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Aim Hypothyroxinemia might be easily ignored, due to attention is typically paid to individuals with elevated thyroid stimulating hormone (TSH). In this study, we aimed to evaluate the association of oxcarbazepine use as adjuvant for treatment of schizophrenia with hypothyroxinemia and central set point of thyroid homeostasis. Methods This retrospective cohort study was conducted in the "Second Affiliated Hospital of Xinxiang Medical University". Inpatients with a diagnosis of schizophrenia admitted between January 2016 and October 2019 with normal thyroid function at admission were included. Oxcarbazepine use was the exposure measure. Newly developed hypothyroxinemia was the primary outcome measure and parameters of thyroid homeostasis central set point as measured by "TSH index" and "thyroid feedback quantile-based index (TFQI)" were the secondary outcome measures. Results A total of 1,207 eligible patients were included. The occurrence of hypothyroxinemia in patients who received oxcarbazepine was higher (35/107, 32.7%) than in those patients who did not (152/1099, 13.8%), with adjusted relative risk of 2.24 and 95% confidence interval of 1.57 and 3.17. Oxcarbazepine use was associated with greater reduction in TSH index (adjusted β -0.33 and 95% confidence interval -0.48, -0.19) and TFQI (adjusted β -0.24 and 95% confidence interval -0.31, -0.16). Conclusion Oxcarbazepine use was independently associated with increased risk of developing hypothyroxinemia, and greater reduction in TSH index and TFQI, suggesting that impaired central set point of thyroid homeostasis might be involved in the mechanism of oxcarbazepine induced hypothyroxinemia.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of early childhood with a poor survival rate, thus there is a requirement for improved treatment strategies. Induced pluripotent stem cells offer the ability to model disease and develop new treatment strategies. JMML is frequently associated with mutations in PTPN11. Children with Noonan syndrome, a development disorder, have an increased incidence of JMML associated with specific germline mutations in PTPN11. We undertook a proteomic assessment of myeloid cells derived from induced pluripotent stem cells obtained from Noonan syndrome patients with PTPN11 mutations, either associated or not associated with an increased incidence of JMML. We report that the proteomic perturbations induced by the leukemia-associated PTPN11 mutations are associated with TP53 and NF-Kκb signaling. We have previously shown that MYC is involved in the differential gene expression observed in Noonan syndrome patients associated with an increased incidence of JMML. Thus, we employed drugs to target these pathways and demonstrate differential effects on clonogenic hematopoietic cells derived from Noonan syndrome patients, who develop JMML and those who do not. Further, we demonstrated these small molecular inhibitors, JQ1 and CBL0137, preferentially extinguish primitive hematopoietic cells from sporadic JMML patients as opposed to cells from healthy individuals.

Hypoxia inducible factor (HIF) is a hypoxia-associated transcription factor that has a protective role against hypoxia-induced damage. Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates HIF targeting it for degradation, therefore, inhibition of the PHD2 enzyme activity acts to upregulate HIF function. This study was to identify novel PHD2 inhibitors.An established fluorescence-based PHD2 activity assay was used for inhibitors screening. Western blot and quantitative real-time PCR was used to detect the protein and mRNA levels respectively. Further animal experiment was carried out.Caffeic acid was screened and identified as a novel PHD2 inhibitor. Caffeic acid treated PC12 and SH-SY5Y neuronal cell lines stabilized endogenous HIF-1α protein levels and consequently increased mRNA levels of its downstream regulated genes VEGF and EPO. Caffeic acid treatment reduced hypoxia-induced cell apoptosis and promoted HIF/BNIP3-mediated mitophagy. Moreover, animal studies indicated that caffeic acid increased the level of HIF-1α protein and mRNA levels of VEGF and EPO in the brain of mice exposed to hypoxia. Conventional brain injury markers including malondialdehyde, lactic acid and lactate dehydrogenase in the caffeic acid treated mice were shown to be reduced to the levels of the control group.This study suggests that caffeic acid inhibits PHD2 enzyme activity which then activates the hypoxia-associated transcription factor HIF leading to a neuroprotective effect against hypoxia.

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.

Background: CD105 is highly expressed on human activated endothelial cells (ECs), is an important component of the TGF-β1 receptor complex and is essential for angiogenesis. CD105 expression is up-regulated in activated ECs and is an important potential marker for cancer prognosis. Materials and Methods: In vitro rat myoblasts transfected with the L-CD105 and S-CD105 transfectants. The transfectants were treated with TGF-β1 for the angiogenesis study. Results: L-CD105 affects cell proliferation in the presence and absence of TGF-β1, and inhibits p-ERK1/2, p-MEK1/2 and p-c-Jun in L-CD105 transfectants compared to controls. The induction of phospho-ERK1/2 following treatment with TGF-β1 remained significantly lower in L-CD105 transfectants compared to controls. Conclusion: L-CD105 inhibits the phosphorylation of ERK1/2, MEK1/2, c-Jun1/2/3, and associated signalling intermediates. CD105 modulates cell growth and TGF-β1 induced cell signalling through ERK-c-Jun expression.

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III–mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box–binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III–directed transcription and shed light on how Sp1 regulates cancer cell proliferation.

