Ceramide induces aSMase expression - The FASEB Journal

Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis HANS-PETER DEIGNER,*,1 RALF CLAUS,* GABRIEL A. BONATERRA,† CHRISTOF GEHRKE,* NILOFAR BIBAK,* MARKUS BLAESS,* MICHAEL CANTZ,‡ ¨ RGEN METZ,† AND RALF KINSCHERF† JU *Institute of Pharmaceutical Chemistry and Clinics of Anaesthesiology and Intensive Care Medicine, Jena, Germany, †Department of Anatomy and Cell Biology III, and the ‡Institute of Pathochemistry and Neurochemistry, University of Heidelberg, 69120 Heidelberg, Germany Sphingomyelinase (SMase) stimulation and subsequent ceramide generation are suggested to be involved in signal transduction of stress-induced apoptosis. We now show that apoptosis of human macrophages (M⌽) and fibroblasts initiated by oxidized low density lipoproteins (minimally modified LDL, mmLDL) is associated with an increase in acid SMase (aSMase, E.C. 3.1.4.12) expression and ceramide concentration. Application of a novel, potent, and specific inhibitor of aSMase expression (NB6) diminished the effects of mmLDL and C6-ceramide treatment by inhibiting transcription via Sp1 and AP-2. Moreover, apoptosis was abolished after mmLDL and C6-ceramide treatment of hereditary aSMase-deficient fibroblasts (from Niemann-Pick patients). We suggest that in mmLDL-initiated apoptosis 1) enhanced ceramide generation via aSMase appears to be required as well as 2) a positive feedback control of aSMase expression by the increase in intracellular ceramide concentration.—Deigner, H.-P., Claus, R., Bonaterra, G. A., Gehrke, C., Bibak, N., Blaess, M., Cantz, M., Metz, J., Kinscherf, R. Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis. FASEB J. 15, 807– 814 (2001) ABSTRACT

Key Words: acid sphingomyelinase 䡠 macrophages 䡠 NiemannPick 䡠 programmed cell death 䡠 C6-ceramide Ceramide is suggested to modify stress response (1) and to function as a common lipid mediator in programmed cell death generated in cells by signaling through CD95-Fas/Apo1 (2), tumor necrosis factor ␣ (3), ionizing radiation (4), and chemotherapeutic agents (5). Oxidized lipoproteins, modified by either transition metal- or by cell-mediated oxidation, undergo increased uptake by M⌽ (6), leading to cell damage. Several cytotoxic effects have been characterized such as the accumulation of cholesterol esters and modified cholesterol, leading to the formation of foam cells, lysosomal destabilization (7), and, finally, apoptosis and postapoptotic necrosis (8 –10). Stimulation of both acid sphingomyelinase (aSMase) and neutral SMase (nSMase) was observed using sphingomyelin substrate 0892-6638/01/0015-0807 © FASEB

from endogenous and exogenous sources (9, 11). Several recent reports implicate ceramide in modified low density lipoprotein (mLDL) -induced apoptosis as a product of sphingomyelin hydrolysis by aSMase/ nSMase (9 –14). The exposure of human M⌽ to mLDL, containing a considerable amount of sphingomyelin, has been shown to cause an increase in the concentration of cell-associated ceramide (10). Langmann and colleagues have suggested that the activity of aSMase is mainly determined by its expression (15). This study was carried out to analyze the role of aSMase and ceramide in minimally modified LDL (mmLDL) -initiated apoptosis in human M⌽ and fibroblasts. In addition, NB6, a novel inhibitor of aSMase expression developed by our group, and hereditary aSMase-deficient Niemann-Pick fibroblasts were used.

