A WNT/�-Catenin Signaling Activator, R-spondin, PlaysPositive Regulatory Roles during Skeletal Myogenesis*□S

Received for publication, July 28, 2010, and in revised form, January 14, 2011 Published, JBC Papers in Press, January 20, 2011, DOI 10.1074/jbc.M110.169391

Xiang Hua Han‡, Yong-Ri Jin‡, Marianne Seto‡§, and Jeong Kyo Yoon‡1

From the ‡Center of Biomedical Research Excellence in Stem Cell Biology and Regenerative Medicine, Center for MolecularMedicine, Maine Medical Center Research Institute, Scarborough, Maine 04074 and §Graduate School of Biomedical Sciences,University of Maine, Orono, Maine 04469

R-spondins (RSPOs) are a recently characterized family ofsecreted proteins that activate WNT/�-catenin signaling. Inthis study, we investigated the potential roles of the RSPO pro-teins during myogenic differentiation. Overexpression of theRspo1 gene or administration of recombinant RSPO2 proteinenhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, inboth C2C12 myoblasts and primary satellite cells, whereasMYOD or PAX7 expression was not affected. RSPOs also pro-moted myogenic differentiation and induced hypertrophicmyotube formation in C2C12 cells. In addition, Rspo2 andRspo3 gene knockdown by RNA interference significantly com-promisedMYF5expression,myogenic differentiation, andmyo-tube formation. Furthermore, Myf5 expression was reduced inthe developing limbs of mouse embryos lacking the Rspo2 gene.Finally, we demonstrated that blocking of WNT/�-catenin sig-naling by DKK1 or a dominant-negative form of TCF4 reversedMYF5 expression, myogenic differentiation, and hypertrophicmyotube formation induced by RSPO2, indicating that RSPO2exerts its activity through the WNT/�-catenin signaling path-way. Our results provide strong evidence that RSPOs are keypositive regulators of skeletal myogenesis acting through theWNT/�-catenin signaling pathway.

WNT signaling plays diverse roles in normal tissue develop-ment during embryogenesis and tissue function in adulthood.The importance of WNT signaling in skeletal myogenesis wasinitially demonstrated in embryonic skeletal myogenesis (1–4).The WNT1 andWNT3A ligands derived from the dorsal neu-ral tube and the surface ectoderm positively regulate skeletalmyogenesis within somites via the canonical WNT/�-cateninsignaling pathway (3, 4). Furthermore, noncanonicalWNT sig-naling, which transmits signals through the RAC/RHO-depen-dent planar cell polarity and calcium-PKC pathways, regulatesPAX3 and subsequently MYOD expression during myogenicdifferentiation (5) and a directional elongation of myofiberswithin the myotome (6).

Recently, progress has beenmade in the understanding of theroles of WNT signaling in postnatal myogenesis. For instance,�-catenin was shown to promote self-renewal of satellite cells,themuscle stem/progenitor cells residing in adult skeletalmus-cle (7). In contrast, WNT/�-catenin signaling was shown toinitiate myogenic differentiation of satellite cells by replacingNOTCH signaling, which is critical for self-renewal of satellitecells, through the inhibition of GSK-3� (8). It was also demon-strated that the activation of the WNT/�-catenin pathwayoccurs within the CD45-positive stem cell population presentin regenerating skeletal muscles but not in satellite cells (9). Inaged skeletal muscle, WNT10B regulates the balance betweenmyogenic and adipogenic lineage determination (10). WNTsignaling was also suggested to induce a fibroblastic phenotypein aged skeletal muscle (11). Furthermore, WNT7A regulatesself-renewal of satellite cells via noncanonical WNT signaling(12). These studies suggest that both the WNT/�-cateninand noncanonical WNT signaling pathways play diverseroles in embryonic and postnatal skeletal muscle develop-ment. However, with the exception of WNT10B andWNT7A, the precise roles and signaling mechanisms of dif-ferent WNT ligands specifically relevant to postnatal myo-genesis remain largely undetermined.R-spondin (RSPO) family proteins activate the canonical

WNT signaling pathway at the receptor level, leading to�-catenin-dependent gene activation (13–16). Additional bio-chemical studies demonstrated that RSPO proteins directlybind with high affinity to the extracellular domain of the LRP62receptor and perhaps the Frizzled (FZD) receptor with a muchlower affinity (14, 16). It was previously demonstrated that thecanonical WNT ligands form a ternary complex with both theFZD and LRP5/6 receptors to activate �-catenin signaling (17,18). Unlike canonical WNT ligands, RSPO fails to form a ter-nary complex with the FZD8 and LRP6 receptors in vitro (16).Our previous studies suggested that the LRP6 receptor alonemay be sufficient to mediate canonical �-catenin activation byRSPOs (16). Thus, the RSPO family proteins are a novel class ofWNT signaling ligands that can activate the canonical WNTpathway via mechanisms distinct from those of WNT ligands.

* This work was supported, in whole or in part, by National Institutes of HealthGrants P20 RR018789 (to D. M. Wojchowski, Program Director and J. K. Y.,Project Principal Investigator) and R01 AR055278 (to J. K. Y., PrincipalInvestigator).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S4.

1 To whom correspondence should be addressed: Maine Medical CenterResearch Inst., 81 Research Dr., Scarborough, ME 04074. Fax: 207-396-8179;E-mail: yoonje@mmc.org.

2 The abbreviations used are: LRP, low density lipoprotein receptor-relatedprotein; RSPO, R-spondin; DKK1, Dickkopf-related protein 1; sFRP1,secreted Frizzled-related protein 1; TCF, T cell factor; MyHC, myosin heavychain; GSK-3�, glycogen synthase kinase-3�; qRT-PCR, quantitative realtime RT-PCR; sTopFlash; Super TopFlash; MSTN, myostatin; FZD, Frizzled;MYOG, myogenin; SHH, sonic hedgehog.

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In this study, we characterized the roles of the RSPO familyproteins in myogenic differentiation using mouse primary sat-ellite cells and C2C12 mouse myoblast cells. We identified thatRSPO positively regulated the expression of the myogenicdetermination factor MYF5 in undifferentiated and differenti-ating C2C12 cells without affecting MYOD or PAX7 expres-sion. Furthermore, RSPO2 promoted myogenic differentiationand hypertrophic myofiber formation. These RSPO2 effectswere mediated through the WNT/�-catenin pathway. Ourstudies identified the RSPO family proteins as novel regulatorsof skeletal myogenesis.

