Targeted Disruption of Ephrin B1 in Cells of Myeloid Lineage Increases Osteoclast Differentiation and Bone Resorption in Mice

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.

Haploinsufficiency of osterix in chondrocytes impairs skeletal growth in mice

“…Heights of the columnar and hypertrophic zones were measured using the OsteoMeasure software (Ostometrics, Atlanta, GA). Tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts were performed as previously described (4). TRAP staining of two fields from primary or secondary spongiosa were measured using the OsteoMeasure software (Ostometrics).…”

“…Immunohistochemistry was carried out according to a previously published procedure (4,10). Osx antibody (ab22552, Abcam) was diluted at 1:500 in blocking solution and incubated at 4°C overnight.…”

“…Quantitative real-time PCR was performed as previously described. (4). Ppia was used as reference gene.…”

Expression and Function of the Eph Receptor Family in Leukemia and Hematopoietic Malignancies: Prospects for Targeted Therapies

“…Ephrin-B1 full knockout mice are prenatally lethal and they have skeletal defects. Studies on disruption of ephrin-B1 on collagen I producing cells result in reduced bone formation and skull defect and studies on ephrin-B1 conditional knockout mice shows defects in osteoblastic mediated bone formation with no increase in osteoclastic bone resorption and this condition results in reduction in bone size and density [101,102].The importance of Eph/ephrin interactions has also been shown in various stem cell niches, including neural, dental and intestinal stem cell compartments [103,104]. More recent studies show their involvement in bone homeostasis and mesenchymal stem cell (MSC) regulation.…”

The role of Eph proteins in haematopoiesis and leukaemia

“…Studies on disruption of ephrinB1 on collagen I producing cells result in reduce bone formation and skull defect and studies on ephrinB1 conditional knockout mice shows defect in osteoblastic mediated bone formation with no increase in osteoclastic bone resorption and this condition results in reduction in bone size and density 138,139 .…”