Cooperation of structurally different aryl hydrocarbon receptor agonists and β-catenin in the regulation of CYP1A expression

Toxicology 325 (2014) 31–41

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Cooperation of structurally different aryl hydrocarbon receptor agonists and b-catenin in the regulation of CYP1A expression Sebastian Vaas 1, Luisa Kreft 1, Michael Schwarz, Albert Braeuning * Institute of Experimental and Clinical Pharmacology and Toxicology, Dept. of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 August 2014 Received in revised form 25 August 2014 Accepted 27 August 2014 Available online 28 August 2014

The ligand-activated nuclear receptor AhR (aryl hydrocarbon receptor) mediates the response of hepatocytes to various exogenous compounds. AhR is classically activated by planar, aromatic hydrocarbons, but also by other, structurally rather unrelated compounds. Recent data show that the canonical Wnt/b-catenin signaling pathway is also involved in the regulation of hepatic zonal gene expression and drug metabolism in mammalian liver. Previous studies indicate that the loss of b-catenin in hepatocytes diminishes the response to the AhR agonists 3-methylcholanthrene (3MC) in vivo and to 2,3,7,8-tetrachlorodibenzo-[p]-dioxin in vitro. The knockout of b-catenin also impairs the zonal pattern of AhR target gene induction by 3MC. However, it is presently unknown whether the chemical nature of the AhR agonist influences the AhR/b-catenin interaction. Moreover, no information is available about the dose–response curves of AhR activation in the absence or presence of Wnt/b-catenin signaling. In the present study, we have analyzed AhR-dependent responses to different concentrations of structurally unrelated AhR agonists in vivo and in vitro. The results demonstrate that b-catenin is essential to obtain the maximum AhR response. Moreover, using transgenic mouse models which allow for the ablation of b-catenin at different age of mice, we demonstrate that the presence of b-catenin, not postnatal developmental effects in b-catenin-deficient livers, is responsible for the observed interplay of b-catenin and the AhR. ã 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cytochrome P450 Liver zonation Perivenous hepatocyte Dioxin receptor TCDD Wnt signaling

1. Introduction The canonical Wnt/b-catenin signaling pathway is an important regulator of adult tissue homeostasis, embryonic development, and tumorigenesis. Extensive reviews of the pathway and its functions can be found in (MacDonald et al., 2009; Nejak-Bowen and Monga, 2008; Takigawa and Brown, 2008). In brief, physiological activation of the pathway by Wnt molecules acting as agonists at so-called Frizzled receptors leads to the stabilization of b-catenin, the crucial protein within this pathway. In the absence of Wnts, free cytosolic b-catenin is phosphorylated by

Abbreviations: AhR, aryl hydrocarbon receptor; BHA, butylated hydroxyanisole; BNF, b-naphthoflavone; CAR, constitutive androstane receptor; CYP, cytochrome P450; DRE, dioxin response element; GS, glutamine synthetase; GST, glutathione Stransferase; 3MC, 3-methylcholanthrene; KO, knockout; tBHQ, tert-butylhydroquinone; TCDD, 2,3,7,8-tetrachlorodibenzo-[p]-dioxin; TCF, T cell factor; WT, wild type. * Corresponding author. Tel.: +49 7071 2974935; fax: +49 7071 292273. E-mail address: [email protected] (A. Braeuning). 1 These authors contributed equally to this work and should both be considered as first authors. http://dx.doi.org/10.1016/j.tox.2014.08.010 0300-483X/ ã 2014 Elsevier Ireland Ltd. All rights reserved.

glycogen synthase kinase 3b and casein kinase 1a in a multiprotein complex. This marks b-catenin for subsequent proteasomal degradation. Upon pathway activation by Wnts, the activity of the b-catenin phosphorylation complex is inhibited and b-catenin will accumulate in the cytosol and translocate into the nucleus. There, the protein acts as a transcriptional co-activator of TCF (T cell factor) transcription factors. During the past few years, an important role of b-catenin in the regulation of gene expression in healthy adult liver has been revealed: transcriptionally active b-catenin is exclusively present in perivenous hepatocytes surrounding the central vein of each liver lobule (Benhamouche et al., 2006; Sekine et al., 2007). The hepatocyte-specific knockout (KO) of the Ctnnb1 gene, encoding b-catenin, leads to a dramatic loss of the expression of perivenous marker genes such as glutamine synthetase, the model hepatic b-catenin target gene, ammonia metabolism-related genes, and especially genes encoding enzymes involved in the metabolism of drugs and xenobiotics, including many important cytochrome P450 (CYP) enzymes from families 1–3 (Benhamouche et al., 2006; Braeuning et al., 2009; Braeuning and Schwarz, 2010; Sekine et al., 2006; Tan et al., 2006). Inversely, the expression of a transgene encoding a mutant, constitutively active version of b-catenin

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induces the expression of perivenous markers in periportal hepatocytes (Schreiber et al., 2011). Drug-metabolizing enzymes such as the CYPs are known to be regulated by a set of nuclear receptors. One of the most extensively studied xenobiotic-sensing receptors is the aryl hydrocarbon receptor (AhR). For a recent review of the AhR and its functions, please refer to (Abel and Haarmann-Stemmann, 2010). The AhR is classically activated by polycyclic aromatic hydrocarbons and dioxins, of which 2,3,7,8,-tetrachloro-[p]-dioxin (TCDD) is the most potent and prominent ligand and a potent inducer of members of CYPs from family 1. However, the receptor is also activated by other classes of chemicals, e.g., flavonoids, endogenous tryptophan derivatives, or by the antioxidant tert-butylhydroquinone (tBHQ; (Schreiber et al., 2006)). Recent studies have demonstrated that b-catenin interacts with the AhR in the induction of drug-metabolizing enzymes: basal expression of the AhR target cytochrome P450 (Cyp) Cyp1a2 is diminished in mice with hepatocyte-specific KO of Ctnnb1 (Braeuning et al., 2009; Sekine et al., 2006; Tan et al., 2006) and the induction of AhR target Cyps by the AhR agonist 3-methylcholanthrene (3MC) is reduced in this mouse model. Both signaling pathways synergize in the induction of AhR targets by TCDD in vitro (Braeuning et al., 2011; Loeppen et al., 2005; Prochazkova et al., 2011). Mechanistic studies indicate that b-catenin and the AhR physically interact and that activation of b-catenin enhances the activity of the AhR at its binding sites on the DNA (Braeuning et al., 2011). Synergistic effects with b-catenin signaling have also been reported for other receptors involved in the induction of drug-metabolizing enzymes, i.e., the constitutive androstane receptor (CAR) and Nrf2 (Braeuning et al., 2009). Interestingly, hepatic zonation of target enzyme induction was lost in mice with KO of Ctnnb1 in that study. By contrast, the zonated response to CAR activation was preserved when b-catenin was not knocked out in the early postnatal phase as in (Braeuning et al., 2009), but not before adulthood, suggesting b-catenin-dependent postnatal priming of perivenous hepatocytes for the susceptibility to CAR agonists (Ganzenberg et al., 2013). The AhR is activated by different substances with considerable differences regarding their chemical structures (see above). These differences and especially the differences regarding downstream biological effects (e.g., perivenous induction of CYP1A by the AhR agonist 3MC but periportal induction by the AhR agonist b-naphthoflavone (BNF)) point towards differences in AhR-related cellular signaling following exposure to these different types of agonists. Up to now, it is not known whether the chemical nature of the AhR agonist has an influence on the interaction of the b-catenin pathway with the activated AhR. Furthermore, the observation that AhR target gene induction at a certain concentration of TCDD is lowered in the absence of b-catenin raises questions about the shape of the corresponding dose–response curves, which cannot be answered with the existing studies. In addition, it is unknown whether the zonation of the AhR response solely depends on the presence of b-catenin at the time point of treatment or whether developmental aspects play a role as in the case of CAR activation. Therefore, in the present study, we examined the response of mice with hepatocyte-specific KO of Ctnnb1 to three different AhR agonists: the polycyclic aromatic hydrocarbon 3MC, the flavonoid BNF, and the antioxidant butylated hydroxyanisole (BHA), the metabolic precursor of tBHQ. Additional studies were conducted in vitro to obtain information about the dose–response relationship of AhR activation in the absence or presence of active b-catenin. Moreover, a comparison of 3MC-induced AhR target gene expression in mice with early postnatal and adult KO of b-catenin was performed.

