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：
8236586
负责人：
BRIAN P. CERESA
金额：
$ 35.31万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
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 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8236586
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 Delivery Systems 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. PUBLIC HEALTH RELEVANCE: The epidermal growth factor receptor (EGFR) promotes cellular changes in the cornea epithelium, such as cell growth, proliferation, and migration, but has clinical limitations in promoting corneal wound healing. The goal of this research is to develop strategies to better use the EGFR as a therapeutic target by 1) identifying the molecular mechanisms that regulate EGFR signaling in the cornea and 2) by-pass those regulatory mechanisms to prolong EGFR signaling, accelerate corneal wound healing, and restore tissue homeostasis.

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）的激活对于角膜上皮的稳态和修复是必要且充分的。尽管有证据表明 EGFR 是角膜上皮伤口愈合的可行靶点，但其治疗用途仍然有限。了解受体信号传导如何调节的分子细节将为利用 EGFR 靶向疗法提供线索。一种这样的调节机制是配体刺激的 EGFR 内吞作用。受体的内化和随后的内吞运输通过将活性受体靶向溶酶体进行降解来负向控制 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中的 EGFR 信号传导。根据我们最近的发现和文献报告，我们提出以下假设：减缓配体 EGFR 的内吞运输将延长 EGFR 信号传导并增强角膜伤口愈合。这一总体假设将通过以下目标进行检验。在目标 1 中，我们将检验以下假设：不同的内源 EGFR 配体具有不同的内吞运输途径和/或动力学。其次，我们将确定那些促进 EGFR 循环的配体是否会增强角膜上皮细胞迁移和伤口愈合。使用永生化和原代人角膜上皮细胞，我们将检查 EGFR 内吞运输响应内源 EGFR 配体的动力学和途径。据报道，这些配体在其他细胞系中具有不同的 EGFR 内吞运输动力学和/或途径。由于 EGFR 表达水平和角膜上皮细胞内在特性的差异，这些配体对角膜上皮细胞中 EGFR 内吞运输的影响尚不清楚。一旦我们确定了这些配体如何影响角膜上皮中的 EGFR 内吞运输，我们将使用体内模型（Sprague-Dawley 大鼠）确定它们如何影响角膜上皮细胞的细胞生物学（细胞迁移）和角膜伤口愈合。在目标 2 中，我们将检验以下假设：抑制 EGFR 内吞运输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。使用上述组织培养细胞，我们将在内吞途径的选定阶段（质膜、早期内体和晚期内体/多泡体）破坏内吞运输，并评估 EGFR 和下游的磷酸化是否效应器延长。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。在目标 3 中，我们将确定其他 EGFR 家族成员（ErbB2、3 和 4）是否在促进角膜伤口愈合方面发挥作用，如果它们比 EGFR 更有效。如果我们能够制定增强 EGFR 信号传导的策略，我们就有可能在治疗上加快角膜伤口愈合的速度，并最大限度地减少患者的不适、感染和失明的风险。公众健康相关性：表皮生长因子受体 (EGFR) 促进角膜上皮的细胞变化，例如细胞生长、增殖和迁移，但在促进角膜伤口愈合方面具有临床局限性。本研究的目标是制定策略，通过 1) 确定调节角膜中 EGFR 信号传导的分子机制，以及 2) 绕过这些调节机制来延长 EGFR 信号传导，加速角膜伤口愈合，从而更好地使用 EGFR 作为治疗靶点，并恢复组织稳态。