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Endocrinology Vol. 147, No. 11 5153-5159
Copyright © 2006 by The Endocrine Society

Neutral Endopeptidase Expressed by Decidualized Stromal Cells Suppresses Akt Phosphorylation and Deoxyribonucleic Acid Synthesis Induced by Endothelin-1 in Human Endometrium

Akira Iwase, Hisao Ando, Tetsuro Nagasaka, Daijiro Shibata, Toko Harata, Yuji Shimomura, Maki Goto and Fumitaka Kikkawa

Departments of Obstetrics and Gynecology (A.I., H.A., D.S., T.H., Y.S., M.G., F.K.) and Maternal and Perinatal Medicine (A.I., T.H.), Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; and Division of Pathology (T.N.), Clinical Laboratory, Nagoya University Hospital, Nagoya 466-8560, Japan

Address all correspondence and requests for reprints to: Dr. Akira Iwase, Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: akiwase{at}med.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin-1 (ET-1) in human endometrium has been proposed to have a potential paracrine role, for its receptors are also present within this tissue. In addition, the expression of ET-1 varies during the menstrual cycle, and therefore, ET-1 may be involved in the cyclic change of the human endometrium, such as proliferation and decidualization. However, neither the inactivation of ET-1 in the endometrium nor the paracrine effect of ET-1 on endometrial cells has been determined. We investigated the production of ET-1 and the presence of neutral endopeptidase (NEP), which cleaves and inactivates ET-1, in primary cultured human endometrial cells. We found primary cultured endometrial epithelial cells, not stromal cells, to be the major source of ET-1. Western blot analysis and RT-PCR demonstrated that NEP was predominantly expressed by endometrial stromal cells. We also demonstrated that ET-1 stimulated the phosphorylation of Akt and DNA synthesis in endometrial stromal cells via the ETA receptor and phospahtidylinositol-3 kinase signaling pathways. The effect of ET-1 was regulated by NEP expressed by stromal cells. We also found that conditioned medium containing ET-1 from endometrial epithelial cell culture stimulated phosphorylation of Akt via the ETA receptor. In conclusion, ET-1 has a paracrine effect of Akt phosphorylation and cell proliferation on endometrial stromal cells, which occurs via the ETA receptor and phospahtidylinositol-3 kinase signaling pathways, and is regulated by cell-surface NEP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN ENDOMETRIUM undergoes a series of developmental changes in response to ovarian steroids that result in defined periods, including implantation and menstruation. This cyclic change consists of the decidualization and regeneration of the endometrium, which is composed of two kinds of epithelia, the luminal and the glandular epithelium, and stroma. Although the growth and regression of the human endometrium is primarily a function of the ovarian hormones, recent studies indicate a potential autocrine/paracrine role of regulatory molecules (1). Growth factors, bioactive peptides, and locally produced prostaglandins fall into this category (2, 3, 4, 5, 6, 7). Endothelin-1 (ET-1), which is a vasoconstrictive peptide with 21 amino acid residues (8, 9), may act as a paracrine or autocrine agent within the endometrium, because both the peptide and its receptors have been found within this tissue (10, 11, 12, 13). ET-1 was originally identified as a long-lasting vasoconstrictor compound in the supernatant of cultured endothelial cells (8). Therefore, a potential paracrine role in the regulation of uterine blood flow has been proposed (10, 14, 15). However, in addition to acting as a vasoconstrictor, ET-1 has been reported to have a range of paracrine actions in other tissues, such as cell proliferation through its mitogenic activity, and the regulation of gene expression (16, 17, 18, 19, 20, 21, 22). Therefore, it seems likely that ET-1 has additional functions in the human endometrium.

In addition, the metabolism of ET-1 in the human endometrium has not been entirely defined, whereas it is known that neutral endopeptidase (NEP; EC 3.4.24.11), a membrane-bound endopeptidase, degrades ET-1 in vitro or in other cell lines (9, 23, 24). More recently, we demonstrated that cell-surface peptidases play important roles in regulating the local function of bioactive peptides in human endometrium (25, 26). These results led us to hypothesize that a cell-surface peptidase, NEP, which has the functions of the cleavage and inactivation of ET-1, regulates ET-1 action in human endometrium.

In the present study, we evaluated the production of ET-1 and the expression of NEP in primary cultured endometrial epithelial and stromal cells. Because we observed the production of ET-1 in endometrial epithelial cells (EECs) and the presence of NEP in decidualized endometrial stromal cells (ESCs), we examined the effects of ET-1 on ESCs and the regulation of ET-1 action with NEP expressed by ESCs. Furthermore, we explored downstream signaling pathways of ET-1 in ESCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary culture of EECs and ESCs and in vitro decidualization of ESCs
EECs and ESCs were separated as described previously (25, 27, 28) based on a modification of the work of Satyaswaroop et al. (29). For all of these samples, informed consent was obtained from each patient before the study. In brief, endometrial tissue in the late proliferative phase was minced into small pieces (~1 mm3), and these pieces were filtered through a cell strainer consisting of 100-µm pore size nylon mesh (Becton Dickinson, Franklin Lakes, NJ) to remove the blood cells. Then, the minced tissue was incubated with stirring at 37 C for 20 min in PBS, 0.5% collagenase (Wako, Osaka, Japan) and deoxyribonuclease (0.1 mg/ml; Sigma Chemical Co., St. Louis, MO). The tissue digest was vigorously pipetted and passed over a cell strainer consisting of 70-µm pore size nylon mesh (Becton Dickinson). The epithelial glands were retained in the nylon mesh sieves, whereas the stromal cells passed through into the receptacle below. The epithelial cells were rinsed and back-flushed out of the sieves onto a 60-mm sterile dish. The stromal cells collected from the lower receptacle were suspended and plated onto a 60-mm sterile dish. The EECs and ESCs were cultured in RPMI 1640 medium (Sigma) containing 10% fetal calf serum (FCS) (Sigma), 100 IU/ml penicillin, and 100 µg/ml streptomycin. The purity of EECs and ESCs was assessed by morphological determination using light microscopy. Each cell population was routinely over 98% pure, as assessed by phase microscopy. The ESCs were cultured with progesterone (10–6 M; Sigma), 17ß-estradiol (10–8 M; Sigma) and dibutyryl cAMP (1 mM; Sigma) for 10–12 d at 37 C in a humidified atmosphere of 5% CO2 in air to induce in vitro decidualization. In vitro decidualization was assessed by evaluating morphological changes and confirming the expression of prolactin mRNA.

ET-1 assay
EECs and ESCs with or without decidualization treatment were seeded at 2 x 105 in 35-mm culture dishes and cultured in RPMI 1640 medium containing 10% FCS for 48 h. Then, the culture medium was replaced with RPMI 1640 medium containing 1% FCS, and the cells were cultured for 48 h before assay. The ET-1 concentration in the medium was determined with a sandwich enzyme immunoassay technique (AN'ALYZA Human Endothelin-1 Immunoassay; TECHNE Corp., Minneapolis, MN). Briefly, 100-µl aliquots of the samples (cell culture supernatants) or ET-1 standards provided with the kit were incubated with horseradish-peroxidase-conjugated anti-ET-1 antibody on a 96-well microplate coated with rat antibody to human ET-1 for 1 h. Then, 100 µl substrate (tetramethylbenzidine) was added to each well after washing and incubated at room temperature for 30 min. The OD at 450 nm was determined using the microplate reader. The intra- and interassay coefficients of variation at approximately 33 pg/ml ET-1 were 4.5 and 5.5%, respectively. The cross-reactivity of the kit was 45% for ET-2, 14% for ET-3, and less than 1% for big-endothelin.

Western blotting and RT-PCR for NEP, ETA receptor, and ETB receptor of EECs and ESCs
Primary cultured EECs and ESCs with or without decidualization were lysed in RIPA buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1.2% aprotinin, 5 µM leupeptin, 4 µM antipain, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM Na3VO4]. Cell lysates were clarified by centrifugation at 13,000 x g at 4 C for 15 min and diluted in 2x sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.2% bromphenol blue, and 4% 2-mercaptoethanol]. The protein extract (10 µg) was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking in PBS containing 5% skim milk for 1 h, the membranes were immunoblotted with mouse anti-NEP antibody (Ab) (NCL; Novocastra Laboratories Ltd., Newcastle, UK; 1:100), rabbit anti-ETA receptor Ab (Calbiochem, San Diego, CA; 1:1000), sheep anti-ETB receptor Ab (Calbiochem; 1:1000), or anti-ß-actin Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:5000). Immunoreactive proteins were stained using the enhanced chemiluminescence system (ECL; Amersham Biosciences Corp., Piscataway, NJ).

Total RNA was isolated from EECs and ESCs in 100-mm dishes using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. A RT reaction with 1 µg total RNA was carried out with a Gene Amp RNA PCR kit (PerkinElmer Corp., Norwalk, CT). Thereafter, 1-µl aliquots of the RT reaction products underwent PCR. The following sets of oligonucleotide primers were used: NEP, 5'-GTG CCC AGC AGT CCA ACT CAT TGA AC-3' (sense) and 5'-CCC CAT TTC TGT GGT GTT GGC AAG TC-3' (antisense), which corresponded to 1697–1722 and 2285–2310 of NEP cDNA (accession number NM_000902), respectively (30, 31, 32); prolactin, 5'-GCC CCC TTG CCC ATC TGT CC-3' (sense) and 5'-AGA AGC CGT TTG GTT TGC TCC T-3' (antisense), which corresponded to 601–616 and 966–986 of prolactin cDNA (accession no. NM_000948), respectively (33); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GGG GAG CCA AAA GGG TCA TCA TCT-3' (sense) and 5'-GAG GGG CCA TCC ACA GTC TTC T-3' (antisense), which corresponded to 399–424 and 614–635 of GAPDH cDNA, respectively (34). Amplification was performed using Taq polymerase (PerkinElmer) over 30 cycles for NEP and 25 cycles for GAPDH. Each cycle consisted of denaturation at 94 C for 1 min, annealing at 54 C for 1 min, and extension at 72 C for 1 min.

Akt phosphorylation with ET-1 or conditioned medium from EECs
For the detection of phosphorylated Akt, ESCs with or without decidualization were cultured in RPMI 1640 medium with 10% FCS in 35 mm dishes to 80% confluent status and the culture medium was replaced with RPMI 1640 medium without FCS. Then, the cells were cultured for 24 h and stimulated with 1 nM, 10 nM, or 1 µM ET-1 (Calbiochem) for 0–20 min. The cells were lysed, resolved by 10% SDS-PAGE, and transferred as described above. Membranes were immunoblotted with anti-Akt Ab (Santa Cruz Biotechnology; 1:500) or anti-phospho-Akt Ab (Ser 473; New England Bio Lab; 1:1000). Recombinant NEP (rNEP), phosphoramidon (NEP inhibitor; Peptide Institute, Osaka, Japan), BQ123 (ETA receptor antagonist; Calbiochem), BQ788 (ETB receptor antagonist; Calbiochem), LY294002 [phospahtidylinositol-3 kinase (PI3K) inhibitor; Promega Corp., Madison, WI), PD98059 (MAPK inhibitor; Promega), and wortmannin (PI3K inhibitor; Wako) were added 2 h before 10 nM ET-1 stimulation for 10 min. EECs were separated and cultured in RPMI 1640 medium containing 10% FCS for 3 d, as described above. The medium was then changed to unsupplemented RPMI 1640 medium, and cultivation was continued for 2 more days. The medium was then collected and used as the EEC-conditioned medium (EEC-CM). ESCs without decidualization were cultured with 1 µM BQ123, 1 µM BQ788, 10 µM LY294002, or 100 µM PD98059 for 2 h, and then the culture medium was replaced with EEC-CM containing 1 µM BQ123, 1 µM BQ788, 10 µM LY294002, or 100 µM PD98059. The cells were lysed with RIPA lysis buffer, as described above, after 10 min stimulation.

