mTOR as a trophoblast folate sensor

描述(由申请人提供)：叶酸对胎儿的正常发育和生长至关重要。妊娠期叶酸缺乏症与神经管畸形有关，母体叶酸水平低与胎儿生长受限有关，但其潜在机制很大程度上尚不清楚。此外，叶酸是DNA甲基化的关键甲基供体，这是一种 表观遗传调控的关键机制。调节和调节胎盘叶酸运输的机制还知之甚少。哺乳动物靶标雷帕霉素(MTOR)信号通路响应营养物质供应和生长因子信号的变化，控制细胞的生长、增殖和代谢。MTOR存在于mTOR复合体1(MTORC1)和mTORC2两个复合体中，它们具有不同的上游调控因子和下游靶点。MTORC1是一种氨基酸感受器，我们最近报道，滋养层细胞的mTOR信号是氨基酸转运的正向调节因子。核心假设是mTOR调节滋养层细胞的叶酸摄取，并通过质子偶联叶酸转运体(PCFT)作为叶酸感受器发挥作用。这项建议的主要焦点是通过使用培养的原代人类滋养细胞(PHT)来验证这一假设，以探索mTOR作为胎盘叶酸感受器的作用。然而，我们进一步提出，在其他类型的细胞中，包括细胞系在内，mTOR也受叶酸供应的调节。为了最大限度地发挥拟议工作的影响，并证明我们的发现在生物学上的广泛适用性，我们将在两个非滋养细胞系中重复一些关键实验。具体目标：(1)确定mTOR信号在调节滋养层细胞叶酸摄取中的作用；(2)确定mTOR信号在滋养层细胞叶酸感知中的作用；(3)确定叶酸可利用性与滋养层细胞mTOR信号之间的联系机制。方法：我们将在PHT细胞和两个细胞系(HEK293和MCF-7细胞)中使用药理学和基因沉默的方法来确定mTOR复合体(MTORC)1和2的抑制或激活对细胞叶酸摄取的影响，并确定相关的机制。我们将进一步探讨叶酸缺乏和重新引入对对照细胞和PCFT沉默细胞中mTORC1和mTORC2信号的影响。MTOR、PCFT和其他蛋白质的亚细胞定位和共定位将使用成像方法和邻近连接分析来确定，这些蛋白质如RAG和Ragator已被证明参与了mTOR的氨基酸感应。意义：这项工作解决了一个主要的知识空白，并将导致确定通过胎盘叶酸运输的调节和调节的机制，以及叶酸的可用性如何调节滋养层细胞的生长和功能。此外，拟议的研究将有助于我们更好地了解母体叶酸状况与胎儿生长发育之间的分子联系。创新：我们的中心假设在概念上是新颖和创新的，因为mTOR和/或PCFT在细胞叶酸感知中的作用尚未在任何细胞类型、细胞系或组织中进行探索。

DESCRIPTION (provided by applicant): Folate is critical for normal fetal development and growth. Periconceptional folate deficiency is associated with neural tube defects and low maternal folate levels are linked to restricted fetal growth, however the underlying mechanisms are largely unknown. In addition, folate is a critical methyl donor for DNA methylation, which is a key mechanism of epigenetic regulation. The mechanisms mediating and regulating placental folate transport are poorly understood. The mammalian target of rapamycin (mTOR) signaling pathway responds to changes in nutrient availability and growth factor signaling to control cell growth, proliferation and metabolism. mTOR exists in two complexes, mTOR Complex 1 (mTORC1) and mTORC2, which have distinct upstream regulators and downstream targets. mTORC1 is an amino acid sensor and we recently reported that trophoblast mTOR signaling is a positive regulator of amino acid transport. The central hypothesis is that mTOR regulates trophoblast folate uptake and functions as a folate sensor mediated by the proton-coupled folate transporter (PCFT). The primary focus of this proposal is to explore the role of mTOR as a placental folate sensor by testing this hypothesis using cultured primary human trophoblast (PHT) cells. However, we further propose that mTOR is regulated by folate availability also in other cell types, including cell lines. To maximize the impact of the proposed work and to demonstrate broad biological applicability of our findings we will repeat some of the critical experiments in two non-trophoblast cell lines. Specific Aims: (1) Determine the role of mTOR signaling in regulating trophoblast folate uptake, (2) Establish the role of mTOR signaling in trophoblast folate sensing and (3) Identify the mechanism linking folate availability to trophoblas mTOR signaling. Approach: We will use pharmacological and gene silencing approaches in PHT cells and two cell lines (HEK293 and MCF-7 cells) to determine the effect of inhibition or activation of mTOR Complex (mTORC) 1 and 2 on cellular folate uptake, and identify the mechanisms involved. We will further explore the effects of folate deficiency and re- introduction on mTORC1 and mTORC2 signaling in control cells and in PCFT silenced cells. Subcellular localization and co-localization of mTOR, PCFT and other proteins, such as Rag and Ragulator that have been shown to participate in amino acid sensing by mTOR, will be determined using imaging approaches and proximity ligation assay. Significance: This work addresses a major gap in knowledge and will lead to the identification of mechanisms by which transplacental folate transport is mediated and regulated and how folate availability modulates trophoblast growth and function. Moreover, the proposed research will help us better understand the molecular links between maternal folate status and fetal growth and development. Innovation: Our cental hypothesis is conceptually novel and innovative because a role of mTOR and/or PCFT in cellular folate sensing has not been explored in any cell type, cell line or tissue.