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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 986-993
Copyright © 2004 by The Endocrine Society

Expression, Localization, and Signaling of Prostaglandin F2{alpha} Receptor in Human Endometrial Adenocarcinoma: Regulation of Proliferation by Activation of the Epidermal Growth Factor Receptor and Mitogen-Activated Protein Kinase Signaling Pathways

Kurt J. Sales, Stuart A. Milne, Alistair R. W. Williams, Richard A. Anderson and Henry N. Jabbour

Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology (K.J.S., R.A.A., H.N.J.), and Department of Pathology, University of Edinburgh Academic Center (A.R.W.W.), Edinburgh, United Kingdom EH16 4SB; and Fujisawa Institute of Neuroscience in Edinburgh, University of Edinburgh (S.A.M.), Edinburgh, United Kingdom EH8 9LE

Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, University of Edinburgh Academic Center, 49 Little France Crescent, Old Dalkeith Road, Edinburgh, United Kingdom EH16 4SB. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin F2{alpha}(PGF2{alpha}) is a bioactive lipid biosynthesized by cyclooxygenase (COX) enzymes and mediates its biological activity via the heptahelical Gq-coupled PGF2{alpha}receptor (FP receptor). This study investigated the expression and molecular signaling of the FP receptor in human endometrial adenocarcinomas. Real-time RT-PCR and Western blot analysis confirmed FP receptor expression in endometrial adenocarcinoma of all grades and differentiation. The expression of FP receptor was up-regulated in all endometrial adenocarcinomas compared with normal endometrium. The site of FP receptor expression was localized by in situ hybridization and immunohistochemistry to the neoplastic epithelial cells in all adenocarcinomas. Treatment of endometrial adenocarcinoma explants with PGF2{alpha} resulted in mobilization of inositol phosphate signaling, indicating functional FP receptor expression. We investigated whether PGF2{alpha} could trans-activate the epidermal growth factor receptor (EGFR) and trigger the MAPK signaling pathway. Treatment of adenocarcinoma explants and endometrial adenocarcinoma cells (Ishikawa) with PGF2{alpha}-phosphorylated EGFR, triggered MAPK signaling and enhanced the proliferation of Ishikawa cells. Inactivation of phospholipase C, EGFR kinase, and MAPK kinase with specific inhibitors abolished PGF2{alpha}-induced trans-activation of EGFR, MAPK signaling, and Ishikawa cell proliferation. These data suggest that PGF2{alpha}-FP receptor promote endometrial tumorigenesis via a phospholipase C-mediated phosphorylation of the EGFR and MAPK signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIAL ADENOCARCINOMA IS the most common gynecological malignancy in the Western world. The incidence of the disease is uncommon under the age of 40 yr and peaks at around 70 yr of age (1, 2, 3). Endometrial carcinomas arise from several cell types, with adenocarcinoma arising from the glandular epithelium being the most common type, accounting for 80–90% of all uterine tumors. The etiology of endometrial adenocarcinoma is unknown.

There is now mounting evidence to support the idea that pathology of the reproductive tract and, more specifically, reproductive tract carcinomas, are regulated in an autocrine/paracrine manner by cyclooxygenase (COX) enzyme products (4, 5, 6, 7, 8, 9, 10). Three isoforms of COX enzyme (COX-1, COX-2, and COX-3) have been reported and catalyze the committed step in prostanoid biosynthesis (11, 12). COX-1 and/or COX-2 expression are up-regulated in numerous solid epithelial tumors, including tumors of the uterus and cervix (4, 5, 6, 7, 8, 9, 10). A functional role for COX-3 remains to be determined.

In the reproductive tract, the E and F series of prostanoids are the most abundantly synthesized, and PGF2{alpha} is a major metabolite of COX enzymes in human endometrium (13, 14, 15). PGF2{alpha} is biosynthesized from arachidonic acid by a series of oxidation steps by COX enzymes and PGF synthase, respectively (16). After biosynthesis, PGF2{alpha} is transported out of the cell by means of a carrier-mediated process (17), where it exerts an autocrine/paracrine function through G protein receptor (GPCR)-mediated interaction. The GPCR for the human PGF2{alpha} (FP) has been cloned, and its activation leads to coupling of the G protein Gq and activation of phospholipase Cß (PLCß) and release of inositol trisphosphate (IP3) and diacylglycerol (18).

Recent data have implicated prostanoid biosynthesis and signaling, specifically PGE2, in malignant change in epithelial cells. This is achieved through immunosuppression by inhibiting T and B cell proliferation and differentiation and accessory monocyte/macrophage function (19), inhibiting apoptosis by prolonging the G1 phase of the cell cycle (20), increasing the metastatic potential of epithelial cells by down-regulating cell surface adhesion proteins (21) and promoting cellular proliferation (22) and angiogenesis (23). However, little is known about the potential role of PGF2{alpha}.

Prostanoid biosynthesis and signaling, including PGF2{alpha}, have been implicated in numerous endometrial pathologies, including excessive menstrual bleeding (menorrhagia), endometriosis, dysmenorrhea, and painful periods (24, 25, 26, 27). The molecular mechanisms underlying the role of prostanoids in the pathology of the reproductive tract remains to be fully elucidated. In this study we investigated a role for PGF2{alpha}-FP receptor in endometrial adenocarcinomas by 1) determining the expression and localization of the FP receptor in human endometrial adenocarcinomas, and 2) investigating the signaling pathways associated with PGF2{alpha}-FP receptor function and its role in the proliferation of endometrial adenocarcinoma cells. Here we provide evidence for a role for PGF2{alpha} and FP receptor in promoting neoplastic endometrial epithelial cell proliferation by mechanisms involving activation of the PLCß, epidermal growth factor receptor (EGFR), and MAPK signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

All culture medium was purchased from Life Technologies, Inc. (Paisley, UK). Penicillin-streptomycin and fetal calf serum (FCS) were purchased from PAA Laboratories Ltd. (Middlesex, UK). EGFR rabbit polyclonal (sc-03), pTYR mouse monoclonal (sc-508), and ERK goat polyclonal (sc-93) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Autogenbiockear, UK). The FP receptor rabbit polyclonal antibody (101802) was purchased from Cayman Chemical Co. (Alexis Corp., Nottingham, UK). Phospho-p42/44 ERK antibody was purchased from Cell Signaling Technologies (New England Biolabs, Hertfordshire, UK). Antigoat, antirabbit alkaline phosphatase secondary antibodies, indomethacin, PBS, BSA, and PGF2{alpha} were purchased from Sigma-Aldrich Corp. (Dorset, UK). The ECF chemiluminescence system was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). PD98059 [18.7 mM stock in dimethylsulfoxide (DMSO)], U73122 (4 mM stock in DMSO), and AG1478 (100 µM stock in DMSO) were purchased from Calbiochem (Nottingham, UK) and stored at -20 C.

Patients and tissue collection

Endometrial adenocarcinoma tissue (n = 24) was collected from women undergoing hysterectomy who had been prediagnosed to have adenocarcinoma of the uterus. Hysterectomy specimens for adenocarcinoma were collected from the operating theater and placed on ice. With minimal delay, the specimens were opened by a gynecological pathologist, and small samples (~5 mm to 3 cm) of adenocarcinoma tissue were collected into neutral buffered formalin and wax embedded (for in situ hybridization and immunohistochemistry studies), snap-frozen in dry ice and stored at -70 C (for RNA extraction), or placed in RPMI 1640 culture medium containing 2 mmol/liter L-glutamine, 100 U penicillin, 100 µg/ml streptomycin, and 8.4 µM indomethacin (to inhibit endogenous COX activity) and transported to the laboratory for in vitro culture. The diagnosis of adenocarcinoma was confirmed histologically in all cases. All women with endometrial adenocarcinoma were postmenopausal. Normal endometrial tissue (control tissues; n = 12) at different stages of the menstrual cycle was collected with an endometrial suction curette (Pipelle, Laboratoire CCD, France) from women with regular menstrual cycles (25–35 d) undergoing hysterectomy for benign gynecological indications and processed as described above. All control subjects reported regular menstrual cycles (cycle length, 25–35 d), and no woman had received a hormonal preparation in the 3 months preceding biopsy collection. Biopsies were dated according to stated last menstrual period and were confirmed by histological assessment according to criteria of Noyes and co-workers (28). Ethical approval was obtained from the Lothian Research ethics committee, and written informed consent was obtained from all subjects before tissue collection. Tissue samples were assigned randomly to the various experiments described herein.

Cell culture

Ishikawa cells (European Collection of Cell Culture, Center for Applied Microbiology, Wiltshire, UK) were routinely maintained in DMEM nutrient mixture F-12 with Glutamax-1 and pyridoxine supplemented with 10% FCS, and 1% antibiotics (stock solution of 500 IU/ml penicillin and 500 µg/ml streptomycin) at 37 C and 5% CO2 (vol/vol).

TaqMan quantitative RT-PCR

Endometrial RNA samples were extracted from endometrial tissue using Tri-Reagent (Sigma-Aldrich Corp.) following the manufacturer’s guidelines. Once extracted and quantified, RNA samples were reverse transcribed using MgCl2 (5.5 mM), deoxy (d)-NTPs (0.5 mM each), random hexamers (2.5 µM), ribonuclease inhibitor (0.4 U/µl), and multiscribe reverse transcriptase (1.25 U/µl; all from PE Applied Biosystems, Warrington, UK). The mix was aliquoted into individual tubes (16 µl/tube), and template RNA was added (4 µl/tube of 100 ng/µl RNA). After mixing by brief centrifugation, samples were incubated for 90 min at 25 C, 45 min at 48 C, and 5 min at 95 C. Thereafter, cDNA samples were stored at -20 C. A tube with no reverse transcriptase was included to control for any DNA contamination.

To measure cDNA expression a reaction mix was prepared containing TaqMan buffer (5.5 mM MgCl2, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, and 400 µM dUTP), ribosomal 18S forward and reverse primers and probe (all at 50 nM), forward and reverse primers for FP receptor (300 nM), FP receptor probe (100 nM), AmpErase UNG (0.01 U/µl), and AmpliTaq Gold DNA polymerase (0.025 U/µl; all from PE Applied Biosystems). After mixing, 48 µl were aliquoted into separate tubes, and 2 µl/replicate (40 ng) cDNA were added and mixed before placing duplicate 24-µl samples into a PCR plate. A no template control (containing water) was included in triplicate. Wells were sealed with optical caps, and the PCR reaction was carried out using an ABI PRISM 7700 (PE Applied Biosystems). FP receptor primers and probe for quantitative PCR were designed using the PRIMER express program (PE Applied Biosystems). The sequences of the FP receptor primers and probe were: forward, 5'-GCA GCT GCG CTT CTT TCA A-3'; reverse, 5'-CAC TGT CAT GAA GAT TAC TGA AAA AAA TAC-3'; and probe (6-carboxyfluorescein-labeled), 5'-CAC TGT CAT GAA GAT TAC TGA AAA AAA TAC-3'. The ribosomal 18S primers and probe sequences were: forward 5'-CGG CTA CCA CAT CCA AGG AA-3'; reverse, 5'-GCT GGA ATT ACC GCG GCT-3'; and probe (VIC-labeled; PE Applied Biosystems), 5'-TGC TGG CAC CAG ACT TGC CCT C-3'. Data were analyzed and processed using Sequence Detector version 1.6.3 (PE Applied Biosystems) as instructed by the manufacturer. The expression of the FP receptor was normalized to RNA loading for each sample using 18S ribosomal RNA as an internal standard. Results are expressed as relative expression to an internal standard of normal endometrial RNA.

Protein extraction

Cells. For EGFR trans-activation studies, 3 x 106 cells were seeded in 10-cm dishes, and for MAPK studies, 1 x 106 cells were seeded in 5-cm dishes and allowed to attach and grow overnight. The following day, culture medium was aspirated, and the cells were washed with PBS and incubated in serum-free culture medium containing penicillin/streptomycin (as described previously) and 8.4 µM indomethacin (a dual COX enzyme inhibitor used to inhibit endogenous prostanoid biosynthesis) overnight. The next day, cells were pretreated with specific inhibitors for PLCß (U73122, 10 µM), EGFR kinase (AG1478, 100 nM), or MAPK kinase (MEK; PD98059, 50 µM) for 1 h before stimulation with 100 nM PGF2{alpha} for 10 min. After stimulation with PGF2{alpha}, cells were washed with PBS. Proteins were extracted from cells by lysis on ice in protein lysis buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 5 mM EGTA, 0.1% sodium dodecyl sulfate containing 2 mM phenylmethylsulfonylfluoride, 1 mM Na3VO4, and 5 µg/ml aprotinin]. Thereafter, insoluble material was pelleted by centrifugation at 14,000 x g for 20 min at 4 C. The clarified lysate was removed to a new tube for protein quantification and SDS-PAGE. The protein content in the supernatant fraction was determined using protein assay kits (Bio-Rad Laboratories, Hemel Hempstead, UK).

Tissue. For EGFR trans-activation studies and MAPK signaling, carcinoma tissue was incubated overnight in serum-free culture medium containing penicillin/streptomycin (as described previously) and 8.4 µM indomethacin. The next day, tissue was pretreated with specific inhibitors for PLCß (U73122, 10 µM), EGFR kinase (AG1478, 100 nM), or MEK (PD98059, 50 µM) for 1 h before stimulation with 100 nM PGF2{alpha} for 10 min. After stimulation with PGF2{alpha}, tissue was washed with PBS, and protein was harvested by homogenization in protein lysis buffer, clarified by centrifugation, and assayed as described above before immunoprecipitation and/or Western blot analysis.

