Kirkland, Lan Jiang and Shalender BhasinWen Guo, John Flanagan, Ravi Jasuja, James

-Catenin Signaling PathwaysWnt/Cross-communication between Smad3 andAre Mediated through Marrow-derived Mesenchymal Stem CellsDifferentiation of Human Bone The Effects of Myostatin on AdipogenicMechanisms of Signal Transduction:

doi: 10.1074/jbc.M708968200 originally published online January 18, 20082008, 283:9136-9145.J. Biol. Chem.

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http://www.jbc.org/content/283/14/9136.full.html#ref-list-1This article cites 56 references, 26 of which can be accessed free at

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The Effects of Myostatin on Adipogenic Differentiation ofHuman Bone Marrow-derived Mesenchymal Stem Cells AreMediated through Cross-communication between Smad3andWnt/-Catenin Signaling Pathways*SReceived for publication,October 31, 2007, and in revised form, January 18, 2008 Published, JBC Papers in Press, January 18, 2008, DOI 10.1074/jbc.M708968200

Wen Guo1, John Flanagan, Ravi Jasuja, James Kirkland, Lan Jiang, and Shalender Bhasin

From the Sections of Endocrinology, Diabetes, and Nutrition and Geriatrics, Boston University School of Medicine,BostonMedical Center, Boston, Massachusetts 02118 and the Karolinska Institute, Stockholm SE-171 77, Sweden

The effects of myostatin on adipogenic differentiation arepoorly understood, and the underlying mechanisms areunknown. We determined the effects of human recombinantmyostatin protein on adipogenesis of bone marrow-derivedhuman mesenchymal stem cells (hMSCs) and adipose tissue-derived preadipocytes. For both progenitor cell types, differen-tiation in the presence of myostatin caused a dose-dependentreduction of lipid accumulation and diminished incorporationof exogenous fatty acid into cellular lipids. Myostatin signifi-cantly down-regulated the expression of adipocyte markersPPAR, C/EBP, leptin, and aP2, but not C/EBP. Overexpres-sion of PPAR, but not C/EBP, blocked the inhibitory effectsof myostatin on adipogenesis. Myostatin induced phosphoryla-tion of Smad3 in hMSCs; knockdown of Smad3 by RNAi or inhi-bition of its upstream kinase by an Alk5 inhibitor blocked theinhibitory effect ofmyostatin on adipogenesis in hMSCs, imply-ing an important role of Smad3 activation in this event. Further-more, myostatin enhanced nuclear translocation of-catenin andformation of the Smad3--catenin-TCF4 complex, together withthe altered expression of a number of Wnt/-catenin pathwaygenes in hMSCs. The inhibitory effects of myostatin on adipogen-esis were blocked by RNAi silencing of -catenin and diminishedby overexpression of dominant-negative TCF4. The conclusion isthatmyostatin inhibited adipogenesis in human bonemarrow-de-rived mesenchymal stem cells and preadipocytes. These effectsweremediated, in part, by activation of Smad3 and cross-commu-nication of the TGF/Smad signal to Wnt/-catenin/TCF4 path-way, leading to down-regulation of PPAR.

Whereas the role of myostatin in the regulation of skeletalmuscle mass in animals has been widely recognized (18), itseffects on adipogenesis are poorly understood. Inactivatingmutations of the myostatin gene in a number of mammalianspecies are associated with hypermuscularity and decreased fat

mass (913). Similarly, myostatin knockout mice are charac-terized by a lower fat mass than wild-type controls (14, 15).These in vivo observations have led to speculation that myosta-tin promotes adipogenesis. However, the data on the effects ofmyostatin on fat mass and metabolism are conflicting. Trans-genic mice that hyperexpress myostatin protein either system-ically or in the skeletal muscle have increased fat mass (5),whereas adipose-specific hyperexpression ofmyostatin leads toreduced fat mass and improved insulin sensitivity (16). Micebearing tumor cells that hyperexpress myostatin experienceloss of lean as well as fat mass (17); it is unclear whether the lossof fat mass is a consequence of myostatin hyperexpression orthe tumor-associated cachexia.In vitro studies using various cell lines also have yielded

inconsistent results. Some studies have reported thatmyostatininhibits adipogenic differentiation of adipocyte precursor celllines ofmurine, bovine, and human origins (16, 18, 19), whereasothers have reported promotion of adipogenic differentiationby recombinant myostatin in a mouse embryo stem cell line(20). Differences in cell lines and culture conditions could havecontributed to these discrepancies.The mechanism by which myostatin affects adipogenic dif-

ferentiation also remain poorly understood. There is evidencethat myostatin activates the TGF2/activin type II receptor,which subsequently activates type I receptor Alk5, leading tophosphorylation of Smad3 (19). It is not known whether thispathway is activated or how it affects adipogenesis in humanprogenitor cells exposed tomyostatin. The downstream signal-ing mechanisms by which the TGF/Smad3 pathway regulatesadipogenesis are also unknown.The Wnt/-catenin signaling pathway plays an important

role in regulating growth and differentiation of mesenchymalstem cells (21, 22). Under normal culture conditions, the Wntsignaling pathway and adipogenic pathway are reciprocally reg-ulated (2326). Factors that activate the Wnt/-catenin path-way, including canonicalWnt ligands, glycogen synthase kinase3 inhibitors, and tumor necrosis factor, are known to inhibit

* This work was supported by National Institutes of Health GrantsR01DK59261 (to W. G.) and R01DK70431 and R01DK49296 (to S. B.). Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertise-ment in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

S The on-line version of this article (available at http://www.jbc.org) con-tains supplemental Fig. S1.

1 Towhom correspondence should be addressed: 670 Albany St., Boston, MA02118. E-mail: wguo@bu.edu.

2 The abbreviations used are: TGF, transforming growth factor ; hMSC,human bonemarrow-derivedmesenchymal stem cells; C/EBP, CCAAT/en-hancer-binding protein; IP, immunoprecipitation; Alk5, activin receptor-like kinase 5; BrdU, bromodeoxyuridine; LAP, liver-activated protein; DM,differentiation medium; PPAR, peroxisome proliferator-activated recep-tor ; eGFP, enhanced green fluorescent protein; RNAi, RNA interference;qPCR, quantitative PCR; dn, dominant-negative.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 14, pp. 91369145, April 4, 2008 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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adipogenesis in vitro (2628). Similar inverse correlationbetween expression of Wnt/-catenin target genes and adipo-genic genes has been found in human fat tissue biopsies (29).Furthermore, adipose tissue-specific hyperexpression ofWnt10b diminishes fat tissue accumulation in obese mice (30).Cross-talk between Smad and Wnt signaling has been

reported in some cell types (3137). Little is known about theinteraction between myostatin/Smad and Wnt signaling path-ways during adipogenic differentiation. Here, we present anintegrated study of the effect of myostatin on adipogenesis, uti-lizing both bone marrow-derived mesenchymal stem cells andfat tissue-derived preadipocytes of human origin. After con-firming a similar inhibition in both progenitor cell types, weused hMSCs to test the hypothesis that the signal generated bymyostatin through theTGF/Smad3 pathway is cross-commu-nicated to the Wnt signaling pathway through -catenin andTCF4, which mediate the effects of myostatin on adipogenicdifferentiation of human adipocyte precursors.

EXPERIMENTAL PROCEDURES

Cell Culture and SupplieshMSC from non-obese youngmale donors (BMI 25 kg/m2, age 2040) were purchasedfrom Lonza (Allendale, NJ). Human preadipocytes fromabdominal subcutaneous, omental, and mesenteric fat depotswere obtained from the Adipocyte Core of Boston Obesity andNutrition Research Center and differentiated as previouslydescribed (38). The effects of myostatin were independentlytested in hMSCs from four different subjects and preadipcotyesfrom three different subjects.Recombinant 110-amino acid human myostatin mature pep-

tide was provided by Amgen (Thousand Oaks, CA), and a mousemonoclonal anti-myostatin (JA16, Ref. 39) byWyeth (Cambridge,MA), respectively. The inhibitor for TGF type I receptor activin-like kinase 5 (Alk5i, cat. 616452) was fromCalbiochem.Adipogenesis was induced by differentiation medium (DM)

that contains IBMX (0.5mM), dexamethasone (1M), and insu-lin (170 nM), as well as proprietary components provided by thevendor. Unless otherwise noted, cells were incubated in DMuntil used for experiments. Differentiation was evaluated bymeasuring the lipid content byOil-red-O staining, lipid synthe-sis rate, and expression of adipocyte differentiation markers.Oil-red-O stained lipids were solubilized in isopropyl alcoholand the relative ratio of lipid contents were quantified by com-paring the corresponding absorbance values at 500 nm. Oil-red-O staining of undifferentiated hMSC grown in parallel cul-ture served as the blank sample for this assay. The results werenormalized to the DM control.The lipid synthesis rate was measured by the incorporation

of a fluorescent Bodipy-fatty acid into triglycerides. hMSCswere differentiated in DM or DM containing different doses ofmyostatin for 21 days.Myostatin was then removed by washingthe cells with phosphate-buffered saline and incubated withfresh DM overnight. Cells were then incubated with 10 MBodipy-fatty acid (Invitrogen, D3382) pre-complexed with 3M bovine serum albumin (Sigma, A2801) in a CO2 incubatorat 37 C. After 4 h of incubation, cells were washed six timeswithwarmphosphate-buffered saline containing 0.1% albumin,solubilized in Me2SO, and analyzed using a Safire fluorescence

plate reader (Durham,NC)with excitation at 485 nm and emis-sion at 550 nm. The blank value for this assay was obtained bytreating the cells with the same protocol except that Bodipy-fatty acid was washed off immediately after contact (10 s).Preliminary studies showed that incorporation of Bodipy-fattyacids into complex lipids is minimal within the first 60 s (datanot shown). The results were normalized to the DM control.Selected samples were subjected to lipid extraction withorganic solvent (CHCl3/CH3OH, v/v, 1:1) and analyzed byTLC.Under our experimental conditions, more than 95% of the flu-orescent components isolated from the cells migrated with asimilar Rf value as natural triglycerides, implying nearly com-plete incorporation into cellular triglycerides.RNAi and Viral VectorsPre-tested duo-pack RNAi oligo-

nucleotides for Smad3, -catenin, and a nonspecific controloligonucleotide were purchased from Invitrogen (Carlsbad,CA) and transfected into hMSC using the Lipofectamine pro-tocol (Invitrogen). Type 5 adenovirus encoding wild-typeTCF4, dominant-negative TCF4, PPAR, eGFP, and eGFP-tagged -catenin (all human genes) were purchased from VectorBioLab (Philadelphia, PA) and transfected into hMSCs as previ-ously described (39). Retrovirus vector encoding a constitutivelyactive C/EBP construct (C/EBP-LAP) and control vector wereprovided by Dr. S. R. Farmer (Boston University School of Medi-cine) and transfected into cells using Lipofectamine.RNA Isolation, Reverse Transcription, and Real-time PCRTo-

tal RNAwas isolated using the RNAeasy isolation kit fromQiagen(Valencia, CA). First-strand cDNAwas synthesizedusing a Super-script cDNA synthesis kit from Invitrogen. Gene probe/primersets for quantitative qPCR of Smad3, -catenin, PPAR, C/EBP,C/EBP, aP2, and leptin were purchased from ABI. A 96-wellqPCR-based macroarray for theWnt signaling pathway and rele-vant Syber Green-based qPCR primers were purchased fromSuperarray (Frederick,MD). The PCR array (APHS-043) was pre-coated with 84 Wnt target genes and 5 housekeeping genes. Allother PCR supplies were purchased fromABI.All real-time qPCR measurements were performed on an

ABI7500 PCR system (ABI) using the standard temperaturecycling protocol for the relative quantification assay. Eachmeasurement was run in duplicate with three independentsamples. Selected samples were run after sequential dilution toconfirm that the detected signals werewithin the linear amplifica-tion range. Results were first normalized to the expression level ofan endogenous housekeeping gene hypoxanthine-guanine phos-phoribosyltransferase (HPRT). Selected samples were testedagainst two additional housekeeping genes, 18S and GAPDH,glyceraldehyde-3-phosphate dehydrogenase, and the results wereno different from the results obtained using HPRT. The finalresults were then normalized to DM control except for thoseshown in Fig. 3, which were normalized to the basal control.Western AnalysisPrimary antibodies for phospho-Smad3,

Smad3, aP2, -tubulin, C/EBP, phospho-C/EBP, PPAR,C/EBP, TCF4, -catenin, and histone-1 were obtainedfrom Cell Signaling (Danvers, MA), Santa Cruz Biotechnol-ogy (Santa Cruz, CA), and Invitrogen. Secondary antibodieswere purchased from Santa Cruz Biotechnology. The cell lysatewas prepared in radioimmune precipitation assay buffer con-taining a protease inhibitormixture and a phosphatasemixture

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(both from Sigma). The protein content was measured by theBradford method, and equal amounts of protein (50 g for thetranscription factors, 10 g for aP2) were loaded for electro-phoresis on a 420% gradient gel. The standardWestern anal-ysis protocol was used thereafter. The protein expression wasdetected by chemiluminescence, quantified by densitometry,and normalized to -tubulin, which is not significantly affectedby differentiation or by myostatin treatment.Nuclear Protein Isolation and ImmunoprecipitationNu-

clear proteins were isolated using a commercial kit (ActiveMotiff, Carlsbad, CA). Protein A/G-Sepharose beads werewashed with the lysis buffer before use. Equal amounts ofnuclear proteins (500 g/reaction) were mixed with 10% (v/v)protein A/G beads preloaded with rabbit anti-Smad3 or mouseanti--catenin and rotated at 4 C overnight. After washing threetimes with phosphate-buffered saline, the proteins bound to thebeads were eluted by boiling in SDS-based sample-loading buffer,separated by SDS-PAGE, transferred to the polyvinylidene difluo-ride membrane, and probed with mouse anti--catenin (IP:Smad3) or rabbit anti-Smad3 (IP: -catenin). Both membraneswere then re-probed using mouse anti-TCF4. Anti-rabbit andanti-mouseTrueBlot fromeBioscience (SanDiego,CA)wereusedas the second antibody for chemiluminescence detection. Equalprotein input for each immunoprecipitation assay was verified byWestern analysis of histone-1 in the input protein mix.

Cell Cycle and DNA SynthesishMSCs were incubated withDM or DM containing myostatin (0.1 g/ml) and harvestedevery 2 h for the first 12 h, every 8 h from 12 to 72 h, and thenonce a week for 3 weeks. Cells were fixed in ethanol, stainedwith propidium iodide, and analyzed by fluorescence-activatedcell sorting, as previously described (40). For the analysis ofDNA synthesis, hMSCs were treated with basal medium, DM,or DM containing myostatin (0.1 g/ml) for different days.BrdU (2 mM) was added 14 h before the termination of incuba-tion. Cells were then fixed, and the incorporation of BrdU wasmeasured using an ELISA kit from Roche Applied Science(Indianapolis, IN). The results were normalized to the value inthe DM control harvested at the same time point.StatisticsAll photomicrographs are representative of at least

three independent experiments. The results are shown as themeanS.E. (n3).Multiple groupcomparisonswereperformedusing theDuncans test, and comparisons between two independ-ent groups were analyzed using the Students t test. The statisticalsignificance was inferred from p values0.05.

RESULTS

Myostatin Inhibits the Differentiation of hMSCs into MatureAdipocytesAdipogenic differentiation in hMSCswas inducedby a hormonemixture (DM) containing graded doses of recom-binant human 110-amino acid mature myostatin protein,

FIGURE 1. Myostatin inhibits adipogenic differentiation of bone marrow-derived hMSC. hMSCs were incubated for 21 days in adipogenic DM with orwithoutmyostatin (myst) and anti-myostatin antibody (JA16). A and B, photomicrograph of Oil-red-O stained hMSCs incubated in DM alone (A, bar 100m)orDMcontaining1.0g/mlmyostatin (B).C, hMSCdistributionamongdifferentdifferentiation stages inDM(openbar) or inDMcontainingmyostatin (1g/ml,dark bar, *, p 0.05, n 3). Stage I: elongated fibroblast-like cells withoutmicroscopically detectable lipid droplets; stage II: flattened cells without detectablelipid droplets; stage III:multiple small lipid droplets ( 12per cell) that are only visible under highmagnification (250); stage IV: fewer but larger lipid droplets(612 per cell) that are detected under lowermagnification (100); stage V: fewer but larger coalescent lipid droplets (36 per cell) that are readily detectableat lowmagnification (40); stage VI: 13 very large coalescent lipid droplet(s) that occupy the majority space within a cell. D, lipid synthesis rate assessed bymeasuring the incorporation of Bodipy-fatty acid into cellular lipids of hMSCs incubated in DM or DM containing myostatin (abc, p 0.05, n 8).E, expression of aP2 and leptinmRNAs in hMSCs (abc, p 0.05, n 3); open bar: DM control; dark gray bar: DM containingmyostatin (1.0g/ml), light graybar: DM containing myostatin (1.0 g/ml) and anti-myostatin antibody JA16 (10 g/ml). F, lipid content of hMSCs measured by Oil-red-O staining andquantification at 500 nm, withmyostatin (0.1g/ml) added at each indicated time point (abc, p 0.05, n 3). G, lipid content in hMSCs treated with DM.Myostatin (0.1g/ml) was added to hMSCs together with DM at time 0 and withdrawn at the indicated time points, and the incubation was continued in DMfor a total of 21 days. Lipid content was measured by Oil-Red-O staining and quantification at 500 nm (abc, p 0.05, n 3).

