Neuropilin-1-mediated Vascular Permeability Factor/Vascular Endothelial Growth Factor-dependent Endothelial Cell Migration

Abstract

Neuropilin-1 (NRP-1) has been found to be expressed by endothelial cells and tumor cells as an isoform-specific receptor for vascular permeability factor/vascular endothelial growth factor (VEGF). Previous studies were mainly focused on the extracellular domain of NRP-1 that can bind to VEGF 165 and, thus, enables NRP-1 to act as a co-receptor for VEGF 165 , which enhances its binding to VEGFR-2 and its bioactivity. However, the exact functional roles and related signaling mechanisms of NRP-1 in angiogenesis are not well understood. In this study we constructed a chimeric receptor, EGNP-1, by fusing the extracellular domain of epidermal growth factor receptor to the transmembrane and intracellular domains of NRP-1 and transduced it into HUVECs with a retroviral expression vector. We observed that NRP-1/EGNP-1 mediates ligand-stimulated migration of human umbilical vein endothelial cells (HUVECs) but not proliferation. Our results show that NRP-1 alone can mediate HUVEC migration through its intracellular domain, and its C-terminal three amino acids (SEA-COOH) are essential for the process. We demonstrate that phosphatidylinositol 3-kinase inhibitor Ly294002 and the p85 dominant negative mutant can block NRP-1-mediated HUVEC migration. NRP-1-mediated migration can be significantly reduced by overexpression of the dominant negative mutant of RhoA (RhoA-19N). In addition, G q family proteins and G␤␥ subunits are also required for NRP-1-mediated HUVEC migration. These results show for the first time that NRP-1 can independently promote cell signaling in endothelial cells and also demonstrate the importance of last three amino acids of NRP-1 for its function.Angiogenesis, the formation of vascular networks by endothelial cells (ECs) 1 sprouting from the vascular bed, occurs in many physiological or pathological processes (1). Vascular permeability factor/vascular endothelial growth factor (VPF/ VEGF) plays a major role in the regulation of angiogenesis; it is regarded as a key contributor to the growth of cancer and vascular disease (2). VPF/VEGF activities are mediated by high affinity receptor tyrosine kinases that are associated primarily with ECs (2). Two important VPF/VEGF binding receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), have been identified, both of which are functionally active during angiogenesis. Recent studies have found a third VPF/ VEGF receptor, neuropilin-1 (NRP-1), that is expressed by ECs and tumor cells (3, 4).NRP-1 is a 130 -135-kDa cell surface glycoprotein. It was originally characterized as a semaphorin III receptor that is important for guiding neural development (5, 6). There is also evidence that NRP-1 mediates angiogenesis. NRP-1-null mice were found to be embryonic lethal and exhibit cardiovascular defects (7). Furthermore, overexpression of NRP-1 in mice resulted in excessive capillary and blood vessel formation and hemorrhaging in embryos (8). NRP-1 also contributes to tumor angiogenesis. Induction of NRP-1 expression in tumor cells in vivo resulted in larger an...

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