Figure 1.
Expression of ephrin B1 is increased during osteoclast differentiation.
[A–B]: Expression of ephrin B1 is increased during osteoclast differentiation. Osteoclast precursors isolated from non-adherent bone marrow of wild-type (WT) mice were treated with 20 ng/ml of M-CSF and 30 ng/ml of RANKL for 0, 2, 4 and 8 days. The differentiated multinuclear cells (MNCs) were monitored by TRAP staining. The parallel cultures were harvested, and total cellular proteins were extracted for Western blot. [C]: Expression of ephrin B1 is more abundant than ephrin B2 in osteoclasts. Mouse splenocytes were treated with M-CSF only or M-CSF plus RANKL for 4 days. Total RNA was extracted for real-time PCR. Values are fold-change over the expression level of ephrin B2 transcript in undifferentiated precursors, and expressed as mean ± SEM (standard error of mean) (n = 3). A star represents statistical significance of expression level of transcript in MNCs as compared to the precursors or between two groups indicated (P<0.01).
Figure 2.
Activation of ephrin B1 reverse signaling suppresses osteoclast differentiation.
[A]: Osteoclast precursors derived from WT mice were differentiated in the presence of 20 ng/ml of M-CSF and 30 ng/ml of RANKL for 8 days. The cells were also treated with clustered EphB2-Fc during day 2–8 or day 5–8 of the culture as indicated in the figure, followed by TRAP staining. [B]: Quantitative data of TRAP positive MNCs. Values are mean ± SEM (n = 8). A star presents statistical significance of MNCs in the differentiated cultures treated with EphB2-Fc as compared to the cultures treated with control Fc (P<0.01).
Figure 3.
Conditional ephrin B1 KO is myeloid-lineage specific.
[A]: Schematic diagram of generation of ephrin B1 conditional KO and control WT. Mice with ephrin B1 deletion in myeloid lineage cells are generated by crossing ephrin B1 loxp mice with Lyz2-Cre knock-in mice. [B]: Ephrin B1 is not expressed in osteoclasts from the myeloid-specific conditional KO mice. Splenocytes derived from WT and KO mice were cultured in the presence of 20 ng/ml of M-CSF and 30 ng/ml of RANKL for 3 days. Bone marrow stromal (BMS) cells isolated from the long bones were also cultured in α-MEM medium containing 10% FBS for 6 days. Osteoclasts (OC) and BMS cells were harvested, respectively, and the cellular proteins were extracted for measurement of ephrin B1 protein, by Western blot.
Figure 4.
Ephrin B1 deletion in cells of myeloid lineage decreases trabecular bone.
[A]: Longitudinal section of μ-CT images of distal femurs of WT and KO mice. The metaphysis of distal femurs were selected for analyses of trabecular bone parameters by μ-CT. [B]: μ-CT images of trabecular bone of the distal metaphysis of the femurs. [C–F]: Quantitative measurements of trabecular bone at the metaphysis of distal femurs. [C]: Percentage change of trabecular bone volume/total bone volume (BV/TV) of the distal femur of KO mice as compared to WT littermate controls. [D]: Percentage change of trabecular number (Tb. N) of the distal femur of KO mice as compared to WT littermate controls. [E]: Percentage change of trabecular thickness (Tb. Th) of the distal femur of KO mice as compared to WT littermate controls. [F]: Percentage change of trabecular separation (Tb. Sp) of the distal femur of KO mice as compared to WT littermate controls. Values are expressed as mean ± SEM (n = 8). A star presents statistical significance (P<0.05) as compared to WT littermate controls.
Figure 5.
Ephrin B1 deletion in cells of myeloid lineage increases bone resorption.
