Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2015/11/03/dmd.115.066308.DC1

1521-009X/44/1/137–150$25.00 http://dx.doi.org/10.1124/dmd.115.066308 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:137–150, January 2016 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/ -Autophagy Pathway s

Junko Sugatani, Yuji Noguchi, Yoshiki Hattori, Masahiko Yamaguchi, Yasuhiro Yamazaki, and Akira Ikari1 Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan

Received July 15, 2015; accepted November 2, 2015

ABSTRACT

The human pregnane X receptor (hPXR) is a xenobiotic-sensing nuclear cytoplasm. Treatment with pifithrin-m and transfection with anti-CHIP Downloaded from receptor that transcriptionally regulates drug metabolism–related small-interfering RNA reduced the levels of CYP3A4 mRNA induced by . The aim of the present study was to elucidate the mechanism rifampicin, suggesting the contribution of and CHIP to the by which hPXR is regulated through threonine-408. A phosphomimetic transactivation of hPXR. Autophagy inhibitor 3-methyladenine accu- mutation at threonine-408 (T408D) reduced the transcriptional activity of mulated YFP-hPXR T408D mutant more efficiently than the WT in the hPXR and its stability in HepG2 and SW480 cells in vitro and presence of proteasome inhibitor lactacystin, and the T408D mutant mouse livers in vivo. Proteasome inhibitors (calpain inhibitor I and colocalized with the autophagy markers, microtubule-associated pro- dmd.aspetjournals.org MG132) and inhibitor geldanamycin, but not Hsp70 inhibitor tein 1 light chain 3 and p62, which were contained in the autophagic pifithrin-m, increased wild-type (WT) hPXR in the nucleus. The trans- cargo. Lysosomal inhibitor chloroquine caused the marked accumula- location of the T408D mutant to the nucleus was significantly reduced tion of the T408D mutant in the cytoplasm. Protein kinase C (PKC) eveninthepresenceofproteasomeinhibitors, whereas the complex of directly phosphorylated the threonine-408 of hPXR. These results yellow fluorescent protein (YFP)-hPXR T408D mutant with heat shock suggest that hPXR is regulated through its phosphorylation at cognate protein 70/ 70 and carboxy terminus Hsp70- threonine-408 by PKC, CHIP/chaperone–dependent stability check, interacting protein (CHIP; E3 ligase) was similar to that of the WT in the and autophagic degradation pathway. at ASPET Journals on September 27, 2021

Introduction sequestered in the cytoplasm through the formation of a complex The pregnane X receptor (PXR) is a member of the nuclear receptor with heat shock protein (Hsp) 90 (Squires et al., 2004). Upon the superfamily and is recognized as a xenobiotic sensor that regulates the binding of PXR ligands to the ligand binding domain, it translocates transcriptional expression of phase I, II, and III metabolic enzymes and to the nucleus, in which it forms a heterodimer with retinoid X transporters involved in the metabolism and elimination of potentially receptor and binds to the xenobiotic-response element of the target toxic substances (reviewed in Timsit and Negishi, 2007; Staudinger , resulting in transcriptional activation (Kawana et al., 2003; and Lichti, 2008). PXR also plays important roles in many biologic Squires et al., 2004). events such as hepatic energy metabolism, immune/inflammatory Post-translational modifications to the human PXR (hPXR) by responses, cell proliferation, bone homeostasis, and the pathogenesis of phosphorylation and through small -related modifier- and inflammatory bowel disease and tumor development (reviewed in ubiquitin-signaling pathways have been shown to influence the Konno et al., 2008; Ihunnah et al., 2011; Zhuo et al., 2014; Rathod et al., functions of hPXR by modulating intracellular localization and 2014; Ma et al., 2015). A previous study demonstrated that PXR was nuclear activation (Lichti-Kaiser et al., 2009; Pondugula et al., 2009; Staudinger et al., 2011; Sugatani et al., 2012, 2014; Smutny et al., 2013; Elias et al., 2014; Cui et al., 2015). Cyclin-dependent kinase 2 has been This work was supported in part by Grant-in-Aid for Scientific Research [Grants shown to phosphorylate hPXR at serine-350 to suppress the binding 21590170 and 22590068] from the Ministry of Education, Culture, Sports, Science with retinoid X receptor by maintaining the acetylation of the hPXR and Technology, Japan. protein, thereby downregulating hPXR activity (Lin et al., 2008; 1Current affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sugatani et al., 2012). We previously reported that phosphomimetic Science, Gifu Pharmaceutical University, Japan. mutations in hPXR at threonine-290 and threonine-408 suppressed the dx.doi.org/10.1124/dmd.115.066308. translocation of hPXR protein to the nucleus even after stimulation with s This article has supplemental material available at molpharm.aspetjournals. roscovitine, resulting in reduced hPXR activities (Sugatani et al., 2012). org.

ABBREVIATIONS: 3-MA, 3-methyladenine; CAR, constitutive androstane receptor; CHIP, carboxy terminus Hsp70-interacting protein; DAPI, 4,6- diamidino-2-phenylindole dihydrochloride; DYRK2, dual-specificity tyrosine-(Y)-phosphorylation–regulated kinase 2; GFP, green fluorescent protein; GR, glucocorticoid receptor; HOP, stress-induced phosphoprotein 1; hPXR, human pregnane X receptor; Hsc70, heat shock cognate protein 70; Hsp, heat shock protein; LC3, microtubule-associated protein 1 light chain 3; MBP, maltose binding protein; PKC, protein kinase C; RBCK1, Ring-B-box-coiled-coil protein interacting with protein kinase C-1; siRNA, small-interfering RNA; UBR5, ubiquitin protein ligase E3 component n-recognin 5; UGT, UDP-glucuronosyltransferase; YFP, yellow fluorescent protein.

137 138 Sugatani et al.

Furthermore, we revealed that Ca2+/calmodulin-dependent protein Materials and Methods kinase II (CaMKII) directly phosphorylated hPXR at threonine-290 Materials. MG132, calpain inhibitor I, lactacystin, and geldanamycin were for its retention in the cytoplasm, and protein phosphatase 1 was purchased from Calbiochem (Darmstadt, Germany). Rifampicin, chloroquine recruited to the hPXR-Hsp90 complex and dephosphorylated hPXR in diphosphate, and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO). ligand-stimulated HepG2 cells (Sugatani et al., 2014). Since the Pifithrin-m was from Cayman Chemical Company (Ann Arbor, MI), adenosine- stability of hPXR is known to be regulated through a proteasome S-triphosphate (ATP) from Roche Diagnostics (Mannheim, Germany), and 32 degradation pathway (Rana et al., 2013; Ong et al., 2014) and the hPXR [g- P]ATP from PerkinElmer (Santa Clara, CA). All other chemicals and T408D mutant accumulates in the cytoplasm in the presence of solvents were of analytical grade and obtained from commercial sources. Plasmids. The hPXR expression vector was generously provided by proteasome inhibitor MG132 (Sugatani et al., 2012), the phosphoryla- Dr. Masahiko Negishi (National Institute of Environmental Health Sciences, tion of hPXR at threonine-408 appears to be involved in the regulation NC). Mutations were induced in hPXR with a QuickChange site-directed of its stability. However, the molecular mechanisms regulating the mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s stability and function of the T408D mutant have not been elucidated in instructions, and expression vectors were prepared as shown in our previous detail (Sugatani et al., 2012). study (Sugatani et al., 2012, 2014). Most cellular in eukaryotic cells are degraded through two Cell Culture Conditions, Transfection, and Luciferase Assays. Human major pathways: the ubiquitin-proteasomal pathway and an autophagic liver-derived cells (HepG2 cells, the RIKEN BioResource Center, Ibaraki, 5 process delivering cytoplasmic proteins and components to the Japan) were seeded on 6-well plates at 2  10 cells (2 ml) in Dulbecco’s lysosome (reviewed in Abounit et al., 2012; Lilienbaum, 2013). Heat modified Eagle’s medium supplemented with 10% fetal calf serum (HyClone, Downloaded from shock proteins Hsc70/Hsp70 assist in the refolding of cytoplasmic GE Healthcare Life Sciences, Logan, UT) and antibiotics (100 mgof streptomycin and 10 IU of penicillin/ml) at 37C in the presence of 5% CO , proteins with cochaperone proteins such as the carboxy terminus 2 unless otherwise stated. Twenty-four hours later, HepG2 cells were transfected Hsp70-interacting protein (CHIP) (Kampinga et al., 2003). Chaperone- with UDP-glucuronosyltransferase (UGT1)A1-luciferase reporter constructs dependent E3 ligase CHIP leads to the ubiquitination of misfolded including the distal (-3570/-3180) and proximal (-165/-1) regions (Sugatani proteins, which is facilitated by chaperone proteins to proteolytic et al., 2001, 2008) (0.2 mg) and expression vectors [pCR3 vector (Invitrogen/ degradation through an autophagic process (Kampinga et al., 2003; Thermo Fisher Scientific, Sunnyvale, CA) (0.2 mg), pCR3-hPXR WT (0.2 mg), Morishima et al., 2008). To elucidate the role of phosphorylation at pCR3-hPXR T408A (0.2 mg), pCR3-hPXR T408D (0.2 mg), and pRL-SV40 dmd.aspetjournals.org threonine-408, we herein systematically characterized and compared (Promega, Madison, WI) (0.2 mg)] using HilyMax reagent (Dojindo Laborato- the expression of the hPXR T408D mutant and its interactions with ries, Kumamoto, Japan) according to the manufacturer’s instructions. The chaperone proteins in the cytoplasm and nucleus with the wild-type medium (0.5 ml per well) was replaced after 12 hours with the same medium.  26 (WT) in the presence and absence of proteasome inhibitors and an Cells were subsequently treated for 24 hours with rifampicin (5 10 M) as  Hsp90 inhibitor, which induced chaperone protein Hsp70 and caused 400 concentrated stocks in dimethylsulfoxide; controls received an equivalent volume of dimethylsulfoxide. The total amount of DNA transfected was held autophagy. We revealed that 1) the hPXR T408D mutant was expressed constant in each transfection by using the corresponding empty vector. Trans- to the same extent as the WT, but was cleared more rapidly from fected cells were washed once in phosphate-buffered saline and harvested in 1  at ASPET Journals on September 27, 2021 cultured cells in vitro and mouse livers in vivo, and that 2) the hPXR passive lysis buffer, and luciferase activity was measured simultaneously with T408D mutant formed complexes with chaperone proteins Hsp90– the Dual-Luciferase reporter assay system according to the manufacturer’s Hsc70/Hsp70, as well as cochaperone proteins such as CHIP, stress- instructions (Promega); firefly luciferase values were normalized to Renilla induced phosphoprotein 1 (HOP), and p23 in the cytoplasm, as did the WT values for each sample. in HepG2 cells. Furthermore, the autophagy inhibitor, 3-methyladenine Real-Time Quantitative PCR. Since PXR is a key transcriptional regulator (3-MA) resulted in an increase in autophagy markers such as of CYP3A (Lehmann et al., 1998) and human UGT1A1 (Sugatani et al., 2004, microtubule-associated protein 1 light chain 3 (LC3) and p62/ 2005), we used CYP3A4 and UGT1A1 as biomarkers of the activation of hPXR. SQSTM1, which were colocalized with the hPXR T408D mutant, and HepG2 cells and SW480 human colon cancer cells (American Type Culture Collection) (2  105 cells) seeded on 6-well plates and cultured for 24 hours in led to the accumulation of higher levels of the hPXR T408D mutant Dulbecco’s modified Eagle’s medium (HepG2 cells) and RPMI 1640 medium than the WT in the presence of lactacystin. Thus, the results presented (SW480 cells), respectively, supplemented with 10% fetal calf serum (Hyclone) here provide novel insights into the molecular mechanisms regulating and antibiotics (100 mg of streptomycin and 10 IU of penicillin/ml) were the stability and function of hPXR through its phosphorylation at transfected with expression vectors [pCR3 (0.2 mg), pCR3-hPXR (0.2 mg), or threonine-408 and the CHIP/chaperone–autophagy system in the pCR3-hPXR mutant (0.2 mg)] using TransIT-LT1 (Mirus Bio, Madison, WI) cytoplasm. according to the manufacturer’s instructions. Cells were given fresh medium

TABLE 1 Sequences of oligonucleotides used

Sequence Gene Accession Number Forward Primer Reverse Primer b-Actin NM_001101 59-TGGCACCCAGCACAATGAA-39 59-CTAAGTCATAGTCCGCCTAGAAGCA-39 UGT1A1 NM_000463 59-AATAAAAAAGGACTCTGCTATGCT-39 59-ACATCAAAGCTGCTTTCTGC-39 CYP3A4 NM_017460 59-GTGGGGCTTTTATGATGGTCA-39 59-GCCTCAGATTTCTCACCAACACA-39 Hsp90 NM_001271969 59-GCAGCTCAAGGAATTTGATGG-39 59-GGTGAAGACACAAGTCTATTGGAGA-39 Hsp70 NM_006597 59-GGAAATTGCAGAAGCCTACCTTG-39 59-CTTTGGTAGCCTGACGCTGAGA-39 Hsc70 NM_005345 59-GGATAACGGCTAGCCTGAGGAG-39 59-CTGGGAAGCCTTGGGACAAC-39 HOP NM_001282652 59-GTGCAGCAGATCATGAGTGACC-39 59-TCACCGAATTGCAATCAGACC-39 CHIP NM_005861 59-AGGCCAAGCACGACAAGTACAT-39 59-CTGATCTTGCCACACAGGTAGT-39 RBCK1 NM_006462 59-CCTTCAGCTACCATTGCAAGACC-39 59- TCCTTGCAGTTCATCTGCTCA-39 UBR5 NM_015902 59-ATTTCAATGAGCAGTCAGTCTCAAG-39 59-GTGCCCAGGCTGAGTTTACA-39 Stability Control of PXR via Chaperone-Autophagy Pathway 139

24 hours after being transfected and were then retransfected with the expression vectors, and cultured with rifampicin (5 Â 1026 M) or vehicle for an additional vectors using the same protocol as that for the first transfection. Ten minutes after 24 hours. The cytoplasmic (530 mg) and nuclear (200 mg) proteins, unless the second transfection, cells were cultured with rifampicin (5 Â 1026 M) or otherwise stated, were precleared with protein G agarose beads and rabbit IgG vehicle in the presence or absence of various inhibitors for an additional (Santa Cruz Biotechnology, Dallas, TX) for 30 minutes at 4C. Yellow 24 hours, unless otherwise stated. Total RNA was extracted using TRIzol reagent fluorescent protein (YFP)-tagged proteins were immunoprecipitated using an from Invitrogen/Thermo Fisher Scientific, and cDNA synthesized from 75 ng of anti-GFP antibody combined with protein G agarose beads. The agarose beads 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. To investigate the stability of the hPXR WT and T408D mutant, plasmids [pCMV-DYKDDDDK-hPXR WT (1 mg) or the pCMV-DYKDDDDK-hPXR T408D mutant (1 mg)] were injected into the tail veins of male imprinting-control-region (ICR) mice (4 weeks old) using the

TransIT-QR Hydrodynamic Delivery Starter Kit (Mirus Bio) according to the Downloaded from manufacturer’s instructions. Twelve and 24 hours later, livers were rapidly excised and weighed. Whole livers were lysed with a freshly prepared lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, 1 mM benzamidine, 1 mg/ml aprotinin, and 1 mg/ml leupeptin at 4C. The lysates were centrifuged at 14,000g for

