Vitamin D stimulates placental L-type amino acid transporter 1 (LAT1) in preeclampsia

Vitamin D insufficiency/deficiency has been linked to an increased risk of preeclampsia. Impaired placental amino acid transport is suggested to contribute to abnormal fetal intrauterine growth in pregnancies complicated by preeclampsia. However, if vitamin D-regulated amino acid transporter is involved in the pathophysiologic mechanism of preeclampsia has not been clarified yet. The aberrant expression of key isoform of L-type amino acid transporter LAT1 was determined by western blot and immunohistochemistry in the placenta from normotensive and preeclamptic pregnancies. The role for vitamin D on placental LAT1 expression was investigated through the exposure of HTR-8/SVneo human trophoblast cells to the biologically active 1,25(OH)2D3 and the oxidative stress-inducer cobalt chloride (CoCl2). Our results showed that placental LAT1 expression was reduced in women with preeclampsia compared to normotensive pregnancies, which was associated with decreased expression of vitamin D receptor (VDR). 1,25(OH)2D3 significantly upregulated LAT1 expression in placental trophoblasts, and also prevented the decrease of mTOR activity under CoCl2-induced oxidative stress. siRNA targeting VDR significantly attenuated 1,25(OH)2D3-stimulated LAT1 expression and mTOR signaling activity. Moreover, treatment of rapamycin specifically inhibited the activity of mTOR signaling and resulted in decrease of LAT1 expression. In conclusion, LAT1 expression was downregulated in the placenta from women with preeclampsia. 1,25(OH)2D3/VDR could stimulate LAT1 expression, which was likely mediated by mTOR signaling in placental trophoblasts. Regulation on placental amino acid transport may be one of the mechanisms by which vitamin D affects fetal growth in preeclampsia.

Preeclampsia (PE) is a pregnancy-specific hypertensive disorder which is associated with substantial morbidity and mortality in mothers and their fetuses worldwide 1 . It also increases the future risk of metabolic and cardiovascular disease later in life 2 . The pathogenesis of preeclampsia remains not fully elucidated, but the impaired remodeling of uterine spiral artery is believed at the root etiology of this disease 3 . This causes reduced blood flow to the placenta and leads to an exposure of the developing fetus to an insufficient oxygen and nutrient supply, which contributes to fetal growth restriction in preeclampsia.
Placental trophoblastic transport of amino acids is vital for the growing fetus in protein synthesis, metabolic and biosynthetic processes. Decreased trophoblastic transfer of amino acids is believed to contribute to fetal growth restriction during pregnancy 4,5 . The L-type amino acid transporter (LAT) is a primary transport system present in the placenta, which is required for the Na + -independent antiport of essential amino acids. The isoform LAT1, as a major accumulative transporter in L-type system, is abundant on the apical surface of syncytiotrophoblast and active in the uptake of specific amino acids from maternal circulation into the placenta 6 . It has been found that trophoblastic LAT1 expression is increased in women with type 2 diabetes mellitus and associated with birth weight and neonatal fat mass 7 , but decreased in the placentas from small-for gestational-age (SGA) fetus 8 . In addition, inhibition of LAT1 transporters is found to be involved in the mouse implantation and placentation by affecting trophoblast differentiation and invasion 9,10 . Placental transport of amino acids is intensively regulated by mammalian target of rapamycin (mTOR) signaling via modulating translocation or global expression of amino acid transporters in trophoblast cells 11 . Jansson et al. demonstrated that mTOR mediated amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells 12 . We previously reported that suppression of mTOR signaling down-regulated the expressions of LAT1 and LAT2 in placental Emerging evidence have shown that insufficient or deficient vitamin D status in pregnancy increases the risk of several pregnancy complications including preeclampsia, fetal growth restriction, and preterm birth 14 . On the contrary, supplementation of vitamin D during pregnancy has been suggested to reduce the risk of preeclampsia 15 . Although the role for vitamin D in placental trophoblasts has not been fully established, it has been clearly demonstrated that vitamin D exerted anti-inflammatory and anti-oxidative stress properties in placental trophoblasts. In our previous studies, we found that the biologically active 1,25(OH) 2 D 3 significantly suppressed the cyclooxygenase-2 activity and the downstream prostaglandin E 2 production in placental trophoblasts in response to hypoxic stimulation 16 , it also attenuated the oxidative stress-induced microparticle shedding from placental trophoblasts 17 . Vitamin D is also implicated to involve in trophoblast proliferation and migration 18 , activation of autophagy 19 , alleviation of insulin resistance 20 in placental trophoblasts. However, there was little data except one study from Southampton Women's survey showed that maternal 25(OH)D 3 and vitamin D binding protein (VDBP) levels were positively related to expression of specific placental amino acid transporters, indicating that vitamin D may be involved in the regulation of placental amino acid transport 21 .
To further study the beneficial properties of vitamin D in placental trophoblasts, we aimed to investigate if vitamin D-regulated amino acid transporter was involved in the pathophysiology of preeclampsia in the present study. To test this, the expression of LAT1 was determined in human placentas from preeclampsia. Then, the role for vitamin D in placental LAT1 expression and underlying mechanisms was investigated through the exposure of HTR-8/SVneo human placental trophoblast cells to 1,25(OH) 2 D 3 and the hypoxia mimic CoCl 2 .

