Cellular Signalling 25 (2013) 848–859

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Cellular Signalling

j ourna l homepage: www.e lsev ie r .com/ locate /ce l l s ig

The dioxin receptor controls β1 integrin activation in fibroblaststhrough a Cbp–Csk–Src pathway

Javier Rey-Barroso a, Georgina P. Colo b, Alberto Alvarez-Barrientos c, Javier Redondo-Muñoz d,1,José M. Carvajal-González e, Sonia Mulero-Navarro f, Angeles García-Pardo b,Joaquín Teixidó b, Pedro M. Fernandez-Salguero a,⁎a Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spainb Departamento de Medicina Celular y Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spainc Servicio de Técnicas Aplicadas a las Biociencias, Universidad de Extremadura, 06071 Badajoz, Spaind Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, CSIC, 28049 Madrid, Spaine Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, 10029 NY, USAf Child Health and Development Institute and Center for Molecular Cardiology, Mount Sinai School of Medicine, 10029 NY, USA

Abbreviations: AMD, accumulated mean distance;Caveolin 1; Cbp/Pag1 (Cbp), C-terminal Src kinase (Cskcellular matrix; EMD, Euclidean mean distance; FAK, fomortalized mouse fibroblasts; siRNA, small interfering R⁎ Corresponding author. Tel.: +34 924289422; fax: +

E-mail address: pmfersal@unex.es (P.M. Fernandez-1 Present address.

0898-6568/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.cellsig.2013.01.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 October 2012Received in revised form 7 January 2013Accepted 9 January 2013Available online 16 January 2013

Keywords:Cbp/Pag1/Src pathwayAhR and β1 integrin activationDirectional cell migration

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Fol-lowing our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathwaycontrols β1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR−/− fibroblasts displayedhigher integrinβ1 activation, revealedby the increasedbinding of the activation reporter 9EG7 anti-β1mAb and ofa soluble fibronectin fragment, as well as by enhanced talin-β1 association. AhR−/− fibroblasts also showed in-creased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression inAhR−/− fibroblasts reduced β1 integrin activation, improved cell migration and rescued wild-type cell morphol-ogy. Cbp over-expression in T-FGMAhR−/− cells enhanced the formation of inhibitory Csk–Cbp complexeswhichin turn reduced c-Src p-Tyr416 activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsiveresidues p-Tyr576 and p-Tyr577. The c-Src target andmigration-related protein Cav1was also hypophosphorylatedat p-Tyr14 in AhR−/− cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fi-broblast migration by modulating β1 integrin activation via Cbp-dependent, Src-mediated signaling.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

The aryl hydrocarbon receptor (AhR) is a basic–helix–loop–helix(bHLH) transcriptional regulator [1]. Despite its role in xenobiotictoxicity [1,2], an increasing number of studies reveal important functionsfor AhR in normal cell functioning and tissue homeostasis [3]. Amongthem, its implication in cell plasticity, adhesion and migration appearsparticularly relevant [4–8]. However, regardless of these experimentalevidences, the mechanisms that integrate AhR in signaling pathwaysgoverning cell adhesion and migration remain mostly unknown.

The effects of AhR on cell migration appear to be dependent on thecell phenotype. While the lack of AhR expression increased epithelialcell migration and acceleratedwound healing in vivo [9], primary endo-thelial cells fromAhR−/−mice had lowermigration rates and impaired

AhR, dioxin receptor; Cav1,)-binding protein; ECM, extra-cal adhesion kinase; FGM, im-NA.34 924289419.

Salguero).

rights reserved.

branching in 3-D cultures [10]. Moreover, embryonic and immortalizedfibroblasts (T-FGM) from AhR−/− mice displayed a spread morpholo-gy and had increased content of focal adhesions (FA) and F-actin stressfibers that led to reduced migration rates, as compared with wild typecells [8,11,12]. A mechanism was proposed by which AhR controls celladhesion and migration through the regulation of the proto-oncogeneVav3 [11]. Several aspects of the adhesive and migratory phenotype ofAhR-null fibroblasts are relevant. First, as compared to wild type cells,AhR−/− fibroblasts had enhanced adhesion on fibronectin, suggestingthe involvement of integrins in the adhesion phenotype. Second, theincreased number of FAs in AhR−/− fibroblasts could result from re-duced FA kinase (FAK) activation, as seen in Fak−/− fibroblasts [13].

Integrins are αβ heterodimeric transmembrane proteins that medi-ate cell adhesion and that activate different signaling pathways follow-ing interaction with their ligands [14,15]. Integrins need to be activatedin order to mediate cell adhesion. This activation takes place followingbinding of talin and kindlins to the cytoplasmic domain of the integrinβ subunits, and involves tension-dependent mechanisms [16,17].Talin binding leads to the separation of the α and β domains and toan extended conformation of the integrin extracellular domain from abent structure [18]. Therefore, the integrin β cytoplasmic domains are

849J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

the recipient of intracellular signaling originated from either inside oroutside cell stimuli, which finally promote integrin activation. Theseprocesses are referred to as inside-out or outside-in activation, respec-tively [16,17].

AT-FGM AhR+/+

Migration distance, μm -400 -200 4002000

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Fig. 1. AhR modulates directional migration of T-FGM fibroblasts and β1 integrin activationsi-AhR were seeded in μ-slide chemotaxis chambers coated with fibronectin. Time-lapse micell migration was recorded towards a FBS chemoattractant stimulus added to the upper chμm (X and Y axis) considering a common starting point in the center of each graph. (B) Thpersistence in migration (EMD/AMD ratio) were measured and quantified using the ImageJcells. (C) The orientation of the F-actin stress fibers was quantified in wounded AhR+/+ andplugin in the ImageJ software. Pseudocolors were used to discriminate the relative orientatiodirection of migration. Quantified data are shown in the right panels. Data are shown as m

The phosphorylation of the Tyr residue in the membrane-proximalNPXYmotif of theβ integrin tails has beenproposed to regulate integrinactivation [19,20]. Thus, the phosphorylation of this Tyr could play anoff signal for integrin activation by interfering with the association of

distance, μm 4002000

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. (A) T-FGM AhR+/+, T-FGM AhR−/− or T-FGM AhR+/+ fibroblasts transfected withcroscopy was used to determine the directional migration of each cell line. Directionalamber (top of the Y axis). Migration of individual cells was tracked and represented ine accumulated median distance (AMD), the Euclidean median distance (EMD) and thechemotaxis plug-in from 40 AhR+/+, 34 AhR−/− and 31 AhR+/+ si-AhR transfectedAhR−/− fibroblast cultures stained with rhodamine–phalloidin using the Orientation Jn of the fibers parallel (0°, blue colors) or perpendicular (−90°/+90°, red colors) to theean±SE.

