Induction of Adherent Activity in Mastocytoma P-815 Cells by
the Cooperation of Two Prostaglandin E2 Receptor
Subtypes, EP3 and EP4*
Noriyuki
Hatae,
Ayumi
Kita,
Satoshi
Tanaka,
Yukihiko
Sugimoto, and
Atsushi
Ichikawa
From the Department of Physiological Chemistry, Graduate School of
Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto
606, Japan
Received for publication, February 6, 2003, and in revised form, March 7, 2003
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ABSTRACT |
In this study, we investigated the role of
PGE2 in mouse mastocytoma P-815 cell adhesion to
extracellular matrix proteins (ECMs) in vitro. We report
that PGE2 accelerated ProNectin FTM (a
proteolytic fragment of fibronectin)-mediated adhesion, which was
abolished by addition of the GRGDS peptide, an inhibitor of the RDG
binding site of ProNectin FTM. We show that the cAMP level
and cAMP-regulated protein kinase (PKA) activity are critical mediators
of this PGE2 effect, because the cell-permeable cAMP
analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin
FTM and the pharmacological inhibitor of PKA, H-89, blocked
PGE2-mediated adhesion. Consistent with mRNA expression
of the Gs-coupled EP4- and Gi-coupled EP3-PGE
receptor subtypes, P-815 cell adhesion was accelerated by treatment
with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3
agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329
and sulprostone resulted in augmentation of both the cAMP level and
cell adhesion. The augmentation of EP3-mediated cAMP synthesis was
dose-dependent, without affecting the half-maximal
concentration for EP4-mediated Gs-activity, which was
inhibited by a Gi inhibitor, pertussis toxin. In
conclusion, these findings suggest that PGE2 accelerates
RGD-dependent adhesion via cooperative activation
between EP3 and EP4 and contributes to the recruitment of mast
cells to the ECM during inflammation.
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INTRODUCTION |
Differentiated mast cells
(MCs),1 which originate from
bone marrow stem cells, traffic throughout the circulation and adhere to the extracellular matrix (ECM) in various tissues. MCs are widely
distributed in tissues throughout the body, especially in connective
tissues, serosal cavities, and on mucosal surfaces under normal
physiological conditions. This characteristic distinguishes MCs from
other bone marrow-derived hematopoietic cells, such as basophils,
neutrophils, and eosinophils. MCs congregate around nerves, blood
vessels, and lymphatic vessels. MCs therefore interact with not only
the ECM but with other cells as well. As well known for rodent
connective tissue-typed MCs and mucosal-typed MCs, the biological
activity of MCs vary with their interactions with the ECM and other cells.
MCs are widely distributed along basement membranes, indicating that
MCs might adhere to laminin. Supporting this fact, mouse bone
marrow-derived mast cells (BMMC) have been reported to adhere to
laminin, when the cells were activated by phorbol 12-myristate 13-acetate (PMA) (1-3) or antigen-stimulated aggregation of Fc
RI (4). In addition to laminin, MCs can adhere to other matrix components
such as fibronectin (5) and vitronectin (6, 7). As with laminin, the
adherence of BMMC to fibronectin has been reported to occur through
activation with PMA or after aggregation of Fc
RI. These adherence
activities required calcium (3). In contrast to BMMC, the mouse PT18
cell line spontaneously adhered to laminin (1), and human skin mast
cells also spontaneously adhered to laminin and fibronectin (8). These
previous findings indicate that the interactions between MCs and matrix
components may depend on the cells involved and the kinds of stimuli.
PGE2, which is involved in inflammation section (9),
affects both differentiation and growth of MCs in vitro.
PGE2 enhances mast cell differentiation from cord blood
mononuclear cells (10) and in the fibroblast co-culture system (11).
Very recently, Dormond et al. (12) reported that a COX-2
inhibitor suppressed
V
3-dependent HUVEC spreading,
migration, and angiogenesis through Rac activation (12), and
PGE2 accelerated
V
3-mediated HUVEC responses in a
cAMP-dependent manner (13). However, no reports have
published the effects of PGE2 on MC adhesion to the
ECM.
The PGE2 receptors (EP) are comprised of four subtypes,
EP1, EP2, EP3, and EP4, which are coupled to different G proteins and
signal pathways (14, 15). Among these subtypes, EP2 and EP4 couple to
Gs, resulting in increases of intracellular cAMP concentrations (16, 17), while EP3 couples to Gi, causing a
decrease in cAMP levels (18-21). Very recently, we and other investigators have reported that activation of the
Gi-coupled EP3 receptor was able to augment adenylyl
cyclase activity via stimulation of a Gs-coupled receptor
(22, 23). We previously reported that the mouse EP3
receptor was
able to augment EP2-induced adenylyl cyclase activity in both EP2- and
EP3-transfected COS-7 cells (22). Southall and Vasko (23) showed that
the simultaneous depletion of rat EP3C and EP4 was essential to abolish
PGE2-stimulated cAMP production and neuropeptide release in
rat sensory neurons (23). These permissive actions between the two
subtypes may be involved in the events of a number of physiological
actions of PGE2 (15).
Here we extended the analyses of the effects of PGE2
on adhesion of mouse mastocytoma P-815 cells to the fibronectin
component in a PKA-dependent manner, and we examined the
correlation between cAMP levels and adhesion in P-815 cells to see
whether EP3 could augment EP4-induced cAMP synthesis.
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EXPERIMENTAL PROCEDURES |
Materials--
Sulprostone was a generous gift from Dr.
M. P. L. Caton of Rhone-Poulenc Ltd. ONO-AE1-259 and ONO-AE1-329 were
generous gifts from ONO Pharmaceuticals (Osaka, Japan). ProNectin
FTM (Pronectin-F) and ProNectin LTM
(Pronectin-L) were generous gifts from Sanyo Chemical Industries, Ltd.
(Kyoto, Japan). The GRGDS peptide was obtained from the PEPTIDE Institute (Osaka, Japan). Collagen I-coated plates were from BD PharMingen Labware, the 125I-labeled cAMP assay system from
Amersham Biosciences. Pertussis toxin (PT) from Seikagaku Kogyo (Tokyo,
Japan), [5,6,8,11,12,14,15-3H]PGE2 (164 Ci/mmol) from Amersham Biosciences and PGE2 from Cayman Chemical (Ann Arbor, MI). Rabbit polyclonal anti-A cyclase II (C-20)
antibody, rabbit polyclonal anti-A cyclase IV (C-20) antibody, and goat
polyclonal anti-A cyclase VII (M-20) antibody were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA).
Reverse Transcription-Polymerase Chain Reaction--
Mouse
mastocytoma P-815 cells (P-815 cells) were maintained in Fischer's
medium with 10% fetal bovine serum under humidified air containing 5%
CO2 at 37 °C. Total RNA was isolated from P-815 cells
using Isogen (Nippon Gene) according to the manufacturer's instructions. The reverse transcription (RT) reaction was performed using Molony murine leukemia virus reverse transcriptase (Invitrogen) in the presence of random hexamers. The polymerase chain reaction (PCR)
