Pregnane X Receptor Target Genes Research Articles

Identification of approved drugs as potent inhibitors of pregnane X receptor activation with differential receptor interaction profiles.

Activation of pregnane X receptor (PXR) results in the induction of first-pass metabolism and drug efflux. Hereby, PXR may cause adverse drug reactions or therapeutic failure of drugs. PXR inhibition is thus an attractive option to minimise adverse effects or to improve therapeutic efficiencies; however, only a limited number of antagonists have been identified so far. We performed a cell-based high-throughput screen to identify PXR antagonists, using a library of approved and investigational drugs. Two approved drugs, pimecrolimus and pazopanib, emerged as novel potent antagonists of PXR activation, with IC50 values of 1.2 and 4.1µM, respectively. We further characterised these with respect to receptor specificity, assembly of the PXR ligand-binding domain (LBD) and interactions with co-factors. In vitro and in silico assays were carried out to identify the site(s) of interaction with the PXR LBD. Primary human hepatocytes were used to investigate antagonism of the induction of endogenous PXR target genes. Pimecrolimus and pazopanib did not affect the transcriptional activity of other nuclear receptors. Both induced the release of co-repressor from PXR and likewise interfered with agonist-induced recruitment of co-activator. Cumulative evidence from cellular and in vitro assays, as well as molecular docking, suggested additional or exclusive binding outside the PXR ligand-binding pocket for both. The compounds differentially antagonised the induction of PXR-regulated genes by rifampicin in primary human hepatocytes. In conclusion, we here have identified two approved drugs as novel potent PXR inhibitors with differential receptor interaction profiles and gene selectivity in primary human hepatocytes.

Acceleration of murine hepatocyte proliferation by imazalil through the activation of nuclear receptor PXR.

The nuclear receptor pregnane X receptor (PXR) plays a major role in the xenobiotic-induced expression of drug-metabolizing enzymes. PXR activation is also associated with several adverse events in the liver. Especially, the receptor enhances hepatocyte proliferation mediated by chemical liver tumor promoters, suggesting that exposure to PXR activators increases the risk of liver cancer. In this study, we have investigated the influences of food additives on PXR to understand their potential adverse effects when they are taken in combination with other chemical compounds. We first screened 25 food additives and related compounds for their PXR-activating ability using reporter assays in HepG2 cells expressing mouse PXR, and found that imazalil dose-dependently activated mouse PXR. Next, to investigate whether imazalil could activate mouse PXR in vivo, mice were treated with imazalil and we found that imazalil treatment increased hepatic mRNA levels of Cyp3a11, a PXR target gene. Finally, to investigate the influence of imazalil exposure on the hepatocyte proliferation induced by nuclear receptor constitutive active/androstane receptor (CAR), mice were treated with imazalil with or without mouse CAR activator TCPOBOP. Although imazalil alone did not induce hepatocyte proliferation, co-treatment with imazalil facilitated the TCPOBOP-dependent proliferation, indicated by the increases in cell proliferation marker levels, Ki-67-positive nuclei and Mcm2 mRNA levels. These results suggest that in mice imazalil activates PXR to enhance hepatocyte proliferation mediated by CAR-activating liver tumor promoters.

PXR mediated induction of CYP3A4, CYP1A2, and P-gp by Mitragyna speciosa and its alkaloids.

Kratom (Mitragyna speciosa), a native herb of Southeast Asia, is widely known for its psychoactive properties. Recent increase in the use of kratom as a recreational drug has increased the risk of its interaction with conventional drugs if taken concomitantly. A few reports are available related to the effects of kratom on the activity of cytochrome P450 enzymes (CYPs), but there are no reports of its effects on pregnane X receptor (PXR), a transcription factor that regulates the expression of CYPs and P-glycoprotein (P-gp). This study was carried out to evaluate the effects of a methanolic extract of kratom leaves, an alkaloid rich fraction and its 5 indole and 4 oxindole alkaloids on PXR activation and the resulting changes in the mRNA expression of PXR target genes (CYP3A4, CYP1A2, and P-gp). A significant activation of PXR was observed by the extract (3-fold), alkaloidal fraction (4-fold) and all 9 alkaloids (4- to 6-fold) that was associated with an increased mRNA expression which resulted into an increase in the activity of CYP3A4, CYP1A2, and P-gp. These results indicate that high consumption of Mitragyna speciosa extract along with the conventional drugs may lead to potential herb-drug interactions due to its effects on PXR.

Modulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation by ursolic acid (UA) attenuates rifampin-isoniazid cytotoxicity

BackgroundInteractions between transcriptional inducers of cytochrome P450 (CYP450) enzymes and therapeutic drugs may be prevented by antagonizing the activation of a nuclear receptor (NR), pregnane X receptor (PXR, NR1I2), thus improving therapeutic efficacy. PurposeIn the present study, we aim to identify that ursolic acid (UA), a widely distributed pentacyclic triterpene, may act as an effective antagonist of PXR and its sister NR receptor, constitutive androstane receptor (CAR, NR1I3). MethodsThe hepatocellular carcinoma cell line, HepG2, was used to evaluate the promoter activity of PXR and CAR target genes, CYP3A4 and CYP2B6, respectively. Catalytic activities, mRNA, and protein expression of CYP3A4 and CYP2B6 were evaluated in a differentiated HepaRG cell line. Coregulation of PXR with coregulators on CYP3A4 promoter response elements was also been characterized. ResultsTransient transfection assays showed that UA effectively attenuated CYP3A4 and CYP2B6 promoter activities mediated by rifampin (RIF, human PXR agonist) and CITCO (human CAR agonist). These inhibitory effects were well correlated with the expression and catalytic activities of CYP3A4 and CYP2B6. Furthermore, the interaction of co-regulators with PXR and the transcriptional complexes in the CYP3A4 promoter activity and CYP3A4 promoter xenobiotic response element (everted repeat 6, ER6), respectively, were disrupted in the presence of UA. UA showed an antagonistic effect against PXR, and reversed the cytotoxic effects of isoniazid (INH) induced by RIF. Taken together, these results show that UA inhibits the transactivation effects of PXR and CAR, and reduces the expression and function of CYP3A4 and CYP2B6. ConclusionThe present study suggests that UA could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.

Profiling of bisphenol S towards nuclear receptors activities in human reporter cell lines

Bisphenol S (BPS) is heat-stable structural analog of bisphenol A (BPA), a known endocrine disruptor. Due to the effort to replace BPA with BPS, it is essential to know if BPS is suitable non-toxic replacement without reported deleterious effects of BPA. Since most of the BPA effects are ascribed to its ability to activate nuclear receptors, we screened some prominent members of this family in order to confirm or refute some recent findings.We found that BPS insignificantly activated aryl hydrocarbon receptor (AhR) in reporter gene assay and no induction of AhR target gene CYP1A1 was observed in human hepatocytes (HH). BPS was able to act like an antagonist of pregnane X receptor (PXR) in reporter gene assay, but the expression of PXR target gene CYP3A4, was only moderately affected in HH. While BPS antagonized dexamethasone-inducible glucocorticoid receptor (GR)-dependent luciferase activity in reporter gene assay (IC50=52μM), it was not able to antagonize dexamethasone effects on GR-target genes, including GILZ, NFKBIA and IL-6. Synergistic effect of BPS (range 0.001–100μM) and DHT (100nM) was observed at androgen receptor (AR) activity level only.In conclusion, we show that BPS had only limited effect on tested nuclear receptors. Moreover, submicromolar concentrations of BPS affected activated AR. Thus, due to the low levels of exposure for humans, BPS is probably of no regulatory concern. However, further investigation should delineate possible impact on male/female development or sexual functions.

Schisandrol B protects against cholestatic liver injury through pregnane X receptors.

Currently, ursodeoxycholic acid and obeticholic acid are the only two FDA-approved drugs for cholestatic liver diseases. Thus, new therapeutic approaches need to be developed. Here we have evaluated the anti-cholestasis effects of Schisandrol B (SolB), a bioactive compound isolated from Schisandra sphenanthera. Hepatoprotective effect of SolB against intrahepatic cholestasis, induced by lithocholic acid (LCA), was evaluated in mice. Metabolomic analysis and gene analysis were used to assess involvement of pregnane X receptor (PXR). Molecular docking, cell-based reporter gene analysis and knockout mice were used to demonstrate the critical role of the PXR pathway in the anti-cholestasis effects of SolB. SolB protected against LCA-induced intrahepatic cholestasis. Furthermore, therapeutic treatment with SolB decreased mortality in cholestatic mice. Metabolomics and gene analysis showed that SolB accelerated metabolism of bile acids, promoted bile acid efflux into the intestine, and induced hepatic expression of the PXR-target genes Cyp3a11, Ugt1a1, and Oatp2, which are involved in bile acid homeostasis. Mechanistic studies showed that SolB activated human PXR and up-regulated PXR target genes in human cell lines. Additionally, SolB did not protect Pxr-null mice from liver injury induced by intrahepatic cholestasis, thus providing genetic evidence that the effect of SolB was PXR-dependent. These findings provide direct evidence for the hepatoprotective effects of SolB against cholestasis by activating PXR. Therefore, SolB may provide a new and effective approach to the prevention and treatment of cholestatic liver diseases.

Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.

A combination of dietary N-3 fatty acids and a cyclooxygenase-1 inhibitor attenuates nonalcoholic fatty liver disease in mice

We sought to determine whether a combination of purified n-3 fatty acids (n-3) and SC-560 (SC), a cyclooxygenase-1-specific inhibitor, is effective in ameliorating nonalcoholic fatty liver disease in obesity. Female wild-type mice were fed a high-fat and high-cholesterol diet (HF) supplemented with n-3 in the presence or absence of SC. Mice treated with SC alone exhibited no change in liver lipids, whereas n-3-fed mice tended to have lower hepatic lipids. Mice given n-3+SC had significantly lower liver lipids compared with HF controls indicating enhanced lipid clearance. Total and sulfated bile acids were significantly higher only in n-3+SC-treated mice compared with chow diet (CD) controls. Regarding mechanisms, the level of pregnane X receptor (PXR), a nuclear receptor regulating drug/bile detoxification, was significantly higher in mice given n-3 or n-3+SC. Studies in precision-cut liver slices and in cultured hepatoma cells showed that n-3+SC enhanced not only the expression/activation of PXR and its target genes but also the expression of farnesoid X receptor (FXR), another regulator of bile synthesis/clearance, indicating that n-3+SC can induce both PXR and FXR. The mRNA level of FGFR4 which inhibits bile formation showed a significant reduction in Huh 7 cells upon n-3 and n-3+SC treatment. PXR overexpression in hepatoma cells confirmed that n-3 or SC each induced the expression of PXR target genes and in combination had an enhanced effect. Our findings suggest that combining SC with n-3 potentiates its lipid-lowering effect, in part, by enhanced PXR and/or altered FXR/FGFR4 signaling.

Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays

Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays.

