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Involvement of the Wnt signaling pathway in experimental and human osteoarthritisProminent role of Wnt-induced signaling protein 1.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 2, February 2009, pp 501–512
DOI 10.1002/art.24247
© 2009, American College of Rheumatology
Involvement of the Wnt Signaling Pathway in
Experimental and Human Osteoarthritis
Prominent Role of Wnt-Induced Signaling Protein 1
Arjen B. Blom,1 Sarah M. Brockbank,2 Peter L. van Lent,1 Henk M. van Beuningen,1
Jeroen Geurts,1 Nozomi Takahashi,1 Peter M. van der Kraan,1 Fons A. van de Loo,1
B. Wim Schreurs,1 Kristen Clements,2 Peter Newham,2 and Wim B. van den Berg1
Objective. Wnt signaling pathway proteins are
involved in embryonic development of cartilage and
bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage.
The present study was undertaken to characterize the
expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected
genes in experimental and human OA.
Methods. Longitudinal expression analysis was
performed in 2 models of OA. Levels of messenger RNA
for genes from the Wnt/␤-catenin pathway were determined in synovium and cartilage, and the results were
validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant
protein, and in vivo by adenoviral overexpression.
Results. Wnt-induced signaling protein 1
(WISP-1) expression was strongly increased in the
synovium and cartilage of mice with experimental OA.
Wnt-16 and Wnt-2B were also markedly up-regulated
during the course of disease. Interestingly, increased
WISP-1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and
chondrocytes with recombinant WISP-1 resulted in
interleukin-1–independent induction of several matrix
metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP-1 in murine knee joints
induced MMP and aggrecanase expression and resulted
in cartilage damage.
Conclusion. This study included a comprehensive
characterization of Wnt and Frizzled gene expression in
experimental and human OA articular joint tissue. The
data demonstrate, for the first time, that WISP-1 expression is a feature of experimental and human OA
and that WISP-1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable
of inducing articular cartilage damage in models of OA.
Supported in part by ZonMW (program Alternatieven voor
dierproeven, project no. 11-400-0082), the Dutch Arthritis Association
(grant NR 08-1-309), and AstraZeneca. Dr. Takahashi’s work was
supported by IOP Genomics (grant IGE02032).
1
Arjen B. Blom, PhD, Peter L. van Lent, PhD, Henk M. van
Beuningen, PhD, Jeroen Geurts, MSc, Nozomi Takahashi, PhD, Peter
M. van der Kraan, PhD, Fons A. van de Loo, PhD, B. Wim Schreurs,
PhD, Wim B. van den Berg, PhD: Radboud University Nijmegen
Medical Center, Nijmegen, The Netherlands; 2Sarah M. Brockbank,
PhD, Kristen Clements, PhD, Peter Newham, PhD: AstraZeneca,
Macclesfield, UK.
Drs. Brockbank, Clements, and Newham own stock or stock
options in AstraZeneca.
Address correspondence and reprint requests to Arjen B.
Blom, PhD, Experimental Rheumatology and Advanced Therapeutics,
Radboud University Nijmegen Medical Center, Geert Grooteplein
26-28, 6525 GA Nijmegen, The Netherlands. E-mail: a.blom@reuma.
umcn.nl.
Submitted for publication May 14, 2008; accepted in revised
form October 13, 2008.
Osteoarthritis (OA) results in the destruction of
cartilage and bone, ultimately leading to loss of joint
function. The cause of the disease is largely unknown,
although obesity, genetic factors, and injury have all
been associated with increased risk of OA (1,2). Although it is likely that in most cases the initial events
leading to OA occur within the cartilage or subchondral
bone (3,4), the synovial tissue of many OA patients
shows a changed morphology, with a marked inflammatory phenotype (5,6).
Loss of articular cartilage extracellular matrix is
thought to be mediated by matrix metalloproteinases
(MMPs) and aggrecanases (ADAMTS-4 and
ADAMTS-5), which are likely the most important
groups of enzymes in the breakdown of extracellular
501
502
cartilage matrix (7). Specifically, MMP-3 (8), MMP-13
(9,10), ADAMTS-4, and ADAMTS-5 (11,12) have been
implicated in OA pathology. The inflammatory cytokine
interleukin-1 (IL-1) potently up-regulates MMP expression in synovial cells and chondrocytes (13,14) and may
drive cartilage damage in OA; however, the role of IL-1
in disease pathology remains unclear (13).
Recently, it has been hypothesized that OA entails a recapitulation of limb development, a process
characterized by chondrocyte hypertrophy and matrix
calcification (15,16). Interestingly, MMP-13 is an early
marker of chondrocyte terminal differentiation, an event
that is of key importance in limb development (17). Wnt
proteins form a family of secreted glycoproteins that
bind to Frizzled (Fz) receptors and regulate embryonic
development, body patterning, and tissue morphogenesis. Moreover, several Wnt family members, such as
Wnt-4, Wnt-14, and Wnt-16, have been shown to be
crucial for development of avian and murine limbs, by
controlling osteogenesis and chondrogenesis (18–20).
Wnt signaling also induces both MMP expression and
cell differentiation, and thus directly regulates tissue
remodeling and also regulates chondrocyte phenotype, a
process critical for bone growth and articular cartilage
formation.
