Received for publication October 11, 2005.
Revised December 9, 2005.
Accepted for publication December 9, 2005.

GLYCOGEN SYNTHASE KINASE-3 PHOSPHORYLATION, T-CELL FACTOR SIGNALING ACTIVATION AND CELL MORPHOLOGY CHANGE FOLLOWING STIMULATION OF THROMBOXANE RECEPTOR

Abstract

Previous reports showed that the activation of the thromboxane receptor (TP) induced some types of cells to proliferate. We report here that TP activates -catenin/ T-cell factor (Tcf)/lymphoid enhancer factor (Lef) pathway through phosphorylation of glycogen synthase kinase (GSK)-3. TP agonist, I-BOP, induced both and forms of GSK-3 phosphorylation in HEK293 cells stably over-expressing TP (HEK293-TP). H-89, a PKA inhibitor, totally blocked the phosphorylation of GSK-3, while wortmannin, a 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. Consistently, I-BOP stimulated about 8 fold increase over basal Tcf/Lef reporter gene activity in HEK293-TP cells. Furthermore, I-BOP-induced Tcf/Lef reporter gene activity was totally inhibited by H-89 and partially inhibited by wortmannin. I-BOP also induced the expression of the Tcf/Lef downstream target gene cyclin D1. Blockade of the -catenin expression by siRNA approach attenuated I-BOP- induced expression of cyclin D1, indicating that the induction was mediated by -catenin/Tcf/Lef pathway. Finally, I-BOP resulted in the morphology change, such as cell rounding and aggregation, in HEK293-TP cells after 1 h incubation. However, HEK293-TP 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 which was related to cell transformation. Taken together, our results showed for the first time that TP agonist-induced phosphorylation of GSK-3 and activation of Tcf/Lef signaling leading to cell proliferation and transformation.

Key words: Beta-catenin, Glycogen synthase kinase-3, Lymphoid enhancer factor, T- cell factor, Thromboxane receptor, protein kinase A

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