Sustained activation of the extracellular signal-regulated kinase pathway is required for extracellular calcium stimulation of human osteoblast proliferation

Elevated levels of [Ca(2+)](o) in bone milieu as a result of the resorptive action of osteoclasts are implicated in promoting proliferation and migration of osteoblasts during bone remodeling. However, mitogenic effects of [Ca(2+)](o) have only been shown in some, but not all, clonal osteoblast-like cells, and the molecular mechanisms underlying [Ca(2+)](o)-induced mitogenic signaling are largely unknown. In this study we demonstrated for the first time that [Ca(2+)](o) stimulated proliferation of primary human osteoblasts and selectively activated extracellular signal-regulated kinases (ERKs). Neither p38 mitogen-activated protein (MAP) kinase nor stress-activated protein kinase was activated by [Ca(2+)](o). Treatment of human osteoblasts with a MAP kinase kinase inhibitor, PD98059, impaired both basal and [Ca(2+)](o)-stimulated phosphorylation of ERKs and also reduced both basal and [Ca(2+)](o)-stimulated proliferation. [Ca(2+)](o) treatment resulted in two distinctive phases of ERK activation: an acute phase and a sustained phase. An inhibition time course revealed that it was the sustained phase, not the acute phase, that was critical for [Ca(2+)](o)-stimulated osteoblast proliferation. Our results demonstrate that mitogenic responsiveness to [Ca(2+)](o) is present in primary human osteoblasts and is mediated via prolonged activation of the MAP kinase kinase/ERK signal pathway.