Identifying Novel Mechanisms and Pathways of Collagen Turnover

Fibrosis is a pathological condition in which the imbalance between collagen production and collagen degradation leads to excessive deposition of collagen-rich matrix in the interstitial space. However, the pathways involved collagen uptake and intracellular degradation are poorly characterized. Through a genome-wide dsRNA-mediated screen in Drosophila S2 cells, we identified 22 candidate genes that decrease efficient collagen uptake in insect cells. one of the 22 candidate genes, Sec61beta, is a subunit of Sec61 translocon complex which allows the transport of secreted and transmembrane proteins across lipid bilayers. Surprisingly, our flow cytometry and microscopic data showed that shRNA-mediated Sec61beta knockdown leads to excessive accumulation of the internalized collagen in macrophages and lung fibroblasts. Encouragingly, in our U937 cell-based screen by targeting the entire human genome using a pooled genomic human shRNA library with collagen uptake analysis, our NGS data revealed the enrichment of sh-Sec61alpha in the U937 cells sorted from the high collagen-uptake group, indicating that Sec61 translocon complex silencing promotes plenty of collagen accumulated in mammalian cells. The results that excessive collagen aggregation in the vesicles inside the cells drives us to speculate (1) whether the excessive accumulation of collagen aggregation directly or indirectly leads to endogenous stress such as ER stress in mammalian cells; (2) whether the collagen endocytic receptor Endo180 expression level would be affected or not when collagen aggregation inside the cells. In addition, we are also 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 high-throughput genome-wide dsRNA-mediated screening in Drosophila S2 cells or a comprehensive screening of collagen turnover in human U937 cells with a pooled lentiviral human genomic shRNA library. Newly designed dsRNAs are successfully generated in our laboratory. Both of these screenings were conducted very successfully to select candidate genes in our laboratory. We will then further characterize the role of candidate genes in collagen turnover in human macrophages and lung fibroblasts. In this manner, we will identify novel molecules that mediate cellular uptake and turnover of collagen. The proposed studies will be critical in establishing an innovative approach to study collagen turnover.

Project ID:PC10507-0105
External Project ID:MOST105-2320-B182-002