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. 2017 Apr;45(4):346–352. doi: 10.1124/dmd.116.074013

Cholic Acid Feeding Leads to Increased CYP2D6 Expression in CYP2D6-Humanized Mice

Xian Pan 1, Rebecca Kent 1, Kyoung-Jae Won 1, Hyunyoung Jeong 1,✉
PMCID: PMC5363697  PMID: 28153841

Abstract

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme, but the factors governing transcriptional regulation of its expression remain poorly understood. Based on previous reports of small heterodimer partner (SHP) playing an important role as a transcriptional repressor of CYP2D6 expression, here we investigated how a known upstream regulator of SHP expression, namely cholestasis triggered by cholic acid (CA) feeding in mice, can lead to altered CYP2D6 expression. To this end, CYP2D6-humanized (Tg-CYP2D6) mice were fed with a CA-supplemented or control diet for 14 days, and hepatic expression of multiple genes was examined. Unexpectedly, CA feeding led to insignificant changes in SHP mRNA but also to significant (2.8-fold) decreases in SHP protein levels. In silico analysis of the SHP gene regulatory region revealed a putative binding site for a microRNA, miR-142-3p. Results from luciferase reporter assays suggest that miR-142-3p targets the SHP gene. Hepatic expression of miR-142-3p was significantly increased in CA-fed mice (∼5-fold), suggesting a potential role of miR-142-3p in the regulation of SHP expression in cholestasis. The decreased SHP protein levels were accompanied by increased expression and activity of CYP2D6 in the liver of CA-fed mice. These results suggest potential roles of differential hepatic levels of bile acids in the transcriptional regulation of CYP2D6 expression.

Introduction

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme, responsible for eliminating ∼20% of marketed drugs such as opioids (e.g., codeine) and antidepressants (e.g., citalopram). Additionally, CYP2D6 expressed in the brain has been implicated in the pathogenesis of Parkinson disease and the synthesis of neurotransmitters (Yu et al., 2003; Lu et al., 2014). Despite its diverse physiologic functions, the regulation of CYP2D6 expression remains poorly understood. Cairns et al. (1996) reported that the CYP2D6 promoter is transactivated by hepatocyte nuclear factor 4α (HNF4α), the most abundant DNA-binding protein in the liver and a master regulator of hepatic functions (Chandra et al., 2013). Recently, we have identified the transcriptional repressor small heterodimer partner (SHP) as a novel regulator of CYP2D6 expression; SHP represses HNF4α transactivation of the CYP2D6 promoter, and SHP knockdown (using small interfering RNA) led to increased CYP2D6 expression in CYP2D6-humanized transgenic (Tg-CYP2D6) mice (Koh et al., 2014). Furthermore, a chemical inducer of SHP expression (GW4064) repressed CYP2D6 expression in Tg-CYP2D6 mice as well as in human hepatocytes (Pan et al., 2015).

SHP is a representative target gene of bile acid sensor farnesoid X receptor (FXR). Along with FXR, SHP serves as a key player in the maintenance of bile acid homeostasis, especially in patients with cholestasis. Cholestasis is a chronic liver disease that is characterized by an interruption of bile flow, enlarged bile acid pool size, and altered bile acid composition. When hepatic bile acid levels rise, bile acids bind to FXR, and the ligand-activated FXR transactivates the SHP promoter via a FXR response element in the proximal promoter region (Goodwin et al., 2000; Chanda et al., 2008). SHP in turn represses the transcription of genes involved in bile acid synthesis (e.g., Cyp7a1 and Cyp8b1) by interfering with the actions of transcriptional activators of the genes (Goodwin et al., 2000; Zhang and Chiang, 2001).

The goal of this study is to define the role of SHP and bile acids in the regulation of CYP2D6 expression by examining the effects of cholestasis on hepatic CYP2D6 expression. As a cholestasis model, Tg-CYP2D6 mice were fed a cholic acid (CA)–supplemented diet for over 1 week (Barone et al., 1996; Fickert et al., 2001; Rost et al., 2003; Teng and Piquette-Miller, 2007). The treatment was known to increase bile acid pool size by 2-fold and to replace ∼90% of bile acids with CA (Fickert et al., 2001), recapitulating the features of cholestatic conditions in humans (van Berge Henegouwen et al., 1976). Our results revealed unexpected directional changes in SHP expression levels and differential regulation of CYP2D6 and Cyp7a1/Cyp8b1 expression in Tg-CYP2D6 mice upon CA feeding.

Materials and Methods

Animals.

Tg-CYP2D6 mice were previously described (Corchero et al., 2001). Adult male mice (8 weeks of age and weighing 20–25 g) were used for the experiments. Mice were fed with normal chow or 1% (w/w) CA-supplemented diet (Teklad Laboratory Animal Diets; Envigo, Indianapolis, IN). After feeding for 14 days, mice were sacrificed, and blood and liver tissue samples were collected. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago.

