Negative Regulation of EGFR/MAPK Pathway by Pumilio in Drosophila melanogaster

In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. Here, we show that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. We observed that loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)–like sequences in their 3’UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. We show that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, our evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila

Crosstalk between the EGFR and other signalling pathways at the level of the global transcriptional corepressor Groucho/TLE

In this minireview, we briefly revisit the Drosophila Notch and epidermal growth factor receptor pathways, and relate to the relationship between them. We then mainly focus on the involvement of Groucho (Gro)/TLE, a global developmental corepressor, in these pathways. In particular, we discuss Gro/TLE's role at the junction between these two signal transduction cascades

NF-κB/Rel-Mediated Regulation of the Neural Fate in Drosophila

Two distinct roles are described for Dorsal, Dif and Relish, the three NF-κB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-κB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. We propose that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis

Drosophila Lipophorin Receptors Mediate the Uptake of Neutral Lipids in Oocytes and Imaginal Disc Cells by an Endocytosis-Independent Mechanism

Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR–like proteins in this process

Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems

The PDZ Protein Canoe/AF-6 Links Ras-MAPK, Notch and Wingless/Wnt Signaling Pathways by Directly Interacting with Ras, Notch and Dishevelled

Over the past few years, it has become increasingly apparent that signal transduction pathways are not merely linear cascades; they are organized into complex signaling networks that require high levels of regulation to generate precise and unique cell responses. However, the underlying regulatory mechanisms by which signaling pathways cross-communicate remain poorly understood. Here we show that the Ras-binding protein Canoe (Cno)/AF-6, a PDZ protein normally associated with cellular junctions, is a key modulator of Wingless (Wg)/Wnt, Ras-Mitogen Activated Protein Kinase (MAPK) and Notch (N) signaling pathways cross-communication. Our data show a repressive effect of Cno/AF-6 on these three signaling pathways through physical interactions with Ras, N and the cytoplasmic protein Dishevelled (Dsh), a key Wg effector. We propose a model in which Cno, through those interactions, actively coordinates, at the membrane level, Ras-MAPK, N and Wg signaling pathways during progenitor specification

Uptake of the Necrotic Serpin in Drosophila melanogaster via the Lipophorin Receptor-1

The humoral response to fungal and Gram-positive infections is regulated by the serpin-family inhibitor, Necrotic. Following immune-challenge, a proteolytic cascade is activated which signals through the Toll receptor. Toll activation results in a range of antibiotic peptides being synthesised in the fat-body and exported to the haemolymph. As with mammalian serpins, Necrotic turnover in Drosophila is rapid. This serpin is synthesised in the fat-body, but its site of degradation has been unclear. By “freezing” endocytosis with a temperature sensitive Dynamin mutation, we demonstrate that Necrotic is removed from the haemolymph in two groups of giant cells: the garland and pericardial athrocytes. Necrotic uptake responds rapidly to infection, being visibly increased after 30 mins and peaking at 6–8 hours. Co-localisation of anti-Nec with anti-AP50, Rab5, and Rab7 antibodies establishes that the serpin is processed through multi-vesicular bodies and delivered to the lysosome, where it co-localises with the ubiquitin-binding protein, HRS. Nec does not co-localise with Rab11, indicating that the serpin is not re-exported from athrocytes. Instead, mutations which block late endosome/lysosome fusion (dor, hk, and car) cause accumulation of Necrotic-positive endosomes, even in the absence of infection. Knockdown of the 6 Drosophila orthologues of the mammalian LDL receptor family with dsRNA identifies LpR1 as an enhancer of the immune response. Uptake of Necrotic from the haemolymph is blocked by a chromosomal deletion of LpR1. In conclusion, we identify the cells and the receptor molecule responsible for the uptake and degradation of the Necrotic serpin in Drosophila melanogaster. The scavenging of serpin/proteinase complexes may be a critical step in the regulation of proteolytic cascades

Gender Differences in Symptoms of Myocardial Ischaemia

Aims Better understanding of symptoms of myocardial ischaemia is needed to improve timeliness of treatment for acute coronary syndromes (ACS). Although researchers have suggested sex differences exist in ischaemic symptoms, methodological issues prevent conclusions. Using percutaneous coronary intervention (PCI) balloon inflation as a model of myocardial ischaemia, we explored sex differences in reported symptoms of ischaemia. Methods and results Patients having non-emergent PCI, but not haemodynamic instability or left bundle branch block or non-acute coronary occlusion, were prospectively recruited. Pre-procedure, descriptions of pre-existing symptoms were obtained using open-ended questioning. Inflation was maintained for 2 min or until moderate discomfort or clinical instability occurred. During inflation, subjects were exhaustively questioned about their symptoms. Concurrent ECG data were collected. The final sample was 305 [39.7% women; mean age 63.9 (±10.6)]. No sex differences were found in rates of chest or typical ischaemic discomfort, regardless of ischaemic status. Women were significantly more likely to report throat/jaw discomfort [odds ratio: 2.91; 95% confidence interval: 1.58–5.37] even after statistical adjustment for clinical and demographic variables. Conclusion This prospective study with ECG-affirmed ischaemia found no statistically significant differences in women\u27s and men\u27s rates of chest and other typical symptoms during ischaemia, although women were more likely to experience throat and jaw discomfort. Currently both popular press and some patient education materials suggest women experience myocardial ischaemia differently from men. Steps to ensure women and health professionals are alert for the classic symptoms of myocardial ischaemia in women, as well as men, may be warranted

