HT-29 colon
carcinoma cells attach to
TNFalpha-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to
E-selectin. This interaction activates, in the
cancer cells, the MAPK
SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of
E-selectin in activating
integrins to modulate adhesion and regulate
integrin-mediated events. Blocking the
integrins from HT-29 cells (alpha2, alpha3, alpha6, alphav/beta5, beta1 and beta4) with specific
antibodies revealed a role for
beta4 integrin in their adhesion to
TNFalpha-treated HUVEC. The
beta4 integrin-dependent adhesion was maximal after 30 min, whereas the-
E-selectin-dependent adhesion was maximal after 15 min.
Integrin beta4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of
E-selectin. Moreover, a recombinant
E-selectin/Fc chimera did not induce the phosphorylation of beta4. The phosphorylation of beta4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of beta4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of beta4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of beta4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of
E-selectin to its receptor on
carcinoma cells and then the binding of beta4 to its own receptor on endothelial cells; 2) that the phosphorylation of
integrin beta4 contributes to enhance the motile potential of
cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic
cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration.