Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix

We screened the small molecule compounds that stimulate osteogenesis by themselves or promote bone morphogenetic protein (BMP)-induced bone formation. We found that a specific inhibitor for MAPK/ extracellular signal-regulated kinase kinase (MEK)-1, promoted the early osteoblastic differentiation and mineralization of extracellular matrix (ECM) in C2C12 pluripotent mesenchymal cells treated with recombinant human BMP-2 (rhBMP-2) and MC3T3-E1 preosteoblastic cells. ALP activity was synergistically increased by the treatment with a specific MEK-1 inhibitor PD98059 and rhBMP-2 in both cell lines. Twenty-five micromolar PD98059 promoted mineralization of ECM in rhBMP-2-treated C2C12 cells and MC3T3-E1 cells. In contrast, PD98059 reduced osteocalcin (OCN) secretion and its transcriptional level in rhBMP-2-treated C2C12 cells but increased its secretion and mRNA level in MC3T3-E1 cells. Stable expression of a dominant-negative MEK-1 mutant in C212 cells represented high ALP activity and low osteocalcin production in the presence of rhBMP-2, while a constitutively active mutant of MEK-1 attenuated both of them. Together, our results indicated that BMP-2-induced mineralization of ECM of pluripotent mesenchymal stem cells and preosteoblastic cells could be controlled by a fine tuning of the MAPK signaling pathway. Further, MEK-1 inhibitors would be useful for the promotion of bone formation, for instance, the treatments for delayed fracture healing or advance of localized osteoporotic change after fracture healing.