An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids

Abstract

Hepatic hydroxylation is an essential step in the metabolism and excretion of bile acids and is necessary to avoid pathologic conditions such as cholestasis and liver damage. In this report, we demonstrate that the human xenobiotic receptor SXR (steroid and xenobiotic receptor) and its rodent homolog PXR (pregnane X receptor) serve as functional bile acid receptors in both cultured cells and animals. In particular, the secondary bile acid derivative lithocholic acid (LCA) is highly hepatotoxic and, as we show here, a metabolic substrate for CYP3A hydroxylation. By using combinations of knockout and transgenic animals, we show that activation of SXR/PXR is necessary and sufficient to both induce CYP3A enzymes and confer resistance to toxicity by LCA, as well as other xenotoxicants such as tribromoethanol and zoxazolamine. Therefore, we establish SXR and PXR as bile acid receptors and a role for the xenobiotic response in the detoxification of bile acids.

Figure 1

Bile acids are SXR/PXR activators and CYP3A inducers. (A) Bile acids activate a reporter gene activity via DNA-bound SXR/LBD. The tk-UAS-Luc reporter was transfected into CV-1 cells together with a chimeric receptor GAL-SXR/LBD alone or in conjunction with VP-ACTR (comprising the receptor interaction domain of ACTR with an amino terminal fusion of the VP16 activation domain). The transfected cells were subsequently treated with indicated compounds. Results represent the average and standard error from triplicate assays. (B) SXR and PXR-mediated activation of CYP3A4 promoter element by bile acids. The SXR/PXR-responsive reporter tk-3A4-Luc or the FXR-responsive reporter tk-EcRE-Luc constructs were transfected into CV-1 cells in the presence of the empty vector or the expression vectors for SXR, PXR, or FXR. The transfected cells were subsequently mock treated or treated with indicated compounds. Results are shown as fold induction over solvent controls and represent the average and standard error from triplicate assays. (C) Induction of CYP3A by LCA in vivo. Total liver RNA isolated from mice of indicated genotypes and treated with LCA (8 mg/day for 4 days) or solvent control were subjected to Northern blot analysis and probed for CYP3A11, CYP7A, and GAPDH mRNA. Similar CYP3A induction by LCA was also seen in mice 24 h after a single dose of 8 mg of LCA, and LCA treatment does not further increase the induction of CYP3A in VPSXR mice (data not shown). Notably, the solvent control is a PXR^+/− mouse; we showed before that the basal expression of CYP3A11 gene remains unchanged independent of PXR genotypes (10).

Figure 2

LCA-mediated liver damage in wild-type, PXR-null, and transgenic mice. Wild-type (A, C, and E), PXR-null (B, D, and F), or Alb-VPSXR transgenic mice (G and H) were given daily treatments of LCA (A, B, E, F, and H) or vehicle (C, D, and G) via gavage for 4 days (24). Wild-type (I) or PXR-null (J) mice were treated with a single i.p. injection of PCN (40 mg/kg) before 4 days of treatment with both LCA (8 mg/day) and PCN (13 mg/kg). (A and B) Photographs of representative livers. (C–J) Liver paraffin sections stained with hematoxylin and eosin. Regions of liver necrosis are marked by arrows in E and F. (×200.)

Figure 3

Loss of PCN-mediated protection against xenotoxicants in PXR-null mice. Tribromoethanol anesthesia tests were first administered in the absence of PCN as described (10). After a recovery period of 3 days, the same groups of mice were treated with PCN (40 mg/kg) by daily i.p. injections for two days and the anesthesia tests then repeated 24 h after the last PCN injection. Results represent the averages and standard error for the indicated numbers of mice. Controls include both PXR^+/+ and PXR ^+/− mice. The statistical analysis was performed by using INSTAT 2.03.

Figure 4

P450-dependent LCA metabolism. (A) Pathways of LCA hydroxylation. (B) Specific inhibition of human liver microsome-catalyzed LCA 6α-hydroxylation and testosterone 6β-hydroxylation by an anti-CYP3A antibody (22). The latter reaction is an established CYP3A-dependent metabolic reaction in human liver microsomes.