Pathogenic role of bone

morphogenetic proteins in

syndesmophytosis of ankylosing

spondylitis

Min-Chan Park

Department of Medicine

The Graduate School, Yonsei University

Pathogenic role of bone

morphogenetic proteins in

syndesmophytosis of ankylosing

spondylitis

Directed by Professor Soo-Kon Lee

The Doctoral Dissertation submitted to the Department of Medicine, the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree

of Doctor of Philosophy

Min-Chan Park

June 2008

This certifies that Doctoral Dissertation of Min-Chan Park is

approved.

-----------------------------------------

Thesis Supervisor: Soo-Kon Lee

-----------------------------------------

[Jong Eun Lee: Thesis Committee Member#1]

-----------------------------------------

[Jin Woo Lee: Thesis Committee Member#2]

-----------------------------------------

[Man-Wook Hur: Thesis Committee Member#3]

-----------------------------------------

[Jongsun Kim: Thesis Committee Member#4]

The Graduate School Yonsei University

June 2008

ACKNOWLEDGEMENTS

First of all, I would like to extend my deep gratitude to my

mentor, Professor Soo-Kon Lee. This study would not have

been finished without his faith and passion. His faith in this

study was great shouts of encouragement for me and his

passion will become a milestone in my life-time career as a

researcher and medical doctor, as well as a man with

soundness. I shall endeavor to return his support with ‘tour

d'ivoire’.

I wish to acknowledge Professor Jong Eun Lee, who helped

me to perform the long-time consuming experiments with

pleasure. I learned research-oriented point of view and

academic attitude from her. I also acknowledge Professor Jin

Woo Lee, Professor Man-Wook Hur, and Professor Jongsun

Kim for their excellent ideas and valuable suggestions. They

gave me prudent advises and helped me to expand my

knowledge and perspective on graduate study.

I wish to thank sincerely to Professor Yong-Beom Park. He

always has been beside me and drives me to pursue the truth.

In the last place, I am very grateful to my parents, my wife

and my children for their never-ending love and support

during my study and life. I feel so lucky to have such a

beautiful family.

TABLE OF CONTENTS

ABSTRACT ································································································1

I. INTRODUCTION ·····················································································3

II. MATERIALS AND METHODS ·································································6

1. Animal experiments ·······································································6

2. Immunohistochemistry ···································································8

3. Western blot analysis ·································································8

4. Statistical analysis ··········································································9

III. RESULTS ······························································································10

1. Differential BMPs expression in sacroiliits ·································10

2. Effect of noggin gene transfer on sacroiliits and arthritis ··············12

3. Effect of noggin gene transfer on BMP signalling ······························20

IV. DISCUSSION ·························································································22

V. CONCLUSION ················································································25

REFERENCES ·····················································································26

ABSTRACT (IN KOREAN) ·········································································30

LIST OF FIGURES

Figure 1.

Smad signaling of BMPs and action mechanism

of noggin ········································································

5

Figure 2. H&E staining of sacroiliac joint section ·······················10

Figure 3.

Immunohistochemistry for BMP expressions in

sacroiliac joint section ···················································

11

Figure 4. Expression of noggin after cDNA transfer ···············13

Figure 5.

Effect of noggin gene transfer on clinical severity

of arthritis ·······································································

16

Figure 6.

The effect of noggin gene transfer on pathology

of sacroiliitis ·····································································

17

Figure 7.

Effect of noggin gene transfer on pathologic

severity of sacroiliac joint ··········································

18

Figure 8.

Effect of noggin gene transfer on BMP signaling

in ankylosing enthesitis ···················································

21

LIST OF TABLES

Table 1. Clinical and histological assessment ·······························7

Table 2.

