Keratinocyte Interactions with Fibronectin during Wound Healing

Larjava H, Koivisto L, Häkkinen L.

Publication Details

Reepithelialization of wounds is critical for survival. After injury, fibronectin-fibrin clot is formed. Keratinocytes become activated and start migrating into the clot. Migration involves coordinate expression of several new extracellular matrix molecules, fibronectin receptors and proteinases. Migrating keratinocytes express alternatively spliced EDA fibronectin while both EDA and EDB fibronectin isoforms are found in the granulation tissue. Fibronectins are assembled into polymerized matrix that is further crosslinked to fibrin, and these multimeric complexes of fibronectins have their own specific effects on cell signaling. Upon wounding, keratinocytes express three new fibronectin receptors, namely α5β1, αvβ6 and αvβ1 integrins. Integrin α5β1 recognizes the RGD sequence and the synergy site of fibronectin and plays a critical role in the early migration. Interestingly, an important role for the synergy site in keratinocyte migration has been proposed. In addition to cell migration, α5β1 integrin also regulates expression of dozens of genes important for a variety of cell functions including cell growth. Integrin αvβ 6 also mediates cell adhesion on fibronectin and it can functionally replace α5β1 integrin. The most important function of αvβ6 may be, however, to activate TGFβ during late wound healing. Laminin-5 and tenascin-C are also expressed by the migrating keratinocytes. These cells have three laminin-5 and two putative tenascin-C receptors, and their function needs to be spatially and temporally coordinated with the three fibronectin receptors. Elucidation of the mechanism of this coordination is crucial for better understanding of reepithelialization.

Reepithelialization: A Controlled Migration/Invasion of Keratinocytes

Tissue loss or damage in adult animals initiates the wound healing process that involves a series of controlled events resulting in tissue repair or scarring. Complete regeneration is only seldom observed, and the site of the original wound is distinguished from the nondamaged tissue even weeks, months or sometimes years after the initial event. In cases where inflammation continues or healing does not follow the ordinary sequence, the wound becomes chronic and healing can lead to localized fibrosis. The roles of epithelial cells in the wound healing process have been expanded from mechanical coverage of the wound to active participation in the control of the inflammatory process. There is some evidence that epithelial cells may contribute to the formation of granulation tissue during the later phase of wound healing and also play a role in the formation of nonhealing wounds. This is evidenced from clinical observations demonstrating that skin wounds that have delayed reepithelialization are more likely to become chronic nonhealing wounds than those in which epithelial migration proceeds at the normal rate. The purpose of this review is to cover the mechanisms of epithelial cell migration in wound provisional matrix with a special emphasis on epithelial cell interactions with provisional matrix fibronectin. Since epithelial (keratinocyte) migration in wound provisional matrix involves several matrix molecules and their receptors, their role in wound healing will also be briefly discussed as the context requires.

After the epithelium has been disrupted by tissue injury, reepithelialization starts rapidly in order to reestablish tissue integrity.1,2 Reepithelialization involves migration of epithelial cells from the edges of the wound or, in the case of skin, from appendages such as hair follicles and sweat glands.1,2 The migratory cells are in close contact with the wound provisional extracellular matrix that is mainly composed of fibrin, fibronectin, and vitronectin.13 Initially, keratinocytes begin moving into the defect about 24 hours after the injury.2 During the early migration, cell proliferation does not contribute to the migration. Rather, epithelial cells dissolve their hemidesmosomes, detach from basement membrane, and move quickly into the wound defect. Proliferation contributes to the reepithelialization process at the later stages when it seeds more cells into the wound. The morphology of keratinocytes changes when they assume the migratory phenotype. Normally polarized basal keratinocytes become flattened and elongated. Long, a-actinin and actin-rich, cytoplasmic processes called lamellipodia, along with ruffling cytoplasmic processes, are observed in migrating keratinocytes in the provisional matrix.1,4,5 Cell-cell connections through desmosomes are also reduced, and there are no hemidesmosomes underneath the migrating keratinocytes. The numbers of cap-junctions, however, increase.6 During the last few years, extensive progress has been made in the understanding of the signaling mechanisms which regulate lamellipodia formation in various cell types.7 Intracellular signaling events are, however, cell and matrix specific, and only scattered information is available about the signals that lead to lamellipodia formation in migrating keratinocytes.8 It is conceivable that the small GTPases are important in the lamellipodia extension, migration and invasion.7 Recent results from our laboratory suggest that the function of the small GTPase Rac is crucial for lamellipodia formation in HaCaT keratinocytes that have been stimulated by staurosporine (Koivisto et al, unpublished). Rac also seems to be essential in cell crawling from edges of wounded MDCK epithelial sheets,8 and it can participate in recruitment of high affinity integrin receptors into the lamellipodia.9 Many cytokines that promote cell migration are able to cause alterations in the organization of cytoskeleton and cell shape. The effects of one such growth factor/cytokine, namely keratinocyte growth factor (KGF), on keratinocyte spreading on fibronectin was recently investigated.10 KGF is a fibroblast-derived polypeptide that was originally found to promote keratinocyte growth. It was shown to promote keratinocyte migration10 and participate in regulation of keratinocyte differentiation, together with β1 integrins.11 KGF was able to promote the organization of actin cytoskeleton and lamella formation on the fibronectin matrix and cause alterations in integrin avidity,10 demonstrating that KGF may have more functions in wound healing than originally anticipated. It can be expected that several wound fluid cytokines may be involved in regulation of lamellipodia formation in migrating keratinocytes in vivo.

