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Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-kappaB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-kappaB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IkappaB-alpha degradation. Here we report the construction of p65/NF-kappaB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,120) --> Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-kappaB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Although a slight inhibition of IkappaB degradation was detected for all SLs, the amount of remaining IkappaB was too low to explain the observed NF-kappaB inhibition.

Investigation of Podachaenium eminens afforded nine sesquiterpene lactones (Sls) from which costunolide, 7-hydroxycostunolide, santamarin as well as 3-chlorodehydroleucodin are new for this plant and 3,4-dehydro-4-dehydroxypodachaenin (= 3-costoyloxydehydroleucodin) is found for the first time in nature. All isolated Sls were studied for their anti-inflammatory activity using the transcription factor NF-kappa B as a molecular target. NF-kappa B is involved in the synthesis of inflammatory mediators, such as cytokines and chemokines. Except for podachaenin, all compounds completely inhibited NF-kappa B DNA binding in an electrophoretic mobility shift assay at concentrations between 5 and 200 microM without showing any cytotoxic effects. 3,4-Epoxydehydroleucodin possessing an alpha-methylene-gamma-butyrolactone and a second reactive structure element by its epoxy ring alpha,beta to a carbonyl group was most active. Although the majority of the Sls tested in this study were monofunctional only low concentrations of 50 microM were often needed for complete inhibition. Possible reasons are discussed for this result.

In Central America aerial parts of the Asteraceae Milleria quinqueflora are used in traditional medicine as a remedy for skin infections. Reinvestigation of this plant afforded thirteen sesquiterpene lactones (Sls), three of them are new. All isolated Sls were studied for their anti-inflammatory activity using the transcription factor NF-kappa B as molecular target. NF-kappa B is involved in the synthesis of inflammatory mediators, such as cytokines and chemokines. NF-kappa B DNA binding was inhibited at micromolar concentrations by all Sls.

The vertebrate transcription factor NF-kappaB is induced by over 150 different stimuli. Active NF-kappaB, in turn, participates in the control of transcription of over 150 target genes. Because a large variety of bacteria and viruses activate NF-kappaB and because the transcription factor regulates the expression of inflammatory cytokines, chemokines, immunoreceptors, and cell adhesion molecules, NF-kappaB has often been termed a 'central mediator of the human immune response'. This article contains a complete listing of all NF-kappaB inducers and target genes described to date. The collected data argue that NF-kappaB functions more generally as a central regulator of stress responses. In addition, NF-kappaB activation blocks apoptosis in several cell types. Coupling stress responsiveness and anti-apoptotic pathways through the use of a common transcription factor may result in increased cell survival following stress insults.

Many sesquiterpene lactones (SLs) possess considerable anti-inflammatory activity. They inhibit the transcription factor NF-kappaB by selectively alkylating its p65 subunit probably by reacting with cysteine residues. Here we assayed 28 sesquiterpene lactones for their ability to inhibit NF-kappaB. The majority of the potent NF-kappaB inhibitors possess two reactive centers in form of an alpha-methylene-gamma-lactone group and an alpha,beta- or alpha,beta,gamma,delta-unsaturated carbonyl group. Based on computer molecular modelling we propose a molecular mechanism of action, which is able to explain the p65 selectivity of the SLs and the observed correlation of high activity with alkylant bifunctionality. A single bifunctional SL molecule can alkylate the cysteine residue (Cys 38) in the DNA binding loop 1 (L1) and a further cysteine (Cys 120) in the nearby E' region. This cross link alters the position of tyrosine 36 and additional amino acids in such a way that their specific interactions with the DNA become impossible. We also created a model for monofunctional SLs.

The sesquiterpene lactone helenalin is a potent anti-inflammatory drug whose molecular mechanism of action remains unclear despite numerous investigations. We have previously shown that helenalin and other sesquiterpene lactones selectively inhibit activation of the transcription factor NF-kappaB, a central mediator of the human immune response. These drugs must target a central step in NF-kappaB pathway, since they inhibit NF-kappaB induction by four different stimuli. It has previously been reported that sesquiterpene lactones exert their effect by inhibiting degradation of IkappaB, the inhibitory subunit of NF-kappaB. These data contradicted our report that IkappaB is not detectable in helenalin-treated, ocadaic acid-stimulated cells. Here we use confocal laser scanning microscopy to demonstrate the presence of IkappaB-released, nuclear NF-kappaB in helenalin-treated, tumor necrosis factor-alpha stimulated cells. These data show that neither IkappaB degradation nor NF-kappaB nuclear translocation are inhibited by helenalin. Rather, we provide evidence that helenalin selectively alkylates the p65 subunit of NF-kappaB. This sesquiterpene lactone is the first anti-inflammatory agent shown to exert its effect by directly modifying NF-kappaB.

