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EXPRESSION AND FUNCTIONAL ANALYSIS OF PROTEIN KINASE C ISOFORMS IN OSTEOCLASTS By Cheng Loon Leong BSc. (Hons) This thesis is presented for the degree of Master of Medical Science of The University of Western Australia 2009 The work presented in this thesis was performed at The University of Western Australia, School of Surgery, Centre for Orthopaedic Research, QEII Medical Centre, Nedlands, Western Australia.

EXPRESSION AND FUNCTIONAL ANALYSIS OF …...1.5 RANK Signalling Pathways in Osteoclasts 11 1.5.1 NF-κB Signalling Pathways 12 1.5.2 MAPK Signalling Pathways 15 1.5.3 PI3K/Akt Signalling

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EXPRESSION AND FUNCTIONAL ANALYSIS OF PROTEIN KINASE C

ISOFORMS IN OSTEOCLASTS

By Cheng Loon Leong BSc. (Hons)

This thesis is presented for the degree of

Master of Medical Science of

The University of Western Australia

2009

The work presented in this thesis was performed at The University of Western

Australia, School of Surgery, Centre for Orthopaedic Research, QEII Medical Centre, Nedlands, Western Australia.

TABLE OF CONTENTS TABLE OF CONTENTS i

DECLARATION v

ABSTRACT vi

ACKNOWLEDGEMENTS viii

ABBREVIATIONS x

Chapter One – Osteoclast Biology

1.1 Introduction 1

1.2 Osteoclast Morphology 2

1.3 Phenotypic Markers of Osteoclasts 5

1.4 Osteoclastogenesis 8

1.5 RANK Signalling Pathways in Osteoclasts 11

1.5.1 NF-κB Signalling Pathways 12

1.5.2 MAPK Signalling Pathways 15

1.5.3 PI3K/Akt Signalling Pathways 16

1.5.4 NFAT Signalling Pathway 16

1.6 Osteoclastic Bone Resorption 18

1.6.1 Cellular Attachment and Polarisation 18

1.6.2 Inorganic Matrix Degradation 19

1.6.3 Organic Matrix Degradation 20

1.6.4 Removal of Degraded Products From The Resorption Lacuna 21

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

2.1 Introduction 23

2.2 PKC Isoforms and Structure 24

2.2.1 The C1 Domain 27

2.2.2 The C2 Domain 27

2.2.3 The Pseudosubstrate Site 28

2.2.4 The Catalytic Domain and V5 Region 28

2.2.5 The Hinge Region 29

2.3 Activation and Localization of PKC 30

2.4 Function of PKCs in Various Cell Types 33

2.5 Signalling Pathways of PKCs 36

2.5.1 PKCs and NF-κB 37

i

2.6 The Role of PKCs in Osteoclast Function 38

Chapter Three – Hypothesis and Aims

3.1 Rationale 40

3.2 Hypothesis and Aims 41

3.3 Outcomes and Significance 42

Chapter Four – Materials and Methods

4.1 Materials 43

4.1.1 Cell Lines 43

4.1.2 Bacterial Strains 43

4.1.3 Commercially Purchased Kits 43

4.1.4 Chemical Reagents 44

4.1.5 Enzymes 47

4.1.6 Antibodies 48

4.1.7 Vectors 49

4.1.8 Other Materials and Equipments 49

4.1.9 Pharmacological Compounds 52

4.1.10 Buffers and Solutions 53

4.1.11 Oligonucleotide Primers 58

4.2 Media and Agar Plates 60

4.2.1 Luria-Bertani (LB) Medium 60

4.2.2 LB/Ampicillin Agar Plates 60

4.3 Cell Culture 61

4.3.1 Cell Culture Media and Reagents 61

4.3.2 Culturing of RAW264.7 and HEK-293 Cells 62

4.3.3 Cryopreservation of Cell Lines 62

4.4 Experimental Methods 63

4.4.1 Extraction of Total RNA and Reverse Transcriptase-Polymerase

Chain Reaction (RT-PCR) 63

4.4.1.1 Reverse Transcription of mRNA 63

4.4.1.2 Polymerase Chain Reaction (PCR) 64

4.4.2 Protein Isolation, SDS-PAGE Protein Analysis, and Western Blot 65

4.4.2.1 Protein Extraction 65

4.4.2.2 SDS-PAGE 66

ii

4.4.2.3 Protein Transfer 66

4.4.2.4 Western Blotting 67

4.4.3 RANKL-Induced Osteoclastogenesis 68

4.4.3.1 Time Course of PKC Isoforms During RANKL-Induced

Osteoclastogenesis in RAW264.7 Cells 68

4.4.3.2 Go6976 Inhibition of RANKL-Induced Osteoclastogenesis in

Bone Marrow Macrophages (BMM) 70

4.4.3.3 TRAP Staining 70

4.4.3.4 Go6976 Inhibition of Osteoclast Markers During

RANKL-Induced Osteoclastogenesis in RAW264.7 Cells 70

4.4.3.5 Go6976 Inhibition of Signalling Pathways During

RANKL-Induced Osteoclastogenesis in RAW264.7 Cells 72

4.4.4 Preparation of pHACE-PKCα Wild-Type (WT) Plasmid Construct

for Luciferase Assay 72

4.4.4.1 Transformation of pHACE-PKCα WT Construct Into

DH5α E. coli Competent Cells 72

4.4.4.2 Miniprep of pHACE-PKCα WT Plasmid From Transformed

DH5α E. coli Cells 73

4.4.4.3 Large-Scale Growth of pHACE-PKCα WT-Transformed

E. coli Cells 74

4.4.4.4 Midiprep of pHACE-PKCα WT Plasmid From Large-Scale

Growth of E. coli Cells 74

4.4.4.5 Glycerol Stock of pHACE-PKCα WT-Transformed

E. coli Cells 75

4.4.4.6 PolyFect Transfection of pHACE-PKCα WT Plasmid Into

HEK-293 Cells 75

4.4.4.7 SuperFect Transfection of pHACE-PKCα WT Plasmid Into

RAW264.7 Cells 76

4.4.4.8 Luciferase Assay 77

4.4.5 Small Interfering RNA (siRNA) Experimental Methods 77

4.4.5.1 Transfection of siRNA into RAW264.7 Cells in 24-Well Plates 77

4.4.5.2 PKCα siRNA Inhibition of RANKL-Induced

Osteoclastogenesis in RAW264.7 Cells 79

iii

iv

Chapter Five – Expression of PKC Isoforms During RANKL-Induced

Osteoclastogenesis

5.1 Introduction 80

5.2 Results 82

5.2.1 PKCα mRNA Expression is Upregulated During

Osteoclastogenesis 82

5.2.2 Go6976 Inhibits The Formation of Osteoclasts in BMM 88

5.2.3 Go6976 Suppresses CTR, V-ATPase d2, and Cathepsin K mRNA

Expression 90

5.3 Discussion 92

Chapter Six – Functional Analysis of PKCα In RANKL-Induced

Osteoclastogenesis

6.1 Introduction 94

6.2 Results 96

6.2.1 Go6976 Has Little To No Effect on p-ERK and IκBα

Protein Expression During Osteoclastogenesis 96

6.2.2 PKCα Wild-Type Construct Potentiates RANKL-Induced

Activation of NF-κB 98

6.2.3 Go6976 Reduces NFATc1 Protein Expression During

Osteoclastogenesis 100

6.2.4 PKCα siRNA Inhibits RANKL-Induced Osteoclastogenesis in

RAW264.7 Cells 102

6.3 Discussion 106

Chapter Seven – Overview of the Thesis

7.1 General Discussion 109

7.2 Future Directions 112

Chapter Eight – Bibliography 114

v  

DECLARATION

This is to certify that all work contained herein was performed by myself, except where

indicated otherwise.

______________________

Cheng Loon Leong

______________________

Professor Jiake Xu

(Supervisor)

______________________

Asst/Prof. Nathan Pavlos

(Co-supervisor)

______________________

Professor Ming-Hao Zheng

(Co-supervisor)

vi

ABSTRACT

Bone is a living tissue that is undergoes a continuous process of remodeling during and

after skeletal development. The process is to change the shape of the bone to

accommodate the growth of the individual as well as to maintain bone integrity

throughout life. It also plays a role in the regulation of calcium homeostasis, as bone is

mainly composed of calcium minerals and thus a major reservoir for calcium. Two

resident bone cell types are primarily responsible for bone remodeling: 1) osteoblasts

and 2) osteoclasts. Osteoblasts are essentially bone-forming cells, whereas osteoclasts

function to resorb bone surfaces. A balance between the activity of bone formation and

bone resorption must be maintained in order to sustain bone structure stability. An

imbalance in the activity of bone formation and bone resorption has been the underlying

cause of many bone diseases such as osteoporosis and Paget’s disease of the bone.

Osteoclasts are large multinucleated cells that are formed by the fusion of mononuclear

progenitors of the monocyte-macrophage family. These cells attach themselves to the

bone surface and form a sealed compartment, known as the resorption lacuna, between

the cell membrane and the bone. H+ ions and osteolytic enzymes are pumped into the

resorption lacuna from the osteoclasts to degrade the bone’s mineral and organic matrix.

Stromal cells and osteoblasts are essential for osteoclast formation (osteoclastogenesis)

as they produce the two essential molecules that are involved in the process: 1)

macrophage colony-stimulating factor (M-CSF) and 2) receptor for activation of nuclear

factor kappa B (NFκB) (RANK) ligand (RANKL). RANKL alone is sufficient to induce

a murine-derived macrophage cell line (RAW264.7) to form bone-resorbing osteoclasts.

RANKL is a transmembrane protein found on stromal cells or osteoblasts that binds to

RANK. RANK is a transmembrane protein expressed on the cell surface of osteoclasts.

The binding of RANKL to RANK stimulates it, inducing osteoclastogenesis,

osteoclastic bone resorption, and osteoclast survival.

Protein Kinase C (PKC) is a family of threonine/serine kinases, which consists of

several different isoforms. These isoforms are grouped into three groups: 1) classical

PKCs (cPKCs), 2) novel PKCs (nPKCs), and 3) atypical PKCs (aPKCs). These groups

are formed on the basis of their different activation requirements. cPKCs are calcium-

dependent and activated by both phosphotidylserine (PS) and diacylglycerol (DAG).

vii

nPKCs are calcium-independent, but are still regulated by PS and DAG. aPKCs are

calcium independent and do not require DAG for activation, although PS can regulate

their activity. PKCs play a crucial role in signal transduction for a variety of

biologically active substances that activate cellular functions and proliferation. The

general importance of PKCs in osteoclast biology has been previously established and

studies have found that PKCβ participates in the ERK signalling pathway during M-

CSF and RANKL-induced osteoclastogenesis, but not the NF-κB pathway. However,

the roles of the other PKC isoforms in RANKL-induced osteoclastogenesis are yet to be

elucidated. Thus, this thesis explores the expression and functional roles of the PKC

isoforms in RANKL-induced osteoclastogenesis.

A time course study was conducted to investigate the expression of PKC isoforms at

different time points during RANKL-induced osteoclastogenesis. PKCα, a classical

PKC, was found to be upregulated during RANKL-induced osteoclastogenesis in both

RAW264.7 cells and bone marrow macrophages (BMM). Inhibition of PKCα by its

selective inhibitor, Go6976, and small interfering RNA (siRNA) specific to PKCα

suppressed RANKL-induced osteoclastogenesis of RAW264.7 and BMM. Inhibition of

PKCα by Go6976 also reduced RANKL-induced expression of vacuolar H+-ATPase

(V-ATPase) d2 and calcitonin receptor (CTR), both markers of osteoclast formation.

NFATc1 protein expression was inhibited by the presence of Go6976. Go6976 had little

to no effect on p-ERK and IκBα protein expression. Interestingly, overexpression of

wild-type PKCα potentiated NF-κB luciferase activity.

Taken together, the results in this thesis suggest that PKCα plays an important role in

RANKL-induced osteoclastogenesis by modulating the NF-κB/NFATc1/V-ATPase d2

pathway. PKCα is a potential molecular target for the discovery of novel bone anti-

resorptive agents for the treatment of lytic bone disorders.

viii

ACKNOWLEDGEMENTS

This study was performed and accomplish at the Department of Orthopaedic Surgery,

UWA. Firstly, I would like to thank Professor Jiake Xu, for supervising me throughout

my time in the lab. Thank you for being patient with me, patiently guiding me and

encouraging me on even when I faced difficulties.

I wish to express my appreciation of the help that Assistant Professor Nathan Pavlos has

provided in my research. Even though I haven’t been communicating much with him,

he has always attempted to provide the help that I need, and the morale boost for

finishing this thesis.

I am extremely grateful to Professor Ming-Hao Zheng for providing his wonderful

insight in conducting research, and ideas on how I should proceed with my research. His

enthusiasm for science and research, and tireless work ethic has been an inspiration to

me.

I would like to specially thank Mr. Jay Steer for providing assistance in performing the

luciferase assay. Thank you for your kindness and patience.

Big thanks to Lesley Gasmier for assisting in the administrative work which I

sometimes find daunting. Your advice has been very helpful.

I am grateful for the assistance and mentorship of Tak and Jamie as I conducted my

experiments. Thank you for showing me how to perform the experiments needed for my

thesis.

I would also like to thank Ee-Cheng for sharing some of his resources with me. I wish

all the best as you continue your work in PKCs.

My heartfelt thanks to everyone else in the lab: Esta, Sky, Jackie, Tamara, Jasreen, Pei

Ying, Qian, Vincent, Jim, Qin-An. Your presence has made the lab an interesting place

to be. I hope I have not left anyone out. If I have, my sincere apologies.

ix

Thanks to my pastor and to all my friends from church who have been praying for me.

Your support is much appreciated.

I would also like to extend my sincere gratitude to those who are sacrificing their time

to mark this thesis. I hope my work will not put you to sleep.

Lastly, but most important of all, I would like to thank my mom and dad for supporting

me emotionally and financially. I would not be here without your support through all

these years. I hope that this thesis will make you proud.

Thank you for reading this thesis.

ABBREVIATIONS

α-MEM Alpha-Minimum Essential Medium

Amp Ampicillin

ATP Adenosine triphosphatase

BMM Bone Marrow Macrophage

BSA Bovine Serum Albumin

bp Base pairs

cDNA Complementary Deoxyribonucleic Acid

CO2 Carbon dioxide

CsA Cyclosporin A

CTR Calcitonin receptor

ddH2O Double distilled water

DMEM Dulbecco’s Modification of Eagles Medium

DMSO Dimethyl sulphoxide

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetracetic acid

ERK Extracellular Signal-Regulated Kinase

FBS Fetal bovine serum

FCS Fetal calf serum

kDa Kilodaltons

LB Luria-Bertani

MAPK Mitogen-Activated Protein Kinase

M-CSF Macrophage Colony Stimulating Factor

MQ ddH2O Milli-Q double distilled water

MW Molecular weight

NFAT Nuclear Factor of Activated T-cells

NF-κB Nuclear Factor Kappa-light-chain-enhancer of Activated

B-cells

x

xi

OPG Osteoprotegrin

PAGE Polyacrylamide Gel Electrophoresis

PCR Polymerase Chain Reaction

PBS Phosphate Buffered Saline

PKC Protein Kinase C

PMSF Phenylmethylsulfonyl fluoride

RANK Receptor Activator of NFκB

RANKL Receptor Activator of NFκB Ligand

rpm Revolutions per minute

RT Reverse Transcriptase

SDS Sodium dodecyl sulphate

siRNA Small interfering RNA

TAE Tris-acetate EDTA

TM Transmembrane

TRAP Tartrate-Resistant Acid Phosphatase

UV Ultraviolet

V Volts

V-ATPase Vacuolar H+-ATPase

WT Wild-type

Chapter One

Osteoclast Biology

Chapter One – Osteoclast Biology

1

1.1 - Introduction

Bone is a dynamic tissue that is constantly remodeled during and after skeletal

development. The process is to change the shape of the bone to accommodate the

growth of the individual as well as to maintain bone integrity throughout life. It also

plays a role in the regulation of calcium homeostasis, as bone is mainly composed of

calcium minerals and thus a major reservoir for calcium. Two resident bone cell types

are primarily responsible for bone remodeling: 1) osteoblasts and 2) osteoclasts.

Osteoblasts are essentially bone-forming cells, whereas osteoclasts function to resorb

bone surfaces. A balance between the activity of bone formation and bone resorption

must be maintained in order to sustain bone structure stability. An imbalance in the

activity of bone formation and bone resorption has been the underlying cause of many

bone diseases such as osteoporosis and Paget’s disease of the bone.

This chapter will broadly discuss the physical and biochemical characteristics of the

osteoclast, its formation (osteoclastogenesis), the main RANK signalling pathways

involved in osteoclast function and differentiation, and how it resorbs bone.

Chapter One – Osteoclast Biology

2

1.2 - Osteoclast Morphology

Osteoclasts are large multinucleated cells (50 – 100 μm2 in diameter) that are

responsible for the resorption of mineralised bone. They usually contain 10 to 20 nuclei

per cell although some osteoclasts can contain up to 100 nuclei (Li et al., 2006).

Osteoclasts are rare cells within the bone with only 2 or 3 cells often observed per μm3

(Roodman, 1996). However, elevated osteoclast numbers are often found at sites of

active bone turnover such as metaphysis of growing bone and under several

pathological conditions including post-menopausal osteoporosis, aseptic loosening and

tumour-mediated osteolysis.

Osteoclasts exist in 2 different states: a) motile and b) resorptive state (Li et al., 2006).

In their motile state, osteoclasts are non-polarized and are characterized by a

lamellipodia and podosome complexes. The lamellipodia are membrane protrusions at

the cell’s leading edge. Podosome complexes are located in a row behind the leading

edge and are comprised of actin filaments associated with several actin-binding proteins

(Li et al., 2006). Osteoclasts migrate by extension of the lamellipodia and retraction of

the membrane at the opposite end of the cell.

Osteoclasts become polarised cells during their resorptive state with the localization of

nuclei towards the apical membrane and the formation of the ruffled membrane border

facing the bone (Feng et al., 2005). The ruffled border is the distinguishing feature of

the osteoclast, and is not seen with macrophage polykaryons. The ruffled border can be

seen by light microscopy but is better visualized by electron microscopy (Roodman,

1996). It is made up of numerous fine finger-like cytoplasmic projections of the plasma

membrane into the space (resorption lacuna) between the osteoclast and bone. The

ruffled border is the result of acidic intracellular vesicles fusing together within the

region of the plasma membrane facing the bone (Li et al., 2006). It is responsible for

bone resorption and only appears during bone resorption. The ruffled border will be

discussed further in Section 1.6.

The osteoclastic cell surface that comes into direct contact with the bone surface forms

what is known as the sealing zone (Feng et al., 2005). The sealing zone does not contain

any organelles but is comprised of bundles of actin-like filaments. When the cell comes

Chapter One – Osteoclast Biology

3

into contact with bone, numerous plasma membrane protrusions organised in rows form

podosomes. These podosomes have αvβ3 integrin associated with talin and vinculin (Li

et al., 2006). The podosomes surround the microfilament cytoskeleton core, providing

the osteoclast with a tight attachment to the bone surface (Li et al., 2006). This forms a

ring around the resorption lacuna beneath the ruffled border, which effectively seals the

resorption lacuna from the outside environment. Thus, a microenvironment favourable

for bone resorption can be formed.

Other physical characteristics of the osteoclast, aside from multinuclei and the ruffled

border, include 1) an abundance of mitochondria, 2) sparse rough endoplasmic

reticulum, 3) many free ribosomes or cluster of ribosomes (Holtrop et al., 1977). Figure

1.1 is a schematic representation of an osteoclast during bone resorption.

Chapter One – Osteoclast Biology

Figure 1.1 Schematic representation of an osteoclast during bone resorption. SZ

= sealing zone; RB = ruffled border; RL = resorption lacuna; FSD = functional

secretory domain; BL = basolateral domain. (Adapted from Vaananen et al., 2000).

4

Chapter One – Osteoclast Biology

5

1.3 - Phenotypic Markers of Osteoclasts

The morphologic characteristics of osteoclasts are not enough to differentiate

osteoclasts from other cells, especially macrophage polykaryons. While osteoclasts are

the primary bone resorbing cells, they are not the only cells that can resorb bone.

