Luca Tamagnone

Plexins Are a Large Family of Receptors for Transmembrane, Secreted, and GPI-Anchored Semaphorins in Vertebrates

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Here we show that the human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. We conclude that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.

Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor.

The extracellular protease urokinase is known to be crucially involved in morphogenesis, tissue repair and tumor invasion by mediating matrix degradation and cell migration. Hepatocyte growth factor/scatter factor (HGF/SF) is a secretory product of stromal fibroblasts, sharing structural motifs with enzymes of the blood clotting cascade, including a zymogen cleavage site. HGF/SF promotes motility, invasion and growth of epithelial and endothelial cells. Here we show that HGF/SF is secreted as a single‐chain biologically inactive precursor (pro‐HGF/SF), mostly found in a matrix‐associated form. Maturation of the precursor into the active alpha beta heterodimer takes place in the extracellular environment and results from a serum‐dependent proteolytic cleavage. In vitro, pro‐HGF/SF was cleaved at a single site by nanomolar concentrations of pure urokinase, generating the active mature HGF/SF heterodimer. This cleavage was prevented by specific urokinase inhibitors, such as plasminogen activator inhibitor type‐1 and protease nexin‐1, and by antibodies directed against the urokinase catalytic domain. Addition of these inhibitors to HGF/SF responsive cells prevented activation of the HGF/SF precursor. These data show that urokinase acts as a pro‐HGF/SF convertase, and suggest that some of the growth and invasive cellular responses mediated by this enzyme may involve activation of HGF/SF.

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.

Induction of epithelial tubules by growth factor HGF depends on the STAT pathway

Hepatocyte growth factor (HGF) induces a three-phase response leading to the formation of branched tubular structures in epithelial cells,. The HGF receptor tyrosine kinase works through a Src homology (SH2) docking site that can activate several signalling pathways. The first phase of the response (scattering), which results from cytoskeletal reorganization, loss of intercellular junctions and cell migration, is dependent on phosphatidylinositol-3-OH kinase and Rac activation,. The second phase (growth) requires stimulation of the Ras–MAP kinase cascade. Here we show that the third phase (tubulogenesis) is dependent on the STAT pathway. HGF stimulates recruitment of Stat-3 to the receptor, tyrosine phosphorylation, nuclear translocation and binding to the specific promoter element SIE. Electroporation of a tyrosine-phosphorylated peptide, which interferes with both the association of STAT to the receptor and STAT dimerization, inhibits tubule formation in vitro without affecting either HGF-induced ‘scattering’ or growth. The same result is obtained using a specific ‘decoy’ oligonucleotide that prevents STAT from binding to DNA and affecting the expression of genes involved in cell-cycle regulation (c-fos and waf-1). Activation of signal transducers that directly control transcription is therefore required for morphogenesis.

The semaphorin 4D receptor controls invasive growth by coupling with Met

Semaphorins are cell surface and soluble signals that control axonal guidance. Recently, semaphorin receptors (plexins) have been discovered and shown to be widely expressed. Their biological activities outside the nervous system and the signal transduction mechanism(s) they utilize are largely unknown. Here, we show that in epithelial cells, Semaphorin 4D (Sema 4D) triggers invasive growth, a complex programme that includes cell–cell dissociation, anchorage-independent growth and branching morphogenesis. Interestingly, the same response is also controlled by scatter factors through their tyrosine kinase receptors, which share striking structural homology with plexins in their extracellular domain . We found that in cells expressing the endogenous proteins, Plexin B1 (the Sema 4D Receptor) and Met (the Scatter Factor 1/ Hepatocyte Growth Factor Receptor) associate in a complex. In addition, binding of Sema 4D to Plexin B1 stimulates the tyrosine kinase activity of Met, resulting in tyrosine phosphorylation of both receptors. Finally, cells lacking Met expression do not respond to Sema 4D unless exogenous Met is expressed. This work identifies a novel biological function of semaphorins and suggests the involvement of an unexpected signalling mechanism, namely, the coupling of a plexin to a tyrosine kinase receptor.

PLEXIN A IS A NEURONAL SEMAPHORIN RECEPTOR THAT CONTROLS AXON GUIDANCE

The Semaphorins comprise a large family of secreted and transmembrane proteins, some of which function as repellents during axon guidance. Semaphorins fall into seven subclasses. Neuropilins are neuronal receptors for class III Semaphorins. In the immune system, VESPR, a member of the Plexin family, is a receptor for a viral-encoded Semaphorin. Here, we identify two Drosophila Plexins, both of which are expressed in the developing nervous system. We present evidence that Plexin A is a neuronal receptor for class I Semaphorins (Sema 1a and Sema 1b) and show that Plexin A controls motor and CNS axon guidance. Plexins, which themselves contain complete Semaphorin domains, may be both the ancestors of classical Semaphorins and binding partners for Semaphorins.

Signalling by semaphorin receptors: cell guidance and beyond

Semaphorins are a large family of secreted or cell-bound signals, known to guide axons in developing nervous tissue. They are expressed in a variety of adult and embryonic tissues and are thought to have a broader spectrum of functions. Recent evidence suggests that semaphorins and their receptors play a key role in the control of cellular interactions, most likely in cell-cell repulsion. A subset of semaphorins interacts with neuropilins - cell-surface molecules lacking a signalling-competent cytoplasmic domain. Another large family of transmembrane molecules, namely plexins, bind specifically to semaphorins. Thus plexins, alone, or in association with neuropilins, behave as fully functional semaphorin receptors. The intracellular responses elicited by plexins are unknown, but their large cytoplasmic moiety, containing the strikingly conserved sex-plexin (SP) domain, is likely to trigger novel signal-transduction pathways.

Functional interaction of VEGF-C and VEGF-D with neuropilin receptors

Lymphatic vascular development is regulated by vascular endothelial growth factor receptor‐3 (VEGFR‐3), which is activated by its ligands VEGF‐C and VEGF‐D. Neuropilin‐2 (NP2), known to be involved in neuronal development, has also been implicated to play a role in lymphangiogenesis. We aimed to elucidate the mechanism by which NP2 is involved in lymphatic endothelial cell signaling. By in vitro binding studies we found that both VEGF‐C and VEGF‐D interact with NP2, VEGF‐C in a heparin‐independent and VEGF‐D in a heparin‐dependent manner. We also mapped the domains of VEGF‐C and NP2 required for their binding. The functional importance of the interaction of NP2 with the lymphangiogenic growth factors was demonstrated by cointernalization of NP2 along with VEGFR‐3 in endocytic vesicles of lymphatic endothelial cells upon stimulation with VEGF‐C or VEGF‐D. NP2 also interacted with VEGFR‐3 in coprecipitation studies. Our results show that NP2 is directly involved in an active signaling complex with the key regulators of lymphangiogenesis and thus suggest a mechanism by which NP2 functions in the development of the lymphatic vasculature.—Kärpänen, T., Heckman, C. A., Keskitalo, S., Jeltsch, M., Ollila, H., Neufeld, G., Tamagnone, L., Alitalo, K. Functional interaction of VEGF‐C and VEGF‐D with neuropilin receptors. FASEB J. 20, 1462–1472 (2006)

Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages

Increased evidence suggests that cancer-associated inflammation supports tumor growth and progression. We have previously shown that semaphorin 4D (Sema4D), a ligand produced by different cell types, is a proangiogenic molecule that acts by binding to its receptor, plexin B1, expressed on endothelial cells (Conrotto, P., D. Valdembri, S. Corso, G. Serini, L. Tamagnone, P.M. Comoglio, F. Bussolino, and S. Giordano. 2005. Blood. 105:4321–4329). The present work highlights the role of Sema4D produced by the tumor microenvironment on neoplastic angiogenesis. We show that in an environment lacking Sema4D, the ability of cancer cells to generate tumor masses and metastases is severely impaired. This condition can be explained by a defective vascularization inside the tumor. We demonstrate that tumor-associated macrophages (TAMs) are the main cells producing Sema4D within the tumor stroma and that their ability to produce Sema4D is critical for tumor angiogenesis and vessel maturation. This study helps to explain the protumoral role of inflammatory cells of the tumor stroma and leads to the identification of an angiogenic molecule that might be a novel therapeutic target.

To move or not to move

Semaphorins were discovered 11 years ago as molecular cues for axon guidance that are conserved from invertebrates to humans. More than 20 semaphorin genes have been identified in mammals and their protein products are now known to be involved in a range of processes from the guidance of cell migration to the regulation of the immune response, angiogenesis and cancer. Plexins, either alone or in association with neuropilins, constitute high‐affinity semaphorin receptors. However, other transmembrane molecules have been implicated in semaphorin receptor complexes, and interactions between plexins and a range of intracellular effectors have been reported. These data indicate that semaphorins might be able to elicit responses through more than one signalling pathway. Interestingly, according to recent findings, the semaphorin‐dependent control of cell migration crucially involves integrin‐based adhesive structures through which polarized cell‐membrane protrusion is coupled to cytoskeletal dynamics. This review focuses on the mechanisms whereby semaphorins are thought to regulate cell migration.