Acetylation of lysine 109 modulates pregnane X receptor DNA binding and transcriptional activity

Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug–drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC–MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXRα dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels

Optical Isomers of Atorvastatin, Rosuvastatin and Fluvastatin Enantiospecifically Activate Pregnane X Receptor PXR and Induce CYP2A6, CYP2B6 and CYP3A4 in Human Hepatocytes.

Atorvastatin, fluvastatin and rosuvastatin are drugs used for treatment of hypercholesterolemia. They cause numerous drug-drug interactions by inhibiting and inducing drug-metabolizing cytochromes P450. These three statins exist in four optical forms, but they are currently used as enantiopure drugs, i.e., only one single enantiomer. There are numerous evidences that efficacy, adverse effects and toxicity of drugs may be enantiospecific. Therefore, we investigated the effects of optical isomers of atorvastatin, fluvastatin and rosuvastatin on the expression of drug-metabolizing P450s in primary human hepatocytes, using western blots and RT-PCR for measurement of proteins and mRNAs, respectively. The activity of P450 transcriptional regulators, including pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR), was assessed by gene reporter assays and EMSA. Transcriptional activity of AhR was not influenced by any statin tested. Basal transcriptional activity of GR was not affected by tested statins, but dexamethasone-inducible activity of GR was dose-dependently and enantioselectively inhibited by fluvastatin. Basal and ligand-inducible transcriptional activity of PXR was dose-dependently influenced by all tested statins, and the potency and efficacy between individual optical isomers varied depending on statin and optical isomer. The expression of CYP1A1 and CYP1A2 in human hepatocytes was not influenced by tested statins. All statins induced CYP2A6, CYP2B6 and CYP3A4, and the effects on CYP2C9 were rather modulatory. The effects varied between statins and enantiomers and induction potency decreased in order: atorvastatin (RR>RS = SR>SS) > fluvastatin (SR>RS = SS>RR) >> rosuvastatin (only RS active). The data presented here might be of toxicological and clinical importance

Chemical structures of enantiopure forms of statins.

<p>Four individual enantiomers of atorvastatin, rosuvastatin and fluvastatin are shown in the figure. Clinically used enantiopure forms are circled.</p

Effects of statin enantiomers on the expression of drug-metabolizing cytochromes P450, PXR and tyrosin aminotransferase TAT at mRNA level in primary human hepatocytes.

<p>Primary human hepatocytes from three different donors (HH59, HH61, HH63) were used. Cells were incubated for 24 h with vehicle (DMSO; 0.1% v/v), dioxin (TCDD; 5 nM), rifampicin (RIF; 10 μM) and individual enantiomers of statins (1 μM, 10 μM, 30 μM). Bar graphs of RT-PCR analyses of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP3A4, PXR and TAT mRNAs are shown. The data are the mean ± SD from triplicate measurements and are expressed as a fold induction over vehicle-treated cells. The data were normalized to GAPDH mRNA levels. Student´s t-test and One-way ANOVA followed by Dunnett's post test were calculated using GraphPad Prism.</p

Effects of statin enantiomers on transcriptional activity of human pregnane X receptor.

<p>LS180 cells, transiently transfected with p3A4-luc reporter, were seeded in 96-well plates and stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10⁻¹⁰ M to 10⁻⁴ M in the absence (agonist mode–upper line) or presence (antagonist mode–lower line) of rifampicin (RIF; 10 μM). The vehicle was DMSO (0.1% v/v). After the treatments, cells were lysed and luciferase activity was measured. Treatments were performed in triplicates. Data are expressed as a fold induction of luciferase activity over control cells (agonist mode) or as a percentage of maximal activation attained by RIF (antagonist mode). The values of EC₅₀ and IC₅₀ from <i>n</i> independent cell passages were calculated where appropriate and the average values are indicated in plots. Representative gene reporter assays are shown. Student´s t-test, One-way ANOVA followed by Dunnett's post test and EC₅₀/IC₅₀ values were calculated using GraphPad Prism.</p

Effects of statin enantiomers on transcriptional activity of human glucocorticoid receptor.

<p>AZ-GR cells were seeded in 96-well plates and stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10⁻¹⁰ M to and 10⁻⁴ M in the absence (agonist mode–upper panels) or presence (antagonist mode–lower panels) of dexamethasone (DEX; 100 nM). The vehicle was DMSO (0.1% v/v). After the treatments, cells were lysed and luciferase activity was measured. Treatments were performed in triplicates. Data are expressed as a fold induction of luciferase activity over control cells (agonist mode) or as a percentage of maximal activation attained by DEX (antagonist mode). The values of EC₅₀ and IC₅₀ from <i>n</i> independent cell passages were calculated where appropriate and the average values are indicated in plots. Representative gene reporter assays are shown. Student´s t-test, One-way ANOVA followed by Dunnett's post test and EC₅₀/IC₅₀ values were calculated using GraphPad Prism.</p

Effect of statin enantiomers on the binding of PXR/RXR complex to the DR3 motif of human CYP3A4 gene promoter.

<p>Nuclear fractions of LS174T cells from three independent cell passages were incubated for 2 h at 30°C with DMSO (0.1% v/v), RIF (10 μM) and individual enantiomers of atorvastatin, rosuvastatin and fluvastatin at concentration 10 μM. Treated nuclear extracts were incubated with a biotin-labeled CYP3A4-DR3 probe and electrophoresed on 5% polyacrylamide gel as described under ‘‘Materials and methods section.” <b>A.</b> The complex formation of CYP3A4 DR3 response element with PXR-RXRα heterodimer. <b>B.</b> Simple western blot showing equal expression levels of PXR nuclear extracts used for EMSA (normalized to β-actin levels).</p

Effects of statin enantiomers on trancriptional activity of human aryl hydrocarbon receptor.

<p>AZ-AHR cells were seeded in 96-well plates and stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10⁻¹⁰ M to 10⁻⁴ M in the absence (agonist mode–upper panels) or presence (antagonist mode–lower panels) of dioxin (TCDD; 5 nM). The vehicle was DMSO (0.1% v/v). After the treatments, cells were lysed and luciferase activity was measured. Treatments were performed in triplicates. Data are expressed as a fold induction of luciferase activity over control cells (agonist mode) or as a percentage of maximal activation attained by TCDD (antagonist mode). The values of EC₅₀ and IC₅₀ from <i>n</i> independent cell passages were calculated where appropriate and the average values are indicated in plots. Representative gene reporter assays are shown. Student´s t-test, One-way ANOVA followed by Dunnett's post test and EC₅₀/IC₅₀ values were calculated using GraphPad Prism.</p

Cytotoxicity of statin enantiomers in human cancer cell lines.

<p>AZ-AHR, AZ-GR and LS180 cells were seeded in 96-well plates, stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10⁻¹⁰ M to 10⁻⁴ M. The vehicle was dimethylsulfoxide (DMSO; 0.1% v/v). After the treatment, a conventional MTT test was performed and absorbance was measured at 540 nm. Treatments were performed in triplicates. The data are the mean ± SD from experiments performed in three consecutive passages of cells and are expressed as percentage of viability of control cells. The values of IC₅₀ were calculated where appropriate and they are indicated in plots. Student´s t-test, One-way ANOVA followed by Dunnett's post test and IC₅₀ values were calculated using GraphPad Prism.</p