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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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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.
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pone-0032887-g004: 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.

Mentions: To study the functions of ephrin B1 expressed in osteoclasts, ephrin B1 gene was deleted in cells of myeloid lineage by Cre/loxp approach (Figure 3A). After 3 generations of breeding, homozygous Lyz2-Cre, loxp homozygous female or hemizygous male mice were generated, and used as experimental KO mice. Both WT alleles of Lyz2 gene, loxp homozygous or hemizygous mice served as controls. To confirm the ephrin B1 expression in the homozygous Lyz2-Cre, loxp homozygous mice, precursors were isolated from the spleen of conditional KO and WT mice, and differentiation was induced by treatments of M-CSF and RANKL. Total cellular extracts from differentiated osteoclasts were used for Western blot. As expected, the expression of the ephrin B1 protein was absent in osteoclasts from homozygous Lyz2-Cre, loxp homozygous mice, but was detected in the cells from Cre- control littermates (Figure 3B). In contrast, bone marrow stromal cells derived from both KO and WT mice expressed high levels of ephrin B1 protein. To characterize the skeletal phenotypes of ephrin B1 conditional KO mice, the femurs were collected from 21 week old mice and analyzed trabecular structures by μ-CT analyses (Figures 4A–B). While there was no significant difference in cortical mineral density at the mid diaphysis of the femur of WT and KO mice (data not shown), the ratio of trabecular bone volume to total volume (BV/TV) was reduced by 40% at the metaphysis of the femur from the mixed genders of KO mice as compared to the littermate controls (Figure 4C). Trabecular number and trabecular thickness were reduced by 23% and 18%, respectively, while trabecular separation was increased by 24% at this site of the femurs isolated from KO mice (Figures 4 D–F). There was no significant difference in the magnitude of trabecular bone volume reduction in the KO mice between the two genders (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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3293909&req=5

pone-0032887-g004: 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.
Mentions: To study the functions of ephrin B1 expressed in osteoclasts, ephrin B1 gene was deleted in cells of myeloid lineage by Cre/loxp approach (Figure 3A). After 3 generations of breeding, homozygous Lyz2-Cre, loxp homozygous female or hemizygous male mice were generated, and used as experimental KO mice. Both WT alleles of Lyz2 gene, loxp homozygous or hemizygous mice served as controls. To confirm the ephrin B1 expression in the homozygous Lyz2-Cre, loxp homozygous mice, precursors were isolated from the spleen of conditional KO and WT mice, and differentiation was induced by treatments of M-CSF and RANKL. Total cellular extracts from differentiated osteoclasts were used for Western blot. As expected, the expression of the ephrin B1 protein was absent in osteoclasts from homozygous Lyz2-Cre, loxp homozygous mice, but was detected in the cells from Cre- control littermates (Figure 3B). In contrast, bone marrow stromal cells derived from both KO and WT mice expressed high levels of ephrin B1 protein. To characterize the skeletal phenotypes of ephrin B1 conditional KO mice, the femurs were collected from 21 week old mice and analyzed trabecular structures by μ-CT analyses (Figures 4A–B). While there was no significant difference in cortical mineral density at the mid diaphysis of the femur of WT and KO mice (data not shown), the ratio of trabecular bone volume to total volume (BV/TV) was reduced by 40% at the metaphysis of the femur from the mixed genders of KO mice as compared to the littermate controls (Figure 4C). Trabecular number and trabecular thickness were reduced by 23% and 18%, respectively, while trabecular separation was increased by 24% at this site of the femurs isolated from KO mice (Figures 4 D–F). There was no significant difference in the magnitude of trabecular bone volume reduction in the KO mice between the two genders (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