IGFBP-1 hyperphosphorylation in IUGR: Role of mTOR and CK2

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。胰岛素样生长因子I（IGF-I）是胎儿生长的关键调节因子，IGF-I的生物利用度通过与IG结合蛋白-1（IGFBP-1）结合来调节。我们和其他人提供的证据表明，IGFBP-1的磷酸化显着增加其结合IGF-I的亲和力，IGFBP-1的过度磷酸化可能构成了一个重要的机制，生长减少IUGR。然而，在IUGR中引起IGFBP-1磷酸化的分子机制在很大程度上是未知的。该机制建议的中心假设是，在胎儿肝脏中抑制mTOR信号传导和激活蛋白激酶CK 2构成了营养剥夺与体外和体内IUGR中IGFBP-1分泌和磷酸化增加之间的关键分子联系。我们的假设是基于我们令人信服的初步数据，包括以下证明：（1）在HepG 2细胞中，mTOR抑制诱导显著的IGFBP-1磷酸化，蛋白激酶CK 2的沉默消除了mTOR抑制诱导的IGFBP-1过度磷酸化;（2）IUGR狒狒肝脏中mTOR活性降低，而CK 2表达和IGFBP-1磷酸化增加。为了验证我们的假设，我们将研究HepG 2细胞，狒狒的原代胎肝细胞，对照和IUGR狒狒胎儿的血液和肝脏组织，有两个具体的目标：在目标1中，我们将在HepG 2细胞和胎儿狒狒原代肝细胞中使用基因沉默方法和药理学抑制剂，将mTOR信号传导与蛋白激酶CK 2和IGFBP-1磷酸化的调节机制联系起来。此外，我们将检验以下假设：由于氨基酸剥夺导致的IGFBP-1磷酸化增加是通过抑制mTOR和激活CK 2介导的。在目标2中，我们将使用一个成熟的狒狒模型IUGR涉及产妇营养限制。我们将确定胎肝样本中mTOR和CK 2的活性、IGFBP-1的表达和磷酸化以及对照和IUGR狒狒胎儿中IGFBP-1的血清浓度和磷酸化。意义：这项工作有可能确定IUGR发展的新分子机制。此外，我们将利用高度相关的非人灵长类动物模型，该模型提供了通过人类研究无法获得的胎儿样本的独特途径。创新：这项工作将提供mTOR和IGF-I信号传导之间的创新机制联系，这是调节细胞生长的两个关键途径。将培养的HepG 2和原代胎狒狒肝细胞中的机制方法与狒狒IUGR模型的肝组织研究相结合是转化和创新的。

DESCRIPTION (provided by applicant): Intrauterine growth restriction (IUGR) increases the risk for perinatal complications and predisposes for adult disease. Insulin-like growth factor I (IGF-I) is a key regulator of fetal growth, and IGF-I bioavailability is modulated by binding to IG binding protein-1 (IGFBP-1). We and others have provided evidence that phosphorylation of IGFBP-1 markedly increases its affinity to bind IGF-I and that IGFBP-1 hyper phosphorylation may constitute an important mechanism by which growth is reduced in IUGR. However, the molecular mechanisms causing IGFBP-1 phosphorylation in IUGR are largely unknown. The central hypothesis in this mechanistic proposal is that inhibition of mTOR signaling and activation of protein kinase CK2 in the fetal liver constitutes a key molecular link between nutrient deprivation and increased IGFBP-1 secretion and phosphorylation in vitro and in IUGR in vivo. Our hypothesis has been formulated based on our compelling preliminary data including the demonstration that (1) mTOR inhibition induces marked IGFBP-1 phosphorylation and silencing of protein kinase CK2 abolishes IGFBP-1 hyper phosphorylation induced by mTOR inhibition in HepG2 cells and (2) mTOR activity is decreased whereas CK2 expression and IGFBP-1 phosphorylation are increased in the liver of IUGR baboons. To test our hypothesis, we will study HepG2 cells, primary fetal hepatocytes from baboons, and blood and liver tissue of control and IUGR baboon fetuses in two specific aims: In Aim 1 we will use gene silencing approaches and pharmacological inhibitors in HepG2 cells and fetal baboon primary hepatocytes to mechanistically link mTOR signaling to regulation of protein kinase CK2 and IGFBP-1 phosphorylation. In addition, we will test the hypothesis that the increased IGFBP-1 phosphorylation due to amino acid deprivation is mediated by inhibition of mTOR and activation of CK2. In Aim 2 we will use a well-established baboon model of IUGR involving maternal nutrient restriction. We will determine the activity of mTOR and CK2, IGFBP-1 expression and phosphorylation in fetal liver samples and IGFBP-1 serum concentrations and phosphorylation in control and IUGR baboon fetuses. Significance: This work has the potential to identify a novel molecular mechanism underlying the development of IUGR. Furthermore, we will utilize a highly relevant non-human primate model that provides unique access to fetal samples not available through human studies. Innovation: This work will provide an innovative mechanistic link between mTOR and IGF-I signaling, two critical pathways in the regulation of cell growth. The combination of mechanistic approaches in cultured HepG2 and primary fetal baboon hepatocytes with studies of liver tissue from a baboon IUGR model is translational and innovative.