ELUCIDATING THE ROLE OF INTEGRIN-EXTRACELLULAR

MATRIX PROTEIN INTERACTIONS IN REGULATING

OSTEOCLAST ACTIVITY

by

Azza Gramoun

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of the Faculty of Dentistry

University of Toronto

© Copyright by Azza Gramoun 2010

ii

Elucidating the Role of Integrin-Extracellular Matrix Protein Interactions in

Regulating Osteoclast Activity

Azza Gramoun

Doctor of Philosophy

Faculty of Dentistry

University of Toronto

2010

ABSTRACT

Millions of people around the world suffer from the debilitating effects of inflammatory bone

diseases characterized by excessive bone loss due to an increase in osteoclast formation and

activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and

disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the

disease progression. The interaction between osteoclasts and the external milieu comprised of

extracellular matrix proteins through integrins is essential for modulating the formation and

activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the

extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the

role of v3 in this process. In primary rabbit osteoclast cultures, blocking the integrin v3

using Vitaxin, an anti-human v3 antibody, decreased osteoclast resorption by decreasing

osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when

osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated

conformation of the integrin v3. Using the RAW264.7 cell line, the effects of the matrix

proteins fibronectin and vitronectin on osteoclast activity were compared to those of

osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed

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compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-

osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast,

fibronectin induced resorption through increasing resorptive activity per osteoclast in

comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an

increase in the pro-inflammatory cytokines nitric oxide and IL-1 Crosstalk between the

signalling pathways of nitric oxide and IL-1was suggested by the ability of the nitric oxide

inhibitor to decrease the level of IL-1 which occurred exclusively on fibronectin. Osteoclasts

on fibronectin also had a compact morphology with the smallest planar area while vitronectin

increased the percentage of osteoclast with migratory morphology and osteopontin induced

osteoclast spreading. The increase in compact morphology on fibronectin was associated with a

decrease in extracellular pH. Low extracellular pH was found to increase the total time

osteoclasts spend in a compact phase. These results show that matrix proteins differentially

regulate osteoclast formation, activity and morphology.

iv

“If we knew what we were doing,

it wouldn't be called research, would it?”

-Albert Einstein

v

ACKNOWLEDGMENTS

Special thanks to Mom and Dad for giving me the chance to embark on this wonderful

adventure seven years ago where not only did I get to learn about science and to explore the

world, but where I also learnt the most about life and ultimately myself. Throughout the years,

their moral and financial support enabled me to continue even through the toughest of times. I

would like to specifically thank Dad who by being the great person and scientist he is, has

taught me to love science. Through his dedication to and perseverance in research, I have learnt

never to give up in the face of obstacles.

I would like to thank Dr. Morris Manolson for being not only a supervisor but also a

mentor for me through the course of my PhD in his lab. I will forever remain indebted for the

leap of faith he took in accepting me as student without ever having met me in person. I am

grateful for all that he has taught me about research and would only hope to be able to put this

knowledge to good use.

To my advisory committee members: Dr. Myron Cybulsky, Dr. Johan Heersche and the

late Dr. Jaro Sodek, I thank you for all your guidance and constructive criticism that helped

shape my research project into something of which I am proud. Special thanks to the late Dr.

Jaro Sodek for the original hypothesis about the effects of extracellular matrix proteins on

osteoclasts, and for always dedicating the time to discuss my results with me.

To my wonderful labmates, I thank you for teaching me all that I know technically in

cell biology. I appreciate your patience and understanding and I hope I was not at any point a

burden on you.

To my beloved friends and family, thank you for believing in me even at the moments

when I had ceased to believe in myself. To Suzie Larouche, my roommate, I thank you for all

vi

your help and support. Your usual words of “You will do it Azza” were at times hard to believe

yet they eventually came true. To my best friend Rania Nada, you are my rock. I could always

find both the encouragement and the nudging I sometimes needed to quit whining and get back

on track. The countless conversations, songs of the day, stories and emails we have exchanged

over the years are precious and I will always hold them dear to my heart. Special thanks to my

trainer Mike Siaflekis. He taught me to endure not only in training, but also in writing.

vii

TABLE OF CONTENTS

Abstract ii

Acknowledgments v

Table of Contents vii

Original Contributions by the Author xii

List of Figures and Tables xiv

Abbreviations xvii

1. Introduction 1

1.1 The Structure and Function of Bone 2

1.2 Bone Remodelling 4

1.2.1 Paracrine Regulation of Bone Remodelling by

Pro-resorptive Factors 5

1.3 Osteoclast Differentiation and Its Associated Signalling Pathways 7

1.3.1 M-CSF Induced Signalling Pathways 8

1.3.2 RANKL Induced Signalling Pathways 9

1.3.3 ITAM-associated Receptor Induced Signalling Pathways 12

1.4 Mechanism of Osteoclastic Bone Resorption 13

1.5 Dynamics of Osteoclast Attachment and Morphological Changes 17

1.6 Matrix/Integrin Interactions and Their Effects on Bone Homeostasis 21

1.6.1 Integrin Structure and Function 21

1.6.2 Integrin v3 and Osteoclasts 25

1.6.3 The Molecular Mechanisms Involved in v3 Signalling 29

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1.6.4 Extracellular Matrix Proteins 32

