Human myostatin negatively regulates human myoblast growth and ...

Am J Physiol Cell Physiol 301: C195–C203, 2011. First published April 20, 2011; doi:10.1152/ajpcell.00012.2011.

Human myostatin negatively regulates human myoblast growth and differentiation Craig McFarlane,1 Gu Zi Hui,2* Wong Zhi Wei Amanda,2* Hiu Yeung Lau,1 Sudarsanareddy Lokireddy,2 Ge XiaoJia,2 Vincent Mouly,3 Gillian Butler-Browne,3 Peter D. Gluckman,1 Mridula Sharma,4 and Ravi Kambadur1,2 1

Growth, Development and Metabolism Program, Singapore Institute for Clinical Sciences, Singapore; 2School of Biological Sciences, Nanyang Technological University, Singapore; 3Université Pierre et Marie Curie-Paris 6, UMR S 974 and UMR S 787, Institut de Myologie, Paris, France; and 4Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Submitted 13 January 2011; accepted in final form 8 April 2011

McFarlane C, Gu ZH, Wong ZW, Lau HY, Lokireddy S, Ge X, Mouly V, Butler-Browne G, Gluckman PD, Sharma M, Kambadur R. Human myostatin negatively regulates human myoblast growth and differentiation. Am J Physiol Cell Physiol 301: C195–C203, 2011. First published April 20, 2011; doi:10.1152/ajpcell.00012.2011.— Myostatin, a member of the transforming growth factor-␤ superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the ␥-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.

growth, a mutation in the human myostatin gene results in muscular hypertrophy (25), and several studies have linked increased expression of myostatin with various human conditions that result in skeletal muscle wasting. For example, human immunodeficiency virus-infected men undergoing skeletal muscle wasting have increased intramuscular and serum levels of a myostatin-immunoreactive protein as compared with healthy controls (12), and human primary myoblasts from Duchenne muscular dystrophy patients show enhanced expression of myostatin (33); increased expression of serum myostatin-immunoreactive protein has also been correlated with advancing sarcopenia (32). In studies of the effect of exogenous addition of myostatin on the growth of human myoblast cultures, Shishkin et al. (28) demonstrated that treatment with increasing concentrations of recombinant myostatin protein results in a dose-dependent decrease in the proliferation rate of cultured human myoblasts. Similarly, Benabdallah et al. (5) showed that inhibition of myostatin signaling with follistatin increases proliferation and differentiation rates in human myoblasts, and that overexpression of follistatin partially rescued recombinant myostatin protein-mediated inhibition of human myogenic differentiation. Here, we investigated the signaling mechanism(s) through which myostatin regulates human myoblast growth and differentiation. Our results indicate that human myostatin potently regulates human myoblast proliferation and differentiation, consistent with previously published reports. Furthermore, we demonstrate for the first time that myostatin signals through the Notch signaling pathway to regulate human myoblast differentiation.

p21; MyoD; Notch; Hes1; Smad3

MATERIALS AND METHODS

growth factor belonging to the transforming growth factor-␤ superfamily. Studies of myostatin function, conducted mostly in rodent (17–21, 29), bovine (14, 23), and chicken models (1, 2), have identified this protein as a negative regulator of myogenesis, but to date, few analyses have been directed to understanding the mechanism(s) underlying the role of myostatin in human skeletal muscle growth. Consistent with the inhibitory effect of myostatin in muscle

MYOSTATIN IS A CIRCULATING

* G. Z. Hui and W. Z. W. Amanda contributed equally to this work. Address for reprint requests and other correspondence: R. Kambadur, School of Biological Sciences, Nanyang Technological Univ., 60 Nanyang Drive, Singapore, 637551 (e-mail: [email protected]). http://www.ajpcell.org

Myostatin purification. Human recombinant myostatin protein (hMstn) was cloned, expressed, and purified from Escherichia coli as described (26). Myostatin-overexpressing Chinese hamster ovary (CHO) cells (kindly provided by Dr. Se-Jin Lee) and purification of murine myostatin (CHO-Mstn) from these cells have been described (22). Cell cultures (hMb5 and C2C12) were treated with CHO-Mstn at a final concentration of 3.5 ng/ml. Cell culture. Two human primary myoblast cultures were used, designated here as hMb5 (isolated from a healthy 5-day-old infant) and hMb15 (isolated from a 15-yr-old healthy subject) (6, 11, 13) and kindly provided by Drs. Vincent Mouly and Gillian Butler-Browne. Mouse C2C12 myoblasts (30) were obtained from American Type Culture Collection (Manassas, VA). Human myoblast cultures and C2C12 cells were maintained as described (20). Proliferation assay and differentiation studies on myoblasts were conducted as described

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MYOSTATIN AND HUMAN MYOGENESIS

