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Distinct Roles of Glycogen Synthase Kinase (GSK)-3� andGSK-3� in Mediating Cardiomyocyte Differentiation inMurine Bone Marrow-derived Mesenchymal Stem Cells*□S

Received for publication, May 8, 2009, and in revised form, September 10, 2009 Published, JBC Papers in Press, October 26, 2009, DOI 10.1074/jbc.M109.019109

Jaeyeaon Cho‡, Pranela Rameshwar§, and Junichi Sadoshima‡1

From the ‡Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, and §Department ofMedicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

The signaling mechanisms facilitating cardiomyocyte (CM)differentiation from bone marrow (BM)-derived mesenchymalstem cells (MSCs) are not well understood. 5-Azacytidine(5-Aza), a DNA demethylating agent, induces expression of car-diac-specific genes, such as Nkx2.5 and �-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regula-tion of glycogen synthase kinase (GSK)-3� anddown-regulationof �-catenin, whereas it stimulated GSK-3� expression onlymodestly. The promoter region of GSK-3� was heavily methyl-ated in controlMSCs, butwas demethylated by 5-Aza. AlthoughoverexpressionofGSK-3�potently inducedCMdifferentiation,that of GSK-3� induced markers of neuronal and chondrocytedifferentiation. GSK-3 inhibitors, including LiCl, SB 216743,and BIO, abolished 5-Aza-induced up-regulation of CM-spe-cific genes, suggesting that GSK-3 is necessary and sufficient forCM differentiation in MSCs. Although specific knockdown ofendogenous GSK-3� abolished 5-Aza-induced expression ofcardiac specific genes, surprisingly, that of GSK-3� facilitatedCMdifferentiation inMSCs. AlthoughGSK-3� is found in boththe cytosol and nucleus in MSCs, GSK-3� is localized primarilyin the nucleus. Nuclear-specific overexpression of GSK-3�failed to stimulate CM differentiation. Down-regulation of�-catenin mediates GSK-3�-induced CM differentiation inMSCs,whereas up-regulation of c-Junplays an important role inmediating CM differentiation induced by GSK-3� knockdown.These results suggest that GSK-3� and GSK-3� have distinctroles in regulating CM differentiation in BM-derived MSCs.GSK-3� in the cytosol induces CM differentiation of MSCsthrough down-regulation of �-catenin. In contrast, GSK-3� inthe nucleus inhibits CM differentiation through down-regula-tion of c-Jun.

Ischemic cardiomyopathy and myocardial infarction areaccompanied by an irreversible loss of cardiomyocytes, endo-thelial cells, and smooth muscle cells, essential components ofthe heart (1). Cell-based cardiac repair offers the promise of

rebuilding the injured heart from its component parts(reviewed in Refs. 2 and 3). Although remarkable progress inthe field has clearly proven the concept of “cell-based cardiacrepair,” initial clinical studies using adult stem cells have shownthat the salutary effects mediated by cell transplantations aregenerally modest (4–7). Amajor challenge for cardiac regener-ation therapy using adult stem cellsmay be to enhance stem celldifferentiation into cardiomyocytes.Among several important signaling mechanisms generally

involved in cardiomyocyte differentiation, Wnt/�-catenin(canonical Wnt) and non-canonical Wnt signaling have beensuggested to have a critical role in cardiogenesis (8). Glycogensynthase kinase (GSK)2-3 is a key component of the canonicalWnt signaling pathway. GSK-3 phosphorylates �-catenin, andphosphorylated �-catenin is then subjected to ubiquitin pro-teasome degradation. However, uponWnt binding to its recep-tors, Frizzled and low-density lipoprotein receptor-related pro-tein, �-catenin phosphorylation by GSK-3� is inhibited and�-catenin is stabilized. Stabilized �-catenin translocates intothe nucleus and induces target gene expression. Although boththe canonical and non-canonical Wnt pathways are importantin mediating cardiomyocyte differentiation in stem cells andcardiac progenitor cells (9–15), the role of downstream com-ponents of the Wnt pathway, and, in particular, the role ofGSK-3, in mediating cardiomyocyte differentiation is not yetfully understood.GSK-3 is a serine/threonine kinase that has a wide variety of

functions in cells. GSK-3 phosphorylatesmany known intracel-lular targets, including �-catenin, glycogen synthase, elF2B�,GATA4, myocardin, c-Jun, cyclin D1, and N-Myc, thereby reg-ulating various cellular functions, including hypertrophy andapoptosis in cardiomyocytes (16). GSK-3 has two major iso-forms, GSK-3� and GSK-3�, which have 97% identical aminoacids in the catalytic domain but differ substantially in the NandC termini. Increasing lines of evidence suggest thatGSK-3�and GSK-3� both have common and non-overlapping func-tions (17). For example, both GSK-3� and GSK-3� phosphory-late/degrade �-catenin in embryonic stem cells, but GSK-3�

* This work was supported, in whole or in part, by National Institutes of HealthGrants HL 059139, HL067724, HL069020, AG023039, AG027211, andHL91469 from the United States Public Health Service and grants from theNew Jersey Commission of Science and Technology Stem Cell ResearchGrant Program.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S7.

1 To whom correspondence should be addressed: 185 S. Orange Ave., MSBG609, Newark, NJ 07103. Fax: 973-972-8919; E-mail: [email protected].

2 The abbreviations used are: GSK, glycogen synthase kinase; 5-Aza, 5-azacy-tidine; BIO, 6-bromo-indirubin-3�-oxime; MSC, mesenchymal stem cell;Dox, doxycycline; Tg, transgenic; NLS, nuclear localization signal; GAPDH,glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription;�-MHC, �-myosin heavy chain; cTnI, cardiac troponin I; cTnC, cardiac tro-ponin C; DN, dominant-negative; shRNA, short hairpin RNA; tTA, tetracy-cline-controlled transactivator; rtTA, reverse tetracycline controlledtransactivator.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 52, pp. 36647–36658, December 25, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

DECEMBER 25, 2009 • VOLUME 284 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36647

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and GSK-3� play distinct roles in cardiac development in mice(18, 19). Importantly, the isoform-specific functions of GSK-3�and GSK-3� during cardiomyocyte differentiation are not wellunderstood in mesenchymal stem cells (MSCs).5-Azacytidine (5-Aza) is a chemical analogue of cytidine that

removes methyl groups from DNA, thereby inducing geneexpression. 5-Aza is a potent inducer of cardiomyocyte differ-entiation in bone marrow-derived MSCs (20). MSCs showpotential for clinical application with evidence of tissue regen-eration, including myocardial regeneration (21). We reasonedthat by studying the function of signalingmolecules modulatedduring cardiac differentiation, we should be able to elucidatethe signaling mechanisms involved in stimulating cardiomyo-cyte differentiation in adult stem cells. Through initial screen-ing of signalingmoleculesmodulated by 5-Aza during cardiom-yocyte differentiation in MSCs, we found that GSK-3 plays animportant role in regulating this process.Thus, the goals in this study were to elucidate whether GSK-

3�/� is affected during differentiation of MSCs into the car-diomyocyte lineage in response to 5-Aza treatment, and if so, toexamine whethermodulation of GSK-3 plays a causative role inmediating cardiomyocyte differentiation in MSCs. Further-more, we evaluated whether GSK-3� and GSK-3� play distinctroles in mediating cardiomyocyte differentiation and whetherregulation of �-catenin is involved in modulation of cardiom-yocyte differentiation by GSK-3�/�.

EXPERIMENTAL PROCEDURES

Transgenic Mice and MSCCulture—MSCs were isolated frombone marrow aspirates of 2–3-week-old C57BL/6 mice. Wild typemice, Tet-inducible GSK-3� trans-genic mice (Tg-Tet-GSK-3�), Tg-Tet-GSK-3� mice cross-bred withCMV-tTA transgenic mice (Tg-Tet-GSK-3�-tTA) (16), and trans-genic mice harboring the mouseNkx2.5 (9.0 kb) promoter-LacZ(see below) were used. Tg-Tet-GSK-3�-tTAmicewere fed doxycy-cline (Dox)-containing chow to sup-pressGSK-3� transgene expression.MSCs were cultured in a 1:1 mix-ture of Dulbecco’s modified Eagle’smedium/F-12 (Invitrogen) andmesenchymal basal medium (StemCell) supplemented with 10% fetalbovine serum (Atlanta Biologicals)and 1% L-glutamine (Invitrogen).At passage three, cells were posi-tive for CD105, CD29, and CD44,and negative for CD45. MSCs pas-saged 3–5 times were used.Plasmid Constructs and Adenovi-

ral Vectors—Adenoviruses harbor-ing GSK-3�, GSK-3�, dominant-negative GSK-3� (DN-GSK-3�),�-catenin, and LacZ have been

described previously (16, 22, 23). To make GSK-3� with anuclear localization signal (GSK-3�-NLS), cDNA encodingGSK-3� was subcloned into pEF/myc/nuc (Invitrogen). cDNAencoding Wnt11 was amplified by reverse transcription-poly-merase chain reaction from mouse heart mRNA, subclonedinto pCR2.1-TOPO (Invitrogen), and sequenced to confirm thecorrect sequence. cDNA encodingWnt3a was purchased fromOrigene. Complementary hairpin sequences for GSK-3�, GSK-3�, �-catenin, c-Jun, and scramble (supplemental Table S1)were commercially synthesized and cloned into pSilencer 2.0under theU6promoter (Ambion). Adenovirus vectors harbor-ing GSK-3�-NLS, Wnt3a, Wnt11, shRNA-GSK-3�, shRNA-GSK-3�, shRNA-�-catenin, shRNA-c-Jun, and shRNA-scram-ble were generated using AdMax (Microbix).5-Aza Treatment—MSCs were treated with 5-Aza (Sigma, 5

�M) for 24 h without serum and then cultured in serum con-taining culture medium. Identically prepared MSCs without5-Aza treatment were used as a control. Inhibition of GSK-3kinase activity was achieved by maintaining MSCs in culturemedium containing LiCl (10 mM), BIO (0.5 �g/ml), orSB216743 (2 �g/ml).GSK-3�Conditional OverexpressionUsing Tet-On or Tet-Off

System—Adenovirus vector harboring tTA or rtTA was intro-duced into Tet-MSCs isolated from Tg-Tet-GSK-3� mice.tTA- or rtTA-transducedMSCs were cultured with or without

FIGURE 1. Basal characteristics of mouse BM-derived MSCs. A, total RNA was prepared from MSCs at the 3rdand 10th passages. mRNA expression of Oct4 and Rex1, pluripotent stem cell markers, was evaluated byRT-PCR. B, protein expression of Oct3/4 (red) in MSCs at the 3rd passage was evaluated by immunostaining.Cells were co-stained with 4�,6-diamidino-2-phenylindole (DAPI). C and D, MSCs were treated with 5-Aza (5 �M)and cultured for 5 days. C, total RNA was prepared and mRNA expression of Nkx2.5, �-MHC, and GAPDH(internal control) evaluated by RT-PCR. D, protein expression of cTnI (green) in MSCs treated with or without5-Aza was evaluated by immunostaining. Cells were co-stained with DAPI. The results are representative of atleast 4 experiments.

Distinct Roles of GSK-3 Isoforms in Cardiac Differentiation

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Dox (0.5 �g/ml, Clontech). Tet/tTA-MSCs isolated fromTg-Tet-GSK-3�-tTAmice were maintained in Dox containingmedium and changed to normal culture medium for inductionof GSK-3� transgene.Western Blots—Total cell extracts were prepared, using cell

lysis buffer containing 20 mM Tris-Cl (pH 7.5), 150 mMNaCl, 1mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodiumpyrophosphate, 1 mM �-glycerophosphate, 1 mM Na3VO4, 0.5�g/ml of leupeptin, and 0.5mM4-(2-aminoethyl)benzenesulfo-nyl fluoride. For immunoblot analyses, polyvinylidene difluo-

ride membranes were incubatedwith 5% nonfat milk buffer contain-ing primary antibody overnight, fol-lowed by incubation with anti-mouse IgG or anti-rabbit IgG (CellSignaling Technology, 1:2500 dilu-tion) for 3 h. The following antibod-ies were used as primary antibodies:GSK-3� (Cell Signaling Technol-ogy, 1:5000), GSK-3� (BD Bio-sciences, 1:5000), phospho-GSK-3�/� (Cell Signaling Technology,1:5000), phospho-GSK-3� (CellSignaling Technology, 1:5000), �-catenin (BD Biosciences, 1:10000),Wnt3a (Santa Cruz Biotechnology,1:2500), Wnt11 (R&D Systems,1:2000), phospho-protein kinase C(Pan) (Cell Signaling Technology,1:2500), sarcomeric�-actinin (Sigma,1:10000), GATA4 (Santa Cruz Bio-technology, 1:5000), c-Jun (Cell Sig-naling Technology, 1:2500), troponinI (Santa Cruz Biotechnology, 1:5000),�-actin (Sigma, 1:10000), andGAPDH (Sigma, 1:5000).Immunocytochemistry—MSCs