850 J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

β integrin tails with talin, reducing the affinity of the interaction. In ad-dition, this Tyr phosphorylation promotes binding of β tails to proteinsthat either do not activate integrins or that compete for talin binding(DOK1 and ICAP-1, respectively), finally resulting in the lack of integrinactivation [21]. The phosphorylation of Tyr at the NPXYmotif ismediat-ed by Src family kinases [22]. Src proteins are non-receptor tyrosine ki-nases that signal to control cell proliferation, cytoskeleton organizationand cell adhesion and migration [14]. Src kinases share conserved con-secutive domainmodules that include SH3 and SH2 and tyrosine kinase(SH1) domains. At their C-terminus, the Src kinases have a negative reg-ulatory element which includes a tyrosine residue (Tyr527 in mousec-Src), whose phosphorylation by the Csk kinase leads to the inhibitionof the Src kinase activity [23,24]. Csk-dependent phosphorylation in-duces the assembly of the SH2, SH3 and kinase domains into a closedautoinhibited conformation that is kept by interactions between thesedomains and which causes inhibition of Src catalytic activity [24]. Theactive form of Src kinases is achieved by autophosphorylation of a tyro-sine residue located in the kinase domain (Tyr416 in mouse c-Src) [25].Active Src localizes to focal adhesions, where it can phosphorylate FAKand paxillin, whereas the inactive form shows a preferential perinuclearand endosomal localization [26]. The cytosolic Csk protein is recruitedto the plasma membrane by several Csk-binding proteins, includingpaxillin, FAK, Cav1, an additional Src substrate, and by the scaffoldtransmembrane protein Cbp/Pag1 (C-terminal Src kinase-binding pro-tein, hereafter Cbp) [27]. A subset of integrins can associate with Cav1to serve as a link to Src kinases and to integrin-dependent control ofcell proliferation and survival [28–30]. Previous studies have shownthe existence of a functional link between AhR and c-Src. Thus c-Srccould be part of the cytosolic AhR complex that becomes released bythe AhR ligand TCDD, as seen in hepatocyte cell extracts, NIH-3 T3

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β-Actin

β-Actin β-Actin

Fig. 2. Modulation of AhR and Cbp/Pag1 expression and activity in T-FGM AhR+/+ and AhRsequences and AhR expression determined by immunoblotting. (B) AhR−/− cells were tra(CA) lacking the PAS-B domain and AhR protein expression determined. (C) Transcriptionalthe Cyp1a1 target gene by real-time RT-PCR. (D) AhR−/− fibroblasts were also transfected wimmunoblotting. β-Actin was used to normalize protein levels. (E) Cbp knock-down did noare shown as mean±SE from three independent cultures.

fibroblasts and human breast MCF-10A cells [31–33]. Therefore, amechanism by which AhR modulates cell adhesion and migrationmight involve c-Src-dependent signaling to integrins.

In this work, we have addressed the potential functional connec-tions between AhR and β1 integrin in the control of fibroblast adhe-sion and migration. We propose a mechanism by which lack of AhRexpression deregulates the Cbp–Csk–Src pathway ultimately leadingto increased integrin activation, which might represent the basis forenhanced adhesion and reduced cell migration.

2. Materials and methods

2.1. Cell culture

Immortalized T-FGM AhR+/+ and T-FGM AhR−/− mouse fibro-blasts [12] were grown in DMEM/F12 medium containing 10% FBS,2 mM L-glutamine, 50 μg/ml gentamycin and 11 mM D-glucose.

2.2. Antibodies, reagents and expression vectors

The Anti-AhR antibody was from Biomol (Plymouth, PA, USA).Polyclonal antibodies for β-actin and talin were from Sigma-Aldrich(St. Louis, MO, USA); anti-β1 integrin (activated form, 9EG7), anti-Cav1, anti-Cav1 p-Tyr14 and anti-Gapdh were from Becton-Dickinson(Franklin Lakes, NJ, USA); protein Sepharose, anti-c-Src and anti-Csk an-tibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); β1integrin clone MB1.2, FITC-labeled β1 integrin and Cbp antibodies werefrom Biolegend (San Diego, CA, USA), Millipore (Billerica, MA, USA)and ExBio (Vestec, Czech Republic), respectively; anti-fibronectin wasfrom Chemicon International (Temecula, CA, USA); antibodies for total

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−/− fibroblasts. (A) AhR+/+ fibroblasts were transfected with a sh-AhR or scramblensfected with a wild type form of the receptor (wt) or with a constitutively active AhRactivity of the CA-AhR formwas confirmed by quantifying the constitutive expression ofith a si-Cbp/Pag1 or scramble sequences (si-scr) and Cbp/Pag1 expression analyzed by

t significantly affect β1 integrin protein levels in AhR+/+ or AhR−/− fibroblasts. Data

851J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

FAK and for the detection of Tyr397, Tyr576 and Tyr577 phosphorylationswere from Transduction Laboratories (Lexington, KY, USA); antibodiesto detect c-Src p-Tyr416 and p-Tyr527 were from Cell Signaling (Danvers,MA, USA); TaqDNApolymerasewas fromEcogen (Barcelona, Spain). Su-perScript II reverse transcriptase and SYBR-Greenmaster mix were fromBio-Rad (Hercules, CA, USA). Rhodamine-labeled phalloidin was fromInvitrogen. Small interfering RNAs (siRNA) and scramble sequenceswere from Dharmacon (Lafayette, CO, USA) (AhR) and Sigma (St. Louis,MO, USA) (Cbp and Xap2). The expression construct for the constitutive-ly active AhR (CA-AhR) was a gift from Dr. Fujii-Kuriyama (University ofTsukuba, Japan) and it was produced from thewild type AhR by deletingthe minimal PAS-B motif [34].

