Roles of Actin Remodeling Proteins, Gelsolin and Flightless-I in Epidermal Wound Healing

ROLES OF ACTIN REMODELING PROTEINS, GELSOLIN AND FLIGHTLESS-I IN EPIDERMAL WOUND HEALING

Huater Chan

Bachelor of Biotechnology (Honours)

Women's and Children's Health Research Institute

And Discipline of Physiology

School of Molecular and Biomedical Science

Faculty of Sciences

University of Adelaide

A Thesis submitted for the degree of

Doctor of Philosophy

April 2011 TABLE OF CONTENTS

DECLARATION ...... I

ACKNOWLEDGEMENTS ...... II

ABBREVIATIONS ...... IV

CHAPTER ONE General Introduction

1.1 BACKGROUND...... 1

1.2 WOUND HEALING ...... 2

1.2.1 The process of wound healing ...... 2 1.2.1.1 Inflammatory phase ...... 6 1.2.1.2 Proliferative phase ...... 7 1.2.1.3 Remodeling phase ...... 10

1.3 THE CYTOSKELETON ...... 11

1.3.1 Types of Cytoskeleton ...... 11 1.3.1.1 Microtubules ...... 13 1.3.1.2 Intermediate filaments ...... 14 1.3.1.3 Microfilaments (Actin) ...... 14 1.3.1.4 Stress fibers ...... 15 1.3.2 Actin dynamics in wound healing ...... 16

1.4 GELSOLIN FAMILY OF ACTIN REMODELING PROTEINS ...... 20

1.4.1 Gelsolin ...... 22 1.4.1.1 Gelsolin in Clinical Settings ...... 23 1.4.1.2 Involvement of Gelsolin in Cellular Apoptosis ...... 24 1.4.1.3 Biological Functions of Gelsolin ...... 25 1.4.1.4 Gelsolin and the sex steroid hormones ...... 26 1.4.1.5 Nuclear hormone receptor signaling ...... 28

1.4.2 Flightless-I ...... 29 1.4.2.1 Biological Functions of Flii ...... 30 1.4.2.2 Protein Interactions are mediated through the Flii LRR domain ...... 32 1.4.2.3 Involvement of Flii in cellular processes ...... 33 1.4.2.4 In Vitro and In Vivo Expression of Flii ...... 35 1.4.2.5 Flii protein is a Negative Regulator of Wound Healing ...... 36

1.5 TRANSFORMING GROWTH FACTOR BETA (TGFΒ) ...... 38

1.5.1 TGFβ superfamily ...... 38 1.5.1.1 TGFβ Activation and Structure ...... 40 1.5.1.2 TGFβ Receptor Signaling ...... 44 1.5.1.3 Smad Effector Signaling ...... 45 1.5.1.4 TGFβs regulation in wound healing ...... 48

1.6 MITOGEN-ACTIVATED PROTEIN KINASE ...... 50

1.6.1 The MAPK Signaling Pathways ...... 50 1.6.1.1 ERK Pathway ...... 53 1.6.1.2 p38Pathway ...... 54 1.6.1.3 JNK/SAPK Pathway ...... 54 1.6.1.4 Phosphoinositide 3-kinase Signaling pathway ...... 54

1.7 INTEGRATION OF MAPK AND PI3K/AKT WITH TGFΒ SIGNALING PATHWAYS ...... 56

1.8 RESEARCH AIM AND RATIONALE ...... 57

CHAPTER TWO Role of Gelsolin in Androgen Mediated Wound Healing

2.1 INTRODUCTION ...... 60

2.1.1 Implications for the gelsolin superfamily in nuclear receptor signaling ...... 60 2.1.2 Gelsolin and AR Nuclear Signaling ...... 61

2.2 MATERIALS AND METHODS ...... 63

2.2.1 Cells, Cell Culture ...... 63

2.2.2 Scratch Assay ...... 63 2.2.3 Protein Extraction ...... 64 2.2.4 Western Blotting ...... 65 2.2.5 Immunocytochemistry ...... 66 2.2.6 siRNA knockdown Assay ...... 68 2.2.7 RNA Extraction ...... 69 2.2.8 DNase Treatment and RNA Quantitation ...... 70 2.2.9 Complementary Deoxyribonucleic Acid (cDNA) Synthesis ...... 70 2.2.10 Real-Time quantitative-Polymerase Chain Reaction (RTq-PCR) ...... 71 2.2.11 Proliferation Assay ...... 71 2.2.12 Wound Closure Assay ...... 72 2.2.13 Statistical Analysis ...... 73

2.3 RESULTS ...... 74

2.3.1 Gelsolin and AR responses to wounding in vitro ...... 74 2.3.2 Effects of DHT addition in gelsolin and AR localization ...... 80 2.3.3 Time-course localization study of gelsolin and AR in the presence of DHT...... 83 2.3.4 Gelsolin gene knockdown using silent RNA technology ...... 91 2.3.5 Response to DHT treatment in gelsolin-ablated HFFs ...... 93

2.4 DISCUSSION ...... 98

CHAPTER THREE Flii Deficiency Improves Wound Healing and is Associated with

Higher Expression Ratio of TGFβ3 to TGFβ1

3.1 INTRODUCTION ...... 103

3.1.1 Flii, a negative regulator of wound healing ...... 103 3.1.2 TGFβ and the wound healing process ...... 104

3.2 MATERIALS AND METHODS ...... 106

3.2.1 Flii Heterozygous and Over-expressing Mice Generation ...... 106 3.2.2 Flii+/- mice generation ...... 106 3.2.3 FliiTg/+ mice generation ...... 109

3.2.4 Murine Incisional Wound Surgery ...... 111 3.2.5 Histological Processing ...... 112 3.2.6 Immunohistochemistry ...... 113 3.2.7 Cell culture ...... 115 3.2.8 siRNA knockdown ...... 116 3.2.9 RNA extraction ...... 116 3.2.10 DNase Treatment and RNA Quantitation ...... 117 3.2.11 Complementary Deoxyribonucleic Acid (cDNA) Synthesis ...... 118 3.2.12 Real-Time quantitative-Polymerase Chain Reaction (RTq-PCR) ...... 119 3.2.13 Statistical Analysis ...... 119

3.3 RESULTS ...... 121

3.3.1 Time-course analyses of TGFβ1 expression in wounded mice skin ...... 121 3.3.2 Time-course analyses of TGFβ2 expression in wounded mice skin ...... 124 3.3.3 Time-course analyses of TGFβ3 expression in wounded mice skin ...... 129 3.3.4 Time-course analyses of TβRI and TβRII in wounded mice skin ...... 134 3.3.5 Flii knockdown alters TGFβ gene expression...... 139

3.4 DISCUSSION ...... 142

CHAPTER FOUR Flii Modulation of TGFβ Expression is Achieved by Direct Association with

Proteins Involved in the Expression and Activity of TGFβ

4.1 INTRODUCTION ...... 147

4.1.1 Binding properties of Flii protein ...... 147 4.1.2 Properties of the LRR motif ...... 148 4.1.3 The relationship between Flii and TGFβ ...... 149

4.2 MATERIALS AND METHODS ...... 151

4.2.1 Cells, Cell Culture ...... 151 4.2.2 Antibodies ...... 151

4.2.3 Immunocytochemistry ...... 152 4.2.4 Nuclear and cytoplasmic fractionation ...... 153 4.2.5 Immunoprecipitation ...... 154 4.2.6 Western Blotting ...... 155

4.3 RESULTS ...... 157

4.3.1 Flii associates with c-Fos and c-Jun ...... 157 4.3.2 Interactions of Flii with TGFβs ...... 160 4.3.3 Flii interacts with nuclear Akt in wounded cells ...... 169 4.3.4 Flii associates with Smad proteins ...... 172

4.4 DISCUSSION ...... 180

CHAPTER FIVE Regulation of TGFβ by Flii Association with the MAPK Signaling Pathway

5.1 INTRODUCTION ...... 187

5.1.1 Interaction of Flii with Ras GTPases ...... 187

5.2 MATERIALS AND METHODS ...... 189

5.2.1 siRNA knockdown ...... 189 5.2.2 RNA extraction ...... 189 5.2.3 DNase Treatment and RNA Quantitation ...... 190 5.2.4 Complementary Deoxyribonucleic Acid (cDNA) Synthesis ...... 191 5.2.5 Real-Time quantitative-Polymerase Chain Reaction (RTq-PCR) ...... 191 5.2.6 Primary Fibroblast Extraction and Culture ...... 192 5.2.7 Immunocytochemistry ...... 194 5.2.8 Collagen Secretion Assay ...... 195 5.2.9 Proliferation Assay ...... 195 5.2.10 Fibroblast Outgrowth Assay ...... 196 5.2.11 Statistical Analysis ...... 197

5.3 RESULTS ...... 198

5.3.1 Flii gene knockdown affects expression of AP-1 and Smad proteins ...... 198

5.3.2 TGFβ1 increases collagen secretion in WT, Flii+/- and FliiTg/+ primary fibroblasts ...... 201 5.3.3 TGFβ1 decreases FliiTg/+ fibroblast proliferation ...... 204 5.3.4 Proliferation of primary fibroblasts treated with MAPK inhibitors ...... 207 5.3.5 TGFβ1 decreases migration in Flii+/- fibroblasts ...... 209 5.3.6 MAPK inhibitors decrease fibroblasts outgrowth ...... 214 5.3.7 Comparison of WT, Flii+/- and FliiTg/+ outgrowths treated with MAPK inhibitors ...... 223

5.4 DISCUSSION ...... 229

CHAPTER SIX General Discussion

6.1 DISCUSSION ...... 238

6.1.1 Gelsolin and the AR ...... 238 6.1.2 Flii and TGFβs ...... 240 6.1.3 Future Directions ...... 244

BLIBLIOGRAPHY ...... 238

DECLARATION

I declare that this thesis does not contain without acknowledgement any work submitted previously for any academic award and that to the best of my knowledge and belief it does not contain any material previously published or written by another person except where otherwise acknowledged.

Huater Chan

May 2010

ACKNOWLEDGEMENTS

I would like to thank my supervisors, Associate Professor Allison Cowin and Associate

Professor Barry Powell for their invaluable advice and support. I deeply appreciate

their patience, encouragement and support over the years. James Waters, Damien

Adams, Xanthe Strudwick, Zlatko Kopecki, Nadira Ruzehaji and Tony Lin of Wound

Healing Laboratory deserve a big thank you for all the assistance and support they have

provided. Special thanks to our collaborators at the Australian National University at

Canberra, Hugh Campbell, Ruth Arkell, Nicole Thomsen and Jane Hooper-Jones for their support and generosity in providing our laboratory and my project with transgenic mice.

I also would like to thank all the staff and students whom I have the honour to meet at the Women's and Child's Health Research Institute. I am particularly grateful to the past and present students in the PhD room for all their support and friendship throughout the years. I am especially grateful to all the intelligent conversations I had over the years. All the mutual support we gave each other will be solely missed.

Lastly, I would like to thank my friends and family for all their support. Thank you to all my friends for your encouragement and patience over the years. Here I would like to express my deepest appreciation and the biggest thank you to my parents, Hing Chan and Boon Hwa Tan, sister, Yee Send Chan and brother Huasheng Chan. I am eternally grateful and honored to be a part of this wonderful family. I wouldn't be what I am today and could not have completed my studies without them. A special thank you to

Yoke Kim Lee who has accompanied me through the years. I am thankful for her care and concern over the years.

iii

ABBREVIATIONS

A Absorbance

α Alpha

ABP Actin binding protein

APS Ammonium persulfate

AR Androgen receptor

β-tubulin Beta-tubulin bp Base pair

β Beta

BCA Bicinchoninic acid

BSA Bovine serum albumin

°C Degrees celcius

Ca2+ Calcium

CaCl2 Calcium chloride

CaMK-II Calcium/calmodulin-dependent protein kinase type II

cDNA Complementary deoxyribonucleic acid

CISK Cytokine-independent survival kinase

CO2 Carbon dioxide

Cy3 Cyanine 3 dATP Deoxyadenocine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythyamine triphosphate

D0 Day 0 post-wounding

D3 Day 3 post-wounding

D7 Day 7 post-wounding

D14 Day 14 post-wounding

D21 Day 21 post-wounding

DEPC Diethylpyrocarbonate

DHT Dihydrotestosterone

DMEM Dulbecco’s modified Eagle’s media

DNA Deoxyribonucleic acid

ECL Enhanced chemical luminescence

ECM Extracellular matrix

EDTA Ethyldiaminetetraacetic acid

EGF Epidermal growth factor

ELISA Enzyme linked immunosorbent assay

FCS Fetal calf serum

FITC Fluorescein isothiocyanate

FGF Fibroblast growth factor

FLAP Flightless-I associated protein

Flii Flightless-I protein

FliiTg/+ Flightless-I transgenic

Flii+/- Flightless-I heterozygous knockout

x g Times the force of gravity

g Grams

GTP Guanosine triphosphate

H&E Hematoxylin and eosin

HFFs Human foreskin fibroblasts hr Hour

HRP Horse radish peroxidase

H2O2 Hydrogen peroxide

IgG Immunoglobulin-G

IL Interleukin

KCl Potassium chloride

kDa Kilo Daltons

KD Knock down

L Litre

LAP Latency associated peptide

λex Lambda (wavelength) of excitation

vii

λem Lambda (wavelength) of emmitance

LRR Leucine-rich repeats

LTBP latent TGFβ binding protein

M Molar

MAPK Mitogen-activated protein kinase

MgCl2 Magnesium chloride

min Minutes

mM Millimolar

MMP Matrix metalloproteinases

mm2 Millimeter square mRNA Messenger ribonucleic acid

NaCl Sodium chloride

NHS Normal horse serum

NLS Nuclear localization signal nM Nano molar

viii

PBS Phosphate buffered saline

PDGF Platelet derived growth factor

PIP2 Phosphatidylinositol 4,5-bisphosphate

PI3K Phosphoinositide 3-kinase

RNA Ribonucleic acid

Rpm Rounds per minute

RTq-PCR Real time quantitative polymerase chain reaction

SDS Sodium dodecylsulphate

SDS-PAGE Sodium dodecylsulphate polyacrylamde gel electrophoresis

SEM Standard error of mean

sec Seconds

siRNA Short interfering ribonucleic acid

TAE Tri(hydroxymethyl)methylamine-acetate-ethylediaminetetraacetic

acid

TβRI Transforming growth factor beta receptor one

TβRII Transforming growth factor beta receptor two

TEMED N,N,N,N-tetramethylethylenediamine

TGFβ1 Transforming growth factor beta one

TGFβ2 Transforming growth factor beta two

TGFβ3 Transforming growth factor beta three

TNF Tumour necrosis factor

Tris Tri(hydroxymethyl)methylamine

TRS Target retrieval solution

µg Microgram

µl Microlitre

µm Micrometer

µM Micromolar

VEGF Vascular endothelial growth factor

WT Wild-type

WST-1 2-(4-iodophenyl)-3-(4-nitropheyl)-5-(2,4-disulfophenyl)-2H-

tetrazolium

x Times

x g Times the force of gravity

% Percent

= Equals

+ Plus

CHAPTER ONE

General Introduction

1.1 Background

Scar contractures arising from abnormal wound healing continue to pose a significant worldwide clinical problem. Elective operations and operations due to trauma in the developed world account for more than 100 million patients acquiring scars each year, which can lead to significant health problems. In addition, burn injury is the most common household injury and results in more than 5 million patients worldwide acquiring burn scars each year, 70% of which occur in children. The problems are not just limited to physical and psychological trauma of the individual but also extend outwards and place a significant burden on families and communities for treatments.

Scarring results in loss of movements, restricted growth and deformity and is particularly important for children whose development often requires regular surgical correction. The long term economic impact involves loss of wages, constant health care cost and loss of skills as a result from scarring deformities. Unfortunately, current wound healing treatments such as garments, dressings or steroidal injections are sub- optimal. As a result, more research into cell based therapies or pharmaceutical drugs to improve wound healing must be conducted.

1.2 WOUND HEALING

1.2.1 The process of wound healing

Wound healing is a restoration process in which the skin repairs itself after injury. In normal skin, the epidermis and dermis act as a protective barrier against the external environment and in the event that this barrier is breached, the systematic physiological process begins to re-establish the integrity of the protective barrier. Molecularly, cutaneous wound healing is a highly complex process involving biochemical reactions and interactions amongst cells and cytokines leading to the restoration, integrity and function of the skin. Cellular migration is one of the most important aspects of wound healing to restore the protective barrier. This process involves the actin cytoskeletal proteins which are regulated by the gelsolin family of actin binding proteins such as gelsolin and Flightless-I, which will be discussed later in this chapter.

The coordination of multiple cellular processes is a very delicate process, which ultimately determines the resultant scar formation from an unnoticeable hairline graze to keloid or hypertrophic scar contractures (Figure 1.1) at the opposite end of the spectrum. The process of wound healing can be divided into 3 distinct but interrelated phases (Figure 1.2), inflammatory phase, proliferative phase and remodeling phase 1.

A C

B D

Figure 1.1

Typical outcomes of abnormal wound healing processes in which tissue repair is non- optimal. (A) Hypertrophic scars are usually raised and limited to the margins of the original wound. (B) Keloid scars infiltrate and invade the surrounding normal tissue area. Histological sections of hypertrophic (C) and keloid (D) scars. Type III collagen bundles are flatter but are predominantly parallel to the epithelial surface in hypertrophic scars. α-SMA (alpha - Smooth Muscle Actin) positive myofibroblasts are

present in hypertrophic scars. However, early collagen fibers in keloid scars are

arranged haphazardly to the epithelial surface possibly due to the absence of α-SMA

positive myofibroblasts.

Adapted from Santucci et al, 2001& Wolfram et al, 2009 6,7

NOTE: This figure is included on page 4 of the print copy of the thesis held in the University of Adelaide Library.

Adapted from Gurtner et al, 2008 8

Figure 1.2

Classic stages of the wound healing process. There are three phases of adult wound healing; inflammation (A), proliferation (B) and remodeling (C). The inflammatory phase, hemostasis, is restored by the formation of fibrin clots and, during this time, potent cytokines and chemokines are released to attract inflammatory cells into the wound site to prevent infection. The proliferative phase occurs about 2 – 10 days post- injury. It begins when fibroblasts migrate into the wound space to proliferate and differentiate into collagen producing myofibroblasts. The production of new ECM

(Extra Cellular Matrix) proteins allows angiogenesis and the subsequent migration of epithelial cells from nearby unwounded tissue into the wound site to re-epithelialize.

The remodeling phase involves the re-organization of the tissue matrix and lasts approximately for a year post-wounding. The contracted and re-epithelialized wound is slightly raised and damaged skin appendages are not restored. Dermal collagens laid down by fibroblasts are disorganized and the collagens begin to mature and are re- organized into thicker bundles that inevitably cause the formation of scar tissue.

1.2.1.1 Inflammatory phase

Wound healing is initiated immediately in response to injury and the first action taken

is the restoration of hemostasis, characterized by the formation of a clot to prevent

further blood loss, and the onset of inflammation. The formation of a clot not only

provides a first barrier against infection but also forms part of the provisional matrix

which is required for incoming inflammatory cells into the wound site. Immediately

upon injury, fibrinogen is first cleaved by thrombin to produce fibrin. Fibrin monomers

then cross-link with one another to form a scaffold which in turn bind directly to

platelets to form a clot 2. The formation of fibrin is integral in the early phase of wound

healing as it binds to different cell types such as monocytes, neutrophils and fibroblasts

via cell surface integrin receptors. Fibrin can also interact with several growth factors

and cytokines such as fibroblast growth factors (FGF) and insulin-like growth factors

(IGF), which affects cellular migration and proliferation, as well as extracellular matrix

(ECM) production 3. Concurrently with the formation of a clot, platelets are also

activated by thrombin upon injury to release granules which contain a range of growth

factors vital in wound healing such as platelet-derived growth factors (PDGF), transforming growth factors (TGF) and epidermal growth factors (EGF) 4.

In addition, platelets also stimulate vasodilation and increase permeability to allow inflammatory cells to enter the wound site 5. This process is regulated by numerous

cytokines and growth factors such as mast cells that produce histamines and

complement factors that assist in vasodilation and act as chemoattractants 9. As a result, vasodilation and increased capillary permeability cause a cascade of inflammatory cell influx and differentiation to supplement the release of more cytokines and growth factors at the wound site 5. Neutrophils are the first population of inflammatory cells that enter the wound site to clear up cell debris and invading bacteria 10. Neutrophils

also produce several proinflammatory cytokines such as interleukins (IL-1α, IL-1β, IL-6) and tumour necrosis factor – α (TNFα) 11. These factors attract circulating monocytes which migrate into the wound site where they are activated and subsequently differentiate into macrophages, where the process of phagocytosis continues in conjunction with the production of chemoattractants, fibronectin, elastin, complement factors and TGFβ 12. These factors together recruit more macrophages and fibroblasts to

the wound site. One of the most important cytokines released by the macrophages is

TGFβ which stimulates dermal fibroblasts to differentiate into myofibroblasts. The myofibroblasts then produce collagen which forms part of the provisional extracellular matrix 3. The migration of fibroblasts into the wound space marks the beginning of the

proliferative phase in wound healing.

1.2.1.2 Proliferative phase

Fibroblasts are important mesenchymal cells which play a dual role in the wound

healing process 10. Firstly, fibroblasts produce collagen based ECM that eventually replaces the fibrin matrix, therefore regulating both growth and function of other cell types. Secondly, differentiated fibroblasts are also involved in wound contraction. The migration of fibroblasts into the wound site occurs in a well coordinated manner by navigation along the provisional matrix fibers and not in a haphazard manner 13. It is

therefore important to note that the arrangement of ECM in wound healing is crucial, as

many complications such as scarring arise out of disorganized ECM arrangement. The

interactions between the fibroblasts and the ECM are mediated by cell surface integrins

that bind to several matrix components such as fibrin and fibronectin 14. IL-1 and TNF-α

production by macrophages also induces the production of matrix metalloproteinases

(MMP) that clear away damaged ECM by removing the inflammatory debris, which

then allows the migration of proliferative cells into the wound site 15. Initiation of

permanent ECM production begins at the arrival of fibroblasts into the wound space.

The new ECM is comprised of mainly collagen which is predominantly synthesized in

fibroblasts as procollagen and exported out of the cell 16. In humans, collagen

production typically starts at day two post-injury and peaks between day five and

seven. The production of collagen matrix and the continuation of fibroblast migration

results in the development of granulation tissue which provides a template for tissue

growth and formation of myofibroblastic cells 17. A range of growth factors and

cytokines including PDGF, EGF, FGF TNF-α, interferon-γ and most importantly TGFβ

are involved in the differentiation of fibroblasts into myofibrobasts which are major

contributors in extracellular matrix deposition. These specialized fibroblasts acquire

properties characteristic of smooth muscle cells and have the ability to generate

mechanical forces which result in lamellipodia formation and subsequent wound

contraction.

The formation of ECM is the first criteria that must be fulfilled before angiogenesis can

occur, as the ECM provides structural support in addition to a repository of important

growth factors for invading capillaries 18. Angiogenesis is the process where new blood

vessels are formed from neighboring intact capillaries. The movement of new capillaries

into the wound site is similar to the migration of fibroblasts that uses the established

ECM as guidance. Multiple growth factors are involved in this entire process including

VEGF, FGF, angiopoietin and TGFβ.

Re-epithelialization involves the migration of epithelial cells from the wound edge using components from the ECM such as collagen and fibronectin as structural foundations which ultimately results in re-establishment of intact epidermis over the newly formed granulation tissue. At the leading edge of the migrating tip are the keratinocytes. As basal keratinocytes migrate over the granulation tissue, they leave a

stratified layer of proliferating keratinocytes and the process continues until

keratinocytes from opposite ends come in contact 1. Rapid re-epithelialization is desirable as it leads to the restoration of the skin’s function as a barrier to defend against micro-organisms, protect from trauma and prevent water loss.

1.2.1.3 Remodeling phase

The remodeling phase involves re-organization of the tissue matrix by means of removal of fibronectin and hyaluronic acid and the replacement of a stronger and more

organized ECM framework composed of collagens 19. Degradation of the initial matrix

and regulation of the new collagen matrix involves the family of matrix

metalloproteinases (MMP), which require controlled expression of exact combinations

of MMPs regulated in part by PDGF, IL-1 and TGFβ 20. Regulation of collagen requires a

balance between collagen production, collagen breakdown and collagen remodeling.

Over time, the proportion of collagen I content in the granulation tissue will increase

with a corresponding decrease of collagen III until it returns to the basal levels of

collagen I to collagen III ratio of 90% : 10% in unwounded skin 19.

Maturation of collagen fibers occurs approximately six to ten days after injury where

the deposited collagens are absorbed and re-organized into thicker bundles parallel to

the skin, correlating with increased tensile strength 12. Studies suggest that new collagen

fibres are orientated in an organized manner which is characteristic of the remodeling

phase of wound healing and results in scar formation, ranging from fine lines to

widespread scars 21. Scar formation is the inevitable outcome in the final remodeling

phase of wound healing in adult humans. One of the causes is the influence of fibrotic

agents, causing fibrosis to take place during the fibro-proliferative response. The main

cause of scarring is the continual presence of fibroblasts that over synthesize collagen

leading to hypertrophic scar morphology 12.

1.3 The Cytoskeleton

1.3.1 Types of Cytoskeleton

The cytoskeleton is a complex and highly dynamic network of microfibers found in the

cell’s cytoplasm. The cytoskeleton was originally thought to provide cellular mechanical

strength, locomotion, support organelles and maintain cell shape. However, it is now well-known that the cytoskeleton is also involved in other processes as well as pathologies arising from cytoskeletal defects 22. The structural integrity of eukaryotic

cells are made up of three molecular building blocks, microtubules, microfilaments and

intermediate filaments (Figure 1.3) differentiated by their sizes from large to small

respectively 23. Each of these cytoskeletal proteins has their own functional roles but

together they function synergistically with each other to withstand higher mechanical

Cell membrane

Nucleus

Centrosome

Microtubule Intermediate filaments Actin

Figure 1.3

Illustrative figure and immunofluorescent staining of microtubules, intermediate

filaments and actin within a typical cell.

Microtubule image obtained from Monton et al, 2009 24

Microtubules 12

stress25,26. The cytoskeletal properties in epithelial cells are especially important in

wound healing as they not only sense the environment but also allow migration from

healthy unwounded tissue into the wound site to re-establish epithelial integrity.

1.3.1.1 Microtubules

Microtubules are large fibrous cytoskeletal structures measuring approximately 25nm in diameter. They are arranged in straight, hollow, cylindrical aggregates made up of alpha and beta tubulin dimers assembled together 27. Microtubules are highly dynamic

and participate in a wide range of cell activities, mostly involving motion but also

including cellular division, morphogenesis and organelle transport. Being an important molecule in cellular motility, the highest concentration of microtubules are found at locomotive structures such as the cilia and flagella of motile cells 27. Cellular

propagation is achieved by the continuous polymerization of tubulin dimers at the plus

end and depolymerization at the minus end of a polarised cell. This process utilizes

energy by means of GTP hydrolysis which is expected since tubulin has GTPase

activity. The movement of cellular components such as vesicles takes place along the

microtubule network. This process requires two microtubule motors, kinesin, which

moves toward the plus end of the microtubules and dynesin, which moves toward the

minus end of the microtubules 28.

1.3.1.2 Intermediate filaments

Intermediate filaments are the second largest cytoskeletal structure after the microtubules, measuring approximately 10nm in diameter 23. Intermediate filaments consist of a conserved central α-helical domain flanked by variable N and C termini that contribute to the diversity of the intermediate filament family 29. The α-helical rod shape arrangement contains seven-residue repeats that interact with other intermediate filament protein to form a coiled-coil dimer 30. This structural configuration allows the intermediate filaments to interact with a range of protein complexes at the cell surface, including desmosomes, hemidesmosomes, focal adhesions and the extracellular matrix

31, 32. The interaction with cell surface molecules suggests that intermediate filaments may act as a sensory mechanism that relays signals from the cell surface to the surface of the nucleus.

1.3.1.3 Microfilaments (Actin)

Microfilaments, also known as actin, are the smallest of the three cytoskeletal proteins and the most important during wound healing. Actin is a highly conserved eukaryotic molecule expressed in animals, plants and fungi. Feuer et al, 1948 33 first identified actin as a major muscle component. Since then, actin has been identified in multiple important cellular processes including cellular motility, cell division and structure.

There are two main isoforms of actin, unpolymerized globular monomeric actin (G-

actin) and polymerized, filamentous actin bundle (F-actin); both exist in equal amounts in vivo 34. F-actin assembly is an energy requiring process in the form of ATP hydrolysis

35. Double-stranded, helical F-actin is always in a constant state of flux with G-actin

monomers, being added to the ‘plus’ end and depolymerized at the ‘minus’ end.

Comparison of different actin structures showed that while their overall configurations

are analogous, they show considerable local differences due to the intrinsic dynamics of

actin filaments modulated by different effector molecules and interactions with other

proteins to form a complex 36.

1.3.1.4 Stress fibers

Stress fibres are contractile actomyosin structures composed of mainly unorganized

actin filament bundles. This is the basic cellular structure that provides the contractile

force required for cell morphogenesis and migration 37. The constant contraction of

stress fibres are in balance with cellular adhesion strength as it results in a more stable

formation of actin bundles that maintain a constant length even under tension 38. There

are three classes of stress fibres in mammals. The first class is the ventral stress fibres

that associate with focal adhesions at both ends and are responsible for tail retraction 39.

The second class is the transverse arcs found from the leading edge to the cell centre

and they do not directly associate with focal adhesions but connect to the substrate via

the dorsal stress fibres which are the third class of stress fibres 39,40. Dorsal stress fibres

associate themselves from the focal adhesion sites at one end to the dorsal section of the cell at the opposite end and therefore efficiently propagate contractile forces from the cell to the substrate 37. Stress fibres highlight the dynamic capabilities of actin filaments and their importance in cell migration.

1.3.2 Actin dynamics in wound healing

In the event of an injury, surrounding motile cells such as fibroblasts migrate towards each other during wound healing. These wound edge motile cells are induced by various extracellular signals to undergo a transition from a non-polarized to a polarized state 35. Once polarized, cells have the ability to migrate by forming and extending protrusive structures, known as lamellipodia and filopodia. Lamellipodia are large sheets which consist of branched actin filaments at the front of a leading edge cell and filopodia are long, parallel actin filaments that protrude beyond the cell membrane 41.

Evidently, actin in this situation develops two different organizations for lamellipodial formation and filopodial protusion. In lamellipodia, small GTPases cdc42 and Rac activate proteins of the Wiskott-Aldrich syndrome family such as Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) which in turn activates Actin-related protein (Arp) 2/3 complex 42. Activated Arp2/3 complex will lead to the nucleation of actin filament branches that results in a broad network of actin filaments 43. Cdc42 in filopodia however, promotes parallel linear actin

polymerization, aided by formins, vasodilator-stimulated phosphoprotein (VASP) and

fascin 44. Clearly, the formation of these protrusion structures is tightly regulated and

cellular migration is dependent on the balance between polymerization on the ‘plus’

end and depolymerization at the ‘minus’ end of F-actin. Therefore to facilitate cellular

motility, the process of polymerization and depolymerization has to be coordinated

such that, on average, the filament moves forward in one direction.

Cell-matrix adhesion is another factor determining migration. During migration, cells

form protrusions that require new attachment sites or focal contacts at the leading edge

to provide traction between the actin cytoskeleton and the underlying substrate 45. The

formation of new focal adhesion contacts is mediated by integrins which are a family of

heterodimeric transmembrane receptors that connect the extracellular matrix to the

actin cytoskeleton 46. Focal complexes act as a "clutch handle" to connect the integrins to

the actin cytoskeleton and in the early stages consist of dynamic proteins such as β3-

integrin, talin and paxillin (Figure 1.4). In addition to providing traction, cell-matrix adhesions also act as extracellular sensors. The force generated by the actomyosin contraction induces focal complex maturation which allows further recruitment of actin binding proteins such as α-actinin, VASP and FAK and further actin polymerization 47-

49. These protein complexes are localized to the leading edge

NOTE: This figure is included on page 18 of the print copy of the thesis held in the University of Adelaide Library.

Adapted from Le Clainche et al, 2008 50

Figure 1.4

Cellular adhesion acts as a "clutch handle" to connect the integrins to the cytoskeleton.

This allows the conversion of the force generated by actin polymerization into protusion. The actin network is represented in grey and newly formed actin is represented in pink. Adhesion molecules are represented in blue. Cellular substrate is represented by the red lines. The cell boundary is represented by the yellow lines. (A)

No connection of the adhesion molecules and the actin cytoskeleton. In this scenario, no protusion occurs as actin polymerization is converted into retrograde flow. (B)

Connection of the polymerizing actin network to the substrate via the adhesion molecules. In this scenario, the connection between the polymerizing actin network and the substrate provides traction that enables cellular protusion.

of cells which assemble and disassemble during cellular migration. These events,

coupled with the forward motion, occur with the simultaneous retraction of attachment

sites at the trailing end of the cell, allowing the cell to pull its rear, therefore driving the

cell in a single direction. This process is known as ‘treadmilling’ which allow cells to

migrate into the wound site (Figure 1.4). ‘Treadmilling’ is a highly complex process that

requires the coordination of a set of proteins including but not limited to actin

depolymerizing factor (ADF) or cofilin, profilin and capping proteins such as the

gelsolin family of actin binding proteins 35.

