Endosomal Nox2 Facilitates Redox-Dependent Induction of NF-κB by TNF-α

Growing evidence suggests that NADPH oxidase (Nox)-derived reactive oxygen species (ROS) play important roles in regulating cytokine signaling. We have explored how TNF-alpha induction of Nox-dependent ROS influences NF-kappa B activation. Cellular stimulation by TNF-alpha induced NADPH-dependent superoxide production in the endosomal compartment, and this ROS was required for IKK-mediated activation of NF-kappa B. Inhibiting endocytosis reduced the ability of TNF-alpha to induce both NADPH-dependent endosomal superoxide and NF-kappa B, supporting the notion that redox-dependent signaling of the receptor occurs in the endosome. Molecular analyses demonstrated that endosomal H2O2 was critical for the recruitment of TRAF2 to the TNFR1/TRADD complex after endocytosis. Studies using both Nox2 siRNA and Nox2-knockout primary fibroblasts indicated that Nox2 was critical for TNF-alpha-mediated induction of endosomal superoxide. Redox-active endosomes that form after TNF-alpha or IL-1 beta induction recruit several common proteins (Rac1, Nox2, p67(phox), SOD1), while also retaining specificity for ligand-activated receptor effectors. Our studies suggest that TNF-alpha and IL-1 beta signaling pathways both can use Nox2 to facilitate redox activation of their respective receptors at the endosomal level by promoting the redox-dependent recruitment of TRAFs. These studies help to explain how cellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane. Antioxid. Redox Signal. 11, 1249-1263.