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First published online 15 July 2003
doi: 10.1242/jcs.00663

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6ß4 integrin regulates keratinocyte chemotaxis through differential GTPase activation and antagonism of 3ß1 integrin

Alan J. Russell^1,*, Edgar F. Fincher¹, Linda Millman¹, Robyn Smith¹, Veronica Vela¹, Elizabeth A. Waterman¹, Clara N. Dey¹, Shireen Guide¹, Valerie M. Weaver² and M. Peter Marinkovich^1,

¹ Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
² Pathology & Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (51K): [in a new window]   Fig. 1. 6ß4 integrin and laminin-5 are required for EGF induction of motility in human keratinocytes. (A) Distribution of HD components in ß4(-) and ß4(+) cells. Cells were cultured for 24 hours on glass coverslips fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100. Immunofluorescence microscopy was performed to identify 6 integrin subunit (with rat mAb GoH3) in combination with the ß4 subunit (mouse mAb 3E1); ß4 integrin (rat mAb 346-11A) in combination with plectin/HD1 (mouse mAb 121) or BP180 (mouse mAb 233), and r ß4 integrin (with mouse mAb 3E1) in combination with laminin-5 (rabbit polyclonal anti-laminin-5 antisera). Mouse antibodies are colored red while rat and rabbit antibodies are colored green, colocalization is therefore represented by a yellow color. Narrow images under each figure represent z-sections of the image above (nuclei are stained blue with Hoechst dye). Scale bar: 10 µm. (B) Effects of 6ß4 expression on keratinocyte monolayer scratch migration. Integrin ß4-deficient EB-PA keratinocytes expressing either LacZ, [ß4(-)] or ß4 cDNA, [ß4(+)] were starved of growth factors for 16 hours before treatment with 10 µg/ml mitomycin C for 3 hours on ice to prevent subsequent proliferation. Cell monolayers were wounded by scraping and migration of the cells into the scrape wound was photographed 48 hours later after incubation in either supplement free medium (top panels) or in medium supplemented with 2 ng/ml EGF (lower panels; Scale bar: 200 µm). (C) Quantification of monolayer scratch assays. Marked areas were photographed at 24 and 48 hours periods and areas between scratch fronts calculated to generate percentage scratch closure in conditions of no EGF (ß4(-), white circles; ß4(+), white triangles) or 2 ng/ml EGF (ß4(-), black circles; ß4(+), black triangles), n=3. (D) Effects of EGF upon induction of motility in transwells coated with collagen IV. Growth factor-starved cells were introduced into the top of transwells coated on the underside with 10 µg/ml collagen IV and incubated for 16 hours without growth factors or with 2 ng/ml EGF in the lower chamber. Migration was quantified by averaging the number of cells per microscopic field on the underside of the filter (3 microscopic fields and 3 filters per condition). Fold induction represents the relative increase in migration observed in transwells with EGF compared to migration without growth factors. Actual induction, ß4(-) 10.3±3.1 cells/field, ß4(+) 94.7±2.6 cells/field. (E) Significance of laminin-5 in EGF-induced chemotaxis. Transwells were prepared as above except cells were incubated with 10 µg/ml laminin-5 inhibitory antibody (BM165) in upper and lower chambers. actual induction, ß4(-) 20.3±5.4, ß4(+) 27.8±3.73 cells/field. (F) Relative contribution of laminin-5 binding integrins to EGF-induced chemotaxis. Transwells were prepared as above and ß4(+) cells incubated for 16 hours with 20 µg/ml IgG1 control, 10 µg/ml inhibitory ß4 integrin antibody ASC-8, 10 µg/ml inhibitory 3 integrin antibody P1B5 or a combination of 10 µg/ml P1B5 and ASC-8 in both upper and lower chambers. Actual induction, IgG 105.7±12.1 cells/field, ASC-8 65.7±13.9 cells/field, P1B5 72.0±13.1 cells/field, ASC-8/P1B5 -2.3±12.7 cells/field.

View larger version (37K): [in a new window]   Fig. 2. Mutation of homologous polar residues within the extracellular domain of ß4 prevents attachment of 6ß4 to laminin-5 and tyrosine phosphorylation after EGF treatment but not recruitment of HD components. (A) Amino acid substitutions to generate an attachment defective ß4 subunit, ß4(AD). Homology analysis was carried out with the extracellular domain of the ß3 integrin subunit. Asterisks indicate residues essential for ligand binding in integrin IIbß3 (Baker et al., 1997). Arrows represent sites of point mutation and alanine substitution. (B) Distribution of HD components in ß4(AD) cells. Cells were cultured for 24 hours on glass coverslips fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100. Immunofluorescence microscopy was performed as described for ß4(-) and ß4(+) cells (see Fig. 1A). Mouse and rabbit antibodies are colored red while rat antibodies are colored green, colocalization is therefore represented by a yellow color. Narrow images under each figure represent z-sections of the image above (nuclei are stained blue with Hoescht dye) Scale bar: 10 µm. (C) Attachment of keratinocytes to laminin-5 in the presence of inhibitory antibodies to 3 integrin (P1B5) and/or cells in suspension were applied to 96-well plates coated with 10 µg/ml laminin-5 and incubated for 60 minutes at 37°C before unattached cells were washed off. Inhibitory antibodies and control IgG were supplemented at 10 µg/ml. Adherent cells were fixed, stained with 0.1% crystal violet and solubilized with 10% acetic acid. Cell number was quantified by measuring optical density at 570 nM (n=4). The bar chart shows adherence of ß4(+) cells (light shading) vs. ß4(AD) cells (dark shading). (D) Attachment of keratinocytes to laminin-5 at 4°C in the presence of ß4 inhibitory antibody (ASC-8). Cells in suspension were applied to 96-well plates coated with 10 µg/ml laminin-5 and incubated for 60 minutes at 4°C before washing off unattached cells. Inhibitory antibodies and control IgG were supplemented at 10 µg/ml. Adherent cells were fixed, stained with 0.1% crystal violet and solubilized with 10% acetic acid. Cell number was quantified by measuring optical density at 570 nM (n=4). The bar chart shows adherence of ß4(+) cells (light shading) compared with ß4(AD) cells (dark shading). (E) Tyrosine phosphorylation of the ß4 subunit after stimulation of cells with EGF. ß4(+) and ß4(AD) keratinocytes were growth factor starved for 16 hours then stimulated with 100 ng/ml EGF. At time intervals indicated (in minutes), cells were lysed and immunoprecipitated with ß4 mAb, 3E1. Western blots show tyrosine phosphorylation (mAb 4G10, upper panels) and total ß4 in immunoprecipitates (with rabbit polyclonal antiserum 1922, lower panels). (F) Laminin-5 secretion and processing by transduced cells. Cells were grown on plastic culture dishes with or without 2 ng/ml EGF. After 24 hours, cells were removed with 20 mM ammonium hydroxide and matrix was extracted with 8 M urea buffer before western blotting with a polyclonal laminin-5 antibody. Symbols to the right of the blot indicate the separate laminin-5 subunits with a (p) indicating a processed subunit. (G) Activation of p44/42 MAP kinase by EGF. Cells were growth factor starved and treated for 5' with 2 ng/ml EGF before lysis and western blotting with phospho-p44/42 MAP kinase antibody (upper panel). Blots were then stripped and reblotted with total p44/42 MAP kinase antibody (lower panel).

View larger version (36K): [in a new window]   Fig. 4. EGF stimulates enhanced chemotaxis and reduced epithelial integrity in ß4(AD) cells dependent upon an alternate pathway utilizing RhoA and integrin 3ß1. (A) Effect of ß4(AD) expression upon monolayer scratch migration. Cells were prepared as in Fig. 1A and incubated without growth factors (top panel) or with 2 ng/ml EGF (lower panel) for 24 hours at 37°C. Scale bar: 300 µm. (B) Effect of ß4(AD) expression upon transwell chemotaxis. Transwell experiments with ß4(AD) keratinocytes were performed with collagen IV-coated transwells and 2 ng/ml EGF. Cells were incubated for 16 hours with media supplemented in upper and lower chambers with IgG1, inhibitory ß4 integrin antibody ASC-8, inhibitory 3 integrin antibody P1B5 or inhibitory laminin-5 antibody BM165. Actual induction IgG 60.0±4.2 cells/field, ASC-8 60.0±4.4 cells/field, P1B5 10.0±1.2 cells/field, BM165 16.1±1.1 cells/field. (C) Effect of GTPase inhibition upon ß4(AD)-dependent chemotaxis. ß4(AD) cells were retrovirally transduced with control LacZ or inhibitory GTPase constructs, N17Cdc42, N17Rac1 or N19RhoA. Chemotaxis experiments were performed with collagen IV-coated transwells and 2 ng/ml EGF. Actual induction, ß4(AD)LacZ 136±23.1 cells/field, ß4(AD)N17Rac1 15.0±3.7 cells/field, ß4(AD)N19RhoA 21.0±3.6 cells/field, ß4(AD)N17Cdc42 8.4±2.7 cells/field. (D) Effect of ß4(AD) expression upon cell scattering. Cells were incubated at low density for 4 days in SFM before being photographed under phase contrast illumination. Scale bar: 40 µm. (E) Effect of EGF upon cell scattering. Cells were grown in growth factor free SFM for 4 days and scattered colonies counted using criteria described in materials and methods (light shaded columns). Cells were then incubated for 16 hours with 2 ng/ml EGF and colony scatter counts repeated (dark shaded columns). For antibody treatments, ß4(+) cells were incubated for 3 days in normal SFM then 1 day in growth factor free SFM with 10 µg/ml mouse IgG or ß4 integrin inhibitor ASC-8, before colony counts (light shaded columns). Colony scattering was measured after 6 hours with 2 ng/ml EGF (dark shaded columns). Each point represents the data from at least 50 colonies (n=3). (F) Effect of N19RhoA expression upon ß4(+) transwell chemotaxis. Transwell experiments with ß4(+)LacZ and ß4(+)N19RhoA keratinocytes were performed with fibronectin-coated transwells and 2 ng/ml EGF. Cells were incubated for 16 hours with media supplemented in upper and lower chambers with IgG1 or inhibitory ß4 integrin antibody ASC-8. Actual induction, ß4(+)LacZ IgG 116.5±18.5 cells/field, ß4(+)LacZ ASC-8 113.5±16.9 cells/field, ß4(+)N19RhoA IgG 77.7±7.5 cells/field, ß4(+)N19RhoA ASC-8 34±21.3 cells/field.

