Molecular mechanisms implicated in bone resorption Dan Georgess To cite this version: Dan Georgess. Molecular mechanisms implicated in bone resorption. Cellular Biology. Ecole normale supérieure de lyon - ENS LYON, 2013. English. NNT : 2013ENSL0838. tel-00954294 HAL Id: tel-00954294 https://tel.archives-ouvertes.fr/tel-00954294 Submitted on 1 Mar 2014 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE en vue de l'obtention du grade de Docteur de l’Université de Lyon, délivré par l’École Normale Supérieure de Lyon Discipline : Sciences de la vie Laboratoire de biologie cellulaire et physiopathologie osseuse École Doctorale de Biologie Moléculaire Intégrative et Cellulaire présentée et soutenue publiquement le 01/10/2013 par Monsieur Dan GEORGESS _____________________________________________ Mécanismes moléculaires impliqués dans la résorption osseuse ______________________________________________ Directeur de thèse : Dr. Pierre JURDIC Après l'avis de : Pr. Stefan LINDER Dr. Isabelle MARIDONNEAU-PARINI Devant la commission d'examen formée de : Dr. Elisabeth GENOT Directeur de recherche, INSERM Membre Dr. Pierre JURDIC Directeur de recherche, INSERM Directeur Pr. Vincent LAUDET Professeur, ENS de Lyon Président Pr. Stefan LINDER Professeur, UKE Rapporteur Dr. Isabelle MARIDONNEAU-PARINI Directeur de recherche, INSERM Rapporteur 2 Acknowledgments Achievement is impossible without opportunity. First and foremost, I would like to express my gratitude to my mentor, Dr. Pierre Jurdic, for opening the gates of research when many would not and for his openness, patience and advice over the past years. Pierre, you have been a formidable boss and an exemplary role model. Thank you for all the discussions, stories and collaboration opportunities. The undeniable cornerstone of my doctoral training and accomplishments was my co- mentor, Dr. Irma Machuca-Gayet. Irma, thank you for your relentless support, constant honesty, thoughtful mentoring and continuously reliable solutions. To Pierre and Irma, I will be forever grateful for sculpting the scientist and person I am today. I would like to extend my thanks to Dr. Marlène Mazzorana for laying down the foundations of my thesis project, sharing her valuable knowledge and taking time to discuss and enlighten. Thanks are also enjoyably due to Mrs. Chantal Domenget for sharing her indispensable expertise and for listening to a student with open ears and, more importantly, an open heart. Over the years, I have greatly appreciated the healthy, friendly and supportive environment created by the members of the Jurdic lab: Dr. Marlène Gallet and the “Marlenettes” Pauline & Margot, Dr. Subramanya Pandruvada, Dr. Romain Daquin, Dr. Justine Bacchetta, Dr. Fabienne Coury, Dr. Kelig Pernelle, Mr. Jean Wach, Mrs. Lise Allard and Ms. Nathalie Demoncheaux. You have all been magnificent colleagues. I have also much enjoyed the dynamic environment of the IGFL which is first owed to our director, Pr. Vincent Laudet, and to young and friendly colleagues. I give my sincere thanks to Ms. Julie Carnesechi, Ms. Juliana Guttierez, Dr. Juliette Sailland, Dr. Cyrielle Billon, Ms. Alexa Sadier and Mr. Damien Curton. If being apart of a superb lab wasn’t in itself a wonderful experience, being apart of a community was beyond amazing. The T3-Net was more than a network of exceptional experts; it was the extended scientific family every student wishes for. I would like to particularly acknowledge Pr. Stefan Linder for his never-ending guidance and encouragement. I would also like to thank Dr. Elisabeth Genot for trusting me as a collaborator, for hosting me in Bordeaux and for her constant support. The T3-Net experience was extremely pleasant also thanks to young peers that have become friends. Thank you Mr. Pascuale Cervero, Mr. Karima Azouzi, Mr. Franco Klingberg, Mr. Nilesh Talele, Dr. Filipa Curado, Ms. Isabel Egana, Dr. Vineetha Vijayakumar, Mr. Paolo Ciufici and Dr. Vinoth Khandelwal. Together with Dr. Linder, I would like to specially acknowledge Dr. Isabelle Maridonneau-Parini, whom I admire for seminal work in the podosome field, for critical revision leading to significant enhancement of this manuscript. I would like to specially acknowledge Pr. Georg Schett, Dr. Jose Terrado-Vicente and Dr. Jean-Christophe Geminard for trusting me with wonderful collaborations. These experiences have not only been pleasant and fruitful, but also giant leaps in my scientific career. Before this thesis, I would only dream of applying for a doctoral position. These dreams became reality thanks to Dr. Andrei Popov who has taught me the essentials in 3 experimental biology along with the proactive perseverance necessary for a professional life. I would also like to thank Pr. Stefan Nonchev for seeing potential in me and for defending it. On a personal level, I would