Pregnane X Receptor Knockout Research Articles

Abstract T P225: Loss of Pregnane X Receptor Impairs Stroke Recovery

Introduction: Inflammation plays an important role in exacerbating injury following stroke, but current understanding of the factors regulating this inflammation is incomplete. Pregnane X Receptor (PXR) is a nuclear receptor which binds many xenobiotic targets including many commonly prescribed medications. Recent work has shown that PXR is crucial for maintaining the integrity of the intestinal epithelial barrier as well repressing inflammatory responses mediated by NFκB. Since both these pathways can modify the peripheral immune response, we hypothesized that PXR would play an important role in limiting inflammation following stroke. Using PXR knockout mice, we show that loss of PXR is detrimental for stroke recovery. Materials & Methods: Male PXR knockout mice and littermate controls underwent sham surgery or 60min middle cerebral artery occlusion (MCAO). Weight loss was monitored post-stroke and mice were sacrificed after 7 days for splenic weights, infarct analysis by cresyl violet staining, and bacterial translocation as assessed by culture of homogenized mesenteric lymph nodes on blood agar plates. Results: Following MCAO, PXR-/- mice lost significantly more weight than wild type littermate controls (n=6 vs. 4 respectively; p<0.05) and also showed greater splenic atrophy 7 days following MCAO (2.14mg/g body weight vs. 3.40mg/g body weight; p<0.05). However, preliminary studies suggest PXR-/- mice do not have larger infarcts (n=3 vs. 2) and did not exhibit more bacterial translocation 7 days following MCAO (5.4 vs. 7 CFU). Discussion: PXR is known to negatively regulate NFκB signaling and maintain intestinal barrier integrity. Both of these mechanisms may contribute to delayed recovery following stroke with PXR acting as a master switch. In summary, this early work begins to explore the importance of a xenobiotic sensor, PXR, in stroke recovery.

Role of Pregnane X Receptor in Obesity and Glucose Homeostasis in Male Mice

Clinical obesity is a complex metabolic disorder affecting one in three adults. Recent reports suggest that pregnane X receptor (PXR), a xenobiotic nuclear receptor important for defense against toxic agents and for eliminating drugs and other xenobiotics, may be involved in obesity. Noting differences in ligand specificities between human and mouse PXRs, the role of PXR in high fat diet (HFD)-induced obesity was examined using male PXR-humanized (hPXR) transgenic and PXR-knock-out (PXR-KO) mice in comparison to wild-type (WT) mice. After 16 weeks on either a control diet or HFD, WT mice showed greater weight gain, whereas PXR-KO mice gained less weight due to their resistance to HFD-induced decreases in adipose tissue peroxisome proliferator-activated receptor α and induction of hepatic carnitine palmitoyltransferase 1, suggesting increased energy metabolism. Interestingly, control-fed PXR-KO mice exhibited hepatomegaly, hyperinsulinemia, and hyperleptinemia but hypoadiponectinemia and lower adiponectin receptor R2 mRNA levels relative to WT mice. Evaluation of these biologic indicators in hPXR mice fed a control diet or HFD revealed further differences between the mouse and human receptors. Importantly, although HFD-fed hPXR mice were resistant to HFD-induced obesity, both PXR-KO and hPXR mice exhibited impaired induction of glucokinase involved in glucose utilization and displayed elevated fasting glucose levels and severely impaired glucose tolerance. Moreover, the basal hepatic levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 were increased in hPXR mice compared with WT mice. Altogether, although the mouse PXR promotes HFD-induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and thus provides a model for exploring the role of human PXR in the metabolic syndrome.