In Central America leaf extracts from the Asteraceae Tithonia diversifolia are used externally for the treatment of haematomas and wounds. Therefore, the main sesquiterpene lactones (Sls) of this species growing in Costa Rica, diversifolin (1), diversifolin methyl ether (2), and tirotundin (3), were studied for their anti-inflammatory activity. We determined whether these compounds inhibit cyclooxygenase-I, phospholipase A2, or the transcription factor NF-kappa B. Here we show that these Sls do not influence the enzymes of the arachidonic acid pathway, but inhibit the activation of NF-kappa B. Thereby, the synthesis of inflammatory mediators such as cytokines and chemokines is reduced. Our results indicate that the inhibitory activity of compounds 1-3 is due to alkylation of cysteine residues, which are probably located in the DNA binding domain of NF-kappa B. The Sls were also studied for their antibacterial activity, but only Sl 1 was moderately active against Bacillus subtilis in the agar plate diffusion test.

Helicobacter pylori is the etiological agent in the development of chronic gastritis, duodenal ulceration, and gastric adenocarcinoma. The difference in virulence between individual strains is reflected in their ability to induce interleukin-8 (IL-8) secretion from gastric epithelial cells. It has been shown that virulence is associated with the presence of a bacterial gene cluster (a pathogenicity island). We have recently demonstrated that H. pylori-mediated IL-8 secretion requires activation of the transcription factor NF-kappaB. Here, we show that NF-kappaB induction requires six membrane proteins encoded within the pathogenicity island.

Helicobacter pylori is an etiologic agent in the development of chronic gastritis, duodenal ulceration, and gastric adenocarcinoma. Exposure of gastric epithelial cells to H. pylori induces secretion of the cytokine IL-8, which plays a pivotal role in the immunopathogenesis of H. pylori infections. Isolated Helicobacter strains differ in their virulence and in their ability to induce cytokine production. High degrees of virulence correlate with enhanced IL-8 production. However, the molecular mechanism of this variance in Helicobacter pathogenicity remains poorly understood. Here we show that H. pylori-mediated IL-8 secretion requires activation of the transcription factor nuclear factor-kappaB (NF-kappaB) in a gastric epithelial cell line. Several H. pylori strains which fail to induce IL-8 secretion do not activate NF-kappaB, while all IL-8-inducing strains activate the transcription factor. Moreover, the antioxidant curcumin, which inhibits NF-kappaB activation, also completely suppresses IL-8 induction by H. pylori. NF-kappaB activation is not mediated by LPSs, since purified H. pylori LPS had no effect on gastric epithelial cells. In contrast, both IL-8 secretion and NF-kappaB activation require a secreted H. pylori product, which is not secreted by strains mutated in picB/cagE, a recently identified putative transport protein.

Alcoholic extracts prepared form Arnicae flos, the collective name for flowerheads from Arnica montana and A. chamissonis ssp. foliosa, are used therapeutically as anti-inflammatory remedies. The active ingredients mediating the pharmacological effect are mainly sesquiterpene lactones, such as helenalin, 11alpha,13-dihydrohelenalin, chamissonolid and their ester derivatives. While these compounds affect various cellular processes, current data do not fully explain how sesquiterpene lactones exert their anti-inflammatory effect. We show here that helenalin, and, to a much lesser degree, 11alpha,13-dihydrohelenalin and chamissonolid, inhibit activation of transcription factor NF-kappaB. This difference in efficacy, which correlates with the compounds' anti-inflammatory potency in vivo, may be explained by differences in structure and conformation. NF-kappaB, which resides in an inactive, cytoplasmic complex in unstimulated cells, is activated by phosphorylation and degradation of its inhibitory subunit, IkappaB. Helenalin inhibits NF-kappaB activation in response to four different stimuli in T-cells, B-cells and epithelial cells and abrogates kappaB-driven gene expression. This inhibition is selective, as the activity of four other transcription factors, Oct-1, TBP, Sp1 and STAT 5 was not affected. We show that inhibition is not due to a direct modification of the active NF-kappaB heterodimer. Rather, helenalin modifies the NF-kappaB/IkappaB complex, preventing the release of IkappaB. These data suggest a molecular mechanism for the anti-inflammatory effect of sesquiterpene lactones, which differs from that of other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and acetyl salicylic acid.

Various conditions that perturb the function of the endoplasmic reticulum (ER) were recently shown to activate the transcription factor NF-kappa B. Activation of NF-kappa B is caused by the accumulation of proteins in the ER membrane, a condition we have called ER overload. Both the release of Ca2+ from the ER and the subsequent production of reactive oxygen intermediates are required for ER-overload-mediated NF-kappa B activation. This novel intracellular signal transduction pathway might be important in antiviral defense and play a role in various diseases as well as in B-cell development.