[A–B]: TRAP staining of osteoclasts at trabecular surface of metaphysis of the femur in WT and KO mice at 21 weeks of age. Bone section was counterstained with methyl green. Arrows denote representative TRAP positive osteoclasts. A: 20× magnification, B: 100× magnification. [C]: Percentage change of TRAP labeled surface/trabecular bone surface (Oc. S/BS) in the bone of ephrin B1 KO mice as compared to WT controls at 21 weeks of age. Values are presented as mean ± SEM (n = 8). [D]: Percentage change of serum TRAP activity of ephrin B1 KO mice as compared to WT controls at 21 weeks of age. Values are presented as mean ± SEM (n = 8). [E–F]: Trabecular bone formation is unaffected in mice with deletion of ephrin B1 in myeloid lineage cells. E: Images of H & E staining of distal femurs of WT and KO mice (20×). F: representative images of calcein double labeled trabecular bone of WT and KO mice (200×). [G–H]: Quantitative data of trabecular bone formation measured at distal femur metaphysis of WT and KO mice at 21 weeks of age. G: Bone formation rate/bone surface (BFR/BS). Values are presented as mean ± SEM (n = 8). H: Mineral apposition rate (MAR). Values are presented as mean ± SEM (n = 8).
Figure 6.
Activation of ephrin B1 reverse signaling inhibits osteoclast formation and bone resorption.
[A]: Interaction of ephrin B1 with EphB2-Fc suppresses osteoclast differentiation. Splenocytes from WT and ephrin B1 KO mice were cultured in the presence of RANKL and M-CSF for 24 hours, and then treated with soluble EphB2-Fc (2 µg/ml) or control Fc for 8 days. TRAP staining was performed after 6 days EphB2-Fc treatment. MNCs were visualized under microscope. [B]: Quantification of MNCs (3 or more nuclei per cell). Values are mean ± SEM (n = 8). A star presents statistical significance (P<0.01) as compared to the cells treated with control Fc. [C]: Interaction of ephrin B1 with EphB2-Fc suppresses bone resorption. Splenocytes from WT and ephrin B1 KO mice were seeded on bone slices, and cultured in the presence of RANKL and M-CSF for 24 hours, and then treated with EphB2-Fc or control Fc for additional 8 days. Resorption pits on bone slices were analyzed after 8 days of EphB2-Fc treatment. [D]: Quantification of resorption pit areas. Values are mean ± SEM (n = 6). A star presents statistical significance (P<0.01) as compared to the cells treated with control Fc.
Figure 7.
Activation of ephrin B1 reverse signaling inhibits phosphorylation of ezrin/radixin/moesin and RANKL target gene expression in osteoclasts.
[A–C]: Treatment of EphB2-Fc inhibits expression of osteoclast differentiation marker genes. Precursors were cultured in the presence of RANKL and M-CSF for 24 hours, and then treated with soluble EphB2-Fc (2 µg/ml) or control Fc for another 4 days. Total RNA was extracted for real-time RT-PCR using specific primers to TRAP, cathepsin D (CatK) and NFATc1. Values are expressed as fold change over WT cells ± SEM (n = 3). A star presents statistical significance (P<0.05) as compared to the cells derived from WT littermate mice. [D]: ERM phosphorylation is increased during RANKL induced osteoclast differentiation. Splenocytes from WT mice were treated with 20 ng/ml of M-CSF and 30 ng/ml of RANKL for 0, 2, 4 and 8 days. Total cellular proteins from osteoclasts were extracted for Western blot. [E]: Ephrin B1 interacts with NHERF1 in osteoclasts. Splenocytes were treated with M-CSF and RANKL for 4 days. Cells were then treated with EphB2-Fc for 5 minutes, and used for immunoprecipitation. [F]: Activation of ephrin B1 reverse signaling inhibits ERM phosphorylation. Splenocytes derived from ephrin B1 KO and WT mice were treated with M-CSF and RANKL for 4 days, and then the cells were treated with 2 µg/ml of EphB2-Fc or Fc in differentiation medium for another 24 hours. Total cellular proteins were extracted for Western blot.
Table 1.
Primer sequences for real time RT-PCR.