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First published online December 31, 2008
doi: 10.1242/10.1242/jcs.029108

Journal of Cell Science 122, 278-288 (2009)
Published by The Company of Biologists 2009
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Integrin {alpha}3β1 inhibits directional migration and wound re-epithelialization in the skin

Coert Margadant, Karine Raymond*, Maaike Kreft, Norman Sachs, Hans Janssen and Arnoud Sonnenberg{ddagger}

Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands


Figure 1
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  		Fig. 1. Skin phenotype of Itga3flox/flox; K14-Cre mice. (A) Cryosections from back skin of neonatal Itga3flox/flox and Itga3flox/flox; K14-Cre mice were stained with antibodies directed against the {alpha}3 subunit and Ln-332 (top row). Scale bar: 100 µm. H&E-stained section showing the structure of the epidermis and the dermis from back skin of 4-month-old Itga3flox/flox and Itga3flox/flox; K14-Cre mice are shown in second row from top. Scale bar, 200 µm. Bottom two rows are EM images and schematic representations of back skin of 1-year-old Itga3flox/flox and Itga3flox/flox; K14-Cre mice, showing the basement membrane and hemidesmosomes (indicated by arrowheads). Scale bar: 200 nm. (B) H&E staining of back skin of a 1-year old Itga3flox/flox; K14-Cre mouse (top). Arrowheads indicate microblisters at the DEJ. Scale bar: 500 µm. Lower panels are cryosections of a region containing microblisters stained with antibodies against Ln-332 and β4. Arrowheads indicate blisters. Scale bar: 50 µm. (C) Ultrastructural analysis and schematic representation of back skin of a 1-year-old Itga3flox/flox; K14-Cre mouse, showing basement membrane duplication. The two basement membranes are marked with solid and dashed lines, and collagen fibers extending throughout the two basement membranes are indicated. Scale bar: 200 nm. (D) Regions of inflammation in a 4-month-old Itga3flox/flox; K14-Cre mouse are denoted by arrows. Back skin sections were stained with an antibody against CD3. Arrows indicate infiltrated lymphocytes. Scale bar in the third panel: 200 nm. (E) Alopecia in a 1-year-old Itga3flox/flox; K5-Cre mouse (right), compared with an Itga3flox/flox mouse of the same age. BM, basement membrane; Col, collagen; D, dermis; E, epidermis.

 

Figure 2
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  		Fig. 2. Deletion of integrin {alpha}3 in the epidermis does not inhibit keratinocyte differentiation. Skin cryosections from neonatal Itga3flox/flox and Itga3flox/flox; K14-Cre mice were stained with antibodies directed against keratin 14, filaggrin or involucrin, and counterstained with Ln-332 and DAPI to visualize the basement membrane and nuclei, respectively. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. Wound closure is accelerated in the absence of {alpha}3β1 integrin. Full-thickness wounds were generated on either side of the dorsal midline in Itga3flox/flox or Itga3flox/flox; K14-Cre mice, and excised 3 or 7 days after injury. (A) Schematic representation of a wound. D, dermis; E, epidermis; Es, eschar; F, fat tissue; G, granulation tissue. (B) H&E-stained sections depicting wound closure 7 days after wounding in Itga3flox/flox (top) and Itga3flox/flox; K14-Cre mice (bottom). Arrows indicate the edges of the migrating epithelium. The number of closed wounds after 7 days of migration is represented in the bar graph. Depicted are the means ± s.e.m. of at least 40 wounds pooled from four independent experiments (*P<0.05). Scale bar, 500 µm. (C) The distance covered by the migrating epidermis (indicated by the dotted line) was quantified 3 days after wounding in Itga3flox/flox (top) and Itga3flox/flox; K14-Cre mice (bottom). The graph indicates the means ± s.e.m. of at least 35 wounds pooled from three experiments (*P<0.05). Scale bar, 250 µm. (D) Proliferation in the re-epithelializing wounds was assessed by injecting BrdU 2 or 4 days after wounding, and determining the ratio of BrdU+ cells over the total number of cells using ImageJ. Depicted are the means ± s.e.m. of at least 30 images. Scale bar: 150 µm.

 

Figure 4
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  		Fig. 4. In vitro adhesion to Ln-332 is rescued by {alpha}6 integrins in the absence of {alpha}3β1 integrin. Keratinocytes were isolated from newborn Itga3flox/flox mice and designated MK{alpha}3+. MK{alpha}3– cells were then obtained by in vitro deletion of Itga3. (A) Immunoblot depicting the expression of {alpha}3 in MK{alpha}3+ and MK{alpha}3– cells. Murine mammary cell line Rac-11P and murine fibroblast cell line NIH3T3 were included as a positive and negative control, respectively. (B) Cell surface expression of integrin subunits {alpha}5, {alpha}2, {alpha}6, {alpha}v, β4 and β1 in MK{alpha}3+ and MK{alpha}3– was determined by FACS analysis. The negative control (only secondary antibody) is indicated by the black graph. (C) MK{alpha}3+ and MK{alpha}3– cells were seeded onto Col-1 or Ln-332 in K-SFM without supplements in the absence or the presence of the {alpha}6-blocking antibody GoH3 (10 µg/ml). After 30 minutes, non-adherent cells were washed away. Values shown represent the average percentages of adherent cells from three experiments performed in triplicate (*P<0.05, ***P<0.0005). (D) Immunoprecipitation of integrin subunits {alpha}6 and β1 was performed on lysates of MK{alpha}3+ and MK{alpha}3– cells. The precipitates were resolved by SDS-PAGE, and analyzed for the presence of the integrins β1 and β4, and the light chains (L) of {alpha}3 and {alpha}6 by western blotting.