Recent genetic data suggest that abnormal Wnt
signaling may contribute to the pathogenesis of OA: an
association between the development of hip OA and a
polymorphism in the FrzB gene has been demonstrated
(21–23). FrzB appears to function as a soluble Fz
receptor and thus can inhibit Wnt/Fz signaling. Interestingly, the FrzB mutation associated with OA exhibits
decreased affinity for Wnt molecules, suggesting a compromised ability to suppress Wnt signaling. In addition,
functional deletion of FrzB in a murine model of OA
results in exacerbation of disease (24). These data,
together with the association of a polymorphism in a
Wnt coreceptor, low-density lipoprotein receptor–
related protein 5, with OA (25), strongly implicate the
involvement of the Wnt signaling pathway in the etiology
or progression of OA.
To explore the role of the Wnt/Fz axis in OA, we
profiled a panel of genes from the Wnt signaling pathway by longitudinal expression analysis in 2 separate
experimental models of OA. Furthermore, we analyzed
the expression of key genes in both experimental and
human OA by quantitative polymerase chain reaction
(PCR) and immunohistochemistry, and assayed the potential of selected gene(s) to induce cartilage breakdown
in vitro and in vivo.
BLOM ET AL
MATERIALS AND METHODS
Animals and models of OA. For all experiments with
induced OA, C57BL/6 mice (Janvier, Le Genest St. Isle,
France) were used. Spontaneous OA was studied in the
STR/Ort strain (originally obtained from Charles River [Sulzfeld, Germany] and bred at our facilities). CBA mice (Charles
River) were used as controls for the STR/Ort strain. IL-1␣/␤–
deficient mice were originally kindly provided by Dr. Y.
Iwakura (Tokyo, Japan) and were bred at our facilities. Mice
were fed a standard diet and tap water ad libitum. All animal
experiments were approved by the local ethics commission.
Instability OA was induced by intraarticular injection
of 1 unit of collagenase on day 0 and day 2. OA pathology
develops gradually through day 42, at which time full-blown
OA is manifested. Knee joints were isolated on day 7, day 21,
and day 42 and processed for histologic analysis. Synovium and
cartilage samples were also obtained and processed for PCR
analysis.
OA develops spontaneously in STR/Ort mice. The first
pathologic changes are observed within 8 weeks after birth.
Tissue from STR/Ort and CBA mice was isolated at ages 4
weeks, 8 weeks, and 16 weeks.
Histologic analysis of murine knee joints. Isolated
knee joints were processed for histologic and immunohistochemistry analysis. Tissue was fixed in 4% buffered formalin
and then decalcified in formic acid and embedded in paraffin.
Six representative sections of each joint from various depths
were mounted on slides. Standard histologic analyses with
Safranin O and hematoxylin and eosin staining were conducted, and studies to detect Wnt-induced signaling protein 1
(WISP-1) and ␤-catenin were performed. Briefly, sections
were deparaffinized and subsequently incubated in buffered
citrate (pH 6.0). Sections were incubated overnight with either
polyclonal rabbit anti–WISP-1 or goat anti–␤-catenin (Santa
Cruz Biotechnology, Santa Cruz, CA) at a concentration of 2
␮g/ml. Thereafter, sections were incubated with biotinylated
polyclonal anti-rabbit IgG and biotinylated ant-goat IgG (Vectastain Elite kit; Vector, Burlingame, CA), respectively. For
staining of the neoepitope VDIPEN the procedure was essentially the same as for WISP-1, but citrate buffer was replaced
with chondroitinase ABC and staining was enhanced using a
nickel-enhancement step and orange G counterstaining; antiVDIPEN was a kind gift from Dr. J. S. Mort (Montreal,
Quebec, Canada). Staining for the neoepitope NITEGE using
an anti-NITEGE antibody (Acris, Hiddenhausen, Germany)
was performed by a process similar to that used for ␤-catenin
staining, with the addition of a 30-minute hyaluronidase treatment after the treatment with citrate buffer.
Human cartilage and synovium. Permission of patients
and the local ethics commission was obtained prior to harvesting of study samples. Human OA synovium and cartilage were
obtained from patients undergoing total knee or hip joint
replacement surgery. Control synovium from patients with
acute knee joint trauma was obtained at the time of arthroscopic examination. Control cartilage was obtained postmortem from subjects with no history of OA.
