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Small Molecules Regulated Bone Resorption and Enzyme Activity in Osseous Cells Zhongyuan Ren

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Small Molecules Regulated Bone Resorption and Enzyme Activity in Osseous Cells Zhongyuan Ren Small molecules regulated bone resorption and enzyme activity in osseous cells Zhongyuan Ren To cite this version: Zhongyuan Ren. Small molecules regulated bone resorption and enzyme activity in osseous cells. Biomolecules [q-bio.BM]. Université Claude Bernard - Lyon I; Jilin University (Changchun, Chine), 2014. English. NNT : 2014LYO10291. tel-01128220 HAL Id: tel-01128220 https://tel.archives-ouvertes.fr/tel-01128220 Submitted on 9 Mar 2015 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. N° d’ordre: 291-2014 Année 2014 THESE EN COTUTELLE Pour obtenir le grade de Docteur délivré par Jilin University et Université Claude Bernard Lyon 1 Ecole Interdisciplinaire Sciences-Santé Spécialité : Biochimie Présentée et soutenue publiquement le 5 décembre 2014 Par Zhongyuan Ren Small Molecules regulated Bone Resorption and Enzyme Activity in osseous cells Rapporteurs : Dr. Joanna Bandorowicz Pikula Pr. Jean-Marie Ruysschaert Pr. Xiao Zha Membres du Jury : Dr. Saida Mebarek (Co-directrice) Pr. Yuqing Wu (Co-directrice) Pr René Buchet (Co-directeur) Pr. Xuexun Fang Dr. Pierre Jurdic Dr. Hong-Wei Li Pr. Xiao Zha 1 UNIVERSITE CLAUDE BERNARD - LYON 1 Président de l’Université M. François-Noël GILLY Vice-président du Conseil d’Administration M. le Professeur Hamda BEN HADID Vice-président du Conseil des Etudes et de la Vie M. le Professeur Philippe LALLE Universitaire Vice-président du Conseil Scientifique M. le Professeur Germain GILLET Directeur Général des Services M. Alain HELLEU COMPOSANTES SANTE Faculté de Médecine Lyon Est – Claude Bernard Directeur : M. le Professeur J. ETIENNE Faculté de Médecine et de Maïeutique Lyon Sud – Charles Directeur : Mme la Professeure C. BURILLON Mérieux Faculté d’Odontologie Directeur : M. le Professeur D. BOURGEOIS Institut des Sciences Pharmaceutiques et Biologiques Directeur : Mme la Professeure C. VINCIGUERRA Institut des Sciences et Techniques de la Réadaptation Directeur : M. le Professeur Y. MATILLON Département de formation et Centre de Recherche en Directeur : Mme. la Professeure A-M. SCHOTT Biologie Humaine COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE Faculté des Sciences et Technologies Directeur : M. F. DE MARCHI Département Biologie Directeur : M. le Professeur F. FLEURY Département Chimie Biochimie Directeur : Mme Caroline FELIX Département GEP Directeur : M. Hassan HAMMOURI Département Informatique Directeur : M. le Professeur S. AKKOUCHE Département Mathématiques Directeur : M. Georges TOMANOV Département Mécanique Directeur : M. le Professeur H. BEN HADID Département Physique Directeur : M. Jean-Claude PLENET UFR Sciences et Techniques des Activités Physiques et Directeur : M. Y.VANPOULLE 2 Sportives Observatoire des Sciences de l’Univers de Lyon Directeur : M. B. GUIDERDONI Polytech Lyon Directeur : M. P. FOURNIER Ecole Supérieure de Chimie Physique Electronique Directeur : M. G. PIGNAULT Institut Universitaire de Technologie de Lyon 1 Directeur : M. C. VITON Ecole Supérieure du Professorat et de l’Education Directeur : M. A. MOUGNIOTTE Institut de Science Financière et d'Assurances Directeur : M. N. LEBOISNE 3 Acknowledgements This thesis was prepared at the University Claude Bernard Lyon 1, UMR 5246 CNRS, UMR 5242 IGFL, in Lyon (France) and at Jilin University, Key Laboratory for Supramolecular Structure and Materials, in Changchun (China) under the co-supervision of Professor René BUCHET, Dr. Saida Mebarek and Professor Yuqing WU. I would like to thank them for their guidance, encouragement, and enthusiasm. I am very grateful Dr. Saida Mebarek. I would never have been able to finish my works and dissertation without her helps. A special thank is also addressed to Prof. Pierre Jurdic for giving me the opportunity to work in his laboratory of IGFL and for his guidance. I wish to thank following people for their suggestions, discussions, teaching and technical help: Jilin University (China): Laboratory ODMB (Lyon): Hongwei LI Anne BRIOLAY Yuan YUE Françoise BESSON Jian SUN Carole BOUGAULT Yang GAO Loïc J. BLUM Shi JIN David MAGNE Xiaoyu YUAN Leyre BRIZUELA Dong PAN Laurence BESUEILLE Jiao LIU Dina ABDALLAH Dongdong ZHENG Le Duy DO Xingpeng WANG Michèle BOSCH Teng ZHANG Maya FAKHRY Dingyi FU Abdelkarim ABOUSALHAM Pengfan GAO Renaud RAHIER Xingfeng REN Aurélie FÉLIZARD Laboratory of IGFL (Lyon): Justine BACCHETTA Fabienne COURY Nathalie DEMONCHEAUX Irma MACHUCA-GAYET Kélig