dorsal

EVOLUTIONARY HOMOLOGS

Table of contents

Transcriptional targets of NF kappa B

Transcriptional activation of the IL-8 gene by several inflammatory mediators, including the cytokines IL-1 and TNF-alpha, is mediated through sequences located between nucleotide -94 and -71 of the IL-8 promoter. Adjacent binding sites for the inducible transcription factors NF-kappa B and NF-IL-6 are located within this region. Maximal transcriptional activation by PMA in Jurkat T lymphocytes was shown to require intact binding sites for both NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis indicates that NF-IL-6, as well as other related members of this family, bind specifically to the NF-IL-6 site in the IL-8 promoter. In addition, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds specifically to the NF-kappa B site. When incubated together, RelA and NF-IL-6/C/EBP form a ternary complex with this region of the IL-8 promoter; this binding is dependent on intact binding sites for both NF-IL-6 and RelA. Transient cotransfection analyses indicate that the cooperative association of NF-IL-6 and RelA with the IL-8 promoter results in synergistic transcriptional activation. Mutational analyses of RelA demonstrate that the C-terminal transactivation domain and the DNA binding domain are required for synergistic activation with NF-IL-6. In addition, overexpression of the NF-kappa B inhibitor molecule, I kappa B, abolished the RelA- and RelA/NF-IL-6-dependent synergistic activation. These data demonstrate that RelA and members of the C/EBP/NF-IL-6 family can functionally cooperate in transcriptional activation of the IL-8 gene and suggest a common mechanism for inducible regulation of cytokine gene expression (Kunsch, 1994).

NF-kappa B induces transcription from the human pro-IL-1 beta (IL-1 beta) gene. A recombinant plasmid containing 4 kb of the IL-1 beta gene upstream regulatory sequence was transactivated by the p65 subunit of NF-kappa B or by treatment of the cells with a combination of NF-kappa B inducers including LPS, PMA, and dibutyryl cyclic AMP (L+P+C). Coexpression of p65 with L+P+C treatment led to a synergistic response, whereas coexpression of the I kappa B alpha/MAD-3 protein, in place of p65, blocked L+P+C induction. A series of 5' deletion mutants of the IL-1 beta promoter were used to define two p65 response regions: region I located between -2800 to -2720 bp and region II located between -512 and -133 bp. Electrophoretic mobility shift assays confirmed that NF-kappa B-like proteins could bind to two consensus binding sites in region II. A site-specific mutation in only one of these NF-kappa B sites (-296/-286 bp) caused a specific loss of induction by p65 or L+P+C. A cyclic AMP response element (CRE) site (-2761/-2753 bp) in region I has been shown previously to be critical for L+P+C induction. Mutation of the CRE in an enhancerless test plasmid containing two copies of region I blocked transactivation by p65. Likewise, coexpression of I kappa B alpha inhibited CRE-dependent L+P+C induction of the wild-type counterpart. These data show that NF-kappa B regulates a nonconsensus CRE site in addition to the consensus binding site at -296/-286 bp and suggest that NF-kappa B may play multiple roles in the induction of IL-1 beta transcription (Cogswell, 1994).

Signal transducer and activator of transcription 6 (Stat6) and NF-kappaB are widely distributed transcription factors that are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-kappaB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathione S-transferase pulldown assays and coimmunoprecipitation techniques, it has been found that NF-kappaB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-kappaB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-kappaB proteins in IL-4-stimulated I.29mu B lymphoma cells. Furthermore, Stat6 and NF-kappaB bind cooperatively to a DNA probe containing both sites; the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-kappaB. It is concluded that the direct interaction between Stat6 and NF-kappaB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-kappaB (Shen, 1998).

Interleukin 4 (IL-4) induces transcription of the germline C epsilon genes in activated B cells and subsequently, cells in this population will undergo switch recombination to immunoglobulin E. Furthermore, transcription of germline C epsilon genes is required for class switching. A 46-bp segment of germline C epsilon gene (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. This segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter (Delphin, 1995).

Rel/NF-kappaB transcription factors and IkappaBalpha function in an autoregulatory network. Avian IkappaBalpha transcription is increased in response to both c-Rel and v-Rel. IkappaBalpha transcription is synergistically stimulated by Rel and AP-1 factors (c-Fos and c-Jun). A 386 bp region of the IkappaBalpha promoter (containing two NF-kappaB and one AP-1 binding sites) is necessary and sufficient for response to both Rel factors alone or Rel factors in conjunction with the AP-1 proteins. In addition, an imperfect NF-kappaB binding site is found to overlap the AP-1 binding site. Mutation of either of the NF-kappaB binding sites or the AP-1 binding site dramatically decreases the response of the IkappaBalpha promoter to Rel proteins alone or Rel and AP-1 factors. Overexpression of c-Rel results in the formation of DNA binding complexes associates with the imperfect NF-kappaB binding site which overlaps the AP-1 site. v-Rel associated with the imperfect NF-kappaB site stronger than c-Rel, and overexpression of v-Rel also results in the formation of a v-Rel containing complex bound to a consensus AP-1 site (Kralova, 1996).

NF-kappa B, which consists of two polypeptides, p50 (M(r) 50K) and p65/RelA (M(r) 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses. Disruption of the relA locus leads to embryonic lethality at 15-16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for I kappa B alpha and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-kappa B-regulated pathways (Beg, 1995).

P-selectin, an adhesion receptor for leukocytes, is constitutively expressed by megakaryocytes and endothelial cells. Synthesis of P-selectin is also increased by some inflammatory mediators. A previously identified A kappa B site (-218GGGGGTGACCCC-207) in the promoter of the human P-selectin gene is unique in that it binds constitutive nuclear protein complexes containing p50 or p52, but not inducible nuclear protein complexes containing p65. Furthermore, the element binds recombinant p50 or p52 homodimers, but not p65 homodimers. Methylation interference analysis indicates that p50 or p52 homodimers contact the guanines at positions -218 to -214 on the coding strand and at -210 to -207 on the noncoding strand. Changes in the three central residues at -213 to -211 altered binding specificity for members of the NF-kappa B/Rel family. Mutations that eliminated binding to NF-kappa B/Rel proteins reduced by approximately 40% the expression the P-selectin promoter. Overexpression of p52 enhances P-selectin promoter activity, and co-overexpression of Bcl-3 further induces promoter activity in a kappa B site-dependent manner. In contrast, overexpression of p50 represses promoter activity; this repression was prevented by co-overexpression of Bcl-3. These data suggest that Bcl-3 differentially regulates the effects of p50 and p52 homodimers bound to the kappa B site of the P-selectin promoter. This site may be a prototype for kappa B elements in other genes that bind specifically to p50 and/or p52 homodimers (Pan, 1995).

