Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

CLINICAL PERSPECTIVES

  • Changes in placental amino acid transport directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. However, the molecular mechanisms regulating placental amino acid transporters are not well established.

  • We demonstrate that mTOR complex 1 modulates amino acid transport in primary human trophoblast cells by regulating Nedd4-2 mediated ubiquitination and plasma membrane trafficking of specific transporter isoforms.

  • Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth. These data advance our mechanistic understanding of common pregnancy complications and may provide a foundation for the development of intervention strategies for fetal growth restriction and overgrowth.

INTRODUCTION

Abnormal fetal growth increases the risk for perinatal complications and predisposes the infant for the development of obesity, diabetes and cardiovascular disease later in life [1,2]. Fetal growth is strongly dependent on nutrient availability, which is determined by placental nutrient transport [3,4]. The activity of key placental amino acid transporters is decreased in intrauterine growth restriction (IUGR) [58] and up-regulated in fetal overgrowth [9,10], suggesting that changes in the activity of placental nutrient transporters may directly contribute to abnormal fetal growth [1113].

The mechanistic target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that responds to nutrient availability and growth factor signalling to control cell growth and metabolism mediated by effects on gene transcription and protein translation [14,15]. mTOR resides in two distinct multiprotein complexes referred to as mTORC1 and mTORC2. mTORC1 phosphorylates S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), which mediates many of the down-stream effects of mTOR [14,15]. mTORC2 phosphorylates Akt, protein kinase Cα (PKCα) and serum and glucocorticoid-regulated kinase (SGK) and influences the actin skeleton and regulates cell metabolism [16,17]. DEPTOR, a protein containing two DEP (Dishevelled, Egl-10, Pleckstrin) domains, is an endogenous inhibitor of both mTORC1 and 2 signalling [18].

We have recently shown that silencing of mTORC1 and/or mTORC2 markedly inhibits trophoblast System A and System L amino acid transport activity by affecting transporter trafficking to the plasma membrane [19]. Both placental mTORC1 and mTORC2 activity is decreased in human IUGR [20,21] as well as in animal models of IUGR such as low protein diet in the rat [22]. In addition, maternal nutrient restriction in the baboon inhibits placental mTOR signalling [23]. These observations are consistent with an important role for placental mTOR signalling in regulating placental function and fetal growth and provide a possible mechanistic link between inhibition of placental mTOR signalling, down-regulation of placental nutrient transport and IUGR. However, the molecular mechanism by which mTOR regulates the cell surface expression of trophoblast amino acid transporters is largely unknown.

System A is a sodium-dependent transporter mediating the uptake of non-essential neutral amino acids into the cell [24]. All three known isoforms of System A, sodium-dependent neutral amino acid transporter 1 (SNAT1; SLC38A1), SNAT2 (SLC38A2) and SNAT4 (SLC38A4) are expressed in the placenta [25]. System L is a sodium-independent amino acid exchanger mediating cellular uptake of essential amino acids including leucine [26]. The System L amino acid transporter is a heterodimer, consisting of a light chain, typically LAT1 (SLC7A5) or LAT2 (SLC7A8), and a heavy chain, 4F2 cell surface antigen heavy chain (4F2hc)/CD98 (SLC3A2). Amino acid uptake via all SLC38 transporters is coupled to the inward movement of sodium down its electrochemical gradient, which helps develop an outwardly directed concentration gradient for System A substrates that can be utilized to drive the exchange uptake of a range of essential amino acids (e.g. leucine) through transporters (such as System L) that function in parallel with SLC38 in the plasma membrane.

Ubiquitination is a post-translational modification that has emerged as an important mechanism regulating the plasma membrane expression of transporters. Ubiquitination requires three sequential enzymatic steps (E1, E2 and E3), through which ubiquitin, a highly conserved 76-aa polypeptide, is attached to proteins. It was originally described as a signal that targets cellular proteins for rapid degradation, but was later found also to regulate numerous other processes in the cell, including protein trafficking [27]. Specificity of ubiquitination in mammals is provided by hundreds of different E3 ubiquitin–protein ligases that recognize relevant target proteins and promote the conjugation of ubiquitin. There are two major E3 classes–RING (really interesting new gene) and HECT (homologous with the E6-AP, carboxyl terminus)–the latter including the neuronal precursor cell-expressed, developmentally down-regulated (Nedd) 4-like proteins Nedd4-1 and Nedd4-2, which interact with target proteins through a specific motif, PY (L/PPxY) [28].

The molecular mechanisms regulating intracellular trafficking of membrane transporters have been established in detail for only two mammalian transporters, the epithelial sodium channel (ENaC) and glucose transporter 4 (GLUT4). Aldosterone and insulin regulate ENaC cell surface expression mediated by phosphorylation of SGK1 and Nedd4-2 [29,30]. Previous studies in mice with tissue specific Nedd4-2 knockout have revealed a critical role for this E3 ubiquitin ligase in regulating salt and fluid transport in the lung and kidney epithelia and in maintaining homoeostasis of systemic blood pressure [31]. Phosphorylation of Nedd4-2 interferes with the ability of the ubiquitin ligase to associate to ENaC, resulting in a decrease in ENaC internalization and, consequently increased cell surface expression of the transporter and increased sodium uptake [29,30,32,33].

