Glycogen synthase kinase-3 phosphorylation, T-cell factor signaling activation, and cell morphology change following stimulation of thromboxane receptor alpha.

Previous reports showed that activation of the thromboxane receptor (TP) induced some types of cells to proliferate. We report here that TPalpha activates beta-catenin/T-cell factor (Tcf)/lymphoid enhancer factor (Lef) pathway through phosphorylation of glycogen synthase kinase (GSK)-3. TP agonist [1S-alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) induced both alpha and beta forms of GSK-3 phosphorylation in human embryonic kidney (HEK)293 cells stably overexpressing TPalpha (HEK293-TPalpha). N-[2-(4-Bromocinnamylamino)ethyl]-5-isoquinoline (H89), a protein kinase A (PKA) inhibitor, totally blocked the phosphorylation of GSK-3, whereas wortmannin, a phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, partially attenuated it, suggesting that PKA as well as PI-3 kinase/Akt pathway were involved in TP-induced phosphorylation of GSK-3. I-BOP consistently stimulated an approximately 8-fold increase over basal Tcf/Lef reporter gene activity in HEK293-TPalpha cells. Furthermore, I-BOP-induced Tcf/Lef reporter gene activity was totally inhibited by H89 and partially inhibited by wortmannin. I-BOP also induced overexpression of Tcf/Lef downstream target gene cyclin D1. Blockade of the beta-catenin expression by small interfering RNA approach attenuated I-BOP-induced expression of cyclin D1, indicating that the induction was mediated by beta-catenin/Tcf/Lef pathway. Finally, I-BOP resulted in the morphology change, such as cell rounding and aggregation, in HEK293-TPalpha cells after 1-h incubation. However, HEK293-TPalpha cells were not able to revert back to normal shape even 24 h after the removal of the agonist, suggesting that the prolonged activation of the Tcf/Lef promoter induced downstream gene expression leading to cell permanent morphology change that was related to cell transformation. Together, our results showed for the first time TP agonist-induced phosphorylation of GSK-3 and activation of Tcf/Lef signaling leading to cell proliferation and transformation.