Mechanistic Target of Rapamycin Complex 2 Regulation of the Primary Human Trophoblast Cell Transcriptome

Abstract

Mechanistic Target of Rapamycin Complex 2 (mTORC2) regulates placental amino acid and folate transport. However, the role of mTORC2 in modulating other placental functions is largely unexplored. We used a gene array following the silencing of rictor to identify genes regulated by mTORC2 in primary human trophoblast (PHT) cells. Four hundred and nine genes were differentially expressed; 102 genes were down-regulated and 307 up-regulated. Pathway analyses demonstrated that inhibition of mTORC2 resulted in increased expression of genes encoding for pro-inflammatory IL-6, VEGF-A, leptin, and inflammatory signaling (SAPK/JNK). Furthermore, down-regulated genes were functionally enriched in genes involved in angiogenesis (Osteopontin) and multivitamin transport (SLC5A6). In addition, the protein expression of leptin, VEGFA, IL-6 was increased and negatively correlated to mTORC2 signaling in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In contrast, the protein expression of Osteopontin and SLC5A6 was decreased and positively correlated to mTORC2 signaling in human IUGR placentas. In conclusion, mTORC2 signaling regulates trophoblast expression of genes involved in inflammation, micronutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions necessary for fetal growth and development.

Oleic acid stimulation of amino acid uptake in primary human trophoblast cells is mediated by phosphatidic acid and mTOR signaling

“…As expected rictor knockdown inhibited trophoblast System A uptake but raptor inhibition failed to decrease System A activity in this series. This was inconsistent with previous results [9,10] but we have no clear explanation for this difference. System L uptake was not impacted by either rictor or raptor knockdown.…”

“…4 For example, mTORC1 activation influences the plasma membrane trafficking of the specific amino acid transporter isoforms SLC38A2 (sodium dependent neutral amino acid transporter 2, a System A transporter isoform) and SLC7A5 (LAT1, a System L transporter isoform) by regulating Nedd4-2 mediated ubiquitination 9 and mTORC2 regulates the actin skeleton responsible for insertion of amino acid transporters in the microvillous plasma membrane. 10 Previous studies in cancer cell lines have demonstrated that mTOR also acts as a lipid sensor and phosphatidic acid (PA), a key intermediate of de novo phospholipid and triglyceride biosynthesis, is the form of lipid that modulates mTOR activity. 11 During pregnancy, maternal fatty acids modulate an array of placental functions including development of extravillous trophoblast cells, lipid droplet formation, and amino acid uptake.…”

“…Both mTOR complexes are modulators of cell surface abundance of amino acid transporters in trophoblast cells mediated by distinct post translational mechanisms 4 . For example, mTORC1 activation influences the plasma membrane trafficking of the specific amino acid transporter isoforms SLC38A2 (sodium dependent neutral amino acid transporter 2, a System A transporter isoform) and SLC7A5 (LAT1, a System L transporter isoform) by regulating Nedd4‐2 mediated ubiquitination 9 and mTORC2 regulates the actin skeleton responsible for insertion of amino acid transporters in the microvillous plasma membrane 10 . Previous studies in cancer cell lines have demonstrated that mTOR also acts as a lipid sensor and phosphatidic acid (PA), a key intermediate of de novo phospholipid and triglyceride biosynthesis, is the form of lipid that modulates mTOR activity 11 …”

Placental Remote Control of Fetal Metabolism: Trophoblast mTOR Signaling Regulates Liver IGFBP-1 Phosphorylation and IGF-1 Bioavailability

“…A well-established method involving sequential trypsin digestion and Percoll centrifugation [ 30 , 79 ] was used to isolate and culture primary human trophoblast (PHT) cells. Briefly, isolated PHT cells were plated at 1.5 × 10 6 per well in six-well plates and cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM, 25 mM glucose) and Ham’s F12 medium (10 mM glucose) with 2 mM glutamine, 50 μg/mL gentamicin, 60 μg/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum.…”

PiggyBac Transposase Mediated Inducible Trophoblast-specific Knockdown of Mechanistic Target of Rapamycin in Mice Decreases Placental Nutrient Transport and Inhibits Fetal Growth

“…In addition, mTOR regulates glucose uptake in trophoblast-derived cell line cultures 33 . Both mTORC1 and mTORC2 signaling regulate trophoblast expression of various genes involved in ribosome biosynthesis, inflammation, micronutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions necessary for fetal growth and development 34,35 . However, data unequivocally demonstrating that changes in trophoblast mTOR signaling regulate placental function in vivo are lacking.…”