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Endocrinology, doi:10.1210/en.2005-1429
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Endocrinology Vol. 147, No. 7 3356-3365
Copyright © 2006 by The Endocrine Society

Seminal Plasma Promotes the Expression of Tumorigenic and Angiogenic Genes in Cervical Adenocarcinoma Cells via the E-Series Prostanoid 4 Receptor

Melissa Muller, Kurt J. Sales, Arieh A. Katz and Henry N. Jabbour

Medical Research Council Human Reproductive Sciences Unit (M.M., K.J.S., H.N.J.), The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom; and Institute of Infectious Disease and Molecular Medicine (M.M., A.A.K.), Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Cape Town, Rondebosch 7701, South Africa

Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, The Queen’s Institute for Medical Research, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
E-series prostanoid (EP)4 receptor is up-regulated in numerous cancers, including cervical carcinomas, and has been implicated in mediating the effects of prostaglandin (PG)E2 in tumorigenesis. In addition to regulation by endogenously biosynthesized PGE2, neoplastic cervical epithelial cells in sexually active women may also be regulated by PGs present in seminal plasma. In this study, we investigated the signal transduction pathways mediating the role of seminal plasma and PGE2 in the regulation of tumorigenic and angiogenic genes via the EP4 receptor in cervical adenocarcinoma (HeLa) cells. HeLa cells were stably transfected with EP4 receptor in the sense orientation. Seminal plasma and PGE2 signaling via the EP4 receptor induced the activation of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) promoters, expression of COX-2 and VEGF mRNA and protein, and secretion of VEGF protein into the culture medium. Treatment of HeLa cells with seminal plasma or PGE2 also rapidly induced the phosphorylation of ERK1/2 via the EP4 receptor. Preincubation of cells with a specific EP4 receptor antagonist (ONO-AE2-227) or chemical inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase or MAPK kinase or cotransfection of cells with dominant-negative mutant cDNA targeted against the EGFR, serine/threonine kinase Raf, or MAPK kinase abolished the EP4-induced activation of COX-2, VEGF, and ERK1/2. Therefore, we have demonstrated that seminal plasma and PGE2 can promote the expression of tumorigenic and angiogenic factors, in cervical adenocarcinoma cells via the EP4 receptor, EGFR, and ERK1/2 signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CANCER OF THE cervix is one of the leading causes of cancer-related morbidity and mortality in women world wide, with a high incidence reported in less developed countries (1, 2). There is strong epidemiological and experimental evidence to support a role for the human papillomavirus (HPV) in cervical neoplastic transformation (3, 4, 5). However, it takes on average 12–15 yr before a persistent HPV infection manifests in cervical carcinoma (6, 7). Thus, there are many other causative factors that are thought to contribute toward the progression of the disease, including sexually transmitted infection, multiple sexual partners, and exposure of neoplastic epithelial cells to seminal plasma (1, 8, 9, 10).

Over the last 10 yr, numerous studies using gene-disruption and gene overexpression systems in cell lines and laboratory animals have provided evidence to support a role for cyclooxygenase (COX) enzymes, prostaglandins (PGs), and PG receptors in pathology (11, 12). We and others have recently determined elevated COX enzyme expression in cervical carcinomas (13, 14, 15, 16). Elevated COX enzyme expression has been shown to promote tumor cell proliferation, reduce apoptosis, and induce angiogenesis in vitro (17, 18, 19). As a result, tumors expressing COX-2 are reported to exhibit a more aggressive phenotype (20). Women whose tumors overexpress COX-2 tend to have a lower response to standard therapy and thus shorter survival times (20). Furthermore, in vitro studies have shown that COX-2 inhibitors can inhibit tumor cell growth and reduce tumor angiogenesis (21). Thus, although HPV infection may be the initiating step in cervical neoplasias, elevated COX enzyme expression and prostanoid biosynthesis and signaling may enhance progression or onset of cervical cancer.

COX enzymes together with specific PG synthase enzymes (such as PGE synthase) catalyze the rate-limiting step in the conversion of arachidonic acid to PGs, including PGE2 (22, 23, 24). After biosynthesis, PGE2 mediates its effect in an autocrine/paracrine manner through interaction with G protein-coupled receptors (GPCRs), which have been pharmacologically classified as E-series prostanoid (EP) receptor, EP1-EP4.

A role for prostanoid receptors in reproductive tract carcinomas has been recently described (12). In cervical and endometrial carcinomas, expression and signaling of EP receptors (namely EP2 and EP4 receptor) is elevated (12, 15, 16, 25), suggesting an autocrine/paracrine regulation of neoplastic cell function by PGE2. Enhanced PGE2-EP2 receptor interaction has also recently been correlated with tumor angiogenesis in knockout mouse and in vitro model systems (15, 25, 26, 27, 28, 29). In addition to the actions of PGE2 in promoting tumorigenesis via the EP2 receptor, a direct role for PGE2 and EP4 receptor in colorectal tumorigenesis has been ascertained. In these studies, enhanced proliferative and tumorigenic effects are mediated by PGE2 after interaction with the EP4 receptor (30). Further evidence for a role for the EP4 receptor in tumor growth has been shown recently by Fujino and colleagues (23, 31) and Pozzi et al. (32), where PGE2 via the EP4 receptor can induce the expression of early growth response factor and enhance cellular proliferation via multiple signaling pathways by activation of protein kinase B/Akt and ERK1/2 (23, 31, 32, 33).