Effect of noggin gene transfer on different features

of ankylosing enthesitis ························································

19

1

<ABSTRACT>

Pathogenic role of bone morphogenetic proteins in

syndesmophytosis of ankylosing spondylitis

Min-Chan Park

Department of Medicine

The Graduate School, Yonsei University

(Directed by Professor Soo-Kon Lee)

Objective. Syndesmophytosis is a major cause of disability in patients with

ankylosing spondylitis (AS) and bone morphogenetic protein (BMP) signaling,

which plays a major part in ossification processes and the related growth and

differentiation factors influence tendon and ligament formation, appears to be

a key molecular pathway involved in this pathological cascade. This study

was performed to investigate the role of BMPs in enthesial inflammation and

syndesmophytosis of proteoglycan (PG)-induced arthritis mice.

Methods. Sacroiliitis was induced in BALB/c mice by immunization with

100 µg of human cartilage PG in complete Freund's adjuvant. Full-length

mouse noggin cDNA, a BMP antagonist was cloned into pcDNA3.1+ vector.

Along with sacroiliitis induction, either 30 or 300 µg of pcDNA3.1+ noggin

or empty vector was injected into the tibialis anterior muscles at week 12, 15,

18, and 21. A group of mice were sacrificed at week 21 to see the findings of

the early stages of the disease and the others were sacrificed at week 35 to see

those of the late stages. Assessments for incidence and severity of sacroiliitis

2

and peripheral arthritis were performed, and differential expressions of

BMP-2, BMP-6, and BMP-7, and expression smad1/5, which are involved in

BMP signaling, were investigated.

Results. On microscopic examination, inflamed synovium invading the

sacroiliac joint surface was seen at the early stages and new bone formation

was seen at the sacroiliac joints surface at the late stages. Different BMP

expressions were detected in distinct stages of sacroiliitis of the mouse model.

BMP-2 was found in fibroblast-like cells at early stages and BMP-7 was seen

in round hypertrophic chondrocytes at later stages. BMP-6 was not detected in

the joint tissues. Both noggin gene transferred groups (30 and 300 µg

pcDNA3.1+ noggin) showed significant reductions in incidence and severity

of sacroiliitis and peripheral arthritis. Noggin gene transfer reduced new bone

formation and resulted in a significant reduction in phosphorylated-smad1/5

compared to those treated with empty pcDNA3.1+.

Conclusion. This study showed that BMP-2 and BMP-7 are differentially

expressed in sacroiliitis of PG-induced arthritis mice and that noggin gene

transfer reduced new bone formation by down-regulating the smad pathway.

These data suggest that blocking BMP signaling might be used as an

alternative therapeutic approach in AS.

Key words: Ankylosing spondylitis, Syndesmophytosis, Bone morphogenetic

proteins, Noggin, Proteoglycan-induced arthritis

3

Pathogenic role of bone morphogenetic proteins in

syndesmophytosis of ankylosing spondylitis

Min-Chan Park

Department of Medicine

The Graduate School, Yonsei University

(Directed by Professor Soo-Kon Lee)

I. INTRODUCTION

Enthesial inflammation and syndesmophytosis, a process of pathologic new

bone formation, are the hallmarks of ankylosing spondylitis (AS), and an

autoimmune mechanism seems to be responsible for this phenomena. A

previous study revealed the presence of T cells and mononuclear cells and

separate areas with high levels of tumor necrosis factor-α and transforming

growth factor (TGF)-β in sacroiliac joint tissue from patients with AS, with

the latter occurring in areas of pathologic new bone formation1. Moreover, the

deposition of matrix proteins and ossification is commonly encountered in

patients with AS, and in advanced stages, syndesmophytosis typically ascends

the spine, resulting in ankylosis of the spine2, 3.

It is well established that bone morphogenetic proteins (BMPs) are

involved in the process of matrix synthesis and turnover. BMPs, members of

the TGF-β superfamily, are primarily synthesized by osteoblasts and

fibroblasts. They initiate a complex cascade of events leading to

post-inflammatory healing and new bone formation 4-6. In varying degrees,

4

BMPs induce chemotaxis, mitosis, and differentiation, all key features of the

cascade of events associated with post-inflammatory healing7. Certain BMPs

play a major part in ossification processes, whereas the related growth and

differentiation factors influence tendon and ligament formation8. It has been

reported that BMP-2 and BMP-6 promote callus during healing processes

after bone fracture and BMP-2 also enhances tendon-to-bone healing. But the

presence of BMP-7 appears to be inappropriate for simple tissue repair and it

strongly induces pathologic new bone formation and bone remodeling9.