E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. Inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers, including E-cadherin. Both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 has been demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibits its binding to both RB and AP-2. Taken together, these results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc (Batsche, 1998).

Gene activation by NF-kappaB/Rel transcription factors is modulated by synergistic or antagonistic interactions with other promoter-bound transcription factors. For example, Sp1 sites are often found in NF-kappaB-regulated genes, and Sp1 can activate certain promoters in synergism with NF-kappaB through nonoverlapping binding sites. Sp1 acts directly through a subset of NF-kappaB binding sites. The DNA binding affinity of Sp1 to these NF-kappaB sites, as determined by their relative dissociation constants and their relative efficiencies as competitor DNAs or as binding site probes, is on the order of that for a consensus GC box Sp1 site. In contrast, NF-kappaB does not bind to a GC box Sp1 site. Sp1 can activate transcription through an immunoglobulin kappa-chain enhancer or P-selectin promoter NF-kappaB sites. p50 homodimers replace Sp1 from the P-selectin promoter by binding site competition and thereby either inhibit basal Sp1-driven expression or, in concert with Bcl-3, stimulate expression. The interaction of Sp1 with NF-kappaB sites thus provides a means to keep an elevated basal expression of NF-kappaB-dependent genes in the absence of activated nuclear NF-kappaB/Rel (Hirano, 1998).

In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets. c-Jun-upregulated cDNA clones homologous to the tenascin-C (see Drosophila Tenascin major) gene have been identified by differential screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix protein involved in cell attachment and migration and in the control of cell growth. Transient overexpression of c-Jun induces tenascin-C expression in primary REF and in FR3T3, an established fibroblast cell line. Surprisingly, tenascin-C synthesis is repressed after stable transformation by c-Jun, as compared to that in the nontransformed parental cells. As assessed by using the tenascin-C (-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at position -210. As demonstrated by gel shift experiments and cotransfections of the reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two transcription factors bind and synergistically transactivate the tenascin-C promoter. Two other extracellular matrix proteins, SPARC and thrombospondin-1, are c-Jun targets. Thus, these results strongly suggest that the regulation of the extracellular matrix composition plays a central role in c-Jun-induced transformation (Mettouchi, 1997).

Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. A nuclear complex from human monocytic cells binds to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex is activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrate that the TF site binds c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. c-Rel and p65 are present and p50 is absent in the TF complex and c-Rel/p65 heterodimers selectively binds to the TF kappa B-like site. Taken together, these results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediate LPS induction of TF gene expression in monocytic cells (Oeth, 1994).

The central developmental event in the human (h)alpha-globin gene cluster is selective silencing of the zeta-globin gene as erythropoiesis shifts from primitive erythroblasts in the embryonic yolk sac to definitive erythroblasts in the fetal liver. Previous studies have demonstrated that full developmental silencing of the hzeta-globin gene in transgenic mice requires the proximal 2.1 kb of its 3'-flanking region. This silencing activity has been localized to a 108 bp segment located 1.2 kb 3' to the zeta-globin gene. Protein(s) in nuclear extracts from cell lines representing the fetal/adult erythroid stage bind specifically to an NF-kappaB motif located at this site. In contrast, this binding activity is lacking in the nuclear extract of an embryonic-stage erythroid line expressing zeta-globin. This complex is quantitatively recognized by antisera to the NF-kappaB p50 and to a lesser extent to p65 subunits. A two-base substitution that disrupts NF-kappaB site protein binding in vitro also results in the loss of the developmental silencing activity in vivo. The data suggest that NF-kappaB complex formation is a crucial component of hzeta-globin gene silencing. This finding expands the roles of this widely distributed transcriptional complex to include negative regulation in mammalian development (Wang, 1999).

Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induce jagged1 gene expression, whereas a mutant defective for transactivation does not. Importantly, jagged1 transcripts are also upregulated by endogenous NF-kappaB activation and this effect is inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers leads to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlates with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 is highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system (Bash, 1999).

In addition to its role in neurogenesis, myogenesis, angiogenesis and retinal cell development, the Notch signaling pathway has also been implicated in hematopoiesis and in immune cell malignancies. Consistent with this notion, the expression of Jagged1 promotes the development of primitive hematopoietic precursor cells, whereas activated forms of Notch1 and Notch2 influence the differentiation of myeloid progenitors in response to different cytokines. Overexpression of an activated form of Notch1 influences T-cell differentiation during thymic development. This process recently has been proposed to involve the silencing of CD4 gene expression by HES-1. The mapping of three human Notch genes to chromosomal locations associated with leukemia, lymphoma and myeloproliferative disorders has also suggested a role in immune cell proliferation and malignancy. The demonstration that constitutively active forms of Notch1 induce T-cell leukemia/lymphoma in mice confirms this prediction. The jagged1 locus has been mapped to human chromosome 20p12. Mutations at this locus are associated with Alagille syndrome. Although alterations in immune or hematopoietic function have not been reported, it remains to be determined whether the disease results from haploinsufficiency or from a dominant negative effect exerted by the mutant protein. The ability of the Notch signaling pathway to influence the differentiation and proliferation of different cell lineages may also depend on different inducing signals and on the cellular microenvironment (Bash, 1999 and references).

