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 TP␣ activates ␤-catenin/T-cell factor (Tcf)/ lymphoid enhancer factor (Lef) pathway through phosphorylation of glycogen synthase kinase (GSK)-3. TP agonist [1S- 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 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.Thromboxane receptor (TP) is a member of the G proteincoupled receptor (GPCR) superfamily. Two isoforms of human TP, TP␣ and TP␤, have been recognized. They differ in their carboxyl-terminal tails and their mechanisms of desensitization and internalization (Kinsella, 2001). TP is known to couple to multiple G proteins, including G q/11 , G 12/13 , G i , G s , and G h , and mediates many cellular responses (Halushka et al., 1995). One of the notable responses is the stimulation of mitogenic and hypertrophic growth of vascular smooth muscle. For example, U-46619, a TP agonist, stimulates the proliferation of rat coronary or aortic smooth muscle cells (Smith, 1992) and enhances the mitogenic effects of classic growth factors such as platelet-derived growth factor and basic fibroblast growth factor (Ali et al., 1993;Zhao and Foegh 1997). I-BOP, another TP agonist, triggers a protein kinase C (PKC)-, pertussis toxin-, Src-, and epidermal growth factor receptor (EGFR)-dependent activation of extracellular

Activation of thromboxane receptor α induces expression of cyclooxygenase-2 through multiple signaling pathways in A549 human lung adenocarcinoma cells

“…Therefore, I-BOP may activate Stat3 through Src, EGFR and JAK pathway since I-BOP may transactivate EGFR. Recently, we showed that I-BOP induced phosphorylation of GSK-3 and cyclin D1 expression in a PKA dependent manner in HEK293-TPα cells [11]. We also observed that I-BOP induced phosphorylation and inactivation of GSK-3 and I-BOP-induced COX-2 expression was slightly increased in the presence of GSK-3 inhibitor in A549-TPα cells as demonstrated in this study.…”

“…JAK inhibitor I totally blocked the phosphorylation of Stat3 as expected. Consistent with the results presented in our recent publication [11], pGSK-3α/β induced by I-BOP was still observed at 40 min (Fig. 4A).…”

“…Rabbit monoclonal antibodies specific to phospho-p38 MAPK, pStat3, phospho-CREB and phospho-NF-κB-p65 (Ser276) were from Cell Signaling Technology Inc. (Beverly, MA). Antibody specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was generated as described [11]. Horseradish peroxidase (HRP)-linked goat anti-mouse and rabbit IgG were supplied by BD Transduction Laboratories (Lexington, KY).…”

“…Activation of TPα also leads to stimulation of adenylate cyclase generating cyclic AMP which activates protein kinase A (PKA), whereas activation of TPβ results in inhibition of adenylate cyclase [7]. Activation of TPs also initiates signaling cascades leading to activation of various kinases known to be involved in mitogenic responses, such as epidermal growth factor receptor (EGFR) [8], extracellular signal-regulated kinase (ERK) [9], Akt/protein kinase B (PKB) [10] and glycogen synthase kinase (GSK) [11]. Apparently, TP signaling is very much related to mitogenesis of target cells.…”

Targeting mediators of Wnt signalling pathways by GnRH in gonadotropes

“…A number of non-FZD GPCRs have been shown to target b-catenin/TCF activity in response to their cognate ligands, including the prostanoid receptors (Fujino & Regan 2001, Fujino et al 2002, M1 muscarinic acetylcholine receptor (Farias et al 2004), lysophosphatidic acid (LPA) receptor (Yang et al 2005) and thromboxane A2/TP a receptor (Yan & Tai 2006). The first demonstration of non-FZD targeting of b-catenin/ TCF-dependent signalling was through the FP B prostanoid receptor (Fujino & Regan 2001).…”