Keratinocyte Invasion into the Clot

It has commonly been described that epithelial cells migrate on the exposed connective tissue matrix underneath the fibrinfibronectin clot. In small oral mucosal wounds, however, the keratinocytes cut their way directly through the clot and may not interact with the connective tissue matrix at all (Fig. 1).12 Migrating keratinocytes are highly phagocytic, allowing them to penetrate through tissue debris or the clot.2 Degradation of the fibrin-fibronectin clot appears to be critical for wound healing since wounds in animals that lack the plasminogen gene do not reepithelialize.13 Interestingly, it was recently demonstrated that keratinocytes migrate though tunnels of digested fibrin-fibronectin in vitro.14 This process requires plasminogen activation at the leading edge. Although it was not addressed in this paper, it seems likely that integrins play a role in fibrin clot removal. Migrating cells must be able to focalize the proteolysis into the leading edge.15 This could be done by activation of proteolytic enzymes at specific sites at the cell membrane. It has been found that urokinase type plasminogen activator receptor (uPAR) is able to associate with integrins in various cell types including keratinocytes.16,17 Plasminogen activator inhibitor type 1 (PAI1) is also induced by wounding,18 and its targeted disruption leads to impaired wound healing in vitro19 suggesting that a well-regulated plasmin activation is essential for epithelial cell migration. This is an example of how a cell is able to focalize fibrinolysis by plasmin and promote subsequent migration by integrins. Formation of cylindrical and helical tunnels into fibrin clot is a novel migratory behavior of normal keratinocytes, and this process appears to speed up the migration.14 Furthermore, wound fluid cytokines such as EGF and TGFβ1 are able to differentially regulate the types of tunnels formed.14 Although these single-cell experiments are highly interesting, it is not known how these findings relate to the in vivo situation in which keratinocytes migrate as a sheet of cells. Also nothing is known yet about the matrix receptors involved. In addition to plasmin, matrix metalloproteinases are also involved in proteolytic degradation of the clot matrix proteins, and this process has recently been reviewed elsewhere.20 Briefly, migrating keratinocytes express MMP-9, MMP-1 (interstitial collagenase), MMP-10 (stromelysin), and MMP-28 (epilysin), of which MMP-9 and -10 could participate in fibronectin-fibrin degradation. MMP-1 degrades collagens, and the function of MMP-28 is not yet known.20,21 MMP-14 (MT1-MMP) is expressed by transformed human epithelial cells22 and by wound epithelial cells in rat cornea,23 but to date it has not been demonstrated in normal human wound epithelial cells. MMP-14 can cleave fibronectin, other glycoproteins, proteoglycans, and collagen,24 and it participates in activation of MMP-2,25 suggesting that it could potentially be used to promote cell migration. Blocking MMP activity prevents keratinocyte migration into the wounds in cell culture.20,26 This proteolytic modulation of the matrix underneath migrating cells must be well controlled since overexpression of MMPs is a common finding in nonhealing chronic wounds. 27 During wound healing, most of the components of the basement membrane zone such as type IV and VII collagens, laminin-1 and heparan sulphate proteoglycan are missing from underneath the migrating keratinocytes.12,28 Fibronectin EDA (see below), tenascin-C and its large molecular weight splicing variant and laminin-5, however, appear to be always deposited against the wound bed matrix by keratinocytes during migration.12,29,30

Figure 1. Keratinocyte migration: through the clot or underneath? In human oral mucosa, wound epithelium migrates through the fibrinfibronectin clot (A).

Figure 1

Keratinocyte migration: through the clot or underneath? In human oral mucosa, wound epithelium migrates through the fibrinfibronectin clot (A). In skin, the migration occurs between the clot and connective tissue (B). E: epithelium; CT: connective tissue; (more...)

Fibronectin is a multidomain glycoprotein that is found in the blood and extracellular matrices of a variety of tissues. Readers are asked to visit an excellent website from Dr. Ingham's laboratory for a detailed review of fibronectin structure and function (www.gwumc.edu/biochem/ingham/fnpage.htm). Fibronectin is a key component in the provisional wound matrix.3 Its main function during wound healing is in mediating cell adhesion and migration but it also promotes cell proliferation, chemotaxis, and cellular signaling.31 In normal skin and mucosa, fibronectin is present underneath the basement membrane. During wound repair, keratinocytes make contact with two types of fibronectins, one from plasma and one of their own. How these fibronectins differ in structure and function will be discussed below. Fibronectin is coded by one gene that has repeating modules called types I, II and III which serve as building blocks for structural domains that have different functions (Fig. 2).32 For example, type I modules are found in fibrin binding domains, type II modules in collagen binding domains, and type III modules in cell-binding domains. The fibronectin gene has three sites for alternative splicing (EDA, EDB, V region) and codes potentially about 20 different variants of fibronectin proteins. Alternative splicing varies in a cell type-specific manner and can modulate the functional properties of the molecule.33,34 Alternative splicing of the fibronectin gene is important in the context of wound repair since alternatively spliced forms are present in the matrix produced by migrating keratinocytes and in the granulation tissue.35,36 Insertion of additional type III repeats EIIIA and EIIIB that flank the major cell adhesion domain of the molecule results in fibronectins termed EDA and EDB, respectively (Fig. 2).33,34 EDA and EDB are present in the so-called cellular fibronectin but absent from plasma fibronectin.33,34 Cellular fibronectins and plasma fibronectin self-assemble but the different fibrils don't mix.37

Figure 2. Molecular structure of fibronectin.

Figure 2

Molecular structure of fibronectin. A. Schematic presentation of the structural and functional domains of fibronectin. B. During fibronectin fibrillogenesis by cells, the folded conformation of the soluble fibronectin molecule becomes stretched, and the (more...)

Plasma contains 300 mg/ml of fibronectin, and it becomes incorporated into the blood clot with fibrin. The role of clot-bound plasma fibronectin has been suggested to be critical for reepithelialization. Rather surprisingly, the wound healing process including the reepithelialization was found to be normal in plasma fibronectin-deficient animals.38 This suggests that cellular fibronectin released from platelets may compensate for the lack of plasma fibronectin. The presence or absence of plasma fibronectin and EDA and EDB variants has spatial and temporal correlations with wound healing (Fig. 3). During migration in human wounds, keratinocytes deposit EDA fibronectin underneath the leading epithelial tongue while EDB is not expressed (Fig. 3). This may also explain the normal reepithelialization in the plasma fibronectin-deficient animals.

Figure 3. Immunolocalization of EDA (A, C, E, G) and EDB (B, D, F, H) fibronectin in human oral mucosal wounds.

Figure 3

Immunolocalization of EDA (A, C, E, G) and EDB (B, D, F, H) fibronectin in human oral mucosal wounds. In 3-day wounds (A and B), EDA fibronectin was expressed under the epithelial cells migrating through the fibrin clot (A; arrowheads) but no expression (more...)

Based on their expression pattern in wound healing, it can be expected that the EDA and EDB domains have important but different functional roles during the repair process. However, the specific functions of these fibronectin variants in keratinocytes are unclear. In CHO cells, EDA-containing fibronectin seemed to be more potent in promoting cell spreading and migration than EDB because of its enhanced binding by α5β1 integrin.39 EDA fibronectin was also more potent than the EDA-negative variant in promoting cell cycle progression and mitogenic signal transduction.40 However, in mesenchymal cells, EDB promoted RGD-mediated cell adhesion and spreading while EDA fibronectin had the opposite effect.41,42

The alternatively spliced sequences of the IIICS region (V region) of fibronectin encode cell type-specific adhesion sites and are found in wounds.3 Although fibroblasts and keratinocytes are not known to use this region of fibronectin for cell adhesion, these modules are important for adhesion of lymphoid cells.3

The ability of fibronectin to bind denatured collagen is likely an important step in wound repair.3 During injury, collagen molecules become denaturated and are no longer recognized by collagen binding integrin receptors.43 During denaturation, cryptic RGD sites of collagen are exposed and recognized by αvβ 3 integrin. Unfortunately, keratinocytes do not express this integrin (see below). Denatured collagen, however, binds fibronectin with high affinity.3 This affinity concentrates fibronectin in the area of injury in which keratinocytes can recognize it with their multiple integrin receptors (see below). It remains to be seen whether fibronectin bound to denaturated collagen also provokes cellular signaling that is different from, e.g., fibronectin bound to fibrin.

Keratinocytes are known to produce and deposit fibronectin in culture.44,45 Wounding of MDCK epithelial cell cultures in vitro down-regulates fibronectin expression but up-regulates the proportion of EDA fibronectin mRNA.46 Cultured keratinocytes leave behind trails of EDA fibronectin (Fig. 4). In vitro, the deposition of EDA fibronectin correlates with migration but is not absolutely essential. By modification of the culture conditions, the deposition of EDA fibronectin can be minimized while the cells are still able to migrate over a scratch wound (unpublished data from our laboratory).

Figure 4. Deposition of EDA fibronectin is induced during keratinocyte migration by TGFβ and becomes removed after migration stops.

Figure 4

Deposition of EDA fibronectin is induced during keratinocyte migration by TGFβ and becomes removed after migration stops. Confluent human skin keratinocyte cultures (HaCaT cells) were wounded and allowed to migrate in the absence (A and B) or (more...)

Cell Adhesive Sites of Fibronectin

Keratinocytes and most other cell types adhere to the cell-binding domain of fibronectins. In fact, the cell-binding domain has similar functions in promoting keratinocyte migration as the intact molecule.47 The cell adhesive sequence in this domain is Arg-Gly-Asp-Ser (RGDS), which is recognized by multiple receptors of the integrin family.3 Peptides containing this RGD sequence have been used both in vitro and in vivo to block cell adhesion and migration. The RGD sequence is, however, present in several extracellular matrix molecules and represents, therefore, a nonspecific approach for manipulation of cell adhesion. Immobilized RGD peptides can support keratinocyte migration although not to the same degree as the cell-binding domain.47 The RGD site in soluble fibronectin may be cryptic since adsorption of fibronectin promotes binding of monoclonal antibodies directed against a sequence flanking the RGD site.48 In addition to the RGD site, fibronectin contains a second so-called synergy site that is needed for maximal adhesion by α5β1 integrin receptor.3The critical sequence in this region is Pro-His-Ser-Arg-Asn (PHSRN) and it is located in the type III module adjacent to the type III that contains the RGD site. In a recent study, a peptide corresponding to this synergy sequence was tested for keratinocyte motility and healing of wounds in an obese diabetic mouse model (C57BL/6Job/ob) in which the healing is known to be compromised.49 Both the 120-kDa cell binding domain of fibronectin and the PHSRN peptide were able to induce keratinocyte motility and invasion through the basement membrane and extracellular matrix of sea urchin embryos. Keratinocytes do not normally invade through the basement membrane matrix but rather use that as a template to establish new, stratified epithelium. On a molar basis, the PHSRN peptide was surprisingly over tenfold more active than the cell-binding domain. Even more surprisingly, this small peptide was able to accelerate wound closure and reepithelialization dramatically in diabetic animals and slightly in normal mice.49 It was speculated that PHSRN is proteolytically released from fibronectin in normal healing wounds, providing chemotactic signals for cells to induce cell migration. This process could be impaired in diabetic animals. Alternatively, PHSRN peptide could become immobilized into the wound and act as a matrix molecule.50 A similar peptide (PHSCN) was able to block metastasis in rats.49 Small peptides like this do not usually possess a strong biological effect because of rapid clearance in circulation.50These papers remain controversial with such dramatic results and need to be confirmed by other investigators.50 If true, the use of this synergy region of the cell-binding domain of fibronectin would be very valuable for millions of people suffering from impaired wound healing. Interpretation of the results of the above study demonstrating improved healing with the synergy peptide in ob/ob mice is even more difficult in light of a recent paper about leptin effects on wound healing in these mice.52 Impaired wound healing in the leptin-deficient ob/ob mice has been explained for years by the diabetic phenotype of the animals. Administration of leptin, either systemically or locally, however, appears to normalize the wound healing defect in these mice.52 The impaired healing response can be, therefore, explained entirely by the genetic background of the animals and not by e.g. keratinocyte chemotaxis towards fibronectin fragments with the synergy region. Leptin effects as such on wound healing are interesting. Leptin seems to enhance the reepithelialization by stimulating keratinocyte proliferation through activation of STAT3 transcription factor by ObRb leptin receptor that is temporarily regulated during wound repair.52 This is in agreement with findings with STAT3-deficient animals that possess an impaired wound repair phenotype.53

