The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling.

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active ß1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active ß1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1.

Integrin signaling and lung cancer.

The poor prognosis of most non small cell lung carcinomas is due to their ability to efficiently invade surrounding tissues and blood vessels, finally metastasizing to distant organs. Integrin mediated adhesive interaction with the surrounding extracellular matrix is a key limiting step in the regulation of the invasive properties of several cancer cell types. Here, we examine the rising evidences about the role that integrins can play in the physiopathology of non small cell lung carcinomas by regulating cell adhesion as well as the activation of growth factors and the traffic of their cognate receptors. Modulation of the signaling pathways controlled by integrins in lung cancer cells might offer the opportunity to design and develop new drugs that might be successfully combined with conventional chemotherapy and radiotherapy.

Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells.

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 alpha5beta1-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 alpha5beta1 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 alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 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 alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.

Angiogenesis: a balancing act between integrin activation and inhibition?

Acquisition of new genes encoding for extracellular matrix (ECM) proteins and their cognate integrin adhesive receptors, as well as secreted pro- and anti-angiogenic factors, proved to be essential for the development of functional vascular networks in the vertebrate embryo. There is now clear evidence that post-natal, pathological tissue neo-vascularization is crucial for cancer growth and therapy as well. Integrins are major ECM receptors that can exist in different functional states with respect to their affinity for ECM proteins. Regulation of integrin activation is crucial for their biological functions. In the embryo, the development of a properly patterned network of blood vessels relies upon the fine modulation of integrin activation by chemoattractant and chemorepulsive cues, such as angiogenic growth factors and semaphorins. Such a fine-tuning of endothelial integrin function is likely to be disrupted in cancer. Here, the vasculature is structurally and functionally abnormal and therefore inadequate for an efficient drug and oxygen delivery, which is a mandatory pre-requisite for successful chemotherapy and radiotherapy. It is thus important to identify the molecular mechanisms that regulate integrin function in normal ECs and which are altered in tumor ECs.

Semaphoring vascular morphogenesis.

In vertebrate embryos, development of an architecturally optimized blood vessel network allows the efficient transport of oxygen and nutrients to all other tissues. The final shape of the vascular system results from vasculogenesis and angiogenesis, during which motile endothelial cells (ECs) modify their integrin-mediated interactions with the extracellular matrix (ECM) in response to pro- and anti-angiogenic factors. There is mounting evidence that different members of the semaphorin (SEMA) family of neural guidance cues participate in developmental and postnatal vessel formation and patterning as well. It turns out that paracrine secretion of class 3 SEMA (SEMA3) by nonendothelial tissues cooperates with vascular endothelial growth factor in regulating EC precursor migration and assembly during vasculogenesis and funnels navigating blood vessel through tissue boundaries during sprouting angiogenesis. Autocrine loops of endothelial SEMA3 instead appears to regulate vascular remodeling, which occurs through blood vessel intussusception and fusion. SEMA3 activity both on the vascular and nervous systems relies upon their ability to hamper the affinity of integrin receptors towards ECM ligands. Indeed, signaling from SEMA-activated plexin receptors negatively regulates cell-ECM adhesive interactions by inhibiting two key integrin activators, such as the small GTPase R-Ras and the focal adhesion protein talin.

Temporal and spatial modulation of Rho GTPases during in vitro formation of capillary vascular network. Adherens junctions and myosin light chain as targets of Rac1 and RhoA.

Endothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of beta-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs beta-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis.

Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function.

The motility and morphogenesis of endothelial cells is controlled by spatio-temporally regulated activation of integrin adhesion receptors, and integrin activation is stimulated by major determinants of vascular remodelling. In order for endothelial cells to be responsive to changes in activator gradients, the adhesiveness of these cells to the extracellular matrix must be dynamic, and negative regulators of integrins could be required. Here we show that during vascular development and experimental angiogenesis, endothelial cells generate autocrine chemorepulsive signals of class 3 semaphorins (SEMA3 proteins) that localize at nascent adhesive sites in spreading endothelial cells. Disrupting endogenous SEMA3 function in endothelial cells stimulates integrin-mediated adhesion and migration to extracellular matrices, whereas exogenous SEMA3 proteins antagonize integrin activation. Misexpression of dominant negative SEMA3 receptors in chick embryo endothelial cells locks integrins in an active conformation, and severely impairs vascular remodelling. Sema3a null mice show vascular defects as well. Thus during angiogenesis endothelial SEMA3 proteins endow the vascular system with the plasticity required for its reshaping by controlling integrin function.