2. Material and methods 2.1. Animal breeding Mice with loxP site-flanked Ctnnb1 (encoding b-catenin) alleles (Huelsken et al., 2001) were interbred either with Alb-Cre or TTRCre mice as previously described (Ganzenberg et al., 2013). This resulted in two different mouse models for Cre recombinasemediated genetic ablation of b-catenin. First, mice with albumin (Alb-Cre model) promoter-driven hepatocyte-specific knockout (KO) of Ctnnb1 (Braeuning et al., 2009), where Cre recombinase accomplishes target gene recombination shortly after birth (Postic and Magnuson, 2000). Second, mice with transthyretin (TTR-Cre model) promoter-driven hepatocyte-specific KO of Ctnnb1 (Ganzenberg et al., 2013), where a modified Cre enzyme can be activated by tamoxifen at the desired point in time. Genotyping was performed by standard PCR using the following primer pairs: Ctnnb1loxP_fwd 50 -ACTGCCTTTGTTCTCTTCCCTTCTG-30 ; Ctnnb1loxP _rev 50 -CAGCCAAGGAGAGCAGGTGAGG-30 ; Cre_fwd 50 -TCCATGAGTGAACGAACCTGGTCG-30 ; Cre_rev 50 -TTTGCCTGCATTACCGGTCGATGC-30 . Only male mice were used in the study. The genetic background of the animals was C3H/He. 2.2. Animal experimentation The group size for treatment was 5–6 mice per genotype. Treatment followed the experimental outline described in (Ganzenberg et al., 2013) and (Braeuning et al., 2009). At 8 weeks of age, mice with Alb-Cre KO of Ctnnb1 (genotype: Ctnnb1loxP/loxP, Alb-Cre+) and age-matched Cre-negative controls (WT; Ctnnb1loxP/loxP, Alb-Cre) were treated with different AhR activators by intraperitoneal injection as follows: 3MC (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 10 or 100 mg/kg body weight (single injection); BNF (Sigma, Taufkirchen, Germany) at 20 or 150 mg/kg body weight (single injection for 20 mg/kg, 2  75 mg in 24 h intervals for 150 mg/kg), or BHA (Sigma) at 350 mg/kg body weight (single injection). Mice with TTR-Cre KO of Ctnnb1 (Ctnnb1loxP/loxP, TTR-Cre+) and age-matched WT (Ctnnb1loxP/loxP, TTR-Cre) controls were treated at 10 weeks of age at five consecutive days by intraperitoneal injections with 1.5 mg tamoxifen per mouse per day. Following a treatment-free interval of 4 weeks, mice were treated with 3MC by single intraperitoneal injection at 10, 25, or 50 mg/kg body weight. All AhR activators were dissolved in corn oil. Control mice received an injection with corn oil alone. Tamoxifen was dissolved in a mixture of 1:7 EtOH/corn oil. Mice were sacrificed 48 h (BHA: 72 h) after start of inducer treatment, between 9 a.m. and 11 a.m. to reduce circadian variation. Livers were excised and tissue aliquots were either immediately frozen in liquid nitrogen or fixed in Carnoy’s solution. Mice had access to tap water and standard chow ad libitum, received humane care, and protocols complied with institutional guidelines. 2.3. Immunostaining Carnoy-fixed liver tissue was embedded in paraffin. Slices of 5 mm thickness were stained for glutamine synthetase (GS) and cytochrome P450 1A (CYP1A) using standard methodology as previously described (Ganzenberg et al., 2013) and antibodies against GS (1:1000 dilution; Sigma) or CYP1A (1:1000; gift from Dr. R. Wolf, University of Dundee, Dundee, UK) in combination with appropriate horseradish peroxidase-conjugated secondary antibodies (1:100; Dako, Glostrup, Denmark) with the substrate 3-amino-9-ethylcarbazole/H2O2. Counterstaining of nuclei was done by hematoxylin staining. An Axio Imager. M1 light microscope (Zeiss, Göttingen, Germany) was used for image acquisition.

