Wnt/�-Catenin and Retinoic Acid Receptor SignalingPathways Interact to Regulate Chondrocyte Function andMatrix Turnover*□S
Received for publication, August 7, 2009, and in revised form, October 22, 2009 Published, JBC Papers in Press, October 26, 2009, DOI 10.1074/jbc.M109.053926
Rika Yasuhara‡1, Takahito Yuasa‡2, Julie A. Williams‡, Stephen W. Byers§, Salim Shah§, Maurizio Pacifici‡,Masahiro Iwamoto‡3, and Motomi Enomoto-Iwamoto‡4
From the ‡Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the§Lombardi Comprehensive Cancer Center and Departments of Oncology and Biochemistry, Molecular and Cellular BiologyDivision, Georgetown University School of Medicine, Washington, D. C. 20007
Activation of theWnt/�-catenin and retinoid signaling path-ways is known to tilt cartilagematrix homeostasis toward catab-olism.Here, we investigated possible interactions between thesepathways. We found that all-trans-retinoic acid (RA) treatmentof mouse epiphyseal chondrocytes in culture did increaseWnt/�-catenin signaling in the absence or presence of exogenousWnt3a, as revealed by lymphoid enhancer factor/T-cell factor/�-catenin reporter activity and �-catenin nuclear accumula-tion. This stimulation was accompanied by increased geneexpression of Wnt proteins and receptors and was inhibited byco-treatment with Dickkopf-related protein-1, an extracellularinhibitor of Wnt/�-catenin signaling, suggesting that RA mod-ulates Wnt signaling at Wnt cell surface receptor level. RA alsoenhanced matrix loss triggered by Wnt/�-catenin signaling,whereas treatment with a retinoid antagonist reduced it. Inter-estingly, overexpression of retinoic acid receptor � (RAR�)strongly inhibitedWnt/�-catenin signaling in retinoid-free cul-tures, whereas small interfering RNA-mediated silencing ofendogenous RAR� expression strongly increased it. Small inter-fering RNA-mediated silencing of RAR� or RAR� had minimaleffects. Co-immunoprecipitation and two-hybrid assays indi-cated that RAR� interacts with �-catenin and induces dissocia-tion of �-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RAR� but notthe C-terminal domain (AF-2) was required for association with�-catenin, whereas bothAF-1 andAF-2were necessary for inhibi-tion of�-catenin transcriptional activity. Taken together, our dataindicate that theWnt and retinoid signaling pathways do interactin chondrocytes, and their cross-talks and cross-regulation playimportant roles in the regulation of cartilagematrix homeostasis.
Growth plate cartilage and hyaline cartilage have essential rolesin skeletal growth and long term function. Growth plate cartilagemediates the formation and elongation of most skeletal elementsduring prenatal and early postnatal life via endochondral ossifica-tion, including vertebral bodies, cranial base, and long bones (1, 2).During this process, growthplate chondrocytes undergo a processof maturation during which the cells first proliferate and thenenlarge and become hypertrophic, mineralize their extracellularmatrix, and are finally replaced by bone andmarrow.As the chon-drocytes mature, they change their production of extracellularmatrix that is mainly composed of aggrecan and collagens II andXI in upper growth plate zones but becomes enriched with colla-gen X and other macromolecules in the hypertrophic zone. Incomparison with the transient nature of growth plate cartilage,hyaline permanent cartilage persists throughout life at importantlocations, such as joints and tracheal rings. This tissue also has avery abundant and unique extracellular matrix that is maintainedin a stable and functional composition and structure by chondro-cyte activity. It is well established that the extracellular matrix inboth hyaline and growth plate cartilage is regulated by a fine bal-ance between synthetic and catabolicmechanisms that are impor-tant for both maintenance of permanent cartilage function andprogression of endochondral bone formation. Indeed, imbalancesin such homeostatic pathways can lead to a variety of pathologicalskeletal conditions that include osteoarthritis and skeletal dyspla-sias (3–5).In recent studies, we found thatWnt/�-catenin signaling can
strongly affect matrix anabolic and catabolic metabolism inchondrocytes (6). Wnt/�-catenin signaling is a major signaltransduction pathway of Wnt proteins (7, 8). Binding of Wntproteins to cell surface receptor complexes composed of Friz-zleds and low density lipoprotein receptor-related protein 5/6(LRP5/6) activates the downstream intercellular proteinDishevelled. This results in inhibition of glycogen synthase 3�(GSK3�) kinase and �-catenin phosphorylation, thus allowing�-catenin to escape the ubiquitin proteasome degradationpathway and to accumulate in the cytoplasm. Non-phosphory-lated �-catenin then translocates to the nucleus, where it inter-acts with lymphoid enhancer factor/T-cell factor (LEF/TCF)5
* This work was supported, in whole or in part, by National Institutes of HealthGrants AR050507 (to M. E. I.), AG025868 (to M. P.), AR056837 (to M. I.), andCA129813 (to S. W. B.).
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.
1 Present address: Dept. of Biochemistry, Showa University School of Den-tistry, Tokyo 142-8555, Japan.
2 Dept. of Orthopaedic Surgery, Juntendo University, Tokyo 113-8421, Japan.3 To whom correspondence may be addressed: 1015 Walnut St., Curtis 501,
Philadelphia, PA 19107. Tel.: 215-955-4076; Fax: 215-955-9159; E-mail:[email protected].
4 To whom correspondence may be addressed: 1015 Walnut St., Curtis 501,Philadelphia, PA 19107. Tel.: 215-955-7624; Fax: 215-955-9159; E-mail:[email protected].
5 The abbreviations used are: LEF, lymphoid enhancer factor; TCF, T-cell fac-tor; RAR, retinoic acid receptor; RA, all-trans-retinoic acid; rWnt3a, recom-binant Wnt3a; ANT, RAR antagonist; HA, hemagglutinin; siRNA, small inter-fering RNA.
transcription factors to regulate target gene expression. Wefound that acute activation ofWnt/�-catenin signaling stronglyinhibited gene expression of aggrecan and collagens II and IXand stimulated gene expression and activity of matrix pro-teases, resulting in matrix loss in vitro and in vivo. Subsequentstudies by several groups, including ours, showed that in addi-tion to matrix homeostasis, Wnt/�-catenin signaling normallyregulates other chondrocyte functions and influences cartilagedevelopment (3, 9–13). What has remained unclear, however,is how Wnt/�-catenin signaling is regulated and modulatedduring cartilage development, skeletal growth, and skeletalhomeostasis and is able to regulate such variety of importantprocesses.Studies have shown that the Wnt/�-catenin pathway can
influence the function of nuclear receptor proteins and affectimportant biological and pathological processes with broadphysiological and clinical relevance (14). Nuclear retinoic acidreceptors (RARs) are also positively or negatively affected byWnt/�-catenin signaling in a variety of cell types, such as colonand breast cancer cells, neuronal cells, and ES cells (15–22).Retinoid signaling has long been known to regulate cartilagedevelopment and skeletogenesis (23–28). Genes encodingRAR�, RAR�, and RAR� display specific spatiotemporal pat-terns of expression during cartilage formation in the developinglimb (29–31). RAR� and RAR� expression is predominant inprecartilaginous mesenchyme and newly formed cartilage, andRAR� is expressed in perichondrium, and chondrogenic celldifferentiation is found to require repressor function of unli-ganded RAR� (29). In good correlation, ablation of genesencoding RARs (32) or enzymes involved in retinoid synthesisor degradation causes a spectrum of skeletal abnormalities(33, 34). In addition, studies in vivo and in vitro have shownthat retinoic acid (RA) has strong effects on cartilage matrixhomeostasis that includes inhibition of matrix synthesis (35,36), stimulation of matrix degradation (37, 38), and stimula-tion of chondrocyte terminal differentiation and calcifica-tion (23–25). In sum, the above studies clearly indicate thatretinoid signaling is very important for skeletogenesis andprogression of endochondral ossification and that dysregu-lation of this signaling pathway can lead to skeletal aberra-tions and cartilage degeneration.Because both retinoid signaling andWnt/�-catenin signaling
strongly affect cartilage development and homeostasis, wetested herewhether these pathways actually interact to regulateand modulate chondrocyte function and phenotype. Ourresults show that this is indeed the case and that RAR� has a keyrole in these mechanisms.
EXPERIMENTAL PROCEDURES
Chondrocyte and Limb Bud Cell Cultures—Mouse primaryepiphyseal chondrocytes were isolated from neonatal C57BL/6mice as previously described (6). Distal cartilaginous ends offemurs and humeri from neonatal mice were digested by 0.25%trypsin and 2 mM EDTA for 15 min, followed by digestion with2 mg/ml collagenase type I (Worthington) for 6 h. Cells wereplated at 40,000 cells/well in 96-well plates and maintainedin Dulbecco’s modified Eagle’s medium containing 10% fetalbovine serum (GEMINI,West Sacramento, CA). Cultures were
treated with recombinant Wnt3a (rWnt3a) (Chemicon,Temecula, CA), recombinant mouse Dickkopf-related protein1 (R&D Systems Inc., Minneapolis, MN), RA (Sigma), RARantagonist (ANT) (synthetic retinoid pan-antagonist AGN/VTP 194310 provided from Allergan Pharmaceuticals andVitae Pharmaceuticals), or 6BIO (BIO, Enzo Life SciencesInternational Inc., Plymouth Meeting, PA). For detection ofsulfated proteoglycans associated with the cell layer, cultureswere fixed with 10% formalin for 10 min and incubated with70% ethanol for 5 min, followed by incubation with 5% aceticacid (pH1.0) for 5min and stainedwith 1%Alcian blue (ElectribMicroscopy Science, Hartfield, PA) for 2 h at room tempera-ture. Staining levels were quantified by Image J software. Limbmesenchymal cells were isolated from the fore and hind limbsof embryonic day 10.5 mouse embryos by incubation with0.25% trypsin and 2 mM EDTA for 15 min. Cells were plated at20,000 cells/well in 96-well plates andmaintained inDulbecco’smodified Eagle’s medium/Ham’s nutrient mixture F-12 con-taining 2% fetal bovine serum. Cells were used for a Wnt/�-catenin reporter assay immediately after isolation and did notexpress chondrogenic characteristics. Freshly isolated limbmesenchymal cells were also inoculated at 2.5 � 105 cells/20 �land cultured in Dulbecco’s modified Eagle’s medium/Ham’snutrient mixture F-12 containing 2% fetal bovine serum and100 ng/ml recombinant BMP2 for 7 days to induce chondro-genic differentiation. The cells were then dissociated by diges-tion with 0.15% collagenase for 15min, replated at 40,000 cells/well in 96-well plates, and used for theWnt/�-catenin reporterassay.Reverse Transfection and Reporter Assays—Reverse transfec-
tion of DNA plasmid was carried out using Lipofectamine 2000(Invitrogen) according to the manufacturer’s protocol. Freshlyisolated chondrocytes or limb bud cells were plated at an initialdensity of 4 � 104/well or 2 � 104/well, respectively, into96-well plates that had been coated with a Wnt/�-cateninreporter plasmid (Super 8x TOPFlash, Addgene Inc., Cam-bridge, MA) in the presence of Lipofectamine 2000. After 24 h,cultures were treated with RA, RAR antagonist rWnt3a, 6BIO,or rDKK-1. To normalize transfection efficiency, we co-trans-fected the Renilla luciferase-expressing plasmid (pRL-TK-luc)as internal control, and luciferase activities of both Super 8xTOPFlash and pRL-TK-luc reporters were measured using adual luciferase assay kit (Promega Corp., Madison, WI). Super8x TOPFlash encodes seven copies of LEF/TCF binding siteslinked to firefly luciferase and reflectsWnt/�-catenin signalingactivity. Super 8x FOPFlash has seven copies of mutated LEF/TCF binding sites and was used as negative internal control. Amammalian two-hybrid system was used to examine protein-protein interactions using the CheckMate Mammaliantwo-hybrid system (Promega) according to the manufacturer’sprotocol. Gal4/�-catenin expression vectors were describedpreviously (16), and VP16/LEF-1 and dominant negative formexpression vector were kindly provided by Dr. A. Hecht(Albert-Ludwigs University). Mouse RAR� (American TypeCulture Collection (ATCC) (Manassas, VA) catalog number10324162, GenBankTM IDNM_009024), RAR� (ATCC catalognumber 10699549, GenBankTM ID NM_011243), or RAR�1(ATCC catalog number MGC-11555, GenBankTM ID
