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Molecular and Cellular Biology, August 2006, p. 6248-6260, Vol. 26, No. 16
0270-7306/06/$08.00+0     doi:10.1128/MCB.00384-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Smad7 Promotes and Enhances Skeletal Muscle Differentiation

Helen D. Kollias, Robert L. S. Perry, Tetsuaki Miyake, Arif Aziz, and John C. McDermott*

Department of Biology, York University, Toronto M3J 1P3, Ontario, Canada

Received 3 March 2006/ Returned for modification 28 March 2006/ Accepted 1 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor ß1 (TGF-ß1) and myostatin signaling, mediated by the same Smad downstream effectors, potently repress skeletal muscle cell differentiation. Smad7 inhibits these cytokine signaling pathways. The role of Smad7 during skeletal muscle cell differentiation was assessed. In these studies, we document that increased expression of Smad7 abrogates myostatin- but not TGF-ß1-mediated repression of myogenesis. Further, constitutive expression of exogenous Smad7 potently enhanced skeletal muscle differentiation and cellular hypertrophy. Conversely, targeting of endogenous Smad7 by small interfering RNA inhibited C2C12 muscle cell differentiation, indicating an essential role for Smad7 during myogenesis. Congruent with a role for Smad7 in myogenesis, we observed that the muscle regulatory factor (MyoD) binds to and transactivates the Smad7 proximal promoter region. Finally, we document that Smad7 directly interacts with MyoD and enhances MyoD transcriptional activity. Thus, Smad7 cooperates with MyoD, creating a positive loop to induce Smad7 expression and to promote MyoD driven myogenesis. Taken together, these data implicate Smad7 as a fundamental regulator of differentiation in skeletal muscle cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Muscle development requires the coordination of multiple cellular events. Myoblasts, muscle precursor cells committed to the skeletal muscle lineage, respond to external signals and activate the program of gene expression, resulting in a terminally differentiated phenotype (34). Muscle regulatory factors (MRFs) MyoD and Myf5 are transcriptional regulators that are intrinsically involved in myoblast commitment and, in concert with other MRFs (MRF4 and myogenin), orchestrate the activation of muscle-specific gene expression (2, 9, 24, 37). The onset and progression of muscle differentiation have proved to be exquisitely sensitive to regulation by a variety of secreted peptide growth factors, such as insulin-like growth factor, fibroblast growth factor, and transforming growth factor ß (TGF-ß) (10, 11, 19, 41, 45).

Interplay between positively and negatively acting growth factors is thus an important determinant of myogenic differentiation. Indeed, recent studies have documented several mechanisms of posttranslational modulation of the transcriptional activity of the MRFs by growth factor-mediated signaling (27, 40, 42, 52). In particular, members of the TGF-ß superfamily demonstrate potent repressive effects on the MRFs and myogenic differentiation in conjunction with their spectrum of activities as pleiotropic cytokines that modulate many cellular processes, such as proliferation, differentiation, and apoptosis (29, 31, 48) The TGF-ß family member myostatin, also known as growth-derived factor 8, is of particular interest since it was observed that naturally occurring mutations of the myostatin gene led to a dramatic enhancement of muscle mass in cattle (33). Further, myostatin-null mice were found to have a pronounced increase in the mass of their skeletal musculature (33).

Evidence to date indicates that myostatin signals through the canonical TGF-ß signaling pathway, which consists of three main components: (i) the ligand, (ii) the receptors, serine/threonine kinases, and (iii) the intracellular mediators, the Smads (26, 50). Transmission of the myostatin signal begins with ligand binding to the type II receptor (activin type II receptors ActRIIA and ActRIIB) (44). The type II receptor translocates to its corresponding type I receptor (ALK-4 and ALK-5), forming an activated receptor complex (44). The activated receptor complex then phosphorylates the receptor-regulated Smads (Smad2 and Smad3), leading to the formation of a complex with a co-Smad (Smad4) (30, 35). The Smad heteromultimer translocates into the nucleus, where it interacts with both DNA and protein targets in a complex manner to confer cellular responsiveness to myostatin. In addition to receptor-regulated Smads and co-Smads, there are also inhibitor Smads, Smad6 and Smad7. Smad6 functions primarily within the bone morphogenic protein signaling pathway, inhibiting its activity through competition with the receptor-regulated Smads (Smad1 and Smad5) for the Smad4 cofactor (14), while, to date, Smad7 has been implicated as a negative modulator of TGF-ß signaling (15, 36).

Smad7 was initially characterized as a factor induced by shear stress in vascular endothelial cells (49). The current view for the mechanism by which Smad7 inhibits TGF-ß-activated responses is by stably associating with the active TGF-ß receptor complexes while being refractory to phosphorylation. Interaction of Smad7 with the receptor inhibits Smad2/Smad3 phosphorylation, resulting in reduced TGF-ß signaling (36). Importantly, TGF-ß1 or myostatin signaling induces Smad7 mRNA, thereby establishing a negative feedback loop to inhibit TGF-ß signaling. Thus, Smad7 is implicated in myostatin as well TGF-ß1 signaling (36, 54).

