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Biomedical subjects

F M Watt

Publications and source records attributed to F M Watt.

At least 91 records · Page 5Linked to original sources
PubMed1993

Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.

The epidermis is believed to contain two types of proliferating cells: stem cells and cells with a lower capacity for self-renewal and higher probability of undergoing terminal differentiation (transit amplifying cells). We report that keratinocytes with characteristics of stem cells can be isolated from cultured human epidermis on the basis of high surface expression of beta 1 integrins and rapid adhesion to extracellular matrix (ECM) proteins. Among keratinocytes there was a log linear relationship between the relative level of beta 1 integrins on the cell surface and proliferative capacity; furthermore, the cells with the highest colony-forming efficiency adhered most rapidly to type IV collagen, fibronectin, or keratinocyte ECM. Proliferating keratinocytes that adhered more slowly had characteristics of transit amplifying cells: after one to five rounds of division, all of their daughters underwent terminal differentiation. Since stem cells can be isolated to greater than 90% purity on the basis of their adhesive properties, it will now be possible to investigate the mechanisms that regulate the fate of their progeny.

Biomarkers↗
PubMed1993

Integrin expression in normal, hyperplastic, dysplastic, and malignant oral epithelium.

We have examined the distribution of a range of integrin subunits in normal and lesional oral mucosa. The alpha 2, alpha 3, alpha 6, beta 1, and beta 4 subunits were highly expressed in normal epithelium, and there was weaker, more variable expression of alpha 5 and alpha v. Expression of all subunits was highest in the basal layer of normal epithelium, but extensive staining above the basal layer was also observed, particularly in the floor of the mouth and the lateral margin of the tongue. In dysplastic lesions and hyperplastic epithelium adjacent to ulcers, suprabasal staining was even more pronounced. Staining patterns in squamous cell carcinomas showed considerable variation, both within and between individual tumours: in some areas there was staining reminiscent of normal epithelium, but uniform staining throughout tumour islands, and patchy and variable cytoplasmic and pericellular staining were also seen. Thirteen out of 17 carcinomas showed some loss of integrin expression: six out of ten moderately well differentiated tumours and all the poorly differentiated tumours. Focal loss of alpha 6 and beta 4 was most commonly observed, but loss of alpha 2 and alpha 3 also occurred. Since integrins regulate not only keratinocyte adhesion, but also the initiation of terminal differentiation, the changes in integrin expression that we have observed may have significance for the behaviour of individual tumours.

Carcinoma, Squamous Cell↗
PubMed1993

Loss of alpha 6 and beta 4 integrin subunits coincides with loss of basement membrane components in oral squamous cell carcinomas.

In oral squamous cell carcinomas, focal or extensive loss of basement membrane components and of integrins has been reported. The purpose of this study was to investigate whether those regions of the tumour-connective tissue interface which lack laminin and type IV collagen coincide with areas of loss of the alpha 6 and beta 4 integrin subunits on basal keratinocytes. Out of a total of 15 poor and moderately or well differentiated squamous cell carcinomas, all showed some loss or fragmentation of basement membrane proteins and in 12 the loss was coincident with loss of alpha 6 and/or beta 4. In three cases, there was loss of basal integrin expression in areas where the basement membrane remained intact. These results provide further evidence that loss of integrins may play an important role in tumour progression and prompt us to speculate about the sequence of events leading to tumour invasion.

Basement Membrane↗
PubMed1993

Towards gene therapy for haemophilia B using primary human keratinocytes.

Haemophilia B might be permanently cured by gene therapy--the introduction of a correct copy of the factor IX gene into the somatic cells of a patient. Here, we have introduced a recombinant human factor IX cDNA into primary human keratinocytes by means of a defective retroviral vector. In tissue culture, transduced keratinocytes were found to secrete biologically active factor IX and after transplantation of these cells into nude mice, human factor IX was detected in the bloodstream in small quantities for one week. This is the first demonstration of a therapeutic protein reaching the bloodstream from transduced primary keratinocytes. This may have implications for the treatment of haemophilia B and other disorders.