PERNELLE 4 Many thanks to Prof. Jean-Marie Ruysschaert, Prof. Joanna Bandorowycz-Pikula and Prof. Xiao Zhafor having accepting to review my Ph. D thesis. I would like to thank to Dr. Geraldine Bechkoff for her pionneering work on infrared enzymatic assay. We thank Dr M.O Platt (Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA) for providing helpful advice on zymography. To my parents, Wei REN and Xiufang BI, My family, my friends, 5 CONTENTS CHAPTER I: Introduction---------------------------------------------------------------------------------------8 1. Bone Metabolism----------------------------------------------------------------------------------------------9 2. Bone resorption and formation------------------------------------------------------------------------------9 3. Communications between osteoclasts and osteoblasts-------------------------------------------------10 4. Cathepsin K is necessary for bone resorption in osteoclasts ------------------------------------------10 5. Inhibition of Cathepsin K for the treatment of Osteoporosis and Osteoarthritis -------------------11 6. Cathepsin K inhibitors --------------------------------------------------------------------------------------11 7. Biomarker of bone formation in osteoblasts: Alkaline phosphatases --------------------------------14 8. State of art of phosphatase activity assays---------------------------------------------------------------15 CHAPTER II: AIMS---------------------------------------------------------------------------------------------17 CHAPTER III: MATERIAL AND METHODS------------------------------------------------------------21 CHAPTER IV: RESULTS AND DISCUSSIONS ---------------------------------------------------------37 Part 1: Effects of Azanitrile Cathepsin K Inhibitors on Mineralization and Bone Resorption ------38 Part 2: Direct determination of pyrophosphate hydrolysis in Matrix Vesicles by Infrared Spectroscopy----------------------------------------------------------------------------------------------------49 Part 3: An infrared assay of the kinetics of phosphate-release from physiological substrates in living cells ------------------------------------------------------------------------------------------------------59 CHAPTER V: CONCLUSIONS AND REMARKS -------------------------------------------------------71 REFERENCES----------------------------------------------------------------------------------------------------74 LIST OF PUBLICATIONS-------------------------------------------------------------------------------------89 LIST OF PRESENTATIONS----------------------------------------------------------------------------------90 SUBSTANTIAL FRENCH SUMMARY-------------------------------------------------------------------- 91 FRENCH ABSTRACT-----------------------------------------------------------------------------------------108 6 ENGLISH ABSTRACT----------------------------------------------------------------------------------------109 7 Abbreviations α-MEM -α-Minimum Essential Media AA -ascorbic acid A-M-P - 2-amino-2-methyl-1-propanol AP -alkaline phosphatase APS - Ammonium persulfate AR-S -alizarin Red-S ATPase -adenosine triphosphatase β-GP -β-glycerophosphate bis-p-NPP -bis-p-nitrophenyl phosphate BCA -bisinchoninic acid assay Cat -Cathepsin CatK -Cathepsin K CKI -Cathepsin K inhibitor CPC - Cetylpyridinium Chloride DMEM -Dulbecco modified Eagles medium DMSO -Dimethyl Sulphoxide DTT -DL-Dithiothreitol E-64 -commercial cathepsin inhibitors ePPi -extracellular pyrophosphate EDTA -Ethylene Diamine Tetraacetic Acid FBS -fetal bovine serum FTIR -Fourier transformed infrared spectroscopy G-1-P -glucose-1-phosphate 8 HA -hydroxyapatite HEPES -2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid solution IC50 -concentration to inhibit half the enzymatic activity IR -infrared spectroscopy Km -Michaelis-Menten constant Ki -Inhibition constant M-CSF -Macrophage Colony Stimulating Factor MMP -matrix metalloprotease MTT -3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MVs -matrix vesicles NADP -Nicotinamide Adenine Dinucleotide Phosphate NPP1 -nucleoside triphosphate pyrophosphatase phosphodiesterase 1 OA -Osteoarthritis OB -Osteoblast OC -Osteoclast ODN -Odanacatib, inhibitor of cathepsin K (actually at clinical phase III for the treatment of osteoporosis) OP -Osteoporosis PAGE -polyacrylamide gel electrophoresis PBS -phosphate-buffered saline p-NPP -p-nitrophenyl phosphate Pi -inorganic phosphate PPi -inorganic pyrophosphate RANK-L -Receptor Activator for Nuclear Factor-κ B Ligand SCL -synthetic cartilage lymph 9 SDS -sodium dodecyl sulfate SEM -standard error
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