2.3. Measurement of β1 integrin activation and binding to solublefibronectin

Τo quantify β1 integrin activation, T-FGM fibroblasts were grown to80% confluence and detached at 4 °C in PBS-2 mMEDTA. After centrifu-gation, cells were resuspended and incubated for 10 min at 4 °C inPBS-1% BSA to block unspecific antibody binding. Subsequently, cellswere incubated with the 9EG7 antibody for 30 min at 4 °C with gentlerotation followed by exposure to Alexa 488 secondary antibody andanalysis in a Cytomics FC-500 flow cytometer (Beckman-Coulter).Total β1 integrin expression was determined in parallel but using theFITC-labeled anti-β1 integrin antibody. Datawere corrected by the fluo-rescences obtained in the absence of primary antibodies. To achieve fullβ1-integrin activation, experiments were performed in the presence of1 mM MnCl2 for 30 min. To determine binding to soluble fibronectin,cells were washed in PBS-2 mM EDTA and resuspended in HBSS-2%fetal bovine serum (FBS) in the presence or absence of 1 mM MnCl2.Then, a solution of 10 μg/ml soluble fibronectin-80 fragment was

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Fig. 3. β1 integrin activation in AhR+/+ and AhR−/− fibroblasts. (A) Cells were detache(β1-FITC) (left) or its activated form (9EG7) (center) in the presence and absence of Mnwith the empty vector and their β1 integrin activation measured in the presence and absenceby the fluorescence values obtained in the absence of primary antibodies. (B) Cell extractslevels by immunoblotting (left), or immunoprecipitated with an anti-β1 integrin antibodyunder gel represent densitometer analyses in arbitrary units. (C) Cells were detached andthe presence or absence of MnCl2. Cell binding was measured by flow cytometry using an ancultures. Data are shown as mean±SE.

added and the mixture incubated for 5 min at 37 °C. Binding wasquenched by incubation for 30 min at 4 °C with 10 μg/ml ofanti-fibronectin-80 antibody. A secondary Alexa-647-conjugated anti-body was added to the suspension and cells were analyzed in aCytomics FC 500 flow cytometer (Beckman-Coulter). PI was used tolabel and discard death cells. As controls, cell stained only with second-ary antibodies were used. Typically, 10.000 events were acquired persample.

2.4. Preparation of extracellular matrices

Culture plateswere treatedwith 1% gelatin for 1 h at 37 °C,washed inPBS and sequentially incubated at room temperature for 20 minwith 1%glutaraldehyde and 1 M glycine. Plates were washed in PBS and DMEMmediumand1×106AhR+/+ orAhR−/− T-FGMfibroblastswere platedand cultured overnight. To improve matrix production and stabilization,cultures were maintained in the presence of 50 μg/ml ascorbic acid.Complete medium was changed every other day for three days. Cellswere removed without affecting the synthesized matrix by gentleincubation in PBS containing 20 mM NH4OH, 0.5% Triton X-100 at37 °C; cultures were checked every 3–5 min for complete removal.Plates were then carefully washed and kept in PBS overnight at 4 °C.Next, T-FGM cells were plated on top of the produced matrices andanalyzed in different assays. The presence of fibronectin in the matriceswas determined by immunofluorescence. Briefly, matrices were fixedin paraformaldehyde for 30 min at room temperature, washed and se-quentially incubated with the anti-fibronectin and the Alexa-488-labeled secondary antibodies. Following a final wash, the fibronectinmatrix was observed in a FluoView FV1000 confocal microscope andquantified by measuring the fluorescence/cell ratio using the ImageJsoftware.

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d from the culture plates and incubated with an antibody to detect total β1 integrinCl2. AhR−/− cells were transiently transfected with an AhR expression construct orof MnCl2 (right). Cells (104) were analyzed by flow cytometry and data were corrected

from T-FGM AhR+/+ and AhR−/− fibroblasts were analyzed for talin and β1 integrinand analyzed for talin co-immunoprecipitation by Western blotting (right). Numbersincubated for 5 min with a solution containing 10 μg/ml of soluble fibronectin-80 inti-fibronectin-80 antibody. Determinations were done in duplicate in two independent

852 J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

2.5. Time-lapse microscopy for directional migration and orientation ofF-actin stress fibers

Time-lapse microscopy was performed as described [35] using che-motaxis chambers (Ibidi, Munchen, Germany) coatedwith 0.5 μg/ml fi-bronectin in DMEM-F12 medium. AhR+/+, AhR−/− or si-AhR+/+T-FGM fibroblastswere seeded into the slide channel and allowed to at-tach. After attachment, reservoirs were filled with culture medium andFBS was added as chemoattractant to the upper reservoir. Afterwards,the chambers were mounted on a live cell Leica AF6000 LX typeDMI6000B imaging microscope. Cell movement was recorded undercontrolled temperature and CO2 using a bright field objective (5 mineach frame for 16 h). Tracks from 30 to 40 cells weremanually analyzedusing the ImageJ software and cell directionality measured with theChemotaxis andMigration Tool (Ibidi and ImageJ softwares). Direction-al migration was quantified as the Euclidean mean distance (EMD) andthe accumulated mean distance (AMD). The orientation of the F-actinstress fibers was quantified essentially as described [36]. In brief,wound were performed in confluent AhR+/+ and AhR−/− fibroblastcultures and after 8 h, cells were fixed in 3.5% paraformaldehyde at4°°C. F-actin fibers were then stained with rhodamine–phalloidin andobserved by confocal microscopy as described [11]. The orientation ofthe F-actin fibers was quantified with the ImageJ software using the

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Fig. 4. T-FGM AhR−/− fibroblasts over-express fibronectin 1. (A) Fibronectin 1 mRNA expreby Gapdh. In some experiments, AhR+/+ and AhR−/− cells were transiently transfected(empty vector as control), respectively (left). Fibronectin protein expression was also de(right). (B) Fibronectin 1 matrices deposited by T-FGM AhR+/+ and AhR−/− fibroblasts wnectin synthesis was quantified as fluorescence intensity per area. (C) T-FGM AhR+/+ and Aof the opposite phenotype after 5 days culturing in the presence of ascorbic acid. Cells weretification was performed by calculating the fluorescence per area ratio using the ImageJ socultures of each genotype.

Orientation J plugin and the Distribution application. Fibers parallel tothe direction of migration (0°) were assigned blue colors while thoseperpendicular to the migration (+90°/−90°) received red colors.

