Plexin-mediated neuronal development and neuroinflammatory responses in the nervous system.

Plexins are a large family of single-pass transmembrane proteins that mediate semaphorin signaling in multiple systems. Plexins were originally characterized for their role modulating cytoskeletal activity to regulate axon guidance during nervous system development. Thereafter, different semaphorin-plexin complexes were identified in the nervous system that have diverse functions in neurons, astrocytes, glia, oligodendrocytes, and brain derived-tumor cells, providing unexpected but meaningful insights into the biological activities of this protein family. Here, we review the overall structure and relevant downstream signaling cascades of plexins. We consider the current knowledge regarding the function of semaphorin-plexin cascades in the nervous system, including the most recent data regarding their roles in neuronal development, neuroinflammation, and glioma.

The involvement of semaphorin 7A in tumorigenic and immunoinflammatory regulation.

Semaphorins, a large group of highly conserved proteins, consist of eight subfamilies that are widely expressed in vertebrates, invertebrates, and viruses and exist in membrane-bound or secreted forms. First described as axon guidance cues during neurogenesis, semaphorins also perform physiological functions in other organ systems, such as bone homeostasis, immune response, and tumor progression. Semaphorin 7A (SEMA7A), also known as CDw108, is an immune semaphorin that modulates diverse immunoinflammatory processes, including immune cell interactions, inflammatory infiltration, and cytokine production. In addition, SEMA7A regulates the proliferation, migration, invasion, lymph formation, and angiogenesis of multiple types of tumor cells, and these effects are mediated by the interaction of SEMA7A with two specific receptors, PLXNC1 and integrins. Thus, SEMA7A is intimately related to the pathogenesis of multiple autoimmune and inflammation-related diseases and tumors. This review focuses on the role of SEMA7A in the pathogenesis of autoimmune disorders, inflammatory diseases, and tumors, as well as the underlying mechanisms. Furthermore, strategies targeting SEMA7A as a potential predictive, diagnostic, and therapeutic agent for these diseases are also addressed.

The Role of Semaphorins in Metabolic Disorders.

Semaphorins are a family originally identified as axonal guidance molecules. They are also involved in tumor growth, angiogenesis, immune regulation, as well as other biological and pathological processes. Recent studies have shown that semaphorins play a role in metabolic diseases including obesity, adipose inflammation, and diabetic complications, including diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic wound healing, and diabetic osteoporosis. Evidence provides mechanistic insights regarding the role of semaphorins in metabolic diseases by regulating adipogenesis, hypothalamic melanocortin circuit, immune responses, and angiogenesis. In this review, we summarize recent progress regarding the role of semaphorins in obesity, adipose inflammation, and diabetic complications.

Real-time QCM-D monitoring of endothelial cells and macrophages adhering and spreading to SEMA4D/heparin surfaces.

In vitro evaluation techniques that address cellular interactions under dynamic circumstances are required for the development of advanced biomaterials. In this study, quartz crystal microbalance and dissipation (QCM-D) provided a surface sensitive technique that enabled real-time monitoring of the adhesion and spreading response of endothelial cells (ECs) and macrophages (MAs) to soluble-semaphoring 4D (SEMA4D)/heparin modified substratum. The high heparin and low SEMA4D of substratum promoted ECs adhering and spreading. However, surfaces with high SEMA4D and low heparin enhanced MA adhering and spreading. Furthermore, DF plots of QCM-D indicated that the adhering and spreading of MAs were mainly mediated via the ligand-receptor interaction of SEMA4D-CD72, while both adhering and spreading of ECs were mainly mediated via heparin-induced integrin binding. This study suggests that QCM-D combined with other appropriate methods can be utilized to explore the mechanisms for cellular interaction with biomaterial surfaces.

Recruitment of Tiam1 to Semaphorin 4D Activates Rac and Enhances Proliferation, Invasion, and Metastasis in Oral Squamous Cell Carcinoma.