EXPERIMENTAL PROCEDURES

Cell Culture—The human embryonic kidney fibroblast cellline 293T was maintained in Dulbecco’s minimal essentialmedium (DMEM) containing 10% fetal bovine serum (FBS) and1% penicillin/streptomycin under 5% CO2 at 37 °C. The mousemyoblast cell line C2C12 was obtained from ATCC (AmericanTissue Culture Collection, Manassas, VA) and maintained ingrowth medium (DMEM containing 10% FBS and 1% penicil-lin/streptomycin) under 5% CO2 at 37 °C. To induce myogenicdifferentiation, C2C12 cells were seeded near confluence andcultured overnight. Growth medium was replaced with differ-entiation medium (DMEM containing 2% heat inactivatedhorse serum). Differentiation medium was changed every 2days. Reserve cells were prepared as described previously (19).Briefly, mononuclear reserve cells were collected by brief incu-bation with trypsin from C2C12 cells differentiated for 7 days.Contaminated myofibers were further removed by filtrationthrough a cell sieve. Primary satellite cells were prepared fromthe hind limb muscle of 12–14-week-old C57BL/6 mice asdescribed previously (8). Cells were maintained in F-10medium supplementedwith 20%FBS, 1%penicillin/streptomy-cin, and 5 ng/ml recombinant basic fibroblast growth factorprotein (Atlanta Biologicals, Lawrenceville, GA).Animals—Mice carrying the Rspo2 null (Rspo2�/�) allele

were described previously (20). A second mutant allele of theRspo2 (Rspo2�ZN) gene in which the LacZ and neo gene cas-settes were removed by Flp-dependent recombinationwas gen-erated.WNT reporter (TopGAL) mice (21) were obtained fromThe Jackson Laboratory (Bar Harbor, ME). The Rspo2 null andRspo2�ZN alleles and TopGAL transgene were genotyped bypolymerase chain reaction (PCR) as described (20) and accord-ing to protocols available fromThe Jackson Laboratory, respec-tively. Mice were housed in a pathogen-free air barrier facility,and animal handling and procedures were approved by theMaine Medical Center Institutional Animal Care and UseCommittee.Whole Mount in Situ Hybridization and �-Galactosidase

Staining—Wholemount in situhybridizationwas performed asdescribed (20). To visualize expression of the LacZ gene encod-ing �-galactosidase (�-GAL), freshly collected embryos werefixed with 0.2% glutaraldehyde for 15min at room temperatureand stained with X-Gal substrate (Invitrogen) overnight at37 °C. The stained embryos were photographed under aStemiSV6 stereomicroscope (Zeiss) using an AxioCam digitalcamera (Zeiss).

Molecular Biology and Reagents—A full-length mouse Rspo1cDNA with C-terminal HA epitope tag was excised frompcDNA3mRspo1HA DNA (16) by appropriate restrictionenzymes. cDNA encoding a dominant-negative form of humanTCF4 (�NTCF4) DNA was PCR-amplified from the CMV�NTCF4 expression vector (22). Rspo1HA and �NTCF4cDNAs were cloned into pWZL retroviral vector (23). siRNApools specific to the mouse Rspo2 (catalog number L-055303-09) and Rspo3 (catalog number L-049342-01) genes were pur-chased from Dharmacon (Lafayette, CO). 6-Bromoindirubin-3�-oxime (BIO), a specific inhibitor for GSK-3� that mimicsWNT/�-catenin signaling activation (24), was purchased fromStemgent (Cambridge, MA). Recombinant RSPO2, DKK1, andsFRP1 proteins were purchased from R&D Systems (Minneap-olis, MN) and used at the indicated concentrations.Retroviral Transduction and DNA/RNATransfection—Con-

trol WZL, WZLmRspo1HA, and WZL�NTCF4 retroviruseswere generated in the ecotropic packaging cell line BOSC23(ATCC). Conditioned medium containing virus particles wasdirectly used to transduce C2C12 cells in the presence of 8�g/ml Polybrene. After overnight incubation, medium con-taining the virus was removed and replaced with fresh growthmedium. After an additional 24 h of culture, the cells werereseeded at low density and selected in the presence of hygro-mycin (300 �g/ml) for 10 days with a medium change every 2days. DNA and siRNA transfection into C2C12 cells was per-formed using Lipofectamine 2000 (Invitrogen) and Lipo-fectamine RNA Max reagent (Invitrogen) according to themanufacturer’s protocol.RNA Isolation, Quantitative Real Time RT-PCR, and Lucif-

erase Assay—Total RNA was isolated from cultured cells andembryonic tissues using TRIzol reagent (Invitrogen). Firststrand cDNA synthesis was performed using a Superscript IIcDNA synthesis kit according to the manufacturer’s protocol(Invitrogen). Normally, 2 �g of total RNA was used for cDNAsynthesis. cDNA (equivalent to 100 ng of total RNA) was usedfor quantitative real time RT-PCR (qRT-PCR). The sequencesof the primers used in qRT-PCR are listed in Table 1. Luciferaseassayswere performedusing theDual-Luciferase assay kit (Pro-mega, Madison, WI) following the provided procedure.Antibodies, Western Blot Analysis, and Immunofluorescent

Staining—Total cell lysates were prepared in a radioimmuneprecipitation assay buffer (10 mM Tris-Cl, pH 7.2, 2 mM EDTA,150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 50 mM NaF, 1%sodium deoxycholate, 1 mM PMSF, 1� protease inhibitor mix-ture set V (EMD Chemicals, Gibbstown, NJ), 0.2 mM sodiumvanadate). Cytoplasmic lysates were prepared in a hypotoniclysis buffer (10 mM Tris, pH 4, 0.2 mM MgCl2, 1 mM PMSF, 1�protease inhibitor mixture set V, 0.2 mM sodium vanadate, 50mMNaF). Equivalent amounts of the protein samples were sep-arated by 10% SDS-PAGE and transferred to PVDF mem-branes. Themembranes were blocked with blocking buffer (5%milk solution). After washing, the membranes were incubatedwith primary antibodies followed by secondary antibodies con-jugated with horseradish peroxidase (HRP). The blots weredeveloped with SuperSignal West Dura Luminol/EnhancerSolution (Thermo Scientific/Pierce) and exposed to x-ray film(EastmanKodakCo.). Antibodies against theHAepitope (clone

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12CA5, 1:1000; Sigma), myogenin (MYOG) (F5D, 1:1000; Devel-opmental StudiesHybridomaBank,University of Iowa, IowaCity,IA), �-catenin (610153, 1:500; BD Biosciences), PAX7 (ab34360,1:250; Abcam, Cambridge, MA), MYF5 (SC-302, 1:1000; SantaCruz Biotechnology, Santa Cruz, CA),MYOD (SC-32758, 1:1000;Santa Cruz Biotechnology), MEF2 (SC-313, 1:1000; Santa CruzBiotechnology), c-MYC (SC-789, 1:1000; Santa Cruz Biotechnol-ogy), and �-tubulin (SC-9104, 1:1000; Santa Cruz Biotechnology)were used at the indicated dilutions.For immunofluorescent staining, C2C12 cells grown on 1%

gelatin-coated coverslips were fixed with 4% formaldehyde for15min, washedwith PBS, permeabilizedwith 1%Nonidet P-40,1�PBS for 5min at room temperature, and preblockedwith 5%serum in 3%BSA, 1� PBS for 30min at room temperature. Thecells were incubated with anti-MYOG (F5D, 1:100; Develop-mental Studies Hybridoma Bank) and anti-myosin heavy chain(MyHC; MF20, 1:100; Developmental Studies HybridomaBank) antibodies overnight at 4 °C and then further incubatedwith anti-mouse IgG secondary antibody conjugated with Cy3(1:800; Jackson ImmunoResearch Laboratories, West Grove,PA). The cells weremounted in the presence of 4�,6-diamidino-2-phenylindole (DAPI) for nuclear staining, and images wereacquired by fluorescence microscopy (Leica DFC340 FX).

RESULTS

Rspo Gene Expression during Myogenic Differentiation—Todetermine whether the Rspo family genes are expressed during

myogenic differentiation, we analyzed theirmRNA levels in themouse myoblast cell line C2C12 using conventional RT-PCR.In undifferentiated, exponentially growing C2C12 cells (main-tained at subconfluent density in growth medium), none of theRspo family genes were expressed at any detectable level (Fig.1A).When the cells were cultured in growthmediumuntil con-fluent and ready to begin differentiation, robust Rspo3 expres-sion was detected.Whenmyogenic differentiation was inducedin confluent cells for 1 day, both Rspo2 and Rspo3 expressionwere detected (Fig. 1A). Their expression was comparable withthat of Myog, a key myogenic basic helix-loop-helix (bHLH)transcription factor tightly associated with the onset of myo-genic differentiation. Interestingly, neither Rspo1 nor Rspo4was expressed in any samples.We further analyzedRspo2 andRspo3 expression by real time

quantitative RT-PCR in a time course (Fig. 1, B and C). Consis-tent with the conventional RT-PCR results, expression of bothgenes was undetectable in undifferentiated cells. Rspo3 wasimmediately induced during differentiation with a peak at 12 hin differentiation medium and gradually declined during laterdifferentiation. The Rspo2 gene was induced with a slightlydelayed manner as its expression peaked at 36 h during differ-entiation and decreased gradually as differentiation progressed.As expected, expression of Myog gradually increased duringdifferentiation (Fig. 1D).We also determined Rspo expression in mouse primary sat-

ellite cells. All Rspo genes but Rspo4 were expressed in satellitecells cultured in growth medium (Fig. 1E). Both Rspo1 andRspo3 expression increased during myogenic differentiation ofsatellite cells, whereas Rspo2 expression remained unchanged(Fig. 1, F–H).RSPO2 Up-regulates Basic Helix-Loop-Helix Class Myogenic

Determination Factor MYF5—To determine the potentialfunctional roles of RSPO during myogenic differentiation, weexamined the expression of several myogenic transcription fac-tors in C2C12 cells cultured in the presence of recombinantRSPO2 protein. In undifferentiated and exponentially growingC2C12 cells, expression of the bHLH class myogenic determi-nation factor MYF5 was significantly increased by RSPO2 (Fig.2A), whereas expression of another bHLH class myogenicdetermination factor, MYOD, was not affected. Expression ofPAX7, a transcription factor critical for early myogenic deter-mination (25), was not affected by RSPO2 protein (Fig. 2A).We next incubated C2C12 cells cultured in differentiation

medium with the RSPO2 protein. Similar to undifferentiatedcells, RSPO2 significantly enhanced MYF5 protein expressionin the early stage of myogenic differentiation (Fig. 2B). MYODand PAX7 expression was also not significantly changed. Addi-tionally, expression of the MEF2 family myogenic differentia-tion factors MEF2A and MEF2C was also not affected. Expres-sion of the differentiation marker MYOG was not changed forthe first 2 days of differentiation and began to be reduced at thelater stage of differentiation (Fig. 2B).All RSPO family members are indistinguishable in their

activity in activating WNT/�-catenin signaling (13, 16, 26). Todetermine whether MYF5 induction is specific to RSPO2, wegenerated C2C12 cell populations constitutively expressing themouse Rspo1 gene (HA epitope-tagged) by retroviral transduc-

TABLE 1DNA sequences of PCR primers used in this study

Rspo2 (qRT-PCR)Sense 5�-ATA GAG GCC GCT GCT TTG-3�Antisense 5�-TGC CGT GTT CTG GTT TCC-3�

Rspo2 (RT-PCR)Sense 5�-TTG CAT AGA GGC CGC TGC TTT-3�Antisense 5�-CTG GTC AGA GGA TCA GGA ATG-3�

Rspo3 (qRT-PCR)Sense 5�-TGG ATA TTA CGG AAC TCG-3�Antisense 5�-CTA ACC CTT CTG GGC AAC-3�

Rspo3 (RT-PCR)Sense 5�-GTA CAC TGT GAG GCC AGT GAA-3�Antisense 5�-ATG GCT AGA ACA CCT GTC CTG-3�

Myf5 (qRT-PCR)Sense 5�-TAT GAA GGC TCC TGT ATC CC-3�Antisense 5�-ACG TGC TCC TCA TCG TCT G-3�

MyoD (qRT-PCR)Sense 5�-CCG CCT GAG CAA AGT GAA TG-3�Antisense 5�-GCG GTC CAG GTG CGT AGA A-3�

Pax7 (qRT-PCR)Sense 5�-AGA GGA CGA CGA GGA AGG AG-3�Antisense 5�-GGG AGG TCG GGT TCT GAT T-3�

Myog (qRT-PCR)Sense 5�-CCT ACA GAC GCC CAC AAT C-3�Antisense 5�-CCC AGC CTG ACA GAC AAT C-3�

Myog (RT-PCR)Sense 5�-GAG CGC GAT CTC CGC TAC AGA GG-3�Antisense 5�-CTG GCT TGT GGC AGC CCA GG-3�

Axin2 (qRT-PCR)Sense 5�-CAG AAG CAG CGG TGC TGC GTG GCC-3�Antisense 5�-TTG GCT CTT TGT GAT CTT CTG GAG-3�

