Myostatin inhibits myoblast differentiation by down-regulating MyoD expression.
Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.
Langley
Brett
B
Animal Genomics, AgResearch, Private Bag 3123, East Street, Hamilton, New Zealand.
Thomas
Mark
M
Bishop
Amy
A
Sharma
Mridula
M
Gilmour
Stewart
S
Kambadur
Ravi
R
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D003001:Cloning, Molecular; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D004926:Escherichia coli / Q000378:metabolism; D007150:Immunohistochemistry; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology; D009132:Muscles / Q000166:cytology*; D017570:MyoD Protein / Q000378:metabolism*; D051787:Myogenic Regulatory Factor 5; D018008:Myogenin / Q000378:metabolism; D055435:Myostatin; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D051900:Smad3 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C525912:Mstn protein, mouse; D009124:Muscle Proteins; C497418:Myf5 protein, mouse; D017570:MyoD Protein; C503801:Myog protein, mouse; D051787:Myogenic Regulatory Factor 5; D018008:Myogenin; D055435:Myostatin; D011994:Recombinant Proteins; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D008156:Luciferases; D014357:Trypsin