Sialic acid-containing glycosphingolipids (gangliosides) have been implicated in the regulation of various biological phenomena such as atherosclerosis. Recent report suggests that exogenously supplied disialoganglioside (GD3) serves a dual role in vascular smooth muscle cells (VSMC) proliferation and apoptosis. However, the role of the GD3 synthase gene in VSMC responses has not yet been elucidated. To determine whether a ganglioside is able to modulate VSMC growth, the effect of overexpression of the GD3 synthase gene on DNA synthesis was examined. The results show that the overexpression of this gene has a potent inhibitory effect on DNA synthesis and ERK phosphorylation in cultured VSMC in the presence of PDGF. The suppression of the GD3 synthase gene was correlated with the down-regulation of cyclinE/CDK2, the up-regulation of the CDK inhibitor p21 and blocking of the p27 inhibition, whereas up-regulation of p53 as the result of GD3 synthase gene expression was not observed. Consistently, blockade of GD3 function with anti-GD3 antibody reversed VSMC proliferation and cell cycle proteins. The expression of the GD3 synthase gene also led to the inhibition of TNF-alpha-induced matrix metalloproteinase-9 (MMP-9) expression in VSMC as determined by zymography and immunoblot. Furthermore, GD3 synthase gene expression strongly decreased MMP-9 promoter activity in response to TNF-alpha. This inhibition was characterized by the down-regulation of MMP-9, which was transcriptionally regulated at NF-kappaB and activation protein-1 (AP-1) sites in the MMP-9 promoter. Finally, the overexpression of MMP-9 in GD3 synthase transfectant cells rescued VSMC proliferation. However, MMP-2 overexpression was not affected by cell proliferation. These findings suggest that the GD3 synthase gene represents a physiological modulator of VSMC responses that may contribute to plaque instability in atherosclerosis.