Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists

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 microM 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 microM 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 10nM. Using a HepG2-based transactivation assay, T0901317 activated human PXR with an EC(50) approximately 20nM, 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.

Fig. 1

Effects of T0901317 or 24(S),25-epoxycholesterol treatments on CYP3A4 and CYP2B6 mRNA levels in primary cultures of human hepatocytes. Five preparations of primary cultured human hepatocytes (HH) were treated for 24 hr with medium containing 0.1% DMSO, 50 μM rifampicin (Rif, in DMSO), 1, 10, 100 or 1000 nM T0901317 (in DMSO, not all concentrations were tested in each hepatocyte preparation), 0.1% ethanol, or 10 or 30 μM 24(S),25-epoxycholesterol [24(S),25-EC, in ethanol]. After treatment, the hepatocytes were harvested for measurement of CYP3A4 (A) and CYP2B6 (B) mRNA levels, using TaqMan Gene Expression Assays. Values are expressed as fold changes relative to the respective DMSO-treated or ethanol-treated controls, and results for each individual hepatocyte preparation are shown. The average fold increases (±sd for n=3 to 5; ±range for n=2) produced by the treatments are shown above the bars.

Fig. 2

Effects of T0901317 and 24(S),25-epoxycholesterol treatments on human PXR-responsive reporter expression. A: Optimization of transactivation conditions. HepG2 cells were transfected with 0 to 200 ng of a plasmid expressing human PXR (pSG5-hPXR1) in combination with 800 ng of a PXR-responsive reporter plasmid (XREM-CYP3A4-Luc), treated for 24 hr with medium containing 0.1% DMSO or 50 μM rifampicin (Rif), and harvested for measurement of luciferase activities. B: HepG2 cells were co-transfected with 50 ng pSG5-hPXR1 and 800 ng XREM-CYP3A4-Luc, treated for 24 hr with medium alone (UT) or containing 0.1% DMSO, 50 μM Rif (in DMSO), 1 nM to 10 μM T0901317 (in DMSO), 0.1% ethanol (EtOH), or 0.3 to 30 μM 24(S),25-epoxycholesterol [24(S),25-EC, in EtOH] (concentrations are shown as log molarities), and harvested for measurement of luciferase activities. Values are presented as mean normalized luciferase activities (firefly/Renilla) ± sd (n=3 wells per treatment group). The average fold-increases produced by selected treatments relative to the appropriate vehicle-treated controls are shown above the bars.

Fig. 3

Concentration-dependent effects of T0901317 and SR-12813 on human PXR-responsive reporter expression, and of T0901317 and 24(S),25-epoxycholesterol on LXR target gene expression. A: HepG2 cells were co-transfected with 50 ng pSG5-PXR1 and 800 ng XREM-CYP3A4-Luc, treated for 24 hr with medium containing 1 nM to 1 μM T0901317 or 10 nM to 30 μM SR-12813 (concentrations shown as log molarities), and harvested for measurement of luciferase activities. Values are presented as mean normalized luciferase activities (firefly/Renilla) ± sd (n=3 wells per treatment group). Fitted sigmoid curves with calculated EC₅₀ values and 95% confidence intervals are shown. B: HepG2 cells were treated for 24 hr with 0.1% DMSO, 1 to 300 nM T0901317 (in DMSO), 0.l% ethanol, or 3 to 30 μM 24(S),25-epoxycholesterol [24(S),25-EC, in ethanol]. After treatment, the cells were harvested for measurement of SREBP1c mRNA levels, using a TaqMan Gene Expression Assay. Values are presented as fold increases [T0901317/DMSO or 24(S),25-EC/ethanol]. The fitted sigmoid curve for the T0901317 data is shown, together with the calculated EC₅₀ value and 95% confidence interval.