muscle cells 12 C 2 synthesis in C Myostatin inhibits cell proliferation and protein

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Lohand glucocorticoid-mediated myostatin expression in skeletal muscle Posttranscriptional mechanisms involving microRNA-27a and b contribute to fast-specific including high resolution figures, can be found at:Updated information and services/content/280/2/E221.full.html can be found at:AJP - Endocrinology and Metabolism about Additional material and information /publications/ajpendo This infomation is current as of March 4, 2011./.20814-3991. Copyright © 2001 by the American Physiological Society. ISSN: 0193-1849, ESSN: 1522-1555. Visit our website at organization. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD publishes results of original studies about endocrine and metabolic systems on any level of AJP - Endocrinology and Metabolism on March 4, 2011 Downloaded fromMyostatin inhibits cell proliferation and protein synthesis in C 2C 12muscle cellsWAYNE E.TAYLOR,1SHALENDER BHASIN,1JORGE ARTAZA,1FRANCES BYHOWER,1MOHD AZAM,2DARRIL H.WILLARD,J R .,3FREDERICK C.KULL,J R .,3AND NESTOR GONZALEZ-CADAVID 11Division of Endocrinology,Metabolism and Molecular Medicine,Charles R.Drew University of Medicine and Science,Los Angeles,California 90059;2ExpressGen,Chicago,Illinois 60612;3Glaxo-Wellcome Laboratories,Research Triangle Park,North Carolina 27709Received 15June 2000;accepted in final form 4October 2000Taylor,Wayne E.,Shalender Bhasin,Jorge Artaza,Frances Byhower,Mohd Azam,Darril H.Willard,Jr.,Frederick C.Kull,Jr.,and Nestor Gonzalez-Cadavid.Myostatin inhibits cell proliferation and protein synthesis in C 2C 12muscle cells.Am J Physiol Endocrinol Metab 280:E221–E228,2001.—Myostatin mutations in mice and cattle are associated with increased muscularity,suggesting that myostatin is a negative regulator of skeletal muscle mass.To test the hypothesis that myostatin inhibits muscle cell growth,we examined the effects of recombinant myostatin in mouse skeletal muscle C 2C 12cells.After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells,the human recom-binant protein was expressed as the full-length (375-amino acid)myostatin in Drosophila cells (Mst375D),or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC).These proteins were identified by immunoblot-ting and were purified.Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay),DNA synthesis ([3H]thymidine incorpora-tion),and protein synthesis ([1-14C]leucine incorporation)in C 2C 12cells.The inhibitory effects of both proteins were greater in myotubes than in myoblasts.Neither protein had any significant effects on protein degradation or apoptosis.In conclusion,recombinant myostatin proteins inhibit cell pro-liferation,DNA synthesis,and protein synthesis in C 2C 12muscle cells,suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.myoblast;myotube;growth differentiation factor 8;sarcope-nia;growth differentiation factorMYOSTATIN ,A MEMBERof the transforming growth fac-tor-␤(TGF-␤)superfamily,is a novel regulator of skel-etal muscle growth (9,19,20).Inactivating mutations of the myostatin gene in cattle (4,10,13,20)and mice (24)are associated with skeletal muscle hypertrophy.Similarly,mice made null for the myostatin gene by homologous recombination (19),or transgenic mice car-rying dominant negative mutations of the myostatin gene,have increased muscle mass (28).The circulatingconcentrations of myostatin protein,measured by an immunoassay,are higher in patients with sarcopenia associated with acquired immunodeficiency (HIV)syn-drome than in healthy young men (9).Furthermore,myostatin expression is increased in the atrophied muscles of rats exposed to hindlimb suspension or microgravity (3,17,26).Collectively,these data led us to hypothesize that the product of the myostatin gene might be an inhibitor of skeletal muscle growth in adult animals and might contribute to the multifacto-rial pathophysiology of sarcopenia associated with chronic illness (16,22)and aging (2).However,the