Immunoprecipitation and Western blot analysis

For immunoprecipitation studies, equal amounts of protein were incubated with specific EGFR antibody immobilized onto protein A-Sepharose overnight at 4 C with gentle rotation. Beads were washed extensively with lysis buffer, and immune complexes were eluted in Laemmli buffer [125 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 20% glycerol, and 0.05% bromophenol blue], boiled for 5 min, and microcentrifuged. For FP receptor expression in carcinoma tissues and MAPK studies, a total of 50 µg protein were resuspended in 20 µl Laemmli buffer, boiled for 5 min, and microcentrifuged. Proteins were resolved on 4–20% Tris-glycine gels (NOVEX, Invitrogen, De Schelp, The Netherlands), transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Watford, UK), and subjected to immunoblot analysis. Membranes were blocked for 1 h at 25 C in 4% BSA diluted in 50 mM Tris-HCl, 150 mM NaCl, and 0.05% (vol/vol) Tween 20 and incubated with specific primary antibodies. After washing and incubating with secondary antibodies, immunoreactive proteins were visualized by the ECF chemiluminescence system following the manufacturer’s instructions. Proteins were revealed and quantified by PhosphorImager analysis and were normalized to total protein using the STORM 860 system (Molecular Dynamics, Amersham Pharmacia Biotech, Little Chalfont, UK). The molecular weights of the respective proteins were determined from the relative mobility on SDS-PAGE compared with molecular weight standards. Where indicated, the membranes were stripped and reprobed with another antibody. Data are presented as the mean ± SEM from four independent experiments.

In situ hybridization

The site of FP receptor mRNA expression was localized in endometrial adenocarcinoma tissues by in situ hybridization. Custom synthesis oligonucleotide fluorescein isothiocyanate (FITC) double-labeled cDNA probes for FP receptor were obtained from Biognostik GmbH (Gottingen, Germany). Sections (5 µm) were cut onto gelatin-coated SuperFrost slides (BDH Laboratory Supplies, Butterworth, UK) from the human adenocarcinoma tissue collected (n = 12). Tissue was dewaxed in xylene, rehydrated using decreasing concentrations of ethanol before proteinase K treatment (100 µg/ml in 100 mM Tris-HCl (pH 7.6) and 50 mM EDTA) for 15 min at 37 C to enhance cDNA probe access. After washing in diethylpyrocarbonate-H2O, sections were prehybridized for 4 h at 30 C with hybridization mixture (50 µl; supplied with probe) before adding cDNA probe (6 U/ml hybridization mix) and incubating overnight at 30 C. Posthybridization washes of 1x saline sodium citrate for 5 min (twice) and 0.1x saline sodium citrate at 42 C for 15 min (twice) were completed before detecting the FITC-labeled probe using standard immunocytochemical reagents (TSA Biotin System, NEN Life Science Products). Endogenous peroxidase activity was first blocked with 3% H2O2 in methanol for 30 min before incubating sections with blocking buffer for 30 min. Conjugated anti-FITC-horseradish peroxidase (Roche, Lewes, UK) was added in blocking buffer, and the sections were incubated for 60 min. After washing, biotinyltyramide amplification reagent was applied to each slide and incubated for 15 min. Streptavidin-horseradish peroxidase (DAKO, Buckinghamshire, UK) was applied after washing and incubated for 30 min, and probe localization was visualized with 3,3'-diaminobenzidine (DAKO). Control oligonucleotide FITC double-labeled cDNA probe containing the same proportion of cysteine (C) and guanine (G) bases as the FP receptor probe was included to assess background hybridization. All treatments were carried out at room temperature unless otherwise specified.

Immunohistochemistry

The site of FP receptor protein expression was localized in endometrial adenocarcinoma tissues by immunohistochemistry (n = 12). Five-micrometer paraffin wax-embedded tissue sections were cut and mounted onto coated slides (TESPA, Sigma-Aldrich Corp.). Sections were dewaxed in xylene, rehydrated in graded ethanol, and washed in water, followed by Tris-buffered saline (50 mM Tris-HCl and 150 mM NaCl, pH 7.4), then blocked for endogenous endoperoxidase (1% H2O2 in methanol). Antigen retrieval was performed by pressure-cooking for 2 min in 0.01 M sodium citrate, pH 6 (29). Sections were blocked using 5% normal swine serum diluted in Tris-buffered saline. Subsequently, the tissue sections were incubated with polyclonal rabbit anti-FP receptor antibody at a dilution of 1:200 at 4 C for 18 h. Control tissue was incubated with rabbit IgG. Thereafter, the tissue sections were incubated with secondary swine antirabbit antibody, followed by streptavidin-peroxidase complex (DAKO) at 25 C for 20 min. Color reaction was developed by incubation with 3,3'-diaminobenzidine (DAKO). The tissue sections were counterstained in aqueous hematoxylin, followed by sequential dehydration using graded ethanol and xylene, before mounting and coverslipping.

Total IP3 assays

PGF2{alpha} stimulation of total IP3 production was measured as previously described (30, 31). Briefly, endometrial adenocarcinoma tissue was incubated with inositol-free DMEM containing 1% dialyzed heat-inactivated FCS and 0.5 µCi/well myo-[3H]inositol (Amersham Pharmacia Biotech) for 48 h. Medium was removed, and the tissue was washed with 1 ml buffer (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM glucose, 1 mM MgCl2, 1 mM CaCl2, and 1 mg/ml BSA) containing 10 mM LiCl. Tissue explants were then incubated for 1 h at 37 C in 1 ml buffer with either PGF2{alpha} or vehicle. Reactions were terminated by the removal of agonist and the addition of 500 µl ice-cold 10 mM formic acid, which was incubated for 30 min at 4 C. Total [3H]IP3 was separated from the formic acid cell extracts on AG 1-X8 anion exchange resin (Bio-Rad Laboratories, Hercules, CA) and eluted with a 1 M ammonium formate/0.1 M formic acid solution. The associated radioactivity was determined by liquid scintillation counting and was plotted relative to protein concentrations in the tissue explants determined using protein assay kits (Bio-Rad Laboratories).

Proliferation assay

The proliferation of Ishikawa cells was determined using a CellTitre 96AQueous One Solution cell proliferation assay (Promega Corp., Madison, WI). Briefly, Ishikawa cells were seeded at 4 x 103 cells/well in a 96-well plate and allowed to adhere overnight. Cells were next starved for 24 h with 8.4 µM indomethacin before the addition of 100 nM PGF2{alpha} or vehicle in serum-free medium. In parallel, wells were treated with serum-free medium containing PGF2{alpha} and indomethacin in the presence or absence of inhibitors for 24 h. Control wells received the same concentration of vehicle alone or vehicle and inhibitor. After 24-h treatment, proliferation was measured by the addition of the CellTitre 96AQueous One Solution reagent according to the manufacturer’s protocol. Cells were then incubated for 3 h at 37 C in 5% CO2 (vol/vol) to reduce the tetrazolium compound to a 490 nm absorbing formazan compound. Data are presented as the mean ± SEM from three independent experiments.

Statistics

Where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference test (StatView 4.0, Abacus Concepts Inc., Berkeley, CA). Statistical significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The expression of FP receptor mRNA in normal and endometrial adenocarcinoma samples was determined by TaqMan quantitative RT-PCR (Fig. 1Go, A and B) and Western blot analysis (Fig. 1CGo). The expression of FP receptor mRNA was significantly up-regulated in all cases of poorly differentiated, moderately differentiated, and well differentiated endometrial adenocarcinomas investigated (Fig. 1AGo) compared with normal endometrium (Fig. 1BGo). No correlation was observed between the different grades of adenocarcinoma; however, a large variance was observed within these tissues, as shown in Fig. 1AGo. The relative expression of endometrial adenocarcinoma (n = 24) and that of normal endometrium across the cycle (n = 12) were determined to be 581.2 ± 318.4 and 0.8 ± 0.2, respectively (Fig. 1BGo; P < 0.001). Western blot analysis confirmed the elevated expression of FP receptor protein in endometrial adenocarcinoma samples (Fig. 1CGo; showing three representative samples for each grade of carcinoma studied) compared with normal endometrium across the menstrual cycle [Fig. 1CGo; showing representative samples across the menstrual cycle for the early secretory-late secretory phase (N1, N2), midproliferative phase (N3, N4), and late proliferative phase (N5, N6)].



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  		FIG. 1. A, The relative expression of FP receptor RNA in poorly differentiated (P), moderately differentiated (M), and well differentiated (W) endometrial adenocarcinomas as determined by real-time quantitative RT-PCR analysis. B, The relative expression of FP receptor RNA in endometrial adenocarcinomas compared with that in normal endometrial tissue was determined by real-time quantitative RT-PCR analysis (*, P < 0.001). C, Western blot analysis of 50 µg total protein isolated from endometrial adenocarcinoma tissue compared with normal endometrium across the cycle. The proteins were loaded onto a 4–20% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and subsequently transferred to a polyvinylidene difluoride membrane. The immunoblot was probed with antibody raised against the COOH terminus of human FP receptor. A band corresponding to 64 kDa was detected in all adenocarcinoma samples (three representative samples for each grade of differentiation are displayed) and normal endometrial samples across the menstrual cycle [two representative samples from the early-late secretory phase (N1, N2), midproliferative phase (N3, N4), and late proliferative phase (N5, N6) are displayed].

 
The site of expression of FP receptor mRNA and protein was determined by in situ hybridization (Fig. 2Go, A-C) and immunohistochemistry (Fig. 2Go, D-F), respectively. FP receptor expression in human adenocarcinoma biopsies was localized to glandular epithelial cells in all adenocarcinoma samples investigated (n = 12). Epithelial cell expression of FP receptor mRNA and protein was observed in all differentiation types, with no discernible change in mRNA and protein pattern among poorly (Fig. 2Go, A and D), moderately (Fig. 2Go, B and E), or well differentiated (Fig. 2Go, C and F) samples, respectively. Incubating sections with the control oligonucleotide (Fig. 2CGo, inset) or nonimmune rabbit IgG (Fig. 2FGo, inset) in place of the cDNA probe or primary antibody abolished the positive staining. In addition, FP receptor mRNA and protein were observed in perivascular cells (arrowhead). A magnified blood vessel showing positive immunoreactive staining of FP receptor in perivascular cells is shown in Fig. 2D.



