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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 8 3928-3935
Copyright © 2002 by The Endocrine Society

Original Article

Expression and Localization of Endothelial Monocyte-Activating Polypeptide II in the Human Endometrium across the Menstrual Cycle: Regulation of Expression by Prostaglandin E2

Sharon Battersby, Sheila C. Boddy, Hilary O. D. Critchley and Henry N. Jabbour

Medical Research Council Human Reproductive Sciences Unit (S.B., S.C.B., H.N.J.) and Department of Obstetrics and Gynaecology (H.O.D.C.), University of Edinburgh, Centre for Reproductive Biology, Edinburgh EH3 9ET, United Kingdom

Address all correspondence and requests for reprints to: Dr. H. N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, United Kingdom. E-mail: . h.jabbour{at}hrsu.mrc.ac.uk

Abstract

A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation, has been established, although the mechanisms by which this is achieved are not fully elucidated. In the present study, cDNA array analysis has identified endothelial monocyte-activating polypeptide II (EMAP II) as a PGE2-regulated gene in endometrial epithelial cells. Incubation of endometrial epithelial cells with 100 nM PGE2 for 4 and 24 h resulted in a 2.3- and 16-fold decrease in EMAP II expression, respectively. In endometrial tissue collected across the menstrual cycle, a significant increase in EMAP II mRNA was observed during the late secretory phase, compared with the proliferative and early-midsecretory phases. The temporal pattern of EMAP II expression was confirmed further by Western blotting; EMAP II protein expression was detected as a 43-kDa band. In situ hybridization and immunohistochemistry localized EMAP II mRNA and protein expression in glandular epithelial, endothelial, and stromal cells in the functionalis and basalis layers of the endometrium. Finally, the role of PGE2 in the regulation of EMAP II expression in the endometrium was assessed. Incubation of fresh endometrial tissue (n = 5) with 3 µg/ml indomethacin resulted in an increase in EMAP II protein expression, compared with control untreated tissue. However, cotreatment of the cells with 100 nM PGE2 resulted in a significant decrease in EMAP II protein expression, compared with tissue incubated with indomethacin alone (P < 0.05). These data confirm temporal variation in EMAP II expression in the human endometrium across the menstrual cycle and localize expression to glandular epithelial, endothelial, and stromal cells. Moreover, EMAP II expression is negatively regulated by PGE2.

MENORRHAGIA IS A MAJOR problem in women’s reproductive health with, in any year, 5% of women in the 30- to 49-yr age group visiting their general practitioners with excessive menstrual bleeding (1, 2). Prostaglandins (PGs) have been implicated in the etiology of menorrhagia, and a positive association has been described between the volume of blood loss at menstruation and PGE2 concentrations measured in menstrual fluid and endometrial tissue (3, 4). Moreover, treatment with nonsteroidal anti-inflammatory drugs results in up to 35% decrease in blood loss during menstruation (1, 5).

In the PG synthesis pathway, the enzyme cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to PGH2, the common intermediate in PG synthesis (6). There are two isoforms of the enzyme, COX-1 and COX-2; COX-1 is constitutively expressed in many cell types and generates PGs for normal cellular function, whereas COX-2 is the inducible form of the enzyme. COX-2 expression is induced in response to mitogens, cytokines, and tumor promoters (6). There are five endogenous prostanoids, PGE2, PGD2, PGF2{alpha}, prostacyclin, and thromboxane A2, and these are synthesized from PGH2 by their respective synthases or isomerases (6). In the human endometrium, COX-2 expression has been localized to epithelial and perivascular cells with maximal expression detected at menstruation (7). In addition, expression of PGE synthase and synthesis of PGE2 has been described recently in the human endometrium (8) and localized to glandular epithelial, endothelial, and stromal cells.

