title: Thioredoxin-interacting protein mediates sustained VEGFR2 signaling in endothelial cells required for angiogenesis.
abstract: OBJECTIVE
  Thioredoxin-interacting protein (TXNIP) is an α-arrestin protein whose function is important for the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling and endothelial cell survival. Because VEGFR2 is critical for angiogenesis, we explored the role of TXNIP in VEGF-induced angiogenesis.
  
  
  RESULTS
  TXNIP knockdown inhibited VEGF-induced endothelial cell tube formation and proliferation in cultured human umbilical vein endothelial cell. To elucidate the mechanism by which TXNIP altered VEGFR2 signaling in human umbilical vein endothelial cell, we studied phosphorylation of VEGFR2, phospholipase C gamma-1 (PLCγ1), endothelial NO synthase, and Akt (known as protein kinase B). TXNIP knockdown significantly decreased phosphorylation of VEGFR2 and PLCγ1 at times >5 minutes, but phosphorylation was unchanged at 2 minutes, as was Akt and endothelial NO synthase phosphorylation. Cell-surface biotinylation assay showed that TXNIP knockdown significantly attenuated VEGFR2 internalization. These results suggested that TXNIP was required for sustained VEGFR2 signaling, which is mediated largely by internalized VEGFR2. Rab5 knockdown to inhibit the trafficking and fusion of early endosomes significantly blocked VEGF-induced VEGFR2 internalization and phosphorylation of VEGFR2 and PLCγ1. Immunofluorescence and coimmunoprecipitation showed that TXNIP was part of a complex that included Rab5 and VEGFR2. Finally, TXNIP knockdown prevented the association of VEGFR2 and Rab5.
  
  
  CONCLUSIONS
  Our results show that TXNIP is essential for VEGFR2 internalization in Rab5 positive endosomes, which is required for endothelial cell growth and angiogenesis.
authors: Aab Cardiovascular Research Institute, University of Rochester, 601 Elmwood Ave, Box CVRI, Rochester, NY 14642, USA.
authors: Shin-Young
authors: SY
authors: Park
authors: Xi
authors: X
authors: Shi
authors: Jinjiang
authors: J
authors: Pang
authors: Chen
authors: C
authors: Yan
authors: Bradford C
authors: BC
authors: Berk
mesh_terms: D019921:Biotinylation; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D018919:Neovascularization, Physiologic; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C486715:TXNIP protein, human; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; C522157:PLCG1 protein, human; D051966:Phospholipase C gamma; D020696:rab5 GTP-Binding Proteins
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