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Osteoactivin and Cd44 : a Novel Interaction Regulating Bone Cell Differentiation and Function

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Osteoactivin and Cd44 : a Novel Interaction Regulating Bone Cell Differentiation and Function OSTEOACTIVIN AND CD44 : A NOVEL INTERACTION REGULATING BONE CELL DIFFERENTIATION AND FUNCTION A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Gregory R. Sondag December 2015 © Copyright All rights reserved Except for previously published materials Dissertation written by Gregory R. Sondag B.S., Edinboro Univeristy of Pennsylvania, Edinboro, PA, USA 2010 M.S., Edinboro Univeristy of Pennsylvania, Edinboro, PA, USA 2011 Approved by Fayez Safadi___________________, Chair, Doctoral Dissertation Committee Walt Horton Jr.___________ ______, Members, Doctoral Dissertation Committee James Hardwick ________________, Werner Geldenhuys _____________, Min-Ho Kim __________________ _, Richard Meindl__________________, Accepted by Ernest Freeman_________________, Director, School of Biomedical Sciences James L. Blank__________________, Dean, College of Arts and Sciences TABLE OF CONTENTS TABLE OF CONTENTS ...................................................................................... III LIST OF FIGURES............................................................................................. VII LIST OF TABLES ............................................................................................ XIII LIST OF ABBREVIATIONS .............................................................................. XIV DEDICATION ..................................................................................................... XV ACKNOWLEDGEMENTS ................................................................................. XVI ABSTRACT ..................................................................................................... XVII CHAPTERS I. CHAPTER 1 INTRODUCTION ........................................................... 1-6 II. CHAPTER 2 LITERATURE REVIEW .............................................. 7-93 2.1 The Skeleton ..................................................................................... 7 2.2 The Composition of Bone ............................................................... 14 2.3 Cellular and Molecular Determinants of Bone................................. 24 2.4 Osteoporosis ................................................................................... 47 2.5 Osteopetrosis .................................................................................. 51 2.6 Bone Fracture ................................................................................. 62 2.7 Current and Future Therapies ......................................................... 66 2.8 Osteoactivin .................................................................................... 75 2.9 CD44 .............................................................................................. 85 2.10 Lipid Raft....................................................................................... 89 III. CHAPTER 3 MATERIALS AND METHODS ............................... 94-109 3.1 Cell Culture and Reagents .............................................................. 94 iii 3.2 Mice ................................................................................................ 95 3.3 MicroCT .......................................................................................... 95 3.4 ELISA ............................................................................................. 96 3.5 Tissue Preparation and Bone Histomorphometry ........................... 96 3.6 Osteoclast Culture .......................................................................... 97 3.7 TRAP Staining and Activity ............................................................. 98 3.8 In Vitro Osteoclast Resorption Assays ............................................ 98 3.9 Calvarial Bone Resorption Assay ................................................... 99 3.10 Immunofluorescence .................................................................. 100 3.11 Mass Spectroscopy Analysis ...................................................... 100 3.12 Immunoprecipitation ................................................................... 101 3.13 Western Blot Analysis ................................................................. 101 3.14 Transfection ................................................................................ 103 3.15 Quantification of Matrix Deposition and Mineralization ............... 103 3.16 RT-qPCR .................................................................................... 104 3.17 Lipid Raft Isolation ...................................................................... 106 3.18 Luciferase Analysis ..................................................................... 107 3.19 Cell Proliferation and Viability Assays ......................................... 107 3.20 Statistical Analysis ...................................................................... 109 IV. CHAPTER 4 RESULTS ............................................................ 110-198 4.1 Mesenchymal Stem Cell and Osteoblast Differentiation ............... 110 4.2 Osteoactivin has no effect on Osteoblast Proliferation or Viability 113 4.3 Osteoactivin enhances Osteloblast Differentiation ........................ 116 iv 4.4 Osteoactivin stimulates Osteoblast Related Gene Expression ..... 119 4.5 Osteoactivin Binds to and Interacts with CD44 in Osteoblasts ..... 123 4.6 CD44 Deficient Mice have Significantly Less Bone Mass in vivo .. 127 4.7 CD44 Deficient Mice have Significantly Less Bone Mass ex vivo . 137 4.8 Osteoactivin Stimulates MAPK signaling through CD44 ............... 141 4.9 Loss of CD44 Prevents Enhancement of Osteoactivin Mediated Osteoblast Differentiation Osteoblast Differentiation .............................................................. 145 4.10 Recombinant Osteoactivin Stimulates Osteoblast Differentiation through the the CD44-ERK Signaling Axis .................................................... 147 4.11 Osteoactivin co-localizes within the Lipid Raft ............................ 152 4.12 Osteoactivin Treatment Stimulates Clustering of CD44 and Caveolin-1 in Lipid Rafts Lipid Rafts .................................................................................. 154 4.13 Disruption of Lipid Raft Prevents CD44 and Caveolin-1 Clustering and Inhibits Inhibits Osteoactivin Mediated ERK Phosphorylation ................. 157 4.14 Recombinant Osteoactivin Stimulates Autophagy in Osteoblasts160 4.15 Osteoactivin Expression during Osteoclastogenesis .................. 163 4.16 Osteoactivin Inhibits Osteoclast Differentiation in a Dose Dependent Manner Manner ....................................................................................... 167 4.17 The Effect of Recombinant Osteoactivin on RANKL Mediated Signaling During OsteoclastogenesisDuring Osteoclastogenesis ......................................................... 171 4.18 Osteoactivin Binds to CD44 on the Osteoclast Cell Membrane .. 177 4.19 Osteoactvin Mediated Inhibition of Osteoclastogenesis is Abrogated in De ficient Osteoclasts CD44 Deficient Osteoclasts ........................................................ 182 v 4.20 Osteoactivin Inhibition of ERK Phosphorylation is Mediated through through CD44 .................................................................... 187 4.21 The Effect of Recombinant Osteoactivin in WT and CD44KO Cells in in vivo ......................................................................................... 190 4.22 Recombinant Osteoactivin Stimulation of Osteoclast Resorption in vitro is Independent Independent of CD44 ................................................................. 193 V. CHAPTER 5 DISCUSSION ........................................................ 199-215 5.1 Aim 1 ............................................................................................ 200 5.2 Aim 2 ............................................................................................ 206 5.3 Aim 3 ............................................................................................ 210 OVERALL SUMMARY AND CONCLUSIONS ......................................... 216-217 REFERENCES .......................................................................................... 218-249 vi LIST OF FIGURES Figure 2.1: Schematic diagram effects of hormones, dietary intake, and mechanical strain on the regulation of bone mass ............................................................ 8 Figure 2.2: Bone histomorphology of the distal femur ........................................ 11 Figure 2.3: Bone histomorphology of the osteoblast and osteoclast .................. 12 Figure 2.4: Diagram of the osteon, Haversian canals, and Volksmann canals ... 13 Figure 2.5: Diagram of the collagen-mineral interface in bone ........................... 17 Figure 2.6: Schematic diagram depicting the different stages and known markers of osteoblast differentiation .............................................................................. 34 Figure 2.7: Schematic diagram indicating the important receptor-ligand interactions critical for osteoblast differentiation and function ......................................... 35 Figure 2.8: Summary of bone remodeling cycle ................................................. 38 Figure 2.9: Diagram depicting the domains and glycosylation sites of full length, recombinant and mutant Osteoactivin (OA) ................................................. 76 Figure 4.1: Schematic diagram showing the potential lineages of mesenchymal stem
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