Research Article Extracellular Signal–Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Pathway Is Involved in Myostatin-Regulated Differentiation Repression 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. Cancer Res 2006; 66: (3). February 1, 2006 1320 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Myostatin Activates Erk1/2 MAPK 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