Figure 6

Reelin-dependent activation of EphB2 induces deadhesive cytoskeletal changes in Cos cells. (A) Expression of components of the Reelin and EphB2 signaling cascades in primary neurons and Cos-1 cells was analyzed by western blotting. Cos-1 cells express EphB2 and ephrin B proteins but do not express detectable levels of VLDLR, ApoER2 or Dab-1. Actin was used as a loading control. (B) Treatment with preclustered ephrin B1 (4 μg/ml), ephrin A5 (20 μg/ml) or Reelin but not with control medium induces deadhesive cytoskeletal changes in Cos-1 cells, resulting in rounding and loss of attachment (arrowheads) after 2.5 h of treatment. The effect of Reelin was not blocked by the lipoprotein receptor antagonist RAP, and treatment with RAP alone has no effect. (C) Quantification of the deadhesive changes in Cos-1 cells shown in B as compared with control (S.E.M., ^***P < 0.001, 3 independent experiments, n = 10 fields of view with a minimum of 1 000 cells per group analyzed). (D) Pharmacological EphB2 kinase inhibition with WHI-P180 (10 μmol/l) blocks the Reelin- or Fc-ephrin B1-induced detachment of Cos-1 cells. Quantification of deadhesive changes in Cos-1 cells (S.E.M., n = 10 fields of view per group analyzed, ^***P < 0.001) is shown. (E) Pretreatment with the EphB2-selective antagonistic 12-mer peptide SNEW (400 μmol/l) blocks the Reelin- or Fc-ephrin B1-induced detachment of Cos-1 cells. Quantification of the deadhesive changes in Cos-1 cells is shown (S.E.M., n = 10 fields of view per group analyzed, ^***P < 0.001). (F) Knockdown of endogenous EphB2 in Cos-1 cells by exogenous siRNA against human EphB2 (siRNA EphB2) prevented the Reelin- and ephrin B1-mediated detachment of Cos-1 cells, as compared with control-treated cells (siRNA control). Quantification of the cell detachment is shown (S.E.M., n = 10 fields of view per group analyzed, ^***P < 0.001, ^**P < 0.01).