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，调解限制长期健康增长和规划的机制仍有待充分建立。我们已经提供了令人信服的证据表明，滋养层哺乳动物雷帕霉素靶标(MTOR)信号作为胎盘营养感受器发挥作用，通过调节胎盘营养转运蛋白间接影响胎儿生长。在这项提案中，我们将测试胎盘mTOR信号直接影响胎儿新陈代谢和生长的创新假设。我们的中心假设是，在营养供应受限的情况下抑制滋养层细胞mTOR信号会改变体液因子的释放，从而导致IGFBP-1的分泌和磷酸化增加，以及胎儿肝脏IGF-I的分泌减少。目的1：确定滋养层细胞mTOR在体外对胎肝细胞IGF-I、IGFBP-1分泌和IGFBP-1磷酸化的调节作用；目的2：建立胎盘特异性mTOR敲除对小鼠胎肝IGF-I、IGFBP-1分泌和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层细胞中获得条件培养液(CM)：1.从正常足月胎盘分离的细胞并转导mTOR信号通路的siRNA或siRNA；2.从IUGR和适宜妊娠年龄(AGA)胎盘分离的细胞。将人的HepG2细胞和原代胎猴肝细胞与滋养层细胞CM孵育，测定IGFBP-1的分泌和磷酸化(Western印迹、双向免疫印迹、ELISA法和质谱法)以及IGF-I的分泌量(ELISA法)。此外，假定的体液因素的候选将使用基于质谱学的定量标记方法来确定。在目标2中，我们将利用含有ERT2-Cre-结构的慢病毒载体，从mTOR-Flobled小鼠的囊胚中导入mTOR基因，建立条件滋养细胞特异性mTOR基因敲除小鼠。用他莫昔芬诱导滋养细胞特异性mTOR后，将研究IGFBP-1在胎肝中的表达和磷酸化、IGF-I的表达、胎儿IGF-I和IGFBP-1的水平以及对胎儿生长的影响。意义：胎儿发育异常是围产期发病率的主要因素，并对长期健康有深远影响。这项工作有可能确定一种分子机制，通过这种机制，胎盘直接影响胎儿的新陈代谢和生长，并为胎儿日后的疾病做好准备。创新：胎盘通过调节胎儿肝脏功能直接调节胎儿生长的假设在概念上是一个新的想法。此外，我们建议开发一种方法，允许在小鼠中进行条件滋养层特异性基因打靶，据我们所知，这在以前没有报道过。因此，拟议的工作在方法上也是创新的。 与公共卫生相关：胎儿发育异常影响到10%-15%的婴儿，增加了分娩时受伤的风险，并在童年和以后的生活中发展为肥胖、糖尿病和心血管疾病。在这个提案中，我们将测试新的假设，即 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异常生长。

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.