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Michael Aviram - One of the best experts on this subject based on the ideXlab platform.
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triglyceride accumulation in macrophages upregulates paraoxonase 2 PON2 expression via ros mediated jnk c jun signaling pathway activation
Biofactors, 2012Co-Authors: Mira Rosenblat, Nina Volkova, Nicole Paland, Michael AviramAbstract:The aim of this study was to analyze the effect and mechanism of action of macrophage triglyceride accumulation on cellular PON2 expression. Incubation of J774A.1 (murine macrophages) with VLDL (0–75 μg protein/mL) significantly and dose-dependently increased cellular triglyceride mass, and reactive oxygen species (ROS) formation, by up to 3.3- or 1.8-fold, respectively. PON2 expression (mRNA, protein, activity) in cells treated with VLDL (50 μg protein/mL) was higher by 2- to 3-fold, as compared with control cells. Similar effects were noted upon using THP-1 (human macrophages). Incubation of macrophages with synthetic triglyceride or triglyceride fraction from carotid lesion resulted in similar effects, as shown for VLDL. Upon using specific inhibitors of MEK1/2 (UO126, 10 μM), p38 (SB203580, 10 μM), or JNK (SP600125, 20 μM), we demonstrated that MEK, as well as JNK, but not p38, are involved in VLDL-induced macrophage PON2 upregulation. VLDL activated JNK (but not ERK), which resulted in c-Jun phosphorylation. This signaling pathway is probably activated by ROS, since the antioxidant reduced glutathione (GSH), significantly decreased VLDL-induced macrophage ROS formation, c-Jun phosphorylation and PON2 overexpression. We conclude that macrophage triglyceride accumulation upregulates PON2 expression via MEK/ JNK/c-Jun pathway, and these effects could be related, at least in part, to cellular triglycerides-induced ROS formation. © 2012 International Union of Biochemistry and Molecular Biology, Inc.
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macrophage endoplasmic reticulum er proteins and reducing elements stabilize paraoxonase 2 PON2
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Michael AviramAbstract:Abstract Objective To analyze the ability of macrophage sub-cellular fractions to stabilize paraoxonase 2 (PON2). Methods Nuclei, mitochondria, lysosomes, endoplasmic reticulum (ER) and cytosol were isolated from J774A.1 macrophage cell line and incubated with recombinant PON2. Results Among the fractions analyzed the ER contains the highest PON2 lactonase activity, and was the most potent one in stabilizing recombinant PON2 (rePON2). Whereas control rePON2 activity was decreased by 40% after 20 h of incubation at 37 °C, in the presence of ER it decreased by only 15%. This effect could be attributed to the ER aqueous phase, and not to the ER lipids. The ER proteins fraction was responsible for PON2 stabilization, since heated ER or proteinase K-treated ER was not able to protect rePON2 from inactivation, while the protein fraction (after ammonium sulfate precipitation) completely prevented rePON2 inactivation. Since in the macrophage ER, there are increased levels of NADPH, secondary to glutathione reductase deficiency, we next studied the effect of the redox environment on PON2 inactivation. Incubation of rePON2 with DTT protected PON2 from inactivation. Similarly, NADPH, but not NADP, significantly increased rePON2 lactonase activity by up to 19%, after 20 h of incubation as compared to control rePON2. Unlike ER from non-treated macrophages, ER harvested from oxidized-, or from cholesterol loaded-macrophages showed a significant lower basal PON2 lactonase activity, and did not protect PON2 from inactivation but rather increased it. Conclusion Under normal conditions macrophage ER stabilizes PON2 activity, and this effect could be attributed to ER proteins and redox status.
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pomegranate juice pj consumption antioxidative properties on mouse macrophages but not pj beneficial effects on macrophage cholesterol and triglyceride metabolism are mediated via pj induced stimulation of macrophage PON2
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Michael AviramAbstract:Abstract Objective To examine whether the beneficial effects of PJ consumption by mice on their macrophages are mediated via PJ-induced increment in serum paraoxonase 1 (PON1) activity and/or in macrophage PON2 expression. Methods and results We performed studies in peritoneal macrophages (MPM) from C57BL/6 control mice, or from PON1KO mice, or from PON2KO mice that consumed PJ (200 μg of gallic acid equivalents/mouse/day, for 1 month period). PJ consumption by C57BL/6 mice resulted in a significant increment, by 36% in serum PON1 catalytic activities, and upregulated MPM PON2 expression. In MPM from C57BL/6 or from PON1KO mice that consumed PJ, the extent of cell-mediated LDL oxidation was decreased by 22%, and that of cellular superoxide release by 20–26%. In contrast, PJ consumption by PON2KO mice resulted in a minimal inhibitory effect on macrophage oxidative stress by only 4–9%. Unlike PJ antioxidative effects in MPM, PJ anti-atherogenic effects on MPM cholesterol and triglyceride metabolism were similar in all mice groups that consumed PJ. After PJ consumption, cellular cholesterol content was decreased by 14–19%, and this could be attributed to a significant inhibition in MPM cholesterol biosynthesis rate by 20–32%, and/or to stimulation of HDL-mediated cholesterol efflux from the cells by 22–37%. Similarly, MPM triglyceride content and triglyceride biosynthesis rate were both significantly decreased after PJ consumption, by 16–27% and by 22–28%, respectively. Conclusion PJ consumption antioxidative properties on mouse macrophages, but not PJ beneficial effects on macrophage cholesterol and triglyceride metabolism, are mediated via PJ-induced stimulation of macrophage PON2 expression. Serum PON1 stimulation by PJ consumption, however, was not involved in PJ-induced effects on macrophages.
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increased macrophage cholesterol biosynthesis and decreased cellular paraoxonase 2 PON2 expression in δ6 desaturase knockout 6 ds ko mice beneficial effects of arachidonic acid
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Manuel Roquetarivera, Manabu T Nakamura, Michael AviramAbstract:Abstract Objective To analyze the possible role of arachidonic acid (AA) in macrophage cholesterol biosynthesis and in PON2 expression. Methods and results We used peritoneal macrophages (MPM) from the 6-DS KO mice that were fed a diet without or with AA. Macrophage cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels were substantially increased, by 98% and 67%, respectively, in MPM from 6-DS KO vs. control (C57BL/6) mice. Furthermore, in the 6-DS KO vs. control mice MPM PON2 expression (mRNA and lactonase activity) was substantially decreased. In line with the above results, AA supplementation to 6-DS KO mice significantly decreased MPM cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels, by 45% and by 4-fold respectively, and increased MPM PON2 lactonase activity and PON2 mRNA, by 119% and 2.3-fold, respectively. Similarly, incubation of control mice MPM or J774A.1 with AA, significantly and dose-dependently decreased cellular cholesterol biosynthesis rate, and increased PON2 expression. These effects were specific for AA since incubation of the cells with docosahexaenoic acid (DHA, another product of 6-DS) had no significant effects on cholesterol biosynthesis rate, and on PON2 activity. Conclusions AA decreased macrophage cholesterol biosynthesis rate, and increased PON2 expression. These effects could protect the cells from cholesterol accumulation and oxidation, and from foam cell formation, the hallmark of early atherogenesis.
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paraoxonase 2 PON2 decreases high glucose induced macrophage triglycerides tg accumulation via inhibition of nadph oxidase and dgat1 activity studies in PON2 deficient mice
Atherosclerosis, 2010Co-Authors: Edna Meilin, Michael Aviram, Tony HayekAbstract:Abstract Objective The present study investigates the role of paraoxonase 2 (PON2) in the attenuation of macrophage triglycerides (TG) biosynthesis, and oxidative stress, under diabetic conditions. Methods Peritoneal macrophages (MPM) from PON2-deficient and from C57BL/6 control mice were harvested and cultured under normal (5 mM) or high glucose concentration (30 mM), and evaluated for cellular TG metabolism as well as for their oxidative stress. Results In PON2-deficient MPM vs. control MPM, under diabetic conditions (high glucose concentration), we observed substantial increment in TG accumulation (3 fold), TG biosynthesis (2.6 fold) and microsomal diacylglycerol acyltransferase1 (DGAT1) activity (+60%). Furthermore, in these cells we have demonstrated increased oxidative stress, as expressed by significant increment in cellular oxidative stress (+25%), macrophage-mediated LDL oxidation (+41%) and expression of the receptor for advanced glycation end products – RAGE (+18%). Apocynin, an NADPH-oxidase inhibitor, abolished the increment in MPM TG accumulation, MPM TG biosynthesis, and microsomal DGAT1 activity, as a result of PON2-deficiency, under diabetic conditions. Conclusion We conclude that PON2 has a significant protective role against macrophage triglyceride accumulation, macrophage TG biosynthesis, microsomal DGAT1 activity and macrophage oxidative stress, under high glucose concentrations. We suggest that this protective effect may be mediated by PON2 through the attenuation of NADPH-oxidase activity. The use of appropriate means to increase macrophage PON2 expression can lead to attenuation in macrophage TG accumulation and in cellular oxidative stress, under diabetic conditions, and thus may contribute to the decrement in macrophage atherogenicity and foam cell formation, attenuating the development of vascular complications in diabetes mellitus.
