ACCELERATED PUBLICATION
FP Prostanoid Receptor Activation of a T-cell Factor/beta -Catenin Signaling Pathway*

Hiromichi Fujino and John W. ReganDagger

From the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-80207

Received for publication, January 24, 2001, and in revised form, February 13, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

FP prostanoid receptors are G-protein-coupled receptors (GPCR) that consist of two known isoforms, FPA and FPB. These isoforms, which are generated by alternative mRNA splicing, are identical except for their carboxyl-terminal domains. Previously we have shown that stimulation of both isoforms with prostaglandin F2alpha (PGF2alpha ) activates the small G-protein Rho, leading to morphological changes consisting of cell rounding and the formation of cell aggregates. Following the removal of PGF2alpha , however, FPA-expressing cells show rapid reversal of cell rounding, whereas FPB-expressing cells do not. We now show that acute treatment of FPB-expressing cells with PGF2alpha leads to a subcellular reorganization of beta -catenin, a decrease in the phosphorylation of cytoplasmic beta -catenin, and persistent stimulation of Tcf/Lef-mediated transcriptional activation. This does not occur in FPA-expressing cells and may underlie the differences between these isoforms with respect to the reversal of cell rounding. The Tcf/beta -catenin signaling pathway is known to mediate the actions of Wnt acting through the heptahelical receptor, Frizzled, and has not been associated previously with GPCR activation. Our findings expand the signaling possibilities for GPCRs and suggest novel roles for FP receptors in normal tissue development and malignant transformation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amino acid sequences of the ovine FPA and FPB prostanoid receptor isoforms are the same throughout their amino termini and seven-membrane-spanning domains, but the FPB isoform is truncated and lacks the last 46 carboxyl-terminal amino acids present in the FPA isoform (1). This is very similar to the EP3 (2) and thromboxane A2 (3) prostanoid receptors in which alternative mRNA splicing gives rise to a variety of isoforms in humans and in other species (4). The physiological significance of these receptor isoforms is not clear, although differences have been shown to exist with respect to second messenger coupling and receptor desensitization. We have found that the FPA and FPB receptor isoforms have similar pharmacological properties and that prostaglandin F2alpha (PGF2alpha )1 stimulates phosphoinositide turnover to a similar extent in cells expressing these isoforms (1). In addition, stimulation of FPA- or FPB-expressing cells with PGF2alpha activates Rho leading to the formation of actin stress fibers, phosphorylation of p125 focal adhesion kinase, and cell rounding (5). Cell rounding involves the retraction of filopodia and a change from an isolated dendritic appearance to one in which the cells are rounded and form small cobblestone-like aggregates (see Fig. 1A). Following the removal of PGF2alpha , however, FPA-expressing cells return to their original dendritic morphology, but the FPB-expressing cells do not and remain rounded (6). We hypothesized that FPB-expressing cells might remain rounded because of prolonged signaling following the removal of agonist. However, a specific mechanism of this prolonged signaling was not established. Here we show that Tcf/beta -catenin-mediated transcriptional activation is elevated 16 h after an initial 1-h treatment of FPB-expressing cells with PGF2alpha . This transcriptional activation is not observed in FPA-expressing cells and suggests that FPB-expressing cells remain rounded because of activation of a Tcf/beta -catenin signaling pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Immunofluorescence Microscopy-- HEK-293 cells stably expressing the ovine FPA and FPB prostanoid receptor isoforms (5) were split and grown in six-well plates containing 22-mm round glass coverslips for 3-4 days. Cells were treated with either vehicle (sodium carbonate, 0.002% final) or 1 µM PGF2alpha and were rapidly washed, fixed, and incubated with a 1:1000 dilution of a mouse monoclonal antibody to beta -catenin (Transduction Laboratories). They were then washed and incubated with a 1:4000 dilution of an fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (Sigma). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma). Cells were visualized by phase contrast and epifluorescence microscopy as described previously (6).

