Identification of Pathways that Regulate Fibrosis through Collagen Resorption

描述（由申请人提供）：肺纤维化是一种常见疾病，无有效的药物治疗。胶原蛋白是纤维化组织的主要成分，其代谢是一个动态过程，胶原蛋白的产生和降解之间的平衡决定了组织结构。负责清除纤维化区域中积累的胶原蛋白的途径知之甚少。目前尚不清楚旨在增加胶原蛋白摄取的疗法是否会使已确诊的纤维化疾病患者受益。我们最近描述了一种调节组织纤维化严重程度的胶原蛋白周转的新途径。我们已经表明，糖蛋白Mfge 8结合胶原蛋白，胶原蛋白已积累在细胞外基质中的纤维化区域。然后，Mfge 8结合的胶原蛋白被巨噬细胞吸收用于细胞内降解。由于体内胶原降解缺陷，Mfge 8缺陷的小鼠在肺损伤后产生过度的纤维化反应。我们计划在这些发现的基础上，通过确定胶原蛋白被细胞吸收的其他新途径。我们设计了一个基于果蝇的胶原蛋白摄取的高通量筛选。在这些研究中，我们计划通过该筛选识别胶原蛋白转换的新型介质，并验证这些基因的人类和直系同源基因的功能。我们将通过两个具体目标来实现这些目标。在第一个目标中，我们将使用基于流式细胞术的高通量筛选果蝇S2细胞胶原蛋白摄取，使用双链RNA文库来识别参与胶原蛋白周转的候选分子。在UCSF创建的文库涵盖了具有人类和/或小鼠直系同源物的果蝇基因。果蝇S2细胞是巨噬细胞样的吞噬细胞，对双链RNA的基因沉默高度敏感。我们随后将重新筛选候选基因与基于流式细胞术和荧光显微镜的胶原蛋白摄取的测定。在第二次筛选后剩下的候选基因将是我们第二个目标的重点。在这些研究中，我们将确认候选分子的人类和小鼠直系同源物在胶原蛋白周转中的功能。我们将选择最引人注目的候选基因确定通过我们的果蝇筛选和评估其在人类和小鼠细胞系和原代细胞中的胶原蛋白周转的作用。我们的方法将包括使用shRNA沉默候选基因和通过添加外源重组蛋白来增加候选基因的蛋白水平。然后，我们将测试这些干预措施对巨噬细胞和成纤维细胞的胶原蛋白摄取和周转的影响。这些研究有可能确定胶原蛋白周转的新介质。更好地理解负责从细胞外基质中去除胶原蛋白的途径的重要性突出了以下事实：没有可用的疗法在治疗患有已确定的纤维化疾病的患者中是有效的。 公共卫生相关性：肺纤维化是一种常见疾病，没有有效的药物治疗选择。旨在发现从肺部去除纤维化瘢痕的途径的研究将提供新的治疗选择。该项目的目标是识别和表征介导瘢痕重塑的正常途径，目的是发现新的治疗肺纤维化的治疗靶点。

DESCRIPTION (provided by applicant): Pulmonary fibrosis is a common disorder without effective pharmacological treatment. The metabolism of collagen, the main component of fibrotic tissue, is a dynamic process with the balance between collagen production and degradation determining tissue architecture. The pathways responsible for removal of collagen that has accumulated in areas of fibrosis are poorly understood. Whether therapies aimed at increasing collagen uptake will benefit patients with established fibrotic disease is unknown. We have recently described a novel pathway of collagen turnover that regulates the severity of tissue fibrosis. We have shown that the glycoprotein Mfge8 binds collagen that has accumulated in the extracellular matrix in areas of fibrosis. Mfge8-bound collagen is then taken up by macrophages for intracellular degradation. Mice deficient in Mfge8 develop an exaggerated fibrotic response after lung injury due to an in vivo defect in collagen degradation. We plan to build upon these findings by identifying other novel pathways by which collagen is taken up by cells. We have designed a Drosophila-based high-throughput screen of collagen uptake. In these studies we plan to identify novel mediators of collagen turnover through this screen and verify the function of human and orthologs of these genes. We will accomplish these goals through two specific aims. In the first aim, we will use a flow cytometry-based high-throughput screen of Drosophila S2 cell collagen uptake using a double stranded RNA library to identify candidate molecules involved in collagen turnover. The library created at UCSF covers Drosophila genes that have human and/or murine orthologs. Drosophila S2 cells are macrophage-like phagocytes that are highly susceptible to gene silencing with double-stranded RNA. We will subsequently rescreen candidate genes with both flow cytometry-based and fluorescent microscopy-based assays of collagen uptake. Candidate genes that remain after the secondary screen will be the focus of our second aim. In these studies we will confirm the function of human and murine orthologs of candidate molecules in collagen turnover. We will select the most compelling candidate genes identified through our Drosophila screen and evaluate their role in collagen turnover in both human and murine cell lines and primary cells. Our approach will involve both silencing candidate genes using shRNA and increasing candidate genes protein levels by adding exogenous recombinant protein. We will then test the effect of these interventions on collagen uptake and turnover by macrophages and fibroblasts. These studies have the potential to identify novel mediators of collagen turnover. The importance of a better understanding of the pathways responsible for collagen removal from the extracellular matrix is highlighted by the fact that no available therapies are efficacious in the treatment of patients with established fibrotic disease. PUBLIC HEALTH RELEVANCE: Pulmonary fibrosis is a common disorder without effective pharmacological treatment options. Research aimed at discovering pathways that remove fibrotic scar from the lung will provide new therapeutic options. The goals of this project are to identify and characterize the normal pathways that mediate scar remodeling with the goal of discovering new therapeutic targets for the treatment of pulmonary fibrosis.