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Analysis of the Role of Wnt6 in Intermediate Filament


Vimentin Expression and the Consequences for
Epithelial Cell Fate in Diabetic Nephropathy

Written by: Shamoon Saljoghei
Supervised by: Dr. John Crean

A thesis submitted in part fulfilment of the requirement for the
degree of BSc. (Honours) in Pharmacology


Pharmacology Research Project
UCD School of Biomedical and Bimolecular Science

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SUMMARY
Diabetic Nephropathy (DN) is the most common cause of ESRD in western societies.
Our understanding of the disease is not complete but recent advances in the molecular
mechanism have increased our knowledge of the pathology of the disease. The major
endpoints of the disease are the irreversible scarring of nephron components such as
glomerulus and renal proximal epithelial tubular cells (PETC). The disease is caused by
an interplay between haemodynamic and metabolic insults on the resident mesangial
and epithelial cells. Transforming Growth Factor ! (TGF!) has been widely implicated
and shown to have a predominant role in the progression of DN by causing a
phenotypic transition through regulation of developmental genes. Multiple strategies
have been formulated to target the signalling pathways have proven ineffective;
subsequently therapeutic targets have been focused on downstream mediators of TGF!
as well as other pathways. Studies from our group and others have implicated Wnt6 in
the progression of DN identifying a role in cell fate specificity and kidney development.
Here, we analyse the effects of Wnt6 on cell polarity through dynamic regulation of the
intermediate filament (IF) Vimentin and the focal adhesion complex protein Paxillin.
We show that Wnt6 can oppose TGF!s effects on cytoskeletal dynamic and implicate
the Polycomb Repressor Complex in modulation of TGF! signaling. Subsequently we
demonstrate that DZNep, a histone methyltransferase inhibitor can reverse TGF!
induced phenotypic changes in epithelial cell lines. These results suggest that
pharmacological intervention directed against the TGF! pathway remains a viable
option and that new therapeutic avenues utilising Wnt6 and targeting the Polycomb
Repressor Complex may prove effective for the treatment of renal injury.

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TABLE OF CONTENTS


INTRODUCTION 4
1.1 Pathophysiology of DN 4
1.2 Molecular mechanisms of DN 5
1.3 The role of TGF! in DN 5
1.4 Wnt signalling and cell fate specificity 6
1.5 Cell migration 7
1.6 Polycomb Repressive Complex and TGF! mediated signalling 9
2. MATERIALS AND METHODS 11
2.1 Cell Culture 11
2.2 Transfections 12
2.3 Confocal Microscopy 12
2.4 Western Blotting 13
2.5 Immunoprecipitation 14
2.5 Polarisation Assay 15
2.6 Effect of DZNep on cell lines treated with TGF! 15
3. RESULTS 17
3.1 Wnt6 causes cell polarisation in HeLa cell lines 17
3.3 Wnt6 induces cell polarity in epithelial cell lines 24
3.2 Wnt6 increases the expression of Cdc42 and Vimentin in HKC8 cells 25
3.4 Wnt6 does not phosphorylate Dvl2 27
3.5 Analysis of EZH2 and TGF! overlapping genes 28
3.6 DZNep reverses the effects of TGF! 30
3.7 IC50 of DZNep 32
4. DISCUSSION 34
5. ACKNOWLEDGMENTS 37
6. REFERENCES 38

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1. INTRODUCTION
1.1 Pathophysiology of DN
Diabetic nephropathy (DN) is the most common cause of End Stage Renal Disease (ESRD)
in western societies which subsequently requires organ transplantation or dialysis
[1]
.
Histological end points of the disease include glomerulosclerosis (irreversible scarring of the
glomerulus) and tubulointerstitial fibrosis
[2]
. Clinical characteristics of the disease begin to
manifest with increased glomerular filtration rate from the onset of diabetes. This is then
followed by thickening of glomerular basal membrane and expansion of mesangium that
results from increased extracellular matrix production and decreased matrix degradation
[3][4]
.
The matrix proteins involved include collagen I, III, IV and Fibronectin
[5]
. Subsequently
fibrosis results in albuminuria and studies have shown that ECM content of glomerulus
directly correlates with severity of albuminuria
[6]
. Mongensen and colleagues observed that
persistent excretion of low amount of albumin results in the progression to proteinuria and
subsequently ESRD
[7]
.
It is now known that disease progression is not limited to the glomerulus but also to renal
epithelial proximal tubular cells (PETC) causing tubulointerstitial fibrosis
[8]
. It has been
shown that disease severity best correlates with tubulointerstial fibrosis, a hallmark of
ESRD
[10]
. Even though primary onset of DN is glomerulosclerosis, data suggests that long-
term loss of function is dependent on the degree of tubulointerstitial fibrosis rather than
glomerulosclerosis. Prior to tubulointerstitial fibrosis, tubular hypertrophy and basement
membrane discrepancies have been implicated. Early stage lesions are characterised by
mononuclear cell infiltrations and fibrosis
[11]
and it is thought that inflammatory cells home
to these regions via intercellular adhesion molecules such as ICAM, V-CAM and MCP-1
[12]

in response to metabolic insults such as hyaloronic acid and D-glucose
[13]
.


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1.2 Molecular mechanisms of DN
It is thought that the disease progresses due to an interplay between haemodynamic and
metabolic stresses on the kidneys
[14].
It has been shown that factors effecting haemodynamic
pathways include systemic hypertension, increases in intraglomerular pressure and activation
of pathways associated with Renin Angiotensin System & Endothelin. These pathways can
potentially increase intraglomerular pressure
[15]
. Haemodynamic activation results in the
induction of pathways associated with PKC, MAPK, NF-kB and various cytokines such as
CTGF/CCN2, TGF!1, VEGF and Angiotensin II
[16][17][18][19][20][21]
. Metabolic insults such as
high glucose result in enhanced reactive oxygen species (ROS), renal polyol formation
[22]
and
production of Advanced Glycosylation End products (AGEs
)[23]
. In cohorts, insults from the
two different pathways on nephron components lead to the clinical progression of the disease.
Central to these processes is the secreted cytokine TGF! that has been shown to mediate the
pathogenic characteristics of the disease
[24]
and whose expression is increased in all tissues of
the kidney in DN
[25]
.

1.3 The role of TGF! in DN
TGF! has a predominant role causing a phenotypic change in cells in vitro called epithelial to
mesenchymal transition (EMT) marked by a loss of the epithelial marker E-cadherin and a
gain of mesenchymal markers Fibronectin and Vimentin
[26]
. Cells that undergo EMT gain
fibroblastic like capabilities of migration and ECM production. In the glomerulus and PETC,
E-cadherin is an important component in creating tight junctions between cells to maintain
epithelial integrity
[27]
. As a result of haemodynamic and metabolic insults cells lose
intercellular connectivity and begin to secrete TGF! and Fibronectin
[28][29]
. TGF! has been
shown to be a strong regulator of development, studies have shown TGF! acts to induce
EMT during gastrulation
[30]
, mesoderm formation and cardiogenesis
[31]
and likely plays a role

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in epithelial dedifferentiation in end stage renal disease Studies in our lab suggest that Wnt6
can reverse TGF! induced expression of Vimentin suggesting that Wnt6 has a role in
maintaining differentiated epithelial cell phenotype.

1.4 Wnt signalling and cell fate specificity
Wnt signalling has been shown to have a role in controlling cell fate specificity and
development. Wnt ligands bind to G-Protein Coupled Receptors (GPCRs) known as Frizzled
(Fzd) constituting LDL receptor-related protein LRP 5/6 to activate canonical signalling
[32]
.
Activation of canonical signalling leads to stabilisation of !-catenin and increased trafficking
to nucleus where it acts to regulate Wnt target genes by co-operating with TCF/LEF
transcriptional factors. These receptors stimulate a signal to multiple intracellular proteins
that include Dishevelled (Dsh), glycogen synthase kinase-3! (GSK-3), Axin and
Adenomatous Polyposis Coli (APC) to regulate the degradation of !-catenin
[33]
. In a non-
canonical pathway a different signalling cascade is activated which does not regulate !-
catenin levels.
Initially studies showed that Wnt 1 induces nephrogenesis
[34]
, although it was not expressed
in kidneys it was found that Wnt 4
[35]
, 2b
[36]
, 7b
[37]
were transiently expressed in developing
kidneys. Other Wnt ligands such as Wnt6
[38]
, 9
[39]
and 11
[40]
have been implicated in ureter
buds in fetal kidneys but have not previously been shown to activate the canonical pathway.
The effects of Wnt6 signalling are not restricted to nephrogenesis and have been implicated
in periodontal development where it has been suggested to promote differentiation of cells
without an effect on proliferation
[41]
. Wnt6 has also been implicated in bone formation and
studies have suggested that knock-downs suffer a loss of cell differentiation and impairment
of osteoblastogenesis
[42]
. Studies in our lab on embryogenesis have suggested high expression
of Wnt6 in the kidneys occuring in the pre and mesonephros during kidney development.

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This suggests that the ligand has a specific role in nephrogenesis and epithelialisation.
Furthermore studies conducted in our lab have shown that Wnt6 induces tubulogenesis in
MDCK cells suspended in matrigel (Beaton et al. J Cell Biol 2014).

1.5 Cell migration
Mesenchymal cells and fibroblasts have migratory behaviour and thus it is valuable to study
the effects of signalling molecules that induce such phenotypic changes. Previous studies in
our lab have suggested that Wnt6 regulates the expression of the mesenchymal marker
Vimentin.
Migrating cells have a polarised morphology consisting of protruding end and a retracting
end formed by cytoskeletal rearrangement
[43]
. Cells polarise in response to gradients of
morphogens and chemoattractants
[44]
that activate a family of G-Protein Coupled Receptors
(GPCRs)
[45].
One of the initial downstream effectors of signalling is the RhoGTPase Cdc42
that has a role in cytoskeletal positioning during cell migration. Cdc42 recruits PAR3/6 to the
leading end of a polarised cell leading to cytoskeletal rearrangement by recruitment of actin
polymerisation machinery to the leading edge
[46]
. Recruitment of PAR3/6 by Cdc42 to
leading edge results in a Par6/PKCzeta complex which phosphorylates GSK-3! resulting in
its inactivation
[47]
. Furthermore the inactivation of GSK-3! kinase activity is thought to
cause an up regulation of APC, a component of microtubule plus ends essential for cell
polarity. Schlessinger et al. showed that non canonical pathways and Cdc42 co-operate to
induce cell polarity at leading edge of a cell
[48]
.
Kupfer et al. showed that microtubule organising centre (MTOC) of the cell closely interacts
with the Golgi Apparatus, thus re-orientation of MTOC in migrating cell also results in re-
orientation of Golgi to the leading edge with respect to the nucleus
[49]
. Furthermore the
wounding of cell monolayer induces this MTOC and Golgi reorientation. Cell polarisation

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assays are based on this principle whereby monolayers are wounded and the degree of cell
polarity is observed by determining orientation of MTOC/Golgi with respect to the nucleus.
As well as its effect on MTOC reorientation, Cdc42 interacts with (MRCK) myotonic
dystrophy kinase-related Cdc42-binding kinase to orient the nucleus towards the lagging
edge
[50].

