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Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice.

Cheng S, Zhao SL, Nelson B, Kesavan C, Qin X, Wergedal J, Mohan S, Xing W - PLoS ONE (2012)

Bottom Line: The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation.Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin.We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Disease Center, Jerry L Pettis VA Medical Center, Loma Linda, California, United States of America.

ABSTRACT
Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

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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.
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pone-0032887-g007: 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.

Mentions: To examine whether activation of ephrin B1 reverse signaling influences expression of osteoclast differentiation marker genes, splenocytes derived from WT mice were cultured in the presence of RANKL and M-CSF for 24 hours, and then treated with soluble EphB2-Fc or control Fc for another 4 days. Total RNA was extracted for RT real-time PCR. As shown in Figure 7A, expression of TRAP gene was reduced by 62% in the differentiated osteoclasts in the EphB2-Fc treated cells as compared to the control cells treated with Fc, but there was no change in TRAP expression between the treatments in the undifferentiated precursors (data not shown). Similarly, EphB2-Fc treatment inhibited the expression of cathepsin K (CatK) and NFAFc1 by 60% and 67%, respectively, in WT osteoclasts, however not in the ephrin B1 deficient cells. There was little change in c-Fos expression in the WT cells treated with EphB2-Fc as compared to the cells treated with Fc (data not shown).


Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice.

Cheng S, Zhao SL, Nelson B, Kesavan C, Qin X, Wergedal J, Mohan S, Xing W - PLoS ONE (2012)

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.
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pone-0032887-g007: 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.
Mentions: To examine whether activation of ephrin B1 reverse signaling influences expression of osteoclast differentiation marker genes, splenocytes derived from WT mice were cultured in the presence of RANKL and M-CSF for 24 hours, and then treated with soluble EphB2-Fc or control Fc for another 4 days. Total RNA was extracted for RT real-time PCR. As shown in Figure 7A, expression of TRAP gene was reduced by 62% in the differentiated osteoclasts in the EphB2-Fc treated cells as compared to the control cells treated with Fc, but there was no change in TRAP expression between the treatments in the undifferentiated precursors (data not shown). Similarly, EphB2-Fc treatment inhibited the expression of cathepsin K (CatK) and NFAFc1 by 60% and 67%, respectively, in WT osteoclasts, however not in the ephrin B1 deficient cells. There was little change in c-Fos expression in the WT cells treated with EphB2-Fc as compared to the cells treated with Fc (data not shown).

Bottom Line: The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation.Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin.We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Disease Center, Jerry L Pettis VA Medical Center, Loma Linda, California, United States of America.

ABSTRACT
Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

Show MeSH
Related in: MedlinePlus