The Affiliated Hospital of Stomatology, Medical College of Zhejiang University, 395 Yan'an Road, Hangzhou, PR China.
Int J Mol Med. 2011 May;27(5):669-77. doi: 10.3892/ijmm.2011.639. Epub 2011 Mar 3.
The EphB4 receptor and ephrin B2 ligand were recently reported to influence the coupling between osteoclasts and osteoblasts in bone biology, but their downstream signaling pathways remain unclear. This study focuses on the preliminary identification of downstream PDZ-domain proteins involved in EphB4/ephrin B2 reverse signaling in osteoclasts. Similarly to primary osteoclast precursors isolated from the bone, we observed that the RAW264.7 cell line, a mouse monocyte/macrophage cell line that is used in conventional assays for osteoclast function, expressed ephrin B2 during RANKL-induced osteoclast differentiation, and that preclustered EphB4 inhibited this osteoclast differentiation. The results demonstrate that RAW264.7 cells provide a good model for further research of EphB4/ephrin B2 signaling in osteoclasts. Immunofluorescence staining and Western blot analysis revealed that all of the eight PDZ-domain proteins previously reported to interact with ephrin B ligands were expressed in the differentiated RAW264.7 osteoclasts. However, in a co-immunoprecipitation assay, only Dishevelled 2 (Dvl2) among eight PDZ-domain proteins tested co-precipitated with ephrin B2 and vice versa, suggesting an endogenous interaction between Dvl2 and ephrin B2 in RANKL-induced osteoclasts. Furthermore, preclustered EphB4 reduced the expression level of Dvl2. Collectively, our results indicate that Dvl2 could be the potential PDZ-domain protein that acts downstream of ephrin B2 in RANKL-induced osteoclast differentiation of RAW264.7 cells, providing a potential novel therapeutic target for bone diseases.
EphB4 受体和 ephrin B2 配体最近被报道影响骨生物学中破骨细胞和骨细胞之间的偶联，但它们的下游信号通路仍不清楚。本研究重点初步鉴定 EphB4/ephrin B2 反向信号在破骨细胞中涉及的 PDZ 结构域蛋白。与从骨中分离的原代破骨细胞前体类似，我们观察到 RAW264.7 细胞系（一种用于破骨细胞功能常规测定的小鼠单核细胞/巨噬细胞细胞系）在 RANKL 诱导的破骨细胞分化过程中表达 Ephrin B2，并且预聚类的 EphB4 抑制了这种破骨细胞分化。结果表明 RAW264.7 细胞为进一步研究 EphB4/ephrin B2 信号在破骨细胞中的作用提供了一个很好的模型。免疫荧光染色和 Western blot 分析显示，以前报道与 Ephrin B 配体相互作用的所有 8 种 PDZ 结构域蛋白均在分化的 RAW264.7 破骨细胞中表达。然而，在共免疫沉淀测定中，在所测试的 8 种 PDZ 结构域蛋白中，只有 Dishevelled 2 (Dvl2) 与 Ephrin B2 共沉淀，反之亦然，表明 Dvl2 和 Ephrin B2 之间存在内源性相互作用在 RANKL 诱导的破骨细胞中。此外，预聚类的 EphB4 降低了 Dvl2 的表达水平。综上所述，我们的结果表明 Dvl2 可能是 Ephrin B2 在 RANKL 诱导的 RAW264.7 细胞破骨细胞分化中的潜在 PDZ 结构域蛋白下游的潜在 PDZ 结构域蛋白，为骨疾病提供了一个潜在的新的治疗靶点。