Human synovial fibroblasts were isolated by enzymatic
digestion of synovial tissue. Synovial tissue was placed in cell
culture medium at ambient temperature and subjected to
enzymatic digestion within 2 hours. Samples were finely
WISP-1 IN EXPERIMENTAL AND HUMAN OA
503
Table 1. Expression patterns of Wnt signaling pathway genes in synovium and cartilage during collagenase-induced OA, and during spontaneous
OA in STR/Ort mice*
Synovium
Collagenase-induced OA
Cartilage
OA in STR/Ort mice
Collagenase-induced OA
OA in STR/Ort mice
Gene
Week 1
Week 3
Week 6
Week 4
Week 8
Week 16
Week 1
Week 3
Week 6
Week 4
Week 8
Week 16
Axin2
Dkk-3
Dishevelled 1
FrzB
Indian hedgehog
LRP-5
LRP-6
sFRP-1
sFRP-2
Siah-2
WISP-1
Fz-3
Fz-4
Fz-5
Fz-6
Fz-7
Fz-8
Fz-10
Wnt-2
Wnt-2B
Wnt-3
Wnt-3A
Wnt-4
Wnt-5A
Wnt-5B
Wnt-6
Wnt-7A
Wnt-7B
Wnt-8A
Wnt-8B
Wnt-9A
Wnt-9B
Wnt-10A
Wnt-10B
Wnt-11
Wnt-16
⫽
1 13
2 2.0
⫽
–
⫽
⫽
1 14
1 6.5
⫽
1 37
⫽
⫽
1 2.1
1 3.5
2 2.3
1 2.3
⫽
⫽
1 82
–
–
2 2.0
⫽
1 2.5
1 4.0
–
–
–
–
⫽
–
⫽
⫽
⫽
1 111
⫽
1 8.7
⫽
⫽
–
⫽
⫽
1 3.2
1 4.9
⫽
1 15
⫽
⫽
⫽
⫽
⫽
1 2.0
⫽
⫽
1 8.6
–
–
⫽
⫽
⫽
⫽
–
–
–
–
⫽
–
⫽
⫽
⫽
1 181
⫽
⫽
⫽
⫽
–
⫽
⫽
1 2.7
1 2.4
⫽
1 9.1
⫽
⫽
⫽
⫽
⫽
1 1.2
⫽
⫽
⫽
–
–
⫽
⫽
⫽
⫽
–
–
–
–
⫽
–
⫽
⫽
⫽
1 104
⫽
⫽
2 1.5
2 1.9
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
2 1.6
2 2.2
⫽
⫽
⫽
⫽
⫽
⫽
⫽
–
⫽
⫽
⫽
2 1.7
⫽
1 4.3
⫽
–
⫽
1 12
⫽
⫽
1 1.4
⫽
2 1.7
⫽
⫽
2 2.4
2 3.2
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
2 1.3
⫽
⫽
⫽
⫽
⫽
1 1.5
1 3.4
–
⫽
⫽
⫽
⫽
⫽
1 7.8
⫽
–
⫽
⫽
⫽
⫽
1 1.4
⫽
2 1.5
1 2.0
2 1.2
2 2.2
⫽
⫽
⫽
⫽
1 1.8
⫽
1 2.6
⫽
⫽
⫽
1 1.8
⫽
⫽
2 4.1
⫽
1 2.8
1 4.2
–
⫽
⫽
⫽
⫽
⫽
⫽
⫽
–
⫽
⫽
⫽
⫽
1 1.5
1 5.6
⫽
⫽
⫽
2 4.3
⫽
⫽
⫽
1 4.2
⫽
⫽
1 2.5
⫽
⫽
2 2.5
1 2.0
2 2.0
2 2.1
⫽
–
⫽
–
–
⫽
⫽
–
⫽
–
⫽
–
–
⫽
–
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
1 2.1
⫽
⫽
1 2.8
⫽
⫽
⫽
⫽
⫽
⫽
⫽
–
⫽
–
–
⫽
⫽
–
⫽
–
⫽
–
–
⫽
–
⫽
⫽
⫽
⫽
⫽
1 2.8
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
–
⫽
–
–
⫽
⫽
–
⫽
–
⫽
–
–
⫽
–
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
–
⫽
–
–
⫽
⫽
⫽
⫽
–
⫽
–
–
⫽
–
⫽
⫽
⫽
⫽
⫽
⫽
2 1.9
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
1 1.7
⫽
⫽
2 3.7
–
⫽
–
–
⫽
⫽
⫽
⫽
–
⫽
–
–
⫽
–
2 2.2
⫽
⫽
⫽
⫽
1 1.9
⫽
⫽
⫽
⫽
1 2.3
⫽
⫽
⫽
1 1.8
⫽
1 2.3
⫽
11.9
⫽
⫽
⫽
–
⫽
–
–
⫽
⫽
⫽
1 1.7
–
⫽
–
–
1 3.4
–
⫽
1 1.8
⫽
1 2.1
* Values are the fold increase (1) or decrease (2) in mice with collagenase-induced osteoarthritis (OA) compared with naive mice, and in STR/Ort
mice (in which OA spontaneously develops) compared with CBA mice. Equal signs represent genes that were not up- or down-regulated; dashes
represent genes that were not detected.
minced and digested for 30 minutes at 37oC in 10 ml phosphate
buffered saline containing 0.1% trypsin (Sigma, St. Louis,
MO). Subsequently, samples were digested in 20 ml 0.1%
collagenase P (Roche Diagnostics, Indianapolis, IN) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal calf serum (FCS; Gibco, Breda, The Netherlands), 1 mM pyruvate, and 40 ␮g/ml gentamicin, for 2 hours at
37°C in 5% CO2. The cell suspension was filtered over a sterile
0.7-␮m filter (BD Falcon, Bedford, MA). Fibroblasts were
purified by negative selection using CD14 magnetic-activated
cell sorting (26) and maintained in DMEM supplemented with
10% FCS, 1 mM pyruvate, and 80 ␮g/ml gentamicin. Cells from
passages 3–6 were used for experiments.
Human articular chondrocytes were obtained by overnight incubation of 1-mm2 cartilage slices with 2 mg/ml colla-
genase P in DMEM supplemented with 10% FCS and 40
␮g/ml gentamicin at 37oC. After resuspension and filtering
over a 0.7-␮m filter, cells were cultured in a 24-well plate at a
concentration of 2 ⫻ 105 cells/ml.
TaqMan quantitative PCR analysis using low-density
arrays and conventional quantitative PCR. To determine
whether expression of genes was altered during experimental
OA, quantitative PCR analysis was performed, either by a
microfluid technique using low-density TaqMan arrays (ABI
Prism low-density arrays) (LDA; Applied Biosystems, Foster
City, CA) or by regular TaqMan analysis. A bespoke LDA of
48 genes was used to perform quantitative PCR analysis.
