Extracellular Signal–Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Pathway Is Involved in Myostatin-Regulated Differentiation Repression

Research Article

Wei Yang,1 Yan Chen,3 Yong Zhang,1 Xueyan Wang,1 Ning Yang,2 and Dahai Zhu1,3

1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; 2Department of Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China; and 3Molecular and Cellular Developmental Biology Laboratory, Harbin Institute of Technology, Harbin, China

Abstract Myostatin knockout mice and cattle with genetic mutations in myostatin gene were characterized by a widespread increase in The cytokines of transforming growth factor B (TGF-B) and its skeletal muscle mass with hyperplasia and hypertrophy (1, 2). superfamily members are potent regulators of tumorigenesis Similarly, the blockade of endogenous myostatin in mdx mice and multiple cellular events. Myostatin is a member of (a Duchenne muscular dystrophy model) resulted in increases of TGF-B superfamily and plays a negative role in the control muscle mass, size, and strength along with a significant decrease in of cell proliferation and differentiation. We now show that muscle degeneration (3, 4). Recently, the reduced level of myostatin myostatin rapidly activated the extracellular signal–regulated protein due to missplicing was identified to be associated with kinase 1/2 (Erk1/2) cascade in C2C12 myoblasts. A more enlarged muscles mass and the development of unusual strength remarkable Erk1/2 activation stimulated by myostatin was in humans (5). On the other hand, systematical administration of observed in differentiating cells than proliferating cells. The exogenous myostatin to adult mice induced severe muscle and fat results also showed that Ras was the upstream regulator loss analogous to human cachexia syndromes (6). Importantly, and participated in myostatin-induced Erk1/2 activation the increased myostatin expression was associated with skeletal because the expression of a dominant-negative Ras prevented muscle degeneration–related diseases, such as chronic illnesses, myostatin-mediated inhibition of Erk1/2 activation and HIV infection, and the aging process (7–9). These genetic and proliferation. Importantly, the myostatin-suppressed myotube clinical analyses showed that myostatin is a negative regulator for fusion and differentiation marker gene expression were skeletal muscle growth. To date, a number of human and animal attenuated by blockade of Erk1/2 mitogen-activated protein disorders are associated with loss of or functionally impaired kinase (MAPK) pathway through pretreatment with MAPK/ muscle tissue. Therefore, therapies based on manipulating the Erk kinase 1 (MEK1) inhibitor PD98059, indicating that biological activities of myostatin would significantly improve the myostatin-stimulated activation of Erk1/2 negatively regulates quality of life for these patients (10). myogenic differentiation. Activin receptor type IIb (ActRIIb) Myostatin was able to arrest the cell cycle at the G phase of was previously suggested as the only type II membrane 1 cultured myoblasts and then induced inhibition of proliferation receptor triggering myostatin signaling. In this study, by using and differentiation. Further investigation showed that the myo- synthesized small interfering RNAs and dominant-negative statin-stimulated cell cycle arrest was probably due to an increased ActRIIb, we show that myostatin failed to stimulate Erk1/2 expression of cyclin-dependent kinase (Cdk) inhibitor p21Waf1,Cip1 phosphorylation and could not inhibit myoblast differentia- and a decreased cyclin E/Cdk2 activity, which further caused the tion in ActRIIb-knockdown C2C12 cells, indicating that hypophosphorylation of Rb protein (11). It was also reported ActRIIb was required for myostatin-stimulated differentiation that myostatin inhibited rhabdomyosarcoma cell proliferation suppression. Altogether, our findings in this report provide the through an Rb-independent pathway (12). MyoD and myogenin first evidence to reveal functional role of the Erk1/2 MAPK were implicated to participate in myostatin-induced differentiation pathway in myostatin action as a negative regulator of muscle suppression (10). Although the biological roles of myostatin on cell growth. (Cancer Res 2006; 66(3): 1320-6) skeletal muscle development were well established (10), the intracellular signaling pathways involved in myostatin function Introduction are poorly defined. Like the other members of the TGF-h Transforming growth factor h (TGF-h) and the signaling superfamily, myostatin was shown to elicit its function by transducers in TGF-h pathway are potent suppressors during regulating a TGF-h-like signaling pathway through activin type tumorigenesis for their regulatory roles on cell proliferation, IIb (ActRIIb), activin type Ib (ActRIb), or TGF-h type I receptors differentiation, and apoptosis. Myostatin, also named growth and (13, 14), followed by activation of the R-Smads. Recently, p38 differentiation factor 8, is a member of the TGF-h superfamily (1). mitogen-activated protein kinase (MAPK) was reported to participate in myostatin-regulated signal transduction (15). Philip et al. have provided evidence that myostatin activated p38 MAPK through the TGF-h-activated kinase 1 and this activation was Note: W. Yang and Y. Chen contributed equally to this work. Requests for reprints: Dahai Zhu, National Laboratory of Medical Molecular independent of Smads. Together, these findings suggest that the Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences complexity of the myostatin signal transduction may be due to and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, P.R. China. the multiple signaling cascades and precise integration of these Phone: 86-01-6529-6949; E-mail: [email protected]. I2006 American Association for Cancer Research. signaling modules plays an important role in regulating cell pro- doi:10.1158/0008-5472.CAN-05-3060 liferation and differentiation.

