RESUMO
Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.
Assuntos
Inflamassomos , MAP Quinase Quinase Quinases , Proteínas NLR , Piroptose , Ribossomos , Estresse Fisiológico , Anisomicina/toxicidade , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamassomos/efeitos da radiação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , MAP Quinase Quinase Quinases/metabolismo , Mutação , Proteínas NLR/genética , Proteínas NLR/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Piroptose/efeitos dos fármacos , Piroptose/efeitos da radiação , Ribossomos/efeitos dos fármacos , Ribossomos/efeitos da radiação , Raios UltravioletaRESUMO
BACKGROUND: Atopic dermatitis (AD) is a common chronic skin condition in children (15-20%) that can significantly impair their quality of life. As a result of its relapsing nature and enrichment of Staphylococcus aureus during flares, clinical management can include eradicating S aureus from the skin of children; however, this does not extend to their healthy caregivers, who are potential reservoirs. OBJECTIVE: Our aim was to understand skin microbiome sharing and microbial features in children with AD and their healthy adult caregivers. METHODS: We utilized whole-metagenome profiling at 4 body sites (volar forearm, antecubital fossae, cheeks, and lesions) in combination with sequencing of S aureus isolates to characterize a cohort of children with AD and their healthy caregivers (n = 30 families) compared to matched pairs from control households (n = 30 families). RESULTS: Metagenomic analysis revealed distinct microbiome configurations in the nonlesional skin of AD children and their healthy caregivers versus controls, which were sufficient to accurately predict case-control status (area under the receiver operating characteristic curve > 0.8). These differences were accompanied by significant microbiome similarity between children and their caregivers, indicating that microbiome sharing may play a role in recurrent disease flares. Whole-genome comparisons with high-quality S aureus isolate genomes (n = 55) confirmed significant strain sharing between AD children and their caregivers and AD-specific enrichment of strains expressing enterotoxins Q and K/K2. CONCLUSION: Our results highlight the distinctive skin microbiome features of healthy caregivers for children with AD and support their inclusion in strategies for the treatment of recurrent pediatric AD.
Assuntos
Dermatite Atópica , Microbiota , Adulto , Cuidadores , Criança , Dermatite Atópica/patologia , Enterotoxinas , Humanos , Recidiva Local de Neoplasia , Qualidade de Vida , Pele/patologia , Staphylococcus aureusRESUMO
BACKGROUND: Wound management is a critical factor when treating patients with the inherited skin fragility disease dystrophic epidermolysis bullosa (DEB). Due to genetic defects in structural proteins, skin and mucous epithelia are prone to blistering and chronic wounding upon minor trauma. Furthermore, these wounds are commonly associated with excessive pruritus and predispose to the development of life-threatening squamous cell carcinomas, underscoring the unmet need for new therapeutic options to improve wound healing in this patient cohort. Vitamin D3 is acknowledged to play an important role in wound healing by modulating different cellular processes that impact epidermal homeostasis and immune responses. In this study, we evaluate the safety and efficacy of low-dose calcipotriol, a vitamin D3 analogue, in promoting wound healing and reducing itch and pain in patients with DEB. METHODS: Eligible DEB patients, aged ≥ 6 years and with a known mutation in the COL7A1 gene, were recruited to a placebo-controlled, randomized, double blind, cross-over phase II monocentric clinical trial. Patients were required to have at least two wounds with a minimum size of 6 cm2 per wound. The primary objective was to evaluate efficacy of daily topical application of a 0.05 µg/g calcipotriol ointment in reducing wound size within a 4-week treatment regimen. Secondary objectives were to assess safety, as well as the impact of treatment on pruritus, pain, and bacterial wound colonization in these patients. RESULTS: Six patients completed the clinical trial and were included into the final analysis. Topical low-dose calcipotriol treatment led to a significant reduction in wound area at day 14 compared to placebo (88.4% vs. 65.5%, P < 0.05). Patients also reported a significant reduction of pruritus with calcipotriol ointment compared to placebo over the entire course of the treatment as shown by itch scores of 3.16 vs 4.83 (P < 0.05) and 1.83 vs 5.52 (P < 0.0001) at days 14 and 28, respectively. Treatment with low-dose calcipotriol did not affect serum calcium levels and improved the species richness of the wound microbiome, albeit with no statistical significance. CONCLUSIONS: Our results show that topical treatment with low-dose calcipotriol can accelerate wound closure and significantly reduces itch, and can be considered a safe and readily-available option to improve local wound care in DEB patients. Trial Registration EudraCT: 2016-001,967-35. Registered 28 June 2016, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001967-35/AT.