effects of myostatin protein on skeletal muscle growth have not been directly studied.Because muscle mass represents the balance be-tween muscle cell replication and protein synthesis and muscle protein breakdown and cell death (5),we considered the possibility that myostatin inhibits mus-cle growth by affecting one or more of these processes.We tested this hypothesis by expressing recombinant human full-length,375-amino acid (aa)myostatin pro-tein and determining its effects on cell proliferation,DNA and protein synthesis,protein degradation,and apoptosis in skeletal muscle cells.Because the 110-aa carboxy-terminal portion of the myostatin protein was proposed (19)to be the mature form of this protein,we also tested the effects of this recombinant protein.We used the C 2C 12skeletal muscle cell line as the in vitro bioassay system,because this model has been used extensively in previous studies of the effects of muscle growth factors on muscle protein synthesis and degra-dation,cell replication,and apoptosis (6,21,23).MATERIALS AND METHODSPreparation of myostatin cDNA constructs in mammalian expression vectors.A human myostatin cDNA plasmid,pPCR-Mst(5a),was cloned and sequenced as described (9).The nucleotide sequence of this 2-kb Mst cDNA is identical to the published sequence (9,19)except for two base changes at the 5Јend (A to G at positions 248and 407,causing conser-Address for reprint requests and other correspondence:W. E.Taylor,UCLA School of Medicine,Division of Endocrinology,Metab-olism,and Molecular Medicine,Charles R.Drew Univ.of Medicine and Science,1731E.120th St.,Los Angeles,CA 90059(E-Mail:wataylor@).The costs of publication of this article were defrayed in part by the payment of page charges.The article must therefore be hereby marked ‘‘advertisement ’’in accordance with 18U.S.C.Section 1734solely to indicate this fact.Am J Physiol Endocrinol Metab280:E221–E228,2001.0193-1849/01$5.00Copyright ©2001the American Physiological SocietyE221on March 4, 2011 Downloaded fromvative amino acid changes of K to R).The Mst cDNA was subcloned for expression in mammalian cells into the cyto-megalovirus promoter (CMV)-driven pcDNA3.1vectors (In-vitrogen,San Diego,CA),creating the following constructs:1)sense clone (pcDNA3-Mst),by insertion of a 2-kb Not I-Eco RI fragment of pPCR-Mst,encoding the entire Mst open reading frame plus some untranslated 3Јsequences,into pcDNA3.1(Ϫ);2)antisense clone [pcDNA3-Mst(A)]by a sim-ilar procedure but with the use of vector pcDNA3.1(ϩ);and 3)sense Myc/His tag clone [pcDNA3-Mst(H)]by ligation of a 1.2-kb fragment for the reading frame of myostatin flanked by 5Ј-Nhe I and 3Ј-Bam HI sites created by PCR with forward primer 5Ј-CTAGCTAGATCATGCAAAAACTGCAACTCT and reverse primer 5Ј-CGGGATCCCATGAGCACCCACAGCGG.This strategy allowed replacement of the stop codon and fusion of myostatin with the Myc/His-6codons in the vector pcDNA3.1/Myc-HisB (Fig.1A ).Cell-free transcription and translation.Plasmids pcDNA3-Mst,pcDNA3-Mst(A),and pcDNA3-Mst(H)were used as templates in the cell-free,coupled transcription and transla-tion system from rabbit reticulocytes (Promega,Madison,WI).In addition to the CMV promoter,these plasmids con-tain the T7promoter for bacterial T7RNA polymerase.Plasmid DNA (1␮m/g),including control plasmids (pRL-TKand pT3-Luc,encoding respectively renilla and luciferase),was transcribed by use of T7(or T3)RNA polymerase and translated with 10␮Ci of [35S]methionine (ICN,Irvine,CA)for 60min at 30°C.Translated proteins in 2-to 5-␮l reaction mix were heated at 98°C for 10min in SDS sample buffer containing mercaptoethanol,separated by electrophoresis on a 12%Tris-glycine Laemmli gel,and detected by autoradiog-raphy.Mammalian cell transfection.Chinese hamster ovary (CHO)cells [American Tissue Culture Collection (ATCC),Atlanta,GA]were grown to 90%confluence in DMEM plus 10%FBS media (GIBCO Life Technologies,Gaithersburg,MD)in T 75plastic flasks at 37°C,and subcultured into 6-well plates.Cultures at 60–80%confluence were transfected with plasmid