Fig. 1.

RM-A induces apoptosis in OCs through inhibition of protein synthesis. (A) Structure of RM-A. Crude OCs prepared from cocultures of bone marrow cells and osteoblasts in the presence of 1α,25(OH)₂D₃ were treated with or without RM-A for 24 h. (B) Cells were fixed and stained for TRAP. (C) Remaining TRAP (+) OCs with more than three nuclei were counted. Data are expressed as means ± SD for four cultures. ∗, P < 0.01; ∗∗, P < 0.001 vs. control. (D) Purified OCs were treated with or without RM-A in the presence of RANKL (100 ng/ml) for 24 h. Remaining TRAP (+) OCs with more than three nuclei were counted. Data represent means ± SD for four cultures. ∗, P < 0.01; ∗∗, P < 0.001 vs. control. (E) Purified OCs were treated with or without RM-A in the presence of RANKL (100 ng/ml) for 12 h. Cells were fixed and stained for TRAP, and nuclei were visualized by Hoechst 33258 staining. OCs with normal and condensed nuclei were counted, and percentages of OCs with condensed nuclei compared with those with normal nuclei are shown as apoptotic OCs. Data are expressed as means ± SD for four cultures. ∗, P < 0.05; ∗∗, P < 0.001 vs. control. (F) Purified OCs were cultured for 1 h with or without zVAD-fmk (50 μM) in the presence of RANKL (100 ng/ml) and then treated for 4 h with or without RM-A (1 μM). Cell lysates were used for measurement of caspase 3-like enzyme activity. Data are expressed as means ± SD for three cultures. (G) Protein synthesis was measured by amounts of incorporated l-[³⁵S]methionine into cellular proteins. OCs, formed from a RAW 264 cell culture, were treated for 4 h with appropriate concentrations of RM-A or cycloheximide (CHX). (H) OCs prepared from bone marrow cell cultures in the presence of macrophage colony-stimulating factor (30 ng/ml) and RANKL (300 ng/ml). In vitro aminoacyl-tRNA synthetase assays were carried out in the presence of 0.015, 0.15, and 1.5 μM RM-A. Data are expressed as means ± SD for three cultures.