Mouse Pregnane X Receptor Research Articles

Activation of pregnane X receptor sensitizes alcoholic steatohepatitis by transactivating fatty acid binding protein 4

Alcoholic steatohepatitis (ASH) is a liver disease characterized by steatosis, inflammation, and necrosis of the liver tissue as a result of excessive alcohol consumption. Pregnane X receptor (PXR) is a xenobiotic nuclear receptor best known for its function in the transcriptional regulation of drug metabolism and disposition. Clinical reports suggested that the antibiotic rifampicin, a potent human PXR activator, is a contraindication in alcoholics, but the mechanism was unclear. In this study, we showed that the hepatic expression of fatty acid binding protein 4 (FABP4) was uniquely elevated in ASH patients and a mouse model of ASH. Pharmacological inhibiting FABP4 attenuated ASH in mice. Furthermore, treatment of mice with the mouse PXR agonist pregnenolon-16α-carbonitrile (PCN) induced the hepatic and circulating levels of FABP4 and exacerbated ASH in a PXR-dependent manner. Our mechanism study established FABP4 as a transcriptional target of PXR. Treatment with andrographolide, a natural compound and dual inhibitor of PXR and FABP4, alleviated mice from ASH. In summary, our results showed that the PXR–FABP4 gene regulatory axis plays an important role in the progression of ASH, which may have accounted for the contraindication of rifampicin in patients of alcoholic liver disease. Pharmacological inhibition of PXR and/or FABP4 may have its promise in the clinical management of ASH.

Rifampicin and its derivatives: stability, disposition, and affinity towards pregnane X receptor employing 2D and 3D primary human hepatocytes

Rifampicin is a model ligand of the pregnane X receptor (PXR), the nuclear receptor involved in the regulation of cytochrome P450 3A4 (CYP3A4). Rifampicin forms several degradation products and metabolites of which 25-desacetylrifampicin is the most abundant in vivo. Here, we aimed to study both the stability and metabolism of rifampicin in media and 2D and 3D primary human hepatocytes (PHHs). Additionally, we analyzed interactions of rifampicin derivatives with PXR. We described that rifampicin gradually degrades by more than 50 % in the medium partly into quinone over 72 h. We observed 25-desacetylrifampicin in 2D PHHs but not in 3D PHHs. Contrary, rifampicin was converted into quinone in a one-direction process in media of 3D PHHs. The potency of rifampicin and its derivatives to activate human PXR was arranged as follows: 3-formylrifamycin SV > rifampicin quinone > rifampicin > rifampicin N-oxide > 25-desacetylrifampicin, respectively, but none activates mouse and rat PXR. The binding differences between rifampicin and 25-desacetylrifampicin were modeled in silico. Finally, we showed that overexpressed uptake organic anion transporting polypeptide 1B1 (OATP1B1) potentiated activation of PXR by rifampicin and rifampicin quinone, but overexpressed efflux multidrug resistance protein 1 (MDR1) decreased PXR activation by all derivatives.

Activation of pregnane X receptor protects against cholestatic liver injury by inhibiting hepatocyte pyroptosis.

Our previous study shows that activation of pregnane X receptor (PXR) exerts hepatoprotection against lithocholic acid (LCA)-induced cholestatic liver injury. In this study we investigated whether PXR activation could inhibit hepatocyte pyroptosis, as well as the underlying mechanisms. Male mice were treated with mouse PXR agonist pregnenolone 16α-carbonitrile (PCN, 50 mg·kg-1·d-1, i.p.) for 7 days, and received LCA (125 mg/kg, i.p., bid) from D4, then sacrificed 12 h after the last LCA injection. We showed that LCA injection resulted in severe cholestatic liver injury characterized by significant increases in gallbladder size, hepatocellular necrosis, and neutrophil infiltration with a mortality rate of 68%; PCN treatment significantly inhibited hepatocyte pyroptosis during LCA-induced cholestatic liver injury, as evidenced by reduced serum lactic dehydrogenase (LDH) levels, TUNEL-positive cells and hepatocyte membrane damage. Furthermore, PXR activation suppressed both the NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and the apoptosis protease activating factor-1 (APAF-1) pyroptosome-induced non-canonical pyroptosis. Inhibition of the nuclear factor kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways was also observed following PXR activation. Notably, dual luciferase reporter assay showed that PXR activation inhibited the transcriptional effects of NF-κB on NLRP3, as well as FOXO1 on APAF-1. Our results demonstrate that PXR activation protects against cholestatic liver injury by inhibiting the canonical pyroptosis through the NF-κB-NLRP3 axis and the non-canonical pyroptosis through the FOXO1-APAF-1 axis, providing new evidence for PXR as a prospective anti-cholestatic target.

