Human pregnane X receptor activation and CYP3A4/CYP2B6 induction by 2,3-oxidosqualene:lanosterol cyclase inhibition

Abstract

The effects of [4'-(6-allyl-methyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071), an inhibitor of 2,3-oxidosqualene:lanosterol cyclase (cyclase), were evaluated on CYP3A4 and CYP2B6 mRNA content in primary cultured human hepatocytes. In seven hepatocyte culture preparations, 24-h treatment with 3, 10, or 30 microM Ro 48-8071 produced median increases in CYP3A4 mRNA content that were 2.2-, 7.1-, and 8.5-fold greater than untreated control, respectively, and produced increases in CYP2B6 mRNA content that were 3.0-, 4.6-, and 3.4-fold greater than control, respectively. Increases in CYP3A4 immunoreactive protein content were also measured in Ro 48-8071-treated hepatocytes. To evaluate the effects of cyclase inhibitor treatments further, a pregnane X receptor (PXR)-responsive transactivation assay in HepG2 cells was used. Ro 48-8071, trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine (BIBX 79), and 3beta-(2-diethylaminoethoxy)androst-5-en-17-one HCl (U18666A) induced luciferase expression from a PXR-responsive reporter with EC(50)s of 0.113, 0.916, and 0.294 microM, respectively. Treatment of the HepG2 system with (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bithiophen-5-yl)methoxy]benzenemethanamine (NB-598), an inhibitor of squalene monooxygenase, at concentrations sufficient to achieve cholesterol biosynthesis inhibition significantly inhibited cyclase inhibitor-mediated, but not rifampicin-mediated, reporter induction. Direct treatment of the HepG2 system with 1 to 10 microM squalene 2,3:22,23-dioxide, but not squalene 2,3-oxide, significantly activated PXR-responsive reporter expression. Also, squalene 2,3:22,23-dioxide bound to human PXR in vitro with an IC(50) of 3.35 microM. These data indicate that cyclase inhibitors are capable of producing CYP3A4 and CYP2B6 induction in primary cultured human hepatocytes, and that an endogenous squalene metabolite is a conserved intracrine activator of PXR.

Fig. 1.

Cholesterol biosynthesis pathway, abridged to highlight the metabolites (regular type), enzymes or receptors (italicized type), and drugs (boldface type) that are featured in this study. Arrows represent metabolic reactions; broken arrows indicate multiple steps; and thick shaded arrows represent receptor activation and induction processes.

Fig. 2.

Effects of Ro 48-8071 treatment on CYP3A4 and CYP2B6 mRNA levels in primary cultured human hepatocytes. Seven preparations of primary cultured human hepatocytes were incubated for 24 h with standard medium alone or containing 3, 10, or 30 μM Ro 48-8071, 0.1% DMSO, or 50 μM rifampicin (Rif). After treatment, hepatocytes were harvested for preparation of total RNA, and CYP3A4 and CYP2B6 mRNA levels were measured as described under Materials and Methods. The Ro 48-8071 data are presented as -fold changes relative to untreated controls, whereas the Rif data are presented as -fold changes relative to DMSO-treated controls. A, data are presented as box and whisker plots, in which the horizontal lines within the boxes represent the median values, the tops and bottoms of the boxes represent the 25th and 75th percentile values, and the upper and lower error bars represent the maximum and minimum values. *, significantly different from the hypothetical value of 1, p < 0.05. B, Ro 48-8071 data from each individual hepatocyte preparation are plotted.

Fig. 3.

Effects of 48-h Ro 48-8071 treatment on CYP3A4 and CYP2B6 expression in primary cultured human hepatocytes. A, four preparations of primary cultured human hepatocytes were treated for 48 h with standard medium alone or containing 3 or 10 μM Ro 48-8071, 0.1% DMSO, or 50 μM rifampicin (Rif). After treatment, hepatocytes were harvested for preparation of total RNA, and CYP3A4 and CYP2B6 mRNA levels were measured as described under Materials and Methods. The Ro 48-8071 data are presented as -fold changes relative to untreated controls, whereas the Rif data are presented as -fold changes relative to DMSO-treated controls. Data are presented as box and whisker plots, in which the horizontal lines within the boxes represent the median values, the tops and bottoms of the boxes represent the 25th and 75th percentile values, and the upper and lower error bars represent the maximum and minimum values. B, hepatocytes from two of the preparations described in A were harvested for preparation of microsomes, and CYP3A immunoreactive protein levels were measured by Western blot hybridization.

Fig. 4.

Concentration-dependent effects of cyclase inhibitor treatments on PXR-responsive reporter expression in HepG2 cells. HepG2 cells were transiently transfected with pSG5-hPXR1, XREM-CYP3A4-Luc, and pRL-CMV and then incubated for 24 h with medium alone or containing 10^–8 to 3 × 10^–5 M Ro 48-8071 (A) or with medium containing 0.1% DMSO or 10^–8 to 10^–5 M BIBX 79 or U18666A (B). After treatment, cells were harvested for measurement of firefly and Renilla luciferase activities. Normalized (firefly/Renilla) values are expressed as -fold changes ± S.D. (n = 3 wells/treatment group) relative to the appropriate negative control group (i.e., medium alone for Ro 48-8071; DMSO-treated for BIBX 79 and U18666A). Concentration-response data were fit to sigmoid relationships, and EC₅₀ values with 95% confidence intervals (CIs) are shown. *, p < 0.05 and **, p < 0.01 versus negative control.

Fig. 5.

Effects of NB-598 cotreatments on cyclase inhibitor-mediated induction of PXR-responsive reporter expression in HepG2 cells. HepG2 cells were transiently transfected with pSG5-PXR, XREM-CYP3A4-Luc, and pRL-CMV and then incubated for 24 h with medium alone or containing 0.1% DMSO or 0.1 to 10 μM NB-598, alone or in combination with 10 μMRo 48-8071 (A), 10 μM BIBX 79 (B), 10 μM U18666A (C), or 50 μM rifampicin (D). After treatment, cells were harvested for measurement of firefly and Renilla luciferase activities. Data are expressed as mean normalized (firefly/Renilla) luciferase values ± S.D. (n = 3 wells/treatment group). Groups not sharing a letter are significantly different from each other, p < 0.05.

Fig. 6.

Effects of NB-598 and Ro 48-8071 treatments on sterol biosynthesis in HepG2 cells. A and B, HepG2 cells were incubated for 24 h with medium containing 0.1% DMSO, 30 μM rifampicin (Rif), or 0.1 to 10 μM NB-598 (A) or with 0.1% DMSO (0, 0), 10 μM Ro 48-8071, 0.1 μM NB-598, or Ro 48-8071 and NB-598 in combination (B). After treatment and metabolic labeling with [¹⁴C]MVA, cells were harvested for evaluation of nonsaponifiable lipid contents by thin-layer chromatography and autoradiography as described under Materials and Methods.

Fig. 7.

Concentration-dependent abilities of squalene 2,3-oxide and squalene 2,3:22,23-dioxide treatments to activate PXR-responsive reporter expression in HepG2 cells and to bind to PXR in vitro. A, HepG2 cells were transiently transfected with pSG5-hPXR1, XREM-CYP3A4-Luc, and pRL-CMV and then incubated for 24 h with (top) medium alone (UT) or containing 0.1% DMSO (DM), 0.1% methanol (MeOH), 0.3 to 10 μM squalene 2,3-oxide (SO), or 0.3 to 10 μM squalene 2,3:22,23-dioxide (SDO) or (bottom) 10 μM Ro 48-8071 (Ro), 0.1 μM NB-598 (NB), Ro and DM, or Ro and NB alone (UT) or in combination with MeOH, SO, or SDO. After treatment, cells were harvested for measurement of firefly and Renilla luciferase activities. Normalized (firefly/Renilla) values are expressed as mean ± S.D. (n = 3 wells/treatment group). Groups not sharing a letter are significantly different from each other, p < 0.05. B, the LanthaScreen TR-FRET PXR (SXR) competitive binding assay was used to evaluate the abilities of SO and SDO (left) and Ro 48-8071 (right) to interact with PXR in vitro. T0901317 was included as a positive control PXR ligand (the same binding data for T0901317 are replicated in each panel). IC₅₀ values and 95% confidence intervals (CIs) are shown. For the Ro 48-8071 data, the IC₅₀ value is an estimate, and CI could not be calculated.