Pregnane X Receptor mRNA Levels Research Articles

Role of nuclear pregnane X receptor in Cu-induced lipid metabolism and xenobiotic responses in largemouth bass (Micropterus salmoides).

The pregnane X receptor (PXR) is a master xenobiotic-sensing receptor in response to toxic byproducts, as well as a key regulator in intermediary lipid metabolism. Therefore, the present study was conducted to investigate the potential role of PXR in mediating the lipid dysregulation and xenobiotic responses under Cu-induced stress in largemouth bass (Micropterus salmoides). Four groups of largemouth bass (52.66 ± 0.03 g) were treated with control, Cu waterborne (9.44 μmol/L), Cu+RIF (Rifampicin, 100 mg/kg, PXR activator), and Cu+KET (Ketoconazole, 20 mg/kg, PXR inhibitor) for 48 h. Results showed that Cu exposure significantly elevated the plasma stress indicators and triggered antioxidant systems to counteract Cu-induced oxidative stress. Acute Cu exposure caused liver steatosis, as indicated by the significantly higher levels of plasma triglycerides (TG), lipid droplets, and mRNA levels of lipogenesis genes in the liver. Liver injuries were detected, as shown by hepatocyte vacuolization and severe apoptotic signals after Cu exposure. Importantly, Cu exposure significantly stimulated mRNA levels of PXR, suggesting the response of this regulator in the xenobiotic response. The pharmacological intervention of PXR by the agonist and antagonist significantly altered hepatic mRNA levels of PXR, implying that RIF and KET were effective agents of PXR in largemouth bass. Administration of RIF significantly exacerbated liver steatosis, and such alterations were dependent on the regulations on pparγ and cd36 rather than srebp1 signaling, which suggested that PXR-PPARγ might be another pathway for Cu-induced lipid deposition in fish. Whereas, KET administration showed reverse effects on lipid metabolism as indicated by the lower hepatic TG levels, suppressed mRNA levels of pparγ and cd36. Activation of PXR stimulated autophagy and inhibited apoptosis, leading to lower hepatic vacuolization; while inhibition of PXR showed higher apoptotic signals, inhibition of autophagic genes and stimulation of apoptotic genes. Taken together, PXR played a cytoprotective role in Cu-induced hepatotoxicity through regulations on autophagy and apoptosis. Overall, our data has demonstrated for the first time on the dual roles of PXR as a co-regulator in mediating xenobiotic responses and lipid metabolism in fish, which implying the potential of PXR as a therapy target for xenobiotics-induced lipid dysregulation and hepatotoxicity.

Adenosine deaminases acting on RNA modulate the expression of the human pregnane X receptor

The pregnane X receptor (PXR) is one of the major transcription factors that regulate the expression of different drug-metabolizing enzymes and transporters. Adenosine-to-inosine RNA editing, the most frequent nucleotide conversion on RNA, which is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, may modulate gene expression and function. Here, we investigated the potential regulation of human PXR expression by adenosine-to-inosine RNA editing. Knockdown of ADAR1 increased PXR mRNA level, and the knockdown of ADAR1 or ADAR2 significantly increased PXR protein level in HepaRG cells. In HepG2 cells, the knockdown of ADAR1 or ADAR2 significantly increased PXR mRNA and protein levels. The increase in the PXR protein by ADAR1 knockdown resulted in increased cytochrome P450 3A4 (CYP3A4) transactivity and CYP3A4 and UDP-glucuronosyltransferase 1A1 (UGT1A1) expression. A reporter assay revealed that the 3′-untranslated region (UTR) of PXR mRNA, especially from +3371 to +3440, is responsible for the ADAR-mediated post-transcriptional control of PXR expression, despite the lack of RNA edited sites in this region. Collectively, we found that PXR is negatively regulated by ADAR1 via an indirect mechanism, which facilitates the degradation of PXR mRNA. We could demonstrate that ADAR1 can cause interindividual variability in hepatic drug metabolism potencies.

Effects of 27 natural products on drug metabolism genes in channel catfish (Ictalurus punctatus) cell line

Pregnane X receptor (PXR) as a ligand dependent transcription factor, is capable of regulating gene expression of cytochromes P450 and transporters involved in xenobiotic/drug metabolism and elimination. Due to the species differences in the regulatory specificity of PXR, gene regulation should not be extrapolated from mammal to fish without research data.The aim of present study was to investigate the effect of 27 natural products on PXR, CYP3A30 and MDR1 genes in channel catfish (Ietalurus punetaus) kidney cells (CC-K). The results showed that bisdemethoxycurcumin, glycyrrhetnic acid, rotenone, artemisinin, dihydroartemisinin, ligustilide and matrine strongly induced the mRNA levels of PXR. Additionally, the up-regulation of CYP3A30 gene ran parallel with PXR gene after the treatment of demethoxycurcumin, glycyrrhetnic acid, artemisinin, matrine, baicalein, schisantherin A, ligustilide, and dihydroartemisinin. Moreover, we found that natural products schisandrin A, schisandrin B, schisandrol A, and schisandrol B significantly up-regulated the mRNA level of MDR1 gene.Our work with a view to provide experimental data support for further research, which will make for the rational application of natural products in channel catfish, such as to avoid adverse herb-drug interactions or accelerating the residue elimination of chemical medicine.

Diurnal Variation of Nuclear Receptors in Mice with or without Fasting

Nuclear receptors such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR) regulate the transcription of transporter and cytochrome P450 (CYP). The diurnal variation was observed in some transporters regulated by nuclear receptors. We investigated whether diurnal variation in PXR and CAR exists in mice. We also examined the effect of food intake on the diurnal rhythm of hepatic PXR and CAR using fed and fasted mice. In liver and small intestine of fed mice, the mRNA levels of PXR and CAR were unchanged between 7:00 AM and 7:00 PM. In contrast to fed mice, fasting mice partly exhibited the diurnal variation in PXR, not in CAR. The mRNA levels of PXR at 7:00 AM were significantly higher than that those at 7:00 PM in liver of fasting mice. These results indicated the different effects of fasting in mice on diurnal variation of PXR in each tissue.

Lower Expression of HNF4α and PGC1α Might Impair Rifampicin-mediated CYP3A4 Induction under Conditions Where PXR Is Overexpressed in Human Fetal Liver Cells

Pregnane X receptor (PXR) mRNA was detected in HepG2 cells by RT-PCR, but not in human fetal liver (HFL) cells. CYP3A4 was induced by rifampicin (RIF), mifepristone (RU486), clotrimazole (CTZ), and dexamethasone (DEX) in HepG2 cells, while these PXR ligands with the exception of DEX did not induce CYP3A4 mRNA expression in HFL cells. Ad-PXR infection increased mRNA levels of PXR and CYP3A4 in both cells despite the absence of PXR ligands. Similar results were observed in reporter gene assays. However, in HFL cells, RIF-mediated CYP3A4 induction was insufficient compared with HepG2 cells, despite PXR overexpression. The expression levels of five coactivators (HNF4α, PGC1α, SRC1, CBP, and P300) related to CYP3A4 expression in HepG2, HFL cells, and human adult liver were analyzed by RT-PCR. Expression levels of HNF4α and PGC1α in HFL cells were downregulated to 20% of those in the human adult liver. On the other hand, the expression level of HNF4α in HepG2 cells was higher than that in HFL cells, although PGC1α expression level was almost the same as that in HFL cells. HNF4α mRNA expression level in HepG2 cells was 57% of that in human adult liver, and the level in HFL cells was 30% of that in HepG2 cells. These results suggested that lower expression of HNF4α and PGC1α may impair RIF-mediated CYP3A4 induction under conditions of PXR overexpression in HFL cells.

Interactions of pharmaceuticals and other xenobiotics on hepatic pregnane X receptor and cytochrome P450 3A signaling pathway in rainbow trout ( Oncorhynchus mykiss)

The pregnane X receptor (PXR) belongs to the nuclear hormone receptor (NR) superfamily and is commonly described as a xenophore or a pharmacophore, as it can be activated by a wide array of xenobiotics, including numerous pharmaceuticals and other environmental pollutants. The PXR regulates expression of e.g. cytochrome P450 3A (CYP3A) and the P-glycoprotein (P-gp) that are involved in excretion of lipophilic xenobiotics and endobiotics. A full length PXR cDNA was isolated from rainbow trout liver and it was expressed in a descending order of magnitude in liver > intestine > kidney > heart. A rainbow trout PXR reporter assay was developed and a suite of pharmaceuticals and other xenobiotics were screened. However, no specific activation of rainbow trout PXR was observed with the substances tested. Interactions of prototypical PXR agonists on PXR signaling in rainbow trout were further investigated in cells of hepatic origin exposed in vitro and in juvenile rainbow trout exposed in vivo. The rainbow trout hepatoma cell line (RTH-149), displayed 600 times lower expression of CYP3A mRNA compared to primary cultures of hepatocytes, and did not respond to treatment with either pregnenolone 16α-carbonitrile (PCN), ketoconazole (KCZ) or rifampicin (RIF), which implies a non-functional PXR in this cell line. Exposure of hepatocytes to PCN and lithocholic acid (LA), resulted in a weak concentration-dependent induction of CYP3A and P-gp mRNA levels, though, exposure to the higher concentration of LA (50 μM) decreased PXR mRNA levels. Exposure to dexamethasone (DEX) resulted in a decrease in PXR mRNA, without affecting CYP3A mRNA levels in hepatocytes in vitro. Injections of rainbow trout in vivo with 1 mg LA/kg fish resulted in a slight (albeit not significant) increase in CYP3A mRNA levels without affecting PXR mRNA levels. Although, injection with 10 mg omeprazole (OME)/kg fish had no effect on PXR and CYP3A mRNA levels, a 60% inhibition of CYP3A enzyme activities was evident. An in vitro screening of the chemicals used showed that OME and RIF acted as weak CYP3A inhibitors whereas LA and DEX did not affect the CYP3A activity. In contrast, PCN acted as an activator of the CYP3A enzyme activity in vitro. Taken together, these data show that some prototypical PXR agonists weakly affect PXR activation in rainbow trout. Besides, some of these agonists have a stronger effect on the CYP3A catalyst. This study demonstrates the importance of investigation effects of pharmaceuticals on the PXR signaling pathway in non-target animals such as fish.

Drug metabolizing enzyme induction pathways in experimental non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a disease that compromises hepatic function and the capacity to metabolize numerous drugs. Aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor-E2 related factor 2 (Nrf2) are xenobiotic activated transcription factors that regulate induction of a number of drug metabolizing enzymes (DMEs). The purpose of the current study was to determine whether experimental NASH alters the xenobiotic activation of these transcription factors and induction of downstream DME targets Cyp1A1, Cyp2B10, Cyp3A11, Cyp4A14 and NAD(P)H:quinone oxidoreductase 1 (Nqo1), respectively. Mice fed normal rodent chow or methionine-choline-deficient (MCD) diet for 8 weeks were then treated with microsomal enzyme inducers beta-naphoflavone (BNF), 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), pregnenolone-16alpha-carbonitrile (PCN), clofibrate (CFB) or oltipraz (OPZ), known activators of AhR, CAR, PXR, PPARalpha and Nrf2, respectively. Results of this study show that (1) Hepatic PXR mRNA levels were significantly increased (1.4-fold) in mice fed MCD diet, while AhR, CAR, PPARalpha and Nrf2 were not affected. (2) The MCD diet did not alter hepatic inducibility of Cyp1A1, Cyp2B10, Cyp3A11 mRNA levels by their respective microsomal inducers. (3) Constitutive levels of Cyp4A14 mRNA were significantly increased in mice fed the MCD diet, yet further induction by clofibrate was not observed. (4) Hepatic Nqo1 mRNA levels were significantly increased by the MCD diet; however, additional induction of Nqo1 was still achievable following treatment with the Nrf2 activator OPZ.

Decreased PXR and CAR inhibit transporter and CYP mRNA Levels in the liver and intestine of mice with collagen-induced arthritis

Nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the transcription of transporters and cytochrome P450s (CYPs). We investigated whether quantitative and functional changes in PXR and CAR affected the transporters and CYPs in a mouse model of chronic arthritis. The mRNA levels of PXR were significantly decreased in the intestine of mice with collagen-induced arthritis (CIA) compared with control mice. The mRNA levels of CAR were significantly decreased in both the liver and intestine of CIA mice. The mRNA levels of Mdr1a/1b, Mrp3, BCRP and Cyp2b10 were decreased in the liver of CIA mice, while little change in the mRNA levels was observed for Cyp3a11 in the liver and the transporters in the intestine. Taken together, the present results reveal that the effects of CAR mRNA suppression on the regulation of transporters and CYPs differ between the liver and intestine in chronic arthritis.

Decreased P-glycoprotein (P-gp/MDR1) expression in inflamed human intestinal epithelium is independent of PXR protein levels

Altered P-glycoprotein expression (P-gp/MDR1) and/or function may contribute to the pathogenesis of gastrointestinal inflammatory disorders. Low intestinal mRNA levels of the pregnane X receptor (PXR) have been linked to low MDR1 mRNA levels in patients with ulcerative colitis (UC). Here we compared intestinal MDR1 mRNA and protein expression in uninflamed and inflamed intestinal epithelium (IE) of patients with gastrointestinal inflammatory disorders to healthy controls. Intestinal mucosal biopsies were obtained from patients with Crohn's disease (CD, n = 20), UC (n = 10), diverticulitis (n = 3), collagenous colitis (n = 3), and healthy controls (n = 10). MDR1, iNOS, MRP1, CYP3A4, and PXR expression was determined using real-time reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting, and/or immunohistochemistry. Furthermore, MDR1 expression was determined in human intestinal biopsies and the human colon carcinoma cell line DLD-1 after exposure to cytokines (TNF-alpha, IFN-gamma, and/or IL-1beta). MDR1 mRNA levels in uninflamed colon of UC patients were comparable to healthy controls, while they were slightly decreased in ileum and slightly increased in colon of CD patients. MDR1 expression, however, was strongly decreased in inflamed IE of CD, UC, collagenous colitis, and diverticulitis patients. A cytokine-dependent decrease of MDR1 expression was observed in human intestinal biopsies, but not in DLD-1 cells. Remarkably, PXR protein levels were equal in uninflamed and inflamed tissue of CD and UC patients despite low PXR mRNA levels in inflamed tissue. MDR1 expression is strongly decreased in inflamed IE of patients with gastrointestinal disorders and this is independent of PXR protein levels. Low MDR1 levels may aggravate intestinal inflammation.

Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response

Expression of P-450 (Cyp) enzymes is reduced in liver during the acute phase response, contributing to the decrease in bile acid levels and drug metabolism during infection. Nuclear hormone receptors CAR and PXR are key transactivators of Cyp2b and Cyp3a genes, respectively. Injection of bacterial lipopolysaccharide (LPS) induced the expected reduction in Cyp2b10 and Cyp3a mRNA levels in mouse liver. These decreases were associated with a marked reduction in CAR and PXR mRNA levels within 4 h following treatment. LPS-induced CAR and PXR repression were dose-dependent and sustained for at least 16 h. LPS treatment also reversed the up-regulation of Cyp3a in mice pre-treated with PXR ligand RU486. In addition, we observed a concomitant decrease in RXR (retinoid X receptor) mRNA levels, the obligatory partner of both CAR and PXR for high affinity binding to DNA. These findings represent one possible molecular mechanism underlying sepsis-induced repression of Cyp enzymes.