1.6.4.1 Osteopontin 32

1.6.4.2 Fibronectin 35

1.6.4.3 Vitronectin 37

1. 7 Rationale and hypothesis 38

2. Effects of Vitaxin®, a Novel Therapeutic in Trial for Metastatic Bone Tumors, on

Osteoclast Functions in vitro 41

2.1 Abstract 42

2.2 Introduction 43

2.3 Materials and Methods 46

2.3.1 Materials 46

2.3.2 Rabbit Osteoclast Isolation 46

2.3.3 Preparation of Devitalized Cortical Bone Slices 47

2.3.4 Attachment Studies 47

2.3.5 Time-Lapse Microscopy 49

2.3.6 Resorption Studies on Bovine Bone Slices 49

2.3.7 Resorption Studies Using the Osteologic Bone Cell Culture System 49

2.3.8 Statistics 50

2.4 Results 51

2.4.1 Vitaxin Inhibits Osteoclast Resorption 51

2.4.2 Vitaxin Decreases the Number of Osteoclasts Attached to Plastic 51

2.4.3 Vitaxin Preferentially Inhibits the Attachment of Small

Osteoclasts (

ix

2.4.5 Vitaxin Inhibits Attachment but Not Early Stages of

Osteoclast Formation 53

2.4.6 Vitaxin Causes Retraction of Osteoclasts Only in the

Presence of M-CSF 53

2.4.7 Vitaxin's Effect on Attachment is Altered by Factors

Known to Change the Conformation of v3 54

2.5 Discussion 62

3. The Extracellular Matrix Protein Fibronectin Enhances Osteoclast Activity via Nitric

Oxide and Interleukin-1β Mediated Signalling Pathways 66

3.1 Abstract 67

3.2 Introduction 69

3.3 Materials and Methods 72

3.3.1 Materials 72

3.3.2 Immobilizing ECM Proteins on Tissue Culture Plates 73

3.3.3 RAW 264.7-Derived Osteoclast Cultures 73

3.3.4 Splenocyte Derived Osteoclast Cultures 75

3.3.5 Tartrate-Resistant Acid Phosphatase (TRAP) Staining 76

3.3.6 TRAP Activity Assay 76

3.3.7 Cell Viability Assay 76

3.3.8 Secreted TRAP5b Activity Assay 77

3.3.9 Nitrite and Nitrate Measurements 77

3.3.10 Resorption Studies 78

3.3.11 IL-1β ELISA 78

x

3.3.12 Flow Cytometry Analysis of Integrin Expression 79

3.3.13 Generation of FN Conditional Knockout Mice 79

3.3.13.1 Transgenic Mice 79

3.3.13.2 Histomorphometry 80

3.3.14 Statistics 80

3.4 Results 81

3.4.1 FN Reduces Osteoclast Formation without Affecting RAW

Cell Proliferation or Initial Attachment 81

3.4.2 FN Inhibits Pre-osteoclast Fusion and/or Migration but Not

Pre-osteoclast Recruitment 81

3.4.3 Assessment of Osteoclast Formation in an FN Conditional

Knockout Mouse Model 83

3.4.4 FN Increases Resorption by Increasing Both the Resorptive

Activity per Osteoclast and the Percentage of Resorbing Osteoclasts 84

3.4.5 FN Increases IL-1β in a NO Dependant Manner 85

3.4.6 Blocking v3 and 51 Has Different Effects on Osteoclast Number 85

3.5 Discussion 101

3.6 Conclusions 107

4. Bone Matrix Proteins and Extracellular Acidification; Potential Co-regulators of

Osteoclast Morphology 109

4.1 Abstract 110

4.2 Introduction 111

4.3 Materials and Methods 114

xi

4.3.1 Materials 114

4.3.2 RAW 264.7-Derived Osteoclast Cultures 114

4.3.3 Rabbit Osteoclast Isolation 115

4.3.4 Tartrate-Resistant Acid Phosphatase (TRAP) Staining 116

4.3.5 Assessment of Osteoclast Morphological Changes Using

Time-lapse Microscopy 117

4.3.6 Morphometrical Analysis of Changes in Osteoclast’s Morphology 117

4.3.7 Scanning Electron Microscopy 118

4.3.8 Intracellular pH Measurements 118

4.3.9 Statistics 119

4.4 Results 120

4.4.1 Osteoclasts Formed on FN, VN and OPN have Distinct

Morphologies and Planar Area 120

4.4.2 M-CSF Induces Osteoclast Spreading on FN but not on OPN 121

4.4.3 Extracellular pH of Cultures on FN and VN are Lower than

that on OPN 122

4.4.4 Osteoclasts Cycle between Spread and Compact Morphologies

and the Rate of these Changes Depends on Osteoclast Size

and Extracellular pH 123

4.5 Discussion 135

5. Summary and General Discussion 141

6. Future Directions 149

Appendix 154

References 161

xii

ORIGINAL CONTRIBUTION BY THE AUTHOR

Publications and submitted manuscripts resulting from this thesis work:

1. Gramoun A*, Shorey S, Bashutski JD, Dixon SJ, Sims SM, Heersche JNM, Manolson MF

(2007) “Effects of Vitaxin®, a novel therapeutic in trial for metastatic bone tumors, on

osteoclast functions in vitro”. The Journal of Cellular Biochemistry; 102(2): 341-352.

*Azza Gramoun wrote the manuscript and performed all experiments except: rabbit osteoclast

resorption on bone slices (figure 2.3) which was performed by Seema Shorey and time-lapse

microscopy of rabbit osteoclasts (figure 2.5) which was performed by Jill Bashutski.

2. Gramoun A*, Azizi N, Sodek J, Heersche JNM, Nakchbandi I, Manolson MF “The

extracellular matrix protein fibronectin enhances osteoclast activity via nitric oxide and

interleukin-1β mediated signalling pathways”. Submitted to the journal Arthritis Research and

Therapy; February 5 2010, Manuscript ID: 3404146443529669.

*Azza Gramoun wrote the manuscript and performed all experiments except:

Histomorphometrical osteoclast measurements on fibronectin conditional knockout mice (table

3.1) which were performed by Inaam Nakchbandi. Natoosha Azizi assisted in the preliminary

experiments conducted to compare the effects of the extracellular matrix proteins.

3. Gramoun A*, Goto T, Nordström T, Rotstein OD, Grinstein S, Heersche JNM, Manolson

MF “Bone matrix proteins and extracellular acidification; potential co-regulators of osteoclast

morphology”. Accepted with revisions in the Journal of Cellular Biochemistry; January 19

2010, Manuscript ID JCB-09-0710.

xiii

*Azza Gramoun wrote the manuscript and performed all experiments except: intracellular pH

measurements and changes in rabbit osteoclast morphology under different pH conditions

(figures 4.5 and 4.6) and (tables 4.1, 4.2 and 4.3) which were performed by Tetsuya Goto.

xiv

LIST OF FIGURES AND TABLES

Figure 1.1 Osteoclast signalling pathways activated during osteoclastogenesis 11

Figure 1.2 Schematic diagram of a bone-resorbing osteoclast 15

Figure 2.1 Vitaxin decreases osteoclast resorption on osteologic slides 56

Figure 2.2 Vitaxin decreases the attachment of small osteoclasts (OCs)

(

xv

Figure 3.5 FN increases resorptive parameters and NO production 92

Figure 3.6 Osteoclasts on FN coated osteologic discs have more sealing zones 95

Figure 3.7 IL-1β and NO production is increased on FN. Inhibition of IL-1β using

the NO synthase inhibitor L-NMMA suggests that NO is upstream of IL-1β 96

Figure 3.8 Exclusive blockade of v3 in osteoclasts differentiated on FN increases

osteoclast number 97

Figure 3.9 Blocking 5but not v3or CD44, decreases osteoclast number on FN

and its expression is highest on FN 98

Figure 4.1 Osteoclasts differentiated on FN, VN and OPN have different morphologies 126

Figure 4.2 Osteoclast morphology and planar area are modulated by the ECM

proteins FN, VN and OPN 127

Figure 4.3 M-CSF treatment causes osteoclast spreading on FN while

osteoclasts on OPN fail to spread 129

Figure 4.4 The effect of ECM proteins on extracellular pH of culture media 130

Figure 4.5 Phase-contrast and scanning electron micrographs demonstrate the

morphological cycling of an osteoclast at pH 7.0 131

Figure 4.6 Time course of morphological cycling of small and large osteoclasts

at pH 7.5 and pH 7.0 132

Figure 6.1 Osteoclasts and pre-osteoclasts degrade fluorescently labelled FN coating 151

Figure 6.2 Osteoclasts on a high density RGD coated nanopattern

exhibit multiple podosome rings 152

Figure 6.3 Osteoclasts on homogenous RGD coated surfaces exhibit normal

podosome arrangement 153

xvi

Figure A.1 Osteoclast formation is enhanced by sFN and suppressed by pFN 157

Figure A.2 Pre-osteoclast velocity and polarity are increased on sFN compared

to pFN and cFN 159

Figure A.3 Osteoclasts formed on sFN exhibit an atypical “sealing zone” like

attachment structure while those on cFN contain a typical podosome ring 160

Table 3.1 Histomorphometric osteoclast parameters in the FN conditional knockout

(cKO) Mx mouse line 100

Table 4.1 The Duration of the Compact and Spread Phases of Osteoclasts

at pH 7.0 and 7.5 133

Table 4.3 The Effects of Bafilomycin A1(BFA), Acetazolamide (AZ), DIDS and

Amiloride (Am) on the Duration of Compact and Spread

Phases of Osteoclasts 134

xvii

ABBREVIATIONS

A domain von Willebrand factor A domain

ADMIDAS Adjacent to MIDAS

Akt RAC-alpha serine/threonine-protein kinase

Am Amiloride

-MEM -Minimum essential medium

ANOVA One way analysis of variance

AP-1 Activator protein-1

Arp 2/3 Actin related protein 2/3

AZ Acetazolamide

BCECF, AM 2',7'-bis- (2-Carboxyethyl)-5 (6)- carboxyfluorescein,

acetoxymethyl ester

Bcl-2 B-cell lymphoma 2

BFA Bafilomycin A1

BL Basolateral domain

BLNK B-cell linker protein

BMM Bone marrow macrophages

BMU Bone metabolic unit

BS Bone surface

BSA Bovine serum albumin

BSP Bone sialoprotein

CAII Carbonic anhydrase II

xviii

CaMK Calcuim/calmodulin-dependent protein kinase

c-Cbl Casitas B-lineage lymphoma

cFN Cellular fibronectin

cKO Knockout

ClC-7 Chloride channel-7

CREB Cyclic-AMP-responsive-element binding protein

CT Control

DAB 3,3'-diaminobenzidine tetrahydrochloride

DAP-12 DNAX activation protein-12

DAPI 4'-6-Diamidino-2-phenylindole

DC-STAMP Dendritic cell specific transmembrane protein

DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid

DMEM Dulbecco’s modified Eagle’s medium

ECM Extracellular matrix

EIIIA Extra type III repeat A

EIIIB Extra type III repeat B

EM Electron microscopy

ERK Extracellular regulated kinase

FBS Fetal bovine serum

FcR Fc receptor

FN Fibronectin

FRET Fluorescence resonance energy transfer

FSD Functional secretory domain

xix

GLA -carboxy glutamic acid

GRB2 Growth-factor-receptor-bound protein 2

HA hydroxyapatite

HEPES 4-[2-hydroxyethyl] piperazine- N'-[ethanesulfonic acid]

