Day and Yingzi Yang
Lilia Topol, Wen Chen, Hai Song, Timothy F.
-Catenin Phosphorylation in the Nucleus βSox9 Inhibits Wnt Signaling by Promoting Developmental Biology:
Molecular Basis of Cell and doi: 10.1074/jbc.M808048200 originally published online December 1, 2008
2009, 284:3323-3333.
J. Biol. Chem.
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Sox9Inhibits Wnt Signaling by Promoting-Catenin Phosphorylation in the Nucleus*□S
Received for publication,October20,2008,and in revid form,December1,2008Published,JBC Papers in Press,December1,2008,DOI10.1074/jbc.M808048200 Lilia Topol,Wen Chen,Hai Song,Timothy F.Day,and Yingzi Yang1
change drum soonFrom the Genetics Dia Rearch Branch,National Human Genome Rearch Institute,National Institutes of Health, Bethesda,Maryland20892
Chondrocyte fate determination and maintenance requires Sox9,an intrinsic transcription factor,but is inhibited by Wnt/-catenin signaling activated by extrinsic Wnt ligands.Here we explored the underlying molecular mechanism by which Sox9 antagonizes the Wnt/-catenin signaling in chondrocyte differ-entiation.We found that Sox9employed two distinct mecha-nisms to inhibit Wnt/-catenin signaling:the Sox9N terminus is necessary and sufficient to promote-catenin degradation, whereas the C terminus is required to inhibit-catenin tran-scriptional activity without affecting its stability.Sox9binds to -catenin and components of the-catenin“destruction com-plex,”glycogen syntha kina3and-transducin repeat con-taining protein,to promote their nuclear localization.Inde-pendent of its DNA binding ability,nuclear localization of Sox9 is both necessary and sufficient to enhance-catenin phospho-rylation and its subquent degradation.Thus,one mechanism whereby Sox9regulates chondrogenesis is to promote efficient -catenin phosphorylati
the viewon in the nucleus.This mechanism may be broadly employed by other intrinsic cell fate determining transcription factors to promptly turn off extrinsic inhibitory Wnt signaling mediated by-catenin.
Differentiation of chondrocytes from menchymal progen-itors is an early critical event in endochondral ossification,a major bone-forming process in vertebrate embryos(1).Chon-drocyte fate determination and maintenance are regulated by both intrinsic and extrinsic factors such as Sox9and Wnt/-catenin signaling,respectively.Sox9is an SRY-box(Sox)con-taining gene required for chondrocyte differentiation(3,4). Heterozygous SOX9mutations cau the human dia cam-pomelic dysplasia(CD),2a form of dwarfism characterized by extreme cartilage and bone malformation and x reversal(5, 6).The Wnt/-catenin pathway,which plays a critical role in regulating many cell proliferation and fate determination pro-cess in embryonic development and oncogenesis(reviewed in Refs.7and8),potently inhibits chondrocyte differentiation and Sox9expression during skeletal development(9,10).Becau Wnt ligand expression is detected around the chondrogenic menchymal condensation,but Wnt signaling activity is dra-matically down-regulated in the differentiating chondrocytes in which Sox9is expresd(11),it is likely that one mechanism by which Sox9promotes chondrocyte differentiation and maintains chondrocyte characters is to inhibit the antichon-drogenic Wnt/-catenin signaling activity.However,the molecular mechanism by which Sox9promotes-catenin deg-radation is still poorly understood.
In the Wnt/-catenin pathway,-catenin protein degrada-tion is regulated by Wnt signaling,which controls-catenin phosphorylation by cain kina I␣(CKI␣)and glycogen syn-tha kina3(GSK3)in the destruction complex asmbled by Axin(7).In the abnce of Wnt signals,-catenin is phospho-rylated in the destruction complex and then degraded in pro-teosomes.Activation of Wnt signaling leads to inhibition of GSK3-mediated-catenin phosphorylation and thus-catenin is stabilized.According to the current model,-catenin degra-dation occurs in the cytoplasm.-Catenin,stabilized by Wnt signaling,then enters the nucleus where it activates down-stream gene expression by binding to LEF/TCF transcription factors.
