Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 884 Syndecan - Regulation and Function of its Glycosaminoglycan Chains ANNA S. ERIKSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-8637-2 UPPSALA urn:nbn:se:uu:diva-197691 2013 Dissertation presented at Uppsala University to be publicly examined in A1:107a, BMC, Husargatan 3, Uppsala, Friday, May 17, 2013 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Abstract Eriksson, A. S. 2013. Syndecan - Regulation and Function of its Glycosaminoglycan Chains. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 884. 54 pp. Uppsala. ISBN 978-91-554-8637-2. The cell surface is an active area where extracellular molecules meet their receptors and affect the cellular fate by inducing for example cell proliferation and adhesion. Syndecans and integrins are two transmembrane molecules that have been suggested to fine-tune these activities, possibly in cooperation. Syndecans are proteoglycans, i.e. proteins with specific types of carbohydrate chains attached. These chains are glycosaminoglycans and either heparan sulfate (HS) or chondroitin sulfate (CS). Syndecans are known to influence cell adhesion and signaling. Integrins in turn, are important adhesion molecules that connect the extracellular matrix with the cytoskeleton, and hence can regulate cell motility. In an attempt to study how the two types of glycosaminoglycans attached to syndecan-1 can interact with integrins, a cell based model system was used and functional motility assays were performed. The results showed that HS, but not CS, on the cell surface was capable of regulating integrin-mediated cell motility. Regulation of intracellular signaling is crucial to prevent abnormal cellular behavior. In the second part of this thesis, the aim was to see how the presentation of glycosaminoglycan chains to the FGF signaling complex could affect the cellular response. When attached to the plasma membrane via syndecan-1, CS chains could support the intracellular signaling, although not promoting as strong signals as HS. When glycosaminoglycans were attached to free ectodomains of syndecan-1, both types of chains sequestered FGF2 from the receptors to the same extent, pointing towards functional overlap between CS and HS. To further study the interplay between HS and CS, their roles in the formation of pharyngeal cartilage in zebrafish were established. HS was important during chondrocyte intercalation and CS in the formation of the surrounding extracellular matrix. Further, the balance between the biosynthetic enzymes determined the ratio of HS and CS, and HS biosynthesis was prioritized over CS biosynthesis. The results presented in this thesis provide further insight into the regulation of HS biosynthesis, as well as the roles of both HS and CS on the cell surface. It is evident, that in certain situations there is a strict requirement for a certain HS structure, albeit in other situations there is a functional overlap between HS and CS. Keywords: heparan sulfate, chondroitin sulfate, integrin, cell motility, fibroblast growth factor signaling, pharyngeal cartilage, biosynthesis regulation Anna S. Eriksson, Uppsala University, Department of Medical Biochemistry and Microbiology, Box 582, SE-751 23 Uppsala, Sweden. © Anna S. Eriksson 2013 ISSN 1651-6206 ISBN 978-91-554-8637-2 urn:nbn:se:uu:diva-197691 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-197691) Fly me to the moon, let me play among the stars Supervisors: Dorothe Spillmann, Senior Lecturer Department of Medical Biochemistry and Microbiology, Uppsala University Ulf Lindahl, Professor Department of Medical Biochemistry and Microbiology, Uppsala University Faculty opponent: Catherine Merry, Senior Lecturer Stem Cell Glycobiology Group, School of Materials, University of Manchester, Manchester, United Kingdom Examining Committee: Anna Dimberg, Associate Professor Department of Immunology, Genetics and Pathology, Uppsala University Karin Forsberg-Nilsson, Professor Department of Immunology, Genetics and Pathology, Uppsala University Anders Hjerpe, Professor Department of Laboratory Medicine, Karolinska Institute Staffan Johansson, Professor Department of Medical Biochemistry and Microbiology, Uppsala University Johan Lennartsson, Associate Professor Ludwig Institute for Cancer Research, Uppsala Chairperson: Lena Kjellén, Professor Department of Medical Biochemistry and Microbiology, Uppsala University The cover picture shows CHO-K1 cells overexpressing uncleavable syndecan-1 in green, the cell nucleus in blue and actin cytoskeleton in red. List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Eriksson, A.S, Spillmann, D. (2012) The mutual impact of syndecan-1 and its glycosaminoglycan chains – A multivariable puzzle. Journal of Histochemistry and Cytochemistry, 60(12):936– 42 II Eriksson, A.S, Reyhani, V, Spillmann, D. (2013) Cell surface hepa- ran sulfate chains are important for integrin-mediated cell motility. Manuscript. III Eriksson A.S, Spillmann, D. (2013) Role of glycosaminoglycans on syndecan-1 in fibroblast growth factor signaling. Manuscript. IV Holmborn, K*, Habicher, J*, Kasza, Z, Eriksson, A.S, Filipek- Gorniok, B, Gopal, S, Couchman, J.R, Ahlberg, P.E, Wiweger, M, Spillmann, D, Kreuger, J*, Ledin, J*. (2012) On the roles and regu- lation of chondroitin sulfate and heparan sulfate in zebrafish pharyn- geal cartilage morphogenesis. Journal of Biological Chemistry, 287(40):33905-16 * These authors contributed equally to the work. Reprints were made with permission from the respective publishers. Contents Introduction ..................................................................................................... 9 Background ................................................................................................... 10 Proteoglycans ............................................................................................ 10 Syndecans ............................................................................................ 11 Glycosaminoglycans ................................................................................. 13 Biosynthesis of glycosaminoglycans ................................................... 14 Regulation of glycosaminoglycan biosynthesis ................................... 19 Integrins .................................................................................................... 20 Integrins and cell migration ................................................................. 21 Fibroblast growth factors and their receptors ........................................... 22 Fibroblast growth factors and heparan sulfate ..................................... 23 Fibroblast growth factor induced signaling ......................................... 23 Glycosaminoglycans in pathology ............................................................ 26 Present Investigations .................................................................................... 29 Aim ........................................................................................................... 29 Model systems .......................................................................................... 30 Chinese hamster ovary cells ................................................................. 30 Zebrafish .............................................................................................. 31 Results and Discussion ............................................................................. 32 Paper I .................................................................................................. 32 Paper II ................................................................................................. 33 Paper III ............................................................................................... 35 Paper IV ............................................................................................... 36 Concluding remarks ...................................................................................... 38 Populärvetenskaplig sammanfattning ........................................................... 40 Acknowledgements ....................................................................................... 41 Thanks/Tack .................................................................................................. 42 References ..................................................................................................... 44 Abbreviations CHO Chinese hamster ovary CS Chondroitin sulfate DS Dermatan sulfate ECM Extracellular matrix Erk1/2 Extracellular-signal-regulated kinase 1 and 2 EXT Exostosin FGF Fibroblast growth factor FGFR Fibroblast growth factor receptor FRS2 Fibroblast growth factor receptor substrate 2 GAG Glycosaminoglycan GalN Galactosamine GalNAc N-acetyl-galactosamine GlcA Glucuronic acid GlcN Glucosamine GlcNAc N-acetyl-glucosamine GlcNS N-sulfated glucosamine HS Heparan sulfate IdoA Iduronic acid KS Keratan sulfate MAPK Mitogen activated protein kinase MMP Matrix metalloproteinase NDST N-deacetylase/N-sulfotransferase OST O-sulfotransferase PAPS 3’-phosphoadenosine 5’-phosphosulfate PG Proteoglycan RGD Arginine-Glycine-Aspartic acid Sulfs Endo-O-sulfatases Introduction A large number of interactions take place at the surface of cells that deter-