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Research ArticleArticle

Identification of Amino Acid Residues in the Ligand-Binding Domain of the Aryl Hydrocarbon Receptor Causing the Species-Specific Response to Omeprazole: Possible Determinants for Binding Putative Endogenous Ligands

Kazuhiro Shiizaki, Seiichiroh Ohsako, Masanobu Kawanishi and Takashi Yagi
Molecular Pharmacology February 2014, 85 (2) 279-289; DOI: https://doi.org/10.1124/mol.113.088856

Abstract

Omeprazole (OME) induces the expression of genes encoding drug-metabolizing enzymes, such as CYP1A1, via activation of the aryl hydrocarbon receptor (AhR) both in vivo and in vitro. However, the precise mechanism of OME-mediated AhR activation is still under investigation. While elucidating species-specific susceptibility to dioxin, we found that OME-mediated AhR activation was mammalian species specific. Moreover, we previously reported that OME has inhibitory activity toward CYP1A1 enzymes. From these observations, we speculated that OME-mediated AhR target gene transcription is due to AhR activation by increasing amounts of putative AhR ligands in serum by inhibition of CYP1A1 activity. We compared the amino acid sequences of OME-sensitive rabbit AhR and nonsensitive mouse AhR to identify the residues responsible for the species-specific response. Chimeric AhRs were constructed by exchanging domains between mouse and rabbit AhRs to define the region required for the response to OME. OME-mediated transactivation was observed only with the chimeric AhR that included the ligand-binding domain (LBD) of the rabbit AhR. Site-directed mutagenesis revealed three amino acids (M328, T353, and F367) in the rabbit AhR that were responsible for OME-mediated transactivation. Replacing these residues with those of the mouse AhR abolished the response of the rabbit AhR. In contrast, substitutions of these amino acids with those of the rabbit AhR altered nonsensitive mouse AhR to become sensitive to OME. These results suggest that OME-mediated AhR activation requires a specific structure within LBD that is probably essential for binding with enigmatic endogenous ligands.

Introduction

Omeprazole (OME), a benzimidazole derivative, is a potent suppressor of gastric acid secretion and has been used to treat gastroesophageal reflux disease and duodenal ulcers (Lind et al., 1983). In human hepatoma cells and liver cells, OME is known to induce CYP1A1 and CYP1A2 (Diaz et al., 1990; Curi-Pedrosa et al., 1994; Krusekopf et al., 1997; Yoshinari et al., 2008). The transactivation of these genes is known to be mediated by a ligand-dependent transcriptional factor, aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) (Burbach et al., 1992; Reyes et al., 1992). The AhR/Arnt heterodimer binds to the specific nucleic acid sequence known as the xenobiotic response element (XRE), which is located in the 5′-flanking region of CYP1A1 gene and other AhR target genes (Denison et al., 1989). Although it is generally accepted that ligand binding is a key determinant for AhR-mediated transcriptional activity, even in the absence of exogenous ligands (Chang and Puga, 1998; Murray et al., 2005; Shiizaki et al., 2005), some reports have shown that OME induces CYP1A1 expression without binding to AhR using competitive binding assays (Daujat et al., 1992; Dzeletovic et al., 1997). Similar to typical AhR ligands, OME induces translocation of the AhR/Arnt complex to the nucleus, and the complex binds to the XRE of the Cyp1a1 enhancer region (Quattrochi and Tukey, 1993; Yoshinari et al., 2008). Therefore, OME is considered to activate AhR through a pathway other than ligand binding. Several reports have shown that the protein tyrosine kinase (PTK) pathway is involved in CYP1A1 induction by OME in rat and human hepatoma cells (Backlund et al., 1997; Kikuchi et al., 1998; Lemaire et al., 2004; Backlund and Ingelman-Sundberg, 2005). OME-mediated AhR signaling has been shown to be attenuated by c-src kinase inhibitors or by expression of the dominant negative c-src protein. Furthermore, it has been suggested that the tyrosine residue at amino acid position 320 in the human AhR is a putative phosphorylation site required for OME-mediated AhR activation (Backlund and Ingelman-Sundbrg, 2004). However, the molecular mechanisms of OME-mediated AhR activation have not been completely elucidated. In addition, OME-mediated AhR activation has been reported to be species-specific (Kikuchi et al., 1995, Dzeletovic et al., 1997). CYP1A1 induction by OME has been demonstrated in human, but not in mouse, primary hepatocytes and hepatoma cell lines. However, the PTK pathway appears to be unrelated to this interspecies difference because the tyrosine 320 residue is conserved in mouse and human AhRs. By fusing mouse and human hepatoma cells, a gene locus was assigned as a human-specific gene responsible for CYP1A1 induction by OME (Kikuchi et al., 2002). However, specific genes exhibiting response to OME have not been identified. Thus, the mechanism underlying species-specific differences in OME-mediated AhR activation remains unclear. Elucidating such a mechanism could lead to a better understanding of OME-mediated AhR activation.

Recent studies have shown that AhR has physiologic roles such as anti-inflammation and T-cell differentiation (Kimura et al., 2008; Quintana et al., 2008). These discoveries led to suggestions of using AhR ligands for chemotherapy. For example, a selective AhR modulator that exhibits anti-inflammatory properties without XRE-dependent xenobiotic gene expression has been proposed (Murray et al., 2010). Because some AhR ligands are already used in human therapy and because OME-mediated AhR activation is unique, OME and other imidazole compounds may be candidates for selective AhR modulators. Elucidation of the molecular mechanism of OME-mediated AhR activation will be important for such applications.

