RESUMEN
Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11ß-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11ß-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11ß-HSD1 inhibition by carbenoxolone (CBX) in mice treated with corticosterone (CORT) or vehicle for 6 weeks. Mice were treated bidaily with topical CBX or vehicle (VEH) 7 days before wounding and during wound healing. CORT mice displayed skin thinning and impaired wound healing but also increased epidermal integrity. 11ß-HSD1 activity was elevated in unwounded CORT skin and was inhibited by CBX. CORT mice treated with CBX displayed 51%, 59%, and 100% normalization of wound healing, epidermal thickness, and epidermal integrity, respectively. Gene expression studies revealed normalization of interleukin 6, keratinocyte growth factor, collagen 1, collagen 3, matrix metalloproteinase 9, and tissue inhibitor of matrix metalloproteinase 4 by CBX during wound healing. Importantly, proinflammatory cytokine expression and resolution of inflammation were unaffected by 11ß-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11ß-HSD1 inhibition can limit the cutaneous effects of GC excess, which may improve the safety profile of systemic steroids and the prognosis of chronic wounds.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Carbenoxolona/uso terapéutico , Corticosterona/envenenamiento , Erupciones por Medicamentos/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Glucocorticoides/envenenamiento , Piel/efectos de los fármacos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Administración Tópica , Animales , Antiinflamatorios/envenenamiento , Carbenoxolona/administración & dosificación , Carbenoxolona/efectos adversos , Corticosterona/sangre , Corticosterona/farmacocinética , Citocinas/antagonistas & inhibidores , Citocinas/genética , Citocinas/metabolismo , Erupciones por Medicamentos/etiología , Erupciones por Medicamentos/metabolismo , Erupciones por Medicamentos/patología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/efectos adversos , Epidermis/efectos de los fármacos , Epidermis/inmunología , Epidermis/metabolismo , Epidermis/patología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/inmunología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/sangre , Glucocorticoides/farmacocinética , Tejido de Granulación/efectos de los fármacos , Tejido de Granulación/inmunología , Tejido de Granulación/metabolismo , Tejido de Granulación/patología , Ratones Pelados , Tamaño de los Órganos/efectos de los fármacos , Piel/lesiones , Piel/metabolismo , Piel/patología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Harlequin Ichthyosis is a severe skin disease caused by mutations in the human gene encoding ABCA12. Here, we characterize a novel mutation in intron 29 of the mouse Abca12 gene that leads to the loss of a 5' splice donor site and truncation of the Abca12 RNA transcript. Homozygous mutants of this smooth skin or smsk allele die perinatally with shiny translucent skin, typical of animal models of Harlequin Ichthyosis. Characterization of smsk mutant skin showed that the delivery of glucosylceramides and CORNEODESMOSIN was defective, while ultrastructural analysis revealed abnormal lamellar bodies and the absence of lipid lamellae in smsk epidermis. Unexpectedly, mutant stratum corneum remained intact when subjected to harsh chemical dissociation procedures. Moreover, both KALLIKREIN 5 and -7 were drastically decreased, with retention of desmoplakin in mutant SC. In cultured wild type keratinocytes, both KALLIKREIN 5 and -7 colocalized with ceramide metabolites following calcium-induced differentiation. Reducing the intracellular levels of glucosylceramide with a glucosylceramide synthase inhibitor resulted in decreased secretion of KALLIKREIN proteases by wild type keratinocytes, but not by smsk mutant keratinocytes. Together, these findings suggest an essential role for ABCA12 in transferring not only lipids, which are required for the formation of multilamellar structures in the stratum corneum, but also proteolytic enzymes that are required for normal desquamation. Smsk mutant mice recapitulate many of the pathological features of HI and can be used to explore novel topical therapies against a potentially lethal and debilitating neonatal disease.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Ictiosis Lamelar/genética , Ictiosis Lamelar/patología , Fenotipo , Piel/metabolismo , Piel/patología , Alelos , Animales , Secuencia de Bases , Ceramidas/metabolismo , Mapeo Cromosómico , Desmosomas/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Epidermis/ultraestructura , Exones , Genes Recesivos , Glucosilceramidas/metabolismo , Ictiosis Lamelar/terapia , Calicreínas/metabolismo , Queratinocitos/metabolismo , Ratones , Modelos Biológicos , Mutación , Permeabilidad , Análisis de Secuencia de ADN , Piel/ultraestructura , Trasplante de PielRESUMEN
