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.