α9β1 Integrin in melanoma cells can signal different adhesion states for migration and anchorage

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Research Article

α9β1 Integrin in melanoma cells can signal different adhesion states for migration and anchorage Magnus C. Lydolph 1 , Marie Morgan-Fisher 1 , Anette M. Høye, John R. Couchman, Ulla M. Wewer, Atsuko Yoneda⁎ Department of Biomedical Sciences, The Faculty of Health Sciences, and Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen Biocenter, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark

A R T I C L E I N F O R M A T I O N

AB S TR AC T

Article Chronology:

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist

Received 16 April 2009

in several activation states regulated in part by ectodomain conformation. The α9 integrin subunit,

Revised version received 17 August 2009

which pairs only with β1, has specific roles in the immune system and may regulate cell migration.

Accepted 23 September 2009

Melanoma cells express abundant α9β1 integrin, and its role in cell migration was assessed.

Available online 29 September 2009

Ligands derived from Tenascin-C and ADAM12 supported α9β1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced

Keywords:

α9β1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting

α9β1 integrin

that the activation status of α9β1 integrin was altered. The effect of manganese ions in promoting

Cell migration

focal adhesion formation was reproduced by β1 integrin activating antibody. The α9β1 integrin

Focal adhesion

translocated to focal adhesions, where active β1 integrin was also detected by conformation-

Cytoskeleton

specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration.

Small GTPase

Endogenous α9β1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an

ADAM12

inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although α9β1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration. © 2009 Elsevier Inc. All rights reserved.

Introduction Cell surface receptor integrins are essential for migratory cells such as malignant melanoma cells to move through the interstitial matrix. Integrins are heterodimers consisting of type-1 α and β transmembrane protein subunits [1] and at least 24 integrins formed by pairing 18 α subunits and 8 β subunits have been identified. Ligand binding triggers signal transduction events that affect many aspects of cell behaviour including cell adhesion,

migration, proliferation, survival, and differentiation [1]. As the integrin cytoplasmic domains lack enzymatic activity, signalling depends on association with cytoplasmic adaptor proteins upon ligand binding. Adapter proteins such as talin directly link to the cytoskeleton whereas signalling molecules including src kinases and focal adhesion kinase (FAK) initiate phosphorylation events or protein-protein interactions [2,3]. The activity (availability) of integrins to interact with ligands is carefully regulated by their affinity and the interaction valency [4].

⁎ Corresponding author. Fax: +45 3532 5669. E-mail addresses: [email protected] (M.C. Lydolph), [email protected] (M. Morgan-Fisher), [email protected] (A.M. Høye), [email protected] (J.R. Couchman), [email protected] (U.M. Wewer), [email protected] (A. Yoneda). 1 These authors contributed equally to the work. 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.09.022

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Evidence from crystal structure and electron microscopic studies of integrin extracellular domains indicate that at least some family members of integrins exist in multiple conformations that may be bent or extended [4]. Furthermore, extended forms can exist with the headpiece open with high affinity for ligands and a closed form with low ligand affinity. Valency of integrin-ligand interactions also affects cell adhesion. The α9 integrin subunit constitutes a small subfamily with α4 integrin, sharing 39% amino acid identity [5], and pairs only with β1 [1,4]. It is widely expressed in tissues, e.g. epithelia, muscle, leukocytes and osteoclasts [5] with potential ligands including tenascin-C [6], ADAMs2 (a disintegrin and metalloprotease) [7,8], the EIIIA segment of tissue fibronectin [9], and vascular cell adhesion molecule-1 [10]. Some of these ligands also bind to α4β1 integrin, though analyses of α9-deficient mice revealed specific roles in lymphatic system development [11], granulocyte maturation [12], and osteoclast differentiation and function [13]. The α9β1 integrin has also been implicated in cell migration and invasion. This integrin and its ligands have been detected at the dermal-epidermal junction in wound healing [14] but additionally on the surface of tumour epithelial cells in primary colorectal and gastric tumours as well as at the invasion front [15]. Very recently α9β1 integrin-dependent glioblastoma migration has been demonstrated [16]. The mRNA levels of this integrin and its ligands tenascin-C and ADAM12 are upregulated in melanoma metastatic xenograft ([17], http://www.oncomine.org) or in metastatic melanoma [18], suggesting that α9β1 integrin could play a role in the cell migration process. The molecular basis of α9β1 integrin-mediated cell adhesion is not well understood, for example, whether or not activity status of α9β1 integrin is regulated and how it relates to functions. In this study, we used melanoma cells that express α9β1 integrin abundantly [8] to investigate the ability of this integrin to regulate cell adhesion/migration. We demonstrate that although α9β1 integrin in melanoma cells has the potential to induce and localise to focal adhesions in a high affinity state, dependent on Rho kinase, it normally supports cell adhesion consistent with migration in a Rac-dependent manner. In the latter case, focal adhesions are sparse, consistent with the migratory phenotype.

Materials and methods Cell culture Clonetics® normal neonatal human epidermal melanocytes (NHEM) were purchased from Lonza Walkersville, Inc. (Walkersville, MD, USA) and maintained to passage 3 according to the manufacturer's protocol. CHO-K1 and human melanoma cells G361, SK-MEL 28 and A375 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). G361 cells were maintained in McCoy's 5A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and Primocin (Amaxia Biosystems). SK-MEL 28 cells and A375 cells were maintained in DMEM with 10% FBS. CHO-K1 and α9-CHO cells, CHO cells stably expressing α9 integrin (a kind gift from Dr. 2 Abbreviation: ADAM, a disintegrin and metalloprotease; NHEM, normal neonatal human epidermal melanocytes; TNfn3, the third fibronectin type III repeat of human tenascin-C; PQ, primaquine.

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Dean Sheppard (Lung Biology Center, University of California, San Francisco, USA) were maintained in DMEM/F12 (1:1) medium supplemented with 10% FBS. Cells were grown at 37 °C in a 5% CO2 humidified atmosphere.

Antibodies Function-blocking antibodies against integrins were: α5 (P1D6) from Calbiochem (La Jolla, CA, USA), α6 (NKI-GoH3) from AbD Serotech (Oxford, UK), α4 (P4C2) from Covance (Berkeley, CA, USA), α4 (HP2/1), αVβ3 (LM609) and α9β1 (Y9A2) [19] from Millipore (Bedford, MA, USA), and AIIB2 targeting β1-integrin which was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences. Monoclonal antibody against β1 integrin (12G10) was from Millipore and TS2/16 from Thermo Scientific. Rabbit polyclonal anti-phospho paxillin at tyrosine 31, rabbit polyclonal anti-phospho FAK at tyrosine 397 were purchased from Invitrogen. Monoclonal antibodies against paxillin, and Rac1 (clone 23A8), rabbit polyclonal anti-phospho paxillin at tyrosine 118 and rabbit polyclonal anti-phospho src at tyrosine 416 were from Millipore, monoclonal antibody against vinculin was from Sigma (St. Louis, MO, USA). Secondary antibodies conjugated to horseradish peroxidase were obtained from Dako A/S (Glostrup, Denmark). Fluorochrome-conjungated (Alexa Fluor 488/546) secondary antibodies were from Invitrogen.

