Osteoclastogenesis: Roles of Filamin A and SBDS, and their Regulation of Rho GTPases during Pre-Osteoclast Migration by Roland Leung A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Faculty of Dentistry University of Toronto © Copyright by Roland Leung 2012 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-79488-3 Our file Notre référence ISBN: 978-0-494-79488-3 NOTICE: AVIS: The author has granted a non- L'auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distrbute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. ii Osteoclastogenesis: Roles of Filamin A and SBDS, and their Regulation of Rho GTPases during Pre-Osteoclast Migration Doctor of Philosophy 2012 Roland Leung Faculty of Dentistry University of Toronto Abstract Osteoclasts are multinucleated, bone resorbing cells that carry out their function using specialized actin-based structures called actin rings and podosomes. Rho GTPases function as molecular switches that regulate the actin cytoskeleton in osteoclasts and many other cell types. Filamin A (FLNa) and SBDS are two proteins that have the potential to interact with both F-actin and Rho GTPases, and thus regulate osteoclast formation, differentiation, or function. We found that in FLNa-null pre-osteoclasts, activation of RhoA, Rac1, and Cdc42 was perturbed, leading to defective pre-osteoclast migration prior to fusion. Ablation of SBDS resulted in the blockage of osteoclast differentiation downstream of RANK and defective RANKL-mediated upregulation of Rac2 that is required for pre-osteoclast migration. Therefore, both FLNa and SBDS are required to coordinate Rho GTPase activation during osteoclastogenesis, in addition to a role for SBDS in osteoclast differentiation downstream of RANK. iii Acknowledgements I would like to take this opportunity to thank my family and friends for supporting me through this long journey. I am indebted to my supervisor and my lab colleagues for guiding me in my research. iv Table of Contents Chapter 1: Literature Review I. Osteoclasts and normal bone physiology…………………………………...……2 a. Origins…………………………………………………………………...……..2 b. Osteoclastogenesis…………………………………………………….……..…3 c. In vitro generation of osteoclasts…………………………………………...…..4 d. M-CSF and RANKL………………………………………………………....…4 e. Other important transcription factors……………………………………..……6 f. Osteoimmunology……………………………………………………………...7 g. NFATc1 – the master regulator of osteoclastogenesis…………………………8 2+ h. Intracellular Ca signalling…………………………………………………….8 i. Genes regulated by NFATc1……………………………………………...……9 j. Pre-osteoclast fusion…………………………………………………………..10 i. DC-STAMP……………………………...……………………………10 ii. Other candidate receptors and enhancers of cell fusion…………........11 k. Bone resorption………………………………………………………………..12 l. Integrin αvβ3……………………………………………………………….….14 m. Actin cytoskeleton and osteoclasts……………………………………………15 i. Podosomes…………………………………………………………….16 ii. Actin Ring………………………………………………………...…...16 iii. Regulation of podosomes and actin ring by phosphoinositide signaling…………………………………………....17 II. Rho GTPases……………………………………………………………………..18 a. Rac, Cdc42, and Rho GTPases…………………………………………..……18 b. Actin filament polymerization and cellular migration…………………….......19 c. Spatio-temporal regulation of Rac/Cdc42/Rho activation…………………….20 d. Rho GTPases and the osteoclast………………………………………………21 e. Post-translational prenylation…………………………………………………22 f. Actin rings, podosomes and Rho GTPases……………………………………22 g. Regulation of osteoclast physiology by Rho GTPases………………………..23 h. Reactive oxygen species and Rho GTPases…………………………………..25 III. Filamin A a. Discovery……………………………………………………………………...25 b. Filamin structure and actin crosslinking………………………………….......26 c. Functions of Filamin A i. Actin cytoskeleton stabilization…………………………………….…27 ii. Cellular migration……………………………………………………..27 iii. Syndromes resulting from FLNa mutations………………………..….28 iv. Filamin A binds actin network regulators………………………..