Based on the potent role of TGF-ß/myostatin signaling in muscle cells and the pivotal role played by Smad7 in modulating these pathways in other cell types, we sought to address the function of Smad7 during skeletal muscle cell differentiation. In this report, we document several novel properties of Smad7 in muscle cells. First, Smad7 abrogates myostatin- but not TGF-ß1-mediated repression of myogenesis, suggesting a muscle-specific role for Smad7. Second, Smad7 accelerates myogenic differentiation, leading to cellular hypertrophy through a positive feedback loop with MyoD in which MyoD activates the Smad7 promoter and Smad7 protein physically associates with and potentiates MyoD transactivation properties. Finally, Smad7 is a prerequisite for myogenesis since "knockdown" of endogenous Smad7 using small interfering RNAs (siRNAs) blocks muscle-specific gene expression and differentiation of cultured muscle cells. Taken together, these data indicate a fundamental role for Smad7 in initiating myogenesis through a Smad7-MyoD positive feedback loop and abrogation of the myostatin signaling pathway.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfections. Mouse C2C12 myoblasts (ATCC CLR-1772; Manassas, Va.) were maintained in growth medium (GM) consisting of 10% fetal bovine serum (HyClone) in high-glucose Dulbecco's modified Eagle's medium supplemented with penicillin and streptomycin (1,000 U/ml; Gibco) at 37°C and 5% CO2. Differentiation medium (DM) consisted of 5% horse serum (Atlanta Biologicals) in high-glucose Dulbecco's modified Eagle's medium supplemented with penicillin and streptomycin (1,000 U/ml; Gibco). Differentiation medium was replenished every 2 days. C3H10T1/2 mouse embryonic fibroblasts (ATCC CCL-226) were maintained in GM. Where indicated, 2 ng/ml of recombinant human TGF-ß1 (R&D Systems), a concentration lower than those used by other groups (25), 1 µg/ml of recombinant mouse myostatin (R&D Systems) (12), or the diluent (0.1% bovine serum albumin and 4 mM HCl) was added to the cells for 2 days.

Transient transfections of C2C12 myoblasts with pMCK-EGFP, pCMV-dsRed2, and appropriate plasmids, as indicated, were performed using Lipofectamine reagents (Invitrogen) according to the manufacturer's instructions. A total of 1 µg of DNA was added to 15,500 cells/cm2. Cells were visualized using a Zeiss Axiovert 35 with the appropriate epifluorescence optics used for enhanced green fluorescent protein (EGFP) or dsRed2.

Plasmids. Expression constructs for pCMV5B-Smad7-HA (15), pCMV5B dominant-negative TGF-ß receptor II (TßRII [K227R]) (4), and pCMV5B dominant-negative ActRIIB (K217R) were kind gifts from J. Wrana (University of Toronto). MRF expression plasmids were constructed in pEMSV as described elsewhere (7). MEF2A and MEF2C were constructed in pMT2 (32, 53). A carboxyl-terminally Myc-His-tagged full-length Smad7 and truncated Smad7 expression plasmids were constructed by inserting full-length PCR-amplified Smad7 (426 amino acids [aa]) or C-terminal truncated Smad7 (409 aa) into pcDNA4.2/TO/myc/his digested with HindIII and XhoI sites.

Transcription reporter assays. Transient transfections of C2C12 myoblasts and C3H10T1/2 fibroblasts were performed using standard calcium phosphate-DNA precipitation with pCMV-ß-galactosidase serving as an internal control of transfection efficiency. Transcription reporter assay constructs pMCK-luc (8), pMEF2-luc (43), p4R SV40-luc (40), and p3TP-lux (51) have previously been described. The pSmad7-luc construct (47) and the pMCK-EGFP construct were kind gifts from S. Dooley (Institut fur Klinische Chemie und Pathobiochemie, Germany) and A. Ferrer-Martinez (Universitat de Barcelona, Spain), respectively. Sixteen hours following transfection, the cells were washed with phosphate-buffered saline and allowed to recover in GM for 24 h. Cells were transferred to DM for 2 days, and cellular extracts were prepared to determine luciferase activity using a 9501 Berthold Lumat luminometer as per the manufacturer's instructions (Promega).

Immunoblot analysis. Protein concentrations were determined by the Bradford assay, and equivalent amounts of protein (25 µg) were electrophoretically resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), 6%, 10%, or 12%, followed by electrophoretic transfer to an Immobilon-P membrane (Millipore, Inc.). Immunoblotting was carried out using {alpha}-myogenin monoclonal antibody (1:5), {alpha}-Myf5 (Santa Cruz) at 1:1,000, {alpha}-MyoD (Dako) at 1:300, {alpha}-MyoD (Santa Cruz) at 1:500, MF20 monoclonal against sarcomeric myosin heavy chain (MHC) at 1:1, {alpha}-MEF2A at 1:1,000 as previously reported (6), {alpha}-Smad2 (Cell Signaling) at 1:1,000, {alpha}-phospho-Smad2 (Cell Signaling) at 1:1,000, {alpha}-Smad3 (Santa Cruz) at 1:1,000, {alpha}-phospho-Smad3 (Cell Signaling) at 1:200, {alpha}-Smad4 (Santa Cruz) at 1:500, and {alpha}-Smad7 (RnD systems) at 1:1,000. Appropriate horseradish peroxidase-conjugated secondary antibody (Bio-Rad) was diluted to 1:1,000 in 5% milk in Tris-buffered saline and 0.1% Tween 20 or in 5% milk in phosphate-buffered saline. Western chemiluminescence reagent (Amersham) was used to detect the secondary antibody on the membranes.

Coimmunoprecipitations. Equal protein amounts were diluted with NP-40 lysis buffer containing protease inhibitors, 1 µg antibody, and 25 µl protein G-conjugated (50% slurry) (Amersham-Pharmacia) Sepharose beads and incubated at 4°C overnight on a rotating platform. The beads were washed with three changes of NETN wash buffer (0.1% NP-40, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl [pH 8.0]). Beads were boiled in sample buffer, and proteins were separated by SDS-PAGE and blotted as described above.

GST pull-down assays. Equal protein amounts were diluted with NP-40 lysis buffer containing protease inhibitors, 5 µg of glutathione S-transferase (GST) fusion proteins as described previously (40), and 25 µl glutathione-Sepharose beads (50% slurry) (Amersham-Pharmacia) and incubated at 4°C overnight on a rotating platform. The beads were washed with three changes of NETN wash buffer (0.1% NP-40, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl [pH 8.0]). Beads were boiled in sample buffer, and proteins were separated by SDS-PAGE and blotted as described above.

RT-PCR. Reverse transcription (RT)-PCR for Smad7 was performed using a 5'-TCCTGCTGTGCAAAGTGTTC-3'forward primer and 5'-TTGTTGTCCGAATTGAGCTG-3'reverse primer targeting Smad7 at 1,906 bp and 2,353 bp, respectively, as previously reported (18). RT-PCR for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was performed as previously described (39). RNA was isolated from C2C12 cells by TRIzol (Invitrogen) as per the manufacturer's instructions.