In addition to activation of EP receptors by endogenously synthesized PGE2, neoplastic cervical epithelial cells in sexually active women can potentially be under direct stimulation of PGE2 present in seminal plasma, in the absence of barrier contraception. Prostaglandin concentration in seminal plasma is 10,000 times higher than that found at a site of inflammation, and PGE2 is the predominant type of PG detected (34, 35). In these women, up-regulation of expression of genes that may modulate tumorigenesis and angiogenesis of neoplastically transformed cervical tissue may be regulated in part by endogenous expression of PGE2, as well as PGE2 present in seminal plasma. Indeed, repeated exposure of neoplastic cervical epithelial cells to seminal plasma has been shown to promote the release of matrix metalloproteinases, which, in turn, can cause degradation of the extracellular matrix and enhance metastases and cervical tumorigenesis (36).

In a previous study, we demonstrated that seminal plasma and PGE2 could promote the expression of the EP4 receptor in HeLa cervical adenocarcinoma cells (37). This lead us to postulate that enhanced prostanoid-EP4 receptor signaling may potentially enhance cervical tumorigenesis and that this may be further exacerbated by exposure of EP4 receptor-overexpressing cells to seminal plasma. In the present study, we investigated the potential role of seminal plasma and PGE2 in modulating intracellular signaling and expression of tumorigenic and angiogenic genes via the EP4 receptor in HeLa cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
All culture medium was purchased from Life Technologies (Paisley, UK). Penicillin-streptomycin and fetal calf serum was purchased from PAA Laboratories Ltd. (Middlesex, UK). The cMyc mouse monoclonal antibody (sc-40) and ERK goat polyclonal antibody (sc-93) were purchased from Santa Cruz Biotechnology (Autogen-Bioclear, Wiltshire, UK). The EP4 receptor rabbit polyclonal antibody (101775) was purchased from Cayman Chemical Co. (Alexis Corp., Nottingham, UK). Anti-phospho-p42/44 ERK (9101) was purchased from Cell Signaling Technologies (New England Biolabs, Hertforshire, UK). Antigoat, antirabbit alkaline phosphatase secondary antibodies, PBS, BSA, and PGE2 were purchased from Sigma Chemical Company (Dorset, UK). An enhanced chemofluorescence system was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). PD98059 (18.7 mM stock in dimethylsulfoxide, used at a final concentration of 50 µM), AG 1478 (10 mM stock in dimethylsulfoxide, used at a final concentration of 200 nM), and Sulprostone (10 mM stock in ethanol, used at a final concentration of 1 µM) were purchased from Calbiochem (Nottingham, UK) and stored at –20 C. The dominant negative Raf, epidermal growth factor receptor (EGFR), and MAPK kinase (MEK) cDNAs were a kind gift from Prof. Zvi Naor (Department of Biochemistry, Tel Aviv University, Tel Aviv, Israel) and have been previously described (38, 39). EP4 antagonist (ONO-AE2-227; 10 mM stock in ethanol, used at a final concentration of 1 µM) was chemically synthesized by Charnwood Molecular Ltd. (Leicester, UK). ONO-AE2-227 has a Ki of 2.7 nM for EP4 receptor and 21 nM for EP3 receptor. The Ki values for the EP1, EP2, T-series prostanoid (TP), D-series prostanoid (DP), F-series prostanoid (FP), and I-series prostanoid (IP) receptor are more than 1000-fold higher than for EP4 receptor (40).

Cell culture
Human cervical adenocarcinoma (HeLa) cells (BioWhittaker, Berkshire, UK) were maintained in DMEM nutrient mixture F-12 with Glutamax-1 and pyridoxine, supplemented with 10% fetal calf serum, and 1% antibiotics (stock 500 IU/ml penicillin and 500 µg/ml streptomycin) at 37 C and 5% CO2 (vol/vol). EP4 stable transfectants were maintained under the same conditions with addition of a maintenance dose of 200 µg/ml G418.

EP4 receptor cell transfections
The EP4 receptor cDNA was kindly supplied by Dr. Mark Abramovitz (Merck Frosst Centre for Therapeutic Research, Quebec, Canada) and was transfected into HeLa cells in the sense orientation using Superfect (QIAGEN, Crawley, UK), and individual populations were selected for with addition of 200 µg/ml G418. Full selection was confirmed by the 100% death of nontransfected control cells, and approximately 15 sense EP4 clones were selected, expanded, and subjected to quantitative RT-PCR and Western blot analysis. Based on the mRNA and protein expression of the EP4 receptor present in wild-type (WT) untransfected cells, three transfectant clones with the highest level of EP4 receptor expression were expanded and stored in liquid nitrogen. All positive clones showed similar physiological, phenotypic, biochemical, and pharmacological characteristics and exhibited similar EP4 receptor expression as we observed for WT cells incubated with seminal plasma and PGE2 in our previous study. The results of our studies using sense clone 1 are presented in this study.

Seminal plasma collection
Semen was collected from healthy male volunteers with Lothian research committee ethics approval. Seminal plasma was isolated from the pooled ejaculate by percoll density gradient centrifugation at 500 x g for 20 min. The seminal plasma was pooled and stored at –70 C. The PGE2 concentration in the pooled seminal plasma was determined by ELISA as described previously (16) to be 43.5 ± 8.7 µg/ml. The seminal plasma was used on the cells at a 1:500 dilution. At this dilution, the PGE2 present in the pooled ejaculate corresponds comparably with the concentration of PGE2 used in this study. At this dilution, seminal plasma has been reported to exert no adverse effect on HeLa cell viability (41).

Laser confocal immunofluorescent microscopy
EP4 receptor expression was visualized in WT and HeLa EP4S cells by laser confocal immunofluorescence microscopy. Approximately 10,000 WT and sense cells were seeded in chamber slides and allowed to adhere overnight, before being fixed in 100% ice-cold methanol. After fixing, cells were washed in Tris-buffered saline [50 mM Tris-HCl, 150 mM NaCl (pH 7.4)] and blocked using 5% normal swine serum diluted in Tris-buffered saline. Subsequently, the cells were incubated with polyclonal rabbit anti-EP4 receptor antibody at a dilution of 1:50 at 4 C for 18 h. Control cells were incubated with rabbit IgG. Thereafter, the cells were incubated with secondary swine antirabbit tetramethyl rhodamine isothyocyanate (Dako Corp., High Wycombe, UK) at 25 C for 20 min. Cells were then mounted in Permafluor (Immunotech-Coulter, Buckinghamshire, UK) and coverslipped. Fluorescent images were visualized and photographed using a Carl Zeiss (Jena, Germany) laser scanning microscope LM510. The Alexafluor 546 and 488 was captured using the helium/neon 1 laser (excitation beam, 543 nm) and an emission band pass filter 560–615.

TaqMan quantitative RT-PCR
EP1, EP2, EP3, EP4 receptor, COX-2, and vascular endothelial growth factor (VEGF) mRNA expression was measured by quantitative RT-PCR analysis. Approximately 5 x 105 cells were seeded in 5-cm dishes and allowed to attach overnight. The following day, cells were harvested in Tri-Reagent (Sigma) for determination of expression of EP receptors as described previously (16), or serum-starved for at least 16 h and treated with vehicle or 1 µM sulprostone or pretreated with specific inhibitors of EGFR kinase (AG1478) or MEK (PD98059) or the EP4 antagonist (ONO-AE2-227) for 1 h and then stimulated with vehicle, 1:500 dilution of seminal plasma, or 300 nM PGE2 (for the time periods specified in the figures). Quantitative RT-PCR was performed as described previously (16, 29). PCR was carried out using an ABI prism 7700 under standard manufacturer’s conditions. EP receptor, COX-2, and VEGF primers and probe for quantitative RT-PCR were designed using the PRIMER express program (PE Biosystems, Warrington, UK) as described (16, 29). Expression of EP receptors/COX-2/VEGF was normalized to RNA loading for each sample using the 18s ribosomal RNA as an internal standard. An additional control cDNA of human endometrium was included in each run to standardize conditions and normalize the data. Fold induction was calculated by dividing the relative mRNA expression of cells treated with seminal plasma or PGE2 by the relative mRNA expression in vehicle-treated cells at the same time point.

Protein extraction
Approximately 1 x 106 HeLa WT and EP4S cells were seeded in 5-cm dishes. The following day, cells were washed in PBS and incubated in serum-free culture medium containing penicillin/streptomycin (as described in Cell culture) for at least 16 h. For inhibitor experiments, cells were pretreated with specific inhibitors of EGFR kinase (AG1478) and MEK (PD98059) or the EP4 antagonist (ONO-AE2-227) for 1 h before stimulation with 1:500 dilution of seminal plasma or 300 nM PGE2 (for the time periods specified in the figures). After stimulation, cells were washed with ice-cold PBS. Proteins were extracted with a protein lysis buffer as described previously (29). The protein content of the clarified cell lysate was determined using a protein assay kit (Bio-Rad, Hemel Hempstead, UK). For Western blot analysis, approximately 20 µg of total protein lysate was loaded on to polyacrylamide gels and immunoblotted as described (29).