were washed with phosphate-buff-ered saline, fixed with 4% para-formaldehyde for 10 min, perme-abilized in 0.3% Triton X-100 for10 min, and blocked in 3% bovineserum albumin for 1 h.The followingantibodies were used as primaryantibodies: Oct3/4 (Santa CruzBiotechnology, 1:200 dilution),troponin I (Santa Cruz Biotech-nology, 1:1000), cardiac troponin I(Abcam, 1:200), sarcomeric �-acti-nin (Sigma, 1:500), GSK-3� (BD Bio-sciences, 1:500), GSK-3� (Abcam,1:500), and c-Jun (Cell SignalingTechnology, 1:500).Reverse Transcriptase-PCR—To-

tal RNA was extracted using TRIzol(Invitrogen) and 1 �g of RNA wasused for cDNA synthesis (Thermo-scriptase�, Ambion). The RT-PCR

mixture (Promega)was incubated at 95 °C for 5min followed by95 °C for 30 s, 59 °C for 1 min, and 72 °C for 30 s for 34 cyclesand then incubated at 72 °C for 7min. PCR primers used in thisstudy are shown in supplemental Table S1.Methylation Specific PCR—Genomic DNA was extracted

from MSCs using the Wizard Genomic DNA Purification Kit(Promega) and then treated with the CpGenome Fast DNAModificationKit (Millipore). CpGenome-modifiedDNA (1�g)was subjected to PCR with methylation- or non-methylation-specific primers (supplemental Table S1) (24).

FIGURE 2. The effect of 5-Aza treatment upon expression of GSK-3�, GSK-3�, and �-catenin in MSCs. MSCswere treated with or without 5-Aza (5 �M) and then cultured for the indicated durations. A, protein expression ofGSK-3�, phosphorylated GSK-3�, GSK-3�, �-catenin, and �-actin (internal control) was evaluated by immunoblotanalyses. B, expression of GSK-3� was quantitated by densitometric analyses of the immunoblots. The level ofGSK-3� was normalized by that in MSCs without 5-Aza at day 1. The results are mean � S.E. from 5 experiments.C, genomic DNA was extracted from MSCs with or without 5-Aza and the methylation status in the GSK-3� andGSK-3� promoter region was examined by methylation-specific PCR. U, demethylated; M, methylated. D, proteinexpression of �-catenin/�-actin was evaluated by densitometric analyses. In B and D, *, p �0.05; **, p �0.01; ***, p �0.001. The experiments were conducted 5 times. Protein expression in MSCs on day 1 without 5-Aza treatment wasset as 1. The error bars show S.E. E, transcriptional activity of TCF/LEF was evaluated by reporter gene assays. MSCswere transfected with the TOP-FOP flash reporter genes and then treated with or without 5-Aza. The luciferaseactivity was evaluated on day 3 and the experiments were conducted 3 times.




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TransgenicMiceHarboringNkx2.5-LacZ—cDNAcontainingmouse Nkx2.5 promoter-LacZ, which directs cardiac specificactivation of luciferase (25), was kindly provided by Dr. K.Yutzey (The Children’s Hospital Research Foundation, Cincin-nati, OH). Transgenic mice harboring the mouse Nkx2.5 (9.0kb) promoter-luciferase were generated on FVB background.Luciferase Assay—Plasmids harboring TOP flash (TCF-lucif-

erase plasmid, Millipore) and FOP flash (mutant TCF-lucifer-ase plasmid,Millipore) were transfected intoMSCs using Lipo-fectamine reagent (Invitrogen), and the luciferase activity wasmeasuredwith the LuciferaseAssay System (Promega) after celllysis with Passive Lysis Buffer (Promega).

Statistical Analyses—All valuesare expressed as mean � S.E. Statis-tical analyses were performed usinganalysis of variance and Newman-Keulsmultiple comparison test witha p � 0.05 considered significant.

RESULTS

Murine Bone Marrow-derivedMSCs Express Pluripotent Markersand 5-Aza Induces Expression ofCardiac Marker Genes in MSCs—MSCs at the third passage expressedOct4 and Rex1 mRNA, pluripotentstem cell markers (Fig. 1A). Ninety-five percent of MSCs at the thirdpassage were Oct3/4 positive (Fig.1B). Although untreated bone mar-row-derived mouse MSCs do notexpress cardiac marker genes,5-Aza treatment (5 �M for 24 h), anestablished method of inducingbone marrow MSC differentiationinto cardiomyocytes (20), inducedmRNA expression of Nkx2.5, one ofthe earliest cardiac markers, and�-myosin heavy chain (�-MHC), acontractile protein (Fig. 1C).Although no cardiac troponin I(cTnI) positive cells were observedin control MSCs, 5-Aza induced apremature but clear striation pat-tern of cTnI in MSCs (Fig. 1D).5-Aza Induces Cardiomyocyte

Differentiation through an Increasein GSK-3� Protein and mRNAExpression—GSK-3� and �-cateninare important components of thecanonical Wnt signaling pathway.To examine the effect of 5-Aza onthe canonical Wnt signaling path-way, protein expression of GSK-3isoforms and �-catenin was evalu-ated in 5-Aza-treated (5�M for 24 h)and control MSCs. Expression ofGSK-3� and GSK-3� was detecta-

ble but low. On the other hand, �-catenin was expressed abun-dantly in unstimulated MSCs. 5-Aza treatment increasedexpression of GSK-3� and GSK-3� in a time-dependent man-ner in MSCs, although induction of GSK-3� by 5-Aza wasmilder than that of GSK-3� (Fig. 2, A and B). Expression ofGSK-3� at day 5 was significantly greater in 5-Aza-treatedMSCs than in untreated MSCs (supplemental Fig. S1A). 5-Azatreatment did not induce up-regulation of GSK-3� in COS-7cells, suggesting that the effect of 5-Aza is cell type-specific(supplemental Fig. S1B). Up-regulation of GSK-3� andGSK-3� by 5-Aza was also observed at the mRNA level (sup-plemental Fig. S1C). The promoter regions of GSK-3� and

FIGURE 3. GSK-3�, GSK-3�, and DN-GSK-3� overexpression in MSCs. A, protein expression of GSK-3�,GSK-3�, �-catenin, and �-actin was evaluated by immunoblot analyses in Ad-LacZ-, Ad-GSK-3�-, and Ad-GSK-3�-transduced MSCs. B, protein expression of �-catenin and �-actin was evaluated by immunoblot analyses inAd-DN-GSK-3�-transduced MSCs. C, mRNA expression of Flk-1, Nkx2.5, atrial natriuretic factor, cTnC, �-MHC,GSK-3�, and GSK-3� was evaluated by RT-PCR. Expression of GAPDH was evaluated as an internal control.D, MSCs were prepared from transgenic mice harboring Nkx2.5-LacZ. MSCs were subjected to 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside (X-gal) staining after GSK-3�, GSK-3�, or DN-GSK-3� overexpression.MSCs without adenovirus transduction were used as a negative control for this experiment. E, MSCs weresubjected to immunostaining with sarcomeric �-actinin and 4�,6-diamidino-2-phenylindole (DAPI). The resultsare representative of at least 4 experiments.




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GSK-3� contain CpG islands (supplemental Fig. S2). The CpGislands are methylated in untreated MSCs, but are demethyl-ated after 5-Aza treatment (Fig. 2C). Although protein expres-sion of �-catenin gradually increased in control MSCs, a pro-gressive decrease in �-catenin was observed in 5-Aza-treatedMSCs (Fig. 2, A andD), accompanied by decreases in TCF/LEFtranscriptional activity as determined by TOP flash/FOP flashreporter gene assays (Fig. 2E).GSK-3� Induces Cardiomyocyte Differentiation in MSCs—

To examine whether expression of GSK-3 isoforms mimics theeffect of 5-Aza upon cardiomyocyte differentiation, GSK-3� orGSK-3� were overexpressed via adenovirus transduction inMSCs (Fig. 3A). Although GSK-3� overexpression reducedexpression of �-catenin in MSCs, GSK-3� overexpression didnot (Fig. 3A, see supplemental Fig. S6B). Adenovirus vectorsharboring LacZ (Ad-LacZ) andDN-GSK-3� (Ad-DN-GSK-3�)were used as controls. As expected, Ad-DN-GSK-3� increasedexpression of �-catenin (Fig. 3B). Transduction of MSCs withadenovirus harboring GSK-3� (Ad-GSK-3�), but not Ad-LacZor Ad-DN-GSK-3�, induced mRNA expression of mesodermmarkers, including Flk-1 (26) (Fig. 3C and data not shown).Ad-GSK-3�-induced mRNA expression of cardiomyocytemarkers, including Nkx2.5, cardiac troponin C (cTnC),�-MHC, and atrial natriuretic factor (Fig. 3C). Although Ad-GSK-3� also slightly inducedmRNAexpression of Flk-1, cTnC,and�-MHC, the extent of their up-regulationwas less than thatby Ad-GSK-3�. Transduction of Ad-LacZ or Ad-DN-GSK-3�did not significantly induce expression of the cardiomyocytemarkers (Fig. 3C). To obtain genetic evidence of cardiomyocytedifferentiation, we generated transgenicmice harboring a LacZgene driven by the Nkx2.5 promoter (9.0 kb), a cardiac specificpromoter (Tg-Nkx2.5-LacZ). Transduction of MSCs isolatedfrom Tg-Nkx2.5-LacZ mice with Ad-GSK-3� increased thenumber of �-galactosidase positive cells, whereas transductionwith Ad-DN-GSK-3�, Ad-GSK-3�, or Ad-LacZ did not (Fig.3D and data not shown). Sarcomeric �-actinin protein expres-sion was observed in MSCs transduced with Ad-GSK-3�, butnot in MSCs transduced with Ad-DN-GSK-3�. On the otherhand, only weak or negligible expression of sarcomeric �-acti-nin was observed inGSK-3�-overexpressingMSCs (Fig. 3E, seealso Fig. 8D). Ad-GSK-3� exhibited stronger induction of�-ac-tinin expression than 5-Aza treatment (supplemental Fig. S3).Taken together, these results suggest that GSK-3� induces car-diomyocyte differentiation in bone marrow-derived MSCs andthat overexpression of GSK-3� induces cardiomyocyte differ-entiation more potently than that of GSK-3�.

Although transduction of MSCs with Ad-GSK-3� inducedmRNA expression of both nestin, a neural marker, and Sox9, achondrocyte marker, Ad-GSK-3� did not induce mRNAexpression of either of them (supplemental Fig. S4). Transduc-tion ofMSCs with Ad-DN-GSK-3� inducedmRNA expressionof Sox9, but not nestin, suggesting that endogenous GSK-3�may inhibit chondrogenic differentiation inMSCs (supplemen-tal Fig. S4).To achieve up-regulation of GSK-3� by an alternative

method,MSCs derived fromTet-GSK-3� transgenicmiceweretransduced with Ad-tTA (the Tet-Off system) or Ad-rtTA (theTet-On system), and then treatedwith orwithoutDox.GSK-3�

expression was induced by withdrawing Dox in the Tet-Offsystem and by adding Dox in the Tet-On system (Fig. 4, A andB). The effect of Dox upon transgene expression was reversible(Fig. 4C), suggesting that GSK-3� expression can be regulatedby Dox treatment in these MSCs. In both the Tet-On and Tet-Off systems, up-regulation of GSK-3� induced expression ofNkx2.5 and �-MHC mRNA in MSCs (Fig. 4D). Alternatively,Tet inducibleGSK-3� transgenicmicewere crossedwith trans-genic mice harboring CMV-tTA, and then MSCs were pre-pared from the bone marrow of bigenic mice. In the absence ofDox, MSCs prepared from the bigenic mice expressed moreGSK-3� and less �-catenin than MSCs from control mice (Fig.4E). Culturing MSCs prepared from Tet-GSK-3� and CMV-tTAbigenicmice inDox-freemedium induced�-MHCexpres-sion, which was completely suppressed in the presence of Dox(Fig. 4F). These results support the notion that cardiomyocytedifferentiation of MSCs can be stimulated by drug-regulatableup-regulation of GSK-3�.

