Mesenchymal Stem Cell Migration Is Regulated by Fibronectin Through A5b1-Integrin-Mediated Activation of PDGFR-B and Potentiation of Growth Factor Signals

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1288 Research Article Mesenchymal stem cell migration is regulated by fibronectin through a5b1-integrin-mediated activation of PDGFR-b and potentiation of growth factor signals

Jennifer Veevers-Lowe, Stephen G. Ball, Adrian Shuttleworth and Cay M. Kielty* Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK *Author for correspondence ([email protected])

Summary 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)-b and a5b1-integrin, which controls the migration of mesenchymal stem (stromal) cells (MSCs). Cell adhesion to fibronectin induced a5b1-integrin-dependent phosphorylation of PDGFR-b in the absence of growth factor stimulation. Phosphorylated PDGFR-b co-immunoprecipitated with a5-integrin and colocalised with a5b1-integrin in the transient tidemarks of focal adhesions. Adhesion to fibronectin also strongly potentiated PDGF-BB-induced PDGFR-b phosphorylation and focal adhesion kinase (FAK) activity, in an a5b1-integrin-dependent manner. PDGFR-b-induced phosphoinositide 3-kinase (PI3K) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin and a5b1-integrin. This synergistic relationship between a5b1-integrin and PDGFR-b is a fundamental determinant of cell migration. Thus, fibronectin-rich matrices can prime PDGFR-b to recruit mesenchymal cells at sites of vascular remodelling.

Key words: Platelet-derived growth factor receptor-b, a5b1-Integrin, Fibronectin, Mesenchymal stem cell, Signalling, Migration

Introduction 2008). Signalling by RTKs such as PDGFR-b is not only regulated Signalling through the receptor tyrosine kinase (RTK) platelet- by growth factors but also by functional collaboration with integrins derived growth factor receptor (PDGFR)-b is essential for the (Eliceiri, 2001; Yamada and Even-Ram, 2002; Giancotti and Tarone,

Journal of Cell Science migration and differentiation of cells during vascular development 2003; Streuli and Akhtar, 2009). Integrins are ab heterodimeric (Yancopoulos et al., 2000; Betsholtz et al., 2001; Kinner et al., cell-surface receptors that act as a transmembrane link between 2002; Lindblom et al., 2003; Ball et al., 2007; Andrae et al., 2008). extracellular matrix (ECM) ligands and the actin cytoskeleton. Knockout of the genes encoding PDGFR-b or PDGF-B in mice They direct inside-out and outside-in signalling that regulates causes death during late embryonic stages from widespread numerous cellular responses, including survival, growth, migration microvascular bleedings caused by deficient mural cell recruitment and differentiation (Hynes, 1992; Danen and Sonnenberg, 2003; (Lindahl et al., 1997; Hellström et al., 1999; French et al., 2008). Askari et al., 2009). b1- and aVb3-integrins can influence PDGFR- PDGF-BB, the main growth factor ligand of PDGFR-b, is a potent b activity (Sundberg and Rubin, 1996; Schneller et al., 1997; stimulant of smooth muscle cell (SMC) recruitment during Woodard et al., 1998; Borges et al., 2000; Minami et al., 2007; neointimal hyperplasia following vascular injury (Andrae et al., Amano et al., 2008; Zemskov et al., 2009), and integrin-linked 2008). PDGFR signalling has also emerged as a predominant kinase (ILK) can control SMC migration in response to PDGF pathway in recruitment of adult perivascular mesenchymal stem (Esfandiarei et al., 2010). However, the molecular mechanisms cells (MSCs), which play crucial roles in angiogenesis, wound underlying crosstalk between PDGFR-b and integrins, and how repair and tissue regeneration (Ferrari et al., 1998; Abedin et al., they coordinate cell recruitment, remain obscure. 2004; Fiedler et al., 2004; Tepper et al., 2005; Ball et al., 2007). We have discovered a fundamental ECM-specific receptor Dimerisation of PDGFR-b, induced by ligating growth factor crosstalk mechanism that controls MSC migration. Adhesion to dimers, stimulates autophosphorylation of specific tyrosine residues fibronectin, through a5b1-integrin, specifically induced MSC within its cytoplasmic domain (Kelly et al., 1991). PDGFR-b is migration by activating PDGFR-b signalling in the absence of mainly activated by PDGF-BB, but also by PDGF-DD and vascular growth factor stimulation. Fibronectin also strongly potentiated endothelial growth factor (VEGF)-A (Fredriksson et al., 2004; Ball growth-factor-mediated receptor activation in an a5b1-integrin- et al., 2007). Autophosphorylation provides docking sites for dependent manner. Phosphorylated PDGFR-b appeared in ruffles downstream signal transduction molecules (Kazlauskas and Cooper, at the leading edge of migratory cells and transiently colocalised 1989), especially phosphoinositide 3-kinase (PI3K), which mediates with a5b1-integrin in the tidemarks of focal adhesions. actin reorganisation and migration, phospholipase C (PLC), Corresponding focal adhesion kinase (FAK)-dependent PI3K which stimulates cell growth and motility, and Src family tyrosine activity, actin reorganisation, membrane ruffling and MSC kinases, which influence cell proliferation (Heldin et al., 1998; migration all exhibited fibronectin- and a5b1-integrin-dependence. Jiménez et al., 2000; Tallquist and Kazlauskas, 2004; Andrae et al., This synergistic relationship between a5b1-integrin and PDGFR- a5b1-integrin controls PDGFR-b 1289

b is thus a fundamental determinant of mesenchymal cell migration. C10) in the same cell lysate. MSCs plated onto a BSA control Fibronectin-rich matrices might therefore act to prime PDGFR-b substrate (basal) for 90 minutes produced no detectable to recruit mesenchymal cells to sites of vascular remodelling. immunoreactivity for any RTKs, but control reactivity against phosphorylated tyrosine was positive. When MSCs were plated Results onto fibronectin for 90 minutes, PDGFR-a and PDGFR-b tyrosine Adhesion to ECM induces PDGFR tyrosine phosphorylation was detected. Fibronectin stimulation also induced phosphorylation tyrosine phosphorylation of other RTKs, notably epidermal growth To evaluate how adhesion to ECM regulates PDGFR activation in factor receptor (EGFR), EphA7, Axl and c-Ret. When MSCs were MSCs, tyrosine phosphorylation of PDGFR-a and PDGFR-b was plated onto fibrillin-1 PF8, collagen type I or collagen type IV for examined in serum-free conditions, after plating onto fibronectin, 90 minutes, PDGFR-b phosphorylation was induced at lower levels laminin, fibrillin-1 PF8 [an Arg-Gly-Asp (RGD)-containing compared with that on fibronectin, but no PDGFR-a fragment that engages the a5b1-integrin] (Bax et al., 2007; Cain phosphorylation was detected. Fibrillin-1 PF8, and collagen types et al., 2005), collagen type I or collagen type IV (all at 10 g/ml) I and IV, also induced tyrosine phosphorylation of EGFR (array for 90 minutes. A human array for phosphorylated RTKs (Fig. 1A), coordinates B1 and B2). By contrast, MSCs plated onto laminin containing 42 different immobilised anti-RTK antibodies, was for 90 minutes showed no detectable RTK phosphorylation, similar utilised; this allowed the simultaneous relative quantification of to that with MSCs plated onto BSA. The percentage of MSCs tyrosine phosphorylation levels for both PDGFR-a (array adhering to the different ECM substrates after 90 minutes was coordinates C7 and C8) and PDGFR-b (array coordinates C9 and similar (supplementary material Fig. S1A), indicating that the Journal of Cell Science

Fig. 1. ECM-induced tyrosine phosphorylation of PDGFR-b.(A) Human phosphorylated RTK arrays were used to examine ECM-induced RTK phosphorylation levels in MSC lysates. PDGFR-a (R-a; coordinates C7 and C8) and PDGFR-b (R-b; coordinates C9 and C10), respectively, were examined in MSC lysates, taken at 90 minutes, from BSA-coated wells or wells coated with 10g/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-b tyrosine phosphorylation was carried out using lysates from cells plated onto BSA control (Con) substrate or 10g/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-b was isolated from equivalent MSC lysates by IP analysis using anti-PDGFR-b 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-b antibody, as a loading control. Quantitative analysis was evaluated by densitometry with Tyr-P normalised to total PDGFR-b 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-b (C) Y751 and (D) Y1021 PDGFR-bphosphorylation was determined using ELISAs for phosphorylated PDGFR-b, with all conditions normalised to the total amount of PDGFR-b. MSCs were plated onto 10g/ml immobilised ECM proteins in serum- free conditions for 90 minutes at 37°C. PDGFR-b phosphorylation is represented as the fold increase above the control BSA-induced PDGFR-b 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. 1290 Journal of Cell Science 124 (8)

ECM-induced increases in RTK phosphorylation was not due to subunits and dimeric integrin receptors. MSC adhesion to differences in cell attachment. fibronectin was greatly reduced by the function-blocking anti-b1- ECM-induced PDGFR-b tyrosine phosphorylation in serum- integrin antibody (mAb13) and was also substantially inhibited by free conditions was also examined by immunoprecipitation and each of three different function-blocking antibodies against the immunoblotting, normalised to the total amount of PDGFR-b (Fig. integrin subunit a5 (mAb16, JBS5 and P1D6) (supplementary 1B). Fibronectin induced the greatest PDGFR-b tyrosine material Fig. S2B). By contrast, non-inhibitory antibodies against phosphorylation activity, but cells cultured on fibrillin-1 PF8 also the integrin subunits b1 (8E3) and a5 (mAb11) had no effect on exhibited an increase in the level of phosphorylated PDGFR-b. By attachment. MSC adhesion to fibronectin was partially reduced by contrast, MSC adhesion to laminin or collagen types I or IV had integrin function-blocking antibodies against the integrin little effect on PDGFR-b activity compared with that in cells on subunitsa4 (HP2/1) or av (17E6), and the avb3-integrin (LM609 the BSA control substrate. and 23C6). As expected, MSC adhesion to laminin was unaffected To confirm that adhesion to ECM in serum-free conditions by function-blocking antibodies against the integrin subunits a5 or differentially activates PDGFR-b, the phosphorylation status of av, and the avb3-integrin (data not shown); however, MSC specific PDGFR-b tyrosine residues was examined using attachment to laminin was almost completely blocked by the immunoassays. Analysis of PDGFR-b tyrosine phosphorylation inhibitory anti-b1-integrin antibody (mAb13) (supplementary revealed that plating MSCs onto fibronectin, fibrillin-1 PF8 and material Fig. S2C). MSC adhesion to laminin was also substantially collagen types I or IV for 90 minutes all triggered phosphorylation reduced by the inhibitory anti-a1-integrin antibody (FB12) and of Y751 (Fig. 1C) and Y1021 (Fig. 1D) above that in the basal partially reduced by the inhibitory anti-a6-integrin antibody BSA control. MSCs plated onto fibronectin had the greatest level (GoH3), but the non-inhibitory anti-b1-integrin antibody (8E3) of PDGFR-b Y751 and Y1021 phosphorylation. By contrast, had little effect. laminin had no more of an effect on PDGFR-b Y751 and Y1021 To investigate whether a5b1- and avb3-integrin, which phosphorylation than exposure to BSA. Because PDGFR-b supported MSC adhesion to fibronectin, modulated fibronectin- phosphorylation was ECM-dependent, we checked whether the induced PDGFR-b activity, PDGFR-b Y751 and Y1021 abundance of PDGFR-b, at protein and mRNA levels, was altered phosphorylation levels were assayed in the presence of function- following the adhesion of MSCs to ECM ligands for 90 minutes blocking antibodies against the integrin subunits a5 and b1, and in serum-free conditions. Immunoblotting, reverse-transcription- the avb3-integrin, or with the cyclic RGD peptide cilengitide that PCR (RT-PCR) and quantitative real-time PCR (qPCR) revealed inhibits av integrins (Fig. 2A). MSCs plated onto fibronectin for that PDGFR-b expression was not increased by adhesion to the 90 minutes in the presence of function-blocking antibodies against ECM over this timeframe (supplementary material Fig. S1B). RT- the a5 (JBS5) or b1 (mAb13) integrin subunits exhibited a PCR and qPCR assays also revealed only trace levels of endogenous substantial reduction in the level of PDGFR-b Y751 and Y1021 PDGF-BB, PDGF-DD and VEGF-A transcripts, which were not phosphorylation compared with the level of fibronectin-induced upregulated by plating MSCs onto fibronectin for 90 minutes phosphorylation (set at 100%). By contrast, the presence of (supplementary material Fig. S1C). Enzyme-linked immunosorbent inhibitory antibodies against avb3-integrin (23C6), cilengitide, assays (ELISAs) showed that adhesion of MSCs to fibronectin or non-functional anti-a5-integrin antibody (mAb11), or non-

