P38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus Via Phosphorylation at Threonine 38 S

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Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/04/12/dmd.116.070235.DC1

1521-009X/44/6/871–876$25.00 http://dx.doi.org/10.1124/dmd.116.070235 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:871–876, June 2016 U.S. Government work not protected by U.S. copyright p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38 s

Takeshi Hori, Rick Moore, and Masahiko Negishi

Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Received February 25, 2016; accepted April 1, 2016

ABSTRACT Nuclear receptor constitutive androstane receptor (CAR, NR1I3), MAPK forms a complex with CAR, enables it to bind to the response which regulates hepatic drug and energy metabolisms as well as cell sequence, phenobarbital-responsive enhancer module (PBREM), growth and death, is sequestered in the cytoplasm as its inactive form within the CYP2B promoter, and thus recruits RNA polymerase II to Downloaded from phosphorylated at threonine 38. CAR activators elicit dephosphory- activate transcription. Subsequently, p38 MAPK elicited rephosphor- lation, and nonphosphorylated CAR translocates into the nucleus to ylation of threonine 38 to inactivate CAR and exclude it from the activate its target genes. CAR was previously found to require p38 nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequen- mitogen-activated protein kinase (MAPK) to transactivate the cyto- tially activating and inactivating CAR-mediated transcription through chrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 phosphorylation of threonine 38. dmd.aspetjournals.org

Introduction been investigated in a given but not in any other nuclear receptors. The nuclear receptor constitutive androstane receptor (CAR, NR1I3), Compared with these regions, much less emphasis has been placed on a member of the thyroid and steroid hormone receptor superfamily, phosphorylation within the DBD. One phosphorylation site resides activates genes encoding for enzymes and transporters that metabolize within a protein kinase C (PKC) motif in the DBD and is conserved in and excrete therapeutic drugs and is activated by these drugs and 41 out of 48 human nuclear receptors. Phosphorylation of this conserved xenobiotics (Kobayashi et al., 2015). With these functions, CAR site is the most well characterized within threonine 38 of CAR (Mutoh at ASPET Journals on October 5, 2021 critically regulates hepatic capability for drug disposition. CAR gains et al., 2009; Mutoh et al., 2013). CAR is, in fact, phosphorylated at its drug responsiveness by suppressing its high constitutive activity threonine 38 in mouse hepatocytes to regulate its drug activation. a by phosphorylation at threonine 38 within the DNA-binding domain Recently, estrogen receptor alpha (ER ) phosphorylated at the corre- (DBD) (Mutoh et al., 2009). Phosphorylation of CAR abolishes its sponding serine 216 was also found in mouse neutrophils and appears to DNA binding ability and is sequestered in the cytoplasm. Phenobarbital, regulate their infiltration into the uterus (Shindo et al., 2013). Although the classic drug that indirectly activates CAR, elicits a cell signal that their phosphorylation in tissues in vivo have not yet been confirmed, stimulates protein phosphatase 2A (PP2A) to dephosphorylate threonine studies with phosphomimetic mutants suggest that these residues in a 38 for activation (Mutoh et al., 2013). Thus, the cell signal–mediated hepatocyte nuclear factor 4 alpha (HNF-4 ), vitamin D receptor (VDR), process of CAR activation as it occurs in the cytoplasm is now well peroxisome proliferator-activated receptor alpha, retinoid X receptor a documented. Here we have investigated p38 mitogen-activated protein alpha (RXR ), and farnesoid X receptor may regulate various function- kinase (MAPK) that regulates CAR in the nucleus. alities of these nuclear receptors, such as cytoplasmic retention, degrada- Phosphorylation has long been investigated in both ligand-dependent tion, and transactivation (Hsieh et al., 1993; Sun et al., 2007; Gineste et al., and -independent regulation of nuclear receptors. It is involved in 2008). Therefore, conserved phosphorylation has provided us with the degradation, cofactor recruitment, and dimerization (Shao and Lazar, opportunity to uniformly investigate nuclear receptors but has only been 1999; Tremblay et al., 1999; Hong et al., 2003; Picard et al., 2008). The studied as the target of PKC or dephosphorylation by PP2A. amino acid residues targeted for studies reside within the activation p38 MAPK is activated in response to various extracellular stimuli, function 1 (AF-1) or ligand-binding domain (LBD) region and have such as growth factors, UV radiation, inflammatory cytokines, oxidative stress, and hyperosmosis (Freshney et al., 1994; Han et al., 1994; Rouse et al., 1994; Huot et al., 1997; Zhang and Jope, 1999). In liver cells, phosphorylated p38 MAPK is accumulated in the nucleus to activate This work was supported by the Intramural Research Program of the National Institutes of Health and National Institute of Environmental Health Sciences [Grant various transcription factors and protein kinases. We have recently Z01ES71005-01]. demonstrated that p38 MAPK is essential for CAR to activate the dx.doi.org/10.1124/dmd.116.070235. cytochrome P450 2B6 (CYP2B6) gene in human hepatoma HepG2- s This article has supplemental material available at dmd.aspetjournals.org. derived cells (Saito et al., 2013b). In the present study, we revealed that

