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Extracellular Superoxide Dismutase (ecSOD) Modulates TNFα Signaling in Human Lung Endothelial Cells
Author(s) -
Miller Michael,
Choi Hyehun,
Lamb Fred
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.s1.03238
Subject(s) - signal transduction , extracellular , phosphorylation , focal adhesion , tumor necrosis factor alpha , microbiology and biotechnology , reactive oxygen species , kinase , mapk/erk pathway , superoxide dismutase , cytokine , chemistry , biology , oxidative stress , immunology , biochemistry
Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O 2 −• ) to be produced extracellularly by NADPH oxidase, but it remains unclear how this redox signal is transduced into activation of intracellular signaling pathways. We previously demonstrated in vascular smooth muscle cells (VSMCs) that TNFα signaling is dependent upon the extracellular isoform of superoxide dismutase (ecSOD). In cultured VSMCs siRNA knockdown of ecSOD decreased downstream MAPK phosphorylation and NFκB activation. In contrast, integrin receptor activation was enhanced, inducing higher levels of focal adhesion kinase (FAK) phosphorylation. These findings hint at a role for ecSOD in cytokine signaling that is independent from an anti‐oxidant influence. Studies in mice have shown that conditional knockout of ecSOD induces a rapid and lethal ARDS phenotype. Due to the critical nature of TNFα and reactive oxygen in the development of acute respiratory distress syndrome (ARDS) we sought to determine the signaling role of extracellular O 2 −• and ecSOD in endothelial cells (ECs). We employed cultured human lung microvascular endothelial cells (HLMVEC) to assess the impact of ecSOD knockdown (siRNA) on TNFα signaling. ecSOD expression was reduced ~ 50% by a 72hr exposure to siRNA. In siRNA treated HLMVECs, phosphorylation of extracellular signal‐regulated kinase (ERK) following a 15 min exposure to TNFα was significantly increased (siControl vs siSOD3 – 0.82 vs 1.93 mean signal intensity; p = 0.016). This short‐term exposure to TNFα also induced a significant reduction in the abundance of ecSOD detected by Western blot (61.6% signal intensity after TNF treatment). This suggests either rapid TNFα‐induced degradation of ecSOD, or dissociation of the protein from the cell surface, where ecSOD is known to bind to either heparin sulfate or Fibulin 5, a component of the extracellular matrix that also binds to integrins. A transendothelial resistance (TEER) assay was employed to assess the role of ecSOD in formation of a tight EC monolayer. HLMVECs treated with ecSOD siRNA were significantly impaired in their ability to develop TEER compared to cells treated with scrambled siRNA. Together, these data suggest that ecSOD plays a critical role in normal endothelial cell function and in signaling in response to TNFα, including a role in regulation of endothelial monolayer integrity. Ongoing work seeks to define the contribution of ecSOD‐dependent signaling to EC inflammation in ARDS. Support or Funding Information This work was supported by R01HL128386 and 5T32HD060554‐10