Proliferation-coupled osteoclast differentiation by RANKL: Cell density as a determinant of osteoclast formation

Although it is widely recognized that the osteoclast differentiation induced by RANKL is linked to the anti-proliferative activity of the cytokine, we report here that RANKL in the presence of M-CSF actually stimulates DNA synthesis and cell proliferation during the early proliferative phase (0–48 h) of osteoclastogenesis ex vivo, while the same cytokine exerts an anti-proliferative activity in the latter half (48–96 h). A tracing of the individual cells using Fucci cell cycle indicators showed that waves of active DNA synthesis in the S phase during the period 0–48 h are followed by cell-cycle arrest and cell fusion after 48 h. Inhibition of DNA synthesis with hydroxyurea (HU) during the first half almost completely inhibited osteoclastogenesis; however, the same HU-treated cells, when re-plated at 48 h at increasing cell densities, exhibited restored osteoclast formation, suggesting that a sufficient number of cells, rather than prior DNA synthesis, is the most critical requirement for osteoclast formation. In addition, varying either the number of bone marrow macrophages at the start of osteoclastogenic cultures or pre-osteoclasts halfway through the process had a substantial impact on the number of osteoclasts that finally formed, as well as the timing of the peak of osteoclast formation. Thus, caution should be exerted in the performance of any manipulative procedure, whether pharmacological or genetic, that affects the cell number prior to cell fusion. Such procedures can have a profound effect on the number of osteoclasts that form, the final outcome of “differentiation”, leading to misinterpretation of the results.