Bromodeoxyuridine (BrdU) cell proliferation assay
The rate of DNA synthesis was determined by the incorporation of BrdU into cells (BrdU Cell Proliferation Assay; Oncogene Research Products, San Diego, CA) according to the manufacturer’s instruction. Briefly, ESCs were seeded at 1 x 104 on a 96-well microplate and cultured with or without ET-1 stimulation for 24 h to allow BrdU incorporation. After fixation, the cells were incubated with anti-BrdU Ab for 1 h, followed by incubation with horseradish-peroxidase-conjugated goat antimouse IgG. Then, 100 µl substrate (tetramethylbenzidine) were added to each well after washing and incubated at room temperature for 30 min. The absorbance at dual wavelengths of 450–540 nm was determined using the microplate reader.

Statistical analysis
Statistical analysis was performed with the one-way ANOVA with Bonferroni correction using SigmaStat (SPSS Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ET-1 production by EECs and ESCs
Before investigating the ET-1 action in human endometrium, we first explored the ET-1 production of EECs and ESCs. There was very little ET-1 released from ESCs, with or without decidualization. On the other hand, the production of ET-1 by primary cultured EECs was significantly higher (Fig. 1Go). These results suggest that most of the ET-1 in human endometrium is produced by EECs, not ESCs.


Figure 1
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  		FIG. 1. ET-1 concentration in the culture medium of primary cultured EECs and ESCs. EECs and ESCs were cultured in RPMI 1640 medium containing 1% FCS for 48 h. The ET-1 concentration in the medium was determined using a sandwich enzyme immunoassay technique. The production of ET-1 by primary cultured EECs was significantly higher than those of ESCs. *, P < 0.001 compared with ESCs D– (without decidualization) and ESCs D+ (with decidualization). Values are the mean ± SD (n = 4). Experiments were repeated twice with similar results.

 
Akt phosphorylation induced with ET-1 in ESCs
Recent studies demonstrated that various G protein-coupled receptor agonists, such as ET-1, can induce the phosphorylation of various signaling molecules (35, 36, 37). We examined whether ET-1 could stimulate the phosphorylation of Akt, which is involved in cell survival/proliferation, in ESCs. Western blotting using an antibody to Ser 473-phospho-specific Akt revealed that the phosphorylation of Akt was induced by ET-1 in ESCs (Fig. 2Go).


Figure 2
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  		FIG. 2. A, Phosphorylation of Akt in ESCs with ET-1 treatment. ESCs were cultured with 10% FCS or without FCS for 24 h and then stimulated with 10 nM ET-1 for 5, 10, and 20 min. B, Phosphorylation of Akt in ESCs with 1 nM, 10 nM, or 1 µM ET-1 stimulation for 10 min. Cells were lysed, subjected to SDS-PAGE (20 µg per lane), and immunoblotted using anti-pAkt Ab (top) and anti-Akt Ab (bottom). The phosphorylation of Akt was induced by ET-1 in ESCs. Experiments were repeated three times with similar results.

 
In vitro expression of NEP in primary cultured EECs and ESCs and the inhibition of ET-1-induced Akt phosphorylation by NEP in ESCs
We next performed Western blotting and RT-PCR using primary cultured EECs and ESCs to assess the expression of NEP in vitro. Western blotting showed that decidualized ESCs expressed NEP, whereas EECs and nondecidualized ESCs possessed negligible NEP (Fig. 3AGo). RT-PCR showed similar results as Western blotting (Fig. 3BGo). Expression of mRNA of prolactin, an endometrial decidualization marker was also confirmed. NEP expressed by the human endometrium has been implicated in the regulation of ET-1. However, there have been few reports that NEP regulates the effects of ET-1 on cells directly. As shown in Fig. 3CGo, ET-1 did not stimulate the phosphorylation of Akt in ESCs incubated with rNEP. Furthermore, ET-1 did not induce the phosphorylation in decidualized ESCs, which express NEP, and phosphoramidon, an NEP inhibitor, abolished this inhibition of ET-1-induced Akt phosphorylation in NEP-positive decidualized ESCs. Phosphoramidon or rNEP alone did not affect the phosphorylation of Akt in ESCs (data not shown). These results indicate that cell-surface NEP expressed by ESCs, which can inactivate ET-1, regulates the effect of ET-1 on ESCs.


