Modulation of EGFR Signaling to Promote Corneal Epithelial Wound Healing

描述（由申请人提供）：我们实验室的长期目标是制定策略来恢复和维持角膜上皮的完整性。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和充分的。尽管有证据表明EGFR是角膜上皮伤口愈合的可行靶点，但其在治疗上的应用受到限制。了解受体信号传导调控的分子细节将为利用egfr靶向治疗提供线索。其中一种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降解来负性地控制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。根据我们最近的发现和文献报道，我们提出以下假设：减缓配体EGFR的内吞运输将延长EGFR信号传导并促进角膜伤口愈合。这一整体假设将通过以下目的进行检验。在目标1中，我们将检验不同内源性EGFR配体具有不同的内吞运输途径和/或动力学的假设。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的迁移和伤口愈合。使用永生化和原代人角膜上皮细胞，我们将研究响应内源性EGFR配体的EGFR内吞运输的动力学和途径。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EGFR的内噬运输，我们将确定它们如何影响角膜上皮细胞的细胞生物学（细胞迁移）和使用体内模型（Sprague- Dawley大鼠）的角膜伤口愈合。在Aim 2中，我们将验证EGFR内吞运输的抑制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的假设。使用上述组织培养细胞，我们将在内吞途径的选定阶段（在质膜、早期内核体和晚期内核体/多泡体）破坏内吞运输，并评估EGFR和下游效应物的磷酸化是否延长。接下来，我们将确定这些变化是否反映在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家族成员（ErbB2、3和4）是否在促进角膜伤口愈合中发挥作用，如果它们比EGFR更有效。如果我们能够开发出增强EGFR信号的策略，我们就有可能在治疗上加速角膜伤口愈合的速度，并最大限度地减少患者的不适、感染风险和失明。

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.