……28 d. Regulation of Filamin………………………………………………………....29 v IV. Shwachman-Diamond syndrome……………………………………….……….29 a. Genetic features……………………………………………………………….30 b. SBDS function i. RNA metabolism and ribosome biogenesis………………………...…31 ii. Cell proliferation and viability…………………………………...……32 iii. Actin dynamics and chemotaxis…………………………………..…..32 c. Skeletal phenotype………………………………………………………..…...33 d. Oral manifestations of SDS…………………………………………………...34 V. Objectives and hypotheses………………………………………..……………..35 Chapter 2: Filamin A regulates monocyte migration through Rho GTPases during osteoclastogenesis…………………………………………………….….37 Chapter 3: Sbds is required for Rac2-mediated monocyte migration and signaling downstream of RANK during osteoclastogenesis………………...…74 Chapter 4: Rho GTPase techniques in osteoclastogenesis…………………………..…….104 Chapter 5: Summary and Conclusions…………………………………………………...122 Chapter 6: References…………………………………………………………………..….129 vi Abbreviations α-MEM α -minimal essential medium ABD Actin binding domain ADAM A disintegrin and metalloprotease Akt Thymoma viral proto-oncogene AP-1 Activator protein-1 BCA Bicinchoninic acid Bcl B-cell leukemia/lymphoma BMC Bone mineral content BMD Bone mineral density BMP Bone morphogenetic protein CA Constitutively-active CaM kinase II Ca2+/calmodulin-dependent protein kinase II DAG Diacylglycerol DAP12 DNAX-activating protein 12 DAPI 4'6,-diamidino-2-phenylinodole DC-STAMP Dendritic cell-specific transmembrane protein DN Dominant-negative DRF Diaphanous-related formin DXA Dual-energy x-ray absorptiometry ECM Extracellular matrix ELISA Enzyme-linked immunosorbent assay ERK Extracellular signal-regulated kinase F-actin Filamentous actin FBE Free-barbed-end FBS Fetal bovine serum FcRγ Fc receptor common γ subunit FITC Fluorescein isothiocyanate FLN Filamin fMLP Formyl-methionyl-leucyl-phenylalanine FOV Field of view FPP Farnesyl diphosphate FSD Functional secretory domain G-actin Globular actin GAP GTPase activating protein GDI Guanine nucleotide dissociation inhibitor GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GGPP Geranylgeranyl diphosphate GGTase Geranylgeranyl transferase vii GM-CSF Granulocygte-macrophage colony stimulating factor GST Glutathione S-transferase GTP Guanosine triphosphate HRP Horse radish peroxidase IFN Interferon Ig Immunoglobulin IL Interleukin IP3 Inositol-1,4,5-trisphosphate IPTG Isopropyl-beta-D-1-thiogalactopyranoside ITAM Immunoreceptor tyrosine-based activation motif JNK c-jun N-terminal kinase LIMK LIM kinase M-CSF Macrophage colony stimulating factor MFR Macrophage fusion receptor MITF Micro-ophthalmia-associated transcription factor MMP Matrix metalloproteinase N-BP Nitrogen-containing bisphosphonate NFATc1 Nuclear factor of activated T cell, cytoplasmic 1 NFκB Nuclear Factor kappa B OCG Osteoclastogenesis ODF Osteoclast differentiation factor OG Octyl-glucoside OPG Osteoprotegerin OPGL Osteoprotegerin ligand OSCAR Osteoclast-associated receptor PAK p21-activated kinase PAMPs Pathogen-associated molecular patterns PBS Phosphate-buffered saline PCR Polymerase chain reaction PFA Paraformaldehyde PI(3,4,5)P3 Phosphatidylinositol 3,4,5 trisphosphate PI(4,5)P2 Phosphatidylinositol 4,5 bisphosphate PI3-K Phosphatidylinositol 3-kinase PI4P-5K Phosphatidylinositol 4-phosphate 5-kinase PKA Protein kinase A PLC Phospholipase C PMSF Phenylmethylsulfonyl fluoride PPAR Peroxisome proliferator-activated receptor PTH Parathyroid hormone PVNH Periventricular nodular heterotopia viii qRT-PCR Quantitative real-time polymerase chain reaction RANK Receptor Activator of Nuclear Factor kappa B RANKL Receptor Activator of Nuclear Factor kappa B ligand RGD Arg-Gly-Asp ROCK Rho kinase ROS Reactive oxygen species SBDS Shwachman-Bodian-Diamond syndrome SDF-1 Stromal cell-derived factor-1 SDS Shwachman-Diamond syndrome SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis TGF Transforming growth factor