BRCA1 is a key regulator of breast differentiation through activation of Notch signalling with implications for anti-endocrine treatment of breast cancers

Here, we show for the first time, that the familial breast/ovarian cancer susceptibility gene BRCA1 activates the Notch pathway in breast cells by transcriptional upregulation of Notch ligands and receptors in both normal and cancer cells. We demonstrate through chromatin immunoprecipitation assays that BRCA1 is localized to a conserved intronic enhancer region within the Notch ligand Jagged-1 (JAG1) gene, an event requiring ΔNp63. We propose that this BRCA1/ΔNp63-mediated induction of JAG1 may be important the regulation of breast stem/precursor cells, as knockdown of all three proteins resulted in increased tumoursphere growth and increased activity of stem cell markers such as Aldehyde Dehydrogenase 1 (ALDH1). Knockdown of Notch1 and JAG1 phenocopied BRCA1 knockdown resulting in the loss of Estrogen Receptor-α (ER-α) expression and other luminal markers. A Notch mimetic peptide could activate an ER-α promoter reporter in a BRCA1-dependent manner, whereas Notch inhibition using a γ-secretase inhibitor reversed this process. We demonstrate that inhibition of Notch signalling resulted in decreased sensitivity to the anti-estrogen drug Tamoxifen but increased expression of markers associated with basal-like breast cancer. Together, these findings suggest that BRCA1 transcriptional upregulation of Notch signalling is a key event in the normal differentiation process in breast tissue.

Bystander Signalling: Exploring Clinical Relevance Through New Approaches and New Models

Classical radiation biology research has centred on nuclear DNA as the main target of radiation-induced damage. Over the past two decades, this has been challenged by a significant amount of scientific evidence clearly showing radiation-induced cell signalling effects to have important roles in mediating overall radiobiological response. These effects, generally termed radiation-induced bystander effects (RIBEs) have challenged the traditional DNA targeted theory in radiation biology and highlighted an important role for cells not directly traversed by radiation. The multiplicity of experimental systems and exposure conditions in which RIBEs have been observed has hindered precise definitions of these effects. However, RIBEs have recently been classified for different relevant human radiation exposure scenarios in an attempt to clarify their role in vivo. Despite significant research efforts in this area, there is little direct evidence for their role in clinically relevant exposure scenarios. In this review, we explore the clinical relevance of RIBEs from classical experimental approaches through to novel models that have been used to further determine their potential implications in the clinic. 

Re-analysis of microarray data reveals insights into altered transcriptional activity of TH17 and TReg signalling in psoriasis

Identifying differential expression of genes in psoriatic and healthy skin by microarray data analysis is a key approach to understand the pathogenesis of psoriasis. Analysis of more than one dataset to identify genes commonly upregulated reduces the likelihood of false positives and narrows down the possible signature genes. Genes controlling the critical balance between T helper 17 and regulatory T cells are of special interest in psoriasis. Our objectives were to identify genes that are consistently upregulated in lesional skin from three published microarray datasets. We carried out a reanalysis of gene expression data extracted from three experiments on samples from psoriatic and nonlesional skin using the same stringency threshold and software and further compared the expression levels of 92 genes related to the T helper 17 and regulatory T cell signaling pathways. We found 73 probe sets representing 57 genes commonly upregulated in lesional skin from all datasets. These included 26 probe sets representing 20 genes that have no previous link to the etiopathogenesis of psoriasis. These genes may represent novel therapeutic targets and surely need more rigorous experimental testing to be validated. Our analysis also identified 12 of 92 genes known to be related to the T helper 17 and regulatory T cell signaling pathways, and these were found to be differentially expressed in the lesional skin samples.

Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

A molecular photoionic AND gate based on fluorescent signalling

MOLECULES that perform logic operations are prerequisites for molecular information processing and computation. We and others have previously reported receptor molecules that can be considered to perform simple logic operations by coupling ionic bonding or more complex molecular-recognition processes with photonic (fluorescence) signals: in these systems, chemical binding (the 'input') results in a change in fluorescence intensity (the 'output') from the receptor. Here we describe a receptor (molecule (1) in Fig. 1) that operates as a logic device with two input channels: the fluorescence signal depends on whether the molecule binds hydrogen ions, sodium ions or both. The input/output characteristics of this molecular device correspond to those of an AND gate.