Jagged1 is highly expressed in the B-cell areas of the spleen, particularly in the splenic marginal zone that is rich in plasma and memory B cells. Consistent with these results, a correlation is found between the expression of Jagged1 and c-Rel in purified mouse splenic B cells. Although the function of Jagged1 in secondary lymphoid organs remains to be determined, both Notch1 and Notch2 are also expressed in the spleen. This raises the possibility of a role for Jagged1-mediated signaling through Notch in the pathways that control the later stages of B-lymphocyte activation, differentiation and/or immune function. The ability of the EBNA-2 protein of Epstein-Barr virus to induce expression of the B-cell activation marker CD23 through its association with the Notch effector CBF1 in transformed B lymphocytes agrees with a role for Notch signaling in B cells. Co-cultivation assays demonstrating that Jagged1-expressing cells induce CD23 gene expression in neighboring B cells is consistent with this hypothesis. Future studies will help to define the role of Jagged1 in the splenic microenvironment, and to clarify whether it signals through Notch in the context of a heterotypic cell-cell interaction or in a cell-autonomous fashion. Recent work suggesting that soluble forms of Notch ligands can trigger signaling through Notch in vivo would be compatible with either possibility (Bash, 1999 and references).

c-rel knock-out mice are impaired for B-cell activation and antibody production. The observation that all known B-cell growth factors fail to rescue the proliferative defect of these cells suggests that c-Rel may regulate the expression of genes, other than those for cytokines and growth factors, which are crucial for the activation and proliferation of B lymphocytes. The finding that c-Rel can trigger the expression of Jagged1 raises the possibility of a connection between the Rel/NF-kappaB and Notch signaling pathways in secondary lymphoid organs. Thus, in addition to controlling the expression of cytokines, immunoregulatory and adhesion molecules, Rel/NF-kappaB factors may also trigger a Notch signaling cascade important for lymphocyte activation and immune function (Bash, 1999 and references).

A number of pathogenic and proinflammatory stimuli, and the transforming growth factor-beta (TGF-beta) exert opposing activities in cellular and immune responses. The RelA subunit of nuclear factor kappaB (NF-kappaB/RelA) is necessary for the inhibition of TGF-beta-induced phosphorylation, nuclear translocation, and DNA binding of SMAD signaling complexes by tumor necrosis factor-alpha (TNF-alpha). The antagonism is mediated through up-regulation of Smad7 synthesis and induction of stable associations between ligand-activated TGF-beta receptors and inhibitory Smad7. Down-regulation of endogenous Smad7 by expression of antisense mRNA releases TGF-beta/SMAD-induced transcriptional responses from suppression by cytokine-activated NF-kappaB/RelA. Following stimulation with bacterial lipopolysaccharide (LPS), or the proinflammatory cytokines TNF-alpha and interleukin-1beta (IL-1beta), NF-kappaB/RelA induces Smad7 synthesis through activation of Smad7 gene transcription. These results suggest a mechanism of suppression of TGF-beta/SMAD signaling by opposing stimuli mediated through the activation of inhibitory Smad7 by NF-kappaB/RelA (Bitzer, 2000).

The nuclear factor-kappaB (NF-kappaB) family of transcription factors is involved in proliferation, differentiation, and apoptosis in a stage- and cell-dependent manner. Recent evidence has shown that NF-kappaB activity is necessary for both chicken and mouse limb development. The NF-kappaB family member c-rel and the homeodomain gene msx-1 have partially overlapping expression patterns in the developing chick limb. In addition, inhibition of NF-kappaB activity results in a decrease in msx-1 mRNA expression. Sequence analysis of the msx-1 promoter reveals three potential kappaB-binding sites similar to the interferon-gamma (IFN-gamma) kappaB-binding site. These sites bind to c-Rel, as shown by electrophoretic mobility shift assay. Furthermore, inhibition of NF-kappaB activity significantly reduces transactivation of the msx-1 promoter in response to FGF-2/-4, known stimulators of msx-1 expression. These results suggest that NF-kappaB mediates the FGF-2/-4 signal regulation of msx-1 gene expression (Bushdid, 2001).

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. A cytokine-responsive enhancer between -5.2 and -6.1 kb in the 5'-flanking hiNOS promoter DNA contains multiple NF-kappa B elements. The role of the IFN-Jak kinase-Stat 1 pathway for regulation of hiNOS gene transcription is described in this study. In A549 human lung epithelial cells, a combination of cytokines TNF-alpha, IL-1 beta, and IFN-gamma function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at -5.8 kb in the hiNOS promoter identifies a bifunctional NF-kappa B/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at -5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-kappa B activates hiNOS promoter-reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-kappa B function in these cells. These results indicate that both Stat 1 and NF-kappa B are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner (Ganster, 2001).

Rel/NF-kappaB transcription factors regulate the division and survival of B lymphocytes. B cells lacking NF-kappaB1 and c-Rel fail to increase in size upon mitogenic stimulation due to a reduction in induced c-myc expression. Mitogen-induced B cell growth, although not markedly impaired by FRAP/mTOR or MEK inhibitors, requires phosphatidylinositol 3-kinase (PI3K) activity. Inhibition of PI3K-dependent growth coincides with a block in the nuclear import of NF-kappaB1/c-Rel dimers and a failure to upregulate c-myc. In addition, PI3K has been shown to be necessary for a transcription-independent increase in c-Myc protein levels that accompanies mitogenic activation. Collectively, these findings establish a role for Rel/NF-kappaB signaling in the mitogen-induced growth of mammalian cells; such growth in B lymphocytes requires a PI3K/c-myc-dependent pathway (Grumont, 2003).

During Drosophila embryogenesis, the Dorsal transcription factor activates the expression of twist, a transcription factor required for mesoderm formation. The mammalian twist proteins (twist-1 and -2), are induced by a cytokine signaling pathway that requires the dorsal-related protein RelA, a member of the NF-kappaB family of transcription factors. Twist-1 and -2 repress cytokine gene expression through interaction with RelA. Mice homozygous for a twist-2 null allele or doubly heterozygous for twist-1 and -2 alleles show elevated expression of proinflammatory cytokines, resulting in perinatal death from cachexia. These findings reveal an evolutionarily conserved signaling circuit in which twist proteins regulate cytokine signaling by establishing a negative feedback loop that represses the NF-kappaB-dependent cytokine pathway (Šošić, 2003).

In Drosophila, the NF-κB-like transcription factor Dorsal activates twist expression through binding to κB sites in the twist promoter. To determine if this aspect of twist regulation might be conserved in vertebrates, whether twist-1 and -2 are regulated by TNFα, a well-known activator of the NF-κB pathway, was tested. Indeed, stimulation of immortalized mouse fibroblasts with TNFα evokes an increase in expression of twist-1 and -2 transcripts. Twist genes are also induced by TNFα in 10T1/2 and NIH3T3 cells. The IκBα and TNFα genes, which are known transcriptional targets of NF-κB, are also induced by TNFα stimulation (Šošić, 2003).

