Mol. Hum. Reprod. Advance Access originally published online on July 2, 2004
Molecular Human Reproduction 2004 10(9):677-684; doi:10.1093/molehr/gah088
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Both mitogen-activated protein kinase and phosphatidylinositol 3-kinase signalling are required in epidermal growth factor-induced human trophoblast migration
1Hormones, Growth and Development Program, Ottawa Health Research Institute, Ottawa, Ontario, Divisions of 2Maternal-Fetal Medicine and 3Reproductive Medicine, Department of Obstetrics and Gynecology, University of Ottawa, The Ottawa Hospital, Ottawa, Ontario, Canada
4 To whom correspondence should be addressed at: Division of Maternal–Fetal Medicine, Department of Obstetrics and Gynecology, The Ottawa Hospital, Ottawa, Room 8420, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6. Email: agruslin{at}ottawahospital.on.ca
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Abstract |
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Adequate extravillous trophoblast (EVT) invasion is an essential step for placental formation. The aim of this study was to examine the possible role of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling in epidermal growth factor (EGF)-induced EVT migration and to determine if the 70 kDa ribosomal S6 kinase (p70S6K) is involved in this process. In this study, EGF significantly stimulated HTR8/SVneo cell migration and the phosphorylation of AKT, ERK1/2 and p70S6K in a concentration-dependent manner. The MAPK inhibitor U0126 decreased cell migration and ERK phosphorylation, but it did not influence p70S6K phosphorylation in response to EGF. In the presence of PI3K inhibitors (Wortmannin), EGF-stimulated trophoblast migration and phosphorylation of AKT and P70S6K (Thr389 and Thr421/Ser424) were decreased, while EGF-induced ERK phosphorylation was not affected. Expression of an activated AKT (Myr-AKT2) increased basal phospho-p70S6K (Thr389 and Thr421/Ser424) content, but failed to stimulate cell migration. However, it induced cell migration in the presence of EGF and Wortmannin, in which both AKT and MAPK pathways were activated. In addition, there was a concentration-dependent inhibition of cell migration and p70S6K phosphorylation (Thr389 and Thr421/Ser424) in the presence of Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR, a downstream of AKT). Taken together, our data suggest that EGF-induced trophoblast migration involves the coordinated regulation of both PI3K/AKT and MAPK signalling pathways. mTOR/p70S6K is important in PI3K- but not MAPK-mediated trophoblast migration in response to EGF.
Key words: MAPK/migration/p70S6K/PI3K/trophoblast
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Introduction |
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Adequate extravillous trophoblast (EVT) invasion is an essential step in placental formation and is important for fetal growth and well-being. The significance of appropriate trophoblast invasion for normal placentation and pregnancy outcome is highlighted by the fact that shallow invasion is a key feature of pre-eclampsia (Kaufmann et al., 2003
), a condition affecting 5–7% of the pregnant population, and associated with restricted placental and fetal growth as well as increases in fetal and maternal morbidity and mortality (Shah, 2001). In addition, inadequate trophoblast invasion is also commonly seen in placentas of women who smoke, a condition known to result in increased first trimester losses, decreased fetal growth and preterm delivery (Genbacev et al., 1995; Shiverick and Salafia, 1999; Zotti et al., 2003). Growth factors or cytokines in the trophoblast microenvironment can regulate trophoblast invasion of the uterus. Cell invasion is a multi-step process involving attachment to, degradation of and subsequent migration through the extracellular matrix. It has been shown that epidermal growth factor (EGF) plays a role in tumour metastasis, wound healing, as well as placental development by inducing the invasion/migration of tumour cells (Pedersen et al., 1994; Kawahara et al., 1999), keratinocytes (Andresen et al., 1997; Haase et al., 2003) and trophoblasts (Bass et al., 1994) respectively. Increased migration could be a key step in EGF-stimulated EVT cell invasiveness.
It is well established in various systems that EGF receptor (EGFR) ligation leads to the activation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathways, resulting in the regulation of several target genes involved in cell survival, proliferation and metabolism (Squires et al., 2003; Thomas et al., 2003). It has been shown that both PI3K and MAPK pathways are involved in cell migration. Several molecules, including insulin-like growth factor-binding protein-1 (IGFBP-1) (Gleeson et al., 2001), insulin-like growth factor-II (IGF-II) (McKinnon et al., 2001) and endothelin-1 (ET-1) (Chakraborty et al., 2003), are known to stimulate migration of human extravillous trophoblast cells (HTR8/SVneo) through MAPK activation, although the possible involvement of PI3K signalling was not examined in these studies. Activation of PI3K is required for insulin-like growth factor-I-induced vascular smooth muscle cell migration (Duan et al., 2000) and for platelet-derived growth factor-mediated migration of mesangial cells (Choudhury et al., 1997). The involvement of PI3K in the SGHPL-4 human trophoblast migration induced by hepatocyte growth factor has also been demonstrated (Cartwright et al., 2002). However, whether both pathways are involved in the regulation of EVT migration independently or coordinately as well as their downstream signalling cascades remains unclear.
The 70 kDa ribosomal S6 kinase (p70S6K) is activated by various mitogens, growth factors and hormones through a complex network of signalling molecules (Avruch et al., 2001; Berven and Crouch, 2000). The primary target of the activated kinase is the 40S ribosomal protein S6, a major component of the protein synthetic machinery in mammalian cells. In addition, recent data indicate that p70S6K may also function in regulating cell motility (Berven and Crouch, 2000). p70S6K has seven phosphorylation sites, including Thr229, Thr389, Ser404, Ser411, Ser418, Thr421 and Ser424 (Pullen and Thomas, 1997). At least two major signalling pathways have been described for the phosphorylation and activation of p70S6K (Chou and Blenis, 1995): a PI3K-dependent pathway in which PI3K activates PDK1, AKT and the mammalian target of rapamycin (mTOR) (Chung et al., 1994; Ming et al., 1994; Filippa et al., 1999), and a protein kinase C-dependent pathway (Iijima et al., 2002). However, the role of the MAPK pathway in the control of p70S6K phosphorylation and function is controversial. Whereas several studies (Chung et al., 1994; Ming et al., 1994; Lenormand et al., 1996) have demonstrated that MAPK family members such as ERK were not involved in p70S6K activation, recent reports have suggested that, under specific physiologic conditions, ERK signalling is required for p70S6K phosphorylation (Eguchi et al., 1999; Iijima et al., 2002). To date, however, the signalling events mediating p70S6K activation by EGF and their possible involvement in the regulation of trophoblast migration have not been studied.
In this study, we have examined the possible role of PI3K and MAPK signalling in the regulation of EVT migration and have determined whether p70S6K is involved in this process. Using pharmacological antagonists and the expression of a constitutively activated AKT, we have demonstrated that both PI3K and MAPK pathways are required in EGF-induced trophoblast migration. Moreover, mTOR/p70S6K is important for EGF-induced EVT migration and is activated by signalling of the PI3K but not of the MAPK pathway.
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Materials and methods |
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Cell lines and culture conditions
The HTR-8/SVneo cells were a gift from Dr Charles H.Graham (Queen' University, Canada). This cell line was developed from explant culture of human first-trimester placenta and immortalized by transfection with a cDNA encoding the simian virus 40 large T antigen (Graham et al., 1993
). These cells exhibit a high proliferation index and share various phenotypic similarities with the parental HTR-8 cells, including in vitro invasive abilities. Extensive phenotypic characterization has shown that HTR-8 cells express all framework antigen W6/32 [a monomorphic epitope, constituting the human MHC class I, HLA-A, B, C], NDOG-5 antigen, IGF-II, urokinase-type plasminogen activator receptor (uPAR), a selective repertoire of integrin subunit, 
1, 3, 5, v and ß1, and the vitronectin receptor vß3/ß5 (Irving and Lala, 1995; Liu et al., 2003). The cells (1.5 x 105/well) were plated overnight (in a 12-well plate) in 1 ml of Roswell Park Memorial Institute 1640 medium/10% fetal bovine serum (FBS), starved overnight in serum-free medium and subsequently incubated in fresh serum-free medium for various durations (5 min to 24 h; EGF = 50 ng/ml; Sigma, Canada) or EGF concentrations (1–50 ng/ml; 15 min). Inhibitors of PI3K (LY294002; 2–50 µmol/l; Sigma), MAPK (U0126; 2–30 µmol/l; Cell Signalling, USA) or mTOR (Rapamycin; 2–50 nmol/l, Sigma) were dissolved in dimethylsulphoxide (DMSO) and added 1 h before treatment with EGF (50 ng/ml). Each experimental group received an equal amount of DMSO (1:1000; vehicle).
