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The transcription factor nuclear factor of kappa-light-chain-enhancer of activated B cells (NF- κ B) is expressed in brown adipocytes, but its role remains largely unknown in the cells. This issue was addressed in current study by examining NF- κ B in brown adipocytes in vitro and in vivo. NF- κ B activity was increased by differentiation of brown adipocytes through elevation of p65 (RelA) expression. The transcriptional activity of NF- κ B was induced by the cold stimulation with an elevation in S276 phosphorylation of p65 protein. Inactivation of NF- κ B in brown adipocytes made the knockout mice [uncoupling protein 1 (Ucp1)–CreER–p65f/f, U-p65-KO] intolerant to the cold environment. The brown adipocytes exhibited an increase in apoptosis, a decrease in cristae density and uncoupling activity in the interscapular brown adipose tissue (iBAT) of p65-KO mice. The alterations became severer after cold exposure of the KO mice. The brown adipocytes of mice with NF- κ B activation (p65 overexpression, p65-OE) exhibited a set of opposite alterations with a reduction in apoptosis, an increase in cristae density and uncoupling activity. In mechanism, NF- κ B inhibited expression of the adenine nucleotide translocase 2 (ANT2) in the control of apoptosis. Data suggest that NF- κ B activity is increased in brown adipocytes by differentiation and cold stimulation to protect the cells from apoptosis through down-regulation of ANT2 expression. Nuclear factor of kappa-light-chain-enhancer of activated B cells (NF- κ B) activity is increased in brown adipocytes by differentiation and cold stimulation to down-regulate adenine nucleotide translocase 2 (ANT2) expression in preservation of cells from apoptosis. Absence of the activity leads to ANT2-mediated apoptosis to impair the thermogenic function of brown fat in uncoupling protein 1 (Ucp1)–p65-KO mice. [Display omitted] [ABSTRACT FROM AUTHOR]

Individual cell sensing of external cues has evolved through the temporal patterns in signaling. Since nuclear factor κB (NF-κB) signaling dynamics have been examined using a single subunit, RelA, it remains unclear whether more information might be transmitted via other subunits. Using NF-κB double-knockin reporter mice, we monitored both canonical NF-κB subunits, RelA and c-Rel, simultaneously in single macrophages by quantitative live-cell imaging. We show that signaling features of RelA and c-Rel convey more information about the stimuli than those of either subunit alone. Machine learning is used to predict the ligand identity accurately based on RelA and c-Rel signaling features without considering the co-activated factors. Ligand discrimination is achieved through selective non-redundancy of RelA and c-Rel signaling dynamics, as well as their temporal coordination. These results suggest a potential role of c-Rel in fine-tuning immune responses and highlight the need for approaches that will elucidate the mechanisms regulating NF-κB subunit specificity. [Display omitted] • Co-imaging of NF-κB RelA and c-Rel captures subunit-specific immune signaling • The correlation of signaling between the two subunits depends on the ligand • Ligand discrimination is more precise through signaling features of both subunits • Mathematical modeling suggests roles of IκBs in subunit-specific ligand encoding Rahman et al. perform quantitative live-cell imaging of macrophages from NF-κB double-knockin reporter mice and show that both RelA and c-Rel subunits contribute to accurate discrimination of pathogen-derived ligands. Signaling features of the two NF-κB subunits are temporally coordinated and non-redundant in individual cells. [ABSTRACT FROM AUTHOR]

To explore the biological function of RELA proto-oncogene, NF-kB subunit (RELA) in hepatocellular carcinoma (HCC) progression, and its potential regulatory effects on the regulators of m6A modification. GEPIA, UALCAN and Human Protein Atlas databases were applied to analyze the expression characteristics of RELA in HCC tissues and non-cancer liver tissues, and its relationship with clinicopathologic indicators and prognosis. Quantitative real-time PCR (qRT-PCR) was used to examine the expression level of RELA mRNA in HCC cells. Cell counting kit-8 (CCK-8) assay, EdU assay and flow cytometry were used to examine cell growth and apoptosis. PROMO database was applied to predict the binding sequence between RELA and methyltransferase like protein 3 (METTL3) promoter region, and this prediction was verified by dual luciferase reporter gene experiment and chromatin immunoprecipitation assay. The effect of RELA on METTL3 expression was examined by Western blot and qRT-PCT, and the regulatory effects of RELA on the other m6A regulators were evaluated by qRT-PCR. RELA was highly expressed in HCC tissues and cell lines, and was closely associated with adverse clinicopathologic indicators and poor prognosis of patients. Overexpression of RELA promoted the growth of HCC cells and inhibited apoptosis; Knocking down RELA had the opposite effects. Overexpression of RELA promoted METTL3 transcription. Knockdown or overexpression of METTL3 reversed the effects of overexpression or knockdown of RELA on HCC cell growth and apoptosis, respectively. RELA also promoted the expression of a series of m6A regulators at mRNA expression level in HCC cell lines. RELA promotes the transcription of METTL3 by binding to METTL3 promoter region, thus promoting the malignancy of HCC cells. This study suggests NF-κB signaling contributes the dysregulation of m6A modification in HCC tumorigenesis. [ABSTRACT FROM AUTHOR]

This study focused on a comprehensive analysis of the canonical activation pathway of the redox-sensitive transcription factor nuclear factor-kappa B (NF-κB) in peripheral blood mononuclear cells, addressing c-Rel, p65 and p50 activation in 28 women at early (T1) and late follicular (T2) and mid (T3) and late luteal (T4) phase of the menstrual cycle, and possible relations with fasting plasma lipids and fatty acids. For the first time, strong inverse relations of c-Rel with apolipoprotein B were observed across the cycle, while those with LDL cholesterol, triglycerides as well as saturated (SFA), particularly C14–C22 SFA, monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) clustered at T2. In contrast, p65 was positively related to LDL cholesterol and total n-6 PUFA, while p50 did not show any relations. C-Rel was not directly associated with estradiol and progesterone, but data suggested an indirect C22:5n-3-mediated effect of progesterone. Strong positive relations between estradiol and individual SFA, MUFA and n-3 PUFA at T1 were confined to C18 fatty acids; C18:3n-3 was differentially associated with estradiol (positively) and progesterone (inversely). Given specific roles of c-Rel activation in immune tolerance, inhibition of c-Rel activation by higher plasma apolipoprotein B and individual fatty acid concentrations could have clinical implications for female fertility. Image 1 • c-Rel activation is inversely related to apolipoprotein B across the menstrual cycle. • c-Rel activation is inversely related to SFA (C14–C22), MUFA and PUFA at T2. • p65 activation is positively related to LDL and total n-6 PUFA (C18:2n-6) at T2. • 18-carbon SFA, MUFA and n-3 PUFA are positively related to estradiol at T1. • C18:3n-3 is positively related to estradiol and inversely to progesterone. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms are critical for hippocampus-dependent memory formation. Building on previous studies that implicate the N-lysine methyltransferase SETD6 in the activation of nuclear factor-κB RELA (also known as transcription factor p65) as an epigenetic recruiter, we hypothesized that SETD6 is a key player in the epigenetic control of long-term memory. Using a series of molecular, biochemical, imaging, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA–mediated knockdown of Setd6 in the rat dorsal hippocampus during memory consolidation. Our findings demonstrate that SETD6 is necessary for memory-related nuclear factor-κB RELA methylation at lysine 310 and associated increases in H3K9me2 (histone H3 lysine 9 dimethylation) in the dorsal hippocampus and that SETD6 knockdown interferes with memory consolidation, alters gene expression patterns, and disrupts spine morphology. Together, these findings suggest that SETD6 plays a critical role in memory formation and may act as an upstream initiator of H3K9me2 changes in the hippocampus during memory consolidation. [ABSTRACT FROM AUTHOR]

More than half of the world's population is infected with persistent bacterial infections, consequently, persisters are gradually becoming a major public health concern. During the persistent phase, bacterial pathogens deploy many regulatory strategies to compensate unfavorable host environmental conditions. The stringent response is one of such gene regulatory mechanisms which is stimulated by nutrient starvation. It is regulated by the synthesis of highly phosphorylated signaling nucleotides, (p)ppGpp or alarmone. (p)ppGpp is synthesized by ppGpp synthetases, and these proteins are classified as RelA/SpoT homolog (RSH) proteins. Subsequently, (p)ppGpp modulate several molecular and biochemical processes ranging from transcription to metabolism. Imperativeness of (p)ppGpp synthetases has been investigated by numerous approaches including microbiology and animal studies, thereby establishing that Rel enzyme deleted strains of pathogenic bacteria were unable to transform in persister form. In this review, we summarize recent findings to corroborate the rationality to consider (p)ppGpp synthetase as a potential target in discovering a novel class of antimicrobial agents to combat persistent infections. Moreover, inhibition studies on Mycobacterium tuberculosis (p)ppGpp synthetase shows that these inhibitors prevent dormant state transition and biofilm formation. Also, we have highlighted the structural biology of (p)ppGpp synthetases, which may provide significant information that could be used in structure-based inhibitor design. Image 1 • Stringent response ensure bacterial pathogens survival during persistent infection. • Stringent response is regulated by synthesis of (p)ppGpp or alarmone. • Structural analysis provide detail information of binding site of (p)ppGpp synthetase. • (p)ppGpp synthetase may be considered as a potential target for drug discovery. [ABSTRACT FROM AUTHOR]

The nuclear organelle the nucleolus and the transcription factor nuclear factor of κ-light-chain-enhancer of activated B cells (NF-κB) are both central to the control of cellular homeostasis, dysregulated in common diseases and implicated in the ageing process. Until recently, it was believed that they acted independently to regulate homeostasis in health and disease. However, there is an emerging body of evidence suggesting that nucleoli and NF-κB signalling converge at multiple levels. Here we will review current understanding of this crosstalk. We will discuss activation of the NF-κB pathway by nucleolar stress and induction of apoptosis by nucleolar sequestration of NF-κB/RelA. We will also discuss the role of TIF-IA, COMMD1, and nucleophosmin, which are key players in this crosstalk, and the therapeutic relevance, particularly with respect to the antitumour effects of aspirin. Nucleoli play a critical role in maintaining cellular homeostasis in health, ageing, and disease, as does the NF-κB transcription factor pathway. Emerging data suggest that interplay between these two cellular factors regulates cell growth and death. An atypical nucleolar stress response pathway, characterized by degradation of the PolI complex component, TIF-IA, and nucleolar enlargement, lies upstream of NF-κB pathway activation. Components of the NF-κB complex accumulate in nucleoli in response to stress. The presence of RelA in nucleoli triggers cytoplasmic relocation of nucleophosmin which mediates apoptosis by transporting BAX to mitochondria. The chemopreventive agent aspirin triggers TIF-IA–NF-κB signalling in human colorectal tumours, which is important for its antitumour activity. [ABSTRACT FROM AUTHOR]

Abstract The type VI secretion system (T6SS) is a versatile molecular machinery widely distributed in Gram-negative bacteria. The activity of the T6SS is tightly regulated by various mechanisms, including quorum sensing (QS), iron concentration, and transcriptional regulators. Here we demonstrated that the stringent response regulator, RelA, contributes to bacterial resistance to multiple environmental stresses in Yersinia pseudotuberculosis. We also revealed that the stress resistance function of stringent response (SR) was partially mediated by the general stress response T6SS4 system. RelA positively regulates the expression of T6SS4 to combat various stresses in response to nutrition starvation collectively mediated by the RovM and RovA regulators. These findings revealed not only the important role of T6SS4 in SR induced stress resistance, but also a new pathway to regulate T6SS4 expression in response to starvation stress. [ABSTRACT FROM AUTHOR]

Abstract Lethal metastasis of primary breast tumors to lymph nodes has been found to be associated with the co-expression of chemokine CXCL13 and its receptor CXCR5. To date, however, the precise molecular events regulating the co-expression of CXCL13 and CXCR5 in the context of breast cancer progression have not been identified. Therefore, to extend our understanding of the drivers of breast cancer metastasis, we undertook a line of investigation in this study in which we demonstrate that the transcriptional regulation of CXCL13 is mediated by the reciprocal activity of RelA and Nrf2, while CXCR5 is transcriptionally silenced by CpG island methylation within its promoter. Critically, we show that intra-tumoral CXCL13 and CXCR5 mRNA expression is positively correlated with intra-tumoral RelA expression within the primary tumor of breast cancer (BCa) patients (n = 98). We demonstrate a role for Nrf2 in the negative transcriptional regulation of cxcl13. Furthermore, using a luciferase assay and deletion analysis of the cxcl13 gene promoter, we demonstrate that RelA and Nrf2 directly act upon the cxcl13 promoter to regulate transcription. Chromatin immunoprecipitation PCR, supported by in silico docking analyses, confirmed that RelA and Nrf2 both occupy multiple positions within the cxcl13 promoter. Collectively, in RelA high conditions, low Nrf2 and lack of cxcr5 promoter DNA-methylation govern CXCL13-CXCR5 co-expression within breast tumors, and thus drive disease progression and metastasis. Graphical abstract Unlabelled Image Highlights • RelA enhances transcription of chemokine ligand-receptor pair cxcl13 and cxcr5. • Nrf2 suppresses cxcl13 transcription, but not cxcr5 transcription. • Methylation of cxcr5 promoter abrogates RelA-induced CXCR5 expression. • Promoter of cxcl13 contains RelA and Nrf2 responsive regions. [ABSTRACT FROM AUTHOR]

Highlights • This paper describes the development and evaluation of a candidate reference measurement procedure (cRMP) for serum urea that uses a LC-MS/MS method. This novel cRMP directly detects urea without derivatization, which makes the pre-processing of samples very simple compared to the reference methods now recognized by the JCTLM. • The performance of this cRMP is well characterized, with good demonstrated precision and accuracy. • This cRMP could facilitate the implementation and standardization of procedures and testing results for serum urea among clinical laboratories. Abstract Urea is an important indicator of liver and kidney disease, and very frequently determined in clinical chemistry. The reference measurement procedures (RMPs) for serum urea recognized by the Joint Committee for Traceability in Laboratory Medicine are spectrophotometry, and isotope dilution (ID)-mass spectrometry (MS) coupled with gas chromatography. This study investigated a candidate RMP (cRMP) for detecting serum urea directly via ID-liquid chromatography (LC)-MS/MS, without derivatization, which simplifies pre-processing samples. The cRMP was developed and evaluated relative to the recognized RMP, inter- and intra-laboratories. The intra-precisions were 1.35%, 1.98% and 1.47% at 4.95, 24.74 and 31.36 mmol/L, respectively; inter-precision was 2.10%, 2.60% and 2.10%. The relative bias for the measurement of standard human serum (SRM 909c) was –0.49%. The relative biases were 0.41% and 0.02% for IFCC-RELA (International Federation of Clinical Chemistry and Laboratory Medicine-External Quality Assessment Scheme [EQAS] for reference laboratories) 2015A and 2015B. The linearity response between 2.4 mmol/L and 53.7 mmol/L was R 2 = 0.9989. No carryover, ion suppression, or interference was detected. Correlation was acceptable with the reference spectrophotometry (R 2 = 0.9985, P < 0.0001). Between our laboratory and other reference laboratories, the absolute deviation range for IFCC-RELA 2016A and 2016B was from –0.63 to 1.52 mmol/L. This well-characterized cRMP for urea can provide a base of accuracy for the traceability of clinical systems. [ABSTRACT FROM AUTHOR]

