Apoptotic Signal of Fas Is Not Mediated by Ceramide

1998 91: 2658-2663

Shu-Ching Hsu, Chia-Cheng Wu, Tien-Yau Luh, Chen-Kung Chou, Shau-Hwa Han and Ming-Zong Lai

Apoptotic Signal of Fas Is Not Mediated by Ceramide

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Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published semimonthly

Apoptotic Signal of Fas Is Not Mediated by Ceramide

By Shu-Ching Hsu, Chia-Cheng Wu, Tien-Yau Luh, Chen-Kung Chou, Shau-Hwa Han, and Ming-Zong Lai

mediating the apoptotic signal for Fas engagement. By using different inhibitors, we demonstrated here that ceramide is unlikely a mediator of Fas-initiated apoptosis. First, cAMP prevented cell death induced by ceramide but not by Fas. Second, ceramide-triggered, but not Fas-triggered, apopto-sis was antagonized by the free radical scavenger C60. Third, the metal chelator pyrrolidinedithiocarbamate suppressed ceramide-initiated DNA fragmentation but had no effect on

the Fas-induced cell death. Fourth, the SAPK/ERK kinase dominant negative mutant, which attenuated ceramide-induced cell death, did not prevent Fas-ceramide-induced apoptosis. Finally, activation of NF-kB inhibited ceramide-induced but not Fas-initiated apoptosis. The fact that many antagonists of ceramide-induced apoptosis could not suppress Fas-mediated cell death clearly indicates that ceramide is not the mediator for Fas-initiated apoptotic signal.

r

F

as (APO-1) IS A 45-kD membrane protein when engaged

by anti-Fas antibody or Fas ligand triggers programmed

cell death (for review, see Nagata

_{). The death pathway initiated}

from Fas activation involves a series of death-induced

mol-ecules.

_{FADD (Fas-associating protein with death domain) or}

MORT1 is recruited to Fas upon its engagement.

_{FADD then}

binds FLICE (FADD-like ICE) or MACH (MORT-1–associated

CED-3 homologue).

_{The association with the Fas}

death-inducing signaling complex activates FLICE,

_{followed by}

eventual activation of ICE and CPP32.

The activation of acidic sphingomyelinase that leads to the

hydrolysis of sphingomyelin and generation of ceramide has

also been suggested as the apoptotic pathway downstream of

Fas ligation.

_{Ceramide is reported as a common}

intermedia-tor for stimulation by tumor necrosis facintermedia-tor (TNF),

interleu-kin-1, nerve growth factor, lipopolysaccharide, ionizing

radia-tion, serum withdrawal, and daunorubicin.

_{The role of}

ceramide in the apoptosis induced by some of these stimuli is

illustrated by the ability of membrane-permeable ceramide to

trigger cell death.

_{In addition, defects in the}

sphingomy-elinase/ceramide pathway confers the resistance to radiation-,

TNF-

a–, and UV-induced apoptosis,

_{supporting the role of}

ceramide in these types of death induction. Ceramide-induced

cell death involves the activation of c-Jun N-terminal kinase

(JNK).

In this study, we compared the sensitivity of Fas and

ceramide to various regulatory reagents. We showed that

ceramide-induced apoptosis was antagonized by cAMP, C

,

and pyrrolidinedithiocarbamate (PDTC). However,

Fas-trig-gered cell death proceeded in the presence of these inhibitors. In

addition, the negative mutant of SAPK/ERK kinase (SEK)

suppressed ceramide-triggered death but did not prevent

Fas-induced apoptosis. Furthermore, activation of NF-

kB

antago-nized ceramide but not Fas-initiated apoptosis. These

observa-tions are not consistent with the model that ceramide acts

downstream of Fas signaling. Together with recent

observa-tions,

_{our results argue against a role of ceramide in}

Fas-mediated apoptosis.

