1 Institute of Cell Biology, Department of Biology, ETH-Zürich, 8093 Zürich, Switzerland
2 Keratinocyte Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, UK
3 Institute of Anatomy, University of Cologne, D-50931 Köln, Germany
4 Department of Experimental Pathology, Lund University, S-221 85 Lund, Sweden
5 Max-Planck-Institute of Biochemistry, D-82152 Martinsried, Germany
* Present address: Viral Carcinogenesis Laboratory, Cancer Research UK, London Research Institute, London WC2A 3PX, UK
The first two authors have equally contributed to this work
Author for correspondence (e-mail: sabine.werner{at}cell.biol.ethz.ch)
Accepted 4 February 2002
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SUMMARY |
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Movies available on-line
Key words: Integrin, Migration, Epidermis, Mouse, Wound
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INTRODUCTION |
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Several genetically modified mouse models have been used to investigate the molecular basis of the re-epithelialisation process. These studies revealed important roles for growth factor signalling, proteases and cell-surface molecules in epithelial repair (reviewed by Grose and Werner, 2002). However, despite a plethora of data on the involvement of integrins in skin development and cell migration in vitro, there is little information on their in vivo functions during wound repair.
Integrins are heterodimeric transmembrane proteins consisting of an and a ß subunit that play crucial roles in cell-cell and cell-matrix interactions. They underpin cell adhesion and migration, as well as being involved in cell proliferation, programmed cell death and differentiation (Brakebusch et al., 1997
). The major integrins in the intact epidermis are
2ß1,
3ß1 and
9ß1, which bind various extracellular matrix proteins, including laminins, collagen I, tenascin C and fibronectin, and
6ß4, an integral component of hemidesmosomes that binds laminin 5 (Palmer et al., 1993
; Yokosaki et al., 1994
; Yamada et al., 1996
). After wounding, the expression profile of integrins on wound margin keratinocytes changes, characterised by suprabasal integrin expression (Hertle et al., 1992
) and induction of specific integrins that recognise proteins of the dermal and provisional matrix. These include the fibronectin receptor
5ß1; the fibronectin and vitronectin receptor
vß5; and
vß6, which binds to fibronectin, vitronectin, tenascin C and transforming growth factor ß (TGFß) (Larjava et al., 1993
; Zambruno et al., 1995
; Clark, 1990
; Yamada et al., 1996
; Plow et al., 2000
).
A series of studies has revealed an essential role of ß1 integrins in the regulation of keratinocyte proliferation and differentiation. Most interestingly, the ß1 integrin subunit has been found to be a marker for stem cells in vitro and in vivo (Jones and Watt, 1993; Jones et al., 1995
). Inhibition of ß1 integrin function by introduction of a dominant-negative ß1 mutant into keratinocytes reduced the activation of mitogen-activated protein kinase and stimulated exit from the stem cell compartment (Zhu et al., 1999
). Furthermore, suprabasal expression of ß1 is a feature of the hyperproliferative epidermis of psoriatic skin, and ectopic expression of the ß1 integrin subunit in the suprabasal epidermal layers of transgenic mice caused keratinocyte hyperproliferation and a psoriasis-like phenotype (Carroll et al., 1995
).
To determine the role of ß1 integrins in skin morphogenesis and homeostasis, we and others generated mice that lack the ß1 integrin subunit specifically in keratinocytes. The characterisation of the ß1-deficient mice revealed crucial roles for the ß1 integrin subfamily for hair follicle development and skin integrity (Brakebusch et al., 2000; Raghavan et al., 2000
). In this study, we used our mouse model to investigate cutaneous wound repair, a process that is reliant on both keratinocyte migration and proliferation, in the absence of epidermal ß1 integrins. We demonstrate an important role for ß1 integrins during wound re-epithelialisation, where ß1-null keratinocytes display a severely compromised epithelial migration.
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MATERIALS AND METHODS |
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Isolation and culture of mouse keratinocytes
Murine epidermal keratinocytes were isolated as described (Roper et al., 2001). Keratinocyte cell lines were generated by repeated subculturing at high density (Romero et al., 1999
).
