Government, and no official endorsement should be inferred. We thank Catrina M. 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 age-dependent decrease in bone mineral density (BMD) and a microarchitectural deterioration of bone tissue, with a ONO-7300243 consequent increase in the risk of developing ONO-7300243 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 ONO-7300243 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 osteoporosis. Ephrin ligands and their receptors have been shown to play key roles in the growth and development of multiple tissues including the skeleton (14,50,53). There are two types of ephrin ligands and their receptors. Ephrin A’s are membrane-anchored proteins, while ephrin B’s are transmembrane proteins. In general, ONO-7300243 ephrin A’s bind to ephrin A receptors (EphA) while ephrin B’s interact with ephrin B receptors (EphB), with few exceptions (29). Mouse monoclonal to ISL1 Ephrin B1 preferentially binds to EphB2 and -B3 receptors with high affinity and interacts with EphB1 and -B4 receptors with low affinity (29). It has been shown that both ephrin B1 and B2 and their receptors (EphB2, -B3, -B4, -B6, and -A4) are expressed in bone cells (58). However, only ephrin B1 and B2 are ONO-7300243 expressed in osteoclasts during osteoclast precursor differentiation, while ephrin B1 and B2 and their receptors are consistently coexpressed during osteoblast differentiation (58). The interaction of ephrin B1 and B2 with their multiple receptors via cell-cell contact leads to the activation of a bidirectional signal in which both the receptor-mediated forward signal and the ligand-mediated reverse signal activate downstream signaling cascades (13,58). In the cells that coexpress both ephrin ligands and their receptors, the ephrin ligands and receptor proteins can be segregated into distinct membrane domains from which they signal biological effects via cell surface interactions (36). Although EphB4 forward signaling and ephrin B2 reverse signaling have been implicated in regulating osteoblastic bone formation and osteoclastic bone resorption processes (58), homozygotes for targeted null mutations of ephrin B2 or EphB4 receptor exhibit severe defects in angiogenesis of both arteries and veins and embryonic lethality (16,18,35). In contrast, total disruption of the ephrin B1 gene in mice results in perinatal lethality and defects in skeletal patterning, while mutations of ephrin B1 in humans have been found to cause craniofrontonasal syndrome (9,13,50,51). Mutation of the cytoplasmic tail of ephrin B1, which allows its extracellular domain to interact with ephrin receptors, produces the same bone phenotypes as those of ephrin B1 knockout (KO) mice (13,14). However, individual KO of EphB1, -B2, -B3, or -A4 receptor, the major receptors.