Antiproliferative Effects of Adiponectin on Human Trophoblastic Cell ...

BIOLOGY OF REPRODUCTION 80, 1107–1114 (2009) Published online before print 25 February 2009. DOI 10.1095/biolreprod.108.070573

Antiproliferative Effects of Adiponectin on Human Trophoblastic Cell Lines JEG-3 and BeWo1 Delphine Benaitreau, Marie-Noe¨lle´ Dieudonne´,2 Esther Dos Santos, Marie-Christine Leneveu, Philippe de Mazancourt, and Rene´ Pecquery Service de Biochimie et Biologie Mole´culaire, UPRES-EA 2493, Faculte´ de Me´decine Paris-Ile de France Ouest, PRES Universud Paris, Centre Hospitalier de Poissy-Saint Germain, Universite´ de Versailles-St-Quentin-en-Yvelines, Poissy, France ABSTRACT

adiponectin, adiponectin receptors, BeWo, cytokines, implantation, JEG-3, placenta, proliferation, signal transduction, trophoblast

INTRODUCTION Embryo implantation and placentation are two critical steps for establishing pregnancy. During placentation, trophoblast cells differentiate into cytotrophoblastic and syncytiotrophoblastic cell types. The syncytiotrophoblast has endocrine activity, whereas cytotrophoblasts have invasive and proliferative properties during placentation. However, unlike cancerous cells, these proliferative and invasive capacities of cytotrophoblast cells are controlled and regulated to ensure successful implantation [1, 2]. Placental functions are regulated by many hormones, growth factors, and cytokines that are produced locally or distally to permit normal placental development and fetal growth [3–5]. Among these, human chorionic gonadotropin (hCG) is produced by the placenta and promotes trophoblast migration and invasion but does not 1 Supported by the Universite´ de Versailles-St-Quentin-en-Yvelines, France. 2 Correspondence: FAX: 33 01 0 139274415; e-mail: [email protected]

Received: 14 May 2008. First decision: 10 June 2008. Accepted: 28 January 2009. Ó 2009 by the Society for the Study of Reproduction, Inc. eISSN: 1259-7268 http://www.biolreprod.org ISSN: 0006-3363

MATERIALS AND METHODS Materials Dulbecco modified Eagle medium (DMEM), DMEM/F12, penicillin, streptomycin, HEPES, leupeptin, aprotinin, 5-aminoimidazole-4-carboxamide 1b-D ribofuranosyl 5 0 monophophate (AICAR), 4-(2-aminoethyl)-benzene-

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During embryo implantation, a complex dialog exists between the mother and the fetus. However, little is known about the molecules that participate in this process. Among various factors secreted at the maternal-fetal interface, the adipose tissuederived leptin is now considered a placental growth factor. Adiponectin is another adipocyte-derived signaling molecule known to exert antiproliferative effects in various cell types. In this work, we studied adiponectin sensitivity and effects on JEG-3 and BeWo choriocarcinoma cell lines. First, we showed that JEG3 and BeWo cells express the specific adiponectin receptors ADIPOR1 and ADIPOR2 and respond to human recombinant adiponectin by AMP-activated protein kinase (PRKA, also known as AMPK) activation. Second, we demonstrated that adiponectin induces a reduction in cell number and in [3H]-thymidine incorporation, demonstrating that adiponectin has antiproliferative effects on trophoblastic cells. Furthermore, these effects of adiponectin seem to be, at least in part, mediated by the mitogenactivated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) signaling pathways. We describe herein the direct effects of adiponectin in the control of trophoblastic cell proliferation.

