κB Sites Research Articles

1,25-Dihydroxyvitamin D3 suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)2D3, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)2D3-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)2D3. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)2D3. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)2D3 effects. 1,25(OH)2D3 blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)2D3 down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.

Beyond the enhanceosome: cluster of novel κB sites downstream of the human IFN-β gene is essential for lipopolysaccharide-induced gene activation

AbstractThe expression of interferon-β (IFN-β) in virus-infected HeLa cells established a paradigm of multifactorial gene regulation, in which cooperative assembly of transcription factors (TFs) at the composite DNA element (enhanceosome), is central for amplification of weak activating signals provided by individual TFs. However, whether the same TFs and the same DNA element are essential for IFN-β induction in response to bacterial stimuli are less well understood. Here we report that rapid and transient transcription of IFN-β in response to TLR4 stimulation with bacterial lipopolysaccharide (LPS) follows nuclear factor-κB (NF-κB) RelA activation and recruitment to the IFN-β genomic locus at multiple spatially separated regulatory regions. We demonstrate that the IFN-β enhanceosome region is not sufficient for maximal gene induction in response to LPS and identify an essential cluster of homotypic κB sites in the 3′ downstream of the gene. The cluster is characterized by elevated levels of histone 3 lysine 4 mono-methylation, a chromatin signature of enhancers, and efficiently binds RelA-containing NF-κB complexes in vitro and in vivo. These findings demonstrate that IFN-β gene activation via multifactorial enhanceosome assembly is potentiated in LPS-stimulated cells by NF-κB interactions with all functional κB sites in the locus.

MiR-301a as an NF-κB activator in pancreatic cancer cells

NF-κB is constitutively activated in most human pancreatic adenocarcinoma, which is a deadly malignancy with a 5-year survival rate of about 5%. In this work, we investigate whether microRNAs (miRNAs) contribute to NF-κB activation in pancreatic cancer. We demonstrate that miR-301a down-regulates NF-κB-repressing factor (Nkrf) and elevates NF-κB activation. As NF-κB promotes the transcription of miR-301a, our results support a positive feedback loop as a mechanism for persistent NF-κB activation, in which miR-301a represses Nkrf to elevate NF-κB activity, which in turn promotes miR-301a transcription. Nkrf was found down-regulated and miR-301a up-regulated in human pancreatic adenocarcinoma tissues. Moreover, miR-301a inhibition or Nkrf up-regulation in pancreatic cancer cells led to reduced NF-κB target gene expression and attenuated xenograft tumour growth, indicating that miR-301a overexpression contributes to NF-κB activation. Revealing this novel mechanism of NF-κB activation by an miRNA offers new avenues for therapeutic interventions against pancreatic cancer.

NF-κB Target Genes Activation Via Displacement of HDACs Bound to NF-κB p50 Homodimers by C/EBPα, Its AML Oncoproteins or C/EBPβ.

AbstractAbstract 3637The NF-κB transcription factors are involved in inflammation, proliferation, and apoptosis. The p50:p65 heterodimer, the most common transcription activating form of NF-κB, is retained in the cytosol and translocates into the nucleus only upon stimulation. In contrast, the p50 homodimer is the primary nuclear NF-κB species in unstimulated cells, and recruit HDACs to repress NF-κB-target gene expression. Dysregulation of NF-κB frequently occurs in AML and lymphoma. C/EBPα is mutated in approximately 10% of patients with AML, resulting in expression of C/EBPαp30 or C/EBPαLZ oncoproteins. We showed that C/EBPα or its oncoproteins, including leucine zipper variants (C/EBPαLZ) that cannot bind DNA directly, protects hematopoietic cells from apoptosis. Through interaction with NF-κB p50, C/EBPα or its AML mutants activate several anti-apoptotic genes including BCL2 and FLIP. C/EBPβ or its truncated LIP isoform are over-expressed in Hodgkin lymphoma and breast cancer. Using Ba/F3 cell lines carrying an estrogen receptor fusion of C/EBPα or C/EBPαLZ, we now show that activation of C/EBPα isoforms results in a significant 90% reduction in the association of endogenous HDAC1 or HDAC3 with the FLIP promoter, within 5 hours. In contrast, activation of the basic region mutant C/EBPαBR that cannot bind p50 did not have an effect. Accordingly, ChIP analysis demonstrated that on the FLIP promoter, the expression of C/EBPα or C/EBPαLZ resulted in a 2.5 or 4-fold increase in histone H3 or H4 acetylation, respectively. To study the specific displacement of HDACs from p50 we used Eμ-C/EBPα transgenic mice which express C/EBPα in splenic lymphoid cells, in contrast to control littermates. The ectopic presence of C/EBPα resulted in a significant reduction of endogenous HDAC1 or HDAC3 on the FLIP promoter to 17 or 4% of the expression in wild type mice. In contrast, in p50-/- mice, the C/EBPα transgene had a minimal effect on HDAC1 or a modest reduction of HDAC3 occupancy on the FLIP promoter, to 90 or 50% of levels seen in p50-/- controls. These findings suggest that on the FLIP promoter C/EBPα displaces primarily p50-bound HDACs. HDAC1 or HDAC3 co-ip with p50 in transiently transfected 293T cells. The co-expression of C/EBPα, its C/EBPαLZ or C/EBPαp30 AML mutants, or C/EBPβ interrupted interaction of p50 with HDAC1 and to a lesser extent with HDAC3. In striking correlation to our previous findings, the basic region mutant C/EBPαBR does not interrupt this interaction. As a control, we confirmed that C/EBPα or C/EBPβ do not disrupt the NF-κB p50:p65 co-ip. We have shown that interaction with p50 is required for induction of bcl-2 and FLIP by C/EBPα and that p50 binds and induces the CEBPA gene. Using ChIP analysis, we now show that in U937 AML cells endogenous C/EBPα, C/EBPβ and p50 specifically bind the proximal segment of the nfkb1 (p50) promoter. In addition, C/EBPαLZ expressed from the metallothionein promoter in Ba/F3 cells binds a proximal site in the nfkb1 promoter. C/EBPα or C/EBPαLZ induced p50 RNA expression 3.5-fold, while C/EBPαBR was not effective. Treating the cells with the translation inhibitor cycloheximide did not change p50 induction, suggesting direct activation. C/EBPα, C/EBPαLZ but not C/EBPαBR activated to a similar degree luciferase reporters containing 1800 or 250 bp fragments of the nfkb1 promoter. In addition, C/EBPβ or its truncated LIP isoform activated these reporters, although LIP cannot activate a (CEBP)2TK-Luc reporter. The murine nfkb1 promoter contains 2 proximal κB sites that are 100% conserved between mouse and human. p50 but not C/EBP avidly binds either of these sites in a gel shift assay. Disruption of either κB site abrogated 65 to 80% of reporter activation, and mutating both resulted in activation of less than 10% by either C/EBPα or C/EBPαLZ. In summary, we show that C/EBPs displace HDAC from NF-κB p50, providing a mechanism whereby C/EBPα or its AML mutants can derepress NF-κB target genes and contribute to their dysregulated expression. In addition, C/EBPα, a C/EBPαLZ AML variant that cannot bind DNA directly, or C/EBPβ bind and activate the nfkb1 promoter through interaction with NF-κB bound to two proximal, highly conserved κB sites. Together these findings lend support to the idea that disrupting the C/EBP:NF-κB p50 complex may offer a complementary approach for targeting NF-κB activity in the nucleus to favor apoptosis in AML or other malignancies in which C/EBP family members are expressed. Disclosures:No relevant conflicts of interest to declare.

C/EBPα, C/EBPα Oncoproteins, or C/EBPβ Preferentially Bind NF-κB p50 Compared with p65 Via Conserved Residues In the C/EBP Basic Region

AbstractAbstract 708Nuclear Factor-κB (NF-κB) is a key mediator of the inflammatory response and also inhibits apoptosis. Activation of NF-κB is prevalent in AML, Hodgkin's lymphoma and subsets of non-Hodgkin's lymphoma, as well as in multiple solid tumors. NF-κB p50 (NF-κB1) and p65 (Rel A) are the most prevalent NF-κB subunits. NF-κB p65 has 30–50 fold higher affinity than p50 for the cytoplasmic protein IκB, and so under basal conditions p50:p65 heterodimers are held in the cytoplasm whereas p50:p50 homodimers are found in the nucleus bound to chromatin where they repress NF-κB target genes via association with HDACs. Canonical NF-κB activation signals lead to IκB proteosome-mediated degradation and translocation of p50:p65 heterodimers into the nucleus to replace inhibitory p50:p50 dimers with activating complexes on chromatin. We postulate an alternative means to activate NF-κB target genes wherein induction or activation of C/EBP proteins allows them to bind chromatin-bound p50 and displace HDACs in cells lacking canonical NF-κB activation. We found that C/EBPα induces bcl-2 and FLIP RNA expression via promoter interaction dependent upon the presence of NF-κB p50, that C/EBPα present in two myeloid leukemia cell lines preferentially binds p50 compared with p65, and that mutation of the C/EBPα basic region prevents bcl-2 induction and weakens interaction with p50 but not p65 (Paz-Priel et al 2005; 2009; Wang et al 2009). Using p50 and p65 variants containing the FLAG epitope at either their N- or C-termini we now demonstrate using reciprocal co-immunoprecipitation that p50 has much higher affinity than p65 for C/EBPα, for N-terminal or leucine zipper mutants of C/EBPα expressed in a subset of AML cases, and for the longer LAP or shorter LIP isoforms of C/EBPβ. The FLAG-tagged p65 proteins retained the ability to bind DNA in a gel shift assay and to activate a reporter containing two κB sites as effectively as untagged p65. Deletion of the p65 trans-activation domain (TAD) did not increase p65 affinity for C/EBPα, indicating that an inhibitory effect of the TAD does not account for specificity of C/EBPα:p50 interaction. Moreover, the increased affinity of the p50 rel homolog domain (RHD) for C/EBPα compared with the p65 RHD suggests that residues that differ between these RHDs rather than those in common make key contacts wih the C/EBP basic region. Germline deletion of the gene encoding p65, by injecting p65(flox/flox); Mx1-CRE mice with pIpC, did not reduce C/EBPα expression, prevent interaction of C/EBPα with the bcl-2 promoter in a ChIP assay, or reduce bcl-2 RNA expression, in contrast to our prior findings with p50 gene deletion. A C/EBPα variant harboring a single point mutation in the basic region, C/EBPα(R300A), has minimal affinity for p50, and further saturating mutagenesis of the C/EBPα basic region identifies an additional block of six amino acids, RERNNI, identical in C/EBPβ and critical for interaction with NF-κB p50. In conclusion, increased affinity of C/EBP proteins for p50 compared with p65 lends support to the hypothesis that C/EBP interaction with p50:p50 complexes provides an alternative means for normal cells or cancer cells to activate NF-κB target genes, for example in response to C/EBP induction or activation. C/EBP interaction with p50:p65 in activated cells may also occur, via p50, to further activate genes regulated by NF-κB. In addition, these findings suggest that targeting the C/EBP:p50 interface rather than C/EBP:p65 interaction will prove more effective in the therapy of inflammatory or malignant conditions, and our mapping of relevant basic region residues and the finding that the p50 RHD retains increased affinity for C/EBPα compared wih the p65 RHD provides a first step towards rational design of such inhibitors. Disclosures:No relevant conflicts of interest to declare.