Abstract Tu149: FDA-Approved Antidepressant Trazodone Potentially Increases the Risk of Dyslipidemia

Cardiovascular disease is the leading cause of death. Many cardiovascular health problems, such as atherosclerosis, are caused by dyslipidemia. One xenobiotic nuclear receptor, Pregnane X Receptor (PXR), plays a significant role in atherosclerosis and dyslipidemia, and is activated by various environmental chemicals, including endocrine-disrupting chemicals (EDCs). EDCs are found in common household items such as plastics, medications, and food. Trazodone is a clinically used medication to treat depression by aiding in restoring the balance of serotonin in the brain. But it is unclear if Trazodone has possible impacts on cardiovascular risk factors such as dyslipidemia. Our preliminary data suggested that Trazodone activated human PXR in both intestinal (LS180) and hepatic (HepG2) cells. We hypothesize that Trazodone could regulate the cholesterol uptake mediated by PXR signaling. In this study we use cell-based transfection assay to evaluate the underlying mechanisms by which Trazodone activates PXR. We found that Trazodone was a more potent agonist of human PXR than mouse PXR. Trazodone could activate PXR more intensely in human liver cells compared with human intestinal cells. Our data suggested that Trazodone was a selective PXR agonist and promoted the dissociation between PXR and its nuclear corepressors. Next, we are to identify the key amino acid residues within PXR ligand binding pocket that interact with Trazodone by using computational docking study along with site-mutagenesis assay. Furthermore, we plan to estimate if Trazodone altered cholesterol uptake by human intestinal cells using fluorescence-labeled cholesterol. In conclusion, we explore the potential molecular mechanisms of how FDA-approved antidepressant Trazodone activates human PXR and increases the possible risk of dyslipidemia, which provides potential evidence on future cardiovascular disease risk assessment for Trazodone as well as other antidepressant drugs.

HNF4α improves hepatocyte regeneration by upregulating PXR.

Hepatocyte nuclear factor 4 alpha (HNF4α) and the pregnane X receptor (PXR) are involved in hepatocyte regeneration. It is not clear whether HNF4α is involved in hepatocyte regeneration through the regulation of PXR. This study aims to explore the regulatory relationship between HNF4a and PXR, and whether it affects hepatocyte regeneration. A mouse PXR gene reporter and an HNF4α overexpression plasmid were constructed and transfected into mouse hepatoma cells (Hepa1-6). Overexpression of HNF4α, detection of the PXR gene reporter fluorescence value, PXR gene, and protein expression analysis were conducted to explore the regulatory relationship between HNF4α and PXR. Apoptosis and cell cycle data were measured to verify whether HNF4α is involved in hepatocyte regeneration through PXR. The luciferase gene reporter assay results indicated when HNF4α was overexpressed, the fluorescence value of the PXR gene reporter was higher than that in the control at 24 h. With increasing HNF4α expression, the PXR gene and protein expression increased, indicating that HNF4α binds to the PXR promoter and upregulates PXR expression. Apoptosis and cell cycle analysis results demonstrated that when the expression of HNF4α increased, the expression of PXR increased, the apoptosis rate decreased, and the proliferation rate increased. Meanwhile, when the upward trend of PXR gene expression was inhibited by ketoconazole, the proliferation rate decreased. By inhibiting HNF4α and creating a partial hepatectomy (PHx), we demonstrated that HNF4α can upregulate PXR to promote liver regeneration invivo. Therefore, HNF4α is shown to improve hepatocyte regeneration by upregulating PXR, which provides a reference for future research on the combined application of drugs for the treatment of liver injury.

Regulation of hepatic xenosensor function by HNF4alpha.

Nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as aryl hydrocarbon receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these 4 major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.

