THE ROLE OF SEMINAL VESICLE SECRETION (SVS) 7 PROTEIN IN BONE HOMEOSTASIS · THE ROLE OF SEMINAL...

THE ROLE OF SEMINAL VESICLE SECRETION (SVS) 7 PROTEIN IN BONE

HOMEOSTASIS

WILLIAM CUNDAWAN, B. Sc B. MedSci (Hons)

This thesis is submitted to fulfil the requirements of the University of Western Australia

for the degree of

DOCTOR OF PHILOSOPHY 2013

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

School of Pathology and Laboratory Medicine and School of Surgery, Centre for

Orthopaedic Research, QEII Medical Centre, Nedlands, Western Australia

Table of Contents Declaration 1

Scientific Abstract 2

Abstracts and Award 6

Acknowledgements 7

List of Figures 9

Abbreviations 12

CHAPTER 1: BONE BIOLOGY 17

1. Introduction to Bone Biology 18

1.1 Osteoclast 20

1.1.1 Osteoclast phenotype 20

1.1.2 Osteoclast differentiation and function 23

1.2 Osteoblast 30

1.2.1 Osteoblast phenotype 30

1.2.2 Osteoblast differentiation and function 32

1.3 Molecular Communication Between Osteoclasts and 37

Osteoblasts

1.3.1 Osteoblast regulation of the osteoclast 39

1.3.2 Osteoclast regulation of the osteoblast 42

1.3.3 Bidirectional signalling between osteoclasts and 44

osteoblasts

1.4 Bone Diseases 45

1.4.1 Osteoporosis 46

1.4.2 Osteopetrosis 47

1.4.3 Paget’s disease of bone (PDB) 48

CHAPTER 2: SEMINAL VESICLE SECRETION (SVS) 7 PROTEIN AND 50

LY-6 PROTEIN FAMILY

2.1 Introduction 51

2.2 Seminal vesicle secretion (Svs) 7 51

2.3 Svs7 as a secreted member of Ly-6 protein family and the 52

biological importance of other Ly-6 proteins

2.4 Identification of Ly-6 family member, Svs7, as a putative 57

osteoclast-derived coupling factor

CHAPTER 3: HYPOTHESIS AND AIMS 61

3.1 Hypothesis 62

3.2 Aims 62

CHAPTER 4: MATERIALS AND METHODS 64

4.1 Materials 65

4.1.1 Cell lines 65

4.1.2 Bacterial strain 65

4.1.3 Chemical reagents 65

4.1.4 Tissue culture reagents 67

4.1.5 Cytokines 67

4.1.6 Enzymes 67

4.1.7 Antibodies 68

4.1.8 Vectors 68

4.1.9 Purchased kits and systems 69

4.1.10 Oligonucleotide primers 69

4.1.11 Other materials 69

4.1.12 Software 71

4.1.13 Equipments 71

4.1.14 Centrifugation 73

4.2 Buffers and Solutions 73

4.2.1 General solutions 73

4.2.2 Media and agar 78

4.3 Animals 79

4.3.1 Svs7 transgenic (TG) mice generation 79

4.3.2 Svs7 knockout (KO) mice generation 79

4.3.3 Mouse tail tip DNA genotyping 79

4.3.4 MicroCT analysis 81

4.3.5 Histology on hindlimbs and prostate of Svs7 TG and KO 81

mice

4.3.5.1 Fixation, decalcification (for hindlimbs), tissue 81

processing, tissue embedding, and sectioning

4.3.5.2 Dewaxing and H&E staining 82

4.3.5.3 Dewaxing and TRAP staining 82

4.3.5.4 Processing, embedding, and sectioning of non- 83

decalcified bone samples

4.3.5.5 Goldner trichrome staining for the non- 84

decalcified bone samples

4.3.6 Double calcein labelling of Svs7 KO mice 84

4.4 General Methods 85

4.4.1 Isolation and culture of primary cells 85

4.4.1.1 Isolation and culture of bone marrow cells 85

4.4.1.2 Isolation and culture of spleen cells 86

4.4.1.3 Isolation and culture of calvarial osteoblasts 86

4.4.2 Cell culture of COS-7 cell line 86

4.4.3 Cryopreservation of cell lines for long-term storage 87

4.4.4 RNA extraction 87

4.4.5 RNA concentration measurement 88

4.4.6 RT reactions 88

4.4.7 Polymerase chain reaction (PCR) and quantitative 89

PCR (qPCR)

4.4.8 DNA agarose gel electrophoresis 92

4.5 Cell Biology 92

4.5.1 In vitro RANKL-induced osteoclastogenesis, TRAP 93

staining, and osteoclasts count

4.5.2 Analysis of osteoclast nuclei and area (osteoclast fusion 93

mechanism)

4.5.3 In vitro osteoclast bone resorption assay 94

4.5.3.1 Preparation of bone slices 94

4.5.3.2 Mature osteoclasts culture on bone slices 94

4.5.3.3 SEM and analysis of osteoclast resorption pits 95

4.5.3.4 Osteoclast bone resorption assays using Corning 95

Osteo Assay plates

4.5.4 CFU alkaline phosphatase (ALP) staining for osteoblast 96

differentiation assay

4.5.5 In vitro bone marrow stromal cells-derived osteoblasts bone 96

nodule formation (mineralization) assay

4.5.6 MTS proliferation assay on Svs7 KO BMM 96

4.6 Cloning Method 97

4.6.1 Restriction enzyme digestion of DNA 97

4.6.2 DNA extraction and purification from agarose gels 97

4.6.3 Cloning strategy and ligation to construct 98

pGEM-T Easy Svs7

4.6.4 Cloning strategy and ligation to construct 98

pcDNA3.1 Svs7-V5 His

4.6.5 Transformation and plating of the transformed cells 98

4.6.6 Bacterial culture and maintenance 98

4.6.7 Plasmid extraction and isolation 99

4.7 Experimental Methods 99

4.7.1 Cell transfection and collection of conditioned medium 99

expressing Svs7 protein

4.7.2 Luciferase assay 100

4.7.3 Flow cytometry analysis on fixed and stained Svs7 KO BM 100

cells

4.7.4 Fluo-4AM intracellular calcium assay on Svs7 KO osteoclast 101

4.7.5 Cell lysis procedure for Western Blot (WB) protein analysis 102

4.7.6 Velocity density gradient centrifugation 102

4.7.7 Measuring protein concentration 103

4.7.8 Western Blot (WB) SDS-PAGE system 103

4.7.8.1 Preparation of the SDS-PAGE system 103

4.7.8.2 Protein transfer to nitrocellulose membrane 105

4.7.8.3 WB reaction 105

4.7.8.4 Stripping the membranes 106

4.8 Statistical Analysis 106

CHAPTER 5: CHARACTERISATION OF SVS7 TRANSGENIC MICE 107

5.1 Introduction 108

5.2 Results 109

5.2.1 Tissue distribution of Svs7 gene 109

5.2.2 Generation of the Svs7 transgenic (TG) mice 109

5.2.3 Histological phenotyping of Svs7 TG mice 112

5.2.4 Body weight and radiographic assessment of Svs7 TG mice 112

5.2.5 MicroCT analysis of tibia, femur, and lumbar vertebrae of 112

Svs7 TG mice

5.2.6 Svs7 TG mice exhibit a normal bone mass 115

5.2.7 Histological assessment of Svs7 TG bones 115

5.2.8 Svs7 TG mice exhibit an unaffected osteoclast differentiation 121

in vitro

5.3 Discussion 121

CHAPTER 6: CHARACTERISATION OF SVS7 KNOCKOUT MICE 128


6.2 Results 129

6.2.1 Genotyping and characterisation of Svs7 KO mice 129

6.2.2 Slow mice breeding process and gross body phenotypic 131

examination of Svs7 KO mice

6.2.3 Gross tissues phenotypic (macro vs. microscopic) 131

assessment of Svs7 KO mice

6.2.4 Svs7 KO mice exhibit a reduced bone mass phenotype 134

6.2.5 Svs7 KO bone histology 134

6.2.6 Bone formation activity in vivo is unaffected in Svs7 KO 139

mice

6.3 Discussion 139

CHAPTER 7: INVESTIGATION OF INTRINSIC CELLULAR 144

MECHANISM IN SVS7 KNOCKOUT MICE

(OSTEOCLAST AND OSTEOBLAST)


7.2 Results – Part 1: Intrinsic cellular mechanism in osteoclasts 146

derived from Svs7 KO mice

7.2.1 Svs7 KO BMM cultures have increased osteoclastogenesis 146

in vitro

7.2.2 Svs7 KO osteoclasts are larger and have more nuclei than 146

WT

7.2.3 WT and Svs7 KO mice have comparable hematopoietic 149

stem cells (HSCs) and osteoclast progenitor cells

7.2.4 Svs7 KO mice exhibit unaltered expression levels of key 152

osteoclast marker genes

7.2.5 Osteoclast bone resorption activity is not affected in Svs7 152

KO mice

7.2.6 Svs7 KO osteoclasts are more sensitive to Ca2+ influx in 157

response to RANKL and extracellular Ca2+ stimulation

7.2.7 Svs7 KO mice exhibit elevated NFATc1 and ATP6v0d2 160

proteins expression during RANKL-induced osteoclastogenesis

7.2.8 Svs7 protein treatment inhibits the activation of NFAT 164

promoter luciferase activity

7.3 Results – Part 2: Osteoblasts from Svs7 KO mice are 166

phenotypically comparable to WT cells

7.3.1 Svs7 KO primary calvarial osteoblasts exhibit an 166

unaffected proliferation rate in vitro

7.3.2 No defect is observed in Svs7 KO alkaline phosphatase 166

(ALP) osteoblast differentiation in vitro

7.3.3 Svs7 KO osteoblasts exhibit an unaffected bone formation 169

activity in vitro

7.3.4 Analysis of osteoblast marker genes expression levels in 169

Svs7 KO mice

7.4 Discussion 172

CHAPTER 8: GENERAL SUMMARY AND FUTURE DIRECTIONS 176

8.1 Overview of thesis and general discussion 177

8.2 Future directions 184

8.2.1 The role of Svs7 in bone homeostasis 184

8.2.2 The role of Svs7 in osteoclast and osteoblast differentiation 185

and activation

CHAPTER 9: BIBLIOGRAPHY 188

APPENDIX 223

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

where indicated otherwise.

William Cundawan

W/Prof Jiake Xu

Assoc Prof Nathan Pavlos

W/Prof Minghao Zheng

Willi C d

W/Prof Minghao Zheng

2

Scientific Abstract Bone, a living tissue that forms our skeletal frame, is continuously remodelled

throughout life by the complementary activities of bone-resorbing osteoclasts and bone-

forming osteoblasts. The cell-to-cell interaction between osteoclasts and osteoblasts is

tightly regulated to achieve bone homeostasis and is governed by the production and

exchange of various coupling factors. However, the nature and identity of these factors

are still largely obscure. In search for novel osteoclast-derived coupling factors, we

have previously employed subtractive hybridization-based differential screening to

probe for differentially expressed genes between osteoclasts and their mononuclear

progenitors. Using this approach, we have successfully identified seminal vesicle

secretion (Svs) 7, known as calcium (Ca2+) transport inhibitor (Caltrin), as a candidate

gene that was robustly upregulated during receptor activator of NF-κB ligand

(RANKL)-induced bone marrow macrophages (BMM) differentiation into mature

osteoclasts (Davey 2008; Phan 2004). Svs7 is exclusively expressed by osteoclasts in

bone and liberated as a secreted factor.

Svs7 is a secreted member of Lymphocyte antigen (Ly)-6 protein family, sharing the

common three fingered proteins (TFP)/Ly-6/urokinase-type plasminogen activator

receptor (uPAR) motif, has been shown to be involved in several cellular signalling

pathways. Furthermore, previous observations by Davey 2008 describe the protective

effects of recombinant purified Svs7 protein (500μg/kg daily injection) against the loss

of trabecular bone volume, trabecular thickness, and trabecular number at the distal

femoral metaphysis of ovariectomised mice (oestrogen deficiency-induced bone loss

mouse model). From here, it was hypothesized that Svs7 might be an important

regulator of bone homeostasis. To address this, a two-armed approach was employed

using genetically modified mouse models. The first approach involved the generation

and characterisation of Svs7 transgenic (TG) mice. Following the confirmation that

Svs7 transgene was successfully incorporated and expressed at both the transcriptional

and protein levels, histological assessment of tissues, known to abundantly express Svs7

(i.e. prostate), from the TG mice was performed. Despite being successfully expressed,

no obvious morphological difference was observed in these prostate tissues between

WT and Svs7 TG mice. To further explore the potential role of Svs7 in bone, X-ray

analysis was performed. The result showed that there was no striking difference in

skeletons between WT and Svs7 TG mice. More precise Svs7 TG bone phenotypic

examination was conducted by performing microCT and histology analysis on

3

calvariae, proximal tibia, distal femur, and vertebral body of the lumbar bone. MicroCT

analysis together with histological assessment revealed that all major bone parameters

(trabecular bone volume, trabecular separation, trabecular thickness, and trabecular

number) were comparable between WT and Svs7 TG mice. Moreover, tartrate-resistant

acid phosphatase (TRAP) histology sections and histomorphometry data further

confirmed that there was no significant difference in the number of TRAP-positive

osteoclasts in the primary epiphysis between WT and Svs7 TG hindlimbs, implying that

Svs7 TG mice exhibit normal bone mass and structure. As Svs7 is exclusively

expressed in osteoclasts in bone, osteoclast formation was assessed by performing in

vitro time course of RANKL-induced osteoclastogenesis. The results indicated that

osteoclast formation in Svs7 TG mice was comparable to that of WT littermates. Thus,

Svs7 overexpression in Svs7 TG mice was insufficient to determine the physiological

role of Svs7 in bone.

Another approach to investigate the potential role of Svs7 in bone involved the

generation and characterisation of Svs7 global knockout (KO) mice. The deletion of

exon 2 and 3 of Svs7 gene, which encode for the TFP/Ly-6/uPAR domain, led to the

complete absence of Svs7 gene and protein in Svs7 KO mice. Despite an unexpectedly

slow mice breeding process, Svs7 KO mice displayed no overt gross body phenotype.

In addition, no obvious difference was observed in the phenotype of major organs and

tissues between WT and Svs7 KO mice, indicating that Svs7 is not a requirement for

organogenesis. To assess the potential bone phenotype of Svs7 KO mice, microCT

analysis on WT and Svs7 KO femurs was performed. The results showed that there

were significant reductions in trabecular bone volume and trabecular number in Svs7

KO mice compared to WT, suggesting that Svs7 KO mice exhibit a phenotype of

osteopenia/osteoporosis. Furthermore, this reduced bone mass phenotype was also

confirmed histologically in sections of Svs7 KO hindlimbs. TRAP histology sections

and histomorphometry data revealed that there was a trend of increase in osteoclast

number near growth plate region in Svs7 KO hindlimbs compared to WT, while no

significant difference in bone formation activity in vivo was observed between WT and

KO mice. Together, these results indicate that Svs7 is a novel regulator of bone

homeostasis, possibly owing to a defect in osteoclast differentiation observed in Svs7

KO mice.

4

To elucidate the intrinsic cellular mechanism underlying the osteoporotic phenotype of

Svs7 KO mice, a series of in vitro assays for osteoclast and osteoblast differentiation

and activity were performed. In vitro time course of RANKL-induced

osteoclastogenesis showed that there was a significant increase in osteoclast number in

Svs7 KO BMM cultures at day 3 and 5 of osteoclastogenesis compared to WT.

Interestingly, relative to WT, Svs7 KO-derived osteoclasts were significantly larger and

possessed more nuclei (i.e. ≥ 11) than WT at day 5 of osteoclastogenesis, while no

detectable defects in Svs7 KO early osteoclast progenitor and hematopoietic stem cell

populations were observed. Despite the increase in osteoclast number and size, there

was no significant difference in osteoclast bone resorption activity or the expression of

known osteoclast marker genes between WT and Svs7 KO mice, indicating that Svs7

has a potential intrinsic role in osteoclast differentiation but not in osteoclast activity.

By performing Fluo-4AM intracellular Ca2+ assays on WT and Svs7 KO osteoclasts, it

was found that KO osteoclasts were more sensitive to Ca2+ influx than WT in response

to RANKL and extracellular Ca2+ stimulation, implying that, together with the observed

larger osteoclast size in Svs7 KO BMM cultures, Svs7 may function as a regulator of

osteoclast fusion. Consistent with this position, western blot analysis on cell lysates of

time course RANKL-induced WT and Svs7 KO BMM differentiation into osteoclasts

showed a significant elevation in nuclear factor-activated T cell cytoplasmic 1

(NFATc1) (at day 3 of osteoclast differentiation) and a trend of increase in d2 subunit of

v0 domain of V-H+ATPase complex (ATP6v0d2) fusogenic protein expression in KO

osteoclasts compared to WT, indicating that Svs7 may regulate osteoclast fusion

through the Ca2+-NFATc1 signaling pathway, which has its main effect on ATP6v0d2

protein expression. On the other hand, no significant differences were observed in

osteoblast differentiation and mineralization activity in vitro between WT and Svs7 KO

mice.

In conclusion, these studies constitute the first comprehensive genetic approach to study

the involvement of Svs7 in bone. Moreover, these studies demonstrate that Svs7 KO

mice exhibit an osteoporotic phenotype, with a significant increase in osteoclast number

and size and no observable defects in osteoblast differentiation and activity in vitro and

in vivo. Through the Ca2+-NFATc1 signaling pathway, Svs7 has shown to regulate

osteoclast differentiation and fusion but not osteoclast activity. Therefore, Svs7 plays an

important regulatory role in the maintenance of bone homeostasis, via an autocrine

5

action on osteoclasts, and thus might represent a potential target for the management

and treatment of osteoclast-related bone diseases.

6

Abstracts and Award 1. Cundawan W, Tickner J, Abel T, Chim SM, Kular J, Zheng MH, Pavlos N, and Xu

J (2012) “Seminal Vesicle Secretion (SVS) VII, upregulated by RANKL in

Mature Osteoclasts, Regulates Osteoclast Precursor Proliferation,

Differentiation, and Bone Homeostasis”. Australian and Scientific Medical

Research (ASMR) Week Symposium, Perth, Western Australia. (Oral Presentation)

2. Cundawan W, Tickner J, Abel T, Chim SM, Kular J, Zheng MH, Pavlos N, and Xu

J (2012) “Seminal Vesicle Secretion (SVS) VII is expressed by mature

osteoclasts and acts to regulate osteoclast precursor proliferation,

differentiation, and bone homeostasis”. 2012 Annual Australian and New Zealand

Orthopaedic Research Symposium (ANZORS), Perth, Western Australia. (Oral

Presentation)




differentiation, and bone homeostasis”. 22nd Annual Australian and New Zealand

Bone and Mineral Society (ANZBMS) Research Meeting, Perth, Western Australia.

(Oral Presentation)




differentiation, and bone homeostasis”. Sir Charles Gairdner Hospital Medical

Research Month Symposium, Perth, Western Australia. (Oral Presentation) 2012

Scientific Young Investigator Award 5. Cundawan W, Tickner J, Abel T, Chim SM, Kular J, Zheng MH, Pavlos N, and Xu

J (2013) “Seminal Vesicle Secretion (Svs) 7 Protein is expressed by Mature

Osteoclasts and Acts to Regulate Osteoclast Fusion”. Combined Biological

Science Meeting (CBSM), Perth, Western Australia. (Poster Presentation)

7

Acknowledgements I would like to thank my coordinating supervisor, W/Prof Jiake Xu (UWA School of

Pathology and Laboratory Medicine) for giving me the chance to do PhD in medical

research and for your excellent supervision and care. My sincere warm appreciation and

thanks go to my co-supervisor, Assoc Prof Nathan Pavlos (UWA Centre for

Orthopaedic Research, School of Surgery) for his patience, guidance, and an excellent

supervision throughout my PhD years. Your unbelievable positive attitude in medical

research and your excellent example of the integrity in life has always been there to

support me to complete my study and to learn to become a better person each day. I

would also like to thank warmly W/Prof Minghao Zheng (UWA Centre for Orthopaedic

Research, School of Surgery) for his support and advices throughout my candidature

years.

My special thanks and huge appreciation go to Dr Tamara Abel (UWA CMCA) for

your tremendous help, technical support, and advices that have contributed a lot to this

study.

My special thanks also go to Dr Jennifer Tickner and Dr Shek Man Chim (both from

UWA School of Pathology and Laboratory Medicine) and Dr Taksum Cheng (UWA

Centre for Orthopaedic Research, School of Surgery) for the friendship, help, technical

support, and advices during my study.

For my special friends, Dr Ee Cheng Khor, Dr Jasreen Kular, Dickson Phiri, and

Chulwon Chang. Thank you and I cannot express it enough to you all for the true

friendship, support, and advice. Even though very few in number, your friendship holds

a special place in my life and has helped me tremendously during my study and will

always be during my life time here in Australia. I am so sorry I haven’t been able to

catch up with you guys for sometimes but I promise I will soon.

To Euphemie, Ying Hua, and Li (UWA Centre for Orthopaedic Research, School of

Surgery) and Jacob (UWA School of Pathology and Laboratory Medicine), thank you

so much for your expertise and assistance in histology. I am really happy to have had

experiences working with you guys during my study. Thank you Jay (UWA School of

Medicine and Pharmacology) for your assistance in using BMG machine during my

8

study and thank you Dr Kathy Heel (UWA School of Pathology and Laboratory

Medicine) for your help in my flow cytometry experiments.

To James Burgess, thank you so much for helping me to get connected to the school

network and printers, with me being the frequent office mover (3 different offices

during 3-years study in the School of Pathology and Laboratory Medicine). Thank you.

To all my lab colleagues from UWA School of Pathology and Laboratory Medicine:

Bay Sie, Vincent, Ben, Dian, Lin, Ryan, and Gaurav, and from UWA Centre for

Orthopaedic Research, School of Surgery: Pei Ying, Su, Zhu Xiang, Qin An, Lin Zhen,

Louis, and Colin, thank you for your friendship, help, supports, and laugh/fun that u

guys have provided during my study.

To my office colleagues, Laura, Li and Dagwin, thank you for your friendship and

support. My loneliness and emptiness in the office during my thesis writing was slowly

disappeared after you guys came in.

For my family, especially Ama, Mum and Dad, and my brothers, thank you so much for

the love, opportunity, supports, and trusts you have given me to complete my study in

Australia. Through your unconditional love, I remember and hold God blessing in my

life every day.

To my dearest love, Audrey Chan, my whole heart fills with gratitude of thankful and

love for you every day, as from nowhere earlier years in my life, now I can picture and

know who my self really is with your extremely strong perseverance and unconditional

love and trust for me. We had been tested really hard through times but we come on top

and God has really answered our prayers. With no doubt at all, my heart and soul want

to dedicate this thesis for you. Without you, who have filled my time with unconditional

love, companion, support, and prayers, I would not able to do it. ‘I Love You So Much

and I will never give up’.

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List of Figures Page

Figure 1.1 The four important domains of polarized, multinucleated 21

osteoclasts

Figure 1.2 Cell-to-cell contact between osteoclast and osteoblast 25

Figure 1.3 Signalling pathways in osteoclast differentiation 26

Figure 1.4 ARF cycle in bone remodelling process 31

Figure 1.5 Osteoblast differentiation from mesenchymal stem cells 33

Figure 1.6 The Wnt/ββ-catenin signalling pathway in osteoblast 38

Figure 2.1 Consensus cysteine residues and a C-terminal consensus 53

sequence motif of CCXXXXCN (TFP/Ly-6/uPAR domain)

of Ly-6 proteins family

Figure 2.2 Identification of Svs7 gene using subtractive hybridization- 58

based differential screening in RAW264.7 cells culture with

RANKL stimulation

Figure 2.3 Svs7 is the most highly expressed Ly-6 gene by mature 59

osteoclasts, shown by the microarray heatmap analysis

Figure 2.4 Svs7 gene expression in osteoclast 60

Figure 4.1 Svs7 transgenic (TG) mice generation 79

Figure 4.2 Svs7 knockout (KO) mice generation 80

Figure 5.1 Tissue distribution of Svs7 mRNA expression 110

Figure 5.2 Generation of the Svs7 TG mice 111

Figure 5.3 Histological examination of prostate of Svs7 TG mice 113

Figure 5.4 Body weight and radiographic assessment of Svs7 TG mice 114

Figure 5.5 2D and 3D microCT images of proximal tibia, distal femurs, 116

and vertebral body of the lumbar bone of Svs7 TG mice

Figure 5.6 Svs7 TG mice exhibit a normal bone mass 118

Figure 5.7 Histological examination on H&E-stained decalcified Svs7 119

TG bone samples

Figure 5.8 Histological examination on goldner trichrome and TRAP- 122

stained non-decalcified femur sections of Svs7 TG mice

Figure 5.9 Bone histomorphometry analysis on NOc/BS (N/mm) of 124

TRAP-stained non-decalcified femur sections of Svs7 TG

mice

Figure 5.10 Svs7 TG mice exhibit an unaffected osteoclastogenesis 125

10

in vitro

Figure 6.1 Svs7 gene and protein were absent in Svs7 KO mice 130

Figure 6.2 Slow mice breeding process and gross body phenotypic 132

examination of Svs7 KO mice

Figure 6.3 Gross tissues phenotypic assessment of Svs7 KO mice 133

Figure 6.4 Svs7 KO mice exhibit a reduced bone mass phenotype 135

Figure 6.5 Svs7 KO bone histology 137

Figure 6.6 Bone histomorphometry analysis on TRAP-stained 140

decalcified femur sections of Svs7 KO mice

Figure 6.7 Svs7 KO mice exhibit an unaffected bone formation 141

activity in vivo

Figure 7.1 In vitro time course RANKL-induced Svs7 KO BMM 147

differentiation into mature osteoclasts

Figure 7.2 Svs7 KO osteoclasts are larger than WT 148

Figure 7.3 Svs7 KO osteoclasts have more nuclei than WT 150

Figure 7.4 Svs7 KO mice exhibit unaltered population of HSCs and 151

early osteoclast precursor

Figure 7.5 The deletion of the Svs7 gene does not affect MAPK and 153

NF-κκB signalling pathways in Svs7 KO osteoclast

precursor

Figure 7.6 Key osteoclast marker genes expression levels are not 154

affected in Svs7 KO mice

Figure 7.7 SEM analysis (on bone slices) for Svs7 KO osteoclast bone 155

resorption activity

Figure 7.8 Corning osteo plate analysis for Svs7 KO osteoclast bone 156

resorption activity

Figure 7.9 Svs7 KO osteoclasts are more sensitive to Ca2+ influx in 158

response to RANKL and extracellular Ca2+ stimulation

Figure 7.10 Svs7 KO osteoclasts have a significant increase in 161

NFATc1 protein expression during RANKL-

induced osteoclastogenesis

Figure 7.11 Svs7 KO osteoclasts have a trend of increase in 162

ATP6v0d2 protein expression during RANKL-

induced osteoclastogenesis

Figure 7.12 DCSTAMP protein expression appears to be unaffected 163

11

in Svs7 KO osteoclasts throughout the time course of

RANKL-induced osteoclastogenesis

Figure 7.13 Svs7 protein treatment inhibits NFAT promoter luciferase 165

activity

Figure 7.14 Svs7 KO primary calvarial osteoblasts exhibit an unaffected 167

proliferation rate in vitro

Figure 7.15 No defect is observed in Svs7 KO ALP osteoblast 168

differentiation in vitro

Figure 7.16 Svs7 KO osteoblasts exhibit an unaffected bone formation 170

activity in vitro

Figure 7.17 Analysis of osteoblast marker genes expression levels in 171

Svs7 KO mice

Figure 8.1 Hypothetical model of the role of Svs7 in the regulation of 182

osteoclast fusion through the Ca2+-NFATc1 signalling pathway

Supplementary Figure 1 Serum biochemistry measurement on calcium 224

and phosphate levels in Svs7 TG mice

Supplementary Figure 2 Svs7 protein detection at the secretory vesicles 225

fraction of osteoclasts

12

Abbreviations ALP Alkaline phosphatase

α-MEM Alpha minimum essential medium

AP-1 Activator Protein 1

ARF Activation (Bone resorption) reversal and formation

ARO Autosomal recessive osteopetrosis

ATF4 Activating transcription factor 4

ATP Adenosine Triphosphate

BFR Bone formation rate

BM Bone marrow

BMC Bone mineral content

BMD Bone mineral density

BMM Bone marrow macrophages

BMP Bone morphogenetic protein

BMSC Bone mesenchymal stem cell

BMU Basic multicellular unit

BV/TV Bone volume per tissue volume

Ca2+ Calcium

CA-II Carbonic anhydrase-II

Caltrin Calcium transport inhibitor

CaMKIV Calcium/Calmodulin-dependent kinase IV

cAMP Cyclic adenosine monophosphate

CCD Cleidocranial dysplasia

cDNA Complementary deoxyribonucleic acid

C/EBP CCAAT/enhancer binding protein

CFU Colony forming unit

ClCN Chloride channel

CLS Coffin-Lowry syndrome

CREB cAMP response element-binding protein

Csk C-terminus Src family kinase

CT-1 Cardiotrophin-1

CTP Cytidine triphosphate

CTR Calcitonin receptor

DAP12 DNAX-activating protein 12

13

DCSTAMP Dendritic cell-specific transmembrane protein

DHT dihydroxytestosterone

Dkk-1 Dickoff-1

DNA Deoxyribonucleic acid

DTT Dithiothreitol

DEXA Dual energy X-ray absorptiometry

ECM Extracellular matrix

EDTA Ethylene diamine tetracetic acid

ELISA Enzyme-linked immunosorbent assay

EMA European medicines agency

ERK Extracellular signal-regulated kinase

FAK Focal adhesion kinase

FBS Fetal bovine serum

FcRγ Fc receptor common gamma chain

FDA United States Food and drug administration

FGF Fibroblast growth factor

GPCR G-protein coupled receptor

GPI Glycosylphosphatidylinositol

H&E Haematoxylin and eosin

HSC Hematopoietic stem cell

HSCT Hematopoietic stem cell therapy

IBSP Integrin-binding sialoprotein

IGF Insulin-like growth factor

IκB Inhibitor of kappa B

IKK Inhibitor of kappa B kinase

IL-1 Interleukin-1

IP3 Inositol-1, 4, 5-triphosphate

IPTG Isoprophyl-β-D-thiogalactopyranoside

ITAM Immunoreceptor tyrosine-based activation motif

JNK c-Jun N-terminal kinase

KO Knockout

LDL Low density lipoprotein

LEF Lymphoid-enhancer factor

LRP LDL receptor-related protein

Ly-6 Lymphocyte antigen-6

14

M-CSF Macrophage colony stimulating factor

MAPK Mitogen-activated protein kinase

MAR Mineral apposition rate

MicroCT Microcomputed tomography

MITF Microphthalmia-associated transcription factor

MMP Matrix metalloproteinase

MRF Myogenic regulatory factor

mRNA Messenger ribonucleic acid

MSC Mesenchymal stem cell

nAChRs Nicotinic acetylcholine receptors

NADH Nicotinamide adenine dinucleotide

NADPH Nicotinamide adenine dinucleotide phosphate

NBF Neutral Buffered Formalin

NF-κB Nuclear factor kappa b

NFATc1 Nuclear factor-activated T-cell cytoplasmic 1

OB Osteoblast

OC Osteoclast

OCN Osteocalcin

OPG Osteoprotegerin

OPN Osteopontin

OPPG Osteoporosis-pseudoglioma syndrome

OSCAR Osteoclast-associated receptor

OSE Osteocalcin-specific element

Osx Osterix

OVX Ovariectomised

PAGE Polyacrylamide gel electrophoresis

PATE Prostate and testes-expressed protein

PBS Phosphate buffered saline

PCR Polymerase chair reaction

PDB Paget’s disease of bone

PIP2 Phosphatidylinositol-4, 5-bisphosphate

PLC Phospholipase C

PMSF Phenylmethylsulphonyl fluoride

PPAR Peroxisome proliferator-activated receptor

PTH Parathyroid hormone

15

Pyk2 Proline-rich tyrosine kinase 2

RANKL Receptor activator of NF-κB ligand

RGD Arginine-glycine-aspartic acid

RNA Ribonucleic acid

Rpm Revolutions per minute

RT Reverse transcription

S1P Sphingosine 1-phosphate

Sca-1 Stem cell antigen-1

SDS Sodium dodecyl sulphate

SEM Scanning electron microscopy

SEM (Statistics) Standard error mean

sFRP Secreted frizzled receptor protein

SLURP Secreted Ly-6/uPAR-related protein

SOCE Store-operated calcium entry

SOST Sclerostin

Sox Sex determining region Y-box

SPARC Secreted protein acidic and rich in cysteine

SQSTM1 Sequestosome 1

STAT Signal transducer and activator of transcription

SVC Seminal vesicle content

Svs7 Seminal vesicle secretion 7

TAE Tris-acetate EDTA

Tb.Sp Trabecular separation

Tb.Th Trabecular thickness

Tb.N Trabecular number

TBS Tris buffered saline

TCF T-cell factor

TCIRG1 T cell immune regulatory 1

TFP Three-fingered protein

TG Transgenic

TGF Transforming growth factor

TNF Tumour necrosis factor

TRAF Tumour necrosis factor receptor-associated factor

TRAP Tartrate-resistant acid phosphatase

TREM2 Triggering receptor-expressed on myeloid cells 2

16

TRPV4 Transient receptor potential vanilloid 4

uPAR Urokinase-type plasminogen activator receptor

V-H+ATPase Vacuolar- H+ATPase

WB Western blot

WT Wild type

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside

17

CHAPTER 1

BONE BIOLOGY

Chapter One – Bone Biology

18

1. Introduction to Bone Biology Bone is a living tissue whose function is to provide a skeletal frame in mammals and an

important organ for the formation of hematopoietic cells and the maintenance of a

normal level of blood calcium (Dvorak-Ewell et al. 2011; Takayanagi 2007). Bone is

continuously remodelled throughout life by two major resident cells, bone-resorbing

osteoclasts and bone-forming osteoblasts. Bone remodelling is an important metabolic

process regulating bone growth, structure and function during adult life (Boyle, Simonet

& Lacey 2003; Teitelbaum 2000a). A highly regulated cell-to-cell interaction between

osteoclasts and osteoblasts is governed by local and systemic factors, such as growth

factors, cytokines, and hormones (Karsenty & Wagner 2002; Zaidi 2007). A balance in

osteoclast and osteoblast activity is critical to maintain healthy bone mass (bone

homeostasis), otherwise perturbations in skeletal structure and function associated with

skeletal diseases may occur (Zaidi 2007). An uncoupled bone formation by the

osteoblast can result in osteopetrosis (an extreme high bone mass), while an excessive

bone resorption activity by osteoclasts underlies skeletal diseases such as osteoporosis

(an extreme low bone mass) and Paget’s disease of bone (PDB) (He et al. 2011; Raisz

2005b).

Osteoclasts are large terminally differentiated multinucleated cells, capable of resorbing

bones (Teitelbaum 2000a; Vaananen et al. 2000). Osteoclasts originate from

monocytes/macrophages lineage of hematopoietic stem cell, in which multinucleated

osteoclasts are formed by the fusion of mononuclear macrophage precursors

(Teitelbaum 2000a). On the other hand, osteoblasts are the only bone cells with the

ability to form bone (Harada & Rodan 2003). Osteoblasts are derived from multipotent

mesenchymal stem cells, which later express specific bone matrix proteins such as

osteopontin (OPN), osteocalcin (OCN), and type I collagen to be incorporated in newly

formed bones (Aubin et al. 2008; Ducy et al. 1997; Yoshida 2012). Fully differentiated

osteoblasts lay down bone matrix proteins in the osteoclasts resorbed compartment,

which are then mineralized to become calcified bone to complete bone remodelling

process.

The balance in activity between osteoclasts and osteoblasts is essential to the

maintenance of bone homeostasis. Disruption to this balanced system will greatly

contribute to the development of many skeletal diseases including osteoporosis,

osteopetrosis, PDB, and rheumatoid arthritis. Osteoporosis is a common skeletal disease


19

occurring worldwide, in which there is excessive bone resorption activity by osteoclasts,

causing a decrease in bone mass and strength and microarchitectural deterioration of the

skeleton and hence having a higher risk of bone fragility fractures (Raisz 2005b). Bone

mineral density (BMD) measurement is used to diagnose osteoporosis and osteopenia

before fractures occur (Raisz 2005a). The current and still most acceptable treatment for

osteoporosis is using anti-resorptive agents such as nitrogen-containing

bisphosphonates, which include alendronate, risedronate, ibrandonate, and zoledronic

acid (Epstein 2006; Lewiecki 2011). These agents bind to the bone surface, causing

inactivation and programmed cell death when they are taken up by the osteoclasts.

Although this treatment is the current frontline therapy, it is unable to reverse the

structural damage caused by osteoporosis (Delmas 2002). More recently, clinical trials

have focused on the use of denosumab, a fully human immunoglobulin G2 monoclonal

antibody specific to Receptor Activator of NF-κB ligand (RANKL) (Kostenuik et al.

2009; Rizzoli, Yasothan & Kirkpatrick 2010). This drug is designed to block the

interaction between RANKL, expressed by osteoblasts, and its receptor RANK,

expressed by osteoclasts and their precursors, hence inhibiting osteoclast differentiation

and activation and causing a net increase in bone formation. The European Commission

and US Food and Drug Administration (FDA) have both approved Denosumab for

clinical use and to date, it shows promising results in treating postmenopausal

osteoporosis and bone loss in men associated with hormone ablation and prostate cancer

(EMA ; FDA ; Rizzoli, Yasothan & Kirkpatrick 2010). On the other hand, defects in

osteoclast differentiation (osteoclast-poor) and function (osteoclast-rich) are the

common cause of osteopetrosis, characterized by an abnormal high bone mass and a

defect in bone-marrow formation (Frattini et al. 2000; Guerrini et al. 2008; Sobacchi et

al. 2007; Takayanagi 2007). Together with PDB, these three skeletal diseases become a

huge burden in our society and they will be discussed later in this chapter.

Basic research examining the regulation of ‘cross-talk’ between osteoclasts and

osteoblasts is fundamental to the discovery of novel genes/proteins, which regulate

intercellular communication, and may help to uncover new therapies for the

management and treatment of many pathological bone diseases. Hence, this project will

focus on a novel osteoclast-secreted protein, Svs7, which has shown to have a role in

regulating bone homeostasis.


20

1.1 Osteoclast

Osteoclasts are multinucleated, giant, bone resorbing cells that are terminally

differentiated and formed by the fusion of mononuclear progenitors of the

monocyte/macrophage lineage of hematopoietic stem cells (Suda et al. 1995;

Takayanagi 2007; Teitelbaum 2000a; Vaananen et al. 2000). Osteoclasts are located on

both endosteal and periosteal surfaces of bone (Li, Kong & Qi 2006). Activated

osteoclasts form resorption pits on bone and dentine slices by acid decalcification and

proteolytic degradation (Boyle, Simonet & Lacey 2003; MacDonald & Gowen 1993;

Suda et al. 1995; Takayanagi 2007) to produce irregular scalloped cavities (Howship

lacunae) on trabecular bone surfaces, or cylindrical Haversian canals in cortical bone

(Raisz 1999).

1.1.1 Osteoclast Phenotype

Osteoclasts exist in two functional states, the motile and the resorptive (active) states.

Motile osteoclasts are flattened, non-polarised cells, having membrane protrusions,

called lamellipodia, at their leading edge and podosome complexes in a row behind the

leading edge to help them to move from bone marrow to their resorptive sites. When

reaching their resorptive sites, osteoclasts become polarized and active by cytoskeletal

reorganization (Li, Kong & Qi 2006).

This cytoskeletal reorganization results in the formation of four important plasma

membrane domains: a ruffled border, sealing zone, functional secretory domain, and

basolateral membrane, as shown in Figure 1.1. The ruffled border, located inside the

sealing zone, presents finger-shaped projections of the osteoclast plasma membrane

juxtaposed to the bone matrix, which contain all acidification resorbing machinery

organelles (Li, Kong & Qi 2006). The sealing zone, which is composed of actin ring

structure, separates the resorptive lacuna from the rest of the cell; hence it is an

osteoclast-specific structure (Lakkakorpi et al. 1999; Li, Kong & Qi 2006). Osteoclastic

bone resorption activity takes place within the sealing zone.


21

Legend

H+

Sealing Zone

Nucleus

V-H+ATPase

Functional Secretory Domain

Ruffled Border

BONE

Basolateral Domain

Resorptive Pit Cathepsin K

Figure 1.1 The four important domains of polarized, multinucleated osteoclasts.

Activated osteoclasts, by having cytoskeletal reorganization, have four crucial domains

when they attach on to bone surface; ruffled border, sealing zone, basolateral domain,

and functional secretory domain. H+ proton and proteases, such as Cathepsin K, are

secreted by the acidification machinery organelles, V-H+ATPase complex, located in

the ruffled border of the osteoclast to degrade bone matrix.


22

Because of this sealed resorptive compartment, acidity maintenance, which is an

essential condition for bone degradation to occur, can be achieved. Osteoclasts

endocytose by-products of resorption through their basolateral domain and excrete them

through the functional secretory domain. During its bone resorption activity, osteoclasts

are dome-shaped and do not display lamellipodias.

Osteoclast precursors and mature osteoclasts express the tyrosine kinase receptor c-fms,

which is the receptor for macrophage-colony stimulating factor (M-CSF), and RANK, a

member of TNF receptor family, which is the receptor for RANKL (Suda et al. 1999;

Takayanagi 2007; Yasuda et al. 1998). Osteoclasts express tartrate-resistant acid

phosphatase (TRAP) (Hayman et al. 1996; Suda et al. 1997), which is widely used as a

specific histochemical marker of osteoclasts. Mice lacking TRAP activity exhibit a mild

osteopetrosis condition due to an intrinsic defect in osteoclast activity (Hayman et al.

