Induction of UGT1A1 and CYP2B6 by an Antimitogenic Factor in HepG2 Cells Is Mediated through Suppression of Cyclin-Dependent Kinase 2 Activity: Cell Cycle-Dependent Expression

Sugatani, Junko; Osabe, Makoto; Kurosawa, Masatoshi; Kitamura, Naomi; Ikari, Akira; Miwa, Masao

doi:10.1124/dmd.109.029785

Cited by 23 publications

(25 citation statements)

References 40 publications

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“…Ten minutes after the second transfection, cells were cultured with rifampicin (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/Thermo Fisher Scientific, and cDNA synthesized from 75 ng of total RNA by reverse transcription with a Prime Script reverse transcription reagent kit (TaKaRa Bio, Otsu, Japan) was subjected to a quantitative real-time PCR as described previously (Sugatani et al, 2012) using SYBR Premix Ex Taq reagent (TaKaRa Bio) according to the manufacturer's specifications for UGT1A1 and CYP3A4 (Sugatani et al, 2010), Hsp90, Hsp70, Hsc70, HOP, RBCK1, and UBR5 (TaKaRa Bio), and CHIP (Kajiro et al, 2009). All primers used for real-time PCR were listed in Table 1 Expression of the Flag-Tagged hPXR WT and T408D Mutant Proteins in Mouse Livers In Vivo.…”

Section: Methodsmentioning

confidence: 99%

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

Sugatani

Noguchi

Hattori

et al. 2015

Drug Metabolism and Disposition

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The human pregnane X receptor (hPXR) is a xenobiotic-sensing nuclear receptor that transcriptionally regulates drug metabolism-related genes. The aim of the present study was to elucidate the mechanism by which hPXR is regulated through threonine-408. A phosphomimetic mutation at threonine-408 (T408D) reduced the transcriptional activity of hPXR and its protein stability in HepG2 and SW480 cells in vitro and mouse livers in vivo. Proteasome inhibitors (calpain inhibitor I and MG132) and Hsp90 inhibitor geldanamycin, but not Hsp70 inhibitor pifithrin-m, increased wild-type (WT) hPXR in the nucleus. The translocation of the T408D mutant to the nucleus was significantly reduced even in the presence of proteasome inhibitors, whereas the complex of yellow fluorescent protein (YFP)-hPXR T408D mutant with heat shock cognate protein 70/heat shock protein 70 and carboxy terminus Hsp70-interacting protein (CHIP; E3 ligase) was similar to that of the WT in the cytoplasm. Treatment with pifithrin-m and transfection with anti-CHIP small-interfering RNA reduced the levels of CYP3A4 mRNA induced by rifampicin, suggesting the contribution of Hsp70 and CHIP to the transactivation of hPXR. Autophagy inhibitor 3-methyladenine accumulated YFP-hPXR T408D mutant more efficiently than the WT in the presence of proteasome inhibitor lactacystin, and the T408D mutant colocalized with the autophagy markers, microtubule-associated protein 1 light chain 3 and p62, which were contained in the autophagic cargo. Lysosomal inhibitor chloroquine caused the marked accumulation of the T408D mutant in the cytoplasm. Protein kinase C (PKC) directly phosphorylated the threonine-408 of hPXR. These results suggest that hPXR is regulated through its phosphorylation at threonine-408 by PKC, CHIP/chaperone-dependent stability check, and autophagic degradation pathway.

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Section: Methodsmentioning

confidence: 99%

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

Sugatani

Noguchi

Hattori

et al. 2015

Drug Metabolism and Disposition

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“…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 ).…”

