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Background: Solar lentigines (SLs) are macular hyperpigmented lesions associated with sun exposure and age. Histopathologically, SLs are defined by a hyperpigmented basal layer and elongated rete ridges. The molecular mechanisms involved in the formation and the development of SLs are not completely understood. Our earlier data show that keratinocyte growth factor (KGF) induces hyperpigmentary lesions with histological resemblance to SLs. Objective: To investigate the association of KGF/KGF receptor (KGFR) and other pigmentary genes with the progression of SL development. To better understand the possible role of KGF in the pathology of SLs. Methods: Archived human skin biopsies (24 SLs and 14 healthy skins) were studied using immunohistochemistry for KGF/KGFR, proliferation marker Ki67, stem cell marker keratin-15 (K15), tyrosinase (TYR), stem cell factor (SCF), and protease-activated receptor-2 (PAR-2). Results: An increase in TYR-positive cells and expression was found throughout SL progression, as compared to normal skin. The levels of KGF, KGFR, SCF, Ki67 and PAR-2 varied during SL progression. Ki67, K15 and KGF/KGFR were significantly upregulated at early-mid SL stages. The latest-stage SLs expressed the lowest levels of KGF, KGFR, SCF, Ki67 and PAR-2. Conclusions: The upregulation of KGF/KGFR might induce the formation of rete ridges and hyperpigmentation. The reduced levels of all examined proteins (except TYR and K15) suggest a possible inactive status (dormancy or quiescence) of advanced lesions.

Often the distinction of primary adnexal carcinoma from metastatic adenocarcinoma to skin from breast, lung, and other sites can be a diagnostic dilemma. Current markers purportedly of utility as diagnostic adjuncts include p63 and D2-40; however, their expression has been demonstrated in 11-22% and 5% of metastatic cutaneous metastases, respectively. Both cytokeratin (CK) 15 and nestin have been reported as follicular stem cell markers. We performed CK15 and nestin, as well as previously reported stains (such as p63, D2-40, and calretinin) on 113 cases (59 primary adnexal carcinomas and 54 cutaneous metastases). Expressions of p63, CK15, nestin, D2-40, and calretinin were observed in 91, 40, 37, 44, and 14% of primary adnexal carcinoma, respectively, and in 8, 2, 8, 4, and 10% of cutaneous metastases, respectively. p63 appeared to be the most sensitive marker (with a sensitivity of 91%) in detecting primary adnexal carcinomas. CK15 appeared to be the most specific marker with a specificity of 98%. Using chi(2) analysis, statistically significant P-values (

Our laboratory discovered nestin-expressing hair follicle stem cells and demonstrated their pluripotency. We have shown that nestin-positive and K15-negative multipotent hair follicle stem cells are located above the hair follicle bulge, and we termed these cells hair follicle pluripotent stem (hfPS) cells. We have previously shown that hair follicle stem cells can regenerate peripheral nerve and spinal cord. In the present study, we describe the embryonic development of the hair follicle stem cell area (hfPSCA), which is located above the bulge and below the sebaceous glands in the adult mouse. At embryonic day 16.5 (E16.5) of nestindriven GFP (ND-GFP) transgenic mice, which express nestin in hfPS cells, the ND-GFP hair follicle stem cells are located in mesenchymal condensates. At postnatal day 0 (P0), the ND-GFP-expressing cells are migrating to the upper part of the hair follicle from the dermal papilla. At P3, keratin 15 (K15)-positive cells, derived from ND-GFP dermal papilla cells, are located in the outer-root sheath and basal layer of the epidermis. By P10, the ND-GFP have formed the K15-positive outer-root sheath as well as the ND-GFP hfPSA. These results suggest that ND-GFP hfPS cells in the dermal papilla form nestin-expressing hair follicle stem cells in the first hair cycle. These observations provide new insight into the origins of hfPS cells and the hfPSCA.

Here we demonstrate that physiological concentrations of the thyroid hormones T3 and T4 enhance the KERATIN 15 promoter activity and expression in epithelial stem cells of adult human scalp hair follicles in situ and in vitro. Additionally, T3 and T4 stimulate expression of the immuno-inhibitory surface molecule CD200. Subsequently, T3 and T4 induce apoptosis and differentiation and inhibit clonal growth of these progenitor cells in vitro. These data suggest that human hair follicle bulge-derived epithelial stem cells underlie profound, previously unknown hormonal regulation by thyroid hormones, and show that primary human keratin 15-GFP+ progenitor cells can be exploited to further elucidate fundamental endocrine controls of human epithelial stem cells.

For most types of cancers, the cell at the origin of tumour initiation is still unknown. Here, we used mouse genetics to identify cells at the origin of basal cell carcinoma (BCC), which is one of the most frequently occurring types of cancer in humans, and can result from the activation of the Hedgehog signalling pathway. Using mice conditionally expressing constitutively active Smoothened mutant (SmoM2), we activated Hedgehog signalling in different cellular compartments of the skin epidermis and determined in which compartments Hedgehog activation induces BCC formation. Activation of SmoM2 in hair follicle bulge stem cells and their transient amplifying progenies did not induce cancer formation, demonstrating that BCC does not originate from bulge stem cells, as previously thought. Using clonal analysis, we found that BCC arises from long-term resident progenitor cells of the interfollicular epidermis and the upper infundibulum. Our studies uncover the cells at the origin of BCC in mice and demonstrate that expression of differentiation markers in tumour cells is not necessarily predictive of the cancer initiating cells.

In this article we show that cloning of the human K15 promoter before a green fluorescence protein (GFP)/geneticin-resistance cassette and transfection of microdissected, organ-cultured adult human scalp hair follicles generates specific K15 promoter–driven GFP expression in their stem cell–rich bulge region. K15-GFP+ cells can be visualized in situ by GFP fluorescence and 2-photon laser scanning microscopy. Vital K15-GFP+ progenitor cells can then be selected by using the criteria of their green fluorescence, adhesion to collagen type IV and fibronectin, and geneticin resistance. Propagated K15-GFP+ cells express epithelial progenitor markers, show the expected differential gene expression profile of human bulge epithelium, and form holoclones. This application of nonretroviral, K15 promoter–driven, GFP labeling to adult human hair follicles facilitates the characterization and manipulation of human epithelial stem cells, both in situ and in vitro, and should be transferable to other complex human tissues. Disclosure of potential conflicts of interest is found at the end of this article.

The role of EphB2 and EphB3 in the organization of thymic epithelial cells has been studied in EphB-deficient fetal thymus lobes grafted under the kidney capsule of WT mice. The deficient lobes, as compared with WT ones, showed altered distribution of medullary areas, shortening of medullary epithelial cell processes and presence of K5(-)K8(-) areas. EphB2 and EphB3 expressed on thymic epithelial cells play an autonomous role in their organization. The relevance of Eph/ephrinB forward and reverse signals for this process was evaluated in grafted fetal thymus lobes from mice expressing a truncated EphB2 receptor capable of activating reverse, but not forward, signaling. These deficient lobes showed important alterations of the thymic epithelial organization as compared with the grafted WT lobes, but a less severe phenotype than the grafted EphB2-deficient thymus lobes, which confirms the relevance of EphB2 forward signal for the thymic epithelial organization but, also, a role of the reverse signaling in determining the final epithelial phenotype.

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Purpose To report the efficacy of human bone marrow-derived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. Methods Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in alpha-modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithelial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air-liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochemistry against K3 and K4. Results MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63alpha, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. Conclusions MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

Contains fulltext : 81146.pdf (Publisher’s version ) (Closed access) BACKGROUND: Immunohistochemistry is an important tool in dermatology but is limited. Certain antigens can only be preserved in formalin-fixed paraffin-embedded sections, while others can only be detected on frozen sections, resulting in situations where two biopsies are needed. We aimed to develop a technique for universal detection of different antigens out of just one biopsy specimen. METHODS: Single biopsies were obtained from lesional skin of patients with psoriasis. Standard sample procedures for frozen and paraffin-embedded sections were used. To convert frozen tissue into paraffin-embedded sections, the biopsy specimen was disposed of the embedding medium and subsequently fixed in 10% neutral buffered formalin. We applied various antigen retrieval techniques with alkaline solutions. The differential expression of keratin 10, keratin 15, CD3, CD26 and human beta defensin-2 (HBD-2) was examined using immunohistochemical staining. RESULTS: We showed that keratin 10 and 15 can be stained on both frozen and paraffin-embedded sections. Staining of paraffin-embedded sections required unmasking with trypsin and Tris-buffered saline Tween solution, respectively. CD3 and CD26 can only be detected on frozen sections, while HBD-2 can only be detected on paraffin-embedded sections. CONCLUSION: We have described a straightforward technique that gives us the opportunity to use just one biopsy specimen to obtain frozen sections as well as paraffin-embedded sections.

Telomeres are heterochromatic structures at chromosome ends essential for chromosomal stability. Telomere shortening and the accumulation of dysfunctional telomeres are associated with organismal aging. Using telomerase-deficient TRF2-overexpressing mice ( K5TRF2 / Terc −/− ) as a model for accelerated aging, we show that telomere shortening is paralleled by a gradual deregulation of the mammalian transcriptome leading to cumulative changes in a defined set of genes, including up-regulation of the mTOR and Akt survival pathways and down-regulation of cell cycle and DNA repair pathways. Increased DNA damage from dysfunctional telomeres leads to reduced deposition of H3K27me3 onto the inactive X chromosome (Xi), impaired association of the Xi with telomeric transcript accumulations (Tacs), and reactivation of an X chromosome-linked K5TRF2 transgene that is subjected to X-chromosome inactivation in female mice with sufficiently long telomeres. Exogenously induced DNA damage also disrupts Xi-Tacs, suggesting DNA damage at the origin of these alterations. Collectively, these findings suggest that critically short telomeres activate a persistent DNA damage response that alters gene expression programs in a nonstochastic manner toward cell cycle arrest and activation of survival pathways, as well as impacts the maintenance of epigenetic memory and nuclear organization, thereby contributing to organismal aging.

Summary In a recent Nature Cell Biology paper, Taddei et al. (2008) reveal that deletion of β1 integrin from K5-expressing mammary epithelial basal cells specifically attenuates ductal stem cell activity, without dramatically altering the basal cell layer, or morphogenesis overall.

While the pathogenesis of most scarring alopecias is poorly understood, one recent study indicates destruction of follicular stem cells as a possible mechanism in lichen planopilaris, the prototypic scarring alopecia. The aim of this cross-sectional study was to ascertain the target of inflammation and to more precisely characterize the inflammatory infiltrate in various stages of primary scarring alopecias. Immunohistochemical studies were performed using a panel of antibodies that included anti-cytokeratin 15, an antibody that specifically targets follicular bulge stem cells and CD4, CD8, CD1a and human leukocyte antigen-DR to characterize the inflammatory infiltrate. Our data showing absence of follicular bulge stem cells in cases with moderate to heavy inflammation suggest involvement of the bulge region in ‘early’ active stages of primary scarring alopecia. The paucity of CD8+ T cells in the inflammatory infiltrate in the majority of these cases argues against a cell-mediated cytotoxic destruction of follicular bulge stem cells. Preservation of CK15+ cells in ‘late’ fibrotic stages of primary scarring alopecia further supports this and implies that the irreversible loss of hair follicles, the sine qua non of primary scarring alopecia, is not necessarily a consequence of T cell-mediated destruction of follicular bulge stem cells.

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34; a middle part that was CD34-positive and K15-negative; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.

Mutations in the transcription factor Foxn1 cause the nude phenotype in mice, which is characterized by a lack of visible hair. New work by Weiner et al. (2007) in this issue of Cell now shows that Foxn1 also contributes to hair color by marking which cells are to receive pigment from melanocytes.

The mineralocorticoid receptor (MR) is a transcription factor of the nuclear receptor family, activation of which by aldosterone enhances salt reabsorption in the kidney. The MR is also expressed in nonclassical aldosterone target cells (brain, heart, and skin), in which its functions are incompletely understood. To explore the functional importance of MR in mammalian skin, we have generated a conditional doxycycline-inducible model of MR overexpression, resulting in double-transgenic (DT) mice [keratin 5-tTa/tetO-human MR (hMR)], targeting the human MR specifically to keratinocytes of the epidermis and hair follicle (HF). Expression of hMR throughout gestation resulted in early postnatal death that could be prevented by antagonizing MR signaling. DT mice exhibited premature epidermal barrier formation at embryonic day 16.5, reduced HF density and epidermal atrophy, increased keratinocyte apoptosis at embryonic day 18.5, and premature eye opening. When hMR expression was initiated after birth to overcome mortality, DT mice developed progressive alopecia and HF cysts, starting 4 months after hMR induction, preceded by dystrophy and cycling abnormalities of pelage HF. In contrast, interfollicular epidermis, vibrissae, and footpad sweat glands in DT mice were normal. This new mouse model reveals novel biological roles of MR signaling and offers an instructive tool for dissecting nonclassical functions of MR signaling in epidermal, hair follicle, and ocular physiology.

We reexamined 11 cases of trichoblastoma, and two cases of trichoblastoma with basal cell carcinoma (BCC)-like foci were found. In these two trichoblastomas with BCC-like foci, the BCC-like foci were often localized in peripheral or deep areas of lesions extending out of the fibrocytic stroma. Immunohistochemistry was performed in five conventional trichoblastomas and in two trichoblastomas with BCC-like foci, using antibodies against CK20 and CK15. No CK20-positive Merkel cells and no expression of CK15 were seen in any neoplastic aggregations of the two trichoblastomas with BCC-like foci. In contrast, increased numbers of Merkel cells and positive staining for CK15 were observed in all five trichoblastomas without BCC-like foci. The five trichoblastomas without BCC-like foci included two trichoblastomas with a popped out or shelled out appearance, which characteristically had a thick fibrous capsule surrounding the fibrotic stroma, demonstrating numerous Merkel cells in the aggregations. Some trichoblastomas may undergo mutations, resulting in the development of foci of BCC and in the loss of the expression of CK15 as well as the disappearance of Merkel cells.

Mice with conditional gene deletions have been extremely valuable in allowing investigators to study the genes of interest in a tissue-specific manner. The Cre-loxP recombination system provides a powerful tool to produce targeted rearrangements of particular genes. The keratin 5-Cre recombinase (K5Cre) transgenic mouse line has been used to generate skin specific gene deletions. We found that the K5Cre mice display a unique phenotype when bred to homozygosity. The K5Cre(+/+) mice have a wavy hair coat and curly whiskers. Histologically, the hair follicles appear disoriented. Over time, the K5Cre(+/+) mice develop patches of alopecia. These mice are also runted when compared to wild-type controls. Fostering the K5Cre(+/+) pups to wild-type mothers results in normal weight gain, suggesting a maternal defect in milk production. When the K5Cre(+/+) mammary glands were examined, we not only found a significant decrease in the number of mammary branches in the virgin females, but also a greater number of quiescent alveoli units in the lactating glands. When the K5Cre(+/+) mice were bred to v-Ha-ras (Tg . AC) transgenic mice, the resulting Tg . AC(+/-) K5Cre(+/+) offspring were utilized in a chemically induced skin carcinogenesis model. The mice were treated with 2.5 microg of 12-O-tetradecanoylphorbol-13-acetate (TPA) weekly for 10 wk. No difference was observed in the time to onset of papilloma formation, the number of papillomas and the average papilloma volume between the Tg . AC(+/-) K5Cre(+/+) mice and their corresponding controls. Surprisingly, however, the K5Cre(+/+) papillomas displayed an accelerated tendency to malignant progression; in addition, the frequency of malignant transformation of the papillomas is significantly enhanced. Although the K5Cre(+/+) mice resemble waved-1 and -2 mutants, the molecular basis for the K5Cre(+/+) phenotype is probably different. In conclusion, we discovered a unique phenotype associated with the K5Cre(+/+) transgenic line.

Recent works demonstrated the presence of a multipotent epithelial cell population in the bulge region of adult human hair follicles. These cells can be cultured in vitro, thus leading to the preparation of dermal-epidermal substitutes which are applicable in the treatment of burns and ulcers. We evaluated the main marker expression in cells obtained from stripped human hair follicles. A pool of hair follicles were incubated at 37 degrees C and 5% CO(2) in a growth medium. The cells were then labelled with antibodies (anti-CD34, anti-CD38, anti-CD45, anti-CD90, anti-CD133, anti-CD146) and analysed by cytometry. We also used hair follicles for immunohistochemical studies, employing antibodies such as CD34, Actin Smooth Muscle, Filaggrin, Desmin, Vimentin, Glial Fibrillary Acidic Protein, Ki-67, PanCytokeratin, CK15, CK19. The cytometry results revealed that a part of bulge cells were CD34+ (1-2%). CD34+ population comprises both large, CD45-, CD133-, CD146- cells and small, CD45+, CD133+, CD146+ cells. Thus, a part of CD34+ cells present a mature endothelial marker (CD146). An expression of the proliferation marker Ki-67 and the stem cell marker CD34 is present in the follicle bulge region. In conclusion, we observed that the stripped hair follicle has the same multipotent cell population as adult and fetal scalp hair follicles.

Background The mechanisms involved in laser-mediated hair removal remain unclear. One means of reducing hair growth is alteration of follicular stem cells. Objective We sought to examine the effects of laser hair removal on the immunohistochemical staining properties of human hair follicles, including the putative stem cells of the bulge region. Methods Treatment of unwanted axillary hair was performed on one side using an 800 nm–wavelength diode laser and on the other side using a 1064 nm–wavelength neodymium:yttrium-aluminum-garnet laser. Serial skin samples were obtained at baseline and various times after treatment and stained using immunohistochemical techniques. Results Hair shafts were thermally altered, but the immunostaining properties of much of the follicle, including the bulge region, remained generally unchanged. Limitations This study only addressed the acute immunohistochemical changes found after a single treatment using specific laser parameters. Conclusions Laser-mediated hair removal does not appear to work by frank destruction of follicular stem cells. Other mechanisms including functional alteration of these cells may underlie the clinical efficacy of the procedure.

We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5 / mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins. r 2006 Elsevier GmbH. All rights reserved.

Humans with selected mutations in the vitamin D receptor (VDR) and mouse models lacking VDR develop alopecia. Mice null for the Vdr gene are born with a normal coat of hair, but fail to initiate normal hair follicle cycling. In this study, we examined the morphology of the hair follicle of the Vdr null mouse during days 13-22 when the hair follicle normally initiates and completes the first catagen. We then explored the possibility that the abnormality in hair follicle cycling was associated with abnormal expression of hairless (Hr), a putative transcriptional regulator known to regulate hair follicle cycling and recently shown to regulate VDR transcriptional activity. Our results demonstrate the progressive deterioration of the hair follicle through catagen. Comparable to VDR, Hr was found in the basal cells of the epidermis and ORS of the hair follicle. However, Hr was also found in the IRS and matrix of the follicle, regions with little or no VDR. Hr levels increased during catagen, reaching a peak by day 19. Levels of Hr were greater in the Vdr null mice compared to wildtype controls, results confirmed by quantitative RT-PCR. We conclude that lack of VDR causes disruption of hair follicle structure during the first catagen resulting in failure of subsequent hair follicle cycling. These changes are associated with increased expression of Hr, suggesting a role for VDR in regulating Hr expression. Both Hr and VDR are required for normal hair follicle cycling.

Development of skin appendages strongly depends on epithelial-mesenchymal interactions. One of the genes involved in this process is p63, a member of the p53 family of transcription factors, essential for ectodermal development, as elucidated by the phenotype of p63 knock-out mice. Surprisingly, no information on p63 expression in tooth and hair is yet available. Here, we show p63 expression during teeth and vibrissae morphogenesis in mouse embryos and we also show a correlation with the expression patterns of the epithelial marker keratin 5 and the proliferation marker Ki67. Our results show that p63 colocalizes with both K5 and Ki67 in the epithelium of developing vibrissae, while in teeth p63 is expressed, together with K5, in the undifferentiated ectoderm (enamel organ), and in ameloblasts, a subpopulation of differentiated ectodermal cells. Moreover, p63 expression in tooth seems not to be fully colocalized with nuclear Ki67 expression.

The epidermis has a great potential as a bioreactor to produce proteins with systemic action. However, the consequences of ectopicepidermalproteinoverexpressionneedtobecarefully addressed to avoid both local and systemic adverse effects. Thus, the long-term effects of leptin on skin physiology have not been studied, and the metabolic consequences of sustained keratinocyte-derived leptin overexpression are unknown. Herein we describe that very high serum leptin levels can be achieved from a cutaneous source in transgenic mice in which leptin cDNA overexpression was driven by the keratinK5generegulatorysequences.Histopathologicalanalysis including the study of skin differentiation and proliferation markers in these transgenic mice revealed that keratinocytederived leptin overexpression appears not to have any impact on cutaneous homeostasis. Although young K5-leptin transgenic mice showed remarkable thinness and high glucose metabolism as shown in other leptin transgenic mouse models, a marked leptin insensitivity become apparent as early as 3–4 months of age as demonstrated by increased weight gain and insulin resistance development. Other signs of leptin/insulin resistance included increased bone mass, organomegaly, and wound healing impairment. In addition, to provide evidence for the lack of untoward effects of leptin on epidermis, this transgenic mouse helps us to establish the safe ranges of keratinocyte-derived leptin overexpression and may be useful as a model to study leptin resistance. (Endocrinology 146: 4167–4176, 2005)

Failure of secondary palate fusion during embryogenesis is a cause of cleft palate. Disappearance of the medial epithelial seam (MES) is required to allow merging of the mesenchyme from both palatal shelves. This involves complex changes of the medial edge epithelial (MEE) cells and surrounding structures that are controlled by several genes whose spatio-temporal expression is tightly regulated. We have carried out morphological analyses and used a semi-quantitative RT-PCR technique to evaluate whether morphological changes and modulation in the expression of putative key genes, such as twist, snail, and E-cadherin, during the fusion process in palate organ culture parallel those observed in vivo, and show that this is indeed the case. We also show, using the organotypic model of palate fusion, that the down-regulation of the transcription factor snail that occurs with the progression of palate development is not dependent on fusion of the palatal shelves. Abbreviations: dsg1, desmoglein1; EMT, epithelial-mesenchymal transition; MEE, medial edge epithelium; MES, medial epithelial seam; RT-PCR, reverse-transcriptase polymerase chain-reaction.

To analyze immunoregulation of autoreactive T cells specific for epidermal skin antigens, we crossed transgenic mice expressing ovalbumin selectively in keratinocytes under the keratin 5 promoter (K5-mOVA) with mice expressing a K(b)-restricted OVA-specific T cell receptor transgene (OT-I). In athymic double-transgenic mice, OT-I cells developed extrathymically and, at 8-12 weeks of age, initiated severe epidermal damage mimicking toxic epidermal necrolysis (TEN). In contrast, euthymic double-transgenic mice showed thymic deletion of OT-I cells, had few of these cells in the periphery, and never developed skin changes mimicking TEN. Adoptive transfer of OT-I cells isolated from euthymic double-transgenic mice induced TEN in athymic K5-mOVA single-transgenic mice. This spontaneous disease in athymic double-transgenic mice was prevented by transferring lymph node cells from euthymic mice, but was not prevented when CD4(+) or CD25(+) cells were depleted from this population. Although purified CD4(+)CD25(+) cells scarcely prevented the skin disease induced by adoptive transfer of OT-I cells, they efficiently prevented the disease when co-transferred with CD11c(+) dendritic cells. These results suggested that thymus-derived regulatory T cells cooperate with CD11c(+) dendritic cells to prevent life-threatening skin damage such as TEN.

Exposure of human fetuses to man-made estrogenic chemicals can occur through several sources. For example, fetal exposure to ethinylestradiol occurs because each year approximately 3% of women taking oral contraceptives become pregnant. Exposure to the estrogenic chemical bisphenol A occurs through food and beverages because of significant leaching from polycarbonate plastic products and the lining of cans. We fed pregnant CD-1 mice ethinylestradiol (0.1 microg/kg per day) and bisphenol A (10 microg/kg per day), which are doses below the range of exposure by pregnant women. In male mouse fetuses, both ethinylestradiol and bisphenol A produced an increase in the number and size of dorsolateral prostate ducts and an overall increase in prostate duct volume. Histochemical staining of sections with antibodies to proliferating cell nuclear antigen and mouse keratin 5 indicated that these increases were due to a marked increase in proliferation of basal epithelial cells located in the primary ducts. The urethra was malformed in the colliculus region and was significantly constricted where it enters the bladder, which could contribute to urine flow disorders. These effects were identical to those caused by a similar dose (0.1 microg/kg per day) of the estrogenic drug diethylstilbestrol (DES), a known human developmental teratogen and carcinogen. In contrast, a 2,000-fold higher DES dose completely inhibited dorsolateral prostate duct formation, revealing opposite effects of high and low doses of estrogen. Acceleration in the rate of proliferation of prostate epithelium during fetal life by small amounts of estrogenic chemicals could permanently disrupt cellular control systems and predispose the prostate to disease in adulthood.

Hypohidrotic ectodermal dysplasia is a human syndrome defined by maldevelopment of one or more ectodermal-derived tissues, including the epidermis and cutaneous appendices, teeth, and exocrine glands. The molecular bases of this pathology converge in a dysfunction of the transcription factor nuclear factor of the kappa-enhancer in B cells (NF-kappaB), which is essential to epithelial homeostasis and development. A number of mouse models bearing disruptions in NF-kappaB signaling have been reported to manifest defects in ectodermal derivatives. In ectoderm-targeted transgenic mice overexpressing the glucocorticoid receptor (GR) [keratin 5 (K5)-GR mice], the NF-kappaB activity is greatly decreased due to functional antagonism between GR and NF-kappaB. Here, we report that K5-GR mice exhibit multiple epithelial defects in hair follicle, tooth, and palate development. Additionally, these mice lack Meibomian glands and display underdeveloped sweat and preputial glands. These phenotypic features appear to be mediated specifically by ligand-activated GR because the synthetic analog dexamethasone induced similar defects in epithelial morphogenesis, including odontogenesis, in wild-type mice. We have focused on tooth development in K5-GR mice and found that an inhibitor of steroid synthesis partially reversed the abnormal phenotype. Immunostaining revealed reduced expression of the inhibitor of kappaB kinase subunits, IKKalpha and IKKgamma, and diminished p65 protein levels in K5-GR embryonic tooth, resulting in a significantly reduced kappaB-binding activity. Remarkably, altered NF-kappaB activity elicited by GR overexpression correlated with a dramatic decrease in the protein levels of DeltaNp63 in tooth epithelia without affecting Akt, BMP4, or Foxo3a. Given that many of the 170 clinically distinct ectodermal dysplasia syndromes still remain without cognate genes, deciphering the molecular mechanisms of this mouse model with epithelial NF-kappaB and p63 dysfunction may provide important clues to understanding the basis of other ectodermal dysplasia syndromes.

Expression and pharmacological studies support a contribution of cyclooxygenase (COX)-2 to mammary gland tumorigenesis. In a recent transgenic study, mouse mammary tumor virus promoter-driven COX-2 expression in mouse mammary glands was shown to result in alveolar hyperplasia, dysplasia, and carcinomas after multiple rounds of pregnancy and lactation. In the study presented here, the effects of constitutive COX-2 overexpression in keratin 5-positive myoepithelial and luminal cells, driven by the keratin 5 promoter in a hormone-independent manner, was investigated. In nulliparous female mice, aberrant COX-2 overexpression correlated with increased prostaglandin (PG) E 2 levels and caused cystic duct dilatations, adenosis, and fibrosis whereas carcinomas developed rarely. This phenotype depended on COX-2-mediated PGE 2 synthesis and correlated with increased expression of proliferation-associated Ki67 in epithelial cells. No changes in the expression of apoptosis-related Bcl-2, caspase 3, or p53 were observed. Hyperproliferation of the mammary gland epithelial cells was associated with increased aromatase mRNA levels in this tissue. The spontaneous pathologies bear analogies to the human breast with fibrocystic changes. Intriguingly, strong COX-2 expression was observed in fibrocystic changes, as compared to low expression in normal breast epithelium. These results show for the first time that aberrant COX-2 expression contributes to the development of fibrocystic changes (FC), indicating that COX-2 and COX-2-mediated PG synthesis represent potential targets for the therapy of this most frequent benign disorder of the human breast.

The location and identity of interfollicular epidermal stem cells of adult human skin remain undefined. Based on our previous work in both adult murine and neonatal human foreskin, we demonstrate that cell surface levels of the alpha6 integrin and the transferrin receptor (CD71) are valid markers for resolving a putative stem cell, transit amplifying and differentiating compartment in adult human skin by flow cytometry. Specifically, epidermal cells expressing high levels of alpha6 integrin and low levels of the transferrin receptor CD71 (phenotype alpha6 (bri)CD71(dim)) exhibit several stem cell characteristics, comprising a minor population (2%-5%) of the K14(bri) fraction, enriched for quiescent and small blast-like cells with high clonogenic capacity, lacking the differentiation marker K10. Conversely, the majority of K14(bri) K10(neg) epidermal cells express high levels of CD71 (phenotype alpha6 (bri)CD71(bri)), and represent the actively cycling fraction of keratinocytes displaying greater cell size due to an increase in cytoplasmic area, consistent with their being transient amplifying cells. The alpha6 (bri)CD71(bri) population exhibited intermediate clonogenic capacity. A third population of K14(dim) but K10 positive epidermal cells could be identified by their low levels of alpha6 integrin expression (i.e. alpha6 (dim) cells), representing the differentiation compartment; predictably, this subpopulation exhibited poor clonogenic efficiency. Flow cytometric analysis for the hair follicle bulge region (stem cell) marker K15 revealed preferential expression of this keratin in alpha6 (bri) cells (i.e., both stem and transient amplifying fractions), but not the alpha6 (dim) population. Given that K15 positive cells could only be detected in the deep rete ridges of adult skin in situ, we conclude that stem and transient amplifying cells reside in this location, while differentiating (K15 negative) cells are found in the shallow rete ridges.

Homozygous mice overexpressing Claudin-6 (Cldn6) exhibit a perturbation in the epidermal differentiation program leading to a defective epidermal permeability barrier (EPB) and dehydration induced death ensuing within 48 h of birth [Turksen, K., Troy, T.C., 2002. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784]. Their heterozygous counterparts are also born with an incomplete EPB; however, barrier formation continues after birth and normal hydration levels are achieved by postnatal day 12 allowing survival into adulthood. Heterozygous Inv-Cldn6 mice exhibit a distinct coat phenotype and histological analysis shows mild epidermal hyperkeratosis. Expression of K5 and K14 is aberrant, extending beyond the basal layer into the suprabasal layer where they are not co-localized suggesting that their expression is uncoupled. There is also atypical K17 and patchy K15 expression in the basal layer with no K6 expression in the interfollicular epidermis; together with marked changes in late differentiation markers (e.g. profilaggrin/filaggrin, loricrin, transglutaminase 3) indicating that the normal epidermal differentiation program is modified. The expression compartment of various Cldns is also perturbed although overall protein levels remained comparable. Most notably induction of Cldn5 and Cldn8 was observed in the Inv-Cldn6 epidermis. Heterozygous Inv-Cldn6 animals also exhibit subtle alterations in the differentiation program of the hair follicle including a shorter anagen phase, and altered hair type distribution and length compared to the wild type; the approximately 20% increase in zig-zag hair fibers at the expense of guard hairs and the approximately 30% shorter guard hairs contribute to coat abnormalities in the heterozygous mice. In addition, the transgenic hair follicles exhibit a decreased expression of K15 as well as some hair-specific keratins and express Cldn5 and Cldn18, which are not detectable in the wild type. These data indicate that Cldn6 plays a role in the differentiation processes of the epidermis and hair follicle and supports the notion of a link between Cldn regulation and EPB assembly/maintenance as well as the hair cycle.

