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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4481-4487
Copyright © 2003 by The Endocrine Society

Prostaglandin E2 Induces Proliferation of Glandular Epithelial Cells of the Human Endometrium via Extracellular Regulated Kinase 1/2-Mediated Pathway

H. N. Jabbour and S. C. Boddy

Medical Research Council Human Reproductive Sciences Unit, The University of Edinburgh Academic Centre, Edinburgh, Scotland EH16 4SB, United Kingdom

Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, The University of Edinburgh Academic Centre, Chancellor’s Building, 49 Little France Crescent, Edinburgh, Scotland EH16 4SB, United Kingdom. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we investigated the effect of prostaglandin E2 (PGE2) on MAPK ERK1/2 protein phosphorylation and on proliferation of epithelial cells of the human endometrium. Treatment of proliferative phase endometrium with PGE2 induced rapid phosphorylation of ERK1/2 proteins in glandular epithelial and endothelial cells. Treatment of human endometrial tissue with PGE2 for 24 h resulted in increased incorporation of 5-bromo-2'-deoxyuridine (a marker of cellular proliferation) in glandular epithelial cells. To investigate further the effect of PGE2 on proliferation of epithelial cells, we used an endometrial epithelial cell line (HES). HES cells express functional EP4 (with absence of expression of EP1, EP2, and EP3) receptors and stimulate cAMP release and rapid phosphorylation of ERK1/2 proteins in response to PGE2 or forskolin. Treatment of HES cells with PGE2 or forskolin alone resulted in a significant increase in HES cell proliferation compared with control untreated cells (P < 0.05). Cotreatment of the cells with PGE2 or forskolin and PD98059 abolished the increase in cellular proliferation. These data demonstrate ERK1/2 phosphorylation in response to PGE2 in the human endometrium and suggest that PGE2 via EP4 receptor may induce glandular epithelial cell proliferation in ERK1/2- dependent manner during the proliferative phase of the menstrual cycle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE PROSTAGLANDIN biosynthetic pathway, cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostaglandin H2 (PGH2), the common intermediate in prostaglandin synthesis (1). Once synthesized, PGH2 serves as a substrate for prostaglandin E (PGE) synthase to produce prostaglandin E2 (PGE2) (2, 3). PGE2 elicits its effects on target cells via G protein-coupled receptors with a typical seven-transmembrane segment architecture. Four receptor subtypes for PGE2, termed EP1, EP2, EP3, and EP4, have been identified. EP1 acts via the phospholipase C/inositol trisphosphate pathway, whereas EP3 activation can inhibit adenylate cyclase and activate phospholipase C. In contrast, EP2 and EP4 receptors activate the cAMP/protein kinase A pathway (3, 4).

In the human endometrium, temporal expression of COX-2/PGE synthase and synthesis of PGE2 have been described. Expression is localized to epithelial, stromal, and perivascular cells with maximal expression detected during the perimenstrual and proliferative phases (5, 6). Similarly, temporal expression and signaling of the cAMP-linked EP4 receptor has been outlined in the human endometrium. Expression is colocalized within epithelial and vascular cells and cAMP generation in response to PGE2 is highest in the proliferative endometrium (6). The site and temporal pattern of expression of the various components of the PGE2 synthesis and signaling pathway have prompted the suggestion that PGE2 may promote epithelial cell proliferation and vascular function in the human endometrium. Expression of COX-2 enzyme and PGE2 synthesis have been associated with various uterine pathologies that involve overproliferation of epithelial cells and/or endothelial cell function. These include carcinoma of the uterus (7, 8, 9), endometriosis (10, 11), and dysfunctional uterine bleeding (12, 13). Recent in vivo and in vitro studies have demonstrated a clear role for COX-2 enzyme and PGE2 in the regulation of epithelial cell growth and angiogenesis. COX-2 expression and PGE2 synthesis are associated with increased cellular proliferation and resistance to apoptosis (14, 15). Moreover, expression of COX-2 and synthesis of PGE2 in epithelial cells enhances the expression of angiogenic factors that act in a paracrine manner to induce endothelial cell migration and microvascular tube formation (16, 17, 18).