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Myogenesis is a complex and highly organized process that TTTTTGTTCGTACATCCAAAAGGCCAG-3V) containing a myc tag and a involves a two-step mechanism. First, precursor cells become ClaI site, and then cloned into retroviral vector pLNCX according to committed to the myogenic lineage; second, after proliferating, standard procedure. myoblasts begin to withdraw from the cell cycle and undergo Retrovirus production and infection. Retrovirus was produced by transient transfection of retroviral constructs into the Phoenix helper-free terminal differentiation by fusing into multinucleated myotubes retrovirus producer cell line (gift from Dr. Gary Nolan, Stanford University, (16, 17). The myogenic regulatory factors (including MyoD, MRF4, Stanford, CA) with calcium phosphate method according to the standard Myf5, and myogenin) and members of the MEF2 family play a key protocol (http://www.stanford.edu/group/nolan/). For infection of C2C12 role during embryonic myogenesis and postnatal skeletal muscle myoblasts, the retroviral supernatant (48 hours posttransfection) was filtered growth and repair (18, 19). It has been well documented that by and added into each C2C12 plate with addition of 3 Ag/mL polybrene and negatively regulating myogenic regulatory factors and their the cells were placed at 37jC with an overnight incubation for infection. coregulators during myogenesis, both basic fibroblast growth Antibodies and Western blot. Antibodies against phospho-MEK1, RSK factor (bFGF) and TGF-h are potent inhibitors for myoblast phospho-Erk1/2, phospho-p90 , phospho-Elk1, Erk1/2, ActRIIb, and Ras h differentiation (20, 21). Although the Ras/MAPK-extracellular were from Cell Signaling; antitubulin, -actin, myosin heavy chain (MyHC), signal–regulated kinase (Erk) kinase 1 (MEK1)/Erk signaling MyoD, myogenin, c-myc, and horseradish peroxidase (HRP)–conjugated second antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). For pathway has been proposed to be involved in myogenic suppression h cell lysate preparation, monolayer cells on a 100-mm plate were lysed with induced by bFGF and TGF- , the molecular basis by which those 1.2 mL of lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5% growth factors elicit their myogenic inhibition remains largely NP40, 50 mmol/L NaF, 1 mmol/L Na3VO4, 5 mmol/L h-glycerophosphate, unknown. The MAPK-mediated signal transduction is crucial 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride]. The lysate for muscle development and maintenance of striated muscle was clarified by centrifugation at 14,000 Â g for 20 minutes. For protein homeostasis (22, 23). The core component of MAPK module is a extraction from tissues, 0.2 g of tissue was rapidly homogenized in 0.5 mL of set of three acting protein kinases that include Erk, c-Jun NH2- homogenization buffer [50 mmol/LTris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5% h terminal kinases/stress-activated protein kinases, and p38 MAPKs. NP40, 50 mmol/L NaF, 1 mmol/L Na3VO4, 5 mmol/L -glycerophosphate, bFGF and TGF-h suppress myogenesis through a common 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride] and the lysate was mechanism of the activation of Ras-MEK-Erk module (23). clarified by centrifugation at 14,000 Â g for 20 minutes. Boiled samples with 2Â SDS loading buffer were loaded onto 12% polyacrylamide gel and, after Myostatin is a member of the TGF-h superfamily whereas TGF-h electrophoresis, the proteins were transferred onto polyvinylidene difluoride inhibits myogenic differentiation by activating multiple signaling membrane (PALL, East Hills, NY). The resulting blots were blocked with pathways including Ras/MEK/Erk and p38. Therefore, we ask 1% BSA for phospho-protein antibodies and with 5% milk for non-phospho- whether the Ras/MEK/Erk pathway is involved in myostatin signaling protein antibodies for 1 hour, and then incubated for another 1 hour at room and the possible role of this pathway in myostatin-regulated temperature with primary antibody. The secondary antibody used in the myogenesis. In this report, we provide biochemical evidence to immunoblot was a 1:5,000 dilution of HRP-linked anti–immunoglobulin G show that myostatin rapidly induced a sustained Erk1/2 activation (IgG). The enhanced chemiluminescence reagent (Amersham Biosciences, in C2C12 cells through ActRIIb. Furthermore, ActRIIb was required Piscataway, NJ) was used as the substrate for detection and the membrane for inhibition of Erk1/2 activation and differentiation in C2C12 was exposed to an X-ray film for visualization. cells in response to myostatin. Our results reveal a new mechanism Immunofluorescence. The cells were fixed with 4% (w/v) paraformal- dehyde in PBS and then washed thrice with permeabilization buffer (0.3% of myostatin-regulated myogenesis inhibition from the involvement Triton X-100 in PBS) for 10 minutes each time and blocked with 3% (w/v) of the Ras/MEK/Erk pathway mediated by ActRIIb. BSA (Calbiochem, San Diego, CA) in PBS. The samples were incubated for 2 hours at room temperature with anti-MyHC antibody (1:100) and for 1 hour with FITC-conjugated secondary antibodies against mouse IgG (Santa Cruz Materials and Methods Biotechnology). Finally, the cells were washed with PBS and mounted onto Cell culture and animals. Mouse C2C12 myoblasts were maintained in microscope glass slides. 4V,6-Diamidino-2-phenylindole (DAPI) staining was DMEM supplemented with 4.5 g/L glucose, 4 mmol/L L-glutamine, 10% fetal done simultaneously to show the position of the nuclei. bovine serum (FBS; Hyclone, Logan, UT), and penicillin/streptomycin at Small interfering RNA. The target sequences of double-stranded 37jC in a 5% CO2 atmosphere. The myogenic differentiation in vitro was nucleotides used for small interfering RNA (siRNA) knockdown are induced by the change of 10% FBS to 2% horse serum (Hyclone) in medium. GGCTCAGCTCATGAACGAC for ActRIIb (Ambion) and ATCCAATGG- The exponential proliferating cells or differentiating cells were treated CACCGTCAAG for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; with purified recombinant myostatin as described (24, 25) at the final RIBOBIO, Guangzhou, Guangdong, China). Cells cultured in a six-well concentration of 500 ng/mL for the indicated time, and the cells treated plate (2 Â 105 per well) were transfected with 70 Amol/L of siRNAs with with the same amount of PBS or with 1 nmol/L bFGF were used as controls. Lipofectimin 2000 (Invitrogen, Carlsbad, CA). Forty-eight hours posttrans- For Erk1/2 MAPK pathway inhibition, cells were pretreated from 30 minutes fection, cells were treated with myostatin, bFGF, or PBS according to to 1 hour with 10 Amol/L of MEK1 inhibitor PD98059 (Cell Signaling, experimental design. Beverly, MA). For animal experiments, 4-week-old C57 female mice were Proliferation assay. About 2 Â 105 cells were seeded in six-well plates given i.m. injections of 0.1 mg/kg recombinant myostatin or equivalent and maintained in DMEM culture medium for retrovirus infection and volume of PBS supplemented with 5% bovine serum albumin (BSA) every myostatin stimulation. During the last 12 hours of each treatment, 5 ACi of 2 days for 2 weeks. The animals were maintained on standard diet in [3H]thymidine were added to the culture medium. The cells were washed accordance with the Animal Care and Use Committee policy of the school thrice with PBS and trypsinized for radioactivity measurement in and killed by cervical dislocation. The gastrocnemius was collected scintillation vials. The assay was done in three replicates and repeated separately and stored at À70jC for protein isolation. thrice for statistical analysis. Constructs. The retroviral construct carrying dominant-negative Ras17N was a gift from Dr. D.M. Tang (McMaster University, Hamilton, Ontario, Results Canada). The dominant-negative ActRIIb (truncated form lacking intracellular kinase domain) was PCR amplified with the forward primer Myostatin activates Erk1/2 MAPK signaling pathway. It has (5V-CCGCTCGAGATGACGGCGCCCTGGG-3V) containing an XhoI site and been recently reported that various MAPK cascades can be the reverse primer (5V-CCATCGATTCACAGATCCTCTTCTGAGATGAG- activated by members of the TGF-h superfamily in several cell www.aacrjournals.org 1321 Cancer Res 2006; 66: (3). February 1, 2006

