RESUMEN
The Cu-ATPase ATP7A (MNK) is localized in the trans-Golgi network (TGN) and relocalizes in the plasma membrane via vesicle-mediated traffic following exposure of the cells to high concentrations of copper. Rab proteins are organelle-specific GTPases, markers of different endosomal compartments; their role has been recently reviewed (Trends Cell Biol. 11(2001) 487). In this article we analyze the endosomal pathway of trafficking of the MNK protein in stably transfected clones of CHO cells, expressing chimeric Rab5-myc or Rab7-myc proteins, markers of early or late endosome compartments, respectively. We demonstrate by immunofluorescence and confocal and electron microscopy techniques that the increase in the concentration of copper in the medium (189 microM) rapidly induces a redistribution of the MNK protein from early sorting endosomes, positive for Rab5-myc protein, to late endosomes, containing the Rab7-myc protein. Cell fractionation experiments confirm these results; i.e., the MNK protein is recruited to the endosomal fraction on copper stimulation and colocalizes with Rab5 and Rab7 proteins. These findings allow the first characterization of the vesicles involved in the intracellular routing of the MNK protein from the TGN to the plasma membrane, a key mechanism allowing appropriate efflux of copper in cells grown in high concentrations of the metal.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Endosomas/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab5/análisis , Adenosina Trifosfatasas/genética , Animales , Biomarcadores/análisis , Células CHO , Proteínas de Transporte de Catión/genética , Compartimento Celular , Fraccionamiento Celular , Cobre/farmacología , ATPasas Transportadoras de Cobre , Cricetinae , Endosomas/metabolismo , Humanos , Microscopía Electrónica , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Transfección , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión a GTP rab7RESUMEN
Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.
Asunto(s)
Enterocitos/metabolismo , Metalotioneína/metabolismo , Zinc/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Polaridad Celular/fisiología , Cromatografía Líquida de Alta Presión , Enterocitos/citología , Enterocitos/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Metalotioneína/biosíntesis , Metalotioneína/genética , Ratones , Modelos Biológicos , Factores de Tiempo , Transfección , Zinc/farmacología , Radioisótopos de ZincAsunto(s)
Proteínas de Unión a Calmodulina , Regulación de la Expresión Génica , Metalotioneína/genética , Animales , Clonación Molecular , Proteínas de Unión al ADN/genética , Células Eucariotas , Humanos , Mamíferos , Metales , Transactivadores/metabolismo , Factores de Transcripción , Transcripción Genética , Zinc , Factor de Transcripción MTF-1RESUMEN
The metal-dependent activation of metallothionein (MT) genes requires the interaction of positive trans-activators (MRFs) with metal-regulatory (MRE) regions of MT promoters. In this report, we examined the role of transition metals in modulating the MRE-binding activities of two different MRE-binding proteins: the metal-regulated factor ZiRF1 and the basal factor SP1. We showed the ability of both proteins to interact with a similar sequence specificity with the cognate target site (MRE-S) of another known MRE-binding protein, mMTF1. We next evaluated the role of metal ions in modulating the MRE-binding activity of recombinant ZiRF1 and basal SP1 proteins by measuring the effect of different metal chelators on DNA interaction. We observed a dose-dependent inhibition of the GST-ZiRF1/MRE-binding activity using three different metal chelators: EDTA, 1,10 PHE and TPEN. Interestingly, EDTA treatment failed to inhibit the recombinant SP1 MRE-binding activity while the effect of 1,10 PHE was comparable to that obtained analyzing 1,10 PHE-treated GST-ZiRF1. The MRE-binding complexes detected in cell extracts showed a response to metal chelator treatment very similar to that displayed by the recombinant ZiRF1 and SP1 proteins. The hypothesis of mutual interactions of both basal and metal-regulated transcription factors with the same metal-regulatory regions is discussed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Metalotioneína/biosíntesis , Metales/farmacología , Podofilino/análogos & derivados , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quelantes/farmacología , Proteínas de Unión al ADN/química , Ácido Edético/farmacología , Etilenodiaminas/farmacología , Glutatión Transferasa , Células L , Metalotioneína/genética , Ratones , Oligodesoxirribonucleótidos , Fenantrolinas/farmacología , Podofilino/química , Podofilino/metabolismo , Podofilotoxina/análogos & derivados , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Factores de Transcripción/química , Transfección , Factor de Transcripción MTF-1RESUMEN
We investigated the injurious effects of reactive oxygen metabolites on the intestinal epithelium and the possible protective role played by two olive oil phenolic compounds, (3,4-dihydroxyphenyl)ethanol and (p-hydroxyphenyl)ethanol, using the Caco-2 human cell line. We induced oxidative stress in the apical compartment, either by the addition of 10 mmol/L H2O2 or by the action of 10 U/L xanthine oxidase in the presence of xanthine (250 micromol/L); after the incubation, we evaluated the cellular and molecular alterations. Both treatments produced significant decreases in Caco-2 viability as assessed by the neutral red assay. Furthermore, we observed a significant increase in malondialdehyde intracellular concentration and paracellular inulin transport, indicating the occurrence of lipid peroxidation and monolayer permeability changes, respectively. The H2O2-induced alterations were completely prevented by preincubating Caco-2 cells with (3,4-dihydroxyphenyl)ethanol (250 micromol/L); when the oxidative stress was induced by xanthine oxidase, complete protection was obtained at a concentration of polyphenol as small as 100 micromol/L. In contrast, (p-hydroxyphenyl)ethanol was ineffective up to a concentration of 500 micromol/L. Our data demonstrate that (3,4-dihydroxyphenyl)ethanol can act as a biological antioxidant in a cell culture experimental model and that the ortho-dihydroxy moiety of the molecule is essential for antioxidant activity. This study suggests that dietary intake of olive oil polyphenols may lower the risk of reactive oxygen metabolite-mediated diseases such as some gastrointestinal diseases and atherosclerosis.