Glucocorticoid regulation of amino acid transport in primary human trophoblast cells

Abstract

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.

Keywords: fetal development; maternal–fetal exchange; mechanistic target of rapamycin; placenta; solute carrier family 38A members 1, 2 and 4.

Fig. 1. 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 hours 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 + SEM. (B) Western blots of cytokeratin 7 (trophoblast marker) and vimentin (mesenchyme marker) in lysates collected from syncytialised PHT cells 90 hours after isolation, compared to crude placental homogenate (Plac. Hom.).

Fig. 2. Effects of glucocorticoids and insulin on REDD1 and insulin receptor abundance.

Representative western blots (A), gene expression (B, D) and protein abundance (C, E) of REDD1 and insulin receptor in syncytialised primary human trophoblast cells treated with vehicle (Con), cortisol (Cort, 1μM) and dexamethasone (Dex, 1μM), ± insulin (concentration, 1nM). Effects of glucocorticoid (P_GC), insulin (P_ins) and interaction (P_GC*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 ± SEM

Fig. 3. Effects of glucocorticoids and insulin on mTORC1, mTORC2 and ERK1/2 signaling.

(A) Representative western blots and (B-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, 1μM) and dexamethasone (Dex, 1μM), ± insulin (concentration, 1nM). Effects of glucocorticoid (P_GC), insulin (P_ins) and interaction (P_GC*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 ± SEM

Fig. 4. Effects of glucocorticoids and insulin on System A and System L amino acid transport.

System A-mediated ¹⁴C-methylaminoisobutyric acid uptake (A, C) and System L-mediated ³H-leucine uptake (B, D) in syncytialised primary human trophoblast cells. (A, B) Time-course of total, non-mediated (+BCH, -Na⁺) and transporter mediated (total – non-mediated) uptake in untreated cells (n=3 placentas). (C, D) Rate of uptake assessed after 8 minutes of tracer incubation in vehicle (Con), cortisol (Cort, 1μM) and dexamethasone (Dex, 1μM) treated cells, ± insulin (concentration, 1nM). Effects of glucocorticoid (P_GC), insulin (P_ins) and interaction (P_GC*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 ± SEM

Fig. 5. Effects of glucocorticoids and insulin on System A transporter gene and protein expression

(A) Representative western blot, (B, D, E) mRNA expression and (C) protein abundance of System A transporters in syncytialised primary human trophoblast cells treated with vehicle (Con), cortisol (Cort, 1μM) and dexamethasone (Dex, 1μM), ± insulin (concentration, 1nM). Effects of glucocorticoid (P_GC), insulin (P_ins) and interaction (P_GC*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 ± SEM