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J. Biol. Chem., Vol. 279, Issue 42, 43386-43391, October 15, 2004

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Prostaglandin E₂ Selectively Antagonizes Prostaglandin F₂-stimulated T-cell Factor/-Catenin Signaling Pathway by the FP_B Prostanoid Receptor^*

Hiromichi Fujino, George A. Vielhauer, and John W. Regan

From the Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721-0207

Received for publication, July 21, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
FP prostanoid receptors are G-protein-coupled receptors that consist of two isoforms named FP_A and FP_B. Both isoforms activate inositol phosphate second messenger signaling pathways by their endogenous ligand prostaglandin F₂ (PGF₂). Previously we have shown that both isoforms undergo Rho-mediated cell rounding following treatment with PGF₂. Following the removal of PGF₂, however, FP_A-expressing cells return to their original morphology, whereas FP_B-expressing cells do not. It was also found that PGF₂-could activate T-cell factor (Tcf)/-catenin signaling in cells expressing the FP_B isoform but not in cells expressing the FP_A isoform. We now show that prostaglandin E₂ (PGE₂) can induce cell rounding and stimulate the formation of inositol phosphates to the same extent as PGF₂ in cells expressing either the FP_A or FP_B isoforms. However, PGE₂ has much lower efficacy as compared with PGF₂ for the activation of Tcf/-catenin signaling in FP_B-expressing cells, and the cell rounding is reversible. Interestingly, pretreatment of FP_B -expressing cells with PGE₂-attenuated PGF₂-stimulated Tcf/-catenin signaling in a dosedependent manner while having no effect on PGF₂-stimulated inositol phosphates formation. Thus, the ratio of endogenous PGE₂ and PGF₂ has the potential to selectively regulate one signaling pathway over another. This represents a novel mechanism for the regulation of cell signaling that is distinct from regulation occurring at the level of the receptor and its effector pathways.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The ovine FP_A and FP_B prostanoid receptors are G-protein coupled receptors that are generated by alternative mRNA splicing of their carboxyl termini (1). Relative to one another, the FP_B isoform represents a FP_A receptor in which the last 46 amino acids of the carboxyl terminus have been removed and replaced with one amino acid. The endogenous ligand of these receptors is considered to be prostaglandin F₂ (PGF₂)¹; however, prostaglandin E₂ (PGE₂) and PGD₂ can bind to these FP receptor isoforms with affinities that are only 10–30-fold less than PGF₂ (1, 2).

The FP_A and FP_B receptor isoforms are both coupled to G_q and following stimulation by PGF₂ both will activate phosphatidylinositol hydrolysis. PGF₂ will also activate the small G-protein, Rho, in cells expressing either the FP_A or FP_B receptors (3). The activation of Rho leads to cell rounding and the formation of cobblestone-like aggregates of cells. Interestingly in FP_A-expressing cells these shape changes show rapid reversal following the removal of PGF₂, but in FP_B-expressing cells the shape changes are persistent and appear to be irreversible following the removal of PGF₂ (4). In FP_B-expressing cells (but not in FP_A-expressing cells) PGF₂ can also produce a marked activation of T-cell factor (Tcf)/-catenin signaling (5). This activation of Tcf/-catenin signaling requires the activation of Rho but again is unique for the FP_B isoform even though both isoforms can activate Rho (6).

As a transcription factor, Tcf regulates the expression of a number of genes, including c-myc, cyclin D1, and cyclooxygenase-2 (COX-2) (7). The regulation of COX-2 expression by Tcf is significant given the ability of the FP_B receptor to activate Tcf/-catenin signaling, and we have recently shown that PGF₂ stimulation of the FP_B receptor leads to a Rho-dependent activation of COX-2 promoter activity (8). One of the hallmarks of colon cancer as well as other cancers is an up-regulation of COX-2 activity and an increased biosynthesis of various prostanoids, most notably PGE₂ and PGF₂ (7, 9–11). Increased levels of PGE₂ and PGF₂ would have the potential to further activate their receptors and thereby establish a positive feedback loop. Experimental evidence supporting the development of such a positive feedback loop involving murine EP₂ receptors and the up-regulation of COX-2 expression has been described (12). Furthermore, the potential of FP and EP prostanoid receptors to form positive feedback loops involving the up-regulation of COX-2 has been described (13).

The phylogeny of the prostanoid receptor family suggests that the primordial prostanoid receptor was an EP subtype whose endogenous ligand is PGE₂. In addition to its high affinity for the EP receptors, PGE₂ has 100 nM affinity for the FP and DP prostanoid receptors. Given that both PGE₂ and PGF₂ are frequently up-regulated in cancer, we were interested in the possible interactions of these compounds at the level of the activation of the FP_A and FP_B receptor isoforms. We were specifically interested in whether both PGE₂ and PGF₂ had the same efficacy for the activation of inositol phosphates hydrolysis and Tcf/-catenin signaling and whether they could both induce changes in cellular morphology. Interestingly, both prostanoids had similar efficacy for the activation of inositol phosphates hydrolysis, but PGE₂ had significantly lower efficacy for the activation of Tcf/-catenin signaling. Furthermore, pretreatment of FP_B-expressing cells with PGE₂ inhibited subsequent PGF₂-stimulated Tcf/-catenin signaling without changing the PGF₂-stimulated accumulation of total inositol phosphates. Both compounds induced changes in cellular morphology, but the effects of PGE₂ on FP_B cells were reversible, whereas the effects of PGF₂ were not.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Total Inositol Phosphates Assay—HEK-293 cells stably expressing the FP_A and FP_B prostanoid receptor isoforms were generated and cultured as described previously (3). These two cell lines have comparable levels of receptor expression judged both by radioligand binding (FP_A, 3.55 ± 0.28 pmol/mg protein; FP_B, 4.09 ± 0.49 pmol/mg protein) and by PGF₂-stimulated inositol phosphate formation (14). Receptor-stimulated total inositol phosphates accumulation was determined by anion exchange chromatography as described previously (14). Cells were plated and incubated overnight with 3 µCi/ml myo-[2-³H]inositol. Cells were trypsinized and centrifuged, and aliquots of 0.5 to 1.0 x 10⁷ cells were resuspended in 500 µl of Dulbecco's modified Eagle's medium (Invitrogen) containing 10 mM LiCl. After drug additions, the cells were incubated for 1 h at 37 °C, and 2.5 ml of chloroform/methanol/water (1:1:0.5) was added. 900 µl of the aqueous phase was removed and mixed with 2 ml of water and applied to a 2.5-ml column of AG1-X8 anion exchange resin. After three washes with 5 ml of water and two washes with 5 ml of 5 mM borax/60 mM sodium formate buffer, the ³H-labeled total inositol phosphates were eluted with 2 ml of 0.2 M ammonium formate/0.1 M formic acid, and radioactivity was determined by liquid scintillation counting.

Cell Imaging—Cells were plated in six-well tissue culture dishes under half-confluent conditions and were grown without any changes of media for 3–4 days as described previously (4). To examine agonist-induced cell rounding and its reversal, either vehicle (0.002% sodium carbonate or 0.1% Me₂SO, final concentration, respectively) or agonist (1 µM PGF₂ or 1 µM PGE₂, final concentration, respectively) were added to the media, and the cells were incubated at 37 °C for 1 h. The cells were then rapidly washed three times each with 1 ml of Opti-MEM (Invitrogen) at 37 °C and were placed in 1 ml of Opti-MEM and incubated for 1 h at 37 °C. The cells were visualized by phase-contrast microscopy using an Olympus IX70 microscope, and images were obtained and processed using a Olympus MagnaFire camera system.

