The Cellular and Molecular Determinants of Naphthoquinone-Dependent Activation of the Aryl Hydrocarbon Receptor

1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) are clinically promising biologically active chemicals that have been shown to stimulate the aryl hydrocarbon receptor (AhR) signaling pathway, but whether they are direct or indirect ligands or activate the AhR in a ligand-independent manner is unknown. Given the structural diversity of AhR ligands, multiple mechanisms of AhR activation of gene expression, and species differences in AhR ligand binding and response, we examined the ability of 1,2-NQ and 1,4-NQ to bind to and activate the mouse and human AhRs using a series of in vitro AhR-specific bioassays and in silico modeling techniques. Both NQs induced AhR-dependent gene expression in mouse and human hepatoma cells, but were more potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation and DNA binding in vitro and was inhibited by AhR antagonists. Ligand binding analysis confirmed the ability of 1,2-NQ and 1,4-NQ to competitively bind to the AhR ligand binding cavity and the molecular determinants for interactions were predicted by molecular modeling methods. NQs were shown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and differences were also observed between species. Mutation of amino acid residues (F289, M334, and M342) involved in critical NQ:AhR binding interactions, decreased NQ- and AhR-dependent gene expression, consistent with a role for these residues in binding and activation of the AhR by NQs. These studies provide insights into the molecular mechanism of action of NQs and contribute to the development of emerging NQ-based therapeutics.

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Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

International Journal of Molecular Sciences ◽

10.3390/ijms19092692 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2692 ◽

Cited By ~ 6

Author(s):

Samantha Faber ◽

Anatoly Soshilov ◽

Sara Giani Tagliabue ◽

Laura Bonati ◽

Michael Denison

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Ligand Binding ◽

Species Differences ◽

In Silico Analysis ◽

Site Directed Mutagenesis ◽

Ah Receptor ◽

Aryl Hydrocarbon ◽

Silico Analysis

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

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Disruption of endogenous regulator homeostasis underlies the mechanism of rat CYP1A1 mRNA induction by metyrapone

Biochemical Journal ◽

10.1042/bj3310273 ◽

1998 ◽

Vol 331 (1) ◽

pp. 273-281 ◽

Cited By ~ 13

Author(s):

Joanna L. HARVEY ◽

Alan J. PAINE ◽

Matthew C. WRIGHT

Keyword(s):

Gene Expression ◽

Glucocorticoid Receptor ◽

Aryl Hydrocarbon Receptor ◽

Culture Medium ◽

Male Rats ◽

Aryl Hydrocarbon ◽

Mrna Induction ◽

Cyp1a1 Gene ◽

Cyp1a1 Mrna

The transcriptional induction of the cytochrome P-450 1A1 (CYP1A1) gene by xenobiotics such as polyaromatic hydrocarbons is dependent on their interaction with the aryl hydrocarbon receptor. Administration of the structurally unrelated compounds metyrapone (a cytochrome P-450 inhibitor) or dexamethasone (a glucocorticoid) to male rats does not induce hepatic CYP1A1 mRNA. However, administration of both metyrapone and dexamethasone to male rats results in the induction of hepatic CYP1A1 mRNA expression. The induction response is mimicked in vitro in cultured rat hepatocytes by the addition of metyrapone and dexamethasone to a serum-free culture medium, suggesting that these compounds act directly on the liver in vivo to effect hepatic CYP1A1 mRNA induction. An examination of the characteristics of CYP1A1 induction by metyrapone and dexamethasone in combination in vitro indicate that at least 6 h of treatment is required for detectable levels of CYP1A1 mRNA to accumulate in hepatocytes. In contrast, β-naphthoflavone, which is known to bind to the aryl hydrocarbon receptor to effect CYP1A1 gene expression, induces detectable levels of CYP1A1 mRNA within 2 h of treatment. CYP1A1 mRNA is also induced when hepatocytes are treated with metyrapone in combination with the protein synthesis inhibitor cycloheximide but not with dexamethasone in combination with cycloheximide, indicating that CYP1A1 mRNA induction is strictly dependent on the presence of metyrapone and suggesting that the metyrapone-associated induction of CYP1A1 mRNA is dependent on a loss of a constitutively expressed protein that functions to suppress CYP1A1 gene expression. The role of dexamethasone in metyrapone-associated induction of CYP1A1 is probably mediated through the glucocorticoid receptor since the glucocorticoid receptor antagonist RU486 reduces the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination. Increasing the levels of the photosensitizer riboflavin present in the culture medium 10-fold and exposure to light increases the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination in vitro, suggesting that photoactivation of inducing medium constituent(s) might be required for induction. Failure to induce CYP1A1 mRNA by co-administration of metyrapone and dexamethasone in hepatocytes cultured in a balanced salt solution with or without photoactivation indicates that induction is dependent on a photoactivated component of the culture medium and not on metyrapone or dexamethasone alone. The addition of tryptophan in the presence of riboflavin to the balanced salt solution restores CYP1A1 mRNA induction by metyrapone alone and induction is increased when medium is exposed to light, indicating that induction is dependent on tryptophan photoactivation in vitro. Metyrapone failed to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for specific binding to the aryl hydrocarbon receptor in rat liver cytosolic fractions. These results suggest that CYP1A1 might be induced in rats by metyrapone through an indirect mechanism associated with an elevation in the level of an endogenously generated inducer such as photoactivated product(s) of tryptophan and not because of metyrapone's interacting with the aryl hydrocarbon receptor. The dependence of CYP1A1 induction on dexamethasone or cycloheximide suggests that derepression by a glucocorticoid receptor-modulated negative-acting factor of CYP1A1 gene expression might be critical to induction by metyrapone.

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The Aryl-hydrocarbon receptor does not require the p23 co-chaperone for ligand binding and target gene expression in vivo

Toxicology Letters ◽

10.1016/j.toxlet.2009.05.002 ◽

2009 ◽

Vol 189 (1) ◽

pp. 57-62 ◽

Cited By ~ 9

Author(s):

Colin Flaveny ◽

Gary H. Perdew ◽

Charles A. Miller

Keyword(s):

Gene Expression ◽

Ligand Binding ◽

Aryl Hydrocarbon Receptor ◽

Target Gene ◽

Target Gene Expression ◽

Aryl Hydrocarbon

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An evidence for regulatory cross-talk between aryl hydrocarbon receptor and glucocorticoid receptor in HepG2 cells

Physiological Research ◽

10.33549/a10.33549/physiolres.931090 ◽

2008 ◽

pp. 427-435

Author(s):

Z Dvořák ◽

R Vrzal ◽

P Pávek ◽

J Ulrichová

Keyword(s):

Glucocorticoid Receptor ◽

Aryl Hydrocarbon Receptor ◽

Cross Talk ◽

Hepg2 Cells ◽

Cellular Signaling ◽

Regulation Of Gene Expression ◽

Future Research ◽

Human Hepatoma Cells ◽

Aryl Hydrocarbon

Aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) play crucial role in the regulation of drug metabolizing enzymes and in many essential physiological processes. Cellular signaling by these receptors shares several functional and regulatory features. Here we investigated regulatory cross-talk between these two receptors. Human hepatoma cells (HepG2) were the model of choice. We analyzed the effects of dexamethasone (DEX) and dioxin (TCDD) on i) expression of AhR and GRα mRNAs; ii) levels of AhR and GR proteins; iii) transcriptional activities of AhR and GR in reporter assays; iv) 7-ethoxyresorufinO-deethylase activity (EROD). We found that both DEX and TCDD affected AhR and GR mRNAs expression, proteins levels and transcriptional activities in HepG2 cells. These effects on cellular signaling by AhR and GR comprised up-/down-regulation of gene expression and ligand-dependent protein degradation. We conclude that interactive regulatory cross-talk between GR and AhR receptors in HepG2 cells defines possible implications in physiology and drug metabolism. Future research should be focused on the investigation of AhR-GR cross-talk in various normal human cells and tissues both in vitro and in vivo.

