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doi: 10.1242/10.1242/jcs.00610

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Regulation of keratinocyte shape, migration and wound epithelialization by IGF-1- and EGF-dependent signalling pathways

¹ Department of Dermatology and Center for Molecular Medicine, University of Cologne (CMMC), Joseph-Stelzmann-Strasse 9, 50924 Cologne, Germany
² Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (32K): [in a new window]   Fig. 1. Effects of IGF-1 and EGF on keratinocyte spreading and rounding. (A) FACS analysis of expression of IGF-1 receptor (grey line) and EGFR (black line) in primary human keratinocytes after suspension culture. (B) Stimulation of keratinocyte spreading by IGF-1. Histograms show size distribution of 200 keratinocytes per sample plated in the presence of 100 ng ml-1 IGF-1 (top), 10 ng ml-1 EGF (middle) or 4 µM cytochalasin D and 100 ng ml-1 IGF-1 (bottom). Grey bars indicate stimulated cells, black bars unstimulated cells. Left panels show microscopic pictures of stimulated keratinocytes at equal magnifications. (C) Stimulation of keratinocyte rounding by EGF. Histograms show size distribution of 200 keratinocytes per sample stimulated with 10 ng ml-1 EGF (top, grey bars) or 100 ng ml-1 IGF-1 (bottom, grey bars). Black bars in both histograms indicate unstimulated control cells.

View larger version (46K): [in a new window]   Fig. 2. Activation of PI-3K and MAPK by IGF-1 and EGF. Western blot with phosphorylation-specific antibodies against MAPK (A, top; B, top) or Akt (B, bottom) showing the effect of growth factor treatment and pharmacological inhibitors on activation of MAPK and Akt. The lower panel in A and the middle panel in B show total (phosphorylated and unphosphorylated) MAPK (ERK2) as loading control. (A) Time course of MAPK activation after stimulation with EGF in single keratinocytes treated with vehicle control (DMSO) or 10 µM PD98059. Keratinocytes were stimulated for the indicated times. (B) Activation of MAPK and Akt in preconfluent cultures of keratinocytes. Cells were pretreated with DMSO or the inhibitors PD98059 (10 µM) and LY294002 (25 µM), respectively, and then stimulated with growth factors for 15 minutes.

View larger version (54K): [in a new window]   Fig. 3. Expression of a constitutively active PI-3K mutant (rCD2p110) in primary human keratinocytes. (A) Western blot analysis with the anti rat CD2 antibody Ox34 of keratinocytes infected with retroviruses encoding rCD2p110 or control virus (neo). Arrow points to rCD2p110 protein. Equal loading of the lanes was checked by Coomassie blue staining. (B,C) Immunofluorescent staining with Ox34 (green) and TRITC-phalloidin (red). Green staining shows localization of rCD2p110 at protruding areas of the cell membrane.

View larger version (28K): [in a new window]   Fig. 4. Activation of PI-3K and MAPK are necessary and sufficient to induce keratinocyte shape changes. Histograms show size distribution of 200 keratinocytes per sample. (A) Keratinocytes plated in the presence of 100 ng ml-1 IGF-1 and pretreated with 25 µM LY294002 (grey bars) or the vehicle control 0.1% DMSO (black bars). (B) Keratinocytes infected with rCD2p110 (grey bars) or empty vector neo (black bars) plated in the absence of growth factors. (C) Keratinocytes stimulated with 10 ng ml-1 EGF and pretreated with 10 µM PD98059 (grey bars) or the vehicle control 0.1% DMSO (black bars). (D) Keratinocytes infected with MAPKK1 (grey bars) or empty vector puro (black bars) in the absence of growth factors.

View larger version (29K): [in a new window]   Fig. 5. Effect of toxin B and PLC inhibition on keratinocyte spreading. (A-F) Intracellular calcium release in primary human keratinocytes loaded with FURA-2. The x axis shows time in seconds and the y axis the level of fluorescence in arbitrary units. Keratinocytes in suspension were stimulated with 10 µM bradykinin (Brad) or 100 ng ml-1 IGF-1 (IGF-1) without preincubation (A,B) or after preincubation with 1 µM (C) or 5 µM (E) U73122, or 1 µM (D) or 5 µM (F) U73343. (G-I) Histograms show size distribution of 200 keratinocytes per sample. Cells represented by black bars were pretreated with the following inhibitors: (G) 20 ng ml-1 toxin B overnight; (H) 3 µM U73122 for 20 minutes; (I) 3 µM U73343 for 20 minutes. Grey bars show cells pretreated for the same time with the relevant vehicle control. All cells were plated in the presence of 100 ng ml-1 IGF-1.

View larger version (26K): [in a new window]   Fig. 6. Effects of MEK-1 activity, IGF-1 and PI-3K activity on keratinocyte motility. (A,B) Trajectories of cells expressing puro (A) or MAPKK1 (B) followed for 24 hours, showing 20 cells per sample. Origin and end point for each cell are indicated by coloured dots (green/red). (C,D) Histograms showing speed of cell movement. Each bar represents a separate experiment, with 20 cells being analysed per experiment. The number of cells moving at the same speed within an experiment is represented by the width of the bar. Red lines connect the means of individual experiments. Mean and standard error of the mean for all replicate experiments are given above the bars. (C) Comparison between cells expressing puro and MAPKK1. (D) Comparison between puro-expressing or uninfected keratinocytes treated with 0.1% DMSO and MANA-expressing or uninfected keratinocytes treated with 25 µM PD98059 in the presence of 10 ng ml-1 EGF. (E) Migration speed (µm h-1) of keratinocytes left untreated or treated with 0.1% DMSO or 25 µM LY294002 and left unstimulated or stimulated with 100 ng ml-1 IGF-1. (F) Keratinocytes were preincubated with 0.1% DMSO and 25 µM LY294002, and left unstimulated or were stimulated with 100 ng ml-1 IGF-1. A similar result was obtained when 50 µM LY294002 were used. Bars in E,F show mean±s.e.m.

View larger version (113K): [in a new window]   Fig. 7. Effect of growth factor signalling on wound epithelialization. Mitomycin-C-treated keratinocytes in FAD low Ca2+ were wounded and incubated for 6 hours in the presence or absence of growth factors. (A-F) Normal human epidermal keratinocytes were left untreated (A) or were treated with: (B) 10 ng ml-1 EGF+ 100 ng ml-1 IGF-1; (C) 100 ng ml-1 IGF-1; (D) 100 ng ml-1 IGF-1 µM and 25 µM LY294002; (E) 10 ng ml-1 EGF; (F) 10 ng ml-1 EGF and 10 µM PD98059. (G-J) primary human keratinocytes infected with retroviral vectors encoding: (G) empty vector puro; (H) MAPKK1; (I) empty vector neo; (J) rCD2p110.

View larger version (114K): [in a new window]   Fig. 8. Effect of toxin B and ML-7 on wound epithelialization in vitro. Mitomycin-C-treated keratinocytes in FAD low Ca2+ were wounded and incubated for 6 hours in the presence of growth factors. Wounded monolayers were stimulated with 100 ng ml-1 IGF-1 (A,B) or 10 ng ml-1 EGF (C-E) in the presence of 20 ng ml-1 toxin B (B) and 5 µM (D) or 10 µM (E) ML-7 or the respective vehicle controls (A,C).

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