Src family kinases play multiple roles in differentiation of trophoblasts from human term placenta
1 Laboratoire de Physiologie materno-ftale, Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Québec, Canada, H3C 3P8
2 Centre de recherche Biomed, Université du Québec à Montréal, Montréal, Québec, Canada, H3C 3P8
3 Département d'Obstétrique-Gynécologie, Hpital St-Luc, Université de Montréal, Montréal, Québec, Canada, H2L 4M1
Abstract
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Tyrosine phosphorylation plays a major role in controlling many biological processes in different cell types. Src family kinases (SFKs) are one of the most studied groups of tyrosine kinases and can mediate a variety of signalling pathways. However, little is known about the expression of SFKs in human term placenta and their implication in trophoblast differentiation. Therefore, we examined the expression profile of SFK members over time in culture and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultured in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that resemble phenotypically mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). In this study, we showed that trophoblasts expressed all SFK members and some of them are expressed as different splice variants. Moreover, using real-time PCR, this study showed two different expression profiles of SFKs in human trophoblasts during culture. In addition, the protein level and phosphorylation status of Src were evaluated using specific antibodies. Src was rapidly phosphorylated at Tyr-416 and dephosphorylated at Tyr-527 after FBS addition. Surprisingly, inhibition of SFKs by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) or herbimycin A had different effects on trophoblast differentiation. While herbimycin A inhibited morphological and hormonal differentiation, PP2 stimulated hormonal differentiation and inhibited cell adhesion and spreading with no effect on cell fusion. In summary, this study showed that SFKs play different roles in trophoblast differentiation, probably depending on SFK members activated. Thus, this study increases our knowledge and understanding of pathology related to impaired trophoblast differentiation such as pre-eclampsia and trophoblast neoplasm.
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Introduction
Tyrosine phosphorylation is a major mechanism controlling many biological process including cell differentiation, shape, proliferation, migration and survival (Hunter, 1998). Protein tyrosine kinases (PTKs) involved in mediating these responses include receptor and non-receptor PTKs. The receptor PTKs share a similar structure consisting of an extracellular binding domain, a hydrophobic transmembrane domain, and an intracellular domain containing regulatory and catalytic sites (Wilks, 1993; Shawver et al. 1995). The non-receptor PTKs transmit signal from membrane proteins and are intermediate conductors of multiple intracellular signalling pathways. Many of them are associated with transmembrane receptors, such as hormone receptors, cytokines and growth factor receptors, and are activated by means of association of receptors with extracellular ligands or cell adhesion components (Bolen, 1993; Taniguchi, 1995). One family of non-receptor PTKs capable of communicating with a large number of different receptors is the Src family kinase (SFK) group (for review see Thomas & Brugge, 1997).
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In 1911, a pathologist, named Peyton Rous, isolated a virus that could induce sarcoma, a form of cancer, in chickens (Rous, 1911). In the middle of the 1970s, the first PTK was identified as the transforming protein (the viral Src, v-Src) of the oncogenic retrovirus, Rous sarcoma virus (RSV) (Brugge & Erikson, 1977; Purchio et al. 1978). V-Src is a mutant variant of a cellular protein (c-Src) ubiquitously expressed and highly conserved through evolution (Stehelin et al. 1976; Brown & Cooper, 1996). These two genes, v-Src and c-Src, were ultimately shown to display some differences in their C-terminal sequences. Shortly thereafter, it was determined that proteins encoded by these genes had protein tyrosine kinase activity (Collett & Erikson, 1978; Levinson et al. 1978; Hunter & Sefton, 1980), and ultimately that v-Src showed increased (uninhibited) tyrosine kinase activity (Brown & Cooper, 1996). SFKs consist of nine proteins, Src, Fyn, Fgr, Lck, Lyn, Hck, Blk, Yes and Yrk. Their molecular weights vary between 52 and 62 kDa and they have a common structure consisting of six domains. These domains are, from the N- to the C-terminus: (i) the SH4 domain or N-terminal membrane-anchoring domain responsible for recruiting SFKs to the membrane; (ii) the unique domain that is distinct for each member; (iii) the SH3 domain which binds proline-rich sequences; (iv) the SH2 domain which binds to short amino acid sequences containing phosphotyrosine (SH2 and SH3 are important for intra- as well as intermolecular interactions that regulate Src catalytic activity, Src localization and recruitment of substrates); (v) the catalytic domain containing an autophosphorylation site at Tyr-416 which is important for the regulation of kinase activity; and finally (vi) a short C-terminal domain containing a negative regulatory tyrosine residue, Tyr-527 (corresponding to Tyr-530 in the human; for review see Brown & Cooper, 1996; Thomas & Brugge, 1997).
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SFKs mediate a variety of signalling pathways (Schwartzberg, 1998). Their implication has been reported in a multitude of intracellular signalling pathways, including responses to UV irradiation and regulation of -adrenergic signalling in response to ethanol consumption (Kabuyama et al. 2002; Ma & Huang, 2002; Cowen et al. 2003). Moreover, they have been implicated in responses to cytokines, growth factors, regulators of apoptosis, adhesive stimulation–integrin signalling and G-protein-coupled receptors (Lowell et al. 1996; Chan et al. 1998; Lowell & Berton, 1999; Gardai et al. 2002; Nijhuis et al. 2002; Rane & Reddy, 2002). Furthermore, the implication of SFKs in the differentiation process of several cell types has been reported. In most cell types, v-Src expression blocked cell differentiation. For example, infection of avian myoblasts, retinoblasts, or chondroblasts with RSV maintained these cells in a proliferative state and blocked differentiation into myotubes, neuroretinal cells, epidermal cells, or chondrocytes, respectively (Muto et al. 1977; Yoshimura et al. 1981; Crisanti-Combes et al. 1982; Alema & Tato, 1987). Kaabeche et al. (2004) showed that degradation of Fyn and Lyn, induced by constitutive fibroblast growth factor receptor-2 activation, supported osteoblast differentiation. In contrast, introduction of v-Src into PC12 cells or immature neurones induced neurite outgrowth and terminal differentiation into neurone-like cells (Alema et al. 1985; Haltmeier & Rohrer, 1990; Hecker et al. 1991). Furthermore, c-Src was implicated in human trophoblast differentiation (Rebut-Bonneton et al. 1993), while Src, Yes and Lyn were activated during rat trophoblast giant cell differentiation (Kamei et al. 1997). Each of the three SFK members exhibited a distinct activation pattern during the transition from proliferation to differentiation in trophoblast cells. Src and Yes were active in proliferating and differentiating trophoblast cells, while Lyn was activated only in differentiating trophoblast giant cells and showed a differentiation-dependent accumulation (Kamei et al. 1997). These results show that the role of SFKs in cell differentiation depends on cell type and expressed or activated SFK member. However, actually, little is known about the expression profile and implication of SFKs in human trophoblast differentiation.
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Human trophoblast differentiation is characterized by fusion and differentiation of cytotrophoblast cells into syncytiotrophoblasts (Midgley et al. 1963). 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).
, http://www.100md.com The aim of the present study was to investigate the expression profile and the role of SFK members in human trophoblast differentiation. Thus, mRNA levels of SFK members were evaluated during the differentiation process of trophoblasts isolated from human term placentas. Moreover, using specific pharmacological inhibitors, our results demonstrate that SFKs play a central role in human trophoblast differentiation.
Methods
Materials
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Hanks' balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (high glucose) (DMEM-HG), trypsin, DNase, Percoll, propidium iodide (PI) and anti-desmosome mouse monoclonal antibody were from Sigma (Oakville, ON, Canada). Calf serum and penicillin–streptomycin–neomycin antibiotic mixture (PSN) were purchased from Invitrogen (Burlington, ON, Canada). SFK inhibitor PP2 and its negative control PP3, genistein and herbimycin A were all purchased from Calbiochem (San Diego, CA, USA). FBS and the ELISA kit for hCG assay were from Medicorp (Montreal, QC, Canada) while the ELISA kit for human placental lactogen (hPL) was from DRG international (Mountainside, NJ, USA). CytoTox 96 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, Nonidet-40, random primers poly(dT), acrylamide, restriction enzymes and anti-cytokeratin 18 neo-epitope (clone M30) were from Roche Applied Science (Laval, QC, Canada). Bicinchoninic acid (BCA) reagent was purchased from Pierce (Brockville, ON, Canada). NucleoSpin RNA II kit for RNA isolation and DNA digestion was from Machery-Nagel (Easton, PA, USA). Omniscript RT, Taq PCR Core and QuantiTect SYBR Green PCR kits were from Qiagen (Mississauga, ON, Canada). Src antibody sampler kit (no. 9935) containing anti-phospho-Src (Tyr-416 or Tyr-527), anti-non-phospho-Src (Tyr-416 or Tyr-527), goat anti-rabbit-IgG and goat anti-mouse-IgG conjugated with horseradish peroxidase was purchased from Cell Signaling (Beverly, MA, USA). Anti-Src rabbit polyclonal antibody (sc-18) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) mouse monoclonal antibody was from Chemicon International (Temecula, CA, USA). Alexa Fluor 488 goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR, 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). The thermal cycler GeneAmp PCR system 2400 was from Perkin Elmer (Markham, ON, Canada), the light cycler (LightCycler System) for quantitative PCR was from Roche Applied Science (Laval, QC, Canada). All other products were from Sigma.
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Cell cultures
The study was approved by the ethical committee of St Luc Hospital of the Centre Hospitalier Universitaire de Montréal (Montréal, QC, Canada) and of Université du Québec à Montréal (Montréal, QC, Canada). Primary human cytotrophoblast cells were prepared from human placentas, obtained from uncomplicated term pregnancies (37–41 weeks). Trophoblasts were isolated using the trypsin-DNase/Percoll method as described by Kliman et al. (1986), with minor modifications (Daoud et al. 2005a,b). Following the isolation of trophoblasts, cells were seeded at a density of approximately 1.5 x 106 cells per well in a 24-well plate or 4.5 x 106 cells per dish in 35 mm dishes, and maintained in DMEM-HG containing 10% FBS, 2 mM glutamine, 25 mM Hepes and PSN. The medium was refreshed daily. Where indicated, cells were kept in 10% FBS overnight to allow attachment and thereafter, were preincubated with PP2 (1, 5, 10 and 20 μM), PP3 (10 μM), herbimycin A (1 μM; see Rebut-Bonneton et al. 1993; Watanabe et al. 2004) and genistein (100 μM; see Watanabe et al. 2004) dissolved in DMSO (< 0.1%) or vehicle alone for 2 h in culture medium (serum free). Following that, 10% FBS was added. These treatments were repeated every day for 4 days and culture media were collected every day for hCG and hPL measurement.
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Secretion of hCG and hPL by trophoblasts
We previously reported that hCG and hPL secretion by primary culture of trophoblasts increased during culture, reaching a maximal value on day 4 and declining thereafter (Daoud et al. 2005a,b). Therefore, trophoblast cells were cultured in 24-well plates for 4 days. For the daily follow-up of hormone release, culture media were collected from control and treated cells as mentioned above, centrifuged and supernatants frozen at –20°C until analysis. The hCG and hPL contents in culture media were evaluated by ELISA following the manufacturer's instructions.
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RNA isolation, reverse transcription, PCR and real-time PCR amplification
Total RNA was isolated from trophoblast cells (day 1 to day 6) and treated with DNase I using the NucleoSpin RNA II kit according to the manufacturer's instructions. After RNA isolation, 2 μg was reverse transcribed into cDNA at 37°C for 1 h using 10 μM random poly(dT) and 4 U of Omniscript reverse transcriptase (Omniscript RT kit) in a 20 μl final volume. For expression screening, PCR reactions were performed in the presence of 0.2 μM dNTP, 0.5 μM of each primer (Table 1) and 2.5 U Taq DNA polymerase (Taq PCR core kit). The real-time PCR reactions were performed in presence of 0.5 μM of each primer (Table 2) according to the manufacturer's instructions (QuantiTect SYBR Green PCR). The specificity of real-time PCR products was evaluated by restriction enzymes according to manufacturer's instructions. In all PCR reactions, a negative control corresponding to RT reaction without the reverse transcriptase enzyme and a blank sample were carried out and showed no PCR product amplification (data not shown). All primer sequences were generated using LightCycler Probe Design Software 2.0 (Roche) and checked for specificity using BLAST analysis. Amplification of the d-glucose-6-phosphate dehydrogenase (G-6PDH) cDNA was used as an internal control to quantify the expression of a given gene in real-time PCRs. For quantification studies, dissociation curves were run on all reactions to ensure amplification of a single product with the appropriate melting temperature (data not shown) and to be used in quantification with the RelQuant software using coefficient files (relative quantification software, version 1.01, Roche). Amplicons were visualized by electrophoresis in a 2% agarose gel, stained with ethidium bromide and photographed under a UV transilluminator.
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Effect of FBS on Src activation in human primary culture of trophoblasts
After the isolation of trophoblasts, 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 were serum-starved overnight. On day 1, cells were preincubated with PP2 or PP3 (10 μM) for 2 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. The lysates were subjected to 12% polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane and blotted with specific antibodies for phosphorylated Src at Tyr-416 or Tyr-527. Blots were then stripped and reprobed using specific antibodies directed against non-phosphorylated Src at Tyr-416 or Tyr-527, respectively, and against total Src.
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Cell lysate preparation
For the daily follow-up of Src protein expression (day 1 to day 6), total trophoblast protein was isolated. Briefly, the medium 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 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μM leupeptin, 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. The protein concentration of the supernatant was determined by spectrophotometric quantification using the BCA reagent with BSA as standard. For Src phosphorylation status (Tyr-416 and Tyr-527), 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 dithiothreitol, 0.25 M sucrose, 0.01 M EDTA-Na2 and 0.075% bromophenol blue) and heated at 95°C for 5 min to denature the proteins. The lysates were resolved in 12% SDS-PAGE and the 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. The membranes were then incubated with the appropriate primary antibody (1/1000 for anti-phospho-Src (Tyr-416), 1/2000 for anti-phospho-Src (Tyr-527), anti-non-phospho-Src (Tyr-416 or Tyr-527) and for anti-Src (total) 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 (1/2500 for anti-rabbit IgG or 1/3000 for anti-mouse 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 a stripping solution containing 25 mM glycine-HCl (pH 2) and 1% SDS at room temperature for 25 min, rinsed twice with PBS (10 mM sodium phosphate (pH 7.2) and 0.9% NaCl) 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 anti-desmosomal antibody as previously described (Daoud et al. 2005a) or with anti-cytokeratin 18 neo-epitope (clone M30) antibodies according to the manufacturer's instructions. Briefly, cells were fixed in methanol at –20°C for 20 min and incubated in PBS containing 2% FBS (v/v) for 45 min to eliminate non-specific binding. Then the cells were rinsed with PBS and incubated in the presence of mouse anti-human anti-desmosomes monoclonal antibody (1/800) or anti-cytokeratin 18 neo-epitope (M30) 1/50 in PBS containing 0.2% BSA for 1 h at room temperature, washed 3 times with PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG (1/1000) for 1 h at room temperature in the dark. For nuclear staining, cells were incubated with PI (50 μg ml–1) for an additional 30 min at room temperature in the dark, washed 3 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 x10 and x40 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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Evaluation of apoptosis and measurement of LDH activity after PP2, herbimycin A and genistein treatments
In order to evaluate cell toxicity, total LDH present in the medium and the cells was measured as previously described (Daoud et al. 2005a,b). Briefly, 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. LDH in the cells and culture medium was measured using a specific kit according to the manufacturer's instructions. For apoptosis evaluation, cells were stained with anti-cytokeratin 18 neo-epitope (M30), an antibody largely used for apoptosis detection in trophoblast cells (Kadyrov et al. 2001, 2003; Reister et al. 2001; Humphrey et al. 2005). Quantification was performed by counting the number of M30-positive cells and nuclei per random field. At least three fields were counted per well. Results were presented as M30-positive cells per 100 nuclei.
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Statistical analyses
Data were expressed as the mean ±S.D. for real-time PCR and protein quantification analysis and as the mean ±S.E.M. for hormonal secretion, and analysed with a one-way ANOVA followed by Tukey's test, two-way ANOVA or t test at a P < 0.05 level of significance, as described.
Results
Trophoblast differentiation
Freshly isolated cytotrophoblast cells differentiated into syncytiotrophoblasts after 4 days of culture, and this phenomenon is characterized by morphological and hormonal differentiation (Midgley et al. 1963; Kliman et al. 1986). We previously reported that hCG and hPL secretion by primary culture of trophoblasts increased during culture, reaching a maximal value on day 4 and declining thereafter (Daoud et al. 2005a,b). Moreover, we showed that most trophoblasts were present as mononucleated cytotrophoblast cells on day 1 while on day 4, more abundant and larger syncytia were observed (Daoud et al. 2005a). In this study, Fig. 1 shows that mRNA expression of -hCG increases with days of culture to reach a maximal value on days 3–4 in accordance with protein secretion in the culture medium as mentioned above (Daoud et al. 2005a,b).
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Primary human trophoblast cells were cultured in the presence of 10% FBS from day 1 to day 6. Two-step real-time PCR was performed on total RNA of trophoblast cells using specific primers for -hCG gene. Data are expressed as relative hCG expression ±S.D. compared to day 4 (100%) of four cell preparations from four different placentas at 37–41 weeks of pregnancy (P < 0.0001, one-way ANOVA; P < 0.05, P < 0.001 compared to day 1 of culture, Tukey's test).
Expression of SFKs in human trophoblast cells
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Several SFK proteins are expressed as two proteins due to alternative splicing patterns or the use of alternative start codons. In order to identify the expression pattern of the SFK and to discriminate between splice variant expression for the same member in trophoblast cells, specific primers (Table 1) were used in RT-PCR. Total RNA used in RT reactions was isolated from cytotrophoblast cells after 24 h of culture. Figure 2A shows the expression of Blk in cytotrophoblast cells. Moreover, Picard et al. (2002) showed that Fyn is alternatively spliced to contain a unique seventh exon, encoding the linker region between the SH2 domain and the kinase domain and that this exon is different in T-cells versus neurones. Using specific primers (named Fyn) surrounding this seventh exon, and depending on the isoforms expressed, amplicon size will differ as mentioned in Table 1. If Fyn-c (NM_153048) lacking exon 7 is expressed, the amplicon size will be 210 bp, whereas if Fyn-b or Fyn-a is expressed, the amplicon size will be 366 or 374 bp, respectively. Using Fyn primers, Fig. 2A shows no expression for Fyn-c (210 bp) while a single band around 366 or 374 was detected. This band could represent the expression of Fyn-a or Fyn-b. Therefore, nested PCR was performed with a new sense primer specific for Fyn-b using the amplicon obtained with the Fyn primers showed in Fig. 2A. Figure 2B shows a single band corresponding to Fyn-b and similar results were obtained when total RNA was used. Moreover, using a specific sense primer localized in the untranslated region of Fyn-a, the expression of Fyn-a was detected in cytotrophoblast cells (Fig. 2A). Figure 2C shows the expression of Fgr, Hck, Lck and two isoforms of Lyn in cytotrophoblast cells. Yi et al. (1991) showed that Lyn is alternatively spliced and generates two isoforms that are co-expressed together; one of them contains an extra 31 amino acids in the N-terminal unique domain. Finally, Fig. 2D shows the expression of Src-1, Src-2 and Yes-1 in cytotrophoblast cells, while Yes-2, a Yes-1 pseudogene, is absent. All amplicons amplified in RT-PCR matched the expected size. The same results were obtained using another set of primers designed to amplify another region of mRNA for all transcripts mentioned above and using total RNA isolated from syncytiotrophoblasts after 4 days of culture (data not shown).
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Primary human trophoblast cells were cultured for 24 h in the presence of 10% FBS. RNA was extracted from the cells and assayed for expression of SFKs as described in Methods. Representative results of experiments done on 3 different cell preparations from 3 different placentas. A, expression of Blk and Fyn. B, nested PCR was performed with amplicon of Fyn in Fig. 2A using antisense primer of Fyn and sense primer specific for Fyn-b. C, expression of Fgr, Hck, Lck and Lyn. D, expression of Src1, Src2 and Yes-1.
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Day-by-day gene expression profile of SFKs in primary culture of trophoblasts
The expression profile of SFKs in the primary culture of trophoblasts was evaluated by real-time PCR using total RNA isolated from day 1 to day 6 of culture. The real-time PCR amplified a single amplicon using specific primers (Table 2) with the expected size. For the SFK members expressed as multiple splice variants (Fyn, Src, Lyn, Fig. 2), primers were designed in order to amplify a common region for all splice variants. Figures 3 and 4 show that the expression profile is modified depending on which isoform is studied. The expression of Fyn, Hck and Lyn is unchanged during culture (Fig. 3), while the expression of Fgr, Lck, Src and Yes increases during culture to reach a maximum on day 6 for Fgr, Lck and Src, while for Yes, the maximum is obtained on day 3 and forms a plateau thereafter (Fig. 4). The expression level of Blk was very low and almost undetectable by real-time PCR, especially in dissociation curves (data not shown). The specificity of all real-time PCR products was verified with restriction enzymes and can be found in Fig. 1 of the Supplemental material.
