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      Inhibition of the phospholipase A 2 activity of peroxiredoxin 6 prevents lung damage with exposure to hyperoxia

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          Abstract

          Lung injury associated with hyperoxia reflects in part the secondary effects of pulmonary inflammation and the associated production of reactive oxygen species due to activation of NADPH oxidase, type 2 (NOX2). Activation of NOX2 requires the phospholipase A 2 (PLA 2) activity of peroxiredoxin 6 (Prdx6). Therefore, we evaluated whether blocking Prdx6 PLA 2 activity using the inhibitor MJ33 would be protective in a mouse model of acute lung injury resulting from hyperoxic exposure. Mice were treated with an intraperitoneal injection of MJ33 (2.5 nmol/g body weight) at the start of exposure (zero time) and at 48 h during continuous exposure to 100% O 2 for 80 h. Treatment with MJ33 reduced the number of neutrophils and the protein content in the fluid obtained by bronchoalveolar lavage, inhibited the increase in lipid peroxidation products in lung tissue, decreased the number of apoptotic cells in the lung, and decreased the perivascular edema associated with the 80 h exposure to hyperoxia. Thus, blocking Prdx6 PLA 2 activity by MJ33 significantly protected lungs against damage from hyperoxia, presumably by preventing the activation of NOX2 and the amplification of lung injury associated with inflammation. These findings demonstrate that MJ33, a potent inhibitor of Prdx6 PLA 2 activity, can protect mouse lungs against the manifestations of acute lung injury due to oxidative stress.

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          Highlights

          • •

            Exposure of mice to sublethal O 2 (100% O 2 for 80 h) resulted in acute lung injury (ALI).

          • •

            Manifestations of ALI included increased PMN, lung edema, tissue lipid peroxidation, and cellular apoptosis.

          • •

            MJ33, an inhibitor of lung acidic, Ca 2+ -independent phospholipase A 2 (aiPLA 2) activity, markedly decreased manifestations of tissue injury with hyperoxia.

          • •

            The proposed mechanism for lung protection is the prevention of NADPH oxidase activation associated with lung inflammation.

          • •

            Possible additional roles for MJ33 have not yet been studied.

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          1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities.

          Michael B. Fisher, Justin Feinstein, Junjun Chen … (2000)
          This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.
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            Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells.

            J. Lambeth, Irina Gorshkova, Peter V. Usatyuk … (2009)
            In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22(phox) compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22(phox), but not Nox2 or p47(phox), are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2(-/-) ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs.
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              Toll-like receptor 4 deficiency causes pulmonary emphysema.

              Jae K. Lee, Lauren Cohn, Peiying Shan … (2006)
              TLRs have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unknown. Given their direct interface with the external environment, TLRs in the lungs are prime candidates to respond to air constituents, namely particulates and oxygen. The mechanism whereby the lung maintains structural integrity in the face of constant ambient exposures is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, usually in the later decades of life and after years of insult, most commonly cigarette smoke. Here we show Tlr4(-/-) mice exhibited emphysema as they aged. Adoptive transfer experiments revealed that TLR4 expression in lung structural cells was required for maintaining normal lung architecture. TLR4 deficiency led to the upregulation of what we believe to be a novel NADPH oxidase (Nox), Nox3, in lungs and endothelial cells, resulting in increased oxidant generation and elastolytic activity. Treatment of Tlr4(-/- )mice or endothelial cells with chemical NADPH inhibitors or Nox3 siRNA reversed the observed phenotype. Our data identify a role for TLR4 in maintaining constitutive lung integrity by modulating oxidant generation and provide insights into the development of emphysema.
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                Author and article information

                Contributors
                Aron B. Fisher
                Journal
                Journal ID (nlm-ta): Redox Biol
                Journal ID (iso-abbrev): Redox Biol
                Title: Redox Biology
                Publisher: Elsevier
                ISSN (Electronic): 2213-2317
                Publication date PMC-release: 16 January 2015
                Publication date Collection: April 2015
                Publication date (Electronic): 16 January 2015
                Volume: 4
                Pages: 321-327
                Affiliations
                [0005]Institute for Environmental Medicine and Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
                Author notes
                [* ]Correspondence to: Institute for Environmental Medicine, University of Pennsylvania, 3620 Hamilton Walk, 1 John Morgan Building, Philadelphia, PA 19104, USA. abf@ 123456mail.med.upenn.edu dawnw@ 123456mail.med.upenn.edu
                Article
                Publisher ID: S2213-2317(15)00012-9
                DOI: 10.1016/j.redox.2015.01.011
                PMC ID: 4803794
                PubMed ID: 25637741
                SO-VID: c10fed16-6231-40b5-b98e-1ec968435d7a
                Copyright © © 2015 The Authors
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 24 December 2014
                Date revision received : 8 January 2015
                Date accepted : 10 January 2015
                Categories
                Subject: Research Paper

                Keywords: acute lung injury,oxidative stress,lipid peroxidation,mj33,inflammation,balf, bronchoalveolar lavage fluid,fox, ferrous oxidation-xylenol orange,ip, intraperitoneal,looh, lipid hydroperoxides,mj33, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol,nox2, nadph oxidase, type 2,pla2, phospholipase a2,ros, reactive oxygen species,tbars, thiobarbituric acid reactive substances,tunel, terminal transferase dutp nick-end-labeling
                Data availability:
                Keywords: acute lung injury, oxidative stress, lipid peroxidation, mj33, inflammation, balf, bronchoalveolar lavage fluid, fox, ferrous oxidation-xylenol orange, ip, intraperitoneal, looh, lipid hydroperoxides, mj33, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol, nox2, nadph oxidase, type 2, pla2, phospholipase a2, ros, reactive oxygen species, tbars, thiobarbituric acid reactive substances, tunel, terminal transferase dutp nick-end-labeling

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