This proposal is a continuation of our ongoing studies on lipid and protein phosphorylation in the cornea, focusing on cell signaling events during wound closure and epithelial cell proliferation. We will test the hypothesis that two paracrine growth factors released after corneal injury, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), modulate corneal repair through activation-deactivation of specific kinases linked to the phosphatidylinositol-3 kinase (PI-3K) and the mitogen-activated protein kinase (MAPK) pathways. We propose experiments to test how HGF protects corneal epithelial cells from apoptosis through activation of PI-3K-protein kinase B/Akt-1 activation. We will investigate the role of three specific protein kinase C (PKC) isoforms, PKCa, PKCe, and PKCu, as modulators of cell proliferation and migration and the relation between two MAPK cascades, ERK1/2 and p38 in the regulation of wound healing. Studies are proposed to investigate the action of specific protein tyrosine phosphatases (PTPs) in the switching-off of PI-3K signals after the wound process has been activated. The debridement of the corneal epithelial layer will be used as a model of wound healing, complemented by in vitro migration and proliferation assays. Selective inhibitors of the different signal pathways will be used to correlate our findings with wound closure. Identification of components will be accomplished by Western blot with specific antibodies, kinase assays using enzyme immunocomplexes, and fluorescence microscopy in live cells using green fluorescent protein tagged to PKC isoforms. The results obtained will allow us to better define the signals activated by PI-3K and MAPK and the involvement of specific PKCs and PTP in corneal wound healing. This knowledge will allow us to characterize drugs that target selective steps of the signaling pathways. ? ?
