Yuichiro Kanno

Activation of the aryl hydrocarbon receptor represses mammosphere formation in MCF-7 cells

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Recent studies have reported the anti-tumor effects of the AhR in breast cancer. In this study, we investigated the anti-tumor effect of AhR activation based on the cancer stem cell hypothesis. We show that AhR activation suppressed mammosphere formation of MCF-7 cells and decreased the proportion of cells with high ALDH-1 (aldehyde dehydrogenase 1) activity. In addition, we also demonstrate that AhR activation regulates self-renewal signaling by down-regulating Wnt/β-catenin and Notch.

Diurnal difference in CAR mRNA expression

BackgroundThe constitutive androstane receptor (CAR, NR1I3) plays a key role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes.ResultsThe expression of CAR mRNA throughout the circadian rhythm is reported for the first time in phase with the clock gene Bmal1 and in antiphase with the clock-controlled gene Rev-erbα mRNAs, with a peak at Zeitgeber time (ZT) 20 and a trough at ZT8, and a peak/trough ratio of 2.0. The diurnal difference in CAR mRNA expression might underlie the 1.7-fold difference in the magnitude of the PB-dependent induction of CYP2B1/2 mRNA.ConclusionThe circadian oscillation of xenosensor gene CAR mRNA expression is partially responsible for chronopharmacokinetics and chronopharmacology in disease.

Suppressive effect of aryl hydrocarbon receptor repressor on transcriptional activity of estrogen receptor alpha by protein-protein interaction in stably and transiently expressing cell lines.

Aryl hydrocarbon receptor repressor (AhRR) suppressed, in a ligand independent manner, the ability of estrogen receptor alpha (ERalpha) to enhance the transcription of heterologous estrogen-responsive reporter plasmids in transient transfection assays, as well as of endogenous estrogen-responsive genes in human breast cancer MCF-7 cells. AhRR repressed ERalpha-mediated trans-activation by interfering allosterically with the ligand-independent function of AF-1. The direct interaction between AhRR and ERalpha at the multipartite binding site of ERalpha, which ranges from a DNA binding domain to a ligand binding domain, but did not include the AF-1 moiety was confirmed by a coimmunoprecipitation assay. The AhRR/ERalpha complex was formed in the nuclear compartment and was entrapped by a cis-element in the promoter of E2-responsive genes, as determined in a chromatin immunoprecipitation assay. AhRR might play a role of co-repressor on the transcriptional activity of the ERalpha homodimer.

HER2 overexpression-mediated inflammatory signaling enhances mammosphere formation through up-regulation of aryl hydrocarbon receptor transcription.

The interaction between HER2 and aryl hydrocarbon receptor (AhR) signaling cascades during mammosphere formation of MCF-7 cells was studied. A newly established clonal MCF-7 cell line (HER2-5), stably overexpressing HER2, showed significantly enhanced levels of AhR mRNA and protein compared with MCF-7 cells. AhR was required for the HER2-mediated induction of interleukin-6 mRNA and for mammosphere formation in HER2-5 and MCF-7 cells. Mammosphere forming efficiency was suppressed by an AhR antagonist in a dose-dependent manner, as well as by knockdown of AhR. Taken together, these results indicate that AhR enhances mammosphere formation by human HER2-overexpressing breast cancer cells.

DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1α, therefore it might act as mediator between hCAR and appropriate co-activators.

Dependence on the Microtubule Network and 90-kDa Heat Shock Protein of Phenobarbital-Induced Nuclear Translocation of the Rat Constitutive Androstane Receptor

The role of the microtubule network in the constitutive androstane receptor (CAR)-mediated transactivation of CYP2B induced by phenobarbital (PB) in rat primary hepatocytes was investigated using the microtubule-disrupting agent nocodazole (NCZ). In human hepatocytes, it was reported that CAR mRNA expression was decreased by a microtubule-disrupting agent through the inhibition of glucocorticoid receptor (GR)-mediated transactivation. However, in the present study, we show that the rat CAR gene was unaffected by the GR-mediated pathway in rat primary hepatocytes treated with NCZ. The PB-induced expression of CYP2B mRNA was repressed in the presence of NCZ for 2 h before and during 4 h of PB treatment, whereas the CAR mRNA and protein expression levels were not affected. Furthermore, the transactivation of the PB-responsive enhancer module-luciferase reporter gene and the nuclear transport of CAR induced by PB were also repressed in the presence of NCZ. Based on these findings, microtubular integrity might be required for PB-induced nuclear translocation of CAR in rat primary hepatocytes. In the same procedures, except that NCZ was replaced with radicicol, the CYP2B mRNA expression induced by PB was also repressed. Taking these into consideration, PB-mediated nuclear translocation of rCAR might be dependent on the 90-kDa heat shock protein as well as the microtubule network.