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  		FIG. 2. Localization of FP receptor mRNA (A–C) and protein expression (D–F) by in situ hybridization and immunohistochemistry, respectively, in poorly differentiated (A and D), moderately differentiated (B and E), and well-differentiated (C and F) endometrial adenocarcinomas. Blood vessels are shown by the arrowhead, and perivascular staining is shown surrounding the magnified blood vessel in D. Insets are shown for negative controls for in situ hybridization (C) and immunohistochemistry (F), respectively. Scale bar, 100 µm.

 
To assess functional activation of the FP receptor, the release of IP3 from endometrial carcinoma tissue explants was measured after treatment with 100 nM PGF2{alpha} (Fig. 3Go). This concentration of PGF2{alpha} was determined previously as the most effective dose (31). PGF2{alpha} induced a significant (P < 0.01) increase in total IP3 release in carcinoma tissue compared with control tissue treated with vehicle alone (24.5 ± 1.6 vs. 10 ± 0.4 counts/µg protein; n = 6).



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  		FIG. 3. Total [3H]IP3 release in endometrial adenocarcinoma explants treated with 100 nM PGF2{alpha} or vehicle for 1 h at 37 C (n = 6). *, P < 0.01 compared with control. Data are presented as the mean ± SEM.

 
As activation of EGFR expression and signaling is up-regulated in most neoplasms (32), and trans-activation of EGFR expression has been observed in colon cancer cells by PGE2 (33), we next investigated whether PGF2{alpha}-induced signaling in endometrial adenocarcinomas and endometrial adenocarcinoma cells (Ishikawa cells) involved cross-talk with the EGFR signaling system. Endometrial adenocarcinoma explants (Fig. 4AGo) and Ishikawa cells (Fig. 4BGo) were treated with 100 nM PGF2{alpha} or vehicle alone for 10 min, and EGFR phosphorylation was assessed by Western blotting. A significant increase in tyrosine phosphorylation of EGFR was observed in endometrial adenocarcinoma explants (Fig. 4AGo, lane 2; P < 0.05) and Ishikawa cells (Fig. 4BGo, lane 2; P < 0.05) in response to stimulation with PGF2{alpha} compared with control tissue (Fig. 4AGo, lane 1; P < 0.05) and Ishikawa cells stimulated with vehicle alone (Fig. 4BGo, lane 1; P < 0.05). Treatment of endometrial adenocarcinoma explants and Ishikawa cells with the EGFR kinase inhibitor AG1478 and the PLCß inhibitor U73122 abolished this action of PGF2{alpha} (P < 0.05). Data are shown as the mean ± SEM from four independent experiments.



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  		FIG. 4. PGF2{alpha} trans-activates EGFR signaling in endometrial adenocarcinoma explants (A) and Ishikawa cells (B). Endometrial adenocarcinoma explants and Ishikawa cells were pretreated for 1 h with inhibitors or vehicle, followed by stimulation with vehicle alone (control; lane 1), 100 nM PGF2{alpha} (lane 2), PGF2{alpha} and U73122 (lane 3), or PGF2{alpha} and AG1478 (lane 4) for 10 min. After lysis, EGFR was immunoprecipitated (IP) with anti-EGFR antibody, and tyrosine-phosphorylated EGFR was detected by immunoblotting (WB) with antiphosphotyrosine antibody (A and B, top panel). The total amount of EGFR in immunoprecipitates was determined by reprobing the same blot with anti-EGFR antibody (A and B, lower panel). Quantitative analysis of EGFR phosphorylation was determined from four independent experiments by determining the ratio between EGFR protein and tyrosine phosphorylation levels. Data are presented as the mean ± SEM. *, P < 0.05.

 
The effect of PGF2{alpha} on the downstream ERK2 signaling pathway was determined after treatment of endometrial adenocarcinoma explants (Fig. 5AGo) or Ishikawa cells (Fig. 5BGo) with 100 nM PGF2{alpha}. Treatment of carcinoma explants and Ishikawa cells resulted in a significant increase in phosphorylation of ERK after stimulation with 100 nM PGF2{alpha} for 10 min (Fig. 5Go, A and B, lane 2; P < 0.05). Administration of specific inhibitors of PLCß (U73122; P < 0.01), MEK (PD98059; P < 0.01), and EGFR kinase (AG1478; P < 0.05) abolished phosphorylation of ERK in endometrial adenocarcinoma explants as well as Ishikawa cells. Data are shown as the mean ± SEM from four independent experiments.



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  		FIG. 5. PGF2{alpha} activation of MAPK signaling in endometrial adenocarcinoma explants (A) and Ishikawa cells (B). Endometrial adenocarcinoma explants and Ishikawa cells were pretreated with inhibitors or vehicle (control) for 1 h, followed by stimulation with vehicle (lane 1), 100 nM PGF2{alpha} (lane 2), PGF2{alpha} and U73122 (lane 3), PGF2{alpha} and PD98059 (lane 4), or PGF2{alpha} and AG1478 (lane 5) for 10 min. After lysis and quantification, 50 µg total protein were electrophoresed and subjected to immunoblot analysis with antiphospho-ERK antibody. Blots were stripped and reprobed with total ERK antibody. Quantitative analysis of ERK phosphorylation was determined from the ratio between phosphorylated protein and total protein. Data are presented as the mean ± SEM from four independent experiments. *, P < 0.05.

 
The effect of PGF2{alpha} on the proliferation of endometrial adenocarcinoma cells was determined in Ishikawa cells. Treatment of Ishikawa cells with 100 nM PGF2{alpha} resulted in a 30.4 ± 2.1% increase in proliferation compared with vehicle-treated cells (Fig. 6Go; P < 0.05). Treatment of cells with specific inhibitors of PLCß (U73122; P < 0.05), EGFR kinase (AG1478; P < 0.05), and MEK (PD98059; P < 0.05) abolished the PGF2{alpha}-induced increase in cell proliferation. Incubation of cells with the specific inhibitors on their own (control cells) did not result in any significant alteration in cellular proliferation over the 24-h period at the concentrations used compared with cells treated with vehicle alone.



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  		FIG. 6. The effect of PGF2{alpha} on Ishikawa cell proliferation. Generation of formazan salt from tetrazolium compound at 492 nm after 3 h of incubation with the CellTitre 96AQueous One Solution reagent. Ishikawa cells were treated for 24 h with vehicle, 100 nM PGF2{alpha}, PGF2{alpha} and U73122, PGF2{alpha} and AG1478, or PGF2{alpha} and PD98059 ({blacksquare}) or with vehicle and inhibitor alone ({square}; control) before addition of the CellTitre 96AQueous One Solution reagent. Data are presented as the mean ± SEM from three independent experiments. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous in vitro studies have demonstrated a role for COX enzymes, prostanoids, and prostanoid receptors in reproductive tract dysfunction (4, 5, 7, 8, 24, 34, 35). In the reproductive tract, the E and F series of prostanoids are the bioactive lipids most abundantly synthesized by COX enzymes. Recent studies have demonstrated that COX enzyme and PGE2 receptor expression and signaling are elevated in endometrial carcinomas (4, 5). However, little has been reported about the role of PGF2{alpha} in reproductive tract pathology. The present study has demonstrated elevated FP receptor expression in human endometrial adenocarcinomas of all grades and differentiation, as confirmed by quantitative RT-PCR and Western blot analysis. The site of FP receptor expression was localized to the neoplastic glandular epithelial cells, with some diffuse perivascular expression in all adenocarcinomas investigated. These data suggest an autocrine/paracrine regulation of epithelial cell function by PGF2{alpha} in endometrial adenocarcinomas. Moreover, the perivascular expression of FP receptor in endometrial adenocarcinomas suggests a possible paracrine regulation of tumor vascular function by PGF2{alpha}. A relationship between PGF2{alpha} and bleeding disorders of the endometrium has been previously described (24, 34, 36). Elevated PGE2 and PGF2{alpha} concentrations have been reported and are associated with excessive menstrual blood loss in patients diagnosed with menorrhagia (24, 34, 35).

The FP receptor is a Gq-coupled heptahelical transmembrane receptor, which, when bound with PGF2{alpha}, activates PLCß signaling, giving rise to the release of IP3 and diacylglycerol (18). In this study the functionality of FP receptor in endometrial adenocarcinomas was demonstrated by the release of IP3 in endometrial adenocarcinoma explants in response to treatment with PGF2{alpha}. These data confirm that PGF2{alpha} mediates an autocrine/paracrine effect in endometrial adenocarcinoma tissue via interaction with the FP receptor and triggers intracellular signaling by activation of the PLCß pathway.

We examined whether mitogenic signaling in endometrial adenocarcinoma explants and Ishikawa endometrial adenocarcinoma cells involved cross-communication with the PGF2{alpha} and EGFR signaling systems. Treatment of adenocarcinoma explants and Ishikawa cells with PGF2{alpha} resulted in a significant tyrosine phosphorylation of EGFR. Cotreatment with specific inactivators of PLCß and EGFR kinase activity abolished EGFR activation, indicating that EGFR phosphorylation was mediated by the PLCß pathway, which was activated by PGF2{alpha}. Several mechanisms are proposed for the transduction of EGFR by G proteins (37, 38). One of these mechanisms involves the activation of transmembrane matrix metalloproteinase and cleavage of EGF-ligand bound in close proximity to the EGFR. Once cleaved, the EGF ligand can associate with and activate the EGFR. Alternatively, several reports have demonstrated that activation of the Src family of nonreceptor tyrosine kinases is involved in GPCR-mediated EGFR trans-activation by activation of intracellular scaffold proteins and protein phosphorylation cascades (37, 38). The mechanism behind the PGF2{alpha}-induced trans-activation of EGFR in endometrial adenocarcinomas remains to be fully elucidated and may involve one or more of these pathways acting in concert. Studies in our laboratory are currently underway to determine this mechanism.

The integrated response to activation of EGFR kinase results in numerous downstream signaling events being initiated. These include activation of small GTPases such as Rho, Rac, Ras, and STAT (signal transducer and activator of transcription), leading to activation of MAPK (39). Activation of GPCR and that of MAPK signaling are known to be potent regulators of cell growth, differentiation, and development (40). Activation of MAPK signaling by PGF2{alpha} has been observed in other cells of the reproductive tract, such as luteal (41) and granulosa cells (42), although the exact role of PGF2{alpha}-induced ERK activation in these systems has not been clarified. We examined whether activation of EGFR by PGF2{alpha} stimulates the downstream MAPK/ERK pathway. PGF2{alpha}-induced activation of EGFR in endometrial adenocarcinoma explants and Ishikawa cells was accompanied by a significant increase in ERK activation, which was inhibited by cotreatment with inhibitors of PLCß, EGFR kinase, and MEK, respectively.

To determine the effect of EGFR kinase signaling initiated by PGF2{alpha}-FP receptor interaction on proliferation of neoplastic endometrial epithelial cells, we used the Ishikawa endometrial epithelial cell line. We have demonstrated that PGF2{alpha} promotes the proliferation of Ishikawa cells, which is mediated by the EGFR and MAPK signaling pathways, after activation of PLCß, as inhibition of PLCß, EGFR kinase, and MEK abolished the proliferative effects of PGF2{alpha} on Ishikawa cells. In addition, as incubation of Ishikawa cells with inhibitors on their own had no significant effect on cell growth and proliferation, the observed increase in proliferation due to FP-PGF2{alpha} ligand interaction and intracellular signal transduction was thus due to the actions of PGF2{alpha} alone. These data suggest that PGF2{alpha}-FP receptor interaction and activation of EGFR and MAPK signaling may promote tumorigenesis in endometrial adenocarcinomas by enhancing the cellular machinery involved in cell growth. Interestingly, we determined previously that FP receptor expression and signaling are increased in normal endometrium during the proliferative phase of the menstrual cycle (31). This phase of the cycle is characterized by rapid and intense proliferation of the glandular epithelium. Whether the proliferation of epithelium in normal cycling endometrium and that in endometrial adenocarcinoma share common autocrine/paracrine signaling pathways remains to be elucidated.

In summary, these data demonstrate elevated and functional FP receptor expression in human endometrial adenocarcinomas of all grades and differentiation. In addition, as summarized schematically in Fig. 7Go, PGF2{alpha} induces the proliferation of neoplastic endometrial epithelial cells by activation of kinase signaling via the PLCß and MAPK pathways by a mechanism involving trans-activation of the EGFR. These data suggest that inhibition of PGF2{alpha} biosynthesis with COX enzyme inhibitors, FP receptor antagonists, or inactivators of EGFR signaling may be efficacious as medical intervention therapies for women with endometrial adenocarcinoma, as they would block the proliferative effects brought about by elevated FP-PGF2{alpha} interaction in epithelial cells.



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  		FIG. 7. A summary diagram showing the molecular signaling events triggered by PGF2{alpha} in endometrial adenocarcinomas. An elevated FP-PGF2{alpha} interaction in endometrial adenocarcinomas enhances endometrial epithelial cell proliferation by mechanisms involving activation of the PLCß, EGFR, and MAPK signaling systems.

 


   Acknowledgments
 
We thank Dr. A. Pawson, Dr. S. Maudsley, and Ms. S. Boddy for helpful discussion and technical assistance during this study.


   Footnotes
 
Abbreviations: COX, Cyclooxygenase; d, deoxy; DMSO, dimethylsulfoxide; EGF, epidermal growth factor; EGFR, EGF receptor; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; FP, GPCR for prostaglandin F2{alpha}; GPCR, G protein receptor; IP3, inositol trisphosphate; MEK, MAPK kinase; PGF2{alpha}, prostaglandin F2{alpha}; PLC, phospholipase C.

Received August 19, 2003.

Accepted November 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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