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

项目总结 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。这个 本应用的总体目标是阐明通过高密度脂蛋白识别2个基因的机制。 胶原周转、脂肪酸合成酶和细胞分裂周期酶相关蛋白的吞吐量筛选 激酶(CDC7)，调节细胞介导的胶原转化，并研究其在胶原转化中的作用 在人类细胞中，通过筛选确定的候选基因，当沉默时，会增加胶原的摄取。 中心假说是细胞介导的胶原碎片清除调节肺损伤的严重程度。 纤维化症。具体地说，Fas介导的脂质介质的产生促进了而CDC7对胶原的调节 内吞机制抑制细胞对胶原的摄取。另一种假设是，有遗传因素 防止细胞参与基质降解和沉默这些基因的控制可以增强 胶原蛋白的吸收，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)RNAi介导的Fas和CDC7在果蝇和哺乳动物细胞中的沉默导致 减少和增加胶原摄取；(2)药物抑制CDC7导致增加 胶原摄取和胶原内吞机制表达增加；以及(3)已发表的工作 脂肪酸治疗通过增加胶原蛋白逆转已建立的小鼠肺纤维化 周转；以及(4)RNAi介导的果蝇一些基因的沉默增加了细胞介导的胶原 营业额。这些假说将通过3个具体的目标来检验：1)确定Fas介导的作用 棕榈酰化在调节胶原内吞中的作用；确定Flotillin-2在解决肺纤维化中的作用 促进胶原摄取；2)研究CDC7是否通过抑制表达来调节胶原周转 胶原蛋白降解途径；3)研究是否沉默候选基因的哺乳动物同源基因 在果蝇筛选中发现的基因会导致细胞介导的胶原蛋白摄取增加。目标1将研究 Fas介导的泡囊转运蛋白Flotillin 1和Flotillin 2的棕榈酰化在胶原调节中的作用 胶原摄取所需的内吞机制以及体内转基因缺失Flotillin-2的影响 关于博莱霉素性肺纤维化的严重程度。目标2将研究CDC7在调控中的作用 Endo180等胶原转换介质的表达及CDC7的体内抑制作用 巨噬细胞介导的胶原摄取。目的3将研究在哺乳动物细胞胶原代谢中的作用 通过果蝇筛选确定的候选基因，当沉默时增加胶原蛋白的摄取。这个 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-- 调节胶原蛋白的周转。这项拟议的研究具有重要意义，因为它有可能为 治疗已确定的纤维性疾病的治疗方法的发展。

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.