Identification of circulating neuropilin-1 and dose-dependent elevation following anti-neuropilin-1 antibody administration

Neuropilin-1 (NRP1) acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factor and is an attractive angiogenesis target for cancer therapy. In addition to the transmembrane form, naturally occurring soluble NRP1 proteins containing part of the extracellular domain have been identified in tissues and a cell line. We developed ELISAs to study the existence of circulating NRP1 and to quantify it in serum. As measured by ELISAs, circulating NRP1 levels in mice, rats, monkeys and humans were 427 ± 77, 20 ± 3, 288 ± 86 and 322 ± 82 ng/ml (mean ± standard deviation; n ≥ 10), respectively. Anti-NRP1^B, a human monoclonal antibody, has been selected from a synthetic phage library. A 4-fold increase in circulating NRP1 was observed in mice receiving a single dose of 10 mg/kg anti-NRP1B antibody. In rats and monkeys receiving single injections of anti-NRP1^B at different dose levels, higher doses of antibody resulted in greater and more prolonged increases in circulating NRP1. Maximum increases were 56- and 7-fold for rats and monkeys receiving 50 mg/kg anti-NRP1B, respectively. In addition to the soluble NRP1 isoforms, for the first time, a ~120 kDa circulating NRP1 protein containing the complete extracellular domain was detected in serum by Western blot and mass spectrometry analysis. This protein increased more than the putative soluble NRP1 bands in anti-NRP1^B treated mouse, rat and monkey sera compared with untreated controls, suggesting that anti-NRP1^B induced membrane NRP1 shedding.     

Long-term systemic administration of human recombinant interleukin-1beta induces a dose-dependent fall in circulating parathyroid hormone in rats.

The synergism/antagonism between interleukin (IL)-1beta and parathyroid hormone (PTH) has been the subject of in vitro and in vivo work, but a possible direct action of the cytokine on PTH release has not been reported. We have investigated the effect of a continuous infusion of human recombinant IL-1beta (rIL-1beta) on circulating PTH during a 14-day period in 7-week-old female rats. This time interval was chosen in order to exclude initial hypercalcemia and to enable data collection under steady-state conditions. Five groups of 20 animals each had miniosmotic pumps (Alzet 2002, 200 microl) implanted subcutaneously and primed to release either distilled water (controls) or 100, 500, 1,000 and 2, 000 ng/24 h of rIL-1beta. Blood was drawn on days 1 and 14 for PTH, corticosterone and Ca2+ determinations. Adequate biological activity of the infused rIL-1beta was supported by elevated rectal temperature records and significant elevations of plasma corticosterone on day 14. The 100-ng dose had no effect but 500-2, 000 ng rIL-1beta/24 h significantly reduced plasma PTH in a dose-dependent manner down to 54% of basal value (20.4 +/- 1.1 vs. 15.3 +/- 1.4 pg/ml for 500 ng, p < 0.005; 20.5 +/- 1.3 vs 12.3 +/- 1.1 for 1,000 ng, p < 0.001, and 19.5 +/- 2.0 vs. 10.6 +/- 1.1 pg/ml for 2,000 ng, p < 0.0008). Despite these findings, no differences in blood Ca2+ could be detected between treated animals and controls. The following conclusions can be inferred from the foregoing: Systemic administration of rIL-1beta to rats induced a dose-dependent fall in circulating PTH without altering calcemia, calling into question the biological relevance of the former finding. Although the recorded PTH depression may indeed not have been severe enough to cause hypocalcemia, it can be hypothesized that osteoclast activation by rIL-1beta would enhance bone mineral release into the pool compensating for depressed PTH activity.     

Analysis of dose-dependent antibody titration curves

Several types of dose-response titration curves were considered. It was demonstrated that the use of the so-called coordinates of dilution suggested earlier by us allows one to analyze the titration curves, obtained either by ELISA, or by agglutination. Theoretical curves, obtained by the developed theory are very similar to those obtained in experiments. It was shown, that the analysis of the titration curves could give important information concerning antibody-blocking factors in titration sera or other samples of studied antibodies.     

Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins.

Neuropilin-1 (np1) and neuropilin-2 (np2) are receptors for class-3 semaphorins and for several isoforms of VEGF. We have cloned and characterized two chick isoforms of np2 cDNA. Expression patterns of np1, np2, and ephrin-B2 were compared in the developing vascular system of 24-72 h old chick embryos. We show for the first time that np2 is expressed in blood vessels in vivo from the earliest stages of their formation. In contrast to ephrin-B2, both np1 and np2 are expressed in blood islands of 24 h old chick embryos. At 48-72 h, np1 expression is localized preferentially in arteries with an expression pattern that resembles that of ephrin-B2. In contrast, np2 is expressed preferentially in veins. Thus, neuropilins may play a role in determining the arterial or venous identity of blood vessels.     