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Hydroquinone Exposure Worsens Rheumatoid Arthritis through the Activation of the Aryl Hydrocarbon Receptor and Interleukin-17 Pathways

Antioxidants ◽

10.3390/antiox10060929 ◽

2021 ◽

Vol 10 (6) ◽

pp. 929

Author(s):

Cintia Scucuglia Heluany ◽

Paula Barbim Donate ◽

Ayda Henriques Schneider ◽

André Luis Fabris ◽

Renan Augusto Gomes ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Gene Expression ◽

Aryl Hydrocarbon Receptor ◽

Cigarette Smoke ◽

Cigarette Smoke Exposure ◽

Interleukin 17 ◽

Aryl Hydrocarbon ◽

Tnf Α

Rheumatoid arthritis (RA) development is strongly associated with cigarette smoke exposure, which activates the aryl hydrocarbon receptor (AhR) as a trigger for Th17 inflammatory pathways. We previously demonstrated that the exposure to hydroquinone (HQ), one of the major compounds of cigarette tar, aggravates the arthritis symptomatology in rats. However, the mechanisms related to the HQ-related RA still remain elusive. Cell viability, cytokine secretion, and gene expression were measured in RA human fibroblast-like synoviocytes (RAHFLS) treated with HQ and stimulated or not with TNF-α. Antigen-induced arthritis (AIA) was also elicited in wild type (WT), AhR −/− or IL-17R −/− C57BL/6 mice upon daily exposure to nebulized HQ (25ppm) between days 15 to 21. At day 21, mice were challenged with mBSA and inflammatory parameters were assessed. The in vitro HQ treatment up-regulated TNFR1, TNFR2 expression, and increased ROS production. The co-treatment of HQ and TNF-α enhanced the IL-6 and IL-8 secretion. However, the pre-incubation of RAHFLS with an AhR antagonist inhibited the HQ-mediated cell proliferation and gene expression profile. About the in vivo approach, the HQ exposure worsened the AIA symptoms (edema, pain, cytokines secretion and NETs formation) in WT mice. These AIA effects were abolished in HQ-exposed AhR −/− and IL-17R −/− animals though. Our data demonstrated the harmful HQ influence over the onset of arthritis through the activation and proliferation of synoviocytes. The HQ-related RA severity was also associated with the activation of AhR and IL-17 pathways, highlighting how cigarette smoke compounds can contribute to the RA progression.

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EFFECT OF ARYL HYDROCARBON RECEPTOR AGONISTS AND LIPOPOLYSACCHARIDE ON BENZO(A)PYRENE GENOTOXICITY MARKERS

Токсикологический вестник ◽

10.36946/0869-7922-2019-3-19-25 ◽

2019 ◽

pp. 19-25 ◽

Cited By ~ 1

Author(s):

V. N. Babakov ◽

N. Yu. Rogovskaya ◽

I. D. Kurdyukov ◽

P. P. Beltyukov ◽

S. A. Dulov ◽

...

Keyword(s):

Dna Repair ◽

Aryl Hydrocarbon Receptor ◽

P53 Protein ◽

Control Level ◽

Repair System ◽

Human Hepatoma Cells ◽

Kinase Activation ◽

Receptor Agonists ◽

Checkpoint Kinases ◽

Aryl Hydrocarbon

The effect of aryl hydrocarbon receptor agonists (FICZ and ITE), as well as lipopolysaccharide under the toxic action of benzo(a)pyrene in HepaRG human hepatoma cells was evaluated. Active forms of the key stress-activated kinase cascades and DNA repair system proteins were used as markers of the genotoxic action of benzo(a)pyrene. A mixture of lipopolysaccharide with benzo(a)pyrene increases benzo(a)pyrene cytotoxicity and reduces the activation of DNA repair system proteins below the control level. Aryl hydrocarbon receptor agonists (FICZ and ITE) exhibit a cytoprotective effect against benzo(a) pyrene, enhance Akt1 kinase activation, and downregulate activation of the p53 protein and Chk1 and Chk2 checkpoint kinases. Thus, FICZ and ITE reduce the genotoxicity of benzo(a)pyrene.

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