title: Myostatin stimulates, not inihibits, C2C12 myoblast proliferation.
abstract: The immortal C2C12 cell line originates from dystrophic mouse thigh muscle and has been used to study the endocrine control of muscle cell growth, development, and function, including those actions regulated by myostatin. Previous studies suggest that high concentrations of recombinant myostatin generated in bacteria inhibit C2C12 proliferation and differentiation. Recombinant myostatin generated in eukaryotic systems similarly inhibits the proliferation of primary myosatellite cells, but consequently initiates, rather than inhibits, their differentiation and is bioactive at far lower concentrations. Our studies indicate that 2 different sources of recombinant myostatin made in eukaryotes stimulate, not inhibit, C2C12 proliferation. This effect occurred at different cell densities and serum concentrations and in the presence of IGF-I, a potent myoblast mitogen. This stimulatory effect was comparable to that obtained with TGFβ1, a related factor that also inhibits primary myosatellite cell proliferation. Attenuating the myostatin/activin (ie, Acvr2b) and TGFβ1 receptor signaling pathways with the Alk4/5 and Alk5 inhibitors, SB431542 and SB505142, respectively, similarly attenuated proliferation induced by serum, myostatin or TGFβ1 and in a dose-dependent manner. In serum-free medium, both myostatin and TGFβ1 stimulated Smad2 phosphorylation, but not that of Smad3, and a Smad3 inhibitor (SIS3) only inhibited proliferation in cells cultured in high serum. Thus, myostatin and TGFβ1 stimulate C2C12 proliferation primarily via Smad2. These results together question the physiological relevance of the C2C12 model and previous studies using recombinant myostatin generated in bacteria. They also support the alternative use of primary myosatellite cells and recombinant myostatin generated in eukaryotes.
authors.affiliation: Department of Animal Sciences (B.D.E., K.E.H.), School of Molecular Biosciences (B.D.R., M.F.J.), School of Biological Sciences (B.D.W.), Washington Center for Muscle Biology, Washington State University, Pullman, Washington 99164-7620; and Department of Molecular Genetics, Biochemistry and Microbiology (R.G.W., T.B.T.), University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Buel D
authors.initials: BD
authors.lastname: Rodgers
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Wiedeback
authors.forename: Knut E
authors.initials: KE
authors.lastname: Hoversten
authors.forename: Melissa F
authors.initials: MF
authors.lastname: Jackson
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Walker
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Thompson
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008024:Ligands; D051379:Mice; D032446:Myoblasts / Q000166:cytology*; D055435:Myostatin / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D008024:Ligands; D055435:Myostatin; D011994:Recombinant Proteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D053773:Transforming Growth Factor beta1; D007334:Insulin-Like Growth Factor I