(17, 29). To block the Notch signaling pathway, human myoblasts were cultured and induced to differentiate under low-serum conditions in DMEM-2% horse serum (HS) in the absence (0.05% DMSO) or presence of the ␥-secretase inhibitor L-685,458 (Sigma) at a final concentration of 1 ␮M, with fresh inhibitor added daily. To study the role of the Notch signaling pathway in myostatin-mediated inhibition of differentiation, human myoblasts were cultured with or without the ␥-secretase inhibitor L-685,458, as described above, in the absence or presence of 4 ␮g/ml hMstn for 72 h. Cells were harvested for protein isolation and Western blot analysis or fixed and stained with Gill’s hematoxylin and eosin. To assess human myotube formation during differentiation, cultures were grown on Thermanox coverslips (Nunc) under low-serum conditions (DMEM 2% HS) with the respective treatments, fixed with ethanol:formaldehyde:glacial acetic acid (20: 2:1) and stained with Gill’s hematoxylin and 1% eosin. Cultures were photographed and assessed for myotube number, area, and myonuclei content. FACS analysis. Human myoblasts cultured for 48 h in proliferation medium (DMEM 20% FBS, 10% HS, 1% chicken embryo extract) with or without 8 ␮g/ml hMstn were harvested by trypsinization, and 1 million cells were centrifuged for 5 min, washed twice with cold PBS, and fixed with 1 ml of ice-cold 70% ethanol. Cells were then centrifuged for 5 min with the 70% ethanol removed and replaced with 1 ml of DNA staining solution (0.1 mg/ml propidium iodide, 0.05 mg/ml RNaseA in PBS). After a 45-min incubation at 4°C, cells were washed twice with cold PBS, resuspended in 1 ml of cold PBS, and examined by flow cytometry (FACSCalibur, Becton-Dickinson) using ModFit software (Becton-Dickinson). Transient cotransfection. Transfections were performed on C2C12 myoblasts to test the effect of hMstn treatment on Smad3 and Notch1 intracellular domain (ICD) interaction. C2C12 myoblasts were seeded on 10-cm2 plates at a density of 10,000 cells/cm2. Following a 24-h attachment period, the cells were transfected with 12.5 ␮g of each plasmid DNA (Smad3-Flag and Notch1-ICD-Myc) using Lipofectamine 2000 (Invitrogen) reagent, as per the manufacturer’s guidelines; this was followed by incubation overnight at 37°C/5% CO2. The Smad3-Flag plasmid (Addgene plasmid 11742) was provided by Dr. Liliana Attisano (16). The Notch1-ICD-Myc plasmid (Addgene plasmid 15131), provided by Dr. Constance L. Cepko (3), was subcloned into the pCDNA3 expression vector before use. Immunoprecipitation studies. For Notch1-ICD-Myc immunoprecipitation, Smad3-Flag and Notch1-ICD-Myc transfected C2C12 myoblasts (see above) were allowed to differentiate in the presence or absence of hMstn for 24 h, harvested in 200 ␮l of RIPA buffer (50 mM NaF, 0.5% Na deoxycholate, 0.1% SDS, 1% IGEPAL, 1.5 mM Na3VO4, and complete protease inhibitor; Roche Molecular Biochemicals), and centrifuged to remove cell debris. Bradford reagent (BioRad, Hercules, CA) was used to estimate total protein content to ensure equal loading. Before immunoprecipitation, 400 ␮g of total protein was precleared using 50 ␮l of a 50% protein A-agarose slurry for 1 h at 4°C. Immunoprecipitation of Notch1-ICD-Myc was per-

formed by incubating the precleared lysate with 2 ␮l of purified mouse monoclonal anti-Myc antibody (no. 2276; Cell Signaling) overnight at 4°C. Protein A-agarose (Invitrogen) (50 ␮l of 50%), washed twice with RIPA buffer, was added for 1 h at 4°C, followed by centrifugation to pellet immunoprecipitated complexes. Pellets were washed four times with cold PBS, resuspended in 50 ␮l of 1⫻ NuPAGE sample buffer (Novex), and boiled for 5 min. Immunoprecipitation samples were fractionated by SDS-PAGE and transferred to nitrocellulose membrane by electroblotting. Western blot analysis. Preparation of protein extracts from myoblasts and subsequent Western blot analysis have been described in detail (29). Details on antibodies used for Western analysis and immunoprecipitation studies are available on request. RNA isolation. RNA was isolated from hMb5 and hMb15 human myoblasts using TRIzol reagent (Invitrogen) as per the manufacturer’s protocol. Real-time PCR analysis. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. Real-time PCR was performed using the CFX96 system (Bio-Rad). Each reaction mix (20 ␮l) consisted of 2 ␮l of 5-fold diluted cDNA, 10 ␮l of 2⫻ SsoFast Evagreen (Bio-Rad) and primers at a final concentration of 100 nM. All reactions were performed using the following thermal cycle conditions: 95°C for 3 min followed by 45 cycles of a two-step reaction; denaturation at 95°C for 3 s and annealing/extension at 60°C for 20 s, followed by a denaturation curve from 60 to 95°C in 5-s increments of 0.5°C to ensure amplification specificity. Transcript levels were normalized to GAPDH, with relative gene expression fold-change calculated using the ⌬⌬CT method [threshold cycle (CT) values]. PCR primers used for real-time PCR analysis are available on request. Statistics. Results of the myoblast proliferation analysis are presented here as means ⫾ SE of eight replicates. Results presented in this paper are representative of at least two independent experiments. Total myotubes were counted in 10 random images per coverslip, and mean myotube number (⫾ SE) from three coverslips per treatment was calculated. Individual myotube area was assessed for all myotubes present in 10 random images taken from three coverslips per treatment. Myotube nuclei and total nuclei were counted in 20 random images per coverslip and mean percentage fusion index (⫾ SE) from three coverslips per treatment was calculated. Fusion index was calculated as the number of nuclei within myotubes and is expressed as a percentage of total nuclei present in each image. All differences were compared using one-way ANOVA, with P ⬍ 0.05 considered significant. RESULTS