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research types, indicating that activation of these pathways by TGF-h exerts critical roles in mediating growth, differentiation, and apoptosis (26–28). To determine whether the Erk1/2 MAPK pathway is involved in myostatin signaling, the proliferating and differentiating C2C12 cells were treated with recombinant myostatin for the time periods of 5 minutes to 24 hours, and bFGF was used as a positive reference for activation of Erk1/2 MAPK in myoblasts. As shown in Figs. 1A and 2A, myostatin activated Erk1/2 within 5 minutes and this activation persisted for up to 24 hours of treatment in both proliferating and differentiating C2C12 cells. A more obvious and rapid myostatin-induced Erk1/2 activation was more evident in differentiating cells than in proliferating cells (Figs. 1A and 2A), which suggests that in low-serum condition, myostatin had more potency to activate Erk1/2. At least 10-fold induction of Erk1/2 phosphorylation by myostatin was detected and the total levels of Erk1/2 proteins remained unchanged. As expected, myostatin-induced Erk1/2 activation was further supported by comparably increased phosphorylation levels of MEK1 and p90RSK, the upstream and downstream molecules in this pathway, in a manner similar with Erk1/2 (Figs. 1A and 2A). Myostatin plays a very important role in regulating postnatal muscle growth by controlling the satellite cell quiescence (29). We

Figure 2. Myostatin activates Erk1/2 signaling pathway in proliferating C2C12 cells and skeletal muscle. A, the proliferating C2C12 cells were stimulated with myostatin (500 ng/mL) for different time intervals and cell lysates were immunoblotted with anti-phospho-MEK1, phospho-Erk1/2, and phospho-p90RSK antibodies. Equal loading was monitored by blotting the membranes with antibodies against Erk1/2 total protein and tubulin, respectively. B, the relative activation of Erk1/2 by myostatin in proliferating C2C12 cells was quantitated by densitometry. C, four-week-old C57 female mice were systematically given i.m. injections of 0.1 mg/kg recombinant myostatin or equivalent volume of PBS supplemented with 5% BSA every 2 days for 2 weeks. The gastrocnemius was homogenized and the phosphorylation level of MEK1, Erk1/2, and Elk1 was measured by Western blots. D, mouse fibroblast NIH 3T3 cells were stimulated with myostatin (500 ng/mL) or PBS for 12 hours and the phosphorylation level of Erk1/2 was measured with anti-phospho-Erk1/2 antibody.

then tested the activation of Erk1/2 by myostatin in adult mouse skeletal muscle tissue in vivo. Four-week-old C57 mice were systematically administrated with myostatin as described in Materials and Methods. The immunoblots showed that myostatin induced an f3-fold activation of Erk1 and the upstream kinase MEK1 was also activated by myostatin (Fig. 2C). Interestingly, Figure 1. Myostatin induces the activation of Erk1/2 signaling pathway in RSK differentiating C2C12 cells. A, C2C12 cells were cultured in differentiation instead of p90 activation by myostatin observed in the cultured medium (DMEM, 2% horse serum) for 1 day and stimulated with 500 ng/mL cells, another Erk1/2-regulated downstream transcription factor, myostatin (Mstn) for different time intervals (0 minute, 5 minutes, 15 minutes, Elk1, was activated by myostatin in skeletal muscle tissue (Fig. 2C). 1 hour, 6 hours, 12 hours, and 24 hours). Stimulation with 1 nmol/L bFGF for 12 hours was used as a positive control for Erk1/2 activation. Cell lysates were In addition, to understand the specificity of the Erk1/2 MAPK immunoblotted with anti-phospho-MEK1, phospho-Erk1/2, and phospho-p90RSK activation stimulated by myostatin, we treated the mouse NIH 3T3 antibodies as described in Materials and Methods. Equal loading was monitored by blotting the membranes with antibodies against Erk1/2 total protein and cells with myostatin for 12 hours; the result shown in Fig. 2D tubulin, respectively. The Western blots presented are representative of three indicates that myostatin has no potency to activate Erk1/2 in NIH independent experiments. B, the relative activation of Erk1/2 was quantitated 3T3 fibroblasts. These data suggest a clear and strong activation by densitometry. The fold increase in phosphorylated Erk1/2 was derived by dividing the total Erk1/2 band intensity at each time point with the phosphorylated of Erk1/2 MAPK pathway by myostatin in both cultured myogenic Erk1/2 band intensity. cells and adult skeletal muscle tissues.

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Inhibition of Ras blocks myostatin-induced Erk1/2 activation and proliferation inhibition. To determine whether Ras was required for the ability of myostatin to activate Raf/MEK/Erk cascade, we used a retrovirus-mediated overexpression of the dominant-negative Ras (Ras17N) in C2C12 cells. Compared with the green fluorescent protein (GFP) control virus infection, overexpression of Ras17N dramatically reduced the basal level of Erk1/2 phosphorylation (Fig. 3A), indicating that the activity of Ras was functionally blocked by Ras17N in C2C12 cells. Therefore, these cells were used for assaying the requirement of Ras in Erk1/2 activation in response to myostatin. The C2C12 cells overexpressing Ras17N were treated with myostatin or the same volume of PBS as control for 12 hours. Western blot analyses showed that Erk1/2 MAPK cannot be activated by myostatin in the absence of a functional upstream Ras (Fig. 3B), suggesting that myostatin- mediated Erk1/2 activation was through the Ras/MEK1 signaling pathway. Then, the next question was whether Ras activity was required for myostatin-mediated proliferation inhibition. To answer this question, we measured the relative proliferation rate by using a [3H]thymidine incorporation assay in both C2C12- and Ras17N-overexpressing C2C12 cells in the presence or absence of myostatin, respectively. As shown in Fig. 3C, an almost 40% reduction of [3H]thymidine incorporation was observed in myostatin-treated C2C12 cells than control cells. However, we found that levels of [3H]thymidine incorporation were indistin- guishable between myostatin-treated and untreated C2C12 cells, which functionally expressed Ras17N. These results indicated that inactivation of Ras by dominant-negative Ras in C2C12 cells essentially abolished the growth inhibitory effect of myostatin.

