RESUMEN

The Wnt/ß-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway's activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/ß-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.

Asunto(s)

Factores de Transcripción TCF , beta Catenina , Animales , Animales Modificados Genéticamente , Desarrollo Embrionario , Codorniz/metabolismo , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismo

RESUMEN

Podocyte depletion is sufficient for the development of numerous glomerular diseases and can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus). Commonly used methods to quantify podocyte depletion introduce bias, whereas gold standard stereologic methodologies are time consuming and impractical. We developed a novel approach for assessing podocyte depletion in whole glomeruli that combines immunofluorescence, optical clearing, confocal microscopy, and three-dimensional analysis. We validated this method in a transgenic mouse model of selective podocyte depletion, in which we determined dose-dependent alterations in several quantitative indices of podocyte depletion. This new approach provides a quantitative tool for the comprehensive and time-efficient analysis of podocyte depletion in whole glomeruli.

Asunto(s)

Recuento de Células/métodos , Tamaño de la Célula , Glomérulos Renales/citología , Podocitos/citología , Animales , Imagenología Tridimensional , Ratones

RESUMEN

The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

Asunto(s)

Activinas/farmacología , Proteína Morfogenética Ósea 7/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Podocitos/metabolismo , Tretinoina/farmacología , Animales , Agregación Celular , Permeabilidad de la Membrana Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/citología , Células Mesangiales/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Podocitos/fisiología , Albúmina Sérica/metabolismo , Técnicas de Cultivo de Tejidos

RESUMEN

Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kappaB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice-induced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

Asunto(s)

Células Epiteliales/inmunología , Bacterias Gramnegativas/inmunología , FN-kappa B/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Peptidoglicano/metabolismo , Vesículas Secretoras/metabolismo , Animales , Femenino , Células HeLa , Helicobacter pylori/inmunología , Humanos , Masculino , Ratones , Neisseria/inmunología , Peptidoglicano/inmunología , Pseudomonas aeruginosa/inmunología , Vesículas Secretoras/inmunología

RESUMEN

We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cis-diaminedichloroplatinum (II) (CDDP). In this study, we found that ATP7A transfection of Chinese hamster ovary cells (CHO-K1) and fibroblasts isolated from Menkes disease patients enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10-hydroxy-camptothecin (SN-38), etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more resistant to SN-38 than ATP7A-negative cells. Thus, ATP7A confers resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Adenosina Trifosfatasas/metabolismo , Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Proteínas de Transporte de Catión/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Resistencia a Múltiples Medicamentos , Adenocarcinoma/genética , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Animales , Antineoplásicos Fitogénicos/farmacocinética , Brefeldino A/farmacología , Células CHO , Camptotecina/farmacocinética , Camptotecina/farmacología , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/genética , Membrana Celular/metabolismo , Cisplatino/farmacocinética , Cisplatino/farmacología , Neoplasias del Colon/genética , Cobre/farmacocinética , Cobre/farmacología , ATPasas Transportadoras de Cobre , Cricetinae , Cricetulus , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Aparato de Golgi/metabolismo , Humanos , Irinotecán , Monensina/farmacología , Transfección

RESUMEN

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusión , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Sitios de Unión/fisiología , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas Transportadoras de Cobre , ATPasas Transportadoras de Cobre , Humanos , Cinética , Metalochaperonas , Mutagénesis/fisiología , Fragmentos de Péptidos/metabolismo , Técnicas del Sistema de Dos Híbridos , Levaduras

RESUMEN

The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

Asunto(s)

Adenosina Trifosfatasas/biosíntesis , Proteínas de Transporte de Catión/biosíntesis , Cobre/metabolismo , Proteínas de la Membrana/biosíntesis , Síndrome del Pelo Ensortijado/metabolismo , Adenosina Trifosfatasas/genética , Animales , Western Blotting , Proteínas de Transporte de Catión/genética , Células Cultivadas , Radioisótopos de Cobre , Transportador de Cobre 1 , ATPasas Transportadoras de Cobre , Fibroblastos , Humanos , Proteínas de la Membrana/genética , Microscopía Fluorescente , Ovinos , Transfección