Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone.

Pseudomonas aeruginosa is an opportunistic bacterium which causes serious infections in immunocompromised and cystic fibrosis patients (10). As with many gram-negative bacteria, P. aeruginosa produces acyl-homoserine lactone (AHL) quorum-sensing (QS) signaling molecules termed autoinducers which allow the single-celled organisms to coordinate their actions (36). N-(3-Oxododecanoyl)-l-homoserine lactone (3OC12-HSL) is a key autoinducer synthesized by P. aeruginosa which regulates the expression of extracellular virulence factors and biofilm formation (5, 36). Rats and mice experimentally infected with P. aeruginosa mutants deficient in the ability to produce or respond to 3OC12-HSL exhibited…

Protective effect of paraoxonase-2 against endoplasmic reticulum stress-induced apoptosis is lost upon disturbance of calcium homoeostasis

PON2 (paraoxonase-2) is a ubiquitously expressed antioxidative protein which is largely found in the ER (endoplasmic reticulum). Addressing the cytoprotective functions of PON2, we observed that PON2 overexpression provided significant resistance to ER-stress-induced caspase 3 activation when the ER stress was induced by interference with protein modification (by tunicamycin or dithiothreitol), but not when ER stress was induced by disturbance of Ca2+ homoeostasis (by thapsigargin or A23187). When analysing the underlying molecular events, we found an activation of the PON2 promoter in response to all tested ER-stress-inducing stimuli. However, only tunicamycin and dithiothreitol resulted i…

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

ABSTRACT 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 acc…

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 sign…

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, vascula…

Differential roles of PKCα and PKCɛ in controlling the gene expression of Nox4 in human endothelial cells

NADPH oxidases are major sources of superoxide in the vascular wall. This study investigates the role of protein kinase C (PKC) in regulating gene expression of NADPH oxidases. Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 endothelial cells with phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate led to a PKC-dependent biphasic expression of the gp91phox homolog Nox4. A downregulation of Nox4 was observed at 6 h and an upregulation at 48 h after phorbol ester treatment. The early Nox4 downregulation was associated with a reduced superoxide production, whereas the late Nox4 upregulation was accompanied by a clear enhancement of superoxi…

Reciprocal regulation of endothelial nitric-oxide synthase and NADPH oxidase by betulinic acid in human endothelial cells.

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is a protective principle in the vasculature. Many cardiovascular diseases are associated with reduced NO bioactivity and eNOS uncoupling due to oxidative stress. Compounds that reverse eNOS uncoupling and increase eNOS expression are of therapeutic interest. Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, an extract of ZSS increased eNOS promoter activity, eNOS mRNA and protein expression, and NO production in a concentration- and time-dependent manner. Maj…

One Enzyme, Two Functions

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 specif…

Paraoxonase-2 alters hematopoietic stem cell differentiation through redox signalling

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 CHO…

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, oxi…

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 proi…

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…

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another famil…

Activation of the Proapoptotic Unfolded Protein Response in Plaques of the Human Carotid Artery

Objective To analyze expression of keystone markers of apoptosis and the proapoptotic signaling pathway “unfolded protein response” (UPR) in rupture-prone plaques of the human carotid artery. Methods Plaque specimens were obtained during endarterectomy for high-grade carotid stenosis, and were formalin-fixed. Ten specimens were identified that exhibited criteria of advanced rupture-prone atherosclerotic plaques, and histological and immunohistological analysis of markers of apoptosis (cleaved Caspase-3, TUNEL) and UPR (KDEL, ATF3, CHOP, CHAC-1) was performed. In addition, co-localization of apoptosis and UPR-activation was assessed by double-immunohistochemistry. Results The mean size of th…

Urokinase activates macrophage PON2 gene transcription via the PI3K/ROS/MEK/SREBP-2 signalling cascade mediated by the PDGFR-β

Aims We have recently shown that urokinase plasminogen activator (uPA) increases oxidative stress (OS), cholesterol biosynthesis, and paraoxonase 2 (PON2) expression in macrophages via binding to its receptor, the uPAR. Since PON2 is regulated by both OS and cholesterol content, we hypothesized that uPA elicits a cascade of signal transduction events shared by NADPH oxidase and cholesterol biosynthesis that culminates in PON2 gene expression. Here, we investigated the signalling pathway that leads to the expression of PON2 in macrophages in response to uPA. Methods and results The increase in macrophage PON2 mRNA levels in response to uPA was shown to depend on PON2 gene promoter activation…

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…

Paraoxonase-2 Reduces Oxidative Stress in Vascular Cells and Decreases Endoplasmic Reticulum Stress–Induced Caspase Activation

Background— In the vascular system, elevated levels of reactive oxygen species (ROS) produce oxidative stress and predispose to the development of atherosclerosis. Therefore, it is important to understand the systems producing and those scavenging vascular ROS. Here, we analyzed the ROS-reducing capability of paraoxonase-2 (PON2) in different vascular cells and its involvement in the endoplasmic reticulum stress pathway known as the unfolded protein response. Methods and Results— Quantitative real-time polymerase chain reaction and Western blotting revealed that PON2 is equally expressed in vascular cells and appears in 2 distinct glycosylated isoforms. By determining intracellular ROS, we…

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 ev…