Role of endogenous TGF-β family in myogenic differentiation of C2C12 cells.
The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis.
Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto, Japan.
Yuuma
Y
Furutani
Takenao
T
Umemoto
Masaru
M
Murakami
Tohru
T
Matsui
Masayuki
M
Funaba
D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta