Betulinic acid stimulates the differentiation and mineralization of osteoblastic MC3T3-E1 Cells: Involvement of BMP/Runx2 and β-Catenin Signals

In the field of osteoporosis, there has been growing interest in anabolic agents that enhance bone formation. Here, we examined the effects of betulinic acid on cell proliferation, differentiation, and mineralization of MC3T3-E1 osteoblasts. Then, the impact of betulinic acid on signaling pathways known to be implicated in osteoblastogenesis was explored. Betulinic acid (1-20 μM) markedly increased alkaline phosphatase (ALP) activity and calcium nodule formation, although without a notable effect on cell proliferation. Stimulation with betulinic acid not only increased the osteopontin level and osteocalcin mRNA expression but also upregulated the osteoprotegerin (OPG)/RANKL ratio. Noggin, but not ICI 182780, significantly repressed betulinic acid-induced ALP activity, suggesting a possible action of betulinic acid through the bone morphogenetic protein (BMP) pathway. This was strengthened by the induction of BMP-2 expression, increases in Smad1/5/8 phosphorylation, and Runx2 expression. Furthermore, betulinic acid increased the nuclear level of the active form β-catenin. These results suggested that betulinic acid has the potential to enhance osteoblastogenesis probably through the activation of BMP/Smad/Runx2 and β-catenin signal pathways. Furthermore, upregulation of the OPG/RANKL ratio to repress bone catabolism may also indirectly contribute to the bone anabolic effect of betulinic acid.