Modulation Of NF-kappaB Activity Research Articles

Macrophages modulate migration and invasion of human tongue squamous cell carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.

Comprehensive whole-genome sequencing of an early-stage primary myelofibrosis patient defines low mutational burden and non-recurrent candidate genes

In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: i) a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; ii) a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and iii) a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).

Whole Genome Sequence Analysis of Primary Myelofibrosis.

AbstractAbstract 2863 Background:JAK2 V617F is a recurrent, activating mutation in patients (pts) with BCR-ABL1-negative MPNs. Mutations in codon 515 of MPL occur in 1–5% and 5–10% of ET and PMF pts, respectively, and similar to JAK2 V617F, lead to constitutive JAK-STAT signaling. The acquisition of multiple mutational events affecting the JAK-STAT axis or components of the epigenetic machinery is common in MPNs and likely contributes to phenotypic diversity, including progression to acute myeloid leukemia. Mutated genes thus far implicated in MPN initiation and/or progression include TET2, CBL, SH2B3, ASXL1, DNMT3A, IDH1/2, IKZF1, EZH2, SRSF2, and TP53. In order to identify novel somatic mutations associated with classic BCR-ABL1-negative MPNs, we performed whole genome sequencing of DNA extracted from peripheral blood granulocytes and cultured skin fibroblasts from a patient with PMF and a known MPL W515K mutation. Methods:Whole genome sequencing (WGS) was undertaken in a 55 year-old man with untreated PMF four years after initial diagnosis. His DIPSS Plus risk group was low (score 0). His karyotype was normal, and molecular testing revealed wild-type JAK2 in addition to the MPL W515K mutation. WGS of purified granulocytes and paired cultured skin fibroblasts was performed using both Illumina HiSeq and Complete Genomics (CGI) platforms. The resulting data were analyzed using multiple independent aligners and variant callers. Amplicon-based targeted resequencing with the Illumina MiSeq platform was used to evaluate additional patient samples for recurrent mutations. Stanford institutional review board approval and informed pt consent was obtained for these analyses. Results:The PMF genome was sequenced to 88X (Illumina) and 128X (CGI) average fold coverage, and the cultured skin fibroblast genome was sequenced to 47X (Illumina) and 126X (CGI). The PMF genome had a low somatic mutation rate, consistent with that observed for other sequenced hematopoietic tumor genomes, with a low number of true somatic mutation calls. To definitively identify true mutations among various sequencing artefacts and germline variants, we use cultured skin fibroblasts which can be prepared with no contamination by neoplastic cells. In addition to re-identification of the MPL W515K mutation, this approach identified six additional somatic mutations that alter gene coding regions, splice sites, or known regulatory regions: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a splice-site mutation in CAP2; three nonsynonymous point mutations in KIAA0355, SOX30, MFRP; and a 19-base pair (bp) deletion involving a regulatory region in the 5'-untranslated region (5'-UTR) of BRD2, a bromodomain-containing protein implicated in transcriptional regulation (Table). CARD6, BRD2, and KIAA0355, an uncharacterized protein, are expressed by the granulocytes derived from this patient, supporting a potential role in the development of PMF in this pt. Using massively parallel sequencing, we are currently examining the transcribed region of BRD2 and the coding region of the other five genes in additional pt samples. Analysis of the first 87 samples (MF=47, PV=20, ET=20) out of a cohort of 180 MPN pts has thus far not identified recurrent somatic mutations in these six genes. Conclusion:High-coverage genome sequencing of neoplastic and germline cells from a patient with MPL-mutated PMF identified six additional somatic mutations of potential functional significance. Work is ongoing to determine if somatic mutations in these genes are found in other patients with BCR-ABL1-negative MPNs, and their pathogenetic relevance to MPN biology.GeneChrStartEndNT changeFunctional effectExpressed in pt granulocytesCARD6540,841,71740,841,718T>ACys>STOPyesBRD2632,939,86632,939,88519-bp deldeletion in 5'UTRyesKIAA03551934,838,86234,838,863G>AArg>HisyesSOX305157,078,635157,078,636C>GGly>ArgnoCAP2617,514,07817,514,079G>Adisrupts splice sitenoMFRP11119,216,847119,216,848C>AArg>LeunoChromosomal coordinates are given relative to hg19Chr: chromosome; NT: nucleotide; pt: patient Disclosures:Snyder:Illumina: Consultancy; GenapSys: Membership on an entity's Board of Directors or advisory committees; Personalis: Consultancy, Founder Other.

