09-10-26 thesis
TRANSCRIPT
Interactions between the Wnt Pathway and Inflammatory
mediators in mesenchymal stem cells
Nadene FangTing Tam
243454
Thesis submitted to the
University of Melbourne for the
Degree of BSc with Honours
The University of Melbourne
Department of Medicine
Royal Melbourne Hospital
26th October 2009
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Acknowledgements
I would like to thank my supervisor Dr Derek Lacey for his tremendous support he provided
throughout the year and especially during the weeks prior to handing in my thesis. Dr Lacey
passed on many different laboratory techniques and his guidance was fundamental to this project.
Many thanks to Prof Gary Anderson, Prof John Hamilton and Dr Derek Lacey for providing me
with such a great opportunity by allowing me to undertake this honours project in such a
fantastic department.
I would also like to thank the Hamilton group especially Agnieszka Swierczak, Jarrad Pobjoy,
Dominic Stipanov and Thao Nguyen for being such great company and providing much needed
laughs throughout the year. Special thanks to Thao and Dr Glen Scholz for passing on their
expertise in laboratory techniques and advice on data presentation.
To my fellow honours students, Shalini Maran and Zohra Miazoi, thank you for being such
amazing friends, this year would not have been the same without your friendship and support.
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Declaration
DECLARATION BY SCHOLAR:
I, Nadene FangTing Tam
certify that
- the thesis comprises only my original work, except where indicated in the
accompanying Acknowledgement statement
- the thesis conforms to the specifications outlined in the Honours Handbook
Signature: (Nadene Tam)
Date:
DECLARATION BY SUPERVISOR:
I confirm that the declaration above of Nadene FangTing Tam thesis are a true and fair representation of
the student’s work.
Signature: (Dr Derek Lacey)
Date:
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Table of Contents
TITLE PAGE ............................................................................................................................................................. 1
ACKNOWLEDGEMENTS .......................................................................................................................................... 2
DECLARATION ........................................................................................................................................................ 3
TABLE OF CONTENTS .............................................................................................................................................. 4
ABSTRACT .............................................................................................................................................................. 6
ABBREVIATIONS .................................................................................................................................................... 8
CHAPTER 1: INTRODUCTION ................................................................................................................................ 10
1.1 STEM CELLS ........................................................................................................................................................... 10
1.1.1 Embryonic Stem Cells ................................................................................................................................ 10
1.1.2 Mesenchymal Stem cells (MSCs) ............................................................................................................... 11
1.1.4 Identification of MSCs ............................................................................................................................... 12
1.2 MSC TO OSTEOBLAST ............................................................................................................................................. 13
1.3 OSTEOGENIC GENES ................................................................................................................................................ 14
1.4 EXTRACELLULAR FACTORS ........................................................................................................................................ 15
1.5 MARKERS OF DIFFERENTIATION ................................................................................................................................. 15
1.6 WNT PATHWAY ..................................................................................................................................................... 16
1.6.1 Wnt proteins ............................................................................................................................................... 16
1.6.2 Wnt signalling pathways ............................................................................................................................ 17
1.6.3 Inhibitors of the Wnt pathway .................................................................................................................... 19
1.6.4 Role of the Wnt pathway in MSCs .............................................................................................................. 20
1.7 HYPOTHESIS AND AIMS ............................................................................................................................................ 22
CHAPTER 2: MATERIALS AND METHODS .............................................................................................................. 23
2.1 MATERIALS ........................................................................................................................................................... 23
2.2 CELL CULTURE ........................................................................................................................................................ 23
2.3 TRANSFECTION OF PLASMIDS .................................................................................................................................... 24
2.4 DUAL-LUCIFERASE REPORTER ASSAY .......................................................................................................................... 24
2.5 NUCLEAR EXTRACTION ............................................................................................................................................. 25
2.6 OSTEOBLAST DIFFERENTIATION .................................................................................................................................. 25
2.7 RNA EXTRACTION, REVERSE TRANSCRIPTION AND REAL-TIME POLYMERASE CHAIN REACTION ................................................. 26
2.8 WESTERNBLOT ANALYSIS .......................................................................................................................................... 27
CHAPTER 3: RESULTS ........................................................................................................................................... 28
3.1 DETERMINING THE OPTIMAL CONCENTRATION OF TOPFLASH AND FOPFLASH PLASMIDS ..................................................... 28
3.2 EFFECTS OF ∆45, IL-1Β AND TNF-Α ON NUCLEAR Β-CATENIN ACTIVITY ............................................................................. 28
3.3 LICL INCREASES Β-CATENIN ACTIVITY ........................................................................................................................... 29
3.4 THE EFFECTS OF WNT3A, IL-1Β AND TNF-Α ON Β-CATENIN ACTIVITY ................................................................................ 29
3.5 ∆45 DID NOT INCREASE Β-CATENIN LEVELS IN WHOLE CELL LYSATES .................................................................................. 30
3.6 DETERMINING THE OPTIMAL STIMULATION PERIOD PRIOR TO NUCLEAR EXTRACTION ............................................................ 31
3.7 NUCLEAR Β-CATENIN LEVELS IN CELLS TREATED WITH LICL WERE NOT INFLUENCED BY IL-1Β OR TNF-Α .................................... 31
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3.8 NUCLEAR Β-CATENIN LEVELS – TREATMENTS WITH IL-1Β OR TNF-Α AND WNT3A ............................................................... 31
3.9 DETERMINING THE INCUBATION TIME PERIOD FOR OPTIMAL ALP STAINING ........................................................................ 32
3.10 IL-1Β AND TNF-Α DECREASE ALP STAINING IN ∆45-TRANSFECTED CELLS ........................................................................ 32
3.12 WNT3A INHIBITS OSTEOBLAST DIFFERENTIATION ......................................................................................................... 33
3.13 CHANGE IN OSTEOBLASTIC GENE EXPRESSION WITH LICL OR WNT3A AND IL-1Β OR TNF-Α ................................................. 33
CHAPTER 4: DISCUSSION ...................................................................................................................................... 35
REFERENCES ......................................................................................................................................................... 42
FIGURES ............................................................................................................................................................... 46
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Abstract
Rheumatoid arthritis a chronic inflammatory disease, consisting of activated leukocytes,
inflamed synovium and the production inflammatory mediators such as IL-1β and TNF-α. These
cytokines have been implicated in the pathology seen in this autoimmune disease such as
cartilage destruction, the loss of bone mass, stiffness and swelling of the joints. Wnt molecules
are morphogens which are expressed at multiple sites, including sites of inflammation. There are
19 known mammalian Wnt family members which signal via binding to frizzled and LRP5/6
receptors. The canonical Wnt signalling pathway has been shown to promote osteogenesis and
has a role in the differentiation of MSCs into osteoblasts.
IL-1β and TNF-α have been shown to block MSC differentiation to osteoblasts. This project
aims to investigate the mechanism by which MSCs are blocked from differentiating in
osteoblasts. It is hypothesised that IL-1β and TNF-α are blocking Wnt/β-catenin signalling and
therefore blocking Wnt driven osteogenesis.
In this project, the effects of IL-1β and TNF-α have been investigated at multiple levels. Firstly,
dual luciferase reporter assays were conducted to determine if β-catenin activity is affected by
the cytokines. Inducing the overexpression of β-catenin with the plasmid ∆45 was used to
determine which part of the Wnt pathway IL-1β and TNF-α regulate. Initial results indicate that
IL-1β and TNF-α are able to inhibit Wnt/β-catenin signalling by reducing the activity of β-
catenin as measured by a reporter assay. However, later experiments using cells that had
undergone prolonged passaging contradict these results. Following reporter assays with cells
transfected with an axin-RFP plasmid failed to show a reduction firefly luciferase activity despite
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other studies showing that the plasmid induces the formation of the β-catenin destruction
complex.
To specifically identify β-catenin levels in cells that have been treated with IL-1β and TNF-α in
the presence or absence of LiCl or Wnt3a, western blots were conducted using whole cell lysates,
cytoplasmic and nuclear extracts. Results did not indicate any detectable change in the levels of
β-catenin regardless of the treatment.
Osteoblast differentiation assays measuring alkaline phosphatase (ALP) expression, which is a
osteoblast marker, seemed to show that IL-1β and TNF-α have been able to block the Wnt
pathway with reduced ALP staining.
In conjunction to the results investigating the effects of IL-1β and TNF-α on the osteoblast
differentiation, there seemed to be a correlation with the cell passage number to the effects seen
by the cytokines on osteoblast differentiation and Wnt signalling in MC3T3-E1 cells. Repetitions
of experiments using cells that had been passaged a number of times over a prolonged period
gave conflicting results to initial experiments using cells that had been freshly cultured after
removal from liquid nitrogen. These results suggest that the effects seen by IL-1β and TNF-α on
osteoblast differentiation are affected by the number of times MC3T3-E1 cells have been
passaged.