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 (9). EP1 acts via the PLC/inositol trisphosphate pathway, and EP3 activation can inhibit adenylate cyclase and activate PLC. In contrast, EP2 and EP4 receptors activate the cAMP/PKA pathway. Expression of the cAMP-linked EP2/EP4 receptors has been confirmed in the human endometrium and is localized to epithelial and vascular cells. Moreover, expression and signaling of the EP4 receptor subtype is up-regulated during the proliferative phase, suggesting a further role for PGE2 in glandular epithelial cell function and angiogenesis (8).

As part of an ongoing study of the mechanisms by which PGs may regulate endometrial function, cDNA array technology was implemented to identify genes whose expression was modulated by PGE2 and that may be relevant in the pathways involved in the mechanisms of menstruation. The aims of the present study were to investigate the spatial and temporal expression of one of these identified genes, endothelial monocyte-activating polypeptide II (EMAP II), within endometrial tissue. In addition, the effect of PGE2 on the regulation of EMAP II synthesis in the human endometrium was investigated.

Materials and Methods

Patients and tissue collection

Endometrial biopsies (n = 35) at different stages of the menstrual cycle were obtained from women with regular menstrual cycles (25–35 d) who had not received a hormonal preparation in the 3 months preceding biopsy collection. Samples were collected either with an endometrial suction curette (Pipelle, Laboratoire CCD, Paris, France) or as full-thickness endometrial biopsies from women undergoing hysterectomy for benign gynecological indications. Shortly after collection, tissue was either snap frozen in dry ice and stored at -70 C (for RNA extraction), fixed in neutral buffered formalin and wax embedded (for immunohistochemical analyses) or placed in RPMI 1640 (containing 2 mM L-glutamine, 100 U penicillin, and 100 µg/ml streptomycin) and transported to the laboratory for in vitro culture.

Biopsies were dated according to stated last menstrual period and confirmed by histological assessment according to criteria of Noyes et al. (10). Furthermore, circulating E2 and progesterone concentrations at the time of biopsy were consistent for both stated last menstrual period and histological assignment of menstrual cycle stage. Samples obtained on d 1–13, 15–18, and 23–28 of the menstrual cycle were staged as proliferative, early-midsecretory, and late secretory phases, respectively. Ethical approval was obtained from Lothian Research Ethics Committee, and written informed consent was obtained from all subjects before tissue collection.

cDNA array analysis

Analysis of the effect of PGE2 on target genes was assessed using the human endometrial epithelial (HES) cell line (a gift from Dr. D. A. Kniss, Ohio State University, Columbus, OH). Cells were grown in Medium 199 containing glutamax and supplemented with 10% FBS. For cDNA array analysis, cells were grown to approximately 70% confluence and starved overnight in serum-free Medium 199 supplemented with 0.5% BSA and 3 µg/ml indomethacin (Sigma, Poole, UK). Following overnight starvation, cells were either left untreated (control) or treated with PGE2 for 4 or 24 h. Following treatment, cells were harvested by trypsinization for 2 min at 37 C in trypsin/EDTA (PAA Laboratories, Linz, Austria), resuspended in PBS, pelleted by centrifugation for 3 min at 650 x g, snap frozen on dry ice, and stored at -70 C. Cell pellets were sent to CLONTECH Laboratories, Inc. (Palo Alto, CA) for custom cDNA array analysis using the Atlas human 1.2 nylon array membranes (containing 1176 human cDNAs). The hybridization results were analyzed using AtlasImage software (CLONTECH Laboratories, Inc.), and the data were normalized and presented as a comparison of 4- and 24-h PGE2 treatments with control untreated cells.

