Inhibitor of DNA binding/differentiation (ID) helix-loop-helix proteins mediate BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells

Bone formation is a well-orchestrated process of osteoblast lineage-specific differentiation. As members of the TGFbeta superfamily, BMPs play an important role in regulating osteoblast differentiation and subsequent bone formation. Cheng et al. and Kang et al. conducted a comprehensive analysis of the osteogenic activity of the 14 human BMPs, and found that BMP2, BMP6, and BMP9 induced the most potent osteogenic differentiation of mesenchymal stem cells.; Although significant progress has been made in elucidating the transcriptional control of osteoblast differentiation, detailed molecular events underlying the osteogenic process remain to be elucidated. Through the microarray analysis of approximately 12,000 genes in pre-osteoblast progenitor cells stimulated by osteogenic BMPs (i.e., BMP2, BMP6, and BMP9) vs. negative/non-osteogenic BMPs (i.e., BMP3 and BMP12), I found that the expression level of 203 genes (105 up-regulated and 98 down-regulated) was altered >2-fold upon osteogenic BMP stimulation. Gene ontology analysis revealed a general trend in the early stage of BMP-mediated osteogenic signaling: an increase in proliferative potential towards osteoblast lineage and a decrease in myogenic potential, which is consistent with the notion that osteogenesis and myogenesis are opposite and divergent processes. Moreover, Id1, Id2, and Id3 are among the most significantly upregulated genes upon BMP2, BMP6, or BMP9 stimulation.; Next, I sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation, and found that the three Ids were significantly induced at the early stage of BMP9 stimulation and returned to basal levels at three (for C2C12 cells) or five (for C3H10T1/2 cells) days after stimulation. Both RNAi-mediated knockdown of Id expression and constitutive expression of Id genes significantly inhibited the BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. Furthermore, I demonstrated that BMP9-regulated Id expression is Smad4 dependent. Overexpression of the Id genes was shown to promote cell proliferation which was coupled with an inhibition of osteogenic differentiation. Thus, these findings suggest that the Id HLH proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells, and a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells.