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Mol. Biol. Cell 12 (11): 3618-3630

Copyright © 2001 by The American Society for Cell Biology.

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

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

Atef N. Hanna,*dagger Luc G. Berthiaume,*Dagger Yutaka Kikuchi,*dagger David Begg,§ Sylvain Bourgoin,|| and David N. Brindley*dagger

 *Signal Transduction Research Group and  dagger Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2;  Dagger 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-alpha (TNF-alpha ) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha -induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha , 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-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C2-ceramide. TNF-alpha , 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-alpha - and C2-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha , 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-alpha -induced stress fiber formation and cytoskeletal reorganization.

Corresponding author. E-mail address: david.brindley{at}

Molecular Biology of the Cell
Vol. 12, 3618-3630, November 2001
Copyright © 2001 by The American Society for Cell Biology

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