The Discovery of Gut Microbial Metabolites as Modulators of Host Susceptibility to Acetaminophen-Induced Hepatotoxicity.

The mammalian gut microbiota plays diverse and essential roles in modulating host physiology. Key mediators determining the outcome of the microbiota-host interactions are the small molecule metabolites produced by the gut microbiota. The liver is a major organ exposed to gut microbial metabolites, and it serves as the nexus for maintaining healthy interactions between the gut microbiota and the host. At the same time, the liver is the primary target of potentially harmful gut microbial metabolites. In this review, we provide an up-to-date list of gut microbial metabolites that have been identified to either increase or decrease host susceptibility to acetaminophen (APAP)-induced liver injury. The signaling pathways and molecular factors involved in the progression of APAP-induced hepatotoxicity are well-established, and we propose that the mouse model of APAP-induced hepatotoxicity serves as a model system for uncovering gut microbial metabolites with previously unknown functions. Furthermore, we envision that gut microbial metabolites identified to alter APAP-induced hepatotoxicity likely have broader implications in other liver diseases. SIGNIFICANCE STATEMENT: This review provides an overview of the role of the gut microbiota in modulating the host susceptibility to acetaminophen (APAP)-induced liver injury. It focuses on the roles of gut bacterial small molecule metabolites as mediators of the interaction between the gut microbiota and the liver. It also illustrates the utility of APAP-induced liver injury as a model to identify gut microbial metabolites with biological function.

Syntheses and studies of deuterated Imdiazo[1,2-a]pyridine-3-carboxamides with potent anti-tuberculosis activity and improved metabolic properties.

The imidazo[1,2-a]pyridine-3-carboxyamides (IAPs) are a unique class of compounds endowed with impressive nanomolar in vitro potency against Mycobacterium tuberculosis (Mtb) as exemplified by clinical candidate Telacebec (Q203). These compounds target mycobacterial respiration through inhibition of the QcrB subunit of cytochrome bc1:aa3 super complex resulting in bacteriostatic efficacy in vivo. Our labs have had a long-standing interest in the design and development of IAPs. However, some of these compounds suffer from short in vivo half-lives, requiring multiple daily dosing or the addition of a cytochrome P450 inhibitor for murine efficacy evaluations. Deuteration has been shown to decrease metabolism as the C-D bond is stronger than the CH bond. Herein we describe our efforts on design and synthesis of potent deuterated IAPs and the effect that deuteration has upon metabolism through microsomal stability studies.

A Family of Small Intrinsically Disordered Proteins Involved in Flagellum-Dependent Motility in Salmonella enterica.

By screening a collection of Salmonella mutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF, STM14_1829, and yciG) required for wild-type flagellum-dependent swimming and swarming motility. The ymdF, STM14_1829, and yciG genes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829 mutant due to the downregulation of the flhDC operon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829 strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829 mutant, suggesting the indirect repression of the flhDC operon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, including Escherichia coli and Pseudomonas aeruginosa We showed that the inactivation of STM14_1829 homologs in E. coli and P. aeruginosa also alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility.IMPORTANCE This study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility in Salmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.

Repression of hepatocyte nuclear factor 4 alpha by AP-1 underlies dyslipidemia associated with retinoic acid.

All-trans retinoic acid (atRA) is used to treat certain cancers and dermatologic diseases. A common adverse effect of atRA is hypercholesterolemia; cytochrome P450 (CYP) 7A repression is suggested as a driver. However, the underlying molecular mechanisms remain unclear. We investigated CYP7A1 expression in the presence of atRA in human hepatocytes and hepatic cell lines. In HepaRG cells, atRA increased cholesterol levels dose-dependently alongside dramatic decreases in CYP7A1 expression. Lentiviral-mediated CYP7A1 overexpression reversed atRA-induced cholesterol accumulation, suggesting that CYP7A1 repression mediated cholesterol accumulation. In CYP7A1 promoter reporter assays and gene-knockdown studies, altered binding of hepatocyte nuclear factor 4 α (HNF4α) to the proximal promoter was essential for atRA-mediated CYP7A1 repression. Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) and ERK pathways attenuated atRA-mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP-1 (c-Jun/c-Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression. In DNA pull-down and chromatin immunoprecipitation assays, AP-1 exhibited sequence-specific binding to the proximal CYP7A1 promoter region overlapping the HNF4α binding site, and atRA increased AP-1 but decreased HNF4α recruitment to the promoter. Collectively, these results indicate that atRA activates JNK and ERK pathways and the downstream target AP-1 represses HNF4α transactivation of the CYP7A1 promoter, potentially responsible for hypercholesterolemia.

