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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3973-3982
Copyright © 2003 by The Endocrine Society

Enhancement of Aminopeptidase A Expression during Angiotensin II-Induced Choriocarcinoma Cell Proliferation through AT1 Receptor Involving Protein Kinase C- and Mitogen-Activated Protein Kinase-Dependent Signaling Pathway

Kazuhiko Ino, Carlos Uehara, Fumitaka Kikkawa, Hiroaki Kajiyama, Kiyosumi Shibata, Takahiro Suzuki, Ei Ei Khin, Mitsuaki Ito, Mikihito Takeuchi, Atsuo Itakura and Shigehiko Mizutani

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (Ang II) is a bioactive peptide of the renin-angiotensin system, exerting its actions not only as a vasoconstrictor, but also as a growth promoter. In human placenta, type 1 Ang II receptors (AT1R) are predominantly expressed in trophoblasts, and we previously reported that aminopeptidase A (APA), a cell surface peptidase that converts Ang II to Ang III, is also expressed in both normal and neoplastic trophoblasts. However, the roles of Ang II and APA in trophoblast function remain to be clarified. In the present study we examined the effects of Ang II on proliferation and APA expression in trophoblast-like BeWo choriocarcinoma cells. Treatment of BeWo cells with Ang II significantly increased DNA synthesis in a dose-dependent manner. Ang II also enhanced APA mRNA and cell surface expression in BeWo cells analyzed by Northern blotting, flow cytometry, and enzyme activity assay. The Ang II-induced proliferation and APA up-regulation were blocked by the AT1R antagonist candesartan, but not by the AT2R antagonist PD123319. Furthermore, these Ang II effects were abolished by the protein kinase C inhibitor bisindolylmaleimide I and the MAPK inhibitor PD98059. Immunohistochemistry using choriocarcinoma tissues demonstrated that APA was expressed on the cell surface of AT1R-positive cytotrophoblastic cells in vivo. With these findings we demonstrate that Ang II stimulates the proliferation of trophoblastic cells via AT1R that are linked to protein kinase C /MAPK-dependent signaling pathways, and that the Ang II-degrading enzyme APA is up-regulated during Ang II-induced cell proliferation. These observations suggest the possible regulatory mechanism by the local renin-angiotensin system, especially the Ang II-AT1R-APA system, for the growth of human choriocarcinoma cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (ANG II), a major bioactive octapeptide of the renin-angiotensin system (RAS), plays a fundamental role as a vasoconstrictor in controlling cardiovascular function and renal homeostasis. Ang II also acts as a potent growth factor for various cell types, such as vascular smooth muscle cells (VSMC) (1, 2). The cellular effects of Ang II are mediated through specific receptor-linked, highly complex, intracellular signaling pathways (3, 4). Ang II binds at least two high affinity G protein-coupled receptors, AT1R and AT2R. Although most of the vascular effects of Ang II, such as vasoconstriction and growth, are mediated via AT1R, AT2R may oppose these effects (5).

In addition to the classical RAS for the regulation of the systemic blood pressure or sodium/water homeostasis, the concept of local RAS in the female reproductive tract has evolved (6, 7). Recent studies have shown that the components of RAS, including renin, angiotensinogen, angiotensin-converting enzyme (ACE), Ang II, and its receptors, are expressed in the utero-placental unit (8, 9, 10, 11). In the human placenta, AT1R is predominantly expressed and localized in both cytotrophoblasts and syncytiotrophoblasts in the chorionic villi as well as in fetal vascular endothelium (12, 13, 14, 15). As Ang II receptors are present in trophoblasts, it is possible that Ang II may act in an autocrine or paracrine fashion on trophoblasts. However, there have been few reports on the direct effects of Ang II on human trophoblastic cells (15, 16, 17, 18, 19). Although a variety of growth factors have been shown to be associated with various aspects of placental growth, its regulatory mechanisms remain obscure. Thus, it is interesting to study the role of Ang II in trophoblast functions, especially in trophoblast growth.

The local concentration of Ang II is dependent not only on the rate of its synthesis, but also on the rate of its metabolism. However, less attention has been paid to Ang II degradation in the regulation of the Ang II levels within tissues. One key enzyme involved in Ang II degradation is aminopeptidase A (APA; angiotensinase, EC 3.4.11.7), which can convert Ang II to Ang III by cleavage of the N-terminal aspartyl acid residue (20). Human APA has been cloned to be a 140- to 160-kDa type II membrane-bound metallopeptidase, which is identical to the mouse B cell differentiation antigen BP-1 (21) or the human kidney differentiation antigen gp160 (22). We previously purified APA from human placenta (23) and showed that it is expressed in placental trophoblasts, implicating this peptidase in the maintenance of homeostasis during pregnancy (24, 25). As the specific inhibitor of APA reduced the metabolism of Ang II and abolished the formation of Ang III in vitro and in vivo (26, 27), APA is considered an essential and highly specific enzyme to metabolize/inactivate Ang II and convert it to Ang III. Recently, we have shown that placental APA activity was higher in patients with preeclampsia than in normal pregnant women and that APA activity was increased by the addition of Ang II to primary culture of normal placental trophoblasts (25). We also demonstrated that APA was highly expressed in malignant trophoblasts using choriocarcinoma cell lines and tissues (28). These findings prompted us to hypothesize that APA could be involved in regulating normal and neoplastic trophoblast functions via degradation of Ang II.

The present study examined the effects of Ang II on the growth of human trophoblastic cells in vitro using a cytotrophoblast-like BeWo choriocarcinoma cell line and to clarify the involvement of Ang II receptors and its signal transduction pathway in their proliferation. Furthermore, we investigated the effect of Ang II on APA expression in BeWo cells and found a possible role for this peptidase during the Ang II-mediated proliferation of choriocarcinoma cells.


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

Human Ang II was purchased from the Peptide Institute (Osaka, Japan). The AT1R antagonist candesartan was donated by Takeda Chemical Industries (Osaka, Japan). The AT2R antagonist PD123319 was obtained from Sigma-Aldrich (St. Louis, MO). The selective MAPK kinase inhibitor PD98059 and the intracellular Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA) were purchased from Sigma-Aldrich. The specific protein kinase C (PKC) inhibitor, bisindolylmaleimide I (Bis I; GF109203X), and the specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, tyrphostin AG1478, were purchased from Calbiochem (San Diego, CA). Both rabbit polyclonal antibody against AT1R (306) and goat polyclonal antibody against AT2R (C-18) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-ERK1/2 and antiphosphorylated ERK1/2 antibodies were purchased from BioSource (Camarillo, CA). Mouse monoclonal antibody S4 (URO-2) specific to human APA was obtained from Signet Laboratories (Dedham, MA). Rabbit polyclonal antibody specific to human APA was raised against a synthetic peptide composed of the 18-amino acid sequence from Ser257 to Thr274 of human APA as previously described (28).

Cell line and culture

The human cytotrophoblast-like choriocarcinoma cell line, BeWo, was purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS; Sigma-Aldrich), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C in a 5% CO2 atmosphere.

Cell proliferation assay in vitro

Cell proliferation was evaluated based on the measurement of bromodeoxyuridine (BrdU) uptake during DNA synthesis in proliferating cells. Cells (2 x 104 /well) were seeded in 96-well microtiter plates and grown in culture medium containing 10% FCS. When the cells reached near subconfluence, the culture medium was replaced with serum-free medium for 12 h to render the cells quiescent. Then the cells were treated with various concentrations of Ang II at 37 C for 24 h. DNA synthesis was evaluated by measuring the incorporation of BrdU into DNA using the Cell Proliferation ELISA System (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s instructions. In some experiments cells were pretreated for 30 min with the receptor antagonists or inhibitors for intracellular signaling before the addition of Ang II. For the time-course assay, cells were seeded in 96-well microtiter plates and grown in culture medium for 24 h. The medium was replaced with serum-free medium containing various concentrations of Ang II, then cells were refed every 24 h with the same Ang II-containing medium up to 72 h of incubation. Cell growth was evaluated by the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) protocol using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) tetrazolium compound (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega Corp., Madison, WI).

Assay for enzymatic activity of APA

Cell surface APA activity was measured spectrophotometrically as previously reported (28). After treatment with various concentrations of Ang II, cells were collected by trypsinization and washed with PBS. Thereafter, intact cells (5 x 105 /well) were resuspended in 96-well microtiter plates in 200 µl substrate solution consisting of 1.5 mM {alpha}-L-glutamic acid-p-nitroanilide (Peptide Institute) in 0.1 M Tris-HCl (pH 7.0) containing 2.5 mM CaCl2. After incubation at 37 C for 30 min with continuous agitation, the reaction was terminated by the addition of ice-cold PBS. APA enzymatic activity was measured at 405 nm by a microplate reader (Labsystems, Helsinki, Finland). Cell-free and substrate-free blanks were run in parallel.