Sox9is a transcription factor that contains three highly con-rved domains:the high mobility group(HMG)domain that binds and bends DNA in a quence specific manner(12–14), the C-terminal PQS transactivation domain and the PQA domain,both required for maximum transcriptional activity of Sox9(15,16).Sox9has two nuclear localization signals(NLS) and a nuclear export signal(NES)located in the HMG domain (17,18).Sox9protein is localized either exclusively in the differentiated chondrocytes)or in both nucleus and arly differentiating chondrocytes).In addi-tion to cartilage,Sox9is expresd in other tissues such as neu-ral crests(19,20),pancreatic progenitors(21,22),and intestinal epithelial cells(23).It has been reported tha
t in the developing cartilage and intestinal epithelium,Sox9inhibits the Wnt/-catenin signaling activities(24,25).Sox9is also expresd in the gonad where it is required for male x determination(26).Like what has been obrved in Sox9loss of function mutants,acti-vation of-catenin in otherwi normal XY mice effectively
*This work was supported,in whole or in part,by the National Institutes of Health,NHGRI intramural rearch program.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked“advertiment”in accordance with18U.S.C.Section1734solely to indicate this fact.
□S The on-line version of this article(available at www.jbc)contains supplemental Figs.S1–S6.
1To whom correspondence should be addresd:49Convent Dr.,MSC4472, Bethesda,MD20892.Tel.:301-402-2034;Fax:301-402-2170;E-mail: yingzi@v.
2The abbreviations ud are:CD,campomelic dysplasia;CKI␣,cain kina I␣;GSK3,glycogen syntha kina3;LEF,lymphoid enhancer factor;TCF, T-cell factor;HMG,high mobility group;NLS,nuclear localization signal;
NES,nuclear export signal;GFP,green fluorescent protein;HA,hemagglu-tinin;GST,glutathione S-transfera;IP,immunoprecipitate;CHO,Chine hamster ovary;DAPI,4Ј,6-diamidino-2-phenylindole;TrCP,-transducin repeat containing protein.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284,NO.5,pp.3323–3333,January30,2009
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disrupts the male program and results in male-to-female x
reversal(27).
Here we demonstrate that Sox9inhibits Wnt signaling by
two distinct mechanisms,which requires different domains of
Sox9.We showed that the N terminus of Sox9including the
HMG domain promoted-catenin degradation,whereas the C-terminal transactivation domain inhibited the transcrip-
tional activity of-catenin,but it was not required for-cate-nin degradation.In addition,we found that Sox9bound to com-
ponents of the-catenin destruction complex and relocated them into the nucleus.Sox9nuclear localization,not the HMG
domain per ,was both necessary and sufficient to induce
nuclear localization of the-catenin destruction complex com-ponents and enhance-catenin phosphorylation for its sub-quent degradation.Furthermore,our results suggest that more
efficient-catenin phosphorylation and degradation in the nucleus may be a general mechanism whereby transcription
factors effectively antagonizing inhibitory Wnt/-catenin sig-naling in cell fate determination and maintenance. EXPERIMENTAL PROCEDURES
Plasmids,Virus,and Antibodies—Mou Sox9cDNA(pro-
vided by Dr.Yoshi Yamada)was tagged with FLAG and sub-
cloned into modified pIRES-hrGFP-1(pIRES),expression vec-
tor(Stratagene)where GFP was removed.The Sox9mutants
were generated by standard PCR-bad mutagenesis proce-
dures(Stratagene)and subcloned into modified pIRES-
wbl
倒装句讲解hrGFP-1vector in-frame with the FLAG tag.To generate NLS
fusion,the NLS5Ј-PKKKRKVE-3Јfrom the SV40T antigen
get along with(28)were added to the C-terminal end of the Sox9mutant pro-
teins.The coding region of rat ck1␣and human-CATENIN were HA-tagged and subcloned into pHM6(2).The wild type
human-CATENIN was Myc-tagged and inrted into the pcDNA-3A(Invitrogen)expression vector.⌬N-CATENIN, provided by Dr.F.McCormick(University of California,San
Francisco,CA)was subcloned into the pIRES vector.The Sox9,
Wnt14,GFP,and CRE-adenovirus were generated according to
the manufacturer’s instructions(Clontech)and concentrated
viral stocks were produced by SAIC(SAIC,Frederick,MD).-Catenin was fud in-frame to the GST quences of the pGEX-4T1bacterial vector(Amersham Bioscience).Recombi-
nant GST-tagged-catenin was expresd in BL21Escherichia-coli cells and purified using glutathione-Sepharo columns
(Amersham Biosciences).Anti-Sox9(H-90,Santa Cruz),anti-
Axin(Zymed Laboratories Inc.),anti--catenin(C19220,BD Transduction Laboratories),anti-tubulin(ICN),anti-GSK-3(Cell signaling and Abcam),anti-FLAG(M2,Sigma),anti-phos-pho-specific--catenin(Thr41/Ser45and Ser33/37)(Cell Signal-ing),anti-HA(cl3F10,Roche Applied Science),anti-Myc tag (9B11,Cell Signaling),anti-TCF4(6H5–3,Upstate),anti-actin (Sigma),and anti-LRP5(Santa Cruz)antibodies were ud in Western blots,IP,or immunohistochemistry.
