www.biomolther.org DOI: 10.4062/biomolther.2010.18.3.262
*Corresponding author
Tel: +82-2-820-5616 Fax: +82-2-825-5616 E-mail: yjchun@cau.ac.kr
Roles of Neutral Sphingomyelinase 1 on CD95-Mediated Apoptosis in Human Jurkat T Lymphocytes
Hyun Min LEE, Bo Young SURH, and Young-Jin CHUN*
College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea (Received May 24, 2010; Revised June 22, 2010; Accepted June 22, 2010)
Abstract － CD95 receptor is a member of tumor necrosis factor receptor family that mediates apoptosis in many cell types when bound by CD95 ligand or cross-linked by agonistic anti-CD95 antibodies. To determine the role of neutral sphingomyelinase (nSMase) on CD95-mediatd apoptosis, human Jurkat T lymphocytes were exposed to recombinant human CD95 ligand. Treatment with CD95 ligand induced cell death in a concentration and time-dependent manner. CD95-induced cell death was suppressed by inhibitors of SMase such as AY9944 or desipramine. Transfection with human nSMase1 siRNA plasmid into CD95 ligand-treated cells significantly prevented CD95-mediated cell death. CD95-mediated elevation of intracellular ceramide level detected by FACS analysis with anti-ceramide antibody was also decreased by nSMase1 siRNA.
Knock-down of nSMase1 expression also blocked cytochrome c release into cytosol and caspase-3 cleavage in CD95-treated cells. Taken together, these results suggest that nSMase1 may play an important role in CD95-mediated apoptotic cell death in Jurkat T cells.
Keywords: CD95 ligand, nSMase1, Jurkat T cell, siRNA, Apoptosis
INTRODUCTION
CD95 (Fas or Apo-1) receptor is a member of tumor ne- crosis factor receptor family that mediated apoptosis in many cell types when bound by the CD95 ligand or cross- linking with agonistic anti-CD95 antibodies (Mitsumasa et al., 2004; Inna et al., 2005). Activation of CD95 receptor re- sults in the formation of the death-inducing signaling com- plex and activation of initiator caspase-8, which then trig- gers a variety of regulated downstream events, which may finally result in apoptotic cell death (Martin et al., 1995; De Maria et al., 1998; Oliver et al., 2000). Many studies also showed that CD95-inducing cell death is related with intra- cellular ceramide generation (Obeid and Hannun, 1995;
Cock et al., 1998; Norma et al., 2004).
Ceramide is one of various sphingolipid metabolites known to have biological activities, serving as a lipid- derived second messenger that modulates the induction of cell differentiation, cell cycle arrest, and/or cell death (Ram et al., 2004; Sawada et al., 2004). Intracellular ceramide accumulation results from multiple stimuli, such as tumor
necrosis factor-α (TNF-α), irradiation, heat shock, CD95 cross linking, and other anticancer drugs (Nieves et al., 2000). These stimuli have been observed to initiate ceramide-mediated signaling cascade and the stimulation of caspase activity, which ultimately leads to DNA fragmentation and cell death (Chalfant et al., 2001; Kondo et al., 2002).
Generation of ceramide through sphingomyelinase (SMase) activation has been shown to play a role in cell death (Jarvis and Grant, 1998). SMase are enzymes that cleave the phosphodiester linkage of sphingomyelin into ceramide and phosphocholine, and they are implicated several signaling transduction and cell regulation (Norma et al., 2003). Activation of acidic SMase (aSMase) has been observed after treatment with anti-CD4 antibodies in vivo (Kirschnek et al., 2000), although the involvement of aSMase in apoptosis induced by TNFα or CD95 is some- what controversial (Segui et al., 2000; Bezombes et al., 2001). Attention has also focused on the neutral SMase (nSMase) for its role of mediating a variety of cellular re- sponses including differentiation, cell cycle regulation, and apoptosis through the generation of the ceramide (Hannun and Luberto, 2000). NSMase activation has been ob- served after ligation of CD95 and TNF receptor and UV ir-
radiation, and also upon heat stress and serum starvation (Hirofumi et al., 1999).
Based on similarity searches with bacterial SMases, two putative nSMases such as nSMase1 (Tomiuk et al., 1998) and nSMase2 (Hofmann et al., 2000) were identified. The recombinant nSMase1 has been found to efficiently hydro- lyze phosphatidylcholine and lyso-platelet-activating factor in addition to SM and to be localized in the endoplasmic re- ticulum (Hirofumi et al., 1999; Tomiuk et al., 2000). In con- trast nSMase1, nSMase2 specifically catalyzes the hydrol- ysis of sphingomyelin and is activated by phosphatidylser- ine, both of which are characteristics of the mammalian plasma membrane SMase (Norma et al., 2003). Recently nSMase3 gene has been cloned and expressed in MCF7 cells (Krut et al., 2006). However, the role of nSMases on CD95-mediated apoptosis in cells is still unclear. In these studies, the roles of nSMase1 on CD95-mediated apop- totic cell death in Jurkat T lymphocyte cells were studied using small interference RNA (siRNA)-mediated gene si- lencing and we suggest that nSMase1 may play an im- portant role in CD95-mediated apoptotic cell death in Jurkat T cells.
