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In response to phenobarbital (PB) and other PB-type inducers, the nuclear receptor CAR translocates to the mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318-6322, 1999). To define the translocation mechanism, fluorescent protein-tagged human CAR (hCAR) was expressed in the mouse livers using the in situ DNA injection and gene delivery systems. As in the wild-type hCAR, the truncated receptor lacking the C-terminal 10 residues (i.e., AF2 domain) translocated to the nucleus, indicating that the PB-inducible translocation is AF2 independent. Deletion of the 30 C-terminal residues abolished the receptor translocation, and subsequent site-directed mutagenesis delineated the PB-inducible translocation activity of the receptor to the peptide L313GLL316AEL319. Ala mutations of Leu313, Leu316, or Leu319 abrogated the translocation of CAR in the livers, while those of Leu312 or Leu315 did not affect the nuclear translocation. The leucine-rich peptide dictates the nuclear translocation of hCAR in response to various PB-type inducers and appears to be conserved in the mouse and rat receptors.

The nuclear receptor constitutive active receptor (CAR) translocates into liver nuclei after phenobarbital (PB) treatment, and activates the conserved enhancer called the PB-response element module (PBREM) found in CYP2B genes. We have examined whether CAR regulates the dimorphic induction by PB of the CYP2B1 gene in Wistar Kyoto (WKY) rats. Northern blot analysis showed that PB induced CYP2B1 mRNA in male WKY rats but not female rats. An in situ injected PBREM-luciferase reporter gene was activated by PB only in the male livers. Western blot analysis revealed extremely low levels of CAR in the cytosols of female livers compared with male counterparts. CAR was accumulated in the liver nucleus of male rats in response to PB treatment, whereas the receptor was barely detectable in the liver nuclei of PB-induced females. These sexually dimorphic responses of PBREM and CAR to PB treatment were not observed with Fisher 344 rats, in which CYP2B1 mRNA was induced in both sexes. Thus, these results indicate that CAR is a regulatory factor that leads to the sexual dimorphic induction of CYP2B1 gene in WKY rats.

Mouse abdominal B–like Hoxa genes are expressed and functionally required in the developing reproductive tracts. Mice lacking either Hoxa-10 or Hoxa-11, two of the AbdB Hoxa genes, exhibit abnormal uterine development similar to that induced by in utero diethylstilbestrol (DES) exposure. Indeed, uterine Hoxa-10 and Hoxa-11 expression is potently repressed by perinatal DES exposure, providing a potential molecular mechanism for DES-induced reproductive tract malformations. We have shown previously that DES can permanently alter uterine lactoferrin gene expression through modulation of the lactoferrin promoter methylation pattern. Here we ask whether a similar mechanism also functions to deregulate uterine Hoxa-10 or Hoxa-11 expression during neonatal DES exposure. We mapped the Hoxa-10 promoter by cloning a 1.485 kb DNA fragment 5′ of the Hoxa-10 exon1a. A 5′ rapid amplification of cDNA ends (RACE) experiment revealed a transcription start site for the a10-1 transcript. Functional analysis of the proximal 200-bp sequences demonstrated significant promoter activity, confirming the location of the Hoxa-10 promoter. Moreover, methylation assays performed on eight CpGs in Hoxa-10 and 19 CpGs in Hoxa-11 proximal promoters demonstrated that all these CpGs were highly unmethylated in both control and DES-dosed mice from postnatal day 5 to day 30. Significant methylation around Hoxa-10 and Hoxa-11 promoters was only observed in DES-induced uterine carcinomas in 18-mo-old mice. Our results suggest that DES-induced downregulations of Hoxa-10 or Hoxa-11 gene expression are not associated with methylation changes in their proximal promoters and that gene imprinting by developmental DES exposure may be a gene-specific phenomenon. Published 2001 Wiley-Liss, Inc.

Heparan sulfate N -deacetylase/ N -sulfotransferase (NDST) catalyzes the deacetylation and sulfation of N -acetyl-d-glucosamine residues of heparan sulfate, a key step in its biosynthesis. Recent crystallographic and mutational studies have identified several potentially catalytic residues of the sulfotransferase domain of this enzyme (Kakuta et al., 1999, J. Biol. Chem. 274:10673–10676). We have used the x-ray crystal structure of heparan sulfate N -sulfotransferase with 3′-phosphoadenosine 5′-phosphate to build a solution model with cofactor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and a model heparan sulfate ligand bound, and subsequently performed a 2-ns dynamics solution simulation. The simulation results confirm the importance of residues Glu 642 , Lys 614 , and Lys 833 , with the possible involvement of Thr 617 and Thr 618 , in binding PAPS. Additionally, Lys 676 is found in close proximity to the reaction site in our solvated structure. This study illustrates for the first time the possible involvement of water in the catalysis. Three water molecules were found in the binding site, where they are coordinated to PAPS, heparan sulfate, and the catalytic residues.

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.

Phenobarbital (PB) increases metabolic capability of hepatocytes by its ability to activate numerous genes encoding various xenochemical-metabolizing enzymes such as cytochrome P450s and specific transferases. More than 35 years since PB induction was first reported, the key nuclear receptor CAR that mediates the induction has now been identified, and the molecular/cellular mechanism involving multiple signal transduction pathways has begun to be unraveled. In response to PB exposure, CAR in the cytoplasm translocates into the nucleus, forms a heterodimer with the retinoid X receptor, and activates the PB response enhancer element leading to the concerted induction of numerous genes.

The crystal structure of SULT2A3 human hydroxysteroid sulfotransferase has been solved at 2.4 Å resolution in the presence of 3′-phosphoadenosine 5′-phosphate (PAP). The overall structure is similar to those of SULT1 enzymes such as estrogen sulfotransferase and the PAP binding site is conserved, however, significant differences exist in the positions of loops Pro14–Ser20, Glu79–Ile82 and Tyr234–Gln244 in the substrate binding pocket. Moreover, protein interaction in the crystal structure has revealed a possible dimer-directed conformational alteration that may regulate the SULT activity.

Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks.

We have studied the roles of estrogen receptor-alpha (ERalpha) and the Stat5b form of STAT (signal transducers and activators of transcription) in sex-specific expression of Cyp2a4 (steroid 15alpha-hydroxylase) and Cyp2d9 (steroid 16alpha-hydroxylase) genes using ERalpha-deficient mice. ERalpha deficiency resulted in the repression of the female-specific Cyp2a4 and expression of the male-specific Cyp2d9 genes, respectively in females. In ERalpha-deficient males, the Cyp2d9 gene continued to be expressed. Nuclear localization of Stat5b occurs in both sexes of ERalpha-deficient mice, although it is normally observed in only wild-type males. Nuclear localization of Stat5b correlates with the repression of Cyp2a4 and expression of Cyp2d9, respectively. Because Stat5b was not detectable in liver nuclear extracts prepared from hypophysectomized ERalpha-deficient females, the regulation by ERalpha appeared to be mediated through a pituitary hormone (i.e., growth hormone). Thus, ERalpha appears to play a key role in the mechanism that inhibits nuclear localization of Stat5b in female mice, leading to feminization of a ERalpha-GH-Stat5b pathway and Cyp expression. Defaulting to this ERalpha-dependent mechanism results in localization of Stat5b to nuclei, which masculinizes the expression of Cyp genes in male mice.

HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAc-->R, is uniquely enriched in neural cells and natural killer cells and is thought to play important roles in cell-cell interaction. HNK-1 glycan synthesis is dependent on HNK-1 sulfotransferase (HNK-1ST), and cDNAs encoding human and rat HNK-1ST have been recently cloned. HNK-1ST belongs to the sulfotransferase gene family, which shares two homologous sequences in their catalytic domains. In the present study, we have individually mutated amino acid residues in these conserved sequences and determined how such mutations affect the binding to the donor substrate, adenosine 3'-phosphate 5'-phosphosulfate, and an acceptor. Mutations of Lys(128), Arg(189), Asp(190), Pro(191), and Ser(197) to Ala all abolished the enzymatic activity. When Lys(128) and Asp(190) were conservatively mutated to Arg and Glu, respectively, however, the mutated enzymes still maintained residual activity, and both mutant enzymes still bound to adenosine 3',5'-diphosphate-agarose. K128R and D190E mutant enzymes, on the other hand, exhibited reduced affinity to the acceptor as demonstrated by kinetic studies. These findings, together with those on the crystal structure of estrogen sulfotransferase and heparan sulfate N-deacetylase/sulfotransferase, suggest that Lys(128) may be close to the 3-hydroxyl group of beta-glucuronic acid in a HNK-1 acceptor. In contrast, the effect by mutation at Asp(190) may be due to conformational change because this amino acid and Pro(191) reside in a transition of the secondary structure of the enzyme. These results indicate that conserved amino acid residues in HNK-1ST play roles in maintaining a functional conformation and are directly involved in binding to donor and acceptor substrates.