Cardiomyocyte Regulation of Systemic Lipid Metabolism by the Apolipoprotein B-Containing Lipoproteins in Drosophila

We thank our colleagues Linda Thompson and Luke Szweda at OMRF, and Gary Struhl at Columbia University for critical comments on the manuscript. We are grateful to Rolf Bodmer (Sanford-Burnham-Presby Medical Discovery Institute), Joquim Culi (CSIC-UPO), Susan Abmayr (Stowers Institute) and Laurent Perrin (TAGC) for fly stocks and antibodies. We also thank the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and the TRiP at Harvard Medical School for fly stocks. We acknowledged the Imaging Core Facility at OMRF for excellent technical assistance.Author Summary The heart is increasingly recognized to serve an important role in the regulation of whole-body lipid homeostasis; however, the underlying mechanisms remained poorly understood. Here, our study in Drosophila reveals that cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body (insect liver and adipose tissue). We found that apoB-lipoproteins generated by the Drosophila cardiomyocytes serve an equally significant role as their fat body-derived counterparts in maintaining systemic lipid homeostasis on normal food diet. Importantly, on high fat diet (HFD), the cardiomyocyte-derived apoB-lipoproteins are the major determinants of whole-body lipid metabolism, a role which could be attributed to the HFD-induced up-regulation of apoB-lipoprotein biosynthesis genes in the cardiomyocytes and their down-regulation in the fat body. Taken together, our results reveal that apoB-lipoproteins are new players in mediating the heart control of lipid metabolism, and provide first evidence supporting the notion that HFD-induced differential regulation of apoB-lipoprotein biosynthesis genes could alter the input of different tissue-derived apoB-lipoproteins in systemic lipid metabolic control.Yeshttp://www.plosgenetics.org/static/editorial#pee

Integrin affinity modulation and survival signalling.

Integrins are heterodimeric transmembrane proteins that provide a bi-directional link between the cell’s internal biological mechanisms and the extracellular environment. During inside-out signalling, intracellular messages converge on the integrin cytoplasmic domain to induce a conformational change. This is transmitted to the extracellular domain where it results in an alteration in affinity for integrin ligands such as fibronectin and laminin. In this way the cell has developed the ability to modulate the critical functions of adhesion and cell movement. In outside-in signalling, the integrin performs a more complex function than simple adhesion; upon binding to ligand, the integrin extracellular domain undergoes a conformational change which is transmitted to the cytoplasmic domain. This alters the integrin’s cytoplasmic domain affinity for intracellular signalling proteins and results in the activation of intracellular second messenger pathways. In this way, the extracellular milieu is able to influence intracellular signalling including those involved in apoptosis. This thesis demonstrates data which provide original insights into bi-directional integrin signalling: Inside-out signalling: Constitutively active Notch1 increases β3-integrin affinity and abrogates Hras-mediated integrin suppression without increasing expression of β3- integrin. Dominant-Negative Rras blocks Notch-mediated integrin activation and Notch1-mediated reversal of Hras and Raf-mediated integrin suppression and this is independent of erk phosphorylation. Notch1 induces Rras activation. Functional adhesion assays confirm that Notch1IC increases K562 adhesion in a β1-integrin dependent manner and this is abrogated by Dominant-Negative Rras. This data supports a mechanism in which Notch1 increases integrin affinity via activation of Rras. Outside-in signalling: Evidence is presented demonstrating that extracellular matrix proteins, laminin and fibronectin, activate β1-integrins to protect SCLC cells against the apoptotic effects of etoposide and ionizing radiation via PI3Kinase activation. This occurs in two ways: 1) PI3Kinase-dependent β1-integrin signalling resulting in phosphorylation of Bad and reduced caspase-9 cleavage and 2) a β1-integrinmediated over-riding of etoposide and radiotherapy-induced cell cycle S phase delay and G2/M arrest. β1-integrin-mediated outside-in survival signalling was investigated further in the in vivo setting; MatrigelTM, a basement membrane product rich in extracellular matrix proteins, promoted SCLC xenograft survival and growth in a β1-integrin and tyrosine kinase-dependent manner. This data provides novel insights into the critical functions that integrins play in adhesion and survival signalling