Original Article

Inhibitory Role of 6ß4-associated erbB-2 and Phosphoinositide 3-Kinase in Keratinocyte Haptotactic Migration Dependent on 3ß1 Integrin

Correspondence to: Vito Quaranta, Department of Cell Biology, SBR12, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Tel:(858) 784-8793 Fax:(858) 784-2246 E-mail:quaranta{at}scripps.edu.

	Abstract

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Keratinocytes and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins 3ß1 and 6ß4. While 3ß1 mediates adhesion, spreading, and migration (Kreidberg, J.A. 2000. Curr. Opin. Cell Biol. 12:548–553), 6ß4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 112:411–418). We investigated a possible regulatory interplay between 3ß1 and 6ß4 in cell motility using HaCaT keratinocytes as a model. We found that 6ß4 antibodies inhibit 3ß1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by 3ß1 activation), and not when chemotactic (i.e., triggered by epidermal growth factor receptor). Inhibition of migration by 6ß4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti–6ß4 antibodies, activation of PI3-K is mediated by 6ß4-associated erbB-2, as indicated by erbB-2 autophosphorylation and erbB-2/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-2 abolishes inhibition of haptotaxis by anti–6ß4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of 3ß1 and 6ß4 integrins, (b) 6ß4-associated erbB-2 and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by 6ß4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM.

Key Words: erbB-2, integrin, keratinocytes, laminin-5, phosphoinositide 3-kinase

	Introduction

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Epithelial cells are separated from the connective tissue by the basement membrane (BM),¹ a network of extracellular matrix (ECM) polymers consisting of several laminin isoforms and type IV collagen, and connected by glycoproteins such as nidogen (Timpl 1996 ; Burgeson and Christiano 1997 ). Keratinocytes are the dominant epithelial cell type in the epidermis, a complex squamous epithelium that forms the outer surface of the skin (Priestley 1993 ) and that is separated from the underlying dermis by the BM. Contact of basal keratinocytes with the BM and their cell–cell interactions are essential for proper function by modulating cell polarity, proliferation, migration, and differentiation (Adams and Watt 1993 ; Burgeson and Christiano 1997 ; Fuchs et al. 1997 ).

Cell–ECM or cell–cell adhesion are mediated by integrins, /ß-heterodimeric transmembrane glycoprotein receptors (Hynes 1992 ). To reach high mechanical stability and resist the frictional stresses the skin is subjected to, the epidermal BM contains specialized anchoring complexes, in addition to conventional integrin-mediated cell–ECM linkages. Such anchoring complexes consist of hemidesmosomes, anchoring fibrils, and anchoring filaments with laminin-5 (Ln-5) as a major component (Burgeson and Christiano 1997 ). Basal keratinocytes express two Ln-5 integrin receptors, 3ß1 and 6ß4, which are recruited to distinct cell adhesion structures (Carter et al. 1990 ; Fuchs et al. 1997 ). 6ß4 is a component of hemidesmosomes, linking Ln-5 anchoring filaments on the outside of the cell with the keratin filament network inside the cell (Borradori and Sonnenberg 1999 ), thus anchoring keratinocytes to the BM. In contrast, 3ß1 is recruited to focal contacts and thereby links the ECM to components of the actin cyto-skeleton, mediating cell spreading and migration (Carter et al. 1990 ; Fuchs et al. 1997 ).

These two types of integrin-mediated adhesive junctions are likely to transmit distinct molecular signals to cells. Since integrins are not equipped with enzymatic activity, they need to associate with signaling molecules at the cell surface (Schwartz et al. 1995 ; Giancotti 1997 ; Porter and Hogg 1998 ; Giancotti and Ruoslahti 1999 , Schwartz and Baron 1999 ). 3ß1 is a typical integrin in terms of its structure, containing a short (50 amino acid) cytoplasmic ß1 tail (Sastry and Horwitz 1993 ) implicated in activation of focal adhesion kinase (FAK) (Schlaepfer et al. 1999 ; Sieg et al. 2000 ). This latter event is coupled to the turnover of focal adhesions and modifications of the cytoskeleton (Giancotti and Ruoslahti 1999 ; Schlaepfer et al. 1999 ; Ren et al. 2000 ), both critical in cell migration (Horwitz and Parsons 1999 ). Furthermore, 3ß1 is associated with transmembrane-4 superfamily proteins such as CD81- or CD151-forming complexes, which may regulate cell migration (Yauch et al. 1998 ; Testa et al. 1999 ).

In contrast, 6ß4 contains a unique ß4 cytoplasmic domain (1,000 amino acids) with no homology to other known ß subunits, which mediates association with the hemidesmosome cytoskeleton (Gil et al. 1994 ; Spinardi et al. 1995 ) and contains a tyrosine activation motif that upon phosphorylation can act as docking site for signaling molecules containing Src homology 2 domains. In primary keratinocytes, ligation of 6ß4 caused tyrosine phosphorylation of this motif, which recruited the adapter proteins Shc and Grb2 and sequentially activated mitogen-activated protein kinase (MAP kinase) pathways, indicating a role for 6ß4 in the regulation of keratinocyte proliferation (Mainiero et al. 1995 , Mainiero et al. 1997 ). Furthermore, in breast and colon carcinoma cells, 6ß4 was shown to activate phosphoinositide 3-kinase (PI3-K), leading to increased Matrigel invasion (Shaw et al. 1997 ).

Integrins not only use adapter proteins to interact with signaling pathways, but they are also in direct physical interaction with growth factor receptors (Miyamoto et al. 1996 ). For example, vß3 integrin was reported to be associated with activated insulin and PDGFß receptors (Schneller et al. 1997 ), and with vascular endothelial growth factor receptor-2 (Soldi et al. 1999 ). Furthermore, coimmunoprecipitation between ß1 integrin and the receptor for epidermal growth factor was demonstrated (Moro et al. 1998 ). Concerted action of integrins and growth factor receptors may be crucial to tightly control many biological processes, including cell motility during wound repair, inflammation, and organogenesis. Cell migration triggered by adhesion receptors is referred to as haptotaxis/haptokinesis, whereas cytokine and growth factor receptor-controlled motility is defined as chemotaxis/chemokinesis. Ligands for these receptors may occur either in a gradient (-taxis) or at a constant concentration (-kinesis) (Wells 2000 ).

During re-epithelialization of wounds, keratinocytes dissolve their stable attachment with the underlying BM and migrate over a provisional matrix, continuously expressing and depositing Ln-5 (Larjava et al. 1993 ; Yamada et al. 1996 ). These observations suggested a role for Ln-5 as a migratory substrate for keratinocytes. On the other hand, Ln-5 was also reported to inhibit keratinocyte migration and to promote establishment of cell anchoring hemidesmosomal complexes in quiescent BM zones (Yamada et al. 1996 ; O'Toole et al. 1997 ; Goldfinger et al. 1999 ). How can the same ECM component mediate two such different cell behaviors like migration and anchorage? In spite of a large body of information gathered by many laboratories (Carter et al. 1990 ; Xia et al. 1996 ; DiPersio et al. 1997 ; Fuchs et al. 1997 ; Mainiero et al. 1997 ; De Arcangelis et al. 1999 ; Goldfinger et al. 1999 ; Nguyen et al. 2000 ; Raghavan et al. 2000 ), there is still no satisfactory answer to this question. Investigating this problem may shed light on important processes such as wound healing and may also provide insight on how cells in general regulate static adhesion versus migration.

