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NFkappaB, stress and inflammation

During the inflammatory response, endothelial cells (EC) transiently upregulate a set of genes encoding, among others, cell adhesion molecules and chemotactic cytokines that together mediate the interaction of the endothelium with cells of the immune system. Gene upregulation is mediated predominantly at the transcriptional level and in many cases involves the transcription factor nuclear factor (NF) kappaB. The inflammatory response can be specifically inhibited by overexpression of a specific inhibitor of NF-kappaB, IkappaBalpha. A recombinant adenovirus expressing IkappaBalpha was constructed and used to infect EC of human and porcine origin. Ectopic expression of IkappaBalpha results in marked, and in some cases complete, reduction of the expression of several markers of EC activation, including vascular cell adhesion molecule 1, interleukins 1, 6, 8, and tissue factor. Overexpressed IkappaBalpha inhibits NF-kappaB specifically since (a) in electrophoretic mobility shift assay, NF-kappaB but not AP-1 binding activity is inhibited, and (b) von Willebrand factor and prostacyclin secretion that occur independently of NF-kappaB, remain unaffected. Functional studies of leukocyte adhesion demonstrate strong inhibition of HL-60 adhesion to IkappaBalpha-expressing EC. These findings suggest that NF-kappaB could be an attractive target for therapeutic intervention in a variety of inflammatory diseases, including xenograft rejection (Wrighton, 1996).

Dendritic cells (DC) derived from bone marrow are critical in the function of the immune system, for they are the primary antigen-presenting cells in the activation of T-lymphocyte response. Their differentiation from precursor cells has not been defined at a molecular level, but recent studies have shown an association between expression of the relB subunit of the NF-kappa B complex and the presence of DC in specific regions of normal unstimulated lymphoid tissues. RelB expression also correlates with differentiation of DC in autoimmune infiltrates in situ, and a mutation disrupting the relB gene results in mice with impaired antigen-presenting cell function, and a syndrome of excess production of granulocytes and macrophages. Thymic medullary epithelial cells from normal mice show striking similarities to DC and, interestingly, these cells are also absent in relB mutant mice. Taken together, these results suggest that relB is critical in the coordinated activation of genes necessary for the differentiation of two unrelated but phenotypically similar cells (DC and thymic medullary epithelial cells) and is therefore a candidate for a gene determining lineage commitment in the immune system (Burkly, 1995).

IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) results in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Death at 7 to 10 days of age is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin (Klement, 1996).

Biosynthesis of tumor necrosis factor-alpha (TNF-alpha) is carried out predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-alpha promoter in cells of monocytic lineage. In one region [-182 to -37 base pairs (bp)] the TNF-alpha promoter possesses enhancer elements that are required for optimal transcription of the TNF-alpha gene in response to LPS. Two regions were identified: region I (-182 to -162 bp) contains an overlapping Sp1/Egr-1 site, and region II (-119 to -88) contains CRE and NF-kappaB (designated kappaB3) sites. The following were all found to bind to the CRE site: unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes. LPS stimulation increases the binding of c-Jun-containing complexes. In addition, LPS stimulation induces the binding of cognate nuclear factors to the Egr-1 and kappaB3 sites, which were identified as Egr-1 (Drosophila homolog: Stripe) and p50/p65, respectively. The CRE and kappaB3 sites in region II together confer strong LPS responsiveness to a heterologous promoter, whereas individually they fail to provide transcriptional activation. Increasing the spacing between the CRE and the kappaB3 sites completely abolishes LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-alpha promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements (Yao, 1997).

Tumor necrosis factor initiates a cytolytic signaling cascade that leads to increased levels of reactive oxygen intermediates and the subsequent apoptosis of some tumor cell lines and virally infected cells. To combat the lethal effect of reactive oxygen intermediates, eukaryote cells have evolved several reactive oxygen-scavenging enzymes, including superoxide dismutase. Manganese superoxide dismutase (MnSOD), a TNF-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identify a 238-bp region of intron 2 that is responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) has the properties of a traditional enhancer element that functions in an orientation- and position-independent manner. IVGF of the TNFRE reveals that TNF- and IL-1-induces factor occupancy of sites that can bind NF-kappaB and C/EBP. The 5' portion of the TNFRE binds C/EBP-beta in vitro and is both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE binds both NF-kappaB and C/EBP but is not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE is required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter (Jones, 1997).