Taqman quantitative RT-PCR

RNA was extracted from endometrial biopsies obtained from across the menstrual cycle (n = 12) using Tri-Reagent (Sigma) following the manufacturer’s instructions. RNA samples were quantified and reverse transcribed using 5.5 mM MgCl2, 0.5 mM each deoxynucleotide triphosphates, 2.5 µM random hexamers, ribonuclease inhibitor (0.4 U/ml), and 1.25 U/ml Multiscribe reverse transcriptase (all from PE Applied Biosystems, Warrington, UK). RNA (400 ng) was added to each reverse transcription reaction, and samples were incubated for 90 min at 25C, 45 min at 48C, and 5 min at 95C. The reaction mix for the PCR consisted of 1x mastermix, ribosomal 18S forward and reverse primers, Ribosomal 18S probe (50 nM, all from PE Applied Biosystems), forward and reverse primers for EMAP II (300 nM) and EMAP II probe (200 nM) (all from Biosource UK, Nivelles, Belgium). The reaction mix (48 µl) was aliquoted into tubes and 2 µl cDNA was added. Duplicate 24-µl samples plus positive and negative controls were placed in a PCR plate, and wells were sealed with optical caps. The PCR reactions were carried out using an ABI Prism 7700 (PE Applied Biosystems). EMAP II primers and probe were designed using the PRIMER express program (PE Applied Biosystems). The sequences of the EMAP II primers and probe were: forward, 5'-GAATAGCCCCAAGGACAGTTG-3'; reverse, 5'-CACCATCCGATTTTGCATCTG-3'; and probe (6-carboxy fluoroscein labeled) 5'-AGTGGCCTGGTGAATCATGTTCCTCTTG-3'. The sequences of 18S primers and probe have been described previously (8). Data were analyzed and processed using Sequence Detector version 1.6.3 (PE Applied Biosystems), according to the manufacturer’s instructions. Results were expressed relative to an internal positive standard included in all reactions.

Ribonuclease protection assay

An antisense cRNA probe for EMAP II was prepared by amplifying a 303-bp fragment of EMAP II mRNA (between bp 493 and 795 of the mRNA sequence) from endometrial epithelial cells by RT-PCR using forward primer 5'-GCCAATAGATGTTTCCCGTCTGG-3' and reverse primer 5'-TCTCCAGGAACAGACCCATTTGG-3'. The amplicon was cloned into the TOPO vector using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands), and its identity was confirmed by DNA sequencing using an ABI 373 automated sequencer (PE Applied Biosystems). The plasmid was linearized by Bam H1 restriction digestion and was transcribed using an Ambion, Inc. Maxiscribe kit [AMS Biotechnology (Europe) Ltd., Witney, UK]. Briefly, linearized DNA was incubated with T7 RNA polymerase enzyme in the presence of [32P] UTP (800 Ci/mmol, Amersham International, Amersham, UK). The full-length radiolabeled cRNA antisense probe was gel purified, and ribonuclease protection assay (RPA) was carried out using an RPA III kit from Ambion, Inc. (AMS Biotechnology), according to the manufacturer’s instructions. RNA samples (20 µg), extracted from the same endometrial biopsies used for RT-PCR, were precipitated with the volume of probe required to give 2 x 105 cpm, mixed with hybridization buffer, heated to 90 C for 4 min, and hybridized overnight at 42 C. Samples were digested with RNase A and RNase T1 at 37 C for 30 min. The protected RNA was resolved on a 5% polyacrylamide gel under denaturing conditions. Gels were dried and exposed to a phosphorescent screen (Molecular Dynamics, Inc., Chesham, UK). The integrity of RNA and the relative amount of total RNA in each reaction was determined by including radiolabeled cRNA prepared from an 18S ribosomal standard cDNA, 2 x104 cpm in each reaction. EMAP II mRNA and 18S ribosomal RNA were counted on a Storm PhosphorImager (Molecular Dynamics, Inc.) and quantified using ImageQuant Software (Molecular Dynamics, Inc.).