1.4 Gelsolin family of Actin Remodeling Proteins

Cytoskeletal rearrangement is fundamental in wound healing to facilitate cell

movement and its process involves a wide range of proteins. Among these is the

gelsolin superfamily of actin binding proteins (ABP). The gelsolin superfamily is a

conserved family of proteins present in mammalian cells and is characterized by having either three or six homologous gelsolin-like structural domains known as G1-G6

segmental domains (Figure 1.5A). Early studies of gelsolin, the founding member of the

family, identified three actin binding regions, a calcium independent strong monomer

binding fragment (G1), a calcium independent filament binding fragment (G2-3) and a calcium dependent monomer binding fragment (G4-6) 51.

NOTE: This figure is included on page 21 of the print copy of the thesis held in

the University of Adelaide Library.

Figure 1.5

(A) Schematic representation of human plasma gelsolin domain structure and residue

G1-G6. Ca2+, phosphatidylinositol 4,5-bisphosphate (PIP2) and actin binding segments are shown. (B) Gelsolin superfamily members include villin, supervillin and flightless-I that have additional unique domains beyond gelsolin G1-G6 domains. CapG which has only 3 gelsolin domains is also a member of the superfamily. ABD, actin binding protein. NLS, nuclear localization signal.

Adapted and modified from Sun et al, 1999 52

Other members of the family such as villin and supervillin have additional actin

binding domains that provide a unique function to each member 53. Supervillin has an

N-terminal addition which is capable of protein-protein interactions and nuclear

localization. CapG has undergone evolutionary truncations and has only the G1-G3

gelsolin domains but still retains full actin severing properties 54. The Flightless-I protein

(Flii) contains an N-terminal leucine rich repeat (LRR) region that is capable of protein- protein interactions. Taken together, members of the gelsolin family have roles other than just actin remodeling owing to their specific additional domains.

Previously, it was thought that there was a potential for redundancy due to the homology between members of the gelsolin family. This is true to some extent owing to the conserved gelsolin-like domains. However, research has shown otherwise. For example, expression patterns of gelsolin and CapG are complementary to one another indicating that they have distinct in vivo functions 55. In fact, a CapG and gelsolin double

knockout study demonstrated that CapG is highly essential in actin-based macrophage

motility that is distinct from gelsolin and its functions include macrophage receptor mediated ruffling, IgG complement and phagocytosis 56.

1.4.1 Gelsolin

Gelsolin is the founding member of the family and regulates the dynamics of

filamentous actin by means of binding, severing and capping actin filaments 52. This

process is initiated by Ca2+ which causes a conformational change that allows gelsolin to

bind to actin and severing it while remaining bound to the barbed end. Uncapping of

actin occurs in the presence of phosphatidylinositol lipids which bind to gelsolin thereby uncapping and exposing the barb ends for further actin polymerization 57.

Other modes of action that are also known to affect activity include phosphorylation

and binding to adenosine triphosphate (ATP) 58,59.

1.4.1.1 Gelsolin in Clinical Settings

Recent research has implicated gelsolin in various diseases including a systemic

disorder known as Hereditary gelsolin amyloidosis 60,61, Meretoja's syndrome (lattice

corneal dystrophy) 62 and a role in Alzheimer's disease 63-65. Furthermore, over-

expression of gelsolin is linked to a negative prognosis in a subset of breast tumours

which show higher expression of tyrosine receptor kinase erbB-2 and EGFR 66.

Interestingly, excluding the above subset of carcinomas, gelsolin is generally considered

to be a tumour suppressor 67. It is reported that tumour progression is correlated with

decreased expression of gelsolin and that over-expression of gelsolin inhibits carcinogenesis. These observations suggest that gelsolin possesses complex multi-role

properties other than as a downstream actin remodeling molecule.

1.4.1.2 Involvement of Gelsolin in Cellular Apoptosis

Apart from being a simple cytoskeletal actin modulator, gelsolin is also involved in

other cellular processes such as being a contributor to cellular apoptosis 68 and the

regulation of Rac proteins 69. Gelsolin is a substrate for caspase-3 which is the core effector caspase activated during apoptosis 70. Gelsolin severing of the actin

cytoskeleton is calcium dependent under normal cellular conditions. However, when

cleaved by caspase-3 to produce the amino-terminal cleavage product (residues 1-352),

gelsolin severing of the actin cytoskeleton becomes calcium independent and results in

the disassembly of the membrane cytoskeleton, which is characteristic of apoptosis 68.

Over-expression of this gelsolin fragment in cells results in apoptosis whereas gelsolin

knockout neutrophils exhibited a delay in the onset of apoptosis more slowly than wild-

type neutrophils 71. These findings highlighted the role of gelsolin in mediating the

apoptotic pathway.

Conversely, a pro-apoptotic role of gelsolin has also been identified. This is supported

by the fact that most cancer cells expressed significantly lower levels of gelsolin 72-74. In

addition, research has shown that over-expression of gelsolin inhibits apoptosis 71. This

is consistent with findings from another group 75 showing the inhibition of apoptosis in

stimulated Jurkat cells (human T-cell line) following gelsolin over-expression at several

times the normal levels. Furthermore, over-expression of a gelsolin mutant form that is

more sensitive to phosphoinositide, suppresses Ras transformation 76. Currently, the conflicting role of gelsolin in apoptosis remains to be elucidated. However, one possible explanation for the conflicting observations is that gelsolin may interact with other intracellular proteins such as phosphoinositides, which as a complex, function as a competitive inhibitor of caspase-3 77. These findings which implicate gelsolin in apoptosis and carcinogenesis highlight the complexity in the balance between the multiple regulatory role of gelsolin.

1.4.1.3 Biological Functions of Gelsolin

Genetic knockout of gelsolin display a mild phenotype allowing the mouse to develop into adulthood 78. Regardless of the mild phenotype accompanied with genetic knockouts, the effect of manipulating gelsolin expression is significant at the cellular level. Gelsolin null skin tissue has pronounced actin stress fibers due to the limited ability to sever and remodel actin filaments 78. As a result, the lack of gelsolin causes poor ruffling of actin filaments in response to growth factors 69 as well as the restriction of platelet activation by actin severing, subsequently hindering the clotting mechanism

78. In addition, cellular motility is heavily reliant on actin cytoskeletal rearrangement.

For example, the formation of lamellipodial and filopodial protusions of neuronal growth cones are induced mainly by adseverin and the presence of Ca2+ but in gelsolin null mice the retractions of the protusions are severely delayed, slowing cellular

motility 79. Furthermore, the motility of migratory cells such as fibroblasts is also

negatively affected in gelsolin null mice 78. Gelsolin-null macrophages also exhibit

impaired IgG-mediated phagocytosis 80. As a consequence, gelsolin null mice exhibit

defective chemotaxis and slower wound healing, consistent with reduced cellular

motility. These findings indicate the importance of gelsolin in the regulation of actin

dynamics.

1.4.1.4 Gelsolin and the sex steroid hormones

Previous studies have implicated gelsolin in the hormone mediated wound healing

process 53,81,82. It is known that androgens and estrogens are important regulators of

wound repair, highlighted by the improvement of healing in young females versus elderly males and females 83. However, sex steroid hormones are better understood in the development of primary and secondary sexual characteristics. In humans, adrenal

cortex and the primary sexual organs, ovaries and testes, are major production sources

of sex steroid hormone precursors. Both estrogen and androgen are synthesized from

an inactive common steroidal precursor, dehydroepiandrosterone that is secreted in

large amounts by the adrenal cortex 84. The formation of the inactive form allows target

tissues to control the formation and metabolism of the sex steroid hormones according

to their requirements. Activation requires the initial conversion of

ehydroepiandrosterone to androstenedione by 3β-hydroxysteroid dehydrogenase.

Several enzymes namely, 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid

dehydrogenase, 5α-reductase and aromatase subsequently convert androstenedione to

the naturally more potent bioactive estrogen, 17β-estradiol and androgen,

Dihydrotestosterone (DHT) which are involved in downstream biological processes.

Downstream effects of the sex hormones are mediated through the nuclear hormone

receptors which regulate the expression of target genes. Post-menopausal women suffer

from impaired wound healing and this is correlated with lower physiological levels of

estrogen. This is highlighted by a prolonged inflammatory response, higher protease levels and reduced matrix deposition 85. Closer studies revealed that topical estrogen is

able to repair skin atrophy, wrinkles and dryness in post-menopausal women and is

associated with accelerated wound repair, highlighted by a reduced inflammatory

response 86 and enhanced extracellular matrix deposition 83. Topical treatment with

estrogen was also found to be beneficial by reversing the reduced healing rate in elderly

women 87. Male counterparts also benefit from this treatment which led to significantly

lower inflammatory responses during wound healing.

In contrast, androgens impede wound healing and are associated with increased

inflammation and wound immune cell dysfunction 88. Furthermore, murine studies

done byAshcroft et al, 2002 89 and Gilliver et al, 2006 90 reported that castration of male

mice or treatment with AR antagonists resulted in a marked improvement of wound repair. This is in part due to the decrease in overall inflammation by inhibiting the upregulation of proinflammatory cytokines by endogenous androgens. As a result, target inhibition of 5α-DHT expression could be used therapeutically for the acceleration and improvement of wound healing.

Taken together, the findings show that while estrogen positively regulates the wound healing process, androgens showed detrimental effects. Therefore, being male is considered to be a risk factor for abnormal wound healing particularly in the elderly 91.

Clearly the complexities of sex hormones are not restricted to the sole purpose of regulating and maintaining sexual characteristics, it also significantly affects the process of wound healing. Therefore, it is safe to assume that while the underlying wound healing process in males and females are similar, there are differences in the mechanism due to the variation in hormonal makeup in males and females. The implications for sex hormones in wound healing have only been established recently and the mechanism of their actions remains unclear.

1.4.1.5 Nuclear hormone receptor signaling

Androgens (testosterone or its metabolite DHT) act through binding to the Androgen

Receptor (AR), which is a ligand inducible nuclear receptor. The AR is an 110kDa

protein belonging to the nuclear receptor family which upon binding to a ligand, typically DHT, translocates into the nucleus and binds to androgen response elements

(AREs). In addition to its primary role, AR also plays important roles in the sex hormone mediated wound repair process.

Nuclear receptors (NR) constitute a family of transcription factors that is regulated by diverse ligands ranging from steroid hormones, lipids to retinoids. NRs mediate their effects by binding to response elements at their target promoter regions (reviewed in 92.

NRs have a centrally located, highly conserved DNA-binding domain (DBD) characteristic of all NRs. Located in the C-terminus of the hormone binding domain and the N-terminus lies the activator function 1 (AF-1) and activator function 2 (AF-2) that are primarily responsible for transcription activation by hormone-activated bound NR.

Their full activity is dependent on ligand binding and the target gene promoter. The majority of ligands bind NRs as dimers that either regulate transcription directly by binding to enhancer elements at the gene promoter, or indirectly by potentiating

nuclear translocation and DNA binding efficiency.

1.4.2 Flightless-I

Flii is a member of the gelsolin superfamily and has highly conserved homologues

sharing 52% and 69% protein sequence similarity between Caenorhabditis elegans and

human respectively 53. The human Flii gene spans 14kb of genomic DNA and contains more introns than human gelsolin or villin 54. The carboxy terminal half of human Flii

has 31% identity and 52% similarity to human gelsolin (Figure 1.5B) 93. The Flii gene is

located in the subdivision 19F on the X-chromosome which encodes a 1256 amino acid peptide with a molecular weight of 143, 672 Da 94.

1.4.2.1 Biological Functions of Flii

The name Flii was given because point mutations in the gene disrupt the structural

organization of Drosophila melanogastor’s indirect flight muscle myofibrils and result in a

flightless phenotype 95. Drosophila melanogastor eggs lacking maternally supplied Flii

showed incomplete cellularization and impaired gastrulation, associated with a

disorganized actin cytoskeleton (Figure 1.6) 96. The flightlessness and the incomplete

cellularization phenotypes indicated that Flii is required for proper actin organization

during myogenesis and embryogenesis respectively. In humans, the Flii locus has been

mapped to a region deleted in the Smith-Magenis Syndrome which is associated with

developmental and psychological abnormalities 97.

Homozygous knockout studies of Flii revealed more important roles of Flii during

development. In Drosophila, homozygous knockout of Flii resulted in the disruption of

the syncytial blastoderm during cellularization leading to early embryonic

Figure 1.6

Cross-sections showing abnormal cellularization of Flii mutant Drosophila embryos. (a, c, e) Wild-type embryo. (b, d, f) Flii mutant embryo. No significant differences between wild-type and Flii mutant embryos are visible during syncytial blastoderm stages (a-b).

However, when cellularization occurs and the nuclei begin to elongate, the layer of nuclei in Flii mutant embryos begins to appear disorganized (c-d). During gastrulation, nuclei align themselves to face the outside of the embryo (e). In Flii mutant embryos, the nuclei move out of the incompletely cellularized peripheral layer of cytoplasm into the interior of the egg. Bar = 50µm in (b).

Obtained from Straub et al, 1996 96.

developmental arrest 98. In murine models, homozygous knockout is embryonic lethal,

with development arresting at a stage preceding gastrulation. Using a human Flii

transgene, normal development of Flii homozygous knockout mouse embryos can be

restored 98. Coupled with the fact that Flii heterozygous mice exhibit no impediments during development, this indicates that a single copy of Flii gene is adequate for normal gene function 98. A homolog of Flii has been implicated in the establishment of the

anterior-posterior polarity in Caenorhabditis elegans 99. It was found that the Flii

homolog also regulates the cytokinesis of somatic cells and the development of

germline. These findings indicated the importance of Flii in performing essential

functions during early embryogenesis in Drosophila, C. elegans and mammals.

1.4.2.2 Protein Interactions are mediated through the Flii LRR domain

The Flii protein represents an interesting fusion of two functionally distinct protein

families’ evolutionary gene insertion events. Flii is a functional filamentous actin

severing protein but, unlike gelsolin, it functions independently of Ca2+ 100. The 5’ end of

Flii protein consists of 16 tandem repeats of 23 amino acid LRR fused to the gelsolin-like

G1-G6 segmental domain by a linker of approximately 100 amino acid residues, making

it a unique member of the gelsolin superfamily (Figure 1.5B). Like many other LRR

containing proteins, the Flii LRR domain may form a hydrophobic, curved solenoid

structure 101 that is particularly suitable for protein-protein as well as protein-lipid

interactions involved in signal transduction, either by binding directly as a ligand or as

a regulator to mediate receptor-ligand binding affinity 102. The first binding partner of

Flii discovered is known as the Flightless-I LRR associated protein (FLAP) of size 626

amino acids 103.

1.4.2.3 Involvement of Flii in cellular processes

As the functions of Flii are still largely unknown, interactions of Flii in cellular

processes continue to be revealed. Recent research has reported that Flii directly and

preferentially interacts with the active form of calcium/calmodulin-dependent protein

kinase type II (CaMK-II), a protein kinase involved in the progression of the cell cycle

104. When CaMK-II was inhibited, Flii over-expression suppressed the transcription of β- catenin dependent transcriptional reporters but the suppression of Flii expression enhanced β-catenin transcription. Furthermore, the mammalian Flii has been identified to be involved in the nuclear receptor signaling by directly associating with estrogen and thyroid hormone receptors as well as their co-activating transcriptional factors,

CARM1 and GRIP1 105. In this case, Flii localizes to the promoter region and functioned as a co-activator protein that actively recruit and coordinate other transcriptional factors

such as p160, mediator and the SWI/SNF complex 106. This allows the assembly of a

larger estrogen receptor associated co-activator complex onto the promoter region of an

estrogen inducible gene. On the other hand, a cytokine-independent survival kinase

(CISK) is a downstream phosphoinositol 3-kinase target has been identified to interact with Flii to regulate the estrogen receptor 107. In the study, Flii was found to be a CISK

substrate where it was shown that CISK could phosphorylate Flii at residues Ser436 and

Thr818. This indicated that Flii could be post-translationally modified and is of

importance in determining its multifunctional properties. These findings suggest a role

of Flii in the regulation of the cell cycle.

