Expression and function of the keratinocyte integrins

The process of differentiation is not confined to the embryo: throughout adult life new differentiated cells are produced to replace cells lost through death or tissue damage. In stratified squamous epithelia such as the epidermis dead, terminally differentiated cells are continually shed from the outermost layers of the tissue and replaced through proliferation of a stem cell population in the basal layer. Human epidermal keratinocytes can be grown in culture under conditions in which they form stratified sheets with the same basic organisation as epidermis in vivo and these can be used to study stem cell proliferation, terminal differentiation and tissue assembly (reviewed by Watt, 1988, 1989, 1991).

Recent work has highlighted the importance of the integrin family of adhesive receptors in regulating keratinocyte behaviour. Each integrin is a heterodimer α and one β subunit, which are non-covalently associated (reviewed by Albelda and Buck, 1990; Hemler, 1990; Ruoslahti, 1991; Hynes, 1992). Each subunit has a large extracellular domain, a transmembrane domain and a cytoplasmic domain which is usually short and usually associates with the actin cytoskeleton. Binding of ligands which are extracellular matrix proteins or counter-receptors of the immunoglobulin superfamily, requires both subunits; the ligand binding sites appear to be intimately associated with cation binding domains on the α subunits. So far over 7 different β subunits and 13 different α subunits have been identified and a growing number have been found to exist as two or more splice variants. An individual β subunit can potentially partner several different α subunits and vice versa: ligand binding specificity depends, to a large extent, on heterodimer composition. In addition, some integrins can bind several apparently structurally unrelated ligands and a given integrin expressed in two different cell types can show different ligand binding properties.

Integrins mediate attachment of keratinocytes to the basement membrane that separates the epidermis from the dermis; this specialised extracellular matrix is rich in a number of proteins and proteoglycans, including laminin, type IV collagen and epiligrin (which may be identical to kalinin) (Yurchenco and Schittny, 1990; Rousselle et al., 1991; Watt and Hotchin, 1992). The most abundant keratinocyte integrins are α₂ β₁, α₃ β₁ and α₆ β₄ (see Table 1 for summary). α₂ β₁ is a receptor for collagen and laminin and α₃ β₁ is a receptor for laminin and epiligrin (Carter et al., 1990a,b, 1991; Staquet et al., 1990; Adams and Watt, 1991). α₆ β₄ is a component of hemidesmosomes; its ligand has yet to be established unequivocally but may be laminin (Stepp et al., 1990; De Luca et al., 1990; Lee et al., 1992).

Two other keratinocyte integrins that are well characterised are α₅ β₁, a fibronectin receptor (Adams and Watt, 1990, 1991; Carter et al., 1990a), and α_v β₅, a vitronectin receptor (Adams and Watt, 1991; Marchisio et al., 1991). Although α₅ β₁is readily detected in cultured keratinocytes it is either weakly expressed (Hertle et al., 1991, 1992) or undetectable (Peltonen et al., 1989; Nazzaro et al., 1990; Pellegrini et al., 1992) in mature epidermis. There is a low level of fibronectin in mature basement membrane (Stenman and Vaheri, 1978; Fleischmajer and Timpl, 1984) and vitronectin is reported to be absent (Reilly and Nash, 1988). However, in wounds in which the basement membrane is destroyed, fibronectin forms the provisional matrix over which keratinocytes migrate (Clark, 1990) and when placed in culture keratinocytes become adhesive to fibronectin (Grinnell, 1992).

Other integrin subunits reported to be expressed by keratinocytes include α₁, which forms a heterodimer with β₁ and is expressed in trace amounts (Belkin et al., 1990; Buck et al., 1990; Hertle et al., 1991 ; but see also De Luca et al., 1990; Nazzaro et al., 1990; Zambruno et al., 1991) and α_s, which is moderately abundant in chick embryonic epidermis and forms a heterodimer with the β₁ subunit (Bossy et al., 1991). A β₇-related mRNA has been identified in cultured mouse keratinocytes; however, its small size suggests that it is unlikely to encode full-length β₇ protein (Yuan et al., 1992). The β₆ integrin subunit forms a heterodimer with α_v and acts as a fibronectin receptor in a range of epithelial cells (Sheppard et al., 1990; Busk et al., 1992); it is not known whether keratinocytes express β₆, but antibodies to α_v do not block kératinocyte adhesion to fibronectin (Adams and Watt, 1991).

Variant forms of integrin subunits are believed to arise through alternative splicing; in each case the classical form is referred to as the A form. In addition to β_1A, keratinocytes express a variant known as β_1B in which a unique 12 amino acid sequence replaces the carboxy-terminal 21 amino acids of β_1A (Altrudaet al., 1990; Balzac et al., 1993). α_6A and α_6B have unique cytoplasmic domains of 36 and 54 amino acids respectively; keratinocytes only express α_6A (Hogervorst et al., 1993); Tamura et al., 1991). Splice variants of the α₃ (Tamura et al., 1991) and β₄ (Hogervorst et al., 1990; Suzuki and Naitoh, 1990; Tamura et al., 1990) subunits have also been described. The β₄ cDNA originally cloned from keratinocytes (Hogervorst et al., 1990) contains a 53 amino acid insert in the cytoplasmic domain not found in p4 cDNA from retinal pigment epithelial cells (Suzuki and Naitoh, 1990) or carcinoma cells (Tamura et al., 1990).

The cytoplasmic domains of integrins not only interact with cytoskeletal proteins but also play a role in signal transduction and can regulate the ligand binding ability of the extracellular domains (Hynes, 1992; Damsky and Werb, 1992; Adams and Watt, 1993; Juliano and Haskell, 1993). It therefore seems likely that the variant integrin subunits will have different cellular functions from the classical forms. Evidence for this has come from transfection experiments which demonstrate that although the ligand binding properties of β_1A and β_1B are similar, β_1Bdoes not localise in focal adhesions (Balzac et al., 1993).

Within the epidermis, integrin expression is largely confined to the basal layer of keratinocytes that are attached to the underlying basement membrane (see, for example, Wayner et al., 1988; De Strooper et al., 1989; Hertle et al., 1991, 1992; Fig. IE). The integrin subunits tend to have a pericellular distribution, although the α₆ and α₄ subunits show a relative concentration at the basement membrane zone, consistent with their association with hemidesmosomes. The α₃ and β₁ subunits are expressed in embryonic epidermis prior to the initiation of stratification and do not change in abundance or distribution during subsequent development; however the other subunits (including α₂; see Fig. 1) show spatial or temporal changes in expression and it is tempting to speculate that integrins may play a role in establishing the spatial organisation of the epidermis (Hertle et al., 1991).

The distribution of integrins in stratified cultures of human keratinocytes resembles their distribution within the epidermis: expression is largely restricted to the basal layer (Nicholson and Watt, 1991; Adams and Watt, 1991). Flow cytometry can be used to analyse the relationship between integrin levels and differentiation status in individual keratinocytes, using binding of peanut lectin (PNA) as a marker of terminal differentiation (Watt and Jones, 1992). Fig. 2 shows that the β₃, β₅and β₆ subunits are markedly downregulated in cells that bind PNA; β₂ expression is also decreased, but to a lesser extent.

Although integrin expression is normally confined to basal keratinocytes, altered expression patterns have been observed in situations in which the normal balance between proliferation and terminal differentiation is perturbed. During wound healing and in psoriatic lesions integrins are expressed suprabasally by keratinocytes that have initiated terminal differentiation (Ralfkiaer et al., 1991 ; Hertle et al., 1992). In squamous cell carcinomas there is considerable variation in integrin expression both within and between tumours (see, for example, Peltonen et al., 1989; Wolf et al., 1990; Jones et al., 1993), but focal loss of the β₂, β₃, β₆ and β₄ subunits is a common feature of poorly differentiated oral squamous cell carcinomas (Jones et al., 1993). Reduced expression of specific integrin subunits is observed in some kératinocyte lines derived from oral squamous cell carcinomas (Sugiyama et al., unpublished data) and experiments are underway to investigate whether ‘repair’ of the lines with appropriate integrin expression vectors has any effect on their proliferative potential and differentiation capacity (cf. Giancotti and Ruoslahti, 1990).

