DOCSLIB.ORG

Type Xviii Collagen

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

TYPE XVIII COLLAGEN HARRI Characterization of the primary structure and expression ELAMAA pattern of different variants in Xenopus laevis, characterization of the human gene structure and analysis of transgenic mice Faculty of Medicine, expressing endostatin Department of Medical Biochemistry and Molecular Biology, Biocenter Oulu, University of Oulu OULU 2004 HARRI ELAMAA TYPE XVIII COLLAGEN Characterization of the primary structure and expression pattern of different variants in Xenopus laevis, characterization of the human gene structure and analysis of transgenic mice expressing endostatin Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Pharmacology and Toxicology, on December 3rd, 2004, at 1 p.m. OULUN YLIOPISTO, OULU 2004 Copyright © 2004 University of Oulu, 2004 Supervised by Professor Taina Pihlajaniemi Reviewed by Docent Klaus Elenius Doctor Takako Sasaki ISBN 951-42-7568-3 (nid.) ISBN 951-42-7569-1 (PDF) http://herkules.oulu.fi/isbn9514275691/ ISSN 0355-3221 http://herkules.oulu.fi/issn03553221/ OULU UNIVERSITY PRESS OULU 2004 Elamaa, Harri, Type XVIII collagen. Characterization of the primary structure and expression pattern of different variants in Xenopus laevis, characterization of the human gene structure and analysis of transgenic mice expressing endostatin Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology, and Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland 2004 Oulu, Finland Abstract In this work the type XVIII collagen has been studied by using several approaches, such as different animal models. The primary structure of frog, Xenopus laevis, type XVIII collagen and the expression pattern of its variants during early embryogenesis have been elucidated. The gene structure of human type XVIII collagen was characterized and the localization and processing of its longest variant was studied by generated antibodies. In addition, the function of the proteolytically released C-terminal part of type XVIII collagen, endostatin, was studied by generating transgenic mice expressing endostatin. The primary structure of X. laevis type XVIII collagen is comprised of three N-terminal variants resembling their mammalian counterparts. The sizes of the polypeptides are 1285, 1581, and 1886 residues. The most conserved regions are the C-terminal endostatin region and the cysteine-rich domain in the N-terminus. Whole-mount in situ hybridization reveals different expression patterns for variants during embryogenesis. The short variant is the most abundant, whereas the two longest variants exhibit more restricted expression. The gene structure of human type XVIII collagen reveals an exon-intron organization that is conserved with mouse. The length of the human gene is about 105 kb and contains 43 exons. The third variant of type XVIII collagen has a conserved cysteine-rich domain with homology to the extracellular part of frizzled proteins. This third variant is localized to developing muscle and lung, and is also found in serum. In cell culture, the proteolytic fragments of the N-terminus, including the cysteine-rich motif, are also detected. Endostatin function was studied by generating mouse lines expressing endostatin under the keratin-14 promoter, which drives the expression mainly in the skin. Three independent transgenic mouse lines were achieved with varied expression levels. The phenotype was seen in the eye with lens opacity and abnormal morphology of epithelial cells in the lens. In the skin, a broading of the basement membrane in the epidermis dermis junction was detected. Immunoelectron microscopy analysis revealed a polarized orientation of type XVIII collagen in the basement membrane. In transgenic mice, altered localization of endogenous type XVIII collagen was seen, suggesting displacement of the endogenous type XVIII collagen with transgenic endostatin leading to disorganized basement membrane. Keywords: basement membrane, collagen type XVIII, endostatins, protein isoforms Acknowledgements This research was carried out at the Department of Medical Biochemistry and Molecular Biology. I wish to thank my supervisor, Professor Taina Pihlajaniemi, for giving me the opportunity to work in her group and introducing me to the field of matrix biology. I wish to express my appreciation and respect for Research Professor emeritus Kari Kivirikko for creating, together with Professor Taina Pihlajaniemi, excellent conditions for many scientists. I also wish to thank Professor emeritus Ilmo Hassinen, Professor Leena Ala- Kokko, Docent Johanna Myllyharju, and Professor Peppi Karppinen for their contributions to department management. I am very grateful to Docent Klaus Elenius and Dr. Takako Sasaki for their accurate review and valuable