GM3 Signals Regulating TNF-Alpha Expression Are Mediated by Rictor and Arhgdib in Mouse Melanoma B16 Cells

Abstract

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.

Sphingolipids and expression regulation of genes in cancer

“…Deletion of glycosphingolipid by inhibition of GCS with D-PDMP (12.5 μM, six days) increases TNF-α expression in FBJ cell variants by protein kinase N1 (Pkn1) that is a serine/threonine kinase, like PKC, and mediates cellular response to stress [73]. In contrast to GD1a, GM3 upregulates TNF-α expression via PI3K, Rictor/mTOR, Akt and also the Rho GDP-dissociation inhibitor 2 (Arhgdib) in mouse melanoma B16 cells [74-75]. GM3 (25 μM, 24 hr) induces the levels of mRNA and protein in mouse B16, B11 and CAH-3 melanoma cell lines; silencing of St3gal 5 gene, an enzyme responsible for GM3 synthesis represses TNF-α expression.…”

Frontier of Epilepsy Research - mTOR signaling pathway

“…Astrocyte-specific TSC2 hGFAP knockout mice showed enlarged cells, megalencephaly and astrocytosis, and start dying after 3 weeks old (Way et al, 2009). Disturbed balance between excitatory and inhibitory synaptic transmission might be linked to seizure incidents in tissues from patients as well as genetically manipulated mouse models (Uhlmann et al, 2002;Meikle et al, 2007;Wang et al, 2007). A rat model carrying a spontaneous TSC2 mutation (Eker rat, TSC2 +/-) showed improved performance in episodic-like memory test, and impaired LTP and LTD in the hippocampus (Von der Brelie et al, 2006;.…”

“…ASK1 is activated by rapamycin treatment via reducing PP5 activity and it interacts with FIP200, an autophagic molecules regulated by mTOR (Huang et al, 2004;Gan et al, 2006). TNFα is increased after kainate injection in rodents and its expression has been shown to be regulated by mTORC2 in melanoma B16 cells (de Bock et al, 1996;Wang et al, 2007). TSC2 inhibits the phosphorylation of a pro-apoptotic molecule, BAD on Ser136, and this phosphorylation facilitates BAD/BCL-2 and BAD/BCL-X L interactions which lead to apoptosis (Freilinger et al, 2006).…”

Changing the Nature of Melanoma Cells by Manipulation of Ganglioside Expression

“…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 mTOR-Rictor and Akt (Ser473), leading to TNF α stimulation. Finally, Ly-GDI, the tumor suppressor gene, whose expression is associated with GM3, was shown to be upstream of TNF α (Wang et al, 2007b). Thus, the GM3 signal is transduced in B16 cells through a PI3K, mTOR-Rictor, Akt, Ly-GDI pathway, leading to stimulated expression of TNF α.…”

“…1. GM3 has been shown to regulate TNF α both at the transcriptional and translational levels in murine melanoma B16 cells (Wang et al, 2007b;Wang et al, 2007c). TNF α expression was increased by addition of GM3 to the B16 transfectants and decreased after treatment with D-PDMP, an inhibitor of glucosyl-ceramide synthesis.…”