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.

Figure 1. Neuropilin expression and glycosylation in human VSMCs and endothelial cells

(A) Whole-cell lysates of HUVECs, HCAECs, HCASMCs and HAoSMCs were immunoblotted for NRP1, NRP2, VEGFR2, synectin, PDGFRα, PDGFRβ and GAPDH. NRP1 bands of approximately 130 kDa and >250 kDa are indicated. (B) Cell-surface expression of NRP1 was examined in HUVECs and HCASMCs using flow cytometry as described in the Supplementary Online Data at http://www.BiochemJ.org/bj/435/bj4350609add.htm. KDR, kinase insert domain-containing receptor (VEGFR2); PE, phycoerythrin. (C) Confluent cultures of the cells indicated were pre-treated with (+) or without (−) 5 μg/ml tunicamycin for 16 h. Lysates were then prepared and immunoblotted with an antibody against NRP1 or NRP2. (D) Top and middle: HCASMCs were treated with chondroitinase, heparitinase or both enzymes combined (each at 1 unit/ml) for 4 h, and lysates were prepared and immunoblotted with an antibody against NRP1 or GAPDH. Bottom: NRP1 immunoprecipitates prepared from HCASMCs were incubated for 4 h with chondroitinase or heparitinase, and then immunoblotted with anti-NRP1 antibody. WB, Western blot; IP, immunoprecipitate. Results shown in (A)–(D) are representative of at least three independent experiments. Molecular masses are indicated in kDa. (E) Amounts of GAG-modified NRP1 after chondroitinase or heparitinase treatments in the Western blots shown in (D) were quantified by scanning densitometry, and used to calculate the relative levels of unmodified NRP1, HS-GAG–NRP1 and CS-GAG–NRP1 in HCASMCs. Results are mean percentages of total NRP1 immunoreactivity (non-GAG-modified 130 kDa NRP1 plus GAG-modified >250 kDa NRP1).

Figure 2. NRP1 plays a role in PDGF-induced VSMC chemotaxis

HCASMCs were transfected with siRNA to NRP1, NRP2 or synectin (syn), or with control non-targeted scrambled siRNA (Scr), and 3 days later were either transferred to transwells and allowed to migrate for 4 h in response to 30 ng/ml PDGF-BB (A and B), 30 ng/ml PDGF-AA (B) or serum-free medium (Control), or were used to assess expression of targeted proteins by immunoblotting of whole-cell lysates (A, bottom). Results are mean+S.E.M. (n=3) numbers of migrated cells obtained from multiple independent experiments. *P<0.05 compared with Scr siRNA plus PDGF-BB or PDGF-AA. Representative transwell filters showing stained migrated cells are shown above the histogram. Protein expression 3 days after siRNA transfection was quantified by scanning densitometry of blots from three independent experiments (A, bottom right).

Figure 3. Overexpression of NRP1 lacking the cytosolic domain inhibits VSMC chemotaxis

HCASMCs were either uninfected (Control) or infected with Ad.GFP, Ad.NRP1WT or Ad.NRP1ΔC. At 3 days after infection, cell lysates were prepared and immunoblotted with antibodies specific for the NRP1 C- or N-terminus (A), or were transferred to transwells and allowed to migrate for 4 h in response to serum-free medium (control) or 30 ng/ml PDGF-AA or PDGF-BB (B). Results are mean+S.E.M. (n=3) numbers of migrated cells obtained from multiple independent experiments. *P<0.05 compared with Ad.NRP1WT plus PDGF-AA or PDGF-BB. Molecular masses in kDa are indicated in (A).

Figure 4. NRP1 associates with PDGFRα in HCASMCs

(A) HCASMCs were incubated in serum-free medium overnight, then treated with (+) or without (−) 30 ng/ml PDGF-BB for 10 min. Cells were then lysed and PDGFRα was immunoprecipitated (IP). In parallel, lysates were incubated with either secondary antibody alone (IgG), or agarose beads alone. Immunoprecipitates were then immunoblotted (WB) with anti-NRP1 or anti-PDGFRα antibodies. Results are representative of at least three independent experiments. Molecular masses are indicated in kDa. (B) HCASMCs were fixed, permeabilized and then immunostained for NRP1 (green) and PDGFRα (red). The lower left-hand panel shows a merge of immunostaining for NRP1 and PDGFRα, and an enlarged image of the region in the green square is shown in the lower right-hand panel. Areas of co-localization at the cell membrane are indicated by white arrowheads. The results shown are representative of at least three independent experiments.

Figure 5. NRP1 knockdown inhibits PDGFRα activation

(A) HCASMCs were transfected with control scrambled siRNA (Scr) or with NRP1 siRNA, and 3 days later, cells were incubated overnight in serum-free medium and then treated for 10 min with serum-free medium (control), 30 ng/ml PDGF-AA or 30 ng/ml PDGF-BB. Cells were then lysed, and the activity of either PDGFRα (upper) or PDGFRβ (lower) was measured using a specific ELISA. Results are means+S.E.M. obtained from three independent experiments each performed in triplicate. *P<0.05 compared with Scr siRNA plus PDGF-BB or PDGF-AA. (B) HCASMCs were transfected with siRNA as indicated, and 3 days later, intact cells were surface labelled with biotin. Biotinylated surface membrane proteins were isolated and then immunoblotted as indicated. Molecular masses are indicated in kDa.

Figure 6. PDGF-stimulated p130^Cas phosphorylation is mediated via NRP1

(A and B) HCASMCs were transfected with control scrambled siRNA (Scr) or with NRP1 siRNA, and 3 days later, cells were incubated overnight in serum-free medium (SF) and then treated for 10 min with 30 ng/ml PDGF-BB or 30 ng/ml PDGF-AA. Cells were then lysed and immunoblotted with the antibodies indicated. Results are representative of at least three independent experiments. p130^Cas tyrosine phosphorylation in three independent experiments similar to those in (A) were quantified by scanning densitometry (B). *P<0.05 compared with Scr siRNA plus PDGFs. (C and D) HCASMCs were infected with Ad.GFP, Ad.NRP1WT or Ad.NRP1ΔC. At 3 days after infection, cells were incubated overnight in serum-free medium and then treated for 10 min with either serum-free medium or with 30 ng/ml PDGF-BB for 10 min. Cells were then lysed and immunoblotted with antibodies specific for the NRP1 N-terminus, total and phosphorylated p130^Cas and GAPDH as indicated. Results from three independent experiments similar to those in (C) were used for quantification of p130^Cas tyrosine phosphorylation using scanning densitometry (D). Molecular masses in kDa are indicated in (A) and (C). RU, relative units.

Figure 7. PDGF-BB-induced VSMC chemotaxis is mediated via p130^Cas

HCASMCs were transfected with control scrambled siRNA (Scr) or with p130^Cas siRNA, and 3 days later, cells were transferred to transwells and allowed to migrate for 4 h in response to 30 ng/ml PDGF-BB, 30 ng/ml PDGF-AA or serum-free medium (Control), or were used to assess expression of targeted proteins by immunoblotting of whole-cell lysates (right). Migration of transfected cells was measured as described in the Materials and methods section. Results are mean+S.E.M. numbers of migrated cells obtained from three independent experiments, each performed in triplicate. *P<0.05 compared with Scr siRNA plus PDGF-BB. Immunoblots are representative of three independent experiments.

Figure 8. Role of NRP1 O-linked glycosylation in PDGF-induced VSMC chemotaxis

(A and B) HCASMCs were incubated in serum-free medium overnight and then treated with chondroitinase, heparitinase or both enzymes combined (each at 1 unit/ml) for 2 h. Cells were then either transferred to transwells and allowed to migrate for 4 h (A) in response to either 30 ng/ml PDGF-BB or serum-free medium (Control), or were stimulated with PDGF-AA or PDGF-BB for 10 min and lysates were immunoblotted with the indicated antibodies (B). (C) HCASMCs were infected with Ad.GFP, Ad.NRP1WT or AD.NRP1S612A. At 3 days after infection, cell lysates were prepared and immunoblotted with antibodies against NRP1 and GAPDH (upper), or were transferred to transwells and allowed to migrate for 4 h in response to serum-free medium (Control) or 30 ng/ml PDGF-AA or PDGF-BB (lower). The values shown are mean+S.E.M. (n=3) numbers of migrated cells obtained from multiple independent experiments. *P<0.05 compared with Ad.NRP1WT plus PDGF-AA or PDGF-BB. Molecular masses are indicated in kDa.