Inhibition of the phospholipase A2 activity of peroxiredoxin 6 prevents lung damage with exposure to hyperoxia

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.

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.

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.