Induction of c-Fos can rescue the osteoclastogenesis failure by GM-CSF.

(A) The c-Kit⁺c-Fms⁺RANK⁻ cells prepared from wild-type littermates (■) or Mx–c-fos mice (▪) were cultured in the presence of 100 ng/mL M-CSF and 25 ng/mL sRANKL with (200 or 1000 U/mL) or without IFN-α/β for 6 days, and a TRAP activity assay was performed. Osteoclastogenesis was strongly inhibited by 1000 U/mL IFN-α/β in both Mx–c-fos and wild-type littermates. (B) The c-Kit⁺c-Fms⁺RANK⁻ cells prepared from Mx–c-fos mice were cultured in the presence of 100 ng/mL M-CSF, 25 ng/mL sRANKL, and 1 ng/mL GM-CSF with IFN-α/β (2, 20, or 200 U/mL) for 6 days, and a TRAP staining was performed. The formation of TRAP-positive cells was observed in a dose-dependent manner. (C) Osteoclast differentiation was not affected by addition of 200 U/mL IFN-α/β in both wild-type littermates and Mx–c-fos mice, and multinuclear TRAP-positive cells were observed in the absence of GM-CSF (i,iii). The multinuclear TRAP-positive cells were formed when c-Fos was induced by the addition of IFN-α/β in Mx–c-fos mice in the presence of GM-CSF (ii), while no TRAP-positive cells were detected in wild-type littermates (iv). Bar = 100 μm. (D) The c-Kit⁺c-Fms⁺RANK⁻ cells were cultured in the presence of M-CSF, sRANKL, and GM-CSF. IFN-α/β was present over the culture period (days 0-6), during the first 3 days (days 0-3), and the last 3 days (day 4-6). After 6 days of culture, the number of TRAP-positive cells were counted. The early expression of c-Fos is more effective than that of late expression in the rescue of osteoclastogenesis.