The reversal of PXR or PPARα activation-induced hepatomegaly

The activation of pregnane X receptor (PXR) or peroxisome proliferator-activated receptor α (PPARα) can induce liver enlargement. Recently, we reported that PXR or PPARα activation-induced hepatomegaly depends on yes-associated protein (YAP) signaling and is characterized by hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. However, it remains unclear whether PXR or PPARα activation-induced hepatomegaly can be reversed after the withdrawal of their agonists. In this study, we investigated the regression of enlarged liver to normal size following the withdrawal of PCN or WY-14643 (typical agonists of mouse PXR or PPARα) in C57BL/6 mice. The immunohistochemistry analysis of CTNNB1 and KI67 showed a reversal of hepatocyte size and a decrease in hepatocyte proliferation after the withdrawal of agonists. In details, the expression of PXR or PPARα downstream proteins (CYP3A11, CYP2B10, ACOX1, and CYP4A) and the expression of proliferation-related proteins (CCNA1, CCND1, and PCNA) returned to the normal levels. Furthermore, YAP and its downstream proteins (CTGF, CYR61, and ANKRD1) also restored to the normal states, which was consistent with the change in liver size. These findings demonstrate the reversibility of PXR or PPARα activation-induced hepatomegaly and provide new data for the safety of PXR and PPARα as drug targets.

Pregnane X receptor activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/β-catenin signaling.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) with various etiologies, which seriously affects the structure and function of the kidney. Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily and plays a critical role in regulating the genes related to xenobiotic and endobiotic metabolism in mammals. Previous studies show that PXR is expressed in the kidney and has protective effect against acute kidney injury (AKI). In this study, we investigated the role of PXR in CKD. Adenine diet-induced CKD (AD) model was established in wild-type and PXR humanized (hPXR) mice, respectively, which were treated with pregnenolone-16α-carbonitrile (PCN, 50 mg/kg, twice a week for 4 weeks) or rifampicin (RIF, 10 mg·kg-1·d-1, for 4 weeks). We showed that both PCN and RIF, which activated mouse and human PXR, respectively, improved renal function and attenuated renal fibrosis in the two types of AD mice. In addition, PCN treatment also alleviated renal fibrosis in unilateral ureter obstruction (UUO) mice. On the contrary, PXR gene deficiency exacerbated renal dysfunction and fibrosis in both adenine- and UUO-induced CKD mice. We found that PCN treatment suppressed the expression of the profibrotic Wnt7a and β-catenin in AD mice and in cultured mouse renal tubular epithelial cells treated with TGFβ1 in vitro. We demonstrated that PXR was colocalized and interacted with p53 in the nuclei of tubular epithelial cells. Overexpression of p53 increased the expression of Wnt7a, β-catenin and its downstream gene fibronectin. We further revealed that p53 bound to the promoter of Wnt7a gene to increase its transcription and β-catenin activation, leading to increased expression of the downstream profibrotic genes, which was inhibited by PXR. Taken together, PXR activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/β-catenin signalingpathway.

Phenobarbital in Nuclear Receptor Activation: An Update.

Phenobarbital (PB) is a commonly prescribed anti-epileptic drug that can also benefit newborns from hyperbilirubinemia. Being the first drug demonstrating hepatic induction of cytochrome P450 (CYP), PB has since been broadly used as a model compound to study xenobiotic-induced drug metabolism and clearance. Mechanistically, PB-mediated CYP induction is linked to a number of nuclear receptors, such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and estrogen receptor α, with CAR being the predominant regulator. Unlike prototypical agonistic ligands, PB-mediated activation of CAR does not involve direct binding with the receptor. Instead, dephosphorylation of threonine 38 in the DNA-binding domain of CAR was delineated as a key signaling event underlying PB-mediated indirect activation of CAR. Further studies revealed that such phosphorylation sites appear to be highly conserved among most human nuclear receptors. Interestingly, while PB is a pan-CAR activator in both animals and humans, PB activates human but not mouse PXR. The species-specific role of PB in gene regulation is a key determinant of its implication in xenobiotic metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In this review, we summarize the recent progress in our understanding of PB-provoked transactivation of nuclear receptors with a focus on CAR and PXR. SIGNIFICANCE STATEMENT: Extensive studies using PB as a research tool have significantly advanced our understanding of the molecular basis underlying nuclear receptor-mediated drug metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In particular, CAR has been established as a cell signaling-regulated nuclear receptor in addition to ligand-dependent functionality. This mini-review highlights the mechanisms by which PB transactivates CAR and PXR.