MATERIALS AND METHODS Human M⌽ and fibroblasts Human peripheral blood mononuclear cells from venous blood of healthy volunteers were routinely prepared by Ficoll-Paque (Sigma, Deisenhofen, Germany) density gradient centrifugation according to the manufacturer’s instructions. Human skin fibroblasts were derived by biopsy from healthy individuals (controls) or from patients with NiemannPick disease (type B). Niemann-Pick fibroblasts were kindly provided by Dr. K. Harzer (University of Tu¨bingen, Germany) and had been biochemically diagnosed on the basis of aSMase deficiency (enzymatic activity) and clinical data. In vitro conditions M⌽ or fibroblasts were cultured in RPMI 1640 medium supplemented with fetal calf serum (FCS), glutamine, and penicillin/streptomycin. Nonadherent cells were removed by washing with supplemented medium. Cells were cultured with lipoprotein-deficient serum (prepared by ultracentrifugation of FCS, ␳⬎1.21) (16) for 24 h prior to experimentation. LDL was isolated according to Havel et al. (16) or Himber 1 Correspondence: Clinics of Anaesthesiology and Intensive Care Medicine, University of Jena, 07740 Jena, Germany. E-mail: [email protected]

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et al. (17); concentrations refer to protein concentrations (apoB, 500 kDa) determined by the modified Lowry method (18). LDL was pooled from three donors for each preparation, dialyzed against Tris (pH 7.4, 20 mM, NaCl 150 mM, 0.1 mM EDTA), sterilized by filtration through a 0.2 ␮m membrane filter, and stored at 4°C under argon (up to 2 wk). mmLDL was obtained by Fe-catalyzed oxidation as described by Watson et al. (19). After incubation with 20 ␮M Fe2⫹ at 20°C for 48 h, the peroxide content as determined according to El-Saadani et al. (20) typically increased from 30 nmol/mg to 800-1000 nmol/mg; electrophoretic mobility was only marginally enhanced by this procedure. M⌽ or fibroblasts were exposed to mmLDL (27 ␮g/ml), C6-ceramide (10 ␮M), or H2O2 (30 ␮M) and apoptotic cells were identified by YOPRO-1 staining (21) and/or by the TUNEL technique (Quantum Appligene, Heidelberg, Germany). The percentage of apoptotic cells was counted using a microscope fitted with mercury light source and fluorescence assembly in combination with a computer-assisted morphometry system developed by our group (22). ␤-Hexosaminidase and ␤-glucosidase activities were determined as described previously (23). Preparation of the inhibitor of aSMase expression, NB6 ((3-carbazol-9-yl-propyl)-[2-(3,4-dimethoxy-phenyl)-ethyl)methyl-amine), will be reported elsewhere. Cytotoxicity was determined by measurement of lactate dehydrogenase (LDH) released from the cytosol of damaged human M⌽ or fibroblasts into the supernatant after treatment with NB6. Cells were incubated in 96-well plates in phenol red-free RPMI (for 18 h, 37°C; 5% CO2 at concentrations up to 10 ␮M) according to the manufacturer’s instructions (Cytotoxicity Detection Kit-LDH, Roche Molecular Biochemicals, Mannheim, Germany) using an ELISA plate reader (Bio-Rad 450) at 490 nm.

14,000 g, 4°C) the supernatants containing nuclear proteins were harvested, protein concentrations determined by the Bradford method, and samples stored at ⫺80°C for later use in the binding assays. EMSA was performed as described by Suzuki et al. (28): reaction mixtures (final volume 16 ␮l) containing 8 ␮g of nuclear extracts, 0.5 ␮g poly(dI-dC) (Gibco-BRL, Karlsruhe, Germany), and 60,000 cpm [32P]labeled probe in binding buffer were incubated at 30°C for 30 min. Samples were then analyzed by native 5% polyacrylamide gels and imaged by autoradiography (48 h). Doublestranded oligonucleotide containing consensus binding sides for SP-1, AP-2, and oct-1 (MWG-Biotech, Ebersberg, Germany) were labeled with ␣-[32P]-dCTP (Amersham Pharmacia, Freiburg, Germany) using Klenow fragment (GibcoBRL) and purified by centrifugation (735 g, 2 min) in MicroSpin娂 G-25 columns (Amersham/Pharmacia). The following oligo sequences were used: Sp-1: 5⬘-TGG AAC CGG GCG GGC GGG CTA CCG GGC GGG CT-3⬘; 5⬘-TGG AAG CCC GCC CGG TAG CCC GCC CGC CCG GT-3⬘. AP-2: 5⬘-TGG ATC GAA CTG ACC GCC CGC GGC-3⬘; 5⬘-AAG GGC CGC GGG CGG TCA GTT GGA-3⬘. oct-1: 5⬘-TGG ATG TCG AAT GCA AAT CAC TAG AA-3⬘; 5⬘-TGG ATT CTA GTG ATT TGC ATT CGT CA-3⬘. Statistical analyses Results are presented as means ⫹ se. Statistical procedures were performed by the Mann Whitney U-Wilcoxon Rank Sum W Test or the impaired Student’s t test using the SIMSTAT program (Provalis Research, Montreal Canada). A P value of 0.05 or less was chosen for statistical significance.