10 minutes. Protein concentrations were determined using a DC protein assay dmd.aspetjournals.org (Bio-Rad, Hercules, CA). Western Blot Analysis. HepG2 cells (2  106 cells) seeded on 75-cm2 flasks and cultured for 24 hours were transfected with expression vectors [pCR3-hPXR WT (10 mg), the pCR3-hPXR T408D mutant (10 mg), pCMV-DYKDDDDK- hPXR WT (10 mg), the pCMV-DYKDDDDK-hPXR T408D mutant (10 mg), pEYFP-hPXR WT (10 mg), or the pEYFP-hPXR T408D mutant (10 mg)] using TransIT-LT1 (Mirus Bio) according to the manufacturer’s instructions. Cells were given fresh medium 24 hours after transfection, were further transfected at ASPET Journals on September 27, 2021 with the expression vectors, and then cultured with rifampicin (5  1026 M) or vehicle for an additional 24 hours unless stated otherwise. Treated and untreated cells were washed three times with ice-cold phosphate-buffered saline and lysed with a freshly prepared lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, 1 mM benzamidine, 1 mg/ml aprotinin, and 1 mg/ml leupeptin at 4C. The lysates were centrifuged at 14,000g for 10 minutes. Nuclear extracts and cytoplasmic fractions were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA). Protein Fig. 1. Transactivation capacity of the hPXR T408D mutant in HepG2 and SW480 concentrations were determined using the DC protein assay (Bio-Rad). cells (A–C), and its protein stability in vitro in HepG2 cells (D) and in vivo in mouse Cytoplasmic fractions, nuclear extracts, cell lysates, or liver lysates (20 mg) were livers (E). (A–C) HepG2 and SW480 cells cultured for 24 hours were transfected resolved by electrophoresis on an SDS 5–20% polyacrylamide gel (ATTO with expression vectors encoding the hPXR WT or T408D mutant. The medium was Corporation, Tokyo, Japan), and electroblotted onto a polyvinylidene difluoride replaced 24 hours after transfection, and cells were retransfected and then cultured with rifampicin (5  1026 M) or vehicle (DMSO) for an additional 24 hours. membrane (Merck Millipore, Darmstadt, Germany). Membranes were blocked at UGT1A1 reporter activity assessed by the expression of a reporter gene encoding 4 C overnight in Blocking One (Nacalai Tesque, Kyoto, Japan) and probed for firefly luciferase (A), and CYP3A4 mRNA levels in HepG2 (B) and SW480 (C) 1 hour with primary antibodies, including the anti-DDDDK (Flag) tag (M185- cells treated with rifampicin or vehicle were expressed by taking the control value 3L), anti-green fluorescent protein (GFP) (598), anti-LC3 (M152-3), and anti- obtained from hPXR WT-expressing and vehicle-treated cells as one. Data presented p62/SQSTM1 (M162-3) from Medical Biologic Laboratories (Nagoya, Japan), are the average 6 S.E. of three experiments. **P , 0.01; ***P , 0.001 versus the # , ## , ### , anti-Hsp90 (#4874) from Cell Signaling Technology (Danvers, MA), anti- hPXR WT in cells treated without rifampicin; P 0.05; P 0.01; P 0.001 versus the hPXR WT in cells stimulated with rifampicin. (D, E) A Western blot Histone H1 (ab125027), anti-Hsp70 (ab2787), anti-HOP (ab56873), anti-CHIP analysis of the Flag-tagged hPXR WT and T408D mutant proteins in HepG2 cells (ab109103), and anti-p23 (ab2814) from Abcam (Cambridge, England), anti- after the in vitro transfection (D) and in the mouse livers 12 hours and 24 hours after Hsc70 (SMC-151A/B) from StressMarq Bioscience (Victoria, BC, Canada), the in situ DNA injection (E). HepG2 cells cultured for 33 hours were transfected anti-ubiquitin (MAB701) from R&D Systems (Minneapolis, MI), anti-PXR with expression plasmids for hPXR. The medium was replaced 24 hours after  26 (AB10302) from Merck Millipore, and anti-a-tubulin (CP06) from Calbiochem/ transfection, and cells were cultured with MG132 (1 10 M) or vehicle (DMSO) Merck Millipore. Antigen-antibody complexes were detected using an appro- for an additional 15 hours. Protein levels in cells before the treatment with MG132 were taken as the level at time 0. Flag-hPXR proteins in HepG2 cell lysates (D) or in priate secondary antibody conjugated to horseradish peroxidase [horseradish liver lysates (E) were determined by Western blotting with the indicated antibodies peroxidase-conjugated anti-rabbit, anti-goat, or anti-mouse immunoglobulin [anti-Flag and anti-a-tubulin (loading control) antibodies]. (E) Band intensities were (Jackson Immuno Research)] and visualized with an enhanced chemilumines- measured with ImageJ software, and each quantified value of the Flag-hPXR bands cence system (GE Healthcare, Little Chalfont, England). was corrected for that of a-tubulin. Relative levels are expressed by taking the Coimmunoprecipitation. HepG2 cells (2  106 cells) seeded on 75-cm2 protein levels obtained from mouse livers expressing Flag-hPXR WT 12 hours after the in situ DNA injection as one. Data presented are the average 6 S.E. of three to flasks and cultured for 24 hours were transfected with expression vectors five experiments. *P , 0.05 versus mouse livers expressing Flag-hPXR WT [pEYFP-hPXR WT (10 mg) or the pEYFP-hPXR T408D mutant (10 mg)] using 24 hours after the in situ DNA injection. Western blottings were performed twice TransIT-LT1 according to the manufacturer’s instructions. Cells were given with similar results, and representative data are shown. WT, wild-type hPXR; fresh medium 24 hours after transfection, further transfected with the expression T408A, T408A-mutated hPXR; T408D, T408D-mutated hPXR. 140 Sugatani et al.

were washed three times with Dulbecco’s phosphate buffer and subjected to SDS-PAGE on a 5–20% gradient gel (ATTO Corporation). The proteins were transferred to an Immobilon-P transfer membrane (Merck Millipore), and the blots were probed with antibodies as indicated. Small-Interfering RNA–Mediated Protein Knockdown. Small-Interfering RNAs (siRNAs) targeting human CHIP (NM_005861_stealth_725; sense 59- CAGCUGGAGAUGGAGAGCUAUGAUG-39 and antisense 59-CAU- CAUAGCUCUCCAUCUCCAGCUG-39) and the human CHIP control (NM_005861_stealth_control_725; sense 59-CAGAGGUAGAGGGAGAUC- GUCUAUG-39 and antisense 59-CAUAGACGAUCUCCCUCUACCUCUG- 39) were obtained from Invitrogen/Thermo Fisher Scientific. HepG2 cells cultured for 24 hours were transfected with siRNA duplexes (100 nM) using TransIT-siQUEST (Mirus Bio) according to the manufacturer’s instructions. Cells were further transfected with expression vectors [the pCR3-hPXR WT, pCR3-hPXR T408D mutant, pCMV-DYKDDDDK-hPXR WT, or pCMV- DYKDDDDK-hPXR T408D mutant] 12 hours after siRNA transfection, and 12 hours after the second transfection were cultured with rifampicin (5 Â 1026 M) or vehicle for an additional 24 hours, unless stated otherwise. Treated and untreated

cells were washed three times with ice-cold phosphate-buffered saline and lysed with Downloaded from a freshly prepared lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenyl-

methylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, 1 mM benzamidine, 1 mg/ml aprotinin, and 1 mg/ml leupeptin at 4C. The lysates were centrifuged at 14,000g for 10 minutes. Total RNA was extracted using TRIZOL reagent from Invitrogen/ Thermo Fisher Scientific, and cDNA synthesized from 75 ng of total RNA was

subjected to real-time quantitative PCR as described previously (Sugatani et al., 2012). dmd.aspetjournals.org In Vitro Ubiquitination Assays. The maltose-binding protein (MBP)-hPXR WT and T408D mutant fusion proteins were expressed in NEB Express Competent E. coli (High Efficiency) and purified using amylose resin according to the manufacturer’s protocols (New England BioLabs, Boston, MA). Recombinant hPXR prepared by the cleavage of MBP-hPXR with Factor Xa, the recombinant His-tagged hPXR ligand-binding domain (138-434) (Sigma- Aldrich), or recombinant Hsc70 (StressMarq Biosciences) was incubated with

