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Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona (M.D.M., J.P.J., L.M.A., C.D.F., N.J.C.); Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island (A.L.S.); Tsukuba Advanced Research Alliance Center, University of Tsukuba Tennoudai, Tsukuba-shi, Ibaraki, Japan (J.M.M.); Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas (W.H., D.D.M.); and Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas (Y.Z., C.D.K.)
(Received February 7, 2008; Accepted May 7, 2008)
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Abstract |
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; Ansher et al., 1986; Davidson et al., 1990; Morel et al., 1993; Primiano et al., 1996).
Induction of these metabolic enzymes by OPZ has been linked to the transcription factor nuclear factor E2–related factor 2 (Nrf2) and its activation of the antioxidant response element/electrophile response element (ARE/EpRE). Under normal conditions, Nrf2 is sequestered in the cytosol and marked for proteasomal degradation by the protein Kelch-like ECH-associated protein 1 (Keap1). This protein acts as an adapter for Cullin 3-based ubiquitin ligase (E3 ligase), as well as a "receptor" for electrophiles (Cullinan et al., 2004). In the absence of electrophiles/oxidative stress, Nrf2 is ubiquitinated and targeted for degradation by the 26S-proteasome, keeping cellular levels of Nrf2 low and preventing activation of its response element. Electrophilic binding to Keap1 is thought to disrupt the ubiquitination complex and inhibit Nrf2 degradation (Zhang et al., 2004). The increased cellular concentration of unbound Nrf2 in turn leads to ARE/EpRE activation (Zhang, 2006). It has been shown in Keap1 knockout mice that the loss of this Nrf2-Keap1 complex leads to Nrf2 nuclear accumulation and activation (Itoh et al., 2004). Further evidence of the role of Nrf2 in OPZ gene induction of detoxication enzymes was provided through experiments showing that loss of expression of Nrf2 abrogates both the chemoprotective activity of OPZ, as well as the inducibility of many known OPZ-induced genes (Kwak et al., 2001; Ramos-Gomez et al., 2001).
Although early investigation into the activity of OPZ focused on induction of detoxication enzymes like NQO1 and GST, OPZ also alters the expression and activity of certain cytochrome P450 enzymes. OPZ has been shown to induce the expression of CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 (Mahéo et al., 1998; Cherrington et al., 2003; Cho and Kim, 2003; Merrell et al., 2008). The induction of at least one of these isoforms, CYP1A1, has been characterized as aryl hydrocarbon receptor-dependent (Le Ferrec et al., 2002), which suggests that OPZ may activate multiple transcription factors.
Little has been reported previously regarding the strong induction of the CYP2B genes by OPZ. The mechanism by which CYP2B genes are induced is well documented (Sueyoshi and Negishi, 2001). CYP2B inducers activate the constitutive androstane receptor (CAR), which is normally sequestered in the cytoplasm by the cytoplasmic CAR retention protein (Kobayashi et al., 2003). Treatment with CAR activators appears to induce nuclear accumulation through one of two mechanisms: 1) some CAR activators are ligands, such as TCPOBOP in mice and CITCO in humans, and act as direct activators of CAR nuclear translocation; and 2) other CAR activators, such as phenobarbital (PB) and phenytoin, do not appear to be actual ligands and are thought to promote CAR nuclear translocation indirectly (Jackson et al., 2004, 2006; Stanley et al., 2006). In both direct and indirect activation, CAR translocates to the nucleus where it heterodimerizes with the retinoid X receptor (RXR) 
. The CAR/RXR heterodimer then binds to a PB responsive enhancer module to drive expression of CYP2B genes, including Cyp2b1 in rats, Cyp2b10 in mice, and CYP2B6 in humans (Honkakoski and Negishi, 1997; Honkakoski et al., 1998). We have previously shown that OPZ induction of Cyp2b10 mRNA expression is significantly less in RXR-deficient mice (Cherrington et al., 2003).
The present study was developed to determine whether, in addition to activating Nrf2, OPZ also activates the nuclear receptor CAR. We report here that OPZ strongly induces Cyp2b10 in a CAR-dependent manner, that OPZ treatment increases CAR nuclear accumulation, and that OPZ appears to be an indirect activator of CAR. This study shows an uncharacterized mechanism for OPZ-mediated gene induction through activation of the CAR.
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Materials and Methods |
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-Androstan-3-ol (androstanol) was purchased from Steraloids (Newport, RI). PB was purchased from Mallinckrodt, Inc. (Paris, KY). Ketamine HCl and xylazine injectables were purchased from Associated Medical Supply (Scottsdale, AZ). Luciferin was obtained from Molecular Imaging Products Company (Ann Arbor, MI). All the other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO).
Animals. All the animals were housed and acclimated in a temperature-, light-, and humidity-controlled environment in cages with hardwood chips and were given Harlan Teklad Rodent Diet W (Harlan Laboratories, Madison, WI) and water ad libitum. All the animals were acclimated for at least 1 week before the experiments and were allowed water and standard chow ad libitum. Housing and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals as determined by the U.S. National Institutes of Health. Male C57BL/6 mice were treated with either corn oil (5 ml/kg), OPZ (150 mg/kg, i.p. in corn oil), or PB (80 mg/kg, i.p. in saline) with an injection volume of 5 ml/kg. The choice of these doses was based on previous work from our laboratory (Cherrington et al., 2002), as well as the preponderance of published literature.
For the dose response, adult male C57BL/6 mice were treated with vehicle or OPZ once daily for 4 days (50, 100, 150, 200, and 250 mg/kg). Male Nrf2-/- mice (Aleksunes et al., 2006), CAR-/- mice (Wei et al., 2000), or C57BL/6 mice (n = 5 in each group) were treated with either corn oil (5 ml/kg), OPZ (150 mg/kg, p.o. in corn oil), or PB (80 mg/kg, i.p. in saline, 24 h) for 4 days with an injection volume of 5 ml/kg. For mRNA analysis, livers were excised 24 h following the final treatment, snap-frozen in liquid nitrogen, and stored at -80°C until use. For nuclear extraction, livers were excised 3 h after OPZ treatment, and nuclear protein was isolated as previously described (Sueyoshi et al., 1995) and stored at -80°C until Western blot analysis of CAR accumulation. To assess the degree of cytosolic contamination in the nuclear extracts, Western blotting was performed against glyceraldehyde-3-phosphate dehydrogenase protein, with minimal cytosolic contamination detected (data not shown).
RNA Isolation. Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) as per the manufacturer's protocol. RNA concentration was determined by UV spectrophotometry, and its integrity was examined by ethidium bromide staining after agarose gel electrophoresis.
Branched DNA Signal Amplification Assay. Mouse Cyp2b10 and Nqo1 probe sets were used as previously described (Cherrington et al., 2003; Aleksunes et al., 2005). These oligonucleotide probes were diluted in lysis buffer supplied in the Quantigene HV Signal Screen Kit (Panomics, Inc., Freemont, CA). Reagents for analysis were supplied with the kit (i.e., lysis buffer, amplifier/label probe buffer, and substrate solution) or prepared in the laboratory (capture hybridization buffer). Total RNA (1 µg/µl, 10 µl) was added to each well of a 96-well plate containing 50 µl of capture hybridization buffer and 50 µl of diluted probe set. mRNA was allowed to hybridize to each probe set overnight at 53°C. Subsequent hybridization steps were carried out as per the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 branched DNA (bDNA) luminometer (Bayer Diagnostics, Tarrytown, NY) interfaced with Quantiplex Data Management Software version 5.02 for analysis of luminescence from 96-well plates.
In Vivo Transcription Assay. Male C57BL/6 mice (20–25 g; Charles River Laboratories, Raleigh, NC) were administered 10 µg of naked plasmid DNA (CYP2B6 promoter luciferase reporter construct) as a rapid (5-s) tail vein injection in sterile saline at a dose volume equal to 10% of body weight. Eighteen hours later, animals were anesthetized with a mixture of ketamine (72 mg/kg) and xylazine (6 mg/kg). Mice were injected i.p. with luciferin (70 µl of a 50-mg/ml stock solution) 5 min before imaging. A VersArray 1300B camera (Princeton Instruments, Trenton, NJ) thermoelectrically cooled to -100°C, with an aperture of f1, connected to a light-sealed imaging chamber was used for all the images. Images were acquired in gray scale, and pseudocolor maps were created with the WinView 32 program (Princeton Instruments). Color maps were superimposed over the light image of the mouse using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA), and these images were considered time 0. Each animal (n = 3) was administered a single i.p. dose of either OPZ (150 mg/kg) or corn oil at a dose volume of 5 ml/kg, and imaging was repeated 24 h after dosing.
Western Blot Analysis. Nuclear extracts from control, PB-, and OPZ-treated C57BL/6 mice were resolved on a SDS-10% polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). Blots were incubated 1 h at room temperature in blocking buffer containing 5% nonfat milk in Tris-buffered saline/Tween 20. After 3-h incubation with a rabbit anti-CAR polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), the membrane was incubated 1 h with a goat anti-rabbit IgG-horseradish peroxidase conjugate (Santa Cruz Biotechnology). Western blots were visualized using enhanced chemiluminescence advanced reagents (Amersham, Arlington Heights, IL) as per manufacturer's protocol.
