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Biomedical subjects

F M Watt

Publications and source records attributed to F M Watt.

At least 55 records · Page 3Linked to original sources
PubMed1998

Role of integrins in mouse eyelid development: studies in normal embryos and embryos in which there is a failure of eyelid fusion.

Eyelid fusion normally occurs between E15.5 and E16.5 of mouse embryonic development and results from the migration of a population of periderm-derived epithelial cells over the corneal surface. Cell migration is known to depend on extracellular matrix receptors of the integrin family and to be regulated by growth factors. We were therefore interested that a failure of eyelid fusion has been reported in mice that are homozygous null for the transforming growth factor alpha (TGF-alpha) gene and in mice (invalpha5beta1) in which a transgenic alpha5beta1 integrin under the control of the involucrin promoter is misexpressed in differentiating keratinocytes. We examined expression of the alpha2beta1, alpha3beta1, alpha5beta1 and alpha6beta4 integrins during eyelid fusion in wild-type embryos and found selective upregulation of the alpha5beta1 integrin and its ligand, fibronectin, in the migrating eyelid tip cells. In TGF-alpha null embryos, the failure of eyelid fusion was correlated with a failure to upregulate the alpha5beta1 integrin and fibronectin in the tip cells. Using beta-galactosidase as a reporter gene in transgenic mice, we observed specific activity of the involucrin promoter in the eyelid tip cells. In invalpha5beta1 mice the transgenic human integrin was overexpressed not only in the tip cells but throughout the eyelid epidermis. In contrast, the endogenous, murine, alpha5beta1 integrin was only weakly expressed in the tip cells. We speculate that selective and coordinated expression of the alpha5beta1 integrin and fibronectin in eyelid tip cells is required for eyelid fusion and may be under the control of growth factors that include TGF-alpha.

Animals↗
PubMed1998

Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice.

Previous attempts to achieve long-term gene expression in retrovirally transduced human epidermal keratinocytes in vivo have been largely unsuccessful. This has been variously attributed to a failure to target epidermal stem cells, suboptimal grafting conditions or inactivation of the retroviral vector. In an attempt to overcome these problems we expressed the chick beta 1 integrin subunit in primary human epidermal keratinocytes, which allowed us to monitor retroviral gene expression on a cell-by-cell basis. We describe optimised methods for selecting high-titre amphotropic packaging cells and for infecting keratinocytes in culture. When transduced cells were grafted into mice, graft survival was comparable in nude and SCID mice, but it was essential to combine the keratinocytes with a dermal substrate. Using these methods the majority of keratinocytes expressed the chick beta 1 integrin subunit for at least 16 weeks after grafting. We conclude that epidermal keratinocytes are attractive recipient cells for gene therapy.

3T3 Cells↗
PubMed1998

Lack of intrinsic polarity in the ligand-binding ability of keratinocyte beta1 integrins.

Within the basal layer of the epidermis the beta1 integrins have a pericellular distribution. Two monoclonal antibodies, 15/7 and 12G10, that detect a conformation of the beta1 integrin subunit that is induced following cation or ligand occupancy selectively recognized beta1 integrins at the basement membrane zone in vivo and in focal adhesions of cultured keratinocytes; they did not recognize integrins on the apical and upper lateral membranes of basal keratinocytes nor integrins on the suprabasal keratinocytes of hyperproliferative epidermis. Inhibition of intercellular adhesion did not induce the 15/7 epitope on the lateral and apical membrane domains. The surface distribution of the epitopes was consistent with the antibodies acting as reporters of ligand-binding; in addition, the 15/7 epitope was exposed on unglycosylated, immature beta1 integrins. Although the apical membrane of basal keratinocytes is not normally in contact with extracellular matrix proteins, we found that it was capable of binding fibronectin-coated beads and that the 15/7 epitope was exposed on plasma membrane in contact with the beads. When a chimeric molecule consisting of the extracellular domain of CD8 and the cytoplasmic domain of the beta1 integrin subunit, used to mimic a constitutively active beta1 heterodimer, was introduced into keratinocytes it localized to the basal, lateral and apical membrane domains. We conclude that although the conformation of the keratinocyte beta1 integrins differs between the basal and the lateral/apical membrane domains there is no intrinsic polarity in the ligand binding potential of the receptors.

Binding Sites↗
PubMed1998

Calcium-induced intercellular adhesion of keratinocytes does not involve accumulation of beta 1 integrins at cell-cell contacts and does not involve changes in the levels or phosphorylation of catenins.

On initiation of terminal differentiation human epidermal keratinocytes detach from the underlying basement membrane as a result of inactivation and subsequent loss of integrins from the cell surface. Assembly of keratinocytes into multilayered sheets requires functional E- and P-cadherin and when stratification is inhibited in low calcium medium differentiating keratinocytes continue to express functional integrins. Using immunofluorescence microscopy, we found that on addition of calcium ions to keratinocyte monolayers there was colocalisation of the beta 1 integrins and E-cadherin along the lateral membranes except for a zone close to the substratum which exclusively contained integrins. Quantitative immunoelectron microscopy showed that on induction of stable cell-cell contacts the density of beta 1 integrins was the same on the apical and lateral membranes, suggesting that the accumulation of integrins on the lateral membranes observed by immunofluorescence microscopy is due to the increased area of contact between adjacent cells and not to an increase in receptor density. There were no changes in the levels of catenins and their degree of phosphorylation after induction of cell-cell contacts. These observations provide new sights into the mechanism of calcium-dependent intercellular adhesion of keratinocytes.

Cadherins↗
PubMed1997

Periplakin, a novel component of cornified envelopes and desmosomes that belongs to the plakin family and forms complexes with envoplakin.

The cornified envelope is a layer of transglutaminase cross-linked protein that is assembled under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We have determined the cDNA sequence of one of the proteins that becomes incorporated into the cornified envelope of cultured epidermal keratinocytes, a protein with an apparent molecular mass of 195 kD that is encoded by a mRNA with an estimated size of 6.3 kb. The protein is expressed in keratinizing and nonkeratinizing stratified squamous epithelia and in a number of other epithelia. Expression of the protein is upregulated during the terminal differentiation of epidermal keratinocytes in vivo and in culture. Immunogold electron microscopy was used to demonstrate an association of the 195-kD protein with the desmosomal plaque and with keratin filaments in the differentiated layers of the epidermis. Sequence analysis showed that the 195-kD protein is a member of the plakin family of proteins, to which envoplakin, desmoplakin, bullous pemphigoid antigen 1, and plectin belong. Envoplakin and the 195-kD protein coimmunoprecipitate. Analysis of their rod domain sequences suggests that the formation of both homodimers and heterodimers would be energetically favorable. Confocal immunofluorescent microscopy of cultured epidermal keratinocytes revealed that envoplakin and the 195-kD protein form a network radiating from desmosomes, and we speculate that the two proteins may provide a scaffolding onto which the cornified envelope is assembled. We propose to name the 195-kD protein periplakin.

Amino Acid Sequence↗
PubMed1997

Antinuclear autoantibodies and lupus nephritis in transgenic mice expressing interferon gamma in the epidermis.

Systemic lupus erythematosus (SLE) is a potentially fatal non-organ-specific autoimmune disease that predominantly affects women. Features of the disease include inflammatory skin lesions and widespread organ damage caused by deposition of anti-dsDNA autoantibodies. The mechanism and site of production of these autoantibodies is unknown, but there is evidence that interferon (IFN) gamma plays a key role. We have used the involucrin promoter to overexpress IFN-gamma in the suprabasal layers of transgenic mouse epidermis. There was no evidence of organ-specific autoimmunity, but transgenic animals produced autoantibodies against dsDNA and histones. Autoantibody levels in female mice were significantly higher than in male transgenic mice. Furthermore, there was IgG deposition in the glomeruli of all female mice and histological evidence of severe proliferative glomerulonephritis in a proportion of these animals. Our findings are consistent with a central role for the skin immune system, acting under the influence of IFN-gamma, in the pathogenesis of SLE.