Plasmid construction and protein purification The cDNA encoding the third fibronectin type III repeat of human tenascin-C (TNfn3) was constructed as previously described [20]. Briefly, total RNA was isolated from G361 cells using TRIzol reagent (Invitrogen). The isolated RNA was subjected to DNase I digestion using the RNase-Free DNase Set (Qiagen, Maryland, USA) and then was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cheshire, UK). The target cDNA was amplified by polymerase chain reaction (PCR) using the cDNA library of G361 as template, primers C24: 5′-AGG GTG ACC ACC ACA CGC TTG GAT GCC and C25: 5′-GGG AGC ATC GAG GCC TGT TGT GAA GG, and the KOD Hot Start DNA polymerase (Novagen, Madison, WI, USA) according to manufacturer's protocol. Site directed mutagenesis (TNfn3 848RGD to RAA, designated as TNfn3RAA) was carried out by the overlap extension method. The PCR to create 5′ terminal fragment was performed using primers C26: 5′-ATG GAT CCA TAG GGT GAC CAC CAC ACG CTT GGA TGC C and C31: 5′-GGT TGC TTG ACA TGG CAG CTC TGC GGG AG. To create a 3′ terminal fragment the primers C27: 5′-GCG AAT TCG GGA GCA TCG AGG CCT GTT GTG AAG G and C30: 5′-CTC CCG CAG AGC TGC CAT GTC AAG CAA CC were used. The PCR for the second step was carried out using primers C26 and C27 and PCR products of the first step as template. The cDNA encoding TNfn3RAA was subcloned into the BamHI and EcoRI sites of the pGEX-3X vector (GE Healthcare, Piscataway, NJ, USA) and the construct was verified by DNA sequencing. Recombinant TNfn3RAA protein was expressed in Escherichia coli BL21 and purified using glutathione agarose (Sigma). As a control, GST protein alone was also expressed in BL21 transformed by pGEX3X and purified. The purified proteins were pooled and dialysed against PBS.

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Recombinant human ADAM12 disintegrin domain (A12-Dis, #1347) and cysteine-rich domain (A12-Cys, #1053) were purified from E. coli by metal affinity resin chromatography as described previously [21].

Cell attachment assays Cell attachment assays were performed as described previously with minor modification [8]. Briefly, NUNC-Immuno1™ 96-well plates with MaxiSorp™ surface (NUNC, Roskilde, Denmark) were coated with 10 μg/ml of A12-Dis or 10 μg/ml of TNfn3RAA or 7.2 μg/ml of GST in PBS unless stated, 10 μg/ml human fibronectin (Calbiochem, San Diego, CA, USA) in PBS or 40 μg/ml bovine type I collagen (BD Biosciences, Erembodegem, Belgium) in 0.1 M acetic acid overnight at 4 °C and blocked with 1 mg/ml heatdenatured BSA for 2 h at 37 °C. NSC23766 (10–200 μM, Calbiochem), or C3 transferase (2 μg/ml, Cytoskeleton) was administered to cells 16 h before adhesion assays. Following this treatment, cells were trypsinised, and then suspended in serumfree medium containing 0.1 mg/ml BSA and 0.25 mg/ml soybean trypsin inhibitor (Sigma) to inactivate trypsin and collected by centrifugation. Cells were incubated for 30 min at 37 °C in serum-free medium with 0.1 mg/ml BSA. Cell surface receptors were treated for 30 min on ice with 10 μg/ml of blocking antibodies, or 10 μg/ml of heparin (Sigma). Intracellular signalling inhibitors were incubated with cells for 30 min at 37 °C before seeding onto coated substrates. Inhibitors were 1–20 μM src inhibitor I (Calbiochem), 1–20 μM SU6656 (Invitrogen), 1–20 μM NA-PP1 (Calbiochem), 1–20 μM PP1 inhibitor (Alexis Biochemicals, Lausen, Switzerland), 1–50 μM EHT1864 (Sigma) 1–10 μM SB203580 (Calbiochem), 2 μM Gö6976 (Calbiochem), 10 μM Y27632 (Calbiochem), or 250 μM primaquine (Sigma). Cells (3.0 × 104 cells) were plated in the absence or presence of manganese chloride (Mn2+, 1 mM). Cells were incubated for 90 minutes at 37 °C.

Western blotting Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% skim milk in TBS with 0.1% Tween20, membranes were incubated at 4 °C with primary antibody and subsequently with HRPconjugated secondary antibody. An enhanced chemiluminescence kit (Biological Industries Ltd, Kibbutz Beit Haemek, Israel) was used for product detection.

Measurement of GTP-Rac1 The level of GTP-Rac1 was analysed by pull down assay using GSTPAK CRIB [22]. Briefly, serum-starved cells were trypsinised and suspended in 0.25 mg/ml soybean trypsin inhibitor and 0.1 mg/ml BSA in serum-free medium. After centrifugation, cells were suspended in serum-free medium with 0.1 mg/ml BSA and kept in suspension for 30 min at 37 °C. Cells (1.5 × 106 cells) were plated on 10 cm dishes coated with A12-Dis. After 2 h, attached cells were lysed, GTP-Rac1 was pulled down with GST-PAK CRIB. The signals from western blotting were analysed by densitometric analysis using the TL100 software (Nonlinear Dynamics). The ratio of GTPRac1 to total Rac1 was calculated from immunoblots probed with Rac1 antibody.

Immunocytochemistry Cells were seeded in serum-free conditions on glass coverslips coated with substrate and inhibitors were added as described above. Cells were incubated on substrates for 90 min, fixed with 4% paraformaldehyde (PFA) for 10–20 min. To stain cytoplasmic proteins, cells were permeabilised with 0.1% Triton X-100 in PBS for 10 min. Cells were stained with primary antibodies (Y9A2 (1:50), P4C2 (1:100), pY416 src (1:50), paxillin (1: 800), 12G10 (1:100), vinculin (1:400), p397 FAK (1:100), p118 paxillin (1:50), p31 paxillin (1:50)) described above at 4C overnight, followed by fluorochrome-conjungated (Alexa Fluor 488) secondary antibodies or Alexa Fluor 546-phalloidin (Invitrogen). Controls for nonspecific cross-reacting of secondary antibodies were included and gave no staining above background. To examine integrin requirements in focal adhesion formation, cells were allowed to spread on substrate at 37 °C for 30 min in the presence of Mn2+, then inhibitory integrin antibodies were added into culture medium as described above. After a further 1 h incubation, cells were fixed and processed for staining. Fluorescence was viewed on a Zeiss Axioplan-2 microscope equipped with a Cool SNAP camera (Photometrics) using a Zeiss Plan-APOCHROMAT 63 × NA 1.4 oil objective. Images were processed using AxioVision software, MetaMorph software and Adobe Photoshop. In order to analyse focal adhesion formation, cells with more than 10 focal adhesions per cell were counted as positive.

Fluorescence-activated cell sorting (FACS) Cell surface integrin expression was measured with a FACS Calibur and analysed with CellQuestPro software (Becton Dickinson). Cells stained with secondary antibody only were used as a negative control. Briefly, NHEM, G361, and SK MEL-28 cells were fixed with 4–6% PFA in PBS for 7 min after harvesting with dissociation buffer. Only A375 cells were trypsinised since these cells could not be harvested with dissociation buffer treatment. FACS analysis of live G361 cells harvested with trypsin or dissociation buffer showed similar cell surface levels of integrins tested (data not shown). Since cells used in attachment assays were prepared by trypsinisation, cell surface integrin levels were assessed in trypsinised live G361 cells with or without treatment with manganese ions or primaquine. After washing twice with cold FACS buffer (PBS including 1% BSA), cells were incubated with primary antibody (10 μg/ml) in chilled FACS buffer for 20 min on ice. After one wash with FACS buffer, Alexa488-conjungated secondary antibody in FACS buffer was incubated with cells for 20 min on ice. Cells were resuspended in FACS buffer with 1 μg/ml propidium iodide and 1 × 104 cells per sample were analysed. Cell surface syndecan expression was analysed in live G361 cells harvested by dissociation buffer without fixation.

