DOCSLIB.ORG

The Ephrin-A1/EPHA2 Signaling Axis Regulates Glutamine Metabolism in HER2-Positive Breast Cancer Victoria M

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Ephrin-A1/EPHA2 Signaling Axis Regulates Glutamine Metabolism in HER2-Positive Breast Cancer Victoria M Published OnlineFirst February 1, 2016; DOI: 10.1158/0008-5472.CAN-15-0847 Cancer Molecular and Cellular Pathobiology Research The Ephrin-A1/EPHA2 Signaling Axis Regulates Glutamine Metabolism in HER2-Positive Breast Cancer Victoria M. Youngblood1, Laura C. Kim1, Deanna N. Edwards2, Yoonha Hwang2,3, Pranav R. Santapuram1, Steven M. Stirdivant4, Pengcheng Lu5, Fei Ye5, Dana M. Brantley-Sieders2,6, and Jin Chen1,2,3,6,7 Abstract Dysregulation of receptor tyrosine kinases (RTK) contributes to bolic pathway that generates intermediates for lipogenesis. Phar- cellular transformation and cancer progression by disrupting key macologic inhibition of glutaminase activity reduced tumor metabolic signaling pathways. The EPHA2 RTK is overexpressed growth in both ephrin-A1–depleted and EPHA2-overexpressing þ in aggressive forms of breast cancer, including the HER2 subtype, tumor allografts in vivo. Mechanistically, we show that the and correlates with poor prognosis. However, the role of EPHA2 enhanced proliferation and glutaminolysis in the absence of in tumor metabolism remains unexplored. In this study, we used ephrin-A1 were attributed to increased RhoA-dependent gluta- in vivo and in vitro models of HER2-overexpressing breast cancer to minase activity. EPHA2 depletion or pharmacologic inhibition investigate the mechanisms by which EPHA2 ligand–indepen- of Rho, glutaminase, or fatty acid synthase abrogated the dent signaling promotes tumorigenesis in the absence of its increased lipid content and proliferative effects of ephrin-A1 prototypic ligand, ephrin-A1. We demonstrate that ephrin-A1 knockdown. Together, these findings highlight a novel, unsus- loss leads to upregulated glutamine metabolism and lipid accu- pected connection between the EPHA2/ephrin-A1 signaling axis mulation that enhanced tumor growth. Global metabolic profil- and tumor metabolism, and suggest potential new therapeutic ing of ephrin-A1–null, HER2-overexpressing mammary tumors targets in cancer subtypes exhibiting glutamine dependency. revealed a significant increase in glutaminolysis, a critical meta- Cancer Res; 76(7); 1–12. Ó2016 AACR. Introduction tion occur upon Eph receptor binding to their ligands, known as ephrins, Eph receptors can also be activated by other cell-surface Receptor tyrosine kinases (RTK) are key regulators of signal receptors, such as EGFR and ERBB2 (3, 5). Thus, cumulative transduction pathways that promote cell growth, survival, and evidence in breast cancer supports two modes for EPHA2 signal- motility during malignant progression of solid tumors. Recent ing. In the ligand-dependent mode, EPHA2 can engage in ligand- advances in the analysis of tumor genomes revealed that EPHA2 dependent forward signaling that suppresses tumor cell prolifer- RTK is frequently overexpressed in aggressive human breast can- ation and invasiveness (6, 7). In the second mode, EPHA2 can cers and correlates with poor patient survival and resistance to signal in a ligand-independent manner that promotes tumor therapeutic agents (1–4). EPHA2 belongs to the Eph family of malignancy—a mechanism highly dependent upon phosphory- RTKs, which contain distinct regions for ligand binding, receptor lation of S897 (6). This model, however, has not been directly clustering, and signaling. Though receptor clustering and activa- tested in a transgenic mammary tumor model, and the mecha- nism by which ephrin-A1 exerts its tumor-suppressive role has yet 1Department of Cancer Biology, Vanderbilt University, Nashville, to be fully elucidated. Tennessee. 2Division of Rheumatology and Immunology, Department Dysregulation of RTK signaling can result in aberrant metab- 3 of Medicine, Vanderbilt University, Nashville, Tennessee. Veterans olism through disruption of critical metabolic pathways. Recent Affairs Medical Center,Tennessee Valley Healthcare System, Nashville, fi Tennessee. 4Metabolon Inc., Durham, North Carolina. 