Characterization of Vitamin A Metabolome in Human Livers With and Without Nonalcoholic Fatty Liver Disease.

Retinoids are essential endogenous compounds involved in regulation of critical biologic processes, including maintenance of metabolic homeostasis in the liver. Much of the knowledge of altered retinoid homeostasis in human disease states is derived from changes in indirect markers such as mRNA expression of retinoid-related genes and circulating concentrations of retinol or its binding protein RBP4. We hypothesized that in the human liver, concentrations of the active retinoid all-trans-retinoic acid (atRA) correlate with the concentrations of retinyl palmitate (RP), the storage form of atRA, retinol, the inactive vitamin A, and the mRNA expression of retinoid-related genes. On the basis of existing knowledge of altered vitamin A homeostasis in metabolic syndrome, we also predicted that in human livers with nonalcoholic fatty liver disease (NAFLD) retinoid concentrations would be decreased. Using novel liquid chromatography-tandem mass spectrometry methods, the hepatic vitamin A metabolome was quantified in normal human livers (n = 50) and 22 livers from donors with NAFLD. The hepatic concentrations of RP, atRA, 13-cisRA, and 4-oxo-atRA were significantly decreased in NAFLD samples in comparison with normal liver samples, whereas retinol levels remained unchanged. The concentrations of atRA were positively correlated with RP and 13-cisRA but not with retinol or the relative mRNA expression of LRAT, ALDH1A1, CYP26A1, RARα, and RARß An active metabolite of atRA, 4-oxo-atRA was, for the first time, detected in human tissues at comparable concentration with RA isomers, suggesting this retinoid may contribute to retinoid signaling in humans. SIGNIFICANCE STATEMENT: This study shows that in NAFLD liver vitamin A homeostasis is disrupted potentially contributing to disease progression. The results show that interpretation of retinoid homeostasis on the basis of indirect markers such as retinol concentrations or mRNA data is probably misleading when evaluating human disease processes, and analysis of the broader retinoid metabolome is needed to characterize disease effects on retinoid signaling.

Commensal Gut Bacteria Convert the Immunosuppressant Tacrolimus to Less Potent Metabolites.

Tacrolimus exhibits low and variable drug exposure after oral dosing, but the contributing factors remain unclear. Based on our recent report showing a positive correlation between fecal abundance of Faecalibacterium prausnitzii and oral tacrolimus dose in kidney transplant patients, we tested whether F. prausnitzii and other gut abundant bacteria are capable of metabolizing tacrolimus. Incubation of F. prausnitzii with tacrolimus led to production of two compounds (the major one named M1), which was not observed upon tacrolimus incubation with hepatic microsomes. Isolation, purification, and structure elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy indicated that M1 is a C-9 keto-reduction product of tacrolimus. Pharmacological activity testing using human peripheral blood mononuclear cells demonstrated that M1 is 15-fold less potent than tacrolimus as an immunosuppressant. Screening of 22 gut bacteria species revealed that most Clostridiales bacteria are extensive tacrolimus metabolizers. Tacrolimus conversion to M1 was verified in fresh stool samples from two healthy adults. M1 was also detected in the stool samples from kidney transplant recipients who had been taking tacrolimus orally. Together, this study presents gut bacteria metabolism as a previously unrecognized elimination route of tacrolimus, potentially contributing to the low and variable tacrolimus exposure after oral dosing.

Physiological Regulation of Drug Metabolism and Transport: Pregnancy, Microbiome, Inflammation, Infection, and Fasting.