2.6. Immunoprecipitation, c-Src activity and Western blotting

• Immunoprecipitation from T-FGM AhR+/+ and AhR−/− cultureswas performed essentially as described [37]. Briefly, cells were lysedand 1 mg total protein was incubated with 2 μg of the correspondingspecific antibody and protein-A Sepharose overnight at 4°°C. Immunecomplexes were collected by centrifugation, washed 3–5 times andused for immunoblotting or enzyme activity assays. To quantify c-Srcactivity, cell extracts were immunoprecipitated with a specific c-Srcantibody as described above. Freshly prepared immunoprecipitateswere incubated for 20 min at 30 °C with a peptide containing thec-Src target sequence (cell signaling) in the presence of [γ32P]-ATP.Following precipitationwith trichloroacetic acid, sampleswere spottedon P81 phosphocellulose paper, which was washed 5 times in 0.75%phosphoric acid. After a final wash in acetic acid, the radioactivityremaining in the paper was measured in a Beckman LS 3801 scintilla-tion counter. SDS-PAGE and Western blotting were performed as de-scribed [11,12].

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ssion was quantified in T-FGM AhR+/+ and AhR−/− cells by RT-qPCR and normalizedwith an AhR siRNA (scramble siRNA as control, si-scr) or an AhR expression constructtermined in both cell types by immunoblotting. β-Actin was used as loading controlere observed by fluorescence microscopy using an anti-fibronectin-80 antibody. Fibro-hR−/− fibroblasts were seeded on their ownmatrix or on the matrix produced by cellsdetached and their β1-integrin activation (9EG7) measured by flow cytometry. Quan-ftware. Data are shown as mean±SE. The experiment was repeated in three different

AhR+/+ AhR-/-

No cells AhR+/+

Fibronectin matrix

Cells spreading on fibronectin matrices

A

B

Fig. 5. Production of T-FGM AhR+/+ and AhR−/− derivedmatrices for exchange assays.(A) Cells were grown for matrix formation and cultures treated with Triton X100. Aftercell digestion, thefibronectin synthesized and deposited on the plate surfacewas revealedby immunofluorescence. A negative control plate was used in the absence of cells.(B) Cells of both genotypes were re-seeded in their own matrix for 2 h to confirm attach-ment and spreading. Cells were stained with crystal violet for visualization. Assays weredone in three cultures of each genotype.

853J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

2.7. Measurement of cell area

Cell area and the minor/major axis ratio were measured by blindedanalysis in different random fields for each cell culture using the ImageJsoftware [11].

2.8. Transient transfections and RNA interference

The AhR-EYFP fusion vector was cloned essentially as indicated [38].T-FGM cells were transiently transfected with 1 μg wild type or consti-tutively active (CA) AhR using LipofectAMINE Reagent (Life Technolo-gies, Grand Island, NY, USA) or nucleofection (MicroPorator MP-100,Digital-Bio) as described [39]. RNA interference for AhR or Cbp wasperformed by transient transfection of specific small interfering RNAs(siRNAs). Concentrations of 20 nM or 100 nM of siRNAs for AhR orCbp, or unspecific scramble RNAs were used.

2.9. Reverse transcription and real-time PCR

Total RNA was isolated from T-FGM fibroblasts using the RNeasykit (Qiagen, Valencia, CA, USA). Reverse transcription was performedusing random hexamers and Super Script II transcriptase as indicated[39]. For real-time the following primers were used: Cbp 5′-TACTGAGCAGTGGGCAGATG-3′ (forward) and 5′-GGAAGGCACATTCATCAGT-3′ (re-verse). For fibronectin 5′-AAGCCGGGGTTTTAACTGCGAG-3′ (forward)and 5′-CCCCGTTTGAATTGCCACCATAAG-3′ (reverse). For Cyp1a1 5′-ACAGACAGCCTCATTGAGCA-3′ (forward) and 5′-GGCTCCACGAGATAGCAGTT-3′ (reverse). For Xap2 5′ AAGCTGGTGGCTCAGGAGTA 3′(forward) and 5′ GCAGGGTCTAGCTCCAACAC 3′ (reverse). For Gapdh5′-TGAAGCAGGCATCTGA. GGG-3′ (forward) and 5′-CGAAGGTGCAAGACTCGGAG-3′ (reverse). SYBR-Green I/QTaq DNA polymerase mix wasused on the iCycler equipment (Bio-Rad), as described [11,39].

2.10. Chromatin immunoprecipitation (ChIP)

ChIP to analyze AhR binding to the Cbp promoter was performed es-sentially as described [11,40,41] using T-FGM AhR+/+ fibroblasts andan AhR specific antibody. The putative XRE sequence for AhR bindingidentified by ChIP-on-chip experiments on the Cbp gene promoter indioxin-treated mouse liver was analyzed [42]. PCR amplification wasperformedusing the oligonucleotides: 5′-CCAAGAGAATGTGCTGGTTG-3′(forward) and 5′-CATAGTGGATACGCCTTGGA-3′ (reverse). Positive con-trols were performed using input DNAs whereas negative controls in-cluded binding reactions in the absence of AhR antibody.

2.11. Statistical analyses

Statistical comparison between experimental conditions was doneusing GraphPad Prism 4.0 software (GraphPad). The student's t testwas applied. For the analyses of cell area and minor/major axis ratio,the Mann–Whitney non-parametric median statistical analysis wasemployed.

3. Results

3.1. AhR-null fibroblasts have defective directional migration andincreased β1-integrin activation

Previous studies from our laboratory using embryonic and immortal-ized fibroblasts have shown that AhR is needed to maintain cell migra-tion and adhesion in the absence of xenobiotics [11,12]. However,whether the reduced migration of AhR−/− fibroblasts involves defec-tive directional motility was not studied. To address this possibility, wetracked the migratory pattern of AhR+/+ and AhR−/− cells onfibronectin-coated surfaces, using in vivo time-lapse microscopy(Fig. 1A). While AhR+/+ fibroblasts exhibited a sustained migratory

pattern in response to serum (left panel), AhR−/− cells had reducedmi-gratory directionality (central panel) that was statistically significant asdetermined by quantifying both the accumulated mean distance(AMD) and the Euclidean mean distance (regular linear distance, EMD)(Fig. 1B). Moreover, down-modulation of AhR levels by small interferingRNAs (AhR+/+ si-AhR) (Fig. 2A) significantly impaired directed migra-tion of AhR+/+ cells (right panel) reducing AMD and EMD values closeto those present in AhR-null cells (Fig. 1B). The persistence in directionalmigration,measured as the EMD/AMD ratio, was also significantly small-er in AhR−/− and si-AhR AhR+/+ cells than in wild type AhR express-ing fibroblasts (Fig. 1B, right panel). The impaired directional migrationof AhR−/− fibroblasts was associated to an altered orientation of theirF-actin stress fibers (Fig. 1C). Whereas wild type cells oriented theirF-actin fibers mainly parallel to the direction of migration in woundhealing assays (peaks between −40 and +40°, upper right panel),AhR−/− fibroblasts had a predominant perpendicular disposition ofF-actin fibers (peaks at −90/+90°, lower right panel).

