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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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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.
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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.

Mentions: NRP1 is expressed by neuronal cells and is a receptor for members of the collapsin-1/semaphorin III family (Luo et al. 1993; He and Tessier-Lavigne 1997; Kolodkin et al. 1997). To determine whether NRP1 can also function as a receptor for collapsin/semaphorins in nonneuronal cells, PAEC lines expressing NRP1, KDR, or both receptors (Soker et al. 1998) were incubated with 125I-collapsin-1 (Fig. 1). A 100-kD protein corresponding to the size of collapsin-1, and larger size proteins possibly representing collapsin-1/NRP1 complexes, were detected in PAEC/NRP1 (Fig. 1, lane 3) and PAEC/KDR/NRP1 (Fig. 1, lane 4), but not in parental PAEC (Fig. 1, lane 1) or PAEC/KDR (Fig. 1, lane 2). These results indicated that the binding of 125I-collapsin-1 to PAEC was totally dependent on the expression of NRP1. In addition, 125I-collapsin also bound to MDA-MB-231 breast carcinoma cells (Fig. 1, lane 5), previously shown to express abundant NRP1 (Soker et al. 1996, Soker et al. 1998).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199727&req=5

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.
Mentions: NRP1 is expressed by neuronal cells and is a receptor for members of the collapsin-1/semaphorin III family (Luo et al. 1993; He and Tessier-Lavigne 1997; Kolodkin et al. 1997). To determine whether NRP1 can also function as a receptor for collapsin/semaphorins in nonneuronal cells, PAEC lines expressing NRP1, KDR, or both receptors (Soker et al. 1998) were incubated with 125I-collapsin-1 (Fig. 1). A 100-kD protein corresponding to the size of collapsin-1, and larger size proteins possibly representing collapsin-1/NRP1 complexes, were detected in PAEC/NRP1 (Fig. 1, lane 3) and PAEC/KDR/NRP1 (Fig. 1, lane 4), but not in parental PAEC (Fig. 1, lane 1) or PAEC/KDR (Fig. 1, lane 2). These results indicated that the binding of 125I-collapsin-1 to PAEC was totally dependent on the expression of NRP1. In addition, 125I-collapsin also bound to MDA-MB-231 breast carcinoma cells (Fig. 1, lane 5), previously shown to express abundant NRP1 (Soker et al. 1996, Soker et al. 1998).

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