Eph receptor tyrosine kinases are involved in nervous system development. Eph
ligands, termed
ephrins, are transmembrane
proteins that bind to
Eph receptors, the mutual activation of which causes repulsive effects in reciprocally contacting cells. Previously, we showed that overexpression of EphB2 in
glioma cells increases cell invasion. Here, expression profiles of
ephrin-B family members were determined in four
glioma cell lines and in invading
glioblastoma cells collected by
laser capture microdissection.
Ephrin-B3 mRNA was up-regulated in migrating cells of four of four
glioma cell lines (1.3- to 1.7-fold) and in invading
tumor cells of eight of eight biopsy specimens (1.2- to 10.0-fold). Forced expression of
ephrin-B3 in low expressor cell lines (U87, T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant with
tyrosine phosphorylation of
ephrin-B3. In high expressor cell lines (U251, SNB19),
ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type cells. Cells transfected with
ephrin-B3 small interfering RNA (
siRNA) showed significant morphologic change and decreased invasion in vitro and ex vivo. Depletion of endogenous
ephrin-B3 expression abrogated the increase of migration and invasion induced by EphB2/Fc, indicating increased invasion is dependent on
ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed that
ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by
siRNA negated the increased invasion by addition of EphB2/Fc. In human
glioma specimens,
ephrin-B3 expression and phosphorylation correlated with increasing
tumor grade. Immunohistochemistry revealed robust staining for phosphorylated
ephrin-B and
ephrin-B3 in invading
glioblastoma cells. These data show that
ephrin-B3 expression and signaling through Rac1 are critically important to
glioma invasion.