Immunoprecipitation and Blotting-- Cells were scraped and sonicated in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 2 mM EGTA, 2 mM phenylmethylsulfonylfluoride, 0.1 mg/ml leupeptin, and 2 mM sodium vanadate. Samples were centrifuged (16,000 × g) for 15 min at 4 °C, the supernatant (cytosolic fraction) was removed, and the pellet (particulate fraction) was solubilized with lysis buffer containing 0.2% Triton X-100 and then centrifuged again to remove insoluble debris. For immunoprecipitation, samples were rotated for 2 h at 4 °C with antibodies to beta -catenin followed by the addition of protein G-Sepharose (Amersham Pharmacia Biotech) and rotation for another hour. The Sepharose was washed with lysis buffer and then resuspended with SDS-polyacrylamide gel electrophoresis sample buffer and boiled. Samples were electrophoresed on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and incubated with either antibodies to beta -catenin or a mixture of mouse monoclonal antibodies to phosphoserine (Sigma) and phosphothreonine (Sigma). The membranes were washed, incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies, and visualized by enhanced chemiluminescence (SuperSignal, Pierce). The resulting films were scanned at high resolution (300 dpi) as positive transparencies (Microtek ScanMaker4) and saved as TIFF files. Quantitation was performed using the Gelplot2 macro in Scion Image for Windows (beta version 4.02). Nuclear extracts were prepared according to the method of Dignam as modified by Westin et al. (7).

RT-PCR-- RT was done using the Superscript Preamplification System (Life Technologies, Inc.) and 1 µg of RNA/sample that had been pretreated with DNase I. This was followed by PCR using an initial incubation at 94 °C for 5 min, followed by 20 cycles of 94 °C, 60 °C, and 68 °C each for 2 min, and a final incubation at 68 °C for 10 min. The human beta -catenin and GAPDH primer pairs were exactly according to Rezvani and Liew (8). Product sizes were 521 base pairs for beta -catenin and 737 base pairs for GAPDH and were resolved by electrophoresis on 1.5% agarose gels. Preliminary experiments were done to find the optimal conditions for quantitative amplification of beta -catenin and GAPDH mRNA.

Tcf/Lef Reporter Gene Assay-- Cells were split into 10-cm dishes and the next day were transiently transfected using FuGENE-6 (Roche Molecular Biochemicals) and either 10 µg/dish of the wildtype Tcf/Lef reporter plasmid TOPflash or the mutant plasmid FOPflash. FOPflash differs from TOPflash by the mutation of its Tcf binding sites and serves to differentiate Tcf/beta -catenin-mediated signaling from background (Upstate Biotechnology, Inc.). Cells were incubated overnight and were treated for 1 h at 37 °C with either vehicle or 1 µM PGF2alpha . They were then rapidly washed three times each with 2 ml of Opti-MEM (Life Technologies, Inc.) as described previously (6) and incubated for 16 h at 37 °C in 10 ml of Opti-MEM containing 250 µg/ml geneticin, 200 µg/ml hygromycin B, and 100 µg/ml gentamicin. Cells were placed on ice and rinsed twice with ice-cold phosphate-buffered saline, and extracts were prepared using the Luciferase Assay System (Promega). Luciferase activity in the extracts (~500 ng protein/sample) was measured using a Turner TD-20/20 luminometer and was corrected for background by subtraction of FOP-FLASH values from corresponding TOP-FLASH values.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fig. 1A shows phase contrast microscopy of HEK cells stably expressing either the ovine FPA prostanoid receptor (panels a and b) or the ovine FPB prostanoid receptor (panels c and d) following a 1-h treatment with either vehicle (panels a and c) or 1 µM PGF2alpha (panels b and d). It can be appreciated that in both FPA- and FPB-expressing cells treatment with PGF2alpha resulted in morphological changes consisting of a loss of filopodia and formation of cell aggregates. We have previously shown that these morphological changes involve the activation of Rho and phosphorylation of p125 focal adhesion kinase (5). However, following the removal of PGF2alpha the FPA-expressing cells show a rapid (within 1 h) reversal of these morphological changes, whereas the FPB-expressing cells remain rounded even after 48 h (6). To investigate the possible role of other adhesion proteins in this process, we used immunofluorescence microscopy to examine the localization of E-cadherin and beta -catenin in HEK cells stably expressing either the FPA or FPB isoforms following treatment with 1 µM PGF2alpha . Although the effects on E-cadherin localization were not apparent (data not shown), Fig. 1B shows that PGF2alpha treatment resulted in a marked accumulation of beta -catenin in regions of cell-to-cell contact in FPB-expressing cells (panels c and d) but not in FPA-expressing cells (panels a and b). Both cell lines, however, showed agonist-dependent cell rounding following treatment with PGF2alpha (Fig. 1A), indicating that the process of cell rounding itself was not responsible for the increased contiguous accumulation of beta -catenin in the FPB-expressing cells.


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Fig. 1.   A, phase contrast microscopy (×225) of FPA- and FPB-expressing cells after treatment with either vehicle (panels a and c) or 1 µM PGF2alpha (panels b and d) for 1 h at 37 °C. B, beta -catenin fluorescein isothiocyanate immunofluorescence (green) and nuclear DAPI fluorescence (blue) microscopy (×225) of FPA and FPB cells after the same treatment. Cells were labeled and prepared for microscopy as described under "Experimental Procedures." The results are representative of more than three experiments.

In addition to its role in cell adhesion, beta -catenin is well recognized as a signaling molecule that undergoes stimulus-dependent translocation from the cytosol to the nucleus where it is involved in the regulation of Tcf/Lef-mediated gene transcription (9-11). We, therefore, used immunoblotting to examine both particulate and cytosolic fractions for changes in beta -catenin expression following treatment of FPA- and FPB-expressing cells with PGF2alpha . Fig. 2A shows that the expression of beta -catenin is ~3-fold higher in the particulate fraction and ~2-fold higher in the cytosolic fraction from FPB-expressing cells compared with FPA-expressing cells. Furthermore, treatment with PGF2alpha caused a slight increase the levels of cytosolic beta -catenin in both the FPA- and FPB-expressing cells but had little effect on the levels of beta -catenin in the particulate fraction. Reverse transcription (RT) followed by polymerase chain reaction (PCR) was used to determine whether there were any differences in beta -catenin mRNA levels under these same experimental conditions. Fig. 2B shows that beta -catenin and GAPDH mRNA levels were the same for both cell lines and were not affected by PGF2alpha , indicating that the observed differences in beta -catenin expression appear to be the result of changes in translation and/or protein turnover.


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Fig. 2.   A, immunoblot of beta -catenin in particulate and cytosolic fractions prepared from FPA- and FPB-expressing cells after treatment with either vehicle (lanes a and c) or 1 µM PGF2alpha (lanes b and d) for 1 h at 37 °C. B, RT-PCR of beta -catenin and control GAPDH mRNA from FPA- and FPB-expressing cells after the same treatment. Immunoblotting (10 µg of protein/sample) and RT-PCR were done as described under "Experimental Procedures." Results for both the immunoblotting and RT-PCR are representative of three independent experiments.

Serine/threonine phosphorylation of beta -catenin by glycogen synthase kinase-3beta (GSK-3beta ) marks beta -catenin for degradation and is a critical factor in the regulation of its signaling activity (12, 13). Thus, under most conditions cytosolic beta -catenin is phosphorylated, leading to an association with the tumor suppressor protein, adenomatous polyposis coli (APC), and the scaffolding protein, axin, which is then followed by ubiquitination and proteasomal degradation (14). Using immunoprecipitation and immunoblotting, we examined serine/threonine phosphorylation of beta -catenin following treatment of either FPA- or FPB-expressing cells with PGF2alpha . Fig. 3 shows that in FPA-expressing cells the vehicle control levels of cytosolic beta -catenin are very low (lane a, middle panel) and there is little detectable phosphorylation (lane a, upper panel). Following treatment of FPA-expressing cells with PGF2alpha , the levels of cytosolic beta -catenin increase 6-fold (lane b, middle panel) and there is a 19-fold increase in phosphorylation (lane b, upper panel). In FPB-expressing cells the vehicle control levels of cytosolic beta -catenin are already higher than in the FPA-expressing cells (cf, lanes c and a, middle panel) and so is phosphorylation (cf, lanes c and a, upper panel). This would be expected to reflect endogenous GSK-3beta activity and tight coupling to the elevated levels of cytoplasmic beta -catenin. After treatment of FPB-expressing cells with PGF2alpha there is a further 2-fold increase in cytosolic beta -catenin (lane d, middle panel) but an unexpected 12-fold decrease in phosphorylation (lane d, upper panel). The ratio of phosphorylated to total beta -catenin in the cytoplasm, therefore, shows dramatic differences following activation of these two FP receptor isoforms. Thus, in FPA-expressing cells this phosphorylation ratio increases from 0.5 to 1.6 with agonist treatment, whereas in FPB-expressing cells it falls from 2.3 to 0.1. It would therefore be expected that degradation of cytosolic beta -catenin would be favored at the expense of nuclear translocation in FPA-expressing cells, whereas the opposite would be true in FPB-expressing cells. This appears to be confirmed in Fig. 3 where immunoblotting of nuclear extracts shows a 3-fold higher level of beta -catenin in FPB-expressing cells following treatment with PGF2alpha (lane d, bottom panel) as compared with FPA-expressing cells (lane b, bottom panel).


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Fig. 3.   Immunoblots (IB) of beta -catenin (beta -cat) in cytosolic fractions and nuclear extracts and of serine/threonine phosphorylated (PS/PT) cytosolic beta -catenin from FPA and FPB-expressing cells after treatment with either vehicle (lanes a and c) or 1 µM PGF2alpha (lanes b and d) for 1 h at 37 °C. Cytosolic fractions were prepared as described under "Experimental Procedures," and samples (100 µg of protein) immunoprecipitated (IP) with antibodies to beta -catenin were first probed with antibodies to phosphoserine and phosphothreonine (upper panel); and then stripped and reprobed with antibodies to beta -catenin (middle panel). Immunoblotting of nuclear extracts (lower panel) was done with 10 µg of protein/sample without prior immunoprecipitation. Results are representative of three independent experiments.

Following nuclear translocation, beta -catenin is known to interact with members of the Tcf/Lef family of transcription factors (15). Because of this signaling potential, we were interested in the possibility that the failure of FPB-expressing cells to return to their original dendritic morphology following removal of PGF2alpha might represent a beta -catenin-mediated switch in gene expression. To examine an effect on gene epression, we transiently transfected either FPA- or FPB-expressing cells with a Tcf/Lef-responsive reporter plasmid (16) and measured luciferase reporter gene activity following treatment with 1 µM PGF2alpha . Initially we found that basal levels of luciferase activity were elevated (~3-fold) in FPB-expressing cells as compared with FPA-expressing cells and that luciferase activity was not stimulated immediately following a 1-h treatment with PGF2alpha in either cell line (data not shown). However, as shown in Fig. 4A, the morphological effects of PGF2alpha on FPB-expressing cells persist long after its removal. Thus, when cells are examined 16 h after an initial 1-h treatment with PGF2alpha (followed by washout and replacement with fresh media), the FPA-expressing cells show a return to their original dendritic morphology (panel b), whereas the FPB-expressing cells remain rounded and aggregated (panel d). We therefore examined Tcf/Lef reporter gene activity at the same time point. As shown in Fig. 4B, FPB-expressing cells show a persistent activation of luciferase activity (column d) that is roughly 6.5-fold higher than either the vehicle control (column c) or PGF2alpha -treated FPA cells (column b). The failure of FPB-expressing cells to show reversal of cell rounding is not because of changes in the kinetics of PGF2alpha binding or in its removal during the washout procedure (6).


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Fig. 4.   A and B, phase contrast microscopy (×225) (A) and stimulation of Tcf/Lef-responsive luciferase reporter gene activity (B) after FPA- and FPB-expressing cells were treated with either vehicle or 1 µM PGF2alpha for 1 h, then washed extensively in drug-free media, and incubated for an additional 16 h at 37 °C in drug-free media. The transfection conditions, drug washout, and luciferase assay are described under "Experimental Procedures." Luciferase data are normalized to the vehicle-treated FPA cells and are the means ± S.E. of three independent experiments each performed in duplicate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We show that FPB-expressing cells differ in several important regards from FPA-expressing cells in terms of their potential for activation of Tcf/beta -catenin-mediated signaling. First, FPB-expressing cells show PGF2alpha -stimulated accumulation of beta -catenin at their contiguous cell boundaries that is not evident in FPA-expressing cells. Second, although both FPA and FPB-expressing cells show PGF2alpha -stimulated increases in cytosolic beta -catenin, in FPA-expressing cells this is accompanied by increased beta -catenin phosphorylation and in FPB-expressing cells by decreased beta -catenin phosphorylation. Third, FPB-expressing cells show a stimulation of Tcf/Lef reporter gene activity 16 h after agonist removal that is essentially absent in FPA-expressing cells. A key control point could be in the differential phosphorylation of beta -catenin. Thus, it is possible that the agonist-stimulated accumulation of beta -catenin at the cell boundaries of FPB cells results in enhanced interactions with E-cadherin. In turn, this could initiate E-cadherin outside-in signaling leading to the sequential activation of phosphatidylinositol 3-kinase and Akt kinase (17). This is meaningful because phosphorylation of GSK-3beta by Akt kinase is inhibitory (18) and could lead to the decreased phosphorylation of beta -catenin found in agonist-treated FPB cells.