Bioimaging Real-Time PXR-Dependent mdr1a Gene Regulation in mdr1a.fLUC Reporter Mice

The MDR1 gene encodes P-glycoprotein, a transmembrane drug efflux transporter that confers multidrug resistance in cancer cells and affects drug pharmacokinetics by virtue of its expression in the liver, kidney, and colon. Nuclear receptors human steroid and xenobiotic receptor (SXR) and constitutive androstane receptor (CAR) are possible master regulators of xenobiotic-inducible MDR1 expression in drug processing organs, but the mechanism of MDR1 regulation has yet to be directly demonstrated in vivo. Moreover, it has previously been impossible to determine the sustained or cumulative effect of repeated doses of xenobiotics on in vivo MDR1 expression. We previously reported a mouse model containing firefly luciferase (fLUC) knocked into the mdr1a genomic locus, allowing noninvasive bioimaging of intestinal mdr1a gene expression in live animals. In the current study, we crossed mdr1a.fLUC mice into the pxr knockout (pxr(-/-)) genetic background and injected mice with pregnenolone-16α-carbonitrile (PCN), a strong mouse pregnane X receptor (PXR) ligand, and two therapeutically relevant taxanes, paclitaxel and docetaxel. All three agents induced mdr1a.fLUC expression (bioluminescence), but only PCN and docetaxel appeared to act primarily via PXR. Luminescence returned to baseline by 24-48 hours after drug injection and was reinducible over two additional rounds of drug dosing in pxr(+/+) mice. TCPOBOP, a CAR ligand, modestly induced mdr1a.fLUC in pxr(+/+) and pxr(-/-) strains, consistent with CAR's minor role in mdr1a regulation. Collectively, these results demonstrate that the mdr1a.fLUC bioimaging model can capture changes in mdr1 gene expression under conditions of repeated xenobiotic treatment in vivo and that it can be used to probe the mechanism of gene regulation in response to different xenobiotic agents.

Role of pregnane X receptor (PXR) in diet‐induced obesity

Clinical obesity is a complex metabolic disorder that affects 1 in 3 adults. While a high‐fat diet (HFD) causes obesity and insulin resistance, the specific genes that determine sensitivity to obesity are not known. Recent reports suggest that the nuclear receptor pregnane X receptor (PXR), a xenobiotic receptor that defends against toxic agents, may also play a role in obesity: PXR ligands alter patient plasma lipid levels and affect lipid homeostatic and energy metabolic gene expression. Because of differences in ligand specificity between human and mouse PXRs, we examined the role of PXR in diet‐induced obesity using male PXR‐humanized (hPXR) transgenic and PXR‐knockout (PXR‐KO) vs. wild type (WT) mice. After 16 weeks of high (45%) or normal (12%) fat diets, WT mice had a higher susceptibility to obesity compared to hPXR and PXR‐KO mice. PXR‐KO mice showed the smallest gain in white adipose tissue mass. In contrast, only HFD‐fed hPXR mice had elevated serum glucose and triglycerides and reduced hepatic adiponectin, even though all three genotypes on a HFD displayed insulin resistance, hyperleptinemia and hepatotoxicity. Taken together, our data indicate that while the mouse PXR promoted HFD‐induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and can be used to explore the role of human PXR in metabolic syndrome. [Supported by NIH grants RO1 HL064761, R25HL059868, P20 MD000175, and SC1 HL099139]

Epithelial expression of the orphan nuclear receptor PXR is critical for the maintenance of gut mucosal barrier function

The gut epithelium is exposed to a significant and diverse microbial flora and in cooperation both provide a first line of innate immune defense. Logically, breach in the epithelium is likely to contribute significantly to gut pathophysiology (e.g., inflammatory bowel disease (IBD)). Clinical, epidemiological and experimental evidence supports an association between IBD and host environment. Recent literature suggests that orphan nuclear receptors (e.g. LXR, FXR and PPAR-γ) may serve as a mechanistic link between host environments and gut immunity. Indeed, activation of pregnane X receptor (PXR), an adopted orphan nuclear receptor has been shown to inhibit inflammation in a mouse colitis model. We hypothesize that PXR is an important and essential candidate that acts as a xenosensor and translates environmental cues into a protective epithelial phenotype. While PXR knock-out (pxr-/-) mice demonstrate no obvious gross gut abnormality, ultra-structural and functional studies of these mice gut reveal a distinctly “leaky” pathology. These mice show up-regulation of several Toll-like receptors (specifically TLR4) and pro-inflammatory signaling. Experiments with bone marrow-chimeras and pxr-/-/tlr4-/- double knock-out mice show that the barrier defect in PXR knock-out mice is due to epithelium-specific aberrant TLR4 signaling. We have also shown that PXR activation represses TLR4 expression and signaling and found that gut commensal-derived indoles can act as an endogenous PXR ligand. Our results suggest that gut commensals maintain immune homeostasis through PXR mediated TLR4 gene regulation in the epithelium and novel therapeutics may utilize methods to target such interactions in IBD.

Deficiency of PXR decreases atherosclerosis in apoE-deficient mice

The pregnane X receptor (PXR, also known as SXR) is a nuclear hormone receptor activated by xenobiotics as well as diverse sterols and their metabolites. PXR functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Recent evidence indicates that PXR may also play an important role in lipid homeostasis and atherosclerosis. To define the role of PXR in atherosclerosis, we generated PXR and apoE double knockout (PXR(-/-)apoE(-/-)) mice. Here we show that deficiency of PXR did not alter plasma triglyceride and cholesterol levels in apoE(-/-) mice. However, PXR(-/-)apoE(-/-) mice had significantly decreased atherosclerotic cross-sectional lesion area in both the aortic root and brachiocephalic artery by 40% (P < 0.01) and 60% (P < 0.001), respectively. Interestingly, deficiency of PXR reduced the expression levels of CD36, lipid accumulation, and CD36-mediated oxidized LDL uptake in peritoneal macrophages of PXR(-/-)apoE(-/-) mice. Furthermore, immunofluorescence staining showed that PXR and CD36 were expressed in the atherosclerotic lesions of apoE(-/-) mice, and the expression levels of PXR and CD36 were diminished in the lesions of PXR(-/-)apoE(-/-) mice. Our findings indicate that deficiency of PXR attenuates atherosclerosis development, which may result from decreased CD36 expression and reduced lipid uptake in macrophages.

Abstract 701: A mechanistic study of mdr1a gene regulation in vivo using bioimaging

Abstract The human P-glycoprotein (Pgp) encoded by the MDR1 gene is as a transmembrane drug transporter that mediates the efflux of drugs from cells. It confers multidrug resistance in cancer cells that overexpress MDR1. In addition, basal expression of MDR1 in organs such as liver, kidney, and colon affects the pharmacokinetics of drug uptake and excretion for agents that are substrates for Pgp transport. Nuclear receptors such as SXR and CAR have been suggested as possible master regulators of xenobiotic- and drug-inducible expression of MDR1 and of other genes involved in drug metabolism in drug processing organs. However, the mechanism of MDR1 regulation in tumors or in normal, non-malignant tissue, particularly in response to xenobiotics, has yet to be demonstrated in vivo. To facilitate the mechanistic study of MDR1 gene regulation in vivo, we previously reported a mouse model that allows non-invasive bioimaging of the mdr1 gene expression in vivo and in real time by inserting a firefly luciferase (fLUC) gene into the murine mdr1a genetic locus by homologous recombination.1 To determine the role of PXR, the murine homologue of SXR, in drug-mediated induction of mdr1a, we crossed mdr1a.fLUC mice with PXR knockout mice. Pregnenolone-16α-carbonitrile (PCN), a known SXR substrate, induced intestinal mdr1a.fLUC expression in the PXR wild type background but not the PXR knockout background. Notably, mdr1a.fLUC was re-inducible over three rounds of drug dosing, which could be measured longitudinally in our real-time assessment of gene expression. TCPOBOP, a known CAR ligand, induced mdr1a.fLUC in both the wild type and knockout background, consistent with the idea that mdr1a is also regulated by the CAR nuclear receptor. The chemotherapeutic agent docetaxel induced mdr1a.fLUC in the PXR wild type background but induction was dramatically reduced in the PXR KO background; paclitaxel gave only low-level induction in both genetic backgrounds. This result is consistent with the reported ligand specificity of mouse PXR. Future studies in a CAR-null background will determine if the observed effect of paclitaxel on mdr1a expression is mediated through CAR. These studies demonstrate the utility of our murine imaging model for the in vivo study of mdr1 gene regulation in real time. Studies with additional chemotherapeutic drugs and genetic backgrounds will establish the variety of agents capable of inducing mdr1a at the transcriptional level and the trans-acting factors that are involved in such induction. 1Gu et al. Proc Natl Acad Sci 106:5394-5399 (2009) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2011-701

Gestational and Pregnane X Receptor-Mediated Regulation of Placental ATP-Binding Cassette Drug Transporters in Mice

The ATP-binding cassette (ABC) drug transporters in the placenta are involved in controlling the exchange of endogenous and exogenous moieties. Pregnane X receptor (PXR) is a nuclear receptor that regulates the hepatic expression of several key ABC transporters, but it is unclear whether PXR is involved in the regulation of these transporters in the placenta. This study explores the role of PXR in the regulation of placental drug transporters. The placental mRNA expression of Mdr1a, Bcrp, and Mrp1, 2, and 3 was examined in PXR knockout (-/-), heterozygote (+/-), and wild-type (+/+) mice by quantitative PCR. The impact of PXR activation was examined in pregnant pregnane-16α-carbonitrile (PCN)-treated mice. Compared with that in controls, the basal expression of Mdr1a, Bcrp, Mrp1, and Mrp2 was significantly higher in (+/-) and (-/-) mice. Alterations in the expression of mdr1a, bcrp, and mrp1, 2, and 3 between gestational day (GD) 10 and GD 17 was dissimilar between (+/+) and (-/-) mice. Although PCN treatment induced maternal and fetal hepatic expression of Cyp3a11; placental expression of transporters were not significantly changed. Overall, our results suggest a repressive role of PXR in the basal expression of several placental transporters and a tissue-specific induction of these target genes after PXR activation.

Pregnane X receptor knockout mice display osteopenia with reduced bone formation and enhanced bone resorption

The steroid and xenobiotic receptor (SXR) and its murine ortholog pregnane X receptor (PXR) are nuclear receptors that are expressed mainly in the liver and intestine where they function as xenobiotic sensors. In addition to its role as a xenobiotic sensor, previous studies in our laboratories and elsewhere have identified a role for SXR/PXR as a mediator of bone homeostasis. Here, we report that systemic deletion of PXR results in marked osteopenia with mechanical fragility in female mice as young as 4 months old. Bone mineral density (BMD) of PXR knockout (PXRKO) mice was significantly decreased compared with the BMD of wild-type (WT) mice. Micro-computed tomography analysis of femoral trabecular bones revealed that the three-dimensional bone volume fraction of PXRKO mice was markedly reduced compared with that of WT mice. Histomorphometrical analysis of the trabecular bones in the proximal tibia showed a remarkable reduction in bone mass in PXRKO mice. As for bone turnover of the trabecular bones, bone formation is reduced, whereas bone resorption is enhanced in PXRKO mice. Histomorphometrical analysis of femoral cortical bones revealed a larger cortical area in WT mice than that in PXRKO mice. WT mice had a thicker cortical width than PXRKO mice. Three-point bending test revealed that these morphological phenotypes actually caused mechanical fragility. Lastly, serum levels of phosphate, calcium, and alkaline phosphatase were unchanged in PXRKO mice compared with WT. Consistent with our previous results, we conclude that SXR/PXR promotes bone formation and suppresses bone resorption thus cementing a role for SXR/PXR as a key regulator of bone homeostasis.

Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice

The nuclear hormone receptor pregnane X receptor (PXR; also called SXR) functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Although many clinically relevant PXR ligands have been shown to affect cholesterol levels, the role of PXR in cholesterol homeostasis and atherosclerosis has not been thoroughly investigated. Here, we report that activation of PXR by feeding the PXR agonist pregnenolone 16alpha-carbonitrile (0.02%) for 2 weeks to wild-type (WT) mice significantly increased total cholesterol levels and atherogenic lipoproteins VLDL and LDL levels, but had no effect in PXR knockout (PXR(-/-)) mice. Chronic PXR activation in atherosclerosis prone apolipoprotein E deficient (ApoE(-/-)) mice was found to decrease HDL levels and increase atherosclerotic cross-sectional lesion area at both the aortic root and in the brachiocephalic artery by 54% (P < 0.001) and 116% (P < 0.01), respectively. PXR activation significantly regulated genes in the liver involved in lipoprotein transportation and cholesterol metabolism, including CD36, ApoA-IV, and CYP39A1, in both WT and ApoE(-/-) mice. Furthermore, PXR activation can increase CD36 expression and lipid accumulation in peritoneal macrophages of ApoE(-/-) mice. In summary, PXR activation in WT mice increases levels of the atherogenic lipoproteins VLDL and LDL, whereas in ApoE(-/-) mice, PXR increases atherosclerosis, perhaps by diminishing levels of the antiatherogenic ApoA-IV and increasing lipid accumulation in macrophages.

The pregnane X receptor: from bench to bedside

Background: The pregnane X receptor (PXR; NR1I2), a member of the nuclear receptor superfamily, regulates the expression of metabolic enzymes and transporters involved in the response of mammals to their chemical environment. Objective: To summarize the functions and clinical implications of PXR. Methods: In the current review, the clinical implications of PXR are discussed, and the use of genetically engineered PXR mouse models is highlighted. Results/conclusion: Recent advances in mouse models, including Pxr-null and PXR-humanized mice, provide in vivo tools for evaluating the physiological functions of PXR and its role in controlling xenobiotic metabolism and transport. By using the PXR knockout and humanized mouse models, PXR was found to influence drug–drug interactions, hepatic steatosis, and the homeostasis of vitamin D, bile acids, and steroid hormones. PXR was also shown to influence inflammatory bowel diseases.

Gender Dictates the Nuclear Receptor-Mediated Regulation of CYP3A44

The CYP3As are broad-spectrum drug-metabolizing enzymes that are collectively responsible for more than 50% of xenobiotic metabolism. Unlike other CYP3As, murine CYP3A44 is expressed predominantly in the female liver, with much lower levels in male livers and no detectable expression in brain or kidney in either gender. In this study, we examined the role of nuclear hormone receptors in the regulation of Cyp3a44 gene expression. Interestingly, we observed differential effects of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) -mediated activation of Cyp3a44 gene expression, which was gender-specific. For example, activation of PXR by pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone (DEX) induced CYP3A44 mRNA levels in a PXR-dependent fashion in male mice, whereas no induction was detected in female mice. In contrast, PCN and DEX down-regulated CYP3A44 expression in female PXR null animals. Similar to PXR, CAR activation also showed a male-specific induction with no effect on CYP3A44 levels in females. When PXR knockout mice were challenged with the CAR activator phenobarbital, a significant up-regulation of male CYP3A44 levels was observed, whereas levels in females remained unchanged. We conclude that gender has a critical impact on PXR- and CAR-mediated effects of CYP3A44 expression.

Hepatic Cytochrome P450 Gene Regulation during Endotoxin-Induced Inflammation in Nuclear Receptor Knockout Mice

Inflammatory agents such as lipopolysaccharide (LPS) down-regulate the hepatic expression of many cytochrome P450 (P450) mRNAs and proteins. Previous studies suggested that suppression of some P450 mRNAs could involve the regulation or modulation of the nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) or pregnane X receptor (PXR). To determine the involvement of these receptors in P450 down-regulation, PPARalpha knockout (KO), PXR KO, and appropriate wild-type (WT) mice were administered either saline or 1 mg/kg LPS. Hepatic mRNA and protein expression of several P450 isoforms, interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF) alpha, alpha1-acid glycoprotein (AGP), and fibrinogen (FBG) were examined 16 h later. LPS administration significantly decreased the hepatic expression of CYP1A2, 2A5, 2C29, 2E1, 3A11, 4A10, and 4A14 mRNAs in both groups of PPARalpha and PXR mice, whereas CYP3A13 mRNA was increased slightly in PPARalpha WT and KO mice, but not in PXR mice. Effects of LPS administration on mouse hepatic P450 proteins (probed using rat P450 2C, 3A, 4A, and 2E antibodies) were consistent with mRNA results in most cases. LPS treatment significantly increased IL-1beta, IL-6, TNFalpha, AGP, and FBG mRNA in both PPARalpha and PXR mice, with the greatest effect observed with TNFalpha. Because decreases in P450 mRNA expression were essentially identical in both WT and KO mice for both nuclear receptors, these data indicate that down-regulation of P450 during inflammation does not require the nuclear receptors PPARalpha and PXR.

The Ratio of Constitutive Androstane Receptor to Pregnane X Receptor Determines the Activity of Guggulsterone against the Cyp2b10 Promoter

Guggulsterone is the active ingredient in gugulipid, an organic extract of the Commiphora mukul plant. Gugulipid has been used for nearly 3000 years in Ayurvedic medicine, mainly as a treatment for arthritis. Herbal practitioners currently use gugulipid therapy in conditions as diverse as rheumatism, coronary artery disease, arthritis, hyperlipidemia, acne, and obesity. The active ingredient in gugulipid is guggulsterone, a plant sterol compound recently identified as a pregnane X receptor (PXR; NR1I2) ligand. We show herein that guggulsterone treatment represses the expression of cytochrome P450 2b10 (Cyp2b10) gene expression by inhibiting constitutive androstane receptor (CAR; NR1I3) activity in hepatocytes lacking functional PXR (PXR-knockout). We also show that PXR-CAR cross-talk determines the net activity of guggulsterone treatment toward Cyp2b10 gene expression. Using mammalian two-hybrid assays, we show that treatment with guggulsterone differentially affects protein cofactor recruitment to these two nuclear receptors. These data identify guggulsterone as an inverse agonist of the nuclear receptor CAR. When viewed together with the data showing that PXR and CAR expression is highly variable in different ethnic populations and that CAR expression is under the control of a circadian rhythm, our data provide important insight into the molecular mechanism of interindividual variability of drug metabolism. These data, together with the recent resolution of the crystal structures of PXR and CAR, will likely aid in the rational design of more specific CAR inverse agonists that are currently viewed as potential antiobesity drugs.

Nuclear receptors constitutive androstane receptor and pregnane X receptor ameliorate cholestatic liver injury

Cholestasis is associated with accumulation of bile acids and lipids, and liver injury. The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic nuclear receptors that coordinate protective hepatic responses to potentially toxic stimuli, including bile acids. We investigated the role of these receptors in the regulation of bile acid and lipid metabolism in a bile duct ligation (BDL) model of cholestasis applied to receptor knockout mice. Hepatic damage from bile acid accumulation was increased in both CAR knockout (CARKO) and PXR knockout mice, but bile acid concentrations were lower in CARKO mice. High-density lipoprotein (HDL) cholesterol was elevated in CARKO mice, and serum total cholesterol increased less in CARKO or PXR knockout mice than WT mice after BDL. Gene expression analysis of the BDL knockout animals demonstrated that, in response to cholestasis, PXR and CAR both repressed and induced the specific hepatic membrane transporters Oatp-c (organic anion transporting polypeptide C) and Oatp2 (Na+-dependent organic anion transporter 2), respectively. Induction of the xenobiotic transporter multidrug resistance protein 1 in cholestasis was independent of either PXR or CAR, in contrast to the known pattern of induction of multidrug resistance protein 1 by xenobiotics. These results demonstrate that CAR and PXR influence cholesterol metabolism and bile acid synthesis, as well as multiple detoxification pathways, and suggest their potential role as therapeutic targets for the treatment of cholestasis and lipid disorders.