Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor ?1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation

Abstract

The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.

Gene expression changes during mouse skeletal myoblast differentiation revealed by transcriptional profiling

“…Several of these genes function in osteoblasts: the transcription factor, Osf2 (cluster 1,2) (9) and the osteoblast matrix proteins, osteomodulin/osteoadherin (Omd; cluster 1,1) (34) and osteoglycin (Ogn; cluster 1,3) (32). Osf2 (Cbfa1) mRNA expression is increased by BMP treatment of C2C12 cells, which causes a transformation from myogenic to osteoblast differentiation (23). The induction of Osf2 transcripts in differentiating C2C12 myoblasts that was observed in our study has not been observed by others.…”

runt Homology Domain Transcription Factors (Runx, Cbfa, and AML) Mediate Repression of the Bone Sialoprotein Promoter: Evidence for Promoter Context-Dependent Activity of Cbfa Proteins

“…Studies have shown that Cbfa1 is necessary but not sufficient for osteogenic differentiation (32). For example, both BMP-2 and TGF-␤ can induce expression of Cbfa1 and homeodomain proteins in the nonosseous myogenic cell line C2C12 (6,32), but only BMP-2 can support a change to the osteogenic phenotype with concomitant expression of OC and other osteoblast markers (27). Thus Cbfa1 may be inducing or interacting with an unknown factor in response to BMP-2 that functions as a transcriptional activator, either directly as a DNA binding protein or indirectly as a Cbfa partner protein.…”

“…1. The upper strands (200 ng) of the oligonucleotides were labeled with 32 P for 1 h at 37°C in a 50-l volume using T4 Polynucleotide Kinase (New England BioLabs, Beverly, Mass.) as indicated by the manufacturer.…”

“…Blots were hybridized with randomly primed (Prime-It kit; Stratagene, La Jolla, Calif.), 32 P-labeled cDNA probes for Gallus BSP at 42°C overnight. The radiographic signal was quantitated by using a STORM Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.).…”

Epigenetic Control of the Bone-master Runx2 Gene during Osteoblast-lineage Commitment by the Histone Demethylase JARID1B/KDM5B

“…In the presence of BMP2, proliferating mesenchymal multipotent C2C12 cells can differentiate to the osteoblast lineage, as reflected by expression of early and late bone-related markers (44,48,49). In contrast, when pre-confluent C2C12 cells are grown in the presence of low serum media (e.g.…”