Marker genes ADAMTS4, BMP4, COL10A1, COL1A2,
COL2A1, IL1B, IL6, IL18, MMP13, and TGFB3 were added
to confirm whether the kinetics of the experiment were
504
BLOM ET AL
representative of the OA models; 18S and HPRT were used
for normalization. Results were expressed as fold change, by
comparing OA knee joints with normal joints after correction
for housekeeping genes, using the threshold cycle (Ct) method
and the 2⌬⌬Ct formula. Comparison of the regulation of the
above-mentioned marker genes indicated that in the current
experiments, regulations were representative of those observed
during collagenase-induced OA and OA in STR/Ort mice in
previous experiments (data not shown).
Stimulation of macrophages and chondrocytes by recombinant WISP-1. RAW 264.7 cells (as a model for synovial
macrophages; American Type Culture Collection, Rockville,
MD), H4 chondrocytes (27), and freshly isolated human cells
(fibroblasts, chondrocytes) were tested by in vitro stimulation
with recombinant human WISP-1 (PeproTech, Rocky Hill,
NJ). After stimulation of cells for 24 hours with WISP-1 at 10,
50, 100, 500, or 1,000 ng/ml, RNA was isolated and used for
quantitative PCR analysis. In some cultures polymyxin B
sulfate (Sigma, St. Louis, MO) was added to the medium at a
concentration of 10 ␮g/ml. Macrophages, human fibroblasts,
and human chondrocytes were cultured in DMEM with Glutamax, 10% FCS, and gentamicin. The murine chondrocyte
cell line H4 was generated in our laboratory and cultured in
DMEM/Ham’s F-12 with 10% FCS and gentamicin.
Generation of adenoviral WISP-1 vector. An adenoviral vector to induce overexpression of WISP-1 was generated
using a previously described method (28). Briefly, the coding
sequence of WISP-1 was cloned with the primers 5⬘TTTTGTCGACACCGCCATGAGGTGGCTCCTGCCC-3⬘
(forward) and 5⬘-TTTTCTAGACTAATTGGCAATCTCTTC-3⬘ (reverse). These primers contained restriction sites
for cloning into an adenoviral vector. WISP-1 was cloned from
murine OA synovial tissue complementary DNA. Viral vectors
were E1A, B, and E3 deleted, and produced according to the
method described by Chartier et al (29). For in vivo overexpression, 107 plaque-forming units of the WISP-1 adenovirus
or of an empty control vector (Ad5del70-3) was injected
intraarticularly, and total knee joints, synovial tissue, and
cartilage were isolated after 2 days or 7 days for analysis.
RESULTS
Up-regulation of ␤-catenin in experimental OA.
To investigate whether Wnt signaling might be relevant
in experimental OA, we tested for the accumulation of
␤-catenin, as a reporter of Wnt/Fz signaling in the knee
joints of naive and osteoarthritic mice, using immunohistochemical staining (results available online at http://
www.rheumaresearch.nl/BlomAnRFigureS1.pdf). Staining for ␤-catenin in naive mouse cartilage was present
and was restricted to the deeper cartilage chondrocyte
layers. However, 21 days after intraarticular collagenase
injection, ␤-catenin staining intensity was increased, and
greater numbers of superficial chondrocytes were positive. Staining for ␤-catenin was widespread in the synovium of naive mice, but was strongly increased 21 days
after induction of OA.
Regulation of several Wnt- and Frizzled-related
genes during experimental OA. Since intracellular
␤-catenin accumulation could represent evidence of
Wnt-dependent signaling, we used an LDA method to
determine the kinetics of Wnt/Fz family gene expression
in 2 murine models of OA: intraarticular collagenase–
induced OA and spontaneous OA in STR/Ort mice.
Gene expression was determined in synovium and cartilage of naive knee joints and compared with expression
in tissue from knee joints 1, 3, and 6 weeks after
induction of OA by administration of collagenase. Similarly, the expression of Wnt family genes was measured
in the cartilage and synovium of 4-, 8-, and 16-week-old
STR/Ort mice. In the collagenase-induced OA model we
found that many Wnt/Fz family genes were up- or
down-regulated in OA synovium compared with synovium from naive mice (Table 1). However, none of the
Wnt genes was regulated at the messenger RNA
(mRNA) level in the cartilage in this model. Expression
of WISP-1 mRNA is shown in Figures 1A and B.
WISP-1 was up-regulated 37-fold in the synovium in the
collagenase-induced OA model after 1 week, and 9-fold
after 6 weeks. In the cartilage, maximal up-regulation of
WISP-1 was found on day 21 (3-fold).
In the STR/Ort mouse model of spontaneous
OA, Wnt genes were similarly up-regulated in the
synovium, although the magnitude of gene expression
change was smaller than in the collagenase-induced OA
model (Table 1). In contrast with the findings in the
cartilage of mice with collagenase-induced OA, in STR/
Ort mouse cartilage some of the Wnt genes were
regulated as well. Wnt-16 expression in synovium was
up-regulated 181-fold and 5.6-fold in mice with collagenase-induced OA and STR/Ort mice, respectively, and
Wnt-2B 82-fold and 2.8-fold, respectively (Table 1).
Consistent with findings in the collagenase-induced OA
model, WISP-1 was found to be significantly upregulated (⬃2-fold) in both the synovium and the cartilage of the STR/Ort mice at age 16 weeks.