Figure 3
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  		FIG. 3. A, Expression of NEP in primary cultured EECs and ESCs. A 10-µg sample was subjected to SDS-PAGE and immunoblotting using anti-NEP Ab (top) or anti-ß-actin Ab as an internal control (bottom). B, RT-PCR for NEP. Each PCR product corresponds to 1697–2310 of NEP cDNA (top), 601–986 of prolactin (PRL) cDNA (middle), and 399–635 of GAPDH cDNA (bottom), respectively. Three independent experiments gave similar results. Western blotting and RT-PCR showed that decidualized ESCs expressed NEP, whereas EECs and nondecidualized ESCs possessed negligible NEP. C, Phosphorylation of Akt in ESCs. ESCs without decidualization were cultured with FCS (lane 1), without serum (lane 2), without serum and then with 10 nM ET-1 for 10 min (lane 3), and without serum and then with 100 µg/ml rNEP for 2 h followed by 10 nM ET-1 (lane 4). Decidualizd ESCs were also cultured with FCS (lane 5), without serum (lane 6), without serum and then with 10 nM ET-1 for 10 min (lane 7), and without serum and then with 100 µg/ml of rNEP and 2 µM of phosphoramidon for 2 h followed by 10 nM ET-1 (lane 8). ET-1 did not induce the phosphorylation in decidualized ESCs, which express NEP. Phosphoramidon, an NEP inhibitor, abolished this inhibition of ET-1-induced Akt phosphorylation in NEP-positive decidualized ESCs. Decidualization was confirmed by expression of prolactin mRNA using RT-PCR (bottom). Experiments were repeated three times with similar results.

 
Effects of endothelin receptor antagonists on the phosphorylation of Akt induced with ET-1 and conditioned medium from EECs
The effects of endothelin receptor antagonists on ET-1-induced Akt phosphorylation were determined. ETA receptor and ETB receptor were expressed by ESCs without decidualization (Fig. 4AGo). The ET-1-induced Akt phosphorylation was completely blocked by treatment with BQ123, an ETA receptor antagonist, whereas BQ788, an ETB receptor antagonist, had no significant effect, indicating that the ETA receptor might be involved in the ET-1-induced Akt phosphorylation in ESCs (Fig. 4BGo). Next, to investigate the effect of EEC-CM containing ET-1 on the phosphorylation of Akt in ESCs, we added EEC-CM at 50% of the final concentration to serum-free RPMI 1640 after incubation with BQ123 or BQ788 for 2 h. As shown in Fig. 4CGo, BQ123, not BQ788, partially inhibited the EEC-CM-induced Akt phosphorylation. Taken together, ET-1 produced by EECs stimulates the phosphorylation of Akt via the ETA receptor, not the ETB receptor.


Figure 4
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  		FIG. 4. A, Expression of ETA receptor (ETAR) and ETB receptor (ETBR) in ESCs without decidualization. B, Inhibition of ET-1-induced Akt phosphorylation in ESCs with endothelin receptor antagonists. ESCs without decidualization were cultured with 1 µM BQ123 (ETA receptor antagonist) or 1 µM BQ788 (ETB receptor antagonist) for 2 h and then stimulated with 10 nM ET-1 for 10 min. The ET-1-induced Akt phosphorylation was completely blocked by treatment with BQ123. C, Phosphorylation of Akt in ESCs with conditioned medium (CM) from EEC culture and the inhibition of Akt phosphorylation with endothelin receptor antagonists. ESCs without decidualization were cultured with 1 µM BQ123 or 1 µM BQ788 for 2 h, and then the culture medium was replaced with CM. The CM-induced Akt phosphorylation was partially blocked by treatment with BQ123. Three independent experiments gave similar results.

