Osteoclast progenitor cells present in significant amounts in mouse calvarial osteoblast isolations and osteoclastogenesis increased by BMP-2.

Abstract

Enzymatically released cells from neonatal mouse calvarial bones are frequently used as primary mouse osteoblasts for in vitro studies. We, here, report that although these cells lack mRNA expression of the osteoclastic genes Calcr, Acp5 and Mmp-9 at early time points after their isolation, these transcripts are gradually upregulated when the calvarial osteoblast cultures are differentiated to more mature osteoblasts in long term cultures. Similarly, Calcr, Acp5, Mmp-9, as well as Rank and Nfatc1 mRNA expressions are robustly increased when the osteoblast cultures were induced to differentiate by treatment with bone morphogenetic protein (BMP-2). The increased Calcr mRNA resulted in functionally active calcitonin receptors. Enhanced osteoblastic differentiation was associated with increased Rankl mRNA expression and decreased Opg and Cfs1 mRNA expression. Treatment of the osteoblastic cells with BMP-2 or RANKL, either on plastic dishes or bone slices, resulted in the formation of multinucleated tartrate-resistant acid phosphatase positive cells, which were able to excavate resorption pits and release CTX from the bones. In contrast, increased osteoblastic differentiation induced by BMP-2 in the mouse calvarial osteoblastic cell line MC3T3-E1 was not associated with increased mRNA expression of Calcr, Acp5, Rank, c-Fms or Oscar. Interestingly, Ctsk mRNA was increased during osteoblastic differentiation in both mouse calvarial osteoblast cultures and in MC3T3-E1 cultures. Also osteoblasts isolated from mouse long bones by outgrowth from explant cultures were contaminated with osteoclast progenitors able to differentiate into bone resorbing osteoclasts. These data indicate that mouse calvarial osteoblast cultures contain osteoclast progenitor cells, which will be differentiated along the osteoclastic lineage during osteoblastic differentiation. Moreover, the data show that BMP-2 not only stimulates osteoblastic differentiation but can also induce osteoclastogenesis through increased RANKL.