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Prolyl 4-Hydroxylases, Key Enzymes Regulating Hypoxia

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Prolyl 4-Hydroxylases, Key Enzymes Regulating Hypoxia D 1197 OULU 2013 D 1197 UNIVERSITY OF OULU P.O.B. 7500 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA SERIES EDITORS DMEDICA Ellinoora Aro ASCIENTIAE RERUM NATURALIUM Aro Ellinoora Senior Assistant Jorma Arhippainen PROLYL 4-HYDROXYLASES, BHUMANIORA KEY ENZYMES REGULATING University Lecturer Santeri Palviainen CTECHNICA HYPOXIA RESPONSE AND Docent Hannu Heusala COLLAGEN SYNTHESIS DMEDICA Professor Olli Vuolteenaho THE ROLES OF SPECIFIC ISOENZYMES IN SCIENTIAE RERUM SOCIALIUM THE CONTROL OF ERYTHROPOIESIS AND E SKELETOGENESIS University Lecturer Hannu Heikkinen FSCRIPTA ACADEMICA Director Sinikka Eskelinen GOECONOMICA Professor Jari Juga EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR Publications Editor Kirsti Nurkkala UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE, ISBN 978-952-62-0082-8 (Paperback) INSTITUTE OF BIOMEDICINE, ISBN 978-952-62-0083-5 (PDF) DEPARTMENT OF MEDICAL BIOCHEMISTRY AND MOLECULAR BIOLOGY; ISSN 0355-3221 (Print) BIOCENTER OULU; ISSN 1796-2234 (Online) CENTER FOR CELL-MATRIX RESEARCH ACTA UNIVERSITATIS OULUENSIS D Medica 1197 ELLINOORA ARO PROLYL 4-HYDROXYLASES, KEY ENZYMES REGULATING HYPOXIA RESPONSE AND COLLAGEN SYNTHESIS The roles of specific isoenzymes in the control of erythropoiesis and skeletogenesis Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Auditorium A101 of the Department of Anatomy and Cell Biology (Aapistie 7 A), on 1 March 2013, at 12 noon UNIVERSITY OF OULU, OULU 2013 Copyright © 2013 Acta Univ. Oul. D 1197, 2013 Supervised by Professor Johanna Myllyharju Reviewed by Doctor Erinn Rankin Docent Anna-Marja Säämänen ISBN 978-952-62-0082-8 (Paperback) ISBN 978-952-62-0083-5 (PDF) ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2013 Aro, Ellinoora, Prolyl 4-hydroxylases, key enzymes regulating hypoxia response and collagen synthesis. The roles of specific isoenzymes in the control of erythropoiesis and skeletogenesis University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute of Biomedicine, Department of Medical Biochemistry and Molecular Biology; Biocenter Oulu; Center for Cell-Matrix Research, P.O. Box 5000, FI-90014 University of Oulu, Finland Acta Univ. Oul. D 1197, 2013 Oulu, Finland Abstract Oxygen deprivation (hypoxia) is related to many disease conditions, such as anemia, but is also a critical regulatory signal during normal development. Cellular responses to hypoxia are largely mediated through alterations in gene regulation brought about by the transcription factor known as hypoxia inducible facor (HIF). One of the most extensively studied systemic consequences of hypoxia is the induction of red blood cell production, erythropoiesis, which occurs through a HIF- dependent increase in erythropoietin (EPO) gene expression. The amount of HIF in cells is regulated by three HIF prolyl 4-hydroxylases (HIF-P4Hs) while a fourth P4H possessing a transmembrane domain (P4H-TM) is able to act on HIF at least in vitro. The putative role of P4H- TM in regulating erythropoiesis is studied here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm null and wild-type mice. By comparing the observed effects with those seen in FG-4497 treated hypomorphic Hif-p4h-2 and Hif-p4h-3 null mice, it is demonstrated for the first time that P4H-TM is involved in the regulation of Epo production in the mammalian kidney, but not in the liver. Long bones are formed via endochondral ossification, in which a cartilaginous template, the growth plate, is first laid down and then replaced with bone. The growth plate is rich in extracellular matrix (ECM) and contains a hypoxic central region in which HIF has been shown to regulate chondrocyte function. Importantly, growth plate chondrocytes are highly active in collagen synthesis. Collagen prolyl 4-hydroxylases (C-P4Hs I-III) provide collagen molecules with thermal stability and are thus necessary for the formation of a proper ECM. Through an in vitro approach it is demonstrated that hypoxia increases the amount and activity of C-P4H in primary mouse epiphyseal growth plate chondrocytes in a HIF-1-dependent manner. Lastly, it was set out to characterize mouse lines with complete inactivation of C-P4H-II with or without partial inactivation of C-P4H-I. A significant reduction in the total amount of C-P4H and its activity was found to result in mild chondrodysplasia and altered bone properties. The above mouse models provided new information on the specific in vivo roles of the C-P4H isoenzymes I and II. Keywords: cell hypoxia, chondrocytes, erythropoietin, hypoxia-inducible factor, prolyl 4-hydroxylase