Positive feedback control of neutral sphingomyelinase activity by ceramide

^a CJF INSERM 9503, Centre Claudius Régaud, Toulouse 31052, France
^b Laboratoire de Biochimie Médicale, INSERM 466, Centre Hospitalier Universitaire Rangueil, Toulouse 31403, France
^c Service d'Hématologie, Centre Hospitalier Universitaire Purpan, Toulouse 31059, France

	ABSTRACT

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While ceramide has emerged as a potent signal transducer, inconsistencies in the kinetics of ceramide generation, or its absence, in response to stimuli have led to confusion and skepticism as to its potential role in apoptosis or proliferation. Here we show that in U937 and HL60 myeloid leukemia cells and in normal skin fibroblasts, cell-permeant ceramides can trigger neutral sphingomyelinase activation, sphingomyelin hydrolysis, and endogenous ceramide generation regardless of Bcl2 overexpression. These observations identify neutral sphingomyelinase as a novel target for ceramide and show that this positive feedback mechanism is responsible for signal propagation, as exemplified by mitogen-activated protein kinase activation in daunorubicin-treated cells. This study provides insight into a fundamental process of cell biology. Indeed, such a sustained ceramide-mediated signal throughout the apoptotic process would ensure self-destruction, perhaps by overriding evolutionary conserved primal cell survival mechanisms.—Jaffrézou, J.-P., Maestre, N., de Mas-Mansat, V., Bezombes, C., Levade, T., Laurent, G. Positive feedback control of neutral sphingomyelinase activity by ceramide. FASEB J. 12, 999–1006 (1998)

Key Words: ERK • daunorubicin • TNF • C6-ceramide

	INTRODUCTION

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CERAMIDE HAS EMERGED as a potentially important mediator of a number of natural and pharmacological agents that affect cell growth, viability, and differentiation (1, 2). This lipid second messenger is the breakdown product of sphingomyelin (SM),³ which is generated by the activation of a sphingomyelinase (SMase). Agonists of the SM-ceramide cycle include cytokines and growth factors such as interleukin 1ß, tumor necrosis factor (TNF-), nerve growth factor, -interferon, antibodies directed against functional molecules such as Fas/APO-1 or CD28 proteins, as well as stress-inducing agents such as ionizing radiation, hydrogen peroxide, ultraviolet light, and antileukemic agents. Ceramide has been shown to exert a wide range of biological effects depending on the cellular model, including cell activation, mitogenic signaling, a survival promoting effect, growth inhibition, and, most notably, apoptosis. For example, ceramide has been described to promote proliferation in fibroblasts, cell cycle arrest in Molt-4 cells, and apoptosis in many other cellular models such as U937, lymphoblastoid, and endothelial cells. The molecular effects of ceramide are multiple and include phosphorylation events, oxidative balance, and proteolytic activities, most of which are blocked by Bcl2. However, Bcl2 does not inhibit ceramide generation induced by cytotoxic agents (1, 2).

The discordance that exists in the described kinetics of ceramide generation, or absence thereof, in response to extracellular stimuli has led to confusion and uncertainty as to the potential role of ceramide in cell signaling. To reconcile these observations, Hannun (2) recently speculated on the existence of several cycles, or phases, of ceramide generation, both acute and more sustained. Indeed, we previously described two distinct cycles of SM hydrolysis triggered by TNF- and two cycles of neutral SMase activation, SM hydrolysis, and ceramide generation in daunorubicin (DNR) -treated cells (3, 4). These observations raised the possibility that sequential ceramide generation was mediated by a ceramide-activated pathway or feedback mechanism.

	MATERIALS AND METHODS

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Drugs and reagents
Daunorubicin was obtained from the NCI Drug Repository. Ceramides were purchased from Sigma Chemical Co. (St. Louis, Mo.). Silica gel 60 thin-layer chromatography plates were from Merck (Darmstadt, Germany). All other drugs and reagents were purchased from Sigma, Carlo Erba (Rueil-Malmaison, France), or Prolabo (Paris, France).

