Pellino3 targets RIP1 and regulates the pro-apoptotic effects of TNF-∝

Tumour necrosis factor-a (TNF) can activate NF-?B to induce pro-inflammatory genes but can also stimulate the caspase cascade to promote apoptosis. Here we show that deficiency of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis without inhibiting the NF-?B pathway. Suppressed expression of Pellino3 leads to enhanced formation of the death-induced signalling complex, complex II, in response to TNF. We show that Pellino3 targets RIP1, in a TNF-dependent manner, to inhibit TNF-induced complex II formation and caspase 8-mediated cleavage of RIP1 in response to TNF/cycloheximide co-stimulation. Pellino3-deficient mice also show increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and an important determining factor in dictating whether TNF induces cell survival or death.

Distinct poor prognostic subgroups of acute myeloid leukaemia, FLT3-ITD and P-glycoprotein-positive, have contrasting levels of FOXO1

Regulation of ABCB1 (P-glycoprotein/Pgp) in AML was investigated. In a historical cohort with Pgp and transcriptional regulator expression profiling data available (n=141), FOXO1 correlated with Pgp protein expression. This was confirmed in an independent cohort (n=204). Down-regulation (siRNA) or hyperactivation (nicotinamide) of FOXO1 led to corresponding changes in Pgp. Low FOXO1 expression correlated with FLT3-ITDs (p

The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia

Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1–CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3 (rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05). Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia cenocepacia

Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic resistant bacterium Burkholderia cenocepacia protects less resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sub-lethal concentrations of PmB and other bactericidal antibiotics induce reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS such as superoxide ion and hydrogen peroxide was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sub-lethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation, and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study exposes BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance, and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

Investigation of the mechanism of repression of rRNA synthesis by an anticancer drug

Ribosome biogenesis is a fundamental cellular process which is tightly regulated in normal cells. A number of tumour suppressors and oncogenes could affect the production of ribosomes at different levels and an upregulation could lead to increased protein biosynthesis which is one of the characteristic features of all cancer cells. Ribosome biogenesis is a very complex process which requires coordinated transcription by all three nucleolar polymerases and the first event in this process is synthesis of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I). Importantly, recent data has pictured rRNA transcription as a key regulator of whole ribosome biogenesis and therefore makes it a valid and very attractive target for anticancer therapy, as well as a perspective biomarker. However, at the moment there is only one known specific inhibitor of Pol I transcription (at stage one of clinical trials) and this makes it very difficult for the development of drugs which would target rRNA transcription and consequently ribosome biogenesis. We have recently discovered that antitumor alkaloid ellipticine (isolated in 1959 from the plant species Ochrosia) is a potent inhibitor of Pol I transcription (both in vitro and in vivo). Ellipticine and its derivatives are known as efficient topoisomerase II inhibitors and inhibitors of some kinases, however we have shown that these inhibitory activities and the ability of ellipticine to repress Pol I activity are unrelated. Moreover, our preliminary data suggests that ellipticine specifically targets Pol I transcription and it has no effect on transcription by Pol II and Pol III at the same time scale. The possible mechanisms of inhibition of Pol I transcription by ellipticines will be discussed.

qPCR utilization for the study of RNA Polymerase I transcription

Ribosome biogenesis is a fundamental cellular process tightly linked to cell growth and proliferation, which requires the coordinated transcription of all three nuclear polymerases. Synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I) has been suggested as a key regulator of ribosome biogenesis, and there is a strong link between transcription of ribosomal RNAs and cellular proliferation. This makes Pol I transcription a valid and attractive target for anticancer therapy. At the moment however there are only a small number of compounds that act as specific inhibitors of Pol I transcription and this makes it very difficult for the development of drugs which would target rRNA transcription and consequently ribosome biogenesis. Therefore, to aid in the development of new inhibitors of Pol I, high-throughput methods to monitor and detect changes in Pol I activity need to be developed. This current study aimed to address the question of whether or not quantitative PCR (qPCR) could be used to detect changes in rRNA production in cells under different conditions that repress Pol I activity i.e. serum starvation and drug treatment. Our results have shown that using primers and a hydrolysis probe designed for the 5’ETS region of the pre-rRNA molecule, rRNA levels in both treated and untreated cells could be determined by using qPCR.
Amplification resulted in formation of a single product and S1 nuclease protection assay confirmed the down-regulation of Pol I transcription. Following serum-starvation and drug treatment there was a dramatic reduction in the amount of 5’ETS transcript quantitated by both Sybr Green chemistry and the use of a fluorescently labelled hydrolysis probe. The optimization of the qPCR strategy will be discussed.