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NM_011244) were tagged with hemagglutinin (HA) by sub-cloning at the EcoRI andKpnI sites of pCMV-HA vector (Clon-tech/Takara, Mountain View, CA). pCMV-HA empty vectorwas used as control.Construction of RAR� Mutants—Mouse RAR�1 cDNA was
purchased from the ATCC, and deletion mutants were gener-ated by PCR. PCR primer pairs for the deletion mutant of theAF-1 domain (718–1833 of NM_011244) were 5�-CCGAATT-CACAAGCCATGCTTTGTATGCAATGAC-3� and 5�-GGG-GTACCTCAGGGCCCCTGGTCAGGT-3�, and those for thedeletion mutant of AF-2 domain (457–1701 of NM_011244)were 5�-CCGAATTCACATGGCCACCAATAAGGAGAG-ACT-3� and 5�-GGGGTACCCTACATCTCTCGGATCAGG-GGTGG-3�. The resulting PCR products were cloned intopCMV-HA vector at EcoRI and KpnI sites.Silencing of RARs—MouseRAR�, -�, and -� siRNApools and
non-targeting siRNA (control siRNA) were purchased fromDharmacon (Chicago, IL). siRNA for each RAR (1 pmol) andcontrol siRNA (1 pmol) (to exclude potential off-target effectscaused by siRNA) were co-transfected with Super 8x TOPFlashreporter plasmid and pRL-TK-luc into freshly isolated mousechondrocytes seeded at an initial density of 4 � 104/well in a96-well plate by reverse transfection using Lipofectamine 2000.RNA Isolation and Gene Expression Assay—Total RNA was
isolated by the guanidine isothiocyanate method as previouslydescribed (24). The resulting reverse transcriptionmixture wasused for a reverse transcription-PCR or quantitative PCR assay.Real-time PCR was performed with an Applied Biosystems7900HT sequence detection system running SDS 2.1 softwareusing SYBR Green (Applied Biosystems, Foster City, CA) fol-lowing the manufacturer’s instructions. The average thresholdcycle value (Ct value) was calculated from 4-fold reactions.Averaged Ct values were then normalized to the averaged Ctvalue of the housekeeping gene glyceraldehyde-3-phosphatedehydrogenase. Standard curves were generated using 10-foldserial dilutions of cDNA of each gene with a correlation coeffi-cient of �0.98. Relative expression levels were calculated basedon standard curves and represented ratios of experimental overcontrol values. Primer sequences for real-time PCR amplifica-tion were as follows: 5�-CTG AGG ACT TTC CAGGTG TTGACT CAA G-3� and 5�-TGG TTC TGC CAT AGC ACA TGCTGA AC-3� for 1362–1623 of mouse matrix metalloprotease 3(Mmp3) 5�-TCAGTT TCT TTA TGGTCCAGGCGATG-3�and 5�-TCA GTC TCT TCA CCT CTT TTG GGA ATC C-3�for 799–1160 ofmouseMmp13, 5�-TCTGGAAATGACAACCCC AAG CAC A-3� and 5TGG CGG TAA CAG TGA CCCTGGAACT-3� for 5463–5939 ofmouse aggrecan, and 5�-AAGCCC ATC ACC ATC TTC CAG GAG-3� and 5�-ATG AGCCCTTCCACAATGCCAAAG-3� for 258–568 of glyceralde-hyde-3-phosphate dehydrogenase, using proper filters to visu-alize fluorescent markers.To profile changes in gene expression of Wnts and Wnt-
related molecules by RA treatment, we carried out a PCRarray using the RT2 Profiler PCR array for the Wnt signalingpathway (Superarray, Frederick, MD) following the manu-facturer’s protocol. The average threshold cycle value (Ctvalue) was calculated from 4-fold reactions and normalizedto that of the housekeeping gene glyceraldehyde-3-phos-
phate dehydrogenase. These experiments were repeatedthree times independently.Immunoblot and Co-immunoprecipitation—Cellular levels
of �-catenin were analyzed by immunoblotting. Cell lysatescontaining equal amounts of proteins were separated on 10%SDS-polyacrylamide gels and electrotransferred onto Immo-bilon-P membranes (Millipore, Billerica, MA). Membraneswere blocked with 5% nonfat dry milk in 20 mM Tris-HCl (pH7.4) containing 150mMNaCl and 0.1%Tween 20 and subjectedto immunoblotting with antibodies against dephosphorylated�-catenin (Alexis, Lausen, Switzerland) or �-tubulin (Sigma),followed by incubation with secondary antibody conjugatedwith horseradish peroxidase. Protein bands were detected bymeans ofWestern Lightning Chemiluminescence Reagent Plus(PerkinElmer Life Sciences). For co-immnoprecipitation, 3 �106 cells COS7 cells were plated into a 100-mm dish and trans-fected with HA-tagged RAR� or deletion mutants of RAR�1.After 48 h, cultures were washed twice with ice-cold phos-phate-buffered saline and lysed in 1% Nonidet P-40 lysis buffer(20 mM HEPES, pH 7.5, 10 mM EGTA, 2.5 nM MgCl2, 40 nM�-glycerophosphate, 1%Nonidet P-40, 2mMVaO4, 1mMphen-ylmethylsulfonyl fluoride, 20 mg/ml aprotinin, 20 mg/ml leu-peptin, 1 mM dithiothreitol). Cell lysates were incubated on icefor 20min and then centrifuged at 150,000 rpm for 10min, andsupernatants were kept at �80 °C until use. Cell lysates wereincubated with IgG� beads (Roche Applied Science) at 4 °Covernight and then incubated with anti-HA antibody (RocheApplied Science) for 1 h at 4 °C, followed by incubation withIgG� beads at 4 °C for 1 h. Bound proteins were released byincubation with 3� SDS sample buffer for 5 min at 70 °C.Immunoprecipitated and co-immunoprecipitated proteinswere analyzed by immunoblotting.Immunofluorescence Staining—Cells were plated at 30 �
104/well onto 12-mm round glass coverslips (Hecht Assistent,Sondheim, Germany) coated with 1% gelatin solution in a24-well plate. After 24 h, cells were fixed with 10% neutralizedformalin, permeabilizedwith 0.5%TritonX-100, and incubatedwith primary �-catenin antibody (BD Biosciences) overnight at4 °C, followed by incubation with Alexofluor 594 dye-conju-gated second antibody (Invitrogen) for 30 min. Cultures werealso stained with 0.1 mg/ml 4�,6-diamidino-2-phenylindole(Sigma) for nuclear staining. Samples weremounted with GEL/MOUNT (Biomeda, Foster City, CA) and analyzed under a flu-orescent microscope (Nikon Eclipse TE2000-U). Images werecaptured and analyzed using ImagePro Plus 5.0 software.Statistical Analysis—One-way analysis of variance followed
by Boneferroni/Dunn post hoc multiple comparison tests(Prism 5, GraphPad Software Inc., La Jolla, CA) was used todetermine statistical significance between groups. p values lessthan 0.01 were considered significant (*, p � 0.01 as indicatedby brackets).
