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Endocrinology, doi:10.1210/en.2003-1166
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Endocrinology Vol. 145, No. 6 2795-2803
Copyright © 2004 by The Endocrine Society

Differential Response to Exogenous and Endogenous Myostatin in Myoblasts Suggests that Myostatin Acts as an Autocrine Factor in Vivo

Ramón Ríos1, Susana Fernández-Nocelos, Isabel Carneiro, Víctor M. Arce and Jesús Devesa

Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, A Coruña 15782, Spain

Address all correspondence and requests for reprints to: Víctor M. Arce, M.D., Ph.D., Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, San Francisco 1, 15782 Santiago de Compostela, Spain. E-mail: fsvarce{at}usc.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Myostatin is a member of the TGF-ß superfamily that is essential for proper regulation of skeletal muscle growth. As do other TGF-ß superfamily members, myostatin signals into the cell via a receptor complex that consists of two distinct transmembrane proteins, known as the type I and type II receptors. Vertebrates have seven distinct type I receptors, each of which can mix and match with one of five type I receptors to mediate signals for all the TGF-ß family ligands. Accumulating evidence indicates that myostatin shares its pair of receptors with activin, and therefore, the question arises about how specificity in signaling is achieved. Our hypothesis is that a mechanism has to exist to restrict myostatin actions to the muscle cells. To investigate this possibility, we compared the effect of endogenous myostatin (myostatin overexpressed by myoblasts) and exogenous myostatin (recombinant myostatin added to the culture medium) in cultured myoblasts. As opposed to exogenous myostatin, endogenous myostatin induced the transcription of a reporter vector in cultured myoblasts. Notably, the myostatin concentrations that failed to induce a response in myoblasts were effective in MCF-7 cells (human mammary carcinoma) and in HepG2 cells (human hepatic carcinoma). Based on our observations, we propose that a mechanism exists that differentially regulates the bioavailability of endogenous and exogenous myostatin to muscle cells. This is consistent with a model in which myostatin actions are exerted in vivo in an autocrine fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TGF-ß SUPERFAMILY of growth and differentiation factors (GDFs) comprises over 35 members that play an essential role in the regulation of embryonic development and tissue homeostasis in adults. Myostatin, formerly known as GDF-8, is a member of the TGF-ß superfamily that is important for proper regulation of skeletal muscle growth (1). Homozygous disruption of the myostatin (Mstn) gene in mice produces a dramatic and widespread increase in skeletal muscle mass, resulting from a combination of muscle cell hyperplasia and hypertrophy (1, 2, 3). Similarly, naturally occurring mutations of the Mstn gene produce the compact phenotype in mice (4) and the double-muscled phenotype in cattle (5, 6), indicating that myostatin function has been remarkably well conserved through evolution.

Expression of Mstn gene is restricted almost exclusively to cells of the skeletal muscle lineage (1). Initially, Mstn gene expression is detected in the myotome compartment of developing somites, and the expression is continued in adult axial and paraxial muscles (1, 7). Although to a lower extent, myostatin expression has been also reported in adipose tissue (1), mammary gland (8), cardiomyocytes and Purkinje fibers of the heart (9), and hemopoietic cells (10). As occurs with other members of the TGF-ß superfamily, myostatin is synthesized as a 375-amino acid precursor protein that is proteolytically processed at an internal dibasic site. Proteolysis of myostatin precursor gives rise to an N-terminal propeptide, and a 12.5-kDa C-terminal fragment that is the mature myostatin ligand (1, 11, 12, 13, 14). After cleavage, dimers of the mature myostatin and the myostatin propeptide remain tightly associated through noncovalent bonds. This latent complex avoids the binding of the mature myostatin to its receptor and thus prevents myostatin signaling (13, 14).

Myostatin regulates muscle mass by acting directly on muscle cells. Myostatin exerts several effects on muscle cells, including induction of cell arrest (11, 12, 15) (also, see Ref. 18), inhibition of differentiation of myoblasts into myotubes (16, 17, 18), and regulation of cell survival (15, 18). Although considerable interest has focused on the identification of a bona fide myostatin receptor, accumulating evidence suggests that myostatin actions are exerted after binding to activin (Act) receptor (ActR)IIB (13). By analogy with Act, activation of ActRIIB would lead to the phosphorylation of one of the type I receptors for Act, designated ActR-like kinases (ALKs). Once activated, the type I receptors will serve as docking sites for the intracellular mediators of myostatin signaling.

Given that myostatin shares a receptor pair with other members of the TGF-ß superfamily, questions arise about how specificity in signaling is accomplished. To gain insight into the mechanism(s) that regulates specificity in myostatin signaling, we compared, in the present work, the effects of endogenous and exogenous myostatin in cultured myoblasts. For the purpose of this study, we use the term endogenous myostatin for myostatin overexpressed by transfected myoblasts, and exogenous myostatin for recombinant myostatin added to myoblasts in culture. In this model, exogenous myostatin is equivalent to paracrine/endocrine myostatin in vivo, whereas endogenous myostatin is equivalent to autocrine-derived myostatin in vivo. We report here that endogenous myostatin induces the phosphorylation of Smad2 and stimulates the transcription of a reporter vector in cultured myoblasts. These results contrast with the absence of an effect when cultured myoblasts are treated with exogenous myostatin. In all, our findings are consistent with a model in which muscle cells respond to autocrine but not endocrine/paracrine myostatin in vivo. Therefore, we propose that myostatin regulates muscle mass in vivo by acting as an autocrine factor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of recombinant myostatin
Recombinant myostatin was produced in Sf21 cells (BD Biosciences, Madrid, Spain), derived from the Spodoptera frugiperda line S9. Cells were grown at 27 C, in Falcon culture dishes containing semisynthetic medium (SF900-II, Invitrogen S.A., Barcelona, Spain) supplemented with 10% fetal bovine serum (Invitrogen), 100 IU/ml penicillin, 100 mg/ml streptomycin, and 25 µg/ml amphotericin B (Sigma Química, Madrid, Spain).

The cloning of the murine myostatin cDNA into the pBluescript KS+ vector (Stratagene, La Jolla, CA) has been previously described (15). The myostatin cDNA was further subcloned into a pBacPak9 vector (BD Biosciences, Madrid, Spain) through the BamHI/EcoRI sites, and this vector was used to transfer the full-length encoding sequence of myostatin to the genome of the baculovirus Autographa californica (AcMNPV, BD Biosciences) by homologous recombination, as described in the manufacturer’s protocol. Briefly, 500 ng of the transference vector were incubated for 1 h at 27 C with 5 µl AcMNPV DNA, and 4 µl of the Bacfectin reagent (BD Biosciences), in a total vol of 100 µl. Sf21 cells were seeded in 35-mm dishes (106 cells/dish) and incubated with the transfection mixture for 5 h at 27 C. After this time, 1.5 ml complete medium was added to each dish, and the cells were cultured for 72 h. Finally, the conditioned medium was collected, precleared by centrifugation, and stored at –80 C. The pCMV-mFur plasmid, containing the full-length encoding sequence of the murine proprotein convertase furine, was provided by Dr. K. Nakayama (University of Tsukuba, Ibaraki, Japan). The insert was subcloned into the pBacPack9 vector and used to coinfect Sf21 cells.

The presence of recombinant myostatin in the conditioned medium of infected cells was confirmed by Western blotting using a polyclonal antimyostatin antibody (dilution 1:1000) provided by Dr. N. González-Cadavid (Charles R. Drew University of Medicine and Science, Los Angeles, CA). Proteins collected from conditioned medium of infected cells were measured using the Bradford reagent (Bio-Rad Laboratories, Hercules, CA), and fractionated by SDS-PAGE under reducing conditions, using 15% separating gels. For Western blot analysis, proteins were electrotransfered onto nitrocellulose membranes (Potran, Schleicher & Schuell, Dassel, Germany) and blocked overnight in Tris saline buffer containing 0.1% Tween 20 (Sigma Química) and 0.2% casein (Sigma Química). Incubation with the primary antibody was carried out at room temperature for 1 h. Detection of the primary antimyostatin antibody was achieved with protein-A coupled to horseradish peroxidase (Amersham Pharmacia Biotech, Barcelona, Spain; dilution 1:1000). Chemiluminescence was detected with an enhanced chemiluminescent substrate for horseradish peroxidase (ECL, Amersham Pharmacia Biotech) and photographed (Hyperfilm, Amersham Pharmacia Biotech). To detect the presence of glycosylated species of myostatin, the conditioned medium was incubated with the lectin concanavaline A coupled with Sepharose, as previously described (19), and centrifuged. The presence of myostatin immunoreactivity in both the supernatant and the precipitate was detected by Western blot as described above.

Mammalian cell cultures and treatments
The following cell lines were used: HepG2 (human hepatic carcinoma), MCF-7 (human mammary carcinoma), C2C12 (mouse myoblasts), and L6C10 (rat myoblasts). All cell lines were grown in Falcon culture dishes containing DMEM (Invitrogen) supplemented with 10% FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. All cultures were maintained at 37 C in a humidified atmosphere with 95% CO2. For recombinant myostatin treatments, 100 µl heat-activated conditioned medium from Sf21 cells or purified recombinant mouse myostatin (R&D Systems, Minneapolis, MN) were employed as indicated. Human recombinant TGF-ß1 and human recombinant Act A were also purchased from R&D Systems and used at 1 ng/ml and 20 ng/ml, respectively.

Smad2 phosphorylation was determined by Western blotting, using an antiphospho-Smad2 antibody (Santa Cruz Biotechnology, Heidelberg, Germany; dilution 1:1,000). Total cell extracts were prepared using ice-cold lysis buffer (10 mM Tris-HCl, pH 7.6; 5 mM EDTA; 150 mM glycerol; 10% Triton X-100; 30 mM Na4P2O7; 50 mM NaF; 1 M Na3VO4; 5 U/liter aprotinin; and 10 µM leupeptin). Cells were collected in 1 ml lysis buffer and centrifuged (15,000 x g, 30 min, 4 C) to eliminate cellular debris, and protein concentrations were determined with the Bradford reagent. Protein extracts were fractionated by SDS-PAGE, using 7.5% separating gels, and Western blot was performed as described above. For loading control, membranes were stripped and incubated with an anti-Smad2 antibody (Cell Signaling, Beverly, MA; dilution 1:1,000).

Expression of ActRIIA and ActRIIB, ALK4, and follistatin (FS) genes was analyzed by RT-PCR. The housekeeping gene hipoxanthine guanine phosphoribosyl transferase (HPRT) was used as a load control. The sequences of the oligonucleotide primers used for the respective amplifications, as well as the predicted size of the amplimers, are shown in Table 1Go. Because the cell lines investigated are originated from three different species (human, mouse, and rat), the oligonucleotide primers were designed to hybridize with DNA regions conserved among orthologues, using the CLUSTAL W program (20). Because four isoforms of ActRIIB have been reported (21), originated by alternative splicing of the RNA, the predicted size of the amplimer is not indicated in this case, because it may vary depending on the isoform expressed in each cell type. Total RNA was extracted with the TRIZOL method (Invitrogen) and reverse-transcribed as described elsewhere (15). One tenth of the reverse reaction product (3 µl) was used for amplification with 0.4 µM of the respective sense and antisense primers, 1.25 U Taq DNA polymerase (Invitrogen), and 0.2 mM of each deoxynucleotide triphosphate in 10 mM Tris-HCl (pH 8.4), 20 mM KCl, and 2 mM MgSO4. PCR was performed through 25 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min, followed by a final amplification step of 10 min at 72 C. After verifying their identity by sequencing, amplimers were resolved in a 2% agarose gel, stained with ethidium bromide, and visualized in a GelDoc 1000 (Bio-Rad Laboratories).


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  		TABLE 1. Sequences of the different primers used for RT-PCR analysis of the expression of ActRIIA, ActRIIB, ALK4, and FS genes, and predicted size of the amplimers

 
Luciferase reporter assays
The signaling pathways activated by myostatin were investigated using the p3TP-luc reporter vector (provided by Dr. J. Massagué, Memorial Sloan-Kettering Cancer Center, New York, NY). This construct contains response elements derived from the promoter of the TGFß-responsive gene pai-1. Plasmids encoding the murine ActRIIB2 (pCMV5-ActRIIB2), and ALK4 (pCMV5-ALK4), were also provided by Dr. J. Massagué. The plasmid encoding murine myostatin (pcDNA-MSTNWT) was generated in our laboratory by subcloning the murine myostatin cDNA sequence into the mammalian expression vector pcDNA3.1-Zeo (Invitrogen), using the BamHI/EcoRI sites (15).

For transient transfections, cells were seeded in 24-well plates at a density of 5 x 104 cells/well. Fugene 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) was used as described in the manufacturer’s protocol. Each well received 1000 ng DNA, including 300 ng pCMV-ßGal, used as an internal control for transfection efficiency. Cells were incubated for 6 h with the DNA/lipid complexes in serum-free medium. The medium was then replaced by DMEM supplemented with 10% FBS in the presence of myostatin, Act A, or TGFß1. Twenty-four hours after transfection, total cell extracts were prepared using 1x reporter lysis buffer (Roche Molecular Biochemicals) and precleared by centrifugation. Luciferase activity was measured in a luminometer (Nichols Institute, San Clemente, CA) for 10 sec after automatic addition of luciferase assay buffer (Roche Molecular Biochemicals) and D-luciferine solution (Roche Molecular Biochemicals). ß-galactosidase activity was measured spectrophotometrically, using 2-nitrophenyl-ßD-galactopyranoside as substrate. Results are shown as relative values based on normalized luciferase activity (i.e. luciferase activity/ß-galactosidase activity) and expressed as the mean ± SEM of at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of recombinant myostatin in Sf21 cells
The recombinant full-length mouse myostatin protein was produced in Sf21 insect cells, a system that allows for faithful processing of recombinant animal proteins. Immunoblotting of the conditioned medium with antibodies raised against myostatin revealed the presence of a band with an apparent molecular weight of about 60 (Fig. 1AGo). This species likely represents the unprocessed myostatin, although its apparent molecular weight is higher than that predicted by the amino acid sequence. This discrepancy may be due to the presence of carbohydrate moieties linked to the N-glycosylation consensus sequence (Asn-Ile-Ser) present in the N-terminal region of the myostatin molecule. In keeping with this hypothesis, almost all the myostatin present in the conditioned medium was precipitated after incubation with concanavalin A-Sepharose (Fig. 1BGo).



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  		FIG. 1. Sf21 insect cells produce active mouse recombinant myostatin. A, Western blot (WB) of conditioned medium from infected Sf21 cells fractionated on 15% SDS-PAGE under reducing conditions and probed with a polyclonal antimyostatin antibody. Sf21 cells were infected with a viral vector carrying the full-length cDNA of wild-type myostatin or with an empty vector (control). Myostatin expression was detected as a 60-kDa band, although a faint 12.5-kDa band can also be observed. This 12.5-kDa species likely represents the proteolytic processing of myostatin by resident proteases. B, Binding of recombinant myostatin to the lectin concanavaline A. The conditioned medium from Sf21 cells was precipitated with concanavaline A-Sepharose, fractionated as indicated above, and probed with the antimyostatin antibody. Almost all the myostatin immunoreactivity present in the conditioned medium was precipitated with this procedure, indicating that myostatin is glycosylated in Sf1 cells. CM, Conditioned medium; P, concanavaline A-Sepharose-precipitated medium; SN, supernatant of concanavaline A-Sepharose precipitation. C, Proteolytic processing of myostatin by furine. Sf1 cells were coinfected with a viral vector carrying the full-length cDNA of myostatin, and with a viral vector containing the sequence of the murine proprotein convertase furine. Conditioned medium from coinfected Sf21 cells was fractionated on 15% SDS-PAGE under reducing conditions, and probed with a polyclonal anti myostatin antibody. The arrow indicates the position of the 12.5-kDa N-terminal mature myostatin originated by proteolytic processing. D, Activation of the 12.5-kDa N-terminal mature myostatin. MCF-7 cells were transiently transfected with the reporter vector p3TP-luc, and treated with 100 µl conditioned medium containing the 12.5-kDa N-terminal mature myostatin before (CM) or after activation by physicochemical methods (heating at 80 C or transient acidification with HCl). Control cells (open bars) were treated with the conditioned medium obtained from Sf21 cells infected with an empty vector. Results shown are relative values based on normalized luciferase activity (i.e. luciferase activity/ß-galactosidase activity) and expressed as the mean ± SEM of at least three independent transfection experiments.

 
As occurs with other members of the TGF-ß superfamily, myostatin is produced from a precursor protein by proteolytic processing. Proteolysis of myostatin results in the generation of a 36-kDa N-terminal propeptide, and a 12.5-kDa C-terminal fragment that is the mature form of myostatin (13). Because only minute amounts of myostatin were processed into its mature form by resident proteases in our system (see Fig. 1AGo), we coinfected Sf1 cells with a vector encoding the murine furine cDNA. Furine is a member of the subtilisin-like proprotein convertase family that is responsible for the proteolytic processing of several members of the TGF-ß superfamily (22, 23, 24). As shown in Fig. 1CGo, coexpression of furine increased the processing of myostatin, giving rise to the appearance of the 12.5-kDa mature myostatin. Notice that almost 100% of the myostatin precursor was processed under our experimental conditions; using densitometric analysis of Coomasie-stained gels, we estimated the 12.5-kDa myostatin concentration present in the conditioned medium to be about 60–180 ng/ml.

There is strong evidence indicating that myostatin signals through ActRIIs (13). To determine the biological activity of the mature myostatin produced in Sf1 cells, we employed MCF-7 cells transiently transfected with the p3TP-luc reporter that is specifically activated by TGF-ß1 and Act. Treatment of MCF-7 cells with the conditioned medium containing the 12.5-kDa myostatin protein barely increased the transcription of the reporter vector (Fig. 1DGo). Only a slight activation was found, likely due to the presence of nonprocessed myostatin, which retains some of the biological activity of the molecule (12). In keeping with this hypothesis, a similar degree of activation of the reporter vector is observed when MCF-7 cells are treated with a myostatin mutant that cannot be cleaved by furine (unpublished observation).

The lack of effect of the mature myostatin is likely due to the presence of the released propeptide noncovalently bound, forming a complex that prevents myostatin binding to its receptor (13, 14). This mechanism of regulation has been also described for other members of the family, including TGF-ß, which forms a small latent complex that can be activated via both physicochemical and proteolytic methods (23). In accord with this, removal of the propeptide moiety by either transient acidification or heating resulted in a clear-cut increase of the transcriptional activity of the p3TP-luc reporter (Fig. 1DGo). All together, our results demonstrate that Sf21 cells can produce biologically active myostatin. Generation of this active myostatin requires the coinfection of Sf21 cells with a vector encoding the subtilisin-like proprotein convertase furine, and the removal of the propeptide by physicochemical methods.

Differential effect of exogenous and endogenous myostatin in cultured myoblasts
Once demonstrated that Sf21 cells are capable of producing active recombinant myostatin, we tested its effects in cultured myoblasts transiently transfected with the p3TP-luc reporter vector. As in MCF-7 cells, recombinant myostatin was able to increase the transcription of the p3TP-luc vector in HepG2 cells (Fig. 2AGo) but, surprisingly, did not elicit any effect in either C2C12 or L6C10 myoblasts. Interestingly, identical results were obtained when cells were challenged with Act: Act treatment increased the transcriptional activity of the p3TP-luc reporter in both MCF-7 and HepG2 cells but failed to do so in cultured myoblasts (Fig. 2BGo). In contrast with these findings, TGF-ß1 stimulated the transcriptional activity of the reporter in all the cell lines investigated, thus ruling out the possibility that the lack of response of myoblasts to either myostatin or Act is due to deficiencies in the signal transduction or DNA-binding machinery necessary to activate the p3TP reporter (Fig. 2CGo). Notably, the transcription of the p3TP reporter was also stimulated in C2C12 when the myostatin concentration was increased up to 1 µg/ml, as shown in the study displayed in Fig. 2DGo. This result is in keeping with previous reports in which elevated concentrations of recombinant myostatin (more than 2 µg/ml) were shown to inhibit C2C12 proliferation (11, 12). However, these myostatin concentrations are clearly higher than those usually required for biological effects in other related members of the TGF-ß family, and also higher (10-fold) than the myostatin concentrations required to activate the transcription vector in nonmyoblast cells. Therefore, as we will further discuss, we consider these myostatin concentrations to be supraphysiological and, therefore, capable of dysregulating the mechanisms that control myostatin latency under normal conditions.



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  		FIG. 2. Myostatin differentially increases the transcriptional activity of the p3TP-luc reporter in myoblasts and nonmyoblast cells. A, Cells were transiently transfected with the p3TP-luc reporter vector and treated with 100 µl myostatin or with 100 µl conditioned medium from Sf21 cells infected with an empty vector. B, Cells were treated with Act A or vehicle. C, Cells were treated with TGF-ß1 or vehicle. D, C2C12 cells were treated with increasing amounts of myostatin. HepG2 cells were used as a positive control. All results shown are relative values based on normalized luciferase activity (i.e. luciferase activity/ß-galactosidase activity) and expressed as the mean ± SEM of at least three (A–C) or two (D) independent transfection experiments.

 
By analogy with Act, we presumed that activation of ActRIIs would lead to activation of the ActRI ALK4, and phosphorylation of Smad2 and Smad3 (25, 26). In accordance with this hypothesis, myostatin was able to induce the phosphorylation of Smad2 in both MCF-7 and HepG2 cells (Fig. 3Go). This result indicates that Smad2 is one of the transcription factors used by myostatin to transduce its signals into the cell nucleus, but also suggests that ALK4 is the type I receptor involved in myostatin signaling. Strikingly, myostatin treatment did not affect Smad2 phosphorylation in C2C12 cells, a finding that is consistent with the aforementioned lack of effect of myostatin on the activity of the p3TP reporter in myoblasts. The absence of an effect on Smad2 phosphorylation was also observed when C2C12 cells were treated with Act A. In contrast, treatment with TGF-ß1 stimulated the phosphorylation of Smad2 in these cells (Fig. 3Go).



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  		FIG. 3. Myostatin induces the phosphorylation of Smad2 in HepG2 cells but not in myoblasts. C2C12 (A) or HepG2 (B) cells were treated for 15 min with either myostatin, Act A, or TGF-ß1, and cell lysates were subjected to immunoblotting with antiphosphospecific Smad2 (pSmad2) antibodies. Total Smad proteins were detected by anti-Smad2 antibodies. All gels were run with the same marker (Kalidascope, Bio-Rad Laboratories). Molecular masses are indicated on the left.

 
Overall, our findings indicate that cultured myoblast, but not MCF-7 or HepG2 cells, are unresponsive to both myostatin and Act. Given that these growth factors signal through the same pair of receptors (ActRIIB/ALK4), we analyzed their expression levels by RT-PCR. Additionally, we also investigated the expression of ActRIIA, which also binds myostatin, although with lower affinity (13). All the cell lines investigated displayed similar levels of expression of ActRIIB (Fig. 4Go). The only remarkable difference among myoblasts and the other cell lines was the subtype of ActRIIB expressed: C2C12 and L6C10 cells expressed the ActRIIB1 isoform, whereas the ActRIIB2 isoform was found in MCF-7 and HepG2 cells. These two mRNA species arise from an alternative splicing of the ActRIIB RNA, which results in the loss of 8 amino acids of a proline-rich region located in the extracellular domain of the receptor (21). We also found that all cell lines expressed ALK4, the type I receptor that links ActRII with Smad2 phosphorylation, and three of them (C2C12, MCF-7 and HepG2) also expressed ActRIIA. The lack of expression of ActRIIA in L6C10 cells appears to have limited significance because C2C12 cells, which are unresponsive to either myostatin or Act, do express this ActRII.



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  		FIG. 4. Expression of ActRI and ActRII. Total RNA was extracted with Trizol, and the expression of ActRIIB, ActRIIA, and ALK4 was analyzed by RT-PCR in C2C12, L6C10, MCF-7, and HepG2 cells. The housekeeping HPRT gene was used as loading control. Amplimers were resolved in 2% agarose gels and ethidium bromide-stained. Lane 1, C2C12; lane 2, L6C10; lane 3, MCF-7; lane 4, HepG2. DNA sizes are indicated on the right.

 
To rule out that differences in the pattern of expression of ActRIIs could account for the lack of response of cultured myoblasts to either myostatin or Act, we transiently transfected C2C12 and L6C10 myoblasts with the p3TP-luc reporter vector, and with two plasmids encoding the ActRIIB2 or the ALK4 receptors. HepG2 cells were used as a positive control to test the effect of the pCMV5-ActRIIB2 and pCMV5-ALK4 constructs on the transcription of the reporter vector. As shown in Fig. 5Go, A and B, transient transfection of both types of receptors in either C2C12 or L6C10 cells failed to increase the transcriptional activity of the reporter vector in response to myostatin treatment. In contrast, a clear-cut increase in the transcriptional response of the reporter to myostatin was observed in HepG2 cells after transient transfection of the pair of receptors (Fig. 5CGo). Again, identical results were obtained when cells were treated with Act A instead of myostatin: the transient transfection of ActRIIB2 and ALK4 receptors was unable to induce myolasts to respond to Act, but increased the response of HepG2 cells to the growth factor (Fig. 6Go). In all, these findings demonstrate that the reason for the lack of response of cultured myoblasts to either myostatin or Act is not located at the receptor level.



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  		FIG. 5. Effect of overexpression of ActRIIB2 and ALK4 on the response of cultured myoblasts to myostatin. A and B, C2C12 and L6C10 myoblasts were transiently transfected with the reporter vector p3TP-luc and with plasmids encoding the murine ActRIIB2 (pCMV5-ActRIIB2) and the murine ALK-4 (pCMV5-ALK4), and treated with myostatin. C, HepG2 cells were used as controls. All results shown are relative values based on normalized luciferase activity (i.e. luciferase activity/ß-galactosidase activity) and expressed as the mean ± SEM of at least three independent transfection experiments.

 


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  		FIG. 6. Effect of overexpression of ActRIIB2 and ALK4 on the response of cultured myoblasts to Act. Panels A and B, C2C12 and L6C10 myoblasts were transiently transfected with the reporter vector p3TP-luc and with plasmids encoding the murine ActRIIB2 (pCMV5-ActRIIB2) and the murine ALK-4 (pCMV5-ALK4), and treated with Act. Panel C, HepG2 cells were used as controls. All results shown are relative values based on normalized luciferase activity (i.e. luciferase activity/ß-galactosidase activity) and expressed as the mean ± SEM of at least three independent transfection experiments.

 
The absence of an effect of exogenous Act has been previously reported in an ovarian cell line (27, 28). In these cells, the reason for the lack of response to exogenous Act is the presence of FS in the cell surface, which acts as a barrier for exogenous, but not for endogenous, Act. To ascertain whether a similar mechanism could explain the absence of response of myoblasts to exogenous myostatin, we transiently transfected C2C12 myoblasts with a plasmid containing the full-length myostatin cDNA. Interestingly, overexpression of endogenous myostatin was able to stimulate both the transcription of the p3TP-luc reporter and the phosphorylation of Smad2 in C2C12 myoblasts (Fig. 7Go, A and B). This differential response of cultured myoblasts to endogenous and exogenous myostatin suggests the existence of a mechanism that prevents the effect of exogenous myostatin. Although we do not presently know the nature of this mechanism, it is intriguing that RT-PCR analysis revealed the presence of abundant FS transcripts in both C2C12 and L6C10 myoblasts, whereas FS expression was not detected in the myostatin- and Act-responsive cells (MCF-7 and HepG2) (Fig. 7DGo).



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  		FIG. 7. Overexpression of myostatin increases the transcription of the p3TP-luc reporter vector and induces the phosphorylation of Smad2 in C2C12 cells. A, C2C12 cells and HepG2 cells were transiently transfected with the p3TP-luc reporter vector and with a plasmid encoding the full-length myostatin cDNA (pcDNA3.1-MSTNWT). Control cells were transfected with the p3TP-luc reporter vector and with an empty plasmid (pcDNA3.1-ø). Results are relative values based on normalized luciferase activity (i.e. luciferase activity/ß-galactosidase activity) and expressed as the mean ± SEM of three (C2C12) or two (HepG2) independent transfection experiments. B, C2C12 and HepG2 cells were transiently transfected with pcDNA3.1-MSTNWT or with an empty vector (pcDNA3.1-ø), and cell lysates were subjected to immunoblotting with antiphosphospecific Smad2 (pSmad2) antibodies. Total Smad proteins were detected by anti-Smad2 antibodies. C, Expression of FS was analyzed by RT-PCR in C2C12, L6C10, MCF-7 and HepG2 cells. The housekeeping HPRT gene was used as loading control. Total RNA was extracted with Trizol, and the amplimers were resolved in 2% agarose gels and ethidium bromide-stained. Lane 1, C2C12; lane 2, L6C10; lane 3, MCF-7; lane 4, HepG2. DNA sizes are indicated on the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Members of the TGF-ß superfamily transduce their signals through two related, but distinct, serine/threonine kinases, known as the type I and the type II receptors (29, 30, 31). Binding of the dimeric ligand results in the formation of a heterotetrameric receptor complex, in which type II receptors transphosphorylate the type I receptors, thereby activating its kinase domain. The activated type I receptors subsequently phosphorylate the Smad proteins that transduce the signal from the cytoplasm to the nucleus. Although the TGF-ß superfamily comprises over 35 members in vertebrates, only seven type I receptors and five type II receptors have been identified. Therefore, members of the TGF-ß superfamily are characterized by the existence of a high level of promiscuity, regarding nonspecifity in ligand binding and flexibility in type I and type II receptor association (32, 33).