 

Figure 5
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  		Fig. 5. The integrin {alpha}6β1 mediates cell spreading on endogenous Ln-332 when {alpha}3β1 is absent. MK{alpha}3+ and MK{alpha}3– cells were seeded on Ln-332 (A) or Col-1 (B), allowed to spread, and then incubated with {alpha}6-blocking antibody GoH3 (10 µg/ml). After 3 hours, the number of spread cells was scored and expressed as a percentage of the total number of cells. In each independent experiment, approximately 500 cells were counted for each condition. The graphs depict the averages of three experiments (*P<0.05, ***P<0.0005). Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. Ln-332 synthesis and deposition are not affected by the deletion of the integrin {alpha}3 subunit. (A) MK{alpha}3+ and MK{alpha}3– cells were seeded on Col-I and then detached with EDTA at the indicated time points after which the ECM was dissolved in sample buffer, subjected to SDS-PAGE, and the {gamma}2 chain of Ln-332 was detected by western blotting. (B) Immunofluorescence images demonstrating patches of deposited Ln-332 in spread MK{alpha}3+ cells (left panel) and MK{alpha}3– cells (right panel). Scale bar: 10 µm. (C) Low-magnification immunofluorescence images demonstrating Ln-332 trails left behind by MK{alpha}3+ cells (left), and MK{alpha}3– cells (right) migrating over Col-I. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. Loss of {alpha}3 enhances directionality and velocity of keratinocyte migration. (A) Confluent MK{alpha}3+ and MK{alpha}3– cells were deprived of growth factors, incubated for 2 hours with mitomycin C (10 µg/ml), and scratched with the tip of a pipette prior to EGF stimulation. The black bars indicate the wound edges at t=0. Scale bar: 100 µm. Wound areas were measured using ImageJ, and the ratio of the wound area after overnight migration over the wound area at t=0 was calculated and expressed in the bar graph. Values shown represent the means ± s.e.m. of three independent experiments (***P<0.0005). (B) EGF-induced phosphorylation of ERK1/2 was monitored by western blotting in lysates of MK{alpha}3+ and MK{alpha}3– cells that were deprived of growth factors prior to EGF stimulation for the indicated time points. (C) Cells were sparsely seeded on Col-I and monitored in time-lapse recordings for 16 hours. An image was captured every 5 minutes. Cell tracks were then determined using ImageJ. The migration plots indicate tracks from ten individual cells from four independent experiments. To quantify the average velocity and directionality (D/T ratio), data from four independent experiments were pooled. The graphs represent the means ± s.e.m. from ~50 cells (*P<0.05, **P<0.005, ***P<0.0005). To determine whether polarization was stable, cells were sparsely seeded on Col-I and monitored by time-lapse recordings. An image was captured every 5 minutes. A cell was considered stably polarized when maintaining a leading lamellipodium for 1 hour. The number of polarized cells was counted and expressed as a percentage of the total number of cells. The graph shows the means ± s.e.m. from 250 cells pooled from four independent experiments (*P<0.05). (D) Cells were sparsely seeded on Ln-332 or Col-I, serum-starved, and then stimulated with 5 ng/ml EGF at the indicated time points. An image was captured every 5 minutes. Cell tracks were determined using Matlab (Mathworks), and average velocity was plotted over time. The graphs depict the average velocity of ~50 cells from a representative experiment.

 

Figure 8
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  		Fig. 8. The phenotype of MK{alpha}3– cells is reversed by reconstitution with human {alpha}3A. (A) MK{alpha}3– cells were transduced with the cDNA encoding human {alpha}3A, and cell surface expression was verified by FACS analysis using monoclonal antibody J143 against human {alpha}3. Negative control (secondary antibody only) is indicated by thin black line. (B) MK{alpha}3R cells were seeded on Ln-332 (left panel) or Col-I (right panel), allowed to spread, and then incubated for 3 hours with GoH3 (10 µg/ml). Scale bar: 20 µm. (C) The number of spread cells was scored and expressed as a percentage of the total number of cells. In each independent experiment, approximately 500 cells per condition were counted. The graphs depict the averages of three experiments (***P<0.0005). (D) Lysates of MK{alpha}3+, MK{alpha}3– and MK{alpha}3R cells were analyzed by SDS-PAGE, and expression of integrin {alpha}3 was determined by western blotting using mAb 29A3 recognizing both human and murine {alpha}3 (L, light chain). Confluent MK{alpha}3+, MK{alpha}3–, and MK{alpha}3R cells were deprived of growth factors, incubated for 2 hours with mitomycin C (10 µg/ml), and scratched with the tip of a pipette prior to EGF stimulation. Wound areas were determined using Image J, and the ratio of the wound area after overnight migration over the wound area at t=0 was calculated and expressed in a bar graph. Values shown represent the means ± s.e.m. of three independent experiments (*P<0.05, ***P<0.0005).

 

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© The Company of Biologists Ltd 2009