Figure 4. Myostatin inhibits myogenic differentiation via activation of MEK1/ Erk1/2. A, C2C12 cells were cultured in differentiation medium for 24 hours and then treated with myostatin (500 ng/mL) or the same volume of PBS for another 3 days in the presence or absence of 10 Amol/L PD98059 (PD). Western blots were done to assay for expression of the differentiation-related genes, MyoD, myogenin, and MyHC. Phosphorylated and total Erk1/2 proteins were also measured as indicated. B, differentiating C2C12 cells treated as described in (A) were analyzed for myosin protein expression using anti-MyHC antibody and a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining.

Taken together, our findings suggest that not only is Ras, as a relative upstream intracellular regulator of Erk1/2, potentially required for Erk1/2 activation by myostatin in C2C12 cells but also that the Ras/MEK1/Erk1/2 cascade plays a critical signaling transduction role in myostatin-mediated proliferation suppression. Myostatin inhibits myogenic differentiation by down- regulating differentiation related genes via MEK1/Erk1/2 pathway. It has been well established that the Ras/MEK/Erk1/2 activation participates in myogenic differentiation (30). Both sustained activation and overactivation of Erk1/2 MAPK are sufficient to repress myogenesis. Myostatin has been shown with inhibitory function on myogenic differentiation. And in this report, Figure 3. Dominant-negative Ras prevents myostatin-mediated Erk1/2 we have shown that myostatin activated the Ras/MEK/Erk1/2 activation and proliferation inhibition. A, duplicate experiments were done as pathway in C2C12 cells. Therefore, these two lines of evidence described in Materials and Methods. C2C12 cells were infected with viruses carrying a dominant-negative form of Ras (Ras17N) and GFP as control. suggest that myostatin-induced Erk1/2 activation may contribute Thirty-six hours postinfection, cell lysates were prepared for measurements of to inhibition of myoblast differentiation. To test this possibility, the expression of Ras and Erk1/2 phosphorylation by Western blots. B, C2C12 cells overexpressing Ras17N were treated with myostatin (500 ng/mL) or subconfluent C2C12 cells were cultured in differentiation medium PBS for 12 hours. The activation of Erk1/2 was detected with anti-phospho-Erk1/ for 24 hours and then treated with myostatin or the same volume 2 and antitubulin antibodies. C, to determine whether Ras17N is able to block of PBS as control for another 3 days in the presence or absence of myostatin-dependent growth inhibition, [3H]-thymidine incorporation assay was done in both wild-type and Ras17N-overexpressing C2C12 cells in the 10 Amol/L PD98059. Subsequently, total cell lysates were prepared presence or absence of myostatin. and analyzed by Western blots for total and phosphorylated Erk1/2 www.aacrjournals.org 1323 Cancer Res 2006; 66: (3). February 1, 2006

MAPK expression as well as myogenic differentiation markers, such as MyHC, MyoD, and myogenin. The results from Fig. 4A indicated that the levels of total Erk1/2 proteins were unchanged in all of the experimental groups. However, we found a significant correlation between Erk1/2 phosphorylation and reduced levels of proteins for MyHC, myogenin, and MyoD in response to myostatin. The results showed that the myostatin-induced reduction of MyHC, MyoD, and myogenin proteins was rescued in the C2C12 cells pretreated with PD98059 (Fig. 4A), which was also morphologically evidenced by an immunofluorescence staining of MyHC with anti-MyHC antibody (Fig. 4B). Altogether, our findings indicate that myostatin may inhibit myoblast differentiation by down-regulating MyoD and myogenin gene expression via MEK/Erk1/2 pathway and that MEK/Erk1/2 MAPK may play a very important role in myostatin- mediated myogenic differentiation suppression. ActRIIb mediates myostatin-stimulated Erk1/2 activation and differentiation suppression. TGF-h receptor type I and ActRIIb are suggested receptors for myostatin action. As the type II receptor, ActRIIb is important and specific for myostatin binding and signaling transduction (14). To determine whether myostatin-stimulated Erk1/2 activation was through ActRIIb, the synthesized siRNA duplexes against mouse ActRIIb mRNA were generated in vitro and transfected into C2C12 cells to knock down ActRIIb expression. A siRNA duplex for mouse GAPDH mRNA was used as nonspecific control in this experiment. As shown in Fig. 5A, the protein level of ActRIIb was knocked down dramatically by transfection of the ActRIIb siRNA compared with the GAPDH control. Thus, the Erk1/2 activation was assayed in the C2C12 cells with a loss of ActRIIb protein expression in response to the myostatin and bFGF treatment. We found that Erk1/2 could not be activated by myostatin when the ActRIIb