Flii has also been implicated in the immune response. It was revealed that Flii regulated

pro-inflammatory caspase-1 and capase-11 by direct association and modulation of their intracellular localization and activity 108. The regulation of the pro-inflammatory

caspases impact on the maturation of interleukin-1β 108, therefore potentially affecting

its activity in processes such as wound healing. The role of Flii in the immune response

is also reported by Wang et al (2006). The authors showed that Flii directly interacts

with MyD88 which is an immediate downstream adaptor protein of Toll-like receptors

(TLR) involved in the regulation of signaling specificity in the innate immune system.

This interaction prevents the formation of TLR4-MyD88 complex which subsequently inhibited the activation of NFkB.

1.4.2.4 In Vitro and In Vivo Expression of Flii

Flii can be induced by serum to translocate from the nucleus to the leading edge of the cell, membrane ruffles and actin arcs where Flii colocalizes with Ras and GTPases 109.

The co-localization of Flii with Ras is consistent with findings using kinetic analysis based on competitive inhibition of Ras-dependent adenylyl cyclase activity which showed the association of the LRR domain of Flii with Ras 94,100. Ras proteins are involved in the regulation of the actin cytoskeleton where the interaction with Raf-1 connects actin to the mitogen-activated protein kinase (MAPK) pathway 100. Therefore, the interaction of Flii with Ras proteins provided a link to the MAPK pathway. The connection of Flii to a signaling pathway is further strengthened by a study which revealed the interaction of Flii to the phosphoinositol-signaling pathway in the regulatory events during ovulation 99.

In Swiss 3T3 fibroblasts, Flii located in the nuclear periplasmic region colocalizes with microtubules 109. In general, Flii protein localizes to actin based structures both in vitro and in vivo 94. The significance of this localization pattern remains unknown but suggests a possible function for Flii in actin regulation. However, the nuclear shuttling trait is not only limited to Flii as supervillin, CapG and gelsolin are also capable of nuclear translocation, and is the first indication that Flii may be involved in roles that are distinct from an actin remodeling protein.

1.4.2.5 Flii protein is a Negative Regulator of Wound Healing

Recent wound healing studies using mice heterozygous Flii knockout and mice over-

expressing the Flii gene demonstrated that Flii is an important negative regulator of the

wound healing process 110,111. The study showed that Flii regulates wound healing by

affecting cellular proliferation, motility and collagen I production in both epidermal

keratinocytes and dermal fibroblasts. Flii heterozygous mice displayed smaller and

more contracted wounds, coupled with an increase in α-smooth muscle actin-positive

myofibroblasts (Figure 1.7A). Conversely, Flii over-expressing transgenic mice

displayed increased wound area and dermal gape, reduced cell proliferation and

delayed epithelial migration (Figure 1.7C) compared to wild-type (Figure 1.7B) 111. It

was also found that Flii deficiency was associated with reduced collagen I production,

as evident from in vivo mouse wounds and in vitro siRNA knockdown fibroblasts. As

collagen I is the most highly produced collagen in response to wounding, the regulation

of it greatly affects the extent and outcome of cutaneous fibrosis and scar formation 112.

In burn wounds, pro-scarring TGFβ1 protein and gene expression were significantly

lower while anti-scarring TGFβ3 was higher in mice heterozygous for the Flii gene.

Furthermore, the addition of Flii neutralizing antibodies to in vitro incisional wounds

(Figure 1.7D) 111 as well as burn wounds 110 significantly improves wound healing, which indicated the possibility of modulating Flii activity to improve wound repair.

Flii+/-

FliiTg/+

Figure 1.7

In vivo incisional wounding of WT, Flii heterozygous (Flii+/-) and Flii over-expressing mice (FliiTg/+) at day 3 post-wounding. (A-C) Incisional wounds showing improved healing in Flii+/- mice whereas FliiTg/+ mice show impaired healing compared to WT. (D)

Comparison of incisional wounds treated with Flii neutralizing antibodies in WT mice.

Images obtained from Cowin et al, 2007 111

1.5 TRANSFORMING GROWTH FACTOR BETA (TGFβ)

1.5.1 TGFβ superfamily

TGFβ is an important cytokine that is ubiquitously expressed in mammals and governs a substantial number of wound healing activities and processes in terms of cellular growth, differentiation, proliferation and adhesion. The TGFβ superfamily (Table 1.1) consists of highly conserved growth regulatory proteins that include bone morphogenenic proteins (BMP), activins, inhibins and Mullerian inhibitory factor (MIF) characterized by their sequence homology 113. Here, we are focusing on three isoforms of TGFβ in mammals; TGFβ1, TGFβ2 and TGFβ3.

All TGFβ isoforms are expressed in the skin and play important roles in wound healing

114,115. In general, TGFβs act as chemoattractants during injury to direct an immune response towards the invading pathogens. TGFβ1 can stimulate the production of ECM such as collagen and therefore contribute to scarring. Studies using neutralizing antibodies to TGFβ1 and TGFβ2 showed improve healing with reduced scarring, indicating the detrimental effects of TGFβ1 and TGFβ2 isoforms in wound healing 116,117.

However, the exogeneous addition of TGFβ3 promoted wound healing indicating that

TGFβ3 is a positive regulator of wound healing 116,118.

TGFβs BMPs/GDFs Activins/Inhibins Others

TGFβ1 BMP-2 Activin A MIS TGFβ2 BMP-4 Activin B Lefty A TGFβ3 BMP-5 Activin C Lefty B BMP-6 Activin D BMP-7 Activin E BMP-8 BMP-9 BMP-10 BMP-15 Nodal GDF-1 GDF-3 GDF-5 GDF-6 GDF-7 GDF-9

Table 1.1

TGFβ superfamily members. The mammalian members of the TGFβ superfamily are sub-divided into (i) TGFβs, (ii) BMPs/GDFs (growth and differentiation factors), (iii)

Activins/Inhibins and (iv) other more distally related members, based on their structural characteristics.

TGFβs are potent cytokines with a range of regulatory receptors 119. The mature

peptides share 70-80% amino acid sequence identity and interact with the same

receptors 120,121. The regulation of TGFβ involves multiple checkpoints, from its

activation to signal transduction and these will be discussed below. Although extensive

research has been done on TGFβ, the full effects of TGFβ are still being elucidated as the

multifunctional nature of TGFβ is greatly influenced by a range of conditions including

cell type, growth conditions and presence of other growth factors.

1.5.1.1 TGFβ Activation and Structure

TGFβs are initially synthesized as large precursor polypeptides where they undergo

further proteolytic processing in the Golgi apparatus 119. The newly processed, mature

TGFβs non-covalently bind to the latency associated peptide (LAP) located at the N-

terminal domain, rendering it inactive by preventing interaction with its receptor. The

activation of TGFβ occurs via a multistep process involving different cell types,

proteolytic enzymes and the current micro-environment. There are many ways in which

TGFβ can be activated. These include acidification of microenvironments, action by

oxygen reactive species and steroid hormones such as anti-estrogens, retinoids, vitamin

D and glucocorticoids 122. However, during wound healing, TGFβ can be activated by secreted proteases such as plasmin and thrombospondin which can cleave off the LAP.

In the wound site, reactive oxygen species and an acidic environment can both disrupt

the interactions between LAP and TGFβ to release bioactive TGFβ 123.

Another protein called latent TGFβ binding protein (LTBP) serves as a further control

during TGFβ activation. It covalently associates with LAP through two disulphide

bonds at the C-terminus 124. LTBP is important in the processing and secretion of mature

TGFβ such that its own expression is co-regulated with TGFβ 125. This also suggests a feedback loop between the expression of LTBP and TGFβ. Studies have shown that in the absence of LTBP, latent TGFβ secretion declines and it is mostly retained in the cis-

Golgi apparatus but, in the presence of LTBP, the association of LTBP with LAP

accelerates the secretion of mature TGFβ 126,127. This study has shown that LTBP

expression and TGFβ secretion are inter-connected and therefore serve as a regulatory

control over TGFβ activation.

The bioactive TGFβ released resembles a butterfly knot structure made up of two

12.5kDa molecules held together by four intrachain disulfide bonds formed by eight

cysteines. An additional sulphydryl bond on the last cysteine residue links another

monomer to form a 25kDa dimeric molecule (Figure 1.8 A, B). In most cases, TGFβs are

homodimeric molecules but heterodimeric TGFβ such as β1:β2, β1:β3 or β2:β3 can also

Adapted from Groppe et al, 2008 & Weiskirchen et al, 2009 128,129

Figure 1.8

Domain and three dimensional structure of mature TGFβs and interactions with TβRs.

(A) Within each TGFβ1 monomer, there are four intrachain disulphite bonds including

one conserved intrachain disulphite bond indicated as speckled line. TGFβ1 individual

monomers are linked together by a disulphite bond at position 77 of the mature

peptide. (B) The 4 intrachain disulphite bonds and the intermolecular disulphite bond

folds the dimer into a butterfly-like tertiary structure typical of all TGFβ isoforms. (C)

Ribbon and molecular surface representation of TGFβ ligand-receptor interaction in a ternary complex. TGFβ3 monomers are shown in blue and red. TβRI shown in yellow and TβRII shown in green.

be formed and this contributes to the extensive repertoire of biological processes

involving TGFβ 130. Mature TGFβ dimers will subsequently bind to their respective

target receptors to induce expression of downstream genes.

1.5.1.2 TGFβ Receptor Signaling

TGFβ signals transduce through heteromeric complexes of type I (65-70kDa) and type II

(85-110) serine/threonine kinase receptors (Figure 1.8C). These receptors are characterized by a cysteine-rich extracellular domain, a hydrophobic transmembrane domain and a C-terminal serine/threonine kinase domain. Functions of larger type III

(300kDa) receptors such as betaglycan and endoglin remain unclear but are not essential to the biological activities of TGFβ, instead they facilitates TGFβ interactions with the receptor signaling complex 131. It has been suggested that TGFβ receptors (TβR) are pre-formed even before binding to TGFβ ligand 132. The subsequent binding of the ligand causes a re-orientation rotation between the receptor chains, followed by simultaneous kinase activity. This may facilitate TGFβ signal transduction as a whole but its real purpose is not clear. However, this mechanism of receptor activation is also found in erythropoietin signaling pathways 133.

There are 11 different type I and six different type II receptors identified that bind to their respective ligands with different affinity. For example, TβRII has a higher affinity

of TβRII for TGFβ1 and TGFβ3 134,135. All type I (TβRI) and type II (TβRII) receptors have

similar structures and exist as homodimers in the absence of ligand. Upon binding to

TGFβ, TβRII recruits and phosphorylates serine and threonine residues in TβRI forming

an active heterotetrameric complex 136. This activated complex in turn initiates the

activation of downstream effectors, the Smad proteins (Figure 1.9).

1.5.1.3 Smad Effector Signaling

Smads are major TGFβ classical signaling transducers, mediating signals from the cell surface receptor to target genes in the nucleus (Figure 1.9). Smads are small molecular weight (42-60kDa) effectors. To date, there are a total of nine Smads that have been

characterized and divided into three groups based on their function. Receptor-activated

Smads (RSmads) that includes Smad 1-3, 5 and 8 make up the receptor-regulated Smad family and all consist of a conserved C-terminal SSXS motif. The other two groups are the common-partner Smad (co-Smad) that comprises Smad 4 and the inhibitory Smads,

6 and 7 137. All Smad proteins consist of two sequence homologies; Mad homology 1

(MH-1) at N-terminus and MH2 at C-terminus. Activation of the TGFβ receptor

heterotetrameric complex binds to RSmads 2/3 and phosphorylates amino acids SSXS

located at the C-terminus of RSmads which leads to a conformational change. Common

Smad 4 and inhibitory Smads 6 and 7 lack this SSXS motif. The phosphorylated RSmads

then dissociate from the receptor complex and form a heteromeric complex with co-

NOTE: This figure is included on page 46 of the print copy of the thesis held in the University of Adelaide Library.

Adapted from Derynck et al, 2003 138

Figure 1.9

Representation of TGFβ receptor and Smad signaling. At the cell membrane, TGFβ ligand binds to TβRII which then recruits and phosphorylates (P) serine and threonine residues in TβRI, forming an active heterotetrameric complex. The active heterotetrameric type I receptor consequently phosphorylate RSmads (Smad 2/3) which then associate with common Smad 4 and translocate into the nucleus where target gene transcription are regulated through additional interaction with DNA binding co-factors and other transcriptional factors. Phosphorylation of RSmads can be inhibited by Smads

6/7. X, DNA binding co-factor.

Smad (Smad 4) which then translocates into the nucleus and either directly or indirectly

associates with other transcriptional factors and binds DNA to induce target gene

expression 139. Rsmads 1, 5 and 8 also mediate signaling but more specifically further downstream of BMPs and their receptors.

Smad activities are also tightly regulated to serve as a layer of control for the signaling of TGFβ. It has been reported before that oncogenic Ras can inhibit the formation of

TGFβ induced Smad 2/3 complex as well as Smad 4 expression, therefore preventing nuclear translocation and accumulation to initiate gene expression 140. The stopping of

nuclear translocation can also be achieved by other proteins such as epidermal growth

factors (EGFs) which phosphorylates Smad linker regions to prevent nuclear

translocation. Smad can also be targeted by Smurf 1 (Smad uibiqutination regulatory

factor) which is an E3 ubiquitin ligase that recognizes and binds Smad 1 and Smad 5

(Zhu et al, 1999). Upon binding, Smurf 1 mediates the ubiquitination and subsequent

degradation of Smads which effectively arrests TGFβ signaling through the Smad

pathway.

1.5.1.4 TGFβs regulation in wound healing

TGFβ can also be regulated by TGFβ binding proteins such as the proteoglycan family

which includes decorin and fibromodulin that are also involved in wound healing by

regulating collagen formation and tensile strength 141,142. These small, leucine-rich

proteoglycans are components of the extracellular matrix which consist of a LRR motif

central protein core covalently linked to glycosaminoglycan side chains 143. These molecules belong to a distinctive protein class of the LRR superfamily 144. Decorin itself

interacts with TGFβ directly to modulate its activity 145 and a study 146 has substantiated

the fact reporting that decorin knockout cells have diminished TGFβ responsiveness.

Fibromodulin, another proteoglycan, also interacts with TGFβ to affect its activity. This

is of more relevance in wound healing because fibromodulin may potentially determine

the outcome of adult scarring by regulating TGFβ activity 142. Many members of the

proteoglycan family such as decorin, fibromodulin, biglycan and asporin directly

interact with TGFβ 147,148 via the LRR motifs 149. Therefore, it is likely that other proteins which contain LRR motifs are also able to bind to TGFβs.

The rate of TGFβ activation is also a major contributing factor to wound healing. As

previously discussed, proteases work to cleave off LAP to release mature TGFβ.

However, proteases do not work at the same efficiency. For example, matrix

metalloproteinase 2 and 9, are cell surface proteases which activates TGFβ and promote

tumor invasion and they work with different efficiency in activating TGFβ1, TGFβ2 and

TGFβ3 150. As a result, TGFβ1, TGFβ2 and TGFβ3 can be sequentially regulated at

different wound healing phases to affect the outcome of the wound. Furthermore, TGFβ

can be differentiated into two groups during blood clotting; the larger latent TGFβ complex (including LTBP, LAP and TGFβ) is released into the serum while the smaller latent TGFβ complex (LAP and TGFβ only) in the clot is activated gradually by proteases as the clot dissolves away 151. Therefore, this gradual release of TGFβ may be a means of maintaining TGFβ activity throughout the wound healing process.

1.6 Mitogen-activated Protein Kinase

1.6.1 The MAPK Signaling Pathways

The MAPK pathway is another common signaling pathway governing multiple cellular processes. The MAPKs are highly conserved protein enzymes responsible for the intracellular signaling cascades that transduce cell-surface receptor signals to the nucleus to induce target gene expression. The MAPK signaling pathways control many cellular processes such as cellular survival, proliferation, migration and adhesion which are also crucial during the wound repair process 152-154. Cell surface signals are transduced through the MAPK pathway through a three-tiered phosphorylation cascade, consisting of first tier MAPKK kinase, second tier MAPK kinase and the third tier MAPK 155. The main components of MAPK signaling cascades are shown in Figure

1.10. Given the complexity of the MAPK signaling cascade and to ensure specificity and maximize efficiency in order to prevent undesired activation of genes. Cells have to

NOTE: This figure is included on page 51 of the print copy of the thesis held in the University of Adelaide Library.

Adapted from Zhang et al, 2002 153

developed a mechanism known as protein scaffolding to regulate the MAPK pathway

156. The main function of protein scaffolds are to assemble multiple components of a particular MAPK signaling cascade such that it brings them to close proximity to operate efficiently. Protein scaffolds can also localize specific signaling molecules to a specific site in a cell and coordinate feedback signals to modify the MAPK cascade 157.

Additionally, protein scaffolds also protect activated signaling molecules from inactivation by other proteins 157. Using this method, cells are able to efficiently facilitate signal propagation from the cell membrane into the nucleus to induce target gene expression.

There are 5 distinctly regulated groups of MAPKs identified in mammals. These are the extracellular signal related kinase (MEK/ERK1/2), cJun N-terminal kinase (JNK), p38 proteins, ERK 3 and ERK 5 156, which are activated by their respective MAPKKs. For instance, MEK1/2 activates ERK1/2, MKK3/6 activates p38, JNKK1/2 activates JNKs and

MEK5 activates ERK5 158. More importantly, each second tier MAPKK can be activated by more than one first tier MAPKKK which greatly increases the complexity and dynamics of the MAPK signaling pathway 158.

1.6.1.1 ERK Pathway

The Raf/MEK/ERK cascade (reviewed in 156) is the most characterized pathway of the

MAPK signaling pathway which is mainly activated by protein tyrosine kinases such as

the epidermal growth factor (EGF) receptor and the vascular endothelial

growth factor (VEGF) receptor 159. Ligand bound receptors undergo a conformational change that induces phosphorylation. Src homology 2 domain-containing proteins such as Brg-2 are then recruited to the membrane receptors and interact with the phosphotyrosine residues. Brg-2 becomes activated when recruited to the membrane and in turn activates Ras GTPase which will then hydrolyze guanosine triphosphate

(GTP) to guanosine diphosphate (GDP). GTP bound Ras consequently results in the activation of downstream effector proteins. The activation of Ras is regulated by two classes of proteins known as the GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) 160. GAPs reduce the availability of GTP-bound Ras by enhancing GTPase activity in Ras which increases the rate of GTP hydrolysis.

Conversely, GEFs facilitate the exchange of GDP for GTP therefore increasing the

numbers of GTP bound Ras. GTP bound Ras complex recruits and activates Raf at the

cell membrane which results in Raf catalyzing the phosphorylation of MEK1/2 which

subsequently activates ERK1/2 where they either continue activating downstream

targets or translocate into the nucleus to phosphorylate transcription factors 161.

1.6.1.2 p38Pathway

The mammalian p38 kinase consist of four members, p38α, p38β, p38γ and p38δ 162.

These p38 kinases can be activated by a range of extracellular stimuli such as UV

irradiation and cellular stress or by hormones and inflammatory cytokines. p38 kinases

are activated by MEK3 and MEK6 by phosphorylation. Once phosphorylated, p38 then

activates MAPK interacting kinases (Mnk1 and Mnk2).

1.6.1.3 JNK/SAPK Pathway

The cJun N-terminal kinase (JNK) family consist of three members, JNK1, JNK2 and

JNK3 161. JNK is activated by cytokines, growth factors or cellular stress. Upon

activation, JNK is phosphorylated by MEK4 or MEK7 and translocates into the cell

nucleus and further activates transcription factors including cJun, ATF2, STAT3 and

HSF1 161. Transactivated cJun results in increased expression of genes which promoters

consist of AP-1 sites such as TGFβ 153.

1.6.1.4 Phosphoinositide 3-kinase Signaling pathway

Phosphoinositide 3-kinase (PI3K) pathway (Figure 1.11) is another major signaling

pathway with many key regulatory roles in cellular processes including cellular

survival, proliferation and differentiation 163. PI3K/Akt is known to be involved in various TGFβ regulated processes including apoptosis and cell cycle arrest 164,165.

Ligand

Receptor Tyrosine Kinase

Ras PI3K LY294002

PIP2 PIP3

PTEN Akt

Response e.g cell proliferation

Figure 1.11

PI3K/Akt signaling pathway. Dotted lines represent activation of PI3K pathway via a

MAPK signaling protein Ras. Red lettering represent PI3K inhibitor.

PI3K are targets of receptor tyrosine kinases and G-protein coupled receptors as well as

Ras proteins. Integrins are also known to be able to activate the PI3K signaling pathway

166. Activated PI3K converts phosphatidylinositol-4-5-bisphosphate (PIP2) to phosphatidylinositol-3-4-5-triphosphate (PIP3) which subsequently activates Akt, a

serine threonine protein kinase 167. Regulation of the PI3K/Akt signaling pathway is

mediated by the tumour suppressor protein PTEN (protein phosphatase) which

dephosphorylate the 3' end of PIP3 and block Akt activation 167. It is also known that

Smad 3 is involved in TGFβ signal transduction and is a target of inhibition by

PI3K/Akt signaling cascade, which suggests the presence of cross-talks between the

signaling pathways.

1.7 Integration of MAPK and PI3k/Akt with TGFβ Signaling

Pathways

In any cellular signaling networks, cross-talk between related or different signaling

networks always exist as regulatory controls to achieve efficiency in cellular physiology.

As Ras signals through the MAPK pathway, the effects of TGFβ are mediated through

the Smad proteins. One convergence between these two signaling pathways is the

activation of p53 168. The Ras/MAPK cascade lies downstream of TGFβ receptor tyrosine

kinase (RTK) signaling. The activation of the RTK/Ras/MAPK signaling pathway

phosphorylates p53 which in turn interacts with TGFβ-activated Smads to target gene

expression. Studies have shown that ERK or JNK activation by RTKs can phosphorylate

Smad 3 at Thr 178, Ser 203 and Ser 207 protein residues in the linker region of Smad 3.

In addition, MAPKs can also regulate Smad 4 and Smad 7 by direct phosphorylation

140,169. In fact, ERK, JNK as well as p38 are all involved in the regulation of Smad 7170,171.

These findings indicated that TGFβ signaling pathway can be indirectly affected

through signaling cross-talk. On the other hand, PI3K/Akt signaling pathway can also

affect TGFβ signaling though its modulation of Smad 3. However, the regulation of

Smad 3 by the PI3K/Akt signaling pathway still remains unclear as it can either

potentiate or inhibit TGFβ responses172,173. It must be noted that TGFβ can also regulate

PI3K/Akt signaling, as evident by the increase in Akt activity in response to TGFβ 174.

1.8 RESEARCH AIM AND RATIONALE

Wound healing is undoubtedly complex with numerous molecular pathways affected

by a range of different factors. Multiple cross-talks exists within the molecular signaling pathways that are yet to be completely understood. This may contribute to the extensive control mechanism of the wound healing process hence providing a specific and effective approach in the regulation of wound healing. Whether or not all the different pathways converge together at some point remains to be elucidated.

Undisputedly, actin cytoskeletal proteins play a fundamental role during wound

healing, without which a lack of cell movement would lead to no wound healing. As a

result, it is very important that the actin cytoskeleton is regulated and this is

fundamentally achieved by the gelsolin family of actin remodeling proteins. In this

thesis, we describe two interconnected pathways affecting the cytoskeleton and wound

healing; first is the gelsolin androgen linked mediated pathway and second is the Flii

linked TGFβ signaling pathway. The underlining common denominator between these

pathways is the gelsolin family of actin remodeling proteins. This is highlighted by

recent findings on the role of gelsolin as a novel transcription regulator in AR-mediated wound healing. The implications of Flii in affecting TGFβ expression significantly expands the boundaries of potential roles for both gelsolin and Flii not just in the modulation of actin dynamics but also other biological roles.

In the first part of the thesis, we explored the relationship of gelsolin and the androgen receptor (AR) in androgen mediated wound healing. At present, steroid hormone mediated wound healing has been investigated and differences in wound repair has been observed. It is now known that hormone receptors can act as transcription factors which require co-regulators to activate target genes. This led to the identification of several co-regulators which includes gelsolin amongst other family members. Since gelsolin also plays important roles in wound healing, we hypothesize that gelsolin may

have a role in androgen mediated wound repair. The aim of this part of the thesis was

therefore to determine if gelsolin plays a part in androgen-mediated wound repair.

This thesis forms two main parts. The first section investigates the role of gelsolin in androgen mediated wound repair. Due to the inconclusive results obtained in part one of the thesis, the major focus of the thesis became the functional role of Flii in mediating

TGFβ gene expression and activity which forms the second part of the thesis.

TGFβ is a major cytokine during wound healing which functions to attract immune cells to prevent infection while also inducing gene expression. Flii, on the other hand, is a negative regulator of wound repair. Manipulation of Flii was observed to coincide with changes in TGFβ expression levels. As a result, our hypothesis was that Flii may be an important regulator of TGFβ expression. Therefore, in this part of the thesis, we aim to determine if Flii association with cell signaling molecules provide a potential mechanism for modulating TGFβ gene transcription and signaling.

CHAPTER TWO

Role of gelsolin in androgen mediated wound healing

2.1 Introduction

2.1.1 Implications for the gelsolin superfamily in nuclear receptor

signaling

The expression of AR target genes inevitably requires the recruitment and interaction of

nuclear co-activators which cooperate together as a complex to initiate expression. The

identification of AR co-activators have revealed many different protein classes

including, β-catenin, breast cancer gene BRCA-1, cyclin E and more importantly, members of the gelsolin family 175. It was first reported in 2001 that supervillin from the

gelsolin family of ABP associates with the AR to enhance its transactivational activity

176,177. Since then, more members of the gelsolin family have been identified to be

involved in nuclear receptor signaling. Flii was also recently identified as a coactivator

in estrogen and thyroid hormone receptor transcription 105. More importantly, Gelsolin itself is identified to be a co-regulator for the AR 81. These findings implicate members of

the gelsolin family in the co-regulation of hormone nuclear receptor signaling. As a

result, there is a possible link for gelsolin in androgen mediated nuclear receptor

signaling during wound healing, given the relationship between gelsolin and AR in

which androgens also play an important part during the wound healing process.

2.1.2 Gelsolin and AR Nuclear Signaling

Looking more closely at the connection between AR and gelsolin, it has been previously

reported that gelsolin enhances the transcriptional activity of AR in the presence of an

agonist, either androgens or hydroxyflutamide 81. The evidence indicated that gelsolin

facilitates the nuclear translocation efficiency of AR during signaling which enhances

AR activity. Further investigation revealed two regions within AR in which gelsolin

interacts in the presence of a ligand; a central DNA binding domain and a ligand

binding domain in the COOH-terminal domain 81. Additionally, most AR co-regulators

have FXXMF or FXXFF peptide motifs which are present not just in gelsolin but also in

members of the gelsolin family including Flii, supervillin and advillin 178. This result

further implicates the involvement of the gelsolin family in nuclear receptor function.

Regardless, the enhanced transcriptional activity of AR is highly dependent on two factors, exposure to AR ligand and the co-expression of gelsolin. In fact, gelsolin has been demonstrated to colocalize with AR during nuclear translocation in the presence of ligand but had no effect if AR was absent 81. In other words, gelsolin remained in the

cytoplasm if AR expression is abrogated even in the presence of ligands. The gelsolin

behavior is expected and predictable quite simply because gelsolin lacks a nuclear

translocation signal. Therefore, it is only possible for gelsolin to co-translocate into the

nucleus while binding to other proteins which most likely in this case is AR. These findings suggest whilst the role of gelsolin in AR mediated gene expression may be to facilitate nuclear translocation, possibly through it's actin binding capabilities, its role in androgen mediated wound healing remains unknown.

Considering that Gelsolin plays an important role in cellular migration and androgens have an important role in the wound healing process, we sought to determine if gelsolin may be an important link between AR and androgen mediated wound healing.

2.2 Materials and Methods

2.2.1 Cells, Cell Culture

Human Foreskin Fibroblasts (HFFs) were cultured in Dulbecco’s modified Eagle’s

Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics (100U

penicillin and 100ug/500ml streptomycin). Cell cultures were incubated at 37°C and 5%

CO2. Cells were serum starved in DMEM containing antibiotics for at least 3 hours or

otherwise stated prior to experimenting.

2.2.2 Scratch Assay

Cells were seeded onto sterile glass coverslips at a density of 2 x 105 cells/well, in a 6

well tissue culture plate and cultured until confluence. Cells were serum starved for 3

hours which were then linearly scratched multiple times using a P200 yellow pipette tip

producing approximately 20mm x 3mm wounds before treatment reagents were added.

Protein samples were obtained at time-points 0, 1, 3, 5, 10 and 24 hours after initial

wounding and analyzed. For immunocytochemical staining, samples grown on sterile

coverslips were fixed in ice-cold acetone for 10 seconds and placed in 1x PBS and stored

at 4°C.

2.2.3 Protein Extraction

For monolayer cell cultures, the cells were washed twice with ice-cold 1x PBS before

5ml of ice-cold 1x PBS was added and a cell scraper was used to gently lift the cells off.

The cell suspension was then centrifuged at 1000rpm for 5mins at 4°C and the supernatant discarded. 500µl of lysis buffer (50mM Tris pH 7.5, 1mM EDTA, 50mM

NaCl, 0.5% Triton-X100, protease inhibitor cocktail tablet (1 per 10ml - Complete Mini

(Roche, NSW, Australia) was then added to resuspend the cell pellet by pipetting up and down several times before incubating at 4°C for 30mins. The samples were then centrifuged at 14,000g at 4°C for 30mins and supernatant collected.

Bicinchoninic Acid (BCA) Quantification Kit (Pierce, Cat# 23227, Illinois, USA) was used as instructed by the manufacturer to quantify protein concentrations. Standards of

Bovine Serum Albumin (BSA) concentration from 20µg/ml to 2000µg/ml were prepared to obtain a standard curve against which the protein samples were quantified. Working reagent was made in the ratio of 50:1 solution A to solution B respectively. Both standards and protein samples were then transferred to 96-well microtitre plate at a volume of 10µl per well in duplicates which working reagent was then added. The samples were then incubated at 37°C for 30mins and absorbance measured at a wavelength of 570nm in a microplate reader.

2.2.4 Western Blotting

Protein samples were equalized by dilution with lysis buffer and loading buffer added before heating at 95°C. Protein fractions were then electrophoresed on a 10% separating

(3.35ml 30% Acrylamide-Bis Solution (37.5:1, 2.6% C, BioRad Laboratories, CA, USA),

1.25ml 3M Tris pH 8.9, 5.25ml distilled water, 125ul 10% SDS, 100ul Ammonium

Persulfate (APS) and 6.25ul TEMED (N,N,N’,N’ – Tetramethylethylene-diamine, Sigma

Aldrich, Sydney, Australia)) and 4% stacking (0.5ml 30% Acrylamide, 0.276ml 0.5M Tris pH 6.8, 4.104ml distilled water, 50µl 10% SDS, 40ul 10 % APS and 4µl TEMED) SDS-

PAGE gels at 100V for 90 mins and then transferred onto 0.2µm pore nitrocellulose membrane (Advantec MFS Inc, CA, USA) by wet transfer (Bio-Rad Laboratories,

Regents Park, NSW, Australia) using standard wet transfer - Towbin’s buffer (25mM

Tris, 192mM Glycine, 20% Methanol and 0.05% SDS) at 100V for 1 hour. Membranes were stained in Ponceau Red Staining Solution (Sigma Aldrich, Sydney, Australia) for 5 minutes to ensure equal protein loading and was later destained in distilled water and washed in PBS Tween (0.3% Tween/1x PBS).

Membranes were blocked in 5% milk blocking buffer (5% skimmed milk powder and

0.3% Tween-20 diluted in 1x PBS) for 1 hour and primary antibodies diluted in blocking buffer added to the membrane and incubated overnight at 4°C. Stringent washes with blocking buffer were performed every 15 minutes for an hour before appropriate

secondary antibodies conjugated with horse radish peroxidise (HRP) was added for 1

hour at room temperature. Washes were then performed before signal detection using

Super Signal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology,

Rockford, USA) and signal capture using GeneSnap analysis program (Syngene,

Maryland, USA). Membranes were stripped and re-probed with β-tubulin as a loading control (Sigma Aldrich, Sydney, Australia).

