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Background and Purpose. There are increasing evidences that show that the prognosis of patients with acute ischemic stroke (AIS) is closely related to the inflammatory response. In the inflammation caused by AIS, plasma C-reactive protein (CRP) will increase and is associated with prognosis in these patients; few studies have looked at the relationship between CRP and large artery atherosclerosis- (LAA-) type AIS. We aim to investigate the role of CRP in predicting the functional outcome of LAA-type AIS patients. Methods. We prospectively included 200 patients with LAA-type AIS and tested their CRP levels on admission. We followed these patients consecutively. The primary outcome was an adverse event, defined as a modified Rankin Scale score of 2-6 at months 3, 6, and 12 after discharge. A logistic regression model was used to analyze the relationship between CRP and the functional outcome of LAA stroke. Results. We divided 200 patients into 3 groups evenly based on CRP level. After adjustment for gender, age, smoking history, drinking history, history of hyperlipidemia, history of diabetes, lipid levels, and blood glucose levels, logistic regression showed that the incidence of LAA-type AIS poor outcome was positively associated with CRP level at admission, whether it was 3 months, 6 months, or 12 months after discharge, respectively (OR: 2.574, 95% CI: 1.213-5.463; OR: 2.806, 95% CI: 1.298-6.065; OR: 2.492, 95% CI: 1.167-5.321. In the highest tertile vs. the lowest tertile as a reference), and both were statistically different. Conclusions. High CRP level predicts poor functional outcome in LAA-type AIS patients, which provides a strong basis for clinicians to make treatment decisions for these patients.

The sustained increase in atmospheric [CO2] over the past two centuries has brought a series of global challenges in plant-environment interactions. However, genetic mechanisms in botanical adaption and feedback to environmental alteration remain elusive. Here we collected and analysed transcriptome sequencing data from leaf tissues of maize Zea mays conditioned under high [CO2] stress for 0, 7 or 14 d. A total of 1390 genes, either up- or down-regulated, differed significantly in these conditions. Gene Ontology (GO) enrichment terms and KEGG metabolism pathways included protein phosphorylation, protein ubiquitination, oxidation-reduction, plant organ development, cellular response to endogenous response and MAPK signaling. We also identified three densely connected gene clusters: TCP/PYE, WRKYs and MYC/JAZ from significant DEGs. In conclusion, we provided a genetic database for biologists in botany, agronomy and ecology to further investigate the molecular machinery by which C4-plants respond to elevated atmospheric [CO2] and accompanying global climate change.

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.

© 2018 Caprio, Badimon, Di Napoli, Fang, Ferris, Guo, Iemma, Liu, Zeinolabediny and Slevin. Circulating C-reactive protein (CRP) is a key acute-phase protein and one of the main clinical biomarkers for inflammation and infection. CRP is an important upstream mediator of inflammation and is associated with the onset of a number of important disease states including cardiovascular disease and neurodegenerative disorders such as Alzheimer's disease. This pentraxin exerts pro-inflammatory properties via dissociation of the pentamer (pCRP) to a monomeric form (mCRP). This dissociation is induced by binding of pCRP to cell surface phosphocholine residues exposed by the action of phospholipase A 2 (PLA 2 ). Given the association of CRP with the onset of a range of serious disease states this CRP dissociation process is a tempting drug target for the development of novel small-molecule therapeutics. This review will discuss potential targets for chemotherapeutic intervention elucidated during studies of CRP-mediated inflammation and provide an up-to-date summary of the development of small molecules, not only targeted directly at inhibiting conversion of pCRP to mCRP, but also those developed for activity against PLA 2 , given the key role of this enzyme in the activation of CRP.

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock “gated” pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic ClockΔ19 mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.

Transcription factor activity is often controlled through the dynamic use of post-translational modifications. Two such modifications are acetylation and sumoylation, which both occur on lysine residues, providing the opportunity for cross-talk at the molecular level. Here, we focussed on the ETS-domain transcription factor PEA3 and studied the potential interplay between these two modifications. We demonstrate that PEA3 is acetylated in a p300-dependent manner. ERK MAPK pathway signalling potentiates acetylation of PEA3, and enhances its trans-activation capacity. However, the major acetylation and sumoylation events take place on the same sites in PEA3 making simultaneous modification impossible. Indeed, manipulation of either the sumoylation or acetylation pathways causes reciprocal changes in PEA3 acetylation and sumoylation respectively. However, despite the mutually exclusive nature of these modifications, both contribute to the trans-activation capacity of PEA3, implying that a dynamic series of modification events occurs during the activation process.

