Disruption of the PXR gene in ES cells and mice. (A) Schematic representation of the PXR locus segment, the targeting vector and the targeted PXR allele. Open boxes indicate exons and labeled boxes indicate the PGK-tk and PGK-neo cassettes. Selected restriction endonuclease sites are indicated: B, BamHI; K, KpnI; N, NheI; S, SpeI; and X, XhoI. A novel BamHI site is introduced into the PXR locus by the homologous recombination event allowing the targeted locus to be distinguished from the wild-type allele by Southern analysis of BamHI digested DNA with the indicated probe (shown at the right). (B) Northern blot analysis of RNA isolated from the livers of wild-type and PXR^−/− mice by using the probes indicated in the schematic of the PXR cDNA. Reverse transcription (RT)–PCR was performed using the primers indicated in the schematic. Amplification of the wild-type and disrupted PXR mRNAs yielded products of 111 bp and 363 bp, respectively. (C) Total RNA was prepared from the livers of three wild-type and PXR^−/− mice treated with PCN, PB, dexamethasone, or vehicle alone. RNA samples were pooled before Northern blot analysis with probes for Cyp3a11. Bands were quantitated as described in Materials and Methods and represent the mean obtained from three animals in each treatment group. Values are normalized to β-actin and are expressed as fold change relative to wild-type mice receiving vehicle alone.

PXR represses Cyp7a1 and induces Oatp2 expression in response to PCN. Total RNA was prepared from the livers of PXR^+/+ and PXR^−/− mice treated with PCN or vehicle alone. RNA from three animals was pooled and subjected to Northern blot analysis with probes for (A) Cyp7a1, (B) Oatp2, and β-actin. Quantitation of bands was performed as described in Fig. 1.

Bile acids bind and activate PXR. (A) CV-1 cells were transfected with expression plasmids for mouse PXR or human PXR and the reporter plasmid (Cyp3a23)₂-tk-CAT, and the cells treated with 100 μM concentrations of the indicated bile acids: CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; 3-keto-LCA, 3-keto-lithocholic acid. PCN and rifampicin (RIF) were used as positive controls for mPXR and hPXR, respectively, at 10 μM concentration. Cell extracts were subsequently assayed for CAT activity. Data represent the mean ± SD of assays performed in triplicate. Activation of mPXR by LCA is highly statistically significant as measured by the Student's t test (P < 0.001). (B) Scintillation proximity competition binding assays were performed with human PXR ligand binding domain and 10 nM [³H]SR12813 in the presence of increasing concentrations of LCA and 3-keto-LCA. A single-point negative control (tauro-β-muricholic acid, open triangle) is also shown.

Effects of treating wild-type and PXR^−/− mice with LCA. (A) Wild-type and PXR^−/− mice were fed a diet supplemented with 0.5% LCA for 8 days. Urine was collected on day 8 of the study and LCA concentrations were determined. Data represent the mean ± SEM of assays performed on urine from eight different mice. ND, not detected; *, Statistically significantly difference between wild-type and PXR^−/−, P < 0.05. (B) Total RNA was isolated and pooled from the livers of six wild-type and PXR^−/− mice injected for 4 days with LCA or vehicle alone and subject to Northern blot analysis with probes for Cyp3a11, Oatp2, and β-actin. (C) Model for PXR as a bile acid sensor. Activation of PXR by LCA and/or LCA metabolites results in repression of Cyp7a1, which blocks bile acid biosynthesis, and induction of Oatp2 and Cyp3a expression, which promote bile acid uptake and metabolism. The net effect is to prevent the accumulation of bile acids to toxic levels.

PXR activation protects against LCA-induced hepatotoxicity. Wild-type (A, C, and E) and PXR^−/− (B, D, and E) mice were treated with vehicle alone (A and B), LCA (C and D), or LCA and PCN (E and F). Liver sections were prepared for histology and stained with hematoxylin and eosin. Areas of severe necrosis in panels C, D, and E are indicated with arrows. (G) Serum ALT (open bars) and SDH (closed bars) levels were measured in wild-type and PXR^−/− mice treated with either LCA and vehicle (Vehicle) or LCA and PCN (PCN). ALT and SDH values in untreated animals were 28 ± 10 units/l and 150 ± 20 units/ml, respectively. Data represent the mean ± SEM of assays performed with serum from six different animals. Statistical differences compared with the same genotype treated with LCA and vehicle: *, P < 0.005; **, P < 0.001.