In conclusion, the effects of IL-1β and TNF-α seen in this project on the Wnt pathway and
osteoblast differentiation are conflicting. Though further investigation into how cell passage
number affects their Wnt/β-catenin signalling within cells could provide more a better
understanding of the relationship between the Wnt pathway and inflammation.
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Abbreviations
ALP Alkaline phosphatase
APC Adenmatous polyposis coli
BMPs Bone morphogenic protein
BSP Bone sialoprotein
CBP CREB binding protein
Cbfa1 Core binding factor a1
CFU-F Colony forming unit fibroblasts
CIA Collagen-induced arthritis
CKIα Casein kinase Iα
Colla1 Type I Collagen
CRD Cysteine-rich domain
CREB cAMP response element binding
DKK Dickkopfs
Dsh Dishevelled
ECM Extracellular matrix
ES Embryonic stem
FGFs Fibroblast growth factors
FZD Frizzled
GSK 3β Glycogen synthase kinase 3β
HDAC Histone deacetylases
hMSCs Human mesenchymal stem cells
IL-1β Interleukin-1β
Int Integration
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Lef lymphoid enhancer factor
LiCl Lithium chloride
LIF Leukaemia inhibitory factor
LDL Low-density lipoprotein
LRP5/6 Low-density lipoprotein receptor-related proteins 5/6
MAP Mitogen-activated protein
MSCs Mesenchymal stem cells
mMSCs Murine mesenchymal stem cells
NLK Nemo-like kinase
Osx Osterix
PCR Polymerase chain reaction
RA Rheumatoid arthritis
RT-PCR Real-time polymerase chain reaction
Runx2 Runt related transcriptional factor 2
sFRPs Secreted frizzled-related proteins
Tcf T-cell factor
TAZ Transcriptional co-activator with PDZ-binding motif
TNF-α Tumour necrosis factor-α
TrCP Transducin repeat-containing protein
TBP TATA binding protein
Wg Wingless
WISP Wnt-1 induced secreted proteins
Wnt Wingless/Int
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Chapter 1: Introduction
1.1 Stem Cells
Due to their unique characteristics, stem cells have been hailed as a potential cure to
multiple diseases and offer hope to thousands of patients. Early experiments initially described
stem cells as cells that are able to rapidly proliferate and form colonies of cells, which were later
determined to be clones generated from single cells (Till and Mc, 1961). Two important
characteristics distinguish stem cells from other cell types. Firstly, they are non-specialised cells
that have the ability to renew themselves through cell division. Secondly, they have the ability to
differentiate into any type of cell in the body (pluripotency) and thereby replace and repair
damaged tissue. Most crucially they are also able to undergo indefinite self-renewal, without
losing their differentiation potential. There are a number of different types of stem cells such as
embryonic stem cells, haematopoietic stem cells, mesenchymal stem cells, and tissue specific
stem cells, and they all have different degrees of pluripotency. Tissue-specific stem cells, for
example, have a limited range of cells they can differentiate into and are known to be multipotent.
Stem cells adjust their responses according to the signals received from the surrounding
microenvironment. This microenvironment that promotes stem cells longevity and enables their
differentiation is known as the stem cell niche (Watt and Hogan, 2000). Under appropriate
extracellular signals, tissue-specific stem cells differentiate into committed progenitors which are
committed to differentiating into a specific cell type (Weissman, 2000).
1.1.1 Embryonic Stem Cells
Embryonic stem (ES) cells develop after fertilization of the oocyte (and before the
development of the foetus), upon which the blastocyst forms first, then there is the formation of
the three germ layers – endoderm, mesoderm and ectoderm. ES cells can be isolated from the
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inner cell mass of the blastocyst (Evans and Kaufman, 1981). However, there is much debate
about the ethical considerations with regards to the use of embryonic stem cells for research and
possible therapeutic use. Furthermore, the research conducted using animal models have shown
that there are many difficulties in maintaining ES cells in their undifferentiated state (Martin et
al., 2005).
The three germ layers mature and develop into cells specific to each post-natal tissue
present in the adult. These post-natal tissues also contain tissue-specific stem cells and are also
termed adult stem cells. Adult stem cells can be broadly classified as haematopoietic, epithelial,
mesenchymal and neural stem cells.
1.1.2 Mesenchymal Stem cells (MSCs)
Mesenchymal stem cells (MSCs) are fibroblast-looking cells that were first observed and isolated
from the bone marrow (Friedenstein et al., 1966). Recent studies however, have shown that
MSCs can be found in most organs and tissues throughout the body (da Silva Meirelles et al.,
2006). They are typically characterised as cells that are able to undergo continual self-renewal,
extensive proliferation and can differentiate into multiple cell types – including osteoblasts,
chondrocytes and adipocytes (Caplan, 1991; Dominici et al., 2006; Pittenger et al., 1999). MSCs
can be distinguished from haematopoietic cells by their adherence to culture dishes in vitro
(Dominici et al., 2006). Friendenstein et al. first coined the term colony forming unit fibroblasts
(CFU-F) to describe the phenomenon of a single cell forming a colony of morphologically
similar cells isolated from bone marrow (Friedenstein et al., 1970).
As mentioned above, stem cells are located within a niche that is conducive to their self
renewal and differentiation. Exposure and stimulation by extracellular signalling factors that are
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secreted into the microenvironment promotes differentiation of MSCs into various non-
haematopoietic cell types (Caplan, 1991). A network of signalling interactions between stem
cells and their daughter cells or surrounding cells are also part of this niche which eventually
regulates their differentiating and proliferation potentials. There are several cytokines and growth
factors such as leukaemia inhibitory factor (LIF) and fibroblast growth factors (FGFs) that have
been shown to be involved in the self-renewal of MSCs, maintaining a pool of undifferentiated
MSCs, but also have been shown to be important in MSC differentiation (Ito et al., 2008; Ng et
al., 2008; Pruijt et al., 1997; Szilvassy et al., 1996; Whitney et al., 2009). Another family of
proteins that have been implicated in the differentiation of MSCs into numerous cell types
including osteoblasts is the wingless/int (Wnt) family of proteins and will be elaborated on in the
following sections.
1.1.4 Identification of MSCs
Much of the early work on MSCs has concentrated on the identification and isolation of stem
cells using a combination of cell surface markers. However, as there is no single cell surface
antigen that exists to positively identify a MSC, a combination of markers is the only means of
phenotypic identification of MSCs. Human MSCs (hMSCs) can be isolated based on the
expression of Stro-1, CD105, CD73 and CD90 while lacking the expression of CD34+, CD-14 or
CD79α or CD19 (Dominici et al., 2006). MSCs isolated from mice are based on the expression
of Sca-1 and CD44 while failing to express CD45, CD 31 or CD11b. Both mouse and human
MSC consistently do not express CD45 and CD11b on their cell surface (Dominici et al., 2006;
Tropel et al., 2004). In comparison, hMSCs are better characterised compared to murine MSCs
(mMSCs) (Tropel et al., 2004).
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Essentially, MSCs are phenotypically identified due to the lack of expression of
haematopoietic markers. Isolating MSCs from different species such as rabbits and rats has
shown that the expression of these markers are different but there has been widespread
agreement that MSCs, regardless of their source, lack the expression of CD45. Due to the
variable expression of cell surface markers depending on the tissue sources, this has made for the
isolation of MSCs for research challenging.
MSC isolation has remained a challenge for researchers. The utilisation of stem cell lines
or precursor lines has helped overcome some of these issues, but this however has created their
own issues.
1.2 MSC to osteoblast
It was first noticed by Friedenstein et al. that by transplanting bone marrow cells to a
bone graft, these cells were able to differentiate into osteoblasts and eventually the formation of
new bone would occur (Friedenstein et al., 1966). It would later be realised that these bone
marrow cells conferred a population of MSCs. The ability of MSCs to differentiate into
osteoblasts is important for the maintenance of bone health, with damaged bone being replaced
by the formation of new bone, for example, when there is a bone fracture. Experiments using
hMSCs have shown that there is an increase in alkaline phosphatase activity when hMSCs are
cultured in osteogenic media which consists of ascorbate, β-glycerol and dexamethasone (Majors
et al., 1997; Pittenger et al., 1999; Tropel et al., 2004) and similarly with mMSCs (Tropel et al.,
2004).
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1.3 Osteogenic genes
There are several osteogenic genes that have been shown to be associated in the process
of MSC differentiation. The transcription of these genes has been shown to be upregulated in
osteoblasts and they include Runx2 (runt related transcriptional factor 2)/Cbfa-1(core binding
factor a1) and Osterix (Osx). These transcription factors are of great interest as they could be
crucial for furthering our understanding of chronic bone diseases such as rheumatoid arthritis and
osteoarthritis.
For MSCs to differentiate into pre-osteoblasts, the gene Runx2/Cbfa-1 is required and
experiments conducted by Komori et al. using mice have shown that the deletion of the
Runx2/Cbfa1 gene results in the inhibition of osteoblast differentiation. The Runx2/Cbfa1 gene
encodes for the Runx2/Cbfa1 transcription factor which is expressed in osteoblasts. In mutant
mice that did not express the Runx2/Cbfa1 gene, there was a lack of ossification with the mice
developing short legs and some showing signs of dwarfism. Furthermore, using x-ray
examinations, these mutant mice, compared to wild type mice of the same embryonic age,
showed weak calcification of various skeletal components such as the skull, ribs and mandibula
(Komori et al., 1997). Using Alazarin Red staining, this result has been replicated in the same
study as well as by other groups (Komori et al., 1997; Otto et al., 1997). The newborn Runx2-
null mice died shortly after birth due to the developmental failure of the ribs resulting in their
suffocation. These studies highlight the importance of the Runx2/Cbfa1 gene in bone formation.
Another gene that was found to be important for osteoblast development is Osterix (Osx)
which is specifically expressed in osteoblasts and is necessary to promote osteoblast
differentiation from preosteoblast prescursors (Ducy and Karsenty, 1995; Ducy et al., 1997;
Towler et al., 1994). As a consequence of the inactivation of Osx, there was little mineralisation
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of the facial and skull bones and deformation of other parts of the skeletal structure including the
ribs and limb bones (Nakashima et al., 2002). The absence of mineralisation and lack of
ossification in these animal models have shown that Runx2/Cbfa1 and Osx genes are essential
for the maturation of osteoblasts. In the same study by Nakashima et al., they were able to
determine that Osx and Runx2/Cbfa1 genes operate in the same osteoblast differentiation
pathway, with Osx gene downstream of Runx2/Cbfa1 (Nakashima et al., 2002). Furthermore,
through staining of the skeletons of Runx2/Cbfa1 null mice, they were also able to determine that
the expression of Osx was dependent on the presence of Runx2/Cbfa1 (Nakashima et al., 2002).
1.4 Extracellular Factors
It has also been shown that a group of growth factors known as bone morphogenetic
proteins (BMPs) promote osteogenesis of MSCs (Zhang et al., 2009). Binding to cell surface
receptors, BMPs are able to induce β-catenin signalling and promote bone formation (Chen et al.,
2007). This is an important and area of research, however it is outside the scope of this thesis.
1.5 Markers of Differentiation
There are several osteoblast markers that can be used in determining the progression of
the differentiation of MSCs into osteoblasts and these markers are commonly used in research
regarding osteoblast differentiation.
Proteins such as alkaline phosphatase (ALP), bone sialoprotein (BSP), osteonectin, and
type I collagen (Col1a1) can be considered early markers of osteoblast differentiation (van
Straalen et al., 1991).
During the proliferation of osteoblasts, there is an increased secretion of extracellular
matrix (ECM) and Col1a1 is one of the ECM proteins (Owen et al., 1990). This increased
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expression of Colla1 makes it a suitable marker for early osteoblast differentiation. Eventually
the proliferative stage in osteoblasts wanes and an increase in the expression of ALP proceeds,
with increased mineralization occurring in MSC cultures. Staining for ALP expression will be
used to determine the effects of Wnt ligands which will be discussed later in this thesis, on the
differentiating potential of pre-osteoblast cells into osteoblasts.
Other proteins such as osteocalcin, osteopontin and bone sialoprotein (BSP) can be found
in mature osteoblasts with osteocalcin being the most cell specific late marker for osteoblast
differentiation. It is expressed in osteoblasts, not in any other ECM producing cells, and can be
measured by real-time polymerase chain reaction (RT-PCR) (Ducy and Karsenty, 1995).
Several of these osteoblastic genes will be used in RT-PCR experiments to determine how
activation of the Wnt pathway affects MSCs differentiating into osteoblasts.
1.6 Wnt Pathway
1.6.1 Wnt proteins
Initial experiments in drosophila identified a gene that was required for wing formation
and was termed the wingless (Wg) gene as flies without this gene did not develop wings
(Nusslein-Volhard and Wieschaus, 1980). Around the same time, cancer researchers identified a
gene at the integration region of a tumour and normal tissue which was termed Int (integration)
(Wainwright et al., 1988). Both genes were found to be homologous and led to the coining of the
Wnt proteins through the combination of both gene names. These Wnt proteins are a family
cysteine-rich secreted glycoproteins, normally ranging from 350 – 400 amino acids in length,
and to date there are 19 known mammalian Wnt proteins.
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At the cellular level, the Wnt pathway is thought to have many roles including the
regulation of cell motility and polarity. In addition, the signalling pathway is also known to be
crucial in the causation of diseases such as cancer. This project focuses on the importance of the
Wnt signalling pathway in chronic bone related disease such as rheumatoid arthritis.
Our skeletal structure undergoes continual remodelling and Wnt proteins are thought to
play an important role in the regulation of bone mass. Through the Wnt signalling pathway, Wnt
proteins are thought to regulate bone formation, by controlling differentiation, proliferation and
cell death.
1.6.2 Wnt signalling pathways
1.6.2.1 Canonical and Non-canonical
There are at least 2 intra-cellular signalling pathways involved in Wnt signalling – the
canonical and non-canonical pathways (Figure 1). Most of the 19 Wnt proteins are known to
activate either the canonical Wnt pathway or the non-canonical Wnt pathway. The non-canonical
pathway does not utilise β-catenin, which is the central molecule in the canonical pathway, and is
not as well-studied. This project focuses the canonical pathway and β-catenin signalling in
osteoblast precursors and therefore the following is a description of the canonical pathway.
It is understood that in the steady state the Wnt/β-catenin pathway is not activated, and a
destructive complex forms in the cytoplasm. This complex results in stabilising of cytoplasmic
β-catenin in a complex, with adenmatous polyposis coli (APC) and Axin, allowing glycogen
synthase kinase 3β (GSK3β) and casein kinase Iα (CKIα) to phosphorylate β-catenin (Nakamura
et al., 1998). Phosphorylation of β-catenin results in its degradation via the β-TrCP (transducin
repeat-containing protein) mediated proteasome pathway (Hart et al., 1999; Liu et al., 1999).
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Biochemical experiments have shown that Wnt proteins interact with cell surface
receptors Frizzled (FZD) and low-density lipoprotein (LDL) receptor-related proteins 5 and 6
(LRP5/6) to initiate the canonical signalling pathway. FZD receptors are 7 transmembrane
molecules with a N-terminal cysteine-rich domain (CRD) while its co-receptor LRP5/6 is a
single-pass transmembrane protein. Wnt proteins bind directly to the CRD of the FZD receptor.
Upon the binding of Wnt proteins to FZD and LRP5/6 receptors, CKIα hyperphosphorylates
Dishevelled (Dsh) causing it to have an increased affinity for the C-terminal domain of the FZD
receptor and Frat-1 (Cong et al., 2004; Klein et al., 2006). Simultaneously, both GSK3β and
CKIα cause the phosphorylation of the LRP5/6 receptor, in turn resulting in the recruitment of
Axin to the cytoplasmic domain of the LRP5/6 receptor (Hino et al., 2003). Furthermore, at the
cell surface, Dsh, Frat-1 and Axin are thought form a complex which results in the disassembly
of the β-catenin destructive complex (Fagotto et al., 1999; Li et al., 1999). Therefore, an
accumulation of β-catenin within the cytoplasm occurs and is able to translocate to the nucleus
via directly interacting with the nuclear pore components (Yokoya et al., 1999).
Accumulation and translocation of β-catenin into the nucleus results in the physical
displacement of Groucho, transiently inducing the conversion of T-cell factor (Tcf)/Lymphoid
enhancer factor (Lef) into a transcriptional activator (Daniels and Weis, 2005). Thus, the
transcriptional complex of β-catenin and Tcf/Lef bind to the promoter and induce the
transcription of Wnt target genes. In the absence of Wnt signalling, Tcf/Lef normally acts as a
transcriptional repressor preventing the transcription of Wnt target genes due its association with
transcriptional inhibitor Groucho which together with histone deacetylases (HDAC) which
prevent the unwinding of DNA (Cavallo et al., 1998; Waltzer and Bienz, 1998). The shuttling of
β-catenin to and from the nucleus has been shown to be regulated by Axin and Tcf/Lef, either
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inducing cytoplasmic or nuclear retention by the 2 respective proteins (Cong and Varmus, 2004;
Tolwinski and Wieschaus, 2001).
There are two other important nuclear proteins, Bcl9/Legless and Pygopus which are
thought to be implicated in the nuclear retention of β-catenin. Pygopus associates with
Bcl9/Legless which in turn binds to the N-terminus of β-catenin (Kramps et al., 2002; Parker et
al., 2002). Simultaneously, there is recruitment of transcriptional co-activators such as histone
acetylase CBP/p300 and Brg-1, part of the SWI/SNF chromatin remodelling complex, to the C-
terminus of β-catenin (Barker et al., 2001; Hecht et al., 2000), facilitating the transcription of
osteoblastic genes.
1.6.3 Inhibitors of the Wnt pathway
There are a number of inhibitors of the canonical Wnt signalling pathway including
secreted frizzled-related proteins (sFRP) 1 – 4 which resembles the CRD of the FZD receptor.
Binding of sFRP to Wnt proteins physically prevents Wnt proteins from binding to FZD
receptors. Using β-catenin specific luciferase reporter gene assay, it has been shown in human
osteoblasts that there is complete suppression of Wnt signalling when Tcf and Wnt plasmids are
co-transfected with sFRP.
Wnt proteins are also able to bind to an unusual Wnt receptor known as the
Derailed/RYK class of tyrosine kinase transmembrane receptors. The inhibitor Wnt inhibitory
factor (WIF) has similarities with the extracellular domain of the Derailed/RYK receptors which
is involved in binding Wnt proteins – both of which are thought to be members in the non-
canonical pathway.
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The Wnt signal transduction pathway has also been shown to be inhibited by Dickkopfs
(Dkk) 1 – 5, secreted glycoproteins, binding to the LRP5/6 receptor (Bafico et al., 2001; Mao et
al., 2001; Semenov et al., 2001). Dkk-1 is able to bind to another transmembrane protein known
as Kremens and this association with Kremens and LRP5/6 receptors, possiblely induces the
internalisation of LRP5/6 receptors (Mao et al., 2002). Since the Wnt canonical signalling
cascade can only be initiated upon the binding of Wnt ligands to LRP5/6, Wnt signalling cannot
occur due to Dkk-1 and LRP5/6 binding.
Another inhibitor of the Wnt pathway is the mitogen-activated protein (MAP) kinase-
related protein Nemo-like kinase (NLK). Unlike the other inhibitors mentioned above, NLK
inhibits the Wnt pathway by phosphorylating Tcf/LEF thus causing a decrease in DNA-binding
affinity of the β-catenin-Tcf/LEF complex (Ishitani et al., 2003).
1.6.4 Role of the Wnt pathway in MSCs
1.6.4.1 Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a chronic inflammatory, auto-immune disease of the joints
resulting in the activation of leukocytes and the excessive production of inflammatory cytokines.
Synovial membrane lining the joints plays an essential role in regulating inflammation and
changes to this thin tissue results in the pathology seen in patients with RA. The synovial
membrane is normally only a few cells thick, containing synoviocytes which produce
extracellular matrix and synovial fluid. In patients with RA, there is an increase in the number
and types of cells that are found in the synovial lining – including activated B and T cells,
macrophages, mast cells and plasma cells and extensive angiogenesis is also observed. These
activated leukocytes produce cytokines that act in an auto- and paracrine fashion to induce the
production of inflammatory mediators and tissue degrading enzymes. This chronic induction of
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cytokines by these pro-inflammatory cells results in the perpetual inflammation seen in
rheumatoid arthritis. This leads to the destruction of bone and cartilage, as well as swelling and
redness of the joints due to the increased numbers of synoviocytes at the joint.
One of the most commonly studied cytokines implicated in the pathogensis of RA is
tumour necrosis factor (TNF)-α. TNF-α was first discovered to have similar necrosing effects on
tumour cells as endotoxin. Later studies showed that TNF-α causes the inhibition of bone
formation and stimulates bone resorption (Bertolini et al., 1986). Since the identification of the
presence of TNF-α in the synovial membrane and synovial fluid of patients with RA, TNF-α has
been implicated in the pathogenesis seen in RA (Chu et al., 1992; Hopkins and Meager, 1988).
Currently, anti-TNF-α therapy is the most successful biological therapeutic used in the treatment
of RA.
Another pro-inflammatory cytokine involved in the pathogenesis of RA is interleukin
(IL)–1 with early experiments detecting its activity in the synovial fluid of patients with RA
(Fontana et al., 1982) and its ability to induce bone resorption (Gowen et al., 1983). Initial
studies using mice with collagen-induced arthritis (CIA), which is a widely used animal model
for arthritis, have shown that early treatment with anti-IL-1 antibodies can delay the onset of RA
(van den Berg et al., 1994) and others indicating that bone damage is abolished with these
antibodies (Joosten et al., 1999).
The mechanism of action of IL-1β and TNF-α on stem cells is unknown but there seems
to be an inter-relationship between TNF-α and IL-1β. Previous studies have shown that anti-
TNF-α antibodies reduce the level of IL-1β production in cell cultures from patients with RA
(Brennan et al., 1989), while others show that TNF-α is able to induce the production of IL-1β
22
(Dinarello et al., 1986). Both IL-1βand TNF-α have also been shown to have additional
inhibitory effects on MSC differentiation into osteoblasts (Lacey et al., 2009).
The implications of these two pro-inflammatory cytokines in the pathogenesis of RA,
leads to the hypothesis that IL-1β and TNF-α blocks the Wnt pathway preventing MSCs from
differentiating into osteoblasts, thus causing the reduction in bone mass seen in patients with RA.
1.7 Hypothesis and Aims
The hypothesis to be tested is that IL-1β and TNF-α inhibit the differentiation of
mesenchymal stem cells into osteoblasts by antagonising the Wnt signalling pathway. To
determine at which level these cytokines interact with the Wnt pathway, the specific aims were
1. To determine, at the cytoplasmic level, how accumulation of β-catenin is affected by
IL-1β and TNF-α
2. To determine how nuclear levels of β-catenin are affected by IL-1β and TNF-α after
stimulation of the Wnt pathway
3. To test the functional importance of IL-1β and TNF-α in osteoblast differentiation
23
Chapter 2: Materials and Methods
2.1 Materials
Alpha-modified minimum essential medium (MEM), Sodium pyruvate, Penicillin Streptomycin,
(Invitrogen – Gibco); 10% Tris Glycine Gel, 1.5mm × 10/15 well (Invitrogen, USA); Purified
Mouse Anti-β-Catenin (BD Biosciences Pharmingen, USA); Monoclonal Anti-β-tubulin clone
TUB 2.1 (Sigma-Aldrich, Inc., USA); Histone H3 antibody (Abcam, Sapphire Bioscience Pty
Ltd, Australia); Polyclonal Swine Anti-rabbit immunoglobulin HRP, polyclonal rabbit Anti-
mouse immunoglobulin HRP ( Dako, Denmark); Detection Reagent (GE Healthcare Limited,
UK); Immobilon Western HRP Substrate Luminol Reagent (Millipore Corporation, USA);
Complete, EDTA-free Protease Inhibitor cocktail tablets, FuGENE HD Transfection Reagent
(Roche Diagnostics GmbH, Germany); Bio-Rad Protein Assay Dye Reagent concentrate (Bio-
Rad Laboratories Inc., USA); Dual-Luciferase Reporter Assay System (Promega Corporation,
USA); 0.05% Trypsin-EDTA 1× (Gibco, Invitogen Australia); Recombinant mouse Wnt-3A,
Recombinant mouse IL-1β, Recombinant mouse TNFα (R&D Systems, Inc., USA); TaqMan
Universal PCR Master Mix (Applied Biosystems, USA); SYBR GREEN PCR Master Mix
(Applied Biosystems, UK); Axin-RFP, TOPFlash/FOPFlash (gift from Dr Maree Faux, Ludwig
Institute for Cancer Research, Victoria, Australia); Renilla (gift from Dr Glen Scholz, University
of Melbourne, Victoria, Australia).
2.2 Cell culture
Mouse pre-osteoblast MC3T3-E1 cells were obtained from Riken – Osaka, Japan. Cells were
maintained in α-modified minimum essential medium (α-MEM) with L-glutamine,
ribonucleosides, deoxyribonucleosides, and supplemented with 5% inactivated fetal calf serum
24
(FCS), penicillin (10,000units/ml), streptomycin (10,000μg/ml) and sodium pyruvate (100mM)
in a humidified atmosphere of 5% CO2 in air at 37°C. After reaching confluence, MC3T3-E1
cells were detached and subcultured by treating the cells with trypsin.
2.3 Transfection of plasmids
MC3T3-E1 cells were seeded the day prior to transfection and incubated in the α-MEM culture
media overnight. A master mix of diluted FuGENE HD Reagent was made up by diluting it at a
ratio of 1:15.5 in serum-free α-MEM and allowed to incubate for 5 minutes at room temperature.
Plasmid DNA was mixed with a suitable amount of diluted FuGENE HD reagent, dependent on
the culture plate used, and incubated at room temperature for 15 minutes. After placing fresh
culture medium on the cells, the FuGENE HD Reagent/plasmid DNA transfection cocktail was
added drop wise to the cells. The cells were incubated overnight and the media changed the
following morning. Cells were lysed 48 hours post-transfection.
2.4 Dual-Luciferase Reporter Assay
MC3T3-E1 cells seeded in 12-well plates at a cell density of 2 × 104 cells per well and
transfected with the TOPFlash and FOPFlash plasmids, a gift from Dr Maree Faux from the
Ludwig Institute, the following day. As previously described, a cocktail of transfection reagent
Fugene HD transfection reagent/plasmid and 25μl per well was added drop-wise to 1ml of
culture media. The concentration of the plasmids used is as follows: Fopflash – 0.5μg/well,
Topflash – 0.5μg/well, Renella – 0.01μg/well, ∆45 – 0.5μg/well. 48 hours post transfection, the
cells were first washed twice with PBS then lysed with 200μl/well of Passive Lysis Buffer which
was diluted 1:4 with distilled water and placed in the ˗20°C freezer overnight. Using the rubber
policeman from a syringe, the cells were scraped from the bottom of the well and the lysates
collected. 20μl of each sample was transferred to luminometer tube and the luciferase activity
25
measured using Luciferase Assay Substrate (LAR) and Stop & Glo Reagent following
manufacturer’s instructions.
2.5 Nuclear extraction
Cells were subcultured onto 10cm Falcon culture plates at a cell density of 1 × 106 cells per plate
and allowed to grow overnight. At various time points, the cells were stimulated with LiCl (1mM)
or Wnt3a (20ng/ml), or were not treated with either stimulus. Cells were washed twice with ice
cold phosphate buffer solution (PBS) prior extraction of nuclear and cytoplasmic proteins. To
obtain the cytoplasmic proteins, the cells were lysed on ice with a hypotonic solution – 5mM
HEPES pH 7.9, 0.25% (v/v) IGEPAL, 25% (v/v) glycerol, 500mM NaCl, 1.5mM MgCl2, and
0.2mM EDTA. To this ice cold solution 10% (v/v) IGEPAL and 1× CompleteTM
Protease
Inhibitor were added. To harvest the nuclei, the solution was centrifuged at 10,000g, 4°C for 30
seconds. The cytoplasmic supernatant is removed and the nuclear pellet washed with the
hypotonic solution. The nuclear pellet is resuspended in a hypertonic lysis buffer made up of
5mM HEPES pH 7.9, 10mM KCl, and 1.5mM MgCl2, and the nuclear proteins are extracted by
rotating the sample on a rotary mixer for 2 hours at 4°C.
2.6 Osteoblast differentiation
Cells were seeded into 12-well plates at a cell density of 2 × 104 cells per well and incubated
overnight. To induce osteoblastic differentiation, the culture media was removed and replace
with osteogenic media (OM) consisting of α-MEM supplemented with 10% FCS,
dexamethasone (0.1mM), β-glycerophosphate (0.1μM) and ascorbate acid (10μM). The
osteogenic media was changed every 3 days. Cells were also treated with murine IL-1β (1ng/ml)
or murine TNF-α (10ng/ml). The cells were washed 3 times with PBS followed by fixing the
cells with 10% ice cold buffered formalin for 40 minutes. Cells were washed twice with distilled
26
water and covered with a filtered alkaline phosphatase stain made up of 0.1M Tris-HCL, napthol
ASMX-PO4 dissolved in N,Ndimethylformamide and Red Violt LB Salt. The plates were placed
in the dark for 30 minutes prior to washing the cells with distilled water and subsequently air
dried and pictures taken by scanning the plates.
2.7 RNA extraction, reverse transcription and real-time polymerase chain reaction
Cells were seeded at a concentration of 0.5 × 106 cells/well in 6 well plates and incubated
overnight before the culture media was replaced with OM. The cells were also treated with LiCl
(1mM), IL-1 (1ng/ml), TNF-α (10ng/ml) and Wnt3a (20ng/ml). After the 3 or 7 day stimulation,
the cells were washed twice with PBS and 350μl of Buffer RLT added to each well to lyse the
cells. The wells were scraped, the cellular extract collected and passed through a 20-gauge
needle using an RNase-free syringe 5 times. The rest of the RNA extraction process was carried
out as per RNeasy Mini Handbook 04/2006 with the removal of any potential DNA
contamination by RNase-free DNase treatment.
Concentrations of RNA extracts were measured and a maximum of 2μg of template RNA was
added to the master mix. The components of the master mix were as set out in the Omiscript
Reverse Transcription Handbook 05/2004 with the following changes – 1μl/reaction Oligo-dT
primer (10μM) and 1μl/reaction Random Primer (3μg/μl). cDNA was synthesised by placing the
RNA/master mix solutions in an incubator at 37°C for 2 hours before being stored at -20°C.
RT-PCR was run for the transcripts of the following genes: ALP, Runx2, Osx, and control gene
TBP. On a 384 well plate, each well was loaded with 5μl of SYBR Green Master mix and 1μl of
the primers. 4μl of diluted cDNA, 1/20 dilution, was transferred into each well. PCR reactions
were conducted on an ABI 7900HT real time PCR machine (Applied Biosystems).
27
2.8 Westernblot analysis
To measure the protein concentration of each sample, 5 × Protein Assay Dye Reagent
concentrate was diluted with Milli-Q water to make up 1 × Protein Assay Dye Reagent. 10μl of
each sample and 7.5μl of bovine serum albumin (BSA) standard was added to 1.5ml of 1 ×
Protein Assay Dye Reagent. The final concentration of BSA in the Protein Assay Dye Reagent is
5μg/μl. The absorbance of each sample was determined using the Bio-Rad SmartSpec 3000 and
the BSA standard was used to determine the concentration for each sample.
10%, 1.5mm Tris Glycine gels (Invitrogen) were loaded with a total of 40μg of protein/well and
15μl of each sample into each well. The gels were run at 150V for 1.5 hours, till the dye front
was close to the bottom of the gel and the gels were transferred to PDF membranes at 100V for
40 minutes. After blocking the membrane for 1 hour with 3% BSA in TBST, the membrane was
incubated overnight at 4°C with the primary antibody which was diluted in 5ml of 1% BSA in
TBST. The dilutions for each primary antibody are as follows: anti β-catenin – 1/5000, anti β-
tubulin – 1/106, anti histone H3 – 1/1000, anti actin – 1/5000. The membrane was washed 3
times, for 15 minutes each, with 1 × TBST then incubated for 1 hour with the appropriate
secondary antibody in 10ml of 1% BSA in TBST. After the membrane was washed another 3
times in 1 × TBST, the membrane was covered with a mixture of equal amounts of Detection
Reagents 1 and 2 (GE Healthcare Limited, UK) or Immobilon Western HRP Substrate Luminol
Reagents ( Millipore Corporation, USA) – depending on the strength of the primary antibody.
28
Chapter 3: Results
3.1 Determining the optimal concentration of TOPFlash and FOPFlash plasmids
Initial experiments were performed to determine the optimal ratio of TOPFlash plasmid to the
Renilla control plasmid. TOPFlash is a plasmid that has 7 Tcf/Lef binding sites upstream to a
thymidine promoter that drives the transcription of the firefly luciferase gene. Hence, the firefly
luciferase gene will only be transcribed in the presence of β-catenin, which binds to Tcf/Lef. The
FOPflash plasmid is similar to the TOPFlash plamid, but with mutated Tcf/Lef binding sites and
thus acts as a negative control. Renilla is also another control plasmid which induces the
constitutive transcription of the Renilla luciferase gene, acting as a transfection efficiency control.
The Dual Luciferase Reporter Assay was used to measure firefly and Renilla luciferase activity,
with the firefly activity standardised against the Renilla control luciferase activity. All treatments
were either transfected with TOPFlash or FOPFlash, together with Renilla, and the data for
FOPFlash is not shown as luciferase/Renilla ratios were consistently low (as expected). The
following concentrations of TOP and FOPFlash plasmids were tested – 0.5μg, 0.25 μg and 0.1μg
per well. The optimal amount of 0.01μg of the Renilla plasmid that was used to transfect cells in
each well had been determined by previous experiments conducted by other members of the
laboratory. A mutant β-catenin overexpression plasmid called ∆45, which encodes a mutant form
of β-catenin that cannot be degraded by the Axin-Gsk3β destructive complex, was used to
specifically activate the TOPFlash reporter plasmid. Figure 2 shows that the optimal amount of
TOP and FOPFlash per well was 0.5μg.
3.2 Effects of ∆45, IL-1β and TNF-α on nuclear β-catenin activity
Preliminary reporter assays conducted, using a range of ∆45 plasmid concentrations in MC3T3-
E1 cells, indicate that the greatest fold change in firefly luciferase activity was obtained by
29
transfecting cells with 0.25μg of the ∆45 plasmid (Figure 3). All subsequent reporter assays used
this concentration for ∆45. IL-1β and TNF-α treatment caused a reduction in the firefly luciferase
activity induced by ∆45 (Figure 4).
3.3 LiCl increases β-catenin activity
Lithium chloride (LiCl) is a non-specific inhibitor of GSK3β and therefore non-specifically
activates the Wnt/β-catenin pathway. LiCl treatment resulted in an increased fold change in the
luciferase activity indicating that there was an overall increase in β-catenin binding to the
Tcf/Lef binding sites (Figure 5). Initial experiments with cells treated with either IL-1β or TNF-α,
in the presence of LiCl, showed that there was a decrease in TOPFlash reporter activity when
compared to cells treated with LiCl alone (Figure 5A). However, later experiments, using cells
that had undergone serial passaging, seem to contradict these results. The cells treated with LiCl
and IL-1β showed an increased fold change in the luciferase activity (Figure 5B), while the
effects of TNF-α remained the same.
3.4 The effects of Wnt3a, IL-1β and TNF-α on β-catenin activity
Wnt3a constantly induced the accumulation of β-catenin in the nucleus resulting in the increased
firefly luciferase activity when compared to the control (Figure 6). This shows that the Wnt3a
ligand is able to activate the canonical Wnt pathway, which requires β-catenin for signalling, in
these cells.
Similar to earlier LiCl results, early reporter assays showed IL-1β and TNF-α were able to
decrease firefly luciferase activity when cells were co-treated with Wnt3a and either of the
cytokines. TNF-α reduced the Wnt3a-induced firefly luciferase activity to the greatest extent –
resulting in similar firefly luciferase activity seen in the control (Figure 6A). These results
30
seemed to indicate that these pro-inflammatory cytokines are able to inhibit the activation of the
β-catenin specific reporter, accumulation of β-catenin within the nucleus and therefore blocking
the Wnt pathway. However, subsequent reporter assays carried out to confirm these results
contradicted the initial findings – TNF-α caused no change in firefly luciferase activity while
treatment with IL-1β seemed to cause a 5 fold increase the firefly luciferase activity compared to
cells treated with Wnt3a (Figure 6C).
However, in the later experiments the axin-RFP plasmid was used as a positive control for the
inhibition of the Wnt/ β-catenin pathway, and over expression of axin-RFP initially caused a
decrease in β-catenin activity (Figure 6B). Axin is crucial for the formation of the β-catenin
destructive complex. By treating the cells with Wnt3a after transfection with the axin-RFP
plasmid, it was determined that the optimal transfection amount of axin-RFP plasmid for the
reporter assay was 0.05μg per transfection. These results indicated that overexpression axin-RFP
was able to inhibit Wnt-induced firefly luciferase activity (Figure 6B). Despite these preliminary
experiments indicating the inhibition of the Wnt/β-catenin pathway using axin-RFP, later
experiments conducted showed an increase in firefly luciferase activity in axin-transfected cells,
suggesting possible errors had occurred in one or more aspects of the experiment (Figure 6C).
3.5 ∆45 did not increase β-catenin levels in whole cell lysates
To confirm the results from luciferase assays showing an increase in β-catenin when MC3T3-E1
cells are transfected with ∆45 (mutant β-catenin plasmid) (Figures 3 and 4), western blots were
conducted using whole cell lysates from MC3T3-E1 cells that had been transfected with
increasing amounts of ∆45 (Figure 7). Results indicate no detectable change in β-catenin levels
as band intensity remained constant.
31
3.6 Determining the optimal stimulation period prior to nuclear extraction
Determining changes in the levels of β-catenin in whole cell lysates is very difficult therefore β-
catenin levels were measured in nuclear extracts from treated cells to observe changes in the
levels of nuclear β-catenin. To determine the best time to obtain maximal change in nuclear β-
catenin levels, cells were stimulated over a range of time points – 1,2,4,6,and 24 hours, with
either LiCl or Wnt3a (Figure 8). The band intensity was greatest for the 2 hour stimulation when
compared to untreated cells and was thus used as one of the suitable time points to obtain
maximal β-catenin stimulation. The 6 hour time point was also chosen as another time point.
3.7 Nuclear β-catenin levels in cells treated with LiCl were not influenced by IL-1β or TNF-α
Initial western blots using nuclear lysates from cells treated with LiCl showed an increase in
nuclear β-catenin levels with stronger band intensity when cells were stimulated for 2hrs (Figure
8A). However, subsequent western blots using nuclear extracts from cells treated with either IL-
1β or TNF-α, in the presence of LiCl, showed no detectable change in nuclear β-catenin levels
(Figure 9) when compared to extracts from cells treated with LiCl alone. Furthermore, LiCl
alone failed to increase β-catenin levels in the nucleus.
3.8 Nuclear β-catenin levels – treatments with IL-1β or TNF-α and Wnt3a
Western blots show that the band intensity of nuclear extracts from cells treated with Wnt3a
alone, for 2 hours, was similar to bands using nuclear extracts from cells treated with either IL-
1β or TNF-α, in the presence of Wnt3a, indicating there was no detectable change in nuclear β-
catenin levels despite treatment with the pro-inflammatory cytokines (Figure 9). In addition,
Wnt3a alone failed to increase β-catenin levels in the nucleus. Similar results were obtained
using cytoplasmic and nuclear extracts from cells treated for 6 hours (Figure 9).
32
3.9 Determining the incubation time period for optimal ALP staining
Alkaline phosphatase (ALP) is a marker of osteoblast differentiation. To determine whether IL-
1β and TNF-α were inhibiting the biological effects of Wnt pathway activation, ALP expression
was measured as a marker of MC3T3-E1 differentitation from a pre-osteoblast state to a
differentiated osteoblast state by staining for ALP.
In the process of determining the effects of IL-1β and TNF-α, each osteoblast differentiation
assay was conducted in duplicate. Initially, 2 sets of the same assay would be differentiated after
being incubated in osteogenic media for 1 week or 2 weeks. Upon the realisation that ALP
staining had reached maximal intensity after 1 week, subsequent assays were differentiated at
either 5 days or 1 week. The 5 day incubation in ostegenic media gave the best results –
maximum difference in staining intensity between osteogenic media and normal media.
3.10 IL-1β and TNF-α decrease ALP staining in ∆45-transfected cells
Earlier reporter assays using cells transfected with ∆45 and treated with either IL-1β or TNF-α
showed a decreased in firefly luciferase activity, indicating a reduction in the accumulation of β-
catenin activity (Figure 4). Osteoblast differentiation assays also support this finding with the
transfected cells treated with IL-1β or TNF-α showing a decrease in ALP staining when
compared to wells treated ∆45 alone. However, wells treated with ∆45 alone failed to show an
additive effect on ALP staining, showing similar staining intensity to the control wells (Figure
10A). 3.11 IL-1β and TNF-α decreased ALP staining in LiCl-treated cells
Both IL-1β and TNF-α were able to reduce the intensity of ALP staining seen in wells treated
with LiCl (Figure 10B). However, LiCl treated cells had no additional effect on ALP staining
compared to osteogenic media alone.
33
3.12 Wnt3a inhibits osteoblast differentiation
Cells were treated with osteogenic media for 7 days in the presence or absence of Wnt3a, axin-
RFP plasmid, IL-1β and TNF-α. Wnt3a appeared to inhibit ALP staining while the axin-RFP
plasmid had no effect on ALP staining (Figure 11). Both IL-1β and TNF-α inhibited ALP
staining alone and in the presence of Wnt3a (Figure 11B).
3.13 Change in osteoblastic gene expression with LiCl or Wnt3a and IL-1β or TNF-α
The expression of several known osteoblastic marker genes can be quantitatively measured via
real-time-PCR (RT-PCR). RNA was extracted from cells treated for 3 or 7 days in osteogenic
media treated with a combination treatments with LiCl, Wnt3a, IL-1β or TNF-α. The expression
of different osteoblast genes – Runx2, Osterix (Osx) and ALP was measured. The TATA binding
protein (TBP) was used as a control gene and was used in analysis of the results to determine
differential expression of the genes.
Treatment of the cells for 3 days in LiCl did not show a significant change in gene expression for
Runx2, Osx or ALP when compared to negative control. However, a general pattern showing a
moderate decrease when co-treated with IL-1β and a greater reduction with TNF-α was observed
in the expression of all 3 genes (Figure 12).
As with the treatment with LiCl, Wnt3a treatment for 3 days did not induce a significant change
in any of the osteoblastic genes. Unlike the results observed in cells treated with LiCl, a
reduction in the expression was only observed in ALP when cells were treated with either IL-1β
or TNF-α in the presence of Wnt3a. Expression of Runx2 and Osx after treatment with Wnt3a
for 3 days did not seem to be affected, though a minor increase in their expression was observed
when co-treated with IL-1β. Furthermore, there was no difference seen in the expression in either
34
of the 2 genes when comparing treatments with IL-1β or TNF-α. However, Osx expression was
lowered to the same extent by both IL-1β and TNF-α. Interestingly, treatment with TNF-α on its
own induced a greater reduction in ALP expression compared to treating cells only with IL-1β.
As seen with the expression of the osteoblastic genes using cells that have been treated for 3 days,
there did not seem to be a significant change in any of the genes using cells that had been treated
for 7 days with either LiCl or Wnt3a. Treatment of the cells for 7 days in osteogenic media with
either IL-1β or TNF-α, in the presence of LiCl, resulted in similar fold changes in the expression
of Runx2, Osx and ALP (Figure 13). This was also observed for Runx2 and Osx in cells treated
with Wnt3a. However, in cells treated with Wnt3a, the expression of ALP seemed to be reduced
to a greater extent when treated with IL-1β as compared to co-treatment with TNF-α.
