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