Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2

Abstract

Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyzes the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mTORC1 decreases amino acid uptake in primary human trophoblast cells (PHT) by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signaling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

Inhibition of placental mTOR signaling provides a link between placental malaria and reduced birthweight

“…As expected, DEPTOR silencing significantly increased mTORC1 signaling activity (rps6: +44%, P  = 0.008; 4E-BP1: +60%, P  = 0.004; Figs 3b, c) and upregulated System A activity (+66%, P  = 0.009; Fig. 3d) [13]. This is consistent with previous reports that silencing of DEPTOR activates mTORC1 [21].…”

“…System A activity was assessed by measuring the Na + -dependent uptake of 14 C-methyl-aminoisobutyric acid (MeAIB) as previously described [13]. …”

“…It exists as two protein complexes: mTOR complex 1 (mTORC1), the master regulator of protein translation and cell growth and proliferation; and mTOR complex 2 (mTORC2), which regulates cytoskeletal organization and cellular metabolism. mTOR is expressed in the placental syncytiotrophoblast where it regulates amino acid uptake by influencing the trafficking of transporters to the plasma membrane [13]. Upstream signals such as amino acids, growth factors, free fatty acids, oxygen, and cytokines have been shown to influence placental mTOR signaling activity [14, 15].…”

Essential Roles of L-Type Amino Acid Transporter 1 in Syncytiotrophoblast Development by Presenting Fusogenic 4F2hc

“…It is, however, still possible that the amino acid transport activity of LAT1 has a functional impact on the cell fusion process in addition to the proposed chaperone-like function supporting 4F2hc expression. The trafficking of LAT1 to the plasma membrane is positively regulated downstream of mTORC1 (and probably of mTORC2 as well) in primary human trophoblasts (27,28). Because mTORC1 is activated by amino acids (34), the amino acid uptake mediated by LAT1-4F2hc may increase the cell surface amount of LAT1-4F2hc itself, thereby further promoting the cell fusion of the chorionic trophoblasts.…”

“…In the human term placenta, LAT1 has been colocalized with 4F2hc at two polarized plasma membrane domains of the syncytiotrophoblast, i.e., the maternally facing apical membrane and the fetally facing basal membrane (22,25,26), implying the importance of LAT1-4F2hc heterodimer in the maternofetal transfer of amino acids. Based on studies using primary human trophoblasts, it has been proposed that abrogated cell surface localization of LAT1 may be implicated in intrauterine growth restriction (IUGR) (27,28), in which placental amino acid transport is impaired (29,30). To further understand the physiological relevance of LAT1 in vivo, we constructed a LAT1 knockout mouse strain.…”