Expression Of NF-κB Target Genes Research Articles

The PPARß Activation Mechanism Which Suppresses the Constitutive NF-kB Activity in Human Myeloma Cell Lines.

[Purpose] It is considered that human myeloma cells have the constitutively high NF-kB activity involved in survival and proliferation. PPAR (Peroxisome proliferator-activated receptor) ß is ubiquitously expressed in all cells and considered to be involved in the lipid metabolism and regulating the inflammatory response and cell proliferation. We already have found that adrenal cortex hormones (DHEA and DHEA-S etc), dexamethasone (Dex) and baicalein augmented the expression of reporter gene. However, it remain to be clarified the role of PPARß in human myeloma cells. We focus on the mechanism of PPARß suppressed NF-kB activity.[Method]1.To know NF-kB activity of human myeloma cells, we performed EMSA with NF-kB consensus oligo.2.To investigate NF-kB and PPARß after stimulation of PPAR agonist, we did EMSA with NF-kB and PPAR consensus oligo.3.To confirm whether it is repression according the NF-kB activity suppression accompanying PPARß activation to the interaction of PPARß and NF-kB, we conducted immunoprecipitation - western blot analysis.4.To check whether the expression of NF-kB target genes (cIAP1, Bcl-xL, etc) were suppressed after stimulation of PPAR agonist, we performed RT-PCR analysis.[Result and discussion] It was suggested that human myeloma cell lines have constitutive NF-kB activity, and its activity mainly regulated p50. NF-kB activity and its target genes were repressed by stimulation of PPAR agonist. From the above, it was suggested that the activated PPARß interacted NF-kB and then its activity was suppressed.

Glutathione Depletion Down-regulates Tumor Necrosis Factor α-induced NF-κB Activity via IκB Kinase-dependent and -independent Mechanisms

Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-kappaB target genes induced by tumor necrosis factor alpha (TNFalpha) and sensitize primary cultured mouse hepatocytes to TNF-mediated apoptotic killing. Here we demonstrate in the same system that GSH depletion down-regulates TNF-induced NF-kappaB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (approximately 50%), the down-regulation is IkappaB kinase (IKK)-independent and likely acts on NF-kappaB transcriptional activity because TNF-induced IKK activation, IkappaBalpha phosphorylation and degradation, NF-kappaB nuclear translocation, NF-kappaB DNA binding in vitro, and NF-kappaB subunit RelA(p65) recruitment to kappaB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (approximately 80%), the down-regulation also is IKK-dependent, and a timeline is established linking the inhibition of polyubiquitination of receptor-interacting protein 1 in TNF receptor 1 complex to partial blockage of IKK activation, IkappaBalpha phosphorylation and degradation, and NF-kappaB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-kappaB nuclear translocation but fails to restore expression of NF-kappaB target genes, revealing both IKK-dependent and -independent inhibition. These findings provide new insights into the complex effects of oxidative stress and redox perturbations on the NF-kappaB pathway.

Smad7 Sensitizes Tumor Necrosis Factor–Induced Apoptosis through the Inhibition of Antiapoptotic Gene Expression by Suppressing Activation of the Nuclear Factor-κB Pathway

Although tumor necrosis factor (TNF) induces apoptosis and cell death in many tumor cells, some cancer cells are still resistant to the TNF-induced death signal. In this report, we showed that Smad7, an inhibitory Smad of transforming growth factor-beta (TGF-beta) signaling, can overcome the TNF resistance in human breast and gastric cancer cells. Overexpression of Smad7 induces the degradation of poly(ADP-ribose) polymerase and the activation of caspase cascade. Although c-Jun NH2-terminal kinase (JNK) signaling is involved in TNF-induced cell death, the expression of Smad7 does not synergize the activation of JNK. However, the activation of nuclear factor-kappaB (NF-kappaB), the cell survival factor, is markedly decreased in Smad7-stable cells. Furthermore, the expression of antiapoptotic target genes of NF-kappaB is significantly reduced in accordance with the level of Smad7. In addition, Smad7 mediates the inhibitory activity of TGF-beta on TNF-induced NF-kappaB activation and the synergistic activity of TGF-beta on TNF-induced apoptosis. These findings suggest that Smad7 sensitizes the tumor cells to TNF-induced apoptosis through the inhibition of expression of antiapoptotic NF-kappaB target genes.

Deficiency of the NF-κB Inhibitor Caspase Activating and Recruitment Domain 8 in Patients with Rheumatoid Arthritis Is Associated with Disease Severity

Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-kappaB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-kappaB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-kappaB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-alpha and the p65 subunit of NF-kappaB. Moreover, TNF-alpha and overexpression of p65 induced the formation of NF-kappaB-CARD8 promoter complexes. Thus, CARD8 may control NF-kappaB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-kappaB transcriptional activity, and abrogates the binding of NF-kappaB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-kappaB target genes (cIAP, A1), in response to TNF-alpha. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-kappaB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.

NF-κB activation in inflammatory breast cancer is associated with oestrogen receptor downregulation, secondary to EGFR and/or ErbB2 overexpression and MAPK hyperactivation

Activation of NF-κB in inflammatory breast cancer (IBC) is associated with loss of estrogen receptor (ER) expression, indicating a potential crosstalk between NF-κB and ER. In this study, we examined the activation of NF-κB in IBC and non-IBC with respect to ER and EGFR and/or ErbB2 expression and MAPK hyperactivation. A qRT–PCR based ER signature was evaluated in tumours with and without transcriptionally active NF-κB, as well as correlated with the expression of eight NF-κB target genes. Using a combined ER/NF-κB signature, hierarchical clustering was executed. Hyperactivation of MAPK was investigated using a recently described MAPK signature (Creighton et al, 2006), and was linked to tumour phenotype, ER and EGFR and/or ErbB2 overexpression. The expression of most ER-modulated genes was significantly elevated in breast tumours without transcriptionally active NF-κB. In addition, the expression of most ER-modulated genes was significantly anticorrelated with the expression of most NF-κB target genes, indicating an inverse correlation between ER and NF-κB activation. Clustering using the combined ER and NF-κB signature revealed one cluster mainly characterised by low NF-κB target gene expression and a second one with elevated NF-κB target gene expression. The first cluster was mainly characterised by non-IBC specimens and IHC ER+ breast tumours (13 out of 18 and 15 out of 18 respectively), whereas the second cluster was mainly characterised by IBC specimens and IHC ER− breast tumours (12 out of 19 and 15 out of 19 respectively) (Pearson χ2, P<0.0001 and P<0.0001 respectively). Hyperactivation of MAPK was associated with both ER status and tumour phenotype by unsupervised hierarchical clustering using the MAPK signature and was significantly reflected by overexpression of EGFR and/or ErbB2. NF-κB activation is linked to loss of ER expression and activation in IBC and in breast cancer in general. The inverse correlation between NF-κB activation and ER activation is due to EGFR and/or ErbB2 overexpression, resulting in NF-κB activation and ER downregulation.

High expression of RelA/p65 is associated with activation of nuclear factor-κB-dependent signaling in pancreatic cancer and marks a patient population with poor prognosis

Activation of nuclear factor-κB (NF-κB) signaling was observed in pancreatic adenocarcinoma cell lines and tumours. However, information on the expression of RelA/p65, the major transcription activating NF-κB subunit, in these carcinomas and possible correlations thereof with NF-κB activation and patient survival is not available. To provide this missing translational link, we analysed expression of RelA/p65 in 82 pancreatic adenocarcinomas by immunohistochemistry. Moreover, we measured activation of the NF-κB pathway in 11 tumours by quantitative PCR for NF-κB target genes. We observed strong cytoplasmic or nuclear expression of RelA/p65 in 42 and 37 carcinomas, respectively. High cytoplasmic and nuclear expression of RelA/p65 had negative prognostic impact with 2-year survival rates for patients without cytoplasmic or nuclear RelA/p65 positivity of 41 and 40% and rates for patients with strong cytoplasmic or nuclear RelA/p65 expression of 22 and 20%, respectively. High RelA/p65 expression was correlated to increased expression of NF-κB target genes. The observation that high expression of RelA/p65 is correlated to an activation of the NF-κB pathway and indicates poor patient survival identifies a patient subgroup that might particularly benefit from NF-κB-inhibiting agents in the treatment of pancreatic cancer. Based on our findings, this subgroup could be identified by applying simple immunohistochemical techniques.