Assuntos
Epidermólise Bolhosa Distrófica , Calcitriol/análogos & derivados , Colágeno Tipo VII , Método Duplo-Cego , Humanos , Pomadas , Dor/tratamento farmacológico , Dor/etiologia , Prurido/tratamento farmacológico , Prurido/etiologia , CicatrizaçãoRESUMO
We report a case of junctional epidermolysis bullosa with pyloric atresia (JEB-PA) with minimal skin involvement but severe protein-losing enteropathy and airway involvement. Genetic analysis revealed heterozygous mutations in the ITGB4 gene encoding integrin ß4 protein. Parental testing confirmed inheritance of frameshift variant (c.794dupC) as maternal and splice site variant (c.1608C>T/p.Cys536Cys) as paternal. Immunofluorescence mapping of her skin revealed a subepidermal blister with decreased and frayed integrin ß4 at both the floor and the roof of the blister, while the intestinal mucosa showed complete absence of integrin ß4. We review the literature and discuss the differential expression of integrins in the skin and gastrointestinal tract, as well as the role of chronic inflammation in the pathogenesis of EB.
Assuntos
Displasia Ectodérmica , Epidermólise Bolhosa , Epidermólise Bolhosa/diagnóstico , Epidermólise Bolhosa/genética , Feminino , Humanos , Integrina beta4/genética , Mutação , PiloroRESUMO
Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.
Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Células Epidérmicas/metabolismo , Espermina/metabolismo , Adenosilmetionina Descarboxilase/genética , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epidérmicas/patologia , Técnicas de Silenciamento de Genes , Humanos , Fator 4 Semelhante a Kruppel/metabolismo , Camundongos , Poliaminas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Self-improving dystrophic epidermolysis bullosa is a rare subtype of dystrophic epidermolysis bullosa (DEB) characterized by significant improvement in skin fragility within the first few years of life. Genetic inheritance has previously been reported as autosomal dominant or recessive with both forms harboring mutations in COL7A1. To date, there have been no reports of this rare clinical entity from various Southeast Asian ethnicities. Here, we describe the clinical and molecular features of five patients from the Southeast Asia region who presented with predominantly acral-distributed blisters and erosions in the first few days of life. Blistering resolved over several months, without appearance of new blisters. By immunofluorescence, intraepidermal retention of Type VII collagen was observed in all patient skin biopsies when investigated with antibody staining. Genetic analysis of four patients revealed pathogenic variants in COL7A1 which have not been previously reported. The clinical diagnosis in these rare patients is confirmed with molecular histology and genetic characterization.
Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Predisposição Genética para Doença , Anormalidades da Pele/genética , Sudeste Asiático/epidemiologia , Biópsia , Pré-Escolar , Epidermólise Bolhosa Distrófica/diagnóstico , Epidermólise Bolhosa Distrófica/fisiopatologia , Epidermólise Bolhosa Distrófica/terapia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Anormalidades da Pele/diagnóstico , Anormalidades da Pele/fisiopatologia , Anormalidades da Pele/terapiaRESUMO
The skin microbial community is a multifunctional ecosystem aiding prevention of infections from transient pathogens, maintenance of host immune homeostasis, and skin health. A better understanding of the complex milieu of microbe-microbe and host-microbe interactions will be required to define the ecosystem's optimal function and enable rational design of microbiome targeted interventions. Malassezia, a fungal genus currently comprising 18 species and numerous functionally distinct strains, are lipid-dependent basidiomycetous yeasts and integral components of the skin microbiome. The high proportion of Malassezia in the skin microbiome makes understanding their role in healthy and diseased skin crucial to development of functional skin health knowledge and understanding of normal, healthy skin homeostasis. Over the last decade, new tools for Malassezia culture, detection, and genetic manipulation have revealed not only the ubiquity of Malassezia on skin but new pathogenic roles in seborrheic dermatitis, psoriasis, Crohn's disease, and pancreatic ductal carcinoma. Application of these tools continues to peel back the layers of Malassezia/skin interactions, including clear examples of pathogenicity, commensalism, and potential protective or beneficial activities creating mutualism. Our increased understanding of host- and microbe-specific interactions should lead to identification of key factors that maintain skin in a state of healthy mutualism or, in turn, initiate pathogenic changes. These approaches are leading toward development of new therapeutic targets and treatment options. This review discusses recent developments that have expanded our understanding of Malassezia's role in the skin microbiome, with a focus on its multiple roles in health and disease as commensal, pathogen, and protector.
Assuntos
Malassezia , Psoríase , Ecossistema , Humanos , Pele , SimbioseRESUMO
We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Filamentos Intermediários/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Proteínas Filagrinas , Imunofluorescência , Heterozigoto , Humanos , Repetições de Microssatélites/genética , Mycoplasma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/genéticaRESUMO
We have generated an induced pluripotent stem cell (iPSC) line KCLi002-A (iOP107) from a female donor, heterozygous for the loss-of-function mutation p.R2447X in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization of KCLi002-A line included molecular karyotyping, mutation screening using restriction enzyme digestion Sanger sequencing and next generation sequencing (NGS), whereas pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and in vivo teratoma assay.
Assuntos
Heterozigoto , Células-Tronco Pluripotentes Induzidas , Mutação com Perda de Função , Mutação de Sentido Incorreto , Proteínas S100 , Substituição de Aminoácidos , Linhagem Celular , Feminino , Proteínas Filagrinas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismoRESUMO
Yes-associated protein (YAP) is a mechanosensor protein and a downstream effector of the Hippo kinase pathway, which controls organ growth, cell proliferation, survival, maintenance and regeneration. Unphosphorylated YAP translocates to the nucleus where it acts as a cofactor of primarily the TEAD transcription factors to activate target gene transcription and cell proliferation. Perturbed YAP activation results in tumorigenesis. The pathways downstream of activated YAP that drive cell proliferation remain relatively unexplored. In this study, we employed YAP2-5SA-∆C transgenic mice, which overexpress a mildly activated YAP mutant protein in basal keratinocytes leading to increased proliferation of the epidermal stem/progenitor cell populations. We performed massively-parallel sequencing of skin biopsy mRNA (RNA-Seq) and found dysregulation of 1491 genes in YAP2-5SA-∆C skin, including many with roles in cell activation and proliferation. Furthermore, we found that 150 of these dysregulated genes harbored YAP/TEAD binding motifs in the 3' UTR, suggesting that these may be direct YAP/TEAD target genes in the control of epidermal regeneration. Further validation and functional characterization assays identified Plau and Tgfbr3 as prime candidate genes that may be activated by epidermal YAP activity in the mouse skin in vivo to promote keratinocyte proliferation. This study provides novel insights into the mechanisms regulated by YAP that control tissue homeostasis, and in particular in conditions where YAP is aberrantly activated such as in neoplastic and regenerative skin disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Queratinócitos/metabolismo , Proteoglicanas/genética , RNA Mensageiro/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Transcriptoma , Ativador de Plasminogênio Tipo Uroquinase/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Epiderme/metabolismo , Epiderme/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Queratinócitos/patologia , Camundongos , Camundongos Transgênicos , Motivos de Nucleotídeos , Ligação Proteica , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas de Sinalização YAPRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) patients suffer from chronic and repeatedly infected wounds predisposing them to the development of aggressive and life-threatening skin cancer in these areas. Vitamin D3 is an often neglected but critical factor for wound healing. Intact skin possesses the entire enzymatic machinery required to produce active 1-alpha,25-dihydroxyvitamin D3 (calcitriol), underscoring its significance to proper skin function. Injury enhances calcitriol production, inducing the expression of calcitriol target genes including the antimicrobial peptide cathelicidin (hCAP18), an essential component of the innate immune system and an important wound healing factor. We found significantly reduced hCAP18 expression in a subset of RDEB keratinocytes which could be restored by calcipotriol treatment. Reduced scratch closure in RDEB cell monolayers was enhanced up to 2-fold by calcipotriol treatment, and the secretome of calcipotriol-treated cells additionally showed increased antimicrobial activity. Calcipotriol exhibited anti-neoplastic effects, suppressing the clonogenicity and proliferation of RDEB tumor cells. The combined wound healing, anti-microbial, and anti-neoplastic effects indicate that calcipotriol may represent a vital therapeutic option for RDEB patients which we could demonstrate in a single-patient observation study.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Calcitriol/análogos & derivados , Fármacos Dermatológicos/farmacologia , Epidermólise Bolhosa/metabolismo , Queratinócitos/efeitos dos fármacos , Cicatrização , Idoso , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Calcitriol/farmacologia , Linhagem Celular , Células Cultivadas , Epidermólise Bolhosa/patologia , Humanos , Queratinócitos/metabolismo , Masculino , CatelicidinasRESUMO
Wound healing is a dynamic process involving gene-expression changes that drive re-epithelialization. Here, we describe an essential role for polyamine regulator AMD1 in driving cell migration at the wound edge. The polyamines, putrescine, spermidine, and spermine are small cationic molecules that play essential roles in many cellular processes. We demonstrate that AMD1 is rapidly upregulated following wounding in human skin biopsies. Knockdown of AMD1 with small hairpin RNAs causes a delay in cell migration that is rescued by the addition of spermine. We further show that spermine can promote cell migration in keratinocytes and in human ex vivo wounds, where it significantly increases epithelial tongue migration. Knockdown of AMD1 prevents the upregulation of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor on wounding and results in a failure in actin cytoskeletal reorganization at the wound edge. We demonstrate that keratinocytes respond to wounding by modulating polyamine regulator AMD1 in order to regulate downstream gene expression and promote cell migration. This article highlights a previously unreported role for the regulation of polyamine levels and ratios in cellular behavior and fate.
Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Movimento Celular/genética , Epiderme/fisiologia , Queratinócitos/fisiologia , Cicatrização , Ferimentos e Lesões/metabolismo , Citoesqueleto de Actina/metabolismo , Adenosilmetionina Descarboxilase/genética , Biópsia , Sinalização do Cálcio , Células Cultivadas , Humanos , RNA Interferente Pequeno/genética , Reepitelização/genética , Espermina/metabolismo , Regulação para Cima , Ferimentos e Lesões/genéticaRESUMO
Cole disease is a genodermatosis of pigmentation following a strict dominant mode of inheritance. In this study, we investigated eight patients affected with an overlapping genodermatosis after recessive inheritance. The patients presented with hypo- and hyperpigmented macules over the body, resembling dyschromatosis universalis hereditaria in addition to punctuate palmoplantar keratosis. By homozygosity mapping and whole-exome sequencing, a biallelic p.Cys120Arg mutation in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was identified in all patients. We found that this mutation, like those causing dominant Cole disease, impairs homodimerization of the ENPP1 enzyme that is mediated by its two somatomedin-B-like domains. Histological analysis revealed structural and molecular changes in affected skin that were likely to originate from defective melanocytes because keratinocytes do not express ENPP1. Consistently, RNA-sequencing analysis of patient-derived primary melanocytes revealed alterations in melanocyte development and in pigmentation signaling pathways. We therefore conclude that germline ENPP1 cysteine-specific mutations, primarily affecting the melanocyte lineage, cause a clinical spectrum of dyschromatosis, in which the p.Cys120Arg allele represents a recessive and more severe form of Cole disease.
Assuntos
Hipopigmentação/genética , Ceratodermia Palmar e Plantar/genética , Melaninas/biossíntese , Melanócitos/metabolismo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Biópsia , Cisteína/genética , Análise Mutacional de DNA , Feminino , Fibroblastos , Mutação em Linhagem Germinativa , Células HEK293 , Homozigoto , Humanos , Hipopigmentação/diagnóstico , Hipopigmentação/patologia , Queratinócitos/metabolismo , Ceratodermia Palmar e Plantar/diagnóstico , Ceratodermia Palmar e Plantar/patologia , Masculino , Linhagem , Diester Fosfórico Hidrolases/metabolismo , Cultura Primária de Células , Pirofosfatases/metabolismo , Índice de Gravidade de Doença , Pele/citologia , Pele/patologia , Sequenciamento do ExomaRESUMO
The nuclear lamina consists of A- and B-type lamins. Mutations in LMNA cause many human diseases, including progeria, a premature aging syndrome, whereas LMNB1 duplication causes adult-onset autosomal dominant leukodystrophy (ADLD). LMNB1 is reduced in cells from progeria patients, but the significance of this reduction is unclear. In this paper, we show that LMNB1 protein levels decline in senescent human dermal fibroblasts and keratinocytes, mediated by reduced transcription and inhibition of LMNB1 messenger ribonucleic acid (RNA) translation by miRNA-23a. This reduction is also observed in chronologically aged human skin tissue. To determine whether altered LMNB1 levels cause senescence, we either increased or reduced LMNB1. Both LMNB1 depletion and overexpression inhibited proliferation, but only LMNB1 overexpression induced senescence, which was prevented by telomerase expression or inactivation of p53. This phenotype was exacerbated by a simultaneous reduction of LMNA/C. Our results demonstrate that altering LMNB1 levels inhibits proliferation and are relevant to understanding the molecular pathology of ADLD.
Assuntos
Proliferação de Células , Senescência Celular , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Lamina Tipo B/metabolismo , Diferenciação Celular , Células Cultivadas , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Fibroblastos/patologia , Genótipo , Humanos , Queratinócitos/patologia , Lamina Tipo A/metabolismo , Lamina Tipo B/genética , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Lâmina Nuclear/metabolismo , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/metabolismo , Doença de Pelizaeus-Merzbacher/patologia , Fenótipo , Interferência de RNA , RNA Mensageiro/metabolismo , Envelhecimento da Pele , Telomerase/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Fermitin genes are highly conserved and encode cytocortex proteins that mediate integrin signalling. Fermitin 1 (Kindlin1) is implicated in Kindler syndrome, a human skin blistering disorder. We report the isolation of the three Fermitin orthologs from Xenopus laevis embryos and describe their developmental expression patterns. Fermitin 1 is expressed in the skin, otic and olfactory placodes, pharyngeal arches, pronephric duct, and heart. Fermitin 2 is restricted to the somites and neural crest. Fermitin 3 is expressed in the notochord, central nervous system, cement gland, ventral blood islands, vitelline veins, and myeloid cells. Our findings are consistent with the view that Fermitin 1 is generally expressed in the skin, Fermitin 2 in muscle, and Fermitin 3 in hematopoietic lineages. Moreover, we describe novel sites of Fermitin gene expression that extend our knowledge of this family. Our data provide a basis for further functional analysis of the Fermitin family in Xenopus laevis.
Assuntos
Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animais , Embrião não Mamífero/anatomia & histologia , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/genética , Distribuição Tecidual , Proteínas de Xenopus/genética , Xenopus laevis/anatomia & histologiaRESUMO
BACKGROUND: Keratitis-ichthyosis-deafness (KID) syndrome is a rare ectodermal dysplasia characterized by generalized erythrokeratotic plaques, sensorineural hearing loss, and vascularizing keratitis. Cutaneous changes and hearing loss typically present in early childhood, whereas ocular symptoms present later. Mutations in the connexin (Cx) 26 gene, GJB2, are now established to underlie many of the affected cases, with the majority of patients harboring the p.D50N mutation. METHODS: A rare patient demonstrating features of incomplete KID syndrome associated with an uncommon Cx26 gene mutation is described. RESULTS: The patient presented late in adolescence with partial features of KID syndrome. There was limited cutaneous involvement and the rare association of cystic acne. Both hearing impairment and ophthalmic involvement were mild in severity. Genetic mutation analysis revealed a previously described, rare mutation in GJB2, resulting in a glycine to arginine change at codon 12 (p.G12R). CONCLUSIONS: This report describes a patient exhibiting characteristics suggestive of a late-onset, incomplete form of KID syndrome with the GJB2 mutation (p.G12R). The p.G12R mutation has only been described in one other patient with KID syndrome, whose clinical presentation was not characterized.
Assuntos
Conexinas/genética , Surdez/genética , Ictiose/genética , Ceratite/genética , Mutação , Conexina 26 , Humanos , Masculino , Síndrome , Adulto JovemRESUMO
The initiation, growth, recurrence and metastasis of solid tumours, including squamous cell carcinoma of the head and neck region, have been related to the behaviour of a small subpopulation of 'tumour-initiating' cells. Cells with stem cell characteristics have also been identified in cell lines derived from cancers and the aim of the present work was to extend examination of such cells. Established cell lines were examined for their patterns of colony morphologies and staining, the presence of a Hoechst dye-excluding 'side population', expression of the putative stem cell markers CD44, CD133 and CD29, and their ability to grow as 'cancer spheroids'. Two cell lines, CaLH2 and CaLH3, recently generated from HNSCC tumour biopsies, were similarly examined. All cell lines showed a holoclone/meroclone/paraclone series of colony morphologies and cell sorting indicated that CD44 marker expression was related to clonogenicity. FACS analysis after exposure to Hoechst dye indicated that the CA1, H357 and UK1 cell lines contain a dye-excluding 'side population', a property associated with stem-like subpopulations. When held in suspension, all cell lines formed spheroids that could be re-passaged. These observations indicate that cell lines derived from HNSCC contain cells with stem cell properties and that such cell lines may provide experimental systems relevant to the behaviour of stem cells present in the tumours of origin and to their responses to therapy.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Antígenos CD/biossíntese , Benzimidazóis/metabolismo , Caderinas/biossíntese , Linhagem Celular Tumoral , Separação Celular , Células Clonais/patologia , Corantes Fluorescentes/metabolismo , Glicoproteínas/biossíntese , Humanos , Receptores de Hialuronatos/biossíntese , Integrina beta1/biossíntese , Células-Tronco Neoplásicas/metabolismo , Peptídeos , Esferoides Celulares/patologia , Ensaio Tumoral de Célula-TroncoRESUMO
Connexin 30 (Cx30) is a component of the gap junction complex. Dominant and recessive mutations in the GJB6 gene encoding Cx30 are associated with a variety of human inherited diseases primarily affecting the epidermis, hair, nail, and/or the inner ear. The underlying mechanism of disease associated with different GJB6 mutations such as the disruption of gap junction mediated intercellular communication is unknown. Towards understanding these disease mechanisms, transfection studies were performed in a keratinocyte cell line and in HeLa cells using EGFP tagged wildtype Cx30 and mutant Cx30 constructs harbouring dominant disease-associated GJB6 mutations. For all three of the skin disease-associated Cx30 mutations investigated, impaired trafficking of the protein to the plasma membrane was observed thus preventing the formation of functional Cx30 gap junctions. In contrast, the deafness-associated mutation T5M-Cx30/EGFP trafficked to the membrane but defective channel activity was observed following dye transfer studies.
Assuntos
Conexinas/genética , Surdez/genética , Mutação , Dermatopatias/genética , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Conexina 30 , Conexinas/metabolismo , DNA/genética , Surdez/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Junções Comunicantes/metabolismo , Genes Dominantes , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fenótipo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Dermatopatias/metabolismoRESUMO
Distinct germline mutations in the gene (GJB3) encoding connexin 31 (Cx31) underlie the skin disease erythrokeratoderma variabilis (EKV) or sensorineural hearing loss with/without peripheral neuropathy. Here we describe a number of functional analyses to investigate the effect of these different disease-associated Cx31 mutants on connexon trafficking and intercellular communication. Immunostaining of a biopsy taken from an EKV patient harbouring the R42P mutation revealed sparse epidermal staining of Cx31, and, when present, it had a perinuclear localization. Transfection and microinjection studies in both keratinocytes and fibroblast cell lines also demonstrated that R42P and four other EKV-associated mutant Cx31 proteins displayed defective trafficking to the plasma membrane. The deafness/neuropathy only mutant 66delD had primarily a cytoplasmic localization, but some protein was visualized at the plasma membrane in a few transfected cells. Both 66delD- and R32W-Cx31/EGFP proteins had significantly impaired dye transfer rates compared to wild-type Cx31/EGFP protein. A striking characteristic feature observed with the dominant skin disease Cx31 mutations was a high incidence of cell death. This was not observed with wild-type, R32W 66delD Cx31 proteins. In conclusion, we have identified some key cellular phenotypic differences with respect to disease-associated Cx31 mutations.