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
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ABSTRACT |
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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 F2 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 F2 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 PGF2 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
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 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/ 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
PGF2 (PGF2
) activates the small G-protein Rho, leading to
morphological changes consisting of cell rounding and the formation of
cell aggregates. Following the removal of PGF2
, 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 PGF2
leads to a subcellular reorganization of
-catenin, a decrease in the
phosphorylation of cytoplasmic
-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/
-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
(PGF2
)1
stimulates phosphoinositide turnover to a similar extent in cells expressing these isoforms (1). In addition, stimulation of FPA- or FPB-expressing cells with
PGF2
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 PGF2
, 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/
-catenin-mediated transcriptional activation is elevated 16 h after an initial 1-h treatment of FPB-expressing cells
with PGF2
. This transcriptional activation is not
observed in FPA-expressing cells and suggests that
FPB-expressing cells remain rounded because of activation of a Tcf/
-catenin signaling pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and were rapidly washed, fixed,
and incubated with a 1:1000 dilution of a mouse monoclonal antibody to
-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).
-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
-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).
-catenin and GAPDH primer pairs were
exactly according to Rezvani and Liew (8). Product sizes were 521 base
pairs for
-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
-catenin and GAPDH mRNA.
-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 PGF2
. 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
(panels b and d). It can be
appreciated that in both FPA- and FPB-expressing cells treatment with PGF2
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 PGF2
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
-catenin in HEK cells stably
expressing either the FPA or FPB isoforms following treatment with 1 µM PGF2
.
Although the effects on E-cadherin localization were not apparent (data
not shown), Fig. 1B shows that PGF2
treatment
resulted in a marked accumulation of
-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 PGF2
(Fig. 1A), indicating that the
process of cell rounding itself was not responsible for the increased
contiguous accumulation of
-catenin in the
FPB-expressing cells.
View larger version (64K):
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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 PGF2 (panels b and
d) for 1 h at 37 °C. B,
-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, -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
-catenin
expression following treatment of FPA- and
FPB-expressing cells with PGF2
. Fig. 2A shows that the expression
of
-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
PGF2
caused a slight increase the levels of cytosolic
-catenin in both the FPA- and FPB-expressing cells but had little effect on the levels of
-catenin in the particulate fraction. Reverse transcription (RT) followed by polymerase chain reaction (PCR) was used to determine whether there were any
differences in
-catenin mRNA levels under these same
experimental conditions. Fig. 2B shows that
-catenin and
GAPDH mRNA levels were the same for both cell lines and were not
affected by PGF2
, indicating that the observed
differences in
-catenin expression appear to be the result of
changes in translation and/or protein turnover.
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Serine/threonine phosphorylation of -catenin by glycogen synthase
kinase-3
(GSK-3
) marks
-catenin for degradation and is a
critical factor in the regulation of its signaling activity (12, 13).
Thus, under most conditions cytosolic
-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
-catenin following treatment of
either FPA- or FPB-expressing cells with
PGF2
. Fig. 3 shows that in
FPA-expressing cells the vehicle control levels of
cytosolic
-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 PGF2
, the levels of cytosolic
-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
-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-3
activity and tight coupling to
the elevated levels of cytoplasmic
-catenin. After treatment of
FPB-expressing cells with PGF2
there is a
further 2-fold increase in cytosolic
-catenin (lane d, middle
panel) but an unexpected 12-fold decrease in
phosphorylation (lane d, upper panel). The ratio of
phosphorylated to total
-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
-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
-catenin in FPB-expressing cells following treatment with PGF2
(lane d, bottom
panel) as compared with FPA-expressing cells
(lane b, bottom panel).
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Following nuclear translocation, -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 PGF2
might represent a
-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
PGF2
. 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
PGF2
in either cell line (data not shown). However, as
shown in Fig. 4A, the
morphological effects of PGF2
on
FPB-expressing cells persist long after its removal. Thus, when cells are examined 16 h after an initial 1-h treatment with PGF2
(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
PGF2
-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 PGF2
binding or in its removal during the washout
procedure (6).
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DISCUSSION |
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We show that FPB-expressing cells differ in several
important regards from FPA-expressing cells in terms of
their potential for activation of Tcf/-catenin-mediated signaling.
First, FPB-expressing cells show
PGF2
-stimulated accumulation of
-catenin at their contiguous cell boundaries that is not evident in
FPA-expressing cells. Second, although both FPA
and FPB-expressing cells show PGF2
-stimulated increases in cytosolic
-catenin, in
FPA-expressing cells this is accompanied by increased
-catenin phosphorylation and in FPB-expressing cells by
decreased
-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
-catenin. Thus, it is
possible that the agonist-stimulated accumulation of
-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-3
by Akt kinase is
inhibitory (18) and could lead to the decreased phosphorylation of
-catenin found in agonist-treated FPB cells.
Recently Meigs et al. (19) reported that constitutively
active mutants of G12 and
G
13 interact with E-cadherin resulting in a
release of
-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/
-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/
-catenin signaling was observed only for
cells expressing the FPB isoform.
We believe that activation of Tcf/-catenin signaling by
PGF2
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 PGF2
, and following its removal the cells
revert back to their original morphology. In contrast, although shape
change in FPB-expressing cells is initiated by
PGF2
, its maintenance is independent of further
PGF2
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 PGF2 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 PGF2
(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 PGF2
.
Another condition that might involve the FPB isoform or a
homologue is in colorectal cancer. It is known that aberrant activation of Tcf/-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 PGF2
would decrease
Tcf/
-catenin signaling by FPB prostanoid receptors. This
conclusion is supported by animal models of skin carcinogenesis in
which PGF2
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/
-catenin signaling. The possible role of FPB
receptors in these and other physiological processes is intriguing and
awaits future studies.
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ACKNOWLEDGEMENTS |
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We thank Ron Heimark, Pat Hoyer, and Qin Chen and the members of their laboratories for helpful discussions and use of equipment.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are:
PGF2, prostaglandin F2
;
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-3
, glycogen synthase
kinase-3
;
APC, adenomatous polyposis coli;
NSAID, nonsteroidal
anti-inflammatory drug.
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