RESUMEN
OBJECTIVE: Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells. APPROACH AND RESULTS: We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites. CONCLUSIONS: We demonstrate that cingulin participates in the modulation of endothelial barrier function both in human cultured cells in vitro and in mouse brains in vivo. Understanding the role of cingulin in maintaining tight barriers in endothelia may allow developing new strategies for the treatment of vascular leak syndromes.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar , Células Endoteliales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Área Postrema/metabolismo , Proliferación Celular , Células Cultivadas , Claudina-5/metabolismo , Impedancia Eléctrica , Genotipo , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Fenotipo , Células de Purkinje/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Uniones Estrechas/metabolismo , Factores de Tiempo , Transfección , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR-induced non-epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR-induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR-induced DNA damage. IR partly reversed the pro-apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR-induced DNA damage and thereby might contribute to melanomagenesis.
Asunto(s)
Apoptosis/efectos de la radiación , Rayos Infrarrojos , Melanocitos/efectos de la radiación , Melanoma/etiología , Rayos Ultravioleta/efectos adversos , Daño del ADN/efectos de la radiación , Humanos , Recién Nacido , Cultivo Primario de CélulasRESUMEN
WFDC proteins such as peptidase inhibitor 3 and SLPI inhibit proteases in the epidermis and other tissues. In this study, we tested the hypothesis that further WFDC protein family members might contribute to epidermal homeostasis. We found that in addition to peptidase inhibitor 3 and SLPI, WFDC5 and WFDC12 were expressed in human epidermis. In contrast to WFDC5, the expression of WFDC12 was induced during the late differentiation of keratinocytes and was restricted to the outermost layer of live cells. Single-cell RNA sequencing demonstrated that WFDC12-positive keratinocytes were characterized by the upregulation of LCE mRNA expression and downregulated the expression of keratins and claudins. Immunogold-electron microscopy revealed the colocalization of WFDC12 with corneodesmosomes in the lower stratum corneum. WFDC12 was elevated in the affected skin of patients with psoriasis, atopic dermatitis, and Darier disease. By contrast, WFDC12 expression was strongly upregulated not only in the affected but even more so in clinically normal-appearing skin of patients with Netherton syndrome. Finally, functional analysis showed distinct inhibitory activity of WFDC12 on neutrophil elastase and epidermal kallikreinârelated peptidase. Altogether, our study identified WFDC12 as a marker of the last stage of epidermal keratinocyte differentiation and suggests that WFDC12 contributes to the control of protease activity in the stratum corneum.
Asunto(s)
Epidermis/enzimología , Queratinocitos/fisiología , Proteínas/fisiología , Inhibidores de Serina Proteinasa/fisiología , Diferenciación Celular , Células Cultivadas , Humanos , Queratinocitos/química , Queratinocitos/citología , Proteínas/análisis , Serina Proteasas/metabolismoRESUMEN
A patient with painful erosions of the oral cavity and the labia minora developed multifocal blisters in inter-triginous areas. These blisters eroded and evolved into papillomatous erosive vegetations. Histopathology and immunopathological investigations confirmed the diagnosis of pemphigus vegetans, mediated by IgG autoantibodies. The circulating IgG1 and IgG4 autoantibodies were exclusively directed against desmoglein 3, as shown by ELISA and indirect immunofluorescence studies. These IgG1 and IgG4 isotypes were also in vivo bound, as demonstrated with immunoperoxidase staining of perilesional skin. Our clinical, biochemical and immunopathological observations confirm the hypothesis that pemphigus vegetans is a variant of pemphigus vulgaris.
Asunto(s)
Pénfigo/clasificación , Pénfigo/diagnóstico , Anciano de 80 o más Años , Diagnóstico Diferencial , Femenino , HumanosRESUMEN
Paraneoplastic pemphigus (PNP) is a rare life-threatening autoimmune bullous skin disease which is an obligate paraneoplasma. A 34-year-old woman presented with recalcitrant stomatitis and a generalized lichenoid rash. A diagnosis of PNP was established based on clinical findings, immunofluorescence, histopathology and biochemistry. A localized mediastinal mass was found with CT imaging and excised. The histologic diagnosis was dendritic cell sarcoma. Despite removal of tumor and immunosuppressive therapy, the PNP progressed rapidly and the patient died of septic multiorgan failure.
Asunto(s)
Neoplasias del Mediastino/complicaciones , Neoplasias del Mediastino/diagnóstico , Síndromes Paraneoplásicos/complicaciones , Síndromes Paraneoplásicos/diagnóstico , Pénfigo/complicaciones , Pénfigo/diagnóstico , Adulto , Resultado Fatal , Femenino , HumanosRESUMEN
To investigate the role of the angiogenic cytokine vascular endothelial growth factor (VEGF) during pregnancy and lactation, we used mice in which VEGF had been inactivated in mammary gland epithelial cells. Pups born to mutant mothers failed to thrive, displaying little milk in their stomachs. However, when they were transferred to control mothers they developed normally. Investigation of the mammary gland morphology revealed that lobulo-alveolar expansion into the fat pad was not complete in lactating mutant glands, and an accumulation of fat globules was evident in their secretory epithelium. In contrast to control glands, lactating mutant glands failed to up-regulate mRNAs for genes involved in milk secretion. Blood vessel density was comparable in pregnant mice of both groups but was only half that of controls in lactating mutant mice. FITC-labeled albumin injected intravenously (i.v.) into lactating mice extravasated rapidly and accumulated in the mammary gland epithelial cells in control animals, but was almost completely retained within the vessels in the mutants. Injection of recombinant VEGF i.v. reversed this effect. These findings demonstrate that mammary epithelium-derived VEGF is partially dispensable for angiogenesis during pregnancy and lactation, and by regulating vascular function during lactation, this factor is crucial to mammary gland differentiation and milk production.
Asunto(s)
Células Epiteliales/metabolismo , Silenciador del Gen/fisiología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Diferenciación Celular/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Lactancia/fisiología , Masculino , Glándulas Mamarias Animales/fisiología , Ratones , Ratones Transgénicos , Leche/metabolismo , Embarazo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Adult wild-type mice are not supposed to be proper models for ultraviolet radiation (UVR)-induced melanoma since melanocytes are confined to hair follicles and cannot be sufficiently reached by UVR. On the other hand, in mutated mouse models used for melanoma research limitations, including an altered immune system and selection of affected pathways, lead to tumors phenotypically quite different from naturally occurring melanomas. We compared the distribution of epidermal melanocytes in UVR and not-UVR-exposed wild-type C57BL/6 mice. Starting at the age of 8 weeks, mice were exposed to physiologic doses of UVR three times weekly over 16 weeks. Back skin biopsies were taken 4, 8, 12 and 16 weeks after initiation of exposure, and stained for Melan-A, representing a highly selective marker for melanocytes. Surprisingly, after exposure to UVR, Melan-A positive cells were detected also in the interfollicular epidermis of C57BL/6 mice. We conclude that UVR is capable of inducing interfollicular epidermal melanocytes in wild-type mice.
Asunto(s)
Epidermis/efectos de la radiación , Antígeno MART-1/análisis , Melanocitos/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Animales , Biomarcadores/análisis , Biopsia , Modelos Animales de Enfermedad , Células Epidérmicas , Epidermis/metabolismo , Femenino , Folículo Piloso/citología , Folículo Piloso/efectos de la radiación , Humanos , Melanocitos/metabolismo , Melanoma/etiología , Melanoma/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
Epidermolytic hyperkeratosis (EHK) (OMIM 113800) is a generalized skin disease with mostly autosomal dominant inheritance, caused by mutations in keratin 1 or keratin 10. These genes are expressed in suprabasal epidermal layers, resulting in abnormal keratin-intermediate filament cytoskeleton. We present a male patient with generalized hyperkeratosis involving palms and soles. In lesional skin massive hyperkeratosis and cytolysis in the suprabasal layers of the epidermis were observed. Immunohistochemistry staining for keratin 1 (and keratin 10) showed abnormal clumping in suprabasal keratinocytes. By electron microscopy perinuclear intermediate filament clumps were detected in the keratinocytes. A heterozygous missense mutation, designated L187F, was identified in exon 1 of the keratin 1 gene by direct sequencing. This mutation was not detected in his unaffected parents, indicative of a de novo mutational event. The homologous mutation (L187F, also designated L7F) in basal keratin genes keratin 5 or -14 causes epidermolysis bullosa simplex. The amount of keratin 1-mRNA in the patient's skin was not altered compared to controls. We propose that the severe EHK phenotype observed in our patient results from a dominant negative effect of the L187F mutant Keratin 1 allele exerted on keratin 10, the associated partner-keratin. These findings should be helpful for genetic counseling, prenatal diagnosis and studying molecular structure-function relationship in EHK.
