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F M Watt

Publications and source records attributed to F M Watt.

At least 37 records · Page 2Linked to original sources
PubMed2000

Structure and regulation of the envoplakin gene.

Envoplakin, a member of the plakin family of proteins, is a component of desmosomes and the epidermal cornified envelope. To understand how envoplakin expression is regulated, we have analyzed the structure of the mouse envoplakin gene and characterized the promoters of both the human and mouse genes. The mouse gene consists of 22 exons and maps to chromosome 11E1, syntenic to the location of the human gene on 17q25. The exon-intron structure of the mouse envoplakin gene is common to all members of the plakin family: the N-terminal protein domain is encoded by 21 small exons, and the central rod domain and the C-terminal globular domain are coded by a single large exon. The C terminus shows the highest sequence conservation between mouse and human envoplakins and between envoplakin and the other family members. The N terminus is also conserved, with sequence homology extending to Drosophila Kakapo. A region between nucleotides -101 and 288 was necessary for promoter activity in transiently transfected primary keratinocytes. This region is highly conserved between the human and mouse genes and contains at least two different positively acting elements identified by site-directed mutagenesis and electrophoretic mobility shift assays. Mutation of a GC box binding Sp1 and Sp3 proteins or a combined E box and Krüppel-like element interacting with unidentified nuclear proteins virtually abolished promoter activity. 600 base pairs of the mouse upstream sequence was sufficient to drive expression of a beta-galactosidase reporter gene in the suprabasal layers of epidermis, esophagus, and forestomach of transgenic mice. Thus, we have identified a regulatory region in the envoplakin gene that can account for the expression pattern of the endogenous protein in stratified squamous epithelia.

Amino Acid Sequence↗
PubMed2000

The alpha2 and alpha5 integrin genes: identification of transcription factors that regulate promoter activity in epidermal keratinocytes.

We analysed the activity of the proximal promoters of the alpha2 and alpha5 integrin genes in human keratinocytes. An AP-1 site, found in the alpha5 but not the alpha2 promoter, bound c-Jun/c-Fos dimers and contributed strongly to promoter activity. Both promoters had a CCAAT/enhancer binding protein (C/EBP) binding site: the alpha5 C/EBP element enhanced activity, while the alpha2 site was a negative regulatory element. C/EBP overexpression repressed the activity of both promoters, but the effect was independent of occupancy of the identified C/EBP binding sites, suggesting interactions with additional transcription factors. We propose that upregulation of C/EBPs contributes to the inhibition of integrin transcription during keratinocyte terminal differentiation, while AP-1 factors play a role in the selective induction of the alpha5 gene during wound healing.

Antigens, CD↗
PubMed2000

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters.

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.

Cell Differentiation↗
PubMed2000

Out of Eden: stem cells and their niches.

Stem cells are currently in the news for two reasons: the successful cultivation of human embryonic stem cell lines and reports that adult stem cells can differentiate into developmentally unrelated cell types, such as nerve cells into blood cells. Both intrinsic and extrinsic signals regulate stem cell fate and some of these signals have now been identified. Certain aspects of the stem cell microenvironment, or niche, are conserved between tissues, and this can be exploited in the application of stem cells to tissue replacement therapy.

Animals↗
PubMed2000

beta1B integrin subunit contains a double lysine motif that can cause accumulation within the endoplasmic reticulum.

Human epidermal keratinocytes are one of the few cell types that express the beta1B splice variant of the beta1 integrin subunit. Although in transfection experiments beta1B acts as a dominant negative inhibitor of cell adhesion, we found that beta1B was expressed at very low levels in keratinocytes, both in vivo and in culture, and had a predominantly cytoplasmic distribution, concentrated within the endoplasmic reticulum. To examine why beta1B accumulated in the cytoplasm, we prepared chimeras between CD8alpha and the beta1A and beta1B integrin cytoplasmic domains. In transfected HeLa cells, both constructs reached the cell surface but the rate of maturation of the beta1B chimera was considerably retarded relative to beta1A. The beta1B cytoplasmic domain contains two lysine residues that resemble the double lysine motif characteristic of many proteins that are resident within the endoplasmic reticulum. Mutation of each lysine individually to serine had no effect on CD8beta1B maturation, but when both residues were mutated the rate of CD8beta1B maturation increased to that of CD8beta1A. Further analysis of beta1B function in keratinocytes must, therefore, take into account the low abundance of the isoform relative to beta1A and the potential for beta1B to accumulate in the endoplasmic reticulum.

Alternative Splicing↗
PubMed2000

Expression of involucrin in normal, hyperproliferative and neoplastic mouse keratinocytes.

Involucrin is a protein precursor of the epidermal cornified envelope. Although expression of the human protein has been documented extensively, studies in the mouse have been hampered by a shortage of good antibodies. We describe the production of recombinant mouse involucrin and preparation of rabbit antisera to the protein that work well by immunohistochemistry and Western blotting. We confirm that in normal mouse epidermis the onset of involucrin expression is in the upper spinous layers and inner root sheath of the hair follicle. Involucrin was also detected in the differentiating epithelial cells of normal tongue, oesophagus and bladder. Involucrin was expressed in a subpopulation of mouse keratinocytes cultured in standard or low calcium medium and the proportion of involucrin-positive cells increased during suspension-induced terminal differentiation. Western blotting of keratinocytes from several inbred mouse strains revealed a remarkable heterogeneity in the electrophoretic mobility of involucrin, reflecting inter-strain variation in the number of tandem repeats in the protein. In the hyperproliferative epidermis of healing wounds involucrin was expressed in most of the suprabasal layers. In epidermal papillomas and carcinomas involucrin expression correlated well with degree of histological differentiation. The sites of expression of the mouse protein were thus the same as those previously reported for human involucrin. With the development of the new antibodies we anticipate that involucrin will become as widely used a marker of keratinocyte differentiation in the mouse as it is in the human.

Animals↗
PubMed2000

Proliferative heterogeneity in the human prostate: evidence for epithelial stem cells.

Clonal analysis of human prostate epithelial cells was undertaken in order to identify stem cells. Two types of colony were distinguished, termed type I and type II. Type I colonies were relatively small and irregular and contained a loose mixture of differentiated and undifferentiated cells. In contrast, type II colonies were large, round, and homogeneous, consisting almost exclusively of small undifferentiated and dividing cells. The colony-forming efficiency was 5.8% +/- 1.8 for freshly isolated epithelial cells. There were approximately 10 times as many type I as type II colonies and about 1 in 200 of the plated cells was capable of forming a type II colony. In three-dimensional culture on Matrigel, the type II colonies produced structures reminiscent of prostate epithelium, with luminal cells expressing markers of prostate epithelial differentiation, including the androgen receptor. On the basis of their proliferative characteristics and pluripotency, the type II colonies may be the progeny of stem cells and the type I colonies of a more differentiated transit-amplifying population.

Aged↗
PubMed2000

beta1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms.

In keratinocytes, the beta1 integrins mediate adhesion to the extracellular matrix and also regulate the initiation of terminal differentiation. To explore the relationship between these functions, we stably infected primary human epidermal keratinocytes and an undifferentiated squamous cell carcinoma line, SCC4, with retroviruses encoding wild-type and mutant chick beta1 integrin subunits. We examined the ability of adhesion-blocking chick beta1-specific antibodies to inhibit suspension-induced terminal differentiation of primary human keratinocytes and the ability of the chick beta1 subunit to promote spontaneous differentiation of SCC4. A D154A point mutant clustered in focal adhesions but was inactive in the differentiation assays, showing that differentiation regulation required a functional ligand-binding domain. The signal transduced by beta1 integrins in normal keratinocytes was "do not differentiate" (transduced by ligand-occupied receptors) as opposed to "do differentiate" (transduced by unoccupied receptors), and the signal depended on the absolute number, rather than on the proportion, of occupied receptors. Single and double point mutations in cyto-2 and -3, the NPXY motifs, prevented focal adhesion targeting without inhibiting differentiation control. However, deletions in the proximal part of the cytoplasmic domain, affecting cyto-1, abolished the differentiation-regulatory ability of the beta1 subunit. We conclude that distinct signaling pathways are involved in beta1 integrin-mediated adhesion and differentiation control in keratinocytes.

Amino Acid Motifs↗
PubMed1999

Envoplakin, a possible candidate gene for focal NEPPK/esophageal cancer (TOC): the integration of genetic and physical maps of the TOC region on 17q25.

Focal nonepidermolytic palmoplantar keratoderma (NEPPK), or tylosis, is an autosomal, dominantly inherited disorder of the skin that manifests as focal thickening of the palmar and plantar surfaces. In three families studied, the skin disorder cosegregates with esophageal cancer and oral lesions. New haplotype analysis, presented here, places the tylosis esophageal cancer (TOC) locus between D17S1839 and D17S785. Envoplakin (EVPL) is a protein component of desmosomes and the cornified envelope that is expressed in epidermal and esophageal keratinocytes and has been localized to the TOC region. Mutation analysis of EVPL in the three affected families failed to show tylosis-specific mutations, and haplotype analysis of three intragenic sequence polymorphisms of the EVPL gene placed it proximal to D17S1839. Confirmation of the exclusion of EVPL as the TOC gene by location was obtained by integration of the genetic and physical mapping data using radiation hybrid, YAC, BAC, and PAC clones. This new physical map will allow further identification of candidate genes underlying NEPPK associated with esophageal cancer, which may also be implicated in the development of sporadic squamous cell esophageal carcinoma and Barrett's adenocarcinoma.

Base Sequence↗
PubMed1999

A central role for alpha beta T cells in the pathogenesis of murine lupus.

We have previously shown that female transgenic mice expressing IFN-gamma in the epidermis, under the control of the involucrin promoter, develop inflammatory skin disease and a form of murine lupus. To investigate the pathogenesis of this syndrome, we generated female IFN-gamma transgenic mice congenitally deficient in either alpha beta or gamma delta T cells. TCR delta-/- transgenics continued to produce antinuclear autoantibodies and to develop severe kidney lesions. In contrast, TCR beta-/- IFN-gamma transgenic mice failed to produce antinucleosome, anti-dsDNA, or antihistone autoantibodies, and kidney disease was abolished. Both alpha beta- and gamma delta-deficient transgenics continued to develop IFN-gamma-associated skin disease, lymphadenopathy, and splenomegaly. The data show that the autoantibody-mediated pathology of murine lupus in IFN-gamma transgenic mice is completely alpha beta T cell dependent and that gamma delta T cells cannot drive autoantibody production. These results imply that production of antinuclear autoantibodies in IFN-gamma transgenic animals is Ag driven, and we identified clusters of apoptotic cells in the epidermis of the mice as a possible source of self Ags. Our findings emphasize the relevance of this murine lupus model to the human disease.

Animals↗
PubMed1999

Signaling via beta1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.

Human epidermal stem cells express higher levels of beta1 integrins and are more adhesive than keratinocytes that are destined to differentiate. To investigate whether high beta1 integrin expression and adhesiveness are essential for maintaining keratinocytes in the stem cell compartment, we introduced a dominant-negative beta1 integrin mutant, CD8beta1, into cultured human keratinocytes, thereby interfering with beta1 integrin function. Surface beta1 integrin levels, adhesiveness, and mitogen-activated protein (MAP) kinase activation on fibronectin were reduced, and exit from the stem cell compartment was stimulated. Adhesiveness and proliferative potential were restored by overexpressing wild-type beta1 integrin or by constitutive MAP kinase activation. Conversely, a dominant-negative MAP kinase kinase 1 mutant decreased adhesiveness and stem cell number in the absence of CD8beta1. MAP kinase activation by alpha6beta4-mediated adhesion and mitogens was normal in CD8beta1 cells, and constitutive MAP kinase activation did not affect adhesion and proliferation of control keratinocytes. We conclude that beta1 integrins and MAP kinase cooperate to maintain the epidermal stem cell compartment in vitro.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Analysis of cultured keratinocytes from a transgenic mouse model of psoriasis: effects of suprabasal integrin expression on keratinocyte adhesion, proliferation and terminal differentiation.

Many important transgenic mouse models of benign and neoplastic skin diseases have been generated through the use of promoters that target transgene expression to the different epidermal layers. However, more mechanistic studies of the specific effects of the transgenes on keratinocytes have been hampered by difficulties in culturing keratinocytes from adult mouse epidermis and by the low differentiation potential of many established mouse keratinocyte lines. We have used the Rheinwald & Green technique to cultivate primary adult keratinocytes and to generate keratinocyte lines from transgenic mice which have a sporadic psoriatic phenotype due to expression of human integrin subunits under the control of the involucrin promoter. We show that the transgenes are induced when keratinocytes are placed in suspension and that the transgenic integrins are capable of clustering in focal adhesions and mediating cell adhesion and spreading. We also show that suprabasal integrin expression has no direct effect on proliferation of cells in the underlying basal layer, ruling this out as a possible explanation for the epidermal hyperproliferation observed in the transgenic mice.

Animals↗
PubMed1999

Evidence that apoptosis and terminal differentiation of epidermal keratinocytes are distinct processes.

Although there are clear parallels between apoptosis and epidermal terminal differentiation it is unclear whether terminal differentiation of keratinocytes is a form of apoptosis. We found that apoptosis was rare in adherent cultures of normal keratinocytes, even when growth factors were removed. When keratinocytes were placed in suspension for 24-96 h the majority of cells were induced to undergo terminal differentiation, as assessed by involucrin expression and cornified envelope assembly, but few cells underwent apoptosis, as assessed by morphological examination, TUNEL labelling and by DNA laddering. Withdrawal of serum and growth factors stimulated apoptosis of suspended keratinocytes but led to some reduction in the number of cells that underwent terminal differentiation. At 96 h the majority of cells retained their nuclei in the presence or absence of serum and growth factors. In normal epidermis only occasional cells within the granular layer had apoptotic nuclei, determined by TUNEL labelling and light and electron microscopy. In affected epidermis of psoriasis, Darier's disease and pityriasis rubra pilaris, diseases characterized by perturbation of growth, differentiation or adhesion, light microscopy revealed no higher proportion of apoptotic nuclei than in normal epidermis. However, the majority of viable epidermal layers in diseased skin were positive by TUNEL labelling, suggesting that TUNEL is not always a specific marker of apoptosis in keratinocytes. We conclude that in vivo and in culture keratinocyte terminal differentiation and apoptosis are distinct cellular events, subject to different stimuli.

Adult↗
PubMed1999

beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion.

We found that cultured human keratinocytes with high proliferative potential, the putative epidermal stem cells, expressed a higher level of noncadherin-associated beta-catenin than populations enriched for keratinocytes of lower proliferative potential. To investigate the physiological significance of this, a series of beta-catenin constructs was introduced into keratinocytes via retroviral infection. Full-length beta-catenin and a mutant containing only nine armadillo repeats had little effect on proliferative potential in culture, the full-length protein being rapidly degraded. However, expression of stabilised, N-terminally truncated beta-catenin increased the proportion of putative stem cells to almost 90% of the proliferative population in vitro without inducing malignant transformation, and relieved the differentiation stimulatory effect of overexpressing the E-cadherin cytoplasmic domain. Conversely, beta-catenin lacking armadillo repeats acted as a dominant negative mutant and stimulated exit from the stem cell compartment in culture. The positive and negative effects of the beta-catenin mutants on proliferative potential were independent of effects on cell-cycle kinetics, overt terminal differentiation or intercellular adhesion, and correlated with stimulation or inhibition of transactivation of a TCF/LEF reporter in basal keratinocytes. We conclude that the elevated level of cytoplasmic beta-catenin in those keratinocytes with characteristics of epidermal stem cells contributes to their high proliferative potential.

Animals↗
PubMed1999

The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.

In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.

Biomarkers↗
PubMed1998

Epidermal stem cells: markers, patterning and the control of stem cell fate.

Within the epidermis, proliferation takes place in the basal layer of keratinocytes that are attached to an underlying basement membrane. Cells that leave the basal layer undergo terminal differentiation as they move towards the tissue surface. The basal layer contains two types of proliferative keratinocyte: stem cells, which have 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. Stem cells express higher levels of the beta 1-integrin family of extracellular matrix receptors than transit amplifying cells and this can be used to isolate each subpopulation of keratinocyte and to determine its location within the epidermis. Variation in the levels of E-cadherin, beta-catenin and plakoglobin within the basal layer suggests that stem cells may also differ from transit amplifying cells in intercellular adhesiveness. Stem cells have a patterned distribution within the epidermal basal layer and patterning is subject to autoregulation. Constitutive expression of the transcription factor c-Myc promotes terminal differentiation by driving keratinocytes from the stem cell compartment into the transit amplifying compartment.

Animals↗
PubMed1998

Comparison of integrin, cadherin, and catenin expression in squamous cell carcinomas of the oral cavity.

In addition to their role in maintenance of tissue integrity, cell adhesion molecules regulate the growth and differentiation of stratified squamous epithelia. Reduced expression of E-cadherin and the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins is already reported to correlate with poor histological differentiation in oral squamous cell carcinomas. However, it is not clear how closely cadherin and integrin loss are related in any given tumour, nor whether cadherin loss is correlated with changes in expression of the cytoplasmic regulatory proteins known as catenins. Double-label immunofluorescence has been used to stain a panel of 22 oral squamous cell carcinomas with antibodies to ten proteins, including E- and P-cadherin, the major keratinocyte integrin subunits, and alpha-, beta- and gamma-catenin. Overall, E-cadherin expression and integrin expression correlated well with tumour grade, while P-cadherin staining was more variable. All tumours, regardless of differentiation status, showed reduced staining for at least two of the catenins, implying that the adhesive function of E- and P-cadherin could be impaired even when cadherin expression is normal. It is concluded that in all squamous cell carcinomas, regardless of degree of histological differentiation, there is some perturbed expression of cell adhesion molecules and that integrin and E-cadherin loss are closely related.

Adult↗