EMSA. The DNA binding assays and extract preparation were carried out as previously described (38). Oligonucleotides were synthesized with an Applied Biosystems synthesizer and annealed together (see Fig. 4H for sequences). For the DNA binding assays with different cell extracts, the incubation reaction mixture comprised of equivalent amounts of protein (1 to 3 µg total protein), 0.2 ng of probe, 0.45 µg of poly(dI-dC), and 100 ng of single-stranded oligonucleotide in a total volume of 20 µl. The electrophoretic mobility shift assay (EMSA) binding reaction buffer consisted of 10 mM HEPES [pH 7.6], 3 mM MgCl2, 20 mM KCl, 1 mM dithiothreitol, and 5% glycerol. After the addition of the radiolabeled probe, the reaction mixture was incubated at room temperature for 20 min. The reaction mixture was then resolved using electrophoresis on a 4.5% nondenaturing polyacrylamide gel to separate the bound from the free fraction. The core nucleotide sequences used in the binding assays are listed in Fig. 4H. Antibody supershifts were performed by preincubating the cell extract with 1 µl of MyoD (Dako) and 0.5 µl or 2 µl of microphthalmia transcription factor (MiTF; Stratagene) added to the preincubation reaction mixture (20 min on ice). Following electrophoresis, the gels were dried and DNA-protein complexes were visualized by autoradiography after overnight exposure to X-ray film at –80°C.


Figure 4
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  		FIG. 4. MyoD directly binds to the Smad7 promoter via an E box, and its binding is critical for Smad7 promoter activity in myogenic cells. (A) Smad7 mRNA accumulation during C2C12 differentiation. Cell cultures at 50 to 60% subconfluent (SC), 80% confluent (C), 12 h in DM, 24 h in DM, and 48 h in DM were harvested, and Smad7 expression was determined by RT-PCR. (B) Cell cultures of C2C12s at SC, C, 12 h, 24 h, 48 h, and 4 days in DM were harvested, and the Smad7 protein level was determined by Western immunoblot analysis; ERK1/2 was used as a loading control. Overexpressed tagged Smad7 was used as a positive control (+), and * denotes a potential degradation product of Smad7. (C) Rat Smad7 promoter region (GenBank accession no. AF156727) containing an SBE, an E box, and an overlapping AP-1 binding site. (D) Smad7 promoter activity in myogenic cells was examined by a luciferase transcription assay. C2C12 myoblasts were transfected with either pGL3-Basic vector, pSmad7-luc (wild type [wt]), pSmad7-SBE* (mutated SBE), or pSmad7-E-box*-luc (mutated E box). Each data point is the mean of triplicate samples from a single experiment, and the error bars represent standard errors of the means. The graph is representative of three separate experiments. (E) MRF effect on Smad7 promoter activity was examined by a luciferase transcription assay. C3H10T1/2 fibroblasts were transfected with pSmad7-luc and either the control, MyoD, myogenin, or Myf5. Each data point is the mean of triplicate samples from a single experiment, and the error bars represent standard errors of the means. The graph is representative of data from three separate experiments. (F) Extracts were made from C2C12 as myotubes (MT), lanes 1 to 6, or myoblasts (MB), lanes 7 to 11, and analyzed for the composition of the binding complex of the Smad7 promoter. The Smad7 binding complex is indicated as an arrow with a "B." The specificity binding to the 32P-labeled E-box and AP-1 oligonucleotides (lanes 1 and 6) was demonstrated by the addition of a 100-fold molar excess of cold competitor, wt, (lanes 6 and 11), or mutants. Mutant A contained an E-box mutation (lanes 3 and 8), mutant B contained an AP-1 mutation (lanes 4 and 9), and mutant C contained double mutations of the E box and AP-1 (lanes 5 and 10). (G) Where indicated, the extracts were preincubated with antibodies directed against MyoD or MiTF. (H) Summary of competition EMSA mutant oligonucleotides. Mutations are indicated by boldface.

 
Small interfering RNA. The small interfering RNA expression vector, pSilencer 3.1-H1 puro (Ambion)containing a Smad7 target sequence, was engineered. Specifically, the target sequence was GAGAAGCATTCTCATTGGA directed against mouse Smad7 mRNA. C2C12 cells were transfected using calcium phosphate-DNA precipitation with pSilencer-siSmad7 or pSilencer-negative control, supplied by Ambion and selected with 4 µg/ml of puromycin (Sigma) for 2 days. Cells were visualized using a Zeiss Axiovert 35 with the appropriate epifluorescence optics used for EGFP or dsRed2.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Smad7 rescues myostatin inhibition. Initially, we sought to determine whether Smad7 could modulate the signaling of myostatin or TGF-ß1 since both growth factors potently inhibit muscle differentiation. We confirmed the inhibition of C2C12 muscle differentiation by myostatin (1 µg/ml) and TGF-ß1 (2 ng/ml) (Fig. 1A). Next, we assessed whether Smad7 could reverse the repressive effects of myostatin and TGF-ß1 by transiently transfecting C2C12 muscle cells with Smad7. Surprisingly, we observed that differentiation of C2C12 cells, as indicated by a muscle-specific EGFP reporter gene (MCK-EGFP) and phenotypic myotube formation, was restored when Smad7 was expressed in the presence of myostatin but not TGF-ß1 (Fig. 1B). Reporter gene analysis using pMCK-luc (Fig. 1C) further confirmed that Smad7 can rescue and enhance differentiation in the presence of myostatin but not TGF-ß1. Western immunoblot analysis of muscle differentiation markers sarcomeric MHC, myogenin, and MEF2A were lower in control C2C12 cells treated with TGF-ß1 or myostatin compared to diluent. Ectopic expression of Smad7 restored these muscle differentiation markers but only in the myostatin-treated C2C12s (Fig. 1D). In contrast to the abrogation of myostatin effects, we observed that ectopic expression of Smad7 in myoblasts treated with TGF-ß1 showed no difference in MHC, myogenin, and MEF2A expression levels (Fig. 1D).


Figure 1
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  		FIG. 1. Smad7 rescues myostatin inhibition but not TGF-ß1. C2C12 myoblasts were transiently transfected with (A) the control (pCMV5B-empty) or (B) Smad7 (pCMV5B-hSmad7-HA), pMCK-EGFP, and pCMV-dsRed2 and transferred to DM with diluent, TGF-ß1 (2 ng/ml), or myostatin (1 µg/ml). Phase-contrast and fluorescence images were obtained after 2 days in DM. Bar, 200 µm. (C) C2C12 myoblasts were transiently transfected with pMCK-luc, pCMV-ß-galactosidase, and either the control or Smad7 and transferred to DM with diluent, TGF-ß1 (2 ng/ml), or myostatin (1 µg/ml). Cells were harvested after 2 days in DM, luciferase activity was assayed and corrected for ß-galactosidase activity, and ß-galactosidase activity was assayed. (D) C2C12 myoblasts were transfected with either the control or Smad7 and PGK-puromycin, selected with puromycin (4 µg/ml) for 2 days, and then transferred to DM for 3 days and harvested. Diluent, TGF-ß1, and myostatin were replenished after 2 days. Expression myogenic factors were assessed by Western immunoblot analysis. (E) C2C12 myoblasts were transiently transfected with the TGF-ß reporter 3TP-lux and either the control or Smad7 and transferred to DM with diluent or TGF-ß1 (2 ng/ml). After 2 days in DM, the cells were harvested and 3TP-lux activity and ß-galactosidase activity were assayed. (F) C2C12 myoblasts were transiently transfected with pMCK-luc and either the control or Smad7, transferred to DM with a TGF-ß1 concentration of 0 ng/ml, 0.5 ng/ml, 1 ng/ml, or 2 ng/ml, and harvested 2 days later to assay luciferase activity. For all luciferase reporter assays, each data point is the mean of triplicate samples from a single experiment and the error bars represent standard errors of the means. The graphs are representative of three separate experiments.

 
The concentrations of myostatin used were based on dose-response data for repression of myogenesis and published data in the literature (12), while concentrations of TGF-ß1 were lower than those previously reported (25), 2 ng/ml but sufficient to inhibit myogenesis. Smad7's ability to overcome canonical TGF-ß signaling was determined by the TGF-ß reporter gene 3TP-lux, which showed that Smad7, in the presence of 2 ng/ml of TGF-ß1, was able to inhibit TGF-ß signaling, as indicated by a reporter gene analysis (Fig. 1E). Further, the TGF-ß1 dose-response assay shows that exogenous Smad7 was unable to rescue TGF-ß1 inhibition of myogenesis at reduced levels (Fig. 1F) whereas supra-physiological levels of myostatin were potently reversed by exogenous Smad7 (Fig. 1C). Thus, the repressive effects of TGF-ß1 are clearly mediated through multiple signaling pathways that may be independent of the canonical Smad pathway.

A potential mechanism for the differential effects of Smad7 on TGF-ß1- compared to myostatin-treated cells could be MyoD expression levels. Both TGF-ß1 and myostatin decreased the levels of MyoD expression compared to the control (Fig. 1D). However, TGF-ß1-treated cells had a greater reduction in MyoD expression than myostatin-treated cells. Interestingly, Smad7 had no effect on MyoD expression in either the TGF-ß1- or the myostatin-treated cells, suggesting that Smad7 is unable to rescue MyoD expression.

Taken together, these data support the conclusion that myostatin but not TGF-ß1 inhibition of myogenesis is negated by Smad7.

Smad7 accelerates differentiation of C2C12 cells. We next examined whether Smad7 was able to exert an effect on C2C12 differentiation without the addition of exogenous myostatin or TGF-ß1. Figure 2A shows that C2C12 cells transfected with pCMV5B-Smad7-HA were EGFP positive and formed myotubes within 2 days in DM in contrast to the controls, which showed minimal differentiation at this time. Smad7-expressing cells after 4 days in DM maintained their accelerated differentiation phenotype compared to the controls, exhibiting extensive myotube networks that were substantially larger than those of the corresponding controls without Smad7 expression (Fig. 2A).


Figure 2
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  		FIG. 2. Smad7 enhances myogenic differentiation. C2C12 myoblasts were transiently transfected with pMCK-EGFP, pCMV-dsRed2 (as a marker of transfection efficiency), and either pCMV5B-empty (control) or pCMV5B-Smad7-HA (Smad7). The cells were then transferred to DM. (A) Phase-contrast and fluorescence images were obtained after 2 days in DM. Bar, 200 µm (B) After 2 days in DM, the number of GFP-positive cells was counted. Values are means and standard errors of the means calculated from nine fields of view in three separate experiments performed in triplicate. (C to E) Enhancement of myogenic activity by Smad7 was examined by transiently transfecting myoblasts with either the control or Smad7 and by a pMCK-luc (C), p4R SV40-luc (multimerized E box) (D), or pMEF2-luc (E) transcription assay. ß-Galactosidase activities were used to normalize for transfection efficiency. Each data point is the mean of triplicate samples from a single experiment, and the error bars represent standard errors of the means. The graph is representative of three separate experiments. (F) Expression of the Smads and the myogenic factors in C2C12 myoblasts transfected with either the control or Smad7 and PGK-puromycin was assessed by Western immunoblot analysis. Cells were selected with puromycin (4 µg/ml) for 2 days and transferred to DM for 2 days prior to being harvested.

 
To provide a quantitative summary of these data, the average number of pMCK-EGFP-positive cells/field from nine fields in three different experiments was counted (Fig. 2B). The pCMV5B-Smad7-HA-expressing cells had more than five times the number of EGFP-positive, differentiated cells compared to the control condition after 2 days in DM. Confirmation of the expression of hemagglutinin (HA)-tagged Smad7 was determined by Western immunoblotting (Fig. 2F).

