Department of Clinical Biochemistry and Laboratory Diagnostics, Institute of Medical Sciences, University of Opole, Oleska 48, 45-052, Opole, Poland.
Sci Rep. 2019 Dec 27;9(1):20165. doi: 10.1038/s41598-019-56781-5.
Under physiological and pathological conditions, elastin is degraded to produce elastin-derived peptides (EDPs). EDPs are detected in the healthy human brain, and its concentration significantly increases after ischemic stroke. Both elastin and EDPs contains replications of the soluble VGVAPG hexapeptide, which has a broad range of biological activities. Effects of VGVAPG action are mainly mediated by elastin-binding protein (EBP), which is alternatively spliced, enzymatically inactive form of the GLB1 gene. This study was conducted to elucidate the activation and role of the N-methyl-D-aspartate receptor (NMDAR) in elastin-derived VGVAPG peptide-dependent calcium homeostasis in mouse cortical astrocytes in vitro. Cells were exposed to 10 nM VGVAPG peptide and co-treated with MK-801, nifedipine, verapamil, or Src kinase inhibitor I. After cell stimulation, we measured Ca level, ROS production, and mRNA expression. Moreover, the Glb1 and NMDAR subunits (GluN1, GluN2A, and GluN2B) siRNA gene knockdown were applied. We found the VGVAPG peptide causes Ca influx through the NMDA receptor in mouse astrocytes in vitro. Silencing of the Glb1, GluN1, GluN2A, and GluN2B gene prevented VGVAPG peptide-induced increase in Ca. Nifedipine does not completely reduce VGVAPG peptide-activated ROS production, whereas MK-801, verapamil, and Src inhibitor reduce VGVAPG peptide-activated Ca influx and ROS production. These data suggest the role of Src kinase signal transduction from EBP to NMDAR. Moreover, the VGVAPG peptide affects the expression of NMDA receptor subunits.
在生理和病理条件下，弹性蛋白会降解产生弹性蛋白衍生肽（EDP）。EDP 在健康人的大脑中被检测到，其浓度在缺血性中风后显著增加。弹性蛋白和 EDP 都包含可溶性 VGVAPG 六肽的复制物，该六肽具有广泛的生物学活性。VGVAPG 作用的影响主要通过弹性蛋白结合蛋白（EBP）介导，EBP 是 GLB1 基因的选择性剪接、无酶活性形式。本研究旨在阐明 N-甲基-D-天冬氨酸受体（NMDAR）在体外小鼠皮质星形胶质细胞中弹性蛋白衍生的 VGVAPG 肽依赖性钙稳态中的激活和作用。细胞暴露于 10 nM VGVAPG 肽并与 MK-801、硝苯地平、维拉帕米或Src 激酶抑制剂 I 共同处理。细胞刺激后，我们测量 Ca 水平、ROS 产生和 mRNA 表达。此外，还应用了 Glb1 和 NMDAR 亚基（GluN1、GluN2A 和 GluN2B）siRNA 基因敲低。我们发现 VGVAPG 肽在体外引起小鼠星形胶质细胞中 NMDA 受体的 Ca 内流。Glb1、GluN1、GluN2A 和 GluN2B 基因沉默阻止了 VGVAPG 肽诱导的 Ca 增加。硝苯地平不能完全减少 VGVAPG 肽激活的 ROS 产生，而 MK-801、维拉帕米和 Src 抑制剂减少了 VGVAPG 肽激活的 Ca 内流和 ROS 产生。这些数据表明 Src 激酶信号转导从 EBP 到 NMDAR 的作用。此外，VGVAPG 肽影响 NMDA 受体亚基的表达。
