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-alpha 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 beta (Arhgdib).


RESULTS
PI3K inhibitors LY294002 and LY303511 were shown to suppress TNF-alpha 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-alpha expression, indicating mTOR to be involved in the pathway. Either siRNA Raptor or siRNA Rictor suppressed TNF-alpha expression, but the latter suppressed the effects of GM3 on TNF-alpha expression and Akt phosphorylation at Ser(473), indicating the GM3 signal to be transduced via Rictor/mTOR and Akt (Ser(473)), leading to TNF-alpha stimulation. Finally, Arhgdib, the tumor suppressor gene whose expression is associated with GM3, was shown to be upstream of TNF-alpha.


CONCLUSIONS
The GM3 signal is thus transduced in B16 cells through a PI3K, Rictor/mTOR, Akt, Arhgdib pathway, leading to stimulated expression of TNF-alpha.
Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang, China.
Pu
P
Wang
Xiaoyan
X
Yang
Peixing
P
Wu
Jinghai
J
Zhang
Toshinori
T
Sato
Sadako
S
Yamagata
Tatsuya
T
Yamagata
D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002417:Cattle; D002867:Chromones / Q000494:pharmacology; D017931:DNA Primers; D004195:Disease Models, Animal; D005679:G(M3) Ganglioside / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D008546:Melanoma, Experimental / Q000235:genetics* / Q000503:physiopathology; D051379:Mice; D010879:Piperazines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D020741:rho GTP-Binding Proteins
D002352:Carrier Proteins; D002867:Chromones; D017931:DNA Primers; D005679:G(M3) Ganglioside; D010879:Piperazines; D034741:RNA, Small Interfering; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; C489577:Rhog protein, mouse; D014409:Tumor Necrosis Factor-alpha; C525637:rictor protein, mouse; C093555:LY 303511; D020558:GTP Phosphohydrolases; D020741:rho GTP-Binding Proteins