Extracellular Superoxide Dismutase (SOD3) mediates extracellular superoxide‐dependent signaling by TNFalpha.

Abstract

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2−•) to be produced extracellularly by NADPH oxidase 1 (Nox1). It remains unclear how this redox signal is transduced into activation of intracellular signaling pathways. We sought to identify steps in the TNFα signaling cascade that are directly O2−•‐dependent. We analyzed TNFα receptor endocytosis and activation of MAPKs and NF‐κB in cultured vascular smooth muscle cells (VSMC). JNK, p38 and ERK1/2 were activated with a similar concentration dependence. TNFα receptor endocytosis, MAPKs (p‐JNK and p‐ERK1/2) and NF‐κB activation were all inhibited by unmodified superoxide dismutase (SOD, 500 U/ml) added extracellularly, but not by catalase (1000 U/ml), or by overexpression of cytoplasmic CuZnSOD or catalase with adenovirus. This suggested a paramount importance of extracellular O2−•. We next sought to determine if endogenous, extracellular SOD (SOD3) modulated these responses by employing siRNA‐mediated downregulation. Surprisingly, siSOD3 blocked TNFα‐induced endocytosis (37 % reduction) and rather than enhancing MAPK and NF‐κB activation, these effects were blocked (Relative NF‐κB activation; siControl+TNFα 10.8 ± 1.0, siSOD3+TNFα 4.0 ± 0.3 fold, n=7, p<0.05). Similar results were obtained by pharmacologic inhibition of SOD3 using DETC (100 μM). These results demonstrate that enzymatic activity of endogenous SOD3 is required for signaling, however, the lack of effect of extracellular catalase calls into question the role of H2O2, the product of SOD3 activity. In order to directly assess the signaling impact of H2O2 we performed a dose‐response to this oxidant. MAPK activation occurred at a lower concentration (1 μM) while a higher concentration (10 μM) potently inhibited signaling (Figure 1). It remains to be determined how SOD3 modulates local H2O2 concentrations in the extracellular matrix following Nox1 activation by TNFα, and whether the H2O2 produced by SOD3 is accessible to catalase placed into that compartment. Taken together, these data suggest a novel role for SOD3 in transducing Nox1‐dependent inflammatory signaling activated by TNFα in VSMC.Support or Funding InformationThis work was supported by R01 HL128386 (FSL) and by a Scientist Development Award from the AHA (HC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.