Recently Meigs et al. (19) reported that constitutively active mutants of Galpha 12 and Galpha 13 interact with E-cadherin resulting in a release of beta -catenin and stimulation of Tcf/Lef reporter gene activity in a mutant cell line lacking APC. This link between heterotrimeric G-proteins and the Tcf/beta -catenin signaling pathway is novel, but its physiological relevance might be questioned because of the altered nature of the model. In light of the present findings, however, it appears likely that both GPCRs and heterotrimeric G-proteins will be involved with activation of this signaling pathway. We have shown that FP receptors activate Rho through the probable activation of G12 and/or G13 (5). Both receptor isoforms were equally effective in this regard, and therefore it appears unlikely that activation of G12 and/or G13 could be solely responsible for the present findings because activation of Tcf/beta -catenin signaling was observed only for cells expressing the FPB isoform.

We believe that activation of Tcf/beta -catenin signaling by PGF2alpha in cells expressing the FPB receptor is involved with a phenotypic transformation that is morphologically similar to, but fundamentally different from, the cell rounding observed in agonist-treated FPA cells. Thus, maintenance of shape change in FPA-expressing cells depends on continuous stimulation by PGF2alpha , and following its removal the cells revert back to their original morphology. In contrast, although shape change in FPB-expressing cells is initiated by PGF2alpha , its maintenance is independent of further PGF2alpha stimulation. In this manner the FPB prostanoid receptor functions as one would expect of a trigger in a developmental or malignant transformation pathway.

The present findings have significance for the signaling potential of FP prostanoid receptors and possibly for other GPCRs as well. For example, in sheep it is known that PGF2alpha is the physiological signal for regression of the corpus luteum but only during a short window of the luteal cycle. Thus, if pregnancy occurs the corpus luteum is maintained and loses sensitivity to the luteolytic actions of PGF2alpha (20). Interestingly, the expression of FPA receptors does not change during this transition (21). Brief expression of a small population of FPB receptors during the sensitive phase of the luteal cycle could explain the luteolytic actions of PGF2alpha .

Another condition that might involve the FPB isoform or a homologue is in colorectal cancer. It is known that aberrant activation of Tcf/beta -catenin signaling is associated with the development of this disease (22-24) and that inhibition of cyclooxygenase by NSAIDs can slow tumor progression (25). However, the specific mechanism of this beneficial effect is vague because of the large number of prostanoid metabolites that are affected. Our findings support a mechanism in which NSAID-mediated decreases in PGF2alpha would decrease Tcf/beta -catenin signaling by FPB prostanoid receptors. This conclusion is supported by animal models of skin carcinogenesis in which PGF2alpha reversed the anti-tumor-promoting activity of indomethacin (26). Although a human homologue of the ovine FPB receptor has not yet been identified other mechanisms could give rise to functional FPB isoforms. Thus, much like the known mutations of APC, truncation of the human FPA receptor by allelic variation, somatic mutations or proteolytic cleavage could give rise to receptors capable of producing activation of Tcf/beta -catenin signaling. The possible role of FPB receptors in these and other physiological processes is intriguing and awaits future studies.

    ACKNOWLEDGEMENTS

We thank Ron Heimark, Pat Hoyer, and Qin Chen and the members of their laboratories for helpful discussions and use of equipment.

    FOOTNOTES

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

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

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.C100039200

    ABBREVIATIONS

The abbreviations used are: PGF2alpha , prostaglandin F2alpha ; Tcf, T-cell factor; Lef, lymphoid enhancer factor; DAPI, 4',6-diamidino-2-phenylindole; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK-3beta , glycogen synthase kinase-3beta ; APC, adenomatous polyposis coli; NSAID, nonsteroidal anti-inflammatory drug.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.