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2.4. Gene expression analysis RNA was isolated from tissue samples using Trizol reagent (Invitrogen, Karlsruhe, Germany) and reverse transcribed by avian myeloblastosis virus reverse transcriptase (Promega, Mannheim, Germany) and a mix of oligo(dT)20 and random (dN)6 primers. Quantification of mRNA expression was performed on a LightCycler using the Fast Start DNA MasterPLUS SYBR Green I kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Gene-specific PCR primers were as follows: Axin2_fwd 50 -CGACGCACTGACCGACGATT-30 ; Axin2_rev 50 -TCCAGACTATGGCGGCTTTCC30 ; Cyp1a1_fwd 50 -TGTCCTCCGTTACCTGCCTA-30 ; Cyp1a1_rev 50 -GTGTCAAACCCAGCTCCAAA-30 ; Cyp1a2_fwd 50 -GAGCGCTGTATCTACATAAACCA-30 ; Cyp1a2_rev 50 -GGGTGAACATGATAGACAC0 0 TATTGT-3 ; Gpr49_fwd 5 -AATCGCGGTAGTGGACATTC-30 ; Gpr49_rev 50 -GATTCGGAAGCAAAAATGGA-30 ; GS_fwd 50 -GCGAAGACTTTGGGG`TGATA-30 ; GS_rev 50 -GTGCCTCTTGCTCAGTTTGTC-30 ; GSTa_fwd 50 -TATGGGAAGGACATGAAGGAGAGAG-30 ; GSTa_rev 50 -AGGCTTTCTCTGGCTGCCAGG-30 ; 18s rRNA_fwd 50 -CGGCTACCACATCCAAGGAA-30 ; 18s rRNA_rev 50 -GCTGGAATTACCGCGGCT-30 . Fold changes in expression were calculated based on crossing point differences and PCR efficiencies according to (Pfaffl, 2001), using 18s rRNA as a housekeeping gene. PCR efficiencies were always >1.8, with the exception of Gpr49 (>1.7). 2.5. Cell culture and treatment Mouse hepatoma cells from line 55.1c (Braeuning et al., 2011) and human colon carcinoma cells from line HCT116 (purchased from ATCC) were cultivated in D-MEM:F12 medium containing 10% fetal bovine serum (FBS) and antibiotics (Invitrogen) at 37  C in a humidified atmosphere. For mRNA analysis, cells were seeded at a density of 350,000 (55.1c) or 500,000 (HCT116) cells/well on 6-well plates 24 h prior to treatment with different concentrations of the AhR agonists TCDD (Ökometric, Bayreuth, Germany), BNF, or tert-butylhydroquinone (tBHQ; Sigma) for additional 24 h, in the presence or absence of the b-catenin inhibitor FH535 (Merck, Darmstadt, Germany). All compounds were dissolved in DMSO; final solvent concentration in the medium was always below 0.2%. For reporter gene analysis, cells were seeded at 75,000 (55.1c) or 100,000 (HCT116) cells/well on 24-well plates 24 h prior to transfection. Using Lipofectamine 2000 (Invitrogen), cells were transfected with Firefly luciferase reporters driven either by 8xTCF/b-catenin binding sites (SuperTopflash reporter) (Braeuning et al., 2007) or by 3xAhR/Arnt binding sites (dioxin response elements, DREs) (Schreiber et al., 2006). Plasmid pRL-CMV, encoding Renilla luciferase under the control of the constitutively active cytomegaly virus promoter, was co-transfected. Cells were treated as described above, starting 24 h after transfection. Primary mouse hepatocytes from 8 to 10 weeks old mice with Alb-Cre KO of Ctnnb1 were prepared by standard collagenase perfusion and seeded on collagen-coated 6-well plates at a density of 500,000 cells/well in D-MEM: F12 medium containing 10% FBS and antibiotics. After 6 h, medium was changed to 1% FBS and cells were treated with TCDD and/or FH535 for 24 h. In all experiments, the absence of treatmentrelated toxicity was checked by means of the Alamar Blue assay. 2.6. Luciferase reporter assay Cells were lysed by 1 passive lysis buffer (Promega, Mannheim, Germany) for 15 min at room temperature. Activities of Firefly and Renilla luciferases were measured in 96-well format using a dual-luciferase assay as recently described (Braeuning and Vetter, 2012). Firefly luciferase activity was normalized to Renilla luciferase activity. All test substances were checked for direct

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interference with the Firefly luciferase enzyme by measuring the activity of commercially available Firefly luciferase (Roche) in the presence or absence of the respective compound. 3. Results and discussion 3.1. Induction of AhR target genes in mice with Alb-Cre-mediated b-catenin KO Mice with Alb-Cre-mediated KO of Ctnnb1 (i.e., early postnatal KO of b-catenin) and corresponding WT controls were treated with three structurally different AhR agonists: the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3MC), the flavonoid b-naphthoflavone (BNF), and the antioxidant butylated hydroxyanisole (BHA). Gene expression analysis for the model b-catenin target genes Axin2, Gpr49, and GS confirmed the KO of b-catenin (Fig. 1A; see also GS immunostaining in Fig. 2). Starting from similar basal expression levels, the expression of the model AhR target Cyp1a1 was significantly induced by treatment of mice with the lower and the higher doses of BNF (20 and 150 mg/kg body weight) or 3MC (10 and 100 mg/kg body weight) in mice from both genotypes. However, the response was more pronounced in WT mice, resulting in significantly higher levels of the transcript in inducer-treated WT mice, as compared to equally-treated KO animals (Fig. 1B). BHA slightly but significantly induced Cyp1a1 in WT mice, whereas no response was observed in KO mice (Fig. 1B). In the absence of exogenous AhR activators, Cyp1a2 was significantly more abundant in WT mice (Fig. 1C). AhR activation by 3MC, BNF, or BHA led to elevated levels of the transcript in both genotypes (Fig. 1C). Again, significantly higher levels of the AhR target gene were observed in WT mice, as compared to the KO groups treated with the same substances in equal doses (Fig. 1C). Similarly, glutathione-S-transferase (GST) a was increased up to higher levels in WT mice. No significant genotype differences in GSTa expression were present in WT and KO mice treated with the maximum dose of 3MC (Fig. 1D). These findings are in line with a previous study on the effects of 2  50 mg/kg body weight 3MC in the same b-catenin-deficient mouse strain (Braeuning et al., 2009). Present data, however, go beyond published data by demonstrating that the decreased response of b-catenin KO mice is independent of the chemical nature of the AhR agonist. Moreover, the differential effects exerted by the lower doses of 3MC and BNF in WT and KO mice indicate that the differences in enzyme induction observed at high doses are not merely based on an earlier saturation of AhR activation in KO mice, which might be assumed at high doses due to the fact that these mice express lower AhR mRNA levels that their WT counterparts (Braeuning et al., 2009). Rather, the responsiveness of b-catenin KO mice to AhR activation seems generally lowered. 3.2. Zonation of CYP1A in mice with Alb-Cre-mediated b-catenin KO By activating the AhR, 3MC preferentially induces perivenous expression of AhR target genes in the liver (Braeuning et al., 2009), whereas BNF has been reported to induce rather panlobular or periportal expression (Wolf et al., 1984). In WT mice, basal CYP1A expression was preferentially perivenous, and induction by 3MC was also preferentially observed in perivenous and midzonal hepatocytes, as expected (Fig. 2A). By contrast, no zonation was detectable in KO mice, neither with regard to basal nor with regard to 3MC-induced CYP1A expression (Fig. 2B). Immunohistochemical staining of BNF-treated WT livers revealed a rather panlobular CYP1A expression pattern, indicative of an induction of the enzyme by BNF in WT mice in periportal hepatocytes (in addition to the existing basal perivenous expression; Fig. 3A). Interestingly, the periportal induction pattern of CYP1A was preserved in b-catenin

