Laboratory of Pathology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2010 Dec 10;285(50):38923-32. doi: 10.1074/jbc.M110.172304. Epub 2010 Oct 5.
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.
血小板反应蛋白 1（TSP1）可以通过与 VEGF 相互作用直接抑制血管生成反应，也可以通过与几种内皮细胞 TSP1 受体相互作用间接抑制血管生成反应。我们现在描述了一种更有效的机制，即 TSP1 通过与其受体 CD47 结合来抑制 VEGF 受体-2（VEGFR2）的激活。已知 CD47 的配体结合会抑制 VEGFR2 的下游信号靶标，包括内皮型一氧化氮合酶和可溶性鸟苷酸环化酶，但尚未检查对 VEGFR2 的直接影响。基于荧光共振能量转移（FRET）和共免疫沉淀实验，CD47 与 VEGFR2 持续相关。TSP1 通过与 CD47 的相互作用，消除了与 VEGFR2 的共振能量转移，并抑制了 VEGFR2 的磷酸化及其下游靶标 Akt，而不抑制 VEGF 与 VEGFR2 的结合。该蛋白的含有 CD47 结合位点的重组结构域以及源自该结构域的 CD47 结合肽可复制 TSP1 在大血管和微血管内皮细胞中的抑制活性，但 TSP1 的 CD36 结合结构域则不能。当在人内皮细胞和小鼠 CD47 缺失细胞中抑制 CD47 的表达时，VEGFR2 磷酸化的抑制作用丧失。这些结果表明，通过 CD47 的抗血管生成信号传导高度冗余，并扩展到抑制一氧化氮信号传导之外，还包括对 VEGFR2 信号传导的全面抑制。