A protein present in Aplysia neurons has many characteristics of the transcription factor NF-kappaB. The protein binds a radiolabeled DNA probe containing the kappaB-binding sequence from the human interferon-beta gene enhancer element (PRDII), and the binding is not affected by PRDIV, an ATF-2 enhancer sequence from the same gene. Binding is efficiently inhibited, however, by nonradioactive oligonucleotides containing H2, the kappaB site from the major histocompatibility complex I gene promoter. Recombinant mammalian IkappaB-alpha, which associates specifically with the P65 subunit of NF-kappaB, inhibits the binding to the PRDII probe in a dose-dependent manner. The nuclear form of the Aplysia protein is constitutively active. Axoplasm, however, contains the constitutively active form as well as a latent form. The latter is activated by treatment with deoxycholate under the same conditions as mammalian NF-kappaB. Based on these findings, it is believed that the protein is a homolog of NF-kappaB. To investigate the role of apNF-kappaB in the axon, the peripheral nerves were crushed to the body wall. Surprisingly, there is a rapid loss of apNF-kappaB binding at the crush site extending as far as 2.5 cm along the axon within 15 min. In contrast, exposing either the intact animal or the nervous system in situ to levels of 5-HT that induce synaptic facilitation did not affect apNF-kappaB activity (Povelones, 1997).

Selected clones of the sympathetic precursor-like cell line PC12 (rCl8) are resistant to oxidative cell death induced by the Alzheimer's disease-associated amyloid beta protein (Abeta) and hydrogen peroxide (H2O2). The transcriptional activity and DNA binding activity of the redox-sensitive transcription factor NF-kappaB and its nuclear expression are constitutively increased in rCl8 cells, as compared with their nonresistant parental PC12 cell (PC12p) counterpart. Suppression of the transcriptional activity of NF-kappaB in rCl8 cells with the synthetic glucocorticoid dexamethasone or by direct overexpression of a super-repressor mutant form of IkappaBalpha, a specific inhibitor of NF-kappaB, reverses the oxidative stress resistance phenotype of these cells and ultimately leads to increased cell death after the challenge with H2O2. Dexamethasone treatment also causes an increase in the protein level of IkappaBalpha. These data show that an increased baseline of NF-kappaB activity may mediate the resistance of these cells of neuronal origin to oxidative stress. Therefore, the presented model may help to identify possible neuronal target genes of NF-kappaB and to further elucidate the molecular basis of the differential sensitivity of neurons in neurodegenerative conditions associated with an increased oxidative burden, such as in Alzheimer's disease (Lezoualc'h, 1998).

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) detected a specific complex formed with the recombination signal sequence binding protein Jkappa (RBP-Jkappa). Single-base exchanges within the oligonucleotide sequence defines 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. It was found that the constitutive occupancy of the IL6-kappaB site by the RBP-Jkappa protein is responsible for the low basal levels of IL-6 promoter activity in L929sA fibrosarcoma cells and that RBP-Jkappa partially blocks access of NF-kappaB complexes to the IL-6 promoter. It is proposed that such a mechanism could be involved in the constitutive repression of the IL-6 gene under normal physiological conditions (Plaisance, 1997).

The cellular interleukin-6 (IL-6) gene contains a target site for the mammalian transcriptional repressor RBP (see Drosophila Suppressor of Hairless). The target site is contained within the interleukin response element (ILRE), which mediates IL-6 activation by NF-kappa B. RBP represses activated transcription from the IL-6 gene. The presence and position of the RBP target site are both crucial in mediating repression by RBP. While RBP binds within the ILRE, it does not target NF-kappa B alone; nonetheless, NF-kappa B binding to the ILRE is required for repression. These results indicate that RBP represses coactivation by NF-kappa B and another cellular transcription factor, C/EBP-beta (Kannabiran, 1998).

Poly (ADP-ribose) polymerase-1 is a nuclear DNA-binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. PARP-1-deficient cells are defective in NF-kappaB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-alpha. Treating mice with lipopolysaccharide (LPS) results in the rapid activation of NF-kappaB in macrophages from PARP-1+/+ but not from PARP-1-/- mice. PARP-1-deficient mice are extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-kappaB-dependent accumulation and of TNF-alpha in the serum and a down-regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-kappaB, with consequences for the transcriptional activation of NF-kappaB and a systemic inflammatory process. The molecular dissection of the interaction between PARP-1 and the transcriptional machinery of NF-kappaB still remains to be elucidated. Efforts to show a physical association between PARP-1 and NF-kappaB p65 or p50 subunit by immunoprecipitation experiments were not conclusive. One possibility might be that PARP-1 and NF-kappaB contact through a third protein involved in the architectural regulation of transcription, the high mobility group-I protein [HMG-I(Y)], which is a well known coactivator of NF-kappaB-dependent transcription and a substrate for PARP-1. This is also an attractive possibility since PARP-1 has a high binding affinity for DNA bends such as those induced in the NF-kappaB enhanceosome by HMG-I(Y) (Oliver, 1999).

Inhibition of NF-kappa B by glucocorticoid

NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. The ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription (Scheinman, 1995).

Repression of NFkappaB-dependent gene expression is one of the major elements of immunosuppression by glucocorticoids. Protein-protein interactions between the glucocorticoid receptor and NFkappaB have been characterized and shown to be a possible mechanism of mutual inhibition of transactivation properties. More recently, glucocorticoid-mediated induction of IkappaBalpha, an inhibitor of NFkappaB, has been described in monocytes and lymphocytes; an increase in IkappaBalpha mRNA and protein resulted in inactivation and cytosolic retention of NFkappaB. Thus, rather than the physical interaction between the glucocorticoid receptor and NFkappaB, the up-regulation of IkappaBalpha was presented as the key element in immunosuppression by glucocorticoids. However, this paper shows that the IkappaBalpha pathway is not involved in glucocorticoid-mediated inhibition of NFkappaB activity in endothelial cells. Although transcriptional activation by NFkappaB was significantly reduced in the presence of glucocorticoids, induction was not detected of IkappaBalpha protein that could prevent nuclear translocation of NFkappaB upon stimulation with lipopolysaccharide or tumor necrosis factor alpha. Furthermore, treatment with glucocorticoids did not seem to affect the transcription rate or mRNA stability of IkappaBalpha. It is therefore conclude that, although induction of IkappaBalpha expression by glucocorticoids seems to be of importance in monocytes and lymphocytes, it cannot explain inhibition of NFkappaB-dependent gene expression in endothelial cells. These results emphasize the relevance of physical interaction between the glucocorticoid receptor and NFkappaB in endothelial cells and thus in suppression of inflammation by glucocorticoids (Brostjan, 1996).

Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive agents. They inhibit synthesis of almost all known cytokines and of several cell surface molecules required for immune function, but the mechanism underlying this activity has been unclear. Here it is shown that glucocorticoids are potent inhibitors of nuclear factor kappa B (NF-kappa B) activation in mice and cultured cells. This inhibition is mediated by induction of the I kappa B alpha inhibitory protein, which traps activated NF-kappa B in inactive cytoplasmic complexes. Because NF-kappa B activates many immunoregulatory genes in response to pro-inflammatory stimuli, the inhibition of its activity can be a major component of the anti-inflammatory activity of glucocorticoids (Auphan, 1995).

Glucocorticoids are efficient antiinflammatory agents, and their effects include transcriptional repression of several cytokines and adhesion molecules. Whereas glucocorticoids down-regulate the expression of genes relevant during inflammation, nuclear factor (NF)-kappa B/Rel proteins function as important positive regulators of these genes. The expression of intercellular adhesion molecule-1 (ICAM-1), which plays an essential role in recruitment and migration of leukocytes to sites of inflammation, is also down-regulated by glucocorticoids. A functional NF-kappa B site in the ICAM-1 promoter, which can be activated by either phorbol or tumor necrosis factor-alpha (TNF alpha), is also the target for glucocorticoids. The ligand-activated glucocorticoid receptor (GR) is able to repress RelA-mediated activation of the ICAM-1 NF-kappa B site. Conversely, transcriptional activation by GR via a glucocorticoid response element is specifically repressed by RelA, but not by other NF-kappa B/Rel family members. Mutational analysis of GR demonstrates that the DNA binding domain and the ligand binding domain are required for the functional repression of NF-kappa B activation. Despite the importance of the DNA binding domain, transcriptional repression of NF-kappa B, mediated by GR, is not caused by binding of GR to the ICAM-1 NF-kappa B element, but by a physical interaction between the GR and RelA protein. The repressive effect of GR on NF-kappa B-mediated activation was not shared by other steroid/thyroid receptors. Only the progesterone receptor, which belongs to the same subfamily as GR and which possesses high homology with GR, is able to repress NF-kappa B-mediated transcription. These studies highlight a possible molecular mechanism that can explain the antiinflammatory effects of glucocorticoid treatment during inflammation (Caldenhoven, 1995).

Table of contents

dorsal continued: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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