mTOR as a trophoblast folate sensor

描述（由申请人提供）：叶酸对胎儿正常发育和生长至关重要。妊娠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

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.