In this study, we attempted to characterize the signaling network that may regulate migration versus anchorage of keratinocytes on Ln-5, via the two Ln-5 binding integrins, 3ß1 and 6ß4. As a model, we used the nontumorigenic, spontaneously immortalized human keratinocyte cell line, HaCaT. We report that integrin 3ß1 mediates both haptotactic and chemotactic migration on Ln-5 in HaCaT keratinocytes. However, integrin 6ß4 may inhibit haptotaxis on Ln-5, but not chemotaxis, via a pathway that involves erbB-2 and PI3-K. Our results define distinct types of keratinocyte migration on Ln-5, and point to possibly general mechanisms whereby 3ß1 and 6ß4 are predominantly a migratory or an anchoring integrin, respectively, for epithelia in contact with Ln-5.

	Materials and Methods

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Cell Lines, Constructs, and Retroviral Infections
HaCaT (Boukamp et al. 1988 ) and A431 cells (American Type Culture Collection) were cultured in DMEM (4.5 g/liter glucose) containing 10% FCS. Primary human keratinocytes were purchased from Clonetics and cultured in completely defined medium (KGMD) according the manufacturer's protocol. Passages 3 and 4 were used for migration assays. Constructs encoding dominant-negative PI3-K (p85iSH2-N and p85SH2-C; Rodriguez-Viciana et al. 1997 ) were from J. Downward (Imperial Cancer Research Fund, London, UK) and the cDNA for the constitutive-active PI3-K (MM72cp3kFL; Jiang et al. 2000 ) was a gift from P.K. Vogt (The Scripps Research Institute). Construct HER2VEK753A (Messerle et al. 1994 ) encoding a dominant-negative erbB-2 variant was from N.E. Hynes (Friedrich Miescher Institute, Basel, Switzerland). All cDNAs were subcloned into the retroviral vector pLNCX (CLONTECH Laboratories, Inc.). Virus production in PT67 packaging cells (CLONTECH Laboratories, Inc.) and infection of HaCaT cells was performed as described in the manufacturer's protocol. A retroviral vector encoding enhanced green fluorescent protein was used to assess infection efficiency, which was at least 95% in each experiment.

Antibodies, Extracellular Matrix Molecules, and Reagents
The anti–CD151 monoclonal antibody (mAb) 1A5 (Testa et al. 1999 ) was provided by J.P. Quigley (The Scripps Research Institute). mAbs 5C11 (anti–CD151; Yauch et al. 1998 ), TS2/16 (anti–ß1; Hemler et al. 1984 ), and A3-X8 (anti–3; Weitzman et al. 1993 ) were gifts from M.E. Hemler (Dana-Farber Cancer Institute, Boston, MA). mAb 12F1 (anti–2; Pischel et al. 1987 ) was provided by V.L. Woods, Jr. (University of California, San Diego, San Diego, CA). Anti–ß4 mAbs AA3 and S3-41 and rabbit anti–6 IgG 6845 were produced in our laboratory (Tamura et al. 1990 ; Domanico et al. 1997 ). Commercially available integrin mAbs were ASC-1 (anti–3; Chemicon), P1B5 (anti–3; GIBCO BRL), GoH3 (anti–6; BD PharMingen), and P4C10 (anti–ß1; GIBCO BRL). Rabbit anti–FAK IgG (BD PharMingen) was used for immunoprecipitations and mAb anti–FAK and anti–P-Tyr mAb PY20 (Transduction Laboratories) for Western blotting. PI3-K subunit p110 was immunoprecipitated with mAb N-20 (Santa Cruz Biotechnology, Inc.) and mAb to PI3-K p85 subunit for Western blotting was purchased from Transduction Laboratories. Goat anti–AKT1 IgG (C-20) was from Santa Cruz Biotechnology, Inc. and rabbit IgG to phosphorylated AKT was from New England Biolabs, Inc. erbB-2 was immunoprecipitated with mAb c-neu (Ab-2; Oncogene Research Products) and analyzed in Western blots with mAb erbB-2 (Transduction Laboratories). Rabbit anti–ERK1/2 IgG was from Santa Cruz Biotechnology, Inc. and mAb to phosphorylated ERK1/2 was from New England Biolabs, Inc. Anti–FLAG mAb M2 was from Sigma-Aldrich. Fab fragments were generated by digestion of mAbs with 0.02 mg/ml papain (Sigma-Aldrich). Human collage IV and bovine fibronectin were from Sigma-Aldrich and Ln-5 deposited by the rat bladder carcinoma cell line 804G was purified in our laboratory. LY294002, PD98059, and tyrphostin AG 825 were from Calbiochem and EGF was from Sigma-Aldrich.

Migration and Adhesion Assays
In Transwell migration assays, the underside of the filters (8.0 µm, pore size; Costar) was coated at 4°C overnight (ON) with 0.25 µg/ml Ln-5, 1 µg/ml collagen IV, or 10 µg/ml fibronectin in PBS. Filters were washed twice with PBS containing 0.2% Tween-20 (PBST), and then blocked with 5% dry milk in PBST at room temperature (RT) for 2 h. Cells (HaCaT: 1.2 x 10⁵ cells/filter; A431: 6 x 10⁴ cells/filter, primary keratinocytes: 8 x 10⁴ cells/filter) in migration medium, MM (culture medium without FCS) were preincubated with antibodies, reagents or vehicle for 30 min at RT before plating on filters that were washed twice with PBS after blocking. EGF was added to the lower chamber only, whereas antibodies, reagents, or vehicle were present in both chambers. Cells were maintained at 37°C for 5 h (primary keratinocytes for 15 h), and were then fixed and stained using the LeukoStat kit (Fisher Scientific). The uncoated side of each filter was wiped with a cotton swap to remove cells that had not migrated through the filter. Filters were viewed under bright-field optics and stained cells were counted in eight fields (using a 20x objective) from each of two filters for each condition, determining the mean number of cells counted per field. Each experiment was done at least three times and results are expressed as mean ± SD of relative cell migration with nonstimulated cells set as 1.

Scratch assays were performed in 24-well plates coated ON at 4°C with 0.25 µg/ml Ln-5 in PBS. HaCaT cells (6 x 10⁵/well) in MM were seeded and incubated at 37°C for 2 h. Then, cell layers were wounded with a plastic pipet tip and washed three times with MM. The denuded surfaces were recoated with 0.25 µg/ml Ln-5 in MM for 1 h at 37°C. Cell layers were washed again, TS2/16 (40 µg/ml) and/or EGF (1 ng/ml) were added, and cells were incubated for 14 h at 37°C. Photographs of identical locations within each scratch were taken before, and 14 h after, addition of TS2/16, EGF, or both stimuli.

Adhesion assays were performed as described by Goodwin and Pauli 1995 , with minor modifications. Microtiter 96-well plates were coated with 1 µg/ml Ln-5, 1 µg/ml collagen IV, or 10 µg/ml fibronectin ON at 4°C, washed twice with PBS, and blocked with 5% dry milk in PBS for 2 h. Cells (6 x 10⁴ cells/100 µl per well) in MM were preincubated with antibodies for 30 min at RT before plating in wells that were washed twice with PBS after blocking. EGF was added at the time of plating. Cells were maintained at 37°C for 30 min, and then 2 x 100 µl of Percoll floatation medium [73 ml Percoll (density 1.13 g/ml; Amersham Pharmacia Biotech), 27 ml distilled water, and 900 mg NaCl] were added to each well. Adherent cells were fixed for 15 min with 50 µl/well of 25% glutaraldehyde (Sigma-Aldrich), washed with PBS, and stained with crystal violet (0.5% in 20% MeOH) for 10 min. Excess dye was washed off with water and absorbance was measured at 595 nm. Bars represent mean absorbance ± SD of each condition tested in triplicates. All values have had background substracted that represents cell adhesion to wells blocked with milk. Experiments were done three times.

Biochemical Methods

Analysis of AKT-, erbB-2-, ERK1/2-, and FAK Phosphorylation, and erbB-2/p85 Coimmunoprecipitation.
Cell culture dishes (6 cm) were coated with 1 µg Ln-5/dish in PBS at 4°C ON, and then prewarmed to 37°C for 1 h. Serum-starved cells (2–4 x 10⁶) in MM were preincubated with antibodies for 30 min at 37°C in suspension before plating onto ligand-coated dishes. (If cells were costimulated with TS2/16 and a second antibody, the latter was added 5 min before TS2/16). After 30–60 min at 37°C, the attached cells were rinsed in PBS and lysed for 1 h on ice in 0.5 ml lysis buffer containing 40 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 6 mM EDTA, 100 mM NaF, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF, 10 µg/ml leupeptin, and one tablet/50 ml of complete protease inhibitor cocktail (Roche Diagnostics Corp.). When FAK was analyzed, the lysis buffer was supplemented with 10% glycerol, 1% sodium deoxycholate, and 0.1% SDS. Lysates were centrifuged at maximum speed for 10 min in a microcentrifuge and antibodies were added to the supernatant for 3 h at 4°C on a rotatory shaker. Then, antibodies were collected with protein-G Sepharose for 1 h at 4°C on a rotatory shaker. The protein complexes were washed three times with ice-cold lysis buffer before boiling in SDS-PAGE loading buffer. Proteins were separated on acrylamide SDS-PAGE gels, blotted, and blots were incubated with the primary antibodies diluted 1:2,000 in 5% milk in PBS containing 0.1% Tween-20. For PY20, milk was replaced by 4% BSA. Each sample was divided in two and analyzed for total AKT, FAK, or erbB-2 content or for phosphorylation or p85 content using the ECLplus system (Amersham Pharmacia Biotech) and a STORM 860 Fluorimeter.

Integrin-erbB-2 Coimmunoprecipitations.
Per sample, 3 x 10⁷ cells were lysed in lysis buffer as described above. Lysates were centrifuged at maximum speed for 10 min and supernatants were precleared for 1 h with protein-G Sepharose. Precleared supernatants were subjected to immunoprecipitation, SDS-PAGE, and Western blotting as described above.

PI3-K Enzyme Assay.
PI3-K activity was measured according to Jiang et al. 1998 . In brief, cells and cell culture dishes were treated as described above. Per dish, 4 x 10⁶ cells were seeded and incubated for 1 h at 37°C. Then, cells were washed with PBS and lysed in lysis buffer for 30 min on ice. Lysates were cleared by centrifugation and total protein concentration was determined. Equal amounts of protein were incubated with precoupled protein-G Sepharose (precoupling: 2 µg anti–p110 antibody were incubated with 50 µl protein-G Sepharose slurry for 1 h at RT) for 2 h at 4°C. Protein complexes were washed twice with lysis buffer and three times with 50 mM Hepes, pH 8.0, containing 160 mM NaCl and 10 mM EDTA. Immunoprecipitates were resuspended in 50 µl kinase buffer [50 mM Hepes, pH 8.0, 5 mM MgCl₂, 0.2 mg/ml phosphatidylinositol (1 mg/ml stock, sonicated), and 60 µM ATP] and kinase reactions were started by adding 20 µCi -P³² ATP. After 10 min at RT, 60 µl of 1 N HCl were added to stop the reactions, and lipids were extracted twice with 160 µl of chloroform:methanol (1:1). Pooled extracts were evaporated in a Speedvac, resuspended in 10 µl chloroform, and subjected to thin layer chromatography for 4 h at RT, using as running solvent a mixture of 65 ml MeOH, 48 ml chloroform, 36 ml pyridine, 60 µl ethoxyquin, 6 ml H₂O, 2.4 ml formic acid, 9.6 g boric acid, and 300 mg BHT. Before sample application, thin layer chromatography plates were pretreated for 10 s in a mixture of 81 ml H₂O, 3 ml 5 N NaOH, 165 ml EtOH, and 2.27 g CDTA, and then incubated at RT for 30 min and at 100°C for 10 min. Assays were analyzed using a PhosphorImager.

	Results

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Integrin 3ß1 Drives Haptotactic and Chemotactic Keratinocyte Migration on Ln-5
To study whether there is an interplay between 3ß1 and 6ß4 integrins in regulating keratinocyte motility on Ln-5, we first set up conditions under which haptotaxis and chemotaxis of keratinocytes could be analyzed. In Transwell chamber assays (Fig 1 A), HaCaT cells showed spontaneous migration towards Ln-5 that was small but highly reproducible (Fig 1 A). We consider this spontaneous migration haptotactic since it appears to be dependent on adhesion receptor/substrate interaction, with no soluble factor added. Addition of TS2/16, an "activating" anti–ß1 integrin antibody (Humphries 1996 ) caused a fivefold increase in migration (Fig 1 A). We consider this effect an enhancement of haptotactic migration and used TS2/16 in most subsequent measurements of haptotaxis since it amplifies signal-to-noise ratio in the assay.

View larger version (113K): [in this window] [in a new window]   Figure 1. Haptotactic migration of HaCaT cells on Ln-5 can be enhanced by the stimulatory anti–ß1 integrin antibody TS2/16. (A) Cell migration assays were performed in Transwell chambers coated with 0.25 µg/ml Ln-5. HaCaT cells were incubated in suspension at RT for 30 min in serum-free culture medium with or without TS2/16 (anti–ß1, 40 µg/ml). Then, aliquots were seeded and the plates were incubated at 37°C for 5 h. EGF (1 ng/ml) was present in the lower chamber only, whereas TS2/16 was in both chambers. Migration was quantified by counting cells migrated through filters (eight microscopic fields on each of two filters for each condition). In absence of any stimuli, 12 cells were counted per microscopic field. In the bar graph, results are expressed as mean ± SD (n = 3) of relative cell migration with nonstimulated cells set at 1. (Bottom) Micrographs of fixed and stained HaCaT cells migrated onto the lower surfaces of Transwell filters (bar, 50 µm). (B) Effects of TS2/16 and EGF in scratch assays. In this assay, cells migrate from the edges of "scrape-wounded" monolayers (arrows) to cover the denuded surface (space between arrows). Scrape wounds were made in serum-free HaCaT cultures plated on Ln-5. Denuded surfaces were recoated with Ln-5 and the wounded cultures were allowed to re-epithelialize for 14 h at 37°C in the presence of TS2/16 (anti–ß1, 40 µg/ml), EGF (1 ng/ml), or both stimuli. Micrographs of wounded, nonfixed cell layers at 0 and 14 h after treatment (bar, 100 µm).

With EGF (1 ng/ml), a well-documented chemoattractant (Wells 2000 ), there was an 25-fold increase in migration (Fig 1 A). By definition, this increase is due to chemotaxis. Combined exposure to TS2/16 and EGF resulted in an additive effect (Fig 1 A).

We further tested TS2/16 and EGF in the scratch assay (Fig 1 B), considered to be an in vitro model for keratinocyte migration occurring during wound healing (Wells 2000 ). Scratch closure after 14 h was enhanced by treatment with either TS2/16 or, more markedly, EGF (Fig 1 B). With both agents together, an additive enhancing effect was detectable on keratinocytes migrating from the edges (Fig 1 B). These results support a distinction between TS2/16-enhanced and EGF-induced migration, indicated by the Transwell assay.

In the Transwell assay, both TS2/16- and EGF-induced HaCaT migration on Ln-5 required integrin 3ß1 since migration was inhibited by antibodies to 3 (P1B5, A3-X8) and ß1 (P4C10) integrins, but not by control anti–2 (12F1) antibody (Fig 2A and Fig B). While the effect of P1B5 and P4C10 antibodies may be indirect (i.e., a consequence of adhesion inhibition; Fig 2 C), A3-X8 antibody is known to block migration but not adhesion (Weitzman et al. 1993 ), indicating that HaCaT migration on Ln-5, under our conditions, is carried out by integrin 3ß1. Furthermore, antibodies to CD151, a tetraspanin stoichiometrically associated with 3ß1 (Yauch et al. 1998 ; Testa et al. 1999 ) also blocked migration (Fig 2 A), but had little effect on adhesion (C).

View larger version (19K): [in this window] [in a new window]   Figure 2. Integrin 3ß1 mediates both TS2/16-stimulated haptotaxis and EGF-stimulated chemotactic migration in HaCaT keratinocytes. (A) Haptotactic migration of HaCaT cells was stimulated with the anti–ß1 antibody TS2/16, as in Fig 1 A. Where indicated, cells were preincubated at RT for 15 min with P1B5 (anti-3, 10 µg/ml), P4C10 (anti–ß1, 1:50 dilution), A3-X8 (anti–3, 10 µg/ml), 5C11 (anti–CD151, 20 µg/ml), 1A5 (anti–CD151, 50 µg/ml), or 12F1 (anti–2, 40 µg/ml) antibodies. Migration was quantified as described in Fig 1 A. (B) Inhibitory effect of A3-X8 antibody (anti–3, 10 µg/ml) on HaCaT cell migration stimulated either by EGF alone or by EGF and TS2/16 together. Migration assays were performed as described in Fig 1 A. (C) Adhesion assays of HaCaT cells pretreated, as indicated, either with antibodies (as described in A) or with EGF (1 ng/ml added at the time of seeding). (Bottom, gray bars) Cells were TS2/16 stimulated in addition to treatment with indicated antibodies or EGF. Tests were performed with aliquots seeded in triplicates in Ln-5–coated (1 µg/ml) 96-well plates. Cells were allowed to adhere for 30 min at 37°C, and then nonadherent cells were removed, and adherent cells were fixed and stained. Results of no less than three representative experiments are expressed as mean absorbance at 595 nm ± SD.

Preincubation with TS2/16 increased adhesion to Ln-5 approximately twofold, indicating that TS2/16 induces an increase in 3ß1 avidity for Ln-5. Furthermore, an antibody to 3 (P1B5) almost completely blocked this enhanced adhesion (Fig 2 C), supporting 3ß1 dependence. On the other hand, no effect of EGF in adhesion assays was observed (Fig 2 C), suggesting no EGF influence on 3ß1 avidity.

To further characterize TS2/16-induced migration, we tested the involvement of two possible 3ß1 effectors, FAK and extracellular signal-regulated kinase (ERK) MAP kinase. All ß1 integrins share the ability to promote assembly of focal adhesions and to activate FAK (Giancotti 1997 ). Indeed, in cells plated on Ln-5, tyrosine phosphorylation of FAK was increased when compared with cells plated on plastic (Fig 3 A). This phosphorylation was amplified in the presence of TS2/16, correlating with integrin activation and stimulation of adhesion and migration by this antibody. TS2/16-induced FAK phosphorylation was also seen in cells kept in suspension. This is consistent with the finding that TS2/16 is an activating antibody that induces changes in integrin shape in a ligand-independent manner (Humphries 1996 ). As a control, antibodies to 6ß4 had no influence on FAK phosphorylation (Fig 3 A). A3-X8, the anti–3 antibody that blocked migration but had no influence on adhesion, was without effect on FAK phosphorylation, neither when added alone nor together with TS2/16. Therefore, inhibition of migration by A3-X8 was not due to decreased phosphorylation of FAK.

View larger version (30K): [in this window] [in a new window]   Figure 3. Analyses of FAK and ERK1/2 as candidate downstream signaling molecules in 3ß1-dependent haptotaxis. (A) FAK phosphorylation. Changes in FAK phosphorylation state relative to cells plated on Ln-5 are observed after TS2/16, but not A3-X8 or anti–6ß4 antibody treatment. Cells were incubated at 37°C in suspension for 30 min with TS2/16 (anti–ß1, 40 µg/ml) and/or GoH3 (anti–6, 15 µg/ml), AA3 (anti–ß4, 40 µg/ml), and A3-X8 (anti–3, 10 µg/ml) before seeding in noncoated or coated (1 µg Ln-5/dish) dishes and incubation for 30 min at 37°C. Total cell lysates were subjected to immunoprecipitation with an anti–FAK antibody and Western blotting with antibodies to phosphotyrosine (P-Tyr) or FAK. Ratios of phosphorylated FAK to total FAK protein is quantified in the bar graph below. Mean ± SD (n = 3) of relative FAK phosphorylation intensity and one representative experiment is depicted. (B) Inhibition of TS2/16-induced (gray bars) HaCaT haptotaxis by MEK inhibitor PD98059 (PD) on Ln-5–coated filters. Cells were pretreated with PD (50 µM) or vehicle (DMSO). Migration assays were performed and results expressed as in Fig 1 A. (C) Analysis of ERK1/2 phosphorylation. ERK1/2 phosphorylation relative to cells plated on Ln-5 alone is increased by TS2/16, and this stimulation is blocked by A3-X8. Antibodies to 6ß4 have no influence on ERK1/2 activation. Phosphorylated ERK1/2 bands (P-ERK1/2) are shown above total ERK1/2 protein bands (ERK1/2), and their ratio is quantified in the bar graph below. Mean ± SEM (n = 3) of relative ERK1/2 phosphorylation intensity and one representative experiment is depicted. Assay conditions as in A, except that anti–ERK1/2 or anti–phospho-ERK1/2 antibodies were used.

MAP kinases, such as ERK1 and ERK2, are known to play a stimulatory role in regulation of cell migration (Lauffenburger and Horwitz 1996 ; Klemke et al. 1997 ). Therefore, we tested for the involvement of these enzymes in our system. The ERK kinase (MEK)-specific inhibitor PD98059 prevented TS2/16-stimulated HaCaT migration on Ln-5 (Fig 3 B), suggesting that ERK1 and ERK2 are possible mediators of haptotaxis. In contrast, PD98059 had no effect in adhesion assays (data not shown), indicating that ERK1/2 are not involved in regulating cell adhesion to Ln-5. Next, ERK1/2 phosphorylation was analyzed in HaCaT cells plated on plastic or on Ln-5 in the presence of anti–integrin antibodies. An increase in ERK1/2 phosphorylation was detected on Ln-5 when compared with the plastic control (Fig 3 C). Addition of TS2/16 enhanced phosphorylation of ERK1/2 further, whereas A3-X8 had a slightly inhibitory effect. If cells were treated with TS2/16 and A3-X8 together, the stimulatory effect of TS2/16 was blocked by A3-X8. Therefore, inhibition of TS2/16-induced migration by A3-X8 may be due to decreased ERK1/2 activation in the presence of A3-X8. As a control, antibodies to 6ß4 had no influence on ERK1/2 phosphorylation, neither when added alone nor in the presence of TS2/16.

These results suggest an involvement of ERK1/2, in the regulation of 3ß1 controlled keratinocyte haptotaxis on Ln-5.

6ß4 Inhibits Haptotactic Cell Migration via Stimulation of PI3-K
We then tested the role of 6ß4 in haptotactic keratinocyte migration, using chemotactic migration as a comparison. Spontaneous haptotactic migration on Ln-5 was readily blocked by antibodies to the integrin subunits 6 (GoH3) or ß4 (AA3) (Fig 4 A). TS2/16-enhanced migration was equally inhibited by antibodies to 6ß4, including S3-41, which recognizes the 6ß4 heterodimer. This inhibition was induced also by the Fab fragments of S3-41 and AA3, suggesting it did not require 6ß4 clustering but simply binding of the antibodies. This 6ß4 inhibitory effect was Ln-5 specific since TS2/16 was also able to enhance migration on collagen IV, but in this case it was not inhibited by GoH3 (Fig 4 A).

View larger version (23K): [in this window] [in a new window]   Figure 4. Integrin 6ß4 antibodies inhibit 3ß1-dependent haptotactic, not chemotactic migration, and have no effect on adhesion in HaCaT cells. (A) HaCaT haptotaxis was stimulated with TS2/16 as described in Fig 1 A. Where indicated, cells were also pretreated as in Fig 2 A with S3-41 antibody (anti–6ß4, 40 µg/ml), S3-41 Fab fragments (13.4 µg/ml), GoH3 (anti–6, 15 µg/ml), AA3 (anti–ß4, 40 µg/ml), or AA3 Fab fragments (13.4 µg/ml). Transwell filters were coated with either 0.25 µg/ml Ln-5 or 1 µg/ml collagen IV. Migration assays performed and results expressed as in Fig 1 A. (B) Anti–6 antibody GoH3 (15 µg/ml) has no effect on HaCaT chemotactic migration stimulated by EGF (1 ng/ml), but inhibits partially migration of cells stimulated with a combination of EGF and TS2/16 (40 µg/ml). Migration assays performed and results expressed as in Fig 1 A. (C) Anti–6ß4 antibodies (GoH3, 15 µg/ml, or AA3, 40 µg/ml) have no effect on HaCaT cell adhesion. (Bottom, gray bars) Cells pretreated with TS2/16 in addition to indicated antibodies (as in Fig 2 C). Antibody treatment of cells, adhesion assays, and result analyses were performed as described in Fig 2 C.

In contrast to haptotaxis, EGF-induced chemotactic migration was not affected by GoH3 (Fig 4 B). If both EGF and TS2/16 were added, GoH3 showed partial inhibition (Fig 4 B), presumably corresponding to that part of migration that was TS2/16 induced. Thus, 6ß4 can influence 3ß1 controlled migration, but only when it is haptotactic.

Neither anti–6ß4 antibodies (AA3 and GoH3) had any influence on adhesion to Ln-5, nor did they inhibit the increased adhesiveness induced by TS2/16 (Fig 4 C). Therefore, like for A3-X8, the observed decrease in migration may be a direct effect of 6ß4 on signals regulating motility in HaCaT cells.

Next, we were therefore interested in identifying a candidate signaling molecule responsible for 6ß4-linked downmodulation of haptotaxis on Ln-5. PI3-K is one such likely candidate because, in breast carcinoma cells, PI3-K was shown to be activated by the anti–6 antibody GoH3 (O'Connor et al. 1998 ). Wortmannin, a PI3-K blocker, abolished inhibition of Ln-5 migration by anti–6ß4 antibodies (data not shown). LY294002, a more stable and specific PI3-K blocker, showed stronger effects (Fig 5 A) and was preferred in further experiments. This finding suggests that downstream of 6ß4, PI3-K may mediate inhibition of 3ß1-dependent haptotactic migration. In contrast, a decrease in migration by A3-X8 is not PI3-K dependent since LY294002 did not overcome the inhibitory effect of this antibody (Fig 5 A).

View larger version (25K): [in this window] [in a new window]   Figure 5. PI3-K blocker LY294002 releases inhibition of HaCaT haptotaxis by anti–6ß4 antibodies. Anti–6ß4 antibodies activate PI3-K in HaCaT cells. (A) Haptotactic migration was stimulated in HaCaT cells by treatment with TS2/16 (as in Fig 1 A). Where indicated, cells were pretreated with the PI3-K blocker LY294002 (LY) (20 µM) or vehicle (DMSO), GoH3 (anti–6, 15 µg/ml), AA3 (anti–ß4, 40 µg/ml), or A3-X8 (anti–3, 10 µg/ml). Migration assays performed and results expressed as in Fig 1 A. (B) PI3-K activity assay. HaCaT cells were allowed to adhere to dishes coated with Ln-5 (1 µg) or indicated antibodies (GoH3, 30 µg; AA3, 50 µg; 1A5, 50 µg; TS2/16, 50 µg; ASC-1, 30 µg) or were kept in suspension (control) for 1 h. Total cell lysates were prepared and equivalent amounts of total proteins were subjected to immunoprecipitation and kinase assay. Phosphorylated lipids were resolved by thin layer chromatography. Mean ± SEM (n = 3 or 4) of percentage of control activity and one representative chromatogram are depicted. PI(3)P, phosphatidylinositol 3-phosphate. (C) AKT phosphorylation. After pretreatment with antibodies (GoH3, 15 µg/ml, or AA3, 40 µg/ml) for 30 min, cells were seeded in dishes coated with Ln-5 (1 µg/dish). After incubation for 30 min at 37°C, total cell lysates were prepared and subjected to immunoprecipitation with an anti–AKT antibody and Western blotting with antibodies to phosphorylated AKT (P-AKT) or AKT (AKT). Mean ± SEM (n = 3) of relative AKT phosphorylation intensity and one representative experiment is shown.

To confirm that 6ß4 is capable of activating PI3-K in HaCaT cells, we used a lipid kinase assay to detect 6ß4-induced PI3-K activation. Endogenous PI3-K isolated with a p110-specific antibody showed increased enzymatic activity in cells plated on anti–6 (GoH3) or anti–ß4 (AA3) antibodies, but not on anti–CD151 antibody 1A5, anti–3 (ASC-1), or anti–ß1 (TS2-16) integrin antibodies (Fig 5 B), suggesting 6ß4 integrin-specific PI3-K activation. Production of PI3-P was also increased in cells plated on Ln-5 compared with cells in suspension (Fig 5 B).

