Glucocorticoid regulation of amino acid transport in primary human trophoblast cells

in Journal of Molecular Endocrinology
Correspondence should be addressed to O R Vaughan: orv20@cantab.net
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Excess maternal glucocorticoids reduce placental amino acid transport and fetal growth, but whether these effects are mediated directly on the syncytiotrophoblast remains unknown. We hypothesised that glucocorticoids inhibit mechanistic target of rapamycin (mTOR) signaling and insulin-stimulated System A amino acid transport activity in primary human trophoblast (PHT) cells. Syncytialised PHTs, isolated from term placentas (n = 15), were treated with either cortisol (1 μM) or dexamethasone (1 μM), ± insulin (1 nM) for 24 h. Compared to vehicle, dexamethasone increased mRNA expression, but not protein abundance of the mTOR suppressor, regulated in development and DNA damage response 1 (REDD1). Dexamethasone enhanced insulin receptor abundance, activated mTOR complex 1 and 2 signaling and stimulated System A activity, measured by Na+-dependent 14C-methylaminoisobutyric acid uptake. Cortisol also activated mTORC1 without significantly altering insulin receptor or mTORC2 read-outs or System A activity. Both glucocorticoids downregulated expression of the glucocorticoid receptor and the System A transporter genes SLC38A1, SLC38A2 and SLC38A4, without altering SNAT1 or SNAT4 protein abundance. Neither cortisol nor dexamethasone affected System L amino acid transport. Insulin further enhanced mTOR and System A activity, irrespective of glucocorticoid treatment and despite downregulating its own receptor. Contrary to our hypothesis, glucocorticoids do not inhibit mTOR signaling or cause insulin resistance in cultured PHT cells. We speculate that glucocorticoids stimulate System A activity in PHT cells by activating mTOR signaling, which regulates amino acid transporters post-translationally. We conclude that downregulation of placental nutrient transport in vivo following excess maternal glucocorticoids is not mediated by a direct effect on the placenta.

 

      Society for Endocrinology

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    Characterisation of primary human trophoblast cells. (A) Daily rate of hCG secretion into the culture medium by PHT cells at 18, 42, 66 and 90 h after isolation. Overall effect of time point determined by one-way ANOVA with Holm–Sidak post hoc. Differing superscripts a, b indicate significantly different time points. n = 9 placentas. Mean + s.e.m. (B) Western blots of cytokeratin 7 (trophoblast marker) and vimentin (mesenchyme marker) in lysates collected from syncytialised PHT cells 90 h after isolation, compared to crude placental homogenate (Plac. Hom.).

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    Effects of glucocorticoids and insulin on REDD1 and insulin receptor abundance. Representative Western blots (A), gene expression (B, D and F) and protein abundance (C, E and G) of REDD1 and insulin receptor in syncytialised primary human trophoblast cells treated with vehicle (Con), cortisol (Cort, 1 μM) and dexamethasone (Dex, 1 μM), ± insulin (concentration, 1 nM). Effects of glucocorticoid (PGC), insulin (Pins) and interaction (PGC*Ins) were determined by two-way ANOVA with repeated measures and significances (P < 0.05) are provided in the figure. Differing superscripts a, b indicate significantly different glucocorticoid treatment groups, * indicates significant simple effect of insulin and † indicates significant simple effect of dexamethasone by Holm–Sidak post hoc. n = 9 placentas (gene expression), n = 6 placentas (protein). Mean ± s.e.m.

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    Effects of glucocorticoids and insulin on mTORC1, mTORC2 and ERK1/2 signaling. (A) Representative Western blots and (B, C, D, E, F and G) phosphorylated and total protein abundance for readouts of mTORC1, mTORC2 and ERK1/2 signaling in syncytialised primary human trophoblast cells treated with vehicle (Con), cortisol (Cort, 1 μM) and dexamethasone (Dex, 1 μM), ± insulin (concentration, 1 nM). Effects of glucocorticoid (PGC), insulin (Pins) and interaction (PGC*Ins) were determined by two-way ANOVA with repeated measures and significances (P < 0.05) are provided in the figure. Differing superscripts a, b indicate significantly different glucocorticoid treatment groups, * indicates significant simple effect of insulin by Holm–Sidak post hoc. n = 6 placentas. Mean ± s.e.m.