The Eukaryotic Translation Initiation Factor eIF4E, a Recently Identified Therapeutic Target In AML, Is a Direct Transcriptional Target of NFkB

AbstractAbstract 3146It has been well observed that NFκB activity is elevated in a wide range of hematopoietic malignancy including acute myeloid leukemia (AML). However, the downstream effectors that mediate the biological effects of NFκB are not well described. In the present study, we demonstrate that the eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NFκB. Elevated levels of eIF4E correlate with oncogenic transformation in in-vitro and in-vivo models and are associated with poor prognosis in several cancer types including AML. In a Phase II clinical trial in poor prognosis M4 and M5 AML patients, we demonstrated that the anti-viral drug, ribavirin, targeted eIF4E activity in patients correlating with clinical benefit including remissions and blast responses. Below, we describe the first steps in understanding the dysregulation of eIF4E expression in hematological malignancy.Examination of the human eIF4E promoter proximal region revealed the presence of putative κB sites proximal to the transcriptional start region. Given this, we hypothesized that eIF4E was an inducible target of NFκB. First, we tested whether eIF4E expression was modulated in response to NFκB stimulation by several agents including TNFα and PMA. We carried these studies out in multiple cell lines including BJAB, Jurkat and fibroblasts. We found that PMA or TNFα treatment led to substantial increases in eIF4E levels versus untreated controls. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated p65 loading on the eIF4E promoter. This binding correlated with a transcriptionally active complex. Importantly, eIF4E levels were not elevated upon stimulation in p65-/- fibroblasts whereas elevation was observed in control fibroblasts. Further, expression of a dominant negative form of the inhibitor of NFκB, IκB (IκB2NΔ4), substantially increased the expression of eIF4E. Luciferase reporter assays confirm the element is necessary and sufficient for activation of eIF4E. Importantly, this p65 binding element in the eIF4E gene is evolutionarily conserved in mammals. Finally, using EMSA, we observed that this eIF4E promoter element is in active NFκB complexes in M4 and M5 primary AML specimens.We examined the physiological relevance of these findings in primary M4/M5 AML specimens and AML cell lines. The use of certain demethylase inhibitors such as azacytidine have been reported to have undesirable off target effects, including the activation of NFκB. Consistent with these reports, we observe that treatment with 3 uM azacytidine leads to elevated p65 levels and furthermore, elevated eIF4E levels. Consistently, this led to the upregulation of several eIF4E target genes including myeloid cell leukemia protein Mcl-1 and anti-apoptotic proteins such as mdm2. The addition of the eIF4E inhibitor ribavirin led to reduction in these eIF4E targets with no change in eIF4E levels. Consistently, in primary colony assays, azacytidine (3 uM) alone did not substantially reduce colonies in M4/M5 AML specimens relative to untreated cells, whereas the addition of a combination of ribavirin and azacytidine reduced growth 2 to 10 fold. These findings strongly suggest a physiological link between NFκB and eIF4E and furthermore, have implications for the design of novel therapeutic modalities. For instance, ribavirin could substantially dampen unwanted proliferative effects of azacytidine; thus improving its efficacy.This report is the first direct link ever observed between the NFκB pathway and eIF4E and thus, between NFkB and protein translation & mRNA export. Remarkably, most of the genes regulated at the level of mRNA export or translation by eIF4E are targets at the transcriptional level by NFκB. These include VEGF, cyclin D1, mdm2 and Mcl-1. In this way, the NFκB pathway is powerfully positioned to amplify the effects of eIF4E on post-transcriptional gene expression by both promoting the expression of eIF4E and its downstream effectors. Thus, NFκB can amplify the proliferation and survival pathways governed by eIF4E at multiple levels. Disclosures:No relevant conflicts of interest to declare.

Pervanadate Induces The Expression Of Specific Atrophy-related Genes In C2C12 Myotubes

In skeletal muscle NF-κB activation is sufficient to cause skeletal muscle atrophy, and required for muscle atrophy during both muscle disuse and cancer. Both cytokines and oxidants are potent activators of NF-κB, however the pathways of their NF-κB activation appear to be distinct. Cytokines activate the classic, or canonical, pathway characterized by IκB kinase (IKK) activation. Active IKK phosphorylates the inhibitor of κB, IκBα, on serines 32 and 36 causing its proteasomal degradation and release of previously bound NF-κB family members which translocate to the nucleus and bind to κB sites in the regulatory region of target genes. Oxidants appear to activate an alternative NF-κB pathway characterized by tyrosine kinase activation which phosphorylate IκBα on tyrosine 42 and/or 305, causing its release of previously bound NF-κB family members which, again, translocate to the nucleus and bind to κB sites in the regulatory region of target genes. While there is clear evidence on the ability of specific cytokines to increase the transcription of genes associated with skeletal muscle atrophy, there is limited data when these cytokines are used in combination. Further, there is no data to suggest whether activation of the tyrosine kinase-activated alternative pathway increases the transcription of specific atrophy genes. Therefore the PURPOSE of the current study was to determine the ability of pervanadate (to activate tyrosine kinases and the alternative NF-κB pathway) and cytokines (to activate the classic NF-κB pathway) to induce the expression of specific atrophy-related genes. METHODS: C2C12 myotubes were treated for 6 hours with 200 μM pervanadate or for 3 hours with 20 ng/ml TNF-α, 20 ng/ml IL-1β, 20 ng/ml IFNγ, individually and combined. Cells were harvested in Trizol, RNA isolated and cDNA generated for measurement of atrogin-1, MuRF1 and cathepsin L mRNA expression using qRT-PCR. RESULTS: Pervanadate and all cytokines and cytokine combinations increased atrogin-1 mRNA levels 3 to 4-fold, except TNF-α + IL-1β which increased atrogin-1 mRNA almost 10-fold. Pervanadate, IFNγ and TNF-α + IL-1 β increased MuRF1 mRNA levels ∼2.5-fold. Cathepsin L mRNA levels were increased 2.7-fold, 3-fold and 2.5-fold by pervanadate, IFNγ, and TNF-α + IL-1β, respectively, but not changed in any other group. CONCLUSIONS: These findings suggest that pervanadate is sufficient to significantly increase atrogin-1, MuRF1 and cathepsin L transcription. The findings also suggest that of the cytokines, the combination of TNF-α and IL-1β provide the greatest stimuli for increased transcription of these atrophy related genes.

Opposite Action of Peroxisome Proliferator-activated Receptor-γ in Regulating Renal Inflammation: FUNCTIONAL SWITCH BY ITS LIGAND

Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, a new class of antidiabetic agents, have been shown to possess antiinflammatory activity. In this study, we investigated the molecular mechanism by which PPARγ agonists inhibit proinflammatory cytokine expression in rat glomerular mesangial cells. Both natural and synthetic PPARγ agonists potently inhibited RANTES (regulated upon activation, normal T cell expressed and secreted) and monocyte chemoattractant protein-1 expression induced by TNF-α in mesangial cells, which was dependent on NF-κB signaling. However, PPARγ agonists had little effect on TNF-α-triggered IκBα phosphorylation and its subsequent degradation, p65 phosphorylation, and nuclear translocation. In the absence of PPARγ ligand, TNF-α induced a physical interaction between nuclear p65 and PPARγ, as demonstrated by co-immunoprecipitation. Such an interaction was mediated by the C-terminal region of p65. Activation of PPARγ by its agonist prevented PPARγ·p65 complex formation. Chromatin immunoprecipitation assay revealed that TNF-α induced p65 binding to the cis-acting κB elements in rat RANTES promoter, whereas disruption of PPARγ·p65 by its agonist blocked p65 interaction with its cognate κB sites. Knockdown of PPARγ via siRNA strategy completely abolished TNF-α-mediated p65 binding to κB sites and negated RANTES induction, suggesting that unliganded PPARγ is obligatory for NF-κB signaling. Consistently, overexpression of PPARγ in the absence of its ligand sensitized mesangial cells to TNF-α stimulation. These results uncover a paradoxical action of the unliganded and ligand-activated PPARγ in regulating NF-κB signaling and demonstrate PPARγ ligand as a molecular switch that controls its ability to modulate inflammatory responses in opposite directions.

IL-10 inhibits transcription elongation of the human TNF gene in primary macrophages

IL-10 plays a central nonredundant role in limiting inflammation in vivo. However, the mechanisms involved remain to be resolved. Using primary human macrophages, we found that IL-10 inhibits selected inflammatory genes, primarily at a level of transcription. At the TNF gene, this occurs not through an inhibition of RNA polymerase II (Pol II) recruitment and transcription initiation but through a mechanism targeting the stimulation of transcription elongation by cyclin-dependent kinase (CDK) 9. We demonstrated an unanticipated requirement for a region downstream of the TNF 3′ untranslated region (UTR) that contains the nuclear factor κB (NF-κB) binding motif (κB4) both for induction of transcription by lipopolysaccharide (LPS) and its inhibition by IL-10. IL-10 not only inhibits the recruitment of RelA to regions containing κB sites at the TNF gene but also to those found at other LPS-induced genes. We show that although IL-10 elicits a general block in RelA recruitment to its genomic targets, the gene-specific nature of IL-10’s actions are defined through the differential recruitment of CDK9 and the control of transcription elongation. At TNF, but not NFKBIA, the consequence of RelA recruitment inhibition is a loss of CDK9 recruitment, preventing the stimulation of transcription elongation.

DNA-dependent activator of IFN-regulatory factors enhances the transcription of HIV-1 through NF-κB

Pattern recognition receptors (PRRs) play a pivotal role in host innate immune responses against microbial infection. Viruses are primarily recognized by PRRs such as Toll-like receptor 3, 7, 8 and 9, and RIG-I-like receptors. Recent studies have demonstrated that DNA-dependent activator of IFN-regulatory factors (DAI) is a cytosolic sensor molecule for dsDNA, and is implicated in antiviral responses to some DNA viruses. Soon after infection, human immunodeficiency virus type-1 (HIV-1) synthesizes viral dsDNA in the cytoplasm by reverse transcriptase. In addition, an immune compromised state due to chronic HIV-1 infection results in opportunistic infection with some microbes that potentially activate DAI. However, it has not been elucidated whether DAI affects HIV-1 replication, or its possible mechanisms. Here, we showed that forced expression of DAI markedly enhanced HIV-1 replication, which was largely impaired by mutations at κB sites in HIV-1 LTR or by suppressing activation of NF-κB. Moreover, intact structure around the D3 region (174–232 aa) and two RIP homotypic interaction motifs (198–214 aa and 256–272 aa) within DAI were critical for its activity. These results suggest that activation of DAI might contribute to augment HIV-1 replication through DAI- NF-κB pathway.

P52–Bcl3 complex promotes cyclin D1 expression in BEAS-2B cells in response to low concentration arsenite

Arsenic is a well-recognized human carcinogen that causes a number of malignant diseases, including lung cancer. Previous studies have indicated that cyclin D1 is frequently over-expressed in many cancer types. It is also known that arsenite exposure enhances cyclin D1 expression, which involves NF-κB activation. However, the mechanism between cyclin D1 and the NF-κB pathway has not been well studied. This study was designed to characterize the underlying mechanism of induced cell growth and cyclin D1 expression in response to low concentration sodium arsenic (NaAsO 2) exposure through the NF-κB pathway. Cultured human bronchial epithelial cells, BEAS-2B, were exposed to low concentration sodium arsenite for the indicated durations, and cytotoxicity, gene expression, and protein activity were assessed. To profile the canonical and non-canonical NF-κB pathways involved in cell growth and cyclin D1 expression induced by low concentration arsenite, the NF-κB-specific inhibitor-phenethyl caffeate (CAPE) and NF-κB2 mRNA target sequences were used, and cyclin D1 expression in BEAS-2B cells was assessed. Our results demonstrated that exposure to low concentration arsenite enhanced BEAS-2B cells growth and cyclin D1 mRNA and protein expression. Activation and nuclear localization of p52 and Bcl3 in response to low concentration arsenite indicated that the non-canonical NF-κB pathway was involved in arsenite-induced cyclin D1 expression. Moreover, we further demonstrated that p52/Bcl3 complex formation enhanced cyclin D1 expression through the cyclin D1 gene promoter via its κB site. The up-regulation of cyclin D1 mediated by the p52–Bcl3 complex in response to low concentration arsenite might be important in assessing the health risk of low concentration arsenite and understanding the mechanisms of the harmful effects of arsenite.

Modulation of the human polymorphic hs1,2 enhancer within the 3′IgH regulatory region by TCDD

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a potent environmental toxin known to inhibit immunoglobulin (Ig) gene expression. Transcriptional regulation of the Ig heavy chain (IgH) involves the 3′IgH regulatory region (3′IgHRR) and its enhancers (hs3, hs1,2, and hs4) which contain DNA binding sites for several transcription factors including NF‐κB and dioxin response elements (DRE). TCDD induces binding of the aryl hydrocarbon receptor complex to DREs within the 3′IgHRR and inhibits murine 3′IgHRR activation in a well characterized mouse B‐cell line (CH12.LX). In humans, a polymorphism of the hs1,2 enhancer resulting in varying numbers of a 53 bp sequence has been correlated with autoimmune diseases like IgA nephropathy and Celiac disease. The repeated sequence contains κB and DRE binding sites. The objective of this study was to comparatively evaluate the effect of TCDD and LPS on human and mouse hs1,2 enhancers in the CH12.LX model. In transient luciferase studies, an increased number of repeats in the human hs1,2 enhancer increased the sensitivity to LPS. Interestingly, TCDD also markedly enhanced human hs1,2 activity and even augmented LPS‐induced activation. This starkly contrasted with TCDD‐induced inhibition of the mouse hs1,2 and 3′IgHRR in LPS‐stimulated CH12.LX cells suggesting a species difference in IgH transcriptional regulation.(Supported by NIEHS R01ES014676)

Histamine modulation of peripheral CRH receptor type 1α expression is dependent on Ca 2+ signalling and NF-κB/p65 transcriptional activity

Histamine promotes immune complex-induced vascular leakage in vivo, a critical and early event that leads to joint-specific autoimmune damage. Initial assessment, using explanted human synovial tissue (ST), indicates that histamine can modulate local expression of type 1 alpha CRH receptors (CRH-R1α). The objective of this study was to elucidate the signalling events and transcriptional mechanism(s) controlling histamine-dependent regulation of CRH-R1α expression in human inflammatory arthritis. Histamine significantly promotes CRH-R1α mRNA and protein expression in a time- and concentration-dependent manner in human endothelial and synoviocyte cells. Transactivation of the human CRH-R1 promoter is significantly enhanced by histamine which can be mimicked by treatment with a Ca 2+ ionophore and completely diminished in the presence of a Ca 2+ chelator. Histamine-mediated responses involve enhanced activation and nuclear localisation of transcription factors including CREB, NF-κB and NR4A2. Functional consequences of enhanced CREB, NF-κB and NR4A2 activity confirm that NF-κB/p65 selectively controls CRH-R1 promoter activity. Co-transfection of NF-κB/p65 potently transactivates the CRH-R1 promoter while co-expression of a dominant negative IκBα kinase inhibits endogenous and histamine-induced promoter activity. Bioinformatic analysis identifies three putative κB consensus binding sites at proximal and distal positions and 5′ deletional analysis identifies promoter region(s) required for activation by histamine and NF-κB/p65. We observe direct NF-κB/p65 interaction within the promoter region and site-directed mutagenesis reveals that all three κB sites are required to mediate histamine and NF-κB/p65 regulation of CRH-R1 promoter activity. These findings confirm that histamine, via enhanced Ca 2+ signalling and NF-κB/p65 activity, contributes to changes in ST inflammation by promoting CRH-R1α-mediated responses.

Xanthohumol inhibits IL-12 production and reduces chronic allergic contact dermatitis

Xanthohumol (XN) and its related compounds were evaluated for their effects on modulating the production of interleukin (IL)-12, the most important factor driving T helper 1 immune responses. XN showed the strongest inhibitory effect on IL-12 production in macrophages stimulated by lipopolysaccharide (LPS) or LPS/interferon-γ. Xanthohumol 4′- O-β-D-glucopyranoside (XNG) inhibited IL-12 production less effectively than XN. Isoxanthohumol and 8-prenylnaringenin showed comparatively lower inhibitory effects on IL-12 production than XNG. (2 S)-5-methoxy-8-prenylnaringenin 7- O-β-D-glucopyranoside did not exert any effect on IL-12 production. We then tested how these compounds affected NF-κB binding activity to the κB site in the nucleus. The compounds inhibited κB binding in macrophages with the same potency order as IL-12 inhibition. Furthermore, we investigated whether XN, which showed the most effective reduction of IL-12 production, attenuated skin inflammation. Chronic allergic contact dermatitis, an experimental model for psoriasis, was used to determine the anti-inflammatory effects of XN in vivo. XN treatment reduced the degree of ear thickening induced by oxazolone. Taken together, XN might be effective as an anti-inflammatory agent to reduce skin inflammation by inhibiting IL-12 production.

