DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation
Introduction
Hepatic cytochrome P450s (CYPs) display diverse functions in the metabolism of biological compounds, including endogenous molecules such as steroid hormones (You, 2004) and xenochemicals such as drugs and environmental pollutants (Iwase et al., 2006, Arlt et al., 2006). Phenobarbital (PB) has been identified as a potent inducer of the CYP 2B subfamily in both human and rodent primary hepatocytes (Wang et al., 2004, Li et al., 2006). Increased CYP 2B activity induced by xenochemicals may result in the accelerated metabolism of co-administered drugs and lead to decreased therapeutic efficacy and increased toxicity (Lin, 2006). PB-responsive enhancer module (PBREM), which responds to numerous PB-type inducers, has been identified in the promoter of human and rat CYP 2B genes (Sueyoshi et al., 1999). PBREM contains a nuclear factor-1 (NF-1) binding site flanked by two DR-4 nuclear receptor-binding motifs, NR-1 and NR-2 (Honkakoski and Negishi, 1998). Specific mutation of these NR motifs results in a complete loss of responsiveness to PB induction (Honkakoski et al., 1998).
The constitutive androstane receptor (CAR), which is present mostly in the cytosol of noninduced mammalian liver and hepatocytes, has been recognized as a transcription factor. It plays an important role not only in the regulation of hepatic fatty acid oxidation, gluconeogenesis (Miao et al., 2006), and the metabolism of steroid hormones and bilirubin (Yamamoto et al., 2003), but also in the modulation of xenochemical-metabolizing gene expression, including that of the phase I enzymes, such as CYPs 2B, 2C, and 3A, and the phase II enzymes, such as glutathione S-transferase alpha 1 (GSTA1) and UDP-glucuronosyltransferase 1A1 (UGT1A1) (Pascussi et al., 2003a, Reschly and Krasowski, 2006). Upon activation by PB-type inducers, CAR translocates from the cytosol to the nucleus and forms a heterodimer with retinoid X receptor (RXR). Subsequently, the heterodimer binds to NR-1 and trans-activates the target genes (Kawamoto et al., 1999, Kim et al., 2001).
N-6 and n-3 polyunsaturated fatty acids (PUFAs) have been identified to play an important role in energy metabolism and in endogenous hormone synthesis (Spector and Yorek, 1985). N-6 and n-3 PUFAs have also been shown to regulate the expression of many genes, such as acetyl-CoA carboxylase (Suchankova et al., 2005), leptin (Reseland et al., 2001), and fatty acid synthetase (Kim et al., 2004). PUFAs have also been studied for their role in the regulation of gene expression involved in the prevention of cardiovascular diseases (Le Jossic-Corcos et al., 2005), tumor development (Trombetta et al., 2007), and neurodegenerative disorders (Kim and Takahashi, 2006). However, studies of the roles of PUFAs in the expression of detoxifying genes are relatively scarce. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 PUFAs, especially docosahexaenoic acid (DHA) (Li et al., 2006). However, the mechanism for this down-regulation by DHA was not fully understood. In the present study, we used the rat primary hepatocyte culture system to study whether CAR plays a crucial role in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression.
Section snippets
Materials
Cell culture medium (RPMI-1640) was purchased from Gibco-BRL (Gaithersburg, MD); collagenase type I was from Worthington Biochemical (Lakewood, NJ); Matrigel and ITS+ (insulin, transferrin, selenium, bovine serum albumin, and linoleic acid) were from Collaborative Biomedical Products (Bedford, MA); and arachidonic acid (AA), linoleic acid (LA), eicosapentaenoic acid (EPA), and DHA were from Cayman Chemical (Ann Arbor, MI). Anti-CAR antibody (sc-13065) and anti-RXRα antibody (sc-553) were
Effect of PB on the nuclear accumulation of CAR in rat primary hepatocytes
Transactivation of the nuclear orphan receptor CAR was identified as a crucial event in PB-inducible CYP 2B gene expression in mouse primary hepatocytes (Kawamoto et al., 1999). To determine the effect of PB on the nuclear accumulation of CAR in rat primary hepatocytes, cells were treated with increasing concentrations of PB (5–500 μM) for 1 h. As shown in Fig. 1A, the nuclear accumulation of CAR was increased by PB in a dose-dependent manner. Also, the nuclear accumulation of CAR with 50 μM PB
Discussion
The nuclear orphan receptor CAR activates the transcription of detoxifying genes, such as CYP 2B (Faucette et al., 2006). CAR accumulates in the nucleus in response to PB and PB-type inducers in rat liver (Pustylnyak et al., 2005a) and mouse primary hepatocytes (Hosseinpour et al., 2006). To demonstrate whether PB triggers the nuclear accumulation of CAR in our rat primary hepatocyte culture system, we subjected the cell nuclear extracts to Western blotting. We showed that a basal level of
Acknowledgments
This work was supported by NSC 94-2320-B-040-033 and NSC-95-2320-B-040-029-MY2.
References (48)
- et al.
Bioactivation of 3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone: evidence for DNA adduct formation mediated by cytochrome P450 enzymes and peroxidases
Cancer Lett.
(2006) - et al.
Characterization of DNA complexes formed by the nuclear receptor constitutive androstane receptor
J. Biol. Chem.
(2003) - et al.
Fatty acid regulation of hepatic gene transcription
J. Nutr.
(2005) - et al.
Phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital
Biochem. Biophys. Res. Commun.
(1999) - et al.
Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU
J. Biol. Chem.
(2001) - et al.
Suppression of hepatic fatty acid synthase by feeding alpha-linolenic acid rich perilla oil lowers plasma triacylglycerol level in rats
J. Nutr. Biochem.
(2004) - et al.
N-6 and n-3 polyunsaturated fatty acids down-regulate cytochrome P-450 2B1 gene expression induced by phenobarbital in primary rat hepatocytes
J. Nutr. Biochem.
(2006) - et al.
Docosahexaenoic acid enhances cyclooxygenase-2 induction by facilitating p44/42, but not p38, mitogen-activated protein kinase activation in rat vascular smooth muscle cells
J. Pharm. Sci.
(2005) - et al.
Down-regulation of astroglial CYP2C, glucocorticoid receptor and constitutive androstane receptor genes in response to cocaine in human U373 MG astrocytoma cells
Toxicol. Lett.
(2005) - et al.
Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha
J. Biol. Chem.
(2006)
CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers
Toxicology
In vivo effects of protein kinase and phosphatase inhibitors on CYP2B induction in rat liver
Toxicology
Reduction of leptin gene expression by dietary polyunsaturated fatty acids
J. Lipid Res.
Preparation of isolated rat liver cells
Methods Biol.
Membrane lipid composition and cellular function
J. Lipid Res.
The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene
J. Biol. Chem.
Dietary polyunsaturated fatty acids enhance hepatic AMP-activated protein kinase activity in rats
Biochem. Biophys. Res. Commun.
Arachidonic and docosahexaenoic acids reduce the growth of A549 human lung-tumor cells increasing lipid peroxidation and PPARs
Chem. Biol. Interact.
Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin
J. Biol. Chem.
The role of the nuclear receptor CAR as a coordinate regulator of hepatic gene expression in defense against chemical toxicity
Arch. Biochem. Biophys.
Extracellular ATP induces anchorage-independent expression of cyclin A and rescues the transformed phenotype of a ras-resistant mutant cell line
J. Biol. Chem.
Steroid hormone biotransformation and xenobiotic induction of hepatic steroid metabolizing enzymes
Chem. Biol. Interact.
Transcriptional regulation of CYP2B1 induction in primary rat hepatocyte cultures: repression by epidermal growth factor is mediated via a distal enhancer region
Mol. Pharmacol.
Docosahexaenoic acid induces proteasome-dependent degradation of {beta}-catenin, down-regulation of survivin and apoptosis in human colorectal cancer cells not expressing COX-2.
Carcinogenesis
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