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Flohe L; Brigelius-Flohe R; Saliou C; Traber MG; Packer L
Redox regulation of NF-kappa B activation.
Department of Molecular and Cell Biology,
University of California, Berkeley, CA 94720-3200
Free Radic Biol Med 1997;22(6):1115-26
Cytosolic reactions of the nuclear factor kappa B/inhibitor (NF- kappaB/IkappaB) complex leading to its activation, NF-kappaB translocation into the nucleus, DNA binding, and transactivation have been described with some degree of clarity, but the upstream processes that stimulate those cytosolic reactions remain obscure. These processes definitely involve multiple protein serine/threonine kinases, as proximal modifiers of IkappaB, as well as the corresponding phosphatases, upstream kinases, and phosphatases, including those acting on tyrosine residues. This complex cascade of phosphorylation and dephosphorylation is modulated by redox reactions of unknown nature in the sense that the oxidant status of the cytosol increases the phosphorylation and degradation of IkappaB. NF-kappaB action, however, requires a thioredoxin-dependent reduced status in the nucleus. Upstream kinase(s) and or phosphatase(s) prone to thiolation or oxidation of vicinal SH groups are at present considered the best candidates mediating the redox regulation of NF-kappaB.

Blackwell TS; Christman JW
The role of nuclear factor-kappa B in cytokine gene regulation.
Department of Veterans Affairs, Nashville, Tennessee
Am J Respir Cell Mol Biol 1997 Jul;17(1):3-9
Transcription factors are DNA-binding proteins that regulate gene expression. Nuclear factor-kappa B (NF-kappa B) is a critical transcription factor for maximal expression of many cytokines that are involved in the pathogenesis of inflammatory diseases, such as adult respiratory distress syndrome (ARDS) and sepsis syndrome. Activation and regulation of NF-kappa B are tightly controlled by a group of inhibitory proteins (I kappa B) that sequester NF-kappa B in the cytoplasm of immune/inflammatory effector cells. NF-kappa B activation involves signaled phosphorylation, ubiquitination, and proteolysis of I kappa B. Liberated NF-kappa B migrates to the nucleus, where it binds to specific promoter sites and activates gene transcription. The activation of NF-kappa B initiates both extracellular and intracellular regulatory events that result in autoregulation of the inflammatory cascade through modulation of NF-kappa B activation. Recently, activation of NF-kappa B has been linked to ARDS and has been shown to be a critical proximal step in the initiation of neutrophilic inflammation in animal models. Activation of NF-kappa B can be inhibited in vivo by treatment with antioxidants, corticosteroids, and the induction of endotoxin tolerance. Identification of more specific and efficacious inhibitors of NF-kappa B activation might prove beneficial for the treatment of cytokine-mediated inflammatory diseases.

Adcock IM
Transcription factors as activators of gene transcription: AP-1 and NF-kappa B.
Division of Thoracic Medicine, Imperial College, School
of Medicine at NHLI, National Heart & Lung Institute, London, UK
Monaldi Arch Chest Dis 1997 Apr;52(2):178-86
Cells respond to a range of cytokines and other inflammatory stimuli by selectively expressing a wide range of genes. These proinflammatory signals bind to receptors and initiate intracellular signalling cascades. This results in the activation of proinflammatory deoxyribonucleic acid (DNA)-binding proteins or transcription factors such as activator protein-1 (AP-1) or nuclear factor-kappa B (NF-kappa B). Following activation, these factors bind to specific recognition sequences in the control regions (promoters) of target genes causing modulation of gene transcription. Furthermore, numerous sites for regulation of cytokine and cytokine receptor genes by these transcription factors are found in their promoter regions. Many factors affect the formation and activity of AP-1 dimers (Fos and Jun heterodimers) through protein specific interactions or by the modulation of pre-existing complexes by phosphorylation. These complexes can vary markedly in their ability to stimulate gene transcription. Thus, induction of AP-1 activity is regulated by stimuli that either induce the de novo synthesis of AP-1 subunits or increase the activity of previously formed AP-1 dimers. NF-kappa B is activated by a number of agents including tumour necrosis factor-alpha (TNF- alpha), interleukin-1 beta (IL-1 beta), lipopolysaccharide (LPS) and viruses. NF-kappa B plays a central role in a range of immunological responses due to its ability to switch on inflammatory genes which lead to further activation of NF-kappa B is a heterodimer of proteins which vary in their ability to bind to DNA and/or to activate gene transcription. NF-kappa B activation is primarily regulated by sequestration of heterodimers within the cytoplasm as inactive complexes with inhibitory molecules (inhibitory-kappa B (I-kappa B)). Treatment of cells with inducing agents results in the phosphorylation of the I-kappa B molecule which is targeted for rapid degradation. This causes a rapid dissociation of the cytoplasmic NF-kappa B/I-kappa B complexes and allows translocation of the active NF-kappa B to the nucleus. Cross-coupling between NF-kappa B and AP-1 has been described which results in a synergistic increase in activity at both AP-1 and NF- kappa B sites. Elevation of both AP-1 and NF-kappa B, as reported in asthma, may therefore lead to far greater inflammation than would be present if either transcription factor alone were activated.