UbcH5c (UBE2D3, E2 ubiquitin-conjugating enzyme; Abcam) and CHIP (E3 at ASPET Journals on September 27, 2021 enzyme; R&D Systems) at 37C for 30 and 60 minutes using an in vitro ubiquitin kit (Abcam) according to the manufacturer’s instructions. The amounts of each substrate used were 21–108 ng in 50 ml of the reaction solution. Ubiquitinated proteins were determined by Western blotting using an anti-ubiquitin antibody. Colocalization of the Fluorescent Protein–Tagged hPXR WT or T408D Mutant and Autophagy Markers P62 and LC3 in HepG2 Cells. To detect the colocalization of the hPXR WT or T408D mutant and the autophagy markers p62 and LC3, HepG2 cells (3.5 Â 104 /well) cultured on four-well glass slides (Laboratory-TekII Chamber Slide, cat. no. 154526; Thermo Fisher Scientific) for 24 hours were transfected with expression vectors [the pEYFP-hPXR WT (0.5 mg) or pEYFP-hPXR T408D mutant (0.5 mg)] using TransIT-LT1 according to the manufacturer’s instructions. Twelve hours after transfection, cells were incubated with 3-methyladenine (5 mM) for 12 hours, and then further cultured with rifampicin (5 Â 1026 M) or vehicle in the presence or absence of lactacystin (5 mM) for an additional 24 hours. Cells were blocked with methanol and incubated with the anti-LC3 (1:250) or anti-p62 (1:250) antibody at room temperature for 30 minutes. These cells were counterstained at room temperature for 30 minutes with an Alexa Fluor 555-conjugated anti-mouse IgG (H + L) antibody (1:1000) (Thermo Fisher Scientific). Coverslips were mounted with Vectashield antifade mounting medium (Vector Laboratories Inc., Burlingame, CA) with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector Labo- ratories) to visualize nuclei, and images were acquired with a Zeiss LSM510 confocal microscope (Carl Zeiss, Oberkochen, Germany). In Vitro Kinase Assays. The MBP-hPXR WT and T408A mutant fusion proteins were expressed in NEB Express Competent E. coli (High Efficiency)

Fig. 2. Effects of proteasome inhibitors on the transactivation capacity of the hPXR WT and T408D mutant in HepG2 cells. HepG2 cells cultured for 24 hours were transfected with expression vectors encoding the hPXR WT or T408D mutant. The medium was replaced 24 hours after transfection, and cells were retransfected and the average 6 S.E. of three experiments. *P , 0.05; **P , 0.01; ***P , 0.001 then cultured with rifampicin (5  1026 M) or vehicle (DMSO) in the presence or versus the hPXR WT in vehicle-treated control cells; ##P , 0.01; ###P , 0.001 absence of calpain inhibitor I (1  1025 M) (A), MG132 (1  1026 M) (B), versus the hPXR WT in rifampicin-stimulated control cells; ^P , 0.05 versus the lactacystin (5  1026 M) (C), or vehicle (DMSO, control) for an additional hPXR T408D mutant in vehicle-treated control cells; +++P , 0.001 versus the 24 hours. CYP3A4 mRNA levels were expressed by taking the value obtained from hPXR T408D mutant in rifampicin-stimulated control cells. WT, wild-type hPXR; hPXR WT-expressing and vehicle-treated control cells as one. Data presented are T408D, T408D-mutated hPXR. Stability Control of PXR via Chaperone-Autophagy Pathway 141 and purified using amylose resin according to the manufacturer’s protocol (New ANOVA or unpaired t test. The level set for significant differences was England Biolabs, Boston, MA). Kinase assays for protein kinase C (PKC; P , 0.05. Promega) and DYRK2 (SignalChem Lifesciences, Richmond, BC, Canada) were performed according to the manufacturer’s specifications. PKC consisted primarily of the a, b, and g isoforms with lesser amounts of the d and z isoforms Results (Walton et al., 1987). Briefly, kinase reactions were performed in 30 ml of the kinase buffer [for PKC, 20 mM HEPES (pH 7.4), 3.4 mM CaCl2, and 10 mM Phosphomimetic Mutation of hPXR at T408 Reduced the MgCl2; for DYRK2, 25 mM MOPS (pH 7.2), 12.5 mM b-glycerol-phosphate, Induction of CYP3A4 mRNA by Rifampicin. We previously 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA, and 0.25 mM dithiothreitol] reported that the phosphomimetic mutation of hPXR at T408 attenuated containing 0.75–4.5 mg of the substrate protein, 100 mM ATP, and 0.12 MBq of the induction of UGT1A1 mRNA expression by roscovitine and 32 [g- P]ATP at 30C for 10 and 20 minutes, unless otherwise stated. Reactions rifampicin in HepG2 cells (Sugatani et al., 2012). The present study  were stopped by adding the same volume of 2 Laemmli sample buffer [125 mM also revealed that the phosphomimetic T408D mutation attenuated Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.002% Bromophenol blue, and 10% UGT1A1 reporter activity by rifampicin, whereas the phosphodeficient 2-mecaptoethanol] and samples were resolved by SDS-PAGE on a 5–20% gradient gel (ATTO Corporation) after heat denaturation. Phosphorylation of the T408A mutation exhibited similar activity to that of the WT (Fig. 1A). substrate was visualized by autoradiography. Furthermore, the T408D mutation reduced the induction of CYP3A4 Statistical Analysis. Values are expressed as the mean 6 standard error. mRNA by rifampicin not only in HepG2 but also in SW480 cells (Fig. All data were analyzed using a one-way analysis of variance (ANOVA). 1, B and C). We then determined whether the phosphomimetic mutation