MATERIALS AND METHODS Cell culture
Human acute leukemic T-cell Jurkat, clone E6-1 cells were cultured in RPM 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml strep- tomycin. Cells were maintained at 37oC in a humidified at- mosphere of 5% CO2. For treatment, 5×105 cells were plat- ed in 1 ml of culture medium and incubated for 24-48 h, as indicated.
Cell viability assay
Cells were plated onto 96-well plates and incubated at 37oC in a 5% CO2 atmosphere. After incubation for the designated time, cells were treated with 10 μl of CCK-8 solution (Dojindo, Kumamoto, Japan). After 210 min in- cubation, formazan generation was quantified by using a GENios Pro microplate reader (Tecan, Switzerland) at 450 nm. The percentages of cells surviving from each group relative to control, defined as 100% survival, were cal- culated.
Construction of siRNA plasmids and transfection For design of small interference RNA oligonucleotides targeting human SMases, DNA sequence for the human nSMase1 (5'-AATCGACAGAAGGACATCTAC-3') was se-
lected using BLAST search. Double stranded siRNA oligo- nucleotides were synthesized (Genotech, Daejeon, Korea) and annealed at 15oC for about 12 h. Annealed oligonu- cleotides were ligated with pSilencer 2.0-U6 plasmid (Ambion, Austin, TX) digested with BamHI and HindIII. A nonspecific scrambled siRNA was used as a negative control. Con- structed siRNA plasmids were transfected in cells using DMRIE-C transfection reagent as recommended by the manufacturer (Invitrogen). Briefly, cells suspended in se- rum and antibacterial reagent-free medium were incubated in opti-MEM medium along with 5 μg of plasmid DNA and 12.5 μl of DMRIE-C for 5 h at 37oC. Cells were maintained in RPMI medium containing 10% FBS for 24 h after transfection.
Subcellular fractionation
Cells were harvested at the end of treatment and wash- ed with ice-cold PBS. Cells were resuspended in 0.5 ml of buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol) containing 0.25 M sucrose and a mixture of protease in- hibitors (1 mM PMSF, 1% aprotinin, 1 mM leupeptin, and 1 μg/ml chymostatin). To lyse the cells, the cell suspension was passed 10 times through a 26-gauge needle fitted to a syringe. Unbroken cells, large plasma membrane piece, and nuclei were removed by centrifuging the homogenates at 1,000×g at 4oC for 10 min. The supernatant was sub- jected to 10,000×g centrifugation at 4oC for 20 min. The pellet fraction (i.e., mitochondria) was first washed with the buffer A containing 0.25 M sucrose and then solubilized in 50 μl of 10 mM Tris-acetate buffer (pH 8.0) containing 0.5% NP-40, and 5 mM CaCl2. The supernatant was cen- trifuged at 100,000×g at 4oC for 1 h to generate cytosol.
Determination of intracellular ceramide
After treatment, cells were resuspended in 0.3 ml of PBS containing 6 μg of mouse anti-ceramide 15B4 anti- body (Alexis, Grunberg, Germany) and stained for 40 min at room temperature. After incubation, cells were centri- fuged at 1,000×g for 5 min and washed with ice-cold PBS.
Cells were incubated in 1 ml of PBS containing 15 μg of Cy-3 conjugated anti-mouse antibody (Jackson Immuno- Researsh laboratories, West Grove, PA) and stained for 40 min in a dark condition at room temperature. Cells were centrifuged and washed again with ice-cold PBS once.
Pellets were suspended in 1 ml of PBS, and cells (104 cells/well) were analyzed on a FACSCaliber (BD Biosciences, San Jose, CA). The intensity of Cy-3 fluorescence was measured with excitation at 550 nm and emission at 570 nm.
Fig. 1. Growth inhibition by recombinant CD95 ligand in Jurkat T cells. (A) Cells were incubated with increasing doses of CD95 ligand (0, 1, 2, 4, or 8 ng/ml) for 24 h and the cell viability was determined by CCK assay. Results are the mean ± S.D. of three separate experiments. *Significantly different from vehicle- treated cells (p＜0.05). (B) Cells were incubated with CD95 ligand (4 or 8 μM) for the designated time (0, 6, 12, 24 or 48 h) and the cell viability was determined by CCK assay. Results are the mean ± S.D. of three separate experiments. *Significantly different from vehicle-treated cells (p＜0.05).
RT-PCR
Total RNA was extracted using Trizol reagent (Gibco BRL, Gaithersburg, MD). RT-PCR was performed using an Access RT-PCR system purchased from Promega (Madison, WI) according to the manufacturer’s instructions. Human nSMase1 cDNA was amplified by PCR using a sense pri- mer (5’-GGCTGCTGCCTGCTGAA-3’) and an antisense primer (5’-TAGAGCTGGGGTTCTGCTGT-3’) with 30 cy- cles by denaturation at 94oC for 20s, annealing at 58oC for 20s, and extension at 72oC for 40s (Zhou et al., 2004).
Human β-actin cDNA was amplified using a sense primer (5’-CTACAATGAGCTGCGTGTGG-3’) and an antisense primer (5’-TAGCTCTTCTCCAGGGAGGA-3’) with 30 cy- cles by denaturation at 94oC for 20s, annealing at 52oC for 20s, and extension at 72oC for 40s. The amplified PCR products were analyzed by 1.5% agarose gel electro- phoresis and ethidium bromide staining.
Western blotting
Cells were solubilized with ice-cold lysis buffer contain- ing 25 mM HEPES (pH 7.4), 1% Triton X-100, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin. Insoluble materials were removed by centrifugation at 10,000×g for 10 min. Extracted proteins were fractionated by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to polyvinylidene difluoride membrane.
For nonspecific binding, blocking was performed in Tris- buffered saline (TBS) containing 5% skim milk powder and 0.1% Tween-20 at 4oC overnight. The membranes were washed with three changes of TBS and incubated with an- tibodies against cytochrome c (1.5 μg/ml in TBS-T, Phar- mingen, San Diego, CA), and caspase-3 (1:1,000 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h at room temperature. After blotting with a primary antibody, the membranes were washed three times with TBS. The membranes were incubated for 90 min with TBS-T con- taining HRP-conjugated goat anti-rabbit or anti-mouse IgG (1:5,000 dilution). The visualization of the blots was carried using an enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ). Protein content was determined by bicinchoninic acid (BCA) method (Pierce, Rockford, IL) using bovine serum albumin (BSA) as a standard.
Statistical analysis
Data are expressed as the mean ± S.D. and the stat- istical differences between means were analyzed using one-way analysis of variance (ANOVA), followed by Dunnett’s Pairwise Multiple Comparison t-test. The difference was
considered statistically significant at p＜0.05.
RESULTS
Effect of CD95 ligand on cell viability in human Jurkat T lymphocytes
To examine the effect of CD95 ligand on cell viability in Jurkat T cells, cells were treated with human recombinant CD95 ligand for 24 h, and cytotoxicity of CD95 ligand was determined using CCK-8. Treatment with CD95 ligand ex- erted a concentration-dependent inhibition of cellular pro- liferation in the cultured cells (Fig. 1A). At 8 ng/ml, cell death effect was significant and this concentration was se- lected for the further studies. As shown in Fig. 1B, treat-
Fig. 2. Effects of SMase inhibitors on CD95-mediated cell death in T cells. Cells were pre-treated with 10 μM AY9944 (A) or 20 μM desipramine (B) for 30 min, and then incubated with CD95 ligand (8 ng/ml) for 24 h. The cell viability was determined by CCK assay. Results are the mean ± S.D. of three separate experiments. *Significantly different from vehicle-treated cells (p
＜0.05), #Significantly different from the CD95 ligand-treated cells (p＜0.05).
Fig. 3. Involvement of nSMase1 in CD95-mediated T cell death.
(A) RT-PCR analyses. Cells were transfected with nSMase1- specific siRNA or with control scrambled siRNA for 24 h. Total RNA was isolated and the partial coding sequence of human nSMase1 was amplified with specific primers. Expression of β-actin mRNA was determined as a RNA control. (B) After transfection with nSMase1-specific siRNA or with control scram- bled siRNA for 24 h, cells were incubated with CD95 ligand (8 ng/ml) for 24 h and the cell viability was determined by CCK assay. Results are the mean ± S.D. of three separate experi- ments. *Significantly different from vehicle-treated cells (p＜
0.05), #Significantly different from the CD95 ligand-treated cells (p＜0.05).
ment of cells with CD95 ligand (8 ng/ml) significantly caused cell death effect up to 48 h.
Blockage of CD95 ligand-induced cell death by SMase inhibitors
To investigate the role of SMase on CD95 ligand-in- duced cell death, the effect of SMase inhibitors such as AY9944 and desipramine was determined (Yoshida et al., 1985) (Fig. 2). Cells were preincubated with AY9944 (10 μM) or desipramine (20 μM) for 30 min, and then were treated with 8 ng/ml CD95 ligand for 24 h. Treatment with inhibitors of SMases significantly recovered the decreased cell viability by CD95 ligand. The data suggest that SMases may be involved in the pathway of CD95 receptor-medi- ated cell death.
Transfection with nSMase1 siRNA results in recovery of cell death by CD95 ligand
To determine whether nSMase1 siRNA is able to sup- press nSMase1 mRNA expression in T cells, cells were transfected with 5 μg of nSMase1 siRNA plasmids for 24 h.
