title: Regulation of NAD(P)H oxidase by associated protein disulfide isomerase in vascular smooth muscle cells.
abstract: NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.
authors.affiliation: Vascular Biology Laboratory, Heart Institute (Instituto do Coração), School of Medicine, University of São Paulo, Av. Eneas Carvalho Aguiar, 44-subsolo, São Paulo, CEP 05403-000 Brazil.
authors.forename: Mariano
authors.initials: M
authors.lastname: Janiszewski
authors.forename: Lucia Rossetti
authors.initials: LR
authors.lastname: Lopes
authors.forename: Alípio O
authors.initials: AO
authors.lastname: Carmo
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Pedro
authors.forename: Ralf P
authors.initials: RP
authors.lastname: Brandes
authors.forename: Célio X C
authors.initials: CX
authors.lastname: Santos
authors.forename: Francisco R M
authors.initials: FR
authors.lastname: Laurindo
mesh_terms: D000166:Acridines; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D001414:Bacitracin / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D006706:Homeostasis; D006801:Humans; D008163:Luminescent Measurements; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D019255:NADPH Oxidases / Q000032:analysis / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D017510:Protein Folding; D021122:Protein Subunits / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D013481:Superoxides / Q000378:metabolism; D014162:Transfection
chemical_list: D000166:Acridines; D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D013481:Superoxides; D000804:Angiotensin II; D001414:Bacitracin; D049452:Green Fluorescent Proteins; C033472:10,10'-dimethyl-9,9'-biacridinium; D019255:NADPH Oxidases; D019704:Protein Disulfide-Isomerases