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henceforth referred to simply as myostatin. Myostatin dimin-ished total lipid accumulation comparedwith cells incubated inthe DM control (Fig. 1, A and B, dark clusters represent the

stained lipids on a grayscale). Cultures treated with myostatinalso had a higher fraction of smaller adipocytes and a lowerfraction of larger adipocytes (Fig. 1C), lower rate of lipid syn-thesis, assessed by the incorporation of an exogenous fluores-cent fatty acid into cellular lipids (Fig. 1D), and reducedmRNAexpression of adipogenic markers, aP2 and leptin (Fig. 1E). Co-incubation with a myostatin-neutralizing antibody (41) par-tially reversed the inhibition of adipocyte gene expression (Fig.1E) and lipid-filling (not shown), implying that the inhibitoryeffects were myostatin-specific.Suppression of adipogenesis was maximally achieved when

myostatin was added early during differentiation. hMSCs incu-bated inDMcontainingmyostatin from day 0 to day 21 showeda 62 8% reduction of cellular lipid content, whereas cellsexposed tomyostatin starting day 3 or laterwere less responsive(Fig. 1F). In separate experiments, myostatin was added tohMSC at the initiation of differentiation, removed at differenttime points thereafter, and replaced with myostatin-free DM.The presence of myostatin in the first 4896 h was sufficient tocause sustained suppression of lipid accumulation throughoutthe 21-day differentiation program, whereas exposure to myo-statin for only the first 024 h did not affect subsequent lipidaccumulation (Fig. 1G). Thus, early exposure to myostatin isboth required and sufficient to inhibit adipogenesis in hMSCs.Myostatin Inhibits Differentiation and Lipid Accumulation in

Human PreadipocytesHuman subcutaneous preadipocyteswere differentiated with and without myostatin. The latter wasassociated with decreased lipid filling (Fig. 2, AC) and reducedaP2 and leptin mRNA expression (Fig. 2D). Similar inhibitory

effects were also observed in humanpreadipocytes derived from omentalandmesenteric fat depots (not shown).Myostatin Did Not Affect Early

Induction of C/EBPThe rapidand transient induction of tran-scription factors C/EBP and - isone of the earliest events in adipo-genesis. These transcription factorsbind to specific sequences in thepromoter of C/EBP and PPAR toinduce their expression, which thenactivates the full differentiation pro-gram required for adipocyte matu-ration (42, 43). Incubation ofhMSCs in DM up-regulated mRNAexpression of C/EBP (Fig. 3A) andC/EBP (not shown). This effectwas attenuated by myostatin in thefirst hour but not beyond this timepoint (Fig. 3A). Western analysisrevealed a similar early and tran-sient induction of C/EBP proteinand phospho-C/EBP (Thr-235)within a 314-h incubation withDM, and the effect was also insensi-tive to myostatin (not shown).After 24 h of incubation in DM,

expression of PPAR and C/EBP

FIGURE 2.Myostatin inhibits adipogenic differentiation of human prea-dipocytes. A and B, phase-contrast photomicrographs of human preadipo-cytes incubated in differentiatingmedium (DM) alone (A) or inDMcontaining1.0g/mlmyostatin (B, bar 100m) for 21 days; C, lipid content wasmeas-ured by Oil-red-O staining on day 21; D, mRNA expression levels of aP2 andleptin in cells treated for 21 dayswithDMcontrol (open bar) or DMcontainingmyostatin (1.0 g/ml, dark bar).

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in hMSCswas induced 56-fold compared with the cells main-tained in basal medium. This induction, however, was bluntedbymyostatin (Fig. 3A, lower panel). Together, these results sug-gested C/EBP was not, whereas C/EBP and PPAR weredown-regulated by myostatin, in association with suppressionof adipogenesis.We then determined whether ectopic expression of C/EBP

reverses the inhibitory effects of myostatin on adipogenic dif-ferentiation. We transfected the cells with a retroviral vectorexpressing a constitutively active C/EBP construct (C/EBP-LAP). After selection with puromycin, the cells were incubatedin DM with or without myostatin. As shown in Fig. 3B, even inthe presence of ectopic C/EBP-LAP, myostatin still signifi-cantly down-regulated the expression of PPAR and aP2 pro-teins. In contrast, when hMSCs were infected with adenovirusencoding PPAR, the inhibitory effects of myostatin on theexpression of adipogenic markers, C/EBP and aP2, were nolonger detected (Fig. 3C).Smad3 Mediates the Inhibitory Effect of Myostatin on

AdipogenesisTo investigate the mechanisms that mediatemyostatin effect on adipogenesis, we began with theupstream steps in myostatin signaling. Myostatin has beenshown to activate TGF/Smad3 signaling pathway in myo-blasts and clonal rodent preadipocytes (19, 44). Consistently,we show that myostatin induced a rapid increase in phos-

pho-Smad3 in hMSCs (Fig. 4A), a process which is usuallycorrelated with Smad3 activation (19). To determinewhether activation of Smad3 is essential for myostatin-me-diated suppression of adipogenesis, we first assessed the cel-lular response to a pharmacological inhibitor of its upstreamkinase, TGF type I receptor Alk5. Incubation with the Alk5inhibitor (Alk5i) decreased phospho-Smad3 (Fig. 4A),increased PPAR, C/EBP, and aP2 expression, and blockedthe inhibitory effects of myostatin on each of these adipocytemarkers (Fig. 4B). The cells treated with Alk5i also hadhigher expression of PPAR and C/EBP than controls andgreatly increased expressionof aP2 (Fig. 4B) aswell as cellular lipidaccumulation (not shown). These data suggest that factors otherthan myostatin may restrain adipogenesis through the Alk5/Smad3 pathway under our control conditions.To evaluate further the role of Smad3 inmediatingmyostatin

effects on adipogenesis in hMSCs, we used two separate RNAioligonucleotides to reduce endogenous Smad3 expression.Each of the two RNAi oligonucleotides reduced Smad3 expres-sion by80% (Fig. 4C, upper left panel). Knockdown of Smad3by each of the two RNAi or their combination blocked myosta-tin-mediated inhibition of mRNA expression of PPAR,C/EBP, and aP2 (Fig. 4C). By Western analysis, we confirmedthat Smad3 RNAi also completely reversed myostatin-medi-ated inhibition of aP2 expression (Fig. 4D).

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-Catenin Is Activated byMyostatin through Smad3 and Is aKey Player in Suppression of AdipogenesisWe tested thehypothesis that Smad3 mediates the inhibitory effects of myo-statin by facilitating -catenin nuclear translocation (45) andactivating the Wnt/-catenin/TCF4 pathway (31, 45, 46). Toevaluate the physical interaction between Smad3, -catenin,andTCF4, we immunoprecipitated nuclear proteins using anti-bodies for Smad3 and -catenin, respectively. Equal proteininput in IP experiments was verified by Western analysis ofhistone-1 (Fig. 5A). The immunoprecipitated protein complexwas separated by SDS-PAGE, transferred to polyvinylidenedifluoride membrane, and immunoblotted with mouse anti--catenin (IP Smad3) or rabbit anti-Smad3 (IP -catenin). Bothmembranes were then stripped and re-probed using mouseanti-TCF4. As shown in Fig. 5A, the protein complex precipi-tated by anti-Smad3 contained -catenin and TCF4. Similarly,the protein complex precipitated by anti--catenin containedSmad3 and TCF4. Incubation of hMSCs with myostatinincreased the amount of Smad3 co-precipitated with anti--catenin and TCF4 that was co-precipitated using either anti-Smad3 or anti--catenin. Co-treatment with Alk5i reduced

the myostatin-mediated association between -catenin andSmad3, as well as between these two proteins and TCF4(Fig. 5A).To confirm the effects of myostatin on the nuclear translo-

cation of -catenin in live cells, we transfected hMSCs witheGFP-tagged-catenin.Myostatin increased-catenin-associ-ated green fluorescence in the nuclei of hMSCs (Fig. 5B). Wefurther performedWestern analyses of nuclear protein extractsfrom hMSCs maintained in DM for 24 or 48 h with or withoutmyostatin. As shown in Fig. 5C, incubation in DM alonedecreased the amount of -catenin in both nuclear and cytoso-lic compartments (47). Similarly, we found that hMSCs treatedwith myostatin retained strong -catenin immunoreactivity inboth the nuclear and cytosolic compartments after an extendedincubation inDM (2448 h, Fig. 5C), suggesting thatmyostatinstabilizes -catenin in differentiating hMSCs.To determine whether -catenin is essential for suppression

of adipogenesis by myostatin, we inhibited endogenous -cate-nin expression by RNAi. As shown in Fig. 5D, each RNAi effi-ciently suppressed -catenin expression by more than 70%.Inhibition of -catenin by each RNAi was associated with

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increased expression of aP2, PPAR, and C/EBP (Fig. 5D).Silencing of -catenin blocked the inhibitory effect of myosta-tin on each of these adipocyte marker genes (Fig. 5D) and lipidaccumulation (not shown). Western analysis confirmed theRNAi effect on the -catenin protein and the reversal of myo-statin-mediated suppression of aP2 expression (Fig. 5E). Thus,-catenin plays an essential role in mediating the inhibitoryeffects of myostatin on adipogenesis in hMSCs.To evaluate whether the increase in nuclear -catenin and

the formation of -catenin /TCF4 complex causes functionalactivation of the Wnt/-catenin signaling pathway, we per-formed a real-time PCR-based macroarray analysis of Wnt/-catenin pathway genes (Superarray APHS-043). Myostatinaltered the expression of a number of Wnt/-catenin pathwaygenes in hMSCs during differentiation. Among these, PITX2,DKK1, andWnt4were found to be themost responsive tomyo-statin (2-fold) after confirmation with real-time PCR (datanot shown). Thus, myostatin-induced nuclear translocation of-catenin and its interaction with TCF4 were associated withchanges in Wnt pathway activity. The increased expression ofPITX2, a progrowth transcription factor, raised the possibilitythat myostatin might inhibit adipogenesis by preventinggrowth arrest, as previously shown in differentiating myoblasts(4850). Accordingly, we evaluate the effect of myostatin oncell cycle distribution using fluorescence-activated cell sorting(FACS). As shown in Fig. 6A, the majority of the cells fell in theGo/G1 phasewith a small fraction remaining in theG2/Mphase,in a pattern similar to that found in differentiatingmouse clonalpreadipocytes (51). This distribution of cells in different phasesof cell cycle was not significantly different in DM control andmyostatin-treated hMSCs at different time points (from hoursto days, not shown).We then assessed DNA synthesis in differentiating hMSCs

bymeasuring the incorporation of bromodeoxyuridine (BrdU).As shown in Fig. 6B, BrdU incorporationwasmarkedly reducedwithin the first 24 h of exposure toDM.Myostatin had no effecton BrdU incorporation in hMSCs at least within the first 10days of the differentiation program. Hence, myostatin-medi-ated activation ofWnt/-catenin/TCF4 pathway was not asso-ciated with altered cell cycle regulation under our experimentalconditions. Furthermore, our results show that, unlike murinepreadipocyte cell lines, differentiating hMSCs do not experi-ence early phase clonal expansion.Inactivation of TCF4 Attenuates Myostatin-mediated Sup-

pression of AdipogenesisAs a central component of the Wntsignaling pathway, TCF4 has been shown to be an importantregulator of adipogenesis (26). To determine whether TCF4 is akey player downstream of -catenin, we transfected hMSCswith adenovirus vector encoding a dominant-negative TCF4(dn-TCF4) and treated the cells in DMwith or withoutmyosta-tin. Ad5 empty virus was used as control. Adenovirus encodingwild-type TCF4 was transfected in parallel cultures. The func-tionality of dn-TCF4 to block TCF4 activity was confirmed byluciferase activity tested inCOS-7 cells transfectedwith aTCF4reporter gene (TOPFLASH) and a control Renilla luciferasegene (Fig. 7D).Transfection with dn-TCF4 increased lipid accumulation