Increased levels of WISP-1 protein in murine OA
knee joints. Our data indicated that a panel of Wnt/Fz
genes is regulated during the progression of experimental OA; thus, Wnt/Fz signaling via ␤-catenin may play a
role in OA pathology. We also found that a small subset
of genes (WISP-1, Dkk-3, and Fz-6) were significantly
up-regulated in both the synovium and the cartilage in
both experimental models of OA; of these, WISP-1 was
the most highly regulated gene (Figure 1; further results
available online at www.rheumaresearch.nl/
BlomAnRFigureS2.pdf). WISP-1, a known Wnt/␤catenin–regulated gene, is a member of the CCN family
WISP-1 IN EXPERIMENTAL AND HUMAN OA
505
Figure 1. Expression of Wnt-induced signaling protein 1 (WISP-1) in synovium and cartilage in 2 separate models of
osteoarthritis (OA) and in humans with OA. A and B, WISP-1 expression in synovium (A) and cartilage (B) of mice with
collagenase-induced OA. Strong up-regulation of WISP-1 was found in the synovium throughout the course of disease. WISP-1
was also significantly up-regulated in cartilage on day 7 and day 21. C and D, WISP-1 expression in synovium (C) and cartilage
(D) of STR/Ort mice compared with CBA controls. WISP-1 was up-regulated in both the synovium and the cartilage of
16-week-old mice. Threshold cycle (Ct) was determined, and results in A–D are expressed as the ⌬⌬Ct (OA joint – naive joint
after correction for housekeeping gene). Each point represents the expression in pooled samples of synovium or cartilage from
4 mice; bars show the mean and 95% confidence interval. E, Relative expression (2⌬Ct) of WISP-1 mRNA in synovial biopsy
specimens from 5 patients with OA and 5 patients with trauma (controls). WISP-1 expression was compared by microarray
analysis, and was found to be significantly higher in the OA patients. Values are the mean and SEM. F, Relative expression
(2⌬Ct) of WISP-1 mRNA in cartilage from 5 patients with OA and 5 postmortem control specimens. Bars show the means.
of growth factors; the biologic properties of CCN proteins include stimulation of cell proliferation, migration,
adhesion, and extracellular matrix formation. We therefore focused on the role of WISP-1 as a prototypical
Wnt/␤-catenin target gene in experimental and human
OA.
To confirm that the increased expression of
WISP-1 mRNA in synovium and cartilage had consequences regarding expression at the protein level, we
assessed the presence of WISP-1 protein in knee joints
of mice after the onset of collagenase-induced OA, using
immunohistochemical methods. Synovial WISP-1 staining increased strongly over time, both in the synovial
lining and in the subsynovium (Figures 2A, C, and E). At
2 different locations in the joint, i.e., near the patellofemoral junction and near the tibiofemoral junction,
this expression of WISP-1 increased rapidly up to day 7
after induction of OA, and slowly subsided thereafter.
WISP-1 protein expression in the cartilage after
induction of OA was scored and was found to increase
up to day 21; thereafter, expression diminished in conjunction with the loss of chondrocytes from the damaged
cartilage (Figures 2B, D, and F). WISP-1 expression was
similar in all layers of cartilage, and this even distribution did not change notably during the development of
OA.
Increased WISP-1 mRNA levels in human OA
tissue. We next examined WISP-1 mRNA expression in
5 human OA synovial tissue specimens and compared
this with expression in synovial tissue from 5 patients
with acute trauma (controls). Expression of WISP-1 in
human OA synovium was 2.7-fold higher than in control
synovium (P ⬍ 0.01) (Figure 1E). In addition, expression
of WISP-1 in the cartilage of OA patients (n ⫽ 12) was
compared with that in control cartilage (n ⫽ 11).
WISP-1 expression was ⬎2-fold higher in OA cartilage
compared with control cartilage (Figure 1F).
Immunohistochemical detection of WISP-1 in
human OA cartilage. In order to determine whether
WISP-1 protein levels were also increased in OA cartilage, immunohistochemical staining was performed.
Cartilage samples from 3 OA patients, obtained during
joint replacement surgery, were compared with 3 control
cartilage samples. WISP-1 was undetectable in 2 control
506
BLOM ET AL
Figure 2. Immunohistochemical detection of WISP-1 in synovium (A, C, and E) and cartilage (B, D, and F) of mice with
collagenase-induced OA. Synovium from naive mice (A) stained less strongly than synovium from mice 42 days after OA
induction (C) (arrows). JS ⫽ joint space. Calculation of the percentage of WISP-1–positive cells in the synovium of mice with
collagenase-induced OA (E) showed that in both the synovial lining and the subsynovium, WISP-1 expression increased rapidly
after OA induction, and slowly subsided after day 7. Expression of WISP-1 was relatively low in cartilage from naive mice (B),
but increased until day 21 in cartilage from mice with collagenase-induced OA (D). Scoring of WISP-1 staining in the cartilage
of mice with collagenase-induced OA (F) showed that by day 42, staining had decreased to below pretreatment levels, due to
the loss of cellularity of cartilage. A–D show representative results (original magnification ⫻ 200). Values in E and F are the
mean ⫾ SEM. See Figure 1 for other definitions.
Figure 3. Results of immunohistochemistry analysis for WISP-1 in knee cartilage obtained postmortem from 3 donors with no
history of musculoskeletal disorders (A–C) and in cartilage obtained during total knee joint replacement in 3 patients with OA
(D–F). OA cartilage showed significantly higher expression of WISP-1 compared with control cartilage. Insets in D and F
demonstrate strong staining for WISP-1 in chondrocyte clusters. (Original magnification ⫻ 200; ⫻ 400 in insets.) See Figure
1 for definitions.
WISP-1 IN EXPERIMENTAL AND HUMAN OA
507
Figure 4. In vitro effect of human Wnt-induced signaling protein 1 (WISP-1) on murine macrophage and chondrocyte cell
lines and on human synovial cells and chondrocytes. A and B, Reactivity with WISP-1 was assayed by stimulation of the
macrophage cell line RAW 264.7 (A) and the murine chondrocyte cell line H4 (B) with human WISP-1 protein for 24 hours.