In order to successfully migrate, cells produce extensions of the plasma membrane by
protrusion. Under normal circumstance protrusions point to the direction of chemoattractants
but in absence of such stimuli cell extend in protrusions in a probing manner
[51]
. These
protrusions extend cell membrane in direction of movement and adhere to a substrate, where
this is unsuccessful membrane ruffling is observed. Cdc42 is involved in formation of
probing protrusions called filopodia consisting of long unbranched actin bundles
[52]
. Cdc42
induces Rac a RhoGTPase involved in formation of protrusions of branched actin bundles
called lamellipodia
[53]
. Some effectors of Rac are N-WASP and WAVE which function in
controlling Arp2/3
[54]
. Furthermore Cdc42 has been shown to participate in the formation of
large multi protein focal adhesion (FA) complexes consisting of adaptor proteins Vinculin,
Paxillin and Focal Adhesion Kinase (FAK) that connect cell cytoskeleton with ECM for
anchorage
[55]
. The formations of these adhesions are important to allow for the effective
adhesion of protrusive forces to ECM or substrates.









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1.6 Polycomb Repressive Complex and TGF! mediated signalling
Gene expression is regulated by chromatin remodelling such as lysine and arginine
methylation, lysing acetylation, serine/threonine phosphorylation and lysine ubiquitination at
N-terminus of core histone proteins
[56]
. In chromatin, DNA is packaged around a functional
unit of chromatin known as the nucleosome which consists two of each type of core proteins
(H2A, H2B, H3 and H4). Via further proteins such as H1 the nucleosomes are packaged into
a spiral arrangement to form 30nm fibres. The N-terminus of core histone proteins (positively
charged) are exposed for modification which interacts negative charge on DNA, an
interaction which promotes chromatin compaction
[56]
. In order to allow for transcription this
machinery needs to unfold to a 11nm fibre exposing DNA for effective binding of
transcriptional machinery. It is thought that this occurs by acetylation of core histone
proteins
[56][57]
. Trimethylation of core histone proteins promotes deacetylation of histones
subsequently resulting in silencing of gene expression. Lysine methylation is well
documented and is thought to occur on lysine 4, 9, 27 and 36 of H3 and lysine 20 of H4 by
methyltransferases
[58][59]
.
Polycomb Repressive Groups (PcG) are multi-protein complexes which function to regulate
gene expression through chromatin remodelling with a key function of maintaining cell
phenotype
[60]
. Predominantly in mammals two complexes exists Polycomb Repressive
Complex 1 and 2 (PRC1 / PRC2)
[61].
PRC1 acts to compact chromatin to silence gene
expression through the ubiquitination of H2A. Whereas, PRC2 silences gene expression
through dimethylation or trimethylation of lysine 27 on H3 through its histone
methyltransferase componenets (EZH1 and EZH2)
[62][63]
. Furthermore PRC2-EZH2 are more
active and establish H3K27me2/3 levels whereas PRC2-EZH1 complexes are less active and
restore lost H3K27me2/3 by histone demethylation
[64][65]
. The PRC2 complex comprises of
four core proteins EZH1/2, SUZ12, EED and RbAp46/48
[66]
. The PRC1 complex comprises

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of two core proteins Ring1A/B and Bmi-1
[67]
. The enzymatic activity of PRC2 is dependant
on the presence on other polypeptides known as AEBP2, PCLs and JARID2 which function
to enhance the recruitment and enzymatic activity of the complex. PcG have been implicated
in development and PRC2 has been shown to target and silence developmental regulators and
Hox genes
[68][69]
.
TGF! is a key regulator of EMT and it has been shown to induce the expression of Snail1
[71]
.
Snail1 is a transcriptional factor, which mediates repression of E-cadherin a phenotypic
change that is required for EMT. Studies showed that PRC2 is a key component in regulating
E-cadherin expression, by recruiting the PRC2 complex to CDH1 E-cadherin gene promoter,
Snail1 interacts with EZH2 to promote silencing of thr CDH1 gene
[73]
. Bmi-1 (component of
PRC1) has been shown to regulate Vimentin and Fibronectin expression by an unknown
mechanism since the over expression of PRC1 components results in increased Fibronectin
and Vimentin
[72]
.
Experiments have shown that following activation of TGF! signalling pathway Smad-2/4 can
displace EZH2 and prevent repression of TGF! target genes by H3K27me3
[73]
. 3-
Deazaneplanocin (DZNep) is a global histone methyltransferase inhibitor which functions by
down regulating the expression of methyltransferases associated with the PcGs. It has been
shown that DZNep reduces the expression of PRC1 and PRC2 core components EZH1,
EZH2, Bmi-1, Eed, Suz12, Mel18
[74]
. Preliminary data suggests an interaction between
Smad-3 and EZH2 that we hypothesis may be critical in regulating Vimentin expression. This
suggests that targeting the PcG complex may have therapeutic benefit by inhibiting TGF!
mediated Vimentin expression.



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2. MATERIALS AND METHODS
2.1 Cell culture
Cell cultures were maintained in T75 tissue culture flasks in an incubator at 37C, 5% CO2
where they adhere to surface and form monolayers. Cultures were implemented in 10%
Foetal Calf Serum(FCS) Gibco Dulbeccos Modified Eagle Medium constituting glutamine
and streptomycin antibiotic. The medium of flasks were changed every one to two days to
maintain normal cell conditions. Cultures were split from one flask to three flasks prior to
becoming 70% confluent i.e. occupying 70% of the flask surface area.
Prior to splitting, cells were washed with phosphate buffered saline. Cell monolayer and
adherence to surface was disrupted using trypsin to bring cells into suspension. Cell
suspension was then diluted by a factor of six using 10% FCS medium and transferred to
three new T75 flasks and placed in an incubator to allow cell adherence to surface and
monolayer formation.
For confocal microscopy, GFP-actin HeLa cells were seeded onto Ibidi "-Slide 8 well
when stock flasks were confluent. Cell monolayer was disturbed using trypsin and
resuspended in 10% FCS medium. A 1:250 split of confluent cell line was made and 250l of
final cell suspension was added to each well. Slides were then placed in an incubator to allow
for cell monolayer formation and growth to the desired confluency.
HKC8 Renal Tubular Epithelial Cells were seeded onto 6cm plates taking into account cell
confluency and surface area of the plates. Also seeded onto 3.8cm 12 well plates with in a
similar fashion. The cell monolayer was the allowed to grow to the required confluency for
the proceeding experiment such as transfections or drug treatments.



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2.2 Transfections
Cells on seeded plates were transfected or co-transfected with the required plasmids coding
for Wnt6, Dvl2, pSM Orange Paxillin and pSM Orange Vimentin using FuGENE HD
Transfection Reagent and Optimem. Seeded wells or plates were transfected when they had
reached 60-70% confluency. For each condition, 1g of plasmid DNA was suspended in 20l
of Optimem. 3l of Fugene was then added bellow the meniscus of mixture gently, and
allowed to form micelles for 15 minutes at RT. In the case of co-transfections 1g of each
DNA was added to Optimem followed by careful addition of 3l of Fgene for every g of
DNA (1g DNA : 3l Fugene). Reaction mixtures were then added to the corresponding
wells or plates and then were left to transfect, undisturbed in an incubator for 24 hours.
Post 24 hour transfection, old media from the wells or plates were removed and replaced with
0% FCS media to starve cells and prevent further growth for 24 hours. Plates or wells were
then ready for further analysis or harvest.

2.3 Confocal Microscopy
GFP-actin Helas seeded on Ibidi "-Slide 8 well and transfected were ready for imaging
following 24 hours of starvation in 0% FCS media. Nikon Eclipse Ti spinning disc confocal
microscope was used to analyse fluorescence from cells using Bitplane Imaris software.
The effects of Wnt6 on de novo expression of photo switchable pVimentin and pPaxillin
Plasmids coding for Vimentin protein and Paxillin protein code for manipulated forms of the
proteins that contain a photo switchable fluorescent protein PSmOrange. De novo expression
of these proteins following transfection with Fugene reagent allowed for the real time visual
analysis of Vimentin and Paxillin dynamics in response to co-transfection with Wnt6 when
compared to controls.



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2.4 Western Blotting
Cells from plates or wells to be harvested were washed with phosphate buffered saline twice
while maintaining all conditions throughout the protocol at 4
o
C to prevent enzymatic
degradation of proteins. Scrapers were then used to physically detach cells from surface and
samples were then centrifuged to pellet. The pellet was the resuspended in RIPA lysis buffer
(50mM Tris-HCl pH 7.4, 0.5% NP-40, 250mM NaCl, 5mM EDTA, 50mM NaF, protease and
phosphatase inhibitors) for 30 minutes with a vortex every 10 minutes on ice. Samples were
then centrifuged at 12000 RPM for 12 minutes resulting in a supernatant containing cell
lysates.
Cell lysates were then quantified for protein content by the Bradford Assays using Bio-Rad
Protein Assay reagent. Concentration of each sample was the spectrometrically determined
using SoftMax Pro software reading absorbance 595nm. Concentration of samples were
automatically calculated by the software using BSA standards.
25g of a protein sample was then transferred to eppendorfs containing 15l of 3X SDS
(gives protein a negative charge) loading buffer and 1l of B-mercaptoethanol (to reduces
dislphide bonds). Volumes were then all equalised to 50l with ddH2O. Samples were then
boiled for 5 minutes to denature proteins and expose antigen sites. Samples were either kept
at 4
o
C or stored at -20
o
C.
Proteins were then separated by (PAGE) Polyacrylamide Gel Electrophoresis. Samples were
loaded onto polyacrylamide gels consisting of a 8% stacking and 10% resolving
compartments. Initially gels were run at 80V until the samples reach the resolving stack
(indicated by the blue dye). Once reached resolving stack, voltage was increased to 120V.
Protein on gels were then transferred to polyvinylidene difluoride (PVDF) membranes at
120V for one hour.

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PVDF membranes were then blocked in 5% milk in PBS-T (0.1% Tween) for one hour at
room temperature to reduce non-specific binding of proteins. The membranes were then
probed for target protein using primary antibody. Membranes were left rolling in 1:1000
dilution of primary antibody in 5% milk in PBS-T for one hour at room temperature or
overnight at 4
o
C. Membranes were then washed with PBS-T for 15 minutes, 3 times to
remove non-bound antibodies. Secondary antibodies linked HRP (horseradish peroxidase)
specific to primary antibody species were allowed to bind on membranes. Membranes were
then placed in 5% milk in PBS-T containing 1:2000 dilution of secondary antibody as before.
Membranes were washed with PBS-T as before.
To enhance signal from HRP; West Dura was used on membranes and allowed to react for 2-
3 minutes. Membranes were then developed on X-ray films in a dark room with variable
exposure time depending on signal intensity.

2.5 Immunoprecipitation
To determine phosphoryation status of Dvl2 in response to Wnt6
HKC8 cell lines were seeded onto 6cm plates and allowed to grow to 60-70% confluency.
Cell lines were then transfected for four conditions; control, Dvl2 transfected, Wnt6
transfected, Wnt6 and Dvl2 co-transfected. Wnt6 plasmids code for a manipulated form of
protein containing a V-5 tag and similarly Dvl2 plasmids code for a protein containing a Flag
tag.
Following normal transfection protocol cells were harvested, lysed and protein content of
lysates were quantified as noted above. Protein samples were then analysed by western blot
to confirm successful transfection of HKC8 with Wnt6 and Dvl2 using V-5 and Flag directed
primary antibodies respectively.