Although ubiquitination is well established to control trafficking of amino acid permeases in yeast [34], little is known about the mechanisms involved in regulating the trafficking of amino acid transport in mammalian cells. We have previously reported that both mTORC1 and mTORC2 regulate amino acid uptake in cultured primary human trophoblast (PHT) cells by influencing the plasma membrane trafficking of specific transporter isoforms: the System A isoform SNAT 2 and the System L transporter isoform LAT1 [19]. Membrane SNAT2 stability is regulated by the ubiquitin proteasome system (UPS) [35,36]. The aim of the present study was to explore the mechanism by which mTOR regulates plasma membrane trafficking of SNAT 2 and LAT1 in PHT cells, with special focus on mTORC1 and Nedd4-2 mediated ubiquitination. We hypothesized that inhibition of mTORC1 decreases amino acid uptake in PHT cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane.

MATERIALS AND METHODS

Isolation and culture of primary human trophoblast (PHT) cells

Cytotrophoblast cells were isolated from normal term placentas and cultured in vitro [37,38]. Tissue collection was approved by the Institutional Review Board of the University of Colorado Anchutz Medical Campus. Cells were plated in either 60 mm culture dishes (∼7.5×106 cells/dish for Western blot analysis) or six-well plates (for amino acid uptake experiments; ∼3.75×106 cells/well for RNAi-mediated gene silencing) and cultured in 5% CO2, 95% atmosphere air at 37°C for 90 h. Cell culture medium (DMEM/Hams F-12, supplemented with L-glutamine, penicillin, streptomycin, gentamycin and 10% fetal bovine serum) was changed daily. We have previously reported that our cultured primary human trophoblast cells have a high expression of cytokeratin-7, a trophoblast specific marker, with no detectable expression of vimentin, a marker for mesenchyme-derived cells [19,39], confirming the high purity of our trophoblast cell population.

Assessment of biochemical differentiation and viability

To confirm that trophoblast cells were undergoing biochemical differentiation, and to assess their viability with time in culture, the release of human chorion gonadotropin (hCG) by trophoblast cells into the culture medium 18, 42, 66 and 90 h after plating was measured using a commercial ELISA kit, which detects the β-subunit of hCG (Immuno Biological Labs).

RNA interference-mediated silencing

Dharmafect 2 transfection reagent (Thermo Scientific) and siRNAs (Sigma–Aldrich), targeting raptor (100 nM; sense, 5′CAGUUCACCGCCAUCUACA′3), rictor (100 nM; sense, 5′ CGAUCAUGGGCAGGUAUUA′3), DEP domain-containing mTOR-interacting protein (DEPTOR) (SASI_1297010-H/5582, 1297011-H) or Nedd4-2 (100 nM; 5′CCCUAUACAUUUAAG-GACU′3) were used. Control cells were transfected with a non-coding scrambled sequence (100 nM; sense: 5′GAUCA-UACGUGCGAUCAGATT). siRNAs were added to cultured PHT cells (∼3.75×106 cells/well in six-well plate; ∼7.5×106 cells in 60 mm dish) after 18 h in culture, incubated for 24 h [40] and subsequently removed and replaced by fresh medium. At 90 h in culture, efficiency of target silencing was determined at the protein and functional levels using Western blot.

System A and System L amino acid uptake assay

Amino acid uptake in PHT cells was determined at 90 h in culture. The activity of System A and System L amino acid transporters was assessed by measuring the Na+-dependent uptake of [14C] methyl-aminoisobutyric acid (MeAIB) and the 2-amino-2-norbornane-carboxylic acid (BCH)-inhibitable uptake of [3H] leucine, respectively, as described in detail previously [19,41].

Isolation of MVM from trophoblast cells

Microvillous plasma membranes (MVMs) were isolated from total homogenates of cultured PHT cells using differential centrifugation and Mg2+ precipitation as described previously [19]. In brief, cells were lysed, scraped off the plate, homogenized and centrifuged. The pelleted crude membrane fraction was resuspended and 12 mM MgCl2 was added. The mixture was stirred slowly for 20 min on ice and then centrifuged. The supernatant containing MVM was centrifuged at 125000 g for 30 min and the final pellet was resuspended. Protein concentration was determined using the Bradford assay. MVM enrichment was assessed using the MVM/homogenate ratio of alkaline phosphatase protein expression as determined using Western blot after loading equal amounts of protein of MVM and cell lysates.

Western blotting

For immunoblotting, cells were lysed in buffer containing phosphatase and protease inhibitors. Subsequently, cells were scraped, collected and sonicated. Proteins in cell lysates and MVM were separated by electrophoresis. Western blotting was carried out as described [41]. Protein expression of Nedd4-2, ubiquitin and total and phosphorylated S6K (T-389), S6 (S-235/236), Akt (S-473), SGK (S-422) and PKCα (S-657), was determined in cell lysates using commercial antibodies (Cell Signaling Technology). Protein expression of the System A amino acid transporter isoform (SNAT2) and the System L amino acid transporter isoform (LAT1) was analysed in total cell lysates and MVM preparations. A polyclonal SNAT2 antibody generated in rabbits [42] was received as a generous gift from Dr. Prasad at University of Georgia, Augusta. The specificity of the SNAT2 antibody was validated by blocking peptide. An antibody targeting the LAT1 was produced in rabbits as described previously [43]. LAT1 antibody specificity was validated using gene-silencing approaches [44]. Anti-beta actin was from Sigma–Aldrich. The expression of actin was used as loading control. For each protein target the mean density of the control sample bands was assigned an arbitrary value of 1. All individual densitometry values were expressed relative to this mean.