35
Chapter 4: Discussion
The Wnt pathway is thought to play a role in the differentiation of MSCs into osteoblasts
based on studies showing Wnt pathway activity in MSCs and osteoblasts. However, many
studies do not use specific Wnt ligands to investigate Wnt signalling. In this project, a specific
Wnt ligand – Wnt3a was used to stimulate murine calvaria pre-osteoblast MC3T3-E1 cells to
study the effects that pro-inflammatory cytokines, IL-1β and TNF-α have on the Wnt pathway.
Given that IL-1β and TNF-α can inhibit stem cells differentiation into osteoblasts (Lacey et al.,
2009), it was hypothesised that IL-1β and TNF-α may inhibit the Wnt/β-catenin pathway and
thereby block stem cell differentiating into osteoblasts.
To test this hypothesis, the Wnt/β-catenin pathway was activated using either a β-catenin
overexpression plasmid (∆45), LiCl or Wnt3a, while the pathway was specifically inhibited
using the axin-RFP overexpressing plasmid. The Wnt/β-catenin pathway was measured under the
above the conditions in the presence or absence of IL-1β or TNF-α.
The results from the dual luciferase reporter assay which measures β-catenin activity are
inconclusive as early experiments showed increases in β-catenin activity in the presence of LiCl,
Wnt3a and the β-catenin overexpressing plasmid ∆45. While the axin-RFP plasmid, which is
crucial to the formation of the β-catenin destructive complex, showed the expected decrease in β-
catenin activity, IL-1β and TNF-α also showed similar decreases. However, later experiments
using cells transfected with the axin-RFP plasmid and treated with both IL-1β and TNF-α did not
show the expected decrease in the firefly luciferase activity. This was surprising as the axin-RFP
plasmid, which was a gift from Dr Maree Faux, was also used by Faux et al. and they
successfully showed the destruction of β-catenin by the expression of axin-RFP (Faux et al.,
36
2008). However, our early experimental testing using different axin-RFP plasmid concentrations
also showed a decrease in Wnt activity (Figure 6B). As axin-RFP is a control for reducing Wnt
activity, this suggests possible experimental errors occurred as the axin-RFP plasmid should
inhibit Wnt activity.
A possible explanation for these differing results could be due to differentiation of the
pre-osteoblast cells that has attributed to the number of passages the cells had undergone before
they were used in these experiments. While the passage number for these cells was not noted, the
morphology of the cells was observed before the plating of the cells. In experiments where IL-1β
was able to reduce the nuclear β-catenin levels in the presence of LiCl or Wnt3a (Figure 5A, 6A),
the cells were observed to be round. However, cells used in experiments where there was an
increase in the fold change by both IL-1β and TNF-α in the presence of LiCl or Wnt3a (Figure
5B, 6C), the cells appeared to be differentiated, adopting an elongated, more fibroblast-like
shape. These observations seem to indicate that the cells were at different stages of
differentiation and are similar to the cell morphology seen in other studies using early and late
passage MC3T3-E1 cells (Chung et al., 1999). Furthermore, the study by Chun et al. suggests
that serial passaging of MC3T3-E1 cells reduced osteoblastic function with lowered alkaline
phosphatase (ALP) activity and osteocalcin secretion. Other studies using human osteoblasts
indicate ALP mRNA levels from samples obtained from older donors were lower than those
from younger donors (Sutherland et al., 1995).
The lack of inhibition by the axin-RFP plasmid could be due to the plasmid not being
expressed in the cells, though this is unlikely as both the TOPFlash and Renilla plasmids were
expressed. However, as our positive control for the inhibition of the Wnt pathway did not work,
37
it suggests that all the results from these experiments are not valid. Additional measures such as
using green fluorescent protein (GFP) to transfect the cells could be taken to avoid transfection
inefficiency speculation.
TNF-α consistently blocks the Wnt/β-catenin pathway in all experiments – in LiCl treated
cells this is likely due to cell death, as there was an increase in dead cells seen in wells co-treated
with LiCl and TNF-α. In other studies using TNF-sensitive cells, LiCl has been shown to cause
an increase in TNF-cytotoxicity in a dose-dependent manner, while the concentrations of LiCl
used did not affect cell survival when the cells were only treated with LiCl (Beyaert et al., 1989).
This effect of LiCl on the MC3T3-E1 cells line used for this project was similar to that seen by
Beyaert et al. In observations of cells co-treated with Wnt3a and TNF-α, there did not appear to
be an increase in cell death, however this was not specifically measured. Therefore although
TNF-α blocked Wnt3a activation of the TOPFlash reporter, cell survival would need to be
measured to ensure that this inhibition is not due to possible cytotoxic effects of TNF-α on this
cell line.
Given that both IL-1β and TNF-α inhibited ∆45-induced increase of firefly luciferase
activity in the reporter assay and if this result is real, then these cytokines are possibly up
regulating a transcriptional regulator of the β-catenin/Tcf transcription. Any inhibition of the
Wnt/β-catenin pathway above the level of β-catenin should have no effect on the ∆45 expressing
cells as ∆45-β-catenin cannot be degraded by the destructive complex. Therefore, in order for IL-
1β and TNF-α to inhibit the β-catenin signal in these cells, they would need to up-regulate a
Tcf/Lef antagonist such as NLK or groucho. Therefore further experiments need to be done to
confirm these results and to determine the possible mechanism of inhibition. However, it is still
38
possible that IL-1β and TNF-α could regulate this pathway at multiple levels, and thus upregulate
Wnt antagonists such as Dkk-1.
To further investigate the effects of Wnt3a, IL-1β and TNF-α on the Wnt pathway,
western blots using whole cell lysates, cytoplasmic and nuclear extracts were conducted. Initial
western blots using whole cells lysates from cells that had been transfected with increasing
amounts of ∆45 (a plasmid that causes the overexpression of β-catenin) did not show a detectable
increase in β-catenin. This observation could be due to the fact that there are large pools of
different β-catenin isoforms found within cells, and that most commercial anti- β-catenin
antibodies detect all isoforms of β-catenin. Furthermore, both phosphorylated β-catenin and
active β-catenin are known to interact with cadherins that are found at cell adhesions (Maher et
al., 2009), as such any small changes in β-catenin levels that signal to the nucleus would be very
difficult to detect using whole cell lysates. Hence, to detect any changes in β-catenin levels, it
was decided that nuclear extracts could provide better results with the majority of β-catenin
sequestered within the cytoplasmic fraction.
After establishing that stimulation with LiCl or Wnt3a for a period of 2 hours results in a
greater increase in the amount of β-catenin detected via western blot (Figure 8), subsequent
western blots conducted used cytoplasmic and nuclear extracts from cells that had been
stimulated for 2 and 6 hours. However, detection using anti-β-catenin antibodies failed to show
any significant increase in band intensity in nuclear lysates from cells that were treated with
either LiCl or Wnt3a (Figure 9). While there could be speculation that there could be residual
cytoplasmic proteins in the nuclear extracts obtained, the anti-β-tubulin control antibody has
clearly shown no detection of any β-tubulin. β-tubulin is required for the assembly of
39
microtubules which is involved in cell motility, and as such is used a loading control for
cyotplasmic proteins. Likewise, a control for the nuclear extracts, anti-histone H3 antibodies,
was used to ensure that there was no cross-contamination of proteins of the 2 separate fractions
during the extraction process. The observed changes in band intensity in the preliminary
westernblots using the nuclear extracts ascertain the best time point for nuclear extraction could
have been arbitrary as the anti-histone H3 loading control had not been used. Without the loading
control, there is no means to determine if the amount of sample loaded in each well was
consistent and that the observed changes in band intensity with the anti-β-catenin were real.
However, based on the subsequent western blots, which indicate no change in β-catenin levels
regardless of the treatment, this could indicate that the initial change in band intensity seen was
due to inconsistent loading of samples. Repeating western blots, with loading controls, using
nuclear extracts from cells stimulated over various time points would provide an indication as to
whether further investigation in this direction would be worthwhile.
Wnt3a has been implicated in promoting skeletal development and playing a role in the
differentiation of MSCs into osteoblasts. ALP expression is used as an osteoblastic marker and
many studies use osteoblast differentiation assays to determine the extent of ALP expression by
cells that have been subjected to different stimulus. The results from the osteoblast
differentiation assays conducted as part of this project appear to show that Wnt3a is inhibiting
the differentiation of osteoblasts (Figure 11). This is seen by the reduced ALP staining intensity
when the MC3T3-E1 cells are transfected with the axin-FRP plasmid and treated with Wnt3a
when compared to the wells with cells only transfected with axin-RFP. This osteoblast inhibition
by Wnt3a has also been seen in other studies staining for ALP expression (De Boer et al., 2004).
However, since the cells are cultured in osteogenic media, there should be a decrease in the
40
intensity of ALP staining in the cells transfected with the axin-FRP plasmid alone when
compared to the control wells, which was not observed in these experiments. This could indicate
that the effect of β-catenin destructive complex is minimal in cells that have already begun to
differentiate as the cells used in these experiments had been passaged a number of times. This is
in concordance with results seen in experiments previously conducted on hMSCs treated with
conditioned media from Wnt3a-secreting cells (Boland et al., 2004).
The osteogenic genes Runx2/Cbfa1, osterix (Osx) and ALP were measured in cells
treated for 3 days with LiCl, Wnt3a, IL-1β and TNF-α. Real-time PCR (RT-PCR) results suggest
that there was no significant time-dependent effect on the change in osteoblastic gene expression,
while there seemed to be a descending fold change in the expression of all genes when treated
with LiCl for 3 days. A small increase in fold change was observed when the cells were treated
with Wnt3a and IL-1β reflecting the reduction in firefly luciferase activity seen with the same
treatment in the reporter assays as mentioned above. However, the fold changes seen in gene
expression were minimal and not significantly higher or lower than the expression of these genes
in the control.
There is a possibility that the rate of differentiation the MC3T3-E1 cells had influenced
the outcomes of the experiment conducted in this project. Experiments conducted by Khatiwala
et al. to determine how the rigidity of the extracellular matrix affects osteoblast differentiation
have shown that MC3T3-E1 cells grown on polystyrene had a greater extent of differentiation
compared to cells grown on less rigid collagen substrates (Khatiwala et al., 2006). In the same
study, they also managed to show that, when cells were grown on polystyrene, cell density was
almost double the density of cells grown on the hydrogels with the least collagen density, over
41
the same period of time. The greater ALP staining observed in this project, using MC3T3-E1
cells that had been passaged a greater amount of times, could partly be due to the increased
differentiation and rapid proliferation of the cells on plastic wells.
Results obtained from the experiments conducted so far are inconclusive with regards to
the effects of IL-1β and TNF-α on the Wnt pathway. However, it has been observed that the cell
passage number of the MC3T3-E1 cells and hence their differentiated state, seems to play an
important role in the osteoblast phenotype observed, and the effects seen by Wnt3a, IL-1β and
TNF- α on these cells. These experiments suggest that using MC3T3-E1 cells that have been
serially passaged, over an extended period of time, result in their spontaneous differentiation
towards osteoblasts. Experiments conducted with MC3T3-E1 cells that have been freshly
cultured from liquid nitrogen, and hence closer to a stem cell line state yield different results,
suggesting that cytokines have different effects on the stages of differentiation. In addition, using
a member of different stem cell lines would also be advantageous.
Further investigation into the effects of IL-1β and TNF-α on the Wnt pathway using cells
with different passage numbers could provide an insight to the effectiveness of current anti-
cytokine therapy at different stages of severity in rheumatoid arthritis and other autoimmune
diseases.
42
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46
Figures
Figure 1: The canonical and non-canonical Wnt signalling pathway. In the absence of Wnt
ligands, a β-catenin destructive complex consisting of Axin, APC and GSK 3β forms and
phosphorylation of β-catenin results in its ubiquination. Upon the binding of Wnt ligands to both
the frizzled (FZD) and LRP5/6 receptors, signalling though the canonical pathway is transduced
by β-catenin. The key difference between the canonical and non-canonical pathway is the
requirement of β-catenin for signal transduction. Adapted from Ling, L., Nurcombe, V., and
Cool, S.M. (2009). Wnt signaling controls the fate of mesenchymal stem cells. Gene 433, 1-7.
47
Figure 2: Dual-Luciferase Reporter Assay – The optimal TOPFlash firefly luciferase plasmid
to Renilla luciferase plasmid ratio, was measured using the mutant β-catenin overexpressing ∆45
plasmid (0.25μg) to specifically activate the TOPFlash promoter. Cells were treated with 0.1,
0.25 or 0.5μg/well of either TOPFlash or FOPFlash control (not shown). Experiments were
performed in triplicate and the firefly luciferase activity standardised against the Renilla
luciferase activity. The firefly luciferase/Renilla ratio is expressed as fold change, normalised
against samples that had not been transfected with ∆45.
48
Figure 3: Dual-Luciferase Reporter Assay – The optimal ∆45 plasmid concentration was
determined by co-transfecting cells with TOPFlash plasmid and 0.05, 0.1 or 0.25μg/well of ∆45
plasmid. Each treatment was performed in triplicate and the firefly luciferase activity
standardised against the Renilla luciferase activity. The firefly luciferase/Renilla ratio is
expressed as fold change with the ratio normalised to control samples.
49
Figure 4: Dual Luciferase Reporter Assay – Effect of IL-1β and TNF-α on β-catenin
activity. MC3T3-E1 cells transfected with ∆45, a mutant β-catenin overexpressing plasmid and
treated with 1ng/ml IL-1β or 1ng/ml TNF-α for 48hrs before luciferase activity was measured.
Each treatment was performed in triplicate and the firefly luciferase activity standardised against
the Renilla luciferase activity. The firefly luciferase/Renilla ratio is expressed as fold change with
the ratio normalised to control samples. The cells were treated with the cytokines for 48hrs.
50
Figure 5: Effects of LiCl,IL-1β and TNF-α on β-catenin activity. MC3T3-E1 cells were
treated in the presence or absence of LiCl (10mM) and treated with IL-1β or TNF-α for 48hrs
before luciferase activity was measured. A: Experiments performed with less differentiated cells.
B: Experiments performed with more differentiated cells. Each treatment was performed in
triplicate and the firefly luciferase activity standardised against the Renilla luciferase activity.
The firefly luciferase/Renilla ratio is expressed as fold change with the ratio normalised to
control samples. Cells were treated with LiCl, IL-1 and TNF-α for 48 hrs.
51
Figure 6: Effects of Wnt3a, IL-1β and TNF-α on β-catenin activity. A: MC3T3-E1 cells were
treated with Wnt3a in the presence or absence of IL-1β and TNF-α for 48hrs before firefly
luciferase activity was measured. B: The optimal concentration of the axin-RFP plasmid (which
inhibits Wnt/β-catenin activity) was determined by transfecting cells with either 0.05, 0.1,
0.25μg of plasmid and treating the cells for 48hrs with Wnt3a before firefly luciferase activity
was measured. C: Cells were treated with Wnt3a, IL-1β, TNF-α and Axin-RFP and firefly
luciferase activity was measured 48hrs later. Wnt3a results in a 60 fold increase in firefly
luciferase activity. Each treatment was performed in triplicate and the firefly luciferase activity
standardised against the Renilla luciferase activity. The firefly luciferase/Renilla ratio is
expressed as fold change with the ratio normalised to control samples.
52
Figure 7: β-catenin western blot. Western blot using whole cell lysates from cells that had been
transfected with ∆45 at 0.1, 0.5 and 1.0μg/well. Increasing amounts of the plasmid failed to
induced an increase in the band intensities using anti-β-catenin 1° antibodies. Actin was used as
a loading control.
53
Figure 8: β-catenin nuclear extract western blot. Western blot of nuclear extracts from cells
that had been stimulated with LiCl (10mM) (A) and Wnt3a (2ng/ml) (B) at various time points –
1,2,4,6, and 24hrs, to determine the best time point to obtain maximal change in nuclear β-
catenin levels.
54
Figure 9: Effect of cytokines on β-catenin. Western blot of cytoplasmic and nuclear extracts
from cells stimulated for 2 or 6 hours with either LiCl or Wnt3a and IL-1β or TNF-α. β-tubulin
and histone H3 were used as loading controls for the cytoplasmic and nuclear extracts
respectively.
55
Figure 10: Osteoblast differentiation assay staining for ALP. Cells were transfected with ∆45
(0.02μg/well) prior to treatment with LiCl and IL-1β or TNF-α. Cells were also treated with LiCl
and either IL-1β or TNF-α. Cells were treated for 5 days in osteogenic media.
56
Figure 11: Osteoblast differentiation assay staining for ALP expression. Wnt3a seemed to
inhibit osteoblast inhibition. Determining the effect of IL-1β and TNF-α in the absence and
presence of Wnt3a on osteoblast differentiation. Cells were treated for 5 days osteogenic media
with IL-1β and TNF-α in the presence of Wnt3a.
57
Figure 12: Real time-PCR using cDNA from cells that had been treated in osteogenic media for
3 days with LiCl, Wnt3a, IL-1β and TNF-α. Results are expressed as fold change and normalised
to TBP expression. The results are the average of 3 separate experiments performed in triplicate.
58
Figure 13: RT-PCR using cDNA from cells that been treated in osteogenic media for 7 days
with LiCl, Wnt3a, IL-1β and TNF-α. Results are expressed as fold change and normalised to
TBP expression. The results are the average of 3 separate experiments performed in triplicate.