Abstract
Previous reports showed that activation of the thromboxane receptor (TP) induced some types of cells to proliferate. We report here that TPα activates β-catenin/T-cell factor (Tcf)/lymphoid enhancer factor (Lef) pathway through phosphorylation of glycogen synthase kinase (GSK)-3. TP agonist [1S-α,2α(Z),3β(1E,3S),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) induced both α and β forms of GSK-3 phosphorylation in human embryonic kidney (HEK)293 cells stably overexpressing TPα (HEK293-TPα). 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-TPα 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 β-catenin expression by small interfering RNA approach attenuated I-BOP-induced expression of cyclin D1, indicating that the induction was mediated by β-catenin/Tcf/Lef pathway. Finally, I-BOP resulted in the morphology change, such as cell rounding and aggregation, in HEK293-TPα cells after 1-h incubation. However, HEK293-TPα 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.
Footnotes
-
This work was supported in part by National Institutes of Health Grant HL46296.
-
doi:10.1124/jpet.105.096826.
-
ABBREVIATIONS: TP, thromboxane receptor; GPCR, G protein-coupled receptor; U-46619, 9α,11α-methanoepoxy-PGF2α; I-BOP, [1S-α,2α(Z),3β(1E,3S),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; PKC, protein kinase C; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; PKA, protein kinase A; COX, cyclooxygenase; Tcf, T-cell factor; Lef, lymphoid enhancer factor; EP, prostaglandin E2 receptor; HEK, human embryonic kidney; FP, prostaglandin F2α receptor; GSK-3, glycogen synthase kinase-3; PKB, protein kinase B; FBS, fetal bovine serum; SQ-29548, 7-[(1S,2R,3R,4R)-3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-, (5Z)-(9CI); H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; pERK, phosphorylated extracellular signal-regulated kinase; siRNA, small interfering RNA; pGSK, phosphorylated glycogen synthase kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; PG, prostaglandin.
- Received October 7, 2005.
- Accepted December 9, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|