RNA-Seq reveals common and unique PXR- and CAR-target gene signatures in the mouse liver transcriptome

The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known xenobiotic-sensing nuclear receptors with overlapping functions. However, there lacks a quantitative characterization to distinguish between the PXR and CAR target genes and signaling pathways in the liver. The present study performed a transcriptomic comparison of the PXR- and CAR-targets using RNA-Seq in livers of adult wild-type mice that were treated with the prototypical PXR ligand PCN (200mg/kg, i.p. once daily for 4days in corn oil) or the prototypical CAR ligand TCPOBOP (3mg/kg, i.p., once daily for 4days in corn oil). At the given doses, TCPOBOP differentially regulated many more genes (2125) than PCN (212), and 147 of the same genes were differentially regulated by both chemicals. As expected, the top pathways differentially regulated by both PCN and TCPOBOP were involved in xenobiotic metabolism, and they also up-regulated genes involved in retinoid metabolism, but down-regulated genes involved in inflammation and iron homeostasis. Regarding unique pathways, PXR activation appeared to overlap with the aryl hydrocarbon receptor signaling, whereas CAR activation appeared to overlap with the farnesoid X receptor signaling, acute-phase response, and mitochondrial dysfunction. The mRNAs of differentially regulated drug-processing genes (DPGs) partitioned into three patterns, namely TCPOBOP-induced, PCN-induced, as well as TCPOBOP-suppressed gene clusters. The cumulative mRNAs of the differentially regulated DPGs, phase-I and -II enzymes, as well as efflux transporters were all up-regulated by both PCN and TCPOBOPOP, whereas the cumulative mRNAs of the uptake transporters were down-regulated only by TCPOBOP. The absolute mRNA abundance in control and receptor-activated conditions was examined in each DPG category to predict the contribution of specific DPG genes in the PXR/CAR-mediated pharmacokinetic responses. The preferable differential regulation by TCPOBOP in the entire hepatic transcriptome correlated with a marked change in the expression of many DNA and histone epigenetic modifiers. In conclusion, the present study has revealed known and novel, as well as common and unique targets of PXR and CAR in mouse liver following pharmacological activation using their prototypical ligands. Results from this study will further support the role of these receptors in regulating the homeostasis of xenobiotic and intermediary metabolism in the liver, and aid in distinguishing between PXR and CAR signaling at various physiological and pathophysiological conditions. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie

MicroRNA-30c-1-3p is a silencer of the pregnane X receptor by targeting the 3′-untranslated region and alters the expression of its target gene cytochrome P450 3A4

The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. Recently, activation of PXR has also been linked to the development of many disease conditions such as metabolic disorders and malignancies. MicroRNAs (miRs) emerge as important molecular species involved in these conditions. This study was undertaken to test a large number of miRs for their ability to regulate PXR expression. As many as 58 miRs were tested and miR-30c-1-3p was identified to suppress PXR expression. The suppression was achieved by targeting the 3′-untranslated region, 438 nucleotides from the stop codon. The suppression was detected in multiple cell lines from different organ origins. In addition, miR-30c-1-3p altered basal and induced expression of cytochrome P450 3A4 (CYP3A4), a prototypical target gene of PXR. The alteration varied depending on the time and amounts of miR-30c-1-3p. CYP3A4 is responsible for the metabolism of more than 50% medicines. The interconnection between miR-30c-1-3p and PXR signifies a role of miRs in drug–drug interactions and chemosensitivity. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Cross-talk between EPAS-1/HIF-2α and PXR signaling pathway regulates multi-drug resistance of stomach cancer cell

EPAS-1/HIF-2α (Endothelial PAS domain-containing protein 1/hypoxia-inducible transcription factors 2α) is a transcription factor expressed in a wide range of human cancers, including stomach cancer. Although EPAS-1 has been studied for years, its function in oncogenic transformation processes needs to be further investigated. In this study, we found that EPAS-1 would promote the growth of stomach cancer cell line BGC-823. Our results revealed that EPAS-1 interacts with Pregnane X Receptor (PXR), a nuclear receptor that regulates multiple genes’ transcription involved in multi-drugs resistance (MDR) process. Protein-protein interaction between EPAS-1 and PXR was identified by co-immunoprecipitation and GST-pull down assays. By this interaction, EPAS-1 recruited PXR to its response elements in promoter/enhancer regions of CYP3A4, a PXR target gene. Over-expression of EPAS-1 increased the expression of PXR responsive genes, enhanced the proliferation of BGC-823 cells and boosted the resistance of BGC-823 cells against the cytotoxicity of chemotherapeutic drugs, e.g. Mitomycin C and Paclitaxel. Reduction of EPAS-1 level via its siRNA disrupted the proliferation, and enhanced the susceptibility of BGC-823 cells to those chemotherapeutic drugs. Our findings suggested that EPAS-1 and PXR may cooperatively participate in development and especially MDR process of stomach cancer. These findings may contribute to more effective targeted drugs discovery for the stomach cancer therapy.

