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Original Studies |
Departments of Medicine and Clinical Science (M.K., I.T., Y.O., T.S., S.M., Y.Ya., A.S., Y.Yo., K.N.), Gynecology and Obstetrics (T.M., N.S.), and Pharmacology (S.N.), Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan
Address all correspondence and requests for reprints to: Issei Tanaka, M.D., Ph.D., Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: isseitnk{at}cam.hi-ho.ne.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies demonstrated that EP3 has multiple alternatively spliced isoforms with different C-terminal tails (4) that are expressed in a variety of tissues in a tissue-specific manner (5). It is notable that the different C-terminal tails of EP3 isoforms determine their signaling pathways, inhibition and/or stimulation of cAMP production, and stimulation of phosphoinositide turnover in bovines and humans (4, 5). We also elucidated the complete structural organization of the human EP3 gene and demonstrated that the C-terminal tails are created by complicated alternative messenger ribonucleic acid (mRNA) splicing mechanisms; the human EP3 gene forms at least nine EP3 mRNAs encoding eight distinct isoforms, named EP3-I, EP3-II, EP3-III, EP3-Iv, EP3-V, EP3-VI, EP3e, and EP3f (6, 7). During the course of that study, two partial amino acid sequences of novel human EP3 isoforms, EP3-V and EP3-VI, were obtained by RT-PCR experiments using polyadenylated [poly(A)+] RNA from human uterus (6). Counterparts of EP3-V and EP3-VI have not been isolated in other species to date.
In the present study we cloned the whole coding regions of the two EP3 complementary DNAs (cDNAs) and characterized their intracellular signaling pathways and tissue distributions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human tissue samples were obtained at autopsy or at the time of hysterectomy for myoma. Total RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). Poly(A)+ RNA was isolated using Poly A Tract (Promega Corp., Madison, WI). The present study was conducted with informed consent and was approved by the ethical committee on human research of Kyoto University Graduate School of Medicine.
Isolation of the human EP3 isoform cDNAs
Approximately 40 ng poly(A)+ RNA from
human uterus were reverse transcribed by oligo(deoxythymidine) priming
using SuperScript II (Life Technologies, Inc.). Two
EP3 cDNA fragments of 265 and 238 bp and
EP3f cDNA (7) of 335 bp were
obtained by RT-PCR with a sense primer (P-5;
5'-CTGAACCAGATCTTGGATCC-3') and an antisense primer (EPR-f2;
5'-ATCTGCTG TCAAAATAGTTC-3'; Fig. 1
).
RT-PCR was also performed using the LA PCR Kit version 2 (Takara Shuzo
Co., Otsu, Japan) with a sense primer (LEPR-u5; 5'-ACCAGAG
GTTTCCCAGAGAGGAAGGCGTGG-3'), which contains the nucleotide sequence of
the 5'-untranslated regions of the EP3 gene, and
an antisense primer (LEPR-7; 5'-TCAG CTTAGCTGGACACTGCAGGGTTCTCTAT-3'),
which contains nucleotide sequences of exons 7 and 9, to obtain the
entire coding regions of the two novel EP3
isoform cDNAs (Fig. 1). Two DNA fragments of 1440 and 1413 bp were
subcloned into the pCR vector (Invitrogen, San Diego, CA)
and designated pCR-V and pCR-VI, respectively. The plasmid clones,
pCR-V and pCR-VI, were sequenced by both strands using universal T7 and
SP6 primers and cDNA-specific primers.
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RT-PCR analysis was performed to examine the expression of the
two novel EP3 isoform mRNA (Fig. 2). Two hundred nanograms of total RNA
were reverse transcribed and subjected to PCR with a sense primer (P-5)
and an antisense primer (EPR-f7; 5'-TATCATGAGAACTGCACCAA-3'; Fig. 1
).
The reaction profile was as follows: denaturation at 94 C for 30
s, annealing at 55 C for 30 s, and extension at 72 C for 60
s, for 35 cycles. RT-PCR for human ß-actin was also performed as an
internal control as previously described (5). Five
microliters of the reaction mixture were electrophoresed and
transferred onto a Biodyne A nylon membrane (Pall, Glen Cove, NY). The
membrane was hybridized with a 32P-labeled
internal oligonucleotide probe (5'-TCCTGGGGAC CTGCCTCCATGCATGACAAA-3')
or a 32P-labeled human ß-actin genomic DNA
fragment (Wako Pure Chemical Co., Osaka, Japan) (8) in a
solution containing 6 x SSC (standard saline citrate), 1% SDS,
and 100 µg/mL salmon sperm DNA at 37 C. The membrane was washed in
6 x SSC and 0.1% SDS at 50 C and exposed to a film with
intensifiers for 16 h.
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Ten-micron-thick human uterus sections were preincubated in a
solution containing 50% formamide, 0.6 mol/L NaCl, 10 mmol/L Tris-HCl
(pH 7.6), 0.06% BSA, 1 mmol/L ethylenediamine tetraacetate, 0.025%
sodium pyrophosphate, 0.12% Ficoll, 0.12% polyvinylpyrrolidone, 0.5
mg/mL salmon sperm DNA, and 10 µg/mL dithiothreitol for 6 h at
37 C. Hybridization was performed by adding a
32P-labeled antisense DNA (EPR-7 in
Fig. 1; 5'-ACTGCACCAAGTCCTGGGGACCTGCCTCCA-3') specific for
EP3-V and EP3-VI mRNAs.
Hybridization was also performed by further adding the unlabeled
antisense DNA to 20-fold the concentration of the labeled DNA as
a negative control. Washing was performed in 0.5 x SSC containing
5 mmol/L dithiothreitol. The sections were exposed to ß-Max film
(Amersham Pharmacia Biotech, Aylesbury, UK) at 4 C for 1
week, then dipped in a nuclear track emulsion (NTB-2, Eastman Kodak Co., Rochester, NY). After 2-week exposure, sections were
developed and counterstained with hematoxylin.
Construction of stable cell lines
To express proteins encoded by pCR-V and pCR-VI, the HindIII and XbaI fragments of pCR-V and pCR-VI were subcloned into an eukaryotic expression vector, pRc/RSV (Invitrogen, San Diego, CA) and designated pRc-pCR-V and pRc-pCR-VI, respectively. These plasmids were transfected into Chinese hamster ovary (CHO) cells using Transfectam (BioSepra, Inc., Marlborough, MA). The CHO cells were cultured in Ham’s F-12 medium (ICN Biomedicals, Inc., Tokyo, Japan) containing 800 µg/mL of the antibiotic G-418 (Life Technologies, Inc.) and cloned as previously described (5).
Crude membrane preparation and ligand binding assay
The crude membranes of CHO cells were prepared as previously
described (9). For saturation experiments, 40 µg crude
membrane protein were used in 1–16 nmol/L
[5,6,8,11,12,14,15-3H]PGE2
(154 Ci/mmol; NEN Life Science Products, Boston, MA) as
previously described (9). For displacement experiments, 40
µg membrane protein were used with 4 nmol/L
[3H]PGE2 and unlabeled
PGE1, PGE2,
PGF2
(Nacalai Tesque, Kyoto, Japan),
PGD2 (Sigma, St. Louis, MO),
M&B28767 (an EP3 receptor agonist), butaprost (an
EP2 receptor agonist; gifts from Dr. M.
P. L. Caton, Rhone-Poulenc Ltd., Anthony, France), or
iloprost (a prostacyclin receptor agonist; Amersham Pharmacia Biotech).
cAMP measurements
CHO cells transfected with pRc-pCR-V or pRc-pCR-VI were cultured in 24-well plates (1 x 105 cells/well) at 37 C for 12 h and further cultured with or without 20 ng/mL pertussis toxin (PTX; Sigma) for another 12 h. After cells were preincubated in 500 µL Ham’s F-12 containing 1 mmol/L 3-isobutyl-1-methylxanthine (Nacalai Tesque) for 10 min at 37 C, they were stimulated by several concentrations of M&B28767 in the presence of 1 µmol/L forskolin or vehicle at 37 C for 15 min. The reactions were terminated by adding an equal volume of 12% trichloroacetic acid (10). The cAMP concentrations were measured using a commercially available RIA kit (Yamasa Corp., Tokyo, Japan) (11).