5Department of studies in tumor metabolism have led to signi cant advances in Biostatistics, Vanderbilt University, Nashville, Tennessee. 6Vanderbilt- our understanding of how tumor cells utilize metabolic switches Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee. to produce intermediates for cell growth and division (8, 9). One 7Department of Cell and Developmental Biology, Vanderbilt Univer- sity, Nashville, Tennessee. hallmark for proliferating tumor cells is high levels of glucose influx and subsequent aerobic glycolysis with lactate production, Note: Supplementary data for this article are available at Cancer Research even in the presence of adequate oxygen (termed the "Warburg Online (http://cancerres.aacrjournals.org/). effect"; ref. 10). Although most cancers depend on a high rate of L.C. Kim and D.N. Edwards contributed equally to this article. aerobic glycolysis, some cancer cells also display addiction Corresponding Authors: Jin Chen, Vanderbilt University School of Medicine, to glutamine (11–13). Glutamine, an abundant amino acid, T-3207E, 1161 21st Ave South, Nashville, TN 37232. Phone: 615-343-3819; Fax: 615- can be utilized for energetics in rapidly proliferating cells and is 343-8648; E-mail: [email protected]; or Dana M. Brantley-Sieders, Phone: first catabolized to glutamate by the enzyme, glutaminase 615-343-4783; E-mail: [email protected] (GLS). Oxidative deamination by glutamate dehydrogenase in doi: 10.1158/0008-5472.CAN-15-0847 the mitochondrion, or the transamination of glutamate, then Ó2016 American Association for Cancer Research. generates a-ketoglutarate (a-KG; ref. 14). In this capacity, www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 1, 2016; DOI: 10.1158/0008-5472.CAN-15-0847 Youngblood et al. glutamine provides nitrogen for biosynthesis of nucleotides and Kaplan–Meier survival curves for 936 patients with lymph node– amino acids, and serves as a mitochondrial substrate (11). positive breast cancer. Tumors were ranked according to gene a-KG can be further metabolized to citrate, which donates expression values of EPHA2 and EFNA1, scored as high or low. acetyl-CoA groups for lipid biosynthesis. This pathway is used Statistical significance was determined by log-rank P value, and for de novo lipogenesis under hypoxic conditions and is linked HRs with 95% confidence intervals are displayed. to tumor cell proliferation, motility, and survival (15). Although RTKs have been shown to regulate these metabolic Cell culture and in vitro studies pathways in tumors, the role of EPH RTKs in tumor metabolism Human cell lines were purchased from the ATCC and main- has yet to be investigated. tained as described (3, 4). BT474 cell lines were authenticated by In this study, we demonstrate a novel role for ephrin-A1 as a the ATCC cell authentication services utilizing short-tandem regulator of mammary tumor growth and tumor metabolism. repeat profiling. Other cell lines were used at low passage ephrin-A1 Efna1 Gene deletion of ( ) increased the growth of and were not authenticated. The mouse MMTV-Neu cell line was endogenous mammary tumors in a mouse model of breast generated and provided by Rebecca Cook (Vanderbilt University). NeuT cancer driven by activated ERBB2 (MMTV- ). RNAi-medi- These cells were maintained in DMEM/F12 media supplemented ated silencing of ephrin-A1 increased lipid accumulation and with estrogen (5 ng/mL), progesterone (5 ng/mL), insulin (0.5 cell proliferation in both mouse and human breast cancer mg/mL), EGF (5 ng/mL), L-glutamine (2 mmol/L), penicillin fi models. Furthermore, metabolic pro ling of tumors revealed (100 U/mL), streptomycin (100 U/mL), and 10% FBS. siRNA- – increased glutaminolysis in ephrin-A1 null tumors. Mechanis- mediated knockdown, BrdU proliferation, three-dimensional fi tically, we identi ed a signaling pathway involving EPHA2- (3D) Matrigel culture, Rho activity assay, and Western blot anal- – RhoA-glutaminase that mediates ephrin-A1 dependent sup- ysis are as described (3, 4, 39) and in Supplementary Materials. pression of glutaminolysis and lipid accumulation in tumor cells. Collectively, these studies provide in vivo evidence for a