DNA by liposomes (Lipofectamine,GIBCO).After 3–5days,cultures were harvested,and the proteins were analyzed by gel electrophoresis and Western blotting,as described below.Preparation of myostatin cDNA constructs in Drosophila and Escherichia coli expression vectors.For expression of full-length human myostatin with a 3ЈHis-6tag in insect S2-DES cells (13),a 1.2-kb fragment (nt 1–1,125)was sub-cloned into the Drosophila expression vector pMT/V5-HisB (Invitrogen,Carlsbad,CA)with a strong metallothionine promoter and with addition of a His tag at the 3Јend of the gene.A 123-bp sequence containing the V5and polyhistidine epitopes was eliminated from the vector by Eco RI and Pme I digestion followed by religation.The 1.2-kb myostatin frag-ment was prepared from pPCR-Mst by use of primers with suitable overhangs,so that the original myostatin stop codon was replaced by an in-frame sequence encoding a 6-histidine stretch at the carboxy end of the fusion protein.The 5Јforward primer is 5Ј-GGACTAGTATCATGCAAAAACTG-CAACTC,and the 3Јreverse primer is 5Ј-GGAATTCTC-AATGGTGATGGTGATGATGTGAGCACCCACAGCGGTC.These primers contained Spe I and Eco RI restriction sites at the 5Јand 3Јends,respectively,allowing for ligation into similar sites of the modified vector.The resulting construct was named pDES-Mst.For expression of the 110-aa COOH-terminal protein in Escherichia coli (E.coli ;Mst110EC),a PCR-amplified DNA fragment (nt 799–1,213)of the human Mst cDNA was subcloned into vector pRSET encoding aa MKKG followed by HHHHHHG (His tag)preceding the last 110aa of myostatin.Preparation of recombinant,full-length,375-aa myostatin protein.Plasmids pDES-Mst along with pCoHygro were transfected by CaPO 4into Drosophila S2cells (15)(Schnei-der cells,Invitrogen),and stable transfectants were selected by growth for 6wk in medium containing 300-␮g/ml hygro-mycin B (GIBCO Life Technologies).To induce myostatin expression,the cells were grown for 24h in medium contain-ing 0.5mg/ml CuSO 4.Recombinant full-length myostatin protein,Mst375D,was purified from the insect cell pellet (5g)by lysis in 5vol of lysis buffer (8M urea,20mM sodium phosphate,500mM NaCl,pH 7.8)for 30min.After centrifugation at 10,000g for 20min,the clarified lysate was adsorbed onto Ni nitrilo triacetic acid agarose beads for 2h in lysis buffer and washed with a buffer containing 6M urea,20mM sodium phosphate,500mM NaCl,and 30mM imidazole,pH 6.0.Bound protein was eluted with a buffer containing 6M urea,20mM sodium phosphate,500mM NaCl,and 50mM EDTA,pH 5.3.Frac-tions containing the highest amounts of Mst375D protein,as assessed by SDS-PAGE,Coomassie blue staining,and West-ern blots,were pooled and concentrated on Centricon-10(YM-10,Millipore)and were purified by preparative HPLC [Delta Pack C 18PrepLC,15␮m,300Å(Waters)],withtheFig.1.Expression of human myostatin gene in vitro and in vivo .A :schematic representation of the human myostatin cDNA constructs subcloned in mammalian vector pcDNA3.1in sense (Mst),antisense [Mst(A)],and as 3Јfusion with His tag [Mst(H)],adjacent to strong CMV/T7promoter.B :autoradiogram of proteins labeled with [35S]methionine by in vitro coupled transcription and translation of the plasmids containing Mst,Mst(A),Mst(H),or no DNA,fraction-ated by 12%SDS-PAGE.The positive control (left lane ,R ϩLuc)had template plasmids pRL-TK and pT3-Luc,which encode proteins of 36kDa (renilla)and 62kDa (luciferase),respectively,shown relative to the marker proteins.C :Western immunoblot of myostatin proteins expressed in Chinese hamster ovary (CHO)cells,transfected for 3days with pcDNA3-constructs Mst,Mst(H),or vector pcDNA3.1plasmid.RSM,rat skeletal muscle protein extract.Antibodies were against the following proteins:top ,myostatin [Mst(B)];middle ,poly-histidine tag;bottom ,Myc epitope.Protein sizes (kDa)are indicated for markers (M)on left and myostatin bands on right .E222MYOSTATIN INHIBITS C 2C 12MUSCLE CELL GROWTHon March 4, 2011 Downloaded fromuse of 0.1%trifluoroacetic acid (TFA).Myostatin eluted in flow through,and smaller