Molecular Mechanisms Regulating Placental Nutrient Transporters

描述（由申请人提供）：胎儿生长异常增加围产期并发症的风险，并易患成人疾病。胎儿的生长强烈依赖于营养的可用性，这是由胎盘营养转移决定的。关键胎盘氨基酸转运蛋白的活性在宫内生长受限（IUGR）中降低，在胎儿过度生长中上调，表明胎盘营养转运蛋白活性的变化直接导致胎儿生长异常。然而，目前缺乏胎盘营养转运蛋白的调节机制信息。我们最近报道了哺乳动物雷帕霉素靶蛋白（mTOR）信号转导是滋养层氨基酸转运蛋白的关键调节因子，但其潜在的分子机制尚不清楚。中心假设：mTOR复合物1（mTORC 1）和2（mTORC 2）通过影响转运蛋白的质膜运输来调节胎盘氨基酸转运蛋白活性。我们进一步提出，所涉及的分子机制是不同的，mTORC 1激活磷酸化E3泛素连接酶Nedd 4 -2，这减少了转运蛋白泛素化，导致增加的氨基酸转运蛋白在细胞表面的表达，而mTORC 2激活刺激由PKC 1介导的肌动蛋白骨架。具体目标：（1）确定mTOR 1和2在调节胎盘氨基酸转运蛋白活性中的作用，（2）确定mTOR信号传导对滋养层氨基酸转运蛋白运输的影响，（3）确定mTOR调节质膜运输和滋养层氨基酸转运蛋白活性的机制;（4）确定IUGR和胎儿过度生长中连接mTOR与氨基酸转运蛋白的信号通路的活性。方法：研究培养的人原代滋养层细胞，测量系统A和系统L氨基酸转运蛋白和葡萄糖转运蛋白的活性，并使用荧光成像、亚细胞分级和蛋白表达研究来确定转运蛋白亚型的细胞分布。通过测量磷酸化蛋白质的表达来确定特定信号传导途径的激活。使用siRNA介导的沉默，我们将实验性地操纵mTORC 1和mTORC 2信号通路，并直接确定信号分子在介导mTOR对营养转运蛋白运输和活性的影响中的机制作用。此外，这些信号通路以及营养转运蛋白的活性和运输将在正常胎儿生长、IUGR和胎儿过度生长的妊娠胎盘中确定。重要性：这项工作解决了知识上的一个主要空白，并将导致确定调节胎盘营养转运和胎儿生长的关键分子机制，这将增加我们对重要妊娠并发症发展的理解。创新：我们将探索连接mTOR和营养转运蛋白的分子机制，这些机制以前在任何哺乳动物组织中都没有得到证实。此外，我们提出了一个新的模型，在人类胎盘的氨基酸转运蛋白的调节。

DESCRIPTION (provided by applicant): Abnormal fetal growth increases the risk for perinatal complications and predisposes for adult disease. Fetal growth is strongly dependent on nutrient availability, which is determined by placental nutrient transfer. The activity of key placental amino acid transporters is decreased in intrauterine growth restriction (IUGR) and up- regulated in fetal overgrowth, suggesting that changes in the activity of placental nutrient transporters directly contribute to abnormal fetal growth. However, mechanistic information on the regulation of placental nutrient transporters is currently lacking. We recently reported that mammalian target of rapamycin (mTOR) signaling constitutes a key regulator of trophoblast amino acid transporters; however the underlying molecular mechanisms are unknown. Central hypothesis: Both mTOR Complex 1 (mTORC1) and 2 (mTORC2) regulate placental amino acid transporter activity by affecting the plasma membrane trafficking of transporters. We further propose that the molecular mechanisms involved are distinct in that mTORC1 activation phosphorylates the E3 ubiquitin ligase Nedd4-2, which decreases transporter ubiquitination resulting in increased amino acid transporter expression at the cell surface whereas mTORC 2 activation stimulates the actin skeleton mediated by PKC1. Specific Aims: (1) Determine the role of mTORC1 and 2 in regulating placental amino acid transporter activity, (2) Establish the effect of mTOR signaling on trophoblast amino acid transporter trafficking, (3) Identify the mechanisms by which mTOR regulates plasma membrane trafficking and activity of trophoblast amino acid transporters and (4) Determine the activity of the signaling pathway linking mTOR to amino acid transporter trafficking in IUGR and fetal overgrowth. Approach: To study cultured human primary trophoblast cells and measure the activity of System A and System L amino acid transporters and glucose transporters, and determine the cellular distribution of transporter isoforms using fluorescence imaging, subcellular fractionation and protein expression studies. Activation of specific signaling pathways will be determined by measurement of the expression of phosphorylated proteins. Using siRNA mediated silencing we will experimentally manipulate mTORC1 and mTORC2 signaling pathways and directly determine the mechanistic roles for signaling molecules in mediating the effects of mTOR on nutrient transporter trafficking and activity. In addition, these signaling pathways as well as nutrient transporter activity and trafficking will be determined in placentas from pregnancies with normal fetal growth, IUGR and fetal overgrowth. Significance: This work addresses a major gap in knowledge and will lead to the identification of key molecular mechanisms regulating placental nutrient transport and fetal growth, which will increase our understanding of how important pregnancy complications develop. Innovation: We will explore molecular mechanisms linking mTOR and nutrient transporters that have not been demonstrated previously in any mammalian tissue. Furthermore, we propose a novel model for the regulation of amino acid transporters in the human placenta.