RelA Subunit Research Articles

Abstract POSTER-BIOL-1344: Epigenetic regulation of SPARC in ovarian cancer

Abstract Background: We have previously reported the tumor suppressor effect of Secreted protein acidic and rich in cysteine (SPARC) in ovarian cancer in vitro and in vivo. We reported that SPARC suppresses ovarian cancer cells interactions with the surrounding environmental cues within the peritoneal milieu. We also reported that the expression of SPARC was decreased in established ovarian cancer cell lines and micro-dissected ovarian cancer cells from advanced tumors consistent with hypermethylation of its promoter. However, the mechanisms of epigenetic regulation of SPARC in ovarian cancer (and other morbidities) are unraveled. Experimental Procedures: In silico analysis, human ovarian cancer tissue microarrays, a panel of established human ovarian cancer cell lines and in vitro ovarian cancer cell models were used. Genetic and pharmacologic manipulation of NFκB subunits and DNA methyl transferases (DNMTs) were used to investigate their involvement in the transcriptional regulation of SPARC expression and function. Results: In silico mapping of SPARC promoter identified consensus binding sites for subunits (RelA and NFκB1). Pharmacologic inhibition of NFκB as well as knockdown of p65-RelA and p50 subunits restored SPARC expression and function in ovarian cancer cells. Treatment of ovarian cancer cells with demethylating agent 5’-azacytidine and knockdown of DNMTs (DNMT1, DNMT3a and DNMT3b) indicated that NFκB-mediated SPARC repression is due to promoter methylation through DNMT3a. In ovarian cancer tissue microarrays, SPARC expression in cancer cells significantly and progressively decreased in advanced stage. The expression of SPARC in cancer cells inversely correlated with nuclear p65-RelA and DNMT3a expression. Conclusion: Our findings indicate that activated NFκB integrates inflammatory signals to exert epigenetic pressure suppressing tumor-SPARC through promoter methylation. Pharmacologic inhibitors of NFκB as well as anti-inflammatory agents may provide a significant therapeutic opportunity. Citation Format: Dylan P. Matthews, Sherine Taylor, Neveen A. Said. Epigenetic regulation of SPARC in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1344.

Abstract 2112: A novel mechanism of NF-κB regulation in bladder cancer

Abstract Protein methyltransferases (PMTs) methylate protein lysine and arginine residues and play an important role in regulating chromatin remodeling and gene transcription. Deregulation of SET-domain-containing PMTs has a crucial role in carcinogenesis (1). SETD6 is a novel methyltransferase that has been shown to monomethylate p65/RelA (a principal subunit of NF-κB) at Lys310. Monomethylated p65 recruits GLP (G9A-like protein), a histone dimethyl transferase to the NF-κB target genes, which results in transcriptional repression (2). The role of SETD6, in carcinogenesis in general and bladder cancer in particular is unknown. We identified SETD6 as a significantly upregulated gene in a screen of chromatin modifying genes that are differentially regulated in bladder cancer cells compared with normal urothelial cells. Subsequently qPCR and immunoblotting showed upregulation of SETD6 at the mRNA and protein level in bladder cancer cells and tissues relative to their normal counterparts. Surprisingly, we found that overexpression of SETD6 increased nuclear level of NF-κB, phosphorylation of NF-κB at Ser536 and the transcriptional activity of NF-κB. Overexpression of wild type SETD6 increased viability and proliferation of non-tumorigenic urothelial cells while overexpression of mutant SETD6 had no effect. Furthermore, knocking down SETD6 transiently with si-SETD6 significantly decreased viability and proliferation of bladder cancer cells suggesting that SETD6 may have a growth promoting role in bladder cancer. Our data shows for the first time that non-histone protein lysine methylation of NF-κB-p65 by SETD6 may be a novel way to regulate NF-κB pathway in bladder cancer. The lack of curative strategies for bladder cancer leads to recurrence rates of 45-70% with >15% of patients developing muscle invasive disease (3). These observations underscore the need to understand survival mechanism in bladder cancer. In this regard our findings are highly significant and could lead to targeting SETD6/ NF-κB axis in bladder cancer in the future.

Comprehensive Evaluation of Nuclear Factor-κΒ Expression Patterns in Non-Small Cell Lung Cancer.

Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC.

Transcriptome profiles of whole blood in Italian Holstein and Italian Simmental lactating cows diverging for genetic merit for milk protein

The aim of the research was to investigate the variation of gene expression in the blood of lactating cows in relation to the genetic merit for productive traits. For the study, 24 Italian Holstein (IH) cows from a single farm and 24 Italian Simmental (IS) cows from a second farm were selected. Cows were in mid-lactation, and farms had similar management and feeding conditions. For each breed, cows were separated into 3 classes of estimated breeding value (EBV) for milk protein yield (EBVp), namely, 8 cows with low EBVp (low group, LG), 8 cows with high EBV (high group, HG), and the 8 cows closest to the median (medium group, MG). Gene expression was measured on blood with a whole-transcriptome bovine microarray, and data of LG and HG were expressed relative to MG. The number of differentially expressed genes between the low and high EBVp groups were 443 for IS and 281 for IH cows. The IS cows had a greater number of genes (398 vs. 241 in IH cows) with opposite expression in the high and low EBV groups, compared with the MG. In IS cows, the largest number of pathways affected were within the KEGG subcategories of “energy metabolism,” “lipid metabolism,” and “metabolism of cofactors and vitamins.” Furthermore, the “glutathione metabolism” pathway was markedly affected, with GSTM1 (from glutathione-S-transferase family), GSS (glutathione synthetase), and G6PD (glucose-6-phosphate dehydrogenase) upregulated in the IS HG but downregulated in the IS LG group. The “intestinal immune network for IgA production” pathway had 2 bovine leukocytes antigen (BoLA-DQ) genes, and the B-cell activating factor of the TNF superfamily (TNFSF13B) upregulated in the IS LG group and downregulated in the IS HG group. The IH cows had the highest number of affected pathways in the “metabolism” and “organismal systems” categories, the latter with 5 out of 10 subcategories relative to the immune system. Within the “T cell receptor signaling pathway,” the IH HG cows had upregulation of the CD1d, CD4, and CD3 antigens and downregulation of the NFkB subunit ReLa gene. This study revealed that specific metabolic and immunological pathways are directly related to genetic merit, and could be considered a signature of quantitative selection. Starting from these preliminary observations, the comparison of differentially expressed genes among animals with different EBVp seems a promising approach to unravel molecular response at a blood level associated with productive traits.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

PWE-182 The effect of nfΚb pathway deregulation on the progression to oesophageal adenocarcinoma through aberrant BCL3 expression

Introduction NFκB pathways have been implicated in many diseases and has been shown to be the link between inflammation and cancer. This pathway is regulated via inhibitor molecules from the IκB family, which BCL3 forms part of. BCL3 functions as an inhibitor to the nuclear translocation of some of the NFκB subunits (p50, p65, RelB, RelA and p52). Not much is known about the role of BCL3 in the regulation of NFκB in upper GI cancer aetiopathogenesis. In this study, we present preliminary data showing that BCL3 has different expression pattern in the Barrett’s metaplasia-dysplasia-carcinoma sequence. Method Paraffin embedded oesophageal specimens were selected from 72 patients, containing normal squamous (n = 14), non-dysplastic Barrett’s (n = 15), low grade dysplasia (LGD) (n = 17), high grade dysplasia (HGD) (n = 17) and invasive OA (n = 7). Immunohistochemistry was performed to assess BCL3 expression within the sub-groups. Intensity (0–3+) and extent (0; 66%=5) for staining were scored by 2 expert GI pathologists. The mean for each group was calculated. The relationships between groups were analysed using Mann-Whitney test. Results 30% (5/17) of patients in the LGD group had no BCL3 expression at all, probably leading to alternative pathways activated as a result of constitutive NFκB activation. Statistically, BCL3 expression was higher in the HGD group compared to the LGD (p = 0.02). Conclusion This novel study shows variation in BCL3 expression patterns across the groups studied. The LGD group has most cases with BCL3 negative expression. This raises the question as to whether alterations in the NFκB pathway are relevant to the early development of Barrett’s neoplasia. Since BCL3 functions as an inhibitor of the nuclear translocation to the NFκB subunits, low expression would lead to the activation of different transcriptomic pathways. Larger sample sizes are needed to improve the statistical power. Further molecular transcriptomic analysis of cases with BCL3 positive and negative expression is warranted. This may shed light on the molecular mechanism of upper GI neoplasia progression and lead to the identification of key therapeutic targets. Disclosure of interest None Declared.

Alcohol Drinking Pattern and Alcohol-Related Liver Disease in India

Background and Aims: TNFa plays a pivotal role in pathophysiology of ALD. High level of this cytokine is often associated with the response to cellular insults. Recently, in a case–control study we have shown that ‘‘A’’ allele of TNFa rs361525 is significantly associated with ALD in ethnic ‘‘Bengali’’ population from Eastern India. This study had aimed further to determine the functional significance of this SNP in the Development of ALD. Methods: TNFa promoter with either ‘‘A’’ or ‘‘G’’ allele was cloned in pGL3-Basic vector, transfected in HepG2 cells and promoter activities were determined by dual luciferase-reporter assay. Extensive bioinformatics analysis was executed to identify allele-specific binding of transcription factors using ‘‘Alibaba’’ and ‘‘Promoserach’’. To study the role of TNFa, expression levels of cytokines were determined by qRT-PCR. Apoptosis and neutrophil migration assay was performed. Result: In accordance to our previous observation of over-representation of ‘‘A’’ allele in ALD patients, a significantly higher (2 fold, P < 0.05) luciferase activity was noted with ‘‘A’’ allele positive TNFa promoter construct compared to ‘‘G’’ allele. Similar pattern in luciferase activities was observed after LPS treatment. Subsequently, by using bioinformatics analysis, NFkb was identified as putative transcription factor, which is specifically binding to 239 position of TNFa promoter. Luciferase reporter assay with allele-specific TNFa promoter construct and p65/ RelA subunit of NFkb revealed that luciferase activity was significantly higher in ‘‘A’’ allele positive construct. This was further verified by luciferase assay with increasing doses of p65/RelA plasmid and in time-course experiment. Furthermore, over-expression of TNFa was found to be associated with high level of downstream pro-inflammatory cytokine IL1b and chemokine IL8 followed by increased apoptosis and neutrophil migration suggesting enhanced inflammation, necrosis and fibrosis progression. Conclusion: This study highlights that subtle nucleotide changes have the potential for genetic modulation and hence progression of diseases.

Inhibition of Oncogenic Transcription Factor REL by the Natural Product Derivative Calafianin Monomer 101 Induces Proliferation Arrest and Apoptosis in Human B-Lymphoma Cell Lines.

Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.

Oxidative stress shapes breast cancer phenotype through chronic activation of ATM-dependent signaling.

Reactive oxygen species (ROS) are thought to be among the initiating insults that drive carcinogenesis; however, beyond the mutagenic properties of ROS, it is unclear how reactive oxygen species and response to redox imbalance may shape cancer phenotype. We have previously observed that basal activity of the powerfully oncogenic transcription factor NF-κB in cultured breast cancer and other tumor cell lines is dependent upon the DNA damage-responsive kinase ATM. Here we show that, in MDA-MB-231 and HeLa cells, basal ATM-dependent NF-κB activation occurs through a canonical DNA damage-responsive signaling pathway as knockdown of two proteins involved in this signaling pathway, ERC1 and TAB1, results in loss of NF-κB basal activity. We further show that knockdown of ATM in MDA-MB-231, a breast cancer line with a pronounced mesenchymal phenotype, results in the reversion of these cells to an epithelial morphology and gene expression pattern. Culture of MDA-MB-231 and HeLa cells on the antioxidant N-acetyl cysteine (NAC) blunted NF-κB transcriptional activity, and long-term culture on low doses of NAC resulted in coordinate reductions in steady-state ROS levels, acquisition of an epithelial morphology, as well as upregulation of epithelial and downregulation of mesenchymal marker gene expression. Moreover, these reversible effects are attributable, at least in part, to downregulation of ATM-dependent NF-κB signaling in MDA-MB-231 cells as RNAi-mediated knockdown of the NF-κB subunit RelA or its upstream activator TG2 produced similar alterations in phenotype. We conclude that chronic activation of ATM in response to persistent ROS insult triggers continual activation of the oncogenic NF-κB transcriptional complex that, in turn, promotes aggressive breast cancer phenotype.

Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB:DNA complex studied by agarose gel electrophoresis.

The nuclear factor κB (NF-κB) is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB – DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB–DNA complex. The most active compounds are substituted at C-3 (α-carbonyl), in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

Methods to detect NF-κB acetylation and methylation.

Posttranslational modifications of NF-κB, including acetylation and methylation, have emerged as an important regulatory mechanism for determining the duration and strength of NF-κB nuclear activity as well as its transcriptional output. Within the seven NF-κB family proteins, the RelA subunit of NF-κB is the most studied for its regulation by lysine acetylation and methylation. Acetylation or methylation at different lysine residues modulates distinct functions of NF-κB, including DNA-binding and transcription activity, protein stability, and its interaction with NF-κB modulators. Here, we describe the experimental methods to monitor the in vitro and in vivo acetylated or methylated forms of NF-κB. These methods include radiolabeling the acetyl or methyl groups and immunoblotting with pan- or site-specific acetyl- or methyl-lysine antibodies. Radiolabeling is useful in the initial validation of the modifications. Immunoblotting with antibodies provides a rapid and powerful approach to detect and analyze the functions of these modifications in vitro and in vivo.

Mechanisms of Immune Signaling in Colitis-Associated Cancer

The inflammatory bowel diseases ulcerative colitis and Crohn’s disease are associated with an increased risk for the development of colorectal cancer. During recent years, several immune signaling pathways have been linked to colitis-associated cancer (CAC), largely owing to the availability of suitable preclinical models. Among these, chronic intestinal inflammation has been shown to support tumor initiation through oxidative stress–induced mutations. A proinflammatory microenvironment that develops, possibly as a result of defective intestinal barrier function and host–microbial interactions, enables tumor promotion. Several molecular pathways such as tumor necrosis factor/nuclear factor-κB or interleukin 6/signal transducer and activator of transcription 3 signaling have been identified as important contributors to CAC development and could be promising therapeutic targets for the prevention and treatment of CAC.

Curcumin, COX-2, and Protein p300/CBP.

Recently, curcumin has received great interest for its emerging role in pain modulation and management [1]. Its anti-nociceptive mechanism is not clear; however, it is part of numerous mechanisms involved in CX3CR1 expression, Mu and Delta opioid receptors, 5-HT (1A) receptors, TNF-α, etc [1]. Recently, I read a report on curcumin which attenuated the pain behavior and serum COX-2 concentration in a rat model of neuropathic pain [2]. That report was of great interest. I have some additional comments about that study. First, you explained that the decreased COX-2 level after curcumin treatment is associated with the down regulation of the expression of the NF-κB-regulated gene products such as COX-2 [3]. However, interaction of the RelA subunit of NF-κB with the general co-activator protein p300/CBP is vital for RelA-dependent gene transcription [4]. Moreover, disruption of this interaction deregulates the NF-κB pathway by interfering with its negative feedback loop. Another recent study also showed that treatment with 60 mg/kg of curcumin increased the mechanical threshold, as in your study, and reduced COX-2 gene expression [5]. That study revealed that curcumin treatment downregulated the recruitment and altered the binding of protein p300/CBP at the BDNF and COX-2 promoters. Curcumin seems to alleviate neuropathic pain by inhibiting p300/CBP which acts as a vital co-activator of NF-κB instead of direct down regulation of the expression of NF-κB. Second, curcumin was used 24 hours before making the CCI model and was continued daily to day 7 post-ligation. However, in a clinical situation, neuropathic pain cannot be easily expected and usually treatment starts after neuropathic pain has developed. Thus, further studies on curcumin as a therapy for neuropathic pain is necessary for application in clinical settings.

Abstract 1836: Erlotinib induces NF-kappa B dependence that promotes EGFR tyrosine kinase inhibitor resistance in lung adenocarcinoma

Abstract The vast majority of patients with lung adenocarcinomas harboring activating mutation in EGFR respond to EGFR tyrosine inhibitors (TKI) (i.e. erlotinib). However, the magnitude of tumor regression is variable and responses are short-lived with a median duration of 9-12 months. We recently identified activation of the NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway as a key mediator of de novo resistance to EGFR TKI therapy in cell lines and tumor xenograft models of lung adenocarcinoma. However, the role of NF-κB activation in mediating EGFR TKI acquired resistance in lung adenocarcinoma is not well defined. We have found that erlotinib treatment of EGFR TKI sensitive lung adenocarcinoma cell lines promotes the rapid phosphorylation and degradation of IκB (inhibitor of NF-κB) and subsequent activation of the NF-κB subunit RelA. RelA activation is accompanied by transcriptional activation of downstream NF-κB target genes, including IL6. Prolonged exposure of EGFR TKI sensitive lung adenocarcinoma cell lines to erlotinib leads to EGFR TKI acquired resistance that is accompanied by sustained NF-κB activation and IL6 expression. EGFR TKI acquired resistance can be overcome by treating cells with erlotinib in combination with PBS-1086 (rel∼MD, Inc.), a direct Rel inhibitor. Furthermore, concomitant treatment of EGFR TKI sensitive lung adenocarcinoma cells with erlotinib + PBS-1086 prevents the development of EGFR TKI acquired resistance. Together, these results demonstrate the molecular basis for the synthetic lethality of combined EGFR and NF-κB inhibition and provide mechanism-based rationale for polytherapies against both EGFR and NF-κB to enhance response in lung adenocarcinoma patients. Broadly, our findings provide novel insights into the biological and clinical consequences of the context-specific and dynamic functional interplay between EGFR and NF-κB signaling. Citation Format: Collin M. Blakely, Evangelos Pazarentzos, Saurabh Asthana, Victor Olivas, Irena Tan, Timothy Fouts, Jeffrey Meshulam, Trever G. Bivona. Erlotinib induces NF-kappa B dependence that promotes EGFR tyrosine kinase inhibitor resistance in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1836. doi:10.1158/1538-7445.AM2014-1836

120: PRMT5 dimethylates R30 of the p65 subunit to activate NF-κB

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ∼85% of the NF-κB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.

Germinal center B cell maintenance and differentiation are controlled by distinct NF-κB transcription factor subunits.

Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-κB signaling pathway has been implicated; however, the distinct roles of the individual NF-κB transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-κB subunits c-REL or RELA, which are both activated by the canonical NF-κB pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-κB pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-κB in GC lymphomagenesis.

The NF-κB Genomic Landscape in Lymphoblastoid B Cells

The nuclear factor κB (NF-κΒ) subunits RelA, RelB, cRel, p50, and p52 are each critical for B cell development and function. To systematically characterize their responses to canonical and noncanonical NF-κB pathway activity, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) analysis in lymphoblastoid B cell lines (LCLs). We found a complex NF-κB-binding landscape, which did not readily reflect the two NF-κB pathway paradigms. Instead, 10 subunit-binding patterns were observed at promoters and 11 at enhancers. Nearly one-third of NF-κB-binding sites lacked κB motifs and were instead enriched for alternative motifs. The oncogenic forkhead box protein FOXM1 co-occupied nearly half of NF-κB-binding sites and was identified in protein complexes with NF-κB on DNA. FOXM1 knockdown decreased NF-κB target gene expression and ultimately induced apoptosis, highlighting FOXM1 as a synthetic lethal target in B cell malignancy. These studies provide a resource for understanding mechanisms that underlie NF-κB nuclear activity and highlight opportunities for selective NF-κB blockade.