was performed with Taq DNA polymerase (TOYOBO) using the first strand template. The primers were designed to be selective for
the prostanoid receptors, and the sequences used were as follows: EP1
(base pairs (545 bp)), 5'-GCC ACT GAT CCA CGC GGC GCG CGT ATC TGT GGC
C-3' (forward) and 5'-CGA TGG CCA ACA CCA CCA ACA CCA GCA GGG-3'
(reverse); EP2 (604 bp), 5'-TTC ATA TTC AAG AAA CCA GAC CCT GGT GGC-3'
(forward) and 5'-AGG GAA GAG GTT TCA TCC ATG TAG GCA AAG-3' (reverse);
EP3 (608 bp), 5'-ATC CTC GTG TAC CTG TCA CAG CGA CGC TGG-3' (forward)
and 5'-TGC TCA ACC GAC ATC TGA TTG AAG ATC ATT-3' (reverse); EP4 (601 bp), 5'-GAC TGG ACC ACC AAC GTA ACG GCC TAC GCC-3' (forward) and 5'-ATG
TCC TCC GAC TCT CTG AGC AGT GCT GGG-3' (reverse); IP (602 bp), 5'-CCT
GCA GTG TTT GTG GCC TAT GCT CGA AAC-3' (forward) and 5'-CTG CTG TCT GGG
GCG ATG GCC TGA GTG AAG-3' (reverse); DP (604 bp), 5'-AAA GGA ACT GCT
GCC TGC CTC AGG CAA TCA-3' (forward) and 5'-GTT CTC AAG TTT AAA GGC TCC
ATA GTA CGC-3' (reverse); FP (603 bp), 5'-GCA TAG CTG TCT TTG TAT ATG
CTT CTG ATA-3' (forward) and 5'-GTG TCG TTT CAC AGG TCA CTG GGG AAT
TAT-3' (reverse); TP (607 bp), 5'-TGC CTT GTT GGA CTG GCG AGC CAC TGA
CCC-3' (forward) and 5'-CAG GTA GAT GAG CAG CTG GTG CTC TGT GGC-3'
(reverse). The thermal cycle programs used were as follows: EP1,
94 °C for 1 min and then 94 °C for 1 min, 72 °C for 3.5 min
for the first 10 cycles, 94 °C for 1 min, 69 °C for 2 min,
72 °C for 1.5 min for the next 10 cycles, 94 °C for 1 min,
66 °C for 2 min, 72 °C for 1.5 min for the last 15 cycles,
followed by 72 °C for 7 min; EP2, EP3, EP4 and DP, 94 °C for 1 min and then 30 cycles of 94 °C for 0.8 min, 60 °C for 0.7 min,
72 °C for 1.5 min, followed by 72 °C for 7 min; IP, 94 °C for
1 min and then 30 cycles of 94 °C for 0.8 min, 65 °C for 0.7 min,
72 °C for 1.5 min, followed by 72 °C for 7 min; FP and TP,
94 °C for 1 min and then 94 °C for 1 min, 72 °C for 3.5 min
for the first 10 cycles, 94 °C for 1 min, 67 °C for 2 min,
72 °C for 1.5 min for the next 10 cycles, 94 °C for 1 min, 63 °C for 2 min, 72 °C for 1.5 min for the last 15 cycles,
followed by 72 °C for 7 min. Samples were visualized on a 1.5%
agarose gel.
[3H]PGE2 Binding Assay--
P-815
cells were homogenized with a Potter-Elvehjem homogenizer in 20 mM Tris-HCl (pH 7.5), containing 10 mM
MgCl2, 1 mM EDTA, 20 µM
indomethacin, and 0.2 mM phenylmethylsulfonyl fluoride. After centrifugation at 250,000 × g for 20 min, the
pellet (membrane fraction) was washed, suspended in 10 mM
Mes-NaOH (pH 6.0) containing 10 mM MgCl2, and 1 mM EDTA, and was used for the
[3H]PGE2 binding assay. The membrane fraction
(50 µg) was incubated with 4 nM
[3H]PGE2 at 30 °C for 1 h, and
[3H]PGE2 binding to the membrane fraction was
determined as described previously (24). Nonspecific binding was
determined using a 2,500-fold excess of unlabeled PGE2 in
the incubation mixture.
Measurement of cAMP Formation--
Cyclic AMP levels in P-815
cells were determined as reported previously (25). P-815 cells (2 × 106 cells/assay) were suspended with 10 µM
indomethacin in HEPES-buffered saline containing 140 mM
NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM
KH2PO4, 11 mM glucose, and 15 mM HEPES (pH 7.4), and preincubated for 10 min at 37 °C.