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In the case of successful transfection, Dvl2 was then separated from cell lysates by
immunoprecipitation. EZview Red Anti-Flag M2 Affinity Gel was used to separate Flag
tagged Dvl2. 200g of cell lysates for each conditioned was used. Immunoprecipitation
protocol was carried out as directed by product manual. Initially the IgG beads suspended in
slurry suspension were aliquoted with Tris Buffered Saline (TBS), vortex and centrifuged
8200g for 30 seconds and repeated twice. Samples were then analysed by western blotting
and probed for phosphotyrosine to determine phosphorylation status of Dvl2.

2.5 Polarisation Assay
To investigate the effect of Wnt6 on cell migration and polarity
A scratch-wound assay was conducted. 12 well plates were seeded with HKC8 cells and
transfected with Wnt6 as described in transfections above. Following 24 hour starvation cells
were washed with PBS twice, scratch wounded laterally using pipette and supplied with 10%
FCS media for two hours to allow for growth. Cells were fixated using 3.7%
paraformaldehyde, washed twice with PBS and permeabilised with Triton. Cells were
blocked with 5% BSA to prevent antibody non-specific binding. Cells were subsequently
stained for GM130 (protein component of Golgi Apparatus) using anti GM-130 and nuclear
associated proteins for 48 hours.

2.6 Effect of DZNep on cell lines treated with TGF!
Effect of DZNep on expression of Fibronectin, Vimentin and E-cadherin
To determine the effect of DZNep on the expression of Fibronectin, Vimentin and E-cadherin
HKC8 cells were seeded onto 6cm plates and allowed to reach 60-70% confluency. Plates
were then serum starved for 24 hours. Subsequently the four conditions were set up control,
TGF! treated (5ng/ml), DZNep (1M), co-treatment with TGF! (5ng/ml) and DZNep (1M).
Cells were allowed to treat for two intervals of 24 hours and 72 hours. Cells were then

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harvested and analysed by western blotting and probed for Fibronectin, Vimentin and E-
cadherin.
Determining the IC50 of DZNep
To determine the IC50 of DZNep in ameliorating TGF! induced regulation of E-cadherin,
Fibronectin and Vimentin. HKC8 cell lines were seeded onto 6cm plates and allowed to reach
confluency for 48 hours. Plates were then starved with 1% FCS media for 24 hours. Plates
were then treated with DZNep concentration range of 0 to 20M. Plates were then allowed to
incubate in presence of treatment for 1 hour and then treated with 5ng/ml of TGF! and were
then allowed to incubate for 48 hours. Finally cells were harvested and analysed by Western
Blotting (as above). Membranes were then probed for Vimentin, Fibronectin and E-cadherin.














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3. RESULTS
3.1 Wnt6 causes cell polarisation in HeLa cell lines








Fig. 1.1 Wnt6 induces cell polarisation. Live images of GFP-actin HeLa cells were taken by
spinning disc confocal microscopy. Cells were grown in 8 well glass slides and transfected at
60-70% confluency for 24 hours and then serum-starved for 24 hours prior to imaging. (A)
Control, (B) Cells transfected with Wnt6 plasmid. Images are representative of three
experiments.








(A) Actin (B) Actin A

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Fig. 1.2 Wnt6 induces formation of prominent paxillin containing focal adhesions. Images of
GFP-actin HeLa cells expressing Paxillin taken by spinning disc confocal microscope. (A)-
(C); control cells, (D)-(F); cells co-transfected with Wnt6 plasmid. Cells were transfected at
60-70% confluency for 24 hours and then serum-starved for a further 24 hours prior to
imaging. Images are representative of three experiments.





(A) Actin
(B) Vimentin
(C) Combined
(D) Actin
(E) Vimentin
(F) Combined

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Fig. 1.3 Wnt6 induces cell polarity in GFP-Actin HeLa cells. Time lapse amages of (A)-(C);
controls transfected with Vimentin and (D)-(F); cells co-transfected with Vimentin and Wnt6.
Cells were transfected at 60-70% confluency for 24 hours and subsequently serum deprived
for 24 hours prior to imaging. Images are representative of three experiments.


(A) Actin
(B) Vimentin
(C) Combined
(D) Actin
(E) Vimentin
(F) Combined

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Fig. 1.4 Wnt6 induces formation of filopodia and membrane ruffling. Time lapse images
ofGFP-actin HeLa cell lines taken by spinning disc confocal microscope. (A)-(P); cells
transfected with Wnt6 plasmid, (Q)-(U); controls. Cells were transfected for 24 hours
followed by 24 hours of serum deprivation. Images are representative of three experiments.





(A) (B) (C) (D)


(E) (F) (G) (H)

d






(I) (J) (K) (L)


(M) (N) (O) (P)


(Q) (R) (S) (T)

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Fig. 1.5 Wnt6 causes Vimentin disassembly during lamellipodium formation. Time lapse
images of GFP-actin HeLa cells taken by spinning disc confocal microscope. (A)-(D); cells
transfected with Wnt6 plasmid expressing Vimentin, (E)-(H) control cells transfected with
Vimentin. Cells were transfected for 24 hours at 60-70% confluency and subsequently serum
deprived for 24 hours prior to imaging. Images are representative of three experiments.







(A)


Actin Vimentin
(B)


(C)



(D)
(E)


Actin Vimentin
(F)


(G)



(H)

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Fig. 1.6 Wnt6 induces cell proliferation in serum starved GFP-actin HeLa cell lines. Time-
lapse images taken by spinning disc confocal microscope. (A)-(C) and (D)-(E) time-lapse
images of cells transfected with Wnt6. Cells were transfected at 60-70% confluency with
Wnt6 plasmid for 24 hours followed by 24 hour serum starvation prior to imaging. Images
are representative of three experiments.