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Primary human trophoblast cells were cultured in the presence of 10% FBS from day 1 to day 6. Two-step real-time PCR was performed on total RNA of trophoblast cells using specific primers for each gene as described in Methods. A, relative expression of Fyn. B, relative expression of Hck. C, relative expression of Lyn. Data are expressed as relative expression ±S.D. compared to day 4 (100%) of 4 cell preparations from 4 different placentas at 37–41 weeks of pregnancy. Statistical analysis revealed no difference in Fyn, Hck and Lyn expression during culture.
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Primary human trophoblast cells were cultured in the presence of 10% FBS from day 1 to day 6. Two-step real-time PCR was performed on total RNA of trophoblast cells using specific primers for each gene as described in Methods. Data are expressed as relative expression ±S.D. compared to day 4 (100%) of 4 cell preparations from 4 different placentas at 37–41 weeks of pregnancy. A, relative expression of Fgr (P < 0.0001, one-way ANOVA; P < 0.01 compared to day 1 of culture, Tukey's test). B, relative expression of Lck (P < 0.0001, one-way ANOVA; P < 0.05, P < 0.001 compared to day 1 of culture, Tukey's test). C, relative expression of Src (P < 0.0001, one-way ANOVA; P < 0.05, P < 0.001 compared to day 1 of culture, Tukey's test). D, relative expression of Yes (P < 0.01, one-way ANOVA; P < 0.05, P < 0.01 compared to day 1 of culture, Tukey's test).
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Protein expression of Src during the differentiation of trophoblasts in culture
The protein expression of Src was evaluated by Western blot using total protein isolated from the trophoblasts. Figure 5A shows representative Western blots of Src from trophoblasts cultured from day 1 to day 6. Following normalization with the GAPDH protein level, our results show an increase in the expression of Src with days of culture to reach a maximum on day 6 (Fig. 5B) in accordance with its respective mRNA level (Fig. 4).
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A, Western blot analysis was performed using Src or GAPDH antibody on total proteins (30 μg) isolated from primary culture of trophoblasts cultured from day 1 to day 6. Blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of Src protein level after normalization with GAPDH protein level. Data are expressed as relative expression ±S.D. compared to day 4 (100%). Independent experiments were performed on cells isolated from 6 different placentas (P < 0.0001, one-way ANOVA; P < 0.001 compared to day 1 of culture, Tukey's test).
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Effect of PP2 and PP3 on the differentiation process of trophoblast primary culture
In order to investigate the role of SFKs on trophoblast differentiation, cells were treated with PP2, a specific SFK inhibitor (Hanke et al. 1996; Salazar & Rozengurt, 1999; Obara et al. 2004) from day 1 to day 4, as described in Methods. PP2 titration experiments in primary culture of trophoblasts showed an increase in hCG (Fig. 6A) and hPL (Fig. 6B) secretion, in a dose-dependent manner. Following 5, 10 or 20 μM PP2 treatments, hCG secretion significantly increased by 176, 330 and 294%, respectively (Fig. 6A). In the same way, hPL secretion increased by 297, 459 and 427% after 5, 10 or 20 μM PP2 treatments (Fig. 6B). However, treatment with 1 μM PP2 had no effect on hCG or hPL secretion. Therefore, subsequent experiments were performed using 10 μM PP2. Thereafter, cells were treated with 10 μM PP2, 10 μM PP3 or control medium for 4 days of culture. Figure 7A and B shows that the presence of PP2 in the culture medium during each day of culture increased hCG and hPL secretion, respectively, while PP3 had no effect. In the same way, mRNA expression of hCG and hPL increased after PP2 treatment. These results can be found in Fig. 2 of the Supplemental material. Moreover, morphological differentiation was evaluated by syncytium formation. Surprisingly, PP2 had no effect on cell fusion (Fig. 7C, 40x). Syncytium formation is similar in control or PP2-treated cells. It should be noted that cells treated with PP2 showed less adhesion and spreading capacity compared to control as shown by more empty space in the well (Fig. 7C, 10x). Cells treated with the vehicle alone showed no effect on hCG or hPL secretion nor morphological differentiation (data not shown).
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Cells were treated from day 1 to day 4 with different concentrations of PP2 (1, 5, 10 and 20 μM) or complete control medium (CTL) as described in Methods. Supernatants from day 4 were then assayed for hCG (A) or hPL (B) secretion. Data are expressed as relative secretion of hCG or hPL ±S.E.M. compared to complete control medium (CTL) hCG or hPL secretion on day 4 of culture from 3 different placentas (P < 0.05, P < 0.01; treated compared to control, t test).
Cells were treated from day 1 to day 4 with 10 μM PP2, 10 μM PP3 or complete culture medium (CTL). Data are expressed as the relative secretion of hCG (A) or hPL (B) ±S.E.M. compared to hCG or hPL secretion of complete culture medium (CTL) on day 4 of culture. Independent experiments were performed on cells isolated from 6 different placentas (P < 0.001; PP2 compared to control for hCG and hPL secretion; two-way ANOVA) (P < 0.05; P < 0.01; PP2 compared to CTL for the same day of culture; t test). C, cells were treated from day 1 to day 4 with 10 μM PP2 or complete culture medium (CTL). On day 4, cells were fixed and stained with propidium iodide (PI) for nuclei (red) and with a specific antibody for plasma membrane desmosomes (green) and observed by confocal microscopy at x10 or x40 magnification (scale bars, 100 μm). Independent experiments were performed on cells isolated from 3 different placentas.
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Effect of FBS and PP2 on Src activation
In Src, two tyrosine residues are present, Tyr-416 in the kinase domain and Tyr-527 in the C-terminal tail (the numbering is based on chicken Src), whose phosphorylation is important in regulating kinase activity. Autophosphorylation at Tyr-416 leads to increased kinase activity, whereas phosphorylation of Tyr-527 represses kinase activity. Conversely, dephosphorylation of Tyr-527 can activate Src (Brown & Cooper, 1996; Abram & Courtneidge, 2000). Thus, following the isolation of cells, trophoblasts were incubated for 5 h in the presence of 10% FBS, and 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 induces a rapid activation of Src (Tyr-416) after 2 min that increases up to 30 min (Fig. 8A (i)). Moreover, using specific antibody that recognizes non-phosphorylated Tyr-416, our results show a decrease of the non-phosphorylated Tyr-416 in accordance with the activation status (Fig. 8A (ii)). In the same way, phosphorylation status at Tyr-527 was also evaluated after FBS addition. Figure 8B (i) shows a decrease in Tyr-527 phosphorylation after 20 and 30 min in accordance with the non-phosphorylated Tyr-527 which increases after 10 min to reach a maximum after 30 min (Fig. 8B (ii)).
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Following cytotrophoblast cells isolation, cells were kept in complete culture media for 5 h and serum-starved overnight. After stimulation with FBS, cell lysates were prepared and subjected to 12% SDS-PAGE. Where indicated, cells were preincubated with PP2 (10 μM) for 2 h. Activated Src was detected by Western blot using specific antibodies for phospho- and non-phospho-Src at Tyr-416 or Tyr-527. First, blots were probed with antiphospho-Src at Tyr-416 or Tyr-527, then stripped and reprobed using an anti-non-phospho-Src at Tyr-416 or Tyr-527, respectively. Finally, blots were stripped one more time and probed using anti-Src (total) antibody. A, Src phosphorylation status at Tyr-416 after FBS stimulation. Cells were stimulated with 10% FBS for indicated time. B, Src phosphorylation status at Tyr-527 after FBS stimulation. Cells were stimulated with 10% FBS for indicated time. Independent experiments were performed on cells isolated from 3 different placentas.
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In order to evaluate the effect of PP2 on Src activation, cells were preincubated for 2 h with PP2 (10 μM) prior to FBS addition. As shown in Fig. 8A (iv), PP2 inhibits Src phosphorylation at Tyr-416 in accordance with non-phosphorylated Tyr-416 (Fig. 8A (v)). In the same way, Tyr-527 phosphorylation was evaluated after PP2 preincubation. As shown in Fig. 8B (iv), PP2 inhibits the dephosphorylation of Src at Tyr-527 and indeed increases its phosphorylation. PP3 does not change the effect of FBS on the phosphorylation status of Src (data not shown).
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Effect of genistein and herbimycin A on the differentiation process of trophoblast primary culture
It was reported that PP2 and herbimycin A inhibit Src activation using two different mechanisms (Yan et al. 1999; Sunohara et al. 2001; Franch et al. 2002). Therefore, herbimycin A and genistein, a broad range PTK inhibitor, were used to compare their effects with those of PP2. In contrast to PP2, herbimycin A and genistein inhibited hCG and hPL secretion by trophoblasts (Fig. 9A and B). Following herbimycin A treatment, hCG and hPL secretion are significantly reduced by 96 and 85%, respectively, on day 4. In the same way, genistein inhibited hCG and hPL secretion by 70 and 60%, respectively, on day 4. To substantiate the effect of the two inhibitors on trophoblast differentiation, morphological differentiation was evaluated by syncytium formation. As shown in Fig. 9C, cells treated with herbimycin A or genistein for 4 days were present as mononucleated cytotrophoblast cells, while the control cells consisted of abundant and large syncytia. Taken together, the results with PP2 (Figs 6 and 7), herbimycin A and genistein (Fig. 9) showed that SFKs play multiple roles in trophoblast differentiation, probably depending on the isoform activated.
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Cells were treated from day 1 to day 4 with 100 μM genistein, 1 μM herbimycin A or complete culture medium (CTL). Data are expressed as the relative secretion of hCG (A) or hPL (B) ±S.E.M. compared to hCG or hPL secretion on day 4 of culture. Independent experiments were performed on cells isolated from 3 different placentas (P < 0.01 and P < 0.05; genistein compared to control for hCG and hPL secretion, respectively; P < 0.001; herbimycin A compared to control for hCG and hPL secretion; two-way ANOVA) (P < 0.05; P < 0.001; genistein or herbimycin A compared to CTL for the same day of culture; t test). C, cells were treated from day 1 to day 4 with 100 μM genistein, 1 μM herbimycin A or complete culture medium (CTL). On 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 bars, 100 μm). Independent experiments were performed on cells isolated from 3 different placentas.
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Evaluation of apoptosis and measurement of LDH activity in treated cells
We studied the cytotoxicity effect of PP2, PP3, genistein and herbimycin A by measuring LDH activity in cells and culture medium. As shown in Table 3, no differences were observed between control and treated cells, demonstrating the non-toxicity of treatments. In the same way, the effect of PP2, herbimycin A and genistein on apoptosis was measured by immunocytochemistry using M30 antibody. As shown in Table 4, no differences were observed between control and treated cells. The immunostaining with M30 antibody can be found in Fig. 3 of the Supplemental material.
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Discussion
In the present study, we demonstrate for the first time the expression profile of SFK members during the culture of primary trophoblasts from human term placenta and their implication in the differentiation process. Moreover, we show that c-Src is rapidly activated upon addition of FBS and this activation increases up to 30 min. Finally, using different pharmacological inhibitors, we show that SFK members play different roles in trophoblast differentiation.
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Regarding trophoblast differentiation, we previously reported an increase in hCG (Daoud et al. 2005a,b) and hPL (Daoud et al. 2005a) secretion. Moreover, we showed that cytotrophoblast cells differentiated morphologically into syncytiotrophoblasts after 4 days of culture (Daoud et al. 2005a). In the present study, we show that mRNA expression of hCG is modest during the first 24 h of culture, suggesting that isolated cytotrophoblast cells are highly purified, without syncytial fragments. Moreover, we show that mRNA transcription and protein synthesis of hCG match perfectly with the secretion pattern. Therefore, hormonal and morphological statuses of primary cultures of trophoblasts were used to evaluate trophoblast differentiation.
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The SFK can be divided into two groups based on their general pattern of expression. Src, Fyn and Yes are ubiquitously expressed; however, some SFKs are highly expressed in certain cell types and as alternatively spliced in other specific cell types (Thomas & Brugge, 1997). The second group of SFKs, Blk, Fgr, Hck, Lck and Lyn, are primarily found in haematopoietic cells (Bolen & Brugge, 1997). However, Hck, Fgr and Lyn were also detected in neurones (Lowell, 2004), suggesting that these kinases may be expressed in many additional cell types. Moreover, Src is expressed as a different isoform in brain containing six additional amino acids between exons 3 and 4, but not expressed in lung, liver or placenta (Pyper & Bolen, 1989). It was reported that primary cultures of trophoblasts expressed Src and that its expression profile was unchanged during culture up to day 4 (Rebut-Bonneton et al. 1993). Moreover, multiple genes are expressed differentially in trophoblast cells with time in culture. In many previous studies, we reported the differential expression of mitogen-activated protein kinases (MAPKs: ERK1/2 and p38) (Daoud et al. 2005a), calcium channels (Moreau et al. 2002a,b), adenylyl cyclases (Bernatchez et al. 2003), cytosolic fatty acid binding protein and peroxysome proliferator-activated receptor (Daoud et al. 2005b) with days of culture of primary trophoblasts. Moreover, Getsios et al. (2001) reported a differential expression of -, - and -catenins during trophoblast differentiation. These observations prompted us to evaluate the expression level of SFK members in trophoblasts isolated from human term placenta on different days of culture and thereafter, verify their implication in trophoblast differentiation. In our present study, we show that trophoblasts express all members of SFK and some of them are expressed as different splice variants (Fyn, Lyn, Src). To our knowledge, we are the first to report mRNA expression of all SFK members in human trophoblasts and the mRNA expression of all SFK members in the same cell type. Moreover, SFKs show two expression profiles as cytotrophoblast cells differentiate into syncytiotrophoblasts. Fyn, Hck and Lyn show no differences in mRNA expression profile while Fgr, Lck, Src and Yes show an increase in mRNA expression during culture. However, Src protein expression was unchanged from day 1 to day 4 as previously described (Rebut-Bonneton et al. 1993) and increased thereafter. Moreover, our results show that mRNA and protein expression of Src matched completely indicating the absence of post-transcriptional regulation. Lannutti et al. (2003) demonstrated the presence of six members of SFK in megakaryocytes and showed that only Fyn is up-regulated during megakaryocyte differentiation. In the same way, Kamei et al. (1997) showed that rat trophoblasts express Src and Yes, which were activated in proliferating and differentiating trophoblast cells, and Lyn which was activated only in differentiating trophoblast giant cells and showed a differentiation-dependent accumulation (Kamei et al. 1997). Moreover, it was reported that Lck and Fyn play different roles in T cell differentiation, survival and activation (Zamoyska et al. 2003). Our results and all these observations indicate that most cells are likely to express multiple SFKs and probably each SFK accomplishes a specialized role in cell physiology.
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The SFKs control many cellular events such as adhesion and spreading, focal adhesion formation/disassembly, lamellipodia, migration, apoptosis, cell cycle progression, gene transcription and cell differentiation (Thomas & Brugge, 1997). Furthermore, many SFK members are implicated in the differentiation of different cell types. Fyn and Lck play major and different roles in T cell differentiation and maturation (Zamoyska et al. 2003). A possible role of Src in myoblasts, retinoblasts and chondrocytes differentiation was demonstrated using v-Src (Muto et al. 1977; Yoshimura et al. 1981; Crisanti-Combes et al. 1982; Alema & Tato, 1987). Fyn and Lyn were implicated in osteoclast differentiation (Kaabeche et al. 2004), while v-Src promotes PC12 differentiation into neurone-like cells (Alema et al. 1985; Haltmeier & Rohrer, 1990; Hecker et al. 1991). Therefore, we evaluated the expression of Src in trophoblasts and its activation after FBS addition, a factor known to induce trophoblast differentiation (Daoud et al. 2005a). In this study, we report a rapid and sustained activation of Src in primary culture of trophoblasts after FBS addition. Previously, Rebut-Bonneton (Rebut-Bonneton et al. 1993) showed the expression of Src in human trophoblasts and that the kinase activity of Src increased significantly on day 2, when cells were completely aggregated and started to fuse, and remained elevated up to day 4, while cells underwent further differentiation. It should be noted that Src activation depends on its conformation. Both the SH3 and SH2 domains lie on the side of the kinase domain opposite to the catalytic cleft. The SH3 and SH2 domains repress the kinase activity by interacting with amino acids within the catalytic domain, as well as with residues N-terminal and C-terminal, respectively (Brown & Cooper, 1996; Thomas & Brugge, 1997). Dephosphorylation of Tyr-527, which is the first site of phosphorylation in vivo, induces a change in Src conformation and increases its activity by autophosphorylation at Tyr-416 in the catalytic domain (Brown & Cooper, 1996; Thomas & Brugge, 1997). Conversely, phosphorylation of Tyr-527 by Csk (C-terminal Src kinase) inhibits Src activation (Nada et al. 1991). In our study, FBS induced a rapid phosphorylation at Tyr-416 after 2 min which was accompanied by a dephosphorylation at Tyr-527 after 20 min. This delay between Tyr-527 dephosphorylation and Tyr-416 phosphorylation could be explained by the sensitivity of antibodies used or by the activation of Src independently of Tyr-527. Some SFKs are not always phosphorylated at this negative regulatory tyrosine, yet remain relatively inactive. For example, in B cells, tyrosine phosphorylation of the C-terminal tail of Lyn is barely detectable even though the catalytic activity of Lyn is not elevated. However, loss of Csk results in activation of Lyn (Nada et al. 1993; Hata et al. 1994).
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To further demonstrate the implication of SFKs in trophoblast differentiation, we used two SFK inhibitors (PP2 and herbimycin A) and a broad range tyrosine kinase inhibitor (genistein). Surprisingly, PP2 and herbimycin A showed different effects on trophoblast differentiation. Herbimycin A and genistein showed hormonal and morphological inhibition of trophoblast differentiation, while PP2 showed an induced hormonal differentiation, inhibition of cell adhesion and spreading and no effect on cell fusion. PP2 seems to repress some inhibitory effects of some members of SFK implicated in hormonal trophoblast differentiation. Moreover, PP2 increased Src phosphorylation at Tyr-527 and inhibited Tyr-416 phosphorylation. It should be noted that SFKs play critical roles in downmodulating or inhibiting signalling pathways in immune cells; a concept that was confirmed through studies on mutant models (Lowell, 2004). Maruyama et al. (2004) showed that PP1 and PP2 promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro. Moreover, they showed that herbimycin A had no effect on the differentiation process compared to PP1 or PP2. Although herbimycin A was widely used as a tyrosine kinase inhibitor with Src selectivity, several studies demonstrate that it behaves in a manner different from PP1 and PP2 (Yan et al. 1999; Sunohara et al. 2001; Franch et al. 2002). PP1 and PP2 act as competitive inhibitors of ATP for SFK (Liu et al. 1999; Schindler et al. 1999), which, however, became controversial (Karni et al. 2003). Herbimycin A covalently interacts with sulfhydryl groups on protein tyrosine kinases (Uehara et al. 1989; Fukazawa et al. 1994; Senga et al. 2000). These differences in their inhibition mode may account for a differential selectivity and various actions of these two inhibitors as presented herein and also previously described (Yan et al. 1999; Sunohara et al. 2001; Franch et al. 2002; Maruyama et al. 2004). Thus, PP2 and herbimycin A may act on different and distinct SFK members which in turn, may play different roles in hormonal and morphological trophoblast differentiation. Further studies will be required to elucidate the molecular basis for the effect of PP2 and herbimycin A on trophoblast differentiation.
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Finally, SFKs are known to regulate downstream signalling pathways that influence nuclear events (Brown & Cooper, 1996) and can stimulate MAPKs and phosphatidylinositol 3-kinases, resulting in changes in cell survival, division, differentiation and gene transcription (Thomas & Brugge, 1997). We previously reported the implication of ERK1/2 and p38 in trophoblast differentiation (Daoud et al. 2005a). Moreover, SFKs can phosphorylate focal adhesion kinases (FAKs) which are very important for cell adhesion, spreading, migration, shape and differentiation (Thomas & Brugge, 1997). Thus, since the implication of FAKs in placental implantation, cytotrophoblast cell migration, invasion and differentiation was already reported (Shiokawa et al. 1998; Gleeson et al. 2001; Ilic et al. 2001; MacPhee et al. 2001), it would be of interest to verify if PP2 and herbimycin A act differentially on MAPKs and FAKs during trophoblast differentiation.
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In conclusion, this study demonstrated for the first time the expression profile of SFKs in human trophoblasts with days in culture, and that SFKs play multiple roles in trophoblast differentiation. Thus, this study increases our knowledge of the trophoblast differentiation process and could be very useful in the understanding of pathology relating to abnormal placental differentiation, such as pre-eclampsia and trophoblastic neoplasm.
Supplemental material
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The online version of this paper can be accessed at:
DOI: 10.1113/jphysiol.2005.102285
http://jp.physoc.org/cgi/content/full/jphysiol.2005.102285/DC1
and contains supplemental material consisting of three figures and legends entitled Figure 1A and B: Specificity of real-time PCR products shown by digestion with restriction enzymes; Figure 2: Effect of PP2 on hCG or hPL mRNA expression in human primary culture of trophoblasts; Figure 3: Effect of PP2, herbimycin A and genistein on trophoblasts apoptosis as monitored by M30 staining.
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This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com
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2 Centre de recherche Biomed, Université du Québec à Montréal, Montréal, Québec, Canada, H3C 3P8
3 Département d'Obstétrique-Gynécologie, Hpital St-Luc, Université de Montréal, Montréal, Québec, Canada, H2L 4M1
Abstract
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Tyrosine phosphorylation plays a major role in controlling many biological processes in different cell types. Src family kinases (SFKs) are one of the most studied groups of tyrosine kinases and can mediate a variety of signalling pathways. However, little is known about the expression of SFKs in human term placenta and their implication in trophoblast differentiation. Therefore, we examined the expression profile of SFK members over time in culture and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultured in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that resemble phenotypically mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). In this study, we showed that trophoblasts expressed all SFK members and some of them are expressed as different splice variants. Moreover, using real-time PCR, this study showed two different expression profiles of SFKs in human trophoblasts during culture. In addition, the protein level and phosphorylation status of Src were evaluated using specific antibodies. Src was rapidly phosphorylated at Tyr-416 and dephosphorylated at Tyr-527 after FBS addition. Surprisingly, inhibition of SFKs by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) or herbimycin A had different effects on trophoblast differentiation. While herbimycin A inhibited morphological and hormonal differentiation, PP2 stimulated hormonal differentiation and inhibited cell adhesion and spreading with no effect on cell fusion. In summary, this study showed that SFKs play different roles in trophoblast differentiation, probably depending on SFK members activated. Thus, this study increases our knowledge and understanding of pathology related to impaired trophoblast differentiation such as pre-eclampsia and trophoblast neoplasm.
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Introduction
Tyrosine phosphorylation is a major mechanism controlling many biological process including cell differentiation, shape, proliferation, migration and survival (Hunter, 1998). Protein tyrosine kinases (PTKs) involved in mediating these responses include receptor and non-receptor PTKs. The receptor PTKs share a similar structure consisting of an extracellular binding domain, a hydrophobic transmembrane domain, and an intracellular domain containing regulatory and catalytic sites (Wilks, 1993; Shawver et al. 1995). The non-receptor PTKs transmit signal from membrane proteins and are intermediate conductors of multiple intracellular signalling pathways. Many of them are associated with transmembrane receptors, such as hormone receptors, cytokines and growth factor receptors, and are activated by means of association of receptors with extracellular ligands or cell adhesion components (Bolen, 1993; Taniguchi, 1995). One family of non-receptor PTKs capable of communicating with a large number of different receptors is the Src family kinase (SFK) group (for review see Thomas & Brugge, 1997).
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In 1911, a pathologist, named Peyton Rous, isolated a virus that could induce sarcoma, a form of cancer, in chickens (Rous, 1911). In the middle of the 1970s, the first PTK was identified as the transforming protein (the viral Src, v-Src) of the oncogenic retrovirus, Rous sarcoma virus (RSV) (Brugge & Erikson, 1977; Purchio et al. 1978). V-Src is a mutant variant of a cellular protein (c-Src) ubiquitously expressed and highly conserved through evolution (Stehelin et al. 1976; Brown & Cooper, 1996). These two genes, v-Src and c-Src, were ultimately shown to display some differences in their C-terminal sequences. Shortly thereafter, it was determined that proteins encoded by these genes had protein tyrosine kinase activity (Collett & Erikson, 1978; Levinson et al. 1978; Hunter & Sefton, 1980), and ultimately that v-Src showed increased (uninhibited) tyrosine kinase activity (Brown & Cooper, 1996). SFKs consist of nine proteins, Src, Fyn, Fgr, Lck, Lyn, Hck, Blk, Yes and Yrk. Their molecular weights vary between 52 and 62 kDa and they have a common structure consisting of six domains. These domains are, from the N- to the C-terminus: (i) the SH4 domain or N-terminal membrane-anchoring domain responsible for recruiting SFKs to the membrane; (ii) the unique domain that is distinct for each member; (iii) the SH3 domain which binds proline-rich sequences; (iv) the SH2 domain which binds to short amino acid sequences containing phosphotyrosine (SH2 and SH3 are important for intra- as well as intermolecular interactions that regulate Src catalytic activity, Src localization and recruitment of substrates); (v) the catalytic domain containing an autophosphorylation site at Tyr-416 which is important for the regulation of kinase activity; and finally (vi) a short C-terminal domain containing a negative regulatory tyrosine residue, Tyr-527 (corresponding to Tyr-530 in the human; for review see Brown & Cooper, 1996; Thomas & Brugge, 1997).
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SFKs mediate a variety of signalling pathways (Schwartzberg, 1998). Their implication has been reported in a multitude of intracellular signalling pathways, including responses to UV irradiation and regulation of -adrenergic signalling in response to ethanol consumption (Kabuyama et al. 2002; Ma & Huang, 2002; Cowen et al. 2003). Moreover, they have been implicated in responses to cytokines, growth factors, regulators of apoptosis, adhesive stimulation–integrin signalling and G-protein-coupled receptors (Lowell et al. 1996; Chan et al. 1998; Lowell & Berton, 1999; Gardai et al. 2002; Nijhuis et al. 2002; Rane & Reddy, 2002). Furthermore, the implication of SFKs in the differentiation process of several cell types has been reported. In most cell types, v-Src expression blocked cell differentiation. For example, infection of avian myoblasts, retinoblasts, or chondroblasts with RSV maintained these cells in a proliferative state and blocked differentiation into myotubes, neuroretinal cells, epidermal cells, or chondrocytes, respectively (Muto et al. 1977; Yoshimura et al. 1981; Crisanti-Combes et al. 1982; Alema & Tato, 1987). Kaabeche et al. (2004) showed that degradation of Fyn and Lyn, induced by constitutive fibroblast growth factor receptor-2 activation, supported osteoblast differentiation. In contrast, introduction of v-Src into PC12 cells or immature neurones induced neurite outgrowth and terminal differentiation into neurone-like cells (Alema et al. 1985; Haltmeier & Rohrer, 1990; Hecker et al. 1991). Furthermore, c-Src was implicated in human trophoblast differentiation (Rebut-Bonneton et al. 1993), while Src, Yes and Lyn were activated during rat trophoblast giant cell differentiation (Kamei et al. 1997). Each of the three SFK members exhibited a distinct activation pattern during the transition from proliferation to differentiation in trophoblast cells. Src and Yes were active in proliferating and differentiating trophoblast cells, while Lyn was activated only in differentiating trophoblast giant cells and showed a differentiation-dependent accumulation (Kamei et al. 1997). These results show that the role of SFKs in cell differentiation depends on cell type and expressed or activated SFK member. However, actually, little is known about the expression profile and implication of SFKs in human trophoblast differentiation.
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Human trophoblast differentiation is characterized by fusion and differentiation of cytotrophoblast cells into syncytiotrophoblasts (Midgley et al. 1963). 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).
, http://www.100md.com The aim of the present study was to investigate the expression profile and the role of SFK members in human trophoblast differentiation. Thus, mRNA levels of SFK members were evaluated during the differentiation process of trophoblasts isolated from human term placentas. Moreover, using specific pharmacological inhibitors, our results demonstrate that SFKs play a central role in human trophoblast differentiation.
Methods
Materials
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Hanks' balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (high glucose) (DMEM-HG), trypsin, DNase, Percoll, propidium iodide (PI) and anti-desmosome mouse monoclonal antibody were from Sigma (Oakville, ON, Canada). Calf serum and penicillin–streptomycin–neomycin antibiotic mixture (PSN) were purchased from Invitrogen (Burlington, ON, Canada). SFK inhibitor PP2 and its negative control PP3, genistein and herbimycin A were all purchased from Calbiochem (San Diego, CA, USA). FBS and the ELISA kit for hCG assay were from Medicorp (Montreal, QC, Canada) while the ELISA kit for human placental lactogen (hPL) was from DRG international (Mountainside, NJ, USA). CytoTox 96 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, Nonidet-40, random primers poly(dT), acrylamide, restriction enzymes and anti-cytokeratin 18 neo-epitope (clone M30) were from Roche Applied Science (Laval, QC, Canada). Bicinchoninic acid (BCA) reagent was purchased from Pierce (Brockville, ON, Canada). NucleoSpin RNA II kit for RNA isolation and DNA digestion was from Machery-Nagel (Easton, PA, USA). Omniscript RT, Taq PCR Core and QuantiTect SYBR Green PCR kits were from Qiagen (Mississauga, ON, Canada). Src antibody sampler kit (no. 9935) containing anti-phospho-Src (Tyr-416 or Tyr-527), anti-non-phospho-Src (Tyr-416 or Tyr-527), goat anti-rabbit-IgG and goat anti-mouse-IgG conjugated with horseradish peroxidase was purchased from Cell Signaling (Beverly, MA, USA). Anti-Src rabbit polyclonal antibody (sc-18) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) mouse monoclonal antibody was from Chemicon International (Temecula, CA, USA). Alexa Fluor 488 goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR, 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). The thermal cycler GeneAmp PCR system 2400 was from Perkin Elmer (Markham, ON, Canada), the light cycler (LightCycler System) for quantitative PCR was from Roche Applied Science (Laval, QC, Canada). All other products were from Sigma.
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Cell cultures
The study was approved by the ethical committee of St Luc Hospital of the Centre Hospitalier Universitaire de Montréal (Montréal, QC, Canada) and of Université du Québec à Montréal (Montréal, QC, Canada). Primary human cytotrophoblast cells were prepared from human placentas, obtained from uncomplicated term pregnancies (37–41 weeks). Trophoblasts were isolated using the trypsin-DNase/Percoll method as described by Kliman et al. (1986), with minor modifications (Daoud et al. 2005a,b). Following the isolation of trophoblasts, cells were seeded at a density of approximately 1.5 x 106 cells per well in a 24-well plate or 4.5 x 106 cells per dish in 35 mm dishes, and maintained in DMEM-HG containing 10% FBS, 2 mM glutamine, 25 mM Hepes and PSN. The medium was refreshed daily. Where indicated, cells were kept in 10% FBS overnight to allow attachment and thereafter, were preincubated with PP2 (1, 5, 10 and 20 μM), PP3 (10 μM), herbimycin A (1 μM; see Rebut-Bonneton et al. 1993; Watanabe et al. 2004) and genistein (100 μM; see Watanabe et al. 2004) dissolved in DMSO (< 0.1%) or vehicle alone for 2 h in culture medium (serum free). Following that, 10% FBS was added. These treatments were repeated every day for 4 days and culture media were collected every day for hCG and hPL measurement.
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Secretion of hCG and hPL by trophoblasts
We previously reported that hCG and hPL secretion by primary culture of trophoblasts increased during culture, reaching a maximal value on day 4 and declining thereafter (Daoud et al. 2005a,b). Therefore, trophoblast cells were cultured in 24-well plates for 4 days. For the daily follow-up of hormone release, culture media were collected from control and treated cells as mentioned above, centrifuged and supernatants frozen at –20°C until analysis. The hCG and hPL contents in culture media were evaluated by ELISA following the manufacturer's instructions.
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RNA isolation, reverse transcription, PCR and real-time PCR amplification
Total RNA was isolated from trophoblast cells (day 1 to day 6) and treated with DNase I using the NucleoSpin RNA II kit according to the manufacturer's instructions. After RNA isolation, 2 μg was reverse transcribed into cDNA at 37°C for 1 h using 10 μM random poly(dT) and 4 U of Omniscript reverse transcriptase (Omniscript RT kit) in a 20 μl final volume. For expression screening, PCR reactions were performed in the presence of 0.2 μM dNTP, 0.5 μM of each primer (Table 1) and 2.5 U Taq DNA polymerase (Taq PCR core kit). The real-time PCR reactions were performed in presence of 0.5 μM of each primer (Table 2) according to the manufacturer's instructions (QuantiTect SYBR Green PCR). The specificity of real-time PCR products was evaluated by restriction enzymes according to manufacturer's instructions. In all PCR reactions, a negative control corresponding to RT reaction without the reverse transcriptase enzyme and a blank sample were carried out and showed no PCR product amplification (data not shown). All primer sequences were generated using LightCycler Probe Design Software 2.0 (Roche) and checked for specificity using BLAST analysis. Amplification of the d-glucose-6-phosphate dehydrogenase (G-6PDH) cDNA was used as an internal control to quantify the expression of a given gene in real-time PCRs. For quantification studies, dissociation curves were run on all reactions to ensure amplification of a single product with the appropriate melting temperature (data not shown) and to be used in quantification with the RelQuant software using coefficient files (relative quantification software, version 1.01, Roche). Amplicons were visualized by electrophoresis in a 2% agarose gel, stained with ethidium bromide and photographed under a UV transilluminator.
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Effect of FBS on Src activation in human primary culture of trophoblasts
After the isolation of trophoblasts, 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 were serum-starved overnight. On day 1, cells were preincubated with PP2 or PP3 (10 μM) for 2 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. The lysates were subjected to 12% polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane and blotted with specific antibodies for phosphorylated Src at Tyr-416 or Tyr-527. Blots were then stripped and reprobed using specific antibodies directed against non-phosphorylated Src at Tyr-416 or Tyr-527, respectively, and against total Src.
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Cell lysate preparation
For the daily follow-up of Src protein expression (day 1 to day 6), total trophoblast protein was isolated. Briefly, the medium 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 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μM leupeptin, 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. The protein concentration of the supernatant was determined by spectrophotometric quantification using the BCA reagent with BSA as standard. For Src phosphorylation status (Tyr-416 and Tyr-527), 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 dithiothreitol, 0.25 M sucrose, 0.01 M EDTA-Na2 and 0.075% bromophenol blue) and heated at 95°C for 5 min to denature the proteins. The lysates were resolved in 12% SDS-PAGE and the 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. The membranes were then incubated with the appropriate primary antibody (1/1000 for anti-phospho-Src (Tyr-416), 1/2000 for anti-phospho-Src (Tyr-527), anti-non-phospho-Src (Tyr-416 or Tyr-527) and for anti-Src (total) 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 (1/2500 for anti-rabbit IgG or 1/3000 for anti-mouse 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 a stripping solution containing 25 mM glycine-HCl (pH 2) and 1% SDS at room temperature for 25 min, rinsed twice with PBS (10 mM sodium phosphate (pH 7.2) and 0.9% NaCl) 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 anti-desmosomal antibody as previously described (Daoud et al. 2005a) or with anti-cytokeratin 18 neo-epitope (clone M30) antibodies according to the manufacturer's instructions. Briefly, cells were fixed in methanol at –20°C for 20 min and incubated in PBS containing 2% FBS (v/v) for 45 min to eliminate non-specific binding. Then the cells were rinsed with PBS and incubated in the presence of mouse anti-human anti-desmosomes monoclonal antibody (1/800) or anti-cytokeratin 18 neo-epitope (M30) 1/50 in PBS containing 0.2% BSA for 1 h at room temperature, washed 3 times with PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG (1/1000) for 1 h at room temperature in the dark. For nuclear staining, cells were incubated with PI (50 μg ml–1) for an additional 30 min at room temperature in the dark, washed 3 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 x10 and x40 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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Evaluation of apoptosis and measurement of LDH activity after PP2, herbimycin A and genistein treatments
In order to evaluate cell toxicity, total LDH present in the medium and the cells was measured as previously described (Daoud et al. 2005a,b). Briefly, 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. LDH in the cells and culture medium was measured using a specific kit according to the manufacturer's instructions. For apoptosis evaluation, cells were stained with anti-cytokeratin 18 neo-epitope (M30), an antibody largely used for apoptosis detection in trophoblast cells (Kadyrov et al. 2001, 2003; Reister et al. 2001; Humphrey et al. 2005). Quantification was performed by counting the number of M30-positive cells and nuclei per random field. At least three fields were counted per well. Results were presented as M30-positive cells per 100 nuclei.
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Statistical analyses
Data were expressed as the mean ±S.D. for real-time PCR and protein quantification analysis and as the mean ±S.E.M. for hormonal secretion, and analysed with a one-way ANOVA followed by Tukey's test, two-way ANOVA or t test at a P < 0.05 level of significance, as described.
Results
Trophoblast differentiation
Freshly isolated cytotrophoblast cells differentiated into syncytiotrophoblasts after 4 days of culture, and this phenomenon is characterized by morphological and hormonal differentiation (Midgley et al. 1963; Kliman et al. 1986). We previously reported that hCG and hPL secretion by primary culture of trophoblasts increased during culture, reaching a maximal value on day 4 and declining thereafter (Daoud et al. 2005a,b). Moreover, we showed that most trophoblasts were present as mononucleated cytotrophoblast cells on day 1 while on day 4, more abundant and larger syncytia were observed (Daoud et al. 2005a). In this study, Fig. 1 shows that mRNA expression of -hCG increases with days of culture to reach a maximal value on days 3–4 in accordance with protein secretion in the culture medium as mentioned above (Daoud et al. 2005a,b).
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Primary human trophoblast cells were cultured in the presence of 10% FBS from day 1 to day 6. Two-step real-time PCR was performed on total RNA of trophoblast cells using specific primers for -hCG gene. Data are expressed as relative hCG expression ±S.D. compared to day 4 (100%) of four cell preparations from four different placentas at 37–41 weeks of pregnancy (P < 0.0001, one-way ANOVA; P < 0.05, P < 0.001 compared to day 1 of culture, Tukey's test).
Expression of SFKs in human trophoblast cells
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Several SFK proteins are expressed as two proteins due to alternative splicing patterns or the use of alternative start codons. In order to identify the expression pattern of the SFK and to discriminate between splice variant expression for the same member in trophoblast cells, specific primers (Table 1) were used in RT-PCR. Total RNA used in RT reactions was isolated from cytotrophoblast cells after 24 h of culture. Figure 2A shows the expression of Blk in cytotrophoblast cells. Moreover, Picard et al. (2002) showed that Fyn is alternatively spliced to contain a unique seventh exon, encoding the linker region between the SH2 domain and the kinase domain and that this exon is different in T-cells versus neurones. Using specific primers (named Fyn) surrounding this seventh exon, and depending on the isoforms expressed, amplicon size will differ as mentioned in Table 1. If Fyn-c (NM_153048) lacking exon 7 is expressed, the amplicon size will be 210 bp, whereas if Fyn-b or Fyn-a is expressed, the amplicon size will be 366 or 374 bp, respectively. Using Fyn primers, Fig. 2A shows no expression for Fyn-c (210 bp) while a single band around 366 or 374 was detected. This band could represent the expression of Fyn-a or Fyn-b. Therefore, nested PCR was performed with a new sense primer specific for Fyn-b using the amplicon obtained with the Fyn primers showed in Fig. 2A. Figure 2B shows a single band corresponding to Fyn-b and similar results were obtained when total RNA was used. Moreover, using a specific sense primer localized in the untranslated region of Fyn-a, the expression of Fyn-a was detected in cytotrophoblast cells (Fig. 2A). Figure 2C shows the expression of Fgr, Hck, Lck and two isoforms of Lyn in cytotrophoblast cells. Yi et al. (1991) showed that Lyn is alternatively spliced and generates two isoforms that are co-expressed together; one of them contains an extra 31 amino acids in the N-terminal unique domain. Finally, Fig. 2D shows the expression of Src-1, Src-2 and Yes-1 in cytotrophoblast cells, while Yes-2, a Yes-1 pseudogene, is absent. All amplicons amplified in RT-PCR matched the expected size. The same results were obtained using another set of primers designed to amplify another region of mRNA for all transcripts mentioned above and using total RNA isolated from syncytiotrophoblasts after 4 days of culture (data not shown).
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Primary human trophoblast cells were cultured for 24 h in the presence of 10% FBS. RNA was extracted from the cells and assayed for expression of SFKs as described in Methods. Representative results of experiments done on 3 different cell preparations from 3 different placentas. A, expression of Blk and Fyn. B, nested PCR was performed with amplicon of Fyn in Fig. 2A using antisense primer of Fyn and sense primer specific for Fyn-b. C, expression of Fgr, Hck, Lck and Lyn. D, expression of Src1, Src2 and Yes-1.
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Day-by-day gene expression profile of SFKs in primary culture of trophoblasts
The expression profile of SFKs in the primary culture of trophoblasts was evaluated by real-time PCR using total RNA isolated from day 1 to day 6 of culture. The real-time PCR amplified a single amplicon using specific primers (Table 2) with the expected size. For the SFK members expressed as multiple splice variants (Fyn, Src, Lyn, Fig. 2), primers were designed in order to amplify a common region for all splice variants. Figures 3 and 4 show that the expression profile is modified depending on which isoform is studied. The expression of Fyn, Hck and Lyn is unchanged during culture (Fig. 3), while the expression of Fgr, Lck, Src and Yes increases during culture to reach a maximum on day 6 for Fgr, Lck and Src, while for Yes, the maximum is obtained on day 3 and forms a plateau thereafter (Fig. 4). The expression level of Blk was very low and almost undetectable by real-time PCR, especially in dissociation curves (data not shown). The specificity of all real-time PCR products was verified with restriction enzymes and can be found in Fig. 1 of the Supplemental material.
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Primary human trophoblast cells were cultured in the presence of 10% FBS from day 1 to day 6. Two-step real-time PCR was performed on total RNA of trophoblast cells using specific primers for each gene as described in Methods. A, relative expression of Fyn. B, relative expression of Hck. C, relative expression of Lyn. Data are expressed as relative expression ±S.D. compared to day 4 (100%) of 4 cell preparations from 4 different placentas at 37–41 weeks of pregnancy. Statistical analysis revealed no difference in Fyn, Hck and Lyn expression during culture.
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Primary human trophoblast cells were cultured in the presence of 10% FBS from day 1 to day 6. Two-step real-time PCR was performed on total RNA of trophoblast cells using specific primers for each gene as described in Methods. Data are expressed as relative expression ±S.D. compared to day 4 (100%) of 4 cell preparations from 4 different placentas at 37–41 weeks of pregnancy. A, relative expression of Fgr (P < 0.0001, one-way ANOVA; P < 0.01 compared to day 1 of culture, Tukey's test). B, relative expression of Lck (P < 0.0001, one-way ANOVA; P < 0.05, P < 0.001 compared to day 1 of culture, Tukey's test). C, relative expression of Src (P < 0.0001, one-way ANOVA; P < 0.05, P < 0.001 compared to day 1 of culture, Tukey's test). D, relative expression of Yes (P < 0.01, one-way ANOVA; P < 0.05, P < 0.01 compared to day 1 of culture, Tukey's test).
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Protein expression of Src during the differentiation of trophoblasts in culture
The protein expression of Src was evaluated by Western blot using total protein isolated from the trophoblasts. Figure 5A shows representative Western blots of Src from trophoblasts cultured from day 1 to day 6. Following normalization with the GAPDH protein level, our results show an increase in the expression of Src with days of culture to reach a maximum on day 6 (Fig. 5B) in accordance with its respective mRNA level (Fig. 4).
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A, Western blot analysis was performed using Src or GAPDH antibody on total proteins (30 μg) isolated from primary culture of trophoblasts cultured from day 1 to day 6. Blot was stripped and reprobed using an anti-GAPDH antibody. B, densitometric analysis of Src protein level after normalization with GAPDH protein level. Data are expressed as relative expression ±S.D. compared to day 4 (100%). Independent experiments were performed on cells isolated from 6 different placentas (P < 0.0001, one-way ANOVA; P < 0.001 compared to day 1 of culture, Tukey's test).
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Effect of PP2 and PP3 on the differentiation process of trophoblast primary culture
In order to investigate the role of SFKs on trophoblast differentiation, cells were treated with PP2, a specific SFK inhibitor (Hanke et al. 1996; Salazar & Rozengurt, 1999; Obara et al. 2004) from day 1 to day 4, as described in Methods. PP2 titration experiments in primary culture of trophoblasts showed an increase in hCG (Fig. 6A) and hPL (Fig. 6B) secretion, in a dose-dependent manner. Following 5, 10 or 20 μM PP2 treatments, hCG secretion significantly increased by 176, 330 and 294%, respectively (Fig. 6A). In the same way, hPL secretion increased by 297, 459 and 427% after 5, 10 or 20 μM PP2 treatments (Fig. 6B). However, treatment with 1 μM PP2 had no effect on hCG or hPL secretion. Therefore, subsequent experiments were performed using 10 μM PP2. Thereafter, cells were treated with 10 μM PP2, 10 μM PP3 or control medium for 4 days of culture. Figure 7A and B shows that the presence of PP2 in the culture medium during each day of culture increased hCG and hPL secretion, respectively, while PP3 had no effect. In the same way, mRNA expression of hCG and hPL increased after PP2 treatment. These results can be found in Fig. 2 of the Supplemental material. Moreover, morphological differentiation was evaluated by syncytium formation. Surprisingly, PP2 had no effect on cell fusion (Fig. 7C, 40x). Syncytium formation is similar in control or PP2-treated cells. It should be noted that cells treated with PP2 showed less adhesion and spreading capacity compared to control as shown by more empty space in the well (Fig. 7C, 10x). Cells treated with the vehicle alone showed no effect on hCG or hPL secretion nor morphological differentiation (data not shown).
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Cells were treated from day 1 to day 4 with different concentrations of PP2 (1, 5, 10 and 20 μM) or complete control medium (CTL) as described in Methods. Supernatants from day 4 were then assayed for hCG (A) or hPL (B) secretion. Data are expressed as relative secretion of hCG or hPL ±S.E.M. compared to complete control medium (CTL) hCG or hPL secretion on day 4 of culture from 3 different placentas (P < 0.05, P < 0.01; treated compared to control, t test).
Cells were treated from day 1 to day 4 with 10 μM PP2, 10 μM PP3 or complete culture medium (CTL). Data are expressed as the relative secretion of hCG (A) or hPL (B) ±S.E.M. compared to hCG or hPL secretion of complete culture medium (CTL) on day 4 of culture. Independent experiments were performed on cells isolated from 6 different placentas (P < 0.001; PP2 compared to control for hCG and hPL secretion; two-way ANOVA) (P < 0.05; P < 0.01; PP2 compared to CTL for the same day of culture; t test). C, cells were treated from day 1 to day 4 with 10 μM PP2 or complete culture medium (CTL). On day 4, cells were fixed and stained with propidium iodide (PI) for nuclei (red) and with a specific antibody for plasma membrane desmosomes (green) and observed by confocal microscopy at x10 or x40 magnification (scale bars, 100 μm). Independent experiments were performed on cells isolated from 3 different placentas.
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Effect of FBS and PP2 on Src activation
In Src, two tyrosine residues are present, Tyr-416 in the kinase domain and Tyr-527 in the C-terminal tail (the numbering is based on chicken Src), whose phosphorylation is important in regulating kinase activity. Autophosphorylation at Tyr-416 leads to increased kinase activity, whereas phosphorylation of Tyr-527 represses kinase activity. Conversely, dephosphorylation of Tyr-527 can activate Src (Brown & Cooper, 1996; Abram & Courtneidge, 2000). Thus, following the isolation of cells, trophoblasts were incubated for 5 h in the presence of 10% FBS, and 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 induces a rapid activation of Src (Tyr-416) after 2 min that increases up to 30 min (Fig. 8A (i)). Moreover, using specific antibody that recognizes non-phosphorylated Tyr-416, our results show a decrease of the non-phosphorylated Tyr-416 in accordance with the activation status (Fig. 8A (ii)). In the same way, phosphorylation status at Tyr-527 was also evaluated after FBS addition. Figure 8B (i) shows a decrease in Tyr-527 phosphorylation after 20 and 30 min in accordance with the non-phosphorylated Tyr-527 which increases after 10 min to reach a maximum after 30 min (Fig. 8B (ii)).
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Following cytotrophoblast cells isolation, cells were kept in complete culture media for 5 h and serum-starved overnight. After stimulation with FBS, cell lysates were prepared and subjected to 12% SDS-PAGE. Where indicated, cells were preincubated with PP2 (10 μM) for 2 h. Activated Src was detected by Western blot using specific antibodies for phospho- and non-phospho-Src at Tyr-416 or Tyr-527. First, blots were probed with antiphospho-Src at Tyr-416 or Tyr-527, then stripped and reprobed using an anti-non-phospho-Src at Tyr-416 or Tyr-527, respectively. Finally, blots were stripped one more time and probed using anti-Src (total) antibody. A, Src phosphorylation status at Tyr-416 after FBS stimulation. Cells were stimulated with 10% FBS for indicated time. B, Src phosphorylation status at Tyr-527 after FBS stimulation. Cells were stimulated with 10% FBS for indicated time. Independent experiments were performed on cells isolated from 3 different placentas.
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In order to evaluate the effect of PP2 on Src activation, cells were preincubated for 2 h with PP2 (10 μM) prior to FBS addition. As shown in Fig. 8A (iv), PP2 inhibits Src phosphorylation at Tyr-416 in accordance with non-phosphorylated Tyr-416 (Fig. 8A (v)). In the same way, Tyr-527 phosphorylation was evaluated after PP2 preincubation. As shown in Fig. 8B (iv), PP2 inhibits the dephosphorylation of Src at Tyr-527 and indeed increases its phosphorylation. PP3 does not change the effect of FBS on the phosphorylation status of Src (data not shown).
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Effect of genistein and herbimycin A on the differentiation process of trophoblast primary culture
It was reported that PP2 and herbimycin A inhibit Src activation using two different mechanisms (Yan et al. 1999; Sunohara et al. 2001; Franch et al. 2002). Therefore, herbimycin A and genistein, a broad range PTK inhibitor, were used to compare their effects with those of PP2. In contrast to PP2, herbimycin A and genistein inhibited hCG and hPL secretion by trophoblasts (Fig. 9A and B). Following herbimycin A treatment, hCG and hPL secretion are significantly reduced by 96 and 85%, respectively, on day 4. In the same way, genistein inhibited hCG and hPL secretion by 70 and 60%, respectively, on day 4. To substantiate the effect of the two inhibitors on trophoblast differentiation, morphological differentiation was evaluated by syncytium formation. As shown in Fig. 9C, cells treated with herbimycin A or genistein for 4 days were present as mononucleated cytotrophoblast cells, while the control cells consisted of abundant and large syncytia. Taken together, the results with PP2 (Figs 6 and 7), herbimycin A and genistein (Fig. 9) showed that SFKs play multiple roles in trophoblast differentiation, probably depending on the isoform activated.
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Cells were treated from day 1 to day 4 with 100 μM genistein, 1 μM herbimycin A or complete culture medium (CTL). Data are expressed as the relative secretion of hCG (A) or hPL (B) ±S.E.M. compared to hCG or hPL secretion on day 4 of culture. Independent experiments were performed on cells isolated from 3 different placentas (P < 0.01 and P < 0.05; genistein compared to control for hCG and hPL secretion, respectively; P < 0.001; herbimycin A compared to control for hCG and hPL secretion; two-way ANOVA) (P < 0.05; P < 0.001; genistein or herbimycin A compared to CTL for the same day of culture; t test). C, cells were treated from day 1 to day 4 with 100 μM genistein, 1 μM herbimycin A or complete culture medium (CTL). On 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 bars, 100 μm). Independent experiments were performed on cells isolated from 3 different placentas.
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Evaluation of apoptosis and measurement of LDH activity in treated cells
We studied the cytotoxicity effect of PP2, PP3, genistein and herbimycin A by measuring LDH activity in cells and culture medium. As shown in Table 3, no differences were observed between control and treated cells, demonstrating the non-toxicity of treatments. In the same way, the effect of PP2, herbimycin A and genistein on apoptosis was measured by immunocytochemistry using M30 antibody. As shown in Table 4, no differences were observed between control and treated cells. The immunostaining with M30 antibody can be found in Fig. 3 of the Supplemental material.
