Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9-and 10-nitro-9-cis-octadecenoic acid; OA-NO 2 ), structurally characterized and determined to be identical to OA-NO 2 found in plasma, red cells, and urine of healthy humans. These regioisomers of OA-NO 2 were quantified in clinical samples using 13 C isotope dilution. Plasma free and esterified OA-NO 2 concentrations were 619 ؎ 52 and 302 ؎ 369 nM, respectively, and packed red blood cell free and esterified OA-NO 2 was 59 ؎ 11 and 155 ؎ 65 nM. The OA-NO 2 concentration of blood is ϳ50% greater than that of nitrated linoleic acid, with the combined free and esterified blood levels of these two fatty acid derivatives exceeding 1 M. OA-NO 2 is a potent ligand for peroxisome proliferator activated receptors at physiological concentrations. CV-1 cells co-transfected with the luciferase gene under peroxisome proliferator-activated receptor (PPAR) response element regulation, in concert with PPAR␥, PPAR␣, or PPAR␦ expression plasmids, showed dose-dependent activation of all PPARs by OA-NO 2 . PPAR␥ showed the greatest response, with significant activation at 100 nM, while PPAR␣ and PPAR␦ were activated at ϳ300 nM OA-NO 2 . OA-NO 2 also induced PPAR␥-dependent adipogenesis and deoxyglucose uptake in 3T3-L1 preadipocytes at a potency exceeding nitrolinoleic acid and rivaling synthetic thiazolidinediones. These data reveal that nitrated fatty acids comprise a class of nitric oxide-derived, receptor-dependent, cell signaling mediators that act within physiological concentration ranges.The oxidation of unsaturated fatty acids converts lipids, otherwise serving as cellular metabolic precursors and structural components, into potent signaling molecules including prostaglandins, leukotrienes, isoprostanes, and hydroxy-and hydroperoxyeicosatetraenoates. These enzymatic and auto-catalytic oxidation reactions yield products that orchestrate immune responses, neurotransmission, and the regulation of cell growth. For example, prostaglandins are cyclooxygenase-derived lipid mediators that induce receptor-dependent regulation of inflammatory responses, vascular function, initiation of parturition, cell survival, and angiogenesis (1). In contrast, the various isoprostane products of arachidonic acid auto-oxidation exert vasoconstrictive and pro-inflammatory signaling actions via receptor-dependent and -independent mechanisms (2). A common element of these diverse lipid signaling reactions is that nitric oxide ( ⅐ NO) 6 and other oxides of nitrogen significantly impact lipid mediator formation and bioactivities.The ability of ⅐ NO and ⅐ NO-derived species to oxidize, nitrosate, and nitrate biomolecules serves as the molecular basis for how ⅐ NO influences the sy...

Macrophages are key defenders of the lung and play an essential role in mediating the inflammatory response. Critical to this is the activation of the NADPH oxidase. Through receptor-mediated interactions, extracellular stimuli activate pathways that signal for the phosphorylation and assembly of the NADPH oxidase. Once the NADPH oxidase is activated, it produces superoxide and H2O2 in a process known as the respiratory burst. The involvement of O2.- and H2O2 in the antimicrobicidal function of macrophages has been assumed for many years, but it is now clear that the H2O2 produced by the respiratory burst functions as a second messenger and activates major signaling pathways in the alveolar macrophage. Both the nuclear factor-kappaB and activator protein-1 transcription factors are activated by H2O2 produced by the respiratory burst, and, since these control the inducible expression of genes whose products are part of the inflammatory response, this may be a critical link between the respiratory burst and other inflammatory responses. The c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK) pathways, two members of the mitogen-activated protein kinase family, are also activated by the respiratory burst. JNK is activated by both exogenous and endogenously produced H2O2. Studies with ERK have shown that specific agonists of the respiratory burst, but not bolus H2O2, can activate this pathway. The ERK pathway also modulates the expression of genes via phosphorylation of the transcription factor Elk-1 that controls the production of the c-Fos transcription factor. Although an understanding of the mechanism of redox signaling is in its infancy, it is becoming clear that the reactive oxygen species produced by the respiratory burst have a profound effect on intracellular signaling pathways and ultimately in modulating gene expression.

Dietary use of curcumin, the active component of tumeric, one of the most widely used spices, is linked to several beneficial health effects, although the underlying molecular mechanisms remain largely unknown. Correlations have been established between curcumin exposure and increases in enzymes for glutathione synthesis, particularly glutamate-cysteine ligase (GCL), and metabolism as well as glutathione content, suggesting the eliciting of an adaptive response to stress. In this study, using HBE1 cells, we found that the mechanism of curcumin-induced GCL elevation occurred via transcription of the two Gcl genes. Gcl transcription has been shown in several systems to be mediated through binding of transcription factor complexes to TRE and EpRE elements. Studies herein showed that curcumin caused modest but sustained increases in binding of proteins to DNA sequences for both cis elements but, more importantly, altered the compositions and nuclear content of proteins in these complexes. Curcumin exposure increased JunD and c-Jun content in AP-1 complexes and increased JunD while decreasing MafG/MafK in EpRE complexes. Thus, the beneficial effects elicited by curcumin appear to be due to changes in the pool of transcription factors that compose EpRE and AP-1 complexes, affecting gene expression of GCL and other phase II enzymes.

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Recently, it has been observed that reactions between NO-derived species, unsaturated fatty acids, and lipid oxidation intermediates yield fatty acid oxidation and nitration products (2, 3). Nitroalkene derivatives of all principal unsaturated fatty acids are present in human blood and urine and represent an abundant pool of bioactive oxides of nitrogen, with tissue levels of these species influenced by endogenous redox reactions and dietary intake of nitrated lipids. Nitrolinoleic acid (10-and 12-nitro-9,12-octadecadienoic acid, LNO 2 ) is present in plasma and red blood cell membranes at concentrations of Ϸ500 nM (3). Nitroalkene fatty acid derivatives induce adaptive antiinflammatory responses by means of induction of cGMPdependent and cGMP-independent cell signaling reactions that result in inhibition of platelet and neutrophil activation (4, 5), vessel relaxation (6), and activation of peroxisome proliferatoractivated receptor (PPAR)-dependent gene expression (7).Vascular tissues maintain viability during inflammation by eliciting adaptive and protective responses. Recent studies have shown that heme oxygenase 1 (HO-1) plays a central role in vascular inflammatory signaling reactions and mediates a protective response in several inflammatory diseases, including atherosclerosis, acute renal failure, vascular restenosis, transplant rejection, and sepsis (reviewed in ref. 8). HO-1, a 32-kDa enzyme, is the ratelimiting step in the degradation of heme, yielding biliverdin, carbon monoxide, and iron (9). Biliverdin is subsequently converted to bilirubin by biliverdin reductase. All heme catabolites can contribute to integrative protective responses to oxidative stress (10). Specifically, HO activity reduces levels of prooxidative heme (11), with the iron released from heme catabolism rendered largely redox-inactive by ferritin sequestration. Ferritin is often coinduced with HO-1 and also displays cytoprotective functions (12). Heme degradation serves as the major endogenous source of carbon monoxide, a gas isoelectronic with NO that displays antiinflammatory, antiapoptotic, vasodilatory, and immune modulatory functions (13-15). The porphyrin metabolites biliverdin and bilirubin also scavenge reactive oxygen species (16,17). Finally, HO-1 induction is associated with concomitant up-regulation of the cell-cycle regulatory protein p21, which mediates antiapoptotic signaling during oxidative injury (18). Biliverdin, bilirubin, and carbon monoxide, acting individually or in concert, exert protective effects in vitro and in vivo in animal models of inflammatory injury (19).Because both nitroalkenes and HO-1 are emerging as key mediators of adaptive inflammatory responses, the impact of LNO 2 on HO-1 gene expression was examined. Herein, the marked up-regulation of HO-1 gene expression by LNO 2 in vascular cells is reported, with this induction occurring primarily at the transcriptional level. We conclude that LNO 2 mediates the induction of HO-1 by means of PPAR␥-independent and both NO-dependent and NO-independent mec...

Transforming growth factor (TGF)-beta upregulates plasminogen activator inhibitor type 1 (PAI-1) in a variety of cell types, and PAI-1 is considered to be an essential factor for the development of fibrosis. Our previous studies demonstrated that TGF-beta decreased intracellular glutathione (GSH) content in murine embryonic fibroblasts (NIH/3T3 cells), whereas treatment of the cells with GSH, which restored intracellular GSH concentration, inhibited TGF-beta-induced collagen accumulation by blocking PAI-1 expression and enhancing collagen degradation. In the present study, we demonstrate that GSH blocks TGF-beta-induced PAI-1 promoter activity in NIH/3T3 cells, which is associated with an inhibition of TGF-beta-induced JNK and p38 phosphorylation. Interestingly, although exogenous GSH does not affect phosphorylation and/or nuclear translocation of Smad2/3 and Smad4, it completely eliminates TGF-beta-induced binding of transcription factors to not only AP-1 and SP-1 but also Smad cis elements in the PAI-1 promoter. Decoy oligonucleotides (ODN) studies further demonstrate that AP-1, SP-1, and Smad ODNs abrogate the inhibitory effect of GSH on TGF-beta-induced PAI-1 promoter activity and inhibit TGF-beta-induced expression of endogenous PAI-1. Furthermore, we show that GSH reduces TGF-beta-stimulated reactive oxygen species (ROS) signal. Blocking ROS production with diphenyleneiodonium or scavenging ROS with a superoxide dismutase and catalase mimetic MnTBaP dramatically reduces TGF-beta-induced p38 and JNK phosphorylation as well as PAI-1 gene expression. In composite, these findings suggest that GSH inhibits TGF-beta-stimulated PAI-1 expression in fibroblasts by blocking the JNK/p38 pathway, probably by reducing ROS, which leads to an inhibition of the binding of transcription factors to the AP-1, SP-1, and Smad cis elements in the PAI-1 promoter.

Adaptation to oxidative and nitrosative stress occurs in cells first exposed to a nontoxic stress, resulting in the ability to tolerate a toxic challenge of the same or a related oxidant. Adaptation is observed in a wide variety of cells including endothelial cells on exposure to nitric oxide or oxidized lipids, and lung epithelial cells exposed to air-borne pollutants and toxicants. This acquired characteristic has been related to the regulation of a family of stress responding proteins including those that control the synthesis of the intracellular antioxidant glutathione. The focus of this article, which includes a review of recent results along with new data, is the regulation and signaling of glutathione biosynthesis, especially those relating to adaptive mechanisms. These concepts are illustrated with examples using nitric oxide and oxidized low density lipoprotein mediated adaptation to oxidative stress. These data are discussed in the context of other adaptive mechanisms relating to glutathione synthesis including those from dietary constituents such as curcumin.