GM3 has been shown to suppress TNF alpha expression in blood monocytes. However, we found that GM3 and TNF alpha were expressed in parallel in mouse melanoma B16 cells that were transfected with UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase cDNA in a sense or antisense direction or CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase siRNA. TNF alpha expression was increased by addition of GM3 to the B16 transfectants and decreased after treatment with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis. These results clearly indicate that GM3 positively regulates TNF alpha expression in B16 cells. Phosphoinositide 3-kinase inhibitors, wortmannin and LY294,002, suppressed TNFa expression and Akt phosphorylation. GN13 was shown to increase phosphorylation of Akt in B 16 cells and the B16-derived transfectants. Treatment of B 16 cells with siRNA targeted to Akt1/2 resulted in TNF alpha suppression, indicating that Akt plays an important role in regulation of TNF alpha expression. Suppression of Akt1/2 rendered cells insensitive to GM3, suggesting that the GM3 signal may be transduced via Akt. (c) 2007 Elsevier Inc. All rights reserved.