Meyer M; Pahl HL; Baeuerle PA
Regulation of the transcription factors NF-kappa B and AP-1 by redox changes.
Laboratory for Molecular Biology, Gene Center, Martinsried, Germany.
Chem Biol Interact 1994 Jun;91(2-3):91-100
There is increasing evidence that reactive oxygen intermediates (ROIs) play an important role in cellular processes such as signal transduction and the control of gene expression. The activity of transcription factors like AP-1 and NF-kappa B is modulated by the redox state of the cell. NF-kappa B is activated by a prooxidant state in the cell and is therefore potentially inhibited by antioxidants. In contrast, AP-1 is strongly activated by antioxidants and shows reduced activity in the presence of oxidants. The antioxidant-mediated activation of AP-1 relies on the de novo synthesis of c-fos and c-jun mRNAs. Induction of c-fos expression is primarily mediated by transcription factors binding to the SRE element in the c-fos promoter. Thus SRE binding factors can be considered as primary antioxidant response factors, while AP-1 itself is a secondary response factor.

Toledano MB; Leonard WJ
Modulation of transcription factor NF-kappa B binding
activity by oxidation-reduction in vitro.
Cell Biology and Metabolism Branch,
National Institute of Child Health and Human Development,
National Institutes of Health, Bethesa, MD 20892.
Proc Natl Acad Sci U S A 1991 May 15;88(10):4328-32
NF-kappa B is a widely used regulator of inducible and tissue-specific gene control. In the cytosol, when complexed to an inhibitory molecule, I kappa B, NF-kappa B is in an inactive form and cannot bind DNA. Activation of cells with appropriate stimuli results in the dissociation of NF-kappa B from I kappa B and its translocation to the nucleus as an active binding protein. We now demonstrate that NF-kappa B binding in vitro can be inhibited by agents that modify free sulfhydryls. Binding is eliminated after treatment with N-ethylmaleimide, an alkylating agent, and diamide, an oxidizing agent. The diamide effect can be reversed by 2-mercaptoethanol. Further, 2-mercaptoethanol acts synergistically with deoxycholate plus Nonidet P-40 in converting inactive cytosolic NF-kappa B to an active DNA-binding form. It is therefore possible that modulation of the redox state of NF-kappa B could represent a post-translational control mechanism for this factor.

Galter D; Mihm S; Droge W
Distinct effects of glutathione disulphide on the nuclear
transcription factor kappa B and the activator protein-1.
Division of Immunochemistry, Deutsches Krebsforschungszentrum,
Heidelberg, Germany.
Eur J Biochem 1994 Apr 15;221(2):639-48
Oxidative conditions potentiate the activation of the nuclear transcription factor kappa B (NF kappa B) and the activator protein-1 (AP-1) in intact cells, but inhibit their DNA binding activity in vitro. We now show that both the activation of NF kappa B and the inhibition of its DNA binding activity is modulated in intact cells by the physiological oxidant glutathione disulphide (GSSG). NF kappa B activation in human T lineage cells (Molt-4) by 12-O-tetradecanoyl-phorbol 13-acetate was inhibited by dithiothreitol, and this was partly reversed by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or by hydrogen peroxide, indicating that GSSG may be required for NF kappa B activation. These effects of BCNU and hydrogen peroxide were not seen in glutathione-depleted cells. However, NF kappa B and AP-1 activation were potentiated by dithiothreitol if added to cell cultures 1 h after the phorbol ester, indicating that a shift of redox conditions may support optimal oxidative activation with minimal inhibition of DNA binding. The elevation of intracellular GSSG levels by BCNU before stimulation suppressed the chloramphenicol acetyltransferase expression dependent on NF kappa B but increased that dependent on AP-1. This selective suppression of NF kappa B was also demonstrable by electrophoretic mobility shift assays. In vitro, GSSG inhibited the DNA binding activity of NF kappa B more effectively than that of AP-1, while AP-1 was inhibited more effectively by oxidized thioredoxin.

Schreck R; Albermann K; Baeuerle PA
Nuclear factor kappa B: an oxidative stress-responsive
transcription factor of eukaryotic cells (a review).
Laboratory for Molecular Biology,
Ludwig-Maximilians-University, Martinsried, Germany.
Free Radic Res Commun 1992;17(4):221-37
NF-kappa B is a multiprotein complex that can activate a great variety of genes involved in early defence reactions of higher organisms. In nonstimulated cells, NF-kappa B resides in the cytoplasm in an inactive complex with the inhibitor I kappa B. Pathogenic stimuli cause release of I kappa B and allow NF-kappa B to enter the nucleus, bind to DNA control elements and, thereby, induce the synthesis of mRNA. A puzzling feature of NF-kappa B is that its activation is triggered by a great variety of agents. These include the cytokines interleukin-1 and tumor necrosis factor, viruses, double-stranded RNA, endotoxins, phorbol esters, UV light and ionizing radiation. We recently found that also low concentrations of H2O2 activate NF-kappa B and that various antioxidants prevent the induction by H2O2. Subsequent analysis revealed that antioxidants not only suppress the activation of NF-kappa B by H2O2 but by all other inducers tested so far. In this review, we will discuss the evidences that NF-kappa B is an oxidative stress-responsive transcription factor of higher eukaryotic cells.

Droge W; Schulze-Osthoff K; Mihm S; Galter D; and others
Functions of glutathione and glutathione disulfide
in immunology and immunopathology.
Department of Immunochemistry, Deutsches Krebsforschungszentrum,
Heidelberg, Germany.
FASEB J 1994 Nov;8(14):1131-8
Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NF kappa B, whereas high GSSG levels inhibit the DNA binding activity of NF kappa B. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fyn are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox-regulated enzymes trigger signal cascades for NF kappa B activation and transduce signals from the T cell antigen receptor, from CD4 and CD8 molecules, and from the IL-2 receptor beta-chain. The effector phase of cytotoxic T cell responses and IL-2-dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well-known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV-infected patients and SIV-infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.

Lee JS; Kahlon SS; Culbreth R; Cooper AD Jr
Modulation of monocyte chemokine production
and nuclear factor kappa B activity by oxidants.
Pulmonary Sections, Birmingham V.A.M.C., AL 35233
J Interferon Cytokine Res 1999 Jul;19(7):761-7B
Reactive oxygen species can directly damage tissue. In this setting, amplification of tissue damage also occurs through infiltration of inflammatory cells either acutely or chronically. Several recent studies suggest that reactive oxygen species stimulate production of certain chemokines, which are potent chemoattractants for inflammatory cells. In the present study, we examined whether oxidants, generated by the combination of xanthine and xanthine oxidase (X/XO), alter chemokine production by monocytes and U937 cells. Our findings demonstrate that X/XO stimulates monocytes, but not U937 cells, to produce increased amounts of interleukin-8 (IL-8) and monocyte chemoattractant protein. This effect is attenuated by pretreatment with dimethylsulfoxide (DMSO), a scavenger of hydroxyl radicals, but is not affected by superoxide dismutase or catalase. In contrast, X/XO-induced cytotoxicity, evidenced by lactate dehydrogenase release, is mediated primarily by hydrogen peroxide, as catalase reverses this effect. Finally, exposure to X/XO causes an increase in nuclear factor kappa B (NF-kappaB), and this effect is attenuated by DMSO. These studies suggest that reactive oxygen species can induce production of molecules that amplify inflammation through attraction of inflammatory cells. It appears the hydroxyl radical is the principal oxidant species involved in stimulation of chemokine production.

Desai A; Huang X; Warren JS
Intracellular glutathione redox status modulates MCP-1
expression in pulmonary granulomatous vasculitis.
Department of Pathology, University of Michigan, Medical School, Ann Arbor 48109
Lab Invest 1999 Jul;79(7):837-47
A wide spectrum of human lung diseases is characterized by the presence of granulomas. Although understanding of the pathways leading to their development remains incomplete, data from in vitro studies suggest that neutrophils, monocytes, and their secreted products (eg, hydrogen peroxide, H2O2) influence the pathogenesis of pulmonary granulomatous disease through the regulation of local chemokine and cytokine production. Using a well-characterized rat model of glucan-induced pulmonary granulomatous vasculitis, we sought to determine the role of intracellular glutathione (GSH) redox status in the expression of monocyte chemoattractant protein-1 (MCP-1). Previous studies have revealed that vascular wall MCP-1 expression is obligatory for granuloma development and that both neutrophils and hydrogen peroxide are required for MCP-1 induction. Because in vitro expression of MCP-1 is in part mediated by the redox-sensitive transcription factors nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1), we studied their activation as a function of varying intracellular GSH redox status in the pathogenesis of glucan-induced pulmonary granulomatosis. Infusion of particulate yeast cell wall glucan into rats resulted in a rapid decrease in intracellular GSH concentrations which was accompanied by the activation of NF-kappaB and AP-1. The pattern of AP-1 and NF-kappaB activation in turn correlated temporally with the expression of MCP-1. Administration of L-buthionine-S, R- sulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase, resulted in a significant reduction in intracellular GSH pools. GSH depletion resulted in a more than 100% increase in pulmonary MCP-1 concentrations and increased cytosolic to nuclear translocation of NF-kappaB while having no effect on AP-1 levels. These observations suggest that in the pathogenesis of pulmonary granulomatous disease, intracellular glutathione redox status modulates the expression of MCP- 1 through redox-sensitive transcription factors.

