THE UBIQUITIN PATHWAY IN CORNEAL SCARRING

角膜疤痕中的泛素通路

• 批准号: 9482016
• 负责人: AUDREY M BERNSTEIN
• 金额: $ 40.31万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9482016
• 关键词: Anterior; Apoptosis; Binding; Biological Assay; Blindness; Bone Marrow; Cell Adhesion; Cell surface; Cells; Chemicals; Cicatrix; Code; Collagen; Complex; Contracts; Cornea; Corneal Injury; Coupled; Deubiquitinating Enzyme; Deubiquitination; Development; Disease; Enzyme-Linked Immunosorbent Assay; Excision; Extracellular Matrix; Eye; Family suidae; Fibroblasts; Fibronectins; Fibrosis; Focal Adhesions; G3BP1 gene; Gel; Gene Expression; Genes; Genetic; Growth Factor; Human; Infection prevention; Integrins; Lead; Link; Luciferases; MADH3 gene; MAPK14 gene; Maintenance; Mediating; Microscopy; Migration Assay; Monocular Blindness; Myofibroblast; Organ Culture Techniques; Oryctolagus cuniculus; PI3K/AKT; PTK2 gene; Pathologic; Pathway interactions; Peptide Hydrolases; Phenotype; Proteins; Proto-Oncogene Proteins c-akt; RNA Splicing; Recycling; Regulation; Reporter; Research; Role; Savings; Signal Transduction; Small Interfering RNA; System; Testing; Therapeutic; Thick; Time; Tissues; Transforming Growth Factor beta; Ubiquitin; Variant; Vinculin; Vision; Western Blotting; Wound Healing; base; cell motility; corneal scar; extracellular; healing; improved; in vivo; inhibitor/antagonist; insight; knock-down; live cell imaging; migration; novel; novel strategies; overexpression; p120 GTPase Activating Protein; prevent; public health relevance; receptor; regenerative; response; screening; tissue repair; transcriptome sequencing

 DESCRIPTION (provided by applicant): Persistence of myofibroblasts (Mfs) in a healing corneal wound leads to scarring and vision loss. Mfs arise by activation of resident keratocytes and bone marrow-derived fibrocytes, which produce excessive extracellular matrix (ECM) and overly contracted tissue. Stromal Mfs are characterized by excess cell surface expression of integrin αvβ5, which increases cell adhesion and activates the fibrotic growth factor, TGFβ generating fibrosis. Thus, understanding the regulation of αvβ5 may be key to preventing persistent Mfs. Unbiased screening of corneal Mfs revealed that the gene expression of the deubiquitinase (DUB), USP10 was increased, implicating ubiquitin removal as a possible mechanism for αvβ5 accumulation. When we overexpressed USP10 we found a posttranslational increase in αvβ5 protein and fibrotic markers (αSMA and fibronectin-EDA). In corneal organ culture, USP10 increased concomitantly with αSMA, integrin αvβ5, and FNEDA; Conversely USP10 knockdown with targeted USP10 siRNA after wounding reduced these markers to control levels. In the following 3 specific aims we now propose to examine the mechanisms underlying the USP10 function in antiscarring therapy using primary human corneal Mfs, ex-vivo porcine organ culture and in-vivo rabbit studies. In Specific Aim 1 we will test whether USP10 binding to and deubiquitination of αvβ5 results in αvβ5 accumulation on the cell surface, inducing integrin-mediated TGFβ signaling and downstream FAK/AKT/FNEDA (antiapoptotic) signaling that leads to persistent Mfs. In Specific Aim 2 we will test whether interaction with p120RasGap and G3BP1 (shown to bind to integrin and USP10 in other systems) drives integrin αvβ5/USP10 to the cell surface. Furthermore, we will test whether the increased USP10-mediated recycling (reduced degradation) of the internalized integrin/ECM complex contributes to fibrotic ECM accumulation and impacts focal adhesion turnover and cell migration. In Specific Aim 3 we will silence USP10 gene expression in rabbits after wounding by anterior corneal keratectomy. The effect on Mf persistence, corneal clarity and thickness, as well as other fibrotic endpoints will be quantified. Together, our proposed studies will provide a rigorous test that will determine the value of DUB USP10 as a potential novel target for corneal antiscarring therapy.

 描述(由申请人提供)：肌成纤维细胞(MFS)在愈合的角膜伤口中持续存在会导致疤痕形成和视力丧失。MFS是由驻留的角质形成细胞和骨髓来源的纤维细胞激活引起的，它们产生过多的细胞外基质(ECM)和过度收缩的组织。间质间质纤维化的特征是细胞表面过度表达整合素αvβ5，它增加细胞黏附并激活纤维生长因子，转化生长因子β产生纤维化。因此，了解αvβ5的调控可能是预防持续性MFS的关键。角膜基质细胞的无偏筛查显示，去泛素酶(DUB)、USP10的基因表达增加，提示泛素去除可能是αvβ5积聚的机制之一。当我们过表达USP10时，我们发现αvβ5蛋白和纤维化标志物(α、SMA和FN-EDA)在翻译后增加。在角膜器官培养中，随着αSMA、整合素αvβ5和FnEDA;的增加，USP10在创伤后被靶向USP10siRNA敲除后，这些标志物的水平降低到对照水平。在以下三个具体目标中，我们现在建议使用原代人角膜MFS、体外猪器官培养和体内兔研究来研究USP10在抗瘢痕治疗中的作用机制。在具体目标1中，我们将测试USP10与αvβ5的结合和去泛素化是否导致αvβ5在细胞表面积聚，从而诱导整合素介导的转化生长因子β信号和下游的FAK/AKT/FnEDA(抗凋亡)信号导致持久的MFS。在特定的目标2中，我们将测试是否与p120RasGap和G3BP1(显示与其他系统中的整合素和USP10结合)的相互作用将整合素αvβ5/USP10驱动到细胞表面。此外，我们将测试USP10介导的内化整合素/ECM复合体的循环增加(降解减少)是否有助于纤维化的ECM堆积，并影响局灶性黏附周转和细胞迁移。在特定的目的3，我们将沉默USP10基因的表达，在兔创伤后，通过角膜切削术。对MF持续性、角膜清晰度和厚度以及其他纤维化终点的影响将被量化。总之，我们提出的研究将提供一项严格的测试，以确定DUB USP10作为角膜抗瘢痕治疗的潜在新靶点的价值。