contaminating proteins were bound to the column.Eluted material was checked on an SDS gel,concentrated on Centricon-10,and loaded on a Sephacryl S-200HR (Sigma)column equilibrated with 10mM HEPES,pH 7.4,150mM NaCl,and 0.1mM phenylmethylsulfonyl fluoride (PMSF).Eluted protein peaks were analyzed by SDS-PAGE and concentrated on Centricon-10,and glycerol was added to 10%.Myostatin protein fractions were dialyzed extensively at 4°C against PBS buffer.Final protein estima-tion was performed by the microbicinchoninic acid method (Pierce)and by immunoblotting.Preparation of recombinant Mst110EC protein.The con-struct for Mst110EC was expressed in E.coli strain BL21(DE3)as protein contained in inclusion bodies.The cells were lysed in Tris-EDTA (TE)buffer containing 1mM PMSF.After centrifugation at 20,000g for 40min,the pellet was resuspended in 8M urea,0.5M L -arginine,0.1M Tris,10mM EDTA (pH 8.3),and incubated for 30min at 37°C.The proteins were refolded by dilution into 20vol of folding buffer {1M NaCl,0.5M Tris,0.5M L -arginine,0.1%[3-(3-cholami-dopropyl)-dimethylammonio]-1-propane sulfonate,10mM EDTA,5mM reduced glutathione,2.5mM oxidized glutathi-one,pH 8.0},incubated for 3days at 4°C,clarified by centrif-ugation,dialyzed exhaustively against 10mM HCl,and ly-ophilized.The Mst110EC protein was further purified by HPLC on a Poros R2/M column with 0.1%TFA in 0–80%linear acetonitrile gradient.Fractions eluting at 50%aceto-nitrile were pooled and lyophilized.The protein was esti-mated by HPLC and SDS-PAGE to be 99%pure,and its identity was confirmed by NH 3-terminal Edman sequencing and amino acid compositional analysis.The pure lyophilized protein was stored at Ϫ80°C in 50%ethanol ϩ3mM HCl.Protein gel electrophoresis and Western blot.Purified myo-statin proteins (0.3–3␮g)or SDS extracts of cultured cells and skeletal muscle (30␮g protein)were treated as described (9),separated by SDS-PAGE on a 12–20%gradient Tris-glycine gel (ReadyGel,Bio-Rad,Hercules,CA),and detected by Coomassie blue and silver staining (Silver Stain Plus,Bio-Rad).Recombinant myostatin proteins were detected by Western blotting to nitrocellulose with polyclonal antibody B against myostatin (9)(1:1,000)or mouse monoclonal anti-body against the Myc epitopes (1:3,000,Invitrogen)as pri-mary antibodies and antibody to IgG linked with horseradish peroxidase (HRP)as the second antibody.Additionally,a direct staining procedure was applied for the detection of the carboxy-end polyhistidine tag with a monoclonal antibody to polyhistidine (COOH terminal)coupled to HRP (1:2,000,In-vitrogen).Blots were developed with an enhanced chemilu-minescent substrate for HRP and exposed to film (ECL hy-perfilm,Amersham).C 2C 12cell proliferation assay.Mouse skeletal muscle cell line C 2C 12(ATCC)was propagated as myoblasts in DMEM plus (DMEM containing 4mM glutamine and antibiotics)with 10%FBS,and incubated at 37°C at 10–50%confluence on the appropriate plates for each assay.For differentiation into myotubes,the myoblasts were plated at ϳ90-100%con-fluence;after 2days,the medium was changed to DMEM plus with 5%horse serum.The myotubes began to form in 2–4days,and multinucleated muscle fiber cultures were used at 7–10days.C 2C 12cell proliferation was determined in 96-well plates by the Formazan dye assay (Promega).Cells were grown at initial densities of 400,800,1,600,or 3,200cells/well;then,after 1day,they were treated with recombi-nant myostatin proteins in varying concentrations for 72h.After 3days of incubation,Formazan substrate buffer was added to the cultures for 3h at 37°C,and the absorbance at 492nm was read by an ELISA plate reader.For cell counting,the cells were removed by trypsinization,and the number of viable cells was counted in a hemocytometer with the use of trypan blue staining.