Biological Functions of Fibronectin Matricryptins

Fibronectin fragments have been demonstrated in wound fluids from chronic wounds,54,55 and they have several potential functions that are different from the intact fibronectin molecule. This extracellular matrix fragmentation is considered crucially important for wound repair because it produces biologically active polypeptides in which novel cryptic sites are exposed.56 These sites were recently termed as matricryptic sites and the polypeptides matricryptins, respectively.56 Fibronectin has potentially several of these matricryptic sites that function in cell adhesion, cell proliferation, matrix assembly, and matrix degradation (see below).56 Chronic skin and corneal wounds show advanced degradation of fibronectin, probably via the plasmin pathway. Fibronectin supplementation into these nonhealing chronic skin and corneal ulcers seems to improve reepithelialization.2 Fibronectin fragments containing the cell-binding site are involved with regulation of MMP-1 expression in vitro.57 This mechanism has not been proven for keratinocytes but is potentially important when the keratinocytes face the connective tissue collagen matrix because the migration of keratinocytes on type I collagen requires MMP-1 activity.27 The EDA domain, but not the native fibronectin, can also induce IL-1 expression, at least in connective tissue cells, which can lead to increased MMP-1 synthesis.58 The role of RGD and PHSRN-containing peptides and other fibronectin-derived matricryptins in keratinocyte migration and signaling clearly warrants further investigation.

Fibronectin Matrix Assembly and Cross-Linking to Fibrin

The fibronectin matrix assembly is likely to play an important role in cell adhesion and signaling during wound repair. Interaction of cells with the extracellular matrix conveys information about the nature of matrix proteins and how the matrix has been assembled into three-dimensional structures.59 Cells polymerize fibronectin by assembling soluble fibronectin from cells or plasma into insoluble fibrils in a multistep pericellular process. Fibronectin fibrillogenesis requires the integrin α5β1 receptors that bind to the cell-binding domain to initiate the process.60 Contacts of α5β1 integrins with both of the RGD and synergy sites as well as interaction of the so-called matrix assembly site at the cell surface with the amino terminal region (first five type I repeats) of fibronectin are needed for fibril polymerization.6166 The process is completed by fibronectin self-assembly.67 Activation of integrins can overcome the requirement of the synergy site.66 A novel RGD-independent fibronectin assembly site was recently discovered in the alternatively spliced V region of the molecule.68 This assembly site requires α4β1 integrin expression. The heparin binding domain of fibronectin is also important for polymerized fibril formation.69 Polymerization of fibronectin has functional significance because polymerized fibronectin matrix promotes cell growth. This promotion appears to involve cell surface proteoglycens.70

Interestingly, an additional site at the amino terminus of the fibronectin molecule also contributes to polymerization. This first type III module contains a 76-amino acid sequence (termed III1C) that can, when added to soluble fibronectin, polymerize it into high molecular mass multimers, so called superfibronectin fibrils, in vitro.67 The isolated III1C fragments are believed to interfere with the intramolecular interactions that keep fibronectin in solution.71 Alternatively, they could change the conformation of fibronectin, opening cryptic sites essential for fibril selfself assembly.65 Polymerized superfibronectin is much more potent in promoting cell adhesion than the nonpolymerized form.67 The structure of superfibronectin and whether superfibronectin fibrils resemble or compose natural fibrils is not known. Interestingly, systemic admininstration of polymerized superfibronectin has a strong antimetastatic effect.72 The III1C fragment appears also to be a potent inhibitor of tumor growth, angiogenesis, and metastasis.73 The effects of polymerized superfibronectin in keratinocyte adhesion, migration, and growth have not been investigated.

The active role of cells in fibronectin fibrillogenesis directs the deposition of the extracellular matrix in the desired location and prevents unwanted fibrillogenesis in circulation. Although keratinocytes express EDA fibronectin in vitro, they don't deposit it into the traditional fibronectin matrix seen in fibroblast cultures. Migrating keratinocytes leave tracks of cellular fibronectin behind, but these tracks are no longer present when the cells reach confluency (Fig. 4). It is likely that fibronectin that is deposited to support migration is proteolytically removed rather than organized into extensive fibrils when keratinocytes stop migrating. Lack of α5β1 integrin does not explain the inability to make polymerized fibronectin fibrils because this integrin is expressed abundantly by cultured keratinocytes.12,74 The inability of keratinocytes to organize a polymerized fibronectin matrix may also relate to alterations in integrin activity.75 Finally, the small GTPase Rho-mediated cell contractility is needed for fibronectin polymerization, possibly because stretching of the fibronectin molecule by the cells exposes cryptic self-assembly sites necessary for polymerization.76 Structurally, the RGD site resides in a flexible loop, and therefore its exposure may potentially also be modulated by the tension of the fibronectin molecule.77 Visualization of green fluorescent protein-tagged fibronectin in real time in living cell cultures has elegantly demonstrated that fibronectin fibrils are highly elastic and are continuously under tension produced by cells fibrblasts (www.pnas.org78). It is possible that keratinocytes are unable to polymerize fibronectin because they are less contractile cells than fibroblasts.

The turnover rate of integrins may also be implicated in fibronectin fibrillogenesis since acceleration of β1 integrin turnover rates by antisense cDNA construct appears to block fibronectin fibrillogenesis in MG63 cells.79 In human keratinocytes, the turnover rate of β1 integrin is much faster than in fibroblasts.74 Also many tumor cells have rapid integrin turnover rates and fail to assemble the fibronectin matrix.80 It can be hypothesized that with high turnover of integrin receptors, cells fail to initiate the conformational change in fibronectin required for fibrillogenesis. The inability to assemble the fibronectin matrix may be beneficial for keratinocytes that don't normally deposit this type of matrix in vivo. Fibrillar fibronectin matrix could impair the deposition of the basement membrane after migration is complete.

