Pax6 expressed in osteocytes

inhibits canonical wnt signaling

Ajita Jami

Department of Medical Science

The Graduate School, Yonsei University

Pax6 expressed in osteocytes

inhibits canonical wnt signaling

Directed by Professor Sung Kil Lim

The Doctoral Dissertation submitted to

the Department of Medical Science,

the Graduate school of Yonsei University

in partial fulfillment of the requirements for

the degree of Doctor of Philosophy

Ajita Jami

June 2013

ACKNOWLEDGEMENTS

“GURU BRHAMA

GURUUR VISHUNUHU

GURUR DEVO MAHESWARA”

- VEDA

I begin my acknowledgement by thanking almighty Teachers whose

blessing has enabled me to fulfill my thesis work with bundle of joy.

It is my proud privilege to express my deep sense of gratitude to my

respected and esteemed teacher and supervisor, Professor Sung Kil Lim for his

invaluable contributions, guidance and cooperation held up at each and every stage

of this work. I am extremely indebted and grateful to him for his realistic approach,

honest advice and sincere heartfelt motivation of guiding and taking interest in this

work. He guided me right from the protocol preparation and presentation up to the

completion of this research work, ready to solve each and every problem that might

have come on my way while pursuing the work. In spite of his busy and hectic

schedule, he was kind enough to be available for the correction of my thesis work.

Last but no the least, it was with his blessings and I could accomplish my work.

I extend my heartfelt thanks to the committee members Prof. Myoung Hee

Kim, Prof. Han Sung Jung, Prof. Sahng wook park, Prof. Jin Woo Lee for their

valuable suggestions and advices for my thesis work.

I express my deep gratitude for the admirable contribution by Dr.Gadi

Jogeswar, who have provided extremely valuable assistance in many aspects of the

study to made the task so very easy and simple.

I would like to say my sincere thanks to the Brain korea 21(BK21) for

providing me the scholarship.

I am very much thankful to the funding sources which made successful

completion of my P.hD work. This work was supported by a National Research

foundation of Korea (NRF) grant funded by the Korean government (MEST) (No.

20120000486).

I would like to say my heartful thanks to my lab senior, assistant research

professor and very good friend Dr.Eun Jin Kim, who given lot of moral support,

encouragement, valuable suggestions, guidance regarding many aspects of my work.

Many thanks to my sister Mi Jeong Lee for her valuable suggestions, help and to

write my thesis abstract in Korean language. I am very much thankful to Myoung

Jin Lee sister for her friendly support. My heartful thanks to previous members of

our lab, Dr. Sujin Park, Dr.Han Seok Choi, Dr.Kyoung Min Kim, Dr.Kwang Joon

Kim, leelan for their help and support in this lab.

My sincere thanks to Min Jung Lee (Dental College) for her good support

in doing animal experiments.

My special thanks to my brother ankamreddy harinarayana for his

admirable help to me. I am very much thankful to my friend Dr.Kiran and his wife

Dr.Prasanna and Dr.Mahesh for their support, encouragement and suggestions. I

wish to thank all the professors, other members and technical support people. I am

very much thankful to my Korean friends, Dr. Hyo Jin Kang, Moon Min Jeong, Lim

Young Sown and others for their help during my research work.

I am very much thankful to my previous house owners in korea Kim

Kyoung moom uncle and Lee Su Jin aunty for their parental care and for providing

accommodation. My sincere thanks to their children jun and jai for their help. My

sincere thanks to my Chinese friend and sister Jin Hanglian for her help and moral

support and suggestions.

I thank my parents and parent-in-laws, my pedanannagaru, peddamma, my

beloved husband (Dr. Gumma Vidyanidhi) , my son (Sourya Nanda Gopal),

Rajuncle, Janaki aunty as they have been the main source of my energy, support and

encouragement. This work would not have reached its present shape without their

constant inspiration and wishes. I would like to say my heartful thanks to my

brothers, brother-in-laws, sisters, sister-in laws, Aanjaneyulu uncle, Laxmi-aunty,

and all close relatives and family members (especially my grand father, ramana

mamayya, bangaaru mamayya, desanna mamayya, ramu chinnaanna, late rambaabu

mamayyagaru, Rajak uncle, Appala raju uncle, Demudu baabu chinnaanna,) who

given lot of care, support, encouragement towards me from my childhood. I am

very much thankful to my friends Dr.Ruthala kalyani, Dr.Murali Krishna,

Dr.Kumara swamy and his wife Dr.P.V.Suneetha, Satish, Vinod kumar badireddy,

Dr.Prachi anand kumar, Dr.P.Sunil Kumar for their moral support and

encouragement.

My heartful thanks to Professor V.K.Sharma, great teacher and philosopher,

who given lot of encouragement, moral support and valuable suggestions and

guidance to me and to my husband.

My heartful thanks to Late Sri Sri. A. Subba Rao, Late Sri Sri Badireddy

Maakunayudu garu, Great teachers, Philosophers. They always inspires me and tries

to build up good state of mind and strongly insist me to help the people.

