Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165 - PubMed (original) (raw)

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

H Q Miao et al. J Cell Biol. 1999.

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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Figures

Figure 1

Cross-linking of 125I-collapsin-1. 125I-collapsin-1 (10 ng/ml) was bound to subconfluent cultures of PAEC (lane 1), PAEC/KDR (lane 2), PAEC/NRP1 (lane 3), PAEC/KDR/NRP1 (lane 4), and MDA-MB-231 (lane 5) cells in 6-cm dishes. The binding was carried out in the presence of 1 μg/ml of heparin. At the end of a 2-h incubation, 125I-collapsin-1 was cross-linked to the cell surface. The cells were lysed and proteins were resolved by 6% SDS-PAGE. The polyacrylamide gel was dried and exposed to X-ray film. Arrow indicates the position of bound 125I-collapsin-1. In this experiment, noncovalent binding, rather than covalent cross-linking of 125I-collapsin-1 to NRP1, occurred.

Figure 2

Collapsin-1 inhibits the motility of EC expressing NRP1. (A) PAEC, PAEC/NRP1, PAEC/KDR, and PAEC/KDR/NRP1 were seeded in the upper wells of a Boyden chamber. Collapsin-1 (150 ng/ml) was added (gray bars) or not added (white bars) to the lower wells. (B) PAEC/NRP1 and PAEC/KDR/NRP1 were seeded in the upper wells and increasing concentrations of collapsin-1 were added to the lower wells of the Boyden chamber. (C) PAEC/NRP1 and PAEC/KDR/NRP1 were cultured in complete medium and seeded in the upper wells of a Boyden chamber. For each cell type, either 0 (lanes 1 and 2) or 150 ng/ml (lanes 3 and 4) of collapsin-1 was added in the absence (hatched bars) or presence (solid bars) of 30 μg/ml anti-NRP1 antibodies. For each set of experiments, after a 4-h incubation, the numbers of cells that had migrated through the filter in each field were counted. Each data point represents the mean ± SD of four independent wells.

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.

Figure 4

Effect of collapsin-1 on microvessel outgrowth in vitro. Rat aortic rings were embedded in type I collagen gels. Serum-free endothelial growth medium was added and replaced every other day with fresh medium. Microvessel structure was observed by phase microscopy on day 14 at low (A, C, and E) or high (B, D, and F) magnification. A and B, no addition; C and D, collapsin-1 (300 ng/ml) was added on day two and every second day thereafter; E and F, collapsin-1 was added on day two and the medium was replaced on day four without further addition of collapsin-1.

Figure 5

Quantification of microvessel outgrowth in the presence or absence of collapsin-1. The sprouting microvessels shown in Fig. 4, without collapsin-1 (white circles) or with 300 ng/ml collapsin-1 (black circles), were counted under a phase microscope. Each data point represents the mean ± SD of four independent wells.

Figure 6

Collapsin-1 inhibits the motility of RAEC. RAEC were isolated from rat aortas as described in Materials and Methods and were cultured in DME containing 10% FCS and FGF-2 (1 ng/ml). (A) Northern blot analysis of total RAEC RNA with 32P-labeled rat NRP1 cDNA as a probe. Arrow indicates the position of rat NRP1. (B) The RAEC were seeded in the upper wells of a Boyden chamber and increasing concentrations of collapsin-1 were added to the lower wells. After 4 h, the number of migrated cells per field was counted as in Fig. 2. Each data point represents the mean ± SD of four independent wells.

Figure 7

Collapsin-1 alters RAEC morphology. RAEC were seeded in 8-well culture slides precoated with fibronectin. The next day, medium was replaced with fresh medium (A, D, and G), medium containing 300 ng/ml collapsin-1 (B, E, and H), and medium containing 300 ng/ml collapsin-1 preheated at 70°C for 30 min (C, F, and I). After 30 min of incubation with or without collapsin-1, cells were fixed. (A–C) Analysis by DIC optic microscopy. (D–F) Staining with phalloidin-FITC and analysis by fluorescence microscopy. (G–I) The cells in D–F were stained for DNA with DAPI. The cells in A–C are not the same as those in D–F, because different fixation methods were used.

Figure 8

Collapsin-1 alters the morphology only of EC expressing NRP1. PAEC (A and E), PAEC/NRP1 (B and F), PAEC/KDR (C and G), and PAEC/KDR/NRP1 (D and H) were incubated for 30 min in the absence (A–D) or presence (E–H) of 300 ng/ml collapsin-1, fixed, and analyzed by DIC optic microscopy as in Fig. 7A–C.

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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 - PubMed (original) (raw)