Myostatin, a Negative Regulator of Muscle Growth, Functions by Inhibiting Myoblast Proliferation

Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C2C12 myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G1- to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21Waf1, Cip1, a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G1-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C2C12 myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G1- to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21Waf1, Cip1, a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G1-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation. transforming growth factor-β myogenic regulatory factor cyclin-dependent kinase retinoblastoma cyclin-dependent kinase inhibitor polymerase chain reaction minimal essential media Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline polyacrylamide gel electrophoresis horseradish peroxidase dithiothreitol reverse transcriptase latency-associated peptide terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling fluorescence-activated cell sorting The transforming growth factor-β (TGF-β)1 superfamily of genes encode secreted factors that are important for regulating embryonic development and tissue homeostasis in adults. Recently, McPherron et al. (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3300) Google Scholar) described a new member of this family,myostatin, that is expressed in developing and adult skeletal muscle. Myostatin-null mice show a dramatic and widespread increase in skeletal muscle mass due to an increase in number of muscle fibers (hyperplasia) and thickness of fibers (hypertrophy) (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3300) Google Scholar). Subsequently, we (2Kambadur R. Sharma M. Smith T.P. Bass J.J. Genome Res. 1997; 7: 910-916Crossref PubMed Scopus (236) Google Scholar) and others (3McPherron A.C. Lee S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12457-12461Crossref PubMed Scopus (1635) Google Scholar, 4Grobet L. Martin L.J. Poncelet D. Pirottin D. Brouwers B. Riquet J. Schoeberlein A. Dunner S. Menissier F. Massabanda J. Fries R. Hanset R. Georges M. Nat. Genet. 1997; 17: 71-74Crossref PubMed Scopus (1227) Google Scholar) reported that the Belgian Blue and Piedmontese breeds of cattle, which are characterized by an increase in muscle mass (double-muscling), have mutations in the myostatin coding sequence. Hence, the function of myostatin as a regulator of muscle mass is very well established. Myostatin shares several features with other members of the TGF-β superfamily: 1) a hydrophobic core of amino acids near the N-terminus that functions as a secretory signal; 2) a conserved proteolytic processing signal of RSRR in the C-terminal half of the protein; and 3) nine cysteine residues in the C-terminal region to facilitate the formation of a “cysteine knot” structure. Myostatin protein is synthesized in skeletal muscle as a 375-amino acid propeptide, which is proteolytically processed at the RSRR (263) site to give rise to a 26-kDa active processed peptide (5Sharma M. Kambadur R. Matthews K.G. Somers W.G. Devlin G.P. Conaglen J.V. Fowke P.J. Bass J.J. J. Cell. Physiol. 1999; 180: 1-9Crossref PubMed Scopus (361) Google Scholar). This processed mature peptide binds to receptor to elicit biological function (6McPherron A.C. Lee S. Growth Factors Cytokines Health Dis. 1996; 1B: 357-393Crossref Scopus (51) Google Scholar). Myostatin gene expression appears to be developmentally regulated (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3300) Google Scholar, 2Kambadur R. Sharma M. Smith T.P. Bass J.J. Genome Res. 1997; 7: 910-916Crossref PubMed Scopus (236) Google Scholar). Initially, myostatin gene expression is detected in myogenic precursor cells of the myotome compartment of developing somites, and the expression is continued in adult axial and paraxial muscles (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3300) Google Scholar). Different axial and paraxial muscles have been shown to express different levels of myostatin (2Kambadur R. Sharma M. Smith T.P. Bass J.J. Genome Res. 1997; 7: 910-916Crossref PubMed Scopus (236) Google Scholar). Although initial reports describing the expression of myostatin gene suggested that myostatin expression is exclusive to skeletal muscle, more recent publications have shown that myostatin mRNA or protein is detected in other tissues. A report using myostatin-specific antibodies indicates that myostatin protein is present in cardiomyocytes and Purkinje fibers of heart (5Sharma M. Kambadur R. Matthews K.G. Somers W.G. Devlin G.P. Conaglen J.V. Fowke P.J. Bass J.J. J. Cell. Physiol. 1999; 180: 1-9Crossref PubMed Scopus (361) Google Scholar), whereas Jiet al. (7Ji S. Losinski R.L. Cornelius S.G. Frank G.R. Willis G.M. Gerrard D.E. Depreux F.F. Spurlock M.E. Am. J. Physiol. 1998; 275(4 Pt 2),: R1265-R1273Google Scholar) detected myostatin mRNA expression in the mammary gland. Although the functional role of myostatin in control of muscle mass has been well documented by genetic models, the mechanism by which myostatin controls muscle fiber number is not known. Because myostatin expression is detected in somites during embryonic myogenesis, and its expression is continued in postnatal muscle, myostatin may very well play a role at all the stages of myogenesis. During myogenesis, myoblasts proliferate and withdraw from the cell cycle at the first gap phase, G1, and commit to a differentiation pathway to form myotubes (8Olson E.N. Dev. Biol. 1992; 154: 261-272Crossref PubMed Scopus (388) Google Scholar). Work over the past several years on myogenic differentiation has elucidated a hierarchical regulatory mechanism for the control of growth and differentiation of myoblasts. After stimulation in vitro by an appropriate environmental signal, such as serum deprivation, members of the basic helix-loop-helix transcription factor family, termed myogenic regulatory factors (MRFs), initiate a cascade of events leading to the expression of muscle-specific genes. Analysis of mice lacking individual MRFs has revealed a genetic hierarchy for their function. MyoD and Myf-5 play redundant roles in specifying a muscle lineage, that is, the formation of myoblasts (9Rudnicki M.A. Braun T. Hinuma S. Jaenisch R. Cell. 1992; 71: 383-390Abstract Full Text PDF PubMed Scopus (796) Google Scholar, 10Rudnicki M.A. Schnegelsberg P.N. Stead R.H. Braun T. Arnold H.H. Jaenisch R. Cell. 1993; 75: 1351-1359Abstract Full Text PDF PubMed Scopus (1370) Google Scholar, 11Braun T. Rudnicki M.A. Arnold H.H. Jaenisch R. Cell. 1992; 71: 369-382Abstract Full Text PDF PubMed Scopus (581) Google Scholar). Myogenin, by contrast, has been shown to be required for the differentiation of myoblasts (12Hasty P. Bradley A. Morris J.H. Edmondson D.G. Venuti J.M. Olson E.N. Klein W.H. Nature. 1993; 364: 501-506Crossref PubMed Scopus (1059) Google Scholar, 13Nabeshima Y. Hanaoka K. Hayasaka M. Esumi E. Li S. Nonaka I. Nature. 1993; 364: 532-535Crossref PubMed Scopus (747) Google Scholar), whereas MRF4 is thought to be involved in the maturation of myotubes (14Rawls A. Valdez M.R. Zhang W. Richardson J. Klein W.H. Olson E.N. Development. 1998; 125: 2349-2358Crossref PubMed Google Scholar). Coupled with the induction of muscle-specific genes is the permanent withdrawal of proliferating myoblasts from the cell cycle to become terminally differentiated myotubes. The connection between myoblast cell cycle withdrawal and differentiation is established through regulation of cyclin-dependent kinases (Cdks), a family of enzymes that catalyze events required for cell cycle transitions. Primary targets for this regulation are the G1 cyclin·Cdk complexes, cyclin-D·Cdk4 and cyclin-E·Cdk2, which cooperate to control the G1 to S transition through phosphorylation and inactivation of the retinoblastoma (Rb) protein (15Guo K. Walsh K. J. Biol. Chem. 1997; 272: 791-797Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Cyclin-dependent kinase inhibitors (CKIs), of which there are two families, the p16 family and the p21 family, in turn, regulate the kinase activities of the Cdks. The p16 family specifically inhibits Cdk4 and Cdk6, whereas the p21 family inhibits all Cdks involved in G1/S transition (16Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5220) Google Scholar). The loss of functional myostatin leads to hyperplasia and hypertrophy of skeletal muscle. Because increased muscle fibers can result from increased myoblast proliferation and delayed differentiation, we investigated the role of myostatin in controlling myoblast proliferation and cell cycle progression. Using cultured C2C12 myoblast cells and recombinant myostatin protein, we show here that myostatin indeed regulates the cell cycle progression of myoblasts by controlling the G1- to S-phase and G2- to M-phase transition. We also demonstrate that myostatin accomplishes this by increasing the level of p21 and decreasing Cdk2 protein levels and activity, thereby rendering the Cyclin-E·Cdk2 complex inactive. This results in suppression of the Rb protein phosphorylation in vivo and concurrent cell cycle arrest of myoblasts in the G1-phase. The increased number of myofibers seen in cattle and mice with heavy muscling thus appears to be the result of deregulated myoblast proliferation caused by the absence of functional myostatin. The pET protein expression system (Novagen; Madison, WI) was used to express and purify recombinant myostatin. A portion of bovine cDNA spanning amino acids 267–375 was amplified by PCR using a BamHI fragment and cloned into the pET 16-B vector. The myostatin coding sequence was placed in-frame with the ten histidine residues according to the manufacturer's protocol (Novagen). The resulting construct was used to transform the BL 21 bacterial strain. An overnight Escherichia coli culture harboring the recombinant myostatin expression vector was diluted and grown up to an optical density of 0.8 (600 nm) in 1 liter of Lennox L broth (LB) medium plus ampicillin (50 mg/liter). The myostatin fusion protein was induced by adding 0.5 mm isopropyl thio-β-galactoside to the culture, and the induction was continued for 2 h. Bacteria were collected by centrifugation, resuspended in 40 ml of lysis buffer (6m guanidine hydrochloride; 20 mm Tris, pH 8.0; 5 mm 2-mercaptoethanol) and sonicated. The lysate was centrifuged at 10,000 × g for 30 min, and myostatin was purified from the supernatant by Ni-Agarose affinity chromatography (Qiagen, Valencia), according to the manufacturer's protocol. Soluble fractions containing myostatin were pooled and dialyzed against two changes of 50 mm Tris-HCl (pH 8.0) containing 500 mm NaCl and 10% glycerol for 6 h. The AgResearch Ruakura Animal Ethics Committee approved the animal manipulations described in this paper. Standard superovulation and embryo transfer techniques were used to generate fetuses with expected double- and normal-muscled phenotypes as described previously (2Kambadur R. Sharma M. Smith T.P. Bass J.J. Genome Res. 1997; 7: 910-916Crossref PubMed Scopus (236) Google Scholar). Double-muscled fetuses were Belgian Blue, and normal-muscled fetuses were Hereford × Friesian crossbreed. Cows were slaughtered at the Ruakura abattoir when fetuses were at 160 days of gestation. The semitendinosus muscle was excised, cut into small pieces, placed in minimal essential media (MEM) containing 20% fetal bovine serum and 10% Me2SO, and frozen in liquid nitrogen for subsequent myoblast culture generation. Mixed cultures containing both myoblasts and fibroblasts were liberated from thawed Musculus semitendinosus by mincing muscle and then digesting with 0.25% trypsin (Sigma Cell Culture Ltd., St. Louis, MO) for 45 min at 37 °C. Media for subsequent culture consisted of MEM (Life Technologies, Grand Island, NY), buffered with 41.9 mm NaHCO3 (Sigma) and gaseous CO2. 