I dedicate my thesis to my beloved family and to great teachers

Professor V.K.Sharma and Late Sri.A.Subbarao garu

TABLE OF CONTENTS

ABSTRACT 1

I. INTRODUCTION 3

II. MATERIALS AND METHODS 6

1. Animal study 6

2. Cell culture 6

3. Osteoblast differentiation 7

4. Alkaline Phosphatase (ALP) staining 7

5. RT-PCR analysis 7

6. Plasmid constructs and transfection 9

7. Sost promoter plasmid construction 9

8. Transfection of cells with small interfering RNA 10

9. Cell proliferation and cell number assay 11

10. Western blot analysis 11

11. Preparation of Wnt-3a conditioned medium 12

12. Chromatin immunoprecipitaion (Chip) assay 13

13. Luciferase reporter assay for monitoring Sost promoter activity

and Wnt signaling 14

14. Induction of Pax6 expression by Wnt3a treatment 15

15. Immunohistochemistry and HE staining 15

16. Statistical analysis 16

III. RESULTS 17

1. Endogenous expression of Pax6 in various types of bone cells

and tissues 17

2. Effect of Pax6 on Wnt inhibitor Sost and other osteocyte markers 21

3. Effect of Pax6 on Sost promoter activity in MLOY4 cells 24

4. Effect of Wnt3a on Pax6 mRNA expression in MLOY4 cells 29

5. Effect of Pax6 on Wnt signaling in MLOY4 cells 30

IV. DISCUSSION 33

V. CONCLUSION 36

REFERENCES 37

ABSTRACT (IN KOREAN) 45

PUBLICATION LIST 47

LIST OF FIGURES

Figure 1. Endogenous expression of Pax6 in bone cells and tissues 19

Figure 2. Immunohistochemical analysis 20

Figure 3. The effects of Pax6 on osteocyte marker genes 22

Figure 4. Effect of Pax6 on proliferation and cell viability 23

Figure 5. The Sost promoter sequence analysis (2kb region) 25

Figure 6. Chip PCR analysis in MLOY4 cells 26

Figure 7. Schematic diagram of Sost promoter reporter construct 27

Figure 8. The effect of Pax6 on Sost promoter activity 28

Figure 9. Effect of Wnt-3a Pax6 mRNA expression 29

Figure 10. Effect of Pax6 on Wnt signaling 31

Figure 11. Schematic representation of the role of Pax6 in inhibiting

Wnt signaling through Sost 32

LIST OF TABLES

Table 1. Primer sequences for RT-PCR and Real-time PCR 8

1

ABSTRACT

Pax6 expressed in osteocytes inhibits canonical wnt signaling

Ajita Jami

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Sung Kil Lim)

Pax6 is a transcription factor, which belongs to the paired box-containing gene

family. It regulates developmental processes, especially in the eyes, central nervous

tissues and craniofacial structures. However, the role of Pax6 in bone has never

been studied. This study report that Pax6 is expressed at both the mRNA and

protein level in the calvaria and long bones of adult mice as well as osteocyte-like

MLOY4 cells and suppresses the canonical Wnt signaling pathway. Moreover, the

expression levels of Pax6 were much higher in the calvaria than the long bones, and

Pax6 was also expressed at E16 to E18 in both the calvaria and long bones.

2

Knockdown of Pax6 in MLOY4 cells did not affect cell proliferation or survival;

however, the expression of Sost, a late osteocyte marker gene, was significantly

decreased. In addition, the overexpression of Pax6 suppressed the canonical Wnt

signaling pathway by enhancing the expression of Sost. Furthermore, also

demonstrated that Pax6 binds to the Sost promoter and that stimulation of Sost

transcription by Pax6 was dependent on a specific Pax6-binding sequence within

the promoter. In conclusion, the results of the present study suggest that Pax6 is

expressed in bone and may play an important role in osteocyte differentiation by

controlling canonical Wnt signaling.

Key words: Pax6, Osteocyte-like MLOY4 cells, MC3T3E1-cells, Sost, Wnt

signaling.

3

Pax6 expressed in osteocytes inhibits canonical wnt signaling

Ajita Jami

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Sung Kil Lim)

I. INTRODUCTION

Skeleton in vertebrates formed by two tissues namely, bone and cartilage1.

Osteocytes are terminally differentiated osteoblasts, derived from matrix producing

osteoblasts. Osteoblasts move along the bone surface before localizing to a targeted,

pre-determined location where they become embedded in the newly forming osteoid

bone matrix. Once embedded, their dendritic processes can extend and retract, and

the cell bodies are able to undulate within their lacunae2. Osteocytes play

4

multifunctional roles as mechanosensors, regulators of mineral homeostasis and

coordinators of bone remodeling processes carried out by osteoblasts and

osteoclasts3. Osteoblasts are differentiated into osteocytes through several stages

which include osteoblasts residing on the bone surface, osteoblastic osteocytes or

preosteocytes, osteoid osteocytes, and mature osteocytes embedded in a mineralized

matrix4. However, the molecular and cellular mechanisms governing osteoblast and

osteocyte differentiation are largely unknown.

Sclerostin, the product of the Sost gene, was previously identified as an important

negative regulator of bone formation in two rare bone sclerosing dysplasias,

Sclerosteosis and Van Buchem disease5. Sclerostin is a secretory glycoprotein that

plays a key role in the regulation of bone formation through Wnt signaling6.

Moreover, expression and secretion of sclerostin have been reported in terminally

differentiated osteocytes7. Bone formation was regulated by sequence of events

starting by the commitment of osteoprogenitor cells, their differentiation into pre-

osteoblasts and into mature osteoblasts whose function is to synthesize the bone

matrix, which was then mineralized. Recently cellular, molecular and genetic

analyses revealed that several transcription factors involved in the control of

osteoblastogenesis8,9,10,11. In this study, transcription factors that were upregulated

with Sost were analyzed using gene expression omnibus (GEO) analysis (in silico

analysis)12

. Pax6 was identified as one of the transcription factors that were

upregulated with the Sost gene in GEO analysis.

Pax6 belongs to the paired box-containing gene family, and acts as a transcription

factor regulating developmental processes13

. Pax6 is also known to regulate the

5

development of eyes, central nervous tissues and craniofacial structures14-17

. Pax6,

encoded by the Pax6 gene, contains two DNA-binding domains, the paired domain

(PD) and the homeo-domain (HD), as well as a transactivation domain. In

vertebrates, several isoforms of Pax6 are encoded by the Pax6 gene18-20

. Two major

isoforms are produced by the Pax6 gene by alternative splicing, Pax6(+5a) and

Pax6(–5a). Pax6(+5a) differs from Pax6(–5a) by the presence of an exon 5a-

encoded 14 amino acid insertion in its PD. These two isoforms demonstrate distinct

DNA-binding properties to the promoters of their downstream target

proteins13,18,19,21,22

. Pax6(-5a) has been shown to influence both cell proliferation and

neuronal differentiation, while Pax6(+5a) has only been shown to affect

proliferation23-26

. The canonical Wnt signaling pathway is involved in many

developmental processes27

. Moreover, Pax6 is known to play an important role in

lens morphogenesis through the inhibition of canonical Wnt/β-catenin signaling in

the lens surface ectoderm. Previously, Pax6 was shown to regulate the expression of

negative regulators of the Wnt signaling pathway, such as secreted frizzeled-related

protein1 (sfrp1) and secreted frizzeled-related protein 2 (sfrp2)28

. Although the

canonical Wnt signaling is known to play a role in deciding the fate of osteoblast

lineage cells, as well as in the regulation of bone mass and homeostasis29

, the role of

Pax6 in bone has never been reported.