Immunoprecipitation and Western blot analysis
To confirm the role of EGFR and MEK in seminal plasma- and PGE2-mediated ERK1/2 phosphorylation, we used a dominant-negative (DN) mutant EGFR, DNRaf, and DNMEK. HeLa cells were seeded to a density of 5 x 105 per well in 6-cm dishes and then transfected with a c-Myc-tagged ERK1/2 cDNA construct together with either empty vector cDNA (pcDNA3; Invitrogen, de Schelp, The Netherlands) or DNEGFR cDNA, DNRaf cDNA, or DNMEK using Superfect (Qiagen) as per the manufacturer’s protocol. Optimal concentrations of cDNA for transfection was determined by titration and the transfection efficiency of the HeLa cell line by transfection with a pcDNA6/V5/His/lacZ cDNA construct (Invitrogen) and ß-galactosidase assay. Transfection efficiency using the standard manufacturer protocol is 40 ± 2%. The tagged ERK1/2 was immunoprecipitated from whole cell lysate. For immunoprecipitation studies, equal amounts of protein were incubated with specific cMyc antibody preconjugated to protein A-Sepharose antibodies overnight at 4 C with gentle rotation. Beads were washed extensively with lysis buffer and immune complexes solubilized in Laemmli buffer and resolved on polyacrylamide gels and immunoblotted as described (29). Membranes were blocked for 1 h at 25 C in 4% BSA diluted in Tris-buffered saline with Tween 20 (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 alkaline-phosphatase-conjugated secondary antibodies, immunoreactive proteins were visualized by the enhanced chemofluorescence system according to the manufacturer’s instructions. Proteins were revealed and quantified using PhosphorImager analysis using the Typhoon 9400 system (Molecular Dynamics, Amersham Biosciences) and normalized for protein loading using ß-actin or Total ERK. Fold increase was determined by dividing the protein expression of stimulated cells by the relative expression obtained from vehicle-treated cells.

COX-2 and VEGF luciferase reporter assays
The COX-2 promoter reporter plasmid [consisting of a 966-bp fragment of the COX-2 promoter from –917 to +49 as described in Bradbury et al. (42) and kindly supplied by Dr. Robert Newton (BioMedical Research Institute, Department of Biological Sciences, University of Warwick, Coventry, UK) or the VEGF promoter reporter plasmid [2324 kb fragment consisting of VEGF 5' sequences from –2274 to +50 ligated to a firefly luciferase construct and kindly supplied by Prof. Keping Xie (Department of Gastrointestinal Medical Oncology and Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX) (43)] or empty vector pGL3-basic cDNA containing the firefly luciferase reporter was cotransfected into HeLa cells in triplicate with an internal control pRL-TK (containing the renilla luciferase coding sequence; Promega, Southampton, UK) and either control vector (pcDNA3.0) or vector encoding a DN isoform of Raf, EGFR, or MEK. Cells were transfected using Superfect (QIAGEN) for 6 h. The next day, the cells were serum-starved for at leased 18 h before stimulation for 8 h with vehicle, 1:500 dilution of seminal plasma, 300 nM PGE2 or seminal plasma, or PGE2 and ONO-AE2-227. The activity of both firefly and renilla luciferase was determined using the dual luciferase assay kit (Promega). Total activity was determined relative to empty vector (pGL3-basic) luciferase activity by dividing the relative luciferase activity in full-length-promoter transfected experiments by the equivalent experiments conducted with the empty vector (pGL3-basic). Fold increase in luciferase activity was calculated by dividing the total luciferase activity in cells treated with 1:500 dilution of seminal plasma or 300 nM PGE2 by the total luciferase activity in cells treated with vehicle.

VEGF ELISA
Secreted VEGF was measured using an ELISA kit as described previously (29). Approximately 2 x 105 cells were seeded in six-well plates and allowed to adhere overnight and then serum-starved for at least 16 h. Thereafter, cells were pretreated with specific inhibitors or the EP4 antagonist (ONO-AE2-227) for 1 h before stimulation with vehicle, 1:500 dilution of seminal plasma, or 300 nM PGE2 (for the time periods specified in the figures). Culture medium was removed and VEGF protein was measured using a human VEGF ELISA kit as per the manufacturer’s instruction (Oncogene, Beeston, Nottingham, UK).

Statistical analysis
The data in this study were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (Statview 5.0; Abacus Concepts Inc., Berkeley, CA). Data are presented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of the HeLa EP4 receptor stable cell line
EP1, EP2, EP3, and EP4 receptor expression was assessed in WT HeLa cells and HeLa cells stably overexpressing the human EP4 receptor in the sense orientation (EP4S) by quantitative RT-PCR analysis (Fig. 1AGo). The expression of EP4 receptor mRNA in HeLa EP4S cells was determined to be 7.8 ± 0.1-fold greater than WT cells (P < 0.05). No alteration in expression of EP2 receptors was observed between WT and EP4S cells and no detectable levels of EP1 or EP3 receptor were measured in WT or EP4S cells by quantitative RT-PCR analysis (data not shown).


Figure 1
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  		FIG. 1. Expression of EP4 receptor in HeLa cells. A, EP4 receptor mRNA expression in HeLa WT and EP4S cells as determined by TaqMan quantitative RT-PCR analysis. B, A representative Western blot of EP4 receptor expression in WT and EP4S cells. Cells were lysed, and protein was isolated from WT and EP4S cells and subjected to SDS-PAGE and immunoblotting with specific EP4 receptor antibody, normalized for loading against ß-actin on the same blot and quantified by PhosphorImager analysis. C, Confocal immunofluorescence microscopy, performed on HeLa WT and EP4S cells. Cells were seeded in chamber slides, fixed in ice-cold methanol, and incubated with specific EP4 receptor primary and tetramethyl rhodamine isothyocyanate-labeled secondary antibody before visualization under a confocal immunofluorescence microscope. Control cells (C) were incubated with normal IgG in place of primary antibody (representative panel showing EP4S cells incubated with IgG). Data are shown as mean ± SEM from three independent experiments. (b is significantly different from a; P < 0.05).