The biological process of enzymatic sulfuryl group transfer has been studied by ab initio (density-functional and Hartree–Fock) and semiempirical quantum mechanical methods. The active site of estrogen sulfotransferase in ternary complex with a sulfate donor (PAPS) and sulfate acceptor (estradiol) is modeled. The mechanism proposed in a recent X-ray crystal structure paper (Kakuta et al., Nat. Struct. Biol. 4 (1997) 904) serves as the basis for the calculations. We find that the mechanism proposed in the crystallographic paper is reasonable. The sulfonation takes place in several key steps: neutralization of the charge on PAPS, lengthening of the bridging S–O bond with no cost in energy, activation of the attacking oxygen and proton transfer from estradiol to histidine and then to the sulfuryl group.

The function of Lys-59, Arg-141, and Arg-277 in PAPS binding and catalysis of the flavonol 3-sulfotransferase was investigated. Affinity chromatography of conservative mutants with PAPS analogues allowed us to determine that Lys-59 interacts with the 5' portion of the nucleotide, while Arg-141 interacts with the 3' portion, confirming assignments deduced from the crystal structure of mouse estrogen sulfotransferase [Kakuta, Y., Pedersen, L. G., Carter, C. W. , Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908]. The affinity chromatography method could be used to characterize site-directed mutants for other types of enzymes that bind nucleoside 3',5'- or 2',5'-diphosphates. 31P NMR spectra of enzyme-PAP complexes were recorded for the wild-type enzyme and K59R and K59A mutants. The results of these experiments suggest that Lys-59 is involved in the determination of the proper orientation of the phosphosulfate group for catalysis.

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.

Estrogen sulfotransferase (EST) catalyzes transfer of the 5'-sulfuryl group of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to the 3alpha-phenol group of estrogenic steroids such as estradiol (E2). The recent crystal structure of EST-adenosine 3', 5'-diphosphate (PAP)- E2 complex has revealed that residues Lys48, Thr45, Thr51, Thr52, Lys106, His108, and Try240 are in position to play a catalytic role in the sulfuryl transfer reaction of EST (Kakuta Y., Pedersen, L. G., Carter, C. W., Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908). Mutation of Lys48, Lys106, or His108 nearly abolishes EST activity, indicating that they play a critical role in catalysis. A present 2.2-A resolution structure of EST-PAP-vanadate complex indicates that the vanadate molecule adopts a trigonal bipyramidal geometry with its equatorial oxygens coordinated to these three residues. The apical positions of the vanadate molecule are occupied by a terminal oxygen of the 5'-phosphate of PAP (2.1 A) and a possible water molecule (2. 3 A). This water molecule superimposes well to the 3alpha-phenol group of E2 in the crystal structure of the EST.PAP.E2 complex. These structures are characteristic of the transition state for an in-line sulfuryl transfer reaction from PAPS to E2. Moreover, residues Lys48, Lys106, and His108 are found to be coordinated with the vanadate molecule at the transition state of EST.

Three mouse cytosolic sulfotransferases were expressed in Escherichia coli cells in order to study their substrate specificities toward natural as well as synthetic steroid hormones. The K m and V max values confirmed the high substrate specificity of estrogen and hydroxysteroid sulfotransferases toward estradiol and dehydroepiandrosterone, respectively. In sharp contrast, the synthetic estrogen diethylstilbestrol was metabolized efficiently by both enzymes to its disulfate ester. These sulfotransferases display highly stereospecific sulfotransferase activity for sulfating only the trans -isomer of diethylstilbestrol. Crystals suitable for high-resolution structure determination of estrogen sulfotransferase were grown with polyethylene glycol. The crystals belong to the orthorhombic space group P2 1 2 1 2, and diffracted to 2.5 A.

To clarify the mechanism underlying the male preference of liver tumor in transforming growth factor (TGF) alpha transgenic mice, we analyzed the sexually dimorphic expression of two P450s, i.e., female-specific mouse 15 alpha hydroxylase P450 (2A4) and coumarin 7-hydroxylase P450 (2A5). The expression of 2A4 mRNA in the livers of both transgenic and nontransgenic males was low compared with that in females. P450 2A5 mRNA in the transgenic males was slightly elevated in the adjacent non-tumorous tissues and dramatically elevated in the tumor compared with that in nontransgenic male liver. The activity of P450 2A5 was higher in females than in males in control and transgenic mice but the difference was smaller in the transgenic mice. The activity of P450 2A5 was exceptionally high in liver tumors of transgenic males, as indicated by mRNA expression. These results suggest that female-specific P450 2A5 is induced in the livers of TGF alpha transgenic male mice, particularly in liver tumors of transgenic male mice overexpressing TGF alpha, and may be useful as a marker for mouse hepatocarcinogenesis.

Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7alpha, 7beta, and 2alpha). An individual P450, however, exhibits high specificity to one of these products. Using site-directed mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7beta-hydroxylase activity, whereas Val at this position defines the 2alpha-hydroxylase activity. Leu at position 209 is essential for high DHEA 7alpha-hydroxylase activity. The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 also depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.

