Kynurenic acid is a potent endogenous aryl hydrocarbon receptor ligand that synergistically induces interleukin-6 in the presence of inflammatory signaling

Abstract

Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations.

FIG. 1.

KA is the primary tryptophan oxidation product that exhibits potent AHR agonist activity. (A) TCDD and the primary products of the IDO pathway were tested at 10μM for their ability after 4 h to activate the AHR in HepG2 40/6 reporter cell line. (B) Induction of CYP1A1 mRNA levels in HepG2 cells after 3-h exposure to increasing concentrations of KA. (C) Structures of the compounds utilized to determine the structural requirements necessary to mediate AHR activity. (D) Luciferase reporter assays were used to test the ability of quinoline and several derivatives at 10μM to activate the AHR in HepG2 40/6 cells. Experiments were performed in triplicate; error bars denote SD. ** or ***Significance at p < 0.01 or p < 0.001, as compared to respective control-treated samples.

FIG. 2.

KA induces CYP1A1/2 metabolism and differentially activates the human AHR relative to the mouse AHR in reporter cell lines. (A) CYP1A1/2 metabolic activity was measured by CEE luciferase in primary human hepatocytes treated with KA, TCDD, or DMSO for 18 h. Experiment was performed in triplicate; error bars denote SD. * or ***Significance at p < 0.05 or p < 0.001, as compared to respective control-treated samples. (B) HepG2 40/6 and (C) H1L1.1c2 cell lines were exposed to increasing concentrations of KA for 4 h. The EC₂₅ values are defined as the dose of KA that yields 25% of the luciferase activity obtained with 10nM TCDD.

FIG. 3.

KA selectively induces human AHR/ARNT binding to its cognate response element and binds directly to the ligand-binding pocket of the AHR. (A) In vitro translated AHR and ARNT were mixed and treated with ligand as indicated and then subjected to an EMSA analysis. (B) Photoaffinity ligand competition assays were performed with increasing concentrations of ligands and KA and benzo(a)pyrene; both effectively reduced radioligand binding. (C) Quantitative graphic illustration of the results from panel B. The IC₅₀ is defined as the concentration of ligand that results in 50% reduction of photoaffinity ligand binding to the human AHR.

FIG. 4.

KA and IL1B synergistically induce IL6 gene expression and requires AHR expression. (A and B) MCF-7 cells were treated with increasing doses of KA for 2 h with or without IL1B and subjected to quantitative real-time PCR for IL6 and CYP1A1. (C) MCF-7 cells were electroporated with control or AHR siRNA and analyzed 48 h later after a 3-h exposure to IL1B in the presence or absence of KA. Western blot analyses of whole-cell extract, AHR, and p23 protein levels were assessed. (C and D) Real-time PCR analysis was performed for IL6 and CYP1A1 following 2-h KA treatment 48 h after electroporation. Experiments were performed in triplicate; error bars denote SD. * or ***Significance at p < 0.05 or p < 0.001, as compared to respective control-treated samples. Statistical analysis in panel A was performed relative to their respective vehicle controls.