Regulation of NAD(P)H Oxidase by Associated Protein Disulfide Isomerase in Vascular Smooth Muscle Cells

doi:10.1074/jbc.M509255200

Regulation of NAD(P)H Oxidase by Associated Protein Disulfide Isomerase in Vascular Smooth Muscle Cells*

^‡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, the ^§Institut für Kardiovaskuläre Physiologie, J. W. Goethe Universität Frankfurt, D-60596 Frankfurt am Main, Germany, the ^¶Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo CEP 05508-900, Brazil, and the ^∥Hospital Israelita Albert Einstein, São Paulo CEP 05651-901, Brazil

2 To whom correspondence should be addressed. Tel.: 55-(11)-3069-5184; Fax: 55-(11)-3069-5920; E-mail: expfrancisco{at}incor.usp.br.

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.

Footnotes

↵3 The abbreviations used are: AII, angiotensin II; DHE, dihydroethidium; PDIasODN, PDI antisense oligonucleotide; ScbRNase, scrambled RNase; ScrODN, scrambled oligonucleotide; ER, endoplasmic reticulum; VSMC, vascular smooth muscle cell; ODN, oligodeoxynucleotide; YFP, yellow fluorescent protein; GFP, green fluorescent protein; DMPO, 5,5′-dimethylpyrroline-N-oxide; IP, immunoprecipitation.
↵* This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Financiadora de Estudas e Projetos/Programa de Núcleos de Excelência, F. Zerbini, the A. Einstein Society, and a Georg-Forster Grant from the Alexander von Humboldt Foundation (to M. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 Both authors contributed equally to this work.
- Received August 22, 2005.
- Revision received September 7, 2005.
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