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 akey 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.
Folate deficiency in pregnancy is associated with fetal malformations and restricted fetal growth and both low and high folate intake have been linked to cancer. In this work we will explore a previously unknown molecular link between folate availability and cell growth and proliferation that may help us better understand how these diseases develop. This research may generate unique information leading to novel treatments to alleviate restricted fetal growth and cancer.