1996). Osteoclasts also highly express the specific lysosomal cysteine protease,

Cathepsin K, which is the major bone matrix-degrading enzyme at low pH environment

(Drake et al. 1996; Gelb et al. 1996; Raisz 1999; Takayanagi 2007; Wilson et al. 2009).

Cathepsin K is responsible for the degradation of type I collagen during osteoclast bone

resorption activity. Hence because of its importance during osteoclast activity,

Cathepsin K-deficient mice show severe osteopetrosis, while in human, mutation in

Cathepsin K gene has been shown to result in pycnodysostosis, a condition

characterized by osteosclerosis and short stature (Gelb et al. 1996; Rachner, Khosla &

Hofbauer 2011; Saftig et al. 2000). Another specific osteoclast marker is matrix

metalloproteinase (MMP), MMP-9 (Nyman et al. 2011; Teti 2012). It belongs to the

gelatinase subfamily of the MMP and hence its substrate is gelatin or denatured

collagen (Ram, Sherer & Shoenfeld 2006). Together with cathepsin K, MMP-9

degrades the organic matrix of bone. Earlier, Haibo, Z et al., 1997 found that the

expression of MMP-9 gene was higher in osteoporotic bone tissues compared to normal

ones, indicating that MMP-9 plays a crucial role in the development of osteoporosis. In

many autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, the

level of MMP-9 expression is also found to be higher than normal controls (Gruber et

al. 1996; Leppert et al. 1998).

The above mentioned proteolytic enzymes are secreted at the ruffled border of activated

osteoclasts (Figure 1.1), which contains the osteoclast primary acidification machinery,

the vacuolar H+-adenosine triphosphatase (V-H+ATPase) complex (Vaananen et al.


23

2000). This V-H+ATPase pump facilitates the secretion of hydrochloric acid (HCl)/H+

protons into resorption lacunae to create an acidic environment necessary for osteoclasts

to demineralize the hydroxyapatite of bone (Baron et al. 1985; Blair et al. 1989;

Kawasaki-Nishi, Nishi & Forgac 2003; Li, Kong & Qi 2006), followed by the secretion

of the proteolytic enzymes to degrade the organic bone matrix (Vaananen et al. 2000).

Hence, an enhanced bone resorption activity by osteoclasts parallels with a marked

expansion in ruffled border surface.

Osteoclast mononuclear precursors are able to fuse to become multinucleated cells

through an as yet unknown mechanism, which involves the fusogenic genes, Dendritic

cell-specific transmembrane protein (DCSTAMP) (Yagi 2005) and d2 subunit of V-

H+ATPase complex (ATP6v0d2) (Lee et al. 2006). The expression of both of these

fusogenic genes are upregulated during RANKL-induced osteoclastogenesis and are

transcriptionally regulated by Nuclear Factor-Activated T-cell cytoplasmic 1 (NFATc1)

transcription factor, the master regulator of osteoclast differentiation (Feng et al. 2008;

Kim et al. 2008). Osteoclast differentiation and fusion are completely disrupted in both

DCSTAMP and ATP6v0d2-deficient mice despite normal gene expression of other

osteoclast markers, resulting in an osteopetrotic condition (Lee et al. 2006; Yagi et al.

2005).

Another important marker of a mature osteoclast is the high expression of calcitonin

receptor (CTR). Calcitonin has been shown to inhibit osteoclast bone resorption activity

(Wallach et al. 1999; Li, Kong & Qi 2006) through its binding to CTR. Davey, R. A et

al. 2008 has shown that global CTR KO mice had a mild increase in bone formation and

had a normal serum calcium level, as well as unaffected osteoclast surface and activity

indicating a modest role of CTR in the regulation of basal state of bone turnover and

calcium homeostasis (Davey et al. 2008).

1.1.2 Osteoclast differentiation and function

Osteoclasts are differentiated from the monocyte/macrophage lineage of the

hematopoietic stem cells (Suda et al. 1995; Teitelbaum 2000a). Osteoclast

differentiation is induced by cell-to-cell contact between osteoclast precursors and

osteoblasts (Kong et al. 1999; Nakashima & Takayanagi 2011; Suda, Takahashi &

Martin 1992; Takayanagi 2007; Udagawa et al. 1990). M-CSF, secreted by osteoblasts,


24

through binding to its receptor c-fms (Stanley et al. 1997) as shown in Figure 1.2,

promotes proliferation, differentiation, and survival of osteoclast progenitors, as shown

in Figure 1.3 (Suda et al. 1995; Takayanagi 2007), through the activation of

extracellular-signal-regulated-kinase (ERK) 1/2 (p42/44) and phosphoinositide 3-kinase

(PI3K)/AKT signalling pathways (Glantschnig et al. 2003; Oikawa & Yamada 2003;

Ross & Teitelbaum 2005; Sugatani 2005; Tanaka et al. 2003). Downstream signalling

pathway of ERK results in the activation of PU.1, a family member of E-twenty six

(ETS)-domain transcription factors, which then regulates the initial developmental

stages of macrophages and osteoclasts by controlling the expression of c-fms

(Takayanagi 2007; Tondravi et al. 1997). PU.1-deficient mice exhibit osteopetrosis

(Tondravi et al. 1997). Mice having non-functional M-CSF (M-CSF deficiency) have an

osteopetrotic condition and the administration of recombinant human M-CSF to these

mice has been shown to rescue the phenotype (Felix, Cecchini & Fleisch 1990; Yoshida

et al. 1990).

The osteoclast differentiation process is driven primarily by the binding of RANKL,

expressed as a membrane-bound cytokine on osteoblasts, to RANK on the osteoclast

surface (Figure 1.2), which leads to the activation (fusion, polarization and cytoskeletal

rearrangement, as described above in Section 1.1.1) of mature osteoclasts on the bone

surface (Burgess et al. 1999; Kong et al. 1999; Nakashima et al. 2011; Nakashima &

Takayanagi 2011). RANKL activates mature osteoclasts in a dose-dependent manner in

vitro and can also induce rapidly pre-existing osteoclasts to resorb bone in vivo (Boyle,

Simonet & Lacey 2003; Burgess et al. 1999; Fuller et al. 2002).

A number of osteoclast transcriptional factors have been shown to be involved in the

regulation of RANKL-induced osteoclastogenesis, in which one of them is a Nuclear

Factor of Activated T cell cytoplasmic 1 (NFATc1) (Takayanagi et al. 2002). The

NFAT transcription factor family was originally discovered in T cells and has been

shown to be involved in many biological systems (Crabtree & Olson 2002; Hogan et al.

2003).


25

Legend

M-CSF

c-Fms

RANKL

RANK OPG

OSTEOCLAST OSTEOBLAST

OSTEOCLAST DIFFERENTIATION

Precursor cells Fusion Mechanism Osteoclast

Figure 1.2 Cell-to-cell contact between osteoclast and osteoblast. Osteoblasts

express RANKL (membrane-bound cytokine) and secrete M-CSF to induce

osteoclastogenesis. Osteoprotegerin (OPG), decoy receptor for RANKL, is also secreted

by osteoblasts to inhibit osteoclastogenesis and hence it is one of the negative regulators

of osteoclastogenesis.


26

Plasma Membrane

Cytosol ITAM DAP12 /

FcRγ

TREM2 / OSCAR RANKL

RANK

ITAM Co-Stimulatory Pathway RANKL Pathway

M-CSF

c-Fms

TRAF6

Proliferation Cytoskeletal Reorganization Survival NFκB

c-Fos

AP-1

P

PLCγ

Ca2+

CaMKIV

Creb

NFATc1

Auto-amplification

Calcineurin

PU.1 AP-1 NFATc1 Creb MITF

Osteoclastic Genes DC-STAMP, Atp6v0d2, OSCAR, β3integrin, TRAP, Calcitonin receptor, Cathepsin K etc.

Blimp1

Anti-Osteoclastic Genes IRF-8, MafB, Bcl6 etc.

Transcription

IκB IκB P

Ubiquitination and degradation

Nucleus

Figure 1.3 Signalling pathways in osteoclast differentiation. The binding of M-CSF

to its receptor c-fms promotes proliferation, survival, and cytoskeletal organization of

osteoclast precursors and mature osteoclasts. The RANKL-RANK interaction activates

TRAF6, which leads to phosphorylation (degradation) of IκBα and activation of NFκB

through AP1 transcription factor. The co-activation of TREM2/OSCAR, which contains

DAP12/FcRγ domain, by calcium influx into the cell during RANKL signalling causes

the activation of Ca2+ signalling pathway. This pathway signals to PLCγ, which then

results in phosphorylation of calcineurin by calcium-dependent calmodulin kinase IV

(CaMKIV) and subsequent dephosphorylation (activation) of the master regulator of

osteoclastogenesis, NFATc1 by calcineurin. NFATc1 autoamplifies its gene to get a

robust induction, regulating the expression of many osteoclast genes such as

DCSTAMP, ATP6v0d2, Cathepsin K, and OSCAR. NFATc1 has also been shown to

regulate the expression of Blimp1 gene, which in turn regulates the expression of anti-

osteoclastogenic factors, such as MafB and Bcl6. (Figure adapted from Nakashima &

Takayanagi 2011; Kular et al. 2012)


27

RANKL stimulation results in the induction of Ca2+ oscillation through the activation of

co-stimulatory signal of the immunoglobulin-like receptors, such as osteoclast

associated receptor (OSCAR) and triggering receptor-expressed on myeloid cells

(TREM) 2, and the subsequent association of immunoreceptor tyrosine-based activation

motif (ITAM)-containing molecules (DNAX-activating protein (DAP) 12 and Fc

receptor common gamma chain (FcRγ), as shown in Figure 1.3 (Koga et al. 2004; Kular

et al. 2012; Nakashima & Takayanagi 2011). This signalling pathway then leads to the

activation of NFATc1 via a calcium-calmodulin-calcineurin-dependent pathway

(Takayanagi et al. 2002). The NFATc1 promoter contains NFAT binding sites and

NFATc1 specifically auto-regulates (auto-amplifies) its own promoter during

osteoclastogenesis generating a robust induction (Figure 1.3). Activator protein 1

(AP1)-containing c-Fos and the continuous calcium signalling are essential for this

auto-amplification (Matsuo et al. 2004; Takayanagi et al. 2002; Takayanagi 2007).

Common cytokines, such as tumour necrosis factor (TNF), have been shown to induce

Ca2+ oscillations in human macrophages, which subsequently leads to the induction and

activation of NFATc1 transcription factor (Yarilina et al. 2011). NFATc1-deficient

embryonic cells fail to differentiate into mature osteoclasts under RANKL stimulation

and with overexpression of NFATc1, osteoclast precursors are able to differentiate into

active osteoclasts without the need for RANKL stimulation (Takayanagi et al. 2002).

NFATc1 has also been shown to directly regulate the gene expression of fusion-

mediating molecules, which are DCSTAMP and ATP6v0d2, as well as Cathepsin K,

TRAP, CTR, and OSCAR in cooperation with other transcription factors such as AP1,

PU.1, and microphthalmia-associated transcription factor (MITF), as shown in Figure

1.3 (Crotti et al. 2006; Kim et al. 2005; Kim et al. 2008; Lee et al. 2006; Matsumoto et

al. 2004; Nakashima & Takayanagi 2011; Takayanagi et al. 2002; Takayanagi 2007;

Yagi et al. 2005). NFATc1 regulation of these osteoclast genes has also been shown to

require the cooperation between cAMP response element-binding protein (CREB),

which is activated by Ca2+/Calmodulin-dependent kinases IV (CaMKIV), with NFATc1

(Figure 1.3) (Sato et al. 2006; Takayanagi 2007).

NF-κB is another essential transcription factor signalling pathway in osteoclastogeneis.

There are 5 NF-κB subunits, which are cRel (Rel), RelA (p65), RelB, NF-κB1 (p50),

and NF-κB2 (p52) (Karin, Yamamoto & Wang 2004; Takayanagi 2007; Xu et al. 2009).

Upon RANKL binding to its receptor RANK, the trimerization of RANK and TNF

receptor-associated factor (TRAF) 6 begins, which leads to the activation of the NF-κB


28

signalling pathway (Figure 1.3) (Kobayashi et al. 2001). Its classical activation involves

the inhibitor κB (IκB) kinase (IKK) phosphorylation, followed by phosphorylation-

induced proteasomal degradation of IκB and subsequent NF-κB (RelA-p50 dimers)

translocation and activation in the nucleus to regulate a vast number of target genes

(Karin, Yamamoto & Wang 2004; Xu et al. 2009). On the other hand, the alternative

pathway depends on the homodimerization of IKK-α, followed by phosphorylation and

the proteasomal processing of p100 to p52, and the nuclear translocation of RelB-p52

dimers (Franzoso et al. 1997; Karin, Yamamoto & Wang 2004; Xu et al. 2009). Double

knockout (KO) mice for NF-κB subunit p50 and p52 exhibit severe osteopetrosis due to

the defect in osteoclastogenesis (Iotsova et al. 1997).

The AP-1 transcription factor complex is also essential for osteoclastogenesis (Wagner

& Eferl 2005). The family consists of Fos-related (FosB, Fra-1, Fra-2) and Jun-related

(c-Jun, JunB, JunD) genes (Grigoriadis et al. 1994). RANK-RANKL binding induces

AP-1 activation through the induction of its component, c-Fos, which is activated by

CaMKIV/CREB signalling pathway, as shown in Figure 1.3 (Sato et al. 2006;

Nakashima & Takayanagi 2011). Mice with c-Fos deficiency develop osteopetrosis due

to a defect in hematopoietic stem/precursor cells differentiation into osteoclasts

(Grigoriadis et al. 1994). A mutation in c-Jun gene has been shown to inhibit NFATc1-

induced osteoclastogenesis in vitro (Ikeda et al. 2004).

The proto-oncogene c-src, highly expressed by osteoclasts and concentrated on the

ruffled border membranes (Tanaka et al. 1992), is a non-receptor tyrosine kinase, which

regulates osteoclast polarization and activation. C-src-deficient mice show defects in

integrin-mediated intracellular signalling and ruffled border formation and hence

develop osteopetrosis (Boyce et al. 1992; Nakamura et al. 1998; Nakamura et al. 2001).

The c-src tyrosine kinase activity is regulated by phosphorylation and

dephosphorylation of the tyrosine residue located close to the C-terminus region

(Tanaka et al. 2003). Dephosphorylation of c-src induces its activation and C-terminus

Src family kinase (Csk) has been shown to be the negative regulator of c-src kinase

activity by inducing its phosphorylation state (Okada et al. 1991). Downstream

signalling pathway of c-src begins with the activation of αvβ3 integrin, which induces

Pyk2 (another non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family)

tyrosine phosphorylation and subsequent its association with c-src via its SH2 domain

(Duong et al. 1998; Nakamura et al. 2012). This complex is then completed with the


29

binding of p130Cas (Cas, Crk- associated substrate) to Pyk2 via its SH3 domain to

regulate the formation of actin rings (sealing zones), an essential step in osteoclast

activation (Lakkakorpi et al. 1999). Both Pyk2 and p130Cas are localised to the sealing

zones in resorbing osteoclasts and c-src-deficient mice have been shown to have a

decrease in activity of both molecules and subsequent defective ruffled border

formation (resulting in an osteopetrotic phenotype), indicating that both molecules are

downstream modulators of c-src (Boyce et al. 1992; Duong et al. 1998; Lakkakorpi et

al. 1999; Soriano et al. 1991).

Upon attachment to the bone surface, a multinucleated osteoclast polarizes to form 4

important domains; the sealing zone, basolateral domain, functional secretory domain,

and the ruffled border, as described before in Section 1.1.1. This process results in the

creation of an isolated extracellular microenvironment, which will then be acidified to

create a low pH environment. This acidification process is mediated by the secretion of

HCL/H+ protons into the resorptive microenvironment by the V-ATPases complex,

resulting in a pH of ~4.5 (Baron 1985; Teitelbaum 2000b). This first process degrades

the hydroxyapatite of bone, which is then followed by the osteoclast secretion of

Cathepsin K and MMPs to resorb the organic bone matrix (comprises primarily of type

I collagen) (Delaissé et al. 2003; Drake et al. 1996). Bone resorption by-products are

endocytosed through the basolateral domain, transported and excreted to the external

environment through the functional secretory domain of osteoclasts (Vaananen et al.

2000). Following the completion of bone resorption, osteoclasts undergo apoptosis.


30

1.2 Osteoblast

After osteoclasts undergo apoptosis, bone remodelling process continues with the

reversal phase, at which bone resorption activity stops, followed by new bone formation

activity by osteoblasts to lay down new mineralized bone matrix at the resorption

lacunae. Hence, this bone remodelling process is tightly regulated to ensure that there is

an equal amount of bone resorbed and formed (bone homeostasis). Many other cells,

apart from osteoclast and osteoblast, have shown to be involved in bone remodelling

process, such as osteocytes, which are located within the bone matrix, and bone lining

cells, as shown in Figure 1.4 (MacDonald & Gowen 1993; N. A. Sims & J. H. Gooi

2008; Xiong et al. 2011). Together, these cells are commonly known as the basic

multicellular unit (BMU) of bone. This section will focus on the bone forming

osteoblast.

1.2.1 Osteoblast Phenotype

Osteoblasts are cuboidal mononuclear cells (Figure 1.2 and 1.4), which form and

deposit bone matrix (Long 2012). Osteoblasts are usually found in a single layer

adherent to periosteal or endosteal surfaces of bone (sites of active bone formation)

(Baker et al. 1992; MacDonald & Gowen 1993). As discussed before and shown in

Figure 1.2, osteoblasts express RANKL as its membrane-bound osteoclast

differentiation factor (Kong et al. 1999; Yasuda et al. 1998). Osteoblasts are stained

positive for alkaline phosphatase (ALP) and express a number of bone matrix proteins,

such as type I collagen (the major component of the organic bone matrix providing

strength and elasticity to the bone (Mundlos & Olsen 1997)), as well as osteonectin

(SPARC), OCN, OPN, and bone sialoprotein (IBSP) (Aubin & Triffitt 2002; Koga et al.

2005; Termine et al. 1984; Yoshida et al. 2012). These latter non-collagenous proteins

and ALP expression by osteoblasts are essential in the regulation of mineral deposition

and turnover and bone-cell activity (Clarke 2008; Kular et al. 2012). ALP knock out

mice have impaired growth and die before weaning as a consequence of defective bone

mineral deposition and abnormal morphology of osteoblasts (Narisawa, Fröhlander &

Millán 1997). Osteoblasts also express parathyroid hormone (PTH) receptor (PTH1R)

at which PTH exerts its role in regulating bone mass (Goltzman 2008). PTH1R is a

seven trans-membrane spanning receptor, which belongs to the B subfamily of G-

protein-coupled receptors (GPCRs) (Juppner et al. 1991). Mice having mutated PTH1R


31

Bone lining cells Osteoclast

Osteoblast

Resorption Reversal Formation Mineralization

Osteoid

BONE Osteocyte

Figure 1.4 Osteoclast bone resorption activation, reversal, and new bone matrix

formation and mineralization by the osteoblast, known as ARF cycle, in bone

remodelling process. After a regulated bone resorption activity by osteoclasts, there is

then a reversal phase, where there are presumably osteoblast precursors and other cells

modifying the resorbed surface to attract osteoblasts. Then osteoblasts start to secrete

bone matrix protein (unmineralized bone matrix or osteoid), which is then mineralized.


32

gene result in perinatal death with an abnormal skeleton (Soegiarto et al. 2001). PTH

has both anabolic and catabolic effects on bone, which will be discussed further in

osteoblast differentiation and function section.

1.2.2 Osteoblast differentiation and function

Osteoblasts, together with chondrocytes, adipocytes, and myoblasts, are derived from

undifferentiated mesenchymal stem cells (MSCs) (Dennis et al. 1999; Pittenger et al.

1999). This cellular differentiation is highly regulated by specific transcription factors

and growth factors, governing the MSCs to differentiate into each different lineage. Sex

determining region Y-box (Sox) 5, 6, and 9 are essential transcription factors regulating

the MSCs differentiation into chondrocytes (Akiyama et al. 2002; Harada & Rodan

2003), while the induction of CCAAT/enhancer binding proteins (C/EBP) α and the

nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPAR)γ results

in the MSCs differentiation into adipocytes (Rosen et al. 2002). Myogenic regulatory

factors (MRFs), namely MyoD, Myf5, myogenin, and MRF4/Myf6, are required for

myogenic differentiation, as shown in Figure 1.5 (Parker et al. 2012).

The importance of the runt-related transcription factor 2 (Runx2) in osteoblasts

differentiation, as shown in Figure 1.5, has been shown by knockout mice having a

complete absence of osteoblasts/ossification (cartilaginous skeleton) and die shortly

after birth without breathing (Ducy et al. 1997; Komori et al. 1997; Otto et al. 1997).

The complete failure of osteoblast differentiation, shown by the absence of alkaline

phosphatase activity, is accompanied by failure of the bone marrow vascularization in

these mice. Heterozygous mice have a defect in intramembranous ossification,

corresponding to the human condition known as cleidocranial dysplasia (CCD – defect

in the closure of the cranial fontanelles) (Mundlos et al. 1997; Otto et al. 1997). Runx2

regulates osteoblast-specific bone matrix genes expression such as type I collagen,

OPN, OCN, and IBSP, hence it is required for osteoblast differentiation and activity

(Ducy et al. 1999). Runx2 has also been shown to be important for chondrocytes

maturation as there is delayed chondrocytes maturation in Runx2-deficient mice, and

the overexpression of Runx2 causes ectopic hypertrophic chondrocytes differentiation

and endochondral ossification (Inada et al. 1999; Ueta et al. 2001; Takeda et al. 2001).

These findings indicate that Runx2 regulates the differentiation of both hypertrophic

chondrocytes and osteoblasts


33

MESENCHYMAL

STEM CELL

MYOCYTE

CHONDROCYTE

ADIPOCYTE

OSTEOBLAST PRECURSOR

MATURE OSTEOBLAST

Sox9 MRFs (MyoD)

C/EBPα PPARγ

OSTEOCYTE

BMP Osterix Runx2

BMP Osterix Runx2 PTH

BMP PTH

Apoptosis

LINING CELL

Figure 1.5 Osteoblast differentiation from mesenchymal stem cells (MSCs). With

specific transcription factors, mesenchymal stem cells can differentiate into myocytes,

chondrocytes, adipocytes, and osteoblasts. Specific transcription factors, Runx2 and

Osterix together with growth factors (BMP) and hormones (PTH) drive this MSCs

differentiation into osteoblast lineage. After new bone formation activity finishes,

osteoblasts can have one of three fates; they undergo apoptosis, become embedded in

the bone matrix as osteocytes, or become bone-lining cells. (Figure modified from Raisz

1999).


34

(Inada et al. 1999; Ueta et al. 2001). Interestingly, it has been revealed that co-culture

system of ST2 bone marrow-derived mesenchymal pluripotent cell line (expressing

Runx2) with murine splenocytes results in the induction of osteoclast genes expression

and the formation of multinucleated osteoclasts (Baniwal 2012). This result has

successfully correlated with the previous study by Geoffroy et al. 2002, who showed

that overexpression of Runx2 in osteoblasts, under type I collagen promoter, could

cause an impairment in osteoblast maturation and an increase in osteoclastic bone

resorption, resulting in an osteopenic phenotype. Hence, together these studies support

the notion that temporal control of Runx2 expression is necessary for healthy bone

development.

The dual role of Runx2 has suggested that there must be another crucial transcription

factor that specifically drives MSCs differentiation into osteogenesis pathways, which is

now known as osterix (Osx) (Figure 1.5). Osx is a novel zinc finger–containing protein

that was originally identified in C2C12 skeletal muscle stimulated with BMP (a potent

stimulator of bone formation) (Harada & Rodan 2003; Nakashima et al. 2002). Osx-

homozygous null mice have a normal cartilaginous skeleton but lack osteoblast activity,

hence no mineralized bone matrix, and die shortly after birth (Nakashima 2002). In

these mice, chondrocytes are fully differentiated, hinting the specific role of Osx in

osteoblast differentiation (Nakashima et al. 2002). Runx2 expression is normal in these

mice, while Osx is absent in Runx2-deficient mice, indicating that Osx works

downstream of Runx2 signalling pathways to specifically induce osteoblast

differentiation. In this Runx2 downstream signalling pathway, further study on the

inhibition of bone formation activity in NFATc1-deficient osteoblasts has shown that

Osx forms a complex with NFATc1 transcription factor and this interaction regulates

Osx-dependent transcriptional activation of type I collagen in osteoblasts (Koga et al.

2005). Later, Baek, W. Y et al. (2009) showed an osteopenic phenotype with an

increase of trabecular bone but a decrease in cortical bone in Osx conditional KO mice

under the control of a 2.3-kb type I collagen promoter. In these mice, both bone

formation rate (BFR) and mineral apposition rate (MAR) were significantly reduced as

well as the expression of OCN bone matrix protein, indicating that Osx plays an

essential role in regulating osteoblast differentiation and bone formation during early

bone development (Baek et al. 2009). Recently, however it has been revealed that the

overexpression of Osx in primary osteoblasts inhibits the late stage of osteoblast

differentiation as shown by reduced expression of alkaline phosphatase and bone matrix


35

proteins (type I collagen and OCN) and a decrease in mineralization, hence causing

osteopenic phenotype (Yoshida et al. 2012). Together these findings suggest that Osx is

essential for osteoblast differentiation at an early stage (in embryo and postnatal bone

development), but its level of expression at the late stage of osteoblast differentiation

requires a tight regulation for a healthy bone formation and maintenance.

From the study of Coffin-Lowry Syndrome (CLS) (X-linked mental retardation

associated with skeletal abnormalities) by Yang et al. 2004, it is then established that

activating transcription factor 4 (ATF4) is important in the maturation process of

osteoblast cell. ATF4 is critical substrate of RSK2, a gene mutated in CLS. ATF4 has

been shown to directly regulate the expression of osteocalcin in osteoblasts by binding

directly to osteocalcin-specific element (OSE) 1 of the osteocalcin gene promoter

region, hence ATF4 KO mice display delayed bone formation during embryonic

development and low bone mass during postnatal life (Long 2012; Yang et al. 2004).

Furthermore, as Runx2 has shown to bind to OSE2 (Ducy et al. 1997), further study has

illustrated that in the presence of C/EBP, ATF4 could form a complex and synergize

with Runx2 to induce osteocalcin expression (Tominaga et al. 2008).

Cytokines, growth factors, and hormones are also important regulators in osteoblast

differentiation and bone formation. Transforming growth factor (TGF)-β has been

shown to be one of the important regulators for osteoblast differentiation (Janssens et al.

2005; Zuo et al. 2012). This family has three isoforms; TGF-β1, 2, and 3, in which

TGF-β1 is the most abundant isoform in skeletal tissue and has its main role in postnatal

skeletal development and remodelling (Geiser et al. 1998; Janssens et al. 2005; Zuo et

al. 2012). Compared to wild-type (WT) mice, TGF-β1-null mice had significantly

decreased bone mineral content (BMC) and shorter tibia (Geiser et al. 1998). A research

on the role of TGF-β1 in osteoblast regulation shows that TGF-β1 induces early

osteoblast proliferation and bone matrix formation (Hock, Canalis & Centrella 1990).

Furthermore, a recent study has illustrated that TGF-β signal in promoting

osteoprogenitor proliferation, differentiation and to drive the commitment of MSCs to

the osteoblast lineage is through the activation of mitogen-activated protein kinases

(MAPKs), which are ERK1/2, c-Jun N-terminal kinase (JNK), and p38, and Smad2/3

signalling (Matsunobu et al. 2009). BMPs, which belong to TGF-β superfamily, have

also major roles in the skeletal development. BMPs signalling is initiated through

specific type I and II serine/threonine kinase receptors with a specific role in osteoblast


36

differentiation, shown by type IB BMPR (Zhao et al. 2002). Mice overexpressing

truncated dominant-negative BMPR-IB targeted to osteoblasts have been shown to have

severely reduced bone mineral density, bone volume, and bone formation rates as there

were impairment of postnatal bone formation and inhibition of BMP2-induced

osteoblast differentiation (Zhao et al. 2002). Loss of both BMP-2 and -4 in developing

limbs has been shown to result in a severe impairment of osteogenesis (Bandyopadhyay

et al. 2006). Following the binding of BMPs to its receptor, Smad proteins (1, 5 and 8)

are phosphorylated and together with Smad4 then translocate to nucleus to interact with

Runx2 transcription factor to induce osteoblast differentiation (Chen, Zhao & Mundy

2004; Zuo et al. 2012). A recent study has shown that TGF-β1 acts synergistically to

enhance bone formation induced by BMP-2 (Tachi et al. 2011).

PTH has been well-known as a factor that can induce bone remodelling (N. A. Sims &

J. H. Gooi 2008). PTH binds and exerts its signalling through PTH1R, expressed by

osteoblasts (Goltzman 2008). Intermittent PTH treatment in rats has been shown to

increase osteoblast differentiation (increased cellular ALP activity) and hence resulted

in an increase in bone mineral density (Nishida et al. 1994). Kramer, I et al. 2010 have

shown that this PTH bone anabolism action is achieved by the suppression of bone

formation inhibitor, Sclerostin (SOST), which is secreted by osteocytes and embedded

deep in bone matrix. Interestingly, PTH (Keller & Kneissel 2005) and mechanical

stimulation (Robling, Bellido & Turner 2006) have been shown to regulate the

expression of sclerostin. Hence, both environmental and metabolic activities influence

the expression of sclerostin by osteocytes to regulate the anabolic effect of PTH

(Kramer et al. 2010). Interesting studies by Qiu et al. 2010 have shown that TGF-β type

II receptor (TβRII) directly phosphorylates PTH1R to facilitate the endocytosis process

of the TβRII-PTH1R complex, which ultimately leads to the downregulation of PTH

signal. This study indicates that there is an intimate cross talk between growth factors,

hormones, and/cytokines to regulate osteoblast differentiation and activation.

Osteoblast bone formation activity comes into two different processes, endochondral

and intramembranous ossification (Olsen, Reginato & Wang 2000). Endochondral

ossification involves the process of removal of terminally differentiated (hypertrophic)

chondrocytes, the resorption of cartilage matrix, the invasion by both vascular and

hematopoietic cells, and the synthesis of osteoid by migrating osteoblasts. This

ossification process is important for the formation of long bones, such as limbs and ribs


37

(Long 2012; Olsen, Reginato & Wang 2000). On the other hand, intramembranous

ossification describes direct synthesis of bone matrix proteins by osteoblasts. Flat

bones, such as the skull and clavicle bones, are formed by the intramembranous

ossification process (Olsen, Reginato & Wang 2000). The canonical signalling pathway

governing this osteoblast bone formation activity is Wnt/β-catenin signalling pathway

(Kular et al. 2012; Zuo et al. 2012), as shown in Figure 1.6, in which TGF-β, BMP, and

PTH share the same pathway (Lin & Hankenson 2011). Wnt signalling begins with the

ligands binding to a receptor complex, composing low density lipoprotein (LDL)

receptor-related protein (LRP) 5/6 and frizzled receptor. This Wnt ligand binding causes

the activation of Dishevelled, which in turn block phosphorylation-induced

ubiquitination of β-catenin by glycogen synthase kinase (GSK)-3β. β-catenin is then

able to translocate into the nucleus to activate lymphoid enhancer factor (LEF)/T-cell

factor (TCF)-mediated osteoblast genes transcription (Kubota, Michigami & Ozono

2009; Kular et al. 2012). Various secreted inhibitors for Wnt/β-catenin are known,

which include Sclerostin (Li et al. 2005) and Dickoff-1 (Dkk-1) binding to LRP5/6

(Bafico et al. 2001) and secreted frizzled receptor protein (sFRP)-1 binding to Wnt

ligands, as shown in Figure 1.6 (Bodine et al. 2004; Kubota, Michigami & Ozono 2009;

Kular et al. 2012). The importance of Wnt/β-catenin signalling pathway in osteoblast

activity has been shown by a study of loss-of-function mutation in the LRP5 that results

in the development of a rare autosomal recessive osteoporosis-pseudoglioma syndrome

(OPPG) (Gong et al. 2001). The patients of this disease are blind and have early onset

of osteoporosis. Conversely, gain-of-function mutation of LRP5 associates with

autosomal dominant high bone mass (Boyden et al. 2002; Little et al. 2002; Van

Wesenbeeck et al. 2003). Besides forming a new bone matrix, osteoblast also functions

to regulate osteoclast differentiation and activation, through its osteoclastogenic

cytokine expression of RANKL, M-CSF, and OPG, which has been discussed

previously in Section 1.1.2.

1.3 Molecular Communication Between Osteoclasts and Osteoblasts

Intercellular communication between osteoclasts and osteoblasts is critically regulated

to achieve a healthy bone mass (bone homeostasis), as pertubations in this system will

result in the development of many pathological bone diseases (Zaidi 2007), as discussed


38

Plasma Membrane

Cytosol LRP5/6

Wnt

Frizzled

Disheveled

Nucleus

GSK-3

Axin APC

β-catenin

TCF/LEF

β-catenin Target genes

DKK

SOST

Wnt sFRP

Disheveled

GSK-3

Axin APC

β-catenin

P

Proteosomal degradation

Figure 1.6 The Wnt/ββ-catenin signalling pathway in osteoblast. The binding of Wnt

to a receptor complex, LRP5/6 and Frizzled, causes the activation of Dishelved, which

in turn inhibits the phosphorylation-induced ubiquitination of β-catenin by GSK-3

activity. β-catenin is then free to translocate into the nucleus, where it activates

lymphoid enhancer factor (LEF)/T-cell factor (TCF)-mediated osteoblast genes

transcription. Dkk-1 and Sclerostin (SOST) have been shown to inhibit this signalling

by binding to LRP5/6, while sFRP-1 binds to Wnt, hence blocking its binding to

Frizzled receptor. (Figure adapted from Kular et al. 2012)


39

later in this chapter. The communication can occur through a direct cell-to-cell contact

between osteoclasts and osteoblasts with RANKL-RANK signalling as the classical

pathway of osteoblast regulation of the osteoclast (discussed in more details in Section

1.3.1), secondly through diffusible paracrine factors, such as cytokines, chemokines,

growth factors, and proteins secreted by either cell acting on the other, and lastly

through factors that are deposited during osteoblast bone formation activity and

liberated during osteoclast bone resorption with latent and active form of TGF-β being

the classical example for this (Matsuo & Irie 2008).

1.3.1 Osteoblast regulation of the osteoclast

Osteoblasts secrete M-CSF and express RANKL, which are two critical factors driving

osteoclast differentiation and activation. M-CSF promotes osteoclast precursors

(monocytes/macrophages) and mature osteoclasts proliferation, survival, differentiation,

and cytoskeletal organization leading for activation (Glantschnig et al. 2003; Kodama et

al. 1991; Lagasse & Weissman 1997; Nakamura et al. 2001; Oikawa & Yamada 2003;

Ross & Teitelbaum 2005; Suda et al. 1995; Sugatani & Hruska 2005; Takahashi,

Udagawa & Suda 1999; Takayanagi 2007; Tanaka et al. 2003; Yoshida et al. 1990; Zou

et al. 2007). M-CSF signalling has been described previously in Section 1.1.2. Injection

of rhM-CSF two or three times a day for 14 days into osteopetrosis (Op/Op) mice has

been shown to result in an increase in the number of monocytes in the peripheral blood

and hence an increase in osteoclast number seen in the bone trabeculae in metaphyseal

and diaphyseal regions compared to the uninjected mice control (Kodama et al. 1991).

This M-CSF alone however is not sufficient to induce osteoclast differentiation from its

precursors. A physical contact between osteoblast/stromal cells and

monocyte/macrophages is required to drive this osteoclast differentiation (Suda,

Takahashi & Martin 1992). This is achieved by the interaction of RANKL, expressed as

a membrane-bound form on osteoblasts, and its receptor RANK, expressed on

osteoclast precursor cells (Takahashi, Udagawa & Suda 1999; Theill, Boyle &

Penninger 2002). RANKL KO mice have severe osteopetrosis and defective tooth

eruption as osteoclastogenesis is stopped at the pre-osteoclast stage (TRAP-negative

osteoclast precursors) (Kong et al. 1999). The RANKL-RANK interaction subsequently

induces trimerization of RANK and the recruitment of RANK main adaptor molecule,

TRAF6, for the activation of intracellular signalling as described in Section 1.1.2 and

shown in Figure 1.3, which is critical for differentiation and activation of osteoclasts


40

(Kobayashi et al. 2001; Lomaga et al. 1999; Wong et al. 1999). RANKL expression has

been shown to be upregulated by osteoclastogenic factors, such as vitamin D3,

prostaglandin E2, PTH, IL-1, IL-6, IL-11, IL-17, and TNF-α (Horwood et al. 1998;

Kong et al. 1999; Lee & Lorenzo 1999; Nakamura et al. 2012; Theill, Boyle &

Penninger 2002; Yasuda et al. 1998). This RANKL-RANK system is closely regulated

by OPG, secereted by osteoblasts, which acts as a decoy receptor for RANKL (Yasuda

et al. 1998) (Figure 1.2). Mice lacking OPG have been shown to have severe

osteoporosis caused by enhanced osteoclast activity (Bucay et al. 1998; Nakamura et al.

2003).

TNF-α expression and secretion by osteoblasts (Bu et al. 2003; Gowen et al. 1990) have

shown to induce osteoclast differentiation and activity in the presence of M-CSF,

independent of RANKL signalling (Fuller et al. 2002). This signalling mechanism has

been confirmed by the robust induction of osteoclast differentiation and activity by

TNF-α in the presence of OPG. Fuller et al., 2002 has also shown that TNF-α enhances

the RANKL-induced osteoclast differentiation and activation through the formation of

actin ring. Further research by Yarilina, A et al. 2010 has shown the mechanism that

TNF induces a sustained calcium oscillation and subsequently NFATc1 activity in

macrophages to enhance osteoclastogenesis in the presence of RANKL. These results

indicate that TNF-α and RANKL work synergistically to drive osteoclast differentiation

and activation through both NF-κB and NFATc1 (through RANK co-stimulatory

pathway, OSCAR/TREM2, as shown in Figure 1.3) signalling pathways.

TGF-β1, as described previously in Section 1.2.2, is the most abundant TGF-β isoform,

expressed and secreted by osteoblasts during bone formation and stored in bone matrix

in its latent form (Hering et al. 2001; Horner et al. 1998; Huang et al. 2007; Pelton et al.

1991). Osteoclast bone resorption activity, through its lysosomal enzymes as described

in Section 1.1.2, has been shown to release and activate TGF-β1 (Oursler 1994;

Pfeilschifter & Mundy 1987; Zuo et al. 2012), which in turn at its high level TGF-β1

could attenuate or inhibit osteoclast differentiation (Janssens et al. 2005; Takai et al.

1998). The mechanism behind its inhibition in osteoclast differentiation, particularly in

in vivo bone remodelling context and in co-culture system, is through the suppression of

RANKL and enhancement of OPG expression by osteoblasts (Takai et al. 1998).

Besides this consistent result on how TGF-β could inhibit osteoclast differentiation,

many other studies have shown that co-stimulation of RANKL/M-CSF on the M-CSF-


41

stimulated bone marrow cells or extremely pure osteoclast precursor population with

TGF-β could stimulate osteoclastogenesis through the activation of NF-κB pathway

(Kaneda et al. 2000; Koseki et al. 2002). Kim et al., 2005 however have also shown that

apart from RANKL-RANK-TRAF6 signalling pathway, osteoclastogenesis can be

induced through a co-stimulation of TNF-α and TGF-β, indicating that TGF-β indeed

has a regulatory role in osteoclast differentiation and in a big picture, bone remodelling

process, by being involved in the number of important osteoclastogenesis signalling

pathways and its interaction with a wide range of other growth factors, cytokines, and

hormones in bone microenvironment, which needs to be further elucidated. Osteoblast

has also shown to express and secrete BMP2 (Huang et al. 2007), which, together with

RANKL and M-CSF, can induce osteoclast differentiation and survival (Itoh et al.

2001), although the mechanism behind this is still unknown.

Recently, it has been discovered that conditioned medium of OPG-deficient calvarial

osteoblast could inhibit osteoclast formation and at the same time promoted osteoblast

bone formation activity (Hayashi et al. 2012). Through anion-exchange chromatography

and subsequent SDS-PAGE analysis on the protein fractions, the anti-osteoclastogenic

molecule has been found to be the axon guidance molecule, Semaphorin 3A (Sema3A).

It has been found that Sema3A inhibition of osteoclastogenesis before RANKL

stimulation is through its complex with a receptor complex of the ligand-binding

subunit Nrp1, which then binds to the class A plexins 1 (PlxnA1) to suppress its

signalling pathway of PlxnA1-TREM2-DAP12 (Hayashi et al. 2012). This results in the

severely impaired osteoclastic gene expression of Cathepsin K, TRAP, and NFATc1,

and hence the inhibition of osteoclast differentiation in the Sema3A-treated bone

marrow macrophages (BMMs) (Hayashi et al. 2012). In addition, Hayashi et al. 2012

also showed that the migration of BMMs were impaired in Sema3A-treated cells as the

RhoA activation, which is important in actin cytoskeletal rearrangement (Kruger,

Aurandt & Guan 2005), in response to M-CSF was abolished. On the other hand,

Sema3A KO mice have been shown to have a massive decrease in alkaline phosphatase

activity and bone nodule formation, which was also reflected in the suppression of

osteoblastic genes expression, such as Runx2, Osx, and osteocalcin, while at the same

time induced adipocyte differentiation (Hayashi et al. 2012). The mechanism behind

this phenotype was then revealed to be the significant reduction in genes expression of

most of the transcriptional target and nuclear accumulation of β-catenin (Hayashi et al.