(1997). The Structure, Function, and Regulation of Cytochrome P450 2A Enzymes. Drug Metabolism Reviews: Vol. 29, No. 4, pp. 977-996.

Among members of the mouse cytochrome P450 2A family, P450 2A5 is the best catalyst of aflatoxin B1 (AFB1) oxidation to its 8,9-epoxide (Pelkonen, P., Lang, M., Wild, C. P., Negishi, M., and Juvonen, R. O. (1994) Eur. J. Pharmacol., Environ. Toxicol. Pharmacol. Sect. 292, 67-73). Here we studied the role of amino acid residues 209 and 365 of the P450 2A5 in the metabolism and toxicity of AFB1 using recombinant yeasts. The two sites have previously been shown to be essential in the interaction of coumarin and steroids with the P450 2A5. Reducing the size of the amino acid at position 209 or introducing a negatively charged residue at this site increased the 8,9-epoxidation of AFB1 compared to the wild type. In addition, replacing the hydrophobic amino acid at the 365 position with a positively charged lysine residue strongly decreased the metabolism of AFB1. These mutations changed the KM values generally less than the Vmax values. The changes in AFB1 metabolism contrast with the changes in coumarin 7-hydroxylation caused by these amino acid substitutions, since reducing the size of the 209 residue strongly reduced coumarin metabolism and increased the K(M) values. On the other hand, the results with AFB1 are similar to those obtained with steroid hydroxylation. This suggests that the size of the substrate is important when interacting with the residue 209 of the protein. The catalytic parameters of AFB1 correlated generally with its toxicity to the recombinant yeasts expressing the activating enzyme and with the binding of AFB1 to yeast DNA. Furthermore high affinity substrates and inhibitors (e.g., methoxsalen, metyrapone, coumarin 311, 7-methylcoumarin, coumarin, and pilocarpine) of P450 2A5 could efficiently block the toxicity of AFB1. It is suggested that the recombinant yeasts expressing engineered P450 enzymes are a useful model to understand the substrate protein interactions, to study the relationship of metabolic parameters to toxicity, and to test potential inhibitors of metabolism based toxicity.

Polybrominated diphenyl ether BDE-47 (2,2',4,4'-tetrabromodiphenyl ether) is a thyroid hormone disruptor in mice; hepatic induction of various metabolic enzymes and transporters has been suggested as the mechanism for this disruption. Utilizing Car (-/-) and Pxr (-/-) mice as well as human primary hepatocytes, here we have demonstrated that BDE-47 activated both mouse and human nuclear receptor constitutive activated/androstane receptor (CAR). In mouse livers, CAR, not PXR, was responsible for Cyp2b10 mRNA induction by BDE-47. In human primary hepatocytes, BDE-47 was able to induce translocation of YFP-tagged human CAR from the cytoplasm to the nucleus andCYP2B6 and CYP3A4 mRNAs expressions. BDE-47 activated human CAR in a manner akin to the human CAR ligand CITCO (6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) in luciferase-reporter assays using Huh-7 cells. In contrast, mouse CAR was not potently activated by BDE-47 in the same reporter assays. Furthermore, human pregnane X receptor (PXR) was effectively activated by BDE-47 while mouse PXR was weakly activated in luciferase-reporter assays. Our results indicate that BDE-47 induces CYP genes through activation of human CAR in addition to the previously identified pathway through human PXR.

Pregnane X receptor (PXR), an orphan member of the nuclear receptor superfamily, is a major xeno-sensing transcription factor. In response to xenobiotic exposure, PXR regulates genes involved in the metabolism and transport of xenobiotics to protect the body from their harmful effects. Recent progress has revealed that PXR responds not only to such external signals but also to internal signals to help the body adapt to changes in the internal environment, including dysregulation of the immune system. PXR responds to external and internal signals by up- or down-regulating certain metabolic pathways and cellular signals through gene regulation. PXR is a potential therapeutic target for inflammatory as well as metabolic diseases, although its activation may also have unfavorable effects on human health. This review will discuss the recent progress in the understanding of the physiological and pathophysiological roles of PXR and their implications in human diseases and drug therapy by elucidating the molecular mechanisms underlying PXR-mediated gene regulation.

