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. Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K(d)=48nM and the related sFRP-2 with a K(d)=95nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited alpha-3/ beta1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.

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National Cancer Institute Division of Clinical Sciences
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Roberts, David D; Kaur, Sukhbir; Isenberg, Jeffrey S (2017) Regulation of cellular redox signaling by matricellular proteins in vascular biology, immunology, and cancer. Antioxid Redox Signal :
Stein, Erica V; Miller, Thomas W; Ivins-O'Keefe, Kelly et al. (2016) Secreted Thrombospondin-1 Regulates Macrophage Interleukin-1? Production and Activation through CD47. Sci Rep 6:19684
Kaur, Sukhbir; Roberts, David D (2016) Divergent modulation of normal and neoplastic stem cells by thrombospondin-1 and CD47 signaling. Int J Biochem Cell Biol 81:184-194
Soto-Pantoja, D R; Sipes, J M; Martin-Manso, G et al. (2016) Dietary fat overcomes the protective activity of thrombospondin-1 signaling in the Apc(Min/+) model of colon cancer. Oncogenesis 5:e230
Cook, Katherine L; Soto-Pantoja, David R; Clarke, Pamela A G et al. (2016) Endoplasmic Reticulum Stress Protein GRP78 Modulates Lipid Metabolism to Control Drug Sensitivity and Antitumor Immunity in Breast Cancer. Cancer Res 76:5657-5670
Soto-Pantoja, David R; Kaur, Sukhbir; Roberts, David D (2015) CD47 signaling pathways controlling cellular differentiation and responses to stress. Crit Rev Biochem Mol Biol 50:212-30
Kaur, Sukhbir; Schwartz, Anthony L; Miller, Thomas W et al. (2015) CD47-dependent regulation of H?S biosynthesis and signaling in T cells. Methods Enzymol 555:145-68
Miller, Thomas W; Soto-Pantoja, David R; Schwartz, Anthony L et al. (2015) CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation. J Biol Chem 290:24858-74
Lessey-Morillon, Elizabeth C; Roberts, David D (2015) Thrombospondin-1: an extracellular message delivered by macrophages that promotes aortic aneurysms. Circ Res 117:113-5
Roberts, David D; Kaur, Sukhbir; Soto-Pantoja, David R (2015) Therapeutic targeting of the thrombospondin-1 receptor CD47 to treat liver cancer. J Cell Commun Signal 9:101-2

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