The aims of this study were to investigate the effect of PGE2 on proliferation of epithelial cells in the human endometrium and to outline a potential role for the ERK pathway in mediating the proliferative effect of PGE2 on epithelial cells. The data from this study demonstrate rapid ERK1/2 phosphorylation in the human endometrium and the HES endometrial epithelial cell line in response to PGE2. Moreover, PGE2 induces proliferation of endometrial epithelial cells via an ERK1/2-dependent pathway. These data outline a potential for utilization of COX enzyme inhibitors and/or EP receptor antagonists in uterine pathologies that involve overproliferation of epithelial cells.


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

PGE2 (Sigma Aldrich, Poole, UK) was stored at -20 C as a 100 µM stock solution in ethanol. PD98059 (50 µM stock; Calbiochem, Nottingham, UK) was stored at -20 C in dimethylsulfoxide. The endometrial epithelial cell line HES (donated by Dr. Douglas Kniss from Ohio State University, Columbia, OH) was grown in DMEM/HAMS F12 medium with Glutamax (Life Technologies Ltd., Paisley, Scotland, UK) containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (PAA Laboratories Ltd., Yeovil, UK).

Tissue collection

Endometrial biopsies (n = 15) from the proliferative phase of the menstrual cycle were collected with an endometrial suction curette (Pipelle, Laboratoire CCD, Paris, France) from women with regular menstrual cycles (25–35 d) undergoing hysterectomy for benign gynecological indication. Shortly after pipelle suction, the tissue was placed in RPMI 1640 medium (containing 2 mmol/liter L-glutamine, 100 U penicillin, and 100 µg/ml streptomycin) and transported to the laboratory for in vitro culture. All subjects reported regular menstrual cycles (cycle length 25–35 d), and no women had received a hormonal preparation in the 3 months preceding biopsy collection. Biopsies were dated according to stated last menstrual period and confirmed by histological assessment according to criteria of Noyes and co-workers (19). Furthermore, circulating estradiol and progesterone concentrations at the time of biopsy were consistent for both stated last menstrual period and histological assignment of menstrual cycle stage. Ethical approval was obtained from Lothian Research Ethics Committee, and written informed consent was obtained from all subjects before tissue collection.

Treatments with endometrial tissue

Following collection, endometrial tissue was cultured overnight in RPMI 1640 in the presence of 3 µg/ml indomethacin (Sigma Aldrich). To investigate the effect of PGE2 on ERK1/2 phosphorylation, endometrial tissue (n = 5) was stimulated with 100 nM PGE2 for 20 min in the presence or absence of 50 µM PD98059; control tissue was cultured in medium with vehicle alone. PD98059 is a specific inhibitor of MAPK kinase, which is an upstream component of the ERK1/2 signaling pathway (20). Following culture, the tissue was snap-frozen in dry ice until processed by Western blotting. To investigate the site of phosphorylation of ERK1/2, endometrial tissue (n = 5) was stimulated with 100 nM PGE2 for 20 min in the presence or absence of 50 µM PD98059; control tissue was cultured in medium with vehicle alone. To investigate the effect of PGE2 on proliferation, endometrial tissue (n = 5) was stimulated with 100 nM PGE2 in the presence or absence of 50 µM PD98059; control tissue was cultured in medium with vehicle alone.

Treatments with HES cells

To investigate the expression of the EP receptors in HES cells, RNA was extracted from cells following culture in normal medium. For all other experiments, HES cells were cultured in serum-free medium containing 3 µg/ml indomethacin the day before each experiment was started. cAMP response was measured in cells following treatment with 100 nM PGE2 (four independent experiments); control cells were cultured in medium with vehicle alone. ERK1/2 phosphorylation (five independent experiments) and proliferation (five independent experiments) in HES cells was measured following treatment with 100 nM PGE2 or 10 µM forskolin in the presence or absence of 50 µM PD98059; control cells were cultured in medium with vehicle alone. Forskolin is a known activator of adenylate cyclase and promotes cAMP generation independently of ligand receptor interaction. Forskolin was used in our model system to investigate the potential role of the cAMP pathway in ERK1/2 phosphorylation and proliferation of glandular epithelial cells.