Fibrin is cross-linked to fibronectin by factor XIII in the clot. The ratio of fibronectin to fibrin in the clot and the degree of clot cross-linking may also regulate keratinocyte behavior.81 Keratinocytes do not express the αvβ 3 integrin that is usually required for fibrin binding.12,82 While adding fibronectin into fibrin increases keratinocyte spreading, the elevated amounts of fibrin reduces keratinocyte adhesion.81 Cross-linking of fibrin to fibronectin by factor XIII is known to increase cell adhesion in many cell types including keratinocytes.83,84 Organization of the cytoskeleton is also regulated by fibronectin-fibrin cross-linking.85 Fibronectin in the clot can be recognized by the α5β1 integrin, and this will promote clot retraction by various cell types.86 Interestingly, cross-linking fibrin alone by factor XIII appears to promote some adhesion of keratinocytes.81 The mechanism of this induction is not known, but it provides evidence that keratinocytes in vivo are able to adhere not only to fibronectin in the clot but also to cross-linked fibrin. Cellular fibronectin produced by migrating keratinocytes also interacts with fibrin but with different kinetics and interactions from that produced from plasma.87

Expression of alternatively spliced variants of fibronectin is regulated by cytokines. Fibronectin expression in keratinocytes can be greatly stimulated by certain growth factors and cytokines. TGFβ is obviously the key cytokine involved in keratinocyte migration. TGFβ1 is the major isoform of the members of the TGFβ family in wound keratinocytes. TGFβ1 inhibits the growth of keratinocytes in vitro and in vivo, and it seems to stimulate keratinocyte motility by switching the cells from the differentiating to regenerative phenotype.88 TGFβ is able to stimulate the production of fibronectin89 and the expression of its fibronectin receptors (see below)82,90 as well as specifically stimulate the expression of EDA-type fibronectin.91 In the keratinocyte scratch-wound model, TGFβ1 stimulates the expression of EDA fibronectin in cells adjacent to the wound (Fig. 4). Wounding alone appears also to slightly stimulate EDA fibronectin expression. In MDCK cells, TGFβ appears to also regulate the ratio of EDA-containing fibronectin mRNA to EDA-negative fibronectin mRNA.46 In this system, migrating cells seem to express a lesser amount of total fibronectin but relatively more of the EDA-containing variant. Induction of TGFβ1 in migrating keratinocytes is thought to be crucial for the successful reepithelialization of skin wounds because reduced amounts of TGFβ1 are associated with impaired wound healing and administration of TGFβ1 has been shown to promote wound healing.92 Surprisingly, there is no defect in reepithelialization in TGFβ1-deficient animals.93 It is possible that other cytokines can compensate for TGFβ1 when it is absent. Epidermal growth factor and hepatocyte growth factor/scatter factor, that are also abundantly present in wounds, stimulate keratinocyte migration and secretion of cellular fibronectin.89,91Alternatively, TGFβ1 could have its major function in regulation of inflammation and granulation tissue formation (see below).

Fibronectin Receptor Integrin Expression during Re-Epithelialization: α5β1 Integrin

As discussed above, integrins a/b heterodimers are responsible for most cellextracellular matrix interactions in various cell types including epithelial keratinocytes.9496 During wound healing, keratinocytes have to adjust their integrin receptors to be able to adhere and migrate on the extracellular matrix proteins of the provisional matrix. Wounding causes a change in the expression levels and/or distribution of the existing integrins and induces expression of three new fibronectin receptor integrins, namely α5β1, αvβ1 and αvβ 6 (Fig. 5).12,30 These integrins are usually absent from normal healthy adult epithelium.12,96,97 Additionally, wound keratinocytes in vivo express at least syndecan-1 and CD44 heparan sulfate proteoglycans that are able to participate in fibronectin binding.28 Integrin α3β1, that localizes to basal keratinocytes in the stationary epithelium and to migrating keratinocytes in vivo,12,98 has been reported to function as a fibronectin receptor in some cell types, but in keratinocytes it preferably acts as a receptor for laminin-5.99,100 In culture, skin keratinocytes become activated and start to express α5β1 integrin.101 The mechanism of induction of α5β1 expression both in vivo and in vitro in keratinocytes is still largely unknown. It can be speculated that local cytokines such as TGFβ or exposure to serum components during early wound repair could initiate the expression that is further up-regulated when keratinocytes contact fibronectin. For example, expression of α5β1 integrin can be either induced or stimulated by TGFβ1 in keratinocytes.90,82 Alternatively, fibronectin itself could participate in the induction of its own receptor. In corneal epithelial cells, fibronectin is known to up-regulate α5β1 integrin expression through specific transcriptional activation.102

Figure 5. Expression of integrin-type fibronectin receptors by migrating wound epithelial cells.

Figure 5

Expression of integrin-type fibronectin receptors by migrating wound epithelial cells. Frozen sections from human oral mucosal wounds collected three days after wounding were stained with antibodies against αv (A; clone L230, Houghton et al, 1982), (more...)

When expressed on the cell surface, α5β1 integrin has multiple potential roles in keratinocytes including regulation of cell migration, proliferation, matrix degradation and gene expression.103 For example, adhesion of salivary gland epithelial cells to fibronectin alters gene expression of more than 30 proteins and several transcription factors.104 During early reepithelialization, perhaps the most critical function is the promotion of keratinocyte adhesion and motility. Cell adhesion and migration depend on integrin affinity and on the amount of fibronectin. HaCaT keratinocyte adhesion to fibronectin increases with elevated amounts of coated protein.82 In contrast, cell motility of HaCaT and normal human epidermal keratinocytes (NHEK) follows a bell-shaped curve demonstrating an initial dose response and then decline with increased amounts of coated fibronectin (unpublished observations).82 The classical report by Palecek et al.105 demonstrated that changes in cell migration speed are influenced by ligand concentration, integrin expression level, and integrin affinity. In NHEK cells the maximal migration was reached with much lower concentrations of fibronectin which can be explained by higher expression of α5β1 integrin in NHEK compared to HaCaT cells.106 Interestingly, when the cells were treated with TGFβ1, their migration was sustained even when fibronectin concentration was increased. This may be due to TGFβ induced alteration in the expression of fibronectin binding integrins, increase in fibronectin matrix degradation by focalized proteolysis, or induction of other receptors or matrix molecules conducive for cell migration.82,90,106 During reepithelialization, the fibronectin concentration in the clot and the receptor expression level in keratinocytes are high, which keeps the cells migratory. Analyzing the active β1 integrin pool in migrating keratinocytes demonstrates that the integrins facing the provisional matrix are in an active, high affinity form (Fig. 5). An additional mechanism by which interaction of α5β1 integrin with fibronectin can be modulated involves keratinocyte membrane glycosphingolipids/gangliosides.107 Ganglioside GM3 appears to promote while GT1b inhibits α5β1 interaction with fibronectin.106 GT1b appears to interact directly with α5β1 integrin through carbohydrate-carbohydrate binding.108 TGFβ can reduce the responsiveness to GT1b inhibition.106 Because of their ability to modulate α5β1 integrin function, gangliosides or their modifying enzymes have been proposed as targets of pharmacological manipulation in wound repair.106

In addition to cell adhesion and migration, α5β1 integrin mediates cell growth signaling.109 Integrin α5β1 interaction with fibronectin also prevents keratinocyte cell differentiation, and a high level of integrin expression is associated with the stem cell phenotype.110 Thus, one of the functions of α5β1 integrin in wounds is prevention of cell differentiation and support of proliferation in nonmigratory keratinocytes (Fig. 6). Hyperproliferative conditions, such as psoriasis, are characterized by increased and suprabasal integrin expression and unusual presence of EDA fibronectin around the basal keratinocytes.111,112 Forced suprabasal integrin expression can lead to psoriasis-type phenotype in transgenic mice.113 Nonlesional psoriatic skin expresses α5β1 integrin.114These authors propose that for proliferative lesions to form, expression of both α5β1 and EDA fibronectin is required.114 Interestingly, keratinocyte-restricted deletion of β1 integrins leads to induced influx of inflammatory cells into the dermis, suggesting that keratinocyte β1 integrins participate in regulation of skin inflammation.115 In addition, dermis is fibrotic in these animals. Whether that is due to the inflammation or altered regulation of endogeneous cytokine expression remains to be investigated.

Figure 6. A hypothetical model illustrating some of the integrin-mediated cell-matrix interactions regulating keratinocyte function during wound reepithelialization.

Figure 6

A hypothetical model illustrating some of the integrin-mediated cell-matrix interactions regulating keratinocyte function during wound reepithelialization. A) The epithelial cells at the tip of the migrating epithelial cell sheet send out cytoplasmic (more...)

Growth factor receptors and integrins collaborate in promotion of cell proliferation, often through common intracellular signaling events.116 In keratinocytes, the interplay between integrins and growth factor receptors is still poorly understood. Existing information is largely based on experiments on mesenchymal or transfected cell lines. For example, α5β1 integrin ligation to fibronectin can either potentiate epidermal growth factor receptor (EGFR) signaling or directly activate EGFR.117 EGFR is localized into the migrating tip of the advancing epithelium and in the hyperproliferative zone behind it, suggesting that EGFR plays a role both in the migration as well as the proliferation of keratinocytes during wound repair.118 Some EGFR ligands such as heparin-binding EGF (HBEGF) are considered autocrine growth factors for keratinocytes.119 EGFR ligands are membrane bound and released (shed) by enzymatic cleavage from the cells. Expression of HBEGF is rapidly induced at the wound edge of scrape-wounded epithelial monolayers in vitro.120 HBEGF shedding appears to be important for reepithelialization in vivo.121 It is likely, therefore, that α5β1 integrin collaborates with EGFR signaling, not only in regulation of cell growth but also in cell migration during wound repair. This was evidenced by the finding that EGF differentially regulated expression of several gene clusters, depending on whether cells were in contact with fibronectin or laminin.122

Function of αvβ6 Integrin

As discussed above, wound keratinocytes express another fibronectin-binding receptor, αvβ6 integrin, which is not normally present in keratinocytes of resting epithelium (Fig. 5). The expression of αvβ6 integrin can be induced by wounding or placing epithelial cells in culture.97,123,124 The kinetics of expression of α5β1 and αvβ6 integrins is different in wound keratinocytes. Induction of α5β1 fibronectin receptor expression occurs at early stages of wound healing when the epithelium starts to migrate across the wound provisional matrix.12 Expression of αvβ6 integrin is low or undetectable during early migration, and maximal expression coincides with basement membrane reorganization and granulation tissue formation when the migrating edges of wound epithelium have already joined.30,97,125 However, both α5β1 and αvβ6 integrins are sometimes coexpressed in migrating keratinocytes (Fig. 5).97