Cell culture
The human myeloblastic cell line U937 obtained from the ATCC (Rockville, Md.), the HL60, HL60/Neo, and HL60/Bcl2 transfectants (generously provided by Dr. Naumovski, Stanford Medical Center, Calif.), and human normal skin fibroblasts (obtained from our laboratory) were cultured in IMDM medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (all from Eurobio, Les Ulis, France). Cell stocks were screened routinely for Mycoplasma (Stratagene Mycoplasma PCR kit, La Jolla, Calif.).

Lipid quantification
SM quantitation was performed by labeling cells to isotopic equilibrium with 0.4 µCi/ml of [methyl-³H]choline (specific activity 81.0 Ci/mmol, DuPont-NEN, Les Ulis, France) for 48 h in complete medium, as previously described (3, 4). Cells were then washed and resuspended in serum-free medium for kinetic experiments in the presence of the indicated agonist (delivered in ethanolic solution, final concentration of 0.25%). Aliquots were taken for protein determination (5). Radioactive SM was extracted (6, 7) and quantitated by scintillation counting. Similar results for SM quantitation were obtained after thin layer chromatography, where SM spots were identified based on Rf and comigration with authentic standards (data not shown).

Total cellular ceramide quantitation was performed by labeling cells to isotopic equilibrium with 1 µCi/ml of [9, 10-³H]palmitic acid (53.0 Ci/mmol, Amersham, Les Ulis, France) for 48 h in complete medium, as described previously (3, 4). Cells were then washed and resuspended in serum-free medium for kinetic experiments. Lipids were extracted and resolved by thin layer chromatography, and ceramide was scraped and quantitated by liquid scintillation spectrometry. Alternatively, intracellular ceramide was quantitated by using Escherichia coli diacylglycerol kinase (Amersham, kit no. RPN200) and [³³P]-ATP (3300 Ci/mmol, Isotopchim, Ganagobie, France) according to previously published procedures (6). Statistical analyses were performed by Student's t test.

Quantitation of sphingomyelinase activities
SMase activities were determined as previously described, using [choline-methyl-¹⁴C]SM (120,000 dpm/assay, Dupont-NEN) as substrate (4, 8).

ERK1 analysis
Immunoprecipitation of MAP kinase was performed essentially as described previously (9). Cells were washed and resuspended in RIPA lysing buffer [50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X100, 10 mM ß-glycerophosphate, 0.5% sodium desoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM NaF, 5 mM EDTA, 1 mM dithiothreitol, 1 mM Na orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A]. Samples were sonicated and centrifuged at 12,500 x g for 5 min at 4°C. A 5 µl aliquot of the supernatant was used for protein determination (5). The supernatant was precleared for 1 h at 4°C with 5 mg protein A-Sepharose. Three migrograms of anti-phosphotyrosine antibody (clone PY20, Transduction Laboratories, Lexington, Ky.) was added and incubated overnight at 4°C; 25 µg protein G Sepharose 4B (Sigma) was then added for 2 h at 4°C. The immunoprecipitates were centrifuged at 12,000 x g for 5 min, washed twice in RIPA buffer, and resolved on a 12.5% SDS-polyacrylamide gel. The proteins were transferred onto nitrocellulose and immunoblotted with an anti-ERK1 antibody (C16, Santa Cruz Biotech., Santa Cruz, Calif.) and a donkey anti-rabbit secondary antibody (Immunotech, Marseille, France). The signal was visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, U.K.).

PARP cleavage
Analysis of poly(ADP-ribose)polymerase (PARP) proteolysis was performed by resuspending cells in sample buffer (62.5 mM Tris, pH 6.8, 4 M urea, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.04% bromophenol blue). Samples were boiled for 5 min, loaded onto a 10% SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. PARP and its cleaved fragment were detected by using a rabbit polyclonal antiserum (Boehringer-Mannheim, Meylan, France) and a donkey anti-rabbit secondary antibody (Immunotech, Marseille, France). The signal was visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, U.K.).

