Requirement of the expression of 3-phosphoglycerate dehydrogenase for traversing S phase in murine T lymphocytes following immobilized anti-CD3 activation
Introduction
l-Serine is required not only for protein synthesis but also for various metabolic pathways to generate several essential compounds including glycine, cysteine, d-serine, phosphatidylserine, sphingomyelins, and cerebrosides [1]. It is also utilized for synthesis of nucleotide precursors, which is linked to cellular replication, and for redox control, which can contribute to cell survival [2]. Although l-serine is available from dietary sources, it can be endogenously synthesized from glycolytic intermediate by the phosphorylated pathway in mammals. The phosphorylated pathway starts at 3-phosphoglycerate and sequentially proceeds three stages of enzymatic reactions to synthesize l-serine. The 3-phosphoglycerate dehydrogenase (PHGDH) catalyses the transition of 3-phosphoglycerate into 3-phosphohydroxy pyruvate, which is the first and rate-limiting step in the phosphorylated pathway, using NAD+/NADH as a cofactor. Phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP) catalyze the conversion of 3-phosphohydroxy pyruvate to 3-phosphoserine and subsequent dephosphorylation of 3-phosphoserine to l-serine, respectively.
It has been reported that the enzyme activity of PHGDH is elevated eight- to 50-fold in rat hepatoma [3], 10-fold in human colon carcinoma, and 32-fold in rat sarcoma [4] as compared with individual normal control values. It has also been shown that PHGDH expression at the transcription level is upregulated in human colon carcinoma, and in most leukemias and lymphomas of human and murine origin [5]. This elevated activity of PHGDH, which leads to the enhanced capacity of l-serine biosynthesis, has been interpreted as an acquired growth advantage of tumor cells because of the metabolic importance of l-serine in nucleotide biosynthesis [3], [4]. In healthy humans, de novo synthesis of l-serine by the phosphorylate pathway is essential to supply the l-serine required in the brain, because the delivery of l-serine to the central nervous system (CNS) is insufficient due to the blood–brain barrier [6], [7]. In relation to the role of l-serine in the CNS, l-serine and its downstream metabolite glycine serve as the neurotrophic factor to promote survival, dendritogenesis, and electrophysiological development of neurons [8]. In addition, d-serine, which acts as a co-agonist of the N-methyl-d-aspartate (NMDA) receptor along with glutamate and glycine, is directly formed from l-serine by serine racemase [9], [10]. The importance of l-serine synthesis via the phosphorylated pathway in the brain has also been verified by human inborn disorder with enzymic defects in PHGDH, which results in a severe neurological syndrome [11], and by the PHGDH-knockout mice model exhibiting early embryonic lethality associated with multiple neurodevelopmental defects [12], [13].
The importance of l-serine in cellular replication and thus highest capacity of the phosphorylated pathway in S phase of the cell cycle are supported by our previous results, which showed that the level of mRNA specific for both PHGDH and PSAT, abruptly down-regulated in accordance with growth arrest of U937 cells on 12-O-tetradecanoylphorbol 13-acete (TPA)-induced monocytic differentiation, can be recovered when TPA-treated cells restore cell growth by a retrodifferentiation process, and that the level of PHGDH- and PSAT-specific mRNA fluctuates during the cell cycle with a maximum in S phase of human Jurkat T cells [5], [14]. It has also been shown that ectopic overexpression of PSAT in colon cancer cell line results in an enhancement in its growth rate and survival [15]. Although these previous results suggested that the expression of PHGDH gene, which contributes to the phosphorylated pathway, might be a prerequisite for traversing S phase, there has been no direct evidence showing the requirement of PHGDH gene expression for S phase during cell cycle progression.
In the present study, the expression of PHGDH gene was investigated during immobilized anti-CD3 activation of murine splenic resting (G0) T lymphocytes, with focusing on contribution of interleukin-2 receptor (IL-2R) signaling, which is known to induce the signal necessary for the G1/S transition in activated T cells [16], [17], [18], to the induction of PHGDH gene expression. Using antisense PHGDH oligonucleotide, the requirement of PHGDH for DNA synthesis of activated T cells was also investigated. We show that the expression of PHGDH mRNA is not detected in G0 T cells but is induced in G1 phase by IL-2R signaling and reaches a maximal level in S phase of activated T cells, along with a significant reduction in [3H]TdR incorporation into DNA of activated T cells in the presence of antisense PHGDH oligonucleotide that can specifically block the translation of PHGDH transcript. These results first demonstrate that the expression of PHGDH gene depends on IL-2R signaling and is required for S phase during the proliferation of activated T cells.
Section snippets
Animals
C57BL/6 male mice, 4–6 months old, were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained in the Gerontology Research Center Animal Facility (Baltimore, MD, USA) and the Animal Resources Center at Korea Research Institute of Bioscience and Bioengineering (Daejeon, Korea) under specific-pathogen free conditions.
Chemicals, antibodies, reagents, cDNA probes, and medium
Phorbol dibutyrate (PBu2) was purchased from Calbiochem (La Jolla, Ca, USA), and cyclosporine A (CsA) and nocodazole were obtained from Sigma Chemical (St. Louis,
Expression of PHGDH gene in anti-CD3-activated T cells
When murine G0 T cells were activated by immobilized anti-CD3, the cells appeared to enter S phase in 20 h and approximately 34% of the T cells were accumulated in S phase 25 h after activation, at which time the cells in G2/M phase began to be detected, as determined by flow cytometry (Fig. 1A). After activation for 30 h, the cell population was more heterogeneous, with approximately 59% of the cells in S phase, 30% in G1 phase, and 10% in G2/M phase. However, after activation for 40 h, only 3% of
Discussion
The interaction of antigen or mitogenic lectin with specific receptors on G0 T lymphocytes initiates a cascade of biochemical events to induce the expression of a wide variety of genes essential for activation and proliferation, and subsequent immune functions [32], [33], [34], [35]. The IL-2 and IL-2R are known to be critical genes for the proliferation of activated T cells, in that binding of IL-2 to IL-2R expressed on the surface of activated T cells induces the signal necessary for the G1/S
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by a Grant (02-PJ1-PG3-20908-0036) from the Ministry of Health and Welfare, Korea, and a Grant (KRF-2003-005-J00103) from the Korean Research Foundation, the National Research Foundation of Korea Grant funded by the Korean government (NRF-353-2009-2-F00021), and in part by the Intramural Research Program of the National Institute on Aging, National Institutes of Health.
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