Pregnenolone 16α-carbonitrile Research Articles

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.

The Basis for Strain-Dependent Rat Aldehyde Dehydrogenase 1A7 (ALDH1A7) Gene Expression.

Aldehyde hydrogenases (ALDHs) belong to a large gene family involved in oxidation of both endogenous and exogenous compounds in mammalian tissues. Among ALDHs, the rat ALDH1A7 gene displays a curious strain dependence in phenobarbital (PB)-induced hepatic expression: the responsive RR strains exhibit induction of both ALDH1A7 and CYP2B mRNAs and activities, whereas the nonresponsive rr strains show induction of CYP2B only. Here, we investigated the responsiveness of ALDH1A1, ALDH1A7, CYP2B1, and CYP3A23 genes to prototypical P450 inducers, expression of nuclear receptors CAR and pregnane X receptor, and structure of the ALDH1A7 promoter in both rat strains. ALDH1A7 mRNA, associated protein and activity were strongly induced by PB and modestly induced by pregnenolone 16α-carbonitrile in the RR strain but negligibly in the rr strain, whereas induction of ALDH1A1 and P450 mRNAs was similar between the strains. Reporter gene and chromatin immunoprecipitation assays indicated that the loss of ALDH1A7 inducibility in the rr strain is profoundly linked with a 16-base pair deletion in the proximal promoter and inability of the upstream DNA sequences to recruit constitutive androstane receptor-retinoid X receptor heterodimers. SIGNIFICANCE STATEMENT: Genetic variation in rat ALDH1A7 promoter sequences underlie the large strain-dependent differences in expression and inducibility by phenobarbital of the aldehyde dehydrogenase activity. This finding has implications for the design and interpretation of pharmacological and toxicological studies on the effects and disposition of aldehydes.

Epigenetic Memory Is Involved in the Persistent Alterations of Drug-Processing Genes in Adult Mice Due to PCN-Activated PXR During Early Life.

Pregnane X receptor (PXR), which can be activated by xenobiotic chemicals (including pediatric drugs), plays a key role in the regulation of drug-processing genes (DPGs). The induction of DPGs due to PXR activation may reduce therapeutic efficacy or cause toxicity. This work aims to demonstrate the impact of pregnenolone 16α-carbonitrile (PCN)-mediated PXR activation during early life on DPGs expression and drug sensitivity in adulthood, as well as the underlying mechanism. In this study, mice were sacrificed at postnatal day 60 to detect the hepatic expression of selected DPGs and histone modifications in the Cyp3a11 promoter. We found that all doses of PCN treatment (50-200 mg/kg/day) at postnatal days 5-8 resulted in persistently increased CYP2B10 expression, whereas only high doses of PCN treatment (150 and 200 mg/kg/day) persistently induced the expression of CYP3A11, 1A2, and UGT1A1. We also demonstrated that PCN treatment before postnatal day 15 had a long-term impact on the expression of CYP3A11, 2B10, ABCC4, and PAPSS2. Additionally, elevated expression of CYP3A11, SULT2A1, UGT1A1, and PAPSS2 was observed in PCN-treated groups at days 25-28. Attenuated inducibility of CYP3A11 by PCN was seen in the primary hepatocytes derived from PCN-pretreated mice. Moreover, enhanced H3K4me3 level and reduced H3K27me3 level in the PXR response elements (PXREs) of the Cyp3a11 promoter may contribute to the persistent upregulation of CYP3A11 by neonatal PCN treatment. Overall, our study suggests that PXR activation during early life could persistently alter the hepatic expression of DPGs and epigenetic memory may be an underlying mechanism in mice.

Gene Expression Profiling Reveals that PXR Activation Inhibits Hepatic PPARα Activity and Decreases FGF21 Secretion in Male C57Bl6/J Mice.

The pregnane X receptor (PXR) is the main nuclear receptor regulating the expression of xenobiotic-metabolizing enzymes and is highly expressed in the liver and intestine. Recent studies have highlighted its additional role in lipid homeostasis, however, the mechanisms of these regulations are not fully elucidated. We investigated the transcriptomic signature of PXR activation in the liver of adult wild-type vs. Pxr-/- C57Bl6/J male mice treated with the rodent specific ligand pregnenolone 16α-carbonitrile (PCN). PXR activation increased liver triglyceride accumulation and significantly regulated the expression of 1215 genes, mostly xenobiotic-metabolizing enzymes. Among the down-regulated genes, we identified a strong peroxisome proliferator-activated receptor α (PPARα) signature. Comparison of this signature with a list of fasting-induced PPARα target genes confirmed that PXR activation decreased the expression of more than 25 PPARα target genes, among which was the hepatokine fibroblast growth factor 21 (Fgf21). PXR activation abolished plasmatic levels of FGF21. We provide a comprehensive signature of PXR activation in the liver and identify new PXR target genes that might be involved in the steatogenic effect of PXR. Moreover, we show that PXR activation down-regulates hepatic PPARα activity and FGF21 circulation, which could participate in the pleiotropic role of PXR in energy homeostasis.

Comparison of the hepatic metabolism of triazolam in wild-type andCyp3a-knockout mice for understanding CYP3A-mediated metabolism inCYP3A-humanised mice in vivo

1. To investigate cytochrome P450 3A (CYP3A)-mediated metabolism in vivo, plasma concentrations of triazolam (TRZ) are often monitored as a CYP3A marker in CYP3A-humanised mice. However, it has not been determined whether plasma concentrations of TRZ after intravenous administration can reflect hepatic CYP3A activity in CYP3A-humanised mice.2. Firstly, we investigated the pharmacokinetics of TRZ in wild-type and Cyp3a-knockout (Cyp3a-KO) mice. Plasma concentration profiles of TRZ and α-hydroxy (OH) TRZ were very similar in wild-type and Cyp3a-KO mice. On the other hand, AUC of 4-OH TRZ in Cyp3a-KO mice was significantly lower than that in wild-type mice. Pregnenolone 16α-carbonitrile (PCN) decreased the areas under the plasma concentration-time curves (AUCs) of TRZ and α-OH TRZ in both groups. There was no significant effect of PCN on AUC of 4-OH TRZ in Cyp3a-KO mice.3. Next, we verified that AUC of 4-OH TRZ in CYP3A-humanised mice was higher than that in Cyp3a-KO mice, although the difference was not significant.4. In conclusion, plasma concentrations of 4-OH TRZ, but not those of TRZ and α-OH TRZ, might reflect hepatic CYP3A activity in mice in vivo. These results provide important insights for in vivo studies using a CYP3A-humanised model.

Constitutive androstane receptor and pregnane X receptor cooperatively ameliorate DSS-induced colitis

BackgroundNuclear receptor pregnane X receptor (PXR) was shown to be protective in case of dextran sulfate sodium (DSS)-induced colitis. Constitutive androstane receptor (CAR) belongs to the same nuclear receptor subfamily with PXR. The roles of both receptors in DSS-induced colitis were evaluated. MethodsWild-type, Car-null, Pxr-null, and Car/Pxr-null mice were treated with a CAR/PXR agonist or vehicle and administered 2.5% DSS in the drinking water. The typical clinical symptoms, histological scoring, proinflammatory cytokine, and apoptosis were analyzed. ResultsMice treated with the PXR agonist pregnenolone-16α-carbonitrile (PCN) were protected from DSS-induced colitis, as in a previous study. Mice treated with the CAR agonist, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were also protected from DSS-induced colitis. Interestingly, the protective effects of PCN in the Car-null mice and those of TCPOBOP in the Pxr-null mice both decreased. PCN or TCPOBOP pretreatment significantly decreased the macrophage and monocyte infiltration in DSS-induced colitis. PXR and CAR agonists reduced the mRNA expression of several proinflammatory cytokines in a PXR- and CAR-dependent manner, respectively. CAR inhibited apoptosis by inducing Gadd45b. PXR inhibited TNF-α and IL-1b and CAR induced Gadd45b in in vitro cell analyses. ConclusionsWe showed that CAR and PXR cooperatively ameliorate DSS-induced colitis. PXR and CAR protected against DSS-induced colitis by inhibiting proinflammatory cytokines and apoptosis, respectively.

The connection of lateral preferences with the specificity of cardiac rhythm variability in children

Nuclear receptor pregnane X receptor (PXR) was shown to be protective in case of dextran sulfate sodium (DSS)-induced colitis. Constitutive androstane receptor (CAR) belongs to the same nuclear receptor subfamily with PXR. The roles of both receptors in DSS-induced colitis were evaluated.Wild-type, Car-null, Pxr-null, and Car/Pxr-null mice were treated with a CAR/PXR agonist or vehicle and administered 2.5% DSS in the drinking water. The typical clinical symptoms, histological scoring, proinflammatory cytokine, and apoptosis were analyzed.Mice treated with the PXR agonist pregnenolone-16α-carbonitrile (PCN) were protected from DSS-induced colitis, as in a previous study. Mice treated with the CAR agonist, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were also protected from DSS-induced colitis. Interestingly, the protective effects of PCN in the Car-null mice and those of TCPOBOP in the Pxr-null mice both decreased. PCN or TCPOBOP pretreatment significantly decreased the macrophage and monocyte infiltration in DSS-induced colitis. PXR and CAR agonists reduced the mRNA expression of several proinflammatory cytokines in a PXR- and CAR-dependent manner, respectively. CAR inhibited apoptosis by inducing Gadd45b. PXR inhibited TNF-α and IL-1b and CAR induced Gadd45b in in vitro cell analyses.We showed that CAR and PXR cooperatively ameliorate DSS-induced colitis. PXR and CAR protected against DSS-induced colitis by inhibiting proinflammatory cytokines and apoptosis, respectively.

Effect of Pregnenolone 16α-Carbonitrile on the Expression of P-Glycoprotein in the Intestine, Brain and Liver of Mice.

P-Glycoprotein (P-gp), encoded by the MDR1 (ABCB1) gene in humans and by Mdr1a and Mdr1b genes in rodents, is a member of the superfamily of ATP-binding cassette transporters. Since P-gp is constitutively expressed in numerous tissues and exhibits a broad specificity in substrate recognition, it can play a crucial role in limiting the absorption and distribution of xenobiotics by decreasing their intracellular accumulation. The expression of P-gp is regulated by various nuclear receptors such as pregnane X receptor (PXR). Although the characterization of P-gp induction by PXR ligands is a crucial goal for predicting pharmacokinetics of drugs, findings regarding the induction of P-gp by PXR ligands in vivo are still controversial. In this study, we examined the effect of pregnenolone 16α-carbonitrile (PCN), a murine PXR ligand, on the expression of Mdr1a/1b mRNA and P-gp protein in the intestine, brain and liver of mice. The results showed that PCN increased the expression of both Mdr1a/1b mRNA and P-gp protein in the intestine and the brain. The present study provided the first evidence that P-gp is inducible by PCN in the large intestine. The results also showed that P-gp protein was induced by PCN in the cortex but not in the whole brain. On the other hand, PCN increased the expression of Mdr1a/1b mRNA in the liver, although no increase was observed in the expression of P-gp protein. These results suggested different effect of PCN on the expression of P-gp protein in the intestine, brain and liver of mice.

The pregnane X receptor (PXR) modulates NLRP3 inflammasome activation – linking the environment with innate immune signaling

BackgroundThe pregnane X receptor (PXR) is a xenobiotic sensing nuclear receptor that is activated by a diverse array of substances, including environmental toxins, pharmaceutical compounds and metabolites released from the intestinal microbiota. While the PXR's prototypical role is to regulate the expression of the drug metabolizing/detoxifying genes in the liver and intestinal epithelium, we and others have reported that it plays a role in regulating inflammatory signaling. More specifically, the PXR can inhibit NFκB‐dependent inflammatory signaling and regulates the function/expression of innate immune receptors, including TLR4 and NLRP3. In the current study, we sought to characterize the function of the PXR in macrophages. Given the interactions between the PXR and innate immune receptors described in other non‐immune cell types, we hypothesized that its activation would modulate NLRP3 inflammasome activation and the resulting processing/release of IL‐1β.AimsTo evaluate the effect of PXR activation of NLRP3 inflammasome activation and to determine the mechanism(s) whereby the PXR modulates inflammasome activity.MethodsMouse peritoneal macrophages and PMA‐differentiated THP‐1 cells were used to assess NLRP3 inflammasome activation. Due to species‐specific ligand interactions with the PXR, in mouse studies we used pregnenolone 16α‐carbonitrile (PCN), whereas in THP‐1 experiments, we used the human PXR agonists, rifaximin and SR12813. To test if PXR activation modulated NLRP3 inflammasome activation, LPS‐primed macrophages or PMA‐differentiated THP‐1 were pretreated for one hour with PXR agonists and then challenged with ATP (5mM), a known NLRP3 inflammasome activator. Caspase‐1 cleavage and IL‐1β secretion were measured to assess inflammasome activation. In some experiments, extracellular ATP was measured following PXR agonist treatment.ResultsWhile PXR activation had no effect on ATP‐induced NLRP3 inflammasome activation, each PXR agonist alone stimulated the cleavage of caspase‐1 and the secretion of IL‐1ββ, reminiscent of inflammasome activation. In PXR−/− macrophages, these responses were absent, supporting a direct role of the PXR in these responses. Deletion of NLRP3 in mouse macrophages and THP‐1 cells attenuated PXR agonist‐induced caspase‐1 cleavage and IL‐1β secretion, suggesting that PXR activation triggers NLRP3 inflammasome activation. Furthermore, PXR driven responses were attenuated following caspase‐1 inhibition. Mechanistically, NLRP3 activation by the PXR did not involve ROS production, nor was it sensitive to chelation of intracellular calcium. However, treating cells with apyrase, to catabolize extracellular ATP, or selective inhibition of the P2X7 receptor attenuated PXR agonist‐induced caspase‐1 activation and IL‐1β secretion. Interestingly, we subsequently found that PXR activation led to a rapid (within 15 seconds) increase in extracellular ATP, reaching levels previous described in NLRP3 inflammasome activation models. This response was absent in PXR−/− cells, supporting a direct role of the PXR in ATP release.ConclusionsTaken together, our data suggest that activation of the xenobiotic sensing PXR triggers the release of ATP, which in turn causes NLRP3 inflammasome activation in macrophages. These findings suggest a novel mechanism whereby non‐microbial/non‐viral agents of environmental origin can stimulate innate immune responses and contribute to inflammatory disease.Support or Funding InformationCrohn's and Colitis CanadaNSERCLloyd Sutherland Investigatorship in IBD/GI researchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats

The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats).The treatment of WT rats for 7 d with 500 ppm NaPB in the diet and 100 mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats.Male C57BL/6 J (WT) and CAR KO/PXR KO mice were given 1000 ppm NaPB in the diet for 7 d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes.These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.

Combining Genomics To Identify the Pathways of Post-Transcriptional Nongenotoxic Signaling and Energy Homeostasis in Livers of Rats Treated with the Pregnane X Receptor Agonist, Pregnenolone Carbonitrile.

Transcriptomic, proteomic, phosphoproteomic, and metabolomic analyses were combined to determine the role of pregnane X receptor (PXR) in nongenotoxic signaling and energy homeostasis in liver after rats were repeatedly orally dosed with the PXR agonist pregnenolone carbonitrile (PCN) for 7 days. Analyses of mRNAs and proteins in the supernatant, membrane, and cytosolic fractions of enlarged liver homogenates showed diverse expression profiles. Gene set enrichment analysis showed that the synchronous increase in mRNAs and proteins involved in chemical carcinogenesis and the response to drug was possibly mediated by the PXR pathway and proteasome core complex assembly was possibly mediated by the Nrf2 pathway. In addition, levels of proteins in the endoplasmic reticulum lumen and involved in the acute-phase response showed specific increase with no change in mRNA level, and those composed of the mitochondrial inner membrane showed specific decrease. The analysis of phosphorylated peptides of poly(A) RNA binding proteins showed a decrease in phosphorylation, possibly by casein kinase 2, which may be related to the regulation of protein expression. Proteins involved in insulin signaling pathways showed an increase in phosphorylation, possibly by protein kinase A, and those involved in apoptosis showed a decrease. Metabolomic analysis suggested the activation of the pentose phosphate and anaerobic glycolysis pathways and the increase of amino acid and fatty acid levels, as occurs in the Warburg effect. In conclusion, the results of combined analyses suggest that PXR's effects are due to transcriptional and post-transcriptional regulation with alteration of nongenotoxic signaling pathways and energy homeostasis.

Regulation of hepatic abcb4 and cyp3a65 gene expression and multidrug/multixenobiotic resistance (MDR/MXR) functional activity in the model teleost, Danio rerio (zebrafish)

Multidrug/multixenobiotic resistance (MDR/MXR) confers resistance to a diverse range of potentially toxic pharmaceuticals and environmental contaminants through a cellular response that involves the coordinated induction and activity of the ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) and the Phase I metabolizing enzyme cytochrome P450 3A (CYP3A). In mammals, ligand-mediated pregnane X receptor (PXR) transcriptional activity regulates the induction of P-gp and CYP3A; however, this mechanism has not been well-characterized in piscine species. Zebrafish (Danio rerio) treated with the Pxr agonist pregnenolone 16α-carbonitrile (PCN) showed decreased P-gp (zebrafish Abcb4) and CYP3A (zebrafish Cyp3a65) mRNA levels after 48h exposure; however, treatment with PCN also resulted in increased hepatic MDR/MXR functional activity (i.e. increased Rhodamine 123 efflux) in vivo. Consistent with mammalian-like MDR/MXR regulated by PXR, the PCN-mediated modulation of hepatic Abcb4 and Cyp3a65 mRNA levels and MDR/MXR functional activity was attenuated by co-treatment with PCN and the mammalian PXR antagonist, ketoconazole (KTC). These results provide evidence that zebrafish Pxr may play a role in MDR/MXR through transcriptional regulation of abcb4 and cyp3a65 gene expression.

Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[α]-pyrene-induced DNA damage

Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) significantly inhibited BaP-induced DNA damage and this protective effect was abolished in PXR-null hepatocytes. Mechanistically, PXR activation inhibited expression of AhR-target genes for CYP1A1, CYP1B1 and CYP1A2 that are required for BaP biotransformation in cultured liver cells, or in the livers of C57BL/6J mice. Using an AhR-responsive reporter assay as well as chromatin immunoprecipitation assay we found that PXR activation transcriptionally represses AhR-regulated gene expression. Furthermore, we found that PXR directly bound AhR at its DNA-binding domain, and this association may play a role in preventing of the AhR from binding to its target genes as shown in the ChIP assay. Taken together, our study has revealed a novel mechanism by which PXR protects liver cells from BaP-induced DNA damage through inhibiting the BaP biotransformation.

The Microbial Metabolite Sensor Pregnane X Receptor (PXR) Restrains Fibroblasts from Promoting Gastrointestinal Inflammation and Fibrosis in Mice

INTRODUCTIONFibrosis contributes to intestinal strictures and obstruction in 30–50% of Crohn's disease patients. The pathogenesis of intestinal fibrosis is incompletely understood and has virtually no effective treatments. The pregnane X receptor (PXR), a xenobiotic receptor involved in detoxification responses, has recently been identified as a modulator of fibrosis in hepatic stellate cells. Of the number of foreign ligands for the PXR, the tryptophan metabolite indole‐3‐propionic acid (IPA) produced by the intestinal commensal Clostridium sporogenes can bind to the PXR to mediate signalling events that protect intestinal barrier function. The role of the PXR in intestinal fibrosis and if microbial metabolite sensing can affect intestinal fibrotic responses is unknown.AIMSTo examine the role of the PXR and its ability to sense microbial metabolites in the modulation of intestinal fibrosis.METHODSIntestinal inflammation was induced using DSS (3.5%) for 5 days followed by healing for 25 days. Fibrosis was assessed using Masson‐trichrome and Serius Red staining of histological sections. Mouse primary fibroblasts were grown from wild type (WT) and PXR−/minus; mice. Fibroblasts were stimulated with cytomix (TNF‐α, IL‐1β, and IFN‐γ) for 24 hours after which cell supernatants were assessed for cytokine concentrations using Luminex. Fibroblasts were also stimulated with cytomix in the presence of the PXR agonists pregnenolone 16α‐carbonitrile (PCN) or IPA and assessed for gene expression using qPCR. NFκB activity was assessed via Western blot for the p65 subunit. To examine the microbiota's role in fibrosis, the microbiota was depleted after the 5‐day course of DSS for 25 days using an antibiotic cocktail of vancomycin, neomycin, metronidazole and ampicillin.FINDINGSFollowing a 25‐day recovery after DSS, WT mice showed clear evidence of fibrosis as indicated by Masson‐trichrome and Serius Red staining. When compared to WT mice, PXR−/− mice demonstrated significantly greater levels of fibrosis. Mouse primary fibroblasts were found to express the PXR, and PXR−/− fibroblasts displayed morphological differences compared to WT fibroblasts. Following stimulation with cytomix, PXR−/− fibroblasts produced dramatically higher levels of proinflammatory cytokines including eotaxin, CXCL2, GM‐CSF, G‐CSF and IL‐9. Importantly, PCN and the microbial metabolite IPA were able to block the expression of these cytokines. These effects in PXR−/− fibroblasts may be linked to the increased activity of NFκB that was observed both basally and after stimulation with cytomix compared to WT fibroblasts. Depletion of the microbiota led to exacerbated intestinal fibrosis.CONCLUSIONSPXR signaling in intestinal fibroblasts appears to be required to restrain inflammation and fibrosis. Luminal sensing of bacterial derived indoles (ie. IPA) via PXR may be involved in this process, highlighting a xenobiotic receptor‐microbiota axis that could be targeted to prevent the intestinal fibrosis observed in Crohn's disease.Support or Funding InformationCrohn's & Colitis Canada, Dr. Keith Sharkey's CCFC Chair in IBD Research, The Dr. Lloyd Sutherland Investigator in IBD/GI Research, Canadian Foundation for Innovation, Canada Research Chairs Program, US Department of Defense, Beverley Philips Rising Star Fellowship.

Effects of MicroRNA-34a on the Pharmacokinetics of Cytochrome P450 Probe Drugs in Mice.

MicroRNAs (miRNAs or miRs), including miR-34a, have been shown to regulate nuclear receptor, drug-metabolizing enzyme, and transporter gene expression in various cell model systems. However, to what degree miRNAs affect pharmacokinetics (PK) at the systemic level remains unknown. In addition, miR-34a replacement therapy represents a new cancer treatment strategy, although it is unknown whether miR-34a therapeutic agents could elicit any drug-drug interactions. To address this question, we refined a practical single-mouse PK approach and investigated the effects of a bioengineered miR-34a agent on the PK of several cytochrome P450 probe drugs (midazolam, dextromethorphan, phenacetin, diclofenac, and chlorzoxazone) administered as a cocktail. This approach involves manual serial blood microsampling from a single mouse and requires a sensitive liquid chromatography-tandem mass spectrometry assay, which was able to illustrate the sharp changes in midazolam PK by ketoconazole and pregnenolone 16α-carbonitrile as well as phenacetin PK by α-naphthoflavone and 3-methylcholanthrene. Surprisingly, 3-methylcholanthrene also decreased systemic exposure to midazolam, whereas both pregnenolone 16α-carbonitrile and 3-methylcholanthrene largely reduced the exposure to dextromethorphan, diclofenac, and chlorzoxazone. Finally, the biologic miR-34a agent had no significant effects on the PK of cocktail drugs but caused a marginal (45%-48%) increase in systemic exposure to midazolam, phenacetin, and dextromethorphan in mice. In vitro validation of these data suggested that miR-34a slightly attenuated intrinsic clearance of dextromethorphan. These findings from single-mouse PK and corresponding mouse liver microsome models suggest that miR-34a might have minor or no effects on the PK of coadministered cytochrome P450-metabolized drugs.