2.2.5 Immunocytochemistry

Sterile glass coverslips were placed in six well culture plates and cells were seeded at a

5 density of 3 x 10 cells per well and cultured in an incubator at 37°C with 5% CO2. Cells

were serum starved for 3 hours before adding treatments and incubating for a further

30 minutes. This was followed by fixing cells with cold acetone for 10 seconds and

placed back into 1x PBS containing six well plates. Washes were performed after every

treatments using 1x PBS. 3% Normal Horse Serum (NHS) diluted in 1x PBS were used

to block cells for 30 minutes at room temperature before incubating with mouse anti-AR

antibodies overnight at 4°C(Refer to Table 2.1). Secondary anti-rabbit Alexa Fluor 488

were added for 1 hr in the dark at room temperature (Refer to Table 2.1). This was then

followed by incubating with mouse anti-gelsolin antibodies for 1 hr at room

temperature. The respective biotinylated secondary antibodies were added and

coverslips incubated for 1 hr in the dark at room temperature. After this, streptavidin

Concentration used Antibody Manufacturer Catalog # Raised in Western Immunostaining

Androgen Receptor Santa Cruz sc815 Rabbit 1µg/ml 2µg/ml

Gelsolin BD Biosciences 610412 Mouse 1µg/ml 2µg/ml

β-Tubulin Sigma T4026 Mouse 0.01µg/ml N/A

HRP-conjugated DAKO P0448 Goat N/A 2µg/ml 2° Anti-Rabbit IgG HRP-conjugated DAKO P0447 Goat N/A 2µg/ml 2° Anti-mouse IgG 2° Anti-rabbit Invitrogen A11008 Goat N/A 2µg/ml Alexa Fluor 488 Biotinylated Vector BA2000 Horse N/A 2µg/ml 2° Anti-mouse IgG Laboratories Strepdavidin Invitrogen S32355 N/A N/A 2µg/ml Alexa Fluor 555

Table 2.1

Information of antibodies used in western analyses and immunofluorescent staining.

conjugated Alexa Fluor 555 was added for 1 hr in the dark at room temperature. DAPI

was added to the cells before a final wash and then mounted onto a microscope slide

using DAKO Fluorescent Mounting Medium (DAKO, Botany, Australia). Slides were stored in the dark at - 20°C. Integrated fluorescence intensity was determined using

AnalySIS software (Soft-Imaging System GmbH, Munster, Germany). Negative controls

were included to demonstrate antibody staining specificity. Control samples undergo

the exact staining procedure omitting either the primary or the secondary antibody. All

control samples had negligible immunofluorescence.

2.2.6 siRNA knockdown Assay

HFFs were seeded into 6 well tissue culture plates and cultured overnight to achieve

30% to 50% confluence at time of transfection. Small interfering RNA (siRNA) for

gelsolin (M-007775-01, Dharmacon, CO, USA) and negative control siRNA (Cat#4611,

Ambion, TX, USA) were used to knockdown gelsolin expression levels in HFFs. siRNA

were transfected into the cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).

Both siRNA and Lipofectamine 2000 were diluted in Opti-MEM I Reduced Serum

Medium (Invitrogen, Carlsbad, USA). 250µl of siRNA (optimized to 100nM per well)

was mixed with 4µg of Lipofectamine 2000 diluted in 250µl Opti-MEM and were

allowed to complex at room temperature for 20 minutes. 500µl of siRNA:Lipofectamine

2000 complex was then added to each well, mixed and cells incubated for 6 hours before

replacing transfection media with DMEM containing 10% FCS only. Cells were incubated for 24-48 hours prior to gene knockdown assessment by real-time quantitative PCR and western blot analysis.

2.2.7 RNA Extraction

RNA extraction from cell cultures only required scraping cells from culture flasks after adding Trizol Reagent (Invitrogen, Victoria, Australia). Samples were transferred into fresh eppendorf tubes and centrifuged at 12,000g at 4°C for 10mins to remove cell debris. The samples were incubated for 5mins at room temperature before adding 200µl of chloroform to each tube and were mixed thoroughly by hand for 15secs. The samples were kept at room temperature for 3mins and centrifuged at 12,000g for 15mins at 4°C.

The aqueous phase containing RNA was transferred into a fresh tube and 500µl of isopropanol added to precipitate the RNA. The samples were then incubated at room temperature for 10mins before centrifuging at 12,000g for 10mins at 4°C. The supernatant was discarded and the residual pellet washed with 1ml of 75% ethanol.

Finally, samples were centrifuged at 7500g for 5mins at 4°C and supernatant discarded.

The pellet was dried and re-dissolved in 50µl DEPC water.

2.2.8 DNase Treatment and RNA Quantitation

RNA samples were subjected to DNA-free DNase Treatment and Removal Kit

(Ambion, TX, USA) as instructed by the manufacturer to remove any contaminating

genomic DNA. Firstly, RNA samples were treated with 0.1 volume of 10x DNase Buffer and 1µl of DNase I and incubated at 37°C for 30mins. Following this, 0.1 volume of

DNAse Inactivating Reagent was added to the samples and incubated at 2mins at room temperature with occasional mixing. The samples were then centrifuged at 10,000g for

90secs and supernatant transferred to fresh tubes. RNA was quantitated by diluting 1 in

20 with RNase free water and 100µl duplicates were quantified using a Pharmacia

Biotech GeneQuant RNA/DNA Calculator using RNase-free water as a blank.

Absorbance at 260nm and 280nm were measured that quantify RNA absorbance as

µg/µl concentration. Purity of RNA was confirmed by the A260/A280 ratio and a value

between 1.7 to 2.0 indicates good RNA quality.

2.2.9 Complementary Deoxyribonucleic Acid (cDNA) Synthesis

cDNA was synthesized from RNA using reverse transcription. Each reaction contains

1µg of RNA with 4µl 2.5µM dNTPs (dATP, dCTP, dGTP and dTTP, 100mM each,

Promega, WI, USA) and 2µl Oligo(dt)12-18 Primer (25µg at 0.5µg/µl, Invitrogen, Victoria,

Australia). This was heated at 85°C for 3mins and placed in ice immediately. 2µl 10x

Stratascript Buffer (Stratagene, Epson, UK), 1µl RNasin (Promega, WI, USA) and 1µl

Stratascript Reverse Transcriptase (Stratagene, Epson, UK) were added to the mixture

and heated at 42°C for 60mins followed by 92°C for 10mins before cooling it down on

ice. A control sample was prepared with the reagents above with the exclusion of

reverse transcriptase for use in the Real-Time quantitative-Polymerase Chain Reaction

(RTq-PCR) as a negative control.

2.2.10 Real-Time quantitative-Polymerase Chain Reaction (RTq-PCR)

Each PCR reaction tube containing cDNA was set up to a final concentration of 1x SYBR

Green, 1x Amplitag PCR buffer, 3mM MgCl2, 5mM dNTPs, 0.9µM primers (forward

and reverse), 1.25 Units of AmpliTag Gold DNA polymerase in 25µl of H2O. The primer

sequences were generated in Genbank and were as follows; Gelsolin (forward) 5’ CAG

ACA GCC CCT GCC AGC ACC C 3’ and (reverse) 5’-GAG TTC AGT GCA CCA GCC

TTA GGC-3’. Cyclophillin A (forward) 5’-GGT TGG ATG GCA AGC ATG TG-3’ and

(reverse) 5’-TGC TGG TCT TGC CAT TCC TG-3’.

2.2.11 Proliferation Assay

WST-1 proliferation reagent (Cayman Chemical, WI, USA) was used to assess the rate of

proliferation of HFFs and primary cell cultures. This is a tetrazolium salt based reagent

that converts to soluble formazan by dividing cells therefore directly correlates to

cellular metabolic activity and their proliferation rate. Cells were seeded into 96-well

microtitre plates at a density of 105 cells per well in 100µl of 10%FCS DMEM with antibiotics. Cells were then incubated at 37°C CO2 incubator overnight and serum

starved for 3 hours the following day. Cells were then treated with a range of

treatments and incubated for 48 hours or otherwise stated and assayed by adding 10µl

of WST-1 reagent and mixed thoroughly for 1 min on an orbital shaker. The cells were

then incubated at 37°C for 1 hour as per manufacturer’s protocol before measuring dual

absorbance at 450nm and 600nm on a Tecan microplate reader.

2.2.12 Wound Closure Assay

Cells were seeded into 6 well culture plates and cultured until confluent. Cells were

serum starved for 3 hours prior to wounding and addition of treatments. Using a P200

pipette tip, a circular wound was created. Photographs were taken at time-points 0, 24,

36, 48, 60, 72 and 84 hours after initial wounding. Rate of wound closure was

determined, which is defined by the residual area and expressed as a percentage of the

initial wound size.

2.2.13 Statistical Analysis

All statistical differences were determined using the Student’s t-test or ANOVA. P value of less than 0.05 was considered significant.

2.3 Results

2.3.1 Gelsolin and AR responses to wounding in vitro

To determine if gelsolin and AR were affected by wounding, HFFs were subjected to an

in vitro scratch wound assay. Figure 2.1 shows protein extracted at 0, 1, 3, 5, 10 and 24

hours post-wounding and analyzed using western analysis. Both gelsolin and AR

protein expression were increased in response to wounding and had a similar temporal

expression pattern during the 24 hour time-course, albeit a difference in expression peak times (Figure 2.1). Gelsolin expression increased and peaked at 3 hours post- wounding before declining back to basal levels after 24 hours. Similarly, AR expression also increased in response to wounding but AR expression had a lag period before increasing at 3 hours and peaked at 5 hours post-wounding before declining back to baseline levels.

The expression pattern and localization of gelsolin and AR were compared. Cellular localization of gelsolin and AR was determined using immunofluorescence staining

(Figure 2.2). Here, it is shown that AR expression increases in response to wounding.

This is reflected by the increased staining intensity at and after 3 hours post-wounding

(Figure 2.2D, G, J, M) compared to time 0 (Figure 2.2A). During the 24 hour wounding

A IB: 0 1 3 5 10 24 B IB: 0 1 3 5 10 24 Gelsolin AR 88 kDa 110 kDa

β-tubulin β-tubulin 55 55 kDa 55 kDa

C D

300 230 * 210 * 250 190 * * 170 * 200 * * * 150 130 150 110 Percentage Change Percentage Change Percentage 100 90 70 50 50 0 1 3 5 10 24 0 1 3 5 10 24 Hours Hours

Figure 2.1

Western analyses of gelsolin and AR expression in time-course wounding in HFFs.

(A,C) SDS-PAGE. Confluent HFF monolayers were scratched multiple times and total lysate collected at 0, 1, 3, 5, 10 and 24 hours post-wounding and immunoblotted with anti-gelsolin and anti-AR antibodies, giving full length 88 kDa gelsolin and 110 kDa AR protein band respectively. β-tubulin was used as a loading control. Both gelsolin and

AR expression were increased in response to wounding. (B,D) Quantitation of gelsolin and AR protein bands normalized to β-tubulin band and expressed as a percentage to unwounded control (0 hour). Gelsolin expression peaked at 3 hour post-wounding compared to AR which peaked at 5 hour post-wounding before declining back to basal after 24 hours. n = 4, *p < 0.05 compared to time at 0 hours.

time-course, AR expression peaked at 5 hours after wounding before gradually

decreasing at 10 and 24 hours post-wounding. AR staining was also detected in the

cytoplasm and nucleus of both unwounded and wounded HFFs (Figure 2.2A, D, G, J,

M). Gelsolin expression also increased in response to wounding. Upon wounding at

time 0 hour, gelsolin expression was low (Figure 2.2B). The highest gelsolin expression

was observed at 3 hours post-wounding (Figure 2.2E) before decreasing at 5, 10 and 24

hours (Figure 2.2 H, K, N) after wounding. Gelsolin expression was detected in the

cytoplasm and nucleus of both unwounded and wounded HFFs.

Co-localization between AR and gelsolin was also investigated by merging identical

field of view images of AR and gelsolin staining to create composite images (Figure

2.2C, F, I, L, O). Cytoplasmic and nuclear co-localizations are represented by arrowheads and arrows in the merged images respectively. Co-localization of AR and gelsolin were observed throughout the wound repair time-course. Strong co- localization of AR and gelsolin were observed at and after 3 hours post-wounding

(Figure 2.2F, I, L, O). Although cytoplasmic co-localization was observed, the majority

of co-localization was in the nucleus and the nuclear periplasmic region (arrows).

AR Gelsolin Merge w B w C w A

0 Hour

D w E w F w

3 Hours

G w H w I w

5 Hours

J w K w L w

10 Hours

M N O

24 Hours w w w

Figure 2.2

HFFs cultured on sterile glass coverslips were wounded and fixed in cold acetone at 0,

3, 5, 10 and 24 hours post- wounding. Wound (w) is defined by the dotted lines Red

represents AR staining (A, D, G, J, M), green represents gelsolin staining (B, E, H, K, N)

and blue represents nuclear staining. Merge images are composite images of AR and

gelsolin staining (C, F, I, L, O). Cytoplasmic co-localizations (arrow heads) and nuclear co-localizations (arrows) are represented by areas of yellow color staining. n = 6, w = wound. Scale bar = 100µm in (O).

2.3.2 Effects of DHT addition in gelsolin and AR localization

To study the effects of adding the active metabolite DHT on AR and gelsolin expression

in HFFs, immunofluorescent staining was used. DHT is the more active metabolite of

the hormone testosterone and is a ligand to the AR. A physiological concentration of 10-

8M DHT was added to HFFs for a period of 24 hours before immunostaining with anti-

gelsolin and anti-AR antibodies. In the absence of DHT, AR and gelsolin expression was

relatively subdued and localized predominantly to the cytoplasm (Figure 2.3A, B). Little

AR and gelsolin staining was observed in the cell nucleus. Co-localization of AR and

gelsolin was investigated in untreated cells. In the absence of DHT, AR and gelsolin

were predominantly co-localized to the nuclear peripheral region (arrowhead) in the

cytoplasm (Figure 2.3C). Addition of DHT on the other hand resulted in significantly

higher staining intensities for AR and gelsolin (Figure 2.3D, E). The increased

expression of AR and gelsolin were predominantly localized to the nuclear periplasmic

region in the cytoplasm, in addition to the cell nucleus, indicating partial translocation

of both proteins into the cell nucleus. Interestingly, AR and gelsolin co-localization were

observed in the cell nucleus (arrows) in addition to the nuclear peripheral region seen

for the untreated cells (Figure 2.3F).

To investigate the effects of DHT addition on AR and gelsolin expressions, three doses

AR Gelsolin Merge A B C

Untreated

D E F

+DHT

Gelsolin Fluorescent Intensity in HFFs Treated with AR Fluorescent Intensity in HFFs Treated with DHT G DHT H 200 * 140 * 180 * * * 120 160 140 100 120 80 100 60 80 60 40

Fluorescent Intensity 40 Fluorescent Intensity 20 (% Normalized Control)to 20 (% Normalized to Control) 0 0 Untreated 10-9M 10-8M 10-7M Untreated 10-9M 10-8M 10-7M

Figure 2.3

Effects of DHT addition on gelsolin and AR expression and localization in HFFs.

Confluent HFFs were serum-starved then treated with 10-8M of DHT in (A-F) and

varying concentrations of DHT as shown respectively in (G,H). Dual

immunofluorescent staining was then performed on HFFs treated with DHT. Red

represents AR staining (A, D), green represents gelsolin staining (B, E), and blue

represents nuclear staining. Merge images are composite images of AR and gelsolin

staining (C, F). Cytoplasmic co-localizations (arrow heads) and nuclear co-localizations

(arrows) are represented by areas of yellow color staining. n = 3 (A-F), n = 6 (G, H).

*p<0.05 compared to untreated control. Scale bar = 20µm in (F).

of DHT, low (10-9M), physiological (10-8M) and high (10-7M) concentrations were added

to HFFs for a period of 24 hours. The cells were then stained with either AR or gelsolin

antibodies and their fluorescent intensities quantitated. DHT addition slightly increased

gelsolin expression at 10-8M and 10-7M concentrations (Figure 2.3G). DHT at 10-9M

concentration did not significantly affect gelsolin expression as compared to the

untreated cells. On the contrary, DHT significantly increase AR expression in HFFs. All

concentrations of DHT at 10-9M, 10-8M and 10-7M increased AR expression by a

minimum of 50% (Figure 2.3H). No differences were observed between the

concentrations suggesting that AR is highly sensitive to the presence of DHT even in

small amounts.

2.3.3 Time-course localization study of gelsolin and AR in the presence

of DHT.

A time-course localization study of gelsolin and AR was conducted using

immunofluorescent staining. Since nuclear receptor signaling is a rapid response

pathway, it would be expected to show differences in localization of both gelsolin and

AR. The effects of DHT treatment on short-term and long-term localization of gelsolin and AR were investigated. Short-term time-points include 0, 5, 10, 15, 30 and 60 minutes

(Figure 2.4). Long-term time-points include 2, 5, 10, 24, 48 and 72 hours (Figure 2.5).

AR Gelsolin Merge A B C

0 Min

D E F

5 Mins

G H I

10 Mins

J K L

15 Mins

M N O

30 Mins

P Q R

60 Mins

Figure 2.4

HFFs cultured on sterile glass coverslips were fixed in cold acetone at 0, 5, 10, 15, 30 and

60 minutes after the addition of 10-8M DHT. Red represents AR staining (A, D, G, J, M,

P), green represents gelsolin staining (B, E, H, K, N, Q) and blue represents nuclear staining. Merge images are composite images of AR and gelsolin stainings (C, F, I, L, O,

R). Cytoplasmic co-localizations (arrow heads) and nuclear co-localizations (arrows) are represented by areas of yellow color staining. n = 6. Scale bar = 100µm in (R).