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

Neonatal exposure of mice to estrogen (diethylstilbestrol) results in a high incidence (90%) of uterine tumor later in life. In an effort to screen for estrogen-regulated genes in the uterus of the neonatal mouse, we have isolated a murine homologue of the human decay-accelerating factor (DAF), a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein and a member of the regulators of complement activation family of proteins that function to prevent autologous complement-mediated tissue damage. The induced mouse DAF cDNA has a 64% sequence identity with the human counterpart at the nucleotide level and a 50% identity in the deduced amino acid sequence. It consists of 390 amino acids and contains four short consensus repeats of internal homology characteristic of human DAF. It also contains a hydrophobic C-terminal that most likely serves as a signal for GPI anchor attachment. Sequence comparison with the recently reported mouse DAF cDNAs confirmed that the estrogen-inducible gene corresponds to the mouse GPI DAF gene. The induction of mouse DAF by estrogen is tissue specific and can be mimicked by the antiestrogen tamoxifen. Furthermore, the regulation of uterine DAF expression by estrogen is limited to the GPI DAF gene. The transmembrane DAF gene is not expressed in the mouse uterus, either with or without estrogen stimulation. These results suggest that the two mouse DAF genes are differentially regulated, and that the GPI-anchored DAF may play important roles in estrogen responses and other physiologic or pathophysiologic processes of the female reproductive system.

The mouse phenobarbital (PB)-inducible Cyp2b10 gene promoter has been isolated and sequenced, and control of its expression has been characterized. The 1405-base pair (bp) Cyp2bl0 promoter sequence is 83% identical to the corresponding region from the rat CYP2B2 gene. In addition to the lack of CA repeats, differences include insertion of 42 base pairs (-123/-82 bp) into the middle of a consensus sequence to the so-called "Barbie box." In this report, we have developed a primary mouse hepatocyte culture system in which endogenous 2B10 mRNA as well as Cyp2b10-driven CAT activity were induced by PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), but not by the 3-chloro derivative of TCPOBOP. Deletion analysis of the Cyp2b10 promoter identified a basal transcription element at -64/-34 bp and a negative element at -971/-775 bp. Sequences contained within the -1404/-971 bp region are responsible for the induced CAT activity. DNase I protection and gel shift assays detected five major protein binding sites within the -1404/-971 bp fragment, one of which shared high sequence identity with a portion of a regulatory element in CYP2B2 gene (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene 158, 263-268). Our results indicate that sequences important for PB-induced transcription of Cyp2b10 gene are located in the distal promoter.

P450 represents a large group of heme-thiolate enzymes that exhibit remarkably diverse activities for the metabolism of numerous endogenous and exogenous chemicals. Recent site-directed mutagenesis studies indicate that a single mutation at any of the key residues can be enough to alter the substrate and/or product specificities in the P450 activities. Molecular modeling predicts that these key residues are located within the substrate heme pocket. Structural elements involved in diversifying P450 activity appear to correspond to the B' helix, the F helix and the F/G interhelical loop in the bacterial P450s. Structures represented by these regions are extremely variable despite the fact that the core of the P450 substrate pocket is well conserved. A mutation within these regions may result in a significant geometrical alteration of the pocket and lead to diversify the P450 activity. Phylogenetical analysis shows a relatively high rate of nonsynonymous substitution within these substrate binding regions. The functional versatility of P450 can thus be largely accounted for in terms of pocket change brought about by rapid mutations.

Expression of the Cyp 2d-9 (steroid 16 alpha-hydroxylase) gene in mouse liver is male specific in such Mus musculus domesticus strains as FVB/N, whereas the corresponding P450 genes in the wild mouse species Mus spretus are not sex specific in their expression. These parental differences in the gene expressions were independently inherited in F1 offspring from crosses of FVB/N and M. spretus. A 5' flanking sequence (-110CTC CTCCCTATTCCGGGCC-92) was defined as a regulatory element (named SDI-A1) for the domestic Cyp 2d-9 promoter. The nucleotide which corresponds to T at position -99 within SDI-A1 was found to be substituted with C in the wild mouse P450 genes. The placing of C at position -99 abolished the transcriptional activity of SDI-A1 in HepG2 cells as well as the binding of SDI-A1 to a nuclear factor. This factor (designated NF2d9) was purified from mouse nuclear extracts, and its cDNA cloned. The purified NF2d9 bound to SDI-A1 but not to the mutated SDI-A1 with C at position -99. The deduced amino acid sequence revealed that NF2d9 is 72 and 94% identical to mouse CP2 and human LBP-1a, respectively. NF2d9 thus belongs to the CP2 family and is the mouse homolog of human LBP-1a, which modulates human immunodeficiency virus type 1 transcription. Anti-NF2d9, which was raised against the bacterially expressed protein, supershifted the SDI-A1 complex with the liver nuclear extract. Both the bacterially expressed and in vitro-translated NF2d9 inhibited SDI-A1 complex formation, although they did not bind to SDI-A1 directly. The results, therefore, indicate that the domestic Cyp 2d-9 gene can be regulated through a specific association of NF2d9 with SDI-A1.