The significance of differences between groups was analyzed using an of hPXR at T408 altered the expression of hPXR in HepG2 cells in vitro Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 3. Immunoprecipitation and Western blot analyses of the nucleocytoplasmic distribution of the hPXR WT and T408D mutant and their interactions with chaperone and cochaperone proteins in proteasome inhibitor–treated HepG2 cells. HepG2 cells cultured for 24 hours were transfected with expression vectors encoding the YFP-hPXR WT or T408D mutant. The medium was replaced 24 hours after transfection, and cells were retransfected and then cultured with rifampicin (5 Â 1026 M) or vehicle (DMSO) in the presence or absence of calpain inhibitor I (1 Â 1025 M), MG132 (1 Â 1026 M), or vehicle (DMSO, control) for an additional 24 hours. Cytoplasmic, 530 mg (A) and nuclear, 200 mg (B) proteins prepared from these cells and a blank sample (vehicle) were precipitated with anti-GFP antibody. The resulting bound proteins were subjected to a Western blot analysis with the indicated antibodies. Cytoplasmic and nuclear YFP-tagged hPXR and chaperone and cochaperone proteins were determined by Western blotting using the indicated antibodies: anti-a-tubulin (loading control) and anti-histone H1 (loading control) antibodies. Experiments were performed three times with similar results, and representative data are shown. WT, wild-type hPXR; T408D, T408D-mutated hPXR. 142 Sugatani et al. and in mouse livers in vivo or reduced the stability of hPXR, resulting in injection. Whereas no significant difference was observed in the levels impaired transcriptional activity. No significant change was observed in of the hPXR WT and T408D mutant proteins expressed in mouse livers the protein levels of the hPXR WT or T408D mutant expressed 24 hours 12 hours after the in situ DNA injection, the levels of the T408D mutant after transfection with expression plasmids for hPXR, and 39 hours protein were significantly lower than those of the WT protein 24 hours after transfection the level of the T408D mutant protein was reduced in after the in situ DNA injection (Fig. 1E). These results suggested that the absence of MG132 but increased to a level similar to that of the WT the impaired function of the phosphomimetic mutant was not caused by protein in the presence of MG132 (Fig. 1D). We also investigated the its reduced expression, and also that phosphorylation at T408 reduced expression of the hPXR WT and T408D mutant proteins in mouse livers the protein stability of hPXR, thereby impairing its function. after the in situ WT- or T408D mutant-expressing plasmid DNA Effects of Proteasome Inhibitors on the Nucleocytoplasmic Distribution of the hPXR WT and T408D Mutant and Their Interactions with Chaperone and Cochaperone Proteins. Since hPXR stability was previously shown to be regulated via phosphorylation-facilitated ubiquitination through dual-specificity tyrosine-(Y)-phosphorylation–regulated kinase 2 (DYRK2) and Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 4. Effects of proteasome inhibitors on the nucleocytoplasmic distribution of the hPXR WT and T408D mutant (A, B) and their interactions with chaperone and cochaperone proteins (C–F). The band intensities shown in Fig. 3 were measured with ImageJ software. (A, B) Each quantified value of the YFP-hPXR bands was Fig. 5. Effects of geldanamycin (A–C) and pifithrin-m (D, E) on the transactivation corrected for that of a-tubulin (A) or histone H1 (B), and relative levels are capacity of the hPXR WT and T408D mutant (A, D), expression of chaperone and expressed by taking the values obtained from cells expressing the YFP-hPXR WT cochaperone mRNAs in cells expressing the hPXR WT (B, C), and nucleocyto- and treated without rifampicin as one. (C–F) Cytosolic YFP-PXR was immunopre- plasmic distribution of the hPXR WT and T408D mutant (E) in HepG2 cells. HepG2 cipitated using an anti-GFP antibody and other proteins bound to PXR were detected cells cultured for 24 hours were transfected with expression plasmids for hPXR. The by Western blotting. Each quantified value of the cytosolic YFP-hPXR, Hsp90, medium was replaced 24 hours after transfection, and cells were retransfected and Hsp70, Hsc70, and CHIP bands was corrected for that of IgG heavy chain (HC). then cultured with rifampicin (5 Â 1026 M) or vehicle (DMSO) in the presence of Relative levels of the Hsp90, Hsp70, Hsc70, and CHIP to the hPXR were expressed pifithrin-m (1 Â 1025 M), geldanamycin (1 Â 1026 M), or vehicle (DMSO, control) by taking the values obtained from cells expressing the YFP-hPXR WT and treated for an additional 24 hours. (A–D) Messenger RNA levels in control cells treated without rifampicin as one. Data presented are the average 6 S.E. of three without rifampicin were taken as one. Data presented are the average 6 S.E. of three experiments. a, P , 0.05 versus the hPXR WT in vehicle-treated control cells; b, experiments. *P , 0.05; **P , 0.01; ***P , 0.001 versus the hPXR WT in P , 0.05; bb, , 0.01 versus the hPXR WT in rifampicin-stimulated control cells; vehicle-treated control cells; ##P , 0.01; ###P , 0.001 versus the hPXR WT in cc, P , 0.01 versus the hPXR T408D mutant in vehicle-treated control cells; d, rifampicin-stimulated control cells; +++p,0.001 versus the hPXR T408D mutant P , 0.05 versus the hPXR T408D mutant in rifampicin-stimulated control cells; e, in rifampicin-stimulated control cells. (E) Cytoplasmic and nuclear YFP-tagged P , 0.05 versus the hPXR WT in vehicle-treated cells with calpain inhibitor I; hPXR from cells treated with pifithrin-m (1 Â 1025 M) or vehicle were determined f, P , 0.05 versus the hPXR WT in rifampicin-stimulated cells with calpain by Western blotting using anti-GFP, anti-a-tubulin (loading control), and anti- inhibitor I; g, P , 0.05 versus the hPXR WT in vehicle-treated cells with MG132; h, histone H1 (loading control) antibodies. Experiments were performed twice with P , 0.05 versus the hPXR WT in rifampicin-stimulated cells with MG132. WT, similar results, and representative data are shown. WT, wild-type hPXR; T408D, wild-type hPXR; T408D, T408D-mutated hPXR. T408D-mutated hPXR. Stability Control of PXR via Chaperone-Autophagy Pathway 143 ubiquitin protein ligase E3 component n-recognition 5 (UBR5) (Ong Geldanamycin induced the expression of Hsp90 and Hsp70 mRNAs, did et al., 2014) as well as via ubiquitination by the Ring-B-box-coiled-coil not alter Hsc70 or HOP mRNA levels, and reduced CHIP mRNA levels protein interacting with PKC-1 (RBCK1) and its proteasomal degra- (Fig. 5 and Supplemental Fig. 2). Furthermore, it markedly increased dation (Rana et al., 2013), we herein examined the effects of proteasome Hsp70, hPXR WT, and T408D mutant protein levels in the cytoplasm, inhibitors on the stability of the T408D mutant. Calpain inhibitor I at whichthenappearedtoincreaseWTandT408Dmutantproteinlevelsin 10 mM and lactacystin at 5 mM, which did not significantly suppress the nucleus, as well as CYP3A4 mRNA levels (Figs. 5 and 6). The cell growth, enhanced the basic levels of CYP3A4 mRNA and also the immunoprecipitation assay revealed that geldanamycin increased complex CYP3A4 mRNA levels induced by rifampicin, respectively, in HepG2 formation by the hPXR WT and T408D mutant with Hsp70, Hsc70, HOP, cells exogenously expressing the hPXR WT (Supplemental Fig. 1 and and CHIP, but not p23 in the cytoplasm (Fig. 6). The binding of the hPXR Fig. 2). MG132 at 1 mM led to the accumulation of the hPXR WT, and WT and T408D mutant with Hsp90, CHIP, and p23 was not observed in T408D mutant proteins in whole cell lysates appeared to slightly the nucleus of geldanamycin-treated cells, the same as in the control and increase the basic levels of CYP3A4 mRNA of the hPXR WT, and proteasome inhibitor-treated cells (Figs. 3 and 6). In contrast, pifithrin-m at increased those of the T408D mutant, whereas it significantly sup- 10 mM accumulated the hPXR WT and T408D mutant proteins in the pressed cell growth (Supplemental Figs. 1 and 2). On the other hand, cytoplasm but not in the nucleus (Fig. 5). These results suggested that even in the presence of calpain inhibitor I at 10 mM and MG132 at Hsp70 contributed to the folding of client proteins such as hPXR in the 1 mM, the protein levels of the T408D mutant were lower than or similar cytoplasm. to those of the WT in the cytoplasm and were significantly lower in the CHIP Deletion Attenuated hPXR Transcriptional Activity in Downloaded from nucleus (Figs. 2–4). Immunoprecipitation and Western blot analyses HepG2 Cells Treated with and without Lactacystin. Since the revealed that the complexes of the hPXR T408D mutant with Hsp90, knockdown of E3 ligases RBCK1 and UBR5 was previously reported Hsc70/Hsp70, CHIP, HOP, and p23 formed as well as that with the WT to result in the accumulation of cellular hPXR and concomitant in the cytoplasm, whereas interactions between hPXR and Hsp90, increases in the induction of hPXR target genes by rifampicin (Rana CHIP, and p23 were not observed in the nucleus (Figs. 3 and 4). et al., 2013; Ong et al., 2014), we evaluated the effects of calpain These results suggested that the T408D mutant was degraded through inhibitor 1 and geldanamycin on the expression of CHIP, RBCK1, and a proteasome-independent degradation pathway in addition to a UBR5 mRNAs. The inhibition of proteasome and Hsp90 did not dmd.aspetjournals.org ubiquitin/proteasome pathway. markedly affect the expression of CHIP, RBCK1, or UBR5 mRNAs; Effects of Hsp90 and Hsp70 Inhibitors on Transcriptional the calpain inhibitor at 10 mM slightly increased CHIP and UBR5 Activities and Nucleocytoplasmic Distribution of the T408D Mutant, mRNA levels but did not alter those of RBCK1 mRNA (Supplemental and Its Complex Formation with Chaperone and Cochaperone Fig. 2). Furthermore, geldanamycin at 1 mM reduced CHIP and Proteins in HepG2 Cells. We investigated the mechanisms by which the RBCK1 mRNA levels but did not alter those of UBR5 mRNA inhibition of Hsp90 and Hsp70 influenced the transcriptional activity and (Supplemental Fig. 2). To examine the effects of CHIP knockdown, cellular distribution of hPXR using the Hsp90 inhibitor geldanamycin and anti-CHIP siRNA was introduced into HepG2 cells and the hPXR WT at ASPET Journals on September 27, 2021 Hsp70 inhibitor pifithrin-m. Geldanamycin at 1 mM increased the basic and T408D mutant were then exogenously expressed in these cells. levels of CYP3A4 mRNA in nonstimulated cells, and pifithrin-m at Transfection with anti-CHIP siRNA reduced CHIP mRNA levels to 10 mM reduced CYP3A4 mRNA levels in the hPXR WT and T408D 0.29–0.33 fold of the control and CHIP protein levels in whole cells to mutant-expressing rifampicin-stimulated cells, whereas cell growth was 0.30–0.34 fold of the control, and also significantly attenuated the suppressed in cells treated with geldanamycin at 1 mM but not in those levels of CYP3A4 mRNA induced by rifampicin, demonstrating that treated with pifithrin-m at 10 mM (Fig. 5 and Supplemental Fig. 1). CHIP may contribute to the transactivation of hPXR (Fig. 7). The