Flow cytometric analysis

Fluorescence-activated cell sorting (FACS) was performed to quantify APA expression on the cell surface of Ang II-treated BeWo cells. Cells were prepared to 1 x 106/ml in PBS and stained with mouse anti-APA monoclonal antibody S4 (diluted 1:40) or the isotype-matched control mouse IgG (Coulter, Hialeah, FL) for 1 h at 4 C. Then the cells were washed three times and incubated with phycoerythrin-conjugated goat antimouse IgG (Coulter; 1:80 dilution) for 30 min at 4 C. After three washes, FACS data were acquired on a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed using CellQuest software (BD Biosciences). Dead cells and debris were excluded from analysis by the window set. Ten thousand cells were analyzed in one parameter mode.

Immunoblot analysis

Immunoblotting for ERK was performed as previously described (29). Cells were grown in six-well culture plates in culture medium containing 10% FCS to reach subconfluence. The cells were serum-starved for 12 h and treated with Ang II for 1–30 min. The cells were washed with PBS and lysed in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and the protease inhibitor mixture. After centrifugation at 15,000 x g for 30 min, the supernatant was obtained. Thirty micrograms of protein extract were separated by SDS/10% PAGE, transferred onto nitrocellulose membranes, and immunoblotted with anti-ERK1/2 phosphospecific antibody or anti-total ERK1/2 antibody at a 1:1000 dilution. The biotinylated secondary antibody was used at a 1:200 dilution. Finally, immunoreactive proteins were stained using a chemiluminescence detection system (ECL, Amersham Pharmacia Biotech). Immunoblot analysis for AT1R expression was performed using the same procedure.

RNA isolation and Northern blot analysis

Total RNA was extracted from BeWo cells with the RNeasy kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol and was stored at -80 C until use. Northern blot analysis was performed as described previously (11). Briefly, aliquots of 20 µg total RNA were electrophoresed through 1.0% agarose gel containing 20 mM 3-(N-morpholino)-propanesulfonic acid (MOPS), 5 mM sodium acetate, 1 mM EDTA (pH 7.0), 0.66 M formaldehyde, and 2 µg/ml ethidium bromide, then transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech). APA probe was synthesized as described in our previous paper (30) and labeled with [32P]deoxy-CTP using the multiprime DNA labeling system (Amersham Pharmacia Biotech). ß-Actin cDNA, a housekeeping gene, was applied as a control. Hybridization was performed at 68 C for 2 h in hybridization solution. The membranes were washed at 68 C in 2x SSC/0.1% SDS three times and autoradiographed. Each was first hybridized with the APA probe, then deprobed and rehybridized with ß-actin probe. The mRNA levels were calculated after normalization to ß-actin expression with a BAS 2000 Bioimage analyzer (Fuji Photo Film Co., Kanazawa, Japan).

RT-PCR

One microgram of total RNA was used for RT using a Gene Amp RNA PCR kit (PerkinElmer, Norwalk, CT) according to the manufacturer’s protocol. One microliter of a solution of the RT reaction products was applied to PCR as described previously (30). The sense and antisense specific primers for human AT1R were 5'-GGAAACAGCTTGGTGGTGAT-3' and 5'-GCAGCCAAATGATGATGCAG-3', respectively. The sense and antisense primers for ß-actin were 5'-GGCTACAGCTTCACCACCA-3' and 5'-ACGGATGTCCACGTCACAC- 3', respectively. PCR products were separated by electrophoresis on 2% agarose gel and visualized by ethidium bromide staining.