 
Expression of EP4 receptor protein in EP4S cells was determined by Western blot analysis to be 4.1 ± 0.5-fold greater than WT cells (Fig. 1BGo). These expression levels of EP4 receptor in our stable cell line are comparable to those induced by seminal plasma and PGE2 reported in our previous study (37). Confocal immunofluorescence microscopy (Fig. 1CGo) confirmed elevated EP4 receptor immunoreactivity in EP4S cells compared with WT cells. Incubating EP4S cells with nonimmune IgG from the host species in place of antibody abolished the immunoreactivity (Fig. 1CGo, Control).

EP4 receptor activation induces COX-2 and VEGF gene expression
The role of seminal plasma or PGE2 signaling via the EP4 receptor on the expression of COX-2 and VEGF was investigated by quantitative RT-PCR analysis after stimulation of EP4S cells with 1:500 dilution of seminal plasma or 300 nM PGE2 for a time period of 2, 4, 8, 16, or 24 h. Treatment of EP4S cells with seminal plasma (Fig. 2AGo) or PGE2 (Fig. 2BGo) resulted in a 5.0 ± 0.5- and 1.6 ± 0.2-fold increase, respectively, in expression of COX-2 after 8 h of stimulation, compared with vehicle-treated cells (P < 0.05). Similarly, treatment of EP4S cells with seminal plasma (Fig. 2CGo) or PGE2 (Fig. 2DGo) resulted in a 3.7 ± 0.5- and 2.6 ± 0.3-fold increase, respectively, in VEGF expression after 8 h of stimulation, compared with vehicle-treated cells (P < 0.05). Coincubation of EP4S cells with the EP4 receptor antagonist ONO-AE2-227 abolished the seminal plasma- and PGE2-induced expression of COX-2 and VEGF (P < 0.001). Because ONO-AE2-227 can also inhibit the activation of the EP3 receptor, we investigated whether the activation of COX-2 and VEGF by seminal plasma and PGE2 is mediated via the EP3 receptor. Treatment of EP4S cells with 1 µM sulprostone had no effect on COX-2 and VEGF mRNA expression at any of the time points investigated (data not shown). This together with the absence of detectable EP3 mRNA in EP4S cells demonstrates that seminal plasma and PGE2 activation of COX-2 and VEGF is mediated via the EP4 receptor.


Figure 2
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  		FIG. 2. COX-2 and VEGF expression in HeLa EP4S cells. COX-2 (A and B) and VEGF (C and D) expression in HeLa EP4S cells was measured by TaqMan real-time quantitative RT-PCR analysis after treatment of cells for 2, 4, 8, 16, and 24 h with vehicle or 1:500 dilution of seminal plasma (A and C) or 300 nM PGE2 (B and D). In parallel, cells were cotreated with 1:500 dilution of seminal plasma or 300 nM PGE2 in the presence/absence or 1 µM of the EP4 receptor antagonist (ONO-AE2-227) for the same time. Data are presented as mean ± SEM from three independent experiments (b is significantly different from a; P < 0.05 and c is significantly different from a and b; P < 0.001). +, Presence of agent.

 
COX-2 and VEGF expression via the EP4 receptor is dependent on EGFR and ERK1/2 signaling
The role of the EGFR and ERK1/2 signaling pathways, activated by seminal plasma- or PGE2-EP4 receptor interaction, on the expression of COX-2 and VEGF was investigated using luciferase reporter assays, quantitative RT-PCR analysis, Western blot analysis, and ELISA. HeLa EP4S cells were transfected with the COX-2 or VEGF 5' flanking promoter sequence fused to the firefly luciferase coding sequence in the PGL3-basic expression vector (42, 43). In addition, a luciferase reporter without any COX-2 or VEGF promoter sequences (pGL3-basic) was used as a control. EP4S cells were cotransfected with the pRL-TK (renilla luciferase vector as an internal control) with either DNRaf, DNEGFR, DNMEK, or empty vector cDNA (pcDNA3.0). As shown in Fig. 3Go, A and B, treatment of empty vector-transfected cells with 1:500 dilution of seminal plasma (open bars) or 300 nM PGE2 (closed bars) significantly elevated COX-2 (Fig. 3AGo) and VEGF (Fig. 3BGo) promoter activity (P < 0.01). Cotreatment of transfected cells with ONO-AE2-227 or cotransfection of cells with DNEGFR, DNRaf, or DNMEK significantly reduced the seminal plasma- or PGE2-induced activation of the COX-2 or VEGF promoter (P < 0.01).


Figure 3
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  		FIG. 3. COX-2 and VEGF promoter activity in EP4S cells transfected with the COX-2 (A)- or VEGF (B)-firefly luciferase promoter. EP4S cells were transfected with COX-2-luciferase or VEGF-luciferase and pRL-TK (renilla luciferase) and cotransfected with either pcDNA3 (control empty vector cDNA) or cDNA encoding DN isoforms of Raf, EGFR, and MEK. In parallel, control cells were transfected with pGL3-basic empty luciferase cDNA and pRL-TK and cDNA encoding the respective DN mutants described above. After transfection, the cells were incubated for 8 h with either vehicle, 1:500 dilution of seminal plasma (open bars), 300 nM PGE2 (closed bars), 1:500 dilution of seminal plasma, and 1 µM EP4 receptor antagonist (ONO-AE2-227), or 300 nM PGE2 and ONO-AE2-227, and firefly and renilla luciferase activity was measured for the calculation of specific COX-2 and VEGF promoter activity as described in Materials and Methods. Data are presented as mean ± SEM from four independent experiments (b is significantly different from a; P < 0.01 and c is significantly different from a and b; P < 0.01). +, Presence of agent; –, absence of agent.