Identifying Oncogenes

The extensive karyotypic abnormalities and genetic disruption characteristic of epithelial cancers make it difficult to pinpoint which mutations are critical to the development and maintenance of cancer. Boehm et al. used an integrative genomic approach to identify potential oncogenes by determining which signaling pathways downstream of oncogenic Ras contribute to cell transformation. They created 16 human embryonic kidney (HEK) cell lines that expressed single or paired combinations of mutant alleles that activated the MAPK (mitogen-activated protein kinase) or PI3K (phosphatidylinositol 3-kinase) signaling pathways and assessed their ability to recapitulate the tumorigenic phenotype of oncogenic Ras. Among these, the phenotype of a constitutively active mutant of MEK1 (a MAPK kinase) in combination with a myristoylated AKT1 mutant (myr-AKT, which constitutively activates PI3K signaling) most resembled that of oncogenic Ras. The authors then screened a library of activated kinases and identified four that could substitute for myr-AKT in eliciting an oncogenic Ras-like phenotype in combination with the MEK1 mutant. One of these, IKBKE [which encodes inhibitor of κB kinase ε (IKKε)], was amplified and overexpressed in a substantial fraction of human breast cancer cells and breast cancer cell lines. Moreover, shRNA directed against IKBKE inhibited the proliferation and survival of breast cancer cell lines with IKBKE amplification. Cells expressing constitutively active IKBKE showed an increase in NF-κB nuclear translocation and in expression of NF-κB target genes, and inhibition of NF-κB signaling increased doubling time and suppressed colony formation in IKBKE -transformed HEK cells. Furthermore, IKKε was associated with NF-κB activation in breast cancer cell lines and tissue. Thus, the authors conclude that IKBKE is a breast cancer oncogene that may link PI3K signaling to activation of the NF-κB pathway. Agami discusses the work. J. S. Boehm, J. J. Zhao, J. Yao, S. Y. Kim, R. Firestein, I. F. Dunn, S. K. Sjostrom, L. A. Garraway, S. Weremowicz, A. L. Richardson, H. Greulich, C. J. Stewart, L. A. Mulvey, R. R. Shen, L. Ambrogio, T. Hirozane-Kishikawa, D. E. Hill, M. Vidal, M. Meyerson, J. K. Grenier, G. Hinkle, D. E. Root, T. M. Roberts, E. S. Lander, K. Polyak, W. C. Hahn, Integrative genomic approaches identify IKBKE as a breast cancer oncogene. Cell 129 , 1065−1079 (2007). [PubMed] R. Agami, All roads lead to ΙΚΚε. Cell 129 , 1043-1045 (2007). [PubMed]

Activation of peroxisome proliferator-activated receptor-γ protects pancreatic β-cells from cytokine-induced cytotoxicity via NFκB pathway

Diabetes mellitus is characterized by cytokine-induced insulitis and a deficit in β-cell mass. Ligands for peroxisome proliferator-activated receptor-γ (PPAR-γ) have been shown to have anti-inflammatory effects in various experimental models. We questioned whether activation of endogenous PPAR-γ by either PPAR-γ ligands or adenoviral-directed overexpression of PPAR-γ (Ad-PPAR-γ) could inhibit cytokine-induced β-cell death in RINm5F (RIN) cells, a rat insulinoma cell line. Treatment of RIN cells with interleukin-1β (IL-1β) and interferon-γ (IFN-γ) induced β-cell damage through NFκB-dependent signaling pathways. Activation of PPAR-γ by PPAR-γ ligands or Ad-PPAR-γ inhibited IL-1β and IFN-γ-stimulated nuclear translocation of the p65 subunit and DNA binding activity. NFκB target gene expression and their product formation, namely inducible nitric oxide synthase and cyclooxygenase-2 were decreased by PPAR-γ activation, as established by real-time PCR, Western blots and measurements of NO and PGE 2. The mechanism by which PPAR-γ activation inhibited NFκB-dependent cell death signals appeared to involve the inhibition of IκBα degradation, evidenced by inhibition of cytokine-induced NFκB-dependent signaling events by Ad-IκBα (S32A, S36A), non-degradable IκBα mutant. IκBβ mutant, Ad-IκBβ (S19A, S23A) was not effective in preventing cytokine toxicity. Furthermore, a protective effect of PPAR-γ ligands was proved by assaying for normal insulin secreting capacity in response to glucose in isolated rat pancreatic islets. The β-cell protective function of PPAR-γ ligands might serve to counteract cytokine-induced β-cell destruction.