ABBREVIATIONS: CAR, constitutive androstane receptor; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DMSO, dimethyl sulfoxide; ER, estrogen receptor; FBS, fetal bovine serum; GST, glutathione S-transferase; HNF, hepatocyte nuclear factor; HRP, horseradish peroxidase; LBD, ligand-binding domain; MAPK, mitogen-activated protein kinase; PBREM, phenobarbital-responsive enhancer module; PCR, polymerase chain reaction; PGC, peroxisome proliferator-activated receptor -g coactivator; PP2A, protein phosphatase 2A; RXR, retinoid X receptor; SRC, steroid receptor coactivator; TBS, Tris-buffered saline; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; VDR, vitamin D receptor.

871 872 Hori et al. p38 MAPK forms a complex with CAR to promote the CAR-mediated weight) in phosphate-buffered saline (PBS), TCPOBOP (3 mg/kg body weight) in transcription in the nucleus. Moreover, p38 MAPK linked CAR dimethyl sulfoxide (DMSO) in corn oil, or a control solution, was intraperito- transactivation and inactivation by stimulating phosphorylation of neally injected into 7- to 8-week-old male mice for a treatment of 6 hours, from threonine 38. which liver RNAs and nuclei were prepared for real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assays, respectively. Mice were maintained under the standard condition at the National Institute Materials and Methods of Environmental Health Sciences, and animal experiments were conducted Materials. Phenobarbital sodium salt, 1,4-bis[2-(3,5-dichloropyridyloxy)] according to protocols approved by the animal ethics committee at NIEHS/ benzene (TCPOBOP), anisomycin, SB 239063, anti-FLAG M2 affinity gel, National Institutes of Health. anti-FLAG M2-horseradish peroxidase (HRP) antibodies, phosphatase inhibitor Plasmid Construction. Mouse CAR cDNA (GenBank accession no. NM cocktails 2 and 3, and L-glutathione reduced were purchased from Sigma-Aldrich 009803.5) was previously cloned into pGEX-4T-3 vector (GE Healthcare, (St. Louis, MO); anti-p38a (ab7952) and anti-phospho-RNA polymerase II CTD Piscataway, NJ) for glutathione S-transferase (GST)-CAR fusion proteins (ab5131) from Abcam (Cambridge, MA); anti-phospho-p38 MAPK (Thr180/ and pCR3 vector (Invitrogen). CAR Thr48Ala (T48A) and CAR Thr48Asp Tyr182; #4511) and anti-p38 MAPK from Cell Signaling Technology (Danvers, (T48D) mutants were generated by a site-directed mutagenesis method with the MA); anti-RXRa (sc-553 X) and HRP-conjugated anti-mouse or rabbit IgG following primers: 59-GGCTTCTTCAGACGAgCAGTCAGCAAAACCATT-39 antibodies from Santa Cruz Biotechnology (Dallas, TX); and anti-V5 from and 59-AATGGTTTTGCTGACTGcTCGTCTGAAGAAGCC-39 for Invitrogen/ThermoFisher Scientific (Carlsbad, CA). Anti-phospho-Thr38 peptide CAR T48A; 59-GGCTTCTTCAGACGAgatGTCAGCAAAACCATT-39 and antibodies for CAR were produced in our previous work (Mutoh et al., 2009). 59-AATGGTTTTGCTGACatcTCGTCTGAAGAAGCC-39 for CAR T48D. Anti-phospho-Ser51 peptide antibodies (GFFRR-pS-MKRKALFTC) for VDR Mouse p38a cDNA (NM 011951.3) was cloned into pcDNA3.1 vector were produced by AnaSpec Inc. (San Jose, CA). A Lipofectamine 2000 reagent (Invitrogen). The FLAG tag was inserted into the 59-flanking region of CAR in Downloaded from and Dynabeads Protein G were obtained from Life Technologies/ThermoFisher pCR3 or p38a in pcDNA3.1. Scientific (Grand Island, NY); TaqMan Gene Expression Assays (probe and Cell Cultures. Primary hepatocytes were isolated using a collagenase two-step primer sets) for CYP2B6 (AssayID: Hs00167937_m1) (FAM), Cyp2b10 perfusion method as described previously (Honkakoski et al., 1996). Hepatocytes (AssayID: Mm00456591_m1) (FAM), Cyp2c55 (AssayID: Mm00472168_m1) (6 Â 105 cells/ml per well) were seeded on collagen-coated wells and cultured in (FAM), and human and mouse GAPDH (FAM) from Applied Biosystems Williams’ medium E containing 10% fetal bovine serum (FBS) and penicillin/ (Foster, CA); cOmplete mini protease inhibitor cocktail tablets from Roche streptomycin. After 3 hours of seeding, cells were treated with 2 mM phenobarbital Diagnostics Corp. (Indianapolis, IN); recombinant active p38 MAPK (#14-587) or 250 nM TCPOBOP for 12 hours in the presence or absence of 20 mM dmd.aspetjournals.org from Millipore UK Limited (Dundee, UK). SB 239063, and total RNA was extracted for real-time PCR analysis. Huh-7 cells Animals. Both Car+/+and Car2/2 mice in C3H/HeNCrlBR background were or HepG2-derived Ym17 cells were cultured in minimum essential medium produced in house (Yamamoto et al., 2004). Phenobarbital (100 mg/kg body (Invitrogen) or Dulbecco’s modified Eagle’s medium (Invitrogen), respectively, at ASPET Journals on October 5, 2021