Mira Rosenblat - One of the best experts on this subject based on the ideXlab platform.
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triglyceride accumulation in macrophages upregulates paraoxonase 2 PON2 expression via ros mediated jnk c jun signaling pathway activation
Biofactors, 2012Co-Authors: Mira Rosenblat, Nina Volkova, Nicole Paland, Michael AviramAbstract:The aim of this study was to analyze the effect and mechanism of action of macrophage triglyceride accumulation on cellular PON2 expression. Incubation of J774A.1 (murine macrophages) with VLDL (0–75 μg protein/mL) significantly and dose-dependently increased cellular triglyceride mass, and reactive oxygen species (ROS) formation, by up to 3.3- or 1.8-fold, respectively. PON2 expression (mRNA, protein, activity) in cells treated with VLDL (50 μg protein/mL) was higher by 2- to 3-fold, as compared with control cells. Similar effects were noted upon using THP-1 (human macrophages). Incubation of macrophages with synthetic triglyceride or triglyceride fraction from carotid lesion resulted in similar effects, as shown for VLDL. Upon using specific inhibitors of MEK1/2 (UO126, 10 μM), p38 (SB203580, 10 μM), or JNK (SP600125, 20 μM), we demonstrated that MEK, as well as JNK, but not p38, are involved in VLDL-induced macrophage PON2 upregulation. VLDL activated JNK (but not ERK), which resulted in c-Jun phosphorylation. This signaling pathway is probably activated by ROS, since the antioxidant reduced glutathione (GSH), significantly decreased VLDL-induced macrophage ROS formation, c-Jun phosphorylation and PON2 overexpression. We conclude that macrophage triglyceride accumulation upregulates PON2 expression via MEK/ JNK/c-Jun pathway, and these effects could be related, at least in part, to cellular triglycerides-induced ROS formation. © 2012 International Union of Biochemistry and Molecular Biology, Inc.
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macrophage endoplasmic reticulum er proteins and reducing elements stabilize paraoxonase 2 PON2
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Michael AviramAbstract:Abstract Objective To analyze the ability of macrophage sub-cellular fractions to stabilize paraoxonase 2 (PON2). Methods Nuclei, mitochondria, lysosomes, endoplasmic reticulum (ER) and cytosol were isolated from J774A.1 macrophage cell line and incubated with recombinant PON2. Results Among the fractions analyzed the ER contains the highest PON2 lactonase activity, and was the most potent one in stabilizing recombinant PON2 (rePON2). Whereas control rePON2 activity was decreased by 40% after 20 h of incubation at 37 °C, in the presence of ER it decreased by only 15%. This effect could be attributed to the ER aqueous phase, and not to the ER lipids. The ER proteins fraction was responsible for PON2 stabilization, since heated ER or proteinase K-treated ER was not able to protect rePON2 from inactivation, while the protein fraction (after ammonium sulfate precipitation) completely prevented rePON2 inactivation. Since in the macrophage ER, there are increased levels of NADPH, secondary to glutathione reductase deficiency, we next studied the effect of the redox environment on PON2 inactivation. Incubation of rePON2 with DTT protected PON2 from inactivation. Similarly, NADPH, but not NADP, significantly increased rePON2 lactonase activity by up to 19%, after 20 h of incubation as compared to control rePON2. Unlike ER from non-treated macrophages, ER harvested from oxidized-, or from cholesterol loaded-macrophages showed a significant lower basal PON2 lactonase activity, and did not protect PON2 from inactivation but rather increased it. Conclusion Under normal conditions macrophage ER stabilizes PON2 activity, and this effect could be attributed to ER proteins and redox status.