To determine whether the induction of twist expression by TNFα was dependent on NF-κB, twist-1 and -2 expression was measured in immortalized fibroblasts derived from p65^-/- mice. Twist-1 and -2 are not inducible in p65^-/- fibroblasts. Expression of TNFα and IκBα is also reduced in mutant cells, consistent with their known dependence upon NF-κB. These findings demonstrate that twist genes are induced by TNFα in an NF-κB-dependent manner, like other well-characterized TNFα-responsive genes (Šošić, 2003).

The concept of modularity in evolutionary biology is firmly established. Modules are shared in unrelated processes and are continuously moved during evolution, leading to the co-option of genes into new regulatory circuits. The NF-κB-twist partnership is an example of a module that has been conserved from insects to mammals and is utilized during two unrelated biological processes: dorsoventral patterning and the immune response. In both flies and mammals, NF-κB activates twist expression during dorsoventral patterning. During the immune response, NF-κB also induces the expression of peptides with antimicrobial properties. This study shows that in mammals twist has been adopted to negatively modulate NF-κB mediated cytokine activation, thus bridging two branches of NF-κB pathway and completing a negative feedback loop (Šošić, 2003).

Remarkably, a similar negative feedback loop also exists in the Drosophila NF-κB pathway. Upstream of spätzle, in that pathway, is the Easter protease. Mutations in any of the downstream genes required for activity of the pathway lead to an increase in the amount of activated Easter. Similarly, upon blockage of this pathway downstream of spätzle, its active form is accumulated in the embryo. Since mutations in dorsal led to increased Easter activation, it has been proposed that the initial component of the feedback loop is a transcriptional target of Dorsal. The results of this study raise the possibility that the function of twist as a key component of a negative feedback loop in this pathway may have been evolutionarily conserved from flies to mammals (Šošić, 2003).

Cytokines, such as tumor necrosis factor-alpha (TNFalpha), potently inhibit the differentiation of mesenchymal cells and down-regulate the expression of Sox9 and MyoD, transcription factors required for chondrocyte and myocyte development. NF-kappaB controls TNFalpha-mediated suppression of myogenesis through a mechanism involving MyoD mRNA down-regulation. NF-kappaB also suppresses chondrogenesis and destabilizes Sox9 mRNA levels. Multiple copies of an mRNA cis-regulatory motif (5'-ACUACAG-3') are necessary and sufficient for NF-kappaB-mediated Sox9 and MyoD down-regulation. These results suggest that the ACTACAG motifs represent, presumably indirect, NF-kappaB-responsive elements in the MyoD and Sox9 mRNAs. Thus, in response to cytokine signaling, NF-kappaB modulates the differentiation of mesenchymal-derived cell lineages via RNA sequence-dependent, posttranscriptional down-regulation of key developmental regulators (Sitcheran, 2003).

To investigate the determinants of promoter-specific gene regulation by the glucocorticoid receptor (GR), the composition and function of regulatory complexes at two NFkappaB-responsive genes that are differentially regulated by GR were compared. Transcription of the IL-8 and IkappaBalpha genes is stimulated by TNFalpha in A549 cells, but GR selectively represses IL-8 mRNA synthesis by inhibiting Ser2 phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). The proximal kappaB elements at these genes differ in sequence by a single base pair, and both recruited RelA and p50. Surprisingly, GR is recruited to both of these elements, despite the fact that GR fails to repress the IkappaBalpha promoter. Rather, the regulatory complexes formed at IL-8 and IkappaBalpha were distinguished by differential recruitment of the Ser2 CTD kinase, P-TEFb. Disruption of P-TEFb function by the Cdk-inhibitor, DRB, or by small interfering RNA selectively blocks TNFalpha stimulation of IL-8 mRNA production. GR competes with P-TEFb recruitment to the IL-8 promoter. Strikingly, IL-8 mRNA synthesis is repressed by GR at a post-initiation step, demonstrating that promoter proximal regulatory sequences assemble complexes that impact early and late stages of mRNA synthesis. Thus, GR accomplishes selective repression by targeting promoter-specific components of NFkappaB regulatory complexes (Luecke, 2005).

The glutamate transporter gene, EAAT2/GLT-1, is induced by epidermal growth factor (EGF) and downregulated by tumor necrosis factor alpha (TNFalpha). While TNFalpha is generally recognized as a positive regulator of NF-kappaB-dependent gene expression, its ability to control transcriptional repression is not well characterized. Additionally, the regulation of NF-kappaB by EGF is poorly understood. Both TNFalpha-mediated repression and EGF-mediated activation of EAAT2 expression require NF-kappaB. EGF activates NF-kappaB independently of signaling to IkappaB. Furthermore, TNFalpha can abrogate IKKbeta- and p65-mediated activation of EAAT2. These results suggest that NF-kappaB can intrinsically activate EAAT2 and that TNFalpha mediates repression through a distinct pathway also requiring NF-kappaB. Consistently, it was found that N-myc is recruited to the EAAT2 promoter with TNFalpha and that N-myc-binding sites are required for TNFalpha-mediated repression. Moreover, N-myc overexpression inhibits both basal and p65-induced activation of EAAT2. These data highlight the remarkable specificity of NF-kappaB activity to regulate gene expression in response to diverse cellular signals and have implications for glutamate homeostasis and neurodegenerative disease (Sitcheran, 2005).

The ability of NF-kappaB to regulate EAAT2 expression has important implications for the regulation of glutamate homeostasis in the CNS. To prevent the overstimulation of neuronal glutamate receptors that can trigger excitotoxic mechanisms and cell death, extracellular concentrations of excitatory amino acids are tightly controlled by transport systems on both neurons and glial cells. EAAT2 is critical for rapid clearance of synaptically released glutamate for proper neurotransmission. Accumulation of excessive glutamate levels in neuronal synapses can lead to excitotoxic neuronal death, which has been implicated in the pathogenesis of numerous neurodegenerative diseases, as well as CNS injury resulting from stroke and ischemia. Notably, these conditions have been associated with increased NF-kappaB activity, and reduced EAAT2 expression is observed after brain injury and in patients with Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and multiple sclerosis. Interestingly, EAAT2 may also have a role in development, as work in Drosophila demonstrates that this transporter is involved in terminal glial cell differentiation. Moreover, reduced glial cell populations are observed in mice lacking the EGF receptor suggesting that EGF signaling is also important for glial cell differentiation. Based on the work in this study, it is proposed that positive regulation of EAAT2 by NF-kappaB in response to EGF may promote glial cell differentiation and uptake of synaptic glutamate by glial cells, whereas TNF-mediated inhibition of EAAT2 by NF-kappaB may contribute to glutamate toxicity and cell death in neuroinflammation and disease (Sitcheran, 2005).

