Induction and repression of NF-kappa B-driven inflammatory genes.
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Animals↗
Biomedical subjects
W Vanden Berghe
Publications and source records attributed to W Vanden Berghe.
Glucocorticoid repression of AP-1 is not mediated by competition for nuclear coactivators.
Interleukin-6 (IL-6) is a pleiotropic cytokine that is involved in many autoimmune and inflammatory diseases. Transcriptional control of IL-6 gene expression is exerted by various compounds, among which glucocorticoids are the most potent antiinflammatory and immunosuppressive agents currently in use. Glucocorticoids exert their transrepressive actions by negatively interfering with transcription factors, such as nuclear factor-kappaB (NF-kappaB) and AP-1. Both factors make use of the coactivator cAMP response element-binding protein (CREB)-binding protein (CBP) to enhance their transcriptional activities, which led to the hypothesis that a mutual antagonism between p65 or c-Jun and activated glucocorticoid receptor (GR) results from a limited amount of CBP. Recently, we showed that glucocorticoid repression of NF-kappaB-driven gene expression occurs irrespective of the amount of coactivator levels in the cell. In the current study, we extend this observation and demonstrate that also AP-1-targeted gene repression by glucocorticoids is refractory to increased amounts of nuclear coactivators. From results with Gal4 chimeric proteins we conclude that glucocorticoid repression occurs by a promoter-independent mechanism involving a nuclear interplay between activated GR and AP-1, independently of CBP levels in the cell.
Binding, Competitive↗
Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6.
Interleukin (IL)-6 is a multifunctional cytokine that can be induced by a plethora of chemical or physiological compounds, including the inflammatory cytokines tumor necrosis factor (TNF) and IL-1. The molecule TNF has a trimeric configuration and thus binds to membrane-bound, cellular receptors to initiate cell death mechanisms and signaling pathways leading to gene induction. Previously, we showed that induced clustering of the intracellular domains of the p55 TNF receptor, or of their respective 'death domains' only, is sufficient to activate the nuclear factor kappa B (NF-kappa B) and several mitogen-activated protein kinase (MAPK) pathways. NF-kappa B is the exclusive transcription factor for induction of the IL-6 gene in response to TNF and functions as the final trigger to activate a multiprotein complex, a so-called 'enhanceosome', at the level of the IL-6 promoter. Furthermore, the enhanceosome displays histone acetylation activity, which turned out to be essential for IL-6 gene activation via NF-kappa B. However, activation of NF-kappa B alone is not sufficient for IL-6 gene induction in response to TNF, as inhibition of the coactivated extracellular signal-regulated kinase and p38 MAPK pathways blocks TNF-mediated gene expression. Nevertheless, the transactivating NF-kappa B subunit p65 is not a direct target of MAPK phosphorylation. Thus, we postulated that other components of the enhanceosome complex are sensitive to MAPK cascades and found that MAPK activity is unequivocally linked to the histone acetylation capacity of the enhanceosome to stimulate gene expression in response to TNF. In contrast, glucocorticoid repression of TNF-driven IL-6 gene expression does not depend on abrogation of histone acetyltransferase activity, but originates from interference of the liganded glucocorticoid receptor with the contacts between NF-kappa B p65 and the promoter configuration around the TATA box.
Animals↗
Mechanisms of anti-inflammatory action and of immunosuppression by glucocorticoids: negative interference of activated glucocorticoid receptor with transcription factors.
Glucocorticoids are the most widely used anti-inflammatory and immunomodulatory agents, whose mechanism of action is based mainly on interference with the activity of transcription factors, such as nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1). The precise molecular mechanisms of gene repression by glucocorticoids are a controversial matter, due to the existence of many conflicting hypotheses. We discuss the three main paradigms reported in the literature, namely the inhibitor kappaB-alpha (IkappaB-alpha) upregulatory model, the protein-protein interaction model and the competition model.
Animals↗
Glucocorticoids repress NF-kappaB-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell.
