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.

A partnership with the proteasome; the destructive nature of GSK3

“…A phosphorylated form of VEGFR-2 was reduced in the mice by hyperglycemia and VEGFR-2 protein was reduced in cells directly exposed to oxidative stress. This coincided with co-location of β-TrCP and VEGFR-2, and increases in ubiquitination of VEGFR-2 [112] . This reduction of VEGFR-2 protein in response to ROS was ameliorated by β-TrCP siRNA or treatment with the proteasome inhibitor MG132 or GSK3 activity inhibitors (LiCl and SB216763).…”

“… Putatively assigned to Ser183/184 [114] Vegfr-2 (vascular endothelial growth factor receptor) Not experimentally identified. [112] , [113] …”

Roles of miR-4463 in H2O2-induced oxidative stress in human umbilical vein endothelial cells

“…H 2 O 2 is one of the main ROS, which has been reported to decrease cellular proliferation and induce apoptosis, in a process involving several miRNAs ( 28 ). Exposure of endothelial cells to H 2 O 2 has been reported to cause cell dysfunction and promote apoptosis, thus inducing endothelial inflammation.…”

“…Cellular proliferation was evaluated using a Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology), as previously described ( 28 , 29 ). Following treatment with H 2 O 2 for 16 h, HUVECs (1×10 4 cells/well) were seeded in 96-well plates (5 wells/experimental group) in 100 µl serum-free DMEM (Hyclone; GE Healthcare Life Sciences) and 10 µl CCK-8 solution were added to each well and cells were incubated at 37°C for 35 min.…”

pH Sensitive Lipid-Polymer Hybrid Nanoparticles Mediated Delivery of Docetaxel: A Viable Approach for Breast Cancer Therapeutic Intervention Development

“…The absorbance (Abs) of obtained solution was recorded at 570 nm by 96 well plate reader (BioTek instruments, USA). The cell viability was calculated by using the following formula [35]. CalcuSyn software was used to calculate IC 50 values via median-effect plot.…”