RESUMEN
The webinar series and workshop titled "Trust Your Gut: Establishing Confidence in Gastrointestinal Models An Overview of the State of the Science and Contexts of Use" was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and processes occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.
Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from different countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.
Asunto(s)
Alternativas a las Pruebas en Animales , Tracto Gastrointestinal , Humanos , Alternativas a las Pruebas en Animales/métodos , Animales , Modelos Biológicos , Medición de Riesgo/métodos , Pruebas de Toxicidad/métodosRESUMEN
Animal models have historically been poor preclinical predictors of gastrointestinal (GI) directed therapeutic efficacy and drug-induced GI toxicity. Human stem and primary cell-derived culture systems are a major focus of efforts to create biologically relevant models that enhance preclinical predictive value of intestinal efficacy and toxicity. The inherent variability in stem cell-based cultures makes development of useful models a challenge; the stochastic nature of stem cell differentiation interferes with the ability to build and validate reproducible assays that query drug responses and pharmacokinetics. In this study, we aimed to characterize and reduce sources of variability in a complex stem cell-derived intestinal epithelium model, termed RepliGut® Planar, across cells from multiple human donors, cell lots, and passage numbers. Assessment criteria included barrier formation and integrity, gene expression, and cytokine responses. Gene expression and culture metric analyses revealed that controlling cell passage number reduces variability and maximizes physiological relevance of the model. In a case study where passage number was optimized, distinct cytokine responses were observed among four human donors, indicating that biological variability can be detected in cell cultures originating from diverse human sources. These findings highlight key considerations for designing assays that can be applied to additional primary cell-derived systems, as well as establish utility of the RepliGut® Planar platform for robust development of human-predictive drug-response assays.
Animal models are frequently used as tools for studying gastrointestinal (GI) disease, but they inadequately replicate the complexities of the human gut, making them poor predictors of how humans respond to new drugs. Models using human stem cells are closer to human GI physiology, but their responses are not uniform owing to variability in the stem cells. We looked for the sources of this variability in the primary stem-cell derived RepliGut® Planar model. We found that limiting how long the cells were kept in culture reduced their variability and improved the physiological relevance of the model. These findings highlight key assay design considerations that also can be applied to other primary cell-derived systems. Reliable and physiologically relevant cell-based models can reduce animal testing, improve research accuracy, and ensure new treatments are more relevant and effective for patients.
Asunto(s)
Mucosa Intestinal , Humanos , Mucosa Intestinal/citología , Colon/citología , Alternativas a las Pruebas en Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Diferenciación Celular , Modelos Biológicos , Citocinas/metabolismo , Células MadreRESUMEN
Animal models have historically been poor preclinical predictors of gastrointestinal (GI) directed therapeutic efficacy and drug-induced GI toxicity. Human stem and primary cell-derived culture systems are a major focus of efforts to create biologically relevant models that enhance preclinical predictive value of intestinal efficacy and toxicity. The inherent variability in stem-cell-based complex cultures makes development of useful models a challenge; the stochastic nature of stem-cell differentiation interferes with the ability to build and validate robust, reproducible assays that query drug responses and pharmacokinetics. In this study, we aimed to characterize and reduce potential sources of variability in a complex stem cell-derived intestinal epithelium model, termed RepliGut® Planar, across cells from multiple human donors, cell lots, and passage numbers. Assessment criteria included barrier formation and integrity, gene expression, and cytokine responses. Gene expression and culture metric analyses revealed that controlling for stem/progenitor-cell passage number reduces variability and maximizes physiological relevance of the model. After optimizing passage number, donor-specific differences in cytokine responses were observed in a case study, suggesting biologic variability is observable in cell cultures derived from multiple human sources. Our findings highlight key considerations for designing assays that can be applied to additional primary-cell derived systems, as well as establish utility of the RepliGut® Planar platform for robust development of human-predictive drug-response assays.
RESUMEN
Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.