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Discussion
In the present study, we demonstrate for the first time the expression profile of SFK members during the culture of primary trophoblasts from human term placenta and their implication in the differentiation process. Moreover, we show that c-Src is rapidly activated upon addition of FBS and this activation increases up to 30 min. Finally, using different pharmacological inhibitors, we show that SFK members play different roles in trophoblast differentiation.
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Regarding trophoblast differentiation, we previously reported an increase in hCG (Daoud et al. 2005a,b) and hPL (Daoud et al. 2005a) secretion. Moreover, we showed that cytotrophoblast cells differentiated morphologically into syncytiotrophoblasts after 4 days of culture (Daoud et al. 2005a). In the present study, we show that mRNA expression of hCG is modest during the first 24 h of culture, suggesting that isolated cytotrophoblast cells are highly purified, without syncytial fragments. Moreover, we show that mRNA transcription and protein synthesis of hCG match perfectly with the secretion pattern. Therefore, hormonal and morphological statuses of primary cultures of trophoblasts were used to evaluate trophoblast differentiation.
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The SFK can be divided into two groups based on their general pattern of expression. Src, Fyn and Yes are ubiquitously expressed; however, some SFKs are highly expressed in certain cell types and as alternatively spliced in other specific cell types (Thomas & Brugge, 1997). The second group of SFKs, Blk, Fgr, Hck, Lck and Lyn, are primarily found in haematopoietic cells (Bolen & Brugge, 1997). However, Hck, Fgr and Lyn were also detected in neurones (Lowell, 2004), suggesting that these kinases may be expressed in many additional cell types. Moreover, Src is expressed as a different isoform in brain containing six additional amino acids between exons 3 and 4, but not expressed in lung, liver or placenta (Pyper & Bolen, 1989). It was reported that primary cultures of trophoblasts expressed Src and that its expression profile was unchanged during culture up to day 4 (Rebut-Bonneton et al. 1993). Moreover, multiple genes are expressed differentially in trophoblast cells with time in culture. In many previous studies, we reported the differential expression of mitogen-activated protein kinases (MAPKs: ERK1/2 and p38) (Daoud et al. 2005a), calcium channels (Moreau et al. 2002a,b), adenylyl cyclases (Bernatchez et al. 2003), cytosolic fatty acid binding protein and peroxysome proliferator-activated receptor (Daoud et al. 2005b) with days of culture of primary trophoblasts. Moreover, Getsios et al. (2001) reported a differential expression of -, - and -catenins during trophoblast differentiation. These observations prompted us to evaluate the expression level of SFK members in trophoblasts isolated from human term placenta on different days of culture and thereafter, verify their implication in trophoblast differentiation. In our present study, we show that trophoblasts express all members of SFK and some of them are expressed as different splice variants (Fyn, Lyn, Src). To our knowledge, we are the first to report mRNA expression of all SFK members in human trophoblasts and the mRNA expression of all SFK members in the same cell type. Moreover, SFKs show two expression profiles as cytotrophoblast cells differentiate into syncytiotrophoblasts. Fyn, Hck and Lyn show no differences in mRNA expression profile while Fgr, Lck, Src and Yes show an increase in mRNA expression during culture. However, Src protein expression was unchanged from day 1 to day 4 as previously described (Rebut-Bonneton et al. 1993) and increased thereafter. Moreover, our results show that mRNA and protein expression of Src matched completely indicating the absence of post-transcriptional regulation. Lannutti et al. (2003) demonstrated the presence of six members of SFK in megakaryocytes and showed that only Fyn is up-regulated during megakaryocyte differentiation. In the same way, Kamei et al. (1997) showed that rat trophoblasts express Src and Yes, which were activated in proliferating and differentiating trophoblast cells, and Lyn which was activated only in differentiating trophoblast giant cells and showed a differentiation-dependent accumulation (Kamei et al. 1997). Moreover, it was reported that Lck and Fyn play different roles in T cell differentiation, survival and activation (Zamoyska et al. 2003). Our results and all these observations indicate that most cells are likely to express multiple SFKs and probably each SFK accomplishes a specialized role in cell physiology.
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The SFKs control many cellular events such as adhesion and spreading, focal adhesion formation/disassembly, lamellipodia, migration, apoptosis, cell cycle progression, gene transcription and cell differentiation (Thomas & Brugge, 1997). Furthermore, many SFK members are implicated in the differentiation of different cell types. Fyn and Lck play major and different roles in T cell differentiation and maturation (Zamoyska et al. 2003). A possible role of Src in myoblasts, retinoblasts and chondrocytes differentiation was demonstrated using v-Src (Muto et al. 1977; Yoshimura et al. 1981; Crisanti-Combes et al. 1982; Alema & Tato, 1987). Fyn and Lyn were implicated in osteoclast differentiation (Kaabeche et al. 2004), while v-Src promotes PC12 differentiation into neurone-like cells (Alema et al. 1985; Haltmeier & Rohrer, 1990; Hecker et al. 1991). Therefore, we evaluated the expression of Src in trophoblasts and its activation after FBS addition, a factor known to induce trophoblast differentiation (Daoud et al. 2005a). In this study, we report a rapid and sustained activation of Src in primary culture of trophoblasts after FBS addition. Previously, Rebut-Bonneton (Rebut-Bonneton et al. 1993) showed the expression of Src in human trophoblasts and that the kinase activity of Src increased significantly on day 2, when cells were completely aggregated and started to fuse, and remained elevated up to day 4, while cells underwent further differentiation. It should be noted that Src activation depends on its conformation. Both the SH3 and SH2 domains lie on the side of the kinase domain opposite to the catalytic cleft. The SH3 and SH2 domains repress the kinase activity by interacting with amino acids within the catalytic domain, as well as with residues N-terminal and C-terminal, respectively (Brown & Cooper, 1996; Thomas & Brugge, 1997). Dephosphorylation of Tyr-527, which is the first site of phosphorylation in vivo, induces a change in Src conformation and increases its activity by autophosphorylation at Tyr-416 in the catalytic domain (Brown & Cooper, 1996; Thomas & Brugge, 1997). Conversely, phosphorylation of Tyr-527 by Csk (C-terminal Src kinase) inhibits Src activation (Nada et al. 1991). In our study, FBS induced a rapid phosphorylation at Tyr-416 after 2 min which was accompanied by a dephosphorylation at Tyr-527 after 20 min. This delay between Tyr-527 dephosphorylation and Tyr-416 phosphorylation could be explained by the sensitivity of antibodies used or by the activation of Src independently of Tyr-527. Some SFKs are not always phosphorylated at this negative regulatory tyrosine, yet remain relatively inactive. For example, in B cells, tyrosine phosphorylation of the C-terminal tail of Lyn is barely detectable even though the catalytic activity of Lyn is not elevated. However, loss of Csk results in activation of Lyn (Nada et al. 1993; Hata et al. 1994).
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To further demonstrate the implication of SFKs in trophoblast differentiation, we used two SFK inhibitors (PP2 and herbimycin A) and a broad range tyrosine kinase inhibitor (genistein). Surprisingly, PP2 and herbimycin A showed different effects on trophoblast differentiation. Herbimycin A and genistein showed hormonal and morphological inhibition of trophoblast differentiation, while PP2 showed an induced hormonal differentiation, inhibition of cell adhesion and spreading and no effect on cell fusion. PP2 seems to repress some inhibitory effects of some members of SFK implicated in hormonal trophoblast differentiation. Moreover, PP2 increased Src phosphorylation at Tyr-527 and inhibited Tyr-416 phosphorylation. It should be noted that SFKs play critical roles in downmodulating or inhibiting signalling pathways in immune cells; a concept that was confirmed through studies on mutant models (Lowell, 2004). Maruyama et al. (2004) showed that PP1 and PP2 promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro. Moreover, they showed that herbimycin A had no effect on the differentiation process compared to PP1 or PP2. Although herbimycin A was widely used as a tyrosine kinase inhibitor with Src selectivity, several studies demonstrate that it behaves in a manner different from PP1 and PP2 (Yan et al. 1999; Sunohara et al. 2001; Franch et al. 2002). PP1 and PP2 act as competitive inhibitors of ATP for SFK (Liu et al. 1999; Schindler et al. 1999), which, however, became controversial (Karni et al. 2003). Herbimycin A covalently interacts with sulfhydryl groups on protein tyrosine kinases (Uehara et al. 1989; Fukazawa et al. 1994; Senga et al. 2000). These differences in their inhibition mode may account for a differential selectivity and various actions of these two inhibitors as presented herein and also previously described (Yan et al. 1999; Sunohara et al. 2001; Franch et al. 2002; Maruyama et al. 2004). Thus, PP2 and herbimycin A may act on different and distinct SFK members which in turn, may play different roles in hormonal and morphological trophoblast differentiation. Further studies will be required to elucidate the molecular basis for the effect of PP2 and herbimycin A on trophoblast differentiation.
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Finally, SFKs are known to regulate downstream signalling pathways that influence nuclear events (Brown & Cooper, 1996) and can stimulate MAPKs and phosphatidylinositol 3-kinases, resulting in changes in cell survival, division, differentiation and gene transcription (Thomas & Brugge, 1997). We previously reported the implication of ERK1/2 and p38 in trophoblast differentiation (Daoud et al. 2005a). Moreover, SFKs can phosphorylate focal adhesion kinases (FAKs) which are very important for cell adhesion, spreading, migration, shape and differentiation (Thomas & Brugge, 1997). Thus, since the implication of FAKs in placental implantation, cytotrophoblast cell migration, invasion and differentiation was already reported (Shiokawa et al. 1998; Gleeson et al. 2001; Ilic et al. 2001; MacPhee et al. 2001), it would be of interest to verify if PP2 and herbimycin A act differentially on MAPKs and FAKs during trophoblast differentiation.
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In conclusion, this study demonstrated for the first time the expression profile of SFKs in human trophoblasts with days in culture, and that SFKs play multiple roles in trophoblast differentiation. Thus, this study increases our knowledge of the trophoblast differentiation process and could be very useful in the understanding of pathology relating to abnormal placental differentiation, such as pre-eclampsia and trophoblastic neoplasm.
Supplemental material
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The online version of this paper can be accessed at:
DOI: 10.1113/jphysiol.2005.102285
http://jp.physoc.org/cgi/content/full/jphysiol.2005.102285/DC1
and contains supplemental material consisting of three figures and legends entitled Figure 1A and B: Specificity of real-time PCR products shown by digestion with restriction enzymes; Figure 2: Effect of PP2 on hCG or hPL mRNA expression in human primary culture of trophoblasts; Figure 3: Effect of PP2, herbimycin A and genistein on trophoblasts apoptosis as monitored by M30 staining.
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This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com
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