title: 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.
abstract: 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.
authors.affiliation: Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Qinhuai, Nanjing 210006, P.R. China.
authors.forename: Wen
authors.initials: W
authors.lastname: Wu
authors.affiliation: Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Qinhuai, Nanjing 210006, P.R. China.
authors.forename: Daimin
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Qinhuai, Nanjing 210006, P.R. China.
authors.forename: Daorong
authors.initials: D
authors.lastname: Pan
authors.affiliation: Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Qinhuai, Nanjing 210006, P.R. China.
authors.forename: Guangfeng
authors.initials: G
authors.lastname: Zuo
authors.affiliation: Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Qinhuai, Nanjing 210006, P.R. China.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Ren
authors.affiliation: Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Qinhuai, Nanjing 210006, P.R. China.
authors.forename: Shaoliang
authors.initials: S
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism; D018919:Neovascularization, Physiologic; D018384:Oxidative Stress; D059748:Proteolysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D044844:beta-Transducin Repeat-Containing Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2; D000071679:Glycogen Synthase Kinase 3 beta