Sulfation represents a major pathway for the inactivation of steroid hormones such as estrogens and is catalyzed by a group of enzymes called sulfotransferases. Aberrant regulation of an estrogen sulfotransferase has been demonstrated previously in the livers of obese and diabetogenic C57BL/KsJ-db/db strain mice. In this paper, we report the molecular cloning and functional characterization of a full-length complementary DNA for estrogen sulfotransferase from mouse testis. The mouse estrogen sulfotransferase complementary DNA encodes 295 amino acids. It shares 88%, 77%, 75%, and 68% identity in amino acid sequence with the rat liver, human liver, guinea pig adrenal, and bovine placental estrogen sulfotransferase, respectively. The mouse enzyme was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The fusion protein was affinity purified, and milligram quantities of pure enzyme were obtained after cleavage of the fusion protein with thrombin. The expressed enzyme exhibits a high substrate specificity toward estrogens, including estradiol and estrone. Neither dehydroepiandrosterone, pregnenolone, testosterone, nor a simple phenolic compound, 4-nitrophenol appears to be a substrate. Northern hybridization indicates that messenger RNA (1.3 kilobases) for the estrogen sulfotransferase is expressed exclusively in the testes in control C57BL/KsJ mice. However, both the messenger RNA and protein are dramatically induced in the livers of obese and diabetogenic C57BL/KsJ-db/db mice. In contrast to the liver, the constitutive expression of the enzyme in the testis is not affected by the db/db genotype. These results recapitulate the species-specific nature in the tissue distribution of estrogen sulfotransferase and suggest complex regulatory mechanisms in its expression under normal and pathophysiological conditions.

Mouse P450 2a-5 (coumarin 7-hydroxylase) acquires androstenedione (AD) hydroxylase activity by substituting Phe at position 209 with Asn. However, this mutant P450 2a-5 (F209N) and the corresponding mutant P450 2a-4 (L209N) exhibit different regiospecificites of androstenedione (AD) hydroxylase activity. While the former mutant catalyzes both AD 15 alpha- and 7 alpha-hydroxylase activities at similar rates, the latter mutant maintains the original high specificity of AD 15 alpha-hydroxylase activity. The AD hydroxylase activities in chimeric enzymes of the mutants L209N and F209N show that the regiospecificites are determined by the carboxy-terminal halves of the P450 molecules. Mutations at each of the four different residues within the carboxy-terminal halves indicate that the differences in regiospecificity are determined by the Val/Ala mutation at position 481. As the size of the hydrophobic amino acid at position 481 becomes larger (Ala < Val < Ile), the regiospecificities toward the C15 position of the AD molecule are dramatically increased. The regiospecificity is also increased by placing positively-charged Arg at position 481, although the remaining 15 alpha-hydroxylase activity in this mutant is considerably lower than the other P450s. The results indicate that the size of the residue at position 481 is a key factor in regulating the regiospecificity of AD hydroxylase activity in the P450s. Modeling AD in the substrate-heme pocket of bacterial P450 101A provided further support that residue 481 may reside near the steroid molecule so as to possibly affect the AD hydroxylase activity.

Mouse Slp, a duplicate of the fourth complement component (C4) gene, exhibits EDTA-independent complement activity with a hepatic expression that is male specific. To provide an underlying mechanism for the male-specific expression, we have analyzed the promoter activity of the various 5'-flanking sequences and CpG demethylation of the Slp gene. Transient transfections using HepG2 cells indicate that the element TTCCGGGC (nt -124 to -117) regulates the promoter activity. Moreover, CpG at position -121 of this regulatory element is demethylated to a much higher degree in males than in females. This sexually dimorphic DNA demethylation is consistent with the male-specific expression of the Slp gene in DBA/2 males. The regulatory element binds to the different TTCCGGGC-specific nuclear proteins depending on the methylation of the CpG site. In contrast, the corresponding CpG at position -119 of the C4 gene, which is expressed in both males and females, is demethylated at equal and high levels in both sexes. We therefore propose that the DNA demethylation and methylation-sensitive transcription factors may be a part of the regulatory mechanism for the male-specific expression of the Slp gene.

This paper releases a report of the application for the fault tolerant sequential circuit technique against periodical transient faults under highly electromagnetic environment. After implementing the proposed fault tolerant technique on a counter in our previous paper, as an example of how the proposed architecture performs on utility-scale sequential circuits, the result obtained by applying the technique to a processor is illustrated. On the basis of our logic simulation, we confirm that the originally designed 8-bit processor with functional BIST can avoid the periodical multiple transient faults. We further show that sequential circuits with a practical size of a processor can apply the fault tolerant technique with low overhead from the viewpoint of the circuit space.