Recently, a report showed that AhR activation by CYP1A1-inhibiting chemicals is caused by an indirect mechanism involving disruption of the clearance of endogenous ligands (Wincent et al., 2012). We speculated that OME-mediated AhR activation is due to this indirect mechanism because OME is one of the CYP1A1-inhibiting chemicals (Shiizaki et al., 2008). Species-specific response to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is considered to be due to differences in the AhR molecular structure responsible for the ligand binding affinity (Bohonowych and Denison, 2007), although this topic is controversial. If a putative ligand is related to OME-mediated AhR activation, a species-specific OME response would be generated because of the affinity of the putative ligand conferred by the AhR structure, as well as response to TCDD. In addition, the structural differences in AhR will be due to particular amino acid residues.

In this study, we used AhRs derived from various laboratory animals and humans to demonstrate that species-specific activation by OME depends on specific amino acids within the AhR amino acid sequences.

Materials and Methods

Chemicals.

Omeprazole, 3-methylcholanthrene (3MC), ellipticine, kynurenic acid, and 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE) were purchased from Sigma-Aldrich (St. Louis, MO). TCDD (>99.5% purity) was obtained from Cambridge Isotope Laboratories (Andover, MA). β-Naphthoflavone was purchased from Wako Pure Chemical (Osaka, Japan). Indirubin was provided by Dr. Tomonari Matsuda (Kyoto University, Kyoto, Japan). FICZ (6-formylindolo[3,2-b]carbazole) was purchased from Biomol International LP (Plymouth Meeting, PA). All chemicals were dissolved in dimethylsulfoxide and added to media. The final concentration of dimethylsulfoxide was adjusted to 0.1% (v/v) in culture media.

CYP1A1 Activity Determined by Cytochrome P450-Glo Assays.

CYP1A1 activity was determined using a cytochrome P450 (P450)-Glo CYP1A1 Assay (Promega, Madison, WI) according to the manufacturer’s instructions. Sf9 cell microsomes containing recombinant human CYP1A1 were purchased from Becton Dickinson (Franklin Lakes, NJ). In brief, an aliquot (0.5 pmol) of microsomes was mixed with 100 mM KPO4 buffer (25 μl) containing 120 μM of luciferin 6′-chloroethyl ether (luciferin-CEE). Then, OME (final concentration 0.5–100 μM) or ellipticine (final concentration 0.1–10 μM) was added to reaction cocktails and incubated at 37°C for 10 minutes. After preincubation, 25 μl of 2× NADPH-regenerating system solution (2.6 mM NADP+, 6.6 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, and 6.6 mM MgCl2) was added and incubated at 37°C for 30 minutes. Luciferin obtained after conversion of luciferin-CEE by CYP1A1 was detected by adding the Luciferin Detection Reagent included in the assay kit, and luminescence was detected using the Wallac Arvo SX Multi-Label counter (PerkinElmer, Boston, MA).

Plasmid Construction.

The reporter plasmid pX4TK-Luc, which includes the firefly luciferase gene under the control of four copies of XRE and the thymidine kinase promoter, was a gift from K. Sogawa (Tohoku University, Sendai, Japan). The expression plasmids containing AhR cDNAs from six mammalian species were constructed as follows. cDNAs were prepared from mouse (Mus musculus, strain C57BL), human (Homo sapiens), rat (Rattus norvegicus, Holtzman), rabbit (Oryctolagus cuniculus, New Zealand White), hamster (Mesocricetus auratus, Syrian), and guinea pig (Cavia porcellus, Hartley). The open reading frames of each AhR were amplified by reverse-transcription polymerase chain reaction and inserted into the MluI and XhoI sites of the pCI-neo vector (Clontech, Palo Alto, CA). To construct the chimeric mouse/rabbit AhRs, AhR cDNA fragments based on the mouse and rabbit AhR ligand-binding domains (LBDs) (Fukunaga et al., 1995) were individually amplified, joined by overlapping polymerase chain reaction, and subcloned into the pCI-neo vector. Single point mutations were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). All primers used in this study are shown in Supplemental Table 1.

Cell Cultures and Transfection.

The human epithelial carcinoma cell line HeLa and murine hepatoma cell lines Hepa1c1c and its derivative c12 were obtained from the American Type Culture Collection (Rockville, MD). HeLa cells were grown in minimum essential medium (MEM) containing 10% charcoal-stripped fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA). Hepa1c1c and c12 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% charcoal-stripped FBS and 1× Antibiotic-Antimycotic. All cultures were incubated at 37°C in 5% CO2. Transfection was performed by a liposome method. In brief, 1 μg of plasmid DNA and 4 μl of Plus reagent (Invitrogen) were combined in 200 μl of OPTI-MEM (Invitrogen). After incubation for 15 minutes, 2 μl of Lipofectamine reagent (Invitrogen), diluted with 200 μl OPTI-MEM, was added. The cells were plated in six-well tissue culture plates at 30%–40% confluence a day before transfection. Liposomes were added in serum-free medium for 3 hours and then replaced with MEM or DMEM containing 10% charcoal-stripped FBS without 1× Antibiotic-Antimycotic.

Luciferase Assay.

The effects of OME and typical AhR agonists on XRE-dependent transcriptional activity were evaluated by cotransfecting the AhR expression plasmid, the reporter plasmid pX4TK-Luc (Mimura et al., 1999), and the Renilla luciferase (Rluc) expression vector pRL-CMV (Promega) into cells. The transfected cells were washed with cold phosphate-buffered saline and lysed in 25 μl of 1× passive lysis buffer (Promega). Aliquots (10 μl) of the lysates were transferred to 96-well plates, and Luc+ and Rluc luminescence was measured using the Dual-Luciferase Reporter Assay System (Promega) in the Wallac Arvo SX Multi-Label counter. Transfection and translation efficiencies varied between independent experiments, and the results were normalized by calculating Luc+/Rluc ratios. All assays were performed in triplicate.

Immunoblotting.

Immunoblotting was performed to evaluate expressed AhR protein levels after transient transfection experiments. Cells plated in six-well plates were washed with phosphate-buffered saline, lysed in 0.5 ml of SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8) containing Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and sonicated to shear DNA. Protein concentration in the cell lysates was measured by the Bradford method using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich, Germany), and 20-μg aliquot of each sample was separated on an SDS-polyacrylamide gel. Immunoblotting for AhR was performed as described previously (Pollenz et al., 1994) with anti-human AhR antibody (SA-210; Biomol GmbH, Hamburg, Germany). Detection was performed with peroxidase-labeled anti-rabbit antibody using the ECL Plus detection system (GE Healthcare Bio-Sciences, Uppsala, Sweden). The bands were visualized and imaged using ChemiDoc XRS Plus (Bio-Rad Laboratories).

Generation of Stable Cell Lines Expressing Mutant AhRs.

The pCI-neo plasmids containing mouse and rabbit AhR cDNAs with point mutations were transfected into c12 cells. Stable transformants expressing AhRs were selected by using G418 and screened for ethoxyresorufin-O-deethylase activity as described previously (Kennedy and Jones, 1994) after treatment with 10 nM TCDD. We chose and mixed more than six representative clones from transformants obtained with each plasmid and investigated CYP1A1 inducibility by TCDD and OME using real-time reverse-transcription polymerase chain reaction methods. In brief, the cells plated in six-well plates at 80% confluence were exposed to 50 μM OME or 10 nM TCDD. After 12 hours of exposure, total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. An aliquot (2 µg) of total RNA was subjected to RT using MultiScribe RT (Applied Biosystems, Foster City, CA) and oligo-dT primers. An aliquot of cDNA (2 µl) or calibrator plasmid DNA (pCI-neo-mAhR) was amplified with master mixture (SYBR Premix Ex Taq; Takara Bio, Kyoto, Japan) containing gene-specific primers. The primer pair for mouse CYP1A1 cDNA amplification is shown in Supplemental Table 1. The reaction mixture was amplified using the Smart Cycler System (Cepheid; Takara Bio) under the following conditions: an initial incubation at 95°C for 15 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 10 seconds. To confirm amplification specificity, the PCR products were subjected to melting curve analyses. A calibration curve was generated by threshold cycles of calibrators of a known plasmid copy number. The initial quantity of target mRNA in the samples was determined by correlating their threshold cycles to the calibration curve.

Results

OME Inhibits CYP1A1 Activity.

To examine whether OME directly inhibits CYP1A1 activity, recombinant human CYP1A1 proteins expressed by Sf9 cells were used in the inhibition study. In addition to ellipticine, a typical CYP1A1 inhibitor, OME also inhibited CYP1A1 activity. The mean inhibitory concentration value was 6.77 µM in case of OME and 2.75 µM in ellipticine (Fig. 1, data re-examined as reported in our previous study, Shiizaki et al., 2008). CYP1A1 activity was inhibited nearly 90% by more than 20 µM of OME, and this concentration correlated to the concentration of OME required for AhR activation (Diaz et al., 1990; Dzeletovic et al., 1997).

Fig. 1.
Fig. 1.

Inhibition of human CYP1A1 activity by omeprazole. CYP1A1 activity was determined using P450-Glo Assays and Sf9 cell microsomes containing recombinant human CYP1A1. Luciferin-CEE (a specific substrate for CYP1A1) and microsomes were incubated at 37°C for 30 minutes with various concentrations of omeprazole or ellipticine. Each data point represents the mean of duplicate determinations.

We confirmed species-specific induction of CYP1A1 by OME (Supplemental Fig. 1A). Induction of CYP1A1 mRNA by OME occurred not only in human hepatoma HepG2 cells but also in mouse Hepa1c1c cells (Kikuchi et al., 1995). This induction was well correlated with XRE-driven reporter gene induction (Supplemental Fig. 1B). We used this reporter assay system to evaluate OME-mediated AhR activation, which, consequently, led to CYP1A1 induction.

OME Restores XRE-Dependent Transcription in CYP1A1-Overexpressing SKII-1A1 Cells and CYP1A1-Deficient SK-Hep-1 Cells.

As reported in our previous study, in the presence of 10% FBS, the human hepatoma cell line SK-Hep-1, which is deficient in CYP1A1 expression, showed higher basal transcription from the XRE-driven reporter gene without additional exogenous ligands than other human hepatoma cells (Shiizaki et al., 2005). We compared basal transcription levels of CYP1A1-overexpressing cell lines (SKII-1A1 cells) and found that basal transcription levels in these cells were significantly lower than those in wild-type SK-Hep-1 cells. Using this reporter system, OME or ellipticine was added to culture media of the wild-type SK-Hep-1 and SKII-1A1#2 cells (one of the CYP1A1-overexpressing clones). OME restored the transcription in the SKII-1A1#2 cells to a level similar to that in the wild-type cells (Fig. 2). Treatment of CYP1A1 inhibitor ellipticine indicated similar results. These observations suggest that OME does not transactivate XRE-driven reporter genes directly via AhR activation but instead inhibits transcription repression by CYP1A1 activity.

Fig. 2.
Fig. 2.

OME restores XRE-dependent transcription in CYP1A1-overexpressing SKII-1A1 cells and CYP1A1-deficient SK-Hep-1 cells. CYP1A1-deficient SK-Hep-1 (Wt) and its derivative cell lines overexpressing CYP1A1 (SKII-1A1#2) cultured in medium containing 10% FBS were transfected with the 4xXRE-Luc reporter gene and pCMV-Rluc. After transfection, fresh medium containing 100 µM omeprazole (closed column), 5 µM ellipticine (gray column) or dimethylsulfoxide (DMSO) (solvent control, open column) was added. Data are represented as the ratio of firefly luciferase activity to Renilla luciferase activity observed 16 hours after OME was added. (FFluc/Rluc, mean ± S.D., n = 3) (**P < 0.01, n.s., no significant difference to control). FFluc, firefly luciferase.

Charcoal-Stripping of Serum Diminished the Basal Transcription Level and OME-Induced Transcription of the XRE-Driven Reporter Gene in HepG2 Cells.

Next, we tested the effect of FBS on the transcription induction of XRE-driven reporter genes by OME to provide indirect evidence of the presence of substances in FBS that might transactivate AhR. The human hepatoma cell line HepG2 and SK-Hep-1 cells are similar in terms of the expression levels of AhR and Arnt molecules (Shiizaki et al., 2005). The 4xXRE-Luc reporter gene and pCMV-Rluc were cotransfected into HepG2 cells. After transfection, the culture medium was changed to fresh DMEM containing normal FBS or charcoal-stripped FBS. Although OME-induced transcription was clearly observed in DMEM containing normal FBS, significantly less OME-induced transcription was observed in DMEM containing charcoal-stripped FBS (Fig. 3). Charcoal-stripping of FBS did not reduce 3MC (1 μM)-induced transcription. Together with this and the previously mentioned results (Figs. 1 and 2), it is highly suspected that OME-mediated AhR activation of the XRE-dependent gene is due to the inhibition of the activity of CYP1A1 enzymes, which may metabolize putative AhR ligands contained in FBS.

Fig. 3.
Fig. 3.

Effects of FBS on XRE-dependent transcriptional activation. HepG2 cells were transfected with the 4xXRE-Luc reporter gene and pCMV-Rluc. After transfection, the culture medium was changed to fresh DMEM containing normal FBS (nFBS, open column) or charcoal-stripped FBS (cFBS, closed column). Twenty-four hours after medium exchange, dimethylsulfoxide (solvent control; CT), 50 or 100 µM OME was added. Significant reductions in OME-induced transcription was noted with charcoal stripping; however, 3MC (1 μM)-induced transcription was not affected. Data are represented as mean ± S.D. (n = 3) 16 hours after OME addition. (*P < 0.05; **P < 0.01; n.s., no significant difference). FFluc, firefly luciferase.

Comparison of the Responses of AhRs Derived from Various Mammals to OME.

While exploring the genes that cause species-specific susceptibility against dioxin, we prepared six AhR expression plasmids from six mammalian species using pCI-neo vectors. In these experiments, we used HeLa cells, which possess CYP1A1 and express a similar level of ARNT molecules and a lower level of AhR molecules compared with HepG2 or SK-Hep-1 cells. We also used mouse hepatoma Hepa1c1c7 cells, which expressed a sufficient level of AhR. During this project, we used OME in addition to TCDD to examine the species-specific influence on AhR-dependent transcription. XRE-driven reporter gene assays were performed by cotransfection of the previously mentioned six AhR expression vectors and 4xXRE-Luc reporter genes.

The constitutive transcription of XRE-dependent luciferase differed among AhR species expressed in HeLa and Hepa1c1c7 cells (Fig. 4, A and B). Although overexpressed AhR protein levels were almost similar in HeLa cell experiments, the highest basal transcription level was detected in the guinea pig AhR, followed by the rabbit AhR (Fig. 4A). The pattern of basal transcription levels with each AhR in the Hepa1c1c7 cell experiments was similar to those in the HeLa cells; however, the levels did not correlate with the protein levels measured by immunoblotting (Fig. 4B). Of the six AhRs, the mouse AhR transfection caused the least increment in constitutive activity in both cells, as well as no significant induction of genes with the addition of OME (Fig. 4, A and B). Considering the effects of exogenous AhRs transfection, reporter gene induction by OME was highest when the rabbit AhR was used, followed by the guinea pig AhR (Fig. 4, A and B). However, in addition to the mouse AhR, rat and hamster AhRs were not activated by OME in mouse hepatoma cells. These results suggest that transcriptional activation by OME was dependent on the AhR species as well as the species of host cells.

Fig. 4.
Fig. 4.

Comparison of the induction of XRE-dependent transcription by OME through six mammalian AhR species. HeLa (A) and Hepa1c1c (B) cells were transfected with an empty vector (pCI-neo, CT, negative control) or six mammalian AhR expression plasmids (generated from the mouse, rat, human, rabbit, guinea pig, and hamster) together with the XRE-driven pX4TK-Luc reporter gene and pCMV-Rluc. After 24 hours, the cells were exposed to 100 μM OME or 0.1% dimethylsulfoxide (DMSO). After 16 hours of incubation, the cells were lysed, and firefly luciferase and Renilla luciferase activities were measured. Data represent the mean ± S.D. of normalized firefly luciferase (FFluc)/Renilla luciferase activities of three independent experiments. OME-treated groups (closed columns) were compared with DMSO controls (open columns) using one- or two-way analysis of variance, and statistically significant differences are denoted by asterisks (*P < 0.05; **P < 0.01). Total cell lysates were subjected to immunoblotting to confirm the expression level of each AhR (lower panels).

Responses of Chimeric AhRs to OME.

