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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.
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 |
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| INTRODUCTION |
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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 |
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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
-myogenin monoclonal antibody (1:5),
-Myf5 (Santa
Cruz) at 1:1,000,
-MyoD (Dako) at 1:300,
-MyoD (Santa
Cruz) at 1:500, MF20 monoclonal against sarcomeric myosin heavy chain
(MHC) at 1:1,
-MEF2A at 1:1,000 as previously reported
(6),
-Smad2 (Cell
Signaling) at 1:1,000,
-phospho-Smad2 (Cell Signaling) at
1:1,000,
-Smad3 (Santa Cruz) at 1:1,000,
-phospho-Smad3 (Cell Signaling) at 1:200,
-Smad4
(Santa Cruz) at 1:500, and
-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.
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| RESULTS |
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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).
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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.
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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).
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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).
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| DISCUSSION |
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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.
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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 |
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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 |
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| REFERENCES |
|---|
|
|
|---|
2. Buckingham, M. 1992. Making muscle in mammals. Trends Genet. 8:144-148.[Medline]
3. Camoretti-Mercado,
B., D. J. Fernandes, S. Dewundara, J. Churchill, L.
Ma, P. C. Kogut, J. F. McConville, M. S.
Parmacek, and J. Solway. 10 May 2006, posting date. Inhibition of
TGFß-enhanced SRF-dependent transcription by SMAD7.
J. Biol. Chem. [Online.] doi: 10.1074/jbc.M602748200. [Epub ahead of print.]
4. Carcamo, J., A. Zentella, and J. Massague. 1995. Disruption of transforming growth factor beta signaling by a mutation that prevents transphosphorylation within the receptor complex. Mol. Cell. Biol. 15:1573-1581.[Abstract]
5. Choy,
L., J. Skillington, and R. Derynck. 2000. Roles of
autocrine TGF-beta receptor and Smad signaling in adipocyte
differentiation. J. Cell Biol.
149:667-682.
6. Cox,
D. M., M. Du, M. Marback, E. C. Yang, J.
Chan, K. W. Siu, and J. C. McDermott.2003
. Phosphorylation motifs regulating the stability and
function of myocyte enhancer factor 2A. J. Biol.
Chem.
278:15297-15303.
7. Davis, R. L., H. Weintraub, and A. B. Lassar.1987 . Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987-1000.[CrossRef][Medline]
8. Donoviel, D. B., M. A. Shield, J. N. Buskin, H. S. Haugen, C. H. Clegg, and S. D. Hauschka. 1996. Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice. Mol. Cell. Biol. 16:1649-1658.[Abstract]
9. Emerson, C. P., Jr. 1993. Embryonic signals for skeletal myogenesis: arriving at the beginning. Curr. Opin. Cell Biol. 5:1057-1064.[CrossRef][Medline]
10. Ewton, D. Z., and J. R. Florini. 1990. Effects of insulin-like growth factors and transforming growth factor-beta on the growth and differentiation of muscle cells in culture. Proc. Soc. Exp. Biol. Med. 194:76-80.[Medline]
11. Florini, J. R., D. Z. Ewton, and K. A. Magri.1991 . Hormones, growth factors, and myogenic differentiation. Annu. Rev. Physiol. 53:201-216.[CrossRef][Medline]
12. Forbes, D., M. Jackman, A. Bishop, M. Thomas, R. Kambadur, and M. Sharma.2006 . Myostatin auto-regulates its expression by feedback loop through Smad7 dependent mechanism. J. Cell. Physiol. 206:264-272.[CrossRef][Medline]
13. Gonzalez-Cadavid,
N. F., W. E. Taylor, K. Yarasheski, I. Sinha-Hikim,
K. Ma, S. Ezzat, R. Shen, R. Lalani, S. Asa, M. Mamita, G. Nair, S.
Arver, and S. Bhasin. 1998. Organization of the human
myostatin gene and expression in healthy men and HIV-infected men with
muscle wasting. Proc. Natl. Acad. Sci. USA
95:14938-14943.
14. Hata,
A., G. Lagna, J. Massague, and A. Hemmati-Brivanlou.1998
. Smad6 inhibits BMP/Smad1 signaling by specifically
competing with the Smad4 tumor suppressor. Genes Dev.
12:186-197.
15. Hayashi, H., S. Abdollah, Y. Qiu, J. Cai, Y. Y. Xu, B. W. Grinnell, M. A. Richardson, J. N. Topper, M. A. Gimbrone, Jr., J. L. Wrana, and D. Falb.1997 . The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling.Cell 89:1165-1173.[CrossRef][Medline]
16. Hua,
X., Z. A. Miller, H. Benchabane, J. L. Wrana, and
H. F. Lodish. 2000. Synergism between
transcription factors TFE3 and Smad3 in transforming growth
factor-beta-induced transcription of the Smad7 gene.J. Biol. Chem.
275:33205-33208.
17. Javelaud, D., V. Delmas, M. Moller, P. Sextius, J. Andre, S. Menashi, L. Larue, and A. Mauviel. 2005. Stable overexpression of Smad7 in human melanoma cells inhibits their tumorigenicity in vitro and in vivo. Oncogene 24:7624-7629.[CrossRef][Medline]
18. Jonckheere, N., M. Perrais, C. Mariette, S. K. Batra, J. P. Aubert, P. Pigny, and I. Van Seuningen. 2004. A role for human MUC4 mucin gene, the ErbB2 ligand, as a target of TGF-beta in pancreatic carcinogenesis. Oncogene 23:5729-5738.[CrossRef][Medline]
19. Joulia, D., H. Bernardi, V. Garandel, F. Rabenoelina, B. Vernus, and G. Cabello. 2003. Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin. Exp. Cell Res. 286:263-275.[CrossRef][Medline]
20. Kambadur,
R., M. Sharma, T. P. Smith, and J. J. Bass.1997
. Mutations in myostatin (GDF8) in double-muscled
Belgian Blue and Piedmontese cattle. Genome Res.
7:910-916.
21. Kavsak, P., R. K. Rasmussen, C. G. Causing, S. Bonni, H. Zhu, G. H. Thomsen, and J. L. Wrana.2000 . Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol. Cell 6:1365-1375.[CrossRef][Medline]
22. Lalani, R., S. Bhasin, F. Byhower, R. Tarnuzzer, M. Grant, R. Shen, S. Asa, S. Ezzat, and N. F. Gonzalez-Cadavid. 2000. Myostatin and insulin-like growth factor-I and -II expression in the muscle of rats exposed to the microgravity environment of the NeuroLab space shuttle flight. J. Endocrinol. 167:417-428.[Abstract]
23. Langley,
B., M. Thomas, A. Bishop, M. Sharma, S. Gilmour, and R.
Kambadur. 2002. Myostatin inhibits myoblast
differentiation by down-regulating MyoD expression. J.
Biol. Chem.
277:49831-49840.
24. Lassar, A. B., S. X. Skapek, and B. Novitch.1994 . Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr. Opin. Cell Biol. 6:788-794.[CrossRef][Medline]
25. Lee,
K.-S., H.-J. Kim, Q.-L. Li, X.-Z. Chi, C. Ueta, T. Komori, J.
M. Wozney, E.-G. Kim, J.-Y. Choi, H.-M. Ryoo, and S.-C. Bae.2000
. Runx2 is a common target of transforming growth
factor ß1 and bone morphogenetic protein 2, and cooperation
between Runx2 and Smad5 induces osteoblast-specific gene expression in
the pluripotent mesenchymal precursor cell line C2C12. Mol.
Cell. Biol.
20:8783-8792.
26. Lee, S. J. 2004. Regulation of muscle mass by myostatin. Annu. Rev. Cell Dev. Biol. 20:61-86.[CrossRef][Medline]
27. Liu,
D., B. L. Black, and R. Derynck. 2001.
TGF-beta inhibits muscle differentiation through functional repression
of myogenic transcription factors by Smad3. Genes Dev.
15:2950-2966.
28. Martin, J. S., M. C. Dickson, F. M. Cousins, A. B. Kulkarni, S. Karlsson, and R. J. Akhurst.1995 . Analysis of homozygous TGF beta 1 null mouse embryos demonstrates defects in yolk sac vasculogenesis and hematopoiesis.Ann. N. Y. Acad. Sci. 752:300-308.[Medline]
29. Massague, J. 1998. TGF-beta signal transduction. Annu. Rev. Biochem. 67:753-791.[CrossRef][Medline]
30. Massague, J. 2000. How cells read TGF-beta signals. Nat. Rev. Mol. Cell Biol. 1:169-178.[CrossRef][Medline]
31. Massague,
J., S. Cheifetz, T. Endo, and B. Nadal-Ginard. 1986.
Type beta transforming growth factor is an inhibitor of myogenic
differentiation. Proc. Natl. Acad. Sci. USA
83:8206-8210.
32. McDermott,
J. C., M. C. Cardoso, Y. T. Yu, V.
Andres, D. Leifer, D. Krainc, S. A. Lipton, and B.
Nadal-Ginard. 1993. hMEF2C gene encodes skeletal
muscle- and brain-specific transcription factors. Mol. Cell.
Biol.
13:2564-2577.
33. McPherron,
A. C., and S. J. Lee. 1997. Double
muscling in cattle due to mutations in the myostatin gene. Proc.
Natl. Acad. Sci. USA
94:12457-12461.
34. Molkentin, J. D., and E. N. Olson. 1996. Defining the regulatory networks for muscle development. Curr. Opin. Genet. Dev. 6:445-453.[CrossRef][Medline]
35. Moustakas,
A. 2002. Smad signalling network. J.
Cell Sci.
115:3355-3356.