Myostatin negatively regulates human myoblast proliferation. Treatment of hMb5 and hMb15 human myoblasts with E. coliproduced human recombinant myostatin protein (hMstn) (Fig. 1A) resulted in a statistically significant dose-dependent decrease in

Fig. 1. Myostatin inhibits human myoblast proliferation. A: Coomassie blue-stained gel showing purified human recombinant fusion myostatin protein (hMstn). A total of 1 ␮g of Ni-agarose-purified hMstn was resolved on a 4 –12% gradient polyacrylamide gel. B: representative images of proliferating human hMb5 myoblasts at 48 h, treated with (⫹) or without (⫺) increasing concentrations of hMstn (0 –10 ␮g/ml) and fixed and stained with Gill’s hematoxylin and eosin. Scale bars, 50 ␮m. C: proliferation analysis of hMb5 myoblast cultures grown under proliferating conditions in the presence of increasing concentrations of hMstn (0 –10 ␮g/ml) for 96 h and monitored by methylene blue assay. *P ⬍ 0.05 and ***P ⬍ 0.001. D: hMb5 myoblasts were cultured with 4 ␮g/ml hMstn for 96 h [96 h (⫹)] or with hMstn for 48 h followed by a further 48 h without hMstn [48 h (⫹)/48 h (⫺)] and fixed and stained with Gill’s hematoxylin and eosin. Scale bars, 50 ␮m. E: proliferation analysis of hMb5 myoblasts and C2C12 cells grown under proliferating conditions for 96 and 72 h, respectively, in the presence (⫹) or absence (⫺) of eukaryotic-produced Chinese hamster ovary cell-secreted recombinant myostatin protein (CHO-Mstn; 3.5 ng/ml) and monitored by methylene blue assay. **P ⬍ 0.01. F, left: Western blot analyses of p21 and cdk2 expression in hMb5 myoblasts grown under proliferating conditions for 24 and 48 h in the absence (⫺) or presence of 4 ␮g/ml (⫹) or 8 ␮g/ml (⫹⫹) hMstn. Tubulin expression was analyzed to ensure equal loading of samples. F, right: densitometric analysis of protein expression for Western blots (p21 and cdk2), normalized to tubulin expression. ***P ⬍ 0.001; n ⫽ 3. G: Western blot analysis of the phosphorylation state of Rb protein in hMb5 myoblasts grown under proliferating conditions for 24 and 48 h in the absence (⫺) or presence of 4 ␮g/ml (⫹) or 8 ␮g/ml (⫹⫹) hMstn. The hyperphosphorylated (pRbPP) and hypophosphorylated (pRb) forms of pRb protein are indicated. Tubulin expression was used as a loading control. AJP-Cell Physiol • VOL

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the number of myoblasts, with a maximal decrease of ⬃70% as compared with untreated controls in both hMb5 (Fig. 1C) and hMb15 (Supplemental Fig. S1A; Supplemental Material for this article is available at the Journal website) myoblasts, without resulting in overt myogenic differentiation (shown for hMb5 cells in Fig. 1B). The myostatin-mediated inhibition of myoblast proliferation was reversible, since hMb5 cultures treated with hMstn for 48 h followed by a further period of 48 h proliferation in the absence of hMstn showed increased myoblast numbers when compared with cultures treated with hMstn for the total 96 h period (Fig. 1D). Analysis of a eukaryotic-produced CHO cell-secreted recombinant myostatin protein (22) for its ability to inhibit myoblast proliferation showed that treatment of mouse C2C12 and human myoblast cultures with 3.5 ng/ml CHO cell-secreted recombinant myostatin protein (CHO-Mstn) significantly reduced the number of resulting myoblasts, with a ⬃30% reduction in the human hMb5 culture and a ⬃20% reduction in the C2C12 culture (Fig. 1E). Treatment of human primary myoblasts with myostatin leads to accumulation of myoblasts at the G1 boundary of the cell cycle. Flow cytometry (FACS analysis) to assess the effect of myostatin on cell cycle distribution revealed no increase in apoptosis of hMstn-treated human primary myoblast cultures but did reveal an increase in the percentage of cells in the G1 phase of the cell cycle, from 86% to 94% in hMstn-treated hMb15 cells, and from 90% to 93% in hMstn-treated hMb5 cells (Table 1). Concomitant with the increase in cells in the G1 phase, the number of cells in the S phase decreased from 10.5% to 0.9% in hMb15 cells and from 5.8% to 0.9% in hMb5 cells after treatment with hMstn (Table 1). Although we observed a slight increase in the percentage of cells in the G2/M phases, from 3.6% to 5.2% in hMb15 cells and from 3.9% to 5.9% in hMb5 cells (Table 1), statistical significance was only noted in hMstn-treated hMb15 cells, which suggests that the increased percentage of cells in the G2/M phases of the cell cycle may not be biologically significant with respect to hMstn-mediated inhibition of human myoblast proliferation. Myostatin regulates proliferation of human myoblasts through control of key cell cycle regulators. Western blot analysis revealed upregulation of p21 expression and inhibition of cdk2 expression in human myoblasts treated with hMstn (shown for hMb5 cells in Fig. 1F). Consistent with these findings and with myostatin-mediated inhibition of myoblast proliferation, WestTable 1. Cell cycle distribution of hMb15 and hMb5 cells following treatment with hMstn Apoptotic Cells, %

