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Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library– Smarcd3/Bap60 is a mechanosensitive pro‐inflammatory gene
Author(s) -
Kumar Sandeep,
Jo Hanjoong
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.689.1
Subject(s) - gene knockdown , mechanosensitive channels , biology , gene silencing , rna interference , microbiology and biotechnology , inflammation , endothelium , gene , gene expression , immunology , genetics , rna , receptor , ion channel
Disturbed blood flow (d‐flow) induces atherosclerosis by altering the expression of mechanosensitive genes in the arterial endothelium. Previously, we identified >580 mechanosensitive genes in the mouse arterial endothelium, but their role in endothelial inflammation is incompletely understood. From this gene set, we obtained 84 Drosophila RNAi fly lines that silences the target “gene of interest” under the control of upstream activation sequence (UAS) promoter. These lines were crossed with C564‐GAL4 flies expressing GFP under the control of drosomycin promoter, an NF‐κB target gene and a marker of pathogen‐induced inflammation. Using this screening approach, we identified 3 potential candidate genes regulating endothelial inflammation, psmd12, ERN1 and Bap60. Silencing of psmd12 or ERN1 decreased infection‐induced drosomycin expression, while Bap60 silencing significantly increased the drosomycin expression. Interestingly, knockdown of Bap60 in adult flies using temperature‐inducible Bap60 RNAi ( C564 ts ‐GAL4‐Bap60‐RNAi ) flies enhanced drosomycin expression upon Gram‐positive bacterial challenge but the basal drosomycin expression remained unchanged compared to the control. In the mammalian system, smarcd3 (mammalian ortholog of Bap60) expression was reduced in the human‐ and mouse aortic endothelial cells exposed to oscillatory shear in vitro as well as in the d‐flow regions of mouse arterial endothelium in vivo. Moreover, siRNA‐mediated knockdown of smarcd3 induced endothelial inflammation. In summary, we developed an in vivo Drosophila RNAi screening method to identify flow‐sensitive genes that regulate endothelial inflammation. The newly identified mechanosensitive smarcd3 may play a critical role in endothelial inflammation and atherosclerosis. This in vivo functional screening approach may be expanded to identify novel genes that regulate other important signaling pathways such as apoptosis and cell proliferation. Support or Funding Information This work was supported by funding from National Institutes of Health grants HL119798, HL113451, HL095070 and HL124879 to HJ.Overall study design and work flow