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Aggrecanase activity was assessed by measuring in the glycosaminoglycan (GAG) release and ARGSVIL neoepitope formation. Explants were further examined by histology after safranin
O proteoglycans (PG) staining/light green counterstaining, and byimmunohistochemistry for aggrecan NITEGE neoepitope. PG andneoepitope content was then quantiﬁed applying a scoring system
to individual images.
In addition, cytokine-induced matrix degradation was assessedin a new rat chondrosarcoma cell model (‘RCS assay’) in which
the amount of sulphated proteoglycan was quantiﬁed using Alcian
blue staining and image processing.
Results: TNFα+OSM or IL1β+OSM strongly induced ADAMTS4and ADAMTS5 in human cartilage explants. OSM is strictly neededfor induction of aggrecanases but not for MMP13.
A speciﬁc ADAMTS5 inhibitor (cpd A, IC50 80% inhibition of GAG
release, >80% inhibition of neoepitope release at 3μM). In contrast, a selective ADAMTS4 inhibitor (cpd B, IC50 <100 nM) did
not inhibited cartilage degradation by more than 50%. Cpd C,a non-selective 3-OH-3-methylpipecolic hydroxamate type metalloproteinase inhibitor completely inhibited GAG and Neoepitope
release.
Histological evaluation conﬁrmed that cpd A prevented PG loss
and inhibited neoepitope formation in cartilage matrix but not in
the pericellular matrix (PM), while cpd B only inhibited neoepitope formation in PM. Cpd C prevented PG loss and inhibited
neoepitope formation in both cartilage matrix and PM.
In addition, the compound ranking obtained in the explant model
was reproduced with the simple ‘RCS assay’.
Conclusions: This study represents the ﬁrst pharmacological evidence for the importance of ADAMTS5 over ADAMTS4 in humancartilage degradation. Strong induction of ADAMTS5 by cytokines
and signiﬁcant prevention of cartilage degradation by its pharmacological inhibition using a selective inhibitor are demonstrated.
This observation underscores the important role of ADAMTS5 in
the degradation of human cartilage. Furthermore, these ADAMTS5
effects were recapitulated in a new RCS-based assay, which
provides a good substitute for the complex cartilage explantmodel.
Finally, histological analysis delivered the unexpected observationthat matrix aggrecan is subject to different proteolytic events
depending on its localization, providing mechanistic informationon the cartilage degradation process.

Poster Presentations
monolayer until passage one and either cultured in 3D-micromasspellets for 7 days or kept in monolayer. After stimulation for 48hwith 10-6 M α-MSH only (micromass pellet culture) or in presence of 1 ng/ml IL-1β (monolayer culture); the cDNA copy numberof Sox9, collagens, MMPs; TGF-β1,TNF-α and interleukins wasassessed with quantitative RT-PCR. POMC, converting enzymes
and MC-receptor gene expression was determined by end point
RT-PCR and, MC-R1 protein expression in situ on cryosectionsof OA-cartilage by immunohistochemistry. Signal transduction ofα-MSH was evaluated by cAMP ELISA and Ca2+ -assays using
Fura2-AM. Protein concentration of MMP-2, MMP-13, IL-6, TNF-α,
TGF-β1 and collagen I and II in cell culture supernatants and celllysates was determined with ELISA.
Results: Articular OA-chondrocytes express receptors MC-1R,
MC-2R and MC-5R plus POMC and converting enzymes PC1,PACE4 and furin convertase. MC-1R protein was preferentiallyfound on chondrocytes from the middle and deep zones of articularcartilage. The MC-1R detected in human chondrocytes appears tobe functional as stimulation with 10-6 M α-MSH resulted in a moderate but signiﬁcant increase in intracellular cAMP levels but not inchanges of the intracellular Ca2+ level. In vitro application of 10-6 M
α-MSH to articular chondrocytes, kept in micromass pellet culture
induced gene expression of COL1A1, COL2A1 and COL10A1. Of
note, gene expression of MMP-2, MMP-7, MMP-9 and MMP-13
was induced, however, MMP-13 protein concentrations in culture
supernatant revealed profound interpatient ﬂuctuations. MMP-2
protein concentration was consistent within the sample collective
and remained unaltered in the presence of α-MSH. IL-1β and TNFα gene expression was reduced, IL-6 remained mostly unchanged
and Sox9 and TGF-β1 were induced upon stimulation with α-MSH.
IL-6 and TNF-α protein concentration were upregulated. α-MSH
attenuates IL-1β-induced expression of IL-4 and IL 8, MMP-2, -9
and -13 in articular OA-chondrocytes.
Conclusions: Human articular chondrocytes are target cells for
α-MSH. The effects of α-MSH on cytokine- and MMP expression suggest a chondroprotective role of this neuropeptide ininﬂammatory and degenerative processes in cartilage. It is conceivable that inﬂammatory reactions can be mitigated by inductionof endogenous melanocortins or administration of α-MSH to theaffected joints. The induction pattern of regulatory and structural
ECM components as collagens, Sox9, anabolic and catabolic cytokines points towards a function of α-MSH as a trophic factor
in skeletal development during endochondral ossiﬁcation or as
anti-inﬂammatory agent in degenerative joint pathologies.

223
THE MELANOCORTIN SYSTEM IN HUMAN
CHONDROCYTES
S. Grässel 1 , A. Opolka 1 , S. Anders 1 , R.H. Straub 1 , J. Grifka 1 ,T. Luger 2 , M. Böhm 2
1 Univ. of Regensburg, Regensburg, Germany; 2 Westfälische
Wilhelms-Univ., Münster, Germany
Purpose: Melanocortins (MCs) are structurally related peptides
that elicit their actions via binding to melanocortin receptors (MCRs). Originally deﬁned as neurohormones these peptides exhibit
an astonishing spectrum of effects on many peripheral cell types
and tissues. However, our present knowledge on the function of
the MC system in cartilage is scarce. We could show that MC1R is expressed in human articular cartilage and chondrocytes.
α-Melanocyte-stimulating hormone (α-MSH), a prototype of MCs,
is capable of regulating a number of important genes involvedin extracellular matrix (ECM) composition and inﬂammation inchondrocytes. The major goal of this study is to deﬁne the role ofMCs and the MC-1R in the context of osteoarthritis (OA).
Methods: Cartilage slices were aseptically dissected from femoral
condyles of OA patients who underwent total knee arthroplasty. Articular chondrocytes isolated from OA-cartilage were proliferated in224
THE FUNCTION OF p53 ON HUMAN CHONDROCYTEAPOPTOSIS IN RESPONSE TO SHEAR STRESS
S. Hashimoto, T. Nishiyama, S. Hayashi, K. Takebe, N. Kanzaki,T. Fujishiro, R. Kuroda, M. Kurosaka
Kobe Univ. Hosp., Kobe, Japan
Purpose: Chondrocyte apoptosis plays an important role in cartilage degeneration in osteoarthritis (OA), and mechanical injury to
cartilage induces chondrocyte apoptosis. p53 has been identiﬁed
as a 53 kDa cellular protein and ‘ultimate tumor suppressor gene’.
p53 is recognized as a pivotal regulatory protein that responds to avariety of signals and recruits an array of biochemical activities totrigger diverse biological responses, most notably cell cycle arrest
and apoptosis. In response to DNA damage, p53 expression is upregulated, increases transcriptional activity, and initiates apoptosis
signals. p53-regulated apoptosis-inducing protein 1 (p53AIP1) isone of the p53-regulated genes, and is activated in response tosevere DNA damage with the phosphorylation of p53 at Ser-46.
In this study, we speculated that p53 and p53AIP1 might play animportant role in DNA damage-induced apoptosis of articular carti-

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Osteoarthritis and Cartilage Vol. 17, Supplement 1
lage, and analyzed the function of p53 and p53AIP1in chondrocyte
apoptosis by shear stress.
Methods: Shear stress was introduced to NHAC-kn cells (humannormal chondrocytes) with Flexcell system. DNA damage wasdetected and quantiﬁed by comet assay, DNA fragmentation, andphosphorylation of ataxia-telangiectasia mutated kinase (ATM).
To explore the p53 function, NHAC-kn cells were pretreated withpiﬁthrin- alpha, which is speciﬁc inhibitor of p53, or p53 siRNAs
before induction of shear stress. Chondrocyte apoptosis was detected by expression of cleaved caspase-9 with Western blotting
and TUNEL staining. Expressions of p53 and p53AIP1, and phosphorylation of p53 at Ser-15 and -46 were analyzed by Westernblotting.
Results: DNA damage was actually induced by shear stress, withthe results of comet assay, DNA fragmentation, and phosphorylation of ATM. After loading shear stress, TUNEL positive cells
and expression levels of p53, p53AIP1 and cleaved caspase-9
were increased in a force and time dependent manner. Phosphorylation of p53 at Ser-15 and Ser-46 increased, compared withnon-loaded samples. Chondrocyte apoptosis was reduced whenpretreatment with piﬁthrin-alpha or p53 siRNAs. Western blotting
revealed that expressions of p53 and p53AIP1 were still detected
when chondrocytes were pre-treated with piﬁthrin-alpha. Piﬁthrinalpha inhibited the phosphorylation of p53 at Ser-15 comparedwith non-treatment of piﬁthrin-alpha. However, piﬁthrin-alpha did
not inhibit the phosphorylation of p53 at Ser-46 compared with
non-treatment of piﬁthrin-alpha. When p53 siRNA was transfected,
p53, phosphorylation of p53 at Ser-15 and Ser-46, and p53AIP1
expressions decreased.
Conclusions: We demonstrated that expression of p53 andp53AIP1 increased with the activation of apoptotic processes
in human normal chondrocytes following shear stress. However,piﬁthrin-alpha reduced chondrocytes apoptosis after loading shearstress. Furthermore, down regulation of endogenous p53 by transfection of p53 siRNA inhibited chondrocytes apoptosis after loading shear stress. Our results suggest that shear stress induces
apoptosis via p53, mainly phosphorylation at Ser-46, and the mitochondrial pathway. In conclusion, we demonstrated that p53 andp53AIP1 act as an important molecule for chondrocyte apoptosis induced by elongation shear stress, and down regulation ofexogenous or endogenous p53 expression inhibited chondrocyte
apoptosis.

Methods: Accumulation of β-catenin protein was evaluated by IHC
in maturing (1, 2 and 3 week) control, IKKα KD or MMP-13 KD micromasses and gene expression was assessed by Real Time PCR
at 1 week maturation and also in high density monolayers. Sox-9
subcellular distribution was analyzed in one week micromasses
by confocal microscopy, wherein permeabilized cells were stainedwith anti-Sox9 Ab linked to Cy5 in conjunction with Sybr greenlabeling of DNA. Activation of the wnt/β-catenin signaling pathway
was investigated by IHC and WB analysis of active β-catenin inthree week micromasses. Confocal microscopy analysis of Sox9 subcellular localization was also carried out on full thichness
slices of cartilage derived from either normal cartilage/early OA orlate OA cartilage, which were also assessed for ECM integrity by
Safranin-O staining.
Results: Both IKKα KD and MMP-13 KD prevented the accumulation of total β-catenin protein in maturing micromasses and highdensity monolayers, which was also associated with signiﬁcantly
lower (n=5, p=0.043) β-catenin RNA expression. Loss of either
MMP-13 or IKKα expression also blunted the accumulation of activated β-catenin. Moreover, confocal microscopy revealed strong
intranuclear Sox-9 staining in the absence of MMP-13 or IKKα,
while the nuclei of control differentiating chondrocytes presented
a halo-like, peri-nuclear Sox-9 staining pattern. Noteworthy, in cartilage tissue with strong safranin-O staining, Sox-9 was mainly
localized in chondrocyte nuclei while the Sox-9 in chondrocytes
within areas of degraded OA cartilage presented a peri-nuclear
like staining pattern.
Conclusions: IKKα and MMP-13 are both required for ECM
remodeling in differentiating chondrocyte micromass cultures expression. Loss of either MMP-13 or IKKα stabilized the ECM inconjunction with the subcellular localization of Sox-9 to cell nuclei, an event which was associated with a reduction in β-catenin
expression and activity. A similar pattern of Sox-9 subcellular
localization was also associated with the ECM integrity of cartilage tissue. On the basis of these observations, we suggestthat when chondrocytes abandon their natural maturation arrested
state in cartilage, IKKα dependent, MMP-13 initiated ECM remodeling can act as a focal point for functional cross-talk between
early (Sox-9) and late (β-Catenin) positive effectors of chondrocyte differentiation, thereby pushing chondrocytes towards a more
hypertrophic-like state.

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225
A FUNCTIONAL LINK BETWEEN ECM HOMEOSTASIS AND
SOX-9 MEDIATED CONTROL OF β-CATENIN ACTIVITY
R. Borzì 1 , E. Olivotto 1 , S. Pagani 1 , D. Platano 2 , S. Santi 3 ,A. Facchini 4 , F. Flamigni 4 , A. Facchini 5,1 , K.B. Marcu 6,1
1 Istituti Ortopedici Rizzoli, Bologna, Italy; 2 Dipartimento diFisiologia Umana e Gene, Università degli Studi di Bologna,Bologna, Italy; 3 Istituto per i Trapianti d’Organo el’Immunocitologia ITOI - C.N.R., c/o Inst. “Codivilla-Putti” I.O.R,Bologna, Italy; 4 Dipartimento di Biochimica, Università degli Studidi Bologna, Bologna, Italy; 5 Dipartimento di Med. Clinica,Università degli Studi di Bologna, Bologna, Italy; 6 Dept. ofBiochemistry and Cell Biology Stony Brook Univ., Stony Brook, NYPurpose: Our recent ﬁndings have unexpectedly linked IKKαto MMP-13 activity dependent ECM remodeling (Olivotto et. alA&R 2008) in differentiating chondrocytes, revealing a linkage
between ECM stabilization in the absence of IKKα with a block inchondrocyte differentiation towards a hypertrophic-like phenotype.
Here we present evidence that knocking down MMP-13 or IKKα,either of which blocked ECM remodeling, resulted in pronounced
alterations in the pattern of Sox-9 nuclear localization that can belinked to changes in β-Catenin levels and activation status.

CROSSTALK BETWEEN RESIDENT CELLS IN THE JOINT:IMPACT ON CHONDROCYTE METABOLISM
T. Ort, J. Wertheimer, J. Luongo, N. Brauchle, M. Ryan,P. Sivakumar, K. Kavalkovich, K. Picha
Centocor R&D, Inc, Radnor, PA
Purpose: Osteoarthritis (OA) is a multi-factorial disorder that results in a net loss of cartilage from synovial joints, often leadingto pain. Cartilage lesions are a product of blunted extracellularmatrix (ECM) synthesis by chondrocytes, and enhanced enzymemediated ECM degradation. Although often characterized by thesingle pathological feature of cartilage degeneration, OA is increasingly recognized as a disease of the whole joint in whichinterplay between the different cells within the joint are important.
Here we explore the impact of various joint resident cells on ECM
metabolism of chondrocytes in an in vitro co-culture system.
Methods: Primary human chondrocytes were isolated by enzymatic digestion of adult articular cartilage and encapsulated in 3D
alginate beads. Primary human osteoblasts, purchased from PromoCell, were isolated by enzymatic digestion of bone from patients
undergoing knee endoprothesis due to arthrosis of the knee joint.
Human macrophages were differentiated from CD14+ peripheral
blood monocytes (Allcells) by treatment with macrophage colony