RESUMO
The intestinal epithelial layer is susceptible to damage by chemical, physiological and mechanical stress. While it is essential to maintain the integrity of epithelium, the biochemical pathways that contribute to the barrier function have not been completely investigated. Here we demonstrate an aryl hydrocarbon receptor (AHR)-dependent mechanism facilitating the production of the antimicrobial peptide AMP regenerating islet-derived protein 3 gamma (REG3G), which is essential for intestinal homeostasis. Genetic ablation of AHR in mice impairs pSTAT3-mediated REG3G expression and increases bacterial numbers of Segmented filamentous bacteria (SFB) and Akkermansia muciniphila in the small intestine. Studies with tissue-specific conditional knockout mice revealed that the presence of AHR in the epithelial cells of the small intestine is not required for the production of REG3G through the phosphorylated STAT3-mediated pathway. However, immune-cell-specific AHR activity is necessary for normal expression of REG3G in all regions of the small intestine. A diet rich in broccoli, capable of inducing AHR activity, increases REG3G production when compared to a semi-purified diet that is devoid of ligands that can potentially activate the AHR, thus highlighting the importance of AHR in antimicrobial function. Overall, these data suggest that homeostatic antimicrobial REG3G production is increased by an AHR pathway intrinsic to the immune cells in the small intestine.
Assuntos
Anti-Infecciosos , Receptores de Hidrocarboneto Arílico , Animais , Camundongos , Citoesqueleto , Células Epiteliais , Intestino Delgado , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays an important role in gastrointestinal barrier function, tumorigenesis, and is an emerging drug target. The resident microbiota is capable of metabolizing tryptophan to metabolites that are AHR ligands (e.g., indole-3-acetate). Recently, a novel set of mutagenic tryptophan metabolites named indolimines have been identified that are produced by M. morganii in the gastrointestinal tract. Here, we determined that indolimine-200, -214, and -248 are direct AHR ligands that can induce Cyp1a1 transcription and subsequent CYP1A1 enzymatic activity capable of metabolizing the carcinogen benzo(a)pyrene in microsomal assays. In addition, indolimines enhance IL6 expression in a colonic tumor cell line in combination with cytokine treatment. The concentration of indolimine-248 that induces AHR transcriptional activity failed to increase DNA damage. These observations reveal an additional aspect of how indolimines may alter colonic tumorigenesis beyond mutagenic activity.
RESUMO
The aryl hydrocarbon receptor (AHR) mediates intestinal barrier homeostasis. Many AHR ligands are also CYP1A1/1B1 substrates, which can result in rapid clearance within the intestinal tract, limiting systemic exposure and subsequent AHR activation. This led us to the hypothesis that there are dietary substrates of CYP1A1/1B1 that functionally increase the half-life of potent AHR ligands. We examined the potential of urolithin A (UroA), a gut bacterial metabolite of ellagitannins, as a CYP1A1/1B1 substrate to enhance AHR activity in vivo. UroA is a competitive substrate for CYP1A1/1B1 in an in vitro competition assay. A broccoli-containing diet promotes the gastric formation of the potent hydrophobic AHR ligand and CYP1A1/1B1 substrate, 5,11-dihydroindolo[3,2-b]carbazole (ICZ). In mice, dietary exposure to UroA in a 10% broccoli diet led to a coordinated increase in duodenal, cardiac, and pulmonary AHR activity, but no increase in activity in the liver. Thus, CYP1A1 dietary competitive substrates can lead to enhanced systemic AHR ligand distribution from the gut, likely through the lymphatic system, increasing AHR activation in key barrier tissues. Finally, this report will lead to a reassessment of the dynamics of distribution of other hydrophobic chemicals present in the diet.
Assuntos
Citocromo P-450 CYP1A1 , Trato Gastrointestinal , Pulmão , Receptores de Hidrocarboneto Arílico , Animais , Camundongos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Ligantes , Fígado/metabolismo , Pulmão/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Dieta , Trato Gastrointestinal/metabolismoRESUMO
The constitutive androstane receptor (CAR; NR1I3) contributes important regulatory roles in biotransformation, xenobiotic transport function, energy metabolism and lipid homeostasis. In this investigation, global serum and liver tissue metabolomes were assessed analytically in wild type and CAR-null transgenic mice using NMR, GC-MS and UPLC-MS/MS-based metabolomics. Significantly, CAR activation increased serum levels of fatty acids, lactate, ketone bodies and tricarboxylic acid cycle products, whereas levels of phosphatidylcholine, sphingomyelin, amino acids and liver glucose were decreased following short-term activation of CAR. Mechanistically, quantitative mRNA analysis demonstrated significantly decreased expression of key gluconeogenic pathways, and increased expression of glucose utilization pathways, changes likely resulting from down-regulation of the hepatic glucose sensor and bidirectional transporter, Glut2. Short-term CAR activation also resulted in enhanced fatty acid synthesis and impaired ß-oxidation. In summary, CAR contributes an expansive role regulating energy metabolism, significantly impacting glucose and monocarboxylic acid utilization, fatty acid metabolism and lipid homeostasis, through receptor-mediated regulation of several genes in multiple associated pathways.
Assuntos
Metabolismo Energético , Metabolômica/métodos , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Receptor Constitutivo de Androstano , Ácidos Graxos/metabolismo , Gluconeogênese , Glucose/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor orchestrating complex roles in cell and systems biology. Species differences in CAR's effector pathways remain poorly understood, including its role in regulating liver tumor promotion. We developed transgenic mouse models to assess genome-wide binding of mouse and human CAR, following receptor activation in liver with direct ligands and with phenobarbital, an indirect CAR activator. Genomic interaction profiles were integrated with transcriptional and biological pathway analyses. Newly identified CAR target genes included Gdf15 and Foxo3, important regulators of the carcinogenic process. Approximately 1000 genes exhibited differential binding interactions between mouse and human CAR, including the proto-oncogenes, Myc and Ikbke, which demonstrated preferential binding by mouse CAR as well as mouse CAR-selective transcriptional enhancement. The ChIP-exo analyses also identified distinct binding motifs for the respective mouse and human receptors. Together, the results provide new insights into the important roles that CAR contributes as a key modulator of numerous signaling pathways in mammalian organisms, presenting a genomic context that specifies species variation in biological processes under CAR's control, including liver cell proliferation and tumor promotion.
Assuntos
Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Neoplasias Hepáticas/genética , Receptores Citoplasmáticos e Nucleares/genética , Androstanos/química , Androstanos/metabolismo , Animais , Receptor Constitutivo de Androstano , Proteína Forkhead Box O3/genética , Genes myc/genética , Genoma/genética , Fator 15 de Diferenciação de Crescimento/genética , Hepatócitos/metabolismo , Humanos , Quinase I-kappa B/genética , Ligantes , Fígado/química , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , Ligação Proteica/genéticaRESUMO
Humans in industrialized areas are continuously exposed to phthalate plasticizers, prompting concerns of their potential toxicities. Previous studies from our laboratory and others have shown that various phthalates activate several mammalian nuclear receptors, in particular the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), and the peroxisomal proliferator-activated receptors (PPARs), although often at concentration levels of questionable relevance to human exposure. We discovered that di(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DiNP), two of the highest volume production agents, were potent activators of human CAR2 (hCAR2), a unique human CAR splice variant and, to a lesser degree, human PXR (hPXR). These diphthalates undergo rapid metabolism in mammalian systems, initially to their major monophthalate derivatives MEHP and MiNP. Although MEHP and MiNP are reported activators of the rodent PPARs, with lower affinities for the corresponding human PPARs, it remains unclear whether these monophthalate metabolites activate hCAR2 or hPXR. In this investigation, we assessed the relative activation potential of selected monophthalates and other low molecular weight phthalates against hCAR, the most prominent hCAR splice variants, as well as hPXR and human PPAR. Using transactivation and mammalian two-hybrid protein interaction assays, we demonstrate that these substances indeed activate hCARs and hPXR but to varying degrees. MEHP and MiNP exhibit potent activation of hCAR2 and hPXR with higher affinities for these receptors than for the hPPARs. The rank order potency for MEHP and MiNP was hCAR2 > hPXR > hPPARs. Results from primary hepatocyte experiments also reflect the MEHP and MiNP upregulation of the respective human target genes. We conclude that both di- and monophthalates are potently selective hCAR2 activators and effective hPXR activators. These results implicate these targets as important mediators of selective phthalate effects in humans. The striking differential affinities for these compounds between human and rodent nuclear receptors further implies that biological results obtained from rodent models may be of only limited relevance for interpolating phthalate-mediated effects in humans.
