Benderdour, M.

Chabane, N.

Fahmi, H.

Martel-Pelletier, J.

Pelletier, J.-P.

Zayed, N.

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180 PGD2 DOWNREGULATES MMP-1 AND MMP-13 EXPRESSION IN HUMAN OSTEOARTHRITIC CHONDROCYTES 

Osteoarthritis and Cartilage Vol. 15, Supplement C C107
suggest a matrix substrate for MMP-28. We have successfully
generated SW1353 cell lines stably transfected with MMP28 (full
length, EA mutant and pro-cat) and ADAMTS-16 (full-length).
Phenotypic changes to adhesion and proliferation have been
measured, along with altered expression of other metallopro-
teinase genes. A more complete analysis including candidate
protein and proteomic screens of potential substrates is currently
underway.
179
CHARACTERIZING THE FUNCTION OF BONE
MORPHOGENETIC PROTEIN-5 (BMP5) IN
CHONDROCYTES
S. Snelling, P.A. Hulley, J. Loughlin
University of Oxford, Oxford, United Kingdom
Purpose: Bone Morphogenetic Protein-5 (BMP5) is a member
of the TGF-b superfamily of growth factors. Variants within the
BMP5 gene have been found to be associated with OA devel-
opment. Furthermore cis-regulatory polymorphism exists within
the BMP5 gene leading to differential allelic expression of BMP5
in the cartilage and other tissues of the articulating joint. These
observations make BMP5 a compelling protein in the OA ﬁeld.
Our aim is to elucidate the role of BMP5 in chondrocytes to shed
light on its mechanism of action in OA pathogenesis.
Methods: ATDC5 cells are a well characterized pre-chondro-
genic murine cell line. Conﬂuent cells can be stimulated upon
addition of insulin and ascorbic acid to the culture medium to
undergo chondrogenesis in sequential progression over a 21-day
period.
ATDC5 cells were cultured for 0, 3, 10, 15 and 21 days in
chondrogenic medium (culture medium plus insulin and ascor-
bic acid). Cells were serum-starved overnight prior to treatment.
Cells were then treated with 20% FCS, BMP5, BMP2, a no-
treatment control was also used. After 10 or 30 minutes incu-
bation the cells were lysed and proteins harvested for western
blotting. Western blots were carried out using primary antibodies
against the phospho- and pan- forms of ERK1/2, smad1/5/8 and
p38 MAPK.
Conﬂuent ATCD5 cells were cultured in normal medium, chon-
drogenic medium, BMP5 or BMP5 plus chondrogenic medium for
6 days. Cells were lysed and alkaline phosphataste ALP activity
measured and normalised to DNA content.
ATDC5 cells were exposed for 0, 3, 6 or 10 days post-conﬂuency
to no treatment, chondrogenic medium alone, BMP5 alone or
chondrogenic medium plus BMP5. RNA was harvested, cDNA
synthesized and real-time PCR carried out using primers against
COL10A1, AGC1, COL2A1, BMP4, BACT and 18S.
Results: BMP5 was shown to activate smad, ERK and p38
MAPK signalling at distinct stages of chondrogenesis. In early
chondrogenesis only p38 MAPK signalling was activated by
BMP5 and while the smad and ERK pathways were activated
later in the differentiation program. In contrast BMP2 was able
to activate smad signalling throughout the 21-day chondrogenic
differentiation of ATDC5 cells. This highlights the differential func-
tions of the BMPs and emphasizes the importance of elucidating
the individual roles of these cytokines in cartilage development.
ALP activity was seen to be increased by the addition of BMP5
alone and BMP5 plus chondrogenic medium. No such increase
in ALP activity was seen with the addition of chondrogenic
medium alone. It is known that ALP levels are increased in
hypertrophic chondrocytes and the BMP5 result suggests a pos-
sible role for BMP5 in encouraging chondrocytes to enter hy-
pertrophy. BMP5 treatment caused increased expression of the
chondrogenic markers COL2A1 and AGC1, an upregulation was
also seen with chondrogenic medium and chondrogenic medium
plus BMP5. Interestingly the addition of BMP5 to chondrogenic
or standard medium caused an increase in the expression of
COL10A1, a marker of hypertrophy, on day 6 of chondrogenesis.
This upregulated expression was above that seen with chon-
drogenic medium alone. The increase in COL10A1 expression
following BMP5 treatment is in agreement with ALP results, and
suggests that BMP5 may be able to push chondrocytes towards
hypertrophy. A role of BMP5 in regulating the expression of other
family members involved in chondrogenesis may be indicated by
the decrease in BMP4 expression in cells treated with BMP5.
Conclusions: BMP5 has a distinct role in chondrogenesis and
may act to modulate the passage of differentiating chondrocytes
into hypertrophy. This initial study acts not only as a springboard
for furthering understanding of joint biology but also suggests
potential avenues for the exploration of OA therapeutics.
180
PGD2 DOWNREGULATES MMP-1 AND MMP-13
EXPRESSION IN HUMAN OSTEOARTHRITIC
CHONDROCYTES
N. Zayed, N. Chabane, M. Benderdour, J. Martel-Pelletier,
J-P. Pelletier, H. Fahmi
University of Montreal, Montreal, PQ, Canada
Purpose: Prostaglandin (PG) D2, a cyclooxygenase (COX)
metabolite, has been shown to play critical roles in multiple
physiological processes including inﬂammation. Matrix metallo-
proteinases (MMPs), in particular MMP-1 and MMP-13 produced
by chondrocytes, are major proteases responsible for cartilage
degradation during osteoarthritis. This work was designed to
investigate the effects of PGD2 on interleukin-1 (IL-1)-induced
MMP-1 and MMP-13 expression in human chondrocytes.
Methods: Cultured chondrocytes were stimulated with IL-1 in
the absence or presence of PGD2 and MMP-1 and MMP-13
protein production was evaluated by ELISA. mRNA expression
and promoter activity were analyzed by real-time RT-PCR and
transient transfection experiments, respectively. The expression
of PGD2 receptors, DP1 and CRTH2 was investigated using
RT-PCR and Western blotting.
Results: PGD2 dose-dependently decreased IL-1-induced
MMP-1 and MMP-13 protein expression. Moreover, PGD2 pre-
vented IL-1-induced MMP-1 and MMP-13 mRNA expression as
well as MMP-1 and -13 promoter activation, indicating that this ef-
fect occurs at the transcriptional level. We also demonstrated that
chondrocytes expressed both PGD2 receptors: DP1 and CRTH2.
Conclusions: These data indicate that PGD2 inhibits IL-1-
induced MMP-1 and MMP-13 production by human chondro-
cytes. Therefore, PGD2 may play an important role in the chon-
drocyte response to inﬂammatory stimuli and may protect carti-
lage integrity.
181
COLLAGEN FIBRIL ORIENTATION,
GLYCOSAMINOGLYCANS AND OSTEOARTHROSIS IN
ARTICULAR CARTILAGE OF GROWING AND ADULT
HAMSTERS AND EFFECTS OF VOLUNTARY RUNNING
WHEEL EXERCISE
E.P. HalmesmÃd’ki1, T. NÃd’rhi1, J.T. Holopainen1, J. Rieppo1,
M.M. Hyttinen1, J.P. Arokoski2, H.J. Helminen1
1Department of Anatomy, Institute of Biomedicine, University of
Kuopio, Kuopio, Finland; 2Department of Physical and
Rehabilitation Medicine, Kuopio University Hospital, Kuopio,
Finland
Purpose: The aim was to investigate the collagen ﬁbril orienta-
tion, glycosaminoglycan content, and prevalence of osteoarthro-


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180 PGD2 DOWNREGULATES MMP-1 AND MMP-13 EXPRESSION IN HUMAN OSTEOARTHRITIC CHONDROCYTES

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