title: GIT1 mediates VEGF-induced podosome formation in endothelial cells: critical role for PLCgamma.
abstract: OBJECTIVE
  We and others showed that tyrosine kinase receptors (TKRs) such as the epidermal growth factor receptor stimulate G protein-coupled receptor (GPCR) kinase-interacting protein 1 (GIT1) phosphorylation via c-Src, which is required for phospholipase C-gamma (PLCgamma) activation, indicating that GIT1 participates in TKR signaling. VEGF is the most important TKR in endothelial cells (ECs); essential for cell survival, migration, and angiogenesis. Podosomes, actin-rich structures, were found to contribute to EC migration, tissue invasion, and matrix remodeling, suggesting a role for podosomes in angiogenesis. Because GIT1 is a substrate of c-Src, and podosome formation is c-Src dependent, we hypothesized that GIT1 plays an important role in VEGF-induced EC podosome formation and cell migration.
  
  
  RESULTS
  Exposure of ECs to VEGF for 30 minutes stimulated GIT1 colocalization with podosomes. Depletion of GIT1 by siRNA significantly decreased VEGF-induced podosome formation. A key role for PLCgamma was suggested by several experiments. Double staining PLCgamma and actin showed colocalization of PLCgamma with podosomes. Podosome formation was dramatically reduced by PLCgamma inhibitor U73122, Src inhibitor PP2, or expression of dominant negative small GTPases. Therefore, VEGF-induced EC podosome formation is dependent on Src, GIT1, PLCgamma, and small GTPases. In addition, matrix metalloprotease 2 (MMP2) and MT-MMP1 were detected at sites of VEGF-induced podosomes. Depletion of GIT1 by siRNA also significantly inhibited VEGF-induced MMP2 activation and extracellular matrix (ECM) degradation. Therefore, GIT1 mediates VEGF-induced matrix metalloproteinase (MMP) activation and ECM degradation by regulating podosome formation. Finally, depletion of GIT1 by siRNA significantly decreased VEGF-induced cell migration.
  
  
  CONCLUSIONS
  These data indicate that GIT1 is an essential mediator for VEGF-induced EC podosome formation and cell migration via PLCgamma.
authors.affiliation: Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Yoji
authors.initials: Y
authors.lastname: Taba
authors.forename: Jinjiang
authors.initials: J
authors.lastname: Pang
authors.forename: Guoyong
authors.initials: G
authors.lastname: Yin
authors.forename: Chen
authors.initials: C
authors.lastname: Yan
authors.forename: Bradford C
authors.initials: BC
authors.lastname: Berk
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D022081:Cell Surface Extensions / Q000187:drug effects / Q000201:enzymology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051966:Phospholipase C gamma / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; C485647:GIT1 protein, human; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D051966:Phospholipase C gamma; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14; D020559:Monomeric GTP-Binding Proteins