Cell Culture,Transfection,Adenoviral Infection,and Lucifer-
a Assay—CHO,HEK293,COS-1,NCI-H28,SW48,SW480,
SNU475,and L-cells were obtained from American Type Cul-
ture Collection.Primary mou chondrocytes were prepared
according to a previously published protocol(29).Cells were
eded the day before transfection and transfected with the indicated plasmids using Lipofectamine PLUS as specified by the manufacturer(Invitrogen).In some cas,transfection was performed using nucleofection technology(Amaxa GmbH). For lucifera assays,cells were transfected with Super-TOP-FLASH or FOPFLASH plasmids,Renilla Lucifera plasmid (phRL-null,Promega),and the indic
ated plasmids.The total amount of transfected DNA was kept constant by adding empty vector DNA.Lucifera activity was measured according to the Dual Lucifera Reporter Assay System(Promega).The results are shown as relative lucifera activity.The histograms are prented as the averageϮS.D.from three independent trans-fections.All measurements were made using Lumat LB9507 Luminometer(EG&G Berthold).Adenoviral infection of cells was performed at1ϫ109plaque-forming units/ml for3–4h, and the results were analyzed24–72h later.
Co-immunoprecipitation and Immunoblotting Assay—In co-immunoprecipitation assays,cells were lyd in lysis buffer(50 m M Tris-HCl(pH7.5),150m M NaCl,1%Triton X-100,and protea inhibitors(Calbiochem),and Halt TM phosphata inhibitor mixture(Pierce)).Equal amounts of cell lysates were incubated with the indicated antibodies for2h and Protein-G Plus(Santa Cruz)was added and incubated for overnight at 4°C.Immunoprecipitates were washed four times in lysis buffer,resolved by NuPAGE,and subjected to standard West-ern immunoblot analysis with the specific antibodies. Protein Binding Assay—In vitro pull-down assays were per-formed as described(30).Briefly,in-catenin binding experi-ments,HEK293or COS cells were transfected with the indi-cated constructs and24h after transfection,cells were lyd with lysis buffer(50m M Tris-HCl(pH7.5),300m M NaCl,0.5% Nonidet P-40and protea inhibitors).Equal amounts of lysates were
incubated for3h at4°C,with20l(about5g)of either purified GST or GST--catenin bound to agaro beads in lysis buffer adjusted to20%glycerol and1m M dithiothreitol. Agaro beads were washed five times with lysis buffer contain-ing500m M NaCl and bound proteins were eluted in LDS sam-ple buffer(Invitrogen)and detected by Western blot. Indirect Immunofluorescence—Cells were plated in four chambers slides(Lab-Tek),transfected with the indicated plas-mids,and fixed with methanol or3.7%formaldehyde,perme-abilized with0.25%Triton X-100and procesd for indirect immunofluorescence.