Gapdh (qRT-PCR)Sense 5�-AGA AGA CTG TGG ATG GCC CCT C-3�Antisense 5�-GAT GAC CTT GCC CAC AGC CTT-3�

Gapdh (RT-PCR)Sense 5�-GTG GCA AAG TGG AGA TTG TTG CC-3�Antisense 5�-GAT GAT GAC CCG TTT GGC TCC-3�

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tion. Expression of the exogenous RSPO1 protein was con-firmed by Western blot using anti-HA antibody in total celllysate (Fig. 2C). Consistent with the above RSPO2 results,MYF5 protein expression was significantly induced in C2C12cells constitutively expressing the exogenous Rspo1 gene(C2C12/Rspo1HA) when compared with C2C12 cells estab-lished with control viral vector (Fig. 2D). MYOD expressionremained largely unchanged in C2C12/Rspo1HA cells. Expres-sion of MYOG was also reduced at the later stage of differenti-ation in C2C12/Rspo1HA cells.C2C12 cells contains “reserve cells,” a population that

remainsmononucleated and undifferentiated duringmyogenicdifferentiation (19). This population is considered similar tosatellite cells in normal muscle tissue that are critical to main-tenance, growth, and repair of skeletal muscle. We isolated thereserve cell population from fully differentiated C2C12 cell cul-tures. RSPO2 efficiently induced MYF5 expression withoutchanging MYOD and PAX7 protein levels in these cells (sup-plemental Fig. S1). Similar to C2C12 cells, MYF5 proteinexpression was also induced by RSPO2 in primary satellite cellscultured in growth condition, whereas MYOD and PAX7expression was unaffected (Fig. 2E).To determine whether the increase inMYF5 protein level by

RSPO2 is a consequence of increasedMyf5 transcript, we ana-lyzed Myf5 mRNA expression in RSPO2-treated C2C12 andsatellite cells by qRT-PCR (Fig. 3, A and D). Myf5 mRNAexpression was considerably increased in cells treated with theRSPO2 protein in both cells. Consistent with their proteinexpression pattern, MyoD mRNA expression was not affectedby RSPO2 (Fig. 3, B and E). Although Pax7 mRNA expressionwas not changed by RSPO2 in C2C12 cells, RSPO2 mildly butsignificantly inhibited Pax7mRNA expression in satellite cells

FIGURE 1. mRNA expression of Rspo genes in C2C12 myoblast and primary satellite cells. A and E, conventional RT-PCR. mRNA expression of the Rspofamily genes was analyzed in C2C12 (A) and satellite cells (E). RNA samples were isolated from undifferentiated C2C12 cells cultured in growth medium (GM) ata subconfluent (SC) or confluent (C) density, and differentiating C2C12 cells were cultured in differentiation medium for 1 day (D1). RNA samples were isolatedfrom satellite cells cultured in growth medium. cDNAs equivalent to 50 (C2C12) and 25 ng (satellite cells) were used for PCR. PCR products were separated ina 2% agarose gel by electrophoresis and visualized by ethidium bromide staining. B–D and F–H, qRT-PCR analysis of the Rspo family genes. C2C12 (B–D) andsatellite cells (F–H) were harvested for RNA isolation at the indicated time points after changing into differentiation medium. Expression of Myog (D), Rspo1 (F),Rspo2 (B and G), and Rspo3 (C and H) was analyzed. The expression level was normalized by Gapdh expression. The relative expression level was calculatedagainst those at 12 (B–D) or 0 h (F–H). Samples were prepared in triplicate, and qRT-PCR was performed in duplicate. Error bars are presented as S.E.

FIGURE 2. RSPO enhances MYF5 protein expression in C2C12 and satellitecells. A, exponentially growing, undifferentiated C2C12 cells were treatedwith recombinant RSPO2 (200 ng/ml) protein for 24 h. Cell lysates were ana-lyzed for MYF5, MYOD, and PAX7 expression by Western blot. The �-tubulinprotein level was utilized as a loading control. B, C2C12 cells were differenti-ated up to 4 days (D1–D4) in the absence (BSA; 200 ng/ml) or presence ofRSPO2 protein (200 ng/ml). The RSPO2 protein was added daily. In addition toMYF5, MYOD, and PAX7, expression of differentiation markers MYOG andMEF2A/C was analyzed by Western blot. C, cell lysates were prepared fromundifferentiated C2C12 cells stably transduced with control WZL retrovirus(Con.WZL) or retrovirus constitutively expressing the mouse Rspo1HA gene(HA epitope-tagged) and analyzed for RSPO1HA expression by Western blot.D, cell lysates were prepared from undifferentiated (U) and differentiating (D)control and Rspo1HA C2C12 cells. Western blot analysis was performed todetect MYF5, MYOD, MYOG, and �-tubulin protein levels. E, cell lysates wereprepared from exponentially growing satellite cells cultured in the presenceor absence of the RSPO2 protein (200 ng/ml) for 24 h. MYF5, MYOD, PAX7, and�-tubulin protein expression was analyzed by Western blot.

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(Fig. 3F). Similar to protein expression, Myog mRNA expres-sion in RSPO2-treated C2C12 cells was similar to that in con-trol cells until differentiation day 3 (supplemental Fig. S2).Reduced Myog mRNA expression, however, was detected onday 4 (supplemental Fig. S2). Taken together, we conclude thatRSPO2 regulates Myf5 mRNA and protein levels withoutaffecting othermyogenic determination factors such asMYODand PAX7.Inhibition of Myf5 Expression by siRNA Specific to Rspo2 and

Rspo3 Genes in Differentiating C2C12 Cells—Myf5 is expressedin exponentially growing undifferentiated cells, and its expres-sion further increased during the early stage of myogenic dif-ferentiation of C2C12 cells. Because Rspos are not expressed atany detectable level in undifferentiated cells (Fig. 1), it is notlikely that RSPO-dependent signaling controlsMyf5 expressionin undifferentiated C2C12 cells. However, further increasedMyf5 expression upon the induction of differentiation can beregulated by RSPOs that also increase during the early stage ofdifferentiation (Fig. 1).To determine whether endogenous Rspo expression is nec-

essary forMyf5 expression duringmyogenic differentiation, weused an RNA interference strategy.We first tested the specific-ity and efficiency of each Rspo siRNA. Rspo2- and Rspo3-spe-cific siRNAs along with Rspo expression plasmids were tran-siently transfected into 293T cells. Expression of the RSPOproteins was analyzed by Western blot. Non-target controlsiRNA showed no effect on either RSPO2or RSPO3 expression.Rspo2 and Rspo3 siRNAs specifically inhibited expression oftheir matching targets over 90%, and no cross-reactivity wasdetected (Fig. 4A).We then transiently transfected C2C12 cells with Rspo

siRNAs and examined Myf5 mRNA and protein expressionduring myogenic differentiation. Because the Rspo2 and Rspo3genes are coexpressed in a nearly identical pattern during dif-ferentiation (Fig. 1, B and C), it is possible that they act in acompensatory manner. To inhibit both Rspo expression effec-tively, we cotransfected both Rspo2 and Rspo3 siRNAs into

C2C12 cells. BothRspo2 andRspo3 siRNAs effectively inhibitedthe expression of endogenous Rspo2 and Rspo3 genes by �50and 80%, respectively (Fig. 4B). Rspo gene knockdown effec-tively reducedMyf5mRNA and protein expression in differen-tiating C2C12 cells (Fig. 4, B and C), confirming that Myf5expression during myogenic differentiation of C2C12 cells isRSPO-dependent.ReducedMyf5 Expression in Developing Limbs of Rspo2 Gene

Knock-out Mice—To determine whether RSPO positively reg-ulatesMyf5 expression in vivo, we examinedMyf5 expression inmouse embryos lacking the Rspo2 gene by whole mount in situhybridization. Rspo2 gene is expressed in the mesenchymalcells in the first brachial arch and developing limb buds (27)whereMyf5-positive myogenic precursors for the jaw and limbmuscles are located.Myf5mRNAexpressionwas severely com-promised in the developing limbs of Rspo2 null embryos com-pared with wild type littermates (Fig. 4,D and E). Expression ofWNT/�-catenin reporter, TopGAL transgene, was alsoreduced in the limbs of Rspo2 null embryos (Fig. 4, F and G).Reduced expression of Myf5 and WNT signaling target Axin2in the developing limbs of Rspo2 mutant embryos was furtherconfirmed by qRT-PCR (Fig. 4, H and I). Interestingly, Myf5expression in the first branchial arch was not affected in Rspo2mutant embryos (Fig. 4H), whereas Axin2 expression was stillreduced (Fig. 4I). These results suggest that limb-specificMyf5expression is RSPO2- and WNT/�-catenin-dependent. How-ever, branchial arch-specific Myf5 expression is Rspo2-inde-pendent. No major abnormalities of limb muscle were noticedin Rspo2 mutants at the later stage (data not shown). It is pos-sible that reducedMyf5 functionmay delay skeletalmyogenesisin the limbs but is likely compensated byMyoD as seen inMyf5null mutant mice (28, 29).RSPO2 Activates WNT/�-Catenin Signaling Pathway in

Myogenic Cells—The RSPO family proteins activate WNT/�-catenin signaling in human embryonic kidney cell line 293Tand other cell lines (13, 15, 16). To determine whether RSPO2can also activate WNT/�-catenin signaling, we examined the

FIGURE 3. Induction of Myf5 mRNA expression by RSPO2. Undifferentiated (U) and differentiating (D) C2C12 (A–C) and satellite cells (D–F) were incubatedwith RSPO2 protein (200 ng/ml) or BSA (200 ng/ml) for up to 3 days (D1–D3). Total RNA samples were prepared and analyzed for Myf5 (A and D), MyoD (B andE), and Pax7 (C and F) mRNA expression by qRT-PCR. Expression was normalized by Gapdh mRNA expression. Samples were collected in triplicate, and qRT-PCRwas performed in duplicate. Error bars are presented as S.E. p values were calculated by Student’s t test.

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activation of aWNT reporter, Super TopFlash (sTopFlash) andthe level of cytoplasmic �-catenin protein in C2C12 cells stim-ulated with the RSPO2 protein. As expected, the RSPO2 pro-tein robustly activated sTopFlash reporter in a dose-dependentmanner (Fig. 5A). Consistent with this result, the cytoplasmic�-catenin protein level was significantly higher in RSPO2-treated differentiating C2C12 cells than in untreated cells (Fig.5B). In C2C12/Rspo1HA cells, we also detected a significantlyhigher level of cytoplasmic �-catenin than in control C2C12cells (supplemental Fig. S3). Robust activation of the sTopFlashreporter was observed in C2C12/Rspo1HA cells, whereas thesame reporter was not active in control cells (supplemental Fig.S3). Furthermore, RSPO2 treatment increased the �-cateninprotein level in the cytoplasm of satellite cells (data not shown).Taken together, we conclude that RSPOs effectively activateWNT/�-catenin signaling in myogenic cells.MYF5 Induction by RSPO2 Is Mediated by WNT/�-catenin

Signaling—To determine whether RSPO2 activates MYF5expression via the WNT/�-catenin pathway, we examinedMYF5 expression in C2C12 cells in which WNT/�-cateninsignaling was compromised. We used two experimentalapproaches to inhibit WNT/�-catenin signaling.

First, we cotreated C2C12 cells with RSPO2 and DKK1 orsFRP1 protein. DKK1 is an antagonist for WNT/�-catenin sig-

naling by acting on the LRP5/6 receptors (17, 30, 31). BecauseRSPO is a high affinity ligand for the LRP6 receptor, we pre-dicted thatDKK1would antagonize RSPO2-activatedWNT/�-catenin signaling. sFRP1 normally binds to WNT and compet-itively blocks WNT protein binding to the FZD receptor (32).Because RSPO2may bind to the FZD receptor weakly or poorly(14, 16) and no interaction between RSPO and sFRP1 has beenreported, we anticipated that RSPO2 signaling would not beaffected by sFRP1. As anticipated, DKK1, but not sFRP1, effec-tively antagonized RSPO2-induced activation of WNT signal-ing in the sTopFlash reporter assay (Fig. 5C). When DKK1 orsFRP1 was cotreated with RSPO2, DKK1 effectively reducedMYF5 expression induced by RSPO2, whereas sFRP1 failed todo so (Fig. 5D).Second, we examined RSPO2 activity on WNT/�-

catenin signaling and MYF5 expression in C2C12 cells(C2C12/�NTCF4) stably expressing a dominant-negativeform of human TCF4/TCF7L2 (�NTCF4-Myc) (22). BothsTopFlash activity and MYF5 expression induced by RSPO2was reversed by �NTCF4-Myc expression (Fig. 5, E and F).Consistent with these results, BIO, a chemical known toinduce WNT signaling activation through the inhibition ofGSK-3� (24), significantly enhanced both Myf5 mRNA andprotein expression (supplemental Fig. S4). We conclusively

