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

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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.