Detrimental consequences of ultraviolet radiation (UVR) in skin include photoageing, immunosuppression and photocarcinogenesis, processes also significantly regulated by local glucocorticoid (GC) availability. In man, the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) generates the active GC cortisol from cortisone (or corticosterone from 11-dehydrocorticosterone in rodents). 11ß-HSD1 oxo-reductase activity requires the cofactor NADPH, generated by hexose-6-phosphate dehydrogenase. We previously demonstrated increased 11ß-HSD1 levels in skin obtained from photoexposed versus photoprotected anatomical regions. However, the direct effect of UVR on 11ß-HSD1 expression remains to be elucidated. To investigate the cutaneous regulation of 11ß-HSD1 following UVR in vivo, the dorsal skin of female SKH1 mice was irradiated with 50, 100, 200 and 400 mJ/cm(2) UVB. Measurement of transepidermal water loss, 11ß-HSD1 activity, mRNA/protein expression and histological studies was taken at 1, 3 and 7 days postexposure. 11ß-HSD1 and hexose-6-phosphate dehydrogenase mRNA expression peaked 1 day postexposure to 400 mJ/cm(2) UVB before subsequently declining (days 3 and 7). Corresponding increases in 11ß-HSD1 protein and enzyme activity were observed 3 days postexposure coinciding with reduced GC receptor mRNA expression. Immunofluorescence studies revealed 11ß-HSD1 localization to hyperproliferative epidermal keratinocytes in UVB-exposed skin. 11ß-HSD1 expression and activity were also induced by 200 and 100 (but not 50) mJ/cm(2) UVB and correlated with increased transepidermal water loss (indicative of barrier disruption). UVB-induced 11ß-HSD1 activation represents a novel mechanism that may contribute to the regulation of cutaneous responses to UVR exposure.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/biosíntesis , Epidermis/enzimología , Epidermis/efectos de la radiación , Rayos Ultravioleta/efectos adversos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Agua Corporal/metabolismo , Agua Corporal/efectos de la radiación , Proliferación Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Inducción Enzimática/efectos de la radiación , Epidermis/patología , Femenino , Glucocorticoides/metabolismo , Humanos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMEN
Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated clinically with ichthyosis vulgaris and atopic dermatitis and molecular genetically with filaggrin-null mutations. In 20 KP patients and 20 matched controls, we assessed the filaggrin and claudin 1 genotypes, the phenotypes by dermatoscopy, and the morphology by light and transmission electron microscopy. Thirty-five percent of KP patients displayed filaggrin mutations, demonstrating that filaggrin mutations only partially account for the KP phenotype. Major histologic and dermatoscopic findings of KP were hyperkeratosis, hypergranulosis, mild T helper cell type 1-dominant lymphocytic inflammation, plugging of follicular orifices, striking absence of sebaceous glands, and hair shaft abnormalities in KP lesions but not in unaffected skin sites. Changes in barrier function and abnormal paracellular permeability were found in both interfollicular and follicular stratum corneum of lesional KP, which correlated ultrastructurally with impaired extracellular lamellar bilayer maturation and organization. All these features were independent of filaggrin genotype. Moreover, ultrastructure of corneodesmosomes and tight junctions appeared normal, immunohistochemistry for claudin 1 showed no reduction in protein amounts, and molecular analysis of claudin 1 was unremarkable. Our findings suggest that absence of sebaceous glands is an early step in KP pathogenesis, resulting in downstream hair shaft and epithelial barrier abnormalities.
