Retinoids induce cytochrome P450 3A4 through RXR/VDR-mediated pathway

A panel of retinoids and carotenoids was screened as potential inducers of CYP3A4 through the RXR/VDR-mediated signaling pathway. Transient transfection assays revealed that 3 out of 12 retinoids screened transactivated RXR/VDR and induced CYP3A4 reporter activity. These three retinoids are the active metabolites of retinoids, 9-cis-retinal, 9-cis-retinoic acid (9-cis-RA), and all-trans-retinoic acid (all-trans-RA). 9-cis-RA and all-trans-RA preferentially transactivated the RXR/VDR heterodimers and RXR homodimers. Retinoids and VDR agonist 1, 25-dihydroxyvitamin D₃, but not PXR or CAR activator, could induce Cyp3a11 mRNA level in hepatocytes derived from PXR/CAR-double null mouse. Moreover, retinoids induced CYP3A4 enzyme activity in HepG2 human hepatoma and Caco-2 human colorectal adenocarcinoma cells. A direct role of retinoid-mediated CYP3A4 induction through RXR/VDR was proved by the results that 9-cis-retinal, 9-cis-RA, and all-trans-RA recruited RXR and VDR to CYP3A4 regulatory region pER6 (proximal everted repeat with a 6-nucleotide spacer) and dXREM (distal xenobiotic-responsive enhancer module). Thus, using various approaches, we have unequivocally demonstrated that retinoids transactivate RXR/VDR heterodimers and RXR homodimers and induce CYP3A expression at mRNA as well as enzyme activity levels in both liver and intestinal cells. It is possible that retinoids might alter endobiotic metabolism through CYP3A4 induction in vivo.     

Constitutive androstane receptor transactivates the hepatic expression of mouse Dhcr24 and human DHCR24 encoding a cholesterogenic enzyme 24-dehydrocholesterol reductase

Phenobarbital treatment has long been known to influence serum and hepatic cholesterol levels in rodents and humans. Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, mediates various biological actions of phenobarbital. We have thus investigated whether CAR transactivates cholesterogenic genes in livers. Activation of CAR in mouse livers and cultured human hepatocytes increased mRNA levels of mouse Dhcr24 and human DHCR24, both of which encode 24-dehydrocholesterol reductase (DHCR24) catalyzing the last step of cholesterol biosynthesis. CAR transactivated the expression of these genes in reporter assays with cultured hepatoma cells. Furthermore, we have identified a DR4 (direct repeat separated by 4 nucleotides) motif in the human DHCR24 distal promoter as a binding site of CAR/retinoid X receptor α (RXRα) heterodimer. We have also demonstrated that the heterodimer of pregnane X receptor (PXR)/ RXRα binds to the DR4 motif and that human DHCR24 reporter gene is transactivated by the ligand-activated PXR. These results suggest a role of xenobiotic-responsive nuclear receptor CAR, and also possibly PXR, in cholesterol biosynthesis in the liver of mice and humans.     

The CYP2B2 5' flank contains a complex glucocorticoid response unit

Rat CYP2B1 and CYP2B2 and mouse CYP2B10 are dramatically induced by phenobarbital (PB) in liver. PB responsiveness requires the constitutive androstane receptor (CAR). However, dexamethasone treatment can also induce CYP2B genes in both rat and mouse liver. Three regions have been shown to be involved in conferring dexamethasone responsiveness on CYP2B2 reporter constructs. They are the PB response unit, a functional glucocorticoid response element at −1.3 kb in the 5′ flank and a weak element in the basal promoter. We report here the identification, by deletion analysis of the CYP2B2 5′ flank, of new glucocorticoid response elements or accessory factor sites. Moreover, we show that CAR acts as an accessory factor in the dexamethasone response in vivo of CYP2B10 protein in mice, by increasing both the basal and induced levels. We propose a model to explain the dexamethasone responsiveness of the CYP2B2 gene in which induction is mediated by a complex glucocorticoid response unit.     

CAR-mediated up-regulation of CYP3A4 expression in LS174T cells by Chinese herbal compounds

The constitutive androstane receptor (CAR) is an orphan nuclear receptor which has been shown to participate in the activation of human CYP3A4, which metabolizes more than 50% of clinically used drugs. We investigated the effects of an array of compounds isolated from herbal medicines such as Rheum palmatum (Da Huang), Peucedanum praeruptorum Dunn (Qian Hu), Cortex Mori Radicis (Sang Bai Pi), Radix Asteris (Zi Wan), Salvia miltiorrhiza (Dan Shen), Polygonum cuspidatum Sieb. et Zucc (Hu Zhang), and Ginkgo biloba (Yin Xing) on the CAR-mediated transactivation of CYP3A4. The effect of herbal compounds on CYP3A4 expression was measured using a CYP3A4 luciferase reporter gene assay in transiently transfected human intestinal LS174T cells. The gene expression, protein expression, and catalytic activity of CYP3A4 in LS174T cells transfected with CAR were determined by using real-time PCR, Western blot analysis, and LC-MS/MS-based substrate assay. The study found that in CAR-transfected cells, praeruptorin A, C, and D significantly induced CYP3A4 luciferase activity, mRNA expression, and functional activity through the CAR-mediated pathway; conversely, induction was not found in untransfected cells. Our findings suggest that these herbal compounds can significantly up-regulate the CYP3A4 gene via the CAR-mediated pathway, which has important implications in herb-drug interactions.     