According to our already described observations, we speculated that the rabbit AhR must contain a domain responsible for OME-induced XRE-dependent transcription. Six chimeric AhRs were constructed using mouse and rabbit AhR cDNAs (Fig. 5A). Among the chimeric constructs, MR1, RM2, and MRM1, which included the rabbit LBD (amino acids 261–393), showed OME-dependent reporter gene induction. MR2, RM1, and RMR1, which included the mouse LBD (amino acids 257–389), did not respond to OME, despite the other regions of these chimeras were of the rabbit AhR (Fig. 5A). To determine the minimum requirement for the response to OME, chimeric AhRs were constructed with half LBDs; however, no constructs with partial rabbit AhR LBDs responded to OME (Fig. 5B). These data suggest that the response to OME required the complete rabbit AhR LBD and that multiple amino acid combinations specific to the rabbit AhR should be indispensable for OME-mediated AhR activation.

Fig. 5.
Fig. 5.

Construction of chimeric AhRs and their responses to OME and TCDD. (A) Schematic diagrams of the six chimeric constructs (MR1, MR2, RM1, RM2, MRM1, and RMR1) generated from parts of cDNAs of the rabbit (white) and mouse AhRs (gray) for the first screening. HeLa cells were transfected with expression plasmids containing chimeric AhRs or the empty vector (CT, negative control) together with pX4TK-Luc and exposed to 100 μM OME, 10 nM TCDD, or 0.1% dimethylsulfoxide (DMSO). Induction of the reporter gene by these chimeric AhRs after exposure to OME (left) and TCDD (right). Data represent means of the fold induction above the control from two independent experiments. Immunoblotting was performed to confirm the expression level of each AhR (lower panel). (B) The second screening to narrow the responsible domain. Four more chimeric constructs (MRM2, MRM3, RMR2, and RMR3) were generated and subjected to the same assay. Immunoblotting was performed to confirm the expression level of each AhR (lower panel).

Determination of the Amino Acid Residues Responsible for OME-Mediated AhR Activation.

Next, we conducted a study of a single amino acid substitution in the rabbit AhR that would eliminate OME-mediated AhR activation. There are 16 amino acid differences between the rabbit and mouse AhR LBDs (Fig. 6A). As shown in Fig. 6B, the basal transcriptional activity of rabbit AhR mutants was variously changed by a single amino acid substitution. Most of the mutants, except M328I, T353A, and F367L, did not much influence the OME-mediated transcriptional level. These three mutants reduced induction to less than one half of wild-type rabbit AhR, which showed an approximately 3-fold change by OME. The OME-mediated inductive ratio of M328I, T353A, and F367L was 1.7-, 1.2-, and 1.0-fold, respectively. Difference in properties between mouse AhR and rabbit AhR is the basal transcriptional activity and OME-induced transcriptional activity. By plotting these properties to emphasize differences of AhR mutants, we found the three mutant AhRs—M328I, T353A, and F367L—indicated intermediate properties of the mouse AhR and rabbit AhR (Fig. 6C).

Fig. 6.
Fig. 6.

Effects of point mutations on the response of the rabbit AhR to OME. (A) Amino acid alignment of mouse and rabbit AhR LBDs. Identical amino acid residues are indicated by asterisks. Differing amino acids are indicated in bold. Underlined sequences indicate the overlapping sequence for connecting with the mouse and rabbit LBDs shown in Fig. 5B. (B) Amino acids within the rabbit AhR LBD were individually replaced with the corresponding amino acids of the mouse AhR. RMR1 is the construct as shown in Fig. 5A. Asterisks indicate the mutated AhR in which two adjacent amino acids were replaced simultaneously. HeLa cells were transfected with expression plasmids containing mutated AhRs, pX4TK-Luc, and pCMV-Rluc. After 24 hours, the cells were exposed to 100 μM OME or 0.01% dimethylsulfoxide (DMSO) for 16 hours. Data represent the means of normalized firefly luciferase/Renilla luciferase activities of two independent experiments. Total cell lysates were prepared 24 hours after transfection and analyzed using immunoblotting to confirm the expression level of each AhR (lower panels). (C) The relationship between basal transcriptional activity and the OME-mediated transcriptional activity were plotted. ○, wild-type rabbit AhR; ●, rabbit AhR mutants; ▵, wild-type mouse AhR; ▴, RMR1 chimera AhR.

In addition, we constructed double and triple substitutions of these three amino acid residues in the rabbit AhR. As shown in Fig. 7A, the combinations of M328I and T353A or T353A and F367L substitutions impaired OME-mediated transcriptional induction. Moreover, the triple substitutions of M328I, T353A, and F367L exhibited properties that were remarkably similar to those of the mouse AhR (i.e., low constitutive activity and OME unresponsiveness). In contrast, mouse AhRs with single amino acid substitutions of I324M, A349T, or L363F, which are residues equivalent to M328, T353, and F367 in the rabbit AhR, slightly introduced OME responsiveness with increased levels of constitutive activities (Fig. 7B). However, triple substitutions of these three residues in the mouse AhR resulted in an approximately 5-fold induction by OME, which was not observed in wild-type mice.

Fig. 7.
Fig. 7.

Effect of single, double, and triple mutations on the response of rabbit and mouse AhRs to OME. (A) Rabbit AhRs with combinations of the three mutations of M328I, T353A, and F367L were transfected into HeLa cells along with the reporter genes. The combinations of the three mutations are indicated in the bottom table of the graph. I, 328Ile; A, 353Ala; L, 367Leu; CT, empty expression vector; Wt, wild-type rabbit AhR. The cells were exposed to 100 μM OME or 0.01% dimethylsulfoxide (DMSO) for 16 hours. Data represent the means of normalized firefly luciferase (FFluc)/Renilla luciferase activities of two independent experiments. (B) Mouse AhRs with combinations of the three mutations of I324M, A349T, and L363F were prepared, and luciferase assays were performed as described above. The combinations of the three mutations are indicated in the bottom table of the graph. M, 324Met; T, 349Thr; F, 363Phe; Wt, wild-type mouse AhR. Total cell lysates were prepared 24 hours after transfection and analyzed using immunoblotting to confirm the expression level of each AhR (lower panels).

Among the three amino acids, we focused on rabbit F367 because this residue is rabbit-specific (Fig. 8A). In addition, the guinea pig AhR was as responsive to OME as the rabbit AhR, and the amino acid corresponding to rabbit F367 is V368 in the guinea pig AhR, which is different in the other four species (Leu). Therefore, we constructed several mutated mouse and guinea pig AhRs to confirm that Val in this position was effective for the response to OME. As shown in Fig. 8B, the mouse AhR with triple substitutions of L363V, I324M, and A349T responded more to OME compared with double substitutions of I324M and A349T. In contrast, the response of the guinea pig AhR to OME was abolished by the V368L substitution. These results suggest that the combination of three amino acid residues of the AhR (i.e., Met, Thr, and Phe) are required for the response to OME) and also that Phe can be replaced with Val. The importance of these three amino acid residues in the OME response was confirmed by human AhR mutants as well as guinea pig AhR (Supplemental Fig. 2). When two amino acid residues (330M and 355T) in human AhR were changed to mouse-type amino acid (330I and 355A), OME response was attenuated. Contrary to this, 369F, which is particularly in rabbit AhR, represent higher OME-response than wild-type human AhR.

Fig. 8.
Fig. 8.

Effect of single, double, and triple mutations on the response of mouse and guinea pig AhRs to OME. (A) Alignment of the LBD amino acid sequences for six mammalian AhRs. The numbers indicate the amino acid positions in the mouse AhR (top line) and guinea pig AhR (bottom line). The three candidate amino acids that are crucial for the response to OME are in bold font. (B) Mouse AhRs with combinations of the three mutations and the guinea pig AhR with the V368L substitution were transfected into HeLa cells along with the reporter genes. Wt, wild-type mouse AhR or wild-type guinea pig AhR. The cells were exposed to 0.01% dimethylsulfoxide (DMSO) or 100 μM OME for 16 hours. Data represent the means of normalized firefly luciferase (FFluc)/Renilla luciferase activities of two independent experiments. Total cell lysates were prepared 24 hours after transfection and analyzed using immunoblotting to confirm the expression level of each AhR (lower panel).

CYP1A1 Gene Induction by OME Restored the Three Amino Acid Substitutions in the Mouse AhR and Abolished Them in the Rabbit AhR.

To confirm the results that M328, T353, and F367 in the rabbit AhR are responsible for the OME-mediated AhR activation, we established cell lines that stably expressed AhRs, including the triple point mutations. The c12 cell line, which is a Hepa1c1c-derived subline lacking endogenous AhR expression, was used for this purpose and examined for CYP1A1 induction by TCDD or OME exposure. As shown in Fig. 9, CYP1A1 mRNA induction by OME was similar to that observed in the reporter assays (Fig. 7); that is, the induction was limited to the cells expressing the wild-type rabbit AhR or the mouse AhR with the three amino acid substitutions. TCDD-induced CYP1A1 mRNA induction was observed at similar levels in all cell lines expressing both wild-type and mutated AhRs, suggesting that TCDD-induced AhR activation was not influenced by these amino acid substitutions.

Fig. 9.
Fig. 9.

CYP1A1 mRNA induction by OME in stable transformants of mutant AhRs. AhR-deficient mouse hepatoma c12 cells were transfected with the expression vector of the mouse AhR, rabbit AhR, their derivative mutated AhRs. Mouse 3mut, mouse AhR with I324M, A349T, and L363F substitutions or pCI-neo empty vector (CT, negative control). Rabbit 3mut, rabbit AhR with M328I, T353A, and F367L substitutions. After transfection, a heterogeneous population of G418-resistant cells was maintained and exposed to 0.1% dimethylsulfoxide (DMSO) (solvent control), 10 nM TCDD, or 100 μM OME. After 16 hours of incubation, total RNA was isolated, and mouse CYP1A1 mRNA was measured using real-time reverse-transcription polymerase chain reaction as described in Materials and Methods. Data represent mean ± S.D. (n = 3).

Responses of Mutated AhRs to Typical AhR Ligands.

Next, we characterized the responses of the mutated AhRs to typical AhR ligands, including AhRs with substitutions of the three amino acids involved in the response to OME. As shown in Fig. 10 and Table 1, EC50 values calculated from the dose-response curves of three typical chemical ligands were slightly changed (within 2-fold) by these three amino acid substitutions that drastically reversed OME responsiveness. We also tested candidates for endogenous ligands, namely, ITE (Song et al., 2002), FICZ (Wei et al., 1998), indirubin (Adachi et al., 2001), and kynurenic acid (DiNatale et al., 2010), which have induction potencies that differ between mouse and rabbit AhRs. FICZ induced transcriptional activity at a lower concentration with the rabbit AhR than with the mouse AhR, whereas indirubin had a reverse effect. However, as with the chemical ligands, the dose-response curves and EC50 values for the putative endogenous ligands were not much changed by the three amino acid substitutions. (Fig. 11; Table 1). These results show that these three amino acid residues define OME sensitivity but do not influence the responsiveness of the other AhR ligands tested in this study.

Fig. 10.
Fig. 10.

Response of mouse and rabbit AhR mutants to OME, TCDD, 3MC, and β-naphthoflavone (β-NF). HeLa cells were transfected with reporter genes and (A) the mouse wild-type AhR (mouse AhR Wt); (B) the mouse AhR with the triple substitutions I324M, A349T, and L363F (mouse 3mut); (C) the rabbit wild-type AhR (rabbit AhR Wt); and (D) the rabbit AhR with the triple substitutions M328I, T353A, and F367L (rabbit 3mut). Open circles represent 1, 10, 25, 50, and 100 μM OME; solid diamonds represent 0.01, 0.1, 0.3, 1, 3, 10, and 20 nM TCDD; open triangles represent 1, 10, 30, 100, 200, and 1000 nM 3MC; and solid squares with dotted lines represent 1, 10, 100, 300, 1000, and 2000 nM β-NF. Data represent the means of normalized firefly luciferase (FFluc)/Renilla luciferase activities from two independent experiments. EC50 values were calculated and are summarized in Table 1.

View this table:
TABLE 1

Summary of EC50 values of endogenous and synthetic chemical ligands

Fig. 11.
Fig. 11.

Response of mouse and rabbit AhR mutants to known endogenous ligands: indirubin, FICZ, ITE, and kynurenic acid. HeLa cells were transfected with reporter genes and various AhRs: (A) the mouse wild-typeAhR (Mouse AhR Wt); (B) the mouse AhR with the triple substitutions I324M, A349T, and L363F (mouse 3mut); (C) the wild-type rabbit AhR (rabbit AhR Wt); and (D) rabbit AhR with the triple substitutions M328I, T353A, and F367L (rabbit 3mut). Open circles represent 0.1, 1, 10, 30, 100, and 1000 nM indirubin; open diamonds represent 0.1, 1, 10, 30, 100, and 1000 nM FICZ; solid triangles represent 0.1, 1, 10, 30, 100, and 1000 nM ITE; and solid squares represent 0.1, 1, 3, 10, and 30 μM kynurenic acid. Data represent the means of normalized firefly luciferase (FFluc)/Renilla luciferase activities from two independent experiments. EC50 values were calculated and are summarized in Table 1.

Discussion

This study aimed to identify and experimentally validate the factor(s) generating species-specific responsiveness of AhR to OME. First, we speculated that OME-mediated AhR activation is due to increasing amounts of putative AhR ligands resulting from the inhibition of CYP1A1 activity by OME. This hypothesis is based on the following observations: 1) OME-mediated AhR activation required CYP1A1 activity and was influenced by endogenous ligands included in serum. 2) The concentration of OME activating AhR was higher than with that of the typical AhR ligand and was similar to the concentration of OME required for CYP1A1 inhibition. 3) The comparison of AhRs from six mammalian species revealed that transcriptional activation of the reporter gene by OME seemed to correlate with the basal transcription level, reflecting the response of the putative endogenous ligand. If a type of ligand mediates OME-dependent AhR activation, OME responsiveness would be attributable to the AhR itself, independent of host cells. In the present study, we constructed chimeric AhRs from mouse and rabbit AhRs, which indicated that OME responsiveness required multiple amino acid residues present in the LBD of the rabbit AhR. These amino acid residues were not present in the mouse AhR. In conclusion, we demonstrated that M328, T353, and F367 of the rabbit AhR were required for OME responsiveness.

These results are fundamentally different from those of previous reports on cell-specific factors that define OME responsiveness (Kikuchi et al., 1995, 2002; Dzeletovic et al., 1997). Some of the differences may be due to the species used because most of the mentioned studies used the human AhR, whereas we primarily used the rabbit AhR, which is highly sensitive to OME. In a previous report (Dzeletovic et al., 1997), the human AhR transiently expressed in mouse hepatoma c12 cells was not activated by OME. We performed a similar experiment with c12 cells stably expressing the rabbit AhR, and these cells exhibited inducible expression of CYP1A1 by OME (Fig. 9). We thought the stably expressed rabbit AhR altered intracellular conditions (e.g., the basal level of CYP1A1 expression) to respond to the AhR activator. In fact, the c12-derived cells expressing the rabbit AhR exhibited a raised level of constitutive CYP1A1 expression and remarkable CYP1A1 induction (>15-fold) on TCDD exposure.

Several investigations have identified specific amino acid residues that are important for AhR activation. We previously reported that L318 in the mouse AhR is required for the response to β-naphthoflavone (Goryo et al., 2007). An AhR polymorphism (A375V) in C57BL/6 and DBA/2 mice results in a difference in TCDD binding affinity and consequent toxicity (Ema et al., 1994). Two polymorphisms, V325I and A381S, in the common tern and chicken AhRs are responsible for sensitivity to TCDD (Karchner et al., 2006). Interestingly, the V325I polymorphism in the bird species corresponds to the mouse AhR I324, one of the critical amino acids for OME responsiveness. In the tern AhR, Ile is required in this position for high sensitivity to TCDD. The single mutation I324M in the mouse AhR was insufficient for producing a response to OME but instead increased basal transcriptional activity. The amino acid residue at this position could be close to TCDD and may be one of the key amino acids for ligand binding and constitutive activity. As mentioned in Results, F367 is specific to the rabbit AhR. In the guinea pig AhR, the corresponding amino acid is V363, and the L363V substitution was an effective alternative to the phenylalanine required for reconstituting the response to OME in the mouse AhR (Fig. 6). It is interesting that there are differences between Leu and Val despite their structural similarity. Ala at position 349 in the mouse AhR is also conserved in rat and hamster AhRs, which exhibit only a slight response to OME. In human and guinea pig AhRs, which are OME-sensitive, the corresponding amino acid at this position is threonine, identical to the rabbit AhR. Overall, these three amino acid residues correspond strongly to the species differences in the response to OME. The differences in intensity of the response to OME among the animal species can be explained well by this comparative approach.

