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Spontaneous development of synovitis and cartilage degeneration in transgenic mice overexpressing cathepsin K.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 12, December 2005, pp 3713–3717
DOI 10.1002/art.21423
© 2005, American College of Rheumatology
Spontaneous Development of
Synovitis and Cartilage Degeneration in
Transgenic Mice Overexpressing Cathepsin K
Jukka Morko, Riku Kiviranta, Kirsi Joronen, Anna-Marja Säämänen,
Eero Vuorio, and Heli Salminen-Mankonen
hypertrophic synovia were positive for cathepsin K. At
12 months, synovia of control mice revealed only a few
isolated cathepsin K–positive cells and mild changes in
articular cartilage.
Conclusion. Our findings demonstrate that overexpression of the cathepsin K gene under its own
promoter in transgenic mice makes them susceptible to
progressive synovitis, which, upon aging, results in
synovial hyperplasia and fibrosis and subsequent destruction of articular cartilage and bone.
Objective. Several recent studies have demonstrated that cathepsin K, a proteolytic enzyme capable
of degrading native fibrillar collagen, is overexpressed
in osteoarthritic cartilage and inflamed synovial tissue.
However, it is not known whether increased cathepsin K
production is a primary or a secondary event in these
diseases. The availability of transgenic UTU17 mice,
which exhibit constitutive overexpression of the cathepsin K gene, prompted us to study possible arthritic
changes in their knee joints.
Methods. Progression of synovitis and articular
cartilage degeneration in the knee joints of UTU17 mice
and their nontransgenic littermates was monitored by
histologic analyses at 7 and 12 months of age. Distribution of cathepsin K in the knee joints was studied by
immunohistochemistry.
Results. At the age of 7 months, UTU17 mice
exhibited clear signs of synovitis, with strong immunostaining for cathepsin K in the synovial lining and the
stroma, while control knee joints appeared normal. At
12 months, marked synovial thickening and fibrosis and
severe degradation of cartilage and subchondral bone
were observed in UTU17 mouse knee joints. In areas of
cartilage degeneration, both chondrocytes and cells of
Cysteine cathepsins B, H, K, L, and S form a
group of proteolytic enzymes that are capable of degrading native collagens and other components of the extracellular matrix (1). Recent data have suggested that
cathepsin K is the major cysteine cathepsin expressed in
both osteoarthritic (OA) cartilage (2,3) and inflamed
synovial tissue (4,5). Using a transgenic Del1 mouse
model for OA (6), we have demonstrated up-regulation
of cathepsin K expression in articular chondrocytes near
sites of matrix destruction, particularly in clusters of
proliferating cells, and in calcified cartilaginous matrix
(3). Because cathepsin K is one of the few enzymes
capable of degrading the networks of type II and I
collagen fibrils (7) (which provide articular cartilage and
bone, respectively, with structural strength), it is an
obvious candidate for a key enzyme involved in the
degradation of these tissues.
In cultures of synovial fibroblasts, inflammation
mediators interleukin-1␤ and tumor necrosis factor ␣
have been shown to activate cathepsin K gene expression
(8), but the sequence of events leading to activation of
cathepsin K production in arthritic joints remains poorly
understood. We recently produced 3 lines of transgenic
UTU17 mice containing variable numbers of extra copies of the murine gene for cathepsin K (Ctsk) under its
Supported by the Academy of Finland (project 205346), the
Maire Lisko Foundation, the Paulo Foundation, and the Research and
Science Foundation of Farmos. Mr. Morko is recipient of a training
grant from the Finnish Graduate School in Musculoskeletal Problems.
Dr. Kiviranta is recipient of a training grant from Turku Biomedical
Graduate School.
Jukka Morko, MSc, Riku Kiviranta, MD, PhD, Kirsi Joronen,
MD, PhD, Anna-Marja Säämänen, PhD, Eero Vuorio, MD, PhD, Heli
Salminen-Mankonen, PhD: University of Turku, Turku, Finland.
Address correspondence and reprint requests to Eero Vuorio,
MD, PhD, Department of Medical Biochemistry and Molecular
Biology, University of Turku, FI-20520 Turku, Finland. E-mail:
eero.vuorio@utu.fi.
Submitted for publication March 8, 2005; accepted in revised
form August 18, 2005.
