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INTRODUCTION |
The EP2 and EP4 prostanoid receptors are
two of the four primary receptor subtypes for prostaglandin
E2
(PGE2).1 Both of
these receptors couple to G
s and can activate adenylyl cyclase resulting in the increased formation of intracellular cyclic
3,5-adenosine monophosphate (cAMP). Prior to the molecular cloning of
these receptors it was thought that the stimulation of adenylyl cyclase
by PGE2 was mediated by a single receptor subtype that was
defined pharmacologically as the EP2 subtype (1). Thus, the
first adenylyl cyclase stimulatory EP receptor to be cloned was thought
to be the EP2 subtype, but it was subsequently redefined as
the EP4 subtype (2) when a second adenylyl cyclase stimulatory EP receptor was cloned, which had the expected pharmacology of the EP2 subtype (3). Surprisingly the EP2
and EP4 receptors encoded by these cDNAs only shared
~30% amino acid homology and were really no more related to each
other than to other prostanoid receptor subtypes (4). In fact, the
EP2 receptor shows a closer phylogenetic relationship to
the DP and IP receptors than it does to the EP4 receptor.
The human EP4 receptor is larger than the human
EP2 (488 amino acids versus 358), which is
almost entirely due to a significantly longer carboxyl-terminal domain
(155 versus 34 amino acids).
Recently we have shown that PGE2 stimulation of the
EP2 receptors activates a T-cell factor (Tcf) signaling
pathway by a mechanism that mainly involves the activation of
cAMP-dependent protein kinase (PKA) (5). PGE2
stimulation of the EP4 receptors also activates Tcf
signaling, but the mechanism is more complex and involves the
activation of phosphatidylinositol 3-kinase (PI3K) (5). The involvement
of PI3K with EP4 receptor signaling has also been suggested
in studies of human colorectal carcinoma cells (6). Thus,
PGE2 stimulation of endogenous EP4 receptors in LS-174 cells increased cellular proliferation and motility through a
PI3K-dependent pathway. Given the known role of PI3K in
carcinogenesis, it is interesting that a recent study of knockout mice
suggested the potential involvement of EP4 receptors in
colon cancer (7).
Early growth response factor-1 (EGR-1) is a member of the zinc finger
family of transcription factors and plays a key role in cell growth and
differentiation. It is recognized as an immediate early gene product
that regulates the expression of a number of downstream genes, such as
tumor necrosis factor-
(TNF-
), interleukin-2, and p53 (8). The
induction of EGR-1 expression is known to involve members of the family
of mitogen-activated protein kinases (MAPKs). For example, the
induction of EGR-1 expression by growth hormone was shown to depend on
the phosphorylation and activation of extracellular signal-regulated
kinases (ERKs), but not on the activation of either Jun N-terminal
kinase (JNK) or p38 MAPK (9). Similarly the calmodulin antagonist,
trifluoroperazine, induced EGR-1 expression through the activation of
ERKs and the downstream transcription factor, Elk-1 (10).
Previous studies have also shown regulation of EGR-1 expression by
protein kinase C (PKC), possibly involving members of the family of
prostanoid receptors. For example, in Swiss 3T3 fibroblasts increases
in PGE2 were associated with a PKC-dependent
increase in EGR-1 mRNA expression (11). Likewise, in mouse MC3T3
osteoblastic cell lines PGE2 was found to increase EGR-1
mRNA expression. This increase was also PKC-dependent,
but did not appear to involve PKA since the induction of EGR-1 mRNA
expression was not observed following stimulation with forskolin, an
agent which increases the formation of intracellular cAMP (12). These
findings would appear to exclude the participation of the adenylyl
cyclase stimulatory EP2 and EP4 receptors in
the induction of EGR-1 mRNA expression. However, because neither of
these studies included pharmacological characterizations, the
conclusions that can be drawn are limited with respect to the specific
prostanoid receptor subtypes mediating these effects. We now show that
the EP4 prostanoid receptor, but not the EP2,
can induce the functional expression of EGR-1 through a signaling
pathway involving the sequential activation of PI3K and the ERKs. These
results further strengthen the involvement of PI3K with EP4
receptor signaling and clearly differentiate the signaling potential of
the EP2 and EP4 receptors.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Cell lines stably expressing the
EP2 or EP4 receptors were prepared using
HEK-293 EBNA cells and the mammalian expression vector pCEP4
(Invitrogen) as previously described (5). Cells were maintained in
Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal
bovine serum, 250 µg/ml geneticin, 100 µg/ml gentamicin, and 200 µg/ml hygromycin B.
