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。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甲基化的关键甲基供体，DNA甲基化是一种重要的代谢途径。 表观遗传调控的关键机制。调节胎盘叶酸转运的机制还不清楚。哺乳动物雷帕霉素靶蛋白（mTOR）信号通路响应于营养物质可利用性和生长因子信号传导的变化来控制细胞生长、增殖和代谢。mTOR存在于两种复合物中，mTOR复合物1（mTORC 1）和mTORC 2，其具有不同的上游调节剂和下游靶点。mTORC 1是一种氨基酸传感器，我们最近报道滋养层mTOR信号是氨基酸转运的正调控因子。核心假设是mTOR调节滋养层叶酸摄取，并作为质子偶联叶酸转运蛋白（PCFT）介导的叶酸传感器发挥作用。该建议的主要焦点是通过使用培养的原代人滋养层（PHT）细胞测试这一假设来探索mTOR作为胎盘叶酸传感器的作用。然而，我们进一步提出，mTOR也在其他细胞类型（包括细胞系）中受到叶酸可用性的调节。为了最大限度地发挥所提出的工作的影响，并证明我们的研究结果的广泛的生物学适用性，我们将在两个非滋养层细胞系中重复一些关键的实验。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）建立mTOR信号传导在滋养层叶酸传感中的作用，和（3）鉴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（HEK 293和MCF-7细胞）中使用药理学和基因沉默方法来确定mTOR复合物（mTORC）1和2对细胞叶酸摄取的抑制或激活作用，并确定相关机制。我们将进一步探索叶酸缺乏和再引入对对照细胞和PCFT沉默细胞中mTORC 1和mTORC 2信号传导的影响。将使用成像方法和邻近连接测定法确定mTOR、PCFT和其他蛋白质（如已显示参与mTOR的氨基酸传感的Rag和Ragulator）的亚细胞定位和共定位。重要性：这项工作解决了一个主要的知识差距，并将导致识别的机制，通过介导和调节经胎盘的叶酸转运和叶酸的可用性如何调节滋养层细胞的生长和功能。此外，拟议的研究将帮助我们更好地了解母体叶酸状态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mTOR和/或PCFT在细胞叶酸传感中的作用尚未在任何细胞类型、细胞系或组织中探索。

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.