like to thank my parents Hossam and Penka Georgess. I owe everything I am to you. Thank you for your unconditional love and limitless belief in me. Thank you for teaching me hope and installing in me the means to realize my dreams. Samar, thank you for hope, love, support, motivation and inspiration. You are my sunshine in the storm. Aunt Salam, uncle Daher and Heba, thank you for all your support, encouragement and love. The Mediterranean-Far East connection – Olimpia, Imtiaz and Esber – thank you for being my home-away-from-home. The Welcome Committee co-founders – François and Florent – with its star artist Emilien and big sister Delphine, your friendship has brought much needed balance to my life. L’ancien and Super G – Laurent and Audrey – thank you for continuous support, heartfelt friendship and smiles. 4 Contents Abstract 7 Abbreviations 9 List of introductory figures 11 CHAPTER 1: INTRODUCTION I. BONE REMODELING 15 I.1. THE SKELETON: CELLS AND MATRIX 15 I.1.1. OSTEOBLASTS 15 I.1.2. OSTEOCLASTS 15 I.1.3. OSTEOCYTES 16 I.2. BONE PATHOLOGIES: THE PREVALENCE OF OSTEOPOROSIS 18 I.3. OSTEOIMMUNOLOGY 18 I.4. AUTOIMMUNITY IN BONE DISEASES: THE CASE OF RHEUMATOID ARTHRITIS 19 II. OSTEOCLAST DIFFERENTIATION 22 II.1. IDENTIFICATION OF OSTEOCLAST PRECURSORS: DATA FROM HUMAN AND MURINE MODELS 22 II.2. THE SITE OF OSTEOCLAST DIFFERENTIATION 25 II.3. RELEVANT CYTOKINES INVOLVED IN OSTEOCLAST DIFFERENTIATION 26 II.3.1. M‐CSF 26 II.3.2. RANKL/RANK/OPG TRIAD: 27 II.3.3. TNF‐Α/IL‐1 29 II.4. TRANSCRIPTION FACTORS INVOLVED IN OC DIFFERENTIATION AND FUNCTION 30 II.4.1. PU.1 30 II.4.2. PAX5 30 II.4.3. MITF 31 II.4.4. NF‐ΚB 31 II.4.5. C‐FOS AND AP‐1 COMPLEX 31 II.4.6. NFATC1 32 II.5. MATRIX­CYTOKINE CONVERGING PATHWAYS IN OSTEOCLASTOGENESIS 37 II.5.1. OSCAR‐RANKL AXIS: 37 II.5.2. INTEGRIN‐RANK AND INTEGRIN‐ MCSF AXES: 38 II.5.3. IS ADHESION REQUIRED FOR DIFFERENTIATION? 39 III. PODOSOME ORGANIZATION IN OSTEOCLASTS 42 III.1. GENERAL PRINCIPLES OF INTEGRIN ACTIVATION 42 III.2. RECRUITMENT OF ADHESION PLAQUE MOLECULES 44 III.3. THE PODOSOME SUBDOMAINS 45 III.3.1. THE PODOSOME CORE DEFINED BY CD44 45 III.3.2. THE PODOSOME CLOUD DEFINED BY SRC 45 III.3.3. THE PODOSOME CAP 46 III.4. UNCERTAINTY ABOUT SPATIO­TEMPORAL ORDER OF PODOSOME FORMATION 46 III.5. PODOSOME INTERNAL DYNAMICS: INTERPLAY BETWEEN POLYMERIZATION AND CONTRACTILITY 48 III.6. OC­SPECIFIC PODOSOME PROPERTIES 50 III.7. PODOSOME PATTERNING IN OSTEOCLASTS 51 III.7.1. STRUCTURAL AND KINETIC PROPERTIES 51 III.7.2. MOLECULAR MECHANISMS IMPLICATED IN PATTERNING 55 5 IV. BONE RESORPTION PROCESSES 59 IV.1. TRANSMIGRATION 59 IV.2. BONE DEGRADATION: TRAFFICKING, ACIDIFICATION, PROTEOLYSIS 60 IV.3. THE RESORPTION­MIGRATION CYCLE 63 CHAPTER 2: RESULTS 63 I. OC MIGRATION 69 I.1. INTRODUCTION AND RATIONALE: HOW DO PODOSOMES DRIVE OC MIGRATION? 69 I.2. PUBLICATION: PODOSOME RINGS DRIVE SALTATORY OSTEOCLAST MIGRATION 71 II. NOVEL REGULATORS OF BONE RESORPTION: FOCUS ON RHOE 79 II.1. INTRODUCTION AND RATIONALE: FINDING NEW REGULATORS OF THE ACTIN CYTOSKELETON IN OSTEOCLAST­MEDIATED BONE RESORPTION 79 II.2. PUBLICATION: COMPARATIVE TRANSCRIPTOMICS REVEALS RHOE AS A NOVEL REGULATOR OF BONE RESOPTION BY OCS 81 CHAPTER 3: DISCUSSION AND PERSPECTIVES 121 REFERENCES 131 ANNEX PUBLICATION 143 6 Abstract Bone remodeling is a physiological process by which old bone is replaced by new bone. Osteoclasts are multinucleated giant cells of the monocytic lineage. Their function is bone resorption, the first step of bone remodeling. The work of this thesis is in continuity with a theme long developed in our laboratory, that of the actin cytoskeleton organization in bone- resorbing osteoclasts. Our first study investigated the role of the podosome organization in osteoclast spreading, adhesion and migration. Our results showed that podosome patterning into rings exerted outward tension upon the substrate and thereby triggered cell migration. Through cycles of assembly, growth and alternating disassembly, rings promote a saltatory mode of migration universal to all osteoclasts. The main objective of this thesis, however, was dedicated to finding new genes that govern podosome patterning in resorption-related processes such as osteoclast migration and sealing zone formation. To find such new genes, we employed a differential transcriptomic analysis of osteoclasts and osteoclast-like cells that exhibit podosomes but are unable to resorb bone. Among a list of six genes highly and exclusively expressed in osteoclasts, we chose to investigate RhoE, a constitutively active GTP-binding protein known for its regulation of actin structures. We provided evidence, using primary RhoE-deficient osteoclasts, that RhoE activity is essential to bone resorption. We unveiled a new role for RhoE in the control of actin turnover in podosomes through a Rock-antagonistic function.