Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists
Introduction
The liver X receptors, LXRα and LXRβ (NR1H3 and NR1H2), are oxysterol-sensing nuclear receptors that regulate the transcription of genes that function in the maintenance of lipid homeostasis (for recent review see [1]). The oxysterol, 24(S),25-epoxycholesterol, is one of the most potent and efficacious ligand activators of LXRα and LXRβ[2], [3]. 24(S),25-Epoxycholesterol is biosynthesized through a pathway parallel to that for cholesterol biosynthesis, which begins with the diversion of squalene 2,3-oxide to squalene 2,3:22,23-dioxide [4], and appreciable amounts of this molecule are detectable in liver extracts [5]. N-(2,2,2-Trifluoroethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide (T0901317) is a synthetic agonist of LXR that is frequently used as an experimental tool for investigating LXR-mediated processes. In the original study characterizing the pharmacological properties of T0901317, the drug was found to activate LXR potently (EC50 ∼ 20 nM) and selectively [6], although treatment with 1 μM T0901317 also produced some activation of the xenobiotic-sensing pregnane X receptor (PXR, NR1I2).
We previously reported that treatment of primary cultured rat hepatocytes with 24(S),25-epoxycholesterol effectively induced CYP3A mRNA and immunoreactive protein levels [7]. CYP3A mRNA induction also occurred in hepatocyte cultures prepared from wild-type or LXR-null mice, but not in hepatocytes from PXR-null mice, demonstrating that the underlying mechanism was PXR, rather than LXR, activation [7]. The 24(S),25-epoxycholesterol concentrations that produced CYP3A induction in the hepatocyte cultures were in the mid micromolar range, not far above the reported EC50 for LXR activation by this sterol, as determined by transactivation analysis in CV-1 cells (3-4 μM) [2]. These findings were confirmed by Gnerre et al. [8], who reported that 10 μM concentrations of the oxysterols, 24(S),25-epoxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol, activated mouse PXR, also in a CV-1-based transactivation assay [8]. We also found that T0901317 treatment caused CYP3A induction in rat and mouse hepatocyte cultures, and that these effects were also mediated through PXR activation [7].
These findings prompted us to investigate the abilities of 24(S),25-epoxycholesterol and T0901317 to regulate P450 expression and activate PXR in human liver cell systems. As the foundation system, we evaluated treatment effects on CYP3A4 and CYP2B6 mRNA levels in primary cultured human hepatocytes, while as a secondary system we performed transactivation assays in the human hepatoma cell line, HepG2, that was co-transfected with supplemental human PXR and a PXR-responsive reporter. We report that 24(S),25-epoxycholesterol has essentially no ability to activate the human PXR, while T0901317 is among the most potent activators of human PXR yet described.
Section snippets
Materials
T0901317 and rifampicin were purchased from Sigma-Aldrich (St. Louis, MO). 24(S),25-Epoxycholesterol and SR-12813 were purchased from BIOMOL International (Plymouth Meeting, PA). Cell culture media, fetal bovine serum and antibiotics were purchased from Invitrogen (Carlsbad, CA). Recombinant human insulin (Novolin R) was purchased from Novo Nordisk Pharmaceuticals, Inc. (Princeton, NJ). Other materials were obtained from the sources indicated below.
Primary cultured human hepatocytes and HepG2 cell culture
Plated primary cultures of human hepatocytes
Results
The abilities of T0901317 and 24(S),25-epoxycholesterol to regulate CYP3A4 and CYP2B6 mRNA levels were evaluated in five preparations of primary cultured human hepatocytes (Fig. 1). The hepatocyte cultures were treated for 24 h with 1, 10, 100 or 1000 nM T0901317 (not all concentrations were tested in each hepatocyte preparation) or 24(S),25-epoxycholesterol (10 or 30 μM in each preparation), or with 50 μM rifampicin as a positive control treatment. Treatment with 10 nM or higher T0901317 produced
Discussion
24(S),25-Epoxycholesterol is one of the most potent and efficacious endogenous ligand activators of the LXR receptors [2], [3], and, as we and others have previously reported, is capable of activating rodent PXRs [7], [8]. However, when tested in either of two models of human hepatic PXR activation/CYP3A4 induction, 24(S),25-epoxycholesterol treatment had essentially no effect, despite the fact that the sterol caused the expected induction of SREBP1c in HepG2 cells, indicating effective LXR
Acknowledgements
This work was supported by NIH grant HL50710 and by services provided by the Cell Culture and Gene Transfer Technologies, Imaging and Cytometry, and Microarray and Bioinformatics Facility Cores of Environmental Health Sciences Center grant ES06636. Plated primary cultures of human hepatocytes were provided through the Liver Tissue Procurement and Distribution System (DK92310).
References (21)
- et al.
Biosynthesis of 24,25-epoxycholesterol from squalene 2,3:22,23-dioxide
J Biol Chem
(1981) - et al.
Key regulatory oxysterols in liver: analysis as d4-3-ketone derivatives by HPLC and response to physiological perturbations
J Lipid Res
(2001) - et al.
LXR deficiency and cholesterol feeding affect the expression and phenobarbital-mediated induction of cytochromes P450 in mouse liver
J Lipid Res
(2005) - et al.
Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes
J Biol Chem
(2003) - et al.
Expression of CYP3A4, CYP2B6, and CYP2C9 is regulated by the vitamin D receptor pathway in primary human hepatocytes
J Biol Chem
(2002) - et al.
Crystal structure of the PXR-T1317 complex provides a scaffold to examine the potential for receptor antagonism
Biorg Med Chem
(2007) - et al.
T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR
FEBS Lett
(2007) - et al.
Liver X receptor signaling pathways in cardiovascular disease
Mol Endocrinol
(2003) - et al.
Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ
Proc Natl Acad Sci USA
(1999) - et al.
Pharmacophore analysis of the nuclear oxysterol receptor LXRα
J Med Chem
(2001)
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