Chemicals and Reagents.

Debrisoquine, (±)-4-hydroxydebrisoquin, and paraxanthine were purchased from Biomol (Plymouth Meeting, PA). β-NADH was purchased from Sigma-Aldrich (St. Louis, MO).

Immunoprecipitation and Western Blot.

SHP protein was detected by using immunoprecipitation followed by Western blotting. Liver tissues were lysed in radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP40, 1 mM EDTA, complete protease inhibitor cocktail; Roche, Mannheim, Germany). For immunoprecipitation, equal amounts of lysates were incubated with the indicated antibody overnight at 4°C and then incubated with Dynabeads (Invitrogen, Carlsbad, CA) for 2 hours. After washing with phosphate-buffered saline, precipitated proteins were extracted from dynabeads by boiling in 1× sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue). Western blotting was performed as described previously (Koh et al., 2014). CYP2D6 (Corning, Corning, NY) and SHP (Santa Cruz Biotechnology, Dallas, TX) protein expression levels were determined by using the respective antibodies. The results of validating SHP detection by using two SHP antibodies (i.e., H160 and Q14) are shown in Supplemental Fig. 1.

Cell Culture.

HEK293T cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (Gemini, West Sacramento, CA), 10 mM HEPES, 100 μM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA).

Plasmids.

To construct a reporter vector for microRNA (miRNA) binding to the 3′-untranslated region (UTR) of SHP, the putative miRNA-142-3p (miR-142-3p) binding region of mouse SHP (110–129) was cloned into the pMIR-REPORT luciferase vector (Life Technologies, Carlsbad, CA) by using 5′-phosphorylated primers listed in Supplemental Table 1. Additionally, the pMIR-REPORT vector harboring the complementary sequence of miR-142-3p was constructed by using a primer set (Supplemental Table 1). miR-142-3p mimic and the control (i.e., negative control 1) were purchased from ThermoFisher Scientific (Walthman, MA).

Luciferase Reporter Assay.

HEK293T cells were seeded in 24-well plates at a density of 1 × 105 cells/ml. On the next day, the cells were transfected with 250 ng of luciferase construct, 30 nM miR-142-3p mimic or null control mimic, and 10 ng of Renilla vector (Promega, Madison, WI) using Fugene HD transfection reagent (Promega) according to the manufacturer protocol. The transfected cells were grown for 48 hours and were harvested for the determination of luciferase activity using a Dual-Luciferase Reporter Assay Kit (Promega). At least two independent experiments were performed in triplicate.

Measurement of CYP2D6 Activity.

Hepatic S9 fractions were prepared as described previously (Felmlee et al., 2008; Koh et al., 2014). S9 fractions were incubated with debrisoquine (a CYP2D6 probe substrate; 200 μM) based on the report that endogenous mouse CYP2Ds play minor roles in debrisoquine hydroxylation (Koh et al., 2014). The concentration of 4-hydroxydebrisoquine was determined by liquid chromatography-tandem mass spectrometry (1200 HPLC; Agilent; interfaced with Qtrap 3200; Applied Biosystems) using an electrospray ion source. Multiple reaction monitoring data acquisition was employed: m/z 192.3/132.2 for 4-hydroxydebrisoquine and 181.1/124.1 for the internal standard paraxanthine (Koh et al., 2014).

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction.

Total RNA was isolated from mouse liver tissues using TRIzol (Life Technologies) and converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Using the cDNA as template, quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the StepOnePlus Real-Time PCR System (ThermoFisher Scientific) and primers listed in Supplemental Table 1. The results were expressed as fold changes under treatment using the gene expression levels normalized to those of GAPDH (2-ΔΔCt method). For analysis of mature miR-142-3p, total RNA was converted to cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies). The specific primers and probes used for miR-142-3p and snoRNA202 were listed in Supplemental Table 1. The results are expressed as fold changes under treatment using the gene expression levels normalized to those of snoRNA202 (2-ΔΔCt method).

Chromatin Immunoprecipitation Assays.

Chromatin immunoprecipitation (ChIP) assays were performed as previously described, with minor modifications (Koh et al., 2014). Briefly, livers were finely minced and incubated in PBS containing 1% formaldehyde at room temperature for 15 minutes, and glycine was added to stop the crosslinking reaction. Cell pellets were resuspended in hypotonic buffer (15 mM HEPES, pH 7.9, 60 mM KCl, 2 mM EDTA, 0.5% bovine serum albumin, 0.15 mM spermine, 0.5 mM spermidine, 0.32 M sucrose) and lysed by homogenization. Nuclei were pelleted and resuspended in nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1% SDS). The samples were sonicated to shear DNA to the length ranging from 100 to 500 base pairs (bp). After centrifugation, the chromatin sample was immunoprecipitated with 2 μg of antibody [HNF4α, sc-6556x; RNA polymerase II (Pol II), sc-899x; SHP, sc-30169; Santa Cruz Biotechnology] or IgG (normal goat IgG, sc-2028; normal rabbit IgG, sc-2027; Santa Cruz Biotechnology) at 4°C overnight. The immune complex was collected, the magnetic beads were extensively washed, and the bound chromatin was eluted. Genomic DNA was purified by PCR Clean-Up Kit (Promega) and was used as a template for PCR. Primer sequences are listed in Supplemental Table 1.

Alkaline Phosphatase and Alanine Aminotransferase Activities.

Plasma alkaline phosphatase (ALP) and alanine aminotransferase (ALT) levels were measured by chemistry analyzer (AU 680; Olympus, Center Valley, PA).

Cytochrome P450 Reductase Activity.

Cytochrome P450 reductase (CPR) activity in hepatic S9 fractions was measured by using the Cytochrome c Reductase (NADPH) Assay Kit (catalog #CY0100; Sigma-Aldrich) following the manufacturer protocol.

Cytochrome-b5 Concentration.

The contents of cytochrome-b5 (Cyb5) in hepatic S9 fractions were determined by measuring the differential absorbance between NADH-reduced and oxidized S9 fractions as previously described (Venkatakrishnan et al., 2000; Gan et al., 2009).

Statistical Analysis.

Values were reported as the mean ± S.D. Comparison between the control and CA group was made by using Student’s t test.

Results

CA Feeding Decreases SHP Expression Via miR-142-3p in Mice.

To determine the effects of cholestasis on hepatic gene regulation, CA or normal chow (control) was fed to mice for 14 days to establish cholestatic conditions. Plasma levels of ALP (a marker for cholestasis (Krones et al., 2015) and ALT (a marker for liver injury) were significantly increased in CA-fed mice (Fig. 1), as previously reported (Barone et al., 1996; Fickert et al., 2001; Rost et al., 2003; Teng and Piquette-Miller, 2007). CA feeding had insignificant effects on hepatic expression of Hnf4α (data not shown). Interestingly, the expression levels of hepatic Shp mRNA did not differ between the CA-fed and control groups (Fig. 2A), contrary to the expectation that CA feeding and subsequent cholestasis would lead to increased Shp expression. On the other hand, the Western blot results showed that SHP protein was markedly decreased in CA-fed mice compared with the control mice (Fig. 2B).

Fig. 1.

Fig. 1.

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CA feeding induces cholestasis in Tg-CYP2D6 mice. Tg-CYP2D6 mice were fed with normal chow (control) or 1% (w/w) CA–supplemented diet (CA) for 14 days (n = 4 mice/group). ALP and ALT activities were measured in mouse Plasma. *p < 0.05.

Fig. 2.

Fig. 2.

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CA feeding leads to decreased SHP protein level in Tg-CYP2D6 mice. Tg-CYP2D6 mice were fed with normal chow (control) or 1% (w/w) CA-supplemented diet for 14 days (n = 4 mice/group). (A) Hepatic Shp mRNA expression level was determined by qRT-PCR. NS, not statistically significant. (B) SHP protein expression level was determined by immunoprecipitation (using H160 antibody against SHP) followed by Western blotting (using Q14 antibody against SHP). Liver tissue lysate from Shp-null mice was used as a negative control. **p < 0.01. (C) Schematic representation of miR-142-3p putative binding sites in Shp 3′-UTR. (D) Hepatic miR-142-3p expression level was determined by qRT-PCR. **p < 0.01. (E) HEK293T cells were cotransfected with luciferase (LUC) reporter vector, Renilla expression vector, and miR-142-3p expression vector (or negative control vector), and dual luciferase assays were performed. The experiment was performed in triplicate. **p < 0.01, versus negative control-transfected cells.

