RESUMO
OBJECTIVE: The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) continues to rise with the increasing obesity epidemic. Rezdiffra as an activator of a thyroid hormone receptor-beta is the only Food and Drug Administration approved therapy. As such, there is a critical need to improve our understanding of gene expression regulation and signaling transduction in MASLD to develop new therapies. Matrin-3 is a DNA- and RNA-binding protein involved in the pathogenesis of human diseases. Here we examined its previously uncharacterized role in limiting hepatic steatosis and stress response via the constitutive androstane receptor (CAR). METHODS: Matrin-3 floxed and liver-specific knockout mice were fed either a chow diet or 60 kcal% high-fat diet (HFD) for up to 16 weeks. The mice were euthanized for different analysis including liver histology, lipid levels, and gene expression. Bulk RNA-seq, bulk ATAC-seq, and single-nucleus Multiome were used to examine changes of transcriptome and chromatin accessibility in the liver. Integrative bioinformatics analysis of our data and publicly available datasets and different biochemical assays were performed to identify underlying the molecular mechanisms mediating matrin-3's effects. Liver-tropic adeno-associated virus was used to restore the expression of CAR for lipid, acute phase genes, and histological analysis. RESULTS: Matrin-3 expression is induced in the steatotic livers of mice. Liver-specific matrin-3 deletion exacerbated HFD-induced steatosis, acute phase response, and inflammation in the liver of female mice. The transcriptome and chromatin accessibility were re-programmed in the liver of these mice with signatures indicating that CAR signaling is dysregulated. Mechanistically, matrin-3 interacts with CAR mRNA, and matrin-3 deficiency promotes CAR mRNA degradation. Consequently, matrin-3 deletion impaired CAR signaling by reducing CAR expression. Matrin-3 levels positively correlate with CAR expression in human livers. Ces2a and Il1r1 were identified as new target genes of CAR. Interestingly, we found that CAR discords with the expression of its target genes including Cyp2b10 and Ces2a in response to HFD, indicating CAR signaling is dysregulated by HFD despite increased CAR expression. Dysregulated CAR signaling upon matrin-3 deficiency reduced Ces2a and de-repressed Il1r1 expression. CAR restoration partially abrogated the dysregulated gene expression, exacerbated hepatic steatosis, acute phase response, and inflammation in liver-specific matrin-3 knockout mice fed a HFD. CONCLUSIONS: Our findings demonstrate that matrin-3 is a key upstream regulator maintaining CAR signaling upon metabolic stress, and the matrin-3-CAR axis limits hepatic steatosis and stress response signaling that may give insights for therapeutic intervention.
Assuntos
Receptor Constitutivo de Androstano , Fígado Gorduroso , Fígado , Camundongos Knockout , Animais , Camundongos , Fígado/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Transdução de Sinais , Estresse FisiológicoRESUMO
BACKGROUND: Our recent studies showed that prolonged administration of novel atypical antipsychotics affected the expression and activity of cytochrome P450 (CYP), as demonstrated in vitro on human hepatocytes and in vivo on the rat liver. The aim of the present work was to study the effect of repeated treatment with asenapine, iloperidone, and lurasidone on the expression of transcription factors regulating CYP drug-metabolizing enzymes in rat liver. METHODS: The hepatic mRNA (qRT-PCR) and protein levels (Western blotting) of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPARγ) were measured in male Wistar rats after 2 week-treatment with asenapine, iloperidone or lurasidone. RESULTS: The 2-week treatment with asenapine significantly diminished the AhR and PXR expression (mRNA, protein level), and CAR mRNA level in rat liver. Iloperidone lowered the AhR and CAR expression and PXR protein level. Lurasidone did not affect the expression of AhR and CAR, but increased PXR expression. The antipsychotics did not affect PPARγ. CONCLUSIONS: Prolonged treatment with asenapine, iloperidone, or lurasidone affects the expression of transcription factors regulating the CYP drug-metabolizing enzymes. The changes in the expression of AhR, CAR, and PXR mostly correlate with alterations in the expression and activity of respective CYP enzymes found in our previous studies. Since these transcription factors are also engaged in the expression of phase II drug metabolism and drug transporters, changes in their expression may affect the metabolism of endogenous substrates and pharmacokinetics of concomitantly used drugs.