The defectivemigration of AhR−/− cells might arise from increasedcell adhesion due to functional alterations of the cell adhesion machin-ery, including changes in β1 integrin activation. Previous results indi-cated that T-FGM AhR−/− fibroblasts have increased adhesion tofibronectin [11]. Cell adhesion to fibronectin is supported by active β1integrin conformations, which can be detected by the binding of theactivation-reporter monoclonal antibody (mAb) 9EG7 [43]. AlthoughAhR−/− andAhR+/+ cells had constitutive similar levels ofβ1 expres-sion (Fig. 3A, left),β1 integrins on AhR−/− fibroblasts displayed higherdegree of activation, as detected by the binding of 9EG7 in flow cytom-etry assays (Fig. 3A center). Higher activation ofβ1 integrinswas indeedlinked to the absence of AhR, as transient transfection of AhR−/− cells

854 J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

with an AhR construct [11] significantly reduced the levels of β1integrin activation, compared to control cells transfected with theempty vector (Fig. 3A, right panel). Control experiments showed thatAhR+/+ and AhR−/− cells retained similar levels of 9EG7 bindingupon exposure toMnCl2, a positive activator of integrin affinity, indicat-ing that β1 integrins in both cell types were able to respond to activa-tion signals from the extracellular milieu.

Integrin activation correlates with talin interaction with the cyto-plasmic domains of the β integrin subunits [44,45]. Immunoblottinganalyses indicated that talin levels were not significantly affectedby AhR knock-out (Fig. 3B left panel). Interestingly, more talinco-immunoprecipitated with β1 in AhR−/− fibroblasts than in theirwild type counterparts (Fig. 3B, right panel). In addition, transfectionof a si-AhR in T-FGM AhR+/+ fibroblasts enhanced the amount oftalin immunoprecipitated with β1 integrin (data not shown). Further-more, increased β1 integrin activation and talin-β1 association inAhR−/− cells correlated with enhanced cell binding to solublefibronectin-80, an α5β1 integrin-binding fibronectin fragment harbor-ing the RGD sequence [46], as compared to AhR+/+ cells (Fig. 3C).These results suggest that higher β1 integrin activation might be amechanism involved in the increased adhesion/reduced migration ofAhR−/− fibroblasts.

A possible factor contributing to β1 integrin activation in AhR−/−cells could be the extracellular matrix (ECM) produced by these cells. In-deed, exposure to ECM proteins upregulates the affinity and avidity ofintegrins [47]. Notably, T-FGM AhR−/− cells expressed higher levels offibronectin mRNA than AhR+/+ cells (Fig. 4A, left). Moreover, transient

p

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age

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rc a

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T

IP:

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FAK

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Y576/FAK 1.0 0.2

Y577/FAK 1.0 0.3

p-SrcY416

c-Src

AhR+/+ AhR-/-T-FGM

Y416/Src 1.0 0.3

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Ah

Cav1Tyr14/Cav1 1

p-FAKY397

Y397/FAK 1.0 1.3

0

25

50

75

100

A B

D

C

Fig. 6. AhR−/− T-FGM cells have alterations in FAK phosphorylation and impaired c-Src acblotting using total cell extracts. Numbers under gel represent densitometer analyses inimmunoprecipitated with anti-Cav1 antibody and the levels of Cav1 Tyr14 phosphorylationof associated Cav1 and p-Cav1 Tyr14 determined by Western blotting (right). (C) c-Src activactivation residue Tyr416. (D) c-Src activity was also measured by an enzymatic assay u[32P]-γATP. (E) Phosphorylation of c-Src at the inhibitory residue Tyr527 was analyzed by iare shown as mean±SE.

transfection of AhR+/+ cells with si-RNA for AhR resulted in increasedfibronectin expression, while an AhR expression construct transfectedin AhR−/− cells reduced fibronectin mRNA to levels similar to those ofAhR+/+ cells. Consistently, western blotting experiments showedhigher fibronectin protein expression in AhR−/− than in AhR+/+cells (Fig. 4A, right), while immunofluorescence analyses of their matri-ces revealed a denser and apparently more complex fibronectin matrixin AhR−/− than in AhR+/+ cells (Fig. 4B). To determine if the ECMproduced by AhR-null cells could contribute to the increased β1 integrinactivation, matrix exchange experiments were done. These assays indi-cated that AhR+/+ fibroblasts cultured over an ECM generated byAhR−/− cells significantly increased their β1 integrin activation levels(Fig. 4C). AhR−/− fibroblasts grown on wild type ECM, on the contrary,did not display alterations in their basal β1 integrin activation, perhapsindicating that these conditions are not sufficient to reverse the β1 acti-vation state. Control experiments indicated that cells were efficiently re-moved from the ECM, and that the matrices generated were functionaland promoted cell attachment (Fig. 5). These results suggested that in-creased fibronectin production by AhR-null cells contributes to the acti-vation of their β1 integrins, either by direct or indirect mechanisms.