FIGURE 4. Loss or reduction of Rspo gene expression attenuates MYF5 expression. A, 293T cells were cotransfected with siRNA specific to the Rspo2 orRspo3 gene along with Rspo2 or Rspo3 expression plasmid. RSPO protein expression was detected in Western blot by using anti-MYC antibodies. B and C,siRNA-mediated Rspo2 and Rspo3 gene knockdown reduces Myf5 expression in C2C12 cells. C2C12 cells were transiently transfected with siRNAs specific to theRspo2 and Rspo3 genes. Control (Con) non-target siRNA was also used. Transfected cells were cultured in differentiation medium for 2 days and harvested fortotal RNA and cell lysates. B, mRNA expression analysis by qRT-PCR. Gapdh expression was used for normalization. Samples were prepared in triplicate, andqRT-PCR was performed in duplicate. Error bars are presented as S.E. p values were determined by Student’s t test. C, endogenous MYF5 protein levels wereanalyzed by Western blot in C2C12 cells transiently transfected with Rspo-specific siRNAs 2 days after myogenic differentiation was induced. D–G, expressionof Myf5 RNA (D and E) and WNT/�-catenin reporter, TopGAL transgene (F and G), in wild type (WT) and Rspo2 gene knock-out (R2KO) embryos. Rspo2 nullmutants (Rspo2�/�) and wild type littermates at embryonic day 10.5 (E10.5) were used for whole mount in situ hybridization. Rspo2�ZN/�ZN and wild typelittermates at embryonic day 11 (E11) were used for �-galactosidase staining. Left side forelimbs are presented. Red arrowheads indicate reduced Myf5expression. Four to five embryos of each genotype were analyzed. H and I, qRT-PCR analysis of Myf5 and Axin2 expression in the forelimbs (FL) and the firstbranchial arches (BA1) of embryos heterozygous (Rspo2wt/�; n � 5) and homozygous (Rspo2�/�; n � 6) for the Rspo2 null allele at embryonic day 10.5.Expression was normalized by Gapdh mRNA expression. qRT-PCR was performed in duplicate for each sample. Error bars are presented as S.E. p values werecalculated by Student’s t test.

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determined that Myf5 activation by RSPO2 is mediated byWNT/�-catenin signaling.RSPO2 Promotes Myogenic Differentiation and Induces

Hypertrophic Myotube Formation—To determine how RSPO2regulates myogenic differentiation of C2C12 cells, we differen-tiated C2C12 cells in the absence or presence of the RSPO2protein for up to 4 days and immunostained cells for MYOGand MyHC expression. We determined the differentiationindex (a percentage of MYOG- or MyHC-positive nuclei intotal nuclei) and myotube fusion index (a distribution of thenucleus number in MyHC-positive cells).The myogenic differentiation index (MyHC-positive nuclei)

was increased in the presence of RSPO2 at days 3 and 4 (Fig. 6,A–C). Interestingly, a number of nuclei positive for MYOG, amarker for theonsetofdifferentiation,didnot increase in thepres-ence of the RSPO2 protein for the first 2 days of differentiation

(Fig. 6E), consistentwith theWestern blot analysis (Fig. 2B).How-ever, atdifferentiationdays3and4,MYOG-positivecellswerealsoincreased in the presence of RSPO2 (Fig. 6E). Clearly, RSPO2activity in enhancing differentiation ismore potent at days 3 and 4of differentiation. Interestingly, the overall expression level ofMYOG protein detected byWestern blot began to be reduced atthe later stage of differentiation (Fig. 2B), whereas the number ofMYOG-positive nuclei increased. It is likely that the MYOGexpression level in each cell decreased by RSPO2 treatment.In addition to promoting differentiation, RSPO2 induced the

formation of hypertrophic myotubes (Fig. 6, A, B, and D). Themyotubes in RSPO2-treated cultures showed increased sizeand a higher fusion index than those in untreated cultures atdifferentiation days 3 and 4. We conclude that RSPO2 stimu-lated myogenic differentiation and myotube hypertrophy inC2C12 cells.

FIGURE 5. Activation of WNT/�-catenin signaling and WNT/�-catenin-dependent MYF5 induction by RSPO2. A, dose-dependent activation of a WNT/�-catenin signaling reporter, sTopFlash, by the RSPO2 protein in undifferentiated C2C12 cells. sTopFlash reporter DNA construct was transiently transfected intoC2C12 cells, and the cells were stimulated with various concentrations of the RSPO2 protein as well as WNT3A-conditioned medium (CM) for 24 h. Reporterluciferase activity was measured and normalized by the activity of cotransfected control Renilla luciferase construct. B, stabilization of the cytoplasmic�-catenin protein by RSPO2 in differentiating C2C12 cells. C2C12 cells were differentiated for up to 4 days (D1–D4) in the absence (BSA; 200 ng/ml) or presenceof the Rspo2 protein (200 ng/ml). The RSPO2 protein or BSA was added daily. Cytoplasmic fractions were prepared and analyzed for �-catenin and �-tubulinprotein expression by Western blot. C, C2C12 cells were transiently transfected with sTopFlash reporter and stimulated with the RSPO2 protein (200 ng/ml) for24 h. WNT signaling antagonists, the DKK1 and sFRP1 proteins, were simultaneously added at the indicated concentrations (ng/ml). sTopFlash activities werenormalized by Renilla control luciferase activities. D, MYF5 protein expression was analyzed in C2C12 cells costimulated with the RSPO2 (200 ng/ml) and DKK1or sFRP1 proteins by Western blot. E, RSPO2-induced sTopFlash reporter activities were measured in C2C12 cells transiently transfected with a dominant-negative human TCF4 construct (�NTCF4-Myc). F, MYF5 expression in C2C12 cells stably expressing �NTCF4. Expression of �NTCF4 was determined usinganti-MYC antibody by Western blot. Error bars are presented as S.E.