Results
Reduced placental VDR expression is associated with decreased expression of LAT1 in preeclampsia. To explore the involvement of LAT1 in preeclampsia, we first observed the expression of LAT1 in placenta from women with preeclampsia. As shown in Fig. 1A, expressions of LAT1 and VDR were mainly located in the cytoplasm of placental syncytiotrophoblasts. The LAT1 expression was significantly reduced in the placenta from preeclamptic pregnancies compared with that from normotensive pregnancies (Fig. 1B,C). The VDR expression was also decreased in preeclamptic placentas (Fig. 1B,D). Since placental hypoxia is at the root pathology of preeclampsia, we further determined the effects of hypoxia on LAT1 expression in cultured human HTR-8/SVneo trophoblast cells. Our results showed that the expression of LAT1 was dose-dependently decreased when trophoblast cells were cultured with an increasing concentration of CoCl 2 compared to untreated cells ( Fig. 2A). Similar to the effects of CoCl 2 on LAT1, the VDR expression was also reduced in a dose-dependent manner in trophoblast cells treated with CoCl 2 ( Fig. 2A).

1,25(OH) 2 D 3 Stimulates LAT1 expression and prevents hypoxia-induced decrease of LAT1 expression in placental trophoblasts.
The role for vitamin D on LAT1 expression in placental trophoblasts was investigated using cultured HTR-8/SVneo trophoblast cells which were cultured with 1,25(OH) 2 D 3 in the presence or absence of CoCl 2 . The results showed that 1,25(OH) 2 D 3 treatment could stimulate both LAT1 and VDR expression in a dose-dependent way in trophoblast cells (Fig. 2B). Moreover, the LAT1 expression was significantly reduced in the cells cultured with 250 μM of CoCl 2 , and such a CoCl 2 -induced decrease of LAT1 was significantly prevented when the cells were treated with 100 nM of 1,25(OH) 2 D 3 (Fig. 3A). The LAT1 expres- Then, VDR siRNA silencing was performed to knockdown VDR expression to elucidate the specificity of vitamin D-stimulated LAT1 in trophoblast cells. Our results showed that VDR expression was significantly reduced in the cells transfected with VDR siRNA compared with control cells, and 1,25(OH) 2 D 3 was unable to upregulate VDR expression when the cells were treated with VDR siRNA (Fig. 3C). The LAT1 expression was markedly downregulated in the cells transfected with VDR siRNA, and 1,25(OH) 2 D 3 could not upregulate LAT1 expression when the VDR expression was knocked down in trophoblast cells (Fig. 3C).