The results of our study suggest some possibilities for elucidating the underlying mechanism of OME-mediated AhR activation. In our experiments, the response required an OME-sensitive AhR that depended on three amino acid residues located in LBD. Some reports have suggested that PTK is associated with OME-mediated AhR activation (Kikuchi et al., 1998; Backlund and Ingelman-Sundberg, 2005). In one report, the Y320 residue of the human AhR was determined to be a putative phosphorylation site required for OME responsiveness (Lemaire et al., 2004). However, this tyrosine residue and the proximal nine amino acids are conserved in all the AhRs tested in the present study. Furthermore, the three amino acid residues that we found to be essential are not putative phosphorylation sites, nor are they included in the PTK consensus. Hence, AhR phosphorylation by PTK may be influenced by a conformational change resulting from these three amino acids. The mutant AhRs generated in the present study will be useful for further investigations of the relationship between OME and PTK. Other than the involvement of PTK, a possible mechanism for OME-mediated AhR activation is the presence of some type of ligand. A possible type of ligand is an endogenous (intrinsic) ligand in the culture medium or one that is generated intracellularly. In general, an endogenous ligand is considered to be inactivated rapidly by P450 enzymes (Chang and Puga, 1998; Adachi et al., 2001; Spink et al., 2003). In the presence of a P450 inhibitor, the endogenous ligand remains and may activate the AhR. In fact, the inhibitory effects of OME on CYP1A1, 2C19, and 3A4 have been reported previously (Li et al., 2004; Shiizaki et al., 2008). By disrupting an autoregulatory loop consisting of CYP1A1 and FICZ, one of the endogenous ligands, a recent report showed that CYP1A1 upregulation by CYP1A1-inhibiting chemicals was due to an indirect mechanism (Wincent et al., 2012). On the basis of this finding, we confirmed CYP1A1 inhibition by OME (Fig. 1) and demonstrated that CYP1A1 activity was required for OME-mediated AhR activation (Fig. 2). These results were quite similar to the results obtained from CYP1A1 inhibitor ellipticine. Furthermore, OME-mediated AhR activation was partially reduced as a result of charcoal-stripping of serum, which is considered to be a supplier of endogenous ligands (Fig. 3). Thus, we tested four putative endogenous ligands, including FICZ; however, AhR activation by these ligands was not significantly influenced by the three amino acid substitutions. We concluded that these four putative endogenous ligands are not involved in the indirect mechanism of OME-mediated AhR activation. Therefore, an enigmatic ligand may be involved. The possible mechanism related to the indirect activation of AhR by OME is illustrated in Supplemental Fig. 3. Another possible ligand is an unstable and short-lived metabolite of OME, which would be difficult to detect by general experimental procedures. Hu et al. developed a sensitive assay that indicated that OME itself could bind to AhR, although with a very low affinity (Hu et al., 2007). The results shown in Fig. 2 involve the possibility that CYP1A1 produces the putative active metabolite, and we do not have data that deny this hypothesis at present. If some ligand is related to OME-mediated AhR activation, it must differ in its affinity for rabbit and mouse AhRs, and the difference would be attributable to the three amino acid residues identified in this study.

In conclusion, three amino acids in the LBD of AhR define species-specific differences in OME-mediated AhR activation. These findings will be useful for elucidating the molecular mechanism underlying OME-mediated AhR activation.

Acknowledgments

The authors thank Dr. Kazuhiro Sogawa (Tohoku University, Japan) for providing the reporter plasmid pX4TK-Luc and Dr. Tomonari Matsuda (Kyoto University, Japan) for providing indirubin.

Authorship Contributions

Participated in research design: Shiizaki, Ohsako.

Conducted experiments: Shiizaki, Ohsako.

Contributed new reagents or analytic tools: Shiizaki, Ohsako, Kawanishi, Yagi.

Performed data analysis: Shiizaki.

Wrote or contributed to the writing of the manuscript: Shiizaki, Ohsako, Yagi.

Footnotes

    • Received July 29, 2013.
    • Accepted November 21, 2013.

  • This research was supported by Grants-in-Aid for Scientific Research (S) [Grants 18101002, 18101003]; (C) [Grant 23590162] from MEXT Japan; and in part, by the Environmental Technology Development Fund to S.O. from the Ministry of the Environment.

  • dx.doi.org/10.1124/mol.113.088856.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

AhR
aryl hydrocarbon receptor
Arnt
AhR nuclear translocator
DMEM
Dulbecco’s modified Eagle’s medium
FBS
fetal bovine serum
FICZ
6-formylindolo[3,2-b]carbazole
ITE
2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester
LBD
ligand-binding domain
luciferin-CEE
luciferin 6′-chloroethyl ether
3MC
3-methylcholanthrene
MEM
minimum essential medium
OME
omeprazole
P450
cytochrome P450
PTK
protein tyrosine kinase
Rluc
Renilla luciferase
TCDD
2,3,7,8-tetrachlorodibenzo-p-dioxin
XRE
xenobiotic response element

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AhR Amino Acid Residues Required for Response to Omeprazole

Kazuhiro Shiizaki, Seiichiroh Ohsako, Masanobu Kawanishi and Takashi Yagi
Molecular Pharmacology February 1, 2014, 85 (2) 279-289; DOI: https://doi.org/10.1124/mol.113.088856
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