Tcf Reporter Gene Experiments—Cells grown in six-well plates were transiently transfected using FuGENE 6 (Roche Applied Science) with 1 µg/well of either the TOP flash or FOP flash reporter plasmids (Upstate Biotechnology, Inc.) as described previously (5, 6). The cells were incubated at 37 °C with either vehicle (0.002% sodium carbonate or 0.1% Me₂SO), 1 µM PGF₂, or 1 µM PGE₂ for 1 h and were rapidly washed three times each with 1 ml/well Opti-MEM and then incubated for 16 h at 37 °C in 2 ml of Opti-MEM containing 250 µg/ml Geneticin, 100 µg/ml gentamicin. In the pretreatment experiments, cells were pretreated with either vehicle (0.1% Me₂SO) or various concentrations of PGE₂ for 15 min followed by the addition of vehicle (0.002% sodium carbonate) or 10 nM PGF₂ for 1 h at 37 °C. Cell extracts were prepared using the Luciferase assay system (Promega). Luciferase activity was measured using a Turner TD-20/20 luminometer as described previously (5, 6) using 10 µg of protein per sample. Measurements were corrected for background activity by subtraction of the FOP flash values from the corresponding TOP flash values.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The signal transduction properties of PGF₂ and PGE₂ were characterized in untransfected HEK cells and HEK cells stably expressing either the FP_A or FP_B prostanoid receptor isoforms. Fig. 1 shows dose response curves for these prostanoids on the accumulation of total inositol phosphates. PGF₂ stimulated total inositol phosphates accumulation to similar maximal levels and with similar EC₅₀ values in both FP_A- and FP_B-expressing cells (21 nM and 16 nM, respectively). PGE₂ also stimulated total inositol phosphates accumulation to a similar maximal extent as PGF₂ in both FP_A- and FP_B-expressing cells. However, the EC₅₀ values for PGE₂ were higher than for PGF₂ (695 nM and 117 nM, respectively). Treatment of untransfected HEK cells with PGF₂ or PGE₂ had negligible effects on total inositol phosphates formation. Thus, endogenous EP receptors that may be expressed in HEK cells contributed very little to the accumulation of inositol phosphates following treatment with PGE₂.

View larger version (16K): [in this window] [in a new window]   FIG. 1. PGF2-stimulated () and PGE2-stimulated () total inositol phosphates formation in untransfected HEK cells and in HEK cells stably expressing either the FPA or FPB prostanoid receptors. Cells were treated with the indicated concentrations of PGF2 and PGE2 for 1 h and total 3H-inositol phosphates were determined as described under "Experimental Procedures." Data were normalized to vehicle-treated FPB cells and are the means ± S.E. of three independent experiments each performed in duplicate.

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View larger version (101K): [in this window] [in a new window]   FIG. 2. PGF2- and PGE2-induced cell rounding (top) and its reversal following agonist wash-out (bottom) in untransfected HEK cells and in HEK cells stably expressing either the FPA or FPB prostanoid receptor isoforms. Four plates each of untransfected HEK cells and FPA- and FPB-expressing HEK cells were treated with either vehicle (panels a), 1 µM PGF2 (panels b), vehicle (panels e) or 1 µM PGE2 (panels f) for 1 h at 37 °C. Cells were then examined by phase-contrast microscopy for their initial response to these treatments. The plates were then washed by rinsing three times with drug-free media (Opti-MEM) followed by the addition of drug-free Opti-MEM. After a 1 h incubation at 37 °C, the cells were examined by phase-contrast microscopy for reversal of cell rounding. The notation to the left of the panels refers either to the initial treatment with either vehicle or prostaglandin (PGF2 or PGE2) or to the initial treatment followed by wash-out with Opti-MEM. Images were obtained as described under "Experimental Procedures." The results are from one experiment that was repeated three times with virtually identical results.