Pregnane X receptor activation ameliorates DSS-induced inflammatory bowel disease via inhibition of NF-κB target gene expression

Pregnane X receptor (PXR) expression was shown to be protective in inflammatory bowel disease (IBD). However, the mechanism by which PXR provides protection remains unclear. Wild-type and Pxr-null mice were treated with the PXR agonist pregnenolone-16alpha-carbonitrile or vehicle and administered 2.5% dextran sulfate sodium (DSS) in drinking water to induce IBD. Typical clinical symptoms were evaluated on a daily basis. In vivo intestinal permeability assays and proinflammatory cytokine analysis were performed. PXR agonist-treated mice were protected from DSS-induced colitis compared with vehicle-treated mice, as defined by body weight loss, diarrhea, rectal bleeding, colon length, and histology. Pregnenolone-16alpha-carbonitrile did not decrease the severity of IBD in Pxr-null mice. PXR agonist treatment did not increase epithelial barrier function but did decrease mRNA expression of several NF-kappaB target genes in a PXR-dependent manner. The present study clearly demonstrates a protective role for PXR agonist in DSS-induced IBD. The data suggest that PXR-mediated repression of NF-kappaB target genes in the colon is a critical mechanism by which PXR activation decreases the susceptibility of mice to DSS-induced IBD.

Human Cytomegalovirus IE86 Attenuates Virus- and Tumor Necrosis Factor Alpha-Induced NFκB-Dependent Gene Expression

Human cytomegalovirus (HCMV) infection regulates a number of genes involved in the host antiviral response. We have previously reported that HCMV attenuates the expression of beta interferon (IFN-beta) and a number of proinflammatory chemokines, and this attenuation is mediated by the HCMV immediate-early protein IE86. The present study seeks to identify the mechanism by which IE86 blocks IFN-beta expression. We demonstrate that the induction of IFN-beta during HCMV infection requires the activation of both the IRF-3 and the NFkappaB pathways. Therefore, IE86 may target either pathway to block IFN-beta expression. Our results show that IE86 does not block IRF-3 phosphorylation, dimerization, nuclear translocation, or target gene expression. However, using gel shift analysis, we demonstrate that IE86 efficiently inhibits virus-induced binding of NFkappaB to the IFN-beta promoter, resulting in attenuation of IFN-beta and NFkappaB-dependent gene expression. Furthermore, IE86 expression inhibits tumor necrosis factor alpha-induced NFkappaB DNA binding and target gene expression. Together, these results identify IE86 as a NFkappaB antagonist, which results in the suppression of NFkappaB-dependent cytokine and chemokine gene expression.

Inhibitory effect of sesaminol glucosides on lipopolysaccharide-induced NF-κB activation and target gene expression in cultured rat astrocytes

The inflammatory reaction plays an important role in the pathogenesis of the neurodegenerative disorder including Alzheimer's disease (AD). Sesame lignan compounds such as sesaminol glucosides (SG) exhibit a range of pharmacological activities including anti-oxidative and anti-inflammatory action. In this study, we tried to elucidate possible effects of SG on lipopolysaccharide (LPS)-induced inflammatory reaction and its underlying mechanism in cultured astrocytes. SG (10–100 μg/ml) inhibited LPS-induced generation of nitric oxide (NO) and reactive oxygen species (ROS), as well as inhibited LPS-induced cytosolic phospholipase A 2 (cPLA 2), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression dose-dependently. This inhibitory effect of SG on NO and ROS generation was enforced by addition of glutathione (GSH) in culture. In addition, SG prevented LPS-induced DNA binding and transcriptional activity of nuclear factor KappaB (NF)-κB. Consistent with the inhibitory effect on NF-κB activity, SG inhibits phosphorylation and degradation of inhibitory KappaB (IκB), thereby translocation of p50 of NF-κB. These data show that SG has an anti-inflammatory effect through inhibition of NF-κB, and may be a useful agent for prevention of inflammatory disease like AD.