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Fig. 1. Gene expression in livers of mice with albumin promoter-driven hepatocyte-specific KO of Ctnnb1 following treatment with different AhR activators, as determined by real-time RT-PCR. (A) Reduced expression of the model b-catenin targets Axin2, GS, and Gpr49 in KO mice without inducer treatment. Treatment with b-naphthoflavone (BNF; 20 and 150 mg/kg body weight), 3-methylcholanthrene (3MC; 10 and 100 mg/kg), or butylated hydroxyanisole (BHA; 350 mg/kg) differentially induces the expression of Cyp1a1 (B), Cyp1a2 (C), and GSTa (D). Mean + SD of n = 5–6 mice per group are shown relative to control W T mice treated with corn oil (co). Statistical significance (p < 0.05; (A) Student’s t-test; (B–D) 2-way ANOVA): $, genotype effect in corn oil-treated mice (WT vs. KO); *, treatment effect (corn oil vs. inducer-treated of the same genotype); #, combined treatment/genotype effect: (corn oil vs. inducer-treated of the same genotype p < 0.05 AND inducer-treated WT vs. inducer-treated KO mice); x, genotype but no treatment effect in the presence of AhR activators (inducer-treated WT vs. inducer-treated KO mice).

KO mice (Fig. 3B; see also Fig. 4 for higher magnification). Following treatment with BHA, CYP1A was mainly localized in perivenous hepatocytes in WT mice, whereas no zonal differences were observed in KO mice (Fig. 3C and D). The observed basal lack of CYP1A expression and zonation in Ctnnb1 KO mice is in line with previous findings describing a loss of the enzyme(s) at the mRNA and protein levels (Braeuning et al., 2009; Schreiber et al., 2011; Sekine et al., 2006). Diminished induction by a high dose of 3MC has also been reported previously (Braeuning et al., 2009). However, the periportal induction phenotype induced by BNF is preserved after the KO of b-catenin. This is intriguing, since in b-catenin KO mice, the zonation of almost all mRNAs or proteins which have been analyzed so far is lost, with only a few exceptions (glucose-6-phosphatase activity and CYP3A expression; (Braeuning et al., 2009; Ganzenberg et al., 2013)). Physiological activity of the b-catenin pathway is restricted to perivenous hepatocytes (Benhamouche et al., 2006; Sekine et al., 2007). Thus, the observed loss of perivenous CYP1A induction by 3MC and BHA together with the preserved periportal induction of CYP1A by BNF might be explained by the fact that active b-catenin is an important regulator of the AhR in perivenous hepatocytes, whereas the periportal induction by BNF, which is mechanistically still not understood, appears to be rather independent of the perivenous master regulator b-catenin. Induction of AhR target genes by BHA is almost abolished following the KO of b-catenin. Classically, BHA acts not only as an agonist of the AhR, but is especially potent as an inducer of the antioxidant-responsive Nrf2 signaling pathway. Of note, the induction of Nrf2 target

