Mesenchymal stem cell migration is regulated by fibronectin through α5β1-integrin-mediated activation of PDGFR-β and potentiation of growth factor signals

Jennifer Veevers-Lowe, Stephen G Ball, Adrian Shuttleworth, Cay M Kielty, Jennifer Veevers-Lowe, Stephen G Ball, Adrian Shuttleworth, Cay M Kielty

Abstract

Cell migration during vascular remodelling is regulated by crosstalk between growth factor receptors and integrin receptors, which together coordinate cytoskeletal and motogenic changes. Here, we report extracellular matrix (ECM)-directed crosstalk between platelet-derived growth factor receptor (PDGFR)-β and α5β1-integrin, which controls the migration of mesenchymal stem (stromal) cells (MSCs). Cell adhesion to fibronectin induced α5β1-integrin-dependent phosphorylation of PDGFR-β in the absence of growth factor stimulation. Phosphorylated PDGFR-β co-immunoprecipitated with α5-integrin and colocalised with α5β1-integrin in the transient tidemarks of focal adhesions. Adhesion to fibronectin also strongly potentiated PDGF-BB-induced PDGFR-β phosphorylation and focal adhesion kinase (FAK) activity, in an α5β1-integrin-dependent manner. PDGFR-β-induced phosphoinositide 3-kinase (PI3K) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin and α5β1-integrin. This synergistic relationship between α5β1-integrin and PDGFR-β is a fundamental determinant of cell migration. Thus, fibronectin-rich matrices can prime PDGFR-β to recruit mesenchymal cells at sites of vascular remodelling.

Figures

Fig. 1.
Fig. 1.
ECM-induced tyrosine phosphorylation of PDGFR-β. (A) Human phosphorylated RTK arrays were used to examine ECM-induced RTK phosphorylation levels in MSC lysates. PDGFR-α (R-α; coordinates C7 and C8) and PDGFR-β (R-β; coordinates C9 and C10), respectively, were examined in MSC lysates, taken at 90 minutes, from BSA-coated wells or wells coated with 10 μg/ml fibronectin, laminin, fibrillin-1 (PF8), collagen type I or collagen type IV. Each array was identically exposed to detection reagents and film. Quantitative analysis was evaluated by densitometry with phosphorylated PDGFR duplicate RTK spots, normalised against phosphotyrosine-positive control spots [coordinates (A1, A2), (A23, A24), (F1, F2), (F23, F24)], and is represented as the fold increase above that with BSA control substrate (±s.d.) for duplicate spots. A representative example of two independent experiments is shown for each array analysis. (B) Immunoprecipitation (IP) analysis of the levels of PDGFR-β tyrosine phosphorylation was carried out using lysates from cells plated onto BSA control (Con) substrate or 10 μg/ml immobilised fibronectin (Fn), laminin (Lam), fibrillin-1 (PF8) (Fib-1), collagen type I (Col I) or collagen type IV (Col IV) in serum-free conditions for 90 minutes at 37°C. PDGFR-β was isolated from equivalent MSC lysates by IP analysis using anti-PDGFR-β with anti-rabbit IgG antibodies as a control, then tyrosine phosphorylation was detected by immunoblotting (IB) using an antibody against phosphorylated tyrosine (Tyr-P) (supplementary material Table S1). Membranes were re-probed with anti-PDGFR-β antibody, as a loading control. Quantitative analysis was evaluated by densitometry with Tyr-P normalised to total PDGFR-β and is represented as the fold increase above that with BSA control substrate. A representative example of two independent experiments is shown. ECM-induced PDGFR-β (C) Y751 and (D) Y1021 PDGFR-β phosphorylation was determined using ELISAs for phosphorylated PDGFR-β, with all conditions normalised to the total amount of PDGFR-β. MSCs were plated onto 10 μg/ml immobilised ECM proteins in serum-free conditions for 90 minutes at 37°C. PDGFR-β phosphorylation is represented as the fold increase above the control BSA-induced PDGFR-β tyrosine phosphorylation, against which ECM proteins were compared (***P<0.001 by one-way ANOVA). Experiments were performed in triplicate, on the same microtitre plate, and at least three times. Data are means±s.d. for at least three independent experiments.