Cell migration assay Both sides of Transwell inserts (polycarbonate filter, 8 μm pore size, Costar, VWR Albertslund, Denmark) were coated with 10 μg/ml of TNfn3RAA in PBS at 37 °C for 3–7 h. After washing with PBS, the filters were blocked with 1 mg/ml heat-inactivated BSA for 1 h at 37 °C. G361 cells were serum starved at 37 °C for 6 h and then harvested using dissociation buffer. Cell suspensions were prepared as described for cell attachment assays. Cells

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(1 × 105 cells) in 100 μl of serum free medium containing BSA (0.1 mg/ml) were seeded on inserts in the absence of serum. After 90 min, unattached cells were removed from the inserts, and fresh serum free medium was added. Manganese chloride (1 mM), integrin blocking antibodies, integrin activating antibody or chemical inhibitors were added to upper wells in some cases. Following further 60 min incubation, medium in lower chamber was exchanged to 600 μl of medium containing 5% FBS to stimulate migration. After 16 h, cells which did not migrate through filter were removed by cotton swab, and cultures were fixed with 2% glutaraldehyde for 20 min at RT. Filters were washed with PBS and stained with 0.1% crystal violet from the bottom for 20 min at RT. After rinsing with PBS to remove excess dye, phase contrast images (random 5 fields for each filter) were recorded by the Olympus IX71 microscope with a camera Olympus DP50 using an Olympus UplanFl 10× NA 0.30 objective. Images were processed with Viewfinder Lite version 1.0 and Studio Lite version 1.0. Numbers of cells migrated through filter were counted.

Results α9β1 integrin interaction with ligands promotes focal adhesion assembly in the presence of manganese ions Cell adhesion/migration on extracellular matrix can be separated into at least 4 phases, cell attachment, spreading/protrusion formation, adhesion structure formation, and subsequent disassembly of adhesion [23]. To characterise α9β1 integrinmediated cell attachment, human malignant melanoma G361 cells, known to express α9β1 integrin on cell surface [8], were used. Two different known α9β1 ligands were used as substrates. One is recombinant 3rd fibronectin type III repeat of tenascin-C with mutation of the RGD sequence to RAA (TNfn3RAA) [20]. Since this fragment is known to interact with α9β1 integrin as well as those recognising the RGD sequence, this mutation was introduced to obtain an α9β1 integrin-specific ligand. The other ligand is recombinant disintegrin domain of ADAM12 (A12-Dis) [8]. Manganese ions (Mn2+) were also tested to determine if α9β1 integrin is affinity-modulated. TNfn3RAA substrates supported attachment through α9β1 integrin specifically since a specific blocking antibody almost completely inhibited cell attachment in the presence or absence of Mn2+ (Fig. 1A; inhibition by α9β1 integrin inhibitory antibody (p < 0.05)) and contributions by α4 integrin, which forms a subfamily with α9 integrin, were not observed. As controls, GST alone or BSA did not support cell attachment. The variability of data where cultures were treated with Mn2+ may be due to different degrees of response to treatment in different experiments. G361 cells also attached primarily through α9β1 integrin on A12-Dis substrates in the absence of Mn2+ as reported previously [8] (Fig. 1B). G361 cell attachment on A12-Dis in the presence of Mn2+ was substantially diminished by β1 blocking antibody and significant (70%) reduction of attachment was observed after treatment with α9β1 blocking antibody (p < 0.01). There was also some inhibition of attachment with α4 or α5 blocking antibodies alone in the presence of Mn2+ (Fig. 1B) and additive inhibition with α9β1 and β1 blocking antibodies in the presence of Mn2+ (Fig. 1B), consistent with A12-Dis having the potential to support cell attachment through other α integrins paired with β1 integrin

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[24,25]. This indicated that even though all G361 surface integrins might have been fully activated, α9β1 integrin remained the preferred receptor for the A12-Dis ligand. To investigate cellular responses to α9β1 integrin-mediated cell attachment, we analysed cell morphology on α9β1 integrin ligands. Fluorescence staining with phalloidin and antibodies against the focal adhesion marker protein paxillin showed that G361 cells formed stress fibres terminating at focal adhesion structures on α9β1 integrin ligands (Fig. 1C). While only 3% of cells attached to TNfn3RAA formed more than 10 focal adhesions per cell, Mn2+ treatment increased this level to 30% of cells. Approximately 25% and 50% of G361 cells adherent to A12-Dis formed focal adhesions in the absence and presence of Mn2+ respectively (Fig. 1C). Other focal adhesion marker proteins including vinculin, FAK phosphorylated at tyrosine 397, paxillin phosphorylated at tyrosine 31 or tyrosine 118, and src kinases phosphorylated at tyrosine 416 were detected in these focal adhesion structures (Supplement Fig. 1 and shown below). In addition, α9β1 integrin but not α4 integrin was detected in focal adhesion structures formed on TNfn3RAA or A12-Dis (Fig. 1D). The small proportion of cells forming focal adhesions on A12-Dis in the absence of Mn2+ also contained the α9β1 integrin. The capability of α9β1 integrin antibody (Y9A2) to stain cell surfaces was confirmed on α9-overexpressing CHO cells (Supplement Fig. 2). Control staining of parental CHO-K1 for α9β1 integrin (Supplement Fig. 2A) or staining of G361 cells with secondary antibody alone (not shown) gave no fluorescence signal. Furthermore, FACS analyses of live and PFA-fixed cells of two CHO lines showed that α9 integrin staining by Y9A2 was specific (Supplement Fig. 2B). Similar staining was observed across different batches of Y9A2 antibodies. Staining for α4 integrin was confirmed on SK-MEL 28 melanoma cells that express higher levels of α4 integrin than do G361 cells (our unpublished data). Importantly, Mn2+ treatment did not alter cell surface levels of α9β1 integrin as determined by FACS analysis (Fig. 6E). Moreover, no changes in cell surface level of any other integrin tested including αvβ3, α5, α4 and β1 integrin were observed with this cation. These data suggested that α9β1 integrin, while competent for cell attachment and spreading, does not normally exist in a fully activated state in G361 melanoma cells.

α9β1 integrin-dependent focal adhesion formation was commensurate with reduced cell migration To examine whether α9β1 integrin is required for the later stages of focal adhesion and stress fibre formation on α9β1 integrin ligands, cells were allowed to preattach to these substrate for 30 min in the presence of Mn2+. Then the α9β1, α4, or αvβ3 inhibitory antibodies were added into culture medium followed by incubation for further 1 h. Focal adhesion and stress fibre formation induced by Mn2+ in cells preattached on TNfn3RAA or A12-Dis required an α9β1 integrin contribution since the corresponding blocking antibody prevented their formation in all attached cells (Fig. 2A). As a control, αvβ3 blocking antibody did not affect focal adhesion and stress fibre formation on A12-Dis (not shown). More importantly, α4 blocking antibody did not affect focal adhesion and stress fibre formation either (Fig. 2A). Treatment of preattached cells with α9β1 blocking antibody also prevented spreading, suggesting α9β1-dependency not only of spreading but also of the later stage of focal adhesion assembly (Fig. 2A).

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Fig. 1 – α9β1 integrin can localise to focal adhesion structures. (A, B) Cells in suspension were treated with 10 μg/ml of integrin blocking antibodies for 30 min before seeding on TNfn3RAA (A) or A12-Dis substrates (B) for 90 min. Cell attachment in the absence (black columns) or presence of Mn2+ (white columns) was measured as described in Materials and methods. Data from control cells in the absence of Mn2+ were set at 1.0. Data are an average of at least three independent experiments and error bars indicate standard deviations. Asterisks show significant difference (p < 0.05). (C) G361 cells were replated for 90 min on A12-Dis or TNfn3RAA in the absence or presence of Mn2+ and were stained for paxillin (green) and F-actin (red). (D) G361 cells attached to A12-Dis or TNfn3RAA in the presence of Mn2+ for 90 min were stained for α9β1 integrin (left two columns, green) or α4 integrin (right two columns, green) first and then were permeabilised to stain for F-actin (left two columns, red) or phospho-FAK (right two columns, red). Bar, 25 μm.

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Fig. 2 – α9β1 integrin interaction with ligands can promote focal adhesion assembly. (A) G361 cells preattached on A12-Dis or TNfn3RAA for 30 min in the presence of Mn2+ were treated with α9β1 or α4 integrin blocking antibodies for further 1 h. Cells were stained for pY416 Src (green) and F-actin (red). Arrowheads indicate focal adhesion structures. Control cells were untreated with antibodies. (B) Cells in suspension were treated with heparin for 30 min before seeding on A12-Dis substrates for 90 min in the absence (black columns) or presence (white columns) of Mn2+ and cell attachment was assessed. Data from control cells in the absence of Mn2+ were set at 1.0. Data are an average of at least three independent experiments and error bars indicate standard deviations. (C and D) G361 cells preattached on A12-Dis for 30 min in the presence of Mn2+ were treated with heparin (C) or 10 μM Y-27632 (D) for further 1 h. Cells were stained for paxillin in green and for F-actin in red. The insert is the magnified image of boxed area. Arrowheads indicate focal complex structures. Bar, 25 μm.

