From the Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD95 is a potent inducer of apoptosis. It activates the caspase cascade, but also induces ceramide (Cer) production, reportedly involving acid sphingomyelinase (aSMase) activity. A role for Cer as a second messenger for apoptosis induction was proposed, based on the finding that synthetic Cer analogues can induce cell death. We have tested whether aSMase is required for 1) apoptosis induction and 2) Cer production by CD95. For this purpose, we have used cultured Niemann-Pick disease (NPD) lymphoid cells with a defined mutation (R600H) in the aSMase protein. Despite their inherited deficiency of aSMase, we found that these cells readily undergo apoptosis upon CD95 stimulation. After retrovirus-mediated gene transfer of the aSMase cDNA, the transduced (i.e. "corrected") NPD cells showed neither increased levels of apoptosis nor altered kinetics of caspase-8 and caspase-3 activation and apoptosis induction as compared with empty vector-transduced cells. The slow sustained elevation of Cer levels in response to CD95, which we have previously documented for Jurkat T cells (Tepper, A. D., Boesen-de Cock, J. G. R., de Vries, E., Borst, J., and van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 24308-24312), was similarly found in NPD cells. Moreover, the kinetics of Cer formation remained unaffected after aSMase transduction. These results indicate that this Cer does not result from aSMase activity. We conclude that aSMase is not required for and does not facilitate CD95-mediated apoptosis and that it is not responsible for the late Cer response.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD95 (Fas/APO-1) and the tumor necrosis factor receptor p55 (TNF-R1)1 belong to the steadily growing TNF receptor superfamily (1). CD95, TNF-R1, and other apoptosis-inducing members of this family share a cytoplasmic "death domain" (2, 3), an ~80-amino acid dimerization motif that is critical for coupling to the caspase cascade (4). Via the adaptor molecule FADD (5, 6), CD95 directly recruits caspase-8 (FLICE/Mach-1) (7-10) and induces its proteolytic activation (11). Caspase-8, in turn, activates other caspase family members that are involved in the actual execution of the death program (12-14).
CD95 and TNF-R1 also induce elevation of intracellular ceramide (Cer) levels. However, data are conflicting with regard to the kinetics of the Cer response and the sphingomyelinase(s) involved. One group reported that TNF-R1 couples through its death domain via a phosphatidylcholine-specific phospholipase C to acid sphingomyelinase (aSMase), which gives rise to a modest increase in Cer levels within minutes after receptor triggering (15, 16). A more membrane-proximal region in the TNF-R1 cytoplasmic tail was found to couple to a neutral sphingomyelinase, which is also activated within minutes (17, 18). Other investigators, however, found no evidence for such a rapid Cer response and reported that TNF-R1 induces a slow sustained elevation of Cer levels over a period of hours (19). Also, data on Cer generation by CD95 are conflicting. Some authors have found a rapid transient response, within several minutes to 1 h (20, 21), which has been attributed to aSMase (22). We and others, however, have detected only a late, more sustained response, occurring over a period of hours after receptor stimulation (23, 24). This response was attributed to a neutral sphingomyelinase activity (23).
aSMase activity results from one gene product, which is localized in
acidic endosomal/lysosomal compartments and is responsible for
hydrolysis of sphingomyelin to Cer and phosphocholine. While aSMase has
an important role in lipid metabolism, as evidenced from the disease
phenotype of aSMase-deficient Niemann-Pick disease (NPD) type A and B
patients and aSMase/
mice (25-28), its role in
apoptosis is less clear. aSMase has been implicated in apoptosis
induction on the basis of two results: first, mutant CD95 molecules
lacking the death domain do not activate aSMase and do not induce
apoptosis (22); and second, aSMase-deficient human lymphoblasts and
aSMase
/
mice were found to be defective in ionizing
radiation-induced apoptosis (29). Furthermore, Cer has been associated
with apoptosis induction because synthetic, cell-permeable Cer, but not
the dihydro-Cer analogue, can induce caspase activation and apoptosis
in various cell types (20, 21, 23, 24, 30, 31).
To investigate whether aSMase is required for CD95-induced apoptosis and Cer generation, we have used Epstein-Barr virus (EBV)-transformed lymphoblasts from an NPD patient, which have a defined point mutation in the aSMase gene and lack detectable aSMase activity (29). These cells were reconstituted with the aSMase cDNA by retrovirus-mediated gene transfer, and CD95 responses in aSMase-deficient and -proficient cells were compared. From our studies, we conclude that aSMase plays no role in CD95-induced caspase activation and apoptosis induction. Moreover, Cer production took place irrespective of functional aSMase expression.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents-- L-[3-14C]Serine (54.0 mCi/mmol), [N-methyl-14C]sphingomyelin (56.0 mCi/mmol), the enhanced chemiluminescence kit, and protein G-Sepharose beads were purchased from Amersham Pharmacia Biotech. The Bradford protein assay kit was from Bio-Rad. The DOTAP liposomal transfection reagent was from Boehringer Mannheim. Silica Gel 60 TLC plates were from Merck. Puromycin was from CLONTECH (Palo Alto, CA). Nitrocellulose membranes were from Schleicher & Schüll (Dassel, Germany).
