Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA2) 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 PLA2 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 p47phox, 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 PLA2 activity, blocked agonist-induced PLA2 activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA2s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA2 active site (S32A, H26A, and D140A), “rescued” Ang II-induced PLA2 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 PLA2 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 PLA2 activity facilitates assembly of the NOX2 complex and activation of the oxidase. Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA2) 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 PLA2 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 p47phox, 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 PLA2 activity, blocked agonist-induced PLA2 activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA2s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA2 active site (S32A, H26A, and D140A), “rescued” Ang II-induced PLA2 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 PLA2 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 PLA2 activity facilitates assembly of the NOX2 complex and activation of the oxidase. IntroductionReactive oxygen species (ROS) 2The abbreviations used are: ROSreactive oxygen speciesAACOCF3arachidonyltrifluoromethyl ketoneAng IIangiotensin IIBELbromoenol lactoneDCFdichlorofluorosceinH2DCFdihydrodichlorofluoresceinDPPC1-palmitoyl, 2-palmitoyl, sn-glycero-3-phosphocholinefMLFN-formyl-Met-Leu-Phe peptideHEhydroethidiumMJ331-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanolNOXNADPH oxidasepBPBp-bromophenacyl bromidePLA2phospholipase A2cPLA2cytoplasmic PLA2PMAphorbol 12-myristate 13-acetatePMVECpulmonary microvascular endothelial cell(s)PrdxperoxiredoxinH2DCFdihydrodichlorofluoresceinPECAMplatelet endothelial cell adhesion molecule 1PCphosphatidylcholineAbantibodyPMNpolymorphonuclear leukocyte(s). comprising O2⨪, H2O2, ·OH, and others, are now regarded as important signaling molecules in biological systems. For example, H2O2 modulates cell growth, apoptosis, and various other endothelial cell functions (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 2Lassegue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2010; 30: 653-661Crossref PubMed Scopus (480) Google Scholar). NADPH oxidases (NOXs) are a family of seven widely distributed enzymes that enzymatically generate O2⨪ and, through dismutation, H2O2 (3Lambeth J.D. Curr. Opin. Hematol. 2002; 9: 11-17Crossref PubMed Scopus (230) Google Scholar). Of these, NOX2 is the canonical NOX responsible for O2⨪ generation by the respiratory burst in neutrophils. NOX2 is also found in other cell types and is a major source of ROS in endothelial cells (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 2Lassegue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2010; 30: 653-661Crossref PubMed Scopus (480) Google Scholar). In the unstimulated state, NOX2 is quiescent, with the intrinsic membrane subunits (cytochrome b558 comprising gp91phox and p22phox) and cytosolic subunits (Rac1, p47phox, p67phox, and p40phox) confined to their respective compartments (4Pendyala S. Usatyuk P.V. Gorshkova I.A. Garcia J.G. Natarajan V. Antioxid. Redox Signal. 2009; 11: 841-860Crossref PubMed Scopus (94) Google Scholar). Activation of the enzyme complex by stimuli such as angiotensin II (Ang II), phorbol esters, or thrombin leads to translocation of the cytosolic components to the plasma membrane, resulting in assembly of the oxidase complex. Cytochrome b558 with its heme, NADPH, and flavin binding sites functions to transfer electrons from NADPH to oxygen, thereby generating O2⨪ in the extracellular space. Dismutation of O2⨪, either catalyzed or spontaneous, generates H2O2, which is regarded as the primary ROS signaling molecule in endothelium (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 2Lassegue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2010; 30: 653-661Crossref PubMed Scopus (480) Google Scholar).Peroxiredoxins are a family of antioxidants that express peroxidase activity and catalyze the removal of H2O2 and other hydroperoxides (5Rhee S.G. Chae H.Z. Kim K. Free Radic. Biol. Med. 2005; 38: 1543-1552Crossref PubMed Scopus (1132) Google Scholar). Of the six mammalian members of the family, peroxiredoxin 6 (Prdx6) is the only peroxiredoxin with both phospholipase A2 (PLA2) and peroxidase activities (6Fisher A.B. Antioxid. Redox Signal. 