The Journal of Physiology

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1 J Physiol (2017) pp mtor folte sensing links folte vilility to tropholst cell function Fredrick J. Rosrio 1,TheresL.Powell 1,2 nd Thoms Jnsson 1 1 Division of Reproductive Sciences, Deprtment of Ostetrics nd Gynecology, University of Colordo Anschutz Medicl Cmpus, Auror, CO 80045, USA 2 Section of Neontology, Deprtment of Peditrics, University of Colordo Anschutz Medicl Cmpus, Auror, CO 80045, USA The Journl of Physiology Key points Folte deficiency during pregnncy is ssocited with restricted fetl growth, lthough the underlying mechnisms re poorly understood. Here we show tht mechnistic trget of rpmycin (mtor) functions s folte sensor in primry humn tropholst (PHT) cells. Folte sensing y mtor in PHT cells involves oth mtor Complex 1 nd 2 nd requires the proton-coupled folte trnsporter. We report previously unknown moleculr mechnism y which folte regultes tropholst cell function. Becuse mtor is positive regultor of plcentl mino cid trnsport nd mitochondril function, plcentl mtor folte sensing my constitute the mechnistic link etween mternl folte sttus nd fetl growth. These findings provide new insight into how folte influences humn cell physiology nd my hve implictions for our understnding of how ltered folte vilility cuses diseses such s fetl growth restriction, fetl mlformtions nd cncer. Astrct Folte is wter-solule B vitmin tht is essentil for cellulr methyltion rections nd DNA synthesis nd repir. Low mternl folte levels in pregnncy re ssocited with fetl growth restriction, ut the underlying mechnisms re poorly understood. Mechnistic trget of rpmycin (mtor) links nutrient vilility to cell growth nd function y regulting gene expression nd protein trnsltion. Here we show tht mtor functions s folte sensor in primry humn tropholst (PHT) cells. Folte deficiency in PHT cells cused inhiition of mtor signlling nd decresed the ctivity of key mino cid trnsporters. Folte sensing y mtor in PHT cells involves oth mtor Complex 1 nd 2 nd requires the proton-coupled folte trnsporter (PCFT, SLC46A1). The involvement of PCFT in mtor folte sensing is not dependent on its function s plsm memrne folte trnsporter. Incresing levels of homocysteine hd no effect on PHT mtor signlling, suggesting tht mtor senses low folte rther thn high homocysteine. In ddition, we demonstrte tht mternl serum folte is positively correlted to plcentl mtorc1 nd mtorc2 signlling ctivity in humn pregnncy. We hve identified previously unknown moleculr link etween folte vilility nd cell function involving PCFT nd mtor signlling. We propose tht mtor folte sensing in tropholst cells mtches plcentl nutrient trnsport, nd therefore fetl growth, to folte vilility. These findings my hve implictions for our understnding of how ltered folte vilility cuses humn diseses such s fetl growth restriction, fetl mlformtions nd cncer. (Received 11 Mrch 2017; ccepted fter revision 29 Mrch 2017; first pulished online 4 April 2017) Corresponding uthor F. J. Rosrio: Division of Reproductive Sciences, Deprtment of Ostetrics nd Gynecology, University of Colordo Anschutz Medicl Cmpus, Est 19th Avenue, Auror, CO 80045, USA. Emil: fredrick.joseph@ucdenver.edu DOI: /JP272424

2 4190 F. J. Rosrio nd others J Physiol Arevitions BCH, 2-mino-2-norornne-croxylic cid; DEPTOR, DEP domin-contining mtor-intercting protein; DMEM, Dulecco s modified Egle medium; 4E-BP1, 4-eukryotic initition fctor inding protein-1; hcg, humn chorion gondotropin; IUGR, intruterine growth restriction; MeAIB, methyl-minoisoutyric cid; mtor, mechnistic trget of rpmycin; mtorc1 nd 2, mechnistic trget of rpmycin complex 1 nd 2; NCS, neworn clf serum; NTD, neurl tue defect; PCFT, proton coupled folte trnsport; PLA, proximity ligtion ssy; rptor, regultory ssocited protein of mtor; rictor, rpmycin-insensitive compnion of mtor; SGK1, serum nd glucocorticoid-regulted kinse 1; sirna, smll interfering RNA; S6K1, p70 S6 kinse. Introduction Low mternl folte intke nd reduced red lood cell folte levels re linked to restricted fetl growth (Tmur & Piccino, 2006; Fekete et l. 2012; vn Uitert & Steegers-Theunissen, 2013). Periconceptionl folte deficiency is risk fctor for fetl structurl mlformtions such s neurl tue defects (NTDs) (Smithells et l. 1976; Czeizel & Duds, 1992). Conversely, mternl folic cid supplementtion reduces the risk of NTDs (MRC, 1991) nd, t lest in some studies, increses irth weight (Iyengr & Bu, 1975; Iyengr & Rjlkshmi, 1975). However, the mechnisms linking mternl folte levels to fetl growth nd neurl tue closure re poorly understood. Folte is wter-solule B vitmin tht is essentil for the synthesis of purine nd thymidine nucleotides, which re needed for DNA repliction nd repir (Stover, 2004). Furthermore, dequte folte sttus is importnt for the production of S-denosylmethionine (SAM), universl donor of methyl groups for numer of methyltion rections, including DNA methyltion, process centrl to gene silencing (Stover, 2004). Chnges in DNA methyltion nd ltertions in the synthesis of nucleotide precursors hve een implicted in the development of NTDs (Blom et l. 2006; Wilde et l. 2014). Mechnistic trget of rpmycin (mtor) is serine/threonine kinse tht is ctivted y mino cids, glucose, ATP, oxygen nd growth fctor signlling. mtor functions s mster regultor of protein trnsltion, therey controlling cell growth, prolifertion nd metolism (Dennis et l. 2001; Peng et l. 2002; Jcinto & Hll, 2003; Mrtin & Hll, 2005; Dnn et l. 2007; Gulti & Thoms, 2007; Lplnte & Stini, 2012; Wiczer & Thoms, 2012; Shimoyshi & Hll, 2014; Alert & Hll, 2015). mtor exists in two complexes, mtor Complex 1 (mtorc1) nd 2, with the protein rptor (regultory ssocited protein of mtor) ssocited to mtorc1 nd rictor (rpmycin-insensitive compnion of mtor) ssocited to mtorc2. mtorc1 phosphoryltes S6K1 (p70 S6 kinse) nd 4E-BP1 (4-eukryotic initition fctor inding protein-1), resulting in incresed protein trnsltion. mtorc2 phosphoryltes Akt, protein kinse Cα (PKCα) nd serum nd glucocorticoid-regulted kinse (SGK) nd influences the ctin skeleton, regultes metolism nd promotes cell survivl (Jcinto et l. 2004; Alessi et l. 2009; Lplnte & Stini, 2012). We hve provided compelling evidence tht oth mtorc1 nd mtorc2 re powerful positive regultors of plcentl mino cid trnsporters (Roos et l. 2007, 2009; Rosrio et l. 2013, 2016; Chenet l. 2015). Specificlly, mtorc1 regultes tropholst System A nd System L mino cid trnsport t the post-trnsltionl level y influencing the trfficking of specific mino cid trnsporter isoforms to the plsm memrne y Nedd4-2 (neuronl precursor cell-expressed, developmentlly downregulted gene 4 isoform 2)-medited uiquitintion (Rosrio et l. 2016). Plcentl mtorc1 ctivity is inhiited in humn intruterine growth restriction (IUGR) (Roos et l. 2007; Yung et l. 2008; Chen et l. 2015) nd ctivted in plcents of lrge ies orn to oese mothers (Jnsson et l. 2013). Furthermore, plcentl mtorc1 ctivity hs een reported to e decresed in hyperthermi-induced IUGR in sheep (Arroyo et l. 2009), in response to mternllowproteindietinthert(rosrioet l. 2011) nd mternl clorie restriction in the oon (Kvith et l. 2014) nd ctivted in mouse model of mternl oesity ssocited with fetl overgrowth (Aye et l. 2015; Rosrio et l. 2016). Importntly, plcentl mino cid trnsport hs een reported to e chnged in the sme direction s mtor signlling in these clinicl conditions nd niml models of ltered fetl growth (Ross et l. 1996; Anderson et l. 1997; de Vrijer et l. 2004; Rosrio et l. 2011; Jnsson et l. 2013; Kvith et l. 2014; Aye et l. 2015; Chen et l. 2015). Collectively, these dt suggest tht tropholst mtor signlling regultes fetl growth y modulting plcentl nutrient trnsport. The primry mechnism y which mtor controls cell growth is y regulting protein trnsltion (Lplnte & Stini, 2012). mtor is n mino cid sensor (Gulti & Thoms, 2007; Lplnte & Stini, 2012; Efeyn et l. 2015) providing direct link etween mino cid vilility, protein synthesis nd cell growth. Cell growth nd prolifertion lso require sufficient supply of folte, prtly due to its criticl role in DNA repir nd de novo DNA synthesis. Both folte deficiency (Tmur & Piccino, 2006; Fekete et l. 2012; vn Uitert & Steegers-Theunissen, 2013) nd inhiition of mtor re ssocited with restricted fetl growth (Roos et l. 2007; Rosrio et l. 2011; Kvith et l. 2014; Chen et l. 2015). These considertions provided the rtionle to test