	RESULTS AND DISCUSSION

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Activation of neutral sphingomyelinase activity by ceramides in myeloid cells
The aim of this study was to determine whether cell-permeant ceramides could trigger endogenous ceramide production. Treatment of the human myeloid leukemia cell line U937 with cell-permeant C6-ceramide triggered SM hydrolysis in a dose-dependent manner, which was maximal at 25 µM ( Fig. 1a). Similar results were obtained with C2-ceramide (data not shown). The SM cycle (hydrolysis and resynthesis) triggered by 25 µM C2- and C6-ceramide was acute in nature, appearing at 4–12 min, and reached a maximum of -20% ( Fig. 1b). This correlated closely with the dose required for maximal apoptotic effect in U937 cells cultured in complete media (data not shown). Neither the natural ceramide (which is relatively impermeant) nor the inactive analog dihydroceramide had any significant effect on SM hydrolysis. The onset of the SM hydrolysis triggered by C6-ceramide was concurrent with an increase in both endogenous ceramide ( Fig. 1c) and neutral SMase activity ( Fig. 1d). Similar results were observed with C2-ceramide, but natural ceramide and dihydroceramide had no significant effect (data not shown). Acid SMase activity remained unchanged throughout all experiments. We also observed cyclical endogenous ceramide generation in acid SMase-deficient Niemann-Pick lymphoblasts (data not shown).

View larger version (134K): [in this window] [in a new window]   Figure 1. Effect of exogenous ceramides on endogenous ceramide metabolism. a) U937 cells were metabolically prelabeled as described in Materials and Methods and treated with various concentrations of cell-permeant C6-ceramide. At different time points, aliquots were taken for SM quantitation. Results represent peak SM hydrolysis, which occurred at 8 min. b) SM quantitation was performed in U937 cells treated with 25 µM C6-ceramide (), 25 µM C2-ceramide (), natural ceramide (), or 25 µM C6-dihydroceramide () in complete RPMI 1640 medium (basal SM level was 10.4±0.8 nmol/mg protein). c) Endogenous ceramide levels (basal level was 0.4 nmol/mg protein) and d) neutral and acid SMase activities were determined in U937 cells treated with 25 µM C6-ceramide. SMase results represent peak SM hydrolysis, which occurred at 8 min. Basal neutral and acid SMase activities were 37 ± 2 and 870 ± 53 pmol/(h·mg), respectively. Data are mean ± SEM of at least three independent determinations. e, f) ERK1 (p44) tyrosine phosphorylation was analyzed in U937 cells treated with 25 µM C6-ceramide for various times. e) Immunoprecipitation; f) densitometry analysis.

Of course, there is not a strict stoichiometric relationship between SM breakdown and ceramide generation. The reasons for this are unclear. It has recently been suggested that a concomitant ceramidase activation is potentially responsible for such lopsided SM/ceramide ratios (10).

ERK1 activation in ceramide-treated cells
Given the importance of the identification of neutral SMase as a target of ceramide, it was necessary to determine whether the endogenously generated ceramide exerted a cellular function. Of the described targets of ceramide (1, 2), we elected to investigate the activation of extracellular signal-regulated kinase 1 (ERK1). Although there is some controversy as to whether apoptosis induced by cell-permeant ceramides leads to activation or inhibition of members of the mitogen-activated protein kinase (MAPK) family (11–14), we observed in U937 cells a threefold increase in ERK1 activation by 25 µM C6-ceramide after 2–6 min ( Fig. 1e, f). Although the kinetics of ceramide generation and ERK1 activation varied somewhat between experiments, 25 µM C6-ceramide reproducibly led to increased endogenous ceramide production and ERK1 activation within the first 10 min.

Activation of neutral sphingomyelinase activity by ceramides in fibroblasts
Since ceramide is described as a pleiotropic signal transducer not only in apoptosis, but equally in cell proliferation (1, 15), we investigated whether C6-ceramide could also trigger endogenous ceramide generation, SM hydrolysis, and neutral SMase activity in a fibroblast model. Indeed, treatment of normal human fibroblasts by C6-ceramide resulted in concomitant ceramide production and SM hydrolysis at 2–8 min (data not shown). These effects were correlated with an increase in the activity of neutral but not acid SMase ( Fig. 2).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of cell-permeant C6-ceramide on SMase activities in normal human fibroblasts. Human skin fibroblasts obtained from normal individuals were treated with 25 µM C6-ceramide. At various time points, aliquots were taken and both neutral () and acid () SMase activities were determined. Results are representative of at least three independent experiments (mean ± SEM). *P < 0.001 at peak. Basal neutral and acid SMase activities were 180 ± 21 and 1200 ± 92 pmol/(h·mg), respectively.