7.22 nm Phenol Red (Sigma) was used as a pH indicator. 1 × 105 IU/liter penicillin (Sigma), 100 mg/liter streptomycin (Sigma), and 10% fetal bovine serum (Life Technologies Ltd.) were routinely added to media. The method of O'Malley et al. (17O'Malley J.P. Greenberg I. Salpeter M.M. Meth. Cell Sci. 1996; 18: 19-23Crossref Scopus (2) Google Scholar) was used to enrich cultures for myoblasts. A total of 1 × 106 liberated cells were added to Matrigel (Collaborative Biomedical Research, CBR)-coated 10-cm dishes (Nunc, Roskilde, Denmark). Matrigel-coated dishes were prepared by adding 10 ml of 5.0 ml/liter Matrigel in Earle's balanced salt solution (obtained as a 10× concentrate; Life Technologies Ltd.) to each 10-cm dish and then incubating at 37 °C for 16 h. Cultures were grown on Matrigel for 3 days, then digested with 0.5 g/liter Type 1-A collagenase (Sigma) for 10 min to preferentially detach fibroblasts. Cultures were washed two times with Earle's balanced salt solution before and three times after collagenase digestion. Myoblast cultures were then grown in MEM media containing 10% FBS for an additional 24 h before trypsinization and seeding into the methylene blue cell proliferation assay. When these myoblast cultures were cloned and cultured under differentiation inducing conditions, more than 90% of clones stained positive for the muscle-specific marker desmin, indicating a predominance of myoblasts. C2C12 myoblasts (18Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1646) Google Scholar) were grown prior to assay in Dulbecco's modified Eagle's medium (DMEM; Life Technologies Ltd.) with additives stated above. Cell proliferation assays were conducted in either uncoated (C2C12cultures) or gelatin-coated (bovine myoblast primary cultures) 96-well Nunc microtiter plates. Plates were gelatinized by the method of Quinn and Nameroff (19Quinn L.S. Nameroff M. Differentiation. 1983; 24: 111-123Crossref PubMed Scopus (22) Google Scholar). C2C12 cultures were seeded at 1000 cells per well and bovine myoblast cultures at a density of 3000 cells per well in relevant media. After a 16-h attachment period, myostatin test media was added. This consisted of 10% fetal bovine serum media with 0–10 μg/ml recombinant myostatin. The position of the samples on the plate was randomly assigned, and all samples were run in replicates of eight. Results presented in this paper are representative of at least two independent experiments. Plates were then incubated in an atmosphere of 37 °C, 5% CO2 for a further 72 h. After the incubation period, proliferation was assessed using a methylene blue photometric end point assay as described previously (20Oliver M.H. Harrison N.K. Bishop J.E. Cole P.J. Laurent G.J. J. Cell Sci. 1989; 92(Pt 3),: 513-518PubMed Google Scholar). In this assay absorbance at 655 nm is directly proportional to the final cell number. Results are presented here as the mean and standard error of eight replicates. C2C12 myoblasts were seeded into Nunc 96-well plates at a density of 1000 cells/well. After an overnight attachment period (time zero) plates were washed once with DMEM/10% FBS, and test media were added. One set of eight replicate wells received DMEM/10% FBS media without myostatin (control wells), whereas wells due to be subsequently “rescued” from myostatin received DMEM/10% FBS containing 4 μg/ml myostatin. After a further 24-h incubation period, all plates were washed once and DMEM/10% FBS without myostatin was added into control wells and those wells due to be rescued from incubation with myostatin at 24 h. DMEM/10% FBS containing 4 μg/ml myostatin was added back to wells due to be rescued subsequently. All plates were washed at 24-h intervals during the course of the experiment, and