Retroviral infection of keratinocytes
AM12 supernatants or concentrated VSV G-pseudotyped virus was added to subconfluent mouse keratinocyte cultures and incubated for 6-8 hours in the presence of 8 µg/ml polybrene. The following retroviral vectors were used: pBabe chicken ß1 integrin subunit (Levy et al., 1998), pBabe EGFPCre and LZRSpBMN EGFPCre (Cre fusion protein and LZRSpBMN, kind gifts from J. Muller, ICRF).
Time-lapse recording
Frames were taken every 5 minutes for 22 hours using a Zeiss Axiovert 135 TV microscope fitted with a Hamamatsu CCD Orca ER camera (Hamamatsu, Japan). Motility was measured using a cell tracking extension (ICRF) written for IPLab (Signal Analytics, USA) and speed was calculated using a program written in Mathematica by Daniel Zicha (ICRF).
FACS analysis
Single cell suspensions of freshly isolated keratinocytes were incubated with a rat antibody against the mouse ß1 integrin subunit (MB1.2; 1:2 diluted; kind gift from B. Chan), a rat antibody against the ß4 integrin subunit (CD104; 1:100 diluted, BD Pharmingen, Franklin Lakes, NJ) or a hamster antibody against the v integrin subunit (1:100 diluted; RDI, Flanders, NJ), and subsequently with AlexaFluor488-conjugated secondary antibody directed against rat IgG or biotinylated antibody directed against hamster IgG (Jackson ImmunoResearch, West Grove, PA), followed by incubation with RPE-Cy5 conjugated streptavidin (Dako, Hamburg, Germany). Immediately before analysis on a Becton-Dickinson FACScan, TO-PRO-3 (Molecular Probes, Leiden, NL) was added to the sample for viability gating.
Adhesion assays
Keratinocytes isolated from 2-day-old mice were seeded into 96-well plates (5x104 cells/well), pre-coated with fibronectin, laminin 1, poly-D-lysine (PDL), collagen type I or collagen type IV (BD Biocoat Cellware, Franklin Lakes, NJ). After overnight incubation, cells were washed with phosphate-buffered saline (PBS) and adhesion was quantified using a CytoTox 96 colorimetric kit (Promega, Madison, WI).
Wounding and preparation of wound tissue
Mice were anaesthetised by intraperitoneal (i.p.) injection of 100 µl ketamine (10 g/l)/xylazine (8 g/l) solution. Two full-thickness excisional wounds, 3 mm in diameter, were made on either side of the dorsal midline by excising skin and panniculus carnosus. Wounds were left uncovered and harvested 1, 2, 3, 5, 10 or 15 days after injury (n4 mice for each timepoint). For expression analyses, the complete wounds including 2 mm of the epithelial margins were excised and immediately frozen in liquid nitrogen. Non-wounded back skin from 10 days post partum (d.p.p.) and 20 d.p.p. mice served as controls. Alternatively, two full-thickness incisional wounds were made on either side of the dorsal midline, left uncovered and harvested at 3 or 6 days after injury (n=3 mice for each time point). For histological analysis, the complete excisional or incisional wounds were isolated, bisected, fixed overnight in 4% paraformaldehyde in PBS and embedded in paraffin wax. Sections (7 µm) from the middle of the wound were stained with Haematoxylin/Eosin (H/E) or Masson trichrome. All experiments with animals were carried out with permission from the local veterinary authorities.
Labelling with 5'BrdU
BrdU labelling was performed as described (Werner et al., 1994). Sections (7 µm) from the middle of the wound were incubated with a peroxidase-conjugated monoclonal antibody directed against BrdU (Roche Diagnostics, Rotkreuz, Switzerland) and stained with a diaminobenzidine-peroxidase substrate kit (Vector Laboratories, Burlingame, CA).