The structure and specificity of the type III secretion system effector NleC suggest a DNA mimicry mechanism of substrate recognition.

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation.

PARP1 enhances inflammatory cytokine expression by alteration of promoter chromatin structure in microglia.

BackgroundPoly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated enzyme that participates in processes such as transcription and DNA repair through the regulation of chromatin structure. Accumulating evidence suggests an important role for PARP1 enzymatic activity in promoting CNS inflammation by facilitating the expression of inflammatory cytokines in glial cells. However, the molecular mechanisms by which PARP1 enzymatic activity mediates this process are not well understood. In this report we sought to determine the molecular mechanisms by which PARP1 enzymatic activity facilitates the expression of Il1β and TNF in LPS-stimulated BV2 cells.MethodsPARP1 enzymatic activity and histone ADP-ribosylation were measured in LPS-stimulated BV2 cells by radioactive labelling with 32P-NAD+. To assess the effect of histone ADP-ribosylation on nucleosome structure, in vitro nucleosome remodeling, nuclease accessibility and binding assays were performed. These studies were complemented by chromatin immunoprecipitation assays in resting and LPS-stimulated BV2 cells in order to determine the occupancy of PARP1, nucleosomes and the RelA subunit of NF-κB, as well as ADP-ribosylation, at the Il1β and Tnf promoters. Finally, we determined the effect of pharmacological inhibition of PARP1 enzymatic activity on the LPS stimulation-dependent induction of Il1β and Tnf mRNA.ResultsOur results indicate that LPS stimulation induces PARP1 enzymatic activity and histone ADP-ribosylation in the chromatin compartment of BV2 cells. In vitro studies show that nucleosome-bound PARP1 disrupts nucleosome structure histone ADP-ribosylation, increasing the accessibility of nucleosomal DNA. Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells. Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust transcription of these inflammatory cytokines. Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-κB recruitment, and Il1β and Tnf expression in LPS-stimulated microglia.ConclusionsCollectively, our data suggest that PARP1 facilitates inflammatory cytokine expression in microglia by increasing the accessibility of promoter DNA via histone ADP-riboyslation.

Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells

TLR4 signaling induces down-regulation of the bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI), which enhances TGF-β signaling during hepatic stellate cell (HSC) activation. We investigated the mechanism by which TLR4 signaling down-regulates BAMBI expression in HSCs and found that TLR4- and TNF-α-mediated BAMBI down-regulation is dependent on regulation of BAMBI promoter activity through the interaction with NF-κBp50 and HDAC1 in HSCs. Bambi was predominantly expressed in HSCs, at high levels in quiescent HSCs but at low levels in in vivo-activated and LPS-stimulated HSCs. In human HSCs, BAMBI expression was down-regulated in response to LPS and TNF-α. A BAMBI reporter assay demonstrated that the regulatory element to repress BAMBI transcription is located between 3384 and 1560 bp upstream from the transcription start site. LPS stimulation down-regulated BAMBI expression in cells with NF-κBp65 knockdown. However, it failed to down-regulate BAMBI in cells with inactivation of NF-κB or NF-κBp50 silencing, indicating that NF-κBp50 is a factor for BAMBI down-regulation. ChIP analysis revealed that LPS and TNF-α induced binding of the NF-κBp50/p50 homodimer to the BAMBI promoter region. We also found that HDAC1 is bound to this region as part of the NF-κBp50-HDAC1 complex, repressing transcriptional activity of the BAMBI promoter. Finally, we confirmed that LPS does not repress BAMBI reporter activity using a BAMBI reporter construct with a mutation at 3166 bp upstream of the coding region. In summary, our study demonstrates that LPS- and TNF-α-induced NF-κBp50-HDAC1 interaction represses BAMBI transcriptional activity, which contributes to TLR4-mediated enhancement of TGF-β signaling in HSCs during liver fibrosis.