In their canonical pathway, BMPs induce ligand-dependent type I and type

II receptor heterodimerization, leading to phosphorylation of smad-signaling

molecules (smad1/5) that bind smad4 and translocate to the nucleus (Figure 1).

BMP signaling is controlled at many levels, including that of extracellular

antagonists10. The most investigated molecules that are able to influence BMP

signaling extracellularly are noggin, a BMP antagonist. Noggin binds to BMP

receptors competitively and inhibits their effect (Figure 1)11. Activation of

smad-signaling pathways has been suggested to be an essential part of repair

and homeostasis in health and disease12, 13 and, until now, studies on the role

of BMPs have focused primarily on fracture healing, osseous fusion, and

healing of bony defects14, 15. However, untimely or unwanted activation of

signaling cascades fundamental for normal development may promote disease

processes such as AS, but the role of BMPs in syndesmophytosis of AS has

not been investigated.

Therefore, a hypothesis that BMP signaling could play a direct role in

syndesmophytosis of AS was presumed. Thus, the differential expression of

BMPs and their signaling in a proteoglycan (PG)-induced spondylitis model,

which provides a tool for studying the mechanisms of syndesmophytosis with

potential relevance to the human AS16, 17, were studied in this study.

Furthermore, the effects of blockade for BMP signaling on ankylosing

enthesitis and syndesmophytosis by gene transfer of noggin were investigated.

5

Figure 1. Smad signaling of BMPs and action mechanism of noggin. An

extracellular BMP antagonist, noggin, binds to BMP receptors competitively

and inhibits the effect of BMPs.

6

II. MATERIALS AND METHODS

1. Animal experiments

The human cartilage for antigen isolation was obtained during from joint

replacement operations. Human cartilage PG was used for immunization of

24-26 week-old female BALB/c mice. Preparation of cartilage PG was carried

out as described earlier18, 19. As a standard method, the first antigen injection

(100 µg of PG protein) was given in complete Freund's adjuvant and the same

doses of antigen were injected as second and third boosts in incomplete

Freund's adjuvant at week 3 and 6 (n = 20). Control female BALB/c mice

matched for age were or were not immunized with ovalbumin. Full-length

mouse noggin cDNA (a gift from F. P. Luyten, Katholieke Universiteit

Leuven, Leuven, Belgium) was cloned into pcDNA3.1+ vector (Invitrogen,

Carlsbad, CA, USA). Either 300 or 30 µg of pcDNA3.1+ noggin or empty

pcDNA3.1+ was injected into the tibialis anterior muscles at week 12, 15, 18,

and 21. A group of mice were sacrificed at 21 weeks to see the changes of

enthesial inflammation in the early stages and the other were sacrificed at 35

weeks to see the changes for pathologic new bone formation in the late stages.

Clinical and histological assessments for enthesitis are summarized in Table

1. Briefly, mice were scored for clinical signs of arthritis twice a week as

follows20, 21: 0, no signs; 1, redness and/or swelling in 1 toe; 2, redness and/or

swelling in more than 1 toe; 3, stiffness of any toe; and 4, deformity of any toe

or ankle involvement. Both hind paws were evaluated, resulting in a

maximum score of 8. For histological examination, hind paw forefeet were

formalin-fixed, decalcified and a histological score was measured as reported

previously21, 22: 1, infiltration of inflammatory cells; 2, fibroblast-like cell

proliferation; 3, cartilage formation; 4, bone formation; and 5, total ankylosis

of joints.

7

Table 1. Clinical and histological assessment

Clinical assessment for arthritis

0 No signs for arthritis

1 Redness and swelling in one toe

2 Redness and swelling in more than one toe

3 Toe stiffness, any

4 Deformity or ankle involvement

Histological assessment for sacroiliitis and arthritis

0 Normal

1 Inflammatory cell infiltration

2 Fibroblast-like cell proliferation

3 Cartilage formation

4 Bone formation

5 Ankylosis

8

2. Immunohistochemistry

Joint sections were quenched with 3% H2O2/H2O and pre-incubated with

donkey serum [20% in Tris-buffered saline (TBS), R&D Systems,

Minneapolis, MN, USA). Joint sections were incubated with polyclonal

primary antibodies against BMP-2 (Pfizer, New London, CT, USA) (5

µg/mL), BMP-6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (10

µg/mL), BMP-7 (Pfizer) (10 µg/mL), phosphorylated smad1/5 (a gift from F.

P. Luyten) (1/100) overnight at 4°C. Anti-BMP-2 antibody was made against

peptide sequence Ac-REKRQAKHKARKRLKSSC-NH2. Anti-BMP-6

antibody was made against peptide sequence from the amino-terminus of

human BMP-6. Anti-BMP-7 antibody was raised against peptide sequence

Ac-TGSKQRSQNRSKTPKNC-NH2. Anti-phosphorylated smad1/5 antibody

recognizing the phosphorylated C-terminus in smad1/5 was raised by

injection of peptide KKK-NPISSVS containing 2C-terminal phosphoserine

residues coupled to keyhole limpet hemocyanin. Negative controls were

performed using mouse-specific IgG (Jackson ImmunoResearch Laboratories,

West Grove, PA, USA) or pre-incubation of antibodies with blocking peptides.

After washing and a second blocking step, joint sections were incubated with

peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch

Laboratories) (1/100).

3. Western blot analysis

For phosphorylated smad1/5, interphalangeal and sacroiliac joints were

dissected, frozen, thawed twice, and stored in 6 M urea/10 mM Tris/pH 7.8. A

total of 500 ng protein diluted in 4× NuPAGE-LDS buffer (Invitrogen) was

analyzed under reducing conditions (2% mercaptoethanol and 0.1 M DTT) in

NuPAGE-MES-SDS buffer (Invitrogen). Proteins were transferred onto a

PVDF membrane in 0.4 M glycine/0.5 M Tris-base/0.01 M SDS and 200

mL/L of methanol. Blots were probed with antibody PS1 (1/2500 in

9

TBS-Triton/3% milk) and washed with TBS-Triton, and goat anti-rabbit

peroxidase-conjugated secondary antibody (1/5000 in TBS-Triton/3% milk)

(Jackson ImmunoResearch Laboratories) was added.

For noggin, Rt. tibialis anterior muscles were injected with 30 or 300 µg

pcDNA3.1+ noggin or empty vector. After 72 hours, muscles and blood were

collected. Muscles were extracted in 1 M guanidium hydrochloride/50 mM

Na-acetate and were desalted, lyophilized, and redissolved in 200 µL of

immunoprecipitation buffer containing 150 mM NaCl/1% NP-40/2 mM

EDTA/50 mM NaF/1 mM Na4P2O7/20 mM Tris/pH 7.6 (Invitrogen). Samples

were incubated overnight at 4°C with 2 µg of goat anti-noggin antibody.

Protein A/G sepharose solution (Amersham Biosciences, Piscataway, NJ,

USA) was used for precipitation. Pellets were dissolved in loading buffer 8 M

urea. Western blot was performed with goat anti-noggin polyclonal antibody

(R&D Systems) (1/5000) and peroxidase-conjugated mouse anti-goat

antibody (1/20000) (Jackson ImmunoResearch Laboratories). Ten ng of

recombinant noggin-Fc (a gift from F. P. Luyten) was used as a positive

control.

4. Statistical analysis

All statistical analyses were performed using SAS version 8.1 (Cary, NC,

USA). Comparisons between groups were made by Kruskal-Wallis or

Mann-Whitney U test. For incidence, Gehan-Wilcoxon test was used. P

values less than 0.05 were considered statistically significant.

10

III. RESULTS

1. Differential BMPs expression in sacroiliits

The disease process of sacroiliitis was initiated with infiltration of

inflammatory cells and proliferation of enthesial fibroblast-like cells at the

joint surface and was followed by cellular differentiation into chondroblasts

and hypertrophic chondrocytes in accordance with previous reports (Figure

2A)1, 17-19. The cartilage was progressively replaced by bone, which eventually

led to syndesmophytosis (Figure 2B).

Figure 2. H&E staining of sacroiliac joint section. A. In sacroiliac joint

section of mice sacrificed at week 21, inflamed synovium invading sacroiliac

joint surface (arrow) are seen at early stage (10× magnified). B. In mice

sacrificed at week 35, new bone formation (arrow) is seen at sacroiliac joint

surface at late stage (100×).

11

Immunohistochemical staining was performed to identify specific BMP

expressions in sacroiliitis and different BMPs were detected in distinct stages

of sacroiliitis (Figure 1). In sacroiliac joint sections, BMP-2 was seen in

spindle-shaped fibroblast-like cells in the proliferative zones at the early

stages (Figure 3A and 3B). In contrast, BMP-7 was detected to hypertrophic

chondrocytes at the late stages (Figure 3C and 3D). BMP-6 was not found at

either the early stages or late stages of sacroiliitis.

Figure 3. Immunohistochemistry for BMP expressions in sacroiliac joint

section. A. The early stage of sacroiliitis is characterized by enthesial

inflammation and cellular proliferation and immunoreactivity for BMP-2 is

detected (10×). B. Immunoreactivity of BMP-2 was detected in proliferated

spindle-shaped fibroblast-like cells (arrow, 100×). C. New bone formation in

sacroiliac joints was noted at late stage and immunoreactivity for BMP-7 was

seen (10×). D. Immunoreactivity of BMP-7 was noted in hypertrophic

chondrocytes (arrow, 100×).

12

2. Effect of noggin gene transfer on sacroiliitis and arthritis

The effect noggin gene transfer on clinical incidence and severity of

sacroiliitis was evaluated. Mice were treated every 3 weeks (at week 12, 15,

18, and 21) with either 30 µg or 300 µg of pcDNA3.1+ noggin, or empty

pcDNA3.1+. Semiquantitative reverse transcriptase-PCR showed significantly

increased local expression of noggin mRNA 72 hr after intramuscular

injection of 30 and 300 µg of pcDNA3.1+ noggin. However, very discrete

amounts of noggin cDNA were detected in samples injected with empty

pcDNA3.1+ (Figure 4A and 4B).

Western blot detected 26 and 38 kDa bands, which are corresponding to

noggin proteins, in both injected muscle (Rt. tibialis anterior muscle) and

plasma, but not in the opposite muscle (Lt. tibialis anterior muscle) 72 hr after

intramuscular injection of 30 and 300 µg of pcDNA3.1+ noggin. Noggin

protein was not detected locally or systemically after injection of empty

pcDNA3.1+ (Figure 4C).

13

Figure 4. Expression of noggin after cDNA transfer. A. Expression of noggin

was increased 72 hr after intramuscular injection of 30 and 300 µg

pcDNA3.1+ noggin. B. Band intensity for noggin expression shows a

dose-dependent increase of noggin mRNA expression in pcDNA3.1+

noggin-injected muscle (*P < 0.01 versus empty vector-treated controls, **P <

0.01 versus 30 µg of noggin-treated group). C. Noggin (26 and 38 kDa bands)

was found in 2 different mice 72 hours after injection of 30 and 300 µg

14

pcDNA3.1+ noggin in injected muscle (Rt. tibialis anterior muscle, lanes 1

and 3) but it was not detected in Lt. tibialis anterior muscle (lanes 2 and 4).

Noggin was also detected in the serum from 2 different mice treated with

pcDNA3.1+ noggin (lanes 5 and 6) but not in 2 different control mice treated

with empty pcDNA3.1+ (lanes 7 and 8). Lane 9 indicates recombinant

noggin-Fc (a 62 kDa band).

15

Injections of both 30 and 300 µg of pcDNA3.1+ noggin significantly

reduced the incidence of peripheral arthritis compared to injections of empty

pcDNA3.1+ controls (P < 0.05) (Figure 5A). In similar, injection of

pcDNA3.1+ noggin significantly reduced histological severity of arthritis

compared to empty pcDNA3.1+ controls (P < 0.05) (Figure 5B). The effect of

noggin gene transfer on clinical severity was evaluated by calculating the area

under the curve of the clinical severity score and the results showed that both

30 and 300 µg of pcDNA3.1+ noggin injections showed a significant

reduction in time-integrated clinical severity of arthritis (P < 0.05).

