4), 2 mM EDTA, 0.1% (w/v) CHAPS, 5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, and 20 µg/ml leupeptin
(Buffer A). PARP cleavage activity was determined by incubating 10 µg of cytosolic protein from Jurkat T cells, [35S]PARP
(~5 × 104 cpm), 10 mM PIPES-KOH, 2 mM EDTA, 0.1% (w/v) CHAPS, and 5 mM dithiothreitol in a total volume of 25 µl. Samples were incubated at
37 °C for 1 h and reactions terminated by the addition of 25 µl of 2× SDS-PAGE sample buffer containing 4% (w/v) SDS, 4% (v/v) -mercaptoethanol, 10% (v/v) glycerol, 0.125 M Tris-HCl
(pH 6.8), and 0.02% (w/v) bromphenol blue. After heating for 10 min at
95 °C, samples were resolved by 10% SDS-PAGE. Gels were fixed in
20% (v/v) methanol, 5% (v/v) acetic acid for 30 min, equilibrated with 1 M salicylic acid (pH 6.5) for 15 min, and protein
bands visualized by fluorography. Gels were also scanned on a Storm 840 imaging system and the 116- and 89-kDa PARP bands analyzed using
ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Western Blotting-- Cytosolic proteins (20 µg) were denatured by boiling in Laemmli buffer (Bio-Rad) for 3 min, then separated on 15% SDS-polyacrylamide gels, and blotted to nitrocellulose. Nonspecific binding was blocked by incubation of the membranes with 0.5% (v/v) Tween/PBS containing 5% (w/v) nonfat dry milk for 2 h at room temperature. Membranes were incubated for 2 h with rabbit antisera specific for the p17 subunit of caspase-3/CPP32 (gift of Dr. Donald Nicholson), goat antisera specific for the p21 subunit of caspase-6/Mch2 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-lamin B (Calbiochem, San Diego, CA), rabbit antisera specific for the p20 subunit of caspase-7/Mch3, or rabbit antisera specific for the p20 subunit of caspase-8/FLICE (gift of Dr. Edward Gelmann). After washing with 0.5% (v/v) Tween/PBS, membranes were incubated for 2 h with horseradish peroxidase-conjugated secondary antibody. After three additional washes with 0.5% (v/v) Tween/PBS, SuperSignal enhanced chemiluminescent substrate (Pierce, Rockford, IL) was used to detect the proteins. Films were also scanned and analyzed by densitometry.
Fluorogenic DEVD Cleavage Enzyme Assays-- Enzyme reactions were performed in 96-well plates and contained 20 µg of cytosolic proteins in 100 µl of Buffer A, diluted with 100 µl of fresh Buffer A containing 40 µM Ac-DEVD-AMC substrate. Fluorescent aminomethyl coumarin (AMC) product formation was measured over a 30-min period at an excitation wavelength of 360 nm and emission at 460 nm using a Cytofluor II fluorometer plate reader (PerSeptive Biosystems, Framingham, MA). Serial dilutions of AMC (Aldrich) were used as standards.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sphingosine 1-Phosphate and TPA Inhibit Fas- and C2-Ceramide-mediated Apoptosis-- As described previously (7, 18, 20), Jurkat cells treated with Fas antibody underwent extensive cell death within 3 h as measured by the quantitative DNA fragmentation assay (Fig. 1A). Blebbing of cell membrane was observed and cells were fragmented into characteristic condensed nuclei and apoptotic bodies (Fig. 1C). In agreement with our previous study (7), when cells were treated simultaneously with TPA, DNA fragmentation was completely inhibited, while co-treatment with SPP reversed apoptosis by ~50% (Fig. 1, A, D, and E). Ceramide has been proposed to play a role in the Fas signaling pathway (20, 21). Treatment of Jurkat T cells with the cell-permeable C2-ceramide (10 µM) resulted in cell death that was overcome either by co-treatment with TPA or to a lesser extent by SPP (Fig. 1F). Stimulation of sphingosine kinase leading to intracellular accumulation of SPP has been shown to be one of the effects triggered by activation of PKC (6, 7). Moreover, activation of PKC by phorbol esters limits ceramide production (7, 22). In agreement, the PKC inhibitors, staurosporine, chelerythrine chloride, and calphostin C, induce sphingomyelin hydrolysis to generate ceramide (23, 24). These studies suggest that PKC activation may oppose the Fas apoptotic pathway, both by activation of sphingosine kinase and by reducing the levels of ceramide.
|
Sphingosine 1-Phosphate and TPA Inhibit Activation of PARP Cleavage
Induced by Fas Ligation and Ceramide--
Because Fas ligation leads
to proteolytic cleavage of PARP (12, 25-27), it was of interest to
determine whether SPP and TPA could inhibit this cleavage. In agreement
with previous results (25-27), extracts prepared from cells exposed to
anti-Fas for 3 h were able to cleave in vitro
translated [35S]PARP substrate into the apoptotic 89-kDa
fragment (Fig. 2A). Densitometric analysis revealed that almost 70% of the 116-kDa full-length PARP was converted to the 89-kDa fragment, while only 38%
was cleaved by extracts from control cells. SPP treatment reduced
cleavage of PARP induced by Fas ligation by over 50% and as expected,
treatment with TPA completely prevented Fas-induced PARP proteolysis.
Ceramide generation has recently been shown to precede PARP
cleavage in TNF-mediated apoptosis (16). In addition,
treatment of Molt-4 or MCF-7 cells with exogenous
C6-ceramide results in cleavage of PARP (16, 17),
suggesting that ceramide activates a caspase responsible for PARP
cleavage. In accordance with these studies (16, 17), we found that
C2-ceramide also induced PARP cleavage (Fig. 2B)
and co-treatment with TPA fully prevented this cleavage (Fig.
2B), while SPP co-treatment inhibited PARP proteolysis by
60-90% (Fig. 2B). These results clearly indicate that SPP
can inhibit PARP cleavage induced either by Fas ligation or exogenous
ceramide.
|
Sphingosine 1-Phosphate and TPA Prevent Activation of Caspase-3/CPP32 and Caspase-7/Mch3-- Caspase-3/CPP32 is known as the most efficient PARP-cleaving caspase, with a Km of ~10 µM (10, 12). Caspase-7/Mch3, which shows the highest homology to human caspase-3/CPP32, can also cleave PARP, albeit at a slightly lower efficiency (13). We used the fluorogenic substrate, Ac-DEVD-AMC, which corresponds to the motif that is cleaved in PARP (10), to measure the activity of these caspases. Extracts from cells exposed to anti-Fas showed a significant increase in caspase-3-like proteolytic activity (Fig. 3A), as described previously (18, 27). In contrast, co-treatment with TPA completely blocked this activity, while SPP co-treatment markedly reduced Fas-induced caspase activity (Fig. 3A). Proteolytic processing of pro-caspase-3/CPP32 in response to Fas was also examined by Western blotting using antisera specific for the active p17 subunit. This caspase is synthesized as a 32-kDa precursor (p32) that is cleaved to generate the mature form composed of 17-kDa (p17) subunit, through an intermediary 20-kDa (p20) form, and 12-kDa (p12) subunit (10, 12, 28). In agreement with previous studies (25, 27), following Fas activation, the p32 precursor was cleaved into active caspase-3 (Fig. 3B). Caspase-3 maturation was completely blocked by TPA, as active subunit was not detected in cells treated with both anti-Fas and TPA. SPP also prevented Fas-mediated caspase-3/CPP32 processing by over 60% as measured by densitometry (Fig. 3B).
|
|
|
SPP and TPA inhibit FAS- and Ceramide-mediated Caspase-6/Mch2 Activation and Subsequent Lamin B Degradation-- In addition to the breakdown of the nuclear enzyme PARP, another event that is common to apoptosis is the cleavage of lamins, which play major roles in nuclear envelope integrity (11). To date, caspase-6/Mch2 is the only known laminase (14, 15). Engagement of Fas in Jurkat T cells has been shown to activate caspase-6 (14) and consequently trigger the cleavage of lamin B (26). Thus, we determined whether ceramide treatment could also induce caspase-6 activation and lamin B cleavage. Lamin B is cleaved into a characteristic 28-kDa fragment after treatment with Fas or exogenous C2-ceramide (Fig. 6, A and B). Western blotting of extracts from cells co-treated with TPA and Fas antibody (Fig. 6A) or C2-ceramide (Fig. 6B) displayed only intact lamin B, indicative of inhibition of cleavage. When cells were treated with SPP during Fas- (Fig. 6A) or C2-ceramide-mediated apoptosis (Fig. 6B), lamin B proteolysis was markedly reduced. We next determined if caspase-6 was protected by SPP and TPA from activation caused by Fas- and C2-ceramide treatment. In accordance with previous studies (14), Fas antibody was able to trigger caspase-6 cleavage, generating 21-, 18-, and 14-kDa fragments (Fig. 7A). Treatment with C2-ceramide similarly led to processing of caspase-6 (Fig. 7B). When cells were co-treated with TPA or SPP, expression of active forms of caspase-6, especially p18 and p14, was strongly reduced (Fig. 7, A and B). Therefore, SPP, like TPA, can attenuate proteolytic cleavage of caspase-6 and its target, nuclear lamins, during Fas- or C2-ceramide-induced apoptosis.
|
|
Fas-induced Caspase-8/FLICE Activation Is Not Inhibited by SPP or
TPA--
Apoptosis and ceramide accumulation induced by TNF- or Fas
are completely inhibited by CrmA, a product of the cowpox virus, while
exogenous ceramide is able to bypass this block and induce apoptosis by
activating downstream caspases (16). CrmA is a potent inhibitor of ICE
(30). However, the importance of caspase-1/ICE itself in Fas-mediated
apoptosis is controversial (Refs. 31 and 32 versus Refs.
27 and 33). Instead, caspase-8/FLICE, the most upstream caspase
implicated in Fas- and TNF
-mediated apoptosis, appears to be the
target of CrmA in vivo (34). Thus, it was of interest to
determine the involvement of sphingolipid metabolites in activation of
caspase-8. Fas-ligation induced the appearance of a p20 band indicative
of cleavage and corresponding to the mature form of FLICE (Fig.
8). In contrast, C2-ceramide treatment did not induce cleavage of caspase-8 (Fig. 8). This is in
agreement with the concept that ceramide acts downstream of a
CrmA-inhibitable caspase. Finally, neither SPP nor TPA have an
inhibitory effect on Fas-triggered caspase-8/FLICE cleavage (Fig.
8).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Within the last few years, new studies have been reported, which
have enhanced our understanding of how cell surface events are
communicated to the cell suicide machinery (21, 35, 36). Protein-protein interactions link death domains of cell surface receptors for TNF- (TNF receptor-1) or Fas ligand (CD95/Fas/APO-1) to a cascade of ICE/CED-3-homologous proteases. Binding of the adaptor
protein FADD, which also contains a death effector domain, either
directly to CD95/Fas/APO-1 or to TNF receptor-1 via another death
domain-containing protein TRADD (35), recruits caspase-8 to the plasma
membrane, resulting in autocatalytic activation of the pro-apoptotic
proteases (36). Among the caspases identified so far, caspase-3, the
closest relative to C. elegans CED-3, is the pivotal
protease involved in Fas-induced apoptosis (27, 33). Caspase-3 not only
cleaves the nuclear protein PARP (10, 12), it is also capable of
downstream activation of both laminase caspase-6 (37) and caspase-7
(13) (Fig. 9). Once activated, caspase-7
and caspase-6 can process their distinctive targets, PARP and lamins,
respectively. Potential sites for interactions between sphingolipid
metabolites and the caspase cascade were investigated in this
study.
|
Although our understanding is not complete, several lines of evidence
suggest that ceramide plays an important role in apoptosis induced by
cytokines, chemotherapeutic agents, serum withdrawal, or ionizing
radiation, and does not arise merely as a consequence of activation of
the death machinery (reviewed in Ref. 3). First, overexpression of the
proto-oncogene Bcl-2, a CED-9 homolog, or treatment with SPP, both of
which prevent apoptosis, did not interfere with ceramide formation
induced by various insults (7, 16, 38), yet did protect against
apoptosis induced by ceramide (7, 16, 17, 38, 39). Second, ceramide
production and apoptosis induced by Fas ligand or TNF- can be
blocked by overexpression of the viral serpin CrmA (16), whose in
vivo target is most likely caspase-8 (34). In agreement, we show
here that ceramide does not induce activation of caspase-8 (Fig. 8).
Third, the cell-permeable analog C2- or
C6-ceramide induce activation of caspase-3 (18, 40) (Fig.
4) and subsequent PARP cleavage (17) (Fig. 2). Furthermore, our study
demonstrates that exogenous C2-ceramide also activates PARP
protease caspase-7 (Fig. 5), the closest homolog to caspase-3, acting
through caspase-3 itself (13) or independently. Finally, we describe
here for the first time that C2-ceramide induces lamin B
cleavage (Fig. 6), likely mediated by caspase-6 activation (Fig. 7).
The mechanism by which ceramide stimulates proteolytic processing of
caspase-6 is still unclear, but it is tempting to speculate that this
may also be mediated via caspase-3 as proposed by Alnemri and
co-workers (41). Taken together, our observations highlight the
relationship between ceramide and the caspase cascade, and demonstrate,
in agreement with other studies (16), that ceramide can be placed
downstream of the initiator caspase-8 and upstream of the executioner
CPP32-like proteases (Fig. 9).
Although SPP was originally shown to rescue cells from ceramide-mediated cell death (7), it was not clear how it was linked to the deadly caspase cascade. Our results demonstrate that cleavage of the death substrates, PARP and lamins, induced by Fas antibody or exogenous ceramide, is inhibited by co-treatment with SPP. In addition, we found that activation of the caspases directly responsible for the breakdown of these substrates by Fas ligation or ceramide is also inhibited by SPP. Our results establish that SPP functions upstream of caspase-3/CPP32. Nevertheless, because the proximal molecular targets by which ceramide activates CPP32 and related CED-3 subfamily caspases remain largely unknown, further work is necessary to identify the targets for ceramide and SPP.
Several reports have provided evidence that some of the effects of SPP are mediated through cell surface receptors (3, 4). However, the anti-apoptotic effects of SPP appear to be mediated via intracellular targets, as inhibition of sphingosine kinase by N,N-dimethylsphingosine not only eliminates formation of SPP induced by TPA, it also induces apoptosis and prevents the cytoprotective activity of TPA which can be restored by addition of SPP (7).
Recently, it has been suggested that the stress-activated protein kinase (SAPK/JNK) pathway is required for ceramide-mediated apoptosis because overexpression of dominant-negative constituents of the JNK pathway abrogates ceramide-mediated apoptosis (42). It has also been well documented that Fas ligation induces SAPK/JNK activation in Jurkat T cells (43-45). Moreover, we have shown previously that SPP not only stimulates the extracellular signal-regulated kinases Erk1 and Erk2, it also prevents SAPK/JNK activation by ceramide and consequent apoptosis (7). Thus, we have proposed that the formation of distinct sphingolipid metabolites and consequent regulation of different family members of the mitogen-activated protein kinase family is an important factor determining the fate of cells (7). However, the relationships between activation of the caspase cascade and the mitogen-activated protein kinase signaling pathway leading to apoptosis have not yet been clarified. Recent evidence supports a parallel pathway model leading to apoptosis, in which the Fas-binding proteins, FADD and Daxx, activate the SAPK/JNK pathway and the caspase cascade independently (46). It is very intriguing that the sphingolipid metabolites, ceramide and SPP, have opposing effects on these two important apoptotic pathways downstream of Fas. Identification of the proximal intracellular targets of these sphingolipids should provide useful clues to the regulation cell survival.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Donald Nicholson (Merck-Frosst Center for Therapeutic Research, Quebec) and Dr. Edward Gelmann (Georgetown University Medical Center) for generous gifts of reagents.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM43880 and CA61774 and by American Cancer Society Grant DE-275 (to S. S.).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.
Supported by a fellowship from La Ligue Nationale Contre Le Cancer
(France).
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Georgetown University Medical Center, 353 Basic Science Bldg., 3900 Reservoir Rd. N.W., Washington, DC 20007. Tel.: 202-687-1432; Fax: 202-687-0260; E-mail: spiegel{at}biochem1.basic-sci.georgetown.edu.
1
The abbreviations used are: TNF, tumor
necrosis factor
; Ac-DEVD-AMC,
acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin; DEVD, Asp-Glu-Val-Asp; ICE,
interleukin1
-converting enzyme; PARP, enzyme poly(ADP-ribose)
polymerase; PKC, protein kinase C; SPP, sphingosine 1-phosphate; TPA,
12-O-tetradecanoylphorbol-13-acetate; PBS,
phsophate-buffered saline; JNK, c-Jun N-terminal kinase; SAPK,
stress-activated protein kinase; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|