Figure 6. ActRIIb is required for myostatin-induced differentiation suppression. A, C2C12 cells were transfected with 70 Amol/L of siActR2b and siGAPDH. Twenty-four hours posttransfection, the cells were cultured in differentiation medium and treated with myostatin or PBS for another 2 days. Expression of MyHC was measured by immunofluorescence with anti-MyHC antibody. B, C2C12 cell lysates from various treatments as described in (A) were immunoblotted with antibodies against myogenin, MyHC, ActRIIb, and h-actin.

expression was blocked; however, the knockdown of GAPDH expression had no effect on myostatin-stimulated Erk1/2 activation (Fig. 5A) and Erk1/2 activation induced by bFGF was not affected in the cells. As shown in Fig. 5B, the requirement of ActRIIb for myostatin-stimulated Erk1/2 activation was further confirmed by overexpression of a dominant-negative form of ActRIIb (a truncated mutant of ActRIIb lacking intracellular kinase domain). In addition, the effect of ActRIIb knockdown on myogenic differentiation in response to myostatin was also examined. Both morphologic and biochemical evidence show that the negative regulation of myostatin on myogenic differentiation was significantly attenuated in C2C12 cells lacking ActRIIb Figure 5. Myostatin-stimulated Erk1/2 activation is through ActRIIb. A, C2C12 (Fig. 6A and B). Our findings provide molecular and cellular cells were transfected with 70 Amol/L of siRNA duplexes targeting for membrane receptor ActRIIb (siActR2b) and for GAPDH (siGAPDH) as negative control, evidence to indicate that ActRIIb was specific and crucial for respectively. Forty-eight hours posttransfection, the cells were treated with myostatin-mediated inhibition of Erk1/2 activation and differen- myostatin, bFGF, or the same volume of PBS for another 2 hours. Cell lysates tiation in C2C12 cells. were immunoblotted with phospho-Erk1/2 and total Erk1/2 antibodies to detect the MAPK activation and with ActRIIb antibody to evaluate gene knockdown effect. B, retroviruses carrying a myc-tagged dominant-negative form of ActRIIb [ActRIIb (DN)] or a GFP gene were used to infect proliferating C2C12 Discussion myoblasts. Forty-eight hours postinfection, the cells were stimulated with myostatin or PBS for another 2 hours and then cell lysates were immunoblotted In this study, we determined the involvement of Erk1/2 MAPK with antibodies against phospho-Erk1/2, total Erk1/2, and c-myc. cascade in myostatin signaling. Myostatin has a unique effect on