Assuntos
Ácidos Ftálicos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Idoso , Células Cultivadas , Receptor Constitutivo de Androstano , Relação Dose-Resposta a Droga , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Ácidos Ftálicos/química , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/metabolismo , Relação Estrutura-AtividadeRESUMO
The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily that functions as a xenosensor, serving to regulate xenobiotic detoxification, lipid homeostasis and energy metabolism. CAR activation is also a key contributor to the development of chemical hepatocarcinogenesis in mice. The underlying pathways affected by CAR in these processes are complex and not fully elucidated. MicroRNAs (miRNAs) have emerged as critical modulators of gene expression and appear to impact many cellular pathways, including those involved in chemical detoxification and liver tumor development. In this study, we used deep sequencing approaches with an Illumina HiSeq platform to differentially profile microRNA expression patterns in livers from wild type C57BL/6J mice following CAR activation with the mouse CAR-specific ligand activator, 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene (TCPOBOP). Bioinformatic analyses and pathway evaluations were performed leading to the identification of 51 miRNAs whose expression levels were significantly altered by TCPOBOP treatment, including mmu-miR-802-5p and miR-485-3p. Ingenuity Pathway Analysis of the differentially expressed microRNAs revealed altered effector pathways, including those involved in liver cell growth and proliferation. A functional network among CAR targeted genes and the affected microRNAs was constructed to illustrate how CAR modulation of microRNA expression may potentially mediate its biological role in mouse hepatocyte proliferation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.
Assuntos
Redes Reguladoras de Genes , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/genética , MicroRNAs/genética , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Animais , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Receptor Constitutivo de Androstano , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Biblioteca Gênica , Ontologia Genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Injeções Intraperitoneais , Ligantes , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/classificação , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Cultura Primária de Células , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de SinaisRESUMO
Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 µg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 µg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 µg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 µg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα.
Assuntos
Arocloros/toxicidade , Sequência de Bases , Misturas Complexas , Primers do DNA , Células Hep G2 , HumanosRESUMO
The CAR (constitutive androstane receptor; NR1I3) is a critical xenobiotic sensor that regulates xenobiotic metabolism, drug clearance, energy and lipid homoeostasis, cell proliferation and development. Although constitutively active, in hepatocytes CAR is normally held quiescent through a tethering mechanism in the cytosol, anchored to a protein complex that includes several components, including heat-shock protein 90. Release and subsequent nuclear translocation of CAR is triggered through either direct binding to ligand activators such as CITCO {6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime} or through indirect chemical activation, such as with PB (phenobarbital). In the present study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR's interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR (human CAR). Furthermore, a non-proteolytic function was indicated by its interaction with a SUG1 (suppressor for Gal1), a subunit of the 26S proteasome. Taken together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Receptor Constitutivo de Androstano , Primers do DNA , Humanos , UbiquitinaçãoRESUMO