(A)




(B)




(C)




(D)




(E)




(F)

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To investigate the effect of Wnt6 on intermediate filament and focal adhesion dynamics cells
were transiently transfected with Vimentin and Paxillin plasmids coding for photo-switchable
fluorescent protein PSmOrange. In order to determine the effect of Wnt6; Vimentin or
Paxillin plasmids were co-transfected with Wnt6. HeLa cells stably expressing GFP-actin
were employed for this experiment and imaged by confocal disc microscopy. In all
experiments cells were serum deprived to synchronise cell cycle and arrest cell cycle at
G0/G1 phase[75].
One of the first observed changes of cells in response to Wnt6 was the induction of
mesenchymal like cell morphology imitating a leading edge and a retracting edge as analysed
by time-lapse images. Protrusions and extensions of the cell membrane such as ruffling,
lamellipodia and filopodia were suggestive of induced cell polarity as seen in Fig. 1.4. In
2010, Brian T. et al. showed that the disassembly of Vimentin filaments at the leading edge
of cells is crucial for lamellipodia formation. Furthermore he and his colleagues suggested
that Rac1 which is induced by Cdc42 is an important effector in enabling Vimentin
disassembly[76]. Wnt6 transfections suggest Vimentin disassembly in parallel bundles during
the formation of lamellipodia or membrane ruffling (Fig. 1.5). When compared to controls
Vimentin disassembly or formation of lamellipodia and membrane ruffling were absent in
controls. Further analysis also showed that Wnt6 may induce formation of prominent large
focal adhesion complexes as seen in Fig. 1.2 when compared to controls. Fig1.6 suggests an
increase in proliferation of cells transfected with Wnt6 with respect to controls.







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3.3 Wnt6 induces cell polarity in epithelial cell lines









Fig. 2 Wnt6 significantly induces cell polarisation in scratch-wound assays of HKC8 cells.
Immunofluorescent images of (A); empty vectors, (B); cells transfected with Wnt6, (C);
statistical analysis effect of Wnt6 on cell polarisation p<0.05, n=4. HKC8 cells were grown
to confluency and transfected with Wnt6 plasmids for 24 hours followed by 24 hours of
serum deprivation. Cells were then laterally wounded and supplemented with 10% FCS for 2
hours, fixated, permeabilised and subsequently stained for Golgi and Nuclear associated
proteins.





(B) Nuclei
Golgi
(A) Nuclei
Golgi

25 of 40
3.2 Wnt6 increases the expression of Cdc42 and Vimentin in HKC8 cells












Fig. 3 Wnt6 increases Cdc42 and Vimentin expression. Western Blot Analysis of HKC8
treated time dependently with Wnt6 conditioned media. HKC8 cells were implemented with
Wnt6 conditioned media retrieved from Wnt6 transfected cell lines. Subsequently HKC8
cells were treated for periods of 0, 10, 30, 60, 90 and 180 minutes. Cells were then harvested
and protein samples were analysed by Western Blot Analysis. Samples were probed for
Cdc42 and Vimentin. Images are representative of three experiments.






Active Cdc42
Vimentin
B-actin
1
8
0

m
i
n

9
0

m
i
n

6
0

m
i
n

3
0

m
i
n

1
0

m
i
n

0

m
i
n


26 of 40
HKC8 cells were transfected with Wnt6 and a scratch-wound assay was employed to quantify
cell polarization seen in HeLa cells(Fig. 2). HKC8 cells were then fixed and stained for
Nuclear and Golgi associated proteins. Cells were then imaged by fluorescent microscopy
and the orientation of the Golgi with respect to Nucleus was used as a determination of cell
polarity. Analysis of data showed a significant increase in polarisation of cells transfected
with Wnt6 p<0.05, n=4. The data suggests that Wnt6 causes significant polarisation in HKC8
epithelial cells.
As discussed in the introduction above Cdc42 is an important effector in inducing cell
polarity and formation of lamellipodia and filopodia. To determine the status of Cdc42 in
relation to Wnt6, HKC8 cells were treated with Wnt6 conditioned media. Results as in Fig. 3
suggest that Wnt6 increases and sustains the expression of active Cdc42.
















27 of 40
3.4 Wnt6 does not phosphorylate Dvl2








Fig. 4 Wnt6 does not phosphorylate Dvl2. Western Blot Analysis of HKC8 cell lines. HKC8
cells were grown to 60-70% confluency and then transfected for 24 hours with respective
conditions, NT; non-transfected, Dvl2; transfected with Dvl2 plasmid, Wnt6; transfected with
Wnt6 plasmid and Dvl2+Wnt6; co-transfected with Dvl2 and Wnt6 plasmid. Following
transfection cells were serum deprived for 24 hours and harvested for analysis. Samples were
analysed for successful transfection of Wnt6 and Dvl2 in primary samples. Samples were
then immunoprecipitated for Dvl2 and probed for phosphotyrosine to detect phosphorylation
status of Dvl2 in response to Wnt6 co-transfection. Analysis shows no change in
phosphorylation of Dvl2 in response to Wnt. Images are representative of three experiments.

To determine the phosphorylation status of Dvl2 in response to Wnt6; HKC8 cells were
transfected with Dvl2, Wnt6 or co-transfected with Dvl2 and Wnt6. Results as in Fig. 4 show
no change in the phosphorylation status of Dvl2 in response to co-transfection with Wnt6.
This result indicates that Wnt6 may not effect Dvl2 phosphorylation.





Phosphotyrosine
Dvl2 IP (Anti-FLAG)
Wnt6 (Anti-V5)
Dvl2 (Anti-FLAG)
B-actin
D
v
l
2

+

W
n
t
6

W
n
t
6

D
v
l
2

N
T


28 of 40
3.5 Analysis of EZH2 and TGF! overlapping genes





Fig. 5.1 The first 194 of genes regulated by PcG were analysed and cross referenced to
expression analysis of genes regulated by TGF!. 41% of genes were shown to be regulated
by both TGF! and PcG. 39% genes regulated by both were shown to be regulated to some
extent by EZH2 component of PRC2 and TGF!1. 20% genes upregulated by TGF!1 are
counter suppressed by EZH2 to an extent. 0.2% genes that are down regulated by TGF!1 are
counter upregulated by EZH2. This data indicates TGF!1 and PcG act as positive and
negative regulators of half of key developmental genes regulated by Polycomb groups.




41%
39%
20%
0%
Genes regulated by TGF! and PcG
Genes regulated y TGF! and EZH2
Genes upregulated by TGF!1 and suppressed by EZH2
Genes downregulated by TGF!1 and upregulated by EZH2

29 of 40


One of the core components of PRC2 is the methyltransferase EZH2 and has been shown to
have a huge role in development. Bracken et al. generated genome-wide expression analysis
of PcG knock-outs in embryonic fibroblasts to determine genes regulated by EZH2, EED,
SUZ1/2 and Bmi-1[77]. We cross referenced the top 194 genes regulated by PcG with
expression analysis of genes regulated by TGF!1, to determine the extent of genes
suppressed by PcG and genes upregulated by TGF!1 (Fig. 5.1). Our analysis indicated that
41% of genes regulated by PcG may be regulated by TGF!. The data indicated that most of
the population of genes regulated by PcG and TGF!1 are more specifically regulated to some
extent by EZH2 (39%). Some 20% of the genes are upregulated by TGF!1 and suppressed by
EZH2 to an extent and only 0.2% of the genes are down regulated by TGF!1 and upregulated
by EZH2.












30 of 40
3.6 DZNep reverses the effects of TGF!

24hours 72hours






















T
G
F

+
D
Z
N
e
p

D
Z
N
e
p

T
G
F


C
o
n
t
r
o
l

T
G
F


+
D
Z
N
e
p

D
Z
N
e
p

T
G
F


C
o
n
t
r
o
l

E-cadherin


Fibronectin

Vimentin

!-actin
Fig 5.2Western Blot Analysis on the effect of DZNep reversing TGF! induced
phenotypic changes over 24 hour and 72 hour treatment periods. HKC8 cells were
serum deprived at 60-70% confluency for 24 hours and treated respectively in the
presence of TGF! and/or DZNep over 24 hour and 72 hour periods. (A); Analysis of E-
cadherin expression, (B); analysis of Fibronectin expression, (C); analysis of Vimentin
expression.

31 of 40
DZNep is a global histone methyltransferase inhibitor. Recently it had been shown that TGF!
induced Snail1 expression promotes recruitment of PRC2 to E-cadherin gene promoter
whereby it enhances the the repression of E-cadherin. PRC1 over expression has been shown
to increase Vimentin and Fibronectin expression through an unknown mechanism as
discussed in introduction above. To determine the effect of DZNep on reversing TGF!
induced phenotypic changes such as Vimentin, Fibronectin up regulation and E-cadherin
down regulation. Western Blot Analysis of HKC8 cells treated with DZNep in presence of
TGF! indicate that DZNep causes an up regulation of E-cadherin and reverses TGF!1
effects. However, data suggests that DZNep does not reverse TGF! induced Fibronectin
expression over 24 hour or 72 hour periods but does down regulate expression in absence of
TGF!1. This suggests that a higher dose of DZNep may be required to reverse TGF! induced
Fibronectin expression. Western Blot Analysis on the expression of Vimentin indicates
DZNep can down regulate Vimentin expression in presence and in absence of TGF!.















32 of 40
3.7 IC50 of DZNEP











E-cadherin
Fibronectin
Vimentin
!-actin
2
0

M

1
0

M

5

M

2
.
5

M

1

M

0
.
7
5

M

0
.
5

M

0

M


Fig. 5.2 Western Blot Analysis of HKC8
cell lines treated with DZNep 0-20M. Cells
were grown to 60-70% confluency and
subsequently serum deprived for 24 hours in
0.1% FCS. Cells were then treated with
respective DZNep concentration in the
presence of 2ng/ml TGF! for 48 hours. (A);
Dose-response curve of E-cadherin
expression indicates IC50 is between 1.5-
3uM, (B); Dose-response curve of
Fibronectin expression indicates IC50 is
between 0.5-4.5M, p<0.05, (C); Dose-
response analysis indicates DZNep has a bell
shaped dose-response curve. This data is
representative of two experiments.

33 of 40
To determine the IC50 of DZNep in response to TGF! on HKC8 cell lines; cells were treated
in 0-20M concentration of drug in presence of TGF! for 48 hours(Fig. 5.2). Although 72
hour treatment was preferred, multiple attempts resulted in cell death due to serum
deprivation which resulted in loss of samples. Therefore, cells were grown in 0.1% FCS and
treated for 48 hours only. Western Blot Analysis shows the up regulation of E-cadherin and
down regulation of Fibronectin. Log Dose-Response curve put the IC50 of DZNep at
approximately 2M for E-cadherin expression and 4.5M for down-regulation of
Fibronectin. Statistical analysis suggests IC50 is between 0.5 and 4.5M for Fibronectin and
1.5-3M for E-cadherin, p<0.05. No conclusive data based on the Western Blot Analysis
showed data that could be used to interpret an IC50 for Vimentin. Since effects of DZNep for
Vimentin are best documented post 24 hours treatment, it indicates that the effect on
Vimentin is largely lost post 24 hours of treatment. We further analysed dose-response
relationship of DZNep with Fibronectin and E-cadherin and results showed that DZNep has a
bell-shaped dose response curve and causes a preferential loss of Fibronectin and gain of E-
cadherin at IC50 values.