Cell transfection
The cells were plated overnight in 1 ml of RPMI 1640/10% FBS. At 60–80% confluence, cells were transfected with the pcDNA3.1 expression vector containing constitutively active HA-Myr-AKT2 (Liu et al., 1998) (provided by Dr Jin Cheng, University of South Florida, Tampa, Florida) or the empty vector pcDNA3.1 (control) using the Effectene Transfection Reagent (Qiagen, Canada) and according to the manufacturer's instructions. After transfection for 6 h, cells were washed, incubated in serum-free medium or serum-reduced medium (0.5% FBS) for 18 h and cultured in the absence or presence of Wortmannin and EGF in the studies where phospho-protein contents were analysed by western blotting. In cell migration experiments, cells were harvested after the 18 h culture in serum-reduced medium and cultured further with or without Wormannin and EGF, as described under ‘Migration assay’. Transfection efficiency, determined by X-gal staining of the cells transfected with pcDNA3.1 containing LacZ gene for a total 24 h incubation, was 
50%.
Western blot analysis
At the end of the culture period, cells were lysed with lysis buffer (50 mmol/l HEPES pH 7.4, 150 mmol/l sodium chloride, 1 mmol/l EGTA, 10 mmol/l sodium pyrophosphate, 1.5 mmol/l manganese chloride, 100 mmol/l sodium fluoride, 10% glycerol, 1% Triton X-100, 1 mmol/l sodium orthovanadate, 1 mmol/l phenylmethylsulphonyl, 10 µg/ml aprotinin) with sonication. Insoluble material was removed by centrifugation (14 000 g, 4°C, 20 min) and the protein content in the supernatant was determined using the BioRad DC protein kit assay (BioRad, Canada). Aliquots of protein extract (50 µg total protein) were resolved by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and electrotransfered to nitrocellulose membranes. The membranes were blocked (1 h, room temperature) in blotto [Tris-buffered saline at pH 7.6 with 0.05% Tween 20 (TBS-T) and 5% dehydrated non-fat milk]. After rinsing with TBS-T, membranes were immunoblotted with antibodies to phospho-p70S6K (Thr389), phospho-MAPK (ERK1/2) and phospho-P70S6K (Thr421/Ser424) (all from Cell Signalling, USA), phospho-AKT (Upstate, USA) and GAPDH (loading control; Abcam Ltd, UK). HA tag proteins in HA-Myr-AKT transfection experiments were detected using an anti-HA (hemaglutinin) antibody (Roche, Canada). The bands were visualized using ECL reagents (Amersham Pharmacia biotech, Inc., Canada).
Migration assay
Migratory activity of trophoblast cells was determined by assessing the ability of the cells to cross the 8 µm pores of migration chambers under chemokinetic conditions. Briefly, the HTR-8/SVneo cells were starved in serum-reduced medium (SRM: RPMI 1640, 0.5% FBS, 0.5% penicillin/streptomycin) overnight, harvested with 2 mmol/l EDTA/PBS and resuspended in SRM at a concentration of 5 x 105 cells/ml. The cell suspension (0.2 ml) was loaded onto the upper chambers of the 24-well Falcon Cell Culture transwells (6.5 mm diameter, 8 µm pore; VWR, Canada) and 0.6 ml of SRM was pipetted into the lower chamber. EGF (0, 1, 10, 50 ng/ml) was added to both upper and lower chambers. In experiments with pharmacological inhibitors, cells were incubated with either LY294002 (0, 2, 10, 50 µmol/l), Wortmannin (0, 2, 10, 50, 150 nmol/l), U0126 (0, 2, 10, 30 µmol/l) or Rapamycin (0, 2, 10, 50 nmol/l) in upper chambers for 1 h prior to the addition of EGF (50 ng/ml) and 0.6 ml of SRM with 50 ng/ml of EGF were pipetted into lower chambers. In the migration assay of HTR8/SVneo cells expressing constitutively active AKT2, cells were transfected after a total 24 h transfection as described before, the transfected cells were lifted with 2 mmol/l EDTA/PBS, plated into the migration upper chamber and incubated with or without Wortmannin (150 nmol/l) for 1 h before the addition of EGF (50 ng/ml) to both the top and bottom chambers. DMSO levels were the same in all wells within each experiment. Following a 24 h culture period, the cells on the surface of the polycarbonate membrane (non-migrating cells) were removed by scraping with a cotton swab. Cells that migrated through the pores and were present in the distal surface of the membrane were stained for 1 h with 1% Toluidine Blue containing 10% buffered formalin. Following several washes with water, the membranes were removed with a small scalpel blade and mounted on a microscope slide. The stained cells in ten random fields were counted under a light microscope (x40 objective).
MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide) cell proliferation/viability assay
Cellular proliferation/viability was determined utilizing the MTT colorimetric assay, in which the yellow tetrazolium MTT is reduced in metabolically active cells to intracellular purple formazan that can be quantified spectrophotometrically. Briefly, HTR8/SVneo cells (2x104 cells/100 µl/well) were seeded in 96-well flat-bottom tissue culture plates in SRM under the same culture conditions as those used in the migration assay. The cells were treated with EGF (0, 1, 10, 50 ng/ml) or pretreated for 1 h with U0126 (0, 2, 20, 30 µmol/l), LY294002 (0, 2, 10, 50), Wortmannin (0, 10, 50, 150 nmol/l) or Rapamycin (0, 2, 10, 50 nmol/l) and then incubated for 24 h with EGF (50 ng/ml). After 24 h incubation, MTT (0.5 mg/ml) was added into each well and the cells were incubated for another 4 h. Following media removal, 200 µl of DMSO was added to each well and absorbance was read at 550 nm. Cells in all experimental groups received the same amount of vehicle (1:1000 dilution of DMSO).
Statistical analyses
The values are given as the mean±SE of three independent experiments. Data were analysed by analysis of variance (ANOVA) and Newman–Keuls multiple comparison tests. The significance level was set at P<0.05.
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EGF increases EVT phospho-AKT, phospho-ERK1/2 and phospho-p70S6K contents and stimulates cell migration
EGF (10 ng/ml) stimulated phosphorylation of AKT, ERK as well as p70S6K (at Thr389 and Thr421/Ser424) in a time-dependent manner, reaching a maximum at 5–15 min after treatment (Figure 1A). After 2 h of EGF stimulation, contents of phosphorylated AKT and ERK returned to basal level, while those of phospho-p70S6K remained high after 6 h of treatment (Figure 1A). These responses were concentration-dependent, with observable increases at 1 ng/ml EGF (Figure 1B).
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Using transwell plates, we measured the effects of different concentrations of EGF (0, 1, 10, 50 ng/ml) on HTR8/SVneo cell migration during 24 h of incubation. EGF stimulated trophoblast migration in a concentration-dependent manner, as indicated by a concentration-dependent increase in the number of cells present on the distal side of the growth surface of the upper chamber, as the concentration of the growth factor was increased. At a concentration as low as 1 ng/ml, EGF significantly increased trophoblast migration (P<0.05, compared to control, Figure 1C). Since migration can be significantly altered by proliferation and EGF has been shown to promote trophoblast proliferation (Maruo et al., 1995
; Li and Zhuang, 1997), we performed MTT assays following EGF treatment of EVT cells. Under the same specific culture conditions used in our migration assay, EGF had no effect on EVT proliferation after 24 h of incubation (Figure 1C), suggesting that the observed migration is not due to increases in cell proliferation but rather to the stimulation of the migratory process itself.
MAPK inhibitor suppresses EGF-induced ERK phosphorylation and cell migration, but not phosphorylation of p70S6K
The signalling events involved in the regulation of HTR8/SVneo cell migration were dissected using pharmacological inhibitors. At first, the role of the MAPK pathway in the regulation of trophoblast migration was investigated using the MAPK-specific inhibitor U0126. HTR8/SVneo cells were incubated with U0126 (0, 2, 10, 30 µmol/l) 1 h prior to treatment with EGF (50 ng/ml). U0126 partially decreased EGF-induced phospho-ERK protein contents at 2 µmol/l and completely blocked this response at 10 and 30 µmol/l, while exerting no effect on EGF-induced phospho-AKT (Figure 2A). U0126 also appeared to have no influence on p70S6K activation as indicated by the absence of detectable changes in phospho-p70S6K (Thr389 and Thr421/Ser424) contents (Figure 2A). U0126, however, significantly inhibited EGF-induced cell migration at 10 µmol/l (P<0.05) and 30 µmol/l (P<0.01) without influence on cell viability (Figure 2B), suggesting that MAPK signalling is important in the EGF-induced HTR8/SVneo cell migration.
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PI3K inhibitors suppress EGF-induced phosphorylation of both AKT and P70S6K and cell migration
Next, we investigated the possible involvement of PI3K signalling in the regulation of EVT migration using two structurally unrelated PI3K-specific inhibitors, LY294002 and Wortmannin. Addition of LY294002 (0, 2, 10, 50 µmol/l), a well-established PI3K inhibitor, 1 h prior to treatment with EGF (50 ng/ml) resulted in suppression of EGF-induced increases in phospho-AKT and phospho-p70S6K (both Thr389 and Thr421/Ser424) in a concentration-dependent manner and complete suppression at 10 µmol/l (Figure 3A). As expected, EGF-induced phospho-ERK content was not affected (Figure 3A). Wortmannin, another widely used PI3K inhibitor, had similar effects. Although Wortmannin completely inhibited EGF-induced phosphorylation of AKT and p70S6K at concentrations of 50 nmol/l and 150 nmol/l respectively, it had no effect on EGF-induced ERK phosphorylation (Figure 3A). Cell proliferation/viability influences the results of the migration assay (Liu et al., 2003
) and MTT assay revealed that LY294002 inhibited HTR8/SVneo cell viability (data not shown). Therefore, LY294002 is not appropriate to use for dissecting the role of PI3K in the regulation of HTR8/SVneo migration. In contrast, Wortmannin inhibited EGF-induced EVT migration in a concentration-dependent manner without effects on cell viability (Figure 3B), suggesting that PI3K signalling is also involved in the EGF-induced EVT migration.
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Active AKT is essential but not sufficient to stimulate trophoblast migration
To further explore the downstream signalling of PI3K in the regulation of EGF-induced migration, we examined the possible involvement of AKT, one of the major targets of PI3K, using an expression vector containing an activated form of AKT (Myr-AKT2-HA). HA-AKT2 protein expression was confirmed in cells transfected with the vector, but not in those with pcDNA3.1 control vector (Figure 4A). Transfection of Myr-AKT2 resulted in AKT/p70S6K activation, as evident by increases in the protein content of phosphorylated AKT and p70S6K at both Thr389 and Thr421/Ser424 sites, relative to transfection of control vector. Similar effects were observed in the presence of EGF and Wortmannin, suggesting that Wortmannin has no effect on Myr-AKT2 vector-induced AKT/p70S6K activation, although it inhibits EGF-activated PI3K signalling. Expression of constitutively active AKT2 alone did not significantly stimulate cell migration compared with control vector (P>0.05, Fig 4B). Whereas, in the presence of EGF and Wortmannin, the MAPK pathway was activated, as evident by an increase in phosphorylated ERK content, overexpression of active AKT2 increased cell migration compared to Myr-AKT2 transfection in the absence of EGF and Wortmannin (P<0.01) and control vector transfection in the presence of EGF and Wortmannin (P<0.01, Figure 4B), suggesting that HTR8/SVneo cell migration requires both AKT and MAPK pathway activation.
