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Nuclear receptor phosphorylation in xenobiotic signal transduction

Open AccessPublished:August 11, 2020DOI:https://doi.org/10.1074/jbc.REV120.007933
      Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.
      PB was found to induce its own metabolism in rat livers in 1963 (
      • Remmer H.
      • Merker H.J.
      Drug-induced changes in the liver endoplasmic reticulum: association with drug-metabolizing enzymes.
      ). Subsequently, not only PB but also various other drugs and xenobiotic chemicals, such as polychlorobiphenyls, were also shown to induce hepatic drug metabolism (
      • Conney A.H.
      Pharmacological implications of microsomal enzyme induction.
      ,
      • Conney A.H.
      • Burns J.J.
      Stimulatory effect of foreign compounds on ascorbic acid biosynthesis and on drug-metabolizing enzymes.
      ). Over 40 years later, CAR and PXR provided us with the experimental basis to investigate how cells respond to xenobiotic exposures at the molecular level, greatly impacting pharmacology, toxicology, and pathophysiology studies. Since CAR and PXR were discovered, over 70,000 manuscripts (PubMed search) have been published that not only determined the molecular mechanisms of xenobiotic responses but also implicated CAR and/or PXR in disease developments and drug therapies and development. This article focuses on defining CAR and PXR as nuclear receptors that are cell signal–regulated through phosphorylation at conserved motifs and determining their molecular mechanisms. For a more comprehensive summary of the enormously expanded research of drug metabolism, readers are referred to the overwhelming numbers of already published reviews. Here, we begin with describing xenobiotic signals transduced by CAR and PXR.

      Xenobiotic signals

      NIEHS, National Institutes of Health, constructed a small mouse liver cDNA microarray in the late 1990s (
      • Ueda A.
      • Hamadeh H.K.
      • Webb H.K.
      • Yamamoto Y.
      • Sueyoshi T.
      • Afshari C.A.
      • Lehmann J.M.
      • Negishi M.
      Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital.
      ). The first omics study for CAR-regulated genes compared PB-treated livers from CAR WT with CAR KO mice. CAR was found to be essential to transduce drug metabolism and to repress energy metabolism (
      • Ueda A.
      • Hamadeh H.K.
      • Webb H.K.
      • Yamamoto Y.
      • Sueyoshi T.
      • Afshari C.A.
      • Lehmann J.M.
      • Negishi M.
      Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital.
      ). This finding of reciprocal regulation became an experimental avenue to understand the extent and diversity of nuclear receptor–mediated transduction of xenobiotic signals (Fig. 1). Subsequently, various types of omics, such as RNA-Seq, DNA ChIP-Seq, and metabolomics, have expanded our understanding of xenobiotic signaling far beyond drug metabolism.
      Figure thumbnail gr1
      Figure 1Xenobiotic-induced signals and diseases. Shown is a schematic representation of CAR/PXR-mediated signaling and diseases. Arrows, activation; stop bars, repression. Hatched arrows, pathways found in mice but not in humans. CAR- and PXR-mediated pathways are indicated in red and blue, respectively.

      Drug metabolism

      Drugs can be metabolically activated to toxicants or detoxified for excretion, affecting their efficacy and/or causing side effects. Prodrugs are metabolically activated. For example, the anti-cancer drugs 5-fluorouracil and 7-ethyl-10-hydroxycamptothecin (SN-38) are activated from their prodrugs by CYP2A6 and carboxylesterases, respectively (
      • Ikeda K.
      • Yoshisue K.
      • Matsushima E.
      • Nagayama S.
      • Kobayashi K.
      • Tyson C.A.
      • Chiba K.
      • Kawaguchi Y.
      Bioactivation of tegafur to 5-fluorouracil is catalyzed by cytochrome P-450 2A6 in human liver microsomes in vitro.
      ,
      • Satoh T.
      • Hosokawa M.
      • Atsumi R.
      • Suzuki W.
      • Hakusui H.
      • Nagai E.
      Metabolic activation of CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin, a novel antitumor agent, by carboxylesterase.
      ,
      • Rivory L.P.
      • Bowles M.R.
      • Robert J.
      • Pond S.M.
      Conversion of irinotecan (CPT-11) to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), by human liver carboxylesterase.
      ,
      • Humerickhouse R.
      • Lohrbach K.
      • Li L.
      • Bosron W.F.
      • Dolan M.E.
      Characterization of CPT-11 hydrolysis by human liver carboxylesterase isoforms hCE-1 and hCE-2.
      ). Naturally produced chemicals and human-made environmental chemicals also journey through these same metabolic pathways toward their respective excreted forms. Data analysis of the NIEHS gene expression array containing 8,736 genes found that 138 genes were either elevated or repressed after PB treatment, of which CAR regulated about half (
      • Ueda A.
      • Hamadeh H.K.
      • Webb H.K.
      • Yamamoto Y.
      • Sueyoshi T.
      • Afshari C.A.
      • Lehmann J.M.
      • Negishi M.
      Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital.
      ). PB-altered gene expression encoded enzymes related to drug metabolism that included CYP2B10, aldehyde dehydrogenase I, esterase 1, flavin-containing monooxygenase, glutathione S-transferase, PAPS synthase 2, and methyl transferase. PAPS synthase produces the donor substrate required for mammalian sulfotransferases. The δ-aminolaevulinic acid synthase (δ-ALAS) is a rate-limiting step in heme synthesis. Because heme is the prophetic group of CYP enzymes and PB was known to induce δ-ALAS, it was reassuring to observe that PB induced δ-ALAS in these microarray data. Unexpectedly, PB induction of δ-ALAS did not appear to be dependent on CAR WT versus KO status. However, a later study demonstrated that it is, in fact, regulated by CAR (
      • Tojima H.
      • Kakizaki S.
      • Yamazaki Y.
      • Takizawa D.
      • Horiguchi N.
      • Sato K.
      • Mori M.
      Ligand dependent hepatic gene expression profiles of nuclear receptors CAR and PXR.
      ,
      • Columbano A.
      • Ledda-Columbano G.M.
      • Pibiri M.
      • Cossu C.
      • Menegazzi M.
      • Moore D.D.
      • Huang W.
      • Tian J.
      • Locker J.
      Gadd45β is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia.
      ). PB also induces NADPH-cytochrome P450 reductase, which is essential for P450 enzymatic activity. CAR also appeared to be essential for this induction. In addition to enzymes, CAR (and PXR) also regulate many transporters to modulate the hepatic capability to import and excrete drugs and their metabolites (
      • Klaassen C.D.
      • Aleksunes L.M.
      Xenobiotic, bile acid, and cholesterol transporters: function and regulation.
      ). One RNA-Seq analysis identified 2,125 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP)-regulated genes and 212 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN)-regulated genes in mouse liver, among which 147 genes are induced by both (
      • Cui J.Y.
      • Klaassen C.D.
      RNA-Seq reveals common and unique PXR- and CAR-target gene signatures in the mouse liver transcriptome.
      ). Many of these common genes encoded for drug-metabolizing enzymes and drug transporters. Another cDNA microarray analysis used CARKO, PXRKO, and CAR and PXR double-KO mice treated with either TCPOBOP or PCN. CAR regulated about 95% of 554 genes that were affected by TCPOBOP treatment. The Cyp3a11 and Cyp2b10 genes were regulated by both CAR and PXR, whereas the Cyp1a1 and Cyp1a2 genes were regulated by CAR only (
      • Tojima H.
      • Kakizaki S.
      • Yamazaki Y.
      • Takizawa D.
      • Horiguchi N.
      • Sato K.
      • Mori M.
      Ligand dependent hepatic gene expression profiles of nuclear receptors CAR and PXR.
      ). A recent meta-analysis of 22 data sets provided a scope of transcriptomes that can be regulated by CAR and/or PXR (
      • Ochsner S.A.
      • Tsimelzon A.
      • Dong J.
      • Coarfa C.
      • McKenna N.J.
      Research resource: a reference transcriptome for constitutive androstane receptor and pregnane X receptor xenobiotic signaling.
      ).
      It was first demonstrated with two mouse proteins, CYP2A4 and CYP2A5, that a single amino acid mutation can alter the substrate specificity of CYP enzymes (
      • Lindberg R.L.
      • Negishi M.
      Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue.
      ). Subsequently, numerous SNPs were found in human CYP genes, some of which resulted in higher or lower CYP enzyme activities. Therefore, polymorphic mutations affect the hepatic capacity to metabolize drugs, increasing or decreasing either drug efficacy or toxicity (
      • Pinto N.
      • Dolan M.E.
      Clinically relevant genetic variations in drug metabolizing enzymes.
      ,
      • Ingelman-Sundberg M.
      • Sim S.C.
      • Gomez A.
      • Rodriguez-Antona C.
      Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects.
      ). Regulation of CAR and PXR expressions and activities should be critical in drug therapies in humans bearing these polymorphic mutations.

      Energy metabolism

      CAR regulation of energy metabolism genes was first realized by findings with the Pepck1 and Cpt1a genes (
      • Ueda A.
      • Hamadeh H.K.
      • Webb H.K.
      • Yamamoto Y.
      • Sueyoshi T.
      • Afshari C.A.
      • Lehmann J.M.
      • Negishi M.
      Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital.
      ). PXR was found to activate the Fasn and Hmgcs2 genes (
      • Rosenfeld J.M.
      • Vargas Jr., R.
      • Xie W.
      • Evans R.M.
      Genetic profiling defines the xenobiotic gene network controlled by the nuclear receptor pregnane X receptor.
      ). Following these findings, the notion is now accepted that CAR attenuates glucose metabolism, whereas PXR augments lipid metabolism (
      • Kanno Y.
      • Otsuka S.
      • Hiromasa T.
      • Nakahama T.
      • Inouye Y.
      Diurnal difference in CAR mRNA expression.
      ,
      • Mackowiak B.
      • Hodge J.
      • Stern S.
      • Wang H.
      The roles of xenobiotic receptors: beyond chemical disposition.
      ,
      • Wada T.
      • Gao J.
      • Xie W.
      PXR and CAR in energy metabolism.
      ). For CAR, recent metabolomic studies detected metabolites either increased or decreased in serum and/or liver extracts after TCPOBOP treatment in CAR WT but not CAR KO mice; those metabolites include fatty acid, lactate, ketone bodies, and tricarboxylic acid cycle products as well as glucose (
      • Chen F.
      • Coslo D.M.
      • Chen T.
      • Zhang L.
      • Tian Y.
      • Smith P.B.
      • Patterson A.D.
      • Omiecinski C.J.
      Metabolomic approaches reveal the role of CAR in energy metabolism.
      ). Moreover, a genome-wide ChIP-Seq analysis demonstrated that CAR competes with HNF4α, PPARα, and FXR for their binding sites, repressing genes in energy metabolism (
      • Tian J.
      • Marino R.
      • Johnson C.
      • Locker J.
      Binding of drug-activated CAR/Nr1i3 alters metabolic regulation in the liver.
      ).

