Abstract 19023: A Large-Scale, Functional Screening of Mammalian Mechanosensitive Genes Using Drosophila Rnai library- Smarcd3/Bap60 is a Mechanosensitive Pro-Inflammatory Gene
Introduction: Atherosclerosis in a chronic inflammatory disease of the arterial wall that preferentially occurs in regions exposed to disturbed blood flow, in part, due to alterations in gene expression in the endothelium. Disturbed flow triggers pro-atherogenic signaling and induce a number of pro-inflammatory genes in endothelial cells that serves as the foundation for plaque formation in the blood vessels. To understand the relationship between the flow-sensitive genes and inflammation, we performed a large-scale, unbiased in vivo RNAi Drosophila screen for regulatory gene(s) of nuclear factor kappa B (NFκB), which is a well-known mediator of inflammatory processes.
Approach and Results: Previously, we identified >580 mechanosensitive genes in mouse arterial endothelium, but which of these genes regulate endothelial inflammation was unknown. By comparing the murine mechanosensitive gene list to that of Drosophila orthologs, we obtained 84 Drosophila RNAi lines expressing double-stranded hairpin RNA (dsRNA) that silences expression of target gene under the UAS promoter. These were then crossed with C564-GAL4 flies expressing GFP under drosomycin promoter, a Drosophila NFκB target gene as a marker in bacterial-induced inflammation. Initial screening by fluorescence microscopy showed that knockdown of psmd12 or IRE1 inhibited bacteria-induced drosomycin activation, while the Bap60 knockdown robustly increased it under both basal and bacteria-infection conditions. Subsequently, psmd12 and smarcd3 were validated and studied further using endothelial cells exposed to laminar or oscillatory shear in vitro and in the mouse partial carotid ligation model of atherosclerosis in vivo.
Conclusions: These results suggest that smarcd3 is a novel repressor of NFκB in endothelial cells, which might play an important anti-inflammatory role in endothelial inflammation in a flow-responsive manner. Additionally, we demonstrate a novel strategy for the identification and characterization of novel mechanosensitive genes that regulate endothelial inflammation.
Author Disclosures: S. Kumar: None. I. Jang: None. D. Kang: None. C. Kim: None. W. Lee: None. H. Jo: None.
- © 2016 by American Heart Association, Inc.