Assuntos
Antipsicóticos , Sistema Enzimático do Citocromo P-450 , Compostos Heterocíclicos de 4 ou mais Anéis , Isoxazóis , Fígado , Cloridrato de Lurasidona , Receptor de Pregnano X , Ratos Wistar , Receptores Citoplasmáticos e Nucleares , Animais , Antipsicóticos/farmacologia , Masculino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Pregnano X/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Cloridrato de Lurasidona/farmacologia , Isoxazóis/farmacologia , Piperidinas/farmacologia , Receptor Constitutivo de Androstano/metabolismo , Dibenzocicloeptenos/farmacologia , Receptores de Esteroides/metabolismo , PPAR gama/metabolismo , Fatores de Transcrição/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
Triclosan (TCS) is an antimicrobial toxicant found in a myriad of consumer products and has been detected in human tissues, including breastmilk. We have evaluated the impact of lactational TCS on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) neonatal mice. In hUGT1 mice, expression of the hepatic UGT1A1 gene is developmentally delayed resulting in elevated total serum bilirubin (TSB) levels. We found that newborn hUGT1 mice breastfed or orally treated with TCS presented lower TSB levels along with induction of hepatic UGT1A1. Lactational and oral treatment by gavage with TCS leads to the activation of hepatic nuclear receptors constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor alpha (PPARα), and stress sensor, activating transcription factor 4 (ATF4). When CAR-deficient hUGT1 mice (hUGT1/Car-/-) were treated with TCS, TSB levels were reduced with a robust induction of hepatic UGT1A1, leaving us to conclude that CAR is not tied to UGT1A1 induction. Alternatively, when PPARα-deficient hUGT1 mice (hUGT1/Pparα-/-) were treated with TCS, hepatic UGT1A1 was not induced. Additionally, we had previously demonstrated that TCS is a potent inducer of ATF4, a transcriptional factor linked to the integrated stress response. When ATF4 was deleted in liver of hUGT1 mice (hUGT1/Atf4ΔHep) and these mice treated with TCS, we observed superinduction of hepatic UGT1A1. Oxidative stress genes in livers of hUGT1/Atf4ΔHep treated with TCS were increased, suggesting that ATF4 protects liver from excessive oxidative stress. The increase oxidative stress may be associated with superinduction of UGT1A1. The expression of ATF4 in neonatal hUGT1 hepatic tissue may play a role in the developmental repression of UGT1A1.
Assuntos
Fator 4 Ativador da Transcrição , Animais Recém-Nascidos , Bilirrubina , Glucuronosiltransferase , Fígado , PPAR alfa , Triclosan , Animais , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/genética , PPAR alfa/metabolismo , PPAR alfa/genética , Camundongos , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Triclosan/farmacologia , Humanos , Bilirrubina/farmacologia , Bilirrubina/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Camundongos Knockout , Feminino , Receptor Constitutivo de Androstano , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are members of the nuclear receptor superfamily, which regulates various physiologic and pathologic processes. Phase separation is a dynamic biophysical process in which biomacromolecules form liquid-like condensates, which have been identified as contributors to many cellular functions, such as signal transduction and transcription regulation. However, the possibility of phase separation for CAR and PPARα remains unknown. This study explored the potential phase separation of CAR and PPARα The computational analysis utilizing algorithm tools examining the intrinsically disordered regions of CAR and PPARα suggested a limited likelihood of undergoing phase separation. Experimental assays under varying conditions of hyperosmotic stress and agonist treatments confirmed the absence of phase separation for these receptors. Additionally, the optoDroplets assay, which utilizes blue light stimulation to induce condensate formation, showed that there was no condensate formation of the fusion protein of Cry2 with CAR or PPARα Furthermore, phase separation of CAR or PPARα did not occur despite reduced target expression under hyperosmotic stress. In conclusion, these findings revealed that neither the activation of CAR and PPARα nor hyperosmotic stress induces phase separation of CAR and PPARα in cells. SIGNIFICANCE STATEMENT: Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are key regulators of various functions in the body. This study showed that CAR and PPARα do not exhibit phase separation under hyperosmotic stress or after agonist-induced activation. These findings provide new insights into the CAR and PPARα biology and physiology.
