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1.  Role of PON2 in innate immune response in an acute infection model 
Molecular genetics and metabolism  2013;110(3):362-370.
N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a quorum-sensing molecule produced by gram-negative microbial pathogens such as Pseudomonas aeruginosa (PAO1). 3OC12-HSL is involved in the regulation of bacterial virulence factors and also alters the function of the host immune cells. Others and we have previously shown that paraoxonase 2 (PON2), a member of the paraoxonase gene family expressed in immune cells, hydrolyzes 3OC12-HSL. In this study, we examined i) whether macrophage PON2 participates in 3OC12-HSL hydrolysis, ii) the effect of PON2 deficiency in acute PAO1 infection in mice and iii) the effect of 3OC12-HSL on PON2 deficient (PON2-def) macrophages. When compared to wild type macrophages, both intact cells and membrane-enriched protein lysates obtained from PON2-def macrophages show a marked impairment in their ability to hydrolyze 3OC12-HSL. PON2 expression (message and protein) is not altered in response to 3OC12-HSL in macrophages. 3OC12-HSL treated PON2-def macrophages showed i) an increase in ER stress and oxidative stress, ii) defective phosphatidylinositol 3-kinase (PI3 kinase)/AKT activation, and iii) reduced phagocytosis function. Moreover, the nitration to phosphorylation ratio of Tyr458 in p85 protein, the regulatory subunit of PI3-kinase that has been correlated with the phagocytosis function of macrophages, was increased in PON2-def macrophages. Antioxidant treatment reversed the effects of PON2 deficiency in macrophage phagocytosis function. Furthermore, following administration of 1.6×107CFU of PAO1, bacterial clearance was significantly reduced in the lungs (5.7 fold), liver (2.5 fold), and spleen (14.8 fold) of PON2-def mice when compared to wild type mice. Our results suggest that PON2 plays an important role in innate immune defense against PAO1 infection.
PMCID: PMC3800229  PMID: 23911207
Paraoxonase 2; Pseudomonas aeruginosa; Quorum sensing; Mitochondrial oxidative stress; Endoplasmic reticulum stress
2.  DJ-1 Interacts with and Regulates Paraoxonase-2, an Enzyme Critical for Neuronal Survival in Response to Oxidative Stress 
PLoS ONE  2014;9(9):e106601.
Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.
PMCID: PMC4161380  PMID: 25210784
3.  Macrophage Paraoxonase 2 regulates calcium homeostasis and cell survival under Endoplasmic Reticulum stress conditions and is sufficient to prevent the development of aggravated atherosclerosis in Paraoxonase 2 deficiency/apoE−/− mice on a Western diet 
Molecular genetics and metabolism  2012;107(3):416-427.
Paraoxonase 2 deficiency (PON2-def) alters mitochondrial function and exacerbates the development of atherosclerosis in mice. PON2 overexpression protects against ER stress in cell culture. In this paper, we examined the role of PON2 in the unexplored link between ER stress and mitochondrial dysfunction and tested whether restoration of PON2 in macrophages is sufficient to reduce aggravated atherosclerosis in PON2-def/apoE−/− mice on a Western diet. ER stress response genes, intracellular calcium levels, and apoptotic nuclei were significantly elevated in PON2-def/apoE−/− macrophages compared to apoE−/− macrophages in response to ER stressors, but not at the basal level. In contrast, PON2-def/apoE−/− macrophages exhibited greater mitochondrial stress at the basal level, which was further worsened in response to ER stressors. There was no difference in ER stress response genes and apoptotic nuclei between apoE−/− and PON2-def/apoE−/− macrophages when pretreated with xestospongin (which blocks the release of calcium from ER) suggesting that PON2 modulates cell survival and ER stress by maintaining calcium homeostasis. Treatment with a mitochondrial calcium uptake inhibitor, RU360, attenuated ER stressor mediated mitochondrial dysfunction in PON2-def/apoE−/− macrophages. CHOP expression (ER stress marker) and apoptotic nuclei were significantly higher in aortic lesions of PON2-def/apoE−/− mice compared to apoE−/− mice fed a Western diet. Restoration of PON2 in macrophage reduced ER stress, mitochondrial dysfunction and apoptosis in response to ER stressors. Furthermore, restoration of PON2 in macrophages reduced lesional apoptosis and atherosclerosis in PON2-def/apoE−/− mice on a Western diet. Our data suggest that macrophage PON2 modulates mechanisms that link ER stress, mitochondrial dysfunction and the development of atherosclerosis.