The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.

The eukaryotic transcription factor NF-kappaB is activated by a large variety of stimuli. We have recently shown that ER stress, caused by an aberrant accumulation of membrane proteins within this organelle, also activates NF-kappaB. Here, we show that activation of NF-kappaB by ER stress requires an increase in the intracellular levels of both reactive oxygen intermediates (ROIs) and Ca2+. Two distinct intracellular Ca2+ chelators and a panel of structurally unrelated antioxidants prevented NF-kappaB activation by various ER stress-eliciting agents, whereas only antioxidants but not the Ca2+ chelators prevented NF-kappaB activation by the inflammatory cytokine TNF-alpha. Consistent with an involvement of calcium, the ER-resident Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid (CPA), which trigger a rapid efflux of Ca2+ from the ER, also potently activated NF-kappaB. Pretreatment with a Ca2+ chelator abrogated this induction. The Ca2+ chelator BAPTA-AM inhibited ROI formation in response to thapsigargin and CPA treatment, suggesting that the Ca2+ increase preceded ROI formation during NF-kappaB activation. The selective inhibitory effect of the drug tepoxalin suggests that the peroxidase activity of cyclooxygenases or lipoxygenases was responsible for the increased ROI production in response to Ca2+ release by thapsigargin.

Opportunistic infections, such as aspergillosis, are among the most serious complications suffered by immunocompromised patients. Aspergillus fumigatus and other pathogenic fungi synthesize a toxic epipolythiodioxopiperazine metabolite called gliotoxin. Gliotoxin exhibits profound immunosuppressive activity in vivo. It induces apoptosis in thymocytes, splenocytes, and mesenteric lymph node cells and can selectively deplete bone marrow of mature lymphocytes. The molecular mechanism by which gliotoxin exerts these effects remains unknown. Here, we report that nanomolar concentrations of gliotoxin inhibited the activation of transcription factor NF-kappaB in response to a variety of stimuli in T and B cells. The effect of gliotoxin was specific because, at the same concentrations, the toxin did not affect activation of the transcription factor NF-AT or of interferon-responsive signal transducers and activators of transcription. Likewise, the activity of the constitutively DNA-binding transcription factors Oct-1 and cyclic AMP response element binding protein (CREB), as well as the activation of protein tyrosine kinases p56lck and p59fyn, was not altered by gliotoxin. Very high concentrations of gliotoxin prevented NF-kappaB DNA binding in vitro. However, in intact cells, inhibition of NF-kappaB did not occur at the level of DNA binding; rather, the toxin appeared to prevent degradation of IkappaB-alpha, NF-kappaB's inhibitory subunit. Our data raise the possibility that the immunosuppression observed during aspergillosis results in part from gliotoxin-mediated NF-kappaB inhibition.

We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappaB. This defined a novel ER-nuclear signal transduction pathway, which is distinct from the previously described unfolded protein response (UPR). The well characterized UPR pathway is activated by the presence of un- or malfolded proteins in the ER. In contrast, the ER stress signal which activates the NF-kappaB pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappaB-activating signal emitted by the ER. E3/19K resides in the endoplasmic reticulum where it binds to MHC class I molecules, thereby preventing their transport to the cell surface. It is maintained in the ER by a retention signal sequence in its carboxy terminus, which causes the protein to be continuously retrieved to the ER from post-ER compartments. Mutation of this sequence allows E3/19K to reach the cell surface. We show here that expression of E3/19K potently activates a functional NF-kappaB transcription factor. The activated NF-kappaB complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappaB activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF-kappaB. However, activation of both NF-kappaB DNA binding and kappaB-dependent transactivation relied on E3/19K ER retention: mutants, which were expressed on the cell surface, could no longer activate the transcription factor. This identifies the NF-kappaB-activating signal as the accumulation of proteins in the ER membrane, a condition we have termed "ER overload." We show that ER overload-mediated NF-kappaB activation but not TNF-stimulated NF-kappaB induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca(2+) -dependent ATPase, thapsigargin and cyclopiazonic acid, which causes a rapid release of Ca2+ from the ER, strongly activated NF-kappaB. We therefore propose that ER overload activates NF-kappaB by causing Ca2+ release from the ER. Because NF-kappaB plays a key role in mounting an immune response, ER overload caused by viral proteins may constitute a simple antiviral response with broad specificity.