Immunocytochemistry of BeWo cells

Cells were cultured in eight-well chamber glass slides (LabTek, Nunc Inc., Naperville, IL) in culture medium. The cells were fixed with 4% paraformaldehyde for 30 min at 4C. Immunocytochemical staining was performed using the avidin-biotin immunoperoxidase technique (29). Endogenous peroxidase activity was blocked by incubation with 0.3% H2O2. The primary antibody specific to AT1R or AT2R at a dilution of 1:100 was added and the slides were incubated for 2 h at room temperature. Then the slides were rinsed and incubated for 30 min with biotinylated second antibody (Nichirei, Tokyo, Japan). After washes with PBS, they were incubated for 10 min with horseradish peroxidase-conjugated streptavidin (Nichirei), and treated with 3-amino-9-ethylcarbazole (AEC, Nichirei) in PBS containing 0.01% H2O2. The slides were counterstained with Meyer’s hematoxylin. As negative controls, the primary antibody was replaced with normal rabbit or goat IgG at an appropriate dilution.

Immunohistochemistry of normal placenta and choriocarcinoma tissues

Tissue samples of human placenta as well as gestational choriocarcinoma were obtained with informed consent from patients who were surgically treated at Nagoya University Hospital. The use of the samples was approved by the ethics committee and institutional review board of Nagoya University Hospital. All samples were fixed in 10% formalin and embedded in paraffin, and sections were cut at a thickness of 4 µm. For heat-induced epitope retrieval, deparaffinized sections in 0.01 M citrate buffer were treated three times at 90 C for 5 min using a microwave oven. Immunohistochemical staining by the avidin-biotin immunoperoxidase method was performed using anti-AT1R or anti-APA polyclonal antibody as described above.

Statistical analyses

Ang II-mediated effects were determined as the percent increase over the control, with the control normalized to 100%. Each experiment was performed in triplicate, and the results are presented as the mean ± SD of at least three independent experiments. Statistical differences among various treatment groups were determined by ANOVA with post hoc Tukey’s test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ang II receptor expression in BeWo cells

First, we examined the expression of Ang II receptors in human cytotrophoblast-like BeWo choriocarcinoma cells. Immunocytochemical staining using specific polyclonal antibodies against Ang II receptors demonstrated that AT1R was clearly present in BeWo cells, whereas AT2R was absent (Fig. 1AGo). AT1R protein and mRNA expression was also detected in BeWo cells by immunoblotting (Fig. 1BGo) and RT-PCR analysis (Fig. 1CGo), as in the normal placental tissue used as a positive control.



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  		FIG. 1. Ang II receptor expression in BeWo cells. A, Immunocytochemical staining using rabbit anti-AT1R antibody or goat anti-AT2R antibody (original magnification, x200). For negative controls, rabbit IgG and goat IgG were used. AT1R was clearly present in BeWo cells. B, Immunoblot analysis for AT1R protein expression. AT1R was detected as an approximately 60-kDa band in BeWo cells (lane 1), and it was detected as a double band with molecular masses of 60 and 45 kDa in normal placenta (lane 2). C, RT-PCR for AT1R mRNA detection. A signal corresponding to AT1R mRNA (330 bp) was detected in BeWo cells (lane 2). Normal placental tissue was used as a positive control (lane 1). Signals corresponding to ß-actin mRNA (282 bp) were detected in both lanes. Restriction enzyme digestion of the PCR products was performed for confirmation of the AT1R transcript.

 
Ang II-induced proliferation of BeWo cells via AT1R

To examine the effects of Ang II on the growth of trophoblastic cells, cell proliferation assay was performed using BrdU uptake and the MTS assay. Treatment with Ang II at 10-7–10-5 M for 24 h resulted in a significant increase in DNA synthesis of BeWo cells in a dose-dependent manner (Fig. 2AGo). These Ang II-induced growth stimulatory effects were completely blocked by pretreatment with the AT1R antagonist candesartan, but not by the AT2R antagonist PD123319 (Fig. 2BGo). There was no cytotoxic effect observed when the cells were treated with the antagonist alone (no Ang II; Fig. 2BGo). These results clearly demonstrated that Ang II stimulated the proliferation of BeWo cells via AT1R. Further, the time-course assay showed that cell growth was significantly increased by the first 24-h incubation with more than 10-7 M Ang II (Fig. 2CGo). However, the growth rate was gradually decreased despite the additional Ang II treatment every 24 h, and the growth curves almost reached a plateau by 72 h (Fig. 2CGo).



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  		FIG. 2. Effect of Ang II on BeWo cell proliferation. A, Cells were treated with the indicated concentrations of Ang II for 24 h, and DNA synthesis was evaluated by BrdU uptake assay. Ang II-mediated effects were determined as the percent increase over control values (no Ang II), with the control normalized to 100%. Data are expressed as the mean ± SD of three independent experiments. *, P < 0.001; **, P < 0.0001 (vs. control). B, Cells were preincubated with the AT1R antagonist candesartan (10-5 M) or the AT2R antagonist PD123319 (10-5 M) for 30 min before the addition of Ang II (10-6 M). BrdU uptake was determined as a percentage of the control normalized to 100%. Data are expressed as the mean ± SD. *, P < 0.001 vs. control (no Ang II, no antagonists); **, P < 0.001 between Ang II and Ang II plus candesartan. C, Growth curves of BeWo cells during Ang II treatment. Cell growth was evaluated at 24, 48, and 72 h by the MTS assay measuring the OD at 492 nm. Data are expressed as the mean ± SD of three independent experiments. *, P < 0.01 vs. control (no Ang II).

 
Enhancement of APA expression and enzymatic activity by Ang II treatment

We previously showed that an Ang II-degrading enzyme APA was expressed in human placental trophoblasts or choriocarcinoma cells (25, 28). To determine whether APA could be involved in the process of Ang II-mediated trophoblast proliferation, we investigated the effects of Ang II on APA expression and enzymatic activity in BeWo cells. FACS analysis demonstrated that APA was weakly expressed on the cell surface of BeWo cells under steady-state conditions (Fig. 3AGo). Ang II enhanced cell surface expression of APA in a dose-dependent manner, as evaluated by FACS analysis (Fig. 3Go, A and B). APA mRNA was also up-regulated by treatment of BeWo cells with Ang II on Northern blot analysis (Fig. 4AGo). Ang II at 10-6 M induced a significant increase in APA mRNA; this mRNA up-regulation reached a maximum level after 3 h, then continued for 12 h (Fig. 4BGo). Consistent with the enhancement of APA cell surface and mRNA expression, Ang II treatment also increased APA enzymatic activity in a dose-dependent manner (Fig. 5AGo). The Ang II-mediated enhancement of APA activity was blocked by pretreatment with the AT1R antagonist candesartan, but not by the AT2R antagonist PD123319 (Fig. 5BGo). These findings demonstrated that Ang II up-regulated APA expression in BeWo cells via AT1R in parallel with the induction of cellular proliferation.



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  		FIG. 3. Cell surface expression of APA on Ang II-treated BeWo cells by FACS analysis. Cells were treated with Ang II for 24 h, and FACS analysis was performed as described in Materials and Methods. A, The histograms show that APA expression was detected at a low level in steady-state conditions (no Ang II), and it was shifted to higher levels by Ang II treatment. B, Dose-dependent increase in cell surface APA expression evaluated by mean fluorescence intensity with the control normalized to 100%. Representative data from three independent experiments are shown, and similar results were obtained in two other experiments.

 


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  		FIG. 4. Northern blot analysis for APA mRNA expression in Ang II-treated BeWo cells. Cells were treated with Ang II at 10-6 M for 3–12 h, and Northern blot analysis was performed as described in Materials and Methods. A, Representative autoradiogram is shown. APA mRNA was detected as signals corresponding to 4.5 kb. As a control, the filter was rehybridized with ß-actin probe. B, Ang II-induced APA mRNA expression was determined as a percentage of the control (time zero) after normalization to ß-actin mRNA expression. Data are expressed as the mean ± SD of three independent experiments. *, P < 0.05 vs. control.

 


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  		FIG. 5. APA enzymatic activity on Ang II-treated BeWo cells. A, Cells were treated with Ang II for 24 h, and APA activity was measured spectrophotometrically using {alpha}-L-glutamyl-p-nitroanilide. Ang II-mediated effects were determined as a percentage of the control (no Ang II) normalized to 100%. Data are expressed as the mean ± SD of three independent experiments, with each experiment performed in triplicate. *, P < 0.05 vs. control. B, Cells were preincubated with candesartan (10-5 M), PD123319 (10-5 M), or both for 30 min before the addition of Ang II (10-6 M). APA activity was determined as a percentage of the control normalized to 100%. Data are expressed as the mean ± SD. *, P < 0.01 vs. control (no Ang II, no antagonists), vs. Ang II plus candesartan, and vs. Ang II, candesartan, and PD123319.

 
Involvement of PKC and MAPK signaling pathways in Ang II-treated BeWo cells

Next, we attempted to clarify the possible signaling pathways involved in Ang II-induced BeWo cell proliferation and APA expression. As shown in Fig. 6AGo, Ang II treatment rapidly stimulated the phosphorylation of MAPKs, ERK1, and ERK2 in BeWo cells. Ang II-mediated growth stimulatory effects were mostly abolished by pretreatment with the PKC inhibitor Bis I (GF109203X), the intracellular Ca2+ chelator BAPTA, and the MAPK kinase inhibitor PD98059, but not by pretreatment with the EGFR tyrosine kinase inhibitor AG1478 (Fig. 6Go, B and C). Ang II-induced enhancement of cell surface APA expression was also blocked by the PKC inhibitor Bis I and the MAPK inhibitor PD98059 as well as by the AT1R antagonist candesartan (Fig. 7Go). These results suggest that Ang II enhances cell proliferation and APA expression in trophoblastic BeWo cells via activation of AT1R linked to Ca2+-dependent PKC and MAPK signaling pathways, but not mainly through the EGFR transactivation system.



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  		FIG. 6. Signaling pathways involved in Ang II-induced BeWo cell proliferation. A, Cells were treated with Ang II (10-6 M) for 2–15 min, then immunoblot analysis for phosphorylated ERK or total ERK expression was performed. ERK1 (p44) and ERK2 (p42) were rapidly phosphorylated by Ang II treatment. B, Cells were pretreated for 30 min with Bis I (10-6 M), PD98059 (10-5 M), or BAPTA (10-6 M), then treated with Ang II at 10-6 M for 24 h. BrdU uptake was determined as the percentage of control normalized to 100%. Data are expressed as the mean ± SD of three independent experiments. *, P < 0.001 between Ang II and Ang II plus Bis I; **, P < 0.001 between Ang II and Ang II plus PD98059; ***, P < 0.0001 between Ang II and Ang II plus BAPTA. C, Cells were pretreated for 30 min with AG1478, then treated with Ang II at 10-6 M for 24 h. BrdU uptake was determined as a percentage of the control normalized to 100%. Data are expressed as the mean ± SD.

 


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  		FIG. 7. Effects of PKC inhibitor and MAPK inhibitor on Ang II-induced APA cell surface expression. Cells were pretreated for 30 min with candesartan (10-5 M), Bis I (10-6 M), or PD98059 (10-5 M), then treated with Ang II at 10-6 M for 24 h. APA cell surface expression by FACS analysis was determined as a percentage of the control (no Ang II, no inhibitors) normalized to 100%. Data are expressed as the mean ± SD of three independent experiments. *, P < 0.01 between Ang II and Ang II plus Bis I; **, P < 0.001 between Ang II and Ang II plus candesartan or between Ang II and Ang II plus PD98059.

 
Immunohistochemical analysis for AT1R and APA expression in trophoblastic tissues

To confirm the expression of both AT1R and APA in trophoblastic cells in vivo, immunohistochemical staining was performed using formalin-fixed, paraffin-embedded tissue sections. AT1R was markedly expressed in cytotrophoblastic choriocarcinoma cells in uterine choriocarcinoma tissues, as it was in 20-wk normal placenta used as a positive control, where AT1R was localized not only in cytotrophoblasts, but also in syncytiotrophoblasts and fetal vessels (Fig. 8Go, A and B). In the same uterine choriocarcinoma tissue sections, APA was clearly localized on the cell surface of AT1R-expressing cytotrophoblastic choriocarcinoma cells (Fig. 8CGo).