Fig. 6. Effects of geldanamycin on the nucleocytoplasmic distribution of the hPXR WT and T408D mutant and their interactions with chaperone and cochaperone proteins. HepG2 cells cultured for 24 hours were transfected with expression vectors encoding the YFP-hPXR WT and T408D mutant. The medium was replaced 24 hours after transfection, and cells were retransfected and then cultured with rifampicin (5 Â 1026 M) or vehicle (DMSO) in the presence or absence of geldanamycin (1 Â 1026 M) for an additional 24 hours. Cytoplasmic, 500 mg (A) and nuclear, 200 mg (B) proteins prepared from these cells and a blank sample (vehicle) were precipitated with an anti-GFP antibody. The resulting bound proteins were subjected to a Western blot analysis with the indicated antibodies. Cytoplas- mic and nuclear YFP-tagged hPXR and chaperone and cochaperone proteins were determined by Western blotting using the indicated antibodies: anti-a-tubulin (loading control) and anti-histone H1 (loading control) antibodies. Experiments were performed twice with similar results, and representative data are shown. IP, Immunoprecipitate analysis; WB, Western blot analysis; WT, wild-type hPXR; T408D, T408D-mutated hPXR. 144 Sugatani et al. knockdown of CHIP led to the significant accumulation of the hPXR contrast, the T408D mutant protein accumulated more efficiently than WT in cells treated without rifampicin in the presence of lactacystin the WT in the cytoplasm but not in the nucleus in the presence of (Fig. 7B). These results revealed that CHIP may contribute to the chloroquine, resulting in reduced transcriptional activities (Fig. 8). degradation of hPXR as well as its transactivation. These results indicated that the T408D mutant was degraded in the The Lysosome Inhibitor Chloroquine Increased the Stability of lysosome. the hPXR T408D Mutant in the Cytoplasm. In an attempt to explore The Complex of hPXR with Chaperone and Cochaperone Proteins the hypothesis that the hPXR T408D mutant is degraded in lysosomes Was Ubiquitinated in Cells TreatedwithorwithoutProteasome that fuse with an autophagic vacuole, we investigated the effects of Inhibitors. To determine whether hPXR was directly ubiquitinated by chloroquine on the transcriptional activity and nucleocytoplasmic E3 CHIP, we assayed the in vitro ubiquitination of the distribution of the T408D mutant. Chloroquine at 20 mM, which did hPXR WT by CHIP with the E2 ubiquitin-conjugating enzyme UbcH5c not affect cell growth, increased the induction of CYP3A4 mRNA by and compared with Hsc70, because Hsc70 is a major target of CHIP (Jiang rifampicin and a concomitant increase was observed in hPXR WT et al., 2001). As shown in Fig. 9A, CHIP directly ubiquitinated Hsc70 but protein levels in the nucleus (Supplemental Fig. 1 and Fig. 8). In not the hPXR WT. In contrast, when immunoprecipitated proteins with the Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 7. Effects of anti-CHIP siRNA transfection on the expression of CYP3A4 mRNA and hPXR, CHIP, and a-tubulin proteins. HepG2 cells cultured for 24 hours were transfected with anti-CHIP siRNA (1 Â 1027 M) or mock-transfected. Cells were retransfected with expression vectors encoding the Flag-tagged hPXR WT or T408D mutant 12 hours after the first transfection. Twelve hours after the second transfection, cells were cultured with rifampicin (5 Â 1026 M) or vehicle (DMSO) in the presence or absence of lactacystin (5 Â 1026 M) for an additional 24 hours. In cells expressing the hPXR WT and T408D mutant (A) and those expressing the hPXR WT and treated with or without lactacystin (B), CHIP and CYP3A4 mRNA levels in mock-transfected and vehicle-treated control cells without lactacystin were taken as one. Data presented are the average 6 S.E. of three experiments. The resulting proteins were determined by Western blotting using anti-Flag, anti-CHIP, and anti-a-tubulin (loading control) antibodies. Band intensities were measured with ImageJ software, and each quantified value of the Flag-hPXR and CHIP bands was corrected for that of a-tubulin. Relative levels are expressed by taking the values obtained from the mock-treated and vehicle-treated cells expressing the Flag-hPXR WT without lactacystin as one. Western blottings were performed three times with similar results, and representative data are shown. Data presented are the average 6 S.E. of three experiments. *P,0.05; ***P, 0.001 versus the hPXR WT in mock-transfected and vehicle-treated cells without lactacystin; ##P,0.01: ###P , 0.001 versus the hPXR WT in mock-transfected and rifampicin-stimulated cells without lactacystin; ^^^P , 0.001 versus the hPXR T408D mutant in mock-transfected and vehicle-treated cells without lactacystin (A) or the hPXR WT in mock-transfected and lactacystin- and vehicle-treated cells (B); +++P , 0.001 versus the hPXR T408D mutant in mock-transfected and rifampicin-stimulated cells without lactacystin (A) or mock-transfected and lactacystin- and rifampicin-treated cells (B). WT, wild-type hPXR; T408D, T408D-mutated hPXR. Stability Control of PXR via Chaperone-Autophagy Pathway 145 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 8. Effects of chloroquine on transactivation capacities of the hPXR WT and T408D mutant and their nucleocytoplasmic distribution in HepG2 cells. HepG2 cells cultured for 24 hours were transfected with expression vectors encoding the Flag-tagged hPXR WT or T408D mutant. The medium was replaced 24 hours after transfection, and cells were retransfected and then cultured with rifampicin (5 Â 1026 M) or vehicle (DMSO) in the presence or absence of chloroquine (2 Â 1025 M) for an additional 24 hours. (A) CYP3A4 mRNA levels in cells treated with rifampicin or vehicle in the presence or absence of chloroquine (2 Â 1025 M) were expressed by taking the control value obtained from hPXR WT- expressing and vehicle-treated cells without chloroquine as one. Data presented are the average 6 S.E. of three experiments. **P , 0.01 versus the hPXR WT in vehicle-treated cells without chloroquine; ###P , 0.001 versus the hPXR WT in rifampicin-stimulated cells without chloroquine; +++P , 0.001 versus the hPXR T408D mutant in chloroquine-treated and rifampicin-stimulated cells. Cytoplasmic (B) and nuclear (C) proteins were determined by Western blotting using anti-Flag, anti-a-tubulin (loading control), and anti-histone H1 (loading control) antibodies. Experiments were performed twice with similar results, and representative data are shown. WT, wild-type hPXR; T408D, T408D-mutated hPXR. anti-GFP antibody for the cytoplasm of vehicle- and calpain inhibitor I- were markedly smeared and polyubiquitinated for the cytoplasm of MG132- treated cells were immunoblotted for ubiquitin, a protein band near 73 kDa treated cells (Fig. 9B). [bandmarkedwithtwoasterisks(**)inFig.9B]inthehPXR/chaperone Autophagic Cargo Contained hPXR. Wu et al. (2010) reported complex and smeared bands were detected; however, the complex proteins that although 3-MA promoted an autophagic flux (resulting in an 146 Sugatani et al.