Metabolic stress induces the lysosomal degradation of neuropilin-1 but not neuropilin-2

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.     

Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library

Non-immune (na?ve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.     

Anti-BDCA-4 (neuropilin-1) antibody can suppress virus-induced IFN-alpha production of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) in human blood are the main source of virus-induced interferon (IFN)-alpha. They exhibit a lineage-negative phenotype but all express BDCA-4, which is homologous to the neuronal receptor neuropilin-1. Specific staining with anti-BDCA-4 antibody is used for positive isolation of PDC from blood by magnetic cells sorting. Here, it is demonstrated that these positively selected PDC showed reduced or completely abolished IFN-alpha release compared to unstained PDC, which were negatively selected by magnetic depletion of lineage-positive blood mononuclear cells. In addition, treatment of these unstained PDC with anti-BDCA-4 mAb also resulted in at least two-fold lower or reduced virus-induced IFN-alpha production. It is shown that the antibody not only affects cell survival or block virus attachment but also reduces IFN-alpha release induced by non-viral CpG oligodeoxynucleotides. In conclusion, data suggest an immunoregulatory role for BDCA-4 on PDC as demonstrated for IFN-alpha response to virus.     

Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms

Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are both receptors for semaphorins, which regulate neuronal guidance, and vascular endothelial growth factor (VEGF), an angiogenic factor. The two human NRP1 and NRP2 genes were cloned, and the exon-intron boundaries were determined. The NRP1 and NRP2 genes span over 120 and 112 kb, respectively, and are composed of 17 exons. Five of the exons are identical in size in the two genes, suggesting that they arose by gene duplication. Both NRP genes are characterized by multiple alternatively spliced variants. Two NRP2 isoforms, NRP2a and NRP2b, were cloned. A striking feature of these two isoforms is that they have identical extracellular domains but have divergent transmembrane and cytoplasmic domains. In these domains, NRP2a is closer in sequence identity to NRP1 than to NRP2b. As determined by Northern blot analysis, both NRP2a and NRP2b are expressed in a variety of tissues, mostly in a nonoverlapping manner. Within NRP2a and NRP2b, there are several alternatively spliced species: NRP2a(17), NRP2a(22), NRP2b(0), and NRP2b(5). In addition to full-length NRPs, there are truncated NRPs as well, which contain only the extracellular a/CUB and b/coagulation factor domains. These genes encode proteins that are soluble (sNRP) and released by cells. In addition to s12NRP1, which was previously cloned, s11NRP1 and s9NRP2 have now been cloned. These sNRP molecules are characterized by having intron-derived sequences at their C-termini. Altogether, eight NRP isoforms are described in this report. It was concluded that there are multiple NRP1 and NRP2 isoforms including intact and soluble forms. Characterization of these isoforms should help to elucidate the function of NRPs in neuronal guidance and angiogenesis.     

Crystal structure of the human neuropilin-1 b1 domain

Neuropilin-1 (Npn-1) is a type I cell surface receptor involved in a broad range of developmental processes, including axon guidance, angiogenesis, and heterophilic cell adhesion. We have determined the crystal structure of the human Npn-1 b1 domain to 1.9 A. The overall structure resembles coagulation factor V and VIII (F5/8) C1 and C2 domains, exhibiting a distorted jellyroll fold. Details of the structure provide insight to b1 domain regions responsible for ligand binding and facilitate rationalization of existing biochemical binding data. A polar cleft formed by adjacent loops at one end of the molecule in conjunction with flanking electronegative surfaces may represent the binding site for the positively charged tails of semaphorins and VEGF(165). The nature of the cell adhesion binding site of the b1 domain can be visualized in context of the structure.     

Determination of cell adhesion sites of neuropilin-1

Neuropilin-1 is a type 1 membrane protein with three distinct functions. First, it can mediate cell adhesion via a heterophilic molecular interaction. Second, in neuronal cells, neuropilin-1 binds the class 3 semaphorins, which are neuronal chemorepellents, and plays a role in the directional guidance of axons. Neuropilin-1 is expected to form complexes with the plexinA subfamily members and mediate the semaphorin-elicited inhibitory signals into neurons. Third, in endothelial cells, neuropilin-1 binds a potent endothelial cell mitogen, vascular endothelial growth factor (VEGF)(165), and regulates vessel formation. Though the binding sites in neuropilin-1 for the class 3 semaphorins and VEGF(165) have been analyzed, the sites involved in cell adhesion activity of the molecule have not been identified. In this study, we produced a variety of mutant neuropilin-1s and tested their cell adhesion activity. We showed that the b1 and b2 domains within the extracellular segment of neuropilin-1 were required for the cell adhesion activity, and peptides with an 18-amino acid stretch in the b1 and b2 domains were sufficient to induce the cell adhesion activity. In addition, we demonstrated that the cell adhesion ligands for neuropilin-1 were proteins and distributed in embryonic mesenchymal cells but distinct from the class 3 semaphorins, VEGF, or plexins.     

Lithium: evidence for reduction in circulating testosterone levels in mice following chronic administration

Lithium, the widely-used antipsychotic drug, is known to exert adverse effects on a number of endocrine organs. In the present investigations, the effects of chronic lithium administration on circulating levels of testosterone and plasma and pituitary levels of luteinizing hormone (LH) were evaluated in order to examine whether or not the pituitary-gonadal axis is a probable target of lithium action. Adult male C57BL/6 mice, maintained on a fixed photoperiodism (LD 14:10), were administered lithium orally, by being fed on a specially prepared chow containing 0.4% lithium chloride for 15 or 30 days, while their matched controls were maintained on standard laboratory chow. At the termination of the respective experimental schedules, the animals were decapitated, their blood collected, and plasma was separated and stored frozen. Pituitaries were quickly removed, weighed, homogenized, centrifuged and their supernatants were stored frozen. Testosterone in plasma and LH in pituitary and plasma were quantitated by standard RIA methods. Plasma Li concentration was determined by using flame photometric methods. A significant suppression in testosterone levels was noted after both 15 (p less than .01) and 30 (p less than .05) days of lithium treatment, but both pituitary and plasma LH levels remained unchanged at both the periods. It is, therefore, suggested that lithium exerts its effect directly at the level of the Leydig cells rather than through the pituitary-gonadal axis. Since the noted lithium-induced reduction of testosterone was manifested when the plasma lithium levels were within (or around) the therapeutic range, these results may have important clinical implications.     

Evidence for elevation of cytochrome P4502E1 (alcohol-inducible form) mRNA levels in rat kidney following pyridine administration.

The effects of pyridine on renal cytochrome P4502E1 (CYP2E1) expression in rat have been examined by immunoblot and Northern blot analyses. Immunoblot analyses revealed that 2E1 protein levels were elevated from 1.4- to 4.6-fold following pyridine administration in a dose- and time-dependent manner. Northern blot analyses revealed that renal 2E1 poly(A)+ RNA levels increased from 1.4- to 3.8-fold following pyridine treatment and that these increases in 2E1 mRNA paralleled the dose- and time-dependent increases in 2E1 protein content. In contrast, hepatic 2E1 poly(A)+ RNA levels failed to increase following these same dosing regimens, suggesting that metabolic alterations, such as those associated with starvation, were not etiologic factors in renal 2E1 induction. These results show that pyridine induced CYP2E1 in kidney and that elevation of renal 2E1 protein levels accompanying pyridine administration occurred at least partly as a consequence of increased 2E1 poly(A)+ RNA levels. The results of this research reveal that regulatory mechanisms governing CYP2E1 expression may differ in hepatic and renal tissues.     

Localization of heparin- and neuropilin-1-recognition sites of viral VEGFs

VEGF-A165 plays a central role in neovascularization. The biological activities of VEGF-A165 are largely mediated through KDR. VEGF-A165 also binds to cellular coreceptors, neuropilin-1 (NP-1), and heparin, via its C-terminal domain, resulting in functional modulation. Parapoxvirus-encoded VEGFs (PV-VEGFs), which recognize KDR, possess basic amino acid clusters in their C-terminal regions. Some PV-VEGFs may interact with NP-1; however, the NP-1- and heparin-binding properties have not been fully characterized. Here, we demonstrate that the heparin- and NP-1-binding region of PV-VEGFs is located in its C-terminal tail. Furthermore, the two arginine residues adjacent to the C-terminus greatly contribute to both interactions.     

Transmembrane domain interactions control biological functions of neuropilin-1

Neuropilin-1 (NRP1) is a transmembrane receptor playing a pivotal role in the control of semaphorins and VEGF signaling pathways. The exact mechanism controlling semaphorin receptor complex formation is unknown. A structural analysis and modeling of NRP1 revealed a putative dimerization GxxxG motif potentially important for NRP1 dimerization and oligomerization. Our data show that this motif mediates the dimerization of the transmembrane domain of NRP1 as demonstrated by a dimerization assay (ToxLuc assay) performed in natural membrane and FRET analysis. A synthetic peptide derived from the transmembrane segment of NRP1 abolished the inhibitory effect of Sema3A. This effect depends on the capacity of the peptide to interfere with NRP1 dimerization and the formation of oligomeric complexes. Mutation of the GxxxG dimerization motif in the transmembrane domain of NRP1 confirmed its biological importance for Sema3A signaling. Overall, our results shed light on an essential step required for semaphorin signaling and provide novel evidence for the crucial role of transmembrane domain of bitopic protein containing GxxxG motif in the formation of receptor complexes that are a prerequisite for cell signaling.     

Neuropilin-1-VEGFR-2 complexing requires the PDZ-binding domain of neuropilin-1

Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF(165) binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF(165) binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1.