Aro, Ellinoora, Prolyyli-4-hydroksylaasit solujen hypoksiavasteessa sekä kollageenisynteesissä. Eri isoentsyymeiden roolien karakterisointi erytropoieesissa ja luuston muodostuksessa Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Biolääketieteen laitos, Lääketieteellinen biokemia ja molekyylibiologia; Biocenter Oulu; Center for Cell-Matrix Research, PL 5000, 90014 Oulun yliopisto Acta Univ. Oul. D 1197, 2013 Oulu Tiivistelmä Kudosten alentunut happipitoisuus (hypoksia) liittyy osana moniin elimistön patologisiin tiloi- hin, kuten anemiaan. Lisäksi se on tärkeä säätelytekijä normaalin yksilönkehityksen aikana. Jot- ta solut havaitsisivat hypoksian ja reagoidakseen siihen, on niille kehittynyt säätelyjärjestelmä, jossa hypoksiassa indusoituva transkriptiotekijä, HIF, on tärkeässä asemassa. Yksi merkittävin HIF:n indusoima systeeminen vaikutus elimistössä on punasolujen tuotannon, erytropoieesin, kiihtyminen. Sitä tapahtuu erytropoietiinia koodittavan geenin (EPO) lisääntyneen ilmentymi- sen kautta. HIF-tekijän määrää soluissa säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4Ht 1-3). Transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) tiedetään myös vai- kuttavan HIF-tekijän määrään soluissa in vitro, mutta sen vaikutusta nisäkkään erytropoieesiin ei ole aiemmin tutkittu. Käyttämällä hyväksi kolmea eri transgeenista hiirilinjaa (P4h-tm-/-, Hif- P4h-2gt/gt, Hif-p4h-3-/-) ja HIF-P4H entsyymeitä inhiboivaa lääkeainetta, FG-4497, tässä työssä osoitettiin ensimmäistä kertaa, että P4H-TM osallistuu nisäkkään Epo-hormonin tuoton sääte- lyyn. Pitkät luut muodostuvat endokondraalisen luutumisen kautta. Siinä ensin muodostuu rustoi- nen malli, kasvulevy, joka vähitellen korvaantuu luukudoksella. Kasvulevyn sisin kerros on sen soluille, kondrosyyteille, hypoksinen kasvuympäristö. HIF:illä on todettu olevan tärkeä rooli kondrosyyttien toiminnan säätelijänä. Kasvulevyn soluvälitila sisältää runsaasti kollageeneja. Kollageenin prolyyli-4-hydroksylaasit (C-P4Ht I-III) ovat avainasemassa kollageenien biosyn- teesissä ja siten niiden toiminta on välttämätöntä kestävän soluvälitilan muodostumiselle. Käyt- tämällä in vitro menetelmiä, tässä työssä osoitettiin, että hiiren epifyseaalisten kasvulevyjen kondrosyyteissä hypoksia lisää C-P4H:n määrää ja aktiivisuutta HIF-tekijästä riippuvalla meka- nismilla. Eri C-P4H-isoentsyymeiden toiminnasta ja merkityksestä in vivo tiedetään vain vähän. Tässä työssä karakterisoitiin hiirilinja, jossa C-P4H-II on täysin inaktiivinen, ja hiirilinja, jossa lisäksi C-P4H-I on osittain inaktiivinen. Merkittävästi alentuneen C-P4H:n aktiivisuuden todet- tiin aiheuttavan hiirimallissa lievän kondrodysplasian sekä heikentyneet luun ominaisuudet. Asiasanat: erytropoietiini, hypoksiaindusoituva tekijä, kondrosyytit, prolyyli-4- hydroksylaasi, soluhypoksia Acknowledgements This work was carried out at the Institute of Biomedicine, Department of Medical Biochemistry and Molecular Biology, University of Oulu, during the years 2006-2012. I wish to express my deepest gratitude to Professor Johanna Myllyharju for the opportunity to learn the basic skills of science and eventually to prepare my doctoral thesis under her excellent supervision. I thank Professor Peppi Karppinen most warmly for her guidance and her collaboration in the first project and Academy Professor Emeritus Kari Kivirikko for his impact on the projects along the way and his many innovative ideas. I am also grateful to Professor Ernestina Schipani for her pleasant collaboration and help with the second and third project. I am particularly grateful for the opportunity to visit Professor Schipani’s laboratory at Massachusetts General Hospital-Harvard Medical School. Those two visits were not only scientifically productive but also became my most inspiring and unforgettable memories of life as a young medical student. I appreciate the work of Professor Emeritus Ilmo Hassinen, Professor Taina Pihlajaniemi, Professor Seppo Vainio, Docent Minna Männikkö, Docent Aki Manninen and Dr. Lauri Eklund in providing wonderful working conditions, and that of Auli Kinnunen, Pertti Vuokila and Risto Helminen in making everyday practical life in the department so much easier. Antti Salo, Anu Laitala and Joni Mäki deserve thanks for all their advice and help in the lab and also for happy cooperation on the projects. I also thank Professor Eeva-Riitta Savolainen, Professor Juha Tuukkanen, Docent Raija Sormunen, Docent Raija Soininen, Dr. Ilkka Miinalainen, Mikko Finnilä and all the other co-writers for their valuable contributions. I am especially grateful to Minna Siurua for her persistent efforts with the everyday technical laboratory work and for being such great company and a friend for
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