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PGF₂-induced changes in cell morphology can be blocked by pretreatment of the cells with C3 toxin, a specific inhibitor of Rho (3). C3 toxin also inhibits the selective activation of Tcf/-catenin signaling by the FP_B isoform. We have hypothesized that the persistent PGF₂-induced shape change in FP_B-expressing cells may be a requirement for activation of Tcf/-catenin signaling (6, 13). Thus, cell rounding and aggregation can be induced in FP_B-expressing cells by treatment with lysophosphatidic acid via endogenous receptors, but these effects are reversible and do not activate Tcf/-catenin signaling (4, 6). We, therefore, used a Tcf responsive luciferase reporter gene to investigate the potential activation of Tcf/-catenin signaling by PGE₂ in FP_B-expressing cells. As shown in Fig. 3A neither PGF₂ nor PGE₂ had much effect on the activation of Tcf-responsive reporter gene activity in FP_A-expressing cells. In contrast, PGF₂ produced a marked stimulation of reporter gene activity in FP_B-expressing cells, whereas the stimulation produced by PGE₂ was much lower. It is noted that 1 µM PGE₂ produced nearly the same levels of total inositol phosphates formation as PGF₂ in FP_B-expressing cells (Fig. 1). This suggests that relative to PGF₂, PGE₂ is a full agonist with respect to the stimulation of inositol phosphates formation but only a partial agonist with respect to stimulation of Tcf/-catenin signaling.

View larger version (19K): [in this window] [in a new window]   FIG. 3. PGF2 and PGE2 stimulation of Tcf-responsive luciferase reporter gene activity in HEK cells stably expressing either the FPA or FPB prostanoid receptors. A, FPA- and FPB-expressing cells were treated with either vehicle, 1 µM PGF2, or 1 µM PGE2 for 1 h and were washed free of drug with Opti-MEM, incubated for 16 h at 37 °C, and assayed for luciferase activity as described under "Experimental Procedures." B, FPA-expressing () and FPB-expressing () cells were treated with the indicated concentrations of PGF2, for 1 h and were washed, incubated, and assayed as described for A. Luciferase data were normalized to the vehicle-treated FPB cells and are the means ± S.E. of three independent experiments each performed in duplicate.

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View larger version (20K): [in this window] [in a new window]   FIG. 4. PGF2 stimulation of Tcf-responsive luciferase reporter gene activity (upper panels) and total inositol phosphates formation (lower panels) following pretreatment with PGE2 in HEK cells stably expressing either the FPA or FPB prostanoid receptor isoforms. Cells were pretreated for 15 min at 37 °C with either vehicle or the indicated concentrations of PGE2 followed by treatment with either vehicle () or 10 nM PGF2 () for 1 h at 37 °C. Cells were washed free of drug with Opti-MEM, incubated for 16 h at 37 °C, and assayed for luciferase activity or total 3H-inositol phosphates formation as described under "Experimental Procedures." Data were normalized to the vehicle-treated FPB cells and are the means ± S.E. of three independent experiments each performed in duplicate.

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On the other hand, Fig. 4 shows that in FP_B-expressing cells the effects of pretreatment with PGE₂ on PGF₂-activated signaling were markedly different depending upon the second messenger pathway being examined. Thus, in the absence of pretreatment with PGE₂ (0 PGE₂), 10 nM PGF₂ stimulated Tcf-responsive luciferase activity 300% and as the concentration of PGE₂ was increased there was a marked inhibition of PGF₂-stimulated luciferase activity (top right panel). This inhibition of luciferase activity leveled off at pretreatment concentration of 10^–6 M PGE₂, at which concentration the amount of luciferase activity was equal to that obtained by PGE₂ itself (closed circles). The EC₅₀ for PGE₂ with respect to the inhibition of PGF₂-stimulated Tcf signaling was 98 nM, which is comparable with its EC₅₀ for the stimulation of inositol phosphates formation (117 nM, Fig. 1) suggesting that the effects of PGE₂ are mediated by direct interactions with the FP_B receptor. Pretreatment of FP_B-expressing cells with PGE₂, however, did not inhibit the stimulation of inositol phosphates formation by 10 nM PGF₂ (bottom right panel); thus, the pattern of activity was very similar to that observed in FP_A-expressing cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
It has been found in a number of studies that aspirin and other nonsteroidal anti-inflammatory drugs, as well as selective COX-2 inhibitors, can attenuate the size and number of colon adenomas (11). These effects are presumably mediated through the inhibition of PG synthesis, although other mechanisms have been proposed (11). In support of a mechanism involving the PGs and their receptors is evidence from gene knock-out studies that implicate EP₂ and EP₄ receptors in various mouse models of colon cancer (15, 16). In addition, we have suggested the possible involvement of an FP_B- or FP_B-like receptor as a factor in the development of colorectal cancer by virtue of its ability to activate the Tcf/-catenin signaling pathway (5).

It is well established that there is an elevated expression of COX-2 and increased concentrations of PGE₂ and PGF₂ in a variety of human cancers (7, 9–11). PGE₂ and PGF₂ are structurally identical except at the C-9 position in the cyclopentane ring where PGE₂ has a keto substituent and PGF₂ has a hydroxyl. It is not unexpected, therefore, that there is some degree of cross-reactivity for PGE₂ to activate FP receptors and for PGF₂ to activate EP receptors (17). For example, based on radioligand binding PGE₂ is only about 30-fold less potent than PGF₂ at the human FP receptor (IC₅₀ values of 85 nM and 2.8 nM, respectively) (18). In the present study we explored the interactions of PGF₂ and PGE₂ with the ovine FP_A and FP_B alternative mRNA splice variants. We found that in cells expressing the FP_B isoform PGE₂ can markedly inhibit PGF₂ stimulation of Tcf/-catenin signaling without affecting PGF₂ stimulation of total inositol phosphates formation. These findings indicate that the relative composition of PGs in a tissue could be significant with respect to the activation of specific signal transduction cascades by a given prostanoid receptor subtype and its cognate ligand.

These data also lend support to thoughts about the influence of the chemical structure of agonists as it concerns the relative dominance of different conformational states of the receptor that are required for the activation of multiple signal transduction pathways (19). The most straight forward interpretation of our data with respect to the differential activation of inositol phosphates signaling as compared with Tcf/-catenin signaling is the concept that PGF₂ and PGE₂ differ simply in terms of their relative strength to generate an initial signal by the FP_B receptor. This interpretation does not require that the FP_B receptor couple to different G-proteins with respect to the activation of these two signaling pathways, but we would assume this to be the case. Therefore, although it has not been specifically verified experimentally, it is likely that coupling to the inositol phosphates pathway is through the G_q/11 family of G-proteins and that coupling to Tcf/-catenin signaling is through G_12/13 family of G-proteins. Under these circumstances differences in initial signal strength arise from differences in the efficacy of PGF₂ or PGE₂ to cause activation of either the G_q/11 or G_12/13 family of G-proteins. Thus, PGF₂ appears to have higher efficacy for the activation of both families of G-proteins, whereas PGE₂ has lower efficacy and preferentially promotes activation of the most efficiently coupled family of G-proteins, i.e. G_q/11.

The apparent antagonist activity of PGE₂ on PGF₂-mediated Tcf/-catenin signaling by the FP_B isoform, however, is harder to explain by simple differences in the strength of signaling. To explain this, it is possible that stimulation of the FP_B receptor by PGF₂ results in selective trafficking (e.g. ternary complex formation) of the FP_B receptor to both the G_q/11 and G_12/13 families of G-proteins. On the other hand, stimulation of the FP_B receptor by PGE₂ primarily directs the receptor to the G_q/11 family of G-proteins. However, because PGE₂ acts as a very weak partial agonist on the Tcf/-catenin pathway, it is capable of causing a functional antagonism with respect to the ability of PGF₂ to promote ternary complex formation of the FP_B receptor with the G_12/13 family of G-proteins.

One additional possibility is that our findings could be explained by the activation of an endogenous EP receptor, which in the presence of FP_B receptor overexpression could give rise to a unique PGE₂ response, possibly through receptor heterodimerization. We believe this is unlikely for several reasons. First, the data in Fig. 2 show that in untransfected HEK cells there is no cell rounding response to treatment with either 1 µM PGF₂ or PGE_2, so if there are endogenous FP or EP receptors, stimulation of these receptors alone is not sufficient to cause changes in cell morphology. Second we have done radioligand binding with [³H]PGE₂ and have examined PGE₂-stimulated cAMP formation in untransfected HEK cells (20), and in both cases there is little evidence for any significant response resulting from presence of an endogenous EP receptor. We are aware, however, of anecdotal reports of a PGE₂-induced cAMP response in some lines of HEK cells, possibly through an EP₄ receptor; although, as noted above we have no conclusive evidence of this in the cells that we have been using. Nevertheless, as additional control we repeated the experiments depicted in Fig. 2 with 1-hydroxy-PGE₁, an agonist that is relatively selective for the EP₄ receptor as compared with the FP receptor (21). Treatment of untransfected HEK cells and HEK cells expressing either the FP_A or FP_B receptors with 1 µM 1-hydroxy-PGE₁ for 1 h at 37 °C did not cause any obvious changes in cell morphology as compared with treatment with vehicle alone (data not shown). In this same experiment treatment with 1 µM PGF₂ caused cell rounding, aggregation, and loss of filopodia in the FP_A- and FP_B-expressing cells but not in the untransfected HEK cells. Thus, it would appear that potential interactions of PGE₂ with endogenous EP receptors would be unlikely to account for the effects of PGE₂ that we have observed in this study.

Down-regulation of Tcf/-catenin signaling has been associated with the differentiation of Caco-2 cells, a tissue culture cell line derived from human colonic carcinoma (22). Down-regulation of Tcf/-catenin signaling has also been associated with the transition of colorectal carcinoma cells in situ from a mesenchymal-like state to an epithelial-like state (23). Thus, well differentiated colorectal carcinomas are characterized as having two populations of cells. One population consists of differentiated epithelial-like cells with higher proliferative activity that is localized to central areas of the primary tumor and metastases. A second population consists of mesenchymal-like cells with lower proliferative activity that is localized to the invasive front of the tumor and to disseminated cells at metastatic sites. It appears that the tumor cells can undergo reversible transitions between these two states, which is somewhat at odds with a linear model of tumor progression in which the transition to a mesenchymal-like state is fixed by genetic changes (23). For example, colorectal carcinoma cells at the invasive front appear to dedifferentiate from an epithelial-like state to a mesenchymal-like state that is associated with increased metastatic potential and characterized by increased dissociation and migration. Following metastasis there appears to be redifferentiation and return to the epithelial-like condition. The epithelial-like condition is associated with decreased nuclear Tcf--catenin signaling, whereas the opposite is true for the mesenchymal-like state (23). It is believed that the regulation of Tcf/-catenin signaling is influenced by the tumor environment, but the specific factors regulating this are not understood.

Our present findings suggest a mechanism by which the local tumor environment could alter cell signaling in the absence of transcriptional or post-transcriptional changes either in the expression of the receptors or its downstream signaling components. Thus, by relatively rapid changes in the activity of the PG biosynthetic enzymes it would be possible to change the levels of various PGs in a tissue and thereby potentially change the patterns of cell signaling. Using the FP_B receptor as an example, the extent of activation of Tcf/-catenin signaling by a fixed concentration of PGF₂ can be significantly reduced in the presence of PGE₂ as compared with its absence. If a similar mechanism were to operate in colorectal carcinoma tumor cells, one might expect the epithelial-like condition to be associated with a relatively higher ratio of PGE₂ to PGF₂ that would result in decreased Tcf/-catenin signaling. On the other hand, a decreased ratio of PGE₂ to PGF₂ would lead to the activation of Tcf/-catenin signaling and the possible transition to a mesenchymal-like state. Clearly additional work will be needed to test whether such a mechanism might explain the effect of the local tumor environment on the phenotypic expression of tumor cells.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grant EY11291 and a grant from Allergan, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 520-626-2181; Fax: 520-626-2466; E-mail: regan{at}pharmacy.arizona.edu.

¹ The abbreviations used are: PGF₂, prostaglandin F₂; PGE₂, prostaglandin E₂; Tcf, T-cell factor; COX-2, cyclooxygenase-2; HEK, human embryonic kidney.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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