Asunto(s)
Anomalías Múltiples/patología , Enfermedad de Darier/patología , Epidermis/anomalías , Cejas/anomalías , Cabello/anomalías , Proteínas de Filamentos Intermediarios/deficiencia , Glándulas Sebáceas/anomalías , Anomalías Múltiples/genética , Adulto , Anciano , Claudina-1/metabolismo , Enfermedad de Darier/genética , Dermoscopía , Desmosomas/metabolismo , Epidermis/ultraestructura , Cejas/patología , Femenino , Proteínas Filagrina , Genotipo , Cabello/ultraestructura , Humanos , Proteínas de Filamentos Intermediarios/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Permeabilidad , Fenotipo , Glándulas Sebáceas/patología , Glándulas Sebáceas/ultraestructura , Adulto JovenRESUMEN
IMPORTANCE: Secondary infections and impaired desquamation complicate certain inherited ichthyoses, but their cellular basis remains unknown. In healthy human epidermis, the antimicrobial peptides cathelicidin (LL-37) and human ß-defensin 2 (HBD2), as well as the desquamatory protease kallikrein-related peptidase 7 (KLK7), are delivered to the stratum corneum (SC) interstices by lamellar body (LB) exocytosis. OBJECTIVE: To assess whether abnormalities in the LB secretory system could account for increased risk of infections and impaired desquamation in inherited ichthyoses with known abnormalities in LB assembly (Harlequin ichthyosis [HI]), secretion (epidermolytic ichthyosis [EI]), or postsecretory proteolysis (Netherton syndrome [NS]). DESIGN, SETTING, AND PARTICIPANTS: Samples from library material were taken from patients with HI, EI, NS, and other ichthyoses, but with a normal LB secretory system, and in healthy controls and were evaluated by electron microscopy and immunohistochemical analysis from July 1, 2010, through March 31, 2013. MAIN OUTCOME AND MEASURES: Changes in LB secretion and in the fate of LB-derived enzymes and antimicrobial peptides in ichthyotic patients vs healthy controls. RESULTS: In healthy controls and patients with X-linked ichthyosis, neutral lipid storage disease with ichthyosis, and Gaucher disease, LB secretion is normal, and delivery of LB-derived proteins and LL-37 immunostaining persists high into the SC. In contrast, proteins loaded into nascent LBs and their delivery to the SC interstices decrease markedly in patients with HI, paralleled by reduced immunostaining for LL-37, HBD2, and KLK7 in the SC. In patients with EI, the cytoskeletal abnormality impairs the exocytosis of LB contents and thus results in decreased LL-37, HBD2, and KLK7 secretion, causing substantial entombment of these proteins within the corneocyte cytosol. Finally, in patients with NS, although abundant enzyme proteins loaded in parallel with accelerated LB production, LL-37 disappears, whereas KLK7 levels increase markedly in the SC. CONCLUSIONS AND RELEVANCE: Together, these results suggest that diverse abnormalities in the LB secretory system account for the increased risk of secondary infections and impaired desquamation in patients with HI, EI, and NS.
Asunto(s)
Exocitosis , Ictiosis/complicaciones , Enfermedades Cutáneas Infecciosas/etiología , Piel/patología , Péptidos Catiónicos Antimicrobianos/metabolismo , Estudios de Casos y Controles , Humanos , Ictiosis/genética , Ictiosis/patología , Inmunohistoquímica , Calicreínas/metabolismo , Microscopía Electrónica , Enfermedades Cutáneas Infecciosas/patología , beta-Defensinas/metabolismo , CatelicidinasAsunto(s)
Epidermis/efectos de los fármacos , Hesperidina/administración & dosificación , Animales , Proliferación Celular , Epidermis/metabolismo , Hesperidina/química , Homeostasis , Concentración de Iones de Hidrógeno , Inflamación , Lípidos/química , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Permeabilidad , Antígeno Nuclear de Célula en Proliferación/metabolismo , Envejecimiento de la Piel , Fenómenos Fisiológicos de la Piel , Cicatrización de HeridasRESUMEN
Multilayered human keratinocyte cultures increasingly are used to model human epidermis. Until now, studies utilizing human epidermal equivalents (HEEs) have been limited because previous preparations do not establish a normal epidermal permeability barrier. In this report, we show that reducing environmental humidity to 50% relative humidity yields HEEs that closely match human postnatal epidermis and have enhanced repair of the permeability barrier. These cultures display low transepidermal water loss and possess a calcium and pH gradient that resembles those seen in human epidermis. These cultures upregulate glucosylceramide synthase and make normal-appearing lipid lamellar bilayers. The epidermal permeability barrier of these cultures can be perturbed, using the identical tools previously described for human skin, and recover in the same time course seen during in vivo barrier recovery. These cultures will be useful for basic and applied studies on epidermal barrier function.