Fig. 1. CAR recruitment on PBREM and interaction between CAR and p38 MAPK. (A, B) Ym17 cells were treated with 250 nM TCPOBOP or DMSO for 12 hours in the presence or absence of 200 nM anisomycin (ANI). (A) CYP2B6 mRNA expression levels were determined by real-time PCR. Each value is shown as the mean 6 S.D. (n = 3). Statistically significant difference: ***P , 0.001, Tukey’s multiple comparison test. (B) ChIP assays were performed to assess recruitment of V5-tagged CAR to the PBREM of the CYP2B6 promoter. (C–E) Coimmunoprecipitation assays were performed using Huh-7 cells. (C) Cells were transfected with FLAG-CAR/pCR3. Immunoprecipitation of p38 MAPK with anti- p38 MAPK antibodies (Abcam) and Western blot analysis with anti-FLAG or anti-p38 MAPK antibodies (Cell Signaling Technol- ogy) were performed. (D) Cells were transfected with FLAG-CAR/ pCR3 and treated with 1 mM ANI or DMSO for 30 minutes in the absence of FBS. Immunoprecipitation of FLAG-CAR with anti- FLAG M2 affinity gel and Western blot analysis for p38 MAPK, phosphorylated p38 (p-p38) MAPK, or FLAG-CAR were performed. (E) Cells were transfected with FLAG-CAR wild-type (WT)/pCR3, the T48A mutant, or the T48D mutant together with p38 MAPK expression vectors. Mouse CAR T48 corresponds to human CAR T38. Immunoprecipitation of FLAG-CAR with anti- FLAG M2 affinity gel and Western blot analysis for p38 MAPK or FLAG-CAR were performed. p38 MAPK Links CAR Activation and Inactivation 873 supplemented with 10% FBS and penicillin/streptomycin at 37°Cwith5%CO2. from –1863 to –1674; –1863/–1674); 59-GCTAATGCCTGTCTGGAT- Plasmids were transfected into Huh-7 or Ym17 cells with a Lipofectamine 2000 CAGGA-39 and 59-GGAATACTGACCCAAGTTCAGTG-39 (PBREM/ reagent according to the manufacturer’s instructions. Cyp2b10) (–2434/–2232); 59-AAGGGAATGAGGAGTGAGC-39 and 59-CAA- Real-Time PCR. Total RNA was extracted from mouse livers, hepatocytes, or GAAGCCCACAAGGAGAG-39 (TATA box/Cyp2b10) (–149/+75); Ym17 cell using TRIzol reagent (Life Technologies), with which cDNAs were 59-GCTTCTCTTTGCCCTCGATA-39 and 59-ACCCAAGTCCCCTGTACCT- synthesized using a High Capacity cDNA Archive kit (Life Technologies). Real- TAC-39 (direct repeat 4 (DR4)/Cyp2c55) (–1860/–1623); 59-GGCCAGAGTC- time PCR was conducted using a TaqMan Universal PCR Master mix and CATTCAGAAG-39 and 59-GAGCTTCCCTCTCCCAGAGT-39 (TATA box/ TaqMan probes and primers with a 7900HT Fast Real-Time PCR System Cyp2c55) (–115/+114). (Applied Biosystems). In Vitro Kinase Assay. Huh-7 cells, which expressed ectopic FLAG-p38 Immunoprecipitation. Huh-7 cells were lysed in a lysis buffer [20 mM Tris- MAPK, were treated with 1 mM anisomycin for 10 minutes. These cells were