Fig. 2.
Fig. 2.
Fibronectin-induced integrin-mediated PDGFR-β tyrosine phosphorylation. The effects of inhibitory anti-integrin antibodies on fibronectin-induced PDGFR-β (i) Y751 and (ii) Y1021 phosphorylation were determined in (A) MSCs or (B) SMCs, using ELISAs for phosphorylated PDGFR-β, with all conditions normalised to the total amount of PDGFR-β. Cells in serum-free conditions were plated onto 10 μg/ml immobilised fibronectin for 90 minutes in the presence of 10 μg/ml anti-integrin inhibitory mAbs or 10 μM integrin-αv-inhibiting peptide cilengitide. Antibodies were specific for the integrin α5 subunit (JBS5, mAb11), the integrin β1 subunit (mAb13, 8E3) and αvβ3-integrin (23C6). Antibody specificity is indicated in brackets next to the antibody name; NF denotes a non-functional antibody, as a control. All conditions are expressed relative to fibronectin-induced PDGFR-β (i) Y751 and (ii) Y1021 phosphorylation (100%) in the absence of antibody (**P<0.01, ***P<0.001 by one-way ANOVA). Control (Con; broken line) indicates the basal tyrosine phosphorylation of PDGFR-β induced by BSA. All experiments were performed in triplicate, on the same microtitre plate, and at least three times. Data are means±s.d. for at least three independent experiments. (C) Expression of the integrin α5 and integrin αv subunits was examined in MSCs and SMCs plated onto 10 μg/ml immobilised fibronectin, in serum-free conditions, for 90 minutes at 37°C. (i) Protein expression was detected by immunoblotting (IB) equal amounts (10 μg) of cell lysates using anti-integrin-α5 or anti-integrin-αv antibodies. Membranes were reprobed with anti-β-actin antibody, as a loading control. A representative example of two independent experiments is shown. (ii) Quantitative analysis was evaluated by densitometry with data normalised to the level of β-actin. Data are represented as the mean pixel density (±s.d.) for two independent experiments (**P<0.01 by one-way ANOVA).
Fig. 3.
Fig. 3.
Fibronectin-induced association of PDGFR-β and α5β1-integrin. (A) The association of PDGFR-β with integrin subunit α5 was examined by immunoprecipitation (IP). MSCs in serum-free conditions were incubated on tissue culture plastic, as a control (Con), or on 10 μg/ml immobilised fibronectin (Fn) or laminin (Lam), for 90 minutes at 37°C. (i) PDGFR-β was isolated from equivalent MSC lysates by IP using anti-PDGFR-β antibody, with anti-rabbit IgG antibody as a control, then integrin subunit α5 association was detected by immunoblotting (IB) using an antibody against integrin α5. Membranes were re-probed with anti-PDGFR-β as a loading control. (ii) Integrin subunit α5 was isolated by IP using antibodies against integrin α5 or IgG, as a control, then PDGFR-β association was detected by IB analysis using anti-PDGFR-β antibody. Membranes were reprobed with an antibody against integrin α5 as a loading control. A representative example of three independent experiments is shown. (B) Immunoblotting of PDGFR-β Y751 or Y1021 phosphorylation was carried out using MSC lysates, taken at 15, 90 or 240 minutes, from wells coated with 10 μg/ml fibronectin. (i) Levels of PDGFR-β phosphorylation were detected in equal (10 μg) amounts of cell lysates using antibodies against phosphorylated PDGFR-β Y751 or Y1021, or anti-PDGFR-β antibody, as a loading control. A representative example of two independent experiments is shown. (ii) Quantitative analysis, evaluated by densitometry, with data normalised to the total amount of PDGFR-β. Data are means±s.d. for two independent experiments (***P<0.001 compared with other time points).
Fig. 4.
Fig. 4.