Metabolite assays tumor-suppressive role of ephrin-A1 in breast cancer and Intracellular glutamate concentrations were determined using uncover a novel function of ephrin-A1 and EPHA2 in the the enzymatic assay, Glutamate Assay Kit (Sigma), according to regulation of tumor metabolism. the manufacturer's protocol. MCF-10A-HER2 or MMTV-Neu cells 6 Materials and Methods (10 ) were cultured in glutamine-free DMEM/F12 base media for 24 hours. Cells were stimulated with 5% serum, EGF (5 ng/mL), Animal models and in vivo studies and 2 mmol/L of glutamine. Briefly, after treatment, cells were Animals were housed under pathogen-free conditions, and washed with PBS and collected with the provided glutamate assay experiments were performed in accordance with Association, buffer. Lysates were centrifuged, and the supernatants were further Assessment, and Accreditation of Laboratory Animal Care guide- centrifuged in 10 kDa ultrafiltration spin columns. The concen- lines and with Vanderbilt University Institutional Animal Care trated samples were plated in 96-well plates with the supplied À/À and Use Committee approval. Ephrin-A1 mice were crossed glutamate enzyme mix. Absorbance was measured at 450 nm. with MMTV-NeuT mice. MMTV-NeuT--positive mice that were Concentrations were determined from the standard curve. Statis- þ/þ À/À EphrinA1 or Ephrin-A1
Load more

Recommended publications
  • Epha Receptors and Ephrin-A Ligands Are Upregulated by Monocytic
    Mukai et al. BMC Cell Biology (2017) 18:28 DOI 10.1186/s12860-017-0144-x RESEARCHARTICLE Open Access EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/ maturation and promote cell adhesion and protrusion formation in HL60 monocytes Midori Mukai, Norihiko Suruga, Noritaka Saeki and Kazushige Ogawa* Abstract Background: Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/ spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/ macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation. Results: Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16− monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and β2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and β1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand- coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes.
    [Show full text]
  • Ephrin-A1 Expression Contributes to the Malignant Characteristics of A
    843 HEPATOBILIARY DISEASE Ephrin-A1 expression contributes to the malignant Gut: first published as 10.1136/gut.2004.049486 on 11 May 2005. Downloaded from characteristics of a-fetoprotein producing hepatocellular carcinoma H Iida, M Honda, H F Kawai, T Yamashita, Y Shirota, B-C Wang, H Miao, S Kaneko ............................................................................................................................... Gut 2005;54:843–851. doi: 10.1136/gut.2004.049486 Background and aims: a-Fetoprotein (AFP), a tumour marker for hepatocellular carcinoma (HCC), is associated with poor prognosis. Using cDNA microarray analysis, we previously found that ephrin-A1, an angiogenic factor, is the most differentially overexpressed gene in AFP producing hepatoma cell lines. In the present study, we investigated the significance of ephrin-A1 expression in HCC. See end of article for Methods: We examined ephrin-A1 expression and its effect on cell proliferation and gene expression in authors’ affiliations five AFP producing hepatoma cell lines, three AFP negative hepatoma cell lines, and 20 human HCC ....................... specimens. Correspondence to: Results: Ephrin-A1 expression levels were lowest in normal liver tissue, elevated in cirrhotic tissue, and Dr S Kaneko, Department further elevated in HCC specimens. Ephrin-A1 expression was strongly correlated with AFP expression of Cancer Gene (r = 0.866). We showed that ephrin-A1 induced expression of AFP. This finding implicates ephrin-A1 in Regulation, Kanazawa University Graduate the mechanism of AFP induction in HCC. Ephrin-A1 promoted the proliferation of ephrin-A1 School of Medical Science, underexpressing HLE cells, and an ephrin-A1 antisense oligonucleotide inhibited the proliferation of 13-1 Takara-Machi, ephrin-A1 overexpressing Huh7 cells.
    [Show full text]
  • Engrailed and Fgf8 Act Synergistically to Maintain the Boundary Between Diencephalon and Mesencephalon Scholpp, S., Lohs, C
    5293 Erratum Engrailed and Fgf8 act synergistically to maintain the boundary between diencephalon and mesencephalon Scholpp, S., Lohs, C. and Brand, M. Development 130, 4881-4893. An error in this article was not corrected before going to press. Throughout the paper, efna4 should be read as EphA4, as the authors are referring to the gene encoding the ephrin A4 receptor (EphA4) and not to that encoding its ligand ephrin A4. We apologise to the authors and readers for this mistake. Research article 4881 Engrailed and Fgf8 act synergistically to maintain the boundary between diencephalon and mesencephalon Steffen Scholpp, Claudia Lohs and Michael Brand* Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany Department of Genetics, University of Dresden (TU), Pfotenhauer Strasse 108, 01307 Dresden, Germany *Author for correspondence (e-mail: [email protected]) Accepted 23 June 2003 Development 130, 4881-4893 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00683 Summary Specification of the forebrain, midbrain and hindbrain However, a patch of midbrain tissue remains between the primordia occurs during gastrulation in response to signals forebrain and the hindbrain primordia in such embryos. that pattern the gastrula embryo. Following establishment This suggests that an additional factor maintains midbrain of the primordia, each brain part is thought to develop cell fate. We find that Fgf8 is a candidate for this signal, as largely independently from the others under the influence it is both necessary and sufficient to repress pax6.1 and of local organizing centers like the midbrain-hindbrain hence to shift the DMB anteriorly independently of the boundary (MHB, or isthmic) organizer.
    [Show full text]
  • On the Turning of Xenopus Retinal Axons Induced by Ephrin-A5
    Development 130, 1635-1643 1635 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00386 On the turning of Xenopus retinal axons induced by ephrin-A5 Christine Weinl1, Uwe Drescher2,*, Susanne Lang1, Friedrich Bonhoeffer1 and Jürgen Löschinger1 1Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany 2MRC Centre for Developmental Neurobiology, King’s College London, New Hunt’s House, Guy’s Hospital Campus, London SE1 1UL, UK *Author for correspondence (e-mail: [email protected]) Accepted 13 January 2003 SUMMARY The Eph family of receptor tyrosine kinases and their turning or growth cone collapse when confronted with ligands, the ephrins, play important roles during ephrin-A5-Fc bound to beads. However, when added in development of the nervous system. Frequently they exert soluble form to the medium, ephrin-A5 induces growth their functions through a repellent mechanism, so that, for cone collapse, comparable to data from chick. example, an axon expressing an Eph receptor does not The analysis of growth cone behaviour in a gradient of invade a territory in which an ephrin is expressed. Eph soluble ephrin-A5 in the ‘turning assay’ revealed a receptor activation requires membrane-associated ligands. substratum-dependent reaction of Xenopus retinal axons. This feature discriminates ephrins from other molecules On fibronectin, we observed a repulsive response, with the sculpturing the nervous system such as netrins, slits and turning of growth cones away from higher concentrations class 3 semaphorins, which are secreted molecules. While of ephrin-A5. On laminin, retinal axons turned towards the ability of secreted molecules to guide axons, i.e.
    [Show full text]
  • Detection of Pro Angiogenic and Inflammatory Biomarkers in Patients With
    www.nature.com/scientificreports OPEN Detection of pro angiogenic and infammatory biomarkers in patients with CKD Diana Jalal1,2,3*, Bridget Sanford4, Brandon Renner5, Patrick Ten Eyck6, Jennifer Laskowski5, James Cooper5, Mingyao Sun1, Yousef Zakharia7, Douglas Spitz7,9, Ayotunde Dokun8, Massimo Attanasio1, Kenneth Jones10 & Joshua M. Thurman5 Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and infammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were signifcantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-β as the top pathways activated in both CKD groups. Pro-infammatory proteins were signifcantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and infammation are activated in CKD patients’ plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD. Approximately one in 10 individuals has chronic kidney disease (CKD) rendering CKD one of the most common diseases worldwide1. CKD is associated with a high burden of morbidity in the form of end stage kidney disease (ESKD) requiring dialysis or transplantation 2. Furthermore, patients with CKD are at signifcantly increased risk of death from cardiovascular disease (CVD)3,4.