2011; (in press)Google Scholar). The PLA2 activity catalyzes the hydrolysis of the acyl group at the sn-2 position of glycerophospholipids, with special affinity for phosphatidylcholine, to produce free fatty acids and a lysophospholipid; the peroxidase activity uses glutathione as the co-factor for reduction of hydroperoxides, including phospholipid hydroperoxides (6Fisher A.B. Antioxid. Redox Signal. 2011; (in press)Google Scholar). In a previous study, Prdx6 (therein called p29) was found to participate in the activation of NOX2 in human neutrophils (7Leavey P.J. Gonzalez-Aller C. Thurman G. Kleinberg M. Rinckel L. Ambruso D.W. Freeman S. Kuypers F.A. Ambruso D.R. J. Biol. Chem. 2002; 277: 45181-45187Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, the mechanism by which Prdx6 might activate NOX2-mediated ROS generation was not determined. Here, we investigated the role of Prdx6 in NOX2 activation in alveolar macrophages and in the endothelium in situ and in vitro. We found that phosphorylation of Prdx6 through MAPK activity causes its translocation to the cell membrane, and the resultant PLA2 activity leads to assembly of the NOX2 enzyme complex and ROS generation.DISCUSSIONNOX2 has been well established as the major source of ROS generation in PMN and macrophages (3Lambeth J.D. Curr. Opin. Hematol. 2002; 9: 11-17Crossref PubMed Scopus (230) Google Scholar). Although NOX2 was originally considered as a protein complex exclusive to these phagocytic cells, there is compelling evidence to indicate that NOX2 expression is widely distributed, and it is now considered as the major enzyme system for ROS generation in vascular endothelial cells (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 2Lassegue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2010; 30: 653-661Crossref PubMed Scopus (480) Google Scholar). NOX2 consists of membrane and cytosolic components that, in response to a wide variety of agonists, assemble at the plasma membrane, thereby activating the enzyme to produce O2⨪. However, the exact mechanisms that drive this assembly are not known. What is clear is that NOX2 assembly can be initiated by pathways involving either a receptor- or non-receptor-mediated process (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 4Pendyala S. Usatyuk P.V. Gorshkova I.A. Garcia J.G. Natarajan V. Antioxid. Redox Signal. 2009; 11: 841-860Crossref PubMed Scopus (94) Google Scholar). An example of the former is Ang II that works through its receptor subtypes AT1 and AT2 to activate downstream signaling pathways, whereas non-receptor-mediated agonists include the phorbol esters (e.g. PMA) that directly stimulate PKC. In this study, we used both Ang II and PMA to activate NOX2-dependent ROS production by intact perfused lungs or isolated PMVEC. With either agonist, NOX2 activation did not occur in the absence of Prdx6 and was rescued by transfection of Prdx6 null cells with a construct expressing wild-type Prdx6. Thus, we conclude that Prdx6 is essential for NOX2 activation and ROS production in endothelial cells. A similar Prdx6 requirement was demonstrated for agonist-mediated ROS production by alveolar macrophages.A similar conclusion regarding a role for Prdx6 in NOX2 activation was reached in a previous in vitro study of PMN in which ROS production was approximately doubled by the presence of recombinant Prdx6 (also known as p29); this latter study used a reconstituted system consisting of isolated plasma membrane from PMN and recombinant cytosolic proteins (7Leavey P.J. Gonzalez-Aller C. Thurman G. Kleinberg M. Rinckel L. Ambruso D.W. Freeman S. Kuypers F.A. Ambruso D.R. J. Biol. Chem. 2002; 277: 45181-45187Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Although the recombinant Prdx6 (p29) exhibited both PLA2 and peroxidase activities, neither activity could be conclusively associated with NOX2 activation, which raised the possibility that enhancement of NOX2 activity by Prdx6 was unrelated to either enzymatic activity. However, the present study using PMVEC shows conclusively that Prdx6 facilitates NOX2 assembly and activation through its PLA2 activity. Evidence to support this mechanism is provided by the following findings: 1) activation of PLA2 activity with agonist (Ang II, PMA) stimulation in wild-type but not Prdx6 null cells; 2) the rescue of Ang II response by transfection of Prdx6 null cells with constructs leading to the expression of Prdx6 with PLA2 activity, whereas constructs that did not lead to PLA2 activity were ineffective; and 3) the inhibition of activation in the presence of the PLA2 inhibitor, MJ33. Thus, we conclude that the essential role of Prdx6 is associated with its PLA2 activity.Our previous studies have demonstrated that Prdx6 has its peak PLA2 activity at pH 4 with minimal activity at cytosolic pH (20Kim T.S. Dodia C. Chen X. Hennigan B.B. Jain M. Feinstein S.I. Fisher A.B. Am. J. Physiol. 