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Sam68 and SLM2 are paralog RNA binding proteins (RBPs) expressed in the cerebral cortex and display similar splicing activities. However, their relative functions during cortical development are unknown. We found that these RBPs exhibit an opposite expression pattern during development. Sam68 expression declines postnatally while SLM2 increases after birth, and this developmental pattern is reinforced by hierarchical control of Sam68 expression by SLM2. Analysis of Sam68:Slm2 double knockout (Sam68:Slm2dko) mice revealed hundreds of exons that respond to joint depletion of these proteins. Moreover, parallel analysis of single and double knockout cortices indicated that exons regulated mainly by SLM2 are characterized by a dynamic splicing pattern during development, whereas Sam68-dependent exons are spliced at relatively constant rates. Dynamic splicing of SLM2-sensitive exons is completely suppressed in the Sam68:Slm2dko developing cortex. Sam68:Slm2dko mice die perinatally with defects in neurogenesis and in neuronal differentiation, and develop a hydrocephalus, consistent with splicing alterations in genes related to these biological processes. Thus, our study reveals that developmental control of separate Sam68 and Slm2 paralog genes encoding homologous RBPs enables the orchestration of a dynamic splicing program needed for brain development and viability, while ensuring a robust redundant mechanism that supports proper cortical development.

While semaphorins were initially identified as axonal guidance cues for wiring the neural network, it was then recognized their wide relevance in tissue development and homeostasis. Notably, semaphorin activities were also extensively studied in many types of solid tumors; however, their relevance in hematological malignancies is far from understood. In this mini-review, we surveyed the current knowledge about semaphorins and their receptors in leukemias, lymphomas, and multiple myeloma. Noteworthy, current data support a promoting role for Semaphorin 4D and Neuropilin-1 in these tumors, while Semaphorin 3A seems to consistently act as oncosuppressor in leukemias and multiple myeloma. The expression levels and functional activities of SEMA3B, SEMA3F, and Neuropilin-2 have furthermore been investigated in leukemias and lymphoma cells. Herein, we reviewed the state of the art and highlighted some of the open questions to be addressed in the field.

Neuropilins (NRPs) are cell surface glycoproteins, acting as co-receptors for secreted Semaphorins (SEMAs) and for members of the vascular endothelial growth factor (VEGF) family; they have been initially implicated in axon guidance and angiogenesis regulation, and more recently in cancer progression. In addition, NRPs have been shown to control many other fundamental signaling pathways, especially mediated by tyrosine kinase receptors (RTKs) of growth factors, such as HGF (hepatocyte growth factor), PDGF (platelet derived growth factor) and EGF (epidermal growth factor). This enables NRPs to control a range of pivotal mechanisms in the cancer context, from tumor cell proliferation and metastatic dissemination, to tumor angiogenesis and immune escape. Moreover, cancer treatment failures due to resistance to innovative oncogene-targeted drugs is typically associated with the activity of alternative RTK-dependent pathways; and neuropilins' capacity to control oncogenic signaling cascades supports the hypothesis that they could elicit such mechanisms in cancer cells, in order to escape cytotoxic stress and therapeutic attacks. Intriguingly, several studies have recently assayed the impact of NRPs inhibition in combination with diverse anti-cancer drugs. In this minireview, we will discuss the state-of-art about the relevance of NRPs as potential predictive biomarkers of drug response, and the rationale to target these proteins in combination with other anticancer therapies.

The inflammatory and immune response elicited by the growth of cancer cells is a major element conditioning the tumor microenvironment, impinging on disease progression and patients' prognosis. Semaphorin receptors are widely expressed in inflammatory cells, and their ligands are provided by tumor cells, featuring an intense signaling cross-talk at local and systemic levels. Moreover, diverse semaphorins control both cells of the innate and the antigen-specific immunity. Notably, semaphorin signals acting as inhibitors of anti-cancer immune response are often dysregulated in human tumors, and may represent potential therapeutic targets. In this mini-review, we provide a survey of the best known semaphorin regulators of inflammatory and immune cells, and discuss their functional impact in the tumor microenvironment.

Acute myeloid leukemia (AML) is often characterized by the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. The presence of mutant proteins prone to misfolding can render the cells sensitive to endoplasmic reticulum (ER) stress and oxidative stress that could otherwise be overcome. Here, we show that the triple combination of the differentiating agent retinoic acid (RA), the ER stress-inducing drug tunicamycin (Tm), and arsenic trioxide (ATO), able to generate oxidative stress, leads to the death of AML cell lines expressing fusion proteins involving the gene MLL and the internal tandem duplication (ITD) in the FLT3 tyrosine kinase receptor. Importantly, the combination of RA, Tm, and ATO decreased the colony-forming capacity of primary leukemic blasts bearing the FLT-ITD mutation without affecting healthy hematopoietic progenitor cells. We demonstrate in cell lines that combination of these drugs generates ER and oxidative stresses and impairs maturation and causes accumulation of FLT3 protein in the ER. Our data provide a proof of concept that low amounts of drugs that generate ER and oxidative stresses combined with RA could be an effective targeted therapy to hit AML cells characterized by MLL fusion proteins and FLT3-ITD mutation.

Neuropilin-1 and neuropilin-2 form a small family of transmembrane molecules found in vertebrates. In addition to their established function as cell surface co-receptors for semaphorins and vascular endothelial growth factors (VEGFs), in association with plexins and VEGF receptors, neuropilins have been found to interact with many other transmembrane receptor molecules (such as EGFR, Met, IGF1-R and PDGF-R tyrosine kinases, TGFb receptor, Hedgehog, integrins, etc.) and elicit a range of intracellular signaling cascades. Thus neuropilins appear to act as signaling hubs on the cell surface, contributing to diverse

Reproductive competence in mammals depends on the projection of gonadotropin-releasing hormone (GnRH) neurons to the hypothalamic median eminence (ME) and the timely release of GnRH into the hypothalamic-pituitary-gonadal axis. In adult rodents, GnRH neurons and the specialized glial cells named tanycytes periodically undergo cytoskeletal plasticity. However, the mechanisms that regulate this plasticity are still largely unknown. We demonstrate that Semaphorin7A, expressed by tanycytes, plays a dual role, inducing the retraction of GnRH terminals and promoting their ensheathment by tanycytic end feet via the receptors PlexinC1 and Itgb1, respectively. Moreover, Semaphorin7A expression is regulated during the oestrous cycle by the fluctuating levels of gonadal steroids. Genetic invalidation of Semaphorin7A receptors in mice induces neuronal and glial rearrangements in the ME and abolishes normal oestrous cyclicity and fertility. These results show a role for Semaphorin7A signalling in mediating periodic neuroglial remodelling in the adult ME during the ovarian cycle.

Semaphorins are extracellular signals known to guide migrating cells during developmental morphogenesis and in adult tissues. Semaphorin receptors, that is plexins and neuropilins, have been found in association with diverse receptor tyrosine kinases (RTKs), such as Met, ErbB2 and VEGFR2. These receptor complexes are formed in a cell-specific manner and can mediate distinctive signalling cascades, sometimes leading to divergent functional outcomes. This is particularly intriguing in cancer, since the same semaphorin has been found to mediate either tumor-promoting or tumor-suppressing functions, depending on the cancer type and cellular context. We will therefore review the current understanding about the role of RTKs in neuropilin and plexin signalling, putatively accounting for the multifaceted role of semaphorins in cancer.

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate. PMID: 1383237 PMCID: PMC2289675 DOI: 10.1083/jcb.119.3.629

Neuropilins comprise two homologous widely-expressed single-pass plasma membrane receptors (Nrp1 and Nrp2), originally identified for binding secreted Semaphorins and Vascular Endothelial Growth Factors (in association with Plexins and VEGF-Receptors). Semaphorins have been implicated with opposite functions in cancer: either as putative tumor suppressors and anti-angiogenic factors, or mediating tumour angiogenesis, invasion and metastasis. Moreover, due to their implication in VEGF signaling, neuropilins regulate vascular development and tumor angiogenesis. Recent evidence further suggests a role of neuropilins in cancer progression due to their interaction with receptor tyrosine kinases, adhesion molecules, and integrins. Furthermore, neuropilins have been implicated in response to additional growth factors, such as Hepatocyte Growth Factor, Fibroblast Growth Factor, Transforming Growth Factor beta, Galectin, etc. Altogether, these data seem to qualify neuropilins as signaling platforms on the cell surface, potentially capable of regulating cancer cells, as well as cells of the tumor microenvironment. Intriguingly, clinical-pathological data often indicate a correlation between increased expression of neuropilins and advanced stage tumors with poor prognosis. In this article, we will review the current experimental evidence about the functional role of neuropilins in cancer and the underlying molecular mechanisms.

Plexins are transmembrane high-affinity receptors for semaphorins, regulating cell guidance, motility, and invasion. Functional evidences implicate semaphorin signals in cancer progression and metastasis. Yet, it is largely unknown whether plexin genes are genetically altered in human tumors. We performed a comprehensive gene copy analysis and mutational profiling of all nine members of the plexin gene family (plexinome), in melanomas and pancreatic ductal adenocarcinomas (PDACs), which are characterized by high metastatic potential and poor prognosis. Gene copy analysis detected amplification of PLXNA4 in melanomas, whereas copy number losses of multiple plexin genes were seen in PDACs. Somatic mutations were detected in PLXNA4, PLXNB3, and PLXNC1; providing the first evidence that these plexins are mutated in human cancer. Functional assays in cellular models revealed that some of these missense mutations result in loss of plexin function. For instance, c.1613G>A, p.R538H mutation in the extracellular domain of PLXNB3 prevented binding of the ligand Sema5A. Moreover, although PLXNA4 signaling can inhibit tumor cell migration, the mutated c.5206C>T, p.H1736Y allele had lost this activity. Our study is the first systematic analysis of the "plexinome" in human tumors, and indicates that multiple mutated plexins may be involved in cancer progression.

Semaphorins and their receptors, plexins, have emerged as key regulators of various aspects of neuronal development. In contrast to the Plexin-A family, the cellular functions of Plexin-B family proteins in developing neurons are only poorly understood. An activation of Plexin-B1 via its ligand, semaphorin 4D (Sema4D), produces an acute collapse of axonal growth cones in hippocampal and retinal neurons over the early stages of neurite outgrowth. However, the functional role of Sema4D-Plexin-B interactions over subsequent stages of neurite development, differentiation and maturation has not been characterized. Here we addressed this question using morphogenetic assays and time-lapse imaging on developing rat hippocampal neurons as a model system. Interestingly, Sema4D treatment over several hours was observed to promote branching and complexity in hippocampal neurons via the activation of Plexin-B1. The activation of receptor tyrosine kinases and the Rho kinase following Sema4D treatment was found to control dendritic and axonal morphogenesis by differentially regulating branching and extension. Phosphoinositide-3-kinase, but not extracellular signal-regulated kinase 1/2, was observed to be important for the stimulatory effects of Sema4D on dendritic branching. Furthermore, we observed that the mammalian target of rapamycin is activated downstream of Plexin-B1 and contributes to Sema4D-induced effects on dendritic branching. In contrast, glycogen synthase kinase-3 beta, another effector of phosphoinositide-3-kinase signalling, was not involved. Thus, our results show that Sema4D-Plexin-B interactions modulate dendritic and axonal arborizations of developing neurons by co-ordinated and concerted activation of diverse signalling pathways.

Cell migration is pivotal in embryo development and in the adult. During development a wide range of progenitor cells travel over long distances before undergoing terminal differentiation. Moreover, the morphogenesis of epithelial tissues and of the cardiovascular system involves remodelling compact cell layers and sprouting of new tubular branches. In the adult, cell migration is essential for leucocytes involved in immune response. Furthermore, invasive and metastatic cancer cells have the distinctive ability to overcome normal tissue boundaries, travel in and out of blood vessels, and settle down in heterologous tissues. Cell migration normally follows strict guidance cues, either attractive, or inhibitory and repulsive. Semaphorins are a wide family of signals guiding cell migration during development and in the adult. Recent findings have established that semaphorin receptors, the plexins, govern cell migration by regulating integrin-based cell substrate adhesion and actin cytoskeleton dynamics, via specific monomeric GTPases. Plexins furthermore recruit tyrosine kinases in receptor complexes, which allows switching between multiple signaling pathways and functional outcomes. In this article, we will review the functional role of semaphorins in cell migration and the implicated molecular mechanisms controlling cell adhesion.

Semaphorins and their receptors, plexins, have emerged as important cellular cues regulating key developmental processes. B-type plexins directly regulate the actin cytoskeleton in a variety of cell types. Recently, B-type plexins have been shown to be expressed in striking patterns in the nervous system over critical developmental windows. However, in contrast to the well characterized plexin-A family, the functional role of plexin-B proteins in neural development and organogenesis in vertebrates in vivo is not known. Here, we have elucidated the functional contribution of the two neuronally expressed plexin-B proteins, Plexin-B1 or Plexin-B2, toward the development of the peripheral nervous system and the CNS by generating and analyzing constitutive knock-out mice. The development of the nervous system was found to be normal in mice lacking Plexin-B1, whereas mice lacking Plexin-B2 demonstrated defects in closure of the neural tube and a conspicuous disorganization of the embryonic brain. After analyzing mutant mice, which bypassed neural tube defects, we observed a key requirement for Plexin-B2 in proliferation and migration of granule cell precursors in the developing dentate gyrus, olfactory bulb, and cerebellum. Furthermore, we identified semaphorin 4C as a high-affinity ligand for Plexin-B2 in binding and functional assays. Semaphorin 4C stimulated activation of ErbB-2 and RhoA via Plexin-B2 and enhanced proliferation and migration of granule cell precursors. Semaphorin 4C-induced proliferation of ventricular zone neuroblasts was abrogated in mice lacking Plexin-B2. These genetic and functional analyses reveal a key requirement for Plexin-B2, but not Plexin-B1, in patterning of the vertebrate nervous system in vivo.

Because data from the literature suggest a lack of innervation of the placenta, we have investigated placenta, umbilical cord, and uterus to identify the molecules that play a role in regulating innervation in these organs. Neuropilin-1 and Plexin-A1 are cell surface proteins that form a receptor complex for Semaphorin 3A (Sema 3A), a secreted molecule mediating repelling signals for axonal growth cones. We have analyzed the expression of Neuropilin-1, Plexin-A1, and Semaphorin 3A in the above-mentioned tissues on the hypothesis that these molecules could regulate innervation in these organs during gestation. We found that nervous fibers are only present in the proximal part of the umbilical cord, close to the newborn, and in nongestational uterine tissues. In contrast, nervous fibers are not present in the distal segment of the umbilical cord, in the placenta and in the uterine tissues during gestation. We also found that Sema 3A receptors, Neuropilin-1 and Plexin-A1, are expressed by the nervous fibers of the proximal part of the umbilical cord, whereas Sema 3A is secreted in the umbilical cord, in the placenta, and in gestational uterine tissues. We report that a factor secreted in the umbilical cord induces the collapse of neurite growth cones in vitro and provide evidence that this factor is Sema 3A. In summary, our results suggest that the chemorepulsive signals mediated by Sema 3A play an important role in preventing nerve fibers growth in the umbilical cord and in gestational uterine tissues. The inhibition of nerve growth into the myometrium as well as into the placenta could be considered fundamental processes to preserve the fetus from external stressful events.

The Bmx sequence was identified and cloned during our search for novel tyrosine kinase genes expressed in human bone marrow cells. Bmx cDNA comprises a long open reading frame of 675 amino acids, containing one SH3, one SH2 and one tyrosine kinase domain, which are about 70% identical with Btk, Itk and Tec and somewhat less with Txk tyrosine kinase sequences. The amino terminal sequences of these four tyrosine kinases are about 40% identical and each contains a so-called pleckstrin homology domain. The 2.7 kb Bmx mRNA was expressed in endothelial cells and several human tissues by Northern blotting and an 80 kD Bmx polypeptide was detected in human endothelial cells. Immunoprecipitates of COS cells transfected with a Bmx expression vector and NIH3T3 cells expressing a Bmx retrovirus contained a tyrosyl phosphorylated Bmx polypeptide of similar molecular weight. The BMX gene was located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci. Interestingly, chromosome X also contains the closest relative of BMX, the BTK gene, implicated in X-linked agammaglobulinemia. The BMX gene thus encodes a novel nonreceptor tyrosine kinase, which may play a role in the growth and differentiation of hematopoietic cells.

HGF is secreted by mesenchymal cells and regulates motogenesis, mitogenesis, and morphogenesis of epithelial and endothelial cells. HGF is a heterodimer of two glycosylated chains, alpha and beta, bound together by a disulfide bond. The molecule is synthesized as single chain precursor devoid of biological activity (pro-HGF). The critical step in pro-HGF activation is a proteolytic cleavage generating the two chain form. This step occurs in the extracellular environment, and is catalyzed by urokinase. Two alternative transcripts originate two HGF variants. One bears a deletion of five amino acids in the alpha chain, and has the same properties of the full-size protein. The other one contains only the first portion of the alpha chain (two kringle HGF). Two kringle HGF binds the HGF receptor, triggers its tyrosine kinase activity and behaves as a partial agonist, inducing motogenesis but not mitogenesis in target cells. The HGF receptor is the tyrosine kinase encoded by the c-MET pro-oncogene, a tyrosine kinase receptor. This molecule is an heterodimer of an extracellular alpha chain disulfide linked to a transmembrane beta chain. The cytoplasmic portion of the beta chain contains the catalytic domain and critical sites for the regulation of its kinase activity. In the C-terminal tail, a bidentate motif containing two tyrosines associates the transducers responsible for HGF signalling.