To quantitate the effect of Smad7 on the muscle creatine kinase (MCK) enhancer, we next carried out reporter gene assays using an MCK enhancer-luciferase reporter gene. Consistent with our observations using pMCK-EGFP, we observed that differentiating C2C12 cells expressing pCMV-Smad7-HA had an enhanced level of pMCK-luc activity compared to the control (Fig. 2C). Since the MCK promoter is known to be activated by the MRFs and MEF2 (1), the effect of Smad7 on these factors independently was investigated using p4R SV40-luc (multimerized E-box reporter) and pMEF2-luc, respectively. Smad7 enhanced the E-box reporter (Fig. 2D) while Smad7 had no effect on pMEF2-luc (Fig. 2E), suggesting that the Smad7 effect on pMCK-luc is through one or more members of the MRFs and not through the MEF2 family.

Further evidence of the enhancement of differentiation by Smad7 is indicated by Western analysis of myogenin and MEF2A, key regulators of differentiation. We observed that myogenin and MEF2A expression levels were higher in myoblasts overexpressing Smad7 than in the control after 2 days in DM (Fig. 2F). Further, Smad7 ectopically expressing myoblasts showed higher levels of MHC than the control, consistent with our observations of enhanced myotube formation (Fig. 2F). In addition, Smad7 ectopic expression increased mobility of phospho-Smad3 compared to the control (Fig. 2F) but had no effect on total Smad3, Smad2, phospho-Smad2, and Smad4 expression levels.

Smad7 enhancement of myotube formation is recapitulated by dominant-negative activin type IIB receptor. Subsequently, we tested whether the acceleration of differentiation by Smad7 was a common property of inhibiting the canonical TGF-ß pathway. Since Smad7 was able to rescue myostatin inhibition but not TGF-ß1 inhibition, we next tested the effect of dominant-negative receptors of the respective pathways, TßRII and activin type IIB receptor (ActRIIB). Consistent with the idea that Smad7 may function through the myostatin pathway, we found that expression of a dominant-negative ActRIIB (dnActRIIB) elicited extensive myotube formation comparable to that of Smad7 after 3 days in DM (Fig. 3A). Conversely, the dominant-negative TßRII (dnTßRII) had minimal effect on differentiation.


Figure 3
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  		FIG. 3. Smad7 and dnActRIIB enhance myogenic differentiation. (A) C2C12 myoblasts were transiently transfected with pMCK-EGFP and either pCMV5B-empty (control), pCMV5B-Smad7-HA (Smad7), pCMV5B-dominant negative TßRII (dnTßRII), or pCMV5B-dnActRIIB (dnActRIIB). Phase-contrast and fluorescence images were obtained after 3 days in DM. Bar, 200 µm. (B and C) C2C12 myoblasts were transiently transfected with either the control, Smad7, dnTßRII, dnActRIIB, or both dnTßRII and dnActRIIB, luciferase reporter pMCK-luc (B) or E-box responsive construct p4R SV40-luc (C), and transferred to DM for 2 days. Each data point is the mean of triplicate samples from a single experiment, and the error bars represent standard errors of the means. The graph is representative of three separate experiments.

 
To quantitate the effect of dnActRIIB and dnTßRII on the MCK enhancer, we next carried out reporter gene assays using an MCK enhancer-luciferase reporter gene. Consistent with our observations using pMCK-EGFP, we observed that differentiating C2C12 cells expressing pCMV5B-dnActRIIB had nearly eightfold-higher levels of pMCK-luc activity than the control (Fig. 3B). Additionally, dnTßRII did marginally enhance pMCK-luc activity compared to the control (twofold). When both dominant-negative receptors were combined, the effect was comparable to that of the dnActRIIB alone. Results of an E-box reporter assay showed similar effects, as pMCK-luc, Smad7, and dnActRIIB enhanced differentiation; however, dnTßRII had no effect on the E-box reporter (Fig. 3C). Together, these data indicate that dnActRIIB but not dnTßRII showed a phenocopy of the effect of Smad7.

Smad7 promoter activation is regulated by MRFs in myogenic cells. We determined the endogenous Smad7 expression through RT-PCR and Western methodology on a time course of differentiation with C2C12 cells. Smad7 transcript levels are induced early in differentiation, increasing from subconfluent myoblasts to confluent myoblasts and peaking at 24 h in DM (Fig. 4A). Smad7 protein levels mirror the mRNA levels, with Smad7 being induced early during differentiation (Fig. 4B).

Having determined that the levels of Smad7 mRNA and Smad7 protein are regulated during differentiation, we next considered the transcriptional control of the Smad7 promoter. The Smad7 promoter contains a Smad binding element (SBE), making it potentially responsive to the TGF-ß stimulus, and an E-box, a potential target for MRFs (47). Thus, we compared the Smad7 promoter (wild type) with a Smad7 promoter containing mutations in the SBE or E-box element (schematic in Fig. 4C) in order to dissect the contribution of each DNA binding motif to the transcriptional control of Smad7 in muscle cells. The wild-type Smad7 promoter driving a reporter gene is robustly active in C2C12 cells (Fig. 4D). The mutation in the SBE in the Smad7 promoter caused a 25% decrease in activity compared to the wild-type promoter. However, mutation of the E box leads to a pronounced 80% decrease in promoter activity in C2C12 cells, indicating that Smad7 promoter regulation is strongly dependent on this E box in myogenic cells. To substantiate the observation that the E-box element in the Smad7 promoter can be activated by the MRFs, the wild-type Smad7 promoter was transfected into C3H10T1/2 fibroblasts along with each of the MRFs (MyoD, myogenin, and Myf5). Figure 4E shows that MyoD and Myf5 increased Smad7 promoter activity while myogenin had no effect.

To further characterize the transactivation of the Smad7 promoter by the MRFs, we next carried out EMSA analysis to determine whether MyoD directly binds the Smad7 promoter and to determine the importance of the E box in the Smad7 promoter. In these experiments, we document that there is an endogenous binding complex in muscle cells that recognizes the E-box element in the context of the Smad7 promoter. We observed that this binding complex was competed away by an excess of the unlabeled wild-type double-stranded oligonucleotide but not when the canonical E-box motif was mutated (Fig. 4F). We were also interested in the possible role of an AP-1 site, which overlaps the E box in the promoter (Fig. 4C). However, mutation of the AP-1 site, while maintaining the E box intact, was still able to compete away the complex formation with the labeled wild-type oligonucleotides, therefore indicating that the E box and not the AP-1 site is critical for complex formation on this element in myogenic cells. These data confirm the importance of the E box observed in reporter gene assays.

We next performed a supershift analysis with antibodies directed against a number of bHLH factors. These studies indicated that MyoD (Fig. 4G) but not myogenin (data not shown) contributes to this binding complex in muscle cells. Interestingly, even though MyoD antibody supershifts part of the complex in muscle cells, there is still a considerable amount of the binding complex that is unaffected by the antibody, thus indicating that there is an as-yet-unidentified E-box binding factor interacting with this region. We were unable to confirm whether this was Myf5 since a number of antibodies that we used proved to be equivocal in their ability to recognize native Myf5 in supershift analysis (data not shown). Since an earlier report had indicated that in vitro-translated TFE3 (a bHLH leucine zipper transcription factor [16]) can bind to this sequence, we determined whether this might be the other component of the complex in muscle, but the results of a supershift analysis with antibodies against TFE3 (data not shown) and the related MiTF (Fig. 4G) were negative. Taken together, these data suggest that Smad7 promoter is transactivated by an E-box binding complex, with MyoD constituting a part of this complex.

Smad7 physically associates with and potentiates MyoD activity. Further studies were subsequently carried out, indicating that Smad7 can cooperate with MyoD in a myogenic conversion assay in which MyoD was transfected into C3H10T1/2 fibroblasts. Consistent with the enhanced differentiation of cultured muscle cells, we observed that MyoD-mediated myogenic conversion is potentiated by Smad7 in C3H10T1/2 fibroblasts, as shown by staining for the expression of MHC (MF20 antibody) (Fig. 5A). Also, Smad7 potentiated MyoD activation of the myosin light chain and MCK promoters (Fig. 5B and C).


Figure 5
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  		FIG. 5. MyoD physically interacts and cooperates with Smad7. Fibroblasts, C3H10T1/2 cells, were transiently transfected with either pEMSV-empty (control) or pEMSV-MyoD (MyoD), with or without pCMV5B-Smad7-HA (Smad7). (A) Cells were fixed and stained for sarcomeric myosin heavy chain with MF20 after 2 days in DM. Bar, 200 µm. White arrows indicate multinucleated MF20-positive cells. (B) MyoD cooperates with Smad7 on the myosin light chain (MLC2) promoter. Promoter activity was examined by a luciferase transcription reporter assay. C3H10T1/2 fibroblasts were transiently transfected with either the control or MyoD, with or without Smad7. Each data point is the mean of triplicate samples from a single experiment, and the error bars represent standard errors of the means. The graph is representative of three separate experiments. (C) MyoD cooperates with Smad7 on the MCK promoter. Promoter activity was examined by a luciferase transcription assay. C3H10T1/2 fibroblasts were transiently transfected with either the control or MyoD, with or without Smad7. Each data point is the mean of triplicate samples from a single experiment, and the error bars represent standard errors of the means. The graph is representative of data from four separate experiments. RLU, relative light units. (D) C3H10T1/2 fibroblasts were transiently transfected with MyoD, full-length Myc-tagged Smad7 (FL), truncated Smad7-myc (t), or in combination as indicated. Expression of MyoD, full-length Smad7, and truncated Smad7 was confirmed by Western immunoblot analysis. Coimmunoprecipitation of C3H10T1/2 cells transfected with MyoD, full-length Smad7, truncated Smad7, or the appropriate combination was done. Full-length Smad7-myc/his was detected by Western immunoblot analysis after immunoprecipitation (Ip) with MyoD. Truncated Smad7-myc was detected after immunoprecipitation. (E) GST-NT (MyoD, aa 1 to 95), but not GST-CT (MyoD, aa 174 to 318), is sufficient to bind Smad7-myc. Whole-cell extracts from MyoD/Smad7-myc-transfected cells were loaded onto glutathione beads bound with GST-MyoD fusion proteins or GST alone. Western analysis with anti-myc antibody revealed that only GST-NT bound Smad7.

 
Having observed that Smad7 can enhance MyoD activity, we assessed whether Smad7 can directly interact with MyoD in a coimmunoprecipitation assay (Fig. 5D). MyoD was transiently transfected into CH310T1/2 fibroblasts with or without Smad7-myc/his. A C-terminally truncated Smad7-myc/his mutant (Fig. 6A) was also transfected. Expression of the respective proteins was confirmed by Western analyses (Fig. 5D). Figure 5D shows that MyoD directly interacts with full-length Smad7 and, to a lesser extent, with truncated Smad7 in DM. Further, Fig. 5E confirms the specificity of the MyoD-Smad7 interaction by a GST pull-down assay, with the N-teriminal region but not the C-terminal region of MyoD interacting with Smad7-myc.


Figure 6
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  		FIG. 6. C-terminally truncated Smad7 is unable to enhance myogenic differentiation. (A) Myoblasts, C2C12 cells, were transiently transfected with either pcDNA4-empty (control), pcDNA-Smad7 myc/his (full-length Smad7), or pcDNA-Smad7 (409 aa) (truncated Smad7). (B) Expression of full-length Smad7 and trunc.Smad7 was confirmed by Western immunoblot analysis using {alpha}-myc antibody. (C and D) C2C12 myoblasts were transiently transfected with pCMV-ß-galactosidase and either the control, full-length Smad7, or trunc.Smad7. Luciferase reporter p3TP-lux (C) or pMCK-luc (D) was used to assess the activation of the canonical TGF-ß pathway and muscle differentiation, respectively. Cells were harvested after 2 days in differentiation medium, and luciferase and ß-galactosidase activities were assayed. RLU, relative light units.

