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Background: Myelomeningocele or spina bifida is an open neural tube defect that is characterized by protrusion of the meninges and the spinal cord through a deformity in the vertebral arch and spinous process. Myelomeningocele of post-natal tissue is well described; however, pre-natal tissue of this defect has no known previous histologic characterization. We compared the histology of different forms of pre-natal myelomeningocele and post-natal myelomeningocele tissue obtained via prenatal intrauterine and postnatal surgical repairs. Methods: Pre-and post-natal tissues from spina bifida repair surgeries were obtained from lipomyelomeningocele, myeloschisis, and myelomeningocele spina bifida defects. Tissue samples were processed for H&E and immunohistochemical staining (KRT14 and p63) to assess epidermal and dermal development. Results: Prenatal skin near the defect site develops with normal epidermal, dermal, and adnexal structures. Within the grossly cystic specimens, histology shows highly dense fibrous connective tissue with complete absence of a normal epidermal development with a lack of p63 and KRT14 expression. Conclusion: Tissues harvested from prenatal and postnatal spina bifida repair surgeries appear as normal skin near the defect site. However, cystic tissues consist of highly dense fibrous connective tissue with complete absence of normal epidermal development.
Assuntos
Imuno-Histoquímica , Meningomielocele , Disrafismo Espinal , Humanos , Disrafismo Espinal/patologia , Disrafismo Espinal/cirurgia , Feminino , Imuno-Histoquímica/métodos , Meningomielocele/cirurgia , Meningomielocele/patologia , Meningomielocele/metabolismo , Gravidez , Recém-NascidoRESUMO
The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced AEC iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.
Assuntos
Fenda Labial , Fissura Palatina , Displasia Ectodérmica , Animais , Camundongos , Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Queratinócitos , Mutação , Proteínas Supressoras de Tumor/genética , Células-Tronco Pluripotentes Induzidas , Camundongos TransgênicosRESUMO
Heritable conditions known as ectodermal dysplasias are rare and can be associated with marked morbidity, mortality, and a reduced quality of life. The diagnosis and care of individuals affected by one of the many ectodermal dysplasias presents myriad challenges due to their rarity and the diverse phenotypes. These conditions are caused by abnormalities in multiple genes and signaling pathways that are essential for the development and function of ectodermal derivatives. During a 2021 international conference focused on translating discovery to therapy, researchers and clinicians gathered with the goal of advancing the diagnosis and treatment of conditions affecting ectodermal tissues with an emphasis on skin, hair, tooth, and eye phenotypes. Conference participants presented a variety of promising treatment strategies including gene or protein replacement, gene editing, cell therapy, and the identification of druggable targets. Further, barriers that negatively influence the current development of novel therapeutics were identified. These barriers include a lack of accurate prevalence data for rare conditions, absence of an inclusive patient registry with deep phenotyping data, and insufficient animal models and cell lines. Overcoming these barriers will need to be prioritized in order to facilitate the development of novel treatments for genetic disorders of the ectoderm.
Assuntos
Ectoderma , Displasia Ectodérmica , Animais , Qualidade de Vida , Doenças Raras/genética , Doenças Raras/terapia , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/genética , Displasia Ectodérmica/terapia , CabeloRESUMO
An international advisory group met at the National Institutes of Health in Bethesda, Maryland in 2017, to discuss a new classification system for the ectodermal dysplasias (EDs) that would integrate both clinical and molecular information. We propose the following, a working definition of the EDs building on previous classification systems and incorporating current approaches to diagnosis: EDs are genetic conditions affecting the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Genetic variations in genes known to be associated with EDs that affect only one derivative of the ectoderm (attenuated phenotype) will be grouped as non-syndromic traits of the causative gene (e.g., non-syndromic hypodontia or missing teeth associated with pathogenic variants of EDA "ectodysplasin"). Information for categorization and cataloging includes the phenotypic features, Online Mendelian Inheritance in Man number, mode of inheritance, genetic alteration, major developmental pathways involved (e.g., EDA, WNT "wingless-type," TP63 "tumor protein p63") or the components of complex molecular structures (e.g., connexins, keratins, cadherins).
Assuntos
Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Fenótipo , Alelos , Biomarcadores , Bases de Dados Genéticas , Displasia Ectodérmica/metabolismo , Humanos , Transdução de SinaisRESUMO
To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders.