At 0 minutes, co-localization between gelsolin and AR was visible but largely localized

in the cytoplasm (Figure 2.4A-C). Nuclear localization of both gelsolin and AR was also

observed. The reason that nuclear staining was seen was due to the time lag which

occurs when DHT was added to the time the image was taken. As a result, the effects of

DHT had already begun by the time the image was taken, therefore explaining the

observation of nuclear staining at time 0. However as time progressed, both gelsolin

and AR translocated into the nucleus gradually, where staining of both proteins was

either concentrated in or around the nucleus. At 5 minutes, co-localization was

observed clearly in the nucleus, represented as pink which becomes increasingly more

defined as time progressed (Figure 2.4D-F). In addition, at 10 minutes post-treatment, staining for AR were beginning to increase significantly, indicating increased expression (Figure 2.4G-I). At 15 to 60 minutes, localization of gelsolin and AR were not significantly different than at 10 minutes except that at 60 minutes, staining for AR was predominantly nuclear where it also co-localized strongly with gelsolin (Figure 2.4J-R).

At longer time-points, gelsolin and AR expression appeared to be increased compared

to time 0 minutes (Figure 2.5A-R). Strong nuclear staining was observed for AR as well

as gelsolin. Gelsolin staining was also distributed throughout the cytoplasm, in addition

to the nucleus. At 5 hours post-treatment, HFFs appeared to be denser compared

AR Gelsolin Merge A B C

2 Hrs

D E F

5 Hrs

G H I

10 Hrs

J K L

24 Hrs

M N O

48 Hrs

P Q R

72 Hrs

Figure 2.5

Gelsolin and AR long-term response to the addition of 10-8M DHT. (A-R) HFFs were

treated with 10-8M DHT and fixed in acetone at 2, 5, 10, 24, 48 and 72 hours after

treatment and then stained with respective antibodies to gelsolin and AR. Red

represents AR staining and green represents gelsolin staining. Longer treatment with

DHT did not yield significant differences in gelsolin and AR localization. At 2 hours

post-treatment and beyond, gelsolin staining and AR staining were localized predominantly to the nucleus where they co-localized as shown in the merge column.

No translocation from the nucleus to cytoplasm was observed after an extended period.

to the earlier timepoints (Figure 2.5D-F). No other significant effects were observed after

5 hours of treatment with DHT. Regardless, in the presence of DHT, AR staining remains to be strongly localized to the nucleus. Gelsolin on the other hand was most strongly localized to the nucleus at 60 minutes post-treatment but was relatively evenly distributed throughout the cell at other time-points.

Using multiple images gathered from immunofluorescence microscopy, the staining intensities of gelsolin and AR at each time-point were quantitated and graphed in

Figure 2.6. As expected, during the early time-points from 0 to 60 minutes, no increase in staining was observed, which indicated that expression of both gelsolin and AR remained relatively constant when treated with DHT in the first 60 minutes (Figure 2.6

A,B). Looking at longer time-points, an increase in staining intensity was observed for gelsolin at and beyond 2 hours, indicating that expression levels were increasing from 2 hours of treatment with DHT (Figure 2.6C). Expression continued to increase the past fifth hour, peaking at 48 hours, where expression levels were approximately 35% higher than at time 0, before leveling off at 72 hours. AR on the other hand also had a similar pattern of increased expression (Figure 2.6D). Expression was significantly higher at 10 hours and continued to increase before leveling off at 72 hours. AR expression was approximately 60% higher than at time 0.

120 120

100 100

80 80 60 60 40 40

20 20 (%) Intensity AR Staining Gelsolin Staining Intensity (%) 0 0 0 5 10 15 30 60 0 5 10 15 30 60 A B Time (mins) Time (mins)

160 200 * * * 140 * * 180 * 160 * 120 * 140 100 120 80 100 60 80 60 40 40

20 (%) Intensity AR Staining 20

Gelsolin Staining Intensity (%) 0 0 0 2 5 10 24 48 72 0 2 5 10 24 48 72 C D Time (Hours) Time (Hours)

Figure 2.6

Multiple images obtained from immunofluorescence microscopy in figure 2.4 and

figure 2.5 were analyzed. AR and gelsolin staining intensities were quantitated and

results normalized to time 0 hours and expressed as a percentage. (A,B) Short time- course treatment with DHT (0, 5, 10, 15, 30, 60 minutes). (C,D) Long time-course treatment with DHT (0, 2, 5, 10, 24, 48, 72 hours). n = 6 per time-point. *p<0.05 compared to Time 0 hours.

2.3.4 Gelsolin gene knockdown using silent RNA technology

To determine whether gelsolin plays a role in the androgen mediated wound repair

process, functional analyses by reducing gelsolin expression were done. Silent RNAs or

short interfering RNAs (siRNA) were used to knockdown gelsolin expression levels.

Optimization of the technique was carried out prior to knocking down gelsolin

expression (results not shown). Three different concentrations of gelsolin siRNA, 50nM,

100nM and 150nM were used to show that 50nM was sufficient to significantly

knockdown gelsolin expression by more than 85% (Figure 2.7A). Additional siRNA did

not further decrease gelsolin expression. Therefore 50nM of siRNA were used in all

subsequent gelsolin knockdown experiments. After knockdown, gelsolin expression

remained significantly lower compared to the control (Figure 2.7B). Expression

remained at approximately 18% and 21% at 72 hours and 92 hours post-transfection respectively. To further illustrate gelsolin siRNA specificity, a non-specific scrambled siRNA sequence was used to transfect HFFs and the end result showed no significant differences in gelsolin expression compared to the control (Figure 2.7C). However, it was observed that decreasing gelsolin expression had an effect on AR expression as shown in figure 2.7D. Specifically, AR expression increased when gelsolin expression was reduced.

120 120 100 100

80 80

60 60

40 40 * * 20 * 20 Gelsolin Expression (%) * * Gelsolin Expression (%) 0 0 A Control 50nM 100nM 150nM B Control 72 Hours 92 Hours

1.2 350 * 300 1 250 0.8 200 0.6 150

Fold Change 0.4 100 AR Expression (%) 0.2 50 0 0

C Control Scrambled siRNA D Control 50nM 100nM

Figure 2.7

Gelsolin expression was knocked down using silencing RNA technology. HFFs were

treated with gelsolin specific siRNA to decrease gelsolin expression. mRNAs were

collected and levels assessed after 48 hours using RT-qPCR. (A) Gelsolin expression levels using 50nM, 100nM and 150nM of siRNA. (B) Gelsolin expression remained suppressed after 92 hours from initial siRNA treatment. Expression level at 92 hours was approximately 20% of control. (C) Using a non-specific, scrambled siRNA sequence, gelsolin expression showed no significant changes. (D) The effects of gelsolin knockdown caused AR expression to increase significantly. n = 3. *p<0.05 compared to control.

2.3.5 Response to DHT treatment in gelsolin-ablated HFFs

The effects of DHT addition on HFFs proliferation were determined by adding

increasing concentrations of DHT (10-7M, 10-8M and 10-9M) and assessing the effect

using the WST-1 colorimetric assay (Figure 2.8). As expected, DHT increased HFF

proliferation by more than 50% at 10-8M DHT compared to the control (Figure 2.8A). 10-

7M and 10-9M DHT also increased proliferation but by a smaller amount. In gelsolin-

ablated HFFs, proliferation was significantly lower compared to control (Figure 2.8B).

This highlighted the importance of gelsolin in cellular proliferation. However, when

DHT was added to gelsolin knockdown HFFs, proliferation increased to be comparable

to the control. 10-7M DHT treated HFFs remained significantly lower than that of the

control (Figure 2.8M). Although proliferation had risen, DHT did not increase

proliferation as significantly as that of HFFs with normal gelsolin levels.

The DHT effects on HFF migration were also investigated using a wound assay that

measures rate of closure over 0, 24, 36, 48, 60, 72 and 84 hours (Figure 2.9). Addition of

DHT at 10-7M, 10-8M and 10-9M all enhanced wound closure (Figure 2.9A). Gelsolin is an

important actin regulator and plays a significant role in cellular migration. As a result, when gelsolin levels were decreased following siRNA transfection, HFF rate of wound closure fell considerably. Addition of DHT at 10-7M and 10-9M concentrations did not

160 * * 140 * 120 100 80 60 40 Proliferation (% to control) (% to Proliferation 20 0

A Control 10-9M DHT 10-8M DHT 10-7M DHT

120

100 * 80 *

20 Proliferation (% to control) (% to Proliferation

0 Control Gelsolin KD Gelsolin KD + Gelsolin KD + Gelsolin KD + B 10-9M 10-8M 10-7M

Figure 2.8

Proliferation of HFFs treated with gelsolin siRNA and DHT. HFFs were serum starved for a minimum of 3 hours before treatments. The assay is based on WST-1 colormetric detection of metabolic enzymes and measured using a spectrophotometer. (A)

Treatment of DHT (10-9M, 10-8M, 10-7M) on normal confluent HFFs (B) Treatment of

DHT (10-9M, 10-8M, 10-7M) on gelsolin siRNA knocked down HFFs. DHT was added to

HFFs 48 hours after initial gelsolin siRNA transfection. n = 6, *p<0.05 compared to controls.

100 Control *a-b 10-9M 80 10-8M *c-e 60 10-7M

40 *f-h Wound SizeWound (%) *i-k 20

0 0 24 36 48 60 72 A Time (Hours)

100 *a-h Control *i-o Gelsolin KD 80 Gelsolin KD + 10-9M

Gelsolin KD + 10-8M 60 Gelsolin KD + 10-7M *p-u

Wound SizeWound (%) *v-z 20

0 0 24 36 48 60 72 84

B Time (Hours)

Figure 2.9

In vitro wound closure assay. HFFs were serum-starved before wounding and measurements recorded at 0, 24, 36, 48, 60, 72 and 84 hours. Wound closure was measured by taking wound size area and converting to percentage of initial wound size. (A) Effects of DHT addition on rate of wound closure. The treatment of DHT accelerated the rate of wound closure as compared to the controls. Rate of wound closure had no differences between DHT concentrations. (B) Effects of gelsolin ablation on HFF wound closure. Wound closure in gelsolin knockdown HFFs was hindered considerably indicating the importance of gelsolin in cell migration. The 84 hours time- point was included to show complete wound closure. Treatment with 10-9M DHT had

no significant effects compared to gelsolin knockdown HFFs and rate of wound closure

was significantly impaired compared to the control. The addition of 10-8M DHT

however did increase the rate of wound closure but remained slower compared to the

control. Addition of 10-7M did increase rate of wound closure slightly at the first 36 hours of wound closure but was of no difference at 48 to 84 hours post-wounding. n = 4,

p<0.05

change the rate of closure, it remained similar to gelsolin siRNA treatment group.

Although in the early time-points, 10-7M DHT treated HFFs had better closure, the significance was low and was identical to gelsolin knockdown cells towards the later time-points. Conversely, gelsolin knockdown HFFs treated with 10-8M DHT had an

improved rate of wound closure. However, it was not enough to recover to that of the

control group. Even with addition of DHT, the rate of wound closure in gelsolin

knockdown HFFs was still notably decreased.

2.4 Discussion

Defining the relationship between two different proteins, gelsolin and the AR is integral

to further understand the fundamentals of hormone mediated wound healing and

potentially could lead to the development of rational pharmacogenomic based

therapeutics. This study has attempted to characterize the relationship between gelsolin

and AR during in vitro wound healing.

Initial studies were focused on elucidating both the gelsolin and AR expression pattern during an in vitro scratch wound assay. The results from this experiment showed up-

regulation of both gelsolin and AR in response to wounding. More specifically, it

showed a temporal increase of gelsolin and AR expression, peaking at 3 and 5 hours

respectively. This increase in gelsolin expression is consistent with findings from

studies showing motility defects, including pronounced stress fibers in skin fibroblasts

from gelsolin knockdown mice 78. The AR increase in expression was also consistent

with the full time-course wound repair findings by Ashcroft et al, 2002. Furthermore,

quantitation of immunofluorescent staining intensities also supported the findings,

showing up-regulation of gelsolin and AR during wound healing. By using a pure

population of human foreskin fibroblasts (HFF), the localization of both gelsolin and AR

could be visualized. Initially, expression of gelsolin was relatively low and localized

mainly to the cytoplasm, as with AR. As time progressed, staining for both gelsolin and

AR increased before leveling off. Nuclear staining was also observed. Since gelsolin does not have a nuclear localization signal, its presence in the nucleus will most likely be serving as a co-regulator for AR target genes 81. During wound healing, gelsolin expression was highest at the leading edge of migrating cells. This is expected as actin cytoskeletal structures such as filopodia have to be regulated for cellular migration 179.

AR was also up-regulated in the leading edge cells which suggests a role for AR in the wound healing process. Since DHT was absent in the cell populations, the increase in

AR would have to be induced by an alternate pathway and maintained by a positive feedback loop 175.

Stimulation of HFFs using a dose response of DHT showed partial nuclear translocation of both gelsolin and AR. Results from Nishimura et al, 2003 showed complete nuclear translocation using COS-1 cells treated with either DHT or hydroxyflutamide. This is in contradiction to this study where no complete nuclear translocation of gelsolin and AR has been observed throughout the time-course experiment using HFFs. Strongest nuclear staining occurred within the first hour of DHT stimulation. This discrepancy may be attributed to the difference in cell line and most likely the sensitivity of cells to

DHT stimulation. Regardless, the co-translocation of both gelsolin and AR in HFFs would suggest cooperative association to initiate AR response genes for cellular growth.

AR expression was induced by addition of DHT and interestingly, DHT addition also

led to an increase in gelsolin expression. Whether this is an experimental coincidence or due to cell growth is difficult to elucidate based on this data alone.

To further assess the role of gelsolin in androgen mediated wound healing, gelsolin

expression was reduced using gene silencing technology. Multiple controls were put in

place to ensure specificity of gelsolin siRNA. Decreased gelsolin expression was

associated with increased AR expression levels. Given the importance of gelsolin as an

AR co-activator81, it seems possible that the increase in AR expression was

compensatory. To verify this observation, expression of a fellow gelsolin member, Flii

was knocked down in the exact same procedure (data not shown). Interestingly, Flii

ablation was not associated with increase in AR expression. This suggests that the result

may be specific to gelsolin and not another member of the family. It is also noted that

Flii was the only family member tested and not all members of the gelsolin family. As

such, it may still be possible that other members of the family i.e. supervillin, may also

cause an increase in AR expression.

Cells are highly responsive to steroid hormones, which acts as a stimulus for various cellular processes including cell growth. Therefore, adding DHT to HFFs led to an increase in cellular proliferation and migration. To elucidate the importance of gelsolin

100

in AR induced cellular proliferation and migration, gelsolin expression was ablated.

The reduction in gelsolin expression results in significantly less proliferation and

impaired migration. This is in agreement with studies done on gelsolin knockout mice

which have impaired wound healing due to impaired actin depolymerization 78. Given

that gelsolin is also a co-activator of AR, the reduction of gelsolin expression would

have a substantial negative impact on AR activity on cellular proliferation and

migration. Indeed, the addition of DHT to gelsolin knockdown HFFs did not increase

proliferation nor accelerate the rate of wound closure beyond that of normal cells

treated with DHT alone. In other words, DHT was still able to increase proliferation and

accelerate migration but to a lesser degree, suggesting that gelsolin was not essential in

the DHT stimulated cellular proliferation and migration. Although the knockdown of

gelsolin decreased migration, it was probably largely due to its role as an actin

remodeling protein that depolymerizes actin to allow cellular migration.

If gelsolin is a crucial co-activator for AR in wound healing, then the knockdown of

gelsolin expression should arrest cellular proliferation and migration which are the

most important processes during wound healing. However, this was not the case as the

presence of DHT improved cellular proliferation and migration. Taken together, this

study has demonstrated that gelsolin is not directly involved in DHT-stimulated cellular proliferation and migration or that functional redundancy has occurred. This

101

implies that while gelsolin acts as a co-activator for AR, its function can be substituted by other co-activators. Perhaps this may account for the increase in AR expression when gelsolin expression is decreased to compensate for the loss of function. The link between gelsolin and the AR is intriguing. If gelsolin is involved in the androgen mediated wound healing molecular process, then many different biological processes could be affected by manipulation of gelsolin levels in order to control AR activity, which translates into many real-life clinical applications for wound healing.

