| Co-Investigator(Kenkyū-buntansha) |
KIDO Mizuho Kyushu University, Faculty of Dental Science, Associate Professor, 大学院歯学研究院, 助教授 (60253457)
TANAKA Teruo Kyushu University, Faculty of Dental Science, Professor, 大学院歯学研究院, 教授 (60077667)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
In this study, we analyzed the kinetics of the endosomes in osteoclasts with double immunofluorescent microscopy. Osteoclast-like cells (OCLs) were differentiated from bone marrow cells of mice. Several sizes of vesicles showed positive for cathepsin K (CathK) and cystatin C (CysC) that are cystein proteinase and its endogenous inhibitor, respectively, in OCLs. CathK and CysC also localized in the same structures. To identify the phenotypes of CathK/CysC containing vesicles, lysosmal membrane markers, early endosome associate protein 1 (EEA1) and lysosomal associate membrane protein 2 (LAMP2) were used as early and late endosome markers, respectively. CathK/CysC contained in LAMP2-positive endosomes, but not in EEA1-positive compartments. Dinitrophenyl (DNP) is an indicator to indicate a pH condition in endosomes. DNP and CathK/CysC co-localized in the same vesicles. LAMP2-positive endosomes were positive for DNP, but EEA1-positive vesicles did not. Wortomannin (WT) is known as an inhi
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bitor for phosphatydil inositol 3 kinase (PI3-kinase). After treatment with WT, the co-localization patterns of endosome proteins were disappeared and each compartment independently localized in OCLs. Electron microscopic analysis revealed both the storage and vacuolation of endosomes and the weakness of the ruffled border in OCLs. These findings indicate that CathK and CysC are transported to an acidic late endosomes before the secretion on bone surface in OCLs, and suggest that PI3-kinase plays a role in the traffic to the acidic late endosomes in exocytosis within OCLs. A treatment of horseradhish peroxidase (HRP) is used to clarify the kinetics of endosomes in the process of transcytosis. EEA1-positive or LAMP2-positive endosomes, as well as DNP-positive compartments, did not contain HRP after treatment for 30 min. After 60 min, HRP was stored in LAMP2-positive endosomes, but not in EEA1-positive structures. HRR-containing vesicles were also positive for DNP. In WT-treatment caused an independent localization of HRP-containing vesicles from LAMP2/DNP-positive endosomes. From these findings, it was cleared that uptaked HRP transported to an acidic late endosomes in OCLs, and suggested that PI3-kinase takes part in the transportation to the acidic late endosomes in the trascytotic pathway. In summary, our findings indicate that PI3-kinase controls the formation of the acidic late endosomes in the membrane traffic, such as exocytosis and transcytosis, in OCLs, suggesting that the development of regents targeted to PI3-kinase is a powerful tool of therapies for metabolic bone disease, such as osteoporosis. Less
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