Immunofluorescence
To stain keratinocyte cultures for paxillin, cells were fixed with 4% paraformaldehyde in PBS containing 0.1% Triton X-100, washed in PBS, blocked in 2% FCS in PBS and incubated with anti-paxillin antibody (1:100 diluted; BD Transduction Laboratories), followed by AlexaFluor488-conjugated anti-rabbit IgG. To stain for filamentous actin, cultures were incubated in a 1:1000 dilution of rhodamine-conjugated phalloidin (Sigma-Aldrich, St Louis, MO). Wax and cryosections (7 µm) from the middle of the wound were incubated with antibodies directed against the ß1 integrin subunit (a kind gift from Dr Staffan Johansson, Uppsala, Sweden), the ß4 integrin subunit, ß-catenin and E-cadherin (kind gifts from Dr Rolf Kemler, Freiburg, Germany), caspase 3 (Roche Diagnostics Ltd, Rotkreuz, Switzerland), keratin 6 (Babco, Richmond, CA), laminin 5 (2LE4-6, kind gift from Dr Rupert Timpl, Munich, Germany), and PECAM1 (BD Biosciences, Heidelberg, Germany), followed by Cy2- or Cy3- conjugated secondary antibodies (Jackson Immunoresearch). Specimens were mounted with Mowiol (Hoechst, Frankfurt, Germany) prior to photographing on a Zeiss Axioplan fluorescence microscope or confocal imaging on a Leica confocal laser scanning microscope.
Immunohistochemistry
Wax sections (7 µm) from the middle of the wound were incubated with the appropriate primary antibody: neutrophils 1:125 dilution of a biotinylated rat antibody targeted to Ly-6G (BD Biosciences); and macrophages 1:125 dilution of a rat antibody targeted to F4/80 (Serotec, Oxford, UK). Anti-F4/80 was incubated with biotinylated donkey anti-rat IgG (Dianova GMBH, Hamburg, Germany). The VectaStain avidin-biotin-peroxidase complex kit was then used according to the manufacturers instructions, prior to peroxidase detection with a diaminobenzidine-peroxidase substrate kit (both from Vector Laboratories). Sections were counterstained with Mayers Hemalum.
Electron microscopy
Wounds were fixed overnight in 2% paraformaldehyde/2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, rinsed in 0.1 M cacodylate buffer and stained in 1% uranyl acetate. They were then incubated in 70% ethanol for 8 hours, dehydrated through an ethanol series and embedded in araldite resin. Semi-thin sections (1 µm) were cut with a glass knife using an ultramicrotome (Reichert, Bensheim, Germany) and stained with Methylene Blue. Ultra-thin sections (30-60 nm) for electron microscopic observation were processed on the same microtome with a diamond knife and placed on copper grids. The transmission electron microscopy was performed with a Zeiss 902A electron microscope (Zeiss, Oberkochem, Germany).
RNA isolation and RNase protection assay
RNA isolation and RNase protection assays were performed as described by Chomczynski and Sacchi (Chomczynski and Sacchi, 1987) or by Werner et al. (Werner et al., 1994
), respectively. As a loading control, 1 µg of each RNA sample was resolved through a 1% agarose gel and stained with Ethidium Bromide. Alternatively, the RNAs were hybridised with an antisense RNA probe to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. The templates we used have been published previously: IL1ß, IL1
and TNF
(Hübner et al., 1996
); VEGF (Frank et al., 1995
); MMP-10 and MMP-13 (Madlener et al., 1998
); fibronectin (Munz et al., 1999
); and collagen
1 (I) (Bloch et al., 2000
).
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RESULTS |
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Impaired adhesion, spreading, proliferation and migration of ß1-null keratinocytes in culture
To determine the role of ß1-integrins in keratinocyte adhesion, proliferation and migration in vitro, we isolated keratinocytes from 2-day-old mice. As shown by FACS analysis (Fig. 2A), ß1 integrins were almost completely absent on keratinocytes of K5ß1-null mice at this early time point, demonstrating efficient Cre-mediated deletion of the gene. Deletion of only one copy of the ß1 integrin gene did not affect the levels of ß1 integrins on the cell surface. ß4 integrin levels were reduced to about 50% of wild-type control levels in K5ß1-null keratinocytes (Fig. 2B), whereas v integrins were expressed at equally low levels in K5ß1-null and wild-type cells (Fig. 2C).
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To determine whether the lack of ß1 integrins affects migration of keratinocytes, we generated keratinocyte cell lines from mice homozygous for the floxed ß1-integrin allele and deleted this gene in culture using a retrovirus that expressed a fusion protein of enhanced green fluorescent protein (EGFP) and Cre recombinase. Five days after infection, 50% of EGFP-positive cells had lost the integrin ß1 subunit (Fig. 3A). Consistent with the results obtained with keratinocytes isolated from K5ß1-null mice, the ß1-deficient keratinocytes generated by retroviral deletion were not able to spread (Fig. 3B). This defect was rescued by infection of the cells with a retrovirus that expressed the chick ß1 integrin subunit (Fig. 3C,D). The cells were filmed over a period of 24 hours on days 4 and 5 after infection. Interestingly, the migration capacity of ß1-deficient keratinocytes was severely impaired (Fig. 3E and Movies). Thus, the average migration speed of wild-type cells was 21.2 (±0.506) µm/hour, whereas ß1-deficient cells only migrated at a speed of 2.56 (±0.858) µm/hour. These results demonstrate the importance of ß1 integrins for keratinocyte migration in vitro. Although the ß1-null keratinocytes had a defect in spreading and migration, they were still able to undergo cell division (see Movies).
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Cell-cell and cell-matrix interactions during wound repair
To confirm the lack of ß1 integrins in wound keratinocytes, we stained 10-day-old wound sections with an antibody against this integrin subunit. It was expressed in basal cells of the hyperproliferative wound epithelium in control mice (Fig. 6A), but absent in keratinocytes of K5ß1-null animals (Fig. 6B).
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A distinct line of laminin-5, a ligand for 3ß1 and
6ß4 integrins, was observed beneath the newly formed epidermis of 5-day wounds from control mice (Fig. 6E), whereas a diffuse distribution in the granulation tissue was seen beneath the hyperthickened epidermis at the margin of wounds of K5ß1-null mice (Fig. 6F). This finding confirms that ß1 integrins are required for correct localisation of laminin-5 in the basement membrane, as previously reported in studies of unwounded skin architecture in the same conditional knockout mouse (Brakebusch et al., 2000
). By contrast, cadherin-dependent cell-cell contacts appeared normal as demonstrated by the normal expression of E-cadherin (Fig. 6G,H) and of ß-catenin (data not shown) in K5ß1-null keratinocytes, and their appropriate association with cell membranes.
Lack of ß1 integrins in the keratinocytes of the hyperproliferative epithelium causes narrowing of the intercellular spaces
In the hyperproliferative epithelium of 5-day-old wounds from control mice, semi-thin sectioning revealed clear intercellular spaces between the migrating cells (Fig. 7A,C). In comparison, the keratinocytes of K5ß1-null mice were much more closely packed (Fig. 7B,D).
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Defective epidermal architecture in late wounds of K5ß1-null mice
K5ß1-null mice never showed complete re-epithelialisation at day 5-10 after injury (Fig. 4D and data not shown). By contrast, 60% of wounds in control mice had re-epithelialised by 5 days and all were completely healed by 10 days (n5 for each). By 15-days post-injury, wounds from control mice were completely covered with a thin, uniform epithelium (Fig. 8A) (n=5), which was characterised by normal expression of the differentiation-specific keratin 10 in the first suprabasal layer (data not shown). The epidermis was well attached to the underlying granulation tissue. In K5ß1-null mice, the whole wound bed was covered by epidermis, but in two out of five cases the converging epithelial tongues had failed to fuse (Fig. 8B). Where fusion had occurred, the hyperproliferative epithelium was about twice as thick as that in control mice (Fig. 8C), keratin 10 was only expressed in the most upper layer (not shown), and the keratinocytes were poorly attached to the underlying tissue.
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We also observed a slight increase in mRNA levels of fibronectin, collagen 1 (I) and tenascin-C both in non-wounded skin and during wound repair of K5ß1-null mice (Fig. 11 and data not shown). Concomitant with elevated levels of mRNAs encoding matrix proteins was a significant increase in the mRNA levels of matrix metalloproteinase 10 (MMP10; stromelysin 2) and MMP13 (collagenase 3), also both in skin and wounds of K5ß1-null mice (Fig. 11). These MMPs are expressed mainly at the migrating edge of the epidermis in wounds of wild-type mice (Madlener et al., 1998
). Taken together, these findings demonstrate that several major players of the wound repair process are aberrantly expressed in K5ß1-null mice.