36. Nakao, A., M. Afrakhte, A. Moren, T. Nakayama, J. L. Christian, R. Heuchel, S. Itoh, M. Kawabata, N. E. Heldin, C. H. Heldin, and P. ten Dijke. 1997. Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.Nature 389:631-635.[CrossRef][Medline]
37. Olson, E. N., and W. H. Klein. 1994. bHLH factors in muscle development: dead lines and commitments, what to leave in and what to leave out. Genes Dev. 8:1-8.[Medline]
38. Ornatsky,
O. I., and J. C. McDermott. 1996.
MEF2 protein expression, DNA binding specificity and complex
composition, and transcriptional activity in muscle and non-muscle
cells. J. Biol. Chem.
271:24927-24933.
39. Overbergh, L., D. Valckx, M. Waer, and C. Mathieu. 1999. Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11:305-312.[CrossRef][Medline]
40. Perry, R. L., M. H. Parker, and M. A. Rudnicki. 2001. Activated MEK1 binds the nuclear MyoD transcriptional complex to repress transactivation. Mol. Cell 8:291-301.[CrossRef][Medline]
41. Pirskanen, A., J. C. Kiefer, and S. D. Hauschka.2000 . IGFs, insulin, Shh, bFGF, and TGF-beta1 interact synergistically to promote somite myogenesis in vitro. Dev. Biol. 224:189-203.[CrossRef][Medline]
42. Puri, P. L., and V. Sartorelli. 2000. Regulation of muscle regulatory factors by DNA-binding, interacting proteins, and post-transcriptional modifications. J. Cell. Physiol. 185:155-173.[CrossRef][Medline]
43. Quinn,
Z. A., C. C. Yang, J. L. Wrana, and
J. C. McDermott. 2001. Smad proteins
function as co-modulators for MEF2 transcriptional regulatory proteins.Nucleic Acids Res.
29:732-742.
44. Rebbapragada,
A., H. Benchabane, J. L. Wrana, A. J. Celeste, and
L. Attisano. 2003. Myostatin signals through a
transforming growth factor ß-like signaling pathway to block
adipogenesis. Mol. Cell. Biol.
23:7230-7242.
45. Schofield, J. N., and L. Wolpert. 1990. Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation. Exp. Cell Res. 191:144-148.[CrossRef][Medline]
46. Shull, M. M., I. Ormsby, A. B. Kier, S. Pawlowski, R. J. Diebold, M. Yin, R. Allen, C. Sidman, G. Proetzel, D. Calvin, et al. 1992. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359:693-699.[CrossRef][Medline]
47. Stopa,
M., D. Anhuf, L. Terstegen, P. Gatsios, A. M. Gressner, and
S. Dooley. 2000. Participation of Smad2, Smad3, and
Smad4 in transforming growth factor beta (TGF-beta)-induced activation
of Smad7. The TGF-beta response element of the promoter requires
functional Smad binding element and E-box sequences for transcriptional
regulation. J. Biol. Chem.
275:29308-29317.
48. Taylor,
W. E., S. Bhasin, J. Artaza, F. Byhower, M. Azam,
D. H. Willard, Jr., F. C. Kull, Jr., and N.
Gonzalez-Cadavid. 2001. Myostatin inhibits cell
proliferation and protein synthesis in C2C12 muscle cells.Am. J. Physiol. Endocrinol. Metab.
280:E221-E228.
49. Topper,
J. N., J. Cai, Y. Qiu, K. R. Anderson, Y.
Y. Xu, J. D. Deeds, R. Feeley, C. J. Gimeno,
E. A. Woolf, O. Tayber, G. G. Mays, B. A.
Sampson, F. J. Schoen, M. A. Gimbrone, Jr., and D.
Falb. 1997. Vascular MADs: two novel MAD-related genes
selectively inducible by flow in human vascular endothelium.Proc. Natl. Acad. Sci. USA
94:9314-9319.
50. Wrana, J. L. 2000. Regulation of Smad activity.Cell 100:189-192.[CrossRef][Medline]
51. Wrana, J. L., L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X. F. Wang, and J. Massague.1992 . TGF beta signals through a heteromeric protein kinase receptor complex. Cell 71:1003-1014.[CrossRef][Medline]
52. Wu,
Z., P. J. Woodring, K. S. Bhakta, K. Tamura, F.
Wen, J. R. Feramisco, M. Karin, J. Y. Wang, and
P. L. Puri. 2000. p38 and extracellular
signal-regulated kinases regulate the myogenic program at multiple
steps. Mol. Cell. Biol.
20:3951-3964.
53. Yu,
Y. T., R. E. Breitbart, L. B. Smoot, Y.
Lee, V. Mahdavi, and B. Nadal-Ginard. 1992. Human
myocyte-specific enhancer factor 2 comprises a group of
tissue-restricted MADS box transcription factors. Genes
Dev.
6:1783-1798.
54. Zhu, X., S. Topouzis, L. F. Liang, and R. L. Stotish. 2004. Myostatin signaling through Smad2, Smad3 and Smad4 is regulated by the inhibitory Smad7 by a negative feedback mechanism. Cytokine 26:262-272.[CrossRef][Medline]
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