Endogenous ceramide generation in cells treated with cell-permeant ceramides
In light of these observations, we investigated the long-term events triggered by cell-permeant ceramides. If ceramide is indeed responsible for a self-activating pathway, one should expect to identify several cycles of acute endogenous ceramide boost in cell-permeant ceramide-treated cells. Therefore, we measured ceramide levels (as well as SM content and both neutral and acid SMase activities) in 25 µM C6-ceramide-treated U937 cells during a 24 h period ( Fig. 3). We identified at least two cycles of endogenous ceramide generation peaking at about 10 min and a somewhat broader one at 30 min, concomitant with increased neutral SMase activation (data not shown). These cycles were then followed after 6 h by a prolonged and persistent accumulation of ceramide, attaining near 400% at 24 h. At this time, no significant increase in neutral SMase was observed (data not shown). Pretreatment of U937 cells with the ceramide synthase inhibitor fumonisin B1 had no significant effect on ceramide generation before the onset of apoptosis, but did inhibit by about 50% the later sustained ceramide elevation. Kinetic experiments revealed significant nuclear fragmentation and the appearance of classic features of apoptosis only at 6 h and beyond. These events were unaffected by fumonisin B1 treatment (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of cell-permeant C6-ceramide on endogenous ceramide production. U937 were metabolically prelabeled as described in Materials and Methods; cells were then pretreated for 1 h with () or without () 50 µM fumonisin B1 and incubated with 25 µM C6-ceramide in complete RPMI 1640 medium. At different time points, aliquots were taken for ceramide quantitation. Results are representative of at least three independent experiments (mean ± SEM). *P < 0.001 at peak.

Ceramide generation and apoptosis in cells treated with daunorubicin
Because we previously described at least two cycles of ceramide generation in DNR-triggered apoptosis (4), we measured ceramide levels (and both neutral and acid SMase activities and SM content) in 1 µM DNR-treated U937 cells during a 24 h period ( Fig. 4a). We observed at least four cycles of ceramide generation (20–40%) appearing approximately at 4–8 min, 12–18 min, 30–60 min, and 90–120 min. These acute changes in ceramide were concurrent with an increase in neutral SMase activity and SM hydrolysis. Acid SMase activity remained unchanged (data not shown). After the fourth hour, these ceramide cycles were followed by a prolonged and persistent accumulation of ceramide, attaining more than 400% at 24 h. Similar results were observed with TNF- (data not shown). Pretreatment of U937 cells with fumonisin B1 had no significant effect on the initial four cycles of ceramide generation, but did inhibit by greater than 50% the ceramide elevation sustained later. Altogether, these results confirmed that at least within the first 4 h, ceramide is generated via SM hydrolysis mediated by a neutral SMase (4). However, inhibition by fumonisin B1 of the (later) sustained ceramide elevation implicates a ceramide synthase activity at these times. Since fumonisin B1 inhibited by > 90% in situ ceramide synthesis in U937 cells (4), we can conclude that the increase in ceramide levels occurring after the fourth hour is only partly (~50%) due to ceramide synthesis.

View larger version (51K): [in this window] [in a new window]   Figure 4. Effect of DNR on endogenous ceramide production, PARP cleavage, and ERK1 activation. U937 cells were preincubated for 1 h with () or without () 50 µM fumonisin B1 and further incubated with 1 µM DNR in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C. At different time points, aliquots were taken for a) ceramide quantitation, b) PARP cleavage analysis, and c–f) ERK1 (p44) tyrosine phosphorylation. c, e) Immunoprecipitation. d, f) Densitometry analysis. Results are representative of at least three independent experiments (mean±SEM). *P < 0.001 at peak.