Targeted Knockdown of EGR-1 Inhibits IL-8 Production and IL-8-mediated Invasion of Prostate Cancer Cells through Suppressing EGR-1/NF-κB Synergy

IL-8 produced by prostate cancer cells may be responsible for the androgen-independent growth of advanced prostate cancers. Accumulating evidence from microarray analyses and animal genetic models highlights the central involvement of the transcription factor early growth response-1 (EGR-1) in prostate carcinoma progression. It is unknown, however, whether knockdown of EGR-1 inhibits IL-8 production and IL-8-mediated tumor metastasis. Here we show that EGR-1 knockdown by a specific shRNA-Egr1 inhibited gene transcription and production of IL-8 by the human prostate cancer cell line DU145. Conversely, enforced expression of EGR-1 in EGR-1-lacking PC3 prostate cancer cells markedly enhanced IL-8 transcription and secretion. By using wild type and a series of mutant IL-8 promoter luciferase constructs, we found that the NF-kappaB binding site is important for EGR-1 regulation of IL-8. Furthermore, silencing EGR-1 suppressed a synergistically functional interaction between EGR-1 and NF-kappaB. Consequently, knockdown of EGR-1 inhibited IL-8-mediated tumor colony formation and invasion. Thus, targeted knockdown of EGR-1 could be an effective therapeutic approach against prostate cancer.

Roles of IKK-β, IRF1, and p65 in the Activation of Chemokine Genes by Interferon-γ

Activation of chemokine genes in response to interferon (IFN)-gamma or NF-kappaB is an important aspect of inflammation. Using the chemokine gene ip-10 in mouse embryonic fibroblast cells as an example, we show that the response to IFN-gamma is long lasting but secondary: initial STAT1 activation drives IRF1 synthesis, and IRF1 then binds to IFN-stimulated regulatory elements (ISREs) in the ip-10 promoter. The promoters of most IKK-beta-dependent IFN-stimulated genes (ISGs) also contain ISREs. In response to IFN-gamma, inhibitor of NF-kappaB (IkappaB) kinase beta (IKK-beta) is required to activate both newly synthesized IRF1 and the p65 subunit of NF-kappaB, which contributes to ip-10 expression by binding to kappaB sites in the ip-10 promoter, with little or no activation of classical NF-kappaB. In contrast to IFN-gamma, IL-1beta induces ip-10 expression rapidly but transiently, by activating classical NF-kappaB and increasing the synthesis of IRF1. Together, IL-1beta and IFN-gamma induce ip-10 synergistically. IFN-gamma does not affect the transient activation of classical NF-kappaB by IL-1beta and synergistic induction of ip-10 expression by IFN-gamma and IL-1beta occurs even after the activation of classical NF-kappaB has returned to basal levels. Therefore, IKK-beta has a novel role in IFN-gamma-dependent activation of chemokine gene expression through its activation of IRF1 and p65.

C/EBPα or Its Leukemic Mutants Directly Activate nfkb1 Transcription and Displace HDAC1 or HDAC3 From Chromatin-Associated NF-κB p50 to Induce Anti-Apoptotic Genes.

Abstract 76Mutated variants of C/EBPα are expressed in approximately 15% of AML cases. We have previously shown that C/EBPα and its AML mutants interact directly with NF-κB p50 through the basic region. Through binding to a DNA κB site they activate anti-apoptotic genes including BCL2 and FLIP, inhibiting both the extrinsic and intrinsic apoptotic pathways. These effects do not depend on DNA binding but rather on the integrity of the basic region, which is rarely altered in C/EBPα AML oncoproteins. Interaction with p50 is necessary for the C/EBPα induction of bcl-2 and FLIP but not of myeloid differentiation genes. In addition, NF-κB p50 binds and activates the CEBPA gene in myeloid cells. We now report that C/EBPα or its AML mutants, including a leukemia-derived leucine zipper mutant that cannot bind DNA directly (C/EBPαLZ), bind in vivo to the nfkb1 (p50) promoter. Using Ba/F3 cell lines expressing an estrogen receptor fusion of C/EBPα or the C/EBPαLZ mutant we demonstrate induction of p50 mRNA within 7 hrs after the addition of estradiol. A basic region mutant (C/EBPαBR) that cannot bind the p50 protein did not activate nfkb1 transcription. Importantly, the translation inhibitor cycloheximide did not prevent nfkb1 induction by C/EBPα, suggesting direct regulation of nfkb1 transcription by C/EBPα. Remarkably, C/EBPα did not induce p65 RNA expression. A minimal 200 bp nfkb1 promoter, containing κB but not C/EBP binding sites, is trans-activated by C/EBPα 9 fold, or 33 fold by the C/EBPαLZ AML oncoprotein. The variant C/EBPαBR fails to trans-activate the nfkb1 promoter. Together, these data indicate that the gene encoding NF-κB p50 is a direct target of C/EBPα. NF-κB p65 is held in the cytoplasm IκBα and enters the nucleus to bind DNA predominantly as p65:p50 heterodimers upon cell stimulation. In contrast, p50 homodimers are present in the nucleus bound to chromatin in resting cells and inhibit transcription due to the lack of a trans-activation domain and the recruitment of co-repressors and histone deacetylases (HDACs). Therefore, the induction of p50 by C/EBPα would be expected to result in diminished expression of target genes. However, our data indicates that C/EBPα and p50 synergistically activate anti-apoptotic genes. Remarkably, ChIP analysis demonstrated that C/EBPα induction from the metallothionein promoter in Ba/F3 cells diminishes the occupation of the endogenous BCL2 and FLIP promoters by HDAC1 but not HDAC3, although induction of C/EBPα expression did not alter the cellular content of either HDAC. In contrast, occupancy of Bcl-3, another p50 transcriptional cofactor, on these promoters did not change. In transiently transfected 293T cells we confirmed that p50 binds to HDAC1 in a co-immunoprecipitation reaction. Remarkably, interaction between p50 and HDAC1 was disrupted in the presence of co-expressed C/EBPα, C/EBPβ, or the AML mutants C/EBPαLZ, or C/EBPαp30. In a striking correlation with our previous findings, the C/EBPαBR basic region mutant of C/EBPα that does not bind p50 did not interrupt this interaction. As an important control, we confirmed that HDAC1 does not interact directly with C/EBPα or its mutants, indicating that C/EBPα competes with HDAC1 for interaction with p50. Interestingly, the AML-derived C/EBPαLZ or C/EBPαp30 oncoproteins, but not wild-type C/EBPα, interrupt the interaction between HDAC3 and p50. This suggests a greater capacity for these leukemia mutants to de-repress NF-κB target genes compared with C/EBPα itself. In summary, C/EBPα cooperates with NF-κB p50 to induce several anti-apoptotic genes, thereby providing a survival advantage to the leukemic blasts. C/EBPα and p50 reciprocally regulate each other's expression at the transcriptional level, establishing a positive feedback relationship. Moreover, C/EBPα or its AML mutants displaces HDAC1, and the C/EBPα oncoproteins displace HDAC3, from NF-κB p50 homodimers bound to anti-apoptotic genes, contributing to NF-κB dysregulation in leukemia. Our data underscore the concept that the C/EBPα:NF-κB p50 complex is a potential novel therapeutic target in AML, and the C/EBPβ:p50 complex may functional similarly in lymphomas and in other malignancies where C/EBPβ rather than C/EBPα expression predominates. Disclosures:No relevant conflicts of interest to declare.