Journal of Cell Science laminin did not induce secretion of PDGF-BB or VEGF-A above inhibitory anti-b1-integrin antibody (8E3), resulted in no that on the BSA control substrate (supplementary material Fig. substantial change in the level of PDGFR-b phosphorylation. As S1D). In addition, treatment of MSCs with a PDGFR-b-neutralising the same concentration of an anti-avb3-integrin antibody (23C6) antibody (AF385), which prevents PDGF-BB from binding and was shown to reduce MSC adhesion to fibronectin (see activating the receptor, failed to block fibronectin-induced Y751 or supplementary material Fig. S2B), failure to inhibit PDGFR-b Y1021 PDGFR-b activation (see Fig. 5A). phosphorylation was not due to the concentration of antibody used. MSCs plated onto laminin for 90 minutes had no detectable a5b1-integrin mediates fibronectin-induced PDGFR-b change in the level of PDGFR-b Y751 or Y1021 phosphorylation tyrosine phosphorylation compared with that of cells on the control BSA substrate, in either Integrins are the principal receptors for ECM ligands, serving as the absence or presence of blocking antibodies against the integrin transmembrane links between extracellular adhesion molecules a5 or b1 subunits or the avb3-integrin (data not shown). Thus, and the intracellular actin cytoskeleton (Askari et al., 2009). As the fibronectin-induced phosphorylation of PDGFR-b in MSCs is integrin expression profile of a cell will dictate its interactions with dependent upon the a5 and b1 integrin subunits, confirming the the ECM, the expression of integrins by cultured MSCs was fibronectin receptor a5b1-integrin, but not avb3-integrin, as the analysed by flow cytometry (supplementary material Fig. S2A). mediator of fibronectin-induced ligand-independent PDGFR-b Integrin subunits av, a3, a5 and b1, and the avb5-integrin, were phosphorylation. all prominently expressed by MSCs, whereas the avb3-integrin, To determine whether a5b1-integrin-dependent fibronectin- and integrin subunits a1, a2, a4, a6 and b4, had a lower level of induced phosphorylation of PDGFR-b is a generalised mechanism, expression. In comparison with the isotype-matched IgG controls, the effect of integrins in modulating fibronectin-induced PDGFR- a9b1-integrin and avb6-integrin were not detectable. b activity was examined in human aortic SMCs (Fig. 2B). In a Having identified the integrins expressed by MSCs, we manner similar to MSCs, SMCs plated onto fibronectin for 90 investigated which of these receptors mediated adhesion to two minutes had a substantial increase in PDGFR-b Y751 and Y1021 contrasting ECM proteins: fibronectin, which induced the greatest phosphorylation compared with cells on BSA control substrate. level of PDGFR-b phosphorylation in MSCs, and laminin, which Furthermore, SMCs on fibronectin in the presence of function- induced no substantial PDGFR-b phosphorylation (see Fig. 1). blocking antibodies against the a5 (mAb16) or b1 (mAb13) integrin Adhesion assays were performed for 90 minutes in the presence of subunits exhibited a substantial reduction in the level of PDGFR- function-blocking monoclonal antibodies (mAbs) against integrin b Y751 and Y1021 phosphorylation compared with the fibronectin- a5b1-integrin controls PDGFR-b 1291

Fig. 2. Fibronectin-induced integrin-mediated PDGFR-b tyrosine phosphorylation. The effects of inhibitory anti-integrin antibodies on fibronectin-induced PDGFR-b (i) Y751 and (ii) Y1021 phosphorylation were determined in (A) MSCs or (B) SMCs, using ELISAs for phosphorylated PDGFR-b, with all conditions Journal of Cell Science normalised to the total amount of PDGFR-b. Cells in serum-free conditions were plated onto 10g/ml immobilised fibronectin for 90 minutes in the presence of 10g/ml anti-integrin inhibitory mAbs or 10M integrin-av-inhibiting peptide cilengitide. Antibodies were specific for the integrin a5 subunit (JBS5, mAb11), the integrin b1 subunit (mAb13, 8E3) and avb3-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-b (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-b 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 a5 and integrin av subunits was examined in MSCs and SMCs plated onto 10g/ml immobilised fibronectin, in serum-free conditions, for 90 minutes at 37°C. (i)Protein expression was detected by immunoblotting (IB) equal amounts (10g) of cell lysates using anti-integrin- a5 or anti-integrin-av antibodies. Membranes were reprobed with anti-b-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 b-actin. Data are represented as the mean pixel density (±s.d.) for two independent experiments (**P<0.01 by one-way ANOVA).

induced phosphorylation (set at 100%). However, in contrast with Fibronectin induces the association of PDGFR-b and MSCs, fibronectin-induced PDGFR-b activity in SMCs was also a5b1-integrin in serum-free conditions reduced in the presence of an inhibitory antibody against the avb3- Crosstalk mechanisms between PDGFR-b and a5b1-integrin in integrin (23C6) integrin. Thus, fibronectin-induced phosphorylation MSCs might involve physical interactions between the integrin of PDGFR-b in SMCs is mediated by a5b1- and avb3-integrin. and PDGFR-b receptors and/or intracellular signalling events As fibronectin-induced PDGFR- phosphorylation was b leading to PDGFR-b activation. Co-immunoprecipitation differentially mediated by integrins in different cell types, the experiments were conducted in serum-free conditions to examine abundance of the integrin subunits a5 and av at the protein level was determined following the adhesion of MSCs or SMCs to receptor associations in MSCs plated onto fibronectin or laminin fibronectin for 90 minutes in serum-free conditions (Fig. 2C). for 90 minutes (Fig. 3A). Co-immunoprecipitation of PDGFR-b Immunoblotting revealed that integrin a5 protein expression was and integrin subunit a5 occurred in lysates derived from MSCs similar in MSCs and SMCs, whereas integrin av was more plated onto fibronectin. By contrast, no association of PDGFR-b abundantly expressed in SMCs compared with its expression in and integrin subunit a5 was detected in lysates derived from MSCs MSCs. plated onto laminin. 1292 Journal of Cell Science 124 (8)

Fig. 3. Fibronectin-induced association of PDGFR-b and a5b1-integrin. (A)The association of PDGFR-b with integrin subunit a5 was examined by immunoprecipitation (IP). MSCs in serum-free conditions were incubated on tissue culture plastic, as a control (Con), or on 10g/ml immobilised fibronectin (Fn) or laminin (Lam), for 90 minutes at 37°C. (i)PDGFR-b was isolated from equivalent MSC lysates by IP using anti-PDGFR-b antibody, with anti-rabbit IgG antibody as a control, then integrin subunit a5 association was detected by immunoblotting (IB) using an antibody against integrin a5. Membranes were re-probed with anti-PDGFR-b as a loading control. (ii)Integrin subunit a5 was isolated by IP using antibodies against integrin a5 or IgG, as a control, then PDGFR-b association was detected by IB analysis using anti-PDGFR-b antibody. Membranes were reprobed with an antibody against integrin a5 as a loading control. A representative example of three independent experiments is shown. (B)Immunoblotting of PDGFR-b Y751 or Y1021 phosphorylation was carried out using MSC lysates, taken at 15, 90 or 240 minutes, from wells coated with 10g/ml fibronectin. (i)Levels of PDGFR-b phosphorylation were detected in equal (10g) amounts of cell lysates using antibodies against phosphorylated PDGFR-b Y751 or Y1021, or anti-PDGFR-b 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-b. Data are means±s.d. for two independent experiments (***P<0.001 compared with other time points).

