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• W2080596058 abstract "Epidermal thickening is a phenomenon common to many genodermatoses but little is known about the underlying causes. We have recently created a mouse model for the human skin disease bullous congenital ichthyosiform erythroderma by gene targeting. Mice heterozygous for a truncated keratin 10 gene exhibit acanthosis and hyperkeratosis as seen in the human disease. The degree of epidermal thickening is highly variable, offering a novel opportunity to investigate how epidermal homeostasis is modulated in keratin disorders by comparing epidermis from different body regions. We have performed bromodeoxyuridine labeling experiments and detected proliferation antigens by immunohistochemical means to compare proliferation in the epidermis of wild-type and heterozygous mice. These results have been compared with the expression of epidermal differentiation markers and of the “hyperproliferation associated” keratins K6 and K16. These experiments indicated that hyperproliferation is only partly responsible for the morphologic changes and that other mechanisms such as decreased desquamation are likely to be involved. Epidermal thickening is a phenomenon common to many genodermatoses but little is known about the underlying causes. We have recently created a mouse model for the human skin disease bullous congenital ichthyosiform erythroderma by gene targeting. Mice heterozygous for a truncated keratin 10 gene exhibit acanthosis and hyperkeratosis as seen in the human disease. The degree of epidermal thickening is highly variable, offering a novel opportunity to investigate how epidermal homeostasis is modulated in keratin disorders by comparing epidermis from different body regions. We have performed bromodeoxyuridine labeling experiments and detected proliferation antigens by immunohistochemical means to compare proliferation in the epidermis of wild-type and heterozygous mice. These results have been compared with the expression of epidermal differentiation markers and of the “hyperproliferation associated” keratins K6 and K16. These experiments indicated that hyperproliferation is only partly responsible for the morphologic changes and that other mechanisms such as decreased desquamation are likely to be involved. bullous congenital ichthyosiform erythroderma proliferating cell nuclear antigen Linkage and mutation analysis of epidermal keratin genes have shown that point mutations are the underlying cause of many genodermatoses (Corden and McLean, 1996Corden L.D. McLean W.H.I. Human Keratin diseases.Exp Dermatol. 1996; 5: 297-307Crossref PubMed Scopus (185) Google Scholar). In vitro and in vivo studies have indicated that these mutations are capable of altering keratin intermediate filament organization leading to reduced keratinocyte strength and epidermal fragility (Ishida-Yamamoto et al., 1992Ishida-Yamamoto A. McGrath J.A. Judge M.R. Leigh I.M. Lane E.B. Eady R.A.J. Selective involvement of keratins K1 and K10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma).J Invest Dermatol. 1992; 99: 19-26Crossref PubMed Scopus (114) Google Scholar; Lane et al., 1992Lane E.B. Rugg E.L. Navasaria H. Leigh I.M. Heagerty A.H.M. Ishida-Yamamoto A. Eady R.A.J. A mutation in the conserved helix termination peptide of Keratin 5 in hereditary skin blistering.Nature. 1992; 356: 244-246Crossref PubMed Scopus (322) Google Scholar; Letai et al., 1992Letai A. Coulombe P.A. Fuchs E. Do the ends justify the mean? Proline mutations at the end of the keratin coiled coil rod segment are more disruptive than internal mutations.J Cell Biol. 1992; 116: 1181-1195Crossref PubMed Scopus (133) Google Scholar; Rothnagel et al., 1992Rothnagel J.A. Dominey A.M. Dempsey L.D. et al.Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis.Science. 1992; 257: 1128-1130Crossref PubMed Scopus (304) Google Scholar; Wilson et al., 1992Wilson A.K. Coulombe P.A. Fuchs E. The roles of K5 and K14 head, tail and R/KLLEGE domains in keratin filament assembly in vitro.J Cell Biol. 1992; 119: 401-414Crossref PubMed Scopus (107) Google Scholar; Mclean et al., 1995Mclean W.H.I. Rugg E.L. Lunny D.P. et al.Keratin 16 and keratin 17 mutations cause pachyonychia congenita.Nat genet. 