Because both canonical and non-canonical Wnt pathwaysinduce differentiation of stem cells into the cardiomyocyte lin-eage, we compared the effect of GSK-3� upon MSC differenti-ationwith that ofWnt agonists. Using adenovirus transduction,we overexpressed eitherWnt11, an agonist for the non-canon-ical Wnt pathway, or Wnt3a, an agonist for the canonical Wntpathway, in MSCs (Fig. 5, A and B). Wnt11 induced activation

FIGURE 4. GSK-3� overexpression, using Tet-On or Tet-Off systems. A andB, MSCs were prepared from Tg-Tet-GSK-3� mice. The GSK-3� transgene hasa Myc tag. A, MSCs prepared from Tg-Tet-GSK-3� mice were transduced withadenovirus harboring tTA (Ad-tTA) and transgene expression was suppressedby Dox (0.5 �g/ml) treatment. Adenoviral vector LacZ (Ad-LacZ) was used as acontrol. B, MSCs prepared from Tg-Tet-GSK-3� mice were transduced withadenovirus harboring rtTA and transgene expression was induced by Dox (0.5�g/ml) treatment. Ad-LacZ was used as a control. C, GSK-3� expression wasdecreased by Dox and recovered after Dox was washed out in MSCs preparedfrom Tg-Tet-GSK-3�-tTA mice. D, the effect of inducible GSK-3� overexpres-sion in the Tet-On (rtTA) or Tet-Off (tTA) system upon mRNA expression ofNkx2.5 and �-MHC is shown. Ad-LacZ was used as a negative control. E and F,Tg-Tet-GSK-3� mice were crossed with transgenic mice harboring CMV-tTAand then MSCs were prepared from either Tg-Tet-GSK-3� (Tet) or Tg-Tet-GSK-3�-tTA (Tet/tTA) mice. E, protein expression of GSK-3�, �-catenin, and GAPDH(internal control) was evaluated by immunoblots. F, MSCs prepared from Tet/tTA mice were cultured with or without Dox. mRNA expression of �-MHC andGAPDH was evaluated by RT-PCR. Experiments were repeated at least 4 times.




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of protein kinase C and Wnt3a caused a significant accumula-tion of �-catenin, suggesting that the non-canonical andcanonical Wnt pathways were stimulated in MSCs in ourexperimental conditions (Fig. 5C). Up-regulation of GSK-3�

induced the frequent appearance oftubular structures in MSCs (Figs.5D and supplemental S5). Tubularstructures are induced when MSCsare differentiated into the cardiom-yocyte lineage (20, 27). Althoughup-regulation of GSK-3� andWnt11 also induced some tubularstructures, they were not as promi-nent as those induced by GSK-3�.Tubular structures were not ob-served in MSCs treated withAd-LacZ, Ad-Wnt3a, or Ad-DN-GSK-3� (Figs. 5D and supplementalS5). Wnt11 induced mRNA expres-sion of �MHC, but little or noexpression of Flk-1, Nkx2.5, orcTnC, in MSCs (Fig. 5E), whereasWnt3a did not induce mRNAexpression of any of these markergenes (Fig. 5F). These results sug-gest that GSK-3� induces cardiom-yocyte differentiation of MSCsmore potently than the Wntagonists.Expression of GSK-3 Is Required

forCardiomyocyteDifferentiation inMSCs—Because GSK-3 was up-reg-ulated by 5-Aza and up-regulationof GSK-3� potently induced car-diomyocyte differentiation in MSCs,we next examinedwhetherGSK-3 isrequired for induction of cardiom-yocyte differentiation by 5-Aza inMSCs. MSCs were stimulated with5-Aza in the presence or absence ofknown inhibitors of GSK-3. Treat-ment ofMSCs with LiCl suppressed5-Aza-induced down-regulation of�-catenin, suggesting that LiCl sup-presses GSK-3 activity under 5-Azatreatment in MSCs (Fig. 6A).Although treatment of MSCs withLiCl induced expression of GATA4,LiCl alone did not induce expres-sion of other cardiomyocyte markergenes. LiCl did, however, inhibit

5-Aza-induced up-regulation of cardiomyocyte markermRNAs, including Nkx2.5, atrial natriuretic factor, cTnC, andcTnI (Fig. 6B), as well as cTnI protein expression (Fig. 6C).Treatment of MSCs with other GSK-3 inhibitors, including

FIGURE 5. GSK-3� induces cardiomyocyte differentiation more potently than Wnt agonists. MSCs were transduced with adenoviruses harboring LacZ, Wnt3a,Wnt11, GSK-3�, GSK-3�, and DN-GSK-3�. A–C, protein expression of Wnt3a (A), Wnt11 (B), �-catenin (C), and phosphorylated pan-protein kinase C (Phospho-PKC (Pan))(C) was evaluated by immunoblot analyses. The level of �-catenin protein expression was quantitated (C, right). GAPDH expression was evaluated as an internalcontrol. D, the effects of LacZ, Wnt3a, Wnt11, GSK-3�, GSK-3�, and DN-GSK-3� expression upon branched myofibril formation were evaluated. Left, representativephotos are shown. Right, the extent of branched myofibrils/cell surface (%) was quantitated. ***, p � 0.001. Pictures with a higher magnification are shown insupplemental Fig. S5. Error bars in B and D show S.E. E and F, mRNA expression of Flk-1, Nkx2.5, cTnC, and �-MHC in MSCs after transduction with Wnt11 (E) and Wnt3a(F) was evaluated by RT-PCR analyses. mRNA expression of GAPDH is shown as an internal control. Experiments were repeated at least 4 times.

FIGURE 6. GSK-3 is required for 5-Aza-induced cardiomyocyte differentiation in MSCs. MSCs were treated with5-Aza (5 �M) for 24 h and cultured 4 more days in the presence or absence of GSK-3 inhibitors. A, the effect of LiCl (10mM) upon �-catenin and �-actin protein expression was evaluated by immunoblot analyses. B, the effect of LiClupon 5-Aza-induced up-regulation of cardiomyocyte marker mRNA was evaluated by RT-PCR. C, the effect of LiClupon 5-Aza-induced up-regulation of cTnI protein was evaluated by immunocytochemistry. Double staining with4�,6-diamidino-2-phenylindole (DAPI) is also shown. D, the effect of GSK-3 inhibitors, BIO and SB216743, uponexpression of �-catenin and GAPDH (internal control) was evaluated by immunoblot (left) and densitometric anal-yses (right). The experiments were repeated 3 times, and the error bars show S.E. E, MSCs were treated with 5-Aza inthe presence or absence of GSK-3 inhibitors. mRNA expression of Flk-1, Nkx2.5, cTnC, �-MHC, and GAPDH (internalcontrol) was evaluated by RT-PCR. The results are representative of 3 to 4 experiments.




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6-bromo-indirubin-3�-oxime (BIO) and SB216743, reversed5-Aza-induced down-regulation of�-catenin (Fig. 6D). In addi-tion, BIO and SB216743 inhibited 5-Aza-induced up-regula-tion of Flk-1, Nkx2.5, cTnC, and �-MHC mRNA (Fig. 6E) andsarcomeric �-actinin protein expression (supplemental Fig.S3). These results suggest that GSK-3 plays an essential role inmediating 5-Aza-induced cardiomyocyte differentiation inMSCs.The Roles of the GSK-3 Isoforms inMediating Cardiomyocyte

Differentiation in MSCs—To evaluate the roles of endogenousGSK-3� and GSK-3� in mediating cardiomyocyte differentia-tion separately, we generated adenovirus vectors harboringshRNA-GSK-3� (Ad-sh-GSK-3�), shRNA-GSK-3� (Ad-sh-GSK-3�), and shRNA-scramble (Ad-sh-scramble) and con-firmed that Ad-sh-GSK-3� and Ad-sh-GSK-3� selectivelydown-regulate GSK-3� and GSK-3�, respectively (Fig. 7A).Although Ad-sh-GSK-3� increased expression of �-catenin,Ad-sh-GSK-3� did not (Fig. 7A). Down-regulation ofGSK-3� did not induce expression of Flk-1, a mesodermmarker, and the cardiomyocyte-specific genes, including�MHC, Nkx2.5, and cTnC (Fig. 7B). Unexpectedly, however,down-regulation of GSK-3� did induce mRNA expression ofFlk-1 and cardiac specific genes (Fig. 7B). Down-regulationof GSK-3�, but not GSK-3�, also induced protein expressionof �-actinin, cTnI, and GATA4, cardiomyocytemarkers (Fig.7C, see also Fig. 10C). Furthermore, although down-regula-tion of GSK-3� inhibited 5-Aza-induced up-regulation ofmesoderm and cardiomyocyte markers, down-regulation ofGSK-3� enhanced it (Fig. 7D). Essentially the same resultswere obtained with shRNA-GSK-3� targeting a distinct site(data not shown), suggesting that the effect of Ad-sh-GSK-3� was mediated by GSK-3� and not an off-targeteffect. These results suggest that endogenous GSK-3� andGSK-3� have opposite effects upon cardiomyocyte differen-tiation in MSCs. When GSK-3� was down-regulated in thepresence of GSK-3� up-regulation, �-catenin remaineddown-regulated (Fig. 7E), whereas mesoderm and cardiom-yocyte differentiation were enhanced (Fig. 7F), suggestingthat up-regulation of GSK-3� and down-regulation ofGSK-3� utilize distinct cellular mechanisms to induce car-diomyocyte differentiation in MSCs.Differential Subcellular Localization of GSK-3 Isoforms May

in Part Mediate Differential Effects of GSK-3� and GSK-3�upon Cardiomyocyte Differentiation in MSCs—Immuno-staining with isoform-specific antibodies indicated thatGSK-3� is localized primarily in the nucleus in MSCs. In con-trast, GSK-3� is localizedmainly in the cytosol but also partly inthe nucleus (Fig. 8A). Transduction of MSCs with adenovirusharboring LacZ did not significantly change subcellular local-ization of endogenous GSK-3� or GSK-3� (Fig. 8B). Adenovi-rus-mediated overexpression of GSK-3� increased expressionof GSK-3� in both the nucleus and cytoplasm and decreasedexpression of endogenous GSK-3� (Figs. 8B and supplementalS6A). Thus, overexpression of GSK-3� alters the subcellulardistribution ofGSK-3 isoforms.On the other hand, adenovirus-mediated overexpression of GSK-3� increased expression ofGSK-3� in both the nucleus and cytoplasm, without signifi-

cantly changing the subcellular distribution of GSK-3 isoforms(Fig. 8B).To examine the role of GSK-3� in the cytosol in modulating

cardiomyocyte differentiation, GSK-3�-NLS was expressed inMSCs. As expected, GSK-3�-NLS induced expression ofGSK-3� predominantly in the nucleus (Fig. 8B). Overexpres-sion of GSK-3�-NLS failed to induce mRNA and proteinexpression of cardiac markers (Fig. 8, C andD), suggesting thatcytosolic expression is essential for cardiomyocyte differentia-tion ofMSCs by GSK-3�. We also attempted tomake GSK-3�-NES, GSK-3� exclusively expressed in the cytosol, but thus farwe have not been successful.GSK-3� Induces Cardiomyocyte Differentiation of MSCs

through Down-regulation of �-Catenin—The signaling mecha-nisms by which GSK-3� and GSK-3� differentially affect

FIGURE 7. The role of GSK-3� and GSK-3� in mediating cardiomyocytedifferentiation of MSCs. A–D, MSCs were treated with Ad-shRNA-scramble(Ad-sh-scramble), Ad-shRNA-GSK-3� (Ad-sh-GSK-3�), or Ad-shRNA-GSK-3�(Ad-sh-GSK-3�) in the presence or absence of 5-Aza. A, protein expression ofGSK-3�, GSK-3�, �-catenin, and GAPDH (internal control) was examined byimmunoblot assays. A short exposure of an immunoblot is shown for �-cate-nin because the band with Ad-sh-GSK-3� was saturated after longer expo-sures. B, mRNA expression of Flk-1, Nkx2.5, �-MHC, cTnC, and �-actin (internalcontrol) was examined by RT-PCR. C, protein expression of sarcomeric �-acti-nin, cTnI, GATA4, and �-actin (internal control) was examined by immunoblotassays. D, mRNA expression of Flk-1, Nkx2.5, �-MHC, cTnC, and GAPDH (inter-nal control) was evaluated by RT-PCR. E and F, MSCs were transduced withAd-GSK-3� together with Ad-sh-scramble or Ad-sh-GSK-3�. E, protein expres-sion of GSK-3�, GSK-3�, �-catenin, and GAPDH (internal control) was evalu-ated by immunoblot assays. F, mRNA expression of Flk-1, Nkx2.5, �-MHC,cTnC, atrial natriuretic factor, and GAPDH was evaluated by RT-PCR. Pleasenote that a smaller cycle number was used in RT-PCR to show that Ad-sh-GSK-3� enhances the effect of Ad-GSK-3�. In A–F, the results are representa-tive of 3– 4 experiments.




