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PMID: 19023093 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

GIT1 mediates VEGF-induced podosome formation in endothelial cells: critical role for PLCgamma.

Arteriosclerosis, thrombosis, and vascular biology ·Vol. 29 ·No. 2 ·2009-02-00 ·Pages 202-8

Wang J, Taba Y, Pang J, Yin G, Yan C, Berk BC

Abstract

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. 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. These data indicate that GIT1 is an essential mediator for VEGF-induced EC podosome formation and cell migration via PLCgamma.

MeSH Terms
Adaptor Proteins, Signal Transducing/genetics,metabolism Cell Cycle Proteins/genetics,metabolism Cell Movement/drug effects Cell Surface Extensions/drug effects,enzymology Cells, Cultured Endothelial Cells/drug effects,enzymology Enzyme Inhibitors/pharmacology Extracellular Matrix/metabolism Humans Matrix Metalloproteinase 14/metabolism Matrix Metalloproteinase 2/metabolism Monomeric GTP-Binding Proteins/genetics,metabolism Phospholipase C gamma/antagonists & inhibitors,metabolism RNA Interference RNA, Small Interfering/metabolism Signal Transduction/drug effects Transfection Vascular Endothelial Growth Factor A/metabolism
Chemicals
Adaptor Proteins, Signal Transducing Cell Cycle Proteins Enzyme Inhibitors GIT1 protein, human RNA, Small Interfering Vascular Endothelial Growth Factor A Phospholipase C gamma MMP2 protein, human Matrix Metalloproteinase 2 MMP14 protein, human Matrix Metalloproteinase 14 Monomeric GTP-Binding Proteins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Wang Jing
Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Taba Yoji
Pang Jinjiang
Yin Guoyong
Yan Chen
Berk Bradford C
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Article Info
Journal
Arteriosclerosis, thrombosis, and vascular biology
Abbr.
Arterioscler Thromb Vasc Biol
ISSN
1524-4636
DOI
10.1161/ATVBAHA.108.174391
Published
2009-02-00
Epub
2008-00-20
Pages
202-8
Language
English
Region
United States
NLM ID
9505803
PMCID
PMC2735351
Subset
IM
Grants
NHLBI NIH HHS · HL 77789 · United States
NHLBI NIH HHS · R01 HL063462-08 · United States
NHLBI NIH HHS · R01 HL063462-08S1 · United States
NHLBI NIH HHS · HL 63462 · United States
NHLBI NIH HHS · R01 HL063462 · United States
NHLBI NIH HHS · P01 HL077789 · United States
External Links
PubMed source DOI: 10.1161/ATVBAHA.108.174391 PMC Full Text View journal details
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