2012), which is a critical factor in the canonical Wnt signalling pathway, as shown in


42

Figure 1.6. The activation of small G protein Rac1, which has been shown to be

important in promoting β-catenin nuclear translocation (Wu et al. 2008), was also

reduced in Sema3A KO mice (Hayashi et al. 2012). Hence, Sema3A, a novel osteoblast-

secreted factor, has a major role in maintaining bone homeostasis by regulating both

osteoclast and osteoblast differentiation and activation.

Research on how osteoblast controls osteoclast activity still needs to be done. There is

an indication that osteoblast might control osteoclast activity by controlling the

secretion of bone matrix proteins, such as osteopontin (OPN) and bone sialoprotein

(IBSP) (containing arginine-glycine-aspartic acid (RGD) sequences), which are needed

by the osteoclast to attach to bone surface via its αvβ3 integrin (Murphy et al. 2005;

Ross & Teitelbaum 2005; Schaffner & Dard 2003; Yamamoto et al. 1998).

Furthermore, a recent study has shown that OPN can induce Ca2+ oscillation in

osteoclast, hence resulting in an enhanced NFATc1 activation (Tanabe et al. 2011).

However, the mechanism behind this signalling pathway together with other potential

bone matrix proteins in regulating osteoclastogenesis is still unknown and hence, further

study in this area is necessary.

1.3.2 Osteoclast regulation of the osteoblast

As the mechanism on how osteoblasts regulate osteoclast differentiation has been well

studied, the other way of communication, in which osteoclasts express factors to

regulate osteoblastogenesis, is still largely undiscovered. After osteoclast stops its bone

resorption activity, the ‘reversal’ (quiescent) and new bone formation phases begin,

when osteoblasts lay new bone matrix proteins in regulated manner in the resorbed

compartments (Everts et al. 2002; MacDonald & Gowen 1993; Natalie A. Sims &

Jonathan H. Gooi 2008; Zuo et al. 2012). Studies on nanotopography have shown that

osteoclasts might regulate osteoblast bone formation activity by using the size and

shape of its resorbed area (N. A. Sims & J. H. Gooi 2008).

Many cytokines and growth factors are released and activated during osteoclast bone

resorption activity and might stimulate osteoblast activity. One of the classical factors is

the insulin-like growth factor (IGF) I and II (Hayden, Mohan & Baylink 1995), with

IGF-II been the major growth factor stored in bone. IGFs, released in active form from

bone matrix during osteoclast bone resorption, stimulate osteoblast activity to initiate

site-specific bone formation in proportion to the resorbed bone volume (Hayden, Mohan


43

& Baylink 1995). Furthermore, Zhang, M et al. 2002 has shown that OB-specific

deletion of type I IGF receptor (Igf1r) results in a significant decrease of trabecular

bone volume, trabecular number and connectivity, and hence an increase in trabecular

spacing. These mice show an osteoporotic phenotype. In vitro data showed that there

was a striking decrease in bone mineralization activity by the osteoblast of these mice

(Zhang et al. 2002). Study from Power, R. A et al. 2002 showed that IGF-I gene

expression was upregulated in ovariectomised (OVX) mice treated with fibroblast

growth factor (FGF), which mediated at least in part the increase in trabecular bone

volume in these mice compared to the vehicle–treated OVX group. These results show

the importance of osteoclast activity in the release and activation of cytokines and/or

growth factors from bone matrix to initiate and regulate bone formation activity by the

osteoblast.

The other well-known factor that regulates the migration of bone mesenchymal stem

cells (BMSCs) to the resorptive sites to couple bone resorption with formation is TGF-

β1 (Hughes, Aubin & Heersche 1992; Lucas 1989; Oreffo et al. 1989; Tang et al. 2009).

High level of active TGF-β1 has been shown to be present in conditioned medium from

osteoclast cultured on bone slices, hence bone resorption is critical for the release and

activation of TGF-β1 (Tang et al. 2009). By using a mouse model lacking TGF-β1 gene

and the injection of the osteogenic BMSCs with GFP tag into these mice, it was then

proved that in reference to WT TGF-β1 null mice had its transplanted BMSCs dispersed

in the bone marrow and only very few were detected at the bone surface, again showing

the importance of TGF-β1 for the migration of BMSCs to the bone surface (Tang et al.

2009). Furthermore, SMAD3 and 4 have been found to be the main signalling pathway

involved in TGF-β1-induced migration of BMSCs to bone surface (Tang et al. 2009).

Another factor that is secreted by osteoclasts and acts on osteoblasts is sphingosine 1-

phosphate (S1P). S1P enhances RANKL expression, as well as migration and survival

of osteoblast cells (Ryu et al. 2006). Recently, it has been discovered that S1P also has

an autocrine effect, where it regulates the migration of osteoclast precursors to and from

bone surface, hence it is holding a major role in a healthy bone remodelling process

(Ishii et al. 2009). This result has been supported by the osteoporotic phenotype of type

1 S1P receptor (S1PR1) KO mice, as there was an enhanced osteoclast attachment on

the bone surface and poor recirculation of S1P-driven osteoclast precursor to systemic

blood flow (Ishii et al. 2009).


44

A recent study has found an osteoclast-expressed factor that regulates osteoblast

activity, which is now known as semaphorin 4D (Sema4D) (Negishi-Koga et al. 2011).

The gene expression of Sema4D is up regulated during RANKL-induced

osteoclastogenesis and a low expression is detected in osteoblasts (Negishi-Koga et al.

2011). The interaction between Sema4D, as an osteoclast membrane-bound factor, and

its receptor Plexin-B1 on osteoblasts causes the activation of the small GTPase RhoA.

This RhoA activation in turn, through Rho-associated protein kinase (ROCK) pathway,

suppresses IGF-I signalling and induces osteoblast motility, thereby suppressing

osteoblast differentiation and its bone formation activity (Negishi-Koga et al. 2011).

With regards to osteoblast motility in Sema4D KO mice model, they also showed that

there were few quiescent bone surfaces and a cluster of osteoblasts in close distance to

osteoclasts, indicating that Sema4D is required for regulating osteoblast localization,

through IGF-I signalling pathway, and hence the maintenance of quiescent surfaces

between osteoblasts and osteoclasts (Negishi-Koga et al. 2011).

Mature osteoclasts have also been found to express another cytokine called

cardiotrophin-1 (CT-1), which is able to regulate osteoblast differentiation and activity

(Walker et al. 2008). CT-1 treatment on murine stromal Kusa cells had resulted in dose-

dependently elevated ALP activity and bone nodules formation as well as an increase in

both STAT3 and ERK phosphorylation, resulting in an enhanced osteoblast activity

(Walker et al. 2008). This enhanced osteoblast activity has also been supported by the

finding that CT-1 induced 2-fold increase in Runx2 gene transcription activation by

using 6xOSE2 osteocalcin reporter assay. Lastly, by using CT-1 null mice model, in

vitro and in vivo experiments showed that not only there was a reduced osteoblast

activity, but also bigger osteoclasts (an increase in DCSTAMP gene expression) and

impaired bone resorption were also observed in reference to WT (Walker et al. 2008).

Hence CT-1 is another osteoclast-derived coupling factor that regulates both osteoclast

and osteoblast differentiation and activity (Walker et al. 2008).

1.3.3 Bidirectional signalling between osteoclasts and osteoblasts

Recent and promising studies in the coupling system between osteoclast and osteoblast

have shown that cell-to-cell contact between the two cells can directly regulate the

differentiation and activity of both cells. Eph receptors are tyrosine kinase receptors and

together with its ligand, Ephrin, are classified into A and B subgroups (mainly based on


45

their structural homologies). The Eph-Ephrin interaction provides a bidirectional

signalling, producing forward (Eph) and reverse (Ephrin) signals (Irie et al. 2009;

Matsuo 2010; Zuo et al. 2012). EphrinB2, a membrane-bound ligand expressed by

osteoclasts, has shown to bind to its receptor EphB4, expressed by osteoblasts (Zhao et

al. 2006; Martin et al. 2010). This binding has been shown to result in the suppression

of osteoclastogenesis by inhibiting the NFATc1 signalling pathway (as reverse

signalling) and at the same time promotes osteoblast differentiation via the inhibition of

RhoA pathway as well as an enhanced ERK1/2 phosphorylation (as forward signalling)

in mouse (Zhao et al. 2006). Further research has shown that PTH can induce the gene

expression of EphrinB2 in osteoblasts (Allan et al. 2008), suggesting that this

EphrinB2-EphB4 interaction might contribute to the anabolic effect of PTH on

osteoblasts (Irie et al. 2009). Hence this cell-to-cell contact dependence communication

of EphrinB2-EphB4 has been proposed to be a crucial control for the transition from

resorption/osteoclast activation phase to renewal/bone formation phase (Irie et al. 2009).

Other Ephrin-Eph bidirectional signalling has been shown by the interaction of

EphrinA2, expressed by osteoclast precursors, with its receptor EphA2, expressed also

by osteoclast precursors (autocrine signalling) and osteoblasts (Irie et al. 2009).

Overexpression of both EphrinA2 and EphA2 in osteoclast precursors, producing both

forward and reverse signalling, has been shown to result in an enhanced osteoclast

differentiation through the phospholipase Cγ2 signalling pathway (Irie et al. 2009). On

the other end for osteoblasts, the forward signalling of EphA2 has shown to inhibit

osteoblast differentiation through the regulation of RhoA activity (Irie et al. 2009). To

finally conclude, by fine tuning the EphrinB2-EphB4 and EphrinA2-EphA2

bidirectional signalling pathways to inhibit osteoclast bone resorption activity and to

increase osteoblast bone formation activity, the bone remodelling process in

postmenopausal women could be corrected (Zuo et al. 2012).

1.4 Bone Diseases

Osteoclasts and osteoblasts, being the two main players in the bone environment,

coordinate with other bone cells (as shown in Figure 1.4) to achieve a healthy bone

mass that can avoid any structural defects and fractures in our skeleton (Dempster et al.

2012; Uthgenannt et al. 2007). As described earlier, bone remodelling process starts


46

when bone-matrix is resorbed by osteoclasts and osteoblasts then lay down new organic

bone matrix, called osteoid, in the resorbed compartments in a balanced manner, which

eventually gets mineralized to form strong and healthy bone. Disruption in this system

will result in a number of pathological bone diseases, such as osteoporosis,

osteopetrosis, and Paget’s disease of bone (PDB).

1.4.1 Osteoporosis

Osteoporosis is characterised by a net bone loss and a decrease in bone strength, caused

generally by an excessive osteoclastic bone resorption (Kular et al. 2012; Riggs &

Melton 1983). As bone becomes weaker, susceptibility of fractures increases and

usually the signs show up after the initial fractures. The lifetime fracture risk for

osteoporotic patients could be as high as 40% (Burge et al. 2007; Melton et al. 1992;

Rachner, Khosla & Hofbauer 2011; Ray et al. 1997). Early diagnostic strategy for

osteoporotic condition includes the measurement of bone mineral density (BMD) of the

osteoporotic patients, which can be assessed by using dual x-ray absorptiometry (DXA),

and osteoporotic condition has been defined as a condition having a T score of less than

2.5, which is well more than 2.5 standard deviations below the average of a young adult

(Rachner 2011).

The two most important factors in osteoporosis are the loss of gonadal function, seen

mostly in postmenopausal women (Riggs & Melton 1983), and aging (Manolagas &

Jilka 1995). In postmenopausal period, there is an altered (upregulated) regulation of

many cytokines, such as IL-1, TNF-α, IL-6, which are potent inducers for osteoclast

differentiation and bone resorption activity (Horowitz 1993). As research progresses, it

is then known that the mediator of this increase in osteoclast activity is the upregulated

RANKL expression by osteoblasts and other cells (Eghbali-Fatourechi et al. 2003). On

the other hand, there is a continuous decline of osteoblast bone formation activity in

aged people, hence less bone formed in proportion to the demand for them (Manolagas

& Jilka 1995; Riggs & Melton 1983).

Treatment of osteoporosis mainly focuses on the prevention of further fractures. Firstly,

nutrition, life style (exercise), and dietary intake (calcium and Vitamin D) are all

important factors that can maximise our bone mass and can also slow down the rate of

bone loss and reduce fracture risk throughout life (Delmas 2002; Raisz 2005b). Anti-


47

resorptive therapy, such as bisphosphonate (alendronate, risedronate, and ibandronate),

has been shown to reduce the incidence of fractures in both vertebral and non-vertebral

and in hip region (Rosen 2005). Osteoclasts take up bisphosphonate, which binds onto

bone surface, and undergo apoptosis, hence this treatment slows down the bone

remodelling process (Delmas 2002; Fleisch 2000). The anabolic agent, PTH, treatment

is only approved by FDA in 2002 and it has been shown that daily injection of PTH (N-

terminal portion) could increase bone formation, hence an increase in bone mass, while

only affected little on osteoclast bone resorption activity (Neer et al. 2001). Current

therapeutic treatments for osteoporosis have mainly focused on the bone remodelling

process, in which Denosumab, a novel recombinant human monoclonal RANKL-

specific antibody has been shown to suppress bone resorption, hence effectively reduces

vertebral, non-vertebral, and hip fractures (Dempster et al. 2012) in postmenopausal

women. Studies have been planned to look at the efficacy and safety profile of

Denosumab for the treatment of male osteoporosis and glucocorticoid-induced

osteoporosis as well as its long-term use out of 10 years with a bigger participant

(Rizzoli, Yasothan & Kirkpatrick 2010). Other novel treatments include the use of

Cathepsin K-specific inhibitor (another anti-resorptive therapy), odanacatib, and

antibodies against the inhibitors of bone formation (anabolic therapies), sclerostin and

Dkk-1, which all of them have shown promising pre-clinical and clinical results (Bone

et al. 2010; Gauthier et al. 2008; Heath et al. 2009; Ominsky et al. 2010; Rachner,

Khosla & Hofbauer 2011; Stoch et al. 2009).

1.4.2 Osteopetrosis

In contrast to osteoporosis, osteopetrosis is an autosomal recessive disorder

characterised by an abnormal high bone density. This ‘stone bone’ disease varies across

suffering individuals, with the most severe cases causing optical nerve compression and

death in childhood (Quarello et al. 2004).

Several mutations have been identified in human, including in RANKL and RANK

genes, which cause osteoclast-poor autosomal recessive osteopetrosis (ARO) (Guerrini

et al. 2008; Sobacchi et al. 2007). On the other hand, the defect in osteoclast bone

resorption activity, particularly connected to the V-H+ATPase acidification machinery

of osteoclasts, contribute to the infantile malignant osteoclast-rich ARO, which counts

for about 70% of cases (Villa et al. 2009). In 2000, Frattini, A et al has shown that 50%


48

of osteoclast dysfunction cases in ARO was found to have mutation in TCIRG1

(encoding a3 subunit of V0 domain of V-H+ATPase proton pump), which resulted in

the formation of osteoclasts that were unable to resorb bone. Mutation in ClCN7

chloride channel has also been shown to be another cause of osteoclast-rich ARO

(Frattini et al. 2003; Kornak et al. 2001), although there is also a strong link between the

ClCN7 mutation and neurodegeneration (hinting the involvement of ClCN7 in the

nervous system) (Kasper et al. 2005). The less common cause of osteoclast-rich ARO is

carbonic anhydrase (CA)-II mutation, which is easily diagnosed as it is accompanied by

renal tubular acidosis and cerebral calcification (Sly et al. 1983). Specifically in

osteoclasts, loss of CA-II function causes osteoclasts unable to acidify (generating H+)

the intended resorbed bone compartment, hence preventing its bone resorption activity

(Whyte 1993).

The current curative treatment for ARO is total bone marrow transplant (allogeneic

hematopoietic stem cell therapy (HSCT)) (Eapen et al. 1998) and it has shown to cure

for RANK, TCIRG1, and CA-II mutations-caused osteopetrosis (Frattini et al. 2000;

Guerrini et al. 2008; McMahon et al. 2001). Meanwhile, mesenchymal cells (MSCs)

transplant and/or administration of exogenous RANKL administration are still on

clinical trials for the treatment of RANKL mutation-induced ARO (Villa et al. 2009).

1.4.3 Paget’s disease of bone (PDB)

Paget’s disease of bone (PDB) is characterised by a local increase in bone turnover and

usually has no symptoms (Selby 2005). It is a common disease with age, coming second

after osteoporosis. In this PDB pathological condition, there is an accelerated bone

remodelling activity in the affected areas of bone, which includes the presence of giant

and active multinucleated osteoclasts (Kanis 1991). The bone formation is also

increased but in a disorganised manner. These conditions then eventually lead to the

formation of woven bone, which can develop to form bone deformity and increases the

risk of fractures (Kanis 1991; Langston et al. 2007; Ralston & Layfield 2012).

The abnormality in osteoclast is the hallmark of PDB. They have an abnormal increase

in number and size, hence having more nuclei than normal controls (Roodman &

Windle 2005). The osteoclasts of PDB also have unique nuclear inclusions, which are

made up of microcylinders in paracrystalline arrays similar to nucleocapsids of


49

paramyxovirus (Mills, Yabe & Singer 1988; Rebel et al. 1981). Further investigations

into osteoclast biology have shown that in in vitro osteoclastogenesis monocytes from

Paget’s patients were hyper-responsive to RANKL and 1,25-(OH)2 vitamin D3, hence

generating significantly more osteoclasts and having an increase in bone resorption,

compared to normal controls (Neale et al. 2000). This result is in line with the finding

that there was an upregulation of RANKL gene expression in pagetic-lesion-developed

stromal cells and in Paget’s patient bone samples compared to healthy controls (Menaa

2000).

To date, the original cause of PDB is still unknown. However mutation in the SQSTM1

gene has been identified to be the common and familial (being autosomal dominant-

transmitted disease) linkage to the development of PDB (Hocking et al. 2001; Laurin et

al. 2001). The exact recurrent mutation of this gene affects primarily on p62 protein

(Laurin et al. 2002), which binds to the deubiquitinating enzyme CYLD and plays an

important role in NF-κB signalling pathway (Jin et al. 2008). The binding of p62 to

CYLD inhibits RANKL-induced osteoclastogenesis by deubiquitinating TRAF6 (Jin et

al. 2008). Hence, the point mutation at p62 causes CYLD unable to bind to p62, which

eventually leads to TRAF6 ubiquitination and NF-κB signalling pathway activation. In

conclusion, SQSTM1 is the gene most frequently affected in PDB, besides numerous

evidences of involvement of other mutations in genes related to osteoclast

differentiation, such as CSF-1 (M-CSF-coding gene) and TNFRSF11A (RANK-coding

gene) (Albagha et al. 2010). The current management for PDB is by using

bisphosphonate (Kular et al. 2012; Langston et al. 2007; Ralston & Layfield 2012; Reid

et al. 2005).

50

CHAPTER 2

SEMINAL VESICLE SECRETION (SVS) 7 PROTEIN AND LY-6

PROTEIN FAMILY

Chapter Two – Seminal Vesicle Secretion (Svs) 7 Protein and Ly-6 Protein Family

51

2.1 Introduction

Highly regulated cell-to-cell communication between osteoclasts and osteoblasts is a

prerequisite for sustaining a healthy bone mass (Karsenty & Wagner 2002; Zaidi 2007).

As mentioned in Chapter 1, one of the methods to achieve this cell-to-cell

communication is through secretion of diffusible paracrine factors, which can be

cytokines, chemokines, growth factors, or proteins, by either cell acting on the other

(Matsuo & Irie 2008). As osteoblast regulation of the osteoclast differentiation and

activation has been well studied, research on osteoclast-expressed novel factors or

proteins regulating osteoclast (autocrine) and/or osteoblast (paracrine) differentiation

and function remains limited. The identification of novel intercellular coupling factors

will enhance our understanding on this basic regulation of osteoclast and osteoblast in

bone microenviroment and help uncover new targets and/or therapies for the treatment

of pathological bone diseases.

2.2 Seminal vesicle secretion (Svs) 7

Svs7 was originally identified in bovine seminal plasma (Rufo et al. 1982), and later in

reproductive tract fluid and seminal vesicle content (SVC) of guinea pig (Coronel, San

Agustin & Lardy 1988), rat and mouse (Coronel et al. 1992). Svs7 gene has been

identified to be at chromosome 9 in mouse and chromosome 11 in human (Margalit et

al. 2012; Turunen et al. 2011). Further study on the protein composition and

functionality shows that Svs7, a small 8538Da (99 amino acids) protein, is a secreted

protein, harbouring N-terminal sequence, which contains signal peptides, and lacking a

carboxyl-terminal glycosylphosphatidylinositol (GPI) moiety (Adermann et al. 1999;

Levitin et al. 2008; Luo, Lin & Chen 2001; Ploug et al. 1991; Turunen et al. 2011). By

binding to post-acrosomal region of head, midpiece, and tail region of the sperm (Luo,

Lin & Chen 2001; Margalit et al. 2012) and having cysteine residues (Figure 2.1) to

form disulphide bonds (Luo, Lin & Chen 2001), functionally mouse Svs7 has been

shown to inhibit Ca2+ influx across the membrane of the sperm cells during their transit

from vagina to oviduct in order to prevent premature acrosomal reaction and

spermatozoa hyperactivation (Coronel et al. 1992). Hence, Svs7 is also known as

calcium transport inhibitor (caltrin) (Coronel et al. 1992; Rufo et al. 1982). Any

biological modifications (reduction and/or alkylation) on these cysteine residues have


52

been shown to abolish this Ca2+ transport inhibitory activity of Svs7 (Coronel et al.

1992). The nature of the binding partners/sites of Svs7 protein has been shown to be

lipids, which are reported to be phosphotidylethanolamine and phosphotidylserine (Luo,

Lin & Chen 2001). This specific binding of Svs7 to the phospholipid liposomes of the

sperm cells has been shown to enhance sperm motility. However there is still no further

study showing the significances and possible downstream signalling pathways coming

from this binding/interaction.

Additionally, Svs7 is also known as Prostate and testes-expressed protein (PATE)-B,

whose gene has been shown to be highly expressed in prostate and interestingly lower

expression was detected in testes (Turunen et al. 2011). Not surprisingly, Svs7

expression has been shown to be regulated by androgen (Levitin et al. 2008). Castration

enhances the prostate gene expression of PATE-B, while the administration of

dihydroxytestosterone (DHT) has been shown to ablate this induction (Levitin et al.

2008). With the consensus cysteine residues domain, Svs7 /PATE-B has shared this

distinctive pattern with lymphocyte antigen (Ly)-6 family (Levitin et al. 2008; Turunen

et al. 2011). Both protein families share the characteristic of three-fingered proteins

(TFP)/Ly-6/urokinase-type plasminogen activator receptor (uPAR) consensus domain,

which is discussed in Section 2.3 below (Levitin et al. 2008; Turunen et al. 2011).

2.3 Svs7 as a secreted member of Ly-6 protein family and the

biological importance of other Ly-6 proteins

Svs7 is a secreted member of Ly-6 protein family, which shares the TFP/Ly-6/uPAR

common motif consisting of a conserved cysteine (C) residues (8-10 residues forming

disulphide bonds), and a carboxyl (C)-terminal consensus sequence motif of

CCXXXXCN, with X represents any amino acid residue, as shown in Figure 2.1

(Coronel et al. 1992; Southan et al. 2002; Turunen et al. 2011; Ushizawa et al. 2009).

This family also includes membrane-linked proteins, which are anchored to the plasma

membrane through a post-translationally added C-terminal GPI attachment (Margalit et

al. 2012; McKenzie et al. 1977; Ploug et al. 1991).


53

Svs7 SLURP-1 SLURP-2 Ly6a UPAR

Svs7 SLURP-1 SLURP-2 Ly6a UPAR

(Sca-1)

(Sca-1)

Figure 2.1 Consensus cysteine residues and a C-terminal consensus sequence motif

of CCXXXXCN (TFP/Ly-6/uPAR domain) of Ly-6 proteins family. Amino acids

alignment of Svs7 and other Ly-6 proteins (2 secreted proteins, SLURP-1 and -2 and 2

membrane-bound, Ly-6a (Sca-1) and uPAR) showing a consensus cysteine residues

(forming disulphide bonds) and a C-terminal consensus sequence motif of

CCXXXXCN (both features are shown in red-coloured letters), where X represents any

amino acid residue and the N stands for asparagine. These two features represent the

TFP/Ly-6/uPAR consensus domain of Ly-6 protein family.


54

For the purpose of this chapter, only two other secreted Ly-6 proteins named secreted

Ly-6/uPAR-related protein (SLURP)-1 (Adermann et al. 1999) and -2 (Tsuji et al.

2003), and two GPI-anchored proteins, Stem cell antigen (Sca)-1 (Bonyadi et al. 2003)

and uPAR (Ohkura et al. 1994), will be discussed with regard to their known biological

roles in order to reveal the current understanding of the significances of this protein

family in the human biological system, especially in bone biology.

After its identification as the first secreted Ly-6 protein (Adermann et al. 1999), further

research has shown that SLURP mRNAs (both 1 and 2) are expressed in variety of

tissues and organs, including spleen, thymus, and brain (Moriwaki et al. 2007;

Moriwaki et al. 2009). The study of SLURP-1 becomes more and more important since

the finding that mutation in this gene causes an autosomal recessive inflammatory

palmoplantar keratodema, Mal de Meleda (MDM) (Fischer et al. 2001), showing its

important expression in skin and keratinocytes (Arredondo et al. 2005; Chimienti et al.

2003)

These two pioneering studies have shown a crucial role of SLURP-1 as an α7 subunit of

nicotinic acetylcholine receptors (nAChRs) ligand to regulate keratinocytes

differentiation and apoptosis. SLURP-2, which shares substantial protein homology

with SLURP-1, is also expressed in skin and keratinocytes (Tsuji et al. 2003). It has

been reported that SLURP-2 binds to the α-3 nAChRs, which is important in preventing

keratinocytes apoptosis (Arredondo et al. 2006) and hence its gene expression was

shown to be upregulated by 3-fold in psoriatic lesional skin compared to healthy

controls (Tsuji et al. 2003). These results suggest the importance of SLURP-1 and -2 in

maintaining epidermal and keratinocytes homeostasis through their fine-tune regulation

of nAChRs signalling pathway. Because of structural homology within Ly-6 protein

family (shown in Figure 2.1), not surprisingly Svs7 has also been shown to regulate α7

nAChR and has no effect on other nAChR (Levitin et al. 2008). Even though there is

common functionality between these ly-6 proteins in regulating nAChRs, further

investigation, particularly for Svs7 in this signalling, is still required, as only one

published study is currently available in this topic. Furthermore, the detection of

SLURP-1 and -2 expression in T and B cells, bone marrow-derived dendritic cells, and

macrophages prompts future studies to elucidate the possible role of these two proteins

in immune system and possibly in bone homeostasis (Moriwaki et al. 2007).


55

Sca-1 (Ly-6A), an 18kDa GPI-linked Ly-6 protein, has been shown to be an important

factor in bone microenvironment as it is expressed by many different kinds of stem

cells, such as hematopoietic stem cells (HSCs), skeletal muscle stem cells, and

mammary epithelial stem cells, a subpopulation of bone marrow (BM) stromal cells

including osteoblasts, and Sca-1 KO mice exhibit age-dependent osteoporotic

phenotype as a result of defects in stem cell renewal capacity (Bonyadi et al. 2003;

Gussoni et al. 1999; Ito et al. 2003; Trevisan & Iscove 1995). Sca-1 KO mice have

healthy bone during adulthood, but with age they develop a massive decrease in bone

mass by having defects in self-renewal capacity of mesenchymal stem cells (MSCs)

specifically (Bonyadi et al. 2003). Consistent data has shown a significant decrease in

osteoprogenitor population (starting from 7-month old mice and onward) and markedly

reduced bone formation activity in in vitro colony-forming unit (CFU)-alkaline

phosphatase (ALP) and bone nodule formation assay of both Sca-1 KO primary murine

calvarial and BM stromal cell-derived osteoblasts, hence Sca-1 KO mice have a

significantly lower whole body bone mineral density (BMD) than controls (Bonyadi et

al. 2003; Holmes et al. 2007). As MSCs make a key component in HSCs niche (Ito et

al. 2003; Mansour et al. 2012), Sca-1 (an important antigen marker for both MSCs and

HSCs) holds a central role in maintaining the self-renewal capacity of stem cells, hence

it is one of crucial factors in maintaining homeostasis of bone microenvironment. Its

importance in bone homeostasis is further supported by its expression in mature

osteoclasts (>2-fold increase), shown in Figure 2.3, hence encouraging further research

of this gene in bone environment.

Lastly, uPAR is another GPI-linked Ly-6 protein (Ohkura et al. 1994). uPAR has been

well studied as the regulator of the plasminogen activation system by binding to its

ligand uPA and pro-uPA (Smith & Marshall 2010), showing its crucial role in the

degradation and clearance of fibrin clots (fibrinolysis) and the activation of MMP9 and

13 (Carmeliet et al. 1997). Besides its function in this proteolysis system, uPAR has

also been shown to be involved in many signalling pathways, governing cell motility,

invasion, proliferation, and survival (Smith & Marshall 2010), such as in mitogen-

activated protein kinases (MAPK) (Aguirre Ghiso 2002; Kjoller & Hall 2001; Liu et al.

2002; Vial, Sahai & Marshall 2003) and signal transducer and activator of transcription

(STAT) pathways (Degryse et al. 2005; Koshelnick et al. 1997; Lester et al. 2007). With

this involvement of uPAR in many biological systems, extensive studies have shown

that its expression is elevated during inflammation, tissue remodelling, and in many


56

human cancers (Del Rosso et al. 2008; Guerrini et al. 2008; Jacobsen & Ploug 2008).

Being GPI-anchored and therefore lacking transmembrane and intracellular domains,

uPAR must interact with transmembrane receptors to carry its role in regulating

intracellular signalling pathways (Smith & Marshall 2010). Many studies have shown

evidences that integrins are uPAR crucial co-receptor in signalling pathways (Madsen et

al. 2007; Smith & Marshall 2010; Zhang et al. 2003). Amongst many studies

elucidating the interaction of integrins isoforms and uPAR, αvβ3 integrin, which is

highly expressed by osteoclasts (Duong et al. 1998; Nakamura et al. 2012), has been

shown to interact with uPAR (Schiller et al. 2009; Smith, Marra & Marshall 2008; Wei

2008) and this interaction regulates subsequent signalling pathways, notably governing

cell proliferation and survival (MAPK pathway) (Woods et al. 2001) and cell motility

through the activation of Rho family small GTPase Rac (Kjoller & Hall 2001; Lester et

al. 2007; Smith, Marra & Marshall 2008; Vial, Sahai & Marshall 2003). This activation

of Rac has been shown to regulate downstream signalling pathway through p130Cas,

which is important for the formation of F-actin ring (sealing zone), a crucial step for

osteoclast bone resorption activity, as discussed in Chapter 1 Section 1.1.2 (Lakkakorpi

et al. 1999; Smith, Marra & Marshall 2008). With the binding of integrin to

extracellular matrix (ECM) proteins, such as vitronection (Lakkakorpi et al. 1999;

Nakamura et al. 2012), to induce its downstream signalling pathway, further research is

needed to confirm the role of uPAR in its interaction with integrins and ECM proteins

and many molecular details of uPAR signalling pathways, which still remain unclear

(Smith & Marshall 2010).


57

2.4 Identification of Ly-6 family member, Svs7, as a putative

osteoclast-derived coupling factor

Current research in our laboratory has focussed on elucidating novel factors, secreted by

osteoclast and/or osteoblast, which have potential roles in regulating either or both cells,

hence is crucial in maintaining bone homeostasis (Kular et al. 2012). Using subtractive

hybridization-based differential screening method on RAW264.7 cells with (mature

osteoclasts) and without (osteoclast precursors) RANKL stimulation, Phan 2004

identified Svs7 gene, only expressed by mature osteoclasts and not by osteoclast

precursors (Davey 2008), as shown in Figure 2.2. Furthermore, microarray heatmap

analysis on Svs7 and other Ly-6 genes expressions in mature osteoclasts (relative to

osteoclast precursors) shows that Svs7 is the most highly expressed gene by mature

osteoclasts amongst other Ly-6 genes (more than 10-fold increase in Svs7 gene

expression by mature osteoclasts) (Figure 2.3).

The initial study of this Ly-6 family member, Svs7, was then continued by investigating

its gene expression in other key bone cells. It is revealed that Svs7 is only expressed by

osteoclasts but not by osteoblasts or other key bone cells (Figure 2.4A) and its gene

expression is upregulated at day 3 and 5 during RANKL-induced differentiation of bone

marrow macrophages (BMM) into mature osteoclasts, in parallel with other known

osteoclast marker genes, such as TRAP, CTR, and NFATc1 (Figure 2.4B). Hence, Svs7

is a novel osteoclast-expressed factor.

In conclusion, with many Ly-6 genes have shown to be involved in many cellular

signalling pathways, such as uPAR in αvβ3 integrin signalling pathway, which is

crucial for osteoclast bone resorption activity, and Sca-1, being the central player for

stem cell renewal capacity, it was then of interest to elucidate the biological role of

Svs7, a novel osteoclast-expressed factor and shown to be the most highly expressed

Ly-6 gene by mature osteoclasts, in regulating osteoclast-osteoblast differentiation and

activation, hence in maintaining bone homoeostasis.


58

Driver Forward subtraction

Driver: RAW264.7

Svs7

RAW264.7 + RANKL

Figure 2.2 Identification of Svs7 gene using subtractive hybridization-based

differential screening in RAW264.7 cells culture with RANKL stimulation. (Top

panel) RAW264.7 cells were stimulated with RANKL to form multinucleated osteoclasts.

(Bottom panel) messenger RNA (mRNA) was extracted from osteoclasts and osteoclast

precursor RAW264.7 cells and was hybridized. Forward and reverse screening was done

to remove false positives and cDNA probes, labelled with 32p-dCTP (deoxycytidine

triphosphate), were used to isolate the clones. Among the isolated and sequenced

clones, Svs7 was identified, only expressed by mature osteoclasts and not by osteoclast

precursors. (Figure adapted from Davey, 2008)


59

-1 0 1

Precursor (P)

Mature (M)

Osteoclast

Acrv1 Ly6a Ly6c Ly6d Ly6e Ly6f Ly6g5b Ly6g5c Ly6g6c Ly6g6d Ly6g6e Ly6i Ly6k Lynx1 Psca Slurp1

Ratio (M/P)

Svs7

0.85 2.07 0.96 0.79 2.36 1.21 1.06 1.12 0.99 1.81 1.07 0.85 0.92 1.02 1.23 1.25

10.49

Figure 2.3 Svs7 is the most highly expressed Ly-6 gene by mature osteoclasts,

shown by the microarray heatmap analysis. RNA of osteoclast precursors (day 0 of

RANKL stimulation) and mature osteoclasts (day 5 of RANKL stimulation) were

reverse transcribed and hybridised for the microarray genes expression profile. The

relative (mature/precursor) gene expression analysis (expressed as fold

increase/decrease), particularly on Svs7 and some other Ly-6 genes expressions, was

carried out and it is clear that Svs7 is the most highly expressed gene by mature

osteoclast amongst other Ly-6 genes (more than 10-fold increase in Svs7 gene

expression by mature osteoclasts).


60

(A)

(B)

Svs7

β-actin

OB

C2C

12

BM

M

OC

RAW

264.

7

RAW

264.

7 +

RA

NK

L

β-actin

NFATc1

Svs7

ATP6v0d2

0 1 3 5

RANKL-stimulated

WT BMM day

TRAP

CTR

DCSTAMP

Figure 2.4 Svs7 gene expression in osteoclast. (A) RNA from myoblasts (C2C12),

murine stromal cells-derived osteoblasts culture (OB), BMM, day 5 RANKL-stimulated

BMM (osteoclasts (OC)), osteoclast precursor RAW264.7 cells, and RANKL-stimulated

RAW264.7 cells were subjected to reverse transcriptase-PCR (RT-PCR) reactions using

specific primers for Svs7 and β-actin as a house-keeping gene. Svs7 gene is only

expressed by osteoclasts but not by osteoblasts or other key bone cells. (B) RNA from

time course RANKL-induced differentiation of BMM into mature osteoclasts was

subjected to RT-PCR reactions using specific primers for Svs7 and other common

osteoclast marker genes. Svs7 gene expression is upregulated at day 3 and 5 during

RANKL-induced osteoclastogenesis (Svs7 is exclusively expressed by mature

osteoclasts). β-actin was used as a loading control.

61

CHAPTER 3

HYPOTHESIS AND AIMS

Chapter Three – Hypothesis and Aims

62

3.1 Hypothesis Bone is continuously remodelled throughout life via the interplay between key bone

resident cells; namely the bone-resorbing osteoclast, bone-forming osteoblast, and

mechano-sensing osteocytes. Bone homeostasis is thus maintained by the coordinated

activities between these cells. Disruption in the balance between bone resorption and

formation will result in the development of many pathological conditions, including

osteoporosis, osteopetrosis, and PDB, as described previously in Chapter 1 Section 1.4.

However, the complement of factors, crucially regulating this intimate intercellular

communication “coupling” between osteoclasts and osteoblasts, remain largely

unknown.

Svs7, a secreted Ly-6 family protein (Southan et al. 2002), was previously identified in

our laboratory as a candidate osteoclast-secreted “couling” factor (Davey 2008; Phan

2004). Svs7 gene expression is upregulated during RANKL-induced osteoclastogenesis,

indicating that Svs7 is exclusively expressed by mature osteoclasts in bone.

Furthermore, studies by Davey 2008 found that recombinant purified Svs7 protein

treatment could protect against loss of trabecular bone volume, trabecular thickness, and

trabecular number in ovariectomised mice (oestrogen deficiency-induced bone loss

mouse model (Aitken, Armstrong & Anderson 1972; Kalu 1991; Saville 1969)). When

considering these preliminary observations, together with the established importance of

Ly-6 family members in many intracellular signalling pathways (e.g. Sca-1, which is

also expressed by osteoclasts) and their crucial role in maintaining self-renewal capacity

of stem cells (Bonyadi et al. 2003; Holmes et al. 2007), it is conceivable that Svs7 acts

to similarly regulate osteoclast and/or osteoblast differentiation and activity and thus

may play an important role in the maintenance of bone homeostasis. Therefore, the

central hypothesis under investigation in this thesis is that Svs7 is an important

regulator of bone homeostasis via the modulation of osteoclast and/or osteoblast

differentiation and activation.

3.2 Aims To address this, the specific aims of this thesis are:

1. To investigate the physiological importance of Svs7 in bone homeostasis

through the generation and characterisation of:

(i) Mice overexpressing Svs7 (Transgenic)

(ii) Mice lacking Svs7 (Knockout)

Chapter Three – Hypothesis and Aims

63

2. To identify potential intrinsic cellular defects in osteoclasts and/or osteoblasts

derived from Svs7 mouse models

Collectively, these studies will shed light on the as yet unknown role of Svs7 in bone

cells function and uncover its precise role in the maintenance of bone homeostasis.

64

CHAPTER 4

MATERIALS AND METHODS

Chapter Four – Materials and Methods

65

4.1 Materials

4.1.1 Cell lines

Cell line Description Supplier

RAW264.7 (Subclone C4) Murine monocyte cell line Dr. A. I Cassady, Centre

for Molecular and Cellular

Biology, Department of

Biochemistry, University

of Queensland.

