ABSTRACT
Human UDP-glucuronosyltransferase (UGT) 1A1 is a critical enzyme responsible for detoxification and metabolism of endogenous and exogenous lipophilic compounds such as bilirubin. The present study shows how cyclin-dependent kinase (CDK) inhibitor roscovitine stimulated the expression of UGT1A1 in HepG2 cells. Pregnane X receptor (PXR)-mediated transactivation of UGT1A1 reporter gene was more prominently enhanced by roscovitine, compared with the basal-, constitutive androstane receptor (CAR)-, and aryl hydrocarbon receptor-mediated activities. We determined the regulatory mechanism of UGT1A1 expression through PXR's stimulation by roscovitine. Although phosphomimetic mutations at Thr290 and Thr408 retained the PXR protein in cytoplasm and attenuated the induction of UGT1A1 expression by both roscovitine and rifampicin, a mutation at Ser350 specifically reduced the activity of PXR induced by roscovitine. Immunoprecipitation analysis revealed that the T290D but not T408D mutant protein remained in cytoplasm by forming a complex with heat shock protein 90 and cytoplasmic CAR retention protein, whereas treatment with proteasome inhibitor MG-132 accumulated the T408D mutant protein in cytoplasm. Transfection with anti-CDK2 small interfering RNA (siRNA) but not anti-CDK1 or CDK5 siRNA led to enhanced expression of UGT1A1. S350D yellow fluorescent protein-PXR fusion protein could translocate from cytoplasm to nucleus similar to the wild-type protein but was detected as an acetylated protein, whose binding with retinoid X receptor (RXR) and histone deacetylase was impaired. Cotransfection with coactivator steroid receptor coactivator (SRC) 2 but not SRC-1 partly recovered its PXR activity. These results indicate that roscovitine stimulated the expression of UGT1A1 by inhibiting CDK2, which phosphorylated PXR at Ser350 to suppress binding with RXR and coactivator and maintain the acetylation of PXR protein.
Subject(s)
Glucuronosyltransferase/metabolism , Protein Processing, Post-Translational , Receptors, Steroid/metabolism , Acetylation , Basic Helix-Loop-Helix Transcription Factors/metabolism , Constitutive Androstane Receptor , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/metabolism , Dose-Response Relationship, Drug , Down-Regulation , Gene Expression Regulation, Enzymologic , Glucuronosyltransferase/genetics , HSP40 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins , Hep G2 Cells , Humans , Immunoprecipitation , Molecular Chaperones , Mutation , Nuclear Receptor Coactivator 2/metabolism , Phosphorylation , Pregnane X Receptor , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Transport , Purines/pharmacology , RNA Interference , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/drug effects , Receptors, Steroid/genetics , Retinoid X Receptors/metabolism , Rifampin/pharmacology , Roscovitine , Serine , Time Factors , Transcriptional Activation , TransfectionABSTRACT
Rats that consumed a high-fat and high-sucrose diet (HF diet) developed hepatic steatosis. Treatment of HF diet-fed rats with fluvastatin (8 mg/kg) was lethal, followed by an elevation in levels of plasma aspartate aminotransferase and creatine kinase activities and skeletal muscle toxicity. This study was conducted to determine whether nutritional status affects statin-induced adverse effects in rats. Fluvastatin treatment of rats fed the HF diet led to an increase in systemic exposure, suggesting altered metabolism and elimination. In fact, although hepatic multidrug resistance-associated protein (Mrp) 2 and multidrug resistance (Mdr) 1b protein levels were not significantly changed by fluvastatin treatment for 8 days of rats fed a HF diet, the organic anion-transporting protein (Oatp) 1, Mrp3, CYP1A, CYP2C, UDP-glucuronosyltransferase (UGT) 1A1, and UGT1A5 protein levels were moderately decreased and the Oatp2, CYP3A, and UGT2B1 protein levels were markedly suppressed. No significant difference in the baseline level of Oatp1, Oatp2, Mrp2, Mrp3, Mdr1b, CYP1A, CYP2C, CYP3A, UGT1A1, UGT1A5, or UGT2B1 protein was found between the standard diet- and HF diet-fed groups. In addition, the mRNA levels of Oatp2, CYP2C11, and CYP3A1/2 were markedly decreased in HF diet-fed and fluvastatin-treated rats. There was no significant difference in the glucuronidation activities against fluvastatin among the four groups. In liver cell nuclei, levels of constitutive androstane receptor, pregnane X receptor, and hepatocyte nuclear factor 4α proteins were decreased in fluvastatin-treated HF diet-fed rats, which correlated with the decrease in Oatp2, CYP2C, and CYP3A. Taken together, these results indicate that nutritional status may influence adverse effects of fluvastatin by increasing systemic exposure through modulation of hepatic uptake and elimination.