Dichotomous Actions of NF-κB Signaling Pathways in Heart

Despite the substantial progress in heart research over the past two decades heart failure still remains a major cause of morbidity and mortality in North America and is reaching pandemic proportions worldwide. Though the underlying causes are varied, the functional loss of contractile myocytes through apoptosis, necrosis, and autophagy has emerged a central unifying theme to explain diminished cardiac performance in individuals with heart failure. At the molecular level, there has been considerable interest in understanding the signaling pathways that regulate cell death in the heart with specific interest in the extrinsic and intrinsic cell death pathways. The cellular factor nuclear factor-kappaB (NF-kappaB) is a key transcription factor involved in the regulation of a wide range of genes involved in cellular process including inflammation, immune cell maturation, cell proliferation, and, most recently, cell survival. NF-kappaB signaling is important for the normal cellular growth and is a major target of inflammatory cytokines. Several studies have highlighted a protective role of NF-kappaB in the heart under certain circumstances including hypoxic or ischemic myocardial injury. The diverse nature and involvement of NF-kappaB in regulation of vital cellular processes including cell survival notably in the post-mitotic heart has sparked considerable interest in understanding the signaling pathways involved in regulating NF-kappaB in the heart under normal and pathological conditions. However, whether NF-kappaB is adaptive, maladaptive or is a homeostatic response to cardiac injury may simply depend on the context and timing of its activation. In this forum we discuss NF-kappaB signaling pathways and therapeutic opportunities to modulate NF-kappaB activity in heart failure.

Interleukin‐2 induces NF‐κB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells

Deregulation of nuclear factor (NF)-kappaB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF-kappaB activation in extranodal natural killer (NK)-cell lymphoma, nasal type (ENKL) is known to be associated with aberrant nuclear translocation of BCL10. Here we investigated the mechanisms leading to NF-kappaB activation and BCL10 nuclear localization in ENKLs. Given that ENKLs are dependent on T-cell-derived interleukin-2 (IL2) for cytotoxicity and proliferation, we investigated whether IL2 modulates NF-kappaB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2-activated NK lymphoma cells were found to induce NF-kappaB activation via the PI3K/Akt pathway, leading to an increase in the entry of G(2)/M phase and concomitant transcription of NF-kappaB-responsive genes. We also found that BCL10, a key mediator of NF-kappaB signalling, participates in the cytokine receptor-induced activation of NF-kappaB. Knockdown of BCL10 expression resulted in deficient NF-kappaB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2-triggered NF-kappaB signalling pathway. Moreover, the addition of IL2 to NK cells led to aberrant nuclear translocation of BCL10, which is a pathological feature of ENKLs. We further show that BCL10 can bind to BCL3, a transcriptional co-activator and nuclear protein. Up-regulation of BCL3 expression was observed in response to IL2. Similar to BCL10, the expression and nuclear translocation of BCL3 were induced by IL2 in an Akt-dependent manner. The nuclear translocation of BCL10 was also dependent on BCL3 because silencing BCL3 by RNA interference abrogated this translocation. We identified a critical role for BCL10 in the cytokine receptor-induced NF-kappaB signalling pathway, which is essential for NK cell activation. We also revealed the underlying mechanism that controls BCL10 nuclear translocation in NK cells. Our findings provide insight into a molecular network within the NF-kappaB signalling pathway that promotes the pathogenesis of NK cell lymphomas.

