Ephrin B1 Regulates Bone Marrow Stromal Cell Differentiation and Bone Formation by Influencing TAZ Transactivation via Complex Formation with NHERF1

Abstract

Mutations of ephrin B1 in humans result in craniofrontonasal syndrome. Because little is known of the role and mechanism of action of ephrin B1 in bone, we examined the function of osteoblast-produced ephrin B1 in vivo and identified the molecular mechanism by which ephrin B1 reverse signaling regulates bone formation. Targeted deletion of the ephrin B1 gene in type 1␣2 collagen-producing cells resulted in severe calvarial defects, decreased bone size, bone mineral density, and trabecular bone volume, caused by impairment in osterix expression and osteoblast differentiation. Coimmunoprecipitation of the TAZ complex with TAZspecific antibody revealed a protein complex containing ephrin B1, PTPN13, NHERF1, and TAZ in bone marrow stromal (BMS) cells. Activation of ephrin B1 reverse signaling with soluble EphB2-Fc led to a time-dependent increase in TAZ dephosphorylation and shuttling from cytoplasm to nucleus. Treatment of BMS cells with exogenous EphB2-Fc resulted in a 4-fold increase in osterix expression as determined by Western blotting. Disruption of TAZ expression using specific lentivirus small hairpin RNA (shRNA) decreased TAZ mRNA by 80% and ephrin B1 reverse signaling-mediated increases in osterix mRNA by 75%. Knockdown of NHERF1 expression reduced basal levels of osterix expression by 90% and abolished ephrin B1-mediated induction of osterix expression. We conclude that locally produced ephrin B1 mediates its effects on osteoblast differentiation by a novel molecular mechanism in which activation of reverse signaling leads to dephosphorylation of TAZ and subsequent release of TAZ from the ephrin B1/NHERF1/TAZ complex to translocate to the nucleus to induce expression of the osterix gene and perhaps other osteoblast differentiation genes. Our findings provide strong evidence that ephrin B1 reverse signaling in osteoblasts is critical for BMS cell differentiation and bone formation.Osteoporosis is a common disease characterized by an agedependent decrease in bone mineral density (BMD) and a microarchitectural deterioration of bone tissue, with a consequent increase in the risk of developing fragility fractures of the hip, spine, and other skeletal sites (19). The decrease in bone mass occurs when the body fails to form enough new bone to replace the amount of old bone resorbed leading to reduced bone strength. There are two major known causes of osteoporosis: low peak BMD, which is typically achieved by around age 30, and high bone loss rate, which occurs particularly after menopause and during the natural process of aging. The accumulation of peak bone mass depends on bone growth during early skeletal development and the balance between osteoblastic bone formation and osteoclastic bone resorption during the postnatal growth period. Therefore, understanding the regulatory factors that govern bone development, bone size, bone mineralization, and bone quality during active growth periods as well as bone homeostasis during menopause and aging is essential for development of therapeutics to prevent osteoporosi...

Alendronate Affects Osteoblast Functions by Crosstalk through EphrinB1-EphB

“…Neural-crest-cell-derived tissues are the source of the frontal bone osteoprogenitor population, and ephrinB1 and ephrinB2 control migration of these cells (Davy et al, 2004(Davy et al, , 2006. Targeted deletion of the ephrinB1 gene in type 1 alpha 2 collagen-producing cells in vivo led to the conclusion that ephrinB1 stimulates osteoblast differentiation through EphB2 in bone marrow stromal cells (Xing et al, 2010). Although ephrinB1 is expressed in both osteoclasts and osteoblasts, in this study we show that ephrinB1 functions in osteoclasts through Eph receptors in osteoblasts.…”

Ephrin‐B stimulation of calvarial bone formation

“…The recently published study by Xing and colleagues (2010) suggested that endogenously produced EphBs in bone marrow stromal cells would cause paracrine activation of ephrin-B1 reverse signaling, and so to reduce this effect the authors plated their cultures at low density to minimize cellcell contact (Xing et al, 2010). We applied this strategy to our MC4 cultures.…”

“…Indeed, genetic studies have linked a number of mutations in ephrin-B1 to craniofrontonasal syndrome, a disorder characterized by craniosynostosis (Twigg et al, 2004;Wieland et al, 2005). Further, molecular studies by Davy et al (2006) and Xing et al (2010) identified deficiencies in OB precursor differentiation caused by cell-autonomous ablation of ephrin-B1 reverse signaling. We asked whether ephrin-B/EphB forward signaling plays a role in developmental bone formation.…”

Histone Deacetylase Inhibitors Target the Leukemic Microenvironment by Enhancing a Nherf1-Protein Phosphatase 1α-TAZ Signaling Pathway in Osteoblasts

“…Dephosphorylation by PP1␣, a ubiquitously expressed Ser/Thr protein phosphatase, induces TAZ nuclear localization to mediate osteoblast differentiation (20 -26). Nherf1 knock-out mice display bone defects (19,27,28), however, the effects of elevating Nherf1 expression and increasing TAZ localization to the nuclei of osteoblasts were previously unknown.…”

“…HDACi have been previously shown to up-regulate Nherf1 (also known as EBP-50), a scaffold protein mutated in human hypophosphatemic nephrolithiasis/osteoporosis type 2. Nherf1 has been linked with TAZ, a ubiquitously expressed transcriptional modulator, in the regulation of osteoblast differentiation (19). Localization of TAZ to the nucleus, where it increases expression of multiple genes including those controlled by the TEAD and SMAD family of transcription factors, is regulated by phosphorylation.…”