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F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo. [ABSTRACT FROM AUTHOR]

Oxidative stress and inflammation are two major contributing factors to atherosclerosis, a leading cause of cardiovascular disease. Oxidation of phospholipids on the surface of low density lipoprotein (LDL) particles generated under oxidative stress has been associated with the progression of atherosclerosis, but the underlying molecular mechanisms remain poorly defined. We identified a novel series of oxidation products containing the cyclopentenone moiety, termed deoxy-A2/J2-isoprostanes-phosphocholine, from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine in vivo using mass spectrometry and by comparison to a chemically synthesized standard. Transcriptomic analysis (RNA-seq) demonstrated that these compounds affected >200 genes in bone marrow-derived macrophages, and genes associated with inflammatory and anti-oxidative responses are among the top 5 differentially expressed. To further investigate the biological relevance of these novel oxidized phospholipids in atherosclerosis, we chemically synthesized a representative compound 1-palmitoyl-2-15-deoxy-δ-12,14-prostaglandin J2-sn-glycero-3-phosphocholine (15d-PGJ2-PC) and found that it induced anti-inflammatory and anti-oxidant responses in macrophages through modulation of NF-κB, peroxisome proliferator-activated receptor γ (PPARγ), and Nrf2 pathways; this compound also showed potent anti-inflammatory properties in a mice model of LPS-induced systematic inflammatory response syndrome. Additionally, 15d-PGJ2-PC inhibited macrophage foam cell formation, suggesting a beneficial role against atherosclerosis. These properties were consistent with decreased levels of these compounds in the plasma of patients with coronary heart disease compared with control subjects. Our findings uncovered a novel molecular mechanism for the negative regulation of inflammation and positive enhancement of anti-oxidative responses in macrophages by these oxidized phospholipids in LDL in the context of atherosclerosis. [ABSTRACT FROM AUTHOR]

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μm). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca2+ ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca2+ binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca2+ citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity. [ABSTRACT FROM AUTHOR]

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μM). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca(2+) ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca(2+) binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca(2+) citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species. One class of compounds are cyclopentenone neuroprostanes (A(4)/J(4)-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A(4)/J(4)-NP, 14-A(4)-NP (A(4)-NP), potently suppresses lipopolysaccharideinduced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A(4)-NP blocks lipopolysaccharide-induced NF-kappaB activation via inhibition of Ikappa kinase-mediated phosphorylation of IkappaBalpha. Mutation on Ikappa kinase beta cysteine 179 markedly diminishes the effect of A(4)-NP, suggesting that A(4)-NP acts via thiol modification at this residue. Accordingly, the effects of A(4)-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A(4)/J(4)-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which eliminate the bioactivity of A(4)-NP, also abrogate the anti-inflammatory effects of oxidized DHA. Thus, we have demonstrated that A(4)/J(4)-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kappaB signaling and may contribute to the anti-inflammatory actions of DHA. These findings have implications for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation.

A regulated pattern of nuclear factor kappaB (NF-kappaB) activation is essential for normal development of the mammary gland. An increase in NF-kappaB activity has been implicated in breast cancer. We have generated a novel transgenic mouse model to investigate the role of the alternative NF-kappaB pathway in ductal development and identify possible mediators of tumorigenesis downstream of p100/p52. By overexpressing the NF-kappaB p100/p52 subunit in mammary epithelium using the beta-lactoglobulin milk protein promoter, we found that transgene expression resulted in increased overall NF-kappaB activity during late pregnancy. During pregnancy, p100/p52 expression resulted in delayed ductal development with impaired secondary branching and increased levels of Cyclin D1, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and cyclo-oxygenase-2 (COX-2) in the mammary gland. After multiple pregnancies the p100 transgenics exhibited a ductal thickening accompanied by small hyperplastic foci. In tumors from mice expressing the polyoma middle T oncoprotein (PyVT) in the mammary gland, increased levels of p100/p52 were present at the time of tumor development. These results show that increased p100/p52 disrupts normal ductal development and provides insight into the mechanism by which this may contribute to human breast cancer.