Western blotting

Endometrial tissue samples (n = 6) were homogenized in 500 µl lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 10 mM EDTA, 0.6% Nonidet P-40, 10% glycerol and protease inhibitors (1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml pepstatin). Lysates were subjected to two freeze-thaw cycles on dry ice and centrifuged at 14,000 x g for 15 min to clarify the lysate. Aliquots of each sample were removed and protein concentration was assayed by a modification of the method of Lowry, according to manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK). Protein samples (20 µg) were mixed with Laemmli protein sample buffer and resolved on a 4–20% Tris-glycine gel (Invitrogen) under denaturing conditions. Proteins were transferred to polyvinyl difluoride membrane (Immobilon-P, Millipore Corp., Watford, UK) using a semidry transfer apparatus (Bio-Rad Laboratories, Inc.). Nonspecific staining was blocked by treatment of the membranes for 1 h at room temperature with 5% nonfat milk powder (Marvel) in Tris normal saline, 0.5 M NaCl, 20 mM Tris HCl (pH 7.4), containing 0.1% Tween 20 (TNS/Tween), and membranes were then incubated overnight at 4 C with a rabbit polyclonal antibody raised against recombinant human EMAP II (11) at a dilution of 1:1,000 in TNS/Tween. Membranes were washed for at least 3 x 5 min in TNS/Tween and incubated for 1 h at room temperature in antirabbit immunoglobulin conjugated to alkaline phosphatase (Sigma). Following further washing in TNS/Tween, the blots were treated for 20 min at room temperature with ECF substrate reagent (Amersham International). Membranes were then dried and scanned on a Storm PhosphorImager (Molecular Dynamics, Inc.) and images visualized using ImageQuant software (Molecular Dynamics, Inc.). To confirm even protein loading, membranes were stripped in 0.2 M glycine, 1% SDS (pH 2.5) for 30 min and reprobed. Nonspecific binding was blocked as above and membranes were incubated overnight at 4 C in a goat polyclonal antibody to human actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:2,000. Following washing, blots were treated for 1 h at room temperature with antigoat immunoglobulin alkaline phosphatase conjugate (Sigma) at a dilution of 1:30,000 in TNS/Tween and subsequently washed, scanned, and quantified as above.

In situ hybridization

A custom synthesized oligonucleotide double-fluoroscein isothiocyanate (FITC)-labeled cDNA probe for EMAP II was obtained from Biognostik (Gottingen, Germany). Sections (5 µM) from full-thickness human uterine biopsies collected across the menstrual cycle (n = 12) were cut onto gelatin-coated slides. Sections were dewaxed and rehydrated and then treated with proteinase K [100 µg/ml in 100 mM Tris-HCl (pH 7.6), containing 50 mM EDTA] for 15 min at 37 C to enhance cDNA probe access. Sections were washed in diethylpyrocarbonate-treated water and prehybridized for 4 h at 30 C with 25 ml hybridization buffer supplied with the probe, which had been previously heated to 95 C. The sections were then hybridized overnight at 30 C with the cDNA probe at 6 U/ml in hybridization buffer. Following hybridization, sections were washed for 2 x 5 min in 1x standard saline citrate and 2 x 15 min in 0.1x standard saline citrate at 39 C. The FITC-labeled probe was detected using standard immunohistochemical reagents with an additional amplification step (TSA biotin system, NEN Life Science Products, Hounslow, UK). Sections were incubated with blocking buffer for 30 min. Conjugated anti-FITC-horseradish peroxidase (Roche Diagnostics Ltd., Lewes, UK) was added at a dilution of 1:200 in blocking buffer and the sections incubated for 60 min. After washing, biotinyl tyramide amplification reagent (1:50) was applied to each slide and incubated for 15 min. Streptavidin-horseradish peroxidase (1:100) was applied after washing and incubated for 30 min and probe localization visualized with 3,3'-diaminobenzidine substrate. Control sections were treated with a double-FITC-labeled oligonucleotide probe containing the same proportion of cysteine and guanine bases as the EMAP II probe to assess background hybridization. All treatments were carried out at room temperature unless otherwise specified.