In macrophages, WISP-1 stimulation resulted in an up-regulation of matrix metalloproteinases (MMPs) except for MMP-2,
which was not expressed. This up-regulation appeared to be independent of interleukin-1 (IL-1), since up-regulation of that
cytokine was minimal. In chondrocytes, MMP-3 was up-regulated by WISP-1 in a dose-dependent manner. C, Human primary
synovial cells and chondrocytes were stimulated with 1.0 ␮g/ml WISP-1. After WISP-1 treatment, levels of MMP-3 and MMP-9
were increased in both cell types. Results in A–C are expressed as the mean fold change compared with unstimulated cells.
specimens (Figures 3B and C), whereas 1 control specimen exhibited very low levels of WISP-1 staining (Figure 3A). The tissue specimens obtained from all 3 OA
patients demonstrated strong WISP-1 staining, with
especially strong staining in the midzone of the cartilage
and in chondrocyte clusters (Figures 3D–F). Staining
was localized on the cell surface or in the pericellular
space adjacent to the extracellular matrix.
Up-regulation of matrix-degrading enzymes by
WISP-1 on murine and human cells. To determine
whether the increased expression of WISP-1 in both the
synovium and the cartilage could result in cellular reactions consistent with OA pathology, we studied the
effect of WISP-1 on murine and human cells. First,
murine RAW 264.7 macrophages were stimulated with
recombinant WISP-1 for 24 hours and the gene expression of a panel of catabolic enzymes (MMPs/ADAMTS
genes) was monitored. WISP-1 caused a dose-dependent
increase in expression of mRNA for ADAMTS-4,
MMP-3, MMP-9, and MMP-13, but not ADAMTS-5 or
MMP-2 (Figure 4A and data not shown). Since several
MMP genes are responsive to IL-1␤, we also assayed
IL-1␤ expression in response to WISP-1. The results
showed that IL-1␤ expression was not up-regulated by
WISP-1. Stimulation of murine H4 chondrocytes with
WISP-1 resulted in a marked up-regulation of MMP-3
mRNA, whereas other MMPs were unaffected (Figure
4B).
We next investigated the effect of WISP-1 on
primary human cells (human synovial cell cultures [passage 6] and human chondrocytes [passage 1]). Synovial
cells responded to WISP-1 (after 24-hour stimulation),
with MMP-3 and MMP-9 expression increased 2-fold
and 3-fold, respectively. Similarly, WISP-1 stimulation
increased chondrocyte MMP-3 and MMP-9 levels by
5-fold and 2-fold, respectively (Figure 4C). No signifi-
508
BLOM ET AL
Figure 5. In vivo effect of adenoviral overexpression of WISP-1 in naive joints. A and B, Quantitative polymerase chain reaction findings in
synovium (A) and cartilage (B) from knee joints of mice that were transfected with adenoviral WISP-1. Results are presented as the mean fold change
in the quantity of mRNA compared with that in knee joints from mice transfected with control virus, after correction for GAPDH expression. MMP
expression was up-regulated in synovium and cartilage. C–F, Immunohistochemical analysis of the expression of NITEGE (C and D) and VDIPEN
(E and F) in the knee joints of mice that were transfected with adenoviral WISP-1 (D and F) or control virus (C and E). G and H, NITEGE staining
in the knee joint of a wild-type mouse (G) and an IL-1␣/␤⫺/⫺ mouse (H) following adenoviral overexpression of WISP-1. C–H show representative
results (magnification ⫻ 200). See Figure 4 for definitions.
WISP-1 IN EXPERIMENTAL AND HUMAN OA
cant effect on MMP-2 or MMP-13 was detected. Coincubation of cells with WISP-1 and polymyxin B (as a
control for lipopolysaccharide [LPS] contamination) did
not significantly affect WISP-1–induced changes (data
not shown).
Induction of cartilage damage by adenoviral
overexpression of WISP-1 in murine knee joints. Since
we observed that WISP-1 was expressed in synovial and
cartilage tissue in rodent OA models and in human OA
and that WISP-1 was capable of inducing MMP expression, we explored the potential of WISP-1 to induce
cartilage damage, by overexpressing WISP-1 in knee
joints of naive mice. WISP-1 adenovirus or control
adenovirus was delivered to the knee joints of naive
mice. Two days after inoculation, WISP-1 was strongly
up-regulated (700-fold versus control) in the synovium
of WISP-1 adenovirus–infected mice (Figure 5A).
In accordance with the in vitro findings, we
observed increased expression of MMP-3 in the synovium (Figure 5A) and cartilage (Figure 5B) of WISP-1
adenovirus–treated mice (15-fold and 110-fold, respectively); stimulation of MMP-13 was also detected in both
synovium and cartilage (15-fold induction in cartilage).
Enhanced MMP-9 expression was found to be restricted
to the synovium. Expression of ADAMTS-4 and, to a
lesser extent, ADAMTS-5 was also increased in the
synovium of WISP-1 adenovirus–inoculated animals
(Figures 5A and B). Similar increases were not observed
in mice inoculated with control adenovirus (data not
shown).
In addition to analyzing the expression of MMP
and ADAMTS mRNA in the joint compartment, we
examined the histologic features of the joints after
WISP-1 adenovirus infection (Figures 5C–F). Some
synovial changes were induced by WISP-1 overexpression, such as focal loss of cells and the presence of cells
in the synovial cavity. However, no strong inflammatory
response was observed. Markers for enzymatic cartilage
damage were markedly increased in the cartilage of
WISP-1–expressing mice only. Notably, staining for both
the NITEGE and the VDIPEN neoepitopes, indicative
of aggrecanase and MMP activity, respectively, was
observed pericellularly and in the extracellular matrix
(VDIPEN) following WISP-1 adenovirus injection (Figures 5D and F). NITEGE and VDIPEN neoepitope
staining was absent in control adenovirus–treated mice
(Figures 5C and E).
Evidence that the effects of WISP-1 are not
mediated by IL-1. Although WISP-1 did not appear to
induce IL-1␤ release in in vitro cultures, we did observe
some differences in MMP expression profiles following
509
in vivo overexpression of WISP-1. Since IL-1␤ is strongly
associated with expression of proinflammatory and
matrix-degrading genes, we explored the effects of
WISP-1 overexpression in IL-1␣/␤–deficient mice. With
the exception of MMP-13, which was slightly decreased
(1.8-fold) in IL-1␣/␤–null mice, there were no significant
differences in synovial or cartilage MMP or ADAMTS
expression. In accordance with these observations, no
reduction of cartilage damage, proteoglycan depletion as
assessed by Safranin O staining, or NITEGE or
VDIPEN neoepitope expression was observed following
intraarticular injection of WISP-1 adenovirus into IL-1␣/
␤–deficient mice (Figures 5G and H).
DISCUSSION
In a comprehensive study of Wnt/Fz gene expression in 2 experimental models of OA, we found that 1)
a panel of Wnt and Wnt signaling–related genes, including WISP-1 (a Wnt-responsive gene), was up-regulated;
2) WISP-1 mRNA expression was significantly upregulated in murine OA and human OA articular joint
tissue and this expression was mirrored at the histologic
level; 3) recombinant WISP-1 induced the production of
matrix-degrading enzymes in murine and human synovial cells and human chondrocytes; and 4) overexpression of WISP-1 in the articular joints of mice in vivo
resulted in the induction of matrix-degrading enzyme
expression and cartilage extracellular matrix damage,
independently of IL-1␣ or IL-1␤.
Although some alternative factors have been
reported to induce ␤-catenin activation (30), its accumulation and translocation to the nucleus is considered
indicative of activated canonical Wnt signaling (31,32).
We found that ␤-catenin, along with a panel of Wnt/Fzrelated genes, was up-regulated in cartilage and synovium during experimental OA. Fz-6, a Wnt receptor that
activates protein kinase C and probably noncanonical
Wnt signaling (33), was up-regulated in all samples, but
only at very low levels. To our knowledge, no role of
Fz-6 in joint development or OA has been suggested
previously. Dkk-3, an associated Wnt pathway member,
was up-regulated in synovium and cartilage in both
models; however, the expression change was again modest. Interestingly, Dkk-3 has been associated with OA
disease progression in a previous study (34), and therefore merits further investigation.
One of the most pronounced changes we observed was the induction of WISP-1 in both the synovium and the cartilage of mice with collagenase-induced
OA and STR/Ort mice with spontaneous OA. Interest-
510
ingly, cartilage WISP-1 expression was not changed in
streptococcal cell wall–induced arthritis, a model of
inflammatory arthritis (results not shown). Recently,
French et al have shown that WISP-1 inhibits the
differentiation of precursor cells into chondrocytes and
may also affect chondrocyte phenotype (35). Taken
together, these findings may indicate that WISP-1 expression is specific to OA-like processes. We therefore
explored the function of WISP-1 in more detail.
WISP-1 is a member of the CCN family of growth
factors to which connective tissue growth factor
(CTGF), Cyr61, nov, WISP-2, and WISP-3 also belong.
Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing,
and CTGF is also thought to be involved in OA pathology (36–38). Moreover, French and colleagues have
shown that overexpression of WISP-1 in ATDC-5 cells
completely prevented type II collagen expression and
ATDC-5 cell differentiation into a chondrocytic phenotype under the influence of bone morphogenetic protein
2 or growth differentiation factor 5 (35).
Our immunohistochemical analyses revealed
WISP-1 expression in synovium and cartilage in both
experimental models; especially intense staining was
observed in the synovial lining layer and in the upper
zones of cartilage, complementing the ␤-catenin staining
pattern. These findings obtained clinical relevance when
we demonstrated that WISP-1 was also increased in
human OA cartilage and synovium specimens relative to
control tissue. The finding that WISP-1 is expressed in
chondrocyte clusters suggests an association of WISP-1
expression with a phenotype change of the cells; however, additional studies are needed before such correlations can be considered further. Interestingly, very low
expression of WISP-1 was also observed in one of the
postmortem control specimens. This same control sample did exhibit some superficial matrix damage, indicative of early-stage OA, perhaps suggesting that WISP-1
is expressed during the very early stages of OA.
Homology between human and murine WISP-1 is
⬎80%, and studies using recombinant human WISP-1 to
stimulate murine macrophages, synovial fibroblasts, or
human chondrocytes demonstrated that WISP-1 induced several matrix-degrading enzymes, suggesting that
it has the potential to cause cartilage damage. The lack
of effect when WISP-1 was cocultured with polymyxin B
confirmed that the observed effects were not caused by
LPS contamination of the recombinant protein. In addition, WISP-1 did not induce IL-1, which is known to be
regulated by (among other mediators) LPS (39).