1998; 274: L750-L761PubMed Google Scholar, 21Chen J.W. Dodia C. Feinstein S.I. Jain M.K. Fisher A.B. J. Biol. Chem. 2000; 275: 28421-28427Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). This finding correlated with the ability of Prdx6 to bind to native phospholipid substrate (liposomes) at acidic but not neutral pH (22Manevich Y. Reddy K.S. Shuvaeva T. Feinstein S.I. Fisher A.B. J. Lipid Res. 2007; 48: 2306-2318Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 25Manevich Y. Shuvaeva T. Dodia C. Kazi A. Feinstein S.I. Fisher A.B. Arch. Biochem. Biophys. 2009; 485: 139-149Crossref PubMed Scopus (64) Google Scholar). However, lipid binding at pH 7 and subsequent PLA2 activity were markedly enhanced by phosphorylation of the protein (8Wu Y. Feinstein S.I. Manevich Y. Chowdhury I. Pak J.H. Kazi A. Dodia C. Speicher D.W. Fisher A.B. Biochem. J. 2009; 419: 669-679Crossref PubMed Scopus (78) Google Scholar). Prdx6 phosphorylation was shown in lung epithelial cells in response to PMA as determined by immunoprecipitation and was blocked by inhibition of MAPKs (8Wu Y. Feinstein S.I. Manevich Y. Chowdhury I. Pak J.H. Kazi A. Dodia C. Speicher D.W. Fisher A.B. Biochem. J. 2009; 419: 669-679Crossref PubMed Scopus (78) Google Scholar). In the present study, Prdx6 phosphorylation and its association with the endothelial cell plasma membrane (presumably as a result of binding to membrane lipids) were demonstrated subsequent to treatment of PMVEC with Ang II. The plasma membrane association was shown by immunofluorescence, immunoblot, and the Duolink procedure using an antibody to phosphorylated Prdx6. Further, Prdx6 translocation, PLA2 activity, and activation of NOX2 were blocked in the presence of U0126, an inhibitor of ERK MAPK activation and shown previously to inhibit Prdx6 phosphorylation (8Wu Y. Feinstein S.I. Manevich Y. Chowdhury I. Pak J.H. Kazi A. Dodia C. Speicher D.W. Fisher A.B. Biochem. J. 2009; 419: 669-679Crossref PubMed Scopus (78) Google Scholar). The requirement for phosphorylation of Prdx6 was confirmed by transfection with a construct expressing a Thr177 mutant into Prdx6 null cells. Thr177 is the unique site of protein phosphorylation when recombinant protein is treated in vitro with MAPKs (ERK, p38) (8Wu Y. Feinstein S.I. Manevich Y. Chowdhury I. Pak J.H. Kazi A. Dodia C. Speicher D.W. Fisher A.B. Biochem. J. 2009; 419: 669-679Crossref PubMed Scopus (78) Google Scholar). In distinction from transfection with the wild-type construct, transfection with a construct expressing the T177A mutant, which cannot be phosphorylated (8Wu Y. Feinstein S.I. Manevich Y. Chowdhury I. Pak J.H. Kazi A. Dodia C. Speicher D.W. Fisher A.B. Biochem. J. 2009; 419: 669-679Crossref PubMed Scopus (78) Google Scholar), was unable to rescue Ang II activation of NOX2 despite exhibiting basal PLA2 activity that was about 50% of the wild-type activity. Thus, we conclude that Prdx6 phosphorylation is required for NOX2 activation.This study has not investigated which product of PLA2 activity is responsible for NOX2 activation. PLA2 liberates both a free fatty acid and lyso-PC from phosphatidylcholine substrate, and previous studies have provided evidence that either metabolite might be directly or indirectly responsible for initiating the events leading to activation of the NOX2 pathway (30Bostan M. Galatiuc C. Hirt M. Constantin M.C. Brasoveanu L.I. Iordachescu D. J. Cell. Mol. Med. 2003; 7: 57-66Crossref PubMed Scopus (15) Google Scholar, 31Dana R. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 1998; 273: 441-445Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 32Silliman C.C. Elzi D.J. Ambruso D.R. Musters R.J. Hamiel C. Harbeck R.J. Paterson A.J. Bjornsen A.J. Wyman T.H. Kelher M. England K.M. McLaughlin-Malaxecheberria N. Barnett C.C. Aiboshi J. Bannerjee A. J. Leukoc. Biol. 2003; 73: 511-524Crossref PubMed Scopus (87) Google Scholar). Of note, Prdx6 does not show a preference for arachidonate-containing phospholipids so that arachidonic acid would not be preferentially liberated by Prdx6 PLA2 activity (24Akiba S. Dodia C. Chen X. Fisher A.B. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1998; 120: 393-404Crossref PubMed Scopus (55) Google Scholar). Thus, the specific product of PLA2 activity that mediates NOX2 activation remains unresolved.Previous studies have provided evidence that PLA2 activity is important for NOX2 activation in PMN and possibly endothelial and other cell types (30Bostan M. Galatiuc C. Hirt M. Constantin M.C. Brasoveanu L.I. Iordachescu D. J. Cell. Mol. Med. 2003; 7: 57-66Crossref PubMed Scopus (15) Google Scholar, 33Rao G.N. Lassègue B. Alexander R.W. Griendling K.K. Biochem. J. 1994; 299: 197-201Crossref PubMed Scopus (95) Google Scholar, 34Dana R. Malech H.L. Levy R. Biochem. J. 1994; 297: 217-223Crossref PubMed Scopus (128) Google Scholar, 35O'Donnell R.W. Johnson D.K. Ziegler L.M. DiMattina A.J. Stone R.I. Holland J.A. Endothelium. 2003; 10: 291-297Crossref PubMed Scopus (29) Google Scholar). However, assessment of the specific PLA2 involved in activation has been hampered by the relative non-specificity of inhibitors used in many of the earlier studies. Although the member of the PLA2 family that might be responsible for NOX2 activation was not identified precisely, suspicion has focused on cytoplasmic PLA2 (cPLA2) (36Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell. Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar, 37Zhao X. Bey E.A. Wientjes F.B. Cathcart M.K. J. Biol. Chem. 2002; 277: 25385-25392Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). cPLA2 is ubiquitously expressed in cells and demonstrates a preference for arachidonate-containing phospholipids as the substrate; release of arachidonic acid can give rise to the broad spectrum of eicosanoid metabolites. cPLA2 can be phosphorylated by MAPKs and translocates to the cell membrane, similar to the pathway we have described herein for Prdx6 (36Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell. Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar, 38Hazan I. Dana R. Granot Y. Levy R. Biochem. J. 1997; 326: 867-876Crossref PubMed Scopus (77) Google Scholar). Phosphorylation of cPLA2 and release of arachidonic acid were demonstrated in vascular smooth muscle cells stimulated with Ang II, although this was not directly linked to NOX2 activation (33Rao G.N. Lassègue B. Alexander R.W. Griendling K.K. Biochem. J. 1994; 299: 197-201Crossref PubMed Scopus (95) Google Scholar). ROS production by endothelial cells activated with low density lipoprotein was inhibited by the presence of the cPLA2 inhibitor, AACOCF3 (35O'Donnell R.W. Johnson D.K. Ziegler L.M. DiMattina A.J. Stone R.I. Holland J.A. Endothelium. 2003; 10: 291-297Crossref PubMed Scopus (29) Google Scholar). The possible role for cPLA2 in NOX2 activation in PMN and monocytes has been inferred by its presence on the plasma membrane following stimulation and by decreased ROS production when expression of cPLA2 was “knocked down” with antisense oligodeoxyribonucleotides (31Dana R. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 1998; 273: 441-445Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 36Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell. Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar, 37Zhao X. Bey E.A. Wientjes F.B. Cathcart M.K. J. Biol. Chem. 2002; 277: 25385-25392Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). On the other hand, the effects of AACOCF3 on ROS production with PMN stimulated by PMA were unimpressive (30Bostan M. Galatiuc C. Hirt M. Constantin M.C. Brasoveanu L.I. Iordachescu D. J. Cell. Mol. Med. 2003; 7: 57-66Crossref PubMed Scopus (15) Google Scholar). Further, deletion of cPLA2 by gene targeting did not alter NOX2 activation in PMN or peritoneal macrophages treated with various agonists, including opsonized zymosan, PMA, and B.B. A. N. L. E. D.W. C. V. E. D. M. J. S. M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). latter indicate that a role for cPLA2 in activation of NOX2 in is In the present study, AACOCF3 did not the ROS response to Ang II or PMA in PMVEC. Thus, cPLA2 is not involved in Ang or NOX2 activation in endothelial cells, although a role for cPLA2 in response to other agonists cannot be this study has shown that Prdx6 is required for activation of NOX2 in PMVEC stimulated with Ang II or PMA and alveolar macrophages stimulated with A or of PMVEC to agonists in phosphorylation of Prdx6, its translocation to the cell membrane, and activation of phospholipase A2 activity that leads to translocation of NOX2 cytoplasmic components and activation of the enzyme