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Myostatin Activates Erk1/2 MAPK skeletal myogenesis: an initial inhibitory role on cell proliferation cells. Pretreatment with PD98059 completely inhibited myostatin- associated with a cell cycle arrest at G1 phase, followed by stimulated phosphorylation of Erk1/2. In addition, PD98059 was myogenic differentiation deficiency associated with a decreased able to rescue the differentiation suppression phenotype induced expression of differentiation necessary genes (11, 13). All of these by myostatin. These data indicate that the inhibitory effects of effects were thought to be mediated by the membrane receptors myostatin on differentiation were mediated through the MEK1/ (14). However, the mechanisms responsible for myostatin intra- Erk1/2 MAPK signaling pathway. Furthermore, using dominant- cellular signaling, from early inhibition of proliferation to late negative Ras allowed us to show that the myostatin-induced repression of differentiation, remain largely unknown. The MAPK inhibition of Erk1/2 activation and proliferation was via upstream signaling pathway is well known as a mediator of a variety of Ras. It has been recently reported that high level of Raf activity, extracellular stimuli and growth signals to the nucleus, and thus, downstream target of Ras, could inhibit myogenic program to influence cell proliferation, differentiation, and apoptosis. In through both MEK-dependent and MEK-independent signaling light of the diversity of biological responses that can be mediated pathways (35). Very interestingly, TGF-h expression was up- by the Ras/Raf/MEK1/Erk1/2 cascade, it is not surprising that a regulated in differentiation-defective myoblasts caused by high growth inhibitor, such as myostatin, might also be able to activate level of Raf activation (35). Together with our data from this study, this pathway for its function. it is most likely that myostatin functions through an autocrine loop In this report, we showed that myostatin dramatically activated to synergize Raf signaling and repress myogenesis in a more Erk1/2 MAPK in a time-dependent manner both in proliferating effective way. and differentiating C2C12 cells. Furthermore, the phosphorylation It has been well established that TGF-h family members can use levels of upstream kinases and downstream effectors in this path- MAPK signaling pathways to elicit their biological effects and the way (such as MEK1, Elk-1, and p90RSK) were also increased significance of cross talk between those pathways is becoming a following myostatin stimulation. The Erk1/2 MAPK module has central scheme for understanding the specificity and multiplicity of been regarded as a key regulator for cell proliferation (31). How- cellular events induced by a given elicitor (36). Recently, Philip et al. ever, previous studies have also suggested that an elevated degree (15) reported that p38 MAPK was activated by myostatin and Smad of Erk1/2 activity was sufficient to induce the differentiation was not required for myostatin-activated p38 MAPK pathway in inhibition of various cell types, including myogenic and erythroid HepG2, A204, and C2C12 cells. Those findings suggest that multiple differentiation (30, 32). Although it has been known that activated signaling cascades are activated by myostatin and the cross talk Ras or Raf inhibits multinuclear myotube fusion through Erk1/2 between those pathways plays a significant role in myostatin MAPK signaling, the upstream molecules and downstream targets signaling. Therefore, further investigation of the complexity of in muscle cells affected by this pathway have remained unclear the myostatin signal transduction and precise integrations of these for a long time. Recently, MEF2 and myogenin have been considered signaling modules will shed light on understanding the molecular to contribute to inhibition of differentiation through the MAPK mechanisms of myostatin function as a negative regulator of pathway (20, 33, 34). In addition to activation of Erk1/2 by myostatin muscle growth. in cultured muscle cells, we also analyzed the influence of myostatin on Erk1/2 pathway in mature skeletal muscle tissue. The same activation of Erk1/2 pathway by myostatin was observed in vivo. Acknowledgments Thus, in this study, we present experimental evidence to reveal the Received 8/25/2005; revised 10/19/2005; accepted 11/11/2005. activation of Erk1/2 MAPK pathway by myostatin in vitro and Grant support: National Basic Research Program of China grant 2005CB522400 and the National Natural Science Foundation of China grants 30025027, 30400231, in vivo. 30429002, and 30330430. To test the functional roles of Erk1/2 activation induced by The costs of publication of this article were defrayed in part by the payment of page myostatin in muscle cell proliferation and differentiation, the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chemical inhibitor of MEK1 and the dominant-negative form of Ras We thank Dr. Chengyu Jiang for the suggestions during manuscript preparation were used to block the myostatin-activated Erk1/2 in the C2C12 and Dr. Damu Tang for the dominant-negative Ras17N plasmid.

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Wei Yang, Yan Chen, Yong Zhang, et al.

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