relevant media were added back to the wells. Further cultures were rescued from incubation with myostatin at 72- and 120-h time points as described above. Plates were fixed at 24-h intervals throughout the experiment and assayed for cell proliferation as described above. Myoblast cultures derived from a 160-day bovine fetus were seeded (9000 cells/cm2) onto coverslips (Nunc) and, following overnight attachment, incubated with 0 or 8 μg/ml myostatin in growth media for 72 h. Cultures were then fixed with 20 parts of 70% ethanol/2 parts of formaldehyde/1 part of glacial acetic acid for 30 s, followed by three rinses with PBS. Cells were blocked overnight at 4 °C in TBS (0.05 m Tris-HCl, pH 7.6 (Sigma), 0.15 m NaCl) containing 1% sheep serum, then incubated with primary antibody (5 μg/ml anti-Myf-5 (Santa Cruz Biotechnologies, Santa Cruz, CA) in TBS-1% normal sheep plasma) for 1 h at room temperature. Rabbit IgG (5 μg/ml; Dako) was used as a negative control. Following primary antibody incubations, cells were washed three times (5 min each) with TBS-Tween (TBS; 0.05% Tween-20) and incubated with secondary antibody, 1:100 dilution of donkey anti-rabbit IgG (Amersham Pharmacia Biotech), in TBS-1% normal sheep plasma for 30 min. Cells were then incubated with 1:100 dilution of streptavidin-biotin peroxidase complex (Amersham Pharmacia Biotech) for 30 min. Myf-5 immunostaining was visualized using 3,3-diaminobenzidine tetrahydrochloride (Life Technologies) enhanced with 0.0375% CoCl. Myf-5 immunostained cultures were lightly counterstained with Gill's hematoxylin and photographed using an Olympus BX50 microscope (Olympus Optical Co., Germany) fitted with a color camera (DC-330, DAGE-MTI Inc.). To visually assess the effect of myostatin on C2C12 proliferation, cells were seeded onto coverslips (3000 cells/cm2) and allowed to attach overnight. Cultures were then incubated with or without 4 μg/ml myostatin for 24, 48, or 72 h and fixed as above. Cells were then stained with 1:1 Gill's hematoxylin followed by 1% eosin. Cultures were photographed as above. A method by Darzynkiewicz and Li 2Darzynkiewicz, Z., and Li., X. (1996) Techniques in Apoptosis: A User's Guide, Portland Press, London, UK. was used that enabled the analysis of apoptosis in addition to cell cycle analysis. Mouse C2C12myoblast cells were cultured as described above in 100-mm dishes with or without myostatin treatment for 48 h. Cells (∼3 × 106) were harvested using trypsin followed by centrifugation and fixed in 800 μl of 70% ethanol/PBS. The fixed cells were resuspended in 500 μl of PBS + 500 μl of DNA extraction buffer (200 mm NaHPO4, 100 mmcitric acid) for 10 min at room temperature. DNA extraction buffer was replaced with DNA staining buffer (50 μg/ml propidium iodide, 50 μg/ml DNase-free RNaseA in PBS), vortexed briefly to resuspend cells and incubated in the dark at room temperature for 30 min. Cells were then examined for propidium iodide fluorescence using a flow cytometer (FACScan, Becton-Dickinson) and analyzed using CellFit software (Becton-Dickinson). For quantitative CKI, Cdk, and cyclin immunoblot analyses, mouse C2C12 myoblasts were cultured as described above in 100-mm dishes with or without myostatin treatment. Cells (∼3 × 106) were resuspended in 200 μl of lysis buffer (50 mm Tris, pH 7.6; 250 mm NaCl; 5 mm EDTA; 0.1% Nonidet P-40; Complete (Roche Molecular Biochemicals, Germany) protease inhibitor) and sonicated. The cell extracts were centrifuged to pellet the cell debris, and the supernatants were frozen at −80 °C. Bradford reagent (Bio-Rad) was used to estimate total protein content to ensure equal loadings. Precisely 15 μg of total protein was separated by SDS-PAGE (12%) and transferred to nitrocellulose membrane by electroblotting. After blotting the gels were stained in Coomassie Blue to visually ensure equal loadings. The membranes were blocked in TBST/5% milk at 4 °C overnight then incubated with the primary antibody for 3 h at room temperature. The following primary antibodies were used for immunoblotting: p15, 1:400 dilution of purified rabbit polyclonal anti-p15 antibody (sc-613, Santa Cruz Biotechnologies); p16, 1:400 dilution of purified rabbit polyclonal anti-p16 antibody (sc-1207, Santa Cruz); p21, 1:400 dilution of purified mouse monoclonal anti-p21 antibody (SX118, PharMingen); p27, 1:400 dilution of purified mouse monoclonal anti-p27 antibody (sc-1641, Santa Cruz); Cdk2, 1:400 dilution of purified mouse monoclonal anti-Cdk2 antibody (sc-2648, Santa Cruz); Cdk4, 1:400 dilution of purified rabbit polyclonal anti-Cdk4 antibody (sc-601, Santa Cruz); cyclin-D1, 1:400 dilution of purified mouse monoclonal anti-cyclin-D1 antibody (sc-8396, Santa Cruz); cyclin-E, 1:400 dilution of purified rabbit polyclonal anti-cyclin-E antibody (sc-481, Santa Cruz); α-tubulin, 1:3000 dilution of purified mouse monoclonal anti-α-tubulin antibody (DM 1A, Sigma). The membranes were washed (5 × 5 min) with TBST and further incubated with either anti-mouse IgG HRP conjugate, 1:2000 dilution (W402B, Promega Corp.), or anti-rabbit IgG HRP conjugate, 1:1000 dilution (P0448, Dako), secondary antibodies for 1 h at room temperature. The membranes were washed as above, and HRP activity was detected using Renaissance Western blot chemiluminescence (NEL104, PerkinElmer Life Sciences). For pRb phosphorylation studies, rat L6 myoblasts (21Yaffe D. Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 477-483Crossref PubMed Scopus (828) Google Scholar) were cultured as described above in 100-mm dishes with or without myostatin treatment. Cells (5–6 × 106) were counted and lysed at 6 × 104 cells/μl in sample buffer (100 mm Tris, pH 6.8; 4% SDS; 0.2% bromphenol blue; 20% (v/v) glycerol) that was brought to boil for 5 min prior to addition to the cells. Lysates were boiled for a further 5 min and frozen at −80 °C. Dithiothreitol (DTT) was subsequently added to 15 μl of lysate (final concentration, 0.2 m), boiled, fractionated by SDS-PAGE (7.5%), and transferred to polyvinylidene difluoride Immobilon-P membrane (Millipore Corp., MA) by electroblotting. A purified mouse monoclonal anti-pRb antibody (G3-245, PharMingen), which recognizes both a hyperphosphorylated form and hypophosphorylated form, was used at a 1:200 dilution. Subsequent steps of membrane blocking, antibody incubations, washes, and detection were performed as described above. For the Cdk2 immunoprecipitation-kinase assay, mouse C2C12myoblasts were cultured as described above in 100-mm dishes with or without myostatin. Cells were resuspended in lysis buffer (50 mm Tris, pH 7.4; 150 mm NaCl; 0.4% Nonidet P-40; 2 mm EDTA; 50 mm NaF; 10 mmβ-glycerophosphate; 1 mm ATP; 2 mm sodium vanadate; 2 mm DTT; Complete (Roche Molecular Biochemicals, Germany) protease inhibitor). After 10 passages through a 21-gauge needle, cell lysates were cleared by centrifugation. Protein concentrations were determined using Bradford reagent (Bio-Rad). 200 μg of each extract were immunoprecipitated with 1:20 dilution polyclonal anti-Cdk2 (M2, sc-163, Santa Cruz Biotechnologies) in 100 μl for 1 h at 4 °C. Protein A-agarose (Life Technologies, Inc.; 50 μl of 50%, washed twice with lysis buffer) was added for 1 h at 4 °C, followed by centrifugation to pellet immunoprecipitated complexes. After centrifugation, pellets were washed three times with lysis buffer, twice in lysis buffer containing 400 mm NaCl, and twice in kinase buffer (25 mmHEPES, pH 7.4; 25 mm MgCl2; 25 mmβ-glycerophosphate; 50 μm ATP; 0.1 mmNaVO3; 2 mm DTT). Pellets were then resuspended in 20 μl of kinase buffer containing 5 μCi of [γ-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech) and 2 μg of histone H1 (Roche) for 40 min at room temperature. The kinase reactions were stopped by the addition of 4× NuPAGE sample buffer (Novex), boiled for 5 min, and 10 μl was loaded and run on a 4–12% gradient NuPAGE gel. RNA was isolated from cultured cells using TRIZOL reagent (15596, Life Technologies, Inc.) according to the manufacturer's protocol. Northern analysis was performed essentially as described by Sambrook et al. (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Twelve micrograms of total RNA was fractionated by 0.66 m formaldehyde-1% agarose gel electrophoresis. RNA was transferred to Hybond N+ membrane (Amersham Pharmacia Biotech) by capillary transfer using 10× SSC. The membrane was prehybridized in Church and Gilbert hybridization buffer (0.5 m Na2HPO4 (pH 7.2); 7% SDS; 1 mm EDTA) at 60 °C for 1 h, followed by hybridization with 32P-labeled p21 cDNA probe in fresh Church and Gilbert hybridization buffer at 60 °C, overnight. The membrane was washed at 60 °C for 15 min each with 2× SSC + 0.5% SDS, and then 1× SSC + 0.5% SDS. The p21 cDNA was obtained by RT-PCR. First-strand cDNA was synthesized in a 20-μl reverse transcriptase (RT) reaction from 5 μg of total RNA (from bovine skeletal muscle) using a SuperScriptII pre-amplification kit (Life Technologies, Inc.), according to the manufacturer's protocol. PCR was performed with 2 μl of the RT reaction at 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 1 min for 35 cycles. This was followed by a single 72 °C extension step for 5 min. The primers used for amplification were 5′-CTGTTAGGCTGGTCTGCCTC-3′ and 5′-GTCCGATCCTGGTGATGTCC-3′ (443 base pairs). The p21 cDNA was radioactively labeled using [α32P]dCTP (Amersham Pharmacia Biotech) and a RediprimeII labeling kit (Amersham Pharmacia Biotech), according to the manufacturer's protocol. Previous published results show that myostatin expression is detected very early in the myotome of the developing somites of mice and cattle embryos (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3300) Google Scholar, 2Kambadur R. Sharma M. Smith T.P. Bass J.J. Genome Res. 1997; 7: 910-916Crossref PubMed Scopus (236) Google Scholar) and the expression continues into adult muscle. In addition, using myostatin-specific antibodies, we recently demonstrated (5Sharma M. Kambadur R. Matthews K.G. Somers W.G. Devlin G.P. Conaglen J.V. Fowke P.J. Bass J.J. J. Cell. Physiol. 1999; 180: 1-9Crossref PubMed Scopus (361) Google Scholar) that myostatin protein is synthesized and proteolytically processed in adult skeletal muscle. However, the site of myostatin biosynthesis and its processing has not been demonstrated so far. To characterize the site of synthesis, we performed Western blot analyses on protein extracts prepared from bovine primary myoblasts. Anti-myostatin antibodies specifically detect two bands on the Western blot (Fig. 1). These two bands correspond to the unprocessed full-length protein (52 kDa) and the N-terminal LAP (latency-associated peptide) (40 kDa). In addition to the full-length and LAP, the anti-myostatin antibodies also detected the mature processed myostatin (26 kDa) in skeletal muscle extract. However, this 26-kDa mature myostatin is not detectable in myoblast extracts. These data demonstrate that myostatin protein is synthesized in myoblasts and that the precursor myostatin is processed in myoblasts.Figure 1Detection of myostatin protein in skeletal muscle and myoblast protein extracts by Western blot analysis.Fifteen micrograms of total protein from Bovine Musculus biceps femoris and 70-day primary bovine myoblast cultures was resolved by 12% SDS-PAGE, and myostatin protein was detected with rabbit anti-myostatin antibodies. Precursor, processed, and latency-associated peptide (LAP) forms of myostatin are indicated. Molecular masses of the Western positive bands are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Myostatin protein is proteolytically processed, possibly at a conserved RSRR (263) site (5Sharma M. Kambadur R. Matthews K.G. Somers W.G. Devlin G.P. Conaglen J.V. Fowke P