THE UBIQUITIN PATHWAY IN CORNEAL SCARRING

角膜疤痕中的泛素通路

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：愈合角膜伤口中肌纤维细胞（MF）的持久性会导致疤痕和视力丧失。 MF是通过激活居民角膜细胞和骨髓来源的纤维细胞而产生的，这些纤维细胞产生过多的细胞外基质（ECM）和过度收缩的组织。基质MF的特征是整联蛋白αVβ5的过量细胞表面表达，这会增加细胞粘附并激活纤维化生长因子，TGFβ产生纤维化。这就是了解αVβ5的调控可能是防止持续MFS的关键。角膜MF的无偏筛选表明，去泛素酶（DUB），USP10的基因表达增加，隐含的泛素去除是αVβ5积累的可能机制。当我们过表达USP10时，我们发现αVβ5蛋白和纤维化标记物（αSMA和纤连蛋白-EDA）的翻译后增加。在角膜器官培养中，USP10与αSMA，整联蛋白αVβ5和FNEDA同时增加。相反，在绘制靶向USP10 siRNA的USP10敲低后，绘制了这些标记以控制水平。在以下三个特定目标中，我们现在建议使用原代人角膜MF，前体猪器官培养和体内兔子研究来检查USP10功能在抗休息疗法中的机制。在特定目标1中，我们将测试USP10与αVβ5的结合和去泛素化是否会导致αVβ5在细胞表面积累，诱导整联蛋白介导的TGFβ信号传导和下游FAK/AKT/FNEDA（抗凋亡）信号，从而导致MFS持久MFS。在特定目标2中，我们将测试与P120RASGAP和G3BP1的相互作用（显示在其他系统中与整合素和USP10结合）是否将整合蛋白αVβ5/USP10驱动到细胞表面。此外，我们将测试内部整合素/ECM复合物的USP10介导的回收（降解）是否有助于纤维化ECM的积累，并影响局灶性粘合剂的转换和细胞迁移。在特定的目标3中，我们将在前角膜角膜切除术生活后沉默兔子中的USP10基因表达。将量化对MF持久性，角膜清晰度和厚度以及其他纤维化终点的影响。总之，我们提出的研究将提供一项严格的测试，该测试将确定DUB USP10作为角膜抗休息疗法的潜在新目标。