View larger version (44K): [in a new window]   Fig. 3. 6ß4 ligation is required for sustained activation of Rac1, lamellipodia formation and RhoA independent chemotaxis. (A) Effect of 6ß4 expression and ligation upon Rac1 and Cdc42 GTPase activation by EGF. Growth factor-starved cells were stimulated with 2 ng/ml EGF for the indicated times. Cells were lysed, and incubated with GST-PAK and glutathione-sepharose beads. Beads were washed and bound proteins separated on a 12% SDS-polyacrylamide gel. GST-PAK pull down blots were initially probed for Rac1 before stripping and reblotting for Cdc42. Control gels show relative quantities of GTPases present in total lysates. Numbers under each profile represent average optical density of pull down lanes from at least 2 separate blots. (B) Effect of 6ß4 expression and ligation upon RhoA GTPase activation by EGF. Growth factor starved cells were stimulated with 2 ng/ml EGF for the indicated times. Cells were lysed, and incubated with GST-RBD and glutathione-sepharose beads. Beads were washed and bound proteins separated on a 12% SDS-polyacrylamide gel. Control gels show relative quantities of GTPases present in total lysates. Numbers under each profile represent average optical density of pull down lanes from at least 2 separate blots. (C) Effect of EGF treatment upon lamellipodia formation. ß4(-), ß4(+) and ß4(AD) cells were starved of growth factors for 16 hours and stimulated with 2 ng/ml EGF for the indicated times before fixing with 3.4% formaldehyde in PBS and stained with TRITC-phalloidin to identify filamentous actin. The area of lamellipodial projections was measured by tracing around membrane extensions on digital images. The lamellar area was then calculated using NIH image software. The graph shows total lamellar area for ß4(-) cells (open triangles), ß4(+) cells (black triangles) and ß4(AD) cells (black circles). (D) Effect of ß4 inhibitory antibody ASC-8 upon lamellipodia formation. Growth factor-starved NHKs incubated with 10 µg/ml IgG or ASC-8 were stimulated with 2 ng/ml EGF and lamellipodial area calculated as described. The graph shows lamellar area for NHK plus IgG (black circles) and NHK plus ASC-8 (open circles). (E) Effect of GTPase inhibition upon 6ß4-dependent chemotaxis. ß4(+) cells were retrovirally transduced with control LacZ or inhibitory GTPase constructs, N17Cdc42, N17Rac1 or N19RhoA. Transwell chemotaxis experiments were performed with collagen IV-coated transwells and 2 ng/ml EGF stimulation. Actual induction, ß4(+)LacZ 106.4±9.0 cells/field, ß4(+)N17Rac1 23.8±3.7 cells/field, ß4(+)N19RhoA 121.1±3.0 cells/field, ß4(+)N17Cdc42 11.8±9.1 cells/field.

View larger version (129K): [in a new window]   Fig. 5. Expression and ligation of 6ß4 integrin changes the localization and activation state of 3ß1 integrin. Cells were cultured on glass coverslips for 4 days prior to immunofluorescence staining for FA components. Cells were fixed with 3% formaldehyde and solubilized with 0.5% Triton X-100 buffer for 30 minutes at RT. Fixed cells were washed with PBS and blocked with 1% BSA for 1 hour. Cells were stained with FITC-phalloidin for filamentous actin (stained in green A-C) and anti-paxillin mAb (stained in red A-C). Further samples were stained for 3 integrin (D-F) and the conformationally active form of the ß1 integrin subunit with mAb HUTS-4 (G-I). Scale bar; 20 µm.

View larger version (77K): [in a new window]   Fig. 6. Activation of Rac1 and suppression of RhoA through 6ß4 integrin control the localization and activation of 3ß1 integrin. (A) Effect of RhoA inhibition or Rac1 activation on 3ß1 distribution. ß4(AD) cells were transduced with control LacZ (top row), inhibitory N19RhoA (middle row) or activated V12Rac1 (bottom row) and examined by immunofluorescence microscopy. Left panel shows actin distribution with TRITC-phalloidin, center and right panels show 3 integrin and conformationally active ß1 integrin. (B) Effect of Rac1 inhibition on distribution and activation of 3ß1 integrin. ß4(+) cells were retrovirally transduced with control LacZ (upper row) or inhibitory N17Rac1 (lower row) and studied by immunofluorescence microscopy. Left panel shows 3 integrin, center panel shows paxillin and right panel shows distribution of conformationally active ß1 integrin. Scale bar: 20 µm.

View larger version (22K): [in a new window]   Fig. 7. Model explaining the role of 6ß4 integrin in the coordination of migration through EGF. Cells without 6ß4 integrin cannot sustain chemotactic EGFR signals, either from a loss of EGFR/ß4 interactions and/or due to suppression of 3ß1 integrin activity. Upon expression and ligation of 6ß4 integrin, Rac1 activation by EGF is sustained, suppressing 3ß1 integrin and RhoA. 3ß1 integrin is redirected from sites of basal focal contact to sites of cell-cell contact and cells migrate as an integral epithelial sheet.