Asunto(s)
Cromatografía Liquida/métodos , Péptidos/aislamiento & purificación , Proteínas/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Bovinos , Humanos , Laboratorios , Estudios Longitudinales , Proteínas/análisis , Control de Calidad , Reproducibilidad de los Resultados , Encuestas y CuestionariosRESUMEN
The microbiome is being characterized by large-scale sequencing efforts, yet it is not known whether it regulates host metabolism in a general versus tissue-specific manner or which bacterial metabolites are important. Here, we demonstrate that microbiota have a strong effect on energy homeostasis in the colon compared to other tissues. This tissue specificity is due to colonocytes utilizing bacterially produced butyrate as their primary energy source. Colonocytes from germfree mice are in an energy-deprived state and exhibit decreased expression of enzymes that catalyze key steps in intermediary metabolism including the TCA cycle. Consequently, there is a marked decrease in NADH/NAD(+), oxidative phosphorylation, and ATP levels, which results in AMPK activation, p27(kip1) phosphorylation, and autophagy. When butyrate is added to germfree colonocytes, it rescues their deficit in mitochondrial respiration and prevents them from undergoing autophagy. The mechanism is due to butyrate acting as an energy source rather than as an HDAC inhibitor.
Asunto(s)
Autofagia , Butiratos/farmacología , Colon/metabolismo , Metabolismo Energético , Metagenoma , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Colon/citología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Perfilación de la Expresión Génica , Vida Libre de Gérmenes , Glucosa/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Metabolómica , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , NAD/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación Oxidativa , Fosforilación , Transducción de SeñalRESUMEN
Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.
Asunto(s)
Técnicas de Laboratorio Clínico , Proteínas/análisis , Proteómica/métodos , Gonadotropina Coriónica/análisis , Glucógeno Fosforilasa/análisis , Humanos , Antígeno Prostático Específico/análisis , Proteómica/educación , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/análisis , Proyectos de InvestigaciónRESUMEN
Estrogens are potent mitogens for some target organs, such as the uterus, and cancers that develop in this organ might be linked to the proliferative action of these hormones. However, the mechanism by which estrogens influence the cell cycle machinery is not known. We found that a null mutation for the insulin receptor substrate (IRS)-1, a docking protein that is important for IGF1 signaling, compromised hormone-induced mitosis in the uterine epithelium; BrdU incorporation was not affected. This selective effect on mitosis was associated with a reduction in uterine cyclin B-associated kinase activity; cyclin A-associated kinase activity was not changed. The null mutation also reduced the extent of hormone-induced phosphorylation of endogenous uterine histone H1, as determined with phospho-specific antiserum. Uterine epithelial cyclin dependent kinase (cdk)1 was induced in response to hormone, but the level of the kinase protein, as determined by immunoblotting, was noticeably less in the irs1 null mutant than that in the wild-type (WT) mouse, especially around the time of peak mitosis (24âh). Since IRS-1 binds/activates phosphatidylinositol 3-kinase (PI3K), the absence of this docking protein could impair signaling of a known pathway downstream of AKT that stimulates translation of cell cycle components. Indeed, we found that phosphorylation of uterine AKT (Ser473) in irs1 null mutants was less than that in WTs following treatment. Based on earlier studies, it is also possible that an IGF1/IRS-1/PI3K/AKT pathway regulates posttranslational changes in cdk1. This model may provide insights as to how a growth factor pathway can mediate hormone action on cell proliferation.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Estradiol/farmacología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Útero/efectos de los fármacos , Animales , Proteína Quinasa CDC2/genética , Proliferación Celular/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Sustrato del Receptor de Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Útero/citología , Útero/enzimologíaRESUMEN
Population-based variability in protein expression patterns, especially in humans, is often observed but poorly understood. Moreover, very little is known about how interindividual genetic variation contributes to protein expression patterns. To begin to address this, we describe elements of technical and biological variations contributing to expression of 544 proteins in a population of 24 individual human lymphoblastoid cell lines that have been extensively genotyped as part of the International HapMap Project. We determined that expression levels of 10% of the proteins were tightly correlated to cell doubling rates. Using the publicly available genotypes for these lymphoblastoid cell lines, we applied a genetic association approach to identify quantitative trait loci associated with protein expression variation. Results identified 24 protein forms corresponding to 15 proteins for which genetic elements were responsible for >50% of the expression variation. The genetic variation associated with protein expression levels were located in cis with the gene coding for the transcript of the protein for 19 of these protein forms. Four of the genetic elements identified were coding non-synonymous single nucleotide polymorphisms that resulted in migration pattern changes in the two-dimensional gel. This is the first description of large scale proteomics analysis demonstrating the direct relationship between genome and proteome variations in human cells.