3 J Physiol mtor functions s folte sensor 4191 the hypothesis tht tropholst mtor functions s folte sensor, representing novel moleculr link etween folte vilility nd fetl growth. In the current study, we cultured primry humn tropholst (PHT) cells to explore the effect of folte deficiency on mtorc1 nd mtorc2 signlling nd used gene trgeting pproches to determine the role of the proton-coupled folte trnsporter (PCFT) s mechnistic link etween folte vilility nd mtor signlling. Methods Collection of plcentl tissue Term plcentl tissue ws collected from helthy pregnnt women undergoing elective Cesren section fter written informed consent. Smples nd medicl informtion were dded to tissue repository pproved y the Colordo Multiple Institutionl Review Bord ( ) nd susequently study personnel provided de-identified smples nd clinicl informtion used in this study. Isoltion nd culture of PHT cells Primry PHT cells were isolted nd cultured in vitro (Klimn et l. 1986; Rosrio et l. 2016). Cells were plted in either 60 mm culture dishes ( cells per dish for Western lot nlysis or six-well pltes for methyl-tetrhydrofolte (MTHF) uptke experiments; cells per well for RNAi medited gene silencing) nd cultured in 5% CO 2, 95% tmospheric ir t 37 C for 90 h. PHT cells were cultured in norml cell culture medi [Dulecco s modified Egle medium (DMEM)/Hms F-12, supplemented with L-glutmine, penicillin, streptomycin, gentmycin nd 10% fetl ovine serum] or in low folte contining medi, contining no folte except folte present in 10% serum (folte-free DMEM, Ct. No. D2429; Sigm, St. Louis, MO, USA) nd Hms F12 (Ct. No. ME L1; Invitrogen, Crlsd, CA, USA) supplemented with L-glutmine, penicillin, streptomycin, gentmycin nd 10% fetl ovine serum. Cell culture medi were chnged dily. We hve previously reported tht our PHT cells hve high expression of cytokertin-7, tropholst-specific mrker, with no detectle expression of vimentin, mrker for mesenchyme-derived cells (Lger et l. 2013; Rosrio et l. 2013), confirming the high purity of our tropholst cell popultion. Folte deficiency in cultured PHT cells To study the effect of folte deficiency on mtor signlling, PHT cells were cultured in folte-deficient medi for 24, 48, 72 or 90 h s illustrted in Fig. 1A. Effect of homocysteine on mtor signlling PHT cells were cultured in DMEM + F12 medium with or without DL-homocystine(5,20or100μM l 1 ) for 10 h (etween 80 nd 90 h fter plting). At 18, 42, 66, 90 h of culture, the medi were removed nd stored t 20 C for determintion of humn chorion gondotropin (hcg). Folte mesurement in PHT cells PHT cell lystes for determintion of intrcellulr folte concentrtions were prepred y incuting pproximtely 5 million PHT cells per 60 mm dish, which were cultured without ntiiotics for h. Susequently, cells were scrped, collected, lysed in scoric cid (%), sonicted nd freeze thwed. Folte content in PHT cell lystes ws mesured y using commercilly ville kit (ALPCO Dignostic Products, Windhm, NH, USA), ccording to the mnufcturer s instructions. Assessment of PHT cell differentition nd viility To confirm tht tropholst cells were undergoing iochemicl differentition, nd to ssess cell viility, the relese of hcg into the culture medium ws mesured using commercil ELISA kit which detects the β-suunit of hcg (Immuno Biologicl Ls, Minnepolis, MN, USA). Syncytin expression ws mesured s n index of tropholst cell cell fusion nd differentition in culture. Antiodies directed ginst cspse-3 (R&D Systems, Minnepolis, MN, USA) were used to determine the presence of poptosis in culture. RNA interference-medited silencing Dhrmfect 2 trnsfection regent (Thermo Scientific, Rockford, IL, USA) nd smll interference RNAs (sirnas) (Sigm-Aldrich), trgeting PCFT (SASI Hs ), DEPTOR (DEP domin-contining mtor-intercting protein; SASI H/5582, H) or non-coding scrmled sequence (100 nm; sense: 5 GAUCAUACGUGCGAUCAGATT) were dded to cultured PHT cells ( cells per well in six-well pltes; cellsin60mmdish)fter18hin culture nd incuted for 24 h. Susequently, sirnas were removed nd fresh medium ws dded. At 90 h in culture, silencing efficiency ws determined t the protein nd functionl levels using Western lot. System A nd system L mino cid trnsport ssy Amino cid uptke in cultured PHT cells ws determined t 90 h in culture. The ctivity of System A nd System L mino cid trnsporters ws ssessed y mesuring the N + -dependent uptke of [ 14 C]methylminoisoutyric cid (MeAIB; 20 μm) nd the