HCl (pH 7.5), 0.5 mM EDTA, 100 mM NaCl, 1% Triton X-100, 10% glycerol, lysed in the above-mentioned lysis buffer containing 2.5 mM Na4P2O7 and 1 mM and protease inhibitor cocktails 2 and 3]. After sonication and centrifugation, the Na3VO4. Anti-FLAG M2 affinity gels, which were pretreated with dimethyl resulting supernatant was incubated with Dynabeads with a given antibody or an pimelimidate (1 mg/ml) and triethanolamine (100 mM), were incubated with anti-FLAG M2 affinity gel (FLAG gel). Dynabeads or FLAG gel were washed lysates for 3 hours at 4°C and were washed four to five times with TBS. Resulting three to four times in a lysis buffer or Tris-buffered saline [TBS; 25 mM Tris-HCl gels were used as an enzyme source for active p38 MAPK. For substrates, GST- (pH 7.4), 140 mM NaCl, and 2.7 mM KCl], respectively. Washed FLAG gel was CAR wild-type and the T48A mutant were expressed and purified as previously heat-treated in 2Â SDS sample buffer [157 mM Tris-HCl (pH 6.8), 4% SDS, 25% reported (Mutoh et al., 2009). In kinase reaction, GST-CAR (1 mg) and FLAG- glycerol, and 0.01% bromophenol blue]. Washed Dynabeads were incubated p38 MAPK-bound gel were mixed in a kinase buffer consisting of 25 mM Tris- and centrifuged in 0.1 M glycine buffer (pH 2.0) to elute proteins that were HCl (pH 7.5), 2.5 mM Na4P2O7, 1 mM Na3VO4, 10 mM MgCl2, and 1 mM subsequently heat-treated in the above-mentioned 2Â SDS sample buffer. dithiothreitol. Reaction was started by adding 200 mM ATP, incubated for 1 hour Downloaded from ChIP Assays. ChIP assay was performed using ChIP-IT Express kit (Active at 37°C, and stopped by adding 1Â SDS sample buffer. Phosphorylated CAR was Motif, Carlsbad, CA) as described previously (Saito et al., 2013a; Gotoh and detected by Western blot analysis using anti-pThr38 CAR antibodies and anti- Negishi, 2015) with some modifications. Sheared chromatin (10 mg) obtained rabbit IgG (light chain–specific)–HRP antibodies (Jackson ImmunoResearch from mouse livers or Ym17 cells was incubated with the indicated antibodies Laboratories, West Grove, PA). (0.3–1 mg; anti-phospho RNA polymerase II, anti-RXRa, anti-phospho-p38, Bacterially expressed active p38 MAPK (0.36 mg) was incubated with GST- anti-V5, or normal IgG) and magnetic beads. After washing beads, DNA was CAR (0.5 mg) and ATP (250 mM) in the above-mentioned kinase buffer for eluted and purified. DNA fragments were amplified by PCR with the following 6 hours at 37°C, and then Western blot analysis was performed to detect dmd.aspetjournals.org specific primers: 59-TTACTGTGTGTAAAGCACTTC-39 and 59-GACAAA- phosphorylated CAR. CAGTCCTATTTGTAAG-39 [for the phenobarbital-responsive enhancer Western Blot. Proteins were separated in a 10% SDS-polyacrylamide gel and module (PBREM) of the CYP2B6 gene; PBREM/CYP2B6] (the amplicon is were transferred onto a polyvinylidene difluoride membrane. Membranes were at ASPET Journals on October 5, 2021