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cardiomyocyte differentiation in MSCs were investigated.Although overexpression of GSK-3� significantly down-regu-lated�-catenin expression, that of GSK-3� did not significantlyreduce it (supplemental Fig. S6B). Down-regulation of GSK3�significantly increased �-catenin protein expression, whereasthat of GSK3� did not affect it in MSCs (supplemental Fig.S6C). These results suggest that endogenous GSK-3�,

but not GSK-3�, plays an essentialrole in regulating the cellular level of�-catenin in MSCs. To examinewhether down-regulation of �-catenin is sufficient to in-duce expression of cardiomyocytemarker genes, �-catenin was down-regulated by adenovirus harboringshRNA-�-catenin (Ad-sh-�-cate-nin) (Fig. 9A). Down-regulation of�-catenin by Ad-sh-�-cateninpotently induced mesoderm andcardiomyocyte markers, whereasshRNA-scramble had no effect (Fig.9B). Up-regulation of �-catenin byadenovirus harboring �-catenin(Ad-�-catenin) (Fig. 9C) inhibitedGSK-3� induced up-regulation ofmesoderm and cardiomyocytemarkers (Fig. 9D). These resultssuggest that down-regulation of�-catenin plays an important role inmediating induction of cardiomyo-cyte differentiation by GSK-3�. Onthe other hand, because down-reg-ulation of GSK-3� did not affect�-catenin expression, induction ofcardiomyocyte differentiation bydown-regulation of GSK-3� is un-likely to be mediated by �-catenin-dependentmechanisms. Down-reg-ulation of �-catenin inhibited,whereas up-regulation of �-cateninstimulated, expression of Sox9, sug-gesting that �-catenin mediateschondrocyte differentiation inMSCs (Fig. 9, E and F).Down-regulation of GSK-3a

Induces Cardiomyocyte Differentia-tion of MSCs through Up-regulationof c-Jun—c-Jun plays an importantrole in mediating cardiomyocytedifferentiation in bone marrowmononuclear cells (9). Down-regu-lation of GSK-3�, but not GSK-3�,up-regulated c-Jun expression inthe nucleus inMSCs (Figs. 10,A andB, and supplemental S6D). Trans-duction with adenovirus harboringshRNA-c-Jun reversed the up-regu-lation of c-Jun, and mesoderm and

cardiomyocyte markers induced by down-regulation ofGSK-3� inMSCs (Fig. 10,C andD), suggesting that c-Jun playsan important role in mediating cardiomyocyte differentiationof MSCs induced by down-regulation of GSK-3�. Down-regu-lation of c-Jun up-regulated mRNA expression of nestin, sug-gesting that c-Jun negatively regulates neuronal differentiationin MSCs (Fig. 10E).

FIGURE 8. GSK-3� and GSK-3� have distinct subcellular localization in MSCs. A, subcellular localization ofendogenous GSK-3� and GSK-3� in MSCs was evaluated by immunostaining. B–D, MSCs were transduced withAd-LacZ, Ad-GSK-3�, Ad-GSK-3�, or Ad-GSK-3�-NLS. B, MSCs were subjected to staining with anti-GSK-3�antibody, anti-GSK-3� antibody, and 4�,6-diamidino-2-phenylindole (DAPI). C, mRNA expression of Flk-1,Nkx2.5, �-MHC, cTnC, and GAPDH (internal control) was evaluated by RT-PCR. D, protein expression of GSK-3�,GSK-3�, �-actinin, cTnI, and GAPDH (internal control) was evaluated by immunoblots. * indicates GSK-3�-NLS.




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DISCUSSION

Expression of GSK-3 remains low when MSCs are uncom-mitted. However, GSK-3� is up-regulatedwhen cardiomyocytedifferentiation of MSCs is initiated by 5-Aza. Furthermore, up-regulation of GSK-3� is both necessary and sufficient for car-diomyocyte differentiation initiated by 5-Aza in MSCs. Unex-pectedly, down-regulation, rather than up-regulation, ofendogenous GSK-3� stimulated cardiomyocyte differentiationin MSCs. These results suggest that GSK-3� is an endogenousregulator ofMSC differentiation and that GSK-3� andGSK-3�have opposite effects upon cardiomyocyte differentiation inMSCs.5-Aza is a cytosine analogue and a demethylating agent that

induces changes in chromatin structure, gene expression, cel-lular morphology, and survival in mammalian cells. Becausephosphorylation of GSK-3� is not significantly affected by5-Aza, 5-Azamust increase the total activity ofGSK-3�primar-ily through up-regulation of GSK-3� mRNA. The promoterregion of GSK-3� contains a prominent CpG island that ismethylated in unstimulated MSCs, suggesting that 5-Aza

induces up-regulation of GSK-3� through epigeneticmodifica-tion of the promoter. Because GSK-3� stabilizes DNAmethyl-transferases, 5-Aza may initiate a positive feedback loop ofGSK-3� promoter demethylation (28). At present, whether ornot demethylation of the GSK-3� promoter is an endogenousmechanism for differentiation of adult stem cells into the car-diomyocyte lineage remains to be elucidated. In any event,GSK-3� may substitute for 5-Aza for induction of cardiomyo-cyte differentiation in MSCs, because clinical use of 5-Azawould be limited due to its nonspecific effects and obvious ter-atogenic actions.GSK-3� is a central component of the Wnt pathway and

negatively regulates �-catenin through phosphorylation-dependent proteolytic degradation (29). Although previousstudies have shown that stimulation and inhibition of the Wntsignaling mechanism affect cardiomyocyte differentiation (8),up-regulation of GSK-3� induced cardiomyocyte markersmore strongly than stimulation of either the canonical or non-canonical Wnt signaling pathways with Wnt3a and Wnt11,respectively. Because down-regulation of �-catenin alone alsopotently induces cardiomyocyte markers, modulating down-stream components of the Wnt signaling pathway may inducecardiomyocyte differentiationmore efficiently than stimulatingthe Wnt pathways at the receptor level.It should be noted that GSK-3� not only regulates the Wnt

pathway but also modulates a wide variety of signaling path-ways, including other signaling cascades known to regulatestem cell differentiation, such as the Notch (30) and Hedgehog(31) pathways. Thus, up-regulation of GSK-3� may have abroader effect than selective stimulation of theWnt pathway bythe Wnt receptor ligand.Increasing lines of evidence suggest that GSK-3� and

