Editors' ChoiceAngiogenesis

Inhibited from Across the Way

Science Signaling  22 Apr 2014:
Vol. 7, Issue 322, pp. ec107
DOI: 10.1126/scisignal.2005396

Neuropilin-1 (NRP1) is a transmembrane protein that functions as a promiscuous ligand-binding coreceptor. When present on endothelial cells, NRP1 binds vascular growth factor (VEGF) to form a complex with the VEGF receptor 2 (VEGFR2). However, NRP1 is also present on other cell types, including some tumor cells. Thus, NRP1 may function both in cis (on the same cell as that with VEGFR2) and in trans (on a cell adjacent to the one with VEGFR2). Koch et al. examined the differences in cis and trans signaling and responses of cultured vascular endothelial cells, tumor models, and the mouse retina. VEGFR2 and NRP1 were introduced into porcine aortic endothelial cells, which lack endogenous NRP1 and VEGFR2, such that either a single cell expressed both proteins (cis) or cells expressing one or the other protein were cultured together (trans). Coimmunoprecipitation and proximity ligation assay (PLA) experiments of VEGF-stimulated cells revealed that cis presentation resulted in a rapid and transient formation of VEGFR2-NRP1 complexes, whereas the kinetics of complex formation was slower and complexes persisted over a longer period when the proteins were in trans. When presented in cis, ligand-receptor complexes (VEGF-VEGFR2-NRP1) were rapidly internalized and colocalized with endosomal markers, whereas in trans, VEGF localized in patches at the surface of cells expressing either protein, consistent with the ligand-binding ability of both VEGFR2 and NRP1. Evaluation of downstream signaling events showed that, when stimulated with VEGF, both cis and trans presentations activated phospholipase Cγ (PLCγ) with kinetics that were similar to those of complex formation. The cis presentation produced a rapid and transient PLCγ activation, followed by a rapid and transient increase in the abundance of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2). Trans presentation produced a rapid and prolonged PLCγ activation, followed by a prolonged increase in ERK2 phosphorylation and less ERK1 phosphorylation than was observed in the cis presentation. NRP1 with a C-terminal deletion favored a low-affinity VEGF binding conformation and, when introduced in cis or trans in the porcine aortic endothelial cells, resulted in similar kinetics of NRP1-VEGFR2 complex formation and downstream ERK signaling, and VEGF was efficiently endocytosed in both cis and trans settings. Examination of NRP1 in cis and trans in tumor models was achieved by creating melanoma or fibrosarcoma xenografts in host mice with an endothelial cell–specfic tamoxifen-inducible knockdown of NRP1. In the mice lacking NRP1 or in which NRP1 was presented only in trans, the tumors that formed were smaller, and a larger proportion of mice failed to develop palpable tumors, suggesting a delay in the initiation of tumor growth. Implantation of matrigel-embedded tumor cells showed that tumors with NRP1 to produce trans signaling showed less vascularization than tumors lacking NRP1 or tumors with the NRP1 C-terminal deletion mutant. To evaluate the effect of cis and trans NRP1 on vessels in vivo, mosaic expression of NRP1 was induced by exposure to a low dose of tamoxifen. Analysis of retinal vessel growth in this model showed that sprouting occurred at sites where only one of the two adjacent cells had NRP1 and was suppressed both in areas completely lacking NRP1 and where the two adjacent cells both had NRP1. Thus, trans NRP1 suppresses angiogenesis and cis NRP1 promotes angiogenesis.

S. Koch, L. A. van Meeteren, E. Morin, C. Testini, S. Weström, H. Björkelund, S. Le Jan, J. Adler, P. Berger, L. Claesson-Welsh, NRP1 presented in trans to the endothelium arrests VEGFR2 endocytosis, preventing angiogenic signaling and tumor initiation. Dev. Cell 28, 633–646 (2014). [PubMed]