Inositol 1,4,5-trisphosphate (Ip3) measurements
CHO cells transfected with pRc-pCR-V or pRc-pCR-VI were harvested, suspended in Ham’s F-12, and stimulated by 10 nmol/L and 1 µmol/L M&B28767 or vehicle for 30 s at 37 C. The Ip3 concentrations were measured using a RRA kit (NEN Life Science Products) (12) by two independent sets of quadruplicated determinations.
Western blot analysis for mitogen-activated protein kinase (MAPK)
After CHO cells transfected with pRc-pCR-V or pRc-pCR-VI were cultured for 6 h in Ham’s F-12 in the absence of FCS with or without 20 ng/mL PTX, cells were incubated in Ham’s F-12 containing 100 pmol/L and 100 nmol/L M&B28767 or vehicle for 5 min. Total cell lysate was extracted by adding a cell lysis solution (13). The protein concentrations were measured by protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Approximately 30 µg total cell lysates were electrophoresed on 10% SDS-PAGE and transferred onto a polyvinylidene difluoride transfer membrane (PolyScreen, NEN Life Science Products). MAPK phosphorylated at Tyr204 was immunoblotted with phospho-specific p44/42 MAPK Antibody (New England Biolabs, Inc., Beverly, MA) in 10% Block Ace (Yukijirushi Nyugyo Co., Sapporo, Japan) and was detected using enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech).
Statistics
Statistical analyses were performed using ANOVA, and P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 2 shows the entire amino acid sequences of the
EP3 isoforms, EP3-V and
EP3-VI, determined by RT-PCR.
EP3-V and EP3-VI are
composed of 402 and 393 amino acid residues, respectively. The
N-terminal 359 amino acid residues of EP3-V were
identical to those of the previously reported human
EP3 isoforms (5).
EP3-V was identical to
EP3-VI, except for the presence of 9 amino acid
residues in the cytoplasmic tail sequence (boxed in Fig. 2).
The 9 amino acid residues were also included in
EP3-Iv, EP3e, and
EP3f (Fig. 2) (5, 7). The downstream
25 amino acid residues are unique among EP3
isoforms (dotted underline in Fig. 2). The C-terminal 9
amino acid residues were identical to those of
EP3f (Fig. 2) (7).
RT-PCR
RT-PCR was performed with an antisense primer specific for
EP3-V and EP3-VI mRNAs
using total RNAs from various human tissues (Fig. 3). The expression of
EP3-V and EP3-VI mRNAs was
detected abundantly in human uterus. In other tissues examined, weak,
but substantial, bands of EP3-V or
EP3-VI mRNAs were detected in lung and
kidney.
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In situ hybridization analysis was performed to
determine the cellular localizations of EP3-V and
EP3-VI mRNAs in human uterus. As shown in Fig. 4A, signals for
EP3-V and EP3-VI mRNAs were
diffusely hybridized in the uterus. The signals were abolished by
adding the unlabeled antisense DNA (Fig. 4B). In a microscopic (x200)
view (Fig. 4C), signals were densely apparent in the myometrium region.
The signal intensity in the endometrium region was weak, comparable to
the background signals.
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The results of saturation and displacement experiments using CHO
cells that stably express EP3-V and
EP3-VI are depicted in Fig. 5.
[3H]PGE2 specifically
bound to the crude membranes prepared from cells expressing
EP3-V and EP3-VI (Fig. 5A).
On the other hand, specific
[3H]PGE2 binding was not
detected in wild-type CHO cell membrane (Fig. 5A). Scatchard analysis
for EP3-V and EP3-VI
isoforms revealed that the dissociation constant values were 3.9 and
1.4 nmol/L and that the maximal specific
[3H]PGE2 bindings were
4402 and 989 fmol/mg protein, respectively (Fig. 5B). As shown in Fig. 5C, PGE1, PGE2, and
M&B28767 were almost equipotent in the displacement of specific
[3H]PGE2 binding for
EP3-V and EP3-VI. Iloprost,
butaorost, PGD2, and
PGF2 showed approximately 2–3 orders of
magnitude lower affinity than PGE1 and
PGE2 in the displacement of specific
[3H]PGE2 binding for both
EP3-V and EP3-VI.
Butaprost, an EP2 agonist, showed no displacement
of specific [3H]PGE2
binding for both EP3-V and
EP3-VI. The rank order of PGs and the related
agents for EP3-V was M&B28767 =
PGE1 = PGE2 >> iloprost,
PGF2, and PGD2 >
butaprost. The binding characteristics of EP3-VI
were very similar to those of EP3-V. The binding
characteristics of EP3-V and
EP3-VI were consistent with those of the
previously reported EP3 isoforms
(5).
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Agonist-induced cAMP concentrations in CHO cells expressing
EP3-V and EP3-VI were
measured as shown in Fig. 6A. No
significant changes in cAMP concentrations were observed in
untransfected CHO cells treated with M&B28767 up to 10 µmol/L as we
reported previously (5). In
EP3-V-expressing cells, cAMP concentrations did
not change in the presence of M&B28767 within the range from 1 nmol/L
to 10 µmol/L. In contrast, in EP3-VI-expressing
cells, cAMP concentrations were significantly increased in the presence
of M&B28767 concentrations higher than 100 nmol/L compared with the
basal cAMP concentrations and reached approximately 270% of the basal
concentrations at 10 µmol/L. We also examined
EP3 isoform-mediated inhibition of
forskolin-induced cAMP concentrations (Fig. 6B). In
EP3-V-expressing cells, cAMP concentrations
stimulated by forskolin were decreased in the presence of M&B28767
within the range from 10 pmol/L to 100 nmol/L, and at 10 nmol/L, they
were decreased to 76% of the concentration in cells stimulated by
forskolin. In EP3-VI-expressing cells, cAMP
concentrations were also decreased in the presence of M&B28767 within
the range from 1–10 nmol/L. However, M&B28767 at a concentration of
100 nmol/L increased cAMP concentrations by approximately 14% of those
of EP3-VI-expressing cells stimulated by
forskolin. The cAMP concentrations were also measured after cells were
cultured for 12 h with 20 ng/mL PTX (Fig. 6C). In cells expressing
EP3-V or EP3-VI, M&B28767
did not inhibit forskolin-induced cAMP concentrations after treatment
with PTX.
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Ip3 concentrations were measured in CHO cells expressing EP3-V and EP3-VI to elucidate EP3 isoform-mediated changes in phosphoinositide turnover. Ip3 concentrations in cells expressing EP3-V with vehicle and 10 nmol/L and 1 µmol/L M&B28767 were 2.28 ± 0.10, 2.18 ± 0.18, and 2.44 ± 0.20 pmol/105 cells, respectively. Ip3 concentrations in cells expressing EP3-VI with vehicle and 10 nmol/L and 1 µmol/L M&B28767 were 2.44 ± 0.17, 2.34 ± 0.26, and 2.34 ± 0.12 pmol/105 cells, respectively. M&B28767 did not significantly increase Ip3 concentrations in cells expressing either EP3-V or EP3-VI.
Western blot analysis for activated MAPK
Western blot analysis for p44/42 MAPK was performed using total
cell lysates of CHO cells expressing EP3-V and
EP3-VI and untransfected CHO cells (Fig. 7). In untransfected CHO cells, 100
nmol/L M&B28767 did not activate MAPK. In cells expressing
EP3-V and EP3-VI, 100
pmol/L and 100 nmol/L M&B28767 activated p44/42 MAPK. This effect was
almost abolished when CHO cells were treated with 20 ng/mL PTX for
12 h. We also performed Western blot analysis with 44/42 MAPK
antibody that we previously developed (13) and confirmed
that quantities of 44/42 MAPK were equal in each lane (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue distributions of EP3-V and EP3-VI mRNAs are restricted compared with those of previously reported EP3 isoforms (5). EP3 is known to be most widely distributed in tissues of EP subtypes (3). We demonstrated that EP3-I-Iv mRNAs are distributed in various human tissues and that all of them are detected in the brain, kidney, pancreas, and uterus (5). Other investigators also demonstrated that EP3e mRNA is detected in human placenta, brain, and heart (7). In contrast, RT-PCR analysis demonstrated that EP3-V and EP3-VI mRNAs are mainly detected in human uterus and that small amounts of EP3-V and EP3-VI mRNAs are detected in the kidney and lung. In situ hybridization analysis suggested possible specific functions of PGE2 through EP3-V and EP3-VI in human myometrium.
Recent studies demonstrated that some G protein-coupled receptors, such
as the m1 and m2 acetylcholine receptors, lysophosphatidic acid
receptor, endothelin receptor, formyl-methionyl-leucyl-phenylalanine
receptor, thrombin receptor, and EP3A isoform of
PGE receptor subtype EP3, activate MAPK
(16, 17). These receptors are known to be coupled with
Gi and/or Gq class G
proteins. Furthermore, ß
-subunits to be coupled with
Gi-subunit are known to activate MAPK
(18). In the present study, EP3-V
and EP3-VI are suggested to be coupled with
PTX-sensitive G proteins to inhibit cAMP concentrations. The MAPK
activations via EP3-V and
EP3-VI are also influenced by PTX treatment.
These results suggest that at least the Gi class
of G proteins is involved in MAPK activation via
EP3-V and EP3-VI. We also
confirmed that M&B28767 activated MAPK in EP3-I-,
EP3-II-, EP3-III-, and
EP3-Iv-expressing cells (data not shown). The
present study suggests a novel function of PGE2
in the cell growth of human uterus via EP3
isoforms.
The present study demonstrated the functional difference in cAMP concentrations between EP3-V and EP3-VI. M&B28767 increases cAMP concentrations in EP3-VI-expressing cells, whereas it does not in EP3-V-expressing cells, even though EP3-V and EP3-VI share the same sequence except that EP3-V contains 9 amino acid residues in the cytoplasmic tail. On the contrary, in our previous study, M&B28767 increased cAMP concentrations in cells expressing EP3-Iv, which includes this 9 amino acid residue, but did not increase cAMP concentrations in cells expressing EP3-III, which has the same amino acid sequence as EP3-Iv except for the absence of the 9 amino acid residues (5). These results suggest that the presence or absence of the 9 amino acid residues seems to be involved in the modulation of adenylate cyclase function, although the precise analyses of the three-dimensional structure-function relationship is necessary. In other G protein-coupled receptors, the pituitary adenylate cyclase-activating polypeptide type 1 receptor isoforms are known to change their Ki value for cAMP concentrations by combinations of several polypeptide in the third cytoplasmic loop (19). In the EP3 isoforms, the coupling properties may be dependent upon the conformational differences of receptors produced by insertional polypeptides in the C-terminal tails.
In contrast, in both cells expressing EP3-V and those expressing EP3-VI, M&B28767 inhibited forskolin-induced cAMP concentrations. These responses are abolished by PTX treatment. PGE2 and the EP3 agonists also inhibited forskolin-induced cAMP concentrations via all of the previously reported human EP3 isoforms (5, 7). Recently, a site-directed mutagenesis analysis of rabbit EP3 revealed that aspartic acid at position 338 in the seventh transmembrane domain is crucial for Gi activation (20). As the seventh transmembrane domain of human EP3 also includes an aspartic acid residue and is highly homologous compared with that of rabbit EP3, the critical position for inhibiting cAMP concentrations in human EP3 isoforms may also be included in the seventh transmembrane domain.
In conclusion, we have characterized two PGE receptor EP3 isoforms from human uterus. The present study will lead to better understanding of multiple functions of PGE2 during pregnancy and delivery via EP3 isoforms.
Received November 11, 1999.
Revised July 11, 2000.
Accepted July 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated MAP kinase activation by
a common signalling pathway. Nature. 376:781–784.[CrossRef][Medline]
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X174 DNA HaeIII was used as the
size marker. One typical result is shown from three reproducible
results.
, and • denote plots for wild-type CHO cell membranes or CHO
cell membranes transfected with pRc-pCR-V and pRc-pCR-VI, respectively.
C, The PGs and related agents used are PGE1 (•),
PGE2 (
), PGF2
), M&B28767 (
), butaprost (X), and iloprost (
).