comments on my thesis manuscript. Sandra Hänninen, M.Sc. is acknowledged for her careful revision of the language of the manuscript. The collaboration between different laboratories and researchers has been crucial for my thesis. I would like to thank all my co-authors for their invaluable efforts. I wish to express my deepest gratitude to Dr. Olivier Destrée and the members of his research group in the Hubrecht laboratory in Utrecht. Dr. Destrée and his group wished me warmly welcome to their group and introduced me to the frog world during my several visits. I wish to thank Docent Helena Autio-Harmainen for her contribution to this work. I also wish to thank Docent Raija Sormunen and her excellent team for sharing their expertise in ultrastructural analysis. I am grateful for the valuable comments from Docent Raija Soininen, and expert in transgenic technology. Particularly, I am very grateful to Marko Rehn for helping me to take my first steps in the scientific field, and his support and advice during these years. Especially I wish to thank Päivi Tuomaala for her excellent technical assistance, which has been extremely valuable for my thesis. I also wish to thank Outi Laurila, Aira Harju, Sirpa Kellokumpu and Anna-Liisa Oikarinen for their valuable technical assistance. I am also grateful to the people in the laboratory animal center, Liisa Asikkala, Sirpa Tausta and Päivi Moilanen. In addition, special thanks go to Marja-Leena Kivelä and Auli Kinnunen for their kind help in secretarial matters, Seppo Lähdesmäki and Pertti Vuokila their help in a variety of practical matters, and Ari-Pekka Kvist and Risto Helminen for helping me with the computers. I have been privileged to work with pleasant researchers who are willing to offer help and consultation. I would like to thank all members of Taina´s group for their support. I am grateful to Ritva Heljasvaara for her comments on my thesis manuscript. I would especially like to thank my friends Joni Mäki and Lauri Eklund. We have shared a lot during our work and leisure time. I also want to thank all of my colleagues from the old downstairs office, L023, for creating a friendly atmosphere. I owe my deepest gratitude to my parents Maire and Eero for their love and support during these years, and my warmest thanks to my brother Kari and sister Satu and their families for their friendship and many hilarious moments. Above all, I owe my greatest gratitude to my wife Sari for her love and caring, and sharing everyday life. You have encouraged me and reminded me of the existence of intriguing life outside of work. I am deeply indebted to our new family member, the sweet daughter, who came to brighten our family life and cheered up her father during the thesis process. This work was supported by grants from the Emil Altonen Foundation, the Research and Science Foundation of Farmos, and the Finnish Cultural Foundation. Oulu, October 2004 Harri Elamaa Abbreviations BM basement membrane bp basepairs C- carboxy- COL collagenous domain CRD cysteine-rich domain fz Frizzled domain GAG glycosaminoglycan HSPG heparin sulphate proteoglycan IEM immunoelectron microscopy kb kilobase kDa kilodalton K14 keratin 14 N- amino- NC non-collagenous PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction RT-PCR reverse transcription polymerase chain reaction SDS sodium dodecyl sulphate VEGF vascular endothelial growth factor List of original articles This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I Elamaa H, Peterson J, Pihlajaniemi T & Destrée O (2002) Cloning of three variants of type XVIII collagen and their expression patterns during Xenopus laevis development. Mech Dev 114: 109-13. II Elamaa H, Snellman A, Rehn M, Autio-Harmainen H & Pihlajaniemi T (2003) Characterization of the human type XVIII collagen gene and proteolytic processing and tissue location of the variant containing a frizzled motif. Matrix Biol 22: 427-42. III Elamaa H, Sormunen R, Rehn M, Soininen R & Pihlajaniemi T (2004) Endostatin overexpression specifically in the lens and skin leads to cataract and ultrastructural alterations in basement membranes. Am. J. Pathol., In press. Contents Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction ...................................................................................................................13 2 Review of the literature .................................................................................................14 2.1 Cryptic angiogenesis inhibitors ..............................................................................14 2.2 Collagen superfamily..............................................................................................15
Recommended publications
  • New Concepts in Basement Membrane Biology Willi Halfter1, Philipp Oertle2, Christophe A