The scrambled siRNA with no human target mRNAs was used as a transfection control. Transfection with nSMase1 siRNA plasmid significantly reduced nSMase1 mRNA ex- pression in cells although scrambled siRNA-transfected cells showed no difference in nSMase1 expression (Fig.
3A). The effect of nSMase1 knock-down on CD95-medi- ated cell death was investigated. After transfection, cells were incubated with CD95 ligand for additional 24 h.
Transfection with nSMase1 siRNA showed a significant re- covery of cell death by CD95 ligand (Fig. 3B). These re- sults suggest that nSMase1 may play an important role in
Fig. 4. Enhancement of intracellular ceramide by CD95 ligand is mediated by nSMase1. Cells were transfected with nSMase1- specific siRNA or with control scrambled siRNA for 24 h and then were treated with CD95 ligand (8 ng/ml) for 30 min. The amounts of intracellular ceramide were determined by using FACS with Cy-3 conjugated anti-ceramide antibody.
Fig. 5. nSMase1 is required for CD95-mediated apoptotic path- way. (A) Cytochrome c release. Cells were transfected with nSMase1-specific siRNA or with control scrambled siRNA for 24 h. and then were treated with CD95 ligand (8 ng/ml) for 24 h.
Cell lysates were prepared and mitochondrial or cytosolic fraction were isolated from cell lysates. The amounts of cyto- chrome c in each fractions were determined by Western blotting using anti-cytochrome c antibody. (B) Procaspase-3 cleavage.
Cell lysates were prepared and cleaved procaspase-3 levels (19 kDa and 17 kDa bands) were detected by Western blotting.
CD95 receptor-mediated apoptotic cell death pathway.
Knockdown of nSMase1 expression suppresses intracellular ceramide generation
To address a potential involvement of nSMase1 in gen- erating ceramide in T cells, we determined amount of intra- cellular ceramide levels using flow cytometry with anti-ce- ramide antibody. Intracellular ceramide levels were sig- nificantly elevated in CD95 ligand-treated cells and nSMase1 siRNA was effective at reducing intracellular ceramide lev- els although scrambled siRNA showed no difference (Fig.
4). The results revealed that nSMase1 may control intra- cellular ceramide generation in Jurkat cells.
Suppression of nSMase1 prevents apoptotic cell death signals
Based on the results described above, we asked wheth- er knock-down of nSMase1 expression and decrease in ceramide production may suppress apoptotic cell death signaling because ceramide is well-known as an inducer of apoptotic cell death in various cells. Immunoblot analysis of cytochrome c protein showed that treatment with nSMase1 siRNA prevents translocation of cytochrome c from mi- tochondria to cytosol in CD95 ligand-exposed cells (Fig.
5A). In vehicle-treated control cells or scrambled siRNA- transfected cells, release of cytochrome c to cytosol was significantly increased by CD95 ligand. Because activation
of caspase-3 may play a central role in the execution stage of apoptosis, we performed Western blot analyses with an- tibody against caspase-3 to measure cleavage of procas- pase-3. Significant cleavage of procaspase-3 was showed in CD95 ligand-treated cells and these effects were com- pletely blocked by nSMase1 siRNA (Fig. 5B).
DISCUSSION
Ceramide is known to play a key role in the regulation of diverse cellular functions, including cell growth, differenti- ation, stress response and apoptosis (Spiegel and Merrill, 1996; Hannun and Luberto, 2000). Because of the sig- nificant roles of ceramide in cells, the interest in the SMases that initiate the generation of intracellular ceramide rose to identify crucial enzymes that participate in the biochemical pathways leads to ceramide accumulation.
To date, at least five types of SMases have been re- ported, including the Zn2＋-dependent acidic SMase, Zn2＋- independent acidic SMase, the Mg2＋-dependent neutral SMase, Mg2＋-independent neutral SMase, and alkaline SMase (Clarke et al., 2006; Martin et al., 2007). Among these, acidic SMase and Mg2＋-dependent neutral SMase are postulated as candidate enzymes for regulating ce- ramide-mediated cellular processes (Horinouchi et al.,
1995; Liu et al., 1998b; Clarke and Hannun, 2006). Acidic SMase is present in lysosomes/endosomes of all cells where it degrades sphingomyelin of endocytosed mem- branes and acidic SMase activity is deficient in patients af- fected with types A and B Niemann-Pick disease (Brady et al., 1966; Schneider and Kennedy, 1967). A pivotal role of acidic SMase in CD95-triggered apoptosis has been stud- ied using acidic SMase knockout mice and Niemann-Pick disease cell (Kirschnek et al., 2000; Lin et al., 2000; Paris et al., 2001).
Three types of Mg2＋-dependent neutral SMases (nSMase 1, 2, and 3) have been identified so far (Tomiuk et al., 1998; Hofmann et al., 2000; Krut et al., 2006). Human nSMase1 gene was first cloned as Mg2＋-dependent nSMase gene pursuing similarity with gene sequences of bacterial SMases such as Bacillus cereus and Staphylococcus aur- eus (Tomiuk et al., 1998). The nSMase1 is known to ex- press ubiquitously with the highest mRNA and protein amounts in kidney and localized to the endoplasmic retic- ulum (Sawai et al., 1999; Neuberger et al., 2000; Tomiuk et al, 2000). Previous report suggested that in vivo function of nSMase1 might be a lysophospholipase C with lysophos- phatidylcholine and lyso-platelet activating factor as sub- strates (Sawai et al., 1999). Human nSMase2 gene enc- odes proteins of 655 amino acid residues with two putative transmembrane domains at the N terminus. The nSMase2 is mainly expressed in brain colocalizing with a Golgi mark- er in neuronal cells (Hofmann et al., 2000). Stable ex- pression of nSMase2 gene in MCF7 cells causes an in- crease in the levels of ceramide and a decrease in the lev- el of sphingomyelin (Marchesini et al., 2003). Mice defi- cient for nSMase2 develop a novel form of dwarfism and delayed puberty, suggesting that nSMase2 may play an important role in the control of developmental stages (Stoffel et al., 2007). Recently, a new mammalian nSMase gene named nSMase3 was cloned and the high level of nSMase3 expression in heart muscle raises the possibility of role of nSMase3 in the pathogenesis of cardiac disease (Krut et al., 2006).
Ligation of CD95 ligand is known to induce the pro- duction of ceramide that results in apoptosis in a variety of cell types (Ogasawara et al., 1993; Grassme et al., 2001;
Miyaji et al., 2005). Several studies have implicated that CD95 ligation activates acidic SMase to hydrolyze sphin- gomyelin to produce ceramide (Tonnetti et al., 1999; Cremesti et al., 2001; Grassme et al., 2001). However, involvement of other SMases such as neutral SMases on CD95-medi- ated apoptosis should be addressed because ceramide production following CD95 ligation still occurred in mice deficient in the acidic SMase gene and CD95 ligand acti-
vates both acidic and neutral SMases (Tonnetti et al., 1999; Lin et al., 2000).
The present work was designed to elucidate whether nSMase1 plays an important role in apoptotic cell death by controlling ceramide levels in Jurkat T cells treated with CD95 ligand. We have shown that the SMase inhibitors such as AY9944 or desipramine are able to prevent cell death of T cell by CD95 treatment and down-regulation of nSMase1 using specific siRNA significantly recovered apop- totic cell death in CD95-treated cells. Gene knock-down of nSMase1 can also block generation of ceramide. The re- lease of cytochrome c from mitochondria to cytosol by CD95 was suppressed when the cells were transfected with nSMase1 siRNA plasmid although scrambled siRNA showed no changes in cytochrome c release compared to the control cells. Procaspase-3 cleavage by CD95 was al- so blocked by nSMase1 knock-down. All of the data re- ported here indicate that that human nSMase1 is involved in CD95-mediated apoptosis pathway. Several lines of evi- dence suggest that nSMase1 activity is regulated by changes in the glutathione level (Liu and Hannun, 1997; Liu et al., 1998a; Martin et al., 2007). Glutathione could be an in- hibitor of nSMase activity and glutathione depletion is nec- essary for TNFα-stimulated nSMase activation (Liu et al., 1998a). Depletion of glutathione has also been observed in response to CD95 activation (Chiba et al., 1996; van den Dobbelsteen et al., 1996). Because CD95 ligand triggers a depletion of cellular glutathione, rapid formation of reactive oxygen species, and apoptosis induction (Watanabe et al., 2004; Reinehr et al., 2005), increased intracellular ceram- ide by activating nSMase1 through glutathione depletion and transient increases in glutathione disulfide may induce apoptosis.
There are conflicting data on the role of nSMase1 on apoptotic cell death. Stimulation of cells overexpressing nSMase1 with TNF or H2O2 did not elevate ceramide levels or induce apoptosis (Tomiuk et al., 1998). Tepper et al. (2001) showed that overexpression of human nSMase1gene in Jurkat cells increased in vitro nSMase activity. However, basal sphingolipid levels such as ceramide, sphingomyelin or glucosylceramide were not changed by nSMase1 ex- pression. The nSMase1 was predominantly localized at the ER membrane, but not at the plasma membrane in HeLa cells. Interestingly, Jurkat cells expressing nSMase1 failed to undergo apoptosis after CD95 ligation. However, Tonetti et al. (1999) have showed that T cell receptor- mediated ceramide production in T cell hybridoma 3DO is inhibited by expressing antisense RNA complementary to nSMase1 cDNA. Recent study also showed that nSMase1 from zebrafish embryonic cell acts as a mediator of
stress-induced apoptosis (Yabu et al., 2008). These ob- servations were also supported by our data that down- regulation of human nSMase1 prevents CD95-mediated apoptotic response in Jurkat cells. The observed discrep- ancies may come from the fact that we and Tonetti group studied the effect of downregulation of nSMase1 using siRNA instead of overexpression. Suppression of nSMase1 expression in T cells by gene silencing may be able to modulate the action of other unknown factor(s) regulated by nSMase1 that can control intracellular ceramide level and apoptotic responses. Although the role of nSMase1 is still not clear, but the function of nSMase1 as a controller of cell signaling needs to be elucidated in more detail.
