Molecular Mechanisms Linking Placental Nutrient Sensing and Fetal Programming

描述（由申请人提供）：宫内生长受限（IUGR）增加了围产期并发症的风险，并易患成人疾病。然而，调解有限增长和长期保健方案规划的机制仍有待充分建立。我们以前提供了令人信服的证据表明，滋养层哺乳动物雷帕霉素靶蛋白（mTOR）信号传导作为胎盘营养传感器，通过调节胎盘营养转运蛋白间接影响胎儿生长。在本研究中，我们将泰特胎盘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，滋养层mTOR信号的抑制，以响应有限的营养供应改变体液因子的释放，导致增加分泌和磷酸化的IGFBP-1和减少分泌的IGF-I从胎肝。我们提出两个具体目标：目标1：确定滋养层mTOR在体外胎肝细胞中调节IGF-I和IGFBP-1分泌以及IGFBP-1磷酸化中的作用，目的2：确定胎盘特异性mTOR敲低对小鼠胎肝IGF-I和IGFBP-1分泌以及IGFBP-1磷酸化的影响。目的1：从培养的人原代滋养层细胞中获得两种类型的条件培养基：1。从正常足月胎盘分离并用乱序siRNA或靶向mTOR信号传导途径的siRNA转染的细胞和2.从IUGR和适合孕龄（阿加）胎盘中分离的细胞。将人HepG 2细胞和原代胎狒狒肝细胞在滋养层CM中孵育，并测定IGFBP-1的分泌和磷酸化（Western印迹、2-D免疫印迹、ELISA和质谱法）以及IGF-I分泌（ELISA）。此外，将使用基于质谱的定量标记方法鉴定推定体液因子的候选物。在目标2中，我们将使用含有ERT 2-Cre-构建体的慢病毒载体转染来自mTOR-floxed小鼠的囊胚来开发条件性滋养层特异性mTOR敲低小鼠。在通过他莫昔芬诱导滋养层特异性mTOR敲低后，将研究IGFBP-1的表达和磷酸化以及胎儿肝脏中IGF-I的表达和IGF-I和IGFBP-1的胎儿水平以及胎儿生长。意义：胎儿生长异常是围产期发病的主要原因，对长期健康有深远影响。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，并为胎儿日后的疾病做好准备。创新：胎盘通过调节胎儿肝功能直接调节胎儿生长的假设是一个概念上的新想法。此外，我们建议开发一种方法，允许有条件的滋养层特异性基因靶向小鼠，这已经-据我们所知-以前没有报道。因此，拟议的工作在方法上也是创新的。 公共卫生相关性：胎儿生长异常影响10-15%的婴儿，并增加分娩时受伤的风险，并在儿童时期和以后的生活中发展肥胖，糖尿病和心血管疾病。在这个建议中，我们将测试新的假设， 胎盘通过释放影响胎儿生长因子分泌的循环因子直接调节胎儿生长和代谢。这项研究将增加我们对胎盘在决定婴儿短期和长期健康方面的作用的理解，并可能有助于设计新的治疗方法来缓解胎儿生长异常。

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.