Post-transcriptional regulation of gene expression via miRNA is known to cause a disconnection between mRNA and protein levels (van Rooij, 2011), and previous studies have shown that cholestasis leads to differential expression of multiple hepatic miRNAs (Hirota et al., 2008; Rieger et al., 2013). In silico analysis of the 3′-UTR of Shp (by using a combination of DIANAmT, miRanda, miRDB, miRWalk, and Targetscan) revealed putative binding sites for miRNAs (i.e., miR-141-3p and miR142-3p) (miR142-3p shown in Fig. 2C). To determine whether CA feeding alters the expression levels of these miRNAs, their expression levels were measured by using qRT-PCR. The level of miR-142-3p was increased ∼5-fold in the livers of CA-fed mice compared with the control (Fig. 2D), whereas miR-141-3p expression did not differ between the groups (data not shown). To validate whether miR-142-3p regulates Shp post-transcriptionally, we constructed a luciferase reporter vector that contains the putative miR-142-3p binding sequence of the Shp 3′-UTR. HEK293T cells were cotransfected with miR-142-3p mimic, luciferase vector, and Renilla expression vector, followed by measurement of luciferase activity. Overexpression of miR-142-3p led to a significant decrease in the activity of the Shp 3′-UTR-luciferase reporter (Fig. 2E). To examine whether miR-142-3p also modulates basal Shp expression, Raw264.7 cells (a mouse macrophage cell line where miR-142-3p expression was ∼9-fold higher than that in HepG2 cells; data not shown) were cotransfected with the antagomir of miR-142-3p (or negative control), luciferase vector, and Renilla expression vector, followed by measurement of luciferase activity. The results, however, showed that the miR-142-3p antagomir did not enhance the expression of the luciferase reporter (Supplemental Fig. 2), potentially due to differences in gene dose (e.g., much larger copy number of luciferase gene compared with miR-142-3p expression level). Together, our data suggest that CA feeding increases the hepatic miR-142-3p level, and this in turn leads to decreased SHP expression in mice.

Altered SHP Expression Exhibits Differential Effects on SHP Target Gene Expression in CA-Fed Mice.

To determine whether decreased SHP protein level leads to altered CYP2D6 expression, mRNA levels of CYP2D6 were measured in the livers of CA-fed mice. CA feeding led to a slight increase in mRNA expression of CYP2D6 (Fig. 3A). Conversely, contrary to the well-accepted notion that SHP is a transcriptional repressor of genes involved in bile acid synthesis, Cyp7a1 and Cyp8b1 expression was dramatically decreased in CA-fed mice (Fig. 3B). To identify potential mechanisms underlying this finding, the extent of SHP recruitment to the promoter regions of CYP2D6, Cyp7a1, and Cyp8b1 was analyzed via ChIP assays by using antibodies against SHP, HNF4α, and Pol II (a marker of transcription). The results showed that CA feeding increased the recruitment of HNF4α and Pol II to CYP2D6 promoter (Fig. 3C), consistent with elevated CYP2D6 expression upon CA feeding. This was accompanied by decreased recruitment of SHP to the CYP2D6 promoter, although the difference did not reach statistical significance due to large variability (Fig. 3C). Interestingly, despite the decreased SHP protein levels in CA-fed mice (Fig. 2B), CA feeding led to increased SHP (and decreased HNF4α and Pol II) recruitment to the promoters of Cyp7a1 (Fig. 3D) and Cyp8b1 (Supplemental Fig. 3), consistent with the decrease in Cyp7a1 and Cyp8b1 mRNA expression observed with CA feeding.

Fig. 3.

Fig. 3.

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Expression of CYP2D6 and Cyp7a1/Cyp8b1 is differentially regulated in CA-fed Tg-CYP2D6 mice. Tg-CYP2D6 mice were fed with normal chow (control) or 1% (w/w) CA-supplemented diet (CA) for 14 days (n = 4 mice/group). Hepatic mRNA expression levels of CYP2D6 (A) and Cyp7a1/Cyp8b1 (B) were determined by qRT-PCR. Recruitment of SHP, HNF4α, and Pol II to promoters of CYP2D6 (C) and Cyp7a1 (D) was analyzed by ChIP assay using mouse liver tissues. The locations of primers used in qRT-PCR for ChIP are shown. Values are presented as the mean ± S.D. (n = 4 mice/group). *p < 0.05; **p < 0.01. NS, not statistically significant.

CA Feeding Leads to Increased CYP2D6 Activity in Mice.

To determine whether increased CYP2D6 mRNA expression leads to increased protein and enzyme activity levels, S9 fractions were isolated from the livers of CA-fed or control mice. Western blot results showed that CYP2D6 protein levels in the S9 fraction were increased in CA-fed mice (Fig. 4A) to a greater extent than the increase in mRNA levels. The production rate of 4-hydroxylated debrisoquine in the S9 fractions (determined as a marker for CYP2D6 activity) was also significantly higher in CA-fed mice (Fig. 4B) but exhibited large variability. Considering that CYP2D6 activity can be regulated by the rate of electron transfer from CPR and Cyb5 (Henderson et al., 2015), we measured the activity of CPR and the content of Cyb5 in S9 fractions. Neither CPR activity nor Cyb5 content in hepatic S9 fractions differed between the control and CA-fed groups (Fig. 4C). Together, these results indicate that CA feeding increases CYP2D6 activity in Tg-CYP2D6 mice.

Fig. 4.

Fig. 4.

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CA feeding increases CYP2D6-mediated drug metabolism in Tg-CYP2D6 mice. Tg-CYP2D6 mice were fed with normal chow (control) or 1% (w/w) CA-supplemented diet (CA) for 14 days (n = 4 mice/group). (A) S9 fractions were prepared from the liver tissues, and protein levels of CYP2D6 in the S9 fractions were determined by Western blot. A gel image (bottom) and the quantified band intensities (top) are shown. **p < 0.01. (B) CYP2D6 activity was measured in hepatic S9 fractions using debrisoquine (200 μM) as a CYP2D6 probe drug. Data are shown as the metabolite production rate in pmoles per minute per milligram protein. *p < 0.05. (C) CPR activity and Cyb5 contents were measured in hepatic S9 fractions. NS, not statistically significant.

Discussion

We have previously shown that SHP is a transcriptional repressor of CYP2D6 expression (Koh et al., 2014), and activation of the FXR and SHP pathways by using a synthetic FXR agonist leads to decreased CYP2D6 expression and activity (Pan et al., 2015). Bile acids are endogenous activators of FXR that are capable of upregulating SHP within hours (Fang et al., 2007; Miao et al., 2009); however, it remains unclear how chronically elevated concentrations of bile acids (e.g., in cholestatic conditions) affect SHP expression/activity and thus its regulation of CYP2D6 expression. In this study, we employed CA feeding in mice to mimic cholestatic conditions and unexpectedly found that CA feeding decreased SHP protein levels, thus increasing CYP2D6 expression and activity.

In this study, mRNA expression levels of SHP were similar between the control and CA-fed mice, but SHP protein expression was decreased upon CA feeding. The lack of SHP induction by bile acids was also observed in a previous study where CA feeding for 12 weeks had insignificant effects on mRNA levels of SHP in mice (Wang et al., 2003), suggesting that cholestasis by CA feeding may have differential effects on SHP expression depending on the duration of CA feeding. Indeed, SHP is known to repress its own expression by inhibiting the action of transcriptional activators on the SHP promoter (Goodwin et al., 2000). Also, bile acids were shown to phosphorylate CCAAT-enhancer-binding protein α (via activation of phosphatidylinositol 3 kinase signaling), which subsequently binds to the SHP promoter and represses the promoter activity (Benet et al., 2015). It appears likely that the 2-week time point used in this study (to examine long-term cholestasis effects on CYP2D6 expression) allowed negative transcriptional regulators of SHP to be activated. Interestingly, despite the lack of difference in SHP mRNA levels between the CA-fed and control groups, SHP protein levels were significantly decreased in CA-fed mice. This is potentially attributable to the action of miR-142-3p. CA feeding significantly increased hepatic levels of miR-142-3p, which is consistent with a previous report (Rieger et al., 2013). A sequence complementary to miR-142-3p was identified within the 3′-UTR region of Shp mRNA, and the overexpression of miR-142-3p significantly repressed the expression of the luciferase gene that is flanked by the Shp 3′-UTR. Considering the pleiotropic roles of SHP in repressing fibrosis and tumorigenesis in the liver (Zhang et al., 2011), upregulation of miR-142-3p and subsequent downregulation of SHP in cholestatic livers may provide insights for a better understanding of the liver damage that accompanies cholestasis.

Our study showed that CA feeding led to increased CYP2D6 expression and activity in mice. Interestingly, however, the magnitude of the increase in CYP2D6 protein and activity in the S9 fraction was greater than the changes in CYP2D6 mRNA levels (i.e., ∼1.5-fold versus ∼3.4-fold increases in mRNA and protein/activity levels, respectively). CYP2D6 mRNA has a very short 3′-UTR (89 bp). This is much shorter than the average length of the 3′-UTRs of most (if not all) miRNA target genes (i.e., 1730 bp) (Hu, 2009), suggesting that the involvement of miRNAs in CYP2D6 regulation is unlikely. Furthermore, in silico prediction by using several programs (miRanda, picTar, and TargetScan) did not reveal any putative miRNA binding sites in 3′-UTR of CYP2D6 mRNA (data not shown). The underlying mechanisms for the disconnect between CYP2D6 mRNA and protein/activity levels remain to be elucidated.

