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. W hile in some experimental systems, ceramide was shown to mediate Fas-induce d 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 t he caspase pathway, since we observed no ICE-like, CPP32-like and Mch2 acti vities and no PARP proteolysis, Moreover, we were unable to protect these c ells from ceramide-induced apoptosis using caspase inhibitors, while they b locked Fas-induced apoptosis and no Fast induction could be detected follow ing 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-linger transcription factor described to upregulate Fast, antagonizes ceramide-induced apoptosis, but not Fas-induced apoptosis, This further supports the hypothesis that Fas and ceramide death pathways are i ndependent in A20 cells. Ceramide-induced cell death was associated with in creased c-myc, p53, Bar and p27kip1 levels; in cont rast, cells transfected with Nur77 (A20Nur77), resistant to ceramide-induced apoptosis, showed a m arked downregulation of p53 after ceramide treatment, with neither Bar nor p27kip1 induction. In conclusion, our results suggest that, in the A20 B ce ll line, Fas and ceramide trigger two distinct pathways and that Nur77 over expression confers protection against ceramide-mediated apoptosis which cor relates with inhibition of p53, Bar and p27kip1 induction.