Analyses of expression and regulation of ganglioside synthases in melanocytes are important to understand roles of gangliosides in melanomagenesis.,In this study, we analyzed the expression and regulatory mechanisms of glycosyltransferase genes responsible for ganglioside synthesis in normal melanocytes.,We reported previously that culture supernatants of UVB-irradiated keratinocytes induced upregulation of ganglioside GD3 synthase gene in melanocytes, and mainly TNFα was responsible for it.,Then, we found that elimination of dibutyryl cyclic AMP and IBMX from the medium also resulted in upregulation of the GD3 synthase gene.,The addition of α-melanocyte-stimulating hormone which increases cAMP, to the medium led to a significant reduction in the GD3 synthase gene expression level, and a PKA inhibitor enhanced the GD3 synthase gene level.,These results suggest that signals mediated via TNFα and cAMP oppositely regulate GD3 synthase gene expression in melanocytes.,The results of an IKK inhibitor indicate the possibility that TNFα induces GD3 synthase gene expression via NF-κB signaling in melanocytes.,When melanoma cells were treated by these factors, no fluctuation in the GD3 synthase gene expression level was observed, although an IKK inhibitor significantly suppressed it, suggesting that ganglioside synthase genes are regulated in distinct manners between melanocytes and melanomas.
Ganglioside GD3 is widely expressed in human malignant melanomas, and has been reported to be involved in the increased cell proliferation and invasion.,In this study, we established GM3-, GM2-, GM1-, GD3-, or GD2-expressing melanoma cell lines by transfecting cDNAs of glyscosyltransferases, and effects of individual gangliosides on the cell phenotypes and signals were examined.,The phenotypes of established ganglioside-expressing cells were quite different, i.e. cell growth increased as following order; GD2+, GD3+ > GM1+, GM2+, GM3+ cells.,Cell invasion activity increased as GD3+ ≧ GM2+ > GM1+, GM3+, GD2+ cells.,Intensity of cell adhesion to collagen I (CL-I) and spreading increased as GD2+ >> GD3+, GM1+ > GM2+, GM3+ cells.,In particular, cell adhesion of GD2+ cells was markedly strong.,As for cell migration velocity, GD2+ cells were slower than all other cells.,The immunocytostaining revealed close localization of gangliosides and F-actin in lamellipodia.,Immunoblotting of phosphorylated p130Cas and paxillin by serum treatment reveled that these phosphorylations were more increased in GD3+ cells than in GD2+ or GM3+ cells, while phosphorylation of Akt underwent similarly increased phosphorylation between GD3+ and GD2+ cells compared with GM3+ cells.,While GD2 and GD3 enhanced cell growth, GD3 might also contribute in cell invasion.,On the other hand, GD2 might contribute in the solid fixation of melanoma cells at metastasized sites.,These results suggested that individual gangliosides exert distinct roles in the different aspects of melanomas by differentially regulating cytoskeletons and signaling molecules.