      Cell proliferation

      The first CAR-regulated gene related to cell growth, cycle, and apoptosis was GADD45B (
      • Columbano A.
      • Ledda-Columbano G.M.
      • Pibiri M.
      • Cossu C.
      • Menegazzi M.
      • Moore D.D.
      • Huang W.
      • Tian J.
      • Locker J.
      Gadd45β is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia.
      ,
      • Yamamoto Y.
      • Moore R.
      • Flavell R.A.
      • Lu B.
      • Negishi M.
      Nuclear receptor CAR represses TNFα-induced cell death by interacting with the anti-apoptotic GADD45B.
      ). Subsequently, the Cdc20 and Cdk1 genes were shown to be regulated by CAR and PXR, whereas the c-Myc and Gadd45b genes were regulated only by CAR (
      • Tojima H.
      • Kakizaki S.
      • Yamazaki Y.
      • Takizawa D.
      • Horiguchi N.
      • Sato K.
      • Mori M.
      Ligand dependent hepatic gene expression profiles of nuclear receptors CAR and PXR.
      ). A recent genome-wide ChIP-Seq analysis of mouse liver ectopically expressing GFP-tagged mouse or human CAR found ∼1,000 genes differentially regulated by mouse or human CAR, including protooncogene Myc and Ikbke, which were only activated by mouse CAR (
      • Niu B.
      • Coslo D.M.
      • Bataille A.R.
      • Albert I.
      • Pugh B.F.
      • Omiecinski C.J.
      In vivo genome-wide binding interactions of mouse and human constitutive androstane receptors reveal novel gene targets.
      ). Yes-associated protein (YAP) was also characterized as a target directly interacting with CAR or PXR, increasing hepatocyte growth and proliferation (
      • Abe T.
      • Amaike Y.
      • Shizu R.
      • Takahashi M.
      • Kano M.
      • Hosaka T.
      • Sasaki T.
      • Kodama S.
      • Matsuzawa A.
      • Yoshinari K.
      Role of YAP activation in nuclear receptor CAR-mediated proliferation of mouse hepatocytes.
      ,
      • Abe T.
      • Shizu R.
      • Sasaki T.
      • Shimizu Y.
      • Hosaka T.
      • Kodama S.
      • Matsuzawa A.
      • Yoshinari K.
      Functional interaction between pregnane X receptor and Yes-associated protein in xenobiotic-dependent liver hypertrophy and drug metabolism.
      ,
      • Jiang Y.
      • Feng D.
      • Ma X.
      • Fan S.
      • Gao Y.
      • Fu K.
      • Wang Y.
      • Sun J.
      • Yao X.
      • Liu C.
      • Zhang H.
      • Xu L.
      • Liu A.
      • Gonzalez F.J.
      • Yang Y.
      • et al.
      Pregnane X receptor regulates liver size and liver cell fate by Yes-associated protein activation in mice.
      ).
      More recent omics studies provided additional information on the roles of CAR and/or PXR in the regulation of cell growth. A metabolomic analysis of mouse urine found decreases of vitamin E metabolites in response to PXR activation. This suggested that PXR may regulate vitamin E signals. In humans, rifampicin treatment altered steroids and their metabolites in urine samples; sulfate conjugates of dehydroepiandrosterone and androsterone were increased, whereas hydroxylated C-19 androgens were decreased (
      • Kim B.
      • Moon J.Y.
      • Choi M.H.
      • Yang H.H.
      • Lee S.
      • Lim K.S.
      • Yoon S.H.
      • Yu K.S.
      • Jang I.J.
      • Cho J.Y.
      Global metabolomics and targeted steroid profiling reveal that rifampin, a strong human PXR activator, alters endogenous urinary steroid markers.
      ), suggesting involvement of PXR in steroid signals. Various types of noncoding RNAs have been reported to modulate the actions of CAR and PXR (
      • Nakano M.
      • Nakajima M.
      Current knowledge of microRNA-mediated regulation of drug metabolism in humans.
      ). CAR and PXR elicit these xenobiotic signals by direct binding of xenobiotics and indirectly through cellular signals such as phosphorylation or by a combination of both. Hereafter, this review focuses on conserved phosphorylation as a nuclear receptor regulator.

      Conserved phosphorylation motifs

      Nuclear receptors contain many Thr and Ser residues as potential phosphorylation residues within their molecules (
      • Treviño L.S.
      • Weigel N.L.
      Phosphorylation: a fundamental regulator of steroid receptor action.
      ). An early emphasis on phosphorylation studies was observed with steroid hormone receptors, while searching for cell signal regulations (so-called ligand-independent mechanism) alternative to the direct hormone-binding mechanism. Nuclear receptors consist of similar-sized DNA- and ligand-binding domains (DBDs and LBDs) with 350–400 amino acid residues and N-terminal domains (NTDs) of various lengths (Fig. 2). Steroid hormone receptors possess long NTDs, with androgen receptor having the longest at 600 amino acid residues, which includes activation function 1 (AF1). Inevitably, investigations targeted many potential phosphorylation residues that were within the NTD (
      • Treviño L.S.
      • Weigel N.L.
      Phosphorylation: a fundamental regulator of steroid receptor action.
      ). However, those within the NTD are not conserved among steroid hormone receptors, impeding efforts to corroborate findings and conceptualize phosphorylation as a common function in steroid hormone receptors.
      Figure thumbnail gr2
      Figure 2Zinc finger of nuclear receptors. A, domain organization of nuclear receptor. B, two treble clef-fold zinc fingers of human estrogen receptor α (PDB code 1HCP) (
      • Schwabe J.W.
      • Chapman L.
      • Finch J.T.
      • Rhodes D.
      • Neuhaus D.
      DNA recognition by the oestrogen receptor: from solution to the crystal.
      ). Within the N-terminal treble clef zinc finger, one zinc knuckle (noncanonical turn with CXXC, which is enclosed by a broken red line), two β-strands (c and d; corresponding to those shown in ), and one α-helix are depicted. Four cysteine residues functioning as ligands for a zinc ion (gray sphere) are also shown. This image was created on the RCSB PDB website using Mol* (
      • Sehnal D.
      • Rose A.S.
      • Kovca J.
      • Burley S.K.
      • Velankar S.
      Mol*: Towards a common library and tools for web molecular graphics.
      ). C, amino acid sequence of two zinc fingers within the DBD of human CAR. Localizations of the well-conserved Thr38 (in red) and extension of the P-box, D-box, and two α-helices are indicated.
      As suggested (
      • Matalon O.
      • Dubreuil B.
      • Levy E.D.
      Young phosphorylation is functionally silent.
      ,
      • Studer R.A.
      • Rodriguez-Mias R.A.
      • Haas K.M.
      • Hsu J.I.
      • Viéitez C.
      • Solé C.
      • Swaney D.L.
      • Stanford L.B.
      • Liachko I.
      • Böttcher R.
      • Dunham M.J.
      • de Nadal E.
      • Posas F.
      • Beltrao P.
      • Villén J.
      Evolution of protein phosphorylation across 18 fungal species.
      ), the residues with the potential for phosphorylation appeared within the last 1.8 million years and are overwhelming in numbers but not functional. Meanwhile, the DBD and LBD were less emphasized as targets of investigation. Because their functions had been clearly defined in DNA and ligand bindings, DBD and LBD were less emphasized as targets of investigations conducted under the ligand-dependent concept. However, both DBD and LBD contain phosphorylation motifs that have been conserved not only among nuclear receptors but also throughout species from mice to humans that are separated by over 70 million years of evolution. These evolutionarily conserved phosphorylation motifs likely inherit essential functions to regulate nuclear receptors.
      CAR and PXR have short NTDs, with only 9 and 39 amino acid residues, respectively, helping us to not repeat the history of hormone receptor studies and to lead us to the DBD and/or LBD. Nuclear receptors conserve a phosphorylation motif within their DBD or LBD, including CAR and PXR. Our investigations of CAR and PXR have been focused on Thr-38 and Ser-350 within their DBD and LBD, respectively. Because of their high conservation among nuclear receptors and across species, what was found with CAR and/or PXR could be extended to many nuclear receptors far beyond CAR and PXR. The zinc finger of DBD was initially examined as a target of phosphorylation to regulate CAR by focusing on its Thr-38.

      Zinc finger of the DBD

      Structure

      Zinc fingers are small structural motifs that are widely present in proteins throughout the kingdoms of living organisms. Zinc fingers can be divided into different structural types (fold groups) often represented by classic C2H2, zinc ribbon, and treble clef (TC)-folds (
      • Krishna S.S.
      • Majumdar I.
      • Grishin N.V.
      Structural classification of zinc fingers: survey and summary.
      ). A main function of TC-fold zinc fingers is DNA binding. The zinc finger of nuclear receptor comprises the classical NR DNA-binding domain group within the nuclear receptor–like family of the TC-fold (
      • Kaur G.
      • Subramanian S.
      Classification of the treble clef zinc finger: noteworthy lessons for structure and function evolution.
      ), and the domain group presents only in the Metazoan kingdom. In the case of classical NR DNA-binding domains, peptides from 26 to 32 amino acid residues form a β-strand, loop, and α-helix, from which four residues coordinate one zinc atom to form a finger loop (Fig. 2). In other words, two pairs of cysteine residues, one from the zinc knuckle (CXXC) and the other from the N terminus of the α-helix, coordinate one zinc atom to form a TC-fold finger loop (Fig. 2B). Two zinc fingers connected via a short linker form double finger loops within the DBD. The five residues at the α-helix within the N-terminal zinc finger form the proximal box (P-box), which directly interacts with DNA by inserting it into a major groove of the double strands. The C-terminal zinc finger is not a typical TC-fold; an additional three-amino acid residue segment is inserted at the N-terminal region to constitute a D-box (distal zinc finger dimerization residues). As a unique structural feature in nuclear receptors, the D-box is involved in intramolecular interactions, but not direct DNA binding.

      Phosphorylation and regulation

      Amino acid sequences of zinc fingers are well-conserved among nuclear receptors. Many polar residues (threonine and serine) and a positively charged lysine residue have been incorporated into the zinc finger during evolution, creating potential protein kinase C, protein kinase A, and/or p38 MAPK phosphorylation motifs. Among them, Thr/Ser-10, Thr/Ser-18, Tyr-14, and Tyr-38 are relatively well-conserved, as well as residues 44, 37, 43, and 41 in the total 46 human nuclear receptors (Fig. S1). Retinoic acid receptor α (RARα) conserves Thr/Ser-10 at Ser-96. RARα is a constitutively activated nuclear receptor. AKT-phosphorylated Ser-96 in transformed cells prevents RARα from heterodimerizing with retinoid X receptor α (RXRα) to repress the transactivation activity (
      • Srinivas H.
      • Xia D.
      • Moore N.L.
      • Uray I.P.
      • Kim H.
      • Ma L.
      • Weigel N.L.
      • Brown P.H.
      • Kurie J.M.
      Akt phosphorylates and suppresses the transactivation of retinoic acid receptor alpha.
      ). Human FXR conserves it at Ser-135. A phosphorylation-blocking mutation (S135A) prohibits FXR from heterodimerizing with RXRα, abrogating its activity (
      • Gineste R.
      • Sirvent A.
      • Paumelle R.
      • Helleboid S.
      • Aquilina A.
      • Darteil R.
      • Hum D.W.
      • Fruchart J.C.
      • Staels B.
      Phosphorylation of farnesoid X receptor by protein kinase C promotes its transcriptional activity.
      ). PXR retains the Thr/Ser-18, and this residue corresponds to Thr-57. The T57A mutation attenuated PXR's ability to bind and activate the CYP3A4 promoter in cell-based assays (
      • Lichti-Kaiser K.
      • Brobst D.
      • Xu C.
      • Staudinger J.L.
      A systematic analysis of predicted phosphorylation sites within the human pregnane X receptor protein.
      ,
      • Pondugula S.R.
      • Brimer-Cline C.
      • Wu J.
      • Schuetz E.G.
      • Tyagi R.K.
      • Chen T.
      A phosphomimetic mutation at threonine-57 abolishes transactivation activity and alters nuclear localization pattern of human pregnane x receptor.
      ). However, phosphorylation of these residues in vivo has not been confirmed in either animal or human tissues.

      Threonine/serine 29

      This motif, which corresponds to Thr-38 of CAR, is conserved in 41 of 46 human nuclear receptors, as well as their corresponding mouse counterparts (Fig. S1). Only five nuclear receptors do not retain this motif: PXR and four members of the nuclear hormone receptors (NR3C subfamily). Thr/Ser-29 is positioned at the middle of the α-helix of the N-terminal zinc finger, connecting it to the C-terminal zinc finger. Thr/Ser-29 has been investigated with many nuclear receptors. Among them, Thr-38 of CAR is the most extensively investigated (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ,
      • Mutoh S.
      • Sobhany M.
      • Moore R.
      • Perera L.
      • Pedersen L.
      • Sueyoshi T.
      • Negishi M.
      Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.
      ,
      • Negishi M.
      Phenobarbital meets phosphorylation of nuclear receptors.
      ,
      • Osabe M.
      • Negishi M.
      Active ERK1/2 protein interacts with the phosphorylated nuclear constitutive active/androstane receptor (CAR; NR1I3), repressing dephosphorylation and sequestering CAR in the cytoplasm.
      ,
      • Yang H.
      • Garzel B.
      • Heyward S.
      • Moeller T.
      • Shapiro P.
      • Wang H.
      Metformin represses drug-induced expression of CYP2B6 by modulating the constitutive androstane receptor signaling.
      ,
      • Shizu R.
      • Min J.
      • Sobhany M.
      • Pedersen L.C.
      • Mutoh S.
      • Negishi M.
      Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.
      ,
      • Shizu R.
      • Osabe M.
      • Perera L.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.
      ). In addition to CAR, four more endogenous nuclear receptors have been confirmed to be phosphorylated in mouse and human tissues and/or primary hepatocytes, with their biological functions described: estrogen receptor α (ERα) at Ser-216, FXR at Ser-154, RXRα at Thr-167, and retinoid-related orphan receptor α (RORα) at Ser-100 (
      • Negishi M.
      Phenobarbital meets phosphorylation of nuclear receptors.
      ,
      • Shindo S.
      • Moore R.
      • Flake G.
      • Negishi M.
      Serine 216 phosphorylation of estrogen receptor α in neutrophils: migration and infiltration into the mouse uterus.
      ,
      • Hashiguchi T.
      • Arakawa S.
      • Takahashi S.
      • Gonzalez F.J.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylation of farnesoid X receptor at serine 154 links ligand activation with degradation.
      ,
      • Sueyoshi T.
      • Sakuma T.
      • Shindo S.
      • Fashe M.
      • Kanayama T.
      • Ray M.
      • Moore R.
      • Negishi M.
      A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice.
      ,
      • Fashe M.
      • Hashiguchi T.
      • Negishi M.
      • Sueyoshi T.
      Ser100-phosphorylated RORα orchestrates CAR and HNF4α to form active chromatin complex in response to phenobarbital to regulate induction of CYP2B6.
      ,
      • Fashe M.
      • Hashiguchi T.
      • Yi M.
      • Moore R.
      • Negishi M.
      Phenobarbital-induced phosphorylation converts nuclear receptor RORα from a repressor to an activator of the estrogen sulfotransferase gene Sult1e1 in mouse livers.
      ). Besides nuclear receptors, Thr/Ser-29 is conserved in many proteins, such as LIM domain proteins, in the other domain groups of the nuclear receptor–like finger family (
      • Mertins P.
      • Mani D.R.
      • Ruggles K.V.
      • Gillette M.A.
      • Clauser K.R.
      • Wang P.
      • Wang X.
      • Qiao J.W.
      • Cao S.
      • Petralia F.
      • Kawaler E.
      • Mundt F.
      • Krug K.
      • Tu Z.
      • Lei J.T.
      • et al.
      Proteogenomics connects somatic mutations to signalling in breast cancer.
      ,
      • Bian Y.
      • Song C.
      • Cheng K.
      • Dong M.
      • Wang F.
      • Huang J.
      • Sun D.
      • Wang L.
      • Ye M.
      • Zou H.
      An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome.
      ).

      The LBD

      Nuclear receptors conserve the Ser/Thr residue as a phosphorylation motif in the loop between helices 8 and 9 within the LBD of 31 human nuclear receptors and their corresponding mouse counterparts. (Fig. 3). Studies on this motif have so far been limited to PXR at Ser-350. The residue Ser-350 was phosphorylated in response to low glucose in human hepatoma-derived HepG2 and Huh-7 cells (
      • Gotoh S.
      • Miyauchi Y.
      • Moore R.
      • Negishi M.
      Glucose elicits serine/threonine kinase VRK1 to phosphorylate nuclear pregnane X receptor as a novel hepatic gluconeogenic signal.
      ). This motif is conserved in 31 human nuclear receptors and highlights the fact that Ser-350 is conserved in NR3C steroid hormone receptors, which include glucocorticoid receptor (GR), mineralocorticoid receptor, and androgen receptor (AR). In addition, estrogen receptors (NR3B 1 and 2) also conserve this phosphorylation motif. PXR and these NR3C steroid hormone receptors are four of only five nuclear receptors that do not conserve the motif within the DBD, as represented by Thr-38 in CAR.
      Figure thumbnail gr3
      Figure 3Phosphorylation motif conserved within the LBD. Nuclear receptor protein sequence alignment was performed with Clustal Omega (RRID:SCR_001591). The C-terminal portion of helix 8 and the N-terminal portion of helix 9 are indicated by green arrows on the top of this alignment as well as the corresponding sequences of PXR, which are highlighted in green. The conserved Ser/Thr is highlighted in orange. PXR and three steroid hormone receptors are boxed in blue.

      CAR activation signal via phosphorylation

      CAR was characterized as a PB-activated nuclear receptor in 1998 (
      • Honkakoski P.
      • Zelko I.
      • Sueyoshi T.
      • Negishi M.
      The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene.
      ,
      • Kliewer S.A.
      • Moore J.T.
      • Wade L.
      • Staudinger J.L.
      • Watson M.A.
      • Jones S.A.
      • McKee D.D.
      • Oliver B.B.
      • Willson T.M.
      • Zetterström R.H.
      • Perlmann T.
      • Lehmann J.M.
      An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.
      ); 15 years later, the molecular mechanism of this activation was determined (
      • Mutoh S.
      • Sobhany M.
      • Moore R.
      • Perera L.
      • Pedersen L.
      • Sueyoshi T.
      • Negishi M.
      Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.
      ). CAR is inactivated by phosphorylation at Thr-38, the conserved motif within the DBD, whereas its dephosphorylation activated CAR. Whereas this phosphorylation and dephosphorylation are regulated by cell growth signals that are elicited on the cell membrane, PB antagonized these cell signals to induce dephosphorylation, activating CAR indirectly (Fig. 4). Hereafter, how the mechanism was investigated and determined will be described in a step-by-step manner as the experimental processes developed. It also became clear that ligands directly induce dephosphorylation by sharing the same mechanism as observed with the indirect activation by PB.
      Figure thumbnail gr4
      Figure 4The phosphorylation-mediated CAR activation mechanism This schematically summarizes the work done over the last 20 years and presents the molecular mechanism by which phosphorylation regulates CAR activation. Prior to PB activation, CAR is phosphorylated as an inactive homodimer through Surface A in the cytoplasm. EGF stimulates ERK1/2 binding to the XRS of the LBD, stabilizing the homodimer. PB binds EGF receptor to attenuate the EGF signal, resulting in ERK1/2 dephosphorylation and dissociation from XRS to monomerize the CAR homodimer. The resulting phosphorylated monomer is dephosphorylated by PP2A for nuclear translocation and heterodimerization with RXRα.

      Phenobarbital

      PB, an anti-epileptic drug widely used for more than 100 years, was found to induce hepatic CYP enzymes, increasing drug metabolism in the early 1960s (
      • Remmer H.
      • Merker H.J.
      Drug-induced changes in the liver endoplasmic reticulum: association with drug-metabolizing enzymes.
      ,
      • Conney A.H.
      Pharmacological implications of microsomal enzyme induction.
      ,
      • Omura T.
      • Sato R.
      A new cytochrome in liver microsomes.
      ,
      • Yasiry Z.
      • Shorvon S.D.
      How phenobarbital revolutionized epilepsy therapy: the story of phenobarbital therapy in epilepsy in the last 100 years.
      ). PB elicits pleiotropic signals, including abnormal enlargement of the liver, hyperproliferation, and dysregulation of energy homeostasis in addition to drug metabolism. PB coordinately activates hepatic genes to increase CYP-mediated drug metabolism, concomitant with increases in heme biosynthesis, an electron transfer enzyme (NADPH-cytochrome P450 reductase), and the supply of NADPH. PB also activates genes encoding certain transferases, such as sulfotransferases and PAPS synthetase, which produces the donor substrate. However, the molecular mechanism by which PB elicits its signals has been a mystery for the last 50 years.

      CAR

      CAR, one of many constitutively activated nuclear receptors, was characterized as a PB-activated nuclear receptor in the late 1990s (
      • Honkakoski P.
      • Zelko I.
      • Sueyoshi T.
      • Negishi M.
      The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene.
      ,
      • Kawamoto T.
      • Sueyoshi T.
      • Zelko I.
      • Moore R.
      • Washburn K.
      • Negishi M.
      Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene.
      ,
      • Sueyoshi T.
      • Kawamoto T.
      • Zelko I.
      • Honkakoski P.
      • Negishi M.
      The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene.
      ), which was soon confirmed by utilizing CAR KO mice (
      • Wei P.
      • Zhang J.
      • Egan-Hafley M.
      • Liang S.
      • Moore D.D.
      The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.
      ). The finding of CAR paired PB with the CYP gene and allowed us to investigate the long-awaited molecular mechanism of PB induction. This investigation was faced with two obstacles to overcome: CAR's constitutive activity and the fact that PB does not directly bind CAR. This constitutive activity must be repressed for CAR to obtain a PB-response capability. As for PB to elicit an induction signal, its initial binding factor must be identified. Phosphorylation of Thr-38 within the DBD through an epidermal growth factor receptor signaling was suggested as an underlying molecular mechanism for CAR activation by PB, as depicted in Fig. 3.

      Thr-38

      CAR was found in cytosolic fractions of mouse primary hepatocytes and accumulated in nuclear fractions upon PB treatment, which correlated with an increase of CYP2B10 mRNA. Moreover, okadaic acid, a protein phosphatase inhibitor, repressed these PB responses (
      • Kawamoto T.
      • Sueyoshi T.
      • Zelko I.
      • Moore R.
      • Washburn K.
      • Negishi M.
      Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene.
      ). Thus, cytoplasmic-nuclear translocation appeared to be a step in the CAR activation process and protein phosphatase as a cell signal regulating this step. Since this finding, okadaic acid has been used as a reagent to examine CAR activation. Eventually, Thr-38 was identified and characterized as the residue that was dephosphorylated as suggested previously by okadaic acid. Protein phosphatase 2A (PP2A) formed a complex with receptor for activated protein kinase C1 (RACK1) and dephosphorylated Thr-38 in in vitro assays. Moreover, siRNA knockdown of PP2A or RACK1 attenuated PB-induced dephosphorylation and nuclear translocation in mouse primary hepatocytes, confirming that PB elicited a signal for this dephosphorylation (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ). This phosphorylation-dephosphorylation was soon demonstrated with mouse livers as well as human primary hepatocytes and three-dimensional cultures of HepG2 cells (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ,
      • Yang H.
      • Garzel B.
      • Heyward S.
      • Moeller T.
      • Shapiro P.
      • Wang H.
      Metformin represses drug-induced expression of CYP2B6 by modulating the constitutive androstane receptor signaling.
      ,
      • Yokobori K.
      • Azuma I.
      • Chiba K.
      • Akita H.
      • Furihata T.
      • Kobayashi K.
      Indirect activation of constitutive androstane receptor in three-dimensionally cultured HepG2 cells.
      ). An ectopically expressed CAR T38D, a phosphorylation-mimicking mutant was retained in the cytoplasm and did not translocate into the nucleus even after PB treatment in mouse livers (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ). In gel-shift assays, CAR T38D was unable to bind its target DR-4 sequence. A dynamic simulation of the CAR DBD molecule showed that phosphorylation of Thr-38 disrupted the α-helix between the two zinc fingers toward its C terminus (Fig. 5A). This disruption can be a structural basis for phosphorylation regulating the translocation and bindings. Examination with 10 other nuclear receptors besides CAR showed their corresponding aspartic acid mutations disabled their nuclear translocation as well as DNA-binding capabilities (
      • Hashiguchi T.
      • Arakawa S.
      • Takahashi S.
      • Gonzalez F.J.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylation of farnesoid X receptor at serine 154 links ligand activation with degradation.
      ).
      Figure thumbnail gr5
      Figure 5Phosphorylation, dimer interfaces, and diverse interaction. A, dynamic simulation of the helix. This is depicted from previous works (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ) and modified by adding the side chain of phosphorylated Thr-38. The C-terminal portion of the helix is distorted by phosphorylation. B, two different dimer interfaces are pictured. C, modeling an ERα homodimer-CAR-RXRα tetramer. First, CAR homodimer (
      • Shan L.
      • Vincent J.
      • Brunzelle J.S.
      • Dussault I.
      • Lin M.
      • Ianculescu I.
      • Sherman M.A.
      • Forman B.M.
      • Fernandez E.J.
      Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism.
      ) and CAR-RXRα heterodimer (
      • Xu R.X.
      • Lambert M.H.
      • Wisely B.B.
      • Warren E.N.
      • Weinert E.E.
      • Waitt G.M.
      • Williams J.D.
      • Collins J.L.
      • Moore L.B.
      • Willson T.M.
      • Moore J.T.
      A structural basis for constitutive activity in the human CAR/RXRα heterodimer.
      ) are superimposed through their CAR monomers to model the CAR-CAR-RXRα trimer. One monomer of the ERα homodimer (PDB code 1ERE) is superimposed with the first CAR monomer of the CAR-CAR-RXRα trimer. By removing the CAR monomer, an ERα homodimer-CAR-RXRα tetramer is modeled (
      • Yi M.
      • Fashe M.
      • Arakawa S.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.
      ).