RESULTS
Wnt/�-Catenin Signaling Is Modulated by Retinoid Agonistsand Antagonists—We first tested whether Wnt/�-catenin sig-naling in chondrocytes is affected by retinoid agonists and/orantagonists. Chondrocytes freshly isolated from neonatalmouse epiphyseal cartilage were transfected with the Wnt/�-
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catenin reporter plasmid Super 8xTOPFlash and treated with differ-ent concentrations of (i) RA, whichis the most potent natural retinoidagonist; (ii) the retinoid panantago-nist AGN/VTP 194310 (ANT),which blocks the function of allRARs; or (iii) vehicle (EtOH). Re-porter activity was dose-dependentlyincreased by RA treatment (Fig. 1A)but was greatly inhibited by retinoidantagonist treatment (Fig. 1B).Transfection of control Super 8xFOPFlash reporter plasmid thatencodes mutated LEF/TCF bindingsites elicited no response (Fig. 1A,FOPFlash). The effects of RA andretinoid antagonist on Wnt/�-catenin signaling were also examinedby monitoring �-catenin nuclearaccumulation, a well recognized traitof Wnt/�-catenin signaling activa-tion, using immunocytochemistry.Nuclear accumulation of �-cateninwas indeed increased by RA treat-ment and decreased by retinoidantagonist treatment compared withcontrols (Fig. 1C).To determine how rapidly RA
and retinoid antagonist change�-catenin phosphorylation and sta-bilization and Wnt/�-catenin sig-naling activity, the levels of unphos-phorylated �-catenin was examinedat 3 or 24 h of RA treatment. Immu-noblot analysis revealed that RAtreatment did not significantlyincrease �-catenin content by 3 h(Fig. 1D, 3 h, lane 2), althoughWnt3a treatment had done so withor without RA treatment (Fig. 1D,3 h, lanes 4 and 5). RA treatment didincrease �-catenin content by 24 h(Fig. 1D, 24 h, lane 2) and furtherenhanced it duringWnt3a co-treat-ment (Fig. 1D, 24 h, lane 5). Inter-estingly, treatment with retinoidantagonist inhibited �-catenin accu-mulation during Wnt3a treatment(Fig. 1D, 24 h, lane 6).The effects of RA and retinoid
antagonist on Wnt/�-catenin sig-naling were also validated in chon-drocyte cultures isolated fromTOPGAL Wnt/�-catenin reportertransgenic mice that express �-ga-lactosidase linked to LEF/TCFbind-ing site-containing promoter ele-
FIGURE 1. RA stimulates and retinoid antagonist inhibits Wnt/�-catenin signaling. A and B, mouse epiph-yseal chondrocytes were transfected with Super 8x TOPFlash or FOPFlash plasmids and pRL-TK. After 24 h,cultures were treated with the indicated concentrations of RA (A) or ANT (B) for 24 h. Reporter activity wasnormalized by pRL-TK luciferase activity. Values represent averages and S.D. obtained from three independentsamples. *, p � 0.01 versus control. C, cultures were treated with EtOH, 300 nM RA, or 100 nM ANT for 24 h andstained with �-catenin antibodies. The percentage of cells exhibiting �-catenin nuclear staining was calculatedas described under “Experimental Procedures.” *, p � 0.01 versus control. D, epiphyseal chondrocytes weretreated with ethanol (lanes 1 and 4), 300 nM RA (lanes 2 and 5), or 100 nM ANT (lanes 3 and 6) in the absence (lanes1–3) or presence (lanes 4 – 6) of 100 ng/ml rWnt3a. The resulting cell lysates were subjected to immunoblotanalysis of �-catenin 3 or 24 h after treatment. F–H, freshly isolated mouse epiphyseal chondrocytes (F), freshlyisolated mouse limb mesenchymal cells (G), or mesenchymal cells replated after induction of chondrogenesis(H) were transfected with Super 8x TOPFlash or FOPFlash plasmids and pRL-TK. After 24 h, cultures were treatedwith vehicle (EtOH; open column), 300 nM RA (closed column), or 100 nM ANT (shadowed column) with or withoutco-treatment with 100 ng/ml rWnt3a for 24 h, and luciferase activity was then measured. Reporter activity wasnormalized by pRL-TK luciferase activity and is shown as relative ratio to vehicle-treated cultures withoutWnt3a treatment. Values represent averages and S.D. obtained from three independent samples. *, p � 0.01.
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ments (39). TOPGAL epiphyseal chondrocyte cultures weretreated with RA or retinoid antagonist for 24 h and were incu-bated with a fluorescent �-galactosidase substrate to visualizereporter activity. The number of fluorescence-positive cells wasincreased by treatment with RA or Wnt3a and was furtherincreased by RA andWnt3a co-treatment (supplemental Fig. 1,B and H). In contrast, retinoid antagonist treatment decreasedthe number of positive cells in the presence of Wnt3a (supple-mental Fig. 1I). Taken together, the above lines of experimen-tation all indicate that RA stimulates and retinoid antagonistinhibits Wnt/�-catenin signaling.