Protein assay and Western blotting

Endometrial tissue was harvested by centrifugation at 2000 x g, the supernatant discarded, and the tissue homogenized in lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 10 mM EDTA, 0.6% Nonidet P-40, 1 mM Na3VO4, 10% glycerol, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonylfluoride]. HES cells were lysed in 250 µl/well lysis buffer for 30 min. Lysate was collected by scraping the wells and clarified by centrifugation at 15,300 x g for 10 min at 4 C. Protein concentration was determined using the modified Lowry method (Bio-Rad D2 Protein Assay kit; Bio-Rad Laboratories, Hemel Hempstead, UK). For Western analysis, 20 µg of protein in standard loading buffer [25 mM Tris-HCl (pH 6.8), 0.8% sodium dodecyl sulfate, 1% 2-mercaptoethanol, 4% glycerol, 0.01% bromophenol blue] were loaded per lane on a 4–12% Tris glycine gel and separated by SDS-PAGE (40 mA per gel for 90 min). Proteins were transferred to a nitrocellulose membrane (Millipore UK Ltd., Watford, UK) that was then blocked in TNS-Tween (50 mM Tris-HCl, 150 mM NaCl, 0.05% vol/vol Tween-20) containing 5% milk wt/vol for 1 h before probing with antibodies. Sequentially the membrane was probed with anti-phospho-ERK1/2 antibody (p44/p42 MAPK [Tyr 202/Tyr 204]; 1:1000 with 5% milk wt/vol; Cell Signaling, New England Biolabs, Beverly, MA), antirabbit alkaline phosphatase conjugated IgG (1:30000; Sigma Aldrich) and detected using fluorescence detection system (ECF plus; Amersham Biosciences UK Ltd., Little Chalfont, UK). Between all treatments, membranes were washed three times with TNS-Tween. To control for equal loading, the membranes were stripped and reprobed with anti-ERK1/2 (1:2000 with 5% milk wt/vol; Autogen Bioclear) followed by antigoat alkaline phosphatase conjugated IgG (1:30000; Sigma Aldrich) and visualized using fluorescence detection system. Changes in ERK1/2 phosphorylation were then quantified relative to total ERK1/2 expression and plotted as mean fold increase above basal expression ± SEM.

Immunohistochemistry

To investigate the effect of PGE2 on proliferation in the endometrium, endometrial tissue (n = 3) was incubated with 100 nM PGE2 for 24 h in the presence or absence of 50 µM PD98059. 5-Bromo-2'-deoxyuridine (BrdU; 10 µM) was added to the culture medium for the final 4 h. Control tissue was incubated in medium with vehicle alone and 10 µM BrdU was added for the final 4 h of culture. Following culture, tissue was fixed in 4% neutral buffered formaldehyde and prepared as paraffin wax-embedded sections, cut and mounted on slides. Slides were dried overnight at 50 C and dewaxed in xylene. Tissue was rehydrated in graded ethanol and washed in water followed by TBS. Sections were heated in 10 mM sodium citrate for 5 min in a pressure cooker followed by treatment with 3% H2O2 in methanol to quench endogenous peroxidase activity and subsequently washed twice in TBS for 5 min. Nonimmune rabbit serum (20% in TBS) was applied for 1 h before overnight incubation at 4 C with mouse anti-BrdU (Roche Molecular Biochemicals, Lewes, East Sussex, UK) at a dilution of 1:30. Sections were washed twice in TBS for 5 min and incubated with biotinylated rabbit antimouse IgG (Dako Corp. Ltd., Cambridge, UK) diluted 500-fold in 20% normal rabbit serum in TBS. An avidin-biotin peroxidase detection system was then applied (Dako) with 3,3'-diaminobenzidine as the chromagen.

To localize the site of phosphorylation of ERK1/2, endometrial tissue was stimulated with 100 nM PGE2 for 20 min in the presence or absence of 50 µM PD98059. Control tissue was incubated in medium with vehicle alone. Following treatment, tissue was fixed in 4% neutral buffered formaldehyde and processed as above. The sections were then incubated with anti-phospho-ERK1/2 antibody, diluted 100-fold in 20% normal swine serum in TBS, overnight at 4 C. Sections were again washed twice in TBS for 5 min and incubated with biotinylated swine antirabbit IgG (Dako), diluted 500-fold in 20% normal swine serum in TBS. Sections were washed as before and incubated with an avidin-biotin peroxidase detection system (Dako) with 3,3'-diaminobenzidine as the chromagen. Sections were counterstained with hematoxylin, dehydrated, and mounted in Xylene.

PCR

RNA was extracted with Tri-reagent (Sigma Aldrich) following the manufacturer’s guidelines. Once extracted and quantified, RNA samples were reverse transcribed using deoxynucleotide triphosphates (10 mM each), oligo-deoxythymidine (25 µg/ml), ribonuclease inhibitor (2 U/µl), and Superscript reverse transcriptase (10 U/µl; Invitrogen Life Technologies, Calne, Wiltshire, UK). The RT product (5 µl cDNA) was then amplified by PCR using homologous primers designed from the coding region of the various EP receptors. The sequence of the primers was as follows: EP1 forward primer at position 82 bp 5'-CGCTATGAGCTGCAGTACC-3', EP1 reverse primer at position 1148 bp 5'-CAAGAGGCGAAGCAGTTGG-3'. EP2 forward primer at position 527 bp 5'-GCAGTACGTCCAGTACTGCC-3', EP2 reverse primer at position 999 bp 5'TCCGACAACAGAGGAACTGAACG. EP3 forward primer at position 287 bp 5'-ACTCCTACACAGGCATGTGG-3', EP3 reverse primer at position 737 bp 5' ATGTGGCTCGCATACCAGTGC-3'. EP4 forward primer at position 242 bp 5'-CCTTCTACACGCTGGTATGTGG-3', EP4 reverse primer at position 737 bp 5'-ATGAACTGGCGGTGCATGCG-3'.

To amplify by PCR, sample mix was denatured at 94 C for 2 min, subjected to 35 cycles of 94 C for 30 sec, 54 C for 30 sec, and 72 C for 40 sec for EP1 and EP3 and 94 C for 30 sec, 57 C for 30 sec, and 72 C for 40 sec for EP2and EP4 with a final extension step of 72 C for 7 min. After amplification, samples were cooled to 4 C, and 10 µl of the PCR mix visualized on a 1% agarose gel. HES cell RNA transcribed in the absence of reverse transcriptase was used as a negative control. Plasmid pcDNA3 containing the cDNA for human EP receptor (generously donated by Mark Abramovitz, Merck Frost Inc., Quebec, Canada), and proliferative phase endometrium were used as positive controls.

cAMP and BrdU proliferation assays

HES cells (2 x 105) were plated in six-well dishes and allowed to attach overnight. The following day, the cells were incubated in medium containing 3 µg/ml indomethacin and 1 mM 3-isobutyl-1-methylxanthine (Sigma Aldrich) for 1 h at 37 C. Cells were then stimulated with 100 nM PGE2 for 1, 2, 5, 10, or 20 min; control cells were incubated in the absence of PGE2. After stimulation, HES cells were harvested by centrifugation at 2000 x g and the cells lysed with 0.1 M HCl. The lysate was cleared by certrifugation at 600 x g for 15 min. cAMP concentration was quantified by ELISA using a cAMP kit (Biomol; Affiniti, Exeter, UK) as per the manufacturer’s protocol and normalized to protein concentration of the lysate. Protein concentrations were determined using protein assay kits (Bio-Rad).

Proliferation of HES cells in response to PGE2 or forskolin was investigated using a BrdU incorporation ELISA (Roche Diagnostics GmbH, Mannheim, Germany). For each experiment, HES cells were seeded in 96-well plates at 5 x 103 cells/well and treated for 24 h with 100 nM PGE2 or 10 µM forskolin in the presence or absence of 50 µM PD98059 for 24 h; control cells received medium with vehicle alone in the presence or absence of 50 µM PD98059. Cells were then labeled with 10 µM BrdU for 4 h, fixed, and BrdU incorporation assessed by ELISA. BrdU incorporation in HES cells is presented as a percentage of untreated cells and plotted as mean ± SEM.