I-domain Insert domain

I-EGF Integrin epidermal growth factor

IIICS V connecting segment

IL-1 Interleukin-1

ITAM Immunoreceptor tyrosine-based activation motif

LIBS Ligand induced binding site

LIMBS Ligand induced metal binding site

L-NMMA L-NG-monomethyl arginine

MAPK Mitogen activated protein kinase

M-CSF Macrophage colony stimulating factor

MIDAS Metal ion dependent adhesion site

MITF Microphthalmia associated transcriptional factor

MMP-9 Matrix metaloprotiease-9

NFATc1 Nuclear factor of activated T cells c1

NFB Nuclear factor B

NO Nitric oxide

O/N Overnight

OC Osteoclast

Oc. N Osteoclast number

xx

Oc. S Osteoclast surface

ODF Osteoclast differentiation factor

OPG Osteoprotegrin

OPGL OPG ligand

OPN Osteopontin

OSCAR Osteoclast associated receptor

PBS Phosphate buffered saline

pFN Plasma fibronectin

PGE2 Prostaglandin E2

pHi Intracellular pH

PI3K Phosphoinositide 3-Kinase

PIR-A Paired immunoglobulin-like receptor A

PLC- Phospholipase C

PSI Plexin/semaphorin/integrin domain

PTH Parathyroid hormone

PTM Posttransitional modification

PyK2 Proline rich tyrosine kinase

RA Rheumatoid arthritis

RANKL Receptor activator of nuclear factor B ligand

RAW cells RAW264.7 cells

RGD Arg-Gly-Asp

RGDS Arg-Gly-Asp-Ser

RL Ruffled border

xxi

RT Room temperature

SD Standred deviation

SDGRG Ser- Asp-Gly- Arg-Gly

SEM Standard error of mean

sFN Superfibronectin

SH2 domain Src homology 2 domain

SIBLING Small integrin-binding ligands with N-linked glycosylation

SIRPβ Signal regulatory protein β

SL Sealing zone

SLP-76 SH2 domain containing leukocyte protein of 76kDa

Syk Spleen tyrosine kinase

TCP Tissue culture polystyrene

TNF Tumour necrosis factor

TRAF TNF receptor associated factor

TRANCE TNF-related activation-induced ligand

TRAP Tartrate resistant acid phosphatase

TREM-2 Triggering receptor expressed on myeloid cells 2

T-test Student T-test

V-ATPase Vacuolar ATPase

Vitamin D3 1, 25-dihydroxyvitamin D3

VN Vitronectin

Wasp Wiskott-Aldrich syndrome protein

WIP Wasp interacting protein

1

1. INTRODUCTION

Inflammatory bone diseases such as rheumatoid arthritis (RA) are prevalent metabolic

conditions characterized by progressive bone loss in the affected joints (1). Bone destruction in

arthritic joints occurs due to the uncoupling of the two events comprising the ongoing bone

remodelling process; bone formation by osteoblasts and bone resorption by osteoclasts. Both

processes are synchronized by inter and intracellular signalling events involving calcitropic

hormones, cytokines, growth factors and attachment receptors binding to the extracellular

matrix (ECM). Due to the abundance of pro-inflammatory cytokines prevalent in the micro-

environment in affected joints, the unbalance of bone remodelling is specifically due to an

increase in the formation and activity of osteoclasts (2, 3). Despite the recent advances in

arthritis treatments controlling different symptoms of the disease such as pain and inflammation,

bone loss resulting in permanent joint damage remains a more difficult problem to resolve.

The interaction between osteoclasts and the ECM is essential not only for their

attachment and survival but also for their function and it requires the integrin v3 (4). Thus,

functionally blocking the interaction between the integrin v3 and its ligands is one of the

methods utilized to prevent bone loss (reviewed by (5)). Although ECM proteins play a role in

wound healing and tissue repair, their elevation in arthritis is associated with pro-inflammatory

cytokine-like properties that amplify joint damage (6-8). In the first part of my thesis I focused

on studying the effects of an v3 blocking antibody known as Vitaxin and found that it

inhibited osteoclast resorption through impairing their attachment. I was also able to show that

integrin activation increases Vitaxin’s inhibitory effects on osteoclast attachment (chapter 2).

Fibronectin (FN) and vitronectin (VN) are two of the bone matrix proteins that have been shown

2

to be elevated in arthritis, yet their effects on osteoclast formation and function were not

investigated. I decided to ask if FN and VN differentially regulate osteoclast function and I

hypothesized that both proteins promote osteoclast formation and function similar to

osteopontin (OPN). I was able to show that both FN and VN decreased osteoclast formation

compared to OPN; however, FN stimulated osteoclast resorption and cytokine production

(chapter 3 and 4). In the following sections, I will review the topics relevant to bone, osteoclast

function and its interaction with the ECM proteins through integrins.

1.1 The Structure and Function of Bone

Bone is a multifunctional tissue that provides the body with the rigid scaffold essential

for shape and support against gravitational forces. As an integral element of the skeletal

framework, bones protect vital organs, facilitate motility and serve as the major store for

calcium and other minerals; thus contributing to homeostasis. It is estimated that approximately

99% of the body’s calcium content is stored in the skeleton. Through its porous structure, bone

simultaneously achieves the lightness needed for locomotion while providing a niche for bone

marrow cells and haematopoiesis. As a mineralized connective tissue, bone has a highly

complex and intricate structure. It is composed of collagenous and non-collegenous protein

meshwork embedded in a hydroxyapatite (HA) matrix. Bone possesses superior elastic

properties that are necessary to sustain the constant stresses, allowing it to reversibly deform

without reaching its fracture point (9, 10). It is both the structure and composition of this

mineralized extracellular matrix that endows the skeletal tissues with the needed strength and

rigidity without compromising its weight and flexibility.

Based on the mechanism of bone formation during development, bones of the skeleton

can be classified into two major groups; long bones (e.g. tibia and femur) and flat bones (e.g.

3

skull and vertebrae). Endocondoral ossification is the mechanism responsible for long bone

formation and it involves the replacement of a cartilage template by mineralized tissues. In

contrast, flat bones formation occurs directly by mesenchymal cell condensation at ossification

centres; a process known as intramembranous ossification.

The inorganic content of bone, mainly in the form of HA crystals, constitutes up to 50%

of the skeleton’s dry weight and gives it the required stiffness. Meanwhile, the resilience and

toughness of the skeletal tissues is conferred by collagen type I which accounts for almost 90%

of the organic content of bone. In addition to their physical properties, collagen fibres form an

interlaced three dimensional meshwork into which HA crystals are deposited, thus protecting

HA, which is the brittle part of the bone matrix.

The remaining 10% of bone’s organic matrix takes the form of a large number of non-

collagenous proteins composed of four major classes: glycoproteins (~7%), proteoglycans

(~1%), small integrin-binding ligands with N-linked glycosylation (SIBLING) (0.5%) and γ-

carboxy glutamic acid (GLA)-containing proteins. Some of the abundant bone glycoproteins are

osteonectin, tetranectin and the Arg, Gly, Asp (RGD) containing glycoproteins FN and VN.

Decorin and biglycan are chondroitin sulphate-containing proteins representing the

proteoglycans family in bone tissues. The SIBLING proteins are a large family of RGD

containing glycoproteins. Of those, OPN and bone sialoprotein (BSP) are the most relevant bone

sialoproteins. Osteocalcin is the most influential type in the GLA-containing protein category.

Because of HA’s ability to physically adsorb serum proteins, a several fold enrichment of

albumin and α2-HS-glycoprotein is seen in bone. Finally, other classes of proteins including

immunoglobulins, growth factors, cytokines and chemokines synthesized extrinsically and

locally can also be found bound to HA. In terms of function, non-collagenous proteins

contribute to bone quality mechanically, physically and metabolically. Mechanically, non-

4

collagenous proteins, through functioning as glue-like molecules, protect bone during loading

by absorbing and dissipating energy by breaking intrahelical collagen bonds and thus allowing a

microscopic increase in fibre length (11, 12). Physically, the majority of the bone non-

collagenous family members play a central role in apatite crystal nucleation. Through binding to

collagen, these matrix proteins dictate the shape, size and orientation of HA crystals and

consequently fibril formation. Therefore, it was not surprising to find that transgenic mice

deficient in many of these non-collagenous proteins exhibited bone phenotypes where the

quality and quantity of bone were compromised (13-18). To further enhance their functional

adaptability, bone matrix proteins and specifically collagen are differentially expressed between

long bones and flat bones. The expression patterns in both types of bone yield specific

biomechanical properties and render them more suited to their stress profiles (19). In addition to

the above mentioned physiomechanical properties, non-collagenous matrix proteins, and

specifically those belonging to the RGD-containing glycoprotein family, play an integral role in

bone metabolism. The RGD-containing glycoproteins regulate bone homeostasis not only

through providing the basis for many attachment related cell functions and mediating integrin

signalling transduction, but also through to their more recently discovered cytokine-like

properties through other receptors.