Bifunctional chondroitin synthase K4CP catalyzes glucuronic acid and N-acetylgalactosamine transfer activities and polymerizes a chondroitin chain. Here we have determined that an N-terminal region (residues 58-134) coordinates two transfer reactions and enables K4CP to catalyze polymerization. When residues 58-107 are deleted, K4CP loses polymerase activity while retaining both transfer activities. Peptide (113)DWPSDL(118) within this N-terminal region interacts with C-terminal peptide (677)YTWEKI(682). The deletion of either sequence abolishes glucuronic acid but not N-acetylgalactosamine transfer activity in K4CP. Both donor bindings and transfer activities are lost by mutating (677)YTWEKI(682) to (677)DAWEDI(682). On the other hand, acceptor substrates retain their binding to K4CP mutants. The characteristics of these K4CP mutants highlight different states of the enzyme reaction, providing an underlying structural basis for how these peptides play essential roles in coordinating the two glycosyltransferase activities for K4CP to elongate the chondroitin chain.

Mouse steroid 7α- and 15α-hydroxylases (P450c7 and P450c15) and coumarin 7-hydroxylase (P450coh) are structurally similar. To study the structural basis of the substrate specificities of these enzymes, we constructed a series of the mutant P450s, expressed in COS-1 and yeast cells, and studied them spectroscopically as well as enzyme-kinetically. A single amino acid mutation of residue-209 is sufficient to alter the substrate specificity of the P450s from xenobiotics to steroids and subsequently, from testosterone to corticosterone. Moreover, residue-209, when it is asparagine, appears to bind directly to the 11β-hydroxyl of corticosterone. The mutations also after the spin equilibrium of P450 depending on the hydrophobicity and size of residue-209. We conclude, therefore, that residue-209 resides close to the 6th ligand of heme in the mouse 2A subfamily and is located at a critical site of the substrate-binding pocket. As a result, the identity of the residue-209 plays a key role in determining the substrate specificity.

Mouse steroid 15 alpha-hydroxylase P-450 2a-4 is restricted in its substrate specificity to the delta 4, 3-ketone steroids such as androstenedione. As a result, the P-450 exhibits little hydroxylase activity toward delta 5, 3-hydroxysteroids including dehydroepiandrosterone (DHEA). A single amino acid mutation of Ala at position 117 to Val, however, is enough to confer a high DHEA hydroxylase activity to P-450 2a-4 with 7 alpha-OH DHEA as one of the two major hydroxylated metabolites. Mouse coumarin 7-hydroxylase P-450 2a-5 contains Val at position 117, but it exhibits very low DHEA hydroxylase activity. P-450 2a-5 acquires high DHEA hydroxylase activity, however, by a mutation of Phe-209 to Asn. Moreover, the mutant P-450 2a-5 loses its activity when Val is replaced by Ala at position 117. The residue at position 117, therefore, plays the principal role in the determination of the DHEA hydroxylase activity of the P-450s. Conversely, mutations at residue 117 have little effect on the androstenedione hydroxylase activities of the P-450s. Further modeling of the DHEA binding orientation in the substrate-heme pocket of bacterial P-450cam (Iwasaki, M., Darden, T., Pedersen, L., Davis, D. G., Juvonen, R. O., Sueyoshi, T., and Negishi, M. (1993) J. Biol. Chem. 268, 759-762) provides support for the hypothesis that the type of residue at position 117 determines the steroid-substrate specificity of the P-450 depending on the substituent at the C3 position of steroid molecule.

Chemical hepatocarcinogenesis in female mice, induced by 5,9-dimethyl(7H)dibenzo[c,g]carbazole, leads to the overexpression of a cytochrome P-450 of the 2a family. This protein was identified as Cyp2a-5, by the use of immunoblots obtained from isoelectric focusing gels. This method allowed the distinction of Cyp2a-5 from Cyp2a-4, another mouse liver cytochrome P-450, by taking advantage of their slightly different pI values. The theoretical pI values, determined from the amino acid sequence, were pI 9.91 for Cyp2a-4 and pI 10.01 for Cyp2a-5. Other structurally related forms were not detected. In hepatomas from female mice, only the Cyp2a-5 form was overexpressed (2–3 fold). Male mice showed a weak expression of Cyp2a-4 and Cyp2a-5 in control liver samples and in hepatomas. The expression of both forms was increased more than fivefold upon castration. Pyrazole induces specifically the Cyp2a-5 form. The Cyp2a-5 overexpression was correlated with enhanced microsomal coumarin-7-hydroxylase and testosterone-15α-hydroxylase activities. An immunohistochemical study showed that Cyp2a-4 and Cyp2a-5 are expressed uniformly in female livers, but centrilobularly in male livers. In hepatomas, this localisation is perturbed; in females we observed a focal cell localisation, and the Cyp2a-containing cells were often hypertrophic and polyploid. In hepatomas from male mice, the Cyp2a-containing cells became dispersed. From a comparison with other studies, the Cyp2a-5 overexpression appears to be a general feature of hepatocarcinogenesis in mice.

