Vol. 12, Issue 11, 3618-3630, November 2001

Tumor Necrosis Factor- Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase

Atef N. Hanna,^* Luc G. Berthiaume,^* Yutaka Kikuchi,^* David Begg,^§ Sylvain Bourgoin, and David N. Brindley^*¶

 ^*Signal Transduction Research Group and  Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2;  Department of Cell Biology, University of Alberta,  ^§Division of Anatomy, University of Alberta, Edmonton, Alberta, Canada T6G 2S2; and  Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ, Laval University, Québec, Canada G1V 4G2

Tumor necrosis factor- (TNF-) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF--induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-, sphingomyelinase, or C₂-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C₂-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF- effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF- and C₂-ceramide. TNF-, sphingomyelinase, and C₂-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-- and C₂-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for ~70% of TNF--induced stress fiber formation and cytoskeletal reorganization.