Extracellular SOD modulates canonical TNFα signaling and α5β1 integrin transactivation in vascular smooth muscle cells

• Published in: Free radical biology & medicine 2023-11, Vol.209 (Pt 1), p.152-164
• Main Authors: Choi, Hyehun, Miller, Michael R., Nguyen, Hong-Ngan, Surratt, Victoria E., Koch, Stephen R., Stark, Ryan J., Lamb, Fred S.
• Format: Article
• Language: English
• Subjects: Alpha5Beta1 integrin; Animals; Extracellular superoxide dismutase; Fibulin 5; Hydrogen Peroxide - metabolism; Integrins - genetics; Integrins - metabolism; Mice; Muscle, Smooth, Vascular - metabolism; NF-kappa B - genetics; NF-kappa B - metabolism; Signal Transduction; Superoxide Dismutase - metabolism; Superoxide dismutase 3; Transcriptional Activation; Tumor Necrosis Factor-alpha - genetics; Tumor necrosis factor-α
• ISSN: 0891-5849; 1873-4596; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2023.10.397

TNFα activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O2•-) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O2•- to signal transduction remain obscure. Extracellular superoxide dismutase (ecSOD, SOD3 gene) is a secreted protein that binds to cell surface heparin sulfate proteoglycans or to Fibulin-5 (Fib-5, FBLN5 gene), an extracellular matrix protein that also associates with elastin and integrins. ecSOD converts O2•- to hydrogen peroxide (H2O2) which prevents NO• inactivation, limits generation of hydroxyl radical (OH•), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNFα signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNFα signals including MAPK (JNK and ERK phosphorylation) and NF-κB activation (luciferase reporter and IκB phosphorylation), interleukin-6 (IL-6) secretion, iNOS and VCAM expression, and proliferation (Sulforhodamine B assay, PCNA western blot). These effects were associated with significant reductions in the expression of both Type1 and 2 TNFα receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-κB activation by TNFα, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNFα-induced phosphorylation of FAK and of glycogen synthase kinase-3β (GSK3β), a downstream target of integrin linked kinase (ILK). These effects were dependent upon α5β1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNFα-induced increases in sulfenylation. To determine how ecSOD modified TNFα-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNFα (10 ng/ml) in culture for 48 hrs. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNFα signaling while tonically inhibiting α5β1 integrin activation. [Display omitted] •ecSOD (SOD3) knockdown blocks canonical, pro-inflammatory TNFα signaling in VSMCs.•SOD3 knockdown (siSOD3) activates α5 and β1 integrin signaling.•TNFα or siSOD3 increases sulfenylation (oxidation) of α5 and β1 integrins.•The enzymatic region of ecSOD binds to fibulin-5, which modifies TNFα signaling.•Smooth muscle-specific ecSOD KO prevents TNFα-induced vascular dysfunction.