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 ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-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 C(2)-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 C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-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 C(2)-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 approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.
Hanna
A N
AN
Signal Transduction Research Group and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
Berthiaume
L G
LG
Kikuchi
Y
Y
Begg
D
D
Bourgoin
S
S
Brindley
D N
DN
D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism / Q000502:physiology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D022502:Stress Fibers / Q000378:metabolism / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism / Q000502:physiology; D020744:rac GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology; D018631:ras Proteins / Q000378:metabolism / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism / Q000502:physiology; D000099157:Sphingosine Kinase
D003598:Cytoskeletal Proteins; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051419:Paxillin; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein; D000099157:Sphingosine Kinase; C064769:N-acetylsphingosine; C496061:PXN protein, human; C496063:Pxn protein, rat; C496050:PTK2 protein, human; C496052:Ptk2 protein, rat