Histone Deacetylase Inhibitors Suppress the Inducibility of Nuclear Factor-κB by Tumor Necrosis Factor-α Receptor-1 Down-regulation

Recently, the inhibition of histone deacetylase (HDAC) enzymes has attracted attention in the oncologic community as a new therapeutic opportunity for hematologic and solid tumors including non-small cell lung cancer (NSCLC). In hematologic malignancies, such as diffuse large B-cell lymphoma, the HDAC inhibitor (HDI), suberoylanilide hydroxamic acid (SAHA), has recently entered phase II and III clinical trials. To further advance our understanding of their action on tumor cells, we investigated the possible effect of HDI treatment on the functionality of the nuclear factor-kappaB (NF-kappaB) pathway in NSCLC. We found that in the NSCLC cell lines, A549 and NCI-H460, the NF-kappaB pathway was strongly inducible, for example, by stimulation with tumor necrosis factor-alpha (TNF-alpha). Incubation of several NSCLC cell lines with HDIs resulted in greatly reduced gene expression of TNF-alpha receptor-1. HDI-treated A549 and NCI-H460 cells down-regulated TNF-alpha receptor-1 mRNA and protein levels as well as surface exposure, and consequently responded to TNF-alpha treatment with reduced IKK phosphorylation and activation, delayed IkappaB-alpha phosphorylation, and attenuated NF-kappaB nuclear translocation and DNA binding. Accordingly, stimulation of NF-kappaB target gene expression by TNF-alpha was strongly decreased. In addition, we observed that SAHA displayed antitumor efficacy in vivo against A549 xenografts grown on nude mice. HDIs, therefore, might beneficially contribute to tumor treatment, possibly by reducing the responsiveness of tumor cells to the TNF-alpha-mediated activation of the NF-kappaB pathway. These findings also hint at a possible use of HDIs in inflammatory diseases, which are associated with the overproduction of TNF-alpha, such as rheumatoid arthritis or Crohn's disease.

Host and parasite-derived IKK activities direct distinct temporal phases of NF-κB activation and target gene expression followingToxoplasma gondiiinfection

Activation of NF-kappaB by the intracellular pathogen Toxoplasma gondii is associated with the localization of phosphorylated IkappaB alpha to the parasitophorous vacuole membrane (PVM). This is mediated by a parasite-derived IkappaB kinase (TgIKK) activity and is independent of host IKK function. In the present study, we examined the roles of host IKK and parasite-derived TgIKK on the temporal modulation of NF-kappaB activation. Despite the presence of TgIKK activity at the PVM, nuclear translocation of NF-kappaB and subsequent gene expression exhibited a requirement for the host IKK complex. A detailed kinetic analysis of NF-kappaB activation revealed a biphasic, hierarchical and temporally regulated response. We propose a novel paradigm for the modulation of NF-kappaB-dependent gene expression by T. gondii that involves both the host IKK complex and TgIKK activity at different phases of infection. Thus, T. gondii effectively alters gene expression in a temporal dimension by exploiting the NF-kappaB signaling machinery and subsequently rewiring the activation circuits of the infected host cell.

RelA repression of RelB activity induces selective gene activation downstream of TNF receptors

TNF-alpha is a potent proinflammatory cytokine that regulates immune and inflammatory responses and programmed cell death. TNF-alpha stimulation causes nuclear translocation of several NF-kappaB dimers, including RelA/p50 and RelB/p50. However, contrary to RelA, RelB entering the nucleus in response to TNF-alpha cannot bind to DNA in mouse embryonic fibroblasts, strongly suggesting that RelB DNA-binding activity is modulated by additional nuclear mechanisms. Here, we demonstrate that TNF-alpha promotes the association of RelA with RelB in the nucleus and that TNF-alpha-induced RelA/RelB heterodimers do not bind to kappaB sites. Remarkably, we show that RelA serine-276, the phosphorylation of which is induced by TNF receptor ligation, is crucial for RelA/RelB complex formation and subsequent inhibition of RelB DNA binding. In the absence of RelA phosphorylation on serine-276, TNF-alpha stimulation leads to a strong increase in the expression of endogenous NF-kappaB-responsive genes, such as Bcl-xL, whose transcriptional up-regulation is mainly controlled by RelB. Our findings demonstrate that RelA has a major regulatory role serving to dampen RelB activity in response to TNF-alpha and define a previously unrecognized mechanism that represents an essential step leading to selective NF-kappaB target gene expression.