外源化合物对孕烷X受体和组成型雄烷受体介导的细胞色素P4503A4和2B6转录调节共转染体系的优化

目的优化孕烷X受体(hPXR)和组成型雄烷受体(hCAR)介导的细胞色素P450(CPY)3A4和CYP2B6诱导共转染体系,提高检测系统的灵敏度。方法利用invitrogen脂质体2000共同转染表达质粒hPXR/hCAR、报告基因质粒CPY3A4/CYP2B6和内参质粒pRL-TK到HepG-2细胞中。系统以hPXR的激动剂利福平,hCAR的激动剂CITCO为阳性对照组,以二甲基亚砜(DMSO)为溶剂阴性对照组。通过调整3种质粒的转染比例,以利福平/DMSO和CITCO/DMSO的比活值,即阳性药物的诱导倍数作为优化系统灵敏度的指标,分别获得最大比值以表示系统具有最佳灵敏度。结果当共转染体系比例为hPXR/hCAR表达质粒150ng、CPY3A4/CYP2B6报告基因质粒600ng、PLR-TK内参质50ng时,转染体系的检测灵敏度最高。结论针对所使用的转染细胞系和共转染质粒,通过优化质粒的转染比例可提高系统的灵敏度,优化的共转染系统可用于药物代谢酶诱导机制的研究。     

Involvement of CAR and PXR in the transcriptional regulation of CYP2B6 gene expression by ingredients from herbal medicines

Abstract

1.?Induction of hepatic drug-metabolizing enzymes can affect drug efficacy and cause toxicity. However, so far, limited information is available regarding the molecular mechanism how herbal medicines induce human CYP2B6, which metabolizes many of the clinically used therapeutics and activates several pro-carcinogens or toxicants. Accumulated evidence suggests that the human constitutive androstane receptor (hCAR) and the human pregnane X receptor (hPXR) play important roles in trans-activation of CYP2B6. In this study, we investigated the effects of 68 Chinese herbal ingredients on the receptor specificity of hPXR/hCAR-mediated CYP2B6 induction by luciferase reporter gene assays in transiently transfected HepG2 cells and on the expression of CYP2B6 in LS174T cells.

2.?The HepG2 cells were transiently transfected with human CYP2B6 luciferase promoter reporter plasmids along with hPXR or hCAR₃. The results indicated that apigenin (Api), curcumol (Cur) and praeruptorin A (Pra A) were identified as potent activators of hPXR, and Pra A was also a ligand of hCAR.

3.?Furthermore, CYP2B6 mRNA expression in LS174T cells treated with the three herbal ingredients was determined by real-time polymerase chain reaction. By combining western blot and LC–MS/MS, CYP2B6 protein expression and catalytic activity induced by the three herbal ingredients were measured.

4.?Our observation showed Api and Cur up-regulated CYP2B6 expression by transactivation of hPXR, and Pra A acted as the ligand of both hPXR and hCAR to induce CYP2B6 expression.     

Toxaphene‐induced mouse liver tumorigenesis is mediated by the constitutive androstane receptor

Toxaphene was shown to increase liver tumor incidence in B6C3F1 mice following chronic dietary exposure. Preliminary evidence supported a role for the constitutive androstane receptor (CAR) in the mode of action of toxaphene‐induced mouse liver tumors. However, these results could not rule out a role for the pregnane X receptor (PXR) in liver tumor formation. To define further the nuclear receptors involved in this study, we utilized CAR, PXR and PXR/CAR knockout mice (CAR^−/−, PXR^−/− and PXR^−/−/CAR^−/−) along with the wild‐type C57BL/6. In this study CAR‐responsive genes Cyp3a11 and Cyp2b10 were induced in the liver of C57BL/6 (wild‐type) mice by toxaphene (30–570‐fold) (at the carcinogenic dose 320 ppm) and phenobarbital (positive control) (16–420‐fold) following 14 days' dietary treatment. In contrast, in CAR^−/− mice, no induction of these genes was seen following treatment with either chemical. Cyp3a11 and Cyp2b10 were also induced in PXR^−/− mice with toxaphene and phenobarbital but were not changed in treated PXR^−/−/CAR^−/− mice. Similarly, induction of liver pentoxyresorufin‐O‐deethylase (CAR activation) activity by toxaphene and phenobarbital was absent in CAR^−/− and PXR^−/−/CAR^−/− mice treated with phenobarbital or toxaphene. Ethoxyresorufin‐O‐deethylase (EROD, represents aryl hydrocarbon receptor activation) activity in CAR^−/− mice treated with toxaphene or phenobarbital was increased compared with untreated control, but lower overall in activity in comparison to the wild‐type mouse. Liver EROD activity was also induced by both phenobarbital and toxaphene in the PXR^−/− mice but not in the PXR^−/−/CAR^−/− mice. Toxaphene treatment increased 7‐benzyloxyquinoline activity (a marker for PXR activation) in a similar pattern to that seen with pentoxyresorufin‐O‐deethylase. These observations indicate that EROD and PXR activation are evidence, as expected, of secondary overlap to primary CAR receptor activation. Together, these results definitively show that activation of the CAR nuclear receptor is the mode of action of toxaphene‐induced mouse liver tumors. Copyright © 2017 John Wiley & Sons, Ltd.     

DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 microM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.