MEOX2 regulates nuclear factor-κB activity in vascular endothelial cells through interactions with p65 and IκBβ

Tumours secrete proangiogenic factors to induce the ingrowth of blood vessels, the end targets of which are vascular endothelial cells (ECs). The MEOX2 homeoprotein inhibits nuclear factor-kappaB (NF-kappaB) signalling and EC activation in response to serum and proangiogenic factors. We hypothesize that MEOX2 interacts with components of this pathway in vascular ECs to modulate NF-kappaB activity and EC activation and that these interactions depend upon specific domains within the MEOX2 protein. To test our hypothesis, we transduced ECs with MEOX2 expression constructs. MEOX2 protein localized to the nuclear fraction, as did IkappaBbeta and p65. By co-immunoprecipitation, MEOX2 bound to both p65 and IkappaBbeta. Immunofluorescence demonstrated that MEOX2 colocalizes in the nucleus with both p65 and IkappaBbeta and that this colocalization requires the MEOX2 homeodomain and N-terminal domain. Finally, promoter assays revealed that MEOX2 expression has a biphasic effect on NF-kappaB-dependent promoters. At low levels, MEOX2 stimulates NF-kappaB activity, whereas at high levels, it represses, effects that also depend upon the homeodomain and the N-terminal domain. Our results represent the first report of an interaction between a homeobox protein and IkappaBbeta and suggest that MEOX2 modulates the activity of the RelA complex through direct interaction with its components. These observations implicate MEOX2 as a potentially important regulatory gene inhibiting not only the angiogenic response of ECs to proangiogenic factors, but also their response to chronic inflammatory stimulation that normally activates NF-kappaB, suggesting MEOX2 as a possible molecular target for the therapy of angiogenesis-dependent diseases such as cancer.

Abstract LB-69: Targeting CK2 with small molecule CX-4945 modulates NF-kappaB, TP53 and cell survival in head and neck squamous cell carcinoma

Abstract Objective: We previously identified CK2 as an upstream kinase activating NF-kappaB and modulating TP53 mediated signaling pathways. Up regulated CK2 activity is implicated in promoting the malignant phenotype and progression of head and neck squamous cell carcinoma (HNSCC). Here, we investigate the antitumor effects of CX-4945, a selective small molecule inhibitor of CK2. Design: The effects of CX-4945 on cell proliferation were measured by MTT assay in a panel of 9 University of Michigan squamous cell carcinoma (UM-SCC) cell lines. The half maximal inhibitory concentration (IC50) was determined in vitro. CX-4945 inhibition of cell cycle and induction of cell death were measured by DNA flow cytometry. The effects of CX-4945 on the activity of NF-kappaB, BCL-XL, and TP53 promoters were tested by the chemiluminescent reporter gene assay. The anti-tumor activity of orally administered CX-4945 was evaluated in SCID mice bearing UM-SCC-11A xenografts. Results: The IC50 in 9 UM-SCC cell lines ranged from 3.4 µM to 11.7 µM. The UM-SCC-11A cell line used for in vivo studies had an IC50 of 4.07 µM in vitro. CX-4945 showed a concentration and time dependent cell cycle arrest in G1 and G2/M phases accompanied by a significant induction of sub-G0 cells indicating cell death. CK2 inhibition with CX-4945 downregulated the reporter activities of survival-promoting genes such as NF-kappaB and BCL-XL and up regulated the reporter activity of apoptosis-promoting gene TP53 in a concentration and time dependent manner. Furthermore, CX-4945 (75 mg/kg) showed significant tumor growth inhibition (31% (p<0.05)) in UM-SCC-11A xenografts compared to the control group. Conclusions: Inhibition of CK2 via CX-4945 reveals anti-proliferative activity and modulation of NF-kappaB activation in HNSCC cell lines. In vivo studies show significant antitumor activity in a bortezomib-resistant HNSCC xenograft model. CK2 is an unexploited target in HNSCC and CX-4945 demonstrates the potential of a therapeutic agent with this novel mechanism of action for the treatment of head and neck cancers. (Supported by NIDCD intramural project ZIA DC-000016) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-69.