Christman JW; Lancaster LH; Blackwell TS
Nuclear factor kappa B: a pivotal role in the systemic inflammatory
response syndrome and new target for therapy.
Department of Medicine, Vanderbilt University School of Medicine
and the Department of Veterans Affairs, Nashville, TN 37322
Intensive Care Med 1998 Nov;24(11):1131-8
Comment in: Intensive Care Med 1998 Nov;24(11):1129-30
NF-kappaB is an important transcription factor complex that appears to play a fundamental role in regulating acute inflammation through activation of the cytokine cascade and production of other pro- inflammatory mediators. There is increasing evidence that NF-kappaB is important in the pathobiology of disease states such as SIRS, MODS and ARDS; therefore, therapeutic interventions aimed at limiting NF-kappaB activation and down-regulating production of inflammatory mediators could prove to be beneficial in decreasing host-derived tissue injury and organ dysfunction. Specific interventions that hold promise for suppressing NF-kappaB activation include the use of antioxidants, inhibition of NIK and the IKK signalsome, treatment with proteasome inhibitors, induction of endotoxin tolerance and, possibly the use of corticosteroids in selected patients.

Milligan SA; Owens MW; Grisham MB
Differential regulation of extracellular signal-regulated kinase
and nuclear factor-kappa B signal transduction pathways
by hydrogen peroxide and tumor necrosis factor.
Department of Medicine, Overton Brooks Veterans Affairs Medical Center,
Shreveport, Louisiana
Arch Biochem Biophys 1998 Apr 15;352(2):255-62
Reactive oxygen metabolites are increasingly recognized for their ability to stimulate signal transduction pathways. This is important because these oxidants are frequently generated at sites of inflammation. However, little is known about the manner in which reactive oxygen species may selectively stimulate distinct signaling pathways. We have examined this question by stimulating mesothelial cells with hydrogen peroxide (H2O2) as a model oxidant stimulus. The response to H2O2 was examined by measuring the activation of the extracellular signal-regulated kinase (ERK1/2) and the nuclear factor- kappa B (NF-kappa B) signal transduction pathways. We found that H2O2 stimulated activity of the ERK1/2 pathway in a dose- and time-dependent manner. The ability of H2O2 to activate ERK1/2 was similar to that found with tumor necrosis factor (TNF) stimulation. The oxidant effect was inhibited by various reactive oxygen scavengers. An inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase, the upstream kinase that activates ERK1/2, inhibited the oxidant effect. The superoxide anion (O2-) also stimulated ERK1/2 activity. In contrast, H2O2 did not stimulate proteolysis of I kappa B-alpha and induced only a small degree of NF-kappa B nuclear translocation. Stimulation of the cells with O2- also induced a minimal degree of NF- kappa B activation. TNF was a potent stimulus for I kappa B-alpha proteolysis and NF-kappa B activation, demonstrating that the cells did have a functional NF-kappa B pathway. These results suggest that oxidants may selectively stimulate certain pathways, thereby preserving some specificity of the signaling process. Furthermore, different cell types and distinct signaling pathways within cells may demonstrate unique profiles in the manner in which they respond to oxidant stimulation.

Los M; Dröge W; Stricker K; Baeuerle PA; Schulze-Osthoff K
Hydrogen peroxide as a potent activator
of T lymphocyte functions.
Eur J Immunol 1995 Jan; 25(1):159-65
During inflammatory processes infiltrating cells produce large amounts of reactive oxygen intermediates (ROI). Increasing evidence suggests that ROI besides being cytotoxic may act as important mediators influencing various cellular and immunological processes. In this study, we have investigated the effects of hydrogen peroxide on several aspects of lymphocyte activation. In ESb-L T lymphoma cells, micromolar concentrations of hydrogen peroxide rapidly induced activation of the transcription factor NF-kappa B, whereas DNA-binding activity of the transcription factor AP-1 was virtually not affected. In addition, hydrogen peroxide induced early gene expression of interleukin-2 (IL-2) and the IL-2 receptor alpha chain. The stimulation of IL-2 expression was found to be conferred by a kappa B-like cis-regulatory region within the IL-2 gene promoter. In contrast to these activating effects, addition of hydrogen peroxide was largely inhibitory on cell proliferation which is consistent with a general requirement of thiol compounds for lymphocyte proliferation. However, hydrogen peroxide significantly increased T cell proliferation when applied for a short period under reducing conditions. These data indicate that ROI may act as an important competence signal in T lymphocytes inducing early gene expression as well as cell proliferation.

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