4 4192 F. J. Rosrio nd others J Physiol mino-2-norornne-croxylic cid (BCH; 64 μm)- inhiitle uptke of [ 3 H] leucine (125 μm), respectively, s descried in detil previously (Rosrio et l. 2013). MTHF uptke ssy [3,5,7,9-3H]-Methyltetrhydrofolic cid uptke in cultured PHT cells ws determined t 90 h following plting. In rief, trnsport experiments were performed in uffer with the following composition (in mm): 150NCl,10Hepesnd5.6 D- (+) glucose (ph 7.5 or 5.5). Initilly, the culture medium ws removed, nd the cells were wshed with uffer t 37 C; uptke ws then initited y the ddition of 1 ml of uffer t 37 C contining 30 nm [ 3 H] MTHF (Morvek Biochemicls, Bre, CA, USA). Bsed on initil time course studies uptke ws liner up to 120 min, nd 80 min ws therefore used t the time t which incution ws stopped y removing the incution medium. Susequently, the cells were plced on ice nd rinsed with 1 ml of ice-cold PBS (ph 7.4). Non-medited uptke wsdeterminedinthepresenceof1.5mm unlelled MTHF. A Time of culture 0h 24h 48h 72h 90h B 150 kd 90 h Folte deficiency 72 h 48 h 24 h Rptor FD 70 kd S6K T kd S6K 32 kd S6 S-235/ kd S6 18 kd 4E-BP1 T-37/46 18 kd 4E-BP1 T kd 4E-BP1 C kd β-ctin FD Reltive density Rptor S6K (T-389) S6K S6 (S-235/236) S6 4E-BP1 (T-37/46) 4E-BP1 (T-70) 4E-BP1 Figure 1. mtorc1 functions s cellulr folte sensor in primry humn tropholst cells A, study design illustrting the time course of folte deficiency in primry humn tropholst cells. B, time course of mtorc1 signlling in PHT cells cultured for 90 h in low folte medium. Representtive Western lots of rptor, phosphorylted S6 kinse (T-389), totl S6 kinse, phosphorylted S6 riosoml protein (S-235/236) nd totl S6 riosoml protein, phosphorylted 4E-BP1 (T-37/46 nd T-70), nd totl 4E-BP1 expression in cell lystes of control nd folte-deficient (FD) cells. Equl loding ws performed. C, summry of the Western lot dt. PHT mtorc1 ws mrkedly inhiited following 90 h of culture in low folte medium. Vlues re given s men + SEM; P < 5 vs. control; unpired Student s t test; n = 4 6 seprte cell preprtions.

5 J Physiol mtor functions s folte sensor 4193 Western lotting For immunolotting, cells were lysed in PBS with 0.1% SDS contining phosphtse nd protese inhiitors. Susequently, cells were scrped, collected nd sonicted. Western lotting ws crried out s descried (Rosrio et l. 2013). Protein expression of totl nd phosphorylted mtor, S6K1 (Thr-389), 4E-BP1 (Thr-37/46 or Thr-70), S6 riosoml protein (Ser-235/236) nd Akt (Ser-473 or Thr-308) were nlysed in cell lystes using commercil ntiodies (Cell Signling Technology, Boston, MA, USA). Trget nd densities were normlized to loding using et-ctin. For ech protein trget the men density of the control smple nds ws ssigned n ritrry vlue of 1. All individul densitometry vlues were expressed reltive to this men. Proximity ligtion ssy nd confocl microscopy Isolted PHT cells (500,000 cells per well) were grown on chmer slides (L-Tek) for 90 h. The cells were fixed in ice-cold methnol t 20 C for 20 min nd were locked using 5% neworn clf serum (NCS) in PBS for 1 h followed y incution in ntiodies trgeting PCFT (generted in rit) nd mtor (generted in mouse) for 2 h. Proximity ligtion ssy (PLA) proes nti-rit PLUS nd nti-mouse MINUS were diluted in Duolink dilution uffer nd incuted in pre-heted humidity chmer for 1 h. This ws followed y ligtion, mplifiction nd detection ccording to the Duolink In Situ Ornge kit (Sigm-Aldrich) mnufcturer s protocol. Confocl microscopy ws performed using Zeiss LSM 780 microscopet63 mgnifiction using oil immersion. Imges were cptured in the sme lser settings with four Z-steps of 0.4 um. Determintion of plcentl mtor signlling nd mternl serum folte concentrtions Plcents were collected from term uncomplicted pregnncies s descried ove. In ddition, mternl lood smples were collected prior to Cesren section. Serum ws prepred nd frozen t 80 C until nlysis of folte concentrtions using n Immulite 1000 (Siemens Helthcre Dignostics, Deerfield, IL, USA), ccording to the mnufcturer s instructions. Intr- nd inter-ssy coefficients of vrition were 6.7 nd 7.9% t 700 pg ml 1. Within 15 min of delivery, the decidu slis nd chorionic plte from plcent were removed nd villous tissue ws dissected nd rinsed in cold physiologicl sline. The villous tissue ws trnsferred to cold uffer D (250 mm sucrose, 10 mm Hepes, ph 7.4) contining 1:100 dilution of protese nd phosphtse inhiitors (Sigm-Aldrich) nd homogenized on ice with Polytron (Kinemtic, Luzern, Switzerlnd). The plcentl homogentes were frozen in liquid nitrogen nd stored t 80 C until further processing. The phosphoryltion of key proteins in the mtorc1 nd 2 signlling pthwys ws determined using Western lots s descried for cells. Dt presenttion nd sttistics Dt re presented s mens ± SEM or + SEM. The sttisticl significnce of differences etween control nd experimentl groups ws ssessed using n unpired Student s t test, repeted mesures ANOVA with Tukey Krmer multiple comprisons post hoc test. A P vlue <5 ws considered significnt. Results Folte deficiency does not ffect PHT cell viility, differentition or poptosis Culturing PHT cells in low folte medium for up to 90 h did not influence the secretion of hcg, well-estlished iochemicl mrker of syncytiliztion. After 66 h in culture, there ws mrked increse in hcg production y tropholst cells, nd the levels remined high until t lest 90 h fter plting (dt not shown). Becuse hcg is produced predominntly y syncytilized cells, these dt provide evidence of cell differentition. We demonstrte further tht there ws no difference in the protein expression of syncytin ( differentition mrker) or in the expression of poptosis mrkers (totl nd phosphorylted p53 or cspse-3 nd cleved cspse-3; dt not shown) in PHT cells cultured in folte-deficient medi s compred to control cells. Collectively, these dt indicte tht culturing PHT cells in low folte medi up to 90 h did not ffect tropholst cell viility nd differentition. Intrcellulr folte levels Intrcellulr folte concentrtions of PHT cells cultured in folte-deficient medium were decresed y 88% (47 ± 3.4 ng/ cells in control cells to 5.8 ± 3.2 ng/ cells in folte-deficient cells, P < 001; n = 5) s compred to control cells cultured in folte-sufficient medium. Folte deficiency inhiits mtorc1 signlling Time course studies indicted tht 90 h of folte deficiency mrkedly ffected mtorc1 (Fig. 1). We studied the phosphoryltion of S6K1, S6 riosoml protein nd 4E-BP1 s functionl red-outs for mtorc1 ctivity in PHT cell lystes of control nd folte-deficient cell (Fig. 1B, C). Folte deficiency decresed the phosphoryltion of S6K1 t Thr-389 y 75% in PHT

6 4194 F. J. Rosrio nd others J Physiol cell lystes (P = 01; n = 6) s compred to control. Phosphoryltion of riosoml protein S6 (Ser-235/236), component of the 40S riosome nd physiologiclly relevnt S6K1 sustrte, ws inhiited y 63% (P = 004; n = 6) in folte-deficient PHT cells. Phosphoryltion of 4E-BP1 occurs t multiple sites in n ordered mnner. Phosphoryltion y mtor t Thr-37 nd Thr-46 of 4E-BP1 my prime it for susequent phosphoryltion t sites including Ser-65 nd Thr-70. The 4E-BP1 phosphoryltion of Thr t 37/46 ws significntly reduced in cell lystes of folte-deficient cells s compred to control ( 59%, P < 04; n = 6). In contrst, phosphoryltion of 4E-BP1 t Thr-70 ws comprle etween PHT cells of the control nd folte-deficient groups. In ddition, expression of rptor ( 32%, P < 03; n = 6), protein ssocited with mtorc1, decresed in response to folte deficiency. No significnt differences were found in the expression of totl S6K, S6 nd 4E-BP1 etween cell lystes of control nd folte-deficient cells (Fig. 1B, C). Folte deficiency inhiits mtorc2 signlling As shown in Fig. 2, culturing PHT cells in low folte medium for 90 h significntly reduced the expression of rictor ( 68%, P < 01; n = 6), protein ssocited A Folte deficiency 90 hr 72 hr 48 hr 24 hr 200 kd Rictor 62 kd Akt S kd Akt T kd Akt 80 kd SGK S-422 B kd β-ctin FD Reltive density Rictor Akt (S-473) Akt (T-308) Akt SGK (S-422) Figure 2. Activity of mtorc2 is modulted y folte levels in primry humn tropholst cells A, time course of mtorc2 signlling in PHT cells cultured for 90 h in low folte medium. Representtive Western lots of rictor, phosphorylted Akt (S-473 nd T-308), totl Akt nd phosphorylted SGK (S-422) expression in cell lystes of control nd folte-deficient (FD) cells. Equl loding ws performed. B, summry of the Western lot dt. PHT mtorc2 ws mrkedly inhiited following 90 h of culture in low folte medium. Vlues re given s men + SEM; P < 5 vs. control; unpired Student s t test; n = 4 6 seprte cell preprtions.

7 J Physiol mtor functions s folte sensor 4195 with mtorc2. In ddition, folte deficiency inhiited mtorc2 ctivity (Fig. 2) s ssessed y phosphoryltion of Akt t Ser-473 ( 72%, P < 04; n = 4) nd SGK1 t Ser-422 ( 83%, P < 003; n = 4). Importntly, folte deficiency did not influence Akt phosphoryltion t Thr-308 (Fig. 2), well-estlished trget for the insulin/igf-i signlling pthwy, suggesting tht the impct of low folte on cellulr signlling is specific. Returning folte-deficient PHT cells to folte-contining medi normlizes mtor signlling To determine whether the mtor inhiition in response to folte deficiency is reversile, PHT cells were grown in folte-deficient medi for 90 h nd susequently cultured in medi with norml folte concentrtions for 10 h (Fig. 3A). Culturing folte-deficient PHT A B C Time of culture mtorc1 mtorc2 0h 90 h- Folte deficient 90 h- Folte deficient 90h 100h 70 kd 70 kd 18 kd 42 kd FD FR S6K T-389 S6 S-235/236 4E-BP1 T-.37/46 β-ctin 62 kd 80 kd 42 kd FD FR Akt S-473 SGK S-422 β-ctin Folte deficient Folte resupplementtion D 1.5 Reltive density S6K (T-389) S6K S6 (S-235/236) S6 4E-BP1 (T-37/40) 4E-BP1 Akt (S-473) Akt SGK (S-422) SGK Figure 3. Re-supplementtion of folte to folte-deficient cultured primry humn tropholst cells reverses mtorc1 nd mtorc2 signlling A, study design illustrting the time course of folte deficiency/folte re-supplementtion in primry humn tropholst cells. B, representtive Western lots of phosphorylted S6 kinse (T-389), phosphorylted S6 riosoml protein (S-235/236) nd phosphorylted 4E-BP1 (T-37/46) expression in cell lystes of control, folte-deficient (FD) nd folte-resupplemented (FR) cells. Equl loding ws performed. C, representtive Western lots of phosphorylted Akt (S-473), nd phosphorylted SGK (S-422) expression in cell lystes of control, folte-deficient nd folte-resupplemented (FR) cells. Equl loding ws performed. D, summry of the Western lot dt. mtorc1 nd mtorc2 signlling in folte-deficient cells were restored y chnging to medium contining norml folte concentrtions. Vlues re given s men + SEM; n = 6 seprte cell preprtions. Mens without common letter differ significntly (P < 5) y repeted-mesures ANOVA with Tukey Krmer multiple comprisons post hoc test.

8 4196 F. J. Rosrio nd others J Physiol cells in control medi for 10 h returned mtorc1 (phosphoryltion of S6K t Thr-389, S6 t Ser-235/236 nd 4E-BP1 t Thr-37/46; n = 6, Fig. 3B, D)ndmTORC2 (phosphoryltion of Akt t Ser-473 nd SGK t Ser-422; n = 6, Fig. 3C, D) signlling to their control levels. Homocysteine does not ffect mtor signlling Becuse folte is required for the metolic conversion of homocysteine to methionine nd folte deficiency results in ccumultion of homocysteine, we determined whether incution of PHT cells in homocysteine (5 100 μm) for 10 h (80 90 h of culture) inhiits mtorc1 nd mtorc2 signlling. The concentrtions of homocysteine (5 100 μm) used in the present study re comprle to pthophysiologicl levels oserved in sujects with mild hyperhomocysteinemi (16 24 μm) (Girling & de Swiet, 1998). When cultures were susequently ssessed for cell viility nd differentition, we found tht incution in homocysteine (up to 100 μm) for 10 h did not influence syncytiliztion or increse poptosis (dt not shown). AsshowninFigs4nd5,mTORC1(n = 4, Fig. 4) A Homo cysteine Homo cysteine 5 μm 20 μm 100 μm 5 μm 20 μm 100 μm 290 kd 290 kd 32 kd 32 kd 18 kd 18 kd 42 kd mtor S-2448 mtor S6 S-235/236 S6 4E-BP1 T-70 4E-BP1 β-ctin B 1.5 Homocysteine (5μM) Homocysteine (20 μm) Homocysteine (100 μm) Reltive density mtor (S-2448) mtor S6 (S-235/236) S6 4E-BP1 (T-70) 4E-BP1 Figure 4. Effect of homocysteine on mtorc1 signlling in primry humn tropholst cells A, PHT cells were incuted in medi with (5, 20 nd 100 µm) or without homocysteine for 10 h. Representtive Western lots of phosphorylted mtor (S-2448), totl mtor, phosphorylted S6 riosoml protein (S-235/236) nd totl S6 riosoml protein, phosphorylted 4E-BP1 (T-70), nd totl 4E-BP1 expression in cell lystes of control nd homocysteine (5, 20 nd 100 µm) incuted cells. Equl loding ws performed. B, summry of the Western lot dt. Homocysteine did not ffect mtorc1 signlling in PHT cells. Vlues re given s men + SEM; n = 4 seprte cell preprtions.

9 J Physiol mtor functions s folte sensor 4197 nd mtorc2 signlling ctivity (n = 4, Fig. 5) ws unffected following incution in homocysteine. These dt suggest tht in cultured PHT cells, mtorc1 nd mtorc2 re modulted y low folte rther thn high homocysteine. Folte deficiency inhiits system A nd L mino cid trnsporter ctivity To study the effect of folte deficiency on tropholst mino cid uptke, we determined BCH-inhiitle [ 3 H] leucine uptke (System L) nd N + -dependent [ 14 C] MeAIB uptke (System A) t 90 h in culture. As shown in Fig. 6, folte deficiency reduced System L uptke y 34% (n = 5; P < 4) nd System A uptke y 33% (n = 5; P < 3) s compred to control. Involvement of PCFT in folte sensing y mtorc1 nd mtorc2 Sustntil progress hs een mde in identifying the moleculr mechnisms tht form the sis for mtorc1 A B 62 kd 80 kd 42 kd 1.5 Homo cysteine 5 μm 20 μm 100 μm Homo cysteine 5 μm 20 μm 100 μm Akt S-473 SGK S-422 β-ctin Homocysteine (5μM) Homocysteine (20 μm) Homocysteine (100 μm) sensing of mino cids, which requires the recruitment of mtorc1 to the outer lysosoml surfce, medited y Rg GTPse-dependent nd -independent mechnisms nd involves the vcuolr H + -ATPse (Kim et l. 2008; Snck et l. 2008, 2010). Although the moleculr identity of the sensor linking mino cid levels to mtorc1 signlling hs remined elusive, recent reports demonstrte tht the sodium-coupled neutrl mino cid trnsporter 9 (SLC38A9) constitutes key component of the lysosoml mino cid sensing mchinery (Efeyn et l. 2015; Jewell et l. 2015; Wng et l. 2015). We resoned tht folte trnsporter intercts specificlly with folte, therey meeting one of the primry requirements of folte sensor. PCFT is criticl for folte uptke in the intestine (Qiu et l. 2006, 2007). Although PCFT is uiquitously expressed (Zho et l. 2011), including in the syncytiotropholst microvillous plsm memrne (Crter et l. 2011), given its dependence on proton grdient for efficient folte trnsport it is unlikely to contriute to cellulr folte uptke in most cells. We tested the hypothesis tht PCFT is involved in mtor folte sensing. First, we determined the effect of PCFT silencing on mtorc1 nd mtorc2 signlling. To confirm efficient silencing, we performed Western lots on sirna-trnsfected cells t 90 h of culture. PCFT sirna decresed the PCFT protein expression y 80% (P < 02; n = 3, dt not shown) s compred to control cells. Furthermore, hcg secretion profiles were similr in cells in which PCFT hd een silenced s compred to cells incuted in scrmled sirna (n = 3, dt not shown). As shown in Fig. 7, PCFT silencing resulted in mrked decrese in phosphoryltion of mtor t Ser-2448 ( 36%, P < 4; n = 5), S6K t Thr-389 ( 71%, P < 3; n = 4), S6 riosoml protein t Ser-235/236 ( 73%, P < 2; Reltive density Akt (S-473) SGK (S-422) A pmol mg 1 min System L B pmol mg 1 min System A FD Figure 5. Effect of homocysteine on mtorc2 signlling in primry humn tropholst cells A, PHT cells were incuted in medi with (5, 20 nd 100 µm) or without homocysteine for 10 h. Representtive Western lots of phosphorylted Akt (S-473), nd phosphorylted SGK (S-422 expression in cell lystes of control nd homocysteine (5, 20 nd 100 µm) incuted cells. Equl loding ws performed. B, summry of the Western lot dt. Homocysteine did not ffect mtorc2 signlling in PHT cells. Vlues re given s men + SEM; n = 4 seprte cell preprtions. Figure 6. Folte deficiency inhiits system A nd L mino cid trnsport ctivity System L ctivity (A) ws mesured s the BCH-inhiitle uptke of [ 3 H]leucine, nd System A ctivity (B) ws determined s the N + -dependent uptke of [ 14 C]MeAIB. Folte deficiency (FD) decresed the sl ctivity of System L nd System A, trnsporters in cultured humn primry tropholst cells. Vlues re mens + SEM; n = 5. P < 5 vs control; unpired Student s t test. 0