Many transcription factors are controlled through SUMO modification, and in the majority of cases this modification results in enhancements in their repressive properties. In some instances, SUMO modification and its associated repressive activities can be reversed by the action of intracellular signaling pathways, leading to enhanced transcriptional capacities of transcription factors. Here we have investigated sumoylation of the ETS domain transcription factor PEA3 and its interplay with the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling pathway. PEA3 is modified by SUMO in vitro and in vivo on multiple sites in its N-terminal region. Activation of the ERK MAP kinase pathway promotes sumoylation of PEA3. Importantly, sumoylation of PEA3 is required for maximal activation of target gene promoters, including MMP-1 and COX-2. Molecularly, sumoylation is selectively required for synergistic activation of target gene expression with the coactivator CBP. Moreover, sumoylation of PEA3 is required for ubiquitination of PEA3 and promotes its degradation, suggesting that SUMO-mediated recycling of PEA3 plays a role in PEA3-mediated promoter activation. Thus, in contrast to the majority of other transcription factors studied, sumoylation of PEA3 plays a positive role in PEA3-mediated transcriptional activation and the ERK MAP kinase pathway cooperates with rather than antagonizes this process.

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption. © 2014 The Author(s) 2014.

Protein SUMOylation represents an important regulatory event that changes the activities of numerous proteins. Recent evidence demonstrates that polySUMO chains can act as a trigger to direct the ubiquitin ligase RNF4 to substrates to cause their turnover through the ubiquitin pathway. RNF4 uses multiple SUMO interaction motifs (SIMs) to bind to these chains. However, in addition to polySUMO chains, a multimeric binding surface created by the simultaneous SUMOylation of multiple residues on a protein or complex could also provide a platform for the recruitment of multi-SIM proteins like RNF4. Here we demonstrate that multiSUMOylated ETV4 can bind to RNF4 and that a unique combination of SIMs is required for RNF4 to interact with this multiSUMOylated platform. Thus RNF4 can bind to proteins that are either polySUMOylated through a single site or multiSUMOylated on several sites and raises the possibility that such multiSIM-multiSUMO interactions might be more widespread.

The progression of breast cancer depends on the establishment of a neovasculature, by a process called angiogenesis. Angiogenesis is an invasive cellular event that requires the co-ordination of numerous molecules including growth factors and their receptors, extracellular proteins, adhesion molecules, and proteolytic enzymes. TGFbeta has emerged to be a major modulator of angiogenesis by regulating endothelial cell proliferation, migration, extracellular matrix (ECM) metabolism, and the expression of adhesion molecules. It is a potent growth inhibitor of normal mammary epithelial cells and a number of breast cancer cell lines. It seems that TGFbeta exerts pleiotropic effects in the oncogenesis of breast cancers in a contextual manner, i.e., it suppresses tumourigenesis at an early stage by direct inhibition of angiogenesis and tumour cell growth. However, over-production of TGFbeta by an advanced tumour may accelerate disease progression through indirect stimulation of angiogenesis and immune suppression. The cell membrane antigen CD105 (endoglin) binds TGFbeta1 and TGFbeta3 and is preferentially expressed in angiogenic vascular endothelial cells. The reduction of CD105 levels in HUVEC leads to in vitro angiogenesis inhibition and massive cell mortality in the presence of TGFbeta1. CD105 null mice die in utero with impaired vasculature, indicating the pivotal role of CD105 in vascular development. The administration of an immunotoxin-conjugate, mab to CD105, induces long-term and complete regression of breast cancer growth in SCID mice. Therefore, CD105 is a promising vascular target for antiangiogenic therapy.

Background:Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells.Methods:Immunohistochemical patterns of PEA3 expression and active ERK signalling were analysed and mRNA expression levels of PEA3, ER81, MMP-1 and MMP-7 were determined in gastric adenocarcinoma samples.Results:Here, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in gastric adenocarcinomas. PEA3 is upregulated at the protein level in gastric adenocarcinomas and both PEA3/ETV4 and ER81/ETV1 are upregulated at the mRNA level in gastric adenocarcinoma tissues. This increased expression correlates with the expression of a target gene associated with metastasis, MMP-1. Enhanced ERK signalling is also more prevalent in late-stage gastric adenocarcinomas, and the co-association of ERK signalling and PEA3 expression also occurs in late-stage gastric adenocarcinomas. Furthermore, the co-association of ERK signalling and PEA3 expression correlates with decreased survival rates.Conclusions:This study shows that members of the PEA3 subfamily of transcription factors are upregulated in gastric adenocarcinomas and that the simultaneous upregulation of PEA3 expression and ERK pathway signalling is indicative of late-stage disease and a poor survival prognosis. © 2011 Cancer Research UK All rights reserved.

