Corneal Innervation and Epithelial Cell Migration

角膜神经支配和上皮细胞迁移

• 批准号: 6852607
• 负责人: JES K KLARLUND
• 金额: $ 37.99万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6852607
• 关键词: CHO cells; PC12 cells; biological signal transduction; cell cell interaction; cell migration; chemotaxis; cornea ulcer; corneal epithelium; dendrites; fluorescence microscopy; green fluorescent proteins; high performance liquid chromatography; innervation; laboratory mouse; laboratory rat; microinjections; nerve growth factors; neurons; organ culture; phosphatidylinositol 3 kinase; posttranslational modifications; protein binding; receptor expression; western blottings; wound healing

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.

描述（由申请人提供）：维持正常的角膜上皮需要恒定的角膜上皮细胞的中心迁移，这些细胞源自边缘的干细胞。上皮的维持还取决于角膜中的感觉神经纤维的存在以及受伤后的延伸。缺陷的角膜神经通常导致上皮缺陷或神经溃疡和失明。我们的长期目标是了解驱动角膜上角膜上皮细胞迁移和角膜上神经突的延伸的机制，以及角膜上皮和角膜神经元如何相互作用。 当前的证据表明，PI 3激酶在角膜上皮细胞迁移中的关键作用。我们最近分离了PI 3激酶，GRP1和相关蛋白GRSP1的靶标。 GRP1是小型GTP结合蛋白ARF6的激活剂，已证明可以调节片状脂蛋白的延伸和细胞运动。该提议的组织假设是激活后，神经生长因子的受体和趋化刺激激活了PI-3激酶，该激酶会产生脂质Messenger PIP3。结果，将GRP1募集到质膜并激活ARF6，这会导致在迁移角膜上皮细胞和角膜神经纤维的生长锥的前缘形成薄片。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和神经突扩展的迁移的信号传导途径的必要组成部分； 2）GRP1作为与GRSP1和3）ARF6的复合物的一部分，也是信号通路的必要组成部分。这些方法将包括用趋化性和神经源性因素刺激信号分子的激活状态，分析在不同步骤下刺激或阻断途径的效果，并试图通过抑制剂或显性负面构建体引入块后拯救信号传导途径。