Modulation of EGFR Signaling to Promote Corneal Epithelial Wound Healing

调节 EGFR 信号传导促进角膜上皮伤口愈合

基本信息

批准号：
8394916
负责人：
BRIAN P. CERESA
金额：
$ 32.77万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8394916
关键词：
Affect Attenuated Back Binding Biochemical Genetics Biological Assay Blindness Blocking Antibodies Cell Line Cell membrane Cells Cellular biology Clinical Complex Cornea Corneal Injury Corneal Ulcer Development Drug Targeting Early Endosome Effectiveness Endocytosis Epidermal Growth Factor Epidermal Growth Factor Receptor Epithelial Cells Epithelium Eye Family member Film Future Goals Homeostasis Human In Vitro Infection Kinetics Laboratories Ligands Light Literature Lysosomes Mediating Molecular Multivesicular Body Pathway interactions Patients Phosphorylation Play Property Proteins Publications Publishing Reagent Receptor Protein-Tyrosine Kinases Receptor Signaling Recycling Reporting Research Retina Risk Role Route Severities Signal Transduction Sprague-Dawley Rats Staging System Testing Therapeutic Tissues Transforming Growth Factors Translating Work Wound Healing base cell growth cell motility corneal epithelium desensitization in vivo in vivo Model inhibitor/antagonist injured late endosome migration prevent receptor receptor expression receptor internalization receptor recycling repaired research study response restoration small molecule therapeutic target tissue/cell culture tool trafficking

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to develop strategies to restore and maintain the integrity of the corneal epithelium. Activation of the epidermal growth factor receptor (EGFR) is necessary and sufficient for the homeostasis and repair of the corneal epithelium. Despite evidence that the EGFR is a viable target for corneal epithelial wound healing, its use therapeutically has been limited. Understanding the molecular details of how signaling by the receptor is regulated will provide clues for utilizing EGFR-targeted therapies. One such regulatory mechanism is ligand-stimulated EGFR endocytosis. The internalization and subsequent endocytic trafficking of the receptor negatively controls EGFR signaling by targeting the active receptor to lysosomes for degradation. Our studies will determine how endocytosis affects EGFR signaling in the corneal epithelium. Based on our recent findings and reports in the literature, we propose the following hypothesis: slowing the endocytic trafficking of the liganded EGFR will prolong EGFR signaling and enhance corneal wound healing. This overall hypothesis will be tested with the following Aims. In Aim 1, we will test the hypothesis that different endogenous EGFR ligands possess different routes and/or kinetics of endocytic trafficking. Secondarily, we will determine if those ligands that promote EGFR recycling will enhance corneal epithelial cell migration and wound healing. Using immortalized and primary human corneal epithelial cells, we will examine the kinetics and routes of EGFR endocytic trafficking in response to endogenous EGFR ligands. These ligands have been reported to have varying kinetics and/or routes of EGFR endocytic trafficking in other cell lines. The effect of these ligands on EGFR endocytic trafficking in corneal epithelial cells is unknown due to differences in the level of EGFR expression and intrinsic properties of the corneal epithelial cells. Once we determine how these ligands impact EGFR endocytic trafficking in the corneal epithelium, we will determine how they affect the cell biology of corneal epithelial cells (cell migration) and corneal wound healing using an in vivo model (Sprague- Dawley rats). In Aim 2, we will test the hypothesis that the inhibition of EGFR endocytic trafficking will prolong receptor activity and enhance the rate of corneal epithelial cell migration and wound healing. Using the tissue culture cells described above, we will disrupt endocytic trafficking at selected stages in the endocytic pathway (at the plasma membrane, pre-early endosome, and in the late endosome/multivesicular body) and assess whether the phosphorylation of the EGFR and downstream effectors is prolonged. Next, we will determine if such changes are reflected in EGFR-mediated corneal epithelial cell migration and wound healing. In Aim 3, we will determine if other EGFR family members (ErbB2, 3, and 4) play a role in promoting corneal wound healing, if they do so more efficaciously that EGFR. If we are able to develop strategies for enhancing EGFR signaling, we have the potential for therapeutically accelerating the rate of corneal wound healing and minimize patient discomfort, the risk of infection, and blindness.

描述（由申请人提供）：我们实验室的长期目标是制定恢复和维持角膜上皮完整性的策略。表皮生长因子受体（EGFR）的激活对于体内平衡和角膜上皮的修复是必要的，足够。尽管有证据表明EGFR是角膜上皮伤口愈合的可行靶标，但其治疗方法受到限制。了解如何调节受体信号传导的分子细节将为使用EGFR靶向疗法提供线索。这种调节机制是配体刺激的EGFR内吞作用。受体的内在化和随后的内吞运输通过将活性受体靶向溶酶体降解来负面控制EGFR信号传导。我们的研究将确定内吞作用如何影响角膜上皮的EGFR信号传导。根据我们最近在文献中的发现和报告，我们提出了以下假设：放慢配体EGFR的内吞运输将延长EGFR信号传导并增强角膜伤口愈合。该总体假设将以以下目的进行检验。在AIM 1中，我们将检验以下假设：不同的内源性EGFR配体具有内吞交易的不同途径和/或动力学。其次，我们将确定那些促进EGFR回收的配体是否会增强角膜上皮细胞迁移和伤口愈合。使用永生和原发性人角膜上皮细胞，我们将检查EGFR内吞运输的动力学和途径，以响应内源性EGFR配体。据报道，这些配体具有不同的动力学和/或其他细胞系中EGFR内吞运输的途径。这些配体对角膜上皮细胞中EGFR内吞运输的影响尚不清楚，这是由于EGFR表达水平和角膜上皮细胞的内在特性的差异。一旦我们确定了这些配体如何影响角膜上皮的EGFR内吞运输，我们将确定它们如何使用体内模型（Sprague-Dawley大鼠）影响角膜上皮细胞（细胞迁移）和角膜伤口愈合的细胞生物学。在AIM 2中，我们将检验以下假设：EGFR内吞运输的抑制作用将延长受体活性并提高角膜上皮细胞迁移和伤口愈合的速度。使用上述的组织培养细胞，我们将在内吞途径（在质膜上，预性内体和晚期内体/多囊体中）中的选定阶段破坏内吞运输，并评估EGFR的磷酸化和下游效应子的磷酸化。接下来，我们将确定这种变化是否反映在EGFR介导的角膜上皮细胞迁移和伤口愈合中。在AIM 3中，我们将确定其他EGFR家庭成员（ERBB2、3和4）是否在促进角膜伤口愈合方面发挥作用，如果他们这样做的话，EGFR是否更有效。如果我们能够制定增强EGFR信号传导的策略，那么我们就有可能在治疗上加速角膜伤口愈合率，并最大程度地减少患者不适，感染的风险和失明。