    [Show full text]
  • Ephrin-A Binding and Epha Receptor Expression Delineate the Matrix Compartment of the Striatum
    The Journal of Neuroscience, June 15, 1999, 19(12):4962–4971 Ephrin-A Binding and EphA Receptor Expression Delineate the Matrix Compartment of the Striatum L. Scott Janis, Robert M. Cassidy, and Lawrence F. Kromer Department of Cell Biology and Interdisciplinary Program in Neuroscience, Georgetown University Medical Center, Washington, DC 20007 The striatum integrates limbic and neocortical inputs to regulate matrix neurons. In situ hybridization for EphA RTKs reveals that sensorimotor and psychomotor behaviors. This function is de- the two different ligand binding patterns strictly match pendent on the segregation of striatal projection neurons into the mRNA expression patterns of EphA4 and EphA7. anatomical and functional components, such as the striosome Ligand–receptor binding assays indicate that ephrin-A1 and and matrix compartments. In the present study the association ephrin-A4 selectively bind EphA4 but not EphA7 in the lysates of ephrin-A cell surface ligands and EphA receptor tyrosine of striatal tissue. Conversely, ephrin-A2, ephrin-A3, and kinases (RTKs) with the organization of these compartments ephrin-A5 bind EphA7 but not EphA4. These observations im- was determined in postnatal rats. Ephrin-A1 and ephrin-A4 plicate selective interactions between ephrin-A molecules and selectively bind to EphA receptors on neurons restricted to the EphA RTKs as potential mechanisms for regulating the com- matrix compartment. Binding is absent from the striosomes, partmental organization of the striatum. which were identified by m-opioid
    [Show full text]
  • Inhibition of Metastasis by HEXIM1 Through Effects on Cell Invasion and Angiogenesis
    Oncogene (2013) 32, 3829–3839 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Inhibition of metastasis by HEXIM1 through effects on cell invasion and angiogenesis W Ketchart1, KM Smith2, T Krupka3, BM Wittmann1,7,YHu1, PA Rayman4, YQ Doughman1, JM Albert5, X Bai6, JH Finke4,YXu2, AA Exner3 and MM Montano1 We report on the role of hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) as an inhibitor of metastasis. HEXIM1 expression is decreased in human metastatic breast cancers when compared with matched primary breast tumors. Similarly we observed decreased expression of HEXIM1 in lung metastasis when compared with primary mammary tumors in a mouse model of metastatic breast cancer, the polyoma middle T antigen (PyMT) transgenic mouse. Re-expression of HEXIM1 (through transgene expression or localized delivery of a small molecule inducer of HEXIM1 expression, hexamethylene-bis-acetamide) in PyMT mice resulted in inhibition of metastasis to the lung. Our present studies indicate that HEXIM1 downregulation of HIF-1a protein allows not only for inhibition of vascular endothelial growth factor-regulated angiogenesis, but also for inhibition of compensatory pro- angiogenic pathways and recruitment of bone marrow-derived cells (BMDCs). Another novel finding is that HEXIM1 inhibits cell migration and invasion that can be partly attributed to decreased membrane localization of the 67 kDa laminin receptor, 67LR, and inhibition of the functional interaction of 67LR with laminin. Thus, HEXIM1 re-expression in breast cancer has therapeutic advantages by simultaneously targeting more than one pathway involved in angiogenesis and metastasis. Our results also support the potential for HEXIM1 to indirectly act on multiple cell types to suppress metastatic cancer.
