Proper healing of corneal wounds is vital to maintaining a clear, healthy cornea and for preserving vision. The long-term goal of the laboratory has been to obtain basic information about the molecular and cellular biology of corneal wound healing. Recent data demonstrated that epithelial injury induces ectodomain shedding of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). Shed HB-EGF, in turn, acts as an autocrine ligand to activate EGF-receptor (EGFR) and subsequent intracellular signaling pathways necessary for regulating corneal epithelial wound healing. The current proposal will test the hypothesis that EGFR ligand shedding is catalyzed by an ADAM (a disintegrin and metalloprotease) in a tightly regulated manner and that EGFR activation by shed HB-EGF elicits several intracellular signaling pathways that work in concert to regulate corneal epithelial migration and proliferation. (i) The ADAM that is involved in HB-EGF shedding and EGFR activation in corneal epithelial cells in response to wounding will be identified by antisense oligonucleotide inhibition and by expression of dominant negative (dn) and constitutively active (ca) mutants of four ADAMs. (ii) The mechanisms by which wound-induced HB-EGF shedding and sub-sequent EGFR activation is regulated will be elucidated. The protein kinase (PKC) isozyme involved in the PKC-Raf1-MEK-ERK signaling cascade will be identified using enzymatic assays. Its role in EGFR activation and corneal wound healing will be assessed by expression of dn and ca mutants. (iii) The mechanisms by which each EGFR-elicited signaling pathway participates in the regulation of corneal epithelial wound healing will be investigated. Activation of four such pathways, mitogen activated protein kinase, phosphatidylinositol 3-kinase, phospholipase Cg-protein kinase C, as well as focal adhesion kinase, will be determined using biochemical kinase assays; their cross-talk and contribution to regulation of corneal epithelial migration and proliferation will be assessed using pharmacological kinase inhibitors and ca- and dn-mutant expression. An understanding of the molecular events from signal generation to signal transduction during corneal epithelial wound healing should help in the identification of targets for therapeutic interventions of corneal diseases like recurrent erosions and persistent defects of the epithelium.
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