title: Glycogen synthase kinase-3 phosphorylation, T-cell factor signaling activation, and cell morphology change following stimulation of thromboxane receptor alpha.
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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, 40536-0082, USA.
authors.forename: Weili
authors.initials: W
authors.lastname: Yan
authors.forename: Hsin-Hsiung
authors.initials: HH
authors.lastname: Tai
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D001667:Binding, Competitive; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D010766:Phosphorylation; D011869:Radioligand Assay; D017482:Receptors, Thromboxane / Q000819:agonists* / Q000235:genetics; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D051820:TCF Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D007546:Isoquinolines; D017482:Receptors, Thromboxane; D013449:Sulfonamides; D051820:TCF Transcription Factors; C061750:7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid; D038362:Glycogen Synthase Kinase 3; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D000077191:Wortmannin