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，没有有效的药物治疗。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，胶原蛋白生成和降解之间的平衡决定了组织结构。在纤维化区域中积累的胶原蛋白的清除途径尚不清楚。目前尚不清楚旨在增加胶原蛋白摄取的治疗方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。我们已经证明，糖蛋白Mfge8结合胶原蛋白已经积累在细胞外基质的纤维化区域。mfge8结合的胶原蛋白随后被巨噬细胞吸收进行细胞内降解。由于体内胶原降解缺陷，Mfge8缺乏的小鼠在肺损伤后会出现过度的纤维化反应。我们计划在这些发现的基础上，确定胶原蛋白被细胞吸收的其他新途径。我们设计了一种基于果蝇的高通量胶原蛋白摄取筛。在这些研究中，我们计划通过这种筛选来鉴定新的胶原蛋白转换介质，并验证这些基因的人类和同源基因的功能。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细胞胶原摄取，使用双链RNA文库来识别参与胶原转换的候选分子。加州大学旧金山分校创建的基因库涵盖了与人类和/或小鼠同源的果蝇基因。果蝇S2细胞是巨噬细胞样的吞噬细胞，对双链RNA基因沉默非常敏感。随后，我们将通过基于流式细胞术和基于荧光显微镜的胶原摄取检测来重新筛选候选基因。在二次筛选后留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同源物在胶原蛋白转化中的功能。我们将选择通过果蝇筛选鉴定出的最有说服力的候选基因，并评估它们在人和小鼠细胞系和原代细胞中胶原蛋白转化中的作用。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摄取和转换的影响。这些研究有可能发现新的胶原蛋白转换介质。更好地了解细胞外基质中胶原蛋白去除的途径的重要性，因为目前尚无有效的治疗方法来治疗已确定的纤维化疾病。

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.