A novel substitution in keratin 10 in epidermolytic hyperkeratosis.
(1/75)
Epidermolytic hyperkeratosis is characterized by tonofilament clumping, cytolysis, and blister formation in suprabasal keratinocytes. It has been shown that the tonofilament aggregates in these areas are composed of keratin 1 (K1) and keratin 10 (K10), and several K1 and K10 point mutations have been identified as the molecular basis of epidermolytic hyperkeratosis. In this report we identify a novel, single base pair substitution resulting in an amino acid exchange from tyrosine to serine at residue 14 within the conserved 1A region of K10 (Y14S). This A to C transversion in codon 160 was only present in the affected individual and was associated with a very severe disease phenotype. Our observations are in agreement with previous reports documenting that this tyrosine residue, located at the beginning of the rod domain of type I keratins, is particularly sensitive to amino acid substitutions, and that alterations in this residue can have deleterious effects on filament assembly and stability. (+info)
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.
(2/75)
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. (+info)
C/EBPbeta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression.
(3/75)
The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPbeta within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPbeta in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPbeta-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPbeta-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPbeta-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPbeta plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression. (+info)
Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line.
(4/75)
The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts. (+info)
Keratin 10 gene expression during differentiation of mouse epidermis requires transcription factors C/EBP and AP-2.
(5/75)
The epidermis forms a vital barrier composed of stratified keratinocytes and their differentiated products. One of these products, keratin K10, is critical to epidermal integrity, because mutations in k10 lead to abnormal blistering. For the normal expression of k10, differentiation-associated transcription factors C/EBPalpha, C/EBPbeta, and AP-2 are well positioned to play an important role. Here, regulation of the k10 gene is examined in keratinocytes in the skin of normal mice and in transgenic mice carrying targeted deletions of c/ebpbeta and ap-2alpha. In cultured cells, C/EBPalpha and C/EBPbeta are each capable of activating the k10 promoter via three binding sites, identified by site-directed mutagenesis. In a given epidermal cell in vivo, however, the selection of C/EBPalpha versus C/EBPbeta for k10 regulation is determined via a third transcription factor, AP-2. This novel regulatory scheme involves: (1) unique gradients of expression for each transcription factor, i.e., C/EBPbeta and AP-2 most abundant in the lower epidermis, C/EBPalpha in the upper; (2) C/EBP-binding sites in the ap-2alpha gene promoter, through which C/EBPbeta stimulates ap-2alpha; and (3) AP-2 binding sites in the c/ebpalpha promoter, through which AP-2 represses c/ebpalpha. Promoter-analysis and gene-expression data presented herein support a regulatory model in which C/EBPbeta activates and maintains AP-2 expression in basal keratinocytes, whereas AP-2 represses C/EBPalpha in those cells. In response to differentiation signals, loss of AP-2 expression leads to derepression of the c/ebpalpha promoter and activation of k10 as cells migrate upward. (+info)
Topological analysis of p21WAF1/CIP1 expression in esophageal squamous dysplasia.
(6/75)
In the normal stratified squamous epithelium of the esophagus, only the third to the fifth layers of cells express the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21). Using immunohistochemical staining, we examined the topological distribution of cells expressing p21, p53, Ki67, and cytokeratin 10 (CK10), a differentiation marker of esophageal squamous cell carcinoma (SCC), in 25 superficial SCCs and 72 dysplastic lesions of the esophagus. Image analysis of p21, p53, and Ki67 expression was also performed in 48 dysplastic lesions. In superficial SCCs, although Ki67- and p53-expressing cells were mainly distributed in the deep layers of tumors despite tumor differentiation, the distribution of p21 correlated with tumor differentiation. In dysplastic lesions, p53- and Ki67-coexpressing cells tended to locate in the same layers and expand in the lower layers of epithelium with the progression of dysplasia. p21-expressing cells shifted to the upper layers of the epithelium with the progression of dysplasia. However, this change was heterogeneous; in some lesions, p21-expressing cells were confined to the superficial layers of atypical cells (confined type), whereas in others, p21-overexpressing cells were scattered among atypical cells (scattered type). CK10 expression was observed in 25% of dysplastic lesions, and the frequency of CK10 expression was significantly higher in the scattered than in the confined type. Our results suggest that esophageal squamous dysplasia represents the earliest pathological process in esophageal squamous carcinogenesis. Our results also suggest that differentiation of esophageal SCC is determined at the stage of dysplasia, and that p21 plays a critical role in the differentiation process. (+info)
Focal activation of a mutant allele defines the role of stem cells in mosaic skin disorders.
(7/75)
Stem cells are crucial for the formation and maintenance of tissues and organs. The role of stem cells in the pathogenesis of mosaic skin disorders remains unclear. To study the molecular and cellular basis of mosaicism, we established a mouse model for the autosomal-dominant skin blistering disorder, epidermolytic hyperkeratosis (MIM 113800), which is caused by mutations in either keratin K1 or K10. This genetic model allows activation of a somatic K10 mutation in epidermal stem cells in a spatially and temporally controlled manner using an inducible Cre recombinase. Our results indicate that lack of selective pressure against certain mutations in epidermal stem cells leads to mosaic phenotypes. This finding has important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses. (+info)
Up- and down-regulation of granulocyte/macrophage-colony stimulating factor activity in murine skin increase susceptibility to skin carcinogenesis by independent mechanisms.
(8/75)
The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in tumorigenesis is complex. On the one hand, GM-CSF can promote tumor cell growth, survival, and even metastasis. On the other hand, it can stimulate tumor cell rejection. In skin, it is early expressed after topic application of tumor-promoting agents and therefore may be responsible for changes that correlate with skin tumor promotion (e.g., epidermal hyperproliferation and inflammation). To analyze GM-CSF function in skin tumorigenesis, we generated transgenic mice epidermally overexpressing either GM-CSF or a GM-CSF antagonist. Both types of transgenic mice exhibited significantly increased numbers of benign tumors in a two-step skin carcinogenesis experiment using 7',12'-dimethylbenz[a]anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-CSF displayed a significantly elevated carcinoma burden following a single-step carcinogenesis protocol consisting of tumor initiation only. Therefore, endogenous promotion is responsible for elevated tumor development in GM-CSF-overexpressing mice. In antagonist transgenic animals, an increased tumorigenicity of modified B16 tumor cells after cutaneous transplantation as compared with nontransgenic or GM-CSF transgenic mice was observed. Thus, the antitumor activity leading to the repression of tumor cell growth in control mice is GM-CSF dependent and is compromised in mice expressing the antagonist. We suggest that both, up-regulation and down-regulation of GM-CSF activity in skin, increase the incidence and growth of tumors via two independent mechanisms: endogenous tumor promotion in the case of increased GM-CSF activity and compromised tumor cell rejection in the case of decreased GM-CSF activity. (+info)