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• W1987132484 abstract "Myostatin, a transforming growth factor-β superfamily ligand, negatively regulates skeletal muscle growth. Generation of the mature signaling peptide requires cleavage of pro-myostatin by a proprotein convertase, which is thought to occur constitutively in the Golgi apparatus. In serum, mature myostatin is found in an inactive, non-covalent complex with its prodomain. We find that in skeletal muscle, unlike serum, myostatin is present extracellularly as uncleaved pro-myostatin. In cultured cells, co-expression of pro-myostatin and latent transforming growth factor-β-binding protein-3 (LTBP-3) sequesters pro-myostatin in the extracellular matrix, and secreted pro-myostatin can be cleaved extracellularly by the proprotein convertase furin. Co-expression of LTBP-3 with myostatin reduces phosphorylation of Smad2, and ectopic expression of LTBP-3 in mature mouse skeletal muscle increases fiber area, consistent with reduction of myostatin activity. We propose that extracellular pro-myostatin constitutes the major pool of latent myostatin in muscle. Post-secretion activation of this pool by furin family proprotein convertases may therefore represent a major control point for activation of myostatin in skeletal muscle. Myostatin, a transforming growth factor-β superfamily ligand, negatively regulates skeletal muscle growth. Generation of the mature signaling peptide requires cleavage of pro-myostatin by a proprotein convertase, which is thought to occur constitutively in the Golgi apparatus. In serum, mature myostatin is found in an inactive, non-covalent complex with its prodomain. We find that in skeletal muscle, unlike serum, myostatin is present extracellularly as uncleaved pro-myostatin. In cultured cells, co-expression of pro-myostatin and latent transforming growth factor-β-binding protein-3 (LTBP-3) sequesters pro-myostatin in the extracellular matrix, and secreted pro-myostatin can be cleaved extracellularly by the proprotein convertase furin. Co-expression of LTBP-3 with myostatin reduces phosphorylation of Smad2, and ectopic expression of LTBP-3 in mature mouse skeletal muscle increases fiber area, consistent with reduction of myostatin activity. We propose that extracellular pro-myostatin constitutes the major pool of latent myostatin in muscle. Post-secretion activation of this pool by furin family proprotein convertases may therefore represent a major control point for activation of myostatin in skeletal muscle. Regulation of skeletal muscle size is an essential feature of organism development and adult muscle homeostasis. Several circulating factors function to control muscle growth, including the transforming growth factor-β (TGF-β) 2The abbreviations used are:TGF-βtransforming growth factor βECMextracellular matrixLAPlatency associated peptideLTBPlatent TGF-β-binding proteinPCproprotein convertaseHAhemagglutininGFPgreen fluorescent proteinPNGasepeptide:N-glycosidase FConAconcanavalin ANP-40Nonidet P-40. superfamily ligand myostatin, which is a negative regulator of skeletal muscle growth (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3222) Google Scholar). Loss of myostatin function, in knock-out mice or mice treated with inhibitors, results in up to a 2-fold increase in skeletal muscle mass due to an increased number of muscle fibers and increased muscle fiber size (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3222) Google Scholar, 2Bogdanovich S. Krag T.O. Barton E.R. Morris L.D. Whittemore L.A. Ahima R.S. Khurana T.S. Nature. 2002; 420: 418-421Crossref PubMed Scopus (726) Google Scholar, 3Lee S.J. McPherron A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9306-9311Crossref PubMed Scopus (1288) Google Scholar, 4Whittemore L.-A. Song K. Li X. Aghajanian J. Davies M. Girgenrath S. Hill J.J. Jalenak M. Kelley P. Knight A. Maylor R. O'Hara D. Pearson A. Quazi A. Ryerson S. Tan X.-Y. Tomkinson K.N. Veldman G.M. Widom A. Wright J.F. Wudyka S. Zhao L. Wolfman N.M. Biochem. Biophys. Res. Commun. 2003; 300: 965-971Crossref PubMed Scopus (432) Google Scholar). In contrast, ectopic expression of myostatin in adult mice induces cachexia, a systemic wasting syndrome (5Zimmers T.A. Davies M.V. Koniaris L.G. Haynes P. Esquela A.F. Tomkinson K.N. McPherron A.C. Wolfman N.M. Lee S.J. Science. 2002; 296: 1486-1488Crossref PubMed Scopus (748) Google Scholar). Regulation of myostatin production and signaling is essential to achieve a balance between muscle growth and wasting. transforming growth factor β extracellular matrix latency associated peptide latent TGF-β-binding protein proprotein convertase hemagglutinin green fluorescent protein peptide:N-glycosidase F concanavalin A Nonidet P-40. In adults, myostatin activity is regulated at several levels. First, myostatin expression is limited to a few cell types, including skeletal muscle and, to a lesser extent, adipose and heart tissues (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3222) Google Scholar, 6Sharma 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 (356) Google Scholar). Second, myostatin is synthesized as a precursor protein that remains inactive until it is modified by several post-translational events (7Lee S.J. Annu. Rev. Cell Dev. Biol. 2004; 20: 61-86Crossref PubMed Scopus (639) Google Scholar). Third, multiple extracellular inhibitors limit access of myostatin to cell surface receptors (3Lee S.J. McPherron A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9306-9311Crossref PubMed Scopus (1288) Google Scholar, 7Lee S.J. Annu. Rev. Cell Dev. Biol. 2004; 20: 61-86Crossref PubMed Scopus (639) Google Scholar). The myostatin precursor, referred to as pro-myostatin, forms a disulfide-linked homodimer following synthesis and translocation in the endoplasmic reticulum (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3222) Google Scholar, 3Lee S.J. McPherron A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9306-9311Crossref PubMed Scopus (1288) Google Scholar). Like other TGF-β superfamily ligands, pro-myostatin is cleaved into amino- and carboxyl-terminal fragments at a tetrabasic cleavage site by the furin family of proprotein convertases (PCs) (3Lee S.J. McPherron A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9306-9311Crossref PubMed Scopus (1288) Google Scholar, 8Scamuffa N. Calvo F. Chretien M. Seidah N.G. Khatib A.M. FASEB J. 2006; 20: 1954-1963Crossref PubMed Scopus (188) Google Scholar, 9Thies R.S. Chen T. Davies M.V. Tomkinson K.N. Pearson A.A. Shakey Q.A. Wolfman N.M. Growth Factors. 2001; 18: 251-259Crossref PubMed Scopus (217) Google Scholar). This cleavage is thought to occur primarily in the Golgi apparatus, and the COOH-terminal, disulfide-linked product of this cleavage is the mature myostatin ligand. The mature myostatin homodimer remains non-covalently associated with the prodomain in a latent complex that is abundant in mouse serum in vivo (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3222) Google Scholar, 9Thies R.S. Chen T. Davies M.V. Tomkinson K.N. Pearson A.A. Shakey Q.A. Wolfman N.M. Growth Factors. 