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Rapamycin abolishes EGF-induced p70S6K phosphorylation and inhibits trophoblast migration
Studies in other cell systems have suggested that mTOR and p70S6K are involved in the regulation of migration (Gomez-Cambronero, 2003
). As such, we were interested in investigating the role of mTOR/p70S6K in the regulation of EVT migration. Addition of an inhibitor of mTOR (Rapamycin; 0, 10, 50 nmol/l) attenuated EGF-induced p70S6K phosphorylation and HTR8/SVneo cell migration in a concentration-dependent manner (Figure 5). At 10 and 50 nmol/l, Rapamycin inhibited p70S6K phosphorylation (at Thr389 and Thr421/Ser424; Figure 5A) and abolished the stimulatory effects of EGF on HTR8/SVneo cell migration without effect on cell viability (Figure 5B). Taken together, these data suggest the involvement of mTOR/p70S6K in the regulation of EGF-induced HTR8/SVneo cell migration. As expected, 1 h incubation with Rapamycin prior to the addition of EGF (50 ng/ml) had no effect on phospho-AKT and phospho-ERK (Figure 5A), indicating the specificity of this inhibitor.
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Discussion |
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Although the possible roles of several signalling pathways in cell migration have been suggested, the exact mechanisms regulating extravillous trophoblast migration are not well understood. The HTR8/SVneo cell line derived from human invasive extravillous trophoblasts has been well characterized (Graham et al., 1993
) and widely used as model in the study of regulation of EVT migration (Gleeson et al., 2001; McKinnon et al., 2001; Chakraborty et al., 2003; Liu et al., 2003). Using this model, we have demonstrated for the first time that both PI3K and MAPK pathways are involved in the regulation of EVT migration in response to EGF. We have shown that MAPK inhibitor (U0126) inhibited EGF-induced EVT migration (Figure 2B), although it failed to influence activation status of PI3K/AKT signalling, as evident by no change in the protein content of highly phosphorylated AKT induced by EGF (Figure 2A), suggesting that activation of PI3K/AKT signalling is not adequate to induce cell migration, and that the MAPK pathway is important. Whereas the PI3K inhibitor (Wortmannin) also inhibited EGF-induced HTR8/SVneo cell migration, the EGF-induced activation of MAPK signalling was maintained, as evident by increased phosphorylated ERK content (Figure 3A), suggesting that activation of the MAPK pathway alone is not sufficient to regulate HTR8/SVneo cell migration, and that PI3K signalling plays a role in this process. Furthermore, expression of a constitutively active AKT alone (in the absence of EGF) failed to stimulate HTR8/SVneo cell migration (Figure 4B). Myr-AKT2 expression along with EGF and Wortmannin treatment (in which both AKT/p70S6K and MAPK signalling were activated) stimulated cell migration compared with Myr-AKT2 expression alone (in which AKT/p70S6K, but not MAPK, was activated) or control vector transfection in the presence of EGF and Wortmannin (in which MAPK, but not AKT/p70S6K, was activated) (Figure 4B). Taken together, these data suggest that activation of both MAPK and PI3K pathways are required simultaneously for the regulation of HTR8/SVneo migration. The involvement of these signalling pathways in EVT migration is supported by the observations that interaction of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) stimulates migration of this specific cell type and that this is dependent on both MAPK and PI3K pathways (Liu et al., 2003).
Given that both MAPK and PI3K are important in the regulation of EGF-induced EVT migration, we are interested in examining whether common targets exist by cross-talking between the two pathways in this process. Indeed, it has been shown that MAPK is a downstream target of PI3K in the regulation of platelet-derived growth factor (PDGF)-induced mesangial cell migration (Choudhury et al., 1997). When examining this possibility in trophoblasts, we demonstrated that the PI3K inhibitors (Wortmannin and LY294002) had no influence on ERK phosphorylation and that the MEK inhibitor (U0126) had no effect on phospho-AKT in response to EGF, therefore not lending support to a possible cross-talk between these specific pathways upstream of AKT or ERK in HTR8/SVneo cells. In other cell systems, mTOR and p70S6K are believed to be involved in the regulation of migration (Berven and Crouch, 2000) and both PI3K and MAPK signalling may activate p70S6K (Chung et al., 1994; Eguchi et al., 1999). This motivated us to examine whether there is cross-talk at this level. In the present study, both PI3K inhibitors (LY294002 and Wortmannin) decreased EGF-induced phosphorylation of AKT and p70S6K (Thr389 and Thr421/Ser424) content (Figure 3A). Expression of an active form of AKT without any stimulants was sufficient to increase phospho-p70S6K content at both Thr389 and Thr421/Ser424 sites (Figure 4A). These data suggest that phosphorylation of both p70S6K (Thr389) and p70S6K (Thr421/Ser424) are downstream effects of PI3K/AKT signalling. Addition of a MAPK inhibitor had no influence on phospho-p70S6K (Thr389 and Thr421/Ser424) content, but still inhibited cell migration (Figure 2), thereby indicating that regulation of MAPK/ERK-mediated HTR8/SVneo cell migration is p70S6K independent.
Activated AKT has various downstream targets including mTOR/p70S6K, Bad, the forkhead family of transcription factors (Brunet et al., 1999), p21 and p27 (Blagosklonny, 2002), as well as GSK3 (Chan et al., 1999). mTOR/p70S6K and other downstream signalling molecules are distinguished by their sensitivity to Rapamycin. In the present study, we demonstrated that Rapamycin abolished EGF-induced p70S6K phosphorylation and migration, therefore supporting the involvement of mTOR/p70S6K in the regulation of EGF-induced HTR8/SVneo cell migration.
Cytotoxicity of pharmacological inhibitors may interfere with the migration assay. MTT assays suggested that the PI3K inhibitor (Wortmannin) and the MAPK inhibitor (U0126), as well as the mTOR inhibitor (Rapamycin) did not influence cell viability at any of the concentrations used in this study. In contrast, LY294002 appeared to be cytotoxic, as indicated by the MTT assay (data not shown), and was therefore excluded in the present migration study.
We propose that, with respect to EVT migration, PI3K/AKT/p70S6K and MAPK/ERK pathways are both important as they may have two distinct downstream targets, both of which are required for migration. It has been demonstrated that p70S6K plays a role in the regulation of cell migration through remodelling of actin filaments (Chou and Blenis, 1996). Recent data also showed that expression of a constitutively active form of p70S6K was sufficient to induce actin filament remodelling to form lamellipodia and filopodia structures and to decrease actin stress fibres, leading to an increase in cell migration in chick embryo fibroblasts (Qian et al., 2003). Other evidence has suggested that MAPK regulates actin/myosin motor function through myosin light chain kinase activation (Klemke et al., 1997). It is therefore possible that EGF-induced EVT migration requires activation of PI3K/Akt/p70S6K in regulating reorganization of the actin cytoskeleton to develop cell polarity, and MAPK/ERK in regulating actin/myosin motor function to promote cytoskeleton contraction.
In conclusion, we propose that both PI3K and MAPK signalling pathways are involved in EGF-stimulated trophoblast migration (Figure 6). p70S6K is expressed by trophoblasts and activated by PI3K/AKT but not by MAPK/ERK. These two pathways appear to be independent and probably have two distinct downstream targets in invasive trophoblasts.
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Acknowledgements |
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This work was supported by a grant from the Philip Morris USA Inc.external research program. We thank Dr Charles H.Graham (Departments of Pharmacology and Toxicology, Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada) for the providing HTR-8/SVneo cell line and Dr Jin Cheng (University of South Florida, Tampa, Florida, USA) who generously provided the constitutively active AKT2 vectors.
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Submitted on May 16, 2004; resubmitted on June 8, 2004; accepted on June 14, 2004.
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