Proc Natl Acad Sci U S A 98(16): 9306-9311

Regulation of myostatin activity and muscle growth

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

^{To whom reprint requests should be addressed. E-mail:}ude.imhj@eeljs.

Communicated by David L. Garbers, University of Texas Southwestern Medical Center, Dallas, TX

Received 2001 Mar 26; Accepted 2001 May 30.

Abstract

Myostatin is a transforming growth factor-β family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may have applications for promoting muscle growth, we investigated the regulation of myostatin signaling. Myostatin protein purified from mammalian cells consisted of a noncovalently held complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal myostatin dimer was capable of binding the activin type II receptors, Act RIIB and, to a lesser extent, Act RIIA. Binding of myostatin to Act RIIB could be inhibited by the activin-binding protein follistatin and, at higher concentrations, by the myostatin propeptide. To determine the functional significance of these interactions in vivo, we generated transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB by using a skeletal muscle-specific promoter. Independent transgenic mouse lines for each construct exhibited dramatic increases in muscle mass comparable to those seen in myostatin knockout mice. Our findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful agents for enhancing muscle growth for both human therapeutic and agricultural applications.

Abstract

Myostatin is a transforming growth factor-β (TGF-β) family member that plays an essential role in regulating skeletal muscle growth (1). Myostatin is expressed initially in the myotome compartment of developing somites and continues to be expressed in the myogenic lineage throughout development and in adult animals. Mice carrying a targeted deletion of the myostatin gene have a dramatic and widespread increase in skeletal muscle mass. Individual muscles of myostatin null mice weigh approximately twice as much as those of wild-type mice as a result of a combination of muscle fiber hyperplasia and hypertrophy. The myostatin sequence has been highly conserved through evolution (2). Remarkably, the human, rat, murine, porcine, turkey, and chicken myostatin sequences are identical in the biologically active C-terminal portion of the molecule following the proteolytic processing site. The function of myostatin also appears to be conserved across species, as mutations in the myostatin gene have been shown to result in the double muscling phenotype in cattle (2–5).

These findings have raised the possibility that pharmacological agents capable of blocking myostatin activity may have applications for promoting muscle growth in human disease settings as well as in livestock animals. To identify novel strategies for blocking myostatin activity, we investigated the regulation of myostatin signaling. Here, we present evidence that myostatin, like TGF-β, may normally exist in vivo in a latent complex with the propeptide (the portion of the precursor protein upstream of the proteolytic processing site) and that on activation, myostatin may signal by binding to activin type II receptors.

All animals (including controls) represent hybrid SJL/C57BL/6 F₀ mice born from injected embryos.

All animals (including controls) represent 4-month-old offspring of transgenic founders (SJL/C57BL/6) mated with wild-type C57BL/6 mice.

Acknowledgments

We thank Neil Wolfman for assistance with the HPLC, Christine Moss for assistance with the manuscript, and Joan Massague, Peter ten Dijke, Carl-Henrik Heldin, Jeffrey Wrana, Liliana Attisano, Wylie Vale, Sonia Pearson-White, Martin Matzuk, and Monique Davies for providing clones. This work was supported by National Institutes of Health Grants R01HD35887 and R01CA88866 (to S.J.-L.). Myostatin was licensed by the Johns Hopkins University to MetaMorphix (MMI) and sublicensed to American Home Products and Cape Aquaculture Technologies (CAT). The authors are entitled to a share of sales royalty received by the University from sales of this factor. The authors and the University own MMI stock, and the authors also own CAT stock, which are subject to certain restrictions under University policy. S.-J.L. is a consultant to MMI and CAT, and A.C.M. is a consultant to CAT. The terms of these arrangements are being managed by the University in accordance with its conflict of interest policies.

Acknowledgments

Abbreviations

TGF-β	transforming growth factor-β
BMP	bone morphogenetic protein
GDF-11	growth/differentiation factor-11

