Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-Honmachi, Chuo-Ku, Kumamoto, 862-0973, Japan.
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017, Japan.
Pharm Res. 2024 Nov;41(11):2213-2223. doi: 10.1007/s11095-024-03793-0. Epub 2024 Nov 12.
Amino acid transporters are expressed in the brain capillary endothelial cells that form the blood-brain barrier (BBB), and their expression levels change during the neonatal period. This study aimed to investigate the molecular mechanisms regulating amino acid transporter levels in mouse brain capillary endothelial cells.
Capillaries were isolated from the brains of neonatal and adult mice. Activation of eukaryotic translation initiation factor 2α kinase 4 (eIF2αK4) was analyzed in MBEC4 (mouse brain capillary endothelial) cells under amino acid-depleted conditions. Protein expression was determined using western blotting and proteomic analyses.
Phosphorylation of eIF2α, a downstream target of eIF2αK4, was induced in the brain capillaries of neonates compared to adults. In vitro experiments using MBEC4 cells revealed that amino acid depletion induced eIF2α phosphorylation and expression of the amino acid transporter, solute carrier (Slc)-7a1. The eIF2αK4 inhibitor, GCN2iB, inhibited these inductions. Proteomic analysis revealed arginine depletion-dependent induction of amino acid transporters Slc1a4, Slc3a2, Slc7a1, Slc7a5, and Slc38a1. These effects were also inhibited by GCN2iB, suggesting the involvement of eIF2αK4 activation. In contrast, the expression of Slc2a1, Slc16a1, Abcb1b, Abcg2, transferrin receptor, insulin receptor, claudin-1, ZO-1, and Jam1 was not suppressed by the GCN2iB treatment.
Overall, the eIF2αK4 pathway plays a regulatory role in amino acid transporter expression in brain capillary endothelial cells and facilitates the maintenance of amino acid homeostasis in the brain. This study provides new insights into the regulatory mechanisms underlying nutrient transport across the BBB.
氨基酸转运体在形成血脑屏障（BBB）的脑毛细血管内皮细胞中表达，其表达水平在新生儿期发生变化。本研究旨在探讨调节小鼠脑毛细血管内皮细胞中氨基酸转运体水平的分子机制。
从新生和成年小鼠的脑中分离出毛细血管。在氨基酸缺乏的条件下，分析 MBEC4（小鼠脑毛细血管内皮）细胞中真核翻译起始因子 2α 激酶 4（eIF2αK4）的激活情况。使用蛋白质印迹和蛋白质组学分析来确定蛋白质表达。
与成年小鼠相比，新生小鼠脑中的 eIF2αK4 下游靶标 eIF2α 的磷酸化被诱导。使用 MBEC4 细胞的体外实验表明，氨基酸耗竭诱导 eIF2α 磷酸化和氨基酸转运体溶质载体（Slc）-7a1 的表达。eIF2αK4 抑制剂 GCN2iB 抑制了这些诱导作用。蛋白质组学分析显示，精氨酸耗竭依赖性诱导氨基酸转运体 Slc1a4、Slc3a2、Slc7a1、Slc7a5 和 Slc38a1 的表达。这些作用也被 GCN2iB 抑制，表明 eIF2αK4 激活的参与。相比之下，Slc2a1、Slc16a1、Abcb1b、Abcg2、转铁蛋白受体、胰岛素受体、claudin-1、ZO-1 和 Jam1 的表达不受 GCN2iB 处理的抑制。
总体而言，eIF2αK4 途径在脑毛细血管内皮细胞中氨基酸转运体的表达中起调节作用，并促进脑内氨基酸的稳态维持。本研究为营养物质穿过 BBB 转运的调节机制提供了新的见解。
