Investigating the role of cell-mediated collagen turnover in regulating tissue fibrosis

项目摘要 当胶原蛋白的产生超过胶原蛋白的降解时，就会发生肺纤维化。长期目标是 了解细胞介导的胶原降解在调节肺纤维化严重程度中的作用。的 本申请的总体目的是阐明通过高通量测序鉴定的2个基因的机制， 胶原周转、脂肪酸合成酶（FAS）和细胞分裂周期酶样相关蛋白的通量筛选 激酶（CDC 7），调节细胞介导的胶原蛋白周转，并研究在胶原蛋白周转中的作用 在人类细胞中，通过筛选确定的候选基因沉默时会增加胶原蛋白的摄取。 中心假设是细胞介导的胶原碎片清除调节肺损伤的严重程度。 纤维化具体地说，FAS介导的脂质介质的产生促进而CDC 7调节胶原 内吞机制抑制细胞对胶原的摄取。另一个假设是， 控制，防止细胞参与基质降解，这些基因的沉默可以增加 胶原蛋白再吸收，从而促进组织纤维化的消退。这些假设是建立在数据基础上的 证明（1）果蝇和哺乳动物细胞中FAS和CDC 7的RNAi介导的沉默导致 分别减少和增加胶原摄取;（2）药物抑制CDC 7导致增加的 胶原蛋白摄取和胶原蛋白内吞机制表达增加;和（3）已发表的工作 表明用脂肪酸治疗通过增加胶原蛋白逆转小鼠中已建立的肺纤维化， （4）RNAi介导的果蝇中许多基因的沉默增加了细胞介导的胶原蛋白 周转这些假设将通过3个具体目标进行检验：1）确定FAS介导的 棕榈酰化在调节胶原内吞作用中的作用;确定Flotillin-2在通过调节胶原内吞作用来解决肺纤维化中的作用。 2）研究CDC 7是否通过抑制表达来调节胶原蛋白周转 3）研究候选基因的哺乳动物直向同源物的沉默是否会影响胶原降解途径; 在果蝇筛选中鉴定的一种蛋白质，导致细胞介导的胶原蛋白摄取增加。目标1将审查 FAS介导的囊泡转运蛋白Flotillin 1和2棕榈酰化在调节胶原中的作用 胶原蛋白摄取所需的内吞机制以及Flotillin 2体内转基因缺失的影响 博来霉素诱导的肺纤维化的严重程度。目的2将研究CDC 7在调节细胞凋亡中的作用。 Endo 180和其他胶原转换介质的表达以及体内抑制CDC 7对 巨噬细胞介导的胶原摄取。目的3将研究在哺乳动物细胞中胶原周转的作用， 通过果蝇筛选确定的候选基因，当沉默时增加胶原蛋白摄取。的 申请人认为，拟议的研究是创新的，因为它研究了细胞的分子基础， 介导的胶原周转。拟议的研究是重要的，因为它有可能告知 开发治疗已确立的纤维化疾病的疗法。

PROJECT SUMMARY Pulmonary fibrosis occurs when collagen production outpaces collagen degradation. The long-term goal is to understand the role of cell-mediated collagen degradation in regulating the severity of pulmonary fibrosis. The overall objective of this application is to elucidate the mechanisms by which 2 genes identified through a high- throughput screen of collagen turnover, Fatty Acid Synthase (FAS) and Cell Division Cyclase-like Related Protein Kinase (CDC7), regulate cell-mediated collagen turnover coupled with investigating the role in collagen turnover in human cells of candidate genes identified through the screen that when silenced increase collagen uptake. The central hypothesis is that cell-mediated clearance of collagen fragments regulates the severity of pulmonary fibrosis. Specifically, FAS-mediated production of lipid mediators promotes while CDC7 regulation of collagen endocytic machinery inhibits cellular uptake of collagen. An additional hypothesis is that there are genetic controls that prevent cells from engaging in matrix degradation and that silencing of these genes can augment collagen resorption thereby promoting resolution of tissue fibrosis. These hypotheses are built on data demonstrating that (1) RNAi-mediated silencing of FAS and CDC7 in Drosophila and mammalian cells leads to reduced and increased collagen uptake respectively; (2) pharmaceutical inhibition of CDC7 leads to increased collagen uptake and increased expression of collagen endocytic machinery; and (3) published work demonstrating that treatment with fatty acids reverses established lung fibrosis in mice by increasing collagen turnover; and (4) RNAi mediated silencing of a number of genes in Drosophila increases cell-mediated collagen turnover. These hypotheses will be tested through 3 specific aims: 1) Determining the role of FAS-mediated palmitoylation in regulating collagen endocytosis; determining the role of Flotillin-2 in resolving lung fibrosis by promoting collagen uptake; 2) Investigating whether CDC7 regulates collagen turnover by inhibiting expression of collagen degradation pathways; 3) Investigating whether silencing of mammalian orthologs of candidate genes identified in the Drosophila screen leads to an increase in cell-mediated collagen uptake. Aim 1 will examine the role of FAS-mediated palmitoylation of the vesicular transport proteins, Flotillin 1 and 2, in regulating the collagen endocytic machinery required for collagen uptake as well as the effect of in vivo transgenic deletion of Flotillin 2 on the severity of bleomycin-induced pulmonary fibrosis. Aim 2 will examine the role of CDC7 in regulating expression of Endo180 and other mediators of collagen turnover and the effect of in vivo inhibition of CDC7 on macrophage-mediated collagen uptake. Aim 3 will examine the role in collagen turnover in mammalian cells of candidate genes identified through a Drosophila screen that when silenced increase collagen uptake. The proposed research is innovative, in the applicant's opinion, because it investigates the molecular basis of cell- mediated collagen turnover. The proposed research is significant because it has the potential to inform the development of therapeutics that treat established fibrotic disease.