 
Inhibition of ET-1-induced Akt phosphorylation and cell proliferation by PI3K inhibitors
We finally explored the signaling pathways downstream of ET-1 in ESCs and investigated the effects of ET-1 on cell proliferation. Pretreatment with PI3K inhibitors, LY294002, or wortmannin inhibited the ET-1-induced Akt phosphorylation. On the other hand, a MAPK inhibitor, PD98059, showed a negligible effect on the phosphorylation of Akt (Fig. 5AGo). Similarly, pretreatment with LY294002 inhibited the EEC-CM-induced Akt phosphorylation (Fig. 5BGo). A BrdU cell proliferation assay revealed that ET-1 induced DNA synthesis in ESCs, which was inhibited by LY294002 but not PD98059 (Fig. 5CGo).


Figure 5
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  		FIG. 5. A, Inhibition of ET-1-induced Akt phosphorylation in ESCs with PI3K inhibitors. ESCs were cultured with 10 µM LY294002 (PI3K inhibitor), 100 µM PD98059 (MAPK inhibitor), or 100 or 300 nM wortmannin (PI3K inhibitor) for 2 h and then stimulated with 10 nM ET-1 for 10 min. Pretreatment with PI3K inhibitor, LY294002, or wortmannin inhibited ET-1-induced Akt phosphorylation. B, Inhibition of EEC-CM-induced Akt phosphorylation in ESCs with PI3K inhibitors. Pretreatment with LY294002 inhibited the EEC-CM-induced Akt phosphorylation. C, BrdU proliferation assay (24 h). ESCs were cultured with 10 nM ET-1, 10 nM ET-1 and 10 µM LY294002, or 10 nM ET-1 and 100 µM PD98059 for 24 h. The incorporation of BrdU was measured using an ELISA system. ET-1 induced DNA synthesis in ESCs, which was inhibited by LY294002 but not PD98059. *, P = 0.001; **, P = 0.006. Values are the mean ± SD (n = 4). Experiments were repeated twice with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we found that ET-1 is mainly produced in EECs and stimulates the phosphorylation of Akt and DNA synthesis in ESCs. This is the first report to demonstrate the paracrine action of ET-1 in vitro using conditioned medium from EEC culture, although it had been proposed that ET-1 might possess a paracrine function in the human endometrium (5, 6, 10, 11, 12, 14, 15, 38). Previous immunolocalization studies showed that ET-1 is present throughout the cycle in both epithelial cells and stromal cells (6, 10, 11). The inhibition of the passage of proteins through the Golgi apparatus with the ionophore monensin resulted in an accumulation of ET in the epithelium, not the stroma, suggesting that ET synthesis occurs primarily in the endometrial epithelium (10), which supports our results that ET-1 is produced mainly by EECs, and not ESCs, in vitro. A number of reports have been published regarding endothelin and its receptors in the human endometrium. In contrast, the function of endometrial ET-1 has hardly been demonstrated either in vivo or in vitro. Recent studies indicate that ET-1 contributes to various biological functions, such as cell proliferation and migration, in other types of cells (16, 17, 18, 19, 20, 21, 22). In the present study, we also demonstrated that ET-1 induced the phosphorylation of Akt and DNA synthesis in ESCs. Furthermore, we demonstrated that the ETA receptor and PI3K signaling pathways are involved in the downstream ET-1 stimulation in ESCs. PI3K and Akt are not only key molecules for cell survival/proliferation but have also been demonstrated to be involved in gene expression via the regulation of transcription factors (39). In addition, Sumitomo et al. (40, 41) revealed that ET-1 induces the phosphorylation of Akt in prostate cancer cells and, therefore, inhibits apoptosis of the cells. Therefore, these results suggest that endometrial ET-1 may have functions other than vasoconstriction. Taken together, ET-1 might be involved in the proliferation and/or decidualization of ESCs via PI3K/Akt signaling pathways.

