TY  - JOUR
A1  - Janiszewski, Mariano
A1  - Lopes, Lucia Rossetti
A1  - Carmo, Alípio O.
A1  - Pedro, Marcelo A.
A1  - Brandes, Ralf
A1  - Santos, Célio X. C.
A1  - Laurindo, Francisco R. M.
T1  - Regulation of NAD(P)H oxidase by associated protein disulfide isomerase in vascular smooth muscle cells
T2  - Journal of biological chemistry
N2  - 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.
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/76205
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-762056
SN  - 0021-9258
VL  - 280.2005
IS  - 49
SP  - 40813
EP  - 40819
PB  - American Society for Biochemistry and Molecular Biology Publications
CY  - Bethesda, Md
ER  -