This article is a report on a symposium entitled "Physiological Regulation of Drug Metabolism and Transport" sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2017 meeting in Chicago, IL. The contributions of physiologic and pathophysiological regulation of drug-metabolizing enzymes and transporters to interindividual variability in drug metabolism are increasingly recognized but in many cases are not well understood. The presentations herein discuss the phenomenology, consequences, and mechanism of such regulation. CYP2D6 transgenic mice were used to provide insights into the mechanism of regulation of this enzyme in pregnancy, via hepatocyte nuclear factor 4α, small heterodimer partner, and retinoids. Regulation of intestinal and hepatic drug-processing enzymes by the intestinal microbiota via tryptophan and its metabolites was investigated. The potential impact of parasitic infections on human drug metabolism and clearance was assessed in mice infected with Schistosoma mansoni or Plasmodium chabaudi chabaudi AS, both of which produced widespread and profound effects on murine hepatic drug-metabolizing enzymes. Finally, the induction of Abcc drug efflux transporters by fasting was investigated. This was demonstrated to occur via a cAMP, protein kinase A/nuclear factor-E2-related factor 2/Sirtuin 1 pathway via antioxidant response elements on the Abcc genes.

Inhibitory and Inductive Effects of Opuntia ficus indica Extract and Its Flavonoid Constituents on Cytochrome P450s and UDP-Glucuronosyltransferases.

Opuntia ficus indica (OFI) is grown abundantly in arid areas and its fruits are regarded as an important food and nutrient source owing to the presence of flavonoids, minerals, and proteins. The previous report that OFI exerts phytoestrogenic activity makes it plausible for OFI-containing supplements to be used as alternative estrogen replacement therapy. In the case of polypharmacy with the consumption of OFI-containing botanicals in post- or peri-menopausal women, it is critical to determine the potential drug-OFI interaction due to the modulation of drug metabolism. In the present study, the modulating effects on the hepatic drug metabolizing enzymes (DMEs) by OFI and its flavonoid constituents (kaempferol, quercetin, isorhamnetin, and their glycosidic forms) were investigated using the liver microsomal fractions prepared from ovariectomized (OVX) rats, human liver microsomes, and human hepatocarcinoma cell line (HepG2). As a result, the oral administration of extracts of OFI (OFIE) in OVX rats induced hepatic CYP2B1, CYP3A1, and UGT2B1. OFIE, hydrolyzed (hdl) OFIE, and several flavonols induced the transcriptional activities of both CYP2B6 and CYP3A4 genes in HepG2 cells. Finally, OFIE did not inhibit activities of cytochrome P450 (CYPs) or uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), whereas hdl OFIE or flavonol treatment inhibited CYP1A2 and CYP3A1/3A4 in rat and human liver microsomes. Our data demonstrate that OFIE may induce or inhibit certain types of DMEs and indicate that drug-OFI interaction may occur when the substrate or inhibitor drugs of specific CYPs or UGTs are taken concomitantly with OFI-containing products.

High-Fat Diet Feeding Alters Expression of Hepatic Drug-Metabolizing Enzymes in Mice.

Medical conditions accompanying obesity often require drug therapy, but whether and how obesity alters the expression of drug-metabolizing enzymes and thus drug pharmacokinetics is poorly defined. Previous studies have shown that high-fat diet (HFD) feeding and subsequent obesity in mice lead to altered expression of transcriptional regulators for cytochrome P450 CYP2D6, including hepatocyte nuclear factor 4α (HNF4α, a transcriptional activator of CYP2D6) and small heterodimer partner (SHP, a transcriptional repressor of CYP2D6). The objective of this study was to examine whether diet-induced obesity alters CYP2D6 expression by modulating HNF4α and SHP expression. Male CYP2D6-humanized transgenic (Tg-CYP2D6) mice were fed with HFD or matching control diet for 18 weeks. Hepatic mRNA expression of CYP2D6 decreased to a small extent in the HFD group (by 31%), but the differences in CYP2D6 protein and activity levels in hepatic S9 fractions were found insignificant between the groups. Although hepatic SHP expression did not differ between the groups, HNF4α mRNA and protein levels decreased by ∼30% in the HFD group. Among major mouse endogenous cytochrome P450 genes, Cyp1a2 and Cyp2c37 showed significant decreases in the HFD group, whereas Cyp2e1 expression did not differ between groups. Cyp2b10 and Cyp3a11 expression was higher in the HFD group, with corresponding 2.9-fold increases in hepatic CYP3A activities in HFD-fed mice. Together, these results suggest that obesity has minimal effects on CYP2D6-mediated drug metabolism, although it modulates the expression of mouse endogenous P450s in a gene-specific manner.

Transcriptional Regulation of CYP2D6 Expression.

CYP2D6-mediated drug metabolism exhibits large interindividual variability. Although genetic variations in the CYP2D6 gene are well known contributors to the variability, the sources of CYP2D6 variability in individuals of the same genotype remain unexplained. Accumulating data indicate that transcriptional regulation of CYP2D6 may account for part of CYP2D6 variability. Yet, our understanding of factors governing transcriptional regulation of CYP2D6 is limited. Recently, mechanistic studies of increased CYP2D6-mediated drug metabolism in pregnancy revealed two transcription factors, small heterodimer partner (SHP) and Krüppel-like factor 9, as a transcriptional repressor and an activator, respectively, of CYP2D6. Chemicals that increase SHP expression (e.g., retinoids and activators of farnesoid X receptor) were shown to downregulate CYP2D6 expression in the humanized mice as well as in human hepatocytes. This review summarizes the series of studies on the transcriptional regulation of CYP2D6 expression, potentially providing a basis to better understand the large interindividual variability in CYP2D6-mediated drug metabolism.

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

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.

Tryptophan hydroxylase 1 and 5-HT7 receptor preferentially expressed in triple-negative breast cancer promote cancer progression through autocrine serotonin signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) has a high risk of relapse and there are few chemotherapy options. Although 5-hydroxytryptamine (5-HT, serotonin) signaling pathways have been suggested as potential targets for anti-cancer drug development, the mechanism responsible for the action of 5-HT in TNBC remains unknown. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. Cell proliferation was measured using CellTiter 96 Aqueous One Solution. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Levels of 5-HT and vascular endothelial growth factor (VEGF) were measured using ELISA kits. Chick chorioallantoic membrane (CAM) assay and mouse tumor model were used to investigate the in vivo effects of SB269970, a 5-HT7 receptor antagonist, and BJ-1113, a novel synthetic compound. RESULTS: TNBC cell lines (MDA-MB-231, HCC-1395, and Hs578T) expressed higher levels of tryptophan hydroxylase 1 (TPH1) than hormone-responsive breast cancer cell lines (MCF-7 and T47D). In MDA-MB-231 cells, 5-HT promoted invasion and proliferation via 5-HT7 receptor, and interestingly, the stimulatory effect of 5-HT on MDA-MB-231 cell invasion was stronger than its effect on proliferation. Likewise, downstream signaling pathways of 5-HT7 differed during invasion and proliferation, that is, Gα-activated cAMP and Gßγ-activated kinase signaling during invasion, and Gßγ-activated PI3K/Akt signaling during proliferation. Also, 5-HT increased the protein expressions of TPH1 and VEGF in MDA-MB-231 cells. These results provide insight of the stimulatory effect of 5-HT on breast cancer progression; 5-HT was found to act more strongly during the first stage of metastasis (during invasion and migration) than during the later proliferative phase after local invasion. Interestingly, these actions of 5-HT were inhibited by BJ-1113, a 6-amino-2,4,5-trimethylpyridin-3-ol analog. BJ-1113 blocked intracellular signaling pathways initiated by 5-HT7 receptor activation, and exhibited anti-proliferative and anti-invasive activities against MDA-MB-231 cells. Furthermore, the inhibitory effect of BJ-1113 against MDA-MB-231 tumor growth was greater than that of SB269970, a 5-HT7 receptor antagonist. CONCLUSIONS: 5-HT7 receptor which mediates 5-HT-induced cancer progression is a potential therapeutic target in TNBC, and BJ-1113 offers a novel scaffold for the development of anti-cancer agents against TNBC.

Interindividual Variability in Cytochrome P450-Mediated Drug Metabolism.

The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.

Estrogen-Induced Cholestasis Leads to Repressed CYP2D6 Expression in CYP2D6-Humanized Mice.

Cholestasis activates bile acid receptor farnesoid X receptor (FXR) and subsequently enhances hepatic expression of small heterodimer partner (SHP). We previously demonstrated that SHP represses the transactivation of cytochrome P450 2D6 (CYP2D6) promoter by hepatocyte nuclear factor (HNF) 4α. In this study, we investigated the effects of estrogen-induced cholestasis on CYP2D6 expression. Estrogen-induced cholestasis occurs in subjects receiving estrogen for contraception or hormone replacement, or in susceptible women during pregnancy. In CYP2D6-humanized transgenic (Tg-CYP2D6) mice, cholestasis triggered by administration of 17α-ethinylestradiol (EE2) at a high dose led to 2- to 3-fold decreases in CYP2D6 expression. This was accompanied by increased hepatic SHP expression and subsequent decreases in the recruitment of HNF4α to CYP2D6 promoter. Interestingly, estrogen-induced cholestasis also led to increased recruitment of estrogen receptor (ER) α, but not that of FXR, to Shp promoter, suggesting a predominant role of ERα in transcriptional regulation of SHP in estrogen-induced cholestasis. EE2 at a low dose (that does not cause cholestasis) also increased SHP (by ∼ 50%) and decreased CYP2D6 expression (by 1.5-fold) in Tg-CYP2D6 mice, the magnitude of differences being much smaller than that shown in EE2-induced cholestasis. Taken together, our data indicate that EE2-induced cholestasis increases SHP and represses CYP2D6 expression in Tg-CYP2D6 mice in part through ERα transactivation of Shp promoter.

Altered expression of small heterodimer partner governs cytochrome P450 (CYP) 2D6 induction during pregnancy in CYP2D6-humanized mice.

Substrates of a major drug-metabolizing enzyme CYP2D6 display increased elimination during pregnancy, but the underlying mechanisms are unknown in part due to a lack of experimental models. Here, we introduce CYP2D6-humanized (Tg-CYP2D6) mice as an animal model where hepatic CYP2D6 expression is increased during pregnancy. In the mouse livers, expression of a known positive regulator of CYP2D6, hepatocyte nuclear factor 4α (HNF4α), did not change during pregnancy. However, HNF4α recruitment to CYP2D6 promoter increased at term pregnancy, accompanied by repressed expression of small heterodimer partner (SHP). In HepG2 cells, SHP repressed HNF4α transactivation of CYP2D6 promoter. In transgenic (Tg)-CYP2D6 mice, SHP knockdown led to a significant increase in CYP2D6 expression. Retinoic acid, an endogenous compound that induces SHP, exhibited decreased hepatic levels during pregnancy in Tg-CYP2D6 mice. Administration of all-trans-retinoic acid led to a significant decrease in the expression and activity of hepatic CYP2D6 in Tg-CYP2D6 mice. This study provides key insights into mechanisms underlying altered CYP2D6-mediated drug metabolism during pregnancy, laying a foundation for improved drug therapy in pregnant women.

Polymer micelle formulations of proteasome inhibitor carfilzomib for improved metabolic stability and anticancer efficacy in human multiple myeloma and lung cancer cell lines.

Carfilzomib (CFZ) is a second-generation proteasome inhibitor drug approved for the treatment of multiple myeloma. Contrary to its excellent antimyeloma activity, CFZ has shown only limited efficacy in patients with solid malignancies. This lack of efficacy has been attributed in part to rapid degradation of CFZ in the body, possibly hindering the ability of CFZ to access the proteasome target in solid tumors. We hypothesized that polymer micelles, a currently Food and Drug Administration-approved nanoparticle drug delivery formulation, may protect CFZ from metabolic degradation and thus expand the clinical utility of the drug as an anticancer agent. To test our hypothesis, we prepared CFZ-entrapped polymer micelle particles with various compositions and drug release profiles and examined the extent of the CFZ metabolism in vitro using mouse liver homogenates. We also assessed the cytotoxic activities of the CFZ-entrapped micelle formulations in human cancer cell lines derived from B lymphocytes (RPMI-8226) and the lung (H460). Our data indicated that polymer micelle-based formulations can improve metabolic stability and cytotoxic effects of CFZ compared with free CFZ in human cancer cell lines tested. Taken together, these results suggest that polymer micelles may have potential as a delivery system for CFZ with an extended therapeutic utility for nonhematologic malignancies in the future.

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.

Farnesoid X Receptor Agonist Represses Cytochrome P450 2D6 Expression by Upregulating Small Heterodimer Partner.