Abstract 1352: Macrophage specific regulation reveals both pro- and anti-tumor effects of NF-kappaB during mammary tumor progression

Abstract An increased level of macrophages in human tumors is associated with a poorer prognosis. The transcription factor Nuclear Factor-kappaB (NF-kappaB) is an important regulator of gene expression within macrophages. Recent studies point to a pro-tumorigenic role for NF-kappaB activity in macrophages; however NF-kappaB also regulates expression of genes such as inducible nitric oxide synthase which are associated with anti-tumor effects. We believe that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects depending on tumor stage. We have developed novel murine models to determine the effects of modulation of NF-kappaB activity specifically within macrophages during primary mammary tumor growth and metastasis. We have used the cfms promoter to specifically express the reverse tetracycline transactivator (rtTA) protein in macrophages. The rtTA can drive tet operon regulated gene expression in the presence of doxycyline (dox). We have used this with a dominant negative IkappaBalpha to inhibit NF-kappaB signaling in the Polyoma mouse mammary tumor model, this model is termed DNFP. We have also used the cfms-rtTA in combination with a constitutively active IKK2 transgene to activate NF-kappaB signaling, this model is termed IKFM. When NF-kappaB is inhibited in macrophages during primary tumor growth in our DNFP model there is a reduction in primary tumor growth accompanied by a reduced level of lung metastasis. This data suggests that NF-kappaB in macrophages is acting in a pro-tumor manner, in agreement with recently published data. In contrast, we have found that activation of NF-kappaB in macrophages by treating IKFM mice with dox for 1 week before and during two weeks following tail vein injection of polyoma mammary tumor cells leads to a significant reduction in surface lung tumor formation. In additional studies, IKFM mice were pretreated with dox for 1 week but treatment was stopped two days after tumor injection. A similar reduction in tumor number was observed, suggesting an inhibition of cell seeding. Real time PCR analysis of lungs from mice with activation of NF-kappaB in macrophages suggests an anti-tumor “M1” macrophage phenotype. In summary, our data indicates that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects dependent on tumor stage. This data would suggest that cell specificity and timing of treatment are important considerations with regards to the use of NF-kappaB inhibitors in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1352.

Vitamin‐regulated cytokines and growth factors in the CNS and elsewhere

There is a growing awareness that natural vitamins (with the only exception of pantothenic acid) positively or negatively modulate the synthesis of some cytokines and growth factors in the CNS, and various mammalian cells and organs. As natural vitamins are micronutrients in the human diet, studying their effects can be considered a part of nutritional genomics or nutrigenomics. A given vitamin selectively modifies the synthesis of only a few cytokines and/or growth factors, although the same cytokine and/or growth factor may be regulated by more than one vitamin. These effects seem to be independent of the effects of vitamins as coenzymes and/or reducing agents, and seem to occur mainly at genomic and/or epigenetic level, and/or by modulating NF-kappaB activity. Although most of the studies reviewed here have been based on cultured cell lines, but their findings have been confirmed by some key in vivo studies. The CNS seems to be particularly involved and is severely affected by most avitaminoses, especially in the case of vitamin B(12). However, the vitamin-induced changes in cytokine and growth factor synthesis may initiate a cascade of events that can affect the function, differentiation, and morphology of the cells and/or structures not only in the CNS, but also elsewhere because most natural vitamins, cytokines, and growth factors cross the blood-brain barrier. As cytokines are essential to CNS-immune and CNS-hormone system communications, natural vitamins also interact with these circuits. Further studies of such vitamin-mediated effects could lead to vitamins being used for the treatment of diseases which, although not true avitaminoses, involve an imbalance in cytokine and/or growth factor synthesis.

