Evidence for a superoxide permeability pathway in endosomal membranes.
The compartmentalized production of superoxide (*O(2)(-)) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-kappaB following interleukin 1beta (IL-1beta) stimulation. It remains unclear how *O(2)(-) produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated *O(2)(-) movement out of IL-1beta-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-kappaB activation. The relative outward permeability of NADPH-dependent *O(2)(-) from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for *O(2)(-). In these studies, approximately 60% of *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-1beta-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-kappaB following IL-1beta stimulation. Together, these results suggest that *O(2)(-) exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of *O(2)(-) at the endosomal surface may produce the localized H(2)O(2) required for redox-activation of NF-kappaB.
Mumbengegwi
Davis R
DR
Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, 51 Newton Road, Iowa City, IA 52242, USA.
Li
Qiang
Q
Li
Canhui
C
Bear
Christine E
CE
Engelhardt
John F
JF
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D045744:Cell Line, Tumor; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D002713:Chlorine / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D009249:NADP / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009544:Niflumic Acid / Q000494:pharmacology; D010084:Oxidation-Reduction; D010539:Permeability; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D053573:Receptors, Interleukin-1 Type I / Q000032:analysis / Q000378:metabolism; D013482:Superoxide Dismutase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D013481:Superoxides / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000737:chemistry
D018118:Chloride Channels; C508590:IL1R1 protein, human; D053583:Interleukin-1beta; D016328:NF-kappa B; D011510:Proteolipids; D053573:Receptors, Interleukin-1 Type I; C000606290:SOD1 protein, human; C020485:proteoliposomes; D013481:Superoxides; D002713:Chlorine; D009544:Niflumic Acid; D009249:NADP; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D014969:Xanthine Oxidase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid