Gangliosides, glycosphingolipids of the cell membrane, have been shown in vitro to impact several biologic processes and signaling pathways of normal keratinocytes and the keratinocyte-derived SCC12 cells. These cell functions, proliferation and adhesion, spreading, and migration on a fibronectin matrix, activation of the epidermal growth factor receptor and alpha5beta1integrin and are critical for wound healing. Caveolin-l-containing signaling domains in membranes are sites of coupling of receptors and signaling molecules. We have shown that gangliosides co-immunoprecipitate with caveolin-1, alpha5beta1 and the EGFR, and suggest that gangliosides affect caveolin-1 mediated signaling. We have recently demonstrated that ganglioside GM3 shifts caveolin-1 from detergent-insoluble domains into detergent-soluble domains in proximity with the EGFR. Given the known inhibitory effect of caveolin-1 on the EGFR, this finding suggests that ganglioside may serve as a shuttle molecule to facilitate caveolin-1 interaction with receptors. In this competing application for continuing support we will explore the relationships among gangliosides, caveolin-1, and alpha5beta1 integrin, and the role of these interactions in cell motility. We will investigate: a) the colocalization of gangliosides with caveolin-1, receptors, and signaling molecules by co-immunoprecipitation and immunomicroscopy; b) the dependence of ganglioside function on the intact caveolar domain and/or caveolin-l. We will disrupt caveolar domains with by beta-cyclodextrin and will interfere specifically with caveolin-1 function by introducing antisense oligomers and mutant caveolin-1; c) the effect of caveolin-1 phosphorylation on cell motility; and d) the possibility of direct binding of gangliosides and caveolin-l. The in vivo effect of ganglioside modulation on wound healing will be assessed using a transgenic mouse model of epidermis-specific ganglioside depletion. These studies will provide information about the role of gangliosides in caveolin-1 mediated signaling and may lead to novel therapies for patients with abnormal healing responses.
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