Activation and Inhibition of Nuclear Factor Kappa B Activity by Cereal Extracts: Role of Dietary Phenolic Acids

The transcription factor nuclear factor kappa B (NF-kappaB) plays a critical role in stress, immune, and inflammatory responses, and the modulation of its activity can be a potentially effective preventive strategy for controlling certain diseases. Cereal grains contain phenolic compounds in concentrations comparable to those in fruits and vegetables, well-known for their beneficial effect on human health. In this study we aimed to examine the effect of different phenolic extracts from barley, oat, wheat, and buckwheat on the modulation of basal and lipopolysaccharide (LPS)-induced NF-kappaB activity and elucidate the role of phenolic acids in this modulation. Three extracts were prepared: extracts of free phenolic compounds (M1), extracts of free phenolic acids (M2), and extracts of bound phenolic acids (HY). Generally, extracts M2 showed the highest effect on modulation of NF-kappaB activity with strong inhibition of LPS-induced NF-kappaB activity at all concentrations and of the basal NF-kappaB activity at concentrations equal to or lower than 3 mg/mL. Most of extracts M1 and HY slightly increased both the basal and the LPS-induced NF-kappaB activation. However, at the highest concentrations (3 or 15 mg/mL) extracts HY inhibited LPS-induced NF-kappaB activation. Similar experiments with standard solutions of phenolic acids indicated their ability to modulate the NF-kappaB activity.

The anti-inflammatory and anti-cancer properties of epigallocatechin-3-gallate are mediated by folate cycle disruption, adenosine release and NF-κB suppression

To understand the mechanism by which (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, exerts its anti-inflammatory action. To check our hypothesis that the anti-inflammatory properties of EGCG could be related to its antifolate action and whether adenosine and its receptors are involved in EGCG action, we investigated the EGCG-induced suppression of NF-kappaB in Caco-2 cell monolayer, which acted as a model of the human intestinal epithelium. We demonstrate that the anti-inflammatory properties of EGCG are associated with its antifolate activity. By using a natural stable folate we were able to reverse the EGCG suppression of TNF-alpha-induced NF-kappaB activation, the phosphorylation and degradation of IkappaBalpha and the phosphorylation of Akt in this human colon carcinoma cell line. These suppressions were mediated by the release of adenosine following disruption of the folate cycle by EGCG. By binding to its specific receptors, adenosine can modulate the Akt and NF-kappaB pathway. Moreover, EGCG produces a significant increase in a specific adenosine receptor, which could explain the suppression of the constitutive activation of NF-kappaB in colon cancer cells. The data suggest that by modulating NF-kappaB activation, EGCG might not only combat inflammation, but also cancer.

Blockage of interleukin-6 signaling with 6-amino-4-quinazoline synergistically induces the inhibitory effect of bortezomib in human U266 cells

The transcription factor nuclear factor-kappa B (NF-kappaB) regulates the transcription of a number of genes involved in a variety of cellular responses, including cell survival, inflammation, and differentiation. NF-kappaB is activated by a variety of stimuli, proinflammatory cytokines, mitogens, growth factors, and stress-inducing agents. Aberrant NF-kappaB expression is considered to be one of the oncogenic factors of cancer and the constitutive activation of NF-kappaB is observed in several hematologic disorders [classic Hodgkin's lymphoma, diffuse large B cell lymphoma, and multiple myeloma (MM)], and the modulation of NF-kappaB activation is emerging as a promising novel anticancer therapeutic strategy.Therefore, we focused on the regulation of NF-kappaB activation in MM. When U266 cells were treated with 6-amino-4-quinazoline, an NF-kappaB activation inhibitor, we determined that it most effectively blocked the interleukin (IL)-6-induced activation of MAPK and JAK/STAT pathways among different signaling inhibitors. The results of the luciferase assay indicated that 6-amino-4-quinazoline inhibited NF-kappaB activation with diminished NF-kappaB protein bound to NF-kappaB DNA binding sites. In addition, 6-amino-4-quinazoline suppressed the production of IL-6, which affected MM cell proliferation. Furthermore, combined treatment with bortezomib and 6-amino-4-quinazoline effectively inhibited the IL-6 and soluble IL-6R-induced activation of STAT3 and extracellular signal-regulated kinase phosphorylation. Our data showed that the inhibition of NF-kappaB activation abrogated MM cell proliferation induced by the IL-6 pathway, and might represent a promising therapeutic strategy for the treatment of MM.

