title: Regulation of pregnane X receptor (PXR) function and UGT1A1 gene expression by posttranslational modification of PXR protein.
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.
authors: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. sugatani@u-shizuoka-ken.ac.jp
authors: Junko
authors: J
authors: Sugatani
authors: Takahiro
authors: T
authors: Uchida
authors: Masatoshi
authors: M
authors: Kurosawa
authors: Masahiko
authors: M
authors: Yamaguchi
authors: Yasuhiro
authors: Y
authors: Yamazaki
authors: Akira
authors: A
authors: Ikari
authors: Masao
authors: M
authors: Miwa
mesh_terms: D000107:Acetylation; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D000090702:Constitutive Androstane Receptor; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation; D018832:Molecular Chaperones; D009154:Mutation; D051958:Nuclear Receptor Coactivator 2 / Q000378:metabolism; D010766:Phosphorylation; D000077297:Pregnane X Receptor; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport; D011687:Purines / Q000494:pharmacology; D034622:RNA Interference; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011987:Receptors, Steroid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D047488:Retinoid X Receptors / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D000077546:Roscovitine; D012694:Serine; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D000099148:UGT1A1 Enzyme
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D000090702:Constitutive Androstane Receptor; D051357:Cyclin-Dependent Kinase 2; D014453:Glucuronosyltransferase; D050956:HSP40 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D051958:Nuclear Receptor Coactivator 2; D000077297:Pregnane X Receptor; D047428:Protein Kinase Inhibitors; D011687:Purines; D018336:Receptors, Aryl Hydrocarbon; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D047488:Retinoid X Receptors; D012293:Rifampin; D000077546:Roscovitine; D012694:Serine; D000099148:UGT1A1 Enzyme; C111235:AHR protein, human; C485782:DNAJC7 protein, human; C495894:CDK2 protein, human