Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted fetal growth

Authors

  • Sara Roos,

    1. Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
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  • Nina Jansson,

    1. Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
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  • Isabelle Palmberg,

    1. Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
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  • Karin Säljö,

    1. Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
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  • Theresa L. Powell,

    1. Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
    2. Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Cincinnati, OH, USA
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  • Thomas Jansson

    1. Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden
    2. Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Cincinnati, OH, USA
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Corresponding author S. Roos: Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, PO Box 432, SE-405 30 Gothenburg, Sweden. Email: sara.roos@gu.se

Abstract

Pathological fetal growth is associated with perinatal morbidity and the development of diabetes and cardiovascular disease later in life. Placental nutrient transport is a primary determinant of fetal growth. In human intrauterine growth restriction (IUGR) the activity of key placental amino acid transporters, such as systems A and L, is decreased. However the mechanisms regulating placental nutrient transporters are poorly understood. We tested the hypothesis that the mammalian target of rapamycin (mTOR) signalling pathway regulates amino acid transport in the human placenta and that the activity of the placental mTOR pathway is reduced in IUGR. Using immunohistochemistry and culture of trophoblast cells, we show for the first time that the mTOR protein is expressed in the transporting epithelium of the human placenta. We further demonstrate that placental mTOR regulates activity of the l-amino acid transporter, but not system A or taurine transporters, by determining the mediated uptake of isotope-labelled leucine, methylaminoisobutyric acid and taurine in primary villous fragments after inhibition of mTOR using rapamycin. The protein expression of placental phospho-S6K1 (Thr-389), a measure of the activity of the mTOR signalling pathway, was markedly reduced in placentas obtained from pregnancies complicated by IUGR. These data identify mTOR as an important regulator of placental amino acid transport, and provide a mechanism for the changes in placental leucine transport in IUGR previously demonstrated in humans. We propose that mTOR functions as a placental nutrient sensor, matching fetal growth with maternal nutrient availability by regulating placental nutrient transport.

Intrauterine growth restriction (IUGR) and fetal overgrowth (resulting in a large-for-gestational-age (LGA), baby) represent important clinical conditions associated with perinatal morbidity and mortality (Casey et al. 1997; Brodsky & Christou, 2004) and increased risk of adult disease, such as diabetes and cardiovascular disease (Pettitt et al. 1991; Barker, 1995). Fetal growth and development is critically dependent on nutrient supply and therefore closely linked to the transport functions of the placenta. Pregnancies complicated by pathological fetal growth are associated with specific changes in placental transport functions and these changes may directly contribute to altered nutrient supply and fetal growth (Jansson & Powell, 2006). Experimental studies have provided evidence supporting a causative link between altered placental amino acid transporter activity and changes in fetal growth. A trophoblast-specific knock-down of the Igf2 gene in the mouse placenta leads initially to an up-regulation of placental system A compensating for reduced placental size and passive permeability, thereby maintaining fetal growth. However, in late gestation this compensation fails and IUGR develops (Constancia et al. 2002). In addition, we recently demonstrated that down-regulation of the placental system A transporter precedes the occurrence of IUGR in pregnant rats subjected to protein malnutrition (Jansson et al. 2006). However, the mechanisms that regulate placental nutrient transporters are poorly understood. This information is critically needed in order to better understand the pathophysiology of altered fetal growth and the mechanisms underlying fetal programming of adult disease.

The mammalian target of rapamycin (mTOR) signalling pathway has been shown to regulate cell growth in response to nutrients and growth factors by controlling transcription and translation (Wullschleger et al. 2006). mTOR is a large protein (∼280 kDa) which belongs to the phosphatidylinositol kinase-related kinase (PIKK) superfamily. As with all PIKK family members, mTOR contains a carboxy-terminal serine/threonine protein kinase domain. Furthermore, in the amino-terminal direction from the kinase domain is the FKBP12-rapamycin binding domain (FRB domain) (Hay & Sonenberg, 2004). mTOR is present in the cell in two complexes, mTORC1 and mTORC2. mTORC1 is inhibited by rapamycin and regulates temporal aspects of cell growth. In contrast, mTORC2 regulates spatial aspects of cell growth and has been regarded as insensitive to rapamycin (Wullschleger et al. 2006). However, recent studies suggest that mTORC2 is also inhibited by rapamycin if exposure to the inhibitor is prolonged (Sarbassov et al. 2006). The best-characterized downstream targets of mTORC1 are the ribosomal protein S6 kinase 1 (S6K1) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). mTORC1 signals to the translation initiation machinery via activation of the protein kinase S6K1 and inhibition of the eIF4E inhibitor 4E-BP1 (Wullschleger et al. 2006). The protein expression of the phosphorylated forms of these downstream effectors is often used as a measure of the activity in the mTOR signalling pathway.