To determine when within the time course of ceramide generation apoptosis was triggered, we measured one of the earliest events, PARP cleavage (16). A kinetic experiment revealed that PARP cleavage appeared at the fourth hour, fully 1 h before the observed sustained ceramide increase ( Fig. 4b). These observations led us to conclude that the ceramide elevation that appeared after the onset of apoptosis (i.e., > 4 h) was not a mediator of apoptosis, but could simply have represented a dysregulation of ceramide metabolism in dying cells. Indeed, similarly high ceramide levels were also seen in necrotic cells (data not shown), for example, after treatment with 10 µM DNR (17, 18). Hence, the autoregulation of ceramide production within the apoptotic pathway occurs at least within the first hour; the later sustained ceramide levels most likely reflect cell death (metabolic deregulation of postapoptotic necrotic cells).

ERK1 activation in daunorubicin-treated cells
Analysis of p44 tyrosine phosphorylation triggered by 1 µM DNR revealed a complex pattern of ERK1 activation. Indeed, DNR triggered at 2–8 min an initial twofold increase in ERK1 phosphorylation, followed by a slight decrease. ERK1 activation further increased up to threefold approximately after the 8th min, followed by a relative stabilization until the 30th min, further increased to 4.5-fold at 2 h, subsided to threefold at the third hour, and finally increased to >sixfold at 6 h ( Fig. 4c–f). Although the kinetics of ERK1 activation varied somewhat between experiments, 25 µM C6-ceramide reproducibly led to increased ERK1 activation in a wave-like pattern that correlated closely with the observed endogenous ceramide boosts ( Fig. 4a).

Effect of Bcl2 on the ceramide-activated sphingomyelinase pathway
To better define where endogenous ceramide generation is situated within the apoptotic signaling cascade, we investigated the role of Bcl-2 overexpression on the ceramide-activated SMase pathway. Indeed, previous studies have shown that whereas Bcl-2 overexpression blocked apoptosis induced by cell-permeant ceramides, TNF-, ionizing radiation, vincristine, and DNR, Bcl-2 did not inhibit early ceramide generation triggered by agonists of the SM-ceramide pathway (19–23). In the myeloid cell lines HL60/neo and HL60/Bcl2, genetically engineered to overexpress Bcl-2 (24), 25 µM C6-ceramide induced about a 25% increase in endogenous ceramide, which was concomitant with SM hydrolysis and neutral SMase activation ( Fig. 5). Acid SMase activity was unchanged. These results identify the cell-permeant triggered endogenous ceramide elevation as an event situated upstream of Bcl-2.

View larger version (21K): [in this window] [in a new window]   Figure 5. Effect of Bcl2 overexpression on C6-ceramide-triggered endogenous ceramide generation. HL60/Neo (open symbols) and HL60/Bcl2 (filled symbols) transfectants were metabolically prelabeled as described in Materials and Methods and then incubated in complete RPMI media with 25 µM C6-ceramide. At different time points, aliquots were taken and a) ceramide, b) SM, and c) neutral (circles) and acid (squares) SMase activities were quantitated. Results are representative of at least three independent experiments (mean±SEM). *P < 0.001 at peak.

In conclusion, this study demonstrates that cell-permeant ceramides trigger the SM-ceramide signal transduction pathway. In essence, endogenous ceramide generation, triggered by an appropriate stimulus, can perpetuate its own production. The mechanisms involved are unknown. In an in vitro cell-free system, we failed to observe SMase activation by ceramides. However, such experimental models are classically known not to be functional for the evaluation of SMase activation (or inhibition) (25–27). Nevertheless, preliminary studies in our laboratory show that this autoregulation can be blocked by serine protease inhibitors, which suggests a role for proteases within this cascade. Furthermore, this study may help explain the discordance in the literature as to the involvement of ceramide in cell death (or cell proliferation). In fact, depending on the time scale investigated, one can easily miss a ceramide generation cycle. We also show that the late ceramide production mediated by ceramide synthase that occurs post-PARP cleavage does not regulate apoptosis; it probably simply illustrates cell suffering, since similar ceramide synthase activation is observed under necrotic conditions (unpublished data; ref 17). Finally, the observation that successive acute cycles of ceramide generation that enable a sustained ceramide-activated pathway (as in the case of ERK1 activation) may provide insight into a fundamental process of cell biology. One could speculate that such a sustained ceramide-mediated signal throughout the apoptotic process is required to ensure self-destruction, perhaps by overriding primal evolutionary conserved survival mechanisms.

