EphB6 is a unique member in the Eph family of receptor tyrosine kinases in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In the present study, we examined the function of EphB6 in cell adhesion and migration. We demonstrated that EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2 ligand; EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic-positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. EphB6 is constitutively associated with the Src family kinase Fyn. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through an Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the receptor by an Src family kinase acts as the molecular switch for the functional transition.

With aging and pathology, cells of the nucleus pulposus (NP) de-differentiate towards a fibroblast-like phenotype, a change that contributes to degeneration of the intervertebral disc (IVD). Laminin isoforms are a component of the NP extracellular matrix during development but largely disappear in the adult NP tissue. Exposing human adult NP cells to hydrogels made from PEGylated-laminin-111 (PEGLM) has been shown to regulate NP cell behaviors and promote cells to assume a biosynthetically active state with gene/protein expression and morphology consistent with those observed in juvenile NP cells. However, the mechanism regulating this effect has remained unknown. In the present study, the integrin subunits that promote adult degenerative NP cell interactions with laminin-111 are identified by performing integrin blocking studies along with assays of intracellular signaling and cell phenotype. The findings indicate that integrin α3 is a primary regulator of cell attachment to laminin and is associated with phosphorylation of signaling molecules downstream of integrin engagement (ERK 1/2 and GSK3β). Sustained effects of blocking integrin α3 were also demonstrated including decreased expression of phenotypic markers, reduced biosynthesis, and altered cytoskeletal organization. Furthermore, blocking both integrin α3 and additional integrin subunits elicited changes in cell clustering, but did not alter the phenotype of single cells. These findings reveal that integrin- mediated interactions through integrin α3 are critical in the process by which NP cells sense and alter phenotype in response to culture upon laminin and further suggest that targeting integrin α3 has potential for reversing or slowing degenerative changes to the NP cell.

Normal human hematopoietic progenitors as well as leukemia/lymphoma cells express kinase-defective EphB6 receptors. The only unique high affinity ligand for EphB6 among eight known mammalian ephrins, ephrins-B2 is expressed not only on hematopoietic malignancies, but also on mesenchymal stem cells. However, the biological functions of the receptor and its ligand in hematopoietic cells are largely unknown. In the present study, we showed that the interaction between EphB6 and ephrinB2 could initiate forward as well as reverse signaling in vitro. Both pre-clustered and unclustered ligands could trigger the signal transduction, but pre-clustered ones more rapidly down-regulated the signaling. We also examined the EphB6/ephrinB2 function in cell adhesion and migration. Figure Figure HEK-EphB6 cells placed in the upper chamber of a Transwell apparatus, in which the lower side of filter was coated with different concentrations of ephrin-B2-Fc or Fc, were allowed to migrate to the lower side at 37°C overnight. Vector-transfected cells were used as controls. The cells that had migrated to the lower side of filter were stained, photographed. A BSA-coated filter is shown as a control. EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2. EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. We further explored the signal transduction of the biphasic effects. Figure Figure The Src family kinase, Fyn was co-immunoprecipitated with anti-EphB6 antibody in the absence or presence of ephrin-B2 stimulation. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through a Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the kinase-defective EphB6 receptor by a Src family kinase acts as the molecular switch for the functional transition. Thus, EphB6 expressed on hematopoietic cells may play an important role in the regulation of cell homing to hematopoietic tissues as well as leukemia cell infiltration.