At Least Three Transporters Likely Mediate Threonine Uptake Needed for Mouse Embryonic Stem Cell Proliferation

Abstract

Stem cells are at the forefront of current regenerative and biomedical research. Thus, there exists an imperative and urgent need to understand the mechanisms that drive stem cell function in order to exploit their use as a therapeutic tool. Amino acids are potent inducers of signaling cascades that drive stem cell proliferation and differentiation. With a focus on mouse embryonic stem (mES) cells, Threonine (Thr) is the only amino acid required in culture media for mES cell proliferation. Current research associates this need for Thr with threonine dehydrogenase (TDH), which catabolizes Thr to glycine and acetyl-CoA in mES cells. This theory depends, in part, on the ability of 3- hydroxynorvaline (3-HNV) to inhibit both TDH and mES cell proliferation. However, the concentration of 3-HNV needed to inhibit mES cell proliferation is more than an order of magnitude less than its apparent Ki for TDH inhibition. Additionally, 3-HNV inhibits human embryonic stem (hES) cell proliferation, but hES cells do not express a functional tdh gene. Such findings indicate another mechanism for Thr stimulated mES and hES cell proliferation. Since amino acid transporters may be inducers of signaling cascades, we characterized the Thr transport systems in mES cells. We found that there is a Na(+)-dependent and a Na(+)-independent component of substrate-saturable transport, with the Na(+)-dependent component predominating. We also found that of 20 amino acids tested, the amino acids that were the strongest inhibitors of the Na(+)-dependent component of radiolabeled Thr transport were Ser, Cys, 4-OH-Pro, Asn, Met, and non-radiolabeled Thr itself. Such findings are consistent with characteristics of the ASC transport system, suggesting that this ASC system is responsible for the majority of Thr transport in mES cells. We confirmed expression of mRNA encoding the ASC system transporters, ASCT1 and ASCT2, in mES cells using RT-PCR. In conclusion, mES cells likely express at least three transporters of Thr; at least two Na(+)-dependent transporters and one Na(+)-independent one.

Keywords: amino acid transport system ASC; amino acid transport system L; cell proliferation; mES cells; threonine.

Figure 1

Time course of 50 uM [³H]-Thr uptake in the presence of 140 mM NaCl, LiCl, Choline Cl, or KCl, or 280 mM Mannitol. (A) Uptake was measured at 2.5, 5, and 10 min and increased significantly with time (p < 0.0001) (n = 3; 1 replicate experiment for each time point). (B) Time course of 50 uM [³H]-Thr uptake in the presence of 140 mM NaCl at 0, 1, 2, and 3 min. Uptake increased significantly with time (r = 0.99, p = 0.01, 1 replicate experiment for each time point).

Figure 2

Percent uptake of 50 uM [³H]-Thr in the presence of 140 mM NaCl and 10 mM of the amino acid indicated. (A) A 1-sample t-test determined the more complete inhibitors (^**p < 0.0001) and other statistically significant inhibitors (^*p < 0.05) (n = 6 for each column; 3 determinations obtained in each of 2 independent experiments). (B) Percent uptake of 50 uM [³H]-Thr in the presence of 280 mM Mannitol and 10 mM of the amino acid indicated. A 1-sample t-test determined the more complete inhibitor (^**p < 0.0001) and other statistically significant inhibitors (^*p < 0.05) (n = 6 for each column; 3 determinations obtained in each of 2 independent experiments).

Figure 3

Percent uptake of 50 uM [³H]-Thr in the presence of 140 mM NaCl and increasing concentrations of (A) Leu (n = 6 for each point; 3 determinations obtained in each of 2 independent experiments) or (B) Pro (n = 6 for each point; 3 determinations obtained in each of 2 independent experiments). Thr uptake was also measured in the presence of 20 mM non-radioactive Thr. A one-way ANOVA indicated statistically significant differences in [³H]-Thr uptake as the concentration of Leu and Pro increased. An asterisk (^*) signifies significantly more inhibition by a treatment than the one that precedes it (p < 0.05). (C) Percent uptake of 50 uM [³H]-Thr in the presence of 140 mM NaCl and Leu, Pro, or Leu and Pro combined (n = 6 for each point; 3 determinations obtained in each of 2 independent experiments). A one-way ANOVA indicated no statistically significant differences (p > 0.05) in [³H]-Thr uptake except between 5 mM Pro and 10 mM Leu.

Figure 4

Percent Na⁺-dependent uptake of 50 uM [³H]-Thr remaining in the presence of the indicated amino acid (500 uM) (A) after subtraction of uptake in 140 mM Choline Cl. A 1-sample t-test confirmed that all amino acids tested were s significant inhibitors (^*p < 0.05). A one-way ANOVA indicated no statistically significant difference in [³H]-Thr uptake in the presence of the different amino acids tested (p > 0.05) (n = 4 for each column; 2 determinations obtained in each of 2 independent experiments). (B) Percent uptake of [³H]-Thr remaining in the presence of the indicated amino acid (500 uM) but without subtraction of Na⁺-independent transport. A 1-sample t-test confirmed that all amino acids were statistically significant inhibitors (^*p < 0.05). A one-way ANOVA indicated no statistically significant difference in [³H]-Thr uptake in the presence of the different amino acids tested (p > 0.05) (n = 4 for each column; 3 determinations obtained in each of 2 independent experiments).

Figure 5

Percent Na⁺-dependent uptake of 50 uM [³H]-Thr remaining in the presence of the indicated amino acid (500 uM) (A) after subtraction of uptake in 140 mM Choline Cl. A 1-sample t-test confirmed that all amino acids were statistically significant inhibitors (^*p < 0.05). A one-way ANOVA indicated no statistically significant difference in [³H]-Thr uptake in the presence of the different amino acids tested (p > 0.05) (n = 4 for each column; 2 determinations obtained in each of 2 independent experiments). (B) Percent uptake of [³H]-Thr remaining in the presence of the indicated amino acid (500 uM) but without subtraction of Na⁺-independent transport. A 1-sample t-test confirmed that all amino acids were statistically significant inhibitors (^*p < 0.05). A one-way ANOVA indicated 4-OH-Pro inhibited [³H]-Thr uptake significantly more than the other amino acids tested (p < 0.05). (n = 4 for each column; 2 determinations obtained in each of 2 independent experiments).

Figure 6

Percent uptake of 50 uM [³H]-Thr in the presence of 140 mM NaCl and increasing concentrations of (A) Ser or (B) 3-HNV (n = 6 for each point; 3 determinations obtained in each of 2 independent experiments). Thr uptake was also measured in the presence of 20 mM non-radiolabeled Thr. A 2-sample t-test indicated that [³H]-Thr uptake in 20 mM Ser was not significantly different from [³H]-Thr uptake in 20 mM non-radiolabeled Thr (p > 0.05), whereas [³H]- Thr uptake in 20 mM 3-HNV was significantly higher than [³H]- Thr uptake in 20 mM non-radiolabeled Thr (p < 0.05). A one-way ANOVA also indicated that 50 uM [³H]-Thr uptake significantly decreased with increasing concentration of Ser or 3-HNV (p < 0.05).

Figure 7

mRNA expression of ASCT1 and ASCT2 in mES cells as determined by RT-RCR. Lane 1: DNA ladder; Lane 2: ASCT1 primers and no RNA template control; Lane 3: ASCT1 primers and template RNA; Lane 4: ASCT2 primers and no template RNA control; Lane 5: ASCT2 primers and template RNA.