To determine whether the responses of chondrocytes to ret-inoid and Wnt/�-catenin signaling differ from those of othercells, we tested limbmesenchymal cells. The cells were plated ata low density, a condition that allows them to maintain theirmesenchymal character and prevents their chondrogenic dif-ferentiation. Interestingly, RA treatment strongly counteracted
Wnt/�-catenin signaling inductionbyWnt3a in these cultures (Fig. 1G,closed column), whereas RA en-hanced Wnt3a effects in parallelchondrocyte cultures (Fig. 1F,closed column), suggesting thatinteractions and effects of retinoidand Wnt/�-catenin signaling path-ways differ in mesenchymal pre-chondrogenic cells versusdifferenti-ated chondrocytes. To verify thisconclusion, freshly isolated limbbud cells were grown at high celldensity and treated with BMP-2, acombination that greatly stimulatestheir chondrogenic differentiation(40).Once the cultures haddifferen-tiated and produced cartilage nod-ules, the cells were dissociated bycollagenase treatment, replated,and processed for Wnt/�-cateninreporter assays. The cells now ex-hibited responses to RA and reti-noid antagonist similar to those ofepiphyseal chondrocytes. Wnt3atreatment stimulated reporter ac-tivity (Fig. 1H, open column), andco-treatment with RA further stim-ulated it (Fig. 1H, closed column),whereas retinoid antagonist treat-ment decreased reporter activity(Fig. 1H, striped column). Theseresults indicate that the responsesto Wnt/�-catenin and retinoid sig-naling pathways change duringchondrogenic cell differentiation.Retinoid Agonists and Antago-
nists AlterWnt/�-Catenin SignalingEffects on ProteoglycanMetabolism—To determine whether modulationof Wnt/�-catenin signaling by reti-
noid agonists and antagonists alters chondrocyte phenotypicfunction, we treated chondrocyte cultures with RA or antago-nist without or with Wnt3a co-treatment and then monitoredproteoglycan accumulation by Alcian blue staining (Figs. 2,A–F). Treatmentwith RAorWnt3a alone decreased proteogly-can content compared with controls (Fig. 2,A, B, andD). Nota-bly, treatment with retinoid antagonist increased proteoglycancontent in the absence or presence of Wnt3a (Fig. 2, C and F,respectively). Computer-assisted quantification of stainingintensity confirmed visual assessments (Fig. 2G). To relate pro-teoglycan content to proteoglycan synthesis and degradation,we examined gene expression of aggrecan and matrix pro-teases. Both RA andWnt3a inhibited gene expression of aggre-can (Fig. 2H) and up-regulated expression of Mmp13 andMmp3 (Fig. 2, I and J), and co-treatment with RA and Wnt3aenhanced these effects (Fig. 2, H–J). In good agreement withAlcian blue staining data, retinoid antagonist treatment stimu-
FIGURE 2. RA enhances and retinoid antagonist inhibits Wnt/�-catenin signaling effects on matrix accu-mulation and gene expression of aggrecan; Mmp3, and Mmp13. Epiphyseal chondrocytes were treatedwith EtOH (A and D), 300 nM RA (B and E), or 100 nM ANT (C and F) plus 100 ng/ml rWnt3a (D–F) or vehicle for 48 h.Cultures were stained with Alcian blue (A–F), and staining intensity was measured by image analysis (G). TotalRNA was prepared from those cultures and subjected to semiquantitative real-time PCR analysis of aggrecan(H), Mmp13 (I), or Mmp3 (J) transcript levels. Values represent averages and S.D. obtained from three indepen-dent samples. *, p � 0.01.
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lated aggrecan gene expression, reduced Mmp13 and Mmp3gene expression to basal level, and counteracted the effects ofWnt3a (Fig. 2, H–J).Up-regulation of Wnt Proteins, Receptors, and Co-receptors
by RA—To determine how RA stimulates Wnt/�-catenin sig-naling,we examined the gene expression levels ofWnt proteins,receptors, and modulators. PCR array analysis clearly revealedthat RA treatment significantly enhanced gene expressionof Wnt2b and Wnt5a in cultured chondrocytes and slightlyincreasedWnt4,Wnt5b, andWnt9a, whereas expression levelsof Wnt4, Wnt5b, and Wnt9a were very low (Fig. 3A). Interest-ingly, we also found strong up-regulation of Fzd-8, Lrp-5, andLrp-6 by RA treatment (Fig. 3B). Among the Wnts that wereup-regulated by RA, Wnt2b, Wnt4, and Wnt9a are known tostimulateWnt/�-catenin signaling (3, 8, 41); in addition, Fzd-7
and Fzd-8 transmit both �-catenin-dependent canonical and non-ca-nonical signals, and Lrp5 and Lrp6are essential co-receptors for Wnt/�-catenin signaling. To determinethe significance and implications ofthese changes in gene expression,we treated chondrocyte cultureswith Dickkopf-related protein 1(DKK-1), an extracellular inhibitorof the Wnt/�-catenin signalingpathway that binds co-receptorLRP5/6 and disturbs Wnt signaltransduction through Frizzledreceptors (42). Treatment withDKK-1 completely abolished theincrease inWnt/�-catenin signalinginduced by Wnt3a, as revealed byreporter activity (Fig. 3C, Wnt3a).When the cultures were treatedwith the GSK3� inhibitor BIO,reporter activity was stronglyincreased (Fig. 4A, BIO), reflectingthe well known fact that inhibitionofGSK3� favors�-catenin accumu-lation and signaling. Indeed, DKK-1treatment had no effects on BIO-in-duced stimulation of reporter activ-ity (Fig. 3C, BIO), verifying the factthat DKK-1 blocks Wnt/�-cateninsignaling at the cell surface receptorlevel and not the cytoplasmic�-catenin stabilization level. Theinhibition of RA action by DKK-1was also tested by examination of�-catenin nuclear accumulation(Fig. 3, D–I). RA treatmentincreased the number of chondro-cytes displaying strong �-cateninnuclear staining (Fig. 3E), whereascontrol cultures displayed weakstaining (Fig. 3D). DKK-1 co-treat-ment strongly counteracted the
effects of RA treatment (Fig. 3F). Together, the above data sug-gest that RA stimulates Wnt/�-catenin signaling mainly bymodulating expression of Wnt receptors and co-receptors.Inhibition of Wnt/�-catenin Signaling by RAR�—Previous
studies indicated that nuclear receptor proteins interact with�-catenin and can positively or negatively regulate �-catenintranscriptional activity. RAR� was found to physically interactwith �-catenin and inhibit �-catenin signaling in retinoid ago-nist-treated cells (16). We previously found that RAR� is themajor RAR expressed in growth plate chondrocytes (23, 43).Thus, we asked whether RAR� is the main modulator of Wnt/�-catenin signaling in chondrocytes. Chondrocytes were co-transfectedwith aWnt/�-catenin reporter plasmid and expres-sion vectors encoding RAR�, RAR�, RAR�, or empty vector asa control. The cells were maintained in retinoid-free medium
FIGURE 3. Inhibition of RA-stimulated Wnt/�-catenin signaling by DKK-1. A and B, epiphyseal chondro-cytes were treated with or without 300 nM RA for 24 h, and RNA from each population was subjected to PCRarray analysis. Values represent expression levels of Wnt proteins (A) and receptors/co-receptors (B) relative toglyceraldehyde-3-phosphate dehydrogenase. Wnt genes whose expression level was below 0.01 are not listed.C, chondrocyte cultures were transfected with Super 8x TOPFlash plasmid and then treated with EtOH, 300 nM
RA, 20 ng/ml rWnt3a, or 0.5 �g/ml BIO with (closed column) or without (open column) 2 �g/ml DKK-1. DKK-1 wasadded 30 min before treatment with rWnt3a. Reporter luciferase activity was measured 24 h after Wnt3atreatment. Values represent averages and S.D. obtained from three independent samples. *, p � 0.01.D–I, cultures were treated with EtOH (D and G) or 300 nM RA with (F and I) or without (E and H) 2 �g/ml DKK-1for 24 h and stained with �-catenin antibodies to examine the �-catenin nuclear levels (D–F). G–J are the phasecontrast images corresponding to D–F, respectively.