Asunto(s)
Epidermis/crecimiento & desarrollo , Epidermis/fisiología , Humedad , Células Cultivadas , Células Epidérmicas , Epidermis/ultraestructura , Regulación de la Expresión Génica , Humanos , Recién Nacido , Iones , Masculino , Proteínas/metabolismoRESUMEN
Systemic and topical glucocorticoids (GC) can cause significant adverse effects not only on the dermis, but also on epidermal structure and function. In epidermis, a striking GC-induced alteration in permeability barrier function occurs that can be attributed to an inhibition of epidermal mitogenesis, differentiation and lipid production. As prior studies in normal hairless mice demonstrated that topical applications of a flavonoid ingredient found in citrus, hesperidin, improve epidermal barrier function by stimulating epidermal proliferation and differentiation, we assessed here whether its topical applications could prevent GC-induced changes in epidermal function in murine skin and the basis for such effects. When hairless mice were co-treated topically with GC and 2% hesperidin twice-daily for 9 days, hesperidin co-applications prevented the expected GC-induced impairments of epidermal permeability barrier homoeostasis and stratum corneum (SC) acidification. These preventive effects could be attributed to a significant increase in filaggrin expression, enhanced epidermal ß-glucocerebrosidase activity and accelerated lamellar bilayer maturation, the last two likely attributable to a hesperidin-induced reduction in stratum corneum pH. Furthermore, co-applications of hesperidin with GC largely prevented the expected GC-induced inhibition of epidermal proliferation. Finally, topical hesperidin increased epidermal glutathione reductase mRNA expression, which could counteract multiple functional negative effects of GC on epidermis. Together, these results show that topical hesperidin prevents GC-induced epidermal side effects by divergent mechanisms.
Asunto(s)
Clobetasol/efectos adversos , Clobetasol/antagonistas & inhibidores , Epidermis/efectos de los fármacos , Glucocorticoides/efectos adversos , Glucocorticoides/antagonistas & inhibidores , Hesperidina/administración & dosificación , Administración Tópica , Animales , Proliferación Celular/efectos de los fármacos , Clobetasol/administración & dosificación , Epidermis/patología , Epidermis/fisiopatología , Femenino , Proteínas Filagrina , Glucocorticoides/administración & dosificación , Glutatión Reductasa/genética , Proteínas de Filamentos Intermediarios/genética , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Ratones Pelados , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Acute psychological stress (PS) mobilizes metabolic responses that are of immediate benefit to the host, but the current medical paradigm holds that PS exacerbates systemic and cutaneous inflammatory disorders. Although the adverse consequences of PS are usually attributed to neuroimmune mechanisms, PS also stimulates an increase in endogenous glucocorticoids (GCs) that compromises permeability barrier homeostasis, stratum corneum cohesion, wound healing, and epidermal innate immunity in normal skin. Yet, if such PS-induced increases in GC were uniformly harmful, natural selection should have eliminated this component of the stress response. Hence, we hypothesized here instead that stress-induced elevations in endogenous GC could benefit, rather than aggravate, cutaneous function and reduce inflammation in three immunologically diverse mouse models of inflammatory diseases. Indeed, superimposed exogenous (motion-restricted) stress reduced, rather than aggravated inflammation and improved epidermal function in all three models, even normalizing serum IgE levels in the atopic dermatitis model. Elevations in endogenous GC accounted for these apparent benefits, because coadministration of mifepristone prevented stress-induced disease amelioration. Thus, exogenous stress can benefit rather than aggravate cutaneous inflammatory dermatoses through the anti-inflammatory activity of increased endogenous GC.
Asunto(s)
Glucocorticoides/metabolismo , Enfermedades de la Piel/metabolismo , Enfermedades de la Piel/psicología , Piel/metabolismo , Estrés Psicológico/metabolismo , Animales , Permeabilidad de la Membrana Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Homeostasis/fisiología , Inmunidad Innata/fisiología , Inmunoglobulina E/sangre , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos C57BL , Piel/fisiopatología , Enfermedades de la Piel/fisiopatología , Estrés Psicológico/fisiopatología , Cicatrización de Heridas/fisiologíaRESUMEN
Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening.