Immunohistochemistry (IHC)

Endometrial sections (5 µm) from across the menstrual cycle (n = 12) were dewaxed in xylene and rehydrated using decreasing grades of ethanol. Antigen retrieval was performed by treating sections for 5 min in a pressure cooker in boiling 0.1% citrate buffer (pH 3.0). After rinsing in PBS, endogenous peroxidase activity was quenched with 10% H2O2 in methanol at room temperature. Nonimmune swine serum (20% serum in PBS) was applied for 1 h before overnight incubation at 4 C with rabbit antihuman EMAP II at a dilution of 1:250. An avidin-biotin peroxidase detection system was then applied (DAKO Corp. Ltd., Cambridge, UK) with 3,3'-diaminobenzidine as the chromagen (8).

Tissue incubation with PGE2

Endometrial biopsies from across the menstrual cycle (n = 5) were minced finely with scissors and divided into three portions. The tissue was incubated overnight at 37 C in a humidified 5% CO2 incubator in 2 ml RPMI (Sigma) medium containing 2 mmol/liter L-glutamine, 100 IU penicillin, and 100 µg streptomycin. One portion was used as a control and received no further treatment. The two other tissue portions were treated with 3 µg/ml indomethacin. After overnight incubation, the medium was replaced either with fresh serum-free medium (control portion), fresh medium containing indomethacin, or fresh medium containing indomethacin in the presence of 100 nM PGE2. The tissue was incubated for another 24 h at 37 C and subsequently snap frozen on dry ice and stored at -70 C. EMAP II expression following treatment was investigated by Western blot analysis. Bands were quantified using ImageQuant software (Molecular Dynamics, Inc.) as above. EMAP II levels in control untreated tissue were taken as 100% and levels in indomethacin or indomethacin and PGE 2-treated tissue expressed relative to this control.

Statistics

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

Results

The initial aim of this study was to characterize the PGE2-regulated target genes in HES cells. Using cDNA array technology, EMAP II was identified as a PGE2-regulated gene. At 4 and 24 h after PGE2 treatment, there was a 2.3- and 16-fold decrease in EMAP II expression, respectively, compared with untreated cells (Fig. 1Go). To investigate the temporal pattern of EMAP II expression in the human endometrium, RNA was extracted from endometrial biopsy tissue collected at different stages of the menstrual cycle and EMAP II expression was assessed by quantitative RT-PCR and RPA. EMAP II mRNA was detected at all phases of the menstrual cycle by RT-PCR (Fig. 2aGo). However, the relative expression was significantly higher (P < 0.01) in the late secretory phase (3.74 ± 1.15; n = 3), compared with the proliferative (0.65 ± 0.17; n = 5) and early-midsecretory (0.7 ± 0.07; n = 4) phases of the menstrual cycle. EMAP II mRNA expression in the same endometrial RNA samples showed a similar temporal variation by RPA as detected by RT-PCR (Fig. 2bGo). Expression of EMAP II was again significantly elevated (P < 0.01) in the late secretory phase of the menstrual cycle (22.28 ± 3.27; n = 3), compared with the proliferative (4.13 ± 0.78; n = 5) and early-midsecretory phases (4.99 ± 0.52; n = 4).



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  		Figure 1. Atlas human 1.2 cDNA array blots following hybridization with cDNA prepared from HES cells. The circled area corresponds to the position of EMAP II on the array blot. Panel a is a blot following hybridization with RNA from untreated cells, and b and c are from cells treated with PGE2 for 4 and 24 h, respectively.

 


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  		Figure 2. Temporal variation in EMAP II RNA expression in endometrial biopsies from across the menstrual cycle (n = 12). a, Histogram of relative expression of EMAP II as assessed by real time RT-PCR. b, RPA showing EMAP II expression in same samples used for RT-PCR. An internal standard of 18S ribosomal RNA was used in each reaction to calculate relative expression.

 
Western blot analysis confirmed further the expression of EMAP II protein in the human endometrium across the menstrual cycle. EMAP II was encoded by a protein of approximately 43 kDa in size (Fig. 3Go). There was a similar temporal pattern of expression of EMAP II protein to that demonstrated for mRNA, with greater expression detected in endometrial tissue collected during the late secretory phase of the cycle (Fig. 3Go).