In the present study, experimentally showed the expression of Pax6 in bone

tissues and MLOY4 established osteocyte-like cell lines, the regulation of the Sost

gene by Pax6, and its role in inhibiting Wnt signaling.

6

II. MATERIALS AND METHODS

1. Animal study

Embryos used in this study were obtained from time-mated adult imprinting

control region (ICR) pregnant mice. Embryonic day 0 (E0) was designated as the

day on which vaginal plugs were confirmed. Embryos at E16, E18 were used in this

study.

2. Cell culture

Osteocyte-like MLOY4 cells were cultured in alpha- Minimal Essential Medium

(α-MEM) (WelGENE Inc., Daegu, South Korea) containing 5% (v/v) fetal bovine

serum (FBS) (WelGENE Inc., Daegu, South Korea), 5% (5%) neonatal calf serum

(NCS) (WelGENE Inc., Daegu, South Korea) and 1% Penicillin-streptomycin

(WelGENE Inc., Daegu, South Korea) as described previously30,31

. Mouse primary

calvarial cells were cultured as described previously32

. Briefly, primary calvarial

cells (Primary-osteoblasts) were prepared from the calvaria tissue (which contains

primary-osteoblasts, mature osteoblasts, and osteocytes, which are engulfed in the

mineralized matrix) of neonatal mice by serial digestions with Type I collagenase

(Gibco BRL, Grand Island, NY, USA). In these serial digestions 3-5 digestion

fractions were collected, which contains primary calvarial cells. During the culture

period, cells were incubated at 370C in a humidified atmosphere of 5% CO2 and

95% air and the medium was changed for every 2 days. MC3T3-E1 cells were

cultured in α-MEM containing 10% (v/v) FBS and 1% penicillin/streptomycin.

7

3. Osteoblast differentiation

To induce osteoblast differentiation, MC3T3-E1 cells were supplemented with α-

MEM containing 50µg/mL ascorbic acid (Sigma, St. Louis, MO, USA) and 10mM

β-glycerophosphate (Sigma, St. Louis, MO, USA). The differentiation medium was

replaced for every two days.

4. Alkaline phosphatase (ALP) staining

To study the osteoblast differentiation, ALP staining was done using ALP

staining kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s protocol.

5. Reverse transcription-PCR (RT-PCR) analysis

For RT-PCR analysis, total RNA was extracted by using Trizol reagent

(Invitrogen, Grand Island, NY, USA) according to the standard protocol. cDNA

synthesis was performed by using MMLV-RT32

(Promega, Madison, WI, USA))

according to manufacturer’s protocol. In total, 1 µL of cDNA was used as the

template for PCR amplification of β- Actin, Pax6, Pax6 isoform, Sost, DMP1 and

E11 along with 10 pmole of primer and Go-Taq DNA polymerase (Promega,

Madison, WI, USA). Amplification was carried out at 95°C for initial denaturation,

followed by appropriate cycles at 95°C for 30 s (denaturation), 55°C (for β-Actin,

Pax6, Pax6 isoform and E11) or 57°C (for DMP1 and Sost) for 30 s (annealing) and

72°C for 30 s (polymerization). Pax6 isoform primers were designed in exon5a of

Pax6. Amplified products were resolved on a 1.7% agarose gel. Quantitative real-

8

time PCR was performed to measure the amount of Pax6, Sost, E11 and DMP1 with

the Applied Biosystems Real-time PCR system with SYBR green mix (Promega,

Madison, WI, USA). In this study, the same primers were used to amplify Pax6,

E11 and DMP1 for both real-time PCR and RT-PCR. Primer pairs were as follows

(Table 1).

Table 1. Primer sequences for RT-PCR and real-time PCR

Gene name Forward Reverse

RT-PCR

Pax6 5’-AGTTCTTCGCAACCTGGCTA-3’ 5’-GAGCCTCAATCTGCTCTTGG-3’

Pax6isoform 5’-TGCGACATTTCCCGAATT-3’ 5’-TCTGTCTCGGATTTCCCAAG-3’

Sost 5’-CCTATGACGCCAAAGATGTG-3’ 5’-GTTGTGGAAGCGGGTGAG-3’

E11 5’-AGTGTTGTTCTGGGTTTTGG-3’ 5’-GTTGTGGAAGCGGGTGAG-3’

DMP1 5’-AGTGAGGAGGACAGCCTGAA-3’ 5’-TCCCTGTGGAGTTGCTCTCT-3’

β-actin 5’-TTCAACACCCCAGCCATGT-3' 5’-TGTGGTACGACCAGAGGCATAC-3’

Real-time PCR

Gapdh 5’-AATGTGTCCGTCGTGGATCTG-3’ 5’-CAACCTGGTCCTCAGTGTAGC-3’

Sost 5’-TCCTGAGAACAACCAGACCA-3’ 5’-GCAGCTGTACTCGGACACATC-3’

9

6. Plasmid constructs and transfection

To clone Pax6(+5a) and Pax6(–5a), these two isoforms were amplified using

MLOY4 cDNA with the following primer sequences: forward sequence, 5’-

GCGGTGAGCAGATGTGTG-3’, and reverse sequence, 5’-TCTCCTTCTC TCTTTACTGTAATC-3’.

The PCR products were cloned into the T&A cloning vector (RBC Life Sciences,

Real Biotech Corporation, Taiwan) and subcloned into a pcDNA3.1 mammalian

expression vector (Invitrogen, Grand Island, NY, USA) after digestion with HindIII

((Promega, Madison, WI, USA) restriction enzyme to generate expression

constructs (i.e., pcDNA3.1-Pax6(+5a) and pcDNA3.1-Pax6(-5a)). Cells were then

seeded in 6-well plates and transfected with 0.8 µg of pax6 plasmid at 70-80%

confluency using Lipofectamine PlusTM

reagent (Invitrogen, Grand Island, NY,

USA). At 48 h after transfection, the cells were processed to determine mRNA and

protein expression levels of Pax6 and other osteocyte marker genes.

7. Sost reporter plasmid construction

The 1,811bp of the human Sost promoter (spanning nucleotides -1956 to -145bp

of the Sost gene locus relative to the position (+1) of the initiation methionine for

the Sost open reading frame) was amplified from liver cells with the following

primer sequences forward sequence (5’-TCTCCCCCGGGTGTGGATCATTTAGAGTTCAAG-

3’) and reverse sequence (5’-GCCCTAGATCTCCCAAAGACTTCTCCTCTAGCTC-3’). The

promoter was cloned into a pGL3-basic luciferase reporter vector after digestion of

the PCR product with SmaI and BglII (Promega, Madison, WI, USA) to construct

10

the pGL3-Sost promoter (wild type) plasmid. The primers used to generate

mutations in the transcription factor binding site were as follows (mutated

nucleotides in the primer sequence are indicated in bold letters): forward primer-5’-

CTCTGGGTCACCTGGAAGTACAAACAGCAATTTGGAAGTTTGC-3’; and the reverse primer-5’-

GCAAACTTCCAAATTGCTGTTTGTACTTCCAGGTGACCCAGAG-3’. A pGL3-Sost promoter

(mut) plasmid was generated by site direct mutagenesis using expand long range

dNTPack (Roche Diagnostics Corporation - Indianapolis, USA). PCR cycling

conditions were as follows: initial denaturation at 950C for 4min followed by

appropriate cycles (24cycles) at 950C for 45s (denaturation), 60

0C for 45s

(annealing) and 680C for 7min 15s (polymerization) and final extension at 68

0 C for

8 min. After PCR 5 l of the PCR reaction was digested with Dpn1 at 370 C for 1h

and 5 l was transformed into DH5 alpha competent cells to prepare the mutant

construct plasmids. All constructs sequence was confirmed by sequencing

(Cosmogenetech, Seoul, South Korea).

8. Transfection of cells with small interfering RNA

Control siRNA (sc-37007; Santa Cruz Biotechnology Inc., CA, USA) and Pax6

siRNA (sc-36196; Santa Cruz Biotechnology Inc., CA, USA) were used in this

study. MLOY4 cells were seeded into 12-well plates and transfected with 50 pmoles

of control siRNA or Pax6 siRNA using Lipofectamine RNAiMax reagent at 70%

confluency (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s

11

protocol. Cells were harvested 60 h after transfection. The siRNA activity was

evaluated by measuring Pax6 mRNA suppression.

9. Cell proliferation and cell number assay

MLOY4 cells were cultured in 12-well plates and transfected with Pax6 siRNA.

After 60 h, cell proliferation levels were determined using a cell counting kit-

8(Dojindo Molecular Technologies Inc., Kumamoto, Japan) according to the

manufacturer’s instructions. The cell number (viability) was determined by

measuring lactate dehydrogenase (LDH) activity with a Cytotox 96 nonradioactive

cytotoxicity assay (Promega, Madison, WI, USA). MLOY4 cells in 12-well plates

were transfected with 0.6 µg of pcDNA3.1 empty vector, pcDNA3.1-Pax6(–5a) or

pcDNA3.1-Pax6(+5a) plasmids using Lipofectamine PlusTM

reagent. After 48 h, the

effects of the Pax6 expression plasmids on cell proliferation and viability were

studied.

10. Western blot analysis

Calvaria and longbone tissues of adult mouse were collected to check the

expression of Pax6. To extract the proteins from tissues, tissue sections were

washed with PBS and homogenized with lysis buffer (50mM Tris, pH 7.5;

150mMNaCl; 1% Tween 20; 0.2%Nonidet P-40; 1mMphenylmethylsulfonyl

fluoride; 500mM NaF; 1mM Na3VO4; 10 µg/ml aprotinin; 2 µg/ml pepstatin A; and

10 µg/ml leupeptin). Proteins were extracted from the cells using the above lysis

12

buffer. The samples were centrifuged for 10 min at 12000 rpm at 40C and the

supernatant was collected for western blot analysis. Protein concentration from each

sample was determined by using Bradford reagent (Bio-Rad Laboratories, Hercules,

CA, USA) according to manufacturer’s protocol. Equal amount of proteins, about

25 µg were denatured at 1000C for 5 min. The extracted proteins were loaded and

separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes

(Millipore, Billerica, MA, USA). The membrane was blocked in TBST (TBS plus

0.1% Tween-20), containing 5% skim milk for 1hr at room temperature and

incubated overnight at 40C with an anti-Pax6 antibody (sc-53108; Santa Cruz

Biotechnology Inc., CA, USA), anti-Sost antibody (R&D systems, Minneapolis,

MN, USA), and anti-β-Actin antibody (sc-8432; Santa Cruz Biotechnology Inc., CA,

USA). After washing, the membrane was incubated with HRP-conjugated anti-

mouse, anti-goat immunoglobulin (Santa Cruz Biotechnology Inc., CA, USA) for 1

h at room temperature. Immunoreactive proteins on the membrane were visualized

with enhanced chemiluminescence (ECL) detection kits (Santa Cruz Biotechnology,

CA, USA).

11. Preparation of Wnt-3a conditioned medium

Wnt-3a conditioned medium was obtained from established L-Wnt-3a stable cell

lines (CRL-2647), which were purchased from ATCC (ATCC, Manassas, VA, USA).

Wnt-3a conditioned medium was prepared according to manufacturer’s protocol

(ATCC, Manassas, VA, USA).

13

12. Chromatin immunoprecipitation (Chip) assay

Pax6 binding element on Sost promoter (~2kb) was analyzed by using r-vista

software, which showed the putative Pax6 binding site (GGGAGTGCCAG) on the

Sost promoter. This sequence was found to be a conserved region between mice and

humans. For Chip assay, MLOY4 cells were cross-linked with 1% formaldehyde for

10 min at room temperature. Cross linking was stopped by using 0.125M glycine,

followed by washing with ice-cold PBS. Cells were resuspended in 1 ml Chip lysis

buffer. After cell lysis, chromatin extracts were prepared and fragmented by

sonication on ice. Sonication product (approximately 500 bp) was used as input

after reverse cross-linking. Protein A/G beads (Santa Cruz Biotechnology Inc., CA,

USA) were equilibrated with RIPA buffer and added to 50g of chromatin extract

for preclearing. DNA-protein-antibody complexes were prepared using anti-Pax6

antibody (sc-53108; Santa Cruz Biotechnology Inc., CA, USA) or normal mouse

IgG antibody (sc-2026; Santa Cruz Biotechnology Inc., CA, USA) as a control

following the Chip protocol (Abcam, Cambridge, MA, USA) with minor

modifications. After precipitation of the chromatin, bound DNA was resuspended in

water and used as a template for PCR using Sost Chip primers, forward 5’-

TCTGAAAACCACAGCCTGAC-3’; and reverse 5’-GTTTCCTCACCCTCCTCCTC-3’ to amplify

the conserved Pax6 binding site of mouse Sost promoter(-317 to -87 bp upstream to

translation initiation site). For the control, Chip primers were designed in the other

region of mouse Sost promoter (-1910 to -1676 bp), which do not contain binding

element. The control Chip primers are as follows: forward primer 5’-

14

TCTCCCAAGTCTGGAGCAAT-3’, and reverse primer 5’-GCAGACAGAAGTCCCCTCAG-

3’.

13. Luciferase reporter assay for monitoring Sost promoter activity and Wnt

signaling

To study the role of Pax6 on Sost promoter activity MLOY4 cells were plated in

24-well plate at a density of 55000cells/well. When cultures were 70-80% confluent

cells were co-transfected using Lipofectamine PlusTM

reagent with the indicated

combinations of expression and Sost promoter plasmids. To study the role of Pax6

in Wnt signaling, MLOY4 cells were plated in 12-well plate at a density of 1.1×105

cells/well and subsequently co-transfected using Lipofectamine PlusTM

reagent with

the indicated combinations of expression and reporter plasmids. In the Wnt

signaling study, the Super TOP Flash plasmid (S-Top) (Clontech Laboratories, Inc.,

Palo Alto, CA, USA), which is driven by T-cell factor (TCF)/lymphoid enhancer

factor (LEF)-binding elements and is responsive to Wnt signaling was co-

transfected with Pax6 expression plasmids, and 24 h after transfection, the cells

were treated with 100 µL of Wnt-3a conditioned medium. At 48 h after transfection,

cell lysates were prepared, and luciferase assays were performed using the Dual-

Luciferase Reporter Assay system (Promega, Madison, WI, USA) according to the

manufacturer’s protocol. Assays were normalized for transfection efficiency by co-

transfecting with 2.5 ng of pRL-tk renilla luciferase reporter plasmid.

15

14. Induction of Pax6 expression by Wnt-3a treatment

MLOY4 cells were seeded in 12-well plates and treated with 100 µL of Wnt-3a

conditioned media. After treatment Pax6 and Sost mRNA expressions were

measured at different time intervals.

15. Immunohistochemistry and HE staining

For immunohistochemical staining, mandibles from mice at each stage were fixed

with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4)

overnight at 4°C and demineralized with 10% ethylenediaminetetraacetic acid (pH

7.4) for 2 weeks at 4°C. After being embedded in paraffin, these samples were

sectioned to a thickness of 6 μm. Sections were then blocked in 3% hydrogen

peroxide for 15 min. The tissue sections were boiled in 10 mM citrate buffer (pH

6.0) for 10 min and cooled at room temperature for 20 min. The immunostained

sections were then counterstained with hematoxylin. These sections were used for

immunohistochemistry. Specimens were incubated with a primary mouse

monoclonal antibody against Pax6 (Santa Cruz) at 4°C overnight. After washing

with PBS, the specimens were incubated with a secondary antibody at room

temperature. The specimens were visualized using a 3,3’-diaminobenzidine (DAB)

reagent kit (Zymed laboratories, South San Fransisco, CA, USA).

16

16. Statistical analysis

Statistical significance was evaluated using Sigma Plot10 software. The results

were calculated and expressed as the mean ± standard deviation (SD). Statistical

significance was determined using Student’s t-test. P-values ≤0.05 were considered

to be statistically significant.

17

III. RESULTS

1. Endogenous expression of Pax6 in various types of bone cells and tissues

Endogenous expression of Pax6 in bone cells and tissues was studied. The

expression of Pax6 was investigated in primary calvarial cells (early digestive

fractions of calavarial tissue, which also called as primary-osteoblast cells

(represented in materials and methods section)), MC3T3-E1 cells and osteocyte-like

MLOY4 cells at the mRNA level. Pax6 mRNA was expressed at high levels in

osteocyte-like MLOY4 cells compared to those in primary calvarial cells and

MC3T3-E1 cells (Fig. 1A and 1B). Alternative splicing of Pax6 is known to result

in two major Pax6 isoforms, both of which detected in MLOY4 cells (Fig. 1B). In

addition, our western blot analysis demonstrated the in vivo expression pattern of

Pax6 in calvaria and long bone tissues of adult mice (Fig. 1C). Pax6 expression at

the protein level in MLOY4 cell lysates was confirmed by loading Pax6(–5a)- and

Pax6(+5a)- overexpressing cell lysates. The Pax6(–5a) isoform was found in

MLOY4 cells (Fig. 1D). To examine the expression of Pax6 during osteoblast

differentiation, MC3T3-E1 cells were differentiated using osteoblast differentiation

media. Differentiation of osteoblast cells was shown by ALP staining (Fig. 1E). The

expression of Pax6 was gradually increased as the differentiation progressed (Fig.

1F). Immunohistochemical studies showed the expression of Pax6 in osteoblasts

and osteocytes in parietal bone region of calvaria sections at E16 and E18

embryonic stages. Whereas the long bone sections of E16 and E18 showed

18

expression of Pax6 in both osteoblasts and osteocytes. Calvaria at E16 and E18

stages showed comparatively higher expression than long bone (Fig. 2).

19

Figure 1. Endogenous expression of Pax6 in bone cells and tissues.

A) RT-PCR analysis of Pax6 expression in mouse primary calvarial cells,

MC3T3-E1 pre-osteoblast cells and osteocyte-like MLOY4 cells. (B)

RT-PCR analysis of the endogenous expression of the two major

i s o f o r ms o f P a x 6 [ P a x 6 ( + 5 a ) a n d P a x 6 ( – 5 a ) ] . T h e i r r e l a t i v e

expressions are shown in the bar graph [Pax6–5a (black bar), Pax6+5a

(gray bar)]. (C) Endogenous Pax6 expression in calvaria and long bone

tissues was analyzed with western blotting and normalized to beta-actin

expression. (D) Endogenous expression levels of Pax6 in MLOY4 lysates

were compared with the overexpression of Pax6(–5a) and Pax6(+5a). (E)

MC3T3-E1 cells differentiation was confirmed by ALP staining . (F)

Pax6 expression was increased after induction of MC3T3-E1 cells with

differentiation medium.

20

Figure 2. Immunohistochemical analysis of endogenous Pax6 expression.

Immunohistochemical analysis of endogenous Pax6 expression in the calvaria

and long bone tissues at embryonic stage 16 and 18. Brown color stained

positive cells for Pax6 are indicated by arrows in each embryonic stages.

Osteoblasts are indicated by red arrows, osteocytes are indicated by black

arrows. Scale bar=50µm.

21

2. Effect of Pax6 on Wnt inhibitor Sost and other osteocyte markers

The effect of Pax6 on osteocyte marker genes was studied via the knockdown or

overexpression of Pax6 in MLOY4 cells. The gel images and semi-quantitative RT-

PCR and real-time PCR data showed a significant knockdown of Pax6 (Fig. 3A and

3B). Knockdown of Pax6 significantly decreased the expression of Sost (Fig. 3A

and 3C). In contrast, the overexpression of Pax6(–5a) and Pax6(+5a) in MLOY4

cells resulted in an significant increase in the expression of Sost, which is a mature

osteocyte marker gene; meanwhile, there were no changes in the other marker genes,

as observed with RT-PCR and real-time PCR (Fig. 3D-3G). Together, these results

suggest that osteocyte-specific expression of Pax6 might be involved in the

transition of pre-osteocytes to mature osteocytes and may also play an important

role in inhibiting Wnt signaling through enhanced Sost expression. In addition to

these results, studied the effect of Pax6 on the proliferation and cell number

(viability) of MLOY4 cells. Present study showed that neither knockdown nor

overexpression of Pax6 in MLOY4 cells had a significant effect on proliferation or

cell number (Fig. 4A-4D).

22

Figure 3. The effects of Pax6 on osteocyte marker genes.

The expression of osteocyte marker genes was analyzed with RT-PCR or real-time

PCR. (A-C) Effect of siPax6 on osteocyte marker genes. RT-PCR and real-time

PCR analysis showed decreased expression of Sost and increased expression of

DMP-1 and E11 in siPax6 transfected cells compared to control siRNA transfected

cells. (D-G) Effect of Pax6 overexpression on osteocyte marker genes. RT-PCR and

real time PCR analysis showed significantly increased expression of Sost by Pax6

overexpression compared to empty vector transfection.

23

Figure 4. Effect of Pax6 on proliferation and Cell viability.

(A-B) Effect of siPax6 on cell proliferation and cell number. There is no effect on

cell proliferation and cell number in cells treated with siPax6 compared to control

siRNA. (C) Represents the effect of overexpression of Pax6(+5a) or Pax6(–5a)

expression plasmids on cell proliferation compared to empty vector transfection.

(D) Represents the effect of transient transfection of Pax6 expression plasmids on

cell number compared to control vector. *Indicates a p-value ≤0.05 relative to the

control.

24

3. Effect of Pax6 on Sost promoter activity in MLOY4 cells

Pax6 binding site on the Sost promoter (~2kb) was found by insilico analysis (Fig.

5A and 5B). This was further confirmed by Chip-PCR analysis. Pax6-bound

chromatin was isolated from MLOY4 cells and immunoprecipitated with anti-Pax6

antibody. The Pax6-bound DNA fragment shows amplification with specific primer

set (Fig. 6A), whereas the control primers designed in the other region shows no

amplification (Fig.6B). This result confirmed the specificity of Pax6 binding site on

the Sost promoter. Since found that Pax6 regulates the expression of the Sost gene,

further investigated the possible mechanism of Pax6 on Sost expression. To confirm

the regulatory mechanism, 1,811bp of the human Sost promoter containing the

Pax6-binding site (-1956 to -145 bp) was cloned into a pGL3-basic luciferase

reporter vector (Fig. 7) by digestion with SmaI and BglII restriction enzymes, and

evaluated the ability of this promoter fragment to drive luciferase expression in

MLOY4 cells. Co-transfection of Pax6(+5a) or Pax6(–5a) plasmids with the wild

type and mutant pGL3-Sost-promoter vectors led to a significant increase in

promoter activity after overexpression of Pax6 in the wild type promoter and a

decrease in activity in the mutant promoter in the presence of both isoforms of Pax6

(Fig. 8). Since the Pax6 could not bind to the mutated region of the promoter, hence

there was a significant decrease of reporter activity after overexpression of both

forms of Pax6 compared to the wild type promoter (Fig. 8). This result suggested

that Pax6 regulates Sost expression by binding to its promoter region specifically.

25

Figure 5. The Sost promoter sequence analysis (2kb region).

(A) Sequence analysis of the 2 kb upstream regulatory region of human Sost

promoter from transcription start site (2kb region shown here). Putative Pax6

conserved binding site was represented in blue colour. The primers used for cloning

of Sost promoter were represented in brown colour. (B) The conserved Pax6

binding site.

26

Figure 6. Chip PCR analysis in MLOY4 cells.

A) PCR amplification was performed by using Pax6 binding element region specific

primers (primers indicated in blue colour) and B) negative control primers (primers

indicated in violet colour) as described in the materials and methods section. This

result confirms the specificity of Pax6 binding element on Sost promoter.

27

Figure 7. Schematic diagram of the Sost promoter-reporter construct.

The Sost promoter region was cloned into SmaI and BglII sites in pGL3-basic

vector (Promega) to make the Sost promoter-reporter construct.

28

Figure 8. The effect of Pax6 on Sost promoter activity.

MLOY4 cells were transfected with the indicated combinations of the wild type and

mutant Sost promoter and Pax6 expression plasmids. After 48 h, the relative

luciferase activity was measured. *Indicates a p-value ≤0.05 relative to the control.

29

4. Effect of Wnt-3a on Pax6 mRNA expression in MLOY4 cells

Sost is a well known negative regulator of Wnt signaling. Because Pax6

regulates Sost expression, then investigated the effect of Wnt-3a on Pax6 mRNA

expression in MLOY4 cells. Interestingly, found that Wnt-3a stimulated the

expression of Pax6 mRNA at different time points, which further stimulated the

expression of Sost mRNA (Fig. 9).

Figure 9. Effect of Wnt3a on Pax6 mRNA expression.

MLOY4 cells were treated with Wnt-3a conditioned media (100 µL/well), and the

effect on Pax6 and Sost mRNA expression was measured at different time points

with RT-PCR.

30

5. Effect of Pax6 on Wnt signaling in MLOY4 cells

In addition, studied the effects of Pax6 on the S-Top reporter vector, which is

driven by T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding elements

and is responsive to Wnt signaling. However, the co-expression of the S-Top

plasmid with Pax6 isoforms showed an inhibition of Wnt/β-catenin signaling in

MLOY4 cells (Fig. 10A). Here also showed overexpression of pax6 and its effect

on Sost expression in protein level through western blot analysis (Fig. 10B). These

results suggested that Pax6 plays an important role in the inhibition of Wnt

signaling mediated by the Sost gene in MLOY4 cells. Additionally, found that Wnt-

3a enhanced Pax6 expression. In turn, overexpression of Pax6 inhibited Wnt

signaling by increasing the expression of Sost, which is a potent inhibitor of Wnt

signaling. Pax6 expression was increased according to further differentiation of

osteoblasts. The role of Pax6 was represented schematically (Fig. 11).

31

Figure 10. Effect of Pax6 on Wnt signaling.

A) MLOY4 cells were transfected with the indicated combinations of the Super

TOPFlash reporter DNA and Pax6 expression plasmids. After 48 h, the relative

luciferase activity was measured. *Indicates a p-value ≤0.05 relative to the control.

B) Pax6 expression was confirmed by western blot analysis.

32

Figure 11. Schematic representation of the role of Pax6 in inhibiting Wnt

signaling through Sost.

33

IV. DISCUSSION

Canonical Wnt/β-catenin signaling plays important role in skeletal development,

tissue regeneration and bone formation33

. Postnatally, it also regulates bone

homeostasis and bone mass34

. Sost encodes sclerostin, which is specifically

expressed by osteocytes and acts through a network of osteocyte canaliculi to reach

the surface of the bone35,36

. Sclerostin inhibits canonical Wnt signaling by binding

to LRP5,637

.

In our GEO analysis, Pax6 was found to be a transcription factor that was

upregulated with the Sost gene (data not shown). Pax6 acts as an important

regulator in the development of the eye and central nervous tissues16,17

. Pax6 is

abundantly expressed in developing eyes, the forebrain, the ventral spinal card and

the endocrine pancreas8. Homozygous mutant mice died at E6 to E8 before somite

formation. Pax6 heterozygote mice have a small body size17

. A Pax6-homozygous

human fetus with aniridia showed an absence of nasal bones and defects in parietal

bones at necropsy38

. However, the role of Pax6 in bone has never been studied.

Therefore, this study hypothesized that Pax6 might play an important role in bone

development.

In this paper, showed that compared with the expression of Pax6 in cultured

primary calvarial cells or established MC3T3-E1 preosteoblast cells, Pax6 was

exclusively expressed in MLOY4 osteocyte-like cells (Fig. 1A). Two major

isoforms of endogenous Pax6 [Pax6(–5a) and Pax6(+5a)] were detected at the

mRNA level in bone cells (Fig. 1B). However, this study showed that Pax6(–5a)

34

was the major Pax6 protein expressed in bone (Fig. 1D). Pax6 was expressed

exclusively in the calvaria and long bone tissues at E16 and E18 (Fig. 2). Pax6 has

been reported to be essential for the proliferation and the pluripotency of retinal

progenitors39

. The proliferation and apoptosis of human retinoblastoma cells are

regulated by Pax640

. To evaluate the role of Pax6 in bone, overexpression and

knock down of Pax6 in MLOY4 cells was performed. In this study, neither

knockdown nor overexpression of Pax6 exerted any effects on the proliferation or

survival of MLOY4 cells (Fig. 4A-D). Additionally, observed that the knockdown

of Pax6 decreased the expression of Sost mRNA, which is a late osteocyte marker

gene, and increased the expression of E11 and DMP1 (Fig. 3A-3C). After transient

transfection with Pax6(+5a) or Pax6(–5a) DNA, the expression of Sost was

significantly increased (Fig. 3D and 3E). The induction of Sost expression by Pax6

was further supported by the observation of enhanced luciferase reporter activity

flanked by the 1811 bp promoter region of the Sost gene after overexpression of

each isoform of Pax6 (Fig. 8). In other studies, Pax6 was found to play an important

role in lens fiber cell and neuronal cell differentiation41,42

. In this study, observed

that the gradual increase of Pax6 mRNA during the differentiation of pre-osteoblast

cells into mature osteoblasts (Fig. 1E and 1F). All these data suggest that Pax6

might play an important role in the differentiation of osteoblasts and osteocytes.

In chicken telencephalic development, Wnt-3a induced the expression of the

dorsal markers Pax6 and Ngn243

. Moreover, Pax6 was shown to play an important

role in lens morphogenesis through the inhibition of canonical Wnt/ β-catenin

35

signaling in the lens surface ectoderm28

. In this study, also observed the induction of

Pax6 expression in MLOY4 osteocyte-like cells after treatment with Wnt-3a (Fig.

9). In addition, Wnt-3a/β-catenin reporter activity was significantly reduced by the

overexpression of both Pax6(+5a) and Pax6(–5a) along with the co-transfection of

the S-Top reporter vector in osteocyte-like MLOY4 cells (Fig. 10A). After transient

transfection with Pax6(–5a) or Pax6(+5a) DNA, the expression of Sost increased

significantly (Fig. 3D and 3E). Additionally, our in silico analysis showed that

mouse and human Sost promoters exhibited a conserved, putative Pax6-binding

element (GGGAGTGCCAG) at the 2kb upstream region of the Sost promoter.

Overexpression of Pax6(+5a) or Pax6(–5a) along with the pGL3-Sost promoter (wt)

and the pGL3-Sost promoter (mut) DNA revealed that mutant promoter activity was

significantly lower than wild type promoter activity in the presence of both isoforms

of Pax6 (Fig. 8). All of these findings suggested that Pax6 inhibits canonical Wnt

signaling through the stimulation of Sost by binding to its promoter. Canonical Wnt

signaling has been shown to stimulate osteoblast differentiation and inhibit the

terminal differentiation of mature osteoblasts and apoptosis44

. In the present study,

knockdown of Pax6 increased the expression of E11 and DMP1, and decreased the

expression of Sost. In addition, Pax6 expression was increased according to further

differentiation of osteoblasts. Therefore, speculated that Pax6 may be an important

transcription factor in stimulating further differentiation of mature osteoblasts to

osteocytes. The potential role of Pax6 is schematically represented in Fig.10.

36

V. CONCLUSION

The present study showed that the transcription factor Pax6 is expressed in the

osteocyte-like MLOY4 cells, calvaria and long bone tissues. Pax6 expression was

observed during the terminal differentiation of MC3T3E1- cells. Overexpression of

Pax6 increased the expression of Sost, a late osteocyte marker gene. By direct

binding to the putative Pax6 binding sequence of the Sost promoter region, Pax6

suppressed the canonical Wnt signaling pathway. Further exploration of phenotype

in osteocyte-specific Pax6 knockout mice could provide more definitive evidence of

the role of Pax6 in differentiation of mature osteoblasts to osteocytes.

37

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45

ABSTRACT (IN KOREAN)

Osteocyte에서 발현하는 Pax6에 의한 canonical wnt signaling 억제

연세대학교 대학원 의과학과

아지타 자미

Pax6는 paired box-containing domain을 갖는 전사인자로서, 눈, 중추신경조

직, 두개구조의 발달과정을 조절한다. 그러나 뼈에서는 아직까지 Pax6의 역할

이 정확하게 알려지지 않았다. 본 연구에서는 성체 마우스의 두개관(calvaria)

과 대퇴골, MLOY4 세포주에서 Pax6가 발현되며 canonical Wnt 신호전달체계

가 억제됨을 확인하였다. 또한 Pax6는 두개관과 대퇴골의 E16, E18 단계에서

모두 표현되었고, 대퇴골에서보다 두개관에서 Pax6의 발현이 더 높았다.

46

MLOY4 세포주에서 Pax6를 knockdown 하였을 때 세포증식에는 영향을 미치

지 않았으나, late osteocyte marker인 Sost가 현저히 감소함을 확인하였다. 또한,

Pax6를 과발현하였을 때에는 E11및 DMP1표현의 변화는 관찰되지 않았지만,

Sost의 표현이 증가되었고, Sost에 의한 canonical Wnt 신호전달체계가 억제됨

을 관찰할 수 있었다. 본 연구에서는 이것을 증명하기 위해 Pax6가 Sost의 특

정 부위 (-GGGAGTGCCAG-)에 결합함으로써 Sost의 전사과정을 조절함을 보

였다. 이로써 Pax6가 뼈에서도 canonical Wnt신호전달체계를 조절하고 골세포

(osteocyte)의 분화에 중요한 역할을 함을 알 수 있었다.

핵심되는 말 : Pax6, Osteocyte-like MLOY4 cells, MC3T3E1 cells, Sost, Wnt signaling

47

PUBLICATION LIST

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Beniwal, Madan Lal , Gajender S Meena. Prevalence of low glomerular

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2. Kyoung Min Kim, Su Jin Park, Seung-Hyun Jung, Eun Jin Kim,

Jogeswar Gadi, Ajita Jami, Yumie Rhee, Cheol-Hee Kim, Sung-Kil Lim.

miR-182 is a Negative regulator of osteoblast proliferation,

differentiation, and skeletogenesis through targeting FoxO1. JBMR 2012;

27(8):1669-1679.

3. Ajita Jami, Jogeswar Gadi, Min Jung Lee, Eun Jin Kim, Mi Jeong Lee,

Han-Sung Jung, Hong-Hee Kim, and Sung-Kil Lim. Pax6 expressed in

osteocytes inhibits canonical wnt signaling. Mol Cells 2013 April; 35(4):

305-12.