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MORKO ET AL
endogenous promoter (9). These mice exhibit increased
production of cathepsin K and progressively develop
high-turnover osteopenia of trabecular bone (9) and
increased porosity of cortical bone (10). Considering the
recent studies of increased cathepsin K production
during (osteo)arthritic degradation of cartilage and
bone, the availability of these mice provided us with an
obvious possibility to study whether constitutive overexpression of cathepsin K could function as the primary
cause of joint destruction. Our findings clearly demonstrate that overexpression of Ctsk in transgenic mice
makes them susceptible to progressive synovitis, which,
upon aging, results in destruction of articular cartilage
and subchondral bone.
MATERIALS AND METHODS
Experimental animals. This study was conducted on
samples of knee joints collected from 15 transgenic UTU17
male mice that overexpressed Ctsk and from their 19 nontransgenic male littermates, which served as controls. Transgenic
UTU17 mice were produced at the Transgenic Core Facility at
the University of Turku, as previously described (9). The
UTU17 mice harbor several copies of an engineered 14-kb
cathepsin K transgene with a silent mutation in an FVB/N
genetic background. The UTU17 mice studied were homozygous for 1 of the 2 transgene alleles, UTU17.2 or UTU17.3.
The litters were genotyped by Southern blot analysis, as
previously described (9). Mice were killed at the ages of 7 and
12 months by cervical dislocation, and the hind limbs were
collected for histology and immunohistochemistry studies. The
study protocol was approved by the Institutional Committee
for Animal Welfare of the University of Turku.
Histology. For routine histology, dissected hind limbs
were fixed overnight in fresh 4% paraformaldehyde prepared
in phosphate buffered saline (PBS). Samples were then decalcified in 10% EDTA in PBS (pH 7.4) for 5–20 days, dehydrated, embedded in paraffin, and cut sagittally into 5-␮m–
thick sections. Sections of the knee joints were either stained
with hematoxylin and eosin (Merck, Darmstadt, Germany) and
used for subsequent histologic grading or processed for immunohistochemistry.
Histologic grading. Degenerative changes of articular
cartilage were analyzed in sagittal sections of knee joints and
classified into 5 grades based on the depth of erosion, as
previously described (6). Hyperplasia and fibrosis were used as
criteria in the scoring of synovial changes, as previously
described (11). The score of each knee was calculated as the
sum of the highest scores for fibrosis and hyperplasia observed
in the serial sections.
Immunohistochemistry. Tissue distribution of cathepsin K was studied using polyclonal anti-mouse cathepsin K
antibodies (12), which have previously been tested for specificity by Western blotting (10). Sections of knee joints (5 ␮m)
were deparaffinized and rehydrated in a series of descending
ethanol concentrations and digested for 1 hour with bovine
testicular hyaluronidase (2,000 units/ml) in PBS (pH 5). Primary antibodies were detected using streptavidin–biotin and
horseradish peroxidase complex (Histostain-Plus kit; Zymed,
Figure 1. Histologic analysis of knee joints of male UTU17 mice.
Hematoxylin and eosin–stained sagittal sections of knee joints of
control mice at the ages of 7 months (A–C) and 12 months (G–I), and
of UTU17 mice at 7 months (D–F) and 12 months (J–L). Bar in A ⫽
25 ␮m (A, D, G, and J); bar ⫽ 50 ␮m (B, C, E, F, H, I, K, and L). Color
figure can be viewed in the online issue, which is available at http:
//www.arthritisrheum.org.
South San Francisco, CA) and diaminobenzidine (Zymed) for
color development. Tissue sections were counterstained with
hematoxylin. The specificity of the reactions was controlled by
replacing the primary antibodies with normal rabbit serum.
Multinucleated cells in synovium and bone were studied in paraffin-embedded decalcified sections by enzyme histochemistry for tartrate-resistant acid phosphatase (TRAP)
according to the protocol recommended by the supplier of the
kit (Leukocyte Acid Phosphatase kit; Sigma-Aldrich, St. Louis,
MO). The sections were counterstained with hematoxylin.
RESULTS
Histologic evaluation of knee joints. At the age of
7 months, articular cartilage of both the UTU17 mice
(Figures 1D and E) and their nontransgenic control
littermates (Figures 1A and B) appeared normal. Compared with control joints (Figure 1C), the synovial
membrane in UTU17 mice was thickened, comprising
3–5 cell layers (Figure 1F).
At the age of 12 months, little cartilage degeneration was seen in control joints (Figures 1G, H, and I).