Asunto(s)
Hiperqueratosis Epidermolítica/genética , Queratina-1/genética , Queratodermia Palmoplantar/genética , Adulto , Humanos , Hiperqueratosis Epidermolítica/patología , Queratodermia Palmoplantar/patología , Masculino , Mutación Missense , ARN Mensajero/análisisAsunto(s)
Hepatitis Autoinmune/cirugía , Trasplante de Hígado , Penfigoide Benigno de la Membrana Mucosa/diagnóstico , Complicaciones Posoperatorias/diagnóstico , Dermatosis del Cuero Cabelludo/diagnóstico , Diagnóstico Diferencial , Femenino , Humanos , Persona de Mediana Edad , Penfigoide Benigno de la Membrana Mucosa/patología , Complicaciones Posoperatorias/patología , Cuero Cabelludo/patología , Dermatosis del Cuero Cabelludo/patologíaRESUMEN
BACKGROUND: IgA pemphigus is a rare pustular autoimmune disease with exclusive IgA anti-keratinocyte cell surface antibody reactivity. Two subtypes have been discerned: in the subcorneal pustular dermatosis type, desmocollin 1 has been identified as a targeted autoantigen, while in few cases of the intraepidermal neutrophilic type, IgA anti-desmoglein 1 or IgA anti-desmoglein 3 reactivity has been demonstrated. PATIENTS AND METHODS: A 48-year-old white male presented with generalized large confluent pustules. Skin pathology was assessed by histology and direct immunofluorescence analysis. IgG/IgA autoantibodies against desmoglein 1/3 and desmocollin 1 were measured by ELISA and indirect immunofluorescence using desmocollin 1 cDNA-transfected COS7 cells, respectively. RESULTS: Histopathology revealed subcorneal pustules and direct immunofluorescence microscopy exclusively showed in vivo bound IgA with an intercellular pattern in the epidermis. Desmocollin 1 was identified as a target of IgA autoantibodies by indirect immunofluorescence microscopy utilizing desmocollin 1 cDNA-transfected COS7 cells. In addition, IgA anti-desmoglein 1 reactivity was demonstrated by ELISA. Neither IgA anti-desmoglein 3 nor IgG anti-desmoglein 1/3 autoantibodies were present. CONCLUSIONS: Both desmocollin 1 and desmoglein 1 were autoantigens in this patient with IgA pemphigus and a distinct clinical presentation. To our knowledge, this is the first IgA pemphigus case with dual autoantibody reactivity.
Asunto(s)
Desmocolinas/inmunología , Desmogleína 1/inmunología , Inmunoglobulina A , Pénfigo/inmunología , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Antiinfecciosos/administración & dosificación , Antiinfecciosos/uso terapéutico , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Autoanticuerpos/análisis , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Cefamandol/administración & dosificación , Cefamandol/uso terapéutico , Dapsona/administración & dosificación , Dapsona/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulina A/inmunología , Masculino , Persona de Mediana Edad , Pomadas , Pénfigo/tratamiento farmacológico , Pénfigo/patología , Piel/patología , Sulfadiazina/administración & dosificación , Sulfadiazina/uso terapéutico , Factores de Tiempo , Resultado del TratamientoRESUMEN
We show that human melanoma cells produce retrovirus-like particles that exhibit reverse transcriptase activity, package sequences homologous to human endogenous retrovirus K (HERV-K), and contain mature forms of the Gag and Env proteins. We also demonstrate expression of the pol gene and of Gag, Env, and Rec proteins in human melanomas and metastases but not in melanocytes or normal lymph nodes. The data suggest that expression of retroviral genes and production of retroviral particles is activated during development of melanoma.
Asunto(s)
Retrovirus Endógenos/genética , Melanoma/virología , Secuencia de Bases , Retrovirus Endógenos/aislamiento & purificación , Retrovirus Endógenos/metabolismo , Productos del Gen env/biosíntesis , Productos del Gen gag/biosíntesis , Productos del Gen pol/biosíntesis , Humanos , Metástasis Linfática , Melanoma/metabolismo , Melanoma/secundario , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/secundario , Neoplasias Cutáneas/virologíaRESUMEN
The FDA-approved BRAF inhibitor vemurafenib achieves outstanding clinical response rates in patients with melanoma, but early resistance is common. Understanding the pathologic mechanisms of drug resistance and identification of effective therapeutic alternatives are key scientific challenges in the melanoma setting. Using proteomic techniques, including shotgun analysis and 2D-gel electrophoresis, we identified a comprehensive signature of the vemurafenib-resistant M24met in comparison with the vemurafenib-sensitive A375 melanoma cell line. The resistant cells were characterized by loss of differentiation, induction of transformation, enhanced expression of the lysosomal compartment, increased potential for metastasis, migration, adherence and Ca2(+) ion binding, enhanced expression of the MAPK pathway and extracellular matrix proteins, and epithelial-mesenchymal transformation. The main features were verified by shotgun analysis with QEXACTIVE orbitrap MS, electron microscopy, lysosomal staining, Western blotting, and adherence assay in a VM-1 melanoma cell line with acquired vemurafenib resistance. On the basis of the resistance profile, we were able to successfully predict that a novel resveratrol-derived COX-2 inhibitor, M8, would be active against the vemurafenib-resistant but not the vemurafenib-sensitive melanoma cells. Using high-throughput methods for cell line and drug characterization may thus offer a new way to identify key features of vemurafenib resistance, facilitating the design of effective rational therapeutic alternatives.
Asunto(s)
Resistencia a Antineoplásicos/genética , Indoles/farmacología , Indoles/uso terapéutico , Proteoma/genética , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Inhibidores de la Ciclooxigenasa 2/farmacología , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteómica/métodos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Vemurafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Cornification of keratinocytes involves the degradation of intracellular constituents which has led to the hypothesis that autophagy plays a role in this process. Mice, in which essential autophagy-related genes such as Atg7 are deleted systemically, die after birth and have not been characterized for potential epidermal defects. OBJECTIVE: This study tested whether autophagy is essential for epidermal barrier formation and function. METHODS: Atg7 was inactivated in epidermal keratinocytes by the Cre-loxP system under the control of the keratin K14 promoter (Atg7Δepi mice). Autophagic activity was detected using the GFP-microtubule-associated protein light chain 3 (GFP-LC3) reporter construct and Western blot analysis of LC3. Epidermal morphology was examined by histological and ultrastructural analyses, and barrier functions were assessed by dye diffusion and water loss assays. RESULTS: Suprabasal epidermal cells of normal mice contained GFP-LC3-labeled autophagosomes and epidermal lysates of these mice showed an excess of lipidated over non-lipidated LC3. These features of active autophagy were efficiently suppressed in Atg7Δepi epidermis. Atg7Δepi mice survived the perinatal period and were apparently healthy. Histologically, their epidermis was inconspicuous and ultrastructural analysis revealed no significant defect in cornification. There was however, an increase in the thickness of corneocytes in the back skin of mutant mice. Nevertheless, resistance to dye penetration into the skin and transepidermal water loss were normal in Atg7Δepi mice. CONCLUSION: This study demonstrates that autophagy is constitutively active in the epidermis but not essential for the barrier function of the skin.
Asunto(s)
Autofagia , Epidermis/metabolismo , Queratinocitos/metabolismo , Proteínas Asociadas a Microtúbulos/deficiencia , Absorción Cutánea , Animales , Proteína 7 Relacionada con la Autofagia , Diferenciación Celular , Células Cultivadas , Difusión , Epidermis/ultraestructura , Proteínas Fluorescentes Verdes , Queratina-14/genética , Queratinocitos/ultraestructura , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Permeabilidad , Regiones Promotoras Genéticas , Pérdida Insensible de AguaRESUMEN
Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.
Asunto(s)
Epidermis , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos , Rayos Ultravioleta/efectos adversos , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Diferenciación Celular/fisiología , Células Cultivadas , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Difusión , Células Epidérmicas , Epidermis/metabolismo , Epidermis/efectos de la radiación , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Proteínas Filagrina , Colorantes Fluorescentes/farmacocinética , Humanos , Isoquinolinas/farmacocinética , Queratina-1/metabolismo , Queratina-10/metabolismo , Queratina-2/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Queratinas/metabolismo , Metabolismo de los Lípidos , Microscopía Electrónica , Técnicas de Cultivo de Órganos , Permeabilidad , ARN Interferente Pequeño , Solubilidad , Ácido Urocánico/metabolismoRESUMEN
Various exogenous factors (eg, drugs, dietary antigens, trauma, infections, radiographs, and UV radiation) are known to induce or aggravate skin diseases. UV radiation in particular is known to induce or aggravate the autoimmune bullous diseases of pemphigus foliaceus, pemphigus vulgaris, and bullous pemphigoid. Its role in linear IgA dermatosis, however, is not well recognized. We report the second case of linear IgA dermatosis induced by intense sun exposure in which blistering was induced by UVA radiation. Furthermore, a review of the literature on photoinduced autoimmune bullous diseases and the wavelengths responsible for the induction of blistering is presented and several proposed mechanisms of action for the blister induction, including release or unmasking of antigens, promotion of antibody fixation by UV radiation, and launching of an inflammatory process, are discussed. We conclude that linear IgA dermatosis should be added to the list of autoimmune bullous diseases induced and/or aggravated by UV radiation.