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First published online 27 March 2007
doi: 10.1242/jcs.03426

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Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1

¹ Department of Dermatology, University of Cologne and Center for Molecular Medicine, University of Cologne (CMMC), Joseph-Stelzmann-Strasse 9, 50924 Cologne, Germany
² Max Planck Institute of Biochemistry, Department of Molecular Medicine, Am Klopferspitz 18, 82152 Martinsried, Germany
³ University of Southampton, School of Biological Sciences, Bassett Crescent East, Southampton, SO16 7PX, UK
⁴ Department of Pathology, University of Cologne and Center for Molecular Medicine, University of Cologne (CMMC), Joseph-Stelzmann-Strasse 9, 50924 Cologne, Germany
⁵ Institute for Cell Biology, University of Bonn, Ulrich Haberlandstrasse 61a, 53121 Bonn, Germany
⁶ Institute of Biochemistry, University of Cologne, Otto Fischer-Strasse 12-14, 50674 Cologne, Germany
⁷ University of Kopenhagen, Institute of Molecular Pathology, Frederik V's Vej 11, 2100 Kopenhagen, Denmark

View larger version (38K): [in this window] [in a new window]   Fig. 1. Expression of N17Rac1 in the epidermis of mice and in primary murine keratinocytes. (a) Schematic representation of the transgenic construct. (b,c) PCR (b) and Southern blot (c) analysis of tail snips from transgenic (tg) and wild-type (wt) mice. Amplified or excised plasmid fragments were used as positive controls (+). (d,e) Immunostaining of skin samples (d) and western blot analysis of adult primary keratinocytes (e) from tg and wt mice with the anti-Myc antibody 9B11. Note expression of N17Rac1 in basal epidermal and outer root sheath keratinocytes of tg mice (green). Bar, 100 µm. (f) Western blot analysis of protein extracts from cultured tg and wt keratinocytes with a monoclonal antibody against Rac (upper panel). The mutant Rac protein (N17Rac1) is Myc-tagged and, therefore, migrates slower compared with endogenous Rac. Loading controls show actin expression (lower panel). (g) Western blot analysis with the anti-Myc antibody 9B11 of protein extracts from cultured wt and tg keratinocytes with strong (se) and weak (we) expression of the transgene, respectively (upper panel). Loading controls show actin expression (lower panel). (h,i) Results of CFE assays with wt (n=4), strong tg (se; n=4) and weak tg (we; n=3) keratinocytes. Bar graphs show number of cells per colony (h) and colony area (µm2x104) (i); error bars show standard deviation (± s.d.). *P<0.05; **P<0.01; ***P<0.001; ns, not significant.

View larger version (80K): [in this window] [in a new window]   Fig. 2. Delayed wound re-epithelialization in N17Rac1 transgenic mice. (a) Schematic representation of wound re-epithelialization. Epidermis, wound edge epidermis; neo-epidermis, newly formed epidermis after wounding (green). Yellow arrow and black perpendicular line indicate the wound margin. Black arrow indicates the front of the epithelial tongue (neo-epidermis). (b-e) Micrographs of H-E-stained sections of wound-edge samples. Samples were taken from (b,d) wild-type (wt) mice or (c,e) N17Rac1 trangenic (tg) mice (b,c) 2 days and (d,e) 5 days after wounding. Yellow arrows indicate wound margin. Black arrows indicate the front of the neo-epidermis. (f,g) Results of histomorphometric analysis of (f) `length' (length of the epithelial tongue starting from the wound edge to the tip of the tongue) and (g) `area' (area covered by the neo-epidermis) in wounds of wt and N17Rac1 tg mice. Blue bars give the mean ± s.d. of results obtained from seven wt and seven tg mice 2 days after wounding. Red bars give the mean ± s.d. of results from eight wt and ten tg mice 5 days after wounding. Bar, 200 µm. *P<0.05; ***P<0.001.

View larger version (65K): [in this window] [in a new window]   Fig. 3. Impairment of wound re-epithelialization in Rac1E-KO mice. (a,b) Micrographs of H-E-stained sections of wound-edge samples. Samples were taken from (a) Rac1E-HET mice and (b) Rac1E-KO mice 5 days after wounding. Yellow arrows indicate wound margin. Black arrows indicate the front of the neo-epidermis. Bar, 200 µm. (c,d) Results of histomorphometric analysis of (c) `length' and (d) `area' in wounds of Rac1E-HET and Rac1E-KO mice as the mean ± s.d. obtained from eight Rac1E-KO mice and six Rac1E-HET mice. ***P<0.001.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Reduced BrdU incorporation and ERK phosphorylation in Rac1E-KO and N17Rac1 transgenic (tg) keratinocytes in vivo and in vitro. (a-c) Numbers of BrdU-positive keratinocytes in sections of wounded skin (a,c) 5 days or (b) 2 days after wounding as the average number of BrdU-positive keratinocytes per microscopic field ± s.d. representative for (a) five or (b) seven female wild type (wt) mice, (a) six or (b) eight female N17Rac1 tg mice and (c) eight Rac1E-HET versus 11 Rac1E-KO mice (mixed sex groups). (d) Percentage of BrdU-positive nuclei in keratinocyte cultures obtained from wt and N17Rac1 tg mice in serum-free low-Ca2+ FAD medium supplemented with 10 ng/ml EGF (dark gray bars) or 100 ng/ml IGF1 (light gray bars) showing the average number of BrdU-positive nuclei ± s.d. (representative for six different N17Rac1 tg and five wt cell lines; n=6). (e) Top: Micrographs showing immunostaining of sections of skin wounds from Rac1E-KO mice (Rac1E-KO) and wt mice using an antibody against phosphorylated ERK1/2. Yellow lines indicate the wound margin; arrows point towards the tips of the tongues; dashed lines indicate basement membrane. Bar, 40 µm. Bottom: Western blot analysis of phosphorylated ERK (pERK) in N17Rac1 tg and wt keratinocytes plated on fibronectin (fn) and collagen I (col I), respectively. Loading controls show actin expression. (f) Western blot analysis of phosphorylated ERK (pERK) in N17Rac1 tg and wt keratinocytes unstimulated (–) or stimulated (+) with 10 ng/ml EGF. Total ERK (ERK) is shown as loading control. The densitiy of the phosphorylated band relative to the loading control is shown in the lower panel (in %). **P<0.01; ***P<0.001; n=3, six different transgenic cell lines.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Reduced adhesion and spreading of N17Rac1 transgenic (tg) keratinocytes on collagen I in vitro. (a) Rac1-GTP loading in keratinocytes 40 minutes after plating onto collagen I (col I)- or fibronectin (fn)-coated dishes. Active Rac (Rac1GTP) was precipitated using biotin-CRIB peptide and analysed by western blotting with anti-Rac antibody (upper panel). Total (active and inactive) Rac1 protein (Rac1total) is shown in the lower panels as a loading control (n=4). Determination of signal intensity for Rac1-GTP relative to Rac1total using densitometry revealed no consistent difference between the levels of Rac1-GTP in keratinocytes plated onto collagen I compared with fibronectin. (b) Percentage of adherent tg and wild type (wt) keratinocytes on collagen I (gray bars) and fibronectin (white bars) 1 hour after plating. Error bars give the mean ± s.d. of four different tg and wt keratinocyte lines each (n=4). (c) Micrographs showing immunostaining of Myc and polymerized actin in wt and tg keratinocytes 40 minutes after plating onto collagen-I-coated dishes. (d,e) Histograms showing cell spreading of four different N17Rac1 tg (red) and three different wt (black) keratinocyte lines on (d) collagen I and (e) fibronectin. Cell size is given in arbitrary units (n=3). **P<0,01; ns, not significant.

View larger version (52K): [in this window] [in a new window]   Fig. 6. Reduced lamella protrusion persistence and increased ruffle frequency in N17Rac1 transgenic keratinocytes. (a,b) Phase-contrast images of individual (a) N17Rac1 transgenic (tg) and (b)wild type (wt) keratinocytes (top) and time-space plots of membrane segments indicated by white lines in the corresponding cells (bottom). Lamella protrusions appear as ascending bright contours, whereas ruffles appear as descending, darker contours. Note that within 5 minutes three ruffle profiles (thick lines) appeared in tg cells, whereas only one ruffle profile became visible in wt cells. By contrast, persistence of lamella protrusion (thin lines) was short in tg cells but prolonged in wt cells. Bar, 10 µm. (c,d) Results of statistical analysis of (c) persistence of lamella protrusion and (d) ruffle frequency expressed as the mean ± s.d. Results are representative for three N17Rac1 tg and three wt cell lines in passages 3-5; at least 80 individual cells per cell line were analysed. Cells were plated on collagen-I-coated glas coverslips. Statistical significance between data groups was determined using the Whitney U-test. Differences between tg and wt keratinocytes were highly significant for persistence of lamella protrusion and for ruffle frequency (P<0.01). (e,f) Micrographs showing focal contacts in primary murine keratinocytes isolated from (e) wt and (f) N17Rac1 tg mice by immunostaining against vinculin. Note reduced number of focal contacts in the transgenic population. Bar, 20 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Reduced migration and scratch wound re-epithelialization of N17Rac1 transgenic keratinocytes in vitro. (a,b) Results of individual cell migration of N17Rac1 transgenic (tg) and wild type (wt) keratinocytes on collagen I (col I)-coated and fibronectin (fn)-coated dishes. Each dot shows (a) the distance covered by an individual cell (path length) or (b) the average migration speed (speed); n= 5 with three different tg and wt cell lines). Red lines indicate the mean of the respective values for each cell population. (c,d) Re-epithelialization of a scratch wound in vitro. (c) Images of scratch-wounded wt and N17Rac1 tg keratinocyte monolayers on col I 0 minutes (immediately) or 1000 minutes after scratching. (d) Graphic representation of the analysis of scratch-wound re-epithelialization in tg or wt keratinocytes on col-I- or fn-coated dishes. Graphs show the distance between the epithelial fronts (y-axis) at three different time points after scratching (x-axis) in percent. Data points represent the mean of 20 measurements each ± s.d. of the distance in four different N17Rac1 tg and 4 wt keratinocyte lines (n=6). *P<0.05; **P<0,01; *** P<0,001; ns, not significant.

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