Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression*

Subcloning and Expression of Myostatin in Escherichia coli

The subcloning and expression of myostatin protein inE. coli has previously been described (24). Briefly, the pET protein expression system (Novagen) was used to express and purify recombinant myostatin. Bovine myostatin cDNA (amino acids 267–375) was PCR amplified and cloned into the pET 16-B vector (Novagen) in-frame with the 10 histidine residues. An overnight BL21 E. coli culture transformed with the recombinant myostatin expression vector was diluted and grown to an OD of 0.8 (600 nm) in 1 liter of Lennox L broth (LB) medium plus ampicillin (50 mg/liter). The myostatin fusion protein was induced by 0.5 mmisopropyl-1-thio-β-d-galactopyranoside for 2 h. Harvested bacteria were resuspended in 40 ml of lysis buffer (6m guanidine hydrochloride, 20 mm Tris, pH 8.0, 5 mm 2-mercaptoethanol), sonicated, and centrifuged at 10,000 × g for 30 min. Myostatin was purified from the supernatant by Ni-agarose affinity chromatography (Qiagen), according to the manufacturer's protocol. Soluble fractions containing myostatin were pooled and dialyzed against two changes of 50 mmTris-HCl (pH 8.0) containing 500 mm NaCl and 10% glycerol for 6 h.

C₂C₁₂ and Bovine Primary Myoblast Culture and Differentiation

C₂C₁₂ myoblasts were grown prior to assay in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), buffered with 41.9 mm NaHCO₃(Sigma) and 5% gaseous CO₂. 7.22 nm Phenol red (Sigma) was used as a pH indicator. 1 × 10⁵ IU/liter penicillin (Sigma), 100 mg/liter streptomycin (Sigma), and 10% fetal bovine serum (FBS; Invitrogen) were added to media. For assaying, C₂C₁₂ myoblasts were seeded on Thermonox coverslips (Nunc) at a density of 20,000 cells/cm². Following a 16-h attachment period test media consisting of DMEM containing 2% horse serum (DMEM, 2% HS) with or without 0–10 μg/ml recombinant myostatin was added. Plates were incubated in at 37 °C, 5% CO₂ for a further 72 h.

Bovine myoblast cultures were derived and enriched fromMusculus semitendinosus of 160-day fetuses as previously described (24) resulting in cultures of at least 90% myoblast purity. Purified bovine primary myoblast cultures were grown prior to assay in minimal essential medium (MEM; Invitrogen) containing 10% FBS for 24 h before trypsinization and seeding onto coverslips. After a 16-h attachment period, media was replaced with test media consisting of MEM containing 2% HS with or without 8 μg/ml myostatin for 72 h.

Reversibility of Myostatin Inhibitory Effect on Differentiation

C₂C₁₂ myoblasts were seeded onto Nunc Thermonox coverslips at a density of 20,000 cells/cm². After a 16-h attachment period, DMEM, 2% HS media with or without 8 μg/ml myostatin was added to cultures. Cultures were incubated at 37 °C, 5% CO₂ for 72 h, after which the media was replaced with fresh media for a further 72 h. Cultures to have myostatin inhibition ”reversed“ received DMEM, 2% HS media without myostatin for a further 72 h.

Limited Trypsinization to Obtain Myotubes and Reserve Cells

The method of Kitzmann et al. (31) was used to separate myotubes from reserve cells in differentiated C₂C₁₂ cultures. C₂C₁₂myoblasts were seeded onto 10-cm Nunc culture dishes at a density of 20,000 cells/cm² and allowed to attach over a 16-h period. Differentiation media (DMEM, 2% FBS) with or without 5 μg/ml myostatin was added and cultures were maintained at 37 °C, 5% CO₂ for 96 h. Following incubation, a short trypsinization period (5 min, 0.15% trypsin) and 2× phosphate-buffered saline washes was used to preferentially detach myotubes. Reserve cells, which remained attached to the culture dish, were then detached by a 10-min incubation with 0.25% trypsin.

MHC Immunocytochemistry, Cell Staining, and Photography

Myoblasts were fixed with 70% ethanol:formaldehyde:glacial acetic acid (20:2:1) for 30 s, then rinsed three times with phosphate-buffered saline. Cells were blocked overnight at 4 °C in TBS (0.05 m Tris-HCl, pH 7.6 (Sigma); 0.15 m NaCl) containing 1% normal sheep serum. Cells were incubated with primary antibody, 1:100 dilution anti-MHC (MF20; gift of Donald Fischman, Cornell University Medical College), in TBS, 1% normal sheep serum for 1 h. Cells were washed (3 × 5 min) with TBST (TBS; 0.05% Tween 20) and incubated with secondary antibody, 1:100 dilution sheep anti-mouse IgG (AmershamBiosciences), in TBS, 1% normal sheep serum for 30 min. Cells were washed as before and incubated with tertiary antibody, 1:100 dilution of streptavidin-biotin peroxidase complex (AmershamBiosciences), in TBS, 1% normal sheep serum for 30 min. MHC immunostaining was visualized using 3,3-diaminobenzidine tetrahydrochloride (Invitrogen) enhanced with 0.0375% CoCl. MHC-immunostained cultures were lightly counterstained with Gill's hematoxylin and photographed using an Olympus BX50 microscope (Olympus Optical Co., Germany) fitted with a DAGE-MTI DC-330 color camera (DAGE-MTI Inc., Michigan City, IN).

Immunoprecipitations and Western Blot Analysis

For MyoD-V5 immunoprecipitation and quantitative MyoD, Myf5, myogenin, MHC, and p21 immunoblot analyses, mouse C₂C₁₂ myoblasts were cultured as described above in 6-well plates or 100-mm dishes according to treatment. Cells (∼3 × 10⁶) were resuspended in 200 μl of lysis buffer (50 mm Tris, pH 7.6, 250 mm NaCl, 5 mm EDTA, 0.1% Nonidet P-40, Complete protease inhibitor (Roche Molecular Biochemicals) and sonicated or passed through a syringe. The cell extracts were centrifuged to pellet cell debris. Bradford reagent (Bio-Rad) was used to estimate total protein content to ensure equal loadings. Immunoprecipitation of MyoD-V5 was performed by incubating 100 μg of total protein with 2 μg of purified mouse monoclonal anti-V5 antibody (R960-25; Invitrogen) in 100 μl of extraction buffer for 1 h at 4 °C. Protein A-agarose (Invitrogen) (50 μl of 50%, washed twice with lysis buffer) was added for 1 h at 4 °C, followed by centrifugation to pellet immunoprecipitated complexes. After centrifugation, pellets were washed five times with lysis buffer. Pellets were then resuspended in 20 μl of 4× NuPAGE sample buffer (Novex) and boiled for 5 min.

Total protein (15 μg) or immunoprecipitations (5–10 μl) were fractionated by SDS-PAGE (4–12% gradient; Novex) and transferred to nitrocellulose membrane by electroblotting. The membranes were blocked in TBST, 5% milk at 4 °C overnight, then incubated with the primary antibody for 3 h at room temperature. The following primary antibodies were used for immunoblotting: p130, 1:500 dilution of purified rabbit polyclonal anti-p130 antibody (sc317); MyoD, 1:200 dilution of purified rabbit polyclonal anti-MyoD antibody (sc304; Santa Cruz Biotechnology Inc.) or 1:150 dilution of purified mouse monoclonal anti-MyoD antibody (554130; Pharmingen); Myf5, 1:200 dilution of purified rabbit polyclonal anti-Myf5 antibody (sc302; Santa Cruz Biotechnology Inc.); myogenin, 1:200 dilution of purified rabbit polyclonal anti-myogenin antibody (sc576; Santa Cruz Biotechnology Inc.); MHC, 1:2000 dilution of purified mouse monoclonal anti-MHC antibody (MF-20; gift of Donald Fischman); p21, 1:400 dilution of purified mouse monoclonal anti-p21 antibody (SX118; Pharmingen); p-Smad 2/3, 1:150 dilution of purified rabbit polyclonal anti-p-Smad 2/3 antibody (sc11769; Pharmingen); Smad 3-FLAG, 1:500 dilution of purified mouse monoclonal anti-FLAG M2 antibody (F-3165; Sigma); α-tubulin, 1:3000 dilution of purified mouse monoclonal anti-α-tubulin antibody (DM 1A; Sigma). The membranes were washed (5 times for 5 min) with TBST and further incubated with anti-mouse IgG horseradish peroxidase conjugate, 1:2000 dilution (W402B; Promega Corp.), anti-rabbit IgG horseradish peroxidase conjugate, 1:1000 dilution (P0448; DAKO), or anti-goat IgG horseradish peroxidase conjugate, 1:5000 dilution (Jackson ImmunoResearch Laboratories, Inc.), secondary antibodies for 1 h at room temperature. The membranes were washed as above, and horseradish peroxidase activity was detected using Renaissance Western blot chemiluminescence (NEL104; Invitrogen).