OP41 - Methylation at positon 7’ but not 5’ and the isoprenoid chain are required for vitamin E to induce P-glycoprotein protein expression and activity in the human colon carcinoma cell line LS180

The eight congeners of vitamin E differ in their methylation pattern and either possess an alkyl (tocopherols, T) or an isoprenoid (tocotrienols, T3) side chain. The different chemical structures influence the biological activity of the vitamin, like the binding to the nuclear pregnane X receptor (PXR). P-glycoprotein (P-gp), a PXR target gene, is an efflux transporter expressed at various barriers in the body including small intestine, bile ducts and the blood-brain-barrier. We therefore investigated the impact of methylation of the chromanol ring (position 5’ and 7’ = α; position 7’ = γ) and the structure of the side chain on the expression and activity of P-gp. LS180 cells, which have a low basal P-gp expression, are a good model to study the induction of P-gp protein expression and activity. LS180 cells were either treated with the positive control rifampicin (25 µmol/L), a known P-gp inducer, the appropriate solvent control, or the respective vitamin E congener for 48 hours. αT was investigated in the range of 10–100 µmol/L, γT 10-50 µmol/L, αT3 5-50 µmol/L and γT 5-25 µmol/L due to differences in cell toxicity. Protein expression was determined by Western blot analysis and P-gp activity via the efflux of the fluorescent substrate rhodamine 123 in the presence or absence of the P-gp specific inhibitor elacridar. αT, γT and αT3 neither influenced the protein expression nor the activity of P-gp. γT3 induced P-glycoprotein activity and protein expression which is in accordance with the literature. To induce P-glycoprotein, both methylation at position 7’ but not 5’ of the chromanol ring as well as an isoprenoid side chain are required as none of the two structural features individually showed an effect.

Ginsenosides Regulate PXR/NF-κB Signaling and Attenuate Dextran Sulfate Sodium-Induced Colitis.

Pregnane X receptor (PXR) activation exhibits anti-inflammatory effects via repressing nuclear factor-κB (NF-κB); however, its overactivation may disrupt homeostasis of various enzymes and transporters. Here we found that ginsenosides restore PXR/NF-κB signaling in inflamed conditions without disrupting PXR function in normal conditions. The effects and mechanisms of ginsenosides in regulating PXR/NF-κB signals were determined both in vitro and in vivo. Ginsenosides significantly inhibited NF-κB activation and restored the expression of PXR target genes in tumor necrosis factor-α-stimulated LS174T cells. Despite not being PXR agonists, ginsenosides repressed NF-κB activation in a PXR-dependent manner. Ginsenosides significantly increased the physical association between PXR and the NF-κB p65 subunit and thereby decreased the nuclear translocation of p65. Ginsenoside Rb1 and compound K (CK) were major bioactive compounds in the regulating PXR/NF-κB signaling. Consistently, ginsenosides significantly attenuated dextran sulfate sodium-induced experimental colitis, which was associated with restored PXR/NF-κB signaling. This study indicates that ginsenosides may elicit anti-inflammatory effects via targeting PXR/NF-κB interaction without disrupting PXR function in healthy conditions. Ginsenoside Rb1 and CK may serve as leading compounds in the discovery of new drugs that target PXR/NF-κB interaction in therapy for inflammatory bowel disease.

Abstract 531: Intestinal Pregnane X Receptor Links Xenobiotic Exposure and Hypercholesterolemia

Objective: Recent studies have associated endocrine disrupting chemical (EDC) exposure with the increased risk of cardiovascular disease (CVD) in humans, but the underlying mechanisms responsible for these associations remain elusive. Many EDCs have been implicated in activating the nuclear receptor pregnane X receptor (PXR) which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. This study aims to investigate the potential role of PXR in lipid homeostasis. Approach and Results: We identified tributyl citrate (TBC), one of a large group of FDA-approved plasticizers for pharmaceutical or food applications, as a potent and selective PXR agonist. TBC efficiently activated PXR and induced PXR target gene expression in vitro and in vivo . Interestingly, TBC was an intestine-specific PXR ligand but did not affect hepatic PXR activity. Exposure to TBC increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. TBC-mediated PXR activation stimulated the expression of the cholesterol transporter Ni emann-Pick C1-Like 1 (NPC1L1) in the intestine. Promoter analysis revealed a DR-4-type of PXR-response element in the human NPC1L1 promoter and TBC promoted PXR recruitment onto the NPC1L1 promoter. Consistently, TBC treatment significantly increased cholesterol uptake by human and murine intestinal cells and deficiency of PXR blocked TBC-elicited cholesterol uptake. Conclusions: Our results established an important role of intestinal PXR in linking EDC exposure and hyperlipidemia. These findings provide critical mechanistic insight for understanding the adverse effects of EDCs on lipid homeostasis and CVD risk in humans.

Pregnane X Receptor modulation by Mitragyna speciosa and its constituents

Traditionally, the leaves of Mitragyna speciosa which are consumed mainly by oral route are used in treating the withdrawal symptoms associated with opiate addiction and sometimes as an opioid substitute due to their euphoric effects in the brain. Pregnane X Receptor (PXR), a type of nuclear receptor, has been shown to transcriptionally regulate the expression of drug metabolizing enzymes and drug transporters. This study was carried out to evaluate the effects of Mitragyna speciosa methanolic extract, alkaloid rich fraction and its 6 alkaloids constituents on PXR activation. In addition, the changes in the expression (mRNA) of PXR target genes (CYP3A4, 1A2, and P-gp) were also determined. Modulation of PXR activity is monitored in hPXR transfected HepG2 cell line through a reporter gene assay, and quantitative RT-PCR was used to study the changes in the mRNA expression. The total extract, alkaloid rich fraction, and all six constituents showed significant activation of PXR which was comparable to the effects of standard drug, rifampicin. The mRNA expression of P-gp and CYP1A2 was also increased by the extract, alkaloid rich fraction and 4 of the constituents, while the expression of CYP3A4 was increased by the alkaloid rich fraction and 3 of the constituents. These results suggests that chronic intake of Mitragyna speciosa may lead to potential drug interactions as a result of PXR activation and increased expression of target genes thereby affecting the pharmacokinetics and pharmacodynamics of the conventional drugs that are substrates of CYPs and P-gp.

GW4064, An Agonist of Farnesoid X Receptor, Represses CYP3A4 Expression in Human Hepatocytes

Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis, and is recognized as a promising therapeutic target for metabolic diseases. The biological function of FXR is in part mediated by small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, SHP in turn represses expression of genes involved in bile acid synthesis. The objective of this study is to investigate the effect of FXR activation on the expression of the major drug-metabolizing enzyme, cytochrome P450 (CYP) 3A4. Methods. The effects of GW4064, a synthetic agonist of FXR, on expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time PCR and S9 phenotyping. The mechanisms were examined by using transient transfection and promoter reporter assays in HepG2 cells. Results. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in 75% decrease in CYP3A4 mRNA expression and 25% decrease in CYP3A4 activity, accompanied by ~3-fold increases in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor (GR). However, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 was shown to directly transactivate CYP2B6 promoter. Conclusion GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur. This work was supported by the National Institute of Child Health and Human Development (Grant HD065532).

