Role of mitogen-activated protein kinase in osteoblast differentiation.

Local control of osteoblast differentiation and bone formation is not well understood. We have previously seen biphasic effects on cell differentiation in response to the short- and long-term exposure to IL-1β in rat calvarial osteoblasts. To characterize the signaling pathway mechanisms regulating IL-1β biphasic effects, we examined the contribution of mitogen-activated protein kinase (MAPK) family. Cells were pretreated with specific inhibitors to extracellular signal-regulated kinase (ERK, PD98059), p38 (SB203580), and c-JUN N-terminal kinase (JNK, SP600125), then co-cultured with IL-1β for 2, 4, and 6 days. Cell differentiation was determined by measuring bone nodules after 10 days of culture. These inhibitors did not alter biphasic effects of IL-1β on cell differentiation. However, PD98059 and U2016, another inhibitor of ERK activation robustly increased osteoblast differentiation compared to vehicle-treated control in a time- and dose-dependent manner. PD98059 appears to stimulate alkaline phosphatase (ALP) activity to promote cell differentiation, where IL-1β appears to suppress it. Interestingly, continuous ERK inhibition with PD98059, after 2 and 4 days of IL-1β treatment, enhanced the IL-1β anabolic effect by increasing bone nodules formed. These observations provide a potential mechanism involving ERK pathway in osteoblasts differentiation and suggest that MAPK family may not directly regulate IL-1β biphasic effects.