[3H]thymidine incorporation into DNA.Cell cultures in 48-well plates were incubated in T-labeling medium (Leu-labeling medium described below plus 160␮g/ml of unlabeled leucine)containing 2␮Ci/ml [3H]thymidine (ICN no.24066)in the absence or presence of myostatin proteins.The cells were harvested at 24,48,and 72h,washed with PBS,and lysed with 120␮l of buffer with 1%Triton X-100ϩ1%SDS.To complete cell disruption,extracts were frozen and thawed,denatured by NaOH added to 0.1M,incubated for 30min at 25°C,and then neutralized with HCl.For analysis of both 14C-labeled protein and/or T-labeled DNA by TCA precipita-tion,20␮l of cell lysate were spotted onto GF/C filters (Whatman).Filters were dried,washed three times in 5%TCA,rinsed in ethanol,dried,and placed in 3ml of scintil-lation fluid (Scinti-Safe,Fisher)and counted in a ␤-counter (Beckman,Fullerton,CA).Protein synthesis and degradation.For measurement of the rate of protein synthesis,cells in 48-well plates were incubated in Leu-labeling medium (RPM11640medium with no leucine,containing 4mM glutamine,insulin,transferrin,selenium,and 1␮g/ml of added leucine,with 10%dialyzed FBS).Quadruplicate wells containing 0.2–12␮g/ml of myo-statin proteins were incubated for 40h and then pulse-labeled with 0.2␮Ci/ml of [1-14C]leucine (ICN)for 2.5h.Medium containing dialyzed horse serum was used in place of FBS for myotube induction.To measure the rate of protein degradation,cells were first pulse-labeled with [14C]leucine in Leu-labeling medium for 16h.The cells were then trans-ferred into T-labeling medium with and without myostatin protein at 6␮g/ml,but without [1-14C]leucine;excess leucine was added at time 0to prevent further labeling or reutiliza-tion of [14C]leucine.Cells were harvested at 1-day intervals over the 3-day chase period.At the completion of both proce-dures,the cells were treated and counted as above.Apoptosis.The apoptotic index was determined by the TdT-mediated dUTP nick end labeling (TUNEL)method,based on the ability of terminal TdT to catalyze addition of digoxenin-dUTP and dATP to 3Ј-OH ends of cleaved DNA.The cells were placed on removable plastic slides within 8-well chambers and were incubated with or without myo-statin proteins for 1–3days and then fixed in 10%parafor-maldehyde.The slides were treated with proteinase K and H 202,followed by addition of primary and secondary anti-sera.The plates were stained with 3Ј-3-diaminobenzidine (DAB)and counterstained with 0.5%methyl green.The ap-optotic index was calculated by dividing the number of apo-ptotic cells by the number of nuclei in the microscope field.Statistical analyses.The data are presented as means ϩSD.All data points represent the means of 4wells.To determine the effects of myostatin on DNA synthesis (Fig.4)and protein degradation (Fig.6),we used a two-way ANOVA,with incubation time and myostatin (Ϯ)as the two factors.To determine the effects of myostatin on protein synthesis,a one-way ANOVA was used.The cell growth curves (Fig.3)in the absence and presence of graded concentrations of recom-binant myostatin proteins were analyzed by a multifactor ANOVA.If ANOVA revealed significant overall effects,com-parison between groups was performed by t -matrix analysis.The P values were adjusted for multiple comparisons using Boneferroni’s correction.E223MYOSTATIN INHIBITS C 2C 12MUSCLE CELL GROWTHon March 4, 2011 Downloaded fromRESULTSExpression of recombinant full-length myostatin pro-teins.The recombinant full-length Mst375D protein was expressed from a cDNA construct subcloned from our previously reported clone(9)into the expression vector pcDNA3in both the sense(Mst)and antisense [Mst(A)]orientation.In addition,the sense cDNA was fused with a Myc-His tag[Mst(H)]at its carboxy ter-minus to facilitate identification of the recombinant protein with the use of anti-Myc or anti-His antibodies (Fig.1A).Myostatin gene expression from these plas-mids was seen both in vitro and in vivo(Fig.1,B and C).When Mst cDNA constructs were transcribed and translated in a cell-free system,we detected the45-kDa full-length protein,as expected.The translation of the Mst(H)plasmid yielded a slightly longer50-kDa protein with a His tag.No specific protein band was detected from the antisense construct(Fig.1B). When