16

Figure 5. Effect of noggin gene transfer on clinical severity of arthritis. A.

Noggin treatment significantly reduced incidence of arthritis as compared

with control mice. B. Noggin treatment significantly reduced time-integrated

clinical severity, as compared with control mice. *P value < 0.05

17

In sacroiliac joint sections, severe inflammatory reaction and destruction of

joint structures were found in empty pcDNA3.1+ injected mice, but

preservation of joint surfaces was seen in pcDNA3.1+ noggin injected mice.

Injections of both 30 and 300 µg of pcDNA3.1+ noggin resulted in

significantly lesser histological severities compared to those treated with

empty pcDNA3.1+ (Figure 6).

Figure 6. The effect of noggin gene transfer on pathology of sacroiliitis. A.

Severe inflammation and destruction of joint surfaces are seen in sacroiliac

joint of mice treated with empty pcDNA3.1+ (H&E stain, 10×). B. Sacroiliac

joint of noggin gene transferred mice is relatively well preserved (H&E stain,

10×).

18

The calculation of pathologic severity scores of sacroiliac joint surfaces

showed that noggin gene transfer significantly reduced pathological severity

in 300 µg of pcDNA3.1+ noggin- or 30 µg of pcDNA3.1+ noggin-injected

mice compared to empty vector-injected mice. Furthermore, pathological

severity scores of sacroiliitis in mice injected with 300 µg of pcDNA3.1+

noggin were lower than those in mice injected with 30 µg of pcDNA3.1+

noggin (Figure 7).

Figure 7. Effect of noggin gene transfer on pathologic severity of sacroiliac

joint. Noggin gene transfer significantly reduced pathological severity in

gene-transferred mice as compared with empty vector-treated controls (*P <

0.01 versus empty vector-treated controls and **P < 0.05 versus 30 µg of

noggin-treated group).

19

The effect of noggin gene transfer on different stages of sacroiliitis was

analyzed and the results are shown in Table 2. In sacroiliac joint sections from

mice treated with pcDNA3.1+noggin, 6 out of 10 animals remained

unaffected. Proliferation of fibroblast-like cells was found in 4 out of 10 mice,

but progression to cartilage or bone formation was not detected. In contrast, 9

out of 10 mice treated with empty pcDNA3.1+ were affected and cellular

proliferation was found in all affected mice. One animal showed signs of

cartilage and bone formation, and ankylosis of joint surface derived from new

bone formation was seen. A similar pattern was also found in the

interphalangeal joint sections from mice.

Table 2. Effect of noggin gene transfer on different features of ankylosing

enthesitis

Area Treatment Normal Prolifera-

tion

Bone

formation Ankylosis

Empty

vector 1/10 9/10 1/10 1/10

SI joint

Noggin 6/10 4/10 0/10 0/10

Empty

vector 0/10 10/10 2/10 2/10

IP joint

Noggin 4/10 6/10 0/10 0/10

SI joint, sacroiliac joint; IP joint, interphalangeal joint of toe

20

3. Effect of noggin gene transfer on BMP signaling

To investigate the target cell population for BMP signaling,

immunofluorescent staining for phosphorylated smad1/5 was performed, and

the results showed positive smad1/5 staining in fibroblast-like cells and cells

with chondrogenic differentiation (Figure 8A and 8B). To determine whether

noggin gene transfer inhibited BMP signaling, the presence of

phosphorylated-smad1/5 molecules was determined by Western blot. Injection

of noggin led to a significant dose-dependent reduction in

phosphorylated-smad1/5 was found in mice 6 days after noggin gene transfer

compared to that in arthritic but non-transferred joints (P < 0.05) (Figure 8C).