Fig. 2. Effect of p38 MAPK on Cyp2b10 mRNA expression in hepatocytes and recruitment of p-p38 MAPK on the Cyp2b10 promoter in vivo. (A) Mouse primary hepatocytes were treated with 2 mM phenobarbital or 250 nM TCPOBOP for 12 hours in the presence or absence of SB 239063. Cyp2b10 mRNA expression levels were determined by real-time PCR. Each value is shown as the mean 6 S.D. (n = 3). Statistically significant differences: **P , 0.01 and ***P , 0.001, Tukey’s multiple comparison test. (B) Effect of treatment with phenobarbital (100 mg/kg) or TCPOBOP (3 mg/kg) for 6 hours on Cyp2b10 mRNA expression in livers. Each value is shown as the mean 6 S.D. (n = 3). Statistically significant difference: ***P , 0.001, Student’s t test. (C) Recruitment of CAR/RXRa, p-p38 MAPK, and phosphorylated RNA polymerase II (p-RNA pol II) after 6 hours of phenobarbital or TCPOBOP injection. ChIP assays were performed for the PBREM and the TATA box of the Cyp2b10 gene using specific primers as described in Materials and Methods. PB or TC indicates phenobarbital or TCPOBOP, respectively. 874 Hori et al. blocked with 2% skim milk or 5% bovine serum albumin in TBS containing 0.1% SB 239063 (Underwood et al., 2000) and TCPOBOP or phenobarbital to Tween 20 and were incubated with given primary and secondary antibodies. Protein enable examination of expression levels of CYP2B10 (the mouse bands were visualized by Western Bright ECL reagents (Advansta, Menlo Park, CA). homolog of human CYP2B6) mRNA. Whereas SB 239063 did not Immunohistochemistry. Paraffin-embedded liver sections were prepared affect basal levels, it effectively diminished either TCPOBOP- or from C3H/HeNCrlBR males and subjected to immunohistochemistry as described phenobarbital-induced increase of this mRNA (Fig. 2A). Phosphory- previously (Shindo et al., 2013). lated p38 MAPK was found in the nuclei of mouse livers (Supplemental Statistical Analysis. Multiple groups were analyzed by one-way analysis of variance followed by Tukey’s multiple comparison test. Two groups were Fig. 1, A and B). Having these findings, we employed ChIP assays to compared by Student’s t test. These statistical analyses were conducted using a analyze the Cyp2b10 promoter in mouse livers. Mice were treated with software GraphPad Prism 6.07 (GraphPad Software, San Diego, CA). either phenobarbital or TCPOBOP and their liver RNAs and nuclei were prepared, respectively, for real-time PCR to confirm that CYP2B10 mRNA had increased (Fig. 2B) and for ChIP assays to measure binding Results between phosphorylated p38 and PBREM (Fig. 2C). Phosphorylated A p38 MAPK Complex with CAR on PBREM. Anisomycin, a p38 was recruited to PBREM after phenobarbital or TCPOBOP p38 MAPK activator, was first confirmed as a synergist in the increase of treatment in the livers of CAR wild-type but not knockout (KO) mice CYP2B6 mRNA by a CAR ligand TCPOBOP in HepG2-derived Ym17 (Fig. 2C). Nonphosphorylated CAR translocates to the nucleus and cells that constitutively express V5-tagged mouse CAR (Fig. 1A). The heterodimerizes with RXRa (Honkakoski et al., 1998). PBREM binding results of a previous experiment with small-interfering RNA confirmed of a CAR/RXRa complex resembled that of the phosphorylated p38

that this synergy was, in fact, mediated by p38 MAPK (Saito et al., MAPK. These binding patterns correlated with those of phosphorylated Downloaded from 2013b). Upon activation, CAR binds the DNA sequence called PBREM RNA polymerase II to the TATA box (Fig. 2C). Weak bands observed within the CYP2B6 promoter to activate it. In ChIP assays, anisomycin with either PBS or DMSO-treated nuclei in CAR wild-type and/or KO cotreatment with TCPOBOP synergistically increased CAR binding to mice appeared to be nonspecific amplifications. Thus, upon CAR PBREM in Ym17 cells (Fig. 1B). Coimmunoprecipitation assays were activation, phosphorylated p38 MAPK was recruited to PBREM, in turn employed to examine interactions between CAR and p38 MAPK in recruiting active RNA polymerase II to induce transcription. Cyp2 human hepatoma Huh-7 cells. An anti-p38 MAPK antibody coprecipi- The c55 gene is also a CAR-regulated gene in mouse livers dmd.aspetjournals.org tated CAR (Fig. 1C). Anisomycin treatment increased CAR coprecipitated (Konno et al., 2010). As observed with CYP2B10 mRNA, treatment with phosphorylated p38 MAPK (Fig. 1D). The nonphosphomimetic with SB 239063 also repressed an increase in CYP2C55 mRNA by CAR T38A mutant spontaneously accumulated in the nucleus, whereas phenobarbital in mouse primary hepatocytes (Fig. 3A). ChIP assays the phosphomimetic mutant CAR T38D was retained in the cytoplasm revealed that phenobarbital treatment induced recruitment of phosphor- (Mutoh et al., 2009; Osabe and Negishi, 2011). Wild-type CAR was not ylated p38 MAPK and CAR to the previously identified element and of effectively phosphorylated at threonine 38 in Huh-7 cells, thus resembling active RNA polymerase II to the TATA box within the Cyp2c55 the CAR T38A mutant in this respect. FLAG-tagged CAR wild-type, promoter (Fig. 3B). These similarities between the Cyp2b10 and T38A, or T38D mutant was ectopically coexpressed with p38 MAPK in Cyp2c55 promoters support the notion that p38 MAPK may be an at ASPET Journals on October 5, 2021 Huh-7 cells and subjected to coimmunoprecipitation assays (Fig. 1E). The essential factor for CAR to activate a set of its target genes including CAR T38D mutant coprecipitated p38 MAPK far less than either CAR these two. wild-type or T38A mutant did. These observations indicated that CAR p38 MAPK-Stimulated Phosphorylation of Threonine 38. In- needs to form a complex with anisomycin-activated p38 MAPK for active CAR is phosphorylated at threonine 38 and sequestered in the binding to PBREM in the nucleus. cytoplasm (Mutoh et al., 2009), although the protein kinase that CAR-Dependent p38 Recruitment to PBREM. First, mouse phosphorylates it has not been identified. Unexpectedly, CAR was primary hepatocytes were cotreated with a specific p38 MAPK inhibitor found to be phosphorylated at threonine 38 when CAR was coexpressed

Fig. 3. Effect of p38 MAPK on Cyp2b55 mRNA expression in hepatocytes and recruitment of p-p38 MAPK on the Cyp2c55 promoter in vivo. (A) Mouse primary hepatocytes were treated with 2 mM phenobarbital for 12 hours in the presence or absence of SB 239063. Cyp2c55 mRNA expression levels were determined by real-time PCR. Each value is shown as the mean 6 S.D. (n = 3). Statistically significant differences: ***P , 0.001, Tukey’s multiple comparison test. (B) Recruitment of CAR/RXRa, p-p38 MAPK, and p-RNA pol II after 6 hours of phenobarbital injection. ChIP assays were performed for the direct repeat (DR)-4 and the TATA box of the Cyp2c55 gene using specific primers as described in Materials and Methods. p38 MAPK Links CAR Activation and Inactivation 875

Fig. 4. Phosphorylation of CAR at threonine 38 by p38 MAPK. (A) Expression vectors for FLAG-tagged mouse CAR wild-type (WT) or the T48A, corresponding to the T38A in humans, were transfected into Huh-7 cells with or without p38 MAPK expression vectors. After immunoprecipitation with anti-FLAG M2 affinity gel, Western blot analysis with anti-pThr38-CAR antibodies was performed to assess phosphorylation levels of CAR. (B, C) In vitro kinase assays with active p38 MAPK purified from Huh-7 cells (B) or purified from Escherichia coli (C) and GST-CAR wild-type or the T48A mutant were performed to demonstrate phosphorylation of threonine 38 by p38 MAPK under the conditions as described in Materials and Methods. Downloaded from dmd.aspetjournals.org with p38 MAPK in Huh-7 cells, as indicated by Western blot analysis phenobarbital or TCPOBOP. In Ym17 cells, anisomycin activates p38 with an anti-phospho-peptide antibody (Fig. 4A). Ectopic p38 MAPK MAPK. The complexes of CAR with active p38 MAPK promote was similarly phosphorylated as the endogenous counterpart was. binding of CAR to the CYP2B promoter. p38 MAPK is known to Subsequently, in vitro kinase assays were performed to further sub- interact with RNA polymerase II (Alepuz et al., 2003). Moreover, the stantiate phosphorylation of CAR by p38 MAPK. To this end, FLAG- promoter-bound kinase activates RNA polymerase–mediated gene p38 MAPK was expressed in Huh-7 cells, from which kinase was transcription, as indicated by the fact that the LexA fusion–p38 MAPK at ASPET Journals on October 5, 2021 purified and incubated with a recombinant CAR (either CAR wild-type activated a luciferase reporter gene bearing LexA DNA binding sites or CAR T38A mutant) as a substrate. Western blot analysis showed upstream of the luciferase reporter (Ferreiro et al., 2010). Thus, a phosphorylation of CAR wild-type but not the mutant (Fig. 4B). In a reasonable scenario may be that p38 MAPK, as part of the CAR similar kinase assay, a bacterially expressed recombinant p38 MAPK also complex, binds the PBREM and facilitates the TATA box to recruit phosphorylated CAR wild-type (Fig. 4C). Thus, these results strongly active RNA polymerase II for effective promoter activation. The suggested that p38 MAPK directly phosphorylated CAR. In VDR CYP2B6 (the human CYP2B homolog) gene looped to locate the distal (NR1I1), which is another member of the nuclear receptor 1I subfamily, PBREM to the TATA box; early growth response 1 facilitated this serine 51 is the corresponding conserved residue. Previous studies with a phosphomimetic mutant suggested that VDR could be inactivated by phosphorylation at serine 51 (Hsieh et al., 1993) and sequestered in the cytoplasm (unpublished data). As was also seen with CAR, serine 51 was phosphorylated in Huh-7 cells when VDR was coexpressed with p38 MAPK as well as in in vitro kinase assays using FLAG-p38 MAPK as the enzyme (Supplemental Fig. 2, A and B). Since the majority of nuclear receptors conserve this phosphorylation motif, p38 MAPK can be used to phosphorylate them and to regulate their activities.

Discussion CAR is activated by dephosphorylation at threonine 38, which occurs in the cytoplasm where phosphorylated CAR is sequestered (Mutoh et al., 2009). Nonphosphorylated CAR translocates into the nucleus to activate its targeted genes. Here, we have demonstrated that threonine 38 is rephosphorylated to inactivate CAR in the nucleus. p38 MAPK, having formed a complex with CAR to recruit it to and activate the CYP2B promoter, subsequently phosphorylates threonine 38. This Fig. 5. Activation and inactivation of CAR-mediated transcription by p-p38 MAPK. rephosphorylation should inactivate CAR and exclude it from the CAR is phosphorylated at threonine 38 under normal conditions in livers. nucleus. Thus, by regulating this series of reactions, p38 MAPK links Phenobarbital (PB) or TCPOBOP (TC) causes dephosphorylation of CAR and CAR-mediated transactivation and inactivation. translocates it to the nucleus. CAR interacts with p-p38 MAPK in the nucleus, and the CAR/p-p38 MAPK complexes activate transcription of the CYP2B gene. P-p38 Active p38 MAPK resides in the nucleus of mouse hepatocytes and MAPK phosphorylates threonine 38 of CAR and translocates it to the cytoplasm as forms a complex with incoming CAR from the cytoplasm in response to an inactive form. 876 Hori et al. looping, possibly enabling the promoter to recruit RNA polymerase II Hong YH, Varanasi US, Yang W, and Leff T (2003) AMP-activated protein kinase regulates HNF4alpha transcriptional activity by inhibiting dimer formation and decreasing protein sta- for effective promoter activation (Inoue and Negishi, 2008, 2009). In bility. J Biol Chem 278:27495–27501. addition to early growth response 1, various other transcription factors Honkakoski P, Moore R, Gynther J, and Negishi M (1996) Characterization of phenobarbital-inducible mouse Cyp2b10 gene transcription in primary hepatocytes. JBiolChem271:9746–9753. were shown to interact with and activate the CYP2B promoters together Honkakoski P, Zelko I, Sueyoshi T, and Negishi M (1998) The nuclear orphan receptor CAR- with CAR, such as peroxisome proliferator-activated receptor -g retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the a CYP2B gene. Mol Cell Biol 18:5652–5658. coactivator-1 alpha (PGC-1 ), steroid receptor coactivator-1 (SRC-1), Hsieh JC, Jurutka PW, Nakajima S, Galligan MA, Haussler CA, Shimizu Y, Shimizu N, Whitfield HNF-4a, and CCAAT/enhancer-binding protein alpha (Shiraki et al., GK, and Haussler MR (1993) Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site. J Biol Chem 2003; Li et al., 2008; Benet et al., 2010). Since p38 MAPK can activate 268:15118–15126. these factors (Lim et al., 1998; Knutti et al., 2001; Guo et al., 2007; Qiao Huot J, Houle F, Marceau F, and Landry J (1997) Oxidative stress-induced actin reorganization et al., 2006; Fernandez-Marcos and Auwerx, 2011; Antoon et al., 2012), mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80:383–392. it may enable CAR to bind to PBREM and to loop the promoter by Inoue K and Negishi M (2008) Nuclear receptor CAR requires early growth response 1 to activate phosphorylating one or more of them. In addition to the CYP2B genes, the human cytochrome P450 2B6 gene. J Biol Chem 283:10425–10432. Inoue K and Negishi M (2009) Early growth response 1 loops the CYP2B6 promoter for syner- p38 MAPK similarly regulated the Cyp2c55 gene in mouse primary gistic activation by the distal and proximal nuclear receptors CAR and HNF4alpha. FEBS Lett hepatocytes. This indicates that the regulation observed with p38 MAPK 583:2126–2130. Knutti D, Kressler D, and Kralli A (2001) Regulation of the transcriptional coactivator PGC-1 via may be a general mechanism by which CAR activates hepatic genes. MAPK-sensitive interaction with a repressor. Proc Natl Acad Sci USA 98:9713–9718. We now know that CAR can be phosphorylated in the nucleus. Kobayashi K, Hashimoto M, Honkakoski P, and Negishi M (2015) Regulation of gene expression by CAR: an update. Arch Toxicol 89:1045–1055. Phosphorylated CAR moves back to the cytoplasm, possibly being Konno Y, Kamino H, Moore R, Lih F, Tomer KB, Zeldin DC, Goldstein JA, and Negishi M (2010)

retained for reactivation by dephosphorylation. Thus, it is possible that The nuclear receptors constitutive active/androstane receptor and pregnane x receptor activate the Downloaded from Cyp2c55 gene in mouse liver. Drug Metab Dispos 38:1177–1182. CAR can be recycled through phosphorylation, dephosphorylation, and Li L, Chen T, Stanton JD, Sueyoshi T, Negishi M, and Wang H (2008) The peripheral benzodi- rephosphorylation of threonine 38 during drug inductions. Figure 5 azepine receptor ligand 1-(2-chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide is a novel antagonist of human constitutive androstane receptor. Mol Pharmacol 74:443–453. schematizes the hypothesis that p38 MAPK plays dual functions and Lim CP, Jain N, and Cao X (1998) Stress-induced immediate-early gene, egr-1, involves activation links the CAR-mediated activation and inactivation of the CYP2B genes of p38/JNK1. Oncogene 16:2915–2926. Mutoh S, Osabe M, Inoue K, Moore R, Pedersen L, Perera L, Rebolloso Y, Sueyoshi T, through phosphorylation/dephosphorylation of threonine 38. The phos- and Negishi M (2009) Dephosphorylation of threonine 38 is required for nuclear translocation a and activation of human xenobiotic receptor CAR (NR1I3). J Biol Chem 284:34785–34792.

phorylation of ER is also known to link its activation and inactivation dmd.aspetjournals.org (Zhou and Slingerland, 2014). However, unlike CAR, which can be Mutoh S, Sobhany M, Moore R, Perera L, Pedersen L, Sueyoshi T, and Negishi M (2013) Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition reused, phosphorylated ERa is degraded through ubiquitination and, of epidermal growth factor receptor signaling. Sci Signal 6:ra31. therefore, cannot be recycled. Osabe M and Negishi M (2011) Active ERK1/2 protein interacts with the phosphorylated nuclear constitutive active/androstane receptor (CAR; NR1I3), repressing dephosphorylation and se- In conclusion, our present study shed light on phosphorylation within questering CAR in the cytoplasm. J Biol Chem 286:35763–35769. the DBD that has long been ignored. Our present findings with CAR and Picard N, Charbonneau C, Sanchez M, Licznar A, Busson M, Lazennec G, and Tremblay A (2008) Phosphorylation of activation function-1 regulates proteasome-dependent nuclear mobility and p38 MAPK can be used to understand biologic roles of this phosphor- E6-associated protein ubiquitin ligase recruitment to the estrogen receptor beta. Mol Endocrinol ylation among members of the nuclear receptor superfamily. 22:317–330.

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