Abbreviations

References

1. McPherron A C, Lawler A M, Lee S-J. Nature (London) 1997;387:83–90.[PubMed]
2. McPherron A C, Lee S-J. Proc Natl Acad Sci USA. 1997;94:12457–12461. . (First Published October 17, 2000; 10.1073/pnas.220421797)
3. Grobet L, Martin L J R, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Ménissier F, Massabanda J, et al Nat Genet. 1997;17:71–74.[PubMed][Google Scholar]
4. Kambadur R, Sharma M, Smith T P L, Bass J J. Genome Res. 1997;7:910–916.[PubMed]
5. Grobet L, Poncelet D, Royo L J, Brouwers B, Pirottin D, Michaux C, Ménissier F, Zanotti M, Dunner S, Georges M. Mamm Genome. 1998;9:210–213.[PubMed]
6. Frolik C A, Wakefield L M, Smith D M, Sporn M B. J Biol Chem. 1984;259:10995–11000.[PubMed]
7. Franzén P, ten Dijke P, Ichijo H, Yamashita H, Schultz P, Heldin C-H, Miyazono K. Cell. 1993;75:681–692.[PubMed]
8. Donoghue M, Merlie J, Rosenthal N, Sanes J. Proc Natl Acad Sci USA. 1991;88:5847–5851.
9. McPherron A C, Lee S-J. J Biol Chem. 1993;268:3444–3449.[PubMed]
10. Heldin C-H, Miyazono K, ten Dijke P. Nature (London) 1997;390:465–471.[PubMed]
11. Massagué J, Blain S W, Lo R S. Cell. 2000;103:295–309.[PubMed]
12. Attisano L, Cárcamo J, Ventura F, Weis F M B, Massagué J, Wrana J. Cell. 1993;75:671–680.[PubMed]
13. Massagué J. Cell. 1992;69:1067–1070.[PubMed]
14. Wang X-F, Lin H, Ng-Eaton E, Downward J, Lodish H, Weinberg R. Cell. 1991;67:797–805.[PubMed]
15. Miyazono K, Hellman U, Wernstedt C, Heldin C-H. J Biol Chem. 1988;263:6407–6415.[PubMed]
16. Gentry L E, Nash B W. Biochemistry. 1990;29:6851–6857.[PubMed]
17. Böttinger E P, Factor V M, Tsang M L-S, Weatherbee J A, Kopp J B, Qian S W, Wakefield L M, Roberts A B, Thorgeirsson S S, Sporn M B. Proc Natl Acad Sci USA. 1996;93:5877–5882.
18. Gamer L, Wolfman N, Celeste A, Hattersley G, Hewick R, Rosen V. Dev Biol. 1999;208:222–232.[PubMed]
19. Nakashima M, Toyono T, Akamine A, Joyner A. Mech Dev. 1999;80:185–189.[PubMed]
20. McPherron A C, Lawler A M, Lee S-J. Nat Genet. 1999;22:260–264.[PubMed]
21. Matzuk M M, Lu N, Vogel H, Sellheyer K, Roop D R, Bradley A. Nature (London) 1995;374:360–363.[PubMed]
22. Berk M, Desai S, Heyman H, Colmenares C. Genes Dev. 1997;11:2029–2039.
23. Luo K, Stroschein S, Wang W, Chen D, Martens E, Zhou S, Zhou Q. Genes Dev. 1999;13:2196–2206.
24. Stroschein S, Wang W, Zhou S, Zhou Q, Luo K. Science. 1999;286:771–774.[PubMed]
25. Sun Y, Liu X, Ng-Eaton E, Lodish H, Weinberg R. Proc Natl Acad Sci USA. 1999;96:12442–12447.
26. Sun Y, Liu X, Ng-Eaton E, Lane W, Lodish H, Weinberg R. Mol Cell. 1999;4:499–509.[PubMed]
27. Akiyoshi S, Inoue H, Hanai J-i, Kusanagi K, Nemoto N, Miyazono K, Kawabata M. J Biol Chem. 1999;274:35269–35277.[PubMed]
28. Sutrave P, Kelly A, Hughes S. Genes Dev. 1990;4:1462–1472.[PubMed]
29. Hemmati-Brivanlou A, Melton D A. Nature (London) 1992;359:609–614.[PubMed]
30. Schulte-Merker S, Smith J C, Dale L. EMBO J. 1994;13:3533–3541.
31. Hemmati-Brivanlou A H, Thomsen G H. Dev Genet (Amsterdam) 1995;17:78–89.[PubMed]
32. Meno C, Gritsman K, Ohishi S, Ohfuji Y, Heckscher E, Mochida K, Shimono A, Kondoh H, Talbot W S, Robertson E J, et al Mol Cell. 1999;4:287–298.[PubMed][Google Scholar]
33. Link B, Nishi R. Exp Cell Res. 1997;233:350–362.[PubMed]