As additional proof that binding of AA3 or GoH3 to 6ß4 results in activation of PI3-K, we tested activation of the downstream effector of PI3-K, AKT (Kandel and Hay 1999 ). Indeed, phosphorylation of AKT immunoprecipitated from cells plated on Ln-5 was higher than in cells plated on plastic, and was further increased in the presence of AA3 or GoH3 (Fig 5 C).

These results suggest that, in our system, PI3-K is an effector for 6ß4 inhibition of haptotactic migration. To confirm this conclusion, dominant-negative and constitutive-active PI3-K variants were transiently overexpressed in HaCaT cells. To this end, we used a retroviral expression system, since other methods, like calcium phosphate coprecipitation or liposome-mediated transfection, failed because HaCaT cells were not able to migrate after these treatments. In HaCaT cells infected with retrovirus encoding a dominant-negative regulatory subunit p85iSH2-N, migration could still be stimulated by TS2/16. However, inhibition of migration by AA3 was no longer possible (Fig 6). Thus, the anti–ß4 antibody can only act as inhibitor if a functional PI3-K is available in these cells. (Similar results were found with p85iSH2-C and GoH3, respectively, data not shown.) In contrast, overexpression of a constitutive-active catalytic subunit p110myr abolished the stimulatory effect of TS2/16 on migration (Fig 6).

View larger version (25K): [in this window] [in a new window]   Figure 6. Dominant-negative PI3-K p85 subunit abolishes inhibition of haptotactic migration by 6ß4, while constitutive active PI3-K p110 subunit abolishes stimulation of haptotactic migration by TS2/16. HaCaT cells infected with retrovirus encoding a dominant-negative regulatory PI3-K domain (p85 iSH2-N) or a constitutive-active catalytic PI3-K domain (p110myr) or mock infectants were subjected to haptotactic migration assays as described in Fig 1 A. Data are mean ± range of two duplicate experiments. Nonstimulated cells were set as 1. *Not significantly different values, P > 0.05; Student's t test. **Significantly different values, P < 0.05. (Right) Expression of infected cDNAs confirmed by immunoblotting of equal amounts of total cell lysates (antibodies indicated above panels).

Taken together, these results support the concept that, in HaCaT cells, 6ß4-dependent inhibition of haptotactic migration operates via a class IA PI3-K pathway, with p110 as the responsible catalytic subunit.

PI3-K Inhibits Haptotactic Migration, but Plays a Stimulatory Role in Chemotactic Migration
In apparent disagreement with our findings, PI3-K has been invariably associated with a stimulatory role in migration (Derman et al. 1997 ; Keely et al. 1997 ; Gambaletta et al. 2000 ). To the best of our knowledge, though, most reports referred to chemotactic migration (for review, see Wells 2000 ), rather than haptotaxis. Indeed, in our system, PI3-K is involved in stimulating HaCaT cell migration when this is of the chemotactic type, as indicated by inhibition of EGF-induced migration by LY294002 (Fig 7 A). On the other hand, LY294002 had no effect on haptotactic, TS2/16-induced migration (Fig 7 B). On collagen IV, another ECM component, HaCaT cells stimulated with TS2/16 behaved exactly as on Ln-5. In summary, PI3-K can play an alternative role in HaCaT motility: if migration is chemotactic, then PI3-K plays a stimulatory role; if migration is haptotactic, then PI3-K plays an inhibitory role.

View larger version (25K): [in this window] [in a new window]   Figure 7. PI3-K blocker LY294002 inhibits chemotaxis but not haptotaxis in HaCaT cells. (A) Chemotactic migration was stimulated with EGF, and cells were pretreated with PI3-K blocker LY294002 or vehicle (DMSO) as in Fig 1 A and 3 B, respectively. (B) Haptotactic migration was performed as in Fig 1 A on Transwell filters coated with Ln-5 (0.25 µg/ml) or collagen IV (1 µg/ml). HaCaT cells were treated (gray bars) with TS2/16 (anti–ß1, 40 µg/ml) to stimulate haptotaxis. Where indicated, cells were pretreated as in A. Results are expressed as mean ± SD (n = 3) of relative cell migration with nonstimulated cells set as 1.

To ensure that these findings are general to keratinocytes, rather than HaCaT specific, migration experiments were also performed with primary keratinocytes and with A431, an epidermoid squamous carcinoma cell line. Similar to HaCaT, primary keratinocytes showed increased migration in the presence of TS2/16 (Fig 8 A). This stimulation was inhibited by antitetraspanin antibody 1A5 and by GoH3 (anti–6). A431 cells showed an 17-fold higher basal migration than HaCaT (data not shown). Nonetheless, similar to HaCaT, TS2/16 stimulated A431 migration on Ln-5 approximately twofold, and this increase was inhibited by antitetraspanin antibody 1A5, anti–6ß4 integrin antibody S3-41, and by GoH3 (anti–6) (Fig 8 B). The 6ß4-mediated inhibition of A431 motility also appeared to be linked to a PI3-K pathway since LY294002 abolished it (Fig 8 B). (Note that the effect of LY294002 could not be analyzed in primary keratinocytes since they did not survive treatment with this reagent.) Furthermore, like HaCaT, A431 migration in the presence of TS2/16 increased on collagen IV (Fig 8 B).

View larger version (24K): [in this window] [in a new window]   Figure 8. Primary keratinocytes and A431 epidermoid carcinoma cells behave like HaCaT cells: TS2/16 enhances haptotaxis, anti–6ß4 antibodies inhibit TS2/16-induced haptotaxis, and PI3-K blockers abolish the anti–6ß4 antibody-mediated inhibition of haptotaxis. (A) Primary keratinocytes were stimulated to migrate haptotactically with TS2/16 antibody (gray bars) as in Fig 1 A. Where indicated, they were pretreated with antibodies to 6 (GoH3, 15 µg/ml) or CD151 (1A5, 50 µg/ml). Migration was performed on filters coated with Ln-5 (0.5 µg/ml) and quantified after 15 h at 37°C as in Fig 1 A. Results pooled from two experiments are expressed as in Fig 1 A. (B) A431 cells were tested in haptotactic migration assays performed as in Fig 1 A and 4 A, except that half the number of cells/well was seeded (6 x 105 cells/ml).

These results showed that our findings on HaCaT cells are likely to be of general applicability to keratinocytes. We then carried out further investigations on possible links between integrin 6ß4 and PI3-K that may inhibit 3ß1-dependent haptotactic migration.

erbB-2 May Be a Signaling Link between 6ß4 and PI3-K
Class IA PI3-K enzymes are stimulated by receptors with intrinsic protein tyrosine kinase activity (Wymann and Pirola 1998 ). Integrin 6ß4 has no such activity, but it was shown to be associated with the EGF receptor family member erbB-2 in human mammary and ovarian carcinoma cell lines (Falcioni et al. 1997 ). This interaction may provide a signaling link between 6ß4 and PI3-K. To address this possibility, we first tested whether 6ß4 is physically associated with erbB-2 in HaCaT cells. Indeed, in coimmunoprecipitation experiments, erbB-2 was precipitated with the anti–6 antibody GoH3 and with AA3 (anti–ß4), but not with the anti–ß1 antibody TS2/16 (Fig 9 A). In addition, the presence of 6ß4/erbB-2 complexes was supported by the fact that an antibody to erbB-2 precipitated integrin subunit 6 (Fig 9 A).

View larger version (59K): [in this window] [in a new window]   Figure 9. 6ß4-associated erbB-2 becomes phosphorylated and binds PI3-K p85 subunit in the presence of anti–6ß4 antibodies. (A) Coimmunoprecipitation of erbB-2 with 6ß4. (Left) erbB-2 (arrow) is present in lanes corresponding to HaCaT lysates subjected to immunoprecipitation with antibodies to erbB-2, 6-integrin (GoH3), or ß4-integrin (AA3), but not to ß1-integrin (TS2/16). Immunocomplexes were analyzed by Western blotting with an antibody to erB-2. *Background band present in all lanes. (Right) 6 reactivity is present in cell lysates subjected to immunoprecipitation with antibodies to erbB-2 or 6-integrin (GoH3), but not with control anti-MHCI antibody W6/32. Immunocomplexes were analyzed by Western blotting with an antibody to the 6-integrin. (B) erbB-2 phosphorylation in response to Ln-5 and 6ß4 antibodies (GoH3 and AA3), but not anti–ß1 antibody TS2/16. Ratio of phosphorylated bands (P-erbB-2) versus protein bands (erbB-2) is represented in the bar graph below (mean ± SD, n = 3). Assay conditions are as in Fig 3, except that an antibody to erbB-2 was used for immunoprecipitations and Western blots. (C) Coimmunoprecipitation of erbB-2 with p85 PI3-K. The PI3-K subunit p85 is coprecipitated by anti–erbB-2 only in cells that were GoH3 treated. Cells were pretreated with GoH3 (15 µg/ml) for 30 min at 37°C, and then seeded on dishes coated with Ln-5 (1 µg/dish). After incubation for 60 min, total cell lysates were prepared and subjected to immunoprecipitation with an anti–erbB-2 antibody and Western blotting with antibodies to p85 PI3-K or erbB-2. Representative of three experiments.

If erbB-2 is necessary for 6ß4-mediated PI3-K activation, stimulation with AA3 or GoH3 should result in erbB-2 autophosphorylation, leading to subsequent recruitment of the PI3-K regulatory domain p85. Phosphorylation of erbB-2 was higher in cells plated on Ln-5 than on uncoated dishes. This effect was further increased when cells were treated with AA3 or GoH3, whereas TS2/16 was without effect (Fig 9 B). Treatment with GoH3 was also effective in the absence of ECM ligand, as seen in cells stimulated in suspension.

Next, we investigated physical interactions between erbB-2 and PI3-K. Coimmunoprecipitation experiments showed that stimulation with GoH3 leads to increased association of p85 with erbB-2 (Fig 9 C), providing a means for triggering increased PI3-K activity.

To substantiate the role of erbB-2 in our system, the effect of the erbB-2–specific inhibitor Tyrphostin AG 825 (Tsai et al. 1996 ) was investigated in migration assays. This compound abolished the inhibitory effect of AA3 and GoH3 on TS2/16-stimulated migration on Ln-5 (Fig 10 A), indicating that the presence of functional erbB-2 is required for 6ß4-mediated inhibition of TS2/16-induced haptotaxis. Finally, the involvement of erbB-2 was also demonstrated by the finding that TS2/16-stimulated HaCaT cells overexpressing a dominant-negative erbB-2 variant could no longer be blocked with AA3 when migrating on Ln-5 (Fig 10 B).

View larger version (33K): [in this window] [in a new window]   Figure 10. Analyses of erbB-2 as a link between 6ß4 and PI3-K for inhibiting 3ß1-dependent haptotaxis. (A) HaCaT haptotaxis, induced as in Fig 1 A, is blocked by anti–6ß4 antibodies, but not in the presence of AG826 (100 nM), a specific erbB-2 inhibitor. Cells were treated with antibodies, AG826 or vehicle (DMSO) as in Fig 3 B. Assays performed and result analyses as in Fig 1 A. (B) HaCaT cells infected with retrovirus encoding dominant-negative erbB-2 (HER2VEK753A), but not mock infectants, fail to display inhibition of haptotaxis by anti–6ß4 antibodies. Migration assays with TS2/16 stimulation performed as in Fig 6. Data shown are mean values ± range of two duplicate experiments, with nonstimulated cells set at 1. *Significantly different values, P < 0.1, Student's t test. (Right) Expression of infected cDNA confirmed by immunoblotting of equal amounts of total cell lysates with an anti–erbB-2 antibody.

In summary, we provide evidence that erbB-2 mediates 6ß4-controlled stimulation of PI3-K in HaCaT cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

In this paper, we investigated integrin-dependent signaling that regulates haptotactic migration of keratinocytes on one of their natural substrates, Ln-5. We obtained results that may be useful to understand the haptotactic component of migration in epithelial cells in general (e.g., during tissue remodeling and regeneration) or in BM crossing by transformed epithelial cells.

Our conclusions can be summarized as follows: (a) one of two integrins that bind Ln-5, 3ß1, drives haptotactic as well as chemotactic migration of keratinocytes; (b) the other Ln-5–binding integrin, 6ß4, inhibits haptotactic, but not chemotactic migration; (c) 6ß4 interferes with keratinocyte haptotaxis via stimulation of PI3-K; (d) PI3-K inhibits only haptotactic migration, whereas it has a stimulatory role in chemotactic migration; (e) erbB-2 is a signaling link to PI3-K for the inhibition of 3ß1-dependent haptotaxis by 6ß4; and (f) the interplay between integrins 3ß1 and 6ß4 affects migration, but not adhesion.

These conclusions are based on results obtained in Transwell migration assays, in which HaCaT cells showed haptotactic migration spontaneously (to a low level) or after stimulation with the integrin-activating antibody TS2/16 (to a higher level), as well as chemotactic migration after exposure to EGF. In all cases, 3ß1 was the integrin-mediating migration as concluded from antibody blocking experiments. Of particular importance to this conclusion were data accumulated with anti–CD151 and A3-X8 antibodies, which interfere with 3ß1-dependent migration, not adhesion (Weitzman et al. 1993 ; Yauch et al. 1998 ; Testa et al. 1999 ). Interestingly, we found that A3-X8 antibody may block migration by inhibiting 3ß1-dependent ERK stimulation, but not FAK phosphorylation.

The distinction between haptotactic (i.e., controlled by adhesion receptors) and chemotactic (i.e., controlled by growth factor receptors) migration is physiologically relevant, but it is sometimes overlooked. In physiopathological situations such as wound healing and inflammation (Martin 1997 ; Wells 2000 ), chemotaxis induced by chemokine gradients may dominate. On the other hand, haptotaxis may be more relevant when tumor cells traverse the BM (Damsky and Werb 1992 ). In this study, we took advantage of the well-known ß1 integrin-activating antibody TS2/16 to enhance spontaneous haptotactic migration of keratinocytes on Ln-5 (as well as collagen IV), producing Transwell assay results with much better signal-to-noise ratio. While the exact mechanisms underlying activation of integrins by TS2/16 are not well defined, this antibody presumably functions by inducing changes in integrin shape, stabilizing a conformation that resembles the ligand-bound conformation of the integrin (Humphries 1996 ; Bazzoni and Hemler 1998 ). Consequently, avidity for ligand is increased, as measured in adhesion assays.