21

Figure 8. Effect of noggin gene transfer on BMP signaling in ankylosing

enthesitis. A. Immunofluorescent staining for phosphorylated smad1/5

demonstrating positive staining in the enthesial fibroblast-like cells in joint

section from mice treated with empty pcDNA3.1+. B. The negative staining

for phosphorylated smad1/5 was seen in mice treated with pcDNA3.1+noggin.

C. Western blot showed that the expression of phosphorylated smad1/5 was

significantly increased in arthritic joints as compared with healthy

interphalangeal joints and was inhibited by noggin gene transfer (*P < 0.01

versus healthy joints).

22

IV. DISCUSSION

Syndesmophytosis, a process of pathologic new bone formation, is a

hallmark of AS. In advanced stages of the disease, syndesmophytosis

typically results in ankylosis of the entire spine1, 2. Therefore, inhibition of

syndesmophytosis may be an important and specific therapeutic target in

AS23-25.

To address the mechanisms resulting in syndesmophytosis in the present

study, a PG-induced spondylitis mouse model was used, on which various

studies involving microscopic analysis have clearly demonstrated that this

model is characterized by enthesial inflammation of the sacroiliac joint and

syndesmophytosis, which are in accordance with the features of human

AS16-18. Similar to these previous reports, enthesial inflammation at the early

stages of ankylosing enthesitise and pathologic new bone formation at the late

stage were found in the present study. Interestingly, differential expressions of

BMPs in different stages of disease progression were also found in the mouse

model. BMP-2 was expressed in fibroblast-like cells at the site of active

inflammation in the early stages and BMP-7 was found in hypertrophic

chondrocytes at the site of pathologic new bone formation in the late stages.

These findings suggest that BMP affects the pathogenesis of enthesial

inflammation and new bone formation in the mouse model of human AS.

BMPs are potent osteoinductive agents and in varying degrees, they induce

the cascade of events associated with bone formation and post-inflammatory

healing5-8. Previous studies have indicated that BMP-2 enhances

tendon-to-bone healing and promotes callus formation after bone fractures.

Moreover, it has been also indicated that BMP-7 stimulates the synthesis of

proteoglycans and collagen9, 26-28. In this context, it was interesting to note the

trend implicating differential expressions of BMP-2 and BMP-7 in the mouse

model of AS, and its strong relationship to time-integrated differential stages

23

of sacroiliitis. These data support the hypothesis that BMP-2 might be

involved in the pathogenic mechanisms responsible for enthesitis and that

BMP-7 might participate in syndesmophytosis in AS.

The somewhat surprising effect in sacroiliitis highlights the importance of

BMP signaling, which is essential for the complex network regulating skeletal

development29-31. New bone formation is initiated by the condensations of

mesenchymal cells and these cells undergo chondrogenic differentiation,

progressively representing the phenotypes of proliferating and hypertrophic

chondrocytes and in the later stages, the cartilage is replaced by bone26, 32, 33.

These previous studies indicate that the balances between BMPs and noggin,

their antagonist, influence different stages of new bone formation34, 35. Along

with the results of these previous studies, differential expressions of BMP-2

and BMP-7 observed in the present study suggest an early involvement for

BMP-2 and later involvement of BMP-7 in AS.

To our knowledge, the present study is the first reporting significant

associations of BMPs in sacroiliitis of the mouse model of AS and their strong

associations with sacroiliitis and syndesmophytosis. This was further

validated by investigation using noggin gene transfer in the present study. A

significant reduction in the incidence and severity of enthesitis and

preservation of the sacroiliac joint surface was found, as well as prevention of

pathologic new bone formation by noggin gene transfer. These effects were

dependent on the dose of noggin gene transferred, and mediated by

down-regulation of the smad1/5 signaling pathway. These results suggest

circumstantial evidence that BMP expressions are linked to inflammation and

syndesmophytosis in AS and that its blockade might be used as a therapeutic

strategy for the disease.

Various challenges regarding the role of BMPs in AS remain. First,

understanding of the relative contribution of specific BMPs and inhibitors to

the pathological cascade needs to be refined. Data from the present study

24

suggest an early role for BMP-2 and later involvement of BMP-7. Although

the presence of additional ligands has not been studied, noggin has been

demonstrated as an extracellular antagonist of BMPs regardless of their

presence35. Therefore, noggin over-expression is likely to change the overall

balance in BMP signaling in the present study. Second, the identification of

factors leading to the activation of the BMP signaling pathway is important to

understanding the links between inflammation and new bone formation.

Previously, up-regulated specific BMPs, including BMP-2 and BMP-7, were

demonstrated in the sera from patients with AS and levels of BMP-2 and

BMP-7 correlated well with disease activity and radiographic damage in

patients with AS, respectively36. Along with these previous observations, the

results of the present study suggest that BMP expressions are linked to

inflammation and new bone formation in AS.

25

V. CONCLUSION

In this study, differential expressions of BMP-2 and BMP-7 according to

the stage of sacroiliitis were found in the mouse model of AS and noggin gene

transfer is effective as a therapeutic strategy in the model, mechanistically

interfering with enthesitis and syndesmophyotsis. These findings support the

concept that BMP signaling is an attractive therapeutic target for achieving

disease modification in AS. Symptom control by inhibition of inflammation

may not be sufficient to halt the progression of structural damage of disease

and the resulting disability24, 25. Therefore, specific molecular targets involved

in pathologic new bone formation, including BMPs and their signaling

pathways, may provide a complementary or alternative therapeutic approach

in patients with AS.

26

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30

< ABSTRACT (IN KOREAN)>

강직성 척추염의 신생골형성 병인에 있어서

골형태형성 단백의 역할

<지도교수 이 수 곤>

연세대학교 대학원 의학과

박 민 찬

목적목적목적목적. 착부염과 신생골형성은 강직성 척추염의 주된 병리적

특징으로 특히, 척추의 신생골형성은 주된 장애의 원인이 된다.

골형태형성 단백은 염증 후 치유의 과정이나 비정상적 골형성에

관여하는 것으로 알려진 바, 골형태형성 단백이 강직성 척추염의

특징인 신생 골형성에 관여할 가능성이 있다. 본 연구에서는 강직성

척추염의 동물모델인 프로테오글라이칸-유도성 척추염 동물모델을

이용하여 골형태형성 단백이 강직성 척추염의 병인에 관여하는지

알아보고자 하였다.

방법방법방법방법. BALB/c 종의 생쥐에 100 µg의 인간 프로테오글라이칸을

접종한 후, 척추염의 발생을 확인하면서 골형태형성 단백의

길항제인 생쥐 노긴(noggin) 유전자 각각 30 µg 또는 300 µg 을

함유한 플라스미드를 투여한다. 이 후 척추염의 임상적 경과,

조직학적 변화를 조사하고 각각 21주와 35주에 안락사하여

골형태형성 단백-2, 6, 7과 이의 신호전달물질인 smad1/5의

31

발현여부를 조사한다.

결과결과결과결과. 강직성 척추염의 병기에 따라 차별적인 골형태형성 단백의

발현이 확인되었으며 초기의 염증상태에서는 골형태형성 단백-2가,

후기의 신생골형성 부위에서는 골형태형성 단백-7이 각각

발현되었다. 노긴 유전자를 투여한 군에서는 용량에 비례하여

관절염의 발생이 억제되었으며 천장관절의 조직학적 변화 역시

감소되었고 특히, 신생골형성의 억제가 천장관절에서 관찰되었다.

신호전달체계의 변화를 관찰한 결과, 유사하게 노긴 유전자를

투여한 군에서 smad1/5의 발현이 용량에 비례하여 억제되는 것이

관찰되었다.

결론결론결론결론. 본 연구의 결과, 골형태형성 단백이 강직성 척추염

동물모델에서 천장관절염 및 신생골형성의 병인에 관여하며 이의

차단이 신생골형성의 발생을 억제함을 확인한 바, 골형태형성

단백의 차단이 강직성 척추염 치료의 새로운 대안이 될 것으로

생각된다.

핵심되는 말: 강직성 척추염, 신생골형성, 프로테오글라이칸-유도성

척추염, 골형태형성 단백, 노긴