Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death.
We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.
Darios
Frédéric
F
INSERM U289, Hôpital de la Salpêtrière, 47 boulevard de l'Hôpital, 75013 Paris, France.
Lambeng
Nathalie
N
Troadec
Jean-Denis
JD
Michel
Patrick P
PP
Ruberg
Merle
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002518:Ceramides / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology
D051023:BH3 Interacting Domain Death Agonist Protein; C494816:Bid protein, rat; D002352:Carrier Proteins; D002518:Ceramides; D003574:Cytochrome c Group; C064769:N-acetylsphingosine; D017382:Reactive Oxygen Species; D020932:Nerve Growth Factor; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D013110:Sphingosine; D002118:Calcium