Western Blotting--
Sixteen hours prior to the immunoblotting
experiments, cells were switched from their regular culture medium to
Opti-MEM (Invitrogen) containing 250 µg/ml geneticin and 100 µg/ml
gentamicin. Cells were then incubated at 37 °C with this same media
containing 1 µM PGE2 for the times indicated
in the figures. In some cases cells were pretreated with either vehicle
(0.1% Me2SO) or 100 nM wortmannin (Sigma) for
15 min or 10 µM PD98059 (Calbiochem) for 10 min at
37 °C. Cells were scraped into a lysis buffer consisting of 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 1% Nonidet P-40, 0.5% sodium
deoxycholate, 10 mM sodium fluoride, 10 mM
disodium pyrophosphate, 0.1% SDS, 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate,
10 µg/ml leupeptin, and 10 µg/ml aprotinin and transferred to
microcentrifuge tubes. The samples were rotated for 30 min at 4 °C
and were centrifuged at 16,000 × g for 15 min.
Aliquots of the supernatants containing 20~100 µg of protein were
electrophoresed on 7.5 or 10% SDS-polyacrylamide gels and transferred
to nitrocellulose membranes as described previously (5). Membranes were
incubated in 5% nonfat milk for 1 h and were then washed and
incubated for 16 h at 4 °C with primary antibodies using the
following conditions, which varied according to the antibody being
used. For the ERKs, incubations were done in 3% nonfat milk containing
either a 1:1,000 dilution of antiphospho-ERK1/2 antibody (9106, Cell
Signaling); or mixture of a 1:500 dilution of anti-ERK1 antibody and a
1:10,000 dilution of anti-ERK2 antibody (sc-93 and sc-154, Santa Cruz
Biotechnology). For JNK, incubations were done in 3% nonfat milk
containing a 1:1,000 dilution of antiphospho-JNK antibody (9255, Cell
Signaling) or 0.1% nonfat milk containing a 1:1,000 dilution of
anti-JNK antibody (9252, Cell Signaling). For p38 MAPK, incubations
were done in 5% bovine serum albumin containing a 1:1,000 dilution of
antiphospho-p38 MAPK antibody (9211, Cell Signaling); or 1% nonfat
milk containing a 1:1,000 dilution of anti-p38 MAPK antibody (sc-535-G,
Santa Cruz Biotechnology). For EGR-1, incubations were done in 3%
nonfat milk containing a 1:1,000 dilution of anti-EGR-1 antibody
(sc-110, Santa Cruz Biotechnology). After incubating with the primary
antibody, membranes were washed three times and incubated for 1 h
at room temperature with a 1:10,000 dilution of the appropriate
secondary antibodies conjugated with horseradish peroxidase using the
same conditions as described above for each of the primary antibodies.
After washing three times, immunoreactivity was detected by
chemiluminescence as described previously (5). To ensure equal loading
of proteins, the membranes were stripped and re-probed with appropriate
antibodies under the same conditions as described above.
Electrophoresis Mobility Shift Assay--
Cells, grown in 10-cm
plates, were pretreated with either vehicle (0.1% Me2SO)
or 10 µM PD98059 for 10 min at 37 °C followed by
treatment with either vehicle or 1 µM PGE2
for 60 min at 37 °C. Cells were washed, trypsinized, and centrifuged
at 500 × g for 1 min, and the supernatant was removed.
