Figure 7.

CCL19 and CCL21 activate osteoclast migration and resorption through Rho signaling. (a) BMMs were cultured with M-CSF (30 ng ml⁻¹) for 2 days. After serum starvation for 6 h, the cells were treated with CCL19 (10 ng ml⁻¹) or CCL21 (10 ng ml⁻¹) for 0, 5, 15 and 30 min. The cells were lysed, and GTP-bound active Rho was pulled down for western blots. A portion of the cell lysate was used to detect the total RhoA protein. (b) BMMs were cultured with M-CSF (30 ng ml⁻¹) for 2 days. After 6 h serum starvation, the cells were stimulated with CCL19 (10 ng ml⁻¹) or CCL21 (10 ng ml⁻¹). PMLC in the cell lysates was measured by western blots. β-Actin was used as a loading control. (c) BMMs were pretreated with Rho inhibitors (simvastatin and Y27632) for 1 h and loaded into the upper chambers of a transwell plate. CCL19 (10 ng ml⁻¹) or CCL21 (10 ng ml⁻¹) was added to the bottom chamber. After 16 h, the cells were fixed and stained with crystal violet. (d) BMMs were seeded and cultured with M-CSF (30 ng ml⁻¹) and RANKL (100 ng ml⁻¹) on calcium phosphate-coated plates. After 4 days, the cells were treated with Rho inhibitors (simvastatin and Y27632) in addition to CCL19 (10 ng ml⁻¹) or CCL21 (10 ng ml⁻¹). After 2 days, the cells were removed, and calcium phosphate was stained using the von Kossa method. The resorbed area was quantified. BMM, bone marrow-derived macrophage.