4.1.2 Bacterial Strain

Strain Description Supplier

DH5α cDNA insert cloning host,

propagation of plasmid

DNA

Invitrogen, Australia

4.1.3 Chemical reagents

Description Supplier

Acridine Orange Sigma-Aldrich, USA

Acrylamide 30% Bio-Rad Laboratories, USA

Agar Powder Oxoid, UK

Agarose Promega Corp, USA

Alizarin red Raymond A Lamb, UK

Aluminium Sulphate Sigma-Aldrich, USA

Ammonium persulfate Bio-Rad Laboratories, USA

Ampicillin Sigma-Aldrich, USA

(L) Ascorbic acid Sigma-Aldrich, USA

Bacto Yeast Extract Oxoid, UK

Bis-acrylamide Bio-Rad Laboratories, USA

Bovine Serum Albumin (BSA) Hyclone, USA

Chloroform Sigma-Aldrich, USA

Complete Protease Inhibitor Cocktail

Tablets

Roche, Germany

Dexamethasone Sigma-Aldrich, USA


66

Dimethysulfoxide (DMSO) Sigma-Aldrich, USA

Ethanol Thermo Fisher Scientific, USA

Ethylene Diamine Tetra-acetic acid

(EDTA)

Acros, USA

Glacial Acetic Acid Thermo Fisher Scientific, USA

Glycerol Thermo Fisher Scientific, USA

Glycine Sigma-Aldrich, USA

Glutaraldehyde Thermo Fisher Scientific, USA

Haematoxylin Sigma-Aldrich, USA

Hydrochloric acid (HCl) VWR International, Australia

Isopropanol (Propan-2-ol) Thermo Fisher Scientific, USA

Isopropyl-β-D-thiogalactopyranoside

(IPTG)

Promega Corp, USA

β-mercaptoethanol Sigma-Aldrich, USA

Methanol Thermo Fisher Scientific, USA

5x MuLV Reverse Transcriptase Buffer Promega Corp, USA

Phenylmethylsulfonyl Fluoride (PMSF) Boehringer Mannheim Corp, USA

Probenecid Sigma-Aldrich, USA

Skim Milk Powder Standard market brand

Sodium Acetate (NaCH3COO) Merck, Australia

Sodium Chloride (NaCl) Thermo Fisher Scientific, USA

Sodium Dodecyl Sulphate (SDS) AppliChem, Germany

Sodium Hydroxide (NaOH) Sigma-Aldrich, USA

N, N, N’, N’-tetra-methyl-

ethylenediamine (TEMED)

Bio-Rad Laboratories, USA

Trisma Base Thermo Fisher Scientific, USA

Trisma Hydrochloride Thermo Fisher Scientific, USA

Triton X-100 Thermo Fisher Scientific, USA

Trizol Sigma-Aldrich, USA

Trypsin Gibco, Australia

Tryptone Oxoid, UK

Tween-20 Acros, USA

Xylene Thermo Fisher Scientific, USA


67

4.1.4 Tissue culture reagents


α-MEM (α-Modification of Eagle’s

Medium)

Gibco, Australia

D-MEM (Dulbecco’s-Modification of

Eagle’s Medium)-GlutaMaxTM-1

Gibco, Australia

Foetal Bovine Serum (FBS) Gibco, Australia

Hanks buffer (for Fluo-4AM calcium

assay)

Gibco, Australia

Opti-MEM Reduced Serum Medium Gibco, Australia

Penicillin-Streptomycin Gibco, Australia

Trypsin-EDTA (pH 7.0) Gibco, Australia

4.1.5 Cytokines


M-CSF Produced in the laboratory in the form of

conditioned media of CMG 14-12 cells

RANKL-GST Produced in the laboratory as described

previously (Xu et al. 2000)

4.1.6 Enzymes


Collagenase Sigma-Aldrich, USA

DNase I Sigma-Aldrich, USA

2xGoTaq Green Master Mix DNA

polymerase

Promega Corp, USA

M-MuLV Reverse Transcriptase Promega Corp, USA

Restriction Enzymes – HindIII, XhoI, SfuI Promega Corp, USA

RNase inhibitor Promega Corp, USA

SYBR Green Master Mix DNA polymerase Promega Corp, USA

T4 ligase Promega Corp, USA


68

4.1.7 Antibodies

Description Supplier Dilution

Anti-mouse (β) actin

(JLA-20 clone)

Calbiochem 1:5000 (WB 1° Ab)

Anti-DCSTAMP (1A2

clone)

Millipore, USA 1:500 (WB 1° Ab)

Anti-ERK1/2 (V114a) Promega Corp, USA 1:5000 (WB 1° Ab)

Anti-p-ERK (E-4) Santa Cruz, USA 1:1000 (WB 1° Ab)

Anti-IκB-α (C-21) Santa Cruz, USA 1:1000 (WB 1° Ab)

Anti-SAPK/JNK (#9252) Cell Signalling, USA 1:1000 (WB 1° Ab)

Anti-phospho-SAPK/JNK

(#9251)

Cell Signalling, USA 1:1000 (WB 1° Ab)

Anti-NFATc1 BD Pharmingen, USA 1:1000 (WB 1° Ab)

Anti-p38 (#9212) Cell Signalling, USA 1:1000 (WB 1° Ab)

Anti-phospho-p38 (#9211) Cell Signalling, USA 1:1000 (WB 1° Ab)

Anti-Svs7 Institute of Medical and

Veterinary Science

(IMVS), South Australia

1:500 (WB 1° Ab)

Anti-mouse IgG

Peroxidase Conjugate

(A9917)

Sigma-Aldrich, USA 1:5000 (WB 2° Ab)

Anti-rabbit IgG Peroxidase

Conjugate (A0545)

Sigma-Aldrich, USA 1:5000 (WB 2° Ab)

PE Cy.7-conjugated anti-

CD3e

BD Pharmingen, USA 1:1000 (Flow cytometry)

FITC-conjugated CD45R BD Pharmingen, USA 1:1000 (Flow cytometry)

V450-conjugated CD11b BD Pharmingen, USA 1:1000 (Flow cytometry)

PE-conjugated cKit BD Pharmingen, USA 1:1000 (Flow cytometry)

APC-conjugated Sca-1 R&D systems, USA 1:20 (Flow cytometry)

4.1.8 Vectors


pcDNA3.1/V5-His Invitrogen, Australia

pGEM-T Easy Vector Promega Corp, USA


69

pNFAT-Luc2P (Luc2P/NFAT-

RE/Hygro)

Promega Corp, USA

4.1.9 Purchased kits and systems


Alkaline phosphatase (ALP) staining kit Sigma-Aldrich, USA

CellTiter 96 Aqueous One Solution Cell

Proliferation Assay (MTS)

Promega Corp, USA

Fluo-4 AM calcium molecular probe

(F14201)


Lipofectamine-2000 Transfection

Reagent


Live/Dead Fixable Aqua Dead cell stain Invitrogen, Australia

Luciferase assay system (Substrate) Promega Corp, USA

QIAEXII Gel Extraction Kit (500) QIAGEN, Australia

Qiagen Plasmid plus midi kit QIAGEN, Australia

QIAprep spin miniprep QIAGEN, Australia

RNeasy RNA Extraction Kit QIAGEN, Australia

Western Lightening Ultra (Detection kit-

chemiluminescence substrate)

Perkin Elmer, USA

4.1.10 Oligonucleotide primers

Oligonucleotide primers for use in PCR reaction were purchased from GeneWorks,

Australia. Upon receipt, the oligos were resuspended in the recommended amount of

double-distilled water (ddH2O) to make up 100mM stock concentration. An aliquot of

20μl of each stock primer was transferred to a new 0.5ml tube and was diluted with

80μl of ddH2O (1 in 5 dilution) to make a working concentration of 20mM. Both stock

and aliquot primers were stored at -20°C until use.

4.1.11 Other materials


100bp DNA ladder (250μl) Promega Corp, USA

1kb DNA ladder (250μl) Promega Corp, USA

6xDNA Blue/Orange Loading Dye Promega Corp, USA


70

Baxter ddH2O Baxter, Australia

Biocoat collagen 1-coated 6-well plate Becton Dickinson, UK

Carbon steel surgical blade sterile Swann Morton, UK

Cell Culture Flask – T25, 75 Nunc, Denmark

Cell Culture Plates: 6-, 12-, 24-, 48-, and

96-wells

Nunc, Denmark

Cell Dissociation Solution (1x Non-

enzymatic)

Sigma-Aldrich, USA

Cell Scraper Sarstedt, Germany

Cell Strainer 100μm BD Biosciences, USA

Centrifuge Tubes – 5, 15 and 15ml Sarstedt, Germany

Corning Osteo Assay Plates Corning, USA

Cryogenic Vials (2ml) Simport, USA

DAKO aqueous mounting media Invitrogen, Australia

DePeX mounting media BDH Laboratory, UK

Double-sided carbon tape (12mm wide) ProSciTech, Australia

Filter paper Whatmans, GE Healthcare, USA

HybondTM C Nitrocellulose Membrane Amersham Biosciences, USA

Microcentrifuge tubes – 0.5, 1.5, and 2ml Sarstedt, Germany

Neubauer Haemocytometer Optik Labor, USA

Optical Bottom Plate Polymer (black

base)

Nunc, Denmark

Parafilm American National, USA

PCR tubes – 0.2ml Sarstedt, Germany

Petri dishes (90mm) Nunc, Denmark

Pin Type SEM mount (12.6mm diameter) ProSciTech, Australia

Pipette tips Sarstedt, Germany

Precision Plus Protein Standard Bio-Rad Laboratories, USA

Protein Assay Dye Reagent concentrate Bio-Rad Laboratories, USA

SEM paper storage box ProSciTech, Australia

Serological Pipettes – 5 and 10ml Sarstedt, Germany

SYBR safe DNA stain Invitrogen, Australia

Needle 23Gx1.25 (0.65x32mm) BD Biosciences, USA

Syringe 5cc/ml BD Biosciences, USA


71

Transfer pipette – 1ml Sarstedt, Germany

MicroAmp optical adhesive film A&B Applied Biosystems, USA

4.1.12 Software


CT analyser Version 1.10.1.0 Skyscan, Belgium

Dataviewer Version 1.4.3.0 Skyscan, Belgium

FlowJo Tree Star, USA

ImageJ 1.45s Wayne Rasband, NIH, USA

Microsoft Excel 2011 Microsoft Corp, USA

Microsoft Word 2011 Microsoft Corp, USA

NIS-Elements Basic Research Nikon, USA

NRecon Version 1.5.1.4 Skyscan, Belgium

ScanScope Aperio, USA

4.1.13 Equipment


A&D HT-500 balance A&D Corp, Korea

Agarose Gel Comb Bio-Rad Laboratories, USA

Beckman J-6B centrifuge Beckman Instruments, USA

BH120 Gelman Sciences cell culture

hood

Gelman, Singapore

Binder 37°C incubator (not for cell

culture)

VWR international, Australia

Bio-Rad Gel Tray Bio-Rad Laboratories, USA

Bio-Rad power pack HC for Western

Blot


Bio-Rad Spectrophotometer Plus Bio-Rad Laboratories, USA

Canon Camera Canon, Japan

Class II biological safety cabinet Gelaire, Australia

Clemco fume hood Oliphant Pty Ltd, Australia

Clifton Cerastir (magnetic stirrer) Lomb Scientific, Australia

Diamond wafering blade Buehler, USA

Eppendorf Biophotometer plus Eppendorf, Germany


72

Eppendorf 5430R centrifuge (including

for 4°C centrifugation)

Eppendorf, Germany

Eppendorf 5810R centrifuge (for cell

culture)

Eppendorf, Germany

Eppendorf MiniSpin Plus

Microcentrifuge

Eppendorf, Germany

Eppendorf Pipettes: 0.2-2μl, 2-20μl, 20-

200μl, 200-1000μl, multichannel

Eppendorf, Germany

Eppendorf Thermocycler Eppendorf, Germany

FACS Canto BD Pharmingen, USA

Forma Steri-cycle CO2 incubator Thermo Fisher Scientific, USA

FujiFilm ImageQuant LAS-3000 FujiFilm, Japan

FujiFilm ImageQuant LAS-4000 FujiFilm, Japan

Gelman Biological Safety Cabinet Class II Gelman, Singapore

Grant W14 water bath Selby Scientific and Medical, USA

Invitrogen Safe Imager 2.0 Invitrogen, Australia

Isomet low speed saw Buehler, USA

LabServ Incubator Biolab, Australia

Liquid nitrogen bucket Nulgene, USA

MaxQ4450 shaking incubator Thermo Fisher Scientific, USA

MilliQ Advantage A10 Millipore, USA

Mini-PROTEAN III Cell Electrophoresis Bio-Rad Laboratories, USA

Mini Trans-Blot Electrophoretic Transfer

Cell


Model 680 Microplate Reader Bio-Rad Laboratories, USA

Nikon Eclipse 50i Nikon, Japan

Nikon Eclipse Ti Nikon, Japan

PV-1 benchtop vortex Grant-bio Quantum Scientific, USA

Ratek Orbital Mixer Incubator Ratek Instruments, Australia

Ratek Rocking Platform Mixer Ratek Instruments, Australia

Skyscan 1072 microCT scanner Skyscan, Belgium

Skyscan 1174 microCT scanner Skyscan, Belgium

Tomy MicroOne mini centrifuge Tomy Tech, USA

TPS digital pH meter TPS, Australia


73

UV Bench-top Transilluminator UVP, USA

4.1.14 Centrifugation

Centrifugation of 0.2ml PCR tubes at room temperature was performed using Tomy

MicroOne mini centrifuge. Other microcentrifuge tubes (0.5, 1.5, and 2ml) were

centrifuged using Eppendorf MiniSpin Plus Microcentrifuge. Centrifugation at 4°C was

carried out using Eppendorf 5430R centrifuge.

4.2 Buffers and Solutions All solutions were prepared using highly purified ddH2O and where applicable, were

sterilized either by autoclaving or filter sterilization. All chemicals were weighed using

A&D HT-500 balance (A&D Corp). The adjustment of pH was carried out using TPS

digital pH meter, calibrated with appropriate pH standards.

4.2.1 General solutions

Solutions Composition and Preparation

10% Ammonium persulphate 10% (w/v) ammonium persulphate

dissolved in ddH2O. Stored at 4°C.

Ampicilin 100mg/ml stock, dissolved in ddH2O, and

aliquoted to 1ml. Stored at -20°C.

0.2% BSA/PBS 0.2% (w/v) BSA dissolved in 1xPBS.

Stored at 4°C.

dNTPs 100mM stock solution for each dNTP

(dCTP, dATP, dTTP, dGTP). 25% (v/v)

of each constituent was mixed together to

yield a 25mM combined stock of dNTPs,

which was then diluted to 5mM with

ddH2O and stored as 200μl aliquots at -

20°C.

14% EDTA pH 7.4 14% (w/v) EDTA dissolved in ddH2O,

brought to pH 7.4 with NaOH.

Autoclaved and stored at room

temperature.

Eosin (containing phloxine) Stock solution: 1% (w/v) of eosin Y


74

dissolved in ddH2O.

Phloxine stock solution: 1% (w/v)

phloxine B dissolved in ddH2O.

Working stock staining (per litre)

composed of 100ml of Eosin stock

solution, 10ml of phloxine stock solution,

780ml 95% ethanol and 4ml glacial acetic

acid.

Flow Cytometry Wash Buffer 50ml stock solution in 1xPBS; composed

of 1% FBS (500μl) and 0.1% (w/v)

sodium azide (0.05g). Stored at 4°C.

Fluo4-AM 1mM concentration made by dissolving

50μg of the dye in 45.58μl of 20%

pluronic acid.

Fluo4-AM/Hanks solution 4μl of Fluo4-AM/ml of

Hanks/probenecid required to make 4μM

concentration.

Haematoxylin (Gill’s) 1L stock solution made: 250ml of

ethylene glycol dissolved in 750ml of

ddH2O, added 6g of haematoxylin, 0.6g

of sodium iodate, 80g of aluminium

sulphate and 20ml of glacial acetic acid

to the mixture and stirred for an hour at

room temperature. Working

concentration of 1 in 5 of stock solution

used. Filtered and stored at room

temperature.

Hanks/Probenecid buffer Prepared 2mM probenecid in Hanks

buffer.

IPTG/X-gal 0.2M IPTG, 0.1M X-gal in

dimethylformamide. Stored at -20°C in

the dark.

Luciferase Lysis Buffer 500ml stock made: 1.51g of Tris-HCl

(pH 7.8), 0.37g of EDTA, 50ml of


75

glycerol, and 5ml of Triton X-100

dissolved in ddH2O. Stored at 4°C.

10% Neutral Buffered Formalin (NBF) A mixture of 10% (v/v) formaldehyde

(100ml), 900 ml of ddH2O, 33mM

NaH2PO4 (4g), and 45mM Na2HPO4

(6.5g). Autoclaved and stored at room

temperature

4% Paraformaldehyde (PFA) 4% (w/v) in 1xPBS. Stored as 10ml

aliquots at -20°C.

1xPBS pH 7.4 100ml stock solution mixed with 900ml

of ddH2O to give 1x concentration, filter

sterilised and stored at room temperature.

PMSF 17.6mg/ml stock was prepared in

isopropanol. Stored as 1ml aliquots at -

20°C.

Pluronic acid (20%) Dissolving 0.2g of the pluronic in 1ml of

DMSO.

Red Blood Cell Lysis Buffer (Flow

cytometry)

500ml stock solution: 4.15g of

ammonium chloride (NH4Cl), 0.788g

Tris-HCl, 0.9ml 5% EDTA pH 8, mixed

with 450ml of ddH2O, pH adjusted to 7.4,

and the final volume was adjusted to

500ml with ddH2O. Filter sterilized and

stored at 4°C.

RipaRipa Lysis Buffer A mixture of 50mM Tris-HCl pH 7.5

(25ml), 150mM NaCl (15ml), 1%

Nonidet P (NP)-40 (5ml), 0.5% sodium

deoxycholate (2.5g), 0.1% SDS (0.5g),

adjusted to 500ml with ddH2O.

Autoclaved and stored at 4°C.

At the time of cell lysis: 100μg/ml of

PMSF, 1x cocktail protease inhibitor,

1mM Sodium orthovanadate, and

500μg/ml of DNaseI is freshly added to


76

the required amount of the lysis buffer

10% SDS 10% (w/v) SDS dissolved in ddH2O.

Stored at room temperature.

4x SDS-PAGE Loading Buffer 4x stock solution composed of 240mM

Tris-HCl (2.4ml), pH adjusted to 6.8, 8%

(w/v) SDS (0.8g), 40% glycerol (4ml),

0.04% bromophenol blue (4mg), and 5%

β-mercaptoethanol (0.5ml), adjusted to

10ml with ddH2O. Stored at 4°C.

10x SDS-PAGE Running Buffer 10x stock solution; a mixture of 25mM

Trizma base (30.28g), 1.92M Glycine

(144.13g), and 1% (v/v) of 10% SDS

(100ml), adjusted to 1L with ddH2O.

Stored at room temperature.

SDS-PAGE Separating Gel Buffer A mixture of 1.5M Trizma base, 0.4%

SDS, prepared in ddH2O. pH adjusted to

8.8 (before addition of SDS). Stored at

room temperature.

SDS-PAGE Stacking Gel Buffer A mixture of 1M Trizma base, 0.4%

SDS, prepared in ddH2O. pH adjusted to

6.8 (before addition of SDS). Stored at

room temperature.

Solution A (Goldner’s trichrome stain) Prepared by mixing 0.075g of Ponceau

2R, 0.025g of acid fuchsin, 0.01g of

azophloxine, and 0.2ml of acetic acid

with 100ml of ddH2O.

Solution B (Goldner’s trichrome stain) Prepared by mixing 2g of orange G and

4g of phosphomolybdic acid with 100ml

of ddH2O. The solution was left to stand

at room temperature for a couple of days

for any precipitate to settle out and then

filtered before use.

Solution C (Goldner’s trichrome stain) Prepared by mixing 0.2g of light green

and 0.2ml of acetic acid with 100ml of


77

ddH2O.

50x Tris-acetate EDTA (TAE) 50x stock solution made of 2M Trizma

base (242g), 5.71% (v/v) glacial acetic

acid (57.1ml), and 500mM EDTA

(100ml), adjusted to 1L with ddH2O.

Stored at room temperature. (1x TAE was

used by doing dilution of 50x stock

solution)

10x TBS 10x stock solution made of 0.5M Trizma

base (60.57g), 1.5M NaCl (87.66g)

dissolved in ddH2O, and pH adjusted to

7.4 with concentrated HCl and volume

adjusted to 1L. Stored at room

temperature.

1x TBS/Tween 1xTBS with additional of 0.1% (v/v)

Tween-20. Stored at room temperature.

TRAP Stain Solution A A mixture of 100mM sodium acetate

trihydrate (6.8g), 50mM sodium tartrate

dihydrate (5.8g), 0.22% glacial acetic

acid (1.1ml), pH adjusted to 5, and

volume adjusted to 500ml with ddH2O.

TRAP Stain (Complete) 5mg of AS-MX dissolved in 250μl of 2-

Ethoxyethanol (EGME) and 30mg of Fast

Red-Violet-LB salt dissolved in 50ml of

TRAP stain solution A. Stored as 10ml

aliquots at -20°C. Filtered before use.

0.1% Triton X-100/PBS 0.1% (v/v) stock solution (50μl of 100%

Triton X-100) in 50ml 1xPBS. Filter

sterilized and stored at 4°C.

Weigert’s iron haematoxylin Solution A prepared by mixing 1g of

haematoxylin (CI75290) with 100ml of

96% ethanol; Solution B prepared by

mixing 4ml of 30% Iron chloride, 1ml of

37% HCl with 100ml of ddH2O. Equal


78

part of solution A and B were mixed to

make a working solution.

Western Blot Stripping Buffer A mixture of 62.5mM Tris-HCl (3.79g),

2% SDS (10g), pH adjusted to 6.7, and

then 100mM β-mercaptoethanol (3.5ml)

was then added. Volume adjusted to

500ml with ddH2O. Autoclaved and

stored at room temperature.

Western Blot Transfer Buffer A mixture of 25mM Trizma base (6.06g),

192mM glycine (28.8g), and 10%

methanol (200ml). Volume adjusted to

2L with ddH2O and stored at room

temperature.

4.2.2 Media and agar

Media and agar Composition and preparation

Luria Bertoni (LB) Broth A mixture of 10g/L Bacto-Tryptone, 5g/L

yeast extract, and 5g/L NaCl, dissolved in

ddH2O. Autoclaved and stored at room

temperature.

LB/Ampicillin (Amp) After autoclaving, LB was cooled at

room temperature until the temperature

drop to <70°C and then supplemented

with 1ml (100μg/ml) ampicillin.

LB/Amp Agar Plates Stock LB plus 15g/L agar. Autoclaved,

cooled at room temperature to <70°C,

supplemented with 1ml (100μg/ml)

ampicillin, and poured into petri dishes.

Stored at 4°C until use.

LB/Amp/IPTG/X-gal Agar Plates IPTG (0.012%) and X-gal (40μg/ml)

were spread onto LB/Amp plates and

allowed to dry prior to use.


79

4.3 Animals 4.3.1 Svs7 transgenic (TG) mice generation

Svs7 gene was cloned into pcDNA3.1 V5-His (described in more details in cloning

method Section 4.6). After SfuI restriction enzyme digestion (as depicted in Figure 4.1),

the linearized DNA fragment, containing CMV promoter region, Svs7 coding sequence,

and BGH polyadenylation signal, was purified and used in blastocysts microinjection to

create TG mice.

SfuI SfuI

CMV promoter

Svs7 coding sequence V5 6xHis BGH polyadenylation signal

654bp 500bp 442bp

3,597bp 811bp

Svs7 transgene (6,000bp) Figure 4.1 Svs7 TG mice generation

4.3.2 Svs7 knockout (KO) mice generation

Briefly, a targeting vector and Cre-LoxP system (in SpeI and KpnI sites) were used to

excise exon 2 and 3 of Svs7 gene with PGK Neo cassette in 129/Sv embryonic stem

cells (ES) (Hayman et al. 1996; Mansour, Thomas & Capecchi 1988) (Figure 4.2). The

Svs7 KO ES cells were then injected into C57BL/6 blastocysts, which were then

transferred to the uterus of a recipient mouse (acting as a surrogate mother). The male

chimeras were then mated with female C57BL/6 mice and the resulted heterozygous

were then inter-crossed to yield Svs7 KO homozygous mice.

4.3.3 Mouse tail tip DNA genotyping

Mouse tail tip DNA genotyping was performed according to Jackson laboratory’s DNA

isolation protocol. Briefly, each mouse tail tip was transferred to a sterile PCR tube,

followed by the addition of 75μl of 25mM NaOH/0.2mM EDTA. The sample was then

placed in Eppendorf thermocycler at 98°C for an hour incubation, which was

subsequently followed by 15°C incubation until ready for the next step. Each sample

was then mixed with 75μl of 40mM Tris-HCl (pH 5.5) and centrifuged at 4,000rpm for

3 minutes at room temperature. Two μl of each DNA sample (supernatant) was then

used as a template in PCR DNA amplification and the rest of the samples were stored at

-20°C.


80

Figure 4.2 Svs7 KO mice generation

PCR reaction was carried out with three primers detailed below.

Primers Sequence (5’-3’)

769 WT Rev GAC ATG TAG GTT AGA TCT GGG T

769 3’ GTG TGT CTG AGT ACA CTC TTG

GAT G

769 M 5’ TGG ATG CAA GTC AAG ACC TCC

TGT G

Svs7 transgene forward ATGATTCAGTGACGAAAT

Svs7 transgene reverse GAAGCTATTACACAAGTTTT

Each PCR genotyping reaction tube contained 12.5μl of 2xGoTaq DNA polymerase

Green Reaction Mix, 0.5μl of each primer, 9μl of ddH2O, and 2μl of tail tip DNA

sample to yield a final reaction volume of 25μl. The reaction tubes were mixed by

centrifugation using Tomy MicroOne centrifuge and then placed in the Eppendorf


81

thermocycler for initial denaturation at 95°C for 5 minutes, followed by 35 cycles of

95°C for a minute, 58°C for 40 seconds, and 72°C for a minute. The DNA amplification

ended with the extension period at 72°C for 5 minutes.

4.3.4 ΜicroCT analysis

MicroCT analysis was carried out using Skyscan 1174 microCT scanner (Skyscan,

Belgium) by Dr Tamara Davey. Tibia, femur, and lumbar bones were imaged using an

X-ray tube with settings: 50kV, 800μA, with a 0.5mm aluminium filter, and 4000ms

exposure time. The scanning angular rotation was 180° and the angular increment step

was 0.4°. The images were reconstructed using NRecon skyscan software (settings:

level 2 smoothing, 32% beam hardening, and 12 rings artefact) and trabecular bone

distal to the proximal growth plate was selected for analysis. A region of 0.3mm from

the growth plate and 1mm in height was analysed. The parameters included trabecular

bone volume (bone volume over tissue volume (BV/TV)), trabecular number (Tb.N),

trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th).

4.3.5 Histology on hindlimbs and prostate of Svs7 TG and KO mice

4.3.5.1 Fixation, decalcification (for hindlimbs), tissue processing, tissue embedding,

and sectioning

Hindlimbs from WT and Svs7 KO mice were dissected out and fixed in 10% NBF for

24 hours. The bones were then washed twice with 1xPBS and were kept in PBS

overnight to completely wash off the fixation solution. The bones were then transferred

into 70% ethanol and stored at 4°C until ready for decalcification and tissue processing.

Prior to decalcification, all non-bone tissues were removed and tibia and femur were

separated gently. The bones were washed once with PBS and then kept wet in PBS

overnight. The decalcification process began by resuspending the bones in excess of

14% EDTA (pH 7.4) and placed them in 37°C incubator for 5 days (with fresh EDTA

media change every day). When the bones became soft and flexible, they were ready for

tissue processing using the Leica TP1020 tissue processor (as shown in table 4.1

below). Before the tissue processing step, the bones were washed with PBS then 70%

ethanol.

Table 4.1 Tissue processing (Dehydration)

Station Reagent Time (minutes)

1 70% Ethanol 30

2 95% Ethanol 30


82

3 95% Ethanol 45

4 100% Ethanol 60

5 100% Ethanol 60

6 100% Ethanol 60

7 Ethanol/Xylene 60

8 Xylene 90

9 Xylene 120

10 Paraffin wax 90

11 Paraffin wax 120

12 Paraffin wax 120

The processed bones were then paraffin-embedded and cut into 5μm thick sections,

performed by Mr Jacob Kenny from The School of Pathology and Laboratory

Medicine. Sections were adhered to microscopy glass slides and dried overnight at

37°C.

4.3.5.2 Dewaxing and H&E staining

Sections were dewaxed by bringing them into 3 changes of fresh xylene for 2 minutes

each. Sections were then rehydrated in 3 changes of 100% ethanol for a minute each,

followed by 95% and 70% ethanol also for a minute each. Sections were then washed in

running deionised ddH2O for about a minute. Sections were then removed from ddH2O

and immersed in Gill’s haematoxylin stain for 3 minutes. Excess stain was washed off

by rinsing the sections in ddH2O, followed by a minute wash in Scott’s tap water to

balance pH. The sections were then briefly washed in running deionised ddH2O before

bringing them into 2 changes of ethanol (in the order of 70% then 95% ethanol) for a

minute each. One % eosin staining was used to counterstain the section for a minute,

followed by dehydration in 3 changes of 100% ethanol for a minute each. Sections were

then immersed in 50% ethanol/50% xylene for 2 minutes first before going through 3

changes of xylene for 2 minutes each. Sections were mounted with DPX mounting

medium. The images were then taken using 4x objective magnification of Nikon Eclipse

Ti microscope.

4.3.5.3 Dewaxing and TRAP staining

Sections were dewaxed and rehydrated as described above. After washing in ddH2O,

excess liquid was removed from the sections (without causing drying to the section) and


83

filtered TRAP stain was then added drop wise to the sections. The staining process was

then carried out at 37°C incubation in the dark for about 1-2 hours. After TRAP stain

was fully developed, the sections were rinsed in ddH2O before counterstaining them

with Gill’s haematoxylin for about 10 seconds. The sections were then rinsed in ddH2O

for a minute to remove excess of the counterstain, followed by a minute wash in Scott’s

tap water to balance pH. When the counterstain colour was too strong, 1% acid alcohol

(for 3 seconds on the sections) was used to wash off some of the counterstain, which

was then further adjusted by bringing them again into the Scott’s tap water for about 30

seconds. The sections were then briefly washed in running deionised ddH2O before

bringing them into 2 changes of ethanol (in the order of 70% then 95% ethanol) for a

minute each. After a brief wash in running deionised ddH2O, the TRAP-stained sections

were now ready to be mounted in DAKO aqueous mounting medium and DPX for long-

term storage. The images were then taken using 10x objective magnification of Nikon

Eclipse Ti microscope.

4.3.5.4 Processing, embedding, and sectioning of non-decalcified bone samples

After the process of extraction, fixation, and PBS wash of hindlimbs (as described in

Section 4.3.4.1) of WT and Svs7 TG mice, the undecalcified samples were then ready

for tissue processing using the Leica TP1020 tissue processor (as shown in table 4.2

below).

Table 4.2 Undecalcified tissue processing (Dehydration)

Station Reagent Time (minutes)

1 50% Ethanol 120

2 70% Ethanol 120

3 80% Ethanol 120

4 96% Ethanol 120

5 100% Ethanol 180

6 100% Ethanol 180

7 Xylene 60

8 Xylene 720

The samples were then washed twice and immersed in the infiltration reagent (89g of

methylmethacrylate (MMA), 10g of plasticiser, 1g of perkadox 16, and 0.01g of

tinogard tt (scavenger)) for 3-4 days at 4°C in vacuum conditions. Finally, the samples

were embedded in the infiltration reagent and immersed in 29°C water bath for 3 days


84

to complete the polymerization process. Samples were then sectioned at the thickness of

5μm using the Leica RM2255 Automated Microtome. The processing, embedding, and

sectioning of undecalcified femur samples of Svs7 TG mice was performed by Miss

Euphemie Landao (Centre for Orthopaedic research, School of Surgery, UWA).

4.3.5.5 Goldner trichrome staining for the undecalcified bone samples

Prior to staining, the MMA resin was removed from the sections by incubating the

samples in 3 changes of methoxyethyl acetate (MEA) solution for 20 minutes each,

followed by 2 changes of xylene for 10 minutes each. The samples were then ready for

rehydration process by rinsing them briefly (about 10 to 15 seconds for each change) in

a decreasing row of ethanol (2 changes of 100%, 96%, 80%, 70%, and finally in 50%

ethanol). To complete the rehydration process, the samples were then rinsed briefly in 2

changes of ddH2O. The sections were then stained for nuclei using Weigert’s iron

haematoxylin for 20 minutes, followed by washing in ddH2O for a minute. The staining

was differentiated in acid alcohol for no more than 5 seconds, which was followed by

gentle wash in ddH2O. The sections were then incubated in solution A stain for 5

minutes and rinsed with 1% aqueous acetic acid after. The sections were treated with

solution B for approximately 3 minutes until collagen is decolourized, followed by a

brief rinse in 1% aqueous acetic acid. The sections were then finally stained in solution

C for 5 minutes and treated with 1% aqueous acetic acid for 5 minutes. The stained

sections were then dehydrated by a brief rinse in 100% ethanol and were cleared and

mounted. The mineralised bone is stained green, orange/red colour for osteoid,

blue/grey for nuclei, and purple for cartilage. The images were then taken using 10x

objective magnification of Nikon Eclipse Ti microscope.

4.3.6 Double calcein labelling of Svs7 KO mice

To analyse bone formation activity in vivo, 3 pairs of 3 month-old WT and Svs7 KO

mice were injected with calcium-binding fluorochrome dye, calcein, intraperitoneally,

twice (double labelling) seven days apart. The mice were sacrificed 2 days after the

second injection. The hindlimbs were extracted from mice and fixed in 10% NBF

followed by plastic embedding. The processing and sectioning of the plastic-embedded

tissues was carried out by our collaborator, Dr Jerry Feng, in China. Briefly, the

samples were first dehydrated at room temperature in gradual increase of ethanol

content; 50%, 70%, 80%, 96%, and lastly 2 changes of 100% ethanol for 5 hours each

step. Two changes of xylene, for 5 and 25 hours respectively, at room temperature were


85

then used to finalise the dehydration process. The samples were then immersed in

infiltration reagent (89g of methylmethacrylate, 10g of plasticiser, 1g of perkadox 16,

and 0.01g of Tinogard® TT (scavenger)) for 3-4 days. Finally, the samples were

embedded in the infiltration reagent and immersed in 29°C water bath to complete the

polymerization process. Sections were then cut at the thickness of 5μm. The image of

double calcein labelling was taken using confocal microscopy and the distance between

2 labels was measured at 3 points along cortical bone using ImageJ software, which was

also performed by Dr Jerry Feng.

4.4 General Methods 4.4.1 Isolation and culture of primary cells

4.4.1.1 Isolation and culture of bone marrow cells

Bone marrow cells were extracted from tibia, femur, and humerus of WT and Svs7 KO

mice. First, the long bones were dissected out from the mice and placed in the 5ml tube

containing complete α-MEM medium (α-MEM medium supplemented with 10% FBS,

100U/ml Penicillin, and 100μg/ml Streptomycin). After all the samples have been

collected on the sterile bench, the isolation of bone marrow cells was achieved by

flushing the bone marrow cavity with the complete α-MEM medium using a 23G

needle. Bone marrow cells, which was suspended in α-MEM medium, was filtered

through a 100μm cell strainer on top of the 50ml tube. The bone marrow cell suspension

was then centrifuged at 1,500rpm for 5 minutes to pellet the cells. The cell pellet was

then resuspended in fresh complete α-MEM medium.

For osteoclast culture, bone marrow macrophages/monocytes (BMM) were resuspended

in complete α-MEM supplemented with 10ng/ml M-CSF in a T75 flask until reaching

confluency. The cells were then trypsinised (using 1xtrypsin for 15-20 minutes

incubation at 37°C 5% CO2 incubator), seeded at the required cell density into culture

plates (shown in table below), and cultured in the same medium as above with

additional stimulation of 100ng/ml RANKL. Culture medium was fully changed every

two days.

Cell culture plate for osteoclast culture Cell density

6-well (RNA and protein extraction, cell

transfection)

5x104 cells/2ml/well

48-well (Fluo4-AM calcium assay) 1.6x104 cells/500μl/well

96-well (Time-course RANKL-induced 6x103 cells/100μl/well


86

osteoclastogenesis)

For osteoblast culture, bone marrow cells were counted and seeded directly into culture

plates and cultured in complete α-MEM medium. When they reached confluence, the

culture medium was then supplemented with osteoblast differentiation/mineralization

factors (10nM dexamethasone, 2mM β-glycerophosphate, and 50μg/ml ascorbate). Full

medium change was carried out every 3-4 days.

Cell culture plate for osteoblast culture Cell density

24-well 1x106 cells/ml/well

4.4.1.2 Isolation and culture of spleen cells

Spleen was dissected out from WT and Svs7 KO mice and placed into a 5 ml tube

containing complete α-MEM medium. Using the back of syringe, spleen was then

crushed against a cell strainer, which was on top of a 50ml tube, to release the cells.

Complete α-MEM medium was then pipetted into the cell strainer to wash the cells

through. The cell suspension was then centrifuged at 1,500rpm for 5 minutes to pellet

the cells. The cell pellet was then resuspended in complete α-MEM medium

supplemented with 10ng/ml M-CSF and transferred to a T75 culture flask until reaching

confluency and used for experimental purposes. All cell cultures were grown and

maintained in a 37°C cell culture incubator with 5% CO2.

4.4.1.3 Isolation and culture of calvarial osteoblasts

Calvariae from WT and Svs7 KO neonatal mice were dissected out from the head. After

the removal of skin and duramater, calvariae were subjected to four sequential 15

minutes digestions in an enzyme mixture containing 0.05% trypsin and 1.5U/ml

collagenase at 37°C incubation. Cell fractions of 2 to 4 were pooled and enzyme

activity was stopped by the addition of DMEM complete medium. The calvarial

osteoblasts were then plated at a density of 1x104 cells/ml/well in a 12-well plate for

MTS proliferation assay.

4.4.2 Cell culture of COS-7 cell line

The monkey kidney fibroblast-like cell line, COS-7 cells, were cultured in complete

DMEM-Glutamax-1 medium in a 37°C 5% CO2 cell incubator. Culture medium was

fully replenished every two days and cells were passaged upon reaching confluence.


87

4.4.3 Cryopreservation of cell lines for long-term storage

When the cells were not needed for a while and they had reached confluence,

cryopreservation procedure of the cells was conducted. After culture medium had been

discarded, the cells in T75 flask were washed once with sterile 1xPBS and trypsinised

(for COS-7 cells to detach usually takes 3-5 minutes incubation at 37°C 5% CO2

incubator). To deactivate trypsin, about 5ml of fresh complete culture medium was

added to the flask and the cells in suspension was transferred to a sterile 50ml tube. To

pellet the cells, the tube was centrifuged at 1,500rpm for 5 minutes at room temperature.

Supernatant was then removed and the cells were resuspended in 92% FBS and 8%

DMSO and aliquoted into 1ml per sterile cryovial. Cryovials were then stored at -80°C

in ethanol/isopropanol-equilibrated Mr. Frosty freezing container. Cryovials can later be

stored in liquid nitrogen tank to virtually stop all biological activity of the cells and

hence for long-term storage.

4.4.4 RNA extraction

RNA extraction (for osteoclast cells grown in monolayer and for tissues/organs) was

carried out using Trizol RNA isolation protocol. Briefly, culture medium was aspirated

and cells grown in monolayer were washed once with 1xPBS. Cells were then lysed by

directly adding 1ml of trizol reagent/3.5cm diameter dish into the cultured plates,

followed by scraping the cells with the pipette tip. The sample suspension in trizol was

then pipetted up and down to completely homogenize the sample. The complete

homogenized sample was then transferred to a 1.5 ml tube and incubated for 5 minutes

at room temperature to allow complete dissociation of nucleoprotein complexes. The

sample was then centrifuged at 12,000rpm 4°C for 10 minutes to pellet cell debris. The

supernatant was then transferred to a new 1.5ml tube, followed by the additional of

0.2ml of chloroform/1ml of trizol reagent used. The sample was vigorously shaken for

15 seconds and then vortexed for another 15 seconds. The sample was incubated at

room temperature for 2-3 minutes and then centrifuged at 12,000rpm 4°C for 15

minutes to allow RNA release into a colourless upper aqueous phase. The aqueous RNA

was then transferred to a new 1.5ml tube and precipitated by mixing it with 0.5ml of

isopropyl alcohol/1ml of trizol reagent used. The sample was incubated at room

temperature for 10 minutes and centrifuged at 12,000rpm 4°C for 10 minutes to pellet

RNA. The supernatant was then completely discarded and the RNA pellet was washed

with 1ml of 75% ethanol/1ml of trizol reagent used, vortexed, and centrifuged at

7,500rpm 4°C for 5 minutes. The leftover ethanol was then removed and the washing


88

procedure was repeated once more. The RNA pellet was air-dried for 10 minutes at

room temperature and 30μl of nuclease-free water was then added to the tube to

dissolve and resuspend the RNA pellet. RNA samples were then stored at -80°C until

required for reverse transcription (RT) reactions.

For the isolation and RNA extraction from tissues/organs, a pair of WT and Svs7 KO

mice was sacrificed and organs, such as bone, seminal vesicle, brain, heart, thymus,

lung, liver, spleen, stomach, kidney, intestine, skin, fat, and ovary, were extracted and

each of them was then placed in an individual sterile mortar. One full scoop of liquid

nitrogen was then added to the mortar and the frozen organ was subsequently crushed

by grinding it using a pestle. After uniform powder-form of the organ was observed in

the mortar, 1ml trizol solution was then added and the grinding procedure continued to

get a homogenous trizol mixture. The sample (in trizol) was then ready to be processed

as described above.

4.4.5 RNA concentration measurement

RNA concentration was measured before RT reaction was carried out. Eppendorf

Biophotometer Plus was used to measure RNA concentration. RNA samples were taken

out from -80°C storage and put on ice. Firstly, the spectrophotometer was blanked using

1μl of nuclease-free water, followed by 1μl of each RNA sample in concentration unit

of ng/μl. After measurement, the samples were returned to -80°C storage until ready for

RT reactions.

4.4.6 RT reactions

RT reaction was carried out using Promega enzymes. Each RNA concentration was

standardised to 1μg and hence the required volume for each RNA was adjusted to 2μl

with nuclease-free water. For the first reaction, each PCR tube contained 2μl of dNTP

mix (5mM), 0.25μl of Oligo(dT) first strand primer (100μM), 10.75μl of nuclease-free

water, and 2μl of RNA sample (1μg) for a total reaction volume of 15μl. The samples

were then mixed by a short spin (5 seconds) using Tomy MicroOne centrifuge and then

heated in Eppendorf thermocycler at 75°C for 3 minutes to disrupt the formation of

secondary structures. Immediately after, the tubes were centrifuged for another 5

seconds using Tomy MicroOne centrifuge and placed back on ice to prevent the re-

formation of secondary structures. The second reaction was then begun by adding a

reaction mix of 4μl of 5xRT buffer (Promega Corp), 0.5μl of RNase inhibitor (Promega


89

Corp), and 0.5μl of M-MuLV RT (Promega Corp) to each reaction tube to yield the

final reaction volume of 20μl. The samples were then spined briefly for 5 seconds and

then placed in Eppendorf thermocycler for the 42°C incubation for an hour. Finally, the

samples were heated at 92°C for 10 minutes to inactivate the RT. All cDNAs were

stored at -20°C.

