MOLECULAR REGULATION OF CORNEAL WOUND HEALING

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DESCRIPTION: Rapid healing of the corneal epithelium in response to injury is essential for maintenance of its barrier function. The long-term goal of the laboratory is to obtain basic information about the molecular and cell biology of corneal wound healing. Recent data demonstrated that a greatly elevated expression of amyloid b/A4 precursor-like protein (APLP2) correlates with post-wound corneal re-epithelization and that cells overexpressing APLP2 have enhanced migration over extracellular matrix (ECM) proteins. APLP2 is very closely related to the amyloid b/A4 precursor protein of Alzheimer's disease. During corneal wound healing, the ectodomain of APLP2 is shed by migrating epithelial cells, and incorporated into the wound bed around the leading edge. The Kunitz type serine proteinase inhibitor domain (KPI) of APLP2 inhibits an uncharacterized proteinase and blocks epithelial wound closure in corneal organ culture. APLP2 in the cornea is modified by addition of chrondroitin sulfate chains. These data suggest that APLP2 plays a role(s) in corneal wound repair. The current proposal will test the hypotheses that APLP2 controls erine proteinase activity, mediates cell adhesion, and promotes cell migration during corneal re-epithelialization. (i) The mechanism by which growth factors regulate synthesis and shedding of APLP2 in corneal epithelial cells will be evaluated by metabolic labeling and Western blotting; the involvement of both protein kinase C and tyrosine phosphorylation dependent signal transduction pathways in APLP2 shedding will be investigated using specific blocking reagents. (ii) The mechanism by which APLP2 mediates epithelial cell adhesion and migration will be assessed using several methods including purification of APLP2 isoforms, cDNA transfection and expression of chimeric APLP2 proteins that are resistant to proteolytic cleavage, and in vitro cell adhesion and cell migration assays. In particular, the role of ectodomain shedding of APLP2 as a means to break adhesion and to facilitate cell migration, and of ECM-associated APLP2 in epithelial cell adhesion and migration will be investigated. (iii) The identify of proteinase(s) that is inhibited by KPI containing APLP2 will be determined using affinity purification and cDNA cloning, and the role of the interplay of this proteinase and APLP2 in the growth factor processing will be elucidated using organ culture and an in vitro cell-free assay. These experiments will provide the basis to begin constructing a detailed map of the molecular pathways and interconnecting networks of proteins functioning in wound repair and to develop therapeutics for treatment of corneal diseases like recurrent erosions and persistent defects of the epithelium.