Statistics

Where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (Statview 4.0; Abacus Concepts Inc., Piscataway, NJ) and statistical significance accepted when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment of proliferative phase endometrium with 100 nM PGE2 for 20 min resulted in increased phosphorylation of ERK1/2 proteins compared with untreated tissue (ERK1/2 phosphorylation in response to PGE2 was 133.7 ± 12.67% of control untreated tissue; P < 0.05). Treatment of endometrial tissue with PD98059 abolished the basal and PGE2-mediated phosphorylation of ERK1/2 (Fig. 1Go). To localize the site of phosphorylation of ERK1/2 in response to PGE2, endometrial tissue was treated with 100 nM PGE2 for 20 min and subsequently subjected to immunohistochemistry using antibodies raised against the phosphorylated form of ERK1/2. ERK1/2 phosphorylation in response to PGE2 was localized to glandular epithelial cells (Fig. 2BGo) and endothelial cells of the microvasculature within the stromal compartment (Fig. 2CGo). Cotreatment of the tissue with PGE2 and PD98509 abolished the enhanced phosphorylation of ERK1/2 in the endometrial tissue (Fig. 2DGo).



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  		FIG. 1. ERK1/2 phosphorylation in endometrial tissue following treatment with 100 nM PGE2 for 20 min in the absence or presence of 50 µM PD98059; control tissue was cultured in the absence of PGE2. A, Western blot analysis of phosphorylated ERK1/2 and total ERK1/2 expression; B, mean ± SEM increase in phosphorylated ERK1/2 band intensity standardized to total ERK1/2 band intensity (n = 3 for each treatment group; open bars are control or PGE2 treatments alone, and closed bars are control or PGE2 treatments in the presence of PD98059. *, P < 0.05 compared with untreated cells).

 


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  		FIG. 2. Localization of the site of PGE2-induced ERK phosphorylation in human endometrial tissue. Endometrial tissue was cultured in the absence (A) or presence (B–D) of 100 nM PGE2 for 20 min. D, Tissue was treated with both 100 nM PGE2 and 50 µM PD98059. Tissue was fixed in neutral buffered formaldehyde and immunohistochemistry performed using anti-phospho-ERK antibody. Arrows in panel C denote vascular/endothelial cell staining. Bar, 100 µM.

 
The HES endometrial epithelial cell line was used to investigate further the effect of PGE2 on proliferation of endometrial epithelial cells and the potential role of ERK1/2 and cAMP pathways in this PGE2-mediated phenotypic effect. HES cells were shown to express the EP4 receptor by standard PCR techniques (Fig. 3Go). However, no signal was detected for EP1, EP2, or EP3 receptors (data not shown). Functional activation of the EP4 receptor in HES cells was investigated by measuring cAMP generation and ERK1/2 phosphorylation following treatment with PGE2. PGE2 resulted in significant elevation of cAMP (Fig. 4Go; P < 0.05) and phosphorylation of ERK1/2 proteins (Fig. 5Go; ERK1/2 phosphorylation in response to PGE2 was 139.7 ± 11.1% of control level; P < 0.05). Similarly, treatment of HES cells with forskolin resulted in rapid phosphorylation of ERK1/2 (Fig. 6Go; ERK1/2 phosphorylation in response to forskolin was 151.7 ± 7.7% of control level; P < 0.05). Cotreatment of the cells with PGE2 or forskolin and PD98059 abolished the PGE2 mediated phosphorylation of ERK1/2 (Fig. 5Go).



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  		FIG. 3. RT-PCR for EP4 receptor in HES cells. HES cell RNA transcribed in the absence of reverse transcriptase was used as negative (-ive) control. Plasmid pcDNA3 containing the cDNA for the human EP4 receptor and cDNA transcribed from proliferative phase endometrium were used as positive (+ive) controls.