Animals↗
PubMed1997

c-Myc promotes differentiation of human epidermal stem cells.

The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. In a search for factors that regulate exit from the stem cell compartment, we constitutively expressed c-Myc in primary human keratinocytes by use of wild-type and steroid-activatable constructs. In contrast to its role in other cell types, activation of c-Myc in keratinocytes caused a progressive reduction in growth rate, without inducing apoptosis, and a marked stimulation of terminal differentiation. Keratinocytes can be enriched for stem or transit amplifying cells on the basis of beta1 integrin expression and by use of this method to fractionate cells prior to c-Myc activation, we found that c-Myc acted selectively on stem cells, driving them into the transit amplifying compartment. As a result, activation of c-Myc in epidermis reconstituted on a dermal equivalent led to premature execution of the differentiation program. The transcriptional regulatory domain of c-Myc was required for these effects because a deletion within that domain acted as a dominant-negative mutation. Our results reveal a novel biological role for c-Myc and provide new insights into the mechanism regulating epidermal stem cell fate.

3T3 Cells↗
PubMed1997

The plakin family: versatile organizers of cytoskeletal architecture.

Desmoplakin, plectin, bullous pemphigoid antigen 1 and envoplakin are four sequence-related proteins--recently named the plakin family--that localize to intermediate filaments and filament attachment sites at the plasma membrane. New interest in the plakins has been stimulated by the discoveries that they can link different cytoskeletal elements together and that loss of plakin function can cause diseases of the skin and other tissues.

Alternative Splicing↗
PubMed1997

Transgenic mice expressing IFN-gamma in the epidermis have eczema, hair hypopigmentation, and hair loss.

To study the role of IFN-gamma in the pathogenesis of inflammatory skin diseases, we used the involucrin promoter to overexpress IFN-gamma in the suprabasal layers of transgenic mouse epidermis. IFN-gamma mRNA and protein were readily detectable in the skin but not in the blood. Mice exhibited striking hypopigmentation of the hair due to a reduced abundance of DOPA-positive melanocytes. Severely affected mice had reddened skin, growth retardation, hair loss, and flaky skin lesions. Keratinocyte proliferation was increased, and there was epidermal thickening with spongiosis and parakeratosis. Suprabasal beta1 integrin expression and induction of keratin 17 in interfollicular epidermis provided evidence of perturbed differentiation. IFN-gamma receptor expression was reduced, and there was induction of ICAM-1 and MHC class II molecules on the surface of transgenic keratinocytes. The skin of severely affected mice was characterized by a dermal infiltrate of T lymphocytes and macrophages/monocytes, but the epidermis was almost devoid of Langerhans cells and T lymphocytes. The number of Langerhans cells in the lymph nodes was increased in the transgenics, and autoantibodies to keratinocytes were produced. Transgenic mice showed an increased contact hypersensitivity reaction to topical application of DNFB. We conclude that constitutive IFN-gamma expression in the epidermis results in a form of eczema resembling contact dermatitis and in a profound contact hypersensitivity reaction.

Animals↗
PubMed1997

Correlation between hyperproliferation and suprabasal integrin expression in human epidermis reconstituted in culture.

In normal epidermis integrin expression is largely confined to the basal layer. However, during wound healing and in psoriatic lesions suprabasal expression is observed. Although the potential importance of suprabasal integrin expression in the pathogenesis of psoriasis has been established, the cause of suprabasal expression is unknown. We now describe changes in integrin expression that occur with time when normal human keratinocytes are grown on two types of dermal equivalent, de-epidermized dermis and collagen gels containing fibroblasts. We show that suprabasal integrin expression is correlated with suprabasal expression of the EGF receptor, but not with expression of keratin 10 or keratin 16. By quantitating the proportion of basal keratinocytes expressing the proliferation marker Ki-67 we could show that suprabasal integrin expression is correlated with high proliferative activity within the basal layer. Taken together with our earlier work, these results suggest that suprabasal integrin expression is linked to hyperproliferation and not to abnormal terminal differentiation or to inflammation; they also establish dermal equivalent cultures as useful experimental models with which to manipulate keratinocyte integrin expression.

Antibodies, Monoclonal↗
PubMed1997

Changes in the expression of alpha v integrins in oral squamous cell carcinomas.

Integrins are cell surface adhesion molecules that regulate normal cellular interactions; aberrant integrin expression is believed to play a role in tumour invasion and metastasis. The alpha v subunit is capable of forming heterodimers with several beta subunits but not all the heterodimers expressed in oral epithelium have been investigated. We have examined the distribution of alpha v integrins in normal buccal mucosa and seventeen oral squamous cell carcinomas. Antibodies to the alpha v subunit and alpha v beta 5 heterodimer stained normal epithelium, with strong expression in the basal layers and weaker staining in the more superficial layers. The beta 3 and beta 6 subunits were not expressed in normal epithelium. Anti-alpha v and anti-alpha v beta 5 antibodies stained all the squamous cell carcinomas, but the pattern of expression was variable both within and between tumours. Poorly differentiated tumours showed the weakest staining and often had areas showing loss of expression. beta 6 was expressed in all of the carcinomas, indicating new expression of the alpha v beta 6 integrin in malignant oral epithelium. These results suggest that alterations in alpha v integrins may contribute to the behaviour of malignant epithelium and that alpha v beta 6 expression may play a role in tumour progression.

Antigens, Neoplasm↗
PubMed1997

The epidermal stem cell compartment: variation in expression levels of E-cadherin and catenins within the basal layer of human epidermis.

The basal layer of the epidermis contains two types of proliferating keratinocyte: stem cells, with high proliferative potential, and transit amplifying cells, which are destined to undergo terminal differentiation after a few rounds of division. It has been shown previously that two- to three-fold differences in the average staining intensity of fluorescein-conjugated antibodies to beta 1 integrin subunits reflect profound differences in the proliferative potential of keratinocytes, with integrin-bright populations being enriched for stem cells. In the search for additional stem cell markers, we have stained sections of normal human epidermis with antibodies to proteins involved in intercellular adhesion and quantitated the fluorescence of individual cell-cell borders. In the basal layer, patches of brightly labeled cells were detected with antibodies to E-cadherin, beta-catenin, and gamma-catenin, but not with antibodies to P-cadherin, alpha-catenin, or with pan-desmocollin and pan-desmoglein antibodies. In the body sites examined, palm and foreskin, integrin-bright regions were strongly labeled for gamma-catenin and weakly labeled for E-cadherin and beta-catenin. Our data suggest that there are gradients of both cell-cell and cell-extracellular matrix adhesiveness within the epidermal basal layer and that the levels of E-cadherin and of beta- and gamma-catenin may provide markers for the stem cell compartment, stem cells expressing relatively higher levels of gamma-catenin and lower levels of E-cadherin and beta-catenin than other basal keratinocytes.

Cadherins↗
PubMed1996

Chromosomal localisation of the human envoplakin gene (EVPL) to the region of the tylosis oesophageal cancer gene (TOCG) on 17q25.

Envoplakin is a membrane-associated precursor of the epidermal cornified envelope. Envoplakin is homologous to desmoplakin I and desmoplakin II (DPI/II), bullous pemphigoid antigen 1 (BPAG1), and plectin and is proposed to link desmosomes and keratin filaments to the cornified envelope. We describe the isolation of cosmids and yeast artificial chromosomes containing the complete human envoplakin gene (EVPL) and show, by analysis of somatic cell hybrids and chromosomal in situ hybridisation, that the envoplakin gene, unlike the genes encoding BPAG1 and DPI/II, maps to 17q25 and is physically linked to D17S1603. This sequence-tagged site segregates with the autosomal dominant human disease focal nonepidermolytic palmoplantar keratosis (NEPKK; "tylosis"), which is associated with an increased risk of oesophageal cancer. The chromosomal localisation of the envoplakin gene, the homology of the encoded protein to keratin-binding proteins, and its expression in epidermal and oesophageal keratinocytes all raise the possibility that loss of envoplakin function could be responsible for this form of palmoplantar keratoderma.

Chromosome Mapping↗
PubMed1996

Differentiation of embryonal stem cells into keratinocytes: comparison of wild-type and beta 1 integrin-deficient cells.

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture, differentiation of embryonic or simple epithelial cells occurred in both ES cell populations, as detected by expression of keratins 8, 18, and 19. From 21 days, expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast, keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface, consistent with the association of these a subunits with beta 1. Furthermore, alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro, they did form keratinocyte cysts expressing alpha 6 beta 4, keratins 1 and 14, and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore, beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy, extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures, but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane.

Animals↗
PubMed1996

Altered expression of CD44 isoforms in squamous-cell carcinomas and cell lines derived from them.

CD44 is a transmembrane glycoprotein that binds hyaluronan, extracellular matrix proteins and growth factors. Multiple isoforms of CD44 are generated by alternative splicing of 10 separate exons (V1-V10). Expression of the variable exons has been correlated with tumour progression and metastasis in a range of cell types. However, multiple CD44 isoforms are expressed by normal stratified squamous epithelia, such as the epidermis and the lining of the oral cavity. The purpose of our study was to examine CD44 expression in squamous-cell carcinomas (SCC). By immunofluorescence we found reduced expression of one or more of the variant exons in a series of 13 oral SCC, with loss being most common in poorly differentiated tumours. Of the exons we examined, V3 was lost most frequently, but otherwise there was no consistent pattern as to which exons (V4/5, 6, 8) were missing. We also studied CD44 expression in a range of SCC lines, using Western blotting and semi-quantitative RT-PCR. All lines showed reduced expression of the terminal differentiation marker involucrin. Two lines showed selective loss of the largest forms of CD44 and one failed to express any of the variant exons. These cell lines, therefore, provide a useful experimental model with which to study the biological significance of exon loss in SCC.

Alternative Splicing↗
PubMed1996

Restoration of alpha v beta 5 integrin expression in neoplastic keratinocytes results in increased capacity for terminal differentiation and suppression of anchorage-independent growth.

Loss of expression of specific integrins is a feature of poorly differentiated oral squamous cell carcinomas (SCCs) and cell lines derived from them. In order to test whether there is a direct link between loss of integrins and abnormal keratinocyte growth and differentiation, we 'repaired' an SCC line, H357, by transfection of its missing integrin subunit, alpha v. We analysed seven independent alpha v-expressing clones and compared them with four empty vector controls and with the parental cell line. alpha v was expressed on the cell surface as a functional alpha v beta 5 heterodimer. The parental cells expressed beta 5 mRNA and introduction of alpha v had no effect on beta 5 mRNA levels nor on cell surface levels of any other integrins examined. Introduction of alpha v had no consistent effect on the growth rate of cells on tissue culture plastic. However, anchorage-independent growth was strongly suppressed and the alpha v-transfectants showed an increased capacity for terminal differentiation, as assayed by expression of involucrin. These results are consistent with the known role of integrins in regulating keratinocyte terminal differentiation and suggest that integrin loss may be directly responsible for the abnormal behaviour of keratinocytes in a subset of oral SCCs.

Carcinoma, Squamous Cell↗
PubMed1996

Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin.

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5-kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210-kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin-binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."

Amino Acid Sequence↗