4.4.7 Polymerase Chain Reaction (PCR) and quantitative PCR (qPCR)

Briefly, each PCR reaction tube contained 7μl of 2x GoTaq green reaction mix, 0.5μl of

each 20μM primer (forward and reverse), 5μl of nuclease-free water, and 1μl of each

cDNA sample to yield a final reaction volume of 14μl. While for qPCR, 10μl total

reaction composed of 5μl of 2x SYBR green reaction mix (Promega), 0.2μl of a mixture

of 20μM forward and reverse primers (for each gene), 1μl of 1 in 3 diluted cDNA, and

3.8μl of nuclease-free water. Each gene at one time point had triplicate wells to get

average Ct value. After normalization of Ct values of test genes against the house-

keeping gene to get ΔCt, the fold induction of each gene (particularly for time course

RANKL-induced osteoclastogenesis) was calculated using the Livak relative gene

quantification method (as shown in the equation below), where the ΔCt of the test gene

for WT and Svs7 KO mice at each time point was normalized to the ΔCt of the test gene

for WT at day 0 of RANKL stimulation.

= 2-(ΔCt- ΔCt (of WT day 0))

= 2-ΔΔCT

The standard PCR/qPCR cycle was run consisting of initial denaturation at 94°C for 5

minutes, cycled at 94°C for 40 seconds, an annealing temperature (varied depends on

primers), and extension at 72°C for 40 seconds for 30 cycles. The reactions were

subjected to final extension step at 72°C for 7 minutes.

Table 4.3 Osteoclast and osteoblast marker genes primers sequences

Name Primer Sequence PCR Conditions

(Anneling

temperature and

number of cycle)

Size of Amplicon

(bp)

ββ-actin (For)

(House-keeping

gene)

CTG TCG AGT

CGC GTC CAC

CC

60°C 40 seconds,

30 cycles

200

ββ-actin (Rev) CCA CCA TCA


90

(House-keeping

gene)

CAC CCT GGT

GCC

Svs7 (For) GCA GAT GCG

TTG CAA AAC

CTG GTG

60°C 40 seconds,

30 cycles

103

Svs7 (Rev) TCC TTG CAC

TGA GGC GAG

CAC

TRAP (For) CCC AAT GAT

GCA CTT CTG

CCC CAG

60°C 40 seconds,

30 cycles

131

TRAP (Rev) CAG CAC CAC

CCA TGA ATC

CTA CCT G

CTR (For) CAA GTC AGG

AGA GCC AGC

CGC

60°C 40 seconds,

30 cycles

116

CTR (Rev) GGG ATG CCA

ACG GCA GGG

TG

NFATc1 (For) TGA GGC TGG

TCT TCC GAG

TT

60°C 40 seconds,

30 cycles

90

NFATc1 (Rev) CGC TGG GAA

CAC TCG ATA

GG

DCSTAMP (For) CTT GCA ACC

TAT GGG CAA

AG

60°C 40 seconds,

30 cycles

246

DCSTAMP (Rev) TCA ACA GCT

CTG TCG TGA

CC

ATP6v0d2 (For) GTG TCC CAT

TCT TGA GTT

60°C 40 seconds,

30 cycles

110


91

TGA GGC CG

ATP6v0d2 (Rev) CCG CAG GTG

GGG AAG AGG

GT

Runx2 (For) CGC ATT CCT

CAT CCC AGT

AT

60°C 40 seconds,

30 cycles

450

Runx2 (Rev) TGT AGG TAA

AGG TGG CTG

GG

Osterix (For) TTC CCT CAC

TCA TTT CCT

GG

60°C 40 seconds,

30 cycles

351

Osterix (Rev) GTA GGG AGC

TGG GTT AAG

GG

Alkaline

phosphatase /

ALP (For)

AAC TGC TGG

CCC TTG ACC

CCT

60°C 40 seconds,

30 cycles

186

Alkaline

phosphatase /

ALP (Rev)

TCC TGC CTC

CTT CCA CCA

GCA

Osteopontin /

OPN (For)

TCT GAT GAG

ACC GTC ACT

GC

60°C 40 seconds,

30 cycles

349

Osteopontin /

OPN (Rev)

TCT CCT GGC

TCT CTT TGG

AA

Osteocalcin / OCN

(For)

GCG CTC TGT

CTC TCT GAC

CT

60°C 40 seconds,

30 cycles

233

Osteocalcin / OCN

(Rev)

ATA GAT GCG

TTT GTA GGC

GG


92

M-CSF (For) AGC CGA GAT

GTG GTG ACC

AAG CC

60°C 40 seconds,

30 cycles

125

M-CSF (Rev) AAG CCA GCC

AGA GGG GCC

AT

RANKL (For) TCT GTT CCT

GTA CTT TCG

AGC GCA G

60°C 40 seconds,

30 cycles

198

RANKL (Rev) TGT TGC AGT

TCC TTC TGC

ACG GC

OPG (For) GCC GTG CAG

AAG GAA CTG

CAA C

60°C 40 seconds,

30 cycles

126

OPG (Rev) GGT GTG CAA

ATG GCT GGG

CCT

4.4.8 DNA agarose gel electrophoresis

1.5% (w/v) agarose gel was used for PCR product gel electrophoresis in this study.

Briefly, 1.5g of agarose powder (Promega Corp) was melted in 100ml 1xTAE buffer,

followed by the addition of 5μl of SYBR safe DNA stain (after cooling period of melted

agar in room temperature for about 3 minutes). The melted agar was then poured into

the Bio-Rad mini- or wide-sub cell apparatus and allowed to set at room temperature

(15-20 minutes). DNA samples together with 4μl of 100bp or 1kb DNA ladder were

loaded onto the gel and the electrophoresis was run for 30 minutes at 90V. DNA bands

were visualized using FujiFilm LAS-4000. The images were edited by inverting the

original colour.

4.5 Cell Biology This section looks at the methods for in vitro RANKL-induced BMM

osteoclastogenesis, TRAP staining, osteoclasts count, and analysis of osteoclast nuclei

and area to analyse Svs7 KO osteoclast differentiation and fusion mechanism in

reference to WT, and bone resorption assay for osteoclast activity. For osteoblast


93

differentiation and activity, CFU alkaline phosphatase and alizarin red staining would

be detailed.

4.5.1 In vitro RANKL-induced osteoclastogenesis, TRAP staining, and osteoclast

count

WT and KO BMM were cultured in complete α-MEM supplemented with M-CSF and

RANKL, as described previously in Section 4.4.1.1, in 96-well plates for 5-7 days with

full medium change every 2 days. When osteoclasts had been observed at day 5/7

cultured wells, the culture medium was then discarded and the cells were washed twice

with 1xPBS. The cells were then fixed in 4% PFA for 20 minutes at room temperature.

The fixed cells were then washed twice with PBS and TRAP stained. The staining was

carried out for 30-35 minutes incubation at 37°C incubator in order to induce TRAP

activity. The TRAP staining development was then observed under Nikon Eclipse Ti

microscope. When the TRAP stain colour was fully developed, the staining solution

was discarded and the cells were washed twice with PBS. The stained cells were then

kept wet in PBS for long-term storage. Osteoclast count was carried out using Nikon

Eclipse Ti under 10x objective magnification and only cells having 3 or more nuclei

were counted as osteoclasts.

4.5.2 Analysis of osteoclast nuclei and area (osteoclast fusion mechanism)

The analysis of osteoclast nuclei and area for osteoclast fusion mechanism was carried

out on osteoclasts at day 3 and 5 of RANKL-induced osteoclastogenesis. For osteoclast

nuclei analysis, 3 categories system had been chosen (Fujita et al. 2012). The system

consists of a group of osteoclasts having 3 to 5 nuclei, followed by 6 to 10, and 11 or

more nuclei groups. After osteoclasts had been TRAP stained, osteoclast pictures at day

3 and 5 (each time point had at least 3 wells, consisting of at least 6 images per well, for

each WT and KO pair) were taken using 10x objective magnification of Nikon Eclipse

Ti microscope integrated with NIS element software. The analysis of osteoclast nuclei

was done using ImageJ software and the average number of osteoclasts falling into each

category was presented as a ratio to total number of TRAP+ osteoclasts/well and the

final result was a representative of 3 biological repeats experiments. Osteoclast area

analysis was also carried out using ImageJ software, where each osteoclast outline, from

at least 3 wells, consisting of at least 6 images of 10x objective magnification per well,

for each time point (day 3 and 5) for each WT and KO pair, was drawn and its area in

μm2 was measured and analysed. The final result was presented as an average OC area


94

(μm2) for day 3 and 5 of RANKL-induced osteoclastogenesis and from 3 biological

repeats.

4.5.3 In vitro osteoclast bone resorption assay

4.5.3.1 Preparation of bone slices

A small segment of shaft from bovine femur or tibia was used as a source for bone

slices for in vitro osteoclast bone resorption assay. The cortical bone was cut into slices,

having only 0.5mm in depth, using the Isomet low speed saw (Buehler) at a speed

setting of 4 with a diamond wafering blade (Buehler). The bone slices were cut from

those cortical bone slices, having only 5-6mm in diameter, using a hole-punch. The

bone slices were then sterilised and stored in 70% ethanol. Before use, the bone slices

were washed twice with sterile 1xPBS and left overnight in α-MEM medium at 4°C

with gentle shake. The following day the bone slices were ready to be seeded onto 96-

well plates for bone resorption assay.

4.5.3.2 Mature osteoclast culture on bone slices

RANKL-induced WT and Svs7 KO BMM osteoclastogenesis was carried out using

Biocoat collagen 1-coated 6-well plates for 5 days or until osteoclasts were formed.

Culture medium was discarded and osteoclasts were then washed once with sterile

1xPBS and detached from the plates using 500μl of non-enzymatic cell dissociation

solution (Sigma Aldrich)/well at 37°C 5% CO2 incubation (15-20 minutes). The cells

were then gently scraped off the bottom of the plate with a cell scraper and the cell

suspension was then transferred to a new 2ml tube for centrifugation (1,500rpm for 5

minutes) to pellet the cells. The cells were then resuspended in a 1.5ml tube containing

complete α-MEM medium supplemented with M-CSF and RANKL (same

concentration as mentioned in Section 4.4.1.1). One hundred μl of the cell suspension

was then added to each required well of 96-well plates, which already contained

sterilized bone slices. The culture plates were then incubated further for 2 days. After

incubation, the cells on bone slices were washed twice with PBS and fixed in 4% PFA,

followed by TRAP staining as described before at Section 4.5.1. Osteoclast pictures on

bone slices (at least 3 bone slices for each WT and KO) were taken for each WT and

KO pair for osteoclast count. To visualise bone resorption pits, osteoclasts were gently

scraped off bone slices using a toothbrush and ddH2O and bone slices were numbered

accordingly at the non-cell surface and gold coated to get ready for scanning electron

microscopy (SEM) analysis.


95

4.5.3.3 SEM and analysis of osteoclast resorption pits

After coating, bone slices, which were attached on pins using double-sided carbon tape

(both from ProSciTech), were ready for Philip XL30 SEM analysis at the centre for

microscopy, characterization, and analysis (CMCA), The University of Western

Australia. The setting was: set for back-scattered electron (BSE), 200x magnification,

15kV electron speed, 14-15mm working distance, and 6mm spot size. After resizing,

SEM images were saved as TIFF files and ready for pit area measurement using ImageJ

software. The resorption pits were outlined and the area was expressed in μm2. The

average resorbed area was then divided by the average number of osteoclasts on the

bone slice to give the final result as a resorption pit area/osteoclast for WT versus Svs7

KO mice.

4.5.3.4 Osteoclast bone resorption assays using Corning Osteo Assay plates

Another method used in this study for analysis of WT and Svs7 KO osteoclast bone

resorption activity was Corning Osteo Assay plates (Corning). The wells of these plates

are coated with hydroxyapatite mineral surface to mimic the actual bone mineral

surface. Briefly, WT and KO BMM were cultured first in Biocoat collagen 1-coated 6-

well plates in the presence of RANKL and M-CSF for 5 days or until osteoclasts were

formed, as described above in Section 4.5.3.2. Then, instead of using sterilised bone

slices in 96-well plates, each 100μl of WT and KO osteoclast suspensions was seeded

onto Corning Osteo Assay wells (4 wells for TRAP staining and osteoclasts count and

the other 4 (a total of 8 wells or 1 strip for each WT and KO) for resorption pits

visualization) and cultured for additional 6 days with full media change (with same

concentration of M-CSF and RANKL as mentioned in Section 4.4.1.1) every 2 days.

The media was then aspirated and for TRAP-stained wells the procedure was the same

as described in Section 4.5.1. In order to visualise the resorbed pits, the culture wells

were first incubated with 100μl/well of 10% household bleach solution for 5 minutes at

room temperature to remove osteoclasts. The bleach solution was then removed and the

wells were washed twice with 150μl/well of ddH2O, followed by air-drying for 3-5

hours at room temperature. The osteoclast count was carried out on TRAP-stained

osteoclasts images and the pits were visualized under 10x and 20x objective

magnification of Nikon Eclipse Ti microscope. The plates were then stored with 200μl

of ddH2O in each well at 4°C for long-term storage.


96

4.5.4 CFU alkaline phosphatase (ALP) staining for osteoblast differentiation assay

The ALP staining was purchased from Sigma Aldrich and the experiment was carried

out according to the protocol provided. Briefly for making 5ml ALP master mix

staining solution, 100μl of FBB alkaline solution was mixed with 100μl of sodium

nitrite solution and the mix was incubated for 2 minutes at room temperature. Four and

half ml of ddH2O was then added to the mixture, followed by the addition of 100μl of

Naphthol ASBI alkaline solution (kept in dark). The ALP staining was then added drop-

wise to the fixed bone-marrow stromal cell-derived osteoblasts and the plate was

incubated in the dark for 10-15 minutes (until blue/purple colour developed) at room

temperature. The positive colonies were then analysed and counted.

4.5.5 In vitro bone marrow stromal cell-derived osteoblast bone nodule formation

(mineralization) assay

WT and Svs7 KO BM cells were cultured in mineralization/differentiation medium

(complete α-MEM medium supplemented with 10nM dexamethasone, 2mM β-

glycerophosphate, and 50μg/ml ascorbate), as described previously in Section 4.4.1.1,

for 2-3 weeks until calcium depositions were observed. The medium was then discarded

and the cells were washed with 1xPBS. The cells were then fixed in 2.5%

glutaraldehyde for 15 minutes at room temperature. After fixation, the cells were

washed three times with PBS, followed by three times 70% ethanol washes and left to

dry completely at room temperature. One % alizarin red staining was then added to the

cells for 2 minutes, followed by three times 50% ethanol washes. The plates were then

allowed to dry completely at room temperature. To measure the total mineralized area,

the plates were scanned at 600dpi on Epson 3490 Photo scanner. The scanned images

(red stain for calcium deposition) were then converted to black (for the red-stained

calcium) and white colour and thresholded. With binary process on Image J software,

the total mineralized area was measured.

4.5.6 MTS proliferation assay on WT and Svs7 KO BMM

Proliferation assay on WT and Svs7 KO BMM was carried out using CellTiter 96

Aqueous One Solution Cell Proliferation Assay (MTS) (Promega). MTS compound is

bioreduced by metabolically active cells into coloured formazan (soluble in culture

medium), by presumably involving NADPH or NADH produced by dehydrogenase

enzymes. The assay was done by measuring the absorbance of formazan-incorporated

culture medium at 490nm using Bio-Rad spectrophotometer plus. The quantity of


97

formazan (measured by the absorbance at 490nm), is directly proportional to the

number of living cells in culture wells. After reaching confluence in T75 culture flasks,

WT and Svs7 KO BMM were seeded with cell density of 6x103 cells/100μl/well on 96-

well plates and cultured in complete α-MEM medium supplemented with M-CSF.

Three individual 96-well plates were needed for each WT and KO BMM pair. The first

plate was used for standard curve (starting from 0, 6, to 96x103 cells density with an

exponential rise), with the absorbance read the next day after cell seeding procedure and

after the addition of 20μl/well of the MTS reagent, followed by incubation at 37°C 5%

CO2 in the dark for about 2-3 hours. The next 2 plates, which were for 24 and 48 hours

time points (each having quadruplicate wells for each WT and KO BMM), were read on

the next two days accordingly after the standard curve reading. The absorbance for 24

and 48 hours time points were then related back to the standard curve equation to get an

estimated cell number for each WT and KO for each time point.

4.6 Cloning Method 4.6.1 Restriction enzyme digestion of DNA

Restriction enzymes cut double-stranded DNA at the restriction site (specific sequence

bases) to release plasmid DNA, which can be used to check for a correct DNA insert,

for DNA sequence analysis, or for an intermediate step to release DNA insert for the

subsequent ligation reactions with other vectors. Digestion reaction contained 5μl (for

miniprep concentration) or 2μl (for midiprep concentration) of plamid DNA, 1μl of 10x

restriction enzyme buffer B, 0.5μl of each restriction enzyme (HindIII and XhoI), and

ddH2O to make up a final volume of 10μl. The reaction mix was incubated overnight at

37°C water bath. The following day, the reaction mix was mixed with 2μl of 6x loading

dye and electrophoresed together with 1kb and 100bp DNA ladder, and visualized on a

UV transilluminator. The gel was examined for the plasmid DNA insert (295bp for

Svs7 DNA insert, which was amplified by PCR using sense 5’

ATGATTCAGTGACGAAAT 3’ and antisense 5’ GAAGCTATTACACAAGTTTT 3’

primers constructed by Dr Tony Phan PhD).

4.6.2 DNA extraction and purification from agarose gels

The DNA insert was extracted from agarose gels using QIAEX II Gel purification kit

(Qiagen). The DNA band was excised from the gel using a sterile surgical blade and

transferred into a clean 1.5ml tube. Gel fragment was weighed and solubilised


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according to the manufacturer’s instructions. All DNA was eluted using sterile ddH2O

and used immediately or stored at -20°C until required.

4.6.3 Cloning strategy and ligation to construct pGEM-T Easy Svs7

Briefly, 0.5μl of pGEM-T Easy vector (Promega), 9μl of 2x rapid ligation buffer

(Promega), and 1μl of T4 ligase (Promega) were added to a sterile 0.5ml tube,

containing 7.5μl of the eluted Svs7 DNA insert in a total reaction volume of 18μl. The

reaction mix was incubated at 16°C overnight. Following ligation process, the DNA

was transformed into DH5α competent cells the next day.

4.6.4 Cloning strategy and ligation to construct pcDNA3.1 Svs7-V5 His

Svs7 DNA insert was digested from the T Easy vector using HindIII/XhoI restriction

enzymes and ligated into HindIII/XhoI site of the pcDNA3.1 V5 His mammalian

expression vector (Invitrogen). Briefly, 2μl of pcDNA3.1 vector, 1μl of 10x T4 ligase

buffer (Promega), and 1μl of T4 ligase (Promega) were transferred into a 0.5 ml tube,

containing 6μl of the purified Svs7 DNA, for a final reaction volume of 10μl. The final

mixture was incubated at 16°C overnight and transformed into DH5α competent cells

the next day.

4.6.5 Transformation and plating of the transformed cells

The ligation product was transferred to a 2ml tube, containing 100μl aliquot of E. Coli

DH5α competent cells, and mixed gently. The cells were incubated on ice for 30

minutes and then heat shocked at 42°C water bath for 90 seconds. The tube was

immediately cooled on ice for 2 minutes. One ml of LB broth was added to the cells and

incubated at 37°C 225rpm for an hour. The cells were then pelleted by centrifugation at

13,400rpm room temperature for a minute. The supernatant was then discarded and the

cells were resuspended in 100μl of fresh LB. Under aseptic conditions, the cells were

spread on LB/Amp/IPTG/Xgal (for pGEM-T Easy-Svs7 construct) or on LB/Amp

plates (for pcDNA3.1 Svs7-V5 His construct). The plate was dried, inverted, and

incubated overnight at 37°C.

4.6.6 Bacterial culture and maintenance

Briefly, 3 clones (for Svs7-V5 His-expressing clones) were picked from the plate using

a pipette tip per clone and dropped directly into a 10ml tube, containing 4ml LB and 4μl

(100mg/ml) of ampicillin. The cultures were incubated at 37°C at 225rpm for 8 hours


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and then transferred to a culture flask, containing 50ml LB/Amp (at the same

concentration as mentioned above), for every clone, followed by incubation overnight at

37°C at 225rpm in order to yield midiprep plasmids.

4.6.7 Plasmid extraction and isolation

Plasmid DNA was extracted from small-scale DH5α cultures (4ml LB/Amp culture

overnight) using QIAprep spin miniprep (Qiagen). The overnight bacterial culture was

transferred to a 1.5ml tube, cells were pelleted, and the plasmid DNA was extracted

according to the manufacturer’s protocol. Purified plasmid DNA was eluted from the

mini column using 100μl of sterile ddH2O and was stored at -20°C until ready for use.

Isolation of plasmid DNA from 50ml overnight cultures was performed using Qiagen

plasmid plus midiprep (Qiagen) and done according to the manufacturer’s protocol. For

long-term storage of cloned bacterial cultures, 3.2ml of cloned bacterial suspension was

mixed with 800μl of glycerol (20%) and stored at -80°C. The midiprep purified plasmid

was subjected to restriction enzyme digestion reactions to confirm the correct DNA

insert for Svs7 in the HindIII/XhoI site of the pcDNA3.1 V5 His mammalian expression

vector, prior to the transfection experiment into COS-7 cells.

4.7 Experimental Methods 4.7.1 Cell transfection and collection of conditioned medium expressing Svs7 protein

COS-7 cells, grown in a T75 flask until reaching confluency, were transfected with

either empty pcDNA3.1 plasmid (as a control) or pcDNA3.1 Svs7-V5 His (one flask for

each plasmid in order to get mass production of conditioned medium expressing Svs7)

using Lipofectamine-2000 transfection reagent (Invitrogen) in accordance with the

manufacturer’s protocol. A total of 24μg of each plasmid DNA was diluted in 1.5ml of

Opti-MEM reduced serum medium and mixed gently. Sixty μl of Lipofectamine reagent

was also diluted in 1.5ml of the Opti-MEM medium, mixed, and incubated at room

temperature for 5 minutes. The diluted Lipofectamine reagent was then added to each

diluted plasmid DNA to make a total volume of 3ml and the complex was then

incubated further for 20 minutes at room temperature. Each 3ml DNA-Lipofectamine

complex was then added drop-wisely to the corresponding flask using a transfer pipette

and mixed gently by rocking the plate back and forth. After 6 hours incubation at 37°C

5% CO2 cell culture incubator, the transfection medium was discarded from each flask

and the cells were washed twice with fresh Opti-MEM medium to wash away the

transfection reagent. Cells were then incubated in 10ml of fresh Opti-MEM medium and


100

the collection of conditioned medium expressing Svs7 protein and control began the

next day (day 1) and performed daily for up to 6 days to generate plenty of conditioned

medium required for luciferase assay experiments. At the day of collection, each

conditioned medium (control and Svs7 plasmid) was collected and transferred to a clean

50ml tube, ready to be centrifuged for 10 minutes at 3,000rpm to pellet any dead cells.

The clean conditioned medium was then transferred to a clean 15ml tube and a 30μl

aliquot of each medium was transferred to a 1.5ml tube for western blot (WB) protein

analysis to test for the expression of Svs7 protein in the medium.

4.7.2 Luciferase assay

RAW264.7 cells, stably expressing NFAT promoter (stable transfection of RAW264.7 cells

with a GL4.30 (luc2P/NFAT-RE/Hygro) (Promega) (Singh et al. 2012)), were seeded

overnight on a 48-well plate with a cell density of 1.5x105 cells/500μl/well. With the

confirmation from WB analysis that Svs7 protein was successfully expressed in the

medium, the following day some wells were then pre-treated with 50% Svs7

conditioned medium and the rest with the control medium for an hour, followed by

100ng/ml RANKL stimulation overnight. The cells were then lysed using 100μl/well of

luciferase lysis buffer with the addition of 2mM DTT. The lysates were then collected

into 1.5ml tubes accordingly and centrifuged at 14,000rpm at 4°C for 20 minutes to

clear cellular debris. The clear lysates were then transferred to fresh 1.5ml tubes. Fifty

μl of each sample was then put into a clean white-bottom 96-well plate and mixed with

50μl of luciferase assay substrate (Promega). The luminescence reading was performed

using the BMG Polar Star Optima luminescence reader at UWA school of

Pharmacology and the NFAT luciferase activity was expressed as arbitrary unit (this

experiment had been assisted by Mingli Yang, a PhD candidate at Jiake Xu cell

signalling group, UWA School of Pathology and Laboratory Medicine).

4.7.3 Flow cytometry analysis on fixed and stained WT and Svs7 KO BM cells

BM cells were extracted from WT and Svs7 KO long bones as described in Section

4.4.11. The cell suspensions were then centrifuged at 1,500rpm for 5 minutes to pellet

the cells. One ml of red blood cell (RBC) lysis buffer was then added to the cells for a

minute, which was then followed by the addition of 9ml of 1xPBS to inactivate the

buffer activity. The cells were then pelleted and the supernatant was then replaced with

1ml of 1xPBS. The cells were then counted using Optik Labor haemocytometer for 106

cells in 50μl 1xPBS suspension for each WT and KO and also for controls (5 antibodies


101

minus 1), unstained, and live/dead (L/D) cells, which were prepared from WT cells.

Antibodies master mix was then prepared for WT and KO test samples by diluting the

antibodies (FITC-CD45R, V450-CD11b, PE Cy.7-CD3e, PE-c-Kit, and APC-Sca-1) to

0.5μg in 50μl cold flow cytometry wash buffer. The diluted antibodies master mix was

then added to the 50μl of 106 cells suspension for a total volume of 100μl and mixed

gently by tapping. The mixture was then incubated in the dark on ice for 45 minutes.

The samples were washed with 500μl of wash buffer to dilute the antibodies and the

cells were then pelleted at 350 g centrifugation 4 °C for 5 minutes. After the removal of

supernatant, the samples were washed twice with 200μl of cold wash buffer at 350 g

centrifugation 4 °C for 5 minutes. The cells were then resuspended in 500μl wash

buffer. To prepare for L/D control, 250μl of cells suspension was aliquoted out and

snap-freezed in liquid nitrogen and mixed them again with the other 250μl of cells

suspension. One μl of L/D Aqua stain was then added into the 500μl cell suspension and

mixed well by gentle tapping. The L/D control sample was then incubated on ice in the

dark for 30 minutes. All the test and control samples were then spined down and the

cells were then washed once with 500μl of wash buffer. The cells were then

resuspended in 300μl of 1xBD Stabilizing Fixative and stored at 4°C in the dark

overnight for the next day analysis. For preparation of bead samples for compensation,

100μl of wash buffer was added into each labelled tube (1 tube for 1 antibody). One full

drop of each negative and positive beads was then added to the tube, followed by the

addition of the appropriate antibody (with the same concentration as mentioned above).

The 5 samples were then incubated in the dark at room temperature for 30 minutes.

Following the incubation, 1ml of wash buffer was then added to each tube and the beads

were then pelleted by 200 g centrifugation for 10 minutes. The supernatant was

discarded and the pellet was resuspended in 300μl of 1xBD stabilizing fixative and

stored at 4°C overnight together with the rest of samples for analysis the next day. Flow

cytometry analysis was performed using FACS Canto at CMCA, UWA. Lineage

negative (Lin-) population, which is regarded as early osteoclast precursors and

characterised as CD3e- CD45R- CD11blow/-, was gated first, followed by the analysis of

Sca-1+ c-Kit+ markers from Lin- population to deduce hematopoietic stem/progenitor

cells (HSCs) niche in the bone marrow of WT and KO mice.

4.7.4 Fluo-4AM intracellular calcium assay on WT and Svs7 KO osteoclasts

Hanks wash buffer for the assay was purchased from Gibco, Australia, while Fluo-4AM

calcium-binding dye kit was from Invitrogen, Australia. First, 20% pluronic acid was


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made by dissolving 0.2g of the pluronic in 1ml of DMSO. Subsequently, 1mM Fluo-

4AM was made by dissolving 50μg of the dye (provided in the kit) in 45.58μl of 20%

pluronic acid. The wash buffer in this assay was prepared by having 2mM probenecid

(stock of 1M) in Hanks buffer solution. Fluo-4AM working solution (Fluo-4AM/Hanks)

was then made by mixing 4μl of Fluo-4AM stock/ml (4μM) of Hanks/Probenecid

required for loading 250μl per well. Forty eight-well plates were used for this assay.

After aspirating culture medium from the wells upon the observation of osteoclasts on

coverslips, the cells were washed twice with 250μl of Hanks/Probenecid wash

buffer/well, followed by loading and incubating the cells with 250μl of Fluo-

4AM/Hanks/well for 45 minutes at room temperature in the dark. After incubation, the

cells were then washed once with the Hanks/Probenecid wash buffer and the cells were

incubated further for 20 mins in 200μl of the wash buffer at room temperature in the

dark. The cells were then washed 3 times with Hanks/Probenecid wash buffer and kept

in 200μl of the wash buffer for the initial baseline reading of fluorescence intensity

(calcium oscillation). Fluo-4 emits at green fluorescence channel (488nm excitation).

Nikon Eclipse Ti microscope with fluorescent light and incorporated NIS-Element

Basic research software were used in this calcium assay. The baseline reading (calcium

oscillation) was observed and recorded for 2 minutes using timelapse tool of the NIS

software. The assay then incorporated the addition of extracellular calcium (using

calcium chloride (CaCl)) of 5, 10, and 20mM concentrations and each fluorescence

intensity reading was recorded for 3 minutes following the initial baseline reading.

4.7.5 Cell lysis procedure for Western Blot (WB) protein analysis

Briefly, culture medium was aspirated and the cells were washed once with 1xPBS. The

cells were then lysed with 200μl/well (6-well plate) of RIPA lysis buffer, followed by

incubation on ice for 20 minutes. The cells were scrapped off the bottom of the plate

using a pipette tip and the cell lysates were collected into 1.5ml tubes and centrifuged at

12,000rpm at 4°C to pellet cellular debris. The clear cell lysates were then transferred to

fresh 1.5ml tubes and stored at -20°C until ready for protein concentration measurement

and WB analysis.

4.7.6 Velocity density gradient centrifugation

Velocity density gradient centrifugation was performed as previously described (Ng et

al. 2013; Pavlos et al. 2011). Briefly, ~107 osteoclasts were washed with ice-cold

1xPBS and scraped with 500ml of lysis buffer (50mM HEPES-KOH pH 7.2, 150mM


103

NaCl, and 1mM MgCl2) supplemented with protease inhibitors: 1mM PMSF, 1mg/ml

aprotinin, 1mg/ml leupeptin, and 0.7mg/ml pepstatin (all from Sigma Aldrich). The cell

lysate was then homogenized by passing cells through a 25-gauge syringe (at least 10

strokes). Proteins were cleared by centrifugation at 3,000rpm for 15 minutes at 4°C.

Five hundreds ml of clarified supernatant was layered on top of 12ml of 5-20% linear

sucrose density gradient, which was prepared in the lysis buffer. Centrifugation was

then performed at 150,000g for 18 hours in a SW40 rotor (Beckman Coulter), followed

by the collection of 1ml protein fractions. An equal volume of fractions were analysed

in western blot system using rabbit polyclonal anti-Svs7 antibody. α-tubulin was used

as a loading control.

4.7.7 Measuring protein concentration

The Bio-Rad protein assay is a simple colorimetric assay for determining total protein

concentration. The Bio-Rad protein dye reagent was diluted 1 in 5 using ddH2O, while

protein samples were diluted 1 in 10 using 1xPBS. Two hundreds μl of the dye reagent

was then added per well to a 96-well plate. Ten μl of each protein standard (BSA/PBS

with concentration of 0, 0.05, 0.1 to 0.5mg/ml (an increment of 0.1mg/ml)), PBS

control, RIPA lysis buffer control, and protein samples (assayed in duplicate) was then

added to the dye, mixed gently, followed by incubation at room temperature for 5

minutes for colour development. The absorbance was read at 595nm wavelength using

the Bio-Rad Model 680 microplate reader.

4.7.8 Western Blot (WB) SDS-PAGE system

SDS-PAGE system is the most commonly used system to separate and analyze proteins

according to their size. To do that, sodium dodecyl sulfate (SDS), a detergent, denatures

and covers the protein primary structures with many negative charges by its negatively

charged sulfate group. Hence all proteins will migrate towards the positive pole (anode)

in an electric field. Finally polyacrylamide gel, with running electricity, will separate

proteins based on their size (Da). Hence, by using standard known protein samples,

relative molecular weight of unknown proteins and their distribution can be estimated

and examined using this SDS-PAGE system.

4.7.8.1 Preparation of the SDS-PAGE system

There are two different gels in this SDS-PAGE system, a stacking gel, where the protein


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samples are loaded onto, and a separating gel underneath, where the proteins run and

get separated based on their size. Electrophoresis of the SDS-PAGE system was carried

out using the Mini-Protean III Cell Electrophoresis System (Bio-Rad) casting apparatus.

10% and 17.5% separating gels were prepared as follows:

Separating Gel Components 10% 17.5%

MilliQ H2O 2.97ml 1.095ml

1.5M Tris-HCl, pH 8.8 1.875ml 1.875ml

10% SDS 75μl 75μl

30% Acrylamide/Bis 2.5ml 4.375ml

10% Ammonium persulfate

(APS) 75μl 75μl

TEMED 6μl 6μl

Total Volume 7.5ml 7.5ml

The separating gel final mixture was then carefully poured into a glass-gel sandwich to

about 1cm below the level of the well comb. Twenty % ethanol was slowly and evenly

overlaid on top of the separating mixture to remove any bubbles and to avoid oxidation

of the gel. The gel was then allowed to set at room temperature for 15-20 minutes. A

5% stacking gel was then prepared as follows:

5% Stacking Gel Components Volume

MilliQ H2O 2.062ml

1M Tris-HCl, pH 6.8 375μl

10% SDS 30μl

30% Acrylamide/Bis 500μl

10% APS 30μl

TEMED 3μl

Total Volume 3ml

As the separating gel set, a clear line was formed between the gel and the 20% ethanol.

The ethanol was then removed from the cast and the stacking gel mixture was poured

carefully into the sandwich on top of the separating gel. A 10 or 15-well comb was

immediately inserted into the stacking gel mixture and the gel was allowed to set at

room temperature for 15-30 minutes. After the gel polymerised, the comb was carefully


105

removed and the glass-gel sandwich was then transferred into the SDS-PAGE tank

system, which was filled with 1xSDS-PAGE running buffer. Protein samples were then

prepared by adding right amount of 4xSDS-PAGE loading dye, containing 5% ß-

mercaptoethanol (which acts to break down disulphide bonds within and between

protein molecules), and subsequently boiled at 99°C using Eppendorf Thermomixer

Comfort for 5 minutes to denature the proteins. Each protein sample was then loaded

onto the stacking gel. A 10μl volume of Precision Plus protein standard (Bio-Rad) was

also loaded and run together with the protein samples for molecular weight analysis.

The electrophoresis was run at 100V for 120 minutes.

4.7.8.2 Protein transfer to nitrocellulose membrane

The Mini Trans-Blot Electrophoresis Transfer Cell (Bio-Rad) apparatus was set up

according to manufacturer's instructions. Two scotch pads and 6 Whatman 3MM papers

were pre-soaked in WB transfer buffer. Following electrophoresis, the glass-gel

sandwich was removed from the tank. One soaked scotch pad was placed onto the clear

side of the gel holder cassette, which was then overlaid by 3 pieces of the soaked

Whatman papers and a nitrocellulose membrane. The gel was slowly released from the

inside of the sandwich and the stacking gel was carefully separated and discarded away

from the separating gel. Finally, the separating gel was then carefully placed on top of

the nitrocellulose membrane, followed by another 3 soaked Whatman papers and the

second soaked scotch pad (respectively from the middle to the top). The gel holder

cassette was then closed and placed in the WB transfer tank filled with WB transfer

buffer and an ice block, making sure that the gel is facing the black terminal (cathode)

and the membrane is facing the red terminal (anode). The protein standard and samples

were transferred to the nitrocellulose membrane at a current of 0.03A overnight.

4.7.8.3 WB reaction

The next day following WB transfer, the gel cassette was removed from the WB

transfer tank and the membrane was carefully released from the separating gel, placed

on a square dish, and incubated with 10ml of 5% skim milk in 1xTBS-Tween (TBST)

solution for an hour at room temperature on the RATEX rocking platform mixer as a

blocking step in order to reduce non-specific antibody binding (background). After an

hour in blocking step, the skim milk was discarded and the membrane was then washed

once with TBST for 5 minutes at room temperature, followed by overnight incubation

in 10ml 1:1000 or 1:500 dilution of primary antibody (listed in Section 4.1.7


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Antibodies) in 1% skim milk/TBST at 4°C. The following day the membrane was

washed 3 times with TBST for 5 minutes/wash at room temperature, followed by an

hour room temperature incubation with 10ml 1:5000 dilution of goat peroxidase-

conjugated secondary antibody in 1% skim milk/TBST on the RATEX rocking platform

mixer. After incubation and removal of the secondary antibody, the membrane was

washed twice with TBST for 5 minutes/wash, followed by twice TBS washes each for 5

minutes. The detection of proteins by immuno-reaction was achieved using Western

Lightening Ultra Extra Sensitivity (Perkin Elmer) kit. Solution A and B (from the

Detection System) were pre-warmed at room temperature and 1:1 ratio mixture of these

2 solutions was then made in a 1.5ml microcentrifuge tube. The detection reagent

mixture was then incubated with the washed membrane for a minute at room

temperature in the dark. After the removal of excess detection reagent from the

membrane, chemiluminescence detection was performed using the FujiFilm LAS-4000.

4.7.8.4 Stripping the membranes

Briefly, the stripping buffer was pre-warmed to 55°C, while the used membranes were

washed once with TBST for 5 minutes. The wash solution was discarded and the

stripping buffer was then added to the membranes, followed by incubation at 55°C with

a gentle shake for 30-40 minutes. The membranes were washed twice with TBST for 5

minutes each. Finally, the membranes were ready for the next WB reaction starting at

the blocking step.

4.8 Statistical Analysis Experimental data are presented as mean ± SEM of 3 independent samples. Statistics

were performed using two-tailed Student t-test for each biological pair (at least 3

biological repeats), where p values < 0.05 or < 0.01 were considered significant.

107

CHAPTER 5

CHARACTERISATION OF SVS7 TRANSGENIC MICE

Chapter Five – Characterisation of Svs7 Transgenic Mice

108

5.1 Introduction Aforementioned, healthy bone mass is achieved largely by balanced yet complementary

activity between bone-resorbing osteoclasts and bone-forming osteoblasts. This bone

remodelling process is governed by several direct cell-to-cell interaction systems,

including between osteoclasts and osteoblasts, as exemplified by the binding of

EphrinB2, an osteoclast-expressed membrane-bound ligand, to its receptor EphB4,

expressed by osteoblasts (Martin et al. 2010; Zhao et al. 2006), and through the

secretion of growth factors, cytokines, and proteins by both cells (Zaidi 2007). Bone

diseases, such as osteoporosis and osteopetrosis, may therefore occur as a consequence

of the loss of coupling and communication between osteoclasts and osteoblasts

(Guerrini et al. 2008; Riggs & Melton 1983; Sobacchi et al. 2007; Villa et al. 2009).

Osteoporosis, by far the most common bone disease (Lerner 2006), is characterized by a

net bone loss, causing bones to become weaker and have a greater susceptibility of

fractures (Riggs & Melton 1983). Excessive osteoclast bone resorption (at the level

where more bone resorbed than formed) accompanies this pathological bone disease,

which is primarily seen in postmenopausal women (triggered by hormonal change)

(Riggs & Melton 1983) and in the elderly (Manolagas & Jilka 1995). As the cost for

management and treatment of osteoporosis and other bone diseases increases each year

(approximately to be $14 billions/year for 1.3 millions fractures in US (Lerner 2006;

Ray et al. 1997)), research has been focused on identifying novel factors, expressed by

osteoclasts and/or osteoblasts, that influence their differentiation and activation and

hence may be utilised as therapeutic targets for skeletal diseases.

In search of candidate coupling factors from osteoclasts, we have previously employed

subtractive hybridization-based differential screening method and identified Svs7 as a

novel osteoclast-expressed factor. Furthermore, studies from Davey 2008 suggested that

recombinant purified Svs7 protein treatment could enhance trabecular bone/tissue

volume, trabecular thickness, and trabecular number in ovariectomised mice (oestrogen

deficiency-induced bone loss mouse model (Aitken, Armstrong & Anderson 1972; Kalu

1991; Saville 1969)), implying that the administration of recombinant Svs7 protein

might be a potential anabolic factor and therefore be beneficial to the treatment of

osteoporosis. Hence, based on these initial data, the major aim of this thesis was to

explore the physiological role of Svs7 in bone homeostasis. To address this, a two-

armed approach was employed: 1. To generate transgenic (TG) mice overexpressing


109

Svs7 and 2. To genetically ablate Svs7 globally in mice. The generation and

characterisation of mice overexpressing Svs7 are presented herein.