In Vitro Luciferase Assay. Cultured HepG2 cells were transiently transfected with a mouse CAR (mCAR) expression plasmid (mCAR/pCR3.0) (Sueyoshi et al., 1999) and a CYP2B6-tk-luciferase construct. The human CYP2B6 promoter luciferase reporter construct was obtained from Dr. Richard Kim (Vanderbilt University School of Medicine, Nashville, TN). The CYP2B6 promoter luciferase reporter construct contains a 1.7-kilobase fragment that maintains the core promoter (+39/-364) and the distal enhancer region (-1461/-2013) including the PB responsive element (PBREM) (R. Kim, unpublished data).
Briefly, HepG2 cells were cultured in Eagle's minimal essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) according to the protocol provided by American Type Culture Collection (Manassas, VA). In 24-well plates, the CYP2B6-tk-luciferase plasmid (0.1 µg) was cotransfected with pRL-tk transfection control (0.001 µg) (Promega Corp., Madison, WI) into HepG2 cells using Transfectene (Qiagen, Hilden, Germany) with or without a mouse CAR expression plasmid (0.025 µg). Twenty-four hours after transfection, cells were cultured in the presence of dimethyl sulfoxide (DMSO; 0.2%), PB (1 mM), TCPOBOP (250 nM), or OPZ (5, 20, 50, 100 µM) with or without androstanol (10 µM). After 24 h of exposure, the media were removed and cells were lysed with 100 µl of passive lysis buffer. Luciferase activity was determined by the Dual-Glo Luciferase Assay per manufacturer's protocol (Promega Corp.).
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Results |
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Dose Response of Induction of Nqo1 and Cyp2b10 to OPZ Treatment. The increase of Nqo1 mRNA levels by OPZ (Fig. 2) reached significance at a dose of 100 mg/kg and continued to increase through the higher doses, reaching an induction of 6-fold by 250 mg/kg. Although Cyp2b10 induction by OPZ did not reach significance until 200 mg/kg, the induction was more profound, with 47- and 74-fold increases at 200 and 250 mg/kg.
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). The absence of Nrf2 had no effect on the induction of Cyp2b10 mRNA, as the robust induction by PB and OPZ was maintained in Nrf2-/- animals.
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Treatment of wild-type mice with PB or OPZ resulted in a greater than 30- and 10-fold increase in Cyp2b10 mRNA, respectively (Fig. 4). This robust induction was completely absent in the CAR-/- animals treated with PB and OPZ, as has been previously noted for PB (Wei et al., 2000). In contrast, the induction of Nqo1 mRNA was maintained in CAR-/- animals.
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Real-Time in Vivo Activation of the Human CYP2B6 Promoter in Mice. The ability of OPZ to activate transcription of the human CYP2B6 promoter containing the PBREM was determined by transfecting a human CYP2B6 promoter-reporter luciferase reporter via rapid i.v. hydrodynamic injection via the tail vein and a real-time in vivo imaging assay before and after dosing. OPZ treatment resulted in robust activation of the human CYP2B6 promoter as seen in Fig. 5.
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Discussion |
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). The induction of GST and other cytoprotective enzymes has been termed the "antioxidant response" (Jaiswal, 1994) and is central to the proposed mechanism of action of OPZ in cancer prevention. The antioxidant response is an important mechanism for cellular defense and is mediated by the Nrf2/ARE pathway, resulting in the induction of several chemoprotective genes, including NQO1, HO-1, and GSTA1 and GSTA2 (Kensler et al., 1985). It is important to note that the induction of NQO1 by OPZ has been shown to be dependent on Nrf2 activation (Ramos-Gomez et al., 2001). Our current data showing the loss of Nqo1 induction in Nrf2-/- mice support that finding.
Although the induction of these genes has been shown to be ARE/EpRE-dependent, OPZ is also capable of inducing other genes in an Nrf2-independent manner (Le Ferrec et al., 2002; Ebert et al., 2005). For example, CYP2B is induced by OPZ yet is considered a hallmark gene of CAR activation. The mechanism of induction of CYP2B isoforms has been well characterized, and currently no ARE/EpRE consensus sequences have been identified in their 5'-flanking regions. Consistent with these facts, our data show that OPZ activates gene transcription through activation of multiple and distinct transcription factors, including Nrf2 and CAR.
To analyze the role of both CAR and Nrf2 in OPZ gene induction, CAR- or Nrf2-deficient mice were treated with OPZ or the known CAR activator PB. Our data indicate that Nrf2 and CAR are required in the regulation of gene induction by OPZ. As expected, the loss of Nrf2 resulted in loss of Nqo1 induction, whereas induction of Cyp2b10 mRNA was unaffected. These results both confirm a role for Nrf2 in Nqo1 regulation and suggest an Nrf2-independent mechanism for the induction of Cyp2b10 mRNA by OPZ. The complete abrogation of Cyp2b10 mRNA induction in CAR-null mice treated with OPZ strongly implicates CAR as the regulatory mechanism. These data agree with our previously published data, which revealed that loss of the obligatory CAR-binding partner RXR abrogates OPZ induction of Cyp2B (Cherrington et al., 2003).
Two additional lines of evidence in this study characterize OPZ as a CAR activator. First, OPZ treatment results in transcriptional activation of a human CYP2B6 promoter construct in transiently transgenic mice. The promoter region of this construct contains the PBREM, the well characterized CAR DNA-binding sequence. The evident induction of this reporter gene in OPZ-treated mice compared with corn oil controls shows the ability of OPZ to induce CAR-driven genes in vivo. Second, OPZ induces CAR nuclear accumulation in hepatocytes of OPZ-treated mice. Because the main component of CAR activation appears to be nuclear translocation (Kobayashi et al., 2003), these findings firmly establish OPZ as a CAR activator. Interestingly, the OPZ dose used in these in vivo studies did not cause maximal induction of the CAR target gene Cyp2b10 (Fig. 2). This indicates that CAR may have not been fully activated at that dose. The increase in CAR activity by OPZ treatment seen even at this lower dose helps to support OPZ as a CAR activator.
In vivo, CAR can be activated either directly by ligand binding (as in the case of TCPOBOP) or indirectly through the activation of currently unknown pathways (as with PB) (Stanley et al., 2006). In cultured cells lines, however, transfected CAR freely translocates to the nucleus without exposure to CAR activators (Kobayashi et al., 2003). This is apparent in our study by the increase in CYP2B6-promoter driven activity of the luciferase reporter gene on the addition of mCAR. The treatment of these cells with the inverse agonist androstanol repressed the activity of mCAR and the transcriptional activation of the luciferase reporter gene. Androstanol is a CAR ligand, and binding results in a conformational change in CAR that inhibits the recruitment of the transcriptional machinery (Wright et al., 2007). The ability of TCPOBOP to overcome this inhibition has been previously shown and results from the direct interaction of TCPOBOP with CAR, which both displaces androstanol and promotes the recruitment of the transcriptional machinery (Sueyoshi et al., 1999; Tzameli et al., 2000). In the current study, TCPOBOP treatment, but not PB or OPZ treatment, was able to reverse the inhibitory effects of androstanol. Although OPZ cotreatment resulted in a slight increase in luciferase activity, this increase never reached significance (p 0.05). Although these findings suggest that OPZ acts similarly to PB to indirectly activate CAR, further investigation of any OPZ-CAR interactions is necessary to definitively rule out OPZ as a CAR ligand.
The mechanism by which PB indirectly activates CAR remains to be elucidated, although the involvement of a number of kinases and phosphatases has been proposed. The involvement of the extracellular signal-regulated kinase (ERK) in the repression of CAR nuclear translocation is of particular interest. Previous work has shown that epidermal growth factor is able to attenuate CAR-mediated induction of CYP2B by PB treatment (Bauer et al., 2004). Inhibitors of mitogen-activated protein kinase kinase 1/2 (a kinase downstream of epidermal growth factor signaling) have been shown to potentiate the induction CYP2B by PB (Joannard et al., 2006). Most recently, ERK1/2 dephosphorylation (deactivation) was shown to be associated with CAR nuclear translocation (Koike et al., 2007). Our findings that OPZ activates CAR may provide additional support to the importance of ERK1/2 in the regulation of CAR. OPZ has been shown to inhibit the constitutive phosphorylation (activation) of ERK1/2 and has been shown to inhibit the activity of mitogen-activated protein kinase kinase 1 (Kang et al., 2003). Whether this inhibition of ERK signaling by OPZ is responsible for the activation of CAR is unclear and under investigation.
The data presented in this manuscript show that OPZ modulates gene expression through CAR activation, in addition to Nrf2. These results add to previous findings on the dual activation of Nrf2 and CAR. Trans-stilbene oxide and diallyl sulfide have both been identified as activators of both CAR and Nrf2 (Slitt et al., 2006; Fisher et al., 2007). The addition of yet another structurally diverse compound to this group raises the possibility of cross-talk between the two factors, although further studies are needed to determine the extent of any connections.
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: OPZ, oltipraz; GST, glutathione-S-transferase; NQO1, NAD(P)H:quinone oxidoreductase; Nrf2, nuclear factor E2-related factor 2; ARE/EpRE, antioxidant response element/electrophile response element; Keap1, Kelch-like ECH-associated protein 1; CAR, constitutive androstane receptor; TCPOBOP, 1,4-bis-[2-(3, 5-dichloropyridyloxy)] benzene; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; PB, phenobarbital; RXR, retinoid X receptor; bDNA, branched DNA; mCAR, mouse constitutive androstane receptor; PBREM, phenobarbital responsive element; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase.
Address correspondence to: Nathan J. Cherrington, University of Arizona, 1703 East Mabel Street, Tucson, AZ 85721. E-mail: cherrington{at}pharmacy.arizona.edu
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