 
Carboxyl-terminally truncated Smad7 is unable to enhance myogenesis. To test whether the association of Smad7 with its receptor is required for the enhancement of myogenesis, we made a C-terminal deletion (409 to 426 aa) (Fig. 6A and B) mutant from Smad7 which would inactivate its capability to interact with the receptor (5). We first tested whether this truncated Smad7 would lose its ability to abrogate activation of a TGF-ß-responsive reporter gene (3TP-lux). Figure 6C shows that full-length Smad7 represses this promoter while the truncated Smad7 loses this property, indicating the utility of the mutant to abrogate receptor signaling. Next, we performed an assay in which we tested the capability of full-length and truncated Smad7 to enhance myogenesis, as indicated by the MCK-luc reporter gene. The data show that the truncated Smad7 does not retain its myogenic potential (Fig. 6D). Thus, the C-terminal region of Smad7 is critical in the myogenic function of Smad7. Further analysis indicated that truncated Smad7 has reduced capability of directly interacting with MyoD (Fig. 5E). Thus, the interaction of Smad7 with MyoD and Smad7 with type I receptors (TßRI and ActRIB) is required for its myogenic potentiation.

siRNA-targeting Smad7 inhibits differentiation of C2C12 muscle cells. Since we had observed that Smad7 is expressed in myogenic cells and, by using a gain-of-function analysis, potently accelerates differentiation when overexpressed, we next determined the effect of extinguishing the expression of the endogenous Smad7 by using siRNA technology. We assessed the efficiency of the siRNAs by testing their ability to reverse the endogenous Smad7 inhibition of a TGF-ß/myostatin-responsive reporter gene (3TP-lux). In this assay, we observed that siRNA (siSmad7) can enhance 3TP-lux, indicating that the repressive effect of Smad7 was removed by the Smad7 siRNA (Fig. 7C). To further confirm the specificity of the siSmad7 effect, we titrated increasing amounts of a Smad7 construct that is not targeted by siSmad7 (Smad7*) and showed that Smad7* abrogated the effect of siSmad7, supporting the specificity of siSmad7 targeting. Strikingly, loss of Smad7 function using siRNA to inhibit Smad7 expression inhibited differentiation, as determined by pMCK-EGFP and morphological myotube formation (Fig. 7A). Myoblasts transfected with pSilencer-siSmad7 showed markedly reduced rates of differentiation after 4 days in DM compared to the control, pSilencer-negative control. In addition, myogenin, MEF2A, and myosin heavy chain expression levels were all substantively reduced in the Smad7-suppressed cells compared to those in the control cells (Fig. 7B).


Figure 7
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  		FIG. 7. Smad7 is required for myogenic differentiation. C2C12 myoblasts were transiently transfected with pCMV-dsRed2, pMCK-EGFP, and either pSilencer-negative control (control) or pSilencer containing Smad7 siRNA sequence-targeting Smad7 (siSmad7). Transfected cells were selected with puromycin (4 µg/µl) for 2 days in growth medium and transferred to differentiation medium for 4 days. (A) Phase-contrast and fluorescence images were obtained. Bar, 200 µm (B) Selected cells were harvested, and expression of the Smads and myogenic factors was determined by Western immunoblot analysis. (C) The specificity of siSmad7 was assessed using a TGF-ß response assay. siSmad7 derepression of the TGF-ß-responsive 3TP-lux reporter gene is abrogated by Smad7 expression. The Smad7 expression vector encodes Smad7 that is not targeted by the siRNA-targeting construct (Smad7*). The reversal of siSmad7 (2 µg) effects on 3TP-lux by increasing concentrations, 0.5 µg, 1 µg, and 2 µg, of Smad7* overexpression indicates the specificity of the siSmad7-targeting construct and abrogation by a non-siRNA-targeted Smad7 titration. RLU, relative light units.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Smad7 functions as a potent negative feedback inhibitor of the TGF-ß/activin pathway, although the precise role of Smad7 in different cell types has not been extensively characterized (15). In this report, we document a critical cell lineage-specific role of Smad7 in initiating and enhancing myogenic differentiation. Several lines of evidence support this contention: first, exogenous Smad7 expression in muscle cells causes a dramatic enhancement of differentiation, partially through direct interaction with MyoD; second, the repressive effect of myostatin signaling on myogenesis is counteracted by Smad7; third, MyoD directly binds to and activates the Smad7 promoter; lastly, suppression of endogenous Smad7 expression by siRNA technology inhibits the differentiation of cultured muscle cells. Taken together, these observations indicate that Smad7 exerts a potent enhancing effect on muscle-specific gene expression and differentiation.

Even though Smad7 has proven able to inhibit both TGF-ß1 and myostatin signaling in other cellular contexts, our results clearly indicate that in muscle cells, Smad7 rescues myostatin- but not TGF-ß1-mediated myogenic repression. However, even though Smad7 is unable to rescue TGF-ß1 inhibition of muscle differentiation, our data indicate that it can abrogate the canonical signaling pathway as shown by a TGF-ß reporter gene assay (3TP-lux). Our experiments show that Smad7 is able to fully rescue the expression of key myogenic regulatory genes that are suppressed by myostatin signaling (MHC, myogenin, and MEF2A). Conversely, we also document that even though the expression of these regulatory genes is suppressed by TGF-ß1 signaling, there is no analogous rescue of their expression by Smad7. Thus, we unequivocally document that Smad7 counteracts the repressive effect of myostatin but not TGF-ß1 on muscle cells.

