Regulation of Pregnane X Receptor (PXR) Function andUGT1A1Gene Expression by Posttranslational Modification of PXR Protein

Abstract

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.

Threonine-290 Regulates Nuclear Translocation of the Human Pregnane X Receptor through Its Phosphorylation/Dephosphorylation by Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Phosphatase 1

“…In a previous study (Sugatani et al, 2012), we found that the expression of UGT1A1 induced by the CDK2 inhibitor roscovitine was increased in HepG2 cells in the presence of exogenously expressed hPXR, whereas a phosphomimetic mutation at the threonine-57, threonine-290, serine-350, and threonine-408 of hPXR suppressed the activity induced by roscovitine. The phosphomimetic T290D mutant YFP-hPXR fusion protein but not the S350D or T408D mutant proteins was markedly retained in the cytoplasm and suppressed nuclear translocation after the treatment with roscovitine (Sugatani et al, 2012).…”

“…Thus, in the present study, CYP3A4 and UGT1A1 were used as biomarkers of exogenously expressed hPXR activation. In a previous study (Sugatani et al, 2012), we demonstrated that a phosphomimetic mutation at the threonine-290 of hPXR suppressed the ligand-independent translocation of hPXR to the nucleus. We herein identified a phosphomimetic mutant of hPXR that abrogated ligand-induced nuclear translocation.…”

“…The hPXR expression vector was generously provided by Dr. Masahiko Negishi (National Institute of Environmental Health Sciences, Research Triangle Park, NC). Mutations were induced in hPXR with a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions (Sugatani et al, 2012). The primers used were; 59-GTCAACTGAGATTCAACG-CAGTGTTCAACGCGGAG-39, 59-CTGTGTCAACTGAGATTCAAC-GATGTGTTCAACGCGGAGACTGGA-39, and their complements for hPXR T290A and T290D, respectively.…”

“…Ten minutes after the second transfection, the cells were cultured with rifampicin (5 Â 10 26 M), roscovitine (5 Â 10 26 M), or vehicle in the presence or absence of various inhibitors for an additional 24 hours unless otherwise stated. Total RNA was extracted using TRIzol reagent from Invitrogen (Carlsbad, CA), and cDNA synthesized from 75 ng of total RNA was subjected to a quantitative real-time PCR as described previously elsewhere (Sugatani et al, 2012) with a Stratagene Mx3000P Real-Time QPCR System (Agilent Technologies Japan, Tokyo, Japan) using SYBR Premix Ex Taq reagent (TaKaRa Bio, Otsu, Japan) for the intercalation reaction with SYBR Green I according to the manufacturer's specifications for UGT1A1 and CYP3A4, as described previously elsewhere (Sugatani et al, 2010), and the protein phosphatase 1 (PP1) catalytic subunit beta isozyme, PP2 catalytic subunit alpha isozyme, and CaMKIIa (TaKaRa Bio). The thermal cycle conditions used were as follows: hold ).…”

“…Similar to CAR, PXR forms a complex with the cytoplasmic CAR retention protein (designated DNAJC7 in the National Center for Biotechnology Information database) and heat shock protein 90 (Hsp90) to maintain a cytoplasmic localization Squires et al, 2004); however, the molecular mechanism underlying nuclear translocation has not yet been fully elucidated. We previously demonstrated that cyclin-dependent kinase (CDK) 2 phosphorylated human pregnane X receptor (hPXR) at serine-350 to suppress binding with hRXR and the coactivator and maintain the acetylation of the hPXR protein, thereby down-regulating hPXR activity (Sugatani et al, 2012). Because the transfection of anti-CDK2 small-interfering RNA (siRNA) was previously shown to elevate the levels of UDP-glucuronosyltransferase 1A1 (UGT1A1), CYP2B6, and CYP3A4 in HepG2 cells (Sugatani et al, 2010), we concluded that the expression of UGT1A1, CYP2B6, and CYP3A4 may be negatively regulated through a CDK2 signaling pathway linked to cell cycle progression in HepG2 cells.…”