Glucocorticoids (GCs) are used to combat inflammatory diseases. Their beneficial effect relies mainly on the inhibition of NF-kappaB- and/or AP-1-driven proinflammatory gene expression. Previously, we have shown that GCs repress tumor necrosis factor-induced IL-6 gene expression by an NF-kappaB-dependent nuclear mechanism without changing the DNA-binding capacity of NF-kappaB or the expression levels of the cytoplasmic inhibitor of NF-kappaB (IkappaB-alpha). In the present work, we investigate the effect of GC repression on different natural and/or recombinant NF-kappaB-driven reporter gene constructs in the presence of increasing amounts of various coactivator molecules, such as CREB-binding protein (CBP), p300, and SRC-1. We found that GCs maintain their repressive capacities, irrespective of the amount of cofactor present in the cell. Similar results were obtained for the reciprocal transrepression of a GC receptor (GR) element-driven reporter gene by p65. We demonstrate that neither the expression levels of p65 and CBP nor their physical association are affected by activated GR. Using Gal4 chimeras, we show that repression by GCs is specific for p65-mediated transactivation, ruling out competition for limiting nuclear factors as the major underlying mechanism of gene repression. In addition, the transactivation potential of a point-mutated Gal4-p65 variant with a decreased CBP interaction capability is still repressed by GR. Finally, we present evidence that the specificity of GC repression on p65-driven gene expression is codetermined by the TATA box context.
Base Sequence↗
Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1.
Interleukin-6 (IL-6) is a pleiotropic cytokine, whose plasma levels are elevated in inflammatory diseases such as atherosclerosis. We have previously reported that peroxisome proliferator-activated receptor alpha (PPARalpha) ligands (fibrates) lower elevated plasma concentrations of IL-6 in patients with atherosclerosis and inhibit IL-1-stimulated IL-6 secretion by human aortic smooth muscle cells (SMC). Here, we show that aortic explants isolated from PPARalpha-null mice display an exacerbated response to inflammatory stimuli, such as lipopolysaccharide (LPS), as demonstrated by increased IL-6 secretion. Furthermore, fibrate treatment represses IL-6 mRNA levels in LPS-stimulated aortas of PPARalpha wild-type, but not of PPARalpha-null mice, demonstrating a role for PPARalpha in this fibrate action. In human aortic SMC, fibrates inhibit IL-1-induced IL-6 gene expression. Furthermore, activation of PPARalpha represses both c-Jun- and p65-induced transcription of the human IL-6 promoter. Transcriptional interference between PPARalpha and both c-Jun and p65 occurs reciprocally, since c-Jun and p65 also inhibit PPARalpha-mediated activation of a PPAR response element-driven promoter. This transcriptional interference occurs independent of the promoter context as demonstrated by cotransfection experiments using PPARalpha, p65, and c-Jun Gal4 chimeras. Overexpression of the transcriptional coactivator cAMP-responsive element-binding protein-binding protein (CBP) does not relieve PPARalpha-mediated transcriptional repression of p65 and c-Jun. Finally, glutathione S-transferase pull-down experiments demonstrate that PPARalpha physically interacts with c-Jun, p65, and CBP. Altogether these data indicate that fibrates inhibit the vascular inflammatory response via PPARalpha by interfering with the NF-kappaB and AP-1 transactivation capacity involving direct protein-protein interaction with p65 and c-Jun.
Animals↗
The nuclear factor-kappaB engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter.
