Signalling by integrin-mediated cell anchorage to extracellular matrix proteins is co-operative with other receptor-mediated signalling pathways to regulate cell adhesion, spreading, proliferation, survival, migration, differentiation and gene expression. It was observed that an anchorage-independent gastric carcinoma cell line (SNU16) became adherent on TGF-β1 (transforming growth factor β1) treatment. To understand how a signal cross-talk between integrin and TGF-β1 pathways forms the basis for TGF-β1 effects, cell adhesion and signalling activities were studied using an adherent subline (SNU16Ad, an adherent variant cell line derived from SNU16) derived from the SNU16 cells. SNU16 and SNU16Ad cells, but not integrin α5-expressing SNU16 cells, showed an increase in adhesion on extracellular matrix proteins after TGF-β1 treatment. This increase was shown to be mediated by an integrin α3 subunit, which was up-regulated in adherent SNU16Ad cells and in TGF-β1-treated SNU16 cells, compared with the parental SNU16 cells. After TGF-β1 treatment of SNU16Ad cells on fibronectin, Tyr-416 phosphorylation of c-Src was increased, but Ras-GTP loading and ERK1/ERK2 (extracellular-signal-regulated kinases 1 and 2) activity were decreased, which showed a dependence on c-Src family kinase activity. Studies on adhesion and signalling activities using pharmacological inhibitors or by transient-transfection approaches showed that inhibition of ERK1/ERK2 activity increased TGF-β1-mediated cell adhesion slightly, but not the basal cell adhesion significantly, and that c-Src family kinase activity and decrease in Ras/ERKs cascade activity were required for the TGF-β1 effects. Altogether, the present study indicates that TGF-β1 treatment causes anchorage-independent gastric carcinoma cells to adhere by an increase in integrin α3 level and a c-Src family kinase activity-dependent decrease in Ras/ERKs cascade activity.
Abbreviations used: ECM, extracellular matrix; ERK, extracellular-signal-regulated kinase; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; NRK, normal rat kidney; PI3K, phosphoinositide 3-kinase; RBD, Ras-binding domain; TGF-β1, transforming growth factor β1.