Identification of potential PDZ-domain proteins downstream of ephrinB2 reverse signaling during osteoclast differentiation of RAW264.7 cells

Par3 and RGS3 have been reported to bind to ephrinB ligands. In this research, it was studied whether they were the potential PDZ-domain proteins involved in the downstream of ephrinB2 during the osteoclast differentiation in vitro. Western blot analysis and immunofluorescence staining revealed that Par3 and RGS3 were prominently expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells. 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. Western blot analysis and real-time RT-PCR results showed that the protein and mRNA expression level of Par3 in the EphB4-Fc treated group was higher than that in the Fc treated group. However, the protein and mRNA expression level of RGS3 in the EphB4-Fc treated group was lower. Although the expression levels of ephrinB2 and RGS3 were consistent with the expression change of c-Jun/c-Fos/Nfatc1 complex, co-immunoprecipitation results showed that there were no direct interactions between ephrinB2 and endogenously expressed Par3 and RGS3 in the RANKL-induced osteoclasts of RAW264.7 cells in vitro. In summary, we demonstrated that both Par3 and RGS3 were expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells, and ephrinB2 reverse signaling regulated the expression levels of Par3 and RGS3 diversely.