Nuclear shaping is a critical event during sperm development as demonstrated by the incidence of male infertility associated with abnormal sperm ad shaping. Herein, we demonstrate that mouse and rat spermatids assemble in the subacrosomal space a cytoskeletal scaffold containing F-actin and Sak57, a keratin ortholog. The cytoskeletal plate, designated acroplaxome, anchors the developing acrosome to the nuclear envelope. The acroplaxome consists of a marginal ring containing keratin 5 10-nm-thick filaments and F-actin. The ring is closely associated with the leading edge of the acrosome and to the nuclear envelope during the elongation of the spermatid head. Anchorage of the acroplaxome to the gradually shaping nucleus is not disrupted by hypotonic treatment and brief Triton X-100 extraction. By examining spermiogenesis in the azh mutant mouse, characterized by abnormal spermatid/sperm head shaping, we have determined that a deformity of the spermatid nucleus is restricted to the acroplaxome region. These findings lead to the suggestion that the acroplaxome nucleates an F-actin–keratin-containing assembly with the purpose of stabilizing and anchoring the developing acrosome during spermatid nuclear elongation. The acroplaxome may also provide a mechanical planar scaffold modulating external clutching forces generated by a stack of Sertoli cell F-actin–containing hoops encircling the elongating spermatid nucleus.

Keratin is a major protein produced during epithelialization following burn injury and is a useful marker for assessing wound healing. Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the keratin 5 (K5) promoter (K5GFP mice) were used to monitor keratin expression, and thus, re-epithelialization of burn wounds.K5GFP transgenic mice were created using conventional techniques, with PCR and Southern blot confirmation of transgene incorporation, followed by selection of the line with the most intense and consistent basal epithelial EGFP expression. Epi-fluorescent microscopy of 24 K5GFP mouse flanks and 10 negative littermate controls was used to characterize EGFP intensity, before wounding and serially for 30 days after administration of a standardized burn wound and excision. Biopsy sections of K5GFP and negative control mice were stained with K5 antibody and imaged with confocal microscopy to characterize the distribution of EGFP and K5 at baseline and after injury and to examine the correlation between K5 expression and EGFP expression during healing.Green fluorescence intensity increased at the advancing wound margin of burned K5GFP mice, reaching a maximum between days 12 and 15 post-burn and then decreasing as healing completed. K5 and EGFP expression increased in parallel in burned K5GFP mice as demonstrated by confocal microscopy.EGFP expression correlated with K5 expression during wound healing and therefore serves as a good marker of re-epithelialization. This transgenic model allows noninvasive, real-time assessment of in vivo K5 expression and will be useful in the study of wound healing.

To isolate and culture the murine fetal epidermal stem cells (ESCs) and folliculus pili cells (FPCs) in vitro, and to observe the regeneration of hair follicle and epidermis after cografting of ESCs and FPCs.The ESCs were isolated by adhering to murine type IV collagen and were cultured in conditional medium. The expression level of beta1-integrin and keratin 15 in ESCs was detected. At the same time, the cell cycle and clony forming eficiency (CFE) in ESCs were also determined. The FPCs were isolated and cultured and inoculated in fibrin-gel to form FPCs-gel. A full skin equivalent was prepared by combining ESCs with FPCs-gel and was grafted onto total skin loss wounds on the back of BALB/C nude mice. The histological changes of the wounds and the hair follicles were observed at 8 - 10 weeks after the grafting.There were high level expressions of beta1-integrin and keratin 15 in murine fetal ESCs. It was indicated by cell cycle analysis that cells in G1 stage accounted for 94.9% of the cells, while that in S stage, 3.5%, suggesting slow cell cycle. Nevertheless, the keratinocytes in G1 stage accounted for 74.1% and that in S stage, 17.5% of cells in control group. The CFE of ESCs was 15.3%, and it was much higher than that in control group (6.7%). The newly formed hair follicles could be found in the grafted rats but not in the control group 8 - 10 weeks after the wound healing in nude rats.The ESCs could be successfully isolated and cultured in vitro and might participate in the formation of hair follicle structure under the induction of FPCs.

The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464–2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654–39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 – 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin.

Glucocorticoid hormones act through the glucocorticoid receptor (GR) and they affect almost all physiological systems in the organism. We have previously reported that transgenic mice overexpressing GR under the control of the keratin k5 promoter (K5-GR mice) display severe phenotypic alterations in the epidermis and other ectoderm derivatives (Perez et al., 2001). In this work, we aimed to characterize the pathological consequences of GR targeted overexpression in the eyelid and cornea at late developmental stages. Despite glucocorticoids being widely prescribed as a topical treatment in ophthalmology, their potential role during ocular development in the embryo is not well understood. As shown by scanning electron microscopy analysis as well as by our histopathological and immunohistochemical data, long-term and newborn transgenic embryos showed unfused eyelids, along with proptosis of the globe and exposure of the anterior surface. In addition, epithelial defects were evident at the cornea. Our results indicate that GR overexpression affected the proliferation rate of targeted epithelia of the cornea and eyelid, thus demonstrating that GR was responsible for the arrest of epithelial proliferation of the developing eyelid edges, as well as for their destruction. We conclude that constitutive targeted overexpression of GR in the eyelid and corneal epithelium dramatically impairs ocular function in these transgenic mice.

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.

Basal cell carcinomas (BCCs) are hedgehog-driven tumors that resemble follicular and interfollicular epidermal basal keratinocytes and hence long have been thought to arise from these cells. However, the actual cell of origin is unknown. Using cell fate tracking of X-ray induced BCCs in Ptch1(+/-) mice, we found their essentially exclusive origin to be keratin 15-expressing stem cells of the follicular bulge. However, conditional loss of p53 not only enhanced BCC carcinogenesis from the bulge but also produced BCCs from the interfollicular epidermis, at least in part by enhancing Smo expression. This latter finding is consistent with the lack of visible tumors on ears and tail, sites lacking Smo expression, in Ptch1(+/-) mice.

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti-inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable-sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5-GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti-inflammatory role of GR was due at least in part to interference with NF-kB, leading to a strong reduction in the kB-binding activity without altering the transcriptional levels of the inhibitor IkBa.

The genes encoding keratin 5 and 14 are highly expressed in the basal cell layer keratinocytes of the epidermis, but both genes are silenced when keratinocytes move into the suprabasal compartment. The POU homeodomain factors Skn-1a and Tst-1, which are expressed in epidermis, may play a role in the suprabasal repression of the keratin 5 and 14 genes because keratin 14 mRNA expression persists in suprabasal cells in Skn-1/Tst-1 double knockout mice. In transfection experiments, both Skn-1a and Tst-1 repress the keratin 14 promoter, with the POU domain being sufficient for repression. The region of the keratin 14 gene sufficient and required for repression by Skn-1a is a 100-base pair sequence lacking POU-binding sites adjacent to the transcription start site. DNA-binding defective mutants of Skn-1a and Tst-1 are as effective at mediating repression as the wild type proteins, suggesting that protein-protein interactions rather than direct DNA binding are important for repression. We also show that CREB-binding protein (CBP)/p300 co-activators are strong activators of keratin 14 gene expression, acting through sequences close to the keratin 14 promoter. Further, CBP interacts directly with the POU domain of Skn-1a, and increasing concentrations of CBP can overcome Skn-1a-mediated repression, suggesting that POU domain factors may repress keratin 14 gene expression by interfering with the activity of co-activators such as CBP/p300.

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10−/−mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10−/−mice suggests that there is a considerable redundancy in the keratin gene family.

In human patients, a wide range of mutations in keratin (K) 5 or K14 lead to the blistering skin disorder epidermolysis bullosa simplex. Given that K14 deficiency does not lead to the ablation of a basal cell cytoskeleton because of a compensatory role of K15, we have investigated the requirement for the keratin cytoskeleton in basal cells by inactivating the K5 gene in mice. We report that the K5−/−mice die shortly after birth, lack keratin filaments in the basal epidermis, and are more severely affected than K14−/−mice. In contrast to the K14−/−mice, we detected a strong induction of the wound-healing keratin K6 in the suprabasal epidermis of cytolyzed areas of postnatal K5−/−mice. In addition, K5 and K14 mice differed with respect to tongue lesions. Moreover, we show that in the absence of K5 and other type II keratins, residual K14 and K15 aggregated along hemidesmosomes, demonstrating that individual keratins without a partner are stable in vivo. Our data indicate that K5 may be the natural partner of K15 and K17. We suggest that K5 null mutations may be lethal in human epidermolysis bullosa simplex patients.

Human Papillomavirus type 16 (HPV-16) is the cause of both benign lesions and ano-genital cancers. In HPV-associated cancers the transforming properties of the expressed viral E6 and E7 proteins have been revealed by a number of different assays. We have generated transgenic mice expressing HPV-16 E6/E7 genes under the control of the murine keratin 5 gene promoter, which should confer cell-type specific expression in the basal cells of squamous stratified epithelia. Transgenic mice developed thymic hyperplasia and lung neoplasia with 100% frequency, the thymus showing a size increase at 2 months and reaching the maximum dimension at 6 months, when lung carcinomas appeared. After this time the size of hyperplastic thymi decreased, while malignant formations invaded the mediastinal area. Hepatic metastasis could be also observed in some of the animals at the autopsy and death invariably occurred around 10-11 months of age.

Keratin intermediate filaments are the major components of the cytoskeleton in epithelial cells. Mutations in keratin genes have been documented in many disorders of the skin, nails, hair, and mucous membranes. Although no mutations have been described in either keratin 15 or keratin 19, they are good candidates for other as yet uncharacterized genetic disorders of keratinization, particularly as the skin, nails, hair, and conjunctiva are sites of keratin 15 and 19 expression. To facilitate future mutation detection analyses, we have therefore characterized the intron–exon organization of the human keratin 15 and keratin 19 genes. The keratin 15 gene comprises 8 exons spanning approximately 5.1 kb on 17q21, and the keratin 19 gene consists of 6 exons covering approximately 4.7 kb on 17q21. We have also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine which plays an important role in cutaneous wound repair. To gain insight into the mechanisms of action of this growth and differentiation factor in the skin, we searched for genes which are regulated by TGF-beta1 in cultured HaCaT keratinocytes. Using the differential display RT-PCR technology we identified a gene which was strongly downregulated by TGF-beta1. The identified cDNA includes sequences of the keratin 15 (K15) gene which encodes a component of the cytoskeleton of basal cells in stratified epithelia. Surprisingly, our cDNA also included an unknown sequence. Since this cDNA lacks an open reading frame, the corresponding mRNA is likely to be nonfunctional. However, we also demonstrate a strong negative regulation of the expression of the published, functional K15 variant. Expression of K15 was also suppressed by tumor necrosis factor alpha (TNF-alpha) and to a lesser extent by epidermal growth factor (EGF) and keratinocyte growth factor (KGF). By contrast, the major basal type I keratin, K14, was upregulated by TGF-beta1, whereas TNF-alpha, EGF, and KGF had no effect. Consistent with the in vitro data, we found a significant reduction of the K15 mRNA levels after skin injury, whereas K14 expression increased during the wound healing process. Immunostaining revealed the presence of K15 in all basal cells of the epidermis adjacent to the wound, but not in the hyperproliferative epithelium above the granulation tissue. These data demonstrate that K15 is excluded from the activated keratinocytes of the hyperthickened wound epidermis, possibly as a result of increased growth factor expression in injured skin.