Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

Abstract

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.

Neuropilin 1 binds PDGF-D and is a co-receptor in PDGF-D–PDGFRβ signaling

“…The different ligand families bind to different sites of NRP1; SEMA3A binding requires the first three sub-domains of NRP1 (a1, a2, and b1) while binding of VEGF-A requires the b1 and b2 domains (Gu et al, 2002;Mamluk et al, 2002). Despite reports of NRP1 interaction with PDGFs (Ball et al, 2010;Banerjee et al, 2006;Cao et al, 2010;Pellet-Many et al, 2011), a specific binding site for PDGFs in NRP1 is not known. In addition, interactions of NRP1 with PDGFRα or PDGFRβ have been suggested (Pellet-Many et al, 2015), but its potential role in PDGF-PDGFR signaling remains elusive.…”

“…Others have reported that NRP1 can modulate PDGF-A-and PDGF-B-induced receptor signaling (Ball et al, 2010;Pellet-Many et al, 2011). However, whether these effects of NRP1 are direct or indirect remain open questions.…”

“…However, whether these effects of NRP1 are direct or indirect remain open questions. Post-translational modification of NRP1 with chondroitin sulfate has been described to be an important aspect of NRP1-dependent modulation of PDGF-A-and PDGF-B-induced downstream signaling (Pellet-Many et al, 2011). For the protein-binding studies, we used non-modified recombinant NRP1 and thus can conclude that post-translational modification of NRP1 is not important for the interaction of PDGF-D with NRP1, although this could explain why we did not observe an interaction of PDGF-B with NRP1, as was suggested by earlier studies.…”

Critical role for DOK1 in PDGF-BB stimulated glioma cell invasion via p130Cas and Rap1 signalling

“…We recently reported that tyrosine phosphorylation of the adaptor protein p130Cas plays a key role in PDGF-BB-dependent migration of glioma cells and vascular smooth muscle cells Pellet-Many et al, 2011). DOK1 has been reported to associate with p130Cas upon stimulation of type 1 Fce receptors (FceRI) in mast cells, but the nature of this association is unclear, and the effect on p130Cas tyrosine phosphorylation was not determined (Abramson et al, 2003).…”

“…Previously, we reported that tyrosine phosphorylation of p130Cas (also known as BCAR1) plays a key role in the PDGF-BB-induced migration of U87 glioma cells and vascular smooth muscle cells Pellet-Many et al, 2011). Here, we investigated the role of DOK1 in PDGF-BBmediated cell motility and chemotactic p130Cas signalling in malignant glioma cells.…”