Evidence for a superoxide permeability pathway in endosomal membranes

The compartmentalized production of superoxide (center dot O-2(-)) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-kappa B following interleuldn 1 beta (IL-10) stimulation. It remains unclear how center dot O-2(-) produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated center dot O-2(-) movement out of IL-1 beta-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-kappa B activation. The relative outward permeability of NADPH-dependent center dot O-2(-) from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for center dot O-2(-). In these studies, similar to 60% of center dot O-2(-) efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that O-2(-) and Cl- are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1 beta-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-kappa B following IL-1 beta stimulation. Together, these results suggest that center dot O-2(-) exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of center dot O-2(-) at the endosomal surface may produce the localized H2O2 required for redox-activation of NF-kappa B.