    [Show full text]
  • Rabbit Anti-Ephrin-A1 Rabbit Anti-Ephrin-A1
    Qty: 100 µg/400 µl Rabbit anti-ephrin-A1 Catalog No. 34-3300 Lot No. See product label Rabbit anti-ephrin-A1 FORM This polyclonal antibody is supplied as a 400 µl aliquot at a concentration of 0.25 mg/ml in phosphate buffered saline (pH 7.4) containing 0.1% sodium azide. The antibody is epitope-affinity-purified from rabbit antiserum. PAD: ZMD.39 IMMUNOGEN Synthetic peptide derived from the C-terminal end of mouse ephrin-A1 protein. SPECIFICITY This antibody is specific for the ephrin-A1 protein. REACTIVITY Reactivity is confirmed with a chimeric protein consisting of the extracellular domain of mouse ephrin-A1 and the Fc region of human IgG1. Cross-reactivity with human ephrin-A1 is confirmed with IHC experiments on human tissue sections and this reactivity was expected because of the 85% shared amino acid identity in the extracellular domain. Sample Western Immunoprecipitation Immunohistochemistry Blotting (FFPE) Mouse +++ +++ NT Human NT NT ++ (Excellent +++, Good++, Poor +, No reactivity 0, Not tested NT) USAGE Working concentrations for specific applications should be determined by the investigator. Appropriate concentrations will be affected by several factors, including secondary antibody affinity, antigen concentration, sensitivity of detection method, temperature and length of incubations, etc. The suitability of this antibody for applications other than those listed below has not been determined. The following concentration ranges are recommended starting points for this product. Western Blotting: 1-5 µg/mL Immunoprecipitation: 5-10 µg/ IP reaction Immunohistochemistry: 4-10 µg/mL Please note that immunohistochemical assays were optimized on formalin-fixed, paraffin-embedded tissue sections and Heat Induced Epitope Retrieval (HIER) with EDTA, pH 8.0 was required for optimal staining.
    [Show full text]
  • FGF8 Signaling Regulates Growth of Midbrain Dopaminergic Axons by Inducing Semaphorin 3F
    4044 • The Journal of Neuroscience, April 1, 2009 • 29(13):4044–4055 Development/Plasticity/Repair FGF8 Signaling Regulates Growth of Midbrain Dopaminergic Axons by Inducing Semaphorin 3F Kenta Yamauchi,1* Shigeki Mizushima,1* Atsushi Tamada,2 Nobuhiko Yamamoto,1 Seiji Takashima,3 and Fujio Murakami1,2 1Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan, 2Division of Behavior and Neurobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan, and 3Department of Molecular Cardiology, Osaka University Graduate School of Medicine, Suita 565-0871, Japan Accumulating evidence indicates that signaling centers controlling the dorsoventral (DV) polarization of the neural tube, the roof plate and the floor plate, play crucial roles in axon guidance along the DV axis. However, the role of signaling centers regulating the rostrocau- dal (RC) polarization of the neural tube in axon guidance along the RC axis remains unknown. Here, we show that a signaling center located at the midbrain–hindbrain boundary (MHB) regulates the rostrally directed growth of axons from midbrain dopaminergic neurons (mDANs). We found that beads soaked with fibroblast growth factor 8 (FGF8), a signaling molecule that mediates patterning activities of the MHB, repelled mDAN axons that extended through the diencephalon. This repulsion may be mediated by semaphorin 3F (sema3F) because (1) FGF8-soaked beads induced an increase in expression of sema3F, (2) sema3F expression in the midbrain was essentially abolished by the application of an FGF receptor tyrosine kinase inhibitor, and (3) mDAN axonal growth was also inhibited by sema3F. Furthermore, mDAN axons expressed a sema3F receptor, neuropilin-2 (nrp2), and the removal ofnrp-2 by gene targeting caused caudal growth of mDAN axons.
    [Show full text]
  • Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6
    15902 • The Journal of Neuroscience, November 7, 2012 • 32(45):15902–15912 Cellular/Molecular Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-Sulfatases Regulates Fibroblast Growth Factor-Induced Boundary Formation by Glial Cells: Implications for Glial Cell Transplantation Jennifer R. Higginson,1* Sophie M. Thompson,2* Alessandra Santos-Silva,1 Scott E. Guimond,2 Jeremy E. Turnbull,2† and Susan C. Barnett1† 1Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary & Life Sciences, Glasgow Biomedical Research Centre, Glasgow G12 8TA, United Kingdom, and 2Centre for Glycobiology, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7XB, United Kingdom Previously, it has been shown that rat Schwann cells (SCs), but not olfactory ensheathing cells (OECs), form a boundary with astrocytes, due to a SC-specific secreted factor. Here, we identify highly sulfated heparan sulfates (HSs) and fibroblast growth factors (FGFs) 1 and 9 as possible determinants of boundary formation induced by rat SCs. Disaccharide analysis of HS in SC-conditioned and rat OEC- conditionedmediashowedthatSCssecretemorehighlysulfatedHSthanOECs.Thedependenceoftheboundary-formingactivityonhigh levels of sulfation was confirmed using a panel of semisynthetic modified heparins with variable levels of sulfation. Furthermore, extracellular HS 6-O-endosulfatase enzymes, Sulf 1 and Sulf 2, were expressed at a significantly lower level by SCs compared with OECs, and siRNA reduction of Sulfs in OECs was, in itself, sufficient to induce boundary formation. This demonstrates a key role for remodelling (reduction) of HS 6-O-sulfation by OECs, compared with SCs, to suppress boundary formation. Furthermore, specific anti-FGF1 and anti-FGF9 antibodies disrupted SC–astrocyte boundary formation, supporting a role for an HS sulfation-dependent FGF signaling mechanism via FGF receptors on astrocytes.
