Tumor necrosis factor-alpha induces stress fiber formation through ceramide production: Role of sphingosine kinase

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which cer amide is a second messenger for TNF-alpha -induced signaling leading to cyt oskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, o r C-2-ceramide induced tyrosine phosphorylation of focal adhesion kinase (F AK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinos itol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N 17) completely blocked C-2-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber for mation. The TNF-alpha effects were only partially inhibited. Dimethylsphing osine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C2-ceramide. TNF-alpha, sphingomyelinase, and C-2-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activat ed protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C-2-ceramide-induced stress fibe r formation. Our results demonstrate that stimulation by TNF-alpha, which i ncreases sphingomyelinase activity and ceramide formation, activates sphing osine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for similar to 70% of TN-F -alpha -induced stress fiber formation and cytoskeletal reorganization.