In the case of myostatin, there is strong evidence indicating that the type II receptor employed for intracellular signaling is the ActRII. First, analysis of the physicochemical similitude of myostatin amino acids involved in receptor binding reveals that they are consistent with those of ligands that use the ActRIIA/IIB to transduce their signals (25). Second, myostatin binds to ActRII (mainly ActRIIB) in both cross-linking and standard receptor binding assays (13, 26). Finally, transgenic mice expressing high levels of a dominant-negative form of ActRIIB by using a skeletal muscle-specific promoter show a marked increase in muscling that, as occurs in myostatin-null mice, results from increases in both fiber number and fiber size (13).

Depending on the ligand, type II receptors may activate different type I receptors that target specific Smad proteins and, thereby, determine the specificity of biological responses (25, 29, 30). Ligands of type II receptors fall into two major categories: TGF-ß-like ligands, and bone morphogenetic protein (BMP)-like ligands. When type II receptors bind TGF-ß-like ligands, the type I receptors recruited into the complex are ALK4, ALK5, or ALK7, which transduce signals for Acts, TGF-ßs, and nodals and induce the phosphorylation of Smad2 and Smad3. In contrast, when type II receptors bind BMPs, the type I receptors activated are ALK1, ALK2, ALK3, or ALK6, and their activation leads to the phosphorylation of Smad1, Smad5, and Smad8. To date, the signaling pathway activated by myostatin in muscle cells has not been completely delineated. However, because myostatin binds to ActRIIB and induces the phosphorylation of Smad2/Smad3 (Refs. 17 and 26 , and the present work), it is likely that ALK4 is the type I receptor that is recruited and activated during myostatin signaling. Interestingly, the ability of myostatin to bind ALK4 (in the presence of ActRIIB) has been recently demonstrated in COS-1 cells (26). Alternatively, myostatin may also use ALK5 (26), a type I receptor that is mainly involved in TGF-ß signaling (29).

Once established that myostatin signals intracellularly via a membrane receptor complex consisting of ActRIIB and ALK4 (or ALK5), the question arises about how specificity in signaling is achieved. To ensure an appropriate regulation of muscle mass, a mechanism(s) has to exist to restrict myostatin actions to the skeletal muscle, but also to regulate the binding of other members of the TGF-ß superfamily to the ActRIIB/ALK4 complexes located in muscle cells. The first possibility is that additional components are required for proper assembly of the ligand/receptor complexes. The synthesis of these additional components would be restricted to skeletal muscle cells, thus preventing myostatin signaling in other cell types. Such a mechanism has been recently described for cripto, the founding member of the EGF-CFC (epidermal growth factor-cripto, FRL1, Cryptic) family, which is necessary for nodal signaling through ActRs (33). However, this mechanism does not provide a means to regulate the binding of other TGF-ß family members to muscular ActRIIB/ALK4 receptors. Moreover, at odds with this hypothesis, we have found in the present study that myostatin is able to induce biological responses in nonmuscle cells such as MCF-7 cells (derived from human mammary carcinoma) and HepG2 cells (human hepatic carcinoma).

The second plausible mechanism is that different isoforms of the receptor may be restricted in their expression patterns and may differ in their ligand binding affinity. This mechanism has been also described for other members of the TGF-ß superfamily such as TGF-ß2, which has low affinity for the TGF-ßRII but signals through an alternatively spliced variant (TGF-ßRIIB) (34). Interestingly, four ActRIIB isoforms have been described (named ActRIIB1 through ActRIIB4), arising from alternative splicing of the ActRIIB RNA; and although the binding affinity of myostatin for each isoform has not been investigated, they show different dissociation constants when the ligand is Act (21). In the present work, we have found that cultured myoblasts express a different ActRIIB isoform than MCF-7 or HepG2 cells. However, myostatin can transduce its signal through both isoforms, thus making it unlikely that this difference may account for the specificity in myostatin signaling.

Finally, the possibility exists that specificity in myostatin signaling depends on the existence of a mechanism that differentially regulates the bioavailability of endogenous and exogenous myostatin in muscle cells. This kind of regulation has been reported for Act in an ovarian teratocarcinoma-derived cell line (PA-1) (28). When PA-1 cells are stably transfected with a construct encoding the Act ßA-subunit cDNA, there is a decrease in the proliferation rate. In contrast, addition of recombinant exogenous Act has no effect on the proliferation of PA-1 cells (27, 28). Notably, we obtained similar results when comparing the effect of endogenous and exogenous myostatin in cultured myoblasts. When myoblasts are transiently transfected with a vector encoding the full-length myostatin sequence, there is an increase in the phosphorylation of Smad2 and a stimulation of the transcription of a reporter vector. These results contrast with the absence of any effect in myoblasts treated with exogenous myostatin.

In PA-1 cells, the differential response to exogenous and endogenous Act depends on the expression of FS on the cell surface. FS acts as a barrier preventing exogenous Act (but not endogenous Act) from binding to its receptor. Intriguingly, high levels of FS were found in the two myoblast cell lines investigated, whereas no FS transcripts were detected in the nonmuscle cells. Moreover, although the precise role of FS in the regulation of myostatin actions has not been established yet, there are several reports demonstrating that FS can bind to either myostatin or to the highly related BMP-11, and block their activity (13, 35, 36, 37, 38, 39). Further studies are now in progress to ascertain whether FS can also differentially regulate the bioavailability of exogenous and endogenous myostatin in myoblasts.

The relevance of the proposed model for regulation of specificity in myostatin signaling is that myostatin would act in vivo as an autocrine factor, at least under physiological conditions. This mechanism of action is in contrast with the state of the art model for myostatin actions, in which myostatin is considered to act in an endocrine fashion. The possibility that myostatin may act systemically is supported by several findings, including the existence of circulating forms of myostatin in both human and mouse serum (36, 37, 40). However, circulating myostatin is inactive (40) due to its association with inhibitory proteins. The composition of the circulating myostatin complexes in serum has been recently defined, and it involves at least three proteins: the myostatin propeptide, the FS-related gene, and the recently identified GDF-associated serum protein-1 (GASP-1) (36, 37). These three proteins may form distinct multicomponent myostatin complexes that inhibit the activity of circulating myostatin by preventing it from associating with its cellular receptor (32, 33, 34) and, in the case of GASP-1, also through additional mechanisms not completely understood (38). Based on these findings, it is likely that circulating myostatin complexes are merely acting as a sink to deplete extracellular concentrations of (inactive) myostatin.

A second line of evidence supporting the systemic actions of myostatin has been obtained in nude mice bearing myostatin-producing tumors. These animals have increased levels of circulating myostatin and exhibit a dramatic muscle and fat loss, analogous to that seen in human cachexia syndromes (40). However, the fact that myostatin may act systemically when delivered at high levels into the circulation does not necessarily indicate that myostatin acts in this fashion under physiological conditions, because very high levels of circulating myostatin can dysregulate the mechanisms controlling latency. Moreover, even if the extreme cachexia observed can be attributed solely to myostatin, it remains to be demonstrated that myostatin produced by the tumoral cells is acting directly on muscle. As we report here, myostatin can activate ActRIIB/ALK4 in hepatic cells once the propeptide is removed and, reportedly, cachexia can be induced in mice by activation of hepatic ActRIIs (41).

In contrast with the findings obtained by implantation of nonmuscle cells engineered with recombinant myostatin cDNA, cachexia is not found in transgenic mice that overexpress myostatin selectively in the muscle cells under the muscle creatin kinase (MCK) promoter (42). MCK transgenic mice have decreased skeletal muscle mass and cardiac muscle mass that are not accompanied by noticeable impairment of their appearance, activity, or other general indicators of health. Although, the reasons for these differences are not clear, one plausible explanation is that the sarcopenia observed in muscle-specific transgenic mice reflects myostatin actions on muscle cells, whereas cachexia observed in tumor-bearing mice is a consequence of both muscular and nonmuscular actions of myostatin. Interestingly, when myostatin expression is driven by a mutated MCK promoter that completely restricts myostatin expression to skeletal muscle (without ancillary expression of myostatin in the heart), there are no reductions in heart weight. This finding indicates that myostatin overexpressed in skeletal muscle cells is unable to act on cardiac muscle cells, thus reinforcing the consideration of myostatin as an autocrine factor.

In summary, we have demonstrated the existence of a differential response to exogenous and endogenous myostatin in cultured myoblasts. In view of our findings, we propose that myostatin actions are exerted in vivo in an autocrine, but not endocrine, fashion. Because myostatin shares its pair of receptors with other members of the TGF-ß superfamily, this mechanism of regulation of myostatin actions would be useful to restrict myostatin effects to muscular cells. Further studies are now in progress to identify the factors that prevent myostatin from acting systemically.


   Acknowledgments
 
This work is devoted to the memory of Ramon Ríos (Santiago de Compostela, 1972-New York City, 2003). We thank Dr. Nestor González-Cadavid for providing the antimyostatin antibody; Dr. Joan Massague for the pCMV5-ActRIIB2, pCMV5-ALK4, and p3TP-luc vectors; and Dr. Kazuhhisa Nakayama for the pCMV-mFur vector.


   Footnotes
 
This work was supported by grants from Fondo de Investigaciones Sanitarias (PI021458) and Xunta de Galicia (PGIDIT03PXIC20801PN). I.C. is the recipient of a Beca de Formación en Investigación fellowship from Ministerio de Sanidad y Consumo, Spain.

Abbreviations: Act, Activin; ActR, Act receptor; ALK, ActR-like kinase; BMP, bone morphogenetic protein; FS, follistatin; GASP, GDF-associated serum protein; GDF, growth and differentiation factor; MCK, muscle creatin kinase.

1 Deceased on 11 April 2003. Back

Received September 4, 2003.

Accepted for publication February 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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