Previously, we demonstrated that CYP2D6 expression and activity were repressed in an ethinylestradiol (EE2)-induced cholestasis mouse model (Pan and Jeong, 2015). On the other hand, we observed enhanced CYP2D6 expression and activity in this study when CA feeding (to mice) was used as a model for cholestatic conditions. Furthermore, the SHP protein level was higher in the EE2-induced cholestasis model (Pan and Jeong, 2015), whereas it decreased in CA-fed mice, suggesting that the differential regulation of SHP expression may underlie the apparent discrepancy in the directional changes in CYP2D6 expression/activity. While both cholestatic mouse models lead to significant increases in intrahepatic bile acid levels, they are distinct with respect to certain pathologies associated with cholestasis, such as altered bile flow. Bile acids are surfactants by nature (Begley et al., 2005) and also modulate the expression of intestinal genes involved in local innate immune functions (Vavassori et al., 2009) or hepatic nutrient metabolism (Jiang et al., 2015). The flow of bile from the gallbladder to the gut is critical in preventing intestinal bacteria overgrowth (and bacterial translocation to the liver) and maintaining systemic nutrient homeostasis. In intrahepatic cholestasis (including EE2-induced cholestasis) and biliary obstruction, bile flow was decreased (Yamamoto et al., 2006), whereas the oral administration of bile acids (such as ursodeoxycholic acid and CA) induces bile flow (Ding et al., 1993; Fickert et al., 2001). These differences in bile flow may lead to the establishment of a distinct gut microbiota environment and differential hepatic functions (Slocum et al., 1992; Ding et al., 1993). Indeed, our preliminary results showed significant increases in hepatic expression of inflammatory marker genes (i.e., tumor necrosis factor-α, interleukin-1β, and interleukin-6) in the mice with EE2-induced cholestasis (which often accompanies intestinal bacteria overgrowth) but not in CA-fed mice (data not shown). Whether and how the altered gut microbiota modulates hepatic regulation of SHP and CYP2D6 expression remains to be further investigated.

SHP is a transcriptional repressor of genes encoding enzymes mediating the synthesis of bile acids, Cyp7a1, and Cyp8b1. Our results showed that CA feeding for 2 weeks led to decreased SHP protein expression. However, despite decreased SHP protein levels in CA-fed mice, SHP recruitment to the promoter regions of Cyp7a1 and Cyp8b1 (but not of CYP2D6) increased in these mice. This finding is in part consistent with previous reports (Kemper et al., 2004; Fang et al., 2007) that bile acids induce the closure of the chromatin region harboring the Cyp7a1 gene by recruiting histone deacetylase and histone methyltransferase G9a. Indeed, we found increased histone methylation of Cyp7a1 and Cyp8b1 promoter regions in the CA-fed mouse liver (by using ChIP assays; data not shown). The mechanism through which bile acids trigger the chromatin remodeling of promoter regions in a gene-specific manner remains unclear.

In conclusion, CA feeding in Tg-CYP2D6 mice led to decreased SHP protein expression, potentially via the upregulation of miR142-3p. This was accompanied by increased CYP2D6 expression and activity (Fig. 5). The results highlight the important role of SHP in the regulation of CYP2D6 expression and provide a better understanding of factors modulating the basal levels of CYP2D6-mediated drug metabolism.

Fig. 5.

Fig. 5.

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Working model for CYP2D6 regulation in CA feeding-induced cholestasis. CA feeding (for 2 weeks) leads to increased FXR activity on the Shp promoter. SHP is known to negatively regulate its own expression. Together, this causes minor (if any) changes in Shp mRNA levels. CA feeding also enhances the expression of miR-142-3p that targets SHP. CA feeding increases CYP2D6 protein amounts in hepatic S9 fractions by unknown mechanisms, subsequently leading to greater CYP2D6-mediated drug metabolism. Red arrows represent directional changes in its hepatic levels in CA-fed Tg-CYP2D6 mice.

Acknowledgments

We thank Dr. Yoon Kwang Lee for providing liver tissues from Shp-null mice.

Abbreviations

ALP

alkaline phosphatase

ALT

alanine aminotransferase

CA

cholic acid

ChIP

chromatin immunoprecipitation

CPR

cytochrome P450 reductase

Cyb5

cytochrome-b5

CYP2D6

cytochrome P450 2D6

EE2

ethinylestradiol

FXR

farnesoid X receptor

HNF4α

hepatocyte nuclear factor 4α

miRNA

microRNA

Pol II

RNA polymerase II

qRT-PCR

quantitative real-time polymerase chain reaction

SHP

small heterodimer partner

UTR

untranslated region

Authorship Contributions

Participated in research design: Pan, Kent, Won, and Jeong.

Conducted experiments: Pan, Kent, and Won.

Performed data analysis: Pan, Kent, Won, and Jeong.

Wrote or contributed to the writing of the manuscript: Pan, Kent, Won, and Jeong.

Footnotes

This work was supported by the National Institutes of Health [Grant R01-GM-112746].

Inline graphicThis article has supplemental material available at dmd.aspetjournals.org.

References

  1. Barone M, Francavilla A, Polimeno L, Ierardi E, Romanelli D, Berloco P, Di Leo A, Panella C. (1996) Modulation of rat hepatocyte proliferation by bile salts: in vitro and in vivo studies. Hepatology 23:1159–1166. [DOI] [PubMed] [Google Scholar]
  2. Begley M, Gahan CGM, Hill C. (2005) The interaction between bacteria and bile. FEMS Microbiol Rev 29:625–651. [DOI] [PubMed] [Google Scholar]
  3. Benet M, Guzmán C, Pisonero-Vaquero S, García-Mediavilla MV, Sánchez-Campos S, Martínez-Chantar ML, Donato MT, Castell JV, Jover R. (2015) Repression of the nuclear receptor small heterodimer partner by steatotic drugs and in advanced nonalcoholic fatty liver disease. Mol Pharmacol 87:582–594. [DOI] [PubMed] [Google Scholar]
  4. Cairns W, Smith CA, McLaren AW, Wolf CR. (1996) Characterization of the human cytochrome P4502D6 promoter. A potential role for antagonistic interactions between members of the nuclear receptor family. J Biol Chem 271:25269–25276. [DOI] [PubMed] [Google Scholar]
  5. Chanda D, Park JH, Choi HS. (2008) Molecular basis of endocrine regulation by orphan nuclear receptor small heterodimer partner. Endocr J 55:253–268. [DOI] [PubMed] [Google Scholar]
  6. Chandra V, Huang P, Potluri N, Wu D, Kim Y, Rastinejad F. (2013) Multidomain integration in the structure of the HNF-4α nuclear receptor complex. Nature 495:394–398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Corchero J, Granvil CP, Akiyama TE, Hayhurst GP, Pimprale S, Feigenbaum L, Idle JR, Gonzalez FJ. (2001) The CYP2D6 humanized mouse: effect of the human CYP2D6 transgene and HNF4alpha on the disposition of debrisoquine in the mouse. Mol Pharmacol 60:1260–1267. [DOI] [PubMed] [Google Scholar]
  8. Ding JW, Andersson R, Soltesz V, Willén R, Bengmark S. (1993) The role of bile and bile acids in bacterial translocation in obstructive jaundice in rats. Eur Surg Res 25:11–19. [DOI] [PubMed] [Google Scholar]
  9. Fang S, Miao J, Xiang L, Ponugoti B, Treuter E, Kemper JK. (2007) Coordinated recruitment of histone methyltransferase G9a and other chromatin-modifying enzymes in SHP-mediated regulation of hepatic bile acid metabolism. Mol Cell Biol 27:1407–1424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Felmlee MA, Lon HK, Gonzalez FJ, Yu AM. (2008) Cytochrome P450 expression and regulation in CYP3A4/CYP2D6 double transgenic humanized mice. Drug Metab Dispos 36:435–441. [DOI] [PubMed] [Google Scholar]
  11. Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Pojer C, Zenz R, Lammert F, Stieger B, Meier PJ, Zatloukal K, et al. (2001) Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 121:170–183. [DOI] [PubMed] [Google Scholar]
  12. Gan L, von Moltke LL, Trepanier LA, Harmatz JS, Greenblatt DJ, Court MH. (2009) Role of NADPH-cytochrome P450 reductase and cytochrome-b5/NADH-b5 reductase in variability of CYP3A activity in human liver microsomes. Drug Metab Dispos 37:90–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, et al. (2000) A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6:517–526. [DOI] [PubMed] [Google Scholar]
  14. Henderson CJ, McLaughlin LA, Scheer N, Stanley LA, Wolf CR. (2015) Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo. Mol Pharmacol 87:733–739. [DOI] [PubMed] [Google Scholar]
  15. Hirota T, Takane H, Higuchi S, Ieiri I. (2008) Epigenetic regulation of genes encoding drug-metabolizing enzymes and transporters; DNA methylation and other mechanisms. Curr Drug Metab 9:34–38. [DOI] [PubMed] [Google Scholar]
  16. Hu Z. (2009) Insight into microRNA regulation by analyzing the characteristics of their targets in humans. BMC Genomics 10:594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jiang C, Xie C, Lv Y, Li J, Krausz KW, Shi J, Brocker CN, Desai D, Amin SG, Bisson WH, et al. (2015) Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction. Nat Commun 6:10166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kemper JK, Kim H, Miao J, Bhalla S, Bae Y. (2004) Role of an mSin3A-Swi/Snf chromatin remodeling complex in the feedback repression of bile acid biosynthesis by SHP. Mol Cell Biol 24:7707–7719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koh KH, Pan X, Shen HW, Arnold SL, Yu AM, Gonzalez FJ, Isoherranen N, Jeong H. (2014) Altered expression of small heterodimer partner governs cytochrome P450 (CYP) 2D6 induction during pregnancy in CYP2D6-humanized mice. J Biol Chem 289:3105–3113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krones E, Erwa W, Trauner M, Fickert P. (2015) Serum alkaline phosphatase levels accurately reflect cholestasis in mice. Hepatology 62:981–983. [DOI] [PubMed] [Google Scholar]