Asunto(s)
Células Madre Embrionarias/metabolismo , Epidermis/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Metilación de ADN , Células Madre Embrionarias/citología , Transición Epitelial-Mesenquimal , Humanos , Células Madre Pluripotentes Inducidas/citología , Queratina-14/genética , Queratina-14/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Permeabilidad , Análisis de Componente Principal , Teratoma/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Humans with darkly pigmented skin display superior permeability barrier function in comparison with humans with lightly pigmented skin. The reduced pH of the stratum corneum (SC) of darkly pigmented skin could account for enhanced function, because acidifying lightly pigmented human SC resets barrier function to darkly pigmented levels. In SKH1 (nonpigmented) versus SKH2/J (pigmented) hairless mice, we evaluated how a pigment-dependent reduction in pH could influence epidermal barrier function. Permeability barrier homeostasis is enhanced in SKH2/J versus SKH1 mice, correlating with a reduced pH in the lower SC that colocalizes with the extrusion of melanin granules. Darkly pigmented human epidermis also shows substantial melanin extrusion in the outer epidermis. Both acute barrier disruption and topical basic pH challenges accelerate reacidification of SKH2/J (but not SKH1) SC, while inducing melanin extrusion. SKH2/J mice also display enhanced expression of the SC acidifying enzyme, secretory phospholipase A2f (sPLA2f). Enhanced barrier function of SKH2/J mice could be attributed to enhanced activity of two acidic pH-dependent, ceramide-generating enzymes, ß-glucocerebrosidase and acidic sphingomyelinase, leading to accelerated maturation of SC lamellar bilayers. Finally, organotypic cultures of darkly pigmented human keratinocytes display enhanced barrier function in comparison with lightly pigmented cultures. Together, these results suggest that the superior barrier function of pigmented epidermis can be largely attributed to the pH-lowering impact of melanin persistence/extrusion and enhanced sPLA2f expression.
Asunto(s)
Ácidos/metabolismo , Epidermis/metabolismo , Fosfolipasas A2 Grupo II/metabolismo , Homeostasis/genética , Melanocitos/metabolismo , Pigmentación de la Piel/fisiología , Animales , Ceramidas/biosíntesis , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Células Epidérmicas , Femenino , Glucosilceramidasa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Queratinocitos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Masculino , Melaninas/metabolismo , Melanocitos/ultraestructura , Ratones Pelados , Microscopía Electrónica , Técnicas de Cultivo de Órganos , Comunicación Paracrina/fisiología , Permeabilidad , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Glucocorticoid (GC) excess inhibits wound healing causing increased patient discomfort and infection risk. 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activates GCs (converting 11-dehydrocorticosterone to corticosterone in rodents) in many tissues including skin, where de novo steroidogenesis from cholesterol has also been reported. To examine the regulation of 11ß-HSD1 and steroidogenic enzyme expression during wound healing, 5âmm wounds were generated in female SKH1 mice and compared at days 0, 2, 4, 8, 14, and 21 relative to unwounded skin. 11ß-HSD1 expression (mRNA and protein) and enzyme activity were elevated at 2 and 4 days post-wounding, with 11ß-HSD1 localizing to infiltrating inflammatory cells. 11ß-HSD2 (GC-deactivating) mRNA expression and activity were undetectable. Although several steroidogenic enzymes displayed variable expression during healing, expression of the final enzyme required for the conversion of 11-deoxycorticosterone to corticosterone, 11ß-hydroxylase (CYP11B1), was lacking in unwounded skin and post-wounding. Consequently, 11-deoxycorticosterone was the principal progesterone metabolite in mouse skin before and after wounding. Our findings demonstrate that 11ß-HSD1 activates considerably more corticosterone than is generated de novo from progesterone in mouse skin and drives GC exposure during healing, demonstrating the basis for 11ß-HSD1 inhibitors to accelerate wound repair.
Asunto(s)
Glucocorticoides/metabolismo , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones Pelados , Receptores de Glucocorticoides/metabolismo , Piel/enzimologíaRESUMEN
We recently discovered a regulatory mechanism that stimulates the production of the multifunctional antimicrobial peptide cathelicidin antimicrobial peptide (CAMP). In response to subtoxic levels of ER stress, increased sphingosine-1-phosphate (S1P) production activates an NFκBî²C/EBPα-dependent pathway that enhances CAMP production in cultured human keratinocytes. As the multifunctional stilbenoid compound resveratrol (RESV) increases ceramide (Cer) levels, a precursor of S1P, we hypothesized and assessed whether RESV could exploit the same pathway to regulate CAMP production. Accordingly, RESV significantly increased Cer and S1P levels in cultured keratinocytes, paralleled by increased CAMP mRNA/protein expression. Furthermore, topical RESV also increased murine CAMP mRNA/protein expression in mouse skin. Conversely, blockade of Cer-->sphingosine-->S1P metabolic conversion, with specific inhibitors of ceramidase or sphingosine kinase, attenuated the expected RESV-mediated increase in CAMP expression. The RESV-induced increase in CAMP expression required both NF-κB and C/EBPα transactivation. Moreover, conditioned media from keratinocytes treated with RESV significantly suppressed Staphylococcus aureus growth. Finally, topical RESV, if not coapplied with a specific inhibitor of sphingosine kinase, blocked S. aureus invasion into murine skin. These results demonstrate that the dietary stilbenoid RESV stimulates S1P signaling of CAMP production through an NF-κB-->C/EBPα-dependent mechanism, leading to enhanced antimicrobial defense against exogenous microbial pathogens.
