Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a >3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGFR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk Monosialoganglioside GM1 is found largely in synaptosomal plasma membranes of the brain. GM1 enhances the activity of nerve growth factor (NGF) in NGF-responsive cells and stimulates neuronal sprouting both in vitro and in vivo (1-6). However, the precise mechanism of this GM1 function has not been elucidated yet. NGF was the first identified member of a family of neurotrophic factors that function both in vitro and in vivo to promote neuronal survival and differentiation (7). NGF induces differentiation of the rat pheochromocytoma cell line PC12 into cells resembling sympathetic neurons (8), thus providing a well-characterized model for the investigation of the mechanism of action of NGF. Following the application of NGF to PC12 cells, long-term transcription-mediated events occur. These include the extension of neurites and the acquisition of a differentiated phenotype that is characterized by the development of electrical excitability and the biosynthesis of neurotransmitters (8, 9). The biological effects of NGF are mediated by high-affinity binding to cell,-surface glycoprotein receptors called Trk (for tyrosine kinase receptor) encoded by the trk protooncogene [now designated Ntrk (NTRK) for neurotrophic tyrosine kinase receptor in the mouse (human)

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Gangliosides are ubiquitous components of mammalian cells. Their expression is frequently altered in many tumor types. We previously showed that alteration of the ganglioside composition often resulted in changes in cellular morphology and differentiation of cultured cells. In this study, we targeted sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O-acetylated GD3 (OAc-GD3) in the neuroblastoma F-11 cells greatly reduced the tumor growth in nude mice. The sense and antisense vectors containing either a 5' end fragment or the entire sequence of the cDNA coding for GD3-synthase were prepared and used in separate experiments to transfect the F-11 cells which express high levels of gangliosides GD3 and OAc-GD3. Single clones were isolated and expanded. Both the activity of the GD3-synthase and the concentrations of GD3 and OAc-GD3 in the antisense-transfected cells were dramatically decreased as a result of transfection with the antisense expression vectors. Further characterization of the antisense-transfected cells showed reduced rates of cell growth and neurite formation and changes in cellular morphology. When the cells were inoculated in athymic nude mice, the tumor growth rate was remarkably suppressed although the tumor incidence was not affected by the altered ganglioside composition. These results indicate that the tumor-associated ganglioside(s) is(are) involved in regulation of tumor growth, probably through the stimulation of angiogenesis of the tumor.

Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 micrograms/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [3H]thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca2+ in the medium. 125I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.

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