Pregnane X receptor (PXR) protects against cisplatin-induced acute kidney injury in mice

Cisplatin-induced acute kidney injury (CAKI) has been recognized as one of the most serious side effects of cisplatin. Pregnane X receptor (PXR) is a ligand-dependent nuclear receptor and serves as a master regulator of xenobiotic detoxification. Increasing evidence also suggests PXR has many other functions including the regulation of cell proliferation, inflammatory response, and glucose and lipid metabolism. In this study, we aimed to investigate the role of PXR in cisplatin-induced nephrotoxicity in mice. CAKI model was performed in wild-type or PXR knockout mice. Pregnenolone 16α‑carbonitrile (PCN), a mouse PXR specific agonist, was used for PXR activation. The renal function, biochemical, histopathological and molecular alterations were examined in mouse blood, urine or renal tissues. Whole transcriptome analysis was performed by RNA sequencing. We found that PXR activation significantly attenuated CAKI as reflected by improved renal function, reduced renal tubular apoptosis, ameliorated oxidative and endoplasmic reticulum stress, and suppressed inflammatory gene expression. RNA sequencing analysis revealed that the renoprotective effect of PXR was associated with multiple crucial signaling pathways, especially the PI3K/AKT pathway. In vitro study further revealed that PXR protected against cisplatin-induced apoptosis of cultured proximal tubule cells in a PI3K-dependent manner. Our results demonstrate that PXR activation can preserve renal function in cisplatin-induced AKI and suggest a possibility of PXR as a novel protective target for cisplatin-induced nephrotoxicity.

PXR is a target of (-)-epicatechin in skeletal muscle

(-)-Epicatechin (EC) is a flavanol that has shown numerous biological effects such as: decrease risk of cardiovascular dysfunction, metabolism regulation, skeletal muscle (SkM) performance improvement and SkM cells differentiation induction, among others. The described EC acceptor/receptor molecules do not explain the EC's effect on SkM. We hypothesize that the pregnane X receptor (PXR) can fulfill those characteristics, based on structural similitude between EC and steroidal backbone and that PXR activation leads to similar effects as those induced by EC. In order to demonstrate our hypothesis, we: 1) analyzed the possible EC and mouse PXR interaction through in silico strategies, 2) developed an EC's affinity column to isolate PXR, 3) evaluated, in mouse myoblast (C2C12 cells) the inhibition of EC-induced PXR's nucleus translocation by ketoconazole, a specific blocker of PXR and 4) analyzed the effect of EC as an activator of mouse PXR, evaluating the expression modulation of cytochrome 3a11 (Cyp3a11) gen and myogenin protein. (-)-Epicatechin interacts and activates PXR, promoting this protein translocation to the nucleus, increasing the expression of Cyp3a11, and promoting C2C12 cell differentiation through increasing myogenin expression. These results can be the base of further studies to analyze the possible participation of PXR in the skeletal muscle effects shown by EC.

SUN-042 PREGNANE X RECEPTOR (PXR) IS A NOVEL THERAPEUTIC TARGET FOR THE TREATMENT OF CISPLATIN-INDUCED NEPHROTOXICITY IN MICE

