Bisphosphonate induces the mitophagy of osteoblastic cells by forming the chelate with intracellular metal ions

Abstract

Extracting teeth of patients treated with bisphosphonate (BP) occasionally induces the necrosis of jaw, but the cause of disease is still unclear. I have proved that the BPs taken into osteoblastic cells were gradually accumulated in lysosomes. In the present study, I investigated the mechanism of BP-induced cytotoxicity in osteoblast, focusing on mitochondria. MC3T3-E1 cells were used as osteoblastic cells. The uptake of BP into cells was observed by fluorescent BP. The intracellular reactive oxygenspecies (ROS) were evaluated by CM-H₂-DCFDA. Detection of autophagy and mitophagy was used DALGreen and mtphagy dye, respectively. The intracellular Ca²⁺ and mitochondrial Fe²⁺ were measured by Fluo 4-AM and Mito-FerroGreen, respectively. Zoledronate (ZD) impaired cells dose-dependently. BP taken into cells was accumulated into lysosomes. MC3T3-E1 cells were always occurred autophagy flux, but bafilomycin A1(BM), a lysosome inhibitor induced cell death, by inhibiting autophagy flux. ZD slightly suppressed the autophagy flux, however the combination of BM and ZD strongly enhanced cell death. ZD decreased intracellular Ca²⁺and mitochondrial Fe²⁺, and inhibited the response of intracellular ROS generation by oxidative stress, resulting in promotion of mitophagy. These results suggest that BP may form the chelate with Ca²⁺and Fe²⁺, and promote mitophagy of damaged mitochondria. Furthermore, the accumulation of BP into lysosomes indicates to induce cell death by inhibiting the autophagy flux.