Multinucleated giant cells of certain tumors are also known to form resorption pits

(Athanasou et al., 1991; Athanasou et al., 1992). Histochemical methods are frequently

used to identify various cell-types. Osteoclasts are known to have Tartrate-Resistant

Acid Phosphatase (TRAP) activity in its cytoplasm. This activity is used as a

histochemical marker for osteoclasts. TRAP was first described in the cytoplasm of

hairy cells of leukemic reticuloendotheliosis (Li et al., 1970). Mice deficient in TRAP

were shown to have mild osteopetrosis and also defective mineralisation of the cartilage

in the developing bone (Hayman et al., 1996). While TRAP has been widely used as a

marker for osteoclasts, it is also found to be expressed in cells belonging to the

monocyte/macrophage lineage such as dendritic cells and activated macrophages

(Hayman et al., 2000; Hayman et al., 2001; Lee et al., 1995). TRAP is also expressed in

multinucleated giant cells (Liu et al., 2003). However, osteoclasts are still the major

cells that express TRAP (Igarashi et al., 2002).

Cathepsins are members of the endosomal/lysosomal protease family (Goto et al.,

2003). The human cDNA encoding cathepsin K was first cloned by Inaoka et al. (1995).

The homologue of the gene in rabbits was reported to be predominantly expressed in

osteoclasts (Tezuka et al., 1994). This led Tezuka et al. (1994) to suggest that cathepsin

K may play an important role in osteoclastic bone resorption. This is now known to be

true as cathepsin K knockout mice exhibit a phenotype of osteopetrosis (Gowen et al.,

1999; Saftig et al., 1998). Pycnodysostosis (Toulouse-Lautrec disease) is a rare

autosomal recessive trait characterized by osteosclerosis and short stature. The disease

is the result of cathepsin K deficiency (Gelb et al., 1996). Furthermore, osteoclastic

bone resorption was inhibited by cathepsin K antisense oligodeoxynucleotide in vitro

(Inui et al., 1997). Cathepsin K has been confirmed by immunocytochemical studies to

be localized in the vesicles, granules, and vacuoles of multinucleated osteoclasts (Goto

et al., 2003). However, cathepsin K is not an osteoclast-specific cathepsin. It is also

found in macrophages, aortic smooth muscle cells, thyroid epithelial cells, and lung

Chapter One – Osteoclast Biology

6

epithelial cells (Goto et al., 2003). Multinucleated giant cells also express cathepsin K

(Liu et al., 2003).

Carbonic anhydrase II is involved in the generation of H+ and HCO3- by catalyzing the

hydration of H2O and CO2. In osteoclasts, the H+ generated by carbonic anhydrase II is

used to acidify the resorption lacunae. Mutations in the gene encoding carbonic

anhydrase II give rise to an autosomal recessive trait known as carbonic anhydrase II

deficiency syndrome (Shah et al., 2004). One of the characteristics of this syndrome is

osteopetrosis. The importance of carbonic anhydrase II in bone resorption was further

confirmed in vitro when antisense RNA and DNA molecules targeting carbonic

anhydrase II inhibits bone resorption (Laitala et al., 1994). By using Reverse-

Transcriptase Polymerase Chain Reaction (RT-PCR) to measure mRNA levels, carbonic

anhydrase II was found to be expressed during the appearance of TRAP-positive

multinucleated osteoclasts (Katagiri et al., 2006). This confirms their expression as

being predominant in mature osteoclasts, but not in osteoclast precursors. Carbonic

anhydrase II is also expressed in multinucleated giant cells (Liu et al., 2003), and is

therefore not entirely specific.

Calcitonin is a peptide hormone that is a potent inhibitor of osteoclastic bone resorption.

It induces the disruption of the sealing zone and dispersion of V-ATPase throughout the

cytoplasm (Okumura et al., 2006). Their activity is mediated by calcitonin receptors on

the surface of the osteoclasts. These calcitonin receptors are expressed on committed

osteoclast precursors as well as on mature osteoclasts (Lee et al., 1995). By using RT-

PCR amplification, Lee et al. (1995) were able to demonstrate that the expression of

calcitonin receptor mRNA in adherent cells of bone marrow cultures parallels the

appearance of the multinucleated cells. This suggests that calcitonin receptor mRNA is

a marker for differentiated osteoclasts. By comparison, TRAP mRNA was found to be

present in both adherent and non-adherent bone marrow cells at all times (Lee et al.,

1995), which illustrates the non-specific expression of TRAP. Calcitonin down-

regulates the mRNA level of the calcitonin receptor, which might explain a

phenomenon known as “calcitonin escape” (Takahashi et al., 1995). This phenomenon

describes osteoclasts that are no longer responsive to calcitonin after extended treatment

with calcitonin. Calcitonin receptors are not an absolute feature of resorbing osteoclasts

as avian osteoclasts do not express calcitonin receptors and are unable to respond to

Chapter One – Osteoclast Biology

7

calcitonin (Roodman, 1996). It is, however, one of the best markers for distinguishing

mammalian osteoclasts from macrophage polykaryons.

Chapter One – Osteoclast Biology

8

1.4 - Osteoclastogenesis

Osteoclasts are formed by the fusion of mononuclear progenitors of the monocyte-

macrophage family (Teitelbaum, 2000). Tartrate-resistant acid phosphatase (TRAP)-

positive mononuclear and multinucleated cells formed when mononuclear cells obtained

from mouse bone marrow, spleen, thymus, or peripheral blood were cultured with

stromal cells in the presence of 1α,25-dihydroxyvitamin D3 and dexamethasone

(Udagawa et al., 1990). Stromal cells and osteoblasts are essential for

osteoclastogenesis as they produce the two essential molecules that are involved in the

process: 1) macrophage colony-stimulating factor (M-CSF) and 2) receptor for

activation of nuclear factor kappa B (NFκB) (RANK) ligand (RANKL) (Teitelbaum,

2000).

M-CSF is produced by macrophages, stromal cells, endothelial cells, and T lymphocytes

in the marrow microenvironment (Roodman, 1996). It is a hematopoietic growth factor

that induces the clonal growth of hemapoietic progenitors in vitro and in vivo. The M-

CSF gene in op / op osteopetrotic mice is mutated by a single-base pair insertion

(Yoshida et al., 1990). This results in a reduced number of mature macrophages and

osteoclasts. In an experiment by Tanaka et al. (1993), osteoclast-like multinucleated

cells formed when mouse osteoblasts were cocultured with mouse spleen cells as a

source of osteoclast precursors in the presence of 1α,25-dihydroxyvitamin D3. They

noted that osteoclast progenitors mainly proliferated in the first 4 days of culture, and

differentiated into osteoclast-like multinucleated cells in the last 2 days. When anti-M-

CSF and anti-M-CSF receptor was added to these cultures during the first 4 days or last

2 days of culture, osteoclast-like cell formation was inhibited. From this, they inferred

that M-CSF stimulates the proliferation and differentiation of osteoclast precursors.

Although M-CSF is a secreted product, contact between osteoclast precursors and

stromal cells or osteoblasts is necessary for osteoclastogenesis to occur (Udagawa et al.,

1990).

In 1997, a secreted protein was found to block osteoclastogenesis in vitro and bone

resorption in vivo (Simonet et al., 1997). This protein, termed osteoprotegerin (OPG), is

a member of the tumour necrosis factor receptor (TNFR) superfamily (Simonet et al.,

1997). The ligand to OPG (RANKL) was found to bind to the surface of osteoclast

Chapter One – Osteoclast Biology

9

progenitors that were treated with M-CSF (Lacey et al., 1998). RANKL (also known as

OPGL and TRANCE) activates mature osteoclasts and regulates osteoclast formation

from bone marrow precursors in the presence of M-CSF (Lacey et al., 1998). RANKL

is a transmembrane protein found on stromal cells or osteoblasts, that binds to either

RANK or OPG. It is now known that OPG is a soluble “decoy” receptor that competes

with RANK for RANKL (Lacey et al., 1998). The binding of OPG to RANKL inhibits

osteoclastogenesis, whereas binding of RANK stimulates it. The balance between OPG

and RANKL determines the amount of bone resorbed by controlling the activation of

RANK on osteoclasts.

The importance of RANKL in osteoclastogenesis is evident from research done in

animal models. RANKL-knockout mice have a complete lack of osteoclasts leading to

severe osteopetrosis (Kong et al., 1999). This complete lack of osteoclasts was

elucidated to be the result of an inability of stromal cells and osteoblasts to support

osteoclastogenesis, and not from an intrinsic block in osteoclast development.

Furthermore, injection of OPG into normal and ovariectomized rats resulted in an

increased bone density and bone volume associated with a decrease in the number of

active osteoclasts (Suda et al., 1999). Since OPG inhibits RANKL activity by binding to

RANK, the result of injecting OPG into rats further confirms that RANKL is essential

in osteoclastogenesis and bone resorption. Inhibition of RANK by the cell-permeable

inhibitory peptide RANK receptor inhibitor (RRI) blocked RANKL-induced osteoclast

formation from murine bone marrow macrophages (Kim, 2009). The peptide also

inhibited bone resorptive activity of osteoclasts and induced osteoclast apoptosis.

Other factors also play a part in osteoclastogenesis. Factors such as parathyroid

hormone (PTH) and 1α,25-dihydroxyvitamin D3, enhance osteoclast differentiation by

affecting the expression of RANKL on stromal cells and osteoblasts (Teitelbaum,

2000), demonstrating the importance of stromal cells and osteoblasts in

osteoclastogenesis. E-cadherin is a member of the family of homophilic calcium-

dependent cell adhesion molecules. Using monoclonal antibodies, expression of E-

cadherin was detected along the cell membrane of osteoclasts at resorbing bone surfaces

(Mbalaviele et al., 1995). The same study found that the expression of E-cadherin was

highest when TRAP-positive mononuclear cells began to fuse. They also reported that

monoclonal antibodies to E-cadherin decreased the number of TRAP-positive

Chapter One – Osteoclast Biology

10

multinucleated cells by inhibiting fusion. In addition, synthetic peptides containing the

cell adhesion recognition sequence of cadherins also decreased the number of TRAP-

positive multinucleated cell formed in murine marrow cultures (Mbalaviele et al.,

1995). It is reasonable to conclude from these results that E-cadherin plays a role in the

fusion of osteoclast precursors. Some other factors that play a role in osteoclastogenesis

include interleukin (IL)-1, -6, and -11, TNF, and oncostatin M (OSM) (Li et al., 2006;

Roodman, 1999).

Chapter One – Osteoclast Biology

11

1.5 - RANK Signalling Pathways in Osteoclasts As previously described, RANK and its associated ligand RANKL are important

regulators of osteoclastogenic differentiation and function. The binding of RANK to

RANKL initiates several signalling pathways that influence osteoclast activity. RANK-

RANKL binding transduces intracellular signalling by recruiting adaptor molecules

known as the TNFR associated factor (TRAF) family of proteins (Wong et al., 1999;

Wong et al., 1998). The TRAF family contains seven members (TRAFs 1, 2, 3, 4, 5, 6

and 7) and mainly mediates signals induced by TNF family cytokines and pathogen-

associated molecular patterns (PAMPs) (Asagiri et al., 2007). From among these seven

members, TRAF6 was identified as the major adaptor molecule for RANK-mediated

osteoclastogenesis. Evidence for this is found in two independent knockout mice

studies. TRAF6-/- mice developed severe osteopetrosis due to impaired osteoclast

formation (Lomaga et al., 1999; Naito et al., 1999). Binding of TRAF6 to RANK

induces the trimerization of TRAF6, which leads to the activation of various signalling

pathways. TRAF6 is essential for the activation of NF-κB and Jun N-terminal kinase

(JNK) signalling pathways (Kobayashi et al., 2001), both important pathways in

osteoclastogenesis.

The signalling pathways that have been reported to be crucial in osteoclasts include

signalling pathways of nuclear factor-kappa B (NF-κB) (Galibert et al., 1998), mitogen-

activated protein kinases (MAPKs) (Lee et al., 2002), serine/threonine kinase Akt

(Wong et al., 1999), and nuclear factor of activated T cell (NFAT) (Takayanagi et al.,

2002).

Chapter One – Osteoclast Biology

12

1.5.1 - NF-κB Signalling Pathways

NF-κB is a pleiotropic transcription factor family consisting of five members. The ‘Rel’

proteins, Rel A (p65), Rel B, and c-Rel (Rel), contain transcription activation domain

(TAD). The NF-κB proteins consist of NF-κB1 (p105/p50) and NF-κB2 (p100/p52)

(Soysa et al., 2009). NF-κB1 and NF-κB2 are originally synthesized as large precursors,

p105 and p100, respectively, that become shorter after post-translational processing into

p50 and p52. All NF-κB proteins share a Rel-homology domain, which contains a

nuclear localization sequence and is involved in dimerization, DNA binding, and

interaction with IκB inhibitory proteins. The NF-κB proteins (p50 and p52) are not able

to activate transcription by themselves, and have to form dimers with the Rel proteins in

order to do so. NF-κB dimers are held in the cytoplasm of most cells by inhibitory IκB

proteins. Interactions between NF-κB and IκB proteins block the nuclear localization

sequence of the dimers and hence prevent nuclear localization and subsequent DNA

binding.

There are two major NF-κB signalling pathways: canonical (classical) and non-

canonical (alternative) (refer to Figure 1.2). The canonical pathway in pre-osteoclasts is

activated by the binding of RANKL to RANK, which in turn activates the IKK complex

that consists of catalytic subunits IKKα, IKKβ, and the regulatory subunit IKKγ

(NEMO). The activated IKK complex then catalyzes the phosphorylation of the

inhibitory IκB proteins (i.e. IκBα), which are subsequently ubiquitinated and degraded

by 26S proteasome. This releases the NF-κB dimers (mostly p50-Rel A) and allows it to

translocate into the nucleus, bind to its DNA transcription site, and initiate gene

transcription (Soysa et al., 2009). Phosphorylation of Rel A also stabilizes it, and

inhibits it from binding to IκBα.

The non-canonical, or alternative pathway depends only on the IKKα subunit for

activation. Like in the canonical pathway, RANKL binds to RANK, which activates

NF-κ-inducing kinase (NIK). This results in the activation of IKKα homodimers and the

subsequent phosphorylation of p100. Phosphorylated p100 is ubiquitinated for partial

proteasome degradation and hence is cleaved to generate an active p52 product. The

p52-Rel B is then able to translocate into the nucleus and initiate transcription of its

Chapter One – Osteoclast Biology

13

target genes. This process is generally slower than that of the canonical pathway (Soysa

et al., 2009).

The RANK mediated NF-κB signalling pathway is central for osteoclast differentiation.

It is known to be activated by RANKL in RAW264.7 cells as well as monocytes (Hsu et

al., 1999; Lacey et al., 1998). Mice with double knockouts of the NF-κB subunits p50

and p52 failed to develop mature osteoclasts and subsequently lead to osteopetrosis

(Franzoso et al., 1997; Iotsova et al., 1997). The expression of various cytokines

involved in osteoclast differentiation (e.g. IL-1, TNF-α, IL-6, GM-CSF, RANKL) and

other growth factors are dependent on NF-κB (Reddy, 2004). Abbas et al. (2003)

demonstrated that under conditions of NF-κB inactivity, levels of pro-survival factors

are reduced, which in turn facilitates TNF induction of pro-apoptotic factors, leading to

apoptosis.

Chapter One – Osteoclast Biology

Figure 1.2 The main NF-κB signalling pathways in osteoclasts. (Adapted from

Soysa et al., 2009)

14

Chapter One – Osteoclast Biology

15

1.5.2 - MAPK Signalling Pathways

MAPKs are a family of serine/threonine kinases that respond to external stimuli such as

mitogens, cellular stress, reactive oxygen species and cytokines (Abbas et al., 2003;

Givant-Horwitz et al., 2003). MAPKs regulate a variety of cellular processes including

gene expression, cell differentiation, cell survival, and apoptosis. There are six distinct

groups of MAPKs that have been characterized in mammals: 1) Extracellular signal-

regulated kinases (ERK1, ERK2), 2) c-Jun N-terminal kinases(JNKs), 3) p38 isoforms,

4) ERK5, 5) ERK3/4, 6) ERK7/8. When activated by phosphorylation of its

serine/threonine residues, MAPKs can translocate into the nucleus where they activate

numerous transcription factors. MAPKs also regulate its substrates in the cytosol.

ERK, JNK, and p38 are known to be activated by RANK in osteoclasts or osteoclast

precursors (Lee et al., 2002; Li et al., 2002; Wong et al., 1997). Inhibitors of p38

MAPK were shown to suppress osteoclastogenesis, which further confirms the

involvement of p38 in osteoclasts (Lee et al., 2002; Li et al., 2002). However, Li et al.

also suggested that osteoclast function is independent of p38-mediated signalling.

TRAF6 is thought to play a critical role for RANK signalling via the MAPKs

(Kobayashi et al., 2001). ERK1/2 was also shown to be a positive regulator of

differentiated osteoclast survival, but has no effect on osteoclastic bone resorption

(Miyazaki et al., 2000). Lee et al. demonstrated that IL-α promotes osteoclast survival

through the ERK, as well as Akt, signalling pathway to reduce caspase-3 activity

(2002).

Inhibition of JNK in TRAP-positive pre-osteoclasts causes the cells to revert back to

TRAP-negative cells (Chang et al., 2008). This inhibition also reduced NFATc1 and

CaMKII levels, which suggests that JNK is important in maintaining the committed

status of pre-osteoclasts during osteoclastogenesis through the CaMKII-NFATc1

pathway. A study has suggested that JNK plays an anti-apoptotic role in osteoclasts

(Ikeda et al., 2008).

Chapter One – Osteoclast Biology

16

1.5.3 - PI3K/Akt Signalling Pathways

The Akt family is a family of serine/threonine protein kinases. There are currently three

isoforms identified in mammalian cells: Akt1, Akt2, and Akt3. Akt1 and Akt2 are

ubiquitously expressed in various tissues and are highly expressed in bone cells

(Kawamura et al., 2007). Akt is a downstream target of phosphoinositide-3-kinase

(PI3K). RANKL stimulation of RAW264 cells increased the phosphorylation of Akt

(Lee et al., 2002), indicating that Akt is activated by the RANK signalling pathway.

Inhibition of Akt signalling by the PI3K inhibitor, LY294002, reduced RANK-mediated

osteoclast differentiation and bone resorption (Lee et al., 2002). Mice with the Akt1 and

Akt2 genes knocked out exhibited impaired bone development (Peng et al., 2003). Ex

vivo culture of osteoclast-precursor BMMs from Akt1-/- mice had suppressed

osteoclastogenesis, even in the presence of RANKL and M-CSF (Kawamura et al.,

2007). The survival of mature osteoclasts formed from BMM of Akt1-/- mice was also

impaired, thus indicating the role of Akt1 in osteoclast formation and survival.

1.5.4 - NFAT Signalling Pathway

Nuclear factor of activated T cells (NFAT) is a family of transcription factors involved

in various biological systems including the cardiovascular and musculoskeletal system.

As the name suggests, it was originally discovered in the context of the immune system,

specifically in T-cells (Shaw et al., 1988). Nuclear factor of activated T cells,

calcineurin-dependent (NFATc) are dependent on Ca2+ signalling for activation through

the action of calmodulin and calcineurin (Day et al., 2005). Cytoplasmic Ca2+ activates

calcineurin, which, in turn, dephosphorylates NFATc. This activates NFATc and allows

it to translocate into the nucleus where it can initiate transcription of its target genes.

There are four members in the NFATc gene family (NFATc1, c2, c3, c4), in which

NFATc1 was shown to be induced during mouse (Hirotani et al., 2004; Ishida et al.,

2002; Takayanagi et al., 2002) and human (Day et al., 2004) osteoclast development.

Using an oligonucleotide array, NFATc1 was identified as a major downstream target of

RANKL stimulation (Takayanagi et al., 2002). In the same study, NFATc1 protein

expression increased continuously, followed by nuclear translocation during RANKL-

induced osteoclastogenesis of bone marrow macrophages. Osteoclast formation was

Chapter One – Osteoclast Biology

17

blocked by cyclosporin A inhibition of NFATc1 (Day et al., 2004; Ishida et al., 2002).

Osteoclast differentiation is also inhibited by tanshinone IIA, which inhibits c-Fos and

NFATc1 expression (Kwak et al., 2006). The same study showed that retrovirus-

mediated overexpression of c-Fos, in the presence of tanshinone IIA, induced the

expression of NFATc1 and also allowed osteoclast differentiation to occur. Studies

showed that NFATc1 is also an important transcription factor for V-ATPase d2 and DC-

STAMP, which are important markers of osteoclastogenesis (Feng et al., 2009; Kim et

al., 2008). Inhibition of NFATc1 activity by cyclosporin A attenuates the expression of

d2 and DC-STAMP as well as the subsequent fusion process required for osteoclasts to

form. Conditional knockout of NFATc1 in mice resulted in an osteopetrotic phenotype

and inhibition of osteoclastogenesis in vitro and in vivo (Aliprantis et al., 2008). In the

same study, deletion of NFATc1 also protected cherubism mice from systemic bone

loss. Interestingly, over-expression of NFATc1 promoted the differentiation of

osteoclast precursor cells into osteoclasts, even in the absence of RANKL (Ikeda et al.,

2004).

Chapter One – Osteoclast Biology

18

1.6 - Osteoclastic Bone Resorption

The importance of osteoclasts in bone resorption has been established in various studies

as the absence of osteoclasts is accompanied with a lack of bone resorption. The process

of bone resorption by osteoclasts requires the cell to undergo several changes. Firstly,

the osteoclast needs to attach itself to the bone surface. Secondly, the osteoclastic

membrane has to undergo polarisation. It is only after cellular attachment and

polarisation, that the osteoclast is able to fulfil its duty in bone resorption, by the

degradation and resorption of the bone matrix.

Osteoclastic bone resorption can be characterised into 3 stages: 1) attachment of

osteoclasts to the bone surface; 2) polarisation of osteoclastic membrane and; 3)

degradation and resorption of bone matrix.

1.6.1 - Cellular Attachment and Polarisation

In order for bone resorption to occur, the osteoclast must be able to attach to the bone

surface. As was discussed in Section 1.2, osteoclasts form podosomes that contain αvβ3

integrin associated with talin and vinculin (Li et al., 2006). It is thought that integrins

play an important role in the early phases of the resorption cycle (Vaananen et al.,

2000). There are at least four different integrins expressed in the osteoclast: αvβ3, αvβ5,

α2β1, and αvβ1 (Vaananen et al., 2000). Much attention has been directed at αvβ3, as

antibodies against αvβ3, as well as RGD-containing peptides such as echistatin and

kistrin, are effective inhibitors of bone resorption (Vaananen et al., 2000). Not much is

known about the specific function of αvβ3, but it could play a role in adhesion and

migration of the osteoclast, and in the endocytosis of resorption products. The genetic

ablation of the β3 gene in mice caused the osteoclast from these mice to spread in vitro

(Crotti et al., 2006). In vivo, the osteoclasts of these β3 knockout mice fail to form actin

rings and do not form ruffled membranes. However, its most important function might

not be the formation of sealing zone itself. It probably prompts intracellular events such

as cytoskeletal organization to occur by transmitting bone matrix-derived signals

(Teitelbaum, 2000). This results in the podosomes attaching the osteoclast to the bone

surface along the sealing zone.

Chapter One – Osteoclast Biology

19

Three discrete areas of plasma membrane are formed during polarisation:

a) The basolateral membrane that faces the marrow space but is not in contact

with the bone.

b) The tight sealing zone that is in close contact with the bone matrix.

c) The ruffled border that is comprised of fine finger-like cytoplasmic

projections and is surrounded by the sealing zone.

The ruffled border is the osteoclast’s resorptive organelle that appears only when the

cell is attached to the bone (Teitelbaum, 2000). This forms a sealed extracellular

compartment between the osteoclast and the bone known as the resorption lacuna,

which provides a microenvironment for osteoclastic bone resorption.

1.6.2 – Inorganic Matrix Degradation

Osteoclasts are able to produce HCl and osteolytic enzymes to degrade bone matrix

(Roodman, 1996; Teitelbaum, 2000). This is achieved through the dissolution of

crystalline hydroxyapatite and proteolytic cleavage of organic matter. After the

dissolution of the inorganic phase occurs, only then is the matrix actually degraded

(Blair et al., 1986). Organic matter is degraded by the lysosomal protease cathepsin K.

Acidification of the resorption lacunae is essential for bone demineralization to occur.

Protons are generated by carbonic anhydrase II in the cytoplasm of the osteoclast.

Vacuolar H+-adenosine triphosphatase (H+-ATPase or V-ATPase) in the cell’s ruffled

border pumps protons into the resorption lacuna against its concentration gradient,

resulting in an acidic microenvironment that has a pH of ~4.5 (Silver et al., 1988). In

order to maintain intracellular pH while protons are continually being pumped out of the

osteoclast, an energy-independent Cl-/HCO3- exchanger on the cell’s basolateral

membrane transports Cl- ions into the cell while HCO3- is pumped out (Rucci et al.,

2005). A ruffled membrane Cl- ion channel that is charge-coupled to the V-ATPase

helps to maintain electroneutrality in the cell by allowing Cl- ions to flow into the cell

(Schlesinger et al., 1997).

Chapter One – Osteoclast Biology

20

1.6.3 - Organic Matrix Degradation

The degradation of the organic matrix occurs after dissolution of the inorganic bone

matrix. The acidic microenvironment formed during the inorganic matrix degradation

phase provides the optimal condition for the activity of various proteolytic enzymes

involved in degrading organic bone. The major structural protein in bone is type I

collagen, where it accounts for 90% of the organic matrix. The other 10% is made up of

non-collagenous proteins such as osteocalcin, osteonectin, osteopontin, fibronectin,

thrombospondin and bone sialoprotein (Zhao et al., 2009). Cathepsin K is an important

proteolytic enzyme and marker (see Section 1.3 - Phenotypic Markers of Osteoclasts)

for osteoclastic bone resorption. It is a collagenolytic, papain-like, cysteine protease. It

is secreted through lysosomes into the resorption lacuna to degrade proteins, which

include type 1 collagen, osteonectin, osteopontin, etc. In vitro studies suggest that

activation of cathepsin K occurs in the acidic microenvironment of the resorption lacuna

by an autocatalytic process (McQueney et al., 1997). The lower pH of the resorption

lacuna induces a conformational change to procathepsin K, which exposes the active

site and makes the propeptide more vulnerable to endoproteolysis. Catalytic amounts of

newly formed mature cathepsin K would accelerate the activation of procathepsin K in

the resorption lacuna. Further degradation by endoproteolysis results in a fully active

mature cathepsin K.

Chapter One – Osteoclast Biology

21

1.6.4 - Removal of Degraded Products From The Resorption Lacuna

Degraded products (Ca2+ and collagen fragments) are removed from the resorption

lacuna after matrix degradation in order for continuous resorption to occur. This is done

through a transcytotic vesicular pathway from the ruffled border to the functional

secretory domain where they are released at the basolateral membrane into the

extracellular space (Salo et al., 1997; Vaananen et al., 2000). Since the volume of the

resorption pit can easily exceed the volume of the osteoclast, it is reasonable to assume

that large amounts of the degraded material are transported through the resorbing

osteoclast. The degraded products can undergo further degradation in the transcytotic

vesicle through the action of TRAP. TRAP is localized in the transcytotic vesicles of

resorbing osteoclasts and is shown to generate reactive oxygen species that are able to

breakdown collagen (Vaananen et al., 2000).

Chapter One – Osteoclast Biology

22

Figure 1.3 Stages of osteoclastic bone resorption. (Adapted from Xu et al., 2007)

Chapter Two

The Role of Protein Kinase C in Osteoclast Function

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

23

2.1 - Introduction

Protein phosphorylation plays a significant role in regulating cellular processes. It is

involved in the control of the cell cycle, the circadian rhythm, neurotransmission, etc.

Phosphorylation is the addition of a phosphate (PO4) group to a protein or molecule

through enzymes known as kinases. The phosphate group is transferred from the

phosphate donor (ATP) to an acceptor protein. Phosphorylation can be reversed by

phosphatases that remove the phosphate group. Protein kinases are a family of proteins

that phosphorylate other proteins through the phosphorylation of serine/threonine or

tyrosine residues, which provides a molecular switch for altering cellular components

(Cooper, 1998).

The protein kinase C (PKC) family was one of the first protein kinases identified. An

early study defined it as a histone kinase, which could be activated by proteolysis (Takai

et al., 1977) and later on as a multifunctional protein kinase that was Ca2+ and

phospholipid dependent (Takai et al., 1979). Further studies of PKC revealed other

family members that are not Ca2+-dependent. Activation of PKCs results in changes to

their subcellular localization following translocation to specific anchoring proteins

(Mackay et al., 2007).

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

24

2.2 - PKC Isoforms and Structure

At present, PKC consists of at least 12 isoforms. Each isoform has distinct, and in some

cases, opposing roles in cell proliferation, differentiation, apoptosis, and angiogenesis.

PKC isoforms differ in their structure, tissue distribution, subcellular localization, mode

of activation and substrate specificity. Isoforms were classified according to structural

and activation characteristics into 3 groups:

1) Classical or conventional PKCs (cPKC) -- PKC-α, β1, β2 and γ;

2) Novel PKCs (nPKC) -- PKC-δ, ε, η, and θ;

3) Atypical PKCs (aPKC) -- PKC-ι, λ and ζ.

cPKCs are calcium-dependent and activated by both phosphotidylserine (PS) and

diacylglycerol (DAG). nPKCs are calcium independent but are still regulated by PS and

DAG. aPKCs are calcium independent and do not require DAG for activation, although

PS can regulate their activity. Tumour-promoting phorbol esters such as phorbol 12-

myristate 13-acetate (PMA), which are DAG analogues, are known to activate many

PKCs by anchoring PKCs in their active conformations to membranes, suggesting a key

role for PKC in tumour promotion and progression (Mackay et al., 2007; Steinberg,

2008). The differences in the activation requirements of the PKCs are due to structural

differences of the isoforms, specifically in the regulatory domain.

PKCs contain a highly conserved catalytic (kinase) domain, and a regulatory domain

(Steinberg, 2008). The catalytic or kinase domain consists of motifs required for

ATP/substrate-binding and catalysis. The regulatory domains reside in the NH2

terminus of the protein and contain an autoinhibitory pseudosubstrate domain and two

discrete membrane targeting modules, termed C1 and C2. The regulatory domain

maintains the enzyme in an inactive conformation.

The three subfamilies of PKC (classical, novel, atypical) contain differences between

their NH2-terminal regulatory domain structure (Steinberg, 2008). Classical PKCs

contain a C1 domain that functions as a DAG-/PMA-binding motif, and a C2 domain

that binds anionic phospholipids in a calcium-dependent manner. Novel PKCs also have

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

25

twin C1 domains and a C2 domain. The linear sequence of the two domains on the

protein is however different from that of classical PKCs (refer to Figure 2.1). The C2

domain of nPKCs also lacks the calcium-coordinating acidic residues required for

calcium-binding the contrast to cPKCs, thus rendering nPKCs calcium-independent.

Atypical PKCs (aPKC) lack a C2 domain altogether, but contain an atypical C1 domain,

which contains only one cysteine-rich membrane targeting structure that binds PIP3 or

ceramide, and a protein-protein interaction PB1 (Phox and Bem 1) (Steinberg, 2008).

The PB1 domain mediates interactions with other PB1-containing scaffolding proteins.

Activity of aPKC is regulated mainly by protein-protein interactions, and

phosphorylation by phosphoinositide-dependent kinase-1 (PDK-1).

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

Figure 2.1 Structure of PKC isoforms. (Adapted from Steinberg, 2008)

26

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

27

2.2.1 - The C1 Domain

The C1 domains are part of the regulatory domain. They are membrane-targeting

molecules that interact with tumor-promoting phorbol esters and lipid metabolites

generated in response to growth factor stimulation (e.g. DAG, ceramide, arachidonic

acid) (Steinberg, 2008). They are defined by the presence of two repeated zinc-finger

motifs, C1a and C1b, each with a conserved pattern of cysteine and histidine residues

responsible for the coordination of two Zn2+ ions. The Zn2+ ions are essential for proper

protein folding and function. The C1 domain is the binding site for phorbol ester

(Mellor et al., 1998), as demonstrated by mutational and deletion analysis, and

confirmed by solution of the crystal structure of PKCδ forming a complex with PMA

(Zhang et al., 1995). Crystal structure images of PKCα, PKCγ, and PKCδ also show

that these C1 domains adopt similar tertiary structures and function as hydrophobic

switches to anchor PKCs to membranes (Hommel et al., 1994; Xu et al., 1997; Zhang et

al., 1995). DAG competes with PMA for binding to PKC, thus it is presumed that they

interact with PKC at the same site (Sharkey et al., 1985).

There are two classes of C1 domains, based on their ligand binding properties: typical

and atypical. Typical C1 domains are found in conventional and novel PKCs and bind

to DAG and phorbol esters. Atypical C1 domains are so named due to their presence in

the atypical PKCs and do not bind DAG or phorbol esters. This difference highlights the

different activation pathway of the PKCs.

2.2.2 - The C2 Domain

The C2 domain has been found to be present in many other proteins that also bind

phospholipid in a Ca2+-dependent manner (Mellor et al., 1998). They are found in

cPKCs at the C-terminal end of the C1 domain. The isolated C2 domain in PKCβ has

been shown to bind to phospholipid vesicles in a Ca2+-dependent manner (Shao et al.,

1996). NMR-spectroscopic studies show that two Ca2+ ions are coordinated by five

conserved aspartate residues. It has been suggested Ca2+ binding triggers a

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

28

conformational change that opens a cleft to allow binding of a phospholipid headgroup

(Grobler et al., 1996).

2.2.3 - The Pseudosubstrate Site

The pseudosubstrate site is located in the N-terminus of the C1 domain. It’s a sequence

that retains the hallmarks of a PKC phosphorylation site, but has an alanine instead of

serine/threonine at the predicted phosphorylation site (House et al., 1987). This site

suppresses the catalytic activity of PKCs by interacting with the catalytic domain of the

protein in an intramolecular fashion, thereby keeping the protein in an inactive

conformation (Mellor et al., 1998).

2.2.4 - The Catalytic Domain and V5 Region

The catalytic (or kinase) domain is mainly made up of a C3 domain and a C4 domain,

which are highly homologous between many kinases. The C3 domain contains the ATP

binding site, whereas the C4 domain is responsible for substrate binding (Kheifets et al.,

2007).

The V5 region is a short (approximately 50 amino acids) region at the carboxy-terminus

of all PKCs. It contains phosphorylation sites, such as the turn and hydrophobic motifs,

that are vital for kinase processing, localization, and activity (Newton, 2003).

Autophosphorylation of the V5 region is regulated by the balance between HSP70 and

PDK1 binding (Gao et al., 2001). Phosphorylation of the hydrophobic motif in the V5

region increases the affinity of the enzyme to Ca2+ as well as phosphotidylserine

(Edwards et al., 1997). These two interactions are mediated by the C2 domain, thus

demonstrating that the V5 region interacts with the C2 domain. V5 and C2 interaction is

terminated during PKC activation, allowing the regions to participate in inter-molecular

protein-protein, as well as lipid interactions. There are numerous examples of the V5

region being involved in important interactions. The V5 region of PKCβII drives

translocation of PKC to nuclear membranes through binding to phosphatidylglycerol (a

potent and selective PKCβII activator in the nuclear membrane) (Gokmen-Polar et al.,

1998). V5 of PKCα contains a PDZ binding domain that interacts with PICK (a PKCα

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

29

binding protein) for proper localization (Staudinger et al., 1997). V5 plays a key role in

PKC phosphorylation. In cPKCs, two V5 region phosphorylation sites have been

identified with at least one that is the target of an upstream, non-PKC kinase (Mellor et

al., 1998).

2.2.5 - The Hinge Region

The hinge, or V3, region is located between the regulatory domain and the catalytic

domain, and is known for its proteolytic sites. When PKC is activated and undergoes

conformational change, it becomes highly accessible to proteolytic cleavage (Kishimoto

et al., 1989). Cleavage results in the release of a constitutively active catalytic domain,

which suggests that most or all of the inhibitory intra-molecular interactions occur

between the regulatory and catalytic domain. Phosphorylation regulates the proteolytic

cleavage of this region in PKCδ (Steinberg, 2004). Aside from its proteolytic sites,

several studies suggest its importance in protein-protein interactions. For example, the

hinge region is involved in the binding of PKCα to β1-integrin (Parsons et al., 2002), as

well as in the targeting of PKCα and ε to cell-cell contacts (Quittau-Prevostel et al.,

2004). The hinge region is possibly involved in targeting PKCε for ubiquitin-dependent

destruction (Rechsteiner et al., 1996).

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

30

2.3 - Activation and Localization of PKC

In 1982, Kraft et al. discovered that phorbol ester stimulation caused a decrease in PKC

activity in the cytosol. In the subsequent year, they demonstrated that this decrease in

PKC activity is accompanied by a significant increase in PKC activity in the plasma

membrane (Kraft et al., 1983), providing evidence of what we now know is the

hallmark of PKC activation: translocation. Since then, numerous studies have further

confirmed this phenomenon including real-time images. Two mechanisms are important

for regulating the cellular function of PKC: 1) phosphorylation, which primes PKC into

an activatable state; and 2) interaction with targeting proteins, which poise specific

isozymes at defined intracellular locations (Dekker, 2004; Newton, 2004).

Early studies of PKCα show that PKCα is localized in the cytosol of resting cells, and

interacts only weakly/transiently with membranes in the absence of calcium or DAG

(Steinberg, 2008). Mobilization of intracellular Ca2+ leads to the binding of Ca2+ to the

C2 domain, which increases the affinity of the domain for plasma membranes. The

mobilization of intracellular Ca2+ is caused by agonists that promote phosphoinositide

hydrolysis and IP3 generation. At first, the electrostatic interaction of the C2 domain of

PKCα with membranes is relatively low affinity, until PKCα is anchored to the

membrane and a secondary C1A domain interation occurs with DAG. PS is important

for this secondary C1A domain interaction, as PS disrupts the electrostatic C1A/C2

interdomain interaction. This disruption frees the C1A domain which allows PKCα to

diffuse within the plane of the lipid bilayer and bind DAG. The combined energy from

the C1/C2 domain interaction with membranes results not only in a high-affinity

binding of cPKC to membranes, but also a conformational change to the protein. This

conformational change causes the autoinhibitory pseudosubstrate domain to be removed

from the substrate-binding pocket and also aids in PKC activation (Mackay et al., 2007;

Steinberg, 2008). Stimulation of P2X7 receptors is shown to cause PKCα to translocate

to the basolateral membrane of osteoclasts formed from RANKL-stimulated RAW264.7

cells (Armstrong et al., 2009).

PKCs also translocate to lipid rafts or caveolae, which are specialized microdomains of

the plasma membrane (Steinberg, 2008). Lipid rafts aid in signal transduction by

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

31

concentrating signaling complexes into large platforms. Ceramide is an important PKC-

regulated lipid in raft/caveolae, which provides the driving force for raft fusion into

platforms.

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

Figure 2.2 Activation of PKC isoforms and their signaling pathways. (Adapted

from Mackay et al., 2007)

32

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

33

2.4 - Function of PKCs in Various Cell Types

PKCs are known to be involved in a wide array of intracellular signaling pathways that

regulate processes such as cell growth, differentiation, secretion, apoptosis, tumour

development, cytoskeleton rearrangement and cell migration. The function of PKCs

tends to vary among different cell types due to differences in substrate availability and

specific cell signaling pathways. Even the same isoform of PKC can have opposing

roles in a cellular process in different cell types.

PKCθ has been shown to be a critical molecule that regulates T cell function at multiple

stages in T cell-mediated immune responses in vivo. High levels of PKCθ was found in

lymphoid tissue, and further studies determined that PKC θ is selectively expressed in T

cells, but not B cells (Isakov et al., 2002). PKC θ-deficient mice exhibit defects in T cell

activation, survival and activation-induced cell death as PKCθ is essential for mediating

the signals required for activation of NF-kappaB, AP1 and NFAT that are essential for

T cell activation (Pfeifhofer et al., 2003; Sun et al., 2000). Pfeifhofer et al. (2006) has

also determined that PKCα is necessary for T cell-dependent immune responses.

In studies of PKCα-knockout mice, PKCα was identified as a nodal integrator of cardiac

contractility by sensing intracellular Ca2+ and signal transduction events (Braz et al.,

2004). PKC has been implicated in the regulation of contraction of uterine muscle

(myometrium). Inhibition of PKCα with Go6976 as well as PKCα-specific antisense

oligodeoxynucleotides decreased the amplitude of potassium chloride-induced

myometrial contractions in a time-dependent manner (Fomin et al., 2009). PKCα levels

were also observed to be elevated in the pregnant myometrium, indicating that PKCα is

involved in the regulation of human uterine contraction.

PKCε has a well-established role in cell proliferation. The oncogenic potential of PKCε

has been extensively studied. One of the earliest studies was in NIH 3T3 fibroblasts,

where overexpression of PKCε resulted in a faster cell growth rate as well as increased

cell density (Mischak et al., 1993). Nude mice injected with NIH 3T3 fibroblasts

overexpressing PKCε developed tumors, demonstrating the role of PKCε as an

oncogene. In the same year, Cacace et al. (1993) discovered that overexpression of

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

34

PKCε in fibroblasts resulted in disordered growth control, leading to tumor formation

when injected into nude mice.