1,25(OH) 2 D 3 Upregulates mTOR signaling via VDR in placental trophoblasts.
In order to investigate whether mTOR signaling is involved in the vitamin D-regulated LAT1 expression, we determined the effect of 1,25(OH) 2 D 3 on the activity of mTOR signaling using the mTOR downstream effector, 70 kD ribosomal protein S6 kinase 1 (p70S6K1). As shown in Fig. 4A, the expression of phosphorylated p70S6K1 (P-p70S6K1) but not total p70S6K1 was downregulated in the cells cultured with CoCl 2 , and such a CoCl 2 -induced decrease of P-p70S6K1 was attenuated by the treatment of 1,25(OH) 2 D 3 . Moreover, siRNA silence targeting VDR significantly suppressed the expressions of P-p70S6K1 and total p70S6K1 in trophoblast cells (Fig. 4B). The bar graphs show relative protein expression for LAT1 and VDR after normalized against β-actin in each sample from three independent experiments. CoCl 2 induced a dose-dependent decrease in both LAT1 and VDR expression in trophoblasts. *P < 0.05, **P < 0.01, ***P < 0.001, CoCl 2 treated vs control cells. (B) Protein expression for LAT1 and VDR in HTR-8/SVneo trophoblast cells treated with different concentrations of 1,25(OH) 2 D 3 . The bar graphs show relative protein expression for LAT1 and VDR after normalization with β-actin in each sample from four independent experiments. In contrast to CoCl 2 , 1,25(OH) 2 D 3 stimulated LAT1 and VDR expression in a dose-dependent manner in trophoblast cells. # P < 0.05, ## P < 0.01, 1,25(OH) 2 D 3 treated vs control cells.

Suppression of mTOR pathway downregulates LAT1 expression in placental trophoblasts.
We used the specific inhibitor of mTOR signaling, rapamycin, to determine the critical role for mTOR signaling in the regulation of LAT1 expression in trophoblast cells. Treatment of rapamycin significantly inhibited P-p70S6K1 expression in trophoblast cells, suggesting a suppressed activity of mTOR signaling caused by rapamycin. Importantly, LAT1 expression was clearly decreased when the mTOR signaling was inhibited by rapamycin in the presence or absence of CoCl 2 (Fig. 5). The P-p70S6K1 and LAT1 expressions were also suppressed in the cells cultured with CoCl 2 alone.

Material and methods
Placenta tissue collection. Human placentas were collected immediately after delivery at the Second Affiliated Hospital of Harbin Medical University. A total of 10 placentas were used in the study, 5 from normal and 5 from preeclamptic pregnancies. Uncomplicated pregnancy is defined as pregnancy with a blood pressure < 140/90 mmHg, and absence of proteinuria, obstetrical and medical complications. Preeclampsia was defined as systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg with at least two separate readings and coexistence of proteinuria (> 1+) at dipstick or ≥ 300 mg protein/day in urine. Patients complicated with HELLP syndrome, diabetes, and/or renal disease were excluded.     Immunohistochemical staining. Fresh placental tissue was fixed with 10% formalin and embedded in paraffin. Expression of LAT1 and VDR was examined by immunohistochemistry (IHC) staining of paraffinembedded tissue sections. A standard immunohistochemistry staining procedure was performed as previously described 22 . Stained slides with the same antibody were all processed at the same time. Stained slides were reviewed under a microscope, and images were captured with a digital scanning microscopy imaging system (PreciPoint, Germany).