Animals↗
PubMed1993

Comparison of integrin expression and terminal differentiation capacity in cell lines derived from oral squamous cell carcinomas.

Receptors of the integrin family regulate adhesion and terminal differentiation of keratinocytes. In order to investigate the significance of changes in integrin expression associated with malignant transformation we have examined normal human oral keratinocytes and seven oral squamous cell carcinoma (SCC) lines. Cell surface levels of the alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1 and beta 4 integrin subunits were determined by flow cytometry and the distribution of the beta 1 subunit was examined by immunohistochemistry. In normal keratinocytes and one SCC line the beta 1 subunit was most abundant in the basal cell layer, but in other lines anti-beta 1 antibodies stained basal and suprabasal layers uniformly. All lines had reduced surface levels of at least one integrin subunit and in some cell lines distinct subpopulations could be distinguished on the basis of differences in integrin expression. Reduced integrin expression was not, however, generally reflected in reduced adhesion to laminin, fibronectin, type IV collagen or vitronectin in three cell lines examined. Those cell lines with the lowest capacity for terminal differentiation, as measured by involucrin expression, had the lowest levels of the alpha 6 and beta 4 subunits or were completely lacking alpha v. Oral SCC show considerable variation in integrin expression, but focal or extensive loss of the alpha 6 and beta 4 subunits is a common feature of poorly differentiated tumours. The cell lines we have examined therefore provide a relevant experimental model with which to explore the relationship between aberrant integrin expression and impaired terminal differentiation capacity.

Carcinoma, Squamous Cell↗
PubMed1993

Changes in the distribution of actin-associated proteins during epidermal wound healing.

We have examined the distribution of actin filaments and a number of actin-associated proteins during human epidermal wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. Filamentous actin was found in all the living epidermal layers before, during and after wound healing. alpha-actinin was also present in all the living layers of normal epidermis, but diffuse cytoplasmic staining was observed at the leading edge of migrating epidermis. Vinculin and talin were concentrated at the basement membrane prior to wounding, but were absent from the leading edge during wound healing. In normal epidermis, filamin and gelsolin showed a complementary distribution, with filamin most abundant in the basal layer and gelsolin most abundant suprabasally. The abundance of both proteins was reduced at the leading edge of migrating epidermis. All of the changes were transient, as the expression patterns returned to normal by 1 week after wounding, when the epidermis had reformed. The relevance of these changes to the process of keratinocyte migration is discussed.

Actinin↗
PubMed1993

Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions.

Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.

Actins↗
PubMed1993

Functional down-regulation of alpha 5 beta 1 integrin in keratinocytes is reversible but commitment to terminal differentiation is not.

Extracellular matrix receptors of the integrin family have a dual role in the epidermis, regulating both adhesion and differentiation. Loss of contact with the extracellular matrix causes keratinocytes to become committed to terminal differentiation, and results in a decrease in the ability of the alpha 5 beta 1 integrin to bind fibronectin. We have investigated whether the decrease in ligand-binding ability is reversible and, if so, whether commitment to terminal differentiation can also be reversed. Keratinocytes that had been placed in suspension for 5 hours to induce commitment were compared with the starting population (0 hour cells) in the presence or absence of 8A2, an activating anti-beta 1 antibody. 8A2 IgG or FAb fragments increased the amount of alpha 5 beta 1 in cell extracts that bound to fibronectin-Sepharose and in the presence of 8A2 the amount of bound alpha 5 beta 1 in 0 hour and 5 hour extracts was equal. 8A2 also restored alpha 5 beta 1 function in adhesion assays of intact 5 hour cells. Ca2+, Mg2+ and Mn2+ alone, at concentrations of up to 1 mM, did not increase the adhesiveness of 5 hour cells relative to 0 hour cells; however, the effect of 8A2 on keratinocytes was dependent on Ca2+. Although 8A2 restored alpha 5 beta 1 ligand-binding ability it did not prevent committed cells from withdrawing from the cell cycle and expressing involucrin, a differentiation marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Evidence against a major role for integrins in calcium-dependent intercellular adhesion of epidermal keratinocytes.

It is well established that integrins mediate keratinocyte adhesion to extracellular matrix proteins, but, in addition, there is some evidence that they mediate intercellular adhesion. We have investigated the role of integrins in keratinocyte-keratinocyte adhesion by adding anti-integrin antibodies to cells in three assays that differ according to the calcium ion concentration of the medium, the presence or absence of an adhesive substrate (glass or tissue culture plastic) and the timing of antibody addition. As previously reported by Larjava et al., (J. Cell Biol. 110:803-815), a monoclonal antibody to the beta 1 subunit perturbed cell-cell adhesion when added to adherent monolayers in low calcium medium (0.1 mM calcium ions), but did not prevent cell-cell adhesion or stratification induced by raising the level of calcium ions to 1.8mM (the concentration in standard medium). Monoclonal antibodies to both the alpha 3 and beta 1 subunits inhibited the attachment, spreading and motility of keratinocytes in low or standard calcium medium when added at the time of plating; however, they had only a modest effect on the accumulation of cells in adherent clusters. Aggregation of keratinocytes in suspension required a calcium ion concentration of greater than 0.1mM and was not inhibited by any of a large panel of anti-integrin antibodies, including three new antibodies that recognise alpha 2 beta 1. We conclude that any inhibitory effects of individual anti-integrin antibodies on cell-cell adhesion are abrogated by a calcium ion concentration above 0.1mM and that in low calcium medium at least some of the inhibition of cell-cell adhesion is a consequence of the inhibition of cell-substrate adhesion and motility.

Antibodies, Monoclonal↗
PubMed1993

Expression and function of the keratinocyte integrins.

Human keratinocytes express several adhesive receptors of the integrin family. Expression is normally confined to the basal (proliferative) layer of keratinocytes, both in mature epidermis and during development. Altered expression patterns are observed during wound healing, in psoriasis and in squamous cell carcinomas. Keratinocyte integrins are subject to both transcriptional and post-translational regulation and ligand binding ability can be modulated independently of expression. Studies with cultured keratinocytes suggest a variety of functions for the receptors: adhesion to extracellular matrix proteins, intercellular adhesion, stratification, lateral migration and the regulation of terminal differentiation. Three distinct subpopulations of basal keratinocytes, with characteristics of stem cells, transit amplifying cells and cells committed to differentiate, can be distinguished on the basis of differences in integrin expression and function.

Cell Adhesion↗
PubMed1992

Transcriptional and post-translational regulation of beta 1 integrin expression during keratinocyte terminal differentiation.

During suspension-induced terminal differentiation of human epidermal keratinocytes, the alpha 5 beta 1 integrin is down-regulated in two stages: first, the ability of the receptor to bind fibronectin is reduced; later, the receptor is lost from the cell surface, and the level of the subunit mRNAs declines. We have begun to examine the mechanisms that regulate these events. Pulse-chase experiments showed that when keratinocytes were placed in suspension to induce terminal differentiation maturation of the beta 1 subunit and its associated alpha subunits was prevented. The inhibition of maturation was at the stage of N-linked glycosylation in the Golgi, because the immature integrin subunits were sensitive to endoglycosidase H digestion and the inhibition could be mimicked in adherent cells by treatment with 1-deoxymannojirimycin. In 1-deoxymannojirimycin-treated adherent keratinocytes, immature integrin subunits reached the cell surface; however, in keratinocytes induced to differentiate in suspension, no beta 1-integrin precursors were detected on the cell surface. Thus commitment to terminal differentiation results in a block both in integrin glycosylation and transport to the cell surface; down-regulation of receptor function must therefore involve modulation of pre-existing receptor on the cell surface. Although fibronectin or rabbit antiserum to alpha 5 beta 1 can inhibit suspension-induced terminal differentiation they did not overcome the inhibition of glycosylation. Nuclear run-on assays showed that transcription of the alpha 5 and beta 1 genes was switched off during suspension-induced terminal differentiation, and treatment of adherent keratinocytes with actinomycin D suggested that the half-lives of the alpha 5 and beta 1 mRNAs were similar in adherent and suspended cells. Thus, loss of alpha 5 beta 1 from the cell surface reflects both inhibition of transcription of the subunit genes and inhibition of maturation and intracellular transport of newly synthesized subunits.

1-Deoxynojirimycin↗
PubMed1992

Characterisation of eight monoclonal antibodies to involucrin.

Involucrin is a precursor of the insoluble protein envelope that is assembled in the outermost layers of the epidermis. The coding sequence of the protein contains a number of short tandem repeats that have been greatly altered during mammalian evolution. We have characterised eight mouse monoclonal antibodies raised against human involucrin, all of which bind to the protein in immunoprecipitation, immunoblot and immunohistochemical preparations. Each antibody was screened for cross-reactivity with gorilla, owl monkey, dog and pig involucrin and with a fragment of the human protein, expressed in lambda gt 11, that includes the entire early region of the modern segment of repeats. Three antibodies recognised involucrin in all of these assays. Four antibodies recognised primate involucrins and the lambda gt 11 fragment. One antibody, which showed cross-reactivity with lower molecular weight proteins, only recognised primate involucrins and therefore bound outside the early region of the modern segment. Since the antibodies can be used to detect involucrin both biochemically and histologically, in a range of species, they will have applications in further studies of the expression, function and evolution of the protein.

Amino Acid Sequence↗
PubMed1992

Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.

We have examined integrin expression during the remodeling of the epidermis that takes place during wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. By immunofluorescence microscopy, we found that the same integrin subunits were expressed during wound healing as in normal epidermis with very little change in the relative intensity or distribution of staining at the leading edge of the migrating epidermis. However, at the time of wound closure, when the epidermis is still hyperproliferative, alpha 2, alpha 3, alpha 6, and beta 1 were no longer confined to the basal layer, as in normal epidermis, but were also found in all the living suprabasal cell layers, coexpressed with the terminal differentiation markers involucrin, keratin 10, and keratin 16. Strong suprabasal staining for alpha v was also found in one specimen. beta 4, which normally forms a heterodimer with alpha 6, and alpha 5 remained predominantly basal. Three of the integrin ligands, fibronectin, type IV collagen, and laminin, remained largely confined to the basement membrane zone and dermis. By 14 d after wounding, the integrins were once more restricted to the basal layer. Suprabasal integrin expression was also observed in involved psoriatic lesions. Thus, in two situations in which the epidermis is hyperproliferative, there is a failure to downregulate integrin expression on initiation of terminal differentiation. The functional consequences of this aberrant integrin expression remain to be explored.

Blister↗
PubMed1992

Changes in type I keratin expression associated with HPV16 transformation of human epidermal keratinocytes.

We have used immunoblotting to compare expression of type I keratins in two strains of normal human epidermal keratinocytes (v and u) and their HPV16-transformed derivatives (vp and up). The levels of keratins 14 and 17 were similar in all four cell strains, whereas keratins 18 and 19 were more abundant in vp and up than in the normal parental keratinocytes. Keratin 13 was more abundant in the transformed cells than in the parentals; in addition, expression in v was higher than in u, and expression in vp was higher than in up, suggesting strain-specific variation in expression. Keratin 16 was the only keratin to be more highly expressed in v and u than in vp and up; this is consistent with the reduced capacity of the transformants for stratification and terminal differentiation. Double-label immunofluorescence of vp and up showed that more cells expressed involucrin than keratin 16. We conclude that HPV16 transformation results in marked changes in keratin expression. The increased expression of keratin 18, a keratin that is normally expressed in simple epithelia, fits well with reports of increased keratin 18 expression in invasive squamous cell carcinomas of skin and other keratinocyte-derived tumours.

Cell Line, Transformed↗
PubMed1991

Desmosomal glycoprotein DGI, a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules.

Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilament system as in the zonula adherens or with intermediate filaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended family, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of approximately 29 amino acid residues predicted to have an antiparallel beta-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2(+)-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu.

Amino Acid Sequence↗