RESULTS Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting aSMase expression was investigated by Western blotting (24, 25) using polyclonal antibodies (kindly provided by K. Sandhoff and K. Ferlinz, Bonn, Germany) and by RT-PCR. For amplification of aSMase, primers were used under the following conditions: 5⬘-CAG GGT TCC TGG CTG GGC AGC A-3⬘ (forward) and 5⬘-GGT CCT GGA CC ATG AGA CCT AC-3⬘, 94°C, 60°C, 72°C, 1 min each, 40 cycles. Analysis of the sequence of the purified PCR product was performed by Dr. H. Delius (Deutsches Krebsforschungszentrum, Heidelberg, Germany). Determination of SMase activity and of ceramide concentration in cell lysates was performed as described previously (11). Quantification of ceramide concentration was assayed by the DAG method under appropriate conditions (26, 27).

As we assumed a critical role of aSMase in mmLDLinitiated programmed cell death of M⌽, we investigated the expression of this enzyme. Using PCR analysis, we show that mmLDL treatment raised the level of aSMase-mRNA in M⌽ (Fig. 1). Sequence analysis the 687 bp product confirmed the amplification of the respective aSMase fragment. In parallel with stimulation of aSMase expression, a significant induction of apoptosis was found [0 ␮g/ml mmLDL, 7⫾2.1% (apoptotic cells); 27 ␮g/ml, 20.7⫾2.7%; 54 ␮g/ml, 29.3⫾3.8%; 100 ␮g/ml, 37⫾4%; data are means ⫾ se of three experiments]. There is convincing evidence that mLDL-mediated

Electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared by the mini-extraction procedure as described earlier with minor modifications (28): after washing with phosphate-buffered saline, (stimulated) M⌽ were centrifuged for 15 s at 900 g at 4°C in a Hermle Z160 centrifuge (Wehingen, Germany). Then pellets were resuspended in 50 ␮l of buffer A (HEPES/KOH [20 mM], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, pH 7.6), incubated on ice for 10 min, and centrifuged for 10 min at 3000 g. The supernatants were discarded and the pelleted nuclei were resuspended in 50 ␮l of buffer C (HEPES/KOH [20 mM], 0.42 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 15% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.2% Nonidet P-40) and incubated on ice for 15 min. After centrifugation (5 min, 808

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Figure 1. RT-PCR of aSMase in M⌽. Expression of aSMase (687 bp) is shown after incubation (4 h) of M⌽ with increasing concentrations of mmLDL. Standard base pair marker is shown in the right panel.

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Figure 2. Structure of the inhibitor of aSMase transcription, NB6.