PKCα has been implicated in growth regulation and cell cycle arrest as well as tumor

formation and was found to be differentially expressed in human breast carcinomas (Ali

et al., 2009). Studies have shown that overexpression of PKCα promotes proliferation in

many cell types, particularly in mouse fibroblasts, U87 malignant glioma cells, and

MCF-7 breast cancer cells (Mandil et al., 2001; Soh et al., 2003; Soh et al., 2003; Ways

et al., 1995). Proliferation of human cancer cells was suppressed when gene expression

of PKCα was knocked down by siRNA (Yin et al., 2003). Shatos et al. (2009) provided

evidence that PKCα stimulates proliferation of conjunctival goblet cells through

activation of ERK1/2. However, other studies suggest that PKCα has an anti-

proliferative effect and that loss of PKCα function actually increases proliferation

(Nakagawa et al., 2006; Oster et al., 2006).

Table 2.1 further summarizes some of the functions of the different PKC isoforms.

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

35

Table 2.1 Processes in which PKC isotypes have been implicated based upon

analysis of PKC-deficient mice. (Adapted from Dekker, L.V., 2004)

Isotype Function

PKCα Insulin receptor feedbackPKCβ B-cell receptor signalling, feedback regulation

Mast cell activationNeutrophil NADPH oxidase activationGlucose transportTranscription factor activation in hypoxiaFear conditioning

PKCγ Hippocampal LTPLearning, addiction, anxietyNeuropathic pain

PKCδ Smooth muscle cell homeostasis/apoptosisB-cell tolerance

PKCε Acute PainAlcohol addictionMacrophage activation, host defense

PKCηPKCθ T-cell activationPKCζ NF-κB activation

B-cell receptor signallingPKCι

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

36

2.5 - Signalling Pathways of PKCs

PKCs are involved in a large variety of signalling pathways. Experiments in bone

marrow-derived mast cells indicate that classic PKC isoforms are upstream of JNK,

MEKK, and ERK signalling cascades (Redig et al., 2007).

Through the use of pharmacologic inhibitors of cPKCs and nPKCs, Gö6976 and

rottlerin respectively, Hock and Brown (2003) were able to demonstrate that both

cPKCs and nPKCs regulate endogenous mast cell NFAT activity, and NFATc1

transactivation. In their study, overexpression of dominant active PKC induced also

NFATc1.α/β transactivation. PKCθ-deficient T cells had reduced NFAT transactivation,

suggesting a critical role for PKCθ in NFAT activity (Pfeifhofer et al., 2003). Another

study revealed that PKCζ induces transcriptional activation driven by the NFATc2

transactivation domain by synergizing with calcineurin (San-Antonio et al., 2002). In

addition, the study provided evidence of physical interaction between PKCζ and

NFATc1 or NFATc2 isoforms, indicating that PKCζ modulates the activity of the

NFAT transactivation domain in T cells (San-Antonio et al., 2002).

Studies have shown that PKCα is involved in the ERK signalling pathway (Rucci et al.,

2005; Shatos et al., 2009). Rucci et al. (2005) demonstrated that inhibition of PKCα

prevented the phosphorylation of ERK in mature osteoclasts. ERK activation in rat

myometrial cells was shown to involve both DAG-sensitive PKC and DAG-insensitive

PKC (Robin et al., 2002). Bourbon et al. (2001) performed co-immunoprecipitation

studies that revealed that human embryonic kidney 293 cells (HEK-293) treated with

IGF-I induced ERK to associate with PKCε but not with PKCζ. PKCβ is involved in the

ERK pathway in the development and progression of diabetic nephropathy (Haneda et

al., 2001). Kv4.2, a subunit of the K+- channel, was recently found to be a possible site

of PKC and ERK cross-talk (Schrader et al., 2009).

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

37

2.5.1 - PKCs and NF-κB

The NF-κB signalling pathways play critical roles in a variety of cellular processes such

as cell growth and apoptosis, as well as in immune responses, development, and in

human diseases such as cancer and inflammation (Dekker, 2004). Early studies showed

that PKCs can activate NF-κB in vitro (Ghosh et al., 1990), which is consistent with the

fact that PMA is a relatively potent NF-κB activator in several cell types. Wang et al.

(2003) demonstrated the importance of PKCs in regulating the NF-κB in osteoclasts.

This leads to the suggestion that NF-κB works downstream of PKCs to transduce

activation signals from the cytoplasm to the nucleus.

A study provided evidence that PKCδ in involved in the transactivation of p65/RelA

(Lu et al., 2009). The study showed that PKCδ forms a complex with the p65/RelA in

the nucleus following exposure to TNF-α (Lu et al., 2009), which is interesting as PKCs

usually translocate to the cell membrane to exert its effects. This study also showed that

kinase activity of PKCδ is required for p65/RelA transactivation. Furthermore,

inhibition of PKCδ resulted in the reduction of NF-kappaB activity in response to TNF-

α.

In endothelial cells, PKCδ and PI3K/Akt pathways are involved in activating mTOR,

which serves as an endogenous modulator of NF-κB signalling of ICAM-1 expression

(Minhajuddin et al., 2009). The study observed that expression of the constitutively

active form of PKCδ was sufficient to induce NF-κB activation and ICAM-1

expression. In THP-1 cells, activation of PKCα leads to NF-κB activation and increased

β1-integrin expression (Kawakami et al., 2007).

PKCζ was implicated in the NF-κB signalling pathway in monocytes and macrophages.

Monocytes and macrophages that were stimulated with LPS resulted in PKCζ

activation. Inhibition of PKCζ activity lead to the suppression of LPS-stimulated

activation of NF-κB (Huang et al., 2009). There is also evidence that suggests that the

lack of PKCθ abrogates NF-κB activation in mature but not immature T cells (Sun et

al., 2000).

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

38

2.6 - The Role of PKCs in Osteoclast Function

As PKCs are involved in a wide variety of cell types, it is not surprising for them to be

involved in osteoclasts. PKCβ was found to have a role in the regulation of osteoclast

formation and function, potentially by participating in the ERK signaling pathway of M-

CSF and RANKL (Lee et al., 2003). Inhibition of PKCβ abolished ERK and MEK

activation by M-CSF and RANKL in osteoclast precursor cells, whereas cytokine-

induced NF-κB activation was not hampered by the PKCβ inhibition. 12-O-

tetradecanoylphorbol-13-acetate (TPA), a PKC activator, was shown to inhibit

RANKL-induced NF-κB activity, resulting in reduced osteoclast differentiation,

providing evidence of PKC having a role in modulating osteoclast formation (Wang et

al., 2003).

Rucci et al., 2005, has reported that PKCα in osteoclasts and in Chinese hamster ovary

(CHO) cells is recruited by αvβ3 upon the activation of the signal transduction pathway

that is associated with αvβ3, and contributes to the adhesion-dependent ERK 1/2

activation. They also demonstrated that αvβ3 integrin-activated PKCα was involved in

osteoclastic cell migration and bone resorption. The αvβ3 integrin in osteoclasts

recognizes and is activated by specific bone matrix proteins, such as osteopontin and

bone sialoprotein II, to provide adhesion, cytoskeletal re-organization and intracellular

signaling essential for cell survival and bone resorbing activity (Nakamura et al., 1999;

Ross et al., 1993).

Pereverzev et al., (2008) demonstrated that extracellular acidification enhances

osteoclast survival through an NFAT-independent, PKC-dependent pathway. The study

showed that the survival of rat osteoclasts over a period of 18 hours was significantly

enhanced by acidification of the medium. The ability of acid to promote survival was

not altered when NFAT activation was inhibited by a cell-permeable peptide. Instead it

suppressed RANKL-induced osteoclast survival. On the other hand, inhibition of PKC

blocked the effect of acid to promote osteoclast survival. This suggests that PKC plays a

critical role in mediating the effects of acid on osteoclast survival (Pereverzev et al.,

2008).

Chapter Two – The Role of Protein Kinase C in Osteoclast Function

39

P62 binds to TRAF6, which plays an important role in RANKL-induced

osteoclastogenesis (Ang et al., 2009). P62 and TRAF6 are upregulated during RANKL-

induced osteoclastogenesis, and genetic inactivation of p62 in mice leads to impaired

osteoclastogenesis in vitro and in vivo, as well as inhibition of IKK activation and NF-

κB nuclear translocation (Duran et al., 2004). Duran et al. (2004) further demonstrated

that RANKL stimulation leads to the formation of a ternary complex comprising of

TRAF6, p62, and the aPKCs suggesting a role of aPKCs during RANKL-induced

osteoclastogenesis.

P2X7 receptors are ligand-gated ion channels that mediate Ca2+ influx in osteoclasts

when activated by ATP. Benzoylbenzoyl-ATP (BzATP, a P2X7 agonist) induced

transient translocation of PKCα to the basolateral membrane of osteoclasts, whereas

osteoclasts derived from the bone marrow of P2rx7–/– mice failed to promote PKCα

translocation even when induced by BzATP (Armstrong et al., 2009). P2rx7–/– mice

showed deficient periosteal bone formation as well as excessive trabecular bone

resorption (Ke et al., 2003) demonstrating its importance in bone remodeling. Thus,

PKCα translocates to the basolateral membrane of osteoclasts when P2X7 receptors

induce an influx of Ca2+ (Armstrong et al., 2009). PKCα substrates on the basolateral

membrane of osteoclasts might include cell surface transporters and receptors.

Chapter Three

Hypothesis and Aims

Chapter Three – Hypothesis and Aims

40

3.1 - Rationale

Bone is a dynamic tissue that is constantly remodeled throughout skeletal repair and

development. Bone remodeling is a tightly regulated process by which the balance of

bone formation and bone resorption is maintained. Two cell-types are primarily

responsible for bone remodeling: 1) osteoblasts and 2) osteoclasts. Osteoblasts are

responsible for the formation of bone, whereas osteoclasts are responsible for bone

resorption. Bone remodeling is necessary to maintain skeletal structure and strength, as

well as regulate the serum calcium concentration level (Tanaka, 2007).

Osteoclasts are large multinucleated cells that resorb bone. They do so by attaching

themselves to the bone surface and forming a sealed compartment, known as the

resorption lacuna, between the cell membrane and the bone. H+ ions and osteolytic

enzymes are pumped into the resorption lacuna from the osteoclasts to degrade the

bone’s mineral and organic matrix. Understanding the molecular and cellular processes

that regulate the signalling pathways during osteoclast formation and bone resorption

might facilitate the development of selective drugs for the treatment of osteoclast-

related diseases, such as osteoporosis, osteoarthritis and cancer-induced bone loss.

Protein Kinase C (PKC) plays a crucial role in signal transduction for a variety of

biologically active substances that activate cellular functions and proliferation

(Kishimoto, et al., 1989). The PKC family of serine-threonine kinases encompasses

several different isoforms. PKC-mediated cellular processes appear to be both tissue-

and isoform-specific. The general importance of PKCs in osteoclast biology has been

previously established (Su, et al., 1992; Zhu, et al., 2004). More recently, we have also

demonstrated a role for PKCs in RANKL-induced osteoclast signalling in this

laboratory. Other studies have found that PKCβ participates in the ERK signalling

pathway during M-CSF and RANKL-stimulated osteoclastogenesis, but not the NF-κB

pathway (Lee, et al., 2003), suggesting that individual PKC isoforms may play specific

roles in the regulation of RANKL-induced signalling pathway and osteoclastogenesis.

Chapter Three – Hypothesis and Aims

41

3.2 - Hypothesis and Aims Osteoclasts are terminally differentiated, multinucleated cells responsible for the

physiological and pathological degradation of mineralised bone. The receptor activator

of nuclear factor-kappa B ligand (RANKL) is obligatory for osteoclast formation,

activation and survival. RANKL interacts with its receptor RANK, which results in

activation of downstream signalling cascades. Like other cytokine signalling pathways,

various adapter molecules, including TRAF6, p62 and protein kinase C (PKC) are in

place to regulate specific stages of the RANKL signalling cascades. It has been shown

that PKCs are important downstream targets of RANKL/RANK/TRAF6, which regulate

RANKL-induced osteoclastogenesis and signalling pathways. This has led to the

hypothesis that PKCs are important regulators of osteoclast differentiation, function

and signalling.

The specific aims of this project are to:

1) Investigate the gene expression of the various PKC isoforms during RANKL-

induced osteoclastogenesis and identify those isoform(s) that are specifically

regulated by RANKL.

2) Determine the role of various PKC isoforms in osteoclastogenesis and RANKL

signalling pathways in vitro.

3) Knockdown expression of PKCs by siRNA-mediated silencing and examine its

effect on osteoclast differentiation and function.

Chapter Three – Hypothesis and Aims

42

3.3 - Outcomes and Significance

The outcomes of the work proposed will help to elucidate the role of PKC isoforms in

RANKL-induced signalling pathways, osteoclastogenesis, and bone resorption.

Overproduction and activation of osteoclasts underline many lytic bone disorders such

as osteoporosis, osteoarthritis and Paget’s disease. Development of strategies to control

the formation or activities of osteoclasts has been a major focus of bone research. The

RANKL-induced signalling pathway is critical for osteoclastogenesis and osteoclastic

bone resorption and, therefore might represent a potential molecular target for the

discovery of novel bone anti-resorptive agents for the treatment of lytic bone disorders.

This thesis aims to examine the fundamental role of PKC in osteoclast differentiation,

function and RANKL signalling pathways. Understanding the molecular and cellular

mechanism(s) by which PKC regulate osteoclast formation and activation will further

facilitate the development of novel and selective inhibitors for the treatment of lytic

bone disorders.

Chapter Four

Materials and Methods

Chapter Four – Materials and Methods

43

4.1 - Materials

4.1.1 – Cell Lines

CELL LINE DESCRIPTION SUPPLIER

RAW264.7

(Subclone C4)

Mouse

myeloid/monocytes

Kindly provided by Dr

A.I. Cassady, Centre for

Molecular and Cellular

Biology, Department of

Biochemistry, University

of Queensland.

HEK-293 Human embryonic kidney

cells

The American Type

Culture Collection

4.1.2 – Bacterial Strains

STRAIN DESCRIPTION SUPPLIER

DH5α Escherichia coli cloning

strain

4.1.3 – Commercially Purchased Kits

KIT SUPPLIER/MANUFACTURER

Dual-Luciferase® Reporter Assay System Promega Corp., Madison, WI., USA

Chapter Four – Materials and Methods

44

GoTaq® Green Master Mix Promega Corp., Madison, WI., USA

HiPerFect Transfection Reagent QIAGEN GmbH, Australia

QIAEX II Gel Extraction Kit (500) QIAGEN GmbH, Australia

RNeasy® Mini Kit QIAGEN GmbH, Australia

Taq DNA polymerase kit QIAGEN GmbH, Australia

Wizard® Plus SV Minipreps DNA

Purification System

Promega Corp., Madison, WI., USA

Wizard® Plus SV Midipreps DNA

Purification System

Promega Corp., Madison, WI., USA

4.1.4 – Chemical Reagents

SOLUTIONS SUPPLIER/MANUFACTURER

99.9 % Acrylamide Bio-Rad Laboratories,

Hercules, CA., USA

Agarose Promega Corp., Madison,

Wi., USA

Agar powder BDH Laboratory Supplies,

Poole, Dorset, England

Bacto yeast extract Difco Laboratories, Detroit,

Michigan, USA

Chapter Four – Materials and Methods

45

Bacto peptone tryptone Difco Laboratories, Detroit,

Michigan, USA

Calcium chloride (CaCI2) Sigma Chemical Co,. St

Louis, Mo, USA.

Chloroform BDH Laboratory Supplies,

Poole, Dorset, England

Diethyl pyrocarbonate (DEPC) water Sigma Chemical Co,. St

Louis, Mo, USA.

Dihydroethidium (DHE) Molecular Probes, OR, USA

Dimethyl sulphoxide (DMSO) BDH Laboratory Supplies,

Poole, Dorset, England

Dithiothreitol (DTT) Sigma Chemical Co,. St

Louis, Mo, USA.

Ethanol (100%) BHD Laboratory Supplies,

Poole, Dorset, England

Ethylene tetra-acetic acid (EDTA) Boehringer Mannheim Corp.,

Indpl, IN, USA

Ethidium bromide Sigma Chemical Co,. St

Louis, Mo, USA.

Glycerol BDH Laboratory Supplies,

Poole, Dorset, England

Chapter Four – Materials and Methods

46

HEPES Sigma Chemical Co., St

Louis, Mo., USA

Isopropanol (Propan-2-ol) BDH Laboratory Supplies,

Poole, Dorset, England

Lithium acetate BDH Laboratory Supplies,

Poole, Dorset, England

β-Mercaptoethanol Sigma Chemical Co., St

Louis, Mo., USA

Methanol BDH Laboratory Supplies,

Poole, Dorset, England

Paraformaldehyde Merck, Darmstadt, Germany

Phenol red Sigma Chemical Co., St

Louis, MO., USA

PMSF (Phenylmethylsulfonyl fluoride) Boehringer Mannheim Corp.,

Indpl, IN, USA

RNA sample loading buffer Sigma Chemical Co., St

Louis, Mo., USA

Sodium chloride (NaCI) BDH Laboratory Supplies,

Poole, Dorset, England

Sodium dodecyl sulphate (SDS) BDH Laboratory Supplies,

Poole, Dorset, England

Sodium hydroxide (NaOH) BDH Laboratory Supplies,

Chapter Four – Materials and Methods

47

Poole, Dorset, England

Sodium phosphate, dibasic, anhydrous

(Na2HPO4)

Sigma Chemical Co., St

Louis, Mo., USA

Sodium phosphate, monobasic, anhydrous

(NaH2PO4)

Sigma Chemical Co., St

Louis, Mo., USA

TEMED

(N, N, N`, N`-Tetra-methyl-

ethylenediamine)

Bio-Rad Laboratories,

Hercules, CA., USA

Tris (Tris (hydroxymethyl) aminomethane) Boehringer Mannheim Corp.,

Indpl, IN, USA

Tris (hydroxymethyl) methane BDH Laboratory Supplies,

Poole, Dorset, England

Triton X-100

(Iso-octylphenoxypolyethoxyethanol)

Packard Instrument

International, SA., Zurich,

Switzerland

4.1.5 - Enzymes

DESCRIPTION SUPPLIER/MANUFACTURER

Moloney Murine Leukaemia Virus (M-MLV)

reverse transcriptase

Promega Corp., Madison, Wi., USA

Proteinase K Promega Corp., Madison, Wi., USA

Restriction Enzymes Promega Corp., Madison, Wi., USA

Chapter Four – Materials and Methods

48

Taq DNA Polymerase Promega Corp., Madison, Wi., USA

T4 DNA Ligase Promega Corp., Madison, Wi., USA

4.1.6 - Antibodies

DESCRIPTION SUPPLIER/MANUFACTURER

Murine monoclonal anti-NFATc1(7A6)

Santa Cruz Biotechnology Inc.

Murine monoclonal anti-p-Erk Santa Cruz Biotechnology Inc.

Rabbit polyclonal anti-IκBα Santa Cruz Biotechnology Inc.

Rabbit polyclonal anti-p-PKCα/βII

(Thr638/641)

Cell Signaling Technology, Inc.

Rabbit polyclonal anti-PKCδ

Cell Signaling Technology, Inc.

Rabbit polyclonal anti-α-tubulin Sigma Chemical Co., St

Louis, Mo., USA

Murine monoclonal anti-β-actin (JLA20)

Anti-mouse IgG Peroxidase Sigma Chemical Co., St

Louis, Mo., USA

Anti-rabbit IgG Peroxidase Sigma Chemical Co., St

Louis, Mo., USA

Chapter Four – Materials and Methods

49

4.1.7 - Vectors

DESCRIPTION SUPPLIER/MANUFACTURER

pFlag-RANK Generated in our laboratory

pcDNA3.1 Invitrogen Australia

pGL3-Basic Vector Promega Corp., Madison, WI., USA

4.1.8 – Other Materials and Equipments

MATERIAL AND EQUIPMENT

SUPPLIER/MANUFACTURER

Baxter ddH20 Baxter, Sydney,Australia

BigDyeTM reaction mix Promega Corp., Madison, Wi.,

USA

Carbon Dioxide (CO2) gas, food grade Aligal® 2, Air Liquid, WA,

Australia

Cell culture flasks: T-25; T-75 Life Technologies, Australia

Cell culture plates: 6 -, 24- & 96-wells Life Technologies, Australia

Cell Scraper Sarstedt, Germany

Centrifuge tubes: 10 mL, 50 mL Sarstedt, Germany

CKX41 Light Microscope Olympus

Chapter Four – Materials and Methods

50

Cling Film Glad, Australia

Cover slips (22 mm x 22 mm) Knittel Glaser, Germany

Cryogenic vials (sterile) Corning Glassworks, USA

Cuvettes (4 mm gap, sterile) Invitrogen, Australia

DNA Ladder, 100 bp (250 μL) Promega Corp., Madison, Wi., USA

DNA Ladder, 1 kb (250 μL) Promega Corp., Madison, Wi., USA

Eclipse TE2000-S Microscope Nikon

ECL Plus Western Blotting Detection

Reagents

Amersham Life Sciences, Australia

ELB300 Portable Electronic Balance Shimadzu Philippines Manufacturing Inc.,

Philippines

Filter paper Whatmans International, England

Filter units (Bottle-top) (0.45 µm) Whatmans International, England

GeneAmp PCR System 2400 Perkins-Elmer

Gene Pulser II Bio-Rad Laboratories Pty. Ltd.

Glass Slides Knittel Glaser, Germany

Hybridisation Oven (Model HO35) Ratek Instruments Pty. Ltd., Vic., Australia

LAS-3000 mini (luminescent image

analyser)

Fujifilm

Liquid Nitrogen (N2) Air Liquid, WA, Australia

Mastercycler® personal (PCR) Eppendorf

Microcentrifuge tubes: 1.5 mL Sarstedt, Germany

Chapter Four – Materials and Methods

51

Microcentrifuge tubes: 500 μL, 2.0 mL

TRACE Bioscience, Australia

Micropore filters (0.2, 0.45 & 0.8 µm) Crown Scientific, Australia

Mini-PROTEAN® 3 Cell Bio-Rad Laboratories Pty. Ltd.

Mini-Sub Cell Bio-Rad Laboratories Pty. Ltd.

Mini Trans-Blot Electrophoretic Transfer

Cell

Bio-Rad Laboratories Pty. Ltd.

M-MLV RT 5x Reaction Buffer Promega Corp., Madison, Wi., USA

Model 200/2.0 Power Supply Bio-Rad Laboratories Pty. Ltd.

Model J-6B Centrifuge Beckman Coulter, Inc.

Oligo(dT) Primers (100 mM) Promega Corp., Madison, Wi., USA

Orbital Mixer Incubator (Model OM11) Ratek Instruments Pty. Ltd., Vic., Australia

Parafilm Laboratory film American National Can,

Menasha, Wi, USA

PCR 200 μL thin-walled tubes Unimed Australia Pty Ltd,

Australia

PolarStar Optima BMG Labtechnologies,

Offenburg, Germany

PowerPac 300 Power Supply Bio-Rad Laboratories Pty. Ltd.

Precision Plus Protein Standards Bio-Rad Laboratories Pty. Ltd.

Rnasin Rnase Inhibitor Promega Corp., Madison, Wi., USA

Rocking Platform Mixer (Model RPM5) Ratek Instruments Pty. Ltd., Vic., Australia

Sartorius R160D Balance Sartorius GmbH Goettingen, Germany

Serological pipettes: 5 ml, 10 ml Life Technologies, Australia

Chapter Four – Materials and Methods

52

Skim milk powder Diploma, Melbourne, Australia

SmartSpec™ 3000 Spectrophotometer Bio-Rad Laboratories Pty. Ltd.

T4 DNA Ligase 10x Reaction Buffer Promega Corp., Madison, Wi., USA

Thermomixer comfort (heating block) Eppendorf

Transfer pipette, 1 ml Sarstedt, Germany

Tomy MicroOne Capsulefuge (Model

PMC-880)

Tomy Kogyo Co. Ltd.

Waterbath (Model WB14) Ratek Instruments Pty. Ltd., Vic., Australia

Wide Mini-Sub Cell Bio-Rad Laboratories Pty. Ltd.

4.1.9 – Pharmacological Compounds

DESCRIPTION SUPPLIER/MANUFACTURER

Rottlerin Sigma Chemical Co., St. Louis, Mo., USA

Go6976 Sigma Chemical Co., St. Louis, Mo., USA

Chapter Four – Materials and Methods

53

4.1.10 – Buffers and Solutions Solutions and buffers were prepared using highly purified Milli-Q double distilled water

(MQ ddH2O) and, where appropriate, were sterilised by autoclaving and stored at room

temperature, unless otherwise stated. All chemicals were weighed using a Mettler

P1210 balance. Adjustment of pH was made using a pH-2 Scan pH meter, calibrated

with appropriate pH standards (pH 4.0 or pH 7.0).

SOLUTIONS COMPOSITION AND PREPARATION

30% Acrylamide 29 g of acrylamide and 1 g of bisacrylamide were dissolved

in a total volume of 60 mL of ddH2O with mild heating at

37°C to acid solubility. Volume adjusted to 100 mL with

ddH2O, then filtered through two sheets of Whatman #1

filter paper. Stored at 4°C in the dark.

3-aminopropyltriethoxy-

saline

Dissolved in acetone (v/v).

Ammonium persulphate 10% stock prepared in ddH2O and stored in 1 mL aliquots at

-20°C.

Ampicillin 100 mg/mL stock. Dissolved in ddH2O, sterilized by

filtration and stored in 1 ml aliquots at -20°C.

Aprotinin 10 mg/mL stock dissolved in ddH2O. Dispensed into small

aliquots and stored at -20°C.

Adenosine triphosphate

(ATP)

100 mM stock prepared in ddH2O. Dispensed into 0.5 mL

aliquots and stored at -20°C.

BSA/PBS 0.2% stock. Dissolved in 1x PBS, sterilized by filtration and

stored at 4°C.

Chapter Four – Materials and Methods

54

Calcium chloride 100 mM stock prepared from solid CaCl2.2H2O. Stored at

4°C.

Denaturing Buffer 0.25 M HCI stock prepared in ddH2O. Stored at room

temperature.

DEPC water 2 mL of DEPC in 18 mL of ethanol (100%) diluted in

ddH2O to a final volume of 2 L. Solution was left to stand

overnight for DEPC treatment prior to autoclaving.

dNTPs 100 mM stock solution made up of each dNTP constituent

(dATP, dTTP, dCTP and dGTP). An equal volume of these

components mixed together produced a 25 mM combined

stock of dNTPs, which was diluted to 5 mM with ddH2O

and stored as 200 µL aliquots at -20°C.

Dithiothreitol (DTT) Prepared at a concentration of 1 M and stored at -20° C in 1

mL aliquots. Not sterilized.

5x DNA Sample Buffer Composition: 0.25% xylene cyanol, 0.25% bromophenol

blue, 30% glycerol. Stored at room temperature.

EDTA 0.5 M stock, brought to pH 8.0 with NaOH and stored at

room temperature.

Ethidium bromide Prepared as 10 mg/mL stock with sterile ddH2O. Stored in

the dark at 4°C.

Hanks balanced salt

solution (HBSS)

138 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2 1.06 mM

MgCl2, 12.4 mM HEPES and 5.6 mM glucose at pH 7.3

Luciferase cell lysis

buffer

30 mM Tris, 10% glycerol, 1% triton X-100, 2 mM EDTA

Chapter Four – Materials and Methods

55

Paraformaldehyde 10% stock prepared by dissolving 5 g of paraformaldehyde

in 50 mL ddH2O. Solution was warmed to 60°C and the pH

adjusted to 7.4 with 1 M NaOH. Stored as 1 mL aliquots at -

20°C.

Paraformaldehyde (4%) Stock solution diluted in PBS (pH 7.4) to a final working

dilution of 4%. Stored at 4°C

.

Parthenolide

10 mM stock stored at -20°C

Phosphate buffered

saline (PBS)

20x stock prepared from the following components: 10 g

NaH2PO4, 203.6 g Na2HPO4, 170 g NaCl in ddH2O. pH was

equilibrated to 7.4 with HCl.

PBS 1x pH 7.4 solution was prepared by diluting 100 mL of

stock solution in 1 L ddH2O. Solution was filter-sterilized

and stored at room temperature.

PBS-T 10x stock solutions were prepared from the following

components: 80 g NaCl, 2 g KH2PO4, 14.4 g Na2HPO4, 2 g

KCl and 5 mL of Tween 20. Stored at room temperature

without being autoclaved.

Pepstatin A 1.0 mg/mL stock prepared in methanol. Stored as small

aliquots at -20°C.

Phenol Ultrapure phenol (BRL) was equilibrated to a pH > 7.8 as

described by Sambrook, Fritsch and Maniatis (1989). Stored

in aliquots at -20°C.

Phenylmethylsulfonyl-

fluoride (PMSF)

100 mM stock solution prepared fresh in ethanol when

required.

Chapter Four – Materials and Methods

56

Sodium dodecyl sulphate

(SDS)

Prepared as an unsterilized, 10% (w/v) solution, stored at

room temperature.

Sodium acetate Prepared at 3 M sodium ion concentration by dissolving

24.6 g of powder form in 100 mL of ddH2O, adjusting the

pH with glacial acetic acid to 5.2, and making up any

remaining volume requirement with ddH2O. Sterilized by

autoclaving and stored at room temperature.

Sodium chloride 5 M stock solution prepared in ddH2O, autoclaved and

stored at room temperature.

di-sodium hydrogen

orthophosphate

1 M stock solution prepared from powdered anhydrous

form. Autoclaved and stored at 4°C.

Sodium dihydrogen

orthophosphate

1 M stock solution prepared from powdered NaHPO4.2H2O

form. Autoclaved and stored at 4°C.

Sodium hydroxide 5 M stock solution, stored at room temperature. Not

sterilized.

TAE 50x stock solution prepared by mixing 242 g Trizma base,

57.1 mL glacial acetic acid and 100 mL of 0.5 M EDTA pH

8.0, and diluting to 1 L with ddH2O. Stored at room

temperature.

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TE Both 10x (100 mM Tris pH 7.5, 10 mM EDTA) and 1x (10

mM Tris pH 7.5, 1 mM EDTA) stock solutions were

prepared and sterilized by autoclaving. Stock solutions of 1

M Tris and 0.5 EDTA diluted in ddH2O were used to

prepare them.

Tris-HCl buffers 1 M Tris stock solutions were prepared at various pHs

between 7.3 and 8.0. Adjustment of the pH to the desired

value was achieved by adding appropriate amounts of

concentrated HCl after dissolving the Tris powder in a

volume of ddH2O 80% the amount of the total volume. The

final volume was then made up with more ddH2O.

Autoclaved and stored either at room temperature or 4°C.

Tris-buffered saline

(TBS)

15 mL of 5 M NaCl, 25 mL of 1 M Tris-HCl (pH 7.4) in 500

mL ddH2O.

TBS-Tween (0.2%)

(TBST)

TBS containing 0.2% Tween-20 detergent.

Tris-Phosphate buffer The 1 M Tris solutions were adjusted to pH 9.0 with 1 M

sodium diphosphate solution.

Trypsin-EDTA 50 mL of 10x Trypsin-EDTA in 1x PBS to 500 mL. Filter-

sterilised and stored at 4°C.

Chapter Four – Materials and Methods

58

4.1.11 – Oligonucleotide Primers Oligonucleotide primers for PCR amplification were purchased from the following

manufacturers: Proligo LLC and Genset Oligos (Australia). Upon receipt in freeze-dried

form, the primers were stored at -20°C in a constant temperature freezer. Table 3.1 lists

the primers, their sequences, and annealing temperature.

Chapter Four – Materials and Methods

59

Table 4.1 Primers used for PCR amplification (TM = Annealing temperature).

Primers Forward sequence 5’ 3’ Reverse sequence 5’ 3’ TM

d2

GGATCCGAATTCATGCTTGA

GACTGCAGAG

GGTCTAGATTATAAAATTGG

AATGTAGCT 530C

e1

GGATCCGAATTCGCAGGCAT

GGCATACCAC

GAATTCTCTAGATCAAGGCC

AGTGGTACTT 600C

NFATc1

CAACGCCCTGACCACCGATA

G

GGCTGCCTTCCGTCTCATAG

T 550C

Cathepsin K GGGAGAAAAACCTGAAGC ATTCTGGGGACTCAGAGC 550C

Calcitonin

Receptor TGGTTGAGGTTGTGCCCA CTCGTGGGTTTGCCTCATC 620C

TRAP

TGTGGCCATCTTTATGCT

GTCATTTCTTTGGGGCTT

550C

36B4 TCATTGTGGGAGCAGACA TCCTCCGACTCTTCCTTT 580C

PKCα GAAGGTGATGCTTGCTGACA CGTTGACGTATTCCATGACG 600C

PKCβ TCCCTGATCCCAAAAGTGAG AACTTGAACCAGCCATCCAC 600C

PKCγ GCAGCTTCACTCCACCTTTC CCTGTAGATGATGCCCTGGT 600C

PKCδ CAGACCAAGGACCACCTGTT CGTCCCTGTCTAGCATCACA 600C

PKCε

GAGGACTGGATTGACCTGG

A ATCTCTGCAGTGGGAGCAGT 600C

PKCη CATCCCACACAAGTTCAACG ATATTTCCGGGTTGGAGACC 600C

PKCθ

CCAGAAAAAGCCAACCATG

T GGAACATGGTTTCTCGGCTA 600C

PKCζ

AAGTGGGTGGACAGTGAAG

G CAGCTTCCTCCATCTTCTGG 600C

PKCι TATGGCTTCAGCGTTGACTG CCTTTGGGTCCTTGTTGAGA 600C

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4.2 – Media and Agar Plates

4.2.1 – Luria-Bertani (LB) Medium

LB medium was prepared by mixing the following:

Bacto peptone tryptone 10 g

Bacto yeast extract 5 g

Sodium chloride (NaCl) 10 g

Autoclaved MQ ddH2O 950 mL

The mixture was stirred at room temperature until all solutes were dissolved. After

dissolution, the pH was adjusted to 7.0 and the final volume made up to 1 L. The

medium was then sterilised by autoclaving for 20 minutes at 121̊C on the liquid cycle.

The medium was stored at room temperature in a sterilised fumehood until use.

4.2.2 – LB/Ampicillin Agar Plates

Prior to autoclaving, 15 g of agar powder was added to 1 L of LB medium. The medium

was then sterilised by autoclaving for 20 minutes at 121˚C on the liquid cycle. After

sterilisation, the medium was stirred to ensure that the molten agar was evenly

distributed throughout the medium. The medium was then allowed to cool to 50̊C

before ampicillin (Amp) was added to the LB/Agar mixture to a final concentration of

50 mg/mL. 15 mL of the LB/Agar/Amp mixture was poured onto agar plates and

allowed to stand at room temperature until the agar set. The plates were then stored at

4˚C until use.

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4.3 – Cell Culture

4.3.1 – Cell Culture Media and Reagents

DESCRIPTION MANUFACTURER

Alpha-Minimum Essential Medium (α-MEM) TRACE, Sydney, Australia

Dulbecco’s Modification of Eagles Medium

(DMEM)

TRACE, Sydney, Australia

Fetal bovine serum (FBS) TRACE, Sydney, Australia

L-Glutamine Gibco BRL, Life

Technologies, Melbourne,

Australia

Opti-MEM I Reduced Serum Medium Gibco BRL, Life

Technologies, Melbourne,

Australia

Penicillin-Streptomycin Gibco BRL, Life

Technologies, Melbourne,

Australia

Trypsin-EDTA (pH 7) Gibco BRL, Life

Technologies, Melbourne,

Australia

All culture media and reagents were pre-warmed and the outside of their containers

sprayed with 70% ethanol before use.

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4.3.2 – Culturing of RAW264.7 and HEK-293 Cells

RAW264.7 and HEK-293 cells were cultured in T-75 culture flasks and maintained in

α-MEM (for RAW264.7 cells) or DMEM (for HEK-293 cells). The medium was

supplemented with pre-warmed 10% FBS, 2 mmol of L-glutamine, 100 U/mL of

penicillin and 100 μg/mL of streptomycin (complete medium). The cell cultures were

incubated in a humidified atmosphere of 5% CO2 at 37˚C in a water-jacketed incubator.

The cells were subcultured every 2 to 3 days and were seeded to appropriate cell

densities when required for transfection upon reaching confluence.

4.3.3 -- Cryopreservation of Cell Lines

For long-term storage, cell lines were trypsinized, pelleted by centrifugation, and

resuspended in a 90% FBS and 10% DMSO solution in cryogenic vials. The cells were

first resuspended in FBS before addition of DMSO dropwise while shaking the vials to

dissipate heat generated chemically by the DMSO. The vials of cells were then initially

stored overnight in -80˚C freezers in an ethanol-equilibrated cryo-freezing container

before being transferred into liquid nitrogen for long-term preservation.

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4.4 – Experimental Methods

4.4.1 - Extraction of Total RNA and Reverse Transcriptase-

Polymerase Chain Reaction (RT-PCR)

Isolation of total cellular RNA was conducted using an RNeasy Mini Kit from Qiagen

according to the manufacturer’s protocol. Briefly, cells grown as a monolayer on a 6-

well or 24-well plate were lysed directly by the addition of 350 μL of Buffer RLT. Next,

one volume (350 μL) of 70% ethanol was added onto the wells of cell lysate. The

samples (700 μL ) were then transferred to an RNeasy mini column placed on a 2 mL

collection tube and centrifuged at 10,000 rpm for 15 seconds at room temperature. The

flow-through was subsequently discarded and 700 μL of Buffer RW1 was added to the

column. The samples were centrifuged at 10,000 rpm for 15 seconds at room

temperature. The flow-through was again discarded and the RNeasy column transferred

onto a new 2 mL collection tube. The RNeasy column was washed twice with 500 μL

Buffer RPE and centrifuged at 10,000 rpm for 2 minutes to dry the RNeasy silica-gel

membrane. RNA was eluted with 30-50 μL of RNase-free water and collected in a new

1.5 mL eppendorf tube by centrifugation at 10,000 rpm for 1 minute at room

temperature. The RNA was immediately stored at -80ºC until further use.

4.4.1.1 - Reverse Transcription of mRNA

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used to convert the

mRNA to cDNA.

The initial mixture for RT-PCR was prepared in 200 μL PCR tubes on ice as follows:

dNTP (5 mM) 2.00 μL

Oligo(dT) (100 μM) 0.25 μL

ddH2O 11.75 μL

Template RNA 2.00 μL

Total Volume 16.00 μL

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The mixture was then mixed, spun down, and heated for 3 minutes at 75ºC. Then, the

following was added to each reaction tube:

10x RT buffer 2 μL

RNase 1 μL

M-MLV RT 1 μL

Initial mixture 16 μL

Total Volume 20 μL

The mixture was then mixed, spun down, and incubated at 42ºC for 1 hour and then at

92ºC for 10 minutes. The resulting samples contain cDNA, which were stored at -20ºC

for further use.

4.4.1.2 - Polymerase Chain Reaction (PCR)

PCR of the cDNA was carried out using the following protocol:

GoTaq® Green Master Mix (2x

concentration)

12.5 μL

Forward primer (20 μM) 0.5 μL

Reverse primer (20 μM) 0.5 μL

cDNA 2.0 μL

ddH2O 10.5 μL

Total volume 25.0 μL

Briefly, a mastermix was made up by first inoculating a 500 μL tube with ddH2O. Next,

GoTaq® Green Master Mix was added, followed by the forward and reverse primers

(refer to Table 4.1). The mastermix was then gently mixed by flicking the tube, and 23

μL of the mixture was aliquoted into a 200 μL PCR tube. Lastly, the cDNA was added

into the PCR to make up a final volume of 25 μL.

Chapter Four – Materials and Methods

65

Each tube was gently mixed by flicking and centrifuged briefly to ensure that the

sample filled the bottom of the tube. The samples were then amplified in a thermal

cycler (Eppendorf Mastercycler® personal). The cycling condition was as follows:

The initial denaturation was at 94˚C for 5 minutes, followed by 30 cycles of

Denaturing at 94˚C for 40 seconds

Annealing at 60˚C for 40 seconds

DNA synthesis at 72˚C for 40 seconds.

Final extension was at 72̊C for 10 minutes followed by a hold at 4̊C at an indefinite

amount of time.

For semi-quantitative RT-PCR, 10 μL of each PCR product was electrophoresed on a

ethidium bromide-stained agarose gel with concentrations ranging from 1-2%

depending on the predicted PCR product size. The gel was visualized with a UV

transilluminator and photos taken with a digital camera. The density of the bands was

measured from the photos using a Scion Image program. The relative ratios between the

samples were normalized to 36B4.

4.4.2 – Protein Isolation, SDS-PAGE Protein Analysis, and Western

Blot

4.4.2.1 - Protein Extraction

To lyse the cells, the media in the wells were first removed and 200 μL of Ripa-Ripa

lysis buffer containing a cocktail of protease inhibitors (aprotinin 1 μg/mL, sodium

orthovanadate 1 mM, PMSF 50 μg/mL) was added directly into the wells. The plate was

incubated on ice for 20 minutes. After 20 minutes, the cell lysates were transferred into

Eppendorf tubes and centrifuged for 20 minutes at 12,000 rpm at 4˚C to pellet the cell

nuclei and unlysed cells. The supernatant was transferred into fresh Eppendorf tubes

and stored in -20˚C until required.

Chapter Four – Materials and Methods

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4.4.2.2 - SDS-PAGE

Proteins were separated by electrophoresis on discontinuous SDS polyacrylamide gels,

prepared and ran using mini-PROTEAN® 3 Cell casting apparatus according to the

manufacturer’s instructions. Briefly, a 10% separating gel solution was pipetted into a

gel space of 0.5 mm. The top of the separating gel was then overlayed with 20% ethanol

to reduce shrinkage and aid polymerization of the gel. The gel was left to solidify at

room temperature for approximately 30 minutes. Once the gel has solidified, the ethanol

was removed from the surface of the gel and the remaining gel space was rinsed with

ddH2O. The remaining gel space was then filled with 4% stacking gel solution and a

well-forming comb was inserted onto the top of the gel. The gel was again left at room

temperature to solidify for approximately 30 minutes. Once the stacking gel has

solidified, the well-forming comb was removed and the wells were rinsed with ddH2O

to remove any un-polymerized acrylamide. The gel was then loaded into a tank filled

with 1 x SDS-PAGE running buffer. 10-25 μL of each protein sample was combined

with equal amounts of 2 x SDS-PAGE sample loading buffer. The samples were then

denatured at 100̊ C for 5 minutes, briefly centrifuged and loaded in the wells. 6 μL of

Precision Plus Protein Standards from Bio-Rad was run in parallel with the samples for

molecular weight comparisons. All samples were electrophoresed at 160V at room

temperature for approximately one hour.

4.4.2.3 - Protein Transfer

Following SDS-PAGE, the mini-PROTEAN® 3 Cell apparatus was disassembled and

rinsed with water. The gel was transferred onto a Mini Trans-Blot Electrophoretic

Transfer Cell and assembled according to the manufacturer’s instructions. Briefly, two

scotch pads and six Whatman 3MM paper were soaked in transfer buffer. One scotch

pad was placed onto the clear (positive) side of the cassette followed by three of the

Whatman 3MM paper on top of it. A piece of nitrocellulose membrane (Hybond-C) was

placed onto the top of the Whatman 3MM paper followed by the gel. Finally, the

remaining three Whatman 3MM paper and scotch pad were place on top of the gel. The

cassette was closed and loaded into a Western transfer tank filled with transfer buffer

and an ice block. The proteins were transferred from the gel onto the nitrocellulose

membrane at a constant current of 30 mAmp overnight.

Chapter Four – Materials and Methods

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4.4.2.4 - Western Blotting

The nitrocellulose membrane containing protein was blocked with TBS-Tween

containing 5% non-fat skim milk for one hour at room temperature on a rocking

platform mixer. After blocking, the membrane was washed twice with TBS-Tween for 5

minutes each time at room temperature. The proteins of interest were detected using

primary antibodies (diluted in 1% skim milk and TBS-Tween) against p-ERK (1:1000),

IκBα (1:1000), NFATc1 (1:1000), p-PKCα, or β-actin (1:5000). The membrane was

incubated with the antibodies for two hours at room temperature while being rocked.

After that, the membrane was washed three times with TBS-Tween for 5 minutes each

time. This was followed by incubating the membrane with the appropriate secondary

HRP-conjugated antibody (anti-mouse or anti-rabbit) at a 1:5000 dilution for 45 minutes

at room temperature with rocking.

After the secondary antibody incubation, the membrane was washed twice with TBS-

Tween and another two times with TBS for 5 minutes each time. Visualization of the

protein was achieved using ECL Plus detection reagents. Solutions A and B were pre-

warmed to room temperature and combined to a 40:1 ratio, respectively. The combined

solution was incubated with the membrane for 5 minutes while covered to prevent any

contact with light. The membrane was then drained of any excess solution and placed

into a LAS-3000 mini imaging system by Fujifilm to visualize and capture images of

the protein bands on the membrane.

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4.4.3 -- RANKL-Induced Osteoclastogenesis

4.4.3.1 - Time Course of PKC Isoforms During RANKL-Induced

Osteoclastogenesis in RAW264.7 Cells

RAW264.7 cells were seeded onto a 6-well plate at a cell density of 5 x 104 cells/well in

2 mL of complete α-MEM (i.e. Containing serum and antibiotics). The cells were

incubated overnight at 37°C, 5% CO2. To achieve a time course of RANKL-induced

osteoclastogenesis, the cells were stimulated with 200 ng/mL of RANKL according to

Figure 4.1. On the last day, the cells were harvested and the total RNA was extracted

using an RNeasy® Mini Kit by Qiagen as described in Section 4.4.1. RT-PCR was

conducted on the samples using primers listed in Table 4.1

Chapter Four – Materials and Methods

69

Untreated RANKL 200 ng/mL for 5 days

RANKL 200 ng/mL for 1 days

RANKL 200 μg/mL for 3 days

RANKL 200 ng/mL for 4 hours

Figure 4.1 Treatment of RAW264.7 cells by 200 ng/mL of RANKL in a 6-well

plate.

Chapter Four – Materials and Methods

70

4.4.3.2 - Go6976 Inhibition of RANKL-Induced Osteoclastogenesis in Bone

Marrow Macrophages (BMM)

For a 96-well plate, 6 x 103 BMMs were seeded per well in 100 μL of complete α-

MEM. The plate was incubated at 37˚C, 5% CO2 overnight to allow the cells to attach to

the bottom of the well. The cells were then pre-treated with different doses of Go6976

(0.1 μM, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM) for an hour before stimulation by

200 ng/mL RANKL. The plate was incubated again at 37˚C, 5% CO2 for two days. The

cells were re-treated with Go6976 and re-stimulated with RANKL after every two days.

After seven days of treatment, the cells were TRAP-stained for osteoclasts as described

in Section 4.4.3.3 below.

4.4.3.3 - TRAP Staining

TRAP staining of osteoclasts was performed by first washing the cells twice with PBS

and fixed for 15 minutes in fresh 4% paraformaldehyde at room temperature. The fixed

cells were then washed three times with PBS and incubated at 37°C for 30 minutes with

the staining solution. After staining, the cells were washed with PBS, photographed, and

quantified under a light microscope.

4.4.3.4 - Go6976 Inhibition of Osteoclast Markers During RANKL-Induced

Osteoclastogenesis in RAW264.7 Cells

For a 6-well plate, 5 x 104 RAW264.7 cells were seeded per well in 2 mL of complete

α-MEM. The plate was incubated at 37˚C, 5% CO2 overnight to allow the cells to attach

to the bottom of the well. The cells were then pre-treated with either 1 μM or 10 μM of

Go6976 for an hour before stimulation by 200 ng/mL of RANKL as shown in Figure

4.2. The plate was incubated again at 37˚C, 5% CO2. After two days, the media was

replaced with fresh complete α-MEM and the cells were re-treated with Go6976 and re-

stimulated with RANKL. The cells were harvested on the fifth day of treatment and the

total RNA extracted using the RNAeasy mini kit by Qiagen as described in Section

4.4.1. RT-PCR was conducted on the samples using primers listed in Table 4.1.

Chapter Four – Materials and Methods

71

Untreated RANKL 200 μg/mL

RANKL 200 μg/mL+ 10 μM Go6976

RANKL 200 μg/mL+ 1 μM Go6976

1 μM Go6976 10 μM Go6976

Figure 4.2 Treatment of RAW264.7 cells by RANKL and Go6976 in a 6-well

plate.

Chapter Four – Materials and Methods

72

4.4.3.5 - Go6976 Inhibition of Signalling Pathways During RANKL-Induced

Osteoclastogenesis in RAW264.7 Cells

For a 6-well plate, 5 x 105 RAW264.7 cells were seeded per well in 2 mL of complete

α-MEM. The plate was incubated at 37˚C, 5% CO2 overnight to allow the cells to attach

to the bottom of the well. The cells were then pre-treated with either 1 μM or 10 μM of

Go6976 for an hour before stimulation by 200 ng/mL of RANKL (refer to Figure 4.2).

The plate was incubated either for 20 minutes (p-ERK and IκBα) or five days

(NFATc1) at 37˚C, 5% CO2. The cells were then harvested and protein extracted as

described in Section 4.4.2. Western blot was performed using antibodies against p-ERK,

IκBα, and NFATc1.

4.4.4 – Preparation of pHACE-PKCα Wild-Type (WT) Plasmid

Construct for Luciferase Assay

4.4.4.1 - Transformation of pHACE-PKCα WT Construct Into DH5α E.coli

Competent Cells

Transformation onto plates

Frozen DH5α Escherichia coli (E.coli) competent cells were first pre-warmed on ice.

200 μL of the cells was mixed with 3 μL of pHACE-PKCα wild-type (WT) construct in

a 1.5 mL tube. The cells were then incubated on ice for 30 minutes, followed by heat

shock at 42̊C for 90 seconds. After the heat shock, the cells were immediately

incubated on ice for 1 minute and 900 μL of Luria-Bertani (LB) broth was added after

that. The cells were incubated at 37̊C in a Ratek Orbital Mixer Incubator for 1 hour

while being shaken at 225 rpm. After incubation, the cells were centrifuged at 13,400

rpm for 30 seconds. The supernatant was removed, and the pellet was resuspended in

100 μL of LB broth. The cell suspension was then spread out onto a LB ampicillin plate

and incubated at 37̊C in a Ratek Hybridisation Oven overni ght. After the overnight

incubation, the transformed cells were grown in LB broth.

Chapter Four – Materials and Methods

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Growing in LB broth 3 mL of LB and 3 μL of 100 mg/mL ampicillin were added to a 50 mL tube. A colony

was picked off the LB-Amp plate using a pipette tip and inoculated into the 50 mL tube.

The tube was then incubated at 37˚C in a Ratek Orbital Mixer Incubator overnight while

shaken at 225 rpm.

4.4.4.2 - Miniprep of pHACE-PKCα WT Plasmid From Transformed DH5 E.coli

Cells

The plasmid DNA was purified from the DH5α E.coli cells by small-scale purification

(i.e. miniprep). A Wizard® Plus SV Minipreps DNA Purification System kit by

Promega was used for this procedure, following the centrifugation protocol provided

with the kit.

Briefly, the DH5α E.coli cells were pelleted in a 2mL tube and the supernatant

discarded. The cells were resuspended in 250 μL of Cell Resuspension Solution. The

sample was added with 250 μL Cell Lysis Solution, 10 μL Alkaline Protease Solution,

and 350 μL Neutralization Solution, in that order. The sample was inverted 4 times after

each solution was added. The plasmid DNA was collected in a Wizard® Spin Column

by centrifugation at top speed for 1 minute at room temperature. The DNA was then

washed with ethanol and eluted in nuclease-free water into a 1.5 mL tube.

The miniprep of the plasmid DNA from the transformed DH5α E.coli cells was put

through a diagnostic digest. This was to confirm that the plasmid had the correct

construct was inserted.

The following were added into a 500 μL tube for each construct:

10 x Restriction Enzyme Buffer 1.0 μL

First Restriction Enzyme 0.2 μL

Second Restriction Enzyme 0.2 μL

Miniprep product 5.0 μL

ddH2O 4.0 μL

Total Volume 10.4 μL

Chapter Four – Materials and Methods

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The digested products were then visualized on a 1.2% agarose gel with ethidium

bromide under UV light. A 1 kb DNA ladder was used to judge the size of the bands.

4.4.4.3 - Large-Scale Growth of pHACE-PKCα WT-Transformed E.coli Cells

In order to purify larger amounts of plasmid DNA by midiprep, larger amounts of the

transformed DH5α E.coli cells have to be grown. This was done by inoculating 200 mL

of LB broth containing 200 μL of ampicillin (100 mg/mL) with 200 μL of bacterial

culture from the small-scale growth in Section 4.4.4.1. The culture was then left to grow

at 37˚C overnight with shaking.

4.4.4.4 - Midiprep of pHACE-PKCα WT Plasmid From Large-Scale Growth of

E.coli Cells

Plasmid DNA from the large-scale growth of transformed DH5α E.coli cells was

purified using a Wizard® Plus SV Midipreps DNA Purification System kit by Promega,

following the kit protocol.

Briefly, the culture from the large-scale growth was decanted into a centrifuge tube. The

cells were pelleted at 4̊ C for 10 minutes, at 10,000 x g. The cell pellet was resuspended

in 3 mL Cell Suspension Solution. After resuspension, 3 mL of Cell Lysis Solution was

added followed by 3 mL of Neutralization Solution. The sample was centrifuged at 4̊C

for 15 minutes at 14,000 x g. The supernatant, that contains DNA, was then kept and

added with resin. The DNA was purified through a Wizard® Midicolumn by vacuum.

The purified DNA was then washed two times with 15 mL of Column Wash Solution

containing ethanol and dried. DNA was eluted with 300 μL of preheated water into a

1.5 mL tube. The eluate was centrifuged again to pellet the resin and the supernatant

transferred to a new 1.5 mL tube. A diagnostic digest was performed to confirm the

insertion of the correct construct into the vector.

Chapter Four – Materials and Methods

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The concentration of the DNA after purification was determined by measuring the

optical density at a wavelength of 260 nm (OD260), using a SmartSpec™ 3000

Spectrophotometer from Bio-Rad Laboratories Pty. Ltd. For measurement, the sample

was diluted 1/100 in water with a conversion factor of 50.

4.4.4.5 - Glycerol Stock of pHACE-PKCα WT-Transformed E.coli Cells

A portion of the transformed cell culture was kept as a glycerol stock for future

experimental work. This was done by adding 500 μL of the culture to 500 μL of 50%

glycerol in a 1.5 mL tube. The glycerol stock was stored in a -80˚C freezer.

4.4.4.6 - PolyFect Transfection of pHACE-PKCα WT Plasmid Into HEK-293 Cells

Transfection of pHACE-PKC WT into RANK-expressing HEK-293 cells was

performed using PolyFect Transfection Reagent by Qiagen according to the

manufacturer’s instruction. In brief, on the day before transfection, HEK-293 cells were

seeded onto a 6-well plate to a cell density of 6 x 105 cells per well in 3 mL of complete

DMEM. The cells were then incubated at 37°C and 5% CO2 in an incubator overnight.

For single plasmid transfection, 2 μg of plasmid DNA dissolved in TE buffer was

diluted to a final volume of 100 μL in serum-free DMEM. The solution was mixed and

spun down for a few seconds to remove any drops of solution from the top of the tube.

Next, 20 μL of PolyFect Transfection Reagent was added to the plasmid DNA solution

and mixture was mixed by pipetting up and down five times. The samples were

incubated for 5–10 min at room temperature to allow transfection complexes to form.

During complex formation, the DMEM from the plate was removed and replaced with

1.5 mL of fresh complete DMEM (containing serum and antibiotics). After incubation,

600 μL of complete DMEM was added to the transfection complex solution. The

transfection complex solution was mixed by pipetting up and down twice, and the total

volume (600 μL) was immediately transferred to the cells drop-wise while the plate was

gently swirled to ensure uniform distribution of the complexes.

Chapter Four – Materials and Methods

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The transfected cells were incubated at 37°C, 5% CO2 to allow for gene expression. The

cells were washed once with serum-free DMEM and replaced with fresh complete

DMEM after 20-24 hours of transfection to avoid transfection reagent-induced cytotoxic

effect to the cells.

4.4.4.7 – SuperFect Transfection of pHACE-PKCα WT Plasmid Into RAW264.7

Cells

The pHACE-PKCα WT constructs were transfected into RAW264.7 cells using a

SuperFect® Transfection Reagent by Qiagen. Transfection was done according to the

manufacturer’s instructions.

Briefly, 1 x 105 cells were seeded into each well in a 24-well plate. Each well contained

350 μL of complete α-MEM. The cells were then incubated at 37˚C and 5% CO 2

overnight. After overnight incubation, 1 μg/well of the pHACE-PKCα WT construct

was dissolved in serum-free α-MEM to a total volume of 60 μL/well. The solution was

then mixed and spun down to remove droplets from the top of the tube. 5 μL/well of the

SuperFect® Transfection Reagent was added to the tube and was mixed by pipetting up

and down several times. The sample was then left at room temperature for 5 to 10

minutes to allow transfection complexes to form.

While transfection complexes are forming, the growth medium from each well of the

seeded 24-well plate was removed. 175 μL of complete α-MEM (containing antibiotics

and serum) was added into each well. After 5 to 10 minutes of transfection complex

formation, 175 μL/well of complete α-MEM (containing antibiotics and serum) was

added into the tube containing the transfection complexes. The complex solution in the

tube was mixed by pipetting the solution up and down twice. 240 μL of the solution was

immediately transferred into each well.

The cells were then incubated at 37˚C and 5% CO 2 overnight. After incubation, the

medium containing the remaining complexes were removed and another 500 μL of fresh

complete α-MEM (containing antibiotics and serum) was added to each well. The cells

Chapter Four – Materials and Methods

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were incubated at 37̊C and 5% CO 2 for 24 hours before being lysed for the luciferase

assay.

4.4.4.8 – Luciferase Assay

The growth medium was removed from the wells and 150 μL of luciferase assay cell

lysis buffer (containing 0.2% DDT) was added to each well to lyse the cells. The plate

was incubated at room temperature for 10 to 15 minutes with gentle rocking. 50 μL of

the cell lysate from each well was transferred into a well of a 96-well plate.

The luciferase assay was done using a Dual-Luciferase® Reporter Assay System by

Promega, according to the manufacturer’s instructions. The luciferase activity

measurements were read by a PolarStar Optima microplate reader. The machine was set

to detect luminescence, with the time for measurement set at 20 seconds.

4.4.5 – Small Interfering RNA (siRNA) Experimental Methods

4.4.5.1 - Transfection of siRNA Into RAW264.7 Cells in 24-Well Plates

Transfection of siRNA into RAW264.7 cells was performed using Hiperfect

Transfection Reagent from Qiagen. On the day of transfection, 2 x 105 cells were seeded

into a well of a 24-well plate in 100 µL of complete α-MEM. The cells were then

incubated at 37°C, 5% CO2. For single transfection, siRNA was diluted in 100µL of

serum-free culture medium to achieve a final concentration of 100 nM of siRNA in 500

µL. 6µL of HiPerFect Transfection Reagent was added to the diluted siRNA and mixed

by vortexing. The contents were then spun down and the siRNA transfection mix was

incubated for 10 minutes at room temperature to allow for formation of transfection

complexes. After incubation, the transfection complexes were added drop-wise onto the

cells with gentle swirling of the plate to ensure uniform distribution of the transfection

complexes. The plate was then incubated at 37°C for 6 hours before the addition of 300

µL complete α-MEM for a final volume of 500 µL. The plate was incubated at 37°C for

24 hours before harvesting and total RNA or protein was extracted as described in

Section 4.4.1 and Section 4.4.2. Figure 4.3 illustrates how the cells were treated in the

plate.

Chapter Four – Materials and Methods

78

Figure 4.3 Treatment of RAW264.7 cells on a 24-well plate for silencing.

Mock

Mock 100nM GFP siRNA

100nM GFP siRNA

100nM PKCα siRNA

100nM PKCα siRNA

200nM PKCα siRNA

200nM PKCα siRNA

Chapter Four – Materials and Methods

79

4.4.5.2 - PKCα siRNA Inhibition of RANKL-Induced Osteoclastogenesis in

RAW264.7 cells

For a 96-well plate, 2 µL of siRNA was initially spotted onto a well. 0.75 µL of

Hiperfect transfection reagent was diluted in 24.25 µL of serum-free α-MEM and the

total 25 µL was dropped onto the well and mixed with the siRNA. The mixture was

incubated at room temperature for 10 minutes to allow for the formation of transfection

complexes. After 10 minutes, 1.3 x 103 RAW264.7 cells were added into the well in 173

µL of complete α-MEM, bringing the total volume in the well to 200 µL. The plate was

then incubated at 37°C, 5% CO2 overnight. After overnight incubation, the media was

replaced with fresh complete α-MEM (200 µL) and the cells were stimulated with 200

ng/mL of RANKL. The plate was again incubated at 37°C, 5% CO2. Every two days,

the cells were re-transfected with siRNA and re-stimulated with RANKL until

osteoclasts formation was observed under a light microscope. The cells were then

TRAP-stained as described in Section 4.4.3.3.