In 12-month-old UTU17 mice, the articular surfaces
were severely eroded, particularly in the posterior aspect
ARTHRITIS RESULTING FROM CATHEPSIN K OVEREXPRESSION
Figure 2. Scoring changes in articular cartilage and synovial tissue
with age. A, Scoring of degenerative changes of articular cartilage at 7
months (n ⫽ 7 control and 5 UTU17 mice) and at 12 months (n ⫽ 8
control mice and 8 UTU17 mice). Cartilage defects were scored 0–4
according to the depth of penetration. B, Corresponding synovial
hyperplasia and fibrosis, scored 0–3. C, Mean ⫾ SD articular cartilage
erosion and synovial hyperplasia and fibrosis scores. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.01 versus controls, by Mann-Whitney U test.
of the femoral and tibial condyles, where proliferative
synovial tissue was in contact with cartilage (Figures 1J,
K, and L). In addition to cartilage erosion, several other
alterations were observed in the knee joints of UTU17
mice with increasing age. Chondrocyte nuclei were
pyknotic in defect areas (Figure 1K). Chondrocyte clusters were frequently found at the margins of the articular
surface and in menisci (Figures 1K and L). At the age of
12 months, the synovial membrane of control mice was
1–2 cell layers thick (Figure 1I), while in UTU17 mice,
moderate to severe hyperplasia was observed (Figure
1L). Proliferation of synovial cells, fibrosis, angiogenesis,
and some inflammatory cells were observed in the
hyperplastic synovium of UTU17 mice (Figures 1J
and L).
Scoring changes in articular cartilage and synovial tissue. Scoring of degenerative changes in articular
cartilage of knee joints confirmed that only mild OA
changes were observed both in UTU17 and control mice
at 7 months (Figure 2A), whereas at 12 months severe
changes were detected in UTU17 mice (Figure 2A). But
scoring of synovial hyperplasia and fibrosis already revealed significant changes in the knee joints of UTU17
mice at 7 months (Figures 2B and C). At the age of 12
months, both synovial and articular cartilage scores were
significantly higher in UTU17 mice (Figure 2C).
Immunohistochemical detection of cathepsin K
in knee joints. In both control mice (Figure 3A) and
UTU17 mice (Figures 3B and D–F), a fraction of cells
located in the synovium were found to stain positive for
3715
cathepsin K, as shown for 12-month-old mice. In UTU17
mice, intense cathepsin K immunostaining was observed
in mononuclear cells in the lining and sublining areas of
synovial villi (Figures 3D and F). Because the synovial
membrane was thickened in UTU17 mice, the number
of cathepsin K–positive cells was considerably greater
than in control mice. Strong cathepsin K staining was
also observed in mononuclear cells in areas of high
proliferation (Figure 3B) and vascularization (Figure
3E). Cells with multiple nuclei, which were in intimate
contact with bone and positive for TRAP, were also
observed in the synovial tissue of UTU17 mice (Figure 3C).
In control knees at 12 months, a fraction of
chondrocytes in uncalcified articular cartilage exhibited
cathepsin K immunostaining (Figure 3G). In the knee
joints of UTU17 mice, pericellular and intracellular
staining for cathepsin K was observed in a number of
chondrocytes throughout uncalcified cartilage and in the
territorial matrix of calcified cartilage (Figures 3H and
I). The eroded cartilage surface and chondrocyte clusters in the remaining articular cartilage and menisci of
UTU17 mice were also positive for cathepsin K (Figures
3H and I), whereas no immunostaining was observed
when primary antibodies were replaced with normal
rabbit serum (Figure 3L). TRAP-positive osteoclasts
were seen on the surface of endosteal bone in the knee
Figure 3. Immunolocalization of cathepsin K and tartrate-resistant
acid phosphatase (TRAP) activity in the knee joints of UTU17 mice.
A, G, and J, Control mice. B–F, H, I, K, and L, Transgenic UTU17
mice. Immunohistochemistry analysis of TRAP activity (C, J, and K)
was performed on sagittal sections of knee joints obtained from
UTU17 mice at the age of 12 months. Intense cathepsin K immunostaining was seen in the hyperplastic synovium (B–F) and in articular
cartilage at the margins of defect areas (H and I). Cells with multiple
nuclei, which were in intimate contact with bone and positive for
TRAP, are seen in C, J, and K. As a staining control, normal rabbit
serum was used (L). Bar in A ⫽ 25 ␮m (A and B); bar ⫽ 50 ␮m (G–L);
bar ⫽ 100 ␮m (C–F).