Expression of the constitutive androstane receptor (CAR, NR1I3) is enriched in the mature mammalian liver and increasingly recognized for its prominent role in regulating a myriad of processes including biotransformation, chemical transport, energy metabolism and lipid homeostasis. Previously, we demonstrated that CAR levels were markedly enhanced during the differentiation of hepatic-like cells derived from hESCs, prompting the hypothesis that CAR contributes a key functional role in directing human hepatogenesis. Here we demonstrate that over-expression of CAR in human embryonic stem cells (ESCs), transduced by a lentiviral vector, accelerates the maturation of hepatic-like cells, with CAR over-expressing cells exhibiting a 2.5-fold increase in albumin secretion by day 20 in culture differentiation, and significantly enhanced levels of mRNA expression of several liver-selective markers, including hepatic transcription factors, plasma proteins, biotransformation enzymes, and metabolic enzymes. CAR over-expressing cells also exhibited enhanced CITCO-inducible CYP3A7 enzymatic activity. Knockdown of CAR via siRNA attenuated the differentiation-dependent expression programs. In contrast, expression levels of the pregnane X receptor (PXR), a nuclear receptor most similar to CAR in primary sequence, were negligible in human fetal liver tissues or in the differentiating hESCs, and stable over-expression of PXR in hepatic-induced hESCs failed to enhance expression of hepatic phenotype markers. Together, these results define a novel role for human CAR in hepatic lineage commitment.
Assuntos
Diferenciação Celular/fisiologia , Fígado/citologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Sequência de Bases , Linhagem Celular , Receptor Constitutivo de Androstano , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA , Células-Tronco Embrionárias/citologia , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Interferente Pequeno , Células-Tronco/citologiaRESUMO
The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays, obscuring the detection of ligand activators. However, a human splice variant of CAR, termed CAR3, exhibits negligible constitutive activity. In addition, CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study, we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses, thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse, rat, and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays, the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis.
Assuntos
Ligantes , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Substituição de Aminoácidos , Animais , Células Cultivadas , Receptor Constitutivo de Androstano , Cães , Regulação da Expressão Gênica , Hepatócitos/química , Hepatócitos/efeitos dos fármacos , Humanos , Itraconazol/farmacologia , Fígado/metabolismo , Camundongos , Isoformas de Proteínas , Sítios de Splice de RNA , RNA Mensageiro/química , Ratos , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The limited availability of hepatic tissue suitable for the treatment of liver disease and drug discovery research advances the generation of hepatic-like cells from alternative sources as a valuable approach. In this investigation we exploited a unique hepatic differentiation approach to generate hepatocyte-like cells from human embryonic stem cells (hESCs). hESCs were cultured for 10-20 days on collagen substrate in highly defined and serum free hepatocyte media. The resulting cell populations exhibited hepatic cell-like morphology and were characterized with a variety of biological endpoint analyses. Real-time PCR analysis demonstrated that mRNA expression of the 'stemness' marker genes NANOG and alkaline phosphatase in the differentiated cells was significantly reduced, findings that were functionally validated using alkaline phosphatase activity detection measures. Immunofluorescence studies revealed attenuated levels of the 'stemness' markers OCT4, SOX2, SSEA-3, TRA-1-60, and TRA-1-81 in the hepatic-like cell population. The hepatic character of the cells was evaluated additionally by real-time PCR analyses that demonstrated increased mRNA expression of the hepatic transcription factors FOXA1, C/EBPα, and HNF1α, the nuclear receptors CAR, RXRα, PPARα, and HNF4α, the liver-generated plasma proteins α-fetoprotein, transthyretin, transferrin, and albumin, the protease inhibitor α-1-antitrypsin, metabolic enzymes HMGCS2, PEPCK, and biotransformation enzymes CYP3A7, CYP3A4, CYP3A5, and CYP2E1. Indocyanine green uptake albumin secretion and glycogen storage capacity further confirmed acquisition of hepatic function. These studies define an expeditious methodology that facilitates the differentiation of hESCs along a hepatic lineage and provide a framework for their subsequent use in pharmacological and toxicological research applications requiring a renewable supply of human hepatocytes.