MATERIALS AND METHODS

Reagents and cell lines. 12-O-tetradecanoylphorbol 13-acetate (TPA), A23187, N6_{, 28-O-dibutyryladenosine 38,58-cyclic}

monophos-phate (Bt2cAMP), forskolin, and PDTC were purchased from Sigma

Chemical Co (St Louis, MO). Anti-Fas antibody CH-1126 _was

pur-chased from Upstate Biotech Inc (Lake Placid, NY). C2-ceramide and

C6-ceramide were obtained from Biomol (Plymouth Meeting, PA). ICE

inhibitor z-VAD-FK was purchased from Kamiya (Thousand Oaks, CA). The two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60derivatives (carboxylfullerenes) were synthesized

as previously described.27_{Both C}

60 (C3) and C60(D3) are effective

scavengers of oxgen radicals, with complete elimination of hydroxyl radicals and superoxide radicals at concentrations of 5 to 50 µmol/L, and are potent inhibitors of neuronal apoptosis, which is associated with increased intracellular free radical production.27_{H-89 and H-85 were}

purchased from Seikagaku (Tokyo, Japan). Human T lymphoblastomas CEM (ATCC CCL 119) and human T-cell leukemia Jurkat (ATCC TIB 152) were obtained from American Type Culture Collection (Rockville, MD). Recombinant human TNF-a was purchased from R&D (Minne-apolis, MN).

Plasmids. CMV-RelA(p65) andkB-TATA-CAT28_{were kind gifts}

of Dr Warren C. Greene (University of California, San Francisco, CA). HA-JNK129_{was obtained from Michael Karin (University of}

Califor-nia, San Diego, CA). SEK-negative mutants SEK1(K=R) and SEK1(A=L)30 _{were obtained from Dr Leonard I. Zon (Harvard}

Medical School, Boston, MA). Green fluorescence protein expression vector pEGFP-N1 was obtained from Clontech (Palo Alto, CA).

Transfection. T cells (1.63 107_{) were washed once with STBS (25}

mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 5 mmol/L KCl, 0.6 mmol/L Na2HPO4, 0.7 mmol/L CaCl2, 0.5 mmol/L MgCl2) and

incubated with a total of 10 µg DNA in 1.2 mL STBS containing 0.5 mg/mL diethylaminoethyl (DEAE)-dextran for 20 minutes at room temperature. T cells were then treated with 15% dimethylsulfoxide for 3 minutes and washed once with STBS.31,32 _{After 24 to 48 hours,}

From the Graduate Institute of Microbiology, National Taiwan University School of Medicine, Taiwan, China; the Institute of Molecu-lar Biology, Academia Sinica, Taiwan, China; the Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Tai-wan, China; the Department of Chemistry, National Taiwan University, Taiwan, China; and the Department of Medical Research, Veteran General Hospital, Taipei, Taiwan, China.

Submitted October 14, 1997; accepted December 31, 1997. Supported by Grant No. DOH86-HR-508 from the Department of Health, by Grant No. NSC 85-2331-B001-050 M30 from National Science Council, and by a grant from Academia Sinica, Taiwan, Republic of China.

Address reprint requests to Ming-Zong Lai, PhD, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘adver-tisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

r

transfected cells were then stimulated with anti-Fas or ceramide, and the cell death was quantitated.

JNK activity assay. The inhibition of JNK by the dominant negative form of SEK was performed with cotransfection of HA-JNK1. T cells were activated as indicated, washed twice with phosphate-buffered saline (PBS), and lysed in ice-cold lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 1% Triton X-100, 10% glycerol, 137 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 50 mmol/L NaF, 1 mmol/L Na3VO4, and 1

mmol/L phenylmethylsulfonyl fluoride). Detergent-insoluble material was removed by centrifugation at 14,000g for 10 minutes at 4°C. For each immuno-precipitation, 200 µg of cell lysate was mixed with anti-HA 12CA5 (Boehring Mannheim, Mannheim, Germany) and incubated at 4°C for 2 hours. Twenty microliters of protein A-sepharose (Pharmacia, Uppsala, Sweden) was then added and incubated for an additional 2 hours at 4°C. Immune complexes were washed three times with lysis buffer and once with kinase buffer (30 mmol/L Tris-HCl, pH 8.0, 20 mmol/L MgCl2, 2 mmol/L MnCl2). Immune complexes were

then incubated in kinase buffer (30 µL) containing 2 mmol/L ATP, GST-c-Jun(1-79), and 5 µCi of (g-32_{P)ATP for 30 minutes at 30°C.}