The pellet was resuspended in approximately three times the original
pellet volume using buffer A, containing 10 mM HEPES-KOH
(pH 7.9), 1.5 mM MgCl2, 10 mM KCl,
and 0.5 mM dithiothreitol. The sample was transferred into
a new tube, placed on ice for 15 min, and then centrifuged at
16,000 × g for 30 s at 4 °C. The supernatant
was discarded, and the pellet was resuspended in ~2 volumes of buffer
A with seven rapid strokes of a 1-ml syringe with a 25-gauge needle. The sample was centrifuged at 16,000 × g for 5 min at
4 °C, and the supernatant was transferred to another tube (S100
fraction). The pellet was rinsed with buffer A and resuspended with
buffer C, consisting of 20 mM HEPES-KOH (pH 7.9), 25%
glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, and 0.5 mM
dithiothreitol. After a 30-min incubation on ice, the sample was
centrifuged at 16,000 × g for 5 min at 4 °C, and
the supernatant (nuclear extract) was dialyzed for 2 h using a
3,000 molecular weight cutoff membrane (Pierce) against buffer D
containing 20 mM HEPES-KOH (pH 7.9), 20% glycerol, 20 mM KCl, 2 mM MgCl2, 0.2 mM EDTA, and 0.5 mM dithiothreitol.
A double-stranded DNA oligonucleotide probe was designed corresponding
to the GC box-1 and GC box-2 sequences in the human prostaglandin
E2 synthase gene promoter (13). The sense strand of this
oligonucleotide sequence is 5'-GTGGGGCGGGGCGTGGGCGGTGCT-3', where the underlined sequence represents the consensus binding site for
EGR-1 (8). The sense and antisense oligonucleotides were annealed and
labeled with [
-32P]ATP (Amersham Biosciences) using T4
kinase (Invitrogen). The reaction mixture was applied to a spin column
to remove free [
-32P]ATP and run on an 8% native
polyacrylamide gel to separate any single-stranded DNA from the
double-stranded probe. The binding reaction was performed for 15 min at
room temperature using ~10,000 cpm of the probe and 10 µg of
nuclear extract in 15 µl of a buffer containing 4% Ficoll 400, 1 mM EDTA (pH 8.1), 1 mM dithiothreitol, 4 mg/ml
bovine serum albumin, 0.1 M KCl, and 2 µg of
poly-(deoxyinosinic-deoxycytidylic) (Sigma). The mobility shift assay
was done with a 5% native polyacrylamide gel followed by drying and
visualization by autoradiography using Hyperfilm MP (Amersham
Biosciences). The supershift assay was performed using 3 µg of the
anti-EGR-1 antibody in which the antibody was incubated with the probe
and the nuclear extract for an additional 15 min following the initial
binding reaction.
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RESULTS |
PGE2 Stimulated Phosphorylation of ERKs in
EP4 Receptor-expressing HEK Cells--
We have previously
reported that EP4 prostanoid receptor can activate Tcf
signaling by a mechanism involving PI3K (5). Further characterization
of this interaction between the EP4 receptor and PI3K was
of interest given the important role of PI3K in both normal and
aberrant cell signaling pathways. One of the downstream effectors of
PI3K signaling is the activation of members of the MAPKs. To determine
if EP4 receptors could potentially activate MAPK signaling,
we examined the ability of PGE2 to stimulate the phosphorylation of three MAPKs in HEK-293 cells stably transfected with
the human EP2 and EP4 prostanoid receptors. For
these experiments untransfected HEK cells and cells stably expressing
the EP2 and EP4 receptors were incubated with 1 µM PGE2 for various times and were then
immunoblotted with antibodies to the phosphorylated and
nonphosphorylated forms of ERK1 and ERK2, p54 JNK, and p38 MAPK. As
shown in panel A of Fig. 1,
treatment with PGE2 resulted in a
time-dependent phosphorylation of ERKs 1 and 2 in
EP4-expressing cells, but not in EP2-expressing
cells or the control HEK cells. The PGE2-stimulated
phosphorylation of ERKs in EP4 cells was maximal at 5-10
min and by 60 min had nearly returned to the original, unstimulated,
levels (0 min). These same blots were stripped and re-probed with
antibodies to the nonphosphorylated forms of ERK1 and ERK2, and as
shown in panel B of Fig. 1 nearly identical amounts of ERKs
1 and 2 were present throughout the time course of treatment and among
the three cell lines.

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Fig. 1.
Immunoblots of the time course of
PGE2-stimulated phosphorylation of ERKs, JNK, and p38 MAPK
in untransfected HEK-293 cells and in HEK-293 cells stably transfected
with either the human EP2 or EP4 prostanoid
receptors. Cells were incubated with 1 µM
PGE2 for the indicated times and were subjected to
immunoblot analysis as described under "Experimental Procedures."
Panel A, immunoblotting with antibodies against phospho-ERKs
1 and 2 (p-ERK1/2). Panel B, the blots
shown in panel A were stripped and re-probed with antibodies
against ERKs 1 and 2 (ERK1/2). Panel
C, immunoblotting with antibodies against phospho-JNK (p-p54
JNK). Panel D, the blots shown in panel C
were stripped and re-probed with antibodies against p54 JNK.
Panel E, immunoblotting with antibodies against phospho-p38
MAPK (p-p38 MAPK). Panel F, the blots shown in
panel E were stripped and re-probed with antibodies against
p38 MAPK. Results are representative of at least three independent
experiments with each antibody and condition.
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Panels C-F of Fig. 1 show the corresponding results for p54
JNK and p38 MAPK, respectively. In contrast to the results obtained with the ERKs, there were no substantive differences between the EP2- and EP4-expressing cells with respect to
the phosphorylation of either p54 JNK (panel C) or p38 MAPK
(panel E). In both EP2- and
EP4-expressing cells there was significant phosphorylation of p54 JNK even at the 0 time point, which was not observed in the
control HEK cells (panel C). This appears to suggest some constitutive effect of EP2 and EP4 receptors on
the phosphorylation of p54 JNK. In both EP2- and
EP4-expressing cells there was a PGE2-dependent induction of the phosphorylation
of p38 MAPK, which was maximal at ~15 min and which nearly returned
to the original, unstimulated, levels by 60 min (panel E).
As with the ERKs, nearly identical amounts of p54 JNK (panel
D) and p38 MAPK (panel F) were present throughout the
time course of treatment and among the three cell lines.
The PI3K inhibitor, wortmannin, was used to examine the potential
involvement of PI3K on the phosphorylation of ERKs 1 and 2 following
PGE2 stimulation of the EP4 receptor. For these
experiments EP2- and EP4-expressing cells were
pretreated with either vehicle or 100 nM wortmannin for 15 min followed by treatment with either vehicle or 1 µM
PGE2 for 10 min. The cells were then subjected to
immunoblot analysis using antibodies to the phosphorylated and
nonphosphorylated forms of the ERKs. As shown in the upper panel of Fig. 2 stimulation with
PGE2 in the absence of wortmannin pretreatment resulted in
the phosphorylation of ERKs 1 and 2 in EP4 expressing
cells, but not in EP2-expressing cells. However, following
pretreatment with wortmannin there was a complete blockade of
PGE2-stimulated phosphorylation of ERKs 1 and 2 in
EP4-expressing cells, suggesting the direct involvement of
PI3K in this process. The lower panel of Fig. 2 shows that
nearly identical amounts of ERKs 1 and 2 were present under all
conditions in both cell lines.

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Fig. 2.
The effects of wortmannin on
PGE2-stimulated phosphorylation of ERKs in HEK-293 cells
transfected with either the EP2 or EP4
prostanoid receptors. Cells were pretreated with either vehicle or
100 nM wortmannin (wort) for 15 min followed by
treatment with either vehicle (v) or 1 µM
PGE2 (P) for 10 min at 37 °C and were
subjected to immunoblot analysis as described under "Experimental
Procedures." Upper panel, immunoblotting with antibodies
against phospho-ERKs 1 and 2 (p-ERK1/2). Lower panel, the
blots shown in the upper panel were stripped and re-probed
with antibodies against ERKs 1 and 2 (ERK1/2).
Results are representative of three independent experiments with each
antibody and condition.
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Induction of EGR-1 Expression by PGE2 in
EP4-expressing Cells but Not in EP2-expressing
Cells--
Given the phosphorylation of ERKs 1 and 2 following
PGE2 stimulation of the human EP4 prostanoid
receptor, it was of interest to explore the possible modulation of
downstream of effectors that could reflect this selective activation of
the ERK signaling pathway. One such effector that is known to be
induced following the activation of ERKs is the early growth response
factor-1 (EGR-1). Immunoblot analysis was, therefore, used to examine
the time course of expression of EGR-1 following treatment of
EP2- and EP4-expressing cells with 1 µM PGE2. As shown in the upper
panels of Fig. 3, there was a modest
induction of EGR-1 expression in EP4-expressing cells
following 30 min of incubation with PGE2, which was
markedly increased at the 60-min time point. On the other hand, in
EP2-expressing cells the expression of EGR-1 was barely
detectable even at the 60-min time point. To check for the equal
loading of protein these blots were stripped and re-probed with
antibodies to ERKs 1 and 2. As shown in the lower panels of
Fig. 3 nearly identical amounts of ERKs 1 and 2 were present in both
cell lines and throughout the time course of treatment with
PGE2.

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Fig. 3.
Immunoblots of the time course of
PGE2-stimulated expression of early growth response
factor-1 (EGR-1) in HEK-293 cells transfected with either the
EP2 or EP4 prostanoid receptors. Cells
were incubated with 1 µM PGE2 for the
indicated times and were subjected to immunoblot analysis as described
under "Experimental Procedures." Upper panels,
immunoblotting with antibodies against EGR-1. Lower panels,
the blots shown in the upper panels were stripped and
re-probed with antibodies against ERKs 1 and 2 (ERK1/2). Results are
representative of three independent experiments with each antibody and
condition.
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Induction of EGR-1 Expression by PGE2 in
EP4-expressing Cells Involves Activation of PI3K and the
ERKs--
Pretreatment of EP2- and
EP4-expressing cells with either the PI3K inhibitor,
wortmannin, or the MAPK/ERK kinase inhibitor, PD98059, was used to
examine the potential involvement of these kinases on the
PGE2 induced expression of EGR-1. As shown in the upper panel of Fig.
4A, the robust induction of
EGR-1 expression in EP4-expressing cells following 60 min
of exposure to 1 µM PGE2 was completely
blocked by pretreatment with wortmannin. EGR-1 expression in
EP2-expressing cells was not detectable either before or
after treatment with PGE2, or following pretreatment with
wortmannin. The lower panel of Fig. 4A shows that
nearly equal amounts of ERKs 1 and 2 were present under all conditions
and indicates that the observed differences in EGR-1 expression are not
because of differences in overall protein expression or in the amount
of protein loaded on the gels.

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Fig. 4.
Immunoblots of the effects of wortmannin
(A) and PD98059 (B) on
PGE2-stimulated expression of early growth response
factor-1 (EGR-1) in HEK-293 cells transfected with either the
EP2 or EP4 prostanoid receptors. Cells
were pretreated with either vehicle or 100 nM wortmannin
(wort) for 15 min (A) or were pretreated with
either vehicle or 10 µM PD98059 for 10 min (B)
followed by treatment with either vehicle (v) or 1 µM PGE2 (P) for 60 min at
37 °C. The cells were then subjected to immunoblot analysis as
described under "Experimental Procedures." Upper panels,
immunoblotting with antibodies against EGR-1. Lower panels,
the blots shown in the upper panels were stripped and
re-probed with antibodies against ERKs 1 and 2 (ERK1/2). Results are
representative of three independent experiments with each antibody and
condition.
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The MAPK/ERK kinase inhibitor, PD98059, was then used to explore
whether this specific induction of EGR-1 expression in
EP4-expressing cells involved the sequential activation of
PI3K and the ERKs. For these experiments EP2- and
EP4-expressing cell lines were pretreated with either
vehicle or 10 µM PD98059 for 10 min followed by treatment
with either vehicle or 1 µM PGE2 for 60 min.
As shown in the upper panel of Fig. 4B, in the
absence of pretreatment with PD98059 there was a strong
PGE2-mediated induction of EGR-1 expression in
EP4-expressing cells that was completely absent in
EP2-expressing cells. Following pretreatment with PD98059, however, there was a complete block of PGE2-mediated EGR-1
expression in EP4-expressing cells. To confirm equal
loading of protein, this blot was stripped and re-probed with
antibodies to ERKs 1 and 2. The lower panel of Fig.
4B shows that nearly identical amounts of ERKs 1 and 2 were
present under all conditions in both cell lines. Together these results
strongly suggest that the sequential activation of PI3K and the ERKs is
required for the induction of EGR-1 expression by PGE2 in
EP4-expressing cells.
Induction of EGR-1 Expression in EP4-expressing Cells
Leads to Functional Interactions with a DNA Promoter Sequence
Containing an EGR-1 Binding Site--
The potential for
PGE2 to stimulate EGR-1 mediated transcriptional activation
was examined in EP2- and EP4-expressing cells using electrophoresis mobility shift assays. The probe used for these
experiments consisted of an oligonucleotide designed from the promoter
region of the human gene for prostaglandin E2 synthase containing a consensus binding site for EGR-1 (13). EP2-
and EP4-expressing cells were pretreated with either
vehicle or 10 µM PD98059 for 10 min followed by treatment
with either vehicle or 1 µM PGE2 for 60 min.
In Fig. 5 a comparison of the "free" lane with the other lanes shows that the probe migrated with relative mobilities of 0.26 and 0.40 under all conditions in which a nuclear extract was present. Following treatment with PGE2,
however, there was a selective shift in the mobility of the probe in
EP4-expressing cells, but not in EP2-expressing
cells, yielding a unique band with a relative mobility of 0.16. The
identity of the protein causing this shift was investigated in a
supershift assay by an additional incubation with antibodies to EGR-1.
As shown in the last two lanes of Fig. 5, incubation with the
antibodies to EGR-1 diminished the intensity of the 0.16-band following
treatment with PGE2 and resulted in the appearance of a new
band with a mobility of 0.08. This supershift confirms the interaction
of the probe with the EGR-1 following the treatment of EP4
expressing cells with PGE2. Fig. 5 also shows that
pretreatment of EP4-expressing cells with the MAPK/ERK
kinase inhibitor, PD98059, prevented the PGE2 induced
mobility shift and appearance of 0.16 band. Thus, as predicted from our
previous experiments with PD98059, the PGE2-induced functional expression of EGR-1 involves the activation of a MAPK/ERK signaling pathway.

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Fig. 5.
Electrophoresis mobility shift assay and
supershift assay of EP2 and EP4 expressing HEK
cells following treatment with PGE2 in the absence or
presence of pretreatment with PD98059. Cells were pretreated with
either vehicle or 10 µM PD98059 for 10 min followed by
treatment with either vehicle (v) or 1 µM
PGE2 (P) for 60 min at 37 °C. The mobility
shift assay was done as described under "Experimental Procedures"
with a 32P-labeled oligonucleotide probe containing a
consensus binding site for early growth response factor-1
(EGR-1). The supershift assay was done by incubating the
probe and nuclear extract for an additional 15 min with antibodies to
EGR-1 (EGR-Ab). Relative mobility is defined as the distance
traveled from the origin by the bound probe, normalized to
the distance traveled by the free probe. Results are
representative of four independent experiments.
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DISCUSSION |
We have previously shown that stimulation of EP2
prostanoid receptors by PGE2 can activate a Tcf signaling
pathway by a mechanism that mainly involves the activation of PKA (5).
PGE2 stimulation of EP4 prostanoid receptors
can also activate a Tcf signaling pathway, but the mechanism is more
complex and involves the activation of PI3K (5). We now show that
PGE2 stimulation of the EP4 receptor activates
an additional signaling pathway involving PI3K. Thus, PGE2
treatment of cells expressing EP4 receptor, leads to a
PI3K-dependent phosphorylation of ERKs 1 and 2 followed by
a de novo increase in the functional expression of EGR-1.
The activation of this PI3K signaling cascade was unique for the
EP4 receptor and was not observed in cells expressing
EP2 receptors.
One of the ways in which the EP2 and EP4
receptors are known to differ is in the characteristics of their
agonist induced desensitization and internalization. Thus,
EP4 receptors undergo rapid, PGE2-mediated
desensitization (14) and internalization (15); whereas EP2
receptors do not. Since the internalization of some G-protein-coupled
receptors is associated with a transactivation of the MAPK pathway (16,
17), it may be supposed that such a mechanism could explain the present
findings of selective activation of ERK signaling by the
EP4 receptors. However, in a previous study using human
EP4 receptors expressed in HEK-293 cells, it was found that
the phosphorylation of ERKs 1 and 2 was independent of
PGE2-mediated receptor internalization (18).
Another way in which the EP2 and EP4 prostanoid
receptors appear to differ is in their ability to stimulate
intracellular cAMP formation. Thus, despite nearly identical levels of
receptor expression, the maximal levels of PGE2-stimulated
cAMP formation in EP4-expressing cells was only 20% of the
level obtained in EP2-expressing cells (5). However, under
the same conditions the ability of PGE2 to stimulate Tcf
signaling was ~50% greater in EP4-expressing cells as
compared with EP2-expressing cells (5). This indicates that
the lower amounts of PGE2-stimulated cAMP formation in
EP4-expressing cells is because of less efficient coupling
to this pathway and not because of an overall impairment in the
signaling potential of these receptors.
The more efficient coupling of the EP2 receptor to
intracellular cAMP formation; however, may be significant with respect to the present findings. Thus, it has been reported that the
phosphorylation of Raf kinase by PKA inhibits the activity of Raf
kinase and subsequently decreases Raf mediated MAPK signaling (19, 20).
In EP2-expressing cells, therefore, a robust activation of
PKA may inhibit Raf kinase and block the phosphorylation and activation
of ERKs.
Our findings of a PGE2-mediated induction of EGR-1
expression by the EP4 receptor is interesting light of
recent studies with knockout mice that show a potential involvement of
the EP4 receptor with colon cancer and rheumatoid
arthritis. For example EP4 knockout mice, but not
EP2 knockout or control mice, show a reduced formation of
preneoplastic lesions following treatment with azoxymethane, a known
colon carcinogen (7). EP4 knockout mice, but not
EP2 knockout or control mice, also show a significantly
decreased incidence and severity of collagen antibody induced
arthritis, an animal model of rheumatoid arthritis (21). In addition,
in both colon cancer and rheumatoid arthritis prostaglandin levels are
elevated and both conditions benefit to some extent by treatment with
inhibitors of the cyclooxygenases. In colon cancer, the expression of
cyclin D1, a key regulator of cell cycle progression, is known to be
regulated by Tcf signaling (22). However, it has also been reported
that the expression of cyclin D1 is regulated by EGR-1 through a PI3K-
and ERK-dependent pathway (23). Furthermore it has been
shown that PGE2 synthase is up-regulated by the binding of
EGR-1 to the promoter region of the mouse gene encoding
PGE2 synthase (13). Signaling through an EP4
receptor would have the potential, therefore, to increase the
expression of cyclin D1 and PGE2 synthase through a
PGE2-mediated induction of EGR-1 expression. Since the
product of PGE2 synthase is PGE2 itself, the
potential for a positive feedback loop is obvious.
A similar situation could also explain the increased levels of
PGE2 observed in rheumatoid arthritis. However, in this
disease in addition to an up-regulation of the expression of
PGE2 synthase, signaling through EP4 receptors
could also potentially up regulate levels of TNF-
(21) whose
expression is under the control of EGR-1 (24). Inhibitors of TNF-
,
such as etancercept and infliximab, are used therapeutically in the
treatment of rheumatoid arthritis because they slow the progression of
this disease (25). It is possible that the reduced incidence and
severity of collagen antibody induced arthritis in EP4
knockout mice (21) is related to the loss of EP4
receptor-mediated signaling through a PI3K/ERKs/EGR-1 pathway.
Recently we have shown that the FPB prostanoid receptor can
signal through a
-catenin/Tcf signaling pathway (26) and interact with PI3K (27); and thus, has potential to be involved in the pathophysiology of colon cancer. It has also been reported that EP1 receptors (28) and EP2 receptors (29) may
have roles in colon cancer. In addition, gene knockout studies with the
cyclooxygenases indicate that the synthesis of prostaglandins by these
enzymes contributes to the pathophysiology of colon cancer (30).
Interestingly, the expression of cyclooxygenase-2 in intestinal polyps
appears to be under a positive feedback through the EP2
receptor (29). Furthermore, it is clear from the findings of Sonoshita
et al. (29) that the effects of the cyclooxygenase-2
knockout on the number and size of intestinal polyps was greater than
the effects of the EP2 knockout and appear to require the
involvement of another prostanoid receptor. Potential involvement of
the EP4 receptor could work in concert with the
EP2 receptor to increase PGE2 levels by
increased expression of both cyclooxygenase-2 and PGE2
synthase. Our present findings with the EP4 receptors
further strengthen an association of the prostaglandins and their
receptors with cancer and inflammation; further knowledge of which
could have practical therapeutic benefits.