hMb15 Without hMstn With hMstn hMb5 Without hMstn With hMstn

Cells in G1, %

Cells in S, %

Cells in G2/M, %

0.00 ⫾ 0.00 85.88 ⫾ 0.23 10.50 ⫾ 0.40 3.63 ⫾ 0.41 0.00 ⫾ 0.00 93.94 ⫾ 0.95† 0.89 ⫾ 0.17† 5.18 ⫾ 0.79* 0.00 ⫾ 0.00 90.40 ⫾ 0.40 0.00 ⫾ 0.00 93.17 ⫾ 1.75†

5.75 ⫾ 0.18 3.86 ⫾ 0.39 0.91 ⫾ 0.26† 5.93 ⫾ 1.57

Data are averages ⫾ SE of quadruplicate determinations. Treatment with human recombinant myostatin protein (hMstn) resulted in accumulation of cells in the G1/S phase of the cell cycle. Percentage of cells, from 10,000 counts, in apoptotic, G1, S, or G2/M phases of the cell cycle, in hMb15 and hMb5 cells, was determined by flow cytometry analysis. *P ⬍ 0.05 and †P ⬍ 0.001, compared with respective controls (without hMstn). AJP-Cell Physiol • VOL

ern analysis also detected a myostatin dose-dependent decrease in the hyperphosphorylated form of the retinoblastoma protein (pRb), a downstream target of cdk2, with accumulation of the active hypophosphorylated form of pRb (Fig. 1G). Myostatin negatively regulates human myoblast differentiation. Treatment of hMb5 and hMb15 cells with hMstn during differentiation led to a myostatin dose-dependent inhibition of myotube formation in both hMb5 and hMb15 myoblasts (Fig. 2A and Supplemental Fig. S1B, respectively), with lower myostatin concentrations leading to a proportionate increase in myotube number, while increasing concentrations led to a steady decline (maximal ⬃84% and ⬃77% decrease in myotube number in hMb5 and hMb15 cells, respectively). Next we analyzed myotube fusion index, and as shown in Fig. 2B and Supplemental Fig. S1C, addition of hMstn resulted in a dose-dependent reduction in myotube fusion index, with a maximal decrease of ⬃84% and ⬃64% in hMb5 and hMb15 cells, respectively (Fig. 2B and Supplemental Fig. S1C). These data demonstrate that myostatin negatively regulates progression of myogenic differentiation in human primary myoblast cultures. Myostatin inhibition of myoblast differentiation is reversible. hMb5 and hMb15 myoblasts cultured for 72 h with hMstn followed by a period of 72 h without hMstn showed a dramatic ⬃480% and ⬃240% increase in myotube number, respectively, as compared with that observed in human myoblasts cultured for 144 h in the continuous presence of hMstn (8 ␮g/ml) (Fig. 2C and Supplemental Fig. S1D). Thus, myostatin-mediated inhibition of human myoblast differentiation is reversible. Myostatin regulates expression of genes involved in progression of differentiation. Western blot analysis of myogenic gene expression in human myoblasts cultured under differentiating conditions with or without addition of hMstn protein (Fig. 2D and Supplemental Fig. S1E) revealed decreased MyoD expression at all time points analyzed in the myostatin-treated cells, consistent with the inhibited differentiation observed after addition of myostatin, whereas expression of p21 was upregulated in both hMb5 and hMb15 myoblasts following addition of myostatin, consistent with results during myoblast proliferation (Fig. 1F) and with the block in cell cycle progression/ accumulation of cells in the G1 phase (Table 1). Analysis of the gene expression patterns of additional muscle regulatory proteins following treatment with myostatin revealed reduced expression of myogenin, myosin light chain (MyLC) and myosin heavy chain (MyHC) at all time points analyzed (Fig. 2D and Supplemental Fig. S1E), in keeping with downregulation of MyoD and inhibition of differentiation. Enhanced Smad 3 phosphorylation during differentiation of myostatin-treated cells was also observed (Fig. 2E), in agreement with previous data (17) demonstrating that myostatin specifically activates Smad3 during myostatin-mediated inhibition of myogenic differentiation. Myostatin signals through the Notch pathway to negatively regulate differentiation of human myoblast cultures. The activated Notch signaling pathway has been shown to inhibit MyoD, myogenin, and MyLC2 expression, resulting in impaired myotube formation during myogenesis (8, 9, 15, 24). To determine whether myostatin signals through the Notch pathway to regulate myogenesis, human myoblasts were induced to differentiate through serum withdrawal and treated with or without hMstn in the absence (0.05% DMSO) or presence of the ␥-secretase inhibitor L-685,458 (1 ␮M). As shown in Fig. 3A and

