α4β1 integrin associates with VEGFR2 in CLL cells and contributes to VEGF binding and intracellular signaling

Abstract

1. α4β1 integrin and VEGFR2 function as a receptor complex for VEGF in CLL cells.

2. Contribution to VEGF functions in CLL is a novel pathological role for α4β1 integrin in this malignancy.

Figure 1.

Functional association between α4β1 integrin and VEGFR2 in CLL cells. (A) 2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM)–labeled MEC-1 cells (3 independent experiments) or primary CLL cells from 3 patients (P4, P9, P10) were added to wells coated with the indicated concentrations of VEGF. After 60 minutes at 37°C, attached cells were quantified using a fluorescence analyzer. (B) MEC-1 cells (3 independent experiments) or primary CLL cells (P1, P4, P5, P9), with or without previous incubation with the indicated inhibitors, were added to wells coated with 5 μg/mL VEGF and adhesion was quantified as explained. Values represent the percentage of the total number of cells added. (C-D) A total of 15 × 10⁶ MEC-1 cells (C) or primary CLL cells (D; P4) were serum-starved for 2 hours and treated or not with 50 ng/mL soluble VEGF for 15 minutes. Cells were lysed, immunoprecipitated (IP) with anti-α4, anti-VEGFR2 (VR2), or control (Ctrol) Abs and analyzed by western blotting. The total lysate for each condition was also analyzed by western blotting. Numbers indicate the ratio of immunoprecipitated protein with respect to the amount of that protein in the total lysate. (E) Primary CLL cells (P2, P5, P11) were cultured (2 hours, 37°C) on glass coverslips coated with 10 μg/mL poly-l-lysine (control, top panels) or 5 μg/mL VEGF (bottom panels). Cells were fixed and analyzed by confocal microscopy using the indicated primary Abs and Alexa 568– or Alexa 488–labeled secondary Abs. Colocalization of α4 integrin (green) and VEGFR2 (red) was further demonstrated by dot-plot analyses and quantified by the Pearson correlation coefficient. Scale bar, 4 µm. Confocal images for P11 and average values for the 3 patients analyzed ± standard error of the mean (SEM) are shown. *P < .05; **P ≤ .01; ***P ≤ .001.

Figure 2.

α4β1 integrin contributes to VEGF-induced signaling and function in CLL cells. (A) CLL cells (P13, P14, P15, P16, P17) were serum-starved for 2 hours and treated or not with the indicated inhibitors for 30 minutes at 37°C. Cells were added to wells coated with 5 μg/mL VEGF or 0.1% bovine serum albumin (BSA; control) and after 15 minutes at 37°C, cells were lysed and lysates analyzed by western blotting. VEGFR2 phosphorylation (p-VR2) at Y1214 is shown for a representative sample (P17) and quantitated for all 5 patients (P13, P14, P15, P16, P17) studied. (B) CLL cells (P3, P12, P15) were treated as in panel A and the phosphorylation of Akt and FAK (p-FAK) was analyzed by western blotting. (C) CLL cells (P10, P14, P15, P19, P20, P21, P22, P23) were treated with the indicated Abs for 30 minutes and incubated for 48 hours in the absence (blue bar) or presence (red bars) of immobilized VEGF (5 μg/mL). Cell viability was measured by the CCK8 method. The viability of cells treated with control Ab and VEGF was normalized to 1. Average values (arbitrary units [AU]) ± SEM are shown. *P < .05; **P ≤ .01; ***P ≤ .001.