Overexpression of WISP-1 in vivo resulted in the
BLOM ET AL
induction of several MMPs, including MMP-3 and
MMP-13, as well as the aggrecanases ADAMTS-4 and
ADAMTS-5. In studies using macrophage depletion and
MMP-3–deficient mice, we have previously shown that
MMP-3 produced by macrophages in the synovium is a
mediator of cartilage breakdown (40). Apart from its
ability to directly cleave cartilage matrix components,
MMP-3 activates proMMPs such as proMMP-13, further affecting extracellular matrix remodeling. Thus,
WISP-1 may indirectly activate MMP-13 and facilitate
cartilage matrix damage. However, increased MMP-13
activity may also reflect a phenotype change of the
chondrocytes, since MMP-13 is a marker for terminal
differentiation of chondrocytes (41). We anticipated that
WISP-1 would induce a phenotypic change, since it has
been demonstrated to drive progenitor cells toward
osteogenesis and it is expressed in chondrocyte clusters
in OA cartilage. However, apart from MMP-13 upregulation, we failed to detect other signs of chondrocyte
phenotype change, such as down-regulation of SOX9 or
type II collagen or up-regulation of RUNX-2 or types I
and X collagen (data not shown). Since our studies were
essentially short-term stimulation assays, additional
longer-term overexpression experiments may be necessary to address this question.
Recently an association between polymorphisms
of the WISP-1 gene and occurrence of spinal OA was
found (42), which indicates a role of this protein in OA.
Those authors described the absence of a G allele
associated with end plate sclerosis in the spine (odds
ratio 2.91, P ⫽ 0.0069). Due to the study design, hip,
knee, and spinal facet joint OA was not evaluated. For
future studies, it will be interesting to determine the
functional consequences of this polymorphism, in order
to elucidate the mechanism behind the association.
Expression profiling of some Wnt signaling molecules other than WISP-1 revealed other potentially
interesting changes that warrant further study. Wnt-16
and Wnt-2B are 2 proteins that may be involved in OA
pathology and were highly and significantly up-regulated
in experimental OA synovium. Wnt-2B is involved in the
generation of bone, and may therefore have a detrimental effect on chondrocytes, driving them to terminal
differentiation, an event thought to be of importance in
OA (43). Guo et al have implicated Wnt-16 as being
involved in the formation of synovial joints during
embryonic development, by inducing dedifferentiation
of chondrocytes to form the synovial space (18).
We were not able to demonstrate any regulation
of Wnt proteins in the cartilage during collagenaseinduced OA. However, it is likely that Wnt signaling
WISP-1 IN EXPERIMENTAL AND HUMAN OA
does occur in the cartilage, as indicated by ␤-catenin
accumulation. In addition, the prominent expression of
WISP-1 in cartilage suggests that Wnt signaling is activated in this tissue. Possibly, Wnt molecules that are
produced in the synovium diffuse into the cartilage.
In both models of OA we identified many genes
that were up-regulated, especially in the synovium.
These genes may be involved in common OA-inducing
mechanisms. However, several were differentially regulated in collagenase-induced OA compared with spontaneous OA in STR/Ort mice. These genes may be
involved in processes that are specific to the individual
models, and more detailed mechanistic understanding of
the models is needed in order to consider the implications of these differences.
IL-1 is a potent inducer of metalloproteinases
and a strong inducer of cartilage damage during experimental arthritis (14,44). However, the involvement of
IL-1 in OA is unclear, as has been found both in clinical
studies and in experimental OA (45,46). We have identified WISP-1 as a mediator that is capable of inducing
MMPs and ADAMTS-4 and -5, independently of IL-1.
Due to the complex nature of the Wnt signaling pathway, the importance of balanced Wnt signaling for joint
integrity, and the diversity of Wnt proteins that are
regulated in OA, it is arguable that the modulation of
several Wnt family members is necessary for achievement of therapeutic efficacy; this was evidenced by the
FrzB genetic data. An alternative may be to modulate a
key Wnt target gene such as WISP-1. Since several Wnt
proteins have the potential to converge on WISP-1,
suppression of WISP-1 function may represent an attractive therapeutic target for OA disease modification;
however, further work is needed to determine whether
there are any safety liabilities associated with WISP-1
modulation.
In conclusion, we have demonstrated for the first
time that WISP-1 is overexpressed during OA and is
capable of inducing cartilage damage in vivo. Further
studies are needed to determine whether WISP-1 is
critically required for cartilage loss in OA.
ACKNOWLEDGMENTS
We are grateful to all patients involved in the study,
and to the orthopedic surgeons at the King’s Mill Centre for
Healthcare Services (Sutton in Ashfield, UK) for providing
clinical material. We especially thank Dr. David Walsh (University of Nottingham, Nottingham, UK) and Dr. Deborah
Wilson (Kings Mill Hospital, Sutton in Ashfield, UK) for
supplying human cartilage tissue, and Dr. Maarten de Waal
511
Malefijt (Department of Orthopedics, Radboud University
Nijmegen Medical Center) for supplying human synovial tissue.
AUTHOR CONTRIBUTIONS
Dr. Blom had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Blom, Brockbank, van der Kraan, Newham, van den
Berg.
Acquisition of data. Blom, Brockbank, van Beuningen, Geurts, Takahashi, van de Loo, Schreurs, Clements.
Analysis and interpretation of data. Blom, Brockbank, van Lent, van
Beuningen, Takahashi, van der Kraan, van de Loo, Clements, Newham, van den Berg.
Manuscript preparation. Blom, Brockbank, van Lent, van der Kraan,
Newham, van den Berg.
Statistical analysis. Blom, van der Kraan, Newham.
ROLE OF THE STUDY SPONSOR
AstraZeneca applied no restrictions to publication of the
manuscript.
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experimentov, induced, involvement, protein, osteoarthritisprominent, role, signaling, human, pathways, wnt
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