Extracellular SOD modulates canonical TNF signaling and 581 integrin transactivation in vascular smooth muscle cells

TNF alpha activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O-2(center dot-)) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O-2(center dot-) 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 O-2(center dot-) to hydrogen peroxide (H2O2) which prevents NO center dot inactivation, limits generation of hydroxyl radical (OH center dot), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNF alpha signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNF alpha signals including MAPK (JNK and ERK phosphorylation) and NF-kappa B activation (luciferase reporter and I kappa B 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 TNF alpha receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-kappa B activation by TNF alpha, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNF alpha-induced phosphorylation of FAK and of glycogen synthase kinase-3 beta (GSK3 beta), a downstream target of integrin linked kinase (ILK). These effects were dependent upon alpha 5 beta 1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNF alpha-induced increases in sulfenylation. To determine how ecSOD modified TNF alpha-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNF alpha (10 ng/ml) in culture for 48 h. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNF alpha signaling while tonically inhibiting alpha 5 beta 1 integrin activation.