Treatment with Inhibitors—To block protein degradation, cells were treated with MG132(Sigma)for8h before lysis.LiCl (Sigma)and6-bromoindirubin-3Ј-oxime(BIO,Calbiochem) were ud to inhibit GSK3at40m M and5M,respectively. Micromass Cultures—Micromass cultures were prepared according to a procedure described previously(9).Briefly,the Sox9-or Wnt14-adenovirus were added to the limb men-chymal cell suspension(ϳ2.5ϫ106cells/ml)at5ϫ109plaque-forming units/ml,and the cell suspension was gently rocked at 4°C for2h.Ascorbic acid was added to induce chondrocyte differentiation48h after plating the cells.Cells were fixed and stained with Alcian blue48h after ascorbic acid treatment. Immunoprecipitation and CKI␣and GSK3Immune Com-plex Kina Assay—Transfected cells were fractionated as described(31).To asss Axin-associated activity of GSK3or CKI␣,protein complex containing Axin-Myc was immunopre-
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cipitated using an anti-Myc antibody,and the kina activity of the complex was assayed(32,33).CKI␣kina reaction was carried out for10min at37°C in a40-l volume of kina buffer
(50m M HEPES(pH7.5),10m M MgCl
china style
2,20m M-glycerophos-
phate,5m M NaF)containing the immunocomplex on agaro beads,supplemented with100g/ml myelin basic protein (Sigma)and10Ci of[␥-32P]ATP(Amersham Biosciences). Supernatant was parated by NuPAGE and the gel was vacu-um-dried and expod to a film.The activity of GSK3was measured as described(34)with some modification.Briefly, immunocomplex on agaro beads was incubated in40l of
kina buffer(20m M Tris(pH7.5),5m M MgCl
2,1m M dithio-
threitol,250M ATP,10Ci of[␥-32P]ATP),with phosphogly-cogen syntha peptide-2(62.5m M)(Upstate Biothechnology) for30min at30°C.
Rever Transcription-PCR—Total RNA was extracted from mou primary chondrocytes using the TRIzol reagent (Invitrogen)and treated with RNa-free DNa(Roche). cDNA was synthesized with oligo(dT)primers.Semi-quantita-tive analysis was performed using2100Bioanalyzer(Agilent). The primers ud in this experiment were:mou Sox9forward, 5Ј-CCA CGG AAC AGA CTC ACA TCT CTC-3Јand mou Sox9rever,5Ј-CTG CTC AGT TCA CCG ATG TCC ACG-3Ј;mou glyceraldehyde-3-phosphate dehydrogena forward, 5Ј-ACC ACA GTC CAT GCC ATC AC-3Јand
mou glycer-aldehyde-3-phosphate dehydrogena rever,5Ј-TCC ACC ACC CTG TTG CTG TA-3Ј.
Confocal Image Acquiring—Confocal images were acquired at room temperature using either a Zeiss LSM510NLO Meta system mounted on a Zeiss Axiovert200M microscope or on a Zeiss LSM510UV system mounted on a Zeiss Axiovert100M microscope(Carl Zeiss Inc.,Thornwood,NY)both using an oil immersion Plan-Apochromatϫ63/1.4DIC objective lens. Excitation wavelengths of488,561(3),or543nm(UV),633, and740nm(3),or364nm(UV)were ud for detection of condary antibodies conjugated by Alexa Fluor488,Rhoda-mine,Alexa Fluor633(Invitrogen),and DAPI(Vector),respec-tively.Fluorescent emissions were collected in band-pass filters 500–550,575–615,and641–705nm collected in the Meta detector and390–465nm,respectively(UV system filters were comparable).All pinholes were t at1.00Airy units,which corresponds to an optical slice of0.8m(excluding the DAPI channel where a multiphoton lar was ud).All confocal images were collected using the Zeiss AIM software version4.0 sp2(UV system version3.2sp2)of frame size512pixels by512 pixels,scan zoom of1and line averaged4times.mmds
RESULTS
出差工作总结报告Sox9Antagonizes Wnt/-Catenin Signaling during Chondro-cyte Differentiation by Promoting-Catenin Degradation—We have shown previously that inactivation of Wnt/-catenin sig-naling promotes chondrocyte differentiation from menchy-mal progenitors(9,11).Becau Sox9promotes chondrocyte differentiation when ectopically expresd(35),we tested the hypothesis that one mechanism by which Sox9promotes chon-drogenesis is to antagonize Wnt/-catenin signaling.We per-formed micromass cultures with the limb menchymal cells from12.5days post coitum mou embryos.In the micromass cultures,chondrocytes differentiate from menchymal pro-genitors to form cartilage nodules,which are stained by Alcian blue.By infecting menchymal progenitors in the micromass cultures with Sox9-or Wnt14-adenovirus,we found that Sox9 expression incread cartilage nodule formation at the outer rim of micromass culture,whereas Wnt14,which signals through-catenin(9),verely inhibited cartilage nodule for-mation(Ref.9and Fig.1A).Sox9-and Wnt14-adenovirus co-infection rescued cartilage nodule formation inhibited by Wnt14to a large extent(Fig.1A).The weak prochondrogenic effect of Sox9alone is consistent with a previous report,which showed that Sox9needs other cofactor(s)to effectively enhance its transcription activity(35).When-catenin protein levels in the micromass cultures were analyzed by Western blot,we found that enhanced cartilage nodule formation by Sox9over-expression correlated with reduced-catenin protein levels (Fig.1B).In addition,Wnt3a treatment of L cells,which are fibroblast
cells,resulted in elevated-catenin protein levels, and such elevation was dampened by Sox9-adenovirus infec-tion prior to Wnt3a treatment(Fig.1C).In CHO cells Sox9 expression also led to reduced-catenin accumulation pro-moted by Wnt3a treatment(Fig.1D).The data indicate that Sox9inhibits Wnt/-catenin signaling,at least in part,by reducing-catenin protein levels.The results also predict that-catenin protein levels should be incread after Sox9 removal.To test this possibility primary chondrocytes from homozygous Sox9conditional(Sox9c/c)(3)newborn mice were isolated and efficient Sox9removal by Cre-adenovirus infection was confirmed by rever transcripta-polymera chain reac-tion(Fig.1E,panel a).-Catenin protein levels were indeed incread in the Cre-induced Sox9-deficient primary chon-drocytes(Fig.1E,panel b).In addition,Wnt/-catenin signal-ing activity measured by the TOPFLASH reporter(36)was also up-regulated in the Sox9-deficient primary chondrocytes(Fig. 1E,panel c).The data suggest that promoting-catenin deg-radation is one of the mechanisms whereby Sox9inhibits Wnt/-catenin activity in primary mou chondrocytes.
Due to the difficulty of maintaining and efficiently transfect-ing the primary limb menchymal cells or primary chondro-cytes,cell lines such as CHO,COS,and HEK293that can be easily transfected,were chon to investigate the molecular mechanisms by which Sox9regulates the protein levels and activ
ities of-catenin.In all the cell lines,Sox9reduced -catenin protein levels like in the primary limb menchymal cells,primary chondrocytes,or L cells(Fig.1F and data not shown).To disct the molecular mechanism by which Sox9 promotes-catenin degradation,a ries of Sox9deletion mutants were constructed(supplemental Fig.S1).Surprisingly, both the C-terminal deleted mutant(Sox9⌬C)and full-length Sox9reduced-catenin protein levels and inhibited its tran-scriptional activity in the TOPFLASH assay(Fig.1,F and G). The Sox9⌬C induced even more pronounced-catenin protein reduction than the full-length one.The results were different from previously published results showing that the C-terminal part of Sox9was required for the degradation of a mutant -catenin that could not be phosphorylated by GSK3(24).Fur-thermore,treatment with MG132,a proteosome inhibitor,sus-tained-catenin levels in the prence of Sox9or Sox9⌬C(Fig.
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1H ),indicating that Sox9acts through its N terminus,not its C terminus,to promote -catenin degradation by proteosomes.However,our results supported that the Sox9C terminus was required to inhibit -catenin transcription activity as reported (24).Compared with Sox9,Sox9⌬C was more potent in induc-ing -catenin protein degradation (Fig.1F ),but less active in inhibiting -catenin-mediated transcription (Fig.1G ).Thus,Sox9inhibits Wnt/-catenin signaling through two parallel and independent mechanisms:it us the N terminus to pro-mote -catenin protein degradation and the C terminus to inhibit tran-scription activity of -catenin/TCF complex.
