Activation of ephrin B1 reverse signaling inhibits phosphorylation of ezrin/radixin/moesin and RANKL target gene expression in osteoclasts.

<p>[<b>A–C</b>]: 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. [<b>D</b>]: 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. [<b>E</b>]: 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. [<b>F</b>]: 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.