Synergistic effect of BMP2 and retinoic acid on osteogenic differentiation

Objectives: Bone morphogenetic protein 2 (BMP2) is involved in the induction of osteoblast differentiation. It has been known that retinoic acid is also the differentiation factor which affects the osteogenic differentiation. We studied the effects of retinoic acid and BMP on the osteogenic differentiation of C2C12 myoblast with reference to retinoic acid receptor (RAR) family (a, b, g). Methods: We tested the effects of synthetic retinoids AM580, Ch55 and CD666, specific agonists for RARa, RARb and RARg, respectively, on the osteogenic differentiation. For gene silencing of each RAR, small interfering RNA (siRNA) expression vectors were introduced into C2C12 cells before the retinoic acid and BMP2 treatment. The osteogenic differentiation of the cell was judged to measure the cells alkaline phosphatase (ALP) activity of the differentiation marker of osteogenesis, and the induction of ALP mRNA using RT-PCR analysis. Biomineralization as a marker of mature osteoblasts was examined using alizarin red staining and a calcium assay after a 7-day culture in medium supplemented with glycerophsphate and ascorbic acid. Results: Marked induction of the ALP activity was detemined in the cells treated with all-trans retinoic acid (ATRA) or CD666, RARg specific agonist, and was synergistically enhanced by simultaneous treatment of BMP2. The synergetic induction of CD666 and BMP2 was also observed in the induction of ALP mRNA, which was resistant to cycloheximide, an inhibitor of protein synthesis. The synergistic effect of BMP2 and ATRA on the ALP induction was interrupted only with the RARg gene silencing. Alizarin red staining and calcium assay showed that the CD666 was the most effective retinoid for biomineralization in the C2C12 cells. Conclusion: It is suggested that the retinoic acid and BMP2 show a synergistic effect in the osteogenic differentiation, and the effect of retinoic acid in this reaction is mediated by RARg.