Corvol, M.-T.

Dumontier, M.-F.

Presume, M.

Savouret, J.-F.

Tahiri, K.

Widerak, M.

Elsevier - Publisher Connector 

278 EXPRESSION AND FUNCTION OF THE ARYL HYDROCARBON RECEPTOR IN GROWTH PLATE CARTILAGE CELLS IN VITRO 

S124 Poster Presentations / Osteoarthritis and Cartilage 18, Supplement 2 (2010) S45–S256
apoptosis via phosphrylation of p38. Stress-induced chondrocyte apoptosis
was decreased due to inhibition of p38 MAP kinase activation when
chondrocytes were incubated with SB203580 or p38 speciﬁc siRNA trans-
fection. While, the phosphorylation of p38 MAP kinase was increased in
OA chondrocytes. From the point of our results, down regulation p38
MAP kinase activation inhibited chondrocyte death induced by heat stress.
Therefore, evaluation of the interaction between phosphorylation of p38
and chondrocyte apoptosis could be one of the keys to elucidating OA
etiology.
Reference
[1] Hashimoto S, Nishiyama T, Hayashi S, Fujishiro T, Takebe K, Kanzaki N,
et al., Role of p53 in human chondrocyte apoptosis in response to shear
strain. Arthritis Rheum. 2009; 60: 2340-9.
276
CROSS-TALK OF SUBCHONDRAL BONE OSTEOBLASTS AND ARTICULAR
CARTILAGE CHONDROCYTES: A NEW INSIGHT IN UNDERSTANDING
OSTEOARTHRITIS PATHOGENESIS
I. Prasadam, R. Crawford, Y. Xiao
Inst. of Hlth. and bioMed. Innovation, Queensland, Australia
Purpose: The purpose of this study was to address the question of how
subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes
(ACCs) interact with each other with respect to regulation of respective
cells’ phenotypic properties and in particular the involvement of mitogen
activated protein kinase (MAPK) signalling pathways under normal and OA
joint condition. We also endeavoured to test the inﬂuence of cross-talk
between SBOs and ACCs isolated from normal and OA joint on matrix
metalloproteinase (MMP) expression.
Methods: Tissues from the knees of OA patients and normal controls were
collected to isolate SBOs and ACCs. The cellular cross-talk of SBOs and ACCs
were studied by means of both direct and indirect co-culture systems,
which made it possible to identify the role of both membrane bound and
soluble factors. Histology, immunohistochemistry, qRT-PCR, zymography,
ELISA and western blotting were some of the techniques applied to distin-
guish the changes in the co-cultured vs. non co-cultured cells. The MAPK
signalling pathways were probed by using targeted MAPK inhibitors, and
their activity monitored by western blot analysis using phospho MAPK
speciﬁc antibodies.
Results: Our co-culture studies demonstrated that OA ACCs enhanced the
SBOs differentiation compared to normal ACCs. We demonstrated that
OA ACCs induced these phenotypic changes in the SBOs via activating an
ERK1/2 signalling pathway. The ﬁndings from this study thus provided clear
evidence that OA ACCs play an integral role in altering the SBO phenotype.
In the second study, we tested the inﬂuence of normal SBOs and OA SBOs
on ACCs phenotype changes. The results showed that OA SBOs increased
the hypertrophic gene expression in co-cultured ACCs compared to normal
SBOs, a phenotype which is considered as pathological to the health
and integrity of articular cartilage. It was observed that these phenotype
changes occurred via de-activation of p38 and activation of ERK1/2 signal-
ing pathways. These ﬁndings suggest that the pathological interaction of
OA SBOs with ACCs is mediated by cross-talking between ERK1/2 and p38
pathways, resulting in ACCs undergoing hypertrophic differentiation.
Subsequent experiments to determine the effect on MMP regulation, of
SBOs and ACCs cross-talk, revealed that co-culturing OA SBOs with ACCs
signiﬁcantly enhanced the proteolytic activity and expression of MMP-2
and MMP-9. In turn, co-culture of OA ACCs with SBOs led to abundant
MMP-2 expression in SBOs. Furthermore, we showed that the addition of
ERK1/2 and JNK inhibitors reversed the elevated MMP-2 and MMP-9 pro-
duction which otherwise resulted from the interactions of OA SBOs-ACCs.
Thus, this study has demonstrated that the altered interactions between
OA SBOs-ACCs are capable of triggering the pathological pathways leading
to degenerative changes seen in the osteoarthritic joint.
Conclusions: Our results has given clear in vitro evidence that the altered
bi-directional communication of SBOs and ACCs may play a role in OA
development and that this process was mediated by MAPK signalling
pathways. Targeting these altered interactions by the use of MAPK in-
hibitors may provide the scientiﬁc rationale for the development of novel
therapeutic strategies in the treatment and management of OA.
277
CROSS-TALK BETWEEN TGFβ AND IL-1β SIGNALLING PATHWAYS:
INVOLVEMENT IN OSTEOARTHRITIS
C. Bauge1, S. Leclercq2, P. Galera1, K. Boumediene3
1Univ Caen Basse-Normandie, Caen, France; 2Clinique Saint-Martin, CAEN,
France; 3Laboratoire Matrice Extracellulaire et Pathologie - Univ Caen
Basse-Normandie, Caen, France
Purpose: Interleukin-1β (IL1β) activity or its signaling cascade has been
implicated in the pathogenesis of a number of diseases including rheuma-
toid arthritis or osteoarthritis. Dysregulated Transforming Growth Factor
β (TGFβ) signalling is also involved in the osteoarthritis process. These
two cytokines play antagonistic roles in cartilage homeostasis and several
reports have highlighted cross-talk between these two pathways. Here,
we investigated whether IL1β interferes with TGFβ signalling in human
chondrocytes.
Methods: TGFβ1-induced gene expression was analyzed through plas-
minogen activator inhibitor 1 and p3TP-Lux induction in human articular
chondrocytes. Receptor-activated Smad (RSmad) phosphorylation, TGFβ
receptors, and Smad expression were determined by Western blotting
and real-time reverse transcription-polymerase chain reaction techniques.
Signalling pathways were investigated using speciﬁc inhibitors, messenger
RNA (mRNA) silencing, and expression vectors.
Results: IL-1β impaired TGFβ signalling through TGFβ receptor type II
(TβRII) down-regulation and Smad7 upregulation. IL-1β led to inhibition
of TGFβ1-induced gene expression and Smad2/3 phosphorylation. Interest-
ingly, TβRII overexpression restored the TGFβ response of human articular
chondrocytes. However, this effect was transient, suggesting that a sec-
ondary mechanism was responsible for the alteration of the TGFβ response
with long-term exposure to IL-1β.
IL-1β down-regulated TβRII expression at both protein and mRNA levels.
It reduced TβRII transcription by inducing Sp3 via NFκB and its binding
on core promoter at the expense of Sp1. In addition, it increased TRII
protein degradation through receptor-mediated endocytosis by lipid raft
and proteasomal pathway.
Furthermore, IL-1 strongly stimulated the expression of inhibitory Smad7.
This effect was late (after 12h of incubation with IL1). It did not require de
novo synthesis, and is dependent on NFkB pathway.
Conclusions: These ﬁndings clarify the regulatory process of IL-1β on TGFβ
pathway. The IL1β-induced-impairment of TGFβ signaling could explain the
reduced responsiveness of chondrocytes to TGFβ and therefore the cartilage
breakdown during OA. Understanding this process provides new insights
into the molecular mechanisms of osteoarthritis and may facilitate the
identiﬁcation of novel approaches for its treatment.
278
EXPRESSION AND FUNCTION OF THE ARYL HYDROCARBON RECEPTOR IN
GROWTH PLATE CARTILAGE CELLS IN VITRO
M. Widerak, M. Presume, K. Tahiri, M.-F. Dumontier, M.-T. Corvol,
J.-F. Savouret
Univ. Paris Descartes; INSERM UMRS-747, Paris, France
Purpose: Growth and development of the musculoskeletal system is a
complex process requiring intricate controls of cellular activity. Persis-
tant environmental pollutants including ligands of the Arylhydrocarbon
Receptor (AhR) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or
Benzo(a)pyrene (BaP, tobacco tar fraction) have been shown to elicit severe
developmental defects in rodent cartilage and bone as well as growth
retardation in humans. However, the status of AhR in cartilage is currently
unknown. We investigated AhR expression and function in growth plate
(GP) chondrocytes and assessed AhR ligands impact on cartilage processes.
Methods: GP chondrocytes were established in primary culture. Cells were
challenged with Interleukin-1beta (IL-1B) for 6 or 20 hr at concentrations
ranging from 0.05 to 2 ng/ml, then for another 20 hr with TCDD (5 nM)
or BaP at 1 μM. IL-1B and AhR target genes were analyzed by qPCR and
western blot. AhR localization was analyzed by immunocytochemistry
Results: GP chondrocytes expressed minor levels of AhR. AhR translocation
by TCDD into the nucleus required normoxia and the presence of IL-1B.
IL-1B induced AhR expression at the mRNA and protein levels and this
induction was higher (2-fold) in normoxia than hypoxia. The IL-1B effect
on AhR is transcriptional, through the AP-1 pathway.
Upon addition, IL-1B elicited a transient decrease (1h-6h) in Cyp1A1 mRNA
Poster Presentations / Osteoarthritis and Cartilage 18, Supplement 2 (2010) S45–S256 S125
expression, followed by a delayed dose-dependent increase (20h-40h).
Cyp1A1 mRNA expression was synergistically stimulated by TCDD and IL-
1B treatment,. BaP exerted similar effects on Cyp1A1 expression while the
AhR antagonist alpha-naphthoﬂavone (aNF) suppressed the TCDD part of
Cyp1A1 induction but not that of IL-1B. Conversely, AhR ligands synergized
with IL1-B and clearly increased the amount of IL-1B mRNA, but only
moderately that of IL1-RA mRNA.
Conclusions: Cartilage appears as a target for persistent environmental
pollutants acting as aggravating pro-inﬂammatory factors through the AhR.
279
CYCLICAL HYPEROSMOLAR LOADING OF HUMAN ARTICULAR
CHONDROCYTES INCREASES SOX9 GENE EXPRESSION AND P38
SIGNALLING IS REQUIRED
M.J. Peffers, P.D. Clegg, S. Tew, P.I. Milne
Univ. of Liverpool, Neston, United Kingdom
Purpose: SOX9 is a transcription factor required for cartilage formation
and is essential for cartilage extracellular matrix formation (ECM). Os-
teoarthritis (OA) is characterized by a loss of ECM. In human aged OA
cartilage SOX9 gene expression is regulated by osmotic loading. Here we
characterise SOX9 mRNA regulation through cyclical application of human
articular chondrocytes (HAC). The role of the p38 MAPK pathway was
investigated using pharmacological inhibition to identify mechanisms of
SOX9 regulation.
Methods: Cartilage was obtained following total knee arthroplasty with
informed consent and ethical approval. Cultures of freshly isolated HAC
were used in experiments within 24 hours. Osmotic load was either static
or cyclical, where loads were applied by incubating cells for 5 hours in
380 and 550mOsm medium alternating every 30 or 60 minutes, containing
either DMSO, or 20μM p38 MAP kinase inhibitor SB202190. Reverse tran-
scription and quantitative real-time PCR was used to measure the levels
of SOX9, Aggrecan and COL2A1 mRNA. Western blots were probed with
antibodies recognising phospho p38, SOX9, total ERK or GAPDH. Statistical
analysis was performed using mixed effects regression models.
Results: Static hyperosmotic loading for 5 hours signiﬁcantly increased
SOX9 mRNA in freshly isolated HAC; 2.5 fold, p=0.0027. Further experi-
ments investigated how the nature of the osmotic load applied to cells
effected SOX9 expression. In vivo, chondrocytes are subjected to periods
of cyclical loading which will generate ﬂuid ﬂow and osmotic ﬂuctuations
within the tissue. Cyclical loading between 380 and 550mOsm increased
SOX9 to a greater extent than cycling at the same osmolarity. This was
signiﬁcant at both 30 and 60 minute frequencies; 2.5 fold; p=0.001, 2.9 fold
p=0.0009 (Fig. 1).
Figure 1
Western blot analysis using a phosphorylated p38 MAPK speciﬁc antibody
identiﬁed an increase in activation of p38 MAPK at 550mOsm under static
hyperosmolar conditions. Cyclical hyperosmolar loading every 60 minutes
led to an increase in p38 MAPK activation compared to control (Fig. 2a).
In addition p38 MAPK inhibition caused a reduction in SOX9 protein in all
conditions (Fig. 2b). There was also a signiﬁcant decrease in SOX9 mRNA at
c30 and c60 conditions; p=0.001, p=0.003.
Finally we looked at the gene expression of COL2A1 and Aggrecan and
found that application of static hyperosmotic conditions for a 5 hour
period resulted in a signiﬁcant decrease in Aggrecan and COL2A1 mRNA;
Figure 2
p=0.0012, p=0.0008 respectively. No effects were evident during cyclical
hyperosmolar loading.
Conclusions: The regulation of SOX9 in chondrocytes is beginning to be
identiﬁed. One mechanism is through p38 MAPK-dependant signalling
leading to post-transcriptional stabilisation of its mRNA. Furthermore
medium osmolarity, a known regulator of p38 MAPK, controls SOX9 by
this mechanism. We identiﬁed that SOX9 is upregulated by both static and
dynamic hyperosmotic conditions. Whilst these conditions are not meant
to precisely simulate the osmotic conditions experienced by a chondrocyte
in vivo, they do demonstrate that repeated applications of these stimuli can
enhance their production of factors such as SOX9.
We examined Aggrecan and COL2A1 mRNA levels. Although there was no
apparent change in mRNA in cyclical loading of HAC, static hyperosmolar
loading caused a reduction in their expression agreeing with the majority
of studies that have noted a decrease in ECM production under static
hyperosmotic loading.
Understanding the regulation of SOX9 could lead to novel strategies to
improve techniques of cartilage repair
280
Systems Biology Approach and High-throughput Proteomic Analysis
Identiﬁes Toll-Like-Receptor Activators as Major Players of Cartilage
Degeneration
L. Alexopoulos1 , A. Chairakaki1,2, I. Melas1, C. Provatidis1, P. Kolia2,
Z. Dailiana3
1Natl. Technical Univ. of Athens, Athens, Greece; 2Univ. of Athens, Athens,
Greece; 3Univ. of Thessaly, Larissa, Greece
Purpose: The goal of this study is to combine biomechanical and sys-
tems biology approaches to reveal major players of cartilage degeneration.
Cartilage degeneration can be characterized by the imbalance of a com-
plex intracellular network orchestrated by several regulatory proteins.
IL-1α and TNF-α are two well known cytokines responsible for cartilage
degeneration but there are possibly several other pro-catabolic stimuli
equally important but less studied. This project utilizes a multidisci-
plinary approach characterized by the fusion of two seemingly different
areas: Biomechanics and Systems Biology. Systems biology approach has
been used for a “functional” screen of 90 different stimuli. Speciﬁcally,
high-throughput proteomic measurements and computational algorithms
have been used to interrogate the intracellular and extracellular activity
of primary human chondrocytes treated with a large number of stimuli
ranging from interleukins, growth factors, chemokines, toll like receptor
(TLR) activators and other inﬂammatory stimuli. Computational models
such as partial least square, principal component analysis, clustering, and
integer linear programming have been employed to create signaling path-
ways, correlate signaling activity, and identify major catabolic stimuli. To
further conﬁrm the results biomechanical approaches (indentation tests)
have are been used to monitor the degradation of cartilage explants by
measuring alterations on the mechanical properties (Young’s modulus and
the permeability). The multidisciplinary approach of the project sheds
light into the molecular pathways that govern cartilage degeneration in an
unbiased way. Several TLR activators as well as other cytokines show major
catabolic role in cartilage that can be proved equally important as IL-1 and
TNFα.
Methods: Systems Biology approach: Chondrocytes were isolated from hu-
man femoral heads following standard isolation procedures. Cells were
plated in 96 well plates at 35,000 cells per well, stimulated by 90 dif-


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278 EXPRESSION AND FUNCTION OF THE ARYL HYDROCARBON RECEPTOR IN GROWTH PLATE CARTILAGE CELLS IN VITRO

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