3.2. Increased β1 integrin activation in AhR-null cells involvesAhR-dependent reduction in Src activation

Activated integrins are able to bind their ligands and elicit signalingpathways, that have been collectively termed outside-in signaling [47].One of the earliest events in this signaling is the autophosphorylation of

=0.01

+ AhR-/-

-FGM

c-Src

IP: Cav1R+/+

.0 0.1

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c-Src

AhR+/+ AhR-/-T-FGM

Y527/Src 1.0 1.9

E

tivation. (A) FAK phosphorylation at different Tyr residues was analyzed by immuno-arbitrary units. (B) Total cell extracts from T-FGM AhR+/+ and AhR−/− cells weredetermined by Western blotting (left) or with an anti-c-Src antibody and the amountsity was analyzed by Western blotting by determining the phosphorylation status of thesing c-Src immunoprecipitates and a canonical peptide substrate in the presence ofmmunoblotting. The experiments were done in three cultures of each genotype. Data

855J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

FAK at Tyr397, and this is followed by Src-dependent phosphorylation ofFAK at additional Tyr residues, including Tyr576 and Tyr577 [48]. Re-markably, FAK autophosphorylation at Tyr397 moderately increasedwhile phosphorylation at Tyr576 and Tyr577 decreased in AhR−/−fibroblasts without alterations in FAK total levels (Fig. 6A). Cav1 is anadditional Src substrate that regulates cell polarity and directional mi-gration [30,49]. Cav1 also mediates integrin-dependent signaling bybridging integrins to Src kinases and by regulating the activation ofdownstream intermediates [28–30]. Phosphorylation of Cav1 by c-Srcat Tyr14 [50] controls Cav1 activity [28]. Although Cav1 protein amountswere essentially unaffected by AhR knock-out, Cav1/c-Src associationand Cav1 phosphorylation at Tyr14 were significantly inhibited inT-FGM AhR−/− cells compared to wild type cells (Fig. 6B, left). More-over, the lack of AhR expression also decreased the association of Cav1Tyr14 with c-Src (Fig. 6B, right). Together these data raised the possibil-ity that Src activitymight be altered in the absence of AhR, and thereforewe analyzed c-Src activity in AhR−/− cells. c-Src activation is linked tothe phosphorylation of its Tyr416 residue [25]. Indeed, AhR−/− fibro-blasts displayed a significant reduction in their level of c-Src activationcompared to AhR+/+ cells (Fig. 6C). Furthermore, enzyme activityassays using c-Src immunoprecipitates and a consensus peptide forc-Src-dependent phosphorylation further demonstrated that AhR−/−fibroblasts had reduced c-Src activity (Fig. 6D), and this might in factrepresent a mechanism accounting for their lower phosphorylationlevels of FAK at Tyr576 and Tyr577 and of Cav1 at Tyr14.

c-Src activity is negatively regulated by phosphorylation at itsTyr527 by the C-terminal Src kinase (Csk) [51,52]. Accordingly, thelower c-Src activity found in AhR−/− cells correlated with the in-creased levels of c-Src p-Tyr527 (Fig. 6E), suggesting that AhR couldprevent Csk-dependent inactivation of c-Src. Csk is recruited to theplasma membrane by Cbp and Cav1 in order to bind to and inacti-vate c-Src [53,54]. The lack of AhR expression did not significantly

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ocia

tion

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0

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0

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D E

Fig. 7. Cbp levels and Cbp association with Csk are increased in T-FGM AhR−/− cells. Csk (A)and AhR−/− cells byWestern blotting. β-Actin was used as loading control. (C) CbpmRNA eregulating Cbp RNAwas also determined by transfecting AhR+/+ fibroblasts with scramble (or a constitutively active receptor lacking the PAS-B domain (CA-AhR). AhR-null cells wereby the expression of Gapdh and then referred to the expression in AhR+/+ cells. (D) AhR blyzed by ChIP. (E) Co-immunoprecipitation of Csk and Cbp was analyzed under normal celsi-RNA for Cbp or scramble si-RNA (si-scr) were immunoprecipitated with an anti-Cav1 ant(G) Scramble (si-scr) or si-Cbp was transfected into T-FGM AhR−/− and AhR+/+ cells andtibody. Data were corrected by the fluorescences in the absence of primary antibodies. The eas mean±SE.

affect Csk protein levels (Fig. 7A), but a significant increase in Cbpprotein expression was found in AhR−/− fibroblasts (Fig. 7B).Such increase in Cbp protein amounts could be controlled at thetranscriptional level since Cbp mRNA was also upregulated inAhR-null cells (Fig. 7C). Moreover, AhR silencing in AhR+/+ fibro-blasts increased Cbp mRNA levels, whereas the re-expression ofwild type AhR (wt-AhR) or a constitutively active receptor(CA-AhR) in AhR−/− cells reduced Cbp mRNA levels (Fig. 7C). Theefficiency of the AhR siRNAs and the wt- and CA-AhR constructsto control AhR levels and activation were determined by immuno-blotting and quantitative RT-qPCR for the AhR canonical targetgene Cyp1a1 (Fig. 2A–C). Furthermore, Cbp transcriptional regula-tion likely involved direct AhR binding to its promoter becausechromatin immunoprecitation (ChIP) analysis of a putative XREbinding site recently identified in the upstream Cbp promoter byChIP-on-chip studies [42] revealed efficient recruitment of AhR tosuch site in AhR+/+ cells (Fig. 7D). Importantly, the amount ofCsk that associates with Cbp was significantly higher in T-FGMAhR−/− than in AhR+/+ fibroblasts (Fig. 7E), indicating that thelack of AhR favors Cbp binding to Csk, which could in turn reducec-Src activity.

The involvement of Cbp in AhR-dependent Src activity is furthersupported by the fact that Cbp silencing in T-FGM AhR−/− fibro-blasts (Fig. 2D) led to a significant increase in Cav1 Tyr14 phosphory-lation (Fig. 7F). Importantly, Cbp contributes to the increase in β1integrin activation since Cbp knock-down by RNA interference signif-icantly diminished β1 integrin activity in T-FGM AhR−/− fibroblastscompared to wild type cell levels (Fig. 7G). Reduced Cbp levels, on thecontrary, did not significantly affect β1 protein levels in either celltype (Fig. 2E). Together, these results strongly suggest that Cbp medi-ates the effects of AhR on β1 integrin activation most likely throughthe regulation of Csk-dependent activity of Src.

R-/-

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tein

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.