Assuntos
Receptor Constitutivo de Androstano , PPAR alfa , PPAR alfa/metabolismo , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Pressão Osmótica , Separação de FasesRESUMO
The fungicide fluxapyroxad (BAS 700â¯F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000â¯ppm and in female rats at a dietary level of 3000â¯ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000â¯ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000â¯ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250â¯ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000â¯ppm fluxapyroxad for 3 and 7 days and in female rats given 50-3000â¯ppm fluxapyroxad for 7 days and 250-3000â¯ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250-3000â¯ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1-100⯵M fluxapyroxad or 500⯵M sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid ß-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25â¯ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1-100⯵M fluxapyroxad or 500⯵M NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1-16⯵M fluxapyroxad or 500⯵M NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.
Assuntos
Receptor Constitutivo de Androstano , Fungicidas Industriais , Hepatócitos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares , Animais , Masculino , Feminino , Ratos , Fungicidas Industriais/toxicidade , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Humanos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Relação Dose-Resposta a Droga , Tamanho do Órgão/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/metabolismo , Replicação do DNA/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologiaRESUMO
Constitutive androstane receptor (CAR, Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid, and lipid metabolizing enzymes, stimulates liver hypertrophy and hyperplasia, and ultimately, hepatocellular carcinogenesis. The mechanisms linking early CAR responses to later disease development are poorly understood. Here we show that exposure of CD-1 mice to TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene), a halogenated xenochemical and selective CAR agonist ligand, induces pericentral steatosis marked by hepatic accumulation of cholesterol and neutral lipid, and elevated circulating alanine aminotransferase, indicating hepatocyte damage. TCPOBOP-induced steatosis was weaker in the pericentral region but stronger in the periportal region in females compared with males. Early (1 day) TCPOBOP transcriptional responses were enriched for CAR-bound primary response genes, and for lipogenesis and xenobiotic metabolism and oxidative stress protection pathways; late (2 weeks) TCPOBOP responses included many CAR binding-independent secondary response genes, with enrichment for macrophage activation, immune response, and cytokine and reactive oxygen species production. Late upstream regulators specific to TCPOBOP-exposed male liver were linked to proinflammatory responses and hepatocellular carcinoma progression. TCPOBOP administered weekly to male mice using a high corn oil vehicle induced carbohydrate-responsive transcription factor (MLXIPL)-regulated target genes, dysregulated mitochondrial respiratory and translation regulatory pathways, and induced more advanced liver pathology. Overall, TCPOBOP exposure recapitulates histological and gene expression changes characteristic of emerging steatotic liver disease, including secondary gene responses in liver nonparenchymal cells indicative of transition to a more advanced disease state. Upstream regulators of both the early and late TCPOBOP response genes include novel biomarkers for foreign chemical-induced metabolic dysfunction-associated steatotic liver disease.
Assuntos
Receptor Constitutivo de Androstano , Progressão da Doença , Fígado Gorduroso , Piridinas , Receptores Citoplasmáticos e Nucleares , Animais , Masculino , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Feminino , Piridinas/toxicidade , Piridinas/farmacologia , Camundongos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/patologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismoRESUMO
BACKGROUND: Antiretrovirals have the potential to cause drug interactions leading to inefficacy or toxicity via induction of efflux transporters through nuclear receptors, altering drug concentrations at their target sites. RESEARCH DESIGN AND METHODS: This study used molecular dynamic simulations and qRT-PCR to investigate bictegravir's interactions with nuclear receptors PXR and CAR, and its effects on efflux transporters (P-gp, BCRP, MRP1) in rat PBMCs. PBMC/plasma drug concentrations were measured using LC-MS/MS to assess the functional impact of transporter expression. RESULTS: Bictegravir significantly increased the expression of ABC transporters, with Car identified as a key mediator. This suggests that bictegravir's influence on nuclear receptors could affect drug transport and efficacy at the cellular level. CONCLUSIONS: Bictegravir activates nuclear receptors enhancing efflux transporter expression. Understanding these interactions is crucial for preventing drug-drug interactions and reducing toxicity in clinical use. Combining CAR antagonists with bictegravir may prevent drug resistance and toxicity. However, these findings are based on preclinical data and necessitate further clinical trials to confirm their applicability in clinical settings.
Assuntos
Interações Medicamentosas , Compostos Heterocíclicos de 4 ou mais Anéis , Leucócitos Mononucleares , Espectrometria de Massas em Tandem , Animais , Ratos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Piperazinas/farmacologia , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Simulação de Dinâmica Molecular , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Receptor Constitutivo de Androstano , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cromatografia Líquida/métodos , Ratos Sprague-Dawley , Dioxolanos/farmacologia , Dioxolanos/farmacocinética , Dioxolanos/administração & dosagem , Amidas , PiridonasRESUMO
Nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as aryl hydrocarbon receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these 4 major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.
Assuntos
Receptor Constitutivo de Androstano , Fator 4 Nuclear de Hepatócito , Fígado , Camundongos Knockout , PPAR alfa , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares , Animais , Fator 4 Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fígado/metabolismo , Fígado/efeitos dos fármacos , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR alfa/genética , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Camundongos , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores de Esteroides/agonistas , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Pirimidinas/farmacologia , Dibenzodioxinas Policloradas/toxicidade , Piridinas/farmacologiaRESUMO
4-Nonylphenol (4-NP), a para-substituted phenolic compound with a straight or branched carbon chain, is a ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor (ED) with potent activities on estrogen receptors. Constitutive Androstane Receptor (CAR) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in the ED mechanism of action. An NP mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by 4-NP are enigmatic, and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here, we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR variants using molecular dynamics (MD) simulations, cellular experiments with various CAR expression constructs, recombinant CAR LBD in a TR-FRET assay, or a differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type CAR1 and CAR3 variant LBDs in MD simulations. Consistently, branched 4-NPs activated CAR3 and CAR1 LBD more efficiently than linear 4-NP. Furthermore, in HepaRG cells, we observed that all 4-NPs upregulated CYP2B6 mRNA, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and human CAR-LBD, as well as its dominant variant CAR3. The work could contribute to the safer use of individual 4-NPs in many areas of industry.
Assuntos
Fenóis , Humanos , Fenóis/química , Fenóis/metabolismo , Receptor Constitutivo de Androstano/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Disruptores Endócrinos/química , Simulação de Dinâmica MolecularRESUMO
Numerous studies have demonstrated the correlation between human gut bacteria and host physiology, mediated primarily via nuclear receptors (NRs). Despite this body of work, the systematic identification and characterization of microbe-derived ligands that regulate NRs remain a considerable challenge. In this study, we discover a series of diindole molecules produced from commensal bacteria metabolites that act as specific agonists for the orphan constitutive androstane receptor (CAR). Using various biophysical analyses we show that their nanomolar affinities are comparable to those of synthetic CAR agonists, and that they can activate both rodent and human CAR orthologues, which established synthetic agonists cannot. We also find that the diindoles, diindolylmethane (DIM) and diindolylethane (DIE) selectively up-regulate bona fide CAR target genes in primary human hepatocytes and mouse liver without causing significant side effects. These findings provide new insights into the complex interplay between the gut microbiome and host physiology, as well as new tools for disease treatment.
Assuntos
Receptor Constitutivo de Androstano , Microbiota , Camundongos , Animais , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Hepatócitos/metabolismo , LigantesRESUMO
BACKGROUND AND PURPOSE: Gut microbiota and their metabolic activity are important regulators of host immunity. However, the role of gut microbiota and their metabolic activity-mediated osteoimmunity in postmenopausal osteoporosis (PMO) remains unknown. This study aimed to explore the role of gut microbiota and their metabolic activity in PMO. EXPERIMENTAL APPROACH: 16S rDNA sequencing was used for analyzing the gut microbiota diversity of patients with PMO and rat models, and a targeted metabolism study was performed for analyzing metabolite levels. Flow cytometry was used for analyzing the frequency of immune cells. Micro-CT was used for analyzing bone damage in rat models. Fecal microbiota transplantation was performed for exploring the therapeutic effect of the gut microbiota on PMO. CD4+ T cells were co-cultured with bone marrow mesenchymal stem cells for evaluating their molecular mechanisms. KEY RESULTS: Patients with PMO exhibited reduced gut microbiota diversity, and fecal glycolithocholic acid (GLCA) levels correlated with the degree of osteoporosis. GLCA levels in the gut were positively correlated with the frequency of circulating Tregs in ovariectomized rats. Restoration of the gut microbiota alleviated osteoporosis in ovariectomized rats. Circulating GLCA augmented CD4+ T cell differentiation into Tregs via constitutive androstane receptors. The increased frequency of Tregs further promoted the osteogenic differentiation of bone marrow mesenchymal stem cells to alleviate osteoporosis. CONCLUSION AND IMPLICATIONS: GLCA alleviated PMO by increasing the frequency of circulating Tregs, acting via the constitutive androstane receptor. This study reveals a new strategy for the treatment of PMO, with GLCA as a potential drug candidate.