This study investigated recruitment of coactivators (SRC-1, SRC-2, and SRC-3) and corepressors (HDAC1, HDAC2, HDAC3, SMRT, and NCoR) to the IkappaBα gene promoter after NF-kappaB activation by tumor necrosis factor-α. The data from chromatin immunoprecipitation assay suggest that coactivators and corepressors are simultaneously recruited to the promoter, and their binding to the promoter DNA is oscillated in HEK293 cells. SRC-1, SRC-2, and SRC-3 all enhanced IkappaBα transcription. However, the interaction of each coactivator with the promoter exhibited different patterns. After tumor necrosis factor-α treatment, SRC-1 signal was increased gradually, but SRC-2 signal was reduced immediately, suggesting replacement of SRC-2 by SRC-1. SRC-3 signal was increased at 30 min, reduced at 60 min, and then increased again at 120 min, suggesting an oscillation of SRC-3. The corepressors were recruited to the promoter together with the coactivators. The binding pattern suggests that the corepressor proteins formed two types of corepressor complexes, SMRT-HDAC1 and NCoR-HDAC3. The two complexes exhibited a switch at 30 and 60 min. The functions of cofactors were confirmed by gene overexpression and RNA interference-mediated gene knockdown. These data suggest that gene transactivation by the transcription factor NF-kappaB is subject to the regulation of a dynamic balance between the coactivators and corepressors. This model may represent a mechanism for integration of extracellular signals into a precise control of gene transcription (Gao, 2005).

TNF-induced NF-kappaB activity shows complex temporal regulation whose different phases lead to distinct gene expression programs. Combining experimental studies and mathematical modeling, two temporal amplification steps have been detected - one determined by the obligate negative feedback regulator IkappaBα - that define the duration of the first phase of NF-kappaB activity. The second phase is defined by A20 (a ubiquitin-editing protein that is involved in the negative feedback regulation of NF-kappaB signaling), whose inducible expression provides for a rheostat function by which other inflammatory stimuli can regulate TNF responses. These results delineate the nonredundant functions implied by the knockout phenotypes of ikappabα and a20, and identify the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses (Werner, 2008).

As the molecular connectivity within signaling networks has increasingly become a focus of biomedical research, a surprising number of inducible negative regulators have been identified; these are usually categorized as negative feedback regulators. However, remarkably few have been examined to determine what functional roles their inducible expression may play; indeed, little is known about whether inducible expression can even allow for distinct functional roles. This analysis demonstrates distinct functions for IkappaBα and A20, whose expression is driven by similarly inducible promoters. In the case of IkappaBα, negative feedback is required for function; in other words, no value of constitutive IkappaBα expression parameters will provide the degree of NF-kappaB activation and post-induction repression that NF-kappaB-responsive expression of IkappaBα allows for. In contrast, there is a range of constitutive A20 expression values that can functionally replace A20 negative feedback. Hence, a distinction can be made between an obligate (IkappaBα) and a nonobligate (A20) feedback regulator. Indeed, the A20 regulatory mechanism may not fit a narrower definition of a negative feedback regulator (Werner, 2008).

Instead, the inducibility of A20 expression functions to tune a rheostat that controls cellular signaling responsiveness. This is demonstrated most clearly by the fact that A20 mediates signaling cross-talk between inflammatory stimuli when they are administered sequentially. However, A20's rheostat function is not limited to cross-talk between IL-1 and TNF; its promoter is inducible by all NF-kappaB-inducing stimuli tested so far. It provides short-term cellular memory by transiently 'tolerizing' (i.e., reducing the sensitivity of) the TNF signaling pathway. Whereas the dynamics of IkappaBα inducibility, but not the actual protein concentration, critically define NF-kappaB activity, the A20 protein concentration determines its attenuation function, regardless of whether the protein level was the result of inducible or constitutive expression. This distinction between the negative regulators may explain how subtle misregulation of A20 protein levels have been implicated in a range of physiological and pathological processes, including atherosclerosis, T-cell responsiveness, the homeostasis of signaling by pathogen-sensing receptors and of commensual bacteria, and suppression of autoreactive immune responses (Werner, 2008).

What might be the molecular basis for the differential functionality? There are differences in network connectivity (model topology) and rate constants (parameter values) that may be relevant to consider. Although both IkappaBα and A20 are rapidly and highly inducible at the level of mRNA transcripts (which show a similarly short half-life), producing the larger A20 protein takes more time. More importantly, a significantly longer protein half-life allows for not only a gradual build-up of the A20 protein, but also a memory function that was revealed in cross-talk or priming experiments. Whereas the obligate negative feedback regulator IkappaBα functions as a stochiometric binder of the NF-kappaB activator, the nonobligate feedback attenuator A20 reaches back many more reactions into the pathway, making its effect more temporally diffuse at the NF-kappaB level. In addition, A20 possesses an enzymatic function, which further slows its total functional effect. These conclusions are insensitive to alterations of parameter values within the ranges set by experimental constraints. In fact, model topology aspects mirror prior theoretical considerations pertaining to metabolic networks. It is suggested that theoretical modeling work may prove useful in distinguishing between different categories of negative feedback regulators in signaling. In addition, a combined computational and experimental strategy may be applied to other signaling systems to characterize the functional diversity of negative feedback regulators (Werner, 2008).

TNF-induced NF-kappaB dynamics are encoded not only by IkappaBα and A20, but also by an IKK autorepression mechanism that provides powerful negative feedback on a faster scale than mechanisms involving de novo gene expression. Although the described model recapitulates the observed temporal IKK activity profile, it does not describe the actual regulatory mechanism(s) because further molecular characterization is required. Indeed, recent work suggests that the association of the essential IKK scaffold subunit NEMO (see Drosophila Kenny) with catalytic subunits IKK1 and IKK2 is regulated via phosphorylation. Similarly, the mechanism by which K63-linked ubiquitin chains activate IKK, the involvement of A20 in their removal, and whether and how TAK1/Tab2/3 is involved in IKK control remains to be characterized in more detail. New mechanistic insights should lead to a revision of the mathematical model, in turn enabling an investigation of their role in determining NF-kappaB dynamics. The iterative strategy of combined experimental and modeling work promises to result in amply validated and sufficiently detailed models that may function as standalone discovery tools (Werner, 2008).

