Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists

doi:10.1016/j.bcp.2007.07.040

Biochemical Pharmacology

Volume 74, Issue 10, 15 November 2007, Pages 1535-1540

https://doi.org/10.1016/j.bcp.2007.07.040 Get rights and content

Abstract

The liver X receptor (LXR) agonists, 24(S),25-epoxycholesterol and T0901317, were previously shown to be capable of inducing CYP3A expression in primary cultured rodent hepatocytes through activation of the pregnane X receptor (PXR). In this study, the abilities of these two LXR agonists to regulate CYP3A4 and CYP2B6 mRNA expression in primary cultures of human hepatocytes were evaluated. Treatment with 10 or 30 μM of the endogenous oxysterol, 24(S),25-epoxycholesterol, had no effect on CYP3A4 mRNA content in five preparations of primary cultured human hepatocytes, while 30 μM 24(S),25-epoxycholesterol treatment increased CYP2B6 mRNA content by approximately two-fold. By comparison, treatment with the synthetic LXR agonist, T0901317, potently increased CYP3A4 and CYP2B6 mRNA levels in the human hepatocyte cultures, producing multi-fold increases at 10 nM. Using a HepG2-based transactivation assay, T0901317 activated human PXR with an EC₅₀ ∼ 20 nM, which was more than 10-fold lower than that of the potent PXR ligand, SR-12813, while treatment with 24(S),25-epoxycholesterol failed to induce reporter expression in this assay. Therefore, while 24(S),25-epoxycholesterol-mediated PXR activation and CYP3A induction does not appear to be conserved from rodent to human, T0901317 is among the most potent known activators of human PXR.

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).

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Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists

Abstract

Introduction

Section snippets

Materials

Primary cultured human hepatocytes and HepG2 cell culture

Results

Discussion

Acknowledgements

J Biol Chem

J Lipid Res

J Lipid Res

J Biol Chem

J Biol Chem

Biorg Med Chem

FEBS Lett

Liver X receptor signaling pathways in cardiovascular disease

Mol Endocrinol

Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ

Proc Natl Acad Sci USA

Pharmacophore analysis of the nuclear oxysterol receptor LXRα

J Med Chem