Corneal Innervation and Epithelial Cell Migration

描述(申请人提供)：维持正常的角膜上皮需要角膜缘干细胞来源的角膜上皮细胞持续向心迁移。角膜上皮的维持也依赖于角膜中感觉神经纤维的存在及其在损伤后的延伸。角膜神经支配缺陷通常会导致上皮缺陷或嗜神经性溃疡和失明。我们的长期目标是了解创伤后驱动角膜上皮细胞迁移和延伸的机制，以及角膜上皮和角膜神经元是如何相互作用的。 目前的证据表明PI-3K在角膜上皮细胞迁移中起着关键作用。我们最近分离到了PI-3激酶的靶标Grp1和相关蛋白GRSP1。Grp1是小GTP结合蛋白ARF6的激活剂，ARF6已被证明调节板脂扩张和细胞运动。这一提议的组织假设是，一旦激活，神经生长因子和趋化刺激的受体就会激活PI-3激酶，从而产生脂质信使PIP3。结果Grp1被募集到质膜并激活ARF6，导致在迁移的角膜上皮细胞前缘和角膜神经纤维的生长锥体形成片状脂膜。我们将检验以下假设：1)Grp1是从激动剂刺激到角膜上皮细胞迁移和轴突延伸的信号通路的必要组成部分；2)Grp1作为与GRSP1的复合体的一部分；3)ARF6也是该信号通路的必要组成部分。这些方法将包括确定在趋化因子和神经源性因子刺激下信号分子的激活状态，分析在不同步骤刺激或阻断该通路的效果，以及试图在引入抑制剂或显性负结构阻断后挽救该信号通路。

DESCRIPTION (provided by applicant): 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.