L-proline metabolism and the regulation of embryonic stem cell differentiation and programming

\(Objective:\) Proline metabolism has been implied in cancer, with changes found to support the growth and metastasis of cancer cells. Based on this, proline metabolism has been proposed as a safe and specific target for cancer therapy. However, the mechanisms by which proline metabolism alters cell programming is not well-characterised. It is not known about the role of proline metabolism in normal cell growth and how proline metabolism regulates cellular programs. Embryonic stem (ES) cell differentiation is a non-pathological cell system, in which proline metabolism was suggested to play a role. Studying proline metabolism in ES cell differentiation will provide useful information about the metabolic control of cell programming and will identify potential targets for future research in cancers. \(Background:\) Proline metabolism is an important node of a complex metabolic network that connects mitochondrial and cytoplasmic activities. Proline metabolism involves multiple reactions that are divided into three steps: proline uptake, catabolism and biosynthesis. The key reaction is the oxidation of proline, which is performed inside the mitochondria by a single enzyme: proline dehydrogenase (PRODH). PRODH oxidises proline to form pyrroline-5-carboxylate (P5C) and reactive oxygen species (ROS) as a by-product. In mouse ES cells, the primary transporter of proline, Snat2, was found to be involved in proline-induced differentiation. The use of amino acids to compete with proline transport via Snat2 was effective to prevent the formation of differentiated cells, suggesting that proline uptake via Snat2 is essential. In addition, the use of Prodh inhibitors and the neutralisation of ROS by antioxidants resulted in a decreased number of proline-induced differentiated cells. This evidence suggests that Prodh activity and the formation of ROS are required for ES cell differentiation. Furthermore, ROS-activated proteins, such as p38-MAPK and Src, were reported to be involved in proline-induced differentiation of ES cells. However, it is currently not known what connects proline metabolism and ROS production with ES cell differentiation. As a result, the precise mechanism of proline-induced ES cell differentiation is unclear. Taken together, it is hypothesised that ROS derived from proline metabolism play a role in ES cell differentiation. \(Aims\) \(and\) \(Methods:\) To test this hypothesis, two lines of mouse ES cells: D3 and WA30 were investigated in three experiments. The first experiment aimed to examine the expression of the proline metabolic enzyme family to confirm potential proline metabolic reactions. The expression of proline transporter, Snat2, is differentially regulated in two different pluripotent states of ES cells: naïve and primed states. It was questioned if the regulation of Snat2 would affect proline metabolism and proline-induced differentiation in these ES cell states. Thus, the expression of the proline metabolic enzyme family was examined in different pluripotent states of ES cells. In the second experiment, the level of mitochondrial ROS production was used as an indicator of proline oxidation. The changes in ROS levels was also detected in different time courses to investigate the dynamics of proline response in naïve and primed ES cells. The third experiment aimed to correlate ROS production with proline metabolism and ES cell differentiation to examine the precise mechanism of proline-induced differentiation. \(Results:\) The data showed that proline metabolic enzyme family is differentially expressed in different pluripotent states of ES cells. It was also found that naïve and primed ES cells have different distribution of the proline metabolic enzymes. The measurement of mitochondrial ROS production revealed heterogeneous populations of ES cells that produced different ROS levels. Up-regulated expression of proline metabolic enzyme, Prodh, in primed ES cells resulted in increased ROS production in the addition of proline, which is correlated with proline-induced differentiation. However, these mitochondrial ROS were not primarily required for the differentiation process. Proline-induced ROS production may not necessarily be correlated with proline metabolism and the role of Prodh needs further clarification. \(Discussion:\) This is the first study to examine proline metabolism in ES cells following their pluripotent lineage specification and differentiation. The data suggest a proline metabolic switch in ES cell programming and differentiation, which could be used to distinguish ES cells of different pluripotent states. These findings indicate that proline metabolism is an important factor in mitochondrial metabolic regulation, which has been previously demonstrated in ES cell programming. The investigation of ROS production has revealed that mitochondrial metabolism can be used to profile ES cells in different pluripotent states. Even though no firm conclusion can be made on the role of proline metabolism and Prodh in ES cell differentiation, the findings of this study indicate that other proline acceptor sites in the cellular environment should be considered for future investigation of proline-induced changes in ES cells. It is suggested that a model of Prodh knock-out ES cell line can be useful to study the role of Prodh and proline metabolism in ES cell differentiation