Function of fibronectin binding integrins was recently investigated in HaCaT keratinocytes, which are spontaneously transformed keratinocytes that have acquired an immortal but nonmalignant growth type.126,127 Integrin expression pattern in HaCaT cells is similar to that in primary keratinocytes, although they express less α5β1 integrin.106,128,129 Even after TGFβ1 treatment, the main receptor for fibronectin in NHEK cells is α5β1 integrin (unpublished). HaCaT keratinocytes constitutively express αvβ6 integrin which can be further up-regulated by TGFβ1.82 HaCaT cells also used αvβ6 integrin as their main fibronectin receptor for cell spreading. In untreated cells, both α5β1 and αvβ6 integrins were needed for maximal cell spreading, whereas in TGFβ1-treated cells, the increased expression of αvβ6 integrin alone was adequate for supporting maximal cell spreading on fibronectin. This agrees with findings of αvβ6 integrin-transfected cells in which αvβ6 integrin could replace α5β1 in fibronectin binding.124 Treatment of HaCaT cells with TGFβ1 also promoted keratinocyte migration. Interestingly, keratinocyte integrins had different functions, depending on whether or not deformation of cell body was required for locomotion.82 When HaCaT cells were allowed to migrate through fibronectin-coated membranes in a short-term Boyden chamber assay that required cell body deformation for translocation, αvβ6 integrin was found to be the main individual mediator of cell movement. Comparable results were recently obtained using a similar assay by Huang et al.130 When keratinocytes migrated laterally from a cell cluster, migration appeared to be mediated mainly by β1 integrins. Therefore, depending on the mechanics of cell migration, different integrins may be involved.

Although the exogenously provided fibronectin dose-dependently supports keratinocyte migration, it is unlikely that fibronectin is the only ligand for the migrating cells. As detailed above, during wound healing, migrating keratinocytes continuously express laminin-5,12 and inhibitory antibodies against both α3 integrin subunit and laminin-5 are able to reduce keratinocyte migration on fibronectin.131 It is, therefore, possible that, along with the induction of fibronectin receptors and fibronectin isoforms, TGFβ could induce other receptors and matrix molecules conducive for cell migration. Based on our data, keratinocyte migration on fibronectin involved at least α3β1, α5β1, and αvβ6 integrins. In addition, cultured keratinocytes can switch between αvβ6 and α5β1 integrins in fibronectin binding if the other receptor is rendered unfunctional since they are both able to mediate cell migration on fibronectin.82 A reservoir of fibronectin receptors is likely to be evolutionarily beneficial to protect the survival of the organism, and different fibronectin receptors may at least partially replace functions of each other when necessary. Support to the idea that αvβ6 integrin can mediate keratinocyte migration on fibronectin comes from the studies on β6-deficient keratinocytes in culture. Migration on fibronectin was reduced in keratinocytes from β6 −/− mice.130 Interestingly, keratinocytes from β6 −/− mice also demonstrated reduced migration on vitronectin, suggesting that vitronectin can also serve as a ligand for αvβ6 integrin. In the same study, Huang et al.130 suggested a critical role for PKC in enhancement of αvβ6-mediated cell migration. It is not known how keratinocytes in vivo could coordinately use both fibronectin and vitronectin for their migration into the wound clot matrix.

In embryonic cells expressing αvβ1 integrin as their major αv integrin, this receptor mediated cell attachment and spreading on fibronectin when these cells were made deficient of αvβ1 integrin but not when α5β1 integrin was present.132 Integrins α5β1, αvβ1, and αvβ6 all bind to the same RGD sequence in fibronectin molecules.123,133,134 Interestingly, binding of αvβ6 integrin to fibronectin has recently been shown to be inhibited, in addition to the RGD-peptides, by non-RGD peptide (DLXXL) present in several molecules but not in fibronectin itself,135 suggesting that there are distinct structural requirements for interaction of the integrins α5β1 and αvβ6 with fibronectin. This was already proposed after demonstration that αvβ6 integrin does not require the synergy site for its full activity in fibronectin binding.136 Because the cell migration rate is dependent on substratum ligand levels, cell integrin expression, as well as integrin-ligand affinity,105 utilization of a combination of receptors of different affinities can be beneficial to migration versatility. Integrin receptors may also regulate different aspects of cell motility, e.g. migration speed and motile cell phenotype as has been shown for α4β1 and α5β1 integrins.137 In addition, fibronectin binding to different integrins is likely to induce distinct cellular signaling pathways in cells.

Although αvβ6 integrin was the major fibronectin-binding integrin in HaCaT cells, it showed a minor role in lateral cell migration, and its expression mainly at late stages of adult wound healing indeed suggests that αvβ6 integrin does not function exclusively in mediating cell migration. Inactivation of the β6 integrin gene has had no influence on short-term wound healing but resulted in inflammatory changes in the skin and lungs, suggesting a role for αvβ6 integrin in modulation of inflammation.138 Neutrophil recruitment appears to be associated with αvβ6 integrin expression.139 Furthermore, it has recently been shown that in cultured lung epithelial cells αvβ6 integrin can bind latency-associated protein (LAP) of the TGFβ protein complex.140 This binding appears to open the structure and lead to activation of TGFβ that can induce cells in the immediate neighborhood. Integrin αvβ6 binds to LAP with a much higher affinity than to fibronectin.140 β6-deficient mice seem to be protected from experimental lung fibrosis,140 suggesting that αvβ6 integrin could participate in the regulation of connective tissue formation. Because TGFβ also regulates the differentiation of myofibroblasts141 and deposition of extracellular matrix in the granulation tissue,142 it is conceivable that keratinocyte αvβ6 integrin could have much wider functions than just mediating fibronectin binding during wound reepithelialization. Our findings that the expression of αvβ6 integrin in wounds matches with the production of the extracellular matrix bridge by fibroblasts under the fused epithelium,12 differentiation of myofibroblasts (Häkkinen et al, unpublished), and peak expression of biologically active TGFβ in the wound143 support the spatiotemporal requirement for this putative mechanism.

Role of αvβ6 Integrin in Malignant Transformation of Keratinocytes

As reviewed above, normal wound healing is a complex process that needs to be strictly regulated. An impairment in these regulatory processes can potentially lead to formation of hypertrophic scarring or chronic, unhealing wounds and even contribute to epithelial tumorigenesis. Cellular functions in malignant transformation resemble those associated with keratinocyte migration/invasion into the clot, except that the cells have lost the coordination of the process. This failure may lead to unregulated growth, to proteolytic degradation of the basement membrane and to cell invasion through the fibronectin and collagen-containing connective tissue.

We and others have previously shown that αvβ6 integrin is induced in oral squamous cell carcinoma (SCC) in vivo.144146Premalignant oral lesions that express αvβ6 integrin are more likely to progress146 but the role of αvβ 6 integrin in this process has not been established. Adhesion to fibronectin, regulation of cell growth inside collagen matrix,147 activation of TGFβ and modulation of MMP expression are all possible functions for αvβ6 integrins in tumorigenesis. Recently, we examined the role of fibronectin binding integrin receptors in SCC cell lines expressing different amounts of αvβ6 integrin on cell surfaces.148 The cell lines expressing αvβ6 integrin demonstrated β1 integrin-independent cell spreading on fibronectin, unlike the cell line with minimal αvβ6 integrin expression. When β6 integrin was transfected into these cells, their spreading on fibronectin also became partially β1 integrin-independent. The data from these experiments suggest that SCC cells in vitro can cooperatively use αvβ6 as well as α5β1 and α5β1 integrin receptors for interaction with fibronectin matrix and also adapt by switching to another receptor of the same ligand. This may also have some in vivo relevance since both αvβ6 and α5β1 integrins appear to be expressed in the primary SCC tumors.144,149,150 The presence of αvβ1 integrin in SCC tumors in vivo is difficult to show because there are no specific antibodies available recognizing the αvβ1 integrin complex. In contrast to the results with SCC cells, in HaCaT keratinocytes and in primary epidermal keratinocytes also expressing αvβ1 integrin as their major av integrin, this integrin failed to support cell spreading on fibronectin (unpublished results from our laboratory).82 In embryonic cells, αvβ1 integrin was functional in cell attachment and spreading on fibronectin only when these cells were made deficient of α5β1 integrin,132 suggesting that the role of αvβ1 integrin may be different in other cell types and even linked to the malignant cell phenotype. The ability to migrate on fibronectin may be crucial for tumor invasion and metastasis. SCC cells seemed to prefer β1 integrins as their main migration receptors on fibronectin. However, αvβ6, αvβ1, and α5β1 integrins all appeared to act cooperatively and interchangeably in SCC migration on fibronectin. In addition to mediating cell migration on fibronectin, αvβ 6 integrin may have some other important roles in tumor cell invasion and cancer progression, e.g. in mediating migration on tenascin-C151 or in inducing MMP expression.152 Integrin αvβ6 may also preferably serve as a migration receptor for other fibronectin isoforms expressed by SCC tumors.153,154

One possible function for αvβ6 integrin in malignant transformation could be in TGFβ activation. TGFβ has been implicated to play a role in cell transformation and carcinogenesis.155 In transgenic mice, continuous overexpression of TGFβ1 by epithelial cells enhanced the malignant progression rate and phenotype and induced high incidence of particularly malignant fibroblastoid spindle cell carcinomas.156 TGFβ can also directly stimulate reversible epithelial-mesenchymal transformation in cultured keratinocytes.157160

How do Keratinocytes Migrate on a Composite Matrix?

It is obvious that, in addition to fibronectin, keratinocytes can use alternative extracellular matrix molecules for their migration. Tenascin-C is also expressed under the migrating wound epithelial cells, and it may modulate interactions of integrins with their ligands.161 Keratinocytes may use α9β1 and αvβ6 integrins for binding to tenascin-C162164 and both of these integrins are expressed by migrating wound keratinocytes in vivo.30 Laminin-5 is always deposited underneath the migrating cells both in vitro and in vivo, regardless of the wound type.12,29 Some studies provide evidence that laminin-5 is the key extracellular matrix molecule mediating migration, regardless of which matrix the cells have been originally seeded on.165 Laminin-5 is recognized by three integrin receptors of keratinocytes, namely α2β1, α3β1 and α6β4. There is controversial data regarding whether laminin-5 promotes or retards keratinocyte migration and which integrins are involved.165,166 A hypothetical view describing how keratinocytes utilize fibronectin and laminin-5 during migration is presented in Figure 6. Recent evidence points to the role of α2β1 integrin in transient binding of endogenously produced unprocessed laminin-5 which promotes keratinocyte migration.165 In contrast, binding of newly synthesized laminin-5 by α3β1 integrin could retard cell migration.165 Finally, α6β4 integrin could then bind to plasmin-cleaved laminin-5 that could induce hemidesmosome formation.167 Although laminin-5 appears to be a crucial extracellular matrix molecule in regulation of keratinocyte migration and hemidesmosome formation, it is controversial whether it is absolutely essential for reepithelialization in vivo.166 Cutaneous wounds in some individuals with defects in laminin-5 can reepithelialize.166 The wound provisional matrix certainly has several matrix components such as fibronectin that can support keratinocyte migration.96

Conclusions

In recent years, the understanding of functions of various extracellular matrix molecules and their integrin receptors in cell migration has vastly improved. There is still, however, little information available how cells interact with composite matrix made of multimers of several matrix molecules. In wound healing, keratinocytes face a number of provisional matrix molecules and also deposit their own pericellular matrix when they migrate. Although various models can be proposed for mechanisms of epithelial sheet migration (Fig. 6), little is known about the complex three-dimensional interplay between more than six integrin receptors simultaneously with polymerized fibrin-fibronectin matrix linked with endogenously produced EDA fibronectin, laminin-5, and tenascin-C. Also limited data is still available describing how integrins switch function during various phases of wound healing and regarding the integrin-mediated signaling pathways induced by composite wound provisional matrix. Keratinocyte functions are further modified by matricryptins released during focalized proteolysis at the leading edge and collaborative signaling from growth factor receptors and integrins. Keratinocyte migration in wounds is evolutionarily well protected and therefore likely to involve integrins and matrix molecules with overlapping and compensatory functions.

Acknowledgments

Original research findings of our laboratory were supported by grants from the Canadian Institutes of Health Research.

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