Background Many members of the ETS-domain transcription factor family are important drivers of tumourigenesis. In this context, their activation by Ras-ERK pathway signaling is particularly relevant to the tumourigenic properties of many ETS-domain transcription factors. The PEA3 subfamily of ETS-domain transcription factors have been implicated in tumour metastasis in several different cancers. Results Here, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in oesophageal adenocarcinomas and determined their role in oesophageal adenocarcinoma cell function. PEA3 plays an important role in controlling both the proliferation and invasive properties of OE33 oesophageal adenocarcinoma cells. A key target gene is MMP-1. The ERK MAP kinase pathway activates PEA3 subfamily members and also plays a role in these PEA3 controlled events, establishing the ERK-PEA3-MMP-1 axis as important in OE33 cells. PEA3 subfamily members are upregulated in human adenocarcinomas and expression correlates with MMP-1 expression and late stage metastatic disease. Enhanced ERK signaling is also more prevalent in late stage oesophageal adenocarcinomas. Conclusions This study shows that the ERK-PEA3-MMP-1 axis is upregulated in oesophageal adenocarcinoma cells and is a potentially important driver of the metastatic progression of oesophageal adenocarcinomas.

7 p.-5 fig., CD105 is a receptor for transforming growth factor-beta but it is also considered to be involved in cellular functions such as cell adhesion and migration. Using CD105 transfected rat myoblasts, we have investigated the role of CD105 in cell adhesion, spreading, growth and migration. CD105 transfected myoblasts expressed abundant CD105, which was preferentially located within focal adhesion sites. These cells took on a bipolar morphology whereas mock cells remained polygonal or rounded, and when wounded, CD105 expressing cells realigned their long axis prior to migrating and migrated as a cohort of cells. CD105 expression promoted cellular attachment, spreading, survival and growth in serum-free conditions and each of these parameters could be inhibited by a RGD-containing peptide but not a RAD-containing peptide. Mock-transfected cells could not attach, spread or grow under these conditions. Attachment, spreading and growth in CD105 expressing cells could be promoted by the addition of a monoclonal antibody against CD105. Expression of CD105 resulted in the phosphorylation of JNK1 but had no effect on beta1 integrin expression. From this preliminary study, we conclude that in addition to acting as a transforming growth factor-beta receptor, CD105 has an important role in cell adhesion, migration and survival.

Introduction The PEA3 sub family consists of 3 similar transcription factors, PEA3, ER81 and ERM. In gastric cancer cells PEA3 is activated by H pylori and it has been shown to regulate MMP-7 and invasion. PEA3 mRNA expression correlates with an adverse prognosis in gastric adenocarcinomas. We have previously shown that PEA3 and/or ER81 mRNA correlates with adverse the indicator, MMP-1 and MMP-7. Very high PEA3 protein expression in isolation is associated with tumour metastases. However tumours with PEA3 mRNA and lower protein expression did not correlate with prognostic markers. PEA3 is regulated by ERK MAP kinase signalling in oesophageal adenocarcinomas and we investigated if this pathway plays an important role in gastric adenocarcinomas. Methods Patients with gastric adenocarcinoma (37) and controls (11) were selected. Tissue microarrays were constructed for immunohistochemical studies of PEA3 and pERK. The t test and chi square test were used for statistical analysis. A p value Results In patients with gastric adenocarcinoma, active ERK signalling is present in 60%. From these, ERK signalling is associated with an advanced tumour stage (AJCC stage 1 67%, stage 2: 33%, stage 3: 21% stage 4: 80% p=0.04). Active signalling is present in stage 1 and 2 tumours (55%). Survival is significantly reduced in patients with active ERK signalling (41 vs 34 months P=0.05) on univariate analysis. Adenocarcinomas with PEA3 and pERK co-occurrence frequently have distant metastases 60% (T and N stage vs M stage p=0.048). Survival is worse in patients with combined pERK and PEA3 presence compared to PEA3 or pERK presence in isolation (12 vs 30 vs 63 months p=0.071). Conclusion Our results indicate that ERK MAP kinase signalling is an important marker of an advanced tumour stage and prognosis in gastric adenocarcinoma. The signalling pathway is frequently active in early stage (AJCC 1–3) tumours. Previous studies have shown poor results with MEK inhibition treatment in Pancreatic, Colon, Lung and Breast cancers. This may be due to the selection of heterogeneous tumours with an advanced disease stage or tumours with activation of alternative signalling pathways. Our results suggest consideration of trialling MEK inhibition treatment in patients with earlier stage disease in gastric adenocarcinoma. The more adverse outcome in patients with the combination of ERK MAP signalling and PEA3 indicates that a combined approach with inhibition of PEA3, could be considered in selected patients.