Departamento de Fisiología, Facultad de Medicina, Universidad de Santiago de Compostela, San Francisco s/n, 15705 Santiago de Compostela, Spain
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ABSTRACT |
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Myostatin (MSTN), a
transforming growth factor (TGF)- superfamily member, has been shown
to negatively regulate muscle growth by inhibiting muscle precursor
cell proliferation. Here, we stably transfected
C2C12 cells with mouse MSTN cDNA to investigate
its possible role in myoblast differentiation. We found that MSTN cDNA
overexpression reversibly inhibits the myogenic process by downregulating mRNA levels of the muscle regulatory factors myoD and
myogenin, as well as the activity of their downstream target creatine
kinase. Taking into consideration that MSTN expression during
development is restricted to muscle, our results suggest that MSTN
probably regulates myogenic differentiation by an autocrine mechanism.
muscle differentiation; muscle regulatory factors; transforming
growth factor- superfamily
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INTRODUCTION |
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DURING MAMMALIAN
DEVELOPMENT, skeletal muscle cells arise from pluripotential
mesenchymal precursors that become committed to the myogenic lineage
upon expression of the muscle-specific basic helix-loop-helix (bHLH)
transcription factors myoD and myf5. In response to differentiation
signals, myogenin and muscle regulatory factor 4, also belonging to the
bHLH family, execute the differentiation program that leads to the
expression of muscle-specific proteins and myocyte fusion into
multinucleated myotubes (18, 23). During this process,
more cells have to be constantly generated to keep pace with embryonic
growth, so muscle growth results from a balance between proliferation
of precursor cells and their subsequent differentiation into muscle
fibers. This process is tightly regulated in vivo through mechanisms
that involve cell-cell and cell-matrix interactions, as well as
extracellular secreted factors. Among the latter, several members of
the transforming growth factor (TGF)- superfamily of growth and
differentiation factors have been shown to be potent regulators of
muscle growth (1).
One of the TGF- superfamily members that plays an essential role in
regulating skeletal muscle growth is myostatin (MSTN) (15). During skeletal muscle development, MSTN
expression is restricted initially to the myotome compartment of
developing somites and continues to be limited to the myogenic lineage
at later stages of development and in adult animals. Several murine (15, 27, 33) and bovine (4-6, 17)
genetic models have clearly established the role of MSTN as a negative
regulator of muscle fiber number. A reduction in muscle fiber number
can result from either a decrease in myoblast proliferation or a delay
in myoblast differentiation. The ability of MSTN to inhibit myoblast progression through the cell cycle has been recently demonstrated. Recombinant MSTN has been shown to reversibly inhibit
C2C12 murine myoblast proliferation by
arresting cells in the G1 and G2/M stages of
the cell cycle (29). This arrest is probably mediated by the upregulation of the cyclin-dependent kinase (cdk) inhibitor p21cip1. In keeping with these findings, Taylor et al.
(28) showed that MSTN inhibited proliferation,
[3H]thymidine incorporation, and protein synthesis
in C2C12 cells. Using a different
approach, we have shown that transient transfection of
C2C12 myoblasts with an expression vector
encoding mouse MSTN cDNA not only inhibited cell proliferation but also
reduced differentiation-associated cell death (25),
probably by a mechanism involving also the upregulation of
p21cip1, which has been previously shown to dramatically
decrease the apoptotic rate of differentiating myoblasts
(31).
Although MSTN expression has been reported to correlate with
differentiation in several chicken muscles (10) and in
C2C12 myoblasts (25), it is
currently unknown whether MSTN plays any role in the regulation of the
myogenic process. Moreover, such a role has been demonstrated for
several other members of the TGF- superfamily, such as TGF-
1
itself (14), activin (12), and bone
morphogenetic protein (BMP)-2 (7), which have been shown
to inhibit the differentiation of C2C12
myoblasts. An inhibitory effect on muscle development in vivo has been
suggested for BMP-4, a close BMP-2 homolog (24). In all
cases, the underlying mechanism explaining the inhibition of the
myogenic program by TGF-
superfamily members involves the
downregulation of the myogenic bHLH transcription factors myoD and myogenin.
In this report we show that the stable transfection of MSTN cDNA in C2C12 cells efficiently inhibits the formation of multinucleated myotubes, reduces the mRNA levels of myoD and myogenin, and inhibits the activity of the myoD and myogenin downstream target, creatine kinase (CK). Therefore, we propose that MSTN negatively regulates muscle mass not only by decreasing the proliferation rate of myoblasts but also by inhibiting its terminal differentiation.
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MATERIALS AND METHODS |
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Generation of stable clones of C2C12 cells expressing MSTN cDNA. The cloning of murine MSTN cDNA into the pBluescript KS+ vector (Stratagene, San Diego, CA) has been previously described (25). The cDNA was further subcloned into the expression vector pcDNA 3.1 Zeo (Invitrogen, Barcelona, Spain). The generated construct was named pcDNA-MSTN.
The mouse myoblast C2C12 cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2 atmosphere. All cell culture reagents were purchased from Life Technologies (Invitrogen). Cells were transfected by means of the Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol with 1 µg of either the pcDNA-MSTN plasmid or the control plasmid (pcDNA 3.1 Zeo alone). Clones were selected in medium supplemented with 250 µg/ml Zeocine (Invitrogen). MSTN overexpression was assessed by RT-PCR (25). To verify whether MSTN was being processed and secreted properly, conditioned media from control and MSTN cDNA transfected clones were separated by 15% SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane by electroblotting. The membrane was then blocked overnight at 4°C in Tris-buffered saline (TBS) buffer with 0.1% Tween 20 and 0.2% casein and then incubated for 1 h at room temperature with an anti-MSTN antibody (1:500) raised against the COOH terminus of MSTN (C-20 antibody; Santa Cruz Biotechnology, Heidelberg, Germany). The membrane was washed with TBS-Tween 0.1% and further incubated with protein A-horseradish peroxidase (HRP) conjugate (Amersham Pharmacia Biotech, Freiburg, Germany) at 1:1,000 dilution for 1 h at room temperature. After washing, HRP activity was detected by using the enhanced chemiluminescence detection kit (ECL; Amersham Pharmacia Biotech).Cell proliferation and apoptosis assays. For the proliferation assay, 4 × 104 cells were seeded in triplicate in 35-mm diameter dishes. Cells were cultured in DMEM supplemented with 10% FBS. At 24, 48, and 72 h, cells were washed with phosphate-buffered saline buffer (PBS), harvested after a 5-min incubation with 0.25% trypsin (Invitrogen), and counted on a Neubauer chamber.
For the quantification of apoptosis, 6.5 × 104 cells were seeded in 15-mm dishes (n = 6). Cells were incubated for 24 h in DMEM containing 10% FBS and then changed to 1% FBS. After an additional period of 72 h, cells were stained with 50 nM Hoechst 33258 (Sigma, St. Louis, MO). Three random fields of each of the six replicates were photographed at a ×40 magnification with a DP10 microscope digital camera (Olympus Optical, Tokyo, Japan). Hoechst-positive condensed nuclei were counted in each of the fields. A parallel experiment was performed to assay apoptosis with the Cell Death Detection ELISA (Roche Molecular Biochemicals, Mannheim, Germany), following manufacturer's instructions. The assay is based on the quantitative determination of oligonucleosomes released into the cytoplasm of apoptotic cells with monoclonal antibodies directed against DNA and histones.RT-PCR analysis. The effects of MSTN cDNA overexpression on the expression of myoD and myogenin were assayed by RT-PCR. Cells (2 × 106) were seeded in 60-mm plates. After a 24-h incubation in 10% FBS-DMEM, differentiation was induced by shifting the medium to 1% FBS-DMEM. To test whether the effect of MSTN was reversible, we incubated cells in the presence of the antibody raised against the COOH-terminal region of MSTN (C-20; Santa Cruz Biotechnology). An antibody directed against the pro-region of MSTN (N-19; Santa Cruz Biotechnology) was used as a control. Cells were harvested at the indicated times, and total RNA was extracted by means of the Trizol reagent (Invitrogen). Total RNA (1 µg) was reverse transcribed for 1 h at 37°C with 200 units of MMLV reverse transcriptase (Invitrogen), followed by 5 min at 95°C, in a 30-µl reaction mixture containing 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 5.5 mM MgCl2, 0.5 mM each dNTP, 40 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 1.7 µg/µl random primers (Invitrogen). Three microliters of the RT reaction were amplified by PCR with 1.25 units of Taq polymerase (Invitrogen) in fifty microliters of a reaction mixture containing 20 mM Tris · HCl, pH 8.4, 50 mM KCl, 2 mM MgCl2, 0.2 mM each dNTP, and 0.4 µM each oligonucleotide primer. The housekeeping gene hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as a load control. The oligonucleotide sequences (with product length and GenBank accession nos. for murine sequences) were as follows: HPRT (139 bp; NM013556), upper 5'-CAGTCCCAGCGTCGTGATTA-3', lower 5'-AGCAAGTCTTTCAGTCCTGTC-3'; myoD (528 bp; M84918), upper 5'-GATGGCATGATGGATTACAGC-3', lower 5'-GACTATGTCCTTTCTTTGGGG-3'; myogenin (424 bp; M95800), upper 5'-GCTCAGCTCCCTCAACCAG-3', lower 5'-ATGTGAATGGGGAGTGGGGA-3'. All the oligonucleotide primer pairs were designed to amplify a region including at least one intron (assuming conservation of exon-intron junctions between murine myoD and myogenin) so that amplimers arising from genomic DNA contamination could be easily distinguished from those originated from genuine cDNA amplification. The conditions of the PCR reactions were 28 cycles of 94°C for 1 min, 64°C for 1 min, and 72°C for 1 min, followed by a final amplification step of 72°C for 10 min. The PCR products were resolved on 2% agarose gels stained with ethidium bromide.
CK activity.
Cells (3.5 × 105) were seeded in triplicate in 35-mm
plates, incubated for 24 h in 10% FBS-DMEM, and then shifted to
medium containing 1% FBS to induce its differentiation. The cells were harvested by trypsinization at the indicated times, washed with PBS,
centrifuged, and stored as a pellet at 20°C until the assay was
performed. Briefly, the cells were sonicated in saline buffer, and CK
activity was measured by a modification of the spectrophotometric method described by Olivier (22) and Rosalki
(26) in a Dimension Clinical Chemistry System (Dade
Behring, Newark, NJ). The amount of total protein in the samples was
determined with the pyrogallol red/molybdenum method, described by
Fujita et al. (2).
Statistical analysis. A Mann-Whitney test was used to evaluate the statistical significance of the data.
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RESULTS |
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Transfected C2C12 cells overexpress a
biologically active MSTN.
To investigate the autocrine effects of MSTN on the myogenic process in
vitro, we generated stable clones of C2C12
cells overexpressing murine MSTN cDNA. Stable transfectants were tested
for MSTN overexpression by RT-PCR analysis of total RNA. As shown in
Fig. 1A, the amount of MSTN
mRNA is considerably higher in cells transfected with the pcDNA-MSTN
plasmid than in control cells. According to our previously published
results (25), only a faint band can be detected in cells
transfected with the pcDNA 3.1 Zeo control plasmid. Moreover, Western
blot analysis of the conditioned medium of transfected cells showed a
band migrating at the predicted size (12 kDa) for the monomeric
processed form of MSTN (Fig. 1B), thus demonstrating that
C2C12 cells are capable of secreting and
proteolytically processing MSTN. Finally, to further validate our model
we determined whether the MSTN overexpressed in
C2C12 cells was biologically active by
investigating its effects on cell proliferation and cell survival.
Whereas proliferation was inhibited (Fig.
1C), survival was enhanced
(Fig. 1, D and E) in MSTN-transfected
C2C12 cells. Because these effects have been
previously attributed to MSTN (25, 28, 29), it is likely
that they are produced by the overexpressed peptide. Furthermore, the
effect of MSTN on the proliferation rate of the myoblasts is also
consistent with the fact that double-muscled cattle, where the
phenotype for disruption of the mstn gene was first
observed, present increased muscle fiber number.
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Overexpression of MSTN in C2C12 cells
inhibits myogenic differentiation.
The ability of MSTN to inhibit myoblast differentiation is shown in
Fig. 2. Incubation of C2C12 cells transfected
with the control plasmid for 5 days in differentiation medium resulted in their fusion into postmitotic syncytial myotubes. In contrast, when
C2C12 cells transfected with the pcDNA-MSTN
plasmid were incubated under the same conditions, the formation of
multinucleated myotubes was suppressed.
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DISCUSSION |
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To our knowledge, this is the first report in the literature demonstrating the inhibitory effect of MSTN on myogenesis. In the same direction, Oldham et al. (21) recently reported that myoD expression is increased in double-muscled cattle fetuses. This increase is probably caused by the lack of an active biological form of MSTN in these animals, thus suggesting a role for MSTN in the negative regulation of the differentiation process in vivo. Surprisingly, no upregulation of myogenin was found in this case. The reason for this discrepancy is unknown.
Two different ways of inhibiting the myogenic process have been
described. One is the use of mitogens, which tend to delay myogenic
differentiation by retaining the cells in a proliferative state
(19). On the other hand, the factors belonging to the TGF- superfamily, along with MSTN, inhibit both proliferation and
differentiation (14). However, some striking differences exist among members of the superfamily. Although both TGF-
and activin, on one side, and BMP-2 and related factors, on the other, inhibit myotube formation in C2C12 myoblasts,
the latter can also induce this cell line to express osteoblast
phenotypes, such as alkaline phosphatase (ALP) activity
(7). According to our own unpublished observations, MSTN
is unable to induce ALP activity in C2C12
cells, not a surprising finding considering the phenotype of
mstn-null mice, which show no defects in osteogenesis
(15). Differences also exist between TGF-
/activin and
BMP with regard to the regulation of adipogenic differentiation. MSTN
has also been reported to be expressed in adipose tissue
(15) and, along with TGF-
1 and TGF-
2, strongly
inhibited adipogenesis (9, 30). In contrast, both BMP-2
and BMP-4 have been shown to induce adipogenic conversion in the
pluripotential mesenchymal cell line 10T1/2 at lower concentrations
than those needed for transdifferentiation into osteoblasts
(30). Again, these results indicate that the effect of
MSTN is similar to that of TGF-
/activin and different from that of BMPs.
The major difference in signaling between TGF-/activin and BMP
occurs at the level of the receptor-regulated Smad (R-Smad) that is
activated by type I receptors before forming a complex with the common
Smad4 and translocating to the nucleus, where they regulate the
transcription of target genes. R-Smads 2 and 3 transduce TGF-
and
activin signals, whereas BMP signaling uses R-Smads 1, 5, and 8 (reviewed in Ref. 13). This differential activation of
R-Smads seems to mediate the differential effects of both pathways on
myogenic differentiation, since forced expression of R-Smads 1, 5, and
8 or R-Smads 2 and 3 mimics the effects of BMPs and TGF-
/activin,
respectively, on C2C12 cells (8,
32). Although MSTN signaling pathway has not been elucidated at
the biochemical level, several lines of evidence, apart from their common effects on myogenesis and adipogenesis, suggest that MSTN may
share TGF-
/activin signaling pathway. First, MSTN clusters with
TGF-
s and activins in a phylogenetic tree of all known TGF-
superfamily members (20). Second, it has been recently
demonstrated that MSTN is able to bind activin receptor type IIb
(ActRIIb) and that transgenic mice overexpressing a dominant negative
form of ActRIIb under the control of a muscle-specific promoter exhibit a dramatic increase in muscle mass (11), similar to that
of mice lacking MSTN (15). In keeping with this
hypothesis, a high degree of similarity also exists between the
phenotypes of mice either lacking ActRIIb or growth and differentiation
factor (GDF)-11, thus suggesting that GDF-11 and activin may as well
share a common receptor and signaling pathway (16). GDF-11
has a 90% identity with MSTN in its COOH-terminal region, and it has
also been shown to inhibit myogenesis in the developing chick limb by a
mechanism involving downregulation of myoD expression (3).
A major difference exists between MSTN and the rest of the TGF-
superfamily factors with regard to its restricted expression pattern
during development. Whereas MSTN expression is confined to developing
skeletal muscles, other members of the TGF-
superfamily that
negatively regulate myogenesis are expressed in neighboring tissues.
This type of communication, characteristic of BMPs and GDF-11, seems to
be involved in the establishment of boundaries between adjacent
territories. For instance, GDF-11, which inhibits avian limb muscle
differentiation, is expressed in nonmuscle progenitor mesenchymal cells
from the progress zone, which will later differentiate into skeletal
structures (3). Similarly, BMP-4 signals emanate from the
dorsal ectoderm, the neural tube, and the lateral plate mesoderm, and
its negative effect on myoD expression is counterbalanced in the
dorsomedial lip of the dermomyotome (where muscle progenitors first
initiate the expression of myogenic bHLH transcription factors) by
noggin (24). Considering the differences stated above,
together with our findings, it is likely that MSTN regulates myogenic
differentiation via an autocrine/paracrine mechanism. We therefore
propose a model, depicted in Fig. 5, in
which MSTN expressed in the course of myogenic differentiation
regulates the deposit of muscle mass during development by inhibiting
both the proliferation of myoblasts and their differentiation into
multinucleated myotubes.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. José Carlos Tutor and Dr. José Antonio Casal for valuable help in the creatine kinase activity experiments.
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FOOTNOTES |
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This work was supported by grants from Xunta de Galicia (XUGA-1999) and Fondo de Investigaciones Sanitarias [FIS/1180, Ministerio de Educación y Cultura, Spain]. R. Ríos is a recipient of a Formación de Profesorado Universitario fellowship from the Ministerio de Educación y Cultura, Spain. I. Carneiro is a recipient of a Beca de Formación en Investigación fellowship from the Ministerio de Sanidad y Consumo, Spain.
Address for reprint requests and other correspondence: J. Devesa, Departamento de Fisiología, Facultad de Medicina, Universidad de Santiago de Compostela, San Francisco s/n, 15705 Santiago de Compostela, Spain (E-mail: fsrrios{at}usc.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 12, 2001;10.1152/ajpcell.00372.2001
Received 3 August 2001; accepted in final form 6 December 2001.
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