10 4198 F. J. Rosrio nd others J Physiol n = 6 7) nd 4E-BP1 t Thr-70 ( 49%, P < 1; n = 6 7) s compred to cells trnsfected with scrmled sirna. No significnt differences were found in the expression of rptor etween cells trnsfected with scrmled nd PCFT sirna. Furthermore, PCFT silencing decresed the phosphoryltion of SGK t Ser-422 ( 69%, P < 1; n = 6 7) s compred to control. In contrst, PCFT silencing did not influence the expression of rictor, protein component of mtorc2. These dt demonstrte tht PCFT is involved in the regultion of mtorc1 nd mtorc2 signlling. Re-introduction of folte to PCFT-silenced nd folte-deficient PHT cells fils to normlize mtor signlling To mechnisticlly link folte, PCFT nd mtor, PHT cells were trnsfected with PCFT or scrmled sirna, cultured in control or folte-deficient medi until 90 h fter plting, followed y culture in folte-contining control medi for 10 h. As expected, returning folte-deficient cells trnsfected with scrmled sirna to control medi completely normlized mtorc1 nd mtorc2 signlling ctivity. A Scrmle sirna PCFT sirna B 2.0 Scrmle sirna PCFT sirna 150 kd Rptor kd 70 kd 32 kd 18 kd mtor S-2448 S6K T-389 S6 S-235/236 4E-BP1 T-37/46 Reltive density 18 kd 42 kd 4E-BP1 T-70 β-ctin Rptor mtor (S-2448) S6K (T-389) S6 (S-235/236) 4E-BP1 (T-37/46) 4E-BP1 (T-70) C Scrmle sirna PCFT sirna D 1.5 Scrmle sirna PCFT sirna 200 kd 80 kd 42 kd Rictor SGK S-422 β-ctin Reltive density Rictor SGK (S-422) Figure 7. PCFT silencing inhiits mtorc1 nd mtorc2 signlling in primry humn tropholst cells A, Representtive Western lots of rptor, phosphorylted mtor (S-2448), phosphorylted S6 kinse (T-389), phosphorylted S6 riosoml protein (S-235/236), nd phosphorylted 4E-BP1 (T-37/46 nd T-70) expression in cell lystes of scrmle nd PCFT sirna silenced cells. Equl loding ws performed. B, r chrt summrizing the immunolot dt. mtorc1 signlling is inhiited following silencing of PCFT. C, representtive Western lots of rictor nd phosphorylted SGK (S-422) in cell lystes of scrmle nd PCFT sirna silenced cells. Equl loding ws performed. D, r chrt summrizing the immunolot dt. mtorc2 signlling is inhiited following silencing of PCFT. Vlues re given s men + SEM; P < 5 vs. control; unpired Student s t test; n = 3 6 seprte cell preprtions.

11 J Physiol mtor functions s folte sensor 4199 Importntly, in cells with PCFT silencing, re-introduction of norml folte concentrtions to the cell culture medium t 90 h filed to reverse mtorc1 nd mtorc2 ctivity (n = 3, Fig. 8A, B) These dt demonstrte tht PCFT is required for mtor folte sensing. Requirement of PCFT in mtor folte sensing is not relted to its function mediting cellulr folte uptke One possiility is tht the effect of PCFT is indirect due to decresed cellulr folte uptke following PCFT A 290 kd PCFT sirna PCFT sirna Scrmle sirna Scrmle sirna mtor S-2448 B PCFT sirna (, ) PCFT sirna (, +) Scrmle sirna (+, +) Scrmle sirna (, +) 32 kd 18 kd 80 kd S6 S-235/236 4E-BP1 T-70 SGK S-422 Reltive density 42 kd β-ctin Folte + Folte re-supp mtor (S-2448) S6 (S-235/236) 4EBP1 (T-70) SGK (S-422) C D E 32 kd 42 kd Scrmle sirna DEPTOR +PCFT sirna PCFT sirna DEPTOR sirna S6 S-235/236 β-ctin 3 H 5-MTHF uptke t ph 7.4 (pmol mg 1 min 1 ) H 5-MTHF uptke t ph 5.5 (pmol mg 1 min 1 ) Scrmle sirna DEPTOR + PCFT sirna Figure 8. PCFT links folte vilility to mtor signlling in primry humn tropholst cells A, representtive Western lots of phosphorylted mtor (S-2448), phosphorylted S6 riosoml protein (S-235/236) nd phosphorylted 4E-BP1 (T-70) expression in cell lystes of scrmle nd PCFT sirna silenced cells cultured in control/folte-deficient medi. Equl loding ws performed. B, r chrt summrizing the immunolot dt. Re-introduction of folte to folte-deficient cells with PCFT silencing does not reverse the inhiition of mtorc1 nd 2 signlling. Vlues re given s men + SEM; n = 3 seprte cell preprtions. Mens without common letter differ significntly (P < 5) y repeted-mesures ANOVA with Tukey Krmer multiple comprisons post hoc test. C, representtive Western lot of phosphorylted S6 riosoml protein (S-235/236) in cell lystes of scrmle, DEPTOR, PCFT nd DEPTOR + PCFT sirna silenced cells. Silencing of DEPTOR + PCFT mintins sl mtorc1 ctivity. Equl loding ws performed. D, DEPTOR + PCFT silencing does not influence the uptke of 3 H-5-MTHF into PHT cells t ph 7.4. E, decresed uptke of 3 H-5-MTHF t ph 5.5 in PHT with DEPTOR + PCFT silencing. Vlues re given s men + SEM; P < 5 vs. control; unpired Student s t test; n = 3 6 seprte cell preprtions.

12 4200 F. J. Rosrio nd others J Physiol silencing. We hve previously shown tht mtor inhiition decreses the expression of specific mino cid trnsporter (Rosrio et l. 2013) nd folte trnsporter isoforms (Rosrio et l. 2016c) in the plsm memrne. Thus, determining folte uptke following PCFT silencing, which inhiits mtor (Fig. 7), my not provide specific informtion out PCFT-medited trnsport. To circumvent this prolem we mesured folte uptke in PHT cells in which oth PCFT nd DEPTOR, n endogenous inhiitor of oth mtorc1 nd 2 signlling tht contins two DEP (Dishevelled, Egl-10, Pleckstrin) domins, were silenced (Peterson et l. 2009). We hve previously reported tht DEPTOR silencing in PHT cells decreses the protein expression of DEPTOR y 55% (Rosrio et l. 2016). DEPTOR silencing significntly incresed the phosphoryltion of S6 kinse (Thr-389), 4E-BP1 (Thr-37/46), S6 riosoml protein (Ser-235/236), functionl redouts for mtorc1 nd Akt (Ser-473), reflecting the ctivity of mtorc2 signlling (Rosrio et l. 2016). Importntly, in cells tht were trnsfected with sirna for oth PCFT nd DEPTOR, mtor ctivity ws mintined t control levels (Fig. 8C). As shown in Fig. 8D,DEPTOR+ PCFT silencing did not ffect MTHF uptke in PHT cells t ph 7.4. In contrst, in the presence of lrge inwrdly directly proton grdient (ph 5.5 in the culture medium), DEPTOR + PCFT silencing mrkedly inhiited MTHF uptke (Fig. 8E). These results re consistent with reports tht PCFT does not medite significnt MTHF uptke in isolted syncytiotropholst microvillous plsm memrne vesicles t physiologicl ph (Crter et l. 2011). Thus, the requirement of PCFT in mtor folte sensing is independent of its role s plsm memrne folte trnsporter. PCFT silencing inhiits folte-medited mtorc1 ctivity y disrupting mtor-lamp2 lysosoml co-locliztion Recent dvnces in the field of mtorc1 mino cid sensing hve plced this cellulr process t the surfce of the lysosome (Snck et l. 2008, 2010). Specificlly it is elieved tht mino cids regulte the recruitment of mtorc1 to the lysosoml surfce, where mtorc1 is ctivted. Conversely, mtorc1 is rpidly removed from the lysosome in mino cid-strved cells, resulting in inctivtion of mtorc1 signlling (Efeyn et l. 2012). We explored co-locliztion of mtor nd LAMP2, lysosoml mrker, using confocl microscopy nd PLA, n pproch tht llows the in situ detection of intercting endogenous proteins (mtor/lamp2). Specificlly, we tested the hypothesis tht folte promotes co-locliztion of mtor nd LAMP2, which requires PCFT. In PHT cells trnsfected with scrmled sirna, folte deficiency for 90 h resulted in loss of mtor/lamp2 co-locliztion (yellow, Fig. 9). However, chnging to folte-contining control medi t 90 h restored mtor/lamp2 co-locliztion. Importntly, in cells with PCFT silencing, re-introduction of norml folte concentrtions to the cell culture medium t 90 h filed to restore mtorc1 locliztion t the lysosoml surfce. These findings re consistent with the possiility tht mtor folte sensing occurs t the lysosoml surfce, which requires PCFT. Mternl serum folte is ssocited with plcentl mtorc1 nd mtorc2 signlling in humn pregnncy To explore the clinicl relevnce of our findings, we exmined the reltionship etween mternl serum folte nd plcentl mtorc1 nd mtorc2 signlling in term plcent from cohort of helthy women (n = 13) undergoing Cesren section t term. Even in this smll cohort, mternl serum folte ws positively correlted with irth weight (Fig. 10A). Consistent with our findings in cultured primry humn tropholst cells we oserved significnt positive correltion etween mternl serum folte nd functionl redouts of mtorc1 signlling (Fig. 10B) nd mtorc2 signlling (Fig. 10C, D). Discussion WeshowforthefirsttimethtmTORfunctionssfolte sensor, therey identifying novel mechnistic link y which folte regultes cell growth nd function. Folte sensing y mtor in PHT cells involves oth mtorc1 nd C2 nd requires the PCFT (SLC46A1). mtor is trfficked to the lysosome in response to incresed folte vilility, similr to the mechnisms involved in mtor mino cid sensing. Our findings re relevnt for humn physiology ecuse studies were crried out in cultured primry humn tropholst cells. Tropholst mtor folte sensing resulting in mtor inhiition nd down-regultion of key plcentl mino cid trnsporters could e one mechnism underlying the reltionship etween low mternl folte levels nd restricted fetl growth. Indeed, the clinicl relevnce of our dt is supported y the oservtion in smll cohort of women tht mternl serum folte correlted positively with irth weight nd plcentl mtorc1 nd mtorc2 signlling. These findings my hve rod iologicl implictions nd provide new insights into the mechnisms underpinning the ssocitions etween folte vilility nd diseses such s cncer, fetl growth restriction nd fetl mlformtions. mtor nutrient sensing, including regultion of mtor signlling y mino cids, ATP nd oxygen, is function previously scried exclusively to mtorc1. We found tht oth mtorc1 nd mtorc2 re responsive to folte vilility, which is, to the est of our knowledge, the first

13 J Physiol mtor functions s folte sensor 4201 Scrmle sirna Scrmle sirna Scrmle sirna Folte + Folte Folte Folte + supplementtion Folte supplementtion Folte + supplementtion 20 μm 20 μm 20 μm PCFT sirna PCFT sirna Folte Folte Folte supplementtion Folte + supplementtion 20 μm 20 μm Figure 9. Folte promotes co-locliztion of mtor nd LAMP2, which requires PCFT Immunofluorescence confocl microscopy in comintion with in situ PLA, which detects protein protein complexes, ws used to explore interctions etween mtor nd LAMP2 following PCFT inhiition with folte deficiency/folte re-supplementtion. Ech detected complex is represented y yellow dot. DNA ws counterstined y DAPI (lue). Scle r = 20 µm.

14 4202 F. J. Rosrio nd others J Physiol report implicting mtorc2 in nutrient sensing. We lso show tht PCFT is required for mtorc1/2 folte sensing in PHT cells. Intriguingly, lthough PCFT silencing nd folte deficiency, respectively, inhiited mtorc1 nd mtorc2 signlling, PCFT silencing regulted 4E-BP1 t different phosphoryltion site (Thr-70) thn folte deficiency (Thr-37/46). The mechnism underlying this difference remins to e estlished. PCFT is uiquitously expressed, lthough in most norml cells extrcellulr ph is slightly higher thn cytosolic ph, generting smll outwrdly directed proton grdient. Thus, given tht PCFT requires proton grdient to drive folte trnsport, it is highly unlikely tht this trnsporter medites folte uptke in norml cells, with some exceptions such s the intestinl epithelium (Desmoulin et l. 2012). Indeed, we demonstrte in PHT cells tht the requirement for PCFT for mtor folte sensing is not relted to its function s plsm memrne folte trnsporter. Becuse the folte trnsport function of PCFT is dependent on ph grdient (Qiu et l. 2006), it hs een proposed tht PCFT plys n importnt role in endosoml/lysosoml efflux of folte (Pn et l. 2002; Zho et l. 2011). Recent dvnces in the field of mtorc1 mino cid sensing hve plced this cellulr process t the surfce of the lysosome (Snck et l. 2008, 2010). Specificlly it is elieved tht mino cids regulte the recruitment of mtorc1 to the lysosoml surfce, where mtorc1 is ctivted. Interestingly two proton-coupled mino cid trnsporters, PAT1 nd PAT4, locted on intrcellulr memrnes, re required for mtorc1 mino cid sensing (Heulein et l. A C mtorc2 Mternl serum folte level (ng ml 1 ) B S6 S235/236 reltive density Birth weight (g) 4500 P= Mternl serum folte (ng ml 1 ) mtorc1 Mternl serum folte level (ng ml 1 ) P= Mternl serum folte (ng ml 1 ) S6 S-235/236 β-ctin Akt S-473 reltive density SGK S-422 reltive density D P= Mternl serum folte (ng ml 1 ) 1.5 mtorc2 Mternl serum folte level (ng ml 1 ) P= Mternl serum folte (ng ml 1 ) SGK S-422 β-ctin Akt S-473 β-ctin Figure 10. Correltion etween mternl serum folte nd irth weight nd plcentl mtorc1 nd mtorc2 signlling Correltion etween mternl serum folte nd irth weight (A) nd plcentl rps6 t Ser-235/236 (B), SGK t Ser-422 (C) nd Akt t Ser-473 (D) in women with uncomplicted term pregnncies, n = 13.

15 J Physiol mtor functions s folte sensor ; Ogmundsdottir et l. 2012) nd hve een suggested to e n integrl prt of the multiprotein nutrient sensing complex on the lysosoml surfce tht medite mtorc1 mino cid sensing (Jnsson et l. 2012). Thus, itis possile tht PCFT links folte vilility to mtorc1 signlling t the lysosome. However, further studies re needed to explore this possiility. Folte deficiency inhiits cell growth nd prolifertion in HepG2 cells (Hung et l. 1999; Yu et l. 2013), dult hippocmpl progenitor cells (Krumn et l. 2005) nd Chinese hmster ovry cells (Bormn & Brnd, 1989). Although decresed vilility of nucleotide precursors is elieved to contriute to impired cell growth nd prolifertion in response to folte deficiency, the mechnisms re not known in detil. mtor is regulted y growth fctor signlling nd mtorc1 ctivity is modulted y cellulr levels of oxygen, energy, mino cids nd glucose (Dennis et l. 2001; Dnn et l. 2007; Lplnte & Stini, 2012; Wiczer & Thoms, 2012). mtor signlling is mster regultor of protein trnsltion, therey controlling cell metolism, growth nd prolifertion (Gulti & Thoms, 2007; Thoreen et l. 2012). In ddition, mtor constitutes powerful positive regultor of tropholst mino cid trnsporters (Roos et l. 2007, 2009; Rosrio et l. 2013, 2016; Chen et l. 2015). Thus, mtor-signlling plys criticl role mtching cellulr nutrient uptke, metolism nd growth with nutrient vilility, s reflected y nutrient levels on the cellulr level nd circulting concentrtions of metolic hormones nd growth fctors on the orgnism level. mtor folte sensing is consistent with this overll integrting role of mtor signlling ecuse folte is required for the synthesis of purine nd thymidine nucleotides constituting DNA uilding locks nd therefore essentil for DNA synthesis. mtor signlling therefore constitutes mechnistic link etween folte vilility nd cell growth in prolifertive cells. In the humn syncytiotropholst, mtor folte sensing my hve criticl role in mtching plcentl functions such s nutrient trnsport, protein trnsltion nd mitochondril function, nd therefore fetl growth, to folte vilility. Although the literture is not entirely consistent, severl studies demonstrte strong positive reltionship etween mternl red lood cell folte nd irth weight (Tmur & Piccino, 2006). Furthermore, met-nlysis of eight rndomized controlled trils investigting the effect of folte supplementtion on fetl growth clerly shows dose response reltionship etween folte intke nd irth weight (vn Uitert & Steegers-Theunissen, 2013). Aleit very smll cohort, our dt re consistent with positive correltion etween mternl folte levels nd fetl growth. In ddition, the clinicl relevnce of our findings is supported y the oservtion in our study tht mternl serum folte correlted positively with plcentl mtorc1 nd mtorc2 signlling. Fetl growth is lrgely determined y nutrient vilility nd tropholst mtor signlling is positive regultor of plcentl mino cid trnsport (Roos et l. 2009; Rosrio et l. 2013). We demonstrte tht mtor signlling in cultured primry humn tropholst cells is highly responsive to chnges in folte vilility. Furthermore, we show tht folte deficiency in PHT cells cuses mrked down-regultion of mino cid trnsport. Importntly, we hve previously reported tht mtorc1 nd mtorc2 specificlly regulte the plsm memrne trfficking of SNAT 2 (sodium-dependent neutrl mino cid trnsporter 2) nd LAT 1 (lrge neutrl mino cid trnsporter 1), ut no other System A/L isoform, in cultured primry humn tropholst cells (Rosrio et l. 2013). Becuse tropholst nutrient trnsport plys criticl role in determining fetl growth (Gccioli et l. 2013; Jnsson et l. 2013; Jnsson & Powell, 2013), folte sensing y tropholst mtor my represent mechnism y which mternl folte sttus modultes fetl growth nd development. We therefore propose tht inhiition of tropholst mtor signlling, resulting in down-regultion of key plcentl mino cid trnsporters nd decresed fetl nutrient vilility, constitutes one mechnism underlying the ssocition etween low mternl folte levels nd restricted fetl growth. Our findings my hve relevnce eyond tropholst function nd fetl growth given the criticl role of folte in norml cell function nd the wide rnge of disorders tht re cused y folte deficiency. Periconceptionl folte deficiency is risk fctor for fetl structurl mlformtions such s NTDs (Czeizel & Duds, 1992; Blom et l. 2006). Although chnges in DNA methyltion nd ltertions in synthesis of nucleotide precursors hve een implicted (Smithells et l. 1976; Wilde et l. 2014), the mechnism y which folte ffects neutrl tue closure remins poorly understood. Folte deficiency or impired folte metolism is lso ssocited with n incresed risk of developing cncer (Duthie, 2011), which is elieved to e due to impired DNA repir. Intriguingly, emerging evidence suggests tht lso high folte intke promotes crcinogenesis (Glynn & Alnes, 1994; Ulrich, 2007; Wien et l. 2012), lthough the underlying moleculr mechnism remins elusive. mtor ctivtion is common finding in cncer nd it my e speculted tht mtor folte sensing, cusing ctivtion of mtor signlling in response to incresed folte vilility, could e one mechnism underlying the link etween high folte nd incresed cncer risk. In conclusion, we hve identified novel specific moleculr link etween folte vilility nd fetl growth, which involves tropholst PCFT nd mtor. We propose tht mtor folte sensing in tropholst cells mtches plcentl nutrient trnsport nd fetl growth to mternl folte sttus.

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