Considering the cyclic regeneration and decidualization of the human endometrium, the metabolism and inactivation of key molecules regulating the functions and morphology of the endometrium are as important as production and activation. It is well known that NEP cleaves and inactivates ET-1 (9, 23, 24) and is expressed by the human endometrium (42). NEP has been considered to inactivate its peptide substrates, including ET-1, and therefore to be involved in the cyclic change of the human endometrium. However, it was not proven that cell-surface NEP expressed by endometrial cells inactivates ET-1 directly. We demonstrated here for the first time that NEP in decidualized ESCs regulates the response to stimulation with ET-1 in vitro. Decidualization in the human endometrium occurs after the implantation period in the secretory phase due to the effect of progesterone and is followed by menstruation in the nonpregnant cycle. Therefore, the production and inactivation of the molecules in the decidualization process might be involved in the functional and morphological change of the human endometrium toward pregnancy. Our results suggest that the ET-1 may be that released from EECs and that NEP expressed by ESCs regulates the paracrine action of ET-1, which might be implicated in the cyclic regeneration and decidualization of the human endometrium including cell proliferation and decidualization via the phosphorylation of Akt (Fig. 6Go).


Figure 6
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  		FIG. 6. Proposed model of the cooperative effect of EECs and ESCs on the activation/inactivation of ET-1. ET-1 is produced by EECs and might be involved in the proliferation of ESCs via Akt phosphorylation, which is regulated by NEP on the cell surface of ESCs.

 
Our extensive studies on peptidases indicate that various peptidases are expressed in and localized to a certain part of the human endometrium. We previously reported that aminopeptidase A expressed in endometrial stromal cells disappears with decidual change (25) and that placental leucine aminopeptidase/oxytocinase localized to epithelial cells shows a change in the subcellular distribution during the menstrual cycle (26). We also found that adipocyte-derived leucine aminopeptidase, a homologue of placental leucine aminopeptidase, which produces peptides present on the class I major histocompatibility complex, is expressed in the endometrial epithelium (28). It is of interest that various peptidases, which have different substrates and different functions, show different profiles of expression and localization in the human endometrium. These results prompt us to speculate that EECs and ESCs have different characteristics, such as the expression profile of peptidases, and therefore show cooperative action. In the present study, we found that NEP is expressed only by stromal cells in human endometrium and regulates stimulation with ET-1, which is released by epithelial cells in vitro, on ESCs. Our findings indicate that the cooperative action of EECs and ESCs in the regulation of ET-1 might be involved in the cyclic change of the human endometrium, although there is not yet a consensus that ET-1 in endometrial stroma is always derived from EECs. Cells in the human endometrium are under the influences of biologically active substances such as cytokines in vivo. Therefore, there is a possibility that ESCs produce ET-1 under certain conditions as well as NEP and regulate the autocrine action of ET-1 by themselves. However, Arnold et al. (27) recently reported that endometrial stromal cells regulate epithelial cell growth in vitro and therefore proposed a coculture model of EECs and ESCs. All things considered, the cooperative action of EECs and ESCs may regulate the development and function of the endometrium.

In summary, our findings indicate that EECs and ESCs regulate the activation and inactivation of ET-1, which may be involved in cell proliferation, cell differentiation, and gene expression in ESCs via the ETA receptor and Akt/PI3K signaling pathways. Additional study, including that to establish whether ET-1 is secreted basally so that it can interact with stroma in vivo, will help define the influence of the cooperative action of EECs and ESCs on the cyclic developmental change of the human endometrium.


   Acknowledgments
 
We acknowledge Hiroko Sato for her technical support.


   Footnotes
 
This work was supported in part by Grant-in-Aid 17791114 for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (A.I.).

Disclosure statement: The authors have nothing to disclose.

First Published Online August 3, 2006

Abbreviations: Ab, Antibody; BrdU, bromodeoxyuridine; EEC, endometrial epithelial cell; EEC-CM, EEC-conditioned medium; ESC, endometrial stromal cell; ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEP, neutral endopeptidase; PI3K, phospahtidylinositol-3 kinase; rNEP, recombinant NEP.

Received February 9, 2006.

Accepted for publication July 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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