One technique which has been used extensively to study integrin expression in keratinocytes is suspension-induced terminal differentiation (Green, 1977; Watt et al., 1988). Cultured human keratinocytes arc disaggregated and resuspended in medium made viscous by the addition of methyl cellulose: the cells remain rounded and are prevented from adhering to one another or to the culture dish. By about 5 hours in suspension, keratinocytes have withdrawn from the cell cycle and become committed to differentiate; by 24 hours the majority of cells have initialed terminal differentiation and arc expressing involucrin, a precursor of the cornified envelope that is expressed suprabasally in culture and in the upper layers of the epidermis.

When keratinocytes are placed in suspension for 24 hr there is a marked reduction in cell surface levels of integrins. although the α₂ subunit is not downregulalcd to the same extent as the other subunits (Fig. 3; Adams and Watt. 1990; Hotchin and Watt, 1992). There is a corresponding decline in integrin mRNA levels (Nicholson and Watt, 1991) which reflects a shut off of transcription of integrin genes (Hotchin and Watt, 1992). In situ hybridisation of sections of skin (Watt and llcrllc. 1993) and of stratified kératinocyte cultures (Hodivala and Walt, unpublished data) shows that integrin niRNAs arc localised to the basal cell layer. The absence of integrins from the surface of terminally differentiating cells docs not simply reflect transcriptional regulation. however: when keratinocytes arc suspended in methyl cellulose, N-linkcd glycosylation and intracellular transport of newly synthesised β₁ integrins arc inhibited by a mechanism that remains to be elucidated (Hotchin and Watt, 1992).

Functional downregulation of integrins precedes loss of the receptors from the cell surface. When keratinocytes become committed to terminal differentiation, al about 5 hr in suspension, the ability of the β₁ integrins to bind extracellular matrix proteins is substantially decreased although there is no reduction in the level of integrins on the cell surface al this time (Adams and Wall. 1990). The decrease in ligand binding ability reflects functional modulation of receptors on the cell surface (Hotchin and Walt. 1992) which would be consistent with a change in receptor conformation (O’Toole et al., 1990; Failli et al., 1993; Hotchin et al., unpublished data). The decreased ability of committed cells to adhere to extracellular matrix proteins is likely to ensure that those cells arc selectively expelled from the basal layer and migrate into the layer above (Adams and Wall, 1990; Fig. 4).

Cell-cell and cell-extracellular matrix adhesion

The primary function of kératinocyte integrins is adhesive, but the receptors do not act simply as passive attachments to the basement membrane. As described above, functional downregulation of the β₁ integrins ensures the migration of committed cells out of the basal layer (Adams and Watt. 1990). In addition, lateral migration of keratinocytes in vitro is mediated by integrins. implying that they play a role in wound healing in vivo (Kim et al., 1992; Grinnell, 1992).

Integrins are present on the lateral membranes of basal keratinocytes (sec Fig. 1) and there is some evidence that they play a role in intercellular adhesion, perhaps through direct binding of α₂β₁ to α₃β₁ (Symington et al., 1993). The best characterised intercellular receptors of keratinocytes arc the dcsmosomal and nondesmo.somal cadhcrins (reviewed by Takcichi, 1991; Geiger and Ayalon. 1992; Buxton and Magee, 1992) which require extracellular calcium ions in order to function. In low calcium medium cell-cell contacts can be disrupted by an anti-β₁ antibody (Larjava et al., 1990); however individual anti-integrin antibodies do not block calcium-dependent aggregation of keratinocytes in suspension (Tcnchini et al., 1993). Further experiments on the role of integrins in keratinocyle-kcratinocyte adhesion arc clearly necessary, particularly in the light of recent evidence that cadhcrins may play a role in the down regulation of inlegrin expression that occurs during terminal differentiation (Hodivala and Watt, unpublished data).

When keratinocytes are plated on an adhesive substrate that restricts spreading, terminal differentiation is stimulated (Watt et al., 1988) and loss of contact with the extracellular matrix may be the primary differentiation stimulus in suspension (Adams and Watt. 1989). Suspension-induced terminal differentiation can be inhibited by inclusion of fibronectin or antibodies to (he [3i inlegrin subunit in the methyl cellulose al the lime of plating (Adams and Wall. 1989; Wall et al., 1993). However, if fibronectin is added more than 2 hr after the cells have been placed in suspension differentiation is not inhibited, because of the decrease in (Xs[3i ligand-binding ability that occurs on commitment to differentiation (Adams and Wall, 1989, 1990). Recent experiments have shown that laminin and type IV collagen, in combination with a low concentration of fibronectin, can participate in the inhibition of differentiation, as can a cocktail of function blocking antibodies (which presumably mimic receptor-ligand binding) to the α₂, α₃ and α₅ subunits (Watt et al., 1993). These observations suggest that, in vivo, terminal differentiation may be regulated by the total proportion of α₁ helerodimcrs occupied by ligand, a decrease in that proportion acting as a stimulus for differentiation (see Fig. 4). One extension of this hypothesis is that integrins on the apical and lateral membranes of basal keratinocytes may be inactive because they are not in contact with extracellular matrix proteins (see, for example, Tenchini et al., 1993).

The epidermis is believed to contain two types of proliferating cell: stem cells, which retain a high capacity for selfrenewal throughout adult life, and transit amplifying cells, which have a lower capacity for self-renewal and a high probability of undergoing terminal differentiation after a few rounds of division (reviewed by Pollen, 1981; Hall and Watt, 1989). Cells with characteristics of stem cells can be isolated on the basis of high surface expression of α₁ integrins and rapid adhesion to fibronectin, type IV collagen or ker -atinocyte extracellular matrix proteins (Jones and Watt, 1993; Fig. 4). There is a log linear relationship between β₁ receptor density, as measured by How cytometry, and the ability of keratinocytes to form colonies of 32 or more cells by 14 days in culture. Basal cells with the highest level of β₁ integrins have about 4 limes the colony forming efficiency of cells with the lowest level. There is specificity in the relationship between keratinocyte adhesiveness and proliferative capacity, since α₆ expression and rate of adhesion to laminin do not correlate with proliferative ability.

Cells with characteristics of transit amplifying cells adhere more slowly to the matrix proteins and express lower levels of Pi integrins. These cells divide 1-5 times and then initiate involucrin expression. One implication of these results is that if a reduction in the number of Pi integrins with bound ligand is a stimulus for terminal differentiation (Adams and Wall. 1989; Watt et al., 1993) transit cells will be more sensitive to that stimulus than stem cells because they have fewer surface β₁ integrins (see Fig. 4).

In a range of stratified epithelia there is indirect evidence that stem cells occupy a specific location within the basal layer (see. for example. Cotsarelis et al., 1989, 1990). It is difficult to determine the location of the high integrin-expressing putative stem cell population within the basal epidermal layer because in histological sections the staining of lateral membranes reflects integrin levels on adjacent cells. Nevertheless, non-uniform staining of the α₂ subunit has been reported in developing epidermis (Hertle et al., 1991; Fig. 1) and we are currently investigating whether there is variation in integrin levels in the basal layer of mature epidermis.

In this review we have presented evidence that integrins have several important functions within the epidermis. It would be wrong, however, to give the impression that all aspects of keratinoeyte behaviour can be explained in terms of integrins. There is no doubt that proliferation and terminal differentiation are profoundly affected by a variety of soluble agents, including growth factors and retinoids (reviewed by Fuchs. 1990; McKay and Leigh. 1991). In addition, (he cells express other families of adhesive receptor, including the cadherins, which arc undoubtedly important in regulating the adhesive properties of keratinocytes (Wheelock and Jensen. 1992; llodivala and Wall, unpublished data). One priority for future research is to understand the interplay between different regulatory factors: extracellular matrix adhesiveness may determine keratinoeyte responsiveness to growth factors; growth factors may regulate integrin expression (Adams and Watt, 1993) ; and the fate of stem cell progeny may be to some extent predetermined and to some extent regulated by the cellular environment (Hall and Watt. 1989).