Expression and Promoter-reporter Plasmids

The expression construct for the dominant-negative Smad 2 S465A,S467A mutant was a gift from Dr. Serhiy Souchelnytskyi and has been previously described (32). Dominant-negative Smad 3 was generated by a site-directed PCR mutagenesis approach to introduce mutations S423A and S425A, and cloned into the pcDNA3.1 D/V5-His TOPO vector (Invitrogen). The following PCR primers were used for this amplification, 5′-CACCATGTGGTCCATCCTGCCTTTC-3′ and 5′-AGCCACAGCGGAACAGCGG-3′. The MyoD expression construct was generated by the RT-PCR amplification of the coding region of MyoD and its subsequent cloning into the pcDNA3.1 D/V5-His TOPO vector. The following PCR primers were used for this amplification, 5′-CACCATGGAGCTTCTATC-3′ and 5′-AAGCACCTGATAAATCGC-3′. Murine C57 total RNA was used as the RT template. The Smad 3-FLAG expression construct was a gift from Dr. Rik Derynck and has been described by Choy et al. (33). The p3TP-lux reporter construct was a gift from Dr. Joan Massague and the 2.7-kb myoD promoter-luciferase reporter construct (pJH17) was a gift from Dr. John Capone. Both have been previously described (34, 35). The human p21promoter-luciferase construct was a gift from Dr. Bert Vogelstein (36). A 4.3-kb fragment, containing the 2.4-kb p21 genomic fragment containing bases −2,300 to +8 with respect to the transcription start site in pGL3 basic, was subcloned as a NotI/XbaI fragment (to include the luciferase gene) into pcDNA3 (Invitrogen), which had been modified, by NruI/BamHI digestion and religation, to remove the cytomegalovirus promoter.

Promoter-Luciferase Assays

Constructs were transfected into C₂C₁₂ cells using LipofectAMINE 2000 (Invitrogen), according to the manufacturer's protocol. For stably expressing cell lines, stable integrants were selected with Geneticin (Invitrogen) at 0.4 mg/ml for 4 weeks. Cell culture, myoblast differentiation, and myostatin treatment were performed as described above. C₂C₁₂ myoblasts were harvested for protein and the luciferase assay was done using the Luciferase kit (Promega) according to manufacturer's protocol. Luciferase activity in the lysates (10 μl) was measured on a Berthold luminometer, integrating light emission over 20 s. Luciferase activities were normalized to total protein content or β-galactosidase expression from the transfection of the SV40-β-galactosidase control vector, pCH110 (Amersham Biosciences), prior to treatment. Bradford reagent (Bio-Rad) was used, as described above, to determine protein concentration, whereas the β-galactosidase assay system (Promega) was used according to the manufacturer's protocol to determine β-galactosidase activity.

Northern Blot Analysis

RNA was isolated from cultured cells using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. Northern analysis was performed essentially as described by Sambrook et al. (37). Total RNA (12 μg) was fractionated by 0.66 m formaldehyde, 1% agarose gel electrophoresis. RNA was transferred to Hybond N⁺ membrane (AmershamBiosciences) by capillary transfer using 10× SSC. The membrane was prehybridized in Church and Gilbert hybridization buffer (0.5m Na₂HPO₄, pH 7.2, 7% SDS, 1 mm EDTA) at 60 °C for 1 h, followed by hybridization with ³²P-labeled cDNA probes in fresh Church and Gilbert hybridization buffer at 60 °C overnight. The membrane was washed at 60 °C for 15 min each with 2× SSC + 0.5% SDS, and then 1× SSC + 0.5% SDS.

The myoD, myf5, myogenin, and p21cDNAs were obtained by RT-PCR from C₂C₁₂total RNA using SuperScript II (Invitrogen), according to the manufacturer's protocol. PCR was performed with 2 μl of the RT reaction at 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 1 min for 35 cycles. This was followed by a single 72 °C extension step for 5 min. The primers used were: MyoD, 5′-GAGGATCCGATGGAGCTTCTATCG-3′ and 5′-CGGATCCTCTCAAAGCACCTGATA-3′ (974 bp); Myf5, 5′-GAGGATCCGATGGACATGACGGAC-3′ and 5′-CGGATCCTCTCATAATACGTGATA-3′ (786 bp); p21, 5′-CTGTTAGGCTGGTCTGCCTC-3′ and 5′-GTCCGATCCTGGTGATGTCC-3′ (443 bp). Probe cDNAs were radioactively labeled using [α-³²P]dCTP (Amersham Biosciences) and Rediprime II labeling kit (Amersham Biosciences), according to the manufacturer's protocol.

Myostatin Inhibits the Differentiation of Myoblasts

We recently showed that myostatin inhibits myoblast proliferation and the loss of this inhibition is suggested to account for the increased muscle fibers (hyperplasia) because of the increased number of myoblasts. Although these results alluded to the possible role of myostatin during myogenic differentiation, to date no experimental evidence is documented. Thus to determine the effect of myostatin on myoblast differentiation, proliferating C₂C₁₂ myoblasts were cultured in differentiation media (containing 2% HS) with varying concentrations of exogenous myostatin and analyzed for their ability to undergo myogenic differentiation. After 3 days of incubation in differentiation media, control C₂C₁₂ cells that were not treated with myostatin showed extensive formation of multinucleated myotubes and were positive for MHC expression (Fig.1, A, a, andB, a). By contrast, C₂C₁₂cells treated with 8 or 10 μg/ml myostatin showed neither myotube formation nor MHC expression (Fig. 1, A, e andf, and B, b). Treatment of C₂C₁₂ cells with lower concentrations of myostatin permitted some myotube formation (Fig. 1, A,b–d). These results show that myostatin inhibits C₂C₁₂ myoblast differentiation and furthermore, this inhibition is dose-dependent.

To confirm the differentiation inhibitory effect of myostatin on myoblasts, primary bovine myoblasts from 160-day-old fetuses were also cultured in differentiation media with or without myostatin for 3 days. Again nontreated control myoblasts showed extensive formation of multinucleated myotubes that were MHC positive (Fig.1 B, c). Treatment with myostatin, however, inhibited myotube formation and MHC expression (Fig.1 B, d). These results clearly show that myostatin inhibits myoblasts from carrying out the myogenic differentiation program.

Myostatin Inhibition of Myoblast Differentiation Is Reversible

The mature portions of TGF-β family members bind to their respective receptors to initiate a signal cascade and biological response. Hence, in an in vitro bioassay, once the ligand is removed, the biological response, such as the differentiation block by myostatin, should also be removed.

To address the question of whether myoblast differentiation inhibition is reversible, C₂C₁₂ myoblasts were cultured in differentiation media and treated with myostatin (8 μg/ml) for 72 h. As before, C₂C₁₂ myoblasts treated with myostatin were differentiation inhibited compared with nontreated C₂C₁₂ myoblasts (Fig.2, a and b). After 72 h, myostatin was removed and the cells were cultured for a further 72 h in differentiation media. As can be seen in Fig.2 d, the removal of myostatin after 72 h resulted in the myoblasts undergoing myogenic differentiation. By comparison, differentiation of the C₂C₁₂ myoblasts continued to be inhibited when myostatin was added throughout the duration of the experiment (Fig. 2 c). Results of this experiment show that myoblast differentiation is not irreversibly inhibited by myostatin, as myoblasts indeed retain the capacity to differentiate after myostatin is removed.

Myostatin-induced Quiescent Cells Are Different Than Reserve Cells

During myoblast differentiation, a subset of myoblasts, termed reserve cells, remain quiescent and undifferentiated but retain their capacity for proliferation and myogenic differentiation (38). Reserve cells have been characterized by their high expression of p130 and Myf5 and low expression of MyoD, compared with myotubes, which express low levels of p130 and Myf5 and high levels of MyoD (39). Because the treatment of myoblasts in differentiation media with myostatin results in seemingly quiescent nondifferentiated cells, it is possible that myostatin treatment increased the pool of reserve cells. To examine this possibility, myostatin-treated myoblasts in differentiation media were analyzed to see if they resembled reserve cells at the molecular level.

After 4 days of incubation in differentiation media (containing 2% FBS), C₂C₁₂ cells that were not treated with myostatin had undergone myogenic differentiation, fusing to form multinucleated myotubes. The differentiated myotubes were removed by a short and mild trypsinization (limited-trypsinization), leaving a population of undifferentiated reserve cells. As expected, the addition of myostatin to C₂C₁₂ cells in differentiation media resulted in a single population of nondifferentiated, seemingly quiescent cells. As previously reported (38), myotubes from the differentiated culture expressed low levels of p130 and Myf5 and high levels of MyoD, whereas reserve cells from the nontreated differentiated culture expressed high levels of p130 and Myf5 and low levels of MyoD (Fig. 3, A andB). Myogenin and MHC expression was also examined in the myotube and reserve cell populations as a marker of differentiation (Fig. 3 A). By contrast, myostatin-treated C₂C₁₂ cells in differentiation media did not express high levels of either p130 or Myf5 as seen by both Northern and Western blot analyses (Fig. 3, A and B). Interestingly, these cells also appeared to have significantly down-regulated levels of MyoD and other differentiation markers such as myogenin and MHC (Fig. 3, A and B). These results suggest that the differentiation-inhibited, seemingly quiescent myostatin-treated myoblasts are functionally different to the previously characterized reserve cells and that myostatin does not function to increase the reserve cell population.

MyoD Expression Is Inhibited by Myostatin

To further investigate the inhibition of MyoD in myoblasts treated with myostatin, C₂C₁₂ myoblasts were examined for MyoD expression after culture in differentiation media with or without myostatin treatment. After 4 days of incubation in differentiation media, control myoblasts, without myostatin treatment, showed a significant increase in MyoD expression compared with myoblasts in growth media (Fig. 4, A andB). By contrast, MyoD expression was not increased in the myostatin-treated C₂C₁₂ myoblasts cultured in differentiation media (Fig. 4, A and B).