Due to the outcome of this study showing that gelsolin was not a key re-activator for

AR in a fibroblast wound healing model, the project was not continued. However, future work may involve the analysis using a different approach. Genetically, gelsolin knockout mice could be designed for a wound healing trial with treatment of hormones.

It may also be interesting to determine which AR target genes are activated when gelsolin is the co-activator for that particular expression. This will provide a more definitive answer for the involvement of gelsolin as an AR co-activator.

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CHAPTER THREE

Flii deficiency improves wound healing and is associated with

higher expression ratio of TGFβ3 to TGFβ1

3.1 Introduction

3.1.1 Flii, a negative regulator of wound healing

Flii is a relatively novel protein involved in a range of cellular processes including its

functions as an actin remodeling protein and nuclear receptor co-activator (reviewed in

180). The full extent of the biological function of Flii is still not fully elucidated. Recent studies using Flii heterozygous null mice, Flii over-expressing transgenic mice, wild-

type (WT) mice in a well established in vivo wound repair model and in vitro research has revealed that Flii is a negative regulator of the wound healing process 110,111. Flii

affects epidermal keratinocyte and dermal fibroblast cellular proliferation and motility

as well as the ability of fibroblasts to produce collagen I. Flii heterozygous mice exhibit

smaller, more contracted wounds with more α-smooth muscle actin positive (αSMA) myofibroblasts, whereas Flii over-expressing mice showed delayed wound closure with increased dermal gape, reduced cellular proliferation and migration 111. Flii deficiency

also correlates with decreased collagen I expression in both in vivo mice wounds as well

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as in vitro siRNA treated fibroblasts 111. Perhaps the most compelling evidence indicating Flii is detrimental to wound repair is the application of specific Flii neutralizing antibodies to murine incisional wounds. Treatment with Flii neutralizing antibodies improves wound healing, confirming that Flii ablation is beneficial and that manipulation of its activity is possible for therapeutic purposes 111. However, the

underlining mechanisms leading to enhanced healing in Flii heterozygous mice are still

to be determined.

3.1.2 TGFβ and the wound healing process

TGFβ is a powerful multifunctional regulator of cellular growth with effects on

proliferation and differentiation. The most well-known biological function of TGFβ is its

role as a cytokine during the wound healing process, coordinating inflammatory cells as

an initial step to prevent wound infection 181. Even so, the true extent of TGFβ function

is still being unraveled. However, as a cytokine, excessive TGFβ is generally correlated

with increased inflammation. Its influence during the wound healing process is not

only limited to the inflammatory cells but also to epithelial cells such as keratinocytes

and fibroblasts 182-184. TGFβ can affect cellular proliferation and migration, although

various studies have yielded contradictory results indicating that the mechanism of

TGFβ function is still to be completely determined. On the one hand, TGFβ stimulates

the production of integrins (α5β1, αvβ5 and αvβ6) which facilitate the migration of

104

epidermal cells over the provisional wound matrix 185,186. On the other hand, TGFβ

inhibits keratinocyte proliferation in vitro and in vivo 187.

TGFβ is a potential candidate for investigation in the Flii wound healing study for two

main reasons. Firstly, Flii over-expressing mice in the wound healing study exhibit

impaired healing and have increased inflammation. Secondly, 188 reported differential

levels of TGFβ1 in Flii over-expressing wounds, pointing to the involvement of TGFβ in

the wound healing process. As there are three mammalian isoforms of TGFβ, the expression levels of all three isoforms in WT, Flii heterozygous and over-expressing mice wounds will be investigated. It is hypothesized that the impaired healing observed in Flii over-expressing mice is due to excessive expression of TGFβ1.

105

3.2 Materials and Methods

3.2.1 Flii Heterozygous and Over-expressing Mice Generation

The Flii heterozygous (Flii+/-) and over-expressing (FliiTg/+) mice were generated by our

collaborators, Hugh Campbell and Ruth Arkell from Molecular Genetics and Evolution

Group for the Molecular Genetics of Development, Research School of Biological

Sciences, Australian National University, Canberra, ACT, Australia 98. WT mice are of

BALB/c genetic background. The methods of generation/breeding are discussed below

in their respective headings.

3.2.2 Flii+/- mice generation

Flii heterozygous mice (Flii+/-) were generated by loss of function mutation in the Flii

gene via homologous recombination in embryonic stem cells and passage of these cells

through the germ line following chimera production. The generation of these mice and

the resulting mutation is described in 98 and a diagram of the targeting strategy is shown in diagram 1A. Animals homozygous for Flii are embryonic lethal and die in utero at embryonic day 7. The strain was therefore maintained by continuous backcross of heterozygous carriers (Flii+/-) to BALB/c mice. All mice used in this project were

heterozygous carriers from backcross generation 10 or later and thus were BALB/c

congenic. The heterozygous mice were identified using three primer PCR sets that

106

1 kb+ ladder mutant allele (538bp)

NOTE: This figure is included on page 107 wild-type allele of the (210bp) print copy of the thesis held in the University of Adelaide Library.

Campbell et al, 2002 98

107

Figure 1

Targeted disruption of the Flii gene. (A) Illustrative representation of the domain structure of the targeting vector, relevant portion of the Flii gene and the targeted allele after homologous recombination. Restriction enzymes sites, BspEI is denoted by B,

EcoRV by E and NcoI by N. Flii exons are represented by the numbered open boxes. The tk-neo and pgk-thymidine kinase casettes are indicated. (B) Three primer PCR indicating wild-type (210kb) and mutant allele (538bp) products. Animals with one wild-type copy of the Flii gene and one mutant allele expressed no more than 50% of the normal wild-type Flii expression levels.

108

amplified products specific to the WT or targeted allele (Diagram 1B). The PCR was performed on DNA extracted from ear biopsies of potential heterozygotes 189. The animals with one wild-type copy of the Flii gene and one mutant copy are presumed to express no more than 50% of the normal Flii gene expression.

3.2.3 FliiTg/+ mice generation

Mice carrying additional copies of the Flii gene were generated by introducing a cosmid construct into the mouse genome via transgenesis. At the time of strain production, the cosmid contained the Flii gene and surrounding sequences with the extent of the construct being defined via restriction mapping. The availability of the mouse genome sequence allowed us to estimate the extent of the cosmid. It is now known that the cosmid contains all of the neighboring SMCR7 gene and parts of the Topo and LLGL1 genes (see Diagram 2). The transgenic strain was backcrossed to BALB/c animals for 10 generations before being intercrossed and homozygous animals were identified via progeny testing. The mouse colony was subsequently maintained by intercross of animals homozygous for the transgene. These animals carry two copies of the mouse

Flii gene and two copies of the human Flii transgene (Flii+/+; FliiTg/Tg). In some cases,

109

NOTE: This figure is included on page 110 of the print copy of the thesis held in

the University of Adelaide Library.

Figure 2

Domain structure of cosmid containing Flii gene which is used to generate Flii transgenic mice. Cosmid contains SMCR7 gene, parts of TOP3A and LLGL1 genes.

Restriction sites on the cosmid are also shown.

Campbell et al, 2002 98

110

these animals were out-crossed to BALB/c animals to generate progeny that carry two

mouse Flii gene and one copy of human Flii transgene (Flii+/+; FliiTg/+ are denoted as

FliiTg/+ throughout the thesis).

3.2.4 Murine Incisional Wound Surgery

All animal experiments were approved by the Adelaide Women’s and Children’s

Hospital Animal Care and Ethics Committee and the Australian National University

Animal Ethics Committee following the Australian Code of Practice for the Care and

Use of Animals for Scientific Purposes. The mice were housed in the Women’s and

Children’s Hospital animal house and cages were cleaned and food and water replaced

daily.

Surgery was performed on 72 twelve weeks old mice, 24 wild-type (WT), 24 Flii

heterozygous (Flii+/-) and 24 transgenic (FliiTg/+) mice. Four timepoints, 3, 7, 14 and 21 days post-wounding were chosen to represent the different periods in wound healing.

n=6 per timepoint, 2 wounds created per mice. Mice were weighed upon arrival, prior

to surgery and post-surgery at timepoints stated above to monitor their well-being.

Mouse behavior was also observed regularly. Before surgery, the mice were induced

with anesthesia using 5% isofluorane at 2L oxygen per minute. Anesthesia was

maintained using 2% isofluorane at 500ml oxygen per minute throughout surgery. The

111

mice were then shaved. Two equidistant, 1cm full thickness incisions were made

through the skin to the panniculus carnosus on the flanks of the mice extending 3-4 cm from the base of the skull, 1cm either side of the spinal column and left to heal by secondary intention (i.e. wounds left unopposed and not sutured). At designated day of

sacrifice, the mice were weighed and euthanized using CO2 and cervical dislocation.

The mice were then re-shaved and photographs of the wounds were taken prior to

excision. Both left and right wounds were excised and bisected, half was used for

histological analyses and the other half was snap frozen in liquid nitrogen and stored at

-80°C for biochemical analyses. For each mouse group, unwounded skin was collected

from the WT, Flii+/- and FliiTg/+ mice and divided as stated above.

3.2.5 Histological Processing

Tissue collected from surgery was fixed in 10% formalin and processed in a Leica

TP1020 tissue processor which dehydrated the tissues in graded alcohol series (70% for

120mins, 80% for 60mins, 90% for 105mins and 100% for 240mins), cleared in transitional solvent xylene for 180mins followed by 240mins of tissue infiltration with paraffin wax. Tissue sections (4µm) were cut from paraffin-embedded fixed tissue using

Leica RM2235 microtome. Prior to staining, skin sections were dewaxed by a series of xylene (30mins), and graduated ethanol washes (100%, 1min, 70%, 1 min & 30%, 1 min)

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before further processing. Skin sections were either stained with haemotoxylin and

eosin (H&E) or with antibodies listed in Table 3.1.

3.2.6 Immunohistochemistry

Immunohistochemical staining of TGFβ1, TGFβ2, TGFβ3, TβRI and TβRII expression

was performed on all WT, Flii+/- and FliiTg/+ mice skin at day 0, day 3, day 7, day 14 and

day 21 post-wounding and analysis of fluorescence assessed in the dermis of both

wounded and unwounded skin. Antigen retrieval was required for these antigens.

Sections were dewaxed by a series of xylene (30mins), and graduated ethanol washes

(100% for 1min, 70% for 1 min & 30% for 1 min) before rinsing in 1x PBS and pre-treated

with 250ml Target Retrival Solution (TRS) (2.8g Citric Acid, 3.76g Glycine, 0.372g

EDTA, pH 5.9 in 1L 1x PBS) solution. The sections were then microwaved on high

setting for 2mins after which a “ballast” pot of water was added to help absorb some

heat and to prevent damage to sections and pretreatment continued for 2 x 5mins in

microwave with regular “airing” to prevent overheating. Temperature was maintained

at 94°C. Sections were cooled to 50°C on ice before rinsing in 1x PBS and enzymatic

digestion for 3mins with 0.0625g of Trypsin (Sigma Aldrich, Sydney, Australia)

dissolved in pre-warmed 1x PBS at 37°C. Following digestion, sections were washed in

1x PBS and incubated with normal horse serum (NHS, 3% dissolved in 1x PBS) blocking

solution

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Concentration used Raised Antibody Manufacturer Catalog # in Western Immunostaining Santa Cruz TGFβ1 sc-146 Rabbit 1µg/ml 2µg/ml Biotechnology

TGFβ2 R&D Systems AB-12-NA Rabbit 1µg/ml 2µg/ml

AB-244- TGFβ3 R&D Systems Goat 1µg/ml 2µg/ml NA Santa Cruz TGFβ Receptor I sc-399 Rabbit 1µg/ml 2µg/ml Biotechnology Santa Cruz TGFβ Receptor II sc-1700 Rabbit 1µg/ml 2µg/ml Biotechnology 2° Anti-rabbit Invitrogen A11008 Goat N/A 2µg/ml Alexa Fluor 488 Biotinylated Vector 2° Anti-mouse BA-2000 Horse N/A 2µg/ml Laboratories IgG Strepdavidin Invitrogen S32355 N/A N/A 2µg/ml Alexa Fluor 555

Table 3.1

Information of antibodies used in western analyses and immunofluorescent stainings.

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for 30 mins at room temperature. Refer to Table 3.1 for antibody information. This was

followed by another wash in 1x PBS before incubation with primary antibodies (3%

NHS dissolved in 1x PBS) in a moist box overnight at 4°C. Sections were then washed 3

x 2mins in 1x PBS followed by incubation with biotinylated secondary antibodies (1x

PBS) for 1hr at room temperature. Sections were then washed again 3 x 2mins in 1x PBS

and incubated with cy3 Streptavidin (Sigma Aldrich, Sydney, Australia) conjugate (1x

PBS) for 45mins at room temperature. This was followed by final 3 x 2mins washes before mounting sections using Dako Fluorescent Mounting Medium (DAKO, Botany,

Australia). Slides were then stored in dark at -20°C. Integrated fluorescence intensity was determined using AnalySIS software (Soft-Imaging System GmbH, Munster,

Germany). Negative controls were included to demonstrate antibody staining specificity. Control samples undergo the exact staining procedure omitting either the primary or the secondary antibody. All control samples had negligible immunofluorescence.

3.2.7 Cell culture

Human Foreskin Fibroblasts (HFFs) were cultured in Dulbecco’s modified Eagle’s

Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics (100U

penicillin and 100ug/500ml streptomycin). Cell cultures were incubated at 37°C and 5%

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CO2. Cells were serum starved in DMEM containing antibiotics for at least 3 hours or

otherwise stated prior to start of experimenting to synchronize cells.

3.2.8 siRNA knockdown

HFFs were seeded into 6 well tissue culture plates and cultured overnight to achieve

30% to 50% confluence at time of transfection. Sequence of Flii siRNA are as follows;

forward: 5’-GCU GGA ACA CUU GUC UGU GTT-3’, reverse: 5’-CAC AGA CAA GUG

UUC CAG CTT-3’ 105. siRNA were transfected into the cells using Lipofectamine 2000

(Invitrogen, Carlsbad, USA). Both siRNA and Lipofectamine 2000 were diluted in Opti-

MEM I Reduced Serum Medium (Invitrogen, Carlsbad, USA). 250µl of siRNA

(optimized to 100nM per well) was mixed with 4µg of Lipofectamine 2000 diluted in

250µl Opti-MEM and were allowed to complex at room temperature for 20 minutes.

500µl of siRNA:Lipofectamine 2000 complex was then added to each well, mixed and

cells incubated for 6 hours before replacing transfection media with DMEM containing

10% FCS only. Cells were incubated for 48 hours prior to gene knockdown assessment.

3.2.9 RNA extraction

RNA was extracted from Human Foreskin Fibroblasts. RNA extraction from cell

cultures only required scraping cells from culture flasks after adding Trizol Reagent

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(Invitrogen, Victoria, Australia). Samples were transferred into fresh eppendorf tubes

and centrifuged at 12,000g at 4°C for 10mins to remove cell debris. The samples were

incubated for 5mins at room temperature before adding 200µl of chloroform to each

tube and were mixed thoroughly by hand for 15secs. The samples were kept at room

temperature for 3mins and centrifuged at 12,000g for 15mins at 4°C. The aqueous phase

containing RNA was transferred into a fresh tube and 500µl of isopropanol added to

precipitate the RNA. The samples were then incubated at room temperature for 10mins

before centrifuging at 12,000g for 10mins at 4°C. The supernatant was discarded and the

residual pellet washed with 1ml of 75% ethanol. Finally, samples were centrifuged at

7500g for 5mins at 4°C and supernatant discarded. The pellet was dried and re- dissolved in 50µl DEPC water.

3.2.10 DNase Treatment and RNA Quantitation

RNA samples obtained were subjected to DNA-free DNase Treatment and Removal Kit

(Ambion, TX, USA) as instructed by the manufacturer to remove any contaminating

genomic DNA. Firstly, RNA samples were treated with 0.1 volume of 10x DNase Buffer

and 1µl of rDNase I and incubated at 37°C for 30mins. Following this, 0.1 volume of

DNAse Inactivating Reagent was added to the samples and incubated at 2mins at room

temperature with occasional mixing. The samples were then centrifuged at 10,000g for

90secs and supernatant transferred to fresh tubes. RNA was quantitated by diluting 1 in

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20 with RNase free water and 100µl duplicates were quantified using a Pharmacia

Biotech GeneQuant RNA/DNA Calculator using RNase-free water as a blank.

Absorbance at 260nm and 280nm were measured that quantify RNA absorbance as

µg/µl concentration. Purity of RNA was confirmed by the A260/A280 ratio and a value

between 1.7 to 2.0 indicates good RNA quality.

3.2.11 Complementary Deoxyribonucleic Acid (cDNA) Synthesis