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  		FIG. 8. Immunohistochemical analysis for AT1R and APA expression in formalin-fixed, paraffin-embedded sections of human trophoblastic tissues. A, Twenty-week normal placenta. AT1R was clearly expressed in villous trophoblasts and was weakly expressed in fetal vessels. ST, Syncytiotrophoblast (arrows); CT, cytotrophoblast (arrowheads); FV, fetal vessel. Original magnification, x200. B and C, Uterine choriocarcinoma tissues. AT1R was markedly expressed in choriocarcinoma cells (B). APA was clearly expressed on the cell surface of AT1R-expressing choriocarcinoma cells in serial sections (C). CC, Choriocarcinoma cells; SC, stromal cells. Original magnification, x400.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the placental RAS, previous studies focused on the Ang II effect on placental vessels as a vasoconstrictor and suggested its contribution to the regulation of fetoplacental blood flow (8, 12). However, AT1R is localized not only to placental vessels, but also to placental trophoblasts throughout gestation, whereas AT2R is very weakly expressed or is absent in the human placenta (9, 13). Consistent with these studies, our results from immunocytochemistry showed that AT1R, but not AT2R, was expressed in the trophoblast-like BeWo cells. AT1R protein and mRNA expression in BeWo cells was also confirmed by immunoblotting and RT-PCR analysis. Further, our immunohistochemistry demonstrated that AT1R was clearly expressed in normal placental trophoblasts as well as in uterine choriocarcinoma cells in vivo. Based on these evidences, we hypothesized that Ang II has a direct effect on human trophoblastic cells via AT1R.

Only a few reports have shown that Ang II is involved in the secretion of several placental hormones from trophoblasts via AT1R (15, 16, 17, 18). Xia et al. (19) recently reported that Ang II stimulated plasminogen activator inhibitor-1 synthesis in human trophoblasts via AT1R, indicating a role for Ang II in extravillous trophoblast invasion. However, the role of Ang II as a placental growth factor, in particular as a promoter of trophoblast proliferation in a physiological or neoplastic context, has never been investigated. The present study clearly demonstrated that Ang II stimulated the proliferation of BeWo cells, and this effect was shown to be dose dependent and mediated via the AT1R subtype. To our knowledge, this is the first report on the growth-promoting effect of Ang II on human trophoblastic cells. Recently, it has been shown that Ang II acts as a growth factor for various organs and cell types, such as VSMC, cardiac myocytes, and renal mesangial cells (1, 2, 31). Therefore, Ang II may be involved in placental growth in addition to the various previously identified growth factors. Confirmation of this observation in a primary culture of trophoblasts isolated from human placental tissues could definitively determine whether Ang II should be considered a growth factor for normal placental trophoblasts.

Ang II-mediated growth effects via AT1R and associated signaling pathways have been extensively studied in VSMC, whereas AT1R-mediated signaling in trophoblastic cells has not yet been identified. In cultured VSMC, the AT1R couples to a wide variety of signal transduction events, leading to intracellular Ca2+ mobilization, PKC activation, and protein tyrosine phosphorylation of multiple substrates, including ERKs, the main signaling molecules of the MAPK cascades (3, 4, 32). ERK activation by Ang II mediates further transmission of growth signals to the nucleus, which stimulates DNA synthesis and enhances cell proliferation (4, 33). Our results showed that treatment of BeWo cells with Ang II rapidly activated ERK1/2. Furthermore, the Ang II-mediated growth stimulatory effect was abrogated by the selective inhibition of PKC, intracellular Ca2+, and MAPK. These results indicated that Ang II-induced trophoblastic cell proliferation was mediated through the calcium-dependent PKC and MAPK/ERK signaling pathways. In agreement with our results, Zou et al. (34) reported that PKC was critical for Ang II-induced ERK activation in cardiac myocytes. ERK was also shown to be essential for Ang II-induced DNA synthesis and proliferation in VSMC (1, 2, 35). Recent studies have demonstrated that Ang II stimulates ERK via two pathways: one is PKC-dependent Raf-1 and ERK activation, and the other is Ca2+- and c-Src-dependent and involves transactivation of EGFR (36, 37, 38, 39). These two pathways vary between cell types. The latter is observed in VSMC, whereas the former is dominant in other AT1R-expressing cells, although there may exist a further diversity of AT1R signals and a cross-talk between the above two pathways (4, 36, 37). In the present study the EGFR tyrosine kinase inhibitor AG1478 at 0.2–20 µM could not block the Ang II-induced BrdU uptake of BeWo cells, and these concentrations of AG1478 are sufficient to fully inhibit EGFR activation (39). These findings suggest that EGFR transactivation was not mainly involved in the Ang II-dependent proliferation of BeWo cells, although further experiments are required to elucidate more detailed signaling pathways for Ang II-induced trophoblastic cell proliferation.