3716
MORKO ET AL
joints of both control mice (Figure 3J) and UTU17 mice
(Figure 3K).
DISCUSSION
The most interesting finding of the present study
was that constitutive overexpression of Ctsk under its
own promoter in mice makes them susceptible to synovitis, which, upon aging, results in the destruction of
articular cartilage and bone. Previously, cathepsin K was
considered mainly as the major proteinase in osteoclastic bone resorption (9,10,13). However, recent studies of
OA and rheumatic joints have also demonstrated increased expression of cathepsin K in articular cartilage
and synovial tissue both in mice and humans (2–5).
These observations, and the availability of transgenic
UTU17 mice exhibiting constitutive overexpression of
Ctsk, prompted us to perform a systematic study on their
knee joint phenotype and its association with cathepsin
K distribution in these joints.
In synovium, cathepsin K overexpression in
UTU17 mice resulted in a marked proliferative response, including angiogenesis and moderate infiltration
of inflammatory cells. This was associated with widespread distribution of synovial cells expressing cathepsin
K. Especially in the hyperplastic synovial membrane of
UTU17 mice, the number of cathepsin K–expressing
cells was increased. Increased cathepsin K expression
has also been observed in the synovial membrane of OA
Del1 mice and patients with arthritis, and has been
suggested to increase the invasiveness of synovium and
to contribute to cartilage and bone erosion (3–5,8). We
propose that increased cathepsin K production in the
synovial membrane of UTU17 mice may contribute to
the development of cartilage and bone erosion in these
mice.
Strong cathepsin K staining was also detected in
deep zones of synovial tissue in UTU17 mice, particularly in mononuclear synoviocytes, around blood vessels,
and in multinucleated synovial cells. Similar distribution
of cathepsin K has been detected in patients with
arthritis, where it has been suggested to facilitate the
proliferation and movement of cells in the interstitial
matrix, and to contribute to matrix remodeling (4,5,8).
Cathepsin K overexpression may also contribute to the
development and progression of synovial hyperplasia
and fibrosis in UTU17 mice that exhibit features of
chronic fibrosing synovitis and rheumatoid pannus, although the underlying mechanism remains unresolved.
Furthermore, cathepsin K immunostaining was
observed in articular cartilage of control and UTU17
mice. In 12-month-old UTU17 mice, intense cathepsin K
staining was detected in areas of cartilage degeneration,
particularly in chondrocyte clusters. Increased cathepsin
K expression has also been observed in OA cartilage of
Del1 mice and human samples, where it has been
suggested to contribute to the progression of cartilage
degeneration (2,3). It is likely that the high cathepsin K
expression in articular cartilage of 12-month-old UTU17
mice has a similar role. Furthermore, since resorption of
subchondral bone has been linked to cartilage erosion in
arthritic joints (14,15), and cathepsin K expression is
significantly increased in subchondral bone of UTU17
mice (9), bone-derived cathepsin K may also play a role
in the development of cartilage and bone erosion detected in UTU17 mice.
We have previously described increased cathepsin K expression in a transgenic Del1 mouse model for
OA during cartilage degeneration (3). However, striking
differences exist in the development of arthritis between
Del1 mice harboring a deletion mutation in the type II
collagen transgene and UTU17 mice. In the former
model, the primary defect resides in articular cartilage,
and is associated with relatively minor hyperplasia of the
synovial tissue during progression of cartilage degeneration (6). In UTU17 mice, synovitis clearly preceded the
destruction of articular cartilage and subchondral bone.
Because cathepsin K overexpression alone was sufficient
to induce the phenotype in UTU17 mice, increased
cathepsin K expression in arthritic joints could even
serve as the primary event for the disease. However, the
relative importance of cathepsin K overexpression in
synovium, cartilage, and bone remains to be determined.
Further studies are therefore needed to identify the
mechanisms whereby increased production of cathepsin
K by synovial tissue, articular chondrocytes, and osteoclasts triggers the phenotype in UTU17 mice. Whichever the case, pharmaceutical inhibition of excessive
cathepsin K activity might help to prevent or slow the
progression of arthritis.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Dieter Brömme for
providing the mouse cathepsin K antibodies. The expert technical help of Ms Maria Ström, Ms Merja Lakkisto, and Ms
Tuula Oivanen is also gratefully acknowledged.
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development, cathepsin, synovitis, spontaneous, transgenic, degeneration, mice, overexpression, cartilage
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