In A375-SM melanoma cells, it has been shown that α5β1dependent adhesion required cooperative signalling through the heparan sulphate proteoglycan syndecan-4 to form focal adhesions and stress fibres whereas α4β1-dependent adhesion did not [26]. FACS analysis indicated that G361 cells expressed both syndecans-1 and 4 on the cell surface (Supplement Fig. 3). Since α4 and α9 constitute an integrin subfamily, we tested whether α9β1-dependent focal adhesion formation required heparan sulphate proteoglycan. As shown in Figs. 2B and C, heparin did not affect Mn2+-induced cell attachment or focal adhesion formation on A12-Dis. This suggested independence from a role of syndecan proteoglycans and its similarity to α4β1-dependent focal adhesion formation in A375-SM melanoma cells. This heparin concentration completely inhibited G361 cell attachment on A12Cys (not shown), which interacts with syndecan cell surface heparan sulphate proteoglycans [27]. Previously, we showed sensitivity of adhesion to the heparin binding fragment of fibronectin by this heparin concentration [28]. Although no downstream pathway to α4β1-dependent focal adhesion formation has been reported, focal adhesion and stress fibre formation often requires actomyosin contractility induced by Rho -Rho kinase pathway [29]. G361 preattached on A12-Dis in the presence of Mn2+ were treated with Rho kinase inhibitor Y-27632 [30].

While control cells formed focal adhesion and stress fibre (Figs. 1C and 2C), inhibition of Rho kinase activity impaired the extent of α9β1-dependent focal adhesion and stress fibre formation (Fig. 2D), and only small focal complexes, which localised beneath membrane protrusion, were formed. These data suggested that α9β1-dependent focal adhesion and stress fibre formation required Rho kinase signalling. Since focal adhesion and stress fibre formation on TNfn3RAA and A12-Dis were α9β1-dependent, the localisation of α9β1 integrin in G361 cells spread on substrate was further analysed by immunofluorescence microscopy. As shown in Fig. 3A, the localisation of α9β1 integrin was not affected by heparin, again indicating that there is no contribution from heparan sulphate proteoglycan for focal adhesion formation driven by α9β1 integrin. Focal adhesions formed on α9β1 integrin ligands contained α9β1 (Fig. 1D) but neither αvβ3 (Fig. 3B) nor α4 integrins (Fig. 1D). Since active β1 integrin, detected by 12G10 antibody staining, was also detected in focal adhesions (Fig. 3C), the α9β1 in focal adhesion is probably in an active form. Upregulation of active β1 integrin levels by Mn2+ treatment was confirmed by FACS analysis of both live and PFA-fixed G361 cells with 12G10 antibody (Fig. 3D). Analysis of fixed cells was performed to avoid 12G10 antibody activation of β1 integrin

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during the staining procedure in the absence of Mn2+. Alternatively, β1 integrin in G361 cells preattached on α9β1 integrin ligands was activated by β1 activating antibody TS2/16 [24]. This resulted in induction of stress fibres and focal adhesions (Fig. 3E). Altered characteristics of α9β1 integrin-driven adhesion, i.e. focal adhesion formation promoted by Mn2+ and β1 activating antibody, and translocation of α9β1 integrin into focal adhesion in response to Mn2+ suggested that α9β1 integrin was not fully activated originally. However, once fully active, this integrin is capable of inducing focal adhesion formation. Previously we reported that G361 cells migrated through A12Dis coated filters in response to serum in α9β1 integrin-dependent manner [8]. Since full activation of integrins induced α9β1 integrin-dependent focal adhesion formation, its effect on melanoma migration was examined in a transwell assay. As shown in Fig. 3F, G361 cells migrated through filters coated with TNfn3RAA. The presence of manganese ions significantly reduced melanoma migration. The same trend was observed with filters coated with A12-Dis (data not shown). Although β1 integrin activating antibody TS2/16 could induce focal adhesions on α9β1 ligands

(Fig. 3E), its effect on cell migration was not significant (Fig. 3F), suggesting higher capability of Mn2+ to activate integrins compared to activating antibody. Cell migration mediated by TNfn3RAA was inhibited by blocking antibodies to α9β1 but not α4 integrin, again showing TNfn3RAA to be an α9β1 integrinspecific ligand. The doubling time for G361 cells is longer than that of the assay (unpublished data). These data suggested that low levels of migration in the presence of Mn2+ were not coupled to, or the result of, a low proliferation rate but to formation of strong adhesion.

PP1-sensitive protein kinase and small GTPase Rac signalling regulate α9β1 integrin-mediated attachment The ability of integrins to support cell attachment on matrix proteins is dependent on their affinity and valency toward ligands, which can be modulated by inside-out signalling [4]. To investigate regulators of α9β1-mediated cell attachment, inhibitors targeting various signalling molecules were utilised in cell attachment assays. Src family kinases are implicated in integrinmediated adhesion signalling, focal adhesion turnover, and receptor endocytosis [31]. The src family kinase inhibitor PP1 (IC50 = 170 nM for src) [32] completely abolished attachment to TNfn3RAA at 10 μM in the absence of Mn2+ (Fig. 4A, p < 0.01), although high standard deviations were obtained, probably due to instability of PP1 compound in addition to different degree of cellular response to Mn2+. Inhibition of G361 cell attachment to

Fig. 3 – α9β1 integrin-dependent focal adhesion formation was commensurate with reduced cell migration. (A) G361 cells preattached on A12-Dis or TNfn3RAA for 30 min in the presence of Mn2+ were treated with heparin for further 1 h. Cells were stained for α9β1 integrin (green) first and then were permeabilised to stain for F-actin (red). Arrowheads indicate α9β1 integrin localisation at the ends of stress fibres. (B and C) G361 cells on A12-Dis (B and C) or TNfn3RAA (C) in the presence of Mn2+ were stained for αvβ3 integrin (B, green) or active β1 integrin with the 12G10 antibody (C, green) and for F-actin (red). Arrowheads indicate active β1 integrin localisation at the ends of stress fibres. (D) Active β1 integrin levels in live or PFA-fixed G361 cells treated (thick line) or untreated (thin line) with 1 mM Mn2+ for 30 min were analysed by FACS using 12G10 antibody. Controls treated (broken line) or untreated (dotted lines) with Mn2+ are labelled only with secondary antibodies. (E) G361 cells preattached on TNfn3RAA for 30 min in the absence of Mn2+ were treated with β1 integrin activating antibody TS2/16 (20 μg/ml) for further 1 h. Cells were stained for phospho-FAK (green) and F-actin (red). Arrowheads indicate focal adhesion structures induced by TS2/16. (F) G361 cells were seeded on TNfn3RAA-coated transwell filters and cells migrated after 16 h in the presence or absence of indicated reagents were measured as described in Materials and methods. Data are an average of three-independent experiments performed in duplicates and error bars indicate standard deviations. Data from control cells were taken for 100%. Asterisks show significant difference (p < 0.02). Bar, 25 μm.

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Fig. 4 – PP1-sensitive protein kinase regulates α9β1 integrin-mediated attachment. G361 cell attachment to TNfn3RAA (A) or A12-Dis (B and C) was measured in the absence (black columns) or presence (white columns) of Mn2+. Cells were pretreated with src-family kinase inhibitors for 30 min before seeding. Attachment was compared to cells treated with vehicle alone. Data from control cells in the absence of Mn2+ were set at 1.0. Asterisk denotes significant difference to vehicle alone (p < 0.05).

A12-Dis substrates with PP1 was also observed but this could be rescued partially by a treatment with Mn2+ (Fig. 4B). Other src inhibitors such as NA-PP1 (IC50 = 1 μM for src, Supplement Fig. 4A) and src inhibitor 1 ((IC50 = 44 nM for src, Fig. 4C) except for high levels (20 μM in the absence of Mn2+) did not affect cell attachment to A12-Dis, whereas SU6656 (IC50 = 280 nM, Supplement Fig. 4A) partially inhibited cell attachment in the absence of Mn2+. PP1 is inhibitory to other recorded targets such as p38 MAPK [33], c-kit or Bcr-Abl [34]. We further tested whether p38MAPK was required for cell attachment. As shown in Supplement Fig. 4B, p38MAPK inhibitor SB203580 partially inhibited cell attachment in the presence of Mn2+, suggesting that p38MAPK was not the major target of PP1. These results

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suggested that the effect of PP1 may not be through inhibition of a src family kinase but other protein kinases. Mn2+ treatment increased G361 cell spreading on A12-Dis in a phosphatidylinositol (PI) 3-kinase-dependent manner [8]. The small GTPase Rac is known to be activated by integrin engagement [35,36] and act downstream of PI3-kinase. Of the three Rac members (Rac1-3), Rac1 is ubiquitously expressed and is essential for lamellipodia and ruffle formation, which are observed in cell spreading processes [37]. Rac becomes active when loaded with GTP, which guanine nucleotide-exchange factors (GEF) catalyse. GTP loading of Rac1 in G361 cell lysates was measured by pull down assay. Active Rac1 in cells adherent to A12-Dis was compared to that of cells in suspension. Adhesion to A12-Dis triggered a 2.5fold activation of Rac1 in G361 cells (Fig. 5A, p < 0.05) in spite of high standard deviation. Integrin activation by Mn2+ was not accompanied by a further increase in Rac1 activation. The chemical inhibitor NSC23766 prevents the binding of Rac to Rac GEFs Trio and Tiam1 so that Rac can not be activated by these GEFs [38]. However, the compound does not inhibit Rac interaction with Vav, Lbc, and Intersectin GEFs. NSC23766 increased cell attachment on A12-Dis (Supplement Fig. 4C), suggesting that at least Trio or Tiam1-mediated Rac signalling negatively regulate cell attachment. On the other hand, EHT1864 possesses high affinity binding to Rac family members and specifically inhibits Rac-dependent but not Cdc42-dependent cell behaviours by promoting the loss of bound nucleotide [39]. This compound inhibited G361 cell attachment on both TNfn3RAA (Fig. 5B) and A12-Dis (not shown) in a dose-dependent manner, although Mn2+ could partially rescue. These data suggested that Rac signalling is required for α9β1-mediated cell attachment and the Rac activation mechanisms other than by Tiam or Trio may be important here. Importantly EHT1864 inhibited G361 cell migration on TNfn3RAA (Fig. 5C), suggesting that Rac downstream may be crucial for G361 melanoma migration. Cellular cortical (peripheral) contractility regulated by the small GTPase Rho and its downstream Rho kinase signalling is considered to be one regulatory factor for integrin-mediated leukocyte adhesion [40]. In leukocytes, stimuli such as cytokines give rise to signals that trigger cortical actin cytoskeleton relaxation, where integrins are more free to diffuse and interact with ligands, resulting in increased adhesion. In G361 cells, inhibition of the small GTPases Rho A/B/C and Rho kinase with C3 transferase [41] and Y-27632 [37] respectively, did not affect cell attachment on A12-Dis (Fig. 5D). These data suggested that contractility is not directly involved in G361 cell attachment to α9β1 integrin ligands, although Rho kinase activity was required for focal adhesion and stress fibre formation on α9β1 integrin ligands as shown above.

Integrin α9β1-mediated attachment requires vesicle exocytosis Recycling of integrins between plasma membrane and endosomal compartments has been recognised as an important process during cell migration, for example, to retarget integrins to particular regions of the cell membrane, resulting in polarity of receptor distribution [42,43]. To assess the role of vesicle exocytosis in α9β1-mediated attachment, cells were treated with primaquine (PQ), a lysosomotropic amine that blocks transport from endosomes to the plasma membrane and the secretory pathway at the trans-Golgi network [44,45]. PQ has been used to

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Fig. 5 – Rac but not Rho GTPase regulates α9β1 integrin-mediated attachment. (A) G361 cells were plated on A12-Dis. Cell lysates were collected after 2 h adhesion and GTP-Rac1 was pulled down. Relative amounts of GTP-Rac1 were analysed as described in Materials and methods. Average values for 3–6 independent experiments are shown. Statistically significant differences (p < 0.05) in GTP-Rac1 between cells in suspension and cells plated on A12-Dis in the absence of Mn2+ was observed. (B) G361 cell attachment to TNfn3RAA in the absence (black columns) or presence (white columns) of Mn2+ was measured in the presence of Rac inhibitor EHT1864. Data are an average of three-independent experiments and error bars indicate standard deviations. Data from control cells in the absence of Mn2+ were set at 1.0. Asterisk shows significant difference (p < 0.05) compared with controls. (C) G361 cells were seeded on TNfn3RAA-coated transwell filters and cells migrated after 16 h in the presence or absence of indicated reagents were measured as described in Materials and methods. Data are an average of two-independent experiments performed in duplicate and error bars indicate standard deviations. Data from control cells were set at 100%. (D) G361 cell attachment to A12-Dis in the absence (black columns) or presence (white columns) of Mn2+ was measured in the presence of inhibitors of classical PKC (Gö6976-Go) or Rho-kinases (Y27632-Y) or Rho A/B/C (C3 transferase-C3). Cells were treated with C3 transferase for 16 h and with other inhibitors for 30 min before seeding. Attachments were compared to cells treated with vehicle. Data from control cells in the absence of Mn2+ were set at 1.0.

investigate recycling of plasma membrane fractions including integrins [46]. Cell attachment was attenuated in a PQ dosedependent manner (Fig. 6A). PQ (250 μM) completely prevented cell attachment on A12-Dis in the absence of Mn2+ and reduced attachment by 50–70% in the presence of Mn2+ (Figs. 6A and B), implying that vesicle exocytosis was required for cell attachment. Residual cell attachment in the presence of PQ and Mn2+ was still α9β1 integrin-dependent since it was inhibited by α9β1 integrin blocking antibody (Fig. 6C). Attachment to fibronectin, type I collagen or serum, in which vitronectin is a major adhesion molecule, were also blocked by PQ (Fig. 6B). This observation is interesting since these matrix molecules utilise not only integrins but also other types of cell surface adhesion receptors such as the heparan sulphate proteoglycans syndecans, and urokinase type plasminogen activator receptor [47,48]. Our data suggested that vesicle exocytosis perhaps receptor recycling was critical for cell attachment mediated by not only integrins but also other adhesion receptors. To examine whether PQ reduces or Mn2+ treatment alters cell surface levels of integrins, FACS analysis of live cells was performed. All the examined integrins were still detectable on the cell surface after PQ treatment (Fig. 6D) and no changes in their

levels were observed in Mn2+ treatment (Fig. 6E). PQ marginally lowered α9β1-integrin levels on the cell surface compared to untreated controls (Fig. 6D), yet PQ effectively reduced cell attachment on A12-Dis (Figs. 6A–C). PQ (250 μM) led to only 5– 9% cell death compared with control treatments, suggesting that the effect of PQ on attachment was not due to cytotoxicity. Vesicle exocytosis may be necessary to maintain a population of α9β1 integrin in an active conformation on cell surface, or to activate integrin. However, integrin activation with Mn2+ after PQ treatment did not fully restore attachment. In fact, this attachment to A12-Dis was supported solely by α9β1 integrin. Since alternate α integrins paired with β1 integrin contributed to cell attachment on A12-Dis in the presence of Mn2+ and α9β1-integrin blocking antibody (Fig. 1B), these data suggested that vesicle trafficking was also required for the function of these integrins. Two protein kinase C (PKC) family members (α and ɛ) have been suggested to regulate integrin signalling and internalisation [49,50]. To determine their role, the PKC inhibitor Gö6976 that inhibits classical PKC isoforms (α, β, and γ) was employed. However Gö6976 did not affect cell attachment to A12-Dis (Fig. 5D), suggesting that α9β1 integrin may not be regulated by a classical PKC-mediated pathway. On the other hand, attachment

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Fig. 6 – Integrin α9β1-mediated attachment requires vesicle trafficking to the cell membrane. (A-C) G361 cells in suspension were treated with indicated concentrations of PQ (A) or 250 μM PQ (B) for 30 min before adhesion to A12-Dis or other matrix proteins (B) in the absence or presence of Mn2+. (C) G361 cells in suspension were treated with 250 μM PQ at 37 °C for 30 min and then incubated with integrin blocking antibody for 30 min on ice before adhesion to A12-Dis in the absence (black columns) or presence (white columns) of Mn2+. (D and E) Cell surface integrin levels in live G361 cells treated (thick line) or untreated (broken line) with 250 μM PQ (D) or with 1 mM Mn2+ (E) for 30 min at 37 °C were analysed by FACS. Cells were stained with the same concentrations of each integrin antibody (10 μg/ml) on ice for 20 min. Controls treated (thin line) or untreated (dotted line) with reagents are labelled only with secondary antibodies.

to A12-Cys was augmented by Y-27632 and Gö6976, showing that these inhibitors are functional in G361 cells (unpublished data).

Cell surface expression of α9β1 integrin was upregulated in G361 and SK-MEL 28 malignant melanoma cells Our previous studies showed α9β1 integrin cell surface expression in a limited number of cell types by FACS, abundant in melanoma G361 but low levels in breast tumour MDA-MB-231 cells, colon carcinoma RKO cells and osteosarcoma MG63 cells [8]. α9β1 integrin cell surface expression in melanoma cell lines and melanocytes was further examined by FACS analyses. Since normal human epidermal melanocytes (NHEM) were fragile and susceptible to damage in FACS analysis, cells were fixed with PFA after harvesting with non-enzymatic dissociation buffer and then stained with specific antibodies. It was confirmed with G361 cells that fixation with PFA did not deplete epitopes recognised by integrin antibodies. In addition to G361 cells, human malignant melanoma SK-MEL 28 cells and A375 cells as well as NHEM expressed cell surface α9β1 integrin (Fig. 7). Its level on two

melanoma cell lines was 5- to 6-fold higher than that of NHEM (Fig. 7), which was not seen for other integrins tested (α4 and αvβ3, not shown), suggesting that α9β1 integrin may be of particular importance in regulating cell-matrix interactions in this cell type.

Discussion The current study showed that α9β1 integrin appears to be regulated in its affinity for Tenascin-C and ADAM12 ligands in G361 cells. Endogenous α9β1 may be in an intermediate affinity state, capable of supporting migratory behaviour that was GTP-Rac dependent. At intermediate affinity states, treatment of G361 cells with manganese ions, known to “fully activate” many integrins, altered the adhesion and migration characteristics. While remaining mostly α9β1-dependent, focal adhesion formation was promoted, a process dependent on the Rho kinase pathway and independent on heparan sulphate proteoglycan. Moreover the integrin as well as active β1 integrin were detected in these

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Fig. 7 – α9β1 integrin is expressed on the cell surface of melanoma and normal primary melanocytes. Normal human epidermal melanocytes (NHEM), human melanoma G361 and SK-MEL 28 cells were detached with dissociation buffer, and fixed. Cell surface integrins were labelled with anti α9β1 integrin and fluorochrome-conjugated secondary antibodies. Human melanoma A375 cells were harvested by trypsinisation and live cells were stained. Cell surface integrin levels (thick line) were analysed by FACS. Controls are labelled only with secondary antibodies (thin line).

structures on α9β1 ligands. Consistent with increased anchorage, through focal adhesion assembly, transwell migration by the G361 in the presence of Mn2+ cells was reduced. Cell adhesion regulators include GTP-Rac, vesicle trafficking machinery and protein kinase other than src family. Compared to other cell types such as breast tumour cells [8], human malignant melanoma cells expressed α9β1 integrin on cell surface abundantly, suggesting its importance in melanoma migration. Diverse roles for integrins activated to different affinity states is in line with recent literature suggesting that integrin activation is tightly controlled in space and time and ranges from pre-activation to full activation [51]. Studies of T lymphocyte migration indicated distinct roles and distribution for different activation states of integrin αLβ2 [52]. Altogether this demonstrates that integrins are flexible receptors in terms of their ability to modulate their affinity in accordance with function and independently of expression on the cell surface. Mn2+-induced α9β1 integrin localisation in focal adhesion may associate with its activation status. Although no tools to detect activation states of α9β1 integrin exist currently, active β1 integrin was detected by 12G10 antibody staining of Mn2+ treated G361 cells on TNfn3RAA or A12-Dis (Fig. 3C). Activation of β1 integrin in G361 cells preattached on α9β1 integrin ligands with

TS2/16 antibody (Fig. 3) reproduced the Mn2+ effect on focal adhesion formation, whereas 12G10 antibody inhibited cell spreading (unpublished data). It is worth noting that similar effects of TS2/16 and 12G10 on α4β1 integrin-mediated adhesion were reported previously [53]. The molecular basis of morphological changes induced by α9β1 integrin has not been well established, however a recent report showed that EIIIA segment of fibronectin can induce filopodia formation accompanied by upregulation of signalling by the Rho family member, cdc42 using SW480 colon carcinoma cells transfected with α9 integrin cDNA [9]. Although cdc42 activation was not explored in this study, our data revealed that endogenous α9β1 integrin in melanoma could induce Rac1 activation, which also mediated cell migration (Figs. 5A–C). Inhibition of cell attachment with EHT1864 may eliminate a primary contribution of cdc42 pathway as this compound specifically inhibits signalling mediated by Rac but not by cdc42 [39]. Early studies showed paxillin interaction with the cytoplasmic domain of α9 integrin [54,55]. Paxillin may provide scaffolding functions to recruit Rac activators to the α9β1 integrin cytoplasmic domain [56]. However paxillin function in α9β1 integrin signalling needs careful interpretation. The interaction of α9 integrin with paxillin inhibited cell spreading [54,55], whereas Rac activation generally induces lamellipodia leading to cell spreading [35]. Tyrosine phosphorylated paxillin, as well as the protein itself were detected in focal adhesions promoted by the α9β1 integrin, suggesting that the interaction of α9β1 integrin and paxillin is dynamic depending on integrin activation state. Lamellipodia formation required for cell spreading often correlates with cell migration. However, previous studies reported that α9β1 integrin-driven cell migration was paxillin-independent [54] but rather required inward rectifier potassium channel activities [57]. Here we showed α9β1 integrin-driven cell migration is regulated by Rac signalling. Adhesion of G361 cells through α9β1 integrin was strongly inhibited by the PP1 src kinase inhibitor, but not by other inhibitors of these tyrosine kinases (Fig. 4 and Supplement Fig. 4). In addition, treatment of cells with PP1 at 10 μM (which blocked cell attachment) did not result in apparent reduction of either phosphorylation states of src at tyrosine 416 or its major substrate cortactin at tyrosine 421 (data not shown). The data suggest that an alternate kinase is required for adhesion, and was not due to cytotoxic effects of PP1 since it did not affect G361 cell adhesion to A12-Cys (unpublished data). Data on the specificity of kinase inhibitors reported by Cohen's group [33] assist in the interpretation of this apparently conflicting data. Among C3 transferase-sensitive Rho members (RhoA, B and C), Rho B localises to endosomes and regulates vesicle trafficking and growth factor endocytosis [58]. However, since C3 transferase did not affect cell attachment on A12-Dis (Fig. 5D), these three Rho family members are not α9β1 integrin traffic regulators. Cellular contractility and actin polymerisation, which are mediated by Rho and Rho kinase pathway, did not affect cell attachment on A12-Dis (Fig. 5D), suggesting that integrin clustering may not be important here. The use of primaquine provided further insight into α9β1 integrin-mediated adhesion to its ligands. Primaquine functions as an effective inhibitor of vesicle trafficking from Golgi to the cell membrane [44,45]. Blocking of vesicle exocytosis with PQ effectively attenuated cell attachment on A12-Dis although only a small reduction in cell surface α9β1-integrin levels was detected (Fig. 6). This suggested that a small proportion of α9β1-integrin

