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

Abstract

Abstract. 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.

Bidirectional ephrin signaling in bone

“…Intracellular signaling by which ephrins regulate osteoclast development and activity also utilizes non-ephrin molecular pathways. In the RAW264.7 pre-osteoclast cell line, Dishevelled2 (Dvl2) co-precipitation with efnB2 was increased by RANKL treatment and reduced by EphB4 treatment, indicating that Dvl2 could be a downstream effector of reverse signaling that associates with efnB2 and mediates cell migration [59] . It has been also recently shown that EphA4 regulates osteoclast activity by modulating β3-integrin functions [47] , presumably by promoting attachment to the extracellular matrix.…”

Essential roles of EphrinB2 in mammalian heart: from development to diseases

“…However, the exact phosphorylation sites of EphrinB2 remained obscure. The PDZ-dependent signaling was activated by binding proteins with PDZ domain to the PDZ motif, such as PDZ-RGS3 [16] and Dvl2 [17, 18], playing an important role in EphrinB2-mediated embryonic development and angiogenesis despite of the unclarified downstream efforts (Fig. 2).…”

TBC1d24-ephrinB2 interaction regulates contact inhibition of locomotion in neural crest cell migration

“…Dishevelled (Dsh) is a key molecule in Wnt signalling and is also known to be a major player in the Wnt/PCP pathway required for proper neural crest cell migration 18 , 39 . Previous studies demonstrated that Dsh interacts with the C-terminal PDZ-binding motif of ephrinB ligands 40 – 43 , which is also the motif required for the interaction with TBC1d24 (Fig. 1d ).…”

“…Moreover, our data support a mechanistic model for how ephrinB2 may regulate CIL in CNC cells via TBC1d24. TBC1d24 interacts with ephrinB2 through the known association of ephrinB2 and Dsh 40 – 43 , as indicated in Fig. 2 .…”