GSK-3� have distinct cellular functions, despite the fact thatthey share 97% identity in their kinase domains and 36% iden-tity overall. Our results suggest that GSK-3� and GSK-3� havedistinct effects upon cardiomyocyte differentiation. Up-regula-tion of GSK-3� has a stronger effect upon cardiomyocyte dif-ferentiation in MSCs than up-regulation of GSK-3�. Althoughoverexpression of GSK-3� slightly induces expression of car-diomyocyte markers, this effect may be mediated throughpromiscuous phosphorylation of GSK-3� substrates due tooverexpression. In fact, adenovirus-mediated overexpres-sion of GSK-3� substantially altered subcellular localizationof GSK-3� in MSCs (see below). Importantly, althoughdown-regulation of GSK-3� inhibited 5-Aza-induced car-diomyocyte differentiation, down-regulation of GSK-3�stimulated cardiomyocyte differentiation in MSCs. Previousstudies have suggested that GSK-3� and GSK-3� could dif-ferentially affect cardiac development. Although GSK-3�knock-out mice exhibit cardiac defects consisting of malfor-mation of the cardiac outflow tract and markedly thickenedventricular walls, contributing to their early mortality,GSK-3� knock-out mice show no significant cardiac defects(18). However, to our knowledge, the fact that GSK-3� andGSK-3� have opposite effects upon differentiation of stemcells has not been shown previously.One possible explanation for the potential difference in their

functions is that GSK-3� and GSK-3� exist in distinct subcel-

FIGURE 9. Down-regulation of �-catenin plays a critical role in mediatingGSK-3�-induced cardiomyocyte differentiation in MSCs. A and B, MSCswere transduced with Ad-shRNA-scramble (Ad-sh-scramble) or Ad-shRNA-�-catenin (Ad-sh-�-catenin). A, protein expression of �-catenin and GAPDH(internal control) was evaluated by immunoblots. B, mRNA expression ofFlk-1, Nkx2.5, �-MHC, cTnC, and GAPDH (internal control) was evaluated byRT-PCR. C, MSCs were transduced with Ad-LacZ or Ad-�-catenin and proteinexpression of �-catenin and GAPDH (internal control) was evaluated byimmunoblots. D, MSCs were transduced with Ad-LacZ, Ad-�-catenin, Ad-GSK-3�, or Ad-�-catenin plus Ad-GSK-3�. mRNA expression of Flk-1, Nkx2.5,�-MHC, cTnC, and GAPDH (internal control) was evaluated by RT-PCR. E and F,MSCs were transduced with either Ad-sh-scramble or Ad-sh-�-catenin (E), oreither Ad-LacZ or Ad-�-catenin (F). mRNA expression of Nestin, Sox9, andGAPDH (internal control) was evaluated by RT-PCR. In A–F, the results arerepresentative of 3 experiments.




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lular localizations. For example, in adult mouse hearts, GSK-3�is localized primarily in the nucleus, whereas GSK-3� existsprimarily in the cytosol (17). GSK-3� localized in the nucleusphosphorylates and induces nuclear exit/proteolytic degrada-tion ofG1 cyclins, whereas endogenousGSK-3� localized in thecytosol does not induce nuclear exit of G1 cyclins in the mouseheart (17). Immunostaining of the GSK-3 isoforms suggest thatGSK-3� is localized primarily in the nucleus, and GSK-3� pri-

marily in the cytosol but also in thenucleus inMSCs. Forced expressionof GSK-3� in the nucleus did notinduce cardiomyocyte differentia-tion, whereas overexpression ofGSK-3� induces cytosolic expres-sion of GSK-3� and partially stimu-lates cardiomyocyte differentiation.We therefore speculate that endog-enous GSK-3� more efficientlyphosphorylates �-catenin in thecytosol, thereby inducing efficientproteolytic degradation, whereasendogenous GSK-3� primarilylocalized in the nucleus may regu-late other targets, presumably tran-scription factors. Because GSK-3�and GSK-3� equally affect expres-sion of �-catenin in some cell types,such as ES cells (19, 32), we specu-late that subcellular localization ofGSK-3�/� may be cell type- ordevelopmental stage-dependent.Importantly, up-regulation of

GSK-3� and down-regulation ofGSK-3� have additive effects uponcardiomyocyte differentiation inMSCs, consistent with the notionthat they mediate cardiomyocytedifferentiation through distinct cel-lular mechanisms. Our results sug-gest that down-regulation of�-cate-nin plays an important role inmediating the effect of GSK-3�upon MSC differentiation into thecardiomyocyte lineage. On theother hand, down-regulation ofGSK-3� induces cardiomyocyte dif-ferentiation through up-regulationof c-Jun in MSCs. Because GSK-3phosphorylates �-catenin and c-Jun(19, 33), thereby stimulating theirdegradation, it is likely that GSK-3�in the cytosol may phosphorylate�-catenin, whereas GSK-3� in thenucleus may phosphorylate c-Jun,thereby regulating cardiomyocytedifferentiation in MSCs (supple-mental Fig. S7). Interventions toselectively stimulate GSK-3� or

inhibit GSK-3� may be considered independently or in combi-nationwith othermethods to facilitate cardiomyocyte differen-tiation of MSCs for cell-based therapy in vivo.We have successfully engineered MSCs that conditionally

express GSK-3� through either withdrawal or application ofDox. Phasic modulation of theWnt/�-catenin signaling mech-anism effectively stimulates differentiation of progenitor cellsinto the cardiomyocyte lineage (10, 34). Activation of�-catenin

FIGURE 10. Up-regulation of c-Jun plays a critical role in mediating shRNA-GSK-3�-induced cardiomyo-cyte differentiation in MSCs. A and B, MSCs were treated with Ad-shRNA-scramble (Ad-sh-scramble), Ad-shRNA-GSK-3� (Ad-sh-GSK-3�), or Ad-shRNA-GSK-3� (Ad-sh-GSK-3�). C–E, MSCs were transduced with Ad-sh-scramble, Ad-shRNA-c-Jun (Ad-sh-c-Jun), Ad-sh-GSK-3�, or Ad-sh-c-Jun plus Ad-sh-GSK-3�. A and C, proteinexpression of c-Jun and GAPDH (internal control) was evaluated by immunoblots. B, MSCs were subjected tostaining with anti-c-Jun antibody, anti-sarcomeric �-actinin antibody, and 4�,6-diamidino-2-phenylindole(DAPI). D, mRNA expression of Flk-1, Nkx2.5, �-MHC, cTnC, and GAPDH (internal control) was evaluated byRT-PCR. E, mRNA expression of Nestin, Sox9, and GAPDH (internal control) was evaluated by RT-PCR. In A–E, theresults are representative of 3 experiments.




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is required tomaintain and expand cardiac progenitor cells (11,35) but must be repressed to induce cardiomyocyte differenti-ation from cardiac progenitor cells (8). Thus, it would be inter-esting to test whether ex vivo engineered MSCs, in which thetiming of expression of GSK-3� can be regulated by Dox treat-ment, enhance the efficacy of cell therapy in vivo.

Acknowledgment—We thank Daniela Zablocki for critical reading ofthe manuscript.

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Jaeyeaon Cho, Pranela Rameshwar and Junichi SadoshimaStem Cells

Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal in Mediatingβ and GSK-3αDistinct Roles of Glycogen Synthase Kinase (GSK)-3

doi: 10.1074/jbc.M109.019109 originally published online October 26, 20092009, 284:36647-36658.J. Biol. Chem.

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