	ACKNOWLEDGMENTS

 
This study was supported in part by l'Association pour la Recherche sur le Cancer grants 6749, 3002, and 2069 (G.L., T.L., and J.P.J.), Actions Concertées Coordonnées-Sciences du Vivant grant (G.L.), La Fédération Nationale des Centres de Lutte Contre le Cancer 96003115 (J.P.J. and T.L.), le Conseil Régional Midi-Pyrénées (T.L. and J.P.J.), and La Ligue Nationale Contre le Cancer (G. L.). N.M. is the recipient of a LIGUE 82 fellowship.

	FOOTNOTES

 
¹ J. P. Jaffrézou and N. Maestre contributed equally to this work.

² Correspondence: CJF INSERM 9503, Centre Claudius Regaud, 20 rue du Pont st. Pierre, Toulouse Cédex 361052 France.

³ Abbreviations: DNR, daunorubicin; ERK1, extracellular signal-regulated kinase 1; SM, sphingomyelin; SMase, sphingomyelinase; TNF, tumor necrosis factor; EDTA, ethylene diaminetetraacetic acid; C6-ceramide, N-hexanoyl-D-sphingosine; C2-ceramide, N-decanoyl-D-sphingosine; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose)polymerase; SDS, sodium dodecyl sulfate.

