Identification of an endogenous ligand that activates pregnane X receptor-mediated sterol clearance

Abstract

The nuclear receptor PXR (pregnane X receptor) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs and xenobiotic agents. On activation by these compounds, PXR coordinately induces a network of transporters, cytochrome P450 enzymes, and other genes that effectively clear xenobiotics from the liver and intestine. Like PXR, the majority of its target genes also possess a broad specificity for exogenous compounds. Thus, PXR is both a sensor and effector in a well integrated and generalized pathway for chemical immunity. Although it is clear that PXR responds to numerous foreign compounds, it is unclear whether it possesses an endogenous ligand. To address this issue, we noted that there is substantial overlap in the substrate specificities of PXR and its critical CYP3A target gene. This prompted us to ask whether endogenous CYP3A substrates also serve as PXR ligands. We demonstrate that 5beta-cholestane-3alpha,7alpha,12alpha-triol (triol), a cholesterol-derived CYP3A substrate, is a potent PXR agonist that effectively induces cyp3a expression in mice. This defines a critical salvage pathway that can be autoinduced to minimize triol accumulation. In contrast, triol can accumulate to very high levels in humans, and unlike mice, these people develop the severe clinical manifestations of cerebrotendinous xanthomatosis. The reason for these dramatic species differences has remained unclear. We now demonstrate that triol fails to activate human PXR or induce the CYP3A-salvage pathway. This explains why humans are more susceptible to sterol accumulation and suggests that synthetic ligands for human PXR could be used to treat cerebrotendinous xanthomatosis and other disorders of cholesterol excess.

Figure 1

Triol is an mPXR agonist. (a) Triol and tetrol activate mPXR. CV-1 cells were transiently transfected with a GAL–mPXR expression vector, a GAL4 reporter construct, and a β-galactosidase vector as an internal control. Where noted, the bile acid transporter NTCP was added to the transfection to promote transport of membrane-impermeable bile acids. Transfected cells were exposed to the indicated compounds (10 μM), and fold activation was determined. (b) Same as a except that a full-length mPXR expression vector was used along with a reporter construct containing three copies of the rat cyp3a2 PXR response element. (c) Dose–response analysis of triol and tetrol activity. Cells were treated as in a but with multiple concentrations of each sterol. (d–f) Triol does not effectively activate other nuclear receptors. Experimental conditions as in a except that cells were transfected with mCAR (d), mFXR + hRXRα (e), or hVDR (f) and their corresponding reporter constructs. The following ligands were used as indicated: 10 μM triol, 5 μM 5α-androstan-3α-ol (Anol), 100 nM vitamin D₃ (VD₃), and 100 μM chenodeoxycholic acid (CDCA). (g) Triol binds directly to PXR. Bacterially expressed hPXR was incubated with [³H]SR12813 in the absence or presence of the following unlabeled competitors: 5 μM hyperforin, 30 μM triol, and 30 μM tetrol. The amount of [³H]SR12813 associated with hPXR is expressed in cpm. (h) Triol and tetrol activate endogenous mPXR target genes. Primary mouse hepatocytes were treated with PXR ligands (10 μM), and Northern analysis was performed using the indicated probes. Transfections were performed in triplicate, and each triplicate experiment was independently repeated three or more times. Error bars represent the standard error of the mean (SEM) from a representative experiment. In some cases, the error bars are not visible because they are negligible relative to the scale of the figure.

Figure 2

Triol is an endogenous activator of mPXR. (a) The metabolic pathways that underlie the production and elimination of triol are shown schematically. Triol is produced as an intermediate in the hepatic conversion of cholesterol to bile acids. Triol is normally metabolized via CYP27; CYP3A provides an alternate elimination pathway when triol levels are elevated. (b) Elevated hepatic triol levels in the liver of cyp27-null mice. GC-MS was used to measure triol levels in liver extracts from WT or cyp27-null mice (cyp27^−/−). (c) An extract from cyp27-null liver activates mPXR. CV-1 cells were transfected with GAL-mPXR as in Fig. 1a. Cells were treated with equal amounts of organic extracts derived from the liver of WT or cyp27-null mice. (d) PXR target genes are induced in the liver of female cyp27-null mice. Northern analysis was performed as in Fig. 1h. (e) Cyp27-null mice are resistant to a xenobiotic challenge. WT and cyp27-null mice received an i.p. injection of tribromoethanol, and their individual sleep times are plotted. WT animals are represented by gray squares; cyp27-null mice (cyp27^−/−) are represented by black circles. WT male, n = 6; cyp27^−/− male, n = 5; WT female, n = 6; cyp27^−/− female, n = 6. **, P < 0.01; ***, P < 0.001. Error bars represent the SEM and are not visible in cases where they are negligible relative to the scale of the figure.

Figure 3

Triol does not activate hPXR-mediated clearance pathways. (a) Triol is a weak agonist of hPXR. CV-1 cells were transfected as in Fig. 1a with either GAL-mPXR (Left) or GAL-hPXR (Right). Ligands were as follows: 2.5 μM hyperforin and 10 μM PCN, triol, tetrol, and 7α,12α-dihydroxy-4-cholesten-3-one. For mPXR, the data are plotted as percent of maximal fold-activation achieved with PCN; for hPXR, it is relative to hyperforin. (b) Triol is a partial agonist/antagonist of hPXR. Experimental conditions were as in a using hPXR. (c) Triol fails to activate human CYP3A4 expression. Northern analysis was performed as in Fig. 1h but using primary human hepatocytes and the following ligands: 2.5 μM hyperforin and 10 μM rifampicin, triol, and tetrol. Error bars represent the SEM and are not visible in cases where they are negligible relative to the scale of the figure.