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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
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
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
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
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.
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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.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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
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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.
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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.
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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
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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
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
82
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
Chapter Five – Expression of PKC Isoforms During RANKL-Induced Osteoclastogenesis
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.
0
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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.
0
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CTR V-ATPase d2 Cathepsin K
Fold
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RANKL
RANKL + 1μM Go6976
RANKL + 10μM Go6976
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
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.
0.0
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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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Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis
102
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
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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.
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Chapter Six – Functional Analysis of PKCα In RANKL-Induced Osteoclastogenesis
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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
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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
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.
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