Proximity ligation assay (PLA) and confocal microscopy

Isolated cytotrophoblast cells were grown on chamber slides (Lab-Tek) for 90 h. At 66 h, cells were treated with rapamycin (100 nM), a specific mTORC1 inhibitor to induce SNAT2/ubiquitin interaction. The cells were fixed in ice-cold methanol at −20°C for 20 min and were blocked using 5% newborn calf serum (NCS) in PBS for 1 h followed by anti-SNAT2 (anti-rabbit) and anti-ubiquitin (anti-mouse) incubation for 2 h. PLA probes anti-rabbit PLUS and anti-mouse MINUS were diluted in Duolink dilution buffer and incubated in a pre-heated humidity chamber for 1 h. This was followed by ligation, amplification and detection according to the Duolink In Situ Orange kit (Sigma–Aldrich) manufacturer's protocol. Confocal microscopy was performed using a Zeiss LSM 780 microscope at 63× magnification using oil immersion. Images were captured in the same laser settings with four Z-steps of 0.4 um.

Immunoprecipitation

PHT cell lysates were incubated with SNAT2 or LAT1 antibody overnight and antibodies were precipitated with protein G-Sepharose. Immunoprecipitates and aliquots of cell lysates were denatured in sample buffer at 95°C, resolved by electrophoresis, and probed with ubiquitin antibody.

Data presentation and statistics

The number of experiments (n) represents the number of placentas studied. Data are presented as means ± S.E.M or + S.E.M. Differences between two independent groups were tested using unpaired Student's t test. The repeated measures analysis of variance (RMANOVA) with Tukey's post-hoc test was used to assess statistical differences between scramble siRNA, raptor/rictor siRNA, rictor + DEPTOR siRNA or raptor/rictor + Nedd4-2 siRNA groups. P value <0.05 was considered significant.

RESULTS

DEPTOR silencing activates mTORC1 and mTORC2 signalling in PHT cells

We have previously reported that inhibition of mTORC1 or mTORC2 decreases amino acid uptake in PHT cells [19]. To further establish mTOR regulation of PHT amino acid transport we tested the hypothesis that activation of mTOR stimulates cellular amino acid uptake. DEPTOR is a constituent of both mTOR complexes and is considered a negative regulator of mTOR function [18]. Thus, to activate mTORC1 and mTORC2 signalling, we silenced the endogenous mTOR inhibitor DEPTOR in PHT cells. DEPTOR silencing in PHT cells decreased the protein expression of DEPTOR by 55% (P<0.001; n=5, Supplementary Figures S1a and S1b). We also measured the activity of the mTOR pathway in cells with RNAi-mediated DEPTOR silencing. Importantly, DEPTOR silencing significantly increased the phosphorylation of S6 kinase (Thr-389), 4E-BP1 (Thr-37/46), S6 ribosomal protein (Ser-235/236), functional readouts for mTORC1 and Akt (Ser-473), reflecting the activity of mTORC2 signalling (Supplementary Figures S1c and S1d). These results confirm that our DEPTOR RNAi approach efficiently activated both the mTORC1 and mTORC2 signalling pathways in PHT cells.

DEPTOR silencing does not affect PHT cell viability or differentiation

PHT cells form syncytial islands in culture, and this is used as a model for the syncytiotrophoblast, the transporting epithelium of the human placenta. After 66 h in culture, there was a marked increase in hCG production by trophoblast cells, and the levels remained high until at least 90 h after plating (Supplementary Figure S1e). Because hCG is produced predominantly by syncytialized cells, these data provide evidence of cell differentiation. Furthermore, hCG secretion profiles were similar in cells in which DEPTOR had been silenced as compared with cells incubated in scrambled siRNA (Supplementary Figure S1e). Collectively, these data indicate that key components of the trophoblast mTOR signalling pathway can be activated without adversely affecting trophoblast cell viability and differentiation.

DEPTOR silencing increases System A and System L amino acid uptake

To determine if the activation of mTORC1 and 2 signalling resulted in increased amino acid transport, we measured amino acid uptake in DEPTOR deficient cells. DEPTOR silencing caused a pronounced increase in System A (+379%, P<0.04; n=3, Figure 1a) and System L (+175%, P<0.05; n=4, Figure 1b) mediated amino acid uptake as compared with control. These data confirm that both mTORC1 and mTORC2 signalling pathways are positive regulators of trophoblast amino acid uptake.

Activation of mTORC1 and 2 stimulates PHT amino acid transport

Figure 1
Activation of mTORC1 and 2 stimulates PHT amino acid transport

(a and b) Silencing of DEPTOR activates System A and System L amino acid uptake in PHT cells. System A activity (a) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=3–4 for System A and System L. *P<0.05 compared with control; unpaired Student's t test. (c and d) Protein expression of System A (SNAT2) and System L (LAT1) amino acid transporter isoforms in MVMs isolated from control and DEPTOR silenced PHT cells. Representative Western blots are shown for sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) and L-type amino acid transporter (LAT1, 37 kDa) in MVMs of PHT cells transfected with scramble siRNA or siRNA targeting DEPTOR. Equal loading was performed. Histograms (e and f) summarize the data. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=6–7. *P<0.05 compared with control; unpaired Student's t test.

