Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis.
Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.
INSERM UMR1037, Centre de Recherches en Cancérologie de Toulouse, Equipe 4, BP84225, 31432 Toulouse Cedex 4, France.
Elodie
E
Lafont
Romain
R
Dupont
Nathalie
N
Andrieu-Abadie
Toshiro
T
Okazaki
Klaus
K
Schulze-Osthoff
Thierry
T
Levade
Hervé
H
Benoist
Bruno
B
Ségui
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis* / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D053222:Fas Ligand Protein / Q000494:pharmacology*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D019169:Jurkat Cells; D015458:Leukemia, T-Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism
D002518:Ceramides; D053222:Fas Ligand Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D045304:Cytochromes c; C455440:SGMS1 protein, human; D017855:Transferases (Other Substituted Phosphate Groups); D053453:Caspase 9