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    Effects of glucocorticoids and insulin on System A and System L amino acid transport. System A-mediated 14C-methylaminoisobutyric acid uptake (A and C) and System L-mediated 3H-leucine uptake (B and D) in syncytialised primary human trophoblast cells. (A and B) Time-course of total, non-mediated (+BCH, -Na+) and transporter-mediated (total – non-mediated) uptake in untreated cells (n = 3 placentas). (C and D) Rate of uptake assessed after 8 min of tracer incubation in vehicle (Con), cortisol (Cort, 1 μM) and dexamethasone (Dex, 1 μM) treated cells, ± insulin (concentration, 1 nM). Effects of glucocorticoid (PGC), insulin (Pins) and interaction (PGC*Ins) were determined by two-way ANOVA with repeated measures and significances (P < 0.05) are provided in the figure. Differing superscripts a, b indicate significantly different glucocorticoid treatment groups, * indicates significant simple effect of insulin by Holm–Sidak post hoc. n = 9 placentas. Mean ± s.e.m.

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    Effects of glucocorticoids and insulin on System A transporter gene and protein expression. (A) Representative Western blot, (B, D and E) mRNA expression and (C) protein abundance of System A transporters in syncytialised primary human trophoblast cells treated with vehicle (Con), cortisol (Cort, 1 μM) and dexamethasone (Dex, 1 μM), ± insulin (concentration, 1 nM). Effects of glucocorticoid (PGC), insulin (Pins) and interaction (PGC*Ins) were determined by two-way ANOVA with repeated measures and significances (P < 0.05) are provided in the figure. Differing superscripts a, b indicate significantly different glucocorticoid treatment groups, * indicates significant simple effect of insulin by Holm–Sidak post hoc. n = 5 placentas (gene expression), n = 9 placentas (protein). Mean ± s.e.m.

References

  • AudetteMCChallisJRJonesRLSibleyCPMatthewsSG 2011 Antenatal dexamethasone treatment in midgestation reduces system A-mediated transport in the late-gestation murine placenta. Endocrinology 35613570. (https://doi.org/10.1210/en.2011-0104)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • AudetteMCChallisJRGJonesRLSibleyCPMatthewsSG 2014 Synthetic glucocorticoid reduces human placental system A transport in women treated With antenatal therapy. The Journal of Clinical Endocrinology & Metabolism E2226E2233. (https://doi.org/10.1210/jc.2014-2157)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • AudetteMCGreenwoodSLSibleyCPJonesCJPChallisJRGMatthewsSGJonesRL 2010 Dexamethasone stimulates placental system A transport and trophoblast differentiation in term villous explants. Placenta 97105. (https://doi.org/10.1016/j.placenta.2009.11.016)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • BallardPLBallardRA 1995 Scientific basis and therapeutic regimens for use of antenatal glucocorticoids. American Journal of Obstetrics & Gynecology 254262. (https://doi.org/10.1016/0002-9378(95)90210-4)

    • Search Google Scholar
    • Export Citation
  • CramerSBeveridgeMKilbergMNovakD 2002 Physiological importance of system A-mediated amino acid transport to rat fetal development. American Journal of Physiology. Cell Physiology C153C160. (https://doi.org/10.1152/ajpcell.2002.282.1.C153)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • CrowtherCAMckinlayCJMiddletonPHardingJE 2015 Repeat doses of prenatal corticosteroids for women at risk of preterm birth for improving neonatal health outcomes. Cochrane Database Systematic Reviews Cd003935. (https://doi.org/10.1002/14651858.CD003935.pub4)

    • Search Google Scholar
    • Export Citation
  • DoyleLWFordGWDavisNMCallananC 2000 Antenatal corticosteroid therapy and blood pressure at 14 years of age in preterm children. Clinical Science 137142. (https://doi.org/10.1042/cs0980137)