Structure‐Activity Analysis of Efavirenz‐Mediated Modulation of Pregnane X Receptor Target Genes

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor used to treat HIV. EFV increases the expression of drug metabolizing enzymes via activation of pregnane X receptor (PXR) and we aimed to identify the EFV moieties that contribute to this. Hepatocytes isolated from wild-type (WT) male mice were treated with 10µM EFV or 11 EFV analogs and mRNA levels of PXR target genes were measured using qPCR. EFV increased cytochrome P450 3a11 (Cyp3a11) mRNA 2.1 ± 0.1-, 9.0 ± 4.7- and 76.6 ± 17.9-fold as compared to control at 24 h, 48 h and 72 h, respectively, and this was abrogated in hepatocytes isolated from PXR null mice. Using the EFV analogs, PXR activation was observed to be size-dependent as the smallest analog (223 Da) failed to increase Cyp3a11 mRNA, while analogs 290 Da to 424 Da in size increased Cyp3a11 mRNA similarly to EFV. Interestingly, mRNA levels of small heterodimer partner (SHP), which is transcriptionally repressed by PXR, was also increased by EFV treatment of WT mouse hepatocytes to 2.1 ± 0.6- and 2.0 ± 0.3-fold of control at 48 h and 72 h, respectively. Further, this effect was augmented in PXR null mice as the SHP mRNA levels were observed to be 4.3 ± 0.8-greater in EFV-treated mouse hepatocytes than in controls at 72 h. Since SHP is positively regulated by farnesoid X receptor (FXR) activation, hepatocytes isolated from FXR null mice were treated with EFV, however, the EFV-mediated increase in SHP mRNA abundance was not abolished in these mice. In conclusion, using a series of EFV analogs, we have demonstrated size-dependent activation of PXR and, in addition, we have identified EFV as a modulator of SHP mRNA levels. Funding: R01 GM103853 and UM1 AI068613

Intestinal Pregnane X Receptor Links Xenobiotic Exposure and Hypercholesterolemia

Recent studies have associated endocrine-disrupting chemical (EDC) exposure with the increased risk of cardiovascular disease in humans, but the underlying mechanisms responsible for these associations remain elusive. Many EDCs have been implicated in activation of the nuclear receptor pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. Here we report an important role of intestinal PXR in linking xenobiotic exposure and hyperlipidemia. We identified tributyl citrate (TBC), one of a large group of Food and Drug Administration-approved plasticizers for pharmaceutical or food applications, as a potent and selective PXR agonist. TBC efficiently activated PXR and induced PXR target gene expression in vitro and in vivo. Interestingly, TBC activated intestinal PXR but did not affect hepatic PXR activity. Exposure to TBC increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. TBC-mediated PXR activation stimulated the expression of an essential cholesterol transporter, Niemann-Pick C1-like 1 (NPC1L1), in the intestine. Promoter analysis revealed a DR-4 type of PXR response element in the human NPC1L1 promoter, and TBC promoted PXR recruitment onto the NPC1L1 promoter. Consistently, TBC treatment significantly increased lipid uptake by human and murine intestinal cells and deficiency of PXR inhibited TBC-elicited lipid uptake. These findings provide critical mechanistic insight for understanding the impact of EDC-mediated PXR activation on lipid homeostasis and demonstrate a potential role of PXR in mediating the adverse effects of EDCs on cardiovascular disease risk in humans.

GW4064, an agonist of farnesoid X receptor, represses CYP3A4 expression in human hepatocytes by inducing small heterodimer partner expression.

Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The effects of 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), a synthetic agonist of FXR, on the expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time polymerase chain reaction and S9 phenotyping. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in a 75% decrease in CYP3A4 mRNA expression and a 25% decrease in CYP3A4 activity, accompanied by ∼3-fold increase in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor. Interestingly, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 enhanced CYP2B6 promoter activity. In conclusion, GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur.