Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction

Abstract

Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using a(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 3 essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P 5 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved. Reproduction (2006) 131 951-960

Placental nutrient supply and fetal growth

“…SNAT2 has an amino acid response element in its promoter region, possibly explaining these findings (Palii et al, 2004). BeWo protein expression for SNAT2 was unaffected in substrate deprivation experiments but there was increased localisation of this isoform to the plasma membrane (Jones et al, 2006). This suggests a similar mechanism to that present in other cell types is responsible for adaptive regulation of system A in placenta.…”

“…In various cell types, substrate regulation of system A is associated with specific changes in the expression and localisation of SLC38A2/SNAT2 (Franchi-Gazzola et al, 2001;Hyde et al, 2001;Ling et al, 2001). In studies using BeWo choriocarcinoma cells, MeAIB transport has been shown to increase following substrate deprivation and this was associated with upregulation of SLC38A2 mRNA (Jones et al, 2006;Novak et al, 2006b). SNAT2 has an amino acid response element in its promoter region, possibly explaining these findings (Palii et al, 2004).…”

Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4

“…In several cell types, amino acid deprivation results in upregulation of system A amino acid transporter activity (37)(38)(39)(40), including the trophoblast-like human choriocarcinoma cell line BeWo ( 38,40 ). Amino acid deprivation increases SNAT2 expression in BeWo trophoblast-like cells ( 38,40 ).…”

“…Amino acid deprivation increases SNAT2 expression in BeWo trophoblast-like cells ( 38,40 ). In cell types other than trophoblast cells, activation of JNK signaling has been implicated as an important component in the adaptive response to amino acid deprivation ( 37,39 ).…”

Regulation of placental amino acid transporter activity by mammalian target of rapamycin

“…System A is subjected to extensive regulation, and, in the placenta, it has been shown to be inhibited by IL-1␤ (64), hypoxia (48), nitric oxide (NO) formation (37), hyperglycemia (21), and growth hormone (20). Insulin (27,36), IGF-I (35), EGF (5), amino acid deprivation (34), cortisol (33), and leptin (27) all stimulate system A transport. The regulation of placental system L and TAUT is less well established.…”