Assuntos
Osteoporose Pós-Menopausa , Humanos , Feminino , Ratos , Animais , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/genética , Osteogênese , Receptor Constitutivo de Androstano , Diferenciação CelularRESUMO
Scoparone (6, 7 dimethylesculetin) is a biologically active compound derived from the herb Artemisia capillaris having anti-inflammatory, anti-lipemic, and anti-allergic roles. Activation of the constitutive androstane receptor (CAR) in primary hepatocytes of both wild-type and humanized CAR mice by scoparone, accelerates bilirubin and cholesterol clearance in vivo. This can prevent gallstones which is a dreaded gastrointestinal disease. To date, surgery is regarded as the gold standard for treating gallstones. The molecular interactions between scoparone and CAR leading to gallstone prevention are not yet explored. In this study, we have analyzed these interactions through an insilico approach. After extracting the CAR structures (mice and human) from the protein databank and 6, 7-dimethylesuletin from PubChem, energy minimization of both the receptors was done to make them stable followed by docking. Next, a simulation was performed to stabilize the docked complexes. Through docking, H-bonds and pi-pi interactions were found in the complexes, which imply a stable interaction, thus activating the CAR. A similarity search for scoparone was performed and the selected compounds were docked with the CAR receptors. Esculentin acetate and scopoletin acetate interacted with human CAR through pi-alkyl and H-bond respectively. While Fraxidin methyl ether, fraxinol methyl ether, and 6, 7 diethoxycoumarin interacted with mice CAR through H-bond and Pi-Pi T-shaped bonds. The selected complexes were simulated further. Our results are in accordance with the hypothesis in the literature. We have also analyzed the drug likeliness, absorption, non-carcinogenicity, and other properties of scoparone which can support further in vivo studies.Communicated by Ramaswamy H. Sarma.
Assuntos
Cumarínicos , Cálculos Biliares , Éteres Metílicos , Camundongos , Humanos , Animais , Receptor Constitutivo de Androstano , Receptores Citoplasmáticos e Nucleares , AcetatosRESUMO
Constitutive androstane receptor (CAR), a nuclear receptor predominantly expressed in the liver, is activated by diverse chemicals and induces hepatocyte proliferation and hepatocarcinogenesis in rodents. However, the underlying mechanism responsible for CAR-dependent hepatocyte proliferation remains unclear. Importantly, this phenomenon has not been observed in the human liver. This study aimed to investigate the molecular mechanism underlying CAR-induced hepatocyte proliferation and to explore the species differences in hepatocyte proliferation between humans and rodents. Treatment of mice with the CAR activator TCPOBOP induced hepatocyte proliferation and nuclear accumulation of yes-associated protein (YAP), a known liver cancer inducer. This induction was abolished in CAR-knockout mice. Exogenously expressed YAP in cultured cells was accumulated in the nucleus by the coexpression with mouse CAR but not human CAR. Pull-down analysis of recombinant proteins revealed that mouse CAR interacted with YAP, whereas human CAR did not. Further investigations using YAP deletion mutants identified the WW domain of YAP as essential for interacting with CAR and showed that the PY motif (PPAY) in mouse CAR was crucial for binding to the WW domain, whereas human CAR with its mutated motif (PPAH) failed to interact with YAP. A mouse model harboring the Y150H mutation (PPAY to PPAH) in CAR displayed drastically attenuated TCPOBOP-induced hepatocyte proliferation and nuclear accumulation of YAP. CAR induces the nuclear accumulation of YAP through the PY motif-WW domain interaction to promote hepatocyte proliferation. The absence of this interaction in human CAR contributes to the lack of CAR-dependent hepatocyte proliferation in human livers.