1.2 Bone Remodelling

Despite its metabolically static appearance, it is estimated that approximately 10% of the

total bone mass in an adult human is replaced per year through a physiological process known as

bone remodelling. Bone modelling and remodelling are closely related, yet different processes.

While bone modelling is the process by which bone is formed during growth leading to an

increase in the size and shape of bone, bone remodelling is the bone’s unique ability to self

5

repair microdamage. Throughout life, bone is constantly deformed under weight-bearing

stresses, causing the accumulation of microfractures and the deterioration of its biomechanical

properties, ultimately increasing its risk of fracture. In addition to being the mechanism for

microdamage elimination, bone remodelling enables bone to fulfill its functional demands by

adapting to the various dynamically changing biomechanical and physiological stimuli. As

opposed to bone modelling which is specifically linked to bone formation, the bone remodelling

cycle is composed of alternating bone resorption and bone formation cycles. The osteoblast, a

cell of mesenchymal origin, is responsible for bone formation, whereas bone resorption is the

exclusive function of the osteoclast. Together, the osteoblast and osteoclast form the bone

metabolic unit (BMU) (as reviewed by (20)). The osteocyte is the third cell type involved in

bone homeostasis. Events such as the microcrack formation, estrogen deficiency and

corticosteroid therapy signal osteocytes to undergo apoptosis (21, 22). Osteocyte apoptosis is

thought to be the first event triggering osteoclast resorption and consequently bone formation

(23, 24). In a remodelling cycle, the alternating bone resorption and formation cycles are

sequential; yet the molecular and cellular processes regulating those two events are not.

1.2.1 Paracrine Regulation of Bone Remodelling by Pro-resorptive Factors

Bone remodelling is a tightly governed process affected by systemic and local factors as

well as mechanical loading. The uncoupling of the anabolic and catabolic activities of bone cells

as a result of an increase in osteoclast number and/or activity is associated with bone loss and is

the prevalent cause for many bone disorders. The key players orchestrating osteoclast formation

and activation are the macrophage colony stimulating factor (M-CSF) and the tumour necrosis

factor (TNF) family member receptor activator of nuclear factor B (NFB) ligand (RANKL)

6

(25-28). In fact, it is well established that the ratio between RANKL and its decoy receptor

osteoprotegrin (OPG) determines the rate of bone turnover and its disruption is an indicator of

enhanced osteoclast resorption and bone erosion in arthritis (29-31). Systemic and local

osteoclastogenic factors modulate osteoclast function by acting upstream of RANKL and M-

CSF by regulating their expression in osteoblasts, stromal cells and activated T-cells.

Parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (vitamin D3), prostaglandin E2 (PGE2)

and corticosteroids are some of the important systemic factors (32-34). Additionally, the

production of the osteolytic cytokines interleukin-1 (IL-1), IL-6, IL-11, IL-12, IL-17 and IL-23

and growth factors TNF-α, granulocyte-macrophage colony stimulating factor and transforming

growth factor in the local bone environment has a stimulatory effect on osteoclasts (35). The

mechanisms by which these pro-resorptive factors induce osteoclast activation are complicated

and involve both direct and indirect interactions with osteoclasts. The effects of glucocorticoids

on osteoclast formation and resorption demonstrate a simultaneous direct and indirect

modulation by a pro-resorptive hormone. Glucocorticoids activate osteoclasts by increasing M-

CSF and RANKL and decreasing OPG expression via interacting with osteoblasts (30, 36).

Concurrently, they act directly on osteoclasts attenuating apoptotic signal and promoting their

survival (37). In contrast, PTH’s mode of action on osteoclasts is only indirect and is mediated

by an increase in RANKL/OPG ratio (31, 38, 39). Similar to glucocorticoids, IL-1 and TNF-α

act indirectly through osteoblasts upregulating RANKL (40), but they also synergistically

promote osteoclast differentiation and resorption together with RANKL (41, 42). Interestingly,

while osteoclast activation by osteoclastogenic factors can be direct and/or indirect, osteoclast

formation can exclusively occur in the presence of RANKL and M-CSF.

7

1.3 Osteoclast Differentiation and Its Associated Signalling Pathways

Osteoclasts are multinucleated terminally differentiated cells with the unique ability to

dissolve mineralized tissues. They are derived from haematopoietic myeloid precursors and thus

they share many phenotypical characteristics with monocytes and macrophages (43).

Osteoclastogenesis is a multistep process in which osteoclast precursors are recruited before

they fuse to form the mature osteoclast. Osteoclast differentiation is initiated by the contact

between osteoclastogenesis supporting cells (bone marrow stromal cells, osteoblasts and

synovial fibroblasts) (44) and osteoclast precursors (CFU-S) inducing their differentiation into

pre-osteoclasts (CFU-GM) (as reviewed by (45)). The binding of M-CSF and RANKL

expressed by any of these cells to their respective receptors RANK and c-Fms on myeloid

precursors is indispensible to the induction of osteoclast differentiation and resorption (46, 47).

Emerging evidence also identified immunoreceptors and other immunoreceptor tyrosine- based

activation motif (ITAM) associated receptors as the co-stimulatory partners of M-CSF and

RANKL (48). Subsequent to receptor ligation, the RANKL primed pre-osteoclasts fuse and give

rise to a multinucleated cell capable of resorbing bone. The phenotypic markers associated with

osteoclast maturation are tartrate resistant acid phosphatase (TRAP), the calcitonin receptor, the

integrin subunit 3, the chloride channel ClC-7, the cystine protease cathepsin K, matrix

metaloprotiease-9 (MMP-9) and the osteoclast associated receptor (OSCAR) (49-51).

Cell fusion is an integral part of osteoclast formation as multinucleation is a requirement

for efficient bone resorption. Several surface molecules have been implicated in osteoclast

fusion yet the exact mechanism of that process is not completely understood. Among these

molecules that are part of the fusion machinery are the macrophage fusion molecule also known

as signal-regulatory protein-α and its ligand CD47 and the transmembrane glycoproteins CD44

8

and CD200 (52, 53). The dendritic cell specific transmembrane protein (DC-STAMP) and the

d2 isoform of vacuolar (H+) ATPase (V-ATPase) V0 domain (Atp6v0d2) play an eminent role in

facilitating fusion as demonstrated by transgenic mice studies. Mice deficient in DC-STAMP

exhibited a total inhibition of pre-osteoclast fusion accompanied by a reduction in bone

resorption which resulted in an osteopetrotic phenotype, whereas osteoclast differentiation

remained unaffected (54). Similarly, deletion of the Atp6v0d2 led to the abrogation of osteoclast

fusion, attenuated their resorptive capacity and rendered them osteopetrotic (55). Both

molecules are significantly elevated during osteoclast differentiation and are under the

transcriptional regulation of the nuclear factor of activated T cells c1 (NFATc1), the master

transcriptional regulator during osteoclastogenesis (56).

1.3.1 M-CSF Induced Signalling Pathways

M-CSF is a membrane bound osteoclastogenic cytokine required during osteoclast

differentiation and multinucleation (25). This growth factor is expressed by many cells

including endothelial cells (57), however during osteoclastogenesis, cells of

mesenchymal/stromal lineage are its major source (44). In addition to its physiological role, M-

CSF is upregulated by TNF- during inflammatory bone loss and by tumour cells in both

soluble and bound forms (58-60). M-CSF plays a critical role in the initial steps of pre-

osteoclast differentiation from haematopoietic cells and subsequently promotes their

proliferation and survival. Its role in osteoclast formation was demonstrated by the osteopetrotic

phenotype of the op/op mouse. The op/op mouse harbours a mutation in the Csf1 gene which

ablates M-CSF production (26). In addition to its osteopetrotic phenotype, calvarial osteoclasts

from the op/op mouse could not support osteoclast formation in vitro (61). The M-CSF receptor,

9

c-Fms is member of the receptor tyrosine kinase super family present on osteoclast precursor

cells and its expression is regulated by transcriptional factor PU.1 (62). The M-CSF/c-Fms

interaction leads to the autophosphorylation of the receptor and transmits critical downstream

signals. The M-CSF-induced activation of growth-factor-receptor-bound protein 2(GRB2) is

responsible for the activation of the extracellular regulated kinase (ERK) (63), whereas

phosphoinositide 3 kinase (PI3K) acts upstream of Akt (RAC-alpha serine/threonine-protein

kinase) (64), both events enhance osteoclast precursors proliferation and survival respectively.