Assuntos
Microdissecção e Captura a Laser , Melanócitos/metabolismo , RNA/isolamento & purificação , Vitiligo/metabolismo , Estudos de Casos e Controles , Humanos , RNA/metabolismo , Vitiligo/radioterapiaRESUMO
Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare monogenetic disorder that is characterized by severe abnormalities in ectoderm-derived tissues, such as skin and its appendages. A major cause of morbidity among affected infants is severe and chronic skin erosions. Currently, supportive care is the only available treatment option for AEC patients. Mutations in TP63, a gene that encodes key regulators of epidermal development, are the genetic cause of AEC. However, it is currently not clear how mutations in TP63 lead to the various defects seen in the patients' skin. In this review, we will discuss current knowledge of the AEC disease mechanism obtained by studying patient tissue and genetically engineered mouse models designed to mimic aspects of the disorder. We will then focus on new approaches to model AEC, including the use of patient cells and stem cell technology to replicate the disease in a human tissue culture model. The latter approach will advance our understanding of the disease and will allow for the development of new in vitro systems to identify drugs for the treatment of skin erosions in AEC patients. Further, the use of stem cell technology, in particular induced pluripotent stem cells (iPSC), will enable researchers to develop new therapeutic approaches to treat the disease using the patient's own cells (autologous keratinocyte transplantation) after correction of the disease-causing mutations.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Anormalidades do Olho/genética , Pálpebras/anormalidades , Animais , Fenda Labial/patologia , Fissura Palatina/patologia , Modelos Animais de Doenças , Displasia Ectodérmica/patologia , Epiderme/patologia , Anormalidades do Olho/patologia , Pálpebras/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Mutação/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.
RESUMO
Desmocollin 3 (DSC3) is a desmosomal cadherin that is required for maintaining cell adhesion in the epidermis as demonstrated by the intra-epidermal blistering observed in Dsc3 null skin. Recently, it has been suggested that deregulated expression of DSC3 occurs in certain human tumor types. It is not clear whether DSC3 plays a role in the development or progression of cancers arising in stratified epithelia such as the epidermis. To address this issue, we generated a mouse model in which Dsc3 expression is ablated in K-Ras oncogene-induced skin tumors. Our results demonstrate that loss of Dsc3 leads to an increase in K-Ras-induced skin tumors. We hypothesize that acantholysis-induced epidermal hyperplasia in the Dsc3 null epidermis facilitates Ras-induced tumor development. Further, we demonstrate that spontaneous loss of DSC3 expression is a common occurrence during human and mouse skin tumor progression. This loss occurs in tumor cells invading the dermis. Interestingly, other desmosomal proteins are still expressed in tumor cells that lack DSC3, suggesting a specific function of DSC3 loss in tumor progression. While loss of DSC3 on the skin surface leads to epidermal blistering, it does not appear to induce loss of cell-cell adhesion in tumor cells invading the dermis, most likely due to a protection of these cells within the dermis from mechanical stress. We thus hypothesize that DSC3 can contribute to the progression of tumors both by cell adhesion-dependent (skin surface) and likely by cell adhesion-independent (invading tumor cells) mechanisms.
Assuntos
Glicoproteínas de Membrana/genética , Neoplasias Cutâneas/patologia , Alelos , Animais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Desmocolinas , Progressão da Doença , Genes ras , Humanos , Camundongos , Neoplasias Cutâneas/genéticaRESUMO
Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create "designer" iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC-derived keratinocytes (iPSC-K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF™ Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell™ 400 6-well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells.
Assuntos
Células-Tronco Pluripotentes Induzidas , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Humanos , Queratinócitos , PeleRESUMO
To keep pace with the rapid advancements in molecular genetics and rare diseases research, we have updated the list of ectodermal dysplasias based on the latest classification approach that was adopted in 2017 by an international panel of experts. For this purpose, we searched the databases PubMed and OMIM for the term "ectodermal dysplasia", referring mainly to changes in the last 5 years. We also tried to obtain information about those diseases on which the last scientific report appeared more than 15 years ago by contacting the authors of the most recent publication. A group of experts, composed of researchers who attended the 8th International Conference on Ectodermal Dysplasias and additional members of the previous classification panel, reviewed the proposed amendments and agreed on a final table listing all 49 currently known ectodermal dysplasias for which the molecular genetic basis has been clarified, including 15 new entities. A newly reported ectodermal dysplasia, linked to the gene LRP6, is described here in more detail. These ectodermal dysplasias, in the strict sense, should be distinguished from syndromes with features of ectodermal dysplasia that are related to genes extraneous to the currently known pathways involved in ectodermal development. The latter group consists of 34 syndromes which had been placed on the previous list of ectodermal dysplasias, but most if not all of them could actually be classified elsewhere. This update should streamline the classification of ectodermal dysplasias, provide guidance to the correct diagnosis of rare disease entities, and facilitate the identification of individuals who could benefit from novel treatment options.
Assuntos
Displasia Ectodérmica , Humanos , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/genética , Síndrome , PubMed , Doenças RarasRESUMO
The last decade has seen a dramatic increase in innovative ideas for the treatment of genetic disorders for which no curative therapies exist. Gene and protein replacement therapies stand out as novel approaches to treat a select group of these diseases, such as certain tissue fragility disorders. Further, the advent of stem cell approaches, such as induced pluripotent stem cells (iPSC) technology, has led to the development of new methods of creating replacement tissues for regenerative medicine. This coincided with the discovery of genome editing techniques, which allow for the correction of disease-causing mutations. The culmination of these discoveries suggests that new and innovative therapies for monogenetic disorders affecting single organs or tissues are on the horizon. Challenges remain, however, especially with diseases that simultaneously affect several tissues and organs during development. Examples of this group of diseases include ectodermal dysplasias, genetic disorders affecting the development of tissues and organs such as the skin, cornea, and epithelial appendages. Gene or protein replacement strategies are unlikely to be successful in addressing the multiorgan phenotype of these diseases. Instead, we believe that a more effective approach will be to focus on correcting phenotypes in the most severely affected tissues. This could include the generation of replacement tissues or the identification of pharmaceutical compounds that correct disease pathways in specific tissues.
RESUMO
In repigmentation of human vitiligo, the melanocyte (MC) precursors in the hair follicle bulge proliferate, migrate, and differentiate to repopulate the depigmented epidermis. Here, we present a comprehensive characterization of pathways and signals in the bulge that control the repigmentation process. Using biopsies from patients with vitiligo, we have selectively harvested, by laser capture microdissection, MC and keratinocyte precursors from the hair follicle bulge of untreated vitiligo skin and vitiligo skin treated with narrow-band UVB. The captured material was subjected to whole transcriptome RNA-sequencing. With this strategy, we found that repigmentation in the bulge MC precursors is driven by KCTD10, a signal with unknown roles in the skin, and CTNNB1 (encoding ß-catenin) and RHO guanosine triphosphatase [RHO GTPase, RHO], two signaling pathways previously shown to be involved in pigmentation biology. Knockdown studies in cultured human MCs of RHOJ, the upmost differentially expressed RHO family component, corroborated with our findings in patients with vitiligo, identified RHOJ involvement in UV response and melanization, and confirmed previously identified roles in melanocytic cell migration and apoptosis. A better understanding of mechanisms that govern repigmentation in MC precursors will enable the discovery of molecules that induce robust repigmentation phenotypes in vitiligo.
Assuntos
Células-Tronco Adultas/metabolismo , Melanócitos/metabolismo , Pigmentação da Pele/efeitos da radiação , Terapia Ultravioleta , Vitiligo/terapia , Adolescente , Adulto , Células-Tronco Adultas/efeitos da radiação , Idoso , Criança , Feminino , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Folículo Piloso/efeitos da radiação , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Masculino , Melanócitos/efeitos da radiação , Pessoa de Meia-Idade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , RNA-Seq , Transdução de Sinais/efeitos da radiação , Resultado do Tratamento , Vitiligo/patologia , Adulto Jovem , beta Catenina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
During skin development, the single-layered surface ectoderm covering the mouse embryo must initiate stratification and terminal differentiation to develop a functional epidermis. A recent article by Lechler and Fuchs in Nature (Lechler and Fuchs, 2005) suggests that these events are triggered by asymmetric cell division.
Assuntos
Divisão Celular , Células Epidérmicas , Epiderme/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Epiderme/fisiologia , Humanos , Queratinas/metabolismo , CamundongosRESUMO
Nonmelanoma skin cancers (NMSCs) consist of a variety of tumor types including basal cell carcinoma, squamous cell carcinoma, a variety of hair follicle tumors, and sebaceous gland tumors. Genetic alterations that alter the fate of multipotent stem cells are believed to influence NMSC phenotype. We previously generated a transgenic mouse line which constitutively expressed c-myc under the control of the K14 promoter (K14.MYC2). These mice exhibited an increase in size and number of sebaceous glands, suggesting that c-myc diverted multipotential stem cells to a sebaceous lineage. Our goal in the current study was to determine if alterations in the commitment of multipotent stem cells to different cell fates would influence tumor phenotype. To this end, we exposed K14.MYC2 mice to a chemical carcinogenesis protocol and discovered that these mice were predisposed to develop sebaceous adenomas. Our data demonstrate that genetic alterations that alter the fate of multipotent stem cells during embryonic development can markedly influence the phenotype of NMSC that develop following exposure to carcinogens.
Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Células-Tronco/patologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Adenocarcinoma Sebáceo/patologia , Animais , Carcinógenos/toxicidade , Diferenciação Celular/genética , Linhagem da Célula/genética , Cruzamentos Genéticos , Feminino , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Endogâmicos , Camundongos Transgênicos , Células-Tronco Multipotentes/patologia , Papiloma/patologia , Fenótipo , Neoplasias das Glândulas Sebáceas/patologia , Acetato de Tetradecanoilforbol/farmacologia , TransgenesRESUMO
While tattooable nanotechnology for in-skin sensing and communication has been a popular concept in science fiction since the 1990s, the first tattooable intradermal nanosensors have only emerged in the past few years, and none have been demonstrated in human skin. We developed a photochromic tattoo that serves as an intradermal ultraviolet (UV) radiometer that provides naked-eye feedback about UV exposure in real time. These small tattoos, or "solar freckles", comprise dermally implanted colorimetric UV sensors in the form of nanoencapsulated leuco dyes that become more blue in color with increasing UV irradiance. We demonstrate the tattoos' functionality for both quantitative and naked-eye UV sensing in porcine skin ex vivo, as well as in human skin in vivo. Solar freckles offer an alternative and complementary approach to self-monitoring UV exposure for the sake of skin cancer prevention. Activated solar freckles provide a visual reminder to protect the skin, and their color disappears rapidly upon removal of UV exposure or application of topical sunscreen. The sensors are implanted in a minimally invasive procedure that lasts only a few seconds, yet remain functional for months to years. These semipermanent tattoos provide an early proof-of-concept for long-term intradermal sensing nanomaterials that provide users with biomedically relevant information in the form of an observable color change.
Assuntos
Melanose , Tatuagem , Humanos , Radiometria , Pele , Luz Solar , Raios UltravioletaRESUMO
Summary Sentence: Conditional ablation of AP-2 gamma results in a delay in skin development and abnormal expression of p63, K14, K1, filaggrin, repetin and secreted Ly6/Plaur domain containing 1, key genes required for epidermal development and differentiation. The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2 gamma is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2 gamma, which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2 gamma in skin development. Mice deficient for AP-2 gamma exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2 gamma in skin development, and reveal the existence of regulatory factors that can compensate for AP-2 gamma in its absence.
Assuntos
Epiderme/embriologia , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Células Epidérmicas , Epiderme/metabolismo , Feminino , Proteínas Filagrinas , Genes Letais , Proteínas de Filamentos Intermediários/genética , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Camundongos , MutaçãoRESUMO
Epidermal development and differentiation are tightly controlled processes that culminate in the formation of the epidermal barrier. A critical regulator of different stages of epidermal development and differentiation is the transcription factor p63. More specifically, we previously demonstrated elsewhere that p63 is required for both the commitment to stratification and the commitment to terminal differentiation. We now demonstrate that DeltaNp63alpha, the predominantly expressed p63 isoform in postnatal epidermis, also plays a role in the final stages of epidermal differentiation, namely the formation of the epidermal barrier. We found that DeltaNp63alpha contributes to epidermal barrier formation by directly inducing expression of ALOX12, a lipoxygenase which contributes to epidermal barrier function. Our data demonstrate that DeltaNp63alpha directly interacts with the promoter of Alox12 in the developing epidermis. Furthermore, we found that the induction of Alox12 expression by DeltaNp63alpha depends on intact p63 binding sites in the Alox12 promoter. Finally, we found that DeltaNp63alpha can induce Alox12 expression only in differentiating keratinocytes, consistent with the role of ALOX12 in epidermal barrier formation.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Epiderme/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Araquidonato 12-Lipoxigenase/genética , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Imunoprecipitação da Cromatina , DNA/metabolismo , Regulação para Baixo/genética , Indução Enzimática , Epiderme/embriologia , Epiderme/enzimologia , Genes Reporter/genética , Quinase I-kappa B/genética , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mifepristona/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Mutação Puntual/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Elementos de Resposta/genética , Pele/efeitos dos fármacos , Pele/embriologia , Pele/metabolismo , Transativadores/genética , Transdução GenéticaRESUMO
Ankyloblepharon Ectodermal Dysplasia and Cleft Lip/Palate (AEC) or Hay-Wells Syndrome is an autosomal dominant disorder characterized by a variety of phenotypes in ectodermal derivatives, including severe skin erosions, ankyloblepharon, coarse and wiry hair, scalp dermatitis, and dystrophic nails. AEC is caused by mutations in the gene encoding the TP63 transcription factor, specifically in the Sterile Alpha Motif (SAM) domain. The exact mechanism, however, by which these specific TP63 mutations lead to the observed spectrum of phenotypes is unclear. Analysis of individual TP63 target genes provides a means to understand specific aspects of the phenotypes associated with AEC. PERP is a TP63 target critical for cell-cell adhesion due to its participation in desmosomal adhesion complexes. As PERP null mice display symptoms characteristic of ectodermal dysplasia syndromes, we hypothesized that PERP dysfunction might contribute to AEC. Using luciferase reporter assays, we demonstrate here that PERP induction is in fact compromised with some, but not all, AEC-patient derived TP63 mutants. Through analysis of skin biopsies from AEC patients, we show further that a subset of these display aberrant PERP expression, suggesting the possibility that PERP dysregulation is involved in the pathogenesis of this disease. These findings demonstrate that distinct AEC TP63 mutants can differentially compromise expression of downstream targets, providing a rationale for the variable spectra of symptoms seen in AEC patients. Elucidating how specific TP63 target genes contribute to the pathogenesis of AEC will ultimately help design novel approaches to diagnose and treat AEC.
Assuntos
Fenda Labial/fisiopatologia , Fissura Palatina/fisiopatologia , Displasia Ectodérmica/fisiopatologia , Epiderme/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/fisiopatologia , Animais , Células Cultivadas , Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/genética , Células Epidérmicas , Pálpebras/anormalidades , Fibroblastos/metabolismo , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Síndrome , Transativadores/genética , Fatores de Transcrição , Proteínas Supressoras de Tumor/genéticaRESUMO
Dominant mutations in TP63 cause ankyloblepharon ectodermal dysplasia and clefting (AEC), an ectodermal dysplasia characterized by skin fragility. Since DeltaNp63alpha is the predominantly expressed TP63 isoform in postnatal skin, we hypothesized that mutant DeltaNp63alpha proteins are primarily responsible for skin fragility in AEC patients. We found that mutant DeltaNp63alpha proteins expressed in AEC patients function as dominant-negative molecules, suggesting that the human AEC skin phenotype could be mimicked in mouse skin by downregulating DeltaNp63alpha. Indeed, downregulating DeltaNp63 expression in mouse epidermis caused severe skin erosions, which resembled lesions that develop in AEC patients. In both cases, lesions were characterized by suprabasal epidermal proliferation, delayed terminal differentiation, and basement membrane abnormalities. By failing to provide structural stability to the epidermis, these defects likely contribute to the observed skin fragility. The development of a mouse model for AEC will allow us to further unravel the genetic pathways that are normally regulated by DeltaNp63 and that may be perturbed in AEC patients. Ultimately, these studies will not only contribute to our understanding of the molecular mechanisms that cause skin fragility in AEC patients, but may also result in the identification of targets for novel therapeutic approaches aimed at treating skin erosions. (c) 2009 Wiley-Liss, Inc.