Incubations were terminated by adding 15 µL of 33 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiling for 3 minutes. The reaction products were resolved on 15% SDS-PAGE followed by autoradiography and quantitated by Phospho-rimager (Molecular Dynamics, Sunnyvale, CA).33

Cell death measurement. All cultures (except those treated with ceramide) were performed in RPMI with 10% fetal calf serum (both from GIBCO, Grand Island, NY), 10 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 23 1025mol/L 2-ME.34_For

apoptosis induced with ceramide, serum free-medium were used throughout the experiment. The extent of apoptosis was determined by propidium iodide (PI) staining or annexin V staining. Cells were treated with different inducers and/or inhibitors, washed with PBS, and fixed with ethanol. DNA content was determined by staining with 20 µg/mL PI and analyzed by FACScan (Becton Dickinson, Mountain View, CA). The fraction of cells with sub-G1DNA content was assessed with the

CELLFIT program (Becton Dickinson). For annexin V staining, the treated cells were washed, resuspended in annexin V-fluorescein isothiocyanate (FITC) (1 µg/mL; Clontech), incubated at room tempera-ture for 15 minutes in the dark, and analyzed on FACScan. For the ceramide/Fas sensitivity of cell transiently transfected with CMV-p65 or pEBG-SEK(A=L), survival was monitored with green fluorescence protein expression vector pEGFP-N1. Treated cells were examined using a Nikon Diaphot 2000 fluorescence microscope (Tokyo, Japan) 48 hours after transfection.

RESULTS AND DISCUSSION

cAMP prevented ceramide-induced cell death but had no

effect on Fas-initiated apoptosis.

Ceramide is a potent inducer

of cell death in T-lymphoma cells Jurkat and CEM (not shown

for CEM). Cell death was assessed by DNA fragmentation as

represented by sub-G

fraction (Fig 1B). DNA fragmentation

induced by ceramide never exceeded 45%, despite extensive

cell death. A similar observation was also earlier reported.

_We

first identified a few reagents that could block

ceramide-initiated cell death. cAMP was an effective antagonist of

ceramide-induced cell death (Fig 1C). Forskolin (10 µmol/L)

suppressed DNA fragmentation initiated by ceramide by at least

50%. A similar extent of inhibition was observed with dibutyryl

cAMP (Bt

cAMP) at 1 mmol/L (Fig 2A). There was a

dose-dependent inhibition of cAMP on ceramide-triggered cell death.

The inhibition of ceramide-induced cell death could be detected

with Bt

cAMP as low as 100 µmol/L. In contrast, apoptosis

triggered by anti-Fas antibody CH-11 was resistant to cAMP.

Treatment with forskolin up to 50 µmol/L or Bt

cAMP up to 2.5

mmol/L did not reduce the apoptosis triggered by Fas (Fig 2B).

cAMP agonists by itself did not induce any T-cell death at the

highest concentrations used here.

The observation that Fas-initiated cell death cannot be

prevented by cAMP is consistent with an earlier report,

_{yet is}

in direct contrast to the prominent inhibitory effect of cAMP

recently documented.

_{Because DNA fragmentation may not}

well represent actual apoptosis,

_{we also quantitated cell}

death by annexin V staining. Figure 3 shows that Fas-induced

apoptosis was associated with an extensive phosphatidylserine

translocation, which was clearly not inhibited by cAMP. cAMP

is an antagonist for ceramide-induced cell death but not for

Fas-initiated apoptosis.

C

and PDTC prevented ceramide-induced cell death but

had no effect on Fas-initiated apoptosis.

