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.