 


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  		FIG. 4. cAMP production in HES cells following treatment with 100 nM PGE2 for 1, 2, 5, 10, or 20 min; control cells were incubated in the absence of PGE2. Data are presented as mean ± SEM of four independent experiments. *, P < 0.05 compared with untreated cells.

 


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  		FIG. 5. ERK1/2 phosphorylation in HES cells following treatment with 100 nM PGE2 or 10 µM forskolin in the absence or presence of 50 µM PD98059; control cells were cultured in the absence of PGE2. A, Western blot analysis of phosphorylated ERK1/2 and total ERK1/2 expression; B, mean ± SEM increase in phosphorylated ERK1/2 band intensity standardized to total ERK1/2 band intensity (n = 5 for each treatment group; open bars are control, PGE2, or forskolin treatments alone; closed bars are control, PGE2, or forskolin treatments in the presence of PD98059; *, P < 0.05 compared with untreated cells).

 


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  		FIG. 6. Proliferation in endometrial epithelial cells in response to treatment with PGE2. A, Localization of the site of PGE2-induced proliferation in human endometrial tissue. Endometrial tissue was cultured in medium alone (A) or in the presence of 100 nM PGE2 (B and C) for 24 h. C, Tissue was cocultured with 100 nM PGE2 and 50 µM PD98059. BrdU (10 µM) was added to the culture medium for the final 4 h. Tissue was fixed in neutral buffered formaldehyde and immunohistochemistry performed using anti-BrdU antibody. Bar corresponds to 100 µm. B, Proliferation in HES cells following treatment with 100 nM PGE2 or 10 µM forskolin in the absence or presence of 50 µM PD98059 for 24 h; control cells received medium alone in the absence or presence of 50 µM PD98059. Cells were then labeled with 10 µM BrdU for 4 h, fixed, and BrdU incorporation assessed by ELISA. BrdU incorporation in HES cells is presented as a percentage of untreated cells and plotted as mean ± SEM (n = 5 for each treatment group; *, P < 0.05 compared with control cells).

 
Finally, the effect of PGE2/forskolin and the ERK1/2 pathway on proliferation was investigated in human endometrial tissue and HES cells (Fig. 6Go). Endometrial tissue was treated with PGE2 in the presence or absence of PD98059. Treatment of endometrial tissue with PGE2 induced increased BrdU incorporation in glandular epithelial cells. BrdU incorporation was abolished when the cells were cotreated with PGE2 and PD98059 (Fig. 6AGo). Similarly, treatment of HES cells with PGE2 or forskolin alone resulted in significant increases in BrdU incorporation (Fig. 6BGo; BrdU incorporation was 132.3 ± 14.5% and 157 ± 25.3% of control level following treatment with PGE2 or forskolin, respectively; P < 0.05). Whereas cotreatment of the cells with PD98059 abolished the PGE2 and forskolin mediated increase in BrdU incorporation (88.89 ± 11.75% and 95.4 ± 1.12% of control levels, respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies investigating expression of the components of the PGE2 biosynthetic and signaling pathways in the human endometrium have outlined temporal variation across the menstrual cycle. Expression of COX/PGE synthase enzymes and synthesis of PGE2 are highest in the perimenstrual and proliferative phases of the menstrual cycle and the sites of expression/synthesis are localized to multiple cellular compartments within the endometrium including epithelial, stromal and perivascular cells (5, 6). PGE2 induces its effect on target cells via interaction with four different membrane bound receptors termed EP1-EP4 (3). Recent data have outlined a temporal variation in the expression of the EP4 receptor across the menstrual cycle (6). EP4 expression is highest during the proliferative phase and colocalizes within the multiple cellular compartments of the functionalis layer; the functionalis layer is shed at menstruation and regenerated during the proliferative phase of every menstrual cycle. Moreover, EP4 receptor signaling, investigated by measuring cAMP generation in response to PGE2, is highest during the proliferative phase (7). This has prompted the suggestion that PGE2 may act in an autocrine/paracrine manner to promote epithelial cell proliferation and vascular function in the functionalis layer of the human endometrium. The data presented in this study support this notion and demonstrate that, in addition to cAMP, PGE2 induces activation of other signaling pathways such as the ERK1/2 MAPK pathway. The sites of phosphorylation of ERK1/2 in response to PGE2 are localized to the sites of expression of the EP receptors (6), namely the glandular epithelial and endothelial cells of the microvasculature. Activation of ERK1/2 proteins in the human endometrium has also been demonstrated recently in response to other prostanoids such as PGF2{alpha} acting via the PGF2{alpha} receptor, which is elevated during the proliferative phase of the menstrual cycle (21). Interestingly, in the human endometrium both PGE2 and PGF2{alpha} concentrations are elevated during the proliferative phase of the menstrual cycle (22, 23). Hence, it is plausible to suggest that various prostanoids converge at specific stages of the menstrual cycle to maximize ERK1/2 signaling.

