Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes α5β1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with α5β1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice. The vascular system is a hierarchical network of blood vessels lined by endothelial cells that, by means of the transmembrane integrin proteins, bind to the surrounding proteinaceous extracellular matrix (ECM). Integrins are required for proper cardiovascular development and exist in bent (inactive) and extended (active) shapes that are correspondingly unable and able to attach to the ECM. Extracellular guidance cues, such as vascular endothelial growth factor and semaphorins, bind the transmembrane protein neuropilin-1 (Nrp1) and then activate biochemical signals that, respectively, activate or inactivate endothelial integrins. Here, we show that Nrp1, via its short cytoplasmic domain and independently of vascular endothelial growth factor and semaphorins, specifically promotes endothelial cell attachment to the ECM protein fibronectin, which is known to be crucial for vascular development. Notably, Nrp1 favors cell adhesion by associating with fibronectin-binding integrins and promoting the fast vesicular traffic of their extended form back and forth from the endothelial cell-to-ECM contacts. Binding of the Nrp1 cytoplasmic domain with the adaptor protein GIPC1, which in turn associates with proteins required for integrin internalization and vesicle motility, is required as well. It is likely that such an integrin treadmill could act as a major regulator of cell adhesion in general.

Semaphorins, a large family of membrane-bound and secreted proteins, signal through their transmembrane receptors, the plexins. Semaphorins and plexins share structural homologies with scatter factor receptors, a family of tyrosine kinase receptors for which Met is the prototype. Semaphorins have been studied primarily in the developing nervous system, where they act as repelling cues in axon guidance. However, they are widely expressed in several tissues, and their role in epithelial morphogenesis has been recently established. Not much is known about their role in angiogenesis, a key step during embryonic development and adulthood. Here we demonstrate that a semaphorin, Sema4D, is angiogenic in vitro and in vivo and that this effect is mediated by its high-affinity receptor, Plexin B1. Moreover, we prove that biologic effects elicited by Plexin B1 require coupling and activation of the Met tyrosine kinase. In sum, we identify a proangiogenic semaphorin and provide insight about the signaling machinery exploited by Plexin B1 to control angiogenesis.

Purpose: MET, the high-affinity receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Tivantinib (ARQ197; Arqule), a staurosporine derivative that binds to the dephosphorylated MET kinase in vitro, is being tested clinically as a highly selective MET inhibitor. However, the mechanism of action of tivantinib is still unclear. Experimental Design: The activity of tivantinib was analyzed in multiple cellular models, including: cells displaying c-MET gene amplification, strictly ‘addicted’ to MET signaling; cells with normal c-MET gene copy number, not dependent on MET for growth; cells not expressing MET; somatic knockout cells in which the ATP-binding cleft of MET, where tivantinib binds, was deleted by homologous recombination; and a cell system ‘poisoned’ by MET kinase hyperactivation, where cells die unless cultured in the presence of a specific MET inhibitor. Results: Tivantinib displayed cytotoxic activity independently of c-MET gene copy number and regardless of the presence or absence of MET. In both wild-type and isogenic knockout cells, tivantinib perturbed microtubule dynamics, induced G₂/M arrest, and promoted apoptosis. Tivantinib did not rescue survival of cells ‘poisoned’ by MET kinase hyperactivation, but further incremented cell death. In all cell models analyzed, tivantinib did not inhibit HGF-dependent or -independent MET tyrosine autophosphorylation. Conclusions: We conclude that tivantinib displays cytotoxic activity via molecular mechanisms that are independent from its ability to bind MET. This notion has a relevant impact on the interpretation of clinical results, on the design of future clinical trials, and on the selection of patients receiving tivantinib treatment. Clin Cancer Res; 19(9); 2381–92. ©2013 AACR.

Basolateral polymerization of cellular fibronectin (FN) into a meshwork drives endothelial cell (EC) polarity and vascular remodelling. However, mechanisms coordinating α5β1 integrin-mediated extracellular FN endocytosis and exocytosis of newly synthesized FN remain elusive. Here we show that, on Rab21-elicited internalization, FN-bound/active α5β1 is recycled to the EC surface. We identify a pathway, comprising the regulators of post-Golgi carrier formation PI4KB and AP-1A, the small GTPase Rab11B, the surface tyrosine phosphatase receptor PTPRF and its adaptor PPFIA1, which we propose acts as a funnel combining FN secretion and recycling of active α5β1 integrin from the trans-Golgi network (TGN) to the EC surface, thus allowing FN fibrillogenesis. In this framework, PPFIA1 interacts with active α5β1 integrin and localizes close to EC adhesions where post-Golgi carriers are targeted. We show that PPFIA1 is required for FN polymerization-dependent vascular morphogenesis, both in vitro and in the developing zebrafish embryo.