Molecular Mechanisms Regulating Placental Nutrient Transporters

描述(由申请人提供)：胎儿发育异常增加了围产期并发症的风险，并易患成人疾病。胎儿的生长很大程度上依赖于营养物质的供应，而营养物质的供应是由胎盘的营养物质转移决定的。胎儿宫内生长受限(IUGR)时胎盘关键氨基酸转运体活性降低，胎儿过度生长时胎盘关键氨基酸转运体活性上调，提示胎盘营养转运体活性改变直接导致胎儿生长异常。然而，目前缺乏关于胎盘营养转运蛋白调节的机械性信息。我们最近报道了哺乳动物靶标雷帕霉素(MTOR)信号转运蛋白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。中心假说：mTOR复合体1(MTORC1)和mTORC2(MTORC2)都通过影响转运蛋白的质膜转运来调节胎盘氨基酸转运蛋白的活性。我们进一步提出，其中涉及的分子机制是mTORC1激活使E3泛素连接酶Nedd4-2磷酸化，从而减少转运体泛素化导致细胞表面氨基酸转运体表达增加，而mTORC 2激活则刺激由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体活性中的作用；(2)确定mTOR信号对滋养层氨基酸转运体转运的影响；(3)确定mTOR调节质膜转运和滋养层氨基酸转运体活性的机制；(4)确定mTOR与IUGR和胎儿过度生长中氨基酸转运体转运相关的信号通路的活性。方法：以原代培养的人滋养层细胞为研究对象，测定A系统和L系统的氨基酸转运体和葡萄糖转运体的活性，并利用荧光成像、亚细胞分裂和蛋白质表达研究确定转运体亚型在细胞内的分布。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，我们将在实验中操纵mTORC1和mTORC2信号通路，并直接确定信号分子在介导mTOR对营养转运体运输和活性的影响中所起的机制作用。此外，这些信号通路以及营养转运体的活性和运输将在胎儿生长正常、宫内发育迟缓和胎儿过度生长的胎盘中确定。意义：这项工作解决了知识上的一个主要差距，并将导致识别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：我们将探索将mTOR和营养转运蛋白联系起来的分子机制，这是以前在任何哺乳动物组织中都没有展示过的。此外，我们还提出了一个新的人体胎盘氨基酸转运蛋白调控模型。

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.