mTOR as a trophoblast folate sensor

描述（由申请人提供）：叶酸对于胎儿的正常发育和生长至关重要。围孕期叶酸缺乏与神经管缺陷有关，母体叶酸水平低与胎儿生长受限有关，但其潜在机制在很大程度上尚不清楚。此外，叶酸是 DNA 甲基化的重要甲基供体，DNA 甲基化是 表观遗传调控的关键机制。人们对介导和调节胎盘叶酸转运的机制知之甚少。哺乳动物雷帕霉素靶标 (mTOR) 信号通路响应营养物质可用性和生长因子信号传导的变化，以控制细胞生长、增殖和代谢。 mTOR 存在于两种复合物中：mTOR 复合物 1 (mTORC1) 和 mTORC2，它们具有不同的上游调节因子和下游靶标。 mTORC1 是一种氨基酸传感器，我们最近报道滋养层 mTOR 信号传导是氨基酸转运的正调节因子。中心假设是 mTOR 调节滋养层叶酸摄取，并作为质子耦合叶酸转运蛋白 (PCFT) 介导的叶酸传感器发挥作用。该提案的主要重点是通过使用培养的原代人滋养层 (PHT) 细胞测试这一假设，探索 mTOR 作为胎盘叶酸传感器的作用。然而，我们进一步提出，在其他细胞类型（包括细胞系）中，mTOR 也受到叶酸可用性的调节。为了最大限度地发挥拟议工作的影响并证明我们的发现的广泛生物学适用性，我们将在两种非滋养层细胞系中重复一些关键实验。具体目标：(1) 确定 mTOR 信号传导在调节滋养层叶酸摄取中的作用，(2) 确定 mTOR 信号传导在滋养层叶酸传感中的作用，以及 (3) 确定将叶酸可用性与滋养层 mTOR 信号传导联系起来的机制。方法：我们将在 PHT 细胞和两种细胞系（HEK293 和 MCF-7 细胞）中使用药理学和基因沉默方法，以确定 mTOR 复合物 (mTORC) 1 和 2 的抑制或激活对细胞叶酸摄取的影响，并确定所涉及的机制。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 信号传导的影响。 mTOR、PCFT 和其他蛋白质（例如已证明参与 mTOR 氨基酸传感的 Rag 和 Ragulator）的亚细胞定位和共定位将使用成像方法和邻近连接测定来确定。意义：这项工作解决了知识上的重大空白，并将导致确定介导和调节经胎盘叶酸转运的机制，以及叶酸可用性如何调节滋养层生长和功能。此外，拟议的研究将帮助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的和创新的，因为尚未在任何细胞类型、细胞系或组织中探索 mTOR 和/或 PCFT 在细胞叶酸传感中的作用。

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.