Caspase-mediated inhibition of sphingomyelin synthesis is involved in FasL-triggered cell death.
Ceramide can be converted into sphingomyelin by sphingomyelin synthases (SMS) 1 and 2. In this study, we show that in human leukemia Jurkat cells, which express mainly SMS1, Fas ligand (FasL) treatment inhibited SMS activity in a dose- and time-dependent manner before nuclear fragmentation. The SMS inhibition elicited by FasL (1) was abrogated by benzyloxycarbonyl valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor; (2) did not occur in caspase-8-deficient cells and (3) was not affected in caspase-9-deficient cells. Western blot experiments showed SMS1 cleavage in a caspase-dependent manner upon FasL treatment. In a cell-free system, caspase-2, -7, -8 and -9, but not caspase-3 and -10, cleaved SMS1. In HeLa cells, SMS1 was Golgi localized and relocated throughout the cytoplasm in cells exhibiting an early apoptotic phenotype on FasL treatment. zVAD-fmk prevented FasL-induced SMS1 relocation. Thus, FasL-mediated SMS1 inhibition and relocation depend on caspase activation and likely represent proximal events in Fas signaling. FasL-induced ceramide production and cell death were enhanced in cells stably expressing an siRNA against SMS1. Conversely, in cells stably overexpressing SMS1, FasL neither increased ceramide generation nor efficiently induced cell death. Altogether, our data show that SMS1 is a novel caspase target that is functionally involved in the regulation of FasL-induced apoptosis.
Cancer Department, U858 INSERM, Equipe 14, BP84225, 31432 Toulouse cedex 4, France.
E
E
Lafont
D
D
Milhas
S
S
Carpentier
V
V
Garcia
Z-X
ZX
Jin
H
H
Umehara
T
T
Okazaki
K
K
Schulze-Osthoff
T
T
Levade
H
H
Benoist
B
B
Ségui
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein / Q000378:metabolism* / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D007938:Leukemia / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013109:Sphingomyelins / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D002518:Ceramides; D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D013109:Sphingomyelins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C455440:SGMS1 protein, human; D017855:Transferases (Other Substituted Phosphate Groups); D020169:Caspases