Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5) isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.
Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis.
Failure of remyelination of multiple sclerosis (MS) lesions contributes to neurodegeneration that correlates with chronic disability in patients. Currently, there are no available treatments to reduce neurodegeneration, but one therapeutic approach to fill this unmet need is to promote remyelination. As many demyelinated MS lesions contain plentiful oligodendrocyte precursor cells (OPCs), but no mature myelinating oligodendrocytes, research has previously concentrated on promoting OPC maturation. However, some MS lesions contain few OPCs, and therefore, remyelination failure may also be secondary to OPC recruitment failure. Here, in a series of MS samples, we determined how many lesions contained few OPCs, and correlated this to pathological subtype and expression of the chemotactic molecules Semaphorin (Sema) 3A and 3F. 37 % of MS lesions contained low numbers of OPCs, and these were mostly chronic active lesions, in which cells expressed Sema3A (chemorepellent). To test the hypothesis that differential Sema3 expression in demyelinated lesions alters OPC recruitment and the efficiency of subsequent remyelination, we used a focal myelinotoxic mouse model of demyelination. Adding recombinant ®Sema3A (chemorepellent) to demyelinated lesions reduced OPC recruitment and remyelination, whereas the addition of rSema3F (chemoattractant), or use of transgenic mice with reduced Sema3A expression increased OPC recruitment and remyelination. We conclude that some MS lesions fail to remyelinate secondary to reduced OPC recruitment, and that chemotactic molecules are involved in the mechanism, providing a new group of drug targets to improve remyelination, with a specific target in the Sema3A receptor neuropilin-1.
Semaphorin family proteins act on cells to mediate both repulsive and attractive guidance via binding to plexin family receptors, thereby playing fundamental roles in the morphogenesis and homeostasis of various tissues. Although semaphorin-plexin signaling is implicated in various diseases and is thus a target of intensive research, our mechanistic understanding of how semaphorins activate plexins on the cell surface is limited. Here, we describe unique anti-plexin-A1 antibodies that can induce a collapsed morphology in mouse dendritic cells as efficiently as the semaphorin 3A (Sema3A) ligand. Precise epitope analysis indicates that these “semaphorin-mimicking” antibodies dimerize cell-surface plexin-A1 by binding to the N-terminal sema domain of the plexin at sites away from the interface used by the Sema3A ligand. Structural analysis of plexin-A1 fragments using negative stain electron microscopy further revealed that this agonistic capacity is closely linked to the location and orientation of antibody binding. In addition, the full-length plexin-A1 ectodomain exhibited a highly curved “C” shape, reinforcing the very unusual dimeric receptor conformation of this protein at the cell surface when engaged with Sema3A or agonistic antibodies.
The semaphorin proteins are among the best-studied families of guidance cues, contributing to morphogenesis and homeostasis in a wide range of tissue types. The major semaphorin receptors are plexins and neuropilins, however other receptors and co-receptors are capable to mediate signaling by semaphorins. These guidance proteins were originally identified as growth cone “collapsing factors” or as inhibitory signals, crucial for nervous system development. Since those seminal discoveries, the list of functions of semaphorins has rapidly grown. Over the past few years, a growing body of data indicates that semaphorins are involved in the regulation of the immune and vascular systems, in tumor growth/cancer cell metastasis and in neural circuit formation. Recently there has been increasing emphasis on research to determine the potential influence of semaphorins on the development and homeostasis of hormone systems and how circulating reproductive hormones regulate their expression and functions. Here, we focus on the emerging role of semaphorins in the development, differentiation and plasticity of unique neurons that secrete gonadotropin-releasing hormone (GnRH), which are essential for the acquisition and maintenance of reproductive competence in all vertebrates. Genetic evidence is also provided showing that insufficient semaphorin signaling contributes to some forms of reproductive disorders in humans, characterized by the reduction or failure of sexual competence. Finally, we will review some studies with the goal of highlighting how the expression of semaphorins and their receptors might be regulated by gonadal hormones in physiological and pathological conditions.
Our previous study showed that wedelolactone, isolated from Ecliptae herba, enhanced osteoblastogenesis but inhibited osteoclastogenesis through Sema3A signaling pathway. This study aims to investigate the role of other semaphorins in wedelolactone-enhanced osteoblastogenesis and -inhibited osteoclastogenesis. Wedelolactone inhibited RANKL-induced Sema4D and Sema7A production, but had no effect on RANKL-reduced Sema6D expression in osteoclastic RAW264.7 cells. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone reversed osteogenic medium(OS)-reduced Sema7A expression and OS-enhanced Sema3E mRNA expression, but no effect on OS-reduced Sema3B mRNA expression. Addition of Sema4D antibody promoted wedelolactone-reduced TRAP activity and bone resorption pit formation. Wedelolactone combined with Sema4D antibody inhibited the formation of Sema4D-Plexin B1 complex. In co-culture of BMSC with RAW264.7 cells, Sema7A antibody, similar with Sema 3A antibody, reversed wedelolactone-enhanced ALP activity and mineralization level, but promoted wedelolactone-inhibited TRAP activity. However, Sema3E and Sema3B antibodies had no effect. Further, wedelolactone enhanced the binding of Sema7A with PlexinC1 and Beta1, but addition of Sema7A antibody partially blocked this binding. Our data demonstrated that wedelolactone inhibited Sema4D production and Sema4D-PlexinB1 complex formation in RAW264.7 cells, thereafter inhibiting osteoclastogenesis. At the same time, wedelolactone enhanced osteoblastogenesis through promoting Sema7A production and Sema7A-PlexinC1-Beta1 complex formation in BMSC.
Class-3 semaphorin guidance factors bind to receptor complexes containing neuropilin and plexin receptors. A semaphorin may bind to several receptor complexes containing somewhat different constituents, resulting in diverse effects on cell migration. U87MG glioblastoma cells express both neuropilins and the four class-A plexins. They respond by cytoskeletal collapse and cell contraction to sema3A or sema3B but fail to contract in response to Sema3C, Sema3D, Sema3G or sema3E even when class-A plexins are over-expressed in the cells. In-contrast, expression of recombinant plexin-D1 enabled contraction in response to these semaphorins. Surprisingly, unlike sema3D and sema3G, sema3C also induced the contraction and repulsion of plexin-D1 expressing U87MG cells in which both neuropilins were knocked-out using CRISPR/cas9. In the absence of neuropilins the EC-50 of sema3C was 5.5 fold higher, indicating that the neuropilins function as enhancers of plexin-D1 mediated sema3C signaling but are not absolutely required for sema3C signal transduction. Interestingly, in the absence of neuropilins, plexin-A4 formed complexes with plexin-D1, and was required in addition to plexin-D1 to enable sema3C induced signal transduction.
Neurodevelopmental programs are frequently dysregulated in cancer. Semaphorins are a large family of guidance cues that direct neuronal network formation and are also implicated in cancer. Semaphorins have two kinds of receptors, neuropilins and plexins. Besides their role in development, semaphorin signaling may promote or suppress tumors depending on their context. Sema3C is a secreted semaphorin that plays an important role in the maintenance of cancer stem-like cells, promotes migration and invasion, and may facilitate angiogenesis. Therapeutic strategies that inhibit Sema3C signaling may improve cancer control. This review will summarize the current research on the Sema3C pathway and its potential as a therapeutic target.
Semaphorin 4D (Sema4D) is expressed on platelets and T-cells and known to be involved in inflammation. The aims of this study include comparing Sema4D and N terminal pro brain natriuretic peptide (NT-proBNP) serum levels in heart failure (HF) patients to a control group, evaluating the correlation between Sema4D and NT-proBNP levels, and assessing Sema4D serum levels in HF patients during acute exacerbation and remission.
Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis
- FASEB journal : official publication of the Federation of American Societies for Experimental Biology
- Published almost 2 years ago
The axonal guidance proteins semaphorin (Sema)4D and Sema3A play important roles in communication between osteoclasts and osteoblasts. As stimulation of adenosine A2A receptors (A2AR) regulates both osteoclast and osteoblast function, we asked whether A2AR regulates both osteoclast and osteoblast expression of Semas. In vivo bone formation and Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1 protein expression were studied in a murine model of wear particle-induced osteolysis. Osteoclast/osteoblast differentiation were studied in vitro as the number of tartrate-resistant acid phosphatase+/Alizarin Red+ cells after challenge with CGS21680 (A2AR agonist, 1 µM) or ZM241385 (A2AR antagonist, 1 µM), with or without Sema4D or Sema3A (10 ng/ml). Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1, and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) expression was studied by RT-PCR and Western blot. β-Catenin activation and cytoskeleton changes were studied by fluorescence microscopy and Western blot. In mice with wear particles implanted over the calvaria, CGS21680 treatment increased bone formation in vivo, reduced Sema4D, and increased Sema3A expression compared with mice with wear particle-induced osteolysis treated with vehicle alone. During osteoclast differentiation, CGS21680 abrogated RANKL-induced Sema4D mRNA expression (1.3 ± 0.3- vs. 2.5 ± 0.1-fold change, P < 0.001, n = 4). PlexinA1, but not Neuropilin-1, mRNA was enhanced by CGS21680 treatment. CGS21680 enhanced Sema3A mRNA expression during osteoblast differentiation (8.7 ± 0.2-fold increase, P < 0.001, n = 4); PlexinB1 mRNA was increased 2-fold during osteoblast differentiation and was not altered by CGS21680. Similar changes were observed at the protein level. CGS21680 decreased RANKL, increased OPG, and increased total/nuclear β-catenin expression in osteoblasts. Sema4D increased Ras homolog gene family, member A phosphorylation and focal adhesion kinase activation in osteoclast precursors, and CGS21680 abrogated these effects. In summary, A2AR activation diminishes secretion of Sema4D by osteoclasts, inhibits Sema4D-mediated osteoclast activation, and enhances secretion of Sema3A by osteoblasts, increasing osteoblast differentiation and diminishing inflammatory osteolysis.-Mediero, A., Wilder, T., Shah, L., Cronstein, B. N. Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.