Vascular endothelial growth factor A (VEGF-A) induces endothelial and cancer cell migration through direct binding to integrin {alpha}9{beta}1: identification of a specific {alpha}9{beta}1 binding site

Abstract

Integrin α9β1 mediates accelerated cell adhesion and migration through interactions with a number of diverse extracellular ligands. We have shown previously that it directly binds the vascular endothelial growth factors (VEGF) A, C, and D and contributes to VEGF-induced angiogenesis and lymphangiogenesis. Until now, the α9β1 binding site in VEGF has not been identified. Here, we report that the three-amino acid sequence, EYP, encoded by exon 3 of VEGF-A is essential for binding of VEGF to integrin α9β1 and induces adhesion and migration of endothelial and cancer cells. EYP is specific for α9β1 binding and neither requires nor activates VEGFR-2, the cognate receptor for VEGF-A. Following binding to EYP, integrin α9β1 transduces cell migration through direct activation of the integrin signaling intermediates Src and focal adhesion kinase. This interaction is biologically important because it mediates in vitro endothelial cell tube formation, wound healing, and cancer cell invasion. These novel findings identify EYP as a potential site for directed pharmacotherapy.

FIGURE 1.

Selection of peptides derived from VEGF-A165 to determine the α9β1 binding site in VEGF-A. A, general protein structure of VEGF (SS, signal sequence; Hep, heparin binding domain) and in particular the VEGF homology domain (VHD) common to VEGF-A, C, and D. B, exon structure of VEGF-A, highlighting exons 1–5 as the regions encoding the receptor binding domains of the VEGF-A protein. C, amino acid sequence of human VEGF-A165, highlighting the peptide regions encoded by the indicated VEGF-A exons. D, characteristics of the synthesized peptides derived from exons 3–5 used to determine the α9β1 binding site in VEGF-A. MW, molecular weight; AA, amino acid number.

FIGURE 2.

Peptide 1 (P1), synthesized from the protein sequence encoded by exon 3 of VEGF-A, supports α9β1-mediated cell adhesion and migration in SW480 cells. A, flow cytometry analysis of α9β1 expression in mock- (left) and α9- (right) transfected SW480 cells. B, cell adhesion assays using mock- and α9-expressing SW480 cells plated on various ligands as indicated. Left, representative photomicrographs of the differential cell adhesion of α9-SW480 cells. Right, quantitative analysis of three separate adhesion experiments. C, haptotaxis assays using mock- or α9-transfected SW480 cells on various ligands as indicated. Left, representative photomicrographs of the differential cell migration of α9-SW480 cells. Right, quantitative analysis of three separate experiments. D, left, flow cytometry analysis of integrin αvβ3 (top) or α3β1 (bottom) expression in α9-transfected SW480 cells. Right, cell adhesion assays using α9-transfected SW480 cells plated on various ligands as indicated, in the absence or presence of various blocking antibodies (Ab): α9β1 (light gray bars) or αvβ3 (gray bars) or α3β1 (black bars).

FIGURE 3.

Peptide 1 (P1), synthesized from the protein sequence encoded by exon 3 of VEGF-A, specifically supports α9β1-mediated cell adhesion and migration in endothelial cells. A, flow cytometry analysis of integrin α9β1 (top) and VEGFR-2 (bottom) expression in HMVECs (left) and HUVECs (right). B, cell adhesion assay using HMVECs plated on various ligands in the absence (open bars) or presence (filled bars) of blocking antibody (Ab) to integrin α9β1. C, haptotaxis assays using HMVECs in the presence of increasing doses of peptide, P1. D, haptotaxis assays using ligands as indicated, in HMVECs (left; VEGFR-2⁺, α9β1⁺) or HUVECs (right; VEGFR-2⁺, α9⁻) in the absence (open bars) or presence (filled bars) of blocking antibody to integrin α9β1. E, cell adhesion (left) and migration (right) assays using HMVECs plated on various ligands as indicated, in the absence or presence of various blocking antibodies (Ab): α9β1 (α9, light gray bars) or αvβ3 (gray bars) or α3β1 (α3, black bars).

FIGURE 4.

Selective targeting of integrin α9β1 by siRNA inhibits P1-induced endothelial cell adhesion and migration. A, Western blot of integrin subunits α9 and β1 from lysates of HMVECs treated with nontargeted control siRNA or α9-targeted siRNA. β-Actin was used as loading control. B, cell adhesion assays using HMVECs treated with either nontargeting siRNA (control, open bars) or α9-targeting siRNA (filled bars) plated on various ligands as indicated. C, haptotaxis assays using HMVECs treated with either nontargeting siRNA (control, open bars) or α9-targeting siRNA (filled bars) in the presence of various ligands as indicated.

FIGURE 5.

The amino acid sequence EYP, in VEGF-A, is required for binding to integrin α9β1. A, haptotaxis assays using HMVECs in the presence of short overlapping peptides derived from P1, the VEGF-A polypeptide that binds to α9β1. B, cell adhesion (left) and haptotaxis (right) assays of HMVECs on various VEGF-A peptides in the absence (open bars) or presence (filled bars) of α9β1-specific blocking antibody (Ab). C, haptotaxis assays using HMVECs in the presence of EYPD and EYPD peptides where alanine (A) is substituted for glutamic (E) and/or aspartic (D) acid residues (EYPA, AYPD, and AYPA). D, chemotaxis assays using SW480 cells (VEGFR-2⁻/α9β1⁺) in the presence of VEGF-A and pretreated with various ligands: EYPD- or EYP-containing peptides: P1, P1-G, P1-H. E, chemotaxis assays using HMVECs (left; VEGFR-2⁺/α9β1⁺) or HUVECs (right; VEGFR-2⁺/α9β1⁻) pretreated with various ligands: EYPD- or EYP-containing peptides: P1, P1-G, P1-H.

FIGURE 6.

The VEGF-A peptide EYP specifically binds and activates α9β1 but not VEGFR-2. A, top, Western blotting to assess for VEGFR-2 phosphorylation in HMVECs (left) or HUVECs (right) following exposure to VEGF-A or P1 for 20 min; lysates were immunoprecipitated with VEGFR-2 antibody and immunoblotted with anti-phosphotyrosine antibody. Total VEGFR-2 was used as loading control. A, bottom, calcium release assays performed in either HMVEC (left) or HUVECs (right) in the presence of VEGF-A or synthesized VEGF-A peptides P1 (binds integrin α9β1) or P2 (does not bind integrin α9β1). B, immunoblots to assess for β1 integrin subunit activation in lysates from HMVECs (left) and HUVECs (right) exposed to various ligands for 20 min. GAPDH was used as loading control. C, top, immunoblots to assess for phosphorylated Src in lysates from HMVECs (left) or HUVECs (right) exposed to various ligands for 20 min. Total Src was used as loading control. C, bottom, immunoblots to assess for phosphorylated Src in lysates from α9-SW480 at increasing time points following stimulation with 4 μg/ml P1 (left) or after 20 min following stimulation with increasing doses of P1. Src inhibitor, PP1, was used in separate lysates from cells exposed to 4 g/ml P1 (right). Total Src was used as loading control for all experiments. D, haptotaxis assays using HMVECs (left) or HUVECs (right) pretreated with ligands as indicated and in the absence or presence of Src inhibitor (PP1). E, immunoblots to assess for phosphorylated VEGFR-2 (pY996 or pY951), Src or FAK (pY925 or pY397) in lysates from HMVECs (left) or HUVECs (right) exposed to the VEGF-A peptides, P1, EYPD, or AYPD. Total VEGFR-2, Src or FAK, respectively, was used as loading control.

FIGURE 7.

EYP mediates α9β1-dependent endothelial cell tube formation, wound healing and Matrigel invasion. A, endothelial cell tube-forming assays using HMVECs stimulated by VEGF-A or the VEGF-A peptides, P1 or EYPD, and conducted in the presence or absence of integrin α9β1 inhibitor. B, in vitro scratch assays using mock- or α9-transfected SW480 cells exposed to VEGF-A or α9β1-specific (P1 and EYPD) VEGF-A peptides or α9β1 nonspecific VEGF-A peptide, AYPD; in the absence or presence of α9β1 inhibitor. C, Matrigel invasion assays using α9-transfected SW480 cells exposed to VEGF-A or VEGF-A peptides as indicated, in the absence or presence of α9β1 inhibitor.