A novel pathway for tumor necrosis factor-α and ceramide signaling involving sequential activation of tyrosine kinase, p21ras, and phosphatidylinositol 3-kinase

Treatment of confluent rata fibroblasts with C-2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNF alpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C-2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Pas, Activation of PI 3-kinase by TNF alpha, sphingomyelinase, and C-2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI S-kinase by sphingomyelinase and C-2-ceramide was not observed in fibroblasts expressing dominant-negative Pas (N17) and the stimulation by TNF alpha was decreased by 70%. PI 3-kinase activation by C-2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C-2-ceramide It is proposed that activation of Pas and PI 3-kinase by ceramide can contribute to signaling effects of TNF alpha that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.