CERAMIDE ACTIVATES THE STRESS-ACTIVATED PROTEIN-KINASES

Tumor necrosis factor alpha (TNF alpha) activates the stress-activated protein kinases (SAPKs, also known as Jun nuclear kinases or JMKs) re sulting in the stimulation of AP-1-dependent gene transcription and in duces the translocation of NF kappa B to the nucleus resulting in the stimulation of NF kappa B dependent gene transcription, A potential se cond messenger for these signaling pathways is ceramide, which is gene rated when TNF alpha activates sphingomyelinases. We show that treatme nt of HL-60 human promyelocytic cells with exogenous sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an effect not mimicke d by treatment with phospholipase A,, C, or D. Further, JNK/SAPK activ ity is stimulated 2.7- and 2.8-fold, respectively, in cells exposed to C-2-ceramide (5 mu M) or TNF alpha (10 ng/ml). The prolonged stimulat ion of this kinase activity by C-2-ceramide is similar to that previou sly reported for TNF alpha, In contrast, the related mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated following TNF alp ha treatment (1.5-fold) and are inhibited by C-2-ceramide treatment. T NF alpha also potently stimulates NF-KB DNA binding activity and trans criptional activity, but these effects are not mimicked by addition of C-2-ceramide or sphingomyelinase to intact cells. Furthermore, TNF al pha, sphingomyelinase, and C-2-ceramide induce c-jun, a gene that is s timulated by the ATF-S and c-Jun transcription factors, These data sug gest that ceramide may act as a second messenger for a subset of TNF a lpha's biochemical and biological effects.