In conclusion, we demonstrate here that nSMase1 may play a pivotal role in CD95-mediated apoptosis in human Jurkat T cells. Stimulation of T cells with CD95 ligand in- duces nSMase1 activation which subsequently led to ac- cumulation of ceramide, which in turn induces caspase-3 activation and apoptosis. Although the detailed mecha- nism needs to be further studied, our finding may be bene- ficial to understand the mechanism and physiological sig- nificance of SMases in apoptotic signal pathways.
ACKNOWLEDGMENTS
This research was supported by the Chung-Ang Univer- sity Research Scholarship Grants in 2009.
REFERENCES
Bezombes, C., Segui, B., Cuvillier, O., Bruno, A. P., Uro-Coste, E., Gouaze, V., Andrieu-Abadie, N., Carpentier, S., Laurent, G., Salvayre, R., Jaffrezou, J. P. and Levade, T. (2001).
Lysosomal sphingomyelinase is not solicited for apoptosis signaling. FASEB J. 15, 297-299.
Brady, R. O., Kanfer, J. N., Mock, M. N. and Fredrickson, D. S.
(1966). The metabolism of sphingomyelin II. Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc. Natl.
Acad. Sci. U S A 55, 366-369.
Chalfant, C. E., Ogretmen, B., GalaDari, S., Kroesen, B. J., Pettus, B. J. and Hannun, Y. A. (2001). Fas activation induces dephosphorylation of SR proteins: dependence on the de novo generation of ceramide and activation of protein phos- phatase 1. J. Biol. Chem. 276, 44848-44855.
Chiba, T., Takahashi, S., Sato, N., Ishii, S. and Kikuchi, K.
(1996). Fas-mediated apoptosis is modulated by intracellular glutathione in human T cells. Eur. J. Immunol. 26, 1164-1169.
Clarke, C. J. and Hannun, Y. A. (2006). Neutral sphingomyelinases and nSMase2: Bridging the gaps. Biochim. Biophys. Acta.
1758, 1893-1901.
Clarke, C. J., Snook, C. F., Tani, M., Matmati, N., Marchesini, N.
and Hannun, Y. A. (2006). The extended family of neutral sphingomyelinases. Biochemistry 45,11247-11256.
Cock, J. G., Tepper, A. D., de Vries, E., van Blitterswijk, W. and
Borst, J. (1998). CD95(Fas/Apo-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingo- myelinase. J. Biol. Chem. 273, 7560-7565.
Cremesti, A., Paris, F., Grassmé, H., Holler, N., Tschopp, J., Fuks, Z., Gulbins, E. and Kolesnick, R. (2001). Ceramide enables fas to cap and kill. J. Biol. Chem. 276, 23954-23961.
De Maria, R., Rippo, M. R., Schuchman, E. H. and Testi, R.
(1998). Acidic sphingomyelinase (ASM) is necessary for Fas-induced GD3 ganglioside accumulation and efficient apoptosis of lymphoid cells. J. Exp. Med. 187, 897-902.
Grassme, H., Jekle, A., Riehle, A., Schwarz, H., Berger, J., Sandhoff, K., Kolesnick, R. and Gulbins, E. (2001). CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem.
276, 20589-20596.
Hannun, Y. A. and Luberto, C. (2000). Ceramide in the euka- ryotic stress response. Trends Cell Biol. 10, 73-80.
Hirofumi, S., Naochika, D., Narasimhan, N. and Hannun, Y. A.
(1999). Function of the cloned putative neutral sphingo- myelinase as lyso-platelet activating factor-phospholipase C.
J. Biol. Chem. 274, 38131-38139.
Hofmann, K., Tomiuk, S., Wolff, G. and Stoffel, W. (2000).
Cloning and characterization of the mammalian brain- specific, Mg2＋-dependent neutral sphingomyelinase. Proc.
Natl. Acad. Sci. U S A 97, 5895-5900.
Horinouchi, K., Erlich, S., Perl, D. P., Ferlinz, K., Bisgaier, C. L., Sandhoff, K., Desnick, R. J., Stewart, C. L. and Schuchman, E. H. (1995). Acid sphingomyelinase deficient mice: A model of types A and B Niemann-Pick disease. Nat. Genet. 10, 288-293.
Inna, L., Alexander, G. and Peter, H. K. (2005). Death receptor signaling. J. Cell. Sci. 118, 265-267.
Jarvis, W. D. and Grant, S. (1998). The role of ceramide in the cellular response to cytotoxic agent. Curr. Opin. Oncol. 10, 552-559.
Kirschnek, S., Paris, F., Wellwe, M., Grassme, H., Ferliz, K., Riehle, A., Fuks, Z., Kolesnick, R. and Gulbins, E. (2000).
