title: Redox-independent activation of NF-kappaB by Pseudomonas aeruginosa pyocyanin in a cystic fibrosis airway epithelial cell line.
abstract: The roles of the Pseudomonas aeruginosa-derived pigment pyocyanin (PYO) as an oxidant and activator of the proinflammatory transcription factor NF-kappaB were tested in a cystic fibrosis (CF) airway epithelial cell line, CF15. 100 microm PYO on its own had no effect or only small effects to activate NF-kappaB (<1.5-fold), but PYO synergized with the TLR5 agonist flagellin. Flagellin activated NF-kappaB 4-20-fold, and PYO increased these activations >2.5-fold. PYO could have synergized with flagellin to activate NF-kappaB by redox cycling with NADPH, generating superoxide (O(2)*), hydrogen peroxide (H(2)O(2)), and hydroxyl radical (HO*). Cytosol-targeted, redox-sensitive roGFP1 and imaging microscopy showed that 1-100 microm PYO oxidized CF15 cytosol redox potential (Psi(cyto)) from -325 mV (control) to -285 mV. O(2)* (derived from KO(2)*. or xanthine + xanthine oxidase) or H(2)O(2) oxidized Psi(cyto) dose-dependently but did not activate NF-kappaB, even in the presence of flagellin, and 400 microm H(2)O(2) inhibited NF-kappaB. Overexpressing intracellular catalase decreased effects of PYO and H(2)O(2) on Psi(cyto) but did not affect flagellin + PYO-activated NF-kappaB. Catalase also reversed the inhibitory effects of H(2)O(2) on NF-kappaB. The HO* scavenger DMSO did not alter the effects of PYO on Psi(cyto) and NF-kappaB. The synergistic NF-kappaB activation was calcium-independent. Thus, in the presence of flagellin, PYO activated NF-kappaB through a redox- and calcium-independent effect.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Schwarzer
authors.forename: Zhu
authors.initials: Z
authors.lastname: Fu
authors.forename: Horst
authors.initials: H
authors.lastname: Fischer
authors.forename: Terry E
authors.initials: TE
authors.lastname: Machen
mesh_terms: D002374:Catalase / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004357:Drug Synergism; D005408:Flagellin / Q000819:agonists / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D009249:NADP / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000819:agonists / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D011710:Pyocyanine / Q000819:agonists / Q000737:chemistry / Q000494:pharmacology*; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000473:pathology; D013481:Superoxides / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D016166:Free Radical Scavengers; D016328:NF-kappa B; D016877:Oxidants; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D013481:Superoxides; D005408:Flagellin; D019820:Xanthine; D009249:NADP; D011710:Pyocyanine; D006861:Hydrogen Peroxide; D002374:Catalase; D014969:Xanthine Oxidase; D004121:Dimethyl Sulfoxide