T. Katagiri et al., BONE MORPHOGENETIC PROTEIN-2 CONVERTS THE DIFFERENTIATION PATHWAY OF C2C12 MYOBLASTS INTO THE OSTEOBLAST LINEAGE, The Journal of cell biology, 127(6), 1994, pp. 1755-1766
The implantation of bone morphogenetic protein (BMP) into muscular tis
sues induces ectopic bone formation at the site of implantation. To in
vestigate the mechanism underlying this process, we examined whether r
ecombinant bone morphogenetic protein-2 (BMP-2) converts the different
iation pathway of the clonal myoblastic cell line, C2C12, into that of
osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for
6 d almost completely inhibited the formation of the multinucleated my
otubes expressing troponin T and myosin heavy chain, and induced the a
ppearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2
dose dependently induced ALP activity, parathyroid hormone (PTH)depen
dent 3',5'-cAMP production, and osteocalcin production at concentratio
ns above 100 ng/ml. The concentration of BMP-2 required to induce thes
e osteoblastic phenotypes was the same as that required to almost comp
letely inhibit myotube formation. Incubating primary muscle cells with
300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas
induced ALP activity and osteocalcin production. Incubation with 300 n
g/ml of BMP-2 suppressed the expression of mRNA for muscle creatine ki
nase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-r
elated protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-
2 completely inhibited the expression of myogenin mRNA by day 3. By da
y 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was tran
siently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mR
NA was greatly stimulated by BMP-2. When C2C12 cells pretreated with B
MP-2 for 6 d were transferred to a colony assay system in the absence
of BMP-2, more than 84% of the colonies generated became troponin T-po
sitive and ALP activity disappeared. TGF-beta 1 also inhibited myotube
formation in C2C12 cells, and suppressed the expression of myogenin a
nd MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteobla
stic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 po
tentiated the inhibitory effect of BMP-2 on myotube formation, whereas
TGF-beta 1 reduced ALP activity and osteocalcin production induced by
BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically
converts the differentiation pathway of C2C12 myoblasts into that of o
steoblast lineage cells, but that the conversion is not heritable.