      CAR homodimer in the cytoplasm

      X-ray crystal structures of both CAR LBD homodimer and heterodimer with RXRα were determined (
      • Shan L.
      • Vincent J.
      • Brunzelle J.S.
      • Dussault I.
      • Lin M.
      • Ianculescu I.
      • Sherman M.A.
      • Forman B.M.
      • Fernandez E.J.
      Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism.
      ,
      • Xu R.X.
      • Lambert M.H.
      • Wisely B.B.
      • Warren E.N.
      • Weinert E.E.
      • Waitt G.M.
      • Williams J.D.
      • Collins J.L.
      • Moore L.B.
      • Willson T.M.
      • Moore J.T.
      A structural basis for constitutive activity in the human CAR/RXRα heterodimer.
      ). CAR utilizes two different dimer interfaces located on opposite sides of the CAR molecule; three loops comprise a homodimer surface, whereas a heterodimer surface is constituted with α-helices, hereafter referred to as Surface A and Surface B, respectively (Fig. 5B). Human CAR formed its homodimer in cells as the crystal structure indicated (
      • Shizu R.
      • Osabe M.
      • Perera L.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.
      ). Human CAR, which was expressed in the liver of FCKO mice (human CAR transgenic mouse in a CAR KO mouse background), formed this homodimer and dissociated into a monomer after PB or human CAR ligand 6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) treatment (
      • Shizu R.
      • Osabe M.
      • Perera L.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.
      ). As these observations indicated that phosphorylated CAR is retained in the cytoplasm as a homodimer, the CAR T38D mutant formed its homodimer in Huh-7 cells (
      • Shizu R.
      • Min J.
      • Sobhany M.
      • Pedersen L.C.
      • Mutoh S.
      • Negishi M.
      Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.
      ). Homodimerization masked a binding site on Surface A for a PP2A-RACK1 complex, preventing Thr-38 from being dephosphorylated by PP2A. Ectopic CAR T38D mutant remained in the cytoplasm in the mouse livers even after PB treatment (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ). Phosphorylation stabilized the CAR homodimer and retained it in the cytoplasm, setting dephosphorylation as an initial step of CAR activation.

      Cell signal regulation

      CAR is, in principle, a cell signal–regulated nuclear receptor repressing its constitutive activity. PB was expected to utilize cell signals to activate CAR. It all began with the finding that EGF repressed PB-induced CAR binding to the PB-responsive enhancer module (PBREM) within the CYP2B promoter in rat primary hepatocytes (
      • Bauer D.
      • Wolfram N.
      • Kahl G.F.
      • Hirsch-Ernst K.I.
      Transcriptional regulation of CYP2B1 induction in primary rat hepatocyte cultures: repression by epidermal growth factor is mediated via a distal enhancer region.
      ), which correlated with epidermal growth factor (EGF) repression of a PB-induced nuclear translocation of CAR in mouse primary hepatocytes (
      • Koike C.
      • Moore R.
      • Negishi M.
      Extracellular signal-regulated kinase is an endogenous signal retaining the nuclear constitutive active/androstane receptor (CAR) in the cytoplasm of mouse primary hepatocytes.
      ). Subsequently, this EGF regulation associated with homodimerization of phosphorylated CAR. Ectopic CAR T38D appeared as a monomer and converted into a homodimer after treatment with EGF in Huh-7 cells (
      • Shizu R.
      • Osabe M.
      • Perera L.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.
      ). Conversely, inhibition of EGF receptor (EGFR) by erlotinib monomerized the CAR homodimer in Huh-7 cells. Upon EGF activation, an EGFR downstream enzyme ERK1/2 is activated and phosphorylated for subsequent binding to the xenochemical response sequence (XRS) near the C terminus of the LBD (
      • Osabe M.
      • Negishi M.
      Active ERK1/2 protein interacts with the phosphorylated nuclear constitutive active/androstane receptor (CAR; NR1I3), repressing dephosphorylation and sequestering CAR in the cytoplasm.
      ). This active ERK1/2 binding converted CAR into a homodimer and buried the PP2A-RACK1 complex binding site within the homodimer interface, protecting Thr-38 from dephosphorylation.

      PB signal transduction

      Isothermal titration calorimetry detected direct PB-EGFR binding, and dynamic simulation of the binding suggested an EGF-binding site to be one of the PB-binding sites (
      • Mutoh S.
      • Sobhany M.
      • Moore R.
      • Perera L.
      • Pedersen L.
      • Sueyoshi T.
      • Negishi M.
      Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.
      ). Through these bindings, PB antagonizes EGF, inhibits EGFR activity, and reverses the ERK1/2 signal, subsequently dissociating an inactivated (dephosphorylated) ERK1/2 from the XRS. This dissociation converted the phosphorylated CAR homodimer to its monomer, allowing a PP2A-RACK1 complex to dephosphorylate Thr-38 (
      • Mutoh S.
      • Sobhany M.
      • Moore R.
      • Perera L.
      • Pedersen L.
      • Sueyoshi T.
      • Negishi M.
      Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.
      ). Reflecting this nature, the PB activation was defined as an “antagonistic activation” (
      • Meyer S.A.
      • Jirtle R.L.
      Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.
      ). A historical enigma, long before the finding of CAR, PB was found to decrease EGFR levels and activity (
      • Meyer S.A.
      • Gibbs T.A.
      • Jirtle R.L.
      Independent mechanisms for tumor promoters phenobarbital and 12-O-tetradecanoylphorbol-13-acetate in reduction of epidermal growth factor binding by rat hepatocytes.
      ). However, this finding was never followed up, because this repression did not appear to agree with the notion that PB should induce hepatic proliferation.
      In addition to EGFR, PB can also bind the insulin receptor to repress ERK1/2 activation (
      • Yasujima T.
      • Saito K.
      • Moore R.
      • Negishi M.
      Phenobarbital and insulin reciprocate activation of the nuclear receptor constitutive androstane receptor through the insulin receptor.
      ). Insulin has long been known to repress PB induction of CYP2B, and, conversely, diabetic livers increased CYP expressions (
      • Schenkman J.B.
      Induction of diabetes and evaluation of diabetic state on P450 expression.
      ,
      • Yoshida Y.
      • Kimura N.
      • Oda H.
      • Kakinuma A.
      Insulin suppresses the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital in adult rat cultured hepatocytes.
      ,
      • Woodcroft K.J.
      • Novak R.F.
      Insulin effects on CYP2E1, 2B, 3A, and 4A expression in primary cultured rat hepatocytes.
      ,
      • Kawamura A.
      • Yoshida Y.
      • Kimura N.
      • Oda H.
      • Kakinuma A.
      Phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital.
      ). This PB-insulin interaction can now be understood by its competitive antagonism via the insulin receptor. An increasing number of drugs and xenobiotics have now been found to indirectly activate CAR by this antagonistic mechanism (
      • Meyer S.A.
      • Jirtle R.L.
      Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.
      ), opening novel research directions in pharmacology and toxicology.
      CAR can also be activated by direct ligand bindings, such as TCPOBOP and CITCO for mouse and human counterparts, respectively (
      • Sueyoshi T.
      • Kawamoto T.
      • Zelko I.
      • Honkakoski P.
      • Negishi M.
      The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene.
      ,
      • Xu R.X.
      • Lambert M.H.
      • Wisely B.B.
      • Warren E.N.
      • Weinert E.E.
      • Waitt G.M.
      • Williams J.D.
      • Collins J.L.
      • Moore L.B.
      • Willson T.M.
      • Moore J.T.
      A structural basis for constitutive activity in the human CAR/RXRα heterodimer.
      ,
      • Maglich J.M.
      • Parks D.J.
      • Moore L.B.
      • Collins J.L.
      • Goodwin B.
      • Billin A.N.
      • Stoltz C.A.
      • Kliewer S.A.
      • Lambert M.H.
      • Willson T.M.
      • Moore J.T.
      Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes.
      ,
      • Suino K.
      • Peng L.
      • Reynolds R.
      • Li Y.
      • Cha J.Y.
      • Repa J.J.
      • Kliewer S.A.
      • Xu H.E.
      The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization.
      ). The ligand binding dissociated ERK1/2 in its active form from the LBD, monomerizing CAR to undergo its activation process (
      • Shizu R.
      • Osabe M.
      • Perera L.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.
      ). Regardless of whether it is direct or indirect, CAR utilizes the same molecular mechanism for activation (Fig. 4).

      Rephosphorylation in the nucleus

      Whereas CAR translocates into the nucleus in its nonphosphorylated form, Thr-38 can be rephosphorylated in the nucleus, which was essential for CAR to activate the Cyp gene but not for all CAR-targeted genes (
      • Hori T.
      • Moore R.
      • Negishi M.
      p38 MAP kinase links CAR activation and inactivation in the nucleus via phosphorylation at threonine 38.
      ). p38 MAPK was essential for CAR to activate the Cyp2b10 gene. CAR recruited p38 MAP kinase to the PBREM and, subsequently, phosphorylated Thr-38 in response to PB. This rephosphorylation may couple CAR activation and inactivation, recycling CAR (
      • Hori T.
      • Moore R.
      • Negishi M.
      p38 MAP kinase links CAR activation and inactivation in the nucleus via phosphorylation at threonine 38.
      ). In addition to rephosphorylation, CAR required the co-chaperone cytoplasmic CAR retention protein (CCRP) to activate the Cyp2b10 gene, as observed in CCRP-KO mice (
      • Ohno M.
      • Kanayama T.
      • Moore R.
      • Ray M.
      • Negishi M.
      The roles of co-chaperone CCRP/DNAJC7 in Cyp2b10 gene activation and steatosis development in mouse livers.
      ). Apparently, whereas nonphosphorylated CAR has a high constitutive activity, this activity is regulated in the nucleus as well as in the cytoplasm.
      A similar rephosphorylation was demonstrated with FXR in mouse livers; Ser-154 was phosphorylated by vaccinia-related kinase 1 (VRK1), likely dissociating FXR from the promoter for subsequent degradation. RORα was phosphorylated at Ser-100 on the Sult1e1 promoter in response to PB in mouse livers (
      • Fashe M.
      • Hashiguchi T.
      • Yi M.
      • Moore R.
      • Negishi M.
      Phenobarbital-induced phosphorylation converts nuclear receptor RORα from a repressor to an activator of the estrogen sulfotransferase gene Sult1e1 in mouse livers.
      ). Noticeably, this phosphorylation occurred in an FXR ligand–dependent manner in the liver, thereby linking phosphorylation with ligand action. A similar link was observed previously with Nur77 (NR4A1). Ser-351, which is in the LBD and does not correspond to Thr-38 of CAR, was phosphorylated and exported from the nucleus in response to a Nur77 ligand treatment (
      • Han Y.H.
      • Cao X.
      • Lin B.
      • Lin F.
      • Kolluri S.K.
      • Stebbins J.
      • Reed J.C.
      • Dawson M.I.
      • Zhang X.K.
      Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt.
      ). These examples show the ligand-induced phosphorylation of nuclear receptors.

