Abstract
Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.
Highlights
We found that phosphorylation of peroxiredoxin 6 (Prdx6) through MAPK activity causes its translocation to the cell membrane, and the resultant phospholipase A2 (PLA2) activity leads to assembly of the NADPH oxidase 2 (NOX2) enzyme complex and reactive oxygen species (ROS) generation
H2O2 production was diminished in NOX2 null lungs, it was greater than the basal level, suggesting a minor contribution to ROS production from non-NOX2 sources
NOX2 has been well established as the major source of ROS generation in polymorphonuclear leukocyte (PMN) and macrophages [3]
Summary
Materials—Horseradish peroxidase (HRP) and the fluorescent dyes dihydrodichlorofluorescein (H2DCF) diacetate, Amplex Red, and dihydroethidium (HE) were obtained from Invitrogen. PMVEC derived from lungs of Prdx null mice were grown to 50 – 60% confluence, trypsinized from the dish, pelleted by centrifugation, and resuspended in Amaxa Nucleofector endothelial kit solution (Amaxa, Gaithersburg, MD). Cellular Immunofluorescence—To evaluate Prdx phosphorylation in intact endothelial cells, PMVEC were fixed in 4% paraformaldehyde for 10 min at room temperature, washed three times, permeabilized with 1% Triton X-100 for 10 min, blocked, and treated with primary antibody followed by Alexa488-labeled secondary antibody. Cells before and after the Ang II incubation were lysed by sonication, extracted with CH3/MeOH, and analyzed by thin layer chromatography using CHCl3/MeOH/NH4OH as the solvent system, and dpm in the [lyso-3H]PC band was measured by scintillation counting as described previously [18, 19]. Differences between mean values were considered statistically significant at p Ͻ 0.05
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