Normal skin contains a stratifying squamous epithelium attached to the dermis by a basement membrane (BM). Expression of cell adhesion receptors, their ligands/co-receptors and associated cytoskeletal components is vital for skin function. Epidermolysis bullosis (EB) are inherited skin disorders that result in separation of the epidermis from the dermis, suggesting possible adhesion defects. We identified epinectin, a new basement membrane glycoprotein complex that adheres the epidermal strata to the BM by with the two integrin adhesion receptors, alpha 6 beta 4 in hemidesmosomes and alpha 3f beta 1 in focal adhesions (FAs). Epidermal cells also express intrinsic membrane heparan sulfate proteoglycans, CD44 receptors, that are involved in cell adhesion. We will examine tissue and cultured epidermal cells from EB patients for abnormal expression/function of adhesion components. This work may identify the molecular basis of mutations causal in EB and provide knowledge of normal epidermal morphogenesis. I. Skin samples from patients with EB. Punch biopsies of the skin and blister roofs will be obtained from patients suffering from simplex and junctional EB and used for histological examination and for establishing EB cells in culture. These will be compared to normal counterparts. II. Disfunction in adhesion components. Adhesion receptors, ligands, and associated cytoskeletal components will be localized in affected skin and in cultured EB cells with antibodies. Defects in receptor function will be assayed by cell-cell and cell-substrate adhesion assays. ECM from EB cells will be assayed for its ability to induce cell adhesion and receptor reorganization into hemidesmosomes and focal adhesions. III. Biochemical defects in adhesion components. Adhesion components will be immunopurified from normal and EB cells and peptide mapped to identify mutation sites. IV. Molecular basis of the adhesion defect. cDNA encoding the defective adhesion component will be cloned and sequenced from EB cells in order to identify the mutation sequence.
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