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

破骨细胞(Osteoclast，OC)是一种多核细胞，在生理性和病理性骨代谢中都与骨吸收有关。OC前体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avβ3整合素共定位，介导OC附着于骨组织，在骨吸收中起重要作用。然而，OPN在OC分化和激活的哪个阶段发挥作用仍不清楚。为了阐明这一点，我们用激光扫描共聚焦显微镜检测了骨髓单个核前体细胞产生的OC。在前融合型OC和移动型多核OC中，骨桥蛋白与骨桥蛋白受体CD44和β3共定位于细胞突起。在丝状孔膜和褶皱膜中，OPN分别与Cdc42和racl共定位。OPN缺失和CD44缺失的OC的免疫细胞化学分析显示CD44和OPN的膜周染色减弱，这表明这些蛋白在细胞表面的定位是相互依赖的。此外，与野生型对照相比，这些突变小鼠产生的OC显示细胞扩散减少，伪足突起有限，细胞融合受损，这与OPN和CD44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CD44共同定位于破骨细胞突起内，OPN可能在调控CD44介导的与OC迁移、融合和吸收相关的细胞骨架变化中起重要作用。进一步证实OPN在鳞状细胞癌创面愈合过程中起关键作用，因为突起伪足并向细胞层间隙迁移的前线细胞强烈表达OPN和CD44。

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.