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Fig. 2. Myostatin inhibits human myoblast differentiation. A: representative images of hMb5 human myoblasts at 72 h differentiation, treated with (⫹) or without (⫺) increasing concentrations of hMstn (0 –10 ␮g/ml) and fixed and stained with Gill’s hematoxylin and eosin. Scale bars, 50 ␮m. Corresponding graph shows the mean myotube number (⫾ SE) over 3 coverslips per treatment for hMb5 cells; total myotubes were counted in 10 random images per coverslip. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001. B: quantification of fusion index in hMb5 myoblasts at 72 h differentiation following treatment with (⫹) or without (⫺) increasing concentrations of hMstn (0 –10 ␮g/ml). The graph shows the mean percentage fusion index (⫾ SE) over 3 coverslips per treatment for hMb5 cells; myotube nuclei, as well as total nuclei, were counted in 20 random images per coverslip. ***P ⬍ 0.001. C: representative images of hMb5 myoblasts differentiated with either 8 ␮g/ml hMstn for 144 h [144 h hMstn] or with hMstn for 72 h followed by a further 72 h without hMstn [72 h hMstn] and fixed and stained with Gill’s hematoxylin and eosin. Scale bars, 50 ␮m. Corresponding graph shows the average myotube number (⫾ SE) over 3 coverslips per time point for hMb5 cells; total myotubes were counted in 10 random images per coverslip. **P ⬍ 0.01. D: Western blot analysis of MyoD, p21, myogenin, myosin light chain (MyLC), and myosin heavy chain (MyHC) expression in hMb5 cells induced to differentiate and treated with (⫹) or without (⫺) hMstn (8 ␮g/ml) for 48, 72, and 96 h. Tubulin was used as a loading control. E: Western blot analysis of pSmad3 expression in hMb15 cells induced to differentiate and treated with (⫹) or without (⫺) hMstn (8 ␮g/ml) for 48 h. Tubulin was used as a loading control. Graph shows results of densitometric analysis of pSmad3 protein expression, normalized to tubulin expression. ***P ⬍ 0.001, n ⫽ 3.

Supplemental Fig. S2A, i and iii, differentiation of human cultures treated with hMstn was impaired as compared with untreated controls. Consistent with a role for the Notch pathway in inhibition of differentiation, treatment with L-685,458 alone resulted in enhanced differentiation (Fig. 3A and SupAJP-Cell Physiol • VOL

plemental Fig. S2A, ii) and an increase in myotube area (data not shown). Furthermore, addition of L-685,458 to hMstntreated cultures resulted in a rescue of myostatin-mediated inhibition of differentiation (Fig. 3A and Supplemental Fig. S2A, iv), with a 500% increase observed in the number of very

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Fig. 3. Inhibition of Notch signaling alleviates myostatin-mediated inhibition of differentiation. A: representative images of hMb5 cells at 72 h differentiation, treated with (iii and iv) or without (i and ii) hMstn (4 ␮g/ml) in the absence (i and iii) or presence (ii and iv) of the ␥-secretase inhibitor L-685,458. Cells were fixed and stained with Gill’s hematoxylin and eosin. Scale bars, 50 ␮m. B: corresponding graph shows the frequency distribution of myotube area (␮m2), assessed for all myotubes present in 10 random images per coverslip, over 3 coverslips per treatment. C, left: Western blot analyses of myogenin and MyLC expression in cells induced to differentiate and treated with (⫹) or without (⫺) hMstn (4 ␮g/ml) in the absence (⫺) or presence (⫹) of L-685,458. Tubulin was used as a loading control. C, right: densitometric analysis of Western blots (myogenin and MyLC), normalized to tubulin expression, in cells induced to differentiate and treated with (⫹) or without (⫺) hMstn (4 ␮g/ml) in the presence (⫹) or absence (⫺) of L-685,458. ***P ⬍ 0.001, n ⫽ 3.

large (⬎30,000 ␮m2) myotubes in hMb15 cells as compared with cells treated with hMstn alone (Supplemental Fig. S2B). In hMb5 cells, treatment with hMstn alone completely blocked the formation of very large myotubes (⬎30,000 ␮m2), which formed only as a result of L-685,458 treatment (Fig. 3B). Western blot analysis revealed inhibited expression of both myogenin and MyLC expression in hMstn-treated cells (Fig. 3C and Supplemental Fig. S2C), comparable to that shown in Fig. 2, but increased expression of both proteins in L-685,458treated cells, consistent with the enhanced differentiation observed upon treatment with L-685,458 (Fig. 3A and Supplemental Fig. S2A, ii). Furthermore, addition of L-685,458 to hMstn-treated hMb15 cultures restored expression of myogenin and MyLC to levels comparable to those of untreated controls (Supplemental Fig. S2C) and, in hMstn-treated hMb5 cells, enhanced expression of both proteins to levels above those of untreated controls (Fig. 3C). AJP-Cell Physiol • VOL

Real-time PCR analysis of the expression of Notch downstream target genes in human myoblasts treated with hMstn during differentiation indicated increased expression of Hey1 in hMb5 cells (Fig. 4A) and increased expression of Hes1, Hes5, and Hey1 genes in hMb15 cells (Supplemental Fig. S3) at 12 h after treatment with hMstn; at 24 h after hMstn treatment, Hes1, Hes5, and Hey1 expression was increased in hMb5 cells (Fig. 4A), while only Hes5 was upregulated in hMb15 cells (Supplemental Fig. S3). Because myostatin signals through Smad3 to regulate the progression of myogenesis, we tested whether myostatinmediated inhibition of differentiation promotes interactions between Smad3 and Notch1. C2C12 myoblasts were cultured and transiently cotransfected with Smad3-Flag and Notch1-ICD-Myc expression constructs, induced to differentiate through serum withdrawal, and treated with or without hMstn (2.5 ␮g/ml) for 24 h. Notch1-ICD-Myc protein

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Fig. 4. Myostatin regulates expression of Notch downstream target genes. A: hMb5 myoblast cultures were induced to differentiate and were treated with or without hMstn (10 ␮g/ml) for 12 h and 24 h. Real-time PCR analysis was performed to analyze the expression of Hes1, Hes5, and Hey1 in hMb5 cells. Graphs show fold change (⫾ SE) normalized to GAPDH in 2 independent experiments. *P ⬍ 0.05 and **P ⬍ 0.01. B: Western blot analysis (top left) of Smad3-Flag coimmunoprecipitated with Notch1-ICD-Myc from transfected C2C12 myoblasts cultured in differentiation medium for 24 h in the presence (⫹) or absence (⫺) of hMstn (2.5 ␮g/ml). Protein was immunoprecipitated with mouse monoclonal anti-Myc antibody, resolved by 4 –12% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a mouse monoclonal anti-Flag antibody. Western blot analysis (bottom left) confirms comparable levels of Smad3-Flag and Notch1-ICD-Myc in the total protein extracts used for immunoprecipitation. Right: densitometric analysis of Smad3 protein coimmunoprecipitated with Notch1-ICD-Myc from transfected C2C12 myoblasts cultured in differentiation medium for 24 h in the presence (⫹) or absence (⫺) of hMstn (2.5 ␮g/ml), normalized to the total amount of Notch1-ICD-Myc used for immunoprecipitation. **P ⬍ 0.01, n ⫽ 3.

was specifically immunoprecipitated using anti-Myc antibodies and analyzed by Western blot for the coimmunoprecipitation of Smad3-Flag using specific anti-Flag antibodies. As shown in Fig. 4B, 24-h treatment with hMstn resulted in an increase in the amount of Smad3 that coimmunoprecipitated with Notch1-ICD. Western blot analysis on total cell lysates revealed comparable levels of Smad3-Flag and Notch1-ICD-Myc in the extracts used for immunoprecipitation analysis (Fig. 4B). These data suggest that myostatin signaling during differentiation results in enhanced binding of Smad3 to Notch1-ICD, consistent with the increased activation of Smad3 by myostatin (Fig. 2E). DISCUSSION

The present study was conducted in an effort to improve the sparse knowledge base on the function of human myostatin and its mechanism(s) of action in regulating human skeletal muscle myogenesis. A better understanding of this protein assumes particular relevance in contemplating human clinical trials with myostatin antagonists. To study the role of myostatin in regulating human skeletal muscle myogenesis, we have treated human primary myoblast cultures with E. coli-produced human recombinant myostatin protein (hMstn). It is important to highlight that the concentrations of hMstn protein used in this study are higher than previously reported (34). Dynamic Light Scattering analysis suggests that a considerable portion of the E. coli-produced hMstn protein that was used in this study exists in an inactive multimeric form rather than in the active AJP-Cell Physiol • VOL

dimer form (data not shown), therefore a higher concentration of hMstn protein is required to elicit biological function. Using human primary myoblast models and recombinant human myostatin, we show here that human myostatin regulates both steps of myogenesis, i.e., proliferation and differentiation, through independent signaling mechanisms. Consistent with findings in other mammalian myoblast models, human myostatin activates Smad3 signaling in human myoblasts. Myostatin-mediated inhibition of myoblast proliferation is associated with enhanced expression of the cyclin-dependent kinase inhibitor, p21; moreover, addition of myostatin further results in reduced expression of the p21 target, cdk2, with associated accumulation of active hypophosphorylated Rb, which then binds to and inhibits genes critical to G1/S transition, thereby blocking cell cycle progression (4). In fact, we observed increased accumulation of human myoblasts in the G1 phase of the cell cycle. The findings to date raise the possibility that the hyperplasia observed in humans (25) with a mutation in the myostatin gene is due to deregulation of the G1/S transition of the cell cycle, resulting in enhanced myoblast number and, ultimately, increased muscle fiber number (hyperplasia). We also tested for a possible interaction between myostatin and Notch signaling during regulation of human myoblast differentiation. Interestingly, blockade of the Notch pathway through treatment with the ␥-secretase inhibitor L-685,458 rescued myostatin-mediated inhibition of human myoblast differentiation, as determined on the basis of an increase in

