The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action.
The mechanism of action of the anti-apoptotic oncogene Bcl-2 is still largely obscure. We have recently shown that the overexpression of Bcl-2 in HeLa cells reduces the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) by increasing the passive Ca2+ leak from the organelle. To investigate whether this Ca2+ depletion is part of the mechanism of action of Bcl-2, we mimicked the Bcl-2 effect on [Ca2+]er by different pharmacological and molecular approaches. All conditions that lowered [Ca2+]er protected HeLa cells from ceramide, a Bcl-2-sensitive apoptotic stimulus, while treatments that increased [Ca2+]er had the opposite effect. Surprisingly, ceramide itself caused the release of Ca2+ from the endoplasmic reticulum and thus [Ca2+] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca2+]er levels, as well as the buffering of cytoplasmic [Ca2+] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl-2-dependent reduction of [Ca2+]er is an important component of the anti-apoptotic program controlled by this oncogene.
Pinton
P
P
Department of Biomedical Sciences and CNR Center for the Study of Biomembranes, University of Padova, Via Colombo 3, I-35121 Padova, Italy.
Ferrari
D
D
Rapizzi
E
E
Di Virgilio
F
F
Pozzan
T
T
Rizzuto
R
R
D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D037282:Calreticulin; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D019254:Genes, bcl-2; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology*; D014162:Transfection
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; C064769:N-acetylsphingosine; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D049452:Green Fluorescent Proteins; D000331:Aequorin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D013110:Sphingosine; D002118:Calcium