Molecular Mechanisms Regulating Placental Nutrient Transporters

描述（由申请人提供）：胎儿生长异常会增加围产期并发症的风险，并易患成人疾病。胎儿生长在很大程度上依赖于营养可利用性，这是由胎盘营养转移决定的。胎盘关键氨基酸转运蛋白活性在宫内生长受限（IUGR）时降低，在胎儿过度生长时上调，提示胎盘营养转运蛋白活性的改变直接导致胎儿生长异常。然而，关于胎盘营养转运蛋白调控的机制信息目前尚缺乏。我们最近报道了哺乳动物雷帕霉素靶蛋白（mTOR）信号是滋养层氨基酸转运蛋白的关键调节因子；然而，潜在的分子机制尚不清楚。中心假设：mTOR复合物1 （mTORC1）和2 （mTORC2）都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的活性。我们进一步提出，所涉及的分子机制是不同的，mTORC1激活磷酸化E3泛素连接酶Nedd4-2，从而降低转运蛋白泛素化，导致细胞表面氨基酸转运蛋白表达增加，而mtorc2激活刺激PKC1介导的肌动蛋白骨架。具体目的：(1)确定mTORC1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转运的影响；(3)确定mTOR调节质膜转运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mTOR与氨基酸转运蛋白转运的信号通路活性。方法：研究培养的人原代滋养细胞，测定系统A和系统L氨基酸转运体和葡萄糖转运体的活性，并利用荧光成像、亚细胞分离和蛋白质表达研究确定转运体异构体的细胞分布。特定信号通路的激活将通过测量磷酸化蛋白的表达来确定。利用siRNA介导的沉默，我们将通过实验操纵mTORC1和mTORC2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。意义：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物质运输和胎儿生长的关键分子机制，这将增加我们对妊娠并发症发生的重要性的理解。创新：我们将探索连接mTOR和营养转运蛋白的分子机制，这在任何哺乳动物组织中都没有被证实。此外，我们提出了人类胎盘中氨基酸转运蛋白调控的新模型。

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.