In the macrophage, toll-like receptors (TLRs) are key sensors that trigger signaling cascades to activate inflammatory programs via the NF-kappaB gene network. However, the genomic network targeted by TLR/NF-kappaB activation and the molecular basis by which it is restrained to terminate activation and re-establish quiescence is poorly understood. Using chromatin immunoprecipitation sequencing (ChIP-seq), this study has define the NF-kappaB cistrome, which is comprised of 31,070 cis-acting binding sites responsive to lipopolysaccharide (LPS)-induced signaling. In addition, it was demonstrated that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome, and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6-deficient macrophages are acutely hypersensitive to LPS and, using comparative ChIP-seq analyses, it was found that the Bcl-6 and NF-kappaB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6-binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements (Barish, 2010).

NF-kappaB site-specific DNA-binding function

The transcription factor NF-kappaB regulates a wide variety of genes involved in multiple processes. Although the apparent consensus sequence of DNA binding sites for NF-kappaB (kappaB sites) is very broad, the sites active in any one gene show remarkable evolutionary stability. Using a lentivirus-based methodology for implantation of gene regulatory sequences, it has been shown that for genes with two kappaB sites, both are required for activity. Swapping sites between kappaB-dependent genes alters NF-kappaB dimer specificity of the promoters and reveals that two kappaB sites can function together as a module to regulate gene activation. Further, although the sequence of the kappaB site is important for determining kappaB family member specificity, rather than determining the ability of a particular dimer to bind effectively, the sequence affects which coactivators will form productive interactions with the bound NF-kappaB dimer. This suggests that binding sites may impart a specific configuration to bound transcription factors (Leung, 2004).

In a previous study, no direct correlation between the kappaB site sequence and kappaB family member requirements for gene activation could be found (Hoffmann, 2003). In that study, one gene was compared to another but when, in this study, interspecies comparisons were made of the same gene, a remarkable constancy of sequence was found, implying that the individual sequences have important characteristics. This led to an examination the role of the particular sequences found associated with particular genes. To do this a lentiviral system was developed for incorporating regulatory sequences into cellular DNA. Then, by swapping the kappaB site sequences within the MCP-1 promoter to the kappaB site sequences for the IP-10 gene, it was found that IP-10's kappaB family member requirements could be imposed onto the MCP-1 promoter. Both IP-10 kappaB site sequences had to be transferred to change kappaB family member requirements and two kappaB sites were shown to function together as a module to regulate gene activation. This suggests that either MCP-1 site is dominant over the two IP-10 sites. By doing chromatin immunoprecipitation experiments, it was found that even though the I1 site (distal kappaB site of the IP-10 promoter) would not work with the p65 homodimer, the IP-10 kappaB sites did bind the homodimer which, in turn, even bound the coactivator CBP/p300. It was then found that the IP-10 requirement for a kappaB heterodimer for activation by TNFalpha is not evident after LPS stimulation. This suggested that the kappaB site specificity operates by imposing on the DNA bound NF-kappaB a cofactor requirement for activation. In fact, it was found the ML I1I2 transgene requires Bcl-3 when stimulated by TNFalpha and that IRF-3 can play this role in p50/p52-deficient cells where Bcl-3 is not able to function. Because the transcription factor IRF-3 is induced by LPS but not TNFalpha, the role of this kappaB site specificity is explained. The analysis was extended to show that requirement of the IRF-3 for LPS-stimulated ML I1I2 transgene activity applies to p50:p65 heterodimers as well. Therefore, the kappaB site sequence affects the configuration of both heterodimers and p65 homodimers. Finally, it was shown that IRF-3 is recruited to the ML I1I2 promoter. The sequence alteration that imposes the coactivator requirement is a single nucleotide in the sixth position of the kappaB site -- it is quite remarkable that such a change can impose on a gene a new cofactor requirement that is fulfilled only under particular circumstances (Leung, 2004).

There are three models for how the single nucleotide difference in the kappaB site can impose such specificity. One model suggests that there is another protein bound to the DNA site that requires this particular nucleotide. It would most likely bind in conjunction with NF-kappaB, just as HMG I(Y) has been shown to bind to certain kappaB sites along with NF-kappaB. The factor would bind the distal IP-10 kappaB site because that site dominantly imposes the heterodimer restriction. It seems a bit far-fetched but not impossible that a protein could bind to the kappaB site and then dominantly impose a restriction on the functioning of a dimer that would be overcome by a cofactor. IRF-3 and Bcl-3 would be the responsible cofactors in LPS- and TNFalpha-stimulated cells, respectively (Leung, 2004).

A second possibility is that kappaB dimers may oligomerize when bound to their respective sites. The sequence of the kappaB site would determine oligomerization efficiency and precise conformation of the overall structure. The kappaB tetramer would determine which cofactors would be needed for gene activation (Leung, 2004).

A third possibility is that the particular distal IP-10 kappaB site imposes a configuration on the bound heterodimers that establishes a requirement for Bcl-3. In the p50/p52-deficient cells, where Bcl-3 cannot bind the p65 homodimers, the requirement for a coactivator can be supplied by IRF-3, which can bind to p65 (Leung, 2004).

To fully explain these data, the authors return to the observation that two kappaB sites are needed for the MCP-1 promoter to function. The stimulus and subunit specificities have been explained by the role of one kappaB site, but why then are two needed? It is suspected that the two sites serve different and nonredundant roles. This postulate leads to a model that the two kappaB sites serve different and jointly obligate functions (Leung, 2004).

