Figure 2.

1α,25(OH)₂D₃ inhibits osteoclast development through VDR by acting directly on osteoclast precursor cells in bone marrow. (A and B) Osteoclast precursor cells were isolated from the bone marrow of WT C57BL/6J and VDR KO mice (B) as M-CSF–dependent adherent cells, as described in Methods, and were further treated with RANKL (40 ng/ml) in the absence or presence of 10^–7 M 1α,25(OH)₂D₃ for 3 days (A). Note that the development of TRAP-positive multinucleate osteoclasts induced by RANKL was markedly inhibited by cotreatment with 1α,25(OH)₂D₃. (B) The inhibitory effect of 1α,25(OH)₂D₃ on the formation of TRAP-positive multinucleate cells (MNCs) was dose-dependent and was not seen in marrow cultures derived from VDR KO mice, even at the highest dose of 10^–7 M. Data are expressed as a percentage of vehicle-treated cultures. *P < 0.05 versus vehicle group, n = 6. (C) Expression of VDRs in the intestine (lane 1) and osteoclast precursor cells (lane 2) as detected by RT-PCR. EF-1α mRNA served as control for PCR. Lane 3 contained water as a negative control.