mTOR signalling regulates the transcription of genes and translation of proteins related to cell growth. There is some evidence, primarily obtained in cell lines, suggesting that nutrient transporters may be down-stream targets of mTOR signalling. Using human BJAB B-lymphoma cells and murine CTLL-2 lymphocytes Peng et al. (2002) demonstrated that rapamycin treatment down-regulated the expression of five genes, which were classified as amino acid transporters. In FL5.12 cells the mTOR signalling system appears to mediate, at least in part, the internalization of glucose transporters and the 4F2hc protein (a subunit of the heterodimer constituting transporters for leucine and cationic amino acids, respectively) subsequent to growth factor withdrawal (Edinger & Thompson, 2002). Stimulation of vascular smooth muscle cells by platelet-derived growth factor increases the expression of LAT1, a member of the system L amino acid transporter family, in a mTOR-dependent manner (Liu et al. 2004). Furthermore, leucine-stimulated system A activity in L6 myoblasts has been shown to be abolished by pretreatment with rapamycin; however, basal system A activity was not affected by rapamycin treatment (Peyrollier et al. 2000).

Using transcriptome analysis, Nijland et al. (2007) recently provided evidence to suggest that the mTOR pathway plays an important role in the developmental programming of the fetal kidney in response to maternal nutrient restriction. The essential role of the mTOR signalling system for early development is illustrated by the early postimplantation lethality after disruption of the mouse mTOR gene (Gangloff et al. 2004; Murakami et al. 2004). In mice homozygous for the mTOR deletion, proliferation of both inner cell mass and trophoblast was inhibited (Murakami et al. 2004). Similarly, studies in immortalized cell lines originating from human trophoblast suggest a key role for mTOR in the regulation of trophoblast proliferation (Wen et al. 2005) and it is suggested that the mTOR pathway is a regulator of invasive trophoblast differentiation (Pollheimer & Knofler, 2005). In the mature placenta mTOR is expressed at the mRNA level (Kim et al. 2002), however, the cellular localization of mTOR and the functional role of this signalling pathway in the placenta after implantation and early placental development is unknown. We hypothesized that placental mTOR regulates nutrient transporters in the human placenta and may be involved in the altered transport functions observed in pregnancies complicated by pathological fetal growth. The aims of this study were to (1) identify which cells in the human placenta express mTOR, (2) investigate the effect of inhibition of placental mTOR on amino acid uptake in villous explants, and (3) study the protein expression of mTOR and its phosphorylated downstream effectors in placentas of IUGR and LGA fetuses.

Methods

Patient selection and tissue collection

Placental tissue collection was carried out with informed consent and was approved by the Committee for Research Ethics at the University of Gothenburg. Placentas were obtained after vaginal or caesarean delivery at term from women with uncomplicated pregnancies giving birth to babies with normal birth weight (appropriate-for-gestational-age; AGA), as well as from pregnancies complicated by LGA or IUGR. AGA was defined as a birth weight between −2 s.d. and +2 s.d. using intrauterine growth curves for a Scandinavian population based on ultrasonically estimated fetal weight (Marsal et al. 1996). IUGR was defined as a birth weight more than 2 s.d. below the mean for gestational age, corresponding to a birth weight below the 3rd percentile at term (Marsal, 2002). In order to decrease the risk that genetically or constitutionally small babies were included in the IUGR group, the presence of one or more signs of fetal compromise (such as increased umbilical artery pulsatility index, oligohydramnios, low ponderal index, and an intrauterine growth deviation observed with serial ultrasound) were used as additional criteria. Pregnancies with other complications than IUGR (such as preeclampsia), cases with chromosomal abnormalities, and other IUGR pregnancies with an identifiable aetiology to the growth restriction were excluded. Thus, the IUGR cases under study may be characterized as ‘idiopathic’ IUGR, i.e. IUGR without known cause (Ghidini, 1996), and was assumed to primarily be due to uteroplacental insufficiency. LGA was defined as a birth weight more than 2 s.d. above the mean for gestational age (Marsal et al. 1996) and LGA placentas were only obtained from pregnancies without type-1 diabetes or clinical signs of gestational diabetes.

Immunohistochemistry

Immunohistochemistry was performed as described in detail previously (Johansson et al. 2000). Briefly, after fixation of trophoblast tissue in zinc containing solutions, paraffin embedding and sectioning (4 μm), sections were mounted on positively charged slides. Subsequently, sections were incubated overnight at 4°C with a polyclonal antibody against mTOR (Abcam, Ltd, Cambridge, UK) diluted 1: 25 in blotto (4% normal horse serum and 4% non-fat dry milk in PBS). Controls were incubated with blotto only. The secondary biotinylated goat anti-rabbit IgG was diluted 1: 300 in 1.5% normal horse serum in PBS and incubated for 45 min at room temperature. In studies of colocalization of mTOR and cytokeratin 7 (Dako Sweden AB, Stockholm, Sweden), the quantum dot double labelling system (Invitrogen, Carlsbad, CA, USA) was used. Q-dot 655 streptavidin conjugate (emitting red light) was used in combination with biotinylated goat anti-mouse IgG to visualize the tissue binding of mouse anti-human cytokeratin 7, and Q-dot 565 streptavidin conjugate (emitting green light) in combination with biotinylated goat anti-rabbit IgG was used to visualize the tissue binding of rabbit anti-human mTOR. Under these conditions, colocalization of cytokeratin 7 and mTOR was observed as an orange/yellow colour.

Cytotrophoblast cell culture

Cytotrophoblast cells were isolated from freshly delivered human term placentas as previously described (Greenwood et al. 1993). Briefly, the placental tissue was washed in 0.9% saline, roughly minced, and digested in 0.25% trypsin (Roche Diagnostics Scandinavia, Bromma, Sweden) and 0.2 mg ml−1 DNase (Sigma-Aldrich, St Louis, MO, USA). The cytotrophoblast cells were then separated on a discontinuous Percoll density gradient and the cells banding between 35% and 55% were collected and plated at a density of approximately 2 × 106 cells in either plastic six-well plates (3 ml per well, for hCG measurements) or in 25 cm2 flasks (5 × 106 cells in 5 ml per flask, for Western Blot). Cells were maintained in a (1: 1) mixture of Dulbecco's modified Eagle's medium (DMEM, Invitrogen) and Ham's F12 culture medium (Invitrogen) supplemented with 10% fetal bovine serum (Sigma), 25 mm Hepes, 50 μg ml−1 gentamicin, 60 μg ml−1 benzylpenicillin, and 100 μg ml−1 streptomycin. The cells were cultured in a humidified incubator at 37°C in 5% CO2–95% air. On the day after isolation, the cytotrophoblast cells were washed two times with 37°C Dulbecco's PBS with Mg2+ and Ca2+ (Sigma) and fresh medium was added. Thereafter the medium was changed daily. In an additional approach to address the cellular localization of mTOR in the placenta, we isolated primary cytotrophoblast cells and cultured these cells for up to 90 h. Cytotrophoblast cells syncytialize in culture, a process that starts approximately 24 h after isolation and plating, as evidenced by production of hCG, a hormone exclusively produced by syncytiotrophoblasts. Cells were harvested at 18, 24, 42, 66 or 90 h after plating, and mTOR protein expression was measured in cell homogenates using Western blot. Cells were homogenized in buffer D (250 mm sucrose, 10 mm Hepes–Tris, pH 7.4 at 4°C, protease inhibitors (0.7 μm pepstatin A, 1.6 μm antipain, and 80 μm aprotinin), and 1 mm EDTA), using a cell scraper.

Measurement of amino acid transporter activity in primary villous fragments

Amino acid transporter activities were measured in primary villous fragments obtained from normal term placentas. We have previously validated this experimental model for transport studies and demonstrated maintained functional and structural integrity of villous fragments for at least 4 h in explant culture (Jansson et al. 2003; Roos et al. 2004). The villous fragments were incubated in 4 ml 1: 3 DMEM–Tyrode solution with or without rapamycin in 0.02% DMSO for 4 h. Subsequently, amino acid uptake experiments were carried out in Tyrode solution only. System A and taurine transporter activity were measured as previously described using [14C]methylaminoisobutyric acid (MeAIB), an amino acid analogue transported exclusively by system A, and [3H]taurine as tracers (Jansson et al. 2003; Roos et al. 2004). System L activity was measured by determining uptake of [3H]leucine in the presence and absence of 5 mm 2-amino-2-norbornanecarboxylic acid (BCH), transported specifically by system L. After incubation with rapamycin, the fragments were washed in Tyrode solution (with or without Na+) for 2 min under constant agitation and then incubated for 20 min in Tyrode solution (with or without Na+ and 5 mm BCH) containing [3H]taurine and [14C]MeAIB or [3H]leucine, in final concentrations of 25 nm, 10 μm and 50 nm, respectively. Mediated amino acid transport was calculated by subtracting the uptake in the buffer representing the non-mediated uptake (Na+-free Tyrode solution for system A and TAUT and Tyrode solution containing 5 mm BCH for system L) from the uptake in the buffer representing total uptake. [3H]Taurine and [3H]leucine uptakes were calculated as femtomoles of amino acid per milligram of total protein per 20 min, and [14C]MeAIB uptake as picomoles of amino acid per milligram of total protein per 20 min. A time course of leucine uptake in isolated villous fragments was performed. Controls (without rapamycin) were incubated with vehicle only and measured control amino acid uptakes were arbitrarily set to 1 for the purpose of comparison.

Western blotting

Placental tissue was homogenized using a Polytron (15 000 r.p.m., 2 min) on ice in cold buffer D, snap-frozen in liquid nitrogen and stored at −80°C until use. The homogenates were thawed on ice, centrifuged at 12 000 r.p.m. for 15 min at 4°C, and the supernatant, referred to as the cytosolic-enriched fraction, was collected. Protein concentrations were determined according to the method of Bradford using a protein assay procedure (Bio-Rad, Hercules, CA) and BSA as the standard.

mTOR

The proteins were separated by SDS-PAGE as described by Johansson et al. (2000), with minor modifications. Twenty micrograms of protein and prestained SDS molecular weight standard mixture (Sigma) were loaded on a 7% SDS-polyacrylamide gel. After the transfer, the membranes were blocked in 5% milk in PBS-Tween for 3 h. For detection of mTOR, the membranes were incubated with anti-mTOR antibody (no. 2732, Abcam) diluted 1: 2000 in PBS-Tween overnight at 4°C. The secondary peroxidase-labelled goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) was diluted 1: 1000 and used in combination with enhanced chemiluminescence (ECL, GE Healthcare Bio-Sciences, Uppsala, Sweden) to visualize the bands on autoradiographic film (GE Healthcare).

Phospho-4E-BP1 and phospho-S6K1

To study the protein expression of phosphorylated downstream targets of mTOR, equal amounts of protein (20 μg for phospho-S6K1 (Thr-389) and phospho-4E-BP1 (Thr-70) and 15 μg for phospho-4E-BP1 (Thr-37/46)) were separated on a 20% SDS-polyacrylamide gel (phospho-4E-BP1 (Thr-70)) or on NuPAGE Novex (Invitrogen) precast 4–12% Bis–Tris gels (phospho-S6K1 and phospho-4E-BP1 (Thr-37/46)). These three primary antibodies were obtained from Cell Signalling Technology (Beverly, MA, USA). The membranes were blocked with 5% milk in Tris-buffered saline (w/v) plus 0.1% Tween 20 (v/v) (TBST) for 1 h at room temperature and then incubated with anti-phospho-4E-BP1 (Thr-37/46 or Thr-70) diluted 1: 1000 or anti-phospho-S6K1 diluted 1: 500, all in 5% BSA-TBST, overnight at 4°C. Finally, the blot was incubated for 1 h with a peroxidase-labelled goat anti-rabbit antibody (Vector Laboratories) diluted 1: 1000 in 5% milk-TBST for detection of phospho-Thr-70–4E-BP1 and phospho-S6K1 and 1: 2000 for detection of phospho-Thr-37/46–4E-BP1. The signal was detected by the ECL system (GE Healthcare). In order to verify data on phospho-S6K1 (Thr-389) expression, phospho-S6K1 (Thr-389) immunoblots were also performed by Kinexus (Vancouver, BC, Canada, analysis KCSS-1.0).

Relative density of bands was evaluated by densitometry with i.p. Laboratory Gel (Signal Analytics, Vienna, VA, USA) or Image Gauge software (version 3.45, Fuji Film). The mean density of the AGA sample (= control) bands was assigned an arbitrary value of 1, and the mean density of the IUGR and LGA groups are expressed relative to the AGA groups.

Data presentation and statistics

The number of experiments (n) represents the number of different placentas studied. In the amino acid uptake studies, three separate villous fragments were studied for each condition, and data were averaged to obtain a value representing each placenta. Values are mean ±s.e.m. Differences between groups in the Western blot analysis were evaluated statistically using the non-parametric Mann–Whitney U test. In the amino acid uptake study, means were compared by one-way ANOVA followed by Dunnett's post hoc test. Normal distribution was verified using the one-sample Kolmogorov–Smirnov goodness-of-fit test (Control P= 0.806, 22 nmP= 0.830, 100 nmP= 0.988). A correlation test was performed to assess whether the time course for leucine uptake was linear. P= 0.05 was considered significant.

Results

Clinical characteristics

Selected clinical data for samples used in protein expression studies are given in Table 1. The gestational and maternal ages were not different between the AGA and IUGR groups. Fetal weight was 36% lower (P < 0.001) in the IUGR group than in controls. Similarly, placental weight was reduced by 40% in the IUGR group (P < 0.001). Furthermore, IUGR was associated with a reduced ponderal index (P < 0.05) suggesting asymmetric fetal growth. The AGA and LGA groups were comparable with regard to maternal and gestational age. Both placental weight and birth weight were significantly higher in the LGA group (P < 0.001).

Table 1.  Selected clinical data
 AGAIUGRAGALGA
  1. Data are presented as means ±s.e.m. AGA, appropriate-for-gestational age; IUGR, intrauterine growth restriction; LGA, large-for-gestational age; C, caesarean section; V, vaginal delivery. *P < 0.05 versus AGA, ***P < 0.001 versus AGA.

n 149216
Maternal age (years)31.8 ± 1.630.8 ± 2.2   33.1 ± 1.331.7 ± 2.0   
Gestational age (weeks)38.5 ± 0.238.6 ± 0.3   38.5 ± 0.338.2 ± 0.4   
Fetal weight (g)3478 ± 1172209 ± 103***3303 ± 83 4273 ± 117***
Placental weight (g)720 ± 40435 ± 56***659 ± 21980 ± 39***
Ponderal index (g cm−3× 100) 2.67 ± 0.082.32 ± 0.10* 
Mode of delivery (C/V)11/18/120/16/0
Primiparous (n)4372

Cellular localization of mTOR in human placenta

Immunohistochemistry Zinc-fixed tissue sections of term placentas (Fig. 1A, left panel) were incubated with a polyclonal antibody directed against mTOR. Expression of mTOR was primarily detected in the cytoplasm of the syncytiotrophoblast. No significant staining could be detected in the control sections (Fig. 1A, right panel). We proceeded by performing colocalization studies, and Fig. 1B shows that mTOR is coexpressed with cytokeratin 7, a trophoblast cell marker, confirming mTOR expression in the trophoblast.

Figure 1.

Localization and expression of mTOR in the human placenta
A, section of zinc-fixed term human placenta was stained with a polyclonal antibody (1: 25) against mTOR (left panel). mTOR was detected in the cytoplasm of the syncytiotrophoblast (marked by arrow). No significant staining could be detected in control sections (right panel). Scale bar, 20 μm. B, sections of fresh frozen term human placentas were stained with a polyclonal antibody (1: 25) against mTOR (green) and a monoclonal antibody (1: 100) against cytokeratin 7 (red) using the quantum dot double labelling system. mTOR is coexpressed with cytokeratin 7 (yellow–green). The grey-scale picture to the right depicts the main histological structures. Scale bar, 20 μm.

Trophoblast cell culture mTOR protein expression was low or undetectable in freshly isolated cytotrophoblast and in cultured trophoblast the first 18 h (Fig. 2A), but increased markedly by 24 h in culture and remained at this level until 90 h. hCG production increased until 66 h in culture and remained high until at least 90 h (Fig. 2B). Thus, our data show that whereas mTOR protein is not significantly expressed in cytotrophoblasts, mTOR protein expression coincides with the appearance of syncytiotrophoblast cells in culture.

Figure 2.

mTOR expression and hCG production in cytotrophoblast cells
A, cytotrophoblast cells isolated from freshly delivered term human placentas were harvested at different time points, and mTOR protein expression was measured in cell homogenates (15 μg per lane). mTOR protein expression was low or undetectable in freshly isolated cytotrophoblast and in cultured trophoblast the first 18 h, but increased markedly by 24 h in culture and remained at this level until 90 h. B, the secretion of hCG is related to cytotrophoblast cell differentiation. To confirm that our cells were undergoing biochemical differentiation, the release of hCG by cytotrophoblast cells into the culture medium was measured after 18, 42, 66 and 90 h using a commercial ELISA detecting the β-subunit of hCG. Secretion of hCG by the 66 h and 90 h cultured cytotrophoblast cell groups was significantly higher than that produced by cytotrophoblast cells maintained in culture for 18 h. Values are means ±s.e.m.*P= 0.001 versus 18 h, n= 6, Friedman Test.

Effect of inhibition of mTOR on the activity of placental amino acid transporters

Uptake of leucine (50 nm) was linear up to at least 30 min (r= 0.89, P < 0.001, n= 14, data not shown). Subsequently, a 20 min incubation time was chosen. Rapamycin, which blocks mTOR signalling by attenuating the ability of mTOR to phosphorylate downstream targets, was used in two different concentrations, 22 and 100 nm, to assess the effect of the mTOR pathway on amino acid transport. A concentration of 100 nm decreased system L transport by 100% (n= 8; controls n= 15; 22 nmn= 9; P= 0.03 versus control, two-sided one-way ANOVA followed by Dunnett's post hoc test; Fig. 3A) after 4 h of incubation but it did not affect the activity of system A or taurine transporters (n= 4; Fig. 3B and C). Control fragments (without rapamycin) were incubated with vehicle only and measured amino acid uptake in these fragments was arbitrarily set to 1.

Figure 3.

Rapamycin inhibits placental leucine transport
Primary villous fragments were incubated with rapamycin, which inhibits mTOR, at two different concentrations for 4 h. At 100 nm rapamycin decreased leucine transport by 100% (n= 8; 22 nmn= 9; controls n= 15; A). However, rapamycin had no effect on the system A amino acid transport system (n= 4; B) or the transport of taurine (n= 4; C). Controls were incubated with vehicle and arbitrarily set to 1 for purpose of comparison. Values are means ±s.e.m.*P= 0.03 versus control, two-sided one-way ANOVA followed by Dunnett's post hoc test.

Protein expression of mTOR and downstream effectors in relation to fetal growth mTOR

After determining the cellular localization of placental mTOR, we tested the hypothesis that there is a relationship between placental mTOR expression and fetal growth. The anti-mTOR antibody identified a distinct band at approximately 240 kDa in cytosol-enriched fractions of placental homogenates (Fig. 4A). Placental mTOR expression was up-regulated by 51% in IUGR placentas (n= 9; controls n= 12; P < 0.05; Fig. 4B) and in fetal overgrowth, placental mTOR expression was down-regulated by 39% (n= 6; controls n= 21; P < 0.05; Fig. 4C).

Figure 4.

Protein expression of mTOR and down-stream targets in relation to fetal growth
A, the anti-mTOR antibody (1: 2000) identified a distinct band at approximately 240 kDa in cytosol-enriched fractions of placental homogenates (20 μg per lane). B and C, placental mTOR expression was up-regulated by 51% in IUGR placentas (n= 9; controls n= 12; P= 0.047 versus AGA, 2-tailed Mann–Whitney U test; B) and in fetal overgrowth, placental mTOR expression was down-regulated by 39% (n= 6; controls n= 21; P= 0.036 versus AGA, 2-tailed Mann–Whitney U test; C). D and E, Western blots detecting phosphorylated (Thr-70) 4E-BP1 (1: 1000, 20 μg per lane, D) and against phosphorylated (Thr-37/46) 4E-BP1 (1: 1000, 15 μg per lane, E) in cytosol-enriched fractions of human placenta. F, a representative Western blot of phospho-Thr-389 S6K1; G, a representative blot of phospho-Thr-412 S6K1 (1: 500). H, the expression of phospho-Thr-389 S6K1 was down-regulated by 45% in IUGR placentas (n= 8; controls n= 6; P= 0.0517 versus AGA, 2-tailed Mann–Whitney U test). A, AGA (appropriate-for-gestational-age); I, IUGR (intrauterine growth restriction); L, LGA (large-for-gestational-age). Values for protein expression (means ±s.e.m.) are relative, with AGA arbitrarily assigned a value of 1.

Phospho-4E-BP1

Figure 4D and E shows representative Western blots using antibodies directed against 4E-BP1 phophorylated at Thr-70 (Fig. 4D) or at Thr-37/46 (Fig. 4E), the primary residues phosphorylated by mTOR. Densitometry analysis showed no significant difference in the expression of phosphorylated 4E-BP1 in cytosolic fractions from AGA (n= 13) and IUGR placentas (n= 9) or between AGA (n= 14) and LGA placentas (n= 6).

Phospho-S6K1

The phospho-S6K1 antibody detects the two isoforms of the S6K1 protein, p70 S6 kinase when phosphorylated at Thr-389 (Fig. 4F) and p85 S6 kinase when phosphorylated at the analogous site (Thr-412, Fig. 4G). The specificity of the bands was confirmed by preincubation of the primary antibody with its blocking peptide, which abolished the 70 and 85 kDa bands. p70 S6 kinase control cell extracts were used as a positive control.

Expression of p70 S6K was down-regulated by 45% in IUGR placentas (n= 8; AGA n= 6; P= 0.05, Fig. 4H). No significant difference was observed in the expression of p70 S6K in the LGA group compared with control (data not shown). No significant differences were found in the expression of phosphorylated p85 S6K in IUGR or LGA placentas (data not shown).

Discussion

Fetal growth is intimately linked to placental nutrient transport. A large body of evidence demonstrates that human IUGR is characterized by a reduced activity of placental amino acid transporters (Dicke & Henderson, 1988; Mahendran et al. 1993; Glazier et al. 1997; Jansson et al. 1998; Norberg et al. 1998; Jansson et al. 2002b), whereas accelerated fetal growth in pregnancies complicated by diabetes is associated with increased expression and activity of placental glucose and amino acid transporters (Jansson et al. 1999, 2001, 2002a). Furthermore, we recently demonstrated that when pregnant rats are subjected to protein malnutrition, an established model of IUGR, down-regulation of placental system A occurs several days prior to the development of growth restriction (Jansson et al. 2006), suggesting that placental transport alterations are a direct cause of, rather than a consequence of, IUGR. Collectively, these studies show that the activity of placental nutrient transporters is positively correlated with fetal growth in important human pregnancy complications, compatible with the possibility that changes in placental nutrient transport directly contribute to the altered fetal growth. However, the factors that cause the alterations in placental nutrient transporters in IUGR and fetal overgrowth are poorly defined and detailed information on the mechanisms of regulation of placental nutrient transport is important in order to better understand the pathophysiology of altered fetal growth.

There is some information available with respect to the mechanisms of regulation of the l-amino acid transporter system in trophoblast cell lines. Uptake of leucine into JAR human placental choriocarcinoma cells has been reported to be regulated by protein kinase C and extracellular pH (Ramamoorthy et al. 1992; Brandsch et al. 1994), and system L transport in BeWo choriocarcinoma cells has been shown to be regulated by protein kinase C and intracellular calcium-concentrations (Okamoto et al. 2002). The L-system activity in isolated syncytiotrophoblast plasma membranes measured in vitro (Jansson et al. 1998) and placental leucine transport assessed using stable isotope techniques in pregnant women are reduced in IUGR (Paolini et al. 2001). The reduction in placental transport of leucine in IUGR may explain the decreased fetal plasma concentrations of leucine in this pregnancy complication (Cetin et al. 1990). In the current study, we show that the protein expression of phosphorylated S6K1, representing activity of the mTOR signalling pathway (Soliman, 2005), is reduced in placentas of human IUGR fetuses. In contrast, we found that the placental expression of mTOR was increased in IUGR. The mechanism underlying the up-regulation of mTOR expression when down-stream activity is reduced can only be speculated on. One possibility is that there is a negative feed-back loop from S6K1 to the translation of mTOR in analogy to the well described S6K1 inhibition of PI3-kinase, upstream of mTOR (Harrington et al. 2005).

Furthermore, using primary villous fragments we demonstrate for the first time that the mTOR signalling pathway regulates the activity of the placental l-amino acid transporter, the primary pathway for transport of leucine across the syncytiotrophoblast microvillous plasma membrane. These data provide a direct link between decreased mTOR activity and decreased L-system activity in the IUGR placenta. We propose that down-regulation of the placental mTOR signalling pathway in IUGR decreases placental leucine transport, which may directly contribute to the altered fetal growth.

It is well established that the system A amino acid transporter, mediating the transfer of neutral non-essential amino acids, is highly regulated in the human placenta (Jansson et al. 2003; Nelson et al. 2003) in line with findings in other tissues and cells (McGivan & Pastor-Anglada, 1994). In contrast to the marked effect on the L-system amino acid transporter, inhibition of mTOR in primary villous explants did not alter system A activity or taurine transport, suggesting that placental mTOR only regulates specific amino acid transporters. Alternatively, the 4 h incubation of explants in rapamycin may have been too short to observe significant effects on the activity of these transporters, especially if they are subjected to transcriptional regulation (Peng et al. 2002).

There are some observations in the literature suggesting that nutrient transporters may be down-stream targets of mTOR (Peyrollier et al. 2000; Edinger & Thompson, 2002; Peng et al. 2002; Liu et al. 2004). With regard to the L-system, mTOR has been shown to mediate the increase in LAT1 expression induced by stimulation of vascular smooth muscle cells by platelet-derived growth factor (Liu et al. 2004). A recent study shows that leucine auxotrophs of the yeast strain Schizosaccharomyces pombe are sensitive to growth in the presence of rapamycin and that rapamycin inhibits leucine uptake (Weisman et al. 2005). To the best of our knowledge, the current study provides the first direct evidence of mTOR regulating l-amino acid transport activity in mammalian cells. Since the intracellular leucine concentration is a potent activator of the mTOR signalling system (Jacinto & Hall, 2003) the regulation of the l-amino acid transporter by mTOR represents a powerful mechanism for altered cell growth in response to availability of nutrients. Thus, the decreased cell growth mediated by inhibition of mTOR will be reinforced by decreased cellular leucine uptake. The mechanism by which mTOR regulates l-amino acid transporter activity remains to be established but may involve altered translation mediated through mTORC1.

In a situation, such as IUGR, where fetal plasma concentrations of glucose and amino acids are decreased (Cetin et al. 1990) homeostatic principles of cellular physiology might predict placental transporters to be up-regulated in an attempt to increase transport. Similarly, in situations with increased fetal plasma concentrations of glucose and amino acids, such as in GDM (Cetin et al. 2005), a down-regulation of placental nutrient transporters may seem an appropriate biological response. This prediction seems particularly reasonable for the system A transporter, which has been shown to be subject to adaptive regulation (Schneider & Dancis, 1974), i.e. if cells in culture are deprived of amino acids in the medium, system A transporters are up-regulated. However, available data, summarized above, indicate the opposite. This apparent contradiction can be reconciled by invoking the effect of physiological integration of a large number of regulatory signals. The net activity of the highly regulated placental system A transporters will be dependent upon input from numerous sources including maternal hormones, intratrophoblast energy status, oxygenation and nutrient concentrations. Thus, in IUGR the possible stimulating effect on system A activity of the modest decrease in fetal amino acid concentrations is overshadowed by a number of factors that will tend to down-regulate system A, including decreased circulating IGF-I levels (Holmes et al. 1997), inhibition of placental IGF-I signalling (Laviola et al. 2005) and decreased insulin receptor density in the placenta (Potau et al. 1981).

We have proposed that the placenta functions as a nutrient sensor, regulating the expression and activity of placental nutrient transporters and fetal growth according to the capacity of the maternal supply line to deliver nutrients to the placenta (Jansson & Powell, 2000, 2006). In the current study, we provide evidence to suggest that mTOR signalling represents a molecular mechanism for nutrient sensing in the human placenta. First, mTOR was shown to be highly expressed in the syncytiotrophoblast, the transporting epithelium of the placenta. This cellular localization is compatible with a nutrient sensing role of the mTOR signalling system since the apical surface of the syncytiotrophoblast is directly exposed to maternal blood (allowing ‘sensing’ of the maternal supply line) and possible targets for mTOR, e.g. nutrient transporters, are also present in the cell. Second, inhibition of placental mTOR signalling decreased the activity of system L, a key transporter for essential amino acids. Third, the activity of placental mTOR signalling was shown to be reduced in IUGR, a condition characterized by an inability of the maternal supply line to provide adequate nutrients to the placenta. Nutrients, growth factors, oxygen availability and cellular energy status have been shown to regulate mTOR activity in other cell types (Wullschleger et al. 2006), and identification of the upstream regulators of placental mTOR is needed in order to obtain further support for a role for mTOR as a placental nutrient sensor.

In summary, the novel findings in the present work are that the mTOR protein is expressed in the transporting epithelium of the human placenta and that inhibition of placental mTOR signalling in primary human placental villous fragments by rapamycin markedly decreased leucine uptake via the l-amino acid transporter but had no effect on system A and taurine transporters. The current study provides the first direct evidence of mTOR specifically regulating l-amino acid transport activity in mammalian cells. Since the intracellular leucine concentration is a potent activator of the mTOR signalling system, the regulation of the l-amino acid transporter by mTOR represents a powerful switch for altering cell growth in response to availability of nutrients. In addition, the protein expression of phosphorylated S6K1 (Thr-389), a key down-stream target of mTOR and a measure of the activity of the mTOR signalling pathway, was markedly down-regulated in placentas from IUGR pregnancies. Therefore, a decreased placental mTOR activity may represent the mechanism for the previously demonstrated decrease in placental leucine transport in IUGR. Since fetal growth is primarily determined by nutrient supply, the decreased placental mTOR activity in IUGR may represent a direct cause of the development of this pregnancy complication. Collectively, these data are compatible with the hypothesis that mTOR functions as a nutrient sensing pathway in the human placenta and identify mTOR signalling as an important regulator of placental transport of essential amino acids.

Appendix

Acknowledgements

This study was supported by grants from the Swedish Research Council (Grant 10838), the Swedish Diabetes Association, the Frimurare-Barnhus Direktionen, the Magnus Bergvall Foundation, the Åhlens Foundation, the Wilhelm and Martina Lundgren Foundation.

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