Subject(s)
Chemical and Drug Induced Liver Injury/etiology , Fatty Acids, Monounsaturated/adverse effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/adverse effects , Indoles/adverse effects , Liver/drug effects , Muscular Diseases/chemically induced , Nutritional Status , Animals , Cells, Cultured , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/metabolism , Dietary Fats/administration & dosage , Dietary Fats/adverse effects , Dietary Sucrose/administration & dosage , Dietary Sucrose/adverse effects , Fatty Acids, Monounsaturated/blood , Fatty Acids, Monounsaturated/pharmacokinetics , Fluvastatin , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/blood , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacokinetics , Indoles/blood , Indoles/pharmacokinetics , Liver/enzymology , Liver/metabolism , Liver Function Tests , Male , Metabolic Detoxication, Phase I , Metabolic Detoxication, Phase II , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Multidrug Resistance-Associated Protein 2 , Muscular Diseases/blood , Muscular Diseases/metabolism , Organic Anion Transporters/metabolism , Rats , Rats, WistarABSTRACT
Hepatocyte growth factor (HGF), an antimitogenic factor for HepG2 cells, increased mRNA and protein levels of UGT1A1 and CYP2B6, as well as the endogenous cyclin-dependent kinase (CDK) inhibitors p16, p21, and p27 in HepG2 cells but not in HuH6, Caco2, or MCF7 cells. Treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) (an extracellular signal-regulated kinase inhibitor) suppressed the HGF-induced expression of UGT1A1 and CYP2B6, as well as p16, p21, and p27 in HepG2 cells. The CDK inhibitor roscovitine also enhanced the expression of UGT1A1, CYP2B6, and CYP3A4. Transfection of anti-CDK2 siRNA led to elevated levels of UGT1A1, CYP2B6, and CYP3A4 in HepG2 and SW480 cells, whereas anti-CDK4 small interfering RNA (siRNA) did not significantly enhance the expression of these enzymes. In fact, CDK2 activity was decreased in HGF-treated HepG2 cells. In cells arrested in S phase by a thymidine block and then released into a synchronous cell cycle, there was a clear dissociation among the activation of CDK2 and the expression of UGT1A1, CYP2B6, and CYP3A4. Furthermore, the induction of CYP3A4 but not UGT1A1 or CYP2B6 mRNA expression by roscovitine was repressed in pregnane X receptor (PXR) siRNA-transfected HepG2 cells. Transfection with constitutive androstane receptor siRNA or PXR siRNA in HepG2 cells did not repress the HGF-stimulated expression of UGT1A1 mRNA. Taken together, our results show that the expression of UGT1A1 and CYP2B6 is negatively regulated through a CDK2 signaling pathway linked to cell cycle progression in HepG2 and SW480 cells, the mechanism of which may differ from that of CYP3A4 expression through PXR phosphorylated by CDK2.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Cell Cycle/physiology , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , Glucuronosyltransferase/metabolism , Hepatocyte Growth Factor/pharmacology , Oxidoreductases, N-Demethylating/metabolism , Aryl Hydrocarbon Hydroxylases/genetics , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins/genetics , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B6 , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Enzyme Induction/drug effects , Enzyme Induction/physiology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression/drug effects , Gene Expression/genetics , Glucuronosyltransferase/genetics , Hep G2 Cells , Histones/metabolism , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Oxidoreductases, N-Demethylating/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Pregnane X Receptor , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , RNA, Small Interfering/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Roscovitine , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Constitutive androstane receptor (CAR) is a transcription factor regulating the expression of several genes related to drug metabolism. CAR expression was elevated in human HepG2 and SW480 cells by serum starvation. From reporter gene assays, mutagenesis, RNA interference, and chromatin immunoprecipitation assays, we identified the serum response element at -142/-139 in the CAR gene transactivated by Elk-1. Whereas treatment with U0126 (ERK inhibitor) enhanced CAR expression, SP600125 (stress-activated protein kinase inhibitor, SAPK) suppressed the phosphorylation of Elk-1 caused by serum-starvation stress and the elevation of CAR mRNA, suggesting that CAR expression may be mediated by phosphorylated Elk-1 via the SAPK signaling pathway.