Through activating the microphthalmia-associated transcription factor (MITF), M-CSF further

promotes monocyte/macrophage osteoclast precursors survival via stimulating B-cell lymphoma

2 (Bcl-2) (65, 66). Most importantly, M-CSF signalling induces RANK expression by pre-

osteoclasts, an event essential for their proper maturation when primed by RANKL (67). In

collaboration with the integrin v3, M-CSF also modulates other osteoclast functions and

facilitates resorption; such as cytoskeleton reorganization and migration. These effects will be

discussed in section 1.6.3.

1.3.2 RANKL Induced Signalling Pathways

The indispensible differentiation factor RANKL is a type II membrane protein and a

member of the TNF family. RANKL is also known as TNF-related activation-induced ligand

(TRANCE), OPG ligand (OPGL) and osteoclast differentiation factor (ODF). In addition to

being the key regulator of osteoclast development and activation, RANKL is responsible for T-

cell mediated dendritic cell activation, lymph node organgenesis and lactating mammary

development during pregnancy (68). It was the observation that osteoclast formation in vitro

could only be achieved when hematopoietic cells were in contact with stromal/osteoblast lineage

10

cells by Suda’s group that lead to the discovery of this critical factor a year after its soluble

inhibitor the decoy receptor OPG was discovered (43, 44). As previously mentioned, multiple

hormones and osteolytic cytokines upregulate RANKL’s expression such as vitamin D3, PGE2,

PTH, glucocorticoids, IL-1, IL-6, IL-11 and TNF- in a paracrine manner (34). RANKL

deficient mice display severe osteopetrosis accompanied by arrested growth and lack of tooth

eruption. These mice are protected from arthritis associated bone loss and can be rescued by

recombinant RANKL injection (69, 70). All RANKL induced signalling pathways involve the

recruitment of one common adaptor molecule. TNF receptor associated factor (TRAF) 6 (71).

The interaction between RANKL and its receptor RANK causes its trimerization along with its

adaptor TRAF6. This is followed by downstream signalling cascades that activate the

transcription factor NFB, mitogen-activated protein kinses (MAPKs); p38 and the Jun N-

terminal kinase. This results in the activation and association of the two components of the

transcription factor activator protein-1 (AP-1); c-Jun and c-Fos (72, 73). These transcription

factors collaborate to induce the activation and nuclear translocation of the master switch of

osteoclastogenesis NFATc1 which inturn promotes its own transcription, autoamplifying its

own expression and results in the activation of a group of osteoclast-related genes and ultimately

osteoclast differentiation (74, 75). In parallel, Ca2+

signalling downstream of TRAF6 indirectly

causes the phosphorylation of phospholipase C (PLC and is responsible for the c-Fos

activation and its recruitment to the AP-1 complex (48, 76). NFATc1 is responsible for the

transcriptional regulation of the following osteoclast-specific genes: TRAP, calcitonin receptor,

3-integrin subunit, cathepsin K,OSCAR, DC-STAMP and Atp6v0d2 (49, 51, 56). Together

with NFATc1, the transcriptional regulation of these genes is also achieved in collaboration with

the transcriptional factors PU.1, MITF and AP-1 (77) as well as the cyclic-AMP-responsive-

11

Figure 1.1 Osteoclast signalling pathways activated during osteoclastogenesis.

Osteoclastogenesis requires the activation of three main signalling pathways downstream of

RANK, c-Fms and ITAM associated immunoreceptors. For more details please refer to section

1.3.

12

element binding protein (CREB) activated by calcium/calmodulin-dependent protein kinase

(CaMK) IV(78).

1.3.3 ITAM-associated Receptor Induced Signalling Pathways

ITAM-coupled receptors are responsible for co-stimulatory signals participating with

RANKL in initiating Ca2+

fluxes essential for osteoclast differentiation (48). ITAM is a

conserved motif present in the cytoplasmic domain of certain transmembrane adaptor molecules

that can be found coupled with immunoreceptors. These receptors and their associated adaptor

proteins are commonly found in cells of hematopoietic lineage including osteoclasts. In myeloid

cells, there are at least 20 ITAM-associated immunoreceptors such as OSCAR, triggering

receptor expressed on myeloid cells 2 (TREM-2), paired immunoglobulin-like receptor A (PIR-

A) and signal regulatory protein (SIRP). In contrast, there are only two ITAM-containing

adaptor proteins expressed by these cells; the DNAX activation protein-12 (DAP12) and Fc

receptor (FcR (79). More recently, the tyrosine receptor c-Fms and integrins 3 and 2 were

also found to associate with DAP12 and FcR indicating that they play a role in co-stimulatory

osteoclast signalling (80, 81). Despite the extensive studies that have delineated the ITAM

signalling cascades, the ITAM-associated receptor ligands are yet to be identified, with the

exception of FcRs which are known to bind immunoglobulins. The ITAM-associated

receptor/ligand binding phosphorylates the ITAM motif of their coupled adaptor molecules

DAP12 and FcR which activates and sequesters the spleen tyrosine kinase (SyK) (76). Syk

activation by ITAM adaptor proteins occurs simultaneously through RANKL signalling

pathway in collaboration with the immunoreceptors-mediated signalling. Ca2+

dependent

signalling activation through the RANKL and ITAM pathways occurs through PLC. PLC

13

activation is a complicated process that requires the formation of a signalling complex

composed of the Tec and BtK tyrosine kinases in combination with the adaptor proteins B-cell

linker protein (BLNK) and SH2 domain containing leukocyte protein of 76kDa (SLP-76) (82-

85). While BLNK and SLP-76 act downstream of the ITAM-associated receptors,

RANKL/RANK ligation is responsible for the activation of Tec and BtK and is dependent on

the c-Src tyrosine kinase (86). This represents another point where the two signalling pathways

converge. The activation PLC triggers Ca2+

fluxes that act through the calcium dependent

phosphatase calcineurin inducing the dephosphorylation and nuclear translocation of NFATc1

(75). Also, calcium oscillations indirectly mediate NFATc1 in a CaMK/c-Fos dependant

mechanism.

1.4 Mechanism of Osteoclastic Bone Resorption

Bone resorption is dependent on the osteoclast’s ability to polarize thereby creating three

functional membrane domains (figure 1.2). The domain with the most critical function to bone

degradation is the ruffled border (RL); which is a highly convoluted V-ATPase rich membrane

found adjacent to the bone surface and directly above the resorption lacuna (87). Opposite to the

ruffled border is the functional secretory domain (FSD), where transcytosis of bone degradation

products occurs (88, 89). The basolateral domain (BL) is the third osteoclast functional domain

and is located lateral to the functional secretory domain. Bone resorption requires tight

osteoclast attachment and dynamic cytoskeletal reorganization generating the compact or

polarized osteoclast morphology and the three essential functional domains. Although the

factors triggering these events are not clearly understood, matrix recognition by the integrin

v3 was shown to play a central role in this process (4, 90). In a resorbing osteoclast, tight

14

attachment is mediated by a complex structure known as the sealing zone (SL) or actin ring

(91). The sealing zone not only facilitates attachment but also creates a tight seal, isolating the

bone surface to be resorbed and thus creating the proper environment for mineral and organic

components of bone to be removed efficiently (92). Once this environment has been created, the

osteoclast’s resorption machinery consisting of H+ pumping V-ATPases and proteolytic

enzymes containing lysosomes are sequestered to the ruffled border where the H+ and the

enzymes are released creating a resorption lacuna within the sealing zone (93, 94). The

cytoplasmic carbonic anhydrase II (CAII) enzyme generates the protons transported by V-

ATPase at the ruffled border (95, 96). During the process of active proton transport by V-

ATPases, cellular pH homeostasis is maintained through the Cl -/HCO3

- exchanger and Na

+/H

+

antiporter while the Cl- channel ClC-7 works in parallel with V-ATPases retaining the cell’s

electroneutrality (97-101). The critical role of these enzymes and channels in osteoclast activity

is clearly demonstrated in diseases characterized by disruption of their activity. Several human

mutations have been reported in V-ATPase, ClC-7 and CA II, that result in a wide range of

osteopetrotic phenotypes (102-104). In addition to the hormonal and cytokine regulation of

osteoclast activity discussed before, other factors such as osteoclast size (defined by their

number of nuclei) and extracellular acidosis were found to promote resorption (105-108).