Kinetic enhancement of NF-κB·DNA dissociation by IκBα

A hallmark of the NF-κB transcription response to inflammatory cytokines is the remarkably rapid rate of robust activation and subsequent signal repression. Although the rapidity of postinduction repression is explained partly by the fact that the gene for IκBα is strongly induced by NF-κB, the newly synthesized IκBα still must enter the nucleus and compete for binding to NF-κB with the very large number of κB sites in the DNA. We present results from real-time binding kinetic experiments, demonstrating that IκBα increases the dissociation rate of NF-κB from the DNA in a highly efficient kinetic process. Analysis of various IκB mutant proteins shows that this process requires the C-terminal PEST sequence and the weakly folded fifth and sixth ankyrin repeats of IκBα. Mutational stabilization of these repeats reduces the efficiency with which IκBα enhances the dissociation rate.

Cytomegalovirus Promoter Up-regulation Is the Major Cause of Increased Protein Levels of Unstable Reporter Proteins after Treatment of Living Cells with Proteasome Inhibitors

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

Fourth NF-κB site in HIV-1 subtype-C LTR confers functional advantage to viral gene expression

Subtype-C strains of HIV-1, among the various viral subtypes, are responsible for ~50% of the global and 85-99% of Indian infections. Among others, the most significant molecular feature differentially conserved in the subtype-C promoter is the polymorphism within the enhancer region constituted by NF-κB sites. While the viral promoter of majority of the subtypes contains two NF-κB sites, subtype-C promoter consists of three canonical motifs. Notably, a minority of clade-C primary isolates contain κB or κB-like sites, in addition to the canonical κB sites. Previous studies from our laboratory identified nearly 6% (34/609) of the primary isolates from India to demonstrate κB-site polymorphism in C-LTR. The functional importance of additional κB or κB-like sites in C-LTR has not been evaluated. We confirmed the subtype nature of the viral isolates by sequencing and phylogenetic analysis of LTR, Tat and Env in 20 of 34 samples demonstrating κB-site polymorphism. Sequence analysis of the additional κB- or κB-like sites identified extensive variation among the viral isolates. Among them, a particular sequence variation, constituting the κB-like site GGGACTTTCT, with a C-T variation at position 10, was found to be the most common. The functional importance of the κB-like site in C-LTR has been evaluated by reporter gene expression from isogenic LTR promoters and EMSA. We compared gene expression pattern of LTR constructs in Jurkat cells under different conditions of cell activation including TNF-α, PMA, PHA and combinations that activate NF-κB pathway. The data demonstrate the strongest reporter gene expression from the LTRs containing the additional κB-like sites especially under synergistic activation conditions. Gel shift experiments with TNF-α activated Jurkat cell nuclear extracts showed the recruitment of p50/p65 heterodimer to the variant κB-like site. Our data for the first time provide experimental evidence that the κB-like site in C-LTR is an authentic κB-site and this site confers quantitative and qualitative gain of function. Furthermore, we hypothesize that this gain of function may augment viral fitness and hence contribute to global predominance of subtype-C infections.