Glycogen Synthase Kinase-3 Phosphorylation, T-Cell Factor Signaling Activation, and Cell Morphology Change following Stimulation of Thromboxane Receptor α

Abstract

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.