Role of intracellular osteopontin in the osteoclast fusion and tumor cell invasion

破骨细胞（Osteoclasts，OC）是一种多核细胞，在生理和病理性骨代谢过程中参与骨吸收。OC的前体细胞需要迁移融合形成多核成熟细胞，这是OC分化的重要步骤。骨桥蛋白（OPN）与α v β3整合素共定位并介导骨桥蛋白与骨的结合，这一发现首次表明OPN在骨吸收中具有重要作用。然而，OPN在OC分化和激活的哪个阶段起作用仍不清楚。为了澄清这一点，我们研究了OC产生的骨髓单核细胞前体激光扫描共聚焦显微镜。在融合前OC和运动的多核OC中，OPN与CD 44和β3整合素（OPN受体）共定位于细胞突起中。还发现OPN分别与Cdc 42和Racl共定位于丝状伪足和皱褶膜中。免疫细胞化学分析OC来自OPN-空和CD 44-空显示减少膜周染色的CD 44和OPN，分别表明这些蛋白质的相互依赖性，其定位在细胞表面。此外，与野生型对照相比，从这些突变小鼠产生的OC显示出减少的细胞铺展、伪足的有限突出和受损的细胞融合，这与OPN和CD 44在介导细胞骨架重排中的假定作用一致。这些研究表明OPN与CD 44共定位于破骨细胞过程中，OPN可能在调节CD 44介导的与OC迁移、融合和再吸收相关的细胞骨架变化中起重要作用。此外，证实了OPN在鳞状癌细胞的伤口愈合过程中起关键作用，因为伸出伪足并向细胞层之间的空间迁移的前线细胞强烈表达OPN和CD 44。

Osteoclasts (OC) are the multinucleated cells which is responsible for the bone resorption in both physiological and pathological bone metabolism. Precursors of OC need to migrate and fuse together to form the multinucleated mature cells, which is an important step in OC differentiation. That osteopontin (OPN) has a prominent role in bone resorption was first indicated by the demonstration that it colocalizes with the avβ3 integrin and mediates the attachment of OC to the bone. However, it is still unclear at which stages of OC differentiation and activation that OPN functions. To clarify this, we examined the OC generated from bone marrow mononuclear precursors by laser scanning confocal microscopy. In prefusion OC and in motile multinucleated OC, staining for OPN colocalized with CD44 and β3 integrin, which are the OPN receptors, in cell processes. OPN was also found to colocalize with Cdc42 and Racl in filopodia and ruffled membranes, respectively. Immunocytochemical analysis of OC derived from OPN-null and CD44-null showed diminished perimembranous staining for CD44 and OPN, respectively, indicating a mutual interdependency of these proteins for their localization at cell surfaces. Moreover, OC generated from these mutant mice showed reduced cell spreading, limited protrusion of pseudopodia and impaired cell fusion compared to wild-type controls, consistent with the putative role of the OPN and CD44 in mediating cytoskeletal rearrangements. These studies indicate that OPN colocalizes with CD44 within osteoclast cell processes, and that OPN may be important in regulating CD44-mediated cytoskeletal changes associated with OC migration, fusion and resorption. Furthermore, it was confirmed that OPN played a critical role in wound healing process in squamous carcimona cells, since the front-line cells which protrude pseudopods and migrate toward the space between cell layers expressed OPN and CD44 strongly.