As a widely used chemotherapeutic agent for treating malignancies, the clinical application of cisplatin (CP) is limited by the toxicity to several vital human organs, especially nephrotoxicity. The pregnane X receptor (PXR) is a ligand-dependent transcriptional factor belonging to the nuclear receptor superfamily. PXR is highly expressed in the liver, intestine and kidney, where it plays an important role in xenobiotic and endobiotic metabolism. PXR is constitutively expressed in normal kidney with relatively high levels in the proximal tubular cells, which are initially affected in CP-induced nephrotoxicity. In this study, we aimed to investigate the role of PXR in CP-induced nephrotoxicity. In vivo, wild-type male C57BL/6 mice were orally pretreated with pregnenolone 16α-carbonitrile (PCN, 40mg/kg), a well-known mouse PXR agonist, for 3 days. After a single nephrotoxic dose of CP (20mg/kg) was administered at day 4, the mice were treated daily with PCN for another 3 days. Levels of blood urea nitrogen (BUN), serum creatinine (sCr), urinary N-acetyl-D-glucosaminidase (NAG) and urinary albumin were measured by commercial kits, high performance liquid chromatography (HPLC), fluorescence probe examination or enzyme linked immunosorbent assay (ELISA). Renal histopathologic features were evaluated by hematoxylin and eosin (H&E) staining on formalin-fixed kidney sections. Caspase 3 activity was measured in kidney homogenates, and TUNEL staining was performed on formalin-fixed sections. Expression levels of important genes involved in apoptosis, oxidative stress, and inflammation were investigated in kidney homogenates by immunoblot and real-time PCR. Whole transcriptome sequencing was applied to reveal the potential mechanisms of PXR protective effects to CP-induced nephrotoxicity. In vitro, PXR expression and activity were modulated by PCN, overexpression, or knockdown by shRNAs in mouse renal tubular epithelial (MCT) cells. A significant increase in BUN, sCr, urinary NAG and albumin levels induced by CP, was markedly prevented by PCN treatment. Histologically, PXR activation ameliorated CP-induced renal morphologic damage, especially in the proximal tubules, characterized by a large number of apoptotic cells. TUNEL-positive cells and renal activity of caspase 3 were both decreased in PCN treated mice. PXR activation abolished CP-induced p53 transcription and phosphorylation, and significantly reduced p53 protein levels. PXR activation also dramatically prevented oxidative and inflammatory stress in the kidneys of mice treated with CP. Whole transcriptome sequencing revealed that protective effects of PXR were associated with multiple crucial signaling pathways, including fatty acid metabolism and degradation, metabolism of xenobiotics by cytochrome P450, protein processing in endoplasmic reticulum, biosynthesis of unsaturated fatty acids, carbon metabolism, steroid hormone biosynthesis, PI3K-Akt signaling pathway, PPAR signaling pathway. Notably, we discovered that PXR protects against CP-induced nephrotoxicity by translational regulating multidrug and toxin extrusion 1 (MATE1), an important transporter mediating cellular excretion of CP, in the kidney. Our data provide evidence that PXR activation preserves renal function after CP treatment, and suggest the possibility of PXR as a novel therapeutic target for CP-induced nephrotoxicity.

CITCO Directly Binds to and Activates Human Pregnane X Receptor.

The xenobiotic receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are activated by structurally diverse chemicals to regulate the expression of target genes, and they have overlapping regulation in terms of ligands and target genes. Receptor-selective agonists are, therefore, critical for studying the overlapping function of PXR and CAR. An early effort identified 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) as a selective human CAR (hCAR) agonist, and this has since been widely used to distinguish the function of hCAR from that of human PXR (hPXR). The selectivity was demonstrated in a green monkey kidney cell line, CV-1, in which CITCO displayed >100-fold selectivity for hCAR over hPXR. However, whether the selectivity observed in CV-1 cells also represented CITCO activity in liver cell models was not hitherto investigated. In this study, we showed that CITCO: 1) binds directly to hPXR; 2) activates hPXR in HepG2 cells, with activation being blocked by an hPXR-specific antagonist, SPA70; 3) does not activate mouse PXR; 4) depends on tryptophan-299 to activate hPXR; 5) recruits steroid receptor coactivator 1 to hPXR; 6) activates hPXR in HepaRG cell lines even when hCAR is knocked out; and 7) activates hPXR in primary human hepatocytes. Together, these data indicate that CITCO binds directly to the hPXR ligand-binding domain to activate hPXR. As CITCO has been widely used, its confirmation as a dual agonist for hCAR and hPXR is important for appropriately interpreting existing data and designing future experiments to understand the regulation of hPXR and hCAR. SIGNIFICANCE STATEMENT: The results of this study demonstrate that 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is a dual agonist for human constitutive androstane receptor (hCAR) and human pregnane X receptor (hPXR). As CITCO has been widely used to activate hCAR, and hPXR and hCAR have distinct and overlapping biological functions, these results highlight the value of receptor-selective agonists and the importance of appropriately interpreting data in the context of receptor selectivity of such agonists.

CYP3A4 Induction in the Liver and Intestine of Pregnane X Receptor/CYP3A-Humanized Mice: Approaches by Mass Spectrometry Imaging and Portal Blood Analysis.

Induction of cytochrome P450 enzyme 3A (CYP3A) in response to pregnane X receptor (PXR) activators shows species-specific differences. To study the induction of human CYP3A in response to human PXR activators, we generated a double-humanized mouse model of PXR and CYP3A. CYP3A-humanized mice generated by using a mouse artificial chromosome (MAC) vector containing the entire genomic human CYP3A locus (hCYP3A-MAC mouse line) were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR, resulting in double-humanized mice (hCYP3A-MAC/hPXR mouse line). Oral administration of the human PXR activator rifampicin increased hepatic expression of CYP3A4 mRNA and triazolam (TRZ) 1'- and 4-hydroxylation activities, CYP3A probe activities, in the liver and intestine microsomes of hCYP3A-MAC/hPXR mice. The plasma concentration of TRZ after oral dosing was significantly decreased by rifampicin treatment in hCYP3A-MAC/hPXR mice but not in hCYP3A-MAC mice. In addition, mass spectrometry imaging analysis showed that rifampicin treatment increased the formation of hydroxy TRZ in the intestine of hCYP3A-MAC/hPXR mice after oral dosing of TRZ. The plasma concentration of 1'- and 4-hydroxy TRZ in portal blood was also increased by rifampicin treatment in hCYP3A-MAC/hPXR mice. These results suggest that the hCYP3A-MAC/hPXR mouse line may be a useful model to predict human PXR-dependent induction of metabolism of CYP3A4 substrates in the liver and intestine. SIGNIFICANCE STATEMENT: We generated a double-humanized mouse line for CYP3A and PXR. Briefly, CYP3A-humanized mice generated by using a mouse artificial chromosome vector containing the entire genomic human CYP3A locus were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR. Expression of CYP3A4 and metabolism of triazolam, a typical CYP3A substrate, in the liver of CYP3A/PXR-humanized mice were enhanced in response to rifampicin, a typical human PXR activator. Enhancement of triazolam metabolism in the intestine of CYP3A/PXR-humanized mice was firstly shown by combination of mass spectrometry imaging of sliced intestine and liquid chromatography with tandem mass spectrometry analysis of metabolite concentration in portal blood after oral dosing of triazolam.

Mechanistic Insights of Phenobarbital-Mediated Activation of Human but Not Mouse Pregnane X Receptor.

Phenobarbital (PB), a broadly used antiseizure drug, was the first to be characterized as an inducer of cytochrome P450 by activation of the constitutive androstane receptor (CAR). Although PB is recognized as a conserved CAR activator among species via a well-documented indirect activation mechanism, conflicting results have been reported regarding PB regulation of the pregnane X receptor (PXR), a sister receptor of CAR, and the underlying mechanisms remain elusive. Here, we show that in a human CAR (hCAR)-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of hCAR and human PXR (hPXR). In human primary hepatocytes and hCAR-KO HepaRG cells, PB-induced expression of CYP3A4 was markedly repressed by genetic knockdown or pharmacological inhibition of hPXR. Mechanistically, PB concentration dependently activates hPXR but not its mouse counterpart in cell-based luciferase assays. Mammalian two-hybrid assays demonstrated that PB selectively increases the functional interaction between the steroid receptor coactivator-1 and hPXR but not mouse PXR. Moreover, surface plasmon resonance binding affinity assay showed that PB directly binds to the ligand binding domain of hPXR (KD = 1.42 × 10-05). Structure-activity analysis further revealed that the amino acid tryptophan-299 within the ligand binding pocket of hPXR plays a key role in the agonistic binding of PB and mutation of tryptophan-299 disrupts PB activation of hPXR. Collectively, these data reveal that PB, a selective mouse CAR activator, activates both hCAR and hPXR, and provide novel mechanistic insights for PB-mediated activation of hPXR.

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.

Indirect activation of pregnane X receptor in the induction of hepatic CYP3A11 by high-dose rifampicin in mice

Rifampicin (RIF), a typical ligand of human pregnane X receptor (PXR), powerfully induces the expression of cytochrome P450 3A4 (CYP3A4) in humans. Although it is thought that RIF is not a ligand of rodent PXR, treatment with high-dose RIF (e.g. more than 20 mg/kg) increases the expression of CYP3A in the mouse liver. In this study, we investigated whether the induction of CYP3A by high-dose RIF in the mouse liver is mediated via indirect activation of mouse PXR (mPXR). The results showed that high-dose RIF increased the expression of CYP3A11 and other PXR-target genes in the liver of wild-type mice but not PXR-knockout mice. However, the results of reporter gene and ligand-dependent assembly assays showed that RIF does not activate mPXR in a ligand-dependent manner. In addition, high-dose RIF stimulated nuclear accumulation of mPXR in the mouse liver, and geldanamycin and okadaic acid attenuated the induction of Cyp3a11 and other PXR-target genes in primary hepatocytes, suggesting that high-dose RIF triggers nuclear translocation of mPXR. In conclusion, the present study suggests that high-dose RIF stimulates nuclear translocation of mPXR in the liver of mice by indirect activation, resulting in the transactivation of Cyp3a11 and other PXR-target genes.

Differential activation of pregnane X receptor by carnosic acid, carnosol, ursolic acid, and rosmarinic acid

Pregnane X receptor (PXR) regulates the expression of many genes, including those involved in drug metabolism and transport, and has been linked to various diseases, including inflammatory bowel disease. In the present study, we determined whether carnosic acid and other chemicals in rosemary extract (carnosol, ursolic acid, and rosmarinic acid) are PXR activators. As assessed in dual-luciferase reporter gene assays, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, activated human PXR (hPXR) and mouse PXR (mPXR), whereas carnosol and ursolic acid, but not carnosic acid or rosmarinic acid, activated rat PXR (rPXR). Dose-response experiments indicated that carnosic acid, carnosol, and ursolic acid activated hPXR with EC50 values of 0.79, 2.22, and 10.77μM, respectively. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, transactivated the ligand-binding domain of hPXR and recruited steroid receptor coactivator-1 (SRC-1), SRC-2, and SRC-3 to the ligand-binding domain of hPXR. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, increased hPXR target gene expression, as shown by an increase in CYP3A4, UGT1A3, and ABCB1 mRNA expression in LS180 human colon adenocarcinoma cells. Rosmarinic acid did not attenuate the extent of hPXR activation by rifampicin, suggesting it is not an antagonist of hPXR. Overall, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, are hPXR agonists, and carnosic acid shows species-dependent activation of hPXR and mPXR, but not rPXR. The findings provide new mechanistic insight on the effects of carnosic acid, carnosol, and ursolic acid on PXR-mediated biological effects.

PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor.

Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnane X receptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16α-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 invitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression.

Identification of Candidate Target <i>Cyp</i> Genes for microRNAs Whose Expression Is Altered by PCN and TCPOBOP, Representative Ligands of PXR and CAR

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mRNA post-transcriptional regulation. The deregulation of miRNAs affects the expression of drug-metabolizing enzymes, drug transporters, and nuclear receptors, all of which are important in regulating drug metabolism. miRNA expression can be altered by several endogenous or exogenous agents, such as steroid hormones, carcinogens, and therapeutic drugs. However, it is unclear whether hepatic miRNA expression is regulated by nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are indispensable for the expression of the CYPs. Here we investigated the effects of the mouse PXR and CAR ligands pregnenolone-16α-carbonitrile (PCN) and 1,4-bis[(3,5-dichloropyridin-2-yl)oxy]benzene (TCPOBOP) on hepatic miRNA expression in mice. We found that the expression of 9 miRNAs was increased (>2-fold) and of 4 miRNAs was decreased (>50%) in response to PCN, while TCPOBOP treatment led to the up-regulation of 8 miRNAs and down-regulation of 6 miRNAs. Using several miRNA target prediction algorithms, we found that the predicted target genes included several lesser known Cyp genes (Cyp1a1, Cyp1b1, Cyp2b10, Cyp2c38, Cyp2u1, Cyp4a12a/b, Cyp4v3, Cyp17a1, Cyp39a1, and Cyp51). We analyzed the expression of these genes in response to PCN and TCPOBOP and found changes in their mRNA levels, some of which were negatively correlated with the expression of their corresponding miRNAs, suggesting that miRNAs may play a role in regulating Cyp enzyme expression. Further studies will be required to fully elucidate the miRNA regulatory mechanisms that contribute to modulating CYP expression.