Effect of CSF-1 on RANKL mRNA expression in murine primary osteoblasts. A, Dose-dependent effect of CSF-1 on RANKL (upper panel). Cells were placed in SF medium for a total period of 48 h including a 24-h incubation time with or without increasing concentrations of rmCSF-1 before harvesting for total RNA. RT-PCR was performed using murine RANKL or 18S primer sets. B, Time course for the effect of rmCSF-1 on RANKL (upper panel). Cells in SF medium were incubated with or without rmCSF-1 (100 ng/ml) for the indicated time points before harvesting at 48 h. Bar graphs (lower panels) show semiquantitative analysis of RANKL expression shown in upper panels. C, Effect of CSF-1 on osteoprotegerin (OPG) mRNA expression. Experiments were carried out as described in A, and RT-PCR was performed using a murine OPG primer set. D, RANKL protein expression in vivo. Western blot analysis of RANKL expression in bone marrow (left panel) and bone (right panel) lysates from wt and sCSF-1-transgenic (TG) mice are shown. a, P < 0.001; b, P < 0.01; c, P < 0.05; d, P > 0.05 vs. untreated cultures.