Endogenous Bmp4 in myoblasts is required for myotube formation in C2C12 cells

Our previous study revealed the indispensable activity of endogenous bone morphogenetic protein (Bmp) prior to differentiation induction of C2C12 myoblasts for myogenesis. Here we investigated the Bmp isoform responsible for endogenous Bmp activity during differentiation and its role in myogenesis.

Gene expression of Bmp4 during myogenesis was evaluated in C2C12 cells. Effects of inhibition of the Bmp pathway on myogenesis were examined. Cells expressing Bmp4 and regulation of Bmp4 expression in myoblasts were explored.

The expression of Bmp4 increased with the progression of myogenesis, although the extent of the increase after differentiation induction was smaller than that before the induction. Down-regulation of Bmp signal components including Bmp4, Bmpr2, and Alk2/3 inhibited the emergence of positive cells for myosin heavy chain II. The treatments also decreased the Myogenin expression. Treatment with cytosine arabinoside decreased the expression of Bmp4. Also, Bmp4 expression was also lower in isolated myotubes than in residual cells. Expression of Rgm c was higher in the myotube fraction. Transcription of Bmp4 was repressed by the conditioned medium of mixed cells consisting of myoblasts and myotubes.

Bmp4 expressed in myoblasts has a positive role in myotube formation/maturation through myogenin expression. The presence of myotubes inhibits Bmp4 expression in proliferating myoblasts through transcriptional regulation, although the expression is intrinsically increased with time of culture.

Taken previous results on involvement of Bmp in the commitment of osteoblasts and adipocytes with the present results together, Bmp may act as a general promoter of mesenchymal cell differentiation.