EphB4/ephrinB2逆向信号对RAW264．7破骨细胞分化中PDZ结构域蛋白表达变化的研究

Eph受体是酪氨酸蛋白激酶受体家族中最大的亚家族,ephrin(Eph受体相互作用蛋白)是其配体,它们是膜结合蛋白,相互依赖进行信号转导.内居蛋白(syntenin)与Pick1属于PDZ结构域(PSD-95/Dlg-/Zo-1 domain)蛋白,报道称能与ephrinB配体结合,但是否受Eph受体调控尚未见报道.以RAW264.7细胞株为研究对象,通过蛋白质印迹及/或免疫荧光分析显示RAW264.7细胞经RANKL诱导的破骨细胞表达ephrinB2、内居蛋白(syntenin)和Pick1三个蛋白质.将提前成簇的可溶性EphB4蛋白加入培养液,与ephrinB2配体结合,用来研究EphB4/ephrinB2逆向信号对syntenin和Pick1表达水平变化的影响.免疫印迹及Real-time RT-PCR分析结果显示,在EphB4-Fc实验组中Pick1的蛋白质及mRNA水平都有明显增加,然而在EphB4-Fc实验组与Fc对照组别间syntenin的蛋白质及mRNA水平未见明显变化.免疫共沉淀结果显示,syntenin和Pick1不能与ephrinB2共沉淀.以上结果初步探索了体外破骨细胞分化过程中,EphB4/ephrinB2逆向信号对PDZ结构域蛋白(ephrinB2配体潜在的下游信号分子)表达变化的调控.

EphB4/ephrinB2 Reverse Signaling Regulates Expression Levels of PDZ-domain Proteins During Osteoclast Differentiation of RAW264.7 Cells

Syntenin and Pick1 (PDZ-domain proteins) have been reported to bind to ephrinB ligands. However, there is no data related to whether ephrinB ligands, located on the membrance of osteoclasts, regulate expression levels of syntenin and Pick1, following activation by EphB receptors. RAW264.7 cell line was used as osteoclast precursors, which can differentiate into osteoclast induced by RANKL. Western blot analysis and/or immunofluorescence staining revealed that not only syntenin and Pick1, but also ephrinB2 were prominently expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells, thus furtherly illustrating that the model of RANKL-induced osteoclast differentiation of RAW264.7 cells can be used for further investigation. In order to study the effects of reverse signaling on the expression levels of PDZ-domain proteins, soluble EphB4-Fc protein was used to stimulate ephrinB2 because EphB4 exclusively interacts with ephrinB2. In contrast to the similar expression level of syntenin between EphB4-Fc and Fc treated group, the protein and mRNA expression levels of Pick1 were obviously enhanced in EphB4-Fc treated group compared with Fc treated group. However, co-immunoprecipitation results showed that there were no direct interactions between ephrinB2 and endogenously expressed syntenin and Pick1 in the RANKL-induced osteoclasts of RAW264.7 cells in vitro. In summary, it was demonstrated that both syntenin and Pick1 were expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells, and EphB4/ephrinB2 reverse signaling regulates the expression levels of Pick1, but not of syntenin. These data help to preliminarily explore the potential PDZ-domain proteins involved in the downstream of ephrinB2 during the osteoclast differentiation of RAW264.7 cells in vitro.