Abstract
Differentiated embryo chondrocyte-2 (DEC2), also known as bHLHE41 or Sharp1, is a pleiotropic transcription repressor that controls the expression of genes involved in cellular differentiation, hypoxia responses, apoptosis, and circadian rhythm regulation. Although a previous study demonstrated that DEC2 participates in the circadian control of hepatic metabolism by regulating the expression of cytochrome P450, the molecular mechanism is not fully understood. We reported previously that brief exposure of HepG2 cells to 50% serum resulted in 24-h oscillation in the expression of CYP3A4 as well as circadian clock genes. In this study, we found that the expression of CYP2D6, a major drug-metabolizing enzyme in humans, also exhibited a significant oscillation in serum-shocked HepG2 cells. DEC2 interacted with CCAAT/enhancer-binding protein (C/EBPα), accompanied by formation of a complex with histone deacetylase-1, which suppressed the transcriptional activity of C/EBPα to induce the expression of CYP2D6. The oscillation in the protein levels of DEC2 in serum-shocked HepG2 cells was nearly antiphase to that in the mRNA levels of CYP2D6. Transfection of cells with small interfering RNA against DEC2 decreased the amplitude of CYP2D6 mRNA oscillation in serum-shocked cells. These results suggest that DEC2 periodically represses the promoter activity of CYP2D6, resulting in its circadian expression in serum-shocked cells. DEC2 seems to constitute a molecular link through which output components from the circadian clock are associated with the time-dependent expression of hepatic drug-metabolizing enzyme.
Introduction
Most living organisms exhibit behavioral and physiological rhythms with a period length of approximately 24 h. Some of these rhythms are controlled by a self-sustained oscillation mechanism called the circadian clock. Molecular studies of the circadian clock system have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of 24-h rhythms (Gekakis et al., 1998; Kume et al., 1999). In mammals, the core molecular mechanism of the oscillator consists of two transcriptional activators, CLOCK and BMAL1, and their transcriptional targets, PERIOD (PER) and CRYPTOCHROME (CRY). PER and CRY proteins act as negative regulators of CLOCK/BMAL1 activity, thus forming the major circadian autoregulatory feedback loop (Reppert and Weaver, 2002). The expression of clock genes is also modulated by a second oscillation loop composed of two orphan nuclear receptors, REV-ERBα and retinoid-related orphan receptor-α (RORα), which drive the circadian oscillation in Bmal1 transcription (Preitner et al., 2002; Akashi and Takumi, 2005).
The circadian oscillators in hepatic cells drive rhythmic physiology through these transcriptional factors, which in turn regulate the transcription of downstream genes (Gachon et al., 2006). D site-binding protein (DBP) and E4 promoter-binding protein 4 (E4BP4) are examples of such output mediators because they are transcriptionally regulated by core oscillator components (Ripperger et al., 2000; Ueda et al., 2005). Clock genes and clock-controlled output genes are expressed rhythmically not only in the suprachiasmatic nucleus, the center of mammalian circadian clock, but also in other brain regions and peripheral tissues (Sakamoto et al., 1998; Yamamoto et al., 2004). The master clock located in the suprachiasmatic nucleus follows a daily light/dark cycle and, in turn, synchronizes subsidiary oscillators in other brain regions and many peripheral tissues through neural and/or humoral signals (Balsalobre et al., 2000a; Terazono et al., 2003). These subsidiary oscillators coordinate a variety of biological processes, producing 24-h rhythms in physiology and behavior. Such rhythmic expression of clock genes is also observed in cultured cells after brief treatment with various compounds (high concentration serum, forskolin, phorbol-12-myristate-13-acetate, calcimycin, or dexamethasone) (Balsalobre et al., 1998, 2000a,b); therefore, the peripheral oscillator in cultured human cells could constitute an in vitro model for the molecular oscillator in human tissues.
The cytochrome P450 gene superfamily encodes a group of heme-containing monooxygenases, many of which metabolize compounds used as therapeutic drugs. CYP3A4 is the most abundant P450 expressed in the human liver and small intestine, contributing to the metabolism of approximately half of the drugs in clinical use today (Evans and Relling, 1999). The expression of CYP3A4 and its metabolizing activity exhibit significant circadian oscillation in serum-shocked HepG2 cells (Takiguchi et al., 2007). DBP and E4BP4 regulate the circadian expression of the CYP3A4 gene, suggesting a molecular link between the circadian clock and xenobiotic metabolism. Differentiated embryo chondrocyte-2 (DEC2), also known as bHLHE41 or Sharp1, is a basic helix-loop-helix (bHLH) transcriptional repressor and acts as an output component of the circadian oscillator. DEC2 regulates the expression of genes involved in cellular differentiation, hypoxia responses, apoptosis, and circadian rhythms (Honma et al., 2002; Miyazaki et al., 2002). A previous study demonstrated that DEC2 also participates in the circadian control of hepatic metabolism by regulating the expression of P450s (Noshiro et al., 2004); however, the regulation mechanism remains to be fully understood.
Families CYP1, CYP2, and CYP3 encompass the most relevant xenobiotic-metabolizing P450s in humans (Evans and Relling, 1999). Despite the low content of the CYP2D6 isoform in human hepatic cells, a variety of drugs are metabolized by this enzyme (Daly et al., 1993; Wolf and Smith, 1999). However, circadian regulation of Cyp2d genes has been little explored, even in experimental animals. In this study, we found that brief exposure of HepG2 cells to 50% serum also induced a significant 24-h oscillation in the expression of CYP2D6. Although hepatic expression of CYP2D6 is dependent on both hepatic nuclear factor-4α (HNF4α) and CCAAT enhancer binding protein-α (C/EBPα), DEC2 repressed C/EBPα-induced transactivation of the CYP2D6 gene. Therefore, we investigated the underlying mechanism of the circadian expression of CYP2D6 in serum-shocked cells by focusing on the transcriptional interaction between DEC2 and C/EBPα.
Materials and Methods
Cell Culture and Animals.
HepG2 cells were supplied by the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (SAFC Bioscience, Kansas City, MO) at 37°C in a humidified 5% CO2 atmosphere. Male ICR mice were housed under a 12-h light/dark cycle (lights on at Zeitgeber time 0) with food and water ad libitum. They were cared for in accordance with the guidelines established by the Animal Care and Use Committee of Kyushu University. Primary cultures of hepatocytes were prepared by standard techniques, and cells were maintained in hepatocyte maintenance medium supplemented with 5% FBS, 0.1 μM insulin, 0.1 μM dexamethasone, 50 μg/ml gentamicin, and 50 ng/ml amphotericin B.
Experimental Design.
To synchronize the circadian clocks in cultured HepG2 cells, serum shock was performed as follows. Cells were grown to semiconfluence in DMEM supplemented with 10% FBS and then incubated in serum-starved medium for 12 h. On the day of serum shock, 50% FBS or phosphate-buffered saline (control) was added for 2 h, and then cells were changed back to starvation medium. Cells were harvested for RNA extraction at 0, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, and 60 h after serum treatment. The mRNA levels of CYP1A2, -2C9, -2C19, and -2D6 were measured by reverse transcription (RT)-polymerase chain reaction (PCR). To quantify the protein levels of CYP2D6 and its enzymatic activity, microsomes were prepared from HepG2 cells at 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, and 60 h after serum treatment. The protein abundance of CYP2D6 was also determined by Western blotting. The enzymatic activity of CYP2D6 in the microsomal fraction was investigated using 7-methoxy-4-(aminomethyl) coumarin (MAMC). To investigate the influence of HNF4α and clock gene products on the expression of endogenous CYP2D6, HepG2 cells were transfected with expression plasmids encoding HNF4α, C/EBPα, CLOCK, BMAL1, PER2, CRY1, DEC2, DBP, E4BP4, RORα, or REV-ERBα. At 24 h after transfection, mRNA levels of CYP2D6 were determined by RT-PCR. To clarify whether endogenous HNF4α and clock gene products affect the expression of CYP2D6 protein, HepG2 cells were transfected with small interfering RNA (siRNA) against HNF4α, C/EBPα, or DEC2. Twenty-four hours after transfection, the protein levels of HNF4α, C/EBPα, DEC2, or CYP2D6 were assessed by Western blotting. The transcriptional mechanism of CYP2D6 by HNF4α, C/EBPα, and DEC2 was analyzed using luciferase reporter vectors containing the 5′-flanking region of the CYP2D6 gene. The luciferase reporter assay was also performed in the presence or absence of trichostatin A (TSA), a histone deacetylase inhibitor. Interactions between DEC2 and C/EBPα were investigated by immunoprecipitation assay. The binding of endogenous C/EBPα, DEC2, or histone deacetylase-1 (HDAC1) on the CYP2D6 promoter in HepG2 cells was analyzed by chromatin immunoprecipitation. To explore the role of DEC2 in the circadian regulation of CYP2D6 expression, HepG2 cells were transfected with siRNA against DEC2 and thereafter treated with 50% FBS as described above. To explore whether the expression of the Cyp2d gene in the experimental animals exhibits circadian oscillation, the temporal profiles of Cyp2d9 and Cyp2d22 mRNA were assessed using serum-shocked primary culture of hepatocytes. We also investigated the temporal expression profiles of Cyp2d9 and Cyp2d22 mRNA in mouse liver kept under a 12-h light/dark cycle.
Quantitative Reverse Transcription-PCR Analysis.
Total RNA was extracted using RNAiso (Takara Bio Inc., Shiga, Japan). cDNA was prepared via reverse transcription of total RNA using a ReverTra Ace qPCR RT kit (Toyobo Co. Ltd., Osaka, Japan). Diluted cDNA samples were analyzed by real-time or semiquantitative RT-PCR. Real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The sequences of primer pairs are listed in Table 1.
Determination of CYP2D6 Activity.
The enzymatic activity of CYP2D6 in HepG2 cells was determined by assessing the production rate of 7-hydroxy-4-aminomethylcoumarin (HAMC), the O-demethylated metabolite of MAMC (Onderwater et al., 1999). Microsomal fractions prepared from HepG2 cells were incubated in 0.1 M potassium phosphate buffer, pH 7.4, containing 10 μM NADPH and 0.4 mM EDTA. We added the inhibitory antibodies against CYP1A2 to the reaction mixture, because MAMC is also metabolized by CYP1A2 (Onderwater et al., 1999). In fact, antibodies against CYP1A2 decreased the production rate of HAMC in the HepG2 microsomal fraction by approximately 12%, whereas inhibitory antibodies to CYP2D6 reduced the activity by approximately 85%. After equilibration at 37°C, MAMC was added to the reaction mixture at a final concentration of 25 μM, and the real-time increase in fluorescence was recorded by spectrofluorometer with the excitation wavelength set at 405 nm and emission wavelength set at 480 nm. The production rate of HAMC was quantified from the resulting increase in fluorescence. Protein concentration of reaction mixtures was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). The CYP2D6 activity was expressed as picomoles of HAMC during the 1-min incubation per milligram of protein.
Western Blotting.
Samples (20 μg of protein) were separated on SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were reacted with antibodies against human CYP2D6 (Nihon Nosan, Kyoto, Japan), HNF4α, C/EBPα, DEC2, HDAC1, or ACTIN (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Specific antigen-antibody complexes were made visible using peroxidase-conjugated secondary antibodies and Chemi-Lumi One (Nacalai Tesque Inc., Kyoto, Japan).
siRNA.
We designed siRNA for knockdown experiments using BLOCK-iT RNAi Designer (https://rnaidesigner.invitrogen.com/rnaiexpress/). The target sequences of C/EBPα, HNF4α, and DEC2 genes are listed in Table 2. One day before transfection, HepG2 cells were seeded (5 × 105 cells/well) in six-well plates containing serum-free DMEM. siRNA against HNF4α, C/EBPα, DEC2, or scrambled control oligo (200 ng each) was transfected into the cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA).
Construction of Reporter Plasmids and Expression Vectors.
To construct the luciferase reporter vectors of the human CYP2D6 gene [CYP2D6-Luc], an approximately 1.4-kilobase pair fragment (−1399 to +89; +1 indicates the putative transcription start site) derived from the 5′-flanking region of the human CYP2D6 gene (GenBank accession no. DQ211353) was amplified by PCR from genomic DNA of HepG2 cells. The PCR products were purified and ligated into the pGL3 basic vector (Promega, Madison, WI). The mutant construct of CYP2D6-Luc was prepared by changing the sequence from CATTGCACAATG to CAAAGCTTAATG (bases −1231 to −1220) and from AGGGCAAAGGCCA to AGGGTAAGCTTCA (bases −55 to −43), respectively (Fig. 3A). Expression plasmids of HNF4α, C/EBPα, CLOCK, BMAL1, PER2, CRY1, DEC2, DBP, E4BP4, RORα, and REV-ERBα were prepared as follows: the coding regions of the transcriptional regulators were obtained by RT-PCR and used after their sequences had been confirmed. All coding regions were ligated into the pcDNA 3.1 vector (Invitrogen).
Transcriptional Assay.
On the day before transfection, the cells were seeded (2 × 105 cells/well) into six-well plates containing DMEM supplemented with 10% FBS. Cells were transfected with 100-ng reporter constructs and 1-μg (total) expression vectors, using Lipofectamine-LTX reagent (Invitrogen) according to the manufacturer's instructions. To correct for variations in transfection efficiency, 0.5 ng of pRL-SV40 (Promega) was cotransfected in all experiments. The total amount of DNA per well was adjusted to 1.0 μg by addition of pcDNA3.1 vector (Invitrogen). The transfected cells were incubated in the presence or absence of TSA, a histone deacetylase inhibitor. At 48 h after transfection, cell extracts were prepared with 500 μl of passive lysis buffer (Promega), and 50-μl portions of the extracts were used for assays of firefly luciferase and Renilla reniformis luciferase by luminometry. The ratio of firefly luciferase activity (expressed from reporter plasmids) to R. reniformis luciferase activity (expressed from pRL-SV40) in each sample served as a measure of normalized luciferase activity.
Immunoprecipitation Assay.
Nuclear fractions of HepG2 cells were prepared at the indicated time points after 50% serum treatment. The fractions were immunoprecipitated by anti-DEC2 or anti-C/EBPα antibodies on protein G-agarose beads. The specific bound proteins were released by resuspending beads in 20 μl of loading buffer, divided into equal amounts, and resolved by SDS-polyacrylamide gels. One gel was subjected to Western blotting with anti-DEC2, anti-C/EBPα, or anti-HDAC1 antibodies.
Chromatin Immunoprecipitation Assay.
Cells were cross-linked with 1% formaldehyde in phosphate-buffered saline at 4°C for 10 min. Each cross-linked sample was sonicated on ice and then incubated with antibodies against C/EBPα, DEC2, or HDAC1. DNA was isolated from the immunoprecipitates and subjected to PCR using the following primer pairs: for the surrounding C/EBPα binding site in the CYP2D6 promoter (from base pairs −1312 to −1074), 5′-TGGTGAAACCCTATCTCTACTG-3′ and 5′-TCACTGCAGTCTCGACATCA-3′; for CYP2D6 promoter that does not contain C/EBPα binding sites (from base pairs −827 to −570), 5′-CCTGTTGCAAACAAGAAGCCATAG-3′ and 5′-GGACACGATTACACATGCAGAA-AAT-3′. As negative controls, chromatin immunoprecipitations were performed in the absence of antibody or in the presence of rabbit IgG. PCR products from these samples were not detectable by ethidium bromide staining.
Statistical Analysis.
The statistical significance of the differences among groups was analyzed by analysis of variance and Dunnett's test or the Bonferroni multiple comparison test. A 5% level of probability was considered significant.
Results
Rhythmic Expression of CYP2D6 Gene in HepG2 Cells after Serum Treatment.
We demonstrated previously that treatment of HepG2 cells with 50% FBS for 2 h induced the rhythmic expression of CYP3A4 and CYP2E1 (Takiguchi et al., 2007; Matsunaga et al., 2008). Treatment of cells with a high concentration of serum transiently induced the expression of CYP1A2 mRNA but did not affect the mRNA levels of CYP2C9 and CYP2C19 (Fig. 1A). On the other hand, serum-shocked HepG2 cells exhibited a significant oscillation in the expression of CYP2D6 (P < 0.05) (Fig. 1A). The rhythmic phase of mRNA levels for CYP2D6 was similar to that of DBP as reported previously (Takiguchi et al., 2007); however, the oscillation in the levels of CYP2D6 protein was delayed by approximately 8 h relative to its mRNA rhythm (Fig. 1B). The rhythmic pattern of CYP2D6 protein expression resembled the overall decreases and increases in the enzymatic activity of CYP2D6 (Fig. 1C). These in vitro results suggest that oscillation in the expression of the CYP2D6 gene is cell autonomous. The rhythmic change in mRNA levels of CYP2D6 seemed to cause the oscillation of its protein abundance and enzymatic activity.
Effects of Clock and Clock-Controlled Gene Products on the mRNA Levels of CYP2D6.
