St. John's Wort alleviates dextran sodium sulfate-induced colitis through pregnane X receptor-dependent NFκB antagonism.

St. John's wort (SJW), from traditional herbs, activates the pregnane X receptor (PXR), a potential drug target for treating inflammatory bowel disease (IBD). However, how SJW alleviates dextran sodium sulfate (DSS)-induced experimental IBD by activating PXR is unknown. To test this, PXR-humanized, wild-type (WT) and Pxr-null mice, primary intestinal organoids cultures, and the luciferase reporter gene assays were employed. In vivo, a diet supplemented with SJW was found to activate intestinal PXR both in WT and PXR-humanized mice, but not in Pxr-null mice. SJW prevented DSS-induced IBD in PXR-humanized and WT mice, but not in Pxr-null mice. In vitro, hyperforin, a major component of SJW, activated PXR and suppressed tumor necrosis factor (TNF)α-induced nuclear factor (NF) κB translocation in primary intestinal organoids from PXR-humanized mice, but not Pxr-null mice. Luciferase reporter gene assays showed that hyperforin dose-dependently alleviated TNFα-induced NFκB transactivation by activating human PXR in Caco2 cells. Furthermore, SJW therapeutically attenuated DSS-induced IBD in PXR-humanized mice. These data indicate the therapeutic potential of SJW in alleviating DSS-induced IBD in vivo, and TNFα-induced NFκB activation in vitro, dependent on PXR activation, which may have clinical implications for using SJW as a herbal drug anti-IBD treatment.

The Role of Vitamin K in Cholestatic Liver Disease.

Vitamin K (VK) is a ligand of the pregnane X receptor (PXR), which plays a critical role in the detoxification of xenobiotics and metabolism of bile acids. VK1 may reduce the risk of death in patients with chronic liver failure. VK deficiency is associated with intrahepatic cholestasis, and is already being used as a drug for cholestasis-induced liver fibrosis in China. In Japan, to treat osteoporosis in patients with primary biliary cholangitis, VK2 formulations are prescribed, along with vitamin D3. Animal studies have revealed that after bile duct ligation-induced cholestasis, PXR knockout mice manifested more hepatic damage than wild-type mice. Ligand-mediated activation of PXR improves biochemical parameters. Rifampicin is a well-known human PXR ligand that has been used to treat intractable pruritus in severe cholestasis. In addition to its anti-cholestatic properties, PXR has anti-fibrotic and anti-inflammatory effects. However, because of the scarcity of animal studies, the mechanism of the effect of VK on cholestasis-related liver disease has not yet been revealed. Moreover, the application of VK in cholestasis-related diseases is controversial. Considering this background, the present review focuses on the effect of VK in cholestasis-related diseases, emphasizing its function as a modulator of PXR.

Helix 12 stabilization contributes to basal transcriptional activity of PXR.

Pregnane X receptor (PXR) plays an important role in xenobiotic metabolism. While ligand binding induces PXR-dependent gene transcription, PXR shows constitutive transcriptional activity in the absence of ligands when expressed in cultured cells. This constitutive activity sometimes hampers investigation of PXR activation by compounds of interest. In this study, we investigated the molecular mechanism of PXR activation. In the reported crystal structures of unliganded PXR, helix 12 (H12), including a coactivator binding motif, was stabilized, while it is destabilized in the unliganded structures of other nuclear receptors, suggesting a role for H12 stabilization in the basal activity of PXR. Since Phe420, located in the loop between H11 and H12, is thought to interact with Leu411 and Ile414 to stabilize H12, we substituted alanine at Phe420 (PXR-F420A) and separately inserted three alanine residues directly after Phe420 (PXR-3A) and investigated their influence on PXR-mediated transcription. Reporter gene assays demonstrated that the mutants showed drastically reduced basal activity and enhanced responses to various ligands, which was further enhanced by coexpression of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1α. Mutations of both Leu411 and Ile414 to alanine also suppressed basal activity. Mammalian two-hybrid assays showed that PXR-F420A and PXR-3A bound to corepressors and coactivators in the absence and presence of ligands, respectively. We conclude that the intramolecular interactions of Phe420 with Leu411 and Ile414 stabilize H12 to recruit coactivators even in the absence of ligands, contributing to the basal transcriptional activity of PXR. We propose that the generated mutants might be useful for PXR ligand screening.

Regulation of CAR and PXR Expression in Health and Disease.

Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are members of the nuclear receptor superfamily that mainly act as ligand-activated transcription factors. Their functions have long been associated with the regulation of drug metabolism and disposition, and it is now well established that they are implicated in physiological and pathological conditions. Considerable efforts have been made to understand the regulation of their activity by their cognate ligand; however, additional regulatory mechanisms, among which the regulation of their expression, modulate their pleiotropic effects. This review summarizes the current knowledge on CAR and PXR expression during development and adult life; tissue distribution; spatial, temporal, and metabolic regulations; as well as in pathological situations, including chronic diseases and cancers. The expression of CAR and PXR is modulated by complex regulatory mechanisms that involve the interplay of transcription factors and also post-transcriptional and epigenetic modifications. Moreover, many environmental stimuli affect CAR and PXR expression through mechanisms that have not been elucidated.

Associations between Pregnane X Receptor and Breast Cancer Growth and Progression.

Pregnane X receptor (PXR, NR1I2) is a member of the ligand-activated nuclear receptor superfamily. This receptor is promiscuous in its activation profile and is responsive to a broad array of both endobiotic and xenobiotic ligands. PXR is involved in pivotal cellular detoxification processes to include the regulation of genes that encode key drug-metabolizing cytochrome-P450 enzymes, oxidative stress response, as well as enzymes that drive steroid and bile acid metabolism. While PXR clearly has important regulatory roles in the liver and gastrointestinal tract, this nuclear receptor also has biological functions in breast tissue. In this review, we highlight current knowledge of PXR's role in mammary tumor carcinogenesis. The elevated level of PXR expression in cancerous breast tissue suggests a likely interface between aberrant cell division and xeno-protection in cancer cells. Moreover, PXR itself exerts positive effect on the cell cycle, thereby predisposing tumor cells to unchecked proliferation. Activation of PXR also plays a key role in regulating apoptosis, as well as in acquired resistance to chemotherapeutic agents. The repressive role of PXR in regulating inflammatory mediators along with the existence of genetic polymorphisms within the sequence of the PXR gene may predispose individuals to developing breast cancer. Further investigations into the role that PXR plays in driving tumorigenesis are needed.

Nutritional status modifies pregnane X receptor regulated transcriptome.

Pregnane X receptor (PXR) regulates glucose and lipid metabolism, but little is known of the nutritional regulation of PXR function. We investigated the genome wide effects of the nutritional status on the PXR mediated gene regulation in the liver. Mice were treated with a PXR ligand pregnenolone 16α-carbonitrile (PCN) for 4 days and subsequently either fasted for 5 hours or after 4-hour fast treated with intragastric glucose 1 hour before sample collection. Gene expression microarray study indicated that PCN both induced and repressed much higher number of genes in the glucose fed mice and the induction of multiple well-established PXR target genes was potentiated by glucose. A subset of genes, including bile acid synthesis gene Cyp8b1, responded in an opposite direction during fasting and after glucose feeding. PXR knockout abolished these effects. In agreement with the Cyp8b1 regulation, PCN also modified the bile acid composition in the glucose fed mice. Contribution of glucose, insulin and glucagon on the observed nutritional effects was investigated in primary hepatocytes. However, only mild impact on PXR function was observed. These results show that nutritional status modifies the PXR regulated transcriptome both qualitatively and quantitatively and reveal a complex crosstalk between PXR and energy homeostasis.

Glucocorticoids Increase Renal Excretion of Urate in Mice by Downregulating Urate Transporter 1.

Both nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids have been widely used for the treatment of gout, a disease promoted by an excess body burden of uric acid (UA); however, their effects on the homeostasis of UA remain poorly understood. The present study showed that 1-week treatments with three NSAIDs (ibuprofen, diclofenac, and indomethacin) had little effect on UA homeostasis in mice, whereas 1-week low doses (1 and 5 mg/kg) of dexamethasone (DEX) significantly decreased serum UA by about 15%. Additionally, low doses of DEX also resulted in increases in hepatic UA concentration and urinary UA excretion, which were associated with an induction of xanthine oxidoreductase (XOR) in the liver and a downregulation of urate transporter 1 (URAT1) in the kidney, respectively. Neither 75 mg/kg DEX nor 100 mg/kg pregnenolone-16α-carbonitrile altered UA concentrations in serum and livers of mice, suggesting that the effect of DEX on UA homeostasis was not due to the pregnane X receptor pathway. Further in vitro studies demonstrated that glucocorticoid receptor (GR) was involved in DEX-mediated downregulation of URAT1. Knockdown of both p65 and c-Jun completely blocked the effect of DEX on URAT1, suggesting that GR regulates URAT1 via its interaction with both nuclear factor κB and activator protein 1 signaling pathways. To conclude, the present study identifies, for the first time, a critical role of glucocorticoids in regulating UA homeostasis and elucidates the mechanism for GR-mediated regulation of URAT1 in mice. SIGNIFICANCE STATEMENT: This study demonstrates, for the first time, a critical role of glucocorticoid receptor in regulating urate transporter 1 in mouse kidney.

The pregnane X receptor and its microbiota-derived ligand indole 3-propionic acid regulate endothelium-dependent vasodilation.

We proposed that circulating metabolites generated by the intestinal microbiota can affect vascular function. One such metabolite, indole 3-propionic acid (IPA), can activate the pregnane X receptor(PXR), a xenobiotic-activated nuclear receptor present in many tissues, including the vascular endothelium. We hypothesized that IPA could regulate vascular function by modulating PXR activity. To test this, Pxr+/+ mice were administered broad-spectrum antibiotics for 2 wk with IPA supplementation. Vascular function was evaluated by bioassay using aorta and pulmonary artery ring tissue from antibiotic-treated Pxr+/+ and Pxr-/-mice, supplemented with IPA, and using aorta tissue maintained in organ culture for 24 h in the presence of IPA. Endothelium-dependent, nitric oxide(NO)-mediated muscarinic and proteinase-activated receptor 2(PAR2)-stimulated vasodilation was assessed. Endothelial nitric oxide synthase (eNOS) abundance was evaluated in intact tissue or in aorta-derived endothelial cell cultures from Pxr+/+ and Pxr-/- mice, and vascular Pxr levels were assessed in tissues obtained from Pxr+/+ mice treated with antibiotics and supplemented with IPA. Antibiotic-treated Pxr+/+ mice exhibited enhanced agonist-induced endothelium-dependent vasodilation, which was phenocopied by tissues from either Pxr-/- or germ-free mice. IPA exposure reduced the vasodilatory responses in isolated and cultured vessels. No effects of IPA were observed for tissues obtained from Pxr-/- mice. Serum nitrate levels were increased in antibiotic-treated Pxr+/+and Pxr-/- mice. eNOS abundance was increased in aorta tissues and cultured endothelium from Pxr-/- mice. PXR stimulation reduced eNOS expression in cultured endothelial cells from Pxr+/+ but not Pxr-/- mice. The microbial metabolite IPA, via the PXR, plays a key role in regulating endothelial function. Furthermore, antibiotic treatment changes PXR-mediated vascular endothelial responsiveness by upregulating eNOS.

Nuclear Receptors Regulate Intestinal Inflammation in the Context of IBD.

Gastrointestinal (GI) homeostasis is strongly dependent on nuclear receptor (NR) functions. They play a variety of roles ranging from nutrient uptake, sensing of microbial metabolites, regulation of epithelial intestinal cell integrity to shaping of the intestinal immune cell repertoire. Several NRs are associated with GI pathologies; therefore, systematic analysis of NR biology, the underlying molecular mechanisms, and regulation of target genes can be expected to help greatly in uncovering the course of GI diseases. Recently, an increasing number of NRs has been validated as potential drug targets for therapeutic intervention in patients with inflammatory bowel disease (IBD). Besides the classical glucocorticoids, especially PPARγ, VDR, or PXR-selective ligands are currently being tested with promising results in clinical IBD trials. Also, several pre-clinical animal studies are being performed with NRs. This review focuses on the complex biology of NRs and their context-dependent anti- or pro-inflammatory activities in the regulation of gastrointestinal barrier with special attention to NRs already pharmacologically targeted in clinic and pre-clinical IBD treatment regimens.

Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis.

BACKGROUND & AIMS: The most prescribed non-nucleoside reverse transcriptase inhibitor, efavirenz, has been associated with elevated risk of dyslipidemia and hepatic steatosis in HIV-infected patients but the underlying mechanisms remain elusive. Herein, we investigated the role of pregnane X receptor (PXR) in mediating the adverse effects of efavirenz on lipid homeostasis. METHODS: Cell-based reporter assays, primary cell culture, and multiple mouse models including conditional knockout and humanized mice were combined to study the impact of efavirenz on PXR activities and lipid homeostasis in vitro and in vivo. A novel liver-specific Pxr knockout mouse model was also generated to determine the contribution of hepatic PXR signaling to efavirenz-elicited dyslipidemia and hepatic steatosis. RESULTS: We found that efavirenz is a potent PXR-selective agonist that can efficiently activate PXR and induce its target gene expression in vitro and in vivo. Treatment with efavirenz-induced hypercholesterolemia and hepatic steatosis in mice but deficiency of hepatic PXR abolished these adverse effects. Interestingly, efavirenz-mediated PXR activation regulated the expression of several key hepatic lipogenic genes including fatty acid transporter CD36 and cholesterol biosynthesis enzyme squalene epoxidase (SQLE), leading to increased lipid uptake and cholesterol biosynthesis in hepatic cells. While CD36 is a known PXR target gene, we identified a DR-2-type of PXR-response element in the SQLE promoter and established SQLE as a direct transcriptional target of PXR. Since PXR exhibits considerable differences in its pharmacology across species, we also confirmed these findings in PXR-humanized mice and human primary hepatocytes. CONCLUSIONS: The widely prescribed antiretroviral drug efavirenz induces hypercholesterolemia and hepatic steatosis by activating PXR signaling. Activation of PXR should be taken into consideration for patients undergoing long-term treatment with PXR agonistic antiretroviral drugs. LAY SUMMARY: Efavirenz is widely prescribed for HIV-infected patients but has some side effects. It can increase lipid levels in patients' blood and liver. Here we show that efavirenz can activate a unique liver protein called PXR which mediates the adverse effects of efavirenz on lipid levels in mouse models.

Effect of panaxytriol on cytochrome P450 3A4 via the pregnane X receptor regulatory pathway.

Panaxytriol (PXT) is one of the major effective components of red ginseng and Shenmai injection. The present study aimed to explore the effect of PXT on cytochrome P450 3A4 (CYP3A4) based on the pregnane X receptor (PXR)-CYP3A4 regulatory pathway in HepG2 cells and hPXR-overexpressing HepG2 cells treated with PXT for different time periods using quantitative polymerase chain reaction, Western blot, and dual-luciferase reporter gene assays. PXT could upregulate the levels of PXR and CYP3A4 mRNA in HepG2 cells treated with PXT for 1 hr, with no impact on the expression of their protein levels. The expression levels of both PXR and CYP3A4 mRNA and protein in HepG2 cells treated with PXT for 24 hr increased in a concentration-dependent manner. The effects of PXT on the expression of PXR and CYP3A4 mRNA and protein in hPXR-overexpressing HepG2 cells were similar to those in HepG2 cells. Moreover, the influence trend of PXT on CYP3A4 was consistent with that of PXR in HepG2 cells and hPXR-overexpressing HepG2 cells. The dual-luciferase reporter gene assay in HepG2 cells further demonstrated that PXT treatment for specific time periods could significantly induce the expression of CYP3A4 mediated by the PXR regulatory pathway.

Porcine interleukin-6 enhances the expression of CYP2C33 through a constitutive androstane receptor/retinoid X receptor-mediated pathway.

Cytochrome P450, which is expressed in humans and other animals, is a superfamily of drug-metabolizing enzymes that play important roles in the metabolism of endogenous and xenobiotic substrates via oxidation, peroxidation and reduction. Of endogenous substrates, interleukin (IL)-6 is a crucial cytokine involved in inflammation in the liver. The present study aims to elucidate the mechanisms through which IL-6 modulates cytochrome P450 expression. CYP2C33 expression was found to be increased in HepLi cells and primary porcine hepatocytes treated with IL-6 in a concentration-dependent manner. IL-6 treatment also increased the expression of the transcriptional regulators, constitutive androstane receptor (CAR) and pregnane X receptor. Overexpression of CAR promoted CYP2C33 expression at the mRNA and protein levels, whereas knockdown of CAR by small interfering RNA reduced CYP2C33 expression. Luciferase assays showed that IL-6 treatment of HepLi cells and primary porcine hepatocytes increased CYP2C33 promoter activity. Co-immunoprecipitation and western blotting demonstrated that CAR and RXR could form heterodimers. IL-6 affects CYP2C33 expression through CAR/retinoid X receptor (RXR) heterodimers.

Pregnane X Receptor Regulates Liver Size and Liver Cell Fate by Yes-Associated Protein Activation in Mice.

Activation of pregnane X receptor (PXR), a nuclear receptor that controls xenobiotic and endobiotic metabolism, is known to induce liver enlargement, but the molecular signals and cell types responding to PXR-induced hepatomegaly remain unknown. In this study, the effect of PXR activation on liver enlargement and cell change was evaluated in several strains of genetically modified mice and animal models. Lineage labeling using AAV-Tbg-Cre-treated Rosa26EYFP mice or Sox9-CreERT , Rosa26EYFP mice was performed and Pxr-null mice or AAV Yap short hairpin RNA (shRNA)-treated mice were used to confirm the role of PXR or yes-associated protein (YAP). Treatment with selective PXR activators induced liver enlargement and accelerated regeneration in wild-type (WT) and PXR-humanized mice, but not in Pxr-null mice, by increase of cell size, induction of a regenerative hybrid hepatocyte (HybHP) reprogramming, and promotion of hepatocyte and HybHP proliferation. Mechanistically, PXR interacted with YAP and PXR activation induced nuclear translocation of YAP. Blockade of YAP abolished PXR-induced liver enlargement in mice. Conclusion: These findings revealed a function of PXR in enlarging liver size and changing liver cell fate by activation of the YAP signaling pathway. These results have implications for understanding the physiological functions of PXR and suggest the potential for manipulation of liver size and liver cell fate.

ETS-1 induces Sorafenib-resistance in hepatocellular carcinoma cells via regulating transcription factor activity of PXR.

Transcription factor E26 transformation specific sequence 1 (ETS-1) is a primary regulator in the metastasis of human cancer cells, especially hepatocellular carcinoma (HCC) cells; and it would affect the prognosis of HCC patients who received chemotherapies. However, the regulatory role of ETS-1 in the resistance of HCC cells to molecular-targeting agent remains poorly understood. In the present work, we demonstrate that high ETS-1 expression correlates with poor prognosis of advanced HCC patients received Sorafenib treatment. Mechanistically, ETS-1 binds to nuclear Pregnane X receptor (PXR) directly and enhances PXR's transcription factor activity, which further leads to the induction of the PXR's downstream multi-drug resistance related genes. Overexpression of ETS-1 accelerates the metabolic clearance of Sorafenib in HCC cells and leads to the better survival and faster migration of those cells. The therapeutic studies show that ETS-1 promotes the Sorafenib-resistance of HCC tumor models and ETS-1 blockade enhances the anti-tumor capacity of Sorafenib by decreasing PXR activation. Thus, our study suggests that ETS-1 could enhance the activation of PXR and be a potential therapeutic target for overcoming Sorafenib resistance in HCC treatment.

Amelioration of PXR-mediated CYP3A4 induction by mGluR2 modulators.

This work describes the rational amelioration of Cytochrome P450 4/5 (CYP3A4/5) induction through the Pregnane-X Receptor (PXR) pathway in a series of compounds that modulate the metabotropic glutamate Receptor 2 (mGluR2) via an allosteric mechanism. The compounds were initially shown to induce CYP3A4/5 via the gold-standard induction assay measured in primary human hepatocytes. This was followed up by testing the compounds in a PXR assay which correlated well with the assay in primary cells. Further, one of the compounds was crystallized with PXR (pdb code 6DUP). Analysis of this co-crystal structure, together with previously published PXR co-crystal structures, lead to modification ideas. The compounds synthesized based on these ideas were shown not to be CYP3A4/5 inducers. The mGluR2 activity of the resulting compounds was maintained.

PXR: More Than Just a Master Xenobiotic Receptor.

Pregnane X receptor (PXR) is a nuclear receptor considered to be a master xenobiotic receptor that coordinately regulates the expression of genes encoding drug-metabolizing enzymes and drug transporters to essentially detoxify and eliminate xenobiotics and endotoxins from the body. In the past several years, the function of PXR in the regulation of xenobiotic metabolism has been extensively studied, and the role of PXR as a xenobiotic sensor has been well established. It is now clear, however, that PXR plays many other roles in addition to its xenobiotic-sensing function. For instance, recent studies have discovered previously unidentified roles of PXR in inflammatory response, cell proliferation, and cell migration. PXR also contributes to the dysregulation of these processes in diseases states. These recent discoveries of the role of PXR in the physiologic and pathophysiologic conditions of other cellular processes provides the possibility of novel targets for drug discovery. This review highlights areas of PXR regulation that require further clarification and summarizes the recent progress in our understanding of the nonxenobiotic functions of PXR that can be explored for relevant therapeutic applications.