Loss of ephrinB1 in osteogenic progenitor cells impedes endochondral ossification and compromises bone strength integrity during skeletal development

Highlights

• Loss of ephrinB1 restricts bone formation and architecture resulting in weaker and more bendable bones.

• Loss of ephrinB1 inhibits chondrocyte maturation and affects cartilage architecture resulting in a smaller growth plate.

• Loss of ephrinB1 impedes osteoblast differentiation and enhances osteoclast numbers, affecting skeletal homeostasis.

Abstract

The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 (EfnB1^fl/fl) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1^−/− mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4 weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1^−/− mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4 weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4 week old Osx:EfnB1^−/− mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification.

Introduction

The Eph receptor tyrosine kinase (RTK) family consists of Eph receptors and ephrin ligands which can be divided into two subclasses based structure and binding affinity. The ephrin (Efn) ligands are GPI-tethered (A-subclass) or transmembrane (B-subclass) molecules. Unlike most RTKs, both Eph and ephrin molecules can mediate cell signalling. Forward signalling is achieved through receptor classes, while reverse signalling is mediated through the ephrin ligand leading to different biological outcomes (reviewed by [1]). These contact-dependent, cell membrane-associated molecules can mediate inhibitory, repulsive and attractive cellular responses, and are involved in numerous developmental and post-natal biological processes and pathologies. The Eph/ephrin molecules, predominately known for their role in establishing and maintaining cellular migration [2], [3] and boundary formation [4], [5] can also stimulate cellular differentiation [6], [7].

Pivotal studies by Zhao et al. [7] demonstrated the importance of the EphB family in mouse bone homeostasis. Their studies showed that activation of EphB4 signalling in osteoblasts promoted mineral formation, while reverse signalling through the EphB4 cognate ligand, ephrinB2, inhibited osteoclast maturation and function [7]. EphB/ephrinB interactions via EphB4/ephrinB2 and EphB2/ephrinB have been found to mediate cell attachment, migration and osteo/chondrogenic differentiation of human bone marrow derived mesenchymal stem cell (MSC) [8], [9]. More recently, we and others have postulated that ephrinB1 may be a key regulator of skeletal development and bone homeostasis [8], [9], [10], [11], [12]. A global mouse knockout and numerous human mutations of ephrinB1 result in cranial defects such as frontonasal dysplasia and coronal craniosynostosis [13], [14], [15]. The global deletion of ephrin-B1 in mouse also results in perinatal lethality and other defects including abnormal cartilage segmentation and ossification pattern [15], indicating a possible role in perichondrium maintenance [14]. Furthermore, unequal arm span to total height ratio and asymmetrical lower limb shortness has been associated with mutations of human ephrinB1[16]. Utilising the Cre-loxP recombinase system to knockout ephrinB1 in the osteoblast lineage, ephrinB1 was shown to be important for in vivo osteoblast differentiation and mineralisation through reverse signalling and was accompanied by translocation of ephrinB1 to the nucleus where it activated osterix expression [11]. Conversely, transgenic mice over-expressing ephrinB1 in osteoblast progenitors exhibited enhanced bone mass and strength [10].

Taken together, these observations strongly suggest that ephrinB1 may be an important mediator of cell cross-talk during the process of endochondral ossification. In this study we deleted ephrinB1 using the osterix-cre mouse line [17], where osterix is positively regulated by Runx2/cbfa1, but expressed prior to COL1α1. Osterix is expressed by pre-osteoblasts from embryonic day (E) E13.5 and localises predominantly within the perichondrium [17]. Here, we provide new evidence that deletion of ephrinB1 in pre-osteoblasts/osteoprogenitors perturbs normal intramembranous and endochondral ossification, abrogating epiphyseal plate formation, trabecular patterning and cortical thickness, resulting in skeletal fragility.