Assuntos
Receptor Constitutivo de Androstano , Roedores , Animais , Humanos , Camundongos , Proliferação de Células , Hepatócitos/metabolismo , Fígado/metabolismo , Especificidade da EspécieRESUMO
Traditionally, xenobiotic receptors are known for their role in chemical sensing and detoxification, as receptor activation regulates the expression of various key enzymes and receptors. However, recent studies have highlighted that xenobiotic receptors also play a key role in the regulation of lipid metabolism and therefore function also as metabolic sensors. Since dyslipidemia is a major risk factor for various cardiometabolic diseases, like atherosclerosis and non-alcoholic fatty liver disease, it is of major importance to understand the molecular mechanisms that are regulated by xenobiotic receptors. In this review, three major xenobiotic receptors will be discussed, being the aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Specifically, this review will focus on recent insights into the metabolic functions of these receptors, especially in the field of lipid metabolism and the associated dyslipidemia.
Assuntos
Dislipidemias , Receptores de Esteroides , Humanos , Receptor de Pregnano X/metabolismo , Receptor Constitutivo de Androstano , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Xenobióticos/metabolismo , Proteínas de TransporteRESUMO
Activation of the constitutive androstane receptor (CAR, NR1I3) by chemical compounds induces liver hyperplasia in rodents. 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a mouse CAR agonist, is most often used to study chemically induced liver hyperplasia and hepatocyte proliferation in vivo. TCPOBOP is a potent murine liver chemical mitogen, which induces rapid liver hyperplasia in mice independently of liver injury. In recent years, great amount of data has been accumulated on the transcription program that characterizes the TCPOBOP-induced hepatocyte proliferation. However, there are only few data about the metabolic requirements of hepatocytes that divide upon exposure to xenobiotics. In the present study, we have employed liquid chromatography - mass spectrometry technology combined with statistical analysis to investigate metabolite profile of small biomolecules, in order to identify key metabolic changes in the male mouse liver tissue after TCPOBOP administration. Analysis of biochemical pathways of the differentially affected metabolites in the mouse liver demonstrated significant TCPOBOP-mediated enrichment of several processes including those associated with nucleotide metabolism, amino acid metabolism, and energy substrate metabolism. Our findings provide evidence to support the conclusion that the CAR agonist, TCPOBOP, initiates an intracellular program that promotes global coordinated metabolic activities required for hepatocyte proliferation. Our metabolic data might provide novel insight into the biological mechanisms that occur during the TCPOBOP-induced hepatocyte proliferation in mice.
Assuntos
Receptor Constitutivo de Androstano , Receptores Citoplasmáticos e Nucleares , Animais , Masculino , Camundongos , Proliferação de Células , Receptor Constitutivo de Androstano/agonistas , Hepatócitos/metabolismo , Hiperplasia/metabolismo , Hiperplasia/patologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
The constitutive androstane receptor (CAR) regulates enzyme transcription related to drug metabolism; therefore, natural compound clarification in food that interacts with CAR is significant for drug development. We revealed that 13-epimanool, which is a compound found in the common sage, is bound to hCAR based on differential scanning fluorometry (DSF) measurements using recombinant hCAR protein. Similar labdane diterpenoids were examined, which revealed that manool and sclareol, which were both natural compounds contained in herbs, are bound to hCAR. They exhibited different effects for CAR activity in the luciferase assay despite the structural similarity. Manool was a partial agonist, 13-epimanool was a weak partial agonist, and sclareol was an antagonist. The activity of hCAR may be regulated by slight differences in the bound compound.
Assuntos
Receptor Constitutivo de Androstano , Diterpenos , Humanos , Receptores Citoplasmáticos e Nucleares , Diterpenos/farmacologiaRESUMO
Following the discovery of 2-(3-methoxyphenyl)-3,4-dihydroquinazoline-4-one and 2-(3-methoxyphenyl)quinazoline-4-thione as potent, but non-specific activators of the human Constitutive Androstane Receptor (CAR, NR1I3), a series of quinazolinones substituted at the C2 phenyl ring was prepared to examine their ability to selectively modulate human CAR activity. Employing cellular and in vitro TR-FRET assays with wild-type CAR or its variant 3 (CAR3) ligand binding domains (LBD), several novel partial human CAR agonists and antagonists were identified. 2-(3-Methylphenyl) quinazolinone derivatives 7d and 8d acted as partial agonists with the recombinant CAR LBD, the former in nanomolar units (EC50 = 0.055 µM and 10.6 µM, respectively). Moreover, 7d did not activate PXR, and did not show any signs of cytotoxicity. On the other hand, 2-(4-bromophenyl)quinazoline-4-thione 7l possessed significant CAR antagonistic activity, although the compound displayed no agonistic or inverse agonistic activities. A compound possessing purely antagonistic effect was thus identified for the first time. These and related compounds may serve as a remedy in xenobiotic intoxication or, conversely, in suppression of undesirable hepatic CAR activation.
