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Inverse relationship between matrix remodeling and lipid metabolism during osteoarthritis progression in the STRORT mouse.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 9, September 2007, pp 2999–3009
DOI 10.1002/art.22836
© 2007, American College of Rheumatology
Inverse Relationship Between Matrix Remodeling and
Lipid Metabolism During Osteoarthritis Progression in
the STR/Ort Mouse
James W. Watters, Chun Cheng, Maureen Pickarski, Gregg A. Wesolowski, Ya Zhuo,
Tadashi Hayami, Wei Wang, John Szumiloski, Robert L. Phillips, and Le T. Duong
Objective. The biologic changes associated with
osteoarthritis (OA) are incompletely understood. The
aim of this study was to elucidate the molecular mechanisms underlying OA progression in an STR/Ort murine model of spontaneous disease.
Methods. Global patterns of gene expression
were assessed using microarray analysis of articular
cartilage/subchondral bone from the tibial plateaus of
STR/Ort mice at 3, 9, and 12 months of age. The
age-dependent severity of osteophyte formation and
extent of cartilage damage were determined in the
corresponding femurs using microfocal computed
tomography and the Mankin histologic scoring system.
Pathway analysis was used to identify the functions of
genes associated with OA progression, and changes in
gene expression were confirmed using immunohistochemistry.
Results. Six hundred twenty-one genes were
associated with both osteophyte formation and cartilage damage in the STR/Ort joints. Genes involved in
the development/function of connective tissue and in
lipid metabolism were most significantly enriched and
regulated during disease progression. Genes directly
interacting with peroxisome proliferator–activated receptor ␣ (PPAR ␣ )/PPAR ␥ were down-regulated,
whereas those genes involved with connective tissue
remodeling were up-regulated during disease progression. Associations of down-regulation of myotubularinrelated phosphatase 1 (a phosphoinositide 3-phosphatase
involved in lipid signaling) and up-regulation of biglycan (a member of the small leucine-rich protein
family known to modulate osteoblast differentiation and
matrix mineralization) with OA progression were confirmed by immunohistochemistry.
Conclusion. Since adipogenesis and osteogenesis
are inversely related in the developing skeletal tissue,
these results suggest that a shift in the differentiation of
mesenchymal cells from adipogenesis toward osteogenesis is a component of the OA pathophysiologic processes occurring in the tibial plateau joints of STR/Ort
mice.
Osteoarthritis (OA), a disease associated with
reduced synovial joint function and increased pain, is a
major cause of disability in humans (1). There are no
consistently effective methods for preventing OA or
slowing its progression, and symptomatic treatments
provide limited benefit for many patients. Gross changes
in the structure and content of articular cartilage, subchondral bone, synovial membrane, joint ligaments, and
tendons have been described for many years in patients
with OA (2). However, the molecular changes associated
with OA in these tissues have only recently begun to be
elucidated (for review, see refs. 3–8).
Important features of OA include the degradation of articular cartilage and remodeling of subchondral
bone (9). Cartilage damage is thought to be mediated
through excess synthesis and release of catabolic factors
including proinflammatory cytokines, matrix metalloproteinases (MMPs), and nitric oxide, as well as a
reduced synthesis of anabolic factors such as insulin-like
growth factor 1 (IGF-1) (10,11). However, other studies
James W. Watters, PhD, Chun Cheng, PhD, Maureen Pickarski, MS, Gregg A. Wesolowski, MS, Ya Zhuo, PhD, Tadashi
Hayami, MD, Wei Wang, PhD, John Szumiloski, PhD, Robert L.
Phillips, PhD, Le T. Duong, PhD: Merck, West Point, Pennsylvania.
Drs. Watters and Cheng contributed equally to this work.
Drs. Watters, Wesolowski, Wang, Szumiloski, Phillips, and
Duong own stock in Merck.
Address correspondence and reprint requests to Le T. Duong,
MD, Department of Molecular Endocrinology, Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486. E-mail:
le_duong@merck.com.
Submitted for publication September 22, 2006; accepted in
revised form May 16, 2007.
2999
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indicate that OA is likely to be a systemic disease,
involving stromal cell differentiation and lipid metabolism (12). Indeed, generalized changes in many tissues of
the joints have been observed, including increased adiposity (13), muscle weakness (14), and weakening of the
anterior cruciate ligament (ACL) (15). Because adipocytes share a common mesenchymal cell precursor with
chondrocytes, tenocytes, and osteoblasts (16), a biologic
link between lipid metabolism and connective tissue
remodeling may be a central component of OA.
In support of this hypothesis, recent studies have
shown that up-regulation of peroxisome proliferator–
activated receptor ␥ (PPAR␥) signaling is therapeutic in
surgically induced OA or type II collagen–induced arthritis in animal models (17,18). Although PPAR␥ was
originally identified as a key regulator of lipid metabolism and adipocyte differentiation (19,20), more recent
evidence has suggested that activation of PPAR␥ can
also regulate inflammatory responses. For example,
PPAR␥ has been shown to be a negative regulator of
macrophage activation (21). Moreover, PPAR␥ activation inhibits the production of interleukin-1␤ (IL-1␤),
tumor necrosis factor ␣ (TNF␣), and IL-6 in monocytes
(22). In addition, PPAR␥ agonists can inhibit the production of MMP-13 in human chondrocytes and MMP-1
in human synovial fibroblasts (23,24).
It has therefore been suggested that PPAR␥
agonists exert their therapeutic effects on surgically
induced or collagen-induced arthritis through the suppression of these inflammatory mediators (17,18).
However, these animal models involve an external
mechanism of inflammation in the development of
OA, and therefore the disease in these models may
not be representative of generalized OA in humans.
As such, the current level of understanding has not
yielded a satisfactory hypothesis as to how spontaneous OA is initiated or what major signaling mechanisms are involved in the progression of spontaneous,
idiopathic OA.
In order to identify the molecular mechanisms
underlying the progression of spontaneous OA, we
analyzed the changes in gene expression that occur in
affected joints during OA progression in the STR/Ort
mouse, a strain derived from the common inbred strain
STR/1N. STR/Ort animals spontaneously develop histologic lesions resembling those of human OA, with ⬃85%
of male STR/Ort mice developing the disease in the
medial tibial plateau at age 1 year (25). Using genomewide expression profiling and functional analysis, development and function of the connective tissue and lipid
metabolism were shown to be the biologic functions that
WATTERS ET AL
are most significantly up-regulated and down-regulated,
respectively, during OA progression. Furthermore,
genes regulated by PPAR␣ and PPAR␥ were downregulated in a coordinated manner during disease progression. These results suggest that PPAR signaling is
down-regulated during the progression of OA in STR/
Ort animals, and that a shift away from adipocyte
formation and toward osteoblast differentiation in mesenchymal precursor cells is an important component in
this spontaneous, idiopathic model of OA in mice.
MATERIALS AND METHODS
Collection of joint samples and tissue preparation. A
colony of STR/Ort mice was established from 3 original
breeding pairs obtained from Dr. R. M. Mason (Imperial
College of Medicine, London, UK) (25). The joints from both
hind limbs were collected from 3 groups of male STR/Ort
mice at 3, 9, and 12 months of age. After careful disarticulation, the femur and corresponding tibia from each joint were
collected for histology and RNA preparation, respectively. The
femur was fixed in 4% paraformaldehyde (Fisher Scientific,
Fair Lawn, NJ) in phosphate buffered saline (PBS). The
corresponding tibia was carefully cleaned of attached ligaments and muscle.
Tibial articular cartilage and subchondral bone were
microdissected together by collecting a 200-␮m–thick section
measured from the cartilage surface of the tibial plateau. From
each group of mice, the cartilage/bone sections from the left
and the right tibia were quickly frozen and stored in liquid
nitrogen until processing for RNA. In addition, the dorsal
segments of the ribs, from approximately equal lengths (2 mm)
of cartilage and bone, were collected from each mouse. The rib
tissues were pooled for RNA preparation.
Mankin histologic scores of cartilage changes. Semiquantitative histopathologic grading was performed as described previously (9), according to a modified Mankin scoring
system established for grading OA changes (26–28). The 5
subcategories of the Mankin score evaluated were structure,
chondrocyte number, chondrocyte clustering, proteoglycan
content, and subchondral plate and/or tidemark changes. After
being scanned by microfocal computed tomography (microCT), the femurs were decalcified in 0.5M EDTA, pH 7.6, and
embedded in paraffin in the same orientation.
OA develops focally in STR/Ort mice, and it is therefore very difficult to compare the histopathologic characteristics of OA cartilage between mouse joints. This limitation is
frequently attributable to the technical inability to collect
histologic sections at the same depth of tissue in the joints. We
therefore collected 20 sections, each of 5 ␮m in thickness and
50 ␮m apart, from each femur. Three toluidine blue–stained
sections were carefully selected from each sample on the basis
of whether they were of comparable orientation and tissue
depth. After microscopic evaluation of each section of cartilage, the modified Mankin score was assessed, as previously
described (29). Briefly, scores were assessed for the severity of
cartilage surface structural damage (scores 0–10), changes in
cellularity (scores 0–4), cell clustering (scores 0–4), pericellu-
MATRIX REMODELING AND LIPID METABOLISM IN MURINE OA
lar staining (scores 0–4), and matrix proteoglycan staining
(scores 0–4), and the maximum score was 26 (28).
Immunohistochemistry. Tissue sections were deparaffinized in xylene, hydrated in graded ethanol, and then treated
with 500 units/ml testicular hyaluronidase (Sigma, St. Louis,
MO) at 37°C for 15 minutes. Endogenous biotin and biotin
binding activity were blocked with an avidin–biotin blocking kit
(Zymed, Burlingame, CA) followed by serum blocking. Tissue
sections were then incubated with either anti-human biglycan
antibodies (R&D Systems, Minneapolis, MN) or anti-human
myotubularin-related phosphatase 1 (MTMR-1) antibodies
(Abgent, Bioggio-Lugano, Switzerland), overnight at 4°C.
