Ceramide activates the stress-activated protein kinases

Tumor necrosis factor α (TNFα) activates the stress-activated protein kinases (SAPKs, also known as Jun nuclear kinases or JNKs) resulting in the stimulation of AP-1-dependent gene transcription and induces the translocation of NFκB to the nucleus resulting in the stimulation of NFκB- dependent gene transcription. A potential second messenger for these signaling pathways is ceramide, which is generated when TNFα activates sphingomyelinases. We show that treatment of HL-60 human promyelocytic cells with exogenous sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an effect not mimicked by treatment with phospholipase A₂, C, or D. Further, JNK/SAPK activity is stimulated 2.7- and 2.8-fold, respectively, in cells exposed to C₂-ceramide (5 μM) or TNFα (10 ng/ml). The prolonged stimulation of this kinase activity by C₂-ceramide is similar to that previously reported for TNFα. In contrast, the related mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated following TNFα treatment (1.5-fold) and are inhibited by C₂-ceramide treatment. TNFα also potently stimulates NF-κB DNA binding activity and transcriptional activity, but these effects are not mimicked by addition of C₂-ceramide or sphingomyelinase to intact cells. Furthermore, TNFα, sphingomyelinase, and C₂-ceramide induce c-jun, a gene that is stimulated by the ATF-2 and c-Jun transcription factors. These data suggest that ceramide may act as a second messenger for a subset of TNFα's biochemical and biological effects.