34 of 40
4. DISCUSSION
Cell migration is an important aspect of normal cell development, embryogenesis, wound
healing and tissue formation. This involves reading of spatial cues in terms of extracellular
microenvironment that initiates a cascade of signalling events resulting in the recruitment of
migration machinery of the cell towards the cue. During embryogenesis two patterns are seen
in nephrogenesis one of which is ductal branching of ureteric-bud derived collecting tubules.
Both systems, undergo a repeat series of inductive signalling that organises the tissue
architecture and differentiated cell functions by employment of gene programs
[78]
. Wnt
signalling has been widely implicated in nephrogenesis, osteoblastogenesis periodontal
development. Furthermore as discussed in introduction Wnt6 has been implicated during
embryonic nephrogenesis at our lab. Our results suggest that Wnt6 has a role in cell
migration and polarity as confirmed by the analysis of Cdc42 expression and cell polarisation
assays. This effect coupled with other studies that suggest Wnt6 has a role in tubulogenesis
and its increased expression during embryonic nephrogenesis, further implicates importance
of Wnt6 in renal development. Intriguingly, loss of Wnt6 expression is evident in both
patients with diabetic nephropathy and in numerous animal models of renal fibrosis
suggesting that its continued expression in adult tissues is vital for the maintenance of
epithelialisation in renal tissues. . Furthermore treatment of cells with Wnt6 causes an
increase in expression of Vimentin an important structural component of migrating and
polarising cells.
Imuncytochemistry of Paxillin dynamics in cells overexpressing Wnt6 identified an
interesting phenomenon of increased focal adhesion size. Previous studies have suggested
that Wnt ligands can modulate focal dynamics by regulating the phosphorylation of Dvl2.
Our results found no change in the phosphorylation of Dvl2 in response to Wnt6. This
finding suggest that Wnt6 may act via a different pathway to induce Cdc42 expression

35 of 40
leading to cell polarisation. Further research is required as some problems arose since the
transfection with a single plasmid and co-transfection with two plasmids results in a
unbalanced expression of target protein in cells as seen in Fig. 4, i.e. the expression of Dvl2
alone in response to transfection is far more effective than co-transfection with Dvl2 and
Wnt6. This imbalance may effect the results seen on the phosphorylation of Dvl2 due to the
expression dynamics. Schlessinger and colleagues showed that Wnt non canonical pathways
and Cdc42 co-operate to induce cell polarity through recruitment of Dvl and Axin. It is
possible that if transfection protocols are better optimised, analysis may show that Wnt6
induces phosphorylation of Dvl2.
TGF! is recognized as the key profibrotic mediator in the pathogenesis of DN. Some of
TGF!s induced effects on Fibronectin, Vimentin and E-cadherin are thought to be caused by
recruitment of PcG such as EZH2 and Bmi-1, components of PRC2 and PRC1 respectively.
We determined the extent to which TGF! signalling and PcG co-operate or overlap in the
maintenance of E-cadherin, Fibronectin and Vimentin expression. We compared genome-
wide expression analysis data of TGF! and PcG regulated genes. As shown in Fig 5.1; 41%
of the genes which are regulated by TGF! and PcG are more specifically regulated by EZH2
to some extent. Furthermore TGF! is responsible for increasing the expression and EZH2
down regulating expression of half of these genes. Only 0.2% of these genes are up regulated
by EZH2. These results suggested that PcG components and TGF! signalling co-operate to
regulate the expression of key developmental genes. Furthermore, preliminary data from our
group has shown that phosphorylated Smad-3 can bind directly to EZH2 during epithelial
dedifferentiation, linking PcG and TGF! signaling directly for the first time.
From further studies that suggested EZH2 and Bmi-1 recruitment were important in
regulating TGF! induced effects. We hypothesized that by down regulating expression of
key components of PRC1 and PRC2 using DZNep it may be possible to reverse TGF!

36 of 40
induced phenotypic changes. Results suggested that DZNep effectively reverses TGF!
induced changes. Results in Fig. 5.2 suggested that DZNep opposes the effects of TGF! on
E-cadherin and Vimentin and Fibronectin. The observation that Wnt6 can oppose TGF!
induced Vimentin expression, further suggests that this nexus may play a critical role in
establishing and maintaining epithelial cell fate.
In cohorts, data from our group suggests that Wnt6 is an important key mediator in
maintenance and development of kidneys. Our analysis of TGF! and PcG effects on
expression of genes suggests that the two pathways converge to regulate expression of genes.
Furthermore our data suggests that regulation of Vimentin, Fibronectin and E-cadherin may
be a part of this modulatory pathway but the mechanism by which Vimentin is regulated is
largely unknown. The upregulation of Vimentin by Wnt6, TGF!, the reversal of TGF!
induced Vimentin by targeting PcG and the reversal of TGF! induced Vimentin by Wnt6
suggests that there may be a common modulatory process by PcG. Preliminary findings that
suggest Smad-3 interacts with EZH2 further implicates PcG in the regulation of TGF!
targets. Thus, targeting PcG offers a valuable therapeutic target in the treatment of DN by
reversing profibrotic events such as loss of epithelial integrity (E-cadherin), increase in ECM
(Fibronectin) and mesenchymal characterisation (Vimentin) that are thought to be essential in
the progression of the disease.





37 of 40
5. ACKNOWLEDGMENTS
I would like to thank Dr. John Crean for giving me the opportunity to be a part of his research
at Diabetes Complications Research Centre and helping me with laboratory techniques on an
ongoing basis. Working with his research has tremendously broadened my knowledge in
many practical and theoretical aspects of research and discovery and it has proven valuable.
Also would like to thank Andrew Gaffney for teaching me practical techniques such as Cell
Culture, Western Blotting, transfections and imaging and has helped me to master them and
understand theory behind them. Overall researching along with the DCRC has made me
understand the complexity of cell biology and therapeutics research.















38 of 40

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