    • Search Google Scholar
    • Export Citation
  • DriverPMRauzSWalkerEAHewisonMKilbyMDStewartPM 2003 Characterization of human trophoblast as a mineralocorticoid target tissue. Molecular Human Reproduction 793798. (https://doi.org/10.1093/molehr/gag091)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • DuthieLReynoldsRM 2013 Changes in the maternal hypothalamic-pituitary-adrenal axis in pregnancy and postpartum: influences on maternal and fetal outcomes. Neuroendocrinology 106115. (https://doi.org/10.1159/000354702)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • FantusIGSaviolakisGAHedoJAGordenP 1982 Mechanism of glucocorticoid-induced increase in insulin receptors of cultured human lymphocytes. Journal of Biological Chemistry 82778283.

    • Search Google Scholar
    • Export Citation
  • FowdenALForheadAJSferruzzi-PerriANBurtonGJVaughanOR 2015 Review: endocrine regulation of placental phenotype. Placenta (Supplement 1) S50S59. (https://doi.org/10.1016/j.placenta.2014.11.018)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • FowdenALValenzuelaOAVaughanORJellymanJKForheadAJ 2016 Glucocorticoid programming of intrauterine development. Domestic Animal Endocrinology (Supplement) S121S132. (https://doi.org/10.1016/j.domaniend.2016.02.014)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • GlazierJDCetinIPeruginoGRonzoniSGreyAMMahendranDMarconiAMPardiGSibleyCP 1997 Association between the activity of the system A amino acid transporter in the microvillous plasma membrane of the human placenta and severity of fetal compromise in intrauterine growth restriction. Pediatric Research 514519. (https://doi.org/10.1203/00006450-199710000-00016)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • GutlingHBionazMSlobodaDMEhrlichLBraunFGramzowAKHenrichWPlagemannABraunT 2016 The importance of selecting the right internal control gene to study the effects of antenatal glucocorticoid administration in human placenta. Placenta 1922. (https://doi.org/10.1016/j.placenta.2016.05.011)

    • Search Google Scholar
    • Export Citation
  • HitomiHKiyomotoHNishiyamaAHaraTMoriwakiKKaifuKIharaGFujitaYUgawaTKohnoM 2007 Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells. Hypertension 750755. (https://doi.org/10.1161/HYPERTENSIONAHA.107.093955)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • JanssonNGreenwoodSLJohanssonBRPowellTLJanssonT 2003 Leptin stimulates the activity of the system A amino acid transporter in human placental villous fragments. Journal of Clinical Endocrinology & Metabolism 12051211. (https://doi.org/10.1210/jc.2002-021332)

    • Search Google Scholar
    • Export Citation
  • JonesHNAshworthCJPageKRMcardleHJ 2006 Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo). American Journal of Physiology. Endocrinology & Metabolism E596E603. (https://doi.org/10.1152/ajpendo.00359.2005)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • KarlPI 1995 Insulin-like growth factor-1 stimulates amino acid uptake by the cultured human placental trophoblast. Journal of Cellular Physiology 8388. (https://doi.org/10.1002/jcp.1041650111)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • KarlPIAlpyKLFisherSE 1992 Amino acid transport by the cultured human placental trophoblast: effect of insulin on AIB transport. American Journal of Physiology C834C839. (https://doi.org/10.1152/ajpcell.1992.262.4.C834)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • KatiyarSLiuEKnutzenCALangESLombardoCRSankarSTothJIPetroskiMDRonaiZChiangGG 2009 REDD1, an inhibitor of mTOR signalling, is regulated by the CUL4A-DDB1 ubiquitin ligase. EMBO Reports 866872. (https://doi.org/10.1038/embor.2009.93)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • KellyBALewandowskiAJWortonSADavisEFLazdamMFrancisJNeubauerSLucasASinghalALeesonP 2012 Antenatal glucocorticoid exposure and long-term alterations in aortic function and glucose metabolism. Pediatrics e1282e1290. (https://doi.org/10.1542/peds.2011-3175)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • KlimanHJNestlerJESermasiESangerJMStraussJF 3RD 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 15671582. (https://doi.org/10.1210/endo-118-4-1567)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • KnutsonVP 1986 The acute and chronic effects of glucocorticoids on insulin receptor and insulin responsiveness. Transient fluctuations in intracellular receptor level parallel transient fluctuations in responsiveness. Journal of Biological Chemistry 1030610312.

    • Search Google Scholar
    • Export Citation
  • LagerSAyeILMHGaccioliFRamirezVIJanssonTPowellTL 2014 Labor inhibits placental mechanistic target of rapamycin complex 1 signaling. Placenta 10071012. (https://doi.org/10.1016/j.placenta.2014.10.006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LeeMJWangZYeeHMaYSwensonNYangLKadnerSSBaergenRNLoganSKGarabedianMJ 2005 Expression and regulation of glucocorticoid receptor in human placental villous fibroblasts. Endocrinology 46194626. (https://doi.org/10.1210/en.2005-0235)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LiJWangZNChenYPDongYPShuaiHLXiaoXMReichetzederCHocherB 2012 Late gestational maternal serum cortisol is inversely associated with fetal brain growth. Neuroscience & Biobehavioral Reviews 10851092. (https://doi.org/10.1016/j.neubiorev.2011.12.006)

    • Search Google Scholar
    • Export Citation
  • MahendranDDonnaiPGlazierJDD'SouzaSWBoydRDSibleyCP 1993 Amino acid (system A) transporter activity in microvillous membrane vesicles from the placentas of appropriate and small for gestational age babies. Pediatric Research 661665. (https://doi.org/10.1203/00006450-199311000-00019)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MandoCTabanoSPileriPColapietroPMarinoMAAvaglianoLDoiPBulfamanteGMiozzoMCetinI 2013 SNAT2 expression and regulation in human growth-restricted placentas. Pediatric Research 104110. (https://doi.org/10.1038/pr.2013.83)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • RogersonFMDimopoulosNSlukaPChuSCurtisAJFullerPJ 1999 Structural determinants of aldosterone binding selectivity in the mineralocorticoid receptor. Journal of Biological Chemistry 3630536311. (https://doi.org/10.1074/jbc.274.51.36305)

    • Search Google Scholar
    • Export Citation
  • RoosSKanaiYPrasadPDPowellTLJanssonT 2009a Regulation of placental amino acid transporter activity by mammalian target of rapamycin. American Journal of Physiology. Cell Physiology C142C150. (https://doi.org/10.1152/ajpcell.00330.2008)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • RoosSLagerlofOWennergrenMPowellTLJanssonT 2009b Regulation of amino acid transporters by glucose and growth factors in cultured primary human trophoblast cells is mediated by mTOR signaling. American Journal of Physiology. Cell Physiology C723C731. (https://doi.org/10.1152/ajpcell.00191.2009)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • RosarioFJDimasuayKGKanaiYPowellTLJanssonT 2016a Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2. Clinical Science 499512. (https://doi.org/10.1042/CS20150554)

    • Search Google Scholar
    • Export Citation
  • RosarioFJPowellTLJanssonT 2016b Mechanistic target of rapamycin (mTOR) regulates trophoblast folate uptake by modulating the cell surface expression of FR-alpha and the RFC. Scientific Reports 31705. (https://doi.org/10.1038/srep31705)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • RosarioFJKanaiYPowellTLJanssonT 2013 Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells. Journal of Physiology 609625. (https://doi.org/10.1113/jphysiol.2012.238014)

    • Search Google Scholar
    • Export Citation
  • SathishkumarKElkinsRChinnathambiVGaoHHankinsGDYallampalliC 2011 Prenatal testosterone-induced fetal growth restriction is associated with down-regulation of rat placental amino acid transport. Reproductive Biology and Endocrinology : RB&E 110. (https://doi.org/10.1186/1477-7827-9-110)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • SherajeeSJFujitaYRafiqKNakanoDMoriHMasakiTHaraTKohnoMNishiyamaAHitomiH 2012 Aldosterone induces vascular insulin resistance by increasing insulin-like growth factor-1 receptor and hybrid receptor. Arteriosclerosis Thrombosis & Vascular Biology 257263. (https://doi.org/10.1161/ATVBAHA.111.240697)

    • Search Google Scholar
    • Export Citation
  • ShibataEHubelCAPowersRWVon Versen-HoeynckFGammillHRajakumarARobertsJM 2008 Placental system A amino acid transport is reduced in pregnancies With small for gestational age (SGA) infants but not in preeclampsia with SGA infants. Placenta 879882. (https://doi.org/10.1016/j.placenta.2008.07.001)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • ShimizuNYoshikawaNItoNMaruyamaTSuzukiYTakedaSNakaeJTagataYNishitaniSTakehanaK 2011 Crosstalk between glucocorticoid receptor and nutritional sensor mTOR in skeletal muscle. Cell Metabolism 170182. (https://doi.org/10.1016/j.cmet.2011.01.001)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • StirratLIJustGHomerNZMAndrewRNormanJEReynoldsRM 2017 Glucocorticoids are lower at delivery in maternal, but not cord blood of obese pregnancies. Scientific Reports 10263. (https://doi.org/10.1038/s41598-017-10266-5)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • StirratLIO'ReillyJRRileySCHowieAFBeckettGJSmithRWalkerBRNormanJEReynoldsRM 2014 Altered maternal hypothalamic-pituitary-adrenal axis activity in obese pregnancy is associated with macrosomia and prolonged pregnancy. Pregnancy Hypertension 238. (https://doi.org/10.1016/j.preghy.2014.03.028)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • StirratLISengersBGNormanJEHomerNZMAndrewRLewisRMReynoldsRM 2018 Transfer and metabolism of cortisol by the isolated perfused human placenta. Journal of Clinical Endocrinology & Metabolism 640648. (https://doi.org/10.1210/jc.2017-02140)

    • Search Google Scholar
    • Export Citation
  • TanCYHagenT 2013 mTORC1 dependent regulation of REDD1 protein stability. PLOS ONE e63970. (https://doi.org/10.1371/journal.pone.0063970)

  • VaughanORDaviesKLWardJWde BlasioMJFowdenAL 2016 A physiological increase in maternal cortisol alters uteroplacental metabolism in the pregnant ewe. The Journal of Physiology 594 64076418. (https://doi.org/10.1113/JP272301)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • VaughanORFisherHMDionelisKNJefferiesECHigginsJSMusialBSferruzzi-PerriANFowdenAL 2015 Corticosterone alters materno-fetal glucose partitioning and insulin signalling in pregnant mice. Journal of Physiology 13071321. (https://doi.org/10.1113/jphysiol.2014.287177)

    • Search Google Scholar
    • Export Citation
  • VaughanORRosarioFJPowellTLJanssonT 2017 Regulation of placental amino acid transport and fetal growth. Progress in Molecular Biology & Translational Science 217251. (https://doi.org/10.1016/bs.pmbts.2016.12.008)

    • Search Google Scholar
    • Export Citation
  • VaughanORSferruzzi-PerriANCoanPMFowdenAL 2013 Adaptations in placental phenotype depend on route and timing of maternal dexamethasone administration in mice. Biology of Reproduction 80. (https://doi.org/10.1095/biolreprod.113.109678)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • VaughanORSferruzzi-PerriANFowdenAL 2012 Maternal corticosterone regulates nutrient allocation to fetal growth in mice. Journal of Physiology 55295540. (https://doi.org/10.1113/jphysiol.2012.239426)

    • Search Google Scholar
    • Export Citation
  • WangHKubicaNEllisenLWJeffersonLSKimballSR 2006 Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1. Journal of Biological Chemistry 3912839134. (https://doi.org/10.1074/jbc.M610023200)

    • Search Google Scholar
    • Export Citation

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