Section: Methodsmentioning

confidence: 99%

“…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. Lin et al (2008) demonstrated that roscovitine activated hPXR-mediated CYP3A4 gene expression by inhibiting CDKs in a ligand-independent manner, and also that CDK2 negatively regulated the activity of hPXR in HepG2 cells.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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The human pregnane X receptor (hPXR) is recognized as a xenobioticsensing nuclear receptor that transcriptionally regulates the gene expression of drug-metabolizing enzymes and transporters. Our study elucidates the mechanism by which the localization of hPXR is regulated through threonine-290. A phosphomimetic mutation at threonine-290 (T290D) retained hPXR in the cytoplasm of HepG2, HuH6, and SW480 cells in vitro and the mouse liver in vivo even after treatment with rifampicin, and a phosphodeficient mutation (T290A) translocated from the cytoplasm to the nucleus as the wild-type hPXR. The amount of the unphosphorylated wild-type yellow fluorescent protein-hPXR fusion protein but not the T290A mutant increased on Phos-tag gels in response to stimulations with rifampicin and cyclin-dependent kinase 2 inhibitor roscovitine, and a marked increase was observed in the unphosphorylated levels of the T290A mutant in nontreated cells. The Ca CaMKII directly phosphorylated the threonine-290 of hPXR, and the T290A mutant conferred resistance to CaMKII. The protein phosphatase (PP) inhibitor okadaic acid (100 nM) and transfection with anti-PP1 siRNA but not anti-PP2A siRNA led to reduced expression of CYP3A4 mRNA. After the rifampicin and roscovitine stimulations, PP1 was recruited to the wild-type hPXR but not the T290A mutant. These results suggest that phosphorylation at threonine-290 by CaMKII may impair the function of hPXR by repressing its translocation to the nucleus, and dephosphorylation by PP1 is necessary for the xenobiotic-dependent nuclear translocation of hPXR.

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“…A previous study conducted in HepG2 cells showed that siRNA-mediated down-regulation of CDK2 resulted in increased expressed levels of CYP3A4 (Sugatani et al, 2010). Moreover, HepG2 cells treated with an antimitogenic factor resulted in decreased activity of CDK2 and increased expression of UGT1A1, CYP2B6, and CYP3A4 (Sugatani et al, 2010), all of which are regulated via PXR (Lehmann et al, 1998;Fig. 9.…”

Section: Pxr-induced Cyp3a4 Expression In Confluent Huh7 Cellsmentioning

confidence: 99%

Induced CYP3A4 Expression in Confluent Huh7 Hepatoma Cells as a Result of Decreased Cell Proliferation and Subsequent Pregnane X Receptor Activation

et al. 2012

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We have previously shown that confluent growth of the human hepatoma cell line Huh7 substantially induces the CYP3A4 mRNA, protein, and activity levels. Here, the mechanisms behind were investigated, and a transcriptome analysis revealed significant up-regulation of liver-specific functions, whereas pathways related to proliferation and cell cycle were downregulated in the confluent cells. Reporter analysis revealed that the CYP3A4 gene was transcriptionally activated during confluence in a process involving pregnane X receptor (PXR). PXR expression was increased, and PXR protein accumulated in the nuclei during confluent growth. The PXR ligand rifampicin further increased the expression of CYP3A4, and siRNAmediated knock-down of PXR in confluent cells resulted in decreased CYP3A4 expression. Cyclin-dependent kinase 2 (CDK2), a known modulator of the cell cycle and a negative regulator of PXR, was more highly expressed in proliferating control cells. Trypsinization of the confluent cells and replating them subconfluent resulted in a decrease in CYP3A4 and PXR expression back to levels observed in subconfluent control cells, whereas the CDK2 levels increased. Knock-down of CDK2 in proliferating control cells increased the CYP3A4 and PXR protein levels. Moreover, the CDK inhibitor roscovitine stimulated the expression of CYP3A4. A phosphorylationdeficient mutation (S350A) in the PXR protein significantly induced the CYP3A4 transcription. In conclusion, the data strongly suggest that the increased CYP3A4 expression in confluent Huh7 cells is caused by the endogenous induction of PXR as a result of cell-cell contact inhibited proliferation and subsequent decreased CDK2 activities, indicating an endogenous, non-ligand-dependent regulation of PXR and CYP3A4, possibly of physiologic and pharmacological significance.

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Induction of UGT1A1 and CYP2B6 by an Antimitogenic Factor in HepG2 Cells Is Mediated through Suppression of Cyclin-Dependent Kinase 2 Activity: Cell Cycle-Dependent Expression

Cited by 23 publications

References 40 publications

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

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

Induced CYP3A4 Expression in Confluent Huh7 Hepatoma Cells as a Result of Decreased Cell Proliferation and Subsequent Pregnane X Receptor Activation

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Induction of UGT1A1 and CYP2B6 by an Antimitogenic Factor in HepG2 Cells Is Mediated through Suppression of Cyclin-Dependent Kinase 2 Activity: Cell Cycle-Dependent Expression

Cited by 23 publications

References 40 publications

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

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

Induced CYP3A4 Expression in Confluent Huh7 Hepatoma Cells as a Result of Decreased Cell Proliferation and Subsequent Pregnane X Receptor Activation

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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