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Ca^<2+> kinetics in mature osteoclast

成熟破骨细胞中的 Ca^<2 > 动力学

基本信息

批准号：
16591847
负责人：
YAMAKI Mariko
金额：
$ 2.18万
依托单位：
Matsumoto Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16591847/
关键词：
Osteoclast Ca^<2+>Bone matrix Endocytosis Transportation in cell Transcytosis Vacuole Lysosome Ca^<+2>

项目摘要

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 … More 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

破骨细胞通过以下步骤在骨中有机和无机成分的代谢中起重要作用：破骨细胞起源于骨髓细胞，在成骨细胞的帮助下分化，相互融合并形成成熟的多核巨细胞，附着在具有许多podosome的骨表面（即纤维状肌动蛋白（F-肌动蛋白^1）和整合素复合物），形成肌动蛋白环，形成突起结构，（称为皱褶边缘），从皱褶边缘排出包括酸和蛋白水解酶在内的物质，溶解骨骼，并在骨骼表面留下吸收陷窝。肌动蛋白环和皱褶边界是成熟破骨细胞的形态学标志，因此通常用于鉴定成熟破骨细胞。破骨细胞经历有机组分如胶原的“转胞吞作用”，其因此经由以下步骤从骨溶解-细胞经由皱褶边界处的内吞作用吸收有机组分，细胞内转运胶原。 ...更多信息将这些成分释放到细胞的顶膜侧，并且可能将它们从顶膜排出（即排空）到细胞外空间。因此，破骨细胞在骨中有机成分的整个代谢过程中发挥着核心作用-不仅在骨溶解中，而且在转胞吞步骤中（即在皱褶边缘的内吞作用、细胞内运输和从顶膜向细胞外的排空）。虽然破骨细胞在有机成分代谢中的作用已有很好的文献记载，但对破骨细胞在骨中钙代谢的整个过程中的作用的认识是不完整的，因为没有人成功地直接观察破骨细胞中的钙。血管主动吸收破骨细胞从骨中释放的钙。已经提出了三种假设的Ca摄取途径：（i）直接从再吸收陷窝（即直接再吸收途径）;（ii）通过破骨细胞介导的Ca特异性通道（即通道介导的转运途径）;（iii）通过破骨细胞介导的胞吞转运（即胞吞转运途径），这与有机组分的途径类似。虽然目前还没有证据明确确定这些途径中的任何一种或反驳转胞吞途径，但许多研究人员目前认为直接途径是主要机制，通道介导的途径是次要机制。在这三种途径中，转胞吞途径似乎是最不受欢迎的，只是因为没有人成功地直接观察到破骨细胞中来自骨的Ca。为了鉴定在破骨细胞中是否存在转胞吞途径，在此我们开发了一种方法，该方法使用先前在体外分化的固定的或活的破骨细胞样细胞（OCL），使用Ca特异性荧光染料（Fura Red<TM>）（细胞膜不可渗透），并使用共聚焦激光扫描显微镜（LSM）。我们称这种方法为细胞膜不可渗透染料（CMID）方法。我们使用OCL，因为它们被认为是体内破骨细胞的良好模型。因为染料Fura Red<TM>不渗透OCL膜，所以存在于OCL内的染料指示OCL对染料的主动摄取，可能是内吞作用。如果Ca与OCL内的这种染料共存，则通过检测染料-Ca络合物的发射，可以直接和特异性地观察这种Ca。骨源性钙离子可能通过内吞和通道两种途径渗透入OCL。如果这种Ca的量小（即，通过通道的Ca），则Ca将不可检测，而如果量大（即，通过内吞作用的Ca），则其将容易检测并因此可视化。因此，我们期望通过CMID方法通过内吞作用直接且特异性地观察Ca。通过使用这种方法，我们成功地获得了第一个直接的证据，表明大量的钙吸收发生通过内吞作用，钙被运输到顶膜侧的OCL。这一证据还表明，破骨细胞在骨中钙的代谢过程中起着重要作用。少