In contrast, in EGF-treated cells, 3ß1 did not appear to be activated, as indicated by unchanged avidity in adhesion assays. Nonetheless, EGF-stimulated cells migrated more efficiently than TS2/16-stimulated ones, consistent with the fact that EGF acts at several levels within cells, possibly lowering their threshold for motility (Wells 2000 ). Therefore, it appears that integrin activation may be a regulatory step for haptotactic, not chemotactic migration.

Another indication that TS2/16 and EGF induce two distinct types of migration is that they are differentially affected by various inhibitors. In this regard, the difference most relevant to this work, and perhaps keratinocyte biology, concerns sensitivity to anti–6ß4 antibodies. Thus, we found that antibodies to 6ß4 inhibited TS2/16-induced migration, but not EGF-stimulated migration. This finding suggests a role for 6ß4 in downregulating 3ß1-dependent haptotactic migration on Ln-5. Indeed, 6ß4 is well known to play a major role in protein complexes called hemidesmosomes, which anchor basal epidermal cells to the underlying basement membrane (Borradori and Sonnenberg 1999 ). Therefore, it is not surprising that 6ß4 may favor immobilization of cells to the substrate. This seems to be accomplished by inhibiting 3ß1-dependent haptotactic migration at the level of signaling rather than by stabilizing adhesion, since anti–6ß4 antibodies did not increase cell adhesion to Ln-5. Furthermore, anti–6ß4 antibodies block migration via a different pathway than A3-X8, because they do not inhibit ERK stimulation.

Recent studies described 6ß4 as the integrin-mediating carcinoma cell migration on Ln-1 and in Matrigel invasion assays (Rabinovitz and Mercurio 1997 ; Shaw et al. 1997 ; Gambaletta et al. 2000 ). These results seemingly contradict our findings, as well as the anchoring role of 6ß4 in epidermis. However, in those assays, 6ß4-dependent motility was only observed in chemotactic, but not in haptotactic migration assays (O'Connor et al. 1998 ). Furthermore, it depended strictly upon the overexpression of 6ß4 (Shaw et al. 1997 ; O'Connor et al. 1998 ; Gambaletta et al. 2000 ). In our system, we found no evidence that 6ß4 supported motility. Instead, keratinocyte migration (either type) on Ln-5 could be entirely accounted for by 3ß1. Differences in cell types and ECM substrates may be responsible for these discrepancies, which need to be solved by further studies.

As mentioned above, the interference by 6ß4 in 3ß1 migration appears to occur at the level of signaling. The phenomenon that occupancy of one integrin, here 6ß4, can suppress the function of another integrin, here 3ß1, has been observed in several other cell systems and is a concept appreciated as trans-dominant inhibition (Diaz-Gonzalez et al. 1996 ). For example, anti–vß3 antibodies suppress 5ß1-dependent phagocytosis (Blystone et al. 1994 ), ligation of 4ß1 inhibits 5ß1-dependent expression of metalloproteinases (Huhtala et al. 1995 ), and 3ß1 inhibits fibronectin and collagen IV receptor functions (Hodivala-Dilke et al. 1998 ). In general, trans-dominant inhibition involves changes in integrin conformation and requires integrin-linked signal transduction cascades (Sastry and Horwitz 1993 ; Diaz-Gonzalez et al. 1996 , Hughes et al. 1997 ). It remains to be seen which mechanisms apply to the 6ß4-initiated inhibition of 3ß1 migration.

As an initial attempt to identify such mechanisms, we began to analyze signaling pathways downstream of 6ß4 responsible, in our system, for the inhibition of 3ß1 migration. Several lines of evidence implicated PI3-K. First, LY294002, a specific inhibitor of PI3-K, abolished the inhibitory effect of anti–6ß4 antibodies on TS2/16-induced haptotaxis in HaCaT cells, as well as A431. Second, constitutive-active PI3-K prevented TS2/16-induced haptotaxis, and dominant-negative PI3-K prevented inhibition of haptotaxis by anti–6ß4 antibodies. Third, phosphatidylinositol 3-phosphate production by PI3-K, identified as class IA p110 isoform, was exclusively increased in cells stimulated with anti–6ß4 antibodies, whereas anti–3ß1 antibodies had no effect. Fourth, phosphorylation of AKT, a downstream effector of PI3-K (Kandel and Hay 1999 ), was increased by anti–6ß4 antibodies in cells plated on Ln-5. Together, these results strongly indicate that PI3-K mediates 6ß4-initiated inhibition of HaCaT haptotactic migration on Ln-5.

In recent literature, PI3-K was reported to play a stimulatory role in growth factor–initiated cell migration (for review, see Giancotti and Ruoslahti 1999 ; Wells 2000 ). How can the same PI3-K enzyme, in the same cell system, mediate both inhibition and stimulation of migration at the same time? This apparent inconsistency may actually not be difficult to account for, because of the complexity and redundancies of the signaling pathways in which PI3-K may be involved (Ren and Schwartz 1998 ; Wymann and Pirola 1998 ; Rameh and Cantley 1999 ; Vanhaesebroeck and Waterfield 1999 ; Nebl et al. 2000 ). Thus, there are many hypothetical possibilities for envisioning PI3-K operating in pathways that have distinct effects on HaCaT migration. Distinct PI3-K isoforms may also be in play (Zhang et al. 1998 ; Arcaro et al. 2000 ). Distinguishing among these possibilities will have to await further characterization of PI3-K signal transduction pathways.

We detected association of 6ß4 with a class IA PI3-K isoform. Activation of this isoform generally requires translocation to the plasma membrane, mediated by the adapter subunit (50, 55, or 85 kD) that links the p110 catalytic subunit to a cell surface receptor with tyrosine kinase domains (Wymann and Pirola 1998 ). Integrin 6ß4 has no such kinase domain, but was shown to be physically associated with a receptor tyrosine kinase, erbB-2 or Her2/neu (Falcioni et al. 1997 ), a member of the EGF receptor family (Hynes and Stern 1994 ; Alroy and Yarden 1997 ). Here, we demonstrate that binding to Ln-5 induces tyrosine phosphorylation of erbB-2 and that this effect is amplified by anti–6ß4 antibodies. Furthermore, we detected increased association of p85 with this activated erbB-2. Complexing of erbB-2 with p85 has been shown to lead to increased p110 activity (Ram and Ethier 1996 ; Olayioye et al. 1998 ). These data corroborate the idea that erbB-2 may act as a signaling link between 6ß4 and PI3-K. Moreover, we also found that blockage of endogenous erbB-2 by a specific inhibitor or by dominant-negative erbB-2 abolished the inhibitory effect of anti–6ß4 antibodies on 3ß1-linked haptotaxis. These are independent indications that 6ß4 cooperates with erbB-2 to trigger downstream signaling pathways regulating integrin-linked functions.

Recent data on breast cancer cells revealed a stimulatory role for erbB-2 in migration (Spencer et al. 2000 ) that may superficially