Deficiency of Antioxidative Paraoxonase 2 (Pon2) Leads to Increased Number of Phenotypic LT-HSCs and Disturbed Erythropoiesis.

BACKGROUND: Long-term hematopoietic stem cells (LT-HSCs) reside in bone marrow niches with tightly controlled reactive oxygen species (ROS) levels. ROS increase results into LT-HSC differentiation and stem cell exhaustion. Paraoxonase 2 (PON2) has been shown to be important for ROS control. OBJECTIVES: We investigate the effects of inactivation of the PON2 gene on hematopoietic cell differentiation and activity. METHODS AND RESULTS: In young mice with inactivated Pon2 gene (Pon2 -/-, <3 months), we observed an increase of LT-HSCs and a reduced frequency of progenitor cells. In competitive transplantations, young Pon2-/- BM outcompeted WT BM at early time points. ROS levels were significantly increased in Pon2-/- whole BM, but not in Pon2-/- LT-HSCs. In more differentiated stages of hematopoiesis, Pon2 deficiency led to a misbalanced erythropoiesis both in physiologic and stress conditions. In older mice (>9 months), Pon2 depletion caused an increase in LT-HSCs as well as increased levels of granulocyte/macrophage progenitors (GMPs) and myeloid skewing, indicating a premature aging phenotype. No significant changes in ROS levels in old Pon2-/- LT- and short-term (ST-) HSCs were observed, but a significant reduction of spontaneous apoptotic cell death was measured. RNA-seq analysis in Pon2 -/- LT-HSCs identified overrepresentation of genes involved in the C-X-C chemokine receptor type 4 (Cxcr4) signaling, suggesting compensatory mechanisms to overcome ROS-mediated accelerated aging in hematopoietic progenitor cells. CONCLUSIONS: In summary, our current data indicate that PON2 is involved in the regulation of HSC functions.

Paraoxonase-2 regulates coagulation activation through endothelial tissue factor.

Oxidative stress and inflammation of the vessel wall contribute to prothrombotic states. The antioxidative protein paraoxonase-2 (PON2) shows reduced expression in human atherosclerotic plaques and endothelial cells in particular. Supporting a direct role for PON2 in cardiovascular diseases, Pon2 deficiency in mice promotes atherogenesis through incompletely understood mechanisms. Here, we show that deregulated redox regulation in Pon2 deficiency causes vascular inflammation and abnormalities in blood coagulation. In unchallenged Pon2-/- mice, we find increased oxidative stress and endothelial dysfunction. Bone marrow transplantation experiments and studies with endothelial cells provide evidence that increased inflammation, indicated by circulating interleukin-6 levels, originates from Pon2 deficiency in the vasculature. Isolated endothelial cells from Pon2-/- mice display increased tissue factor (TF) activity in vitro. Coagulation times were shortened and platelet procoagulant activity increased in Pon2-/- mice relative to wild-type controls. Coagulation abnormalities of Pon2-/- mice were normalized by anti-TF treatment, demonstrating directly that TF increases coagulation. PON2 reexpression in endothelial cells by conditional reversal of the knockout Pon2 cassette, restoration in the vessel wall using bone marrow chimeras, or treatment with the antioxidant N-acetylcysteine normalized the procoagulant state. These experiments delineate a PON2 redox-dependent mechanism that regulates endothelial cell TF activity and prevents systemic coagulation activation and inflammation.

Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response.

Human neutrophil elastase impacts on atherosclerotic plaque stability by inducing apoptosis in endothelial cells. Our aim was to investigate the proapoptotic mechanism of elastase on endothelial cells and to evaluate the presence of elastase in human plaque material. Human endothelial cells were treated with purified human neutrophil elastase. Apoptosis was assayed by capsase-3/7 activation, TUNEL, and sub-G1 assay. Activation of unfolded protein response (UPR) effector molecules binding Ig protein, soluble X-binding protein-1, protein kinase RNA-like ER kinase (PERK), and C/EBP-homologous protein (CHOP) was analyzed by RT-PCR, immunocytochemistry, and Western blot. Genetic silencing of CHOP was achieved by small interfering RNA. Elastase induces autophagic-apoptotic forms of endothelial cell death in a time- and dose-dependent manner, in conjunction with a significant increase in phosphorylation/expression of the canonical UPR-activation markers PERK and CHOP. By using CHOP knockdown, we identified CHOP as a key mediator of elastase-induced endothelial cell death. Immunohistochemical analysis of human rupture-prone plaque specimens confirmed the presence of elastase and colocalization with apoptosis. We have demonstrated for the first time that the PERK-CHOP branch of the UPR is causally involved in elastase-induced apoptosis of endothelial cells. Ex vivo analysis of human rupture-prone plaques confirmed the presence of elastase and its colocalization with markers of apoptosis. This novel role of elastase underlines the potential of combined targeting of elastase and endoplasmic reticulum stress in the prevention of plaque progression and cardiovascular events.-Grechowa, I., Horke, S., Wallrath, A., Vahl, C.-F., Dorweiler, B. Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response.

From thrombosis to fibrosis in chronic thromboembolic pulmonary hypertension.

The pathomechanisms underlying the development of thrombofibrotic pulmonary artery occlusions in Chronic Thromboembolic Pulmonary Hypertension (CTEPH) are largely unknown. The aim of this study was to allocate distinct cellular processes playing a role in thrombus resolution, such as inflammation, hypoxia, proliferation, apoptosis and angiogenesis, to different stages of thrombofibrotic remodelling. A total of 182 pulmonary endarterectomy (PEA) specimens were collected from 31 CTEPH patients. To facilitate co-localisation, Tissue MicroArrays were prepared and processed for (immuno)-histochemistry and confocal fluorescence microscopy. Murine venous thrombus formation and resolution was examined after inferior vena cava ligation. PEA tissues exhibited five morphologically distinct regions predominantly consisting of either fibrin-, erythrocyte- or extracellular matrix-rich thrombus, myofibroblasts, vessels or fibrotic tissue, and were found to resemble chronological stages of thrombus resolution in mice. Cellularity was highest in vessel-rich regions, and numerous cells were strongly positive for HIF1α or HIF2α as well as markers of activated VEGF signalling, including endothelial nitric oxide synthase. On the other hand, negative regulators of angiogenic growth factor signalling and reactive oxygen species were also highly expressed. Immune cells, primarily macrophages of the M2 subtype and CD117 haematopoietic progenitors were detected and highest in vascularised regions. Our findings demonstrate the simultaneous presence of different stages of thrombus organisation and suggest that hypoxia-induced endothelial, mesenchymal and immune cell activation may contribute to thrombofibrosis in CTEPH. This systematic histological characterisation of the material obstructing pulmonary vessels in CTEPH may provide a valuable basis for further studies aimed at determining causal factors underlying this disease.

The anti-apoptotic PON2 protein is Wnt/ß-catenin-regulated and correlates with radiotherapy resistance in OSCC patients.

Aberrant Wnt signaling and control of anti-apoptotic mechanisms are pivotal features in different types of cancer to undergo cell death programs. The intracellular human enzyme Paraoxonase-2 (PON2) is known to have anti-apoptotic properties in leukemia and oral squamous cell cancer (OSCC) cells. However, the distinct regulating pathways are poorly understood. First, we present a so far unknown regulation of PON2 protein expression through the Wnt/GSK3ß/ß-catenin pathway in leukemia and OSCC cells. This was confirmed via in silico analysis, promoter reporter studies and treatment of multiple cell lines (K562, SCC-4, PCI-13) with different Wnt ligands/inhibitors in vitro. Ex vivo analysis of OSCC patients revealed a correlation between PON2 and ß-catenin expression in tumor tissue. Higher PON2 expression in OSCC is associated with relapse independently of treatment (e.g. surgery/radio-/chemotherapy). These results emphasize the clinical impact of the newly described regulation of PON2 through Wnt/GSK3ß/ß-catenin. More importantly, the study revealed the fundamental finding of an overall Wnt/GSK3ß/ß-catenin dependent regulation of PON2 in different cancers, which was confirmed by systematic and multimethodological approaches. Thus, the herein presented mechanistic insight contributes to a better understanding of tumor specific escape from cell death strategies and suggests PON2 as a new potential biomarker for therapy resistance or as a prognostic tumor marker.

Uncoupling of Endothelial Nitric Oxide Synthase in Perivascular Adipose Tissue of Diet-Induced Obese Mice.

OBJECTIVE: The present study was conducted to investigate the contribution of perivascular adipose tissue (PVAT) to vascular dysfunction in a mouse model of diet-induced obesity. APPROACH AND RESULTS: Obesity was induced in male C57BL/6J mice with a high-fat diet for 20 weeks, and vascular function was studied with myograph. In PVAT-free aortas isolated from obese mice, the endothelium-dependent, nitric oxide-mediated vasodilator response to acetylcholine remained normal. In contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when PVAT was left in place. Adipocytes in PVAT were clearly positive in endothelial nitric oxide synthase (eNOS) staining, and PVAT nitric oxide production was significantly reduced in obese mice. High-fat diet had no effect on eNOS expression but led to eNOS uncoupling, evidenced by diminished superoxide production in PVAT after eNOS inhibition. As mechanisms for eNOS uncoupling, arginase induction and l-arginine deficiency were observed in PVAT. Obesity-induced vascular dysfunction could be reversed by ex vivo l-arginine treatment and arginase inhibition. CONCLUSIONS: Diet-induced obesity leads to l-arginine deficiency and eNOS uncoupling in PVAT. The combination therapy with l-arginine and arginase inhibitors may represent a novel therapeutic strategy for obesity-induced vascular disease.

Novel Paraoxonase 2-Dependent Mechanism Mediating the Biological Effects of the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxo-Dodecanoyl)-L-Homoserine Lactone.

Pseudomonas aeruginosa produces N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12), a crucial signaling molecule that elicits diverse biological responses in host cells thought to subvert immune defenses. The mechanism mediating many of these responses remains unknown. The intracellular lactonase paraoxonase 2 (PON2) hydrolyzes and inactivates 3OC12 and is therefore considered a component of host cells that attenuates 3OC12-mediated responses. Here, we demonstrate in cell lines and in primary human bronchial epithelial cells that 3OC12 is rapidly hydrolyzed intracellularly by PON2 to 3OC12 acid, which becomes trapped and accumulates within the cells. Subcellularly, 3OC12 acid accumulated within the mitochondria, a compartment where PON2 is localized. Treatment with 3OC12 caused a rapid PON2-dependent cytosolic and mitochondrial pH decrease, calcium release, and phosphorylation of stress signaling kinases. The results indicate a novel, PON2-dependent intracellular acidification mechanism by which 3OC12 can mediate its biological effects. Thus, PON2 is a central regulator of host cell responses to 3OC12, acting to decrease the availability of 3OC12 for receptor-mediated effects and acting to promote effects, such as calcium release and stress signaling, via intracellular acidification.

Paraoxonase-2 (PON2) protects oral squamous cell cancer cells against irradiation-induced apoptosis.

PURPOSE: Patients with oral squamous cell carcinomas (OSCC) often receive radiotherapy to preferentially induce apoptosis of cancer cells through generation of overwhelming DNA damage. This is amplified by generation of reactive oxygen species (ROS), thereby causing oxidative stress and cell death. However, tumors resist through different mechanisms, including upregulation of anti-apoptotic factors and enhanced ROS resistance. We recently reported that the antioxidative enzyme PON2 significantly enhances cellular stress resistance by attenuating mitochondrial ROS-mediated apoptosis. Further, PON2 is often upregulated in cancer. This prompted us to investigate its yet unknown role in the protection of OSCC against irradiation-induced cell death. METHODS: PON2 expression was determined after 7 Gy singular irradiation in four OSCC cell lines (PCI-13, PCI-52, SCC-4, SCC-68) accompanied by the detection of caspase 3/7 activity. A direct role of PON2 was tested by siRNA-mediated knockdown. In vivo PON2 expression was tested in five patients with oral carcinoma and compared with healthy mucosa for the evaluation of clinical significance. RESULTS: PON2 is variably expressed in OSCC in vitro and in vivo. Compared with the other cell lines, SCC-4 cells showed twofold more basal PON2 (p ≤ 0.05) and the lowest caspase 3/7 activity after singular irradiation (p ≤ 0.05). Contrarily, irradiation led to 1.2-fold induction of PON2 in PCI-13 with no effect on SCC-4 (≤0.05), suggesting that PON2 levels reflect the cells' irradiation sensitivity. In agreement, PON2 knockdown resulted in significant higher apoptosis rates (p ≤ 0.05). CONCLUSION: Our findings give first evidence that upregulation of PON2 may protect OSCC against irradiation-induced apoptosis.

Vascular oxidative stress, nitric oxide and atherosclerosis.

In the vascular wall, reactive oxygen species (ROS) are produced by several enzyme systems including NADPH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide synthase (eNOS) and the mitochondrial electron transport chain. On the other hand, the vasculature is protected by antioxidant enzyme systems, including superoxide dismutases, catalase, glutathione peroxidases and paraoxonases, which detoxify ROS. Cardiovascular risk factors such as hypercholesterolemia, hypertension, and diabetes mellitus enhance ROS generation, resulting in oxidative stress. This leads to oxidative modification of lipoproteins and phospholipids, mechanisms that contribute to atherogenesis. In addition, oxidation of tetrahydrobiopterin may cause eNOS uncoupling and thus potentiation of oxidative stress and reduction of eNOS-derived NO, which is a protective principle in the vasculature. This review summarizes the latest advances in the role of ROS-producing enzymes, antioxidative enzymes as well as NO synthases in the initiation and development of atherosclerosis.

Breaking the chain at the membrane: paraoxonase 2 counteracts lipid peroxidation at the plasma membrane.

Lipid peroxidation through electrophilic molecules of extracellular origin is involved in the pathogenesis of many inflammatory conditions. To counteract free radical actions at the plasma membrane, cells host a variety of antioxidative enzymes. Here we analyzed localization, membrane topology, and trafficking of PON2 a member of the paraoxonase family of 3 enzymatically active proteins (PON1-3) found to have antiatherogenic properties. Immunohistochemistry localized PON2 to the villous tip of human intestinal epithelial cells. Employing membrane preparations, surface biotinylation experiments, and mutational analyses in HEK 293T and HeLa cells, we demonstrate that PON2 is a type II transmembrane protein. A hydrophobic stretch in the N terminus was identified as single transmembrane domain of PON2. The enzymatically active domain faced the extracellular compartment, where it suppressed lipid peroxidation (P<0.05) and regulated the glucosylceramide content, as demonstrated by mass spectrometry (P<0.05). PON2 translocation to the plasma membrane was dependent on intracellular calcium responses and could be induced to >10-fold as compared to baseline (P=0.0001) by oxidative stress. Taken together, these data identify the paraoxonase protein PON2 as a type II transmembrane protein, which is dynamically translocated to the plasma membrane in response to oxidative stress to counteract lipid peroxidation.

Oxidative stress in vascular disease and its pharmacological prevention.

Cardiovascular risk factors lead to enhanced production of reactive oxygen species (ROS) generated by NADPH oxidase, xanthine oxidase (XO), the mitochondrial electron-transport chain (ETC), and dysfunctional endothelial nitric oxide synthase (eNOS). When the capacity of antioxidant defense systems [e.g., superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), heme oxygenase (HO), paraoxonase (PON)] is exceeded, this results in oxidative stress, which can promote atherogenesis. Therefore, pharmacological means to prevent oxidative stress are of major therapeutic interest. Some established drugs and novel therapeutic approaches can prevent oxidative stress and, presumably, vascular disease. These include angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor type 1 (AT1 receptor) blockers (ARBs), statins, nebivolol, pentaerithrityl tetranitrate (PETN), resveratrol, and mitochondria-targeted antioxidants. Molecular mechanisms involved in the induction of oxidative stress under pathological conditions as well as pharmacological approaches (and their molecular mechanisms) are summarized in this review.

Transcriptional regulation of Nox4 by histone deacetylases in human endothelial cells.

Nox4 is a member of the NADPH oxidase family, which represents a major source of reactive oxygen species (ROS) in the vascular wall. Nox4-mediated ROS production mainly depends on the expression levels of the enzyme. The present study was aimed to investigate the mechanisms of Nox4 transcription regulation by histone deacetylases (HDAC). In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, treatment with the pan-HDAC inhibitor scriptaid led to a marked decrease in Nox4 mRNA expression. A similar down-regulation of Nox4 mRNA expression was observed by siRNA-mediated knockdown of HDAC3. HDAC inhibition in endothelial cells was associated with enhanced histone acetylation, increased chromatin accessibility in the human Nox4 promoter region, with no significant changes in DNA methylation. In addition, we provided evidence that c-Jun played an important role in controlling Nox4 transcription. Knockdown of c-Jun with siRNA led to a down-regulation of Nox4 mRNA expression. In response to scriptaid treatment, the binding of c-Jun to the Nox4 promoter region was reduced despite the open chromatin structure. In parallel, the binding of RNA polymerase IIa to the Nox4 promoter was significantly inhibited as well, which may explain the reduction in Nox4 transcription. In conclusion, HDAC inhibition decreases Nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the Nox4 promoter, most likely because of a hyperacetylation-mediated steric inhibition.

The anti-inflammatory fungal compound (S)-curvularin reduces proinflammatory gene expression in an in vivo model of rheumatoid arthritis.

In previous studies, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as an anti-inflammatory agent using a screening system detecting inhibitors of the Janus kinase/signal transducer and activator of transcription pathway. The objective of the present study was to investigate whether SC is able to decrease proinflammatory gene expression in an in vivo model of a chronic inflammatory disease. Therefore, the effects of SC and dexamethasone were compared in the model of collagen-induced arthritis (CIA) in mice. Total genomic microarray analyses were performed to identify SC target genes. In addition, in human C28/I2 chondrocytes and MonoMac6 monocytes, the effect of SC on proinflammatory gene expression was tested at the mRNA and protein level. In the CIA model, SC markedly reduced the expression of a number of proinflammatory cytokines and chemokines involved in the pathogenesis of CIA as well as human rheumatoid arthritis (RA). In almost all cases, the effects of SC were comparable with those of dexamethasone. In microarray analyses, we identified additional new therapeutic targets of SC. Some of them, such as S100A8, myeloperoxidase, or cathelicidin, an antimicrobial peptide, are known to be implicated in pathophysiological processes in RA. Similar anti-inflammatory effects of SC were also observed in human C28/I2 chondrocyte cells, which are resistant to glucocorticoid treatment. These data indicate that SC and glucocorticoid effects are mediated via independent signal transduction pathways. In summary, we demonstrate that SC is a new effective anti-inflammatory compound that may serve as a lead compound for the development of new drugs for the therapy of chronic inflammatory diseases.

Protectors or Traitors: The Roles of PON2 and PON3 in Atherosclerosis and Cancer.

Cancer and atherosclerosis are major causes of death in western societies. Deregulated cell death is common to both diseases, with significant contribution of inflammatory processes and oxidative stress. These two form a vicious cycle and regulate cell death pathways in either direction. This raises interest in antioxidative systems. The human enzymes paraoxonase-2 (PON2) and PON3 are intracellular enzymes with established antioxidative effects and protective functions against atherosclerosis. Underlying molecular mechanisms, however, remained elusive until recently. Novel findings revealed that both enzymes locate to mitochondrial membranes where they interact with coenzyme Q10 and diminish oxidative stress. As a result, ROS-triggered mitochondrial apoptosis and cell death are reduced. From a cardiovascular standpoint, this is beneficial given that enhanced loss of vascular cells and macrophage death forms the basis for atherosclerotic plaque development. However, the same function has now been shown to raise chemotherapeutic resistance in several cancer cells. Intriguingly, PON2 as well as PON3 are frequently found upregulated in tumor samples. Here we review studies reporting PON2/PON3 deregulations in cancer, summarize most recent findings on their anti-oxidative and antiapoptotic mechanisms, and discuss how this could be used in putative future therapies to target atherosclerosis and cancer.

Paraoxonases-2 and -3 Are Important Defense Enzymes against Pseudomonas aeruginosa Virulence Factors due to Their Anti-Oxidative and Anti-Inflammatory Properties.

The pathogen Pseudomonas aeruginosa causes serious damage in immunocompromised patients by secretion of various virulence factors, among them the quorum sensing N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) and the redox-active pyocyanin (PCN). Paraoxonase-2 (PON2) may protect against P. aeruginosa infections, as it efficiently inactivates 3OC12 and diminishes PCN-induced oxidative stress. This defense could be circumvented because 3OC12 mediates intracellular Ca(2+)-rise in host cells, which causes rapid inactivation and degradation of PON2. Importantly, we recently found that the PON2 paralogue PON3 prevents mitochondrial radical formation. Here we investigated its role as additional potential defense mechanism against P. aeruginosa infections. Our studies demonstrate that PON3 diminished PCN-induced oxidative stress. Moreover, it showed clear anti-inflammatory potential by protecting against NF-κB activation and IL-8 release. The latter similarly applied to PON2. Furthermore, we observed a Ca(2+)-mediated inactivation and degradation of PON3, again in accordance with previous findings for PON2. Our results suggest that the anti-oxidative and anti-inflammatory functions of PON2 and PON3 are an important part of our innate defense system against P. aeruginosa infections. Furthermore, we conclude that P. aeruginosa circumvents PON3 protection by the same pathway as for PON2. This may help identifying underlying mechanisms in order to sustain the protection afforded by these enzymes.

Hyperglycemia and oxidative stress in cultured endothelial cells--a comparison of primary endothelial cells with an immortalized endothelial cell line.

Diabetes mellitus is a major risk factor for the development of cardiovascular disease and oxidative stress plays an important role in this process. Therefore, we investigated the effects of hyperglycemia on the formation of reactive oxygen species (ROS) and nitric oxide/cGMP signaling in two different endothelial cell cultures. Human umbilical vein endothelial cells (HUVEC) and EA.hy 926 cells showed increased oxidative stress and impaired NO-cGMP signaling in response to hyperglycemia. The major difference between the two different cell types was the dramatic decrease in viability in HUVEC whereas EA.hy cells showed rather increased growth under hyperglycemic conditions. Starvation led to an additional substantial decrease in viability and increased superoxide formation in HUVEC. Both endothelial cell types, HUVEC and EA.hy 926, may be used as models for vascular hyperglycemia. However, high growth medium should be used to avoid starvation-induced oxidative stress and cell death.

Assessment of endoplasmic reticulum stress and the unfolded protein response in endothelial cells.

In the vascular wall, the most inner cell layer that separates the blood from organelles is comprised of only a single layer of endothelial cells (ECs). This cell type is fundamental to a large variety of processes, ranging from blood coagulation and interaction with inflammatory cells to cardiovascular diseases such as hypertension, diabetes, and atherosclerosis. Dysfunction of ECs is often causally linked to these processes such that research exploring such events attracted much attention. Damage of ECs and subsequent disruption of the intact endothelial barrier can result not only from oxidative stress, but also from conditions that stress the endoplasmic reticulum (ER) and induce a signaling pathway termed unfolded protein response (UPR). While its primary goal is to alleviate ER stress, the UPR can also induce cell death. Cultured ECs are often used in in vitro approaches to understand various pathophysiological events, but they behave differently from many other cell types such that cell-type-specific procedures are needed. Here, we describe how ER stress can be induced and assessed in cultured ECs and demonstrate their specific responses to classical ER stress conditions.

One enzyme, two functions: PON2 prevents mitochondrial superoxide formation and apoptosis independent from its lactonase activity.

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O(2)(-) dismutase activities and cytochrome c expression unaltered, and it did not oxidize O(2)(-) but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His(114) and His(133) are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys(311), was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.

Paraoxonase 2 is down-regulated by the Pseudomonas aeruginosa quorumsensing signal N-(3-oxododecanoyl)-L-homoserine lactone and attenuates oxidative stress induced by pyocyanin.

Two virulence factors produced by Pseudomonas aeruginosa are pyocyanin and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12). Pyocyanin damages host cells by generating ROS (reactive oxygen species). 3OC12 is a quorum-sensing signalling molecule which regulates bacterial gene expression and modulates host immune responses. PON2 (paraoxonase-2) is an esterase that inactivates 3OC12 and potentially attenuates Ps. aeruginosa virulence. Because increased intracellular Ca2+ initiates the degradation of PON2 mRNA and protein and 3OC12 causes increases in cytosolic Ca2+, we hypothesized that 3OC12 would also down-regulate PON2. 3OC12 and the Ca2+ ionophore A23187 caused a rapid cytosolic Ca2+ influx and down-regulated PON2 mRNA, protein and hydrolytic activity in A549 and EA.hy 926 cells. The decrease in PON2 hydrolytic activity was much more extensive and rapid than decreases in protein, suggesting a rapid post-translational mechanism which blocks PON2's hydrolytic activity. The Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] diminished the ability of 3OC12 to decrease PON2, demonstrating that the effects are mediated by Ca2+. PON2 also has antioxidative properties and we show that it protects cells from pyocyanin-induced oxidative stress. Knockdown of PON2 by transfecting cells with siRNA (small interfering RNA) rendered them more sensitive to, whereas overexpression of PON2 protected cells from, pyocyanin-induced ROS formation. Additionally, 3OC12 potentiated pyocyanin-induced ROS formation, presumably by inactivating PON2. These findings support a key role for PON2 in the defence against Ps. aeruginosa virulence, but also reveal a mechanism by which the bacterium may subvert the protection afforded by PON2.