Downregulation of vascular endothelial growth factor receptor-2 under oxidative stress conditions is mediated by β-transduction repeat-containing protein via glycogen synthase kinase-3β signaling.
Downregulation of vascular endothelial growth factor receptor-2 under oxidative stress conditions is mediated by β-transduction repeat-containing protein via glycogen synthase kinase-3β signaling.
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氧化应激条件下血管内皮生长因子受体 2 的下调是通过糖原合酶激酶 3 β 信号转导含有重复序列的蛋白质介导的
DOI：
10.3892/ijmm.2016.2493
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发表时间：
2016-04
影响因子：
5.4
通讯作者：
Chen S
中科院分区：
文献类型：
--
作者：
Wu W;Zhang D;Pan D;Zuo G;Ren X;Chen S
血管内皮生长因子受体-2(VEGFR-2)是血管生成反应的关键决定因素，在糖尿病小鼠氧化应激下表达降低。β转导重复序列蛋白(β-TrCP)参与了甲状腺癌细胞对VEGFR-2的降解。此外，糖原合成酶激酶-3β(GSK-3β)在β-TrCP诱导的多种蛋白质降解中起中介作用。然而，β-TrCP和β在诱导高血糖的血管内皮细胞降解VEGFR-2中的作用尚不完全清楚。在本研究中，我们旨在通过研究高血糖或葡萄糖氧化酶(GO)诱导的过量活性氧自由基(ROS)来分析VEGFR-2的降解机制。用不同浓度的葡萄糖(6.6、19.8和33 mM)、甘露醇(33 MM)和GO(1U/ml)处理人脐静脉内皮细胞(HUVEC)。观察血管生成功能、ROS水平、血管内皮生长因子受体-2与β-TrCP的共存情况。收集细胞进行RT-qPCR和Western印迹分析。我们注意到，随着葡萄糖浓度的增加，血管生成受到损害。当人脐静脉内皮细胞处于高血糖状态时，ROS的产生增加，与GO的暴露相当；GO催化葡萄糖氧化成H_2O_2和D-葡萄糖酸-δ-内酯。高血糖可使VEGFR-2磷酸化水平降低，而总VEGFR-2水平基本不变。然而，当直接暴露于ROS时，VEGFR-2被减少，结果是β-TrCP和VEGFR-2共定位。通过免疫共沉淀实验，我们注意到泛素化的VEGFR-2被过量的ROS显著增强。β-TrCP siRNA、蛋白酶体抑制剂MG132和GSK-3β活性抑制剂(氯化锂和SB216763)可改善ROS引起的VEGFR-2表达下降。我们认为，冗余的ROS通过β-TrCP介导的VEGFR-2降解而降低VEGFR-2，推测其受β的调节。
Vascular endothelial growth factor receptor-2 (VEGFR-2), which is a key determinant of the angiogenecic response, is decreased in diabetic mice under oxidative stress. β-transduction repeat-containing protein (β-TrCP) has been reported to participate in VEGFR-2 degradation in thyroid cancer cells. Additionally, glycogen synthase kinase-3β (GSK-3β) acts as a mediator in the β-TrCP-induced degradation of several proteins. However, the role played by β-TrCP and GSK-3β in the degradation of VEGFR-2 in endothelial cells where hyperglycemia had been induced was not fully understood. In the present study, we aimed to analyze the mechanisms of VEGFR-2 degradation by studying excess reactive oxygen species (ROS) induced by hyperglycemia or glucose oxidase (GO). Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of glucose (6.6, 19.8 and 33 mM), mannitol (33 mM) and GO (1 U/ml). Angiogenic function, ROS levels, the co-location of VEGFR-2 and β-TrCP were evaluated. Cells were collected for RT-qPCR and western blot analysis. We noted that angiogenesis was impaired upon increasing the glucose concentration. When HUVECs were in a hyperglycemic state, ROS production increased, comparable to exposure to GO; GO catalyzes oxidation of glucose into H2O2 and D-glucono-δ-lactone. Phosphorylated VEGFR-2 was reduced by hyperglycemia while total VEGFR-2 was almost unaltered. However, VEGFR-2 was reduced when directly exposed to ROS, with resultant co-location of β-TrCP and VEGFR-2. Through a co-immunoprecipitation assay, we noted that ubiquitinated VEGFR-2 was significantly augmented by excess ROS. Decreased VEGFR-2 caused by ROS was ameliorated by β-TrCP siRNA, proteasome inhibitor MG132 and GSK-3β activity inhibitor (lithium chloride and SB216763). We suggest that redundant ROS reduces VEGFR-2 through β-TrCP-mediated VEGFR-2 degradation, which is postulated to be regulated by GSK-3β.