Orphan receptor IL-17RD tunes IL-17A signalling and is required for neutrophilia

Interleukin-17A, the prototypical member of the interleukin-17 cytokine family, coordinates local tissue inflammation by recruiting neutrophils to sites of infection. Dysregulation of interleukin-17 signalling has been linked to the pathogenesis of inflammatory diseases and autoimmunity. The interleukin-17 receptor family members (A-E) have a broad range of functional effects in immune signalling yet no known role has been described for the remaining orphan receptor, interleukin-17 receptor D, in regulating interleukin-17A-induced signalling pathways. Here we demonstrate that interleukin-17 receptor D can differentially regulate the various pathways employed by interleukin-17A. Neutrophil recruitment, in response to in vivo administration of interleukin-17A, is abolished in interleukin-17 receptor D-deficient mice, correlating with reduced interleukin-17A-induced activation of p38 mitogen-activated protein kinase and expression of the neutrophil chemokine MIP-2. In contrast, interleukin-17 receptor D deficiency results in enhanced interleukin-17A-induced activation of nuclear factor-kappa B and interleukin-6 and keratinocyte chemoattractant expression. Interleukin-17 receptor D disrupts the interaction of Act1 and TRAF6 causing differential regulation of nuclear factor-kappa B and p38 mitogen-activated protein kinase signalling pathways. © 2012 Macmillan Publishers Limited. All rights reserved.

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.

Induction of signalling in non-erythroid cells by pharmacological levels of erythropoietin

Erythropoiesis is maintained by the hormone erythropoietin (Epo) binding to its cognate receptor (EpoR) on erythroid progenitor cells. The Epo-EpoR interaction initiates a signal transduction process that regulates the survival, growth and differentiation of these cells. Originally perceived as highly lineage-restricted, Epo is now recognised to have pleiotropic effects extending beyond the maintenance of red cell mass. Functional interactions between Epo and EpoR have been demonstrated in numerous cells and tissues. EpoR expression on neoplastic cells leads to concern that recombinant human erythropoietin, used to treat anaemia in cancer patients, may augment tumour growth. Here we demonstrate that EPO, at pharmacological concentrations, can activate three major signalling cascades, viz. the Jak2/STAT5, Ras/ERK and PI3K/Akt pathways in non-small cell lung carcinoma (NSCLC) cell lines. EpoR synthesis is normally under the control of GATA-1, but NSCLC cells exhibit decreased GATA-1 levels compared to GATA-2, -3 and -6, suggesting that GATA-1 is not essential for EpoR production. The increased Epo-induced signalling was not associated with a growth advantage for the NSCLC cells.

Role of ion channels and subcellular Ca2+ signalling in arachidonic acid induced dilation of pressurised retinal arterioles

Purpose: To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Methods: Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacological agents inhibiting Ca2+ sparks and oscillations and K+ channels. Subcellular Ca2+ signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Results: AA dilated pressurised retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. AA-induced dilation was associated with an inhibition of subcellular Ca2+ signals. Interventions known to block Ca2+ sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. AA accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous BK currents, but only at positive membrane potentials. Pharmacological inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. AA suppressed L-type Ca2+ currents. Conclusions: These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca2+ signalling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca2+ channel activity. AA actions on K+ currents are inconsistent with a model in which K+ channels contribute to the vasodilator effects of AA.

Selective Targeting of Glucagon-Like Peptide-1 Signalling As a Novel Therapeutic Approach for Cardiovascular Disease in Diabetes

Glucagon-like peptide-1 (GLP-1) is an incretin hormone whose glucose-dependent insulinotropic actions have been harnessed as a novel therapy for glycaemic control in type 2 diabetes. Although it has been known for some time that the GLP-1 receptor is expressed in the cardiovascular system where it mediates important physiological actions, it is only recently that specific cardiovascular effects of GLP-1 in the setting of diabetes have been described. GLP-1 confers indirect benefits in cardiovascular disease (CVD) under both normal and hyperglycaemic conditions via reducing established risk factors, such as hypertension, dyslipidaemia and obesity, which are markedly increased in diabetes. Emerging evidence indicates that GLP-1 also exerts direct effects on specific aspects of diabetic CVD, such as endothelial dysfunction, inflammation, angiogenesis and adverse cardiac remodelling. However, the majority of studies have employed experimental models of diabetic CVD and information on the effects of GLP-1 in the clinical setting are limited although several large-scale trials are ongoing. It is clearly important to gain a detailed knowledge of the cardiovascular actions of GLP-1 in diabetes given the large number of patients currently receiving GLP-1 based therapies. This review will therefore discuss current understanding of the effects of GLP-1 on both cardiovascular risk factors in diabetes and direct actions on the heart and vasculature in this setting, and the evidence implicating specific targeting of GLP-1 as a novel therapy for CVD in diabetes.

Assessing Climate Effects on Railway Earthworks Using MASW

Many parts of the UK’s rail network were constructed in the mid-19th century long before the advent of modern construction standards. Historic levels of low investment, poor maintenance strategies and the deleterious effects of climate change have resulted in critical elements of the rail network being at significant risk of failure. The majority of failures which have occurred over recent years have been triggered by extreme weather events. Advance assessment and remediation of earthworks is, however, significantly less costly than dealing with failures reactively. It is therefore crucial that appropriate approaches for assessment of the stability of earthworks are developed, so that repair work can be better targeted and failures avoided wherever possible. This extended abstract briefly discusses some preliminary results from an ongoing geophysical research project being carried out in order to study the impact of climate or seasonal weather variations on the stability of a century old railway embankment on the Gloucestershire Warwickshire steam railway line in Southern England.