Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130^Cas, and paxillin when treated with fetal calf serum than GD3− cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin β1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin β1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin β1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.

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.

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2−) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin β1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin β1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin β1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin β1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin β1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2− cells. All these results suggest that GD2 and integrin β1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL‐I). Although stimulation of melanoma N1 cells (GD3+ and GD3−) with either HGF or adhesion to CL‐I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H₂O₂ was examined, only GD3+ cells treated with both HGF and adhesion to CL‐I showed clearly low percentages of dead cells compared with GD3− cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5‐ethynyl‐2‘ deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas.

Gangliosides, sialic acid-containing glycosphingolipids, are highly expressed in nervous systems of vertebrates and have been considered to be involved in the development, differentiation, and function of nervous tissues. Recent studies with gene-engineered animals have revealed that they play roles in the maintenance and repair of nervous tissues. In particular, knockout (KO) mice of various ganglioside synthase genes have exhibited progressive neurodegeneration with aging. However, neurological disorders and pathological changes in the spinal cord of these KO mice have not been reported to date. Therefore, we examined neurodegeneration in double knockout (DKO) mice of ganglioside GM2/GD2 synthase (B4GANLT1) and GD3 synthase (ST8SIA1) genes to clarify roles of gangliosides in the spinal cord.

Acidic glycosphingolipids, i.e., gangliosides, are predominantly and consistently expressed in nervous tissues of vertebrates at high levels. Therefore, they are considered to be involved in the development and function of nervous systems. Recent studies involving genetic engineering of glycosyltransferase genes have revealed novel aspects of the roles of gangliosides in the regulation of nervous tissues. In this review, novel findings regarding ganglioside functions and their modes of action elucidated mainly by studies of gene knockout mice are summarized. In particular, the roles of gangliosides in the regulation of lipid rafts to maintain the integrity of nervous systems are reported with a focus on the roles in the regulation of neuro-inflammation and neurodegeneration via complement systems. In addition, recent advances in studies of congenital neurological disorders due to genetic mutations of ganglioside synthase genes and also in the techniques for the analysis of ganglioside functions are introduced.