Patients with Paget’s disease and end-stage renal acidosis have hyperactive large multinucleated

osteoclasts (109, 110). Following the demineralization of bone surface, matrix degradation

occurs mainly through the proteolytic activity of the cystine protease cathepsin K. Cathepsin K’s

optimal acidic pH and its targeted transport in V-ATPase containing vesicles to the ruffled

border are evidence that it functions as the major collagenolytic enzyme (94, 111).

15

Figure 1.2 Schematic diagram of a bone-resorbing osteoclast. Actively resorbing osteoclasts are

highly polarized cells with three domains; the ruffled border is the most important of all three

and is formed by fusion of exocytotic vesicles containing V-ATPases, cathepsin K and CLC-7.

Targeted vesicular trafficking triggered by matrix/ integrin interaction induces the association of

these vesicles with microtubules and their subsequent delivery to the ruffled border. Bone

degradation products are transcytosed across the osteoclast to be released through the functional

secretory domain. CAII, carbonic anhydrase II; RL, ruffled border; BL, basolateral membrane;

FSD, functional secretory domain; SL, sealing zone.

16

This role is further confirmed by bone scelerosis and pycnodysostosis associated with human

mutations causing cathepsin K deficiency (112). While matrix metalloproteases (MMPs) such as

MMP-9 were implicated in bone matrix degradation, this role is not supported by their neutral

optimal pH and transient osteopetrosis exhibited by MMP-9 knockout mice (113, 114). MMP-9

was, however, shown to participate in the initiation of bone demineralization via removing the

collagenous layer off the bone surface as well as cleaning of lacunae initiating bone formation

(115). It has also been suggested recently that MMP-9’s role varies depending on the origin of

osteoclast population (116, 117). Another enzyme secreted by osteoclasts and correlating with

their resorptive activity is TRAP. Due to the high levels of the enzyme in osteoclasts, TRAP has

been used as a histochemical marker identifying the cells in vivo and in vitro (118). Two TRAP

isoforms are present in serum, TRAP5a and TRAP5b. While TRAP5a is the isoform produced

by macrophages and dendritic cells, TRAP5b is an osteoclast specific isoform that is

proteolytically cleaved and activated by cathepsin K and needs pH 5.8 for optimal activity (119,

120). Although TRAP5b is elevated in patients experiencing excessive bone loss, the exact role

of TRAP in bone resorption is not clear (121, 122). The targeted deletion of the TRAP gene in

bone has demonstrated that TRAP has a role in bone development and bone resorption. TRAP-/-

mice had significant bone phenotypes. These mice had shorter, broader flat and long bones with

thicker cortical bone with disorganized growth plate (123). These effects resulted in age

progressive osteopetrosis due to a defect in collagen metabolism involving both synthesis and

cleavage of collagen (123, 124). The diminished resorptive activity of the TRAP-/- osteoclasts is

mainly due to defects in the structure of the ruffled border and intracellular trafficking (125). It

is speculated that TRAP’s effects on osteoclast resorption are related to its role in transcytosis

and OPN processing (126). After being regarded for a long time as a resorption marker (127,

128), recent evidence indicates that TRAP5b is an osteoclast formation marker rather than a

17

resorption marker and its elevation is associated with an increase in osteoclast number seen in

many bone loss diseases (129-131).

1.5 Dynamics of Osteoclast Attachment and Morphological Changes

In osteoclasts, similar to other cells of hematopoietic origin, podosomes are the basic

unit of attachment (132, 133). This adhesion structure can also be seen in certain human

leukemia cells and other v-Src transformed cells (134, 135). Podosomes partake in cell

attachment, migration, matrix degradation and invasion (136-138). Ultrastructurally, a

podosome is composed of columnar actin filaments surrounding a small tubular invagination of

the plasma membrane perpendicular to the substrate’s surface (139). Numerous focal adhesion

and actin polymerization regulatory proteins including Wiskott–Aldrich syndrome protein

(Wasp), actin related protein 2/3 (Arp 2/3), vinculin, paxillin and talin are present within the

structure of the podosome (140-143). On a molecular level, podosomes share many of these

proteins with focal adhesions, the podosome’s counterpart found in fibroblast-like cells (144). In

addition to those common molecules, podosomes contain certain unique actin-binding proteins

such as gelsolin, dynamin and cortactin (145, 146). Together these proteins provide a highly

dynamic actin cytoskeletal assembly essential for polarization and migration. The arrangement

of podosomes into highly organized adhesion complexes depends on two factors: the degree of

osteoclast differentiation and matrix composition (147, 148). During early osteoclast

differentiation on glass or tissue culture polystyrene (TCP), podosomes are arranged in clusters

that are later organized into multiple short lived podosome rings. In mature osteoclasts, a stable

peripheral podosome belt is found with an average thickness of 2 μm and inter-podosome

distance of 500 ± 140 nm (147, 149). When the podosome belt was examined carefully using 3-

D confocal microscopy and environmental scanning electron microscopy, the F-actin dense

18

podosome core was located inside a less dense actin cloud made of polymerized actin

interconnecting branches (147, 149, 150). The centrifugal patterning and growth of podosome

clusters into rings which then fuse to form the peripheral podosome belt is controlled by the

polymerization of the acetylated microtubules. This was elegantly shown by the nocodazole-

induced depolymerisation of microtubules which was followed by disorganization of the

podosome belt (151-153). Based on these observations, two distinct actin subdomains were

defined in a podosome belt; the podosome or actin core and actin cloud (154, 155). Using the

Wasp interacting protein (WIP) -/- and Src

-/- osteoclasts, the existence of these two separate

domains was confirmed and the distribution of multiple podosome associated proteins and

adhesion receptors was determined. While the podosome core is absent in WIP -/-osteoclasts, no

actin cloud is seen in the Src -/- cells (154, 156, 157). It is worth noting that in Src

-/- osteoclasts

podosome superstructures can be rescued by kinase-dead c-Src expression, indicating that in

podosomes c-Src functions as an adaptor molecule and not as a kinase (153). After further

examination of the podosome belt, a molecular model was proposed where integrin v3 is

central for organizing the actin cloud and for linking the actin cytoskeleton to the extracellular

matrix through the adaptor proteins paxillin, vinculin and talin (157-159). To achieve this

function, the integrin v3 activates and complexes with c-Src, the proline rich tyrosine kinase

(PyK2) and casitas B-lineage lymphoma (c-Cbl) (146, 160). In the podosome core, CD44 which

is a cell surface single pass transmembrane proteoglycan that binds hyaluronan and OPN, plays

the main role in actin nucleation. CD44 fulfills this function by directly binding to and

activating WASP as well as other actin regulating proteins such as Arp2/3 and cortactin (154,

161). Despite their unique molecular makeup and actin organization, the two actin subdomains

play an additive role in osteoclast attachment as indicated by the ability of both WIP -/- and Src

19

-/- osteoclasts to attach (154, 157).

In contrast to the podosome belt seen on glass, osteoclasts on bone exhibit an adhesion

superstructure known as the sealing zone. Many studies have demonstrated that sealing zone

formation is triggered only by the mineral content of the substrate onto which the osteoclasts are

attached and not affected by a substrate’s matrix protein content (147). Using GFP tagged actin

expressing osteoclasts and immunofluorescent microscopy, the sealing zone was shown to be

composed of a thick continuous central actin band surrounded by an inner and outer vinculin

rings (91, 147, 150). Luxenburg et al. have found that the intensity of staining of actin, vinculin

and paxillin in the sealing zones is significantly higher than that measured in individual

podosomes seen in polarized osteoclasts or in a podosome belt on glass (158). Nonetheless,

levels of phosphorylated tyrosine in the sealing zone were significantly less than those measured

in individual podosomes and podosome belts. The origin of the sealing zone is a disputed topic.

Although, Saltel et al. earlier reported de-novo sealing zone formation, studies using high-

resolution electron microscopy by Luxenburg et al. and Geblinger et al. have shown beyond

doubt that the sealing zone has a structure that resembles that of a highly compacted podosome

belt (149, 150). In the study by Geblinger and colleagues, scanning electron micrographs clearly

reveal that the podosome is the building unit of a sealing zone and that they are connected to by

actin fibres (149). The average thickness of the sealing zone is 3-6 μm and the inter-podosome

distance is significantly smaller than it is on glass (250 ± 60 nm vs. 500 ± 140 nm respectively)

(149). The average sealing zone thickness and inter-podosome distance on calcite crystals and

bone were not significantly different. However, the ruffled border was less pronounced on

calcite crystals compared to bone (149). While the molecular composition of the sealing zone is

not significantly different from that of the podosome belt, the distribution of these molecules

and the effects of their deletion on sealing zone formation are different. In a sealing zone, the

20

adaptor molecules talin and vinculin are found encircling the actin condensation core rather than

localizing with it (148). Additionally, c-Src is found in the ruffled border and not the sealing

zone. Even though WIP deletion did not affect sealing zone formation and CD44 localization,

bone resorption by WIP -/- osteoclasts is impaired (154). Demonstrating c-Src’s critical role in

sealing zone formation, Src -/- osteoclasts are devoid of a sealing zone and have fewer

podosomes (157).

During an osteoclast’s life span, the cell goes through several resorption cycles before

undergoing apoptosis (162). A resorption cycle is a multistep process that is initiated when the

osteoclast attaches to the bone surface and undergoes rapid actin repolymerization to become

polarized with a compact cytoplasm (163). These changes result in the generation of the sealing

zone and ruffled border (91, 164). When the osteoclast is finished resorbing at one site, another

series of cytoskeletal rearrangements occur prompting the osteoclast to spread before it migrates

to another location and the cycle is repeated. Osteoclast migration involves attachment and

formation of lamellipodia on the leading edge and cell detachment on the trailing edge (165,

166). These changes result in the characteristic migrating osteoclast phenotype in which the

osteoclast has a dendritic-like morphology with podosome patches at the leading edge where

v3 and F-actin are present but are not localized. Thus, the osteoclast’s resorption cycle

corresponds to a morphological cycle with two main morphologies indicative of the function

performed by the osteoclast at a certain point. These morphologies are the polarized (compact)

and migratory morphologies with occasional transitional spread morphology in between.

Despite the lack of ruffled border in osteoclasts on glass (149), osteoclasts on glass can be seen

alternating between the polarized and migratory morphologies similar to those on mineralized

surfaces (167). This is further confirmed by our results presented in chapter 3. While only

21

matrix/integrin interaction is responsible for the formation of the sealing zone, RhoA GTPase

regulates the organization of the podosomes into a sealing zone or podosome belt (168). While

increased activation of Rho activity is needed for sealing zone formation, expression of

constitutively active RhoA is not sufficient to initiate sealing zone formation on glass (152,

169). Further proof of the importance of Rho GTPases in sealing zone formation in that when

Rho GTPases are inhibited in polarized osteoclasts, osteoclasts immediately depolarize and

spread (148). This is associated with the disappearance of the sealing zone and its replacement

by a podosome belt (148). Crosstalk between cytokine and cytoskeletal signalling pathways

regulates the sealing zone formation and osteoclast activation. Using a washaway –recovery

system, M-CSF, RANKL, IL-1 and TNF were found to directly trigger sealing zone formation

(170).

1.6 Matrix/Integrin Interactions and Their Effects on Bone Homeostasis

1.6.1 Integrin Structure and Function

Integrins are a superfamily of adhesion receptors that act as bidirectional gateways on

the cell surface mediating cell to cell and cell to matrix interactions (171). As heterodimeric

transmembrane proteins, they are composed of non-covalently bonded and chains. In

vertebrates, there are eighteen different subunits and eight different subunits, forming over

twenty four distinct integrins. This makes them the largest class of cell adhesion molecules, with

a highly diverse structure and function (172, 173). Despite their abundance, integrins have

specific non-redundant functions and bind to distinct yet overlapping ligands. Integrins have a

wide range of functions in both health and disease. These functions include embryonic

development, autoimmune responses, leukocyte trafficking, tumour growth and metastasis,

22

blood clot formation and retraction, mechano-transduction, angiogenesis, bone homeostasis,

inflammation and cell survival and apoptosis (173-179). Integrins’ unique roles are evident in

the distinct phenotypes resulting from the deletion of different and subunits. Many of these

transgenic deletions were embryonically lethal (3, 68v, 8 and some caused severe

developmental defects (45v and 8), while others exhibited discrepancies in hemostasis

(IIb2and 3), bone remodelling (3) and angiogenesis (1 and 3) (4, 180-182)(reviewed

and listed by (183, 184)).

The structure of integrin is well adapted to meet the demands of the complex and

dynamic nature of their functions. Structural and topological information have been generated

using X-ray crystal images, nuclear magnetic resonance, fluorescence resonance energy transfer

(FRET), electron microscopy (EM) and site specific mutagenesis. To act as linkers between the

cell cytoskeleton and the matrix, integrins possess a single span transmembrane domain, a short

cytoplasmic domain (40-70 amino acids) and a large and complex extracellular domain (185).

The extracellular domain of the chain (>940 amino acids) contains four or five domains, in

integrins containing the insert (I) domain which is also known as von Willebrand factor A (A)

domain (186, 187). These domains are the -propeller (188), the I/A domain, the thigh, the calf-

1 and calf-2 domains. Only half of the integrins contain the I-domain that is inserted in the -

propeller and in those integrins, the I-domain is the site of ligand binding (as reviewed by

(189)). The subunit on the other hand is shorter (640 amino acids) and contains eight domains

including an I-like domain, hybrid domain, the plexin/semaphorin/integrin (PSI) domain, four

repeating integrin epidermal growth factor–like (I-EGF) domains and T domain (190-192).

Both the I and I-like domains present in the and subunits contain a Rossmann fold with

metal ion dependent adhesion site (MIDAS) (186). From the crystal structure of integrin v3 it

23

was evident that the integrin can exist in a bent confirmation (192). In this confirmation the

most N-terminal fragments of the and chains fold forms what is known as the “headpiece”

while the rest of the extracellular domains forms the “tailpiece” (191-194). Most importantly,

the I like domain contains the MIDAS which is critical for regulation of ligand binding affinity

(187, 192, 195). Ligand binding occurs through a series of conformational changes in the ligand

binding domains induced by the binding of divalent cations and alternations in the MIDAS

(196). In integrins lacking the I domain such as v3, ligand binding occurs in an interface

formed by both the propeller and the I-like domain (197). Due to the conformational changes

associated with ligand binding, the ectodomain of integrin acquires three conformation states

corresponding to its ligand binding (192, 197). Prior to ligand binding, integrins exist in

equilibrium between these three activation states; the bent low affinity conformation, the

extended conformation with closed headpiece and the extended conformation with open

headpiece (198, 199). However, upon ligand binding, the ligand acts as a hatchet locking the

integrin in an activated position in a “switchblade” like extended confirmation. While the

classical outside-in integrin signal transduction occurs through ligand binding to the

ectodomain, inside-out signalling occurs by activation of certain intracellular signalling

pathways. The main extracellular factors regulating ligand binding and outside-in integrin

activation are type and concentration of divalent cations (200-202). While high concentrations

of Mn2+

and low concentrations of Ca2+

synergized with suboptimal Mg2+

are positive regulators

inducing integrin activation, high concentrations of Ca2+

negatively regulate integrin’s activity

(203-205). The effects of divalent cation on integrin activation and ligand binding are mediated

through MIDAS, ligand induced metal binding site (LIMBS) and adjacent to MIDAS

(ADMIDAS) (205, 206). Mg2+

, low concentrations of Ca2+

and high concentrations of Ca2+

24

competed by Mn2+

induce their regulatory effects through MIDAS, LIMBS and ADMIDAS

respectively (192, 197). Another extracellular factor triggering superactivation of integrins is a

low concentration of an integrin antagonist such as an RGD peptide (207). In contrast to

outside-in integrin activation, the mechanism of inside-out signalling transduction relies on

conformational changes and separation of the and cytoplasmic domains of integrins (199,

200). Talin, another integrin activator, plays a central role in this process. Activation of

intracellular signalling pathways downstream of growth factors results in the activation of talin.