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myotube area and rescue of myogenin and MyLC expression. Myostatin-mediated inhibition of human myoblast differentiation also resulted in increased expression of the Notch downstream targets genes Hes1, Hes5, and Hey1 in both the hMb5 and hMb15 human myoblasts. Data from our study as well as from previously published reports (17, 31) demonstrate that myostatin signals through Smad and ERK1/2 MAPK pathways to regulate the progression of differentiation, suggesting that the Notch signaling pathway may work in conjunction with Smad and/or ERK1/2 signaling to facilitate myostatin-mediated inhibition of differentiation. Cross talk between the Notch pathway and other pathways, such as Smad, in response to myostatin seems highly likely, consistent with our findings here that treatment with myostatin during myoblast differentiation enhanced the interaction between Smad3 and Notch1 ICD. Moreover, transforming growth factor-␤ has been shown to upregulate expression of the Notch downstream target Hes1 in a Notch-dependent manner resulting from direct interaction of Smad3 with Notch1-ICD (7). Smad1 interaction with Notch1-ICD is also critical for bone morphogenetic protein-4mediated regulation of Notch downstream target genes and inhibition of myogenic differentiation (10). Notch-mediated inhibition of myogenic differentiation has been reported to occur through two pathways: CSL-independent (27), which appears to be a generic block that is not cell type-specific, and CSL-dependent, which involves a mechanism that targets downstream mediators of Notch signaling resulting in inhibition of critical myogenic regulatory factors including MyoD (24). CSL-dependent signaling results in upregulation of Notch downstream targets such as Hes1, a feature consistent with findings upon myostatin treatment, and leads to inhibition of differentiation through targeting MyoD, which cannot be rescued through further overexpression of MyoD (24). Indeed, we also found that myostatin negatively regulates the expression of MyoD during inhibition of human myoblast differentiation; the inability of forced MyoD expression to alleviate myostatin repression of myogenic differentiation has been documented previously (17). Thus, myostatinmediated inhibition of differentiation might involve Smad3/ Notch1 interactions that result in CSL-dependent upregulation of Notch downstream targets, including Hes1, ultimately leading to inhibition of MyoD and a block in the progression of myogenic differentiation. Together, our data identify a mechanism of action of myostatin in regulating human myogenesis whereby myostatin negatively regulates human myoblast proliferation through targeting of key cell cycle regulators, and regulates myoblast differentiation, through inhibiting the expression of critical myogenic regulatory factors, through a mechanism involving both canonical Smad signaling and the Notch signaling pathway. ACKNOWLEDGMENTS The MF 20 (MyHC) monoclonal antibody developed by Dr. Donald A. Fischman and the T14 (MyLC) monoclonal antibody developed by Dr. Frank E. Stockdale were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AJP-Cell Physiol • VOL