 
Treatment of EP4S cells with seminal plasma or PGE2 resulted in a significant up-regulation of expression of COX-2 protein (Fig. 4Go, A and B, P < 0.01), VEGF mRNA (Fig. 4Go, C and D, P < 0.01) and VEGF protein (Fig. 4Go, E and F, P < 0.01). Whereas, cotreatment of the EP4S cells with seminal plasma or PGE2 and ONO-AE2-227, AG1478, or PD98059 significantly reduced the seminal plasma- and PGE2-induced expression of COX-2 protein (Fig. 4Go, A and B; P < 0.05), VEGF mRNA (Fig. 4Go, C and D; P < 0.05), and VEGF protein (Fig. 4Go, E and F; P < 0.01), confirming that the effect of seminal plasma and PGE2 on COX-2 and VEGF is exerted via the EP4 receptor, EGFR, and ERK1/2 pathways.


Figure 4
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  		FIG. 4. COX-2 protein (A and B) and VEGF mRNA and protein (C, D and E, F, respectively) expression in EP4S cells as measured by Western blot analysis (COX-2) and quantitative RT-PCR analysis and ELISA (VEGF). Cells were pretreated with vehicle, 50 µM PD98059 (MEK inhibitor), 200 nM AG1478 (EGFR kinase inhibitor), or 1 µM ONO-AE2-227 (EP4 receptor antagonist) for 1 h and then treated with vehicle, 1:500 dilution of seminal plasma or 300 nM PGE2 in the absence or presence of ONO-AE2-227, AG1478, or PD98059 for 8 h. Representative Western blots of COX-2 protein expression in EP4S cells are shown. After treatment, cells were lysed and total protein was isolated and subjected to SDS-PAGE and immunoblotting with specific COX-2 antibody (A and B), normalized for loading against ß-actin on the same blot, and quantified by PhosphorImager analysis. VEGF mRNA expression (C and D) was analyzed by quantitative RT-PCR and secreted VEGF (E and F) was measured by ELISA. Data are presented as mean ± SEM from three independent experiments (b is significantly different from a and c is significantly different of a and b; P < 0.05). –, Absence of agent; +, Presence of agent.

 
EP4 receptor stimulation activated MAPK signaling
The effect of seminal plasma or PGE2 on the activation of the MAPK signaling pathways [ERK1/2, p38, and c-Jun N-terminal kinase (JNK)] was determined after treatment of EP4S cells with 1:500 dilution of seminal plasma or 300 nM PGE2. Stimulation of EP4S cells with seminal plasma (Fig. 5AGo) or PGE2 (Fig. 5BGo) resulted in a rapid time-dependent phosphorylation of ERK1/2, which was maximal after 1 min. This agonist-induced phosphorylation of ERK1/2 was abolished by cotreatment of cells with the specific EP4 receptor antagonist ONO-AE2-227 (P < 0.001). No significant phosphorylation of p38 or JNK MAPK was observed in EP4S cells after treatment of cells with seminal plasma or PGE2 within the 10-min time frame (data not shown).


Figure 5
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  		FIG. 5. Seminal plasma- and PGE2-EP4 signaling to ERK1/2 requires EGFR and Raf activity. The effect of seminal plasma and PGE2 on ERK1/2 signaling in HeLa EP4S cells. EP4S cells were stimulated with 1:500 dilution of seminal plasma (A) or 300 nM PGE2 (B) for 1, 5, and 10 min or left unstimulated (0 min). In parallel, cells were treated with seminal plasma or PGE2 and 1 µM of the EP4 receptor antagonist ONO-AE2-227 for the same length of time. C, EP4S cells were pretreated for 1 h with 1 µM EP4 receptor antagonist, inhibitors, or vehicle followed by stimulation with vehicle, 1:500 dilution of seminal plasma or 1:500 dilution of seminal plasma and 1 µM ONO-AE2-227 (EP4 receptor antagonist), 200 nM AG1478 (EGFR kinase inhibitor), or 50 µM PD98059 (MEK inhibitor) for 3 min. D, In parallel, EP4S cells were pretreated for 1 h with EP4 receptor antagonist, inhibitors, or vehicle followed by stimulation with vehicle, 300 nM PGE2, or 300 nM PGE2 and 1 µM ONO-AE2-227 (EP4 receptor antagonist), 200 nM AG1478 (EGFR kinase inhibitor), or 50 µM PD98059 (MEK inhibitor). HeLa EP4S cells were transfected with c-Myc-tagged ERK cDNA together with pcDNA3 (control empty vector) or pcDNA3 cDNA encoding DN-Raf, DNEGFR or DNMEK. E, Cells were pretreated for 1 h with ONO-AE2-227 or vehicle followed by stimulation with vehicle (control), 1:500 dilution of seminal plasma, or 1:500 dilution of seminal plasma and ONO-AE2-227. F, In parallel, cells were also stimulated with vehicle, 300 nM PGE2, 300 nM PGE2, and ONO-AE2-227. Cell lysates were subjected to immunoblot analysis using antibody against phosphorylated ERK1/2. The total amount of ERK in cell lysates was determined by reprobing the same blot with antibody recognizing total protein (lower panel). A representative Western blot is shown for each, with semiquantitative analysis of ERK phosphorylation determined from three independent experiments, by scanning densitometry software, by determining the ratio between total protein and phosphorylated protein. Data are presented as mean ± SEM (b is significantly different from a; P < 0.001); –, Absence of agent; +, presence of agent.

