Role of tyrosine phosphatase SHP-1 in the mechanism of endorepellin angiostatic activity

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 ␣2␤1, the functional endorepellin receptor. The Src homology-2 protein phosphatase-1 (SHP-1) coprecipitated with integrin ␣2 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 ␣2␤1 ؊/؊ mice and by response to endorepellin in cells genetically engineered to express the ␣2␤1 integrin, but not in cells either lacking this receptor or expressing a chimera harboring the integrin ␣2 ectodomain fused to the ␣1 intracellular domain. siRNA-mediated knockdown of integrin ␣2 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 ␣2␤1 ؊/؊ mice. Our results show that SHP-1 is an essential mediator of endorepellin activity and discover a novel functional interaction between the integrin ␣2 subunit and SHP-1. (Blood. 2009; 114:4897-4906) IntroductionThe development and maintenance of an efficient vascular system is vital for organ function and health in all higher organisms. Angiogenesis is abundant during fetal development but in the adult is a rare process mainly restricted to the female reproductive cycle and wound healing. However, deregulated angiogenesis is a prominent factor in many major diseases such as cancer, diabetes, and ischemia occurring after stroke. Controlled angiogenesis is achieved by interplay of proangiogenic and antiangiogenic factors. Proangiogenic growth factors, such as vascular endothelial growth factor (VEGF), signal by their respective receptor tyrosine kinase (RTK) to stimulate endothelial cell migration and proliferation, whereas angiogenic inhibitors counteract these actions. 1 The list of reported negative regulators of angiogenesis is long, ever growing, and truly versatile. Nevertheless, a common theme for many of them is that they are derived from limited proteolysis of extracellular matrix components and that they interact with integrin receptors. 2 The angiostatic protein endorepellin, located within the C-terminus of the heparan sulfate proteoglycan perlecan, consists of 3 laminin globular (LG1-3) domains separated by 4 epidermal growth factor-like repeats. 3 Endorepellin exerts its activity by a noncanonical cation-independent binding of the LG3 domain to the ␣2 I domain of the integrin ␣2␤1, 4-7 a key receptor regulating angiogenesis. [8][9][10] This triggers a signaling cascade that leads to endothelial cell immobilizatio...

Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents

“…We hypothesized that this could occur if DV binds to α5β1 differently from fibronectin, as has been demonstrated for DV and collagen I binding to α2β1 integrin (27,31). Supplemental Figure 9 demonstrates that soluble, but not insoluble, DV is capable of increasing brain endothelial proliferation, even in the presence of fibronectin, a scenario that mimics the post-stroke fibronectin-rich in vivo environment.…”

“…It was next hypothesized that DV could be neuroprotective and restore post-stroke motor function after ischemic stroke by increasing growth factor concentrations. We focused on VEGF for several reasons: DV activity outside of the brain has been linked to the suppression of VEGF signaling (27), perlecan synthesis and release in brain endothelial cells is induced by VEGF (28), and VEGF is known to be neuroprotective (8,9). We found that total VEGF concentrations following post-stroke DV treatment (as compared with PBS treatment)…”

Novel insights into the function and dynamics of extracellular matrix in liver fibrosis

“…Like other mentioned angiogenesis inhibitors, such as arresten, canstatin, endostatin, and tumstatin, endorepellin mediates its antiangiogenesis functions through binding to ␣2␤1-integrin, a key angiogenesis receptor (376). This interaction disintegrates the actin cytoskeleton and focal adhesions (237). The laminin-like globular domain (LG3) of endorepellin can be released by further proteolytic processing with BMP-1 (111).…”

Proteomic Analysis of Candidate Prognostic Urinary Marker for Cervical Cancer

“…Endorepellin blocks endothelial cell migration and capillary morphogenesis, both in vitro and in vivo [26]. Endorepellin interacts specifically with the α2β1 integrin [27], and stimulation with endorepellin induces the interaction and phosphorylation of Src homology-2 protein phosphatase-1 with integrin α2 in endothelial cells [28]. Endorepellin LG3 fragment (amino acids 3687-4391) has potent antiangiogenic properties [29].…”