NOS2 Regulation of NF-κB by S-Nitrosylation of p65

Signal transduction in the NF-kappaB transcription factor pathway is inhibited by inducible nitric oxide synthase (NOS2) activity, although the molecular mechanism(s) are incompletely understood. We have previously shown that nitric oxide (NO), derived from NOS2 consequent upon cytokine stimulation, attenuates NF-kappaB p50-p65 heterodimer DNA binding and have identified the p50 monomer as a locus for inhibitory S-nitrosylation. We now show that the binding partner of p50, NF-kappaB p65, is also targeted by NO following cytokine stimulation of respiratory epithelial cells and macrophages and identify a conserved cysteine within the Rel homology domain that is the site for S-nitrosylation. S-Nitrosylation of p65 inhibits NF-kappaB-dependent gene transcription, and nuclear levels of S-nitrosylated p65 correlate with decreased DNA binding of the p50-p65 heterodimer. NOS2 regulates cytokine-induced S-nitrosylation of p65, resulting in decreased NF-kappaB binding to the NOS2 promoter, thereby inhibiting further NOS2 expression. Collectively, these findings delineate a mechanism by which NOS2 modulates NF-kappaB activity and regulates gene expression in inflammation.

A Role for the IκB Family Member Bcl-3 in the Control of Central Immunologic Tolerance

Bcl-3 is a member of the family of IkappaB inhibitors. Unlike the classical, cytoplasmic IkappaBs, Bcl-3 does not inhibit RelA- or c-Rel-containing NF-kappaB transcription factor dimers. Instead, Bcl-3 can enter the nucleus and modulate NF-kappaB activity, although the underlying mechanism and physiologic function remain largely unknown. Here we identified Bcl-3 as a regulator of immunologic tolerance to self. In parallel with NF-kappaB2, Bcl-3 functions within stroma to generate medullary thymic epithelial cells, which are essential for negative selection of autoreactive T cells. Loss of both NF-kappaB2 and Bcl-3, but not either one alone, led to a profound breakdown in central tolerance resulting in rapid and fatal multiorgan inflammation. These data reveal extensive utilization of the NF-kappaB system to promote central tolerance in the thymus, in apparent contrast with the well-known roles of NF-kappaB to promote inflammation and autoimmunity in the periphery.

Ras effector pathways modulate scatter factor-stimulated NF-κB signaling and protection against DNA damage