(Fig. 7A) and increased the mRNA expression of PPAR,

CEBP, and aP2 (Fig. 7B). At appropriate concentrations, dn-TCF4 largely, but not completely, blocked the inhibitory effectof myostatin on mRNA expression of each of these adipocytegene markers (Fig. 7B).We next tested whether ectopic dn-TCF4, at its optimal con-

centration, might diminish the potency of myostatin in inhib-iting adipogenesis.Myostatin down-regulated the expression ofaP2 and PPAR at a similarly low concentration range (0.010.05 g/ml) in hMSCs transfected with either vector or dn-TCF4. However, at any given concentration of myostatin, theextent of inhibition was consistently attenuated by dn-TCF4.These results suggest that TCF4 is a major component thatmediates the inhibitory effect of myostatin, whereas otherinhibitory pathways are present that can be activated by myo-statin independent of TCF4.

DISCUSSION

Adult hMSC have been well characterized as a renewableprogenitor pool that can differentiate into adipogenic andmul-

FIGURE 6. Under adipogenic differentiation conditions, myostatin doesnot affect cell cycle progression or DNA synthesis. Confluent hMSCs weretreated with basal medium, DM, and DM containing myostatin (0.1 g/ml).A, cells were harvested after 24 h and subjected to fluorescent-activated cellsorting (FACS) analysis. The area under each phase (G0/G1 versus G2/M) rep-resents the relative number of cells residing in the corresponding phase. Themajority of the cells fell in the quiescent G0/G1 phase, regardless of the pres-ence ofmyostatin; B, DNA synthesis assessedbyBrdU incorporation in hMSCsincubated in basal medium, DM, or DM containing myostatin (0.1 g/ml) fordifferent days, as shown.

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tiple other lineages. Adipocytes derived from hMSCs have geneexpression profiles similar to that of primary adipocytes (52),rendering them a good model for studying the effects of myo-statin on adipogenic differentiation. In this study, we providethe first evidence that recombinant human myostatin proteininhibits adipogenic differentiation of hMSCs and human adi-pose tissue-derived preadipocytes. The inhibitory effects ofmyostatin require the participation of the Alk5 receptor andSmad3. Our data show that Smad3 interacts with -catenin toform a complex that includes TCF4. Thus, the signal fromSmad3 is cross-communicated to the Wnt/-catenin pathway,resulting in activation of theWnt/-catenin pathway and inhi-bition of C/EBP and PPAR, the two principal regulators ofterminal adipogenesis.-Catenin plays an obligatory role in thecross-communication between Smad3 signaling and Wnt/TCF4 signaling pathways and in mediating the inhibitoryeffects of myostatin on adipogenesis.Myostatin did not affect the early transient induction of

C/EBP, which has been shown to facilitate mitotic clonalexpansion and to transactivate C/EBP and PPAR in murinepreadipocytes. In hMSC, we did not observe clonal expansion.However, myostatin markedly down-regulated the expressionof PPAR and C/EBP, which was not reversed by ectopic

C/EBP. In contrast, ectopic PPAR completely blocked theinhibitory effects of myostatin.Several lines of evidence demonstrate that activation and

cross-communication of Smad signal to Wnt/-catenin path-way are essential for mediating the effects of myostatin on adi-pogenic differentiation. First, the inhibitory effect of myostatinwas blocked by an Alk5/Smad3 inhibitor and by RNAi ofSmad3. Second, myostatin promotes -catenin associationwith Smad3, its nuclear translocation, and its association withTCF4, a hallmark of Wnt pathway activation. Finally, myosta-tin-mediated suppression of adipogenesis was completelyblocked by RNAi silencing of -catenin and also greatly atten-uated by dn-TCF4, a binding partner of -catenin.The mechanisms by which TCF4 and -catenin interfere

with adipogenesis remain unclear. Both have been shown toassociate with PPAR in different cell systems (23). BecausePPAR is auto-regulated and also cross-regulates C/EBP,inactivation of this transcription factor down-regulates its ownexpression and suppresses essentially all other adipogenicgenes. Other Wnt/TCF4 pathway proteins, such as c-Myc andcyclin D1, have also been shown to bind and inactivate PPAR(23, 53). Although myostatin did not alter the steady-statemRNA levels of these genes (not shown), it is not known

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whether myostatin alters their functions through post-tran-scriptional regulation. In addition, -catenin, with and withoutTCF4, binds to multiple nuclear receptors and transcriptionfactor coactivators, including CBP/p300 (54), which can regu-late the activity of PPAR. TheWnt signaling pathwaymay alsoinactivate PPAR through modulation of histone methylation(55). The precisemechanisms that inactivate PPAR under ourexperimental conditions remain to be investigated.The inhibitory effects ofmyostatin on differentiation ofmes-

enchymal progenitor cells and lipid accumulation reported inthis work are consistent with the observations that transgenicmice with adipose tissue-specific hyperexpression of myostatinare lean and resistant to a high fat diet. It is, however, remark-able that myostatin-null mice and cattle have a lower fat massthan the wild types (13, 14). Similarly, a human child with aninactivating mutation in the myostatin gene was strikingly lean(56). We speculate that increased levels of circulating muscle-derived factors, myokines, associated with hypermuscularity inmyostatin-null mice, may indirectly affect adipose tissue devel-opment. This possibility is now being investigated.The effects of myostatin on the Wnt/-catenin signaling

pathway are highly cell type-specific. While we show that myo-statin activates the Wnt pathway through cross-communica-tionwith its direct target Alk5/Smad3 in hMSCs differentiatinginto the adipogenic linage, an opposite effect was observed dur-ing myogenic differentiation of C2C12 mouse myoblasts. Asshown under supplemental Fig. S1, in contrast to a reduction innuclear-catenin observed during adipogenesis,myogenesis inC2C12 cells greatly increases nuclear -catenin. This increase,however, was inhibited by myostatin. This result is in agree-ment with others who reported higherWnt target gene expres-sion in skeletal muscle of myostatin knock-out than wild-typemice (57). Elucidation of such tissue-specific effects might helpresolve the paradox that activation of myostatin inhibits adipo-genesis, whereas inactivation of myostatin in vivo reduces bodyfat mass.

AcknowledgmentsWe thank Dr. Steven Farmer for critical readingof this manuscript and for the generous gift of the retroviral vector ofC/EBPmutant, andAmgen Inc. andWyeth Inc. for providing recom-binant human myostatin peptide and antibody, respectively. Wethank the Adipocyte Core of the Boston Obesity Nutrition ResearchCenter (DK42600) for providing human preadipocytes.

REFERENCES1. Marcell, T. J., Harman, S. M., Urban, R. J., Metz, D. D., Rodgers, B. D., and

Blackman, M. R. (2001) Am. J. Physiol. Endocrinol. Metab. 281,E1159E1164

2. Rodgers, B. D., Weber, G. M., Sullivan, C. V., and Levine, M. A. (2001)Endocrinology. 142, 14121418

3. Langley, B., Thomas, M., Bishop, A., Sharma, M., Gilmour, S., and Kam-badur, R. (2002) J. Biol. Chem. 277, 4983149840

4. McMahon, C. D., Popovic, L., Oldham, J. M., Jeanplong, F., Smith, H. K.,Kambadur, R., Sharma, M., and Maxwell, L. B. J. (2003) Am. J. Physiol.Endocrinol. Metab. 285, E82E87

5. Reisz-Porszasz, S., Bhasin, S., Artaza, J. N., Shen, R., Sinha-Hikim, I.,Hogue, A., Fielder, T. J., andGonzalez-Cadavid,N. F. (2003)Am. J. Physiol.Endocrinol. Metab. 285, E876E888

6. Wehling, M., Cai, B., and Tidball, J. G. (2000) FASEB J. 14, 1031107. Zhu, X., Hadhazy, M., Wehling, M., Tidball, J. G., and McNally, E. M.

(2000) FEBS Lett. 474, 71758. Nishi, M., Yasue, A., Nishimatu, S., Nohno, T., Yamaoka, T., Itakura, M.,

Moriyama, K.,Ohuchi,H., andNoji, S. (2002)Biochem. Biophys. Res. Com-mun. 293, 247251

9. Shelton, G. D., and Engvall, E. (2007) Neuromuscul. Disord. 17, 72172210. Mosher, D. S., Quignon, P., Bustamante, C. D., Sutter, N. B., Mellersh,

C. S., Parker, H. G., and Ostrander, E. A. (2007) PLoS Genet. 3, e7911. Liang, Y. C., Yeh, J. Y., and Ou, B. R. (2007) J. Exp. Biol. 210, 47748312. Clop, A., Marcq, F., Takeda, H., Pirottin, D., Tordoir, X., Bibe, B.,

Bouix, J., Caiment, F., Elsen, J. M., Larzul, C., Laville, E., Meish, F.,Milenkovic, D., Tobin, J., Charlier, C., and Georges, M. (2006) Nat.Genet. 38, 813818

13. Martyn, J. K., Bass, J. J., andOldham, J. M. (2004)Anat. Rec. A Discov.Mol.Cell Evol. Biol. 281, 13631371

14. McPherron, A. C., and Lee, S. J. (2002) J. Clin. Investig. 109, 59560115. Lee, S. J. (2007) PLoS ONE 2, e78916. Feldman, B. J., Streeper, R. S., Farese, R. V., Jr., and Yamamoto, K. R. (2006)

Proc. Natl. Acad. Sci. U. S. A. 103, 156751568017. Zimmers, T. A., Davies, M. V., Koniaris, L. G., Haynes, P., Esquela, A. F.,

Tomkinson, K.N.,McPherron, A. C.,Wolfman,N.M., and Lee, S. J. (2002)Science 296, 14861488

18. Hirai, S., Matsumoto, H., Hino, N., Kawachi, H., Matsui, T., and Yano, H.(2007) Domest. Anim. Endocrinol. 32, 114

19. Rebbapragada, A., Benchabane, H., Wrana, J. L., Celeste, A. J., and Atti-sano, L. (2003)Mol. Cell. Biol. 23, 72307242

20. Artaza, J. N., Bhasin, S.,Magee, T. R., Reisz-Porszasz, S., Shen, R., Groome,N. P., Meerasahib, M. F., and Gonzalez-Cadavid, N. F. (2005) Endocrinol-ogy 146, 35473557

21. Cai, L., Ye, Z., Zhou, B. Y.,Mali, P., Zhou, C., andCheng, L. (2007)Cell Res.17, 6272

22. Pal, R., and Khanna, A. (2006) Stem Cells Dev. 15, 293923. Mulholland, D. J., Dedhar, S., Coetzee, G. A., and Nelson, C. C. (2005)

Endocr. Rev. 26, 89891524. Kennell, J. A., and MacDougald, O. A. (2005) J. Biol. Chem. 280,

240042401025. Akimoto, T., Ushida, T., Miyaki, S., Akaogi, H., Tsuchiya, K., Yan, Z.,

Williams, R. S., and Tateishi, T. (2005) Biochem. Biophys. Res. Commun.329, 381385

26. Ross, S. E., Hemati, N., Longo, K. A., Bennett, C. N., Lucas, P. C., Erickson,R. L., and MacDougald, O. A. (2000) Science 289, 950953