The effect of PGE2 on intracellular signaling in the endometrial epithelial cells was investigated further using the HES cell line. The HES cell line was shown to express only the EP4 receptor and to respond to PGE2 treatment by increased cAMP generation and ERK1/2 phosphorylation. Moreover, treatment of HES cells with forskolin resulted in rapid activation of ERK1/2, and this phosphorylation was inhibited following cotreatment of the cells with the MEK inhibitor PD98059. The activation of cAMP and ERK1/2 pathways by PGE2 in the human endometrium and the activation of ERK1/2 proteins by PGE2 and forskolin in HES cells would suggest a cross talk between the cAMP/protein kinase A and ERK1/2 pathways in endometrial epithelial cells. G protein-coupled receptors are known to activate, in addition to G-proteins, MAPK cascades including ERK1/2. This is predicted to be mediated via cross talk between signaling systems that is commonly associated with G protein-coupled receptors and members of the receptor tyrosine receptor family. Members of this family are classically associated with activation of the ERK/MAPK phosphorylation cascade (24, 25). Taken together, these data outline a potential cross talk leading to the activation of the ERK/MAPK pathway by cAMP in the human endometrium. Future research in our laboratory is focused on elucidating the exact mechanism of this cross talk and its potential effects on phenotypic changes in target cells of the normal and pathologic human endometrium.

Treatment with PGE2 or forskolin induced proliferation in endometrial epithelial cells. This was observed both in endometrial biopsy tissue and in the HES cell line. Inhibition of the ERK1/2 pathway by cotreatment with PD98059 abolished the PGE2 and forskolin induced proliferation in these cells. This suggests that PGE2 induced proliferation in endometrial epithelial cells may be mediated following activation of the ERK1/2 signaling pathway by cAMP. Both cAMP and ERK1/2 proteins are known to play a central role in mitogenesis in a number of cell types and impeding their function prevents cell proliferation in response to a number of growth-stimulating agents (26). Further support for an in vivo proliferative effect of PGE2 may be deduced from the temporal variation in expression of EP4 receptors and cAMP generation in response to PGE2 across the menstrual cycle. The significant reduction in EP4 receptor expression and cAMP during the secretory phase is suggestive of the loss of the proliferative potential of PGE2 during the latter phase of the menstrual cycle (6).

In summary, the data presented in this manuscript demonstrate activation of the ERK1/2 MAPK pathway by PGE2 in the human endometrium and suggest a potential cross talk between the cAMP and MAPK pathways in glandular epithelial cells. Furthermore, PGE2 induces proliferation of human endometrial epithelial cells via an ERK1/2-dependent pathway.


   Acknowledgments
 
We thank Professor Hilary Critchley for advice, Ms. Catherine Murray for assistance with tissue collection, and Dr. Oliver Gubbay for assistance in establishing the BrdU proliferation assays in human endometrial tissue.


   Footnotes
 
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; COX, cyclooxygenase; EP1-EP4, four receptor subtypes for PGE2; HES, endometrial epithelial cell line; PGE, prostaglandin E; PGH2, prostaglandin H2.

Received February 21, 2003.

Accepted June 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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