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Ceramide activates NFkappaB by inducing the processing of p105
1Department of Biochemistry, Trinity College, Dublin 2, Ireland. mpboland@tcd.ie
The Journal of Biological Chemistry
|June 23, 1998
Summary
Ceramide activates NF-kappaB by promoting p105 processing, but does not drive gene expression or affect TNF-mediated transcription. This suggests ceramide activates non-transactivating NF-kappaB complexes.
Area of Science:
- Cellular signaling
- Molecular biology
- Biochemistry
Background:
- The role of ceramide as a second messenger in tumor necrosis factor (TNF)-mediated signal transduction is debated.
- While some studies suggest ceramide's importance, others indicate it's unnecessary for many TNF-mediated cellular events.
Purpose of the Study:
- To directly compare the effects of N-acetylsphingosine (C2-ceramide) with TNF on NF-kappaB function.
- To investigate ceramide's role in NF-kappaB activation and its relationship with TNF signaling.
Main Methods:
- Comparison of C2-ceramide and TNF effects on NF-kappaB activation in HL60 and Jurkat cells.
- Electrophoretic mobility shift assay (EMSA) to analyze NF-kappaB complex formation.
- Immunoblot analysis to assess IkappaBalpha degradation and p105 processing to p50.
- Scanning densitometry to quantify p105/p50 ratios.
Main Results:
- C2-ceramide did not drive kappaB-linked gene expression or affect TNF-mediated transcription.
- C2-ceramide induced dose-responsive NF-kappaB activation, primarily forming p50 homodimers, unlike TNF which activated p50/p65 complexes.
- C2-ceramide minimally affected IkappaBalpha degradation but strongly promoted p105 processing to p50, with optimal effect at 1 hour.
Conclusions:
- Ceramide promotes the activation of NF-kappaB complexes lacking transactivating potential via enhanced p105 processing.
- TNF signaling involves both IkappaBalpha degradation and p105 processing, with rapid activation, and increases cellular ceramide levels after 1 hour.