affect proliferation [6]. Progesterone and estradiol upregulate trophoblast cell proliferation [7]. Finally, it has been recently demonstrated that IGF1 and IGF2 were able to induce cytotrophoblast proliferation and prevent cell apoptosis [8]. Leptin is mainly expressed in adipose tissue and, to a lesser extent, in placenta [9, 10]. Studies [11, 12] have confirmed leptin involvement in placental development. In particular, leptin promotes trophoblast cell proliferation, survival, and invasion [13–16]. All these results demonstrate that leptin can be considered a placental hormone. Adiponectin is another adipokine shown to have pleiotropic activities (in contrast to leptin) such as anti-inflammatory, antiangiogenic, antiatherosclerotic, and antiproliferative effects [17, 18]. Adiponectin is present at high concentrations (5–15 mg/L) in human circulation and is described as an insulinsensitizing hormone [17, 19]. Adiponectin is synthesized as a simple polypeptide of 30 kDa and is then assembled into an array of complexes composed of multimers of the 30-kDa polypeptide (trimers, middle-molecular-weight hexamers, and an elaborate high-molecular-weight [HMW] complex). The HMW form is predominant in human circulation [20]. The biological actions of adiponectin are carried out through interactions with its specific surface adiponectin receptor subtypes ADIPOR1 and ADIPOR2 [21]. Additional receptors for adiponectin have been recently described such as Tcadherin [22]. Binding of adiponectin to its receptors ADIPOR1 and ADIPOR2 activates various signal transduction pathways such as the PRKA (AMP-activated protein kinase), PIK3 (phosphoinositide-3-kinase), P38/P42/P44 MAPK (mitogen-activated protein kinase), and JUN kinase pathways [23– 25]. ADIPOR1 is abundantly expressed in muscle, and ADIPOR2 is predominantly expressed in liver [21]. Furthermore, ADIPOR1 and ADIPOR2 are both expressed in human endometrium and placenta [26–28]. In the endometrium, expression of ADIPOR1 and ADIPOR2 is increased during the luteal period, which corresponds to the embryo implantation period [28]. These observations suggest that adiponectin could be implicated in the implantation process. Therefore, the objective of this study was to determine whether adiponectin and its receptors are involved in the molecular mechanisms controlling proliferation of trophoblastic cells. The direct effects of adiponectin on growth capacities of JEG-3 and BeWo choriocarcinoma cell lines, commonly used as models for first-trimester trophoblast cells, were investigated.

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sulfonyl fluoride (AEBSF), NaF, and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human adiponectin and leptin were provided by R&D Systems Europe Ltd. (Abingdon, England), Superscript II RNase H-RT by Invitrogen Corporation (Carlsbad, CA), and RNAguard by Pharmacia Biotechnology (Uppsala, Sweden). Mitogen-extracellular kinase (MEK) inhibitor UO126 was from Promega (Madison, WI), and PIK3 (PI3K) inhibitor LY294002 was from Calbiochem (Merck Chemicals, Nottingham, England). Origins of different antibodies used are described in the following paragraphs.

Tissue Preparation and Cell Culture

[3H]-Thymidine Incorporation JEG-3 and BeWo cells were plated into 24-well culture dishes at a density of 1.5 3 104 in 10% and 15% FCS for JEG-3 and BeWo cells, respectively. After 24 h, cells were cultured in medium supplemented with 1% FCS and various concentrations of human recombinant adiponectin, 250 ng/ml of human recombinant leptin, or 50 lM forskolin. The latter was used to inhibit choriocarcinoma cell proliferation [30]. One percent serum in culture medium was chosen because previous experiments performed with 0%, 1%, or 5% serum revealed that 1% serum was the best concentration to avoid possible stimulatory effects of factors present in FCS and to allow the survival of both cell lines. In some experiments, signaling pathways were studied in the presence or absence of adiponectin (250 ng/ml) with or without 10 lM UO126 and 10 lM LY294002, inhibitors of MAPK and PIK3 signaling pathways, respectively, for 24 h and/or 48 h. For the last 6 h, [3H]-thymidine (1 mCi/ml) was added to the culture medium. After washing three times with saline buffer, cells were lysed during 5 min with 1% SDS and were treated with 10% trichloroacetic acid for 45 min at 48C. Radioactivity was counted after filtration on GF/C filters (Whatman, Clifton, NY).

Cell Counting Experimental cell culture conditions used were those already described except that the adiponectin concentration tested was 250 ng/ml only and that (after the washing steps) cells were harvested with calcium-free and magnesium-free Hanks solution containing 0.2% trypsin. Finally, cells were stained for 5 min using crystal violet (0.5 mg/ml) and were counted in a hemocytometer before and after 24, 48, and 72 h of treatment.

Immunohistochemistry To study the effects of adiponectin on placental growth ex vivo, we cultured placental explants (6–9 wk) obtained from eight different placentas on collagen type I-coated dishes (BD Biosciences, Bedford, MA) in F12 medium with 1% FCS at 378C in a 5% CO2 atmosphere during 24, 48, and 72 h in the presence or absence of adiponectin (250 ng/ml). After careful removal, placental explants were fixed in cold phosphate-buffered neutral paraformaldehyde (4%), embedded in paraffin, and cut into 5-lm sections. Deparaffinized sections in 0.01 M citrate buffer were treated three times for 5 min at 908C in a microwave oven for heat-induced epitope retrieval. Endogenous peroxidase activity was blocked by incubation with 1% H2O2, and nonspecific IgG binding was blocked by incubation with normal blocker serum. Samples were incubated with primary monoclonal mouse anti-human MKI67 (also known as Ki67) antibody or anti-human cytokeratin 7 (DAKO, Glostrup, Denmark) for 60 min. The slides were then rinsed and incubated with biotin-conjugated secondary antibody for 10 min at room temperature. Visualization was achieved using a

ADIPOR1 and ADIPOR2 Protein Expression Confluent JEG-3 cells, BeWo cells, and fresh placental tissue were sonicated on ice in buffer containing 50 mM Tris, 120 mM NaCl, 1 mM edetic acid (EDTA), 1% Nonidet P40, 0.5 mM desoxycholate, 0.1% SDS, 1 mM sodium vanadate, 30 mM b-glycerophosphate, 5 lg/ml of aprotinin, 12.5 lg/ml of leupeptin, 100 lg/ml of AEBSF, and 10 mM NaF. Human breast cancer cell line MDA-MB 231 cells were also used as positive controls [31]. After centrifugation at 100 000 3 g for 10 min at 48C, supernatants were diluted in Laemmli buffer (vol/vol). Protein concentration was measured according to the method by Bradford [32] with BSA as standard. Equal amounts of proteins were resolved by SDS-PAGE (10% acrylamide) and were transferred to polyvinylidene fluoride (PVDF) membrane and blocked in buffer A (20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20, pH ¼ 7.6) with 2.5% gelatin during 2 h. Membranes were incubated overnight at room temperature with goat polyclonal anti-ADIPOR1 (sc-46749) or anti-ADIPOR2 (sc-46756) antibodies (1:300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in buffer A with 2.5% gelatin. Finally, an enhanced chemiluminescence kit from Thermo Scientific (Rockford, IL) was used for signal detection.

PRKA Activation The presence of serum in culture medium could induce unwarranted kinase phosphorylation. For these reasons, cells were maintained in a serum-free medium overnight as described by Dos Santos et al. [33] and by Dieudonne et al. [34]. Cells were then exposed during 5, 15, and 30 min and 1 h to human recombinant adiponectin (250 ng/ml) or during 30 min to AICAR 500 lM, a PRKA activator [35]. Thereafter, cells were scraped and sonicated in cold buffer as already described. Equal amounts (30 lg) of cellular extracts were subjected to SDS-PAGE (12.5%). Proteins were transferred to PVDF membrane and were blocked in buffer A with 2.5% gelatin during 2 h. Membranes were incubated overnight at room temperature in buffer A with 2.5% gelatin rabbit polyclonal anti-phospho-PRKA antibody (1:700 dilution, #2531; Cell Signaling Technology, St-Quentin-en-Yvelines, France) or rabbit polyclonal anti-total PRKA antibody (phosphorylated and nonphosphorylated forms) (1:1000 dilution, #2532; Cell Signaling Technology). Incubation with the secondary antibody and signal detection were performed as already described. Control experiments with various protein amounts (10–50 lg) were performed to ensure that the densitometric signal intensity was proportional to the loaded amount of protein.

Quantitative RT-PCR Total RNA (0.1 lg) was extracted and reverse transcribed as previously described [36]. Quantitative PCR was performed using a LightCycler 480 instrument from Roche Diagnostics (Basel, Switzerland) with primer sets listed in Table 1. Second derivative maximum method was used to automatically determine the crossing point (Cp) for individual samples. Three reference genes (TBP, RP13A, and B2M [b2 microglobulin]) have been tested for their expression stability in JEG-3 and BeWo cells. Analysis using geNorm software (PrimerDesign Ltd., Southampton, England) revealed that the TBP gene was the most stable gene to normalize data. For each sample, the concentration ratio (target/TBP mRNA) was calculated using RelQuant software (Roche Diagnostics) and was expressed in arbitrary units. Calibration curves were log linearized over the quantification range with r2 correlation coefficient .0.99 and with efficiency ranging from 1.8 to 2. The intraassay variability of duplicate Cp values never exceeded 0.2 cycle, and the interassay variability (coefficient of variation value) ranged from 1% to 5% for the three or four runs of each transcript. The PCR products were separated on a 2% agarose gel in 90 mM Trisborate and 2 mM EDTA buffer (Tris-borate-EDTA) (pH 8.0). They were visualized by staining with ethidium bromide and by UV transillumination.

Statistical Analysis All values were expressed as means 6 SEM of four to eight separate experiments. Statistical analysis was performed using the nonparametric paired Wilcoxon test or unpaired Mann-Whitney U-test.


Placental tissues (6–12 wk [n ¼ 8]) were obtained from healthy pregnant women aged 18–35 yr when undergoing legal abortions. Placental tissues were placed in saline (NaCl [150 mM]), washed several times, and aseptically dissected to remove decidual tissues and fetal membranes. Intra-abdominal adipose tissues were obtained from patients undergoing elective abdominal surgery (mean 6 SEM age, 59.5 6 4.3 yr). This study was approved by the local ethical committee (Comite´ Consultatif pour la Protection des Personnes dans la Recherche Biomedicale), and informed consent was obtained from each donor before clinical sampling. Human choriocarcinoma cell lines JEG-3 and BeWo obtained from American Type Culture Collection (Manassas, VA) were maintained under standard culture conditions in 5% CO2 atmosphere at 378C. Culture media used were phenol red-free DMEM with HEPES (20 mM) and 10% fetal calf serum (FCS) for JEG-3 cells and phenol red-free DMEM/F12 with 15% FCS for BeWo cells in the presence of streptomycin (0.1 mg/ml) and penicillin (100 U/ ml). The cellular toxicity of various factors was verified by measuring the lactate dehydrogenase (LDH) activity released into the culture medium as described by Lacasa et al. [29].

DAB detection kit (Ventana Medical System, Tucson, AZ). To determine the extent of nonspecific immunostaining, primary antibody was substituted with mouse IgG. The slides were counterstained with hematoxylin before mounting. For each culture condition, at least 1500 nuclei per section were randomly examined using Histolab analysis software (version 5.2.3; Microvision Instruments, Paris, France), and the number of MKI67-positive nuclei relative to the total number of nuclei was determined.

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ADIPONECTIN AND TROPHOBLAST PROLIFERATION TABLE 1. Primers used for RT-PCR. Primer set

PCR product (bp)

5 0 TTC TTC CTC ATG GCT GTG ATG T 3 0 5 0 AAG AAG CGC TCA GGA ATT CG 3 0

71

5 0 ATA GGG CAG ATA GGC TGG TTG A 3 0 5 0 GGA TCC GGG CAG CAT ACA 3 0

76

5 0 GAG AAC TCC GTC CGT TCT GT 3 0 5 0 AGT GCA GTC CAA ATC TTC TGC 3 0

76

5 0 GTC ATT GTC ATT ATC AGC 3 0 5 0 GCT ATG CTC TTC ACC TAT 3 0

153

5 0 CCA AGA TGG ACC AGA CAC TG 3 0 5 0 GCC ACC ACC TCT GTG GAG TA 3 0

220

5 0 TGC ACA GGA GCC AAG AGT GAA 3 0 5 0 CAC ATC ACA GCT CCC CAC CA 3 0

132

5 0 TTG AGG ACC TCT GTG TAT TTG TCA A 3 0 5 0 CCT GGA GGA GAA GAG GAA AGA GA 3 0

125

5 0 TGC TGT CTC CAT GTT TGA TGT ATC T 3 0 5 0 TCT CTG CTC CCC ACC TCT AAG T 3 0

86

RESULTS Expression of Adiponectin and Adiponectin Receptors in JEG-3 and BeWo Cells We aimed to investigate the effects of adiponectin on BeWo and JEG-3 cells. Therefore, we first studied adiponectin and adiponectin receptor expression in both cell lines. Using real-time RT-PCR, we demonstrated that ADIPOR1 and ADIPOR2 mRNAs were expressed in BeWo and JEG-3 cells and in first-trimester placenta (Fig. 1A). Expression of the third receptor for adiponectin, T-cadherin, was also measured

and was expressed in neither cell type. Adipose tissue and MDA-MB 231 were used as positive controls [31, 37]. Using quantitative RT-PCR, we showed that ADIPOR1 mRNA was expressed at a 10-fold higher level than ADIPOR2 mRNA in both trophoblastic cells (Fig. 1C). We also demonstrated that JEG-3 cells expressed a 2-fold higher level of ADIPOR1 mRNA than BeWo cells. Western blot analyses confirmed the presence of the two ADIPOR subtypes in JEG-3 and BeWo cells and in firsttrimester placenta extracts. Human breast cancer cells MDAMB 231 were used as positive controls (Fig. 1B).

FIG. 1. Adiponectin and ADIPOR1 and ADIPOR2 expression in JEG-3 and BeWo cells. A) Total RNA was extracted from BeWo, JEG-3, and MDA-MB 231 cells and human adipose and placental tissue and was analyzed by RT-PCR with the primers listed in Table 1. The PCR products were analyzed by agarose gel electrophoresis. The figure shows results of one representative experiment among six separate experiments. B) Cell lysates (100 lg) were subjected to Western blot analysis using anti-ADIPOR1 and anti-ADIPOR2 antibodies as described in Materials and Methods. One experimental result that is representative of three experiments is shown. C) The RT-PCR relative quantification of ADIPOR1 and ADIPOR2 mRNA expression. Results are means 6 SEM of six separate experiments. *P , 0.05, Wilcoxon test; #P , 0.05, Mann-Whitney U-test; ns, nonsignificant.


ADIPOR1 Sense Antisense ADIPOR2 Sense Antisense CDH13 (T-cadherin) Sense Antisense ADIPOQ (adiponectin) Sense Antisense LEP (Leptin) Sense Antisense TBP Sense Antisense RP13A Sense Antisense B2M (b2 microglobulin) Sense Antisense

Sequence

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As shown in Figure 1A, adiponectin was expressed neither in JEG-3 or BeWo cells nor in first-trimester placenta. However, this adipokine was expressed in human adipose tissue that was used as a control. We verified that leptin (as a positive control) was expressed in both JEG-3 and BeWo cells as described by Magarinos et al. [15]. Activation of PRKA Pathway by Adiponectin Because phosphorylation-dependent activation of PRKA is the major transduction pathway reported for adiponectin

signaling, the phosphorylation of PRKA was investigated in JEG-3 and BeWo cellular extracts. Figure 2 shows the kinetics of PRKA activation resulting from exposure to 250 ng/ml of adiponectin. This concentration was chosen because doseresponse experiments (25–500 ng/ml) revealed that 250 ng/ml was the adiponectin concentration giving maximal PRKA activation (data not shown). In BeWo cells, this activation was maximal after 5-min exposure to adiponectin and returned to basal levels by 60 min (Fig. 2A). In JEG-3 cells, the kinetics of activation by adiponectin showed maximal activation at 5 min, which was maintained at 15 min and then decreased (Fig. 2B).

FIG. 3. Effect of adiponectin on DNA synthesis in BeWo (A) and JEG-3 (B) cells. Cells were exposed to different concentrations (25–500 ng/ml) of human recombinant adiponectin, 250 ng/ml of leptin, or 50 lM forskolin for 24 h in the presence of [3H]-thymidine as described in Materials and Methods. Results are means 6 SEM of five to seven experiments and are normalized as percentages of the control value (untreated). *P , 0.05; ns, nonsignificant (adiponectin, leptin, or forskolin vs. control; Wilcoxon test).


FIG. 2. Phosphorylation of (P-PRKA) by adiponectin in BeWo and JEG-3 cells. Effects of adiponectin on PRKA activation in BeWo (A and C) and JEG-3 (B and D) cells. Cells were maintained in a serum-free culture medium overnight. Cells were then exposed to human recombinant adiponectin (250 ng/ml) or AICAR (500 lg/ml). At the indicated times, cellular extracts were prepared and immunoblotted with anti-phospho-PRKA or with anti-total PRKA. A and B) Western blot analysis from a representative experiment. C and D) Densitometric analysis of PRKA immunoblots. The figure shows means of active phospho-PRKA/total PRKA ratios. Results are means 6 SEM obtained from four to six separate experiments and are expressed as percentages of control value (untreated). *P , 0.05, Wilcoxon test; ns, nonsignificant.

ADIPONECTIN AND TROPHOBLAST PROLIFERATION

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Under our experimental conditions, exposure to 500 lg/ml of AICAR induces strong PRKA activation. Reprobing with antitotal PRKA antibody confirmed the presence of equal amounts of proteins in each lane (Fig. 2).

FIG. 5. Effects of adiponectin on cell proliferation in human firsttrimester placental explants. Placental explants were exposed to adiponectin (250 ng/ml) or FCS (10%) for 48 h. Following incubation, explants were fixed, paraffin embedded, and stained for anti-MKI67 antigen, reflecting proliferation of cells as described in Materials and Methods. Quantitative analysis of cell proliferation was performed by examining at least 1500 nuclei per section. Results are means 6 SEM of eight separate experiments and are expressed as percentages of MKI67 positive nuclei. *P , 0.05, **P , 0.01 (adiponectin or FCS [10%] vs. control, Wilcoxon test).

Effect of Adiponectin on Cell Proliferation As shown in Figure 3A, exposure to adiponectin (25 ng/ml) for 24 h resulted in a significant reduction in [3H]-thymidine incorporation in BeWo cells (38.0% 6 9.2%) but not in JEG3 cells compared with control conditions (Fig. 3B). Adipo-

FIG. 6. Effects of MEK and PIK3 inhibitors on adiponectin-reduced BeWo cell proliferation. Cells were exposed for 24 h to adiponectin (250 ng/ml), the MEK inhibitor UO126 (10 lM) with or without adiponectin (250 ng/ml), or the PIK3 inhibitor LY294002 (10 lM) with or without adiponectin (250 ng/ml) in the presence of [3H]-thymidine as described in Materials and Methods. Results are means 6 SEM of six experiments and are normalized as percentages of the control value (untreated). *P , 0.05; ns, nonsignificant ([a] adiponectin, UO126, or LY294002 vs. control; [b] UO126 þ adiponectin vs. UO126; [c] LY294002 þ adiponectin vs. LY294002; Wilcoxon test).


FIG. 4. Effects of adiponectin on BeWo (A) and JEG-3 (B) cell numbers. Cells were exposed to 250 ng/ml of human recombinant adiponectin, 50 lM forskolin, or control conditions (untreated). Cells were counted before treatment and 24, 48, and 72 h after treatment. Results are means of five to eight experiments. *P , 0.05; ns, nonsignificant (adiponectin or forskolin vs. control, Wilcoxon test).

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Role of MAPK and PIK3 Pathways in Antiproliferative Effects of Adiponectin in BeWo Cells Adiponectin inhibition of cell growth was recently shown to require MAPK and PIK3 signaling in endothelial cells, vascular smooth muscle cells, and breast cancer cells [39–42]. To identify the molecular mechanisms of adiponectin-reduced proliferation in trophoblastic cells, further experiments were performed using selective inhibitors of MAPK and PIK3 (UO126 and LY294002, respectively). Because adiponectin exerts similar effects in JEG-3 and BeWo cells, this experiment was performed in BeWo cells only. As shown in Figure 6, proliferation of BeWo cells was unaltered by either inhibitor. However, adiponectin reduction in [3H]-thymidine incorporation was abrogated when both MAPK and PIK3 pathways were inhibited. DISCUSSION Recent studies [14–16] have demonstrated that leptin increases trophoblastic cell proliferation. Adiponectin and leptin have opposite effects in various cell types [17, 18].

However, there are no data regarding direct effects of adiponectin in trophoblast cells, to our knowledge. We studied the in vitro effects of adiponectin and show herein that adiponectin exerts antiproliferative effects on BeWo and JEG-3 trophoblastic cells. First, we investigated whether BeWo and JEG-3 cells were target cells for adiponectin and demonstrated (for the first time to our knowledge) that ADIPOR1 and ADIPOR2 (but not Tcadherin) were expressed in JEG-3 and BeWo cells. As observed in other cell types [31, 37, 43], ADIPOR1 mRNA expression was far more important than ADIPOR2 mRNA expression. We also verified that ADIPOR1 and ADIPOR2 were functional in JEG-3 and BeWo cells by measuring PRKA activation by human recombinant adiponectin. Because Tcadherin receptors were not expressed in JEG-3 and BeWo cells, the effects of adiponectin may be exclusively mediated by ADIPOR1 and/or ADIPOR2. Moreover, experiments in our laboratory revealed that antiproliferative effects of adiponectin were mediated by these receptors, as they were abrogated by short interfering RNA for both receptors in MDA-MB 231 breast cancer cells [31]. In various cell types such as MDA-MB 231 cells [31], rat placenta [27], and rat ovary [43], ADIPOR1 and ADIPOR2 were found to be downregulated not only by adiponectin [44] but also by leptin and other hormones [45]. Experiments are in progress in our laboratory to determine whether ADIPOR1 and ADIPOR2 expression in trophoblastic cells can be regulated by adiponectin or hormones implicated in pregnancy such as hCG, estrogens, or progesterone. Second, we observed a reduction in cell number and DNA synthesis in trophoblastic cells and in first-trimester placental explants by human recombinant adiponectin. These effects were observed with adiponectin concentrations (25–250 ng/ml) much lower than those found in normal human circulation or in cord blood during gestation [46–48]. The human recombinant adiponectin used in the present study contains mainly the HMW adiponectin form [49–51], known as the active form of adiponectin [52]. Moreover, adiponectin reduced osteoblastic, endothelial, and prostate cell proliferation at subphysiological concentrations [49–51]. Finally, effects of adiponectin were observed in a dose-independent manner as was previously shown in MDA-MB 231 cells [31] and in osteoblastic cells [50]. The antiproliferative effect of adiponectin is sometimes, but not always, associated with apoptotic effects [49]. For example, in breast cancer cell line MCF-7 [34], adiponectin induces cell proliferation reduction and DNA fragmentation, the last step of apoptosis. During placentation, apoptosis is an important process, mediated mainly through caspase 3 activation [53, 54]. Further experiments will be needed to establish whether adiponectin has apoptotic effects in JEG-3 and BeWo cells. To identify the signaling pathways by which adiponectin reduces cell proliferation, we investigated the role of MAPK and PIK3 pathways, which are known to mediate the effects of adiponectin. UO126 and LY 294002, two specific inhibitors of these pathways, suppressed the effects of adiponectin on [3H]thymidine incorporation, demonstrating the importance of MAPK and PIK3 pathways in antiproliferative effects of adiponectin on BeWo and JEG-3 cells. Recent studies [55, 56] emphasize that it is important to consider not only the separate effects of adiponectin or leptin on cancer processes but also the ratios between these adipokines. In breast cancer, the leptin/adiponectin ratio is correlated to tumor size and tumor aggression [57]. Likewise, in colon epithelial cells, adiponectin blocks multiple signaling cascades associated with leptin-induced cell proliferation [56]. Another recent study [55] showed that leptin and adiponectin


nectin at 250 ng/ml induced a significant decrease in [3H]thymidine incorporation in BeWo (40.0% 6 11.1%) and JEG-3 (36.0% 6 6.8%) cells (Fig. 3, A and B). These effects persisted with 500 ng/ml of adiponectin (35.0% 6 6.1% and 18.5% 6 7.6% in BeWo and JEG-3 cells, respectively). Under our experimental conditions, leptin used as a positive control increased [3H]-thymidine incorporation in JEG-3 cells. Forskolin (50 lM) decreased [3H]-thymidine incorporation in BeWo cells (28% 6 15%) as previously described [30, 38]. Moreover, the inhibitory effects of adiponectin could no longer be seen after 48 h in the two cell lines (data not shown). Finally, we observed an inhibition of [3H]-thymidine incorporation in BeWo cells with 250 ng/ml of adiponectin during 24 h independent of the serum concentration in culture medium (61% 6 4%, 36.0% 6 6.8%, and 31.5% 6 35.0% for the 15%, 1%, and 0% FCS conditions, respectively). For the following studies, the intermediate concentration (250 ng/ml) of adiponectin was used. The antiproliferative action of adiponectin was confirmed by direct counting of BeWo and JEG-3 cells (Fig. 4, A and B). After 48 h of adiponectin exposure, a significant decrease in BeWo and JEG3 cell numbers was observed (27.00% 6 3.14% and 15.0% 6 2.0%, respectively) compared with control conditions (without adiponectin). These effects were still observed after 72 h of adiponectin treatment. The BeWo cell number was also reduced after 72 h of 50 lM forskolin exposure (57% 6 13%). Finally, we tested the effects of adiponectin on trophoblast cell proliferation in first-trimester placental explants. Immunostaining for cytokeratin 7 demonstrated all types of trophoblasts, including cytotrophoblasts, syncytiotrophoblast, and extravillous trophoblasts in the explant cultures (data not shown). We then examined immunoreactivity for MKI67 (an antigen indicative of DNA synthesis) to evaluate cell proliferation. The explants cultured with adiponectin (250 ng/ml) during 48 h showed a lower percentage of MKI67-positive nuclei (16.5% 6 2.6%) vs. under control conditions (without adiponectin) (26.0% 6 4.5%) (Fig. 5). Fetal calf serum (10%), used as a positive control, showed a higher percentage of MKI67 positive nuclei for MKI67 compared with control conditions (29.7% 6 1.6%). LDH activity was measured under different FCS culture conditions in BeWo cells 24 h after plating. The LDH values were similar under 15% FCS, 1% FCS, and serum-free conditions (3.85 6 1.50, 4.18 6 1.21, and 5.00 6 1.88 mU/ 15 000 cells, respectively).

ADIPONECTIN AND TROPHOBLAST PROLIFERATION

18. 19.

20.

21. 22.

23.

24.

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interact in the regulation of prostate cancer cell growth. In maternal plasma, adiponectin concentrations remain constant during pregnancy, while those of leptin increase [58, 59]. In cord blood, leptin and adiponectin concentrations increase with gestational age [46–48]. The leptin/adiponectin ratio seems to be modified during pregnancy and should be considered in interpreting the effects of adipokines on the implantation process. Additional experiments will be needed to determine the importance of the leptin/adiponectin balance in trophoblast cell proliferation. Local production of adiponectin in placenta as reported by Caminos et al. [27] and by Chen et al. [26] was not confirmed by Corbetta et al. [46]. We found no adiponectin mRNA in JEG-3 or BeWo cells or in placental extracts. These controversial results suggest that the origin of adiponectin at the maternal-fetal interface may be the endometrium and that adiponectin could intervene as a paracrine signal in the human placenta [28, 46]. In conclusion, we show herein that adiponectin may act as an antiproliferative factor in human trophoblastic cells and provide examples of cells affected by the opposite actions of leptin and adiponectin. Our findings indicate that adiponectin should be considered a newly identified regulator of early placental development.

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