Antibodies-- Anti-CD95 mAb CH-11 was purchased from Immunotech (Marseille, France). Mouse anti-human caspase-3 mAb was purchased from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-conjugated rabbit anti-mouse Ig and swine anti-rabbit Ig were obtained from Dako (Glostrup, Denmark), and horseradish peroxidase-conjugated rabbit anti-goat Ig was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase-8 serum was raised in rabbits against a synthetic peptide comprising amino acids 2-20 of human caspase-8. Specificity of the antiserum was confirmed by immunoblotting of detergent lysates of COS-7 cells transfected with the human caspase-8 cDNA (8), which was kindly provided by Dr. M. Peter (German Cancer Research Center, Heidelberg, Germany). The 12CA5 mAb directed against the hemagglutinin (HA) tag was used as hybridoma supernatant (32). Goat anti-human aSMase serum, which was generated as described (33), was a kind gift from Drs. K. Ferlinz and K. Sandhoff (Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany).
Cells-- The EBV-transformed B cell line derived from NPD patient MS1418 is homozygous for a nucleotide substitution at position 1799 of G to A, which changes the normal arginine at residue 600 in the aSMase protein (25) to a histidine.2 The EBV-transformed B cell line JY was used as a control. Cells were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics at 37 °C and 5% CO2. COS-7 cells were routinely grown in Dulbecco's modified Eagle's medium containing 8% fetal calf serum and antibiotics. COS-7 cells were transfected with cDNA cloned into the pMT2 expression vector according to the DEAE-dextran method (34).
Retroviral Vector Construction-- The LZRS-pBMN-lacZ vector was kindly provided by Dr. G. P. Nolan (Stanford University School of Medicine, Stanford, CA) (35). We have used a modified version of this vector in which the lacZ gene was replaced by a linker region, an IRES sequence, and enhanced green fluorescent protein (eGFP) cDNA as described by Heemskerk et al. (36). The aSMase cDNA (25) was kindly provided by Dr. K. Sandhoff. An HA tag, a new stop codon, and an EcoRV site for blunt cloning were added to the 3'-end of the aSMase cDNA before cloning into the EcoRI and SnaBI sites of the linker. The correct sequence was checked by dideoxy nucleotide sequencing.
Generation of Retrovirus and Gene Transduction--
Plasmid
LZRS-aSMase-IRES-eGFP or empty vector was transfected into the
FNX-Ampho producer cell line (kindly provided by Dr. G. P. Nolan)
by calcium phosphate precipitation (34). Transfected cells were
selected with puromycin (1 µg/ml). Virus-containing supernatants from
the producer cell lines were collected, aliquoted, and stored at
80 °C until further use. MS1418 and JY cells were transduced at a
density of 0.5 × 106/ml of virus-containing
supernatant in the presence of 10 µg/ml DOTAP. After overnight
incubation, the supernatant was removed, and cells were cultured in
fresh medium. eGFP-expressing cells were sorted on a FACStar Plus
(Becton Dickinson Advanced Cellular Biology, San Jose, CA). Experiments
were performed with cell populations consisting of >80% green
fluorescent cells.
Quantitation of aSMase Activity-- In vitro aSMase activity was assayed as described by Wiegmann et al. (16) with some minor modifications. Cells (10 × 106/sample) were collected and washed twice with ice-cold phosphate-buffered saline. Cell pellets were resuspended in 0.2% Triton X-100 and lysed by sonication. Lysates were centrifuged at 4000 × g for 5 min at 4 °C, and 50 µg of protein (as determined with a Bradford protein assay kit) was assayed for aSMase activity using [N-methyl-14C]sphingomyelin at pH 5.0. After a 1-h incubation at 37 °C, lipids were extracted with chloroform/methanol (1:2, v/v). Radioactive phosphocholine in the aqueous phase was quantitated by liquid scintillation counting.
Cer Quantitation-- Cer levels were measured as described previously (24). Briefly, cells (1 × 106/ml) were labeled to equilibrium with L-[3-14C]serine (0.2 µCi/ml) for 48 h in synthetic Yssel's medium (37). Cells were washed twice in Yssel's medium and resuspended at 5 × 106/ml. Following stimulation with 500 ng/ml anti-CD95 mAb CH-11, lipids were extracted with chloroform/methanol (1:2, v/v). Total lipid was spotted on Silica Gel 60 TLC plates. After chromatography, radioactive lipids were visualized and quantitated using a Fuji BAS 2000 TR phosphoimager and identified using external lipid standards. Cer was expressed relative to phosphatidylserine and phosphatidylethanolamine levels, which remained unaltered upon stimulation.
