- Human keratinocyte motility on fibronectin is critical in the reepithelialization of healing wounds and in the spread of cutaneous malignancy. The molecular events that influence this migration are poorly understood. Supplemental GT1b, a polysialyated ganglioside of keratinocytes, inhibits migration of keratinocytes on a fibronectin matrix at nM concentrations. Dr. Paller and colleagues propose that gangliosides modulate the alpha 5 beta 1/fibronectin interaction, probably by direct interaction with alpha 5 beta 1. The hypothesis is supported by the findings that: a)GT1b inhibition of keratinocyte migration occurs only on a fibronectin matrix; b)RGDS peptide competes with GT1b to inhibit cell adhesion to fibronectin; c)Preincubation of GT1b with the fibronectin matrix does not alter keratinocyte binding to the fibronectin; d)GT1b induces apoptosis of keratinocytes only when grown on a fibronectin matrix; e)FAK phosphorylation in response to fibronectin is decreased when keratinocytes are treated with GT1b; f)GT1b fails to inhibit the migration and proliferation of SCC13 and HaCaT cells, which have decreased expression of alpha 5 beta 1; and g)TGF-beta 1 increases HaCaT cell expression of alpha 5 beta 1, and induces cells to respond to the inhibitory effects of GT1b on adhesion to fibronectin. Dr. Paller and colleagues will explore the physiologic significance and mechanism(s) of the ganglioside action on epidermal cells by these specific aims: 1. Modulate the expression of ganglioside biosynthetic pathways by stable transfection of glycosyltransferase genes into a keratinocyte-derived line. In order to shift the endogenous production of gangliosides, including increasing expression GT1b, GalNAc transferase and sialyltransferase genes, driven by a progesterone antagonist-inducible system, will be transfected individually into SCC12 cells, a keratinocyte-derived GT1b-responsive cell line. 2. Examine the effects of changes in ganglioside expression on migration, adhesion, induction of apoptosis, and integrin signalling in cultured cells. The investigators will note changes in morphology, proliferative potential, apoptosis, and wound healing capability in an ex vivo transplantation model. They will also develop transgenic mice to study the effects of altered ganglioside content on proliferation, differentiation, and wound healing of normal keratinocytes in vivo. 3. Assess the mechanism of ganglioside inhibition of the alpha 5 beta 1/fibronectin interaction: The ganglioside/alpha 5 beta 1 interaction will be investigated in both a cell-free system using recombinant alpha 5 and beta 1 proteins, and with alpha 5 beta 1 immunoprecipitated from the transfected SCC12 cells. Dr. Paller will ask the following questions: a)Do gangliosides, including GT1b, bind directly to alpha 5 beta 1?; b)Do gangliosides interfere with alpha 5 beta 1 assembly?; c)Do gangliosides downregulate the expression of alpha 5 beta 1?; and d)Do alterations in ganglioside content affect membrane fluidity and thus the orientation of integrin receptors? Understanding the effect of gangliosides on the fibronectin-integrin interaction may lead to modulation of glycosyltransferase activity in normal or neoplastic keratinocytes as a novel therapeutic modality for treatment of chronic wounds, cutaneous neoplasia, and hyperproliferative epidermal disorders such as psoriasis.
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