In LPS-stimulated p50/p52-deficient cells infected with the ML M1M2 transgene, it is known that p65 homodimers are bound to the kappaB sites along with p300/CBP and drive luciferase expression. The postulate that the two sites serve different functions leads to the supposition that only one kappaB homodimer binds p300 and that the other binds an unknown factor X but other explanations are conceivable. In LPS-stimulated p50/p52-deficient cells infected with the ML I1I2 transgene, it is postulated that the I2 site continues to bind p300/CBP but the I1 kappaB site sequence alters the p65 homodimer conformation such that another cofactor must take the place of X. IRF-3 can serve this function and in LPS-induced cells, it binds and cooperates to drive luciferase expression. In TNFalpha-stimulated p50/p52-deficient cells with the ML M1M2 transgene, the situation is similar to that in LPS-simulated cells. However, in the TNFkappa-stimulated p50/p52-deficient cells with the ML I1I2 transgene, the change in the I1 kappaB site sequence alters the conformation for p65 homodimers such that they require a cofactor of a type that is simply not present in the cells. Instead, only kappaB heterodimers are able to drive transcription because of the requirement for the second activity is supplied by Bcl-3 binding to the p50 subunit (Leung, 2004).

MCP-1 and IP-10 are differentially regulated, presumably because they serve different functions. MCP-1 plays a significant role in innate immunity by bringing macrophages to sites of inflammation. In contrast, IP-10 is important for lymphocytic (adaptive) immunity and regulates T cell proliferation. MCP-1 is also activated by a more diverse set of TLR agonists than IP-10. For example, TLR-2 agonists stimulate MCP-1, but not IP-10 expression. Recent studies reveal that MCP-1 and IP-10 are regulated differently during TLR-4 stimulation. IP-10 is activated through TLR-4 via a Trif-dependent pathway. The Trif-dependent pathway activates the interferon-response pathway and is responsible for a late NF-kappaB activation. It has been shown that this pathway regulates a specific subset of TLR3/TLR4-dependent genes, and that the pathway is evolutionarily diverged from other members of the TLR family. In contrast, MCP-1 is activated by both the MyD88-dependent and Trif-dependent pathways. The MyD88 pathway differs from the Trif pathway in two ways: (1) it does not activate the interferon-response pathway; (2) it activates NF-kappaB much earlier than the Trif pathway (Leung, 2004).

These two pathways normally work together to ready an immune response to a bacterial pathogen. A bacterial pathogen would signal the TLR-4 receptor and activate both downstream pathways. The MyD88 pathway would activate NF-kappaB immediately to drive MCP-1 production and recruit macrophages to the target site. Later, the Trif pathway would activate both the NF-kappaB and IRF-3 pathways to drive IP-10 production and regulate T cell proliferation. By simply changing the sequence of kappaB sites in the MCP-1 promoter, MCP-1's regulation profile was converted into IP-10's. It is speculated that in a mouse this would severely disrupt the delicate balance between innate and adaptive immunity. The results underscore the functional importance of the sequence of the kappaB site and confirm why the sequence of kappaB sites is strictly conserved over time. Not only does the kappaB site sequence determine kappaB dimer specificity, it also determines coactivator requirements (Leung, 2004).

NFkappaB and chromatin

the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by recruitment of the RNA polymerase II transcriptional machinery to the chromatin-repressed basal promoter (Pazin, 1996).

Are transcription factors with diverse DNA binding domains able to exploit nucleosome disruption by SWI/SNF? To test this, an investigation was made into the possible mechanisms by which the SWI/SNF complex differentially regulates different genes. In addition to GAL4-VP16, the SWI/SNF complex stimulates nucleosome binding by the Zn2+ fingers of Sp1, the basic helix-loop-helix domain of USF, and the rel domain of NF-kappaB. In each case SWI/SNF action results in the formation of a stable factor-nucleosome complex that persists after the detachment of SWI/SNF from the nucleosome. Thus, stimulation of factor binding by SWI/SNF appears to be universal. The degree of SWI/SNF stimulation of nucleosome binding by a factor appears to be inversely related to the extent that binding is inhibited by the histone octamer. Cooperative binding of 5 GAL4-VP16 dimers to a 5-site nucleosome enhances GAL4 binding relative to a single-site nucleosome, but this also reduces the degree of stimulation by SWI/SNF. The SWI/SNF complex increases the affinity of 5 GAL4-VP16 dimers for nucleosomes equal to that of DNA but no further. Similarly, multimerized NF-kappaB sites enhance nucleosome binding by NF-kappaB and reduce the stimulatory effect of SWI/SNF. Thus, cooperative binding of factors to nucleosomes is partially redundant with the function of the SWI/SNF complex (Utley, 1997).

Defining the molecular mechanisms that integrate diverse signaling pathways at the level of gene transcription remains a central issue in biology. Interleukin-1ß (IL-1ß) causes nuclear export of a specific N-CoR corepressor complex, resulting in derepression of a specific subset of NF-kappaB-regulated genes, exemplified by the tetraspanin KAI1 that regulates membrane receptor function. Nuclear export of the N-CoR/TAB2/HDAC3 complex by IL-1ß is temporally linked to selective recruitment of a Tip60 coactivator complex. Surprisingly, KAI1 is also directly activated by a ternary complex, dependent on the acetyltransferase activity of Tip60, consisting of the presenilin-dependent C-terminal cleavage product of the amyloid ß precursor protein (APP: Drosophila homolog: ß amyloid protein precursor-like), Fe65, and Tip60, identifying a specific in vivo gene target of an APP-dependent transcription complex in the brain (Baek, 2002).

This work defines a molecular mechanism that links inflammation to derepression of a specific subset of NF-kappaB-regulated genes via control of a previously unknown stable N-CoR complex. This N-CoR/TAB2/HDAC3-containing complex binds to p50-regulated target genes and undergoes a nuclear to cytoplasmic translocation in response to IL-1ß signaling. TAB2 (Drosophila homolog: TGF-ß activated kinase 1) itself enhances N-CoR-dependent repression, but the apparently critical function of TAB2 is to regulate IL-1ß-mediated translocation of the N-CoR complex out of the nucleus. TAB2 thus seems to have dual roles upon activation of the NF-kappaB pathway, serving to both derepress p50-dependent transcription units (nuclear function) as well as to activate the p50/p65 targets (cytoplasmic function) (Baek, 2002).

Evidence is provided indicating that the molecular basis of IL-1ß-dependent nuclear export of the nuclear N-CoR likely represents a MEKK1-dependent phosphorylation of TAB2 in the nucleus, putatively causing an allosteric alteration that exposes the TAB2 nuclear export signal. Thus, MEKK1 might also serve to integrate signal transduction pathways, both in the nucleus and in the cytoplasm (Baek, 2002).

