Pathogenic role of bone morphogenetic proteins in ......Pathogenic role of bone morphogenetic...
Transcript of Pathogenic role of bone morphogenetic proteins in ......Pathogenic role of bone morphogenetic...
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
REFERENCES
1. Braun J, Bollow M, Neure L, Seipelt E, Seyrekbasan F, Herbst H, et al.
Use of immunohistologic and in situ hybridization techniques in the
examination of sacroiliac joint biopsy specimens from patients with
ankylosing spondylitis. Arthritis Rheum 1995; 38: 499-505.
2. Maksymowych WP. Ankylosing spondylitis: at the interface of bone and
cartilage. J Rheumatol 2000; 27: 2295-301.
3. Francois RJ, Braun J, Khan MA. Entheses and enthesitis: a
histopathologic review and relevance to spondyloarthritides. Curr Opin
Rheumatol 2001; 13: 255-64.
4. Urist MR, Mikulski A, Lietze A. Solubilized and insolubilized bone
morphogenetic protein. Proc Natl Acad Sci USA 1979; 76: 1828-32.
5. Reddi AH. Bone morphogenetic proteins: an unconventional approach to
isolation of first mammalian morphogens. Cytokine Growth Factor Rev
1997; 8: 11-20.
6. Sampath TK, Maliakal JC, Hauschka PV, Jones WK, Sasak H, Tucker
RF, et al. Recombinant human osteogenic protein-1 (hOP-1) induces new
bone formation in vivo with a specific activity comparable with natural
bovine osteogenic protein and stimulates osteoblast proliferation and
differentiation in vitro. J Biol Chem 1992; 267: 20352-62.
7. Reddi AH. Bone morphogenetic stromal proteins, bone marrow cells, and
mesenchymal stem cells: Maureen Owen revisited. Clin Orthop 1995;
313: 115-9.
8. Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, et
al. Ectopic induction of tendon and ligament in rats by growth and
differentiation factors 5, 6, and 7, members of the TGF-beta gene family.
J Clin Invest 1997; 100: 321-30.
9. Carlsen S, Hansson AS, Olsson H, Heinegard D, Holmdahl R. Cartilage
27
oligomeric matrix protein (COMP)-induced arthritis in rats. Clin Exp
Immunol 1998; 114: 477-84.
10. Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol
2000; 1: 169-78.
11. Leboy PS. BMP and BMP inhibitors in bone. Ann N Y Acad Sci 2006;
1068: 19-25.
12. Steiling H, Wüstefeld T, Bugnon P, Brauchle M, Fässler R, Teupser D, et
al. Fibroblast growth factor receptor signalling is crucial for liver
homeostasis and regeneration. Oncogene 2003; 22: 4380-8.
13. Reddi AH. Cartilage morphogenetic proteins: role in joint development,
homoeostasis, and regeneration. Ann Rheum Dis 2003; 62: 73-8.
14. Matsushita N, Terai H, Okada T, Nozaki K, Inoue H, Miyamoto S, et al.
Accelerated repair of a bone defect with a synthetic biodegradable
bone-inducing implant. J Orthop Sci 2006; 11: 505-11.
15. Little DG, McDonald M, Bransford R, Godfrey CB, Amanat N.
Manipulation of the anabolic and catabolic responses with OP-1 and
zoledronic acid in a rat critical defect model. J Bone Miner Res 2005; 20:
2044-52.
16. Glant TT, Bárdos T, Vermes C, Chandrasekaran R, Valdéz JC, Otto JM,
et al. Variations in susceptibility to proteoglycan-induced arthritis and
spondylitis among C3H substrains of mice: evidence of genetically
acquired resistance to autoimmune disease. Arthritis Rheum 2001; 44:
682-92.
17. Bárdos T, Szabó Z, Czipri M, Vermes C, Tunyogi-Csapó M, Urban RM,
et al. A longitudinal study on an autoimmune murine model of
ankylosing spondylitis. Ann Rheum Dis 2005; 64: 981-7.
18. Glant TT, Cs-Szabó G, Nagase H, Jacobs JJ, Mikecz K. Progressive
polyarthritis induced in BALB/c mice by aggrecan from human
osteoarthritic cartilage. Arthritis Rheum 1998; 41: 1007-18.
28
19. Glant TT, Mikecz K. Proteoglycan aggrecan-induced arthritis: a murine
autoimmune model of rheumatoid arthritis. Methods Mol Med 2004;
102: 313-38.
20. Matthys P, Lories RJ, De Klerck B, Heremans H, Luyten FP, Billiau A.
Dependence on interferon-gamma for the spontaneous occurrence of
arthritis in DBA/1 mice. Arthritis Rheum 2003; 48: 2983-8.
21. Corthay A, Hansson AS, Holmdahl R. T lymphocytes are not required
for the spontaneous development of entheseal ossification leading to
marginal ankylosis in the DBA/1 mouse. Arthritis Rheum 2000; 43:
844-51.
22. Holmdahl R, Jansson L, Andersson M, Jonsson R. Genetic, hormonal
and behavioural influence on spontaneously developing arthritis in
normal mice. Clin Exp Immunol 1992; 88: 467-72.
23. Zhang X, Aubin JE, Inman RD. Molecular and cellular biology of new
bone formation: insights into the ankylosis of ankylosing spondylitis.
Curr Opin Rheumatol 2003; 15: 387-93.
24. Dougados M, Dijkmans B, Khan M, Maksymowych W, van der Linden
S, Brandt J. Conventional treatments for ankylosing spondylitis. Ann
Rheum Dis 2002; 61: 40-50.
25. Zochling J, van der Heijde D, Burgos-Vargas R, Collantes E, Davis JC Jr,
Dijkmans B, et al. ASAS/EULAR recommendations for the management
of ankylosing spondylitis. Ann Rheum Dis 2006; 65: 442-52.
26. Kronenberg HM. Developmental regulation of the growth plate. Nature
2003; 423: 332-6.
27. Flechtenmacher J, Huch K, Thonar EJ, Mollenhauer JA, Davies SR,
Schmid TM, et al. Recombinant human osteogenic protein 1 is a potent
stimulator of the synthesis of cartilage proteoglycans and collagens by
human articular chondrocytes. Arthritis Rheum 1996; 39: 1896-904.
28. Huch K, Wilbrink B, Flechtenmacher J, Koepp HE, Aydelotte MB,
29
Sampath TK, et al. Effects of recombinant human osteogenic protein 1
on the production of proteoglycan, prostaglandin E2, and interleukin-1
receptor antagonist by human articular chondrocytes cultured in the
presence of interleukin-1beta. Arthritis Rheum 1997; 40: 2157-61.
29. Thomas JT, Kilpatrick MW, Lin K, Erlacher L, Lembessis P, Costa T, et
al. Disruption of human limb morphogenesis by a dominant negative
mutation in CDMP1. Nat Genet 1997; 17: 58-64.
30. Polinkovsky A, Robin NH, Thomas JT, Irons M, Lynn A, Goodman FR,
et al. Mutations in CDMP1 cause autosomal dominant brachydactyly
type C. Nat Genet 1997; 17: 18-9.
31. Storm EE, Huynh TV, Copeland NG, Jenkins NA, Kingsley DM, Lee SJ.
Limb alterations in brachypodism mice due to mutations in a new
member of the TGF beta-superfamily. Nature 1994; 368: 639-43.
32. Olsen BR, Reginato AM, Wang WF. Bone development. Annu Rev Cell
Dev Biol 2000; 16: 191-220.
33. John T, Stahel PF, Morgan SJ, Schulze-Tanzil G. Impact of the
complement cascade on posttraumatic cartilage inflammation and
degradation. Histol Histopathol 2007; 22: 781-90.
34. Pizette S, Niswander L. BMPs are required at two steps of limb
chondrogenesis: Formation of prechondrogenic condensations and their
differentiation into chondrocytes. Dev Biol 2000; 219: 237-49.
35. Balemans W, Van Hul W. Extracellular regulation of BMP signaling in
vertebrates: a cocktail of modulators. Dev Biol 2002; 250: 231-50.
36. Park MC, Park YB, Lee SK. Relationship of bone morphogenetic
proteins to disease activity and radiographic damage in patients with
ankylosing spondylitis. Scand J Rheumatol 2008; 37: 200-4.
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