Scatter factor (SF) (hepatocyte growth factor) is a pleiotrophic cytokine that accumulates within tumors in vivo and protects tumor cells against cytotoxicity and apoptosis due to DNA damaging agents in vitro. Previous studies have established that SF-mediated cell protection involves antiapoptotic signaling from its receptor (c-Met) to PI3 kinase --> c-Akt --> Pak1 (p21-activated kinase -1) --> NF-kappaB (nuclear factor-kappa B). Here, we found that Ras proteins (H-Ras and R-Ras) enhance SF-mediated activation of NF-kappaB and protection of DU-145 and MDCK (Madin-Darby canine kidney) cells against the topoisomerase IIalpha inhibitor adriamycin. Studies of Ras effector loop mutants and their downstream effectors suggest that Ras/PI3 kinase and Ras/Raf1 pathways contribute to SF stimulation of NF-kappaB signaling and cell protection. Further studies revealed that Raf1 positively regulates the ability of SF to stimulate NF-kappaB activity and cell protection. The ability of Raf1 to stimulate NF-kappaB activity was not due to the classical Raf1 --> MEK1/2 --> ERK1/2 pathway. However, we found that a MEK3/6 --> p38 pathway contributes to SF-mediated activation of NF-kappaB. In contrast, RalA, a target of the Ras/RalGDS pathway negatively regulated the ability of SF to stimulate NF-kappaB activity and cell protection. Ras, Raf1 and RalA modulate SF stimulation of NF-kappaB activity, in part, by regulating IkappaB kinase (IKK)-beta kinase activity. These findings suggest that Ras/Raf1/RalA pathways may converge to modulate NF-kappaB activation and SF-mediated survival signaling at the IKK complex and/or a kinase upstream of this complex.

RNAi Screen in Mouse Astrocytes Identifies Phosphatases that Regulate NF-κB Signaling

Regulation of NF-kappaB activation is controlled by a series of kinases; however, the roles of phosphatases in regulating this pathway are poorly understood. We report a systematic RNAi screen of phosphatases that modulate NF-kappaB activity. Nineteen of 250 phosphatase genes were identified as regulators of NF-kappaB signaling in astrocytes. RNAi selectively regulates endogenous chemokine and cytokine expression. Coimmunoprecipitation identified associations of distinct protein phosphatase 2A core or holoenzymes with the IKK, NF-kappaB, and TRAF2 complexes. Dephosphorylation of these complexes leads to modulation of NF-kappaB transcriptional activity. In contrast to IKK and NF-kappaB, TRAF2 phosphorylation has not been well elucidated. We show that the Thr117 residue in TRAF2 is phosphorylated following TNFalpha stimulation. This phosphorylation process is modulated by PP2A and is required for TRAF2 functional activity. These results provide direct evidence for TNF-induced TRAF2 phosphorylation and demonstrate that phosphorylation is regulated at multiple levels in the NF-kappaB pathway.

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-kappaB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-kappaB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of--or without--inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.

Embelin, an Inhibitor of X Chromosome-Linked Inhibitor-of-Apoptosis Protein, Blocks Nuclear Factor-κB (NF-κB) Signaling Pathway Leading to Suppression of NF-κB-Regulated Antiapoptotic and Metastatic Gene Products

Identifying the active chemical ingredients of ancient medicines and the molecular targets of those ingredients is an attractive therapeutic objective. Embelin, identified primarily from the Embelia ribes plant, is one such compound shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities through an unknown mechanism. Because nuclear factor-kappaB (NF-kappaB) regulates several genes associated with inflammation, proliferation, carcinogenesis, and apoptosis, we postulated that embelin might mediate its activity through modulation of NF-kappaB activation. We found that embelin inhibited tumor necrosis factor (TNF) alpha-induced NF-kappaB activation. Both inducible and constitutive NF-kappaB activation were abrogated by embelin. In addition, NF-kappaB activated by diverse stimuli such as interleukin-1beta, lipopolysaccharide, phorbol myristate acetate, okadaic acid, hydrogen peroxide, and cigarette smoke condensate also was suppressed. We found that embelin inhibited sequentially the TNFalpha-induced activation of the inhibitory subunit of NF-kappaBalpha (IkappaBalpha) kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, and p65 phosphorylation and nuclear translocation. Embelin also suppressed NF-kappaB-dependent reporter gene transcription induced by TNFalpha, TNF receptor-1 (TNFR1), TNFR1-associated death domain protein, TNFR-associated factor-2, NF-kappaB-inducing kinase, and IkappaBalpha kinase but not by p65. Furthermore, we found that embelin down-regulated gene products involved in cell survival, proliferation, invasion, and metastasis of the tumor. This down-regulation was associated with enhanced apoptosis by cytokine and chemotherapeutic agents. Together, our results indicate that embelin is a novel NF-kappaB blocker and potential suppressor of tumorigenesis.

