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 (C2-ceramide), with TNF, on NFκB function, a transcription factor whose activation is central to many TNF-mediated effects. We describe how C2-ceramide failed to drive κB-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 NFκB, 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 C2-ceramide, which are known to lack trans-activating activity, was strongly suggested from these data. Further investigations revealed that C2-ceramide had only a marginal effect on IκBα 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 IκBα degradation and p105 processing, in accordance with previous findings. Furthermore, the effect of TNF on NFκB activation was rapid, whereas C2-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 NFκB complexes that lack transactivating activity by enhanced 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 (C2-ceramide), with TNF, on NFκB function, a transcription factor whose activation is central to many TNF-mediated effects. We describe how C2-ceramide failed to drive κB-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 NFκB, 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 C2-ceramide, which are known to lack trans-activating activity, was strongly suggested from these data. Further investigations revealed that C2-ceramide had only a marginal effect on IκBα 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 IκBα degradation and p105 processing, in accordance with previous findings. Furthermore, the effect of TNF on NFκB activation was rapid, whereas C2-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 NFκB complexes that lack transactivating activity by enhanced processing of p105. Tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; NFκB, nuclear factor κ B; C2-ceramide,N-acetylsphingosine; CAT, chloramphenicol acetyltransferase. is a pleiotropic cytokine that induces a variety of cell type-specific events including proliferation, differentiation, necrosis, and apoptosis (1Aggarwal B.B. Natarajan K. Eur. Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar). It is among a range of agents that have been reported to increase levels of the neutral lipid, ceramide, in cells (2Mathias S. Younes A. Kan C.-C. Orlow I. Joseph C. Kolesnick R.N. Science. 1993; 265: 1596-1599Google Scholar, 3Kim M.-Y. Linardic C. Obeid L. Hannun Y. J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar, 4Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. 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Med. 1997; PubMed Scopus Google Scholar). we have the effect of a used cell-permeable analogue of (C2-ceramide), with TNF in of NFκB We have found that C2-ceramide can activate NFκB as by shift treatment are required and only a marginal increase in IκBα degradation is Furthermore, is no effect on reporter gene However, C2-ceramide can p105 processing, with NFκB complexes predominantly p50 In complexes contain both p50 homodimeric and of TNF promotes both IκBα and p105 processing and as induces gene Our may therefore to of the in this in that ceramide not to be in the to IκBα and it may signal p105 processing in to HL60 and Jurkat T cells obtained from the of were in in with and and obtained from was a from was from and the to the κ were from and were from was from to the NFκB and were from and A. Cell. 1992; 71: Full Text PDF PubMed Scopus Google an of the p105 protein also its this were by and to the protein, were from The was a from NFκB was from was from other were from cells in of were in a of for cell and in a of stimulation were by the of and either nuclear or cell were as J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). were the with as The transactivating potential of NFκB complexes was following of cells with a NFκB of a chloramphenicol acetyltransferase reporter gene. Jurkat T cells were as J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). HL60 cells were by H. C. M. 1996; PubMed Scopus Google with cells were with by in with for h. treatment in from cells were for activity J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). was by for data. NFκB was by the electrophoretic mobility shift a the κ which had been with as J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of nuclear protein was with for as J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). NFκB complexes were on and following the of NFκB complexes and the of the analysis and were as J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), to NFκB and NFκB of cell protein were by and and IκBα or immunoblot analysis was as J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). was used a of and for IκBα and The were by to the Ceramide was by the R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (786) Google with as J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The of ceramide in HL60 cells following treatment with or not was by with a with known of ceramide In with C2-ceramide, we to mimic the effect of TNF on an reporter gene HL60 or Jurkat were therefore with a gene NFκB of a reporter gene. cells were stimulated with C2-ceramide or no increase in activity was in either Jurkat 1 or HL60 cells 1 TNF, as stimulated gene a increase in Jurkat T cells. stimulation levels was in HL60 cells. of Jurkat with both C2-ceramide and TNF in a to that with TNF 1 suggested that treatment of cells with ceramide had no effect on gene We ceramide activate NFκB in these cells, as has been reported by (13Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (972) Google Scholar, 14Yang Z. Costanzo M. Golde D.W. Kolesnick R.N. J. Biol. Chem. 1993; 268: 20520-20523Abstract Full Text PDF PubMed Google Scholar, 15Machleidt T. Wiegmann K. Henkel T. Schutze S. Baeuerle P. Kronke M. J. Biol. Chem. 1994; 269: 13760-13765Abstract Full Text PDF PubMed Google Scholar). C2-ceramide NFκB in HL60 cells, as was by the of complexes in nuclear from cells This effect was from 1 and contact of 1 were required to an of HL60 cells with both C2-ceramide and TNF in a of NFκB activation in with that with TNF and The dihydro of C2-ceramide be it a in the was inactive TNF strongly NFκB in both cell and and was found to increase ceramide levels in HL60 a increase after 1 of as shown in 1 C. C2-ceramide also NFκB in Jurkat T cells a range in HL60 cells and a contact time of 1 We the NFκB by C2-ceramide in of complexes from HL60 cells was by the NFκB the of whereas a had no effect We the of the complexes from HL60 cells treated with either C2-ceramide or this analysis was specific to p50 and The complexes were further to that the complexes by TNF from those by C2-ceramide and TNF induces of with the of the whereas both and complexes and a further of the In the C2-ceramide was only by the However, this was