Nigramide J is a novel potent inverse agonist of the human constitutive androstane receptor

The constitutive androstane receptor (CAR, NR1I3) is very important for drug development and for understanding pharmacokinetic drug–drug interactions. We screened by mammalian one hybrid assay among natural compounds to discover novel ligands of human constitutive androstane receptor (hCAR). hCAR transcriptional activity was measured by luciferase assay and mRNA levels of CYP2B6 and CYP3A4 in HepTR‐hCAR cells and human primary hepatocytes were measured by real‐time RT‐PCR. Nigramide J (NJ) whose efficacy is comparable to those of hitherto known inverse agonists such as clotrimazole, PK11195, and ethinylestradiol. NJ is a naturally occurring cyclohexane‐type amide alkaloid that was isolated from the roots of Piper nigrum. The suppressive effect of NJ on the CAR‐dependent transcriptional activity was found to be species specific, in the descending order of hCAR, rat CAR, and mouse CAR. The unliganded hCAR‐dependent transactivation of reporter and endogenous genes was suppressed by NJ at concentrations higher than 5 μmol/L. The ligand‐binding cavity of hCAR was shared by NJ and CITCO, because they were competitive in the binding to hCAR. NJ interfered with the interaction of hCAR with coactivator SRC‐1, but not with its interaction with the corepressor NCoR1. Furthermore, NJ is agonist of human pregnane X receptor (hPXR). NJ is a dual ligand of hCAR and hPXR, being an agonist of hPXR and an inverse agonist of hCAR.

Picrasidine N Is a Subtype-Selective PPARβ/δ Agonist

Recently, growing evidence of the pivotal roles of peroxisome proliferator-activated receptor (PPAR) β/δ in various physiological functions, including lipid homeostasis, cancer, and inflammation, has raised interest in this receptor. In this study, the naturally occurring dimeric alkaloid picrasidine N (1) from Picrasma quassioides was identified as a novel PPARβ/δ agonist from a library consisting of plant extracts and natural compounds using a mammalian one-hybrid assay, and this compound was characterized. Compound 1 activated PPARβ/δ but did not activate or slightly activated PPARα and PPARγ. Furthermore, a peroxisome proliferator response element-driven luciferase reporter gene assay demonstrated that 1 enhanced PPARβ/δ transcriptional activity. Moreover, 1 selectively induced mRNA expression of ANGPTL4, which is a PPAR target gene. This observation is quite different from previously identified synthetic PPARβ/δ agonists, which can induce the expression of not only ANGPTL4 but also other PPAR target genes, such as ADRP, PDK4, and CPT-1. These results demonstrate that 1 is a potent subtype-selective and gene-selective PPARβ/δ agonist, suggesting its potential as a lead compound for further drug development. This compound would also be a useful chemical tool for elucidating the mechanism of PPARβ/δ-regulated specific gene expression and the biological significance of PPARβ/δ.

Identification of Picrasidine C as a Subtype-Selective PPARα Agonist

Picrasidine C (1), a dimeric β-carboline-type alkaloid isolated from the root of Picrasma quassioides, was identified to have PPARα agonistic activity by a mammalian one-hybrid assay from a compound library. Among the PPAR subtypes, 1 selectively activated PPARα in a concentration-dependent manner. Remarkably, 1 also promoted PPARα transcriptional activity by a peroxisome proliferator response element-driven luciferase reporter assay. Furthermore, 1 induced the expression of PPARα-regulated genes involved in lipid, glucose, and cholesterol metabolism, such as CPT-1, PPARα, PDK4, and ABCA1, which was abrogated by the PPARα antagonist MK-886, indicating that the effect of 1 was dependent on PPARα activation. This is the first report to demonstrate 1 to be a subtype-selective PPARα agonist with potential application in treating metabolic diseases, such as hyperlipidemia, atherosclerosis, and hypercholesterolemia.

Nigramide C Is a Natural Agonist of Human Pregnane X Receptor

Pregnane X receptor (PXR) is known as a xenosensor, playing a key role in response to xenochemical stimuli. Activation of PXR enhanced expression of various drug-metabolizing enzymes and transporters such as cytochrome P450 3A4 (CYP3A4). During a screening of a natural compounds library for novel ligands of human xenosensing receptors by the mammalian one-hybrid assay, two cyclohexene-type amide alkaloids were isolated, with nigramide C (NigC) showing the most potent activation of human PXR (hPXR). NigC-mediated hPXR activation was enhanced by overexpression of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor γ, coactivator 1α, and protein arginine methyltransferase 1. A direct interaction between the ligand-binding domain of hPXR and the receptor interaction domain of SRC1 was observed. NigC induced the expression of endogenous CYP3A4 mRNA and protein in both cultured hepatoma cells and primary hepatocytes. However, in primary hepatocytes, the relative agonist activity of NigC was not as potent as that of rifampicin, probably because of lower metabolic stability of NigC in these cells. In conclusion, NigC was found to be an effective agonist of hPXR. NigC is a useful tool for investigation of hPXR function.