Assuntos
Receptor Constitutivo de Androstano , Receptores de Esteroides , Humanos , Receptores Citoplasmáticos e Nucleares , Ligantes , Quinazolinas/farmacologia , Tionas , Receptores de Esteroides/agonistas , Receptores de Esteroides/metabolismoRESUMO
The nuclear receptor, constitutive androstane receptor (CAR), which forms a heterodimer with the retinoid X receptor (RXR), was initially reported as a transcription factor that regulates hepatic genes involved in detoxication and energy metabolism. Different studies have shown that CAR activation results in metabolic disorders, including non-alcoholic fatty liver disease, by activating lipogenesis in the liver. Our objective was to determine whether synergistic activations of the CAR/RXR heterodimer could occur in vivo as described in vitro by other authors, and to assess the metabolic consequences. For this purpose, six pesticides, ligands of CAR, were selected, and Tri-butyl-tin (TBT) was used as an RXR agonist. In mice, CAR's synergic activation was induced by dieldrin associated with TBT, and combined effects were induced by propiconazole, bifenox, boscalid, and bupirimate. Moreover, a steatosis, characterized by increased triglycerides, was observed when TBT was combined with dieldrin, propiconazole, bifenox, boscalid, and bupirimate. Metabolic disruption appeared in the form of increased cholesterol and lowered free fatty acid plasma levels. An in-depth analysis revealed increased expression of genes involved in lipid synthesis and lipid import. These results contribute to the growing understanding of how environmental contaminants can influence nuclear receptor activity and associated health risks.
Assuntos
Doenças Metabólicas , Hepatopatia Gordurosa não Alcoólica , Praguicidas , Animais , Camundongos , Receptor Constitutivo de Androstano , Receptores X de Retinoides/metabolismo , Praguicidas/toxicidade , Dieldrin , Receptores Citoplasmáticos e Nucleares , LipídeosRESUMO
The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that controls the transcription of genes responsible for fatty acid metabolism. We have recently reported a possible drug-drug interaction mechanism via the interaction of PPARα with the xenobiotic nuclear receptor constitutive androstane receptor (CAR). Drug-activated CAR competes with the transcriptional coactivator against PPARα and prevents PPARα-mediated lipid metabolism. In this study, to elucidate the crosstalk between CAR and PPARα, we focused on the influence of PPARα activation on CAR's gene expression and activation. Male C57BL/6N mice (8-12 weeks old, n = 4) were treated with PPARα and CAR activators (fenofibrate and phenobarbital, respectively), and hepatic mRNA levels were determined using quantitative reverse transcription PCR. Reporter assays using the mouse Car promoter were performed in HepG2 cells to determine the PPARα-dependent induction of CAR. CAR KO mice were treated with fenofibrate, and the hepatic mRNA levels of PPARα target genes were determined. Treatment of mice with a PPARα activator increased Car mRNA levels as well as genes related to fatty acid metabolism. In reporter assays, PPARα induced the promoter activity of the Car gene. Mutation of the putative PPARα-binding motif prevented PPARα-dependent induction of reporter activity. In electrophoresis mobility shift assay, PPARα bound to the DR1 motif of the Car promoter. Since CAR has been reported to attenuate PPARα-dependent transcription, CAR was considered a negative feedback protein for PPARα activation. Treatment with fenofibrate induced the mRNA levels of PPARα target genes in Car-null mice more than those in wild-type mice, suggesting that CAR functions as a negative feedback factor for PPARα.
Assuntos
Receptor Constitutivo de Androstano , Fígado , PPAR alfa , Animais , Masculino , Camundongos , Receptor Constitutivo de Androstano/genética , Receptor Constitutivo de Androstano/metabolismo , Ácidos Graxos/metabolismo , Fenofibrato/farmacologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , PPAR alfa/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.