Summary The majority of supratentorial ependymomas (ST-ependymomas) have few mutations but frequently display chromothripsis of chromosome 11q that generates a fusion between C11orf95 and RELA ( RELA FUS ). Neural stem cells transduced with RELA FUS ex vivo form ependymomas when implanted in the brain. These tumors display enhanced NF-κB signaling, suggesting that this aberrant signal is the principal mechanism of oncogenesis. However, it is not known whether RELA FUS is sufficient to drive de novo ependymoma tumorigenesis in the brain and, if so, whether these tumors also arise from neural stem cells. We show that RELA FUS drives ST-ependymoma formation from periventricular neural stem cells in mice and that RELA FUS -induced tumorigenesis is likely dependent on a series of cell signaling pathways in addition to NF-κB. [ABSTRACT FROM AUTHOR]

Many diseases are associated with the abnormal activation of NF-κB and its signaling pathway. NF-κB has become an important target for disease treatment and development of new drugs. Many various NF-κB inhibitors were therefore developed; however, they have difficulties to become clinical drugs due to their adverse side effects resulted from the affected normal physiological functions of this transcription factor. To overcome this limitation, this study construct a transgenic vector that can express an artificial miRNA targeting NF-κB RelA under the control of a NF-κB-specific promoter. The promoter consists of a NF-κB decoy and a minimal promoter. The vector was named as decoy minimal promoter-artificial microRNA (DMP-amiRNA). This study verified that this vector can sense and control the intracellular NF-κB activity upon transfection. Working of the vector forms a perfect feedback loop that realizes the NF-κB self-control. With the vector in cells, the higher NF-κB activity, the higher DMP transcriptional activity, and the more amiR533 expression. DMP-amiRNA can moderately inhibit the intracellular NF-κB activity but exert no significant effect on cell viability. This study therefore develops a new strategy for inhibiting over activity of NF-κB, which should has great potential in clinical application. [ABSTRACT FROM AUTHOR]

This study aimed to build an innovative system to deliver a systemic small interfering RNA (siRNA) treatment for rheumatoid arthritis. We combined arginine-histidine-cysteine based oligopeptide-modified polymer micelles with siRNA targeting the nuclear factor κB subunit, RelA (siRelA). This is a key molecule in the control of inflammation. We tested the cellular uptake of siRNA and its effects on inflammatory cytokine levels in vitro using synoviocytes, and siRNA distribution and therapeutic effects in vivo in mice with collagen-induced arthritis (CIA). These studies showed that arginine-histidine based oligopeptide modified micelles produced effective cellular siRNA uptake and suppressed inflammatory cytokine levels in synoviocytes. In vivo , these micelles produced marked accumulation of siRNAs in arthritic paws in CIA mice, with much less accumulation in healthy mice. The siRelA-polymer micelle complexes also produced more effective suppression of RelA mRNA expression and inflammatory cytokine levels in the arthritic paws of CIA mice and reduced their clinical symptom scores and paw thickness. [ABSTRACT FROM AUTHOR]

The practice of neuropathology underwent major changes due to the discovery of molecular alterations with diagnostic, prognostic, and therapeutic implications. Many of these alterations are incorporated in the updated 2016 edition of the World Health Organization (WHO) Classification of Tumours of the Central Nervous System (CNS), which represents a shift from the principle that neuropathology diagnosis is based entirely on microscopy. Molecularly defined entities were introduced, and a diagnosis that integrates the histology and molecular results is strongly recommended. This complex approach is timely because the neuro-oncologists seek specific diagnoses that can lead to more precise treatment. In the current review, we present the most common molecular alterations with known clinical implications in gliomas, and provide a practical guide to an integrated diagnosis. [ABSTRACT FROM AUTHOR]

In vitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins in vivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled. Overcoming hurdles in simultaneous live-cell imaging of RelA and c-Rel, we show that quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells, and shift toward c-Rel in microglia from aged brains. RelA:c-Rel heterodimer is unexpectedly depleted in the nuclei of stimulated cells. Trajectories of subunit co-expression in immune lineages reveal a reduction at key cell maturation stages. These results demonstrate the power of these reporters in gaining deeper insights into NF-κB biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications. [Display omitted] • NF-κB double knockin mice have fluorescently labeled RelA and c-Rel • Live imaging reveals single-cell signaling features unique to primary cells • NF-κB signaling shifts toward c-Rel in brain-resident microglia from aged animals • RelA and c-Rel protein expression is reduced during specific immune cell maturations Rahman et al. present fluorescent knockin mice that track one or both of the canonical subunits of NF-κB. The power of these reporters is illustrated with several approaches that gain deeper insights into NF-κB biology. The mice enable diverse applications from single-molecule analyses to in situ identification of active inflammatory cells. [ABSTRACT FROM AUTHOR]

Summary ARID1A is frequently mutated in ovarian clear cell carcinoma (OCCC) and often co-exists with activating mutations of PIK3CA. Although induction of pro-inflammatory cytokines has been observed in this cancer, the mechanism by which the two mutations synergistically activate cytokine genes remains elusive. Here, we established an in vitro model of OCCC by introducing ARID1A knockdown and mutant PIK3CA into a normal human ovarian epithelial cell line, resulting in cell transformation and cytokine gene induction. We demonstrate that loss of ARID1A impairs the recruitment of the Sin3A-HDAC complex, while the PIK3CA mutation releases RelA from IκB, leading to cytokine gene activation. We show that an NF-κB inhibitor partly attenuates the proliferation of OCCC and improves the efficacy of carboplatin both in cell culture and in a mouse model. Our study thus reveals the mechanistic link between ARID1A/PIK3CA mutations and cytokine gene induction in OCCC and suggests that NF-κB inhibition could be a potential therapeutic option. [ABSTRACT FROM AUTHOR]

RelA, the most important regulator of NF-kB activity, and its mechanisms in keratoplasty immune rejection have not been fully investigated. In the present study, lentivirus-mediated silencing of RelA expression in a bone marrow-derived dendritic cell (BMDC) model was tested. The BMDCs were transfected with RelA-shRNA to induce an immature, maturation-resistant and tolerogenic phenotype, while not significantly changing IFN-γ, IL-10 and IL-17 expression. A fully allogeneic rat cornea transplant model was established for in vivo studies. The allograft mean survival time (MST) of lv-shRelA-DC injection groups were significantly longer than the untreated BMDC group and control group. The corneal opacity and neovascularization scale of the lv-shRelA-DC injection groups were slight compared to pair control others. Postoperative flow cytometric analysis revealed that the percentage of Treg positive cells was dramatically increased in animals that received an lv-shRelA-DC injection. ELISA and qRT-PCR analyses of serum showed that IFN-γ and IL-17 expression were suppressed by lv-shRelA-DC treatement. In vivo experiments demonstrated that IL-10 induced immunosuppression was partly attributed to injection of lv-shRelA-DC throughout the experiment, differing from the general anti-inflammatory factors. Luciferase and Chromatin IP evaluation showed that RelA knockdown in BMDCs significantly reduces DNA binding to IFN-γ, IL-10 and the IL-17 promoter and inhibited of transcriptional activity. Taken together, this study illustrates a significant role of RelA in mediating the corneal neovascularization by affecting IL-17 expression. Our comprehensive analysis shows that the significant role of RelA provides a novel and feasible therapeutic approach for the prevention of corneal allograft rejection. [ABSTRACT FROM AUTHOR]

Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-κB p65 proteoforms isolated from human cells. We show isotopic resolution of N-terminally acetylated p65 and determined it is the most abundant proteoform expressed following transfection in 293 T cells. We also show MS 1 evidence for monophosphorylation of p65 under similar culture conditions and describe a high propensity for p65 proteoforms to fragment internally during beam-style MS 2 fragmentation; up to 71% of the fragment ions could be matched as internals in some fragmentation spectra. Finally, we used native spray mass spectrometry to measure proteins copurifying with p65 and present evidence for the native detection of p65, 71 kDa heat shock protein, and p65 homodimer. Collectively, our work demonstrates the efficient isolation and top down mass spectrometry analysis of p65 from human cells, and we discuss the perturbations of overexpressing tagged proteins to study their biochemistry. This article is part of a Special Issue entitled: Protein Species. Biological significance Characterizing transcription factor proteoforms in human cells is of high value to the field of molecular biology; many agree that post-translational modifications and combinations thereof play a critical role in modulating transcription factor activity. Thus, measuring these modifications promises increased understanding of molecular mechanisms governing the regulation of complex gene expression outcomes. To date, comprehensive characterization of transcription factor proteoforms within human cells has eluded measurement, owing primarily—with regard to top down mass spectrometry—to large protein size and low relative abundance. Here, we utilized HaloTag technology and recombinant protein expression to overcome these limitations and show top down mass spectrometry characterization of proteoforms of the 60 kDa NF-kB protein, p65. By optimizing the analytical procedure ( i . e . purification, MS 1 , and MS 2 ), our results make important progress toward the ultimate goal of targeted transcription factor characterization from endogenous loci. [ABSTRACT FROM AUTHOR]

Diligent balance of nuclear factor kappa B (NF-κB) activity is essential owing to NF-κB's decisive role in cellular processes including inflammation, immunity and cell survival. Ubiquitin/proteasome-system (UPS)-dependent degradation of activated NF-κB/RelA involves the cullin-RING-ubiquitin-ligase (CRL) ECS SOCS1 . The COP9 signalosome (CSN) controls ubiquitin (Ub) ligation by CRLs through the removal of the CRL-activating Ub-like modifier NEDD8 from their cullin subunits and through deubiquitinase (DUB) activity of associated DUBs. However, knowledge about DUBs involved in the regulation of NF-κB activity within the nucleus is scarce. In this study we observed that USP48, a DUB of hitherto ill-defined function identified through a siRNA screen, associates with the CSN and RelA in the nucleus. We show that USP48 trims rather than completely disassembles long K48-linked free and substrate-anchored Ub-chains, a catalytic property only shared with ataxin-3 (Atx3) and otubain-1 (OTU1), and that USP48 Ub-chain-trimming activity is regulated by casein-kinase-2 (CK2)-mediated phosphorylation in response to cytokine-stimulation. Functionally, we demonstrate for the first time the CSN and USP48 to cooperatively stabilize the nuclear pool of RelA, thereby facilitating timely induction and shutoff of NF-κB target genes. In summary, this study demonstrates that USP48, a nuclear DUB regulated by CK2, controls the UPS-dependent turnover of activated NF-κB/RelA in the nucleus together with the CSN. Thereby USP48 contributes to a timely control of immune responses. [ABSTRACT FROM AUTHOR]

Abstract: Helicobacter pylori colonises the gastric epithelial cells of half of the world's population and represents a risk factor for gastric adenocarcinoma. In gastric epithelial cells H. pylori induces the immediate early response transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) and the innate immune response. We show that H. pylori induces in a type IV secretion system-dependent (T4SS) and cytotoxin associated gene A protein (CagA)-independent manner a transient activation of the inhibitor of NF-κB (IκBα) kinase (IKK)-complex. IKKα and IKKβ expression stabilises the regulatory IKK complex subunit NF-κB essential modulator (NEMO). We provide evidence for an intimate mutual control of the IKK complex by mitogen-activated protein kinase kinase kinase 3 (MEKK3) and transforming growth factor β activated kinase 1 (TAK1). TAK1 interacts transiently with the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). Protein modifications in the TAK1 molecule, e.g. TAK1 autophosphorylation and K63-linked ubiquitinylation, administer NF-κB signalling including transient recruitment of the IKK-complex. Overall, our data uncover H. pylori-induced interactions and protein modifications of the IKK complex, and its upstream regulatory factors involved in NF-κB activation. [Copyright &y& Elsevier]

Introduction: We previously demonstrated that NF-κB may be associated with 18F-FDG PET uptake and patient prognosis using radiogenomics in patients with non-small cell lung cancer (NSCLC). To validate these results, we assessed NF-κB protein expression in an extended cohort of NSCLC patients. Methods: We examined NF-κBp65 by immunohistochemistry (IHC) using a Tissue Microarray. Staining intensity was assessed by qualitative ordinal scoring and compared to tumor FDG uptake (SUVmax and SUVmean), lactate dehydrogenase A (LDHA) expression (as a positive control) and outcome using ANOVA, Kaplan Meier (KM), and Cox-proportional hazards (CPH) analysis. Results: 365 tumors from 355 patients with long-term follow-up were analyzed. The average age for patients was 67±11 years, 46% were male and 67% were ever smokers. Stage I and II patients comprised 83% of the cohort and the majority had adenocarcinoma (73%). From 88 FDG PET scans available, average SUVmax and SUVmean were 8.3±6.6, and 3.7±2.4 respectively. Increasing NF-κBp65 expression, but not LDHA expression, was associated with higher SUVmax and SUVmean (p =0.03 and 0.02 respectively). Both NF-κBp65 and positive FDG uptake were significantly associated with more advanced stage, tumor histology and invasion. Higher NF-κBp65 expression was associated with death by KM analysis (p =0.06) while LDHA was strongly associated with recurrence (p =0.04). Increased levels of combined NF-κBp65 and LDHA expression were synergistic and associated with both recurrence (p =0.04) and death (p =0.03). Conclusions: NF-κB IHC was a modest biomarker of prognosis that associated with tumor glucose metabolism on FDG PET when compared to existing molecular correlates like LDHA, which was synergistic with NF-κB for outcome. These findings recapitulate radiogenomics profiles previously reported by our group and provide a methodology for studying tumor biology using computational approaches. [ABSTRACT FROM AUTHOR]

Abstract: Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response, responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent Response, resulting in the inactivation of long-term bacterial survival strategies, leading to bacterial cell death. Herein, we describe a series of deoxyguanosine-based analogs of the Relacin molecule that inhibit in vitro the synthetic activity of Rel proteins from Gram positive and Gram negative bacteria, providing a deeper insight on the SAR for a better understanding of their potential interactions and inhibitory activity. Among the inhibitors evaluated, compound 2d was found to be more effective and potent than our previously reported Relacin. [Copyright &y& Elsevier]

Abstract: Heat shock transcription factor 1 (HSF1), which has been identified as an endogenous cardioprotective factor, possesses potent anti-inflammatory effects. However, the underlying mechanisms have not been fully understood yet. In this study, we investigated the effects of HSF1-regulated RelA, a subunit of NFκB on cardiomyocyte death. Cultured cardiomyocytes were transfected with HSF1 plasmid before the treatment of TNFα. Cell death ratio was determined by cell staining. Additionally, the expression of RelA in the cytoplasm and cytonucleus as well as its subcellular location was detected, and the expression of heat shock proteins (HSP70 and HSP90) in the cardiomyocytes was also examined. Not only did TNFα remarkably enhanced cardiac cell death, but also elevated the expressions of intracellular RelA and elicited its translocation. Overexpression of HSF1 effectively attenuated cell death induced by TNFα. Although HSF1 didn't significantly inhibit the intracellular activation of RelA induced by TNFα at an early stage, HSF1 decreased the levels of RelA and the translocation of RelA in the cytoplasm and cell nucleus at late stage. Besides, the expression of HSP70 and HSP90 was significantly increased when HSF1 was overexpressed. These results suggested that HSF1 attenuated cardiomyocyte death via inhibiting activation of RelA as well as preventing its translocation from the cytoplasm to the cytonucleus, which was partially associated with HSP70 and HSP90 up-regulated by HSF1 overexpression. [Copyright &y& Elsevier]

Bacteria inhabit enormously diverse niches and have a correspondingly large array of regulatory mechanisms to adapt to often inhospitable and variable environments. The stringent response (SR) allows bacteria to quickly reprogram transcription in response to changes in nutrient availability. Although the proteins controlling this response are conserved in almost all bacterial species, recent work has illuminated considerable diversity in the starvation cues and regulatory mechanisms that activate stringent signaling proteins in bacteria from different environments. In this review, we describe the signals and genetic circuitries that control the stringent signaling systems of a copiotroph, a bacteriovore, an oligotroph, and a mammalian pathogen –Escherichia coli, Myxococcus xanthus, Caulobacter crescentus, and Mycobacterium tuberculosis, respectively – and discuss how control of the SR in these species is adapted to their particular lifestyles. [ABSTRACT FROM AUTHOR]

Background & Aims: Nuclear factor-κB (NF-κB) is activated during early stages of pancreatitis. This transcription factor regulates genes that control many cell activities, including inflammation and survival. There is evidence that activation of NF-κB protects against pancreatitis, and, in other cases, that it promotes this disease. We compared the effects of NF-κB in different mouse models of pancreatitis to understand these complications. Methods: To model constitutive activation of NF-κB, we expressed a transgene that encodes its p65 subunit or the inhibitor of κB kinase (IKK)2 in pancreatic acinar cells of mice. We analyzed effects on pancreatic tissues and levels of NF-κB target genes in these mice and compared them with mice that did not express transgenic p65 or IKK2 (controls). Results: Transgenic expression of p65 led to compensatory expression of the inhibitory subunit IKB-α and, therefore, no clear phenotype. However, p65 transgenic mice given injections of cerulein, to induce acute pancreatitis, had higher levels of NF-κB activity in acinar cells, greater levels of inflammation, and more severe outcomes than control mice. In contrast, constitutive expression of IKK2 directly increased the activity of NF-κB in acinar cells and induced pancreatitis. Prolonged activity of IKK2 (3 months) resulted in activation of stellate cells, loss of acinar cells, and fibrosis, which are characteristics of chronic pancreatitis. Co-expression of IKK2 and p65 greatly increased the expression of inflammatory mediators and the severity of pancreatitis, compared with control mice. Conclusions: The level of NF-κB activation correlates with the severity of acute pancreatitis in mice. Longer periods of activation (3 months) lead to chronic pancreatitis. These findings indicate that strategies to inactivate NF-κB might be used to treat patients with acute or chronic pancreatitis. [ABSTRACT FROM AUTHOR]

Abstract: The exposure of Pseudomonas aeruginosa cells to very low UVA fluences induces a growth delay, a phenomenon proposed in Escherichia coli as an adaptive mechanism related to protection against lethal and mutagenic effects of UVA. This paper reports that the treatment with low UVA irradiation fluences protects P. aeruginosa PAO1 strain from a subsequent lethal exposure. This phenomenon depends on the relA gene, coding for the main (p)ppGpp synthetase, and is unrelated to the induction of quorum sensing or catalase activity, two essential factors involved in the response of P. aeruginosa to UVA. Cross-protection between osmotic stress and UVA is observed when a great protective response to lethal UVA is caused by the induction of resistance to osmotic stress. The increase in resistance to osmotic shock observed in the pre-irradiated PAO1 strain but not in its relA derivative, unable to show photo-protection, leads us to hypothesize that the photo-protection could be attributed to an adaptive response to osmotic stress. It is concluded that the exposure of P. aeruginosa to low UVA doses induces a relA-dependent adaptive response that protects against cell death induced by high doses and causes an increase in the resistance to osmotic stress. [Copyright &y& Elsevier]

Abstract: Multilocus sequence analysis (MLSA) was used to refine the phylogenetic analysis of the genus Kribbella, which currently contains 17 species with validly-published names. Sequences were obtained for the 16S rRNA, gyrB, rpoB, recA, relA and atpD genes for 16 of the 17 type strains of the genus plus seven non-type strains. A five-gene concatenated sequence of 4099 nt was used to examine the phylogenetic relationships between the species of the genus Kribbella. Using the concatenated sequence of the gyrB-rpoB-recA-relA and atpD genes, most Kribbella type strains can be distinguished by a genetic distance of >0.04. Each single-gene tree had an overall topology similar to that of the concatenated sequence tree. The single-gene relA tree, used here for the first time in MLSA of actinobacteria, had good bootstrap support, comparable to the rpoB and atpD gene trees, which had topologies closest to that of the concatenated sequence tree. This illustrates that relA is a useful addition in MLSA studies of the genus Kribbella. We propose that concatenated gyrB-rpoB-recA-relA-atpD gene sequences be used for examining the phylogenetic relationships within the genus Kribbella and for determining the closest phylogenetic relatives to be used for taxonomic comparisons. [Copyright &y& Elsevier]

Abstract: Dimethylcelecoxib, a non-COX-2 inhibiting derivative of celecoxib, inhibits PGE2 synthesis by transcriptional inhibition of mPGES-1. Previously we demonstrated that DMC downregulates EGR1 expression and increases nuclear NF-κB in human cervical cancer cells (HeLa) [1]. Both transcription factors are important regulators of mPGES-1 expression. Here we show that treatment of HeLa cells with DMC inhibits EGR1 promoter activity by influencing the transactivation activity of NF-κB. Mutation of the NF-κB motif as well as downregulation of NF-κB(p65)RelA using siRNA repealed the inhibitory effect of DMC on the EGR1 promoter. The transactivation activity of NF-κB is regulated by various co-activators or co-repressors. One of these co-repressors is HDAC1. DMC did not influence HDAC1 expression, but the HDAC activity was enhanced under DMC influence. After DMC treatment NF-κB co-immunoprecipitated with HDAC1. Electromobility shift assays depicted an increased interaction between NF-κB–HDAC1 and DNA containing NF-κB binding motives. Performing CHIP-assays we finally demonstrated the interaction of NF-κB and HDAC1 at the EGR1 promoter that was in part reversed by the HDAC1 inhibitor trichostatin A. Using siRNA against HDAC1 we could repeal the inhibitory effect of DMC on the EGR1 promoter. In conclusion we demonstrated that treatment of HeLa cells with DMC leads to an enhanced formation of a complex consisting of NF-κB and HDAC1 that binds to the EGR1 promoter resulting in downregulation of EGR1 expression which plays a major role for transcriptional inhibition of mGPES-1 expression. How these effects of DMC may contribute to a potential therapeutical benefit of various diseases is discussed. [Copyright &y& Elsevier]

Abstract: Dysregulation of Maspin expression and constitutive activation of NF-κB subunits are important events in tumorigenesis of prostate cancer. Recent finding points that RelB, which contributes to the alternative NF-κB activity, interferes with carcinogenesis in the prostate. We report here, that both the classical and the alternative NF-κB activities are constitutively present in androgen-insensitive human prostate cancer cells. Maspin and RelB expression is correlated negatively in prostate cancer tissues at the later stage. TNF-α signaling triggers the nuclear accumulation of RelB and the concomitant reduction of Maspin expression in a time-dependent manner. In addition, the proteasome inhibitor-induced Maspin expression is accompanied by the reduction of RelB expression. A successful depletion of RelB expression, but not RelA expression, induces Maspin expression. RelB-deficiency abrogates the proteasome inhibitor-induced Maspin expression. Moreover, we demonstrate that the enforced expression of RelB protein in prostate cancer cells inhibits Maspin expression. We propose that RelB is an essential molecule controlling the endogenous and the proteasome inhibitor-induced Maspin expression. Developing a RelB-targeted therapeutic intervention, which might be coupled with the induction of a tumor suppressor Maspin, is valuable in treating advanced, metastatic prostate cancer. [Copyright &y& Elsevier]

Abstract: Ethyl pyruvate, an aliphatic ester derived from pyruvate, reportedly has anti-inflammatory actions through inhibition of the transcription mediated by nuclear factor-kappa B (NF-κB). It was suggested that ethyl pyruvate inhibited NF-κB/DNA-binding activity through the covalent modification of RelA. However, the interaction of ethyl pyruvate with RelA in vitro has not been reported. In the present study, we confirmed that treatment of cultured alveolar epithelial cells, A549 cells, with tumor necrosis factor α (TNFα) increased the NF-κB/DNA-binding activity. When the nuclear extract of the cells was incubated with ethyl pyruvate, the NF-κB/DNA-binding activity was strongly inhibited. Because we previously found that the NF-κB/DNA complex included RelA and p50, we bacterially expressed a deletion mutant of RelA, RelA (1-220), and a full-length form of p50. Incubation of RelA (1-220) or p50 with ethyl pyruvate induced dramatic changes in mobility in two types of nondenaturing gel electrophoresis. Electrophoretic mobility shift assays revealed that incubation of RelA (1-220) or p50 with ethyl pyruvate inhibited the DNA-binding activity. Furthermore, immunostaining of A549 cells revealed that ethyl pyruvate inhibited the nuclear association of RelA after TNFα treatment. These results suggest that ethyl pyruvate interacts with RelA and p50 to inhibit their functions at multiple points. [ABSTRACT FROM AUTHOR]

Abstract: A prominent feature of the stringent response is the accumulation of two unusual phosphorylated derivatives of GTP and GDP (pppGpp: 5′-triphosphate-3′-diphosphate, and ppGpp: 5′-3′-bis-diphosphate), collectively called (p)ppGpp, within a few seconds after the onset of amino-acid starvation. The synthesis of these ‘alarmone’ compounds is catalyzed by RelA homologues. Other features of the stringent response include inhibition of stable RNA synthesis and modulation of transcription, replication, and translation. (p)ppGpp accumulation is important for virulence induction, differentiation and antibiotic resistance. We have synthesized a group of (p)ppGpp analogues and tested them as competitive inhibitors of Rel proteins in vitro. 2′-Deoxyguanosine-3′-5′-di(methylene bisphosphonate) [compound (10)] was found as an inhibitor that reduces ppGpp formation in both Gram-negative and Gram-positive bacteria. In silico docking together with competitive inhibition analysis suggests that compound (10) inhibits activity of Rel proteins by competing with GTP/GDP for its binding site. As Rel proteins are completely absent in mammalians, this appears to be a very attractive approach for the development of novel antibacterial agents. [Copyright &y& Elsevier]

NF-κB is a dimeric transcription factor that has emerged as an important regulator of liver homeostasis and is mechanistically implicated in a variety of liver pathologies including hepatitis, steatosis, fibrosis, and hepatocellular carcinoma. The question remains as to whether NF-κB can really be exploited for the development of therapeutics for these pathologies in the diseased human liver. This review casts a critical eye on the experimental evidence gathered to date and in particular questions the rationale for the current focus on components of the upstream IKK complex as therapeutic targets. We make the argument that translation of NF-κB biology to new therapies is more likely to emerge from a re-focus of basic research back to the NF-κB/Rel subunit functions and the complexities of their post-translational modifications and context-dependent co-regulator interactions. [Copyright &y& Elsevier]

Summary: Objective: Although SOX9 is a key molecule for chondrogenic differentiation, little is known about the upstream signal. The present study attempted to identify transcription factors to induce SOX9 expression and examined the mechanism. Methods: Sequences of about 1kb of 5′-end flanking regions were compared between human and mouse SOX9 genes. In vivo localization was examined by immunohistochemistry in the limb cartilage of fetal mice. Promoter activities of the SOX9, SOX6, and type II collagen (COL2A1) genes were determined in human non-chondrogenic HeLa cells and mouse chondrogenic ATDC5 cells transfected with a luciferase-reporter gene containing the promoter fragments. Protein–DNA binding was examined by electrophoretic mobility shift and chromatin immunoprecipitation assays. The chondrogenic differentiation was assessed by endogenous SOX9, SOX6, and COL2A1 mRNA levels, and by Alcian blue staining and alkaline phosphatase activity. Results: Among transcription factors whose binding motifs were identified in the highly-conserved regions between human and mouse SOX9 promoters, a nuclear factor kappa B (NF-κB) member RelA strongly activated the promoter activity. RelA and SOX9 were co-localized in the limb cartilage. Deletion, mutagenesis, and tandem-repeat analyses identified the core region responsive to RelA at the NF-κB binding motif to be around −250bp of the human SOX9 promoter, and this was confirmed to show specific binding to RelA. RelA induced the chondrogenic differentiation parameters in HeLa and ATDC5 cells. Conclusion: We have identified RelA as a transcriptional factor for SOX9 induction and chondrogenic differentiation via binding to an NF-κB binding motif in the SOX9 promoter. [Copyright &y& Elsevier]

Abstract: The fine regulation of NF-κB activity is crucial for both resting and stimulated cells and relies on complex balance between multiple activators and inhibitors. We report here that c-mip, a recently identified pleckstrin homology (PH) and leucine-rich repeat (LRR)-domain-containing protein, inactivates GSKβ and interacts with RelA, a key member of the NF-κB family. We show that c-mip inhibits the degradation of I-κBα and impedes the dissociation of the NF-κB/I-κBα complexes. C-mip acts downstream signaling of classical NF-κB pathway and may represent one of the missing links in the control of NF-κB activity. [Copyright &y& Elsevier]

Abstract: Cerebral ischemia triggers inflammation and apoptosis, and the transcription factor NF-κB is a key regulator of both events. Here, we report on the induction of the peptidoglycan recognition protein-S (PGRP-S) in a mouse model of cerebral ischemia. Upregulation was reduced if the NF-κB subunit RelA was conditionally deleted in the brain. Regulation of PGRP-S transcription by RelA was confirmed in vitro. Cotransfection of a RelA expression plasmid stimulated the expression of a PGRP-S luciferase fusion gene. Mutation of two NF-κB response elements in the PGRP-S promoter disrupted stimulation by RelA. To investigate the function of PGRP-S in cerebral ischemia, we subjected PGRP-S−/− mice to cerebral ischemia. However, there was no difference in the infarct size in PGRP-S-deficient mice compared to controls. In summary, the data show that PGRP-S is induced in cerebral ischemia by RelA, but its role in ischemia is unclear. [Copyright &y& Elsevier]

Abstract: Gene regulation in sepsis is known to be controlled by the transcription factor NF-κB. However, the function of neuronal NF-κB in sepsis is not well defined. In a mouse model of sepsis induced by i.p. injection of lipopolysaccharides (LPS), we found an activation of NF-κB in the brain as shown by the induction of a transgenic NF-κB reporter. Inhibition of neuronal NF-κB by cell-specific expression of the NF-κB super-repressor IκBα-SR improved LPS-induced hypothermia and survival but had no effect on body weight or on the humoral response to LPS. In contrast, glial inhibition of NF-κB did not influence body temperature and survival. By immunohistochemistry, we detected the active NF-κB subunit RelA in neuronal nuclei of the organum vasculosum of the lamina terminalis. Our data reveal an important role of neuronal NF-κB in thermoregulation and survival. The upcoming group of NF-κB inhibitors may have a place in the treatment of the acute-phase response. [Copyright &y& Elsevier]

Abstract: Tumor necrosis factor-α (TNF-α) is a potent mediator of inflammation, inducing expression of a gene network mediated by NF-κB. Previously we found that TNF-α-induced reactive oxygen species (ROS) production is required for NF-κB action because antioxidants inhibited TNF-α-inducible IL-8 expression without affecting its nuclear translocation. Here, we further investigated this ROS pathway controlling NF-κB/RelA dependent gene expression. We observed that TNF-α enhanced ROS production ∼2-fold 20 min after stimulation and significantly increased oxidative DNA damage (8-oxoguanine lesions) over controls. Treatment with chemically unrelated antioxidants specifically inhibited expression of TNF-inducible NF-κB-dependent genes without producing detectable cytotoxicity or affecting GAPDH expression. We found that TNF-α-induced NF-κB/RelA Ser276 phosphorylation, a modification critical for its transcriptional activity, was inhibited by abrogation of the ROS signaling pathway, whereas NF-κB/RelA Ser536 phosphorylation was not. Interestingly, antioxidant treatment selectively inhibited TNF-α-induced catalytic activity of cAMP dependent protein kinase A (PKAc) but not mitogen-stress related kinase-1 (MSK1), kinases known to phosphorylate RelA at Ser276. Using PKAc inhibitors and siRNA mediated PKAc knockdown, TNF-α-induced Ser276 phosphorylation and IL-8 expression were both significantly reduced, indicating PKAc is required for RelA Ser276 phosphorylation. Consistently, a site mutation of Rel A (Ser276 to Ala) in RelA-deficient embryonic fibroblasts failed to activate IL-8 Luciferase activity in response to TNF-α. Furthermore, TNF-α-inducible NF-κB/RelA interaction with the co-activator CBP/p300, essential for enhanceosome formation, was attenuated by antioxidant treatment. Using chromatin immunoprecipitation assay (ChIP), we observed that recruitment of p300 and RNA polymerase II (Pol II) to the IL-8 promoter was also abrogated by antioxidant. These results indicate that the ROS-mediated TNF-α-induced IL-8 transcription is regulated by NF-κB/RelA phosphorylation at the critical Ser276 residue by PKAc, resulting in stable enhanceosome formation on target genes. These studies provide insight into a novel antioxidant-sensitive pathway that can be targeted to inhibit NF-κB-mediated inflammation. [Copyright &y& Elsevier]

Abstract: Although the enhancement of amino-acid synthesis by guanosine-3′,5′-tetraphosphate (ppGpp) is well known, the effect of intracellular ppGpp levels on amino-acid overproduction in Escherichia coli has not been investigated. In this study, we demonstrate that overexpression of the relA gene, encoding ppGpp synthetase, increases the accumulation of amino acids, such as glutamate and lysine, in amino-acid-overproducing strains of E. coli. Elevation of intracellular ppGpp levels due to depletion of required amino acids also enhances glutamate overproduction. Moreover, the extent of overproduction is highly dependent on the intracellular ppGpp level. These results demonstrate that amino-acid overproduction in E. coli is closely connected to amino-acid auxotrophy via the accumulation of ppGpp. [Copyright &y& Elsevier]

Abstract: In this investigation, we identify the CodY protein from Streptococcus pyogenes as a pleiotropic transcription regulator with global features. The notion that acquisition of nutrients by this polyauxotrophic organism is the primary event occurring during the establishment of infection and that virulence expression is a result of this quest, led us to study the action of codY and relA genes on transcriptional gene expression under different nutritional conditions using complex and chemically defined media. Real-time reverse transcription PCR was used to determine the extent to which inactivation of codY and relA affects the mRNA levels of selected transcription factors, virulence genes, transporters, and genes encoding metabolic enzymes. The results show that CodY and RelA did not affect the expression of each other but that both exhibited strong negative autoregulatory properties. Genes negatively controlled by the relA-directed stringent response to amino acid starvation included, besides relA itself, transporters, metabolic enzymes, and at least two virulence genes (graB and speH). The expression of many genes of all four groups studied proved to be subject to direct or indirect control by CodY, often in a nutritional status-dependent fashion. One of the most important results implicates CodY in growth phase-dependent positive transcriptional regulation of pel/sagA and mga, loci that themselves positively affect the expression of numerous virulence factors. Increasing the cellular activity of nicotinamidase in both a codY mutant and wild-type background induced extensive transcriptional reprogramming, altering, among others, the growth phase-dependent transcription pattern of the genes for cysteine protease (speB) and several transporters. Inasmuch as CodY influenced the expression of other regulators (pel/sagA, mga, covRS, ropB, pyrR), its action is amplified and expands the complex regulatory network that governs gene expression in S. pyogenes. [Copyright &y& Elsevier]

Abstract: In this report, we identify the CodY protein and the product of the activated RelA protein, (p)ppGpp, from Streptococcus pyogenes as pleiotropic regulatory factors that direct nutritional status-dependent transcriptional expression of specific regulatory genes, virulence factors, transporters, and metabolic enzyme genes. Real-time reverse transcription PCR was used to determine the extent to which inactivation of codY and relA affects the mRNA levels of the target genes under different nutritional conditions using complex and chemically defined media. [Copyright &y& Elsevier]

Abstract: Nuclear factor kappa-B (NF-κB), a key downstream player of the LPS signaling pathway, has been shown to undergo alternative splicing in in vitro studies. In this study, we examined the effect of injury and the role of CD14 on NF-κB alternative splicing using a murine burn model. CD14 knockout and respective wild-type mice were sacrificed after 18% total body surface area burn. RT-PCR and subsequent sequencing analysis revealed that injury induced multiple novel splicing variants of relA, relB, and NF-κB2 in the lungs of CD14 knockout but not wild-type mice. These novel variants resulted either from exon skipping, alternative usage of splicing signals, or intron retention. All but one variant resulted in a frameshift leading to premature termination of translation. These splicing variants encoded for proteins that lacked the domains essential for NF-κB transcription factor functions. Two NF-κB2 variants acquired only minor changes in their C-terminus that might affect their post-translational cleavage into active isoforms. These results suggest that alternative splicing may be one of the mechanisms by which NF-κB activity in the lungs can be regulated after injury. Furthermore, the CD14-mediated LPS signaling pathway may play a role in the regulation of NF-κB alternative splicing in the lungs after injury. [Copyright &y& Elsevier]

α -Conidendrin is a lignan isolated from Taxus wallichiana and other species. In the present study, we demonstrated that α -conidendrin inhibited the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) at an IC 50 value of 40–60 μM in human lung adenocarcinoma A549 cells. α -Conidendrin decreased ICAM-1 protein and mRNA expression levels at concentrations of 40–100 μM in TNF-α-stimulated A549 cells. The TNF-α-induced mRNA expression of vascular cell adhesion molecule-1, E-selectin, and cyclooxygenase-2 was also reduced by α -conidendrin. In the TNF-α-induced nuclear factor κB (NF-κB) signaling pathway, α -conidendrin did not influence the translocation of the NF-κB subunit RelA from the cytoplasm to the nucleus at concentrations up to 100 μM. A chromatin immunoprecipitation assay revealed that α -conidendrin at 100 μM reduced the binding of RelA to the ICAM-1 promoter in response to a stimulation with TNF-α. Collectively, these results indicated that α -conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter, thereby reducing ICAM-1 transcription. • α -Conidendrin is a lignan isolated from Taxus wallichiana and other species. • α -Conidendrin inhibited TNF-α-induced ICAM-1 expression. • α -Conidendrin did not influence the TNF-α-induced nuclear translocation of RelA. • α -Conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter. [ABSTRACT FROM AUTHOR]

• Immune response-related elements are present in the 5′ flanking sequence of Rasgef1b. • Minimal TLR-responsive promoter region harbors Sp1 and NF-κB binding sites. • Rasgef1b promoter is located in an accessible chromatin region. • NF-κB RelA is recruited to Rasgef1b promoter upon LPS stimulation of macrophages. • Optimal TLR-induced expression of Rasgef1b requires NF-κB. Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5′ flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela -deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages. [ABSTRACT FROM AUTHOR]