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme responsible for eliminating approximately 20% of marketed drugs. Studies have shown that differential transcriptional regulation of CYP2D6 may contribute to large interindividual variability in CYP2D6-mediated drug metabolism. However, the factors governing CYP2D6 transcription are largely unknown. We previously demonstrated small heterodimer partner (SHP) as a novel transcriptional repressor of CYP2D6 expression. SHP is a representative target gene of the farnesoid X receptor (FXR). The objective of this study is to investigate whether an agonist of FXR, 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), alters CYP2D6 expression and activity. In CYP2D6-humanized transgenic mice, GW4064 decreased hepatic CYP2D6 expression and activity (by 2-fold) while increasing SHP expression (by 2-fold) and SHP recruitment to the CYP2D6 promoter. CYP2D6 repression by GW4064 was abrogated in Shp(-/-);CYP2D6 mice, indicating a critical role of SHP in CYP2D6 regulation by GW4064. Also, GW4064 decreased CYP2D6 expression (by 2-fold) in primary human hepatocytes, suggesting that the results obtained in CYP2D6-humanized transgenic mice can be translated to humans. This proof of concept study provides evidence for CYP2D6 regulation by an inducer of SHP expression, namely, the FXR agonist GW4064.

Krüppel-like factor 9 promotes hepatic cytochrome P450 2D6 expression during pregnancy in CYP2D6-humanized mice.

Cytochrome P450 2D6 (CYP2D6), a major drug-metabolizing enzyme, is responsible for metabolism of approximately 25% of marketed drugs. Clinical evidence indicates that metabolism of CYP2D6 substrates is increased during pregnancy, but the underlying mechanisms remain unclear. To identify transcription factors potentially responsible for CYP2D6 induction during pregnancy, a panel of genes differentially expressed in the livers of pregnant versus nonpregnant CYP2D6-humanized (tg-CYP2D6) mice was compiled via microarray experiments followed by real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR) verification. As a result, seven transcription factors-activating transcription factor 5 (ATF5), early growth response 1 (EGR1), forkhead box protein A3 (FOXA3), JUNB, Krüppel-like factor 9 (KLF9), KLF10, and REV-ERBα-were found to be up-regulated in liver during pregnancy. Results from transient transfection and promoter reporter gene assays indicate that KLF9 itself is a weak transactivator of CYP2D6 promoter but significantly enhances CYP2D6 promoter transactivation by hepatocyte nuclear factor 4 (HNF4α), a known transcriptional activator of CYP2D6 expression. The results from deletion and mutation analysis of CYP2D6 promoter activity identified a KLF9 putative binding motif at -22/-14 region to be critical in the potentiation of HNF4α-induced transactivation of CYP2D6. Electrophoretic mobility shift assays revealed a direct binding of KLF9 to the putative KLF binding motif. Results from chromatin immunoprecipitation assay showed increased recruitment of KLF9 to CYP2D6 promoter in the livers of tg-CYP2D6 mice during pregnancy. Taken together, our data suggest that increased KLF9 expression is in part responsible for CYP2D6 induction during pregnancy via the potentiation of HNF4α transactivation of CYP2D6.

17ß-Estradiol induces sulfotransferase 2A1 expression through estrogen receptor α.

Sulfotransferase (SULT) 2A1 catalyzes sulfonation of drugs and endogenous compounds and plays an important role in xenobiotic metabolism as well as in the maintenance of steroid and lipid homeostasis. A recent study showed that 17ß-estradiol (E2) increases the mRNA levels of SULT2A1 in human hepatocytes. Here we report the underlying molecular mechanisms. E2 enhanced SULT2A1 expression in human hepatocytes and HepG2-ER cells (HepG2 stably expressing ERα). SULT2A1 induction by E2 was abrogated by antiestrogen ICI 182,780, indicating a key role of ERα in the induction. Results from deletion and mutation assays of SULT2A1 promoter revealed three cis-elements located within -257/+140 region of SULT2A1 that are potentially responsible for the induction. Chromatin immunoprecipitation assay verified the recruitment of ERα to the promoter region. Electrophoretic mobility shift assays revealed that AP-1 proteins bind to one of the cis-elements. Interestingly, SULT2A1 promoter assays using ERα mutants revealed that the DNA-binding domain of ERα is indispensable for SULT2A1 induction by E2, suggesting that direct ERα binding to the SULT2A1 promoter is also necessary for the induction. Taken together, our results indicate that E2 enhances SULT2A1 expression by both the classical and nonclassical mechanisms of ERα action.