Maintenance of the normal corneal epithelium requires constant centripetal migration of corneal epithelial cells derived from stem cells in the limbus. Maintenance of the epithelium is also dependent on presence of sensory nerve fibers in the cornea and their extension after wounding. Defective corneal innervation commonly results in epithelial defects or neurotropic ulcers and blindness. Our long-term objective is to understand the mechanisms that drive corneal epithelial cell migration and extension of neurites in the cornea upon wounding, and how corneal epithelium and corneal neurons interact. ? ? Current evidence points to a critical role for PI 3 kinase in corneal epithelial cell migration. We have recently isolated a target for PI 3 kinase, GRP1, and an associated protein GRSP1. GRP1 is an activator for the small GTP binding protein ARF6, which has been shown to regulate extension of lamellipodia and cell movement. The organizing hypothesis for this proposal is that upon activation, the receptors for nerve growth factor and for chemotaxtic stimuli activate PI-3 kinase, which produces the lipid messenger PIP3. As a result GRP1 is recruited to the plasma membrane and activates ARF6, which causes formation of lamellipodia at the leading edge of migrating corneal epithelial cells and in the growth cones of corneal nerve fibers. We will test the following hypotheses: 1) GRP1 is a necessary component of the signaling pathway leading from agonist stimulation to migration of corneal epithelial cells and neurite extension; 2) GRP1 functions as part of a complex with GRSP1 and 3) ARF6 is also a necessary component of the signaling pathway. The approaches will include determination of the activation states of the signaling molecules upon stimulation with chemotactic and neurogenic factors, analysis of the effects of stimulating or blocking the pathway at different steps, and attempts to rescue the signaling pathway after introduction of a block by inhibitors or dominant negative constructs.
