Thrombospondin-1 Inhibits VEGF Receptor-2 Signaling by Disrupting Its Association with CD47

Jeff S. Isenberg,David D. Roberts,Sukhbir Kaur,Gema Martin-Manso,Michael L. Pendrak,Susan H. Garfield

doi:10.1074/jbc.m110.172304

Abstract

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.

Highlights

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors
Because endogenous TSP1 limits phosphorylation of Akt and Src in retina [23], we further examined the effects of CD47 on upstream signaling through VEGF receptor-2 (VEGFR2)
TSP1 has been recognized as an endogenous angiogenesis inhibitor for 20 years, the receptors and molecular mechanisms that mediate this activity remain controversial [11, 23, 43]

Summary

Introduction

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. Consistent with these studies, VEGF-stimulated VEGFR2 phosphorylation at Tyr1175 in serum-starved BAEC was inhibited in a dose-dependent manner when the cells were pretreated for 20 min with 0.2–2 nM TSP1 (supplemental Fig. S1B). Based on three independent experiments, treatment with 1 ␮g/ml (2.2 nM) TSP1 inhibited VEGF-stimulated VEGFR2 phosphorylation by 3-fold relative to VEGF-stimulated cells (p ϭ 0.0001) (Fig. 1, A and B).

Results

Conclusion