Abstract
Endorepellin, the C-terminal domain of perlecan, is a powerful angiogenesis inhibitor. To dissect the mechanism of endorepellin-mediated endothelial silencing, we used an antibody array against multiple tyrosine kinase receptors. Endorepellin caused a widespread reduction in phosphorylation of key receptors involved in angiogenesis and a concurrent increase in phosphatase activity in endothelial cells and tumor xenografts. These effects were efficiently hampered by function-blocking antibodies against integrin alpha2beta1, the functional endorepellin receptor. The Src homology-2 protein phosphatase-1 (SHP-1) coprecipitated with integrin alpha2 and was phosphorylated in a dynamic fashion after endorepellin stimulation. Genetic evidence was provided by lack of an endorepellin-evoked phosphatase response in microvascular endothelial cells derived from integrin alpha2beta1(-/-) mice and by response to endorepellin in cells genetically engineered to express the alpha2beta1 integrin, but not in cells either lacking this receptor or expressing a chimera harboring the integrin alpha2 ectodomain fused to the alpha1 intracellular domain. siRNA-mediated knockdown of integrin alpha2 caused a dose-dependent reduction of SHP-1. Finally, the levels of SHP-1 and its enzymatic activity were substantially reduced in multiple organs from alpha2beta1(-/-) mice. Our results show that SHP-1 is an essential mediator of endorepellin activity and discover a novel functional interaction between the integrin alpha2 subunit and SHP-1.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Carcinoma, Lewis Lung / blood supply
-
Cell Adhesion / drug effects
-
Endothelium, Vascular / physiology
-
Heparan Sulfate Proteoglycans / pharmacology
-
Heparan Sulfate Proteoglycans / physiology*
-
Humans
-
Integrin alpha1 / genetics
-
Integrin alpha2beta1 / chemistry
-
Integrin alpha2beta1 / deficiency
-
Integrin alpha2beta1 / genetics
-
Integrin alpha2beta1 / metabolism
-
Mice
-
Mice, Inbred C57BL
-
Mice, Knockout
-
Neovascularization, Pathologic / drug therapy
-
Neovascularization, Physiologic / physiology*
-
Peptide Fragments / pharmacology
-
Peptide Fragments / physiology*
-
Phosphorylation / drug effects
-
Phosphotyrosine / analysis
-
Protein Interaction Mapping
-
Protein Processing, Post-Translational / drug effects
-
Protein Structure, Tertiary
-
Protein Tyrosine Phosphatase, Non-Receptor Type 6 / chemistry
-
Protein Tyrosine Phosphatase, Non-Receptor Type 6 / deficiency
-
Protein Tyrosine Phosphatase, Non-Receptor Type 6 / metabolism
-
Protein Tyrosine Phosphatase, Non-Receptor Type 6 / physiology*
-
Receptor Protein-Tyrosine Kinases / antagonists & inhibitors
-
Receptor Protein-Tyrosine Kinases / physiology
-
Recombinant Fusion Proteins / physiology
-
Xenograft Model Antitumor Assays
Substances
-
Heparan Sulfate Proteoglycans
-
Integrin alpha1
-
Integrin alpha2beta1
-
Peptide Fragments
-
Recombinant Fusion Proteins
-
perlecan
-
Phosphotyrosine
-
Receptor Protein-Tyrosine Kinases
-
PTPN6 protein, human
-
Protein Tyrosine Phosphatase, Non-Receptor Type 6
-
Ptpn6 protein, mouse