We have futher

observed that two malonic acid derivatives of C

(carboxy-fullerens)

_{were antagonist of ceramide-induced cell death}

(Figs 1D and 2A). The pure carbon sphere of C

(buckminster-fullerene) is known for its avid reactivity with free radicals, yet

the usage is limited by its water insolublility. The two

regioiso-forms with C3 or D3 symmetry of water-soluble C

derivatives

remain as potent free radical scavengers and are effective

antagonists of apoptotic neronal death induced by serum

deprivation, glutamate receptor agonists, and amyloid peptide.

Fig 1. The inhibition of ceramide-induced cell DNA fragmentation. Jurkat T cells were treated with 5mmol/L of C2-ceramide (C2-C) in the absence or presence of various antagonists for 4 hours, washed with PBS, and fixed with ethanol. DNA content was determined by staining with 20mg/mL PI and analyzed by FACScan (Becton Dickin-son). Fraction of cells with sub-G1DNA content (M1 fraction in the diagram) were assessed with CELLFIT program (Becton Dickinson). CTR, untreated cell control. The inhibitors used were as follows: forskolin (F), 10mmol/L; C60D3 (D3), 100mmol/L; PDTC, 200 mmol/L; Z-VAD-FK (ICEi), 300mmol/L. The sub-G1fractions were less than 6% for cells treated with inhibitors only. The exception was PDTC, in which a slightly elevated background death (8%) was observed.

Ceramide-triggered apoptosis was suppressed by 50% in the

presence of 10 µmol/L C

(D3) and was inhibited by 90% with

100 µmol/L C

(D3). We have repeatedly observed that C

(D3) was more effective than C

(C3) in the prevention of

ceramide-induced cell death (Fig 2A). In contrast, C

(D3) and

C

(C3) (at 100 µmol/L each) had no preventive effect on

Fas-triggered cell death as analyzed by both sub-G

fraction and

annexin V binding (Figs 2B and 3D). The same contrast was

found with metal chelator PDTC. High concentration of PDTC

antagonized ceramide-induced apoptosis (Figs 1E and 2A), but

did not inhibit Fas-initiated cell death (Fig 2B). Of all the

inhibitors analyzed in this study, PDTC was the only reagent

that by itself increased spontaneous cell death at the

concentra-tion used (1% to 3% over control, data not shown). Because

such increase was minute, the differential effect of PDTC on

ceramide- and Fas-initiated apoptosis was still prominent. As a

control, the potent ICE proteases inhibitor z-VAD-FK

com-pletely abolished cell death induced by either ceramide or Fas

(Figs 1, 2, and 3), consistent with the notion that both

ceramide-and Fas-induced cell death are known to involve caspases such

as ICE.

Therefore, we have identified three types of reagents that

antagonized ceramide-induced cell death but did not prevent

Fas-induced cell death. Between 50% and 90% of

ceramide-triggered cell death was suppressed by these inhibitors. If

ceramide is the major apoptotic messenger downstream of Fas,

a lesser but still prominent inhibition on Fas-induced cell death

by these inhibitors should be detectable. The distinct effect on

Fas- and ceramide-induced apoptosis suggest that ceramide is

dissociated from Fas-induced death cascade.

SEK dominant negative mutant did not interfere Fas-induced

cell death.

SEK/JNK activation is shown to be essential for

ceramide-initiated apoptosis.

_{Despite the fact that JNK is}

activated after Fas engagement, its role in apoptosis is less

certain. Lenczowski et al

_{demonstrated that the activation of}

SEK/JNK is downstream of ICE protease and is not required for

Fig 2. Inhibitors that blocked ceramide-induced DNA fragmenta-tion did not prevent Fas-induced DNA fragmentafragmenta-tion. (A) Jurkat cells were treated with ceramide and various inhibitors for 4 hours. The sub-G1DNA content was quantitated as described in Fig 1. Additional inhibitors used were as follows: dbcAMP, 1 mmol/L; C60(C3/100), 100 mmol/L; C60(D3/100), 100mmol/L; C60(D3/10), 10mmol/L. (B) Jurkat cells were stimulated with anti-Fas antibody CH11 (125 ng/mL) in the absence or presence of the inhibitors for 14 hours, and the sub-G1 DNA content was determined. The inhibitors used were as follows: forskolin, 50mmol/L; dbcAMP, 2.5 mmol/L; C60(C3), 100mmol/L; C60 (D3), 100mmol/L; PDTC, 200 mmol/L; Z-VAD-FK (ICEi), 300 mmol/L. None of the inhibitors alone induced DNA fragmentation at the concentrations used, except PDTC, in which an 1% to 2% increase over control was observed. The result is the average of duplicates, with standard deviation shown as an error bar. Those not shown are too small in scale. Experiments were repeated three times with the same results.

Fig 3. Inability of cAMP and C60(D3) to prevent Fas-induced apoptosis as assessed by annexin V binding. Jurkat cells were treated with CH-11 and inhibitors for 14 hours as described in Fig 2. Cells were washed, resuspended in annexin V-FITC (1mg/mL; Clontech), and analyzed on FACScan. M1 is designated as the fraction of apoptotic cells. In data not shown, cells treated with ceramide were similarly assayed. cAMP, (C60)D3, and ICEi were used at the same concentration as in Fig 2B.

Fas-induced apoptosis. On the contrary, the prevention of JNK

activation by SEK dominant negative mutant is shown to block

Fas-induced cell death in L929 and 293 cells.

_{We have thus}

studied the effect of the dominant negative mutant of SEK

_on

the same cell when treated with ceramide and anti-Fas. A

transient transfection analysis was used to assess the effect on

cell death in which SEK mutant was cotransfected with green

fluorescence protein. Expression of the dominant negative

mutants of SEK blocked the activation of the cotransfected

HA-JNK1.

_{Ceramide triggered cell death was suppressed by}

coexpression of SEK (A=L) in Jurkat cells (Fig 4), but the

inhibition was less with SEK (K=R) mutant (data not shown).

This is in contrast to the inability of SEK (A=L) to interfere

with Fas-induced apoptosis (Fig 4). In both Jurkat and CEM

cells (not shown for CEM), the inhibition of SEK activation

prevented ceramide- but not Fas-induced apoptosis. JNK

activa-tion was not essential for Fas-mediated apoptosis at least in

these two T-lymphoma cells.

Activation of NF-

kB did not interfere Fas-induced cell death.

In accordance with the recent finding that activation of NF-

kB

prevents the apoptotic signal of TNF-

a, we also found that

NF-

kB activation antagonized ceramide-induced DNA

fragmen-tation (Fig 5). Transient expression of NF-

kB p65, which led to

activation of

kB-CAT (not shown), prevented ceramide-induced

apoptosis. On the contrary, Fas-induced cell death was not

affected by the activation of NF-

kB in Jurkat cells. These results

further showed that ceramide is not a mediator of Fas-induced

apoptosis, for otherwise Fas would be equally sensitive to the

inhibition by NF-

kB.

In summary, we have presented evidence that ceramide is not

essential for Fas-induced cell death. Ceramide-triggered cell

death was sensitive to the inhibition of cAMP, yet its blockage

did not prevent Fas-initiated apoptosis. Two other antagonists,

C

and PDTC, also demonstrate such selectivity in suppressing

ceramide-induced apoptosis. We have further shown that

block-ing SEK activation diminished ceramide-induced cell death in T

lymphomas Jurkat and CEM, yet had no effect on Fas-triggered

apoptosis on the same cells (Fig 4). A similar distinction was

also found with NF-

kB activation (Fig 5). Therefore, a large

fraction of Fas-triggered death pathways must be

ceramide-independent. Even though ceramide has been suggested as the

mediator for Fas-initiated apoptosis, the linkage between

ce-ramide and Fas-induced cell death is less than confirmative.

Recent biochemical analysis have questioned the role of

ceramide in Fas-induced apoptosis. In one study, the direct

measurement of sphingosine-based ceramide failed to detect

induction of ceramide up to 2 hours after Fas triggering.

_In

another study, ceramide increase was found to be slower than

Fas-induced cell death and can be inhibited by Z-VAD-FMK.

Our results are fully in support of these observations and

suggest that ceramide is not the mediator of Fas-initiated

apoptosis.

