| Co-Investigator(Kenkyū-buntansha) |
OZAWA Hidehiro Graduate School of Oral Science, Hard Tissue Research, Professor, 大学院・歯学独立研究科, 教授 (60018413)
TAKAHASHI Naoyuki Graduate School of Oral Science, Hard Tissue Research, Professor, 大学院・歯学独立研究科, 教授 (90119222)
SAHARA Noriyuki Graduate School of Oral Science, Hard Tissue Research, Professor, 大学院・歯学独立研究科, 教授 (70064699)
HOSOYA Akihiro School of Dentistry, Assistant, 歯学部, 助手 (70350824)
奥村 茂樹 松本歯科大学, 歯学部, 助手 (80350825)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Osteoclasts play an important role in the metabolism of both organic and inorganic components in bone via the following steps-osteoclasts originate from bone marrow cells, differentiate with the aid of osteoblasts, fuse to each other and form mature, multi-nuclear gigantic cells, attach onto a bone surface with many podosomes (i.e. fibrous actin (F-actin^1) and integrin complexes), form actin rings, develop projecting structures (called ruffled borders), excrete substances including acids and proteolytic enzymes from the ruffled borders, solubilize bone, and leave resorption lacunae on the bone surface. Actin rings and ruffled borders are the morphological hallmarks of mature osteoclasts, and are therefore routinely used to identify mature osteoclasts. Osteoclasts undergo ‘transcytosis' of organic components, such as collagen, that are thus solubilized from bone via the following steps-the cells absorb the organic components via endocytosis at the ruffled borders, in-cell transport the
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se components to the apical membrane side of the cell, and perhaps expel (i.e.evacuate) them from the apical membrane toward the extracellular space. Thus, osteoclasts play a central role throughout the metabolism of organic components in bone-not only in the solubilization from bone but also in transcytosis steps (i.e. endocytosis at the ruffled borders, in-cell transportation, and evacuation from the apical membrane toward the extracellular). Although the role of osteoclasts in the metabolism of organic components is well documented, knowledge of the role of osteoclasts in the entire process of metabolism of Ca in bone has been fragmentary because no one has succeeded in directly observing such Ca in osteoclasts. Blood vessels actively absorb Ca released from bone by osteoclasts. Three hypothetical pathways for this uptake of Ca have been propose : (i)directly from resorption lacunae (i.e.the direct resorption pathway) ; (ii)through the intermediary of osteoclasts via Ca-specific channels (i.e.the channel-mediated transport pathway) ; (iii)through the intermediary of osteoclasts via transcytosis (i.e.the transcytosis transport pathway), which is analogous to the pathway for organic components. Although there is yet no evidence that definitively identifies any of these pathways or that refutes the transcytosis pathway, many researchers currently believe that the direct pathway is the main mechanism and that the channel-mediated pathway is a secondary mechanism. Among the three pathways, the transcytosis pathway seems to be least favored, only because no one has succeeded in directly observing Ca originating from bone in osteoclasts. To identify if the transcytosis pathway exists in osteoclasts, here we developed a method uses either fixed or living osteoclast-like cells (OCL) that were previously differentiated in vitro, uses a Ca-specific fluorescent dye (Fura Red^<TM>), to which a cell membrane is impermeable, and uses confocal laser scanning microscopy (LSM). We call this method the cell-membrane-impermeable dye (CMID) method. We used OCL because they are considered a good model of in vivo osteoclasts. Because the dye Fura Red^<TM> does not permeate an OCL membrane, the dye that exists inside OCL indicates active uptake, probably endocytosis, of the dye by OCL. If the Ca co-exists with such a dye inside OCL, direct and specific visualization of this Ca should be possible by detecting the emission from a dye-Ca complex. The Ca originating from bone might permeate OCL via two mechanisms : endocytosis and channels. If the quantity of such Ca is small (i.e.Ca via channels), the Ca would not be detectable, whereas if the quantity is large (i.e.Ca via endocytosis), it would be readily detectable and thus visualized. We therefore expected direct and specific visualization of the Ca via endocytosis by the CMID method. By using this method, we were successful in obtaining the first direct evidence that suggests massive Ca uptake occurs via endocytosis and that the Ca is transported toward the apical membrane side in OCL. This evidence also suggests that osteoclasts play an important role throughout the metabolism of Ca in bone. Less
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