genes by BHA was almost extinguished in mice from the same b-catenin-deficient strain in a previous study (Braeuning et al., 2009). This might indicate a general lack of responsiveness to BHA in Ctnnb1 KO mice. 3.3. Induction of AhR target genes in mice with TTR-Cre-mediated b-catenin KO In a recent study, we have analyzed the role of the time point of Ctnnb1 KO in the induction of drug-metabolizing enzymes by agonists of the constitutive androstane receptor (Ganzenberg et al., 2013). A major finding of this study was that the zone-specificity of CAR target gene induction is lost in mice with an early postnatal, Alb-Cre-mediated KO of b-catenin, whereas a zonated response occurs when the Ctnnb1 gene is recombined at the adult stage via the tamoxifen-inducible TTR-Cre system. We therefore tested whether the loss of zonal induction by AhR agonists in the Alb-Cre Ctnnb1 early postnatal KO model would similarly be observed in Ctnnb1 KO mice with the TTR-Cre-driven KO of Ctnnb1 induced at adulthood. Fig. 5A demonstrates the efficient KO of b-catenin by the tamoxifenactivated Cre enzyme, as documented by reduced expression of model b-catenin target genes. For comparison, see also immunostainings for GS in Fig. 6. Similar to the Alb-Cre model, basal Cyp1a1 levels were not significantly affected by the TTR-Cre-driven KO of Ctnnb1, whereas basal Cyp1a2 levels were significantly reduced (Fig. 5B and C). Also comparable to what had been observed in the other mouse model, 3MC-induced levels of

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Fig. 2. Zonation of CYP1A in livers of mice with albumin promoter-driven hepatocyte-specific KO of Ctnnb1 following treatment with 3MC (10 and 100 mg/kg body weight), as determined by immunohistochemistry. (A) Perivenous CYP1A basal expression and induction in WT mice. (B) Lack of zonation and zonally unspecific CYP1A induction in KO mice. Staining for the perivenous marker and b-catenin target GS is shown for comparison. Original magnification of the images: 5. Please note the few strongly GS- and CYP1A-positive cells marked by arrows in (B). Here, a few hepatocytes have escaped the Cre-mediated KO of Ctnnb1 and therefore display the WT phenotype.

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the two AhR target genes Cyp1a1 and Cyp1a2 were significantly higher in WT mice as compared to the corresponding KO groups, with the one exception of Cyp1a1 at 25 mg/kg body weight, where the genotype difference in expression failed our criteria of statistical significance.

3.4. Zonation of CYP1A in mice with TTR-Cre-mediated b-catenin KO Zonation of basal and 3MC-induced CYP1A expression was analyzed in mice with TTR-Cre-mediated, adult loss of b-catenin. As expected, basal CYP1A expression was confined to the perivenous

Fig. 3. Zonation of CYP1A in livers of mice with albumin promoter-driven hepatocyte-specific KO of Ctnnb1 following treatment with BNF (20 and 150 mg/kg body weight) or BHA (350 mg/kg), as determined by immunohistochemistry. (A) Panlobular induction of CYP1A in WT mice by BNF. (B) Periportal induction of CYP1A in KO mice by BNF. For an enlarged image, see Fig. 4. The arrow indicates a small cluster of remnant Ctnnb1 WT hepatocytes, which express GS due to their vicinity to a central vein. (C) Perivenous induction of CYP1A in WT mice by BHA. (D) Zonally unspecific CYP1A induction in KO mice by BHA. Staining for the perivenous marker and b-catenin target GS is shown for comparison. Original magnification of the images: 5. Please note the few strongly GS- and CYP1A-positive cells marked by arrows in (B). Here, a few hepatocytes have escaped the Cre-mediated KO of Ctnnb1 and therefore display the WT phenotype. For control images of mice treated with corn oil only, please refer to Fig. 2.

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Fig. 4. Preferential periportal localization of CYP1A in livers of mice with albumin promoter-driven hepatocyte-specific KO of Ctnnb1 following treatment with 20 mg/ kg body weight BNF (enlarged version of images in Fig. 3C). Staining for the perivenous marker and b-catenin target GS is shown for comparison. Original magnification of the images: 40.

hepatocyte subpopulation and 3MC-induced CYP1A was also preferentially located in perivenous and midzonal hepatocytes in WT controls (Fig. 6A). By contrast, the TTR-Cre b-catenin KO mice showed no zonated expression of CYP1A and induction of the enzyme by 3MC was diffusely distributed throughout the whole liver lobule without zonal preferences (Fig. 6B). This finding is in agreement with the situationin the Alb-Cre b-catenin KO mouse model(Fig. 2), where there is also no zonation of basal or 3MC-induced CYP1A. However, data reveal an interesting difference between the induction of drugmetabolizing enzymes by AhR and CAR activation in TTR-Cre-driven b-catenin KO mice.On the one hand, the model CAR agonist TCPOBOP induces CAR target genes in this mouse model preferentially in perivenous hepatocytes, notwithstanding the fact that the zonation keeper b-catenin had been removed from the cells weeks ago (Ganzenberg et al., 2013). This preservation of zonal induction is not observed in the early postnatal Alb-Cre model of Ctnnb1 KO (Braeuning et al., 2009; Ganzenberg et al., 2013), indicating that hepatocytes are primed in the postnatal phase for their susceptibility to CAR agonists at an older age. With the AhR agonist 3MC, zonation is visible in neither of the two mouse models. This means that, in contrast to the situation with CAR activators, the susceptibilitytoAhR induction is directly dependenton the presence of functional b-catenin at the time point of treatment with the inducer and not influenced by developmental factors as it has been

Fig. 5. Gene expression in livers of mice with tamoxifen-inducible TTR promoterdriven hepatocyte-specific KO of Ctnnb1 following treatment with 3MC (10, 25, and 50 mg/kg body weight), as determined by real-time RT-PCR. (A) Reduced expression of the model b-catenin targets Axin2, GS, and Gpr49 in KO mice without inducer treatment. Treatment for 48 h with 3MC differentially induces the expression of Cyp1a1 (B) and Cyp1a2 (C). Mean + SD of n = 5–6 mice per group are shown relative to control WT mice treated with corn oil (co). Statistical significance (p < 0.05; (A) Student’s t-test; (B and C) 2-way ANOVA): $, genotype effect in corn oil-treated mice (WT vs. KO); *, treatment effect (corn oil vs. inducer-treated of the same genotype); #, combined treatment/genotype effect: (corn oil vs. inducer-treated of the same genotype p < 0.05 and inducer-treated WT vs. inducer-treated KO mice).

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observed for CAR. This view is substantiated by previous observations, which describe a physical interaction of b-catenin and the AhR in liver cells and the functional synergism of b-catenin- and AhR-dependent signaling in vitro (Braeuning et al., 2011).

3.5. Crosstalk of AhR- and b-catenin-dependent signaling in vitro Available in vitro data have shown that b-catenin activation supports the induction of Cyp1a1 transcription and/or AhR-

Fig. 6. Zonation of CYP1A in livers of mice with tamoxifen-inducible TTR promoter-driven hepatocyte-specific KO of Ctnnb1 following treatment with 3MC (10, 25, and 50 mg/ kg body weight), as determined by immunohistochemistry. (A) Perivenous CYP1A basal expression and induction in WT mice. (B) Lack of zonation and zonally unspecific CYP1A induction in KO mice. Staining for the perivenous marker and b-catenin target GS is shown for comparison. Original magnification of the images: 5.

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Fig. 7. Concomitant regulation of Cyp1a1 expression by different AhR activators and b-catenin in vitro. All cells were treated for 24 h prior to analysis. (A) Down-regulation of the b-catenin-dependent Supertopflash (STF) luciferase reporter by 10 mM of the b-catenin inhibitor FH535 in 55.1c mouse hepatoma cells. (B) Down-regulation of the b-catenin-dependent Supertopflash (STF) luciferase reporter by 20 mM of the b-catenin inhibitor FH535 in HCT116 human colon carcinoma cells. (C) Absence of interference of FH535 with basal and AhR agonist-inducible Cyp1a1 mRNA expression in a b-catenin-free cell system of Ctnnb1 KO hepatocytes. (D) Concomitant regulation of the AhR-responsive 3xDRE luciferase reporter by 10 nM TCDD and 20 mM FH535 in HCT116. (E) Induction of Cyp1a1 mRNA in 55.1c cells by 5 nM TCDD in the absence or presence of 10 mM FH535. (F) Induction of Cyp1a1 mRNA in 55.1c cells by BNF (10 or 40 mM) in the absence or presence of 10 mM FH535. (G) Induction of CYP1A1 mRNA in HCT116 cells by tBHQ (40 mM) in the absence or presence of 20 mM FH535. Induction by 10 nM TCDD is shown for comparison. (H) Dose–response curve of the 3xDRE reporter in 55.1c cells treated with different concentrations of TCDD in the absence or presence of 10 mM FH535. (I) Dose–response curve of the 3xDRE reporter in 55.1c cells treated with different concentrations of BNF in the absence or presence of 10 mM FH535. Please note the repression of luciferase activity at higher BNF concentrations by BNF directly acting on the luciferase enzyme (see Supplemental figure). (J) Dose–response curve of the 3xDRE reporter in HCT cells treated with different concentrations of tBHQ in the absence or presence of 20 mM FH535. Black squares: AhR inducer treatment; open squares: treatment with AhR inducer plus 10 mM FH535 (HCT 116: 20 mM FH535). Mean + SD of n  3 experiments (mRNA) or n  5 (luciferase reporter) are shown. Statistical significance (p < 0.05; mRNA data; (A and B, G (right bar)) Student’s t-test; (C–F, G (left bars)) 2-way ANOVA): $, b-catenin effect in cells w/o AhR inducer treatment (FH vs. DMSO); *, treatment effect (inducer-treated vs. DMSO control or FH, respectively); #, combined treatment/genotype effect (inducer-treated vs. DMSO control AND inducer-treated vs. cells treated with inducer and FH). Statistical significance (p < 0.05; luciferase reporter data; Student’s t-test): *, b-catenin effect: significantly different from cells treated with the same concentration of inducer and with FH535.

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driven reporter gene activity induced by the model AhR activator TCDD (Braeuning et al., 2011; Loeppen et al., 2005). However, these data were obtained by using only one single concentration of the AhR agonist, thus precluding firm conclusions about the shape of the dose–response curve of AhR activation in the absence or presence of b-catenin. In addition, no information is available about b-catenin effects on the AhR in vitro when activated by different classes of AhR inducers. The chemical b-catenin inhibitor FH535 was chosen for in vitro experimentation due to its low cytotoxicity, its highly potent inhibition of b-catenin-dependent signaling (Fig. 7A), and its lack of interference with Cyp1a1 expression and induction in a b-catenin-free cell system consisting of primary hepatocytes isolated from Alb-Cre b-catenin KO mice (Fig. 7C). The latter observation is of special importance, since there are numerous small molecule inhibitors, which act as partial agonists of the AhR, e.g., U0126 (Andrieux et al., 2004) and SB216763 (Braeuning and Buchmann, 2009). Thus, it was necessary to prove that FH535 neither induces basal Cyp1a1 expression nor interferes with AhR agonist-mediated effects. The present data demonstrate that the compound has no remarkable b-cateninindependent influence on Cyp1a1 and therefore prove that FH535 is suited for the analysis of the b-catenin/AhR crosstalk. AhR activity in 55.1c mouse hepatoma cells in vitro was analyzed via Cyp1a1 mRNA analysis and via activity of a luciferase reporter driven by 3xAhR/Arnt binding sites (DREs). TCDD and BNF treatment produced the expected increase in Cyp1a1 mRNA and 3xDRE-driven reporter activity (Fig. 7). Co-incubation with TCDD and the b-catenin inhibitor FH535 significantly reduced Cyp1a1 mRNA levels and luciferase reporter activities (Fig. 7). At higher concentrations of BNF, an unexpected decrease in luciferase activity was observed (Fig. 7I), notwithstanding the strong induction of Cyp1a1 by similar BNF concentrations (Fig. 7F). This effect is caused by the inhibition of the Firefly luciferase enzyme by BNF, independent of the BNF/AhR interaction. As shown in the Supplemental figure, activity of luciferase was reduced in a dose-dependent manner in the presence of BNF, demonstrating that BNF is a direct Firefly luciferase inhibitor. Treatment of 55.1c cells with the major BHA metabolite tBHQ induced cytotoxicity even at low concentrations (data not shown). Therefore, analyses of CYP1A1 mRNA expression and 3xDRE-driven reporter activity in response to tBHQ were measured in human colon carcinoma cells from line HCT116, which are much more tolerant to tBHQ. Inhibition of b-catenin signaling (Fig. 7B), TCDD-mediated 3xDRE reporter activity (Fig. 7D), and CYP1A1 induction by TCDD (Fig. 7G, right bar) were comparable to 55.1c cells. Consistent with the lack of response to BHA in vivo, tBHQ was unable to elicit a pronounced AhR response in b-catenin-inhibited HCT116 cells in vitro (Fig. 7). Data obtained with the 3xDRE reporter and TCDD demonstrate that the dose–response curve of AhR activation is not just shifted towards higher concentrations in the state of b-catenin inhibition. Rather, the two curves start to rise at comparable concentrations of TCDD, thus indicating similar sensitivity of the AhR system under both conditions, but then show strikingly different saturation levels. This suggests that the maximum AhR activity at the DREs in response to TCDD treatment can only be achieved when b-catenin signaling is active. Thus, b-catenin is essential for the full AhR response. This view is supported by the in vivo data from the TTR-Cre model of b-catenin KO. By contrast, the findings with BHA and tBHQ indicate that the sensitivity against this type of inducer is strongly affected by b-catenin signaling. In summary, the present data (i) substantiate the idea that b-catenin is an important factor in the regulation of

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