GM3 signals regulating TNF-alpha expression are mediated by rictor and Arhgdib in mouse melanoma B16 cells

Objective: We have previously shown GM3 to positively regulate TNF-α expression via a PI3K/Akt pathway in mouse melanoma B16 cells [Wang et al.: Biochem Biophys Res Commun 2007;356:438-443]. The GM3 signal was shown to be located upstream of Akt, but whether it is located upstream of PI3K and which molecule is the effector of PI3K remain to be clarified. Methods: We used inhibitors of PI3K and mTOR, and siRNA directed to Rictor, Raptor and Rho-GDP dissociation inhibitor β (Arhgdib). Results: PI3K inhibitors LY294002 and LY303511 were shown to suppress TNF-α expression that is stimulated by GM3 in B16 cells, suggesting that the GM3 signal is located upstream of the PI3K-Akt pathway. Rapamycin suppressed TNF-α expression, indicating mTOR to be involved in the pathway. Either siRNA Raptor or siRNA Rictor suppressed TNF-α expression, but the latter suppressed the effects of GM3 on TNF-α expression and Akt phosphorylation at Ser⁴⁷³, indicating the GM3 signal to be transduced via Rictor/mTOR and Akt (Ser⁴⁷³), leading to TNF-α stimulation. Finally, Arhgdib, the tumor suppressor gene whose expression is associated with GM3, was shown to be upstream of TNF-α. Conclusions: The GM3 signal is thus transduced in B16 cells through a PI3K, Rictor/mTOR, Akt, Arhgdib pathway, leading to stimulated expression ofTNF-α.