  21. Lu Y, Peng Q, Zeng Z, Wang J, Deng Y, Xie L, Mo C, Zeng J, Qin X, Li S. (2014) CYP2D6 phenotypes and Parkinson’s disease risk: a meta-analysis. J Neurol Sci 336:161–168. [DOI] [PubMed] [Google Scholar]
  22. Miao J, Fang S, Lee J, Comstock C, Knudsen KE, Kemper JK. (2009) Functional specificities of Brm and Brg-1 Swi/Snf ATPases in the feedback regulation of hepatic bile acid biosynthesis. Mol Cell Biol 29:6170–6181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pan X, Jeong H. (2015) Estrogen-induced cholestasis leads to repressed CYP2D6 expression in CYP2D6-humanized mice. Mol Pharmacol 88:106–112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pan X, Lee YK, Jeong H. (2015) Farnesoid X receptor agonist represses cytochrome P450 2D6 expression by upregulating small heterodimer partner. Drug Metab Dispos 43:1002–1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rieger JK, Klein K, Winter S, Zanger UM. (2013) Expression variability of absorption, distribution, metabolism, excretion-related microRNAs in human liver: influence of nongenetic factors and association with gene expression. Drug Metab Dispos 41:1752–1762. [DOI] [PubMed] [Google Scholar]
  26. Rost D, Herrmann T, Sauer P, Schmidts HL, Stieger B, Meier PJ, Stremmel W, Stiehl A. (2003) Regulation of rat organic anion transporters in bile salt-induced cholestatic hepatitis: effect of ursodeoxycholate. Hepatology 38:187–195. [DOI] [PubMed] [Google Scholar]
  27. Slocum MM, Sittig KM, Specian RD, Deitch EA. (1992) Absence of intestinal bile promotes bacterial translocation. Am Surg 58:305–310. [PubMed] [Google Scholar]
  28. Teng S, Piquette-Miller M. (2007) Hepatoprotective role of PXR activation and MRP3 in cholic acid-induced cholestasis. Br J Pharmacol 151:367–376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van Berge Henegouwen GP, Brandt KH, Eyssen H, Parmentier G. (1976) Sulphated and unsulphated bile acids in serum, bile, and urine of patients with cholestasis. Gut 17:861–869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. van Rooij E. (2011) The art of microRNA research. Circ Res 108:219–234. [DOI] [PubMed] [Google Scholar]
  31. Vavassori P, Mencarelli A, Renga B, Distrutti E, Fiorucci S. (2009) The bile acid receptor FXR is a modulator of intestinal innate immunity. J Immunol 183:6251–6261. [DOI] [PubMed] [Google Scholar]
  32. Venkatakrishnan K, von Moltke LL, Court MH, Harmatz JS, Crespi CL, Greenblatt DJ. (2000) Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between the approaches. Drug Metab Dispos 28:1493–1504. [PubMed] [Google Scholar]
  33. Wang L, Han Y, Kim CS, Lee YK, Moore DD. (2003) Resistance of SHP-null mice to bile acid-induced liver damage. J Biol Chem 278:44475–44481. [DOI] [PubMed] [Google Scholar]
  34. Yamamoto Y, Moore R, Hess HA, Guo GL, Gonzalez FJ, Korach KS, Maronpot RR, Negishi M. (2006) Estrogen receptor alpha mediates 17alpha-ethynylestradiol causing hepatotoxicity. J Biol Chem 281:16625–16631. [DOI] [PubMed] [Google Scholar]
  35. Yu AM, Idle JR, Byrd LG, Krausz KW, Küpfer A, Gonzalez FJ. (2003) Regeneration of serotonin from 5-methoxytryptamine by polymorphic human CYP2D6. Pharmacogenetics 13:173–181. [DOI] [PubMed] [Google Scholar]
  36. Zhang M, Chiang JY. (2001) Transcriptional regulation of the human sterol 12alpha-hydroxylase gene (CYP8B1): roles of heaptocyte nuclear factor 4alpha in mediating bile acid repression. J Biol Chem 276:41690–41699. [DOI] [PubMed] [Google Scholar]
  37. Zhang Y, Hagedorn CH, Wang L. (2011) Role of nuclear receptor SHP in metabolism and cancer. Biochim Biophys Acta 1812:893–908. [DOI] [PMC free article] [PubMed] [Google Scholar]

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