Arachidonic acid increases activation of NADPH oxidase in monocytic U937 cells by accelerated translocation of p47-phox and co-stimulation of protein kinase C

doi:10.1016/0898-6568(96)00077-0

Cellular Signalling

Volume 8, Issue 5, August 1996, Pages 397-402

https://doi.org/10.1016/0898-6568(96)00077-0 Get rights and content

Abstract

Arachidonic acid (AA) has been implicated as an important amphiphilic co-factor in the activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neutrophils and reconstituted cell-free systems. To assess the role of AA in the activation of O₂- generation in monocytic cells, we studied pre-monocytic U937 cells differentiated with 1,25-(OH)₂-vitamin D₃ plus interferon-γ (IFN-γ). AA dose-dependently enhanced phorbol myristate acetate (PMA)-stimulated O₂⁻ generation, with a maximum increase of 4,5-fold, through: (1) a more than 50% reduction of the lag-phase, defined as the time between addition of PMA and detection of O₂⁻; and (2) a more than 60% increase in the constant rate of O₂⁻ generation. Reduction of the lag phase was associated with increased protein kinase C (PKC)-independent translocation of the cytosolic subunit of NADPH oxidase p47-phox to the cell membrane, whereas increased generation of O₂⁻ correlated with enhanced activation of PKC. The data indicate that AA increases activation of NADPH oxidase by accelerating its assembly and by co-stimulating PKC in monocytic U937 cells.

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Upon IFN-γ stimulation, Hefner et al. (36) demonstrated that cPLA2 is activated through Ser-727 phosphorylation by mitogen- and stress-activated protein kinase 1 (MSK1) involved in the JAK-STAT pathway. Stimulated cPLA2 hydrolyzes membrane phospholipids from the sn-2 position to release arachidonic acid, which in turn translocates p47phox to the cell membrane (37). Concomitantly IFN-γ priming of macrophages triggers phosphorylation of p40phox, p47phox, and p67phox, which are translocated from the cytoplasm to the membrane to associate with cytochrome b558 and form the NADPH oxidase enzyme complex (35).
Protein phosphorylation is a central cell signaling event that underlies a broad spectrum of key physiological processes. Advances in affinity chromatography and mass spectrometry are now providing the ability to identify and quantitate thousands of phosphorylation sites simultaneously. Comprehensive phosphoproteome analyses present sizable analytical challenges in view of suppression effects of phosphopeptides and the variable quality of MS/MS spectra. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of titanium dioxide (TiO₂)-enriched cell extracts. The correlation of retention times of more than 750 phospho- and dephosphopeptide pairs from J774 macrophage cell extracts indicated that removal of the phosphate groups can impart a gain or a loss in hydrophobicity that is partly explained by the formation of a salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity that facilitated peptide sequencing. More importantly, alkaline phosphatase digestion enhanced the overall population of putative phosphopeptides from TiO₂-enriched cell extracts providing a unique approach to profile multiphosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon-γ for 5 min. TiO₂ enrichment enabled the identification of 1143 phosphopeptides from 432 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-γ exposure. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-γ signaling events involved in vesicle trafficking, production of reactive oxygen species, and mRNA translation.
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Atherosclerosis is a multifactorial disease for which the molecular etiology of many of the risk factors is still unknown. As no single genetic marker or test accurately predicts cardiovascular death, phenotyping for markers of inflammation may identify the individuals at risk for vascular diseases. Reactive oxygen species (ROS) are key mediators of signaling pathways that underlie vascular inflammation in atherogenesis, starting from the initiation of fatty streak development through lesion progression to ultimate plaque rupture. Various animal models of atherosclerosis support the notion that ROS released from NAD(P)H oxidases, xanthine oxidase, lipoxygenases, and enhanced ROS production from dysfunctional mitochondrial respiratory chain indeed have a causatory role in atherosclerosis and other vascular diseases. Human investigations also support the oxidative stress hypothesis of atherogenesis. This is further supported by the observed impairment of vascular function and enhanced atherogenesis in animal models that have deficiencies in antioxidant enzymes. The importance of oxidative stress in atherosclerosis is further emphasized because of its role as a unifying mechanism across many vascular diseases. The main contraindicator for the role oxidative stress plays in atherosclerosis is the lack of effectiveness of antioxidants in reducing primary endpoints of cardiovascular death and morbidity. However, this lack of effectiveness by itself does not negate the existence or causatory role of oxidative stress in vascular disease. Lack of proven markers of oxidative stress, which could help to identify a subset of population that can benefit from antioxidant supplementation, and the complexity and subcellular localization of redox reactions, are among the factors responsible for the mixed outcomes in the use of antioxidants for the prevention of cardiovascular diseases. To better understand the role of oxidative stress in vascular diseases, future studies should be aimed at using advances in mouse and human genetics to define oxidative stress phenotypes and link phenotype with genotype.
Arachidonic acid activates tissue transglutaminase and stress fiber formation via intracellular reactive oxygen species
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Citation Excerpt :
These results suggest that tTGase plays a major role in TGase activation by AA in NIH3T3 cells. There have been reports that AA stimulated the generation of intracellular ROS [15,32,33] and AA-induced cellular processes were mediated by the intracellular ROS [16,24]. Thus, we investigated whether the activation of tTGase by AA was mediated by the production of intracellular ROS in NIH 3T3 cells.
We have investigated whether arachidonic acid could regulate tissue transglutaminase (tTGase) via intracellular reactive oxygen species (ROS) in NIH3T3 cells. tTGase was identified in NIH3T3 cells by Western blot and confocal microscopy. Arachidonic acid elevated in situ tTGase activity in dose- and time-dependent manners with a maximal level at 1 h, and ROS scavengers, N-(2-mercaptopropionyl)glycine and catalase, blocked the tTGase activation by arachidonic acid. The activation of tTGase by arachidonic acid was largely inhibited by transfection of tTGase siRNA. The role of intracellular ROS in the activation of in situ tTGase was supported by the activation of in situ tTGase by exogenous H₂O₂. Arachidonic acid stimulated the formation of stress fibers in a dose- and time-dependent manner, and the ROS scavengers suppressed the arachidonic acid-induced formation of stress fibers. These results suggested that the activation of in situ tTGase and stress fiber formation by arachidonic acid was mediated by intracellular ROS in NIH3T3 cells.
Cytosolic phospholipase A<inf>2</inf> (cPLA<inf>2</inf>) regulation of human monocyte NADPH oxidase activity: cPLA<inf>2</inf> affects translocation but not phosphorylation of p67<sup>phox</sup> and p47<sup>phox</sup>
2002, Journal of Biological Chemistry
Citation Excerpt :
Kadri-Hassani et al. (55) reported that 13-methyltetradecanoic acid and AA have bimodal effects on superoxide anion production by human adherent monocytes activated by phorbol 12-myristate 13-acetate or diacylglycerol through the modulation of protein kinase C as well as p47 phox translocation. AA (30–50 μm) also increased NADPH oxidase activity in monocytic U937 cells by accelerating translocation of p47 phox and co-stimulation of protein kinase C (29). Therefore, cPLA2 or AA may have multiple effects on regulation of NADPH oxidase.
The NADPH oxidase of human monocytes is activated upon exposure to opsonized zymosan and a variety of other stimuli to catalyze the formation of superoxide anion. Assembly of the NADPH oxidase complex is believed to be a highly regulated process, and molecular mechanisms responsible for this regulation have yet to be fully elucidated. We have previously reported that cytosolic phospholipase A₂ (cPLA₂) expression and activity are essential for superoxide anion production in activated human monocytes. In this study, we investigated the mechanisms involved in cPLA₂ regulation of NADPH oxidase activation by evaluating the effects of cPLA₂ on translocation and phosphorylation of p67 ^phox and p47 ^phox . We report that translocation and phosphorylation of p67 ^phox , as well as p47 ^phox , occur upon activation of human monocytes and that decreased cPLA₂ protein expression, mediated by antisense oligodeoxyribonucleotides (AS-ODN) specific for cPLA₂mRNA, blocked the stimulation-induced translocation of p47 ^phox and p67 ^phox from the cytosol to the membrane fraction. Inhibition of translocation of both p47 ^phox and p67 ^phox by cPLA₂ AS-ODN was above 85%. Arachidonic acid (AA), a product of cPLA₂ enzymatic activity, completely restored translocation of both of these oxidase components in the AS-ODN-treated, cPLA₂-deficient human monocytes. These results represent the first report that cPLA₂activity or AA is required for p67 ^phox and p47 ^phox translocation in human monocytes. Although cPLA₂ was required for translocation of p47 ^phox and p67 ^phox , it did not influence phosphorylation of these components. These results suggest that one mechanism of cPLA₂ regulation of NADPH oxidase activity is to control the arachidonate-sensitive assembly of the complete oxidase complex through modulating the translocation of both p47 ^phox and p67 ^phox . These studies provide insight into the mechanisms by which activation signals are transduced to allow the induction of superoxide anion production in human monocytes.
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Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin: Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo
1999, Journal of Biological Chemistry
Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O⨪₂ and H₂O₂ generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47^phox and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47^phox and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47^phoxmight be similarly regulated in vivo in a rat aorta balloon injury model and found that p47^phox protein was increased after injury. Immunocytochemistry localized expression of p47^phox to the neointima and media of injured arteries. Our data demonstrate that generation of O⨪₂ and H₂O₂ is required for thrombin-mediated mitogenesis in VSMC and that p47^phox is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.

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^☆: This work was supported by grants from the Bundesministerium für Forschung und Technologie (07 ERG 037), the Deutsche Forschungsgemeinschaft (We 681/6-10), and the August Lenz-Stiftung.

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General paperArachidonic acid increases activation of NADPH oxidase in monocytic U937 cells by accelerated translocation of p47-phox and co-stimulation of protein kinase C☆

Abstract

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Biochem. Sci.

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J. Biol. Chem.

Biochim. Biophys. Acta

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J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Blood

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General paper
Arachidonic acid increases activation of NADPH oxidase in monocytic U937 cells by accelerated translocation of p47-phox and co-stimulation of protein kinase C☆