© 2018, United States & Canadian Academy of Pathology. HIV-associated neurocognitive disorder in HIV patients substantially reduces their quality of life. We previously showed that the HIV matrix protein, p17 could stimulate lymph-angiogenesis in vitro potentially contributing to lymphoma tumour growth and in addition is associated with vascular activation in neuro-degenerating brain tissue; here, therefore, we have investigated the detailed molecular mechanisms of this action. We performed in vitro cell culture, angiogenesis experiments, phospho-protein microarrays and Western blotting to identify cellular signalling induced by p17 within human brain endothelial cells (HbMEC), and inhibitor studies to block p17-induced vascular growth. We also characterised the effects of hippocampal CA1 injection of p17 on epidermal growth factor receptor-1 (EGFR1) expression linked to our murine model of dementia. p17 strongly induced angiogenesis of HbMEC (migration, tube formation and spheroid growth). p17 concomitantly increased phosphorylation of EGFR1 as well as down-stream intermediates ERK1/2, FAK, PLC-γ and PKC-β whilst an inhibitor peptide of EGFR, blocked cell signalling and angiogenesis. Finally, Mice that showed reduced cognitive function and behavioural deficiencies after p17 injection, demonstrated that p17 localised in cortical microvessels and also neurones many of which stained positive for p-EGFR1 by histology/IHC. This work provides strong support that p17 may be involved in initiating and/or perpetuating vascular tissue pathophysiology associated with comorbidity in HIV patients.

© 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.

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.

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.

CD105 (endoglin) is an important component of the transforming growth factor-beta (TGF-beta) receptor complex and is highly expressed in endothelial cells in tissues undergoing angiogenesis such as healing wounds, infarcts and in a wide range of tumours. In an attempt to understand the molecular mechanism by which CD105 exerts its effects on angiogenesis by modulating TGF-beta1 signalling, in this preliminary communication, CD105 transfected rat myoblasts were utilized as an in vitro model. Overexpression of CD105 in these transfectants antagonised TGF-beta1-mediated inhibition of cell proliferation and reduced TGF-beta1-mediated p3TP-Lux (PAI-1 promoter) luciferase activity. It also reduced (CAGA)12-Luc luciferase activity in response to TGF-beta1. The CAGA sequence is specific for Smad3/4 binding, implying that CD105 is involved in inhibition of TGF-beta1/Smad3 signalling. Furthermore, CD105 overexpression reduced serine phosphorylation of Smad3 and inhibited subsequent nuclear translocation of Smad3. CD105 resulted in high phosphorylation of JNK1, which is able to activate c-Jun. c-Jun is known to inhibit Smad3 transcriptional activity on CAGA sites, suggesting that CD105 may also inhibit Smad3 signalling through JNK1.

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.

The vascular endothelium participates in angiogenesis, inflammation and the immune response, which are modulated by vasoactive cytokines such as tumour necrosis factor-alpha (TNF alpha) and transforming growth factor-beta 1 (TGF beta 1). CD105 is a component of the TGF beta receptor complex and is abundantly expressed in activated/injured endothelium where it is implicated in multiple cellular processes. Up-regulation of CD105 in synovial cells of rheumatoid arthritis and psoriatic lesions implies a possible role in the pathogenesis of such inflammatory disorders. The pro-inflammatory cytokine, TNF alpha, and anti-inflammatory cytokine, TGF beta 1, regulate multiple cellular processes such as proliferation, differentiation and apoptosis. Our hypothesis is that CD105 gene expression in endothelial cells is regulated by the multifunctional cytokines TNF alpha and TGF beta 1. By using human dermal microvascular endothelial cells the present study has shown that long-term treatment with TNF alpha (0.1-5 ng/ml) elicited a concentration- and time-dependent significant suppression (over 50% reduction) in CD105 protein levels. The observations that no significant alterations in the CD105 mRNA levels or in the CD105 promoter activity were found and that the potent inhibitor of NF kappa B, PDTC, did not affect the TNF alpha action suggest that CD105 down-regulation by TNF alpha is not at the transcriptional level. In contrast to TNF alpha, TGF beta 1 significantly elevated CD105 protein and mRNA expression (approximately 2-fold increase) through activation of its promoter activity. From these data we conclude that TNF alpha and TGF beta 1 exert opposing effects on CD105 expression in human vascular endothelial cells and that CD105 is enmeshed in the network of signal pathways modulating multiple cellular functions.

To the Editor: The recent paper by Zhu et al1 in this journal provides an insight into the possible mechanism of hypoxia-induced upregulation of CD105 (endoglin). The following 3 points are relevant to their paper. Lately there has been a major debate regarding the relevance of cell culture/animal models for human stroke in general and for angiogenesis in particular. Some authors maintain that the failure of current therapies for stroke in humans based on the use of animal models is possibly owing to the fact that the pathobiology of human stroke is not mirrored by experimentally-induced stroke in animal models. If true, it would limit the value findings of Zhu et al,1 which are based on the use of murine endothelial cell culture and an in vivo mouse model of focal cerebral ischemia. However, there is strong evidence to discount this possibility. First, CD105 has been found …