RESUMEN
Mutations in the type I keratin 16 (Krt16) and its partner type II keratin 6 (Krt6a, Krt6b) cause pachyonychia congenita (PC), a disorder typified by dystrophic nails, painful hyperkeratotic calluses in glabrous skin, and lesions involving other epithelial appendages. The pathophysiology of these symptoms and its relationship to settings in which Krt16 and Krt6 are induced in response to epidermal barrier stress are poorly understood. We report that hyperkeratotic calluses arising in the glabrous skin of individuals with PC and Krt16 null mice share a gene expression signature enriched in genes involved in inflammation and innate immunity, in particular damage-associated molecular patterns. Transcriptional hyper-activation of damage-associated molecular pattern genes occurs following de novo chemical or mechanical irritation to ear skin and in spontaneously arising skin lesions in Krt16 null mice. Genome-wide expression analysis of normal mouse tail skin and benign proliferative lesions reveals a tight, context-dependent coregulation of Krt16 and Krt6 with genes involved in skin barrier maintenance and innate immunity. Our results uncover a role for Krt16 in regulating epithelial inflammation that is relevant to genodermatoses, psoriasis, and cancer and suggest a avenue for the therapeutic management of PC and related disorders.
Asunto(s)
Redes Reguladoras de Genes/inmunología , Inmunidad Innata/inmunología , Queratina-16/metabolismo , Queratina-6/metabolismo , Paquioniquia Congénita/inmunología , Animales , Western Blotting , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Humanos , Inmunidad Innata/genética , Ratones , Análisis por Micromatrices , Microscopía Electrónica de Transmisión , Paquioniquia Congénita/patología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Calcium-mediated signals play important roles in epidermal barrier formation, skin homoeostasis and wound repair. Calmodulin 4 (Calm4) is a small, Ca2+ -binding protein with strong expression in suprabasal keratinocytes. In mice, Calm4 first appears in the skin at the time of barrier formation, and its expression increases in response to epidermal barrier challenges. In this study, we report the generation of Calm4 knockout mice and provide evidence that Calm4 is dispensable for epidermal barrier formation, maintenance and repair.
Asunto(s)
Calmodulina/fisiología , Calpaína/fisiología , Epidermis/metabolismo , Animales , Proteínas de Unión al Calcio/química , Calmodulina/genética , Calpaína/genética , Movimiento Celular , Queratinocitos/citología , Operón Lac , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , Piel/metabolismo , Cicatrización de HeridasRESUMEN
This protocol describes the basic steps involved in conventional plasmid-based cloning. The goals are to insert a DNA fragment of interest into a receiving vector plasmid, transform the plasmid into E. coli, recover the plasmid DNA, and check for correct insertion events.
Asunto(s)
Clonación Molecular/métodos , ADN/aislamiento & purificación , Vectores Genéticos , ADN/genética , Escherichia coli , Plásmidos , Reacción en Cadena de la PolimerasaRESUMEN
To create electro-competent E. coli and transform them with a plasmid of choice via electroporation.
Asunto(s)
Electroporación/métodos , Escherichia coli/genética , Transformación Bacteriana/genética , Plásmidos/genéticaRESUMEN
This protocol describes the preparation of several commonly used liquid growth media for E. coli.
Asunto(s)
Técnicas Bacteriológicas/métodos , Medios de Cultivo , Escherichia coli/crecimiento & desarrollo , Técnicas Bacteriológicas/instrumentación , Medios de Cultivo/químicaRESUMEN
Keratin 16 (KRT16 in human, Krt16 in mouse), a type I intermediate filament protein, is constitutively expressed in epithelial appendages and is induced in the epidermis upon wounding and other stressors. Mutations altering the coding sequence of KRT16 cause pachyonychia congenita (PC), a rare autosomal dominant disorder characterized by hypertrophic nail dystrophy, oral leukokeratosis, and palmoplantar keratoderma (PPK). PPK associated with PC is extremely painful and compromises patient mobility, making it the most debilitating PC symptom. In this study, we show that, although inherited in a recessive manner, the inactivation of Krt16 in mice consistently causes oral lesions as well as PPK-like hyperkeratotic calluses on Krt16(-/-) front and hind paws, which severely compromise the animals' ability to walk. Our findings call into question the view that PC-related PPK arises exclusively as a gain-of-function on account of dominantly acting mutated keratins, and highlight the key role of modifiers in the clinical heterogeneity of PC symptoms.