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The deubiquitinase USP40 promotes microvascular endothelial cell integrity and protects against lung injury
Author(s) -
Miao Jiaxing,
Wei Jianxin,
Taleb Sarah,
Mallampalli Rama,
Zhao Yutong,
Zhao Jing
Publication year - 2020
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.2020.34.s1.07120
Subject(s) - rhoa , proinflammatory cytokine , ubiquitin , chemistry , microbiology and biotechnology , phosphorylation , myosin light chain kinase , downregulation and upregulation , lipopolysaccharide , gene knockdown , transfection , inflammation , biology , signal transduction , immunology , apoptosis , biochemistry , gene
Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks for inflammatory diseases, such as acute lung injury. Ubiquitination is a post‐translational modification. Ubiquitin proteins covalently link to substrate proteins, thereby regulating protein stability and enzyme activity. Deubiquitinating enzymes (DUBs) reserve this process. USP40, a newly recognized DUB, has been shown to regulate glomerular permeability in zebrafish. Our goal is to understand the role of USP40 in microvascular EC integrity. NF‐κB pathway plays a central role in the upregulation of EC activity. V5‐tagged USP40 (USP40‐V5) overexpression attenuated lipopolysaccharide (LPS)‐induced phosphorylation of I‐κB in human lung microvascular endothelial cells (HLMVECs). TNFα treatment increased NF‐κBp65 nuclear translocation, while the effect was attenuated in USP40‐overexpressing cells. Overexpression of USP40 significantly attenuated LPS‐induced ICAM1 expression, while depletion of USP40 promoted ICAM1 levels. RhoA‐mediated phosphorylation of myosin light chain (MLC) promotes EC barrier dysfunction. USP40‐V5 attenuated LPS‐induced activation of RhoA. Further, LPS treatment of HLMVECs increased phosphorylation of MLC, and it was reduced by USP40 overexpression. Overexpression of USP40 attenuated LPS‐reduced transendothelial electrical resistance (TEER), while knockdown of USP40 by USP40 siRNA transfection increased LPS‐induced EC permeability in a transwell leakage assay. Immunostaining with VE‐cadherin showed that USP40 siRNA promoted LPS‐induced gap formation in HLMVECs. We generated USP40 deficient (USP40 −/− ) mice by CRISPR/Cas9 system. USP40 −/− mice showed an increase in ICAM1 in lung tissues and BAL protein levels in intratracheal (IT) LPS (2 mg/kg, 24 h)‐induced murine model of acute lung injury (ALI). To investigate if increases in USP40 levels protect against LPS‐induced ALI, lentiviral‐driven overexpression of USP40 in vessels of mice was performed. LPS increased Evans blue leakage, BAL protein levels, and neutrophil influx, which were significantly decreased in lenti‐USP40 delivered mice. Our observations are the first to reveal that USP40 reduces the severity of ALI through diminishing the activation of NF‐κB‐and RhoA‐mediated inflammation and EC permeability. Support or Funding Information This study is supported by NIH R01 grants