Sox9Promotes -Catenin Degra-dation by Enhancing Its Phos-phorylation —Becau -catenin phosphorylation by CKI ␣and GSK3and its subquent association with TrCP leads to -catenin degrada-tion (38–46),we tested whether -catenin degradation promoted by Sox9requires GSK3and TrCP.First,we found that Sox9expression led to incread -catenin phospho-rylation by CKI ␣(at Ser 45)and GSK3(at Ser 33/37)(Fig.2A ).The rel-ative amount of -catenin phospho-ryla
ted by GSK3and CKI ␣versus the total -catenin protein levels was significantly incread in Sox9expressing cells compared with that in the control cells (Fig.2A ).In addi-tion,when -catenin protein degra-dation was blocked by MG132treatment,more -catenin phosphorylation by GSK3was also obrved in the accumulated -catenin protein in Sox9express-ing cells (Fig.2B ).We then inhibited GSK3activity by LiCl or BIO (47–49)and found that the inhibitors significantly compromid the abil-ity of Sox9and Sox9⌬C to promote -catenin degradation (Fig.2C ).
Sox9and Sox9⌬C still exhibited residual activities to promote -catenin degradation in the pres-ence of LiCl or BIO,possibly due to incomplete inhibition of GSK3.The results indicate that Sox9-promoted -catenin degradation requires GSK3activity.Second,a dominant negative mutant of TrCP (⌬TrCP),which interacts with phosphorylated -catenin but is unable to form an SCF TrCP
-ubiq-uitin liga complex (50),reduced
Sox9-promoted -catenin degrada-tion (Fig.2D ,lanes 4and 6).Third,Sox9was unable to promote degradation of a deleted form of -catenin (⌬N -catenin)(51)that could not be phosphorylated by CKI ␣or GSK3(Fig.2E ).This was further supported by our obrvation that Sox9was not able to promote the degradation of a stabilized mutant -catenin that contains a Ser 33to Tyr misn mut
ation in the SW48cells (supplemental Fig.S2).In addition,expression of Sox9in a colon cancer cell line,SW480,which contains a trun-cated APC protein lacking the Axin binding domain (52),
failed
FIGURE 1.Sox9promotes chondrocytes differentiation by antagonizing the Wnt/-catenin signaling.
A ,micromass cultures using mou limb menchymal cells were infected with the indicated adenovirus and stained with Alcian blue to show cartilage nodules.The boxed areas were enlarged and shown in the ints.
B ,the levels of total -catenin protein in micromass cultures were analyzed by Western blot.Total protein level was normalized using Tubulin.
C ,L cells were infected with Sox9-or GFP-adenovirus and then treated with
Wnt3a protein (100ng/ml)for 1h before harvesting.-Catenin protein levels were analyzed by Western blot.D ,CHO cells were infected with Sox9-adenovirus and then treated with Wnt3a (100ng/ml)for 1h.Total -catenin protein levels were analyzed as in C .Actin was ud as a loading control.E ,mou primary chondro-cytes isolated from the Sox9c/c mice were infected with Cre-adenovirus to remove Sox9.Reduced Sox9expres-sion was confirmed by mi-quantitative rever transcripta-PCR in A .Glyceraldehyde-3-phosphate dehy-drogena (GADPH )was ud to normal
ize the total RNA ud in the rever transcripta-PCR.-Catenin protein levels were analyzed by Western blot (B ).Comparison of TOPFLASH activities with and without Cre -adenovirus infection is shown in C .F ,CHO cells were transiently transfected with Sox9-Flag and Sox9⌬C-Flag and 24h later,cells were treated with Wnt3a (100ng/ml)for 1h,lyd,and analyzed by Western blot.G ,CHO cells were transiently transfected with the indicated plasmid DNA.Lucifera activity was measured 24h after
transfection.Standard deviations (S.D.)of three independent experiments are shown.H ,CHO cells were tran-siently transfected with the indicated plasmid DNA.24h later,cells were treated with MG132(25M /8h)and analyzed by Western blot.Actin was ud as a loading control.Sox9Promotes -Catenin Phosphorylation in the Nucleus
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