Asunto(s)
Variación Genética , Linfocitos/fisiología , Proteoma/análisis , Proteoma/genética , Sitios de Carácter Cuantitativo , Animales , Línea Celular , Electroforesis en Gel Bidimensional , Genotipo , Humanos , Linfocitos/citologíaRESUMEN
The aryl hydrocarbon receptor (AHR) is known for its role in the adaptive and toxic responses to a large number of environmental contaminants, as well as its role in hepatovascular development. The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes. The AHR has also been implicated in signaling events independent of nuclear localization and DNA binding, and it has been suggested that such pathways may play important roles in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs. Using this model, we provide evidence that DNA binding is required AHR-mediated liver development, as Ahr(dbd/dbd) mice exhibit a patent ductus venosus, similar to what is seen in Ahr(-/-) mice. Furthermore, Ahr(dbd/dbd) mice are resistant to TCDD-induced toxicity for all endpoints tested. These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.
Asunto(s)
Carcinógenos Ambientales/toxicidad , ADN/metabolismo , Hígado/anomalías , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Elementos de Respuesta , Transducción de Señal , Células 3T3 , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas Portadoras/metabolismo , Fisura del Paladar/inducido químicamente , Fisura del Paladar/embriología , Citocromo P-450 CYP1A1/metabolismo , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hidronefrosis/inducido químicamente , Hidronefrosis/embriología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Mutantes , Proteínas Asociadas a Microtúbulos , Vena Porta/anomalías , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Timo/efectos de los fármacos , Timo/metabolismo , TransfecciónRESUMEN
Global changes in the phosphorylation state of human H1 isoforms isolated from UL3 cells have been investigated using mass spectrometry. Relative changes in H1 phosphorylation between untreated cells and cells treated with dexamethasone or various CDK inhibitors were determined. The specific cyclin-dependent kinase consensus sites of phosphorylation on the histone H1 isoforms that show changes in phosphorylation were also investigated. Three sites of phosphorylation on histone H1.4 isoforms have been identified.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Histonas/metabolismo , Espectrometría de Masas/métodos , 2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Western Blotting , Línea Celular Tumoral , Dexametasona/farmacología , Histonas/genética , Histonas/aislamiento & purificación , Humanos , Ratones , Datos de Secuencia Molecular , Fosfopéptidos/análisis , Fosfopéptidos/inmunología , Fosfopéptidos/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Conejos , Alineación de Secuencia , Serina Endopeptidasas/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , VacunaciónRESUMEN
Electron-transfer dissociation (ETD) has recently been introduced as a fragmentation method for peptide and protein analysis. Unlike collisionally induced dissociation (CID), fragmentation by ETD occurs randomly along the peptide backbone. With the use of the sequences determined from the protein termini and the parent protein mass, intact proteins can be unambiguously identified. Because of the fast kinetics of these reactions, top-down proteomics can be performed using ETD in a linear ion trap mass spectrometer on a chromatographic time scale. Here we demonstrate the utility of ETD in high-throughput top-down proteomics using soluble extracts of E. coli. Development of a multidimensional fractionation platform, as well as a custom algorithm and scoring scheme specifically designed for this type of data, is described. The analysis resulted in the robust identification of 322 different protein forms representing 174 proteins, comprising one of the most comprehensive data sets assembled on intact proteins to date.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Espectrometría de Masas/métodos , Proteómica/métodos , Automatización , Proteínas de Escherichia coli/químicaRESUMEN
High-throughput genome sequencing continues to accelerate the rate at which complete genomes are available for biological research. Many of these new genome sequences have little or no genome annotation currently available and hence rely upon computational predictions of protein coding genes. Evidence of translation from proteomic techniques could facilitate experimental validation of protein coding genes, but the techniques for whole genome searching with MS/MS data have not been adequately developed to date. Here we describe GENQUEST, a novel method using peptide isoelectric focusing and accurate mass to greatly reduce the peptide search space, making fast, accurate, and sensitive whole human genome searching possible on common desktop computers. In an initial experiment, almost all exonic peptides identified in a protein database search were identified when searching genomic sequence. Many peptides identified exclusively in the genome searches were incorrectly identified or could not be experimentally validated, highlighting the importance of orthogonal validation. Experimentally validated peptides exclusive to the genomic searches can be used to reannotate protein coding genes. GENQUEST represents an experimental tool that can be used by the proteomics community at large for validating computational approaches to genome annotation.
Asunto(s)
Bases de Datos de Proteínas/tendencias , Documentación/métodos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Línea Celular Tumoral , Genoma Humano , Genómica/métodos , Humanos , Focalización IsoeléctricaRESUMEN
Orthogonal analysis of amino acid substitutions as a result of SNPs in existing proteomic datasets provides a critical foundation for the emerging field of population-based proteomics. Large-scale proteomics datasets, derived from shotgun tandem MS analysis of complex cellular protein mixtures, contain many unassigned spectra that may correspond to alternate alleles coded by SNPs. The purpose of this work was to identify tandem MS spectra in LC-MS/MS shotgun proteomics datasets that may represent coding nonsynonymous SNPs (nsSNP). To this end, we generated a tryptic peptide database created from allelic information found in NCBI's dbSNP. We searched this database with tandem MS spectra of tryptic peptides from DU4475 breast tumor cells that had been fractioned by pI in the first-dimension and reverse-phase LC in the second dimension. In all we identified 629 nsSNPs, of which 36 were of alternate SNP alleles not found in the reference NCBI or IPI protein databases. Searches for SNP-peptides carry a high risk of false positives due both to mass shifts caused by modifications and because of multiple representations of the same peptide within the genome. In this work, false positives were filtered using a novel peptide pI prediction algorithm and characterized using a decoy database developed by random substitution of similarly sized reference peptides. Secondary validation by sequencing of corresponding genomic DNA confirmed the presence of the predicted SNP in 8 of 10 SNP-peptides. This work highlights that the usefulness of interpreting unassigned spectra as polymorphisms is highly reliant on the ability to detect and filter false positives.
Asunto(s)
Sustitución de Aminoácidos/genética , Polimorfismo de Nucleótido Simple , Proteínas/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Neoplasias de la Mama/química , Bases de Datos de Proteínas , Humanos , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/genética , Reacción en Cadena de la Polimerasa , Proteínas/genética , Análisis de Secuencia de ADN , Análisis de Secuencia de ProteínaRESUMEN
Disruption of the murine Mop3 (also known as Bmal1 or Arntl) locus results in a loss of behavioral and molecular circadian rhythms. Although Mop3 null mice do not display anomalies in early development, they do display reduced activity as they age. In an effort to explain this decreased activity, we characterized the physiological and anatomical changes that occurred with age. We observed that Mop3 null mice display an increased mortality after 26 weeks of age and a phenotype best described as a progressive noninflammatory arthropathy. Although little pathology is observed prior to 11 weeks of age, by 35 weeks of age essentially all Mop3 null animals develop joint ankylosis due to flowing ossification of ligaments and tendons and almost complete immobilization of weight-bearing and nonweight-bearing joints. This pathology appears to explain the decreased activity of Mop3 null mice and suggests that MOP3 is an inhibitor of ligament and tendon ossification.
Asunto(s)
Envejecimiento/fisiología , Artropatías/genética , Osificación Heterotópica/genética , Factores de Transcripción/fisiología , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Huesos/fisiología , Ritmo Circadiano/genética , Femenino , Artropatías/patología , Ligamentos/patología , Ligamentos/fisiología , Masculino , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Osificación Heterotópica/patología , Fenotipo , Tendones/patología , Tendones/fisiología , Factores de Transcripción/genética , Pérdida de PesoRESUMEN
The aryl hydrocarbon receptor (AHR) is commonly known for its role in the adaptive metabolism of xenobiotics and in the toxic events that follow exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin). Previously, we have demonstrated that the AHR and its heterodimeric partner, the AHR nuclear translocator (ARNT), play a role in the developmental closure of a hepatic vascular shunt known as the ductus venosus (DV). To investigate the mechanism of DV closure, we generated hypomorphic alleles of the Ahr and Arnt loci. Using these models, we then asked whether this vascular defect could be rescued by receptor activation during late development. By manipulating gestational exposure, the patent DV in AHR or ARNT hypomorphs could be efficiently closed by dioxin exposure as early as embryonic day 12.5 and as late as embryonic day 18.5. These findings define the temporal regulation of receptor activation during normal ontogeny and provide evidence to support the idea that receptor activation and AHR-ARNT heterodimerization are essential for normal vascular development. Taken in the broader context, these data demonstrate that similar AHR signaling steps govern all major aspects of AHR biology.
Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/embriología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Vasos Sanguíneos/anomalías , Proteínas de Unión al ADN/efectos de los fármacos , Femenino , Edad Gestacional , Hígado/irrigación sanguínea , Hígado/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Mutantes , Modelos Biológicos , Fenotipo , Dibenzodioxinas Policloradas/administración & dosificación , Embarazo , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Transducción de Señal , Factores de Transcripción/efectos de los fármacosRESUMEN
The Ah receptor nuclear translocator (ARNT) is the dimeric partner of hypoxia-inducible factors and thus plays a pivotal role in cellular adaptation to low oxygen environments. ARNT is also a dimeric partner for the Ah receptor (AHR), and this complex is essential in regulating the adaptive metabolic response to polycyclic aromatic hydrocarbons. Because of the essential role of ARNT in hypoxia-driven developmental events, it has been difficult to study the physiological significance of AHR.ARNT heterodimers in vivo. To address this issue, we developed a hypomorphic Arnt allele that displayed normal development and allowed the examination of the role of ARNT in AHR biology. In this regard, the AHR is also known to mediate two additional biological processes: the toxicological response to compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and the developmental closure of a fetal vascular structure known as the ductus venosus. Although the mechanism of the adaptive pathway has been well described, the mechanism of AHR-mediated signal transduction in the toxic and developmental pathways is not well understood. Liver perfusion studies demonstrated that ARNT hypomorphs have a patent ductus venosus, identical to that observed in the Ahr null mice. Parallel dioxin toxicity studies demonstrated that the ARNT hypomorphs exhibited resistance to the end points of dioxin exposure. Moreover, we observed that toxicity could be segregated from the classical adaptive responses such as P4501A induction. Taken in sum, these experiments demonstrate that ARNT is an essential component of AHR developmental signaling and shed light on the mechanism of dioxin toxicity.
Asunto(s)
Proteínas de Unión al ADN , Dioxinas/toxicidad , Resistencia a Medicamentos/genética , Conducto Arterioso Permeable/genética , Receptores de Hidrocarburo de Aril/genética , Factores de Transcripción/genética , Alelos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Conducto Arterioso Permeable/metabolismo , Ratones , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
The Ah receptor (AHR) mediates the metabolic adaptation to a number of planar aromatic chemicals. Essential steps in this adaptive mechanism include AHR binding of ligand in the cytosol, translocation of the receptor to the nucleus, dimerization with the Ah receptor nuclear translocator, and binding of this heterodimeric transcription factor to dioxin-responsive elements (DREs) upstream of promoters that regulate the expression of genes involved in xenobiotic metabolism. The AHR is also involved in other aspects of mammalian biology, such as the toxicity of molecules like 2,3,7,8-tetrachlorodibenzo-p-dioxin as well as regulation of normal liver development. In an effort to test whether these additional AHR-mediated processes require a nuclear event, such as DRE binding, we used homologous recombination to generate mice with a mutation in the AHR nuclear localization/DRE binding domain. These Ahr(nls) mice were found to be resistant to all 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxic responses that we examined, including hepatomegaly, thymic involution, and cleft palate formation. Moreover, aberrations in liver development observed in these mice were identical to that observed in mice harboring a null allele at the Ahr locus. Taken in sum, these data support a model where most, if not all, of AHR-regulated biology requires nuclear localization.