27. Hammarstedt, A., Isakson, P., Gustafson, B., and Smith, U. (2007) Bio-chem. Biophys. Res. Commun. 357, 700706

28. Cawthorn, W. P., Heyd, F., Hegyi, K., and Sethi, J. K. (2007) Cell DeathDiffer. in press

29. Yang, X., Jansson, P. A., Nagaev, I., Jack,M.M., Carvalho, E., Sunnerhagen,K. S., Cam, M. C., and Cushman, S. W., and Smith, U. (2004) Biochem.Biophys. Res. Commun. 317, 10451051

30. Wright, W. S., Longo, K. A., Dolinsky, V. W., Gerin, I., Kang, S., Bennett,C. N., Chiang, S. H., Prestwich, T. C., Burant, C. F., Susulic, V. S., andMacDougald, O. A. (2007) Diabetes 56, 295303

31. Warner, D. R., Greene, R. M., and Pisano, M. M. (2005) FEBS Lett. 579,35393546

32. Pouponnot, C., Jayaraman, L., and Massague, J. (1998) J. Biol. Chem. 273,2286522868

33. Waltzer, L., and Bienz, M. (1998) Nature 395, 52152534. Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F., and Kemler, R.

(2000) EMBO J. 19, 1839185035. Labbe, E., Letamendia, A., and Attisano, L. (2000) Proc. Natl. Acad. Sci.

U. S. A. 97, 8358836336. Fischer, L., Boland, G., and Tuan, R. S. (2002) J. Biol. Chem. 277,

308703087837. Zhou, S., Eid, K., and Glowacki, J. (2004) J. Bone Miner. Res. 19, 46347038. Tchkonia, T., Giorgadze, N., Pirtskhalava, T., Tchoukalova, Y., Karagian-

nides, I., Forse, R. A., DePonte, M., Stevenson, M., Guo, W., Han, J.,Waloga, G., Lash, T. L., Jensen, M. D., and Kirkland, J. L. (2002) Am. J.Physiol. Regul Integr Comp. Physiol. 282, R1286R1296

39. Guo, W., Pirtskhalava, T., Tchkonia, T., Xie, W., Thomou, T., Han, J.,Wang, T., Wong, S., Cartwright, A., Hegardt, F. G., Corkey, B. E., and

Mechanism ofMyostatin Action on Adipogenic Differentiation of hMSCs

9144 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283NUMBER 14APRIL 4, 2008

at CAPES/M

EC - UFM

T , UN

IR , CEFET/BA on Septem

ber 19, 2014http://w

ww

.jbc.org/D

ownloaded from

Kirkland, J. L. (2007) Am. J. Physiol. Endocrinol Metab. 292,E1041E1051

40. Guo, W., Wong, S., Xie, W., Lei, T., and Luo, Z. (2007) Am. J. Physiol.Endocrinol. Metab. 293, E576E586

41. Hill, J. J., Davies, M. V., Pearson, A. A., Wang, J. H., Hewick, R. M., Wolf-man, N. M., and Qiu, Y. (2002) J. Biol. Chem. 277, 4073540741

42. Darlington, G. J., Ross, S. E., and MacDougald, O. A. (1998) J. Biol. Chem.273, 3005730060

43. Nakamura, T., Shiojima, S., Hirai, Y., Iwama, T., Tsuruzoe, N., Hirasawa,A., Katsuma, S., and Tsujimoto, G. (2003) Biochem. Biophys. Res. Com-mun. 303, 306312

44. Zhou, S., Lechpammer, S., Greenberger, J. S., and Glowacki, J. (2005)J. Biol. Chem. 280, 2268822696

45. Jian, H., Shen, X., Liu, I., Semenov, M., He, X., and Wang, X. F. (2006)Genes Dev. 20, 666674

46. Sato, M. (2006) Acta Derm Venereol. 86, 30030747. Moldes, M., Zuo, Y., Morrison, R. F., Silva, D., Park, B. H., Liu, J., and

Farmer, S. R. (2003) Biochem. J. 376, 60761348. Briata, P., Ilengo, C., Corte, G., Moroni, C., Rosenfeld, M. G., Chen, C. Y.,

and Gherzi, R. (2003)Mol. Cell 12, 1201121149. Kioussi, C., Briata, P., Baek, S. H., Rose, D.W., Hamblet, N. S., Herman, T.,

Ohgi, K. A., Lin, C., Gleiberman, A, W. J., Brault, V., Ruiz-Lozano, P.,

Nguyen, H. D., Kemler, R., Glass, C. K., Wynshaw-Boris, A., and Rosen-feld, M. G. (2002) Cell 111, 673685

50. Joulia, D., Bernardi, H., Garandel, V., Rabenoelina, F., Vernus, B., andCabello, G. (2003) Exp. Cell Res. 286, 263275

51. Xie, W., Hamilton, J. A., Kirkland, J. L., Corkey, B. E., and Guo, W. (2006)Lipids 41, 267271

52. Mackay, D. L., Tesar, P. J., Liang, L. N., and Haynesworth, S. E. (2006)J. Cell. Physiol. 207, 722728

53. Bennett, C. N., Ross, S. E., Longo, K. A., Bajnok, L., Hemati, N., Johnson,K. W., Harrison, S. D., and MacDougald, O. A. (2002) J. Biol. Chem. 277,3099831004

54. Takemaru, K. I., and Moon, R. T. (2000) J. Cell Biol. 149, 24925455. Takada, I., Mihara,M., Suzawa,M., Ohtake, F., Kobayashi, S., Igarashi,M.,

Youn, M. Y., Takeyama, K., Nakamura, T., Mezaki, Y., Takezawa, S., Yo-giashi, Y., Kitagawa, H., Yamada, G., Takada, S., Shibuya, H., Matsumoto,K., and Kato, S. (2007) Nat. Cell Biol. 9, 12731288

56. Schuelke, M., Wagner, K. R., Stolz, L. E., Hubner, C., Riebel, T., Komen,W., Braun, T., Tobin, J. F., and Lee, S. J. (2004) N. Engl. J. Med. 350,26822688

57. Steelman, C. A., Recknor, J. C., Nettleton, D., and Reecy, J. M. (2006)FASEB J. 20, 580582

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