apoptosis involves generation of ceramide (10, 11). It has not been studied whether ceramide itself stimulates the production of the ceramide-delivering enzyme, aSMase. Here we show for the first time that not only mmLDL (see Fig. 1), but also ceramide alone, induced aSMase expression (see Fig. 3). The latter also occurred after H2O2 (30 ␮M; not shown) treatment. To provide further evidence for the role of aSMase in mmLDL or ceramide initiated cell death, we performed inhibition experiments. We selected one of several compounds capable of inhibiting aSMase expression and activity in pretreated M⌽: NB6 (Fig. 2). This agent did not show cytotoxic effects in M⌽ or fibroblasts at concentrations of up to 10 ␮M (18 h treatment) as determined by LDH release; the synthesis and analytical data of NB6 will be reported in more detail elsewhere. We found that NB6 pretreatment of M⌽ abolished ceramide-stimulated aSMase expression, induction of apoptosis (Fig. 3), and inhibited the enhancement of enzymatic activity [IC50⫽4 ␮M (mean of 4 experiments)] in lysates of pretreated cells (30 min) stimulated with 27 ␮g/ml mmLDL (4 h). To confirm a critical role of lysosomal enzymes in mmLDL-induced apoptosis, we investigated two other lysosomal enzymes as well as the effect of modulating the intralysosomal pH. We found that activities of ␤-hexosaminidase and ␤-glucosidase were not significantly different after mmLDL treatment. Untreated

Figure 3. Effect of NB6 pretreatment (2 ␮M, 30 min) on aSMase expression and apoptosis in M⌽. Percentage of apoptotic M⌽, determined by YOPRO-1 staining after incubation with medium or C6-ceramide (10 ␮M) with or without NB6 pretreatment. Data are means ⫾ se of three experiments. *P ⱕ 0.05 vs. control or #P ⱕ 0.05 vs. C6-ceramide (120 min) -treated cells. CERAMIDE INDUCES aSMase EXPRESSION

Figure 4. Apoptosis in fibroblasts and Niemann-Pick fibroblasts. Cell death (determined by YOPRO-1 staining) after incubation (16 h) with medium (control), mmLDL (27 ␮g/ml), C6-ceramide (10 ␮M), H2O2 (30 ␮M) (A) data of fibroblasts. Inset: concentration-dependent increase in mmLDL-induced apoptosis in fibroblasts; B) data of Niemann-Pick fibroblasts are shown as mean ⫹ se of three independent experiments. *P ⱕ 0.05 vs. control.

cells revealed ␤-hexosaminidase and ␤-glucosidase activities of 125.2 ⫾ 7.4 and 0.205 ⫾ 0.025; in mmLDLtreated cells we observed 145.2 ⫾ 10.1 and 0.185 ⫾ 0.005 activities of both enzymes, respectively. Furthermore, we raised the intralysosomal pH in fibroblasts by pretreatment (18 h) with the lysosomotropic agent ammonium chloride (9 mM) inhibiting lysosomal function (29). This treatment impaired the apoptogenic potential of mmLDL, i.e., no significant increase in the number of apoptotic cells could be observed (three experiments) upon addition of 54 ␮g/ml mmLDL. To further analyze the effect of aSMase on apoptosis initiated by mmLDL, we exposed aSMase-deficient human fibroblasts to mmLDL and C6-ceramide. Since significant differences were observed in mmLDLtreated normal human fibroblasts after 16 h (Fig. 4A, inset), this time was chosen. In contrast to the normal fibroblasts, where mmLDL and C6-ceramide significantly induced apoptosis (Fig. 4A), programmed cell death was abolished in Niemann-Pick fibroblasts (Fig. 4B). H2O2 treatment, however, induced apoptosis in Niemann-Pick fibroblasts similar to the control. 809

Figure 5. RT-PCR of aSMase in fibroblasts. Expression of aSMase (687 bp) in comparison with ␤-microglobulin (␤-Mg) is shown after incubation (16 h) of fibroblasts with increasing concentration of mmLDL.