Asunto(s)
Antioxidantes/farmacología , Catelicidinas/metabolismo , Lisofosfolípidos/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Estilbenos/farmacología , Animales , Péptidos Catiónicos Antimicrobianos , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Catelicidinas/inmunología , Línea Celular Transformada , AMP Cíclico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/inmunología , Células Epidérmicas , Epidermis/efectos de los fármacos , Epidermis/inmunología , Femenino , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Ratones , Ratones Pelados , FN-kappa B/metabolismo , Resveratrol , Transducción de Señal/inmunología , Esfingosina/metabolismo , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Infecciones Cutáneas Estafilocócicas/inmunologíaRESUMEN
The beneficial effects of certain herbal medicines on cutaneous function have been appreciated for centuries. Among these agents, chrysanthemum extract, apigenin, has been used for skin care, particularly in China, for millennia. However, the underlying mechanisms by which apigenin benefits the skin are not known. In this study, we first determined whether topical apigenin positively influences permeability barrier homoeostasis, and then the basis thereof. Hairless mice were treated topically with either 0.1% apigenin or vehicle alone twice daily for 9 days. At the end of the treatments, permeability barrier function was assessed with either an electrolytic water analyzer or a Tewameter. Our results show that topical apigenin significantly enhanced permeability barrier homoeostasis after tape stripping, although basal permeability barrier function remained unchanged. Improved barrier function correlated with enhanced filaggrin expression and lamellar body production, which was paralleled by elevated mRNA levels for the epidermal ABCA12. The mRNA levels for key lipid synthetic enzymes also were upregulated by apigenin. Finally, both cathelicidin-related peptide and mouse beta-defensin 3 immunostaining were increased by apigenin. We conclude that topical apigenin improves epidermal permeability barrier function by stimulating epidermal differentiation, lipid synthesis and secretion, as well as cutaneous antimicrobial peptide production. Apigenin could be useful for the prevention and treatment of skin disorders characterized by permeability barrier dysfunction, associated with reduced filaggrin levels and impaired antimicrobial defenses, such as atopic dermatitis.
Asunto(s)
Apigenina/administración & dosificación , Apigenina/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Epidermis/fisiología , Homeostasis/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Administración Tópica , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Chrysanthemum , Relación Dosis-Respuesta a Droga , Células Epidérmicas , Epidermis/efectos de los fármacos , Femenino , Proteínas Filagrina , Homeostasis/fisiología , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Ratones Pelados , Modelos Animales , Piel/citología , Piel/efectos de los fármacos , Piel/metabolismo , beta-Defensinas/metabolismo , CatelicidinasRESUMEN
Systemic antagonists of the histamine type 1 and 2 receptors (H1/2r) are widely used as anti-pruritics and central sedatives, but demonstrate only modest anti-inflammatory activity. Because many inflammatory dermatoses result from defects in cutaneous barrier function, and because keratinocytes express both Hr1 and Hr2, we hypothesized that H1/2r antagonists might be more effective if they were used topically to treat inflammatory dermatoses. Topical H1/2r antagonists additively enhanced permeability barrier homeostasis in normal mouse skin by the following mechanisms: (i) stimulation of epidermal differentiation, leading to thickened cornified envelopes; and (ii) enhanced epidermal lipid synthesis and secretion. As barrier homeostasis was enhanced to a comparable extent in mast cell-deficient mice, with no further improvement following application of topical H1/2r antagonists, H1/2r antagonists likely oppose mast cell-derived histamines. In four immunologically diverse, murine disease models, characterized by either inflammation alone (acute irritant contact dermatitis, acute allergic contact dermatitis) or by prominent barrier abnormalities (subacute allergic contact dermatitis, atopic dermatitis), topical H1/2r agonists aggravated, whereas H1/2r antagonists improved, inflammation and/or barrier function. The apparent ability of topical H1r/2r antagonists to target epidermal H1/2r could translate into increased efficacy in the treatment of inflammatory dermatoses, likely due to decreased inflammation and enhanced barrier function. These results could shift current paradigms of antihistamine utilization from a predominantly systemic to a topical approach.
Asunto(s)
Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/inmunología , Epidermis/efectos de los fármacos , Epidermis/inmunología , Antagonistas de los Receptores Histamínicos/farmacología , Administración Tópica , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Cimetidina/farmacología , Dermatitis por Contacto/tratamiento farmacológico , Dermatitis por Contacto/inmunología , Difenhidramina/farmacología , Modelos Animales de Enfermedad , Epidermis/metabolismo , Femenino , Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Homeostasis/efectos de los fármacos , Homeostasis/inmunología , Irritantes/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/inmunología , Ratones , Ratones Pelados , Permeabilidad/efectos de los fármacosRESUMEN
A variety of external perturbations can induce endoplasmic reticulum (ER) stress, followed by stimulation of epithelial cells to produce an innate immune element, the cathelicidin antimicrobial peptide (CAMP). ER stress also increases production of the proapoptotic lipid ceramide and its antiapoptotic metabolite, sphingosine-1-phosphate (S1P). We demonstrate here that S1P mediates ER stress-induced CAMP generation. Cellular ceramide and S1P levels rose in parallel with CAMP levels following addition of either exogenous cell-permeating ceramide (C2Cer), which increases S1P production, or thapsigargin (an ER stressor), applied to cultured human skin keratinocytes or topically to mouse skin. Knockdown of S1P lyase, which catabolizes S1P, enhanced ER stress-induced CAMP production in cultured cells and mouse skin. These and additional inhibitor studies show that S1P is responsible for ER stress-induced upregulation of CAMP expression. Increased CAMP expression is likely mediated via S1P-dependent NF-κB-C/EBPα activation. Finally, lysates of both ER-stressed and S1P-stimulated cells blocked growth of virulent Staphylococcus aureus in vitro, and topical C2Cer and LL-37 inhibited invasion of Staphylococcus aureus into murine skin. These studies suggest that S1P generation resulting in increased CAMP production comprises a novel regulatory mechanism of epithelial innate immune responses to external perturbations, pointing to a new therapeutic approach to enhance antimicrobial defense.
Asunto(s)
Catelicidinas/inmunología , Inmunidad Innata , Queratinocitos/inmunología , Lisofosfolípidos/inmunología , Piel/inmunología , Esfingosina/análogos & derivados , Animales , Péptidos Catiónicos Antimicrobianos , Proteína alfa Potenciadora de Unión a CCAAT/inmunología , Catelicidinas/genética , Células Cultivadas , Ceramidas/inmunología , Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica , Humanos , Queratinocitos/microbiología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Piel/microbiología , Esfingosina/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/inmunologíaRESUMEN
Corneocyte desquamation has been ascribed to the following: 1) proteolytic degradation of corneodesmosomes (CDs); 2) disorganization of extracellular lamellar bilayers; and/or 3) "swell-shrinkage-slough" from hydration/dehydration. To address the cellular basis for normal exfoliation, we compared changes in lamellar bilayer architecture and CD structure in D-Squame strips from the first versus fifth stripping ("outer" vs. "mid"-stratum corneum (SC), respectively) from nine normal adult forearms. Strippings were either processed for standard electron microscopy (EM) or for ruthenium-, or osmium-tetroxide vapor fixation, followed by immediate epoxy embedment, an artifact-free protocol, which, to our knowledge, is previously unreported. CDs are largely intact in the mid-SC, but replaced by electron-dense (hydrophilic) clefts (lacunae) that expand laterally, splitting lamellar arrays in the outer SC. Some undegraded desmoglein 1/desmocollin 1 redistribute uniformly into corneocyte envelopes (CEs) in the outer SC (shown by proteomics, Z-stack confocal imaging, and immunoEM). CEs then thicken, likely facilitating exfoliation by increasing corneocyte rigidity. In vapor-fixed images, hydration only altered the volume of the extracellular compartment, expanding lacunae, further separating membrane arrays. During dehydration, air replaced water, maintaining the expanded extracellular compartment. Hydration also provoked degradation of membranes by activating contiguous acidic ceramidase activity. Together, these studies identify several parallel mechanisms that orchestrate exfoliation from the surface of normal human skin.
