ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta
1 Laboratoire de Physiologie materno-ftale, Departement des Sciences Biologiques, Universite du Quebec à Montreal, Montreal, Quebec, Canada, H3C 3P8
2 Centre de recherche Biomed, Departement des Sciences Biologiques, Universite du Quebec à Montreal, Montreal, Quebec, Canada, H3C 3P8
3 Departement d'obstetrique-gynecologie, Hpital St-Luc, Universite de Montreal, Montreal, Quebec, Canada, H2L 4M1
, http://www.100md.com Abstract
Mitogen-activated protein kinases (MAPKs) control many cellular events from complex programmes, such as embryogenesis, cell differentiation and proliferation, and cell death, to short-term changes required for homeostasis and acute hormonal responses. However, little is known about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) and p38 in human placenta. Therefore, we examined the expression of ERK1/2 and p38 in trophoblasts from human term placenta, and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultivated in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). This study shows that the level of ERK1/2 and p38 decreases with increasing days of culture, to reach an undetectable level after 5 days of culture. Moreover, pretreatment of cells with an ERK1/2-specific inhibitor (PD98059) and/or a p38-specific inhibitor (SB203580) suppressed trophoblast differentiation. Our results also demonstrate that the p38 pathway is highly solicited as compared to the ERK1/2 pathway in the differentiation process. Furthermore, ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. In summary, this study showed that ERK1/2 and p38 pathways are essential to mediate initiation of trophoblast differentiation.
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Introduction
Mitogen-activated protein kinases (MAPKs) are a family of protein kinases whose functions and regulation have been conserved during evolution from unicellular organisms, such as yeast, to complex organisms including humans (Widmann et al. 1999). In multicellular organisms, there are three well-characterized subfamilies of MAPKs. They included the extracellular signal-regulated kinases (ERK1 and ERK2) (Boulton et al. 1990, 1991), the c-Jun NH2-terminal kinases (JNK 1, JNK 2, and JNK 3) (Derijard et al. 1994; Kyriakis et al. 1994; Gupta et al. 1996), and the four p38 enzymes (p38, p38, p38, and p38) (Han et al. 1994; Jiang et al. 1996; Lechner et al. 1996; Goedert et al. 1997). Moreover, a relatively recent MAPK (ERK5) was identified, and forms the subject of intense studies (Zhou et al. 1995). MAPKs are responsible for the conversion of a large number of extracellular stimuli and environmental conditions into specific cellular responses controlling cell proliferation, differentiation, apoptosis, embryogenesis and regulation of inflammatory and stress responses (for review see Kyriakis & Avruch, 2001; Pearson et al. 2001)). The first mammalian MAPK pathway described was the ERK pathway. ERK1 and ERK2 (ERK1/2) share an 83% amino acid homology, and are expressed to various extents in all tissues (for review see Chen et al. 2001)). They are strongly activated by growth factors, serum, phorbol esters and, to a lesser extent, by ligands of heterotrimeric G protein-coupled receptors, cytokines, osmotic stress and microtubule disorganization (Lewis et al. 1998). In contrast, the p38 pathway is strongly activated by most environmental stresses, pro-inflammatory cytokines, such as interleukin 1 (IL–1) and tumour necrosis factor (TNF-), both playing an important role in the regulation of the inflammatory response. While p38 kinases were originally associated with stress- and inflammation-related kinases, recent evidence involves this kinase in multiple physiological roles in cell cycle control, and in cell proliferation, differentiation and apoptosis (Nebreda & Porras, 2000; Ambrosino & Nebreda, 2001; Pearson et al. 2001). Thus, both the ERK1/2 and p38 pathways play important roles in the differentiation process of several cell types including adipocytes, cardiomyocytes, chondroblasts, erythroblasts, myoblasts and neurones (Nebreda & Porras, 2000; Kohmura et al. 2004; Lee et al. 2004). Moreover, O'Brien et al. (2004) demonstrated that activation of ERK1/2 is essential and sufficient for the initial stage of epithelial tubule development during which cells depolarize and migrate. Thereafter, ERK becomes dispensable for the latter stage, during which cells repolarize and differentiate. ERK1/2 also mediates signalling pathways involved in mesenchyme formation and differentiation in the sea urchin embryo (Fernandez-Serra et al. 2004). Furthermore, Mudgett et al. (2000) demonstrated the requirement of p38 MAPK in mouse diploid trophoblast development and placental vascularization, and suggest a more general role for p38 MAPK signalling in embryonic angiogenesis. However, little is known about the implication of MAPK pathways in human trophoblast differentiation.
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Human trophoblast differentiation is characterized by the formation of a specific multinuclear structure, the syncytiotrophoblast. This structure arises by fusion and differentiation of the relatively undifferentiated, mitotically active cytotrophoblast cells (Midgley et al. 1963). Moreover, throughout pregnancy, the syncytiotrophoblasts become a continuous epithelial layer located at the villous surface of the placenta, floating in maternal blood. Therefore, essential fetal nutrients must cross this placental barrier to reach the fetal circulation. Trophoblast growth and differentiation has been studied in in vitro models by many investigators during the last two decades. Many studies reported that, in vitro, cytotrophoblast cells in serum-free conditions fail to aggregate or fuse, and remain in a mononuclear state (Ringler & Strauss, 1990; Yang et al. 2003). In contrast, when cells are cultivated in medium supplemented with fetal bovine serum (FBS), they spontaneously fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts. The morphological differentiation is defined by the fusion of mononucleated cytotrophoblast cells with adjacent syncytium (Midgley et al. 1963), while the biochemical differentiation is characterized by the production of hormones such as human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) (Kliman et al. 1986; Morrish et al. 1987; Strauss et al. 1992).
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The aim of the present study was to investigate the role of ERK1/2 and p38 in human trophoblast differentiation. Thus, protein levels of ERK1/2 and p38 were evaluated during the differentiation process of trophoblasts isolated from human term placentas. Moreover, using specific inhibitors of both pathways, our results demonstrated for the first time that ERK1/2 and p38 play a central role in human trophoblast differentiation.
Methods
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Materials
Dulbecco's modified Eagle medium (high glucose) (DMEM-HG), Hanks balanced salt solution (HBSS), trypsin, DNAse, Percoll, propidium iodide (PI), SB203580, PD98059 and antidesmosome mouse monoclonal antibody were from Sigma (Oakville, ON, Canada). Calf serum and penicillin, streptomycin, neomycin (PSN; 100X) were purchased from Invitrogen (Burlington, ON, Canada). FBS and an ELISA kit for hCG assay were from Medicorp (Montreal, QC, Canada). An ELISA kit for human placental lactogen (hPL) was from DRG international (Mountainside, NJ, USA). A CytoTox 96R. non-radioactive cytotoxicity assay kit was from Promega (Madison, WI, USA), and was used to measure lactate dehydrogenase (LDH) activity. Bovine serum albumin (BSA), the BM chemiluminescence (POD) system, TRIS-base, Nonidet-40 and acrylamide were from Roche Applied Science (Laval, QC, Canada). Bicinchoninic acid (BCA) reagent was purchased from Pierce (Brockville, ON, Canada). Anti-phospho-Erk, anti-Erk, anti-phospho-p38 and anti-p38 rabbit polyclonal antibodies were all purchased from Cell Signalling (Beverly, MA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody, sheep anti-rabbit-IgG, goat anti-mouse-IgG conjugated with horseradish peroxidase and Re-Blot plus Mild solution were from Chemicon International (Temecula, CA, USA). Alexa Fluor 488 goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR, USA). The fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against cytokeratin-7 was from Accurate Chemical & Scientific Corp (Westbury, NY, USA). Bio-Rad minigel system was from Bio-Rad (Mississauga, Ontario, Canada). Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore (Cambridge, Ontario, Canada). The 24-well plates and 35 mm dishes were obtained from Corning (Acton, MA, USA). BioMax Light autoradiography films were from Eastman Kodak Co. (Rochester, NY, USA). All other products were from Sigma (Oakville, ON, Canada).
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Human placental trophoblast isolation and purity evaluation
The procedure of Kliman et al. (1986), with minor modifications, was used for cell dispersion and cytotrophoblast cell isolation. Human term placentas from 35 to 41 weeks of gestation were obtained immediately after spontaneous vaginal delivery, in accordance with the established guidelines of the ethical committee of St Luc Hospital of the Centre Hospitalier Universitaire de Montreal (Montreal, QC, Canada) and of Universite du Quebec à Montreal (Montreal, QC, Canada). Briefly, placental villi were cut and thoroughly washed to remove blood. Thereafter, they were incubated four times in a digestion medium composed of HBSS, containing trypsin and deoxyribonuclease for 30 min at 37°C in a water bath with continuous shaking. The dispersed cells were layered on top of a discontinuous 5–70% Percoll gradient, and centrifuged for 25 min at 507 g. The intermediate layers (density between 1.048 and 1.062) containing cytotrophoblast cells were removed and washed, and cell viability was determined by trypan blue exclusion. Following trophoblast isolation, cells were seeded at a density of approximately 1.6 x 106 cells well–1 in 24-well plate or 4.5 x 106 cells dish–1 in 35 mm dishes. The complete culture medium, constituted of DMEM-HG, 2 mM glutamine, 10% FBS and PSN (1x), was refreshed daily.
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The purity of cytotrophoblast cells preparations was evaluated by flow cytometry using FITC-conjugated monoclonal antibody against cytokeratin-7 as previously described (Daoud et al. 2005). Only cell preparations having a minimum of 96% of cytotrophoblast cells were used in this work.
Secretion of hCG and hPL by trophoblasts
Trophoblasts cells were cultivated in 24-well plates for 4 or 6 days. Every day, the culture media were collected, centrifuged and supernatants were frozen at –20°C until measurements. The hCG and hPL contents in culture media were evaluated by ELISA following manufacturer's instructions.
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Effect of MAPK inhibitors on hCG and hPL secretion
Following isolation, cells were plated in 24-well plates in complete culture medium, and left overnight prior to treatment. Thereafter, cells were preincubated with PD98059 (75, 50, 25 or 10 μM), SB203580 (20, 10, 5 or 1 μM), PD98059 + SB203580 (50 μM +10 μM, respectively), dissolved in DMSO (<0.1%) or vehicle alone for 1 h in culture medium (serum free). Following that, 10% FBS was added. These treatments were repeated every day for 4 days. Culture media were collected every day for hCG and hPL measurement. In another set of experiements, to investigate the role of ERK1/2 and p38 in hCG synthesis and secretion, cells were cultivated in complete culture medium for 3 days, to allow differentiation of cytotrophoblast cells into syncytiotrophoblasts. Thereafter, cells were rinsed and preincubated for 1 h with MAPK inhibitors, according to the above conditions, before the addition of 10% FBS. The culture media were collected at day four and subjected to hCG measurement.