Clock genes, consisting of core oscillation loops, generate 24-h variations in output physiology through the periodic activation/repression of clock-controlled output genes (Ripperger et al., 2000; Gachon et al., 2006). To explore whether the products of clock genes and/or clock-controlled output genes affect the expression of CYP2D6, we investigated the effects of the transfection of expression plasmids encoding CLOCK, BMAL1, PER2, CRY1, DEC2, DBP, E4BP4, RORα, or REV-ERBα on the mRNA level of CYP2D6 in HepG2 cells. Cells were also transfected with expression vectors coding HNF4α or C/EBPα as a positive control (Cairns et al., 1996; Jover et al., 1998). As shown in Fig. 2A, the mRNA level of CYP2D6 was elevated significantly when cells were transfected with HNF4α or C/EBPα expression plasmids (P < 0.05 for both), whereas transfection of cells with DEC2 significantly suppressed the endogenous expression of CYP2D6 mRNA (P < 0.05). Consistent with these findings, transfection of HepG2 cells with siRNA against HNF4α or C/EBPα resulted in an obvious reduction of CYP2D6 protein levels (Fig. 2B), whereas transfection with DEC2 siRNA increased CYP2D6 protein abundance. Among the products of clock genes and/or clock-controlled output genes, DEC2 seems to act as a repressor of CYP2D6. In this experiment, the protein levels of C/EBPα were decreased by transfection with the siRNA against HNF4α, but transfection of C/EBPα siRNA had little effect on the protein levels of HNF4α. These results also suggest that HNF4α may play a predominant role in hepatic C/EBPα expression.
Transcriptional Regulation of the CYP2D6 Gene by HNF4α and DEC2.
To investigate the transrepression mechanism of the CYP2D6 gene by DEC2, we performed the luciferase reporter assay using native or mutated CYP2D6 reporter constructs. Cotransfection of CYP2D6-Luc with HNF4α resulted in a 6-fold increase in promoter activity (Fig. 3B). A similar increase in promoter activity was also detected when CYP2D6-Luc was cotransfected with C/EBPα. These transactivation effects of HNF4α and C/EBPα were abolished when their binding sites were mutated (Fig. 3B). DEC2 repressed either HNF4α- or C/EBPα-mediated transactivation of CYP2D6. Although cotransfection with 1.0 μg of DEC2 plasmid partially inhibited HNFα-mediated transcription, transfection with the same amount of DEC2 plasmid suppressed the C/EBPα-induced promoter activity of CYP2D6 at the basal level (Fig. 3C). These results suggest that DEC2 represses the transcription of CYP2D6 by mainly interacting with C/EBPα.
HDAC is often involved in transcriptional repression by bHLH transcription factors, and it has been reported that some actions of DEC2 are suppressed by HDAC inhibitors (Sun and Taneja, 2000; Garriga-Canut et al., 2001). We therefore examined the effects of the HDAC inhibitor TSA on DEC2-mediated repression. TSA dose dependently restored the DEC2-mediated repression of C/EBPα-induced CYP2D6 transactivation (Fig. 4A). The results of the immunoprecipitation assay revealed that DEC2 precipitated together with C/EBPα (Fig. 4B). Endogenously expressed HDAC1 proteins in HepG2 cells were also coimmunoprecipitated together with DEC2.
Although transfection of HepG2 cells with DEC2 expression vectors had little effect on the binding amounts of C/EBPα to its binding site in the CYP2D6 promoter, the treatment-enhanced formation of the HDAC1-chromatin complex (Fig. 4C). The DEC2-enhanced HDAC1-chromatin formation seemed to be dependent on the C/EBPα protein, because no DNA bands were detected in HDAC1-immunoprecipitated chromatin by using primer pairs for amplifying the CYP2D6 promoter region that does not contains C/EBPα binding site (Fig. 4C). These results suggest that DEC2 promotes HDAC1 recruitment on the CYP2D6 promoter through the protein-protein interaction with C/EBPα. The correlation between the interaction of these proteins and the transcriptional regulation of CYP2D6 suggests that suppressive action of DEC2 on C/EBPα-mediated transactivation of CYP2D6 is attributable to the sustained recruitment of HDAC1.
Role of DEC2 in the Circadian Regulation of CYP2D6 in Serum-Shocked HepG2 Cells.
The levels of DEC2 mRNA also showed obvious 24-h oscillation in serum-shocked HepG2 cells (Fig. 5A). The rhythmic phase of DEC2 mRNA was similar to Per2 mRNA oscillation (Takiguchi et al., 2007). The oscillation in the expression of DEC2 protein was nearly antiphase to that in the mRNA levels of CYP2D6 (Fig. 5B), but the protein levels of C/EBPα and HDAC1 failed to show obvious circadian oscillation in serum-shocked cells. Immunoprecipitation experiments revealed that in serum-shocked HepG2 cells, the amount of DEC2 associated with C/EBPα increased at the time corresponding to the trough of the CYP2D6 mRNA expression (Fig. 5C), whereas a decrease in the amount of DEC2-C/EBPα complex almost matched the peak of CYP2D6 expression. As shown in Fig. 2B, transfection of cells with siRNA against DEC2 caused an elevation of CYP2D6 protein levels. Furthermore, treatment of cells with DEC2 siRNA also prevented serum shock-induced oscillation in the expression of CYP2D6 mRNA (Fig. 5D). These results indicate that DEC2 protein interacts with C/EBPα in a time-dependent manner. Time-dependent interactions may underlie the circadian expression of CYP2D6 in serum-shocked HepG2 cells.
Rhythmic Expression of Cyp2d9 Gene in Mouse Hepatocytes.
In the final set of experiments, we explored whether the expression of Cyp2d gene in the experimental animals also exhibited circadian oscillation. Computer-aided analysis identified putative C/EBPα-binding sites in the promoter region of the mouse Cyp2d9 gene, the murine homolog to human CYP2D6; however, putative binding sites were not found in the promoter region of the mouse Cyp2d22 gene. The mRNA levels of Cyp2d9, but not of Cyp2d22, showed significant 24-h oscillation not only in the primary cultured mouse hepatocytes (P < 0.05) (Fig. 6A) but also in the liver of mice (P < 0.05) (Fig. 6B). These findings suggest that putative C/EBPα-binding sites in the promoter region of Cyp2d9 gene are also functionally important for rhythmic expression of its mRNA.
Discussion
Members of the bHLH family of transcription factors have been shown to play critical roles in cellular differentiation, growth, apoptosis, hypoxia response, and circadian rhythm regulation (Honma et al., 2002; Miyazaki et al., 2002; Azmi et al., 2004; Takiguchi et al., 2007). DEC2 was originally found in rat brain (Rossner et al., 1997) and was subsequently identified in humans and mice (Rossner et al., 1997; Garriga-Canut et al., 2001). DEC2 is expressed in a variety of tissues (Lu et al., 1999; Fujimoto et al., 2001; Miyazaki et al., 2002); however, its role in the circadian regulation of hepatic metabolism has not been fully evaluated. In this study, we showed that DEC2 acts as a potent repressor of C/EBPα. The repressive action of DEC2 on C/EBPα seemed to be the underlying cause of circadian expression of CYP2D6 in serum-shocked HepG2 cells (Fig. 7). The expression levels of several types of P450s in HepG2 cells have been reported to be lower than those in primary human hepatocytes (Jover et al., 1998; Hara and Adachi, 2002; Westerink and Schoonen, 2007). However, considerable metabolic activity of CYP2D6 was detected in HepG2 cells, and the drug-metabolizing activity also varied in a circadian fashion.
A recent report also demonstrated that DEC2 interacts with C/EBPα to suppress the expression of C/EBPα-target genes (Gulbagci et al., 2009). Protein-protein interaction is dependent on both the bHLH domain and carboxyl-terminal region of DEC2 protein. Furthermore, the suppressive actions of DEC2 on C/EBPα-mediated transactivation are suggested to be caused by sustained recruitment of HDAC1. The results of the immunoprecipitation assay revealed that DEC2 interacted with C/EBPα, accompanied by the association with HDAC1. The formation complex with HDAC1 seemed to underlie the suppressive actions of DEC2 on C/EBPα-mediated transactivation. DEC2 also functions as a corepressor of retinoid X-receptors (Cho et al., 2009). Retinoid X-receptors heterodimerized with pregnane X receptors or constitutive androstane receptors to regulate the expression of CYP3A4 (Pascussi et al., 2000a,b; Chen et al., 2010). Therefore, DEC2 may also contribute to the circadian regulation of CYP3A4 gene in the serum-shocked HepG2 cells (Takiguchi et al., 2007).
CYP2D6, a member of the P450 superfamily, is responsible for the metabolism of approximately 25% of commonly prescribed drugs (Bertilsson and Dahl, 1996). The gene that encodes CYP2D6 has more than 90 variants (Ingelman-Sundberg, 2005; Beverage et al., 2007). Such polymorphism leads to a variety of enzymatic activities and different phenotypes. In fact, the activity of CYP2D6 ranges from complete deficiency to excessive activity, potentially causing medication toxicity or therapeutic failure even at the recommended drug dosage (Zanger et al., 2004; Ingelman-Sundberg, 2005; Beverage et al., 2007). CYP2D6 polymorphism is therefore regarded as the reason for interindividual differences in the pharmacokinetics and pharmacodynamics of drugs. In addition to interindividual variation, the present findings using an in vitro model of the hepatic circadian clock suggested that there was also intraindividual variation in CYP2D6 activity. In serum-shocked HepG2 cells, significant 24-h oscillation was detected not only in the mRNA levels of CYP3A4 but also in its metabolic activity (Takiguchi et al., 2007). Daily variation in CYP3A4 activity in humans has been suggested by the fact that the pharmacokinetics of several drugs, which are mainly eliminated by CYP3A4 metabolism, vary according to their dosing times (Smith et al., 1986; Min et al., 1997). Although the CYP2D6-mediated drug metabolism in human liver may also vary depending on its dosing time, it has not been clarified whether drug-metabolic activity of CYP2D6 in human liver exhibits circadian oscillation. Further studies are required to investigate this point. The mRNA levels of Cyp2d9, the murine homolog to human CYP2D6, exhibited significant circadian oscillation in mouse liver. The oscillation of Cyp2d9 mRNA levels was nearly antiphase to that of DEC2 expression (Noshiro et al., 2004). Because computer-aided analysis identified putative C/EBPα-binding sites in the promoter region of the mouse Cyp2d9 gene, DEC2 may periodically repress the transcriptional activity of C/EBPα, thereby inducing the circadian expression of Cyp2d9 mRNA. Taken together, these findings suggest the possibility that the expression of CYP2D6 oscillates in human liver.
The individualization of pharmacotherapy has been achieved mainly by monitoring drug concentrations. Consequently, dosage adjustment is based on interindividual differences in drug pharmacokinetics; however, intraindividual as well as interindividual variability should be considered to aim for further improvement in rational pharmacotherapy, because the pharmacokinetics of many drugs also vary, depending on rhythmicity in absorption, distribution, metabolism, and elimination (Ohdo et al., 2010). Although the contribution of 24-h variation in CYP2D6 expression to drug metabolism should be clarified, our results suggest a mechanism underlying the dosing time-dependent differences in the pharmacokinetics of drugs and provide a molecular link between the circadian clock and xenobiotic metabolism.
Authorship Contributions
Participated in research design: Matsunaga, Koyanagi, and Ohdo.
Conducted experiments: Matsunaga, Inoue, Kusunose, Kakimoto, Hamamura, Hanada, Toi, and Koyanagi.
Contributed new reagents or analytic tools: Hanada, Toi, Sato, Fujimoto, and Koyanagi.
Performed data analysis: Matsunaga, Inoue, Yoshiyama, and Koyanagi.
Wrote or contributed to the writing of the manuscript: Matsunaga, Inoue, Koyanagi, and Ohdo.
Acknowledgments
We are indebted to Dr. N. Watanabe (Daiichi Sankyo Co., Ltd., Tokyo, Japan) and M. Iwasaki (Daiichi Sankyo RD Novare Co., Ltd., Tokyo, Japan) for technical support.
Footnotes
This study was partially supported by the Japan Society for the Promotion of Science [Grant-in-Aid for Scientific Research (B) 21390047, Grant-in-Aid for Challenging Exploratory Research 21659041]; the Mandom International Research Grants on Alternative to Animal Experiments; and The Cosmetology Research Foundation.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- DBP
- D site-binding protein
- E4BP4
- E4 promoter-binding protein 4
- P450
- cytochrome P450
- DEC2
- differentiated embryo chondrocyte-2
- bHLH
- basic helix-loop-helix
- HNF4α
- hepatic nuclear factor-4α
- C/EBPα
- CCAAT enhancer binding protein-α
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- MAMC
- 7-methoxy-4-(aminomethyl) coumarin
- RORα
- retinoic orphan receptor-α
- siRNA
- small interfering RNA
- TSA
- trichostatin A
- HDAC1
- histone deacetylase-1
- HAMC
- 7-hydroxy-4-aminomethylcoumarin.
- Received October 17, 2011.
- Accepted February 21, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics