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The activity and close regulation of nuclear factor kappaB (NF-κB) transcription factors is critical for a variety of cellular processes including inflammation, immunity, differentiation and cell survival. Thus, dysregulation of the NF-κB system could lead to serious diseases, e.g. uncoordinated growth of the normal tissue during the development of cancer. Transcriptional activity of the NF-κB factor RelA is regulated by a number of mechanisms which comprise ubiquitinylation by a multimeric ubiquitin ligase containing Elongins B and C, cullin-2 (Cul2) and suppressor of cytokine signaling 1 (SOCS1), but also USP48-dependent deubiquitinylation. Further, USP48 promotes cell survival and antagonizes also other E3 ligase functions which are involved in genome stability and DNA repair. The regulation of RelA by USP48 has been investigated in detail, but the domains of USP48 and RelA for direct interaction are not known. In this study we report that USP48 interacts physically with RelA in the nucleus. Further, we show by overexpression of truncated proteins that the catalytic USP domain of USP48 interacts with the N-terminal region of the Rel homology domain (RHD) of RelA. This study provides first evidence that the USP domain of USP48 is important for the physical association with substrate proteins, and a suitable target for small molecule inhibitors for therapeutic intervention strategies. [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]

Background/Aims: Acute pancreatitis contributes to high mortality in pancreatitis patients, and miRNAs play a vital role in the development of acute pancreatitis (AP), however, its precise biological role remains largely elusive. Methods: To clarify the potential mechanisms of miRNAs in AP, we built mouse models of mild acute pancreatitis (MAP) and moderate/ severe acute pancreatitis (SAP). MiRNA microarray analysis and Real-time quantitative PCR (qRT-PCR) were used to analyze the expression of miRNA in MAP/SAP. TargetScan software, dual-luciferase gene reporter assays and Western blotting were used to assess the target genes of miR-155-5p in AP. Results: miR-155-5p was significantly decreased in MAP/SAP mice compared to controls. In pancreatic acinar AR42J cells transfected with miR-155-5p mimic, the expression of Rela and Traf3 notably decreased in both the caerulein- and TLC-S-induced groups compared with the negative control (NC); however, the expression of Rela and Traf3 notably increased after transfection with miR-155-5p inhibitor. Combined analysis using the TargetScan software and dual-luciferase gene reporter assays indicated that Rela and Traf3 were both targeted by miR-155-5p. Meanwhile, the expression of Ptgs2 also decreased after transfection of the AR42J cells with miR-155-5p mimic. The opposite results were found when miR-155-5p inhibitor was transfected into the AR42J cells. In addition, we treated caerulein- and TLC-S-induced AR42J cells with the Rela inhibitor helenalin and found that the expression of Rela, Traf3 and Ptgs2 decreased compared with the NC, while the expression of miR-155-5p did not show any significant difference. Furthermore, we found that miR-155-5p was significantly down-regulated in pancreatitis patients. Conclusion: miR-155-5p inversely regulated AP development through the Rela/Traf3/Ptgs2 signaling pathway. [ABSTRACT FROM AUTHOR]

Background: The epithelium is the first line of defense against pathogens. Notably the epithelial cells lining the respiratory track are crucial in sensing airborne microbes and mounting an effective immune response via the expression of target genes such as cytokines and chemokines. Gene expression regulation following microbial recognition is partly regulated by chromatin re-organization and has been described in immune cells but data from epithelial cells is not as detailed. Here, we report genome-wide changes of the H3K27ac mark, characteristic of activated enhancers and promoters, after stimulation of nasopharyngeal epithelial cells with the bacterial endotoxin Lipopolysaccharide (LPS). Results: In this study, we have identified 626 regions where the H3K27ac mark showed reproducible increase following LPS induction in epithelial cells. This indicated that sensing of LPS led to opening of the chromatin in our system. Moreover, this phenomenon seemed to happen extensively at enhancers regions and we could observe instances of Super-enhancer formation. As expected, LPS-increased H3K27ac regions were found in the vicinity of genes relevant for LPS response and these changes correlated with up-regulation of their expression. In addition, we found the induction of H3K27ac mark to overlap with the binding of one of the NF-kB members and key regulator of the innate immune response, RELA, following LPS sensing. Indeed, inhibiting the NF-kB pathway abolished the deposition of H3K27ac at the TNF locus, a target of RELA, suggesting that these two phenomena are associated. Conclusions: Enhancers' selection and activation following microbial or inflammatory stimuli has been described previously and shown to be mediated via the NF-kB pathway. Here, we demonstrate that this is also likely to occur in the case of LPS-sensing by nasopharyngeal epithelial cells as well. In addition to validating previous findings, we generated a valuable data set relevant to the host immune response to epithelial cell colonizing or infecting pathogens. [ABSTRACT FROM AUTHOR]

Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-κB activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-κB regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into various vascular cell derivatives to study how NF-κB modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating inflammation, survival, vasculogenesis, cell differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon tumor necrosis factor α (TNFα) stimulation. Lastly, further evaluation of gene expression patterns in IκBα knockout vascular cells demonstrated that IκBα acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-κB/RelA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets. [ABSTRACT FROM AUTHOR]

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.

Controlling metastasis is an important strategy in cancer treatment. Nanotechnology and nucleic acids with novel modalities are promising regulators of cancer metastasis. We aimed to develop a small interfering RNA (siRNA) systemic delivery and anti-metastasis system using nanotechnology. We previously reported that polyethylene glycol-polycaprolactone (PEG-PCL) and functional peptide CH2R4H2C nano-micelle (MPEG-PCL-CH2R4H2C) has high siRNA silencing effects, indicated by increased drug accumulation in tumor-bearing mice, and has an anti-tumor effect on solid tumors upon systemic injection. In this study, we aimed to apply our micelles to inhibit metastasis and evaluated the inhibitory effect of anti-RelA siRNA (siRelA), which is a subunit of NF-κB conjugated with MPEG-PCL-CH2R4H2C, via systemic administration. We report that siRelA/MPEG-PCL-CH2R4H2C had a high cellular uptake and suppressed the migration/invasion of cells in B16F10 cells without toxicity. In addition, in a lung metastasis mouse model using intravenous administration of B16F10 cells treated with siRelA/MPEG-PCL-CH2R4H2C, the number of lung nodules in lung tissue significantly decreased compared to naked siRelA and siControl/MPEG-PCL-CH2R4H2C micelle treatments. Hence, we show that RelA expression can reduce cancer metastasis, and MPEG-PCL-CH2R4H2C is an effective siRNA carrier for anti-metastasis cancer therapies.

Metastatic melanoma remains incurable for many due to its aggressive nature. Secreted cathepsins promote metastasis by cleaving matrix and activating pro-invasive proteases. We reported that ABL kinases induce cathepsin secretion and subsequent metastasis by activating ETS1, SP1, and RELA pathways, indicating that ABL inhibitors may serve as novel anti-cathepsin agents.

The present study shows a new connection of protein tyrosine phosphatase interacting protein 51 (PTPIP51) to the nuclear factor κB (NFκB) signalling pathway. PTPIP51 mRNA and protein expression is regulated by RelA. If bound to the PTPIP51 promoter, RelA repress the mRNA and protein expression of PTPIP51. The parallel treatment with pyrrolidine dithiocarbamate (PDTC) reversed the suppression of PTPIP51 protein expression induced by TNFα. Using the intensity correlation analysis PTPIP51 verified a co-localization with RelA, which is also regulated by TNFα administration. Moreover, the direct interaction of PTPIP51 and RelA was established using the DuoLink proximity ligation assay. IκBα, the known inhibitor of RelA, also interacted with PTPIP51. This hints to the fact that in un-stimulated conditions PTPIP51 forms a complex with RelA and IκBα. The PTPIP51/RelA/IκBα complex is modulated by TNFα. Interestingly, the impact on the mitogen activated protein kinase pathway was negligible except in highest TNFα concentration. Here, PTPIP51 and Raf-1 interactions were slightly repressed. The newly established relationship of PTPIP51 and the NFκB signaling pathway provides the basis for a possible therapeutic impact.

Metaflammation in adipose tissue, which is characterized by increased local gene expression and secretion of pro-inflammatory factors, may contribute to the increased risk of metabolic complications in obesity. It has been suggested that IL-1β might induce metaflammation in adipose tissue via modulating the NF-κB signaling pathway. In our study, the mRNA and secretion levels of major pro-inflammatory factors including IL (interleukin)-1β, IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and RANTES (regulated on activation, normal T cell expressed and secreted) were measured as indicators of the inflammatory profile. We herein showed that IL-1β could induce adipose tissue metaflammation by enhancing the inflammatory profile of preadipocytes. Moreover, IL-1β could enhance pro-inflammatory gene expression by increasing the phosphorylation or decreasing the methylation of relA (NF-κB p65) of the NF-κB signaling pathway. VDR (vitamin D receptor) ligands have been shown to have anti-inflammatory properties in adipocytes. Likewise, our study demonstrated that the inflammatory profile of IL-1β-stimulated preadipocytes is significantly attenuated by VDR ligands 1α,25(OH) 2 D 3 , ZK159222 and ZK191785. Importantly, we showed that ZK159222 and ZK191785 could inhibit pro-inflammatory gene expression by decreasing the phosphorylation or increasing the methylation of relA. Furthermore, pro-inflammatory secretion could be reduced by 1α,25(OH) 2 D 3 , ZK159222 and ZK191785 via increasing the phosphorylation of eIF (eukaryotic translation initiation factor)-2α of the UPR (unfolded protein response) pathway. These observations suggest that the VDR ligands may be considered potential anti-inflammatory treatments for obesity associated metabolic complications. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) vFLIP , a latent gene of KSHV, was first identified as a FLICE-inhibitory protein (FLIP) protecting cells from apoptosis. The vFLIP protein has been shown to activate the NF-κB signaling involved in spindle morphology formation both in HUVECs infected with KSHV and Kaposi's sarcoma (KS) itself. In this study, we independently established stably vFLIP -expressing cells and showed that they exhibited upregulated NF-κB family protein expression independent of the ability of IKKs to bind vFLIP. Further, vFLIP induced upregulation of IKKε, phosphorylation of RelA at Ser468 (p-RelA S468) and nuclear localization of Re1A concomitant with spindle morphology formation, and these effects were reversed by knockdown of IKKε and treatment with Bay-11. Overexpression of IKKε alone also showed spindle morphology formation with p-RelA S468. In conclusion, the spindle cell morphology in KS should be induced by RelA activation (p-RelA S468) by IKKε upregulation in vFLIP-expressing EA hy926 cells. [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]

Survival motor neuron (SMN) protein deficiency causes the genetic neuromuscular disorder spinal muscular atrophy (SMA), characterized by spinal cord motoneuron degeneration. Since SMN protein level is critical to disease onset and severity, analysis of the mechanisms involved in SMN stability is one of the central goals of SMA research. Here, we describe the role of several members of the NF-κB pathway in regulating SMN in motoneurons. NF-κB is one of the main regulators of motoneuron survival and pharmacological inhibition of NF-κB pathway activity also induces mouse survival motor neuron (Smn) protein decrease. Using a lentiviral-based shRNA approach to reduce the expression of several members of NF-κB pathway, we observed that IKK and RelA knockdown caused Smn reduction in mouse-cultured motoneurons whereas IKK or RelB knockdown did not. Moreover, isolated motoneurons obtained from the severe SMA mouse model showed reduced protein levels of several NF-κB members and RelA phosphorylation. We describe the alteration of NF-κB pathway in SMA cells. In the context of recent studies suggesting regulation of altered intracellular pathways as a future pharmacological treatment of SMA, we propose the NF-κB pathway as a candidate in this new therapeutic approach. [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]

Histone deacetylation, together with altered acetylation of NF-κB/RelA, encompassing the K310 residue acetylation, occur during brain ischemia. By restoring the normal acetylation condition, we previously reported that sub-threshold doses of resveratrol and entinostat (MS-275), respectively, an activator of the AMP-activated kinase (AMPK)-sirtuin 1 pathway and an inhibitor of class I histone deacetylases (HDACs), synergistically elicited neuroprotection in a mouse model of ischemic stroke. To improve the translational power of this approach, we investigated the efficacy of MS-275 replacement with valproate, the antiepileptic drug also reported to be a class I HDAC blocker. In cortical neurons previously exposed to oxygen glucose deprivation (OGD), valproate elicited neuroprotection at 100 nmol/mL concentration when used alone and at 1 nmol/mL concentration when associated with resveratrol (3 nmol/mL). Resveratrol and valproate restored the acetylation of histone H3 (K9/18), and they reduced the RelA(K310) acetylation and the Bim level in neurons exposed to OGD. Chromatin immunoprecipitation analysis showed that the synergistic drug association impaired the RelA binding to the Bim promoter, as well as the promoter-specific H3 (K9/18) acetylation. In mice subjected to 60 min of middle cerebral artery occlusion (MCAO), the association of resveratrol 680 μg/kg and valproate 200 μg/kg significantly reduced the infarct volume as well as the neurological deficits. The present study suggests that valproate and resveratrol may represent a promising ready-to-use strategy to treat post-ischemic brain damage. [ABSTRACT FROM AUTHOR]

We consider a single-cell network of random transmitters and fixed relays in a bounded domain of Euclidean space. The transmitters arrive over time and select one relay according to a spatially inhomogeneous preference kernel. Once a transmitter is connected to a relay, the connection remains and the relay is occupied. If an occupied relay is selected by another transmitters with later arrival time, this transmitter becomes frustrated. We derive a large deviation principle for the space-time evolution of frustrated transmitters in the high-density regime. [ABSTRACT FROM AUTHOR]

In enteric bacteria such as Escherichia coli, the transcription factor SgrR and the small RNA SgrS regulate the response to glucose phosphate stress, a metabolic dysfunction that results in growth inhibition and stems from the intracellular accumulation of sugar phosphates. SgrR activates the transcription of sgrS, and SgrS helps to rescue cells from stress in part by inhibiting the uptake of stressor sugar phosphates. While the regulatory targets of this stress response are well described, less is known about how the SgrR-SgrS response itself is regulated. To further characterize the regulation of the glucose phosphate stress response, we screened global regulator gene mutants for growth changes during glucose phosphate stress. We found that deleting dksA, which encodes a regulator of the stringent response to nutrient starvation, decreases growth under glucose phosphate stress conditions. The stringent response alarmone regulator ppGpp (synthesized by RelA and SpoT) also contributes to recovery from glucose phosphate stress: as with dksA, mutating relA and spoT worsens the growth defect of an sgrS mutant during stress, although the sgrS relA spoT mutant defect was only detectable under lower stress levels. In addition, mutating dksA or relA and spoT lowers sgrS expression (as measured with a PsgrS-lacZ fusion), suggesting that the observed growth defects may be due to decreased induction of the glucose phosphate stress response or related targets. This regulatory effect could occur through altered sgrR transcription, as dksA and relA spoT mutants also exhibit decreased expression of a PsgrR-lacZ fusion. Taken together, this work supports a role for stringent response regulators in aiding the recovery from glucose phosphate stress. [ABSTRACT FROM AUTHOR]

The nuclear factor (NF)-κB family of transcriptional factors plays a critical role in inflammation, immunoregulation, cell differentiation, and tumorigenesis. This study aims to investigate the role of methylation of genes encoding for the NF-κB family in breast cancer. We analyze the DNA methylation status of the NFKB1 gene and the RELA gene in breast cancer using pyrosequencing. The expression of NF-κB1 and RELA proteins is assessed and the level of RNA transcripts in frozen tissue is determined using RT-PCR. There is no statistically significant difference in the methylation status of the NFKB1 and the RELA genes between tumors and normal tissues. The methylation status of the NFKB1 gene and the RELA gene is not significantly associated with the levels of NF-κB1 transcripts in tumor tissues. However, the methylation level of the RELA gene is significantly associated with the level of tumor necrosis factor (TNF)-α. In addition, the level of NF-κB1 transcripts was associated with the levels of TNF-α and IL-4. In tumors with positive TNF-α, the increased methylation level of the RELA gene is significantly associated with the positive expression of NF-κB1 transcripts. These results demonstrate that the level of the RELA gene methylation is related to the levels of NF-κB1 transcripts under the influence of TNF-α. Further study is needed to determine how TNF-α is involved in the methylation of the RELA gene and the subsequent expression of NF-κB1.

Transcription factors (TFs) are proteins that control the transcription of genetic information from DNA to mRNA by binding to specific DNA sequences either on their own or with other proteins as a complex. TFs thus support or suppress the recruitment of the corresponding RNA polymerase. In general, TFs are classified by structure or function. The TF, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), is expressed in all cell types and tissues. NF-κB signaling and crosstalk are involved in several steps of carcinogenesis including in sequences involving pathogenic stimulus, chronic inflammation, fibrosis, establishment of its remodeling to the precancerous niche (PCN) and transition of a normal cell to a cancer cell. Triggered by various inflammatory cytokines, NF-κB is activated along with other TFs with subsequent stimulation of cell proliferation and inhibition of apoptosis. The involvement of NF-κB in carcinogenesis provides an opportunity to develop anti-NF-κB therapies. The complexity of these interactions requires that we elucidate those aspects of NF-κB interactions that play a role in carcinogenesis, the sequence of events leading to cancer.

Ependymomas are primary neuroepithelial malignancies that mainly occur during childhood, and arise from ependymal cells along the ventricular systems of the CNS. Recently, it was elucidated that two-thirds of supratentorial (ST) ependymomas harbor oncogenic fusions of RELA, whose protein product is the principal effector of canonical NF-κB signaling. RELA fusion proteins activate signaling for tumor proliferation and malignant progression, resulting in poorer prognoses in these patients compared to those in patients with other ST ependymomas. In this study, we encountered a case of C11orf- RelA fusion-positive ST anaplastic ependymoma that was diagnosed in first tumor resection surgery of multi-staged gross total resection with molecular evidence. In ependymomas, regardless of tumor location or pathological grade, subtotal resection is associated with higher rates of mortality compared with GTR. Molecular analysis based on the application of recent molecular knowledge for ST ependymomas performs a role in appropriate and individualized treatment strategies. [ABSTRACT FROM AUTHOR]

Specific protein-protein interaction (PPI) is an essential feature of many cellular processes however, targeting these interactions by small molecules is highly challenging due to the nature of the interaction interface. Thus, screening for PPI inhibitors requires enormous number of compounds. Here we describe a simple and improved protocol designed for a search of direct PPI inhibitors. We engineered a bacterial expression system for the split-Renilla luciferase (RL) complementation assay that monitors PPI. This enables production of large quantities of the RL fusion proteins in a simple and cost effective manner that is suitable for very large screens. Subsequently, inhibitory compounds are analyzed in a similar complementation assay in living cultured mammalian cells to select for those that can penetrate cells. We applied this method to NF-κB, a family of dimeric transcription factors that plays central roles in immune responses, cell survival and aging, and its dysregulation is linked to many pathological states. This strategy led to the identification of several direct NF-κB inhibitors. As the described protocol is very straightforward and robust it may be suitable for many pairs of interacting proteins. [ABSTRACT FROM AUTHOR]

Overwhelming evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity and the potential to prevent cancer, particularly colorectal cancer. However, the mechanisms underlying this effect remain hypothetical. Dysregulation of the nuclear factor-kappaB (NF-κB) transcription factor is a common event in many cancer types which contributes to tumour initiation and progression by driving expression of pro-proliferative/anti-apoptotic genes. In this review, we will focus on the current knowledge regarding NSAID effects on the NF-κB signalling pathway in pre-cancerous and cancerous lesions, and the evidence that these effects contribute to the anti-tumour activity of the agents. The nuclear organelle, the nucleolus, is emerging as a central regulator of transcription factor activity and cell growth and death. Nucleolar function is dysregulated in the majority of cancers which promotes cancer growth through direct and indirect mechanisms. Hence, this organelle is emerging as a promising target for novel therapeutic agents. Here, we will also discuss evidence for crosstalk between the NF-κB pathway and nucleoli, the role that this cross-talk has in the anti-tumour effects of NSAIDs and ways forward to exploit this crosstalk for therapeutic purpose. [ABSTRACT FROM AUTHOR]

The accumulation of macrophages has been observed around lesions of the brain in patients with Minamata disease. In this condition, mercury has been detected histochemically in macrophages throughout the brain. However, the role of macrophages in the neurotoxicity of methylmercury (MeHg) and the molecular mechanisms of their response to MeHg exposure remain to be elucidated. Here, we investigated how MeHg affects the expression of proinflammatory cytokines such as interleukin (IL)-6 and IL-8 in cultured human U937 macrophages. Compared with controls, IL-6 and IL-8 mRNA expression was maximally induced in U937 macrophages after treatment with 10 μM MeHg for 6 h. The protein secretion of IL-6 and IL-8 was significantly stimulated by MeHg in U937 macrophages. Results from luciferase reporter assay indicated functional activation of nuclear factor kappa B and the involvement of subunit RelA and p50 in MeHg-induced IL-6 and IL-8 activation, which was confirmed by siRNA knockdown experiments. MeHg exposure at 4 μM also significantly induced IL-8 expression in U-87 MG cells at mRNA and protein level, indicating that IL-8 induction might be a general mode of action of MeHg treatment among different cell types. These results indicate a possible involvement of an early inflammatory response, including IL-6 and IL-8 expression in the pathogenesis of MeHg. N-acetyl- l-cysteine suppressed MeHg-induced activation of IL-6 and IL-8 mRNA expression in U937 macrophages, indicating the effectiveness of N-acetyl- l-cysteine as a therapeutic drug in MeHg-induced inflammation. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. Results: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. Conclusions: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis. [ABSTRACT FROM AUTHOR]

Background: Most cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. Across many cancer types, patients with advanced stage disease would benefit from a novel therapy preventing or reversing these changes. To this end, we have investigated the unique WR domain of the transcription factor TWIST1, which has been shown to play a role in driving metastasis and drug resistance.Methods: In this study, we identified evolutionarily well-conserved residues within the TWIST1 WR domain and used alanine substitution to determine their role in WR domain-mediated protein binding. Co-immunoprecipitation was used to assay binding affinity between TWIST1 and the NFκB subunit p65 (RELA). Biological activity of this complex was assayed using a dual luciferase assay system in which firefly luciferase was driven by the interleukin-8 (IL-8) promoter, which is upregulated by the TWIST1-RELA complex. Finally, in order to inhibit the TWIST1-RELA interaction, we created a fusion protein comprising GFP and the WR domain. Cell fractionation and proteasome inhibition experiments were utilized to elucidate the mechanism of action of the GFP-WR fusion.Results: We found that the central residues of the WR domain (W190, R191, E193) were important for TWIST1 binding to RELA, and for increased activation of the IL-8 promoter. We also found that the C-terminal 245 residues of RELA are important for TWIST1 binding and IL-8 promoter activation. Finally, we found the GFP-WR fusion protein antagonized TWIST1-RELA binding and downstream signaling. Co-expression of GFP-WR with TWIST1 and RELA led to proteasomal degradation of TWIST1, which could be inhibited by MG132 treatment.Conclusions: These data provide evidence that mutation or inhibition of the WR domain reduces TWIST1 activity, and may represent a potential therapeutic modality. [ABSTRACT FROM AUTHOR]

MicroRNA is an important regulator of glioblastoma. This study aims at validating microRNA-221 (miR-221) as a biomarker for glioblastoma, and understanding how miR-221 regulates glioblastoma progression. Using clinical samples, miR-221 expression was analyzed by quantitative reverse-transcriptase PCR (qPCR). SHG-44 cells were treated with anti-miR-221 or U87MG-derived exosomes followed by monitoring changes in cell viability, migration and temozolomide (TMZ) resistance. Bioinformatics approach was used to identify targets of miR-221. The interaction between miR-221 and its target, DNM3 gene, was studied with dual-luciferase reporter assay, Spearman's correlation analysis, and western blotting. To verify that RELA regulates miR-221 expression, RELA-expressing vector or shRNA was introduced into SHG-44 cells and its effect on miR-221 expression was monitored. Both tissue-level and exosomal miR-221 expression increased with glioma grades. In SHG-44 cells, downregulating miR-221 expression inhibited cell proliferation, migration, and TMZ resistance, whereas incubation with U87MG-derived exosomes exerted tumor-promoting effects. DNM3 gene is a target of miR-221. RELA induced miR-221 expression. In glioma, elevated miR-221 expression is a biomarker for glioma. DNM3 is a target of miR-221 and RELA regulates miR-221 expression. The RELA/miR-221 axis is a target for glioma diagnosis and therapy. [ABSTRACT FROM AUTHOR]

Multiple independent genomic profiling efforts have recently identified clinically and molecularly distinct subgroups of ependymoma arising from all three anatomic compartments of the central nervous system (supratentorial brain, posterior fossa, and spinal cord). These advances motivated a consensus meeting to discuss: (1) the utility of current histologic grading criteria, (2) the integration of molecular-based stratification schemes in future clinical trials for patients with ependymoma and (3) current therapy in the context of molecular subgroups. Discussion at the meeting generated a series of consensus statements and recommendations from the attendees, which comment on the prognostic evaluation and treatment decisions of patients with intracranial ependymoma (WHO Grade II/III) based on the knowledge of its molecular subgroups. The major consensus among attendees was reached that treatment decisions for ependymoma (outside of clinical trials) should not be based on grading (II vs III). Supratentorial and posterior fossa ependymomas are distinct diseases, although the impact on therapy is still evolving. Molecular subgrouping should be part of all clinical trials henceforth. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) has emerged as the most prevailing oral malignancy worldwide, characterized by cervical solid lymph node metastasis and strong local invasiveness. Overexpression of Transcription Factor AP-2 alpha (TFAP2A) is observed in a significant proportion of OSCC cases. In this study, we aimed to elucidate the function of TFAP2A in the progression of OSCC and the related molecular signaling pathways. The role of RELA was predicted using bioinformatics analysis. The mRNA abundances of RELA, TFAP2A, and β-catenin were assessed by Western blot and quantitative real-timePCR. The relationship between RELA, TFAP2A, and β-catenin and their correlation with clinicopathological characteristics of OSCC was evaluated. The target of RELA and TFAP2A was identified by the chromatin immunoprecipitation as well as luciferase reporter assay. The colony formation assay and MTS assay were performed to determine the proliferative level of OSCC cells. OSCC cell motility was determined by Transwell assay and wound-healing assay. The protein expressions of epithelial-mesenchymal transition-associated factors were evaluated by Western blot. The expressions of RELA and TFAP2A were elevated in OSCC, and their expressions displayed a positive correlation. The expression levels of RELA and TFAP2A were found to be associated with TNM staging and lymphatic metastasis of OSCC patients. RELA upregulation promoted OSCC progression, as manifested by increased levels of proliferation, invasion, and migration of OSCC cells. We also demonstrated that RELA was directly bound to the promoter of TFAP2A transcription, which activated multiple malignant and metastatic phenotypes. Furthermore, TFAP2A activated the Wnt/β-catenin signaling by targeting the promoter regions of β-catenin. The study found that RELA is critical for promoting the progression of OSCC via the RELA-TFAP2A-Wnt/β-catenin signaling pathway. The RELA-TFAP2A-Wnt/β-catenin signaling pathway is a potential target for reducing the aggressiveness of OSCC., (© 2023 Wiley Periodicals LLC.)

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]

Ubiquitination and deubiquitination have emerged as critical regulators in cancer. In the present study, the expression pattern of 50 ubiquitin-specific proteases (USPs) was summarized in breast cancer using a bioinformatics approach, and USP21 was identified as the most altered gene in breast cancer. In particular, expression of USP21 in triple negative breast cancer (TNBC) cell lines was greater compared with other subtypes of breast cancer. Knockdown of USP21 in TNBC cells inhibited cell proliferation, migration and invasion. Microarray profiling of the USP21 knockdown cells revealed significant downregulation of multiple genes associated with the NOD-like receptor signaling pathway. The results of the present study suggest that USP21 has a significant role in TNBC progression, and therefore may represent a novel therapeutic target. [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]

The bacterial RelA-dependent stringent response exerts a strong influence over a wide variety of processes. In this work, the impact of the relA gene mutation in E. coli cells was evaluated by a quantitative proteomics analysis, employing stable-isotope labelling and high resolution mass spectrometry. Chemostat cultures of E. coli W3110 and ∆relA mutant strains were performed at two dilution rates (0.1 and 0.2 h-1) to assess the influence of the relA gene mutation in steady-state protein levels. A total of 121 proteins showed significant alterations in their abundance when comparing the proteome of mutant to wild-type cells. The relA gene mutation induced changes on key cellular processes, including the amino acids and nucleotide biosynthesis, the lipid metabolism, transport activities, transcription and translation processes and responses to stress. Furthermore, some of those changes were more pronounced under specific growth conditions, as the most significant differences in protein ratios were observed at one of the dilution rates. An effect of the relA gene mutation in the acetate overflow was also observed, which confers interesting characteristics to this mutant strain that could be useful in the production of recombinant proteins. Overall, these results provide a valuable insight into the E. coli stringent response under defined steady-state conditions, suggesting that this stress response might influence multiple metabolic processes like the acetate overflow or the catabolite repression.

Streptococcus mutans activates multiple cellular processes in response to the formation of a complex between comX-inducing peptide (XIP) and the ComR transcriptional regulator. Bulk phase and microfluidic experiments previously revealed that ComR-dependent activation of comX is altered by pH and by carbohydrate source. Biofilm formation is a major factor in bacterial survival and virulence in the oral cavity. Here, we sought to determine the response of S. mutans biofilm cells to XIP during different stages of biofilm maturation. Using flow cytometry and confocal microscopy, we showed that exogenous addition of XIP to early biofilms resulted in robust comX activation. However, as the biofilms matured, increasing amounts of XIP were required to activate comX expression. Single-cell analysis demonstrated that the entire population was responding to XIP with activation of comX in early biofilms, but only a sub-population was responding in mature biofilms. The sub-population response of mature biofilms was retained when the cells were dispersed and then treated with XIP. The proportion and intensity of the bi-modal response of mature biofilm cells was altered in mutants lacking the Type II toxins MazF and RelE, or in a strain lacking the (p)ppGpp synthase/hydrolase RelA. Thus, competence signaling is markedly altered in cells growing in mature biofilms, and pathways that control cell death and growth/survival decisions modulate activation of comX expression in these sessile populations.

Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 Å wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding.

The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple ‘one modification, one effect’ system.

Nucleoli are emerging as key sensors of cellular stress and regulators of the downstream consequences on proliferation, metabolism, senescence, and apoptosis. NF-κB signalling is activated in response to a similar plethora of stresses, which leads to modulation of cell growth and death programs. While nucleolar and NF-κB pathways are distinct, it is increasingly apparent that they converge at multiple levels. Exposure of cells to certain insults causes a specific type of nucleolar stress that is characterised by degradation of the PolI complex component, TIF-IA, and increased nucleolar size. Recent studies have shown that this atypical nucleolar stress lies upstream of cytosolic IκB degradation and NF-κB nuclear translocation. Under these stress conditions, the RelA component of NF-κB accumulates within functionally altered nucleoli to trigger a nucleophosmin dependent, apoptotic pathway. In this review, we will discuss these points of crosstalk and their relevance to anti-tumour mechanism of aspirin and small molecule CDK4 inhibitors. We will also briefly the discuss how crosstalk between nucleoli and NF-κB signalling may be more broadly relevant to the regulation of cellular homeostasis and how it may be exploited for therapeutic purpose.

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation risk uncertainties. The transcription factor Nuclear Factor κB (NF-κB) plays a fundamental role in the immune response and in the pathogenesis of many diseases. We have previously shown that heavy ions with a linear energy transfer (LET) of 100–300 keV/µm have a nine times higher potential to activate NF-κB compared to low-LET X-rays. Here, chemical inhibitor studies using human embryonic kidney cells (HEK) showed that the DNA damage sensor Ataxia telangiectasia mutated (ATM) and the proteasome were essential for NF-κB activation in response to X-rays and heavy ions. NF-κB’s role in cellular radiation response was determined by stable knock-down of the NF-κB subunit RelA. Transfection of a RelA short-hairpin RNA plasmid resulted in higher sensitivity towards X-rays, but not towards heavy ions. Reverse Transcriptase real-time quantitative PCR (RT-qPCR) showed that after exposure to X-rays and heavy ions, NF-κB predominantly upregulates genes involved in intercellular communication processes. This process is strictly NF-κB dependent as the response is completely absent in RelA knock-down cells. NF-κB’s role in the cellular radiation response depends on the radiation quality.