Asunto(s)
Desmosomas/patología , Epidermis/patología , Epidermis/ultraestructura , Matriz Extracelular/patología , Matriz Extracelular/ultraestructura , Adulto , Deshidratación/metabolismo , Deshidratación/patología , Desmocolinas/metabolismo , Desmogleína 1/metabolismo , Desmosomas/metabolismo , Desmosomas/ultraestructura , Epidermis/metabolismo , Matriz Extracelular/metabolismo , Fijadores , Glicoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Microscopía ElectrónicaRESUMEN
Orange peel extract appears to exhibit beneficial effects on skin whitening, inflammation, UVB protection, as well as keratinocyte proliferation. In the present study, we determine whether topical hesperidin influences epidermal permeability barrier function and its underlying mechanisms. Hairless mice were treated topically with 2% hesperidin or 70% ethanol alone twice daily for 6 days. At the end of treatment, basal transepidermal water loss (TEWL) was measured 2 and 4 h post barrier disruption. Epidermal proliferation and differentiation were evaluated by immunohistochemical staining and Western blot analysis. Additionally, lamellar body density and secretion were assessed by electron microscopy. Although there were no significant differences in basal barrier function, in comparison with control animals, topical hesperidin significantly accelerated barrier recovery at both 2 and 4 h after acute barrier abrogation. Enhanced barrier function in hesperidin-treated skin correlated with stimulation of both epidermal proliferation and differentiation, as well as enhanced lamellar body secretion. These results indicate that topical hesperidin enhances epidermal permeability barrier homeostasis at least in part due to stimulation of epidermal proliferation, differentiation, as well as lamellar body secretion.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Epidérmicas , Hesperidina/administración & dosificación , Hesperidina/farmacología , Administración Tópica , Animales , Biopsia , Diferenciación Celular/fisiología , Permeabilidad de la Membrana Celular/fisiología , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epidermis/fisiología , Epidermis/ultraestructura , Femenino , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Ratones , Ratones Pelados , Modelos AnimalesRESUMEN
Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (AMP; LL-37 and ß-defensin-2) expression. Urea stimulates the expression of, and is transported into, keratinocytes by two urea transporters (UTs), UT-A1 and UT-A2, and by aquaporins 3, 7, and 9. Inhibitors of these UTs block the downstream biological effects of urea, which include increased mRNA and protein levels of (i) transglutaminase-1, involucrin, loricrin, and filaggrin, (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and ß-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and AMP expression in a murine model of atopic dermatitis. Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and AMP expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Dermatitis Atópica/tratamiento farmacológico , Epidermis/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Urea/farmacocinética , Adulto , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Acuaporina 3/genética , Acuaporina 3/metabolismo , Acuaporinas/genética , Acuaporinas/metabolismo , Diferenciación Celular/fisiología , Permeabilidad de la Membrana Celular/fisiología , Dermatitis Atópica/metabolismo , Dermatitis Atópica/fisiopatología , Células Epidérmicas , Femenino , Proteínas Filagrina , Regulación de la Expresión Génica/fisiología , Humanos , Queratinocitos/citología , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Pelados , Persona de Mediana Edad , Cultivo Primario de Células , Agua/metabolismo , Adulto Joven , Transportadores de UreaRESUMEN
Herbal medicines have been used in preventing and treating skin disorders for centuries. It has been demonstrated that systemic administration of chrysanthemum extract exhibits anti-inflammatory properties. However, whether topical applications of apigenin, a constituent of chrysanthemum extract, influence cutaneous inflammation is still unclear. In the present study, we first tested whether topical applications of apigenin alleviate cutaneous inflammation in murine models of acute dermatitis. The murine models of acute allergic contact dermatitis and acute irritant contact dermatitis were established by topical application of oxazolone and phorbol 12-myristate 13-acetate (TPA), respectively. Inflammation was assessed in both dermatitis models by measuring ear thickness. Additionally, the effect of apigenin on stratum corneum function in a murine subacute allergic contact dermatitis model was assessed with an MPA5 physiology monitor. Our results demonstrate that topical applications of apigenin exhibit therapeutic effects in both acute irritant contact dermatitis and allergic contact dermatitis models. Moreover, in comparison with the vehicle treatment, topical apigenin treatment significantly reduced transepidermal water loss, lowered skin surface pH, and increased stratum corneum hydration in a subacute murine allergic contact dermatitis model. Together, these results suggest that topical application of apigenin could provide an alternative regimen for the treatment of dermatitis.