whether the autophagic cargo contained hPXR, we examined the colocalization of the hPXR WT and T408D mutant with LC3 using an anti-LC3 antibody to identify the autophagic structure. Many LC3 puncta contained the hPXR WT and the T408D mutant (Fig. 11). Similar results were obtained for the ubiquitinated substrate adaptor p62 in cells treated with lactacystin and/or 3-MA (Fig. 11 and Supplemental Fig. 3). These results suggested that LC3- and p62-positive autophagic cargo functioned to deliver hPXR to the lysosome. hPXR Was Phosphorylated at Threonine-408 by PKC In Vitro. In our previous study (Sugatani et al., 2014), we demonstrated that the mRNA levels of UGT1A1 induced by rifampicin or roscovitine were slightly higher in HepG2 cells simultaneously treated with Go6983 (PKC inhibitor) but were lower in HepG2 cells simultaneously treated with Rp-8-Br-cAMPS (PKA inhibitor). Since hPXR contained the minimal recognition motif S/T-X-R at amino acids 408–410, which may potentially be phosphorylated by PKC (Kang et al., 2012), and DYRK2 phosphorylates hPXR to facilitate its subsequent ubiquitina- Downloaded from tion by UBR5 (Ong et al., 2014), we performed assays with PKC or DYRK2 as protein serine/threonine kinase, [g-32P]ATP and engineered MBP fusion proteins containing hPXR. In vitro kinase assays revealed that hPXR was phosphorylated by PKC and DYRK2, as assessed by radiography (Fig. 12A). To identify the phosphorylated site(s), we investigated whether the hPXR T408A mutant protein was phosphor- ylated by these kinases and found that mutating the threonine-408 of dmd.aspetjournals.org hPXR to alanine led to significantly lower phosphorylation levels by PKC than by the hPXR WT (0.73 6 0.08 of the WT; P , 0.05 versus the WT), whereas DYRK2 phosphorylated the hPXR T408A mutant more than the hPXR WT (1.26 6 0.10 of the WT; P , 0.05 versus the WT) (Fig. 12). Taken together, these results indicated that the threonine-408 of hPXR in the ligand-binding domain was one of

the PKC-specific phosphorylation sites. at ASPET Journals on September 27, 2021 Fig. 9. Ubiquitination of hPXR by an in vitro assay (A) and the chaperone/hPXR complex in HepG2 cells (B). (A) Recombinant hPXR, recombinant hPXR ligand binding domain, or recombinant Hsc70 was incubated with UbcH5c and CHIP at Discussion 37C for ;30–60 minutes using an in vitro ubiquitin kit (Abcam), as described in Materials and Methods. Ubiquitinated proteins were determined by Western blotting The glucocorticoid receptor (GR), which is a ligand-dependent using an anti-ubiquitin antibody. (B) HepG2 cells cultured for 24 hours were nuclear receptor, is known to be phosphorylated through cell- and transfected with expression vectors encoding the YFP-hPXR WT or T408D mutant. tissue-specific signaling systems, leading to conformational changes, The medium was replaced 24 hours after transfection, and cells were retransfected 2 which in turn modulate its transcriptional responses and stability and then cultured with rifampicin (5 Â 10 6 M) or vehicle (DMSO) in the presence or absence of calpain inhibitor I (1 Â 1025 M) or MG132 (1 Â 1026 M) for an (reviewed in Galliher-Beckley and Cidlowski, 2009). Previous studies additional 24 hours. Cytoplasmic proteins (530 mg) prepared from these cells and a demonstrated that phosphomimetic mutations at the threonine-57, blank sample (vehicle) were precipitated with an anti-GFP antibody. The resulting threonine-290, serine-350, and threonine-408 of hPXR suppressed bound proteins were subjected to a Western blot analysis with an anti-ubiquitin or anti-PXR antibody. Experiments were performed three times with similar results, hPXR activities (Lichti-Kaiser et al., 2009; Pondugula et al., 2009; and representative data are shown. WT, wild-type hPXR; T408D, T408D-mutated Sugatani et al., 2012, 2014; Wang et al., 2015). Therefore, hPXR hPXR. *Nonspecific bands; **see text. functions are also considered to be modulated through the phosphor- ylation of hPXR at specific amino acid residues as well as nuclear increase in autophagic markers) when treated under nutrient-rich receptors such as constitutive active/androstane receptor (CAR) and GR conditions for a prolonged period of time, it was still capable of (Galliher-Beckley and Cidlowski, 2009; Mutoh et al., 2013). We also suppressing starvation-induced autophagy. Since inhibition of the previously reported that the phosphorylation of hPXR at threonine-408 proteasome has been shown to induce macroautophagy (Ding et al., may be associated with its degradation through proteasomal machinery 2007; Tang et al., 2014) and 3-MA blocks the maturation of the because the hPXR T408D mutant markedly accumulated in the autophagophore into a closed autophagosome in the macroautophagic cytoplasm of cells treated with MG-132 at an extremely high 5 mM process (Abounit et al., 2012), we evaluated the effects of lactacystin concentration (Sugatani et al., 2012). However, the results of the and 3-MA on the cellular stability of hPXR and intracellular trafficking present study suggest the presence of a proteasome-independent of the hPXR T408D mutant to the autophagic cargo to further explore degradation pathway in addition to the proteasome-dependent degra- our hypothesis that the hPXR T408D mutant is degraded through dation pathway because the protein level of the T408D mutant was less autophagy. A Western blot analysis revealed that the treatment of than or similar to that of the WT in the cytoplasm and significantly the hPXR WT- or T408D mutant-expressing cells with lactacystin or lower than the WT in the nucleus, even in the presence of proteasome 3-MA alone accumulated not only cellular hPXR WT but also the inhibitors (Figs. 1–4). Thus, we herein examined the molecular T408D mutant (Fig. 10). The hPXR T408D mutant accumulated more mechanisms regulating hPXR functions and stability, especially those efficiently than the WT in cells treated with lactacystin in the presence underlying autophagy for its degradation. of 3-MA. The autophagic markers LC3 and p62 markedly increased in The chaperone proteins Hsc70/Hsp70 assist in the correct protein cells treated with 3-MA and/or lactacystin in full medium. To determine folding of nascent or denatured client proteins for intracellular functions Stability Control of PXR via Chaperone-Autophagy Pathway 147

Fig. 10. Autophagy inhibitor 3-methyladenine (3-MA) led to the greater accumulation of the hPXR T408D mutant than the WT in the presence of proteasome inhibitor lactacystin. HepG2 cells cultured for 24 hours were transfected with expression vectors encoding the Flag-tagged hPXR WT or T408D mutant. Cells were incubated with 5 mM 3-MA or vehicle 12 hours after transfection. Twelve hours later, cells were cultured with lactacystin (5 Â 1026 M) or vehicle for an additional 24 hours. (A) Flag-hPXR, LC3, and p62 proteins in whole cell lysates were determined by Western blotting with the indicated antibodies and anti-a-tubulin antibody (loading control). (B) Band intensities were measured with ImageJ software, and each

quantified value of the Flag-hPXR bands was corrected for that Downloaded from of a-tubulin. Relative levels were expressed by taking the values obtained from vehicle-treated cells expressing the Flag- hPXR WT as one. Western blottings were performed three times with similar results, and representative data are shown. Data presented are the average 6 S.E. of three experiments. *P , 0.05; **P , 0.01; ***P , 0.001 versus the hPXR WT in vehicle-treated cells; ##P , 0.01; ###P , 0.001 versus the hPXR

T408D mutant in vehicle-treated cells; +P , 0.05 versus the dmd.aspetjournals.org hPXR WT in lactacystin- and 3-MA-treated cells. WT, wild- type hPXR; T408D, T408D-mutated hPXR. at ASPET Journals on September 27, 2021

and, in contrast, work with a ubiquitin pathway for the proteolytic T408D mutant with Hsc70/Hsp70 and CHIP was similar to that of the degradation of unstable or misfolded client proteins (Hayer-Hartl et al., WT in the cytoplasm of cells treated with and without the proteasome 1996; Wickner et al., 1999; Meacham et al., 2001; Kampinga et al., inhibitors, and because the hPXR T408D mutant did not easily 2003). CHIP interacts with Hsc70/Hsp70 and Hsp90, which results in translocate to the nucleus and did not accumulate in the cytoplasm, it the refolding of chaperone complexes with client proteins, and is also may have been misfolded by the CHIP/Hsp70 quality check and then involved in the direct ubiquitination of client proteins such as GR as an degraded in the cytoplasm through autophagy (Figs. 3– 6, 7, 10, and E3 ligase for delivery to the proteasome (reviewed in McDonough and 11). Furthermore, the hPXR WT accumulated in CHIP-deleted control Patterson, 2003). HOP has been shown to assist in protein transfer in the cells in the presence of lactacystin (Fig. 7), suggesting that CHIP may Hsp70- and Hsp90-dependent protein-folding pathways (Yamamoto play a key role in the proteasome-independent degradation pathway as et al., 2014). Hsp90, which regulates transcription by maintaining well as chaperone-dependent quality control (Figs. 3, 6, and 7). transcription factors in the cytoplasm, is known to interact with p23 and RBCK1 and UBR5 ubiquitinate target proteins for proteasomal CHIP through different sites, and CHIP interacts with the carboxyl- degradation in innumerable physiologic reactions; for example, terminal domain of Hsc70 (reviewed in McDonough and Patterson, RBCK1 negatively regulates tumor necrosis factor a– and interleukin- 2003). In addition to GR and CAR, hPXR has been identified as an 1–triggered nuclear factor kB activation by targeting transforming Hsp90 client substrate (Sanchez et al., 1985; Honkakoski et al., 1998; growth factor b–activated kinase 1 binding proteins 2/3 for degradation Squires et al., 2004). The present study revealed that hPXR was (Tian et al., 2007), and UBR5 is involved in regulating the stability of involved in the complex of chaperones Hsp90 and Hsc70/Hsp70 with the gluconeogenic rate-limiting enzyme phosphoenolpyruvate car- such cochaperones as CHIP, HOP, and p23 in the cytoplasm (Figs. 3, 4, boxykinase (Jiang et al., 2011). RBCK1 and UBR5 were previously and 6). Proteasome inhibitors (calpain inhibitor I and MG132) and shown to be responsible for the ubiquitination of hPXR to regulate the Hsp90 inhibitor geldanamycin induced Hsp70, which may have hPXR degradation through proteasomal machinery (Rana et al., 2013; resulted in the more effective formation of the chaperone/hPXR Ong et al., 2014), whereas the ubiquitin-proteasomal regulation of complex (Figs. 3, 4, and 6). This study revealed that the Hsp70 cytoplasmic CAR retention protein and Hsp70, but not CAR itself, inhibitor pifithrin-m and CHIP knockdown reduced the transcriptional may be an important contributor to the regulation of cytoplasmic activity of hPXR (Figs. 5 and 7). CHIP has been shown to enhance the CAR levels (Timsit and Negishi, 2014). Unlike RBCK1 and UBR5, folding capacity of Hsp70 in mammalian cells (Kampinga et al., 2003); CHIP functions in chaperone-dependent ubiquitination for substrate therefore, the refolding of hPXR by CHIP/Hsp70 may enhance the delivery to the proteasome (reviewed in McDonough and Patterson, translocation of hPXR from the cytoplasm to the nucleus, resulting in its 2003). Although CHIP ubiquitinated Hsc70 and Hsp70 (Jiang et al., elevated transactivation. In contrast, since the interaction of the hPXR 2001; Soss et al., 2015), but not hPXR, in vitro (Fig. 9), it currently 148 Sugatani et al.

Fig. 13. Schematic model for the regulation of hPXR stability through the Downloaded from phosphorylation of hPXR at threonine-408 and the CHIP/chaperone–autophagy pathway. After the treatment with an activator, the CHIP/Hsp70/Hsc70 complex may check and enable the correct folding of the hPXR WT but not the T408D mutant for transfer into the nucleus through nuclear pores. The chaperone–hPXR T408D mutant complex ubiquitinated by CHIP binds to p62 in the autophagophore Fig. 11. The hPXR T408D mutant was colocalized with LC3 and p62 in HepG2 for delivery to the lysosome for its degradation. cells treated with 3-MA and/or lactacystin. HepG2 cells cultured for 24 hours were

transfected with expression vectors encoding the YFP-tagged hPXR WT or T408D dmd.aspetjournals.org mutant. Cells were incubated with 5 mM 3-MA or vehicle 12 hours after transfection. Twelve hours later, cells were cultured with lactacystin (5 Â 1026 M) Immunocytochemistry revealed that YFP-fused hPXR and p62, which or vehicle for an additional 24 hours. The subcellular localization of YFP-hPXR was acts as a cargo receptor of ubiquitinated substrates, accumulated and examined by confocal microscopy. Blue, DAPI-labeled nuclei; red, Alexa Fluor colocalized in cells treated with 3-MA and lactacystin (Figs. 10 and 11). 555-labeled LC3 or p62; green, YFP-labeled hPXR. Merged images are shown in the rightmost column and the yellow-stained puncta indicated LC3 or p62, Thus, p62 may play a role in the transport of the ubiquitinated colocalized with hPXR. Experiments were performed twice with similar results, and chaperone complex as an acceptor of phagophores in the autophagy- representative data are shown. Scale bar, 50 mm. WT, wild-type hPXR; T408D, lysosome pathway. Autophagy is a process by which cellular compo- T408D-mutated hPXR. nents are delivered to the lysosome for degradation and involves three at ASPET Journals on September 27, 2021 main pathways: macroautophagy, chaperone-mediated autophagy, and remains unclear whether CHIP participates in the delivery of the microautophagy (reviewed in Feng et al., 2014). Inhibitions of the ubiquitinated chaperone complex to the proteasome or autophagic proteasome by its inhibitors and Hsp90 by geldanamycin have been cargo containing p62 or if ubiquitination of the chaperone complex by shown to induce macroautophagy and chaperone-mediated autophagy, CHIP induces substrate recognition and recruitment to the proteasome respectively, and ultimately client degradation in the lysosome (Finn or autophagy. Since Hsc70 ubiquitinated by CHIP is stable (Jiang et al., et al., 2005; Qing et al., 2006; Ge et al., 2009). We herein demonstrated 2001; Soss et al., 2015), it is conceivable that the ubiquitinated protein that inhibition of the lysosome by chloroquine resulted in the accumu- may have been detected in the chaperone complex with hPXR (Fig. 9). lation of the hPXR WT and T408D mutant in the cytoplasm (Fig. 8), whereas proteasome inhibition was linked to the enhanced degradation of the T408D mutant because 3-MA suppressed the degradation of hPXR, particularly that of the T408D mutant, in the presence of lactacystin (Figs. 3 and 10). The inhibition of Hsp90 led to the accumulation of the hPXR WT and T408D mutant in the cytoplasm and nucleus (Fig. 6), indicating that the balance of the hPXR quality check by Hsp90 and Hsp70/Hsc70 affected its stability, and also that the T408D mutant was targeted for degradation in a manner that was largely mediated by macroautophagy but not chaperone-mediated autophagy. PKC cell signaling is known to repress the transcriptional activity of PXR by increasing the strength of the interaction between PXR and the nuclear receptor corepressor protein NCoR (Ding and Staudinger, 2005). We herein demonstrated that PKC directly phosphorylated the threonine-408 of hPXR in vitro (Fig. 12). Although the cellular signaling pathways by which PKC is activated to phosphorylate hPXR Fig. 12. Threonine-408 of hPXR was phosphorylated by PKC in vitro. (A) MBP- in vivo remain unclear, the T408D mutant was degraded faster than the hPXR WT and T408A mutant fusion proteins (0.25–1.5 mg) were incubated with WT in the cytoplasm (Figs. 1, 3, and 6). Thus, PKC may play a key role PKC (0.63–5.0 ng) and DYRK2 (0.75 to 6.0 ng) in the presence of [32P]-labeled ATP at 30C for 10 minutes, then electrophoresed, and exposed to an autoradiographic in regulating the cellular stability of hPXR through phosphorylation at film. (B) The MBP-hPXR WT and T408A mutant fusion proteins (20 mgeach)were threonine-408. The T408D mutant reduced its stability not only in stained with Coomassie Brilliant Blue. Band intensities were measured with ImageJ cultured cancer cells but also in normal cells in mouse livers (Fig. 1E). software. Relative levels were expressed by taking the value obtained from the MBP- Therefore, emerging evidence prompted us to propose a schematic hPXR WT fusion protein as one. Experiments were performed three times with similar results, and representative data are shown. WT, wild-type hPXR; T408A, T408A- model of the regulation of hPXR transactivation and its degradation mutated hPXR. through the CHIP–Hsc70/Hsp70 protein quality check system (Fig. Stability Control of PXR via Chaperone-Autophagy Pathway 149

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