Tumor Necrosis Factor-α-stimulated Cell Proliferation Is Mediated through Sphingosine Kinase-dependent Akt Activation and Cyclin D Expression

Tumor necrosis factor-α (TNF-α) has been shown to activate sphingosine kinase (SphK) in a variety of cell types. The extent to which SphK signaling mediates the pleiotropic effects of TNF-α is not entirely clear. The current study examined the role of SphK activity in TNF-α-stimulated cell proliferation in 1321N1 glioblastoma cells. We first demonstrated that pharmacological inhibitors of SphK markedly decrease TNF-α-stimulated DNA synthesis. Signaling mechanisms through which SphK mediated the effect of TNF-α on DNA synthesis were then examined. Inhibition of Rho proteins with C3 exoenzyme or of Rho kinase with Y27632 attenuated TNF-α-stimulated DNA synthesis. However, RhoA activation by TNF-α was not blocked by SphK inhibition. ERK activation was also required for TNF-α-stimulated DNA synthesis but likewise TNF-α-induced ERK activation was not blocked by inhibition of SphK. Thus, neither RhoA nor ERK activation are the SphK-dependent transducers of TNF-α-induced proliferation. In contrast, TNF-α-stimulated Akt phosphorylation, which was also required for DNA synthesis, was attenuated by SphK inhibition or SphK1 knockdown by small interfering RNA. Furthermore, cyclin D expression was increased by TNF-α in a SphK- and Akt-dependent manner. Additional studies demonstrated that TNF-α effects on DNA synthesis, ERK, and Akt phosphorylation are not mediated through cell surface Gi -coupled S1P receptors, because none of these responses were inhibited by pertussis toxin. We conclude that SphK-dependent Akt activation plays a significant role in TNF-α-induced cyclin D expression and cell proliferation.