Pregnenolone 16α-carbonitrile ameliorates concanavalin A-induced liver injury in mice independent of the nuclear receptor PXR activation

The pregnane X receptor (PXR) is well-known as a key regulator of drug/xenobiotic clearance. Upon activation by ligand, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters. Recent studies have revealed that PXR also plays a role in regulating immune/inflammatory responses. Specific PXR activators, including synthetic ligands and phytochemicals, have been shown to ameliorate chemically induced colitis in mice. In this study, we investigated an anti-inflammatory effect of pregnenolone 16α-carbonitrile (PCN), a prototypical activator for rodent PXR, in concanavalin A (Con A)-induced liver injury, a model of immune-mediated liver injury, using wild-type and Pxr−/− mice. Unexpectedly, pretreatment with PCN significantly ameliorated Con A-induced liver injury in not only wild-type but Pxr−/− mice as well, accompanied with lowered plasma ALT levels and histological improvements. Pretreatment with PCN was found to significantly repress the induction of Cxcl2 and Ccl2 mRNA expression and neutrophil infiltration into the liver of both wild-type and Pxr−/− mice at the early time point of Con A-induced liver injury. Our results indicate that PCN has unexpected immunosuppressive activity independent of PXR activation to protect mice from immune-mediated liver injury induced by Con A.

Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD.

Development of a New Practical Approach to Perform Single‐Mouse Pharmacokinetic Study

Single‐animal pharmacokinetic (PK) study has largely been limited to rats and larger animals. However, given the increasingly large body of research using transgenic and immunocompromised mouse models, and an economical advantage, a single‐mouse PK model is highly warranted. Given a high sensitivity of LC‐MS/MS in quantification of drugs and metabolites, we have established a practical and standard protocol for series blood samplings from single mouse via tail vein at multiple time points towards a complete PK profile. CD‐1 Mice were given a cocktail of cytochrome P450 (CYP) isoform‐specific probe substrates per os, consisting of midazolam (MDZ), dextromethorphan (DXM), phenacetin (PHE), diclofenac (DIC), and chlorzoxazone (CHL). CYP inhibitors, ketoconazole (KTZ) and α‐napthoflavone (α‐NF), were administered by intraperitoneal injection 1 hour prior to dosing; CYP inducers, pregnenolone 16α‐carbonitrile (PCN) and 3‐methyl cholanthrene (3‐Me), were administered 3 days prior to dosing. Blood (10–20 μl per time point) was collected by tail vein puncture at 7–10 different time points over 3 hours and plasma was prepared for LC‐MS/MS quantification of MDZ, DXM, PHE, DIC, and CHL, and CYP isoform‐specific metabolites 1′‐OH‐MDZ, DXO, ACE, 4′‐OH‐DIC, and 6′‐OH‐CHL, respectively.A small volume (3 μl) of plasma at each time point was found to be sufficient to produce a complete PK profile in a single mouse. Visual inspection and non‐compartmental PK modeling showed that formation of 1′‐OH‐MDZ and ACE was sharply and significantly reduced in mice pre‐treated with KTZ and α‐NF, respectively, suggesting that these compounds preferentially inhibit MDZ 1′‐hydroxylase and PHE O‐deethylase, respectively, in mice in vivo. Additionally, plasma MDZ concentrations and all PK parameters (Cmax, AUC, t1/2) were significantly altered in mice administered PCN. Interestingly, this was not reflected by an increase of 1′‐OH‐MDZ levels, yet by a decrease in plasma 1′‐OH‐MDZ and Cmax and AUC values, suggesting that formation of other metabolites may be induced. Furthermore, plasma PHE and all PK parameters were significantly reduced to near negligible concentrations in mice administered 3‐Me, but plasma ACE, Cmax, and AUC were also slightly attenuated relative to control, suggesting that other metabolic pathways may be induced. Lastly and surprisingly, plasma MDZ concentrations were also reduced in mice administered 3‐Me, but to a much lesser extent, suggesting that 3‐Me is a promiscuous inhibitor to MDZ 1′‐hydroxylation in mice.This approach provides a useful and practical means to conduct single mouse PK research and may prove useful for studying drug metabolism in widely‐used laboratorial mouse models. Additionally, our data suggest alternative roles for classical human CYP inhibitors and inducers in a mouse model.Support or Funding InformationJ.L.J., Y.T., and A.‐M. Y. were supported by a National Institutes of Health National Cancer Institute grant [U01CA175315].

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.