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.
Highlights
Redox-dependent signal transduction, a central aspect of vascular physiology and pathophysiology, converges on enzyme systems generating reactive oxygen species, required to act as second messengers of cell stimulators such as angiotensin II (AII)3 [1]
We focused into the thioredoxin superfamily enzyme protein disulfide isomerase (PDI), given its abundance, multiple biological effects, versatile redox behavior, and known interaction with other proteins [10, 12, 13]
PDI Inhibition in vascular smooth muscle cell (VSMC) Membrane Fraction Decreases NAD(P)H Oxidase Activity—To assess functional dependence of NAD(P)H oxidase activity on PDI, NADPH-driven superoxide generation was assessed by EPR spectroscopy in VSMC membrane fraction incubated after its separation with known PDI inhibitors (Fig. 2)
Summary
Redox-dependent signal transduction, a central aspect of vascular physiology and pathophysiology, converges on enzyme systems generating reactive oxygen species, required to act as second messengers of cell stimulators such as angiotensin II (AII)3 [1]. PDI is mainly located at endoplasmic reticulum (ER) lumen, where it assists in redox protein folding, which involves both oxidation and multiple intramolecular thiol-disulfide exchanges, i.e. isomerase, activities. Such properties stem from its structural configuration, which consists of five modules ordered as a-b-bЈ-aЈ-c, in which a is a thioredoxin domain bearing the redox-active WCGHCK motif, b a thioredoxin structural fold without the redox motif, possibly related to peptide recognition [12, 13], and c a putative Ca2ϩ-binding C-terminal domain [12, 14]. Despite bearing the C-terminal ER retention sequence KDEL, PDI displays active intracellular traffic to the cell surface, where it likely acts as a reductase due to the high local reducing potential [10, 12, 13] This property led to further exploration of other cellular PDI effects. NAD(P)H Oxidase Regulation by Protein Disulfide Isomerase and that such interaction affects redox-dependent consequences of AII exposure
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