CD95-mediated apoptosis in vivo involves acid sphingo- myelinase. J. Biol. Chem. 275, 27316-27323.
Kondo, T., Kitano, T., Iwai, K., Watanabe, M., Taguchi, Y., Yabu, T., Umehara, H., Domae, N., Uchiyama, T. and Okazaki, T.
(2002). Control of ceramide-induced apoptosis by IGF-1:
involvement of PI-3 kinase, caspase-3 and catalase. Cell Death Differ. 9, 682-692.
Krut, O., Wiegmann, K., Kashkar, H., Yazdanpanah, B. and Krönke, M. (2006). Novel tumor necrosis factor-responsive mammalian neutral sphingomyelinase-3 is a C-tail-anchored protein. J. Biol. Chem. 281, 13784-13793.
Lin, T., Genestier, L., Pinkoski, M. J., Castro, A., Nicholas, S., Mogil, R., Paris, F., Fuks, Z., Schuchman, E. H., Kolesnick, R. N. and Green, D. R. (2000). Role of acidic sphingomye- linase in Fas/CD95-mediated cell death. J. Biol. Chem. 275, 8657-8663.
Liu, B., Andrieu-Abadie, N., Levade, T., Zhang, P., Obeid, L. M.
and Hannun, Y. A. (1998a). Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death. J. Biol. Chem. 273, 11313-11320.
Liu, B. and Hannun, Y. A. (1997). Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione. J.
Biol. Chem. 272, 16281-16287.
Liu, B., Hassler, D. F., Smith, G. K., Weaver, K. and Hannun, Y.
A. (1998b). Purification and characterization of a membrane bound neutral pH optimum magnesium-dependent and phosphatidylserine-stimulated sphingomyelinase from rat brain. J. Biol. Chem. 273, 34472-34479.
Marchesini, N., Luberto, C. and Hannun, Y. A. (2003). Bio- chemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism. J. Biol. Chem. 278, 13775-13783.
Martin, S. F., Sawai, H., Villalba, J. M. and Hannun, Y. A.
(2007). Redox regulation of neutral sphingomyelinase-1 activity in HEK293 cells through a GSH-dependent mechanism.
Arch. Biochem. Biophys. 459, 295-300.
Martin, S. J., Newmeyer, D. D., Mathias, S., Farschon, D. M., Wang, H. G., Reed, J. C., Kolesnick, R. N. and Green, D. R.
(1995). Cell-free reconstitution of Fas-, UV radiation-, and ceramide-induced apoptosis. EMBO J. 14, 5191-5200.
Mitsumasa, W., Toshiyuki, K., Tadakazu, K., Takeshi, Y., Yoshi- mitsu, T., Masaro, T., Hisanori, U., Naochika, D., Takashi, U.
and Toshiro, O. (2004). Increase of nuclear ceramide through caspase-3-dependent regulation of "sphingomyelin cycle" in Fas-induced apoptosis. Cancer Res. 64, 1000- 1007.
Miyaji, M., Jin, Z. X., Yamaoka, S., Amakawa, R., Fukuhara, S., Sato, S. B., Kobayashi, T., Domae, N., Mimori, T., Bloom, E.
T., Okazaki, T. and Umehara, H. (2005). Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis. J. Exp. Med. 202, 249-259.
Neuberger, Y., Shogomori, H., Levy, Z., Fainzilber, M. and Futerman, A. H. (2000). A lyso-platelet activating factor phos- pholipase C, originally suggested to be a neutral-sphingo- myelinase, is located in the endoplasmic reticulum. FEBS Lett. 469, 44-46.
Nieves, E., Pilar, F. V., Salvador, A., Eduardo, L. C. and Juan, C. L. (2000). Apoptosis induced by rac GTPase correlates with induction of FasL and ceramides production. Mol. Biol.
Cell 11, 4347-4358.
Norma, M., Chiara, L. and Hannun, Y. A. (2003) Biochemical properties of mammalian neutral sphingomyelinase2 and its role in sphingolipid metabolism. J. Biol. Chem. 278, 13775- 13783.
Norma, M., Walid, O., Jacek, B., Chiara, L., Lina, M. O. and Hannun, Y. A. (2004). Role of mammalian neutral sphingo- myelinase 2 in confluence-induced growth arrest of MCF7 cells. J. Biol. Chem. 279, 25101-25111.
Obeid, L. M. and Hannun, Y. A. (1995). Ceramide: a stress signal and mediator of growth suppression and apoptosis. J.
Biol. Chem. 258, 191-198.
Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., Matsu- zawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S. (1993). Lethal effect of the anti-Fas antibody in mice. Nature 364, 806-809.
Oliver, C., Lisa, E. and Sarah, S. (2000). Involvement of sphingosin in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells. J. Biol. Chem. 275, 15691-15700.
Paris, F., Grassmé, H., Cremesti, A., Zager, J., Fong, Y., Haimovitz- Friedman, A., Fuks, Z., Gulbins, E. and Kolesnick, R. (2000).