Protein expression by western blot.
Placental and trophoblastic protein expressions for LAT1, VDR, p70S6K1, and phospho-p70S6K1 were examined by western blot. Antibody against human LAT1 (sc-134994) was purchased from Santa Cruz Biotechnology (CA, USA), antibodies against human VDR (AF6159), p70S6K1 (AF6226), and phospho-p70S6K1 (AF3228) were all obtained from Affinity Biosciences (Jiangsu, China). An aliquot of 10 μg of tissue of cellular protein was subject to electrophoresis. The bound antibody was visualized with an enhanced chemiluminescencent detection kit (Yeasen, Shanghai, China). The bands for LAT1, VDR, and p70S6K1 were detected at 55KD, 48KD and 70KD, respectively. The band density was analyzed by ImageJ software (National Institutes of Health, USA). β-actin expression was determined and used to normalize relative protein expression in each sample.

Immunofluorescent staining. HTR-8/SVneo cells were cultured on glass coverslips in 24-well plates
(1 × 10 5 cells/well) and fixed with ice-cold methanol and permeabilized with Triton X-100. Cells were incubated with primary anti-human LAT1 antibody followed by matched secondary antibody. After staining, coverslips were mounted on glass slides with 30% glycerol with 4,6-diamido-2-phenylindole (DAPI) and reviewed under a fluorescent microscope (Nikon corporation, Tokyo, Japan). Images were captured with a digital camera linked to a computer with imaging software (PreciPoint, Germany).
Data presentation and statistics. All data are presented as mean ± SEM. Statistical analysis was performed with unpaired t test or One-way ANOVA using GraphPad Prism 8 software. A Tukey test was used as post hoc test. A value of P < 0.05 was considered statistically significant.

Discussion
Emerging studies have shown that low maternal vitamin D levels in pregnancy was associated with increased risk of preeclampsia and preterm birth, suggesting that vitamin D deficiency is a risk factor of preeclampsia 23,24 . However, the underlying mechanisms remain unclear. In the current study, we demonstrate that placental LAT1 expression is reduced in women with preeclampsia, and 1,25(OH) 2 D 3 stimulates LAT1 expression through VDR in cultured placental trophoblasts. Since altered placental transport of amino acids has been implicated in the fetal growth restriction in preeclampsia 4 , our findings suggest that the role for vitamin D in placental amino acid transporter is one possible mechanism underlying the linkage between maternal vitamin D levels and fetal growth in preeclampsia. The fetal growth is largely dependent on placental transport of nutrients. The studies mentioned above and other studies have indicated that an altered placental transport of amino acids is intimately associated with fetal growth in pregnancy 4,5,25 , but controversial data are present regarding the modifications of placental amino acid transport in pregnancy disorders. For example, using IHC, Aiko et al. found that L-type amino acid transporter 4F2hc and LAT1 was increased in placental syncytiotrophoblast from pregnancies complicated by preeclampsia or intrauterine growth restriction (IUGR), suggesting an adaptive response to help maintaining growth in both of pregnancy complications 26 . Using real-time PCR, Malina et al. reported that mRNA levels for placental A-type amino acid transporters is not different between preeclampsia and normal pregnancies 27 . Interestingly, Shibata et al. found that A-type amino acid transporter activity was not reduced in the placentas of SGA infants from preeclamptic pregnancies, but significantly reduced in the placenta from SGA pregnancies without preeclampsia 28 , suggesting that growth restriction in the two disorders may be largely different. A recent study using multiple approaches including bioinformatic analysis, molecular biology, and mathematical www.nature.com/scientificreports/ diagramming demonstrated that amino acid transporter y + LAT1 was significantly increased in placentas associated with PE, but decreased in IUGR placentas 29 . This opposite but significant changes between preeclampsia and IUGR may suggest a different function of y + LAT1 in these two diseases. y + LAT1 is responsible for the transport of Na + -independent cationic amino acids, such as lysine, ornithine and arginine 30 . High expression of placental y + LAT1 could result lowered maternal levels of lysine, ornithine and arginine during pregnancy, which may elicit similar symptoms as reported in women complicated with lysinuric protein intolerance. Nevertheless, the pregnancy with lysinuric protein intolerance has been associated with an increased risk of serious complications, including preeclampsia 31 .
In the present study, the findings regarding the reduced placental LAT1 expression might be associated with fetal growth restriction observed in preeclampsia. LAT1 is a major Na + independent transporter for indispensable amino acids, such as lysine, leucine, and histidine et al. Low expression of placental LAT1 associated decreased transport of amino acids across the placenta might cause high maternal plasma amino acid concentrations, which is in agreement with the previous findings as reported by Powers et al. 32 . The differentially expression patterns of placental LAT1 in preeclampsia in the above studies might result from the differing methods, sample size, or criteria of preeclampsia sample selection.
Information for the regulation of vitamin D on amino acid transport in placental trophoblasts is very scarce. Only Jansson et al. reported that bioactive 1,25-dihydroxy vitamin D 3 markedly increased mRNA expression of the A-type isoform SNAT2 and the activity of A-type transporters in primary human placental trophoblasts, but had no effect on L-type and did not affect mTOR signaling 33 . On the contrary, our results showed that 1,25-dihydroxy vitamin D 3 could significantly stimulate the protein expression of L-type transporter LAT1 under normoxic condition in cultured placental trophoblasts, and more importantly attenuate the decrease of LAT1 caused by CoCl 2 -induced hypoxia. We further demonstrated that knockdown of VDR by siRNA silencing prevented 1,25-dihydroxy vitamin D 3 -stimulated LAT1 expression in placental trophoblasts. Our findings suggest that vitamin D-stimulated LAT1 may make up for the shortage of decreased placental LAT1 in preeclampsia, which helps to maintain fetal growth. Those opposite findings between our and Jasson's study indicate that the role for vitamin D-regulated LAT1 in preeclampsia should be further investigated.
Regarding the role for vitamin D in mTOR signaling, we found a similar trend as the vitamin D-regulated LAT1 that 1,25-dihydroxy vitamin D 3 could prevent CoCl 2 -induced decrease of mTOR activity. However, 1,25-dihydroxy vitamin D 3 had no effect on mTOR signaling in placental trophoblasts under normoxic condition, which was consistent with the results in Jasson's study 33 . The VDR knockdown produced by siRNA silencing blocked the effects of vitamin D on CoCl 2 -induced mTOR activity. Since the mTOR signaling is directly implicated in the expression of LAT1 which is also confirmed in the present study, we propose that vitamin D-stimulated LAT1 might be mediated by mTOR signaling in placental trophoblasts. There was a study revealed that the role for vitamin D in mTOR signaling involved occupancy of vitamin D response elements (VDREs) of the gene for DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mTOR signaling 34 . They found that DDIT4 was a direct target for 1,25-dihydroxy vitamin D 3 and could be induced by 1,25-dihydroxy vitamin D 3 treatment in osteoblasts, which might be resulted from the competition for the binding to DDIT4 gene promoter by VDR and VDRE binding protein 34 .
In the past decade, several novel vitamin D metabolites including 20(OH)D 3 , 22(OH)D 3 , 20,22(OH) 2 D 3 and 20,23(OH) 2 D 3 were recognized in placenta, adrenal glands, and epidermal keratinocytes, suggesting 1,25(OH) 2 D 3 is not solely bioactive form of vitamin D in vivo 35,36 . And their action may not through the genomic site of the VDR 37 . However, the biological functions of these vitamin D hydroxyderivatives in placenta and preeclampsia are still unknown (Supplementary Figures).
In summary, our study shows that placental LAT1 expression is reduced in preeclamptic pregnancies and vitamin D stimulated-LAT1 expression may be mediated by mTOR signaling in placental trophoblasts. Our results provide evidence for the vitamin D supplementation during pregnancy may be beneficial for reducing the risk of fetal growth restriction observed in preeclampsia probably by increasing essential amino acid transport across the placenta.

Data availability
The datasets are available from the corresponding author on reasonable request.