CHO cells were transfected with the pcDNA3-Mst(H)plasmid,the full-length myostatin protein with the Myc-His tag could be detected by Western blotting by use of antibodies against myostatin(Fig.1C,top), the His tag(Fig.1C,middle),and the Myc epitope(Fig. 1C,bottom).Myostatin proteins were seen in both monomeric(50–55kDa)and dimeric(100–110kDa) forms,expressed from the Mst(H)plasmid.As ex-pected,full-length myostatin protein lacking the His tag was detected only with anti-myostatin antibody. No comparable bands were detected in the vector con-trol with anti-myostatin,anti-His,or anti-Myc antibod-ies(Fig.1C).To produce the full-length375-aa myostatin protein with a His tag(Mst375D),the cDNA coding region was subcloned into an insect expression vector.This DESculture system allows for a more faithful processing of recombinant animal proteins in S2insect cells than in bacterial systems.Myostatin expression was detected as a45-to50-kDa band in induced,but not in unin-duced,S2cells and was verified by Western immuno-blotting with antibodies against myostatin(B)and the poly-His tag(Fig.2A).Recombinant myostatin was purified by nickel affinity columns,reverse phase HPLC,gelfiltration,and dialysis and was character-ized by gel electrophoresis and staining with Coomas-sie blue(Fig.2B,left)and silver staining(Fig.2B, middle)and Western blot analysis with anti-myostatin antibody(Fig.2B,right).We detected a45-kDa mono-mer and a90-kDa dimer by Coomassie blue and silver staining and by Western blot with anti-myostatin an-tibody.In addition,a22-kDa myostatin peptide band was also detected by silver staining and Western blot with anti-myostatin antibody(Fig.2B).One silver-stained55-kDa band,which did not react with the anti-Mst(B)antibody,may be a nonmyostatin contam-inant.Expression of the110-aa carboxy-terminal myostatin protein in E.coli.The recombinant110-aa carboxy-terminal protein was produced in E.coli(Mst110EC) by use of the Mst110coding region fused with a start codon and NH3-terminal polyhistidine tag.The Mst110EC protein was expressed in E.coli,refolded, and purified by HPLC.On SDS gel electrophoresis,this protein was visualized as a15-to17-kDa monomer and a30-kDa dimer by silver staining and immunoblotting with the anti-myostatin antibody(Fig.2B).On the basis of the intensity of the major Coomassie-stained 15-kDa band,this Mst110EC protein isϾ95%pure. Effects of Mst375D and Mst110EC proteins on mus-cle cell replication and DNA synthesis.To measure myostatin effects on cell number(Fig.3A),C2C12myo-blasts were grown in media containing recombinant Mst375D(top)or Mst110EC(bottom)protein at in-creasing concentration from0to6␮g/ml.The wells were harvested each day for3days,and the cell num-ber per well was counted.In the absence of myostatin protein,the C2C12myoblast cell number increased pro-gressively from days1to3(Fig.3A).Graded concen-trations of both the Mst375D and the Mst110EC pro-teins dose dependently inhibited the growth of C2C12 myoblasts.After3days,the increase in cell number was inhibited by55Ϯ5%in the presence of4␮g/ml Mst375D(Fig.3A,top)and by37Ϯ5%in the presence of4␮g/ml Mst110EC(Fig.3A,bottom).In C2C12myoblasts,cell proliferation,as assessed by the Formazan assay,was also inhibited in a dose-dependent manner by recombinant myostatinproteins Fig.2.Analysis of human recombinant myostatin proteins produced in Drosophila(375aa,Mst375D)and Escherichia coli cells(110aa, Mst110EC).A:Western blot of lysates from Drosophila S2cells transfected with plasmid pDES-Mst containing a poly-His tag.Ly-sates from noninduced(Ϫ)or induced(ϩ)cells were fractionated in duplicate on12%SDS-PAGE.Left:anti-myostatin antibody B;MSM, mouse skeletal muscle extract.Right:anti-polyhistidine antibody.B: gel electrophoretic analysis of purified recombinant myostatin pro-teins by staining and Western immunoblot.The recombinant Mst375D and Mst110EC were fractionated on12%SDS-PAGE(3␮g/lane).Left:Coomasie blue stain;middle:silver staining;right: Western immunoblotting with anti-myostatin Ab-Mst(B).Gel lanes