complex. IntroductionReactive oxygen species (ROS) 2The abbreviations used are: ROSreactive oxygen speciesAACOCF3arachidonyltrifluoromethyl ketoneAng IIangiotensin IIBELbromoenol lactoneDCFdichlorofluorosceinH2DCFdihydrodichlorofluoresceinDPPC1-palmitoyl, 2-palmitoyl, sn-glycero-3-phosphocholinefMLFN-formyl-Met-Leu-Phe peptideHEhydroethidiumMJ331-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanolNOXNADPH oxidasepBPBp-bromophenacyl bromidePLA2phospholipase A2cPLA2cytoplasmic PLA2PMAphorbol 12-myristate 13-acetatePMVECpulmonary microvascular endothelial cell(s)PrdxperoxiredoxinH2DCFdihydrodichlorofluoresceinPECAMplatelet endothelial cell adhesion molecule 1PCphosphatidylcholineAbantibodyPMNpolymorphonuclear leukocyte(s). comprising O2⨪, H2O2, ·OH, and others, are now regarded as important signaling molecules in biological systems. For example, H2O2 modulates cell growth, apoptosis, and various other endothelial cell functions (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 2Lassegue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2010; 30: 653-661Crossref PubMed Scopus (480) Google Scholar). NADPH oxidases (NOXs) are a family of seven widely distributed enzymes that enzymatically generate O2⨪ and, through dismutation, H2O2 (3Lambeth J.D. Curr. Opin. Hematol. 2002; 9: 11-17Crossref PubMed Scopus (230) Google Scholar). Of these, NOX2 is the canonical NOX responsible for O2⨪ generation by the respiratory burst in neutrophils. NOX2 is also found in other cell types and is a major source of ROS in endothelial cells (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 2Lassegue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2010; 30: 653-661Crossref PubMed Scopus (480) Google Scholar). In the unstimulated state, NOX2 is quiescent, with the intrinsic membrane subunits (cytochrome b558 comprising gp91phox and p22phox) and cytosolic subunits (Rac1, p47phox, p67phox, and p40phox) confined to their respective compartments (4Pendyala S. Usatyuk P.V. Gorshkova I.A. Garcia J.G. Natarajan V. Antioxid. Redox Signal. 2009; 11: 841-860Crossref PubMed Scopus (94) Google Scholar). Activation of the enzyme complex by stimuli such as angiotensin II (Ang II), phorbol esters, or thrombin leads to translocation of the cytosolic components to the plasma membrane, resulting in assembly of the oxidase complex. Cytochrome b558 with its heme, NADPH, and flavin binding sites functions to transfer electrons from NADPH to oxygen, thereby generating O2⨪ in the extracellular space. Dismutation of O2⨪, either catalyzed or spontaneous, generates H2O2, which is regarded as the primary ROS signaling molecule in endothelium (1Frey R.S. Ushio-Fukai M. Malik A.B. Antioxid. Redox Signal. 2009; 11: 791-810Crossref PubMed Scopus (313) Google Scholar, 2Lassegue B. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2010; 30: 653-661Crossref PubMed Scopus (480) Google Scholar).Peroxiredoxins are a family of antioxidants that express peroxidase activity and catalyze the removal of H2O2 and other hydroperoxides (5Rhee S.G. Chae H.Z. Kim K. Free Radic. Biol. Med. 2005; 38: 1543-1552Crossref PubMed Scopus (1132) Google Scholar). Of the six mammalian members of the family, peroxiredoxin 6 (Prdx6) is the only peroxiredoxin with both phospholipase A2 (PLA2) and peroxidase activities (6Fisher A.B. Antioxid. Redox Signal. 2011; (in press)Google Scholar). The PLA2 activity catalyzes the hydrolysis of the acyl group at the sn-2 position of glycerophospholipids, with special affinity for phosphatidylcholine, to produce free fatty acids and a lysophospholipid; the peroxidase activity uses glutathione as the co-factor for reduction of hydroperoxides, including phospholipid hydroperoxides (6Fisher A.B. Antioxid. Redox Signal. 2011; (in press)Google Scholar). In a previous study, Prdx6 (therein called p29) was found to participate in the activation of NOX2 in human neutrophils (7Leavey P.J. Gonzalez-Aller C. Thurman G. Kleinberg M. Rinckel L. Ambruso D.W. Freeman S. Kuypers F.A. Ambruso D.R. J. Biol. Chem. 2002; 277: 45181-45187Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, the mechanism by which Prdx6 might activate NOX2-mediated ROS generation was not determined. Here, we investigated the role of Prdx6 in NOX2 activation in alveolar macrophages and in the endothelium in situ and in vitro. We found that phosphorylation of Prdx6 through MAPK activity causes its translocation to the cell membrane, and the resultant PLA2 activity leads to assembly of the NOX2 enzyme complex and ROS generation.