These data indicate that KAI1/CD82 is an IL-1ß-induced NF-kappaB target gene based on binding of p50 homodimer. Under unstimulated conditions, p50, but not p65, was detected on the KAI1 promoter, while after IL-1ß stimulation, the level of promoter-associated p50 remained constant, without any binding by p65. Bcl3 occupies the KAI1 promoter in the presence or absence of IL-1ß, and several independent studies have suggested that Bcl3 can act as a bridging factor linking NF-kappaB to nuclear coregulators. Tip60 has been suggested to be a binding partner of Bcl3, enhancing Bcl3/p50-activated transcription through a NF-kappaB binding site (Baek, 2002).

Thus, the recruitment of Tip60 to KAI1/CD82 promoter after IL-1ß treatment, possibly requiring Bcl3, appears to be of functional importance to activation of the gene, which is accompanied by acetylation of histones H3/H4. Tip60 has been identified as a component of a multimeric protein complex containing histone acetylase, ATPase, DNA helicase activity, and structural DNA binding activity, which links it to DNA repair function. In the case of KAI1/CD82 promoter, Tip60 appears to be recruited as a component of a TRRAP-containing complex, likely distinct from the purified repair complex, although the precise complement of corecruited factors remains to be defined. The Tip60 HAT function appears to be required, directly or indirectly, for effective gene activation, because Tip60 HAT_mut abolishes histone H3 and H4 acetylation and recruitment of Pol II. The observed acetylation of histones H3/H4 during IL-1ß-stimulated KAI1 transcription could reflect direct acetylation by Tip60 in a promoter-specific fashion, or it could reflect recruitment of an as yet unidentified histone acetyltransferase. In contrast to KAI1, examination of two other NF-kappaB-regulated genes that recruit p50/p65 heterodimers revealed no recruitment of the N-CoR/TAB2/HDAC3 complex. The identification of a large number of transcriptional coactivators and corepressors, capable of interacting with distinct DNA bound transcription factors, has raised questions regarding their potential specificity and complementarity in gene regulation events. The NF-kappaB-regulated genes examined appear to recruit distinct coactivator machinery during gene activation events in response to IL-1ß, which is in contrast to the apparently more uniform, ligand-dependent recruitment of many coactivator complexes in estrogen receptor-regulated genes (Baek, 2002).

gamma-Secretase cleavage of APP releases not only Aß from the membrane but also the intracellular fragment AICD, which was identified only very recently because of its instability. A number of investigators have speculated that by analogy to signaling by NICD derived from Notch 1 receptor, the corresponding AICD may also function in signal transduction. In this manuscript, it has been shown that transgenic mice overexpressing APP, which develop age-related amyloid deposits and associated pathologic changes, unexpectedly exhibit increased expression of both Fe65 and Tip60 in the CNS. All three components of the AICD/Fe65/Tip60 complex are unexpectedly induced, forming a complex binding to the KAI1/CD82 promoter. This complex is capable of displacing the N-CoR/TAB2/HDAC3 complex in the absence of an IL-1ß signal and causing target gene activation. KAI1 itself provides a potentially intriguing transcriptional target of APP overexpression. As many cell surface receptors and cell adhesion molecules that regulate cytoskeletal functions are impacted by the tetraspanin KAI1/CD82, it is tempting to speculate that expression of KAI1 might contribute to later pathological events (Baek, 2002).

Remarkably, the acetyltransferase function of Tip60 is required to form the ternary complex that can displace an N-CoR/TAB2/HDAC3 corepressor complex. In addition to autoacetylation of Tip60, increased levels of acetylated Fe65 are found in the trimeric complex, suggesting that acetylation of Fe65 might be a regulatory component of ternary complex formation. These data provide a striking example of acetylation as a critical regulatory aspect of coactivator complex assembly, required for specific gene activation events. Since the transcriptional activation of KAI1 by AICD/Fe65/Tip60 is abolished by both the NSAIDs ibuprofen and naproxen with restoration of the binding of the NCoR/TAB2/HDAC3 complex on the promoter, it is postulated that NSAIDs may act at some step(s) distal to generation of AICD by an as yet unknown mechanism (Baek, 2002).

Together, these data are consistent with a model in which IL-1ß acts physiologically to cause dismissal of a specific N-CoR corepressor complex and recruitment of a Tip60-containing coactivator complex resulting in activation of p50 target genes. The AICD/Fe65/Tip60 trimeric complex can similarly displace the N-CoR complex, derepressing gene targets such as KAI1/CD82, providing a potential transcriptional activation strategy that may underlie specific aspects of APP function, both in normal physiology and in Alzheimer's disease (Baek, 2002).

NF-kappaB is a principal transcriptional regulator of diverse cytokine- mediated processes and is tightly controlled by the IkappaB kinase complex (IKK-alpha/beta/gamma). IKK-beta and IKK-gamma are critical for cytokine-induced NF-kappaB function, whereas IKK-alpha is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-alpha in NF-kappa B-dependent gene expression in response to cytokine treatment1. Nuclear accumulation of IKK-alpha after cytokine exposure is demonstrated, suggests a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-alpha is recruited to the promoter regions of NF-kappaB-regulated genes on stimulation with tumor-necrosis factor-alpha. Notably, NF-kappaB regulated gene expression is suppressed by the loss of IKK-alpha and this correlates with a complete loss of gene-specific phosphorylation of histone H3 on serine 10, a modification associated with positive gene expression. Furthermore, IKK-alpha is shown to directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. It is proposed that IKK-alpha is an essential regulator of NF-kappaB-dependent gene expression through control of promoter-associated histone phosphorylation after cytokine exposure. These findings provide additional insight into the role of the IKK complex in NF-kappaB regulated gene expression (Anest, 2003).

Signaling downstream of NF-kappaB

IKKβ-dependent NF-κB activation plays a key role in innate immunity and inflammation, and inhibition of IKKβ has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKβ deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1β as a result of increased pro-IL-1β processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1β processing depends on caspase-1, whose activation is inhibited by NF-κB-dependent gene products. In neutrophils, however, IL-1β secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-κB gene products. Prolonged pharmacologic inhibition of IKKβ also augments IL-1β secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKβ-dependent NF-κB signaling in the negative control of IL-1β production and highlight potential complications of long-term IKKβ inhibition (Greten, 2007).

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