Natural ceramide reverses Fas resistance of acid sphingo- myelinase-/- hepatocytes. J. Biol. Chem. 276, 8297-8305.
Ram, K., ManLin, J., Maria-A, G. and David, R, H. (2004).
Ceramide-induced apoptosis: role of catalase and hepatocyte growth factor. Free Radical Biol. Med. 37, 166-175.
Reinehr, R., Becker, S., Eberle, A., Grether-Beck, S. and Häus- singer, D. (2005). Involvement of NADPH oxidase isoforms and src family kinases in CD95-dependent hepatocyte apoptosis. J. Biol. Chem. 280, 27179-27194.
Sawada, M., Kiyono, T., Nakashima, S., Shinoda, J., Naganawa, T., Hara, S., Iwama, T. and Sakai, N. (2004). Molecular mechanisms of TNF-α-induced ceramide formation in human glioma cells: p53-mediated oxidant stress-dependent and -independent pathways. Cell Death Differ. 11, 997-1008.
Sawai, H., Domae, N., Nagan, N. and Hannun, Y. A. (1999).
Function of the cloned putative neutral sphingomyelinase as lyso-platelet activating factor-phospholipase C. J. Biol. Chem.
274, 38131-38139.
Schneider, P. B. and Kennedy, E. P. (1967). Sphingomyelinase in normal human spleens and in spleens from subjects with Niemann-Pick disease. J. Lipid Res. 8, 202-209.
Segui, B., Bezombes, C., Uro-Coste, E., Medin, J. A., Andrieu- Abadie, N., Auge, N., Brouchet, A., Laurent, G., Salvayre, R., Jaffrezou, J. P. and Levade, T. (2000). Stress-induced apop- tosis is not mediated by endolysosomal ceramide. FASEB J.
14, 36-47.
Spiegel, S. and Merrill, A. H. Jr. (1996). Sphingolipid metabolism and cell growth regulation. FASEB J. 10, 1388-1397.
Stoffel, W., Jenke, B., Holz, B., Binczek, E., Günter, R. H., Knifka, J., Koebke, J. and Niehoff, A. (2007). Neutral sphingomyelinase (SMPD3) deficiency causes a novel form of chondrodysplasia and dwarfism that is rescued by Col2A1- driven smpd3 transgene expression. Am. J. Pathol.
171, 153-161.
Tepper, A. D., Ruurs, P., Borst, J. and van Blitterswijk, W. J.
(2001). Effect of overexpression of a neutral sphingomy- elinase on CD95-induced ceramide production and apoptosis.
Biochem. Biophys. Res. Commun. 280, 634-639.
Tomiuk, S., Hofmann, K., Nix, M., Zumbansen, M. and Stoffel, W. (1998). Cloned mammalian neutral sphingomyelinase:
functions in sphingolipid signaling? Proc. Natl. Acad. Sci. U S A 95, 3638-3643.
Tomiuk, S., Zumbansen, M. and Stoffel, W. (2000). Charac- terization and subcellular localization of murine and human magnesium-dependent neutral sphingomyelinase. J. Biol.
Chem. 275, 5710-5717.
Tonnetti, L., Veri, M. C., Bonvini, E. and D’Adamio, L. (1999). A role for neutral sphingomyelinase-mediated ceramide pro- duction in T cell receptor-induced apoptosis and mitogen- activated protein kinase-mediated signal transduction. J.
Exp. Med. 189, 1581-1589.
van den Dobbelsteen, D. J., Nobel, C. S. I., Schlegel, J., Cotgreave, I. A., Orrenius, S. and Slater, A. F. G. (1996).
Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/Apo-1 antibody. J. Biol. Chem.
271, 15420-15427.
Watanabe, M., Kitano, T., Kondo, T., Yabu, T., Taguchi, Y., Tashima, M., Umehara, H., Domae, N., Uchiyama, T. and Okazaki, T. (2004). Increase of nuclear ceramide through caspase-3-dependent regulation of the "sphingomyelin cycle"
in Fas-induced apoptosis. Cancer Res. 64, 1000-1007.
Yabu, T., Imamura, S., Yamashita, M. and Okazaki, T. (2008).
Identification of Mg2＋-dependent neutral sphingomyelinase 1 as a mediator of heat stress-induced ceramide generation and apoptosis. J. Biol. Chem. 280, 29971-29982.
Yoshida, Y., Arimoto, K., Sato, M., Sakuragawa, N., Arima, M.
and Satoyoshi, E. (1985). Reduction of acid sphingomyeli- nase activity in human fibroblasts induced by AY-9944 and other cationic amphiphific drugs. J. Biochem. 98, 1669-1679.
Zhou, Q., Band, M. R., Hernandez, A., Liu, Z. L. and Kummerow,
F. A. (2004). 27-Hydroxycholesterol inhibits neutral sphingo- myelinase in cultured human endothelial cells. Life Sci. 75, 1567-1577.