Chapter Five

Expression of PKC Isoforms During RANKL-Induced

Osteoclastogenesis

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

80

5.1 - Introduction

As reviewed in Section 1.4, RANKL is an essential cytokine for osteoclast formation

and function. RANKL binds to its receptor RANK at the cell surface which leads to the

activation of various signalling pathways that influence osteoclast activity. As was

discussed in Chapter 2, PKCs are expressed in a variety of cell types and studies have

shown PKCs to be expressed in osteoclasts. PKCβ was found to be upregulated in

differentiated osteoclasts (Lee et al., 2003). PKCα was also shown to be expressed in

mature osteoclasts (Rucci et al., 2005). 12-0-tetradecanoyl phorbol-13-acetate (TPA),

an activator of PKCs, was shown to decrease osteoclastic bone-resorbing activity (Mano

et al., 2000). In addition, TPA was shown to suppress RANKL-induced signalling

pathways and osteoclastogenesis (Wang et al., 2003). However, the precise role of

PKCs during RANKL-induced osteoclastogenesis still remains an open question.

Protein Kinase C (PKC) plays a crucial role in signal transduction of a variety of

biologically active substances which activate cellular functions and proliferation

(Nishizuka, 1984). The PKC family of serine-threonine kinases encompasses a number

of different isoforms including PKC α, β1, β2, γ , δ, ε, η, ζ, ι and θ. (Liu et al., 1998).

On the basis of overall amino acid sequence similarity, the PKC family has been

divided into subfamilies. The first subfamily is commonly referred to as the classical

PKCs or conventional PKCs (cPKCs), and consists of PKC-α, β1, β2 and γ. The second

subfamily is called the novel PKCs (nPKCs), and comprises PKC-δ, ε, η, and θ.

Atypical PKCs (aPKCs) consist of PKC-ι, λ and ζ. To be more specific, the PKCs

isoenzymes are classified according to their activator/cofactor requirements. The

conventional PKCs require both Ca2+ and diacylglycerol or phorbol esters such as

phorbol 12-myristate 13-acetate (PMA) as cofactor. Novel PKCs can be stimulated by

diacylglycerol or PMA alone, independent of Ca2+. Atypical PKCs are independent of

both stimuli and the PKC-related kinases are even structurally different.

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

81

One method used to dissect the function of PKCs is to inhibit their activity. To do this, a

compound called Go6976 (indolocarbazole) is routinely employed. Go6976 is a cell-

permeable inhibitor of Ca2+- dependent PKCs α and β1. It has no effect on the kinase

activity of Ca2+-independent PKCs (Martiny-Baron et al., 1993). Its IC50 (half maximal

inhibitory concentration) for PKCα is three times lower than PKCβI (0.0023 μM vs.

0.006 μM) thus making it relatively more specific to PKCα. It is also known to inhibit

PKCμ at higher concentrations (IC50 = 0.02 μM) (Gschwendt et al., 1996). It has been

suggested that it inhibits the catalytic activity of the PKCs by interacting with the

nucleotide binding site of the enzyme (Martiny-Baron et al., 1993).

Molecular structure of Go6976

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

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5.2 - Results

5.2.1 - PKCα mRNA Expression is Upregulated During Osteoclastogenesis

The purpose of this study was to examine the role of PKCs in osteoclasts. Specifically,

to determine which PKC isoform(s) play a distinct role in the process of

osteoclastogenesis. To achieve this, specific primers for PKCα, β, ε, γ, ι, δ, η, ζ, and θ

were constructed for reverse transcriptase polymerase chain reaction (RT-PCR)

analyses. As an initial source of osteoclastic cells, a RANKL-differentiated RAW264.7

cells pro-osteoclastogenic system was employed. RAW264.7 cells are a murine

macrophage cell-line that has the capacity to differentiate into bone-resorbing

osteoclasts when stimulated only with RANKL (Hsu et al., 1999; Xu et al., 2000).

Therefore, RAW264.7 cells were used to examine the mRNA expression of PKC

isoforms during osteoclastogenesis. RAW264.7 cells were treated at different time

points (4 hours, 1 day, 3 days, and 5 days) with RANKL and then harvested for mRNA

isolation. The mRNA was then subjected to RT-PCR with the previously mentioned

primers. By comparing the different time points, we were able to observe specific

changes in mRNA expression as the cells undergo osteoclastic differentiation.

RAW264.7 cells formed large osteoclasts at day 5 when observed under a light

microscope. Bone marrow macrophages (BMM) also form bone-resorbing osteoclasts

when stimulated with M-CSF and RANKL. M-CSF is essential for the osteoclast

precursor cells’ proliferation and survival, as well as upregulate RANK (receptor for

RANKL) expression (Asagiri et al., 2007). BMM were used to further confirm the

result in RAW264.7 cells. BMMs were harvested from mice bone marrow and treated

the same way as the RAW264.7 cells apart from the addition of M-CSF. Representative

image of RAW264.7 cells forming osteoclasts after seven days of RANKL treatment

are shown in Figure 5.1. Osteoclasts are the large TRAP-positive (pink colored) cells.

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

Figure 4.1 Representative images of RAW264.7 cells forming osteoclasts. RAW264.7 cells were seeded onto a 96-well plate and stimulated with 200 ng/mL of RANKL for seven days before being TRAP-stained. Cells in the mock well were not stimulated over the same period (seven days). Cells stained for TRAP are pink in colour. Osteoclasts are the large multinucleated TRAP-stained cells.

83

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

84

The results show that PKCα is upregulated by more than 2-fold after stimulation by

RANKL for 5 days (Figure 5.2). PKCβ was similarly upregulated by almost 2-fold;

however, expression of PKCβ was not as high as that of PKCα. It is interesting to note

that PKCα was expressed slightly prior to RANKL stimulation whereas PKCβ was not

expressed until 3 days post-stimulation. By comparison, PKCζ and θ did not show any

expression throughout the 5 day time course. In addition, the other PKC isoforms

examined did not show much change in mRNA expression during osteoclastogenesis.

CTR and V-ATPase d2 were used as markers of osteoclastogenesis whereas 36B4

served as an internal control.

Although RAW264.7 cells are known to reliably form bone-resorbing osteoclasts, they

are merely experimental substitutes and not the actual osteoclast-forming cells in bone.

As such, to further confirm the gene expression result in Figure 5.2, a similar

experiment was performed on BMM derived from mice. Figure 5.3 shows

representative images of BMM undergoing osteoclastogenesis during a 7 day period

after stimulation by M-CSF and RANKL.

The result in Figure 5.4 shows that PKCα is upregulated during BMM

osteoclastogenesis by over 2-fold after3 days of MCSF and RANKL stimulation. This is

similar to the RAW264.7 result. PKCβ on the other hand was much more highly

expressed than PKCα but did not show much change in expression (expression is

slightly decreased after 5 days).

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

PKCβ

PKCγPKCε

PKCι

PKCζ

PKCδ

PKCθ

PKCη

0 4 hours 1 Day 3 Days 5 Days

PKCα

36B4

CTR

V-ATPase d2

Figure 4.2 PKCα and β are upregulated during RAW264.7 cell osteoclastogenesis. RAW264.7 cells were treated at different time points (4 hours, 1 day, 3 days, 5 days) with RANKL and then harvested for mRNA. The mRNA was then subjected to RT-PCR with the appropriate primers and visualized on an agarose gel with ethidium bromide under UV light. V-ATPase d2 and CTR are osteoclast markers that are known to be upregulated during osteoclastogenesis. 36B4 is an internal control. The density of the bands was quantified using Scion Image software, normalized to 36B4, and presented as fold induction relative to the earliest detectable expression. PKCα and β showed the highest fold induction by RANKL stimulation when compared to the other PKC isoforms.

85

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

Figure 4.3 Representative images of bone marrow macrophages (BMM) undergoing osteoclastogenesis. BMM were seeded onto separate wells on a 96-well plate and each well was stimulated with RANKL at different time points before being TRAP-stained together. For example, the Day 7 well was stimulated on the first day of the experiment whereas the Day 5 well was stimulated two days after the Day 7 well. This allowed the cell density in all the wells to be consistent as the cells proliferate throughout the seven days. Cells stained for TRAP are pink in colour. Osteoclasts are the large multinucleated TRAP-stained cells.

86

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

0 1 Day 3 Days 5 Days 7 Days

36B4

PKCα

PKCβ

DCSTAMP

NFATc1

CTR

Cathepsin K

TRAP

Figure 4.4 PKCα is upregulated in BMM osteoclastogenesis. BMMs were treated at different time points (1 day, 3 days, 5 days, 7 days) with RANKL and then harvested for mRNA. The mRNA was then subjected to RT-PCR with the appropriate primers and visualized on an agarose gel with ethidium bromide under UV light. TRAP, NFATc1, DCSTAMP, Cathepsin K, and CTR are osteoclast markers that are known to be upregulated during osteoclastogenesis. 36B4 is an internal control. The density of the bands was quantified using Scion Image software, normalized to 36B4, and presented as fold induction relative to the earliest detectable expression. PKCα showed a similar fold induction by RANKL stimulation when compared to the mRNA expression profile in RAW264.7 cells (Figure 4.2). PKCβ is constitutively expressed in BMMs which is dissimilar that in RAW264.7 cells.

87

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88

5.2.2 - Go6976 Inhibits The Formation of Osteoclasts in BMM

If PKCα does play a role in osteoclastogenesis, then inhibition of its activity should

affect osteoclastogenesis in some manner. Therefore, Go6976 (a cPKC inhibitor) was

used to inhibit the activity of PKCα. It should be noted that Go6976 affects PKCα, β,

and μ. However, the IC50 of Go6976 on α is greater than β and μ. With this in

consideration, and the fact that other PKC inhibitors affect a wider range of PKCs and

are therefore less specific, Go6976 was considered the best available inhibitor for

PKCα.

BMM were pre-treated with differing concentrations of Go6976 (0, 0.1, 0.25, 0.5, 0.75,

1, 2μM) for an hour before stimulation by RANKL for 8 days. As shown in Figure 5.5,

Go6976 dose-dependently inhibits osteoclastogenesis in BMM from the concentration

of 0.1μM onwards.

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

Figure 4.5 Go6976 dose-dependently inhibits BMM osteoclastogenesis. BMMs were seeded onto a 96-well plate and pre-treated with differing concentrations of Go6976 for an hour before being stimulated with RANKL for several days until osteoclasts were observed to be formed in the RANKL well (no Go6976 treatment). Mock cells were not treated with Go6976 and RANKL. Cells were TRAP-stained and osteoclasts counted under a light microscope. 0.1 μM of Go6976 effectively inhibited osteoclast formation and at 0.25 μM, few osteoclasts could be found.

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89

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

90

5.2.3 - Go6976 Suppresses CTR, V-ATPase d2, and Cathepsin K mRNA

Expression

To further confirm that Go6976 inhibits osteoclastogenesis, we examined its effect on

the mRNA expression of osteoclastogenic markers calcitonin receptor (CTR), V-

ATPase d2, and cathepsin K in RAW264.7 cells treated with RANKL for 5 days. Semi-

quantitative RT-PCR was performed on the mRNA isolated from the cells. The results

demonstrate that CTR mRNA expression was reduced by 10μM of Go6976 whereas d2

was already affected by 1μM (Figure 5.6). Cathepsin K also appeared to show reduced

expression when treated with 1 and 10 μM of Go6976, after the density of the bands

was normalized to 36B4.

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

Cathepsin k

CTRV-ATPase d2

+++-- 10- 1

RANKLGo6976 (μM)

36b4

Figure 4.6 Go6976 inhibits mRNA expression of CTR, V-ATPase d2, and cathepsin K in RAW264.7 cell osteoclastogenesis. RAW264.7 cells were pre-treated with 1 or 10 μM of Go6976 an hour before stimulation with 200 ng/mL of RANKL over a period of 5 days. Cells were harvested and mRNA extracted. The mRNA was then subjected to RT-PCR with the appropriate primers and visualized on an agarose gel with ethidium bromide under UV light. V-ATPase d2 and Cathepsin K are osteoclast markers that are known to be upregulated during osteoclastogenesis. 36B4 is an internal control. The density of the bands was quantified using Scion Image software, normalized to 36B4, and presented as fold induction relative to the earliest detectable expression. The results demonstrate that CTR mRNA expression was reduced by 10μM of Go6976 whereas d2 was already affected by 1μM of Go6976. Cathepsin K also appeared to show reduced expression when treated with 1 and 10 μM of Go6976, after the density of the bands was normalized to 36B4.

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91

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

92

5.3 - Discussion

RANKL-induced osteoclastogenesis is accompanied by the upregulation of several

genes that were later studied and found to play major roles in osteoclastogenesis as well

as osteoclast function. These genes are now used as markers of osteoclastogenesis, and

they include TRAP, cathepsin K, calcitonin receptor (CTR), V-ATPase d2 etc. (see

Section 1.3). Therefore, a good indicator of activity for a particular molecule is

upregulation of mRNA expression during a certain process. In this case, mRNA

expression of PKC isoforms was examined to see if any of them were upregulated

during RANKL-induced osteoclastogenesis. Absence of change in expression does not

necessarily mean that the isoform has no function in osteoclasts. As long as they are

expressed in the cells, they each have a role to play. However, research resources are

limited, and therefore, upregulated genes were focused on to narrow the field of

research.

Using Western blot analysis, previous studies have shown that RAW 264.7 cells express

PKCα, βI, βII, δ, ε, μ, λ, and ζ and not γ, θ, η, and ι (Larsen et al., 2000; Lin et al.,

1998). In this study, RT-PCR revealed that PKCα, δ, ε, γ, ι, and η were expressed in

unstimulated RAW264.7 cells while PKCβ, θ, and ζ were not expressed. The presence

of PKCβ and ζ in the Western blot and not in the RT-PCR result is probably because the

cells have already synthesized the proteins and have stopped DNA transcription. It is

uncertain as to why PKCγ, η, and ι were not detected by Western blot from the two

studies collectively, whereas their mRNA expression was detected in this study. It is

possible that although the mRNA has been transcribed, the proteins themselves were

never fully synthesized or undergone the post-translational modifications necessary to

form mature proteins.

The upregulation of PKCα and β during RANKL-induced osteoclastogenesis in

RAW264.7 cells (Figure 5.2) suggests that the two PKC isoforms might play a role in

osteoclastogenesis. However, in BMM, PKCβ does not appear to be upregulated during

osteoclastogenesis (Figure 5.4). This result does not agree with the study done by Lee et

al. (2003) which demonstrated that not only is PKCβ upregulated during BMM

osteoclastogenesis, but that it also plays role in regulation of osteoclast formation and

Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis

93

function. The result in Figure 5.4 further confirms that PKCα might play a role in

osteoclastogenesis. While Rucci et al. (2005) may have provided insights into the

involvement of PKCα in mature osteoclast function, the role of PKCα in RANKL-

induced osteoclast formation remains unclear. While the other PKC isoforms do not

show any change in mRNA expression during RANKL-induced osteoclastogenesis, it

does not mean that they do not affect osteoclastogenesis and osteoclast function. The

proteins of these isoforms might still be active in osteoclasts and pre-osteoclasts; but for

now, the focus of the research is on PKCα.

PKCα and β belong to the classical PKC (cPKC) subgroup and therefore are activated

by Ca2+, phosphotidylserine (PS), and diacylglycerol (DAG). The fact that they are

activated by Ca2+ is interesting as calcium signalling is essential for osteoclastogenesis.

Inhibition of CaMKII, a downstream mediator of Ca2+, suppressed osteoclast

differentiation (Ang et al., 2007). Ca2+ is also important for the activation of NFATc1

by calcineurin which is an essential transcription factor in osteoclasts (Aliprantis et al.,

2008).

While it is promising that BMM osteoclastogenesis is inhibited by the presence of

Go6976, there is a degree of uncertainty as to whether the effect is due solely to the

inhibition of PKCα, or if other PKCs such as PKCβ was affected by the drug as well.

The IC50 for PKCβ is much lower than the doses used in the experiment. As such, more

specific methods of suppressing PKCα were explored and the decision was to use small

interfering RNA (siRNA) to knock down gene expression in the cells. The use of

siRNA will be discussed in the next chapter (Chapter 5).

It is interesting that V-ATPase d2 mRNA expression was reduced by Go6976 (Figure

5.6). Since V-ATPase d2 plays an important role in cell fusion during

osteoclastogenesis (Lee et al., 2006), it is possible that PKCα operates upstream of V-

ATPase d2, and disruption of PKCα activity could lead to the disruption of d2

expression, thus reducing cell fusion and subsequent osteoclast formation.

Chapter Six

Functional Analysis of PKCα in RANKL-Induced

Osteoclastogenesis

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

94

6.1 - Introduction

Numerous signalling pathways are involved in osteoclastogenesis. Most of these

pathways function downstream of RANKL as discussed in Section 1.5. PKCs are

known to participate in various signalling pathways such as ERK, NF-κB, and NFAT in

other cell types (Hock et al., 2003; Manicassamy et al., 2006; Rucci et al., 2005). These

signalling pathways are involved in osteoclast formation, function, and survival.

ERK1/2 was shown to be a positive regulator of differentiated osteoclast survival, but

has no effect on osteoclastic bone resorption (Miyazaki et al., 2000). Lee et al. (2002)

demonstrated that IL-α promotes osteoclast survival through the ERK, as well as Akt,

signalling pathway to reduce caspase-3 activity. Hotokezaka et al. (2002) suggested that

ERK1/2 negatively regulates osteoclastogenesis in RAW264.7 cells. NF-κB is known to

be activated by RANKL in RAW264.7 cells as well as monocytes (Hsu et al., 1999;

Lacey et al., 1998). Mice with double knockouts of the NF-κB subunits p50 and p52,

which are involved in the classical NF-κB pathway, failed to develop mature osteoclasts

and subsequently lead to osteopetrosis (Franzoso et al., 1997; Iotsova et al., 1997).

NFATc1 is an important transcription factor for V-ATPase d2 and DC-STAMP, which

are important markers of osteoclastogenesis (Feng et al., 2009; Kim et al., 2008).

Go6976 was used as a PKCα inhibitor as it was the most specific inhibitor that is

currently available and has been used in to study PKCα several other studies (Fomin et

al., 2009; Le et al., 2001). However, it is known to also affect PKCβ, and might not be

as specific as hoped. For this reason, PKCα siRNA was used as a more specific means

to knock down PKCα expression in osteoclasts precursors.

The knock down of gene expression by small interfering RNA (siRNA) has been widely

used to examine the function of a gene. SiRNAs are double-stranded RNA molecules

that are notably involved in the RNA interference (RNAi) pathway. RNAi is a cellular

system that helps to control the activation of genes within the cell. For example, binding

of siRNA to a specific mRNA would prevent translation of the mRNA into a protein. In

this chapter, synthetic siRNA was used to specifically knock down PKCα gene

expression, thus allowing a more precise examination of the role of PKCα in

osteoclastogenesis.

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

95

The results from the previous chapter have shown that PKCα is upregulated during

osteoclastogenesis, and inhibition of its activity by the drug Go6976 inhibits

osteoclastogenesis in BMM culture. Go6976 was also found to reduce the expression of

CTR and d2 during osteoclastogenesis. It is now known that d2 is important for cell

fusion during osteoclastogenesis. From this, it can be suggested that inhibition of PKCα

leads to the inhibition of d2 expression, which leads to reduced cell fusion and hence the

inhibition of osteoclastogenesis.

This chapter examines the signalling pathways that PKCα might be involved in and to

further confirm the role of PKCα in osteoclastogenesis through the use of siRNA.

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

96

6.2 - Results

6.2.1 - Go6976 Has Little To No Effect on p-ERK and IκBα Protein Expression

During Osteoclastogenesis

To examine whether PKCα is involved in the ERK and NF-κB signalling pathways

during osteoclastogenesis, RAW264.7 cells were treated with Go6976 for an hour prior

to stimulation by RANKL for 20 to 30 minutes. The cells were then harvested and the

total protein content was extracted. Western blot analysis was used to look at the protein

expression of phosphorylated ERK (p-ERK) and IκBα (an inhibitory molecule of NF-

κB). The results show that, as expected, RANKL stimulation increased the expression

of p-ERK and decreased the expression of IκBα (Figure 6.1). However, addition of

Go6976 has little to no effect on p-ERK protein expression and IκBα protein expression

(Figure 6.1).

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

p-ERK

IκBα

β-actin

β-actin

- + + +RANKL

- - 1 10Go6976 (μM)

Figure 6.1 Go6976 has little effect on p-ERK and IκBα protein expression during RANKL stimulation. RAW264.7 cells were pre-treated with Go6976 at 1μM or 10μM concentration for an hour before being stimulated by RANKL. The cells were then harvested and protein extracted. Proteins were separated by SDS-PAGE and Western blot performed with antibodies to p-ERK and IκBα. Go6976 does not affect p-ERK and IκBα protein expression. β-actin was probed for as an internal loading control.

97

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

98

6.2.2 - PKCα Wild-Type Construct Potentiates RANKL-Induced Activation of NF-

κB

To fu rth er examin e if PKCα p lays a ro le in th e NF-κB signalling pathway, plasmid

constructs of wild-type PKCα (PKCα-WT) were co-transfected with a NF-κB luciferase

reporter construct into HEK-293 cells expressing RANK (RANK-293) and RAW264.7

cells. The cells were stimulated with RANKL and luciferase activity was measured. The

results show that cells transfected with PKCα-WT had a significantly higher NF-κB

promoter activity after RANKL stimulation compared to cells transfected with an empty

vector (pcDNA3.1) in both RANK-293 and RAW264.7 cells (Figure 6.2).

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

Figure 6.2 PKCα WT potentiates RANKL-induced activity of NF-κB. pHACE-PKCα-WT or pcDNA3.1 was co-transfected with NF-κB luciferase reporter constructs into RANK-293 cells (A) and RAW264.7 cells (B). Cells were then stimulated with RANKL and luciferase activity measured. The graphs show NF-κB activity fold increase after RANKL stimulation. (A) PKCα-WT had a significantly higher fold increase in NF-κB activity after RANKL stimulation compared to the empty vector (pcDNA3.1) control (p = 0.0061) in RANK-294 cells. (B) A similar effect is also observed in RAW264.7 cells. Student’s t-Test was performed using Microsoft Excel.

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Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

100

6.2.3 - Go6976 Reduces NFATc1 Protein Expression During Osteoclastogenesis

From the previous chapter, V-ATPase d2 was implicated in the role of PKCα on

osteoclastogenesis. NFATc1 is an important transcription factor for V-ATPase d2 (Feng

et al., 2009; Kim et al., 2008). Therefore, Western blot analysis was used to see if

Go6976 has any effect on NFATc1 activity during RANKL-induced osteoclastogenesis.

RAW264.7 cells were treated with Go6976 for an hour prior to stimulation by RANKL

for 24 and 48 hours. The cells were then harvested and the total protein content was

extracted. NFATc1 protein expression was upregulated by RANKL stimulation. This

upregulation was observed to be inhibited by Go6976 dose-dependently (Figure 6.3).

The inhibitory effect was more pronounced after 48 hours of drug treatment and

stimulation by RANKL.

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

24 hours

48 hours

- + + +

NFATc1

NFATc1

β-actin

β-actin

RANKL

- - 1 10Go6976 (μM)

Figure 6.3 Go6976 reduces NFATc1 protein expression during RANKL stimulation. RAW264.7 cells were pre-treated with Go6976 at 1μM or 10μM concentration for an hour before being stimulated by RANKL for 24 and 48 hours. The cells were then harvested and protein extracted. Proteins were separated by SDS-PAGE and Western blot performed with antibodies to NFATc1 Go6976 reduces NFATc1 protein expression. β-actin was probed for as an internal loading control.

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6.2.4 - PKCα siRNA Inhibits RANKL-Induced Osteoclastogenesis in RAW264.7

Cell

To further confirm that PKCα affects RANKL-induced osteoclastogenesis, and to

achieve a more specific knockdown of PKCα, siRNA was used to silence PKCα gene

expression. RAW264.7 cells were seeded onto a 24-well plate and treated with 100nM

of PKCα for 24 hours before total RNA and protein extraction. The RNA was subjected

to semi-quantitative RT-PCR, whereas the protein was subjected to Western blot. Figure

6.4 shows that 100nM of the PKCα siRNA reduced mRNA expression of PKCα by

around 50% in RAW264.7 cells. Figure 6.5 shows PKCα siRNA reducing the protein

expression of p-PKCα as well in RAW264.7 cells. GFP siRNA was used as a negative

silencing control.

After optimizing the conditions for knockdown of the gene, we proceeded to confirm

that PKCα knockdown would inhibit osteoclastogenesis. RAW264.7 cells were silenced

with PKCα siRNA for 24 hours before the addition of RANKL. PKCα siRNA was

added every 2 days to ensure that PKCα expression does not recover from suppression.

Cells were observed under light microscopy to determine the formation of osteoclasts

after 5 to 6 days of RANKL stimulation. The cells were then TRAP-stained to visualize

the osteoclasts better. Figure 6.6 shows that PKCα siRNA reduced the number of

osteoclasts formed compared to the Hiperfect and GFP siRNA negative controls. It was

also observed that the PKCα siRNA transfected cells were less than the mock control

cells.

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

Mock100nM

GFP siRNA

200nM

PKCαsiRNA

100nM

PKCαSiRNA

PKCα

36B4

Figure 6.4 PKCα siRNA reduces PKCα mRNA expression in RAW264.7 cells. RAW264.7 cells were transfected with 100nM GFP siRNA, 100nM PKCα siRNA, and 200nM PKCα siRNA for 24 hours. The cells were then harvested for mRNA. Semi-quantitative RT-PCR was performed to examine PKCα gene expression. Roughly 50% knockdown of PKCα was achieved with 100nM of PKCα siRNA while 200nM of PKCα siRNA knocked down PKCα by about 40%.

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Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

100nM PKCα siRNA

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Figure 6.5 PKCα siRNA reduces phosphorylated PKCα protein expression in RAW264.7 cells. RAW264.7 cells were transfected with 100nM GFP siRNA and 100nM PKCα siRNA for 24 hours. The cells were then harvested for protein. The protein was separated by SDS-PAGE and Western blot performed to probe for p-PKCα. 100nM PKCα siRNA reduced p-PKCα protein expression.

104

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

Mock RANKL + Hiperfect

RANKL + GFP siRNA RANKL + PKCα siRNA

Figure 6.6 PKCα siRNA inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells. RAW264.7 cells were transfected with 100nM GFP siRNA and 100nM GFP siRNA for 24 hours before RANKL stimulation. Cells were restimulated with RANKL and re-transfected with the appropriate siRNA every two to three days to ensure osteoclastogenesis occurs, as well as maintain the knockdown of PKCα. Cells transfected with PKCα siRNA showed a marked reduction in osteoclast formation as well as total cell number.

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Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

106

6.3 - Discussion

The results from the previous chapter suggest that PKCα plays an important role in

RANKL-induced osteoclastogenesis. PKCs are involved in a number of signalling

pathways in cells, several of which are known to be important in RANKL-induced

osteoclastogenesis. The three pathways examined in this chapter were the ERK

pathway, the classical NF-κB pathway, and the NFATc1 pathway. The activity of ERK

was determined by probing for phosphorylated ERK (p-ERK) as ERK is activated by

phosphorylation. In the classical NF-κB pathway, IκBα is an inhibitory molecule of NF-

κB that is degraded by proteasomes to allow the NF-κB molecule to translocate into the

nucleus and promote gene transcription. Inactive NFATc1 is normally phosphorylated

and located in the cytoplasm. Upon dephosphorylation by calcineurin, NFATc1

translocates into the nucleus to promote gene transcription.

Go6976 was observed to have little to no effect on p-ERK protein expression (Figure

6.1), which suggests that PKCα might not be critically involved in ERK signalling

during osteoclast formation, although studies have shown that PKCα is involved in the

ERK pathway in other cells types (Rucci et al., 2005; Shatos et al., 2009). Rucci et al.

(2005) demonstrated that inhibition of PKCα prevented the phosphorylation of ERK in

mature osteoclasts. Shatos et al. (2009) provided evidence that PKCα stimulates

proliferation of conjunctival goblet cells through activation of ERK1/2. It is possible

PKCα only affects the ERK pathway once osteoclasts are fully formed and not during

the early stages of osteoclast formation. The ERK pathway was suggested to negatively

regulate RAW264.7 osteoclastogenesis, whereas p38 is a positive regulator

(Hotokezaka et al., 2002). It might be of interest to examine the p38 pathway in relation

to PKCα in future studies.

Go6976 had little effect on IκBα protein expression (Figure 6.1). However, PKCα-WT

was shown to significantly potentiate RANKL-induced NF-κB promoter activity in

RANK-293 and RAW264.7 cells (Figure 6.2). In addition, previous studies in our

laboratory utilizing luciferase assay demonstrated that Go6976 inhibits NF-κB

luciferase activity (unpublished data). These results suggest that PKCα is involved in

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

107

the activation NF-κB gene transcription, but has little affect the degradation of the

inhibitory molecule IκBα.

Figure 6.3 indicates that during osteoclastogenesis, PKCα has an effect on NFATc1

signalling. From the previous chapter, V-ATPase d2 gene expression was affected by

the inhibition of PKCα by Go6976. NFATc1 is now known to be a major transcription

factor for V-ATPase d2 (Feng et al., 2009). This suggests that PKCα affects RANKL-

induced osteoclastogenesis by influencing the activity of NFATc1, thus affecting V-

ATPase d2 expression, and subsequently cell fusion of pre-osteoclasts.

From the siRNA experiment, the results further confirm that PKCα in involved in

RANKL-induced osteoclastogenesis (Figure 6.2). However, in contrast to the Go6976

experiments on osteoclastogenesis, PKCα siRNA transfected cells were observed to

have fewer cells than the un-treated mock cells. It is possible that PKCα siRNA caused

the cells to detach from the bottom of the well, or even undergo apoptosis. Another

possibility would be that knock down of PKCα inhibits the proliferation of RAW264.7

cells. PKCα has been implicated in growth regulation and cell cycle arrest as well as

tumor formation and was found to be differentially expressed in human breast

carcinomas (Ali et al., 2009). Other studies have shown that the overexpression of

PKCα promotes proliferation in many cell types, particularly in mouse fibroblasts, U87

malignant glioma cells, and MCF-7 breast cancer cells (Mandil et al., 2001; Soh et al.,

2003; Soh et al., 2003; Ways et al., 1995). Additionally, knock down of PKCα gene

expression by siRNA suppressed proliferation of human cancer cells (Yin et al., 2003).

However, other studies suggest that PKCα has an anti-proliferative effect and that loss

of PKCα function actually increases proliferation (Nakagawa et al., 2006; Oster et al.,

2006). It is possible that PKCα plays a dual-role in this aspect. Depending on the cell

type, differences in cell signalling, and availability of substrates, the function of PKCα

can change. The results in this chapter would suggest that PKCα promotes proliferation

in RAW264.7 cells. This raises the question of whether inhibition of PKCα specifically

inhibits the proliferation of osteoclast precursors, and thus there are not enough cells to

form osteoclasts; or if inhibition of PKCα directly inhibits the osteoclastogenic process

itself.

Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis

108

To summarize, these results suggest that PKCα regulates RANKL-induced

osteoclastogenesis through the NF-κB and NFATc1 signalling pathway, but not through

the ERK pathway. It has been shown that NF-κB is an upstream regulator of NFATc1

(Yamashita et al., 2007), and that NFATc1 is an important transcription factor for V-

ATPase d2 (Feng et al., 2009). Taken together, one can speculate that PKCα regulates

RANKL-induced osteoclastogenesis by modulating NF-κB activity, which leads to

activation of NFATc1. NFATc1 then initiates transcription of V-ATPase d2 that

promotes cell fusion to form osteoclasts.

Chapter Seven

Overview of the Thesis

Chapter Seven – Overview of the Thesis

109

7.1 – General Discussion

Osteoclasts are large multinucleated cells formed from mononuclear progenitors of the

monocyte-macrophage family (Teitelbaum, 2000). The formation of osteoclasts requires

the presence of RANKL, which binds to its receptor (RANK) on the surface of

osteoclast progenitors. The stimulation of RANK leads to the initiation of several

downstream signalling pathways such as MAPK, NF-κB, NFATc1, and p38.

Protein kinase C (PKC) is a family of protein kinases that were first identified as Ca2+

and phospholipid dependent (Takai et al., 1979). Since their discovery, several other

PKC isoforms have also been identified that are Ca2+-independent. At present, there are

12 known isoforms, each with their own distinct roles, cellular localization, mode of

activation, and substrate specificity. These differences are due to structural differences

between the isoforms. Operationally, PKCs can be classified into 3 groups: classical,

novel, and atypical. Classical PKCs (cPKCs) are activated by Ca2+, diacylglycerol

(DAG) and phosphotidylserine (PS). Novel PKCs (nPKCs) are those not activated by

Ca2+, but are still activated by DAG and PS. Atypical PKCs (aPKCs) are not activated

by either Ca2+ or DAG but are still activated by PS. These differences in mode of

activation contribute to their functional differences.

In this dissertation, the results demonstrate that PKCα, δ, ε, γ, ι, and η are endogenously

expressed in unstimulated RAW264.7 cells, whereas PKCβ, θ, and ζ were not. Upon

RANKL stimulation of the RAW264.7 cells and osteoclast formation, PKCα and β

(both cPKCs) were upregulated, pointing to a role for these two PKC isoforms in

osteoclastogenesis. Interestingly, PKCα but not PKCβ was upregulated during RANKL-

induced osteoclastogenesis of BMM. This is in contrast to the previous studies of Lee et

al. (2003), which not only found that PKCβ was upregulated during BMM

osteoclastogenesis, but that it is also implicated in the regulation of osteoclast formation

and function. By comparison, PKCζ and θ were not expressed throughout RANKL-

induced osteoclastogenesis. Although the other PKC isoforms did not show any obvious

change in mRNA expression during RANKL-induced osteoclastogenesis, at this point

in time, one cannot exclude the possibility that they are not involved in

osteoclastogenesis and/or osteoclast function.

Chapter Seven – Overview of the Thesis

110

Next, the functional role of PKCα in RANKL-induced osteoclastogenesis was

investigated. To this end, Go6976 (indolocarbazole) was employed to examine the

effects of PKCα inhibition on RANKL-induced osteoclastogenesis. Go6976 is a cell-

permeable inhibitor of Ca2+- dependent PKCs α and β1 and has no effect on the kinase

activity of Ca2+-independent PKCs (Martiny-Baron et al., 1993). Its IC50 (half maximal

inhibitory concentration) for PKCα is three times lower than that for PKCβI (0.0023 μM

vs. 0.006 μM) thus making it relatively more selective to PKCα than to PKCβI. At

higher concentrations however, Go6976 has been shown to inhibit PKCμ (IC50 = 0.02

μM) (Gschwendt et al., 1996). It has also been suggested that it confers its inhibitory

activity on PKCs by interacting with the nucleotide binding site of the enzyme

(Martiny-Baron et al., 1993).

In the present study, BMM osteoclastogenesis was inhibited by the presence of Go6976.

Previous work in our laboratory has similarly demonstrated an inhibitory role for

Go6976 on RANKL-induced osteoclastogenesis in RAW264.7 cells (unpublished data).

However, whether the effect is due solely to the inhibition of PKCα, or if other PKCs

such as PKCβ were affected by the drug is uncertain. This lead to the use of small

interfering RNA (siRNA) as a more specific means of suppressing PKCα. Knockdown

of PKCα expression by siRNA inhibited RANKL-induced osteoclastogenesis in

RAW264.6 cells, further confirming that PKCα in involved in RANKL-induced

osteoclastogenesis.

Interestingly, PKCα siRNA transfected cells were observed to have fewer cells than the

un-treated mock cells. There are several possibilities as to why this was so. PKCα

siRNA might have caused the cells to detach from the bottom of the well, or even

undergo apoptosis. Another possibility would be that knock down of PKCα inhibits the

proliferation of RAW264.7 cells. Studies have shown that the overexpression of PKCα

promotes proliferation in many cell types, particularly in mouse fibroblasts, U87

malignant glioma cells, and MCF-7 breast cancer cells (Mandil et al., 2001; Soh et al.,

2003; Soh et al., 2003; Ways et al., 1995). Additionally, knock down of PKCα gene

expression by siRNA suppressed proliferation of human cancer cells (Yin et al., 2003).

On the other hand, other studies suggest that PKCα has an anti-proliferative effect and

Chapter Seven – Overview of the Thesis

111

that loss of PKCα function actually increases proliferation (Nakagawa et al., 2006;

Oster et al., 2006). These studies implicate a potential dual-role of PKCα in regulating

cell proliferation. The results presented in this thesis suggest that PKCα promotes

proliferation in RAW264.7 cells. It must be noted, however, that we cannot guarantee

whether the inhibition of PKCα specifically inhibits the proliferation of osteoclast

precursors, and thus there are not enough cells to form osteoclasts; or if inhibition of

PKCα directly inhibits osteoclastogenic process itself.

Unexpectedly, Go6976 was observed to have little to no effect on p-ERK protein

expression during RANKL-induced osteoclastogenesis in RAW264.7 cells, despite

previous studies showing that PKCα regulates ERK phosphorylation (Rucci et al., 2005;

Shatos et al., 2009). For instance, Rucci et al. (2005) demonstrated that inhibition of

PKCα prevented the phosphorylation of ERK in mature osteoclasts. While these studies

are difficult to reconcile with the findings of the present study, it is possible that PKCα

only affects the ERK pathway in mature osteoclasts and not during the early stages of

osteoclast formation.

Interestingly, V-ATPase d2 mRNA expression was suppressed by Go6976 treatment of

RANKL stimulated RAW264.7 cells. V-ATPase d2 is now known to play an important

role in cell fusion during osteoclastogenesis (Lee et al., 2006). Inhibition of PKCα by

Go6976 also suppresses the protein expression of NFATc1 (a major transcription factor

for V-ATPase d2) in RANKL-induced RAW264.7 cells. Furthermore, PKCα-WT was

able to potentiate RANKL-induced NF-κB promoter activity in RANK-293 and

RAW264.7 cells. In addition, previous studies in our laboratory demonstrated that

Go6976 inhibits NF-κB luciferase activity, despite Go6976 having no effect on IκBα

protein expression, which suggest that PKCα is involved in the activation NF-κB gene

transcription but does not affect the degradation of the inhibitory molecule IκBα

(unpublished data). Taken together, it is tempting to speculate that PKCα regulates

RANKL-induced osteoclastogenesis by modulating NF-κB activity, which leads to

activation of NFATc1. NFATc1 might then initiate transcription of V-ATPase d2 which

would, in turn, promote cell fusion to form osteoclasts.

Chapter Seven – Overview of the Thesis

112

7.2 – Future Directions

The collective results from this thesis have opened up several new avenues of research

that could be pursued for a better understanding of the role of PKCs in RANKL-induced

osteoclastogenesis. Of the several PKC isoforms that were examined, PKCα was chosen

to be the main focus of this study. Whether or not the other PKC isoforms play

additional and/or overlapping roles with PKCα in osteoclast function and formation

clearly warrants future examination.

This study has provided promising new evidence to hypothesize that PKCα participates

in the regulation of the NF-κB/NFATc1/V-ATPase d2 pathway which is important for

cell fusion of pre-osteoclasts. However, further studies are required to confirm this

position. For example, experiments are required to determine whether inhibiting PKCα

activity would specifically block cell fusion from occurring. Cell-cell fusion assay

would be one such approach to address this issue.

The ERK pathway was suggested to negatively regulate RAW264.7 osteoclastogenesis,

whereas p38 is a positive regulator (Hotokezaka et al., 2002). It therefore would be of

interest to examine the p38 pathway in relation to PKCα in future studies. It would also

be important to investigate the subcellular localization of PKCα during RANKL-

induced osteoclastogenesis by employing immunofluorescence analysis. This could also

be used to examine if PKCα co-localizes with any proteins of interest such as NF-κB or

NFATc1.

Unfortunately, due to time constraints, this study was also unable to conduct further

PKCα siRNA experiments. Other possible uses of the siRNA might have been to

examine its effects on the ERK, NF-κB, and NFATc1 signalling pathways to further

confirm the results of the Go6976 experiment.

A wild-type PKCα construct was used to investigate NF-κB activity in this thesis. A

constitutively active and a dominant-negative PKCα constructs were also obtained but

were unable to be utilized in time for this study. Future work will involve using these

constructs to investigate NFATc1 luciferase activity. Introduction of these constructs

Chapter Seven – Overview of the Thesis

113

into an osteoclast culture system can provide further insight into understanding the role

of PKCα in osteoclast function and formation.

It is uncertain as to whether PKCα directly regulates the proliferation of pre-osteoclasts.

A first step would be to determine if PKCα siRNA would induce the same effect on

BMM as it did on RAW264.7 cells. This would determine the cell-specificity of the

effect. Next, one could perform cell proliferation and apoptotic assays to quantify the

affect of PKCα inhibition on cell proliferation, and also to determine if the effect is

reduced cell proliferation and not apoptosis.

A more comprehensive approach to the study of PKCα would be to examine its role in

PKCα knockout mice. A look at the bone phenotype of such mice would provide a

major clue into its overall function in bone biology. The bones derived from such mice

would be analysed by microCT, histomorphometry, and loading studies to characterize

its physical, micro-architectural, and biochemical properties. Knockout mice are also

essential to examine whether PKCα-deficient osteoclasts exhibit any defects in bone

resorption or osteoclastogenesis in vivo.

Chapter Eight

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