Figure 1
Activation of mTORC1 and 2 stimulates PHT amino acid transport

(a and b) Silencing of DEPTOR activates System A and System L amino acid uptake in PHT cells. System A activity (a) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=3–4 for System A and System L. *P<0.05 compared with control; unpaired Student's t test. (c and d) Protein expression of System A (SNAT2) and System L (LAT1) amino acid transporter isoforms in MVMs isolated from control and DEPTOR silenced PHT cells. Representative Western blots are shown for sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) and L-type amino acid transporter (LAT1, 37 kDa) in MVMs of PHT cells transfected with scramble siRNA or siRNA targeting DEPTOR. Equal loading was performed. Histograms (e and f) summarize the data. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=6–7. *P<0.05 compared with control; unpaired Student's t test.

DEPTOR silencing increases MVM System A and System L amino acid isoform expression

To study changes in cell surface transporter expression we isolated a MVM fraction from cultured primary trophoblast cells and determined isoform protein expression in response to activation of mTORC1 and 2 signalling. The justification for determining protein expression of transporters in MVM rather than in cell lysates is that trophoblast nutrient transporters mediate cellular uptake and transfer across the placental barrier only if localized in the syncytiotrophoblast plasma membranes. Thus, data on amino acid transporter protein expression in MVM are much more informative than determination of protein expression in cell lysates. Furthermore, we demonstrated that mTOR regulates placental System A and System L amino acid transporter isoform expression in MVM [19]. The enrichment of alkaline phosphatase, a MVM marker, was determined by the ratio of alkaline phosphatase expression in MVM over total cell lysates. Alkaline phosphatase enrichment in MVM isolated from control cells was 6.7±0.5, n=3, which was not different from MVM isolated from DEPTOR silenced cells (7.0±0.6, n=3). These data confirm significant MVM enrichment. We found that silencing of DEPTOR caused a marked increase in the expression of the System A transporter isoform SNAT2 (+73%, P<0.0005; n=6–7, Figures 1c and 1e) and the System L transporter isoform LAT1 (+69%, P<0.005; n=6–7, Figures 1d and 1f) in the MVM fraction. These data indicate that mTOR signalling regulates System A and System L transport by post-translational mechanisms.

mTORC 1 inhibition increases SNAT2 and LAT1 ubiquitination

Ubiquitination is a post-translational modification that has been shown to be involved in regulating the plasma membrane expression of mammalian transporters, in particular ENaC [45]. To study the molecular mechanisms linking mTORC1 signalling to System A and System L amino acid transporter intracellular trafficking, we measured System A (SNAT2) and System L (LAT1) transporter isoform ubiquitination in PHT cell lysates following mTORC1 inhibition. To this effect, we immunoprecipitated proteins in the cell lysate using an anti-SNAT2 or LAT1 antibody. The proteins in the immunoprecipitates were then separated by SDS-PAGE and subsequently immunoblotted with an anti-ubiquitin antibody. As shown in Figures 2(a) and 2(b), raptor silencing (mTORC1 inhibition) resulted in increased ubiquitination of SNAT2 and LAT1 as compared with control.

Increased ubiquitination of SNAT2 and LAT1 in response to mTORC1 inhibition

Figure 2
Increased ubiquitination of SNAT2 and LAT1 in response to mTORC1 inhibition

PHT cell lysates was used for IP with an anti-SNAT2 (a) or LAT1 antibody (b). Immunoprecipitated proteins were separated by SDS-PAGE and blotted (IB) with an anti-ubiquitin (Ub) antibody. The data are from a representative experiment, and similar results were obtained from two other experiments.

Figure 2
Increased ubiquitination of SNAT2 and LAT1 in response to mTORC1 inhibition

PHT cell lysates was used for IP with an anti-SNAT2 (a) or LAT1 antibody (b). Immunoprecipitated proteins were separated by SDS-PAGE and blotted (IB) with an anti-ubiquitin (Ub) antibody. The data are from a representative experiment, and similar results were obtained from two other experiments.

To further confirm that inhibition of mTORC1 increases the interaction between SNAT2 and ubiquitin, we used PLA (proximate ligation assay) and confocal microscopy, which allows the in situ detection of interacting endogenous proteins. The principle for PLA is described in supplementary Figure S2. A few SNAT2/ubiquitin complexes (yellow) were co-localized in the control/scramble cells (Figure 3). After rapamycin (a mTORC1 inhibitor) treatment and raptor siRNA silencing the number of SNAT2/ubiquitin complexes increased markedly (Figure 3), suggesting increased ubiquitination of SNAT2.

Interaction of SNAT2 and ubiquitin following mTORC1 inhibition

Figure 3
Interaction of SNAT2 and ubiquitin following mTORC1 inhibition

Immunofluorescence confocal microscopy in combination with in situ PLA, which detects protein–protein complexes, was used to explore interactions between SNAT2 and ubiquitin following mTORC1 inhibition with rapamycin or raptor siRNA. Each detected complex is represented by a yellow dot. DNA was counterstained by DAPI (blue). Scale bar represents 20 μm.

