CD95/Fas-induced Ceramide Formation Proceeds with Slow Kinetics and Is Not Blocked by Caspase-3/CPP32 Inhibition

The current confusion regarding the relevance of endogenous ceramide in mediating CD95/Fas-induced apoptosis is based mainly on (i) discrepancies in kinetics of the ceramide response between different studies using the same apoptotic stimulus and (ii) the observation that late ceramide formation (ho...

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Bibliographic Details
Published in:The Journal of biological chemistry 1997-09, Vol.272 (39), p.24308-24312
Main Authors: Tepper, Annemiek D., Cock, Jeanine G. R. Boesen-de, de Vries, Evert, Borst, Jannie, van Blitterswijk, Wim J.
Format: Article
Language:English
Subjects:
Apoptosis
Caspase 3
Caspases
Ceramides - biosynthesis
Cysteine Endopeptidases - metabolism
Cysteine Proteinase Inhibitors - pharmacology
Enzyme Activation
fas Receptor - metabolism
Humans
Jurkat Cells
Kinetics
Signal Transduction
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Summary:The current confusion regarding the relevance of endogenous ceramide in mediating CD95/Fas-induced apoptosis is based mainly on (i) discrepancies in kinetics of the ceramide response between different studies using the same apoptotic stimulus and (ii) the observation that late ceramide formation (hours) often parallels apoptosis onset. We investigated CD95-induced ceramide formation in Jurkat cells, using two methods (radiolabeling/thin layer chromatography and benzoylation/high performance liquid chromatography), which, unlike the commonly used diglyceride kinase assay, discriminate between ceramide species and de novoformed dihydroceramide. We demonstrate that ceramide accumulates after several hours, reaching a 7-fold increase after 8 h, kinetics closely paralleling apoptosis induction. No fast response was observed, not even in the presence of inhibitors of ceramide metabolism. The majority (∼70%) of the ceramide response remained unaffected when apoptosis was completely inhibited at the level of caspase-3/CPP32 processing by the inhibitor peptide DEVD-CHO. Exogenous cell-permeable C2-ceramide induced the proteolytic processing of caspase-3, albeit with somewhat slower kinetics than with CD95. DEVD-CHO dose-dependently inhibited C2-ceramide- or exogenous sphingomyelinase-induced apoptosis. The results support the idea that ceramide acts in conjunction with the caspase cascade in CD95-induced apoptosis.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.272.39.24308