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Effect of FBS and MAPK inhibitors (PD98059 and SB203580) on ERK1/2 and p38 activation in human primary culture trophoblasts
After trophoblast isolation, cells were seeded in 35 mm dishes in the presence of complete culture medium for 5 h at 37°C to allow attachment, and thereafter, cells were serum-starved overnight. At day one, cells were preincubated with 50 μM PD98059 or 10 μM SB203580 for 1 h, prior the addition of 10% FBS for different periods of time (0, 2, 5, 10, 20 and 30 min). The reaction was stopped by aspiration of cell culture, and cell lysates were prepared. Thereafter, lysates were subjected to 12% polyacrylamide gel electrophoresis (SDS-PAGE) using a Bio-Rad minigel system. Activated ERK and p38 were detected by immunoblotting using antiphospho-Erk and antiphospho-p38 antibodies. Blots were then stripped and reprobed using specific antibodies directed against total ERK or p38.
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Cell lysate preparation
For every day of follow-up of ERK1/2 and p38 protein expression (day 1 to day 4), total trophoblast proteins were isolated. For this set of experiments, cells were seeded in 35 mm dishes. For protein preparation, media was aspirated, cells were rinsed twice with ice-cold PBS, solubilized with ice-cold radioimmunoprecipitation (RIPA) buffer (150 mM NaCl, 9.1 mM Na2HPO4, 1.7 mM NaH2PO4 (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS, containing freshly added 200 μM Na3VO4, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μM leupeptine, 1.46 μM pepstatin and 2 μg ml–1 aprotinin) and harvested. Cell lysates were clarified by centrifugation at 14 000 g for 10 min at 4°C. Protein concentration of the supernatant was determined by spectrophotometric quantification using the BCA reagent with BSA as standard. For MAPK activation, protein extracts were prepared in the same way.
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Western blots and densitometric analysis
Cellular proteins (30 μg) were solubilized in sample buffer (4% SDS, 30 mM dithiotreitol, 0.25 M sucrose, 0.01 M EDTA-Na2 and 0.075% bromophenol blue), and heated at 95°C for 5 min to denature proteins. The lysates were resolved in 12% SDS-PAGE and proteins were electroblotted on a PVDF membrane at 1.7 mA cm–2 for 2 h. The membranes were blocked for 1 h at room temperature in TBS-T (20 mM Tris (pH 7.6), 137 mM NaCl and 0.05% Tween-20) containing 5% skimmed milk. Membranes were then incubated with the appropriate primary antibody (1/2000 for antiphospho-ERK1/2, 1/500 for antiphospho-p38, 1/1000 for anti-ERK1/2 and antip38 or 1/4000 for anti-GAPDH in TBS-T/5% BSA) overnight at 4°C, washed three times with TBS-T, and probed with horseradish peroxidase-conjugated secondary antibodies (1/1500 for antirabbit-IgG or 1/3000 for antimouse-IgG) for 2 h at room temperature. Blots were washed three times with TBS-T and the detection was performed using the BM Chemiluminescence system and visualized by autoradiography (BioMax Light film). In certain instances, the PVDF membranes were stripped with Re-Blot plus Mild solution at room temperature for 15 min, rinsed twice with blocking buffer, and blocked for 1 h before reprobing with another antibody. The band intensity after exposure and development was digitized and analysed by Quantity One software (Bio-Rad Laboratories, Mississauga, ON, Canada).
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Immunofluorescence analysis
Primary trophoblasts were stained with antidesmosomal antibody as previously described for anticytokeratin-7 antibody (Daoud et al. 2005). Briefly, cells were washed twice with PBS, fixed in methanol at –20°C for 20 min, washed again with PBS (twice), and then incubated in PBS containing 2% FBS (v/v) for 45 min, to eliminate non-specific binding. Thereafter, cells were rinsed with PBS and incubated in the presence of mouse antihuman antidesmosomes monoclonal antibody (1/800) in PBS containing 0.2% BSA for 1 h at room temperature, washed three times with PBS, and incubated with Alexa Fluor 488 goat antimouse IgG (1/1000) for 1 h at room temperature in the dark. For nuclear staining, cells were incubated with propidium iodide (50 μg ml–1) for an additional 30 min at room temperature in the dark, washed three times with PBS, and viewed using a Nikon Eclipse TE300 camera (Nikon, Tokyo, Japan) equipped with a confocal laser-scanning microscope (Bio-Rad MRC1024, CA, USA). All observations were performed at 10x magnification on monolayer cells. A syncytium was defined as three or more nuclei in the same cytoplasm without intervening surface desmosomal membrane staining.
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Effect of MAPK inhibitors on ERK1/2 and p38 expression
Following isolation, cells were plated in 24-well plates in complete culture medium, and left overnight prior to treatments. Thereafter, cells were preincubated with 50 μM PD98059, 10 μM SB203580 dissolved in DMSO (<0.1%), or vehicle alone for 1 h in culture medium (serum free), and then 10% FBS was added. The same treatment was repeated every day, for four days. Thereafter, cells were harvested, proteins were isolated and protein levels of ERK1/2 and p38 were evaluated by western blot.
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MAPK inhibitors delayed trophoblast differentiation
Following isolation, cells were plated in 24-well plates in complete culture medium and left overnight. At day 1, cells were preincubated with 50 μM PD98059, 10 μM SB203580 dissolved in DMSO (<0.1%), or vehicle alone for 1 h in culture medium (serum free), and then 10% FBS was added. The same treatment was repeated for 2 days. On day 3, culture medium was refreshed with the complete culture medium, and cells were cultivated for an additional 3 days. The culture media were collected every day for hCG measurement.
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Measurement of LDH activity after PD98050 or/and SB203580 treatment
After 4 days of culture, the culture medium was collected and the attached cells were washed with PBS and suspended in lysis buffer containing 0.1% Triton X-100. As an assay of cell toxicity, total LDH present in the medium and the cells was measured with a LDH kit, according to the manufacturer's instructions. Color change was quantified by reading the absorbance at 490 nm, and the cell toxicity was calculated as (OD in the supernatant – OD of medium (background))/(OD of Triton X-100-lysed cells – OD of background), where OD is the optical density.
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Statistical analyses
Data were expressed as the mean ± S.E.M., and analysed with a one-way ANOVA followed by the Tukey test, two-way ANOVA or t test at a P < 0.05 level of significance.
Results
Trophoblast differentiation
To evaluate the differentiation process of isolated cytotrophoblast cells into functional syncytiotrophoblasts, the levels of secreted hCG and hPL, two well-known makers of differentiation, were assayed in the culture medium over a period of six days (Kliman et al. 1986). Figure 1A and B shows the profile of hCG and hPL secretion. In both panels, the secretion was modest during the first 24 h of culture. After that, the secretion increased with increasing days of culture, reaching a maximal value at 3–5 days of culture for hCG and 4–5 days for hPL, and declined thereafter. The hCG secretion values were calculated as mU (mg protein)–1 (24 h)–1, while the hPL values were calculated as μg (mg protein)–1 (24 h)–1. Moreover, since these values were variable between placentas of different weeks of pregnancy, our data were expressed as relative hCG or hPL secretion compared to the maximal value obtained at day 4 of culture. To substantiate the effect of days of culture on trophoblast differentiation, we examined the syncytium formation as an indicator of morphological differentiation. As shown in Fig. 1C, most trophoblasts were present as mononucleated cytotrophoblast cells at day 1, while at day 4, more abundant and larger syncytia were observed. Therefore, subsequent experiments were performed up to day 4 of culture, when differentiation occurred.
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A, cells were cultivated from day 1 to day 6 in complete culture medium. Data are expressed as relative secretion of hCG ± S.E.M. compared to secretion at day 4 of culture of six cell preparations from six different placentas. ***P < 0.001, day 1 to day 6, one-way ANOVA; **P < 0.01; *** P < 0.001 compared to day 1 of culture, Tukey test. B, cells were cultivated from day 1 to day 6 in complete culture medium. Data are expressed as relative secretion of hPL ± S.E.M. compared to secretion at day 4 of culture of five cell preparations from five different placentas. ***P < 0.001, day 1 to day 6, one-way ANOVA; *P < 0.05; **P < 0.01 compared to day 1 of culture, Tukey test. C, cells were cultivated for 1 or 4 days in complete culture medium, fixed and stained with PI for nuclei (red) and specific antibody for plasma membrane desmosomes (green), and observed by confocal microscopy. Independent experiments were performed on cells isolated from three different placentas. Scale bar = 100 μm.
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Expression of ERK1/2 and p38 along the differentiation of trophoblasts in culture
The expression of ERK1/2 and p38 was evaluated by western blot using total protein isolated from trophoblasts. Figures 2 and 3 show representative western blots of the expression of ERK1/2 and p38, respectively, from trophoblasts cultivated for days 1–4. Following normalization with the GAPDH protein level, our results show that the expression of ERK1/2 declined by 33, 51 and 78% for days 2, 3 and 4, respectively (Fig. 2A and B), while the expression of p38 declined by 15%, 45% and 68% for days 2, 3 and 4, respectively (Fig. 3A and B). Interestingly, after 5 and 6 days of culture, no signal was detected for either ERK1/2 or p38 by western blot (data not shown).
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A, western blot analysis was performed using ERK1/2 or GAPDH antibody on total proteins (30 μg) isolated from primary culture of trophoblasts cultivated from day 1 to day 4. The blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of ERK1/2 protein level after normalization with GAPDH protein level. Independent experiments were performed on cells isolated from four different placentas. P < 0.001, day 1 to day 4, one-way ANOVA; *P < 0.05; ***P < 0.001 compared to day 1 of culture, Tukey test. Error bars show S.E.M.
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A, western blot analysis was performed using p38 or GAPDH antibody on total proteins (30 μg) isolated from primary culture of trophoblasts cultivated from day 1 to day 4. The blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of p38 protein level after normalization with GAPDH protein level. Independent experiments were performed on cells isolated from four different placentas. P < 0.05, day 1 to day 4, one-way ANOVA; *P < 0.05 compared to day 1 of culture, Tukey test. Error bars show S.E.M.
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Effect of MAPK inhibitors on the differentiation of trophoblasts primary culture
In order to investigate the role of ERK1/2 and p38 pathways on trophoblast differentiation, cells were treated with either PD98059, an ERK1/2-specific inhibitor, or SB203580, a p38-specific inhibitor (Davies et al. 2000; Chen et al. 2001; Otis et al. 2004). First, we titrated the inhibitors in trophoblast cultures. As shown in Fig. 4A and B, SB203580 inhibited hCG and hPL secretion in a dose-dependent manner. Following 5, 10, or 20 μM SB203580 treatment, hCG secretion was significantly reduced by 65%, 80% and 92%, respectively (Fig. 4A), while 1 μM SB203580 had no effect on hCG secretion. In the same way, hPL secretion was reduced by 44%, 81%, 79% and 87% after 1, 5, 10 or 20 μM SB203580 treatments (Fig. 4B). The same results were obtained with PD98059 (Fig. 4C and D). Following 10, 25, 50 or 75 μM PD98059 treatment, hCG secretion was significantly reduced by 49%, 58%, 76% and 82%, while hPL secretion was reduced by 55%, 53%, 68% and 73%, respectively. Therefore, subsequent experiments were performed using 10 μM SB203580 or 50 μM PD98059, and hCG secretion was used to evaluate trophoblasts' biochemical differentiation, while morphological differentiation was evaluated by syncytium formation. Figure 5A shows that the presence of PD98059 (50 μM) and/or SB203580 (10 μM) in the culture medium during all days of culture inhibited the hCG secretion. This inhibition was more important under SB203580 treatment compared to that with PD98059. However, cells treated with both inhibitors conjointly showed an additional inhibiting effect on hCG secretion. To substantiate the effect of the two inhibitors on trophoblast differentiation, we examined syncytium formation. As shown in Fig. 5C, cells treated with SB203580 or PD98059 for 4 days were present as mononucleated cytotrophoblast cells, while the control cells consisted of abundant and large syncytia. Taken together, these results show that the suppression of both ERK1/2 and p38 pathways significantly impaired trophoblast differentiation. Furthermore, Fig. 5B shows that neither pathway is involved in the synthesis of hCG by syncytiotrophoblasts, since the addition of these inhibitors in the culture medium when trophoblasts were already differentiated did not alter hCG secretion. Cells treated with the vehicle alone showed no effect on hCG secretion or morphological differentiation (data not shown).
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A and B, cells were treated from day 1 to day 4 with different concentrations of SB203580 (1, 5, 10 and 20 μM) or complete control medium (CTL). Supernatants from day 4 were then assayed for hCG (A) or hPL (B) secretion. C and D, cells were treated from day 1 to day 4 with different concentrations of PD98059 (10, 25, 50 and 75 μM) or complete control medium (CTL). Supernatants from day 4 were then assayed for hCG (C) and hPL (D) secretion. Data are expressed as relative secretion of hCG or hPL ± S.E.M. compared to control hCG or hPL secretion at day 4 of culture. *P < 0.05, **P < 0.01, ***P < 0.001; treated compared to control, t test.
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A, cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059, 10 μM SB203580 + 50 μM PD98059 or complete culture medium (CTL). Data are expressed as the relative secretion of hCG ± S.E.M. compared to hCG secretion at day 4 of culture. *P < 0.05; **P < 0.01; SB203580 or SB203580 + PD98059 compared to PD98059; t test, P < 0.05; 50 μM PD98059 compared to control, P < 0.005; 10 μM SB203580 compared to control, and P < 0.001; 50 μM PD98059 + 10 μM SB203580 compared to control; two-way ANOVA. B, cells were cultivated in complete culture medium for 3 days. Thereafter, cells were treated with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL) for another 24 h. After that, culture medium was collected and hCG secretion was measured. Data are expressed as the relative secretion of hCG ± S.E.M. compared to control. C, cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL). At day 4, cells were fixed and stained with PI for nuclei (red), and with a specific antibody for plasma membrane desmosomes (green) and observed by confocal microscopy. Scale bar = 100 μm. Independent experiments were performed on cells isolated from three different placentas.
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Effect of FBS and MAPK inhibitors on ERK1/2 and p38 activation
Following isolation of cells, trophoblasts were incubated for 5 h in the presence of 10% FBS, then serum-starved overnight. Following starvation, cells were treated with 10% FBS for different periods of time (1–30 min). Our results show that treatment with FBS induced a transient activation of ERK1/2 that reached a peak at 5 min and declined thereafter; however, the phosphorylation level did not return to the basal value (Fig. 6A and B). In the same way, p38 activation was also transient (Fig. 7A and B), but the kinetics were slightly different, since the maximal activation (phosphorylation) was reached between 5 and 10 min, and declined thereafter to reach the basal level after a 30 min incubation with FBS. The same results were obtained using 4-day cultivated syncytiotrophoblasts (data not shown).
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Following isolation of cytotrophoblast cells, cells were kept in complete culture media for 5 h and serum-starved overnight. After stimulation, cell lysates were prepared and subjected to 12% SDS-PAGE. Activated ERK1/2 were detected by western blot using antiphospho-ERK1/2 antibodies. The blot was then stripped and reprobed using an anti-ERK1/2 (total) antibody. A, cells were stimulated with 10% FBS for the indicated time. C, cells were preincubated with SB203580 (10 μM) for 1 h and then stimulated with 10% FBS for the indicated time. E, cells were preincubated with PD98059 (50 μM) for 1 h and then stimulated with 10% FBS for indicated time. B, D and F, densitometric analysis of activated ERK1/2 protein level after normalization with total ERK1/2 protein level. Independent experiments were performed on cells isolated from three different placentas. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control time (0 min); t test. Error bars show S.E.M.
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Following isolation of cytotrophoblast cells, cells were kept in complete culture medium for 5 h and serum-starved overnight. After stimulation, cell lysates were prepared and subjected to 12% SDS-PAGE. Activated p38 was detected by western blot using antiphospho-p38 antibodies. The blot was then stripped and reprobed using an antip38 (total) antibody. A, cells were stimulated with 10% FBS for the indicated time. C, cells were preincubated with SB203580 (10 μM) for 1 h and then stimulated with 10% FBS for the indicated time. E, cells were preincubated with PD98059 (50 μM) for 1 h and then stimulated with 10% FBS for the indicated time. B, D and F, densitometric analysis of activated p38 protein level after normalization with total p38 protein level. Independent experiments were performed on cells isolated from three different placentas. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (time 0); t test. Error bars show S.E.M.
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Specific inhibitors of both MAPKs were also used to verify their specific effects on the activation of ERK1/2 and p38 induced by FBS. Thus, when cytotrophoblast cells were pretreated with SB203580 (Fig. 6C and D), ERK1/2 phosphorylation was stimulated as compared to control (time 0 in control cells compared to time 0 in SB203580-treated cells), while the profile of p38 phosphorylation remained unchanged (Fig. 7C and D). However, pretreatment of cytotrophoblast cells with PD98059 for 1 h completely blocked FBS-induced ERK1/2 activation (Fig. 6E and F), while p38 activation was stimulated compared to control (time 0 in control cells compared to time 0 in PD98059-treated cells; Fig. 7E and F). Cell treatment with the vehicle alone showed no effect on ERK1/2 or p38 activation (data not shown).
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Effect of MAPK inhibitors on ERK1/2 and p38 protein level
As previously shown (Fig. 5A and C), both ERK1/2 and p38 inhibitors inhibit trophoblast differentiation. Therefore, the effect of the presence of SB203580 or PD98059 in the culture medium for 4 days was evaluated on protein levels of ERK1/2 and p38. Figure 8 shows that the presence of SB203580 increased the expression of ERK1/2 and p38 1.64- and 2-fold, respectively. In the same way, the presence of PD98059 increased the expression of ERK1/2 1.2-fold, while for p38 a tendency was observed, but this increase was not significant (Fig. 8).
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Cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL). A, western blot analysis was performed using total ERK1/2 or p38 antibodies on total proteins (30 μg) isolated from treated or control trophoblasts after 4 days of culture. The blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of ERK1/2 and p38 protein level after normalization with GAPDH protein level. Independent experiments were performed on cells isolated from three different placentas. **P < 0.01, *P < 0.05; SB203580 or PD98059 compared to control, t test. Error bars show S.E.M.
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MAPK inhibitors delayed trophoblast differentiation
According to previous results (Fig. 5) on the effects of MAPK inhibitors on trophoblast differentiation, we evaluated the hypothesis that these inhibitors could delay trophoblast differentiation. Thus, cells were cultivated in the presence of MAPK inhibitors; either SB203580 or PD98059, for 3 days, and after that the culture medium was switched to complete culture medium for the next 3 days. Figure 9A shows that the hCG secretion remained low for the first 4 days in the presence of MAPK inhibitors, but when the inhibitors were removed, the hCG secretion increased to reach its control value at day 5, while at day 6, this level exceeded its relevant control value. Finally, our results demonstrate that the levels of hCG secretion, at days 5 and 6, in cells pretreated with MAPK inhibitors, reached the same value as that observed at day 4 in the control cell culture conditions, when trophoblast differentiation occurred (Fig. 9A). Moreover, morphological differentiation was confirmed at day 6 as shown in Fig. 9B. These results confirm that the suppression of the activation of ERK1/2 or p38 delayed the differentiation process in trophoblasts.
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Cells were treated from day 1 to day 3 with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL). After that, culture medium was switched to complete culture medium and preparation cultured for three additional days. A, data are expressed as the relative secretion of hCG ± S.E.M. compared to hCG secretion at day 4 of culture. Independent experiments were performed on cells isolated from four to five different placentas. **P < 0.01; SB203580 and PD98059 on day 4 compared to control on day 4, t test. B, at day 6, cells were fixed and stained with PI for nuclei (red) and with a specific antibody for plasma membrane desmosomes (green), and observed by confocal microscopy. Scale bar = 100 μm. The experiment was performed on cells isolated from one placenta.
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Measurement of LDH activity in treated cells
We studied the cytotoxicity effect of SB203580 and PD98059 on trophoblasts by measuring the activity of an enzyme marker, lactate dehydrogenase, in cells and culture medium. Figure 10 shows no difference between control and treated cells (10 μM SB203580 and/or 50 μM PD98059), demonstrating the non-toxicity of treatments. The same results were obtained with 1, 5, 20 μM SB203580 or 10, 25, 75 μM PD98059 (data not shown).
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Cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059, 10 μM SB203580 + 50 μM PD98059 or complete culture medium (CTL). As an assay of cell toxicity, total LDH present in the medium and the cells was measured as described in Methods. Independent experiments were performed on cells isolated from eight different placentas. Error bars show S.E.M.
Discussion
In the present study, we have reported for the first time the expression of ERK1/2 and p38 along the days of culture of trophoblasts from human term placenta, and their implication during the differentiation process. Moreover, we have shown that the p38 pathway is highly solicited in trophoblast differentiation compared to the ERK1/2 pathway. Furthermore, we have shown that ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. Finally, we have shown that the inhibition of ERK1/2 or p38 pathways delays trophoblast differentiation.
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In vitro, freshly isolated cytotrophoblast cells differentiated into syncytiotrophoblasts at 4 days of culture, and this phenomenon is characterized by morphological and hormonal differentiation. The morphological differentiation is defined by the fusion of mononucleated cytotrophoblast cells with adjacent syncytium (Midgley et al. 1963), while the biochemical differentiation is characterized by the production of hormones such as hCG and hPL (Kliman et al. 1986; Morrish et al. 1987; Strauss et al. 1992). In this study, we showed that hPL and hCG secretion was modest during the first 24 h of culture, signifying that isolated cytotrophoblast cells were highly purified, without syncytial fragments. After that, the secretion increased progressively to reach a maximum value after 4 days of culture, and decreased thereafter. This decrease has been previously observed (Morrish et al. 1987; Kato & Braunstein, 1989), but explanation remains obscure although apoptosis could explain such decrease (Mayhew et al. 1999). Moreover, we showed that cytotrophoblast cells differentiated morphologically into syncytiotrophoblasts after 4 days of culture. Therefore, in this study, morphological and hormonal status of cells were used to evaluate trophoblast differentiation.
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The MAPKs control many cellular events from complex programmes, such as embryogenesis, cell differentiation, cell proliferation, and cell death, to short-term changes required for homeostasis and acute hormonal responses (Lewis et al. 1998). Mudgett et al. (2000) showed, through gene targeting in mouse, that homozygosity for a null mutation in p38 resulted in embryonic lethality at mid-gestation stages, probably as a consequence of defective placental development characterized by a severe reduction in the spongiotrophoblast layer, as well as a near absence of the labyrinth layer. Moreover, Kita et al. (2003) showed the expression of total ERK1/2 in the villous human cytotrophoblast cells throughout pregnancy, but not in the syncytiotrophoblasts. Furthermore, they reported that phosphorylated ERK1/2 was immunolocalized in the villous cytotrophoblast cells up to 12 weeks of gestation, and suggested a role of ERK1/2 in the proliferation and invasion of these cells (Kita et al. 2003). These observations led us to evaluate the expression level of ERK1/2 and p38 in trophoblasts isolated from human term placenta over increasing days of culture, and verify their implication in trophoblast differentiation. The protein levels of ERK1/2 and p38 drastically decreased with increasing days of culture, to reach an undetectable level on days 5 and 6, indicating a possible role of ERK1/2 and p38 in the initiation process of trophoblast differentiation. Furthermore, we have reported a rapid and transient activation of both ERK1/2 and p38 in primary culture of trophoblasts upon addition of FBS, a factor know to be essential for trophoblast differentiation. The role of MAPK in initiation of differentiation following FBS treatment was evaluated using two specific MAPK inhibitors for ERK1/2 and p38. The presence of SB203580 did not modify p38 activity, but it should be kept in mind that SB203580 inhibits p38 activity by acting directly on p38 by binding to its ATP site, not on an upstream event in this pathway (Young et al. 1997; Davies et al. 2000). Moreover, Frantz et al. (1998) reported that SB203580 bind either unphosphorylated or phosphorylated p38. However, the activation of ERK1/2 was inhibited in the presence of PD98059, because this specific inhibitor acts upstream of ERK1/2 by selectively inhibiting MEK1/2, suppressing the phosphorylation of ERK1/2 and subsequent events in this pathway (Alessi et al. 1995; Dudley et al. 1995).
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In this study, we report that the suppression of ERK1/2 and/or p38 activity early in the differentiation process leads to an impaired trophoblast differentiation process, since the levels of hCG and hPL secretion were reduced as compared to classical culture conditions. Peptide secretion by endocrine cells occurs by at least two distinct pathways, described as constitutive and regulated pathways (Kelly, 1985). In the latter, peptides are packaged and stored in moderate- and high-density secretory granules, which are released in response to the appropriate external stimuli (Burgess & Kelly, 1987). Peptides conveyed by the constitutive secretory pathway are packaged into secretory vesicles of low density, and released at a constant rate at the cell surface (Burgess & Kelly, 1987). Thus, to confirm that the reduction of hCG secretion observed following cell culture in the presence of MAPK inhibitors is due to impaired trophoblast differentiation, rather than an inhibition of either of the peptide secretory pathways mentioned above or reduced hCG synthesis, the effect of PD98059 and SB203580 on hCG secretion was evaluated on syncytiotrophoblasts (trophoblasts that are already differentiated). In this condition, the hCG secretion was not modified, indicating that the reduction of hCG observed during the 4 days of culture is mainly linked to an impaired trophoblast differentiation process. These findings suggest that, in cytotrophoblast cells, ERK1/2 and p38 are essential for the initiation of trophoblast differentiation.
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Although numerous studies have suggested essential requirements for p38 MAPKs in inflammatory and environmental stress responses, the role of the p38 MAPK pathway in normal development and cell differentiation is unclear (for review see Kyriakis & Avruch, 2001). However recently, many investigators have reported that the p38 pathway plays a role in the differentiation of several cell types, including preadipocytes (Engelman et al. 1998, 1999), myoblasts (Cuenda & Cohen, 1999; Galbiati et al. 1999; Zetser et al. 1999), neurones (Morooka & Nishida, 1998; Iwasaki et al. 1999), chondroblasts (Nakamura et al. 1999), erythroblasts (Nagata et al. 1998), cardiomyocytes (Kolodziejczyk et al. 1999; McKinsey & Olson, 1999) and mouse trophoblasts (Mudgett et al. 2000). Moreover, the ERK1/2 pathway plays a role in the differentiation of several cell types including neurones (Morooka & Nishida, 1998), is essential for the initial stage of epithelial tubule development (depolarization and migration) (O'Brien et al. 2004), and mediates the signalling pathway in mesenchyme formation and differentiation in the sea urchin embryo (Fernandez-Serra et al. 2004). In this study we have reported the implication of ERK1/2 and p38 in the initiation of differentiation of human trophoblasts, while a decline of the expression of both MAPKs is observed as differentiation progresses. This conclusion was reached by the evaluation of the protein levels of ERK1/2 and p38 following 4 days of treatment using specific inhibitors for each pathway, demonstrating the induction of their protein expression compared to control. This induction was more important when the p38 pathway was inhibited (SB203580), confirming that this pathway is highly utilized compared to the ERK1/2 pathway in the differentiation process. In addition, SB203580 was more effective than PD98059 in inhibiting hCG and hPL secretion as shown in Figs 4 and 5. Moreover, when trophoblasts were cultivated in the presence of either SB203580 or PD98059 for 3 days, and that culture medium was switched to a complete culture medium for days 4–6, the level of hCG secretion and associated morphological differentiation increased as differentiation progressed, to reach the level observed in differentiated trophoblasts that were cultivated for 4 days in control conditions. These results demonstrate unequivocally the importance of MAPKs in the initiation of the differentiation process in trophoblasts, since the presence of MAPK inhibitors only delays the differentiation process.
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Interestingly, the inhibition of either one of these MAPKs, ERK1/2 or p38, seems to induce a cross-activation of the other, since each inhibitor is very selective and shows no cross effect on the other pathway (Davies et al. 2000; Otis et al. 2004). Thus, it is possible that a compensatory mechanism, working to maintain the normal trophoblast differentiation process could explain such results. However, the potential compensatory mechanism seems to be insufficient to initiate the trophoblast differentiation process. Similar results were obtained in PC12 neuronal cells, where the employment of both pathways (ERK1/2 and p38) was demonstrated in neuronal differentiation (Morooka & Nishida, 1998; Iwasaki et al. 1999). Effectively, treatment of PC12 cells with SB203580, or transfection with the kinase-inactive MKK6 or p38 mutants resulted in a marked inhibition in nerve growth factor (NGF)-induced neurite outgrowth (Morooka & Nishida, 1998), suggesting an important role for p38 in NGF-induced neuronal differentiation of PC12 cells. By contrast, previous reports based on the induction of PC12 differentiation (in the absence of NGF) by a constitutively active mutant of MEK, a MAPK kinase that specifically activates ERKs, suggested that sustained activation of the ERK pathway was sufficient for NGF-induced PC12 differentiation (Morooka & Nishida, 1998). Surprisingly, it now appears that expression of the constitutively active MEK in PC12 cells can activate p38, and that SB203580 can block MEK-induced neurite outgrowth, even though ERK was still active (Morooka & Nishida, 1998). However, the sustained activation of p38 (by transfection of p38 and MKK6) was not sufficient to induce neurite outgrowth in PC12 cells, except when combined with EGF stimulation (which induces transient activation of both ERK and p38), and could in this case be blocked by SB203580. Based on these results, these investigators suggested that neuronal differentiation of PC12 cells might require sustained activation of either ERK or p38 MAPKs, combined with a transient activation of the other (Morooka & Nishida, 1998).
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In conclusion, this study has demonstrated for the first time the expression of ERK1/2 and p38 in human trophoblasts with increasing days of culture, and that ERK1/2 and p38 are essential to mediate initiation of trophoblast differentiation. We also observed a possible cross-talk between both pathways. Moreover, the understanding of cellular events that control trophoblasts differentiation could be very useful in the comprehension of much pathology related to abnormal placental differentiation, such as pre-eclampsia and trophoblastic neoplasm. Therefore, other studies are necessary to verify the implication of other kinases acting upstream and/or downstream of ERK1/2 and p38 in the signalling cascade controlling trophoblast differentiation.
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2 Centre de recherche Biomed, Departement des Sciences Biologiques, Universite du Quebec à Montreal, Montreal, Quebec, Canada, H3C 3P8
3 Departement d'obstetrique-gynecologie, Hpital St-Luc, Universite de Montreal, Montreal, Quebec, Canada, H2L 4M1
, http://www.100md.com Abstract
Mitogen-activated protein kinases (MAPKs) control many cellular events from complex programmes, such as embryogenesis, cell differentiation and proliferation, and cell death, to short-term changes required for homeostasis and acute hormonal responses. However, little is known about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) and p38 in human placenta. Therefore, we examined the expression of ERK1/2 and p38 in trophoblasts from human term placenta, and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultivated in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). This study shows that the level of ERK1/2 and p38 decreases with increasing days of culture, to reach an undetectable level after 5 days of culture. Moreover, pretreatment of cells with an ERK1/2-specific inhibitor (PD98059) and/or a p38-specific inhibitor (SB203580) suppressed trophoblast differentiation. Our results also demonstrate that the p38 pathway is highly solicited as compared to the ERK1/2 pathway in the differentiation process. Furthermore, ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. In summary, this study showed that ERK1/2 and p38 pathways are essential to mediate initiation of trophoblast differentiation.
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Introduction
Mitogen-activated protein kinases (MAPKs) are a family of protein kinases whose functions and regulation have been conserved during evolution from unicellular organisms, such as yeast, to complex organisms including humans (Widmann et al. 1999). In multicellular organisms, there are three well-characterized subfamilies of MAPKs. They included the extracellular signal-regulated kinases (ERK1 and ERK2) (Boulton et al. 1990, 1991), the c-Jun NH2-terminal kinases (JNK 1, JNK 2, and JNK 3) (Derijard et al. 1994; Kyriakis et al. 1994; Gupta et al. 1996), and the four p38 enzymes (p38, p38, p38, and p38) (Han et al. 1994; Jiang et al. 1996; Lechner et al. 1996; Goedert et al. 1997). Moreover, a relatively recent MAPK (ERK5) was identified, and forms the subject of intense studies (Zhou et al. 1995). MAPKs are responsible for the conversion of a large number of extracellular stimuli and environmental conditions into specific cellular responses controlling cell proliferation, differentiation, apoptosis, embryogenesis and regulation of inflammatory and stress responses (for review see Kyriakis & Avruch, 2001; Pearson et al. 2001)). The first mammalian MAPK pathway described was the ERK pathway. ERK1 and ERK2 (ERK1/2) share an 83% amino acid homology, and are expressed to various extents in all tissues (for review see Chen et al. 2001)). They are strongly activated by growth factors, serum, phorbol esters and, to a lesser extent, by ligands of heterotrimeric G protein-coupled receptors, cytokines, osmotic stress and microtubule disorganization (Lewis et al. 1998). In contrast, the p38 pathway is strongly activated by most environmental stresses, pro-inflammatory cytokines, such as interleukin 1 (IL–1) and tumour necrosis factor (TNF-), both playing an important role in the regulation of the inflammatory response. While p38 kinases were originally associated with stress- and inflammation-related kinases, recent evidence involves this kinase in multiple physiological roles in cell cycle control, and in cell proliferation, differentiation and apoptosis (Nebreda & Porras, 2000; Ambrosino & Nebreda, 2001; Pearson et al. 2001). Thus, both the ERK1/2 and p38 pathways play important roles in the differentiation process of several cell types including adipocytes, cardiomyocytes, chondroblasts, erythroblasts, myoblasts and neurones (Nebreda & Porras, 2000; Kohmura et al. 2004; Lee et al. 2004). Moreover, O'Brien et al. (2004) demonstrated that activation of ERK1/2 is essential and sufficient for the initial stage of epithelial tubule development during which cells depolarize and migrate. Thereafter, ERK becomes dispensable for the latter stage, during which cells repolarize and differentiate. ERK1/2 also mediates signalling pathways involved in mesenchyme formation and differentiation in the sea urchin embryo (Fernandez-Serra et al. 2004). Furthermore, Mudgett et al. (2000) demonstrated the requirement of p38 MAPK in mouse diploid trophoblast development and placental vascularization, and suggest a more general role for p38 MAPK signalling in embryonic angiogenesis. However, little is known about the implication of MAPK pathways in human trophoblast differentiation.
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Human trophoblast differentiation is characterized by the formation of a specific multinuclear structure, the syncytiotrophoblast. This structure arises by fusion and differentiation of the relatively undifferentiated, mitotically active cytotrophoblast cells (Midgley et al. 1963). Moreover, throughout pregnancy, the syncytiotrophoblasts become a continuous epithelial layer located at the villous surface of the placenta, floating in maternal blood. Therefore, essential fetal nutrients must cross this placental barrier to reach the fetal circulation. Trophoblast growth and differentiation has been studied in in vitro models by many investigators during the last two decades. Many studies reported that, in vitro, cytotrophoblast cells in serum-free conditions fail to aggregate or fuse, and remain in a mononuclear state (Ringler & Strauss, 1990; Yang et al. 2003). In contrast, when cells are cultivated in medium supplemented with fetal bovine serum (FBS), they spontaneously fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts. The morphological differentiation is defined by the fusion of mononucleated cytotrophoblast cells with adjacent syncytium (Midgley et al. 1963), while the biochemical differentiation is characterized by the production of hormones such as human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) (Kliman et al. 1986; Morrish et al. 1987; Strauss et al. 1992).
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The aim of the present study was to investigate the role of ERK1/2 and p38 in human trophoblast differentiation. Thus, protein levels of ERK1/2 and p38 were evaluated during the differentiation process of trophoblasts isolated from human term placentas. Moreover, using specific inhibitors of both pathways, our results demonstrated for the first time that ERK1/2 and p38 play a central role in human trophoblast differentiation.
Methods
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Materials
Dulbecco's modified Eagle medium (high glucose) (DMEM-HG), Hanks balanced salt solution (HBSS), trypsin, DNAse, Percoll, propidium iodide (PI), SB203580, PD98059 and antidesmosome mouse monoclonal antibody were from Sigma (Oakville, ON, Canada). Calf serum and penicillin, streptomycin, neomycin (PSN; 100X) were purchased from Invitrogen (Burlington, ON, Canada). FBS and an ELISA kit for hCG assay were from Medicorp (Montreal, QC, Canada). An ELISA kit for human placental lactogen (hPL) was from DRG international (Mountainside, NJ, USA). A CytoTox 96R. non-radioactive cytotoxicity assay kit was from Promega (Madison, WI, USA), and was used to measure lactate dehydrogenase (LDH) activity. Bovine serum albumin (BSA), the BM chemiluminescence (POD) system, TRIS-base, Nonidet-40 and acrylamide were from Roche Applied Science (Laval, QC, Canada). Bicinchoninic acid (BCA) reagent was purchased from Pierce (Brockville, ON, Canada). Anti-phospho-Erk, anti-Erk, anti-phospho-p38 and anti-p38 rabbit polyclonal antibodies were all purchased from Cell Signalling (Beverly, MA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody, sheep anti-rabbit-IgG, goat anti-mouse-IgG conjugated with horseradish peroxidase and Re-Blot plus Mild solution were from Chemicon International (Temecula, CA, USA). Alexa Fluor 488 goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR, USA). The fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against cytokeratin-7 was from Accurate Chemical & Scientific Corp (Westbury, NY, USA). Bio-Rad minigel system was from Bio-Rad (Mississauga, Ontario, Canada). Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore (Cambridge, Ontario, Canada). The 24-well plates and 35 mm dishes were obtained from Corning (Acton, MA, USA). BioMax Light autoradiography films were from Eastman Kodak Co. (Rochester, NY, USA). All other products were from Sigma (Oakville, ON, Canada).
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Human placental trophoblast isolation and purity evaluation
The procedure of Kliman et al. (1986), with minor modifications, was used for cell dispersion and cytotrophoblast cell isolation. Human term placentas from 35 to 41 weeks of gestation were obtained immediately after spontaneous vaginal delivery, in accordance with the established guidelines of the ethical committee of St Luc Hospital of the Centre Hospitalier Universitaire de Montreal (Montreal, QC, Canada) and of Universite du Quebec à Montreal (Montreal, QC, Canada). Briefly, placental villi were cut and thoroughly washed to remove blood. Thereafter, they were incubated four times in a digestion medium composed of HBSS, containing trypsin and deoxyribonuclease for 30 min at 37°C in a water bath with continuous shaking. The dispersed cells were layered on top of a discontinuous 5–70% Percoll gradient, and centrifuged for 25 min at 507 g. The intermediate layers (density between 1.048 and 1.062) containing cytotrophoblast cells were removed and washed, and cell viability was determined by trypan blue exclusion. Following trophoblast isolation, cells were seeded at a density of approximately 1.6 x 106 cells well–1 in 24-well plate or 4.5 x 106 cells dish–1 in 35 mm dishes. The complete culture medium, constituted of DMEM-HG, 2 mM glutamine, 10% FBS and PSN (1x), was refreshed daily.
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The purity of cytotrophoblast cells preparations was evaluated by flow cytometry using FITC-conjugated monoclonal antibody against cytokeratin-7 as previously described (Daoud et al. 2005). Only cell preparations having a minimum of 96% of cytotrophoblast cells were used in this work.
Secretion of hCG and hPL by trophoblasts
Trophoblasts cells were cultivated in 24-well plates for 4 or 6 days. Every day, the culture media were collected, centrifuged and supernatants were frozen at –20°C until measurements. The hCG and hPL contents in culture media were evaluated by ELISA following manufacturer's instructions.
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Effect of MAPK inhibitors on hCG and hPL secretion
Following isolation, cells were plated in 24-well plates in complete culture medium, and left overnight prior to treatment. Thereafter, cells were preincubated with PD98059 (75, 50, 25 or 10 μM), SB203580 (20, 10, 5 or 1 μM), PD98059 + SB203580 (50 μM +10 μM, respectively), dissolved in DMSO (<0.1%) or vehicle alone for 1 h in culture medium (serum free). Following that, 10% FBS was added. These treatments were repeated every day for 4 days. Culture media were collected every day for hCG and hPL measurement. In another set of experiements, to investigate the role of ERK1/2 and p38 in hCG synthesis and secretion, cells were cultivated in complete culture medium for 3 days, to allow differentiation of cytotrophoblast cells into syncytiotrophoblasts. Thereafter, cells were rinsed and preincubated for 1 h with MAPK inhibitors, according to the above conditions, before the addition of 10% FBS. The culture media were collected at day four and subjected to hCG measurement.
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Effect of FBS and MAPK inhibitors (PD98059 and SB203580) on ERK1/2 and p38 activation in human primary culture trophoblasts
After trophoblast isolation, cells were seeded in 35 mm dishes in the presence of complete culture medium for 5 h at 37°C to allow attachment, and thereafter, cells were serum-starved overnight. At day one, cells were preincubated with 50 μM PD98059 or 10 μM SB203580 for 1 h, prior the addition of 10% FBS for different periods of time (0, 2, 5, 10, 20 and 30 min). The reaction was stopped by aspiration of cell culture, and cell lysates were prepared. Thereafter, lysates were subjected to 12% polyacrylamide gel electrophoresis (SDS-PAGE) using a Bio-Rad minigel system. Activated ERK and p38 were detected by immunoblotting using antiphospho-Erk and antiphospho-p38 antibodies. Blots were then stripped and reprobed using specific antibodies directed against total ERK or p38.
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Cell lysate preparation
For every day of follow-up of ERK1/2 and p38 protein expression (day 1 to day 4), total trophoblast proteins were isolated. For this set of experiments, cells were seeded in 35 mm dishes. For protein preparation, media was aspirated, cells were rinsed twice with ice-cold PBS, solubilized with ice-cold radioimmunoprecipitation (RIPA) buffer (150 mM NaCl, 9.1 mM Na2HPO4, 1.7 mM NaH2PO4 (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS, containing freshly added 200 μM Na3VO4, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μM leupeptine, 1.46 μM pepstatin and 2 μg ml–1 aprotinin) and harvested. Cell lysates were clarified by centrifugation at 14 000 g for 10 min at 4°C. Protein concentration of the supernatant was determined by spectrophotometric quantification using the BCA reagent with BSA as standard. For MAPK activation, protein extracts were prepared in the same way.
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Western blots and densitometric analysis
Cellular proteins (30 μg) were solubilized in sample buffer (4% SDS, 30 mM dithiotreitol, 0.25 M sucrose, 0.01 M EDTA-Na2 and 0.075% bromophenol blue), and heated at 95°C for 5 min to denature proteins. The lysates were resolved in 12% SDS-PAGE and proteins were electroblotted on a PVDF membrane at 1.7 mA cm–2 for 2 h. The membranes were blocked for 1 h at room temperature in TBS-T (20 mM Tris (pH 7.6), 137 mM NaCl and 0.05% Tween-20) containing 5% skimmed milk. Membranes were then incubated with the appropriate primary antibody (1/2000 for antiphospho-ERK1/2, 1/500 for antiphospho-p38, 1/1000 for anti-ERK1/2 and antip38 or 1/4000 for anti-GAPDH in TBS-T/5% BSA) overnight at 4°C, washed three times with TBS-T, and probed with horseradish peroxidase-conjugated secondary antibodies (1/1500 for antirabbit-IgG or 1/3000 for antimouse-IgG) for 2 h at room temperature. Blots were washed three times with TBS-T and the detection was performed using the BM Chemiluminescence system and visualized by autoradiography (BioMax Light film). In certain instances, the PVDF membranes were stripped with Re-Blot plus Mild solution at room temperature for 15 min, rinsed twice with blocking buffer, and blocked for 1 h before reprobing with another antibody. The band intensity after exposure and development was digitized and analysed by Quantity One software (Bio-Rad Laboratories, Mississauga, ON, Canada).
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Immunofluorescence analysis
Primary trophoblasts were stained with antidesmosomal antibody as previously described for anticytokeratin-7 antibody (Daoud et al. 2005). Briefly, cells were washed twice with PBS, fixed in methanol at –20°C for 20 min, washed again with PBS (twice), and then incubated in PBS containing 2% FBS (v/v) for 45 min, to eliminate non-specific binding. Thereafter, cells were rinsed with PBS and incubated in the presence of mouse antihuman antidesmosomes monoclonal antibody (1/800) in PBS containing 0.2% BSA for 1 h at room temperature, washed three times with PBS, and incubated with Alexa Fluor 488 goat antimouse IgG (1/1000) for 1 h at room temperature in the dark. For nuclear staining, cells were incubated with propidium iodide (50 μg ml–1) for an additional 30 min at room temperature in the dark, washed three times with PBS, and viewed using a Nikon Eclipse TE300 camera (Nikon, Tokyo, Japan) equipped with a confocal laser-scanning microscope (Bio-Rad MRC1024, CA, USA). All observations were performed at 10x magnification on monolayer cells. A syncytium was defined as three or more nuclei in the same cytoplasm without intervening surface desmosomal membrane staining.
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Effect of MAPK inhibitors on ERK1/2 and p38 expression
Following isolation, cells were plated in 24-well plates in complete culture medium, and left overnight prior to treatments. Thereafter, cells were preincubated with 50 μM PD98059, 10 μM SB203580 dissolved in DMSO (<0.1%), or vehicle alone for 1 h in culture medium (serum free), and then 10% FBS was added. The same treatment was repeated every day, for four days. Thereafter, cells were harvested, proteins were isolated and protein levels of ERK1/2 and p38 were evaluated by western blot.
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MAPK inhibitors delayed trophoblast differentiation
Following isolation, cells were plated in 24-well plates in complete culture medium and left overnight. At day 1, cells were preincubated with 50 μM PD98059, 10 μM SB203580 dissolved in DMSO (<0.1%), or vehicle alone for 1 h in culture medium (serum free), and then 10% FBS was added. The same treatment was repeated for 2 days. On day 3, culture medium was refreshed with the complete culture medium, and cells were cultivated for an additional 3 days. The culture media were collected every day for hCG measurement.
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Measurement of LDH activity after PD98050 or/and SB203580 treatment
After 4 days of culture, the culture medium was collected and the attached cells were washed with PBS and suspended in lysis buffer containing 0.1% Triton X-100. As an assay of cell toxicity, total LDH present in the medium and the cells was measured with a LDH kit, according to the manufacturer's instructions. Color change was quantified by reading the absorbance at 490 nm, and the cell toxicity was calculated as (OD in the supernatant – OD of medium (background))/(OD of Triton X-100-lysed cells – OD of background), where OD is the optical density.
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Statistical analyses
Data were expressed as the mean ± S.E.M., and analysed with a one-way ANOVA followed by the Tukey test, two-way ANOVA or t test at a P < 0.05 level of significance.
Results
Trophoblast differentiation
To evaluate the differentiation process of isolated cytotrophoblast cells into functional syncytiotrophoblasts, the levels of secreted hCG and hPL, two well-known makers of differentiation, were assayed in the culture medium over a period of six days (Kliman et al. 1986). Figure 1A and B shows the profile of hCG and hPL secretion. In both panels, the secretion was modest during the first 24 h of culture. After that, the secretion increased with increasing days of culture, reaching a maximal value at 3–5 days of culture for hCG and 4–5 days for hPL, and declined thereafter. The hCG secretion values were calculated as mU (mg protein)–1 (24 h)–1, while the hPL values were calculated as μg (mg protein)–1 (24 h)–1. Moreover, since these values were variable between placentas of different weeks of pregnancy, our data were expressed as relative hCG or hPL secretion compared to the maximal value obtained at day 4 of culture. To substantiate the effect of days of culture on trophoblast differentiation, we examined the syncytium formation as an indicator of morphological differentiation. As shown in Fig. 1C, most trophoblasts were present as mononucleated cytotrophoblast cells at day 1, while at day 4, more abundant and larger syncytia were observed. Therefore, subsequent experiments were performed up to day 4 of culture, when differentiation occurred.
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A, cells were cultivated from day 1 to day 6 in complete culture medium. Data are expressed as relative secretion of hCG ± S.E.M. compared to secretion at day 4 of culture of six cell preparations from six different placentas. ***P < 0.001, day 1 to day 6, one-way ANOVA; **P < 0.01; *** P < 0.001 compared to day 1 of culture, Tukey test. B, cells were cultivated from day 1 to day 6 in complete culture medium. Data are expressed as relative secretion of hPL ± S.E.M. compared to secretion at day 4 of culture of five cell preparations from five different placentas. ***P < 0.001, day 1 to day 6, one-way ANOVA; *P < 0.05; **P < 0.01 compared to day 1 of culture, Tukey test. C, cells were cultivated for 1 or 4 days in complete culture medium, fixed and stained with PI for nuclei (red) and specific antibody for plasma membrane desmosomes (green), and observed by confocal microscopy. Independent experiments were performed on cells isolated from three different placentas. Scale bar = 100 μm.
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Expression of ERK1/2 and p38 along the differentiation of trophoblasts in culture
The expression of ERK1/2 and p38 was evaluated by western blot using total protein isolated from trophoblasts. Figures 2 and 3 show representative western blots of the expression of ERK1/2 and p38, respectively, from trophoblasts cultivated for days 1–4. Following normalization with the GAPDH protein level, our results show that the expression of ERK1/2 declined by 33, 51 and 78% for days 2, 3 and 4, respectively (Fig. 2A and B), while the expression of p38 declined by 15%, 45% and 68% for days 2, 3 and 4, respectively (Fig. 3A and B). Interestingly, after 5 and 6 days of culture, no signal was detected for either ERK1/2 or p38 by western blot (data not shown).
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A, western blot analysis was performed using ERK1/2 or GAPDH antibody on total proteins (30 μg) isolated from primary culture of trophoblasts cultivated from day 1 to day 4. The blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of ERK1/2 protein level after normalization with GAPDH protein level. Independent experiments were performed on cells isolated from four different placentas. P < 0.001, day 1 to day 4, one-way ANOVA; *P < 0.05; ***P < 0.001 compared to day 1 of culture, Tukey test. Error bars show S.E.M.
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A, western blot analysis was performed using p38 or GAPDH antibody on total proteins (30 μg) isolated from primary culture of trophoblasts cultivated from day 1 to day 4. The blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of p38 protein level after normalization with GAPDH protein level. Independent experiments were performed on cells isolated from four different placentas. P < 0.05, day 1 to day 4, one-way ANOVA; *P < 0.05 compared to day 1 of culture, Tukey test. Error bars show S.E.M.
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Effect of MAPK inhibitors on the differentiation of trophoblasts primary culture
In order to investigate the role of ERK1/2 and p38 pathways on trophoblast differentiation, cells were treated with either PD98059, an ERK1/2-specific inhibitor, or SB203580, a p38-specific inhibitor (Davies et al. 2000; Chen et al. 2001; Otis et al. 2004). First, we titrated the inhibitors in trophoblast cultures. As shown in Fig. 4A and B, SB203580 inhibited hCG and hPL secretion in a dose-dependent manner. Following 5, 10, or 20 μM SB203580 treatment, hCG secretion was significantly reduced by 65%, 80% and 92%, respectively (Fig. 4A), while 1 μM SB203580 had no effect on hCG secretion. In the same way, hPL secretion was reduced by 44%, 81%, 79% and 87% after 1, 5, 10 or 20 μM SB203580 treatments (Fig. 4B). The same results were obtained with PD98059 (Fig. 4C and D). Following 10, 25, 50 or 75 μM PD98059 treatment, hCG secretion was significantly reduced by 49%, 58%, 76% and 82%, while hPL secretion was reduced by 55%, 53%, 68% and 73%, respectively. Therefore, subsequent experiments were performed using 10 μM SB203580 or 50 μM PD98059, and hCG secretion was used to evaluate trophoblasts' biochemical differentiation, while morphological differentiation was evaluated by syncytium formation. Figure 5A shows that the presence of PD98059 (50 μM) and/or SB203580 (10 μM) in the culture medium during all days of culture inhibited the hCG secretion. This inhibition was more important under SB203580 treatment compared to that with PD98059. However, cells treated with both inhibitors conjointly showed an additional inhibiting effect on hCG secretion. To substantiate the effect of the two inhibitors on trophoblast differentiation, we examined syncytium formation. As shown in Fig. 5C, cells treated with SB203580 or PD98059 for 4 days were present as mononucleated cytotrophoblast cells, while the control cells consisted of abundant and large syncytia. Taken together, these results show that the suppression of both ERK1/2 and p38 pathways significantly impaired trophoblast differentiation. Furthermore, Fig. 5B shows that neither pathway is involved in the synthesis of hCG by syncytiotrophoblasts, since the addition of these inhibitors in the culture medium when trophoblasts were already differentiated did not alter hCG secretion. Cells treated with the vehicle alone showed no effect on hCG secretion or morphological differentiation (data not shown).
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A and B, cells were treated from day 1 to day 4 with different concentrations of SB203580 (1, 5, 10 and 20 μM) or complete control medium (CTL). Supernatants from day 4 were then assayed for hCG (A) or hPL (B) secretion. C and D, cells were treated from day 1 to day 4 with different concentrations of PD98059 (10, 25, 50 and 75 μM) or complete control medium (CTL). Supernatants from day 4 were then assayed for hCG (C) and hPL (D) secretion. Data are expressed as relative secretion of hCG or hPL ± S.E.M. compared to control hCG or hPL secretion at day 4 of culture. *P < 0.05, **P < 0.01, ***P < 0.001; treated compared to control, t test.
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A, cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059, 10 μM SB203580 + 50 μM PD98059 or complete culture medium (CTL). Data are expressed as the relative secretion of hCG ± S.E.M. compared to hCG secretion at day 4 of culture. *P < 0.05; **P < 0.01; SB203580 or SB203580 + PD98059 compared to PD98059; t test, P < 0.05; 50 μM PD98059 compared to control, P < 0.005; 10 μM SB203580 compared to control, and P < 0.001; 50 μM PD98059 + 10 μM SB203580 compared to control; two-way ANOVA. B, cells were cultivated in complete culture medium for 3 days. Thereafter, cells were treated with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL) for another 24 h. After that, culture medium was collected and hCG secretion was measured. Data are expressed as the relative secretion of hCG ± S.E.M. compared to control. C, cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL). At day 4, cells were fixed and stained with PI for nuclei (red), and with a specific antibody for plasma membrane desmosomes (green) and observed by confocal microscopy. Scale bar = 100 μm. Independent experiments were performed on cells isolated from three different placentas.
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Effect of FBS and MAPK inhibitors on ERK1/2 and p38 activation
Following isolation of cells, trophoblasts were incubated for 5 h in the presence of 10% FBS, then serum-starved overnight. Following starvation, cells were treated with 10% FBS for different periods of time (1–30 min). Our results show that treatment with FBS induced a transient activation of ERK1/2 that reached a peak at 5 min and declined thereafter; however, the phosphorylation level did not return to the basal value (Fig. 6A and B). In the same way, p38 activation was also transient (Fig. 7A and B), but the kinetics were slightly different, since the maximal activation (phosphorylation) was reached between 5 and 10 min, and declined thereafter to reach the basal level after a 30 min incubation with FBS. The same results were obtained using 4-day cultivated syncytiotrophoblasts (data not shown).
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Following isolation of cytotrophoblast cells, cells were kept in complete culture media for 5 h and serum-starved overnight. After stimulation, cell lysates were prepared and subjected to 12% SDS-PAGE. Activated ERK1/2 were detected by western blot using antiphospho-ERK1/2 antibodies. The blot was then stripped and reprobed using an anti-ERK1/2 (total) antibody. A, cells were stimulated with 10% FBS for the indicated time. C, cells were preincubated with SB203580 (10 μM) for 1 h and then stimulated with 10% FBS for the indicated time. E, cells were preincubated with PD98059 (50 μM) for 1 h and then stimulated with 10% FBS for indicated time. B, D and F, densitometric analysis of activated ERK1/2 protein level after normalization with total ERK1/2 protein level. Independent experiments were performed on cells isolated from three different placentas. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control time (0 min); t test. Error bars show S.E.M.
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Following isolation of cytotrophoblast cells, cells were kept in complete culture medium for 5 h and serum-starved overnight. After stimulation, cell lysates were prepared and subjected to 12% SDS-PAGE. Activated p38 was detected by western blot using antiphospho-p38 antibodies. The blot was then stripped and reprobed using an antip38 (total) antibody. A, cells were stimulated with 10% FBS for the indicated time. C, cells were preincubated with SB203580 (10 μM) for 1 h and then stimulated with 10% FBS for the indicated time. E, cells were preincubated with PD98059 (50 μM) for 1 h and then stimulated with 10% FBS for the indicated time. B, D and F, densitometric analysis of activated p38 protein level after normalization with total p38 protein level. Independent experiments were performed on cells isolated from three different placentas. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (time 0); t test. Error bars show S.E.M.
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Specific inhibitors of both MAPKs were also used to verify their specific effects on the activation of ERK1/2 and p38 induced by FBS. Thus, when cytotrophoblast cells were pretreated with SB203580 (Fig. 6C and D), ERK1/2 phosphorylation was stimulated as compared to control (time 0 in control cells compared to time 0 in SB203580-treated cells), while the profile of p38 phosphorylation remained unchanged (Fig. 7C and D). However, pretreatment of cytotrophoblast cells with PD98059 for 1 h completely blocked FBS-induced ERK1/2 activation (Fig. 6E and F), while p38 activation was stimulated compared to control (time 0 in control cells compared to time 0 in PD98059-treated cells; Fig. 7E and F). Cell treatment with the vehicle alone showed no effect on ERK1/2 or p38 activation (data not shown).
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Effect of MAPK inhibitors on ERK1/2 and p38 protein level
As previously shown (Fig. 5A and C), both ERK1/2 and p38 inhibitors inhibit trophoblast differentiation. Therefore, the effect of the presence of SB203580 or PD98059 in the culture medium for 4 days was evaluated on protein levels of ERK1/2 and p38. Figure 8 shows that the presence of SB203580 increased the expression of ERK1/2 and p38 1.64- and 2-fold, respectively. In the same way, the presence of PD98059 increased the expression of ERK1/2 1.2-fold, while for p38 a tendency was observed, but this increase was not significant (Fig. 8).
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Cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL). A, western blot analysis was performed using total ERK1/2 or p38 antibodies on total proteins (30 μg) isolated from treated or control trophoblasts after 4 days of culture. The blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of ERK1/2 and p38 protein level after normalization with GAPDH protein level. Independent experiments were performed on cells isolated from three different placentas. **P < 0.01, *P < 0.05; SB203580 or PD98059 compared to control, t test. Error bars show S.E.M.
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MAPK inhibitors delayed trophoblast differentiation
According to previous results (Fig. 5) on the effects of MAPK inhibitors on trophoblast differentiation, we evaluated the hypothesis that these inhibitors could delay trophoblast differentiation. Thus, cells were cultivated in the presence of MAPK inhibitors; either SB203580 or PD98059, for 3 days, and after that the culture medium was switched to complete culture medium for the next 3 days. Figure 9A shows that the hCG secretion remained low for the first 4 days in the presence of MAPK inhibitors, but when the inhibitors were removed, the hCG secretion increased to reach its control value at day 5, while at day 6, this level exceeded its relevant control value. Finally, our results demonstrate that the levels of hCG secretion, at days 5 and 6, in cells pretreated with MAPK inhibitors, reached the same value as that observed at day 4 in the control cell culture conditions, when trophoblast differentiation occurred (Fig. 9A). Moreover, morphological differentiation was confirmed at day 6 as shown in Fig. 9B. These results confirm that the suppression of the activation of ERK1/2 or p38 delayed the differentiation process in trophoblasts.
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Cells were treated from day 1 to day 3 with 10 μM SB203580, 50 μM PD98059 or complete culture medium (CTL). After that, culture medium was switched to complete culture medium and preparation cultured for three additional days. A, data are expressed as the relative secretion of hCG ± S.E.M. compared to hCG secretion at day 4 of culture. Independent experiments were performed on cells isolated from four to five different placentas. **P < 0.01; SB203580 and PD98059 on day 4 compared to control on day 4, t test. B, at day 6, cells were fixed and stained with PI for nuclei (red) and with a specific antibody for plasma membrane desmosomes (green), and observed by confocal microscopy. Scale bar = 100 μm. The experiment was performed on cells isolated from one placenta.
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Measurement of LDH activity in treated cells
We studied the cytotoxicity effect of SB203580 and PD98059 on trophoblasts by measuring the activity of an enzyme marker, lactate dehydrogenase, in cells and culture medium. Figure 10 shows no difference between control and treated cells (10 μM SB203580 and/or 50 μM PD98059), demonstrating the non-toxicity of treatments. The same results were obtained with 1, 5, 20 μM SB203580 or 10, 25, 75 μM PD98059 (data not shown).
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Cells were treated from day 1 to day 4 with 10 μM SB203580, 50 μM PD98059, 10 μM SB203580 + 50 μM PD98059 or complete culture medium (CTL). As an assay of cell toxicity, total LDH present in the medium and the cells was measured as described in Methods. Independent experiments were performed on cells isolated from eight different placentas. Error bars show S.E.M.
Discussion
In the present study, we have reported for the first time the expression of ERK1/2 and p38 along the days of culture of trophoblasts from human term placenta, and their implication during the differentiation process. Moreover, we have shown that the p38 pathway is highly solicited in trophoblast differentiation compared to the ERK1/2 pathway. Furthermore, we have shown that ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. Finally, we have shown that the inhibition of ERK1/2 or p38 pathways delays trophoblast differentiation.
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In vitro, freshly isolated cytotrophoblast cells differentiated into syncytiotrophoblasts at 4 days of culture, and this phenomenon is characterized by morphological and hormonal differentiation. The morphological differentiation is defined by the fusion of mononucleated cytotrophoblast cells with adjacent syncytium (Midgley et al. 1963), while the biochemical differentiation is characterized by the production of hormones such as hCG and hPL (Kliman et al. 1986; Morrish et al. 1987; Strauss et al. 1992). In this study, we showed that hPL and hCG secretion was modest during the first 24 h of culture, signifying that isolated cytotrophoblast cells were highly purified, without syncytial fragments. After that, the secretion increased progressively to reach a maximum value after 4 days of culture, and decreased thereafter. This decrease has been previously observed (Morrish et al. 1987; Kato & Braunstein, 1989), but explanation remains obscure although apoptosis could explain such decrease (Mayhew et al. 1999). Moreover, we showed that cytotrophoblast cells differentiated morphologically into syncytiotrophoblasts after 4 days of culture. Therefore, in this study, morphological and hormonal status of cells were used to evaluate trophoblast differentiation.
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The MAPKs control many cellular events from complex programmes, such as embryogenesis, cell differentiation, cell proliferation, and cell death, to short-term changes required for homeostasis and acute hormonal responses (Lewis et al. 1998). Mudgett et al. (2000) showed, through gene targeting in mouse, that homozygosity for a null mutation in p38 resulted in embryonic lethality at mid-gestation stages, probably as a consequence of defective placental development characterized by a severe reduction in the spongiotrophoblast layer, as well as a near absence of the labyrinth layer. Moreover, Kita et al. (2003) showed the expression of total ERK1/2 in the villous human cytotrophoblast cells throughout pregnancy, but not in the syncytiotrophoblasts. Furthermore, they reported that phosphorylated ERK1/2 was immunolocalized in the villous cytotrophoblast cells up to 12 weeks of gestation, and suggested a role of ERK1/2 in the proliferation and invasion of these cells (Kita et al. 2003). These observations led us to evaluate the expression level of ERK1/2 and p38 in trophoblasts isolated from human term placenta over increasing days of culture, and verify their implication in trophoblast differentiation. The protein levels of ERK1/2 and p38 drastically decreased with increasing days of culture, to reach an undetectable level on days 5 and 6, indicating a possible role of ERK1/2 and p38 in the initiation process of trophoblast differentiation. Furthermore, we have reported a rapid and transient activation of both ERK1/2 and p38 in primary culture of trophoblasts upon addition of FBS, a factor know to be essential for trophoblast differentiation. The role of MAPK in initiation of differentiation following FBS treatment was evaluated using two specific MAPK inhibitors for ERK1/2 and p38. The presence of SB203580 did not modify p38 activity, but it should be kept in mind that SB203580 inhibits p38 activity by acting directly on p38 by binding to its ATP site, not on an upstream event in this pathway (Young et al. 1997; Davies et al. 2000). Moreover, Frantz et al. (1998) reported that SB203580 bind either unphosphorylated or phosphorylated p38. However, the activation of ERK1/2 was inhibited in the presence of PD98059, because this specific inhibitor acts upstream of ERK1/2 by selectively inhibiting MEK1/2, suppressing the phosphorylation of ERK1/2 and subsequent events in this pathway (Alessi et al. 1995; Dudley et al. 1995).
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In this study, we report that the suppression of ERK1/2 and/or p38 activity early in the differentiation process leads to an impaired trophoblast differentiation process, since the levels of hCG and hPL secretion were reduced as compared to classical culture conditions. Peptide secretion by endocrine cells occurs by at least two distinct pathways, described as constitutive and regulated pathways (Kelly, 1985). In the latter, peptides are packaged and stored in moderate- and high-density secretory granules, which are released in response to the appropriate external stimuli (Burgess & Kelly, 1987). Peptides conveyed by the constitutive secretory pathway are packaged into secretory vesicles of low density, and released at a constant rate at the cell surface (Burgess & Kelly, 1987). Thus, to confirm that the reduction of hCG secretion observed following cell culture in the presence of MAPK inhibitors is due to impaired trophoblast differentiation, rather than an inhibition of either of the peptide secretory pathways mentioned above or reduced hCG synthesis, the effect of PD98059 and SB203580 on hCG secretion was evaluated on syncytiotrophoblasts (trophoblasts that are already differentiated). In this condition, the hCG secretion was not modified, indicating that the reduction of hCG observed during the 4 days of culture is mainly linked to an impaired trophoblast differentiation process. These findings suggest that, in cytotrophoblast cells, ERK1/2 and p38 are essential for the initiation of trophoblast differentiation.
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Although numerous studies have suggested essential requirements for p38 MAPKs in inflammatory and environmental stress responses, the role of the p38 MAPK pathway in normal development and cell differentiation is unclear (for review see Kyriakis & Avruch, 2001). However recently, many investigators have reported that the p38 pathway plays a role in the differentiation of several cell types, including preadipocytes (Engelman et al. 1998, 1999), myoblasts (Cuenda & Cohen, 1999; Galbiati et al. 1999; Zetser et al. 1999), neurones (Morooka & Nishida, 1998; Iwasaki et al. 1999), chondroblasts (Nakamura et al. 1999), erythroblasts (Nagata et al. 1998), cardiomyocytes (Kolodziejczyk et al. 1999; McKinsey & Olson, 1999) and mouse trophoblasts (Mudgett et al. 2000). Moreover, the ERK1/2 pathway plays a role in the differentiation of several cell types including neurones (Morooka & Nishida, 1998), is essential for the initial stage of epithelial tubule development (depolarization and migration) (O'Brien et al. 2004), and mediates the signalling pathway in mesenchyme formation and differentiation in the sea urchin embryo (Fernandez-Serra et al. 2004). In this study we have reported the implication of ERK1/2 and p38 in the initiation of differentiation of human trophoblasts, while a decline of the expression of both MAPKs is observed as differentiation progresses. This conclusion was reached by the evaluation of the protein levels of ERK1/2 and p38 following 4 days of treatment using specific inhibitors for each pathway, demonstrating the induction of their protein expression compared to control. This induction was more important when the p38 pathway was inhibited (SB203580), confirming that this pathway is highly utilized compared to the ERK1/2 pathway in the differentiation process. In addition, SB203580 was more effective than PD98059 in inhibiting hCG and hPL secretion as shown in Figs 4 and 5. Moreover, when trophoblasts were cultivated in the presence of either SB203580 or PD98059 for 3 days, and that culture medium was switched to a complete culture medium for days 4–6, the level of hCG secretion and associated morphological differentiation increased as differentiation progressed, to reach the level observed in differentiated trophoblasts that were cultivated for 4 days in control conditions. These results demonstrate unequivocally the importance of MAPKs in the initiation of the differentiation process in trophoblasts, since the presence of MAPK inhibitors only delays the differentiation process.
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Interestingly, the inhibition of either one of these MAPKs, ERK1/2 or p38, seems to induce a cross-activation of the other, since each inhibitor is very selective and shows no cross effect on the other pathway (Davies et al. 2000; Otis et al. 2004). Thus, it is possible that a compensatory mechanism, working to maintain the normal trophoblast differentiation process could explain such results. However, the potential compensatory mechanism seems to be insufficient to initiate the trophoblast differentiation process. Similar results were obtained in PC12 neuronal cells, where the employment of both pathways (ERK1/2 and p38) was demonstrated in neuronal differentiation (Morooka & Nishida, 1998; Iwasaki et al. 1999). Effectively, treatment of PC12 cells with SB203580, or transfection with the kinase-inactive MKK6 or p38 mutants resulted in a marked inhibition in nerve growth factor (NGF)-induced neurite outgrowth (Morooka & Nishida, 1998), suggesting an important role for p38 in NGF-induced neuronal differentiation of PC12 cells. By contrast, previous reports based on the induction of PC12 differentiation (in the absence of NGF) by a constitutively active mutant of MEK, a MAPK kinase that specifically activates ERKs, suggested that sustained activation of the ERK pathway was sufficient for NGF-induced PC12 differentiation (Morooka & Nishida, 1998). Surprisingly, it now appears that expression of the constitutively active MEK in PC12 cells can activate p38, and that SB203580 can block MEK-induced neurite outgrowth, even though ERK was still active (Morooka & Nishida, 1998). However, the sustained activation of p38 (by transfection of p38 and MKK6) was not sufficient to induce neurite outgrowth in PC12 cells, except when combined with EGF stimulation (which induces transient activation of both ERK and p38), and could in this case be blocked by SB203580. Based on these results, these investigators suggested that neuronal differentiation of PC12 cells might require sustained activation of either ERK or p38 MAPKs, combined with a transient activation of the other (Morooka & Nishida, 1998).
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In conclusion, this study has demonstrated for the first time the expression of ERK1/2 and p38 in human trophoblasts with increasing days of culture, and that ERK1/2 and p38 are essential to mediate initiation of trophoblast differentiation. We also observed a possible cross-talk between both pathways. Moreover, the understanding of cellular events that control trophoblasts differentiation could be very useful in the comprehension of much pathology related to abnormal placental differentiation, such as pre-eclampsia and trophoblastic neoplasm. Therefore, other studies are necessary to verify the implication of other kinases acting upstream and/or downstream of ERK1/2 and p38 in the signalling cascade controlling trophoblast differentiation.
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