Figure 3
Interaction of SNAT2 and ubiquitin following mTORC1 inhibition

Immunofluorescence confocal microscopy in combination with in situ PLA, which detects protein–protein complexes, was used to explore interactions between SNAT2 and ubiquitin following mTORC1 inhibition with rapamycin or raptor siRNA. Each detected complex is represented by a yellow dot. DNA was counterstained by DAPI (blue). Scale bar represents 20 μm.

mTORC1 but not mTORC2 silencing increases Nedd4-2 expression

Nedd4-2 is one of the E3 ubiquitin ligases that catalyses ubiquitination of plasma membrane transporter and controls cell surface expression of transporters [45]. Membrane trafficking of the System A amino acid transporter isoform SNAT 2 in 3T3-L1 adipocytes is regulated by Nedd4-2 dependent ubiquitination [36]. Moreover, aldosterone and insulin regulate ENaC cell surface expression mediated by phosphorylation of SGK1 and Nedd4-2 [29,30]. Because SGK1 is a downstream target of mTORC2, these data implicate mTORC2 in Nedd4-2 mediated transporter ubiquitination. This provides the rationale for studying the regulation of Nedd4-2 by mTORC1 and mTORC2 in PHT cells. We found that silencing of raptor, but not rictor silencing (specifically inhibiting mTORC2), increased the expression of Nedd4-2 (+81%, P<0.05; n=3, Supplementary Figures S3a and S3b) as compared with PHT cells transfected with scramble siRNA. Because SGK1 has been shown to regulate Nedd4-2 we explored the possibility that mTORC1 signals through SGK1 in PHT cells. However, mTORC1 inhibition by raptor silencing did not alter the phosphorylation of SGK1 in PHT cells (Supplementary Figures S3c and S3d). Similarly, silencing of raptor did not alter the phosphorylation of PKCα, another mTORC2 target (Supplementary Figures S3c and S3d).

Nedd4-2 silencing increases System A and System L amino acid transport

Next, we determined the effect of Nedd4-2 silencing on amino acid transport activity. To confirm efficient silencing, we performed Western blots on siRNA-transfected cells at 90 h of culture (Supplementary Figures S4a and S4b). Nedd4-2 siRNA decreased the Nedd4-2 protein expression by 70% (P<0.0005; n=3) as compared with control cells. Furthermore, hCG secretion profiles were similar in cells in which Nedd4-2 had been silenced as compared with cells incubated in scrambled siRNA (Supplementary Figure S4c). As shown in Figure 4, Nedd4-2 silencing resulted in a marked increase in System A (+77%, P<0.05; n=5, Figure 4a) and System L (+126%, P<0.001; n=5, Figure 4b) amino acid uptake as compared with control, suggesting that Nedd4-2 is involved in the regulation of amino acid transporter activity.

Nedd4-2 silencing promotes trafficking of SNAT2 and LAT1 to the plasma membrane

We found that Nedd4-2 silencing caused a marked increase in the expression of the System A transporter isoform SNAT2 [+120%, P<0.05; n=3, Figures 4(c) and 4(d)] and System L transporter isoform LAT1 [+135%, P<0.005; n=3, Figures 4(c) and 4(d)] in the microvillous plasma membrane fraction. Collectively, these data support the hypothesis that Nedd4-2 regulates System L and System A amino acid transporter activity by modulating transporter trafficking through ubiquitination of specific transporter isoforms.

Nedd4-2 silencing stimulates PHT amino acid transport

Figure 4
Nedd4-2 silencing stimulates PHT amino acid transport

System A activity (a) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=5 for System L and System A. *P<0.05 compared with control; unpaired Student's t test. Representative Western blots are shown for L-type amino acid transporter (LAT1, 37 kDa) and sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) in MVM of control and Nedd4-2 silenced cells (c). Equal loading was performed. Histogram (d) summarizes the data. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=6–7. *P<0.05 compared with control; unpaired Student's t test.

Figure 4
Nedd4-2 silencing stimulates PHT amino acid transport

System A activity (a) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=5 for System L and System A. *P<0.05 compared with control; unpaired Student's t test. Representative Western blots are shown for L-type amino acid transporter (LAT1, 37 kDa) and sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) in MVM of control and Nedd4-2 silenced cells (c). Equal loading was performed. Histogram (d) summarizes the data. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=6–7. *P<0.05 compared with control; unpaired Student's t test.