For immunostaining to detect MTMR1, sections were
rinsed in PBS with 0.3% Tween 20 and then incubated with
biotin-conjugated anti-rabbit antibodies (Vector, Burlingame,
CA) for 30 minutes, followed by streptavidin–horseradish
peroxidase conjugate (Zymed) for 10 minutes. These sections
were again rinsed with PBS, developed using the aminoethylcarbazole chromogen of the Histostain SP kit (Zymed), and
counterstained with hematoxylin. For immunostaining to detect biglycan, sections were rinsed in PBS with 0.3% Tween 20
and then incubated with biotin-conjugated anti-goat antibodies
(Vector) for 30 minutes, followed by high-sensitivity
streptavidin–horseradish peroxidase (R&D Systems) for 30
minutes. Sections were rinsed and developed to a brown color
using 2.5% 3,3⬘-diaminobenzidine. For controls, the same
procedures were carried out in the absence of the primary
antibodies.
Osteophyte score. The left and right femurs were fixed
in 4% paraformaldehyde for 24 hours, and immersed in 70%
ethanol. The distal regions were scanned by micro-CT (␮CT40; Scanco Medical, Bassersdorf, Switzerland) at a resolution
of 12 ␮m. Osteophytes were scored by 2 independent observers
(MP and GAW). A grading system was developed whereby
joints were assigned an osteophyte score of 0–5 depending on
the number and size of the osteophytes observed.
Gene expression profiling. Total RNA was isolated
from mouse tissues and converted to fluorescently labeled
complementary RNA (cRNA) that was hybridized to DNA
oligonucleotide microarrays as described previously (30,31).
Briefly, 4 ␮g of total RNA from each tissue sample was used to
synthesize double-stranded DNA through reverse transcription. The cRNA was produced by in vitro transcription and
labeled postsynthetically with Cy3 or Cy5. The cRNA derived
from individual tibial plateaus (experimental sample) were
hybridized against pools of cRNA from the rib tissue derived
from the same individuals (reference sample). Separate reference pools were constructed for each time point to account for
age-induced changes in gene expression. This hybridization
scheme was used to 1) remove the majority of age-related
genes, 2) enrich for genes locally regulated in weight-bearing
joints (the knee), and 3) normalize the reference values for
different rates of disease progression between the left and the
right knee joints and between animals within the same age
group.
Two hybridizations were done with each pair of cRNA
samples, using a fluorescent dye reversal strategy. The microarrays contained 23,564 probes that were representative of
genes or expressed sequence tags. Probe sequences were
chosen to maximize gene specificity and minimize the 3⬘replication bias inherent in reverse transcription of messenger
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RNA (mRNA). In addition, the microarrays contained ⬃100
control probes for quality control purposes. All probes on the
microarrays were synthesized in situ with inkjet technology
(Agilent Technologies, Palo Alto, CA) (30). After hybridization, arrays were scanned, and fluorescence intensities for each
probe were recorded. Ratios of transcript abundance (experimental to control) were obtained following normalization and
correction of the array intensity data. Gene expression data
were analyzed using Rosetta Resolver gene expression analysis
software (version 5.1; Rosetta Biosoftware, Seattle, WA).
Identification of genes associated with disease phenotypes. In order to identify genes associated with the osteophyte
score, joints were placed into 2 groups (group 1 comprising
samples with osteophyte scores ⱕ1; group 2 comprising samples with osteophyte scores ⱖ4), and an analysis of variance
(ANOVA) calculation was performed to identify probes differentially expressed between groups. Correlation analysis was
used to identify genes associated with the Mankin score. An
estimated false discovery rate (FDR) of 5%, based on 500
permutations of the data, was used to determine the thresholds
for significant values in the ANOVA and correlation analyses.
The significance of the size of overlap between genes associated with each disease phenotype was calculated using the
hypergeometric distribution.
Gene function and network analysis. Genes identified
as being positively or negatively associated with the Mankin
score and the osteophyte score were used for network and gene
function analyses. These genes comprised the seed set. Locus
identification numbers were imported into the Ingenuity Pathway Analysis (IPA) system (Ingenuity Systems, Mountain
View, CA), and genes were then categorized based on the
published findings regarding biochemical, biologic, or molecular functions. Calculations of the P value for enrichment of
gene functions were based on the hypergeometric distribution.
The identified genes were also mapped to interaction
networks as described previously (32). Briefly, the construction
of interaction networks involves 1) overlay of genes identified
as significant from the experimental data onto the IPA interactome, 2) determination of the specificity of connections
between genes by calculating the percentage of each gene’s
connections to other significant genes (networks are grown
from genes with the highest specificity connections), and 3)
assessment of the significance of the identified networks by
determining the probability that a collection of genes with a
sample size equal to or greater than the number in the network
could be achieved by chance alone. The resulting networks are
ranked by score, with a score of 3 indicating that there is a
lower than 1 in 1,000 chance that the focus genes are in a
network due to random chance. Networks with a ranking score
⬎3 were considered significant.
RESULTS
Gross morphologic and histologic features of
individual joints. Male STR/Ort mice were killed for
morphology and histology. OA-like pathologic features
were surveyed by histologic evaluation of the individual
joints from mice at 3, 9, and 12 months of age (n ⫽ 15–18
samples per group). Typically, the normal femoral con-
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WATTERS ET AL
Figure 1. Evaluation of the progression of osteoarthritis (OA) in STR/Ort mice. A, The pathogenesis of age-dependent OA progression was
assessed histologically in normal femoral condyles of 3-month-old STR/Ort animals, revealing typical intact cartilage integrity (Mankin scores 0–1)
accompanied by limited penetration of calcified tissue into the articular cartilage (arrows) (a). Inset, Higher magnification view of the boxed area
in a. As early as 3 months of age, joints developed more advanced disease, with focal cartilage surface damage (Mankin score 8) and small
chondro-osteophytes (arrows) (b). Inset, Higher magnification view of the boxed area in b. At 12 months of age, many joints had significant cartilage
thinning (arrows) and osteophytosis, or developed either severe bone sclerosis associated with reductions in bone marrow (BM) space or complete
bone (B) eburnation (Mankin score 22) (c). Original magnification ⫻ 4 in A (a–c); ⫻ 20 in insets of a and b. B, Osteophyte growth (arrows) was
assessed by microfocal computed tomography of the coronal (a and c) and axial (b and d) planes of the femur of a 12-month-old CBA mouse as
control (a and b) or an age-matched STR/Ort mouse (c and d). The osteophyte score (range 0–5) was assigned based on the overall size and extent
of the osteophytes as assessed on all axial plane images for each femur. Asterisks in d indicate subchondral sclerosis.
dyles (Figure 1A, panel a) and tibial plateaus (results not
shown) of the joints of 3-month-old STR/Ort mice had
intact cartilage integrity (Figure 1A, panel a, inset),
occasionally accompanied by limited penetration of calcified tissue from the subchondral surface (Figure 1A,
panel a, arrows). In contrast, OA-associated lesions
could be readily observed in joints from mice as early as
3 months of age (Figure 1A, panel b, inset), including a
loss of cartilage cellularity and proteoglycan staining,
increase in focal surface damage, bone growth into the
cartilage, and growth of small osteophytes (Figure 1A,
panel b, arrows). As the animals aged, surface erosion
became more severe, accompanied by increases in proteoglycan loss, osteophyte formation, and subchondral
bone sclerosis (Figure 1A, panel c). By 12 months of age,
complete loss of cartilage and bone eburnation could be
observed in the animals (Figure 1A, panel c).
At each time point, joint damage was assessed by
measuring the presence and severity of osteophytes and
extent of cartilage damage. Micro-CT scans were performed on individual joints, and presence/severity of
osteophytes was scored by 2 independent observers. A
grading system was developed whereby joints were assigned a score of 0–5 depending on the number and size
of the observed osteophytes. Figure 1B shows micro-CT
images of the coronal and axial planes of a typical
normal femur from a 12-month-old CBA mouse (Figure
1B, panels a and b) compared with those from an
age-matched STR/Ort mouse, the latter of which
showed severe osteophyte development (Figures 1B,
panels c and d).
Cartilage damage was assessed using the modified Mankin scores for various joint histologic features
(as described in Figure 1 and in Materials and Methods).
As illustrated in Figures 2A and B, the osteophyte scores
and Mankin scores of individual joints showed an agedependent increase, and the disease progression in
individual joints from the same animal appeared to
behave in an independent manner. Two-way ANOVA
calculations revealed that both the osteophyte score and
the Mankin score of joints from mice at 9 months of age
and at 12 months of age were significantly increased
compared with these scores at 3 months of age (Figures
2A and B).
MATRIX REMODELING AND LIPID METABOLISM IN MURINE OA
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Figure 2. Assessment of osteoarthritis-related pathologic markers as a function of increasing age in STR/Ort mice. Osteophyte scores determined
by microfocal computed tomography (␮CT) (A) and Mankin scores of histologic changes to the cartilage (B) were calculated as described in
Materials and Methods. Scores for individual joints are shown separately for the left (L) and right (R) femurs. Broken lines connect data points
(open circles) from the same animal. Solid circles with bars represent the mean and SEM for each age group. Triangles represent the scores for
animals in which only 1 joint was phenotyped. Raw P values (by analysis of variance) for differences between age groups were calculated as follows:
in A, P ⫽ 0.004, P ⫽ 6 ⫻ 10⫺4, and P ⫽ 0.037 for age 3 months versus 9 months, age 3 months versus 12 months, and age 9 months versus 12 months,
respectively; in B, P ⫽ 0.012, P ⫽ 9 ⫻ 10⫺5, and P ⫽ 0.044 for age 3 months versus 9 months, age 3 months versus 12 months, and age 9 months
versus 12 months, respectively.
Interestingly, disease progression was not observed in all of the joints assessed. No osteophytes were
observed in 4 of 18 joints from 3-month-old animals, 2 of
18 joints from 9-month-old animals, and 1 of 15 joints
from 12-month-old animals (Figure 2A). This observation is consistent with previous reports of incomplete
disease penetration at 1 year of age (25). Whereas 4 of
the 18 joints from 3-month-old animals showed absence
of osteophyte development, all 18 joints from the
3-month-old animals showed Mankin scores ⱖ1 (Figure
2B), indicating that mild cartilage damage precedes
osteophyte development in the STR/Ort murine disease
model. This is consistent with the observations in the rat
ACL transection model of OA, in which cartilage damage has also been shown to precede the development of
osteophytes (29).
Joint anabolic and catabolic factors. Data on
gene expression were generated from the same joints
used to assess disease phenotypes. Joint tissue, comprising articular cartilage and subchondral bone, was obtained from the mouse tibial plateaus for profiling on
Agilent Technologies oligonucleotide-based DNA microarrays. Due to the small size of rodent joints and the
inherent difficulties in separating articular cartilage
from bone, the tibial plateaus that contained both
articular cartilage and subchondral bone tissue were
microdissected together in a 200-␮m section. The tibial
tissue section from each animal was processed for profiling studies.
To assess the catabolic and anabolic processes
that occur in STR/Ort joints, we first analyzed the
expression of candidate genes involved in biologic functions thought to be important for the initiation or
progression of OA. We chose candidate genes that are
commonly described in the literature, from 4 general
categories: 1) cartilage components (Col2a1, Agc1,
Hapln1, and Comp), 2) cartilage catabolism (Adamts5,
Il1b, and Il6), 3) osteoclast function (Ctsk and Mmp9),
and 4) bone anabolism (Igf1 and Tgfb1). Figure 3 shows
the regulation of these genes in the STR/Ort joints by
age groups.
The expression levels of cartilage components,
cartilage catabolic factors, and genes indicative of osteoclast function were significantly higher in the joints
of mice at 3 months of age compared with 12 months of
age (P ⬍ 0.05). This is again consistent with the observations in the rat ACL transection model of OA, in
which subchondral bone resorption and cartilage loss are
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Figure 3. Regulation of candidate genes involved in murine osteoarthritis. Genes were assigned to 1 of 4 categories: 1) cartilage components (Col2a1, Agc1, Hapln1, and Comp), 2) cartilage catabolism
(Adamts5, Il1b, and Il6), 3) osteoclast function (Ctsk and Mmp9), and
4) bone anabolism (Igf1 and Tgfb1). Data represent the log10 of the
ratio of expression in each joint sample relative to the pooled reference
sample from mouse rib tissue. Genes that were up-regulated relative to
the reference sample are shown in magenta, while genes that were
down-regulated relative to the reference sample are shown in cyan.
Asterisks denote genes whose average expression in the joints from
mice at age 3 months was significantly higher than that in mice at age
12 months (P ⬍ 0.05 by analysis of variance).
early events during disease progression (9,29). Igf1 and
Tgfb1, which are genes that have been previously implicated in osteophyte development in experimentally induced OA in the mouse and rat (9,33), were not
up-regulated with increasing age in the STR/Ort joints.
Since these joints clearly showed an increase in the
presence and severity of osteophytes at 12 months of age
relative to the findings at 3 months of age (Figure 2A),
these data suggest that other molecular mechanisms are
contributing to osteophyte development in STR/Ort
animals.
Genes significantly associated with joint pathologic changes. We then performed a genome-wide analysis to identify the genes associated with disease progression in STR/Ort joints. In order to identify the genes
associated with osteophyte formation, joints were placed
into either group 1 (osteophyte score ⱕ1) or group 2
(osteophyte score ⱖ4). An ANOVA calculation was
WATTERS ET AL
performed, and 2,214 genes were identified as being
significantly differentially expressed between groups
(P ⬍ 0.005); this significance threshold was selected so
that the FDR in 500 random permutations was lower
than 5%. In order to identify the sequences associated
with cartilage damage, we performed a correlation analysis, and 3,624 genes were identified as being significantly correlated with the Mankin histologic score (Pearson’s r ⬎0.45, FDR ⬍5%).
Six hundred twenty-one genes were identified as
being associated with both the osteophyte score and the
Mankin score (P for overlap ⬍ 0.001). The expression
levels of 331 genes were up-regulated with increasing
age, while 290 genes were down-regulated with increasing age (results not shown). These findings suggest that
the development of OA in STR/Ort animals involves
both the up-regulation and the down-regulation, coinciding with increasing age, of similar numbers of genes
associated with disease pathology.
Functions of genes in relation to disease progression. In order to gain insights into the biologic processes
and signaling networks involved in OA progression, we
performed analyses of the biologic functions and contributory pathways in the STR/Ort joints, in order to
uncover the relationships among genes associated with
disease progression in this model. Instead of focusing on
individual genes or a group of candidate genes that were
previously demonstrated to change with disease progression, we utilized a different approach to allow a more
unbiased assessment of the biologic processes underlying disease progression in the STR/Ort model. This
approach involved use of the IPA software tool as
described in Materials and Methods (see the Ingenuity
Systems Web site at http://www.ingenuity.com), which
enabled us to identify the gene functions that were
statistically significantly enriched among the 621 genes
described as being associated with the osteophyte and
Mankin histologic scores. Note that others have previously used the IPA tool to identify biologic networks
involved in complex processes, including inflammation,
glucocorticoid receptor signaling, and cancer (32,34,35).
The general function most significantly enriched
among the 331 genes up-regulated during disease progression was the development and function of the connective tissue (P ⫽ 9.32 ⫻ 10⫺5–4.55 ⫻ 10⫺2). The
specific function most significantly associated with the
group of up-regulated genes was patterning of bone
(P ⫽ 9.32 ⫻ 10⫺5 for the genes Bgn, Cdx1, Enpp1, Ggt1,
Nog, Ptn, Ptprv, Src, and Wnt3a), which is consistent with
the development of osteophytes with increasing age in
STR/Ort animals (Figure 2A). The general function
MATRIX REMODELING AND LIPID METABOLISM IN MURINE OA
Figure 4. Inverse age-dependent regulation of murine genes involved
in connective tissue development/function and lipid metabolism during
osteoarthritis progression. Data represent the log10 of the ratio of
expression in each tibial sample relative to the pooled reference
sample of rib tissue derived from age-matched individual mice. Genes
that were up-regulated relative to the reference sample are shown in
magenta, while genes that were down-regulated relative to the reference sample are shown in cyan.
most significantly enriched among the group of 290
down-regulated genes was lipid metabolism (P ⫽ 8.08 ⫻
10⫺7–4.93 ⫻ 10⫺2), and modification of fatty acid was
the most significantly enriched specific function (P ⫽
8.08 ⫻ 10⫺7 for the genes Acas2, Acox1, Amacr, Ech1,
Gpam, Hadha, Hadhb, Phyh, and Slc27a2). These findings suggest that a general down-regulation of genes
involved in lipid metabolism may play a key role in OA
pathogenesis. The inverse time-dependent regulation of
genes identified as being involved in the development
and function of the connective tissue and in lipid metabolism is summarized in Figure 4.
Interaction networks. In order to further assess
the biologic processes associated with lipid metabolism
during OA progression, we investigated, with the use of
the IPA platform, interaction networks formed by the
above-described 290 genes identified as being downregulated in association with disease progression. This
system identifies biologic interaction networks among
genes of interest by mining published findings regarding
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biochemical, biologic, or molecular interactions (see
Materials and Methods).
Interestingly, 2 networks that contain PPAR transcription factors as central nodes were observed to be
highly significant. As shown in Figure 5A, PPAR␣ was
identified as a central node of the most significant
interaction network identified among the downregulated genes (significance score of 23). This network
includes 3 transcriptional targets of PPAR␣ (Acox1, Fgg,
and Acas2, as highlighted in Figure 5A). In addition, a
second significant interaction network (significance
score of 14) contained PPAR␥ as a central node (Figure
5B). This network includes 4 transcriptional targets of
PPAR␥ (Fasn, Vnn1, Cyp4b1, and Tpm2, as highlighted
in Figure 5B) and 1 gene known to physically interact
with PPAR␥ (Smarcd3). These results suggest that reduction of PPAR signaling is an important mechanism
underlying the progression of OA in the STR/Ort
mouse. This is supported by the results of previous
studies that have suggested a therapeutic role for
PPAR␥ activation in animal models of surgically induced OA (17).
Relative protein expression of MTMR-1 and biglycan. To provide an additional level of validation of
these changes in mRNA expression, we performed a
qualitative assessment of the protein levels in the STR/
Ort joints by immunohistochemistry. We chose to evaluate protein expression of the signature genes for which
immunohistochemistry reagents are available for use in
a murine system. Among the signature genes that correlated with OA progression in the STR/Ort mice, the
protein expression of MTMR-1 and biglycan, as assessed
by immunohistochemical methods, was further verified
(Figure 6).
MTMR-1, a phosphoinositide 3-phosphatase involved in lipid signaling, was found to be highly expressed in the joints of 3-month-old mice compared with
12-month-old mice (Figures 6A and B, brown stain). In
the joints of 3-month-old mice, MTMR-1 appeared to be
highly expressed in the hypertrophic chondrocytes and,
to a lesser extent, in chondrocytes near the articular
surface (results not shown). MTMR-1 expression was
not detected in the ligaments and muscles surrounding
the joints. The regulation of MTMR-1 protein expression was consistent with our findings of the general
down-regulation of genes involved in lipid metabolism
occurring in conjunction with OA progression (as shown
in Figure 4).
Conversely, biglycan, a small leucine-rich proteoglycan that plays a critical role in the formation of
collagen fibrils, was highly expressed in the joints of
3006
WATTERS ET AL
Figure 5. Networks involving peroxisome proliferator–activated receptor ␣ (PPAR␣) (A) and PPAR␥ (B) formed by genes down-regulated during
osteoarthritis progression in mice. Genes indicated in green were identified by microarray analysis (comprising the seed set as described in Materials
and Methods), while all other genes were brought into the network based on their known interactions with genes in the seed set. The intensity of
the color represents the mean level of down-regulation in mice at age 12 months relative to that in mice at age 3 months. Direct PPAR␣ or PPAR␥
interactions are denoted by blue arrows.
12-month-old mice compared with 3-month-old mice
(Figures 6D and E). In the joints of 12-month-old mice,
we detected high levels of biglycan protein in the
hypertrophic cartilage/bone interphase and in cells lining the surfaces of articular cartilage and subchondral
trabecular bone. This increase in protein expression of
biglycan supports our observations of the up-regulation
of genes involved in connective tissue development and
function occurring in parallel with OA progression (also
shown in Figure 4).
Figure 6. Immunohistochemical analysis of the relative protein expression levels of myotubularin-related phosphatase 1 (MTMR-1) and
biglycan, to illustrate the inverse relationship between adipogenesis
and bone matrix formation in mice. A and B, Expression of MTMR-1,
a protein involved in lipid signaling, was found to be higher in cartilage
of a typical 3-month-old mouse (A) compared with the levels in
cartilage of a typical 12-month-old mouse (B), suggesting a downregulation in lipid signaling. MTMR-1 expression was primarily found
in articular cartilage. D and E, Expression of biglycan, a marker of
bone formation, increased with age-dependent disease progression,
and higher levels of biglycan were detected in the joints of a typical
12-month-old mouse (E) compared with levels in the joints of a typical
3-month-old mouse (D). Biglycan was primarily found in articular
cartilage and in cells lining the articular cartilage and the subchondral
trabecular bone surface. C and F, Controls for analyses of MTMR-1
(C) and biglycan (F) comprised nonspecific staining in the absence of
primary antibodies.
DISCUSSION
OA is currently considered to be a complex joint
disease in which all tissues in the joints play an important
role in disease initiation and/or progression. It has long
been suggested that the progression of articular cartilage
degeneration is concomitant with intense remodeling of
the subchondral bone and increased bone stiffness,
leading to abnormal mechanical stress across the overlying cartilage (36,37). Indeed, increased subchondral
bone activity in OA patients, as determined by enhanced
uptake of scintigraphic technetium-labeled disphosphonate, has been shown to precede detectable cartilage loss
(38), and the bone formation marker osteocalcin was
MATRIX REMODELING AND LIPID METABOLISM IN MURINE OA
reported to be higher in synovial fluid from patients with
severe scintigraphic signals compared with that from
patients with mild alterations on knee scans (39). Unlike
patients with osteoporosis, OA patients tend to have a
high body mass index together with an elevated rate of
bone turnover, resulting in increased bone density (40).
This suggests that the process of new bone synthesis
exceeds degradation in susceptible individuals.
Studying the pathogenesis of OA in humans is
hampered by inherent difficulties such as a lack of
availability of normal and diseased tissue at early stages
of the disease. Therefore, animal models of OA are
essential for understanding disease etiology and for the
development of effective therapies. Although numerous
animal models have been developed and characterized
(for review, see ref. 41), many of these models involve
surgical intervention or inflammatory stimuli to induce
disease. In models of OA involving surgically induced
joint instability in several species, both bone and cartilage changes occur concomitantly (29). In contrast, in
animal models in which a spontaneous OA-like disease
develops (resembling human disease), increased bone
density and osteoid volume are often more severe than
cartilage changes. For example, Dunkin-Hartley guinea
pigs (42) and cynomolgous macaques (43) have agerelated changes in bone that precede those in cartilage.
Evidence demonstrating that subchondral bone remodeling is linked to cartilage destruction in both humans
and animals is well accepted; however, the mechanisms
by which the changes in subchondral bone influence
articular cartilage are incompletely understood.
In this study, we sought to elucidate the molecular mechanisms underlying disease progression in the
spontaneous STR/Ort mouse model. STR/Ort mice
share physical characteristics similar to those in human
subjects with OA, such as high body weight and high
bone mineral density. In addition, a number of biochemical features of OA in STR/Ort mice are similar to the
changes observed in human disease, such as matrix
proteoglycan depletion by MMPs and aggrecanase (25).
Due to recent progress in mouse genomics, the STR/Ort
model provides a unique opportunity to investigate the
events associated with the initiation and progression of
spontaneous OA.
We used gene expression profiling to understand
how biologic pathways change with disease progression
in the STR/Ort model. By associating gene expression
changes with cartilage damage and micro-CT scores in
SRT/Ort joints, we were able to identify the genes that
were up- or down-regulated with disease progression.
Rather than focusing on individual candidate genes, we
analyzed biologic functions in an unbiased manner by
3007
leveraging informatics tools to identify ascribed biologic
functions that were statistically significantly associated
with the disease. In this way, we can build a more
pathway-centric view of disease biology that leverages
information across many genes and is not reliant on
individual genes that are chosen for analysis based on
preexisting knowledge of other disease models.
This study is the first to provide evidence that
altered lipid metabolism through a reduction in PPAR
signaling is an important component of spontaneous,
idiopathic OA. While PPAR␥ and PPAR␣ themselves
were not found to be significantly altered at the mRNA
level, multiple direct targets of the PPARs were altered.
We speculate that there is a non–mRNA-based means of
down-regulating the activity of the PPARs, for example,
a posttranslational modification or a binding to other
coactivators or corepressors that alters the activity of
PPAR signaling without actually altering the mRNA for
PPARs. The PPAR␥ agonist rosiglitazone has recently
been shown to be therapeutic for surgically induced OA
in guinea pigs (17), an effect hypothesized to be mediated through the inhibition of proinflammatory signals
in the affected joint.
While no direct link between PPAR␣ and OA
has been reported, PPAR␣ has also been shown to have
antiinflammatory properties (44,45). Therefore, one
possible explanation for the relationship between PPAR
signaling and OA progression is that decreased PPAR
signaling leads to an elevation of inflammation in the
joint microenvironment that favors catabolic signals over
anabolic signals, resulting in the observed cartilage
damage and osteophyte formation. An alternative explanation involves the developmental link between adipogenesis, chondrogenesis, and osteogenesis. These cells
share a common mesenchymal cell precursor that can be
induced to differentiate into one of these cell types in
vitro by adjustment of the culture microenvironment
(46,47). It is thus possible that an early event in the
initiation and progression of OA is a preferential shift
toward osteoblastogenesis resulting from the downregulation of PPAR signaling. This is supported by the
observed increase in subchondral bone formation and
sclerosis associated with disease progression in humans
and animal models of OA.
Cumulative evidence suggests that the dysregulation of subchondral bone metabolism is different in OA,
possibly due to an altered osteoblast phenotype. Indeed,
osteoblasts isolated from the subchondral bone of patients with OA demonstrated altered phenotypes
(48,49). In comparison with normal osteoblasts, OA
osteoblasts produce more alkaline phosphatase, osteocalcin, IGF-1, urokinase plasminogen activator, cyto-
3008
WATTERS ET AL
kines, and eicosanoids, including IL-1␤, IL-6, prostaglandin E2, and leukotrienes. All of these factors could
promote subchondral bone remodeling and are also
involved in deposition and turnover of matrix. Because
of the development of microcracks, vascular channels or
neovascularization may provide a link between subchondral bone tissue and cartilage, potentially enabling these
factors to influence the abnormal metabolism of articular chondrocytes and remodeling of OA cartilage.
Currently, no single affector responsible for
osteoblast-induced cartilage degradation has been identified. Unlike in other animal models of experimentally
induced OA, IGF-1 and transforming growth factor ␤1
(TGF␤1) were not associated with disease phenotypes in
our STR/Ort model, as determined by microarray profiling and reverse transcription–polymerase chain reaction analysis on the same RNA samples used in profiling
studies (results not shown). This suggests that alternative mechanisms are important for the development of
spontaneous OA in the STR/Ort model. Nevertheless,
even with the lack of observed changes in IGF-1 and
TGF␤1 mRNA levels during disease progression, we
cannot rule out the possibility that posttranscriptional
modifications or storage of these growth factors in the
extracellular matrix and release via osteoclastic bone
resorption during subchondral bone remodeling are
alternative mechanisms through which IGF-1 and
TGF␤1 might affect OA. However, their lack of association with disease progression is consistent with recent
reports that IGF-1 and TGF␤1 mRNA levels are not
different between OA and non-OA human bone (50).
In summary, the involvement of bone formation
in OA initiation and progression has been recognized for
many years (36), and our previous work in the rat ACL
transection model of OA supports an early role of bone
remodeling in disease development (9,29). Based on the
data presented herein, we hypothesize that a shift of
mesenchymal cell differentiation from adipogenesis toward osteogenesis in the subchondral region is an important component of the pathogenesis of spontaneous
OA. Due to the observed inverse relationship between
lipid metabolism and matrix remodeling in this spontaneous disease model, it is likely that up-regulation of
PPAR signaling abrogates OA progression by inhibiting
early bone formation, which constitutes a therapeutic
strategy that could be applicable to human OA.
AUTHOR CONTRIBUTIONS
Dr. Duong 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. Watters, Cheng, Zhuo, Hayami, Phillips, Duong.
Acquisition of data. Watters, Pickarski, Wesolowski, Zhuo, Hayami,
Wang.
Analysis and interpretation of data. Watters, Cheng, Pickarski, Wesolowski, Zhuo, Hayami, Wang, Szumiloski, Phillips.
Manuscript preparation. Watters, Pickarski, Duong.
Statistical analysis. Watters, Cheng, Szumiloski, Phillips.
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