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

Abstract

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-α-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-α, 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-α effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-α and C2-ceramide. TNF-α, 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-α- and C2-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-α, 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-α-induced stress fiber formation and cytoskeletal reorganization.

PKC-dependent Activation of Sphingosine Kinase 1 and Translocation to the Plasma Membrane

“…Several studies suggest that sphingomyelinases and ceramidases are activated in response to the same inducers that stimulate SK (11,(25)(26)(27)(28)(29), thus raising the possibility that sphingosine could be generated in or close to the plasma membrane concomitant with SK activation. These studies are consistent with S1P being generated in a location conducive for swift secretion out of the cell.…”

Cell-permeable ceramides increase basal glucose incorporation into triacylglycerols but decrease the stimulation by insulin in 3T3-L1 adipocytes

“…36,50,51 Our work with Rat2 fibroblasts demonstrated that TNFa, sphingomyelinase and C 2 -ceramide increase Ras-GTP concentrations, the binding of Ras to activated PI 3-K 23 cause the subsequent activation Rac, Cdc42 and the physical interaction of Cdc42 with activated PAK. 24 The involvement of PI 3-K, pp70 S6K and ERK in increasing GLUT1 concentrations is confirmed by the partial inhibition of the ceramide effect by Ly 294002, rapamycin and PD 98059, which inhibit PI 3-K activity and the activations of pp70 S6K and ERK, respectively. Ly294002 could also have inhibited the mammalian target of rapamycin (mTOR).…”

“…23 Therefore, the effects of short-chain ceramides are mainly direct rather than involving metabolism to sphingosine or sphingosine 1-phosphate. 23,24 TNFa and ceramides stimulate PI 3-K in 3T3 L1 adipocytes, 22,25 fibroblasts, 23,24 smooth muscle cells, 26 osteoclasts 45 and macrophages. 46 These increases in PI 3-K are associated with protection from apoptosis and increased division rather than cell death.…”

“…11 Increased tyrosine phosphorylation of IRS-1 and binding to PI 3-K occurred after 20 -60 min in 3T3-L1 adipocytes. 25 In Rat2 fibroblasts, TNFa, sphingomyelinase and C 2 -ceramide increased PI 3-K transiently after 10 -20 min, 23,24 whereas longer incubation times were required in 3T3-L1 adipocytes. 22 Furthermore, in Rat2 fibroblasts the stimulation of ERK was inhibited almost completely by Ly 294002, indicating the involvement of PI 3-K. 23 PI 3-K is thought to be particularly important for ERK activation in cells that do not exhibit large stimulations in response to growth factors.…”

“…22 In fact, ceramides stimulated rather than inhibited PI 3-K activity associated with IRS-1 and the ceramide-induced inhibition of insulin-stimulated glucose probably resulted from decreased insulin-induced transport of vesicles containing GLUT1 and GLUT4 to the cell surface. 22 Our later studies with Rat2 fibroblasts demonstrate that TNFa, sphingomyelinase and C 2 -ceramide rapidly stimulate tyrosine kinases, production of Ras-GTP and its association with activated PI 3-K. 23,24 Furthermore, incubation of 3T3-L1 adipocytes with TNFa increases the tyrosine phosphorylation of IRS-1 and its binding to PI 3-K. 25 Ceramides also activate Src and PI 3-K in smooth muscle cells. 26 In L6 myocytes, ceramides inhibit insulin-stimulated glucose uptake, but this did not depend upon decreased IRS-1 phosphorylation, or PI 3-K activity.…”