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was competent for ligand-binding. In other words, these data also demonstrated that different activation states of α9 integrin exist. Vesicle exocytosis may enable active integrins to redistribute to functional membrane domains such as rafts to support adhesion. It was suggested that integrin internalisation in keratinocytes was associated with their redistribution to regions of cell-cell contact [59]. Another possibility of the PQ effect is that vesicle transport may facilitate integrin activation as suggested previously [46]. However, Mn2+ treatment expected to activate all integrins could only partially overcome inhibition of attachment by PQ (Fig. 6). This supports the idea that Mn2+ alone is insufficient for conversion of integrins to a high affinity state, but requires a vesicle transport process to activate integrin by an unknown mechanism. An attractive speculation is that vesicle exocytosis provides regulatory factors or integrin modifiers for activation. α9β1 integrin supports migration in melanoma cells, but is dependent on a vesicle traffic component. Future understanding of the specific molecular requirements for melanoma migration may provide new opportunities for intervention.

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[8]

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[10]

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Acknowledgments UMW is supported by the Danish Cancer Society, the Danish Medical Research Council, the Lundbeck Foundation, and the Novo Nordisk Foundation. JRC is supported by the Danish National Research Foundation, the Danish Medical Research Council, Vilhelm Pedersen and Haensch Foundations. AY is supported by the Danish Cancer Research Foundation, the Danish Medical Research Council and the Novo Nordisk foundation. We would like to thank Dr. Dean Sheppard (Lung Biology Center, Univ. of California, San Francisco, USA) for providing us with α9-CHO cells, and Ms. Anna B. Fossum (BRIC, Univ. of Copenhagen, Denmark) for assistance with FACS analysis.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2009.09.022.

[13]

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REFERENCES [18] [1] R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, Cell 110 (2002) 673–687. [2] E.G. Arias-Salgado, S. Lizano, S. Sarkar, J.S. Brugge, M.H. Ginsberg, S.J. Shattil, Src kinase activation by direct interaction with the integrin β cytoplasmic domain, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13298–13302. [3] S. Liu, D.A. Calderwood, M.H. Ginsberg, Integrin cytoplasmic domain-binding proteins, J. Cell Sci. 113 (2000) 3563–3571. [4] B.H. Luo, C.V. Carman, T.A. Springer, Structural basis of integrin regulation and signaling, Annu. Rev. Immunol. 25 (2007) 619–647. [5] E.L. Palmer, C. Rüegg, R. Ferrando, R. Pytela, D. Sheppard, Sequence and tissue distribution of the integrin α9 subunit, a novel partner of β1 that is widely distributed in epithelia and muscle, J. Cell Biol. 123 (1993) 1289–1297. [6] Y. Yokosaki, E.L. Palmer, A.L. Prieto, K.L. Crossin, M.A. Bourdon, R. Pytela, D. Sheppard, The integrin α9β1 mediates cell attachment to a non-RGD site in the third fibronectin

[19]

[20]

[21]

[22]

[23]

3323

type-III repeat of tenascin, J. Biol. Chem. 269 (1994) 26691–26696. X. Lu, D. Lu, M.F. Scully, V.V. Kakkar, Structure-activity relationship studies on ADAM protein-integrin interactions, Cardiovasc. Hematol. Agents Med. Chem. 5 (2007) 29–42. C.K. Thodeti, C. Fröhlich, C.K. Nielsen, P. Holck, C. Sundberg, M. Kveiborg, Y. Mahalingam, R. Albrechtsen, J.R. Couchman, U.M. Wewer, Hierarchy of ADAM12 binding to integrins in tumor cells, Exp. Cell Res. 309 (2005) 438–450. A.V. Shinde, C. Bystroff, C. Wang, M.G. Vogelezang, P.A. Vincent, R.O. Hynes, L. Van De Water, Identification of the peptide sequences within the EIIIA (EDA) segment of fibronectin that mediate integrin α9β1-dependent cellular activities, J. Biol. Chem. 283 (2008) 2858–2870. Y. Taooka, J. Chen, T. Yednock, D. Sheppard, The integrin α9β1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1, J. Cell Biol. 145 (1999) 413–420. X.Z. Huang, J.F. Wu, R. Ferrando, J.H. Lee, Y.L. Wang, R.V. Farese Jr., D. Sheppard, Fatal bilateral chylothorax in mice lacking the integrin α9β1, Mol. Cell. Biol. 20 (2000) 5208–5215. C. Chen, X. Huang, A. Atakilit, Q.S. Zhu, S.J. Corey, D. Sheppard, The Integrin α9β1 contributes to granulopoiesis by enhancing granulocyte colony-stimulating factor receptor signaling, Immunity 25 (2006) 895–906. H. Rao, G. Lu, H. Kajiya, V. Garcia-Palacios, N. Kurihara, J. Anderson, K. Patrene, D. Sheppard, H.C. Blair, J.J. Windle, S.J. Choi, G.D. Roodman, α9β1: a novel osteoclast integrin that regulates osteoclast formation and function, J. Bone Miner. Res. 21 (2006) 1657–1665. P. Singh, C.L. Reimer, J.H. Peters, M.A. Stepp, R.O. Hynes, L. Van De Water, The spatial and temporal expression patterns of integrin α9β1 and one of its ligands, the EIIIA segment of fibronectin, in cutaneous wound healing, J. Invest. Dermatol. 123 (2004) 1176–1181. M. Gulubova, T. Vlaykova, Immunohistochemical assessment of fibronectin and tenascin and their integrin receptors α5β1 and α9β1 in gastric and colorectal cancers with lymph node and liver metastases, Acta Histochem. 108 (2006) 25–35. M.C. Brown, I. Staniszewska, P. Lazarovici, G.P. Tuszynski, L. Del Valle, C. Marcinkiewicz, Regulatory effect of nerve growth factor in α9β1 integrin-dependent progression of glioblastoma, Neuro Oncol. 10 (2008) 968–980. L. Xu, S.S. Shen, Y. Hoshida, A. Subramanian, K. Ross, J.P. Brunet, S.N. Wagner, S. Ramaswamy, J.P. Mesirov, R.O. Hynes, Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases, Mol. Cancer Res. 6 (2008) 760–769. S. Ilmonen, T. Jahkola, J.P. Turunen, T. Muhonen, S. Asko-Seljavaara, Tenascin-C in primary malignant melanoma of the skin, Histopathology 45 (2004) 405–411. A. Wang, Y. Yokosaki, R. Ferrando, J. Balmes, D. Sheppard, Differential regulation of airway epithelial integrins by growth factors, Am. J. Respir. Cell Mol. Biol. 15 (1996) 664–672. A.L. Prieto, G.M. Edelman, K.L. Crossin, Multiple integrins mediate cell attachment to cytotactin/tenascin, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 10154–10158. B.J. Gilpin, F. Loechel, M.G. Mattei, E. Engvall, R. Albrechtsen, U.M. Wewer, A novel, secreted form of human ADAM 12 (meltrin α) provokes myogenesis in vivo, J. Biol. Chem 273 (1998) 157–166. X.D. Ren, W.B. Kiosses, M.A. Schwartz, Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton, EMBO J. 18 (1999) 578–585. A.J. Ridley, M.A. Schwartz, K. Burridge, R.A. Firtel, M.H. Ginsberg, G. Borisy, J.T. Parsons, A.R. Horwitz, Cell migration: integrating signals from front to back, Science 302 (2003) 1704–1709.

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[24] K. Eto, W. Puzon-McLaughlin, D. Sheppard, A. Sehara-Fujisawa, X.P. Zhang, Y. Takada, RGD-independent binding of integrin α9β1 to the ADAM-12 and -15 disintegrin domains mediates cell-cell interaction, J. Biol. Chem. 275 (2000) 34922–34930. [25] J. Huang, L.C. Bridges, J.M. White, Selective modulation of integrin-mediated cell migration by distinct ADAM family members, Mol. Biol. Cell 16 (2005) 4982–4991. [26] Z. Mostafavi-Pour, J.A. Askari, S.J. Parkinson, P.J. Parker, T.T. Ng, M.J. Humphries, Integrin-specific signaling pathways controlling focal adhesion formation and cell migration, J. Cell Biol. 161 (2003) 155–167. [27] K. Iba, R. Albrechtsen, B. Gilpin, C. Fröhlich, F. Loechel, A. Zolkiewska, K. Ishiguro, T. Kojima, W. Liu, J.K. Langford, R.D. Sanderson, C. Brakebusch, R. Fässler, U.M. Wewer, The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to β1 integrin-dependent cell spreading, J. Cell Biol. 149 (2000) 1143–1156. [28] Y. Mahalingam, J.T. Gallagher, J.R. Couchman, Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin require heparan sulfate with specific properties, J. Biol. Chem. 282 (2007) 3221–3230. [29] K. Riento, A.J. Ridley, Rocks: multifunctional kinases in cell behaviour, Nat. Rev. Mol. Cell Biol 4 (2003) 446–456. [30] T. Ishizaki, M. Uehata, I. Tamechika, J. Keel, K. Nonomura, M. Maekawa, S. Narumiya, Pharmacological properties of Y-27632, a specific inhibitor of Rho-associated kinases, Mol. Pharmacol. 57 (2000) 976–983. [31] M.C. Frame, Newest findings on the oldest oncogene; how activated src does it, J. Cell Sci. 117 (2004) 989–998. [32] J.H. Hanke, J.P. Gardner, R.L. Dow, P.S. Changelian, W.H. Brissette, E.J. Weringer, B.A. Pollok, P.A. Connelly, Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation, J. Biol. Chem. 271 (1996) 695–701. [33] J. Bain, L. Plater, M. Elliott, N. Shpiro, C.J. Hastie, H. Mclauchlan, I. Klevernic, J.S. Arthur, D.R. Alessi, P. Cohen, The selectivity of protein kinase inhibitors: a further update, Biochem. J. 408 (2007) 297–315. [34] L. Tatton, G.M. Morley, R. Chopra, A. Khwaja, The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases, J. Biol. Chem. 278 (2003) 4847–4853. [35] L.S. Price, J. Leng, M.A. Schwartz, G.M. Bokoch, Activation of Rac and Cdc42 by integrins mediates cell spreading, Mol. Biol. Cell 9 (1998) 1863–1871. [36] M.A. Del Pozo, W.B. Kiosses, N.B. Alderson, N. Meller, K.M. Hahn, M.A. Schwartz, Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI, Nat. Cell Biol. 4 (2002) 232–239. [37] L. Vidali, F. Chen, G. Cicchetti, Y. Ohta, D.J. Kwiatkowski, Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor, Mol. Biol. Cell 17 (2006) 2377–2390. [38] Y. Gao, J.B. Dickerson, F. Guo, J. Zheng, Y. Zheng, Rational design and characterization of a Rac GTPase-specific small molecule inhibitor, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 7618–7623. [39] A. Shutes, C. Onesto, V. Picard, B. Leblond, F. Schweighoffer, C.J. Der, Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases, J. Biol. Chem. 282 (2007) 35666–35678. [40] L. Liu, B.R. Schwartz, N. Lin, R.K. Winn, J.M. Harlan, Requirement for RhoA kinase activation in leukocyte de-adhesion, J. Immunol. 169 (2002) 2330–2336. [41] M. Vogelsgesang, A. Pautsch, K. Aktories, C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins, Naunyn-Schmiedebergs Arch. Pharmacol. 374 (2007) 347–360.

[42] M.C. Jones, P.T. Caswell, J.C. Norman, Endocytic recycling pathways: emerging regulators of cell migration, Curr. Opin. Cell Biol. 18 (2006) 549–557. [43] A.G. Ramsay, J.F. Marshall, I.R. Hart, Integrin trafficking and its role in cancer metastasis, Cancer Metastasis Rev. 26 (2007) 567–578. [44] R.R. Hiebsch, T.J. Raub, B.W. Wattenberg, Primaquine blocks transport by inhibiting the formation of functional transport vesicles. Studies in a cell-free assay of protein transport through the Golgi apparatus, J. Biol. Chem. 266 (1991) 20323–20328. [45] A.W. van Weert, H.J. Geuze, B. Groothuis, W. Stoorvogel, Primaquine interferes with membrane recycling from endosomes to the plasma membrane through a direct interaction with endosomes which does not involve neutralisation of endosomal pH nor osmotic swelling of endosomes, Eur. J. Cell Biol. 79 (2000) 394–399. [46] M. Roberts, S. Barry, A. Woods, P. van der Sluijs, J. Norman, PDGF-regulated rab4-dependent recycling of αvβ3 integrin from early endosomes is necessary for cell adhesion and spreading, Curr. Biol. 11 (2001) 1392–1402. [47] M.R. Morgan, M.J. Humphries, M.D. Bass, Synergistic control of cell adhesion by integrins and syndecans, Nat. Rev. Mol. Cell Biol. 8 (2007) 957–969. [48] C.D. Madsen, N. Sidenius, The interaction between urokinase receptor and vitronectin in cell adhesion and signalling, Eur. J. Cell Biol. 87 (2008) 617–629. [49] T. Ng, D. Shima, A. Squire, P.I. Bastiaens, S. Gschmeissner, M.J. Humphries, P.J. Parker, PKCα regulates β1 integrin-dependent cell motility through association and control of integrin traffic, EMBO J. 18 (1999) 3909–3923. [50] J. Ivaska, K. Vuoriluoto, T. Huovinen, I. Izawa, M. Inagaki, P.J. Parker, PKCɛ-mediated phosphorylation of vimentin controls integrin recycling and motility, EMBO J. 24 (2005) 3834–3845. [51] K. Clark, R. Pankov, M.A. Travis, J.A. Askari, A.P. Mould, S.E. Craig, P. Newham, K.M. Yamada, M.J. Humphries, A specific α5β1-integrin conformation promotes directional integrin translocation and fibronectin matrix formation, J. Cell Sci. 118 (2005) 291–300. [52] P. Stanley, A. Smith, A. McDowall, A. Nicol, D. Zicha, N. Hogg, Intermediate-affinity LFA-1 binds α-actinin-1 to control migration at the leading edge of the T cell, EMBO J. 27 (2008) 62–75. [53] J.D. Humphries, N.R. Schofield, Z. Mostafavi-Pour, L.J. Green, A.N. Garratt, A.P. Mould, M.J. Humphries, Dual functionality of the anti-β1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors, J. Biol. Chem. 280 (2005) 10234–10243. [54] B.A. Young, Y. Taooka, S. Liu, K.J. Askins, Y. Yokosaki, S.M. Thomas, D. Sheppard, The cytoplasmic domain of the integrin α9 subunit requires the adaptor protein paxillin to inhibit cell spreading but promotes cell migration in a paxillin-independent manner, Mol. Biol. Cell 12 (2001) 3214–3225. [55] S. Liu, M. Slepak, M.H. Ginsberg, Binding of paxillin to the α9 integrin cytoplasmic domain inhibits cell spreading, J. Biol. Chem. 276 (2001) 37086–37092. [56] M.C. Brown, C.E. Turner, Paxillin: adapting to change, Physiol. Rev. 84 (2004) 1315–1339. [57] G.W. deHart, T. Jin, D.E. McCloskey, A.E. Pegg, D. Sheppard, The α9β1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 7188–7193. [58] A.J. Ridley, Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking, Trends Cell Biol. 16 (2006) 522–529. [59] J.C. Adams, F.M. Watt, Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes α5β1-integrin loss from the cell-surface, Cell 63 (1990) 425–435.