were cut out and spliced together from1gel/panel;3similar gels were run in parallel with identical markers(BioRad,Kalidascope). Molecular masses of Mst proteins are indicated on right and protein size markers on left.E224MYOSTATIN INHIBITS C2C12MUSCLE CELL GROWTHon March 4, 2011Downloaded from(Fig.3B ).After 3days’growth in the presence of 4␮g/ml recombinant proteins,the cell titer in wells with initial cell density of 3,200cells/well was inhibited by 63Ϯ9%by Mst375D (Fig.3B ,top ),and 34Ϯ3%by Mst110EC (Fig.3B ,bottom ).In CHO cells,a nonmyo-genic cell line,Mst375D,had significantly less effect,inhibiting cell titer by Ͻ25%at 4␮g/ml concentration.To determine the effects of recombinant myostatin on DNA synthesis,C 2C 12cells,grown as 35%confluent myoblasts or differentiated myotubes,were treated with or without 6␮g/ml of Mst375D protein in medium containing [3H]thymidine.As expected,the rate of [3H]thymidine incorporation into DNA was higher in proliferating myoblasts than in differentiated myo-tubes (Fig.4).DNA synthesis,measured by [3H]thymi-dine incorporation,was inhibited in both myoblasts and myotubes by myostatin protein.The inhibition of DNA synthesis by 6␮g/ml of full-length myostatin protein after 3days of incubation was more pro-nounced in myotubes (63Ϯ8%)than in myoblasts (33Ϯ13%)(Fig.4).Effects of recombinant myostatin proteins on protein synthesis.The full-length Mst375D protein inhibited protein synthesis,measured by incorporation of [14C]leucine,in a dose-dependent manner in both myo-blasts and myotubes (Fig.5).The inhibition of the rate of protein synthesis was 59Ϯ6%in myoblasts incu-bated with 6␮g/ml of full-length Mst375D protein for 40h;in myotubes,the inhibition was 75Ϯ1%at this concentration (Fig.5,top ).The Mst110EC protein also inhibited protein synthesis in differentiated myotubes,as measured by [14C]leucine incorporation.The inhibi-tion was 39Ϯ8and 71Ϯ3%,respectively,in the presence of 2and 6␮g/ml of Mst110EC (Fig.5,bottom ).Effects of recombinant myostatin proteins on muscle cell protein degradation and apoptosis.To determine the effects of recombinant myostatin proteins on mus-cle protein breakdown,we prelabeled cell proteins with [14C]leucine and then prevented further incorporation of [14C]leucine into protein by addition of excess leucine in the medium.The remaining 14C counts in the protein fraction were chased during 3days ofin-Fig.3.Effects of recombinant myostatin proteins on C 2C 12myoblast cell proliferation.A :cell number measured by direct count of viable cells in a hemocytometer.C 2C 12myoblasts were grown for 24,48,or 72h in media containing 0,0.5,2,4,and 6␮g/ml Mst375D (top )or Mst110EC (bottom ).Data for cell count/well are means ϮSD (n ϭ4wells at each time point and concentration).ANOVA for Mst concentrations of 2,4,and 6␮g/ml vs.none (0)at 48and 72h show a significant effect of P Ͻ0.001.B :cell titer measured by Formazan dye assay.C 2C 12cells were plated at various initial densities of cells/well (x -axes)for 1day and then incubated for 3days in media containing increasing concentrations of Mst375D (top )or Mst110EC (bottom ),followed by Formazan assay.Data are means ϮSD (n ϭ4wells at each point).ANOVAs for Mst375D concentrations of 2,4,and 6␮g/ml vs.none (0)at 800or 1,600initial cells show a significant effect of P Ͻ0.001,and at 3,200cells show P Ͻ0.04.Fig.4.Effects of myostatin on DNA synthesis in C 2C 12myoblasts and myotubes.C 2C 12cells were grown as 35%confluent myoblasts (top )or 9-day differentiated myotubes (bottom ).At time 0,media were replaced with media containing [3H]thymidine with (Œ)or without (F )6␮g/ml Mst375D protein.Cells were harvested at time 0and every 24h for 3days,for determination of [3H]thymidine incorporation into DNA.Data in counts/min (cpm)of [3H]thymidine are means ϮSD,n ϭ4wells/dose and time point.ANOVAs for Mst375D concentrations of 6␮g/ml vs.none (0)at 48and 72h have P Ͻ0.0001for myotubes and P Ͻ0.013for myoblasts.E225MYOSTATIN INHIBITS C 2C 12MUSCLE CELL GROWTHon March 4, 2011 Downloaded from。