Rictor + DEPTOR silencing efficiency

Our data suggest that mTORC1 inhibition (raptor silencing) increased the Nedd4-2 expression (Supplementary Figures S2a and S2b) and increased the ubiquitination of SNAT2 and LAT1 isoforms (Figure 2). As expected, silencing of raptor decreased the phosphorylation of mTORC1 functional readouts S6K and S6 but had no effect on the phosphorylation of Akt at Ser-473, a functional readout for mTORC2 (Supplementary Figure S5). This observation suggests that there is no significant cross-talk between mTORC1 and mTORC2 in PHT cells. To further confirm that Nedd4-2 is involved in mTORC1-mediated membrane trafficking of transporters, we studied Nedd4-2 expression, amino acid transporter activity and expression in PHT cells with activation of mTORC1. To selectively activate mTORC1, we silenced DEPTOR (activates mTORC1 and mTORC2) and rictor (inhibits mTORC2). Silencing of PHT cells with rictor + DEPTOR siRNA increased the phosphorylation of S6K (Thr-389) and S6 (Ser-235/236) but did not affect the phosphorylation of Akt at Ser-473 (Supplementary Figure S5), consistent with specific activation of mTORC1.

mTORC1 activation increases System A and System L amino acid transport activity and isoform expression

Confirming our previous studies [19], inhibition of mTORC1 by silencing of raptor decreased System A (−69%, P<0.05; n=4, Figure 5a) and System L (−67%, P<0.05; n=4, Figure 5b) amino acid transporter activity as compared with cells transfected with scramble siRNA. Activation of mTORC1 by silencing rictor + DEPTOR decreased Nedd4-2 protein expression (data not shown) and increased System A (+64%, P<0.05; n=4, Figure 5a) and System L (+48%, P<0.05; n=4, Figure 5b) activity. Furthermore, inhibition of mTORC1 decreased the expression of the System A transporter isoform SNAT2 (−56%, P<0.05; n=3, Figures 5c and 5d) and the System L transporter isoform LAT1 (−76%, P<0.05; n=4, Figures 5c and 5e) in the microvillous plasma membrane fraction as compared with cells transfected with scramble siRNA. In contrast, activation of mTORC1 by silencing rictor + DEPTOR increased SNAT 2 (+64%, P<0.05; n=3, Figures 5c and 5d) and LAT1 expression in MVM (+107%, P<0.01; n=3, Figures 5c and 5e). These data are consistent with the model that mTORC1 activation stimulates PHT amino acid uptake and cell surface expression of key amino acid transporter isoforms by down-regulation of Nedd4-2.

Activation of mTORC1 increases System A and System L amino acid uptake in PHT cells

Figure 5
Activation of mTORC1 increases System A and System L amino acid uptake in PHT cells

System A activity (a) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=3–4 for System L and System A. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test. Representative Western blots are shown for L-type amino acid transporter (LAT1, 37 kDa) and sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) in MVMs of scramble siRNA, raptor siRNA and rictor + DEPTOR silenced PHT cells (c). Equal loading was performed. Histograms (d and e) summarize the results. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=4. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test.

Figure 5
Activation of mTORC1 increases System A and System L amino acid uptake in PHT cells

System A activity (a) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=3–4 for System L and System A. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test. Representative Western blots are shown for L-type amino acid transporter (LAT1, 37 kDa) and sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) in MVMs of scramble siRNA, raptor siRNA and rictor + DEPTOR silenced PHT cells (c). Equal loading was performed. Histograms (d and e) summarize the results. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=4. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test.

Nedd4-2 is required for mTORC1 but not mTORC2 regulation of System A and System L amino acid transport activity and isoform plasma membrane trafficking

To mechanistically link mTOR, Nedd4-2 and amino acid transport activity, PHT cells were transfected with raptor or rictor siRNA or raptor + Nedd4-2 or rictor + Nedd4-2 siRNA, and System A and System L amino acid transport activity were measured at 90 h. As shown in Figure 6, silencing of raptor (System A, −57%, P<0.01; System L, −55%, P<0.01; n=5, Figures 6a and 6b) or rictor (System A, −59%, P<0.01; System L, −58%, P<0.02; n=5, Figures 6c and 6d) markedly inhibited basal levels of System A and System L amino acid transporter activity in cultured primary human trophoblast cells as compared with cells transfected with scramble siRNA. These data confirm our previous observations that mTORC1 and mTORC2 independently regulate trophoblast amino acid transport [19]. Raptor + Nedd4-2 silencing prevented the inhibitory effect of mTORC1 silencing on System A (n=5, Figure 6a) and System L (n=5, Figure 6b) amino acid transport activity. In contrast, Nedd4-2 silencing did not influence the ability of mTORC2 silencing to inhibit System A (Figure 6c) and System L (Figure 6d) amino acid transport activity.

Nedd4-2 is required for mTORC1 but not mTORC2 regulation of System A and System L amino acid transport activity and isoform plasma membrane trafficking

Figure 6
Nedd4-2 is required for mTORC1 but not mTORC2 regulation of System A and System L amino acid transport activity and isoform plasma membrane trafficking

System A activity (a and c) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b and d) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=5 for System L and System A. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test. Representative Western blots are shown for sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) and L-type amino acid transporter (LAT1, 37 kDa) in MVM of scramble, raptor/rictor and raptor or rictor + Nedd4-2 silenced cells (e and h). Equal loading was performed. Histograms (f, g, i and j) summarize the data. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=3. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test.

Figure 6
Nedd4-2 is required for mTORC1 but not mTORC2 regulation of System A and System L amino acid transport activity and isoform plasma membrane trafficking

System A activity (a and c) was determined as the Na+-dependent uptake of [14C] MeAIB, and System L activity (b and d) was measured as the BCH-inhibitable uptake of [3H] leucine. Values are means + S.E.M.; n=5 for System L and System A. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test. Representative Western blots are shown for sodium-coupled neutral amino acid transporter (SNAT2, 52 kDa) and L-type amino acid transporter (LAT1, 37 kDa) in MVM of scramble, raptor/rictor and raptor or rictor + Nedd4-2 silenced cells (e and h). Equal loading was performed. Histograms (f, g, i and j) summarize the data. Expression in control cells for each isoform was arbitrarily assigned a value of one. Values are means + S.E.M.; n=3. Means without a common letter differ significantly (P<0.05) by RMANOVA with Tukey–Kramer multiple comparisons post hoc test.

Inhibition of mTORC1 or mTORC2 caused a marked decrease in the expression of the System A transporter isoform SNAT2 (raptor siRNA: −67%, P<0.05; n=3, Figures 6e and 6f; rictor siRNA: −71%, P<0.01; n=3, Figures 6h and 6i) and System L transporter isoform LAT1 (raptor siRNA: −65%, P<0.05; n=3, Figures 6e and 6g; rictor siRNA: −47%, P<0.01; n=3, Figures 6h and 6j) in the MVM, as compared with cells treated with scramble siRNA. Nedd4-2 silencing prevented the decrease in SNAT2 (Figures 6e and 6f) and LAT1 (Figures 6e and 6g) MVM expression in response to mTORC1 inhibition. In contrast, Nedd4-2 silencing did not influence the decrease in SNAT2 (Figures 6h and 6i) and LAT1 (Figures 6h and 6j) MVM expression following mTORC2 inhibition. These data demonstrate that Nedd4-2 is required for the regulation of plasma membrane trafficking of amino acid transporter isoforms by mTORC1 but not mTORC2.

DISCUSSION

The novel finding in this report is that Nedd4-2 is required for the regulation of plasma membrane trafficking of amino acid transporter isoforms by mTORC1. First, silencing of mTORC1 increased Nedd4-2 expression and caused ubiquitination of the System A and System L isoforms SNAT2 and LAT1. Second, Nedd4-2 inhibition stimulated System A and System L amino acid transporter activity by increasing SNAT2 and LAT1 expression in MVM. Third, Nedd4-2 silencing prevented the decrease in System A and System L activity in response to mTORC1 inhibition. These findings are consistent with the model that inhibition of placental mTORC1 in human IUGR [20,21] increases the ubiquitination of specific System A and System L amino acid transporter isoforms mediated by activation of Nedd4-2 (Figure 7), resulting in decreased placental amino acid transport and fetal amino acid availability [58].

A proposed model linking mTORC1 inhibition to decreased placental amino acid transport in human IUGR

Figure 7
A proposed model linking mTORC1 inhibition to decreased placental amino acid transport in human IUGR

The findings of the present study are consistent with the model that inhibition of placental mTORC1 in human IUGR increases the ubiquitination of specific System A and System L amino acid transporter isoforms mediated by activation of Nedd4-2, resulting in decreased placental amino acid transport and fetal amino acid availability.

Figure 7
A proposed model linking mTORC1 inhibition to decreased placental amino acid transport in human IUGR

The findings of the present study are consistent with the model that inhibition of placental mTORC1 in human IUGR increases the ubiquitination of specific System A and System L amino acid transporter isoforms mediated by activation of Nedd4-2, resulting in decreased placental amino acid transport and fetal amino acid availability.

DEPTOR binds to both mTORC1 and mTORC2, as evident from co-immunoprecipitation (IP) experiments [18]. Its precise function is not fully elucidated, but Peterson et al. [18] have shown in a series of elegant experiments that knocking down DEPTOR leads to activation of signalling through mTORC1 and mTORC2. We have previously shown that inhibition of mTORC1 by raptor silencing or inhibition of mTORC2 using silencing of rictor causes a marked decrease in System A and System L amino acid transport, mediated by altered plasma membrane trafficking of SNAT2 and LAT1 [19]. Here, we extend these studies and demonstrate that activation of mTORC1 and mTORC2 by DEPTOR silencing promotes trafficking of SNAT2 and LAT1 to the plasma membrane, which increases System A and System L amino acid transporter activity. This is the first report showing up regulation of amino acid transport by mTORC1 and 2 activation, in any cell type, and confirms an important role of mTOR in regulating trophoblast amino acid transport. The ubiquitin proteasome pathway is involved in the degradation of plasma membrane transporters [46]. Nedd4-2 belongs to the family of homologous with E6-AP COOH-terminus (HECT) E3 ligases that catalyse the final step in the ubiquitination cascade, the conjugation of ubiquitin to lysine residues of their target proteins, thus targeting them for degradation [27,47]. Nedd4-2 ubiquitinates the ENaC [48], the chloride channel ClC-5, voltage-gated potassium and the cardiac potassium channel hERG1 [49]. In the present study we found that mTORC1 silencing increased Nedd4-2 expression and SNAT2 ubiquitination, which is associated with a decreased translocation of amino acid transporter SNAT2 from an intracellular pool to the MVM. Furthermore, silencing Nedd4-2 increases SNAT2 and LAT1 expression in MVM and prevented mTORC1 inhibition of System A and System L activity. Because we previously have reported that mTORC1 regulates the plasma membrane trafficking of specific isoforms of System A (SNAT 2 but not SNAT 1) and System L (LAT1 but not LAT2) [19], mTORC1-mediated ubiquitination is likely to specifically target a small group of proteins. Taken together, these data are consistent with an important role in Nedd4-2 mediated ubiquitination in mediating the effect of mTORC1 on transporter isoform plasma membrane trafficking and transport activity.

Our data in primary human trophoblast cells are in general agreement with the observation by Hatanaka et al. [36] that membrane trafficking of the System A amino acid transporter isoform SNAT 2 in 3T3-L1 adipocytes is regulated by Nedd4-2 dependent ubiquitination. It is therefore possible that Nedd4-2 dependent ubiquitination is a mechanism by which key amino acid transporters are regulated in many cell types. The current study extends previous knowledge by demonstrating that mTORC1 inhibition increases Nedd4-2 protein expression and that Nedd4-2 is required for mTORC1 regulation of System A and System L activity. The mechanism involved in mTORC1 modulation of Nedd4-2 remains to be established. Although it was reported previously that mTORC2 regulates ENaC via SGK1 phosphorylation [50], it is unknown whether mTORC1 regulates amino acid transporter trafficking mediated by this signalling pathway. However, our data show that mTORC1 does not signal though SGK in PHT cells.

Although both mTORC1 and mTORC2 signalling regulates System A and System L amino acid uptake by governing the cell surface expression of SNAT2 and LAT1, the current study demonstrates that distinct molecular mechanisms are involved. Specifically, whereas Nedd4-2 is required for mTORC1-mediated regulation of plasma membrane trafficking of amino acid transporters, mTORC2 regulation of amino acid transport in PHT cells is independent of Nedd4-2. Given the involvement of SGK1, a mTORC2 target, and Nedd4-2 in ENaC trafficking [51] this finding was unexpected. However, this may reflect that primary human cells behave differently than transformed cell lines. Alternatively, the biology of human trophoblast cells may differ from primary cells isolated from other tissues. This possibility is supported by the observation that full length adiponectin, which is well known to promote insulin sensitivity in, for example, muscle and liver, has the opposite effect in trophoblasts in vitro [52,53] and the placenta in vivo [54]. Nevertheless, the mechanisms linking mTORC2 to increased cell surface expression of amino acid transporter remains to be established.

Abnormal fetal growth affects 15% of all babies and increases the risk for injuries at delivery and to develop obesity, diabetes and cardiovascular disease in childhood and later in life [1,2]. No specific strategies to treat these conditions are currently available. Altered placental nutrient transport is believed to directly contribute to changes in fetal growth [1113] and in order to better understand the underlying causes of these conditions identification of key mechanisms regulating the transfer of nutrients in the human placenta is critically needed. Placental mTORC1 signalling is inhibited in human IUGR [20,55] and activated in association with fetal overgrowth [10]. Furthermore, observations from our laboratory show that placental Nedd4-2 protein expression and SNAT2 ubiquitination are increased and SNAT2 expression in the syncytiotrophoblast plasma membranes is decreased in IUGR [56]. These findings together with the data reported in the current study suggest that placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

AUTHOR CONTRIBUTION

Fredrick Rosario, Thomas Jansson and Theresa Powell made contribution to conception and design of the experiments. Fredrick Rosario, Thomas Jansson and Theresa Powell performed collection, analysis and interpretation of data; Kris Genelyn Dimasuay performed confocal analysis; Fredrick Rosario, Thomas Jansson, Yoshikatsu Kanai and Theresa Powell wrote the manuscript. All authors approved the final version of the manuscript.

Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advance Light Microscopy Core.

FUNDING

This work was supported by the National Institutes of Health/National Center for Advancing Translational Sciences Colorado Clinical & Translational Sciences Institute [grant number UL1 TR001082]; and the National Institutes of Health [grant number HD068370].

Abbreviations

     
  • 4E-BP1

    eukaryotic initiation factor 4E binding protein 1

  •  
  • BCH

    2-amino-2-norbornane-carboxylic acid

  •  
  • DEPTOR

    DEP domain-containing mTOR-interacting protein

  •  
  • ENaC

    epithelial sodium channel

  •  
  • hCG

    human chorion gonadotropin

  •  
  • HECT

    homologous with the E6-AP, carboxyl terminus

  •  
  • IP

    immunoprecipitation

  •  
  • IUGR

    intrauterine growth restriction

  •  
  • LAT

    large neutral amino acid transporter

  •  
  • MeAIB

    methyl-aminoisobutyric acid

  •  
  • mTOR

    mechanistic target of rapamycin

  •  
  • mTORC1 and 2

    mechanistic target of rapamycin complex 1 and 2

  •  
  • MVM

    microvillous plasma membrane

  •  
  • Nedd4-2

    neuronal precursor cell-expressed, developmentally down-regulated gene 4 isoform 2

  •  
  • PHT

    primary human trophoblast

  •  
  • PKCα

    protein kinase Cα

  •  
  • PLA

    proximity ligation assay

  •  
  • raptor

    regulatory associated protein of mTOR

  •  
  • rictor

    rapamycin-insensitive companion of mTOR

  •  
  • RMANOVA

    repeated measures analysis of variance

  •  
  • S6K1

    p70 S6 kinase

  •  
  • SGK1

    serum and glucocorticoid-regulated kinase 1

  •  
  • SNAT

    sodium-dependent neutral amino acid transporter

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Supplementary data