Ceramide Activates NFκB by Inducing the Processing of p105

The role of ceramide as a second messenger in tumor necrosis factor (TNF)-mediated signal transduction has been much debated. It is supported by recent reports describing an expanding number of potential targets for this lipid, but is opposed by those describing how ceramide is not necessary for many TNF-mediated cellular events. In this paper, we directly compare the effects of the cell-permeable ceramide analogue, N-acetylsphingosine (C sub(2)-ceramide), with TNF, on NF Kappa B function, a transcription factor whose activation is central to many TNF-mediated effects. We describe how C sub(2)-ceramide failed to drive Kappa B-linked chloramphenicol acetyltransferase gene expression in either HL60 promyelocytic or Jurkat T lymphoma cells. Furthermore, it had no effect on TNF-mediated transcription of this reporter gene. However, electrophoretic mobility shift analysis following cell stimulation with this ceramide analogue revealed a dose-responsive activation of NF Kappa B, which was not apparent following cell treatment with the inactive dihydro form. Activated complexes from treated cells were shown to contain predominantly the p50 subunit, in contrast to complexes from TNF-treated cells, where both p50 and p65/RelA subunits were present. The specific activation of p50 homodimeric complexes by C sub(2)-ceramide, which are known to lack trans-activating activity, was strongly suggested from these data. Further investigations revealed that C sub(2)-ceramide had only a marginal effect on I Kappa B alpha degradation but strongly promoted the processing of p105 to its p50 product as revealed by immunoblot analysis. The increase in p50 arising from the processing of its p105 precursor was further established from p105/p50 ratios obtained by scanning densitometric analysis of bands from immunoblots. TNF, on the other hand, stimulated both I Kappa B alpha degradation and p105 processing, in accordance with previous findings. Furthermore, the effect of TNF on NF Kappa B activation was rapid, whereas C sub(2)-ceramide required an optimal treatment time of 1 h. Interestingly, TNF was found to increase ceramide in cells but only after a 1-h contact time. Our data therefore suggest that ceramide promotes the activation of NF Kappa B complexes that lack transactivating activity by enhanced processing of p105.