REFERENCES 1. Amthor H, Huang R, McKinnell I, Christ B, Kambadur R, Sharma M, Patel K. The regulation and action of myostatin as a negative regulator of muscle development during avian embryogenesis. Dev Biol 251: 241– 257, 2002. 2. Amthor H, Otto A, Macharia R, McKinnell I, Patel K. Myostatin imposes reversible quiescence on embryonic muscle precursors. Dev Dyn 235: 672–680, 2006. 3. Bao ZZ, Cepko CL. The expression and function of Notch pathway genes in the developing rat eye. J Neurosci 17: 1425–1434, 1997. 4. Beijersbergen RL, Bernards R. Cell cycle regulation by the retinoblastoma family of growth inhibitory proteins. Biochim Biophys Acta 1287: 103–120, 1996. 5. Benabdallah BF, Bouchentouf M, Rousseau J, Tremblay JP. Overexpression of follistatin in human myoblasts increases their proliferation and differentiation, and improves the graft success in SCID mice. Cell Transplant 18: 709 –718, 2009. 6. Bigot A, Jacquemin V, Debacq-Chainiaux F, Butler-Browne GS, Toussaint O, Furling D, Mouly V. Replicative aging down-regulates the myogenic regulatory factors in human myoblasts. Biol Cell 100: 189 –199, 2008. 7. Blokzijl A, Dahlqvist C, Reissmann E, Falk A, Moliner A, Lendahl U, Ibanez CF. Cross-talk between the Notch and TGF-beta signaling pathways mediated by interaction of the Notch intracellular domain with Smad3. J Cell Biol 163: 723–728, 2003. 8. Buas MF, Kabak S, Kadesch T. Inhibition of myogenesis by Notch: evidence for multiple pathways. J Cell Physiol 218: 84 –93, 2009. 9. Buas MF, Kabak S, Kadesch T. The Notch effector Hey1 associates with myogenic target genes to repress myogenesis. J Biol Chem 285: 1249 – 1258. 10. Dahlqvist C, Blokzijl A, Chapman G, Falk A, Dannaeus K, Ibanez CF, Lendahl U. Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation. Development 130: 6089 –6099, 2003. 11. Furling D, Coiffier L, Mouly V, Barbet JP, St Guily JL, Taneja K, Gourdon G, Junien C, Butler-Browne GS. Defective satellite cells in congenital myotonic dystrophy. Hum Mol Genet 10: 2079 –2087, 2001. 12. Gonzalez-Cadavid NF, Taylor WE, Yarasheski K, Sinha-Hikim I, Ma K, Ezzat S, Shen R, Lalani R, Asa S, Mamita M, Nair G, Arver S, Bhasin S. 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, 1998. 13. Jacquemin V, Furling D, Bigot A, Butler-Browne GS, Mouly V. IGF-1 induces human myotube hypertrophy by increasing cell recruitment. Exp Cell Res 299: 148 –158, 2004. 14. Kambadur R, Sharma M, Smith TP, Bass JJ. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res 7: 910 –916, 1997. 15. Kuroda K, Tani S, Tamura K, Minoguchi S, Kurooka H, Honjo T. Delta-induced Notch signaling mediated by RBP-J inhibits MyoD expression and myogenesis. J Biol Chem 274: 7238 –7244, 1999. 16. Labbe E, Silvestri C, Hoodless PA, Wrana JL, Attisano L. Smad2 and Smad3 positively and negatively regulate TGF beta-dependent transcription through the forkhead DNA-binding protein FAST2. Mol Cell 2: 109 –120, 1998. 17. Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem 277: 49831–49840, 2002. 18. McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R. Myostatin negatively regulates satellite cell activation and self-renewal. J Cell Biol 162: 1135–1147, 2003. 19. McCroskery S, Thomas M, Platt L, Hennebry A, Nishimura T, McLeay L, Sharma M, Kambadur R. Improved muscle healing through enhanced regeneration and reduced fibrosis in myostatin-null mice. J Cell Sci 118: 3531–3541, 2005. 20. McFarlane C, Hennebry A, Thomas M, Plummer E, Ling N, Sharma M, Kambadur R. Myostatin signals through Pax7 to regulate satellite cell self-renewal. Exp Cell Res 314: 317–329, 2008. 21. McFarlane C, Plummer E, Thomas M, Hennebry A, Ashby M, Ling N, Smith H, Sharma M, Kambadur R. Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism. J Cell Physiol 209: 501–514, 2006.

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MYOSTATIN AND HUMAN MYOGENESIS 22. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387: 83–90, 1997. 23. McPherron AC, Lee SJ. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94: 12457–12461, 1997. 24. Nofziger D, Miyamoto A, Lyons KM, Weinmaster G. Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts. Development 126: 1689 –1702, 1999. 25. Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, Komen W, Braun T, Tobin JF, Lee SJ. Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350: 2682–2688, 2004. 26. Sharma M, Kambadur R, Matthews KG, Somers WG, Devlin GP, Conaglen JV, Fowke PJ, Bass JJ. Myostatin, a transforming growth factor-beta superfamily member, is expressed in heart muscle and is upregulated in cardiomyocytes after infarct. J Cell Physiol 180: 1–9, 1999. 27. Shawber C, Nofziger D, Hsieh JJ, Lindsell C, Bogler O, Hayward D, Weinmaster G. Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 122: 3765–3773, 1996. 28. Shishkin SS, Krokhina TB, Akhunov VS, Makarov AA, Popov VO. [Effects of myostatin and other growth factors on cultured human cells]. Prikl Biokhim Mikrobiol 40: 630 –633, 2004. 29. Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R. Myostatin, a negative regulator of muscle growth,

AJP-Cell Physiol • VOL

30.

31.

32.

33.

34.

C203

functions by inhibiting myoblast proliferation. J Biol Chem 275: 40235–40243, 2000. Yaffe D, Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270: 725–727, 1977. Yang W, Chen Y, Zhang Y, Wang X, Yang N, Zhu D. Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway is involved in myostatin-regulated differentiation repression. Cancer Res 66: 1320 –1326, 2006. Yarasheski KE, Bhasin S, Sinha-Hikim I, Pak-Loduca J, GonzalezCadavid NF. Serum myostatin-immunoreactive protein is increased in 60 –92 year old women and men with muscle wasting. J Nutr Health Aging 6: 343–348, 2002. Zanotti S, Saredi S, Ruggieri A, Fabbri M, Blasevich F, Romaggi S, Morandi L, Mora M. Altered extracellular matrix transcript expression and protein modulation in primary Duchenne muscular dystrophy myotubes. Matrix Biol 26: 615–624, 2007. Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tomkinson KN, McPherron AC, Wolfman NM, Lee SJ. Induction of cachexia in mice by systemically administered myostatin. Science 296: 1486 –1488, 2002.

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