To determine whether the stimulatory effect of fibronectin on PDGFR-a and EGFR, which were induced in a fibronectin-

Journal of Cell Science PDGFR-b activation was attributable to a physical interaction dependent manner (see Fig. 1A), but the phosphorylation remained between the extracellular regions of PDGFR-b and a5b1-integrin, constant over the time period examined (supplementary material surface plasmon resonance (BIAcore) analysis was performed Fig. S4A). Although the level of PDGFR-a phosphorylation (supplementary material Fig. S3A,B). No direct interaction was was of lower magnitude compared with that of PDGFR-b detected between the extracellular domains of either PDGFR-b phosphorylation under the same conditions, an ELISA for and a5b1-integrin using either immobilised PDGFR-b or phosphorylated PDGFR-a showed that MSCs plated onto immobilised a5b1-integrin, although positive controls PDGF-BB fibronectin for 90 minutes in the presence of function-blocking and fibronectin (50 kDa adhesion fragment) bound PDGFR-b and antibodies against integrin subunits a5 (JBS5), b1 (mAb13) or av a5b1-integrin, respectively. However, both the extracellular (17E6), or cilengitide, exhibited a substantial reduction in the level domains of a5b1-integrin and PDGFR-b (Kd19 nM) strongly of PDGFR-a Y742 phosphorylation compared with the fibronectin- bound immobilised heparin (supplementary material Fig. S3C), induced phosphorylation (set at 100%) (supplementary material and fibronectin-induced co-immunoprecipitation of PDGFR-b and Fig. S4B). Thus, whereas fibronectin–a5b1-integrin engagement integrin subunit a5 was prevented by excess heparin and treatment mediates PDGFR-b activity in MSCs, fibronectin-induced with K5 heparan lyase (supplementary material Fig. S3D). Thus, phosphorylation of PDGFR-a is mediated by a5b1-integrin and cell surface heparan sulphate proteoglycans mediate the association av integrins. between PDGFR-b and a5b1-integrin. To investigate further the relationship between PDGFR-b and a5b1-integrin in MSCs following adhesion to fibronectin, the Fibronectin induces temporal phosphorylation of cellular distribution of phosphorylated PDGFR-b and integrin PDGFR-b and colocalisation with a5b1-integrin subunit a5 was examined by immunofluorescence microscopy. in serum-free conditions PDGFR-b phosphorylated on Y751 and Y1021 was detected at the Maximal fibronectin-induced Y751 and Y1021 PDGFR-b cell periphery following a 15-minute incubation on fibronectin phosphorylation was detected at 90 minutes by immunoblotting, (Fig. 4A,Bi), whereas MSCs had not adhered to laminin at this compared with that upon exposure for 15 or 240 minutes (Fig. 3B). timepoint (data not shown). After MSCs had been plated for 90 Temporal phosphorylation of PDGFR-b was confirmed by minutes on fibronectin (Fig. 4A,Bii), but not laminin phosphorylated RTK array analysis (supplementary material Fig. (supplementary material Fig. S5A), PDGFR-b Y751 and Y1021 S4A). Fibronectin also induced tyrosine phosphorylation of other phosphorylation was detected at the leading edge and in a tidemark RTKs throughout the timecourse of 15, 90 and 240 minutes, notably of integrin-a5-containing focal adhesions behind the leading edge. a5b1-integrin controls PDGFR-b 1293

Fig. 4. Fibronectin-induced temporal colocalisation of phosphorylated PDGFR-b with a5b1-

Journal of Cell Science integrin. Following adhesion to fibronectin, the cellular distribution of phosphorylated PDGFR-b and integrin subunit a5 was examined over time using immunofluorescence microscopy. MSCs in serum-free conditions were plated onto 10g/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-b (red) and integrin subunit a5 (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m.

Fibronectin-induced colocalisation of phosphorylated PDGFR-b Fibronectin and PDGF-BB additively contribute to PDGFR- and integrin subunit a5 at 90 minutes was supported by b tyrosine phosphorylation colocalisation analysis (Fig. 4C). After 4 hours of incubation on Given that MSC adhesion to fibronectin in serum-free conditions fibronectin (Fig. 4A,Biii), PDGFR-b phosphorylated on Y751 and was shown to induce ligand-independent phosphorylation of Y1021 was detected predominantly at the leading edge of MSCs, PDGFR-b and association with a5b1-integrin, we investigated whereas integrin subunit a5 was present towards their trailing the effect of fibronectin on PDGF-BB-stimulated PDGFR-b edge. Thus, in serum-free conditions, fibronectin first induces phosphorylation. PDGF-BB, the major ligand of PDGFR-b, is an PDGFR-b phosphorylation at the leading edge, followed by important mitogen and chemoattractant for mesenchymal cells transient colocalisation of phosphorylated PDGFR-b with a5b1- (Betsholtz et al., 2001). ELISA analysis demonstrated that, integrin in focal adhesions, then dissociation and translocation of compared with ligand-independent fibronectin-induced PDGFR-b a5b1-integrin towards the rear of the cells. activity, exposure to 50 ng/ml PDGF-BB increased the levels of 1294 Journal of Cell Science 124 (8)

Fig. 5. Fibronectin enhances PDGF-BB-induced PDGFR-b tyrosine phosphorylation. (A)The effects of fibronectin (Fn) or laminin (Lam) on PDGF-BB (BB)- stimulated PDGFR-b (i) Y751 and (ii) Y1021 phosphorylation were examined using an ELISA for phosphorylated PDGFR-b with all conditions normalised to the total amount of PDGFR-b. MSCs in serum-free conditions were plated onto 10g/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-b induced by BSA. The involvement of integrin subunit a5 in fibronectin-regulated PDGF-BB stimulation of PDGFR-b (i) Y751 or (ii) Y1021 phosphorylation levels was also determined. MSCs in serum-free conditions, treated with either 10g/ml anti-integrin-a5 inhibitory antibody (JBS5) or anti-PDGFR-b neutralisation antibody (AF385), or both, were plated onto 10g/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-b (i) Y751 or (i) Y1021 phosphorylation (100%) in the absence of PDGF-BB, and are compared against the respective fibronectin-induced phosphorylation of PDGFR-b in the absence or presence of PDGF-BB (***P<0.001 by one-way ANOVA), or in the presence of both JBS5 and AF385 (P<0.001 by one-way ANOVA). Laminin-induced phosphorylation of PDGFR-b in the presence of PDGF-BB is

Journal of Cell Science 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-b (i) Y751 and (ii) Y1021 phosphorylation levels were determined using an ELISA for phosphorylated PDGFR-b with all conditions normalised to the total amount of PDGFR-b. MSCs in serum-free conditions were plated onto 10g/ml immobilised fibrillin-1 (PF8) in the presence of 10g/ml anti-integrin inhibitory mAbs or 50 ng/ml PDGF-BB for 90 minutes at 37°C. Antibodies were specific for the integrin a5 subunit (mAb16, mAb11), integrin b1 subunit (mAb13, 8E3) and avb3-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-b (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-b 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.

Y751 (Fig. 5Ai) and Y1021 (Fig. 5Aii) phosphorylation by 78% PDGFR-b phosphorylation, to near the basal levels induced by and 83%, respectively. The addition of PDGF-AA, which binds BSA, whereas AF385 had no detectable effect. However, when only the PDGFR-a homodimer, was used as a negative control and MSCs plated onto fibronectin were stimulated with PDGF-BB, did not induce PDGFR-b activity above that induced by fibronectin addition of either JBS5 or AF385 resulted in a substantial reduction (data not shown). MSCs cultured on laminin did not induce receptor in PDGFR-b Y751 and Y1021 phosphorylation levels, compared phosphorylation above that with BSA, although PDGF-BB did with the levels in MSCs in the absence of inhibitory antibodies. induce a substantial increase in PDGFR-b activity (Fig. 5A). These Phosphorylation of Y751 and Y1021 was further reduced when results show that fibronectin and PDGF-BB both contribute to MSCs were cultured on fibronectin and stimulated with PDGF-BB PDGFR-b phosphorylation, whereas on laminin PDGFR-b in the presence of both JBS5 and AF385. Taken together, these phosphorylation is only induced in the presence of PDGF-BB. antibody blocking experiments show that fibronectin a5b1-integrin- To confirm that the fibronectin-enhanced PDGF-BB stimulation dependent regulation of PDGFR-b enhances PDGF-BB-stimulated of PDGFR-b Y751 and Y1021 phosphorylation in MSCs was PDGFR-b Y751 and Y1021 phosphorylation. We also found that a5b1-integrin-dependent, the effects of the function-blocking PDGF-BB directly binds a5b1-integrin (supplementary material antibody against integrin subunit a5 (JBS5) and/or a neutralising Fig. S3B), suggesting that receptor co-clusters of a5b1-integrin anti-PDGFR-b (AF385) antibody, which inhibits receptor with PDGFR-b might enhance the interaction of PDGF-BB with phosphorylation, were examined (Fig. 5A). In the absence of the its receptor. However, although PDGFR-b was also shown to be PDGF-BB ligand, only JBS5 substantially reduced the level of activated by the a5b1-integrin-binding fragment (PF8) of fibrillin- a5b1-integrin controls PDGFR-b 1295

Fig. 6. PDGFR-b and a5b1-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g/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 (P<0.001 by one-way ANOVA). (B)The involvement of integrin subunit a5 in PDGF-BB-induced MSC migration towards fibronectin was also determined. MSCs in serum-free conditions in the upper chamber treated with either 10g/ml anti-integrin-a5 inhibitory antibody (JBS5), anti-PDGFR-b neutralisation antibody (AF385), or both, were cultured for 4 hours at 37°C in

Journal of Cell Science Boyden chambers pre-coated on the underside with 10g/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 (P<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m.

1 (see Fig. 1) in an a5b1-integrin-dependent (but not integrin whereas the anti-PDGFR-b antibody had no detectable effect. avb3-dependent) manner (Fig. 5B), in contrast with fibronectin- MSCs exposed to PDGF-BB in the presence of JBS5 or AF385 enhanced PDGF-BB stimulation of PDGFR-b phosphorylation, resulted in a substantial reduction in MSC migration towards PDGF-BB had no further effect on PDGFR-b Y751 and Y1021 fibronectin, compared with the amount of migration in the absence phosphorylation when MSCs were cultured on PF8 (Fig. 5B). of inhibitory antibodies. MSC migration towards fibronectin was further reduced upon exposure to PDGF-BB in the presence of PDGFR-b and a5b1-integrin crosstalk mediates MSC both JBS5 and AF385. By contrast, incubation of MSCs with a migration function-blocking antibody against avb3-integrin (LM609) had no We next investigated the functional importance of the crosstalk detectable effect on fibronectin-induced MSC migration in the between a5b1-integrin and PDGFR-b for MSC migration. In absence or presence of PDGF-BB (supplementary material Fig. serum-free conditions, MSC migration in fibronectin-coated S5B). Thus, a5b1-integrin-dependent regulation of PDGFR-b Boyden chambers was increased by ~2.8-fold above that on BSA- enhances PDGF-BB-stimulated MSC migration towards coated inserts, whereas laminin had no effect above that with the fibronectin. basal BSA control (Fig. 6A). PDGF-BB substantially increased MSC migration towards fibronectin, laminin and BSA above that Fibronectin–a5b1-integrin engagement and FAK activation induced by serum-free conditions (Fig. 6A). To confirm that the mediates PDGFR-b-induced PI3K activity and actin fibronectin-enhanced PDGF-BB stimulation of MSC migration arrangement was a5b1-integrin-dependent, the effects of JBS5 and/or AF385 Having established that fibronectin- and a5b1-integrin-dependent antibody on MSC migration were examined (Fig. 6B). In the regulation of PDGFR-b enhances PDGF-BB-stimulated PDGFR- absence of PDGF-BB ligand, only JBS5 inhibited MSC migration, b phosphorylation and MSC migration, we examined the 1296 Journal of Cell Science 124 (8)

Fig. 7. a5b1-Integrin and FAK activation mediates PDGFR-b-induced PI3K activation. (A)MSCs in serum-free conditions treated with 10g/ml anti- integrin-a5 inhibitory antibody (JBS5) or non-functional (NF) anti-integrin-a5 antibody (mAb11), were plated onto tissue culture plastic, as a control (basal), or 10g/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) PLC-1 were detected in equal (10g) amounts of cell lysates by immunoblotting (IB) using antibodies against (i) phosphorylated FAK (Y397), (ii) phosphorylated Akt (S473) or (iii) phosphorylated PLC-1 (Y783). Membranes were reprobed with anti-b-actin antibody as a loading control. Quantitative analysis was performed by densitometry with (i) phosphorylated FAK, (ii) phosphorylated Akt or (iii) phosphorylated PLC-1 normalised to the level of b-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-b Y751 and Y1021 phosphorylation levels was determined using an ELISA for phosphorylated PDGFR-b, with all conditions normalised to that with total PDGFR-b. MSCs transfected with 3g of siRNA FAK or scrambled siRNA, as a control, were plated onto 10g/ml immobilised fibronectin for 90 minutes at 37°C. Conditions are expressed relative to fibronectin-induced PDGFR-b Y751 or Y1021 phosphorylation (100%) in the

Journal of Cell Science 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g of siRNA FAK or scrambled siRNA, as a control, were plated onto 10g/ml immobilised fibronectin for 90 minutes at 37°C. (i)Phosphorylation levels of Akt were detected in equal (10g) amounts of cell lysates using an antibody against phosphorylated Akt S473 (S473). The membrane was re-probed with anti-b-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 b-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.

involvement of specific intracellular signalling pathways. FAK is was a5b1-integrin-dependent, the effect of JBS5 was examined an important receptor-proximal link between PDGFR and integrin (Fig. 7Ai). In the absence or presence of PDGF-BB ligand, FAK signalling pathways (Sieg et al., 2000). Therefore to determine phosphorylation was markedly reduced by adding JBS5. whether FAK integrates the signalling pathways activated by both Furthermore, partial knockdown of FAK by small interfering RNA fibronectin–a5b1-integrin engagement and PDGFR-b, FAK (siRNA) was sufficient to substantially reduce fibronectin-induced activation at the major autophosphorylation Y397 site was PDGFR-b Y751 and Y1021 phosphorylation (Fig. 7B). Thus, FAK examined by immunoblotting (Fig. 7Ai). In a manner similar to is a mediator of fibronectin- and a5b1-integrin-dependent ECM-induced PDGFR-b phosphorylation, MSCs plated onto regulation of PDGFR-b. fibronectin for 90 minutes induced an increase in FAK Y397 As FAK was shown to mediate fibronectin-induced PDGFR-b phosphorylation, whereas MSCs plated onto laminin or BSA had Y751 and Y1021 phosphorylation, we examined the activity of no detectable FAK Y397 phosphorylation. MSCs stimulated with PI3K and PLC, which are known to associate with these specific PDGF-BB showed markedly increased fibronectin-induced tyrosine residues within the PDGFR-b cytoplasmic domain, phosphorylation of FAK. By contrast, however, MSCs exposed to respectively. PI3K and PLC are important regulators of motogenic PDGF-BB when plated on laminin did not display an increase in signal transduction (Kundra et al., 1994; Wennström et al., 1994), FAK Y397 phosphorylation, suggesting that MSC adhesion to and cells expressing a PDGFR-b mutant lacking the PI3K-binding fibronectin, and engagement of a5b1-integrin, is necessary for site are unable to migrate directionally towards PDGF (Ruusala et PDGF-BB-induced FAK phosphorylation. To confirm that al., 1998). Phosphorylation of Akt at S473, as an indicator of PI3K fibronectin-induced PDGF-BB-stimulated phosphorylation of FAK activity, and phosphorylation of PLC-1 at Y783, was examined by a5b1-integrin controls PDGFR-b 1297 Journal of Cell Science

Fig. 8. PI3K mediates PDGFR-b-induced actin arrangement and migration. (A)The cellular distribution of F-actin and phosphorylated PDGFR-b Y751 was examined by immunofluorescence microscopy. MSCs in serum-free conditions treated with 10g/ml anti-integrin a5 inhibitory antibody (JBS5) or non-functional (NF) anti-a5-integrin antibody (mAb11), as a control, were plated onto 10g/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-b (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-b distribution (1–500 pixels) or PDGFR-b 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-b clustering in the absence of JBS5). Images are representative of at least three independent experiments. Scale bar: 20m. (B)The cellular distribution of F-actin and integrin subunit a5 was examined by immunofluorescence microscopy. MSCs in serum-free conditions treated with 20M LY294002, or DMSO as a control, were plated onto 10g/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 a5 (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m. 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M 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g/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m. (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).

immunoblotting. MSCs plated onto fibronectin for 90 minutes increased fibronectin-induced phosphorylation of both Akt and PLC- induced S473 phosphorylation of Akt (Fig. 7Aii) and Y783 1. Fibronectin-induced S473 Akt phosphorylation in the absence or phosphorylation of PLC-1 (Fig. 7Aiii). By contrast, MSCs plated presence of PDGF-BB, was abolished by adding JBS5 (Fig. 7Aii), onto laminin or BSA, displayed neither Akt nor PLC-1 indicating that a5b1-integrin regulates PDGFR-b-ligand-induced phosphorylation. MSC stimulation with PDGF-BB markedly PI3K activity in MSCs. By contrast, incubation of MSCs with JBS5 1298 Journal of Cell Science 124 (8)

chemical inhibitor LY294002 on MSC F-actin organisation and migration. Inhibition of PI3K activity ablated both fibronectin- and PDGF-BB-induced F-actin membrane ruffles (Fig. 8B), and substantially attenuated MSC migration towards fibronectin in the absence or presence of PDGF-BB (Fig. 8C). In summary, our results show that the adhesion of MSCs to fibronectin through a5b1-integrin both directly stimulates PDGFR- b and potentiates PDGF-BB-stimulated PDGFR-b signalling. In turn, PDGFR-b-mediated Akt-PI3K activation drives the cytoskeletal changes that regulate cell migration.

Discussion The crucial importance of PDGFR-b in directing vascular cell behaviour is well documented, yet ECM-dependent mechanisms that regulate its signalling are not well understood. Using human MSCs, we have shown that adhesion to fibronectin specifically induces PDGFR-b signalling in an a5b1-integrin-dependent manner (Fig. 9). Fibronectin regulates PDGFR-b-dependent actin reorganisation and cell migration in the absence of growth factor ligand and also potentiates PDGF-BB-induced PDGFR-b signalling. Thus, fibronectin-rich matrices are crucial regulators of Fig. 9. MSC migration is regulated by fibronectin through a5b1-integrin- mediated activation of PDGFR-b. Schematic model depicting the synergistic PDGFR-b-mediated mesenchymal cell migration during vascular connection between a5b1-integrin-mediated adhesion to fibronectin and remodelling. PDGFR-b, which controls Akt-PI3K activity and MSC migration. Adhesion to Nascent blood vessels contain many ECM molecules, including fibronectin promotes receptor clustering and association, which is mediated by fibronectin, laminin, collagens and fibrillins. Our study shows that heparan sulphate proteoglycans (HSPG), induces a5b1-integrin- and FAK- PDGFR-b signalling is differentially affected by cell adhesion to dependent PDGFR-b phosphorylation in the absence of growth factor and specific ECM ligands. In particular, adhesion to fibronectin and strongly potentiates PDGF-BB-stimulated PDGFR-b signalling. The fibrillin-1, but not laminin, induces tyrosine phosphorylation of potentiation of PI3K-Akt activity by crosstalk between a5b1-integrin and PDGFR-b in a manner independent of growth factor ligand. The PDGFR-b is an essential event in the cascade that induces actin reorganisation stimulatory effect of fibronectin (a key regulator of cell adhesion, and cell motility. migration and survival during tissue formation, and a regulator of wound repair) is integrin-dependent as blocking a5b1-integrin but did not alter PLC-1 phosphorylation (Fig. 7Aiii). Furthermore, not avb3-integrin prevented PDGFR-b activation. Thus ECM, partial knockdown of FAK by siRNA was sufficient to substantially through specific integrins, directly stimulates PDGFR-b.

Journal of Cell Science reduce fibronectin-induced Akt S473 phosphorylation levels (Fig. PDGFR-b activation induces dynamic changes in the 7C). Thus, FAK is a mediator of fibronectin- and a5b1-integrin- cytoskeleton and membrane ruffles during cell migration dependent regulation of Akt-PI3K activity. (Mellström et al., 1988; Ruusala et al., 2008). We found that, in PDGF-BB induces cell migration in conjunction with marked MSCs on fibronectin, crosstalk with a5b1-integrin regulates reorganisation of actin filaments and the appearance of F-actin PDGFR-b-mediated cell adhesion, spreading and migration. clusters at membrane protrusions (Meima et al., 2009), whereas Adhesion to fibronectin induces a rapid movement of mutant PDGF-stimulated cells expressing the non-phosphorylatable phosphorylated PDGFR-b to the leading edge of MSCs, and a Tyr-to-Phe (Y751F) PDGFR-b lack ruffled edges (Wennström et tidemark of phosphorylated PDGFR-b transiently colocalises with al., 1994). Our finding that a5b1-integrin engagement of fibronectin a5b1-integrin-containing focal adhesions behind the leading edge. regulates PDGFR-b-mediated Akt-PI3K activity in MSCs led us to Thus, PDGFR-b activity is a crucial element of a5b1-integrin- examine the effect of a5b1-integrin inhibition on F-actin mediated adhesion and migration in these cells. Although co- organisation and the localisation of Y751 phosphorylated PDGFR- immunoprecipitations showed that PDGFR-b and integrin subunit b (Fig. 8A). Plating MSCs on fibronectin in serum-free conditions a5 associate in complexes, even in the absence of PDGF, we did induced Y751 phosphorylated PDGFR-b clustering and membrane not detect a direct interaction between the extracellular domains of ruffling; these cellular changes were more pronounced in the PDGFR-b and a5b1-integrin. However, we did find that both presence of PDGF-BB, with intensely stained patches of actin at receptor ectodomains strongly bound heparin, and fibronectin- the rim of the cells. Inhibition of a5b1-integrin using the JBS5 induced co-immunoprecipitation of PDGFR-b and integrin subunit function-blocking antibody substantially reduced the clustering of a5 was prevented by excess heparin and treatment with K5 heparan fibronectin-induced Y751-phosphorylated PDGFR-b and F-actin lyase. Thus, cell surface heparan sulphate proteoglycans mediate in membrane ruffles, both in the absence and presence of PDGF- the association between PDGFR-b and a5b1-integrin. The receptor BB (Fig. 8A). By contrast, when MSCs were plated onto laminin, crosstalk could also involve molecules such as pericellular tissue Y751-phosphorylated PDGFR-b and F-actin clustering were only transglutaminase, which binds to PDGFR-b (Zemskov et al., 2009), induced in the presence of PDGF-BB (supplementary material Fig. or tetraspanins, which form membrane complexes with integrin S5C). receptors and are implicated in integrin-mediated cell migration As a5b1-integrin engagement of fibronectin regulated PDGFR- (Berditchevski and Odintsova, 1999). b-mediated Akt-PI3K activity (Fig. 7Aii) and F-actin organisation Previous studies have indicated that the b1 integrin subunit and (Fig. 8A), we investigated the effect of PI3K inhibition using the aVb3-integrin can influence PDGFR-b activity (Sundberg and a5b1-integrin controls PDGFR-b 1299

Rubin, 1996; Schneller et al., 1997; Woodard et al., 1998; Borges 2005) or a recombinant 50-kDa fibronectin fragment comprising type III repeats 6– et al., 2000; Minami et al., 2007; Amano et al., 2008; Zemskov et 10 (a gift from Martin Humphries, University of Manchester, Manchester, UK). All growth factors and the anti-PDGFR-b neutralising antibody (AF385) were obtained al., 2009). Our study is the first to identify the major fibronectin from R&D Systems. The cyclic RGD-blocking peptide cilengitide (Nisato et al., a5b1-integrin as a key partner in PDGFR-b-mediated mesenchymal 2003) was a gift from Simon L. Goodman, Merck, Garching, Germany, and the cell migration, both in the absence of growth factors and in PI3K inhibitor LY294002 was from Merck. For the antibodies used, see supplementary material Table S1. potentiating PDGF-BB-mediated MSC migration. Integrin- mediated activation of PDGFR-b could occur as a consequence of Phosphorylated RTK array integrin clustering, which is known to alter the spatial distribution A human phosphorylated RTK array kit (R&D Systems) was used to simultaneously detect the relative tyrosine phosphorylation levels of 42 different RTKs, as previously of PDGFR-b within focal adhesions (Burridge et al., 1988; described (Ball et al., 2007). Miyamoto et al., 1996; Boudreau and Jones, 1999) enhancing PDGFR-b dimerisation and activation. This hypothesis is supported Immunoblotting by our finding that 5 1-integrinengagement of fibronectin and Isolated proteins were resolved using pre-cast Tris-acetate (3–8%) or Bis-Tris (4– a b  12%) gels (Invitrogen), transferred onto nitrocellulose, incubated with primary PDGF-BB can induce both clusters of phosphorylated PDGFR-b antibody overnight and then incubated with horseradish peroxidase (HRP)-conjugated and membrane ruffles, and that PDGF-BB can bind a5b1-integrin, secondary antibody for 2 hours. Detection of proteins was performed with enhanced which might further enhance the activity of PDGFR-b. Interactions chemiluminescence (ECL) western blotting reagent, as previously described (Ball et al., 2007). The pixel density of bands was determined with the Gene Tools v3 of phosphorylated PDGFR-b with cytoskeletal proteins have been software. reported (Schneller, 2001), but it is not known whether PDGFR-b can directly bind actin. However, EGFR binds actin (den Hartigh Immunoprecipitation Cell lysates (500 g) were pre-cleared using 10% protein-A–Sepharose, et al., 1992; Tang and Gross, 2003) and localises in signalling immunoprecipitated with primary antibody overnight, and then the immunocomplexes complexes at membrane ruffles (Diakonova et al., 1995), thereby were isolated by incubation with 10% protein-A–Sepharose for 2 hours, as previously enhancing signalling efficiency (Gronowski and Bertics, 1993). described (Ball et al., 2007). We have found that crosstalk between PDGFR- and 5 1- b a b siRNA transfection integrininduce synergistic signalling responses. FAK and Akt-PI3K MSCs (5ϫ105 cells), together with 3 g of small interfering RNA (siRNA), were activity was cooperatively stimulated by fibronectin and PDGF- transfected by electroporation using a human Amaxa Nucleofector kit (Lonza), then BB in an a5b1-integrin-dependent manner, whereas FAK cultured in growth medium overnight. Validated siRNA, functionally tested to provide ≥70% gene knockdown for FAK, was obtained from Qiagen (SI02622130), knockdown reduced fibronectin-induced PDGFR-b and Akt and scrambled siRNA was used as a control (Qiagen). phosphorylation, thereby linking FAK and Akt-PI3K signals. This is in agreement with a previous study that found FAK to be an Phosphorylated PDGFR immunoassays important receptor-proximal link between PDGFR and integrin Phosphorylated PDGFR-b immunoassays (R&D Systems) were used to measure the levels of tyrosine residue (Y751 and Y1021) phosphorylation for PDGFR-b, signalling pathways (Sieg et al., 2000). Inhibition of Akt-PI3K according to the manufacturer’s instructions. Assays were modified to measure inhibited both fibronectin- and PDGF-induced actin reorganisation phosphorylated PDGFR-a (Y742) utilising an antibody against phosphorylated and migration of MSCs. These findings are consistent with previous PDGFR-a (Y742) (R&D Systems). reports that show a PDGFR-b mutant that is unable to bind and Immunofluorescence analysis activate PI3K fails to mediate actin reorganisation and chemotaxis Cells were fixed using 4% paraformaldehyde for 20 minutes, permeabilised with

Journal of Cell Science (Wennström et al., 1994). Moreover, in the absence of PDGFR-b- 0.5% Triton X-100 for 4 minutes, blocked with 2% fish-skin gelatin (Sigma–Aldrich) for 1 hour and then incubated with primary antibody overnight at 4°C. Alexa-Fluor- driven PI3K signalling, epicardial cells adopt an irregular actin 488 or -594-conjugated secondary antibodies (Invitrogen), and Rhodamine-conjugated cytoskeleton, leading to aberrant migration into the myocardium phalloidin (Invitrogen) to stain filamentous actin, were added for 2 hours, then and defective coronary artery formation (Mellgren et al., 2008). coverslips were mounted using Vectashield containing DAPI (Vector Laboratories). ϫ In summary, we have identified a novel synergistic connection Images were collected on a wide-field microscope (Leica DM RXA) using a 60 oil objective and captured using a Coolsnap EZ camera (Photometrics) driven by between a5b1-integrin-mediated adhesion to fibronectin and MetaVue Software (Molecular Devices). For colocalisation analysis, images were PDGFR-b that controls receptor signalling and mesenchymal cell analysed using ImageJ software and the colocalisation analysis plugin. For receptor migration. We envisage that the functional interaction between distribution and clustering analysis, images were analysed using the ‘analyse particles’ function of ImageJ software (Chen et al., 2010). Similar best-fit lower threshold a5b1-integrinand PDGFR-b promotes receptor clustering, which values were determined for each image to reduce signal background, with the upper increases PDGFR-b affinity for its growth factor ligands and threshold always set at 255. Particle sizes for colocalisation were set at a minimum enhances PDGFR-b activity. The physical proximity of a5b1- of 1 pixel and maximum of 500 pixels, and colocalisation is represented by a yellow integrinand PDGFR- might facilitate the recruitment of numerous image. Particle sizes for receptor distribution and clustering were set at a minimum  b of 1 or 10 pixels, respectively, and a maximum of 500 pixels, and are represented shared signalling and adapter proteins; the potentiation of FAK and by a black and white image. Akt-PI3K activity by crosstalk between a5b1-integrinand PDGFR- b is an essential event in the cascade that induces cell motility. Cell migration assay Modified Boyden chamber assays were conducted using filter inserts of 8-m pore Thus, ECM controls mesenchymal cells through crosstalk with the size and 6.5-mm diameter (BD Biosciences), as previously described (Ball et al., potent vascular receptor PDGFR-b. 2007). Migratory cells were imaged by phase-contrast microscopy (Leica DM RXA brightfield) and captured using a Coolsnap EZ camera driven by MetaVue software.

Materials and Methods Statistical analysis Cells and reagents In all quantification experiments, results are expressed as means±s.d. Statistical Human MSCs derived from the normal bone marrow of five different individuals differences between two sets of data were determined using an unpaired Student’s t- (CD44, CD73 and CD105 positive and CD11b, CD14, CD34 and CD45 negative; test, and differences between more than two data sets were determined by one-way from 28- and 34-year-old females and 19-, 25- and 33-year-old males; Lonza) were ANOVA using the Tukey comparison test. P<0.05 was considered statistically tested for their ability to differentiate into osteogenic, adipogenic and chondrogenic significant. All statistical calculations were performed using the GraphPad Prism 5 lineages. MSCs were maintained as previously described (Ball et al., 2007). Human software. aortic SMCs (32-year-old female; Lonza), were maintained in SMC growth medium (Invitrogen). Cells were cultured on human plasma fibronectin (Chemicon), murine laminin (a1b11), bovine collagen type I, murine collagen type IV (BD Biosciences), This study was funded by the Medical Research Council (UK). recombinant fibrillin-1 protein fragment (PF8) (prepared in-house) (Cain et al., We thank Stuart Cain for assistance with the BIAcore experiments, 1300 Journal of Cell Science 124 (8)

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