(Verbatim) Reepithelialization is a fundamental requirement for successful wound healing and restoration of epidermal barrier function. Cells at the leading edge of the wound margin undergo a phenotypic conversion characterized by a dramatic re- organization of the cytoskeleton and associated junctional complexes. These include retraction of intermediate filaments from the cell surface, dissolution of most desmosomes and hemidesmosomes and partial or complete dissolution of the basement membrane. Desmosomes have been reported to disappear during the migratory phase of reepithelialization and reassembly of these intercellular junctions marks the end of keratinocyte migration; however, the mechanisms underlying dynamic regulation of desmosome assembly and disassembly are unknown. A role for the epidermal growth factor receptor (EGFR) in wound healing has been proposed. In particular, there is evidence that the EGFR and ligands are transiently up-regulated during the reepithelialization phase of wound repair. We have established stable cell lines to test the hypothesis that elevation of the EGFR confers changes in EGF-dependent cellular responses. We have determined that increased EGFR levels foster increased rate of cell migration and elevation of matrix degrading proteinase production. Importantly, we have found that EGF-dependent dissolution of desmosomal junctions is greatly augmented in EGFR overexpressing cells when compared to the control cell lines. In this proposal we will test the central hypothesis that EGF-dependent cellular response is not only modulated by the concentration and availability of ligand, but also at the level of receptor abundance. We will focus on studies to 1) identify the mechanisms involved in EGF-dependent desmosomal dissolution that are differentially regulated as a function of EGFR abundance and to 2) investigate EGF-stimulated signal transduction cascades that are required for loss of desmosomal integrity. In particular, we will address the underlying biochemical basis for EGF-dependent regulation of sustained extracellular signal regulated kinase/mitogen activated protein kinase (ERK/MAPK) which we have identified as a requirement for EGF-dependent colony dispersion, metalloproteinase-9 induction and desmosomal dissolution. We will also identify the functional contributions of a growth-factor induced transcription factor (Slug) known to modulate desmosomal integrity in adult epithelium. The proposed studies should provide insights into factors that govern keratinocyte outgrowth from a wound edge and the regulatory mechanisms involved in modulation of desmosome
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