Regulation of Lung Fibrosis by Collagen Turnover

Lung fibrosis is a common disorder without effective pharmacological treatment. Fibrosis is a pathological tissue response characterized by extracellular deposition of collagen-rich matrix that disrupts normal alveolar architecture preventing effective gas exchange. Excessive deposition of collagen-rich matrix is due to the imbalance between collagen production and collagen degradation. Several identified pathways are mainly focused on the collagen production preceding fibrosis. Therapies aimed at inhibiting these pathways prevent the establishment of fibrotic disease in animal models. Even if these therapies prove successful in human subjects, they will not treat a significant number of patients that have established fibrosis at diagnosis. These patients would benefit from therapies that target remodeling of fibrotic tissue through resorption of collagen. Degradation of collagen is mediated through an extracellular proteolytic pathway and intracellular pathway of cellular uptake and lysosomal digestion. Recent studies demonstrate that disruption of the intracellular pathways can exacerbate fibrosis, but these pathways are poorly characterized. We recently identified novel mediators of collagen turnover through a genome-wide RNA interference screen in Drosophila S2 cells. Screening of 7505 Drosophila genes conserved among human and murine identified 22 genes that are required for efficient internalization of type I collagen. We show further that the flotillin, one of the 22 selected genes, have a conserved and central role in collagen uptake in Drosophila and human cells. Flotillin knockdown in human monocyte and fibroblasts impaired collagen uptake by promoting lysosomal degradation of the endocytic collagen receptor Endo180. The Drosophila high-throughput screening successfully provides an initial characterization of intracellular pathways of collagen turnover and identifies the flotillin genes as critical regulators of this process. However, flotillin knockdown in human monocyte and fibroblasts decreases collagen uptake by 60 - 70 % and 50 - 60 %, respectively. These results indicate that the extracellular collagen could be internalized by additional mechanisms or pathways regulated by other candidate genes in addition to flotillins. We are now focused on identifying additional pathways through which collagen is targeted for cellular turnover. In the proposed experiments, we will identify more candidate genes with roles in collagen turnover through our dsRNA-mediated screening in Drosophila S2 cells or a comprehensive screening in human U937 cells with a pooled lentiviral human genomic shRNA library. In this manner, we will identify novel molecules that mediate cellular uptake and turnover of collagen. These candidates will then serve as the basis of future grant proposals focused on in vivo analysis of their functions in murine models of fibrosis. The proposed studies will be critical in establishing an innovative approach to study fibrotic disease. The overall goal of this proposal is to offer a better understanding of the pathways that normally regulate collagen turnover, and to shed light on novel targets for the treatment of established fibrotic disease in the future.

Project ID:PC10404-0010
External Project ID:MOST104-2320-B182-003-MY2