by that p50 was much p65/RelA in the NFκB whereas both were in the HL60 cells treated with C2-ceramide or TNF that in NFκB activation were for degradation of and degradation was following stimulation with TNF as by The of a to was of the of this IκBα to be by IκBα levels to the of the cells. In C2-ceramide treatment in a marginal degradation of degradation not We the effect of TNF and C2-ceramide on p105 and p50 levels in time revealed that treatment of HL60 for with TNF in a increase in p50 levels with a in p105 1 and effect was with C2-ceramide, where a degradation of p105 was This effect can be the of p105 to p50 is for by the of from the by This to for and the precursor for increase in p50 with a in p105 can be for both TNF and for C2-ceramide The effect of ceramide was also shown to be with both and an increase in p50 The degradation of p105 was these data suggest that in HL60 cells, the lack of effect of C2-ceramide on by NFκB, the of NFκB can be by p105 processing in of IκBα This shift the of transcription in the of of Ceramide has as a second messenger in cellular as cell and in Y.A. Science. 1996; PubMed Scopus Google Scholar). its role is an of ceramide may as a of the cell effects of TNF, in to other cellular events in to this of the that the role of this neutral in TNF-mediated we to compare the events TNF and ceramide that in the activation of the transcription factor, In we the of C2-ceramide to κB-linked gene C2-ceramide has been reported to and mimic TNF in this (13Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (972) Google Scholar, 14Yang Z. Costanzo M. Golde D.W. Kolesnick R.N. J. Biol. Chem. 1993; 268: 20520-20523Abstract Full Text PDF PubMed Google Scholar, 15Machleidt T. Wiegmann K. Henkel T. Schutze S. Baeuerle P. Kronke M. J. Biol. Chem. 1994; 269: 13760-13765Abstract Full Text PDF PubMed Google Scholar). We were to κB-linked activity with this This was in with an where failed to in a reporter gene in Jurkat T cells either or in with TNF J.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The of stimulation can be by that activation of p50 in to C2-ceramide and not its inactive a dose-responsive activation of NFκB in the cell it was revealed that the of subunits in complexes was from that in with p50 The p50 was for p50 homodimeric of NFκB, and the data obtained that this be in to We p50 from the processing of its precursor as a of ceramide, an that both the protein and its the p50 subunit, and data with that obtained with We found a for p105 processing IκBα degradation by ceramide, in contrast to TNF, which of p105 processing also a degradation of IκBα by an apparent in with the of NFκB Our data therefore that ceramide NFκB, the from that with number of other have ceramide can activate NFκB and the Ceramide analogues have been found to activate NFκB (13Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (972) Google Scholar, 14Yang Z. Costanzo M. Golde D.W. Kolesnick R.N. J. Biol. Chem. 1993; 268: 20520-20523Abstract Full Text PDF PubMed Google Scholar, 15Machleidt T. Wiegmann K. Henkel T. Schutze S. Baeuerle P. Kronke M. J. Biol. Chem. 1994; 269: 13760-13765Abstract Full Text PDF PubMed Google Scholar), to the activation in to TNF Obeid Hannun Y.A. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), or to have no effect Dbaibo B. Obeid Hannun Y.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Obeid Hannun Y.A. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, J.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google M. S. J.-P. B.B. J. 1996; Google Scholar). The for are that not effect were in Jurkat Dbaibo B. Obeid Hannun Y.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, J.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), which we found to be HL60 cells. We also found that were to an effect and that treatment of 1 were may not be in cell where ceramide may the used in no apoptosis was Dbaibo B. Obeid Hannun Y.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google that C2-ceramide not IκBα degradation in Jurkat T cells. This also found no effect of this on NFκB as by shift the cells were for only a C2-ceramide of We found that C2-ceramide NFκB in Jurkat T cells but that a 1-h contact time was was obtained in cells M. S. J.-P. B.B. J. 1996; Google Scholar), where a contact time of was required to NFκB The that a 1-h contact time was required may be TNF was found to increase ceramide levels in cells after a 1-h reported Dbaibo B. Obeid Hannun Y.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. J.-P. B.B. 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The of ceramide to increase p50 in the of may have for gene is a transcription activation subunit, whereas p50 is of the of a D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Henkel T. 1994; PubMed Scopus Google Scholar). In where p50 as of M. Science. 1992; PubMed Scopus Google Scholar). the of to inactive complexes is in gene in to the of NFκB for these transcription R. D. J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, p105 processing may also to by that with as has been suggested F. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, G. S. M. K. U. 1992; PubMed Scopus Google Scholar). We found no effect on the expression of the reporter that the of TNF to increase effect of p50 levels of p50 may be required to as has been shown in on the in T cells M. Science. 1992; PubMed Scopus Google Scholar). The of C2-ceramide to increase p50 for of the reported for ceramide and as of transcription R.T. Bielawska A. Obeid Hannun Y.A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In of this a in the of gene transcription has been directly to a increase in the of p50 which to the M. M. H. Cell. Biol. 1996; PubMed Scopus Google Scholar). of expression has been shown to in to and apoptosis in G. E. A. H. B. M. R. Biol. Sci. 1994; PubMed Scopus Google Scholar), and ceramide has been shown to of levels an protein phosphatase R.T. Bielawska A. Obeid Hannun Y.A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). C2-ceramide has also been shown to the expression of the gene J. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The protein product of this gene is a of a of whose has been found to in cells and Ceramide is to this a is not the gene for is NFκB we that its may in p50 The effects of ceramide also the of p50 NFκB activation has been shown to be D. Science. 1996; PubMed Scopus Google Scholar), the of increase in p50 by ceramide the expression of and In that the activation of NFκB by ceramide p105 processing in to IκBα This may be in that of NFκB activation by TNF that the of p50 IκBα degradation by of the to the NFκB p50 and its p105 precursor were by and to the protein was a from of We are to the and for and to this