Fibronectin-induced temporal colocalisation of phosphorylated PDGFR-β with α5β1-integrin. Following adhesion to fibronectin, the cellular distribution of phosphorylated PDGFR-β and integrin subunit α5 was examined over time using immunofluorescence microscopy. MSCs in serum-free conditions were plated onto 10 μg/ml immobilised fibronectin for (i) 15 minutes, (ii) 90 minutes or (iii) 240 minutes, at 37°C, before fixation with paraformaldehyde. Representative examples of MSCs double-labelled for (A) Y751- or (B) Y1021-phosphorylated PDGFR-β (red) and integrin subunit α5 (green), and merged images, are shown. Nuclei appear blue owing to DAPI staining. To quantify receptor colocalisation, images were analysed using the ImageJ colocalisation plugin (see Materials and Methods). Merged particle analysis images are shown, with red and green channels having similar threshold values and the same particle size range (1–500 pixels), together with colocalisation events represented in yellow. The white asterisks highlight the leading edge and the boxed area highlights the transient tidemark of receptor colocalisation. The mean number of colocalised particles (±s.d.) derived from five different single-cell images is denoted in yellow and is represented graphically (C) (***P<0.001 compared with other timepoints). Images are representative of at least three independent experiments. Scale bar: 20 μm.
Fig. 5.
Fig. 5.
Fibronectin enhances PDGF-BB-induced PDGFR-β tyrosine phosphorylation. (A) The effects of fibronectin (Fn) or laminin (Lam) on PDGF-BB (BB)-stimulated PDGFR-β (i) Y751 and (ii) Y1021 phosphorylation were examined using an ELISA for phosphorylated PDGFR-β with all conditions normalised to the total amount of PDGFR-β. MSCs in serum-free conditions were plated onto 10 μg/ml immobilised fibronectin or laminin in the absence or presence of 50 ng/ml PDGF-BB for 90 minutes at 37°C. The broken line indicates basal tyrosine phosphorylation of PDGFR-β induced by BSA. The involvement of integrin subunit α5 in fibronectin-regulated PDGF-BB stimulation of PDGFR-β (i) Y751 or (ii) Y1021 phosphorylation levels was also determined. MSCs in serum-free conditions, treated with either 10 μg/ml anti-integrin-α5 inhibitory antibody (JBS5) or anti-PDGFR-β neutralisation antibody (AF385), or both, were plated onto 10 μg/ml immobilised fibronectin in the absence or presence of 50 ng/ml PDGF-BB for 90 minutes at 37°C. Antibody specificity is shown in brackets next to the antibody name. Conditions are expressed relative to fibronectin-induced PDGFR-β (i) Y751 or (i) Y1021 phosphorylation (100%) in the absence of PDGF-BB, and are compared against the respective fibronectin-induced phosphorylation of PDGFR-β in the absence or presence of PDGF-BB (***P<0.001 by one-way ANOVA), or in the presence of both JBS5 and AF385 (ΔΔΔP<0.001 by one-way ANOVA). Laminin-induced phosphorylation of PDGFR-β in the presence of PDGF-BB is compared with that of laminin in the absence of PDGF-BB (***P<0.001 by one-way ANOVA). Experiments were performed in triplicate, on the same microtitre plate, and at least three times. Data are means±s.d. for at least three independent experiments. (B) The effects of inhibitory anti-integrin antibodies and PDGF-BB on fibrillin-1 PF8-induced PDGFR-β (i) Y751 and (ii) Y1021 phosphorylation levels were determined using an ELISA for phosphorylated PDGFR-β with all conditions normalised to the total amount of PDGFR-β. MSCs in serum-free conditions were plated onto 10 μg/ml immobilised fibrillin-1 (PF8) in the presence of 10 μg/ml anti-integrin inhibitory mAbs or 50 ng/ml PDGF-BB for 90 minutes at 37°C. Antibodies were specific for the integrin α5 subunit (mAb16, mAb11), integrin β1 subunit (mAb13, 8E3) and αvβ3-integrin (23C6). Antibody specificity is indicated in brackets next to the antibody name; NF denotes a non-functional antibody, as a control. All conditions are expressed relative to PF8-induced PDGFR-β (i) Y751 and (ii) Y1021 phosphorylation (100%) in the absence of antibody (***P<0.001 by one-way ANOVA). Control (Con; broken line) indicates the basal tyrosine phosphorylation of PDGFR-β induced by BSA. All experiments were performed in triplicate on the same microtitre plate at least three times. Data are means±s.d of at least three independent experiments.
Fig. 6.
Fig. 6.
PDGFR-β and α5β1-integrin crosstalk mediates MSC migration towards fibronectin. (A) MSC migration towards fibronectin with or without PDGF-BB (BB) stimulation was evaluated using a Boyden chamber migration assay. BSA (basal) and laminin were used as control substrates. MSCs in serum-free conditions in the upper chamber were cultured for 4 hours at 37°C in Boyden chambers pre-coated on the underside with BSA as a basal control, or 10 μg/ml fibronectin or laminin, with or without 50 ng/ml PDGF-BB in the lower chamber. Conditions are compared against the respective MSC migration in the absence of PDGF-BB (**P<0.01; ***P<0.001 by one-way ANOVA), or against basal in the absence of PDGF-BB (ΔΔΔP<0.001 by one-way ANOVA). (B) The involvement of integrin subunit α5 in PDGF-BB-induced MSC migration towards fibronectin was also determined. MSCs in serum-free conditions in the upper chamber treated with either 10 μg/ml anti-integrin-α5 inhibitory antibody (JBS5), anti-PDGFR-β neutralisation antibody (AF385), or both, were cultured for 4 hours at 37°C in Boyden chambers pre-coated on the underside with 10 μg/ml fibronectin, with or without 50 ng/ml PDGF-BB in the lower chamber. Conditions are compared against the respective MSC migration in the absence or presence of PDGF-BB (***P<0.001 by one-way ANOVA), or in the presence of both JBS5 and AF385 (ΔΔΔP<0.001 by one-way ANOVA). All data are the mean (±s.d.) optical density (OD 570 nm) of stained migratory cells from two readings in an individual experiment, repeated three times. Images above each bar graph are representative of the migratory cells per field on the membrane underside of three independent experiments. Scale bar: 100 μm.
Fig. 7.
Fig. 7.
α5β1-Integrin and FAK activation mediates PDGFR-β-induced PI3K activation. (A) MSCs in serum-free conditions treated with 10 μg/ml anti-integrin-α5 inhibitory antibody (JBS5) or non-functional (NF) anti-integrin-α5 antibody (mAb11), were plated onto tissue culture plastic, as a control (basal), or 10 μg/ml immobilised fibronectin or laminin in the absence or presence of 50 ng/ml PDGF-BB for 90 minutes at 37°C. Phosphorylation levels of (i) FAK, (ii) Akt and (iii) PLCγ-1 were detected in equal (10 μg) amounts of cell lysates by immunoblotting (IB) using antibodies against (i) phosphorylated FAK (Y397), (ii) phosphorylated Akt (S473) or (iii) phosphorylated PLCγ-1 (Y783). Membranes were reprobed with anti-β-actin antibody as a loading control. Quantitative analysis was performed by densitometry with (i) phosphorylated FAK, (ii) phosphorylated Akt or (iii) phosphorylated PLCγ-1 normalised to the level of β-actin. Data are the fold increase above that with basal control substrate. A representative example of two independent experiments is shown. (B) The effect of FAK siRNA knockdown on fibronectin-induced PDGFR-β Y751 and Y1021 phosphorylation levels was determined using an ELISA for phosphorylated PDGFR-β, with all conditions normalised to that with total PDGFR-β. MSCs transfected with 3 μg of siRNA FAK or scrambled siRNA, as a control, were plated onto 10 μg/ml immobilised fibronectin for 90 minutes at 37°C. Conditions are expressed relative to fibronectin-induced PDGFR-β Y751 or Y1021 phosphorylation (100%) in the presence of scrambled siRNA (***P<0.001 by one-way ANOVA). Experiments were performed in triplicate, on the same microtitre plate, and at least three times. Data are the means±s.d. for at least three independent experiments. The knockdown efficiency of the FAK siRNA as assessed by immunoblotting is shown below the graph. (C) The effect of FAK siRNA knockdown on fibronectin-induced Akt S473 phosphorylation levels was determined by immunoblotting. MSCs transfected with 3 μg of siRNA FAK or scrambled siRNA, as a control, were plated onto 10 μg/ml immobilised fibronectin for 90 minutes at 37°C. (i) Phosphorylation levels of Akt were detected in equal (10 μg) amounts of cell lysates using an antibody against phosphorylated Akt S473 (S473). The membrane was re-probed with anti-β-actin antibody as a loading control. A representative example of two independent experiments is shown. (ii) Quantitative analysis, as determined by densitometry, with the levels of phosphorylated Akt normalised to those of β-actin. Data are the means±s.d. for two independent experiments (**P<0.01 by Student's t-test). The knockdown efficiency of the FAK siRNA is shown in B.
Fig. 8.
Fig. 8.
PI3K mediates PDGFR-β-induced actin arrangement and migration. (A) The cellular distribution of F-actin and phosphorylated PDGFR-β Y751 was examined by immunofluorescence microscopy. MSCs in serum-free conditions treated with 10 μg/ml anti-integrin α5 inhibitory antibody (JBS5) or non-functional (NF) anti-α5-integrin antibody (mAb11), as a control, were plated onto 10 μg/ml immobilised fibronectin in the absence or presence of 50 ng/ml PDGF-BB for 90 minutes at 37°C, before fixation with paraformaldehyde. Representative examples of MSCs double-labelled for phalloidin (white and red) and Y751 phosphorylated PDGFR-β (green), and merged images are shown. Nuclei appear blue owing to DAPI staining. F-actin membrane ruffling is highlighted by a white asterisk. To quantify receptor distribution and clustering (Chen et al., 2010), images were analysed using the ImageJ ‘analyse particles’ function (as described in the Materials and Methods). Corresponding particle analysis images of PDGFR-β distribution (1–500 pixels) or PDGFR-β clustering (10–500 pixels) are shown next to each original image. The mean number of particles (±s.d.) derived from five different single-cell images is denoted in black (**P<0.01; ***P<0.001 compared with the fibronectin-induced PDGFR-β clustering in the absence of JBS5). Images are representative of at least three independent experiments. Scale bar: 20 μm. (B) The cellular distribution of F-actin and integrin subunit α5 was examined by immunofluorescence microscopy. MSCs in serum-free conditions treated with 20 μM LY294002, or DMSO as a control, were plated onto 10 μg/ml immobilised fibronectin in the absence or presence of 50 ng/ml PDGF-BB for 90 minutes at 37°C, before fixation with paraformaldehyde. Representative examples of MSCs double-labelled for phalloidin (white and red) and integrin α5 (green), and merged images are shown. Nuclei appear blue after DAPI staining. F-actin membrane ruffling is highlighted by white asterisks. Images are representative of at least three independent experiments. Scale bar: 20 μm. Inhibition efficiency of LY294002 on Akt phosphorylation by immunoblotting is shown to the right of the image panel. (C) The involvement of PI3K in MSC migration towards fibronectin was evaluated using a Boyden chamber migration assay. MSCs in serum-free conditions in the upper chamber treated with 20 μM LY294002 (LY) or DMSO (−), as a control, were cultured for 4 hours at 37°C in Boyden chambers pre-coated on the underside with 10 μg/ml fibronectin, with or without 50 ng/ml PDGF-BB in the lower chamber. (i) Images are representative of the migratory cells per field on the membrane underside of three independent experiments. Scale bar: 400 μm. (ii) Data are represented as mean (±s.d.) optical density (OD 570 nm) of stained migratory cells of two readings of an individual experiment, repeated three times. Conditions are compared against their respective MSC migration in the absence or presence of PDGF-BB (***P<0.001 by one-way ANOVA).
Fig. 9.
Fig. 9.
MSC migration is regulated by fibronectin through α5β1-integrin-mediated activation of PDGFR-β. Schematic model depicting the synergistic connection between α5β1-integrin-mediated adhesion to fibronectin and PDGFR-β, which controls Akt-PI3K activity and MSC migration. Adhesion to fibronectin promotes receptor clustering and association, which is mediated by heparan sulphate proteoglycans (HSPG), induces α5β1-integrin- and FAK-dependent PDGFR-β phosphorylation in the absence of growth factor and strongly potentiates PDGF-BB-stimulated PDGFR-β signalling. The potentiation of PI3K-Akt activity by crosstalk between α5β1-integrin and PDGFR-β is an essential event in the cascade that induces actin reorganisation and cell motility.

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