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and Cbp (B) protein levels were determined in total cell extracts from T-FGM AhR+/+xpression in wild type and AhR-null cells was quantified by RT-qPCR. The role of AhR insi-scr) or specific siRNA (si-AhR) or the AhR−/− cells with either a wild type (wt-AhR)also transfected with the empty vector (E.V.) as control. mRNA levels were normalizedinding to the putative XRE site identified in the upstream Cbp promoter [42] was ana-l conditions. (F) Total cell extracts from T-FGM AhR−/− fibroblasts transfected with aibody and the amounts of total Cav1 and p-Cav1 Tyr14 determined by Western blotting.the activation levels of β1 integrin determined by flow cytometry using the 9EG7 an-xperiments were done in duplicate in three cultures of each genotype. Data are shown

AhR-/-

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ratio

n di

stan

ce, A

.U.

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Circularity Major/Minor

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mal

ized

val

ues

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0 h

48 h

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Fig. 8. Cbp regulates β1 integrin activation and cell morphology and migration. (A) T-FGM AhR−/− cells were transfected with scramble (si-scr) or Cbp siRNA and used in woundhealing experiments at 48 h. Migration distance was calculated for each experimental condition in different wounds in triplicate experiments. (B) The effects of Cbp siRNA on themorphology of T-FGM AhR−/− fibroblasts were determined by measuring circularity and major/minor axis ratio. Scramble si-RNA (si-scr) was used as control. Data are shown asmean±SE. Experiments were performed in duplicate in two different cultures.

Fibronectin/β-Actin 1.0 0.9

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si-scr si-CbpAhR-/-A

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100

150

Fig. 9. Effect of Cbp knock-down on fibronectin expression in T-FGM AhR−/− fibroblasts. (A) AhR−/− fibroblasts were transfected with a si-Cbp or scramble sequences (si-scr) andfibronectin expression determined by immunoblotting. β-Actin was used to normalize fibronectin expression. (B) Extracellular fibronectin was also analyzed in parallel cultures byimmunofluorescence. Quantification was done using the ImageJ software and represented as fluorescence intensity per area (left panel). Data are shown as mean±SE. Experimentswere performed in duplicate in three different cultures.

856 J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

857J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

Cbp knock-down in T-FGM AhR−/− fibroblasts significantly in-creased their migration in wound healing experiments as comparedwith cells transfected with scramble siRNA (Fig. 8A). Likewise, interfer-ence of Cbp expression rescued altered relevant parameters to resemblethose of the AhR-null morphology [11] and that included a reduction incircularity and an increase in themajor/minor axis ratio (Fig. 8B). Inter-estingly, Cbp down-modulation in T-FGM AhR−/− fibroblasts did notsignificantly reduce cellular fibronectin protein levels (Fig. 9A) nor itsdeposition in the extracellular matrix (Fig. 9B), suggesting that theCbp- and fibronectin-dependent pathways independently act to regu-late cell adhesion and migration in these cells.

3.3. Down-modulation of the AhR cytosolic chaperone Xap2 inhibitsfibronectin and Cbp expression but does not significantly affect β1integrin and Src activation

Cytosolic localization of the AhR represents a mechanism thatlimits the amount of transcriptionally active nuclear receptor. The arylhydrocarbon receptor-interacting protein (Aip), also known as Ara9(AHR-associated protein 9) and Xap2 (hepatitis B virus X-activatingprotein 2) is a relevant component of the cytosolic AhR complex [55]known to retain the receptor in the cytoplasmic compartment [56,57].Xap2 could contribute to AhR-dependent signaling in cell adhesionand migration because it targets phosphodiesterases to the cytosoliccomplex [58], and phosphodiesterases have been shown to modulateintegrin activation and cell migration [59,60]. We have thereforeinvestigated if Xap2 knock-down in T-FGM AhR+/+ cells (Fig. 10A)alters the expression/activity of signaling molecules of the AhRpathway. Consistently with the increased expression of fibronectinand Cbp found in AhR-null cells, Xap2 down-modulation significantly

si-scr si-Xap2

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Fig. 10. Effect of Xap2 knock-down on the expression and activation of signaling intermediatewith a si-Xap2 or with scramble sequences (si-scr) and the mRNA levels of Xap2 (A), fibronectwith scramble sequenceswere also used (D) T-FGMAhR+/+ cells transfectedwith si-XAp2 or sindicated. (E) Protein levels of Cbp, fibronectin, p-SrcY416, p-SrcY527, p-Cav1Y14 and total Cav1 wscramble sequences. β-Actin was used to normalize protein levels. Data are shown as mean±

reduced fibronectin and Cbp mRNA levels and, to a lesser extent, theirprotein amounts (Fig. 10 B,C). However, β1 integrin activationwas only marginally reduced in Xap2-interfered cells (Fig. 10D). Addi-tionally, Xap2 knock-down did not significantly alter Src (p-Src Tyr416

and p-SrcTyr527) or Cav1 (p-Cav1 Tyr14) activation (Fig. 10E).

4. Discussion

Among the recently discovered functions of AhR [61], the control ofcell adhesion and migration has attracted significant interest. We havepreviously shown that AhR is needed to maintain fibroblast cell migra-tion, and that lack of AhR led to increased cell adhesion to fibronectinand enhancement in the number and size of focal adhesions [11,12]. Itwas then suggested that one of the mechanisms influencing fibroblastsadhesion and migration involved the proto-oncogene Vav3 [11]. Addi-tional sets of data have since proposed the existence of other regulatorymechanisms, including the implication of β1 integrins. Additionally, thelower level of total FAK phosphorylation present in AhR−/− fibroblasts[12] could involve diminished c-Src activity since c-Src appears to be as-sociated to the cytosolic AhR complex [32,33].

Here, we describe a mechanism that integrates AhR in cell adhesionand migration by regulating c-Src activity that impinges on β1 integrinactivation. Our results show that AhR transcriptionally regulates thelevels of Cbp, a membrane protein that modulates Csk-dependent Srcactivity [27,62]. We also provide experimental evidence demonstratingthat fibronectin production is regulated by AhR, representing anotherpossible mechanism for the modulation of β1 integrin activation. Alto-gether, we propose a model in which AhRmediates signaling to plasmamembrane-associated β1 integrins controlling cell adhesion andmigra-tion (Fig. 11).

si-Xap2 si-scr+/+ AhR-/-

01

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integrin activation

β-Actin

β-Actin

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β-Actin

s in the AhR-dependent control of cell migration. T-FGM AhR+/+ cells were transfectedin (B) and Cbp (C) quantified by real-time RT-PCR. T-FGM AhR−/− fibroblasts transfectedcramble sequenceswere also analyzed for theirβ1 integrin activation byflowcytometry asere determined by immunoblotting in T-FGM AhR+/+ cells transfected with si-XAp2 orSE. Experiments were performed in two different cultures of each genotype.

Nucleus

AhR

Focal adhesionsAdhesionMigration

Cav1

Cbp/Pag1

Cbp

c-Src CskFAK

ECM Fn

β1

Fig. 11. Scheme depicting the proposed model for the AhR-dependent control of celladhesion and migration. Cbp is expressed in an AhR-dependent manner. Transmem-brane Cbp recruits Csk with a concomitant reduction in c-Src activation. In turn,c-Src controls FAK and Cav1 phosphorylation. AhR also modulates β1 integrin activa-tion by a pathway involving Cbp and probably ECM-deposited fibronectin. The involve-ment of AhR in these pathways will ultimately contribute in maintaining the dynamicsof focal adhesions and the extent of cell adhesion and migration.

858 J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

Evidence for enhanced β1 integrin activation in AhR-null cellscame from three experimental findings: (1) increased binding to theactivation-reporter 9EG7 mAb; (2) increased binding to solublefibronectin-80; and (3) enhanced talin-β1 subunit association. Theseobservations correlated with decreased Src activity in AhR−/− cells, re-vealed by the reduced phosphorylation of the Src activation residueTyr416 and by the increased phosphorylation of the Src negative regula-tory Tyr527 residue. This was associated with reduced phosphorylationof the Src substrates FAK Tyr576–Tyr577 and Cav1 Tyr14.

Importantly, our findings also demonstrate the involvement ofCbp, a transmembrane protein that recruits Csk to the plasma mem-brane in order to inactivate c-Src [27,53] in the regulation of Src activityin AhR−/− fibroblasts. Indeed, Cbp protein and mRNA wereoverexpressed in T-FGM AhR−/− fibroblasts and this is likely responsi-ble for the increased Cbp–Csk association found in AhR-null fibroblastimmunoprecipitates. Additionally, our knock-down and rescue experi-ments strongly suggest that Cbp transcription could be constitutivelyregulated by AhR. Consistently with a recent ChIP-on-chip study indioxin-treated mouse liver identifying a functional XRE element in theupstream Cbp promoter [42], we have also found that AhR binds tosuch XRE site in AhR+/+ fibroblasts, thus further supporting anAhR-dependent transcriptional regulatory mechanism. Since Cbpupregulation in metastatic colon cancer cells reduced c-Src activationand blocked migration [63], we suggest that enhanced Cbp expressionin AhR-null fibroblasts promotes the formation of Cbp–Csk inhibitory

complexes that reduce c-Src activation which, in turn, will decreasephosphorylation of its downstream target FAK and Cav1.

Our present data therefore support amodel inwhich Cbp plays a cen-tral role in the inside-out activation of β1 integrins in AhR−/− cells,leading to Csk recruitment and subsequent c-Src inactivation. In thiscontext, reduced c-Src activation could maintain β1 integrins in ahypophosphorylated and active conformation that will re-inforce celladhesion preventing cell migration. Cpb could be at the cross-roads ofinside-out and outside-in signaling that controls integrin activationand cell adhesion and migration. Whether the Cbp- or fibronectin-mediated effects on integrin activation are connected, it is not knownat present. Although additional studies have to be performed to solvethat question, the possibility exists that both pathways act independent-ly since Cbp knock-down did not significantly reduce fibronectin deposi-tion in the extracellular matrix. Other members of the AhR complexpresumably involved in cell migration could contribute to the mecha-nism proposed here. While the decrease in fibronectin and Cbp mRNAsproducedbyXap2 knock-downagreeswith an increased repressor activ-ity of transcriptionally active AhR, and with the over-expression of bothproteins in T-FGM AhR−/− cells, the verymild effects onβ1 integrin, Srcand Cav1 activities suggest that, at least for the chaperone Xap2, it doesnot have a major role in AhR-dependent signaling in cell adhesion andmigration.

Altogether, our results indicate that the expression of AhR plays animportant role in the regulation of β1 integrin activation by controllingCbp–Csk–Src activity (Fig. 11). Therefore,β1 integrin collects inside-outsignals from Cbp/c-Src and outside-in signals from its extracellular li-gand fibronectin, ultimately modulating fibroblast adhesion andmigra-tion. Collectively, these novel regulatory functions of AhR might beimportant for the study of cell migration during physiological processessuch as wound healing, and for the analysis of tumormetastasis involv-ing β1 integrin and c-Src activities.

5. Conclusions

The dioxin receptor is a novel partner in the regulation ofβ1 integrinactivation and in the control of cell adhesion andmigration. At least twoconverging mechanisms appear to be involved: (1) an inside-outsignaling through the transcriptional control of the docking proteinCbp/Pag1 ultimately controlling Csk-c-Src and FAK activity; and (2) anoutside-in mechanism through the regulation of its extracellular ligandfibronectin. Together both mechanisms could integrate AhR in cell mi-gration under normal and pathological conditions.

Acknowledgments

This workwas supported by grants from the SpanishMinistry of Sci-ence and Innovation to P.M.F-S. (SAF2008-00462 and BFU2011-22678),to J. T. (SAF2008-00479 and SAF2011-24022) and to A.G.P. (SAF2009-07035), and from the Junta de Extremadura to P.M.F-S. (GR10008).Research at P.M.F-S., J.T. and A.G.P. laboratories are also funded by theRed Temática de Investigación Cooperativa en Cáncer (RTICC), Fondode Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministryof Health (RD06/0020/1016 to P.M.F-S. and RD06/0020/0011 to J.T. andA.G.P.). J.R.B. was a F.P.U. program fellow from the Spanish Ministry ofEducation and Sciences. All Spanish funding is co-sponsored by theEuropean Union FEDER program. The support and help of the Serviciode Técnicas Aplicadas a la Biociencia, Universidad de Extremadura aregreatly acknowledged.

We are very grateful to Dr. Piero Crespo and to Dr. Miguel A.Alonso-Lebrero for assistance with sucrose density gradients and toDr. Miguel A. del Pozo for the protocol to prepare fibronectin-enrichedextracellular matrices. The technical support of Eva Barrasa is greatlyacknowledged.

859J. Rey-Barroso et al. / Cellular Signalling 25 (2013) 848–859

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