Apoptosis Assays-- Cells were incubated overnight in Yssel's medium, seeded at 1 × 106/ml, and stimulated with anti-CD95 mAb CH-11 at the indicated concentrations at 37 °C and 5% CO2 for different time periods. All assays were performed in duplicate. To measure nuclear fragmentation, cells were harvested, washed with phosphate-buffered saline, and lysed in a hypotonic buffer containing 0.1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml propidium iodide. In this lysis buffer, the nuclei remain intact. Fluorescence intensity of propidium iodide-stained DNA was determined on a FACScan (Becton Dickinson Advanced Cellular Biology), and data were analyzed using Lysys software. Segmented apoptotic nuclei are recognized in this assay by subdiploid DNA content (38). To measure propidium iodide uptake, cells were stimulated, washed, and suspended in 10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2 containing 1 mg/ml glucose and 0.5% bovine serum albumin. Propidium iodide (final concentration of 5 µg/ml) was added immediately before analysis, and propidium iodide-stained cells were analyzed on the FACScan.
Immunoblotting-- After overnight incubation in Yssel's medium, cells were seeded at 10 × 106/ml and incubated with 500 ng/ml anti-CD95 mAb CH-11 for the indicated time periods at 37 °C and 5% CO2. Cells were lysed in 1% Nonidet P-40-containing lysis buffer (0.01 M triethanolamine HCl, pH 7.8, 0.15 M NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.02 mg/ml trypsin inhibitor, and 0.02 mg/ml leupeptin). Lysates were centrifuged at 13,000 × g for 15 min, and supernatants were mixed with concentrated reducing SDS sample buffer. Equivalents of 106 cells/lane were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes, which were subsequently blocked with 5% nonfat dry milk in Tris-buffered saline and 0.05% Tween 20 (TBST) and incubated with anti-caspase-3 mAb at 10 µg/ml or with anti-caspase-8 serum at a 1:500 dilution in TBST with 1% nonfat dry milk. After subsequent incubation with a 1:7500 dilution of horseradish peroxidase-conjugated rabbit anti-mouse Ig or swine anti-rabbit Ig, respectively, proteins were visualized by enhanced chemiluminescence. For immunoprecipitation, Nonidet P-40 lysates were cleared with normal goat serum and protein G-Sepharose beads and incubated with 2 µl of goat anti-human aSMase serum and protein G-Sepharose beads. Protein isolates were separated by SDS-polyacrylamide gel electrophoresis, and immunoblotting was performed as described above with 12CA5 hybridoma supernatant (anti-HA tag) or anti-aSMase serum at a dilution of 1:500. Antibodies were detected with horseradish peroxidase-conjugated rabbit anti-mouse Ig (1:7500) or horseradish peroxidase-conjugated rabbit anti-goat Ig (1:2000), respectively.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of NPD and aSMase-reconstituted Lymphoblasts-- To determine the contribution of aSMase to the CD95 response, we used an EBV-transformed B cell line derived from NPD patient MS1418 (29), with or without the retrovirally transduced human aSMase cDNA. As a control, we used the EBV-transformed B cell line JY. Fig. 1 shows that the aSMase gene transfer was effective. The HA-tagged cDNA product could readily be detected in anti-aSMase immunoprecipitates from aSMase-transduced MS1418 and JY cells as well as in whole cell lysates of transfected COS-7 cells. Its size of ~70 kDa corresponds to that of the mature protein as described (33). We also immunoblotted anti-aSMase immunoprecipitates with anti-aSMase serum to detect endogenous protein. Endogenous aSMase levels appeared to be very low. No signal was observed in JY cells, whereas a very weak band migrating at the same position as the cDNA product was detected in the MS1418 cells (Fig. 1). Since MS1418 carries a point mutation in the aSMase gene, these cells may well express a nonfunctional protein. From the immunoblot analysis, we conclude that gene transduction elevates aSMase protein levels by at least a factor of 10 in both NPD and JY cells.
|
|
|
No Difference in CD95-induced Apoptosis and Caspase Activation in the Presence or Absence of a Functional aSMase-- To assess the contribution of aSMase to CD95-induced apoptosis, we compared the sensitivity of NPD and JY cells to anti-CD95 mAb CH-11. It appeared that aSMase-deficient MS1418 cells readily undergo apoptosis in response to CD95 ligation. Propidium iodide uptake (Fig. 3A) and nuclear fragmentation (Fig. 3B) profiles of NPD cells were very similar to those obtained with JY cells. Apparently, functional aSMase is not an absolute requirement for CD95-induced apoptosis. To examine whether aSMase facilitated CD95-induced apoptosis, we compared the sensitivity of parental and aSMase-reconstituted MS1418 cells (Fig. 4). The presence of aSMase had no effect on the kinetics of apoptosis induction (Fig. 4, upper panels) or on the sensitivity of the cells to CD95 stimulation (lower panels). Similar results were found in JY cells, where the apoptotic response was also not altered by aSMase overexpression (Fig. 4, upper and lower panels). From these experiments, we conclude that aSMase activity is not required for and does not influence CD95-induced apoptosis in lymphoblasts.
|
|
|
CD95 Stimulation Generates Cer in aSMase-deficient Cells-- We have previously shown that in Jurkat cells, CD95 stimulation induces a slow and sustained increase in Cer levels (24). This "late" Cer response remains largely unaffected upon inhibition of caspase function and apoptosis induction by the tetrapeptide inhibitor DEVD-CHO (24), consistent with the possibility that Cer may participate in the apoptotic pathway. Therefore, it was relevant to investigate Cer formation upon CD95 ligation in aSMase-deficient NPD cells. Fig. 6 shows that upon CD95 stimulation, Cer levels gradually increased to the same extent and with similar kinetics in both MS1418 and JY cells, irrespective of the presence of a functional aSMase. We conclude that CD95 induces late Cer formation in which aSMase is not involved.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
While aSMase is important for sphingolipid turnover in cells, its
contribution to signal transduction is questionable. It was suggested
that Cer resulting from aSMase activity signals to the NF-B
transcription factor upon TNF-R1 stimulation (15, 16). However, it has
recently been established that in aSMase-deficient fibroblasts from NPD
patients or gene-targeted mice, NF-
B induction by TNF-R1 occurs
normally, clearly ruling out a function for aSMase in this signaling
pathway (39-41).
With the study reported here, we also question the role of aSMase in signaling to apoptosis by CD95. Consistent with our previous work in Jurkat T cells (24), the EBV-transformed B cells studied here have a late Cer response, but lack the CD95-induced "rapid Cer response," which has been attributed to aSMase (22). Moreover, CD95-induced activation of caspase-8 and caspase-3 and apoptosis is the same in the presence or absence of aSMase, clearly excluding aSMase from the CD95 signaling pathway leading to apoptosis, at least in lymphoid cell. Whether aSMase plays a role in CD95-induced apoptosis in other cell types should be evaluated in aSMase-deficient mice.
In contrast to CD95-mediated apoptosis (this work and Refs. 23 and 24),
radiation-induced apoptosis has been reported to involve rapid Cer
formation (29, 42). In irradiated aSMase-deficient human lymphoblasts
and aSMase/
mice, both this Cer response and apoptosis
induction were severely impeded, whereas aSMase reconstitution restored
responsiveness (29). Apparently, radiation-induced apoptosis, unlike
CD95-induced apoptosis, requires aSMase. In radiation-induced
apoptosis, Cer has been reported to signal to c-Jun kinase (42), which
may couple to the caspase cascade. CD95, however, directly activates the caspase cascade. This may explain differences in the requirement for secondary signals.
We have previously reported that CD95-induced Cer formation is not blocked by caspase-3 inhibition (24). Therefore, it is possible that this slow Cer response contributes to signaling to apoptosis. We now know that this Cer formation does not involve aSMase. Apparently, it results either from a neutral sphingomyelinase activity or from de novo biosynthesis. In either case, evaluation of the role of Cer in apoptosis induction is important and can be accomplished by attenuation of Cer levels using pharmacological or genetic approaches.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. K. Ferlinz, K. Sandhoff, G. P. Nolan, and H. Spits for gift of reagents; Drs. E. Hooijberg and M. Heemskerk for expert advice on gene transduction; E. Noteboom for excellent assistance with the fluorescence-activated cell sorting analysis; and Dr. E. H. Schuchman for sharing unpublished observations.
![]() |
Note Added in Proof |
---|
We are aware that others have found apoptosis resistance in MS1418 cells. We have been informed that the MS1418 line is subject to phenotypic changes upon continuous in vitro culture. However, we have confirmed the aSMase gene mutation (R600H) and unambigously shown the aSMase deficiency of the cells used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported by the Dutch Cancer Society.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. Tel.: 31-20-5121972; Fax: 31-20-5121989; E-mail: jborst{at}nki.nl.
1 The abbreviations used are: TNF-R1, tumor necrosis factor receptor 1; Cer, ceramide; aSMase, acid sphingomyelinase; NPD, Niemann-Pick disease; EBV, Epstein-Barr virus; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate; mAb, monoclonal antibody; HA, hemagglutinin; eGFP, enhanced green fluorescent protein.
2 E. H. Schuchman, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|