2001; 18: 251-259Crossref PubMed Scopus (217) Google Scholar, 10Hill J.J. Davies M.V. Pearson A.A. Wang J.H. Hewick R.M. Wolfman N.M. Qiu Y. J. Biol. Chem. 2002; 277: 40735-40741Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). The BMP-1/tolloid family of metalloproteinases can activate this latent complex by proteolytic cleavage between Arg-75 and Asp-76 of the myostatin prodomain (11Wolfman N.M. McPherron A.C. Pappano W.N. Davies M.V. Song K. Tomkinson K.N. Wright J.F. Zhao L. Sebald S.M. Greenspan D.S. Lee S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15842-15846Crossref PubMed Scopus (363) Google Scholar). Following this activation of the latent complex, mature myostatin activates the Smad2/Smad3 signal cascade (12Langley B. Thomas M. Bishop A. Sharma M. Gilmour S. Kambadur R. J. Biol. Chem. 2002; 277: 49831-49840Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 13Rebbapragada A. Benchabane H. Wrana J.L. Celeste A.J. Attisano L. Mol. Cell. Biol. 2003; 23: 7230-7242Crossref PubMed Scopus (464) Google Scholar). Several additional myostatin inhibitors have been identified in serum, including follistatin, GASP-1, and FLRG. It is still unclear, however, to what extent these inhibitors act individually or in conjunction with the inhibitory prodomain (10Hill J.J. Davies M.V. Pearson A.A. Wang J.H. Hewick R.M. Wolfman N.M. Qiu Y. J. Biol. Chem. 2002; 277: 40735-40741Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 14Hill J.J. Qiu Y. Hewick R.M. Wolfman N.M. Mol. Endocrinol. 2003; 17: 1144-1154Crossref PubMed Scopus (181) Google Scholar). Thus, the current model for myostatin activation suggests that the latent myostatin complex is constitutively secreted into circulation from myostatin-producing cells, and that the activity of this complex is regulated by BMP-1/tolloid metalloproteinases and secreted inhibitors. Like myostatin, canonical TGF-β ligands are retained in a latent complex containing a mature peptide homodimer and a non-covalently associated inhibitory prodomain. The latter is commonly referred to as latency associated peptide (LAP) (15Miyazono K. Hellman U. Wernstedt C. Heldin C.H. J. Biol. Chem. 1988; 263: 6407-6415Abstract Full Text PDF PubMed Google Scholar). TGF-β latent complexes also covalently associate with latent TGF-β-binding proteins (LTBPs), which are required for efficient folding and secretion of the ligands (16Rifkin D.B. J. Biol. Chem. 2005; 280: 7409-7412Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Four LTBPs have been identified and are designated LTBP 1-4 (17Kanzaki T. Olofsson A. Moren A. Wernstedt C. Hellman U. Miyazono K. Claesson-Welsh L. Heldin C.H. Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 18Moren A. Olofsson A. Stenman G. Sahlin P. Kanzaki T. Claesson-Welsh L. Dijke ten P. Miyazono K. Heldin C.H. J. Biol. Chem. 1994; 269: 32469-32478Abstract Full Text PDF PubMed Google Scholar, 19Saharinen J. Taipale J. Monni O. Keski-Oja J. J. Biol. Chem. 1998; 273: 18459-18469Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 20Yin W. Smiley E. Germiller J. Mecham R.P. Florer J.B. Wenstrup R.J. Bonadio J. J. Biol. Chem. 1995; 270: 10147-10160Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Of the four LTBPs identified, LTBPs 1, 3, and 4 form a disulfide linkage with cysteine 33 of the TGF-β LAP (15Miyazono K. Hellman U. Wernstedt C. Heldin C.H. J. Biol. Chem. 1988; 263: 6407-6415Abstract Full Text PDF PubMed Google Scholar, 21Saharinen J. Taipale J. Keski-Oja J. EMBO J. 1996; 15: 245-253Crossref PubMed Scopus (190) Google Scholar, 22Wakefield L.M. Smith D.M. Flanders K.C. Sporn M.B. J. Biol. Chem. 1988; 263: 7646-7654Abstract Full Text PDF PubMed Google Scholar). The domain of LTBP that forms this disulfide bond with LAP is an 8-Cys motif that is unique to the LTBP/Fibrillin family (21Saharinen J. Taipale J. Keski-Oja J. EMBO J. 1996; 15: 245-253Crossref PubMed Scopus (190) Google Scholar, 23Gleizes P.E. Beavis R.C. Mazzieri R. Shen B. Rifkin D.B. J. Biol. Chem. 1996; 271: 29891-29896Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Once secreted, the TGF-β·LTBP complex, called large latent complex, covalently associates with the extracellular matrix (ECM) through LTBP (24Nunes I. Gleizes P.E. Metz C.N. Rifkin D.B. J. Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (348) Google Scholar). TGF-β ligands must be released from this latent complex to signal (25Koli K. Saharinen J. Hyytiainen M. Penttinen C. Keski-Oja J. Microsc. Res. Tech. 2001; 52: 354-362Crossref PubMed Scopus (226) Google Scholar). The binding of latent TGF-β to LTBPs tethers the latent complex to the ECM, providing a mechanism for local activation of TGF-β signaling. In the case of latent myostatin in serum, however, how local activation in tissues might be regulated is not clear. Despite our detailed understanding of how LTBPs interact with canonical TGF-β ligands, little is known about interactions between LTBPs and other ligands in the TGF-β superfamily. In Xenopus, LTBP-1 (xLTBP-1) has been shown to synergize with the TGF-β superfamily ligands activin and nodal to induce mesoderm, but the basis for this synergy is not known (26Altmann C.R. Chang C. Munoz-Sanjuan I. Bell E. Heke M. Rifkin D.B. Brivanlou A.H. Dev. Biol. 2002; 248: 118-127Crossref PubMed Scopus (24) Google Scholar). To what extent LTBPs interact generally with TGF-β superfamily ligands, and whether these interactions share common mechanisms or functions with those reported for LTBPs and TGF-β, have not been elucidated. The maturation of TGF-β superfamily ligands by furin-like proteases is thought to occur constitutively in the Golgi apparatus. In the case of the TGF-β superfamily ligand nodal, however, it has recently been demonstrated that the PCs furin and PACE4 act extracellularly to cleave secreted pro-nodal into mature ligand, thus localizing nodal activity near cells that secrete PCs (27Beck S. Good Le J.A. Guzman M. Haim Ben N. Roy K. Beermann F. Constam D.B. Nat. Cell Biol. 2002; 4: 981-985Crossref PubMed Scopus (180) Google Scholar). This provides an intriguing example of localization of ligand activity by extracellular localization of maturation activity, but whether this example has broader relevance to regulation of TGF-β superfamily ligands is not known. In this article we demonstrate that the predominant form of myostatin detectable in muscle is the pro-form, that this pro-myostatin is present extracellularly, and can be cleaved extracellulary by furin proteases. We also find that pro-myostatin associates with LTBPs, and that the major LTBP expressed in skeletal muscle, LTBP-3, sequesters pro-myostatin in the ECM. This retention of pro-myostatin by LTBP-3 limits myostatin signaling, as demonstrated by LTBP-3-dependent reduction of myostatin-induced Smad2 activation. In addition, ectopic expression of LTBP-3 in adult mouse skeletal muscle increases fiber area, consistent with the local inhibition of myostatin activity. These observations point to local maturation of secreted pro-myostatin in skeletal muscle by furin-like PCs as a significant new point of regulation of myostatin activation, and suggest new approaches to the therapeutic inhibition of myostatin function for the treatment of muscle-wasting diseases. Expression Constructs—pBS(KS+) mouse myostatin was a gift from Se-Jin Lee (John Hopkins University School of Medicine, Baltimore, MD). Mouse myostatin was subcloned into pCS4+ using the restriction sites StuI and XhoI. FLAG myostatin and HA myostatin were constructed by inserting codons 23-376 of mouse myostatin into a pCS4+ vector containing the Xnr1 signal peptide (codons 1-12) followed by either 3-FLAG or 3-HA epitope tags. FLAG myostatin prodomain/mature domain was constructed with 3 FLAG epitope tags following the Xnr1 signal peptide, as above, followed by 3 FLAG epitope