Cobrotoxin Inhibits NF-κB Activation and Target Gene Expression through Reaction with NF-κB Signal Molecules

Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.

Avoiding Too Much of a Good Thing

The transcription factor nuclear factor κB (NF-κB) plays a critical role in mediating inflammatory responses to bacterial pathogens and proinflammatory cytokines. Proinflammatory stimuli lead to activation of IKK [inhibitor of NF-κB α (IκBα) kinase], a complex that contains two catalytic subunits--IKKα and IKKβ. Whereas IKKβ phosphorylates IκBα, enabling NF-κB activation, the role that IKKα plays in the inflammatory response has been less clear. Lawrence et al. discovered that mice expressing inactivatable IKKα ( Ikkα AA/AA mice) not only showed reduced bacterial titers when challenged with group B Streptococcus (GBS) but, paradoxically, had reduced survival. When injected with Escherichia coli lipopolysaccharide (LPS), Ikkα AA/AA mice exhibited increased expression of NF-κB target genes and enhanced susceptibility to septic shock. Experiments in which the hematopoietic system of wild-type mice was reconstituted with bone marrow from Ikkα AA/AA mice revealed that this exaggerated inflammatory response depended on cells of the hematopoietic system. Macrophages from Ikkα AA/AA mice were resistant to GBS-induced apoptosis and displayed enhanced in vitro bactericidal activity and increased expression of proinflammatory and antiapoptotic genes after exposure to LPS. Although NF-κB DNA binding activity and nuclear translocation of the NF-κB subunits RelA and c-Rel were initially comparable in Ikkα AA/AA and wild-type mice, increased nuclear content of RelA and c-Rel persisted in Ikkα AA/AA mice, as did their association with a target gene promoter. Moreover, phosphorylation of RelA was reduced in macrophages from Ikkα AA/AA mice, and the half-life of RelA and c-Rel was prolonged. Thus, the authors propose that, in contrast to IKKβ, the role of IKKα is to terminate the inflammatory response. T. Lawrence, M. Bebien, G. Y. Liu, V. Nizet, M. Karin, IKKα limits macrophage NF-κB activation and contributes to the resolution of inflammation. Nature 434 , 1138-1143 (2005). [PubMed]

The Met-196 → Arg Variation of Human Tumor Necrosis Factor Receptor 2 (TNFR2) Affects TNF-α-induced Apoptosis by Impaired NF-κB Signaling and Target Gene Expression

Tumor necrosis factor-alpha (TNF-alpha)-induced signaling is pivotally involved in the pathogenesis of chronic inflammatory diseases. A polymorphism in the TNF receptor 2 (TNFR2) gene resulting in a juxtamembrane inversion from methionine (TNFR2(196MET)) to arginine (TNFR2(196ARG)) has been genetically associated with an increased risk for systemic lupus erythematosus and familial rheumatoid arthritis. Albeit the mutation does not affect the TNF binding kinetics of TNFR2, the present study provides evidence that the mutation results in a significantly lower capability to induce TNFR2-mediated NF-kappaB activation. Pretriggering of TNFR2 with a receptor-specific mutein leads to an enhancement of TNFR1-induced apoptosis, which is further increased in cells carrying the TNFR2(196ARG) variant. A diminished induction of NF-kappaB-dependent target genes conveying either anti-apoptotic or pro-inflammatory functions, such as cIAP1, TRAF1, IL-6, or IL-8 is observed. The mutated form TNFR2(196ARG) shows a reduction of inducible TRAF2 recruitment upon TNF-alpha stimulation. The findings suggest a common molecular mechanism for the involvement of the TNFR2(196ARG) variant in the etiopathogenesis of different chronic inflammatory disorders.

Neuronal Activation of NF-κB Contributes to Cell Death in Cerebral Ischemia

The transcription factor NF-kappaB is a key regulator of inflammation and cell survival. NF-kappaB is activated by cerebral ischemia in neurons and glia, but its function is controversial. To inhibit NF-kappaB selectively in neurons and glial cells, we have generated transgenic mice that express the IkappaBalpha superrepressor (IkappaBalpha mutated at serine-32 and serine-36, IkappaBalpha-SR) under transcriptional control of the neuron-specific enolase (NSE) and the glial fibrillary acidic protein (GFAP) promoter, respectively. In primary cortical neurons of NSE-IkappaBalpha-SR mice, NF-kappaB activity was partially inhibited. To assess NF-kappaB activity in vivo after permanent middle cerebral artery occlusion (MCAO), we measured the expression of NF-kappaB target genes by real-time polymerase chain reaction (PCR). The induction of c-myc and transforming growth factor-beta2 by cerebral ischemia was inhibited by neuronal expression of IkappaBalpha-SR, whereas induction of GFAP by MCAO was reduced by astrocytic expression of IkappaBalpha-SR. Neuronal, but not astrocytic, expression of the NF-kappaB inhibitor reduced both infarct size and cell death 48 hours after permanent MCAO. In summary, the data show that NF-kappaB is activated in neurons and astrocytes during cerebral ischemia and that NF-kappaB activation in neurons contributes to the ischemic damage.

Small Molecule Inhibitors of IκB Kinase Are Selectively Toxic for Subgroups of Diffuse Large B-Cell Lymphoma Defined by Gene Expression Profiling

Abstract Constitutive activation of the NF-κB pathway is required for survival of the activated B cell–like (ABC) subgroup of diffuse large B-cell lymphoma (DLBCL). Here we show that a small molecule IκB kinase (IKK) inhibitor, PS-1145, and related compounds are toxic for ABC DLBCL cell lines but not for cell lines derived from the other prevalent form of DLBCL, germinal center B cell–like DLBCL. Treatment of ABC lines with these inhibitors rapidly induced a series of gene expression changes that were attributable to cessation of constitutive IKK activity, similar to changes induced by acute expression of genetic inhibitors of NF-κB, confirming the effectiveness and specificity of this compound. Before cell death, inhibition of IKK also induced features of apoptosis and an arrest in the G1 phase of the cell cycle. To test further the specificity of this toxicity, an inducible form of NF-κB was created by fusing the p65 NF-κB subunit with the ligand-binding domain of the estrogen receptor (p65-ERD). In the presence of tamoxifen, p65-ERD reversed the toxicity of IKK inhibition and restored expression of many NF-κB target genes. Another subgroup of DLBCL, primary mediastinal B-cell lymphoma (PMBL), also expresses NF-κB target genes, and treatment of a PMBL cell line with an IKK inhibitor was toxic and induced gene expression changes of a distinct group of NF-κB target genes. These studies validate the NF-κB pathway as a promising therapeutic target in ABC DLBCL, PMBL, and other lymphomas that depend on the activity of NF-κB for survival and proliferation.

Activation of Classical and Alternative Nuclear Factor-kappaB (NF-kB) Pathways in Diffuse Large B-Cell Lymphomas.

Activation of the NF-kB pathway is involved in many human neoplasms. In this study we examined the status of the NF-kB canonical (IkB, p50/p65) and non-canonical (p52, RelB) pathways in diffuse large B-cell lymphomas (DLBCL), which are a common and heterogeneous group of lymphoid malignancies. DLBCL have been divided into activated B-cell (ABC) like, and germinal center B-cell (GCB) like subgroups, which have been reported to have high and low NF-kB activity, respectively. However, the nature of the NF-kB complexes in this lymphoma entity has not been previously evaluated. Therefore we performed Western blotting, electrophoretic mobility shift assays (EMSA) and real time quantitative RT-PCRs on nuclear and cytoplasmic protein and total RNA extracts in 20 primary tumor samples and 9 different DLBCL cell lines. In the cell lines, presence of NF-kB proteins in nuclear extracts correlated with expression of NF-kB target genes (CCR7, IkBa, CCND2, BCL-2, IRF4) as determined by RT-PCR. Therefore, these could be assigned into GCB and ABC-like categories. In primary DLBCLs, EMSA showed NF-kB binding in all but one case. The same cases (19/20) had high p52 in the nucleus indicating activation of the alternative pathway. TNF family ligand BAFF was also found to be expressed in all primary samples and most cell lines. The classical pathway, as determined by nuclear p50, was also present in these cases. Levels of p65 and RelB expression were variable, but did not correlate with the mRNA expression of NF-kB target genes. In conclusion, while DLBCL cell lines may be divided into two distinct categories, the primary samples represented a spectrum of NF-kB target gene activity, while levels of NF-kB expression were high. BAFF expression and activation of the alternative pathway may be important in the pathogenesis of DLBCL.