Comparable to M⌽, mmLDL increased the level of aSMase-mRNA in fibroblasts in a concentration-dependent manner (Fig. 5), whereas NB6 inhibited the ceramide- and mmLDL-mediated induction of aSMase expression (Figs. 6A, B). Pretreatment of fibroblasts with NB6 (10 ␮M) prevented the rise of aSMase activity [pmol/(h ⫻ mg)]: control: 910 ⫾ 58, mmLDL: 1597 ⫾ 88, ceramide: 1466 ⫾ 111, and H2O2: 2154 ⫾ 164 vs. control ⫹ NB6: 780 ⫾ 69; mmLDL ⫹ NB6: 712 ⫾ 118, ceramide ⫹ NB6: 843 ⫾ 123, and H2O2 ⫹ NB6: 2213 ⫾ 241. NB6 pretreatment inhibited not only an increase in aSMase activity after stimulation, but also reduced its basal activity; no direct effect on acid/neutral SMase activity was found in cell lysates. To study potential changes in the transcriptional level, we investigated the effects of mmLDL, ceramide and hydrogen peroxide on Sp1 and AP-2 activities and their modulation by NB6. mmLDL, but ceramide in particular, enhanced the binding capacity of both transcription factors Sp1 and AP-2 in the nuclear extracts of fibroblasts (Fig. 7). Pretreatment of these cells with NB6 reduced the mmLDL- or ceramide-mediated increase in Sp1 and AP-2 affinity (Fig. 7). A minor increase in transcription factor activity was observed in response to the plain compound NB6. Specificity of binding was confirmed by the effective competition of a 40-fold molar excess of unlabeled probe. Furthermore, NB6 inhibited the mmLDL- and C6-ceramide-induced apoptosis in control fibroblasts similar to aSMase-deficient NiemannPick fibroblasts (Fig. 8). As depicted in Fig. 9, the cell-associated ceramide level, which roughly corresponded to the respective aSMase expression, increased after mmLDL, ceramide or H2O2 treatment.

amide. Inhibition of aSMase by NB6, a novel aSMase transcription inhibitor synthesized in our lab, abolished the effects of mmLDL treatment. In aSMase-deficient (Niemann-Pick) fibroblasts, mmLDL treatment did not initiate apoptosis. mmLDL is known to be internalized mainly via the LDL receptor pathway when exposed to M⌽ (30). The resulting endocytotic vesicles do not necessarily fuse with lysosomes because signaling via aSMase was demonstrated not to depend on ingestion and processing of mLDL in these organelles (31). mmLDL was shown to damage the lysosomal membranes of M⌽ (7), suggesting a leakage of ceramide into the cytosol under these conditions. Although exchange of ceramide over intact membrane bilayers requires days and thus likely is too slow to participate in signaling, a trans-bilayer transport (flip-flop) is expected to occur much faster (32). Several possibilities have been proposed as to how ceramide may interact with its targets: 1) potential target molecules might diffuse to the site of ceramide generation, 2) ceramide might act by changing the physical properties of the membrane where it has been synthesized, and 3) ceramide could be delivered by lipid transfer proteins (33). Furthermore, endosomal/lysosomal cathepsin D, an

DISCUSSION We and others have shown recently that stimulation of SMases is involved in the mLDL-mediated program of cell death (9 –14). Our results now demonstrate that initiation of apoptosis by treatment of M⌽ with mmLDL leads to an increase in both the expression of aSMase and the intracellular concentration of cer810

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Figure 6. RT-PCR and Western blot of aSMase in fibroblasts. The effect of NB6 pretreatment (30 min) in fibroblasts after addition of C6-ceramide (A) or mmLDL (B) is demonstrated. Experimental conditions as described in legend to Fig. 4.

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Figure 8. Effect of NB6 pretreatment on apoptosis in fibroblasts. Cells were incubated with mmLDL (27 ␮g/ml) or C6-ceramide (10 ␮M) with or without NB6 (2 ␮M, 30 min) pretreatment. Representative data of three experiments are shown as means ⫹ se. *P ⱕ 0.05 and ⫹P ⱕ 0.01 vs. control.