Gene and protein expression of the glucose transporter GLUT6 are elevated in multiple cancers, including endometrial cancer. However, the extrinsic and intrinsic mechanisms that regulate GLUT6 expression in this malignancy are unknown. Herein we investigate the potential mechanisms regulating GLUT6 expression in endometrial cancer. Data mining of the GLUT6 gene (SLC2A6) in The Cancer Genome Atlas (TCGA) PanCan datasets was performed in cBioPortal. A transcriptome PCR array was used to identify regulators of GLUT6 expression. The role of RELA in regulating GLUT6 gene and protein expression was investigated by overexpressing constitutively active and dominant-negative RELA in endometrial cells. Endometrial cells were treated with the pro-inflammatory cytokine TNFα and the expression of RELA, IκBα, TNFα, and GLUT6 were examined by Western blotting and RT-qPCR. GLUT6 is altered in 1% of all cancer samples (157 of 10, 967 samples) within TCGA datasets including 4.7% of uterine (endometrial) cancers. GLUT6 expression was positively co-expressed with multiple members of the NF-κB signalling pathway including NFKB2 , RELB , NFKBIE, and TNF in endometrial cancer samples. A transcriptome PCR array identified RELA as the top potential transcriptional regulator of GLUT6 expression. Overexpression of constitutively active RELA increased GLUT6 gene expression in normal endometrial epithelial cells (hUE-Ts), while overexpression of dominant-negative RELA decreased GLUT6 expression in cancerous RL95–2 endometrial cells. TNFα treatment activated canonical NF-κB signalling and increased the expression of GLUT6, but not that of other glucose transporters (GLUTs 1, 3, 4, 8, 10, or 12) in endometrial cells. TNFα is a cytokine that is commonly increased in obesity-related endometrial cancer and the findings herein support a potential mechanism whereby TNFα may contribute to endometrial cancer initiation or progression by increasing GLUT6 expression. Furthermore, we identified RELA, an important downstream mediator of the TNFα signalling cascade, as a regulator of GLUT6 expression in endometrial cells. Future studies are warranted to determine how GLUT6 expression affects endometrial tumourigenesis or cancer progression. • Mutations or amplifications in GLUT6 occur more frequently in endometrial cancer than all other malignancies • GLUT6 gene expression positively correlates with genes of the NF-κB signalling pathway in endometrial cancer • A transcription factor PCR array identifies RELA as a positive regulator of GLUT6 gene expression • Constitutively-active RELA increases, while dominant-negative RELA decreases GLUT6 expression in endometrial cells • TNFα stimulation of endometrial cells exclusively increases GLUT6 expression without altering other glucose transporters [ABSTRACT FROM AUTHOR]

Helicobacter pylori infection represents a major risk factor for the development of gastric diseases and gastric cancer. The capability of H. pylori to inject the virulence factor cytotoxin-associated gene A (CagA) depends on a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cag PAI). Further, infection by H. pylori activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in a T4SS-dependent manner but CagA-independent manner. Here we investigated the role of host cell receptors carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and the bacterial adhesin HopQ in the activation of non-canonical NF-κB and CagA translocation into gastric epithelial cells. AGS cells express six of twelve CEACAMs found in humans. In HeLa cells, only CEACAM19 is expressed. We showed that deletion of hopQ attenuates the activation of non-canonical NF-κB only in AGS but not in HeLa cells. CagA translocation was in both cell lines affected by HopQ depletion, although to a much lesser extent in HeLa cells. Moreover, we observed a possible redundancy between the three HopQ-binding CEACAMs 1, 5 and 6 and their capacity to support non-canonical NF-κB activation. Our results illustrate that the interaction between HopQ and CEACAMs could promote the efficiency of the T4SS. [ABSTRACT FROM AUTHOR]

Dysregulated activity of the transcription factors of the nuclear factor κb (NF-κB) family has been implicated in numerous cancer types, inflammatory diseases, autoimmune disease, and other disorders. As such, selective NF-κB pathway inhibition is an attractive target to researchers for preclinical and clinical drug development. A plethora of commercially and clinically available inhibitors claim to be NF-κB specific; however, such claims of specificity are rarely quantitative or benchmarked, making the biomedical literature difficult to contextualize. This imprecision is worsened because some NF-κB reporter systems have low signal-to-noise ratios. Herein, we use a robust, defined, commercially available reporter system to benchmark NF-κB agonists and antagonists for the field. We also functionally characterize a RELA fusion-positive ependymoma cell culture with validated NF-κB inhibitor compounds. [ABSTRACT FROM AUTHOR]

• High level of MLL2 was significantly associated with early-stage ESCC lymph node metastasis. • MLL2 positively regulates gene expression programs associated with ESCC cell migration. • MLL2 interacts with RelA to enhance transcription of stanniocalcin-1 (STC1) and to facilitates cancer metastasis. • MLL2 can be a marker for early prediction of lymph node metastasis of ESCC. Esophageal squamous cell carcinoma (ESCC) presents with lymph node metastasis in the early stages, limiting the opportunities for curative local resection, including endoscopic submucosal dissection (ESD). ESD is regarded as the standard treatment for early-stage ESCCs. However, radical surgery is recommended when lymph node metastasis risk exists. More efforts are needed to find the markers for early prediction and clarify the molecular mechanism underlying the pathogenesis of lymph node metastasis. Recently, aberrant regulation of gene expression by histone methylation modifiers has emerged as an important mechanism for cancer metastasis. Herein, we demonstrated that mixed-lineage leukemia 2 (MLL2) positively regulates gene expression programs associated with ESCC cell migration. MLL2 interacts with RelA in the nucleus to enhance transcription of stanniocalcin-1 (STC1) and to facilitate cancer metastasis. Meanwhile, MLL2 knockdown resulted in a significant decrease in the migration of ESCC cells. Clinically, high level of MLL2 was significantly associated with early-stage ESCC lymph node metastasis. In summary, these findings discovered a previously unidentified molecular pathway underlying the coordinated regulation of metastasis-related STC-1 expression by MLL2 and RelA and highlighted the critical role of MLL2 in ESCC. [ABSTRACT FROM AUTHOR]

Studies with Mycobacterium avium subsp. paratuberculosis (Map) in cattle revealed deletion of relA , a global regulator gene, abrogated ability of the mutant to establish a persistent infection, attributed to development of an immune response that cleared infection. Analysis of the recall response demonstrated presence of CD8 cytotoxic T cells that kill intracellular bacteria. Replication of the primary response demonstrated the CTL response could be elicited with the Δ Map/relA mutant or the target of the immune response, a 35 kD membrane protein. Follow up comparative studies with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and a BCG relA (ΔBCG/ relA) deletion mutant revealed deletion of relA enhanced the CTL response compared to BCG. Analysis of the cytokine profile of cells proliferating in response to stimulation with BCG or BCG/ relA showed increased expression of IFN-γ, TNF-α, and IL-17 by cells stimulated with ΔBCG/ relA in comparison with BCG. The proliferative and CTL responses were markedly reduced in response to stimulation with heat killed BCG or ΔBCG/ relA. Intracellular bacterial killing was mediated through the perforin, granzyme B (GnzB), and the granulysin pathway. The data indicate relA is the Achilles' heel for pathogenic mycobacteria and deletion may be key to improving efficacy of attenuated vaccines for mycobacterial pathogens. • RelA is Achilles' heel for mycobacterial pathogens. • CD8 cytotoxic T cells kill Intracellular mycobacteria. • Macrophages are no safe haven for mycobacterial pathogens. [ABSTRACT FROM AUTHOR]

The phenomenon of colistin dependence in Acinetobacter baumannii has been described in a situation in which colistin is now considered as the last resort for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. In this study, we aimed to reveal a gene associated with colistin dependence in A. baumannii. The colistin-dependent A. baumannii H08-391D strain was isolated from a patient, and target gene-inactivation mutants were constructed. We investigated the effects of target gene on colistin dependence with quantitative real-time PCR and endotoxin assay. Also, we observed the change of cell morphology by electron microscopy. The expression of ACICU_02898, encoding a soluble lytic transglycosylase associated with cell-wall degradation and recycling, was increased by eight-to 42-fold in colistin-dependent mutants, and deletion of ACICU_02898 in a colistin-dependent strain led to colistin susceptibility (MIC = 8 mg/L). Endotoxin activity was significantly low in a colistin-dependent derivative ACICU_02898-inactivated mutant and a complemented mutant. In addition, the ACICU_02898-inactivated mutant showed a highly reduced growth rate. The colistin-dependent derivative and ACICU_02898-inactivated mutant showed clearly distinguished absorption profiles in the red/green fluorescence dot blot with regard to their membrane potential. Electron microscopy revealed that the deletion mutant cells were elongated compared to the colistin-susceptible wild-type strain and colistin-dependent strain. A colistin-dependent A. baumannii strain exhibited a deficiency in its outer membrane integrity and high expression of lytic transglycosylase was required for survival. This study reveals why the colistin-dependent mutant can tolerate high antibiotic concentrations. [ABSTRACT FROM AUTHOR]