The present study demonstrated that Ang II enhanced the expression of APA while enhancing cellular proliferation in BeWo cells. Not only FACS analysis and enzyme assay, but also Northern blot analysis showed that Ang II clearly up-regulated APA mRNA and cell surface expression. This is consistent with our previous report showing that APA activity was increased by the addition of Ang II in a primary culture of normal placental trophoblast (25). It is of interest that APA expression could be regulated by its specific peptide substrate, Ang II, in trophoblastic cells. Furthermore, our results showed that enhancement of APA expression by Ang II was mediated through the AT1R and its downstream PKC- and MAPK-dependent signaling pathways, similar to the signaling system of Ang II-induced cell proliferation. It is known that cell surface peptidases, including APA, have a key role in the control of growth, differentiation, and signal transduction of many cellular systems by modulating the activity of peptide substrates and regulating their access to the receptors (40). Thus, up-regulation of APA expression during the process of Ang II-induced cell proliferation may lead to down-regulation of Ang II receptor binding and signal transduction through the degradation/inactivation of Ang II on the cell surface. This may contribute to regulation of Ang II-mediated trophoblastic cell growth. The time-course study showed that the growth rate was down-regulated by Ang II treatment for more than 24 h. These data may be due at least in part to increased Ang II degradation by APA on the BeWo cell surface. Consistent with our findings, Wolf et al. (31) recently reported that overexpression of APA abolished the growth-promoting effect of Ang II in mouse mesangial cells, and the APA inhibitor amastatin restored the proliferative effect of Ang II, indicating the regulatory role of APA in Ang II-dependent mesangial cell proliferation. However, down-regulation of AT1R expression and its desensitization were observed by continuous Ang II exposure in VSMC and mesangial cells (41, 42). In addition, regulation of AT1R by internalization in trophoblasts was reported (14). Thus, it appeared that APA could play some role in the regulation of Ang II-mediated signal transduction and proliferation in trophoblastic cells, although the change in the level of AT1R and its relation to APA during Ang II treatment remain to be elucidated.

Finally, the clinical significance of APA in human trophoblastic tissues should be discussed. In the feto-placental unit, we previously found that ACE, the essential enzyme processing Ang I to generate Ang II, was localized to endothelial cells of the fetal vessels, but not to trophoblasts (11). We also showed that ACE expression and activity in preeclamptic placenta were higher than those in normal placenta, suggesting overproduction of Ang II in preeclamptic placenta through the activated local RAS (11). Although the circulating Ang II levels in the umbilical artery or vein were lower (~10-9–10-10 M) than the effective concentrations used in our in vitro studies (43), trophoblasts might be exposed to the higher concentrations of locally generated Ang II within the placental tissues. Interestingly, we and others showed that placental APA activity was higher in patients with preeclampsia than that in normal pregnant women (25, 44). We also demonstrated that APA was highly expressed in malignant trophoblasts in human choriocarcinoma tissues (28). Our immunohistochemistry revealed that APA was localized to the membrane of choriocarcinoma cells that were coexpressing AT1R. The existence of both AT1R and APA might reflect the functional role of APA in trophoblastic tissues via degradation of locally generated Ang II. Taken together, it is possible to speculate that up-regulation of APA in trophoblastic tissues under conditions where local Ang II levels are elevated would have a regulatory effect on Ang II-mediated trophoblast proliferation. In support of our hypothesis, Healy and Song (45, 46) reported that APA could be regulated by Ang II in vivo, because rats exposed to increased levels of Ang II have elevated expression of APA in the kidney.

In conclusion, the present study provides the first demonstration that functional AT1R are present in human trophoblastic BeWo cells and that through the activation of AT1R and its downstream PKC/MAPK signaling pathways, Ang II stimulates trophoblastic cell proliferation. Furthermore, Ang II enhances its degrading enzyme APA during Ang II-induced proliferation. These data suggest new implications of the local RAS, especially the Ang II-AT1R-APA system, in regulating the growth of human placenta or the progression of trophoblastic tumors.


   Footnotes
 
Abbreviations: ACE, Angiotensin-converting enzyme; Ang II, angiotensin II; APA, aminopeptidase A; AT1R, angiotensin type 1 receptor; AT2R, angiotensin type 2 receptor; BAPTA, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetic acid tetraacetoxymethyl ester; Bis I, bisindolylmaleimide I; BrdU, bromodeoxyuridine; EGFR, epidermal growth factor receptor; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PKC, protein kinase C; RAS, renin-angiotensin system; VSMC, vascular smooth muscle cell.

Received October 10, 2002.

Accepted May 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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