PMCID: PMC3483415  PMID: 22864055
Paraoxonase 2; Mitochondria; Endoplasmic reticulum; Macrophages; Oxidative stress
4.  Impaired hepatic insulin signalling in PON2-deficient mice: a novel role for the PON2/apoE axis on the macrophage inflammatory response 
The Biochemical journal  2011;436(1):91-100.
Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)−/− mice, relative to apoE−/− mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE−/− mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE−/− mice is directly associated with altered production of macrophage proinflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE−/− mice is mediated by a shift in the balance of NO and ONOO− (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.
PMCID: PMC3743554  PMID: 21361875
apolipoprotein E (apoE); insulin receptor substrate-1 (IRS-1); liver; macrophage; paraoxonase; paraoxonase 2 (PON2)
5.  Decreased Obesity and Atherosclerosis in Human Paraoxonase 3 Transgenic Mice 
Circulation research  2007;100(8):1200-1207.
Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4- to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.
PMCID: PMC3740095  PMID: 17379834
atherosclerosis; obesity; genetics
6.  Paraoxonase-2 modulates stress response of endothelial cells to oxidized phospholipids and a bacterial quorum-sensing molecule 
Chronic infection has long been postulated as a stimulus for atherogenesis. Pseudomonas aeruginosa infection has been associated with increased atherosclerosis in rats, and the bacteria produce a quorum-sensing molecule 3-oxo-dodecynoyl-homoserine lactone (3OC12-HSL) that is critical for colonization and virulence. Paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL and also protects against the effects of oxidized phospholipids thought to contribute to atherosclerosis. We now report the response of human aortic endothelial cells (HAEC) to 3OC12-HSL and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) in relation to PON2 expression.
Methods and Results
Using expression profiling and network modeling, we identified the unfolded protein response (UPR), cell cycle genes, and the MAPK signaling pathway to be heavily involved in the HAEC response to 3OC12-HSL. The network also showed striking similarities to a network created based on HAEC response to Ox-PAPC, a major component of minimally-modified LDL. HAEC in which PON2 was silenced by siRNA showed increased pro-inflammatory and UPR responses when treated with 3OC12-HSL or Ox-PAPC.
3OC12-HSL and Ox-PAPC influence similar inflammatory and UPR pathways. Quorum sensing molecules such as 3OC12-HSL contribute to the pro-atherogenic effects of chronic infection. The anti-atherogenic effects of PON2 include destruction of quorum sensing molecules.
PMCID: PMC3244174  PMID: 21836061
Atherosclerosis; Chronic Infection; Inflammation; Oxidative Stress; Unfolded Protein Response
7.  Paraoxonase 2 Deficiency Alters Mitochondrial Function and Exacerbates the Development of Atherosclerosis 
Antioxidants & Redox Signaling  2011;14(3):341-351.
Increased production of reactive oxygen species (ROS) as a result of decreased activities of mitochondrial electron transport chain (ETC) complexes plays a role in the development of many inflammatory diseases, including atherosclerosis. Our previous studies established that paraoxonase 2 (PON2) possesses antiatherogenic properties and is associated with lower ROS levels. The aim of the present study was to determine the mechanism by which PON2 modulates ROS production. In this report, we demonstrate that PON2-def mice on the hyperlipidemic apolipoprotein E−/− background (PON2-def/apolipoprotein E−/−) develop exacerbated atherosclerotic lesions with enhanced mitochondrial oxidative stress. We show that PON2 protein is localized to the inner mitochondrial membrane, where it is found associated with respiratory complex III. Employing surface-plasmon-resonance, we demonstrate that PON2 binds with high affinity to coenzyme Q10, an important component of the ETC. Enhanced mitochondrial oxidative stress in PON2-def mice was accompanied by significantly reduced ETC complex I + III activities, oxygen consumption, and adenosine triphosphate levels in PON2-def mice. In contrast, overexpression of PON2 effectively protected mitochondria from antimycin- or oligomycin-mediated mitochondrial dysfunction. Our results illustrate that the antiatherogenic effects of PON2 are, in part, mediated by the role of PON2 in mitochondrial function. Antioxid. Redox Signal. 14, 341–351.
PMCID: PMC3011913  PMID: 20578959

Results 1-7 (7)