The Research On The Modulation Of RANKL-induced Osteoclast Differentiation Of Raw264.7 Cells By EphB4/ephrinB2 Reverse Signaling

Osteoclastic bone resorption and osteoblastic bone formation are coordinated as a coupled mechanism to maintain bone homeostasis. This coupling is of great importance because a large majority of the diseases of bone that result in changes in bone mass, such as osteoporosis and osteopetrosis, are due to disruption of the coupling. The molecular mechanisms that are responsible for osteoclast-osteoblast communication remain one of the central issues in bone cell biology. Over the past decades, the pivotal roles of the receptor activator of NF-κB (RANK), its ligand RANKL. and the natural decoy receptor for RANKL, osteoprotegerin (OPG) have been well-documented. The binding of RANKL, which is released by osteoblasts and bone marrow cells, to its receptor RANK has an essential role in the differentiation and fusion of precursors into mature osteoclasts. There is recent evidence to suggest a role for the Eph receptors and their ephrin ligands in bone biology, a role that may affect the coupling between osteoblasts and osteoclasts.An important characteristic of interactions between Eph receptors and ephrin ligands is the bidirectional signaling, due to activation of signaling pathways in both the receptor-expressing and the ligand-expressing cells. Activation of the EphB receptors by the ephrinB ligands is designated as "forward signaling". Conversely, activation of the ephrinB ligands by the EphB receptors is referred to "reverse signaling". The reverse inhibitory effect in osteoclasts is dependent on the cytoplasmic domain of ephrinB2, while the C-terminal YKV motif is critical for signal transduction, indicating that downstream PDZ-domain proteins are involved.This pilot_study was designed to preliminarily identify the potential PDZ-domain proteins involved downstream of ephrinB2 during the osteoclast differentiation of Raw264.7 cells in vitro, and to find out whether the expression levels of potential PDZ-domain proteins are regulated by EphB4/ephrinB2 reverse signaling.Part I Establishing the osteoclastogenesis model that EphB4/ephrinB2 reverse signaling inhibits RANKL-induced osteoclast differentiation of Raw264.7 cellsPurpose:The first step of our study was to select the type of osteoclastic precursors to be used for further research. However, inter-individual variability and insufficient material are disadvantages of working with primary cell cultures, and cell lines are being increasingly recognized as standard experimental reagents and as essential components to ensure reproducible and reliable results in life science research. Our study used Raw264.7 cells, a macrophage/monocyte lineage cell line, as osteoclast precursors, and to examine the effect of EphB4/ephrinB2 reverse signaling on the RANKL-induced osteoclastogenesis and the gene expression of critical transcriptional factors such as c-Fos. c-Jun and NFATc 1.Methods:Raw264.7 was used in this study and could be differentiated into osteoclast-like cells on activation by RANKL. The detection of ephrinB2 protein was examined by immunofluorescence techniques and the western blot. The EphB4 protein was added to nutrient solution to find out the effect of EphB4/ephrinB2 reverse signaling on the osteoclast differentiation of Raw264.7 cells. After 4 days, the cultured cells were stained by a leukocyte acid phosphatase kit to analyze the cytochemical tartrate-resistant acid phosphatase (TRAP). The Expression of the marker enzyme gene of osteoclast differentiation of Raw264.7 cells, and transcriptional factors such as c-Fos, c-Jun and NFATc1, were assessed by quantitative real-time RT-PCR.Results:By immunofluorescence techniques and the western blot, the expression of ephrinB2 protein was detected in the RANKL induced osteoclast differentiation of Raw264.7 cells, but was not detected in the absence of RANKL. After four days in the conditioning culture, TRAP staining was performed, and TRAP-positive MNCs in each well were counted as osteoclasts. The number of TRAP-positive MNCs was significantly lower in the EphB4-Fc treated group than that in the Fc treated group (P<0.01). Total RNA was collected and analyzed, and the results showed that the EphB4-Fc treatment reduced the mRNA expression levels of osteoclast specific genes such as TRAP, cathepsin K and calcitonin receptor compared with the Fc treatment (P0.01). The mRNA level of these transcription factors was also reduced in the EphB4-Fc treated group compared with the Fc treated group (P<0.01).Conclusions:These results show that EphB4/ephrinB2 reverse signaling inhibits RANKL-induced osteoclast differentiation of Raw264.7 cells.PartⅡA preliminary identification of potential PDZ-domain proteins downstream of ephrinB2 during osteoclast differentiation of Raw264.7 cellsPurpose:The intracellular domain of ephrinB ligands, particularly the last 33 C-terminal amino acids, is highly conserved and contains multiple tyrosine residues, and the C-terminal YKV motif is a binding site for PDZ (postsynaptic density protein, disks large, zona occludens) domain-containing proteins. This pilot study was designed to preliminarily identify the potential PDZ-domain proteins involved downstream of ephrinB2 during the osteoclast differentiation of Raw264.7 cells in vitro.Methods:The immunofluorescence staining and western blot analysis were used to identify potential PDZ-domain proteins (PDZ-RGS3. Dv12, Pickl/PHIP, Syntenin, GRIP1, GRIP2, FAP-1 and Par3) expressed during the RANKL-induced osteoclast differentiation of Raw264.7 cells. To investigate whether ephrinB2 ligands associate with any of these potential PDZ-domain proteins, we immunoprecipitated ephrinB2 proteins from the cell lysis buffer with a goat polyclonal antibody, and the precipitate was assayed for coimmunoprecipitated PDZ-domain proteins by immunoblotting. Finally, to find out whether the EphB4-ephrinB2 reverse signaling affects the expression level of potential PDZ-domain proteins, preclustered soluble EphB4-Fc or Fc fragments were added to the RANKL-induced osteoclastogenic cultures. After four days in the conditioning culture, the expressions of the potential PDZ-domain proteins were assessed by the western blot and quantitative real-time RT-PCR.Results:The immunofluorescence staining and western blot analysis revealed that the eight PDZ-domain proteins were all expressed in the RANKL+ group after four days in the conditioning culture. However, among the eight proteins, only Dv12 showed the expected size coprecipitated with ephrinB2, and the reverse experiment (immunoprecipitation by the Dv12 antibody and immunoblotting by the ephrinB2 antibody) confirmed the relationship. The other PDZ-domain proteins were not detected at the expected sizes in the precipitate after immunoprecipitation with the ephrinB2 antibody in three independent experiments. After four days in the conditioning culture, the western blot showed that the band for Dv12 in the EphB4-Fc treated group was weaker than that in the Fc treated group (P<0.01), and the mRNA level of Dvl2 in the EphB4-Fc treated group was a little lower than in the Fc treated group, albeit with no statistical significance (P>0.05). What is more, we studied the effects of reverse signaling on the expression levels of Pickl and syntenin, PDZ-domain proteins which could not coprecipitate with ephrinB2. The western blot and quantitative real-time RT-PCR results showed that, in contrast to the similar expression level of syntenin between EphB4-Fc treated group and Fc treated group (P>0.05), the protein and mRNA expression levels of Pickl were obviously enhanced in EphB4-Fc treated group compared with Fc treated group (P<0.01).Conclusions:Dv12 may be the potential downstream PDZ-domain protein that is the binding partner for the C-terminal YKV motif of ephrinB2.