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Molecular and Cellular Biology, June 2008, p. 3700-3712, Vol. 28, No. 11
0270-7306/08/$08.00+0     doi:10.1128/MCB.02038-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evidence for a Superoxide Permeability Pathway in Endosomal Membranes{triangledown}

Davis R. Mumbengegwi,1 Qiang Li,1 Canhui Li,3 Christine E. Bear,3 and John F. Engelhardt1,2*

Department of Anatomy and Cell Biology,1 Center for Gene Therapy, College of Medicine, University of Iowa, Iowa City, Iowa 52242,2 Programme in Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario MG5 1X8, Canada3

Received 12 November 2007/ Returned for modification 4 January 2008/ Accepted 24 March 2008

The compartmentalized production of superoxide (·O2–) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-{kappa}B following interleukin 1β (IL-1β) stimulation. It remains unclear how ·O2– produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated ·O2– movement out of IL-1β-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 ·O2– from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for ·O2–. In these studies, ~60% of ·O2– 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 ·O2– and Cl– are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1β-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-1β stimulation. Together, these results suggest that ·O2– exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of ·O2– at the endosomal surface may produce the localized H2O2 required for redox-activation of NF-{kappa}B.

* Corresponding author. Mailing address: Room 1-111 BSB, Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, 51 Newton Road, Iowa City, IA 52242. Phone: (319) 335-7744. Fax: (319) 335-6581. E-mail: john-engelhardt{at}uiowa.edu

{triangledown} Published ahead of print on 31 March 2008.

Molecular and Cellular Biology, June 2008, p. 3700-3712, Vol. 28, No. 11
0270-7306/08/$08.00+0     doi:10.1128/MCB.02038-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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