Ceramide-induced cell death is independent of the Fas/Fas ligand pathway and is prevented by Nur77 overexpression in A20 B cells.
The role of ceramide in triggering apoptosis is still a matter of debate. While in some experimental systems, ceramide was shown to mediate Fas-induced cell death, in other instances it was claimed to induce the expression of Fas ligand (FasL), killing cells in a caspase-dependent fashion. We found that, in mature A20 B cells, ceramide-induced apoptosis is independent of the caspase pathway, since we observed no ICE-like, CPP32-like and Mch2 activities and no PARP proteolysis. Moreover, we were unable to protect these cells from ceramide-induced apoptosis using caspase inhibitors, while they blocked Fas-induced apoptosis and no FasL induction could be detected following ceramide treatment. These results suggest that ceramide does not induce apoptosis through the Fas/FasL pathway. We also found that overexpression of Nur77, a zinc-finger transcription factor described to upregulate FasL, antagonizes ceramide-induced apoptosis, but not Fas-induced apoptosis. This further supports the hypothesis that Fas and ceramide death pathways are independent in A20 cells. Ceramide-induced cell death was associated with increased c-myc, p53, Bax and p27kip1 levels; in contrast, cells transfected with Nur77 (A20Nur77), resistant to ceramide-induced apoptosis, showed a marked downregulation of p53 after ceramide treatment, with neither Bax nor p27kip1 induction. In conclusion, our results suggest that, in the A20 B cell line, Fas and ceramide trigger two distinct pathways and that Nur77 overexpression confers protection against ceramide-mediated apoptosis which correlates with inhibition of p53, Bax and p27kip1 induction.
Brás
A
A
Departamento de Microbiologia e Imunologia, Centro de Citologia Experimental, Universidade do Porto, 4100 Porto, Portugal.
Albar
J P
JP
Leonardo
E
E
de Buitrago
G G
GG
Martínez-A
C
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins; D002518:Ceramides / Q000494:pharmacology*; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D015870:Gene Expression; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D016160:Retinoblastoma Protein / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D019014:fas Receptor / Q000378:metabolism*
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D002518:Ceramides; D019925:Cyclin A; D019927:Cyclin E; D004268:DNA-Binding Proteins; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; C064769:N-acetylsphingosine; C539818:Nr4a1 protein, mouse; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D016271:Proto-Oncogene Proteins c-myc; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C109343:dihydroceramide; D019014:fas Receptor; D050760:Cyclin-Dependent Kinase Inhibitor p27; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1; D013110:Sphingosine