STAT3 activation in circulating immune cells is related to neovascular age-related macular degeneration

Purpose:The Signal Transducer and Activator of Transcription 3 (STAT3) pathway is known to play an important role in inflammation and angiogenesis. STAT3 can be activated by IL-6 family cytokines through the receptor IL-6R/gp130. Increased IL-6 has been detected in the plasma and vitreous in neovascular age-related macular degeneration (nAMD) patients. The aim of this study was to investigate the role of the STAT3 pathway in the pathogenesis of nAMD.

Methods:Blood cells from nAMD patients (n = 11) and age-, gender-matched healthy controls (n = 13) were stimulated with IL-6 for 20 minutes. The expression of the activated form of STAT3 (p-STAT3) was examined by flow cytometry. The mRNA levels of gp130, IL-6R and the suppressor of cytokine signalling 3 (SOCS3, a negative regulator of p-STAT3) were evaluated by real-time RT-PCR. Laser-induced choroidal neovascularisation (CNV) was performed in WT C57BL/6J mice as well as in the myeloid cell specific SOCS3 deficiency mice i.e., the LysMCre-SOCS3fl/fl mice. STAT3 activation in CNV lesions was examined by western blot. The size of CNV at different times after laser treatment was measured by confocal microscopy of RPE/choroidal flatmounts.

Results:The expression of p-STAT3 in CD11b+ monocytes was significantly increased in nAMD patients compared to healthy controls, although mRNA expression of gp130, IL-6R and SOCS3 did not differ between patients and controls. The expression of p-STAT3 in the retinal and RPE/choroidal tissues was increased at 1 and 3 days after laser treatment. The administration of a STAT3 inhibitor LLL12 significantly suppressed CNV. CD11b+ monocytes from LysMCre-SOCS3fl/fl mice expressed higher levels of p-STAT3 compared to the cells from WT mice. Laser induced CNV developed earlier and were larger in LysMCre-SOCS3fl/fl mice compared to WT C57BL/6J mice.

Conclusions:Our results suggest that STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD, and targeting the STAT3 pathway may have therapeutic potential in nAMD.

Molecular basis of non-mutational derepression of ramA in Klebsiella pneumoniae

Objectives: The ram locus, consisting of the romA–ramA genes, is repressed by the tetracycline-type regulator RamR, where regulation is abolished due to loss-of-function mutations within the protein or ligand interactions. The aim of this study was to determine whether the phenothiazines (chlorpromazine and thioridazine) directly interact with RamR to derepress ramA expression.

Methods: Quantitative real-time PCR analyses were performed to determine expression levels of the romA–ramA genes after exposure to the phenothiazines. Electrophoretic mobility shift assays (EMSAs) and in vitro transcription experiments were performed to show direct binding to and repression by RamR. Direct binding of the RamR protein to the phenothiazines was measured by fluorescence spectroscopy experiments and molecular docking models were generated using the RamR crystal structure.

Results: Exposure to either chlorpromazine or thioridazine resulted in the up-regulation of the romA–ramA genes. EMSAs and in vitro transcription experiments demonstrated that both agents reduce/abolish binding and enhance transcription of the target PI promoter upstream of the ramR–romA genes in Klebsiella pneumoniae compared with RamR alone. Fluorescence spectroscopy measurements demonstrated that RamR directly binds both chlorpromazine and thioridazine with micromolar affinity. Molecular docking analyses using the RamR crystal structure demonstrated that the phenothiazines interact with RamR protein through contacts described for other ligands, in addition to forming unique strong polar interactions at positions D152 and K63.

Conclusions: These data demonstrate that phenothiazines can modulate loci linked to the microbe–drug response where RamR is an intracellular target for the phenothiazines, thus resulting in a transient non-mutational derepression of ramA concentrations.

Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.

Impact of vitamin D3 on cutaneous immunity and antimicrobial peptide expression

Antimicrobial peptides (AMPs) are effectors of cutaneous innate immunity and protect primarily against microbial infections. An array of AMPs can be found in and on the skin. Those include peptides that were first discovered for their antimicrobial properties but also proteins with antimicrobial activity first characterized for their activity as chemokines, enzymes, enzyme inhibitors and neuropeptides. Cathelicidins were among the first families of AMPs discovered in skin. They are now known to exert a dual role in innate immune defense: they have direct antimicrobial activity and will also initiate a host cellular response resulting in cytokine release, inflammation and angiogenesis. Altered cathelicidin expression and function was observed in several common inflammatory skin diseases such as atopic dermatitis, rosacea and psoriasis. Until recently the molecular mechanisms underlying cathelicidin regulation were not known. Lately, vitamin D3 was identified as the major regulator of cathelicidin expression and entered the spotlight as an immune modulator with impact on both, innate and adaptive immunity. Therapies targeting vitamin D3 signalling may provide novel approaches for the treatment of infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions through AMP regulation.

Control of cutaneous antimicrobial peptides by vitamin D3

Constant exposure to a wide variety of microbial pathogens represents a major challenge for our skin. Antimicrobial peptides (AMPs) are mediators of cutaneous innate immunity and protect primarily against microbial infections. Cathelicidins were among the first AMPs identified in human skin and recent evidence suggests that they exert a dual role in innate immune defense: At first, due to their antimicrobial activity they kill pathogens directly. In addition, these peptides initiate a potent host response to infection resulting in cytokine release, inflammation and a cellular response. Disturbed cathelicidin expression and function was observed in several common inflammatory skin diseases, such as psoriasis where cathelicidin peptide converts inert self-DNA and self-RNA into an autoimmune stimulus. In atopic dermatitis decreased levels of cathelicidin facilitating microbial superinfections have been discussed. Furthermore, abnormally processed cathelicidin peptides induce inflammation and a vascular response in rosacea. Until recently, the molecular mechanisms underlying cathelicidin regulation were unknown. Recently, the vitamin D3 pathway was identified as the major regulator of cathelicidin expression. Consequently, vitamin D3 entered the spotlight as an immune modulator with impact on both innate and adaptive immunity. Therapies targeting vitamin D3 signaling may provide new approaches for infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions.

FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1

NAD(P)H quinone oxidoreductase 1 is involved in antioxidant defence and protection from cancer, stabilizing the apoptosis regulator p53 towards degradation. Here, we studied the enzymological, biochemical and biophysical properties of two cancer-associated variants (p.R139W and p.P187S). Both variants (especially p.187S) have lower thermal stability and greater susceptibility to proteolysis compared to the wild-type. p.P187S also has reduced activity due to a lower binding affinity for the FAD cofactor as assessed by activity measurements and direct titrations. Native gel electrophoresis and dynamic light scattering also suggest that p.P187S has a higher tendency to populate unfolded states under native conditions. Detailed thermal stability studies showed that all variants irreversibly denature causing dimer dissociation, while addition of FAD restores the stability of the polymorphic forms to wild-type levels. The kinetic destabilization induced by polymorphisms as well as the kinetic protection exerted by FAD was confirmed by measuring denaturation kinetics at temperatures close to physiological. Our data suggest that the main molecular mechanisms associated with these cancer-related variants are their low binding affinity for FAD and/or kinetic instability. Thus, pharmacological chaperones may be useful in the treatment of patients bearing these polymorphisms.

Factors influencing European consumer uptake of personalised nutrition. Results of a qualitative analysis.

The aim of this research was to explore consumer perceptions of personalised nutrition and to compare these across three different levels of "medicalization": lifestyle assessment (no blood sampling); phenotypic assessment (blood sampling); genomic assessment (blood and buccal sampling). The protocol was developed from two pilot focus groups conducted in the UK. Two focus groups (one comprising only "older" individuals between 30 and 60 years old, the other of adults 18-65 yrs of age) were run in the UK, Spain, the Netherlands, Poland, Portugal, Ireland, Greece and Germany (N = 16). The analysis (guided using grounded theory) suggested that personalised nutrition was perceived in terms of benefit to health and fitness and that convenience was an important driver of uptake. Negative attitudes were associated with internet delivery but not with personalised nutrition per se. Barriers to uptake were linked to broader technological issues associated with data protection, trust in regulator and service providers. Services that required a fee were expected to be of better quality and more secure. An efficacious, transparent and trustworthy regulatory framework for personalised nutrition is required to alleviate consumer concern. In addition, developing trust in service providers is important if such services to be successful. (C) 2013 Elsevier Ltd. All rights reserved.

Up-regulated MSI2 is associated with more aggressive chronic myeloid leukemia

A better understanding of events triggering chronic myeloid leukemia progression are critical to optimised clinical management of chronic myeloid leukemia (CML). We sought to validate that increased Musashi 2 (MSI2), a post transcription regulator, expression is associated with progression and prognosis. Screening of 152 CML patients showed MSI2 was significantly decreased among CML patients in CP at diagnosis (p<0.0001), but found no significant difference between the normal control group and treated CML patients in CP. Moreover it was significantly increased (p<0.0001) in advance disease (AD) CML patients. Furthermore, our human hematopoietic cell line data imply MSI2 and BCR-ABL1 mRNA expression correlate. However, these data cast a doubt on earlier reports that MSI2 effects HES1 expression via NUMB-NOTCH signaling.

MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalised medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation towards ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.

Methods and Results: In this study we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in VEGF transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation towards SMCs. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.

Conclusions: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses.