Preliminary identification of potential PDZ-domain proteins downstream of ephrin B2 during osteoclast differentiation of RAW264.7 cells

Abstract

The EphB4 receptor and ephrinB2 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/ephrinB2 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 ephrinB2 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/ephrinB2 signaling in osteoclasts. Immunofluorescence staining and Western blot analysis revealed that all of the eight PDZ-domain proteins previously reported to interact with ephrinB ligands were expressed in the differentiated RAW264.7 osteoclasts. However, in a co-immunoprecipitation assay, only Dishevelled2 (Dvl2) among eight PDZ-domain proteins tested co-precipitated with ephrinB2 and vice versa, suggesting an endogenous interaction between Dvl2 and ephrinB2 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 ephrinB2 in RANKL-induced osteoclast differentiation of RAW264.7 cells, providing a potential novel therapeutic target for bone diseases.