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conditions in which RARs are largely unliganded. Overexpres-sion of RAR�, RAR�, and RAR� was comparable in each cul-ture (data not shown). Treatment with Wnt3a strongly
increased Wnt/�-catenin reporter activity in control cultures(Fig. 4A), but this response was reduced by nearly 50% by RAR�overexpression (Fig. 4A). Overexpression of RAR� or RAR�was far less effective (Fig. 4A).To test whether endogenous RAR� acts in a similar manner,
we measured Wnt reporter activity during siRNA-mediatedsilencing of RAR�. Wnt/�-catenin reporter activity was dra-matically increased in RAR� siRNA-treated cultures with orwithout Wnt3a co-treatment (Fig. 4B), whereas silencing ofRAR� or RAR� hadminimal effects (Fig. 4B). Effectiveness andspecificity of siRNA on RAR expression were confirmed bysemiquantitative PCR (Fig. 4C).RAR� Associates with �-Catenin and Inhibits �-Catenin
Association with LEF—Next, we examined the mechanism bywhich RAR� inhibits Wnt/�-catenin signaling. Because RAR�was found to associate with�-catenin during RA treatment andto affect �-catenin transcriptional activity (16), we askedwhether RAR� also associates with �-catenin. COS7 cell cul-tures were transfected with HA-tagged RAR� and treated withBIO to increase �-catenin levels; some of the cultures wereco-treated with RA. Cell homogenates were immunoprecipi-tated with anti-HA antibodies (IP:HA in Fig. 5) or control IgGs(IP: IgG) , followed by immunoblot with anti-�-catenin (WB:�-catenin) or anti-HA antibodies (WB: HA). In cultures notreceiving RA, RAR� associated with �-catenin as indicated byco-immunoprecipitation with anti-HA antibodies (Fig. 5A,lane 2), but not control IgGs (Fig. 5A, lane 1). Interestingly, incultures treated with RA, the RAR���-catenin complexes wereundetectable (Fig. 5A, lane 3), suggesting that complex forma-tion occurred only in retinoid-free cultures and involved unli-ganded RAR�. Note that BIO treatment was necessary toobserve RAR���-catenin complex formation (Fig. 5C, lanes 2and 3), indicating that detection of such complexes in untreatedcultures may require more sensitive methods.To examine the domains of RAR� that are important for
association with �-catenin, we tested RAR� mutants lackingthe N-terminal domain (AF-1) or C-terminal domain (AF-2)(Fig. 5B) and carried out similar co-immunoprecipitationexperiments. Deletion of AF-1 completely hampered the abilityof RAR� to associate with �-catenin (Fig. 5C, lane 4), whereasdeletion of AF-2 domain had no effect (Fig. 5C, lane 5).Next, we askedwhether the association of RAR�with�-cate-
nin affects �-catenin interactions with LEF/TCF. We carriedout two-hybrid assays using a constitutively active form of�-catenin linked to Gal4 (Gal4-CA-�-catenin; ca in Fig. 6) andLEF1 linked to VP16 (VP16-LEF; LEF in Fig. 6) (16). Whenchondrocytes were transfected with these two plasmidstogether with a Gal4-sensitive luciferase reporter plasmid,luciferase activity was markedly increased as expected (Fig. 6A,column 4), reflecting interactions of Gal4-CA-�-catenin withGal4 binding sites on the luciferase VP16 reporter and tran-scriptional trans-activation. Companion cells transfected withcontrol VP16 empty vector (con) or a dominant-negative formof LEF fused to VP16 (dnLEF) exhibited background luciferaseactivity (Fig. 6A, columns 1–3 and 5), verifying trans-activationactivity of Gal4-CA-�-catenin. Chondrocytes were then trans-fected with Gal4-CA-�-catenin and VP16-LEF plasmids plusplasmids encoding full-length RAR� (� full in Fig. 6) or ��AF1
FIGURE 4. Effects of RARs on Wnt/�-catenin signaling activity. A, epiphy-seal chondrocytes were transfected with Super 8x TOPFlash and RAR�, RAR�,or RAR� expression plasmids and were then treated with 100 ng/ml rWnt3a24 h after transfection; luciferase activity was measured after an additional24 h. B, chondrocyte cultures were transfected with siRNA for RAR�, -�, or -�or control random siRNA (Con) and Super 8x TOPFlash plasmid and treatedwith 100 ng/ml of rWnt3a 24 h after transfection; luciferase activity was mea-sured after an additional 24 h. C, total RNAs were prepared from culturestransfected with siRNA for RAR�, -�, or -� or control random siRNA and sub-jected to semiquantitative real-time PCR analysis of RAR�, RAR�, and RAR�transcript levels. Values are means � S.D. obtained from three independentsamples. *, p � 0.01; **, p � 0.05.
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or ��AF2 deletion mutants. RAR� overexpression diminishedreporter activity elicited by Gal4-CA-�-catenin and VP16-LEF(Fig. 6A, column 6). In contrast, expression of ��AF1 deletionmutant did not inhibit reporter activity but actually enhanced it(Fig. 6A, column 7), and expression of ��AF2 partiallydecreased reporter activity moderately (Fig. 6A, column 8).These results indicate that RAR� interferes with association of�-catenin with LEF and that both AF1 and AF2 domains arerequired for inhibition. Thus, we asked whether the AF1 andAF2 domains are actually involved in inhibition ofWnt/�-cate-nin signaling. We found that both deletion mutants failed to
inhibitWnt3a-induced reporter activity (Fig. 6B, columns 7 and8), whereas full-length RAR� did so (Fig. 6B, column 6).
DISCUSSION
In this study, we show that the Wnt/�-catenin and retinoidsignaling pathways interact and affect matrix homeostasis andphenotypic expression in chondrocytes. We find that RA stim-ulates Wnt/�-catenin signaling, increases gene expression ofWnt proteins and receptors, and enhances the inhibitoryeffects of Wnt/�-catenin signaling on matrix accumulation(Fig. 7A). In contrast, overexpression of RAR� inhibits Wnt/�-catenin signaling in chondrocyte cultures maintained underretinoid-free conditions, and silencing of endogenous RAR�expression strongly stimulates �-catenin transcriptional activ-ity (Fig. 7B). Furthermore, we present evidence that RAR�
FIGURE 5. Interaction of RAR� with �-catenin. A, COS7 cells were trans-fected with expression vector encoding full-length HA-tagged RAR� (�full) orpCMV-HA empty vector (ev) and were then treated with 0.5 �g/ml BIO, a GSK3inhibitor, in the presence or absence of 300 nM RA. Cell lysates were collectedand subjected to immunoprecipitation with anti-HA antibodies (IP: HA) orcontrol IgGs (IP: IgG). Immunoprecipitated material was analyzed by immu-noblot with anti-HA antibodies (WB: HA), and whole lysates were analyzed byimmunoblot using �-catenin (WB: �-catenin) antibodies. B, schematic of RAR�deletion mutants. RAR� has an AF-1 domain, a DNA binding domain (DBD),and an AF-2 domain (RAR� Full). Mutants lacking AF-1 or AF-2 were con-structed by adding an HA tag (RAR� �AF1 and RAR� �AF2, respectively).C, COS7 cells were transfected with expression vectors encoding full-lengthHA-tagged RAR� (�full), RAR� �AF-1 (��AF1), RAR� �AF-2 (��AF2) orpCMV-HA empty vector (ev) and then treated with 0.5 �g/ml of BIO and 300nM RA (only for RAR�). Immunoprecipitation and immunoblot were carriedout as described above.
FIGURE 6. RAR� inhibits �-catenin association with LEF-1. A, epiphysealchondrocytes were transfected with Gal4-Luc, Gal4 vector, VP16 vector, andeither empty vector (ev) or the indicated RAR� mutant vector. The Gal4 vectoris encoding either control Gal4 (con) or Gal4-contsitutively active �-catenin(ca). The VP16 vector is encoding control VP16 (con), VP 16-LEF1 (LEF), orVP16-dominant negative LEF1 (dnLEF), and RAR� mutant vector is RAR� full(�full), RAR� �AF1 (��1), or RAR� �AF2 (��2). Gal4 luciferase activity wasmeasured 24 h after transfection. B, epiphyseal chondrocytes were trans-fected with TOPFlash plasmid and vectors encoding RAR� full (�), RAR� �AF-1(��1), RAR� �AF-2 (��2), or empty vector (ev) and treated with 100 ng/mlrWnt3a for 24 h after the transfection. Luciferase activity was measured afteran additional 24 h. Values are means and S.D. obtained from three indepen-dent samples. *, p � 0.01.
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interacts with �-catenin in retinoid-free cultures but does notdo so in RA-treated cultures. Treatment with retinoid antago-nist counteracts the ability ofWnt3a to inhibit matrix accumu-lation and aggrecan expression. Taken together, our findingssuggest that RAR� can regulate Wnt/�-catenin signaling inchondrocytes positively or negatively depending on retinoidligand availability. RAR� enhances Wnt/�-catenin signalingin retinoid-free conditions but inhibits it in RA-treated cells(Fig. 7).Regulation in Chondrocyte Differentiation and Maturation—
Studies have indicated that unliganded RAR� represses tran-scriptional activity of target genes and promotes chondrogenicdifferentiation ofmesenchymal cells in the developing limb (44,45). Our findings indicate that RAR� interacts with �-cateninin retinoid-free cultures and inhibits the antichondrogeniceffects of Wnt/�-catenin signaling. We recently reported thatconditional ablation of RAR� andRAR� genes in cartilage leadsto growth retardation (43). In these compound mutants, theprehypertrophic zone of growth plate displayed lower amountsof proteoglycans compared with wild type growth plates. Genesilencing of RAR� also inhibited proteoglycan synthesis inchondrocyte cultures maintained under retinoid-free condi-tions (43). Analysis of RARE reporter mice previously showedthat retinoid signaling is undetectable in upper zones of growthplate but increases in the hypertrophic zone (46). Togetherwithfindings in this study, it is likely that unliganded RAR�decreasesWnt/�-catenin signaling in upper growth plate zonesand sustains strong proteoglycan expression and accumulationtypical of those zones. The molecules participating in thesemechanisms could include direct and indirect targets of Wnt/�-catenin signaling, such as aggrecan,MMPs, twist-1, and othermatrix and regulatory genes (13, 47).
It was suggested recently thatWnt/�-catenin signaling is involvedin pathologies of articular cartilage.Genetic studies showed that muta-tion of FRZB, a Wnt antagonist, islinked to incidence of hip arthritis incertain patient cohorts (48, 49).Increased Wnt/�-catenin signalingactivity has been documented inhuman osteoarthritic cartilage (50,51) and OA animal models (6). Inaddition, acute experimental acti-vation of Wnt/�-catenin signalingstimulates catabolic action and car-tilage matrix degradation (6) andinduces degeneration of articularcartilage (51). Thus, increasedWnt/�-catenin signaling could enhancearticular cartilage degeneration,and inhibition of this pathway couldrepresent a new way to prevent ortreat joint degenerative diseases.Our results suggest that a retinoidantagonist may inhibitWnt/�-cate-nin signaling at the level ofWnt pro-teins and receptors. Indeed, sys-
temic application of a retinoid antagonist was found to decreasethe degree of joint degenerative changes in collagen- or bacte-ria-induced arthritis (52).Stimulation of Wnt/�-Catenin Signaling by Retinoid
Signaling—Retinoid signaling has been found to be a potentinhibitor for Wnt/�-catenin signaling in colon and breast can-cer cells (14–17). Such inhibition is believed to occur throughphysical interaction of �-catenin with RAR� and to occur pref-erentially when retinoid ligands are present in the tissues (15–17). Our results show that retinoid signaling also inhibits Wnt/�-catenin signaling inmesenchymal cell cultures but stimulatesit in chondrocyte cultures, indicating that interrelationsbetween these two signaling pathways are differentia-tion-dependent and change during the transition from undif-ferentiated mesenchymal cells to differentiated chondrocytes.This transition is known to involve changes in RAR expression;mesenchymal cells mostly express RAR� and RAR�, whereaschondrocytesmainly express RAR� (43, 53). Thus, our data andprevious studies (16) suggest that RAR� and RAR� may haveopposite effects on Wnt/�-catenin signaling in prechondro-genic cells and chondrocytes, respectively.Further insights into the unique roles of retinoid and Wnt/
�-catenin signaling pathways in chondrocytes come from ourglobal gene expression analyses.We observe that RA treatmentup-regulates gene expression of several Wnt proteins, recep-tors, and co-receptors. Among the Wnt genes up-regulated byRA, Wnt2b, Wnt4, and Wnt9a all stimulate the �-catenin sig-naling pathway in several cell types (3, 8, 41), whereas Wnt5acan stimulate the non-canonical pathway (3) and inhibits�-catenin signaling by directing �-catenin for degradation bythe ubiquitin proteasome system (8).However,Wnt5a has beenreported to have the ability to activate�-catenin signaling in the
FIGURE 7. Schematic of possible cross-talk and cross-regulation between Wnt/�-catenin and retinoidsignaling pathways. A, liganded RAR� (represented as RA-RAR�) would stimulate gene expression of Wntproteins, receptors, and co-receptors and would enhance Wnt/�-catenin signaling activity through thesechanges in gene expression. B, in conditions in which retinoid ligands are not available to chondrocytes,unliganded RAR� would associate with �-catenin and inhibit �-catenin interactions with LEF/TCF, therebyresulting in overall inhibition of Wnt/�-catenin signaling and decreases in �-catenin target gene expression.
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presence of appropriate Frizzled receptors (54). In our chon-drocyte cultures, Wnt5a showed no effect on Wnt reporteractivity in absence or presence of Wnt3a, but activity signifi-cantly increased in cells co-treated with RA and Wnt3a (datanot shown). Thus, Wnt5a could also participate in stimulationof Wnt/�-catenin signaling by RA. Stimulation of Wnt2band Wnt5a gene expression was observed within 6 h after RAtreatment (data not shown), and these genes have several can-didate sites for RAR/RXR binding in their 3-kb upstream pro-moter regions as determined by in silico analysis, suggestingthat these genes might be direct targets of retinoid signaling. Inaddition toWnt genes, we also observe up-regulation of Fzd-7,Fzd-8, Lrp-5, and Lrp-6. TheseWnt receptors and co-receptorscould also enhance responsiveness to Wnt proteins (3, 55).These changes are probably responsible for enhancement ofWnt3a action by RA treatment.Inhibition of Wnt/�-Catenin Signaling by RAR�—In cells
maintained in retinoid-free conditions, RAR� associates withco-repressors, such as N-CoR and R2/SMRT, and inhibits genetranscription (33, 56, 57). Our findings indicate that in chon-drocytes maintained in similar retinoid-free conditions, RAR�also functions as a repressor of �-catenin�LEF/TCF complexaction. The mechanisms of this inhibition may involve compe-tition of �-catenin recruitment/binding to LEF/TCF proteins.In addition, we observe that treatment with a retinoid antago-nist decreases �-catenin levels, suggesting that unligandedRAR� modulates cytosolic �-catenin degradation. There hasbeen increased attention to novel non-genomic mechanism ofsignal transduction through nuclear hormone receptors (14,58). For example, RAR� interactswith the subunits of phospha-tidylinositol 3-kinase and activates the phosphatidylinositol3-kinase/Akt pathway, depending on ligand availability intumor cells (58). Interestingly, activation of the Akt pathwayhas also been reported to stimulate Wnt/�-catenin pathway inseveral systems (59, 60). Thus, RAR�might affect the Akt path-way or other signaling pathways that are directly or indirectlyinvolved in �-catenin stabilization.The finding that the inhibition of Wnt/�-catenin signaling
by RAR� is reversed by treatment with RA differs with previousreports on RAR� action in tumor cells (14–17). The differencecould be explained by the diversity of RAR isoforms involved.For example, we showed earlier that inhibition of Wnt/�-cate-nin signaling by RAR� is ligand-dependent (16), whereas weshow here that inhibition ofWnt/�-catenin signaling by RAR�is ligand-independent and involves AF1 and AF2 domains thatdiffer significantly from analogous domains in other RARs.Our results suggest that the AF1 domain of RAR� is neces-sary for interaction with �-catenin and inhibition of �-cate-nin transcriptional activity and that the AF2 domain is notinvolved in protein complex formation but is required forinhibition of �-catenin signaling activity. It is also likely thatregulation of Wnt/�-catenin signaling by RA could varyaccording to cell type. For example, RA inhibits �-cateninsignaling in colon and breast cancer cells (15–17), whereasRA stimulates signaling in ES cells (19) and neuronal cells(21) just as we show here in chondrocytes. These differenttypes of cells probably respond differently to RA in terms of
induction or repression of Wnt proteins and receptors andother coactivators and corepressors.In sum, the findings in our study indicate that the Wnt/�-
catenin and retinoid signaling pathways are tightly connectedand cooperatively affect chondrocyte function and phenotype.This cross-regulation could be quite important in the regula-tion of chondrogenesis and proper progression of endochon-dral ossification during skeletal growth. It will be interesting todetermine whether the cross-talks and interactions betweenthese two signaling pathways may be altered in pathologies ofpermanent cartilage, such as joint disease and disc degenera-tion, and whether modulation of these interactions could betargeted for therapy.
Acknowledgments—We thankA. Hecht (Albert-Ludwings University)for kindly providing the VP-16 fused LEF and VP16-dominant nega-tive LEF1 plasmids. We also thank A. Harget and D. Pilchak for tech-nical assistance and T. Nakamura (Tohoku University) for helpfuldiscussions and suggestions.We thankAllergan Pharmaceuticals andVitae Pharmaceuticals for providing the retinoid antagonist.
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