Am J Physiol Cell Physiol 282: C993–C999, 2002. First published December 12, 2001; 10.1152/ajpcell.00372.2001. Myostatin is an inhibitor of myogenic differentiation RAMO´ NRI´OS, ISABEL CARNEIRO, VI´CTOR M. ARCE, AND JESU´ S DEVESA Departamento de Fisiologı´a, Facultad de Medicina, Universidad de Santiago de Compostela, San Francisco s/n, 15705 Santiago de Compostela, Spain Received 3 August 2001; accepted in final form 6 December 2001 Rı´os, Ramo´ n, Isabel Carneiro, Vı´ctor M. Arce, and the myotome compartment of developing somites and Jesu´ s Devesa. Myostatin is an inhibitor of myogenic differ- continues to be limited to the myogenic lineage at later entiation. Am J Physiol Cell Physiol 282: C993–C999, 2002. stages of development and in adult animals. Several First published December 12, 2001; 10.1152/ajpcell.00372. murine (15, 27, 33) and bovine (4–6, 17) genetic models 2001.—Myostatin (MSTN), a transforming growth factor have clearly established the role of MSTN as a negative (TGF)-␤ superfamily member, has been shown to negatively regulate muscle growth by inhibiting muscle precursor cell regulator of muscle fiber number. A reduction in mus- cle fiber number can result from either a decrease in proliferation. Here, we stably transfected C2C12 cells with mouse MSTN cDNA to investigate its possible role in myo- myoblast proliferation or a delay in myoblast differen- blast differentiation. We found that MSTN cDNA overexpres- tiation. The ability of MSTN to inhibit myoblast pro- sion reversibly inhibits the myogenic process by downregu- gression through the cell cycle has been recently dem- lating mRNA levels of the muscle regulatory factors myoD onstrated. Recombinant MSTN has been shown to and myogenin, as well as the activity of their downstream reversibly inhibit C2C12 murine myoblast proliferation target creatine kinase. Taking into consideration that MSTN by arresting cells in the G1 and G2/M stages of the cell expression during development is restricted to muscle, our cycle (29). This arrest is probably mediated by the results suggest that MSTN probably regulates myogenic dif- upregulation of the cyclin-dependent kinase (cdk) in- ferentiation by an autocrine mechanism. hibitor p21cip1. In keeping with these findings, Taylor muscle differentiation; muscle regulatory factors; transform- et al. (28) showed that MSTN inhibited proliferation, ing growth factor-␤ superfamily [3H]thymidine incorporation, and protein synthesis in C2C12 cells. Using a different approach, we have shown that transient transfection of C2C12 myoblasts DURING MAMMALIAN DEVELOPMENT, skeletal muscle cells with an expression vector encoding mouse MSTN arise from pluripotential mesenchymal precursors that cDNA not only inhibited cell proliferation but also become committed to the myogenic lineage upon ex- reduced differentiation-associated cell death (25), prob- pression of the muscle-specific basic helix-loop-helix ably by a mechanism involving also the upregulation of (bHLH) transcription factors myoD and myf5. In re- p21cip1, which has been previously shown to dramati- sponse to differentiation signals, myogenin and muscle cally decrease the apoptotic rate of differentiating myo- regulatory factor 4, also belonging to the bHLH family, blasts (31). execute the differentiation program that leads to the Although MSTN expression has been reported to expression of muscle-specific proteins and myocyte fu- correlate with differentiation in several chicken mus- sion into multinucleated myotubes (18, 23). During cles (10) and in C2C12 myoblasts (25), it is currently this process, more cells have to be constantly generated unknown whether MSTN plays any role in the regula- to keep pace with embryonic growth, so muscle growth tion of the myogenic process. Moreover, such a role has results from a balance between proliferation of precur- been demonstrated for several other members of the sor cells and their subsequent differentiation into mus- TGF-␤ superfamily, such as TGF-␤1 itself (14), activin cle fibers. This process is tightly regulated in vivo (12), and bone morphogenetic protein (BMP)-2 (7), through mechanisms that involve cell-cell and cell- which have been shown to inhibit the differentiation of matrix interactions, as well as extracellular secreted C2C12 myoblasts. An inhibitory effect on muscle devel- factors. Among the latter, several members of the opment in vivo has been suggested for BMP-4, a close transforming growth factor (TGF)-␤ superfamily of BMP-2 homolog (24). In all cases, the underlying mech- growth and differentiation factors have been shown to anism explaining the inhibition of the myogenic pro- be potent regulators of muscle growth (1). gram by TGF-␤ superfamily members involves the One of the TGF-␤ superfamily members that plays downregulation of the myogenic bHLH transcription an essential role in regulating skeletal muscle growth factors myoD and myogenin. is myostatin (MSTN) (15). During skeletal muscle de- In this report we show that the stable transfection of velopment, MSTN expression is restricted initially to MSTN cDNA in C2C12 cells efficiently inhibits the Address for reprint requests and other correspondence: J. Devesa, The costs of publication of this article were defrayed in part by the Departamento de Fisiologı´a, Facultad de Medicina, Universidad de payment of page charges. The article must therefore be hereby Santiago de Compostela, San Francisco s/n, 15705 Santiago de Com- marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 postela, Spain (E-mail: [email protected]). solely to indicate this fact. http://www.ajpcell.org 0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society C993 C994 MYOSTATIN INHIBITS C2C12 MYOGENESIS formation of multinucleated myotubes, reduces the RT-PCR analysis. The effects of MSTN cDNA overexpres- mRNA levels of myoD and myogenin, and inhibits the sion on the expression of myoD and myogenin were assayed activity of the myoD and myogenin downstream target, by RT-PCR. Cells (2 ϫ 106) were seeded in 60-mm plates. creatine kinase (CK). Therefore, we propose that After a 24-h incubation in 10% FBS-DMEM, differentiation MSTN negatively regulates muscle mass not only by was induced by shifting the medium to 1% FBS-DMEM. To test whether the effect of MSTN was reversible, we incubated decreasing the proliferation rate of myoblasts but also cells in the presence of the antibody raised against the by inhibiting its terminal differentiation. COOH-terminal region of MSTN (C-20; Santa Cruz Biotech- MATERIALS AND METHODS nology). An antibody directed against the pro-region of MSTN (N-19; Santa Cruz Biotechnology) was used as a Generation of stable clones of C2C12 cells expressing MSTN control. Cells were harvested at the indicated times, and cDNA. The cloning of murine MSTN cDNA into the pBlue- total RNA was extracted by means of the Trizol reagent script KSϩ vector (Stratagene, San Diego, CA) has been (Invitrogen). Total RNA (1 ␮g) was reverse transcribed for previously described (25). The cDNA was further subcloned 1hat37°C with 200 units of MMLV reverse transcriptase into the expression vector pcDNA 3.1 Zeo (Invitrogen, Bar- (Invitrogen), followed by 5 min at 95°C, in a 30-␮l reaction celona, Spain). The generated construct was named pcDNA- mixture containing 50 mM Tris⅐HCl (pH 8.3), 75 mM KCl, MSTN. 5.5 mM MgCl2, 0.5 mM each dNTP, 40 units of RNaseOUT The mouse myoblast C2C12 cell line was cultured in Dul- recombinant ribonuclease inhibitor (Invitrogen), and 1.7 becco’s modified Eagle’s medium (DMEM) containing 10% ␮g/␮l random primers (Invitrogen). Three microliters of the fetal bovine serum (FBS), 2 mM glutamine, 100 IU/ml peni- RT reaction were amplified by PCR with 1.25 units of Taq cillin, and 100 ␮g/ml streptomycin in a humidified 5% CO2 polymerase (Invitrogen) in fifty microliters of a reaction mix- atmosphere. All cell culture reagents were purchased from ture containing 20 mM Tris⅐HCl, pH 8.4, 50 mM KCl, 2 mM Life Technologies (Invitrogen). Cells were transfected by MgCl2, 0.2 mM each dNTP, and 0.4 ␮M each oligonucleotide means of the Lipofectamine reagent (Invitrogen) according to primer. The housekeeping gene hypoxanthine guanine phos- the manufacturer’s protocol with 1 ␮g of either the pcDNA- phoribosyl transferase (HPRT) was used as a load control. The MSTN plasmid or the control plasmid (pcDNA 3.1 Zeo alone). oligonucleotide sequences (with product length and GenBank Clones were selected in medium supplemented with 250 accession nos. for murine sequences) were as follows: HPRT ␮g/ml Zeocine (Invitrogen). MSTN overexpression was as- (139 bp; NM013556), upper 5Ј-CAGTCCCAGCGTCGTGATTA- sessed by RT-PCR (25). To verify whether MSTN was being 3Ј, lower 5Ј-AGCAAGTCTTTCAGTCCTGTC-3Ј; myoD (528 bp; processed and secreted properly, conditioned media from M84918), upper 5Ј-GATGGCATGATGGATTACAGC-3Ј, lower control and MSTN cDNA transfected clones were separated 5Ј-GACTATGTCCTTTCTTTGGGG-3Ј; myogenin (424 bp; by 15% SDS-PAGE under reducing conditions and trans- M95800), upper 5Ј-GCTCAGCTCCCTCAACCAG-3Ј, lower ferred onto a nitrocellulose membrane by electroblotting. The 5Ј-ATGTGAATGGGGAGTGGGGA-3Ј. All the oligonucleotide membrane was then blocked overnight at 4°C in Tris-buff- primer pairs were designed to amplify a region including at ered saline (TBS) buffer with 0.1% Tween 20 and 0.2% casein least one intron (assuming conservation of exon-intron junc- and then incubated for1hatroom temperature with an tions between murine myoD and myogenin) so that am- anti-MSTN antibody (1:500) raised against the COOH termi- plimers arising from genomic DNA contamination could be nus of MSTN (C-20 antibody; Santa Cruz Biotechnology, easily distinguished from those originated from genuine Heidelberg, Germany). The membrane was washed with cDNA amplification. The conditions of the PCR reactions TBS-Tween 0.1% and further incubated with protein were 28 cycles of 94°C for 1 min, 64°C for 1 min, and 72°C for A-horseradish peroxidase (HRP) conjugate (Amersham Phar- 1 min, followed by a final amplification step of 72°C for 10 macia Biotech, Freiburg, Germany) at 1:1,000 dilution for 1 h min.