Mixed effect of cAMP and C

on TNF-

a–induced apoptosis.

We also examined the effect of cAMP, C

, and PDTC on

TNF-

a–induced apoptosis, which is reported to be mediated by

ceramide.

_{The study was performed on L929 cells because}

TNF-

a alone does not induce cell death in Jurkat cells. The

effect of these inhibitors on TNF-

a–induced cell death was not

as unequivocal as on Fas-mediated apoptosis. PDTC (5 µmol/L)

induced spontaneous cell death (70%) in L929 cells, and its

inhibitory effect on TNF-

a–induced cell death cannot assessed.

TNF-

a–triggered apoptosis was completely prevented by 50

µmol/L of C

(C3) and C

(D3), yet was resistant to treatment

with forskolin and Bt

cAMP (Fig 6). In contrast to the

NF-

kB–independent Fas-mediated apoptosis (Fig 5), TNF-a–

induced cell death is also known to be suppressed by the

activation of NF-

kB.

_{Some of the discrepancy may be}

attributed to the difference in apoptotic signaling between Fas

and TNF-

a previously reported.

_{For example, the different}

sensitivity to C

(Figs 2 and 6) is well correlated with the

involvement of reactive oxgen radicals in TNF-

a–induced, but

not in Fas-induced, cell death.

_{It may also be noted that the}

mixed effect of the inhibitors on TNF-

a–mediated apoptosis is

Fig 4. Dominant negative mutant of SEK prevented ceramide- but not Fas-induced apoptosis. Jurkat cells were transfected with 5mg of either SEK1(A=L) or pEBG vector, together with 5mg of pEGFP-N1 (Clontech). Thirty-six hours later, cells were untreated (Ctr) or treated with either C2-ceramide (5mmol/L) or anti-Fas antibody CH11 (100 ng/mL) (Induction). Ceramide-induced cell death was analyzed 6 hours later, whereas Fas-induced cell death were quantitated 12 hours later. Data are the average of duplicates.

Fig 5. Differential sensitivity of ceramide- and Fas-triggered cell death to activation of NF-kB. Jurkat cells were transfected with 5 mg of either CMV-RelA or CMV vector, together with 5mg of pEGFP-N1 (Clontech). Thirty-six hours later, cells were untreated or treated with either C2-ceramide (5mmol/L) or anti-Fas antibody CH11 (100 ng/mL). Ceramide-induced cell death was analyzed 6 hours later, whereas Fas-induced cell death were quantitated 14 hours later. Data are the average of duplicates.

consistent with the observation that ceramide contributes to a

fraction, but not all, of TNF-

a–induced cell death.

The distinction between the Fas-induced apoptotic cascade

and ceramide-induced death pathway shows that death

pro-cesses induced by different stimulation can be clearly

indepen-dent of each other. The combination of different stimulations

may hence be more effective in the induction of apoptosis,

especially in tumor cells for therapeutic purposes, notably,

ceramide-mediated cell death initiated by TNF-

a, daunorubicin,

and

g-irradiation, which are reagents widely used in tumor

treatment.

_{The dissociation of Fas-induced death signals}

from ceramide may indicate an additional dimension in

manipu-lation of apoptosis.

ACKNOWLEDGMENT

The authors thank Dr Leonard Zon for SEK(A=L) and SEK(K=R), Dr Warren C. Greene forkB-TATA-CAT, and Dr Michael Karin for HA-JNK1. We also thank Douglas Platt for editorial correction of the manuscript.

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Fig 6. Mixed effect of cAMP and C60 on TNF-a–induced apoptosis. L929 cells were treated with recombinant TNF-a (250 mg/mL) in the absence or presence of the indicated inhibitors for 18 hours. The sub-G1DNA content was quantitated as described in Fig 1. The concentration of the inhibitors used were as follows: forskolin, 10 mmol/L; dbcAMP, 1.0 mmol/L; C60 (C3), 50 mmol/L; C60 (D3), 50 mmol/L; Z-VAD-FK (ICEi), 100 mmol/L.

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