Though there have been little differences demonstrated between TGF-ß1 and myostatin in cultured muscle cells, there are striking differences in their roles in vivo as illustrated by gene targeting experiments. Gene targeting of TGF-ß1 in mice results in multifocal inflammatory disease in half of the conceptuses, while the remainder do not reach parturition as a result of defective hematopoiesis and endothelial cell differentiation (28, 46). Conversely, homozygous null mutation of the myostatin gene in mice results in a dramatic enhancement of skeletal muscle mass (33), consistent with a prominent role of myostatin in skeletal muscle growth. Congruent with this role, inactivating mutations of myostatin in cattle and humans results in hypermuscularity (20, 33).

We observed that siRNA inhibition of Smad7 blocks myogenesis, and it is thus tempting to speculate that Smad7 may be required for abrogation of myostatin signaling and for initiation of the myogenic differentiation program. This is supported by the expression pattern of Smad7, which is increased in the early phases of differentiation. This result suggests that abrogation of the TGF-ß signaling pathway is a fundamental checkpoint that has to be traversed in order for skeletal muscle cells to activate the differentiation program.

Since both myostatin and TGF-ß1 are known to function, at least in part, through the canonical Smad pathway, our observations indicate a difference in Smad independent TGF-ß1 signaling in muscle that is not shared with myostatin. Interestingly, there is growing precedence for the uncoupling of the canonical TGF-ß pathway from phenotypic effects exerted by TGF-ß. In a recent study in which Smad7 inhibited Smad3/Smad4 activity, as shown by a reporter gene assay [(CAGA)9-luc], it was also observed that TGF-ß1-induced growth inhibition was unaffected (17). Our conclusion is that there are noncanonical (Smad-independent) aspects of TGF-ß1 signaling in muscle that is not shared with myostatin that profoundly contribute to its repressive effects on myogenesis. One example of these differential effects that we documented is that TGF-ß1 has a much more potent repressive effect on MyoD expression than myostatin. Interestingly, while MyoD overexpression can rescue TGF-ß-mediated myogenic repression, a report in the literature shows that MyoD overexpression does not rescue myostatin-mediated repression of myogenesis (23). Thus, clear differences in the effects of these two cytokines are becoming apparent which are supported by their divergent roles in vivo.

An interesting aspect of our observations is the duality of the interaction between MyoD and Smad7. Smad7 has been shown to have several interacting partners (15, 21, 3). While we observe a direct cooperative effect of Smad7 on MyoD in enhancing its function, we also document that the Smad7 promoter is activated through a canonical E box in its proximal promoter region. These observations constitute a novel mechanistic explanation for the potentiating effect of Smad7 on myogenesis. The muscle specificity of the Smad7-MyoD cooperativity is exemplified in our reporter gene assays in which Smad7 can activate the MCK enhancer in C2C12 cells but not in C3H10T1/2 fibroblasts unless they are converted to a muscle phenotype by cotransfection with MyoD (Fig. 5). These observations, albeit based on overexpression of Smad7 and MyoD, lend support to our contention that Smad7 cooperates through direct interaction with MyoD in exerting its effect on myogenic cells.

To reconcile our observations, we posit a model based on our data for Smad7 effects in skeletal muscle which explains a number of observations concerning myostatin signaling in skeletal muscle (Fig. 8). In this model, we propose a positive feedback loop between MyoD and Smad7 during differentiation. This model predicts that when MyoD activity is enhanced at the onset of differentiation, one of its target genes is Smad7, and when Smad7 protein levels increase, it directly cooperates with MyoD to activate the early phase of muscle gene expression. In parallel, the increase in Smad7 also abrogates the canonical myostatin signaling pathway, lessening its repressive effect on myogenesis. The cumulative result of these promyogenic stimuli is a strong impetus toward differentiation. Conversely, when MyoD activity is low in proliferating myoblasts, Smad7 levels are not enhanced by MyoD, and the absence of Smad7 protein allows the myostatin pathway to functionally repress the differentiation program.


Figure 8
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  		FIG. 8. Model of the role of Smad7 on myogenesis. Smad7 enhances myogenesis in two ways: (i) through inhibiting myostatin signaling and (ii) through enhancing MyoD activity.

 
One final consideration is that the counterregulatory role of Smad7 in negating the growth-suppressive role of myostatin in muscle could have potential therapeutic implications, since myostatin levels are elevated in a variety of clinical conditions associated with muscle loss. A correlation between enhanced myostatin levels and muscle atrophy has been observed in severe human immunodeficiency virus infection (13), aging, and exposure to microgravity (22). Thus, if this association proves causal, then Smad7 expression could be an important therapeutic target molecule for conditions involving muscle wasting.

These studies underscore a critical role for Smad7 in the control of skeletal muscle growth and differentiation. Our observations point toward abrogation of myostatin signaling and cooperation with MyoD as the underlying mechanism for Smad7's growth-promoting effects. Also, an unforeseen requirement for Smad7 in myogenic differentiation has been observed by loss-of-function analysis, suggesting a physiological requirement for Smad7 in the initiation of the myogenic program. These data raise interesting questions concerning the function of Smad7 in the hierarchy of molecular control of myogenesis and also highlight a potential therapeutic use of Smad7 in counteracting muscle wasting in a variety of pathological myopathies.


   ACKNOWLEDGMENTS
 
We thank J. Wrana and S. Dooley for the plasmids provided. We thank Nikky Soora and Hareem Ilyas for their technical assistance.

These studies were made possible by a grant from the Canadian Institutes of Health Research (CIHR) to J.C.M. Salary support for R.L.S.P. was in part provided by a postdoctoral fellowship from the Muscular Dystrophy Association of Canada (MDAC) and CIHR.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, 327 Farquharson, LSB, York University, 4700 Keele St., Toronto M3J 1P3 Ontario, Canada. Phone: (416) 736-2100, ext. 30337. Fax: (416) 736-5698. E-mail: jmcderm{at}yorku.ca. Back


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Molecular and Cellular Biology, August 2006, p. 6248-6260, Vol. 26, No. 16
0270-7306/06/$08.00+0     doi:10.1128/MCB.00384-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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