    New Concepts in Basement Membrane Biology Willi Halfter1, Philipp Oertle2, Christophe A

    REVIEW ARTICLE New concepts in basement membrane biology Willi Halfter1, Philipp Oertle2, Christophe A. Monnier2,*, Leon Camenzind2, Magaly Reyes-Lua1, Huaiyu Hu3, Joseph Candiello4, Anatalia Labilloy5,†, Manimalha Balasubramani6, Paul Bernhard Henrich1 and Marija Plodinec2,7 1 Department of Ophthalmology, University Hospital Basel, Switzerland 2 Biozentrum and the Swiss Nanoscience Institute, University of Basel, Switzerland 3 Department of Neurobiology and Physiology, Upstate University Hospital, SUNY University, Syracuse, NY, USA 4 Department of Bioengeneering, University of Pittsburgh, PA, USA 5 Department of Renal Physiology, University of Pittsburgh, PA, USA 6 Proteomics Core Facility of the University of Pittsburgh, PA, USA 7 Department of Pathology, University Hospital Basel, Switzerland Keywords Basement membranes (BMs) are thin sheets of extracellular matrix that basal lamina; basement membrane; outline epithelia, muscle fibers, blood vessels and peripheral nerves. The biomechanical properties; collagen IV; current view of BM structure and functions is based mainly on transmis- laminin; membrane asymmetry; nidogen; sion electron microscopy imaging, in vitro protein binding assays, and phe- perlecan notype analysis of human patients, mutant mice and invertebrata. Correspondence Recently, MS-based protein analysis, biomechanical testing and cell adhe- W. Halfter, Department of Ophthalmology, sion assays with in vivo derived BMs have led to new and unexpected University Hospital Basel, Mittlere insights. Proteomic analysis combined with ultrastructural studies showed Strasse 91, 4031 Basel, Switzerland that many BMs undergo compositional and structural changes with Fax: +41 61 267 21 09 advancing age. Atomic force microscopy measurements in combination Tel: +49 7624 982528 with phenotype analysis have revealed an altered mechanical stiffness that E-mail: [email protected] M.
  • Undenatured Type II Collagen Mechanism of Action

    Undenatured Type II Collagen Mechanism of Action

    Undenatured Type II Collagen Mechanism of Action Page Executive Summary .................... 2 Animal Research ......................... 3 In Vitro Research ........................ 4 For Professional Use Only UC-II® Undenatured Type II Collagen (Mechanism of Action) Executive Summary UC-II® undenatured type II collagen is a patented form of collagen with undenatured (native) type II collagen for joint health support. A small amount (40 mg/day) is believed to work by inducing a process known as oral tolerance that ultimately engages the immune system in the repair of its own joint cartilage. Oral tolerance is an immune process that allows the body to distinguish between innocuous compounds such as dietary proteins and intestinal bacteria and potentially harmful foreign invaders. In the digestive tract, oral tolerance occurs in the gut-associated lymphoid tissue (GALT), considered to be the body’s most abundant lymphoid tissue. The GALT is primarily composed of mesenteric lymph nodes and patches of lymphoid tissue surrounding the small intestine called Peyer’s patches. It is in the Peyer’s patches, which contain an organized collection of immune cells, where most of the immune responses in the digestive tract are generated. Through a cascade of immunological events, Peyer’s patches take in and screen compounds from the gut lumen and, depending on the compound, turn the body’s immune response on or off (Weiner et al1). Researchers believe UC-II® undenatured type II collagen induces a form of oral tolerance that results from exposure to a compound in small amounts. More specifically, UC-II® undenatured type II collagen is believed to be transported across the gut epithelial cells to the underlying immune cells in the Peyer’s patches where it activates naive T cells to become T regulatory (Treg) cells that specifically target type II collagen.
  • Ophthalmological Features Associated with COL4A1 Mutations

    Ophthalmological Features Associated with COL4A1 Mutations

    OPHTHALMIC MOLECULAR GENETICS SECTION EDITOR: JANEY L. WIGGS, MD, PhD Ophthalmological Features Associated With COL4A1 Mutations Isabelle Coupry, PhD; Igor Sibon, MD, PhD; Bruno Mortemousque, MD; Franc¸ois Rouanet, MD; Manuele Mine, PharmD, PhD; Cyril Goizet, MD, PhD Objective: To investigate the wide variability of ocular Conclusions: The COL4A1 mutations may be associ- manifestations associated with mutations in the COL4A1 ated with various ophthalmologic developmental anoma- gene that encodes collagen IV␣1. lies of anterior segment dysgenesis type, which are reminiscent of Axenfeld-Rieger anomalies (ARA). Cere- Methods: We clinically evaluated 7 patients from 2 un- brovascular disorders should be added to the list of signs related families in whom ocular features segregated with potentially associated with ARA. COL4A1 mutations that were identified by direct se- quencing. Clinical Relevance: These data suggest that cerebral magnetic resonance imaging may be recommended in Results: The G2159A transition (c.2159GϾA) that leads the clinical treatment of patients with apparently iso- tothemissensemutationp.Gly720Aspwasidentifiedinfam- lated ARA, even when neurological symptoms or signs ily A. An ocular phenotype of variable severity was observed are lacking. in all affected relatives. The missense mutation c.2263GϾA, p.Gly755Arg was identified in family B. One patient from family B also displayed notable ocular features. Arch Ophthalmol. 2010;128(4):483-489 NEW FORM OF HEREDITARY constellation of ocular findings that in- cerebrovascular disorder clude anomalies of the anterior chamber was recently associated angle and aqueous drainage structures (iri- with mutations in the dogoniodysgenesis), iris hypoplasia, ec- COL4A1 gene that en- centric pupil (corectopia), iris tears (poly- Acodes collagen IV␣1.1,2 Mutations in coria), and iridocorneal adhesions COL4A1 were initially associated with ce- traversing the anterior chamber.
  • Native Type II Collagen for Joint Health: Small Dose, Big Benefits

    Native Type II Collagen for Joint Health: Small Dose, Big Benefits

    Print this Document! Native Type II Collagen for Joint Health: Small Dose, Big Benefits By Cai Berg Collagens are the most abundant family of proteins in the extracellular matrix of connective tissues. They perform a variety of biological functions, the best known of which is providing the structural framework for tissues throughout the body. Based on their supramolecular organization, 26 different types of collagen have been identified, each with its own role and position in the body. Type II collagen is the main structural protein in the cartilage. It provides tensile strength and toughness to the tissue. Joint disorders involving inflammation and cartilage erosion, such as arthritis, are often characterized by an autoimmune component in which the immune system acts against the body's own type II collagen. Studies have shown that supplementing native (undenatured) type II collagen can help modulate the destructive immune response against endogenous type II collagen to support joint health and comfort.1 Not All Collagens Are Equal Collagen and collagen derivatives have been investigated and employed for joint nourishment for decades. Collagen products are defined by their degree of hydrolyzation and their molecular weight as either undenatured (native) collagen, collagen hydrolysate (hydrolyzed collagen) or gelatin. Undenatured (native) collagen has the highest molecular weight (300 kDA), while hydrozyled collagen ranges from 2 to 9 kDa. Unlike gelatin and hydrolyzed collagen supplements, which are intended to be absorbed to augment the collagen needs of various cells and tissues, native type II collagen supplements work through a mechanism known as oral tolerization. This immune-mediated process involves ingestion of an autoantigen (in this case, type II collagen) in order to suppress the immune response against endogenous tissue.
  • A Collagen Glucosyltransferase Drives Lung Adenocarcinoma Progression in Mice

    A Collagen Glucosyltransferase Drives Lung Adenocarcinoma Progression in Mice

    ARTICLE https://doi.org/10.1038/s42003-021-01982-w OPEN A collagen glucosyltransferase drives lung adenocarcinoma progression in mice Hou-Fu Guo 1, Neus Bota-Rabassedas 1, Masahiko Terajima 2, B. Leticia Rodriguez1, Don L. Gibbons 1, Yulong Chen1, Priyam Banerjee1, Chi-Lin Tsai 3, Xiaochao Tan1, Xin Liu1, Jiang Yu1, Michal Tokmina-Roszyk4, Roma Stawikowska4, Gregg B. Fields4, Mitchell D. Miller 5, Xiaoyan Wang3, Juhoon Lee6,7, Kevin N. Dalby6,7, Chad J. Creighton 8,9, George N. Phillips Jr 5,10, John A. Tainer 3, Mitsuo Yamauchi2 & ✉ Jonathan M. Kurie 1 Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and 1234567890():,; is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen’s amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interac- tions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH- independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyl- transferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A.
  • Collagen Type XVIII (H-140): Sc-25720

    Collagen Type XVIII (H-140): Sc-25720

    SANTA CRUZ BIOTECHNOLOGY, INC. Collagen Type XVIII (H-140): sc-25720 The Power to Question BACKGROUND APPLICATIONS Type XV and XVIII collagens form the new subgroup MULTIPLEXIN, within the Collagen Type XVIII (H-140) is recommended for detection of Collagen α1 diverse family of collagens, which contains nineteen distinct types of collagens Type XVIII of mouse, rat and human origin by Western Blotting (starting found in vertebrates. Both type XV and XVIII collagens are characterized by dilution 1:200, dilution range 1:100-1:1000), immunoprecipitation [1–2 µg extensive interruptions in their collagenous sequences. Members of the MUL- per 100–500 µg of total protein (1 ml of cell lysate)] and immunofluores- TIPLEXIN subgroup contain polypeptides with multiple triple-helical domains cence (starting dilution 1:50, dilution range 1:50-1:500). separated and flanked by non-triple-helical regions. Type XV is predominantly Suitable for use as control antibody for Collagen Type XVIII siRNA (h): expressed in internal organs such as adrenal gland, kidney and pancreas. Type sc-43072 and Collagen Type XVIII siRNA (m): sc-43073. XVIII encodes two different α1 chains, which have different signal peptides and variant N-terminal non-collagenous NC1 domains of 495 and 303 amino Molecular Weight of Collagen Type XVIII: 20-22 kDa. acids. The long variant NC1-434 Type XVIII mRNAs are prominently expressed Positive Controls: rat C6 glioblastoma, human PBL or rat lung extract: sc-2396. in liver, while the variant NC1-303 mRNAs are predominantly expressed in heart, kidney, placenta, prostate, ovaries, skeletal muscle and small intestine. RECOMMENDED SECONDARY REAGENTS Endostatin is a fragment of the C-terminal domain NC1 of collagen XV and XVIII that inhibits angiogenesis and tumor growth.
  • Characterization of Collagen Fibers (I, III, IV) and Elastin of Normal and Neoplastic Canine Prostatic Tissues

    Characterization of Collagen Fibers (I, III, IV) and Elastin of Normal and Neoplastic Canine Prostatic Tissues

    veterinary sciences Article Characterization of Collagen Fibers (I, III, IV) and Elastin of Normal and Neoplastic Canine Prostatic Tissues Luis Gabriel Rivera Calderón 1, Priscila Emiko Kobayashi 2, Rosemeri Oliveira Vasconcelos 1, Carlos Eduardo Fonseca-Alves 3 and Renée Laufer-Amorim 2,* 1 Department of Veterinary Pathology, School of Agricultural and Veterinarian Sciences, São Paulo State University (Unesp), Jaboticabal, São Paulo 14884-900, Brazil; [email protected] (L.G.R.C.); [email protected] (R.O.V.) 2 Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, São Paulo State University (Unesp), Botucatu, São Paulo 18618-681, Brazil; [email protected] 3 Department of Veterinary Surgery and Anesthesiology, School of Veterinary Medicine and Animal Science, São Paulo State University (Unesp), Botucatu, São Paulo 18618-681, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-14-3880-2076 Received: 7 January 2019; Accepted: 25 February 2019; Published: 2 March 2019 Abstract: This study aimed to investigate collagen (Coll-I, III, IV) and elastin in canine normal prostate and prostate cancer (PC) using Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Coll-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle, and 5% surrounding blood vessels, in both normal prostate and PC. There was a higher median area of Coll-III in PC when compared to normal prostatic tissue (p = 0.001 for PSR and p = 0.05 for IHC).
  • List of Publications Taina Pihlajaniemi

    List of Publications Taina Pihlajaniemi

    List of publications Taina Pihlajaniemi 1 April, 2016 A Peer-reviewed scientific articles Journal article (refereed), original research; review article, literature review, systematic review; book section, chapters in research books; conference proceedings A.1 Savolainen E-R, Kero M, Pihlajaniemi T, and Kivirikko KI. Deficiency of galactosylhydroxylysyl glucosyltransferase, an enzyme of collagen synthesis, in a family with dominant epidermolysis bullosa simplex. N Engl J Med 304: 197-204, 1981 A.2 Pihlajaniemi T, Myllylä R, Alitalo K, Vaheri A, and Kivirikko KI. Post-translational modifications in the biosynthesis of type IV collagen by a human tumor cell line. Biochemistry 20: 7409-7415, 1981 A.3 Anttinen H, Puistola U, Pihlajaniemi T, and Kivirikko KI. Differences between proline and lysine hydroxylations in their inhibition by zinc or by ascorbate deficiency during collagen synthesis in various cell types. Biochim Biophys Acta 674: 336-344, 1981 A.4 Pihlajaniemi T, Myllylä R, Kivirikko KI, and Tryggvason K. Effects of streptozotocin diabetes, glucose, and insulin on the metabolism of type IV collagen and proteoglycan in murine basement membrane-forming EHS tumor tissue. J Biol Chem 257: 14914-14920, 1982 A.5 de Wet WJ, Pihlajaniemi T, Myers J, Kelly TE, and Prockop DJ. Synthesis of a shortened pro- 2(I) chain and decreased synthesis of pro- 2(I) chains in a proband with osteogenesis imperfecta. J Biol Chem 258: 7721-7728, 1983 A.6 Oikarinen J, Pihlajaniemi T, Hämäläinen L, and Kivirikko KI. Cortisol decreases the cellular concentration of translatable procollagen mRNA species in cultured human skin fibroblasts. Biochim Biophys Acta 741: 297-302, 1983 A.7 Myllylä R, Koivu J, Pihlajaniemi T, and Kivirikko KI.
  • Binding and Endothelial Tube Formation

    Binding and Endothelial Tube Formation

    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 7275–7280, June 1998 Biochemistry Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation SHAWN M. SWEENEY†,CYNTHIA A. GUY‡,GREGG B. FIELDS§, AND JAMES D. SAN ANTONIO†¶ †Department of Medicine and the Cardeza Foundation for Hematologic Research, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107; ‡Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455; and §Department of Chemistry and Biochemistry, and the Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, FL 33431 Edited by Darwin J. Prockop, MCP-Hahnemann Medical School, Philadelphia, PA, and approved April 14, 1998 (received for review January 12, 1998) ABSTRACT Cell surface heparan sulfate proteoglycan To localize more precisely the heparin-binding regions on type (HSPG) interactions with type I collagen may be a ubiquitous I collagen, we studied complexes between collagen monomers cell adhesion mechanism. However, the HSPG binding sites on and heparin–albumin–gold particles by electron microscopy type I collagen are unknown. Previously we mapped heparin (6), and we observed heparin binding primarily to a region on binding to the vicinity of the type I collagen N terminus by the triple helix near the procollagen N terminus. In collagen electron microscopy. The present study has identified type I fibrils, heparin–gold bound to the a bands region within each collagen sequences used for heparin binding and endothelial D-period, which is consistent with an N-terminal heparin- cell–collagen interactions. Using affinity coelectrophoresis, binding site on its monomers. The resolution of the mapping we found heparin to bind as follows: to type I collagen with technique was insufficient to assign heparin-binding function high affinity (Kd ' 150 nM); triple-helical peptides (THPs) to any particular protein sequence.
  • Human Bronchial Fibroblasts Exhibit a Mesenchymal Stem Cell Phenotype and Multilineage Differentiating Potentialities

    Human Bronchial Fibroblasts Exhibit a Mesenchymal Stem Cell Phenotype and Multilineage Differentiating Potentialities

    Laboratory Investigation (2005) 85, 962–971 & 2005 USCAP, Inc All rights reserved 0023-6837/05 $30.00 www.laboratoryinvestigation.org Human bronchial fibroblasts exhibit a mesenchymal stem cell phenotype and multilineage differentiating potentialities Federica Sabatini1,3, Loredana Petecchia1, Manuela Tavian2, Vanina Jodon de Villeroche´3, Giovanni A Rossi1 and Danie`le Brouty-Boye´3 1Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy; 2INSERM U506, IFR Andre´ Lwoff, Universite´ Paris XI, Villejuif, France and 3INSERM U602, IFR Andre´ Lwoff, Universite´ Paris XI, Villejuif, France Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate along different pathways including chondrogenic, osteogenic and adipogenic lineages. MSCs with a fibroblast-like morphology have been identified in human fetal lung. However, their frequency and characterization in human adult lung have not been yet evaluated. Therefore, we analyzed the mesenchymal phenotype and differentiation ability of cultured human adult bronchial fibroblast-like cells (Br) in comparison with those of mesenchymal cell progenitors isolated from fetal lung (ICIG7) and adult bone marrow (BM212) tissues. Surface immunophenotyping by flow cytometry revealed a similar expression pattern of antigens characteristic of marrow-derived MSCs, including CD34 (À), CD45 (À), CD90/Thy-1 ( þ ), CD73/SH3, SH4 ( þ ), CD105/SH2 ( þ ) and CD166/ALCAM ( þ ) in Br, ICIG7 and BM212 cells. There was one exception, STRO-1 antigen, which was only weakly expressed in Br cells. Analysis of cytoskeleton and matrix composition by immunostaining showed that lung and marrow-derived cells homogeneously expressed vimentin and nestin proteins in intermediate filaments while they were all devoid of epithelial cytokeratins. Additionally, a-smooth muscle actin was also present in microfilaments of a low number of cells.
  • Premature Aggregation of Type IV Collagen and Early Lethality in Lysyl Hydroxylase 3 Null Mice

    Premature Aggregation of Type IV Collagen and Early Lethality in Lysyl Hydroxylase 3 Null Mice

    Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice Kati Rautavuoma*†‡, Kati Takaluoma*†‡, Raija Sormunen§, Johanna Myllyharju*†, Kari I. Kivirikko*†, and Raija Soininen†¶ *Collagen Research Unit and Departments of †Medical Biochemistry and Molecular Biology and §Pathology, Biocenter Oulu, University of Oulu, 90014 Oulu, Finland Edited by Erkki Ruoslahti, The Burnham Institute, La Jolla, CA, and approved August 17, 2004 (received for review July 9, 2004) Collagens carry hydroxylysine residues that act as attachment sites LH3 is essential for the synthesis of type IV collagen during early for carbohydrate units and are important for the stability of development and, hence, for the stability of basement mem- crosslinks but have been regarded as nonessential for vertebrate branes (BMs). survival. We generated mice with targeted inactivation of the gene for one of the three lysyl hydroxylase isoenzymes, LH3. The null Materials and Methods embryos developed seemingly normally until embryonic day 8.5, Targeting of the Plod3 Gene and Generation of Mutant Mice. The but development was then retarded, with death around embryonic genomic clone used to clone the targeting construct was isolated day 9.5. Electron microscopy (EM) revealed fragmentation of base- from a 129SV mouse genomic library with human LH3 cDNA ment membranes (BMs), and immuno-EM detected type IV collagen (5) as a probe. The construct contains 1.2- and 5-kb genomic within the dilated endoplasmic reticulum and in extracellular arms and the ␤-gal-PGK-neo cassette inserted in-frame into exon aggregates, but the typical BM staining was absent. Amorphous 1. Embryonic stem cells were targeted, and mice were generated intracellular and extracellular particles were also seen by collagen by standard techniques.
  • Matrix Cross-Linking–Mediated Mechanotransduction Promotes Posttraumatic Osteoarthritis

    Matrix Cross-Linking–Mediated Mechanotransduction Promotes Posttraumatic Osteoarthritis

    Matrix cross-linking–mediated mechanotransduction promotes posttraumatic osteoarthritis Jin-Hong Kima,b, Gyuseok Leea, Yoonkyung Wona, Minju Leea, Ji-Sun Kwaka, Churl-Hong Chunc, and Jang-Soo Chuna,1 aSchool of Life Sciences, Cell Dynamics and Integrative Aging Research Centers, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; bDepartment of Biological Sciences, Seoul National University, Seoul 151-747, Korea; and cDepartment of Orthopedic Surgery, Wonkwang University School of Medicine, Iksan 570-711, Korea Edited by Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 23, 2015 (received for review March 22, 2015) Osteoarthritis (OA) is characterized by impairment of the load- In this study, we sought to determine molecular mechanisms bearing function of articular cartilage. OA cartilage matrix un- leading to ECM remodeling over the course of OA development dergoes extensive biophysical remodeling characterized by de- and to investigate how mechanical alterations in cartilage matrix creased compliance. In this study, we elucidate the mechanistic affect chondrocyte metabolism and regulate OA pathogenesis. origin of matrix remodeling and the downstream mechanotrans- duction pathway and further demonstrate an active role of this Results mechanism in OA pathogenesis. Aging and mechanical stress, the Aging-Associated Accumulation of Advanced Glycation End-Products two major risk factors of OA, promote cartilage matrix stiffening Drives Matrix Stiffening. Aging is one of the most prominent risk through the accumulation of advanced glycation end-products factors contributing to OA (4, 5). Aging processes have been and up-regulation of the collagen cross-linking enzyme lysyl implicated in elevating ECM stiffness in various tissues (9).