Molecular Mechanisms Linking Placental Nutrient Sensing and Fetal Programming

描述（由申请人提供）：宫内生长受限（IUGR）会增加围产期并发症和成人疾病的风险。然而，调节限制生长和规划长期健康的机制仍有待充分确立。我们之前已经提供了令人信服的证据，证明哺乳动物滋养细胞雷帕霉素靶（mTOR）信号作为胎盘营养传感器，通过调节胎盘营养转运体间接影响胎儿生长。在本研究中，我们将验证胎盘mTOR信号直接影响胎儿代谢和生长的创新假设。我们的中心假设是，滋养细胞mTOR信号的抑制是对营养供应受限的反应，改变了体液因子的释放，从而导致胎儿肝脏中IGFBP-1分泌和磷酸化增加，igf -1分泌减少。我们提出了两个具体目的：目的1：确定滋养细胞mTOR在体外调节胎儿肝细胞IGF-I和IGFBP-1分泌及IGFBP-1磷酸化中的作用；目的2：建立胎盘特异性mTOR敲低对小鼠胎儿肝IGF-I和IGFBP-1分泌及IGFBP-1磷酸化的影响。在目标1中，我们将从培养的两种类型的人原代滋养细胞中获得条件培养基（CM）： 1。从正常足月胎盘中分离的细胞，转染了混乱的siRNA或靶向mTOR信号通路的siRNA。从IUGR和适合胎龄（AGA）胎盘中分离的细胞。人HepG2细胞和原代胎狒狒肝细胞将在滋养细胞CM中孵育，并测定IGFBP-1的分泌和磷酸化（Western blot， 2d免疫印迹，ELISA和质谱法）以及IGF-I的分泌（ELISA）。此外，候选的假定体液因素将被确定使用质谱为基础的定量标记方法。在Aim 2中，我们将利用含有ert2 - cre构建体的慢病毒载体转染mTOR- floxxed小鼠的囊胚，开发条件性滋养层特异性mTOR敲低小鼠。在他莫昔芬诱导滋养细胞特异性mTOR下调后，研究胎儿肝脏中IGFBP-1的表达和磷酸化、IGF-I的表达、胎儿IGF-I和IGFBP-1的水平以及胎儿的生长情况。意义：胎儿生长异常是围产期发病的重要因素，对长期健康有着深远的影响。这项工作有可能确定胎盘直接影响胎儿代谢和生长的分子机制，并使胎儿在以后的生活中患上疾病。创新：胎盘通过调节胎儿肝功能直接调节胎儿生长的假说在概念上是新颖的。此外，我们建议开发一种方法，允许条件滋养细胞特异性基因靶向小鼠，这是-据我们所知-以前没有报道过的。因此，拟议的工作在方法上也是创新的。

DESCRIPTION (provided by applicant): Intrauterine growth restriction (IUGR) increases the risk for perinatal complications and predisposes for adult disease. However, the mechanisms mediating the restricted growth and programming of long-term health remain to be fully established. We have previously provided compelling evidence that trophoblast mammalian target of rapamycin (mTOR) signaling functions as a placental nutrient sensor, indirectly influencing fetal growth by regulating placental nutrient transporters. In this proposal we will tet the innovative hypothesis that placental mTOR signaling directly influences fetal metabolism and growth. Our central hypothesis is that inhibition of trophoblast mTOR signaling in response to restricted nutrient availability alters the release of humoral factors that cause increased secretion and phosphorylation of IGFBP-1 and decreased secretion of IGF-I from the fetal liver. We propose two specific aims: Aim 1: Determine the role of trophoblast mTOR in the regulation of IGF-I and IGFBP-1 secretion and IGFBP-1 phosphorylation in fetal liver cells in vitro and Aim 2: Establish the effect of placental specific mTOR knock down on fetal liver IGF-I and IGFBP-1 secretion and IGFBP-1 phosphorylation in the mouse. In Aim 1, we will obtain conditioned media (CM) from cultured human primary trophoblast cells of two types: 1. Cells isolated from normal term placentas and transfected with scrambled siRNA or siRNA targeting the mTOR signaling pathway and 2. Cells isolated from IUGR and Appropriate-for-Gestational-Age (AGA) placentas. Human HepG2 cells and primary fetal baboon liver cells will be incubated in trophoblast CM and secretion and phosphorylation of IGFBP-1 (Western blot, 2-D immunoblotting, ELISA and mass spectrometry approaches) as well as IGF-I secretion (ELISA) will be determined. In addition, candidates for the putative humoral factors will be identified using mass spectrometry based quantitative labeling approaches. In Aim 2, we will develop a conditional trophoblast specific mTOR knock down mouse using transfection of blastocysts from mTOR-floxed mice by lentiviral vectors containing ERT2-Cre-constructs. The expression and phosphorylation of IGFBP-1 and expression of IGF-I in fetal liver and fetal levels of IGF-I and IGFBP-1 as well as fetal growth will be studied after induction of trophoblast specific mTOR knock down by tamoxifen. Significance: Abnormal fetal growth is a major contributor to perinatal morbidity and has profound impact on long-term health. This work has the potential to identify a molecular mechanism by which the placenta directly influences fetal metabolism and growth and programs the fetus for disease later in life. Innovation: The hypothesis that the placenta directly regulates fetal growth by modulating fetal liver function is a conceptually novel idea. Furthermore, we propose to develop an approach allowing conditional trophoblast specific gene targeting in the mouse, which has - to the best of our knowledge - not previously been reported. Thus, the proposed work is also methodologically innovative. PUBLIC HEALTH RELEVANCE: Abnormal fetal growth affects 10-15% of all babies and increases the risk for injuries at delivery and to develop obesity, diabetes, and cardiovascular disease in childhood and later in life. In this proposal we will test the novel hypothesis that the placenta directly regulates fetal growth and metabolism by the release of circulating factors affecting fetal growth factor secretion. This research will increase our understanding of the role of the placenta in determining the short- and long-term health of the baby and may help design novel treatments to alleviate abnormal fetal growth.