SHIBATA Mitsue Nagasaki University, Graduate School of Biomedical Sciences, Instructor, 大学院・医歯薬学総合研究科, 助手 (20274665)
OKAMOTO Kuniaki Nagasaki University, Graduate School of Biomedical Sciences, Associate Professor, 大学院・医歯薬学総合研究科, 助教授 (10311846)
KATO Yuzo Nagasaki University, Graduate School of Biomedical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (20014128)
|Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2003: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2002: ¥2,400,000 (Direct Cost: ¥2,400,000)
To investigate the, molecular events involved in osteoclastogenesis induced by macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), we analyze gene expression using cDNA microarray techniques. Significant induction of a number of mRNAs including annexins 2,4,5were observed during, osteoclast differentiation. We confirmed protein expression of annexins 2,4,5 by immunostaining techniques using specific antibody for each. All of them were expressed in differentiated osteoclasts. Annexin expressions were enhanced by M-CSF without RANKL, suggesting annexins are not necessary for osteoclastgenesis. It has been known that annexins are distributed in lipid raft in some cells. Previous studies show that many signaling molecules localize in microdomains of the plasma membrane, lipid rafts. A number of subsequent studies indicate that glycosphingolipid and cholesterol enriched membrane fraction are clustered with receptors and signal transducing molecules to form lipid rafts and can be recovered as low density, triton X-100 insoluble membrane fractions. We found that RANK, c-Src, TRAF2, and TRAF6, but not c-fins, integrin a_v, or annexins were localized in lipid rafts. Depletion of endogenous glycosphingolipids by 2.5 to 10mM of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glucosylceramide synthase inhibitor, led to depletion of endogenous glycosphingolipids, treatment-induced suppression of osteoclastgenesis, and elimination of RANK and c-Src from lipid rafts. Further, exogenous glycosphingolipids induced the translocation of RANK and c-Src to rafts, indicating that glycosphingolipids in general are required for the association of RANK and c-Src with rafts. Thus, the lipid raft structure plays important roles in RANK signaling events in osteoclasts. Although the function of annexin is still unclear, it may participate intracellular vesicle transport out side of lipid rafts of differentiated osteoclasts.