The semaphorins and the plexins are a family of large, cysteine-rich proteins originally identified as regulators of axon growth and lymphocyte activation that are now known to provide motility and positional information for a number of cell and tissue types. For example, our group and others have shown that some malignancies over express Semaphorin 4D (S4D), which acts through its receptor Plexin-B1 (PB1) on endothelial cells to attract blood vessels from the surrounding stroma for the purpose of supporting tumor growth. While plexins are the known functional receptors for the semaphorins, there is evidence that transmembrane semaphorins may transmit a signal themselves through their short cytoplasmic tail, a phenomenon known as 'reverse signaling.' We used computational methods based upon correlated evolution of sequences of interacting proteins, mutational analysis and in vitro and in vivo measurements of tumor aggressiveness to show that when bound to PB1, transmembrane S4D associates with the Rac GTPase exchange factor T lymphoma invasion and metastasis (Tiam) 1, which activates Rac and promotes proliferation, invasion and metastasis in oral squamous cell carcinoma (OSCC) cells. These results suggest that not only can S4D production by tumor cells affect the microenvironment, but engagement of this semaphorin at the cell surface activates a reverse signaling mechanism that influences tumor aggressiveness in OSCC.

The regulation of Sema4D exodomain shedding by protein kinase A in platelets.

We have previously shown that Sema4D expressed on the platelet plasma membrane can be cleaved by the metalloprotease ADAM17, producing a 120-kDa exodomain fragment that retains biological activity and remnant fragments of 24-28 kDa that remain associated with the platelet membrane. This process is modulated by calmodulin. Here we investigated the potential role of protein kinase A (PKA) in these events. Using a pharmacological approach, we now show that inhibition of PKA by H89 is sufficient to induce Sema4D exodomain shedding, while activation of PKA inhibits agonist-initiated shedding. Studies on the regulatory mechanism show that the shedding induced by PKA inhibition is mediated by ADAM17, but, unlike agonist-induced shedding, does not involve the dissociation of calmodulin from the Sema4D cytoplasmic domain. In attempt to identify the cleavage sites for shedding, we found that ADAM17 mediates variable cleavages in the juxtamembrane region. Therefore, our data reveal a potential regulatory mechanism for the shedding of Sema4D in platelets.

Understanding cell signalling systems: paving the way for new therapies.

The cell-to-cell signalling mechanisms of multi-cellular organisms orchestrate human development during embryogenesis and control homeostasis in adult tissues. These are mechanisms vital to human health and perturbation of cell-to-cell signalling is a contributing factor in many pathologies including cancer. The semaphorin cell guidance cues and their cognate plexin receptors exemplify a cell-to-cell signalling system for which insights into mechanistic principles are emerging. X-ray crystallographic data from Diamond beam lines have enabled us to probe the inner workings of semaphorin-plexin signalling to atomic-level resolutions. Importantly, we can complement protein crystallographic results with biophysical and cellular studies to dovetail structural information with functional impact. The signature seven-bladed ß propeller 'sema' domain of the semaphorins forms a dimer; in contrast the equivalent domain in the plexins is monomeric. The generic architecture of a semaphorin-plexin complex is characterized by the dimeric semaphorin cross-linking two copies of the plexin receptor. For specific family members, the co-receptor neuropilin serves to bolster this architecture, but in all cases, the dimeric interaction lies at the core of the ligand receptor complex, providing the essential trigger for signalling.

Viral semaphorin inhibits dendritic cell phagocytosis and migration but is not essential for gammaherpesvirus-induced lymphoproliferation in malignant catarrhal fever.

UNLABELLED: Viral semaphorins are semaphorin 7A (sema7A) mimics found in pox- and herpesviruses. Among herpesviruses, semaphorins are encoded by gammaherpesviruses of the Macavirus genus only. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus that persistently infects wildebeest asymptomatically but induces malignant catarrhal fever (MCF) when transmitted to several species of susceptible ruminants and the rabbit model. MCF is caused by the activation/proliferation of latently infected T lymphocytes. Viral semaphorins have been suggested to mediate immune evasion mechanisms and/or directly alter host T cell function. We studied AlHV-sema, the viral semaphorin encoded by the A3 gene of AlHV-1. Phylogenetic analyses revealed independent acquisition of pox- and herpesvirus semaphorins, suggesting that these proteins might have distinct functions. AlHV-sema showed a predicted three-dimensional structure very similar to sema7A and conserved key residues in sema7A-plexinC1 interaction. Expression analyses revealed that AlHV-sema is a secreted 93-kDa glycoprotein expressed during the early phase of virus replication. Purified AlHV-sema was able to bind to fibroblasts and dendritic cells and induce F-actin condensation and cell retraction through a plexinC1 and Rho/cofilin-dependent mechanism. Cytoskeleton rearrangement was further associated with inhibition of phagocytosis by dendritic cells and migration to the draining lymph node. Next, we generated recombinant viruses and demonstrated that the lack of A3 did not significantly affect virus growth in vitro and did not impair MCF induction and associated lymphoproliferative lesions. In conclusion, AlHV-sema has immune evasion functions through mechanisms similar to poxvirus semaphorin but is not directly involved in host T cell activation during MCF. IMPORTANCE: Whereas most poxviruses encode viral semaphorins, semaphorin-like genes have only been identified in few gammaherpesviruses belonging to the Macavirus genus. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus carried asymptomatically by wildebeest but induces a latency-associated lymphoproliferative disease of T lymphocytes in various ruminant species, namely, malignant catarrhal fever (MCF). Viral semaphorins have been hypothesized to have immune evasion functions and/or be involved in activating latently infected T cells. We present evidence that the viral semaphorin AlHV-sema inhibits dendritic cell phagocytosis and migration to the draining lymph node, both being indispensable mechanisms for protective antiviral responses. Next, we engineered recombinant viruses unable to express AlHV-sema and demonstrated that this protein is dispensable for the induction of MCF. In conclusion, this study suggests that herpesvirus and poxvirus semaphorins have independently evolved similar functions to thwart the immune system of the host while AlHV-sema is not directly involved in MCF-associated T-cell activation.

Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X.

Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4A(V78M) demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.

Crucial role of Plexin C1 for pulmonary inflammation and survival during lung injury.

Acute pulmonary inflammation during lung injury is initiated by the migration of neutrophils into the alveolar space. The severity of these inflammatory changes within the pulmonary tissue determines the severity of lung injury and ultimately patient outcome. Recent work has demonstrated that the guidance protein Semaphorin 7A propagates the infiltration of neutrophils into an hypoxic tissue site, yet the role of its target receptor Plexin C1 (PLXNC1) during lung injury is to date unknown. We demonstrate here that PLXNC1(+) neutrophils are present within the alveolar space and that PLXNC1 is induced in vitro and in vivo during lung injury. In a model of high-pressure ventilation PLXNC1(-/-) animals show decreased signs of alveolar inflammation and improved survival compared with wild-type controls. Studies employing chimeric animals identified the hematopoietic expression of PLXNC1 to be of crucial importance for the observed results. Functional inhibition of PLXNC1 resulted in improved survival and ameliorated the signs of inflammation within the lung. Furthermore, the injection of a peptide binding to PLXNC1 resulted in improved survival and attenuated pulmonary inflammation. As such we demonstrate here, that previously unknown PLXNC1 holds significant importance for degree of pulmonary inflammation and determines outcome during experimental lung injury.

Semaphorin 3E suppresses tumor cell death triggered by the plexin D1 dependence receptor in metastatic breast cancers.

The semaphorin guidance molecules and their receptors, the plexins, are often inappropriately expressed in cancers. However, the signaling processes mediated by plexins in tumor cells are still poorly understood. Here, we demonstrate that the Semaphorin 3E (Sema3E) regulates tumor cell survival by suppressing an apoptotic pathway triggered by the Plexin D1 dependence receptor. In mouse models of breast cancer, a ligand trap that sequesters Sema3E inhibited tumor growth and reduced metastasis through a selective tumor cytocidal effect. We further showed that Plexin D1 triggers apoptosis via interaction with the orphan nuclear receptor NR4A1. These results define a critical role of Sema3E/Plexin D1 interaction in tumor resistance to apoptosis and suggest a therapeutic approach based on activation of a dependence receptor pathway.

Analysis of SEMA6B gene expression in breast cancer: identification of a new isoform.

BACKGROUND: SEMA6B is a member of the semaphorins axon-guidance family. A growing body of evidence has been accumulated describing the role of semaphorin molecules in cancer development and the involvement of SEMA6B in cancer progression has recently been proposed. METHODS: Our analysis, based on real-time PCR, focused on the expression of SEMA6B in a panel of breast cancer tissues, compared to the normal counterpart. RESULTS: In cancer tissues we found a significantly strong down-modulation of this transcript. Moreover we identified and characterized a novel SEMA6B isoform, named SEMA6Ba. This isoform has a novel splice junction, created by the usage of alternative donor and acceptor splice sites internal to the exon 17. By in silico analysis we found that the new transcript 3' UTR lacks some highly-conserved miRNA binding sites, suggesting possible consequences on both spatial and temporal expression of SEMA6Ba. The translated sequence of SEMA6Ba lacks the cytoplasmic tail, crucial for triggering the reverse signaling described for the transmembrane semaphorins. We also demonstrated, by immunofluorescence analysis of endogenous and overexpressed SEMA6Ba, that the protein clearly localized to the endoplasmic reticulum and plasma membrane. In conclusion, SEMA6B gene products are strongly down modulated in breast cancer tissues and a new isoform named SEMA6Ba has been described and characterized. GENERAL SIGNIFICANCE: Our work states a clear relation among breast cancer and SEMA6B expression; moreover we describe for the first time the SEMA6Ba protein and report here the analysis of SEMA6Ba RNA messenger, the protein expression and the cellular localization.

A point mutation in Semaphorin 4A associates with defective endosomal sorting and causes retinal degeneration.

Semaphorin 4A (Sema4A) has an essential role in photoreceptor survival. In humans, mutations in Sema4A are thought to contribute to retinal degenerative diseases. Here we generate a series of knock-in mouse lines with corresponding mutations (D345H, F350C or R713Q) in the Sema4A gene and find that Sema4A(F350C) causes retinal degeneration phenotypes. The F350C mutation results in abnormal localization of the Sema4A protein, leading to impaired endosomal sorting of molecules indispensable for photoreceptor survival. Additionally, protein structural modelling reveals that the side chain of the 350th amino acid is critical to retain the proper protein conformation. Furthermore, Sema4A gene transfer successfully prevents photoreceptor degeneration in Sema4A(F350C/F350C) and Sema4A(-/-) mice. Thus, our findings not only indicate the importance of the Sema4A protein conformation in human and mouse retina homeostasis but also identify a novel therapeutic target for retinal degenerative diseases.

Class 3 semaphorins as a therapeutic target.

INTRODUCTION: The semaphorins were initially described as axon guidance molecules that play important roles in the development of nervous system. Recent studies suggest that semaphorins and their receptors also exert such diverse functions as immune response, control of vascular endothelial cell motility and invasion of many types of cancer cells. AREAS COVERED: The available results concerning application of class 3 semaphorins and their inhibitors for the treatment in animal disease models. EXPERT OPINION: Because semaphorins are now recognized as key players in immune, cardiovascular, bone metabolism and neurological system, semaphorins and their receptors are most promising therapeutic targets for various disease states. As semaphorins exert their diverse or even opposing activities in vivo, more elaborate studies on pathophysiology and signal transduction mechanisms of semaphorins are required.

Plexin structures are coming: opportunities for multilevel investigations of semaphorin guidance receptors, their cell signaling mechanisms, and functions.

Plexin transmembrane receptors and their semaphorin ligands, as well as their co-receptors (Neuropilin, Integrin, VEGFR2, ErbB2, and Met kinase) are emerging as key regulatory proteins in a wide variety of developmental, regenerative, but also pathological processes. The diverse arenas of plexin function are surveyed, including roles in the nervous, cardiovascular, bone and skeletal, and immune systems. Such different settings require considerable specificity among the plexin and semaphorin family members which in turn are accompanied by a variety of cell signaling networks. Underlying the latter are the mechanistic details of the interactions and catalytic events at the molecular level. Very recently, dramatic progress has been made in solving the structures of plexins and of their complexes with associated proteins. This molecular level information is now suggesting detailed mechanisms for the function of both the extracellular as well as the intracellular plexin regions. Specifically, several groups have solved structures for extracellular domains for plexin-A2, -B1, and -C1, many in complex with semaphorin ligands. On the intracellular side, the role of small Rho GTPases has been of particular interest. These directly associate with plexin and stimulate a GTPase activating (GAP) function in the plexin catalytic domain to downregulate Ras GTPases. Structures for the Rho GTPase binding domains have been presented for several plexins, some with Rnd1 bound. The entire intracellular domain structure of plexin-A1, -A3, and -B1 have also been solved alone and in complex with Rac1. However, key aspects of the interplay between GTPases and plexins remain far from clear. The structural information is helping the plexin field to focus on key questions at the protein structural, cellular, as well as organism level that collaboratoria of investigations are likely to answer.

Structural basis of semaphorin-plexin signalling.

Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed ß-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity.

Semaphorins and their receptors in stem and cancer cells.

There is a growing body of evidence that links cancer with genes and pathways that are required for normal embryonic development, increasing the possibility that cancer cells with stem cell properties, particularly self-renewal and multipotentiality, are primarily involved in tumor formation and progression. One novel pathway that is important in regulating the morphogenesis, proliferation, survival and growth in a variety of adult and embryonic tissues is the semaphoring signaling pathway. Semaphorins are a large family of secreted, transmembrane and GPI-linked proteins with a broad spectrum of functions. Semaphorin signaling is transduced by plexins which, in the case of most class 3 semaphorins, require high affinity neuropilin receptors. The neuropilins also function as receptors for VEGF and other growth factors, and their expression is abnormal in tumors. Various semaphorins can either promote or inhibit tumor progression through the promotion or inhibition of processes such as tumor angiogenesis, metastasis and tumor cell survival. In normal tissues, semaphoring signaling is mainly active in precursor cells. This increases the possibility of tumors being derived from such cells, possibly even stem cells, which are unable to differentiate and/or to stop proliferating. In this review, we summarize the molecular mechanisms of semaphorin signal transduction involved in the stem cell compartment, and describe the evidence that links semaphorins to the control of tumor progression.

Ethnic differences and functional analysis of MET mutations in lung cancer.

PURPOSE: African Americans have higher incidence and poorer response to lung cancer treatment compared with Caucasians. However, the underlying molecular mechanisms for the significant ethnic difference are not known. The present study examines the ethnic differences in the type and frequency of MET proto-oncogene (MET) mutation in lung cancer and correlated them with other frequently mutated genes such as epidermal growth factor receptor (EGFR), KRAS2, and TP53. EXPERIMENTAL DESIGN: Using tumor tissue genomic DNA from 141 Asian, 76 Caucasian, and 66 African American lung cancer patients, exons coding for MET and EGFR were PCR amplified, and mutations were detected by sequencing. Mutation carriers were further screened for KRAS2 and TP53 mutations. Functional implications of important MET mutations were explored by molecular modeling and hepatocyte growth factor binding studies. RESULTS: Unlike the frequently encountered somatic mutations in EGFR, MET mutations in lung tumors were germline. MET-N375S, the most frequent mutation of MET, occurred in 13% of East Asians compared with none in African Americans. The frequency of MET mutations was highest among male smokers and squamous cell carcinoma. The MET-N375S mutation seems to confer resistance to MET inhibition based on hepatocyte growth factor ligand binding, molecular modeling, and apoptotic susceptibility to MET inhibitor studies. CONCLUSIONS: MET in lung cancer tissues contained nonsynonymous mutations in the semaphorin and juxtamembrane domains but not in the tyrosine kinase domain. All the MET mutations were germline. East Asians, African-Americans, and Caucasians had different MET genotypes and haplotypes. MET mutations in the semaphorin domain affected ligand binding.

Semaphorins in vascular development and head and neck squamous cell carcinoma-induced angiogenesis.

The semaphorins belong to a family of proteins originally shown to be important in axon migration and proper central nervous system development. Recent evidence has demonstrated that semaphorins and their receptors are expressed in tissues outside of the nervous system and represent a widespread signaling system that is involved in regulation of motility in many different cell types. This review specifically addresses the effects of the semaphorins on physiological and pathological angiogenesis and will discuss the most recent findings on the role of semaphorins in head and neck squamous cell carcinoma-induced angiogenesis.

Targeting endothelial and tumor cells with semaphorins.

Neuropilins (NRP) are receptors for the class 3 semaphorin (SEMA3) family of axon guidance molecules and the vascular endothelial growth factor (VEGF) family of angiogenesis factors. Although the seminal studies on SEMA3s and NRPs first showed them to be mediators of axon guidance, it has become very apparent that these proteins play an important role in vascular and tumor biology as well. Neuronal guidance and angiogenesis are regulated similarly at the molecular level. For example, SEMA3s not only repel neurons and collapse axon growth cones, but have similar effects on endothelial cells and tumor cells. Preclinical studies indicate that SEMA3F is a potent inhibitor of tumor angiogenesis and metastasis. In addition, neutralizing antibodies to NRP1 enhance the effects of anti-VEGF antibodies in suppressing tumor growth in xenograft models. This article reviews NRP and SEMA3 structural interactions and their role in developmental angiogenesis, tumor angiogenesis and metastasis based on cell culture, zebrafish and murine studies.