INHIBITORS FOR VACUOLAR THPE HィイD1+ィエD1-ATPaseINDUCES APOTOSIS IN OSTEOCLASTS

破骨细胞是多核骨吸收巨细胞。破骨细胞通过酸吸收骨矿物质。破骨细胞产酸受碳酸酐酶和空泡型三磷酸腺苷酶控制。近年来，我们发现空泡型atp酶抑制剂可引发破骨细胞凋亡。在本研究项目中，我们研究了atp酶抑制剂诱导的细胞凋亡事件的细胞机制。从小鼠骨髓培养物中制备纯化破骨细胞。空泡型atp酶抑制剂、康那霉素A和巴非霉素可诱导纯化的破骨细胞在24 h内死亡。Hoechst染色和FITC-TUNEL显示了这种破骨细胞死亡的凋亡特征。caspase活性测定显示，在破骨细胞凋亡事件中，ATPase抑制剂激活caspase-1和caspase-3。此外，还检测到caspase-8和caspase-9的活化。用MitAlert对破骨细胞线粒体膜进行荧光标记，发现线粒体膜被atp酶抑制剂破坏。提示空泡型atp酶在骨吸收和破骨细胞存活中起重要作用。

Osteoclasts are multinucleated bone resorbing giant cells. Osteoclasts resorb bone minerals by acid. Production of acid by osteoclast is controlled by carbonic anhydrase and vacuolar type ATPase. Recently, we found that the inhibitors of vacuolar type ATPase trigger apoptotic cell death of osteoclasts. In this research project, we investigate the cellular mechanism of apoptotic events induced by the ATPase inhibitors.Purified osteoclasts were prepared from mouse bone marrow cultures. Vacuolar type ATPase inhibitors, concanamycin A and bafilomycin induced cell death of purified osteoclast within 24 h. Hoechst stain and FITC-TUNEL revealed apoptotic features of this cell death of osteoclasts. Measurement of caspase activities revealed that ATPase inhibitors activated caspase-1 and caspase-3 during apoptotic events of osteoclasts. Furthermore, activation of caspase-8 and caspase-9 also detected. Fluorescenece labeling of osteoclastic mitochondorial membrene by MitAlert revealed that the mitochondoria membrane was destroyed by the ATPase inhibitors. These results suggested that vacuolar type ATPase plays an important role in bone resorption as well as survival of osteoclasts.