      Nongenomic signals

      CAR transduces stress signals by directly interacting with signal molecules, such as GADD45B. TCPOPOP treatments repressed transforming growth factor-α–induced death of mouse primary hepatocytes. This cell death was not observed with primary hepatocytes of CAR KO or GADD45B KO mice (
      • Yamamoto Y.
      • Moore R.
      • Flavell R.A.
      • Lu B.
      • Negishi M.
      Nuclear receptor CAR represses TNFα-induced cell death by interacting with the anti-apoptotic GADD45B.
      ). CAR directly interacted with GADD45B, augmenting MKK6 to phosphorylate c-Jun and apoptosis. PB treatment attenuated nuclear levels of active (phosphorylated) p38 MAPK, a tumor suppressor in mouse livers (
      • Hori T.
      • Moore R.
      • Negishi M.
      p38 MAP kinase links CAR activation and inactivation in the nucleus via phosphorylation at threonine 38.
      ). GADD45B scaffolds MKK6 to phosphorylate p38 MAPK, stimulating its antiproliferation signal. CAR repressed this signal by directly binding GADD45B and dissociated a p38-MKK6 complex, attenuating apoptosis. PB induced male-predominant hepatocyte proliferation in GADD45B WT but not GADD45B KO mice (
      • Hori T.
      • Saito K.
      • Moore R.
      • Flake G.P.
      • Negishi M.
      Nuclear receptor CAR suppresses GADD45B-p38 MAPK signaling to promote phenobarbital-induced proliferation in mouse liver.
      ). GADD45B may play a role as a promotion signal for PB to develop hepatocellular carcinoma.

      Scope beyond PB

      CAR ligands like CITCO dissociate ERK1/2 from phosphorylated CAR to induce dephosphorylation of Thr-38 for direct activation. This suggests that, in principle, all CAR activators utilize, at least in part, the same mechanism by which PB activates CAR. There are many xenobiotics and therapeutic drugs that do not activate CAR in transformed cell–based reporter assays but activate CAR in primary cells, rodents, and/or humans in vivo, as indirect CAR activators. Although the concept of indirect activation is widely accepted (
      • Li L.
      • Welch M.A.
      • Li Z.
      • Mackowiak B.
      • Heyward S.
      • Swaan P.W.
      • Wang H.
      Mechanistic insights of phenobarbital-mediated activation of human but not mouse pregnane X receptor.
      ), no experimental groups were available to investigate the mechanism of this indirect activation. The PB-EGFR-ERK1/2-Thr-58 signal provided us with the first molecular basis to study the indirect activation mechanism (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ,
      • Mutoh S.
      • Sobhany M.
      • Moore R.
      • Perera L.
      • Pedersen L.
      • Sueyoshi T.
      • Negishi M.
      Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.
      ). An increasing number of CAR activators have been associated with the function of EGFR. EGFR inhibitors such erlotinib and alflutinib activated CAR (
      • Shizu R.
      • Osabe M.
      • Perera L.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.
      ,
      • Liu X.Y.
      • Guo Z.T.
      • Chen Z.D.
      • Zhang Y.F.
      • Zhou J.L.
      • Jiang Y.
      • Zhao Q.Y.
      • Diao X.X.
      • Zhong D.F.
      Alflutinib (AST2818), primarily metabolized by CYP3A4, is a potent CYP3A4 inducer.
      ). These include (but are limited to) endocrine disruptors, pesticides, herbicides, natural products, and therapeutic drugs (
      • Hardesty J.E.
      • Al-Eryani L.
      • Wahlang B.
      • Falkner K.C.
      • Shi H.
      • Jin J.
      • Vivace B.J.
      • Ceresa B.P.
      • Prough R.A.
      • Cave M.C.
      Epidermal growth factor receptor signaling disruption by endocrine and metabolic disrupting chemicals.
      ,
      • Hardesty J.E.
      • Wahlang B.
      • Falkner K.C.
      • Clair H.B.
      • Clark B.J.
      • Ceresa B.P.
      • Prough R.A.
      • Cave M.C.
      Polychlorinated biphenyls disrupt hepatic epidermal growth factor receptor signaling.
      ,
      • Carazo Fernández A.
      • Smutny T.
      • Hyrsová L.
      • Berka K.
      • Pavek P.
      Chrysin, baicalein and galangin are indirect activators of the human constitutive androstane receptor (CAR).
      ). A cell-based assay system was used to screen indirect CAR activators and identified a number of chemicals (
      • Pinne M.
      • Ponce E.
      • Raucy J.L.
      Transactivation assays that identify indirect and direct activators of human pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3).
      ). This system can also be used to screen indirect PXR activators. A human primary hepatocyte–based system was also developed to identify CAR-activating chemicals, including indirect activators (
      • Li L.
      • Welch M.A.
      • Li Z.
      • Mackowiak B.
      • Heyward S.
      • Swaan P.W.
      • Wang H.
      Mechanistic insights of phenobarbital-mediated activation of human but not mouse pregnane X receptor.
      ). In addition, repression or activation of EGFR affected not only the functions of CAR, but also those of PXR (
      • de Boussac H.
      • Gondeau C.
      • Briolotti P.
      • Duret C.
      • Treindl F.
      • Römer M.
      • Fabre J.M.
      • Herrero A.
      • Ramos J.
      • Maurel P.
      • Templin M.
      • Gerbal-Chaloin S.
      • Daujat-Chavanieu M.
      Epidermal growth factor represses constitutive androstane receptor expression in primary human hepatocytes and favors regulation by pregnane X receptor.
      ), possibly causing drug-drug interactions. Since they were published, the two original papers (
      • Mutoh S.
      • Osabe M.
      • Inoue K.
      • Moore R.
      • Pedersen L.
      • Perera L.
      • Rebolloso Y.
      • Sueyoshi T.
      • Negishi M.
      Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
      ,
      • Mutoh S.
      • Sobhany M.
      • Moore R.
      • Perera L.
      • Pedersen L.
      • Sueyoshi T.
      • Negishi M.
      Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.
      ) have been cited over 210 times, suggesting the degree of their impact on the research field of xenobiotic metabolism.

      Biology of conserved phosphorylation

      Sporadic studies of Thr-38–corresponding residues first suggested that these residues are conserved as a phosphorylation motif through which nuclear receptors can be regulated (
      • Gineste R.
      • Sirvent A.
      • Paumelle R.
      • Helleboid S.
      • Aquilina A.
      • Darteil R.
      • Hum D.W.
      • Fruchart J.C.
      • Staels B.
      Phosphorylation of farnesoid X receptor by protein kinase C promotes its transcriptional activity.
      ,
      • Sun K.
      • Montana V.
      • Chellappa K.
      • Brelivet Y.
      • Moras D.
      • Maeda Y.
      • Parpura V.
      • Paschal B.M.
      • Sladek F.M.
      Phosphorylation of a conserved serine in the deoxyribonucleic acid binding domain of nuclear receptors alters intracellular localization.
      ,
      • Hsieh J.C.
      • Jurutka P.W.
      • Galligan M.A.
      • Terpening C.M.
      • Haussler C.A.
      • Samuels D.S.
      • Shimizu Y.
      • Shimizu N.
      • Haussler M.R.
      Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function.
      ). In fact, this motif is conserved in 41 of a total of 46 human nuclear receptors and their corresponding mouse nuclear receptors (
      • Negishi M.
      Phenobarbital meets phosphorylation of nuclear receptors.
      ). This conservation suggests that the motif is one of the early evolved phosphorylation motifs in nuclear receptors and is expected to have biological functions. Two hypotheses as to how this phosphorylation regulates nuclear receptors have been examined. The first one is whether phosphorylation confers a novel function over nonphosphorylated nuclear receptors. The second hypothesis is the possibility that phosphorylation can be utilized as communication language for nuclear receptors to integrate their functions.

      Novel gains by phosphorylation

      ERα at residue Ser-216 was the second nuclear receptor confirmed to be phosphorylated endogenously in tissues in vivo after CAR. Phosphorylated ERα was expressed in neutrophils infiltrating into the mouse uterus from the bloodstream (
      • Shindo S.
      • Moore R.
      • Flake G.
      • Negishi M.
      Serine 216 phosphorylation of estrogen receptor α in neutrophils: migration and infiltration into the mouse uterus.
      ). Phosphorylated ERα was also found in mouse microglia, the resident macrophage in the brain. To examine the in vivo role of this phosphorylation, a phosphorylation-blocking ERα S216A KI mouse was generated. No apparent defect in the reproductive function of KI mice was observed. Inflammation and apoptosis were stimulated in the brain and/or isolated microglia from ERα KI mice. This phosphorylation conferred the anti-inflammatory and anti-apoptotic capability to ERα (
      • Shindo S.
      • Chen S.-H.
      • Gotoh S.
      • Yokobori K.
      • Hu H.
      • Ray M.
      • Moore R.
      • Nagata K.
      • Martinez J.
      • Hong J.-S.
      • Negishi M.
      Estrogen receptor α phosphorylated at Ser216 confers inflammatory function to mouse microglia.
      ).
      RXRα was found to be phosphorylated at Thr-167 in mouse adipose tissues in response to fasting (
      • Sueyoshi T.
      • Sakuma T.
      • Shindo S.
      • Fashe M.
      • Kanayama T.
      • Ray M.
      • Moore R.
      • Negishi M.
      A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice.
      ). Fasting increased blood glucose levels in phosphorylation-blocking RXRα T167A KI over WT mice. In fasting mutant mice, gene expression in lipid synthesis in white adipose tissue was attenuated. These observations have widened what was found with Thr-38 of CAR to the other nuclear receptors and provided the experimental basis for further investigations.

      Communication

      The CAR molecule has two different dimerization interfaces: A and B (Fig. 5B). Both androgen and glucocorticoid receptors utilized Surface A to form their homodimers (
      • Nadal M.
      • Prekovic S.
      • Gallastegui N.
      • Helsen C.
      • Abella M.
      • Zielinska K.
      • Gay M.
      • Vilaseca M.
      • Taulès M.
      • Houtsmuller A.B.
      • van Royen M.E.
      • Claessens F.
      • Fuentes-Prior P.
      • Estébanez-Perpiñá E.
      Structure of the homodimeric androgen receptor ligand-binding domain.
      ,
      • Shizu R.
      • Yokobori K.
      • Perera L.
      • Pedersen L.
      • Negishi M.
      Ligand induced dissociation of the AR homodimer precedes AR monomer translocation to the nucleus.
      ,
      • Bledsoe R.K.
      • Montana V.G.
      • Stanley T.B.
      • Delves C.J.
      • Apolito C.J.
      • McKee D.D.
      • Consler T.G.
      • Parks D.J.
      • Stewart E.L.
      • Willson T.M.
      • Lambert M.H.
      • Moore J.T.
      • Pearce K.H.
      • Xu H.E.
      Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition.
      ), suggesting that Surface A can be a general dimer interface for nuclear receptors to interact with each other. A superimposed model structure reveals that ERα homodimer and CAR-RXRα heterodimer form an (ERα)2-CAR-RXRα tetramer through such interactions (Fig. 5C). Therefore, two different dimer interfaces can provide nuclear receptors with the structural basis for functional integration and diversification. Moreover, phosphorylation of the conserved motif may provide nuclear receptors with signals to regulate their interactions. To this end, experimental examinations began demonstrating that PB activates CAR and integrates additional nuclear receptors, such as ERα and RORα, into hepatic gene regulation (
      • Fashe M.
      • Hashiguchi T.
      • Negishi M.
      • Sueyoshi T.
      Ser100-phosphorylated RORα orchestrates CAR and HNF4α to form active chromatin complex in response to phenobarbital to regulate induction of CYP2B6.
      ,
      • Fashe M.
      • Hashiguchi T.
      • Yi M.
      • Moore R.
      • Negishi M.
      Phenobarbital-induced phosphorylation converts nuclear receptor RORα from a repressor to an activator of the estrogen sulfotransferase gene Sult1e1 in mouse livers.
      ,
      • Yi M.
      • Fashe M.
      • Arakawa S.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.
      ).

      CAR-RORα

      PB activated the Sult1e1 gene that encodes estrogen sulfotransferase (SULT1E1), which sulfates estrogens to metabolically inactivate them in mouse livers. For this activation, CAR integrated RORα activity through phosphorylation. RORα repressed the Sult1e1 gene, as indicated by an increase of SULT1E1 mRNA in RORα KO mice, as well as a proximal promoter (−168/+67) of this gene in cell-based reporter assays. This promoter contains an RORα–binding motif (−113/−102). On the other hand, RORα S100A, a phosphorylation-blocking mutant, activated this promoter. Subsequent examination found RORα on the promoter in the liver of CAR WT and CAR KO mice prior to PB treatment in ChIP assays. CAR was not involved in this repression by RORα. After PB treatment, RORα remained on the promoter but was phosphorylated at Ser-100 in only CAR WT mice where the promoter was activated. Whereas CAR did not regulate RORα repressing the promoter, CAR signaled RORα phosphorylated at Ser-100, converting RORα from a transcriptional repressor to an activator in response to PB. Ser-100 phosphorylation strengthened RORα interaction with CAR. It appears that this binding with the nonphosphorylated RORα is to initiate phosphorylation of Ser-100.

      CAR-ERα-RORα

      It is reasonable to expect that estrogen and/or ERα are involved in the regulation of the Sult1e1 gene, because SULT1E1 is an enzyme that metabolically maintains estrogen homeostasis. CAR, ERα, and RORα sequentially interacted via phosphorylation at their conserved motifs, which may be the molecular mechanism integrating xenobiotic and hormonal signals (Fig. 6). It was first found that a PB-induced increase of SULT1E1 mRNA was severely attenuated in the liver of ERα S216A KI mice (
      • Yi M.
      • Fashe M.
      • Arakawa S.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.
      ). This observation indicated that phosphorylated ERα is essential for CAR to activate the Sult1e1 gene. RORα binding to the promoter was examined by ChIP assays using nuclear extracts prepared from livers of ERα Ser-216 KI and CAR KO and their WT mice. Results showed that CAR recruited ERα to the promoter after PB treatment, and subsequently, ERα was phosphorylated at Ser-216. CAR and a phosphorylation-mimicking ERα S216D, but not ERα S216A mutant, effectively co-precipitated, showing that phosphorylation strengthened ERα interaction with CAR. Because ERα is known to form its homodimer through Surface B, Surface A is available for the interaction with the CAR of the CAR-RXRα heterodimer (Fig. 5B) (
      • Yi M.
      • Fashe M.
      • Arakawa S.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.
      ). CAR mediated the PB signal to ERα by eliciting Ser-216 phosphorylation. Phosphorylation strengthened CAR's interaction with ERα to form a CAR-phosphorylated ERα complex promoting transcription, when the Sult1e1 gene was activated.
      Figure thumbnail gr6
      Figure 6Nuclear receptor integration via conserved phosphorylation. Shown is a schematic representation of the PB-induced process utilized to integrate CAR, ERα, and RORα on the Sult1e1 promoter. ERα homodimerizes and CAR heterodimerizes with RXRα through Surface B, allowing CAR to interact with ERα through Surface A. Having two Surfaces A, the phosphorylated ERα homodimer can interact with RORα, sandwiched between RORα and the CAR-RXRα heterodimer, and forms a hexamer. This interaction with phosphorylated ERα regulates RORα phosphorylation, converting RORα from a transcriptional repressor to an activator.
      Unlike when phosphorylated RORα activated the Sult1e1 promoter in cell-based reporter assays, ERα did not affect the activity of the same promoter. What is the role of phosphorylated ERα in this gene regulation? With the information that RORα and ERα bound the same promoter after PB treatment, their interaction was examined in ERα S216A KI mice using ChIP assays. RORα was found on the promoter and remained not phosphorylated even after PB treatment, indicating that phosphorylated ERα is required for RORα to be phosphorylated. One scenario may be that phosphorylated ERα mediates PB-induced phosphorylation of RORα, interacting with and conferring to phosphorylated RORα the ability to activate the Sult1e1 promoter, converting RORα from a transcriptional repressor to activator. If so, ERα may be a sensor but not a transcription factor. It is highly likely, but not yet confirmed, that estrogen senses ERα to feedback its metabolism. CAR senses therapeutics and growth signals, whereas ERα may sense an estrogen signal. Together, multiple signals can be integrated through these nuclear receptors using phosphorylation of the conserved motif as a communication language. SULT1E1 is up-regulated in the liver of diabetic mice, both males and females (
      • Yi M.
      • Fashe M.
      • Arakawa S.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.
      ,
      • Song W.C.
      • Moore R.
      • McLachlan J.A.
      • Negishi M.
      Molecular characterization of a testis-specific estrogen sulfotransferase and aberrant liver expression in obese and diabetogenic C57BL/KsJ-db/db mice.
      ). This same mechanism that integrates CAR and ERα was utilized to activate the Sult1e1 gene in diabetic livers.
      CAR initiated a chain reaction to phosphorylate ERα and then RORα on the promoter. Because this reaction occurs on chromatin, protein kinases associated with chromatin can be candidates that catalyze this phosphorylation, such as VRK1 and casein kinases. Identification and characterization of protein kinases should give insights into understanding the molecular mechanism and its regulation of how phosphorylation integrates nuclear receptors transducing xenobiotic signaling in the future.

      The CYP2B6 gene

      The human CYP2B gene's promoter contains the distal PBREM (−1733/−1683), where CAR binds, and the proximal okadaic acid response element (OAREKI, −268/−217) that provides the hepatocyte-enriched nuclear factor 4α (HNF4α)–binding site. Upon PB activation, CAR binds PBREM, looping it toward the OARE, enabling CAR to interact with HNF4α to promote transcription (
      • Inoue K.
      • Negishi M.
      Nuclear receptor CAR requires early growth response 1 to activate the human cytochrome P450 2B6 gene.
      ). In addition to PBREM and OARE, there is a RORα-binding motif (RORE, −662/−649) in a region between PBREM and OARE in the promoter. In human primary hepatocytes, RORα constitutively resides on the RORE in its phosphorylated Ser-100 form (
      • Fashe M.
      • Hashiguchi T.
      • Negishi M.
      • Sueyoshi T.
      Ser100-phosphorylated RORα orchestrates CAR and HNF4α to form active chromatin complex in response to phenobarbital to regulate induction of CYP2B6.
      ). Upon CAR binding to PBREM, phosphorylated RORα interacted with the CAR, bringing it close to and interacting with HNF4α, synergistically activating the CYP2B6 promoter. Differing by target gene and signaling, phosphorylation of RORα appears to be a common signal that activates both Sult1e1 and CYP2B6 genes.

      LBD phosphorylation

      PXR conserves this phosphorylation motif at Ser-350. Ser-350 was investigated as a target for PXR to regulate CYP3A4 expression in human hepatoma–derived Huh-7 cells (
      • Sivertsson L.
      • Edebert I.
      • Palmertz M.P.
      • Ingelman-Sundberg M.
      • Neve E.P.
      Induced CYP3A4 expression in confluent Huh7 hepatoma cells as a result of decreased cell proliferation and subsequent pregnane X receptor activation.
      ). CYP3A4 was spontaneously induced in confluent Huh-7 cells (Huh-7 cells were grown fully to cover a Petri dish), and this induction correlated with the repression of CDK2. The hypothesis raised was that CDK2 phosphorylated Ser-350 to attenuate PXR to activate the CYP3A4 gene; therefore, CDK2 repression resulted in Ser-350 dephosphorylation and restored PXR's ability to activate the gene in confluent Huh-7 cells. A phosphorylation-mimicking PXR S350D mutant was unable to heterocomplex with RXRα and activate the CYP3A4 promoter (
      • Gotoh S.
      • Miyauchi Y.
      • Moore R.
      • Negishi M.
      Glucose elicits serine/threonine kinase VRK1 to phosphorylate nuclear pregnane X receptor as a novel hepatic gluconeogenic signal.
      ,
      • Bulutoglu B.
      • Mert S.
      • Rey-Bedón C.
      • Deng S.L.
      • Yarmush M.L.
      • Usta O.B.
      Rapid maturation of the hepatic cell line Huh7 via CDK inhibition for PXR dependent CYP450 metabolism and induction.
      ). Consistent with this hypothesis, treatment with a CDK inhibitor up-regulated CYP3A4 expression (
      • Bulutoglu B.
      • Mert S.
      • Rey-Bedón C.
      • Deng S.L.
      • Yarmush M.L.
      • Usta O.B.
      Rapid maturation of the hepatic cell line Huh7 via CDK inhibition for PXR dependent CYP450 metabolism and induction.
      ). Moreover, a phosphorylation-mimicking single mutation of Ser-350 to Asp disabled PXR induction of drug-metabolizing enzymes in mouse livers (
      • Wang Y.M.
      • Chai S.C.
      • Lin W.
      • Chai X.
      • Elias A.
      • Wu J.
      • Ong S.S.
      • Pondugula S.R.
      • Beard J.A.
      • Schuetz E.G.
      • Zeng S.
      • Xie W.
      • Chen T.
      Serine 350 of human pregnane X receptor is crucial for its heterodimerization with retinoid X receptor alpha and transactivation of target genes in vitro in vivo.
      ). However, whereas CDK2 mediated this confluence-response signal to regulate PXR, a direct phosphorylation of Ser-350 by CDK2 has not been demonstrated. Instead, vaccinia-related kinase 1 (VRK1) is now known to phosphorylate PXR at Ser-350 in HepG2 cells cultured in low-glucose medium (
      • Gotoh S.
      • Miyauchi Y.
      • Moore R.
      • Negishi M.
      Glucose elicits serine/threonine kinase VRK1 to phosphorylate nuclear pregnane X receptor as a novel hepatic gluconeogenic signal.
      ). As to the mechanism of this phosphorylation (Fig. 7A), CDK2 dephosphorylated at Thr-14 represses VRK1's ability to phosphorylate PXR at Ser-350 in HepG2 cells cultured in high-glucose medium. In response to low glucose, CDK2 was phosphorylated and lost its capability to repress VRK1's phosphorylation of PXR (Fig. 7A). As a result, PXR became phosphorylated in response to low glucose and activated gluconeogenic genes in HepG2 cells (
      • Gotoh S.
      • Miyauchi Y.
      • Moore R.
      • Negishi M.
      Glucose elicits serine/threonine kinase VRK1 to phosphorylate nuclear pregnane X receptor as a novel hepatic gluconeogenic signal.
      ). Phosphorylated PXR scaffolded protein phosphatase 2Cα to dephosphorylate serum and glucocorticoid–regulated kinase 2 (SGK2), thereby disabling SGK2 to repress these genes. Phosphorylation repressed PXR to directly bind promoters as a transcription factor, converting PXR to a cell signal transducer (Fig. 7).
      Figure thumbnail gr7
      Figure 7Function of conserved phosphorylation motif in the LBD. A, PXR phosphorylated at Ser-350 is a low-glucose response signal. This model is depicted from previous work (
      • Gotoh S.
      • Miyauchi Y.
      • Moore R.
      • Negishi M.
      Glucose elicits serine/threonine kinase VRK1 to phosphorylate nuclear pregnane X receptor as a novel hepatic gluconeogenic signal.
      ) and modified. CDK2 regulates phosphorylation of PXR at Ser-350 by VRK1 in response to low glucose. Phosphorylated PXR binds PP2Cα that dephosphorylates SGK2, regulating genes. Similarly, ligand-activated PXR heterodimerizes with RXR to regulate dephosphorylation of SGK2. B, intramolecular interactions of Ser-673 in GR molecule. Left, cartoon diagram of the crystal structure (PDB code 1M2Z) of hGR (pink) with bound dexamethasone (cyan). The C-terminal region is colored gray, and the Ser-673 side-chain hydroxyl group is shown in red. Right, Ser-673 forming a side-chain hydrogen bond with Leu-670 and a backbone hydrogen bond with Leu-772.
      VRK1 has long been known to inherit high autophosphorylation activity, whereas residues that are phosphorylated have only recently been determined (
      • Yokobori K.
      • Miyauchi Y.
      • Williams J.G.
      • Negishi M.
      Phosphorylation of vaccinia-related kinase 1 at threonine 386 transduces glucose stress signal in human liver cells.
      ). Among them, Thr-386 was phosphorylated in response to low glucose in Huh-7 cells, and this phosphorylation enabled VRK1 to phosphorylate c-Jun and p53. Therefore, Thr-386 phosphorylation is a general signal for VRK1 to regulate various stress-induced factors. In addition to a glucose signal, VRK1 was phosphorylated at Ser-376 in response to UV exposures in Huh-7 cells, suggesting that VRK1 utilized different motifs to transduce different cell stresses and damages.
      The NR3C steroid hormone receptors conserve this phosphorylation motif in LBD (e.g. GR at Ser-673 and AR at Ser-815) (Fig. 3). In fact, AR was found to be phosphorylated at Ser-815 in mouse prostates in vivo (
      • Yokobori K.
      • Negishi M.
      Androgen receptor phophorylated at serine 815 in mouse and human prostates.
      ). AR is known to form its homodimer in the cytoplasm and to dissociate to a monomer in response to androgens, translocating into the nucleus in prostate cancer–derived PC-3 cells (
      • Shizu R.
      • Yokobori K.
      • Perera L.
      • Pedersen L.
      • Negishi M.
      Ligand induced dissociation of the AR homodimer precedes AR monomer translocation to the nucleus.
      ). Thus, phosphorylation appeared to confer upon AR novel functions over nonphosphorylated AR. One feature of this phosphorylation motif is its position in the structure of steroid hormone receptors. Residues such as Ser-673 and Ser-815 interact with a random coil called the C-terminal extension after the activation function 2 (AF2) in helix 12 (Fig. 7B). For this residue to be phosphorylated, either the loop in which it resides or this random coil should move away, exposing the residue for phosphorylation. It may be that hormone binding alters a configuration of the helix 12 to open the residue for phosphorylation, or it can be that the loop moves by other means of cellular stimuli in a hormone-independent manner. Although the molecular mechanism remains to be determined in future investigations, this conserved phosphorylation motif should provide steroid hormone receptors with the molecular basis to diversify their functional capability and may enable them to communicate among themselves to integrate hormone actions in physiology as well as pathophysiology.

      Diseases

      Phosphorylation enabled PXR to transduce low-glucose signal to induce hepatic gluconeogenesis. CAR underwent phosphorylation-dephosphorylation to elicit signals for disease developments. We begin this section by summarizing current knowledge regarding CAR- and/or PXR-mediated disease developments caused by xenobiotic exposures (Fig. 1). Subsequent discussion implicates phosphorylation in these functions. Moreover, phosphorylation is debated as a tool for CAR and additional nuclear receptors to integrate their signals in disease developments.

      Diabetes

      Type 2 diabetes is a chronic disease characterized by uncontrolled hyperglycemia caused by insulin resistance. Clinical studies indicated that CAR and PXR play opposite roles in the regulation of blood glucose levels. PB treatment decreased blood glucose levels and improved insulin sensitivity in epileptic patients (
      • Lahtela J.T.
      • Arranto A.J.
      • Sotaniemi E.A.
      Enzyme inducers improve insulin sensitivity in non-insulin-dependent diabetic subjects.
      ). CAR aberration worsened hepatic glucose tolerance in Type 2 diabetic (ob/ob) mice or high-fat diet–induced obese mice following treatment with mouse CAR ligand TCPOBOP (
      • Dong B.
      • Saha P.K.
      • Huang W.
      • Chen W.
      • Abu-Elheiga L.A.
      • Wakil S.J.
      • Stevens R.D.
      • Ilkayeva O.
      • Newgard C.B.
      • Chan L.
      • Moore D.D.
      Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease.
      ,
      • Gao J.
      • He J.
      • Zhai Y.
      • Wada T.
      • Xie W.
      The constitutive androstane receptor is an anti-obesity nuclear receptor that improves insulin sensitivity.
      ). CAR repressed hepatic expression of gluconeogenic enzymes (
      • Ueda A.
      • Hamadeh H.K.
      • Webb H.K.
      • Yamamoto Y.
      • Sueyoshi T.
      • Afshari C.A.
      • Lehmann J.M.
      • Negishi M.
      Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital.
      ,
      • Yasujima T.
      • Saito K.
      • Moore R.
      • Negishi M.
      Phenobarbital and insulin reciprocate activation of the nuclear receptor constitutive androstane receptor through the insulin receptor.
      ,
      • Kodama S.
      • Koike C.
      • Negishi M.
      • Yamamoto Y.
      Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-metabolizing and gluconeogenic enzymes.
      ). A study with HepG2 cells showed that CAR directly interacted with and prevented the insulin response transcription factor FOXO1 from activating the insulin response sequence (IRS) of the promoter (
      • Kodama S.
      • Koike C.
      • Negishi M.
      • Yamamoto Y.
      Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-metabolizing and gluconeogenic enzymes.
      ). Whereas insulin signaled FOXO1 phosphorylation, dissociating it from IRS and repressing gluconeogenic genes (
      • Guo S.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence.
      ), direct FOXO1 binding may be a molecular mechanism by which CAR repressed gluconeogenesis in the liver. CAR also repressed gluconeogenic genes by stimulating a Cullin1 E3 ligase–mediated ubiquitin degradation of PGC1α (
      • Gao J.
      • Yan J.
      • Xu M.
      • Ren S.
      • Xie W.
      CAR suppresses hepatic gluconeogenesis by facilitating the ubiquitination and degradation of PGC1α.
      ).
      Treatment with rifampicin or statins, human PXR activators, increased blood glucose levels during oral glucose tolerance tests in tuberculosis patients and in healthy volunteers (
      • Rysä J.
      • Buler M.
      • Savolainen M.J.
      • Ruskoaho H.
      • Hakkola J.
      • Hukkanen J.
      Pregnane X receptor agonists impair postprandial glucose tolerance.
      ,
      • Sukhija R.
      • Prayaga S.
      • Marashdeh M.
      • Bursac Z.
      • Kakar P.
      • Bansal D.
      • Sachdeva R.
      • Kesan S.H.
      • Mehta J.L.
      Effect of statins on fasting plasma glucose in diabetic and nondiabetic patients.
      ). Recent meta-analysis confirmed that new-onset diabetes risk was higher in statin users than in nonusers (
      • Casula M.
      • Mozzanica F.
      • Scotti L.
      • Tragni E.
      • Pirillo A.
      • Corrao G.
      • Catapano A.L.
      Statin use and risk of new-onset diabetes: a meta-analysis of observational studies.
      ). Because of this, the United States Food and Drug Administration (FDA) warned that statins increase the risk of developing diabetes (FDA Drug Safety Communication, Important Safety Label Changes to Cholesterol-lowering Statin Drugs; https://www.fda.gov/drugs/drug-safety-and-availability/fda-drug-safety-communication-important-safety-label-changes-cholesterol-lowering-statin-drugs). Statin-activated PXR activated gluconeogenic genes by derepressing the SGK2-mediated repression of the promoter, which includes IRS in HepG2 cells (
      • Gotoh S.
      • Negishi M.
      Serum- and glucocorticoid-regulated kinase 2 determines drug-activated pregnane X receptor to induce gluconeogenesis in human liver cells.
      ). This may be a molecular basis for PXR to exert its side effect. Phosphorylated PXR also utilized the SGK2-mediated mechanism to activate the promoter in response to low glucose in HepG2 cells (
      • Gotoh S.
      • Negishi M.
      Statin-activated nuclear receptor PXR promotes SGK2 dephosphorylation by scaffolding PP2C to induce hepatic gluconeogenesis.
      ). However, whether this phosphorylation signal regulates gluconeogenesis in the liver remains to be investigated in the future. In our previous work, PXR, as observed with CAR, interacted with FOXO1 to repress the IRS activity in HepG2 cells (
      • Kodama S.
      • Koike C.
      • Negishi M.
      • Yamamoto Y.
      Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-metabolizing and gluconeogenic enzymes.
      ). Although this repression is apparently inconsistent with the fact that the genes were activated by drug treatments, the possibility remains that the liver utilizes this PXR-FOXO1 interaction to control gluconeogenesis in physiology and/or pathophysiology. In addition, PXR activators translocated glucose transporter 2 (GLUT2) from the plasma membrane to the cytoplasm in the liver, disrupting their glucose-sensing capability (
      • Ling Z.
      • Shu N.
      • Xu P.
      • Wang F.
      • Zhong Z.
      • Sun B.
      • Li F.
      • Zhang M.
      • Zhao K.
      • Tang X.
      • Wang Z.
      • Zhu L.
      • Liu L.
      • Liu X.
      Involvement of pregnane X receptor in the impaired glucose utilization induced by atorvastatin in hepatocytes.
      ,
      • Hassani-Nezhad-Gashti F.
      • Rysä J.
      • Kummu O.
      • Näpänkangas J.
      • Buler M.
      • Karpale M.
      • Hukkanen J.
      • Hakkola J.
      Activation of nuclear receptor PXR impairs glucose tolerance and dysregulates GLUT2 expression and subcellular localization in liver.
      ).
      Phosphorylated RXRα regulated a fasting-induced triglyceride homeostasis in mouse adipose tissues (
      • Sueyoshi T.
      • Sakuma T.
      • Shindo S.
      • Fashe M.
      • Kanayama T.
      • Ray M.
      • Moore R.
      • Negishi M.
      A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice.
      ). Blood glucose levels in fasted condition were elevated in phosphorylation-blocked RXRα KI mice (
      • Sueyoshi T.
      • Sakuma T.
      • Shindo S.
      • Fashe M.
      • Kanayama T.
      • Ray M.
      • Moore R.
      • Negishi M.
      A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice.
      ). However, the role of this phosphorylation in diabetes development either in mice or humans remains to be investigated.
      Type 1 diabetes can be mimicked in AKITA mice with mutations of the insulin gene, nonobese diabetic mice with defective insulin secretion, or streptozotocin-treated mice. In the liver of AKITA mice, CAR spontaneously accumulated in the nucleus, and SULT1E1 (also CYP2B10) mRNA was induced. Moreover, this induction was not observed in AKITA-CAR KO mice (
      • Yi M.
      • Fashe M.
      • Arakawa S.
      • Moore R.
      • Sueyoshi T.
      • Negishi M.
      Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.
      ). Hepatic levels of CYP2B10 and CYP3A11 mRNAs were induced in nonobese diabetic and streptozotocin-treated mice, and CAR, but not PXR, regulated this induction (
      • Dong B.
      • Qatanani M.
      • Moore D.D.
      Constitutive androstane receptor mediates the induction of drug metabolism in mouse models of type 1 diabetes.
      ). Thus, CAR regulated these Cyp genes in Type 1 diabetic mice. However, whether CAR is directly involved in diabetes remains to be investigated.

      Hepatic steatosis

      Hepatic steatosis is an intrahepatic fat (triglyceride) accumulation in at least 5% of hepatocytes containing lipid vacuoles and is a risk factor for further advancing diseases, such as nonalcoholic steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Although no clinical observation has been reported, CAR prevented liver from developing steatosis in mice. TCPOBOP treatments improved fatty liver by decreasing hepatic triglycerides in ob/ob mice and in mice fed with a high-fat diet, which is explained by the CAR-dependent repression of lipogenesis; sterol regulatory element binding protein-1c (SREBP-1c) and its downstream targets fatty acid synthase and stearoyl-CoA desaturase 1 (
      • Dong B.
      • Saha P.K.
      • Huang W.
      • Chen W.
      • Abu-Elheiga L.A.
      • Wakil S.J.
      • Stevens R.D.
      • Ilkayeva O.
      • Newgard C.B.
      • Chan L.
      • Moore D.D.
      Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease.
      ,
      • Gao J.
      • He J.
      • Zhai Y.
      • Wada T.
      • Xie W.
      The constitutive androstane receptor is an anti-obesity nuclear receptor that improves insulin sensitivity.
      ). TCPOBOP attenuated liver cell apoptosis and inflammation in the nonalcoholic steatohepatitis model induced by a methionine- and choline-deficient diet (
      • Baskin-Bey E.S.
      • Anan A.
      • Isomoto H.
      • Bronk S.F.
      • Gores G.J.
      Constitutive androstane receptor agonist, TCPOBOP, attenuates steatohepatitis in the methionine choline-deficient diet-fed mouse.
      ), but it worsened hepatic injury and fibrosis by elevation of lipid peroxidation (
      • Yamazaki Y.
      • Kakizaki S.
      • Horiguchi N.
      • Sohara N.
      • Sato K.
      • Takagi H.
      • Mori M.
      • Negishi M.
      The role of the nuclear receptor constitutive androstane receptor in the pathogenesis of non-alcoholic steatohepatitis.
      ).
      PXR activation potentiated steatosis in mice, repressing β-oxidation through FOXA2 (
      • Nakamura K.
      • Moore R.
      • Negishi M.
      • Sueyoshi T.
      Nuclear pregnane X receptor cross-talk with FoxA2 to mediate drug-induced regulation of lipid metabolism in fasting mouse liver.
      ). Treatment with the triazole propiconazole or tebuconazole led to accumulated triglycerides in HepaRG cells (
      • Knebel C.
      • Buhrke T.
      • Süssmuth R.
      • Lampen A.
      • Marx-Stoelting P.
      • Braeuning A.
      Pregnane X receptor mediates steatotic effects of propiconazole and tebuconazole in human liver cell lines.
      ), whereas treatment with efavirenz, a widely prescribed antiretroviral drug, induced hepatic steatosis in PXR-humanized mice as well as in human primary hepatocytes (
      • Gwag T.
      • Meng Z.
      • Sui Y.
      • Helsley R.N.
      • Park S.H.
      • Wang S.
      • Greenberg R.N.
      • Zhou C.
      Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis.
      ). PXR activated by rifampicin induced the expression of the transporter SLC13A5 gene in human primary hepatocytes (
      • Li L.
      • Li H.
      • Garzel B.
      • Yang H.
      • Sueyoshi T.
      • Li Q.
      • Shu Y.
      • Zhang J.
      • Hu B.
      • Heyward S.
      • Moeller T.
      • Xie W.
      • Negishi M.
      • Wang H.
      SLC13A5 is a novel transcriptional target of the pregnane X receptor and sensitizes drug-induced steatosis in human liver.
      ). Because SLC13A5 functions to uptake citrate, a precursor in the biosynthesis of fatty acids and cholesterol, an increased SLC13A5 expression could contribute to lipid accumulations (
      • Li L.
      • Li H.
      • Garzel B.
      • Yang H.
      • Sueyoshi T.
      • Li Q.
      • Shu Y.
      • Zhang J.
      • Hu B.
      • Heyward S.
      • Moeller T.
      • Xie W.
      • Negishi M.
      • Wang H.
      SLC13A5 is a novel transcriptional target of the pregnane X receptor and sensitizes drug-induced steatosis in human liver.
      ).

      HCC

      Hepatocellular carcinoma is the most common type of primary liver cancer and has a high rate of death worldwide. HCC development is a multistage process involving initiation by genotoxic carcinogens and promotion by nongenotoxic carcinogens toward spontaneous progression. PB or methionine- and choline-deficient diet promotes HCC development initiated by genotoxic carcinogen (i.e. nitrosamines or diethylnitrosamine) in a CAR-dependent manner in rodents (
      • Yamamoto Y.
      • Moore R.
      • Goldsworthy T.L.
      • Negishi M.
      • Maronpot R.R.
      The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice.
      ,
      • Takizawa D.
      • Kakizaki S.
      • Horiguchi N.
      • Yamazaki Y.
      • Tojima H.
      • Mori M.
      Constitutive active/androstane receptor promotes hepatocarcinogenesis in a mouse model of non-alcoholic steatohepatitis.
      ). CAR directly interacts with GADD45B, which regulates hepatic proliferation signals (
      • Columbano A.
      • Ledda-Columbano G.M.
      • Pibiri M.
      • Cossu C.
      • Menegazzi M.
      • Moore D.D.
      • Huang W.
      • Tian J.
      • Locker J.
      Gadd45β is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia.
      ,
      • Yamamoto Y.
      • Moore R.
      • Flavell R.A.
      • Lu B.
      • Negishi M.
      Nuclear receptor CAR represses TNFα-induced cell death by interacting with the anti-apoptotic GADD45B.
      ,
      • Hori T.
      • Saito K.
      • Moore R.
      • Flake G.P.
      • Negishi M.
      Nuclear receptor CAR suppresses GADD45B-p38 MAPK signaling to promote phenobarbital-induced proliferation in mouse liver.
      ). This PB-induced tumor promotion has not been observed in patients chronically treated by PB (
      • La Vecchia C.
      • Negri E.
      A review of epidemiological data on epilepsy, phenobarbital, and risk of liver cancer.
      ). In a chimeric mouse liver replaced with human hepatocytes, PB did not induce replicative DNA synthesis in the human hepatocytes and attenuated the Wnt/β-catenin signaling network (
      • Yamada T.
      • Okuda Y.
      • Kushida M.
      • Sumida K.
      • Takeuchi H.
      • Nagahori H.
      • Fukuda T.
      • Lake B.G.
      • Cohen S.M.
      • Kawamura S.
      Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.
      ,
      • Ohara A.
      • Takahashi Y.
      • Kondo M.
      • Okuda Y.
      • Takeda S.
      • Kushida M.
      • Kobayashi K.
      • Sumida K.
      • Yamada T.
      Candidate genes responsible for early key events of phenobarbital-promoted mouse hepatocellular tumorigenesis based on differentiation of regulating genes between wild type mice and humanized chimeric mice.
      ). Given the caveat of whether this complex mouse model mimicked normal liver physiology and no experiments with chronic PB treatment, it may mean that PB regulated this HCC signal differently in mice and humans and that PB could act to prevent HCC development. It was also reported that PB prevented HCC development in mouse neonates (
      • Lee G.H.
      • Ooasa T.
      • Osanai M.
      Mechanism of the paradoxical, inhibitory effect of phenobarbital on hepatocarcinogenesis initiated in infant B6C3F1 mice with diethylnitrosamine.
      ). However, whether this possible prevention is CAR-regulated remains currently unknown. PXR induces human CYP3A4 that metabolically activates many procarcinogens, including aflatoxin B1 (
      • Guengerich F.P.
      • Johnson W.W.
      • Shimada T.
      • Ueng Y.F.
      • Yamazaki H.
      • Langouët S.
      Activation and detoxication of aflatoxin B1.
      ,
      • Shimada T.
      • Guengerich F.P.
      Evidence for cytochrome P-450NF, the nifedipine oxidase, being the principal enzyme involved in the bioactivation of aflatoxins in human liver.
      ,
      • Shimada T.
      • Iwasaki M.
      • Martin M.V.
      • Guengerich F.P.
      Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA 1535/pSK1002.
      ).

      Inflammatory diseases

      Inflammatory bowel disease (IBD) is a chronic inflammation of the intestinal tract. Therapeutic agents targeting PXR and CAR may be useful for treatment of IBD, but via different mechanisms. Clinical PXR activation by drugs such as rifaximin eased IBD symptoms in patients as well as in a humanized PXR mouse model (
      • Prantera C.
      • Lochs H.
      • Campieri M.
      • Scribano M.L.
      • Sturniolo G.C.
      • Castiglione F.
      • Cottone M.
      Antibiotic treatment of Crohn's disease: results of a multicentre, double blind, randomized, placebo-controlled trial with rifaximin.
      ,
      • Cheng J.
      • Shah Y.M.
      • Ma X.
      • Pang X.
      • Tanaka T.
      • Kodama T.
      • Krausz K.W.
      • Gonzalez F.J.
      Therapeutic role of rifaximin in inflammatory bowel disease: clinical implication of human pregnane X receptor activation.
      ). PXR decreased NF-κB activities by directly interacting with NF-κB or indirectly through repressing its gene expression (
      • Zhou C.
      • Tabb M.M.
      • Nelson E.L.
      • Grün F.
      • Verma S.
      • Sadatrafiei A.
      • Lin M.
      • Mallick S.
      • Forman B.M.
      • Thummel K.E.
      • Blumberg B.
      Mutual repression between steroid and xenobiotic receptor and NF-κB signaling pathways links xenobiotic metabolism and inflammation.
      ). The role of CAR in the pathogenesis of IBD is not understood. However, it has been shown that CAR expression was reduced in intestinal mucosal biopsies obtained from IBD patients as well as in the colitic mouse model (
      • Hudson G.M.
      • Flannigan K.L.
      • Erickson S.L.
      • Vicentini F.A.
      • Zamponi A.
      • Hirota C.L.
      • Alston L.
      • Altier C.
      • Ghosh S.
      • Rioux K.P.
      • Mani S.
      • Chang T.K.
      • Hirota S.A.
      Constitutive androstane receptor regulates the intestinal mucosal response to injury.
      ). CAR activation accelerated intestinal epithelial wound healing by enhancing cell migration in human colonic epithelial Caco-2 cells (
      • Hudson G.M.
      • Flannigan K.L.
      • Erickson S.L.
      • Vicentini F.A.
      • Zamponi A.
      • Hirota C.L.
      • Alston L.
      • Altier C.
      • Ghosh S.
      • Rioux K.P.
      • Mani S.
      • Chang T.K.
      • Hirota S.A.
      Constitutive androstane receptor regulates the intestinal mucosal response to injury.
      ). CAR also protected against colitis by inhibiting apoptosis in mice (
      • Uehara D.
      • Tojima H.
      • Kakizaki S.
      • Yamazaki Y.
      • Horiguchi N.
      • Takizawa D.
      • Sato K.
      • Yamada M.
      • Uraoka T.
      Constitutive androstane receptor and pregnane X receptor cooperatively ameliorate DSS-induced colitis.
      ).
      ERα was phosphorylated in mouse immune cells, such as neutrophils and brain microglia (
      • Shindo S.
      • Moore R.
      • Flake G.
      • Negishi M.
      Serine 216 phosphorylation of estrogen receptor α in neutrophils: migration and infiltration into the mouse uterus.
      ,
      • Shindo S.
      • Chen S.-H.
      • Gotoh S.
      • Yokobori K.
      • Hu H.
      • Ray M.
      • Moore R.
      • Nagata K.
      • Martinez J.
      • Hong J.-S.
      • Negishi M.
      Estrogen receptor α phosphorylated at Ser216 confers inflammatory function to mouse microglia.
      ). Phosphorylated ERα regulated microglia to exert its anti-inflammatory and apoptotic functions in mouse brains, thus simulating inflammation in phosphorylation-blocking ERα KI mice. Phosphorylation may enable ERα to prevent inflammatory neurodegeneration.

      Conclusion

      It has been over a half-century since PB was found to induce drug metabolism and the concept that “drugs and xenobiotics induce their metabolism” was developed. 22 years have passed since CAR and PXR were identified for their ability to activate CYP genes and induce the metabolism. Now, collectively, CAR and PXR are two of the most studied nuclear receptors outside steroid hormone receptors (
      • Becnel L.B.
      • Ochsner S.A.
      • Darlington Y.F.
      • McOwiti A.
      • Kankanamge W.H.
      • Dehart M.
      • Naumov A.
      • McKenna N.J.
      Discovering relationships between nuclear receptor signaling pathways, genes, and tissues in Transcriptomine.
      ). Their function and molecular mechanisms as ligand-activated transcription factors to regulate numerous genes, in response to therapeutics as well as xenobiotics, including both human-made and naturally produced chemicals, are well-established. In addition to transcription factors, both CAR and PXR have become well-known for their response to xenobiotics as cell signal transducers either by binding to xenobiotics directly or indirectly through phosphorylation. Studies of CAR and PXR by utilizing the conserved phosphorylation motifs discovered a unique mechanism by which cell signals regulate CAR and PXR and conversely how they regulate cell signals. CAR and PXR were found as orphan receptors, and their research history had largely copied that of steroid hormone receptors. The concept generated by investigating CAR and PXR through the conserved phosphorylation motifs should not only take the xenobiotic response studies to new frontiers, but also return to steroid hormone receptors to redefine and diversify their functions. Nuclear receptors, including steroid hormone receptors, are widely known to possess constitutive activity and so-called “ligand-independent activity.” Investigation to understand the molecular mechanism of these activities has been largely ignored in the research history.

      Acknowledgments

      We thank Drs. Satoru Kakizaki (Gunma University, Japan) and Ryota Shizu (Shizuoka Prefectural University, Japan) and Yuu Miyauchi (Sojo University, Japan) for comments that helped us to write this review. Paavo Honkakoski (University of North Carolina) is deeply acknowledged for comments and editing. Our sincerest thanks to Drs. Lars Pedersen and Lalith Perera at NIEHS, National Institutes of Health, for modeling nuclear receptor structures and complexes. We also thank Mack Sobhany (NIEHS, National Institutes of Health) for editing the manuscript. Foremost, all members of the Pharmacogenetic Section are deeply appreciated for contributions that helped us achieve our scientific accomplishments over the past 37 years.

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