Citrobacter rodentium‐induced NF‐κB activation in hyperproliferating colonic epithelia: role of p65 (Ser536) phosphorylation

1. The transcription factors of the NF-kappaB/Rel family form dimeric complexes that control expression of various genes involved in inflammation and proliferation. 2. During transmissible murine colonic hyperplasia (TMCH) induced by Citrobacter rodentium, nuclear translocation of NF-kappaB in isolated colonic crypts increased 3 day's post-infection and continued over 12 days paralleling peak hyperplasia. Antibody supershifts for both p65/p50 hetero- and p50/p50 homodimers occurred. Expression levels of both p50 and p65 subunits increased in cytosolic/nuclear extracts and correlated with NF-kappaB activation kinetics. IkappaB alpha levels decreased during this time. 3. Phosphorylation of IKK alpha (at Ser(176/180)) and -beta (at Ser(177/181)) increased significantly during TMCH suggesting activation in vivo. 4. p65-Ser536 (p65(536)) exhibited increased phosphorylation on immunoblotting and immunohistochemistry (IHC) both at day 6 and 12 TMCH. p65(536) translocated to nucleus and interacted with transcriptional coactivator CREB binding protein (CBP). 5. Proteasomal inhibitor bortezomib (Velcade) caused accumulation of Ser(32/36)-phosphorylated IkappaB alpha and significant inhibition of NF-kappaB activity in vivo. Velcade also blocked nuclear translocation of activated p65: both immunoblotting and IHC failed to detect p65(536) nuclear immunoreactivity. Velcade, however, did not abrogate TMCH. 6. p65 interacted strongly with ribosomal S6 kinase 1 (RSK-1) during coimmunoprecipitation but not with IKK alpha or -beta. 7. Thus, NF-kappaB activation during TMCH involves both IkappaB alpha degradation and p65-Ser536 phosphorylation. p65/RSK-1 interaction and concomitant increase in p65(536) complexed with CBP may be important in modulating NF-kappaB activity in vivo. Activated NF-kappaB, besides modulating proliferation, may aid in providing protective immunity against C. rodentium infection in vivo.

Human immunodeficiency virus‐encoded Tat activates glycogen synthase kinase‐3β to antagonize nuclear factor‐κB survival pathway in neurons

The pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated dementia is mediated by neuronal dysfunction and death, brought about by the action of soluble neurotoxic factors that are released by virally infected macrophages and microglia. Paradoxically, many candidate HIV-1 neurotoxins also possess the ability to activate nuclear factor-kappa B (NF-kappaB), which has a potent pro-survival effect in primary neurons. The present study explored this conundrum and investigated why NF-kappaB might fail to protect neurons that are exposed to candidate HIV-1 neurotoxins. Here, we evaluated the ability of virus-depleted conditioned medium produced by HIV-1-infected human macrophages (HIV-MCMs) to modulate NF-kappaB activity in neurons. We demonstrated that HIV-MCMs inhibit the normal signaling pathways that lead to NF-kappaB activation in neurons. This inhibitory effect of HIV-MCM is dependent upon the presence of HIV-1 Tat, which activates glycogen synthase kinase (GSK)-3beta in neurons. Activation of GSK-3beta, in turn, results in modification of the NF-kappaB subunit RelA at serine 468, thereby regulating the physical interaction of RelA with histone deacetylase-3 corepressor molecules. Furthermore, neutralization of Tat or inhibition of GSK-3beta activity prevents neuronal apoptosis induced by HIV-MCM. We conclude that HIV-1 Tat may compromise neuronal function and fate by interfering with normal survival pathways subserved by NF-kappaB. These findings may have important therapeutic implications for the management of HIV-1-associated dementia.