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 preeclampsia23,24. 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)2D3 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 preeclampsia4, 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 pregnancy4,5,25, 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 complications26. 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 pregnancies27. 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 preeclampsia28, 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 diagramming demonstrated that amino acid transporter y+LAT1 was significantly increased in placentas associated with PE, but decreased in IUGR placentas29. 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 arginine30. 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 preeclampsia31.
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 D3 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 signaling33. On the contrary, our results showed that 1,25-dihydroxy vitamin D3 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 CoCl2-induced hypoxia. We further demonstrated that knockdown of VDR by siRNA silencing prevented 1,25-dihydroxy vitamin D3-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 D3 could prevent CoCl2-induced decrease of mTOR activity. However, 1,25-dihydroxy vitamin D3 had no effect on mTOR signaling in placental trophoblasts under normoxic condition, which was consistent with the results in Jasson’s study33. The VDR knockdown produced by siRNA silencing blocked the effects of vitamin D on CoCl2-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 signaling34. They found that DDIT4 was a direct target for 1,25-dihydroxy vitamin D3 and could be induced by 1,25-dihydroxy vitamin D3 treatment in osteoblasts, which might be resulted from the competition for the binding to DDIT4 gene promoter by VDR and VDRE binding protein34.
In the past decade, several novel vitamin D metabolites including 20(OH)D3, 22(OH)D3, 20,22(OH)2D3 and 20,23(OH)2D3 were recognized in placenta, adrenal glands, and epidermal keratinocytes, suggesting 1,25(OH)2D3 is not solely bioactive form of vitamin D in vivo35,36. And their action may not through the genomic site of the VDR37. 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.