We next examined if myostatin treatment can down-regulate MyoD expression when it is added to the culture media after MyoD expression has been induced by the switch to differentiation media, or if myostatin can only inhibit the induction of MyoD at the switch. To this end, cultured C₂C₁₂ cells were induced to differentiate in differentiation media and myostatin was added 0, 6, 12, 24, or 48 h after the switch. MyoD expression was analyzed by Northern and Western blot analyses. As can be seen in Fig.5, A–C, without myostatin treatment, MyoD is up-regulated as early as 6 h after the switch to differentiation media. This up-regulation of MyoD continued throughout the course of the experiment. When myostatin was added at the switch to differentiation media, as seen previously, MyoD expression was not induced (Fig. 5, A–C). The comparison of the level of MyoD expression at the time of myostatin treatment (after 6, 12, or 24 h) with the level of MyoD expression after treatment shows that MyoD can be down-regulated by myostatin even after it has been induced and the myogenic differentiation program has been initiated.

Because myoD is a key regulator of genes involved in the initiation and maintenance of differentiation in muscle, it is likely that myostatin, through MyoD, inhibits the muscle gene expression program. To test this we examined the expression of MRFs and muscle-specific genes, which function downstream of myoD, in response to myostatin signaling. Similar to MyoD expression, after 4 days of incubation in differentiation media, control cells showed a significant increase in Myf5 myogenin and MHC expression (Fig.4, A and B). In the myostatin-treated C₂C₁₂ myoblasts cultured in differentiation media, Myf5, myogenin, and MHC were all repressed (Fig. 4, Aand B).

Myf5, myogenin, and MHC expression was also examined in cultured C₂C₁₂ cells that were switched from growth media to differentiation media and myostatin was added 0, 6, 12, or 24 h after the switch. Western blot analyses of control cells show that Myf5, like MyoD was up-regulated as early as 6 h after the switch to differentiation media, whereas myogenin was up-regulated slightly later at 12 h. This up-regulation was maintained throughout the course of the experiment (Fig. 5, A andC). The addition of myostatin at all time points after the switch to differentiation media down-regulated both Myf5 and myogenin compared with controls suggesting that, like MyoD, Myf5 and myogenin expression can be inhibited even after induction. Western blot analysis or MHC immunostaining of control C₂C₁₂myoblasts showed that in control C₂C₁₂myoblasts, not treated with myostatin, MHC was also up-regulated with differentiation. MHC expression was detected after 24 h of culture in differentiation media and increased at 48 h (Fig. 5,A and D, a–e). By contrast, the addition of myostatin at 0, 6, 12, and 24 h after the switch to differentiation dramatically reduced MHC expression after 48 h in differentiation media (Fig. 5, A and D, f–i). Taken together, these results suggest that myostatin inhibits both the up-regulation and expression of MRFs and MHC in myoblasts cultured under differentiating conditions.

Myostatin Inhibits p21 Expression

The initiation of C₂C₁₂ differentiation is also accompanied by changes in expression of cell cycle regulatory factors, including the up-regulation of the cyclin-dependent kinase inhibitor p21. Previously we have shown that proliferating myoblasts in growth media are arrested by myostatin treatment and concomitant with this arrest is the up-regulation of p21. Because the up-regulation of p21 is part of the myogenic differentiation program and moreover is thought to be up-regulated by MyoD during differentiation we investigated p21 expression in C₂C₁₂ myoblasts cultured in differentiation media with myostatin treatment. After 4 days of incubation in differentiation media, control cells, without myostatin treatment, showed a significant increase in p21 expression consistent with myogenic differentiation (Fig. 4, A and B). C₂C₁₂ cells cultured in differentiation media with myostatin treatment, by contrast, showed no up-regulation of p21. In fact p21 expression appeared to be down-regulated by myostatin treatment (Fig. 4, A and B).

The expression of p21 was also examined in cultured C₂C₁₂ cells that were switched from growth media to differentiation media and myostatin was added 0, 6, 12, or 24 h after the switch. Western blot analyses of control cells show that like myogenin, p21 was up-regulated after ∼12 h after the switch to differentiation media. In contrast to this, myostatin treatment resulted in the down-regulation of p21 expression after 48 h of culture in differentiation media, even after p21 expression had been up-regulated (Fig. 5, A and C).

To further investigate the response of p21 to myostatin treatment, C₂C₁₂ myoblast cells were stably transfected with p21 promoter-luciferase gene constructs and analyzed for luciferase expression after culture in growth or differentiation media, with or without myostatin treatment for 48 h. As can be seen in Fig. 6 A, luciferase activity in the cells was increased by myostatin treatment when the cells were cultured in growth media. This finding supports the previously published report that myostatin up-regulates p21 in myoblasts cultured in growth media (24). In contrast to this, and in support of the above findings, luciferase activity was decreased by myostatin in differentiation media (Fig. 6 B).

Inhibition of Myogenic Differentiation by Myostatin Is Not Overcome by Forced MyoD Expression

As reported above, myostatin inhibits the myogenic differentiation program concomitant with a reduction in MyoD, Myf5, myogenin, and p21. Because MyoD appears to be a central regulator of myogenesis, the loss of its expression or activity may be responsible for the loss of myogenic differentiation. To determine whether the down-regulation of MyoD was required for the inhibition of myoblast differentiation, myostatin-treated C₂C₁₂ myoblasts were transiently transfected with a construct containing myoD under the control of a constitutive cytomegalovirus promoter and examined for the expression of differentiation markers. Western analyses showed that after 48 h of incubation in differentiation media, vector-only transfected control cells showed high levels of MyoD, myogenin, p21, and MHC expression compared with myostatin-treated cells (Fig.7). Western blot analysis also confirmed ectopic MyoD expression in the C₂C₁₂ myoblasts transfected with the MyoD expression vector. This expression was specific to myoD-transfected myoblasts and independent of myostatin treatment (Fig. 7). The expression of this exogenous MyoD did not rescue myogenin, p21, or MHC expression in the C₂C₁₂ myoblasts treated with myostatin (Fig.7). This result shows that the forced expression of MyoD is not able to rescue myostatin-inhibited myogenic differentiation.

The Down-regulation of MyoD Is Mediated through Smad 3

Myostatin has recently been shown to be capable of binding activin type II receptors, Act RIIB and Act RIIA (30). By analogy with other TGF-β members, this binding could result in the activation of Smads 2 and/or 3 (40). Moreover, TGF-β-activated Smad 3 has been shown to physically interact with and antagonize the activity of MyoD, inhibiting myogenic differentiation (41). Because myostatin inhibited myogenic differentiation in the presence of constitutive levels of MyoD, we investigated if Smad 2 and/or 3 are activated by myostatin.

Cultured C₂C₁₂ myoblasts were induced to differentiate in differentiation media for 6 h followed by treatment with or without myostatin. Myoblasts were harvested after 0 and 3 h of treatment and the levels of phosphorylated Smad 2 and 3 were detected by Western blot analysis of total lysates using phosphorylated Smad 2/3-specific antibodies. As can be seen in Fig.8 A, the amount of phosphorylated Smad 2 detected was relatively unchanged between 0 and 3 h culture in differentiation media. The amount of phosphorylated Smad 2 detected was also unchanged with myostatin treatment, suggesting that myostatin does not activate Smad 2 under differentiating conditions. Similar to Smad 2, the amount of phosphorylated Smad 3 detected was relatively unchanged between 0 and 3 h culture in differentiation media (Fig. 8 A). In contrast to this, however, the culture of myoblasts in differentiation media with myostatin for 3 h resulted in an increase in phosphorylated Smad 3 detection (Fig. 8 A). This result suggests that myostatin specifically activates Smad 3.

Because TGF-β has been shown to inhibit muscle differentiation through the repression of MRFs via Smad 3 interacting with the helix-loop-helix domain of MyoD (41), we examined the effect of myostatin on MyoD and Smad 3 interactions by co-immunoprecipitation analysis. Cultured C₂C₁₂ myoblasts were transfected with MyoD-V5 and Smad 3-FLAG expression constructs, induced to differentiate in differentiation media for 24 h followed by treatment with or without myostatin for 6 or 12 h. MyoD-V5 protein was specifically immunoprecipitated using anti-V5 antibodies and analyzed by Western blot for the co-immunoprecipitation of Smad 3-FLAG using anti-FLAG antibodies. As can be seen in Fig. 8 B, myostatin treatment increased the level of Smad 3 that co-immunoprecipitated with MyoD after 6- and 12-h time points. Western blot analysis also confirmed comparable levels of Smad 3-FLAG and MyoD-V5 in the total protein extracts used in the immunoprecipitation studies (Fig. 8 B). This result suggests that myostatin signaling increases Smad 3 binding to MyoD and is consistent with the increased activation of Smad 3 by myostatin shown in Fig.8 A.

To investigate if Smad 3 and/or 2 are involved in mediating the inhibition of myogenic differentiation by myostatin, we examined the effect of altered Smad activity on the MyoD promoter activity under differentiation conditions with or without myostatin. The transfection of dominant-negative Smad 2 or dominant-negative Smad 3 expressing constructs was used to alter Smad signaling. Dominant-negative Smad 2 has previously been described (32). To demonstrate that dominant-negative Smad 3 is effective, a well characterized p3TP-Lux promoter-reporter assay (34) was performed on myoblast cells transfected with dominant-negative Smad 3 or empty vector and treated or untreated with TGF-β1. As can be seen in Fig.8 C, i, transfection with the dominant-negative Smad 3 reduced the p3TP-Lux reporter activity in TGF-β1-treated myoblasts to near untreated levels. The dominant-negative Smad 2, dominant-negative Smad 3, or empty vector were then co-transfected with the myoD promoter-luciferase reporter construct and analyzed for luciferase expression after culture in differentiation media, with or without myostatin treatment for 24 h. As can be seen in Fig.8 C, ii, luciferase activity in the control vector-transfected C₂C₁₂ myoblasts decreased 34.3 ± 2.7% with myostatin treatment, supporting that myostatin signaling results in decreased myoD expression under differentiating conditions. The co-transfection of dominant-negative Smad 2 with the myoD promoter-luciferase gene construct resulted in a similar decrease (35.9 ± 1.9%) in luciferase activity with myostatin treatment (Fig. 8 C, ii). In contrast to the vector only and dominant-negative Smad 2, however, the co-transfection of dominant-negative Smad 3 with the MyoD promoter-luciferase gene construct resulted in a 13.7 ± 3.6% decrease in luciferase activity with myostatin treatment (Fig.8 C, ii). These results support that, like TGF-β, myostatin inhibition of myogenic differentiation is, in part, mediated by Smad 3.

Myostatin Induces “Quiescent Myoblasts” That Are Different than “Reserve Cells”

Previously we (24), and others (42), have shown that addition of myostatin to myoblasts in culture results in growth inhibition. This growth inhibition was accompanied with an up-regulation of p21 expression and concomitant down-regulation of Cdk2 expression and activity resulting in hypophosphorylated Rb and cell cycle arrest (24). Despite being reminiscent of myoblast entry into the differentiation pathway (5, 14-16, 43, 44), myostatin-treated myoblasts displayed no morphological properties of a differentiated phenotype. This has prompted us to address the role ofmyostatin in myogenic differentiation. Myoblasts in culture can be induced to differentiate by mitogen withdrawal when myoblasts are transferred from high to low serum media (21, 43, 45). However, under these conditions the differentiation of primary bovine and C₂C₁₂ myoblasts were inhibited by the presence of myostatin, as determined by morphology and MHC staining (Fig. 1,A, a–f, and B, a–d). The degree of inhibition of myoblast differentiation was dose-dependent (Fig. 1, A, a–f) and reversible (Fig. 2, A–D). The lack of either proliferation and/or differentiation in the presence of myostatin suggests that myostatin signaling results in a quiescent myoblast similar to reserve cells.

Reserve cells are a subset of myoblasts that under differentiation-inducing conditions, such as serum deprivation, withdraw from the cell cycle but do not commit to a differentiation pathway. Instead, reserve cells retain their myogenic potential for subsequent proliferation and differentiation in the appropriate media (38, 39). Reserve cells, characterized as having high expression of p130 and Myf5 and low expression of MyoD compared with myotubes, are suggested to have exited the cell cycle to a G₀ phase (39). The myostatin-induced quiescent cells did not express either p130 or Myf5; two markers that define reserve cells (Fig. 3, A andB). Taken together these results suggest that the myoblasts, inhibited for both proliferation and differentiation by myostatin, are different at the molecular level from reserve cells.

Myostatin Negatively Regulates Differentiation by Down-regulating MyoD and Other Differentiation Inducing Genes

Skeletal myogenesis is a highly ordered process of temporally separable events and coordinated gene expression. Tissue culture experiments and targeted deletion of genes in mice have yielded a great deal of information on this coordinated event of myogenesis. Initial up-regulation of MyoD in the late G₁ phase followed by myogenin expression signals the commitment of mononucleated myoblasts to differentiation (3-5). This is followed by up-regulation of the cyclin-dependent kinase inhibitors, p21 and p57, and cell cycle withdrawal, resulting in differentiation of postmitotic myoblasts into multinucleated myotubes (12, 13, 44). Several molecules that interfere with the expression of one or more differentiation genes have been shown to block differentiation. The expression of constitutively activated Rac1, for example, is suggested to repress the induction of p21 such that myoblasts fail to completely withdraw from the cell cycle and undergo myogenic differentiation (46). Similarly the down-regulation of MyoD, Myf5, or myogenin expression by the overexpression of the ATBF1-A isoform of the multiple homeodomain zinc finger transcription factor (47) or SOX15 (48) has been shown to block the myogenic differentiation of myoblasts. Furthermore, inhibition of myogenic differentiation by Delta-induced notch (49, 50) or peroxisome proliferator-activated receptor γ signaling (35) has also been shown to involve the loss of MyoD, Myf5, or myogenin expression and activity. The activity of the MyoD family of transcription factors have also been shown to be repressed by TGF-β-induced Smad 3 (41). Smad 3-mediated repression occurs by Smad 3 physically interacting with the basic helix-loop-helix domain of MyoD, interfering with the formation of an active MyoD·E protein complex (41).

In myostatin-treated C₂C₁₂ cells switched to differentiation media, a dramatic decrease in MyoD, Myf5, and myogenin levels were noted (Fig. 4, A and B), coinciding with the inhibition of myoblast differentiation. Myostatin treatment at 0 to 24 h after the switch to differentiation media also significantly reduced the levels of induced MyoD family MRFs even after the initial commitment to differentiation such as MyoD and myogenin up-regulation (Fig. 5, A–C). Furthermore, the inhibition of MRF expression could not be rescued by the ectopic expression of MyoD (Fig. 7), suggesting that the activity of MyoD is repressed by myostatin. Like TGF-β signaling, the inhibition of MyoD expression and activity by myostatin signaling appears to be mediated through Smad 3. Myostatin signaling in C₂C₁₂cells cultured in differentiation media specifically induced Smad 3 phosphorylation and increased Smad 3-MyoD interactions (Fig. 8,A and B). Moreover, the expression of a dominant-negative Smad 3 construct in C₂C₁₂cells cultured in differentiation media rescued the expression of amyoD promoter-reporter, normally inhibited by myostatin (Fig. 8 C, ii). By analogy with TGF-β, the increased interaction between Smad 3 and MyoD by myostatin treatment is likely to result in the interference of MyoD·E protein binding and thus transcriptional activation by MyoD (41).

The finding that myostatin regulates the expression of MyoD is independently supported by the observation that double muscled fetuses (Belgian Blue) have higher levels of MyoD expression throughout primary and secondary fiber formation compared with normal muscled fetuses (51). Conversely, the implantation of myostatin-coated beads within the developing somite of chicken was found to significantly reduce the expression of MyoD.2 The role of myostatin regulating myogenic differentiation is also supported by studies analyzing myostatin expression during muscle regeneration. Following muscle injury in rats, no myostatin expression is detected in regenerating regions where satellite cell proliferation is most abundant. Furthermore, no myostatin expression is detected in nascent myotubes and only low levels are detected in the maturing myotubes (53).

Previously, we have demonstrated that myostatininhibits myoblast proliferation through the up-regulation of the cyclin-dependent kinase inhibitor, p21 (24). In similar experiments, TGF-β1 has been shown to induce p21 expression via the Smad pathway involving Smads 3 and 4, which interact with Sp1 (54). TGF-β1 has also been shown to regulate the mitogen-activated protein kinase pathway causing p21 induction with no effect on Smad activity suggesting an independent pathway (55). Because myostatin is capable of binding the activin type II receptors, Act RIIB and Act RIIA (30), and can activate Smad 3, the myostatin induction of p21 under proliferating conditions may be through a similar mechanism as that shown for TGF-β1.

An integral feature of myoblast commitment to myogenic differentiation is the induction of p21. In contrast to the up-regulation of p21 by myostatin in proliferating conditions, we show here that p21 expression is down-regulated under differentiating conditions (Fig. 4, A and B). This down-regulation of p21 by myostatin was also achieved after the initial up-regulation of p21 during differentiation (Fig. 5, A andC). Furthermore, a loss of p21 promoter activity was observed in C₂C₁₂ cells with myostatin treatment (Fig. 6). Quite independent to the Smad or mitogen-activated protein kinase pathway, the induction of p21 in differentiating cells is reported to be directly mediated by MyoD (14-16). Therefore the loss of p21 induction in myostatin-induced differentiation-inhibited myoblasts is likely to be because of the down-regulation of MyoD-MRF expression.

The role of MyoD in myoblast cell cycle exit during differentiation is not just limited to up-regulating p21 expression. MyoD has been shown to bind the family of CIP/KIP Cdk inhibitors, p21, p27, and p57, which is suggested to cooperate in their homodimerization, increasing their levels and subsequent inhibition of Cdks (56). MyoD has also been proposed to facilitate terminal cell cycle arrest by binding to retinoblastoma protein enhancing its expression (57) and inhibiting inactivating phosphorylation by Cdks (52, 58). Thus, the loss of MyoD by the addition of myostatin may also result in the inability of myoblasts to properly exit the cell cycle, adding to their loss of myogenic differentiation.

We conclude that myostatin plays a major role in regulating myogenesis and add to the proposed model (24) of the role of myostatinduring myogenesis, whereby in addition to inhibiting proliferation, myostatin also inhibits myogenic differentiation (Fig.9). The observed muscular hyperplasia and hypertrophy seen in myostatin-null animals is therefore the result of deregulated (increased) myoblast proliferation and differentiation.