Expression of the pleiotropic cytokine interleukin (IL)-6 can be stimulated by the proinflammatory cytokine tumor necrosis factor (TNF) and the microbial alkaloid staurosporine (STS). In this report, the transcriptional mechanisms were thoroughly investigated. Whereas transcription factors binding to the activator protein-1-, cAMP-responsive element-, and CAAT enhancer-binding protein-responsive sequences are necessary for gene activation by STS, nuclear factor (NF)-kappaB alone is responsible and sufficient for inducibility by TNF, which reveals distinct signaling pathways for both compounds. At the cofactor level, cAMP-responsive element-binding protein-binding protein (CBP) or p300 potentiate basal and induced IL-6 promoter activation via multiple protein-protein interactions with all transcription factors bound to the promoter DNA. However, the strongest promoter activation relies on the p65 NF-kappaB subunit, which specifically engages CBP/p300 for maximal transcriptional stimulation by its histone acetyltransferase activity. Moreover, treatment of chromatin-integrated promoter constructions with the histone deacetylase inhibitor trichostatin A exclusively potentiates TNF-dependent (i.e. NF-kappaB-mediated) gene activation, while basal or STS-stimulated IL-6 promoter activity remains completely unchanged. Similar observations were recorded with other natural NF-kappaB-driven promoters, namely IL-8 and endothelial leukocyte adhesion molecule (ELAM). We conclude that, within an "enhanceosome-like" structure, NF-kappaB is the central mediator of TNF-induced IL-6 gene expression, involving CBP/p300 and requiring histone acetyltransferase activity.
Acetyltransferases↗
The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN.
The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression. Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN.
Animals↗
N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-kappaB inhibition.
N-acetyl-L-cysteine (NAC) is an antioxidant molecule endowed with immunomodulatory properties. To investigate the effect of NAC on the induction phase of T cell responses, we analyzed its action on human dendritic cells (DC) derived from adherent PBMC cultured with IL-4 and granulocyte-macrophage CSF. We first found that NAC inhibited the constitutive as well as the LPS-induced activity of the transcription factor NF-kappaB. In parallel, NAC was shown to down-regulate the production of cytokines by DC as well as their surface expression of HLA-DR, CD86 (B7-2), and CD40 molecules both at the basal state and upon LPS activation. NAC also inhibited DC responses induced by CD40 engagement. The inhibitory effects of NAC were not due to nonspecific toxicity as neither the viability of DC nor their mannose receptor-mediated endocytosis were modified by NAC. Finally, we found that the addition of NAC to MLR between naive T cells and allogeneic DC resulted in a profound inhibition of alloreactive responses, which could be attributed to a defect of DC as APC-independent T cell responses were not inhibited by NAC. Altogether, our results suggest that NAC might impair the generation of primary immune responses in humans through its inhibitory action on DC.
Acetylcysteine↗
Dissociated glucocorticoids with anti-inflammatory potential repress interleukin-6 gene expression by a nuclear factor-kappaB-dependent mechanism.
Synthetic glucocorticoids (GCs) remain among the most effective agents for the management of chronic inflammatory diseases. However, major side effects severely limit their therapeutic use. Physiologic and therapeutic activities of GCs are mediated by a nuclear receptor belonging to a superfamily of ligand-inducible transcription factors that, in addition to directly regulating their cognate gene programs, can also mutually interfere with other signaling pathways. We recently identified selective ligands of the glucocorticoid receptor that dissociate transactivation from activator protein 1 transrepression, and most importantly retain in vivo anti-inflammatory activity. To further document the mechanisms of action sustaining the observed in vivo activity, we report here on the interference of dissociated GCs with nuclear factor kappaB (NF-kappaB)-driven gene activation. We show that dissociated GCs repress tumor necrosis factor-induced interleukin-6 gene expression by an NF-kappaB-dependent mechanism, without changing the expression level of inhibitor kappaB. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with the various compounds. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines constitutively expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB, which was able to efficiently repress a Gal4-dependent luciferase reporter gene upon addition of the dissociated compounds. We therefore conclude that, in addition to dissociating transactivation from activator protein 1 transrepression, dissociated GCs mediate inhibition of NF-kappaB signaling by a mechanism that is independent of inhibitor kappaB induction.
Animals↗
p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappaB p65 transactivation mediated by tumor necrosis factor.
Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)-kappaB plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription factors. Even the smallest deletion variant, containing almost exclusively a NF-kappaB-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple kappaB-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a kappaB-dependent reporter gene construct, without affecting the levels of NF-kappaB binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 "one-hybrid" system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation potential of the p65 kappaB subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF-kappaB, the p38 and extracellular signal-regulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery.
Animals↗
Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation.
Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-kappaB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IkappaB molecule, which in turn would bind and remove activated, DNA-bound NF-kappaB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-kappaB-dependent transcription, without changing the expression level of IkappaB. Neither the mRNA of IkappaB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-kappaB p65 subunit.
Animals↗
Recombination signal sequence binding protein Jkappa is constitutively bound to the NF-kappaB site of the interleukin-6 promoter and acts as a negative regulatory factor.
Analysis by electrophoretic mobility shift assays (EMSA) of the different proteins associated with the kappaB sequence of the interleukin-6 (IL-6) promoter (IL6-kappaB) allowed us to detect a specific complex formed with the recombination signal sequence binding protein Jkappa (RBP-Jkappa). Single-base exchanges within the oligonucleotide sequence defined the critical base pairs involved in the interaction between RBP-Jkappa and the IL6-kappaB motif. Binding analysis suggests that the amount of RBP-Jkappa protein present in the nucleus is severalfold higher than the total amount of inducible NF-kappaB complexes but that the latter bind DNA with a 10-fold-higher affinity. A reporter gene study was performed to determine the functional implication of this binding; we found that the constitutive occupancy of the IL6-kappaB site by the RBP-Jkappa protein was responsible for the low basal levels of IL-6 promoter activity in L929sA fibrosarcoma cells and that RBP-Jkappa partially blocked access of NF-kappaB complexes to the IL-6 promoter. We propose that such a mechanism could be involved in the constitutive repression of the IL-6 gene under normal physiological conditions.
Animals↗
The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.
Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.
Amino Acid Sequence↗
A hidden-line algorithm for graphical reconstruction of serially sectioned objects.
An algorithm for hidden-line removal in graphical reconstructions of serially sectioned biological objects is developed for PC-like computer configurations. The aim is to handle complicated structures defined by vast numbers of coordinate pairs and intersections between contours, without requiring excessive computing time. The procedure depends upon a fast searching routine for the localization of intersections and visibility.
Algorithms↗
A microcomputer-based graphical reconstruction technique for serial sectioned objects, with hidden line removal.
The presented software package fulfills the need for processing serial sections with a microcomputer configuration, enabling three-dimensional reconstruction with hidden line removal. The language used is an interpreted BASIC-dialect (HPL). The input is performed via an interactive program. The object can be rotated in space. The Hidden Line algorithm does not depend upon a raster technique. Points of intersection of successive contours are calculated and inserted, thus providing drawings of high resolution and quality. The handling time can be said to be short, especially when considering the capacities of the microcomputer configuration used.
Animals↗
TNF-induced intracellular signaling leading to gene induction or to cytotoxicity by necrosis or by apoptosis.
TNF-induced apoptosis, e.g. in murine PC60 cells, requires the TNF receptor p55 (TNF-R55) and the TNF receptor p75 (TNF-R75); the latter even does not have to be triggered. The intracellular domain of TNF-R55 can be activated in the cytosol by linking it to the trimeric CAT protein; induction of this fusion protein leads to a full TNF response. A new MAP kinase, p38, has been shown to be also activated by TNF. This activation is essential for gene induction, but not for cytotoxicity in L929 cells. TNF treatment of L929 leads to reactive oxygen formation in the mitochondria, resulting in cell death by necrosis. TNF treatment of many other cell types results in apoptosis, and this process involves activation of one or more ICE homologs (IHO). In the mouse, seven cysteine proteases of the IHO family have been cloned and partially characterized. One or more of these IHOs is involved in cell killing by proteolysis of critical substrate(s). One substrate, which may be a key effector molecule in the apoptotic process, is PITSLRE kinase.
Animals↗