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Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165.

Miao HQ, Soker S, Feiner L, Alonso JL, Raper JA, Klagsbrun M - J. Cell Biol. (1999)

Bottom Line: To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined.Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner.These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

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Competition of collapsin-1 and VEGF165 for PAEC/KDR/NRP1 motility, DRG collapse, and binding to NRP1 expressed by COS-7 cells. (A) PAEC/KDR/NRP1 motility at a constant concentration of collapsin-1. Serum-starved PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Increasing concentrations of VEGF165 were added to the lower wells in the absence (white circles) or presence (black triangles) of 150 ng/ml collapsin-1. (B) PAEC/KDR/NRP1 motility at a constant concentration of VEGF165. Serum-starved PAEC/KDR/NRP1 cells were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells in the absence (white circles) or presence (black circles) of 5 ng/ml VEGF165. After 4 h, the numbers of migrated cells per field were counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells. (C) DRG collapse. The biological activity of collapsin-1 on DRG is attenuated by VEGF165. A growth cone collapse assay was performed in the presence (black circles) and absence (white circles) of 100 ng/ml recombinant VEGF165. The percentage of collapsed growth cones extending from explanted DRG are plotted against the concentration of recombinant collapsin-1 added to the culture. Greater concentrations of collapsin-1 are required to achieve the same level of collapse when VEGF165 is present. (D) Competitive binding to COS-7/NRP1. COS-7 cells stably expressing NRP1 were incubated for 1 h with serial dilutions of CM containing AP-VEGF165 in the absence (white circles) or presence of 30 nM (3 μg/ml) collapsin-1 (black triangles) or 50 nM (1.25 μg/ml) VEGF165 (black circles). AP-VEGF165 bound to COS-7/NRP1 cells was measured colorimetrically at OD414.
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Figure 3: Competition of collapsin-1 and VEGF165 for PAEC/KDR/NRP1 motility, DRG collapse, and binding to NRP1 expressed by COS-7 cells. (A) PAEC/KDR/NRP1 motility at a constant concentration of collapsin-1. Serum-starved PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Increasing concentrations of VEGF165 were added to the lower wells in the absence (white circles) or presence (black triangles) of 150 ng/ml collapsin-1. (B) PAEC/KDR/NRP1 motility at a constant concentration of VEGF165. Serum-starved PAEC/KDR/NRP1 cells were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells in the absence (white circles) or presence (black circles) of 5 ng/ml VEGF165. After 4 h, the numbers of migrated cells per field were counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells. (C) DRG collapse. The biological activity of collapsin-1 on DRG is attenuated by VEGF165. A growth cone collapse assay was performed in the presence (black circles) and absence (white circles) of 100 ng/ml recombinant VEGF165. The percentage of collapsed growth cones extending from explanted DRG are plotted against the concentration of recombinant collapsin-1 added to the culture. Greater concentrations of collapsin-1 are required to achieve the same level of collapse when VEGF165 is present. (D) Competitive binding to COS-7/NRP1. COS-7 cells stably expressing NRP1 were incubated for 1 h with serial dilutions of CM containing AP-VEGF165 in the absence (white circles) or presence of 30 nM (3 μg/ml) collapsin-1 (black triangles) or 50 nM (1.25 μg/ml) VEGF165 (black circles). AP-VEGF165 bound to COS-7/NRP1 cells was measured colorimetrically at OD414.

Mentions: Since NRP1 is a receptor for both VEGF165 and collapsin-1, we wanted to test whether they would compete with each other in affecting NRP1-mediated activities and in binding to NRP1. First, we investigated the effects of collapsin-1 on VEGF165 stimulatory activity and the effects of VEGF165 on collapsin-1 inhibitory activity (Fig. 3). VEGF165 is chemotactic for PAEC/KDR/NRP1 (Soker et al. 1998). PAEC/KDR/NRP1 were stimulated with increasing concentrations of VEGF165 in the absence or presence of 150 ng/ml collapsin-1 in a Boyden chamber motility assay (Fig. 3 A). In the absence of collapsin-1, VEGF stimulated PAEC/KDR/NRP1 chemotaxis in a dose-dependent manner with a typical bell-shaped curve. However, in the presence of a constant amount of collapsin-1 (150 ng/ml), the chemotactic activity of VEGF165 was reduced at each concentration of VEGF165 (0.1–50 ng/ml) although never totally abrogated. About five times more VEGF165 was required for half-maximal stimulation of PAEC/KDR/NRP1 chemotaxis when 150 ng/ml of collapsin-1 was present. In a reciprocal experiment, the effect of increasing concentrations of collapsin-1 (0–300 ng/ml) on the inhibition of PAEC/KDR/NRP1 motility at a constant level of VEGF165 was tested (Fig. 3 B). VEGF165 was added at 5 ng/ml, the optimal concentration for stimulating chemotaxis, as shown in Fig. 3 A. Collapsin-1 inhibited both basal motility and VEGF165-induced motility in a dose-dependent manner. However, in the presence of VEGF165, the motility levels were higher, about six- to sevenfold at each concentration of collapsin-1.


Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165.

Miao HQ, Soker S, Feiner L, Alonso JL, Raper JA, Klagsbrun M - J. Cell Biol. (1999)

Competition of collapsin-1 and VEGF165 for PAEC/KDR/NRP1 motility, DRG collapse, and binding to NRP1 expressed by COS-7 cells. (A) PAEC/KDR/NRP1 motility at a constant concentration of collapsin-1. Serum-starved PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Increasing concentrations of VEGF165 were added to the lower wells in the absence (white circles) or presence (black triangles) of 150 ng/ml collapsin-1. (B) PAEC/KDR/NRP1 motility at a constant concentration of VEGF165. Serum-starved PAEC/KDR/NRP1 cells were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells in the absence (white circles) or presence (black circles) of 5 ng/ml VEGF165. After 4 h, the numbers of migrated cells per field were counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells. (C) DRG collapse. The biological activity of collapsin-1 on DRG is attenuated by VEGF165. A growth cone collapse assay was performed in the presence (black circles) and absence (white circles) of 100 ng/ml recombinant VEGF165. The percentage of collapsed growth cones extending from explanted DRG are plotted against the concentration of recombinant collapsin-1 added to the culture. Greater concentrations of collapsin-1 are required to achieve the same level of collapse when VEGF165 is present. (D) Competitive binding to COS-7/NRP1. COS-7 cells stably expressing NRP1 were incubated for 1 h with serial dilutions of CM containing AP-VEGF165 in the absence (white circles) or presence of 30 nM (3 μg/ml) collapsin-1 (black triangles) or 50 nM (1.25 μg/ml) VEGF165 (black circles). AP-VEGF165 bound to COS-7/NRP1 cells was measured colorimetrically at OD414.
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Figure 3: Competition of collapsin-1 and VEGF165 for PAEC/KDR/NRP1 motility, DRG collapse, and binding to NRP1 expressed by COS-7 cells. (A) PAEC/KDR/NRP1 motility at a constant concentration of collapsin-1. Serum-starved PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Increasing concentrations of VEGF165 were added to the lower wells in the absence (white circles) or presence (black triangles) of 150 ng/ml collapsin-1. (B) PAEC/KDR/NRP1 motility at a constant concentration of VEGF165. Serum-starved PAEC/KDR/NRP1 cells were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells in the absence (white circles) or presence (black circles) of 5 ng/ml VEGF165. After 4 h, the numbers of migrated cells per field were counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells. (C) DRG collapse. The biological activity of collapsin-1 on DRG is attenuated by VEGF165. A growth cone collapse assay was performed in the presence (black circles) and absence (white circles) of 100 ng/ml recombinant VEGF165. The percentage of collapsed growth cones extending from explanted DRG are plotted against the concentration of recombinant collapsin-1 added to the culture. Greater concentrations of collapsin-1 are required to achieve the same level of collapse when VEGF165 is present. (D) Competitive binding to COS-7/NRP1. COS-7 cells stably expressing NRP1 were incubated for 1 h with serial dilutions of CM containing AP-VEGF165 in the absence (white circles) or presence of 30 nM (3 μg/ml) collapsin-1 (black triangles) or 50 nM (1.25 μg/ml) VEGF165 (black circles). AP-VEGF165 bound to COS-7/NRP1 cells was measured colorimetrically at OD414.
Mentions: Since NRP1 is a receptor for both VEGF165 and collapsin-1, we wanted to test whether they would compete with each other in affecting NRP1-mediated activities and in binding to NRP1. First, we investigated the effects of collapsin-1 on VEGF165 stimulatory activity and the effects of VEGF165 on collapsin-1 inhibitory activity (Fig. 3). VEGF165 is chemotactic for PAEC/KDR/NRP1 (Soker et al. 1998). PAEC/KDR/NRP1 were stimulated with increasing concentrations of VEGF165 in the absence or presence of 150 ng/ml collapsin-1 in a Boyden chamber motility assay (Fig. 3 A). In the absence of collapsin-1, VEGF stimulated PAEC/KDR/NRP1 chemotaxis in a dose-dependent manner with a typical bell-shaped curve. However, in the presence of a constant amount of collapsin-1 (150 ng/ml), the chemotactic activity of VEGF165 was reduced at each concentration of VEGF165 (0.1–50 ng/ml) although never totally abrogated. About five times more VEGF165 was required for half-maximal stimulation of PAEC/KDR/NRP1 chemotaxis when 150 ng/ml of collapsin-1 was present. In a reciprocal experiment, the effect of increasing concentrations of collapsin-1 (0–300 ng/ml) on the inhibition of PAEC/KDR/NRP1 motility at a constant level of VEGF165 was tested (Fig. 3 B). VEGF165 was added at 5 ng/ml, the optimal concentration for stimulating chemotaxis, as shown in Fig. 3 A. Collapsin-1 inhibited both basal motility and VEGF165-induced motility in a dose-dependent manner. However, in the presence of VEGF165, the motility levels were higher, about six- to sevenfold at each concentration of collapsin-1.

Bottom Line: To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined.Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner.These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

Show MeSH
Related in: MedlinePlus