Activated talin binds to the subunit, separating it from the subunit and causing the extension

and activation of the extracellular domains, locking the integrin in this confirmation and thereby

increases its ligand binding affinity (208). To underscore the specific roles of talin and Src

family kinases in integrin activation, it was found that mutations in the cytoplasmic domain of

3 that prevent the binding of talin abrogated inside-out integrin activation. In contrast,

mutations in the 3 Src binding domain resulted in inhibition of outside-in integrin activation

and the associated cytoskeletal changes (209). Ultimately, integrin activation and bidirectional

signalling result in lateral displacement of integrin and in a process known as integrin clustering

(210). Although the specific mechanism is not yet understood, it has been proposed that integrin

clustering is required to increase the avidity of the integrin (211). Furthermore, the small

GTPase Rap1 is involved in this process as indicated by the inhibition of IIb3 activation in

platelets when the enzyme is deleted (212).

1.6.2 Integrin v3 and Osteoclasts

25

Among the classes of adhesion receptors present in osteoclasts, integrins and most

specifically the integrin v3 play an indispensable role not only in osteoclast attachment but

also in differentiation and function. Several other integrins were identified in human osteoclasts,

among these the 21 collagen/laminin receptor and v1 the fibronectin/vitronectin receptor

(213-215). Bone marrow macrophage derived osteoclast precursors express M1, v5

(another vitronectin receptor) and 41 in vitro (216-218). Integrins v5 and the fibronectin

receptor 51 and potentially were also identified in avian osteoclasts (219, 220). Most

recently, the integrin 91 was found in osteoclasts bound to the matrix metalloproteinase

ADAM8 (221). Despite the presence of multiple integrins in mature osteoclasts, the integrin

v3 is the predominant osteoclast attachment receptor and is highly enriched in osteoclasts.

The integrin v3 is also known as the vitronectin receptor which is a misnomer since the

integrin binds several other matrix proteins such as OPN, FN, BSP, fibrinogen and denatured

collagen type-I (222, 223). Similar to other vcontaining integrins, v3’s interaction with

matrix proteins is through a common RGD domain. While the highest expression of v3 in

vivo is present in osteoclasts, the integrin’s expression is physiologically elevated in the placenta

where it was first isolated, and is present at a lower level in megakaryocytes, kidney, endothelial

cells and vascular smooth muscle cells (224). The expression of v3 is upregulated during

inflammation and bone metastasizing tumours (225, 226). Both aggregation and spreading of

pre-fusion osteoclasts are essential for de novo synthesis and proper surface expression of v3

in culture during osteoclastogenesis (227, 228). Interestingly, during osteoclast differentiation

the integrins v3 and v5 are reciprocally expressed on osteoclast precursors and mature

osteoclasts respectively. v5 is the main integrin in macrophages, however, during

osteoclastogenesis its expression progressively decreases and is gradually replaced by v3

26

(218, 229). This suggests the roles the two integrins play in regulating osteoclast formation are

opposite and that v5 has an inhibitory effect on osteoclastogenesis. This hypothesis was

corroborated when ovariectomized 5 null mice were found to be more prone to bone loss than

the control group due to enhanced osteoclast formation and resorption of the 5 -/- osteoclasts in

vitro and in vivo (230). In contrast, 3 deletion protects mice against ovariectomy-induced bone

loss (231).

Similar to other integrins, v3 exists in two conformations; active and inactive,

regulated by outside in and inside out signalling. In its high affinity (active) state, the integrin

exposes its ligand induced binding site (LIBS) and this state is the result of either ligand binding

extracellularly or growth factor activation intracellularly (232, 233). As previously mentioned,

v3 is found in the actin cloud of podosomes in osteoclasts on glass and on the basal

membrane and around the sealing zone on bone. Careful examination of v3 distribution in

light of its activation state revealed that the v3 present in podosomes on glass is in an inactive

low affinity conformation. Once activated, the integrin translocates from podosomes to the

lamellipodia on the leading edge, mediating and promoting osteoclast migration. On bone, the

activated form of v3 is present on the ruffled border while the inactive form surrounds the

sealing zone (232, 234).

The first evidence of the involvement of v3 in osteoclast activity was provided by the

experiments showing that the monoclonal antibody 13C2 inhibited bone resorption (235).

Further investigations identified v3 as the antigen of 13C2 and it was later shown that in

osteoclasts it is the major integrin (213, 236). Based on these findings, v3 became a novel

target for inhibition of bone loss. Consequently, strategies developed for the prevention of bone

loss revolved around interfering with the interaction between the RGD motif and v3 using

27

different methods such as blocking v3 antibodies, RGD peptides and mimetics and

disintegrins. Studies have confirmed that bone resorption was inhibited when any of these

methods were used in vitro (237, 238). In vivo, the disintegrin echistatin, the anti-rat 3

antibody (mAB F11) and RGD peptidomimetics reduced bone loss and serum calcium levels in

hypercalcemic mice fed low calcium diet, a PTH induced bone loss model and in

ovariectomized rats respectively (217, 239-241). The mechanism by which v3 inhibition

affects bone resorption is still controversial. While in vitro data has shown that RGD peptides

and echistatin impair osteoclast attachment and retraction, inhibition of v synthesis using an

antisense oligodeoxynucleotide demonstrated that the impairment of osteoclast attachment is

associated with induction of apoptosis signalling pathways through reducing the Bcl-2/bax ratio

(242). However, the in vivo findings contradicted the mechanism suggested by the in vitro

experiments. In echistatin treated mice, the number of osteoclasts was increased while

osteoclasts exhibited a normal morphology and no detachment could be seen (217, 241). Further

in vitro investigations revealed that echistatin inhibited bone resorption at concentrations that

did not affect osteoclast attachment and that it interfered with M-CSF induced osteoclast

migration (243, 244). This proposed mechanism is supported by data showing that v3

activation promotes osteoclast haptotactic migration to OPN (218). Other data implicate

specifically the ectodomain of the 3 subunit in this process (245).

Indisputable evidence of v3’s critical and direct role in bone metabolism arises from

the transgenic deletion of 3. 3 knockout mice have increased bone mass which progresses

into late onset osteopetrosis (4). Despite the 3.5 fold increase in their number, 3 null

osteoclasts are dysfunctional due to abnormalities in ruffled border formation. Consistent with

their osteopetrotic phenotype, these mice are also hypocalcemic (4). While mature osteoclasts

28

isolated from 3 -/- mice fail to exhibit a sealing zone, those formed in vitro from 3

-/-

precursors do. Nonetheless, significantly fewer 3 -/- osteoclasts are formed in vitro and the

osteoclasts are not able to form a normal ruffled border and therefore the size and number of

resorption pits they form are substantially diminished (4, 246). In addition to those resorption-

related defects, osteoclasts from 3 null mice fail to spread on RGD-containing substrates,

indicating disruption of the osteoclast’s ability to undergo cytoskeletal reorganization (4). 3

deletion also had an unexpected anti-apoptotic effect on osteoclasts due to lack of caspase-8

signalling that results in cell death (247). Furthermore, the deletion of the cytoplasmic tail of 3

impaired osteoclast’s function in a similar fashion to that seen with the deletion of the full length

of 3, indicating that the effects of 3 on osteoclast activity are mediated through its

cytoplasmic domain (248). Using a series of 3 point mutations, the residue S752

in 3’s

cytoplasmic domain was found to be specifically responsible for regulating osteoclast spreading,

sealing zone formation and bone resorption (248). Interestingly, in humans S752

P is one of the

point mutations identified in some cases of Glanzmann thrombasthenia which is a disease

characterized by disruption of hemostasis due to lack of IIb3 activation in platelets.

Conversely, the double Y747

F/ Y759

F mutation which was shown to inhibit platelet function had

no effect on osteoclasts (248). Despite the definitive osteopetrotic phenotype of 3 ablation in

mice, osteopetrosis was reported only in one case of Glanzmann mutation in humans. The

discrepancy between the two phenotypes related to lack of 3 signalling may be due to the

upregulation of 2 in Glanzmann thrombasthenia patients which partially restores bone

remodelling (249).

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1.6.3 The Molecular Mechanisms Involved in v3 Signalling

Three models have been proposed for the signalling cascade downstream of the outside-

in v3 activation. These models all involve c-Src activation and recruitment to 3. While the

proto-oncogene c-Src is abundantly expressed in osteoclasts, other Src kinase family members

(c-Fyn, c-Yes and c-Lyn) are present at a lower level (250). It was thus not surprising that c-Src

deletion results in substantial bone anomalies and that other Src family kinases could not

compensate for its absence. Similar to the phenotype seen with 3 ablation, c-Src knockout mice

are osteopetrotic despite the increase in osteoclast number (251