Oxygen and derived ROIs became a threat for all organisms more than two billion years ago. Both prokaryotic and higher eukaryotic cells are able to alter their genetic programme in response to changes in the intracellular levels of reactive oxygen intermediates (ROIs). In bacteria and yeast, this response leads to the new synthesis of proteins that protect the induced cells from the consequences of oxidative damage, such as DNA strand breaks, lipid peroxidation and oxidative damage of proteins, thereby ensuring growth in a prooxidant environment. In higher eukaryotic cells, oxidative stress can be the consequence of reoxygenation of ischemic tissus or of exposure to environmental hazards. Intriguingly, multicellular organisms have also evolved cellular mechanisms to actively produce ROIs. In one case, the reactive compounds are needed as weapons against invading microorganisms. Granulocytes, neutrophils and macrophages have specialized in releasing of large amounts of H2O2 and superoxide. However, many other cell types can also inducibly produce ROIs but in amounts insufficient to threat microorganisms. There is increasing evidence that the small increases in ROI levels fulfil a role as second messengers. We propose that these pandemic pathogens have been conserved throughout evolution as universal pathogen messengers turning on genes with important functions in the immune response and cell proliferation. The higher eukaryotic transcription factor NF-kappa B will be described as a protein which is activated by ROIs under a great variety of pathogenic conditions and initiates the new expression of genes with important roles in inflammatory, immune and acute phase responses.

Post-translational activation of the higher eukaryotic transcription factor NF-kappa B requires both phosphorylation and proteolytic degradation of the inhibitory subunit I kappa B-alpha. Inhibition of proteasome activity can stabilize an inducibly phosphorylated form of I kappa B-alpha in intact cells, suggesting that phosphorylation targets the protein for degradation. In this study, we have identified serines 32 and 36 in human I kappa B-alpha as essential for the control of I kappa B-alpha stability and the activation of NF-kappa B in HeLa cells. A point mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. This and various other Ser32 and Ser36 mutants behaved as potent dominant negative I kappa B proteins attenuating kappa B-dependent transactivation in response to OA, phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF). While both endogenous and transiently expressed wild-type I kappa B-alpha were proteolytically degraded in response to PMA and TNF stimulation of cells, the S32/36A mutant of I kappa B-alpha remained largely intact under these conditions. Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kinase system to phosphorylate and thereby destabilize I kappa B-alpha, leading to NF-kappa B activation.

The inducible, higher eukaryotic transcription factor NF-kappa B is activated by a variety of external stimuli including inflammatory cytokines, viral and bacterial infection and UV irradiation. Here we show that internal stress, caused by the accumulation of proteins in the endoplasmic reticulum (ER), also induces NF-kappa B DNA binding as well as kappa B-dependent gene expression. This was observed upon expression of immunoglobulin mu chains in the absence of light chains and by treatment of cells with several agents known to cause ER stress, such as tunicamycin, brefeldin A, 2-deoxyglucose and thapsigsargin. The transcription factor AP-1 was weakly induced under similar conditions. Overexpression of NF-kappa B subunits did not influence expression of the gene encoding grp78/BiP, a protein induced by various forms of ER stress. Likewise, the glucosidase inhibitor castanospermine, which induced grp78/BiP expression, failed to activate NF-kappa B, while the antioxidant dithiothreitol augmented grp78/BiP expression but prevented activation of NF-kappa B. Hence, NF-kappa B participates in a novel ER-nuclear signal transduction pathway distinct from the unfolded-protein-response described previously. We provide evidence that the ER can produce at least two distinct signals in response to a functional impairment. One is emitted by the presence of unfolded proteins, the other in response to overloading of the organelle, for example through the overexpression of secretory proteins.

Influenza virus infection initiates transcription of a variety of genes for cytokines such as tumor necrosis factor alpha (TNF-alpha), TNF-beta, interleukin 1 alpha, (IL-1 alpha), IL-1 beta, IL-2, IL-4, IL-6, IL-10, granulocyte macrophage colony-stimulating factor, and gamma interferon. However, the mechanism by which virus infection elicits cytokine expression remains unknown. Six influenza virus-induced cytokine genes are targets for the inducible transcription factor NF-kappa B, a central regulator of the human immune response. Here, we show that expression of a single influenza virus protein, the virion surface hemagglutinin, strongly activates NF-kappa B DNA binding and transactivation. Activation is inhibited in the presence of the antioxidant dithiothreitol, suggesting that, similar to the findings for previously described inducers of NF-kappa B, hemagglutinin expression generates radical oxygen intermediates which activate the transcription factor. Hemagglutinin is the first secretory and structural viral protein reported to activate NF-kappa B and thus represents a new class of inducers for this transcription factor. We discuss these results in the context of clinical complications of influenza virus infection.

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.