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）：愈合的角膜伤口中肌成纤维细胞（Mfs）的持续存在会导致疤痕和视力丧失。Mfs是由常驻角膜细胞和骨髓源性纤维细胞的活化引起的，其产生过量的细胞外基质（ECM）和过度收缩的组织。基质Mfs的特征在于整合素αvβ5的过量细胞表面表达，其增加细胞粘附并激活纤维化生长因子，TGFβ产生纤维化。因此，了解αvβ5的调节可能是预防持续性Mfs的关键。角膜Mfs的无偏筛选显示，去泛素化酶（DUB）USP 10的基因表达增加，暗示泛素去除是αvβ5蓄积的可能机制。当我们过表达USP 10时，我们发现αvβ5蛋白和纤维化标志物（αSMA和纤连蛋白-EDA）的翻译后增加。在角膜器官培养物中，USP 10与αSMA、整联蛋白αvβ5和FNEDA同时增加，相反，在创伤后用靶向USP 10 siRNA敲减USP 10将这些标志物降低至对照水平。在以下3个具体目标中，我们现在建议使用原代人角膜Mfs、离体猪器官培养和体内兔研究来检查USP 10在抗瘢痕形成治疗中的潜在机制。在特定目标1中，我们将检测USP 10与αvβ5的结合和去泛素化是否导致α v β5在细胞表面蓄积，诱导整合素介导的TGFβ信号传导和下游FAK/AKT/FNEDA（抗凋亡）信号传导，从而导致持续性Mfs。在特定目标2中，我们将测试与p120 RasGap和G3 BP 1（在其他系统中显示与整联蛋白和USP 10结合）的相互作用是否驱动整联蛋白αvβ5/USP 10到达细胞表面。此外，我们将测试是否增加USP 10介导的再循环（降解减少）的内化整合素/ECM复合物有助于纤维化ECM积累和影响局灶性粘附周转和细胞迁移。在特定目标3中，我们将在前角膜角膜切除术受伤后沉默兔中的USP 10基因表达。将量化对Mf持续性、角膜透明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个严格的测试，将确定DUB USP 10作为角膜抗瘢痕治疗的潜在新靶点的价值。