1995; 9: 273-278Crossref PubMed Scopus (279) Google Scholar); however, changes in the epidermis as a result of keratinocyte dysfunction are not clearly understood due to the lack of knowledge of epidermal tissue homeostasis. Epidermal thickening is a clinical feature common to the many keratin disorders, but there have been very limited investigations into changes in keratinocyte proliferation, differentiation, and keratin expression in these disorders. Early studies of epidermal proliferation in ichthyosiform dermatoses showed a 3-fold increase in the number of cells labeled with tritiated thymidine (Frost et al., 1966Frost P. Weinstein G.D. Van-Scott E.J. The ichthyosiform dermatoses. II. Autoradiographic studies of epidermal proliferation.J Invest Dermatol. 1966; 47: 561-567Abstract Full Text PDF PubMed Scopus (101) Google Scholar), and an increase in the number of mitoses in epidermolytic hyperkeratosis, more correctly called bullous congenital ichthyosiform erythroderma (BCIE) (Frost and Van-Scott, 1966Frost P. Van-Scott E.J. Ichthyosiform dermatoses. Classification based on anatomic and biometric observations.Arch Dermatol. 1966; 94: 113-126Crossref PubMed Scopus (154) Google Scholar); however, other workers were unable to observe an increase in mitotic rate in BCIE patient samples using colcemid (Fisher and Wells, 1968Fisher L. Wells G. The mitotic rate and duration in lesions of psoriasis and ichthyosis.Br J Dermatol. 1968; 80: 235-240Crossref PubMed Scopus (20) Google Scholar). More recently, proliferation was investigated in several skin disorders using antibodies to proliferating cell nuclear antigen (PCNA) and immunohistochemical staining of nucleolar organizer region-associated argyrophilic proteins (AgNOR) (Kanitakis et al., 1993Kanitakis J. Hoyo E. Chouvet B. Thivolet J. Faure M. Claudy A. Keratinocyte proliferation in epidermal keratinocyte disorders evaluated through PCNA/cyclin immunolabeling and AgNOR counting.Acta Derm Venereol. 1993; 73: 370-375PubMed Google Scholar). Their results for BCIE were not significant, however. The contribution of proliferation to the acanthosis and hyperkeratosis observed in BCIE is therefore unclear. The mouse model for BCIE generated by gene targeting (Porter et al., 1996Porter R.M. Leitgeb S. Melton D.W. Swensson O. Eady R.A.J. Magin T.M. Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.J Cell Biol. 1996; 132: 925-936Crossref PubMed Scopus (84) Google Scholar) offers an opportunity to investigate how the balance between cell proliferation and cell loss is altered in keratin disorders. These mice express a truncated K10 molecule consisting of the head, coil 1A, and 39 amino acids of coil 1B (Reichelt et al., 1997Reichelt J. Bauer C. Porter R.M. Lane E.B. Herzog V. Magin T.M. Out of balance: consequences of a partial keratin 10 knockout.J Cell Sci. 1997; 110: 2175-2186PubMed Google Scholar). Although the human disease is normally caused by a point mutation in K10 (or K1) (Cheng et al., 1992Cheng J. Syder A.J. Yu Q.-C. Letai A. Paller A.S. Fuchs E. The genetic basis of epidermolytic hyperkeratosis: Adisorder of differentiation-specific epidermal keratin genes.Cell. 1992; 70: 811-819Abstract Full Text PDF PubMed Scopus (272) Google Scholar; Chipev et al., 1992Chipev C.C. Korge B.P. Markova N. Bale S.J. DiGiovanna J.J. Compton J.G. Steinert P.M. A leucine to proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis.Cell. 1992; 70: 821-828Abstract Full Text PDF PubMed Scopus (244) Google Scholar; Rothnagel et al., 1992Rothnagel J.A. Dominey A.M. Dempsey L.D. et al.Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis.Science. 1992; 257: 1128-1130Crossref PubMed Scopus (304) Google Scholar), the mouse model exhibits many of the features characteristic of the human disease. Neonatal mice homozygous for the mutation exhibit severe skin fragility with cytolysis and marked acanthosis and hyperkeratosis. Keratin intermediate filament aggregation is apparent at the electron microscopy level. In heterozygotes, the skin fragility is not obvious at birth but extensive scaling is apparent on the ears, tail, and paws of adult animals due to hyperkeratosis. The particular attraction of this mouse model is that the epidermis from different regions of the mouse exhibits a range of morphologic changes from mild to severe acanthosis and hyperkeratosis. We have compared levels of BrdU incorporation and levels of two proliferation antigens (PCNA and Ki67) in wild-type and heterozygous mice by immunohistochemical techniques, and show that hyperproliferation is unlikely to be wholly responsible for the acanthosis/hyperkeratosis observed. Changes in keratin expression that have been associated with hyperproliferation are the inductions of K6, K16, and K17 (Weiss et al., 1984Weiss R. Eichner R. Sun T.T. Monoclonal antibody analysis of keratin expression in epidermal diseases: A 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes.J Cell Biol. 1984; 98: 1397-1406Crossref PubMed Scopus (435) Google Scholar; Tyner and Fuchs, 1986Tyner A.L. Fuchs E. Evidence for posttranscriptional regulation of the keratins expressed during hyperproliferation and malignant transformation in human epidermis.J Cell Biol. 1986; 103: 1945-1955Crossref PubMed Scopus (122) Google Scholar; Mansbridge and Knapp, 1987Mansbridge J.N. Knapp A.M. Changes in keratinocyte maturation during wound healing.J Invest Dermatol. 1987; 89: 253-263Abstract Full Text PDF PubMed Google Scholar; Stoler et al., 1988Stoler A. Kopan R. Duvic M. Fuchs E. Use of mono-specific antisera and cRNA probes to localise the major changes in keratin expression during normal and abnormal epidermal differentiation.J Cell Biol. 1988; 107: 427-446Crossref PubMed Scopus (285) Google Scholar; Kopan and Fuchs, 1989Kopan R. Fuchs E. The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells.J Cell Biol. 1989; 109: 295-307Crossref PubMed Scopus (116) Google Scholar). Keratin expression studies of our mouse model suggest that all three of these keratins are upregulated in BCIE (Porter et al., 1996Porter R.M. Leitgeb S. Melton D.W. Swensson O. Eady R.A.J. Magin T.M. Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.J Cell Biol. 1996; 132: 925-936Crossref PubMed Scopus (84) Google Scholar; this study). Upregulation of K16 has been observed in patients with the disease (Ishida-Yamamoto et al., 1992Ishida-Yamamoto A. McGrath J.A. Judge M.R. Leigh I.M. Lane E.B. Eady R.A.J. Selective involvement of keratins K1 and K10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma).J Invest Dermatol. 1992; 99: 19-26Crossref PubMed Scopus (114) Google Scholar) and upregulation of K6 has been reported in two conventional transgenic mouse models for BCIE (Fuchs et al., 1992Fuchs E. Esteves R.A. Coulombe P.A. Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis.Proc Natl Acad Sci (USA). 1992; 89: 6906-6910Crossref PubMed Scopus (159) Google Scholar; Bickenbach et al., 1996Bickenbach J.R. Longley M.A. Bundman D.S. Dominey A.M. Bowden P.E. Rothnagel J.A. Roop D.R. A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis.Differentiation. 1996; 61: 129-139Crossref PubMed Google Scholar); however, there is evidence to suggest that expression of K6 and K16 is not directly linked to hyperproliferation, but is indirectly due to altered differentiation (Schermer et al., 1989Schermer A. Jester J.V. Hardy C. Milano D. Sun T.-T. Transient synthesis of K6 and K16 keratins in regenerating rabbit corneal epithelium: keratin markers for an alternative pathway of keratinocyte differentiation.Differentiation. 1989; 42: 103-110Crossref PubMed Scopus (91) Google Scholar). To distinguish between these we compared the expression of K6 and K16 expression to proliferation associated antigens and to K2e, a differentiation associated keratin. Epidermis from 6 mo old wild-type and heterozygous adult mice was fixed overnight in 4% formaldehyde before embedding in paraffin. Sections (5 μm) for morphologic analyses were stained with hematoxylin and eosin and mounted in Corbit-Balsam (Hecht, Hamburg, Germany). Epidermal thickness was calculated from photographs taken at regular intervals (≈100 basal cells) of hematoxylin and eosin stained sections or of sections used for PCNA analysis. PCNA was detected in paraffin sections using the rabbit polyclonal anti-serum 3009 (a gift from S.M. Picksley, University of Dundee, Dundee, U.K.) at 1:500 dilution followed by biotinylated goat anti-rabbit (Sigma, Poole, U.K.) at 1:100 and avidin conjugated to horseradish peroxidase (Vector, Peterborough, U.K.) at 1:500. All dilutions were with phosphate buffered saline containing horse serum (1:60). Color development was carried out using a peroxidase substrate kit (Vector) following instructions provided. Sections were counterstained in 0.5 μg DAPI per ml (which detected negative basal cells only) and sections were mounted in 10% Mowiol/2.5% DABCO (Sigma). Positive nuclei were counted per 100 basal cells. For immunofluorescence studies, samples of epidermis were embedded in OCT compound (BDH, Poole, U.K.) and were frozen in liquid nitrogen. Frozen sections (5 μm) were fixed with acetone or acetone:methanol (1:1) for 10 min at –20°C. Primary antibodies were diluted in PBS at the following dilutions: mouse monoclonal Ki67-MM1 (Novacastra, Newcastle-upon-Tyne, U.K.) 1:50, rabbit polyclonal anti-serum IMP2e (K2e) 1Smith LT, Underwood RA, McLean WHI: Ontogeny and regional variability of keratin 2e (K2e) in developing fetal skin; a unique spatial and temporal pattern of keratin expresion in development. Submitted for publication. 1:100, rat monoclonals MA6 and MB1.2 (α6β1 and β1 integrin; a gift from Bosco Chan, The John P. Robarts Research Institute, London, Ontario) undiluted, rat monoclonals 346–11 A (β4 integrin; Pharmingen, San Diego, CA) 1:200, and GoH3 (α6 integrin; Serotec, Oxford, U.K.) 1:50. Incubation was for 1 h at room temperature except Ki67-MM1 that was incubated overnight at 4°C. Secondary antibodies were fluoroscein isothiocyanate conjugated anti-rabbit IgG (Dianova, Hamburg, Germany), anti-mouse IgG (Sigma) or anti-rat IgG (Sigma), and Texas red conjugated anti-rabbit (Amersham, Bucks, U.K.). Slides were mounted in 10% Mowiol/2.5% DABCO (Sigma). Immunohistochemistry using mouse monoclonals LHK6 A (K6; a gift from Irene Leigh, The Royal London School of Medicine and Dentistry, London, U.K.) and LLO25 (K16) (Wilson et al., 1994Wilson C.L. Dean D. Lane E.B. Dawber R.P.R. Leigh I.M. Keratinocyte differentiation in psoriatic scalp: morphology and expression of epithelial keratins.Br J Dermatol. 1994; 131: 191-200Crossref PubMed Scopus (50) Google Scholar) was carried out using a modified method of indirect immunodetection (Hierck et al., 1994Hierck B.P. Iperen L.V. Gittenbergerdegroot A.C. Poelmann R.E. Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies.J Histochem Cytochem. 1994; 42: 1499-1502Crossref Scopus (53) Google Scholar) that reduced background caused by the fluoroscein isothiocyanate-conjugated anti-mouse secondary antibody. The anti-mouse fluoroscein isothiocyanate secondary was diluted 1:200 in undiluted keratin antibody and preincubated overnight at 4°C. Mouse serum was then added to bind excess secondary antibody for 2 h before immunohistochemistry. Wild-type and heterozygous animals, 6 mo old, were injected intraperitoneally with BrdU (1 ml per 100 g body weight) (Cell proliferation kit, Amersham, Braunschweig, Germany). Biopsies of ear and back skin were taken from one wild-type and one heterozygous animal after 3 and 8 h and tissue from various body sites was also taken when the animals were sacrificed at 24 h. Tissue was fixed for 12 h in 4% formalin before embedding in paraffin. Sections were immunostained according to instructions in the kit, counterstained with Kernechtrot, and mounted with Corbit-Balsam (Hecht, Hamburg, Germany). The number of positive nuclei were counted for 100 basal cells and an average obtained. Statistical analysis was carried out using the Student t test. The morphology of the epidermis from various areas of the mouse was examined from both wild-type and heterozygous 6 mo old littermates. All body sites showed an increase in thickness of the living and dead layers of the epidermis; however, the morphologic changes, including the degree of acanthosis and hyperkeratosis, were clearly dependent on the region from which the epidermis had been taken. The average increase in thickness of both the living layer of the epidermis and the stratum corneum from the different sites is presented in Table 1. Representative photographs of wild-type and heterozygous epidermis are shown in Figure 1 for comparison.Table 1Increase in skin thickness of the various regionsaFigures represent mean values ± SD.SampleEpidermal thickness below stratum corneum (μm)Average increase in thickness ratio WT: HETEpidermal thickness of stratum corneum (μm)Average increase in thickness ratio WT:HETWTHETWTHETBack10.8 ± 5.030.2 ± 13.81:2.82.9 ± 1.210.6 ± 8.61:3.7(p < 0.001)(p < 0.05)Ear9.1 ± 2.634.1 ± 12.61:3.74.8 ± 1.422.4 ± 11.11:4.7(p < 0.001)(p < 0.001)Foot pad71.8 ± 10.5124.6 ± 26.11:1.7ND(p < 0.001)Tail46.5 ± 2.970.4 ± 11.81:1.5ND(p < 0.001)Snout10.6 ± 0.238.7 ± 0.41:3.78.6 ± 0.232.2 ± 7.61:3.7(p < 0.001)(p < 0.001)Oesophagus28.4 ± 3.745.7 ± 10.71:1.610.7 ± 2.918.5 ± 7.61:1.6(p < 0.001)(p < 0.001)a Figures represent mean values ± SD. Open table in a new tab Wild-type adult mice have a very thin back epidermis of only 2–3 cells thick Figure 1a. Heterozygous mice show mild acanthosis and hyperkeratosis with an approximately 2–3-fold increase in the number of cell layers Figure 1b. Occasionally, regions of the back showed dramatic acanthosis (data not shown). Tail skin is thicker than back skin, usually 5–6 layers thick, and showed a 1.5-fold increase in epidermis when the truncated K10 was expressed (see Table 1). The epidermis of the ear is also very thin in the wild-type mouse, with usually only a single layer of suprabasal cells Figure 1c. In contrast, suprabasal epidermis of heterozygote ear was at least 4–5 layers thick Figure 1d and sometimes as much as 10 layers thick. The stratum corneum was comparable in thickness with the living layers and was easily lost from the epidermal surface, consistent with the extensive scale observed on the ears of heterozygous mice. Snout epidermis of heterozygote mice showed acanthosis of both the stratum spinosum and the stratum granulosum and hyperkeratosis of the stratum corneum Figure 1h. Paw (foot pad) epidermis, in wild-type animals, is distinctive in several respects. It has an unusually thick suprabasal compartment (10–12 cells) and the stratum corneum is as thick as the living layers. The cell layers of the stratum corneum are regular and tightly packed Figure 1e. In the heterozygotes, the cells of the suprabasal layer are larger and there are twice as many cell layers as in the wild-type mice. The cell layers of the stratum corneum are densely packed in the inner layers but the integrity of the outer stratum corneum is severely reduced, leading to loss of organization in the outer layers Figure 1f. Hence the extensive scale on the paws of the heterozygotes. Due to the fact that K10 is expressed in the esophagus and forestomach of the mouse (but not humans) (Schweizer et al., 1988Schweizer J. Rentrop M. Nischt R. Kinjo M. Winter H. The intermediate filament system of the keratinising mouse forestomach epithelium: coexpression of keratins of internal squamous epithelia and of epidermal keratins in differentiating cells.Cell Tiss Res. 1988; 253: 221-229Crossref PubMed Scopus (25) Google Scholar), we extended our study to the epithelia of these regions. Acanthosis and hyperkeratosis are clearly present in the esophagus of affected animals (Table 1; Figure 2a, b, but the forestomach shows no morphologic changes Figure 2c, d. To examine the possible contribution of proliferation to the morphologic changes observed in the different regions of the epidermis in the mouse model, we have looked at the expression of cell cycle associated antigens Ki67 and PCNA and the level of incorporation of BrdU. Ki67 staining was restricted to the basal and epibasal cell layers of the epidermis in all sites examined. In ear epidermis, very few basal cells stained with the Ki67 antibody in wild-type animals Figure 3a, but nuclei were frequently stained in the heterozygote Figure 3b. An increase in the number of labeled nuclei was also observed in the snout epidermis of animals expressing the truncated K10 (Table 2). BrdU label incorporation was detected in epidermis taken from animals at 3, 8, and 24 h after injection of the label. Even after 24 h, BrdU labeled nuclei were confined to the basal layer in all sites. In wild-type mice very few nuclei were labeled in the ear epidermis (0.5%–3.4% of basal cells) but more labeling in heterozygote ear indicated a significantly larger number of cycling cells (Table 3). Significant increases in labeling were also observed in the snout and eyelid at 24 h (see Table 3). Very little difference in labeling indices was observed for the other body sites (see Table 3).Table 2Number of PCNA and Ki67 labeled nucleiaFigures represent mean values ± SD.SamplePCNA/100 basal cellsKi67 100 basal cellsWTHETWTHETBack35.8 ± 11.230.8 ± 40.73.8 ± 2.06.1 ± 5.6Ear25.0 ± 8.0210.5 ± 95.01.9 ± 1.5(p > 0.05)25.6 ± 6.2Foot pad156.3 ± 66.2(p < 0.001)121.5 ± 33.932.7 ± 14.6(p < 0.001)24.4 ± 11.5Tail114.5 ± 44.9101 ± 22.115.0 ± 9.6(p > 0.05)19.5 ± 7.5SnoutND6.1 ± 6.5(p > 0.1)27.3 ± 12.7(p < 0.05)a Figures represent mean values ± SD. Open table in a new tab Table 3BrdU labeled nucleiaFigures represent mean values ± SD.Sample3 h8 h24 hWTHETWTHETWTHETBack5.5 ± 1.45.2 ± 2.94.2 ± 3.714.8 ± 7.52.75 ± 3.50(p > 0.1)(p < 0.05)(p < 0.05)Ear3.4 ± 1.712.3 ± 4.00.5 ± 0.3726.3 ± 11.50.5 ± 0.315.5 ± 2.4(p < 0.001)(p < 0.001)(p < 0.001)Foot padNDND4.5 ± 3.515.0 ± 7.1(p > 0.05)SnoutNDND0.44 ± 0.327.0 ± 1(p < 0.001)EyelidNDND6.5 ± 2.522.5 ± 8.5(p < 0.01)OesophagusNDND13.0 ± 3.912.0 ± 4.4(p < 0.1)StomachNDND26.5 ± 5.127.0 ± 7.4(p < 0.1)a Figures represent mean values ± SD. Open table in a new tab PCNA staining was observed in basal cells and many suprabasal cells. Far more cells were labeled than with either of the other two techniques. No significant differences were observed between wild-type and heterozygous mice in the back, tail, and foot pad, but the PCNA positive nuclei of ear epidermis were greatly increased (Figure 3e, f; Table 2). Keratin expression is well documented as varying with changes in epithelial differentiation, including wound healing (Fuchs and Green, 1980Fuchs E. Green H. Changes in keratin gene expression during terminal differentiation.Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (779) Google Scholar; Weiss et al., 1984Weiss R. Eichner R. Sun T.T. Monoclonal antibody analysis of keratin expression in epidermal diseases: A 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes.J Cell Biol. 1984; 98: 1397-1406Crossref PubMed Scopus (435) Google Scholar; Stoler et al., 1988Stoler A. Kopan R. Duvic M. Fuchs E. Use of mono-specific antisera and cRNA probes to localise the major changes in keratin expression during normal and abnormal epidermal differentiation.J Cell Biol. 1988; 107: 427-446Crossref PubMed Scopus (285) Google Scholar). Previously, we demonstrated that the hyperproliferation associated keratins K16 and K17 are upregulated in neonatal heterozygous and homozygous mice (Porter et al., 1996Porter R.M. Leitgeb S. Melton D.W. Swensson O. Eady R.A.J. Magin T.M. Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.J Cell Biol. 1996; 132: 925-936Crossref PubMed Scopus (84) Google Scholar). Using the antibody LHK6a, we are now able to compare the localization of K6 and K16 expression in the epidermis of heterozygous adult mice. In adult wild-type mice, K6/K16 staining was confined to the hair follicles in wild-type ear, snout, and eyelid; however, superficial staining was often present in the suprabasal interfollicular epidermis of both tail and back skin. This additional staining in wild-type mice might be due to recent injury caused by scratching for example. In heterozygous samples we observe an induction of both K6 and K16, but to variable extents. In heterozygous tail samples, there was a strong induction of K6 and K16 in the suprabasal layer Figure 4a, b. Interfollicular expression of these two keratins was induced in the ear of heterozygous mice, but was restricted to certain regions Figure 4d, e. In heterozygote snout epidermis, suprabasal K6 expression was confined to regions close to and including hair follicles, but K16 was induced extensively throughout the suprabasal layer Figure 4f, g. Regions where K16 was expressed in the absence of K6 were also observed in the ear. In situations where K6/K16 are expressed there is a reduction in expression of the normal differentiation-specific keratins (Fuchs and Green, 1980Fuchs E. Green H. Changes in keratin gene expression during terminal differentiation.Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (779) Google Scholar; Weiss et al., 1984Weiss R. Eichner R. Sun T.T. Monoclonal antibody analysis of keratin expression in epidermal diseases: A 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes.J Cell Biol. 1984; 98: 1397-1406Crossref PubMed Scopus (435) Google Scholar; Stoler et al., 1988Stoler A. Kopan R. Duvic M. Fuchs E. Use of mono-specific antisera and cRNA probes to localise the major changes in keratin expression during normal and abnormal epidermal differentiation.J Cell Biol. 1988; 107: 427-446Crossref PubMed Scopus (285) Google Scholar; Schermer et al., 1989Schermer A. Jester J.V. Hardy C. Milano D. Sun T.-T. Transient synthesis of K6 and K16 keratins in regenerating rabbit corneal epithelium: keratin markers for an alternative pathway of keratinocyte differentiation.Differentiation. 1989; 42: 103-110Crossref PubMed Scopus (91) Google Scholar). We therefore looked at K2e expression to see if there was any downregulation of this differentiation associated keratin. In wild-type animals, K2e staining was observed in paw, tail, and ear as described previously (Rentrop et al., 1987Rentrop M. Nischt R. Knapp B. Schweizer J. Winter H. An unusual type II 70 kilodalton keratin protein of mouse epidermis exhibiting postnatal body site specificity and sensitivity to hyperproliferation.Differentiation. 1987; 34: 189-200Crossref PubMed Scopus (20) Google Scholar; Schweizer, 1993Schweizer J. Murine epidermal keratins.in: Darmon M.B.M. Molecular Biology of the Skin: The Keratinocyte. Academic Press, San Diego1993: 33-77Crossref Google Scholar). In heterozygous adult animals, K2e expression persisted and using double immunolabeling we were able to observe coexpression of K2e and K6 or K16 (e.g., see Figure 4b, c. Downregulation of β1 integrin is believed to be an important event in the commitment of keratinocytes to terminal differentiation (Hotchin et al., 1993Hotchin N.A. Kovach N.L. Watt F.M. Functional down-regulation of alpha 5 beta 1 integrin in keratinocytes is reversible but commitment to terminal differentiation is not.J Cell Sci. 1993; 106: 1131-1138PubMed Google Scholar). Also, integrins normally expressed in the basal layer of the epidermis have been observed in the suprabasal layer in wound healing and in psoriasis (Hertle et al., 1992Hertle M.D. Kubler M.-D. Leigh I.M. Watt F.M. Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.J Clin Invest. 1992; 89: 1892-1901Crossref PubMed Scopus (207) Google Scholar). We were therefore interested to see if changes in integrin expression could be involved in BCIE. Immunohistochemistry of integrins was carried out on frozen sections of neonatal wild-type and homozygous back skin, as well as of adult epidermis from both wild-type and heterozygous animals. Antibodies to β4 and α6 integrins detected integrins along the dermal–epidermal junction in all sections examined. Antibodies to β1 and α6β1 integrins stained the basal and lateral cell membranes of basal cells. Some epibasal staining was also occasionally seen but no differences were observed between genotypes (data not shown). We have shown that keratinocyte fragility in a model for BCIE leads to a range of morphologic changes from mild to severe acanthosis and hyperkeratosis. Three commonly used immunohistochemical techniques (detection of the cell cycle associated antigens Ki67 and PCNA and BrdU incorporation) have been used to detect changes occurring in proliferation that might lead to the epidermal thickening. A significant increase in the number of cycling cells in the ear, snout, and eyelid was observed. All other regions of the epidermis (tail, foot pad, and back a" @default.
• W2080596058 created "2016-06-24" @default.
• W2080596058 creator A5001554153 @default.
• W2080596058 creator A5016586166 @default.
• W2080596058 creator A5022377757 @default.
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• W2080596058 creator A5066560261 @default.
• W2080596058 date "1998-06-01" @default.
• W2080596058 modified "2023-09-27" @default.
• W2080596058 title "The Relationship Between Hyperproliferation and Epidermal Thickening in a Mouse Model for BCIE" @default.
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