5.2 Results 5.2.1 Tissue distribution of Svs7 gene

As an initial step towards characterising the influence of Svs7 overexpression in mice,

the global expression pattern of Svs7 was first established in all major mouse tissues,

with osteoclast as a control. For this, organs and tissues were extracted from 12-week

old WT mice, snap frozen, and total RNA was extracted using Trizol method (as

described in Chapter 4 Section 4.4.4). Semi-quantitative reverse transcription PCR (RT-

PCR) reaction was then conducted across each tissue sample using specific primers for

Svs7 and densitometry analysis was performed to quantify the relative ratio of Svs7

expression in each tissue over β-actin, which served as a loading control. As expected,

Svs7 was most abundantly expressed in seminal vesicles (SV), which included prostate

tissue, with little to no expression observed in other tissues examined, including brain,

thymus, lung, liver, and stomach (Figure 5.1). In addition, Svs7 was detectable in

osteoclasts but not in total bone, possibly owing to low level of resident osteoclasts in

vivo (Figure 5.1). Thus, these data indicate that Svs7 is largely restricted to SV tissue

and osteoclasts.

5.2.2 Generation of the Svs7 transgenic (TG) mice

Having established the expression pattern of Svs7 in osteoclast and other peripheral

tissues, we proceeded with the generation of Svs7 TG mice. This was achieved by

microinjecting a linearized and purified pcDNA3.1 Svs7 V5-His transgene (confirmed

by agarose gel electrophoresis (lane 2) and shown in Figure 5.2A) into blastocysts, as

detailed in Chapter 4 Section 4.3.1 Figure 4.1. Following breeding, genotyping was

performed for the first six litters using specific primers for Svs7. The expression of the

Svs7 coding sequence (295bp) in litter number 4 and 5 was confirmed by gel

electrophoresis (Figure 5.2B). In order to confirm the transgenic expression of Svs7 at

the protein level, livers were extracted from 9-week old WT and Svs7 TG mice, snap

frozen, and subsequently crushed using sterile mortars and pestles. Protein extraction

was performed using Ripa lysis buffer. Western blot (WB) analysis on cleared liver

lysates from WT and Svs7 TG mice was conducted using rabbit polyclonal anti-Svs7

antibody. Unexpectedly, the result showed that although the expression of Svs7

transgene was successful, the TG mice only showed a modest increase in Svs7 protein


110

0 0.2 0.4 0.6 0.8 1 1.2

OC Bone

Seminal vesicle Brain Heart

Thymus Lung Liver

Spleen Stomach

Kidney Intestine

Skin Fat

Ovary

Ratio to β-actin

Thym

us

Stom

ach

Intes

tine

Svs7

β-actin

Semina

l Ve

sicle

OC Bon

e

Brain

Heart

Lung

Li

ver

Splee

n

Kidney

Skin

Fat

Ovary

Figure 5.1 Tissue distribution of Svs7 mRNA expression. Selected organs and tissues

were extracted from 12-week old WT mice, snap frozen with liquid nitrogen, and RNA

extraction from these frozen tissues was performed using Trizol method. RT-PCR

reaction on these tissues samples was then performed using specific primers for Svs7.

Osteoclast RNA sample was used as a positive control. Densitometry analysis was

carried out using ImageJ software and the Svs7 gene expression in each tissue was

presented as a ratio to β-actin, which served as a loading control. The result showed that

Svs7 mRNA was abundantly expressed in SV (including prostate tissue) but

undetectable in other tissues, including bone, brain, thymus, lung, liver, and stomach.


111

1 2 3 4 5 6

250bp

500bp Marker

WT TG

Svs7

β-actin

0

0.05

0.1

0.15

0.2

0.25

0.3

WT TG Sv

s7 p

rote

in e

xpre

ssio

n in

live

r (r

atio

to β

-act

in)

pcDNA3.1 Svs7-V5 His

pcDNA3.1 Svs7-V5 His linearised with SfuI

2 1 DNA

Ladder

6000bp

3000bp

(A) (B)

(C)

Figure 5.2 Generation of the Svs7 TG mice. (A) SfuI restriction enzyme digestion on

pcDNA3.1 Svs7 V5-His was performed to get the linearized form of the transgene

plasmid and the product was subsequently run and checked on agarose gel

electrophoresis, together with the undigested plasmid. The result showed a successful

generation of the linearized Svs7 transgene in lane 2 with size of about 6,000bp. The

linearized Svs7 transgene plasmid was then purified and microinjected into blastocysts

for the generation of Svs7 TG mice. (B) Genotyping for the first six litters using

specific primers for Svs7 and gel electrophoresis for the DNA genotyping products

showed that litter number 4 and 5 had successfully expressed Svs7 coding sequence

(295bp). (C) Livers were extracted from 9-week old WT and Svs7 TG mice and after

being snap-frozen with liquid nitrogen and crushed using sterile mortars and pestles, the

protein extraction was performed using Ripa lysis buffer. (Top panel) WB analysis on

these liver lysates using rabbit polyclonal anti-Svs7 antibody and (Bottom panel)

densitometry measurement using ImageJ software (at which Svs7 expression was

expressed as a ratio to β-actin that served as a loading control) revealed the modest

increase in Svs7 protein expression (about 2.5 fold increase) in Svs7 TG mice, relative

to the very low expression of Svs7 in WT.


112

expression (about 2.5 fold increase, shown by densitometry measurement using ImageJ

software (Figure 5.2C)), which was relative to the very low expression of Svs7 in WT

liver lysate. β-actin served as a loading control. Together, these results reveal the

successful albeit modest expression of the Svs7 transgene in Svs7 TG mice.

5.2.3 Histological phenotyping of Svs7 TG mice

To begin to characterise the effect of Svs7 overexpression in TG mice, prostate tissues

were dissected from 9-week old (sex-matched) WT and Svs7 TG mice, fixed,

processed, embedded, sectioned, and Haematoxylin and eosin (H&E) stained for

histological examination. Prostate tissue was selected for closer phenotypic assessment

as it has the highest Svs7 gene expression amongst other organs. As shown in Figure

5.3, the histological assessment revealed no distinguishable differences in the size or

morphology of the prostate between WT and Svs7 TG mice, indicating that

overexpression of Svs7 does not influence prostate development or morphogenesis.

5.2.4 Body weight and radiographic assessment of Svs7 TG mice

To further assess the phenotype of Svs7 TG mice, the total body weight was next

assessed. Body weight measurements of 9-week old WT and Svs7 TG mice were

performed. Mice were sacrificed and each body weight was measured. The result

showed that there was no significance difference in body weight between WT and Svs7

TG mice (Figure 5.4A). Subsequently, the whole body of WT and Svs7 TG mice were

taken for X-ray analysis. The result showed that there was no striking difference in

skeletons between WT and Svs7 TG mice (Figure 5.4B Left panel). To further confirm

this result, the vertebrae were dissected from the pairs and taken for X-ray. Again, the

result showed that there was no difference in the vertebrae between WT and Svs7 TG

mice (Figure 5.4B Right panel). Together, these results indicate that there is no obvious

change in skeletons of Svs7 TG mice.

5.2.5 MicroCT analysis of tibia, femur, and lumbar vertebrae of Svs7 TG mice

To further elucidate the bone phenotype of Svs7 TG mice, the trabecular bone

architecture of the proximal tibia, distal femur, and vertebral body of the lumbar bone of

TG mice was investigated by microCT. For this, 9-week old WT and Svs7 TG mice

were sacrificed and tibia, femur, and lumbar vertebral bones were dissected from both,

fixed, and analysed by microCT. Representative 2D and 3D microCT images showed

no apparent difference in trabecular bone architecture at the proximal tibia, distal femur,


113

WT TG Pr

osta

te

Figure 5.3 Histological examination of prostate of Svs7 TG mice. Prostates were

dissected from 9-week old WT and Svs7 TG mice, fixed, processed, embedded,

sectioned, and H&E stained for histological assessment. The result showed no obvious

difference in the size or morphology of the prostate between WT and Svs7 TG mice.

Hence, the Svs7 transgene expression does not appear to affect prostate development or

morphogenesis. Scale bars, 500μm.


114

WT TG WT TG

WT TG Body

Weight (g) 18.71 ± 0.48 19.26 ± 0.20

(A)

(B)

Figure 5.4 Body weight and radiographic assessment of Svs7 TG mice. (A) Nine-

week old WT and Svs7 TG mice were sacrificed and their body weights measurements

were conducted using the A&D HT-500 balance. The result revealed that there was no

significant difference in body weight between WT and Svs7 TG mice. (B) (Left panel)

The whole body of the 9-week old WT and Svs7 TG mice were taken for X-ray

analysis. The result showed that there was no obvious difference in skeletons between

WT and Svs7 TG mice. (Right panel) To further confirm this result, the vertebrae were

dissected from these mice and taken for X-ray. The result confirmed that there was no

obvious change in skeletons of Svs7 TG mice. Data are presented as mean ± SEM.


115

and at the vertebral body of the lumbar bone between WT and Svs7 TG mice (Figure

5.5), further confirming that there is no obvious change in overall bone architecture of

Svs7 TG mice.

5.2.6 Svs7 TG mice exhibit a normal bone mass

In addition to radiographic and microCT assessments of the bones of Svs7 TG mice, the

solid quantitative bone parameters were also assessed. Bone parameters, including

trabecular bone/tissue volume (BV/TV) (%), trabecular separation (Tb.Sp) (mm),

trabecular thickness (Tb.Th) (mm), and trabecular number (Tb.N) (1/mm), were

assessed from femurs of 9-week old WT and Svs7 TG mice, based on data obtained

from microCT analysis. The results revealed that there were no significant differences

in all bone parameters between WT and Svs7 TG mice (Figure 5.6). Hence, the bone

mass of Svs7 TG mice is comparable to their WT littermates.

5.2.7 Histological assessment of Svs7 TG bones

As a standard procedure for bone phenotype characterisation, histological examination

on both paraffin-embedded decalcified and plastic-embedded non-decalcified bone

samples from 9-week old WT and Svs7 TG mice was performed. First, calvarial, tibia,

femur, and lumbar bones were extracted from both WT and TG mice, fixed, decalcified,

processed, embedded, sectioned, and H&E stained for histological assessment, as

described in Chapter 4 Section 4.3.5. The other femurs from the same WT and TG pairs

of mice described above were not decalcified and processed for plastic embedding, as

described in Chapter 4 Section 4.3.5 starting at point 4.3.5.4. The non-decalcified

sections were then stained with goldner trichrome (bone stained green) and/or TRAP

(an osteoclast marker; red precipitates). By using goldner trichrome stain on non-

decalcified sections, the osteoid, which is the unmineralised organic component of bone

and stained orange/red, can be observed and used in the histomorphometry analysis.

The H&E-stained sections showed that there was no obvious difference in bone volume

architecture and periosteal thickness of calvarial bone between WT and Svs7 TG mice

(Figure 5.7A and B). Furthermore, there were also no apparent changes in trabecular

bone architecture and cortical bone at the metaphyseal region of tibia (Figure 5.7C and

D), femur (Figure 5.7E and F), and lumbar (Figure 5.7G and H) bones of Svs7 TG mice

relative to that of WT littermates. In support of these results, the goldner trichrome non-

decalcified femurs sections depicted normal and healthy appearances of trabecular bone

architecture and cortical thickness of Svs7 TG hindlimbs in comparison


116

WT TG

Tibia

Femur

Lumbar


117

Figure 5.5 2D and 3D microCT images of proximal tibia, distal femurs, and

vertebral body of the lumbar bone of Svs7 TG mice. Tibia, femur, and lumbar

vertebral bones were extracted from 9-week old WT and Svs7 TG mice, fixed, and

analysed using the Skyscan 1072 microCT system. The representative 2D and 3D

microCT images showed that there was no obvious difference in trabecular bone

architecture at the proximal tibia, distal femur, and at the vertebral body of the lumbar

bone between WT and Svs7 TG mice. These results further confirmed that there was no

obvious change in overall bone architecture of Svs7 TG mice. Scale bars, 500μm.


118

WT TG n.s.

0

5

10

15

20

25

30

WT TG

BV

/TV

(%)

0 0.02 0.04 0.06 0.08

0.1 0.12 0.14 0.16 0.18

0.2

WT TG

Tb.

Sp (m

m)

n.s.

0

0.01

0.02

0.03

0.04

0.05

0.06

WT TG

Tb.

Th

(mm

) n.s.

0 0.5

1 1.5

2 2.5

3 3.5

4 4.5

5

WT TG

Tb.

N (1

/mm

)

n.s.

Figure 5.6 Svs7 TG mice exhibit a normal bone mass. Nine-week old WT and Svs7

TG mice were sacrificed and femurs were extracted from both, fixed, and analysed

using the Skyscan 1072 microCT system. The results showed that there were no

significant differences in all bone parameters (BV/TV (%), Tb.Sp (mm), Tb.Th (mm),

and Tb.N (1/mm)) between WT and Svs7 TG mice. Hence, Svs7 TG mice exhibit a

normal bone mass. Data are presented as mean ± SEM. Number of mice (n) = 2-

3/group. n.s. = no significance against WT. Scale bars, 500μm.


119

WT TG

Calvarial

Tibia

Femur

Lumbar

(A) (B)

(C) (D)

(E) (F)

(G) (H)


120

Figure 5.7 Histological examination on H&E-stained decalcified Svs7 TG bone

samples. (A and B) Calvarial, (C and D) tibia, (E and F) femur, and (G and H) lumbar

vertebral bones were extracted from 9-week old WT and Svs7 TG mice, fixed,

decalcified, processed, embedded, sectioned, and H&E stained for histological

examination. The representative images were taken with 10x objective magnification.

The results showed that there were no obvious differences in bone volume architecture

and periosteal thickness of the calvarial bone between WT (A) and Svs7 TG mice (B).

Furthermore, there were also no apparent changes in trabecular bone architecture and

cortical bone at the metaphyseal region of tibia (C and D), femur (E and F), and lumbar

(G and H) bones of Svs7 TG mice relative to that of WT littermates. Scale bars, 100μm.


121

to that of WT (Figure 5.8A). Moreover, TRAP-stained sections and bone

histomorphometry analysis on number of osteoclasts/bone surface (NOc/BS) parameter

further revealed a comparable number of TRAP-positive osteoclasts near the growth

plate region between WT and Svs7 TG hindlimbs (Figure 5.8B and Figure 5.9,

respectively), which indicate that Svs7 TG mice exhibit a characteristic of healthy bone

remodelling process.

5.2.8 Svs7 TG mice exhibit an unaffected osteoclast differentiation in vitro

Having established that the bone architecture of Svs7 TG mice appeared phenotypically

to be comparable to WT littermates and as a final step towards characterising any

potential cell-specific influence of Svs7 overexpression in TG mice, the capacity of

bone marrow (BM) progenitor cells to differentiate into osteoclasts was assessed. For

this, 9-week old WT and Svs7 TG mice were sacrificed, which was followed by

isolation and culture of bone marrow macrophages (BMM) from long bones (tibia and

femur), as described in Chapter 4 Section 4.4.1.1. Upon confluency, the cells were

seeded into 96-well plates, ready for time course RANKL and M-CSF-induced

osteoclastogenesis. The result showed that there was no significant difference in

osteoclastogenesis between WT and Svs7 TG mice (Figure 5.10). Hence, this result

further supported the findings in the TRAP sections histological assessment and

histomorphometry data described above that Svs7 TG mice exhibited an unaffected

osteoclastogenesis in vitro and in vivo.

5.3 Discussion Bone homeostasis is maintained by balanced yet complementary activity between bone-

resorbing osteoclast and bone-forming osteoblast, in which many growth factors,

cytokines, and proteins, secreted by either or both cells, have been shown to have

regulatory roles in maintaining bone homeostasis (Kular et al. 2012; Martin et al. 2010;

Walker et al. 2008; Zaidi 2007; Zhao et al. 2006). Based on previous observations in

our laboratory, Svs7 has been identified to be exclusively expressed in mature

osteoclasts in bone and its gene expression is upregulated during RANKL-induced

osteoclastogenesis (as discussed in Chapter 2 Section 2.4). In addition, studies from

Davey 2008 suggested that recombinant purified Svs7 protein treatment could enhance

trabecular bone/tissues volume, trabecular thickness, and trabecular number in

ovariectomised mice (oestrogen deficiency-induced bone loss mouse model (Aitken,

Armstrong & Anderson 1972; Kalu 1991)).


122

100μm 100μm

WT TG

500μm

500μm

500μm

500μ

500μm

μ

100μm

100μm

Gol

dner

tric

hrom

e

TRA

P (4

x ob

j.)

TRA

P (1

0x o

bj.)

(A)

(B)


123

Figure 5.8 Histological examination on goldner trichrome (A) and TRAP (B)

stained non-decalcified femur sections of Svs7 TG mice. The other femurs from the

same pairs of WT and Svs7 TG mice described above were not decalcified and

processed for plastic embedding procedure. The non-decalcified sections were then

goldner trichrome (bone stained green) and TRAP (osteoclast stained red)-stained. In

support of the H&E sections results, (A) the goldner trichrome-stained sections depicted

normal and healthy appearances of trabecular bone architecture and cortical thickness of

Svs7 TG hindlimbs in comparison to that of WT littermates. (B) Accordingly, from

TRAP sections, it showed a comparable number of TRAP-positive osteoclasts near the

growth plate region between WT and Svs7 TG hindlimbs, indicating that Svs7 TG mice

exhibit a characteristic of healthy bone remodelling process. Together, these results

suggested normal bone phenotype and TRAP-positive osteoclasts number in Svs7 TG

hindlimbs. Scale bars for 4x obj. magnification images, 500μm; for 10x, 100μm.


124

0 2 4 6 8

10 12 14 16 18

WT TG

NO

c/B

S (N

/mm

)

Figure 5.9 Bone histomorphometry analysis on NOc/BS (N/mm) of TRAP-stained

non-decalcified femur sections of Svs7 TG mice. Histomorphometry analysis was

performed on TRAP sections of non-decalcified femurs from WT and Svs7 TG mice

described above. No difference was observed in NOc/BS parameter between WT and

Svs7 TG mice. Data are presented as mean ± SEM. Number of mice (n) = 2/group.


125

RANKL stimulation

WT

TG

n.s.

n.s. nn.s.

0

50

100

150

200

250

300

350

Day 0 Day 1 Day 3 Day 5

Num

ber o

f TR

AP+

mul

tinuc

leat

ed c

ells

(≥

3 n

ucle

i)/w

ell

WT

TG

Figure 5.10 Svs7 TG mice exhibit an unaffected osteoclastogenesis in vitro. BM

cells were isolated from long bones of 9-week old WT and Svs7 TG mice and cultured

in M-CSF supplemented complete medium until reaching confluency. The cells were

then seeded into 96-well plates and cultured for 5-day time course RANKL and M-CSF

stimulation with a fresh media change every 2 days. The result showed that there was

no significant difference in osteoclastogenesis between WT and Svs7 TG mice. Hence,

Svs7 TG mice exhibited an unaffected osteoclastogenesis in vitro. Data are presented as

mean ± SEM and are representative of three independent experiments. n.s. = no

significance against WT.


126

Hence, these results have indicated that Svs7 might have a potential role in the

maintenance of bone homeostasis. In the present study, global tissue distribution of

Svs7 mRNA expression revealed that Svs7 was almost exclusively restricted to SV

(including prostate tissue) and a lesser degree in osteoclasts. In an effort to address this,

mice overexpressing the Svs7 transgene were generated by introducing the purified and

linearized pcDNA3.1 Svs7 V5-His transgene into WT mice by blastocysts

microinjection. Svs7 TG mice generation was confirmed by DNA tail tip genotyping

and agarose gel electrophoresis as well as WB analysis on cleared liver lysates from 9-

week old WT and TG mice to confirm the transgenic protein expression of Svs7.

Unfortunately, immunoblot assessment confirmed that there was only a modest increase

in Svs7 protein expression in Svs7 TG mice (about 2.5 fold increase in liver) relative to

WT. Although several factors, including post transcriptional and translational

regulations as well as hormonal change, could affect the relationship between the gene

and protein expression level (King et al. 2012; Levitin et al. 2008), these observations

suggested that global overexpression of Svs7 protein in Svs7 TG mice was only mildly

overexpressed above the endogenous level, which might have excluded the detection of

a clear tissue/bone phenotype in these TG mice.

Despite the modest increase in Svs7 expression in Svs7 TG mice, the assessment of the

gross tissue morphology and the bone phenotype of these mice were persevered. First,

the tissue histology of the prostate (including SV), which has the highest Svs7 gene

expression, was assessed. The result showed that Svs7 TG prostate appeared to have a

normal phenotype relative to that of WT. Hence, from this global tissue phenotype

characterisation study, the Svs7 transgene expression does not appear to affect organs

and tissues phenotype. Furthermore, the normal organ and tissue phenotype observed in

Svs7 TG mice correlated well with their comparable body weight with WT littermates.

To assess the potential bone phenotype of Svs7 TG mice, X-ray analysis on the whole

body and spine of WT and TG mice were performed. The result revealed that there was

no obvious change in skeletons of Svs7 TG mice. These findings were further supported

by the microCT analysis on trabecular bone architecture at proximal tibia, distal femur,

and vertebral body of the lumbar bone of Svs7 TG mice that showed no striking change

in the bone volume architecture at all three sites in these TG mice in reference to WT

counterparts. Moreover, the quantitative analysis on trabecular bone in the secondary

spongiosa region of distal femurs (Lee et al. 2006; Nishikawa et al. 2010) from WT and


127

Svs7 TG mice clearly indicated that TG mice had a normal bone volume architecture,

by showing comparable statistics on all bone parameters (BV/TV (%), Tb.Sp (mm),

Tb.Th (mm), and Tb.N (1/mm)) to WT littermates. As a second standard procedure in

the bone phenotype characterisation, histological examination on both paraffin-

embedded decalcified (H&E stained) and plastic-embedded non-decalcified (goldner

trichrome and TRAP stained) bone samples of WT and Svs7 TG mice was performed.

Both H&E and goldner trichrome-stained sections demonstrated that there were no

marked differences in bone volume and periosteal thickness of the calvarial bone

between WT and Svs7 TG mice, together with no observable change in trabecular bone

architecture and cortical bone in the metaphyseal region of tibia, femur, and lumbar

bones of the TG mice relative to that of WT littermates. In line with the normal bone

mass and architecture observed in Svs7 TG mice, the TRAP sections and bone

histomorphometry data on number of osteoclasts/bone surface parameter showed that

there was no significant difference in the number of TRAP-positive osteoclasts near the

growth plate region between WT and Svs7 TG hindlimbs. These results, along with the

observed findings of unaltered calcium and phosphate serum levels (Supplementary

Figure 1), indicate the presence of healthy bone remodelling process in Svs7 TG mice.

Finally, in order to probe for potential intrinsic cell-specific influences in Svs7 TG

mice, in vitro RANKL-induced osteoclastogenesis assays on WT and TG BMM was

performed. The result revealed that, in reference to WT, indeed Svs7 TG mice exhibited

an unaffected osteoclastogenesis in vitro. Hence in conclusion, Svs7 TG mice showed

no obvious defects in organs and tissues (especially bone) phenotype. Through microCT

analysis and histological examination, Svs7 TG mice showed to have normal bone

volume and architecture. The indication of normal bone remodelling process in Svs7

TG mice to explain the observed phenotype had been illustrated by the histological

TRAP-stained hindlimbs sections, histomorphometry data, unaltered calcium and

phosphate serum levels, and in vitro osteoclastogenesis results that showed an

unaffected osteoclastogenesis in the TG mice. Thus, the Svs7 transgenic strategy did not

allow an accurate assessment of the potential role of Svs7 in bone homeostasis, likely

owing to the modest transgene protein expression of Svs7 in Svs7 TG mice. Therefore,

an alternative knockout strategy was required to gain better insights into the potential

role of Svs7 in the maintenance of bone homeostasis.

128

CHAPTER 6

CHARACTERISATION OF SVS7 KNOCKOUT MICE

Chapter Six – Characterisation of Svs7 Knockout Mice

129

6.1 Introduction Despite the naturally low gene expression of Svs7 in bone, as shown in Chapter 5

Figure 5.1, and with the initial studies that indicate Svs7 gene is upregulated during

RANKL-induced osteoclastogenesis together with other common osteoclast marker

genes (Chapter 2 Figure 2.4), it would be plausible to hold the hypothesis that Svs7 is

an important regulator of osteoclasts and/or osteoblasts differentiation and activation.

By considering that Svs7 is a secreted member of Ly-6 protein family, sharing common

TFP/Ly-6/uPAR motif (Coronel et al. 1992; Levitin et al. 2008; Southan et al. 2002;

Turunen et al. 2011; Ushizawa et al. 2009), and the importance of other Ly-6 members

in many cellular signalling pathways, such as shown by uPAR in αvβ3 integrin

signalling pathway (Schiller et al. 2009; Smith, Marra & Marshall 2008; Wei et al.

2008) and Sca-1, also expressed by osteoclasts (Chapter 2 Figure 2.3), in stem cell

renewal capacity (Bonyadi et al. 2003; Ito et al. 2003), the initial approach and

investigation using Svs7 TG mice was conducted but it had failed to provide accurate

insights into the potential role of Svs7 in the maintenance of bone homeostasis. Svs7

TG mice showed no obvious change in organs and tissues, especially in bone,

phenotype. Normal bone mass and architecture, with the addition of an unaffected

osteoclastogenesis in vitro, were observed in Svs7 TG mice. This might be partly due to

a weak level of Svs7 transgene protein expression in Svs7 TG mice.

Therefore, as an alternative approach to investigate the potential role of Svs7 in

regulating bone homeostasis, Svs7 global knockout (KO) mouse model was generated.

Hence, this chapter focuses on the characterisation of Svs7 global KO mice, with a

particular emphasis on the bone phenotype.

6.2 Results 6.2.1 Genotyping and characterization of Svs7 KO mice

DNA isolated from mouse tail tips was genotyped according to Jackson laboratory’s

DNA isolation protocol, as discussed in Chapter 4 Section 4.3.3, and DNA products

were PCR amplified using specific primers for Svs7 and run on a 1.5% agarose gel.

Figure 6.1A illustrates the 99 amino acids composition of Svs7 protein (Amino (N) to

carboxyl (C) terminal), encoded by 3 exons (each exon is separated by red dot), and the

importance of exon 2 and 3, which together encode for the common TFP/Ly-6/uPAR

domain, consisting of conserved cysteine residues and the consensus C-terminal

sequence motif of CCXXXXCN (‘X’ represents any amino acid and ‘N’ for asparagine


130

Svs7

β-actin

WT KO

1 2 3 4

Svs7

KO

Het

WT

NE

G

Exon 1 Exon 2 Exon 3

SP C-C-C-C-C

C-C-C-C-CN

Poly (A)

TFP/Ly-6/uPAR domain

Svs7 protein (99 aa):

MNSVTKISTLLIVILSFLCFVE.GLICNSCEKSRDSRCTMSQSRCVAKPGESCSTVS

HFV.GTKHVYSKQMCSPQCKEKQLNTGKKLIYIMCCEKNLCNSF

(A)

(B) (C)

Figure 6.1 Svs7 gene and protein were absent in Svs7 KO mice. (A) (Top panel)

Svs7 amino acids composition, encoded by 3 exons (each exon is separated by red rot),

showing consensus cysteine (red highlighted letter ‘C’) residues and the consensus C-

terminal sequence motif CCXXXXCN (‘X’ for any amino acid and ‘N’ for asparagine),

and (Bottom panel) the structure of Svs7 gene with exon 2 and 3 encoding for TFP/Ly-

6/uPAR domain (The structure of Svs7 gene was illustrated by Levitin et al. 2008). (B)

Agarose gel electrophoresis of mouse tail tip DNA genotyping product showing that the

deletion of exon 2 and 3 of Svs7 gene in Svs7 KO mice (lane 2) had produced a smaller

DNA product compared to WT (lane 1). (C) WB protein analysis on WT and Svs7 KO

seminal vesicle lysates showed that there was no Svs7 protein detected in the KO lysate.

β-actin served as a loading control.


131

residue) (Levitin et al. 2008). Deletion of these exons was confirmed by DNA analysis

using agarose gel electrophoresis. As shown in Figure 6.1B, Svs7 KO (lane 2) produced

a smaller DNA product compared to WT (lane 1), indicating that the exons had been

successfully deleted. To confirm Svs7 deletion at the protein level, western blot (WB)

analysis on seminal vesicle (SV) lysates from WT and Svs7 KO mice was performed.

As anticipated, the loss of exon 2 and 3 within Svs7 gene resulted in the complete

absence of Svs7 protein in Svs7 KO lysate, as shown in Figure 6.1C. β-actin served as a

loading control. Together, these results validate the deletion of Svs7 in Svs7 KO mice.

6.2.2 Slow mice breeding process and gross body phenotypic examination of Svs7

KO mice

Following the generation of heterozygous (Het) mice, the breeding process was

continued by setting up Het x Het crossing to produce WT and Svs7 KO littermates. As

shown in Figure 6.2A, amongst 71 newborn pups (39 for male and 32 for female), the

ratio of getting male WT and female Svs7 KO mice were only 12% and 16%

respectively, which are significantly lower than the expected 25% (Mendelian ratio),

suggesting that there was a slow breeding process and hence difficulty in getting

adequate WT and KO pairs littermates for both male and female pups. The possible

outcome of this slow mice breeding process was then investigated by first examining

the gross body phenotype of age- and sex-matched WT and Svs7 KO pairs littermates.

To achieve this, 12-week old WT and Svs7 KO mice were sacrificed and the two bodies

were laid side-by-side for examination and the representative images were taken. The

result depicted that there was no apparent defect in the gross body phenotype of Svs7

KO mice compared to WT in the same litter (Figure 6.2B). Collectively, although there

was a slow mice breeding process, Svs7 KO mice appeared to be similar in body size as

WT littermates.

6.2.3 Gross tissues phenotypic (macro vs. microscopic) assessment of Svs7 KO mice

To continue with the gross organs and tissues phenotype characterization of Svs7 KO

mice, 12-week old WT and KO pair littermates were sacrificed and general organs and

tissues were dissected from both mice and placed side-by-side for careful macroscopic

examination. The result showed that, in relative to WT, Svs7 KO organs and tissues

were comparable (Figure 6.3A). Furthermore, histology-prepared prostate (including

seminal vesicle) sections from WT and Svs7 KO mice were H&E stained for

microscopic assessment. No obvious difference in prostate tissue phenotype was


132

0

10

20

30

40

50

60

70

WT Het KO

Perc

enta

ge o

f mal

e pu

ps

0 5

10 15 20 25 30 35 40 45 50

WT Het KO

Perc

enta

ge o

f fem

ale

pups

(A)

(B) WT KO

Figure 6.2 Slow mice breeding process and gross body phenotypic examination of

Svs7 KO mice. (A) Amongst 71 newborn pups (39 for male and 32 for female), the

ratio of getting male WT and female Svs7 KO mice were 12% and 16% respectively,

which are well below than the expected 25%, suggesting that there was a slow mice

breeding process and subsequent difficulty in getting adequate WT and KO pairs

littermates for both male and female mice for experiments. (B) Twelve-week old WT

and Svs7 KO mice were sacrificed and laid side-by-side for gross body phenotypic

examination. The result showed that Svs7 KO mice appeared to be similar in body size

as WT littermates.


133

WT KO WT KO WT WT WT WT

WT WT WT WT WT

WT WT WT

KO KO KO KO

KO KO KO KO KO

KO KO KO

Tibia SV

(prostate) Testis Brain Heart Thymus

Lung Spleen Liver Stomach Kidney

Intestine Fat Ovary

WT KO

500μm 500μm

(A)

(B)

Figure 6.3 Gross tissues phenotypic assessment of Svs7 KO mice. (A) Organs and

tissues were extracted from 12-week old WT and Svs7 KO mice and placed side-by-

side for macroscopic examination. The result showed that Svs7 KO organs and tissues

were comparable to that of WT. (B) Prostates (including seminal vesicle) were extracted

from WT and Svs7 KO mice, fixed, processed, embedded, sectioned, and H&E stained

for histological assessment. The result depicted that there was no obvious difference in

prostate tissue phenotype between WT and Svs7 KO mice. Hence, the loss of the Svs7

gene did not appear to affect the organogenesis in Svs7 KO mice. Scale bars, 500μm.


134

observed between WT and Svs7 KO mice (Figure 6.3B). Together, these results

suggested that the deletion of the Svs7 gene did not appear to affect the organogenesis

in Svs7 KO mice.

6.2.4 Svs7 KO mice exhibit a reduced bone mass phenotype

As an initial step toward characterising the potential bone phenotype of Svs7 KO mice,

femurs were extracted from 15- and 30-week old WT and Svs7 KO mice, fixed, and

processed for microCT analysis, as detailed in Chapter 4 Section 4.3.4. Initially, no

striking phenotype was observed in Svs7 KO mice. Mice grew at lower ratio than the

expected without gross abnormality was observed. However, Svs7 KO mice exhibited a

progressive decline in bone mass with age as compared to WT littermates. The 2D and

3D microCT images of distal femurs from WT and Svs7 KO mice showed that Svs7

KO mice appeared to have less trabecular bone volume compared to WT littermates

(Figure 6.4A). Indeed, through the quantitative analysis on trabecular bone in secondary

spongiosa region (Lee et al. 2006; Nishikawa et al. 2010) of both WT and Svs7 KO

femurs, it was then revealed that KO bones had significantly lower bone/tissue volume

(BV/TV (%)) in both age (15- and 30-week old) groups and a significantly less

trabecular number (Tb.N (1/mm)) in the 30-week old group compared to WT (Figure

6.4B). However, there were no significant differences in trabecular separation (Tb.Sp

(mm)) and trabecular thickness (Tb.Th (mm)) in both age groups between WT and Svs7

KO mice (Figure 6.4B). Together, these results imply that Svs7 KO mice exhibit a

phenotype of osteopenia/osteoporosis.

6.2.5 Svs7 KO bone histology

The reduced bone volume in Svs7 KO mice prompted us to conduct a close examination

of the bone phenotype using histology and histomorphometry analysis. For this,

histology sections of decalcified femurs from 12-week old WT and Svs7 KO mice were

H&E and TRAP (as an osteoclast marker; red precipitates) stained. The H&E-stained

sections showed that, indeed, Svs7 KO femurs seemed to have less trabecular bone

volume in the secondary spongiosa region than WT (Figure 6.5A). In accordance with

this result, TRAP-stained sections depicted that Svs7 KO hindlimbs appeared to have a

trend of increase in the number of TRAP-positive osteoclasts near the growth plate

region compared to WT (Figure 6.5B). The bone histomorphometry analysis on TRAP-

stained femurs sections from WT and Svs7 KO mice for the number of


135

WT KO

0.02

0.04

0.06

15wk 30wk

Tb.T

h (m

m)

2

6

10

14

18

15wk 30wk

BV

/TV

(%)

1

2

3

4

5

15wk 30wk

Tb N

(1/m

m)

0.05 0.1 0.15 0.2 0.25 0.3

15wk 30wk

Tb S

p (m

m)

WT KO

n.s. n.s.

n.s. n.s.

n.s.

(A)

(B)


136

Figure 6.4 Svs7 KO mice exhibit a reduced bone mass phenotype. (A) 2D and 3D

(longitudinal and cross sections) microCT images of distal femurs from 15- and 30-

week old WT and Svs7 KO mice showed that the KO mice appeared to have less

trabecular bone volume compared to WT. (B) The quantitative microCT analysis on

bone parameters of femurs from 15- and 30-week old WT and Svs7 KO mice revealed

significantly reduced BV/TV (in both age groups) and trabecular number (Tb.N) (only

in the 30-week old mice) in Svs7 KO mice compared to WT. There were no significant

differences in trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th) between

WT and Svs7 KO mice in both age groups. Hence, Svs7 KO mice exhibit a phenotype

of osteopenia/osteoporosis. Data are presented as mean ± SEM. * p < 0.05, n.s. = no

significance against WT. Scale bars, 500μm.


137

WT KO

H&

E TR

AP

(10x

obj

.) TR

AP

(20x

obj

.) (A)

(B)


138

Figure 6.5 Svs7 KO bone histology. (A) Fixed and decalcified femurs sections from

12-week old WT and Svs7 KO mice were H&E stained. The result showed that Svs7

KO hindlimbs appeared to have less trabecular bone volume in the secondary spongiosa

region compared to WT. (B) (Top panel) 10x and (Bottom panel) 20x objective

magnifications of representative images of TRAP (osteoclasts stained red)-stained WT

and Svs7 KO femurs sections showed a trend of increase in the number of TRAP-

positive osteoclasts near the growth plate region in KO hindlimbs relative to WT. Scale

bars for 4x obj. H&E sections, 500μm; for 10x and 20x obj. TRAP sections, 100μm.


139

osteoclasts/bone surface (NOc/BS (N/mm)) similarly indicated that there was a trend of

increase in TRAP-positive osteoclasts number in the KO hindlimbs compared to WT

(Figure 6.6). Therefore, the bone histology sections of Svs7 KO mice supported the

MicroCT data that there was a reduced bone volume and a trend of increase in the

number of TRAP-positive osteoclasts in Svs7 KO hindlimbs.

6.2.6 Bone formation activity in vivo is unaffected in Svs7 KO mice

To further characterize the Svs7 KO bone phenotype, double calcein labelling was

performed. For this purpose, 12-week old WT and Svs7 KO mice were subjected to the

calcium-binding fluorescent dye, calcein, injection. Calcein is incorporated into the

newly mineralized tissues and hence by giving the mice two injections (two labels)

seven days apart and measuring the distance between two labels using ImageJ software

(after images were taken using confocal microscopy), the difference in bone formation

activity (mineral apposition rate (MAR)) in vivo between WT and Svs7 KO mice can be

deduced. Analysis of plastic-embedded non-decalcified tibia sections from WT and

Svs7 KO mice by confocal microscopy (Figure 6.7A) and MAR measurement (Figure

6.7B) showed that there was no significant difference in bone formation activity in vivo

between WT and KO mice. Hence, Svs7 KO mice exhibit an unaffected bone formation

activity in vivo.

6.3 Discussion This study builds upon our previous observations that Svs7 is exclusively expressed by

mature osteoclasts but not by osteoblasts or other key bone cells and its gene expression

is upregulated during RANKL-induced osteoclastogenesis (as discussed in Chapter 2

Section 2.4 Figure 2.4). Here to directly address the possible role of Svs7 in bone

homeostasis, which had failed to be shown in our first approach and study using Svs7

TG mice, we generated Svs7-deficient mice. Svs7 KO mice generation was achieved by

the deletion of exon 2 and 3 of Svs7 gene. This deletion was then confirmed by DNA

tail tip genotyping and immunoblotting for Svs7 protein in Svs7 KO seminal vesicle

lysate, which demonstrated that Svs7 protein expression was lost in these KO mice.

Assessment of Svs7 KO mice breeding revealed that the ratio of male WT and female

Svs7 KO mice were well below 25% (12% and 16% respectively), which consequently

limited both the sample size and number of assays available for assessment. Although


140

0

2

4

6

8

10

12

WT KO

NO

c/B

S (N

/mm

) n.s.

Figure 6.6 Bone histomorphometry analysis on TRAP-stained decalcified femur

sections of Svs7 KO mice. Fixed and decalcified femurs sections from 12-week old

WT and Svs7 KO mice were TRAP stained and number of osteoclasts/bone surface

(NOc/BS (N/mm)) parameter was analysed. The result revealed that there was a trend of

increase in the number of TRAP-positive osteoclasts in Svs7 KO hindlimbs compared

to WT. Data are presented as mean ± SEM. Number of mice (n) = 3/group. n.s. = no



141

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6 1.8

2

WT KO

MA

R (μ

m/d

ay)

WT KO (A)

(B) n.s.

Figure 6.7 Svs7 KO mice exhibit an unaffected bone formation activity in vivo. (A)

Calcein double labelling confocal microscopy images of plastic-embedded non-

decalcified tibia sections from 12-week old WT and Svs7 KO mice showed that there

was no striking difference in bone formation activity between WT and KO mice. (B)

MAR analysis by measuring the distance between the two calcein labels (in each

section) revealed that there was no significant difference in bone formation activity in

vivo between WT and Svs7 KO mice. Hence, Svs7 KO mice exhibit an unaffected bone

formation activity in vivo. Data are presented as mean ± SEM and are representative of

three independent experiments. n.s. = no significance against WT. Scale bars, 10μm.


142

the precise reason for this is unclear, it may imply an important role of Svs7 in seminal

vesicle and fertility, which has been previously suggested by Coronel et al. 1992 that

Svs7 inhibits calcium (Ca2+) influx across the membrane of sperm cells during

fertilization to prevent premature acrosomal reaction and spermatozoa hyperactivation.

Despite this breeding limitation, the examination of the gross phenotype of body,

organs, and tissues, including histological assessment on prostate (the tissue which has

the highest Svs7 gene expression, as shown in Chapter 5 Figure 5.1), of Svs7 KO mice

relative to WT littermates (age- and sex-matched pairs) was successfully achieved. The

results showed that there were no apparent differences in body, organs, and tissues gross

phenotype between WT and Svs7 KO mice. Hence, the loss of the Svs7 gene does not

appear to affect the gross phenotype (macro- and microscopic) of body, organs, and

tissues of Svs7 KO mice.

Moreover, microCT analysis on trabecular bone volume and architecture in the

secondary spongiosa region of distal femurs from 15- and 30-week old WT and Svs7

KO mice (Lee et al. 2006; Nishikawa et al. 2010) revealed that the KO mice had

significantly reduced trabecular bone/tissue volume (BV/TV) at both age groups

compared to WT littermates. Consistently, there was a significant reduction in

trabecular number (Tb.N) in Svs7 KO hindlimbs, even though only in the 30-week old

mice group. The possibility that Svs7 KO mice exhibit a progressive decline in bone

mass with age, as compared to WT littermates, could play a significant role in this

microCT analysis. In line with these microCT results, H&E-stained sections of

decalcified femurs from 12-week old WT and Svs7 KO mice showed that there was an

obvious decrease in trabecular bone volume in the secondary spongiosa region of KO

hindlimbs relative to that of WT littermates. Subsequently, TRAP-stained sections and

bone histomorphometry analysis on the number of osteoclasts/bone surface (NOc/BS)

indicated that there was a trend of increase in the number of TRAP-positive osteoclasts

near the growth plate region of Svs7 KO hindlimbs compared to WT littermates.

Collectively, these results suggest that Svs7 KO mice exhibit a phenotype of reduced

bone mass (osteopenia/osteoporosis) and a trend of increase in osteoclasts number in

vivo, which could strongly indicate an alteration in osteoclast differentiation and/or

activation in the KO mice.

To further explore the possible cause of the reduced bone mass phenotype in Svs7 KO

mice, osteoblastic bone formation activity in vivo was investigated by performing


143

double calcein labelling (Hayashi et al. 2012) in 12-week old WT and Svs7 KO mice

and measuring the distance between two labels on the images of calcein-labelled plastic

embedded non-decalcified tibia sections to calculate mineral apposition rate (MAR).

The result showed that there was no significant difference in MAR between WT and

Svs7 KO mice. Hence, this finding indicates that Svs7 KO mice exhibit an unaffected

bone formation activity in vivo and that the alteration in osteoclast differentiation and/or

activation might be the major contributor to the osteoporotic phenotype observed in

Svs7 KO mice.

In conclusion, Svs7, a novel osteoclast-secreted factor (Supplementary Figure 2),

appears to be a candidate regulator of bone homeostasis. Svs7 KO mice exhibit an

osteopenic/osteoporotic phenotype and an unaffected bone formation activity in vivo,

but an elevated osteoclast number in KO hindlimbs suggests that Svs7 might influence

bone homeostasis via the regulation of osteoclast differentiation and/or activity.

144

CHAPTER 7

INVESTIGATION OF INTRINSIC CELLULAR MECHANISM IN

SVS7 KNOCKOUT MICE (OSTEOCLAST AND

OSTEOBLAST)

Chapter Seven – Investigation of Intrinsic Cellular Mechanism in Svs7 Knockout Mice (Osteoclast and Osteoblast)

145

7.1 Introduction Bone homeostasis is achieved by balanced activities between bone-resorbing osteoclast

and bone-forming osteoblast. Various cytokines, proteins, and growth factors are

expressed and/or secreted by both osteoclasts and osteoblasts in a regulated manner to

create intercellular communication and regulation, crucial for the differentiation and

activation of each cell. RANKL and M-CSF, produced by osteoblasts, are two essential

cytokines for driving osteoclast differentiation and activation (Kodama et al. 1991;

Takahashi, Udagawa & Suda 1999). On the other hand, osteoclasts release the active

form of IGF-II from bone matrix during its bone resorption activity to stimulate and

initiate site-specific bone formation by osteoblasts (Hayden, Mohan & Baylink 1995).

Thus, an imbalance in this coupling system, which could come from defects in either or

both osteoclasts and osteoblasts, may lead to the development of many pathological

bone diseases, such as osteoporosis (reduced bone mass) and osteopetrosis (increased

bone mass).

The results presented in Chapter 6 demonstrate that Svs7 KO mice exhibit a reduced

bone mass phenotype, consistent with osteopenia/osteoporosis. Since osteoporosis is

attributed to an excessive bone resorption by osteoclasts (Riggs & Melton 1983) and/or

a decline in bone formation activity by osteoblasts (Manolagas & Jilka 1995), these

observations imply that the changes in osteoclasts and/or osteoblasts number and/or

activity might account for the reduced bone mass phenotype observed in Svs7 KO mice.

To address this, the present chapter focuses on the in vitro characterization of

osteoclasts (including their bone marrow macrophages (BMM) progenitors) and

osteoblasts from Svs7 KO mice. Parameters explored include Svs7 KO osteoclasts and

osteoblasts differentiation and activity and osteoclast fusion mechanism.

In addition, it has been well documented that calcium (Ca2+) signalling is essential in

the activation of NFATc1, the master regulator of osteoclastogenesis (Takayanagi et al.

2002). Amongst other osteoclast genes that are known to be transcriptionally regulated

by NFATc1, such as TRAP, cathepsin K, c-Src, and β3 integrin (Crotti et al. 2006;

Ikeda et al. 2006; Matsumoto et al. 2004; Takayanagi et al. 2002), it has been shown

that NFATc1 also crucially regulates fusion-mediating genes activation in osteoclasts,

which are DCSTAMP and ATP6v0d2 genes (Kim et al. 2008; Lee et al. 2006; Yagi et

al. 2005). In general, mononuclear preosteoclasts fuse together to become

multinucleated cells, which takes place around day 3 of RANKL-induced osteoclast


146

differentiation (osteoclastogenesis) and upon further RANKL stimulation they become

active and functional bone resorbing cells (Fujita et al. 2012; Kim et al. 2008). NFATc1

has been shown to bind to the promoter region of both fusogenic genes to induce their

expression. The inactivation of NFATc1 decreases the expression of these fusogenes

and attenuates osteoclast fusion (Kim et al. 2008). Hence, the overexpression of

ATP6v0d2 and DCSTAMP genes has been shown to rescue the NFATc1-deficient

osteoclast fusion defect (Kim et al. 2008). As Svs7 is known to regulate Ca2+ influx

across the membrane of sperm cells during fertilization (Coronel et al. 1992), it was

then plausible to investigate the potential role of Svs7 in Ca2+-NFATc1 signalling

pathway, particularly in the regulation of osteoclast fusion mechanism. Through a series

of in vitro experiments, the findings in this chapter indicate that Svs7 plays a role in

regulating osteoclast fusion through the NFATc1 signalling pathway.

7.2 Results – Part 1: Intrinsic cellular mechanism in osteoclasts

derived from Svs7 KO mice 7.2.1 Svs7 KO BMM cultures have increased osteoclastogenesis in vitro

First, the histomorphometry results obtained from Svs7 KO hindlimbs indicated that

there was a trend of increase in osteoclast number in vivo. Based on this observation, we

speculated that the reduced bone mass phenotype observed in Svs7 KO mice might be

due to an alteration in osteoclast differentiation and/or activation. To assess osteoclast

formation in Svs7 KO mice, in vitro 5-day time course (day 0, 1, 3, and 5) RANKL and

M-CSF-induced osteoclastogenesis assays on WT and KO bone marrow (BM) cells

were performed. At the end of the time course, the cells were fixed, TRAP stained and

osteoclasts (TRAP-positive cells having ≥ 3 nuclei) were counted. The result showed

that at day 3 and 5 of osteoclastogenesis, there were significantly more TRAP-positive

osteoclasts in Svs7 KO cultures compared to WT (Figure 7.1). Hence, in vitro

osteoclastogenesis was increased in Svs7 KO mice.

7.2.2 Svs7 KO osteoclasts are larger and have more nuclei than WT

Further investigation in in vitro osteoclastogenesis assay results showed that Svs7 KO

osteoclasts exhibited significantly larger spread area than WT at day 5 of RANKL-

induced osteoclastogenesis (Figure 7.2). Although there was no significant difference at

day 3 between WT and Svs7 KO osteoclasts (Figure 7.2), these results indicate that the

KO cells have an accelerated fusion process during differentiation. To further assess

this, osteoclasts were separated into three groups based on the number of nuclei per


147

0

20

40

60

80

100

120

140

160

180


Num

ber o

f TR

AP+

mul

tinuc

leat

ed c

ells

(≥

3 n

ucle

i)/w

ell

RANKL stimulation

WT

KO

WT

KO

Figure 7.1 In vitro time course RANKL-induced Svs7 KO BMM differentiation

into mature osteoclasts. BM cells from 12-week old WT and KO mice were cultured

for 5-day time course in the presence of M-CSF and RANKL with a fresh media change

every 2 days. Upon formation of osteoclasts, TRAP stain and osteoclast count were

performed. Svs7 KO cultures showed a significant increase in osteoclastogenesis

compared to WT at day 3 and 5 of RANKL-induced osteoclastogenesis. Data are

presented as mean ± SEM and are representative of three independent experiments. ** p

< 0.01 against WT. Scale bars, 100μm.


148

0

10000

20000

30000

40000

50000

WT KO

Aver

age

OC

are

a (μ

m2 )

at d

ay 5

of

Ost

eocl

asto

gene

sis

0 2000 4000 6000 8000

10000 12000 14000 16000 18000

WT KO

Aver

age

OC

are

a (μ

m2 )

at d

ay 3

of

Ost

eocl

asto

gene

sis

n.s.

Figure 7.2 Svs7 KO osteoclasts are larger than WT. Analysis of osteoclast spread

area at day 3 and 5 of RANKL-induced osteoclastogenesis revealed that Svs7 KO

osteoclasts were significantly larger than WT at day 5 of osteoclastogenesis (Bottom

panel), while there was no significant difference at day 3 between both cells (Top

panel). Data are presented as mean ± SEM and are representative of three independent

experiments. * p < 0.05, n.s. = no significance against WT.


149

osteoclast (having 3-5, 6-10, or ≥ 11 nuclei), which were then added together and

expressed as a ratio to total TRAP-positive multinucleated cells per well. Based on this

quantification method, there were significantly more large osteoclasts, having ≥ 11

nuclei, in Svs7 KO cultures compared to WT at day 5 of osteoclastogenesis (Figure

7.3). These findings suggest that Svs7 might play a role in regulating osteoclast fusion.

7.2.3 WT and Svs7 KO mice have comparable hematopoietic stem cells (HSCs) and

osteoclast progenitor cells

As an increase in osteoclastogenesis was observed in Svs7 KO mice, it was then of

interest to further investigate the early lineage negative (Lin-) osteoclast precursor

(CD45R- CD3e- CD11b-/low) (Jacquin et al. 2006) and subsequent HSCs population (Lin-

Sca-1+ c-Kit+/LSK) (Kollet et al. 2006; Lymperi et al. 2011; Miyamoto et al. 2011) in

BM population of WT and Svs7 KO mice. To explore this, 12-week old WT and KO

mice were sacrificed and BM cells were then isolated from their long bones. Following

red blood cell lysis procedure, the cells were counted, antibodies were prepared in

master mix with the required dilutions, followed by cell incubation with antibodies and

overnight fixation, as detailed in Chapter 4 Section 4.7.3. Flow cytometry analysis was

performed on the following day using FACS Canto at CMCA, UWA. As shown in

Figure 7.4A, both WT and Svs7 KO BM had no significant differences in both HSCs

and early osteoclast progenitor populations.

Next, BM osteoclast progenitor proliferation was assessed. For this purpose, BM cells

were cultured in complete medium supplemented with M-CSF for 48 hours and were

subjected to MTS proliferation assay, which measures the metabolic activity of the

viable cells through the absorbance of the formazan product at 490nm excitation

wavelength. The result showed that there was no significant difference in proliferation

rate between WT and Svs7 KO BM osteoclast precursors over both 24 and 48 hours

time points (Figure 7.4B), which therefore implied that the loss of the Svs7 gene did not

alter the regulation of HSCs and early osteoclast precursor population and proliferation.

As early osteoclast precursor proliferation, growth, and survival is primarily mediated

by the mitogen-activated protein kinases (MAPKs) signalling pathway upon RANKL

stimulation, which includes ERK, c-Jun N-terminal Kinase (JNK), and p38 (Cobb &

Goldsmith 1995; Teitelbaum 2000), cell lysates of WT and Svs7 KO BM osteoclast

precursors, stimulated for an hour (0, 5, 10, 20, 30, and 60 minutes time points) with


150

0 0.2 0.4 0.6 0.8

1

WT KO WT KO WT KO

#3-5 #6-10 #11+ Rat

io to

tota

l num

ber o

f TR

AP+

mul

tinuc

leat

ed c

ells

(≥

3 n

ucle

i) (p

er w

ell)

Nuclei number at day 3 of osteoclastogenesis

0 0.2 0.4 0.6 0.8

1

WT KO WT KO WT KO

#3-5 #6-10 #11+ Rat

io to

tota

l num

ber o

f TR

AP+

mul

tinuc

leat

ed c

ells

(≥

3 n

ucle

i) (p

er w

ell)

Nuclei number at day 5 of osteoclastogenesis

n.s.

n.s. n.s.

n.s.

n.s.

Figure 7.3 Svs7 KO osteoclasts have more nuclei than WT. Analysis of number of

osteoclasts (based on their nuclei number) in ratio to total number of TRAP-positive

multinucleated cells showed that indeed Svs7 KO cultures had significantly more large

osteoclasts, having ≥ 11 nuclei, than WT at day 5 of osteoclastogenesis, which perfectly

correlates with the osteoclast spread area analysis described above. At day 3 of

osteoclast differentiation, both WT and Svs7 KO cultures had similar distribution of

osteoclast sizes. These findings suggest that Svs7 might play a role in the regulation of

osteoclast fusion. Data are presented as mean ± SEM and are representative of three

independent experiments. * p < 0.05, n.s. = no significance against WT.


151

(A)

(B)

0 5

10 15 20 25 30

WT KO

% o

f CD

11blo

w/C

D3e

- /CD

45R- (

Lin- )/

tota

l cel

ls

0

0.5

1

1.5

WT KO

% o

f Lin

- Sca

-1+ c

-Kit+

(LSK

) HSC

s/tot

al ce

lls n.s.

n.s.

0

5

10

15

20

25

30

35

40

24hrs 48hrs

Est

imat

ed c

ell c

umbe

r (x

103 )

Cell Proliferation

WT

KO

n.s.

n.s.

Figure 7.4 Svs7 KO mice exhibit unaltered population of HSCs and early

osteoclast precursor. (A) BM cells were extracted from long bones of 12-week old WT

and KO mice, followed by cell incubation with fluorophore-conjugated antibodies and

overnight fixation. Flow cytometry analysis was performed on the following day, which

revealed that there were no significant differences in Lin- early osteoclast precursor

(Left panel) and Lin- Sca-1+ c-Kit+ (LSK) HSCs population (Right panel) between WT

and Svs7 KO mice. (B) MTS proliferation assays on WT and Svs7 KO BM osteoclast

precursors showed that there was no significant difference in proliferation rate between

WT and KO cells over period of 24 and 48 hours time points. These findings suggested

that the loss of the Svs7 gene did not affect the regulation of HSCs and early osteoclast

precursor population and proliferation. Data are presented as mean ± SEM and are

representative of three independent experiments. n.s. = no significance against WT.


152

RANKL (50ng/ml), were subjected to WB analysis to explore the MAPK and IκB

(inhibitor of NF-κB) α signalling pathways. As shown in Figure 7.5A (WB result) and

B (densitometry analysis), there was no significant difference in the phosphorylation

(activation) of ERK, JNK, or p38 as well as in the IκBα degradation (NF-κB activation)

between WT and Svs7 KO mice, suggesting that the deletion of the Svs7 gene does not

affect early MAPK and NF-κB signalling pathways in osteoclast progenitor.

7.2.4 Svs7 KO mice exhibit unaltered expression levels of key osteoclast marker

genes

Next, the expression of osteoclast marker genes in Svs7 KO mice was examined. WT

and KO BM cells were extracted from long bones and, upon confluency, were cultured

for 5-day time course with RANKL and M-CSF stimulation. RNA was then extracted,

followed by reverse transcription (RT) and conventional semi-quantitative PCR

reactions. Forward and reverse primers for CTR, TRAP, NFATc1, DCSTAMP, and

ATP6v0d2 were run alongside Svs7 and β-actin as a loading control. As shown in

Figure 7.6A (agarose gel electrophoresis) and B (densitometry analysis), both WT and

Svs7 KO mice had comparable expression levels of key osteoclast marker genes, which

indicated that despite the increase in osteoclast number and size, the loss of the Svs7

gene did not alter the expression of key osteoclast marker genes.

7.2.5 Osteoclast bone resorption activity is not affected in Svs7 KO mice

To examine osteoclast activity, WT and Svs7 KO BMM-derived mature osteoclasts

cultured in collagen-coated 6-well plates, were detached and seeded onto bone slices

and cultured for 48 hours in RANKL-supplemented medium to initiate bone resorption.

An independent method using Corning osteo assay plates (containing bone

(hydroxyapatite)-like material), after the initial mature osteoclast culture in the

collagen-coated 6-well plates, was also performed, as described in Chapter 4 Section

4.5.3. After 48 hours, cells on either bone slices or Corning osteo assay wells were

fixed, TRAP stained, and number of osteoclasts per bone slice was counted, the surface

of bone slices was then visualised under scanning electron microscopy (SEM) and for

the Corning osteo wells the Nikon Microscope Ti was used to observe resorption pits.

By dividing the total resorbed area with the total osteoclast number on each sample,

resorbed pit area/cell result was then recorded as a representative of the osteoclast bone

resorption activity in WT and Svs7 KO mice. As shown in Figure 7.7 (SEM analysis)

and Figure 7.8 (Corning osteo plate analysis), there was no significant difference in the


153

0 5 10 20 30 60 0 5 10 20 30 60 WT KO

RANKL Stimulation mins

p-ERK1/2

β-actin

p-p38

p-JNK

IkBα

ERK1/2

p38

JNK

(A)

(B)

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

0 5 10 20 30 60

p-ER

K1/

2 ra

tio to

ER

K1/

2

RANKL stimulation (minute)

WT

KO

n.s. n.s.

n.s.

n.s.

n.s. n.s. WT

O

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 20 30 60

p-p3

8 ra

tio to

p38


WT

KO

n.s. n.s. n.s. n.s. n.s.

n.s.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 5 10 20 30 60

p-JN

K ra

tio to

β-a

ctin


WT

KO

n.s. n.s.

n.s. n.s.

n.s. n.s. WT

O

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 20 30 60

IκB

α ra

tio to

β-a

ctin


WT

KO

n.s. n.s. n.s.

n.s. n.s.

n.s.

Figure 7.5 The deletion of the Svs7 gene does not affect MAPK and NF-κB

signalling pathways in Svs7 KO osteoclast precursor. (A) Cell lysates of WT and

Svs7 KO BM osteoclast precursors, which were given short (an hour) RANKL

stimulation, were subjected to WB analysis and immunoblotted with specific antibodies

against MAPKs (ERK, JNK, and p38) and IκBα. MAPKs activation (phosphorylation)

and IκBα degradation appeared to be unaffected in Svs7 KO mice. (B) Densitometry

analysis was performed using ImageJ software and revealed that there was no

significant difference in the phosphorylation (activation) of ERK, JNK, or p38 as well

as in the IκBα degradation between WT and Svs7 KO mice, suggesting that the deletion

of the Svs7 gene does not affect early MAPK and NF-κB signalling pathways in

osteoclast progenitor. Data are presented as mean ± SEM and are representative of four

independent experiments. n.s. = no significance against WT.


154

β-actin

NFATc1

Svs7

ATP6v0d2

TRAP

CTR

DCSTAMP

0 1 3 5

RANKL-stimulated

BMM day 0 1 3 5

WT KO (A)

(B)

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8


Svs7

gen

e ex

pres

sion

(ratio

to β

-act

in)

RANKL stimulation

WT

KO n.s. n.s.

0

0.1

0.2

0.3

0.4

0.5


CTR

gene

expr

essio

n (ra

tio to

β-a

ctin)

RANKL stimulation

WT

KO n.s. n.s.

n.s.

n.s.

0

0.2

0.4

0.6

0.8

1

1.2


TRAP

gen

e exp

ress

ion

(ratio

to β

-acti

n)

RANKL stimulation

WT

KO n.s.

n.s.

n.s. n.s.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8


NFA

Tc1

gene

expr

essio

n (ra

tio to

β-a

ctin

)

RANKL stimulation

WT

KO

n.s. n.s.

n.s. n.s.

0

0.2

0.4

0.6

0.8

1

1.2

1.4


DCS

TAM

P ge

ne e

xpre

ssio

n (ra

tio to

β-a

ctin

)

RANKL stimulation

WT

KO n.s.

n.s. n.s. n.s.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8


ATP6

v0d2

gen

e exp

ress

ion

(ratio

to β

-acti

n)

RANKL stimulation

WT

KO

n.s. n.s.

n.s. n.s.

Figure 7.6 Key osteoclast marker genes expression levels are not affected in Svs7

KO mice. (A) RNA of 5-day time course RANKL and M-CSF-stimulated WT and Svs7

KO BM cells was extracted and subjected to RT and semi-quantitative PCR reactions

with specific forward and reverse primers for Svs7 and key osteoclast marker genes

(CTR, TRAP, NFATc1, DCSTAMP, and ATP6v0d2). β-actin was used as a house-

keeping gene. The result showed that there were comparable expression levels of the

osteoclast marker genes between WT and Svs7 KO mice. (B) Densitometry analysis

was then performed using ImageJ software and it revealed that there were no significant

differences in the key osteoclast marker genes expression levels between WT and Svs7

KO mice (with the confirmation that Svs7 gene was absent in Svs7 KO mice). Data are

presented as mean ± SEM and are representative of four independent experiments. ** p

< 0.01, n.s. = no significance against WT.


155

0

50

100

150

200

250

300

WT KO

Res

orpt

ion

Pit A

rea

(μm

2 /cel

l)

n.s.

WT KO

Figure 7.7 SEM analysis (on bone slices) for Svs7 KO osteoclast bone resorption

activity. WT and Svs7 KO mature osteoclasts, grown in collagen-coated 6-well plates,

were detached and seeded onto bone slices and cultured for 48 hours in RANKL-

supplemented complete medium to initiate bone resorption. TRAP staining and

osteoclasts counting were subsequently performed, followed by visualization of

resorption pits by SEM. The osteoclast bone resorption activity was expressed as a total

resorbed pit area/cell. No significant difference in osteoclast activity was observed

between WT and Svs7 KO osteoclasts. Hence, osteoclast activity was not affected in

Svs7 KO mice. Data are presented as mean ± SEM and are representative of three

independent experiments. n.s. = no significance against WT. Scale bars, 200μm.


156

0

2000

4000

6000

8000

10000

12000

WT KO

Res

orpt

ion

Pit A

rea

(μm

2 /ce

ll)

n.s.

WT KO

100μm 100μm

Figure 7.8 Corning osteo plate analysis for Svs7 KO osteoclast bone resorption

activity. After the initial WT and KO mature osteoclasts culture in collagen plates, the

cells were detached and seeded onto the Corning wells, each containing bone

(hydroxyapatite)-like material. Four wells were prepared for TRAP stain and osteoclast

count and the other four for resorption pits analysis. The osteoclast bone resorption

activity was expressed as a total resorbed area/cell. The result revealed that both WT

and Svs7 KO osteoclasts had a comparable bone resorption activity. Together, these

findings indicated that the osteoclast bone resorption activity was independent of Svs7.

Data are presented as mean ± SEM and are representative of four independent

experiments. n.s. = no significance against WT. Scale bars, 100μm.


157

amount of resorbed area/cell between WT and Svs7 KO osteoclasts. Together, these

findings indicated that the osteoclast bone resorption activity was independent of Svs7.

7.2.6 Svs7 KO osteoclasts are more sensitive to Ca2+ influx in response to RANKL

and extracellular Ca2+ stimulation

Considering that Ca2+ influx/signalling, induced by RANKL, is a prerequisite for the

robust induction of NFATc1 activation-driven osteoclast differentiation and activation

(Takayanagi et al. 2002), together with the fact that Svs7 acts as a Ca2+ transport

inhibitor on the membrane of sperm cells during fertilization process, it was then of

interest to explore the potential impact of the deletion of the Svs7 gene on Ca2+ influx in

osteoclasts. To this end, WT and Svs7 KO BM were first cultured on coverslips in 48-

well plates with complete medium supplemented with RANKL and M-CSF until

osteoclasts were observed (5 days culture). The cells were then loaded with 4μM Fluo-

4AM intracellular Ca2+-binding dye and the change of fluorescence intensity inside the

cells was observed by time-lapse microscopy. The basal Ca2+ level reading was taken

for 2 minutes with every 2 seconds interval recorded for a total of 60 intervals. As

expected, the result showed that both WT and Svs7 KO osteoclasts had a trend of

increase in Ca2+ influx in response to RANKL stimulation overnight (Figure 7.9A).

Svs7 KO osteoclasts, however, appeared to have a higher increase in basal Ca2+ influx

response compared to WT, with about 0.055 increase in fluorescence intensity (ratio to

baseline reading) for KO and 0.04 fluorescence intensity increase for WT from 0 to 60

interval mark (Figure 7.9A). This finding indicated that Svs7 KO osteoclasts were more

sensitive to Ca2+ influx in response to RANKL and hence Svs7 might have a role in

regulating Ca2+ influx (transport) in osteoclasts. To further investigate this possibility,

the effects of adding extracellular Ca2+ (in the form of CaCl2) on the osteoclast Ca2+

response was assessed. Three concentrations of CaCl2 were used: 5, 10, and 20mM. The

period of this extracellular Ca2+ stimulation was recorded for 3 minutes (with 2 seconds

interval), following the 2 minutes reading of the basal Ca2+ level. In all three

concentrations, the addition of this extracellular Ca2+ at 60 interval mark (120 seconds;

the end of the basal Ca2+ level fluorescence reading) caused an initial sudden increase in

Ca2+ influx, as monitored by the corresponding increase in fluorescence intensity, in

both WT and Svs7 KO osteoclasts, as shown in Figure 7.9B ((i) 5mM, (ii) 10mM, and

(iii) 20mM CaCl2 concentration). The major difference in this initial sudden Ca2+ influx

between WT and Svs7 KO osteoclasts was at the addition of 5mM CaCl2 concentration

(Figure 7.9B(i)), at which the KO cells had a faster and steeper Ca2+ influx response


158

0.94

0.95

0.96

0.97

0.98

0.99

1

1.01

0 10 20 30 40 50 60 70

Fluo

resc

ence

inten

sity

(ratio

to b

aseli

ne)

Intervals

WT

KO

Basal Ca2+ level

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120 140 160

Flu

ores

cenc

e in

tens

ity

(rat

io t

o ba

seli

ne)

Intervals

WT

KO

Addition of 5mM CaCl2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120 140 160 F

luor

esce

nce

inte

nsit

y (r

atio

to b

asel

ine)

Intervals

WT

KO


0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120 140 160

Flu

ores

cenc

e in

tens

ity

(rat

io to

bas

elin

e)

Intervals

WT

KO


Basal Ca2+ level Basal Ca2+ level

Basal Ca2+ level

0

20

40

60

80

100

120

140

160

180

5mM 10mM 20mM

Fina

l Ca2+

upt

ake

by m

atur

e os

teoc

last

s (%

of b

asel

ine)

Extracellular Ca2+ (CaCl2) stimulation

WT

KO

n.s.

n.s. n.s.

(A)

(B)

(C)

(i) (ii)

(iii)

Figure 7.9 Svs7 KO osteoclasts are more sensitive to Ca2+ influx in response to

RANKL and extracellular Ca2+ stimulation. (A) After overnight RANKL stimulation,

Fluo4 intracellular calcium dye was incubated with WT and Svs7 KO osteoclasts and

the basal Ca2+ level was read for 2 minutes with every 2 seconds interval recorded for a

total of 60 intervals. Svs7 KO osteoclasts showed to have a higher increase in basal


159

Ca2+ influx response compared to WT, with 0.055 increase in fluorescence intensity

(ratio to baseline) for KO and 0.04 fluorescence intensity increase for WT from 0 to 60

interval mark. (B) After the 60-interval mark, the cells were stimulated with 3 different

concentrations of CaCl2 (extracellular Ca2+), (i) 5, (ii) 10, and (iii) 20mM and the assay

was continued for another 3 minutes. In all 3 concentrations, the addition of

extracellular Ca2+ caused an initial sudden increase in Ca2+ influx in both WT and Svs7

KO osteoclasts, with the major difference between WT and KO response was observed

at the addition of 5mM CaCl2 concentration, at which KO osteoclasts had a faster and

steeper Ca2+ influx response (0.12 increase in fluorescence intensity from the basal level

(ratio to baseline)) than WT. However, this initial response in Svs7 KO osteoclasts was

disappeared as time progressed with WT and KO cells showed a similar trend of Ca2+

uptake. (C) Final Ca2+ uptake at the end of (each) extracellular CaCl2 stimulation was

expressed as a percentage increase (relative) over the baseline reading. The result

showed that there was no significant difference in final Ca2+ uptake (% of baseline) at

the end of each extracellular Ca2+ stimulation (5, 10, and 20mM) between WT and Svs7

KO osteoclasts. Collectively, these findings indicated that Svs7 KO osteoclasts were

more sensitive to Ca2+ influx in response to RANKL and extracellular Ca2+ stimulation

compared to WT. Data are presented as mean ± SEM. n.s. = no significance against

WT.


160

(0.12 increase in fluorescence intensity from the basal level (ratio to baseline)) than

WT. Following this initial sudden Ca2+ influx response, the addition of extracellular

Ca2+ resulted in the similar and continuous pattern of Ca2+ uptake in both WT and Svs7

KO osteoclasts. Despite a comparable final Ca2+ uptake (percentage of final

fluorescence reading at the end of (each) extracellular Ca2+ stimulation to baseline

reading) between WT and Svs7 KO osteoclasts shown in Figure 7.9C, collectively,

these findings indicated that Svs7 KO osteoclasts were more sensitive to Ca2+ influx in

response to RANKL and extracellular Ca2+ stimulation compared to WT. Thus, it is

tempting to speculate that the dysregulated Ca2+ response might alter the Ca2+-NFATc1

signalling pathway in Svs7 KO osteoclast differentiation.

7.2.7 Svs7 KO mice exhibit elevated NFATc1 and ATP6v0d2 proteins expression

during RANKL-induced osteoclastogenesis

As indicated in Section 7.2.2, osteoclasts from Svs7 KO mice were significantly larger

and possessed more nuclei than WT. These findings indicated the possible involvement

of Svs7 in osteoclast fusion mechanism and, together with the finding that Svs7 KO

osteoclasts were more sensitive to Ca2+ influx in response to RANKL, it then led us to

hypothesize that Svs7 may be a negative regulator of NFATc1 expression and possibly

the fusogenic proteins, DCSTAMP and ATP6v0d2 (Lee et al. 2006; Yagi et al. 2005),

whose expressions have been shown to be regulated by NFATc1 (Kim et al. 2008;

Takayanagi et al. 2002; Takayanagi 2007). Since osteoclasts from Svs7 KO mice had

comparable mRNA expression levels of osteoclast marker genes, which included

NFATc1, DCSTAMP, and ATP6v0d2 genes, with WT during RANKL-induced

osteoclastogenesis (Figure 7.6), we reassured that Svs7 involvement in osteoclast fusion

might be linked to RANKL-induced ITAM co-stimulatory pathway activation, which

involves Ca2+ influx response and the subsequent activation of NFATc1 via

Ca2+/calmodulin-dependent calcineurin pathway (Koga et al. 2004; Takayanagi et al.

2002).

To explore the effects of the loss of the Svs7 gene on NFATc1 and fusogenic proteins

expression in osteoclasts, cell lysates of 5-day time course RANKL-induced WT and

Svs7 KO BMM were extracted and subjected to western blot (WB) analysis and

immunoblotted with specific NFATc1 (Figure 7.10), ATP6v0d2 (Figure 7.11), and

DCSTAMP (Figure 7.12) antibodies. As shown in Figure 7.10, there was a significant


161

0 1 2 3 5 0 1 2 3 5 WT KO

NFATc1

β-actin

RANKL Stimulation day

0

0.2

0.4

0.6

0.8

1

1.2

Day 0 Day 1 Day 2 Day 3 Day 5

Rat

io to

β-a

ctin

RANKL stimulation

WT

KO n.s. n.s.

n.s. n.s.

Figure 7.10 Svs7 KO osteoclasts have a significant increase in NFATc1 protein

expression during RANKL-induced osteoclastogenesis. Cell lysates of 5-day time

course RANKL-induced WT and Svs7 KO BMM were extracted and subjected to WB

protein analysis, immunoblotted with specific NFATc1 antibody. Densitometry analysis

was performed using ImageJ software and the NFATc1 protein expression was

expressed as a ratio to β-actin, which served as a loading control. There was a

significant increase in NFATc1 protein expression in Svs7 KO osteoclasts compared to

WT at day 3 of RANKL-induced osteoclastogenesis. Data are presented as mean ±

SEM and are representative of three independent experiments. ** p < 0.01, n.s. = no



162

0 1 2 3 5 0 1 2 3 5 WT KO

ATP6v0d2

β-actin


0

0.2

0.4

0.6

0.8

1

1.2


Rat

io to

β-a

ctin

RANKL stimulation

WT

KO n.s. n.s.

n.s.

n.s. n.s.

Figure 7.11 Svs7 KO osteoclasts have a trend of increase in ATP6v0d2 protein

expression during RANKL-induced osteoclastogenesis. Cell lysates of 5-day time

course RANKL-induced WT and Svs7 KO BMM were extracted and subjected to WB

protein analysis, immunoblotted with specific ATP6v0d2 antibody. Densitometry

analysis was performed using ImageJ software and the ATP6v0d2 protein expression

was expressed as a ratio to β-actin, which served as a loading control. There was a

consistent trend of increase in ATP6v0d2 protein expression in Svs7 KO osteoclasts

compared to WT particularly at day 3 and 5 of RANKL-induced osteoclastogenesis.

Data are presented as mean ± SEM and are representative of four independent

experiments. n.s. = no significance against WT.


163

0 1 2 3 5 0 1 2 3 5

WT KO

DCSTAMP

β-actin


0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8


Rat

io to

β-a

ctin

RANKL stimulation

WT

KO

n.s.

n.s. n.s. n.s. n.s.

Figure 7.12 DCSTAMP protein expression appears to be unaffected in Svs7 KO

osteoclasts throughout the time course of RANKL-induced osteoclastogenesis. Cell

lysates of 5-day time course RANKL-induced WT and Svs7 KO BMM were extracted

and subjected to WB protein analysis, immunoblotted with specific DCSTAMP

monoclonal antibody. Densitometry analysis was performed using ImageJ software and

the DCSTAMP protein expression was expressed as a ratio to β-actin, which served as a

loading control. There was no significant difference in DCSTAMP protein expression

between WT and Svs7 KO osteoclasts throughout the time course of RANKL-induced

osteoclastogenesis. Data are presented as mean ± SEM and are representative of three

independent experiments. n.s. = no significance against WT.


164

increase in NFATc1 protein expression in Svs7 KO osteoclasts at day 3 of

osteoclastogenesis compared to WT. This finding implied that the dysregulated Ca2+

influx response, observed in Svs7 KO osteoclasts upon RANKL stimulation, might

account for the altered NFATc1 signalling, resulting in its stronger activation.

Subsequently, it was then revealed that the protein expression of ATP6v0d2 also

showed to have a trend of increase in Svs7 KO osteoclasts compared to WT,

particularly at day 3 and 5 of osteoclastogenesis (Figure 7.11). The DCSTAMP protein

expression, however, appeared to be unaffected in Svs7 KO mice, relative to WT,

throughout the time course of RANKL-induced osteoclastogenesis (Figure 7.12). These

findings suggest that Svs7 may play a crucial role in the regulation of osteoclast fusion

mechanism through the Ca2+-NFATc1 signalling pathway via modulation of the

expression of ATP6v0d2 but not DCSTAMP expression.

7.2.8 Svs7 protein treatment inhibits the activation of NFAT promoter luciferase

activity

Considering the increase in NFATc1 protein expression observed in Svs7 KO

osteoclasts (Figure 7.10), it was of further interest to investigate the effects of Svs7

protein treatment on NFAT promoter activation. After the successful cloning process of

Svs7 coding sequence (295bp) into HindIII/XhoI restriction enzyme site of the

pcDNA3.1 V5-His mammalian expression vector, as shown in Figure 7.13A, the

experiment was continued by transiently transfecting COS-7 cells, one group with the

Svs7 DNA construct and the other with pcDNA3.1 V5-His empty plasmid, which

served as a vehicle control. The conditioned medium (CM) was collected each day post

transfection and tested for Svs7 protein expression using WB analysis. Using rabbit

polyclonal anti-Svs7 antibody, Svs7 protein was detected to be successfully expressed

in the CM (Figure 7.13B). Luciferase assay was performed by stimulating RAW264.7

cells, stably expressing NFAT promoter and luciferase reporter gene, with either 50%

Svs7 CM or the vehicle control medium for an hour, followed by overnight RANKL

stimulation. The luminescence reading, which corresponds to the NFAT promoter

luciferase activity, was measured on the following day. The result showed that Svs7

protein treatment could significantly inhibit the activation of NFAT promoter (Figure

7.13C), further suggesting a regulatory role of Svs7 in NFAT promoter activation.


165

pcDNA3.1 V5-His (5.5kbp)

Svs7 (295bp)

1kbp DNA ladder

75 50 36

25 20

15

10

kDa +C 1 2 3 5 6 7

Collected CM in Opti-MEM reduced serum medium (day post-transfection)

Svs7

(A) (B)

0

20000

40000

60000

80000

100000

120000

140000

vehicle SVS7

NFA

T lu

cife

rase

act

ivity

(A

rbitr

ary

unit)

(C)

Figure 7.13 Svs7 protein treatment inhibits NFAT promoter luciferase activity (A)

DNA agarose gel electrophoresis of a midiprep pcDNA3.1 Svs7 V5-His cloning

construct, which had been cut by HindIII/XhoI restriction enzymes, showed that Svs7

coding sequence (295bp) was successfully cloned into the pcDNA3.1 V5-His

mammalian expression vector. (B) After transient transfection of either the Svs7

construct or the vehicle control plasmid into COS-7 cells, WB analysis on the CM using

rabbit polyclonal anti-Svs7 antibody was performed to detect the expression of Svs7

protein. The result showed that Svs7 was detected to be successfully expressed in the

CM collected each day post transfection. (C) RAW264.7 cells, stably expressing NFAT

promoter and luciferase reporter gene, were pretreated with either 50% Svs7 CM or the

vehicle control medium for an hour, followed by overnight RANKL stimulation.

Luciferase assay was then performed on the following day and the result revealed that

Svs7 CM treatment significantly inhibited the activation of NFAT promoter. Data are

presented as mean ± SEM and are representative of three independent experiments. ** p

< 0.01 against vehicle.


166

7.3 Results – Part 2: Osteoblasts from Svs7 KO mice are

phenotypically comparable to WT cells A balance in the cell-to-cell communication between osteoclasts and osteoblasts is the

prerequisite for achieving a healthy bone mass. Following the establishment of

increased osteoclastogenesis in vitro in Svs7 KO mice and the intrinsic role of Svs7 in

the regulation of osteoclast fusion mechanism through the Ca2+-NFATc1 signalling

pathway, in vitro Svs7 KO osteoblast phenotype was next assessed. To this end, Svs7

KO osteoblast differentiation and bone formation (mineralization) assays were then

performed.

7.3.1 Svs7 KO primary calvarial osteoblasts exhibit an unaffected proliferation

rate in vitro

To examine Svs7 KO osteoblast phenotype in vitro, Svs7 KO osteoblast proliferation

was investigated. To achieve this, WT and Svs7 KO neonatal mice were sacrificed and

the calvariae were dissected from both. After going through four series of collagenase

digestion, primary calvarial osteoblast cells were seeded overnight on 12-well plates,

ready for MTS proliferation assay and standard curve reading the next day. Two sets of

plates were incubated for 24 and 48 hours time points after the standard curve reading.

The result showed that at both time points there were comparable proliferation rates

between WT and Svs7 KO primary calvarial osteoblasts (Figure 7.14). Hence, this

finding suggested that Svs7 KO mice exhibited an unaffected osteoblast proliferation

rate in vitro.

7.3.2 No defect is observed in Svs7 KO alkaline phosphatase (ALP) osteoblast

differentiation in vitro

In order to examine if there is an osteoblast phenotype in Svs7 KO mice, ALP

osteoblast differentiation assays were performed. BM cells were extracted from long

bones of 12-week old WT and Svs7 KO mice and cultured in osteoblast differentiation

medium (complete medium supplemented with β-glycerophosphate and ascorbate) for

14 days in vitro. The ALP staining was then performed and the ALP-positive colonies

were analysed and counted. The result revealed that there was no significant difference

in colony forming unit (CFU)-ALP positive colonies formed between WT and Svs7 KO

osteoblast cultures (Figure 7.15), indicating that Svs7 KO mice had no defects in

osteoblast differentiation in vitro.


167

0 2000 4000 6000 8000

10000 12000 14000 16000

Day 1 Day2

Estim

ated

cel

l num

ber

Cell Proliferation

WT

KO

n.s. n.s.

Figure 7.14 Svs7 KO primary calvarial osteoblasts exhibit an unaffected

proliferation rate in vitro. Calvariae were dissected from WT and Svs7 KO neonatal

mice and primary calvarial osteoblast cells were extracted from the calvariae through

serial collagenase digestion method. After the cells were seeded overnight, MTS

proliferation assay for standard curve reading was performed, followed by 24 and 48

hours time points incubation and the absorbance analysis. The result showed that there

were no significant differences in proliferation rate at both time points between WT and

Svs7 KO primary calvarial osteoblasts. Hence, this finding indicated that Svs7 KO mice

exhibited an unaffected osteoblast proliferation rate in vitro. Data are presented as mean

± SEM. n.s. = no significance against WT.


168

0

10

20

30

40

50

60

WT KO

Num

ber o

f CFU

-ALP

+

WT

KO

n.s.

Figure 7.15 No defect is observed in Svs7 KO ALP osteoblast differentiation in

vitro. BM cells were extracted from long bones of 12-week old WT and Svs7 KO mice

and cultured in osteogenic medium (complete medium supplemented with β-

glycerophosphate and ascorbate) for 14 days in vitro. The ALP staining was then

performed and the ALP-positive colonies were analysed and counted. The result

showed that there was no significant difference in CFU-ALP positive colonies formed

between WT and Svs7 KO osteoblast cultures, indicating that Svs7 KO mice had no

defects in osteoblast differentiation in vitro. Data are presented as mean ± SEM and are

representative of three independent experiments. n.s. = no significance against WT.


169

7.3.3 Svs7 KO osteoblasts exhibit an unaffected bone formation activity in vitro

To further confirm a normal bone formation activity in Svs7 KO osteoblasts in vivo,

shown by double calcein labelling experiment and mineral apposition rate (MAR)

analysis in Chapter 6 Figure 6.7, in vitro bone nodule formation (mineralization) assay

was performed. To this end, BM cells were extracted from long bones of 12-week old

WT and Svs7 KO mice and cultured for 14 days in osteogenic medium. Alizarin red

staining, which stains for calcium deposition in culture wells, was then performed.

Following the staining procedure, the culture plates were scanned and the total

mineralized nodules were measured using ImageJ software. The result showed that

there was no significant difference in total mineralized nodules formed between WT

and Svs7 KO osteoblast cultures (Figure 7.16). This finding therefore indicated that

Svs7 KO osteoblasts exhibited an unaffected bone formation activity in vitro.

7.3.4 Analysis of osteoblast marker genes expression levels in Svs7 KO mice

To complete the in vitro Svs7 KO osteoblast phenotype characterization, RT-PCR

reaction was performed on 14-day (0, 7 and 14 day time points) time course WT and

Svs7 KO BM cells differentiation into mature osteoblasts. For this, BM cells were

extracted from long bones of 12-week old WT and Svs7 KO mice and cultured in

osteogenic medium for 14 days. RNA was then extracted, followed by RT and semi

quantitative PCR reactions using specific primers for Runx2, Osterix, ALP, osteopontin

(OPN), osteocalcin (OCN), M-CSF, RANKL, and OPG. β-actin was used as a loading

control. Densitometry analysis was performed using ImageJ software and the expression

of each osteoblast gene was expressed as a ratio to β-actin. The result revealed that

there were no distinguishable differences in the expression levels of key osteoblast

marker genes between WT and Svs7 KO mice (Figure 7.17). This finding suggested

that Svs7 KO mice exhibited unaltered expression levels of osteoblast marker genes.


170

0

2

4

6

8

10

12

WT KO

Min

eral

ized

nod

ules

(mm

2 )

WT

KO

n.s.

Figure 7.16 Svs7 KO osteoblasts exhibit an unaffected bone formation activity in

vitro. BM cells were extracted from long bones of 12-week old WT and Svs7 KO mice

and cultured in osteogenic medium for 14 days. Upon the formation of calcium

deposition, cells were fixed with 4% paraformaldehyde and alizarin red stain was then

applied to the culture wells, followed by total mineralized nodules measurement using

ImageJ software. The result showed that there was no significant difference in total

mineralized nodules formed between WT and Svs7 KO osteoblast cultures. This finding

indicated that Svs7 KO osteoblasts exhibited an unaffected bone formation activity in

vitro. Data are presented as mean ± SEM and are representative of three independent

experiments. n.s. = no significance against WT.


171

ALP

WT KO

0 7 14 0 7 14 Runx2

Osterix

OPN

OCN

β-actin

RANKL

OPG

OB Differentiation day

M-CSF

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Day 0 Day 7 Day 14

Runx

2 gen

e exp

ressio

n (ra

tio to

β-ac

tin)

OB Differentiation

WT

KO

0

0.2

0.4

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(A)

(B)

Figure 7.17 Analysis of osteoblast marker genes expression levels in Svs7 KO mice.

(A) BM cells were extracted from long bones of 12-week old WT and Svs7 KO mice

and cultured in osteogenic medium for 14 days. RNA was then extracted and subjected

to semi quantitative RT-PCR reactions using specific primers for Runx2, Osterix, ALP,

OPN, OCN, M-CSF, RANKL, and OPG. β actin served as a loading control. Agarose

gel electrophoresis depicted that there was no striking difference in the expression of

osteoblast marker genes between WT and Svs7 KO mice, as also shown in (B)

Densitometry analysis using ImageJ software, in which the expression of each

osteoblast gene was expressed as a ratio to β-actin. Data are representative of one

experiment.


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7.4 Discussion As previously shown in Chapter 6, Svs7 KO mice exhibit a low bone mass phenotype,

which could have resulted from either a decrease in bone formation by osteoblasts

(Manolagas & Jilka 1995) and/or an excessive bone resorption activity by osteoclasts

(Riggs & Melton 1983). Therefore, the first part of this chapter explored the potential

intrinsic role of Svs7 in osteoclast differentiation (including osteoclast fusion

mechanism) and activation. First, a five-day time course of RANKL-induced WT and

Svs7 KO BMM revealed that KO cells had a significant increase in osteoclastogenesis

than WT, starting at day 3 and continued to day 5 of osteoclastogenesis. In addition to

this increase in the number of osteoclasts, Svs7 KO osteoclasts had also shown to have

significantly larger spread area and more nuclei than WT, indicating the potential

intrinsic role of Svs7 in the regulation of osteoclast fusion mechanism (Fujita et al.

2012; Lee et al. 2006; Yagi et al. 2005). The potential role of Svs7 in the regulation of

osteoclast fusion gained a huge interest because of the fact that the fusion process of

osteoclast precursors to differentiate into multinucleated osteoclasts starts at day 3

onwards during RANKL-induced osteoclastogenesis (Fujita et al. 2012; Kim et al.

2008), at which Svs7 gene expression is upregulated in osteoclasts (Chapter 2 Figure

2.4). With the additional results that showed the unaffected Svs7 KO BM population of

early osteoclast precursor (Lin-) and LSK HSCs, as well as MAPK (ERK, JNK, and p38

phosphorylation) and NF-κB (IκBα degradation) signalling pathways (Cobb &

Goldsmith 1995; Teitelbaum 2000) in Svs7 KO osteoclast progenitor relative to WT,

these findings suggest that Svs7 has a potential intrinsic regulatory role in osteoclast

fusion and has no effects on early osteoclast progenitor population and proliferation.

Since osteoclasts from Svs7 KO mice had a comparable mRNA expression of osteoclast

marker genes, such as NFATc1, DCSTAMP, and ATP6v0d2 fusogenes, with WT

during RANKL-induced osteoclastogenesis, we are therefore tempted to speculate that

Svs7 involvement in osteoclast fusion might be linked to RANKL-induced ITAM co-

stimulatory pathway activation, which involves Ca2+ influx response and the subsequent

activation of NFATc1 via Ca2+/calmodulin-dependent calcineurin pathway (Koga et al.

2004; Takayanagi et al. 2002).

Although there were more large osteoclasts in Svs7 KO BMM in vitro cultures, the total

resorbed area/cell, as an indication of the osteoclast bone resorption activity, was found

to be comparable between WT and Svs7 KO osteoclasts. Hence, this result indicates


173

that the osteoclast bone resorption activity is independent of Svs7. The study from Lee

et al. 2006 has also shown that despite osteoclast differentiation is affected in

ATP6v0d2-deficient mice, osteoclast bone resorption activity is normal in these mice,

indicating a sole role of ATP6v0d2 in osteoclast fusion process. It is then plausible to

speculate that the nature of increase in in vitro and in vivo osteoclastogenesis itself that

could have contributed to the osteoporotic phenotype observed in Svs7 KO mice.

Considering that Ca2+ influx/signalling, induced by RANKL, is a prerequisite for the

robust induction of NFATc1 activation-driven osteoclast differentiation and activation

(Hwang & Putney 2012; Kim et al. 2008; Takayanagi et al. 2002; Takayanagi 2007),

together with the fact that Svs7 acts as a Ca2+ transport inhibitor on the membrane of

sperm cells during fertilization process, it was then interesting to explore the potential

effect of the loss of the Svs7 gene on Ca2+ influx in osteoclasts. Fluo-4AM intracellular

calcium assays revealed that Svs7 KO osteoclasts were more sensitive to Ca2+ influx

(shown by a higher increase in fluorescence intensity in the basal Ca2+ level (ratio to

baseline)) in response to RANKL and extracellular Ca2+ stimulation (particularly at the

addition of 5mM concentration of CaCl2) than WT. Despite a comparable final Ca2+

uptake (percentage of final fluorescence reading at the end of extracellular Ca2+

stimulation to baseline reading) between WT and Svs7 KO osteoclasts, these findings

indicate that there is a dysregulated Ca2+ influx/transport in Svs7 KO osteoclasts.

NFATc1, a Ca2+-dependent transcription factor and a master regulator of

osteoclastogenesis, has been shown to regulate a number of osteoclast marker genes

expression, such as Cathepsin K, TRAP, CTR, OSCAR, and β3 integrin (Crotti et al.

2006; Kim et al. 2005; Matsumoto et al. 2004; Takayanagi et al. 2002), as well as the

two fusogenic factors, DCSTAMP and ATP6v0d2 (Kim et al. 2008; Lee et al. 2006;

Yagi et al. 2005). With the findings that indicate a dysregulated Ca2+ influx in Svs7 KO

osteoclasts in response to RANKL, there was then a strong indication that NFATc1 and

possibly the two fusogenic proteins expression might be affected in osteoclasts of Svs7

KO mice. To explore the effects of the loss of the Svs7 gene on NFATc1 and fusogenic

proteins expression in osteoclasts, cell lysates of 5-day time course of RANKL-induced

WT and Svs7 KO BMM were extracted and subjected to western blot (WB) analysis

and immunoblotted with specific NFATc1, ATP6v0d2, and DCSTAMP antibodies. The

results revealed that there was a significant increase in NFATc1 (at day 3 of

osteoclastogenesis) and a trend of increase in ATP6v0d2 protein expression in Svs7 KO


174

osteoclasts compared to WT, particularly at day 3 and 5 of RANKL-induced

osteoclastogenesis. Although DCSTAMP protein expression was found to be unaffected

in Svs7 KO osteoclasts, together these findings imply that the dysregulated Ca2+ influx,

observed in KO osteoclasts upon RANKL stimulation, might account for the altered

NFATc1 signalling, resulting in its stronger activation with the subsequent increase in

ATP6v0d2 fusogenic protein expression. Furthermore, the direct regulatory role of Svs7

in NFATc1 activation has been shown by the inhibitory effect of Svs7 protein treatment

on NFAT promoter luciferase activity. Although the possibility of direct regulatory role

of Svs7 in NFATc1 protein expression needs further investigation, collectively, these

results suggest that Svs7 may regulate osteoclast fusion mechanism through the Ca2+-

NFATc1 signalling pathway, which has its primary effect on the expression of

ATP6v0d2 but not DCSTAMP expression.

As bone homeostasis is maintained by the balanced cell-to-cell communication between

osteoclasts and osteoblasts, in vitro Svs7 KO osteoblast phenotype characterization was

next performed. To achieve this, Svs7 KO primary calvarial osteoblast proliferation was

investigated first. Calvariae were dissected from WT and Svs7 KO neonatal mice and

the primary calvarial osteoblast cells were extracted from the calvariae using serial

collagenase digestion method. MTS proliferation assays on standard and two time

points (24 and 48 hours incubation) were then performed. The result showed that at both

time points there were no significance differences in proliferation rate between WT and

Svs7 KO primary calvarial osteoblasts, indicating that Svs7 KO osteoblasts exhibit an

unaffected proliferation rate in vitro. Next, ALP osteoblast differentiation and bone

nodule formation (mineralization) assays were performed on WT and Svs7 KO BM-

derived osteoblasts. Following 14 days culture in osteogenic medium, ALP staining was

conducted to analyse osteoblast differentiation in vitro, while alizarin red staining was

used to stain for calcium deposition in mineralization assays. The results showed that

there were no significant differences in CFU-ALP positive colonies and total

mineralized nodules formed between WT and Svs7 KO osteoblast cultures. With the

previous double calcein labelling experiment and MAR analysis results that showed

Svs7 KO mice had a normal bone formation activity in vivo, together these results

indicate that Svs7 KO mice exhibit unaltered osteoblast differentiation in vitro and

osteoblast bone formation activity both in vitro and in vivo. Furthermore, through semi

quantitative RT-PCR reactions on 14-day time course of WT and Svs7 KO BM cells

differentiation into mature osteoblasts using specific primers for common osteoblast


175

marker genes, it is then revealed that, relative to WT, Svs7 KO mice exhibit unaltered

expression levels of osteoblast marker genes.

So in conclusion, Svs7 has shown to have a role in regulating osteoclast differentiation

but not osteoclast activity. Svs7 KO mice have a significant increase in

osteoclastogenesis and osteoclast size (possessing larger spread area and more nuclei)

than WT, hence osteoclast fusion is affected in the KO mice. By performing

intracellular Ca2+ assays, WB analysis, and NFAT promoter luciferase assays, it is then

revealed that Svs7 has a role in the regulation of osteoclast fusion mechanism through

the Ca2+-NFATc1 signalling pathway and the subsequent ATP6v0d2 fusogenic protein

expression. As Svs7 KO mice have shown to have unaffected osteoblast proliferation,

differentiation, and bone formation activity, we are then tempted to speculate that the

loss of Svs7 regulation in osteoclast differentiation and fusion through the Ca2+-

NFATc1 signalling pathway is the likely cause for the osteoporotic phenotype observed

in the KO mice. Hence, Svs7 is a novel regulator of bone homeostasis, which acts in an

autocrine fashion to regulate osteoclast differentiation and fusion.

176

CHAPTER 8

GENERAL SUMMARY AND FUTURE DIRECTIONS

Chapter Eight – General Summary and Future Directions

177

8.1 Overview of Thesis and General Discussion Healthy bone during adult life is maintained by balanced yet complementary activities

of bone-resorbing osteoclasts and bone-forming osteoblasts and hence is tightly

regulated and governed by many local and systemic factors, such as growth factors,

cytokines, proteins, and hormones (Karsenty & Wagner 2002; Zaidi 2007). The

osteoblast regulation of osteoclast differentiation and activation has been well studied,

while the other way of communication, in which osteoclasts regulate osteoblasts

function, is still poorly understood. In search for novel osteoclast-derived factors,

subtractive hybridization-based differential screening method was employed to probe

for differentially expressed genes between osteoclasts (RANKL-stimulated RAW264.7

cells) and their mononuclear progenitors (RAW264.7 osteoclast precursors) (Phan 2004).

Subsequently, Svs7 gene was identified as a candidate gene, whose expression was

robustly upregulated during RANKL-induced osteoclastogenesis (Davey 2008; Phan

2004). Svs7 is exclusively expressed by osteoclasts in bone and synthesized as a

secreted factor.

Svs7 is a secreted member of Ly-6 family, which shares common TFP/Ly-6/uPAR

motif (a conserved 8-10 cysteine residues and a C-terminal consensus sequence motif of

CCXXXXCN) (Coronel et al. 1992; Southan et al. 2002; Turunen et al. 2011; Ushizawa

et al. 2009). Several Ly-6 genes have been shown to be involved in many cellular

signalling pathways, such as uPAR in αvβ3 signalling pathway (Schiller et al. 2009;

Smith, Marra & Marshall 2008; Wei et al. 2008) and Sca-1 in stem cell renewal

capacity (Bonyadi et al. 2003; Holmes et al. 2007; Ito et al. 2003). With this knowledge

together with the previous studies by Davey 2008, which showed that treatment with

recombinant purified Svs7 protein (daily injection of 500μg/kg protein) could protect

against loss of trabecular bone volume, trabecular thickness, and trabecular number at

the distal femoral metaphysis of ovariectomised mice (oestrogen deficiency-induced

bone loss mouse model (Aitken, Armstrong & Anderson 1972; Kalu 1991; Saville

1969)), it led us to hypothesize that Svs7 is an important regulator of bone homeostasis.

To investigate this potential role of Svs7 in bone homeostasis, Svs7 TG mice were

initially generated and characterised. Following the successful cloning and purification,

the linearized pcDNA3.1 Svs7-V5 His transgene was microinjected into blastocysts to

generate Svs7 TG mice. Assessment of Svs7 tissue distribution revealed that, in normal

WT mice, Svs7 was almost exclusively expressed in seminal vesicle (including


178

prostate) with little to no expression in other tissues, including bone. In liver, the protein

expression of Svs7 in Svs7 TG mice was found to be low, which equalled to only ~2.5

fold increase relative to WT. It is possible that post transcriptional and translational

regulations as well as hormonal change might influence gene and protein expression

levels (King et al. 2012; Levitin et al. 2008) and therefore the use of osteoclast-specific

Svs7 TG mice and the subsequent detection of overexpressed Svs7 protein in bone

tissue of these TG mice should be considered in the future to induce direct bone cells-

specific responses. Global characterisation of Svs7 TG mice was further pursued by

performing histological assessment on TG organs and tissues (prostate). No obvious

change was observed in Svs7 TG prostate tissue phenotype relative to WT littermates,

suggesting that the overexpression of Svs7 does not affect the organogenesis in these

TG mice.

Next, the bone phenotype of Svs7 TG mice was assessed by X-ray, microCT, and

histology/histomorphometry analysis. Whole body X-ray of Svs7 TG mice showed that,

relative to WT, TG mice had phenotypically normal skeletons. Furthermore, microCT

analysis on trabecular bone at distal femoral metaphysis revealed comparable bone

parameters (trabecular bone volume, trabecular separation, trabecular thickness, and

trabecular number) between WT and Svs7 TG mice, indicating that the TG mice have

normal bone mass and architecture. Histology/histomorphometry analysis supported the

microCT data, which showed normal trabecular bone mass and structure at Svs7 TG

distal femurs and a comparable number of TRAP-positive osteoclasts observed near the

growth plate region of WT and TG hindlimbs. To confirm the osteoclast phenotype of

Svs7 TG mice, in vitro osteoclast differentiation assay was performed and the result

revealed that the TG mice exhibited an unaffected osteoclast differentiation, relative to

WT counterparts. Hence, this unaltered osteoclast differentiation is likely to have

contributed to the normal bone mass phenotype observed in Svs7 TG mice.

The normal bone phenotype of Svs7 TG mice therefore highlights two potential limiting

factors to the assessment of the potential role of Svs7 in the maintenance of bone

homeostasis. These factors are: (i) the naturally very low expression of Svs7 gene in

bone and (ii) the modest protein expression of Svs7 achieved in Svs7 TG mice.

Accordingly, the use of osteoclast-specific Svs7 TG mice in the future could potentially

induce a stronger expression of the Svs7 transgene and hence being able to trigger direct

bone cells-specific responses.


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The second approach utilised the generation and characterisation of Svs7 global KO

mice. This was achieved by deleting exon 2 and 3 of Svs7 gene, which together encode

for the TFP/Ly-6/uPAR domain, resulted in the complete absence of Svs7 gene and

protein in the KO mice. Breeding of Svs7 KO mice revealed that the ratio of male WT

and female Svs7 KO mice were well below than the expected mendelian ratio of 25%,

which consequently limited both sample size and number of assays possible for

assessment. As Svs7 has been shown to inhibit Ca2+ influx across the membrane of

sperm cells during fertilization to prevent premature acrosomal reaction and

spermatozoa hyperactivation (Coronel et al. 1992), this slow mice breeding process

could partly be due to a role for Svs7 in fertility process, which warrants investigation

in the future studies. Despite the slow mice breeding process, the gross body phenotype

of Svs7 KO mice appeared to be as normal as WT littermates. The global

characterisation of Svs7 KO mice was continued by performing macro- and microscopic

assessments on the KO mice organs and tissues phenotype. The results depicted that

there was no apparent difference in organs and tissues (including prostate histology

sections) phenotype between WT and Svs7 KO mice, therefore indicating that Svs7 KO

mice have no obvious defects in organs and tissues.

To directly investigate the role of Svs7 in bone, the bone phenotype of Svs7 KO mice

was assessed. To this end, microCT analysis on trabecular bone in the secondary

spongiosa of distal femurs (Lee et al. 2006; Nishikawa et al. 2010) from 15- and 30-

week old WT and Svs7 KO mice revealed that the KO mice had a significantly reduced

trabecular bone/tissue volume at both age groups and hence a significant decrease in

trabecular number (only in 30-week old group) compared to WT littermates. Hence,

these results not only suggest that Svs7 KO mice exhibit an osteoporotic phenotype but

also the possibility that the KO mice have a progressive decline in bone mass with age.

Consistent with these results, histology and histomorphometry analysis showed that

there was an obvious decrease in trabecular bone volume and a trend of increase in the

number of TRAP-positive osteoclasts, respectively, in Svs7 KO hindlimbs compared to

WT. Together, these results gave a strong indication that Svs7 KO mice might have an

alteration in osteoclast differentiation and/or activity, which could be the main

contributor to the osteoporotic phenotype of the KO mice. This position was further

supported by the finding that Svs7 KO mice exhibit an unaffected bone formation

activity in vivo, relative to WT, in double calcein labelling experiment and MAR

analysis. Considering that a naturally low Svs7 gene expression in bone is likely to have


180

contributed to the mild phenotype observed in Svs7 KO mice, this study however has

shown that Svs7 is a potential regulator of bone homeostasis and the increase in

osteoclast number observed in the histomorphometry analysis of TRAP-stained sections

of Svs7 KO hindlimbs therefore hinted for a possible intrinsic role of Svs7 in osteoclast

differentiation and/or activation.

Next was the investigation of the intrinsic cellular mechanism underlying the

osteoporotic phenotype of Svs7 KO mice. In vitro time course RANKL-induced BMM

differentiation into osteoclasts revealed that Svs7 KO cultures had a significant increase

in osteoclast number compared to WT at day 3 and 5 of osteoclastogenesis. Hence,

these in vitro osteoclast differentiation data have correlated well with the

histomorphometry result, demonstrating that Svs7 KO mice have an alteration in

osteoclast differentiation. Furthermore, Svs7 KO osteoclasts were significantly larger

and possessed more nuclei (i.e. ≥ 11) than WT at day 5 of osteoclastogenesis, further

indicating that Svs7 regulates osteoclast fusion mechanism. Other possibility that the

loss of Svs7 gene might affect early bone marrow (BM) osteoclast progenitor

population was then investigated by performing flow cytometry analysis on WT and

Svs7 KO BM population for specific analysis on CD11b-/lowCD45R-CD3e- (Lin-) early

osteoclast precursor and Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem cell population

(Jacquin et al. 2006; Kollet et al. 2006). The results showed that there were no

significant differences in both populations between WT and Svs7 KO mice. By

conducting MTS proliferation assay and MAPK (ERK, JNK, and p38 phosphorylation)

and NF-κB (IκBα degradation) signaling, which are important signaling pathways

governing early osteoclast precursor proliferation, growth, and survival (Cobb &

Goldsmith 1995; Teitelbaum 2000), WB analysis on WT and Svs7 KO early osteoclast

precursors, it was found that the KO mice exhibited unaltered proliferation and survival

of osteoclast precursor. Together, these results confidently suggest a role of Svs7 in

osteoclast fusion mechanism.

Despite the increase in osteoclast number and size in Svs7 KO BMM cultures, no

significant differences were observed in osteoclast bone resorption activity and key

osteoclast marker genes expression, including NFATc1 and fusogenic genes,

DCSTAMP and ATP6v0d2 (Kim et al. 2008; Lee et al. 2006; Yagi et al. 2005),

between WT and Svs7 KO mice. Hence, these results raise two possibilities for the role

of Svs7 in bone homeostasis. First, the osteoclast bone resorption activity is


181

independent of Svs7. Previous studies by Lee et al. 2006 had presented similar results

that in ATP6v0d2-deficient mice, osteoclast differentiation and fusion were affected but

not osteoclast activity. Secondly, since osteoclast marker genes expression levels were

unaffected in Svs7 KO mice relative to WT, it is then possible that Svs7 involvement in

osteoclast fusion is linked to RANKL-induced ITAM co-stimulatory pathway

activation, which involves intracellular Ca2+ release from endoplasmic reticulum (ER)

store upon RANKL stimulation, followed by Ca2+ influx response through Ca2+

permeable channel (causing an increase of intracellular Ca2+ level) to sustain Ca2+

oscillation and the subsequent activation of NFATc1 via Ca2+/calmodulin-dependent

calcineurin pathway (Feske 2006; Hwang & Putney 2012; Kar, Nelson & Parekh 2011;

Koga et al. 2004; Lewis 2001; Takayanagi et al. 2002), as shown in the hypothetical

model of the role of Svs7 in osteoclasts (Figure 8.1).

To elucidate the possible mechanistic involvement of Svs7 in the osteoclast Ca2+

signalling pathway, Fluo-4AM intracellular calcium assay on WT and Svs7 KO

osteoclasts was performed. Upon RANKL stimulation (overnight), Svs7 KO osteoclasts

showed to have a higher increase in basal Ca2+ level than WT, which indicates that KO

osteoclasts are more sensitive to Ca2+ influx than WT in response to RANKL

stimulation. Furthermore, the addition of extracellular Ca2+ (especially at 5mM

concentration of CaCl2) caused a rapid and steep Ca2+ influx response in Svs7 KO

osteoclasts compared to WT littermates, although the final Ca2+ uptake (% of baseline)

between WT and KO osteoclasts was found to be comparable. Collectively, these

findings suggest that there is a dysregulated Ca2+ transport in Svs7 KO osteoclasts in

response to RANKL and extracellular Ca2+ stimulation, hence hinting the regulatory

role of Svs7, a novel osteoclast-secreted factor, in Ca2+ transport system in osteoclasts.

As NFATc1, a master regulator of osteoclastogenesis (Takayanagi et al. 2002), has been

shown to regulate osteoclast fusogens, such as DCSTAMP and ATP6v0d2 (Kim et al.

2008), the possibility that Svs7 regulates osteoclast fusion through the Ca2+-NFATc1

signalling pathway was further elucidated by performing WB analysis on cell lysates of

time course RANKL-induced WT and Svs7 KO BMM differentiation into mature

osteoclasts. Consistent with the possible role of Svs7 in regulating Ca2+ response in

osteoclasts, WB results showed that there was a significant increase in NFATc1 (at day

3 of osteoclastogenesis) and a trend of increase in ATP6v0d2 protein expression in Svs7

KO osteoclasts compared to WT.


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Plasma Membrane

Cytosol ITAM DAP12 /

FcRγ

TREM2 / OSCAR

RANKL

RANK

ITAM Co-Stimulatory Pathway RANKL Pathway

TRAF6

NF-κB

c-Fos

P

PLCγ

Ca2+

NFATc1

Auto-amplification Calcineurin

NFATc1 Transcription

IκB IκB P

Ubiquitination and degradation

Nucleus

ER

IP3

Calmodulin

Ca2+ channel (Orai1 or TRPV4 ??)

Ca2+

Svs7

22+

NFATc

Intracellular [Ca2+] increases

?

Osteoclastic Genes

DC-STAMP, ATP6v0d2, OSCAR, β3 integrin, TRAP, CTR, Cathepsin K

etc.

Figure 8.1 Hypothetical model of the role of Svs7 in the regulation of osteoclast

fusion through the Ca2+-NFATc1 signalling pathway. RANKL-induced late stage of

osteoclast differentiation (fusion) begins with the binding of RANKL to its receptor

RANK on osteoclast surface, which leads to the activation of NF-κB and ITAM co-

stimulatory signalling pathways. Subsequently, the production of IP3 by PLCγ causes

Ca2+ release from intracellular ER stores, which is accompanied by Ca2+ influx response

through Ca2+ permeable channel (Orai1 or TRPV4), to increase intracellular Ca2+ level

and at the same time to induce a long and sustain Ca2+ oscillation, necessary for the

Ca2+/calmodulin/calcineurin-dependent NFATc1 signalling pathway activation, which

has been shown to transcriptionally regulate a number of osteoclast marker genes

expression, such as DCSTAMP, ATP6v0d2, and OSCAR. Svs7, a novel osteoclast-

secreted factor, regulates Ca2+ influx/transport in osteoclasts by its possible

regulation/interaction with Orai1 or TRPV4 on osteoclast surface.


183

With Svs7 KO DCSTAMP was found to be unaffected throughout the time course of

RANKL-induced osteoclastogenesis, these results indicate that Svs7 regulates osteoclast

fusion through the Ca2+-NFATc1 signalling pathway (Figure 8.1), with its main effect

on the ATP6v0d2 protein expression. The fact that only one affected fusogen can alter

osteoclast fusion has also been shown by previous studies from Hwang et al. 2012 that

showed that Orai1 (store-operated Ca2+ entry (SOCE) Ca2+ channel subunit)-deficient

mice had defects in osteoclast fusion, owing to the significant inhibition in NFATc1 and

ATP6v0d2 proteins expression but not DCSTAMP. With the fact that Orai1 provides

the major Ca2+ influx pathway for sustaining Ca2+ oscillation in hematopoietic stem

cells, from which osteoclasts are differentiated (Feske 2006; Hogan et al. 2003; Lewis

2001), and the reduced NFATc1 protein expression in Orai1 KO mice, it is tempting to

speculate that Svs7 regulates Ca2+ transport in osteoclasts through its regulation on

Orai1 (Figure 8.1). As the interaction between Svs7 and Orai1 still needs further

investigation, recent studies on transient receptor potential vanilloid (TRPV) 4 has also

shown its importance in Ca2+ entry for RANKL-induced mature osteoclast

differentiation, with TRPV4 KO mice having an impairment of mature osteoclast

differentiation and subsequent inhibition of bone resorption activity, caused by an

attenuation of Ca2+/calmodulin-NFATc1 signalling pathway (Masuyama et al. 2012;

Masuyama et al. 2008). Together, these results hint a complexity in the molecular

mechanism of Ca2+ entry and intracellular Ca2+ change during osteoclast differentiation

and the necessity for further and thorough investigation in the future (Kajiya 2012).

As bone homeostasis is maintained by balanced cell-to-cell communication between

osteoclasts and osteoblasts, the dissection into the cellular mechanism behind the

osteoporotic phenotype of Svs7 KO mice was further investigated by performing in

vitro osteoblast differentiation and mineralization assays. After confirming that, in

relative to WT, Svs7 KO primary calvarial osteoblasts exhibited an unaffected

proliferation rate, WT and KO BM differentiation into mature osteoblasts and its bone

formation activity after 14 days culture in osteogenic medium were assessed by

performing ALP and alizarin red staining, respectively. The results showed that there

were no significant differences in CFU-ALP positive colonies and total mineralized

nodules formed between WT and Svs7 KO osteoblast cultures. Together with the in

vivo calcein labelling and MAR analysis showing an unaffected bone formation activity

in Svs7 KO mice relative to WT, these results suggest that Svs7 KO mice have normal

osteoblast differentiation in vitro and exhibit an unaltered bone formation activity both


184

in vitro and in vivo. The naturally low Svs7 gene expression in bone is in line with our

observations of a normal osteoblast phenotype in Svs7 KO mice. The use of osteoclast-

specific deletion of Svs7 should be considered in the future to further investigate the

possible paracrine effect of Svs7 in the regulation of osteoblast differentiation and/or

activity, together with the elucidation and confirmation of osteoclast-specific Svs7

autocrine effects.

In conclusion, this study has identified Svs7 as a regulator of bone homeostasis. Svs7

regulates osteoclast differentiation and fusion through the Ca2+-NFATc1 signalling

pathway but not osteoclast activity. With the increasing complexity in the molecular

mechanism of Ca2+ transport in osteoclasts, the regulatory role of Svs7 in this Ca2+

signalling pathway requires further investigation. The loss of Svs7 gene has been

observed not to affect osteoblast differentiation and activity. Collectively, this study

further improves our knowledge in the regulation of cell-to-cell communication

between osteoclasts and osteoblasts and hence Svs7 might represent as a therapeutic

agent against osteoclast-related osteolytic diseases.

8.2 Future Directions 8.2.1 The role of Svs7 in bone homeostasis

The studies presented here have uncovered the potential role of Svs7 in the maintenance

of bone homeostasis. By using Svs7 global KO mouse model, Svs7 has shown to

regulate osteoclast fusion through the Ca2+-NFATc1 signalling pathway. With regard to

the naturally low Svs7 gene expression in bone, including no detectable Svs7 protein

expression in WT osteoclast culture, in both culture medium and cell lysate, and the

modest phenotype observed in this study from both the Svs7 global TG and KO mice

models and still with knowledge that in the previous studies by Davey 2008 suggested

anabolic effects of treatment with recombinant purified Svs7 protein in ovariectomised

(OVX) mice, the possibility that Svs7 might still hold a more specific role in the

regulation and maintenance of bone homeostasis warrants further investigation.

First, to complete the characterisation and to confirm the bone phenotype of both Svs7

global TG and KO mice, extensive bone histomorphometry analysis, such as on

osteoclast and osteoblast area/bone surface (%), number of osteoblasts/bone surface

(N/mm), eroded area/bone surface (%), and osteoid area/bone surface (%), together with


185

other essential bone parameters (trabecular bone/tissue volume, trabecular separation,

trabecular thickness, and trabecular number), should be performed in near future.

Next, to facilitate a more thorough investigation into the specific role of Svs7 in bone

homeostasis, the generation and characterisation of osteoclast-specific Svs7 conditional

TG and conditional KO mice should be performed. By incorporating common

osteoclast marker genes promoter sequence, such as Cathepsin K and TRAP, into the

targeting vectors and the use of Cre-LoxP system (Masuyama et al. 2012; Miyauchi et

al. 2010; Nakamura et al. 2007; Qin et al. 2011; Takeshita et al. 2013), osteoclast-

specific Svs7 TG and KO lines can be generated. Next, the overexpression of Svs7

protein in bone tissue and osteoclast culture (in conditioned medium and/or cell lysate)

of these osteoclast-specific TG mice should be investigated first to confirm the

successful generation of these Svs7 conditional TG mice. MicroCT and

histology/histomorphometry, together with double calcein labelling and MAR analysis,

for the bone phenotype characterisation of these osteoclast-specific Svs7 TG and KO

mice will certainly help to uncover the more specific role of Svs7 in the regulation and

maintenance of bone homeostasis, which might have been overlooked in the current

study.

8.2.2 The role of Svs7 in osteoclast and osteoblast differentiation and activation

Although these studies indicate a role for Svs7 in osteoclast fusion mechanism via the

Ca2+-NFATc1 signalling, this pathway still requires further investigation. Furthermore,

detailed investigation of in vitro osteoclast and osteoblast differentiation and activity for

the osteoclast-specific Svs7 TG and KO mice is required to confirm the regulatory role

of Svs7 in osteoclast differentiation and fusion and at the same time helps to uncover

the possible paracrine effects of Svs7 in osteoblast differentiation and activation. The

potential intrinsic role of Svs7 in the regulation of cell-to-cell communication between

osteoclast and osteoblast also requires further investigation using in vitro osteoclast and

osteoblast coculture systems.

Alternative quantitative measurements to better analyse osteoclast bone resorption

activity, e.g. measuring serum type I collagen cross-linked telopeptide (CTX) through

enzyme-linked immunosorbent assay (ELISA) (Lee et al. 2006), should be performed in

the future to rule out any direct effects of Svs7 in osteoclast activity. Serum

biochemistry measurement on Ca2+ and phosphate concentrations by using colorimetric


186

assays (Masuyama et al. 2012) should also be performed to complement the analysis for

osteoclast bone resorption activity in vitro and in vivo.

The effect of retroviral induction (overexpression/rescue) on BMM-derived osteoclast

differentiation and activation should be tested alongside the characterisation of

osteoclast-specific Svs7 TG and KO mice to confirm the specific role of Svs7 in

osteoclasts. With difficulties in detecting Svs7 protein in culture medium (supernatant)

of RANKL-induced BMM differentiation into osteoclasts, immunofluorescence using

specific Svs7 antibody and imaging method using confocal microscopy on these

retrovirally-induced (overexpressed) osteoclasts (Pavlos et al. 2011; Qin et al. 2011)

should be carried out to locate specific expression of Svs7 in osteoclasts.

Intracellular Ca2+ measurement using fura-2 fluorescent probe on both osteoclasts and

osteoblasts of the osteoclast-specific Svs7 TG and KO mice, in which emission ratio of

alternated 340/380nm excitation wavelength can be recorded (Miyamoto et al. 2012), is

necessary to be performed to repeat the Fluo-4AM calcium assay results in this current

study and to confirm the potential regulatory role of Svs7 in both osteoclasts and

osteoblasts Ca2+ transport system, particularly in response to high extracellular Ca2+,

which remains poorly understood. The incorporation of thapsigargin, an ATPase Ca2+

transport inhibitor at ER store (Hwang & Putney Jr 2011), should also be considered in

the future intracellular Ca2+ study to specifically investigate the potential regulatory role

of Svs7 in Orai1-regulated Ca2+ influx in the store-operated Ca2+ entry (SOCE) system,

which has been shown to have a crucial role in mediating the Ca2+/NFATc1 signalling

pathway activation (Deng et al. 2009; Feske 2006; Feske 2009; Hogan et al. 2003;

Hwang & Putney 2012). Moreover, this future study on the potential role of Svs7 in the

regulation of Ca2+ transport in osteoblasts in response to high extracellular Ca2+ can give

another light of mechanism in the regulation and formation of Ca2+ phosphate

(Ca3(PO4)2) vesicles in osteoblasts, required for Ca2+ deposition on new bone-forming

surfaces (Boonrungsiman et al. 2012).

To further dissect the regulatory role of Svs7 in NFATc1 protein expression,

particularly in osteoclast fusion mechanism, first, cytoplasmic and nuclear fractionation

on cell lysates of time course RANKL-induced osteoclast differentiation should be

conducted and hence the results can therefore clearly demonstrate the effect of Svs7 in

the regulation of NFATc1 protein expression, both in cytoplasm and nucleus. Secondly,


187

immunofluorescence and confocal microscopy should be performed on Svs7

conditional TG and KO-derived osteoclasts to detect and analyse NFATc1 translocation

into the nucleus and hence to confirm the regulatory role of Svs7 in NFATc1 signalling

pathway. The investigation of ATP6v0d2 and DCSTAMP fusogenic proteins

expression, two main players in osteoclast fusion mechanism (Kim et al. 2008; Lee et

al. 2006; Yagi et al. 2005), in the osteoclast-specific Svs7 TG and KO mice still needs

to be carried out to get a conclusive mark for the role of Svs7 in osteoclast fusion.

Finally, the putative binding of Svs7 on key bone cells, such as chondrocytes,

osteoclasts, osteoblasts, and osteocytes, and the potential interacting partner for Svs7 in

bone cells environment are still unknown and therefore need to be investigated. First,

flow cytometry experiments on bone cells, treated with recombinant purified Svs7

protein and stained with specific Svs7 antibody and fluorochrome-conjugated secondary

antibody, should be performed to detect and analyse the putative binding of Svs7 on

major bone cells. Secondly, to investigate the potential interacting partner for Svs7,

random screening using surface plasmon resonance (SPR) should be performed (Canalis

et al. 2010; Schneeweis, Willard & Milla 2005; Theoleyre et al. 2006). In this

technique, Svs7 protein, acting as a ligand, will be placed in the biosensor, while a clear

cell lysate of WT osteoclasts (‘the analyte’) containing numerous proteins, passes

through over the other side of the biosensor site. A protein-protein interaction will cause

a change in the refractive index, which is proportional to the mass added in the sensor

surface, followed by the measurement of dissociation constant (KD) of the interaction.

The protein-protein interaction can later be confirmed by performing co-

immunoprecipitation and/or pull down experiments (Masuyama et al. 2012; Pavlos et

al. 2011). The identification of Svs7 binding partner will lead us to a better

understanding about the role and function of Svs7 in bone microenvironment.

In conclusion, this study documents, for the first time, the role of Svs7 in bone

homeostasis. Svs7 has shown to play a role in regulating osteoclast fusion through the

Ca2+-NFATc1 signalling pathway. Considering the numerous limitations being

encountered in this study, including i) the low Svs7 overexpression in Svs7 TG mice

and ii) the modest phenotype observed in Svs7 KO mice (mild effects of Svs7 in

intracellular Ca2+ study and osteoclast fusion proteins expression), further investigation

is required to fully elucidate the precise contribution of Svs7 in osteoclast fusion and

maintenance of bone homeostasis.

188

CHAPTER 9

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APPENDIX

Appendix

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Serum Biochemistry WT TG Calcium (mg/dL) 1.61 ± 0.31 1.46 ± 0.14

Phosphate (mg/dL) 2.07 ± 0.12 2.18 ± 0.15

Supplementary Figure 1. Serum biochemistry measurement on calcium and

phosphate levels in Svs7 TG mice. Nine-week old WT and Svs7 TG mice were

sacrificed and blood was extracted from both through cardiac puncture and allowed to

clot in room temperature for 20 minutes. The clotted blood was then centrifuged at

3,000rpm in room temperature for 15 minutes to release serum sample. Calcium and

phosphate serum biochemistry measurement was performed at PathWest Laboratory

Medicine of Western Australia and the result showed that there were comparable

calcium and phosphate levels between WT and Svs7 TG mice. Data are presented as

mean ± SEM. Number of mice (n) : 2/group.

Appendix

225

5% 20%

1 2 3 4 5 6 7 8 9 10 11 12 P OC

fraction:

Svs7

V-H+ATPase

α-tubulin

IB:

Supplementary Figure 2. Svs7 protein detection at the secretory vesicles fraction of

osteoclasts. Osteoclast fractions by sucrose gradient centrifugation were subjected to

WB analysis using rabbit polyclonal anti-Svs7 antibody. The result showed that Svs7

was successfully detected at fraction 3 and 4, which correspond to the fraction of

secretory vesicles. Hence, Svs7 is a novel osteoclast-secreted protein. V-H+ATPase

complex served as a positive control for osteoclast-specific marker protein and α-

tubulin was used as a loading control.

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