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1.  Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor 
BMC Immunology  2011;12:30.
Semaphorins were originally identified as molecules regulating a functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins". It is known that Sema4A and Sema4D bind Tim-2 and CD72 expressed on leukocytes and PlexinD1 and B1 present on non-immune cells. These neuroimmune semaphorins and their receptors have been shown to play critical roles in many physiological and pathological processes including neuronal development, immune response regulation, cancer, autoimmune, cardiovascular, renal, and infectious diseases. However, the expression and regulation of Sema4A, Sema4D, and their receptors in normal and allergic lungs is undefined.
Allergen treatment and lung-specific vascular endothelial growth factor (VEGF) expression induced asthma-like pathologies in the murine lungs. These experimental models of allergic airway inflammation were used for the expression analysis of immune semaphorins and their receptors employing immunohistochemistry and flow cytometry techniques. We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes. Surprisingly, under inflammation various cell types including macrophages, lymphocytes, and granulocytes in the lung expressed Tim-2, a previously defined marker for Th2 cells. CD72 was found on lung immune, inflammatory, and epithelial cells. Bronchial epithelial cells were positive for both plexins, whereas some endothelial cells selectively expressed Plexin D1. Plexin B1 expression was also detected on lung DC. Both allergen and VEGF upregulated the expression of neuroimmune semaphorins and their receptors in the lung tissue. However, the lung tissue Sema4A-Tim2 expression was rather weak, whereas Sema4D-CD72 ligand-receptor pair was vastly upregulated by allergen. Soluble Sema4D protein was present in the lung lysates and a whole Sema4A protein plus its dimer were readily detected in the bronchoalveolar (BAL) fluids under inflammation.
This study clearly shows that neuroimmune semaphorins Sema4A and Sema4D and their receptors might serve as potential markers for the allergic airway inflammatory diseases. Our current findings pave the way for further investigations of the role of immune semaphorins in inflammation and their use as potential therapeutic targets for the inflammatory lung conditions.
PMCID: PMC3118960  PMID: 21595947
2.  Role of Semaphorin 7a signaling in TGF-β1 induced lung fibrosis and scleroderma-related interstitial lung disease 
Arthritis and rheumatism  2011;63(8):2484-2494.
Semaphorin (Sema) 7a regulates TGF- β1 induced fibrosis. Using a murine model of pulmonary fibrosis in which an inducible, bioactive form of the human TGF- β1 gene is overexpressed in the lung, we tested the hypothesis that Sema-7a exerts its pro-fibrotic effects in part by promoting the tissue accumulation of CD45+ fibrocytes.
Fibrosis and fibrocytes were evaluated in TGF- β1 transgenic mice in which the Sema-7a locus had been disrupted. The effect of replacement or deletion of Sema-7a on bone marrow derived cells was ascertained using bone marrow transplantation. The role of the Sema-7a receptor β1 integrin was assessed using neutralizing antibodies. The applicability of these findings to TGF-β1-driven fibrosis in humans was examined in patients with scleroderma-related interstitial lung disease.
The appearance of fibrocytes in the lungs in TGF- β1 transgenic mice requires Sema-7a. Replacement of Sema-7a in bone marrow derived cells restores lung fibrosis and fibrocytes. Immunoneutralization of β1 integrin reduces pulmonary fibrocytes and fibrosis. Peripheral blood mononuclear cells from patients with scleroderma-related interstitial lung disease show increased mRNA for Sema-7a and the β1 integrin, with Sema-7a located on collagen producing fibrocytes and CD19+ lymphocytes. Peripheral blood fibrocyte outgrowth is enhanced in these patients. Stimulation of normal human peripheral blood mononuclear cells with recombinant Sema-7a enhances fibrocyte differentiation; these effects are attenuated by β1 integrin neutralization.
Interventions that reduce Sema-7a expression or prevent the Sema-7a - β1 integrin interaction may be ameliorative in TGF- β1-driven or fibrocyte-associated autoimmune fibroses.
PMCID: PMC3651701  PMID: 21484765
3.  Semaphorin 3E, an exception to the rule 
The Journal of Clinical Investigation  2010;120(8):2658-2660.
Class 3 semaphorins (Sema3s) regulate axon guidance, angiogenesis, tumor growth, and tumor metastasis. Neuropilins (NRPs; NRP1 and NRP2) are the cell surface receptors for the Sema3s. However, to signal, interaction of Sema3s and NRPs with plexins is obligatory. In this issue of the JCI, Casazza and colleagues report data that challenge the conventional wisdom about the role of Sema3s in tumor metastasis. As a rule, Sema3B and Sema3F, for example, are inhibitors of tumor angiogenesis, progression, and metastasis. However, Casazza et al. found that Sema3E inhibited tumor growth but atypically promoted invasiveness and metastasis. This metastatic potential was dependent on Plexin D1 expression but was independent of NRP expression. Of clinical importance, Sema3E and Plexin D1 were found to be upregulated in human colon cancer, liver metastasis, and melanoma progression.
PMCID: PMC2912208  PMID: 20664165
4.  Semaphorin 4D cooperates with VEGF to promote angiogenesis and tumor progression 
Angiogenesis  2012;15(3):391-407.
The semaphorins and plexins comprise a family of cysteine-rich proteins implicated in control of nerve growth and development and regulation of the immune response. Our group and others have found that Semaphorin 4D (SEMA4D) and its receptor, Plexin-B1, play an important role in tumor-induced angiogenesis, with some neoplasms producing SEMA4D in a manner analogous to vascular endothelial growth factor (VEGF) in order to attract Plexin-B1-expressing endothelial cells into the tumor for the purpose of promoting growth and vascularity. While anti-VEGF strategies have been the focus of most angiogenesis inhibition research, such treatment can lead to upregulation of pro-angiogenic factors that can compensate for the loss of VEGF, eventually leading to failure of therapy. Here, we demonstrate that SEMA4D cooperates with VEGF to promote angiogenesis in malignancies and can perform the same function in a setting of VEGF blockade. We also show the potential value of inhibiting SEMA4D/Plexin-B1 signaling as a complementary mechanism to anti-VEGF treatment, particularly in VEGF inhibitor–resistant tumors, suggesting that this may represent a novel treatment for some cancers.
PMCID: PMC3733222  PMID: 22476930
Semaphorin 4D; Plexin-B1; VEGF; Head and neck squamous cell carcinoma; Tumor-induced angiogenesis
5.  Roles of Sema4D and Plexin-B1 in tumor progression 
Molecular Cancer  2010;9:251.
Sema4D, also known as CD100, is a protein belonging to class IV semaphorin. Its physiologic roles in the immune and nervous systems have been extensively explored. However, the roles of Sema4D have extended beyond these traditionally studied territories. Via interaction with its high affinity receptor Plexin-B1, Sema4D-Plexin-B1 involvement in tumor progression is strongly implied. Here, we critically review and delineate the Sema4D-Plexin-B1 interaction in many facets of tumor progression: tumor angiogenesis, regulation of tumor-associated macrophages and control of invasive growth. We correlate the in vitro and in vivo experimental data with the clinical study outcomes, and present a molecular mechanistic basis accounting for the intriguingly contradicting results from these recent studies.
PMCID: PMC2955613  PMID: 20858260
6.  Biology and function of neuroimmune semaphorins 4A and 4D 
Immunologic Research  2011;50(1):10-21.
Semaphorins belong to a family of membrane-bound and secreted molecules that regulate the functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins also found to be expressed in immune cells and were, therefore, termed “immune semaphorins”. It is known that Sema4A has three functional receptors, namely Plexin D1, Plexin B1, and Tim-2, whereas Sema4D binds to Plexin B1 and CD72. Recent studies suggest that immune semaphorins play critical roles in many physiological and pathological processes and such. In this review, we summarize the current knowledge on the biology of neuroimmune semaphorins and their corresponding receptors, their distribution in organs and tissues, function in the immune response, and critical regulatory roles in various diseases.
PMCID: PMC3366695  PMID: 21203905
Sema4A; Sema4D; Tim-2; CD72; Plexin B1; Plexin D1; Expression; Lung; Immune response; Diseases
7.  Expression patterns of semaphorin7A and plexinC1 during rat neural development suggest roles in axon guidance and neuronal migration 
Although originally identified as embryonic axon guidance cues, semaphorins are now known to regulate multiple, distinct, processes crucial for neuronal network formation including axon growth and branching, dendritic morphology, and neuronal migration. Semaphorin7A (Sema7A), the only glycosylphosphatidylinositol-anchored semaphorin, promotes axon growth in vitro and is required for the proper growth of the mouse lateral olfactory tract in vivo. Sema7A has been postulated to signal through two unrelated receptors, an RGD-dependent α1β1-integrin and a member of the plexin family, plexinC1. β1-integrins underlie Sema7A-mediated axon growth and Sema7A function in the immune system. Sema7A-plexinC1 interactions have also been implicated in immune system function, but the neuronal role of this ligand-receptor pair remains to be explored. To gain further insight into the function(s) of Sema7A and plexinC1 during neural development, we present here a detailed analysis of Sema7A and plexinC1 expression in the developing rat nervous system.
In situ hybridization revealed select expression of Sema7A and plexinC1 in multiple neuronal systems including: the olfactory system, the hypothalamo-hypophysial system, the hippocampus, the meso-diencephalic dopamine system, and the spinal cord. Within these systems, Sema7A and plexinC1 are often expressed in specific neuronal subsets. In general, Sema7A transcript levels increase significantly towards adulthood, whereas plexinC1 expression decreases as development proceeds.
PlexinC1, but not Sema7A, is strongly expressed by distinct populations of migrating neurons. In addition to neuronal expression, Sema7A and plexinC1 transcripts were detected in oligodendrocytes and ependymal cells, respectively.
Sema7A and plexinC1 expression patterns are consistent with these proteins serving both cooperative and separate functions during neural development. The prominent expression of plexinC1 in several distinct populations of migrating neurons suggests a novel role for this plexin family member in neuronal migration.
PMCID: PMC2008261  PMID: 17727705
8.  Semaphorin 3E Initiates Antiangiogenic Signaling through Plexin D1 by Regulating Arf6 and R-Ras▿ †  
Molecular and Cellular Biology  2010;30(12):3086-3098.
Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.
PMCID: PMC2876686  PMID: 20385769
9.  Semaphorin3a Enhances Endocytosis at Sites of Receptor–F-Actin Colocalization during Growth Cone Collapse 
The Journal of Cell Biology  2000;149(2):411-422.
Axonal growth cone collapse is accompanied by a reduction in filopodial F-actin. We demonstrate here that semaphorin 3A (Sema3A) induces a coordinated rearrangement of Sema3A receptors and F-actin during growth cone collapse. Differential interference contrast microscopy reveals that some sites of Sema3A-induced F-actin reorganization correlate with discrete vacuoles, structures involved in endocytosis. Endocytosis of FITC-dextran by the growth cone is enhanced during Sema3A treatment, and sites of dextran accumulation colocalize with actin-rich vacuoles and ridges of membrane. Furthermore, the Sema3A receptor proteins, neuropilin-1 and plexin, and the Sema3A signaling molecule, rac1, also reorganize to vacuoles and membrane ridges after Sema3A treatment. These data support a model whereby Sema3A stimulates endocytosis by focal and coordinated rearrangement of receptor and cytoskeletal elements. Dextran accumulation is also increased in retinal ganglion cell (RGC) growth cones, in response to ephrin A5, and in RGC and DRG growth cones, in response to myelin and phorbol-ester. Therefore, enhanced endocytosis may be a general principle of physiologic growth cone collapse. We suggest that growth cone collapse is mediated by both actin filament rearrangements and alterations in membrane dynamics.
PMCID: PMC2175148  PMID: 10769032
membrane dynamics; ephrins; dextran uptake; axon guidance; axon repulsion
10.  Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development 
Semaphorins and Plexins are cognate ligand-receptor families that regulate important steps during nervous system development. The Plexin-B2 receptor is critically involved in neural tube closure and cerebellar granule cell development, however, its specific ligands have only been suggested by in vitro studies. Here, we show by in vivo and in vitro analyses that the two Semaphorin-4 family members Sema4C and Sema4G are likely to be in vivo ligands of Plexin-B2. The Sema4C and Sema4G genes are expressed in the developing cerebellar cortex, and Sema4C and Sema4G proteins specifically bind to Plexin-B2 expressing cerebellar granule cells. To further elucidate their in vivo function, we have generated and analyzed Sema4C and Sema4G knock-out mouse mutants. Like Plexin-B2−/− mutants, Sema4C−/− mutants reveal exencephaly and subsequent neonatal lethality with partial penetrance. Sema4C−/− mutants that bypass exencephaly are viable and fertile, but display distinctive defects of the cerebellar granule cell layer, including gaps in rostral lobules, fusions of caudal lobules, and ectopic granule cells in the molecular layer. In addition to neuronal defects, we observed in Sema4C−/− mutants also ventral skin pigmentation defects that are similar to those found in Plexin-B2−/− mutants. The Sema4G gene deletion causes no overt phenotype by itself, but combined deletion of Sema4C and Sema4G revealed an enhanced cerebellar phenotype. However, Sema4C/Sema4G double mutants showed overall less severe cerebellar phenotypes than Plexin-B2−/− mutants, indicating that further ligands of Plexin-B2 exist. In explant cultures of the developing cerebellar cortex, Sema4C promoted migration of cerebellar granule cell precursors in a Plexin-B2-dependent manner, supporting the model that a reduced migration rate of granule cell precursors is the basis for the cerebellar defects of Sema4C−/− and Sema4C/Sema4G mutants.
PMCID: PMC3030677  PMID: 21122816
Cerebellum; lobule; granule cell migration; Semaphorin; Plexin
11.  The Fer tyrosine kinase regulates an axon retraction response to Semaphorin 3A in dorsal root ganglion neurons 
Fps/Fes and Fer are the only two members of a distinct subclass of cytoplasmic protein tyrosine kinases. Fps/Fes was previously implicated in Semaphorin 3A (Sema3A)-induced growth cone collapse signaling in neurons from the dorsal root ganglion (DRG) through interaction with and phosphorylation of the Sema3A receptor component PlexinA1, and members of the collapsin response mediator protein (CRMP) family of microtubule regulators. However, the potential role of the closely related Fer kinase has not been examined.
Here we provide novel biochemical and genetic evidence that Fer plays a prominent role in microtubule regulation in DRG neurons in response to Sema3A. Although Fps/Fes and Fer were both expressed in neonatal brains and isolated DRGs, Fer was expressed at higher levels; and Fer, but not Fps/Fes kinase activity was detected in vivo. Fer also showed higher in vitro kinase activity toward tubulin, as an exogenous substrate; and this activity was higher when the kinases were isolated from perinatal relative to adult brain stages. CRMP2 was a substrate for both kinases in vitro, but both CRMP2 and PlexinA1 inhibited their autophosphorylation activities. Cultured mouse DRG neurons retracted their axons upon exposure to Sema3A, and this response was significantly diminished in Fer-deficient, but only slightly attenuated in Fps/Fes-deficient DRG neurons.
Fps/Fes and Fer are both capable of phosphorylating tubulin and the microtubule regulator CRMP2 in vitro; and their in vitro kinase activities were both inhibited by CRMP2 or PlexinA1, suggesting a possible regulatory interaction. Furthermore, Fer plays a more prominent role than Fps/Fes in regulating the axon retraction response to Sema3A in DRG neurons. Therefore, Fps/Fes and Fer may play important roles in developmental or regenerative axon pathfinding through signaling from Sema3A to the microtubule cytoskeleton.
PMCID: PMC2217550  PMID: 18053124
12.  Hormonal Regulation and Distinct Functions of Semaphorin-3B and Semaphorin-3F in Ovarian Cancer 
Semaphorins comprise a family of molecules that influence neuronal growth and guidance. Class-3 semaphorins, semaphorin-3B (SEMA3B) and semaphorin-3F (SEMA3F) illustrate their effects by forming a complex with neuropilins (NP-1 or NP-2) and plexins. We examined the status and regulation of semaphorins and their receptors in human ovarian cancer cells. A significantly reduced expression of SEMA3B (83 kD), SEMA3F (90 kD), and plexin-A3 was observed in ovarian cancer (OVCA) cell lines when compared to normal human ovarian surface epithelial (HOSE) cells. The expression of NP-1, NP-2 and plexin-A1 was not altered in HOSE and OVCA cells. The decreased expression of SEMA3B, SEMA3F, and plexin-A3 was confirmed in stage 3 ovarian tumors. Treatment of OVCA cells with luteinizing hormone, follicle-stimulating hormone, and estrogen induced a significant upregulation of SEMA3B, whereas SEMA3F was upregulated only by estrogen. Co-treatment of cell lines with a hormone and its specific antagonist blocked the effect of the hormone. Ectopic expression of SEMA3B or SEMA3F reduced soft-agar colony formation, adhesion, and cell invasion of OVCA cell cultures. Forced expression of SEMA3B, but not SEMA3F, inhibited viability of OVCA cells. Overexpression of SEMA3B and SEMA3F reduced focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase (MMP)-2 and -9 expression in OVCA cells. Forced expression of SEMA3F, but not SEMA3B in OVCA cells, significantly inhibited endothelial cell tube formation. Collectively, our results suggest loss of SEMA3 expression could be a hallmark of cancer progression. Furthermore, gonadotropin- and/or estrogen-mediated maintenance of SEMA3 expression could control ovarian cancer angiogenesis and metastasis.
PMCID: PMC2820590  PMID: 20124444
Gonadotropins; estrogens; ovarian surface epithelial cells; cell invasion; angiogenesis
13.  Crystal Structure of the Sema-PSI Extracellular Domain of Human RON Receptor Tyrosine Kinase 
PLoS ONE  2012;7(7):e41912.
Human RON (Recepteur d’Origine Nantais) receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP). RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema), Plexins-Semaphorins-Integrins domain (PSI), and four Immunoglobulins-Plexins-Transcription factor (IPT) domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a seven-bladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors’ exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF), and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPβ binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of RONΔ160 splice variant by the soluble RON splice variant, RONΔ85.
PMCID: PMC3405059  PMID: 22848655
14.  Semaphorin 3A is a marker for disease activity and a potential immunoregulator in systemic lupus erythematosus 
Arthritis Research & Therapy  2012;14(3):R146.
Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen induced arthritis. This study was undertaken to evaluate the role of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients.
Thirty two SLE and 24 rheumatoid arthritis (RA) patients were assessed and compared with 40 normal individuals. Sema3A serum levels were measured and correlated with SLE disease activity. The in vitro effect of sema3A in reducing Toll-like receptor 9 (TLR-9) expression in B cells of SLE patients was evaluated.
Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (55.04 ± 16.30 ng/ml versus 65.54 ± 14.82 ng/ml, P = 0.018) and lower yet than in normal individuals (55.04 ± 16.30 ng/ml versus 74.41 ± 17.60 ng/ml, P < 0.0001). Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, when sema3A was co-cultured with cytosine-phosphodiester-guanine oligodeoxynucleotides (CpG-ODN)-stimulated B cells of SLE patients, their TLR-9 expression was significantly reduced, by almost 50% (P = 0.001).
This is the first study in which a reduced serum level of sema3A was found in association with SLE disease activity. It also raises the possibility that sema3A may have a regulatory function in SLE.
PMCID: PMC3446531  PMID: 22697500
15.  Semaphorin 5B mediates synapse elimination in hippocampal neurons 
Neural Development  2009;4:18.
Semaphorins are known to play an important role in axon guidance and growth by triggering dynamic rearrangements of the actin cytoskeleton in the neuronal growth cone. Intriguingly, some of these guidance molecules are persistently expressed after axonal pathfinding and target recognition are completed. Although their function at these later stages is poorly understood, recent findings suggest a role for these proteins in regulating synaptic connections.
Here we demonstrate that semaphorin 5B (Sema5B) regulates the elimination of synaptic connections in cultured hippocampal neurons. We show that Sema5B is proteolytically processed in neonatal brains and primary hippocampal cultures, resulting in the secretion of Sema5B fragments that include the biologically active semaphorin domain. Overexpression of full-length Sema5B in hippocampal neurons reduces synapse number while expression of a Sema5B construct lacking the semaphorin domain has no effect. Moreover, bath application with the proteolytically processed, secreted fragments containing the semaphorin domain of Sema5B, results in a rapid elimination of synaptic connections as demonstrated by time-lapse imaging. Conversely, depletion of endogenous Sema5B using RNA interference results in a significant increase in synapse number as well as a significant increase in the size of presynaptic and postsynaptic compartments.
Our results demonstrate that in addition to its role as a guidance cue, Sema5B regulates the development and maintenance of synapse size and number in hippocampal neurons. In addition, proteolytic cleavage of Sema5B results in the release of a potentially diffusible semaphorin domain that is a necessary component for its biological function in the regulation of synapse morphology.
PMCID: PMC2696441  PMID: 19463192
16.  Selective requirements for NRP1 ligands during neurovascular patterning 
Development (Cambridge, England)  2007;134(10):1833-1843.
Blood vessels and neurons share several types of guidance cues and cell surface receptors to control their behaviour during embryogenesis. The transmembrane protein NRP1 is present on blood vessels and nerves. NRP1 binds two structurally diverse ligands, the semaphorin SEMA3A and the VEGF164 isoform of vascular endothelial growth factor. SEMA3A was originally identified as a repulsive cue for developing axons that acts by signalling through receptor complexes containing NRP1 and plexins. In vitro, SEMA3A also inhibits integrin function and competes with VEGF164 for binding to NRP1 to modulate the migration of endothelial cells. These observations resulted in a widely accepted model of vascular patterning in which the balance of VEGF164 and SEMA3A determines endothelial cell behaviour. However, we now demonstrate that SEMA3A is not required for angiogenesis in the mouse, which instead is controlled by VEGF164. We find that SEMA3A, but not VEGF164, is required for axon patterning of limb nerves, even though the competition between VEGF164 and SEMA3A for NRP1 affects the migration of neuronal progenitor cells in vitro and has been hypothesised to control axon guidance. Moreover, we show that there is no genetic interaction between SEMA3A and VEGF164 during vasculogenesis, angiogenesis or limb axon patterning, suggesting that ligand competition for NRP1 binding cannot explain neurovascular congruence, as previously suggested. We conclude that NRP1 contributes to both neuronal and vascular patterning by preferentially relaying SEMA3A signals in peripheral axons and VEGF164 signals in blood vessels.
PMCID: PMC2702678  PMID: 17428830
VEGF; Neuropilin; Semaphorin; Mouse
17.  ZEB-1, a Repressor of the Semaphorin 3F Tumor Suppressor Gene in Lung Cancer Cells12 
Neoplasia (New York, N.Y.)  2009;11(2):157-166.
SEMA3F is a secreted semaphorin with potent antitumor activity, which is frequently downregulated in lung cancer. In cancer cell lines, SEMA3F overexpression decreases hypoxia-induced factor 1α protein and vascular endothelial growth factor mRNA, and inhibits multiple signaling components. Therefore, understanding how SEMA3F expression is inhibited in cancer cells is important. We previously defined the promoter organization of SEMA3F and found that chromatin remodeling by a histone deacetylase inhibitor was sufficient to activate SEMA3F expression. In lung cancer, we have also shown that ZEB-1, an E-box transcription repressor, is predominantly responsible for loss of E-Cadherin associated with a poor prognosis and resistance to epidermal growth factor receptor inhibitors. In the present study, we demonstrated that ZEB-1 also inhibits SEMA3F in lung cancer cells. Levels of ZEB-1, but not ZEB-2, Snail or Slug, significantly correlate with SEMA3F inhibition, and overexpression or inhibition of ZEB-1 correspondingly affected SEMA3F expression. Four conserved E-box sites were identified in the SEMA3F gene. Direct ZEB-1 binding was confirmed by chromatin immunoprecipitation assays for two of these, and ZEB-1 binding was reduced when cells were treated with a histone deacetylase inhibitor. These results demonstrate that ZEB-1 directly inhibits SEMA3F expression in lung cancer cells. SEMA3F loss was associated with changes in cell signaling: increased phospho-AKT in normoxia and increase of hypoxia-induced factor 1α protein in hypoxia. Moreover, exogenous addition of SEMA3F could modulate ZEB-1-induced angiogenesis in a chorioallantoic membrane assay. Together, these data provide further support for the importance of SEMA3F and ZEB-1 in lung cancer progression.
PMCID: PMC2631140  PMID: 19177200
18.  Gene Deletion Mutants Reveal a Role for Semaphorin Receptors of the Plexin-B Family in Mechanisms Underlying Corticogenesis ▿  
Molecular and Cellular Biology  2009;30(3):764-780.
Semaphorins and their receptors, plexins, are emerging as key regulators of various aspects of neural and nonneural development. Semaphorin 4D (Sema4D) and B-type plexins demonstrate distinct expression patterns over critical time windows during the development of the murine neocortex. Here, analysis of mice genetically lacking plexin-B1 or plexin-B2 revealed the significance of Sema4D-plexin-B signaling in cortical development. Deficiency of plexin-B2 resulted in abnormal cortical layering and defective migration and differentiation of several subtypes of cortical neurons, including Cajal-Retzius cells, GABAergic interneurons, and principal cells in vivo. In contrast, a lack of plexin-B1 did not impact on cortical development in vivo. In various ex vivo assays on embryonic forebrain, Sema4D enhanced the radial and tangential migration of developing neurons in a plexin-B2-dependent manner. These results suggest that Sema4D-plexin-B2 interactions regulate mechanisms underlying cell specification, differentiation, and migration during corticogenesis.
PMCID: PMC2812242  PMID: 19948886
19.  The vesicular SNARE Synaptobrevin is required for Semaphorin 3A axonal repulsion 
The Journal of Cell Biology  2012;196(1):37-46.
Semaphorin 3A-mediated signaling and axonal repulsion in the mouse brain require Synaptobrevin-dependent vesicular traffic.
Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.
PMCID: PMC3255983  PMID: 22213797
20.  Semaphorin-4D (Sema-4D), the Plexin-B1 ligand, is involved in mouse ovary follicular development 
Human Plexin-B1 is expressed in two truncated forms. The long form encodes a trans-membranal protein, while the short form, which is bound to the cell surface and partially secreted, possibly serves as a decoy receptor. Plexin receptors are trans-membrane proteins. The sema domain, found in the extracellular region, is common to all plexins, semaphorins, and the scatter factor receptors and is crucial for the biological activity and plexin receptor specificity. Semaphorin-4D/Plexin-B1 binding provides attractive and repulsive cues for the navigation of axonal growth cones, and new studies suggest that this system also plays a role in the regulation of the biological functions of endothelial cells, specifically in the control of angiogenesis. In a previous study, we have demonstrated the expression and possible role of Plexin-B1 in the mouse ovary. The present study was designed to test the hypothesis that Plexin-B1 effects are mediated by Semaphorin-4D.
In vivo expression and localization of mouse ovarian Sema-4D were tested by immunohisto-chemistry. The role of Sema-4D in follicular development was examined by in vitro growth of preantral follicles in the presence or absence of Semaphorin-4D, with or without neutralizing antibodies against Plexin-B1. Follicular growth and steroid hormone secretion rates were tested.
Semaphorin-4D is expressed in the mouse ovary in vivo mostly in the granulosa cells and and its expression is modulated by PMSG and hCG. In the presence of Semaphorin-4D, in-vitro constant growth was observed as indicated by follicular diameter during the culture period and elevated steroid hormone secretion rates compared with control. These effects were abolished after addition of neutralizing antibodies against Plexin-B1.
In the ovarian follicle, the effect of Plexin-B1 is mediated by sema-4D.
PMCID: PMC1838422  PMID: 17376242
21.  Expression of Semaphorin 3A and Neuropilin 1 in Asthma 
Journal of Korean Medical Science  2013;28(10):1435-1442.
Neuropilin 1 (NP1) is a part of essential receptor complexes mediating both semaphorin3A (SEMA3A) and vascular endothelial growth factor (VEGF) which is one of important mediators involved in the pathogenesis of asthma. Therefore, it is possible that SEMA3A plays a role in the pathogenesis of asthma through attenuation of VEGF-mediated effects. In the present study, we aimed to evaluate expression levels of SEMA3A and NP1 using induced sputum of asthmatics and a murine model of asthma. Firstly, SEMA3A and NP1 expressions in induced sputum of asthmatics and SEMA3A and NP1 expression on bronchoalveolar lavage (BAL) cells and lung homogenates of asthmatic mice were determined. Then we evaluated the immunolocalization of VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), and NP1 expressions on asthmatic mice lung tissue and their subcellular distributions using fibroblast and BEAS2B cell lines. Sputum SEMA3A and NP1 expressions were significantly higher in asthmatics than controls. Similarly, SEMA3A and NP1 expressions on BAL cells and lung homogenates were significantly elevated in asthmatic mice compared to control mice. Immunohistochemical analysis showed that VEGFR1, VEGFR2, and NP1 expressions were also uniformly increased in asthmatic mice. Our observations suggest that SEMA3A and NP1 may play important roles in the pathogenesis of asthma.
PMCID: PMC3792596  PMID: 24133346
Asthma; Neuropilin; Semaphorin-3A (SEMA3A); Vascular Endothelial Growth Factor
22.  Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II 
Nature immunology  2010;11(7):594-600.
Recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate leukocyte entry into the lymphatics remain unclear. Here we show that plexin-A1, a primary receptor component for class III and class VI semaphorins, is crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that Sema3A, but not Sema6C or Sema6D, is required for DC transmigration, and that Sema3A produced by the lymphatics promotes actomyosin contraction at the trailing edge of migrating DCs. These findings not only demonstrate that semaphorin-signals are involved in DC trafficking but also provide a novel mechanism that induces actomyosin contraction as these cells pass through narrow gaps.
PMCID: PMC3045806  PMID: 20512151
23.  Semaphorin 4D/Plexin-B1–mediated R-Ras GAP activity inhibits cell migration by regulating β1 integrin activity 
The Journal of Cell Biology  2006;173(4):601-613.
Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration (Oinuma, I., Y. Ishikawa, H. Katoh, and M. Negishi. 2004. Science. 305:862–865). We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras–mediated phosphatydylinositol 3-kinase activation, and β1 integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras–specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing β1 integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated β1 integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1–mediated R-Ras GAP activity controls cell migration by modulating the activity of β1 integrins.
PMCID: PMC2063868  PMID: 16702230
24.  Role of Sema4C in TGF-β1-induced mitogen-activated protein kinase activation and epithelial–mesenchymal transition in renal tubular epithelial cells 
Nephrology Dialysis Transplantation  2010;26(4):1149-1156.
Background. The p38 mitogen-activated protein kinase (p38 MAPK) is an important intracellular signal transduction pathway involved in TGF-β1-induced epithelial–mesenchymal transition (EMT). Sema4C, a member of the semaphorin family, was found to be essential for the activation of p38 MAPK. However, the role of Sema4C in promoting TGF-β1-induced EMT is unclear.
Methods. Renal fibrosis was induced by 5/6 subtotal nephrectomy rat model. In vitro, Sema4C was induced in human proximal tubular epithelial cells (HKC) by treatment with TGF-β1, or was inhibited by siRNA or was over-expressed by Sema4C transfection. The selective p38 MAPK inhibitor, SB203580, was administered to inhibit the p38 pathway. The expression of Sema4C, the markers of EMT, p38 phosphorylation and fibronectin secretion were measured by western blotting, immunohistochemistry, immunocytochemistry or enzyme-linked immunosorbent assay.
Results. The expression of Sema4C increased in HKC cells that were treated with TGF-β1. Knockdown of Sema4C potently inhibited phosphorylation of p38 MAPK and reversed TGF-β1-induced EMT. Over-expression of Sema4C via Sema4C transfection elicited p38 MAPK phosphorylation and promoted EMT. The effects of Sema4C during EMT were blocked by a p38-specific inhibitor. In vivo, the expression of Sema4C increased in the tubular epithelia of 5/6-nephrectomized rats and human fibrotic renal tissue, and similar localization of phosphorylated p38 and Sema4C was demonstrated by immunohistochemistry on serial sections.
Conclusions. Our findings suggest that Sema4C plays an important role in TGF-β1-induced EMT through activation of p38 MAPK in proximal tubular epithelial cells.
PMCID: PMC3070071  PMID: 20959347
epithelial to mesenchymal transition; p38 MAPK; Sema4C; TGF-β1
25.  Semaphorin3A, Neuropilin-1, and PlexinA1 Are Required for Lymphatic Valve Formation 
Circulation research  2012;111(4):10.1161/CIRCRESAHA.112.269316.
The lymphatic vasculature plays a major role in fluid homeostasis, absorption of dietary lipids, and immune surveillance. Fluid transport depends on the presence of intraluminal valves within lymphatic collectors. Defective formation of lymphatic valves leads to lymphedema, a progressive and debilitating condition for which curative treatments are currently unavailable. How lymphatic valve formation is regulated remains largely unknown.
We investigated if the repulsive axon guidance molecule Semaphorin3A (Sema3A) plays a role in lymphatic valve formation.
Methods and Results
We show that Sema3A mRNA is expressed in lymphatic vessels and that Sema3A protein binds to lymphatic valves expressing the Neuropilin-1 (Nrp1) and PlexinA1 receptors. Using mouse knockout models, we show that Sema3A is selectively required for lymphatic valve formation, via interaction with Nrp1 and PlexinA1. Sema3a−/− mice exhibit defects in lymphatic valve formation, which are not due to abnormal lymphatic patterning or sprouting, and mice carrying a mutation in the Sema3A binding site of Nrp1, or deficient for Plxna1, develop lymphatic valve defects similar to those seen in Sema3a−/− mice.
Our data demonstrate an essential direct function of Sema3A-Nrp1-PlexinA1 signaling in lymphatic valve formation.
PMCID: PMC3861899  PMID: 22723296
valve; guidance; development; lymphatic vessel; vascular biology; vascular smooth muscle

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