Reactions were started by addition of test agents along with 500 µM 3-isobutyl-1-methyl-xanthine (Sigma). After incubation
for 10 min at 37 °C, reactions were terminated by the addition of
10% trichloroacetic acid. The level of cAMP was measured by
radioimmunoassay with an Amersham Biosciences cAMP assay system.
Expression of the Adenylyl Cyclase Subtypes--
Expression of
adenylyl cyclase II, IV, and VII were determined by immunoblot analysis
of whole cell detergent lysates of P-815 cells using rabbit polyclonal
anti-A cyclase II (C-20) antibody, rabbit polyclonal anti-A-cyclase IV
(C-20) antibody, and goat polyclonal anti-A cyclase (M-20) antibody.
P-815 cells (2 × 108 cells) were washed twice in PBS.
The cell pellet was suspended in 1 ml of radioimmune precipitation
assay buffer containing 30 mM HEPES-NaOH (pH 7.3), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS,
and incubated for 2 h at 4 °C. For protection against proteolytic degradation, a mixture of protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 100 µM
benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml
E-64, and 1 µg/ml pepstatin A) was added. The mixture was then
centrifuged at 10,000 × g for 15 min at 4 °C. The
resulting supernatant was dissolved in Laemmli buffer and heated for 30 min at 60 °C. Aliquots (500 µg of protein) were then subjected to
SDS-PAGE (7.5%) as described by Laemmli (26), and the separated
proteins were transferred electrophoretically onto a polyvinylidene
difluoride membrane in 25 mM Tris base containing 40 mM 6-aminohexanoic acid, 0.02% SDS, and 20% methanol at
room temperature for 30 min at 15 V. The membrane fraction was rinsed in Tris-buffered saline (TBS) containing 20 mM Tris-HCl (pH
7.5) and 150 mM NaCl, and then preincubated overnight in
TBS containing 5% nonfat milk at 4 °C. The membrane fraction was
then incubated with anti-A cyclase antibodies (A cyclase II, IV, 1:200;
A cyclase VII, 1:100) in TBS containing 5% nonfat milk for 1 h at
37 °C. After washing three times with TTBS (TBS containing 0.05%
Tween 20) at room temperature, the membrane fraction was incubated with peroxidase-conjugated anti-rabbit or -goat IgG in TTBS for 1 h at
room temperature, and then detected with the ECL Western blot detection reagent.
Cell Adhesion Assay--
P-815 cells were suspended in
Fischer's medium with test agents along with 10% fetal bovine serum
and 10 µM indomethacin at a density of 5 × 105 cells per ml, and 1 ml per well was dispensed into
12-well tissue culture plates. The 12-well tissue culture plates were
precoated with 10 µg/ml of Pronectin-F or Pronectin-L for 5 min at
room temperature. Nonspecific binding was determined by using wells precoated with bovine serum albumin (3%) for 1 h at room
temperature. The P-815 cells were incubated for 8 h at 37 °C in
a CO2-humidified atmosphere and non-adherent P-815 cells
were suspended in 2 ml of PBS (non-adherent cells). The adherent P-815
cells were treated with PBS containing 0.02% EDTA and 0.25% trypsin
at 37 °C for 30 min, and the collected cells were suspended in 2 ml
of PBS containing 0.02% EDTA and 2% fetal bovine serum (adherent
cells). The numbers of non-adherent and adherent cells were counted
using the COULTER Z1 cell counter (Beckman Coulter). The percentage of
cell adhesion was calculated with the following formula shown in
Equation 1.
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(Eq. 1)
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RESULTS |
P-815 Cell Adhesion to Pronectin-F-coated Plates--
When P-815
cells were cultured in Fischer's medium containing 10% fetal bovine
serum together with 10 µM indomethacin, almost no
attachment to non-coated plates was observed. However, after incubation
with 1 µM PGE2 for 8 h, ~11% of P-815
cells were retained on the non-coated plates as adhered cells, after
being washed three times with PBS containing 0.02% EDTA (Fig.
1A). We next examined the
effect of fibronectin on cell adhesion using Pronectin-F, which
consists of multiple copies of the RGD cell attachment ligand of
fibronectin (27). The number of adhered P-815 cells did not significantly change between the Pronectin-F-coated plates and the
non-coated plates when the cells were incubated without
PGE2, but markedly increased upon incubation with 1 µM PGE2 (Fig. 1B). PGE2-induced adherent activity to Pronectin-F-coated plates
almost completely disappeared with addition of the GRGDS peptide (Fig. 1C), which is a soluble inhibitor for the RDG site of
fibronectin as well as Pronectin-F (28). Furthermore, this
PGE2-treated cell adhesion was not affected by further
incubation with 0.02% EDTA for 30 min, but the cells could be detached
by 0.25% trypsin treatment.

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Fig. 1.
Adhesion of PGE2-stimulated P-815
cells to Pronectin-F-coated plates. A, P-815 cells were
incubated at 37 °C for 8 h with or without 1 µM
PGE2. B, P-815 cells were incubated at 37 °C
for 8 h in wells coated with or without Pronectin-F (10 µg/ml)
in the presence (solid bars) or absence (open
bars) of 1 µM PGE2, and the percentages
of P-815 cell adhesion were determined as described under
"Experimental Procedures." Values are shown as the means ± S.E. of triplicate experiments. *, p < 0.01 versus non-coat. C, P-815 cells were incubated at
37 °C for 8 h in wells coated with Pronectin-F (10 µg/ml) in
the presence (closed circles) or absence (open
circles) of 1 µM PGE2 together with the
indicated concentrations of soluble GRGDS peptide, and the percentages
of P-815 cell adhesion were determined as described under
"Experimental Procedures." Values are means ± S.E. for
triplicate experiments. *, p < 0.01 versus
without the GRGDS peptide.
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Expression of the EP3 and EP4 Subtypes in P-815 Cells--
The
RT-PCR experiment revealed that P-815 cells express mRNAs for EP3,
EP4, and IP (Fig. 2A). We
confirmed the expression of the EP3 and EP4 receptor proteins by the
replacement of the binding of 4 nM
[3H]PGE2 to membrane fractions with 10 µM PGE2, sulprostone (EP1 and EP3 agonist),
or ONO-AE1-329 (EP4 agonist). The order of replacement potency was
found to be PGE2 > sulprostone > ONO-AE1-329 (Fig. 2B). These results indicate that PGE2 can bind
to EP3 and EP4 present in the membrane fractions of P-815 cells.

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Fig. 2.
Detection of prostaglandin receptor mRNAs
and prostaglandin E2 receptor subtype proteins in P-815
cells. A, expression of mRNAs for the prostanoid
receptors was assessed by RT-PCR. Total RNA from P-815 cells was
isolated and subjected to RT-PCR, and PCR products for the EP1, EP2,
EP3, EP4, IP, DP, FP, and TP receptors were detected according to the
procedures described under "Experimental Procedures." B,
expression of the EP subtypes, EP3 and EP4, was assessed by
[3H]PGE2 binding to each subtype. The
membranes of P-815 cells were incubated at 30 °C for 1 h with 4 nM [3H]PGE2 in the presence or
absence (none) of 10 µM PGE2, 0.1 µM sulprostone (EP1, 3 agonist), 0.1 µM
ONO-AE1-329 (EP4 agonist). Values are the means ± S.E. for
triplicate determinations.
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PGE2-induced cAMP and Protein Kinase
A-dependent P-815 Cell Adhesion--
Along with
PGE2, 8-Br-cAMP, and IBMX accelerated the adhesion of P-815
cells to Pronectin-F-coated plates (Fig.
3A). PGE2-mediated cell adhesion was greatly reduced by pretreatment with 10 µM H-89, a PKA inhibitor (Fig. 3B). On the
other hand, phorbol 12-myristate 13-acetate (PMA) had no effect on cell
adhesion in the absence of PGE2, and in addition,
PGE2 did not change the intracellular Ca2+
level (data not shown). These results suggest that the
PGE2-induced adhesion of P-815 cells to Pronectin-F is via
a cAMP-dependent and cAMP-protein kinase
A-dependent pathway.

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Fig. 3.
Adhesion of PGE2-stimulated P-815
cells to Pronectin-F-coated wells mediated by PKA activation.
A, P-815 cells were incubated at 37 °C for 8 h in
wells coated with Pronectin-F (10 µg/ml) in the presence of 1 µM PGE2, 1 mM 8Br-cAMP, or 0.5 mM IBMX, and the percentages of P-815 cell adhesion were
determined as described under "Experimental Procedures." Values are
shown as the means ± S.E. for triplicate experiments. *,
p < 0.01 versus untreated. B,
P-815 cells were preloaded with or without 10 µM H-89 for
30 min, then the cells were incubated for 8 h in the presence or
absence of 1 µM PGE2, with or without 10 µM H-89. The percentages of P-815 cell adhesion were
determined as described under "Experimental Procedures." Values are
shown as the means ± S.E. for triplicate experiments. *,
p < 0.01 versus without H-89
treatment.
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Sulprostone-induced Augmentation of ONO-AE1-329-induced cAMP
Formation in P-815 Cells--
Since P-815 cells express the
Gi-coupled EP3 as well as the Gs-coupled EP4,
it is possible that PGE2 may bind to both subtype receptors
at the same time, and hence the cAMP level may reflect the difference
between Gs- and Gi-activated adenylyl cyclase
activity. We therefore examined the cAMP levels in P-815 cells when
treated with the EP1/EP3 agonist sulprostone and/or the EP4 agonist ONO AE1-329 (Fig. 4A). The cAMP
level was increased by treatment with ONO-AE1-329 and reached a
plateau level at 0.1 µM. However, cAMP accumulation
induced by various concentrations of the EP4 agonist were all augmented
by the simultaneous addition of sulprostone (1 µM),
without affecting the half-maximal concentration, although sulprostone
alone did not stimulate cAMP accumulation (Fig. 4A). The
half-maximal concentration of sulprostone for EP3-mediated augmentation
in EP4-induced cAMP synthesis was calculated to be ~5 × 10
8 M (data not shown). Furthermore,
sulprostone-induced augmentation of ONO-AE1-329-activated cAMP
formation was absolutely inhibited by treatment with PT, and the cAMP
level was almost equal to that observed in the cells stimulated by
ONO-AE1-329 alone (Fig. 4B). These findings suggest that
EP3-mediated augmentation of Gs activity is achieved
through G
subunits resulting from Gi/o protein
activation (29), although EP4-mediated Gs activity is
not.

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Fig. 4.
Sulprostone-induced augmentation of
ONO-AE1-329-stimulated cAMP formation in P-815 cells.
A, P-815 cells were incubated at 37 °C for 10 min with 1 µM sulprostone in the presence of indicated
concentrations of ONO-AE1-329. cAMP formation was determined as
described under "Experimental Procedures." Values are the
means ± S.E. of triplicate experiments. *, p < 0.01 versus stimulation with the indicated concentrations of
ONO-AE1-329 alone. B, P-815 cells were pretreated with 25 ng/ml PT for 7 h, and the treated cells were then incubated at
37 °C for 10 min with 1 µM PGE2, 1 µM sulprostone, 1 µM ONO-AE1-329, or both
1 µM sulprostone and 1 µM ONO-AE1-329, and
cAMP formation was determined as described under "Experimental
Procedures." Values are shown as the means ± S.E. for
triplicate experiments. *, p < 0.01 versus
without PT treatment.
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Sulprostone-induced Augmentation of ONO-AE1-329-stimulated P-815
Cell Adhesion to Pronectin-F--
We next examined whether EP3
stimulation could augment EP4-mediated adhesion of P-815 cells to
Pronectin-F. 1 µM sulprostone further augmented
ONO-AE1-329 induced P-815 cell adhesion to Pronectin-F (Fig.
5), and the level of EP3-mediated
augmentation of this adhesion was almost equal to the level of cAMP
augmentation by the EP3 subtype (Fig. 4B). These findings
suggest that PGE2-stimulated P-815 cell adhesion to
Pronectin-F is mediated through the cooperative action of the EP3 and
EP4 subtypes.

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Fig. 5.
Effect of PGE2 on adhesion of
P-815 cells to Pronectin-F-coated wells. P-815 cells were
incubated at 37 °C for 8 h in wells coated with Pronectin-F (10 µg/ml) in the presence or absence of 1 µM
PGE2, 1 µM sulprostone, 1 µM
ONO-AE1-329, or both 1 µM sulprostone and 1 µM ONO-AE1-329. The percentages of P-815 cell adhesion
were determined as described under "Experimental Procedures."
Values are shown as the means ± S.E. for triplicate experiments.
*, p < 0.01 versus stimulation with
ONO-AE1-329 alone.
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 |
DISCUSSION |
PGE2 was previously reported to affect both
differentiation and growth of mast cells in vitro. For
example, PGE2 is essential for the differentiation of
IL-3-dependent mouse mast cells from spleen cells (30),
PGE2 enhanced human mast cell differentiation from cord
blood mononuclear cells by inhibiting the production of
macrophage-derived GM-CSF (10), and PGE2 enhanced the
growth of differentiated mast cells in a fibroblast co-culture system (11). It is therefore possible that PGE2 plays a role in
the interaction of mast cells with fibroblastic cells and extracellular matrix components. The interaction between mast cells and extracellular matrix components have profound influences on the targeting of mast
cell progenitors to specific locations, the distribution of mast cell
subsets, and the biological responsiveness of mast cells in tissues.
The ability of mast cells to adhere to fibronectin, which involves the
RGD sequence located within the cell-attachment domain of the
fibronectin molecule, may play a role in the migration of mast cells in
various tissues (5). In the present report, we found that
PGE2 is able to stimulate the adhesion of P-815 cells to
Pronectin-F through the RGD cell attachment domain of fibronectin, and
that this adherent activity is mediated via a cAMP-dependent pathway induced by the activation of the EP4
and/or EP3 receptors. It is possible that the cAMP-protein kinase A
pathway may be involved in the induction of these cell attachment
molecules, as PGE2-induced cell attachment was inhibited by
treatment with H-89 (Fig. 3B) and cycloheximide (data not
shown). However, neither PGE2 nor 8Br-cAMP augmented the
expression of VLA-5, one of the fibronectin receptors (data not shown).
Therefore, cycloheximide may affect the signaling pathway of the
fibronectin receptors. Further experiments are required to clarify
these points.
Prostaglandin E2 acts through binding to its specific
receptors, which are comprised of four subtypes, EP1, EP2, EP3, and EP4
(14, 15). Among them, EP3 and EP4 couple to Gi and
Gs, and result in inhibition and stimulation of adenylyl
cyclase activity, respectively (17-21). The mouse EP3 receptor is
comprised of three isoforms, EP3
, EP3
, and EP3
, which differ
in their C terminus. Among them, EP3
, which was used in this
experiment is known to be coupled to the Gi protein (19).
Very recently, activation of a Gi-coupled receptor has been
reported to augment the adenylyl cyclase activity induced by the
stimulation of a Gs-coupled receptor in COS-7 cells. For
example, activation of Gi-coupled receptors such as
2
adrenoreceptor (29) and bradykinin B2 receptor (32) lead to the
augmentation of Gs-stimulated adenylyl cyclase in COS-7
cells. This synergistic effect has not been clearly shown in mammalian
cells. Southall and Vasko (23) showed that the simultaneous depletion
of both rat EP3C and EP4 was essential for abolishing
PGE2-stimulated cAMP production and neuropeptide release in
rat sensory neurons (23). In the current experiment we showed that the
Gi-coupled EP3 agonist sulprostone augmented cAMP formation
induced by the EP4 agonist ONO-AE1-329 in P-815 cells. Similarly, we
found that sulprostone was able to augment cAMP formation in P-815
cells activated by an IP agonist, carbacyclin, suggesting that the
augmentative effects of EP3 can be observed irrespective of
Gs activation. The mechanism underlying these phenomena was
thought to be via G
-mediated activation of type IV adenylyl
cyclase (29), because pretreatment with PT inhibited the augmentation
by the activation of the Gi-coupled EP3 receptor, and P-815
cells express type IV adenylyl cyclase (data not shown). Therefore,
G
-mediated activation of type IV adenylyl cyclase may be involved
in EP3-mediated signaling to augment EP4-stimulated adenylyl cyclase
activity in P-815 cells.
Although the involvement of phosphatidylinositol 3-kinase (PI3K) in
cell adhesion to matrix proteins is shown in a variety of cell types
(33), the role of PI3K in mast cell adhesion is still unknown. Kinashi
et al. (31) reported that the adhesion of platelet-derived
growth factor receptor-expressed bone marrow-derived mast cells was
inhibited by the addition of the PI3K inhibitor wortmannin. To
understand whether PI3K is activated by PGE2 during P-815
cell adhesion, we examined the effect of the PI3K inhibitors, wortmannin (100 nM) and LY294002 (10 µM) on
EP3/EP4-agonist induced cell adhesion to fibronectin. As a result,
these inhibitors showed an additive effect on the augmentation of
PGE2-induced cell adhesion (wortmannin: 81.7 ± 0.1%
and LY294002: 82.4 ± 0.2%, compared with PGE2
stimulation alone: 24.8 ± 0.6%). Furthermore, each inhibitor alone had an effect on cell adhesion (wortmannin: 20.4 ± 0.1% and LY294002: 24.1 ± 0.1%, compared with the absence of these inhibitors (3.3 ± 0.1%)). Therefore, these results indicate that P-815 cell attachment to fibronectin is regulated by two independent signaling pathways involving PKA and PI3K. Further experiments are
necessary to clarify the fundamental differences in the signaling involving PKA and PI3K in P-815 cell attachment to fibronectin. In
summary, this study clearly demonstrates that two subtypes of the
PGE2 receptor, EP3 and EP4, are cooperatively involved in
PGE2-evoked and cAMP-mediated functions of P-815 cell adhesion.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. P. L. Caton of
Rhone-Poulenc Ltd. for the generous gift of sulprostone. We are
grateful to Dr. Manabu Negishi of the Laboratory of Molecular
Neurobiology, Graduate School of Biostudies, University of Kyoto for
valuable advice on this study.
 |
FOOTNOTES |
*
This work was supported in part by a grant-in-aid for
Scientific Research by the Ministry of Education, Science, Sports, and Culture of Japan.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: Dept. of Physiological
Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Tel.: 81-75-753-4527; Fax: 81-75-753-4557; E-mail: aichikaw@pharm.kyoto-u.ac.jp.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M301312200
 |
ABBREVIATIONS |
The abbreviations used are:
MC, mast cell;
PG, prostaglandin;
G protein, heterotrimeric GTP-binding protein;
PT, pertussis toxin;
PMA, phorbol 12-myristate 13-acetate;
PBS, phosphate-buffered saline;
Mes, 4-morpholineethanesulfonic acid;
PI3K, phosphatidylinositol 3-kinase;
PKA, cAMP-dependent protein
kinase;
ECM, extracellular matrix.
 |
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