Extracellular Superoxide Dismutase (SOD3) Links Tumor Necrosis Factor‐alpha Receptor 1 to Integrin Signaling

Abstract

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1). Extracellular superoxide dismutase (ecSOD) can convert this O2•− to hydrogen peroxide (H2O2). ecSOD is anchored to the cell surface by interaction with either heparin sulfate proteoglycan via a heparin binding domain, or by binding to fibulin‐5 (Fib5), an extracellular matrix protein which interacts with integrin receptors. Fib5 binding does not activate integrins, but blocks interaction with other components of the extracellular matrix. The physiologic significance of ecSOD‐Fib5‐integrin interaction has not been well defined. We hypothesized that ecSOD regulates extracellular redox signaling of TNFα/Nox1 by modulating secondary effects on integrin signaling. We analyzed TNFα‐induced interleukin‐6 (IL‐6) production and NF‐κB activation in cultured vascular smooth muscle cells (VSMC). Knockdown of ecSOD by siRNA blocked TNFα‐mediated IL‐6 (siControl+TNFα 2922±354, si ecSOD+TNFα 952.5±99 pg/mL, p<0.05, n=8–9) and NF‐κB activation (siControl+TNFα 10.8±1.0, si ecSOD+TNFα 4.0±0.3 fold change, p<0.05, n=7). Similar inhibitory effects were observed following knockdown of Fib5 (IL‐6; siControl+TNFα 2372±123, siFib5+TNFα 1686±124 pg/mL, p<0.05, n=4. NF‐κB activation; siControl+TNFα 10.8±1.0, siFib5+TNFα 5.4±0.3 fold change, p<0.05, n=7). We next sought to determine how ecSOD affected integrin signaling. Focal adhesion kinase (FAK) and integrin‐linked kinase (ILK) are critical downstream effectors of integrin activation. TNFα induced phosphorylation of both FAK and Akt, which was used as a surrogate for ILK activation. This approach was validated by the ability of ILK inhibition (Cpd22; 3 μM) to prevent Akt phosphorylation. Both FAK and Akt phosphorylation in response to TNFα were inhibited by an anti‐integrin β1 antibody. Knockdown of ecSOD significantly enhanced TNFα‐induced phosphorylation of both FAK and Akt. FAK inhibition (FAK inhibitor 14; 2 μM) blocked while ILK inhibition (Cpd22; 3 μM) markedly potentiated NF‐κB activation. These results suggest that TNFα transactivates β1 integrins and therefore FAK and ILK in VSMCs. We speculate that ecSOD localizes H2O2 production to the vicinity of integrin receptors via physical association with Fib5 and provides tonic suppression of β1integrin receptor activation. Knockdown of either ecSOD or Fib5 would promote ILK‐dependent inhibition of NF‐κB activation and IL‐6 secretion which occurs via direct TNFR1‐dependent signaling.Support or Funding InformationAmerican Heart Association (16SDG30610002)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.