RESUMEN
AIMS: The aim of this study is to determine the physical and functional interplay between fatty acid-binding protein 4 (FABP4) and its membrane receptor-like candidate protein, cytokeratin 1 (CK1), and to determine the effect of hindering CK1-mediated FABP4 cellular uptake on non-disturbed or metabolically stressed endothelial cells. METHODS: We monitored the direct interaction between FABP4 and CK1 using surface plasmon resonance, and the effects of blocking exogenous FABP4 (eFABP4) cellular uptake were determined by using specific siRNA to knock down the expression of CK1 in human umbilical vein endothelial cells (HUVECs). The expression and nuclear translocation of transcription factors involved in oxidative stress (NRF2) and inflammation (p65 subunit of NF-ĸB transcription factor) were determined by Western blotting analysis. RESULTS: Our data showed that FABP4 and CK1 bind to each other and that the putative FABP4 binding domain would be within the 151GIQEVTINQSLLQPLNVEID170 CK1 sequence. We determined that in non-disturbed or metabolically stressed endothelial cells, eFABP4 regulates the cellular response to oxidative stress. In addition, we also found that in the presence of palmitate, eFABP4 increases the pro-inflammatory effects induced by palmitate per se, probably due to an increase in the transport of palmitate inside cells, suggesting that these FABP4-mediated pro-oxidative and pro-inflammatory effects are dependent on CK1 expression. CONCLUSIONS: We demonstrated that CK1 facilitates eFABP4 cellular uptake in endothelial cells. Therefore, the CK1-targeted inhibition of exogenous FABP4 cellular uptake might be a potential therapeutic strategy to protect endothelial cells against FABP4-induced activation of inflammation and oxidative stress.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Queratina-1/metabolismo , Transporte Biológico/fisiología , Células Endoteliales/metabolismo , Proteínas de Unión a Ácidos Grasos/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/metabolismo , Queratina-1/genética , Queratina-1/fisiología , Queratinas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores de Superficie Celular , Transducción de SeñalRESUMEN
Inflammatory bowel disease (IBD) is the result of a chronic intestinal inflammatory response which usually occurred in colon and small intestine. Keratins constitute the intermediate filament cytoskeleton in all epithelia. The present study was intended to explore the role of Keratin 1 (KRT1) in the progress of IBD. In normal intestinal tissue, the expression of KRT1 was detected by RT-PCR and Western blot. The levels of KRT1 protein significantly decreased in serum samples of IBD patients as compared with sera of healthy controls. Immunohistochemistry revealed that the expression of KRT1 decreased in various intestinal diseases, especially in Crohn's disease and ulcerative colitis. Furthermore, down-regulated KRT1 was correlated with the severity of IBD. The overexpression of KRT1 maintained epithelial barrier in Caco-2 cells after IL-1ß treatment. Furthermore, IL-1ß-induced disruption of tight junction became significantly attenuated in KRT1 over-expressing Caco-2 cells as compared with control cells. Thus, KRT1 played an important role of maintaining epithelial barrier and its down-regulation in intestinal tissue was correlated with the progression of IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Queratina-1/fisiología , Uniones Estrechas/genética , Adolescente , Adulto , Anciano , Células CACO-2 , Estudios de Casos y Controles , Células Cultivadas , Niño , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Mucosa Intestinal/metabolismo , Queratina-1/genética , Masculino , Persona de Mediana Edad , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Adulto JovenRESUMEN
Widespread reversion of genetic disease is rare; however, such events are particularly evident in some skin disorders in which normal clones develop on a background of affected skin. We previously demonstrated that mutations in keratin 10 (KRT10) cause ichthyosis with confetti (IWC), a severe dominant disorder that is characterized by progressive development of hundreds of normal skin spots via revertant mosaicism. Here, we report on a clinical and histological IWC subtype in which affected subjects have red, scaly skin at birth, experience worsening palmoplantar keratoderma in childhood, and develop hundreds of normal skin spots, beginning at around 20 years of age, that increase in size and number over time. We identified a causal de novo mutation in keratin 1 (KRT1). Similar to IWC-causing KRT10 mutations, this mutation in KRT1 resulted in a C-terminal frameshift, replacing 22 C-terminal amino acids with an alternate 30-residue peptide. Mutant KRT1 caused partial collapse of the cytoplasmic intermediate filament network and mislocalized to the nucleus. As with KRT10 mutations causing IWC, reversion of KRT1 mutations occurred via mitotic recombination. Because reversion is not observed with other disease-causing keratin mutations, the results of this study implicate KRT1 and KRT10 C-terminal frameshift mutations in the high frequency of revertant mosaicism in IWC.
Asunto(s)
Mutación del Sistema de Lectura , Ictiosis/genética , Queratina-1/genética , Adulto , Edad de Inicio , Secuencia de Aminoácidos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Niño , Preescolar , Cromosomas Humanos Par 12/genética , Citoesqueleto/ultraestructura , Humanos , Ictiosis/patología , Filamentos Intermedios/metabolismo , Queratina-1/fisiología , Queratinocitos/patología , Pérdida de Heterocigocidad , Masculino , Datos de Secuencia Molecular , Mosaicismo , Fenotipo , Polimorfismo de Nucleótido Simple , Transporte de Proteínas , TransfecciónRESUMEN
Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression.
Asunto(s)
Arsenitos/farmacología , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Queratinocitos/efectos de los fármacos , Proteína Morfogenética Ósea 6/antagonistas & inhibidores , Proteína Morfogenética Ósea 6/fisiología , Proteínas Morfogenéticas Óseas/fisiología , Células Cultivadas , Factores de Transcripción Forkhead/antagonistas & inhibidores , Humanos , Queratina-1/fisiología , Queratina-10/fisiología , Queratinocitos/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy of tumors. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cis-diamminedichloroplatinum (CNE2/cDDP) were established from human nasopharyngeal carcinoma (NPC) cell lines CNE2. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and ESI-Q-TOF-MS were performed on protein extracted from CNE2 and CNE2/cDDP cell lines to screen drug resistance-related proteins. Keratin 1 (KRT1), cathepsin D (CTSD) and annexin a5 (ANXA5) were identified as three proteins showing higher expression in CNE2/cDDP compared to CNE2. Furthermore, suppression of KRT1 expression by siRNA resulted in decreased MDR in siRNA-CNE2/cDDP cells. And upregulation of KRT1 could result in increased of drug resistance in NPC cell lines. Taken together, KRT1 protein and its activity levels were higher in cDDP-resistant NPC cell lines compared to their parental cell lines. These data clearly linked KRT1 and cDDP resistance mechanisms. KRT1 could serve as a biomarker for chemotherapy sensitivity of NPC.
Asunto(s)
Cisplatino/uso terapéutico , Resistencia a Antineoplásicos , Queratina-1/fisiología , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma , Línea Celular Tumoral , Cisplatino/farmacología , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Electroforesis en Gel Bidimensional , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Queratina-1/antagonistas & inhibidores , Queratina-1/genética , Queratina-1/metabolismo , Espectrometría de Masas , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , ARN Interferente Pequeño/farmacología , Transfección , Estudios de Validación como AsuntoRESUMEN
During an inflammatory state, functional myeloperoxidase (MPO) is released into the vessel as a result of intravascular neutrophil degradation. One mechanism of resulting cellular injury involves endothelial internalization of MPO, which causes oxidative damage and impairs endothelial signaling. We report the discovery of a protein that facilitates MPO internalization, cytokeratin 1 (CK1), identified using affinity chromatography and mass spectrometry. CK1 interacts with MPO in vitro, even in the presence of 100% human plasma, thus substantiating biological relevance. Immunofluorescent microscopy confirmed that MPO added to endothelial cells can co-localize with endogenously expressed CK1. CK1 acts as a scaffolding protein for the assembly of the vasoregulatory plasma kallikrein-kinin system; thus we explored whether MPO and high molecular weight kininogen (HK) reside on CK1 together or whether they compete for binding. The data support cooperative binding of MPO and HK on cells such that MPO masked the plasma kallikrein cleavage site on HK, and MPO-generated oxidants caused inactivation of both HK and kallikrein. Collectively, interactions between MPO and the components of the plasma kallikrein-kinin system resulted in decreased bradykinin production. This study identifies CK1 as a facilitator of MPO-mediated vascular responses and thus provides a new paradigm by which MPO affects vasoregulatory systems.