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Reduced Rspo Expression Attenuates Myogenic Differentia-tion and Myotube Formation—To determine whether expres-sionof the endogenousRspo genes is essential formyogenic differ-entiation, C2C12 cells were transiently transfected with siRNAspecific to the Rspo2 and Rspo3 genes and examined for the effi-ciency ofmyogenic differentiation andmyotube formation (Fig. 6,F andG).Myogenicdifferentiation inC2C12cells transfectedwithRspo siRNAs was decreased �50% compared with C2C12 cellstransfected with control siRNA (Fig. 6F). Additionally, myotubeformation was also negatively affected as the percentage of myo-tubes containing more than five nuclei in cells transfected withRspo siRNAs was reduced to about 50% of control cells (Fig. 6G).Both results strongly suggest that expressionof endogenousRspo2and Rspo3 is required for myogenesis of C2C12 cells.RSPO2-induced Myogenic Differentiation and Hypertrophic

Myotube Formation Are WNT/�-Catenin Signaling-dependent—To determine whether myogenic differentiation and hyper-

trophicmyotube formation by RSPO2 are alsoWNT/�-cateninpathway-dependent, we examined myogenic differentiationand hypertrophic myotube formation in C2C12 cells overex-pressing �NTCF4. In the absence of RSPO2 treatment, bothcontrol C2C12/WZL andC2C12/�NTCF4 cells showed a similardegreeofmyogenicdifferentiation (Fig.7,A,B, andE).However, inC2C12/�NTCF4 cells, RSPO2 failed to enhance myogenic differ-entiation (Fig. 7E), suggesting thatRSPO2enhancesmyogenic dif-ferentiation in a �-catenin/TCF-dependent manner. When themyotube fusion indexwas analyzed, C2C12/�NTCF4 cells exhib-ited slightly reducedmyotube fusion comparedwith controlWZLC2C12 cells in the absence of RSPO2 (Fig. 7F). However, whenC2C12/�NTCF4 cells were treated with RSPO2, no enhancedmyotube fusion was detected (Fig. 7, C, D, and F). Therefore, weconclude that RSPO2 action on myogenic differentiation andhypertropic myotube formation is mediated by the WNT/�-catenin pathway.

FIGURE 6. Regulation of myogenic differentiation and hypertrophic myotube formation by RSPO2. A–E, C2C12 cells were cultured in differentiation medium inthe absence (BSA; 200 ng/ml) or presence of RSPO2 protein (200 ng/ml) up to 4 days (D1–D4). Cells were immunostained with anti-MyHC (A–D) or anti-MYOG antibody(E). DAPI staining was performed to visualize nuclei. A and B, fluorescent staining images of differentiating C2C12 cells cultured in the absence or presence of RSPO2protein. C, MyHC-positive nuclei were counted and presented as a percentage of total nuclei. D, a distribution of MyHC-positive cells based on nucleus number. Thenumber of MyHC-positive cells containing a single nucleus (Mono), two to five nuclei, and more than five nuclei was counted. The percentage of each group ispresented. E, MYOG-positive nuclei were counted and presented as a percentage of total nuclei. F and G, C2C12 cells were transiently transfected with control (Con) orRspo siRNA at differentiation day 3. MyHC-positive nuclei (F) and the cell fusion index (G) were analyzed. The experiment was performed in duplicate, and more than1500 total nuclei were counted. Error bars are presented as S.E. p values were determined by Student’s t test.

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DISCUSSION

RSPOsAreWNTSignalingActivators in SkeletalMuscleCells—Dynamic expression of the Rspo family genes during myogenicdifferentiationofC2C12 andprimary satellite cells implicate themas attractive candidates for the extracellular WNT signaling acti-vators in skeletalmyogenesis. Indeed, RSPOs,when either overex-pressed or administrated as a recombinant protein to C2C12 andsatellite cells, effectively inducedWNT/�-catenin signaling (Fig. 5andsupplementalFig. S3).3 Furthermore,DKK1, aWNTsignalingantagonist that acts on the LRP5/6 receptors, effectively inhibitedRSPO2 activity inMyf5 induction,whereas the sFRP1protein thatis structurally similar to the extracellular domain of the FZDreceptors was unable to inhibit RSPO2 signaling function (Fig. 5).Therefore, RSPO seems to activate the canonical WNT pathwaythrough the LRP5/6 receptorswithout affecting signaling throughthe FZD receptor in C2C12 cells.Canonical WNT ligands such as WNT3A can simultane-

ously activate both the canonical and noncanonical pathways inthe same cell, whereas noncanonical WNT ligands likeWNT5A can only activate the noncanonical pathway withoutaffecting the canonical pathway. Therefore, when canonicalWNT3A ligand was used to mimic the activation of canonical

WNT signaling, distinguishing its signal transmission throughthe canonical and noncanonical pathways was necessary. Wefailed to detect any activation of RAC1 and RHOA, two keymediators of noncanonical WNT signaling, by the RSPO pro-teins in C2C12 cells.4 Thus, the RSPO family proteins are aunique class of canonical WNT/�-catenin signaling activatordistinct from the WNT proteins. However, it is worth notingthat human RSPO1 synergizes with the specific FZD receptorsin �-catenin activation in human embryonic kidney (HEK)293T cells and Xenopus embryos (14). RSPO3 binds weakly tothe extracellular domain of the FZD8 receptor in vitro (16).Therefore, it remains to be further determined whether RSPOcan activate noncanonical WNT signaling through the specificFZD receptors in different cell contexts.RSPO as Positive Regulator Specific to MYF5—MYF5 and

MYOD are two key myogenic determination factors that initi-ate skeletal myogenesis during embryonic and postnatal myo-genesis in an independent but compensatory manner. Micelacking the Myf5 or MyoD gene do not develop any majordefects in embryonic myogenesis (29, 33). However, mice lack-ing both genes display severe defects in myogenic commitment(34), resulting in a massive loss of skeletal muscle during

3 X. H. Han and J. K. Yoon, unpublished data. 4 Y. R. Jin and J. K. Yoon, unpublished data.

FIGURE 7. Myogenic differentiation and hypertrophic myotube formation induced by RSPO2 are WNT/�-catenin signaling-dependent. A–D, fluores-cent images of C2C12 cells. The percentage of MyHC-positive nuclei of total nuclei (E) and a distribution of MyHC-positive cells containing a single nucleus(Mono), two to five nuclei, and more than five nuclei (F) were determined in differentiating C2C12 cells transduced with control WZL or �NTCF4 retrovirus. Theexperiment was performed in duplicate, and more than 1500 total nuclei were counted. Error bars, S.E. p values were calculated by Student’s t test.

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embryogenesis, indicating that Myf5 and MyoD have a redun-dant role in myogenic commitment. Embryonic Myf5 expres-sionwithin the epaxial domain of the somites is regulated by theWNT1 and WNT3A ligands derived from the dorsal region ofdeveloping neural tube in mice and chicken (1, 3, 4). An effi-cient activation ofMyf5 gene by WNT in the somites is syner-gized by SHH signaling (1, 4). Several TCF binding sites as wellas SHH signaling target, a GLI transcription factor binding site,were identified within the epaxial specific enhancer region ofthe mouseMyf5 gene (1). Mutagenesis analysis of these sites intransgenic mice showed that two TCF and GLI sites arerequired for robustMyf5 expression (1).The Rspo1 and Rspo3 genes are also expressed in the dorsal

neural tube inmouse embryos (27). Therefore, it is highly likelythat these RSPO proteins act as a part of an inducing signal forMyf5 gene activation in the somites. It is possible that Myf5activation by RSPOs derived from the dorsal neural tube ismediated by the TCF sites within the epaxial specific enhancerand can synergize with SHH signaling. Interestingly, no skeletalmuscle phenotypes are reported in Rspo1 or Rspo3 gene knock-out mice (35, 36). Given that both genes are coexpressed in thedorsal neural tube, they likely have a redundant function.Rspo1, Rspo3 double knock-out mice need to be examined forskeletal muscle phenotype in the future.Rspo2 expression is detected within mesenchymal cells in the

developing limbs and branchial arches (27) where myogenic pro-genitors migrating from the hypaxial somites are localized.Reduced Myf5 expression and WNT/�-catenin signaling in thedeveloping limbs ofRspo2 null embryos (Fig. 4,D–I) strongly sug-gests thatMyf5 expression in limb-specificmyogenic cells is regu-lated by RSPO2 via the WNT/�-catenin signaling pathway. Theenhancer region for the limb-specificMyf5 expression was previ-ously identified but does not overlap with the epaxial specificenhancer (37, 38). It will be interesting to investigate whether thisenhancer contains potential TCF binding sites and whetherRSPO2-mediatedMyf5 induction is regulated through this region.In Xenopus, Rspo2 also positively regulates embryonic myo-

genesis (13). Interestingly, expression of both Myf5 and MyoDis inhibited by Rspo2 knockdown inXenopus embryos, suggest-ing bothMyf5 andMyoD can be the targets for RSPO signaling(13). Taken together, RSPOs positively regulates Myf5 expres-sion and skeletal myogenesis in different vertebrates.Possible Roles of RSPOs in AdultMuscle StemCells—Satellite

cells are mononucleated cells residing in adult muscle thatserve as a stem/progenitor pool for skeletal muscle mainte-nance, growth, and repair (39). In adult mice,Myf5 expression,as detected by LacZ expression knocked into the Myf5 genelocus, is generally considered to be associated with quiescentand activated satellite cells (40). Pax7 is also expressed in qui-escent and activated satellite cells (25).MyoD, in contrast, is notexpressed in quiescent satellite cells but is induced in activatedsatellite cells that are committed to the myogenic lineage (41).Expression of both Myf5 and Pax7 declines as satellite cellsinitiate differentiation, and expression is undetectable inMyog-positive cells. In contrast, MyoD is continuously expressed indifferentiated cells (41). AdultMyf5 null mice showed severelydefective muscle regeneration, implicating aMyf5 role in satel-lite cell function duringmuscle regeneration (42, 43). A specific

activation of MYF5 in satellite cells and C2C12 myoblasts byRSPOs strongly suggests a critical role of RSPO signaling, pos-sibly through the�-catenin pathway, in regulating satellite cellsat the stage of activation and myogenic commitment duringmuscle regeneration. Interestingly, PAX7 and MYOD expres-sion is not significantly regulated by RSPOs in C2C12 and sat-ellite cells. Therefore, it is possible that RSPO signaling onlyinduces and regulates MYF5- but not PAX7-positive satellitecells or MYOD-positive myogenic cells derived from satellitecells. It remains to be determined whether RSPO signaling spe-cifically regulates myogenic commitment in satellite cells.RSPO2 in Myogenic Differentiation and Hypertrophic Myotube

Formation—In addition to Myf5 activation, the RSPO2 proteinpromoted myogenic differentiation and induced hypertrophicmyotube formation in differentiating C2C12 cells. It is currentlyunclear whether enhanced Myf5 activation is a primary cause ofthe promoted differentiation and myotube growth at the laterstageof differentiation. EndogenousMyf5 expressionduringmyo-genic differentiation of C2C12 cells is quickly down-regulated asdifferentiation is advanced (Fig. 2B). It is reported that loss ofMyf5induces precocious differentiation in primary myoblasts asincreased MYOG-positive cells were detected in Myf5 null cellculture comparedwithwild typemyoblast culture (44), suggestingthatMYF5 expression is associatedwith commitment to themyo-genic lineage andprevents prematuredifferentiation.Therefore, itseems that increased Myf5 expression may not directly result inenhancing differentiation.When antisense oligomers against the Myf5 gene were deliv-

ered into differentiating C2C12 cells, they showed a strong inhib-itory activity in myoblast fusion (45). This result suggests that, inaddition to myogenic determination, MYF5 may be involved inmyoblast fusion. However, Myf5 overexpression in C2C12 cellsdid not result in an increase of the myotube fusion index (46).Thus, it is possible that prolonged MYF5 expression induced byRSPO2 enhances hypertrophic myotube formation.Two prominent signalingmolecules, insulin-like growth fac-

tor 1 (IGF1) and myostatin (MSTN), were previously impli-cated in regulating skeletal muscle growth (47, 48). IGF1 posi-tively regulates muscle mass through the PI3K and AKTpathway, whereas MSTN acts as a negative regulator by regu-lating the SMAD pathway. In our preliminary observation, theexpression of these two genes was not significantly regulated byRSPO2 treatment in C2C12 cells.3 It is still possible that RSPO2canmodify IGF1 and/orMSTN signaling bymodulating signal-ing components. Overall, our study clearly suggests that RSPOmay play more than one positive role in skeletal myogenesis viathe WNT/�-catenin pathway.

Acknowledgments—We thank Drs. Lucy Liaw and Doug Spicer forcomments and suggestions and Norma Albrecht for proofreading themanuscript. We are grateful to Drs. Masanobu Kawai and DougSpicer for providing reagents. We also thank Nancy Chandler-Conreyin Recombinant Viral Vector Core (supported by National Institutesof Health Grant P20 RR015555 to R. Friesel, Program Director) forproducing Rspo1 and �NTCF4 retroviruses, and Bioinformatics corefor qRT-PCR analyses (supported by P20 RR018789) at Maine Med-ical Center Research Institute.

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R-spondin in Skeletal Myogenesis

MARCH 25, 2011 • VOLUME 286 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 10659

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Xiang Hua Han, Yong-Ri Jin, Marianne Seto and Jeong Kyo Yoonduring Skeletal Myogenesis

-Catenin Signaling Activator, R-spondin, Plays Positive Regulatory RolesβA WNT/

doi: 10.1074/jbc.M110.169391 originally published online January 20, 20112011, 286:10649-10659.J. Biol. Chem. 

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