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pomegranate juice pj consumption antioxidative properties on mouse macrophages but not pj beneficial effects on macrophage cholesterol and triglyceride metabolism are mediated via pj induced stimulation of macrophage PON2
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Michael AviramAbstract:Abstract Objective To examine whether the beneficial effects of PJ consumption by mice on their macrophages are mediated via PJ-induced increment in serum paraoxonase 1 (PON1) activity and/or in macrophage PON2 expression. Methods and results We performed studies in peritoneal macrophages (MPM) from C57BL/6 control mice, or from PON1KO mice, or from PON2KO mice that consumed PJ (200 μg of gallic acid equivalents/mouse/day, for 1 month period). PJ consumption by C57BL/6 mice resulted in a significant increment, by 36% in serum PON1 catalytic activities, and upregulated MPM PON2 expression. In MPM from C57BL/6 or from PON1KO mice that consumed PJ, the extent of cell-mediated LDL oxidation was decreased by 22%, and that of cellular superoxide release by 20–26%. In contrast, PJ consumption by PON2KO mice resulted in a minimal inhibitory effect on macrophage oxidative stress by only 4–9%. Unlike PJ antioxidative effects in MPM, PJ anti-atherogenic effects on MPM cholesterol and triglyceride metabolism were similar in all mice groups that consumed PJ. After PJ consumption, cellular cholesterol content was decreased by 14–19%, and this could be attributed to a significant inhibition in MPM cholesterol biosynthesis rate by 20–32%, and/or to stimulation of HDL-mediated cholesterol efflux from the cells by 22–37%. Similarly, MPM triglyceride content and triglyceride biosynthesis rate were both significantly decreased after PJ consumption, by 16–27% and by 22–28%, respectively. Conclusion PJ consumption antioxidative properties on mouse macrophages, but not PJ beneficial effects on macrophage cholesterol and triglyceride metabolism, are mediated via PJ-induced stimulation of macrophage PON2 expression. Serum PON1 stimulation by PJ consumption, however, was not involved in PJ-induced effects on macrophages.
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increased macrophage cholesterol biosynthesis and decreased cellular paraoxonase 2 PON2 expression in δ6 desaturase knockout 6 ds ko mice beneficial effects of arachidonic acid
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Manuel Roquetarivera, Manabu T Nakamura, Michael AviramAbstract:Abstract Objective To analyze the possible role of arachidonic acid (AA) in macrophage cholesterol biosynthesis and in PON2 expression. Methods and results We used peritoneal macrophages (MPM) from the 6-DS KO mice that were fed a diet without or with AA. Macrophage cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels were substantially increased, by 98% and 67%, respectively, in MPM from 6-DS KO vs. control (C57BL/6) mice. Furthermore, in the 6-DS KO vs. control mice MPM PON2 expression (mRNA and lactonase activity) was substantially decreased. In line with the above results, AA supplementation to 6-DS KO mice significantly decreased MPM cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels, by 45% and by 4-fold respectively, and increased MPM PON2 lactonase activity and PON2 mRNA, by 119% and 2.3-fold, respectively. Similarly, incubation of control mice MPM or J774A.1 with AA, significantly and dose-dependently decreased cellular cholesterol biosynthesis rate, and increased PON2 expression. These effects were specific for AA since incubation of the cells with docosahexaenoic acid (DHA, another product of 6-DS) had no significant effects on cholesterol biosynthesis rate, and on PON2 activity. Conclusions AA decreased macrophage cholesterol biosynthesis rate, and increased PON2 expression. These effects could protect the cells from cholesterol accumulation and oxidation, and from foam cell formation, the hallmark of early atherogenesis.