The key role of aSMase and ceramide in cell death is further supported by 1) the fact that a raise of intralysosomal pH by ammonium chloride diminishes mmLDL-initiated apoptosis (this report) and 2) data of Lepple-Wienhus et al. (36), who found that effects on Ca2⫹ influx after CD95/CD95L interactions could be mimicked by ceramide and sphingosine generated by aSMase. Furthermore, Farina et al. have most recently shown that ceramide accumulation due to a ceramidase deficiency (Farber disease) induces abundant morphological changes associated with apoptosis in colonocytes (37). Overexpression of another ceramide-regulating enzyme in lysosomes, acid ceramidase, was consistently shown to protect from apoptosis (38), and unpublished experiments from our laboratories demonstrate a decreased sensitivity of ceramidase overexpressing fibroblasts (kindly provided by D. Adam, Kiel, Germany) toward apoptosis, induced by mLDL. To further analyze the prominent role of aSMase and

Figure 7. EMSA of A) Sp1 and B) AP-2 in fibroblasts. Nuclear extracts were isolated from untreated-, C6-ceramide-, mmLDL-, or hydrogen peroxide-treated cells without NB6 pretreatment. Lane 8: competition of control; lane 9: competition of ceramide; lane 10: free probe without nuclear extract; lanes 8 –10: with 40-fold excess of unlabeled probe.

enzyme involved in mediation of apoptosis (34) by initiating a proteolytic cascade, has recently been identified as likely to convey downstream signaling of aSMase by ceramide (35). The release of cathepsin D into the cytosol was also found after treatment of M⌽ with mLDL (7), i.e., under conditions where an eventual increase in cellular ceramide might be assumed. CERAMIDE INDUCES aSMase EXPRESSION

Figure 9. Ceramide levels in fibroblasts: effect of NB6. Ceramide concentration in control fibroblasts (100% ⫽ 112 pmol ceramide/mg protein) with and without NB6 pretreatment (10 ␮M, 30 min). For other concentrations/conditions of treatment, see legend to Fig. 4. Values represent the mean from three experiments. Significance is indicated as follows: **P ⬍ 0.01, *P ⬍ 0.05 relative to control and ⫹P ⬍ 0.05 vs. mmLDL-treated cells. 811

subsequent ceramide generation in the initiation of the death program after mmLDL treatment, we tested NB6, a novel inhibitor of aSMase transcription, and Niemann-Pick fibroblasts, which differ from fibroblasts by the lack of aSMase activity. NB6 inhibited mmLDL or ceramide-induced aSMase expression, stimulation of ceramide production, and consequently reduced programmed cell death in M⌽ and fibroblasts. In Niemann-Pick fibroblasts, mmLDL and ceramide (used in concentrations like in control fibroblasts) failed to induce apoptosis. These data provide evidence for a similar antiapoptogenic mechanism in NB6-treated or Niemann-Pick fibroblasts during mmLDL/ceramidemediated apoptosis, involving inhibition of aSMase transcription and, to some extent, ceramide formation. Here we show for the first time that mmLDL and ceramide are both able to stimulate aSMase expression. The recent findings of Langmann and colleagues indicate cooperative regulation of aSMase by redox-sensitive transcription factors AP-2 and Sp1 (15, 39, 40). Our data demonstrate an enhancement of transcription factor activities in response to mmLDL and C6-ceramide treatment. In particular, Sp1 binding activity is markedly increased after exposure to C6-ceramide and is reducible by NB6 pretreatment. Hence, this agent inhibits mmLDL/ceramide-induced expression of aSMase by yet unknown mechanisms. Furthermore, antiapoptotic effects may include the modulation of other factors such as stress-inducible manganese superoxide dismutase (10), known to be controlled by Sp1 (41). The addition of mmLDL as well as plain ceramide further stimulate aSMase-mediated ceramide production and enzyme expression, thus suggesting an autofeedback mechanism being interruptible by NB6 (Fig. 10). This hypothesis is supported by the fact that exogenous ceramide at concentrations applied to fibroblasts did not result in increased apoptosis in NiemannPick fibroblasts. Treatment of fibroblasts with the inhibitor of aSMase transcription, NB6, prior to mmLDL or ceramide exposure also neutralized programmed cell death induction. These results could be explained by the absence of aSMase stimulation via a positive auto-regulative loop, thus preventing the generation of the lipid mediator at sites critical to apoptogenic signaling. Comparable to our findings with aSMase, Jaffre´zou et al. (42) have suggested a positive feedback control of nSMase activity by ceramide in M⌽, but no data about enzyme expression have been provided. Addition of ceramide to fibroblasts raises ceramide concentrations comparable to those obtained after exposure to mmLDL. However, a marginal reduction of the ceramide-induced increase to a level similar to that found after mmLDL addition is associated with a decrease in apoptosis. These observations indicate that the ceramide concentration per se is not critical to apoptosis induction, but rather generation in the lysosomal compartment. The importance of the total amount of cell associated ceramide, however, should not be overestimated as further distribution and local812

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Figure 10. Schematic representation of the suggested autofeedback loop. Results of the present and previous studies (10) support the following scheme regarding the mechanism of ceramide/mLDL-mediated aSMase induction. Stimuli such as exogenous ceramide (or ceramide released on mLDL internalization) enhance transcription of aSMase and thereby amplify further hydrolysis of SM by this enzyme. This pathway is likely to be critical to mLDL-induced apoptosis of M⌽ and FB. Inhibition at the transcriptional level of aSMase is achieved by novel synthetic compounds such as NB6. aSMase, acid SMase; mRNA, messenger RNA; mLDL, modified LDL; SM, sphingomyelin; question mark designates unknown signaling pathways.

ization in subcellular compartments have not been addressed in this study. Thus, exogenous C6-ceramide may not completely mimic ceramide species generated from endogenous sphingomyelin on stimulation. However, there is no experimental option as long-chain analogs added to the medium do not enter the cell or distribute intracellularly. In the context of this study, apoptosis can be induced by passing the aSMase deficiency with higher concentrations of exogenous C6ceramide (50 ␮M) (not shown). Our results agree with previous findings with Niemann-Pick patients, whose cells did not respond to ionizing radiation with ceramide formation and apoptosis (43). Furthermore, irradiated endothelial cells of aSMase knockout mice revealed a 70% reduction of apoptosis (44). Similarly, the induction of oxidative stress by photodynamic therapy was shown to be absent in Niemann-Pick lymphoblasts (36, 45, 46). The combination of both exogenous SMase and photodynamic therapy was required to induce oxidative stress, whereas the addition of the enzyme alone failed to induce apoptosis, despite of an intracellular increase in ceramide. Evidence for stress as an integral element of mLDL/ceramide/aSMase-induced cell death is also supported by our recent observations that mLDL- and ceramide-mediated apoptosis is associated with an increase in manganese superoxide dismutase expression/ activity and a decrease in intracellular glutathione levels in M⌽ (10). Vice versa, stress-induced apoptosis apparently requires the generation of ceramide in U937 cells (47). The latter study indicates that exogenous addition of hydrogen peroxide increases cer-

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amide levels. Stress-induced apoptosis in turn requires the generation of ceramide in U937 cells (47). However, the addition of hydrogen peroxide also induced apoptosis in Niemann-Pick fibroblasts, which may be due to additional or alternative pathways. One possible mechanism might be the direct and concentrationdependent damage of mitochondrial DNA, which was more extensive and persisted longer than nuclear DNA damage in human cells during oxidative stress (48). Until now there were no data available to clarify whether aSMase deficiency (Niemann-Pick patients) inhibits atherosclerosis. Nevertheless, reduction of aSMase activity could be a principle to control mmLDLmediated cell death. This concept may have further implications for various states of human diseases involving ceramide-dependent signaling (49 –51). In atherogenesis, for instance, apoptosis may be detrimental, since it could lead to plaque rupture and thrombosis (52). This study was supported by the Deutsche Forschungsgemeinschaft, by the Ernst und Berta-Grimmke Stiftung, and by the Fonds der Chemischen Industrie. Thanks are due to Dr. K. Harzer for providing the fibroblasts and N. Blenck, T. Ko¨hler, and U. Traut for excellent technical assistance. We wish to thank Drs. K. Sandhoff and K. Ferlinz for providing aSMase antibodies.

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The FASEB Journal

Received for publication April 23, 2000. Revised for publication July 27, 2000.

DEIGNER ET AL.