Proper healing of corneal wounds is vital to maintaining a clear, healthy cornea and preserving vision. Recent data suggest the following model. Upon wounding, members of the erbB ligand family, present in tear film and wounded epithelium, rapidly activate erbB-1 (EGF receptor) across the entire cornea, stimulating both cell proliferation and migration. Wounding also stimulates upregulation of TGF-beta receptor types I and II, with TbetaR-II present only in cells migrating to cover the wound area. Expression of TbetaR-II is stimulated by erbB-1 activation. The enhanced levels of TbetaR-I and -II allow the cells migrating over the wound to be stimulated by TGF-beta, in turn stimulating the upregulation of the cyclin-dependent kinase inhibitor p15INK4b. This inhibitor blocks proliferation in migrating cells, effectively separating spatially the proliferative and migratory responses. The hypothesis to be tested is that spatial separation of migratory and proliferative responses to wounding (1) is generated through erbB activation and TbetaR-II-mediated upregulation of genes such as p15INK4b, and (2) facilitates epithelial wound closure and restoration of the epithelial barrier. Biochemical and molecular biological techniques, including the generation of TAT-fusion proteins (technology allowing transduction of functional proteins directly into cells), will be used to test 3 specific aims. First, activation of the 4 members of the erbB family will be quantified in wounded and unwounded corneas, and heterodimer formation in migrating and proliferating epithelial cells will be compared. The effect of activation of the erbBs on TbetaR-II expression will also be quantified. Second, spatial and temporal expression of the Smad family of transcription factors will be examined to determine if TGF-beta signaling is confined to migratory cells. The effect of erbB ligands on modulating expression of the Smads will also be examined. In addition, the effect of inhibiting TGF-beta signaling on restoration of the epithelial barrier will be quantified by transducing dominant negative Smad proteins into wounded corneas using TAT-fusion proteins Third, it will be determined, using epithelial cell and corneal organ cultures, if overexpression of p15INK4b is sufficient to inhibit cell proliferation, enhance epithelial wound closure, and restore barrier function. TAT-p15INK4b will be used to transduce corneal epithelial cells. Techniques will include immunoprecipitation, western blotting, immunofluorescence microscopy, and functional assays of epithelial wound closure and barrier integrity. These experiments will result in basic information required for developing therapeutics to be used to enhance impaired healing responses.
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