    [Show full text]
  • Migration Dichotomy of Glioblastoma by Interacting with Focal Adhesion Kinase
    Oncogene (2012) 31, 5132 --5143 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase SD Wang1, P Rath1,2, B Lal1,2, J-P Richard1,2,YLi1,2, CR Goodwin3, J Laterra1,2,4,5 and S Xia1,2 Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumors in adults. Uncontrolled proliferation and abnormal cell migration are two prominent spatially and temporally disassociated characteristics of GBMs. In this study, we investigated the role of the receptor tyrosine kinase EphB2 in controlling the proliferation/migration dichotomy of GBM. We studied EphB2 gain of function and loss of function in glioblastoma-derived stem-like neurospheres, whose in vivo growth pattern closely replicates human GBM. EphB2 expression stimulated GBM neurosphere cell migration and invasion, and inhibited neurosphere cell proliferation in vitro. In parallel, EphB2 silencing increased tumor cell proliferation and decreased tumor cell migration. EphB2 was found to increase tumor cell invasion in vivo using an internally controlled dual-fluorescent xenograft model. Xenografts derived from EphB2-overexpressing GBM neurospheres also showed decreased cellular proliferation. The non-receptor tyrosine kinase focal adhesion kinase (FAK) was found to be co-associated with and highly activated by EphB2 expression, and FAK activation facilitated focal adhesion formation, cytoskeleton structure change and cell migration in EphB2-expressing GBM neurosphere cells. Taken together, our findings indicate that EphB2 has pro-invasive and anti-proliferative actions in GBM stem-like neurospheres mediated, in part, by interactions between EphB2 receptors and FAK.
    [Show full text]
  • Epha1 Interacts with Integrin-Linked Kinase and Regulates Cell Morphology and Motility
    Research Article 243 EphA1 interacts with integrin-linked kinase and regulates cell morphology and motility Tohru Yamazaki*, Junko Masuda*, Tsutomu Omori, Ryosuke Usui, Hitomi Akiyama and Yoshiro Maru‡ Department of Pharmacology, Tokyo Womenʼs Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected]) Accepted 7 October 2008 Journal of Cell Science 122, 243-255 Published by The Company of Biologists 2009 doi:10.1242/jcs.036467 Summary dependent on stimulation of the EphA1 ligand ephrin-A1. The Eph-ephrin receptor-ligand system is implicated in cell Activation of EphA1 kinase resulted in a decrease of ILK behavior and morphology. EphA1 is the founding member of activity. Finally, we demonstrated that expression of a kinase- the Eph receptors, but little is known about its function. Here, active form of ILK (S343D) rescued the EphA1-mediated we show that activation of EphA1 kinase inhibits cell spreading spreading defect, and attenuated RhoA activation. These results and migration in a RhoA-ROCK-dependent manner. We also suggest that EphA1 regulates cell morphology and motility describe a novel interaction between EphA1 and integrin-linked through the ILK-RhoA-ROCK pathway. kinase (ILK), a mediator of interactions between integrin and the actin cytoskeleton. The C-terminal sterile α motif (SAM) domain of EphA1 is required and the ankyrin region of ILK is Supplementary material available online at sufficient for the interaction between EphA1 and ILK. The http://jcs.biologists.org/cgi/content/full/122/2/243/DC1 interaction is independent of EphA1 kinase activity but Key words: EphA1, ILK, Ephrin-A1, Migration (Bruckner et al., 1997; Davy et al., 1999; Holland et al., 1996).
    [Show full text]