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  		Figure 3. Western blot analysis of EMAP II expression in protein lysates extracted from endometrial biopsies (20 µg protein per lane) collected at different days of the menstrual cycle (n = 6; numbers represent day of cycle). EMAP II protein was detected as a 43-kDa band. Actin was used as control to confirm equal protein loading in all samples.

 
Localization of the site of expression of EMAP II was investigated by in situ hybridization and IHC. In sections stained by in situ hybridization, EMAP II expression was detected across the menstrual cycle in glandular epithelial cells of the basalis and functionalis layers during both the proliferative (Figs. 4Go, a and b) and secretory (Fig. 4Go, c and d) phases of the menstrual cycle. Expression was generally cytoplasmic, but staining overlying nuclei was also detected (Fig. 4Go, c and d). Stromal cell reactivity was present in a high percentage of cells in the functionalis layer of the uterus (Fig. 4Go, b and d). In contrast, less frequent and less intense staining was seen in stromal cells in the basalis compartment (Fig. 4Go, a and c). EMAP II was localized to endothelial cells lining the microvasculature in all areas of the endometrium (Fig. 4dGo). Immunohistochemical localization of EMAP II (Fig. 5Go) showed a similar temporal and spatial distribution to that detected by in situ hybridization. Intense cytoplasmic immunoreactivity was present in glandular epithelial cells across both layers of the endometrium in the proliferative (Fig. 5aGo) and secretory (Fig. 5bGo) phases of the menstrual cycle. In contrast, stromal reactivity showed spatial differences between the basalis and functionalis regions of proliferative (Fig. 5Go, c and d) and secretory (Fig. 5Go, e and f) endometrium. As with RNA expression, assessed by in situ hybridization, immunohistochemical stromal reactivity was weak and infrequent in the basalis layer of the endometrium (Fig. 5Go, c and e), but intense staining was present in many stromal cells in the functionalis layer (Fig. 5Go, d and f). EMAP II protein was detected in endothelial cells lining microvessels in both the basalis and functionalis layers (Fig. 5Go, c, d, and f).



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  		Figure 4. In situ hybridization of EMAP II probe to sections of full-thickness human endometrial tissue collected in proliferative (a and b) and secretory (c and d) phases of menstrual cycle. EMAP II was expressed in glandular epithelium (G) in basalis and functionalis layers within cytoplasm and overlying nuclei. Stronger stromal expression was detected in functionalis layer (b and d), compared with basalis layer (a and c). EMAP II expression was detected in endothelial cells (outlined by arrow in d). Inset in b is section treated with control oligonucleotide probe. Scale bars, 50 µM.

 


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  		Figure 5. Immunohistochemical localization of EMAP II in full-thickness human endometrial tissue collected in the midproliferative (a, c, and d), midsecretory (b), and late secretory (e and f) phases of the menstrual cycle. Strong cytoplasmic staining was seen in glandular epithelial cells (G) in basalis (B) and functionalis (F) layers and in surface epithelium (SE) during all phases of menstrual cycle (a and b); M, Myometrium. Stronger stromal expression was seen in functionalis layer (d and f), compared with basalis layer (c and e) of proliferative (c and d) or secretory (e and f) endometrium. Immunoreactivity was detected in endothelial cells of the microvasculature (outlined by arrows) in basalis (c) and functionalis (d and f) layers. Inset in b is section treated with nonimmune serum. Scale bars, 50 µM.

 
Finally, to investigate the effect of PGE2 on EMAP II expression, endometrial tissue was cultured with indomethacin (to inhibit endogenous prostaglandin synthesis) in the absence or presence of PGE2; control tissue received no treatment (Fig. 6Go). Indomethacin treatment resulted in an 18.65% ± 5.41% increase in EMAP II expression, compared with control untreated cells. However, cotreatment with 100 nM PGE2 resulted in a significant reduction in EMAP II expression (P < 0.05), compared with indomethacin treatment alone (89.78% ± 9.01% of control vs. 118.65% ± 5.41% of control). It is noteworthy that the magnitude of the down-regulation of EMAP II expression in the in vitro studies was lower than that detected in the cDNA array analysis. This may be a reflection of the differences in the cell populations analyzed between the two experiments. The cDNA array analysis was conducted on a homogeneous endometrial epithelial cell population, whereas the effect of PGE2 on expression of EMAP II in the endometrium was conducted using endometrial biopsy tissue. This tissue contains a mixed population of cells, many of which do not express EMAP II as demonstrated by the in situ hybridization and immunohistochemical data.



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  		Figure 6. The effect of PGE2 on EMAP II expression in human endometrium. Typical Western blot (a) showing EMAP II protein expression in endometrial biopsy tissue incubated in control medium (C), medium containing 3 µg/ml indomethacin (I), or medium containing 3 µg/ml indomethacin and 100 nM PGE2 (I+PGE2). Actin was used as control to confirm even protein loading. Relative expression of EMAP II (b) in endometrial tissue following treatment with 3 µg/ml indomethacin (I) or 3 µg/ml indomethacin and 100 nM PGE2 (I+PGE2). Expression was calculated as a percentage of basal expression detected in tissue incubated with control medium. The values are presented as mean ± SEM of five independent experiments. *, Significantly lower (P < 0.05) expression of EMAP II in tissue treated with I+PGE2, compared with I alone.

 
Discussion

This study confirms the expression of EMAP II in the human endometrium and demonstrates significant up-regulation of EMAP II RNA and protein in the late secretory phase of the menstrual cycle. EMAP II is a proinflammatory mediator, which can induce expression of cytokines and chemokines (11, 12). In the mouse embryo, it has been shown to be most abundant at sites undergoing apoptosis, and it causes apoptosis in vascular endothelial cells (13, 14, 15). This range of function makes EMAP II an excellent candidate gene for a role in the mediation of the inflammatory response demonstrated in the endometrium in the late secretory phase of the menstrual cycle. Responses seen at this time, as part of the initiation of menstruation, include up-regulation in the expression of cytokines and chemokines and infiltration of leukocytes (16, 17). Such cytokines/chemokines include IL-8, together with monocyte chemotactic protein-1, which are potent neutrophil and leukocyte attractants and activators (7, 18). In addition, associated with the process of menstruation, there is an increase in epithelial cell apoptosis, which commences from 2 d before menstruation (peaking at d 2 of menses) (19, 20) and a decrease in proliferation in microvessels, particularly in the basalis layer of the endometrium (21).

Classically, mature EMAP II is detected as a 22-kDa protein. However, EMAP II in the human endometrium was detected as a 43-kDa protein by Western blot analysis using an antibody raised against the mature 22-kDa EMAP II protein (11). Expression of EMAP II as a 43-kDa protein has been detected previously in Meth A fibrosarcoma and 32D myeloid precursor cells (13), and it has been confirmed that the COOH-terminal of the 43-kDa protein is identical to the mature 22-kDa EMAP II (22, 23). It is not established yet whether the 43-kDa protein is a functionally active form of EMAP II or whether it is a precursor, which is converted to the mature 22-kDa protein on the initiation of apoptosis. However, support for a functional activity of p43 in the human endometrium can be substantiated from recent in vitro studies confirming that the p43 protein displays stronger cytokine activity than the 22-kDa EMAP II and can induce an increase in the expression of intracellular signaling proteins (such as ERK1/2, p38, and JNK) and chemokines and cytokines known to be associated in inflammatory pathways (24).

In the present study, it has been demonstrated by cDNA array analysis that EMAP II is a target gene for PGE2 in the human endometrium. Moreover, the cDNA array analysis and the in vitro culture experiments confirm that EMAP II expression is under negative regulation by PGE2. It can also be postulated that the temporal variation in the expression of EMAP II across the menstrual cycle may be a result of the negative regulatory effect of PGE2. EMAP II expression is up-regulated in the late secretory phase of the menstrual cycle, and this up-regulation can be temporally correlated with decreased activity of several components of the prostaglandin system. For example, expression of PGE synthase and synthesis of PGE2 are decreased (8) in the late secretory phase. Moreover, expression and signaling of the EP4 receptor in the human endometrium is lowest in the late secretory phase of the menstrual cycle (8). It is not known whether the effect of PGE2 on the regulation of EMAP II expression is mediated via the EP4 receptor subtype. However, evidence from an in vitro study has demonstrated a possible coregulatory pathway between EMAP II and PGE2; p43 has been shown to induce an up-regulation in EP4 receptor expression (24). It is also plausible that PGE2 may regulate expression of EMAP II via other EP receptor isoforms and alternate signaling pathways such as EP1, which is associated with PLC activation, inositol trisphosphate production, and mobilization of intracellular calcium.

EMAP II expression was localized to epithelial, stromal, and endothelial cells of the human endometrium at different stages of the menstrual cycle. This is the first detailed report of the distribution of EMAP II in endometrial tissue and supports a previous study in which EMAP II was detected in proliferative glandular epithelium (11). EMAP II expression was stronger and more frequent in stromal cells in the functionalis layer, compared with the basalis region of the endometrium, and this difference probably reflects the variability in cell types seen between the two compartments. It might be postulated that EMAP II expression will be more abundant in the functionalis layer because this is the major site that undergoes epithelial and endothelial cell apoptosis, together with the infiltration of leukocytes premenstrually (25, 26). Furthermore, it is the layer of the endometrium that is shed during menstruation. This spatial variation in expression of EMAP II correlates with a similar spatial and temporal pattern of expression in PGE synthase, and EP receptors in the human endometrium (8) and is further evidence for the regulatory effect of PGE2 on EMAP II expression.

As mentioned above, EMAP II, PGE2 and its receptors are expressed both in epithelial and endothelial cells of the endometrium (8). However, PGE2 and EMAP II show a number of opposing effects in vitro, which are consistent with the negative regulation of EMAP II synthesis by PGE2. There is good evidence that PGE2 promotes epithelial cell proliferation and angiogenesis, but EMAP II is proapoptotic and antiangiogenic in its action. In epithelial cells, several studies have demonstrated that PGE2 will promote proliferation and survival both in normal tissue (27) and in carcinomas (28), and EMAP II is correlated with levels of apoptosis and tissue remodeling (13). PGE2 has been shown also to promote angiogenesis in vitro both directly (29) by acting on endothelial cells and indirectly via autocrine/paracrine mechanisms leading to increased expression of angiogenic factors, including vascular endothelial growth factor and basic growth factor (30, 31). In contrast, EMAP II induces cell death in actively proliferating endothelial cells (15) and inhibits tumor angiogenesis (14).

In conclusion, our studies confirm the expression of EMAP II in the human endometrium with increased expression in the late secretory phase of the menstrual cycle. In addition, we have shown a negative regulatory effect of PGE2 on EMAP II expression. These results outline a role for EMAP II in key endometrial functions, such as initiation of apoptosis and the inflammatory reaction associated with menstruation.

Acknowledgments

We thank Teresa Drudy and Catherine Murray for tissue collection. We are also grateful to Dr. Clifford Murray for advice and providing the antibody to EMAP II.

Footnotes

Abbreviations: COX, Cyclooxygenase; EMAP II, endothelial monocyte-activating polypeptide II; FITC, fluoroscein isothiocyanate; HES, human endometrial epithelial cell; IHC, immunohistochemistry; RPA, ribonuclease protection assay.

Received February 5, 2002.

Accepted April 18, 2002.

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H.-Y. Lee, T. J. Acosta, D. J. Skarzynski, and K. Okuda
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