Received for publication December 1, 1997. Accepted for publication March 13, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1. Kolesnick, R., and Fuks, Z. (1995) Ceramide: a signal for apoptosis or mitogenesis? J. Exp. Med. 181, 1949–1952[Free Full Text]
2. Hannun, Y. A. (1996) Functions of ceramide in coordinating cellular responses to stress. Science 274, 1855–1859[Abstract/Free Full Text]
3. Andrieu, N., Salvayre, R., Jaffrézou, J. P., and Levade, T. (1995) Low temperatures and hypertonicity do not block cytokine-induced stimulation of the sphingomyelin pathway but inhibit nuclear factor-kappa B activation. J. Biol. Chem. 270, 24518–24524[Abstract/Free Full Text]
4. Jaffrézou, J. P., Levade, T., Bettaïeb, A., Andrieu, N., Bezombes, C., Maestre, N., Vermeersch, S., Rousse, A., and Laurent, G. (1996) Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis. EMBO J. 15, 2417–2424[Medline]
5. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85[Medline]
6. Van Veldhoven, P. P., Matthews, J. J., Bolognesi, D. P., and Bell, R. M. (1992) Changes in bioactive lipids, alkylacylglycerol and ceramide, occur in HIV-infected cells. Biochem. Biophys. Res. Commun. 187, 209–216[Medline]
7. Levade, T., Tempesta, M. C., and Salvayre, R. (1993) The in situ degradation of ceramide, a potential lipid mediator, is not completely impaired in Farber disease. FEBS Lett. 329, 306–312[Medline]
8. Wiegmann, K., Schhtze, S., Machleidt, T., Witte, D., and Krönke, M. (1994) Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78, 1005–1015[Medline]
9. Ikeda, U., Oguchi, A., Okada, K., Ishikawa, S., Saito, T., Ikeda, M., Kano, S., Takahashi, M., Shiomi, M., and Shimada, K. (1994) Involvement of LDL receptor in the proliferation of vascular smooth muscle cells. Atherosclerosis 110, 87–94[Medline]
10. Nokolova-Karakashian, M., Morgan, E. T., Alexander, C., Liotta, D. C., and Merrill, A. H., Jr. (1997) Bimodal regulation of ceramidase by interleukin-1ß: implication for the regulation of cytochrome P450 2C11 (CYP2C11). J. Biol. Chem. 272, 18718–18724[Abstract/Free Full Text]
11. Raines, M. A., Kolesnick, R. N., and Golde, D. W. (1993) Sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid HL60 cells. J. Biol. Chem. 268, 14572–14575[Abstract/Free Full Text]
12. Westwick, J. K., Bielawska, A. E., Dbaido, G., Hannun, Y. A., and Brenner, D. A. (1995) Ceramide activates the stress-activated protein kinases. J. Biol. Chem. 270, 22689–22692[Abstract/Free Full Text]
13. Yao, B., Zhang, Y., Delikat, S., Mathias, S., Basu, S., and Kolesnick, R. N. (1995) Ceramide-activated protein kinase is a Raf kinase. Nature (London) 378, 307–310[Medline]
14. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Requirement for ceramide initiated SAPK/JNK signalling in stress-induced apoptosis. Nature (London) 380, 75–79[Medline]
15. Olivera, A., Buckley, N. E., and Spiegel, S. (1992) Sphingomyelinase and cell-permeable ceramide analogs stimulte cellular proliferation in quiescent Swiss 3T3 fibroblasts. J. Biol. Chem. 267, 26121–26127[Abstract/Free Full Text]
16. Kaufman, S. H., Desnoyers, S., Ottaviano, Y., Davidson, N. E., and Poirier, G. G. (1993) Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 53, 3976–3985[Abstract/Free Full Text]
17. Bose, R., Verheij, M., Haimovitz-Friedman, A., Scotto, K., Fuks, Z., and Kolesnick, R. (1995) Ceramide synthase mediates DNR-induced apoptosis: an alternative mechanism for generating death signal cell. Cell 82, 405–414[Medline]
18. Quillet-Mary, A., Mansat, V., Duchayne, E., Come, M. G., Allouche, M., Bailly, J. D., Bordier, C., Naval, J., and Laurent, G. (1996) Daunorubicin-induced internucleosomal DNA fragmentation in acute myeloid cell lines. Leukemia 10, 417–425[Medline]
19. Chen, M., Quintans, J., Fuks, Z., Thompson, C., Kufe, D. W., and Weichselbaum, R. R. (1995) Suppression of Bcl-2 messenger RNA production may mediate apoptosis after ionizing radiation, tumor necrosis factor , and ceramide. Cancer Res. 55, 991–994[Abstract/Free Full Text]
20. Martin, S. J., Takayama, S., McGahon, A., Miyashita, T., Cobeil, J., Kolesnick, R. N., Reed, J. C., and Green, D. G. (1996) Inhibition of ceramide-induced apoptosis by Bcl-2. Cell Death Diff. 2, 253–257
21. Smyth, M. J., Perry, D. K., Zhang, J., Poirier, G. G., Hannun, Y. A., and Obeid, L. (1996) prICE: a downstream target for ceramide-induced apoptosis and for the inhibitory action of Bcl-2. Biochem. J. 316, 25–28
22. Zhang, J., Alter, N., Reed, J. C., Borner, C., Obeid, L. M., and Hannun, Y. A. (1996) Bcl-2 interrupts the ceramide-mediated pathway of the cell death. Proc. Natl. Acad. Sci. USA 93, 5325–5378[Abstract/Free Full Text]
23. Allouche, M., Bettaïeb, A., Vindis, C., Rousse, A., Grignon, C., and Laurent, G. (1997) Influence of Bcl-2 overexpression on the ceramide pathway in daunorubicin-induced apoptosis of leukemic cells. Oncogene 14, 1837–1845[Medline]
24. Naumovski, L., and Cleary, M. L. (1994) Bcl-2 inhibits apoptosis associated with terminal differentiation of HL60 myeloid leukemia cells. Blood 83, 2261–2267[Abstract/Free Full Text]
25. Masson, M., Albouz, S., Boutry, J. M., Spezzatti, B., Castagna, M., and Baumann, N. (1989) Calmodulin antagonist W-7 inhibits lysosomal sphingomyelinase activity in C6 glioma cells. J. Neurochem. 52, 1645–1647[Medline]
26. Albouz, S., Vanier, M. T., Hauw, J. J., Le Saux, F., Boutry, J. M., and Baumann, N. (1983) Effect of tricyclic antidepressants in sphingomyelinase and other sphingolipid hydrolases in C6 cultured glioma cells. Neurosci. Lett. 36, 311–315[Medline]
27. Jaffrézou, J. P., Chen, G., Duran, G. E., Muller, C., Bordier, C., Laurent, G., Sikic, B. I., and Levade, T. (1995) Inhibition of lysosomal acid sphingomyelinase by agents which reverse multidrug resistance. Biochim. Biophys. Acta 1266, 1–8[Medline]