Ganglioside GD1a has been reported to suppress metastasis [S. Hyuga, S. Yamagata, Y. Takatsu, M. Hyuga, H. Nakanishi, K. Furukawa, T. Yamagata, Suppression of FBJ-LL cell adhesion to vitronectin by ganglioside GD1a and loss of metastatic capacity, International J. Cancer. 83 (1999) 685-691.] and MMP-9 production in mouse osteosarcoma FBJ cells [D. Hu, Z. Man, P. Wang, X. Tan, X. Wang, S. Takaku, S. Hyuga, T. Sato, X. Yao, S. Yamagata, T. Yamagata, Ganglioside GD1a negatively regulates MMP9 expression in mouse FBJ cell lines at the transcriptional level, Connect. Tissue Res. 48 (2007) 198-205.]. In the present study, TNFalpha increased cell motility and MMP-9 and TNFalpha expression at the transcriptional level. TNFalpha expression was found to be inversely proportional to GD1a content in the FBJ-cell variants. The addition of exogenous GD1a to FBJ-LL cells suppressed TNFalpha expression, and treatment of FBJ-S1 cells with D-PDMP (glucosylceramide synthesis inhibitor) led to an increase in TNFalpha, indicating that TNFalpha is negatively regulated by GD1a in FBJ cells. SiRNA of Pkn1, a Rho-GTPase effecter protein kinase, suppressed TNFalpha levels as well as Pkn1 expression, suggesting that Pkn1 is involved in TNFalpha signaling. Treatment of Pkn1-silenced FBJ-LL cells with GD1a failed to suppress TNFalpha expression, demonstrating that GD1a signals that lead to TNFalpha suppression are transduced through Pkn1.