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DNA AND CELL BIOLOGY Volume 22, Number 2, 2003 © Mary Ann Liebert, Inc. Pp. 119–129 SOX9 Exerts a Bifunctional Effect on Type II Collagen Gene (COL2A1) Expression in Chondrocytes Depending on the Differentiation State MAGDALINI KYPRIOTOU, 1 MAGALI FOSSARD-DEMOOR, 1 CHRISTOS CHADJICHRISTOS, 1 CHAFIK GHAYOR, 1 BENOIT DE CROMBRUGGHE, 2 JEAN-PIERRE PUJOL, 1 and PHILIPPE GALÉRA 1 ABSTRACT As a key transcription factor in cartilage formation, SOX9 is a potent activator of type II collagen expres- sion, a phenotypic marker of articular chondrocytes. This study was designed to determine the potential role of SOX9 on COL2A1 gene transcription during chondrocyte dedifferentiation, a characteristic feature of os- teoarthritic cartilage that can be partially mimicked invitro by subculturing primary chondrocytes. Con- structs containing different regions from the promoter and the first intron of human COL2A1 gene were transfected in differentiated (primary) and dedifferentiated (passaged) rabbit articular chondrocytes (RAC), together with an expression vector containing or not the SOX9 cDNA. As expected, low levels of SOX9 over- expression were capable of enhancing COL2A1 gene transcription in both fully differentiated and slightly phenotypically altered chondrocytes, through the specific intronic enhancer. In contrast, when overexpressed at high levels, SOX9 induced an inhibition of COL2A1 gene expression, mediated by the 2266 bp promoter region, whatever the differentiation state of chondrocytes. However, in the advanced stages of dedifferentia- tion, SOX9, independently of its expression level, depressed COL2A1 transcriptional activity through the 263 bp short promoter. Although SOX9 has a crucial role in chondrocyte differentiation, our findings indicate that this factor cannot restore the phenotype of osteoarthritic chondrocytes by itself. 119 INTRODUCTION S OX9 IS A TRANSCRIPTION FACTOR belongingto the SOX (Sry- type HMG box) proteins family, which are characterizedby a high-mobility-group box (HMG) DNA-binding domain bear- ing 50% homology with that of the sex-determiningfactor SRY (sex-determiningregion of Y chromosome). The implication of SOX9 in campomelic dysplasia (CD), a skeletal malformation syndrome (Foster et al., 1994; de Crom- brugghe et al., 2000), led to the observation that parallel ex- pression of SOX9 and type II collagen occurs in all chondro- cytic cells during mouse embryonic development (Zhao et al., 1997). Then, it was found that SOX9 directly regulates type II collagen gene (COL2A1) expression, mediating the transcrip- tional activity of a 48-bp specific enhancer located in the first intron of the mouse gene (Lefebvre et al., 1997). SOX9 effect is also enhancedby two other cooperativemembers of the same family, L-SOX5 and SOX6 (Lefebvre et al., 1998). Type II collagen is the characteristic fibrillar collagen of ar- ticular cartilage and one of the main macromolecular compo- nents of its extracellular matrix. It forms a fibrillar network throughoutthe cartilagematrix and provides tensile strength by resisting to the swelling pressure resulting from the hydratation of the highlynegativelychargedproteoglycanaggregateswithin the matrix (Poole, 1997). Because type II collagen is considered as a phenotypic marker of cartilage, the above findings suggested the involve- ment of SOX9 in the physiology of this tissue. Indeed, inacti- vation of SOX9 gene by homologous recombination in embry- onic stem (ES) cells in mouse embryos showed that it is required for the differentiationof mesenchymal cells into chondrocytes (Bi et al., 1999). 1 Laboratoire de Biochimie du Tissu Conjonctif, Faculté de Médecine, Caen Cedex, France. 2 Department of Molecular Genetics and Graduate Program in Genes and Development, M.D. Anderson Cancer Center, University of Texas, Houston, Texas.

SOX9 Exerts a Bifunctional Effect on Type II Collagen Gene (COL2A1) Expression in Chondrocytes Depending on the Differentiation State

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DNA�AND�CELL�BIOLOGYVolume�22,�Number�2,�2003© Mary�Ann�Liebert,�Inc.Pp.�119–129

SOX9�Exerts�a�Bifunctional�Effect�on�Type�II�Collagen�Gene(COL2A1)�Expression�in�Chondrocytes�Depending�on�the

Differentiation�State

MAGDALINI�KYPRIOTOU,1 MAGALI�FOSSARD-DEMOOR,1 CHRISTOS�CHADJICHRISTOS,1

CHAFIK�GHAYOR,1 BENOIT�DE�CROMBRUGGHE,2 JEAN-PIERRE�PUJOL,1 and�PHILIPPE�GALÉRA1

ABSTRACT

As�a�key�transcription�factor�in�cartilage�formation,�SOX9�is�a�potent�activator�of�type�II�collagen�expres-sion,�a�phenotypic�marker�of�articular�chondrocytes.�This�study�was�designed�to�determine�the�potential�roleof�SOX9�on�COL2A1�gene�transcription�during�chondrocyte�dedifferentiation,�a�characteristic�feature�of�os-teoarthritic�cartilage�that� can�be�partially�mimicked� in�vitro by� subculturing�primary�chondrocytes.�Con-structs� containing�different� regions�from� the� promoter�and� the� first� intron�of�human�COL2A1�gene�weretransfected�in�differentiated�(primary)�and�dedifferentiated�(passaged)�rabbit�articular�chondrocytes�(RAC),together�with�an�expression�vector�containing�or�not�the�SOX9�cDNA.�As�expected,�low�levels�of�SOX9�over-expression�were�capable�of�enhancing�COL2A1�gene�transcription�in�both� fully�differentiated�and� slightlyphenotypically�altered�chondrocytes,�through�the�specific�intronic�enhancer.�In�contrast,�when�overexpressedat�high�levels,�SOX9�induced�an�inhibition�of�COL2A1�gene�expression,�mediated�by�the�2266�bp�promoterregion,�whatever�the�differentiation�state�of�chondrocytes.�However,�in�the�advanced�stages�of�dedifferentia-tion,�SOX9,�independently�of�its�expression�level,�depressed�COL2A1�transcriptional�activity�through�the�263bp� short�promoter.�Although�SOX9�has�a�crucial�role�in�chondrocyte�differentiation,�our�findings�indicatethat�this�factor�cannot�restore�the�phenotype�of�osteoarthritic�chondrocytes�by�itself.

119

INTRODUCTION

SOX9�IS A TRANSCRIPTION FACTOR belonging�to�the�SOX�(Sry-type�HMG�box)�proteins�family,�which�are�characterized�by

a�high-mobility-group�box�(HMG)�DNA-binding�domain�bear-ing�50%�homology�with�that�of�the�sex-determining�factor�SRY(sex-determining�region�of�Y�chromosome).

The�implication�of�SOX9�in�campomelic�dysplasia (CD),�askeletal�malformation�syndrome�(Foster�et�al.,�1994;�de�Crom-brugghe�et�al.,�2000),�led�to� the�observation�that�parallel�ex-pression�of�SOX9�and�type�II�collagen�occurs�in�all�chondro-cytic�cells�during�mouse�embryonic�development�(Zhao�et�al.,1997).�Then,�it�was�found�that�SOX9�directly�regulates�type�IIcollagen�gene�(COL2A1)�expression,�mediating�the�transcrip-tional�activity�of�a�48-bp�specific�enhancer�located�in�the�firstintron�of�the�mouse�gene�(Lefebvre�et�al.,�1997).�SOX9�effect

is�also�enhanced�by�two�other�cooperative�members�of�the�samefamily,�L-SOX5�and�SOX6�(Lefebvre�et�al.,�1998).

Type�II�collagen�is�the�characteristic�fibrillar�collagen�of�ar-ticular�cartilage�and�one�of�the�main�macromolecular�compo-nents�of� its� extracellular�matrix.� It� forms�a� fibrillar�networkthroughout�the�cartilage�matrix�and�provides�tensile�strength�byresisting�to�the�swelling�pressure�resulting�from�the�hydratationof�the�highly�negatively�charged�proteoglycan�aggregates�withinthe�matrix�(Poole,�1997).

Because� type� II� collagen� is� considered� as� a� phenotypicmarker�of�cartilage,�the�above�findings�suggested�the�involve-ment�of�SOX9�in�the�physiology�of�this�tissue.�Indeed,�inacti-vation�of�SOX9�gene�by�homologous�recombination�in�embry-onic�stem�(ES)�cells�in�mouse�embryos�showed�that�it�is�requiredfor�the�differentiation�of�mesenchymal�cells�into�chondrocytes(Bi�et�al.,�1999).

1Laboratoire�de�Biochimie�du�Tissu�Conjonctif,�Faculté�de�Médecine,�Caen�Cedex,�France.2Department�of�Molecular�Genetics�and�Graduate�Program�in�Genes�and�Development,�M.D.�Anderson�Cancer�Center,�University�of�Texas,

Houston,�Texas.

So�far,�only�few�recent�studies�deal�with�the�role�of�SOX9in� joint� diseases,� such� as� osteoarthritis.� In� an� experimentalmodel�of�osteoarthritis�induced�in�transgenic�mice,�SOX9�wasfound�in�cartilage�in�the�first�steps�of�the�disease�(Salminen�etal.,� 2001).�These� early� stages�are� accompanied�by� a� hyper-trophic� reaction� of� chondrocytes,� suggesting� an� attempt� to�tissue�repair.�It�may�be�hypothesized�that�SOX9�could�be�im-plicated�in�this�process.�During�the�osteoarthritic�process,�chon-drocytes�dedifferentiate�and�overexpress�several�characteristiccomponents�of�the�cartilage�matrix,�together�with�cartilage�nonspecific�molecules,�including�types�I�and�III�collagens.�Modu-lation�of� chondrocyte�phenotype�in� vitro,� by� successive�pas-sages�of�monolayer�cultures�(Benya�et�al.,�1977),�has�shownthat�SOX9�expression�is�reduced�under�these�conditions�(Lefeb-vre�et�al.,�1997;�Stokes�et�al.,�2001;�Kolettas�et�al.,�2001).

In�the�present�study,�we�demonstrate�that�SOX9�exerts�a�bi-functional� effect� on� the� transcriptional� activity� of� humanCOL2A1�gene,�depending�on�its�expression�level�and�the�de-differentiation�process.�We� found�that�this�differential�actionimplicates�two�distinct�regions�of�COL2A1�gene.�Indeed,�acti-vation�of�COL2A1� gene� expression�in� primary�cultures�andslightly�dedifferentiated�chondrocytes�involves�the�first�intronspecific�enhancer,�whereas�inhibition�in�dedifferentiated�chon-drocytes�is�mediated�by�a� short�promoter�region.�These�dataalso�indicate�that�SOX9�overexpression,�by�itself,�is�not�suffi-cient�to�restore�type�II�collagen�expression�in�profoundly�de-differentiated�chondrocytes.

MATERIALS�AND�METHODS

Cell�cultures

RAC�were�released�by�enzymatic�digestion,�seeded�at�a�den-sity�of�1 3 106 cells/55-cm2 dishes�or�3.5 3 106 cells/175-cm2

flasks�and�cultured�as�described�previously�(Ghayor�et�al.,�2000,2001).�For�dedifferentiation,�RAC�cultures�were�passaged�withtrypsin�(0.25%)�after�reaching�confluency.

Transfection�experiments

RAC�at�80%�of�confluency,�in�55-cm2 culture�plates,�weretransiently�transfected�by�the�calcium�phosphate�precipitationmethod�using�HEPES.�The�reporter�plasmids�(15–25�mg)�werecotransfected�with�a�pSV40-b-galactosidase�expression�vector(2�mg),�as�a�control�of�transfection�efficiency,�and�with�15�mgof�a�pcDNA-59UT-FLAG�expression�vector�containing�or�notthe�cDNA�encoding�human�SOX9,�generously�provided�by�B.de�Crombrugghe�(University�of�Texas,�M.D.�Anderson�CancerCenter).�Ten�to�15�h�after�transfection,�the�medium�was�replacedby�a�fresh�one,�and�cells�were�harvested�24�h�later.�Luciferaseand�b-galactosidase�activities,�as�well� as� the�protein�amountwere� determined�(Ghayor�et� al.,� 2000).�Luciferase�activitieswere�normalized�only�to�protein�amounts—because�it�was�ob-served� that� overexpression�of� SOX9� influences�the� b-galac-tosidase�expression�level—and�expressed�in�relative�luciferaseunits�(RLU)�as�the�mean 6 SD�of�three�independent�samples.

DNA�constructions

A�SalI-SphI�DNA�fragment�of�8-kb�of�the�human�COL2A1gene�has�been�cloned�in�pUC19�(Ala-Kokko�et�al.,�1991).�This

clone�contains�approximately�1.8�kb�of�the�promoter�sequence,exon�1,� intron�1,�exon�2,�and�part�of� intron�2� (21840�bp� to15800�bp).

The�DNA�constructs�pGL2-3.774�kb,�pGL2-3.059�kb,�pGL2-2.367�kb,�pGL2-1.167�kb,�and�pGL2-0.387�kb�have�been�gen-erated� from� the� initial� subclone�SalI–SphI� according� to� thecloning�strategies�previously�described�(Ghayor�et�al.,�2000).

The�clone�pGL2-0.110�kb�was�obtained�by�cloning�a�PCRgenerated�insert�of�110-bp�(position�in�the�COL2A1�gene�se-quence:�263/147),�in� the�BglII–HindIII�sites�of�pGL2-basicvector.�This�110-bp�fragment�was�obtained�by�PCR�using�thefollowing� primers:� sense� a1(II):� 59-CCGGAGATCTGCGA-TTCGCCAG-39 and� antisense�a1(II):� 59-CCGGAAGCTTG-GAGCAGGAGGAG-39.�The�PCR�conditions�were:�30�sec�at95°C,�30�sec�at�55°C,�and�1�min�at�72°C.�The�pGL2-0.110�kbhas�been�verified�by�sequencing.

Nuclear�extracts�and�gel�retardation�assays

Nuclear�extracts�were�prepared�by�a�minipreparation�proce-dure�(Andrews�and�Faller,�1991)�and�gel�retardation�assays�wereperformed�with�the�following�nucleotides:�12392/12415�wildtype:� 59-AGCCCCATTCATGAGAGACGAGGT-3 9 (SOX9binding�site�is�indicated�in�italics),�12392/12415�mutant:�59-AGCCCCGGGGGTGAGAGACGAGGT-3 9 (mutation�in�boldcharacters),� and� 12353/12415� wild-type:� 59-GCAGAGAC-CTGTGAATCGGGCTCTGTGTGCGCTCGAGAAAAGCCCCATTCATGAGAGACGAGGT-3 9 (HMG-like� sites� in� italics).They�were�end-labeled�with�[g-32P]�dATP�(NEN�Life�ScienceProducts,�Paris,�France)�using�T4�polynucleotide�kinase�(LifeTechnologies,�Inc.,�Gaithersburg,�MD).�RAC�nuclear�extracts(7�or�15�mg)�were�incubated�for�30�min�at�room�temperaturewith�the�probe�(2�fmol)�in�20�ml�of�a�specific�binding�buffer(Lefebvre� et� al.,� 1996)� and� in� the� presence� of� 1� mg� ofpoly(dGdC)?poly(dGdC)�(Pharmacia,�Piscataway,�NJ)�used�asDNA� nonspecific�competitor.�Samples� were� fractionated�byelectrophoresis�for�1.5�h�at�150�V�on�a�5%�polyacrylamide�gel(acrylamide/bis�at�30:1)�in�0.53 TBE� (45�mM�Tris�borate,�1mM�Na2EDTA)�and�visualized�by�autoradiography.

In�supershift�analysis,�1�ml�of�purified�SOX9�antibody�(gen-erously�provided�by�B.�de�Crombrugghe)�was�added�to�each�re-action�mixture,�15�min�before�addition�of�the�DNA�probe.

Total�RNA�extraction�and�RT-PCR�analysis

Total�RNA�was�isolated�by�the�method�of�Chomczynski�andSacchi�(1987).�For�RT-PCR,�the�primers�used�to�amplify�typeII�collagen�(COL2A1),�glyceraldehyde-3-phosphate�deshydro-genase� (GAPDH)� and� b-actin� cDNAs,� were� the� following:COL2A1:�sense:�59-GACCCCATGCAGTACATG-3 9 and�an-tisense:� 59-GACGGTCTTGCCCCACTT-3 9 (Takaishi� et� al.,1997).�GAPDH:�sense:�59-TGGTATCGTGGAAGGACTCAT-GAC-39 and� antisense:� 59-ATGCCAGTGACGTTCCCGTT-CAGC-39 (Rediske� et� al.,� 1994).� b-actin:� sense:� 59-GTG-GGGCGCCCCAGGCACCA-3 9 and� antisense:� 59-CTCCT-TAATGTCACGCACGATTTC-3 9 (Lupparello� et� al.,� 1993).About�30�PCR�cycles�were�performed�according�to�the�exper-iment� (Hybaid,� OmniGene� thermocycler).� Each� PCR� cyclecomprised:�1�min�at�95°C,�1�min�at�55°C,�and�1�min�at�72°C.RT-PCR�products�were�then�analyzed�on�a�2%�agarose�elec-trophoresis�gel,�in�the�presence�of�ethidium�bromide.�After�pho-

KYPRIOTOU�ET�AL.120

tography,�the� intensities�of� the� COL2A1� cDNA� bands�werequantified�by� densitometric�scanning� using� the� ImageQuantSoftware� (Amersham� Biosciences,�Saclay,� France)� and� nor-malized�to�GAPDH�or�to�b-actin�cDNA�levels.

RESULTS

SOX9�exerts�a�bifunctional�effect�on�human�COL2A1gene�expression�depending�on�its�overexpression�level

SOX9� was� found� to� activate�mouse� COL2A1� gene� tran-scription�through�an�intronic�48-bp�specific�enhancer,�in�chon-drocytes�and�various�fibroblast�and�osteoblast�cell�lines�(Lefeb-vre� et� al.,� 1997,�1998).�Here,� using� transfection�of� primarycultures�of�RAC�with�the�pGL2-3.774�kb�construct,�which�cov-ers�932�bp�of�the�gene�promoter,�the�first�exon�and�approxi-mately�60%�of�the�first�intron�of�the�human�COL2A1�gene,�andincreasing�amounts�of�SOX9�expression�vector,�we�found�thatSOX9�stimulated�COL2A1�gene�transcription�when�it�was�over-expressed�at�low�levels,�as�already�reported.�However,�we�ob-served�an�inhibition�of�COL2A1�expression�when�the�amountof�SOX9�cDNA�transfected�was�above�5�mg�(Fig.�1A).

To�assess�the�relative�level�of�SOX9�overexpression,�nuclearextracts�from�primary�RAC�transfected�with�increasing�amountsof�SOX9�expression�vector�were�used�in�EMSA,� in�the�pres-ence�of� a� radiolabeled�oligonucleotide�representing�the�highaffinity�binding�site�of�SOX9�present�in�the�intronic�COL2A1specific�enhancer�(Lefebvre�et�al.,�1997).�The�binding�activityof�the�complex�was�increased�proportionally�to�SOX9�overex-

pression�level,�thereby�confirming�the�increasing�overexpres-sion�level�of�the�nuclear�factor�in�our�system�(Fig.�1B).

The�specificity�of�SOX9–DNA�complex�was�further�assessedin�gel�retardation�assays,�using�the�unlabeled�oligonucleotideas�a�competitor.�The�binding�activity�of�the�complex�was�com-peted�away,�proportionally�to�the�amount�of�unlabeled�probeadded�(Fig.�2A).�Furthermore,�the�SOX9–DNA�complex�dis-appeared�when�a� labeled�probe�mutated�on� the�high-affinitybinding�site�of�SOX9�on�COL2A1�gene�was�used�(Fig.�2B).�Fi-nally,�EMSA�analysis�with�the�anti-SOX9�antibody�confirmedthat�SOX9�transcription�factor�was�involved�in�that�complex,as�reflected�by�the�formation�of�a�supershifted�complex�(Fig.2C).�Thus,�SOX9�appeared�as�a�positive�or�a�negative�regula-tor�of�COL2A1�gene�expression�in�primary�articular�chondro-cytes,�depending�on�its�overexpression�level,�while�its�bindingactivity�to�the�gene�was�increased�proportionally�to�the�amountoverexpressed.

SOX9�bifunctional�effects�are�mediated�by�twodifferent�regions�of�human�COL2A1�gene

To�delineate�the�regions�of�COL2A1�gene�involved�in�SOX9transcriptional�effects,� primary� RAC� cultures� were� cotrans-fected�with�reporter�constructs�covering�characteristic�regionsof�the�promoter�and/or�the�first�intron�of�the�gene�in�the�pres-ence�of�a�SOX9�expression�vector�(Fig.�3).�An�activating�ef-fect,�mediated�by�the�12384/12842-bp�sequences�of�the�genespecific�enhancer,�was�observed�when�SOX9�was�transfectedat�the�amount�of�1�mg�(Fig.�3A).�However,�low�overexpressionof�SOX9�caused�also�an�inhibition�of�the�transcriptional�activ-

SOX9�IMPLICATION�IN�HUMAN�COL2A1�GENE�EXPRESSION 121

FIG.�1. SOX9�exerts�bifunctional�effect�on�human�COL2A1�gene�expression�in�primary�RAC.�(A)�RAC�primary�cultures,�at80%�of�confluency,�were� transiently�cotransfected,�as�described�in�Materials�and�Methods,�with�25�mg�of� the�pGL2-3.774-kbCOL2A1�gene�construct�and�different�amounts�of�an�expression�vector�containing�the�SOX9�cDNA.�For�each�amount�of�SOX9overexpressed,�the�insertless�pcDNA-59UT-FLAG�vector�was�used�as�a�complement�to�15�mg,�to�transfect�each�sample�with�thesame�amount�of�the�expression�vector.�Relative�luciferase�activities�were�measured�in�cell�extracts�and�normalized�to�protein�amountper�culture�plate.�RLU�represents�the�mean 6 SD�of�three�independent�samples�of�a�representative�experiment.�(B)�SOX9�bindingactivity�on� the�COL2A1� gene-specific�enhancer�was� studied�in� gel� retardation�assays.�The� double-stranded�labeled�wild-type12392/12415�probe�was�incubated�for�30�min�at�room�temperature�with�15-mg�nuclear�extracts�from�primary�RAC�transfectedwith�increasing�amounts�of�the�SOX9�cDNA�expression�vector.�The�arrow�indicates�the�position�of�the�SOX9–DNA�complex.

ities�of�pGL2-0.387�kb�and�pGL2-0.110�kb�constructs,�whichcover�266�and�63�bp�upstream�from�the�transcription�initiationsite,�respectively�(Fig.�3A).�In�parallel,�a�high�level�of�SOX9overexpression�(induced�by�15�mg�of�transfected�SOX9�vector)inhibited�the�expression�of�COL2A1�gene�through�a�region�lo-

cated�between�the�2266�and�the�263�bp�of�the�proximal�pro-moter�(Fig.�3B).�We�concluded�that�SOX9�effects�involve�twodifferent�regions�of�the�human�COL2A1�gene:�the�specific�en-hancer,�which�mediates�the�activating�effect,�and�the�proximalpromoter,�which�is�responsible�for�downregulation.

KYPRIOTOU�ET�AL.122

FIG.�3. Determination�of�the�COL2A1�gene�regions�mediating�SOX9�bifunctional�effects.�RAC�primary�cultures�were�tran-siently�cotransfected,�as�in�Figure�1,�with�25�mg�of�different�COL2A1�reporter�plasmids�in�the�presence�of�1�mg�(A)�or�15�mg(B)�of�an�expression�vector�containing�(SOX9)�or�not�(control)�the�cDNA�encoding�SOX9.�In�the�case�of�1�mg�of�SOX9�over-expressed,�the�insertless�pcDNA-59UT-FLAG�vector�was�used�as�a�complement�to�15�mg,�to�have�the�same�conditions�of�trans-fection�for�each�sample.�RLU�represents�the�mean�6SD�of�three�independent�samples�of�a�representative�experiment.

FIG.�2. SOX9�binds�specifically�to�the�human�12392/12415-bp�sequences�present�in�the�intronic�specific�enhancer.�(A)�Adouble-stranded�labeled�wild-type�12392/12415�probe�was�incubated�for�30�min�at�room�temperature�with�15�mg�of�nuclear�ex-tracts�from�primary�RAC,�in�the�presence�(lanes�3,�4,�and�5)�or�not�(lane�2)�of�25-,�50-,�and�100-fold�molar�excesses�of�the�un-labeled�probe,�respectively.�The�arrow�indicates�the�position�of�the�SOX9–DNA�complex.�(B)�The�double-stranded�labeled�wild-type�12392/12415� probe� (wt)� and� the� respective�mutant� of� the� same� oligonucleotide�were� incubated�for� 30� min� at� roomtemperature�with�15-mg�nuclear�extracts�from�primary�RAC,�transfected�(lanes�2�and�4)�or�not�(lanes�1�and�3)�with�15�mg�of�theSOX9�cDNA�expression�vector.�The�arrow�indicates�the�position�of�the�SOX9–DNA�complex.�(C)�Fifteen�micrograms�of�nu-clear�extracts�from�primary�RAC�transfected�with�15�mg�of�SOX9�expression�vector�were�incubated�with�1�ml�of�SOX9�antibody(lane�2)�and�without�it�(lane�1),�at�room�temperature,�before�addition�of�a�double-stranded�labeled�wild-type�12353/12415�probe.Lane�3�represents�the�migration�pattern�of�the�probe�incubated�without�nuclear�extracts�and�SOX9�antibody.�After�an�incubationof� 30�min�at� room�temperature,�the�samples�were�processed�for� electrophoresis.�The�arrows�indicate�the�SOX9–SOX9� anti-body–DNA�supershifted�complex�(SS)�( )�and�the�SOX9-DNA�complex�( fi ).

SOX9�transcriptional�effects�are�correlated�with�theendogenous�steady-state�amounts�of�type�II�collagenmRNA

Type�II�collagen�mRNA�analysis�was�performed�with�totalRNA�extracted�from�RAC�primary�cultures�transfected�with�1or�15�mg�of�SOX9�expression�vector.�RT-PCR�analysis�showedthat�the�steady-state�levels�of�COL2A1�mRNA�were�reduced,as�expected,�when�15�mg�of�SOX9�expression�vector�were�trans-fected�(Fig.�4A)�while� they�were�enhanced�when�SOX9�wasoverexpressed�at�low�levels�(Fig.�4B).

SOX9�binding�activity�decreases�when�RACdedifferentiate

To� study� SOX9� binding� activity�on� COL2A1� during� thechondrocyte�dedifferentiative�process,� nuclear� extracts� fromprimary�RAC�(P0)�and�from�second�(P2)�or�fifth�(P5)�passageswere�tested�in�EMSA�analysis�using�the�12392/12415�labeledprobe.�As�shown�in�Figure�5A,�the�SOX9–DNA�complex�dis-appeared�with�increasing�passage�number,�indicating�that�SOX9

binding�activity�on� human�COL2A1� gene� is� reduced�duringRAC�dedifferentiation.�In�parallel,�steady-state�levels�of�type�IIcollagen�mRNA�were�also�decreased,�as�a�reflect�of�phenotypemodulation�during�subsequent�passages�(Fig.�5B).

In�addition,�EMSA�experiments�demonstrated�that�a�down-regulation�of�SOX9�binding�activity�to�the�human�COL2A1�genespecific�enhancer�was�induced�by�two�cytokines,�IL-1b and�TGF-b1,�which�are�known�to�play�key�roles�in�the�osteoarthritic�pro-cess�(Fig.�5C).�These�findings�are�consistent�with�the�fact�thatthese�two�factors�were�shown�to�downregulate�type�II�collagenexpression�in�primary�RAC�(Galéra�et�al.,�1992;�Chadjichristoset�al.,�2002),�and�that�IL-1b inhibits�SOX9�expression�in�chon-drocytes�(Murakami�et�al.,�2000;�Kolettas�et�al.,�2001).

SOX9�exerts�a�bifunctional�effect�on�COL2A1�geneexpression�in�slightly�dedifferentiated�RAC,�dependingon�its�level�of�overexpression

RAC�dedifferentiated�by�two�passages�were�transfected�withthe�pGL2-3.774�kb�construct�and�increasing�amounts�of�SOX9

SOX9�IMPLICATION�IN�HUMAN�COL2A1�GENE�EXPRESSION 123

FIG.�4. Correlation�of�SOX9�transcriptional�effects�with�the�endogenous�type�II�collagen�mRNA� levels.�Primary�RAC�weretransfected�with�15�mg�(A)�or�1�mg�(B)�of�an�expression�vector�containing�(SOX9)�or�not�(control)�the�cDNA�encoding�SOX9.Total�RNAs�were�extracted�6,�12,�and�48�h�after�the�replacement�of�the�culture�medium�that�followed�the�transfection�procedure.Three�micrograms�of�total�RNA�were�used�for�the�reverse�transcription�reaction;�19–34�PCR�cycles�were�then�performed�to�am-plify�the�COL2A1�cDNA,�as�well�as�the�GAPDH�cDNA,�by�using�specific�primers,�as�described�in�Materials�and�Methods.�Therelative�expression�of�COL2A1� cDNAs�was�normalized�to�GAPDH� cDNA�levels,�after�densitometric�analysis.�AU:� arbitraryunits.

expression�vector.�When�overexpressed�at�low�concentrations(1� mg� of� transfected� vector),� SOX9� slightly� upregulatedCOL2A1�gene�transcription,�whereas�it�inhibited�gene�expres-sion�when�transfected�at�higher�levels�($5�mg)�(Fig.�6A).

To�delineate�the�gene�regions�mediating�SOX9�effects,�RACdedifferentiated�by�two�passages�(P2)�were�cotransfected�withconstructs�representing�human�COL2A1�gene�characteristic�re-gions�in�the�presence�of�1�(Fig.�6B�i)�or�15�mg�(Fig.�6B�ii)�ofSOX9�expression�vector.�In�the�first�case,�SOX9�exerted�an�ac-tivating�effect�through�the�12384/12842-bp�sequences�of�thegene�specific�enhancer,�whereas�it�inhibited�the�gene�expres-sion�by�a�short�region�of�the�proximal�promoter�covering�the2266/263-bp�sequences�when�overexpressed�using�15�mg�ofthe�vector.�These�results�suggest�that�in�early�stages�of�chon-drocyte�dedifferentiation,�SOX9� can�still�exert�a�bifunctionaleffect�on�COL2A1�gene�expression,�as�it�is�the�case�in�primaryRAC.�Here,�too,�this�effect�is�mediated�by�the�specific�enhancerfor�the�activation,�and�the�proximal�promoter�for�the�inhibition.

SOX9�fails�to�activate�human�COL2A1�genetranscription�in�fully�dedifferentiated�chondrocytes

To�investigate�SOX9�effects�on�human�COL2A1�gene�tran-scription�in�more�advanced�stages�of�chondrocyte�dedifferenti-ation,� transfections�were� performed,�in�RAC� dedifferentiatedby�four�passages�(RAC�P4),�as�previously�described�for�RACP2.

When�overexpressed�in�increasing�amounts,�in�the�presenceof� pGL2-3.774� kb� construct,� SOX9� was� unable� to� activateCOL2A1� transcription�(Fig.� 7A).� In� contrast,� the� SOX9� in-hibitory�effect�became�more�important�for�higher�levels�of�over-expression.�In�experiments�where�RAC�P4�were�cotransfectedwith�different�COL2A1�gene�constructs�and�1�or�15�mg�of�SOX9expression�vector,�an�inhibition�was�observed�in�all�cases,�sug-gesting�that�the�SOX9�effect�was�mediated�by�the�63-bp�ele-ment�of�the�COL2A1�promoter�(Fig.�7B).

It� was� concluded� that� SOX9� loses� its� ability� to� activate

KYPRIOTOU�ET�AL.124

FIG.�5. The�binding�activity�of�SOX9�decreases�during�the�process�of�RAC�dedifferentiation.�SOX9�binding�activity�on�theCOL2A1�gene-specific�enhancer�was�studied�in�gel�retardation�assays.�(A)�A�double-stranded�labeled�wild-type�12392/12415probe�was�incubated�for�30�min�at�room�temperature�with�15�mg�of�nuclear�extracts�from�primary�RAC�(lane�2),�RAC�P2�(twopassages;�lane�3)�or�RAC�P5�(five�passages;�lane�4).�(B)�Total�RNA�(1�mg)�from�primary�RAC�(RAC�P0),�and�RAC�P2�(2�pas-sages)�were�reverse-transcribed�into�cDNA�by�using�specific�antisense�primers�for�COL2A1�and�b-actin�mRNAs,�as�describedin�Material�and�Methods;�15–30�PCR�cycles�were�then�performed�to�amplify�the�COL2A1�cDNA,�as�well�as�the�b-actin�cDNA.The�relative�expression�of�COL2A1�was�normalized�to�b-actin�cDNA�levels,�after�densitometric�analysis.�(C)�The�double-strandedlabeled�wild-type�12392/12415�probe�was�incubated�for�30�min�at�room�temperature�with�15�mg�of�nuclear�extracts�from�pri-mary�RAC�treated�with�TGF-b1�(T)�(5�ng/ml)�(lane�2)�and�IL-1b (I)�(5�ng/ml)�(lane�3)�or�not�treated�(C)�(lane�1).�The�arrowindicates�the�position�of�the�SOX9–DNA�complex.

COL2A1�gene�transcription,�in�fully�dedifferentiated�chondro-cytes.�Thus,�overexpression�of�this�factor�is�not�sufficient�to�re-store�the�chondrocyte�phenotype,�which�is�lost�during�the�phe-notype�alteration.

DISCUSSION

To�better�understand�the�molecular�mechanisms�implicatedin� the�chondrocyte�phenotypic�alteration,�we� investigated�theeffect�of�SOX9,�a�transcription�factor�required�for�cartilage�for-mation,�on� COL2A1� gene� transcription�in� differentiated�anddedifferentiated�articular�chondrocytes�in� culture.�SOX9� hasbeen�initially�described�as�an�activator�of�a�48-bp�specific�en-hancer�in�different�cell�lines�(Lefebvre�et�al.,�1997;�Ng�et�al.,1997).�In�addition,�it�was�demonstrated�that�SOX9�overexpres-sion� can� slightly� increase� type� II� collagen� mRNA� level� in10T1/2� fibroblastic�cells�(Lefebvre�et�al.,�1998).�The�presentdata�are�consistent�with�this�general�effect,�because�SOX9�wasfound�here� to� induce�the� transactivation�of�a� large�constructcovering�the�main�regulatory�regions�of�human�COL2A1�gene

(intronic�enhancer,�promoter,�silencers)�already�reported�(Sav-agner�et�al.,�1990;�Mukhopadhyay�et�al.,�1995;�Krebsbach�etal.,�1996;�Ghayor�et�al.,�2000,�2001).�However,�we�found�thatSOX9�has�a�dual�role�on�type�II�collagen�regulation�of�tran-scription,�depending�on�the�level�of�its�expression�and�the�chon-drocyte�differentiation�state.�Thus,�when�the� transfection�ex-periments�were�performed�with�cDNA�amounts�above�5�mg,�aninhibition�of�the�COL2A1�gene�transcription�was�observed.�Thiseffect�is�mediated�by�the�proximal�promoter,�and�may�be�dueto�a� squelching�mechanism:�when�SOX9� is�overexpressed�inrelatively�higher�amounts,�it�could�disturb�the�cellular�balancebetween�the�transcription�factors,�preventing�the�binding�of�oneof�these�proteins�to�the�gene�specific�sequence,�either�maskingits�binding�domain,�or�blocking�its�interaction�with�the�acces-sory�factors.�Such�a�mechanism�has�already�been�proposed�toexplain�the�dual�effect�of�several�factors,�according�to�their�ex-pression�level�and�their�cellular�localization.�For�example,�high-level�overexpression�of�Stat5b,�a�mediator�of�the�IFN-g trans-duction� signal,� was� found� to� inhibit� IRF-1� (InterferonRegulating�Factor-1)�and�the�NF-kB� signaling�pathway,�pre-venting�the�limiting�factors�p300/CBP�to�act�as�coactivators�for

SOX9�IMPLICATION�IN�HUMAN�COL2A1�GENE�EXPRESSION 125

FIG.�6. Effects�of�SOX9�on�COL2A1�gene�expression�in�slightly�dedifferentiated�RAC.�(A)�RAC�dedifferentiated�by�two�pas-sages�(RAC�P2)�were�transiently�cotransfected�with�25�mg�of�the�pGL2-3.774-kb�COL2A1�gene�construct�and�with�increasingamounts�of�SOX9�expression�vector.�For�each�amount�of�SOX9�overexpressed,�the�insertless�pcDNA-59UT-FLAG�vector�wasused�as�a�complement�to�15�mg,�to�transfect�each�sample�with�the�same�amount�of�the�expression�vector.�Relative�luciferase�ac-tivities�were�measured�in�cell�extracts�and�normalized�to�protein�amount�per�culture�plate.�RLU�represents�the�mean 6 SD�ofthree�independent�samples�of�a�representative�experiment.�(B)�RAC�P2�were�transiently�cotransfected�with�25�mg�of�differentCOL2A1�reporter�plasmids�in�the�presence�of�1�mg�(1)�or�15�mg�(2)�of�an�expression�vector�containing�(SOX9)�or�not�(control)the�cDNA�encoding�SOX9.�In�the�case�of�1�mg�of�SOX9�overexpressed,�the�insertless�pc�DNA-59UT-FLAG�vector�was�used�asa�complement�to�15�mg.�RLU�represents�the�mean 6 SD�of�three�independent�samples�of�a�representative�experiment.

Stat1� transcription� factor—which� activates� IRF-1—via� pro-tein–protein�interactions�(Guoyang�and�Li-yuan,�2000).�It�is�notknown�whether�this�mechanism�could�have�some�physiologicalrelevance,�because�the�amount�of�SOX9�protein�expressed�innormal�or�pathological�cartilage�is�not�precisely�determined�sofar.�Nevertheless,�it�suggests�that�the�effect�of�SOX9�on�typeII�collagen�expression�may�be�modulated�by�the�relative�ratioof�the�protein�to�the�other�factors�cooperating�with�it,�includ-ing�L-SOX5�and�SOX6.

As�an�alternative,�an�indirect�effect�of�SOX9�on�COL2A1gene�could�also�be�suggested.�In�this�respect,�it�must�be�notedthat�the�expression�of�b-galactosidase,�used�here�as�an�internalcontrol�of�transfection�efficiency�in�all�the�experiments,�but�notemployed� for� normalization,�was� constantly� inhibited�whenSOX9�was�transfected�whatever�the�amount�used�(Fig.�8).�SV40promoter�contains�several�binding�sites�for�Sp1/Sp3�factors,�asit�is�also�the�case�for�the�2266/11-bp�sequences�of�COL2A1gene�that�bear�seven�Sp1/Sp3�binding�sites,�one�being�locatedin�the�263/11-bp�segment.�Thus,�we�may�suggest�that�SOX9-

induced�inhibition�is�mediated�by�Sp1/Sp3�transcription�factorsratio�(Ghayor�et�al.,�2000,�2001;�Chadjichristos�et�al.,�2002).We�have�already�demonstrated�that�TGF-b1�exerts�an�inhibi-tion�on�COL2A1�gene�transcription�in�primary�RAC�through�amechanism� that� involves� the� Sp1/Sp3� binding� site� in� the263/11-bp�sequences�and�that�fixation�of�Sp3�displaces�Sp1,therefore�suppressing�its�transactivating�effect�(Chadjichristoset� al.,� 2002).�A� similar� mechanism� could� perhaps� apply� toSOX9�and�explain�the�present�findings.

Furthermore,�the�inhibitory�effect�of�SOX9�was�constantlyobserved�with�the�266-bp�promoter�construct,�which�containsseven�copies�of�Sp1�responsive�elements,�whereas�it�was�notsystematically�found�with�the�63-bp�segment�upstream�fromthe�transcription�start�site�that�contains�only�one�Sp1�site,�sug-gesting�that�SOX9�exerts�its�full�effect�only�in�the�presenceof�the�six�binding�sites�located�upstream�from�the�63-bp�prox-imal� sequence.� Therefore,� SOX9� inhibitory� property� may�depend� on� the� amounts� of� endogenous� factors� (probablySp1/Sp3)�available�in� the�cells�and�on� the�balance�between

KYPRIOTOU�ET�AL.126

FIG.�7. Effects�of�SOX9�on�human�COL2A1�gene�transcription�in�fully�dedifferentiated�RAC.�(A)�RAC�dedifferentiated�byfour�passages�(RAC�P4)�were�transiently�cotransfected�with�25�mg�of�pGL2-3.774-kb�COL2A1�gene�construct�and�with�increasingamounts�of�the�SOX9�expression�vector.�For�each�amount�of�SOX9�overexpressed,�the�insertless�pc�DNA-59UT-FLAG�vectorwas�used�as�a�complement�to�15�mg.�Relative�luciferase�activities�were�measured�in�cell�extracts�and�normalized�to�protein�amountper�culture�plate.�RLU�represents�the�mean 6 SD�of�three�independent�samples�of�a�representative�experiment.�(B)�RAC�P4�weretransiently�cotransfected�with�25�mg�of�different�COL2A1�reporter�plasmids�in�the�presence�of�1�or�15�mg�of�an�expression�vec-tor�containing�(SOX9)�or�not�(control)�the�cDNA�encoding�SOX9.�In�the�case�of�1�mg�of�SOX9�overexpressed,�the�insertless�pcDNA-59UT-FLAG�vector�was�used�as�a�complement�to�15�mg.�The�relative�luciferase�activities�obtained�for�the�samples�trans-fected�with�the�SOX9�expression�vector�are�expressed�as�the�percentage�of�the�relative�luciferase�units�of�the�respective�controlplasmids�receiving�the�empty�expression�vector.�The�values�represent�the�mean 6 SD�of�three�independent�samples�of�a�repre-sentative�experiment.

activating�and� inhibitory�cis elements�present� in� the� trans-fected�construct.

Interestingly,�an�inhibitory�effect�of�SOX9�on�type�II�collagenexpression�was�observed�in�our�experiments�when�chondrocyteswere�fully�dedifferentiated�by�several�culture�passages.�This�ef-fect�was�obtained�for�1�mg�of�SOX9�cDNA�expression�vectortransfected�as�well�as�for�higher�amounts,�suggesting�that�it�can-not�be�due�to�a�squelching�mechanism�or�to�a�nonspecific�bind-ing�of�SOX9.�In�fact,�the�mechanism�of�switch�between�SOX9positive�and�negative�effects�was�found�to�be�progressive�duringthe�phenotype�alteration�process.�SOX9�activating�action�was�re-duced�in�the�proximal�passages,�to�finally�shift�to�a�clear�inhibi-tion�in�fully�dedifferentiated�chondrocytes�(four�to�five�passages).The�inhibition�of�COL2A1�expression�in�the�final�steps�of�ded-ifferentiation�involves�the�263/11-bp�region.�This�clearly�indi-cates�that�SOX9�can�act�on�two�distinct�regions�of�COL2A1�gene,as�previously�reported�for�C-Krox,�a�factor�that�activates�the�genetranscription� through� its� intronic� enhancer� in� primary� RAC,whereas�it�inhibits�through�the�gene�proximal�promoter�in�ded-ifferentiated�chondrocytes�(Ghayor�et�al.,�2000).

The� initial�aim�of�this�study�was� to�assess�whether�SOX9overexpression�in�dedifferentiated�chondrocytes�was�capable�tostimulate�type�II�collagen�expression�and�to�restore�the�chon-drocytic�phenotype.�The�present�data�do�not�support�this�hy-pothesis,�because�SOX9� activating�effect�is� reduced�with� in-creasing�number�of�culture�passages,�to�finally�shift�to�a�clearinhibition�in� highly�dedifferentiated�chondrocytes.�This� sug-gests�that�SOX9�alone�is�not�capable�of�reorienting�the�chon-drocyte�phenotype.

SOX9�IMPLICATION�IN�HUMAN�COL2A1�GENE�EXPRESSION 127

FIG.� 8. SOX9� downregulates� b-galactosidase� expressionlevel�in�a�dose-dependent�way.�Primary�RAC�cultures�were�tran-siently�cotransfected�with�25�mg�of�a�pGL2-3.774-kb�COL2A1gene�construct�and�with�different�amounts�of�an�expression�vec-tor�containing�the�SOX9� cDNA,� in� the�presence�of� 2�mg� ofpSV40–b-galactosidase�expression�vector,�used�as�internal�con-trol�for�transfection�efficiency.�For�each�amount�of�SOX9�over-expressed,�the�insertless�pc�DNA-59UT-FLAG�vector�was�usedas�a�complement�to�15�mg.�b-Galactosidase�activity�was�mea-sured�in�cell�extracts�as�described�in�the�Materials�and�Meth-ods� section� by� measuring� the� absorbance� at� 572� nm� after�incubation�with� the� b-galactosidase�substrate,�resorufin�b-D-galactopyranoside�(Ghayor�et�al.,�2000).�The�values�representthe�mean 6 SD�of�three�independent�samples�of�a�representa-tive�experiment.

FIG.�9. SOX9�has�a�bifunctional�effect�on�human�COL2A1�gene�expression�depending�on�the�amount�overexpressed�and�onthe�chondrocyte�differentiation�state.�Implication�of�two�different�regions�of�the�gene.�SOX9�can�inhibit�(Q)�the�human�COL2A1gene�expression,�independently�of�the�chondrocyte�differentiation�state,�when�it�is�overexpressed�at�high�concentration�(10–20mg),�via�a�region�covering�the�266-bp�of�its�short�promoter.�In�contrast,�SOX9�is�an�activator�(q)�of�COL2A1�gene�expression,when�overexpressed�at�low�concentration�(1�mg),�in�primary�and�slightly�differentiated�articular�chondrocytes�(P2);�this�activat-ing�effect�is�mediated�by�the�specific�enhancer�located�in�the�first�intron�of�the�gene.�However,�this�transcription�factor,�evenwhen�overexpressed�at�a�low�level,�has�no�effect�( rR )�on�the�human�COL2A1�gene�transcription�mediated�by�the�specific�in-tronic�enhancer�in�the�latest�stages�of�RAC�dedifferentiation.

SOX9�has�been�shown�to�require�the�cooperating�effect�ofL-SOX5�and�SOX6�for�its�full�activating�action�on�COL2A1gene� expression.�The� coexpression�of� the� three� proteins� in10T1/2�fibroblastic�cells�(Lefebvre�et�al.,�1997)�and�in�dedif-ferentiated�fetal�bovine�chondrocytes�(Stokes�et�al.,�2001)�pro-duce�a�higher�activation�of�COL2A1�gene�transcription�and�anincreased�mRNA� level,�compared�to�SOX9� alone.�We�couldtherefore�suppose�that�a�group�of�transcription�factors�are�re-quired�to�activate�the�COL2A1�gene�transcriptional�activity�indedifferentiated�cells.

Other�protein�factors�could�play�a�role�in�the�maintenance�orrecovery�of�chondrocyte�phenotype,�including�Sp1�and�C-Krox.These� factors� activate�COL2A1� gene� expression�in� primaryRAC,� and� their�pattern�of�expression�are� similar�to� those�ofCOL2A1�and�SOX9�during�the�mouse�embryonic�development(8.5-9.5�dpc)�(Galéra�et�al.,�1996;�Zhao�et�al.,�1997;�Ng�et�al.,1997;�Marin�et�al.,�1997;�Lefebvre�et�al.,�1998;�Leung�et�al.,1998).�Moreover,�they�are�present�in�great�amounts�in�primaryRAC�and�their�level�is�reduced�with�subsequent�passages�of�thecultures�(Ghayor�et�al.,�2000,�2001).

Supporting�the�potential�role�of�L-SOX5�and�SOX6�duringchondrocyte�dedifferentiation,�it�has�been�reported�that�their�in-teraction�with�the�human�COL2A1�gene�enhancer�was�modi-fied�with�increasing�number�of�passages�(Stokes�et�al.,�2001).mRNA�levels�of�SOX9�were�also�found�to�decrease�in�the�courseof� in�vitro dedifferentiation�of�chondrocytes�(Lefebvre�et�al.,1997;�Kolettas�et�al.,�2001;�Stokes�et�al.,�2001).�Here,�a�de-crease�in�SOX9� binding�activity�on�COL2A1�gene�enhancerwas�observed�with�increasing�the�number�of�passages,�in�agree-ment�with�previous�reports�(Lefebvre�et�al.,�1997;�Stokes�et�al.,2001).�This�suggests�a�decreased�affinity�of�SOX9�for�this�re-gion�of�the�gene�or/and�a�lower�level�of�SOX9�expression�dur-ing�the�dedifferentiation�process�of�RAC.�Interestingly�enough,in�an�animal�model�of�osteoarthritis�realized�on�transgenic�mice,it� was� recently� shown� that� SOX9� was� highly� expressed� inslightly�altered�cartilage�areas,�where�chondrocytes�were�pro-liferating,�whereas�it�was�not�detected�in�profoundly�fibrillatedareas�(Salminen�et�al.,�2001).�This�may�indicate�that�SOX9�isplaying�a�role�in�the�hypertrophic�response�of�cartilage�gener-ally�observed�in�early�stages�of�the�disease.

Taking�into�account�that�SOX9�is�also�required�for�the�ex-pression�of�other�specific�components�of� cartilage,�includingcollagen�types�IX�and�XI�and�aggrecan�core�protein�(Lefebvreet�al.,�1997;�Ng�et�al.,�1997;�Bridgewater�et�al.,�1998;�Bi�et�al.,1999;�Sekiya�et�al.,�2000),�it�appears�as�a�key�factor�for�dif-ferentiated�chondrocytes,�mostly�implicated�in�their�anabolismbut�unable�to�prevent�or�reduce�their�degradative�evolution�inthe�osteoarthritic�process.

This�concept�is�corroborated�by�the�fact�that�SOX9�expres-sion�is�decreased�by�cytokines�such�as�IL-1�and�TNF-a,� twoproinflammatory�factors,�mainly�responsible�for�cartilage�ero-sion�in�joint�diseases�(Murakami�et�al.,�2000;�Kolettas�et�al.,2001).�In�this�study,�it�was�also�demonstrated�that�IL-1b andTGF-b1� decreased�SOX9� binding� activity�on� COL2A1� en-hancer.�Although�TGF-b1� is�generally�considered�as�an�acti-vator�of�collagen�genes,�studies�performed�in�our�laboratory�re-vealed�that�this�cytokine�inhibits�type�II�collagen�expression�inprimary�proliferating�RAC�(Chadjichristos�et�al.,�2002).�In�thisregard,�it� is�worth�mentioning�that�dedifferentiated�chondro-cytes�treated�by�TGF-b1�cannot�recover�their�phenotype,�sug-

gesting� that� TGF-b1� is� not� sufficient� to� antagonize�the� os-teoarthritic�process�(Galéra�et�al.,�1992).�These�observations�arecoherent�with�the�present�findings,�because�they�demonstrate�acorrelation�of�SOX9�and�type�II�collagen�expressions�and�theregulation�of�both�of�them�by�cytokines.

In�conclusion�and,�as�summarized�in�Figure�9,�SOX9,�de-spite�its�importance�for�the�cartilage�formation�and�the�chon-drocyte�differentiation,�is�not�sufficient�to�maintain�the�differ-entiated�chondrocyte�phenotype�because�it� inhibits�COL2A1gene�transcription�in�fully�dedifferentiated�chondrocytes.�How-ever,�the�role�of�L-SOX5�and�SOX6,�which�have�been�shownto�be�equally�important�for�chondrocyte�differentiation,�remainsto� be�elucidated�in� the�course�of�dedifferentiation.�As� far� asthese�in�vitro data�could�be�extrapolated�to�osteoarthritis�phys-iopathology,�they�may�help�to�understand�the�role�of�these�fac-tors,�together�with�Sp1�and�C-Krox,�in�the�alteration�of�chon-drocyte�phenotype�during�the�disease,�and�offer�possibilities�tomanipulate�them�to�prevent�or�reverse�the�process.

ACKNOWLEDGMENTS

We�would�like�to�thank�the�Regional�Council�of�“Basse�Nor-mandie”�for�supporting�this�work.

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Address�reprint�requests�to:Prof.�Philippe�Galéra

Laboratoire�de�Biochimie�du�Tissu�ConjonctifFaculté�de�Médecine

CHU�niveau�3Avenue�de�la�Côte�de�Nacre14032,�Caen�Cedex,�France

E-mail: [email protected]

Received�for�publication�October�16,�2002;�received�in�revisedform�October�30,�2002;�accepted�December�2,�2002.

SOX9�IMPLICATION�IN�HUMAN�COL2A1�GENE�EXPRESSION 129