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paraoxonase 2 attenuates macrophage triglyceride accumulation via inhibition of diacylglycerol acyltransferase 1
Journal of Lipid Research, 2009Co-Authors: Mira Rosenblat, Srinivasa T Reddy, Raymond Coleman, Michael AviramAbstract:This study questioned the role of paraoxonase 2 (PON2) in attenuation of macrophage lipids accumulation. Mouse peritoneal macrophages (MPMs) harvested from PON2-deficient mice versus control C57BL/6 mice, look like foam cells and were larger in size and filled with lipid droplets. Macrophage triglyceride (but not cholesterol) content, biosynthesis rate, and microsomal acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) activity (not mRNA and protein) in PON2-deficient versus control MPM were all significantly increased by 4.6-, 3.6-, and 4.4-fold, respectively. Similarly, microsomal DGAT1 activity and cellular triglyceride content were significantly decreased in human PON2-transfected cells as well as upon incubation of PON2-deficient MPM with recombinant PON2. In all the above experimental systems, PON2 also decreased macrophage oxidative state. Incubation of PON2-deficient MPM with the free radicals generator 2,2'-amidinopropane hydrochloride increased cellular oxidative stress and DGAT1 activity by 2.2- and 3.4-fold, respectively, whereas incubation of microsomes from PON2-deficient MPM with superoxide dismutase decreased DGAT1 activity by 40%. We thus conclude that PON2 attenuates macrophage triglyceride accumulation and foam cell formation via inhibition of microsomal DGAT1 activity, which appears to be sensitive to oxidative state.
Sven Horke - One of the best experts on this subject based on the ideXlab platform.
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the anti apoptotic PON2 protein is wnt β catenin regulated and correlates with radiotherapy resistance in oscc patients
Oncotarget, 2016Co-Authors: Maximilian Krüger, Ines Witte, Petra Wilgenbus, Maximilian Moergel, Julianna Amort, Johanna Helmstadter, Irina Grechowa, Julia Ebert, Stefan Tenzer, Sven HorkeAbstract: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/GSK3β/β-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 β-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/GSK3β/β-catenin. More importantly, the study revealed the fundamental finding of an overall Wnt/GSK3β/β-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.
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Paraoxonase-2 (PON2) protects oral squamous cell cancer cells against irradiation-induced apoptosis
Journal of Cancer Research and Clinical Oncology, 2015Co-Authors: Maximilian Krüger, Sven Horke, Andreas Max Pabst, Bilal Al-nawas, Maximilian MoergelAbstract: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.
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paraoxonase 2 PON2 protects oral squamous cell cancer cells against irradiation induced apoptosis
Journal of Cancer Research and Clinical Oncology, 2015Co-Authors: Maximilian Krüger, Sven Horke, Andreas Pabst, Bilal Alnawas, Maximilian MoergelAbstract: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.
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protectors or traitors the roles of PON2 and pon3 in atherosclerosis and cancer
Journal of Lipids, 2012Co-Authors: Ines Witte, Ulrich Foerstermann, Asokan Devarajan, Srinivasa T Reddy, Sven HorkeAbstract: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.
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one enzyme two functions PON2 prevents mitochondrial superoxide formation and apoptosis independent from its lactonase activity
Journal of Biological Chemistry, 2010Co-Authors: Sebastian Altenhöfer, Ines Witte, Ulrich Förstermann, John F Teiber, Petra Wilgenbus, Andrea Pautz, Andreas Daiber, Heidrun Witan, Albrecht M Clement, Sven HorkeAbstract: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.
Denis A Khotimsky - One of the best experts on this subject based on the ideXlab platform.
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ng PON2 transmission convergence layer a tutorial
Journal of Lightwave Technology, 2016Co-Authors: Denis A KhotimskyAbstract:The first family of multi-wavelength PON systems, 40-gigabit-capable capable passive optical networks (NG-PON2), has been specified by the Telecommunication Standardization Sector of the International Telecommunications Union (ITU-T). This tutorial, which is based on the author's ECOC'2015 presentation on the topic, offers a comprehensive review of the NG-PON2 transmission convergence layer specification, and is addressed to a wide range of readers who are active in the field of optical communications.
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ng PON2 transmission convergence layer a tutorial
European Conference on Optical Communication, 2015Co-Authors: Denis A KhotimskyAbstract:The first multi wavelength PON system, NG-PON2, has been specified by ITU-T (Q2/15). The tutorial presents a comprehensive review of the NG-PON2 trasnmission converegence layer specification and is targeted at development and verification engineers, system engineers, as well as product line management experts. The paper addresses selected key aspect within the tutorial scope.
Nina Volkova - One of the best experts on this subject based on the ideXlab platform.
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triglyceride accumulation in macrophages upregulates paraoxonase 2 PON2 expression via ros mediated jnk c jun signaling pathway activation
Biofactors, 2012Co-Authors: Mira Rosenblat, Nina Volkova, Nicole Paland, Michael AviramAbstract:The aim of this study was to analyze the effect and mechanism of action of macrophage triglyceride accumulation on cellular PON2 expression. Incubation of J774A.1 (murine macrophages) with VLDL (0–75 μg protein/mL) significantly and dose-dependently increased cellular triglyceride mass, and reactive oxygen species (ROS) formation, by up to 3.3- or 1.8-fold, respectively. PON2 expression (mRNA, protein, activity) in cells treated with VLDL (50 μg protein/mL) was higher by 2- to 3-fold, as compared with control cells. Similar effects were noted upon using THP-1 (human macrophages). Incubation of macrophages with synthetic triglyceride or triglyceride fraction from carotid lesion resulted in similar effects, as shown for VLDL. Upon using specific inhibitors of MEK1/2 (UO126, 10 μM), p38 (SB203580, 10 μM), or JNK (SP600125, 20 μM), we demonstrated that MEK, as well as JNK, but not p38, are involved in VLDL-induced macrophage PON2 upregulation. VLDL activated JNK (but not ERK), which resulted in c-Jun phosphorylation. This signaling pathway is probably activated by ROS, since the antioxidant reduced glutathione (GSH), significantly decreased VLDL-induced macrophage ROS formation, c-Jun phosphorylation and PON2 overexpression. We conclude that macrophage triglyceride accumulation upregulates PON2 expression via MEK/ JNK/c-Jun pathway, and these effects could be related, at least in part, to cellular triglycerides-induced ROS formation. © 2012 International Union of Biochemistry and Molecular Biology, Inc.
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macrophage endoplasmic reticulum er proteins and reducing elements stabilize paraoxonase 2 PON2
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Michael AviramAbstract:Abstract Objective To analyze the ability of macrophage sub-cellular fractions to stabilize paraoxonase 2 (PON2). Methods Nuclei, mitochondria, lysosomes, endoplasmic reticulum (ER) and cytosol were isolated from J774A.1 macrophage cell line and incubated with recombinant PON2. Results Among the fractions analyzed the ER contains the highest PON2 lactonase activity, and was the most potent one in stabilizing recombinant PON2 (rePON2). Whereas control rePON2 activity was decreased by 40% after 20 h of incubation at 37 °C, in the presence of ER it decreased by only 15%. This effect could be attributed to the ER aqueous phase, and not to the ER lipids. The ER proteins fraction was responsible for PON2 stabilization, since heated ER or proteinase K-treated ER was not able to protect rePON2 from inactivation, while the protein fraction (after ammonium sulfate precipitation) completely prevented rePON2 inactivation. Since in the macrophage ER, there are increased levels of NADPH, secondary to glutathione reductase deficiency, we next studied the effect of the redox environment on PON2 inactivation. Incubation of rePON2 with DTT protected PON2 from inactivation. Similarly, NADPH, but not NADP, significantly increased rePON2 lactonase activity by up to 19%, after 20 h of incubation as compared to control rePON2. Unlike ER from non-treated macrophages, ER harvested from oxidized-, or from cholesterol loaded-macrophages showed a significant lower basal PON2 lactonase activity, and did not protect PON2 from inactivation but rather increased it. Conclusion Under normal conditions macrophage ER stabilizes PON2 activity, and this effect could be attributed to ER proteins and redox status.
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pomegranate juice pj consumption antioxidative properties on mouse macrophages but not pj beneficial effects on macrophage cholesterol and triglyceride metabolism are mediated via pj induced stimulation of macrophage PON2
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Michael AviramAbstract:Abstract Objective To examine whether the beneficial effects of PJ consumption by mice on their macrophages are mediated via PJ-induced increment in serum paraoxonase 1 (PON1) activity and/or in macrophage PON2 expression. Methods and results We performed studies in peritoneal macrophages (MPM) from C57BL/6 control mice, or from PON1KO mice, or from PON2KO mice that consumed PJ (200 μg of gallic acid equivalents/mouse/day, for 1 month period). PJ consumption by C57BL/6 mice resulted in a significant increment, by 36% in serum PON1 catalytic activities, and upregulated MPM PON2 expression. In MPM from C57BL/6 or from PON1KO mice that consumed PJ, the extent of cell-mediated LDL oxidation was decreased by 22%, and that of cellular superoxide release by 20–26%. In contrast, PJ consumption by PON2KO mice resulted in a minimal inhibitory effect on macrophage oxidative stress by only 4–9%. Unlike PJ antioxidative effects in MPM, PJ anti-atherogenic effects on MPM cholesterol and triglyceride metabolism were similar in all mice groups that consumed PJ. After PJ consumption, cellular cholesterol content was decreased by 14–19%, and this could be attributed to a significant inhibition in MPM cholesterol biosynthesis rate by 20–32%, and/or to stimulation of HDL-mediated cholesterol efflux from the cells by 22–37%. Similarly, MPM triglyceride content and triglyceride biosynthesis rate were both significantly decreased after PJ consumption, by 16–27% and by 22–28%, respectively. Conclusion PJ consumption antioxidative properties on mouse macrophages, but not PJ beneficial effects on macrophage cholesterol and triglyceride metabolism, are mediated via PJ-induced stimulation of macrophage PON2 expression. Serum PON1 stimulation by PJ consumption, however, was not involved in PJ-induced effects on macrophages.
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increased macrophage cholesterol biosynthesis and decreased cellular paraoxonase 2 PON2 expression in δ6 desaturase knockout 6 ds ko mice beneficial effects of arachidonic acid
Atherosclerosis, 2010Co-Authors: Mira Rosenblat, Nina Volkova, Manuel Roquetarivera, Manabu T Nakamura, Michael AviramAbstract:Abstract Objective To analyze the possible role of arachidonic acid (AA) in macrophage cholesterol biosynthesis and in PON2 expression. Methods and results We used peritoneal macrophages (MPM) from the 6-DS KO mice that were fed a diet without or with AA. Macrophage cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels were substantially increased, by 98% and 67%, respectively, in MPM from 6-DS KO vs. control (C57BL/6) mice. Furthermore, in the 6-DS KO vs. control mice MPM PON2 expression (mRNA and lactonase activity) was substantially decreased. In line with the above results, AA supplementation to 6-DS KO mice significantly decreased MPM cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels, by 45% and by 4-fold respectively, and increased MPM PON2 lactonase activity and PON2 mRNA, by 119% and 2.3-fold, respectively. Similarly, incubation of control mice MPM or J774A.1 with AA, significantly and dose-dependently decreased cellular cholesterol biosynthesis rate, and increased PON2 expression. These effects were specific for AA since incubation of the cells with docosahexaenoic acid (DHA, another product of 6-DS) had no significant effects on cholesterol biosynthesis rate, and on PON2 activity. Conclusions AA decreased macrophage cholesterol biosynthesis rate, and increased PON2 expression. These effects could protect the cells from cholesterol accumulation and oxidation, and from foam cell formation, the hallmark of early atherogenesis.
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urokinase activates macrophage PON2 gene transcription via the pi3k ros mek srebp 2 signalling cascade mediated by the pdgfr beta
Cardiovascular Research, 2009Co-Authors: Bianca Fuhrman, Sven Horke, Nina Volkova, Anna Gantman, Jasmin Khateeb, Julia Kiyan, Inna Dumler, Michael AviramAbstract: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 and mRNA transcription. LDL abolished these effects, suggesting a possible role for a transcription factor involved in cellular cholesterogenesis. Indeed, uPA upregulated PON2 expression in a sterol regulatory binding protein-2 (SREBP-2)-dependent manner, since blocking SREBP-2 maturation by 4-(2-aminoethyl)-benzenesulfonyl fluoride abolished uPA-stimulation of PON2, whereas inhibition of SREBP-2 catabolism by N -acetyl-leucyl-norleucinal had an opposite effect. The upstream signalling mechanisms include uPA activation of extracellular signal-regulated kinases (ERK1/2), which was dependent on NADPH oxidase and phosphatidylinositol 3-kinase activation, and these latter effects were mediated by the tyrosine kinase activity of the platelet-derived growth factor receptor-β. Conclusion These findings provide a framework linking interactions among cellular signalling pathways associated with reactive oxygen species production, macrophage cholesterol biosynthesis, and cellular PON2 expression in vascular pathophysiology.