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

Shaohong Cheng,Brittany Nelson,Xuezhong Qin,Subburaman Mohan,Shien Lucy Zhao,Weirong Xing,Chandrasekhar Kesavan,Jon Wergedal,Richard L Eckert

doi:10.1371/journal.pone.0032887

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.

Highlights

Osteoporosis is an aging-related major health problem in women and men
Since little is known on the role of ephrin B1 in regulating osteoclast functions, we examined if activation/ inactivation of ephrin B1 reverse signaling modulates macrophage colony-stimulating factor (M-CSF)/receptor activator of nuclear factor kb ligand (RANKL) signaling and alters mature osteoclast formation in vivo and in vitro
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 treated with soluble EphB2-Fc or control Fc for another 4 days

Summary

Introduction

Osteoporosis is an aging-related major health problem in women and men. There are two major known causes of osteoporosis; low peak bone mineral density that is typically achieved around the age of 30, and high bone loss rate which occurs after menopause and during the natural process of aging. The interaction of ephrin B with its receptors via cell-cell contact leads to the activation of a bidirectional signal in which both the receptors (forward) and the ligand (reverse) activate downstream signaling cascades [5,6,7,8]. Studies in non-bone cells have shown that the PDZ binding motif, and six tyrosine residues within the C-terminal 33 amino acids of ephrin B1 and B2 function as receptor-like signaling molecules which transduce signals into the interior of the cell through tyrosine phosphorylation and interaction with PDZ domain-containing proteins [11,12,13,14]. We have shown that sodium hydrogen exchange regulatory factor 1 (NHERF1) interacts with ephrin B1, recruits other PDZ proteins and mediates transcription factor TAZ dephosphorylation and nuclear transportation leading to increased expression of genes that are critical for osteoblast differentiation [16]

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