tags at codon 272 of the myostatin mature domain. FLAG myostatin prodomain was constructed by PCR-based subcloning. FLAG myostatin prodomain contains the Xnr1 signal peptide (codons 1-12), 3 FLAG epitope tags, and mouse myostatin prodomain (codons 23-266). FLAG myostatin ANAA was constructed by changing codons 39-42 from CNAC to ANAA by mutagenesis PCR using the template FLAG mouse myostatin and the mutagenesis primer 5′-GAGAGAGAAGAAAATGTGGAAAAAGAAGGCCTAGCTAATGCAGCAGCGTGGAGACAAAACACGAGG-3′. HA epitope-tagged human LTBP-2 and mouse LTBP-3 were provided in pcDNA3 vectors by Daniel B. Rifkin (New York University School of Medicine, New York, NY). pCS4+ mouse LTBP-3ΔC HA was constructed by PCR-based subcloning. Mouse LTBP3-ΔC HA contains the mouse LTBP-3 signal peptide (codons 1-22), one HA epitope tag, and mouse LTBP-3 codons 41-883. pCS2 + 6Myc human Smad3 was provided by Jeffrey L. Wrana (Samuel Lunenfeld Research Institute, Toronto, Ontario). pGEM7zf Furin:Flag was provided by Gary Thomas (Vollum Institute, Portland, OR). PCR-based subcloning was used to insert codons 1-802 of Furin:Flag into pCS4+. pCS4+ Furin: FlagΔC was constructed by PCR-based subcloning of codons 1-723 of Furin:Flag into pCS4+. In Vitro Translation—In vitro translation of pCS4+ FLAG myostatin was performed using the Promega TnT® SP6-coupled Reticulocyte Lysate System (L4600, Promega, Madison, WI). Tissue Culture—293T human kidney epithelial cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (10-437-028, Invitrogen), 50 IU/ml penicillin-streptomycin (30-001-C1, Mediatech, Herndon, VA). Cells were transfected using 25-kDa linear polyethylenimine (23966, Polysciences, Warrington, PA) as described previously (28Durocher Y. Perret S. Kamen A. Nucleic Acids Res. 2002; 30: E9Crossref PubMed Scopus (837) Google Scholar). Xenopus Ectoderm Explants—Xenopus embryos were collected, fertilized, and cultured as previously described (29Faure S. Lee M.A. Keller T. Dijke ten P. Whitman M. Development. 2000; 127: 2917-2931Crossref PubMed Google Scholar, 30Lee M.A. Heasman J. Whitman M. Development. 2001; 128: 2939-2952PubMed Google Scholar). Each blastomere of 2-cell stage embryos was injected with synthetic mRNA that was transcribed using the SP6 mMessage mMachine™ Kit (Ambion, Austin, TX). 200 pg of HA myostatin and 1500 pg of LTBP-3 HA RNA were injected as indicated. Ectoderm explants were harvested as previously described (30Lee M.A. Heasman J. Whitman M. Development. 2001; 128: 2939-2952PubMed Google Scholar). Transfection of Mouse Tibialis Anterior Muscle and Fiber Area Measurements—Adult female CD1 mice weighing 34-39 g were used. All mice were housed in the Seeley G. Mudd Animal Facility at Harvard Medical School. Tibialis anterior muscles were transfected as described previously (31Sandri M. Sandri C. Gilbert A. Skurk C. Calabria E. Picard A. Walsh K. Schiaffino S. Lecker S.H. Goldberg A.L. Cell. 2004; 117: 399-412Abstract Full Text Full Text PDF PubMed Scopus (2226) Google Scholar). A 150 mm NaCl solution containing 5 μg of pCS2+ GFP CAAX and 16 μg of pcDNA3 mouse LTBP-3 HA plasmid DNA was injected into the tibialis anterior muscle, as indicated. Electric pulses were applied to the muscle at 50 volts/cm, 5 pulses, 100-ms intervals. Muscles were harvested 7 days later and processed by cryosection. Sections were fixed with 4% paraformaldehyde. Pictures of muscle cross-sections were captured with a ×10 objective using a Zeiss Axio Imager.M1 with AxioVision Release 4.5 software. Images were layered and color was added using Adobe Photoshop Software (San Jose, CA). Muscle fiber area was determined for GFP positive fibers using IMAGE software (Scion, Frederick, MD). At least 725 fibers were counted for each condition in a total of four mice. Precipitation and Western Blotting—Cultured 293T cells were rinsed three times in ice-cold phosphate-buffered saline then lysed in modified RIPA buffer (150 mm NaCl, 50 mm Tris (pH 8), 25 mm β-glycerophosphate, 100 mm sodium fluoride, 2 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 2× Complete EDTA-free protease inhibitor mixture (Roche Applied Science), 1 mm phenylmethylsulfonyl fluoride) plus 2 mm EDTA, 1% Nonidet P-40 (NP-40). Lysates were centrifuged and supernatants collected. NP-40-insoluble cell fraction was obtained by adding modified RIPA buffer plus 2 mm EDTA, 1% SDS to cell pellets that remained after lysis. Xenopus ectoderm explants were homogenized in modified RIPA buffer, plus 2 mm EDTA, 1% NP-40, 0.5% sodium deoxycholate, as above. Skeletal muscle and liver samples were rinsed three times in ice-cold phosphate-buffered saline then lysed in modified RIPA buffer plus 0.5 mm EDTA, 0.5% NP-40, 0.1% SDS, 0.25% sodium deoxycholate. Samples were homogenized using a Polytron tissue homogenizer. Lysates were centrifuged and supernatants collected. For concanavalin A (ConA) precipitation, samples were incubated in ConA-agarose (Sigma), 1 mm MnCl2, 1 mm CaCl2 for 4 h at 4 °C. Samples bound to ConA-agarose were washed twice with cold phosphate-buffered saline, 1% NP-40, 1 mm MnCl2, 1 mm CaCl2; once with cold phosphate-buffered saline, 1% NP-40, 300 mm NaCl, 1 mm MnCl2, 1 mm CaCl2; and once with cold Tris buffer (10 mm Tris (pH 8.0)), 1 mm MnCl2, 1 mm CaCl2. Protein was eluted from the ConA-agarose overnight at 4 °C with modified RIPA buffer plus 0.5 mm EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.75 m methyl α-d-mannopyranoside (Sigma). The following antibodies were used for immunoprecipitations. Anti-HA rat monoclonal antibody (clone 3F10; Roche), followed by incubation with protein A matrix preincubated in rat anti-rabbit IgG (Jackson Laboratories, West Grove, PA); anti-Biotin rabbit polyclonal antibody (Rockland Immunochemicals, Gilbertsville, PA); anti-mouse GDF-8 propeptide sheep polyclonal antibody (AF1539, R&D, Minneapolis, MN), followed by incubation with protein A matrix preincubated in sheep anti-rabbit IgG (Jackson Laboratories); anti-mouse GDF-8 goat polyclonal antibody (AF788, R&D), followed by incubation with protein A matrix preincubated in goat anti-rabbit IgG (Jackson Laboratories). The following antibodies were used for Western blotting. Anti-HA-peroxidase rat monoclonal peroxidase-conjugated antibody (clone 3F10; Roche); anti-FLAG-peroxidase mouse monoclonal peroxidase-conjugated antibody (Sigma); anti-Myc-peroxidase mouse monoclonal peroxidase-conjugated antibody (Roche); anti-actin mouse monoclonal antibody (A4700, Sigma); anti-P-Smad2 rabbit polyclonal antibody (Cell Signaling Technologies, Danvers, MA); anti-mouse GDF-8 propeptide sheep polyclonal antibody (AF1539, R&D); and anti-mouse GDF-8 goat polyclonal antibody (AF788, R&D). Glycosidase Treatment—For PNGase treatment, immunoprecipitated proteins were treated with the glycosidase PNGase F (New England Biolabs, Beverly, MA) in 50 mm sodium phosphate (pH 7.5), 1% NP-40 for 2 h at 37 °C. Sulfo-NHS-Biotin-LC Treatment—For 293T cells, 1 mg/ml EZ Link Sulfo-NHS-LC-Biotin (21335, Pierce) was added to 293T cells in HEPES-buffered saline (HBS) (pH 8.0). Cells were incubated for 30 min at room temperature, then sulfo-NHS-LC-Biotin was quenched with HBS, 100 mm glycine (pH 8.0). For mouse skeletal muscle and liver, 1 mg/ml sulfo-NHS-LC-Biotin was added to dissected skeletal muscle or liver from adult female CD1 mice in Krebs bicarbonate ringer solution (Krebs buffer) (pH 8.0). Tissues were incubated on ice for 30 min, then sulfo-NHS-LC-Biotin was quenched with Krebs buffer, 100 mm glycine (pH 8.0). Myostatin Interacts with Latent TGF-β-binding Proteins—LTBPs assist in the secretion of canonical TGF-β ligands and their retention in the ECM (32Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (423) Google Scholar). To determine whether LTBPs play a role in the regulation of myostatin, we investigated whether myostatin interacts with LTBPs. We compared, in transfected 293T cells, the binding of myostatin to LTBP-3, the most highly expressed LTBP in skeletal muscle, and to LTBP-2, which is expressed at low levels in muscle cells (expression of ectopic LTBP-1 and LTBP-4 was very low in our hands and was not pursued) (18Moren A. Olofsson A. Stenman G. Sahlin P. Kanzaki T. Claesson-Welsh L. Dijke ten P. Miyazono K. Heldin C.H. J. Biol. Chem. 1994; 269: 32469-32478Abstract Full Text PDF PubMed Google Scholar, 33Penttinen C. Saharinen J. Weikkolainen K. Hyytiainen M. Keski-Oja J. J. Cell Sci. 2002; 115: 3457-3468PubMed Google Scholar). Pro-myostatin interacts with LTBP-2 and LTBP-3 in co-immunoprecipitations from 293T cells (Fig. 1A). The prodomain of myostatin alone interacts very poorly with LTBP-3 (Fig. 1, A and B), indicating that the mature region is important for stable interaction of pro-myostatin with LTBP-3. Mature myostatin, however, is not immunoprecipitated by LTBP-3, indicating that myostatin does not interact with LTBP-3 following proteolytic cleavage (Fig. 1C), and that both the mature and pro-domains of myostatin are necessary for stable interaction with LTBP-3. We also observed (Fig. 1C) that LTBP-3 expression results in a decrease in the amount of mature myostatin in lysates, indicating that LTBP-3 can inhibit production of mature myostatin. We noted that pro-myostatin is present in 293T cells as a doublet, and that each LTBP immunoprecipitated different forms of this pro-myostatin doublet; LTBP-2 predominantly precipitated the faster migrating form of pro-myostatin, whereas LTBP-3 almost exclusively precipitated the slower migrating form of pro-myostatin (Figs. 1A and 2A). The most likely basis for altered migration of pro-myostatin is the addition of N-linked glycosylations during passage through the secretory pathway (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3222) Google Scholar, 34Jiang M.S. Liang L.F. Wang S. Ratovitski T. Holmstrom J. Barker C. Stotish R. Biochem. Biophys. Res. Commun. 2004; 315: 525-531Crossref PubMed Scopus (94) Google Scholar). To determine whether the pro-myostatin doublet we observed was a result of N-linked glycosylation, we compared the migration of pro-myostatin synthesized in vitro, in the absence of the secretory apparatus, to pro-myostatin isolated from cells and treated with or without the deglycosylating enzyme PNGase F (Fig. 2A). The faster migrating form of pro-myostatin corresponds in size to pro-myostatin expressed in vitro and to PNGase-treated pro-myostatin, indicating that the faster migrating form of pro-myostatin is the unglycosylated form. The slower migrating form of pro-myostatin is converted to the faster migrating form by PNGase F treatment, indicating that N-linked glycosylations are responsible for the difference in migration. When co-expressed myostatin and LTBP-3 were immunoprecipitated for the HA epitope tag in LTBP-3, PNGase F treatment shifted the LTBP-3-associated pro-myostatin from the slower migrating form to the faster migrating form (Fig. 2A). These data indicate that LTBP-2 preferentially associates with unglycosylated pro-myostatin, whereas LTBP-3 preferentially associates with glycosylated pro-myostatin. Because N-linked glycosylations are typically added as proteins move through the secretory apparatus, these data suggest that LTBP-2 binding is restricted to pro-myostatin that is retained in the endoplasmic reticulum in a form that is not competent for glycosylation or secretion, whereas LTBP-3 binds to pro-myostatin that can be secreted. LTBPs are covalently linked to canonical TGF-βs by a disulfide bond between an 8-cysteine motif in the LTBPs and a cysteine near the N terminus of the TGF-β prodomain (15Miyazono K. Hellman U. Wernstedt C. Heldin C.H. J. Biol. Chem. 1988; 263: 6407-6415Abstract Full Text PDF PubMed Google Scholar, 21Saharinen J. Taipale J. Keski-Oja J. EMBO J. 1996; 15: 245-253Crossref PubMed Scopus (190) Google Scholar, 22Wakefield L.M. Smith D.M. Flanders K.C. Sporn M.B. J. Biol. Chem. 1988; 263: 7646-7654Abstract Full Text PDF PubMed Google Scholar, 23Gleizes P.E. Beavis R.C. Mazzieri R. Shen B. Rifkin D.B. J. Biol. Chem. 1996; 271: 29891-29896Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 35Yin W. Fang J. Smiley E. Bonadio J. Biochim. Biophys. Acta. 1998; 1383: 340-350Crossref PubMed Scopus (8) Google Scholar). The myostatin prodomain contains two cysteines at a similar position as the cysteine in TGF-β that links to LTBPs, raising the possibility that pro-myostatin also forms a disulfide linkage with LTBP. To determine whether myostatin interacts with LTBP-3 in a manner similar to the canonical TGF-β ligands, we created a myostatin in which the cysteines near the N terminus of the prodomain have been changed to alanines (Fig. 1B). Co-expression of pro-myostatin in which the prodomain cysteines have been mutated to alanine has no effect on LTBP-3 binding to myostatin (Fig. 1A), however, indicating that a disulfide linkage is not necessary for stable interaction between these proteins. Furthermore, an LTBP-3 construct that lacks the carboxyl-terminal region containing the TGF-β binding motif, LTBP-3ΔC (Fig. 1B), binds as effectively to pro-myostatin as does wild type LTBP-3 (Fig. 2B), indicating that pro-myostatin binds to a different region of LTBP-3 than does TGF-β. The pro-myostatin-LTBP-3 interaction is not retained during Laemmli gel electrophoresis under non-reducing conditions (not shown), further indicating that this interaction, unlike the binding of TGF-β to LTBPs, does not involve a disulfide linkage. Pro-myostatin Is Retained in the Extracellular Matrix of Cells Expressing Latent TGF-β-binding Protein-3—LTBPs assist in the secretion of canonical TGF-β ligands (32Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (423) Google Scholar). To determine whether LTBPs influence the secretion of myostatin, we monitored myostatin levels in NP-40-solubilized cell lysates, conditioned media, and the NP-40-insoluble cell fraction of cells expressing myostatin in the presence or absence of LTBP-3. In contrast to expectations based on observations of the effect of LTBP on TGF-β secretion, co-expression of LTBP-3 dramatically reduced the amount of pro-myostatin in the conditioned media of 293T cells (Fig. 2B). Concomitant with this reduction of pro-myostatin in conditioned media by LTBP-3 expression, however, we observed an increase in pro-myostatin in the NP-40-insoluble cell pellet. This pellet could reflect either an intracellular NP-40-insoluble fraction (e.g. cytoskeleton) or the NP-40-insoluble ECM. To distinguish between these possibilities, we treated intact 293T cel" @default.
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• W1987132484 date "2008-03-01" @default.
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• W1987132484 title "Identification of a Novel Pool of Extracellular Pro-myostatin in Skeletal Muscle" @default.
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• W1987132484 doi "https://doi.org/10.1074/jbc.m706678200" @default.
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