Nitrogen-containing bisphosphonates inhibit RANKL- and M-CSF-induced osteoclast formation through the inhibition of ERK1/2 and Akt activation

Masanobu Tsubaki,Hirotaka Shimaoka,Shozo Nishida,Naoki Ogawa,Daiichiro Fujiwara,Junji Mukai,Tomoya Takeda,Motohiro Imano,Katsuhiko Sakaguchi,Makiko Komai,Tatsuki Itoh,Kenji Mashimo,Takao Satou,Kotaro Sakamoto

doi:10.1186/1423-0127-21-10

Abstract

BackgroundBisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7.ResultsIt was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF-κB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-κB ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation.ConclusionsThis indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.

Highlights

Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases
Minodronate and alendronate inhibited osteoclast formation through suppression of Geranylgeranyl pyrophosphate (GGPP) biosynthesis We investigated the effect of minodronate and alendronate on osteoclast formation in the presence of 25 ng/mL Receptor activator of NF-κB ligand (RANKL) plus 50 ng/mL macrophage-colony stimulating factor (M-CSF), with or without minodronate and alendronate in C7 cells
Minodronate and alendronate inhibited RANKL- and M-CSF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt activation in C7 cells To investigate the molecular mechanisms of minodronate and alendronate in C7 cells, we examined phosphorylated ERK1/2, Akt and p38MAPK by Western blot analysis

Summary

Introduction

Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. C7 cells were cultured for 12 days and treated with 0.1, 0.25, or 0.5 μM minodronate (C, E) or 0.5, 1, or 2 μM alendronate (D, F). Cells were cultured in the presence of 25 ng/mL RANKL plus 50 ng/mL M-CSF. Cultures were fed every 3 days by replacing with 500 μL of fresh medium with or without minodronate, alendronate, RANKL, and M-CSF. Cultures were fixed and stained for TRAP-positive multinucleated cells (C, D), and TRAP-positive cells (E, F) per well was counted These results are representative of 5 independent experiments. (G, H) Inhibitory effect of minodronate and alendronate on RANKL and M-CSF-induced CTR and cathepsin K mRNA expression. C7 cells were treated with minodronate (G) or alendronate (H) with 25 ng/mL RANKL plus 50 ng/mL M-CSF for 12 days. The results are representative of 4 independent experiments. *P < 0.01 compared to 25 ng/mL RANKL plus 50 ng/mL M-CSF administration

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Results

Conclusion