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DISCUSSION |
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Proliferation of ß1-null wound keratinocytes is reduced in vitro but not in vivo
Within a few hours of cutaneous injury, keratinocytes start to migrate over the injured dermis. Subsequently, the cells behind the migrating epithelial sheet increase their proliferation rate and constitute a pool of extra cells that replace those lost during injury (reviewed by Martin, 1997). Thus, delayed re-epithelialisation can result from either impaired migration or proliferation. Interestingly, the results of the present study revealed no difference in the percentage of proliferating cells in the hyperthickened epidermis at the wound edge in early wounds of K5ß1-null mice and even prolonged keratinocyte hyperproliferation. This was unexpected, as high expression of the ß1 integrin subunit has been shown to be a marker for proliferation competent stem cells (Jones and Watt, 1993
; Jones et al., 1995
). Furthermore, keratinocyte proliferation is reduced in non-wounded skin of K5ß1-null mice (Brakebusch et al., 2000
) and of K14ß1-null mice, which revealed a similar, but more severe, phenotype (Raghavan et al., 2000
). Finally, the proliferation rate of our cultured ß1-deficient keratinocytes was almost halved and terminal differentiation was stimulated in these cells. These differences between the in vitro and in vivo situation could reflect the lack of dermal inflammatory cytokines and growth factors in culture, the presence of a different matrix and the three-dimensional organisation in vivo.
Keratinocyte migration is impaired in the absence of ß1 integrins in vitro and in vivo
Because the total number of wound keratinocytes was reduced in the mutant mice, although the percentage of proliferating cells was unchanged, reduced migration of ß1-null keratinocytes is likely to be the reason for the decrease in the total number of keratinocytes in the wound. This observation is strengthened by the observation that the ß1-null keratinocytes are piling up at the wound edges instead of forming a thin epithelial tongue across the wound. Most importantly, this hypothesis is strongly supported by the impaired migratory capacity of ß1-deficient cultured keratinocytes.
Under normal circumstances, keratinocytes first migrate down over the injured dermis where they contact fibrillar type I collagen (Pilcher et al., 1997). Based on in vivo expression studies and functional in vitro data, it has been suggested that the
2ß1 integrin is required for this migration over the wounded dermis (Pilcher et al., 1997
; Hodivala-Dilke et al., 1998
; Goldfinger et al., 1999
). The results presented in the present study thus provide the first in vivo evidence for this hypothesis.
Although the onset of keratinocyte migration was retarded in K5ß1-null mice, a strongly hyperthickened epidermis was observed at the wound edge, most probably due to the continued keratinocyte hyperproliferation in the absence of migration. Several days after wounding, the keratinocytes finally migrated over the wound bed, but the epidermis was strongly hyperplastic, poorly differentiated and poorly attached to the underlying granulation tissue. Most interestingly, the healed epidermis of incisional wounds in control mice had a V-shaped morphology, whereas it was flat in K5ß1-null mice, suggesting that re-epithelialisation in K5ß1-null mice occurs via an alternative, compensatory mechanism (summarised in Fig. 12). Because ß1-deficient keratinocytes lack the principal adhesive receptors used for ligation to dermal matrix, they must wait until compensatory mechanisms are upregulated. Such mechanisms are probably based on the expression of non-ß1 integrins by keratinocytes and on the presence of the corresponding ligands. The most likely candidates are vß5 or
vß6, receptors for the provisional wound matrix proteins vitronectin, fibronectin and tenascin C (Gailit et al., 1994
; Larjava et al., 1993
; Zambruno et al., 1995
; Haapasalmi et al., 1996
; Huang et al., 1998
). Although
v integrins are weakly expressed in normal and ß1-deficient cultured keratinocytes,
vß5 and
vß6 are upregulated in migrating keratinocytes during cutaneous wound repair (Cavani et al., 1993
; Gailit et al., 1994
; Haapasalmi et al., 1996
).
vß5 on keratinocytes is characteristic of a migratory phenotype (Adams and Watt, 1991
), and
vß6 is important for keratinocyte migration on vitronectin, fibronectin and tenascin C in vitro (Huang et al., 1998
). The period required for the upregulation of these integrins and for the deposition of their ligands in the wound bed is likely to determine the time frame for the onset of re-epithelialisation in K5ß1-knockout animals. The dependence on non-ß1 integrins also provides an explanation for the flat morphology of the healed epidermis. Thus, we propose that K5ß1-null keratinocytes are not able to migrate down the injured dermis, because of the absence of ligands for the dermal substrate, but rather wait until the wound is filled with granulation tissue that contains the ligands for non-ß1 integrins.
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Abnormal epidermal architecture in healed wounds of K5ß1 null mice
Re-epithelialisation was eventually completed in K5ß1-null epidermis, but the wounds had a thicker epidermal covering, reflecting the prolonged period of keratinocyte hyperproliferation and their delayed differentiation. This is unlikely to be a direct result of a lack of ß1 integrins in keratinocytes, but could be due to a prolonged proliferative signal from cells within the granulation tissue, as suggested by the increased expression of epithelial growth factors. In addition, there might be a lack of ß1-dependent contact inhibition when the epithelial fronts meet (Huttenlocher et al., 1998) as supported by the epithelial morphology of some of the wounds where the epidermal edges had not fused and where obviously unlimited growth of the epidermis had occurred.
Finally, K5ß1-null keratinocytes failed to establish a normal basement membrane above the newly formed granulation tissue. This finding is likely to underlie the poor attachment of the new epidermis to the mesenchyme and concurs with the observed blister formation, as well as with the reduced number of hemidesmosomes seen in non-wounded skin of K5ß1-null mice (Brakebusch et al., 2000). Blister formation was also observed in mice that lack the integrin
3 subunit (DiPersio et al., 1997
), suggesting that the lack of
3ß1 in our mice is predominantly responsible for the poor attachment of the epidermis. However, in contrast to K5ß1-null keratinocytes, the exclusive lack of the
3 integrin subunit in this cell type did not cause an altered expression of
6ß4 in vitro or in vivo (Hodivala-Dilke et al., 1998
), suggesting that the additional loss of other ß1 integrins is responsible for the reduction in the number of
6ß4 integrins in K5ß1-null keratinocytes.
Prolonged inflammation in K5ß1-null mice
In K5ß1-null mice, we observed an extended window of neutrophil presence, most likely as a result of the prolonged exposure of K5ß1-null wounds to the external environment. Because re-epithelialisation is so delayed, their wounds are susceptible to pro-inflammatory stimuli such as desiccation and mechanical stress for a longer period. As neutrophils are a major source of pro-inflammatory cytokines in skin wounds (Hübner et al., 1996), the elevated number of these cells is likely to contribute to the increased expression of IL1
, IL1ß and TNF
in the wounds of K5ß1-null mice. These cytokines induce expression of many epithelial growth factors (reviewed by Werner and Smola, 2001
), which could contribute to the increased keratinocyte proliferation rate. Increased expression of pro-inflammatory cytokines was also observed in the unwounded skin of older K5ß1-null mice, but these are thought to be released by macrophages rather than neutrophils (Brakebusch et al., 2000
). However, the expression levels associated with the developmental inflammation and fibrosis of unwounded skin were significantly lower than the transient expression levels seen during the repair process. Thus, the developmental fibrosis should have a rather minor effect on the repair process in K5ß1-null mice, as supported by the normal contraction of the healing wound.
Taken together, our results reveal a strongly impaired migratory capacity of ß1-deficient keratinocytes in vitro and a dramatic delay in epithelial migration during wound repair in K5ß1-null mice. We thus present the first in vivo evidence in support of findings from in vitro studies that have shown ß1 integrins to be key players in cell migration. However, our results also demonstrate that keratinocytes are not totally dependent on this integrin subunit to heal their wounds. Rather, other integrins appear to compensate at least partially for the lack of ß1, leading to complete, although imperfect, re-epithelialisation.
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ACKNOWLEDGMENTS |
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