 
Recent studies conducted in our laboratory (29, 44, 45) and others’ (46, 47, 48) have shown that the EGFR mediates the signaling of prostanoid GPCRs to downstream ERK1/2. We investigated the effect of targeted disruption of EGFR activity on the seminal plasma- and PGE2-mediated signaling to ERK1/2 via the EP4 receptor. HeLa EP4S cells were either incubated with ONO-AE2-227 or specific chemical inhibitor of EGFR tyrosine kinase (AG1478) or MEK (PD98059) (Fig. 5Go, C and D) or cotransfected with cMyc-tagged ERK cDNA together with either empty vector cDNA or DN mutant EGFR, DNRaf or DNMEK cDNA (Fig. 5Go, E and F). EP4S cells were then stimulated with vehicle, 1:500 dilution of seminal plasma (Fig. 5Go, C and E), or 300 nM PGE2 (Fig. 5Go, D and F). The tagged ERK was immunoprecipitated and subjected to Western blot analysis. Seminal plasma (Fig. 5Go, C and E) and PGE2 (Fig. 5Go, D and F) rapidly phosphorylated ERK1/2 in HeLa EP4S cells compared with vehicle-treated cells. This elevation in ERK1/2 phosphorylation in EP4S cells was abolished by cotreatment of cells with ONO-AE2-227, AG1478, or PD98059 (Fig. 5Go, C and D) or by cotransfection of cells with DNEGFR, DNRaf, or DNMEK cDNA (Fig. 5Go, E and F; P < 0.001). No significant alteration in the basal level of phosphorylation of ERK1/2 was observed in EP4S cells in response to treatment with the EP4 receptor antagonist or small molecule chemical inhibitors on their own (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is much emerging evidence to suggest that the introduction of semen into the female reproductive tract during coitus orchestrates molecular and cellular changes to facilitate conception. Seminal plasma is known to contain estrogen, progesterone, cytokines, and growth factors (49), all of which have the capacity to modulate cellular function via their actions on their cognate receptors. Indeed, seminal plasma has been shown to influence the expression of genes in endometrial epithelial and stromal cells, where expression of a number of proinflammatory mediators such as IL-1ß, IL-8, and VEGF is increased after treatment with seminal plasma (50).

In addition to activation of EP receptors by endogenously synthesized PGE2, neoplastic cervical epithelial cells in sexually active women will potentially be under direct stimulation of PGE2 present in seminal plasma. Seminal plasma concentrations of PGs are 10,000-fold greater than that detected at the site of inflammation, and PGE2 is one of the major types detected (35). This has prompted the suggestion that seminal plasma PGs may exacerbate pathologies of the reproductive tract by activating intracellular signaling via the elevated prostanoid receptors.

In a previous study, we showed that seminal plasma and PGE2 can autoregulate the COX-PG axis by elevating the expression of COX-2 and EP4 receptor in WT HeLa cervical adenocarcinoma cells (37); however, the molecular mechanisms mediating the role of seminal plasma or PGE2 on intracellular signaling and target gene transcription in cervical tumor cells via the EP4 receptor remains to be determined.

To elucidate the molecular mechanisms whereby seminal plasma and PGE2, via the EP4 receptor could modulate the expression of tumorigenic and proangiogenic factors such as COX-2 and VEGF, and thereby potentially enhance or sustain cervical tumorigenesis, we elevated EP4 receptor expression in HeLa cells to those levels observed previously in HeLa cells exposed to seminal plasma or PGE2 (37). This was done by stably transfecting the EP4 receptor cDNA into HeLa cells in the sense orientation (HeLa EP4S cells). Elevated EP4 receptor expression in HeLa EP4S cells, compared with WT cells was confirmed by quantitative RT-PCR, Western blot analysis, and confocal immunofluorescence microscopy.

GPCR coupling results in activation of multiple effector signaling pathways, including the MAPK pathway (51). The MAPK pathway is a key signaling mechanism that regulates many cellular functions such as growth, differentiation, and transformation (51, 52). One of the upstream components of the ERK-MAPK pathway is the serine/threonine kinase Raf, which, in turn, phosphorylates and activates MEK and, consequently, ERK1/2 (53, 54). We examined the activation of the downstream MAPK cascades (ERK, p38, and JNK) by seminal plasma and PGE2 acting via the EP4 receptor. We found that within our experimental paradigms, seminal plasma and PGE2 induced a rapid time-dependent increase in ERK1/2 (but not p38 or JNK) phosphorylation via the EP4 receptor, because ERK1/2 phosphorylation in EP4S cells could be abolished by cotreatment of the cells with the specific EP4 receptor antagonist (ONO-AE2-227). Furthermore, we have demonstrated that the seminal plasma- and PGE2-induced phosphorylation of ERK1/2 in EP4S cells occurs in an EGFR- and Raf-dependent manner, becauseERK1/2 phosphorylation could be completely inhibited with specific chemical inhibitors of EGFR kinase (AG1478) and MEK (PD98059) or by cotransfection of EP4S cells with DN mutant isoforms of EGFR, Raf, and MEK. These data are in agreement with other studies that have shown that prostanoid-GPCR-mediated activation of intracellular signaling pathways to ERK1/2 is dependent on EGFR function, indicating that transactivation of EGFR by GPCRs is a recurrent theme in cell signaling (29, 44, 46, 47, 48, 55). Recently, Fujino and Regan (33) have shown that EP4-mediated activation of ERK1/2 in HEK-293 cells is mediated via the pertussis toxin-sensitive inhibitory G protein (Gi). Whether the EP4-mediated activation of ERK1/2 in HeLa EP4S cells is mediated in a cAMP-dependent manner via Gs-coupling or cAMP independent manner via Gi-coupling remains to be determined.

In prostate, colorectal, and endometrial cancer cells, PGE2 and PGF2{alpha} have recently been shown to induce the expression of COX-2, thereby establishing a positive feedback loop for sustaining tumorigenesis (56, 57, 58). In the present study, we have shown that seminal plasma and PGE2 can induce the expression of COX-2 mRNA and protein. These data, together with our previous findings of elevated PGE2 biosynthesis in cervical adenocarcinomas and HeLa cells overexpressing COX enzymes (15, 16), indicate that seminal plasma and PGE2 can also promote a positive feedback loop to perpetuate the role of prostanoids in cervical neoplasias, and that these effects may be mediated via the EP4 receptor. Elevated expression of COX-2 and synthesis and signaling of PGE2 can promote tumor angiogenesis by up-regulating the expression of multiple proangiogenic factors (19, 21, 59, 60), which, in turn, can act on endothelial cells to promote neovascularization (19, 28). In addition to the up-regulation of COX-2 expression observed in the present study, seminal plasma and PGE2 can also promote the expression and release of a potent proangiogenic factor, VEGF, via the EP4-mediated activation of EGFR and ERK1/2 signaling. VEGF is known to be the progenitor angiogenic factor in the formation of new blood vessels (61), by encouraging endothelial cells to sprout and recruit pericytes to form capillaries and smooth muscle cells to form larger vessels (61, 62, 63). In addition to the de novo synthesis of VEGF mRNA and protein in response to EP4-receptor activation, as presented here, VEGF is also known to be present in seminal plasma in concentrations up to 772 ng/ml (50). This may account for the higher basal levels of VEGF present in EP4S cells treated with seminal plasma and EP4 receptor antagonist or inhibitors, compared with EP4S cells treated with PGE2 and EP4 receptor antagonist or inhibitors. Therefore, angiogenesis in cervical carcinomas of sexually active women may be promoted directly by VEGF present in seminal plasma and enhanced by the VEGF produced via the seminal plasma activation of the EP4 receptor signal transduction pathway.

Taken together, our observations highlight a potential risk for the enhancement of tumorigenesis and angiogenesis in sexually active women with cervical neoplasias. In these women, endogenous PGE2 via the EP4-mediated activation of ERK1/2 can establish a positive feedback loop to sustain prostanoid production, by elevating expression of COX-2, while concomitantly elevating expression of potent proangiogenic factors like VEGF to enhance tumor angiogenesis, and this may be further enhanced by the actions of seminal plasma.

Our observations reported herein further highlight the potential advantages of using combinatorial therapeutic approaches of COX inhibitors with receptor antagonists or signaling inhibitors targeted against the ERK1/2 pathway in sexually active women with pathologies of the reproductive tract that are associated with elevated prostanoid receptor expression. These combinatorial approaches have been successfully demonstrated in various model systems where nonselective COX-enzyme inhibitors in combination with an inhibitor of EGFR kinase can reduce polyp formation in APC{Delta}716 mice more effectively than either compound on their own (64). In light of this latter observation and our observations presented herein, an ERK inhibitor or EGFR tyrosine kinase inhibitor in combination with a COX enzyme inhibitor or EP4 receptor antagonist may be of clinical relevance as an efficacious therapy for sexually active women with cervical neoplasias. The effectiveness of these combinatorial approaches as means of therapy will need to be extensively investigated.


   Footnotes
 
Disclosure of potential conflicts of interest: M.M., K.J.S., A.A.K., and H.N.J. have nothing to declare.

First Published Online March 30, 2006

Abbreviations: COX, Cyclooxygenase; DN, dominant-negative; EGFR, epidermal growth factor receptor; EP, E-series prostanoid; GPCR, G protein-coupled receptor; HPV, human papillomavirus; JNK, c-Jun N-terminal kinase; PG, prostaglandin; MEK, MAPK kinase; VEGF, vascular endothelial growth factor; WT, wild type.

Received November 10, 2005.

Accepted for publication March 22, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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