Novel Approaches to Understanding the Nutrient Regulation of Fetal Somatic Growth

描述(由申请人提供)：控制妊娠晚期胎儿肝脏生长和功能分化的机制对胎儿新陈代谢和新生儿的代谢适应至关重要。它们的扰动导致了成人代谢紊乱的胎儿起源。胎儿肝脏发育的生物学也与肝病的细胞治疗、肝癌的发生和控制胎儿的体细胞生长有关。这一建议是基于一个中心假设，即妊娠晚期胎儿肝脏发育的调节机制与控制成人肝脏质量的机制不同。重点介绍了哺乳动物靶基因雷帕霉素(MTOR)调节细胞周期进程、核糖体生物发生、蛋白质合成和基因表达的机制。mTOR是一种营养敏感的丝氨酸/苏氨酸激酶。该项目的长期目标是了解胎儿体细胞的营养调节。 成长。这一目标将通过三个具体目标来实现。前两个将侧重于控制细胞增殖和生长的分子机制。这些目标的结果将应用于第三个目标，该目标将采用增强mTOR信号的新动物模型。具体目的1是确定mTOR通过调节含有细胞周期蛋白E的复合体的活性来控制细胞周期进程的机制。这一目标将检验一种假设，即mTOR介导了细胞周期蛋白E复合体中调节细胞周期蛋白E效应因子--细胞周期蛋白依赖性激酶2(CDK2)活性的翻译后修饰。具体目的2是验证Ser/Thr蛋白磷酸酶PP2A和PP6直接参与mTOR介导的肝细胞增殖、核糖体生物发生和翻译调节的假设。在特定的目标3中，我们将把前两个目标的发现应用于妊娠晚期小鼠的全局mTOR激活模型。我们将使用一个遗传小鼠模型，在该模型中，关键的mTOR抑制物TSC1有条件地缺失。这一目标将检验两个假设。首先，一系列组织中的胎儿细胞通过促进细胞周期进程、核糖体生物发生和全球蛋白质合成，以及通过调节基因表达来对mTOR活性的增强做出反应。第二，mTOR的激活可以将胎儿从母体蛋白限制所致的生长迟缓中解救出来。该项目将结合创新的分析方法，包括稳定的shRNA介导的基因缺失和基于质谱学的磷酸蛋白质组学，以表征涉及mTOR信号的分子机制。这些目标的完成将促进我们对营养物质如何在妊娠晚期调节胎儿生长的理解。我们的结果将为研究肝脏代谢紊乱和肝癌的发病机制和预防提供帮助，同时也有助于发展基于细胞的肝病治疗策略。这项拟议的工作对人类健康和疾病的几个广泛领域也具有重要意义，包括胎儿生长及其随之而来的出生后后果的失调、兼性祖细胞的生物学，以及组织再生和癌症发生。 公共卫生相关性：拟议的工作与公共健康的相关性是基于胎儿生长与一系列疾病之间的关系，这些疾病包括肥胖、代谢综合征、2型糖尿病和癌症。了解胎儿细胞的生长调控对多种疾病的细胞治疗也有重要意义。因此，该项目的目标与NICHD对整个发展过程中的健康和疾病的关注是一致的。

DESCRIPTION (provided by applicant): The mechanisms that control the growth and functional differentiation of the liver in the late gestation fetus are critical to fetal metabolis and metabolic adaptation of the newborn. Their perturbation contributes to fetal origins of adult metabolic disorders. The biology of fetal liver development also has relevance for cell-based therapy for liver diseases, hepatic carcinogenesis, and the control of fetal somatic growth. This proposal is based on the central hypothesis that late gestation fetal liver development is regulated by mechanisms that differ from those that control adult liver mass. It focuses on the mechanisms by which the mammalian Target of Rapamycin (mTOR), a nutrient-sensing Ser/Thr kinase, regulates cell cycle progression, ribosomal biogenesis, protein synthesis, and gene expression. The long term goal of the project is to understand nutrient regulation of fetal somatic growth. This goal will be pursued through three Specific Aims. The first two will focus on molecular mechanisms that control cell proliferation and growth. Results from these aims will be applied to the third aim that will employ a novel animal model of enhanced mTOR signaling. Specific Aim 1 is to determine the mechanism by which mTOR controls cell cycle progression through regulation of the activity of cyclin E-containing complexes. This aim will test the hypothesis that mTOR mediates post-translational modification of components of cyclin E complexes that regulate activity of the cyclin E effector, cyclin- dependent kinase 2 (CDK2). Specific Aim 2 is to test the hypothesis that the Ser/Thr protein phosphatases, PP2A and PP6, are directly involved in the mTOR-mediated regulation of hepatocyte proliferation, ribosomal biogenesis, and translation. In Specific Aim 3, we will apply the findings from the first two aims to a model of global mTOR activation in the late gestation fetal mouse. We will use a genetic mouse model in which there is conditional deletion of a key mTOR inhibitor, Tsc1. This aim will test two hypotheses. The first is that fetal cells in a spectrum of tissues are responsive to augmented mTOR activity through augmented cell cycle progression, ribosomal biogenesis and global protein synthesis, and through modulation of gene expression. The second is that mTOR activation can rescue the fetus from the growth retardation associated with maternal protein restriction. The project will incorporate innovative analytical approaches, including stable shRNA- mediated gene deletion and mass spectrometry-based phosphoproteomics, to characterize molecular mechanisms involved in mTOR signaling. Completion of these aims will advance our understanding of how nutrients regulate fetal growth during late gestation. Our results will provide insight into the pathogenesis and prevention of dysregulated liver metabolism and liver cancer while also contributing to the development of cell- based strategies for therapy of liver disease. The proposed work also has significance for several broad areas of human health and disease, including the dysregulation of fetal growth and its attendant postnatal consequences, the biology of facultative progenitor cells, and tissue regeneration and carcinogenesis. PUBLIC HEALTH RELEVANCE: The relevance of the proposed work to public health is based on the relationship between fetal growth and a spectrum of disorders that includes obesity, metabolic syndrome and type 2 diabetes, and cancer. Under- standing the growth regulation of fetal cells also has implications for cell-based therapy for a variety of dis- eases. Thus, the goas of the project are consonant with the focus of NICHD on health and disease across the developmental continuum.