DISSOCIATION OF ENDOGENOUS CELLULAR CERAMIDE FROM NF-KAPPA-B ACTIVATION

The participation of cell ceramide in tumor necrosis factor (TNF)-alph a-stimulated NF-kappa B activation in Jurkat T cells and HL-60 cells w as studied. TNF-alpha readily stimulated NF-kappa B activity in both c ell lines as assayed by electrophoretic mobility shift assay and the u se of a human immunodeficiency virus-chloramphenicol acetyltransferase reporter construct. However, TNF-alpha stimulation did not increase c ell ceramide levels in either cell line. The exogenous addition of a s hort chain ceramide, N-acetylsphingosine, to Jurkat cells had no effec t on NF-kappa B activity. When Jurkat T cells were exposed to the gluc osylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholino -1-propanol, endogenous ceramide levels increased 4-fold. The increase in ceramide, however, did not result in NF-kappa B activation nor did it potentiate TNF-alpha or phorbol ester-stimulated activity. We conc lude that TNF-alpha-induced NF-kappa B activation occurs in Jurkat and HL-60 cell lines that do not demonstrate an increase in TNF-alpha-ind uced ceramide. Increasing ceramide levels by the addition of short cha in ceramides or the use of a glucosylceramide synthase inhibitor can b e dissociated from activation of NF-kappa B by TNF-alpha.