Estrogen receptor α (ERα) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ERα activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ERα a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ERα and mutants ERα S212A, ERα S212D, ERα S236A and ERα S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ERα. ERα S212D, but not ERα S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ERβ; the ERβ S176D mutant retained its trans-activation activity, but the ERβ S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ERα S212A or ERα S212D, revealed that phosphorylation of serine 212 enabled ERα to regulate a unique set of genes and cellular functions.

The nuclear constitutive active/androstane receptor (CAR) is inactivated and sequestered in the cytoplasm when Thr-38 is phosphorylated. Here, we have demonstrated that activated ERK1/2 interacts with phosphorylated CAR to repress dephosphorylation of Thr-38. The phosphorylation-dependent interaction between CAR and ERK1/2 was examined by co-immunoprecipitation experiments of ectopically expressed FLAG-tagged CAR T38A and CAR T38D mutants with endogenous phospho-ERK1/2 in Huh-7 cells. Phospho-ERK1/2 coprecipitated only the phosphorylation-mimicking CAR T38D mutant; this coprecipitation was mediated by the interaction with the xenochemical response signal peptide near the C terminus of CAR. This interaction increased after EGF treatment and decreased after treatment with the MEK inhibitor U0126 as well as after knockdown of MEK1/2 by shRNA in Huh-7 cells. The phosphorylation levels of Thr-38 of CAR decreased in U0126-treated Huh-7 cells. Thus, activated ERK1/2 interacts with CAR and represses dephosphorylation of Thr-38, providing a cell signal-regulated mechanism for CAR activation.

The nuclear xenobiotic receptor CAR is a phenobarbital (PB)-activated transcription factor. Using a mouse model of two-step liver tumorigenesis, in which tumor growth was initiated by diethyl nitrosamine (DEN) and promoted by chronic treatment with PB, we previously demonstrated that tumors developed only in the presence of CAR. Here, we have identified the FAM84A (family with sequence similarity 84, member A) gene as a CAR-regulated gene that is over-expressed during development of phenobarbital-promoted mouse liver tumors. FAM84A mRNA was induced in the liver of DEN/PB-treated mice prior to the development of liver tumors and this induction continued in the non-tumor as well as tumor tissues of a tumor-bearing liver. Western blotting demonstated that FAM84A protein expression increased in mouse liver after PB treatment; however, the FAM84A protein in liver and liver tumors was not phosphorylated at the serine 38 residue, which has been reported to correlate with morphological changes in cells. Immunohistochemistry analysis revealed the cytoplasmic localization of FAM84A protein and its expression during tumor development in normal tissues (especially in hepatocytes around the central vein), eosinophilic foci, adenomas and carcinomas. HepG2 cell-based reporter assays indicated that CAR activated the FAM84A promoter. Exogenous over-expression of FAM84A in HepG2 cells resulted in increased cell migration. The physiological function of FAM84A remains unknown, but our results suggest that FAM84A is up-regulated by CAR during the development of liver tumors, and may play an important role in the progression of liver cancer by increasing cell migration.

The F209L mutation alters specificity of P450coh from coumarin 7-hydroxylation to 15 alpha-hydroxylation of 11-deoxysteroids such as testosterone and 11-deoxycorticosterone. Neither the wild-type nor F209L exhibits activity toward 11 beta-hydroxysteroids including corticosterone. Mutation of Phe-209 to Asn, however, confers on mutant F209N a high corticosterone 15 alpha-hydroxylase activity. F209V also exhibits low corticosterone 15 alpha-hydroxylase activity; Km and Vmax are 10-fold higher and lower, respectively, than for F209N. The results are consistent with the hypothesis that direct interaction of Asn-209 with 11OH is responsible for high corticosterone 15 alpha-hydroxylase activity. To support this hypothesis, a possible steroid-binding orientation is modeled in the substrate pocket of P450cam. Our weighted homology and constrained alignments map residue 209 of P450coh to Met-184 and Met-191 of P450cam. Energy minimization of corticosterone in the substrate pocket results in the 11OH of the steroid directed toward Met-184 (7 A) and Met-191 (16 A), and in C15 located near the sixth axial position of the heme. The steroid-binding model suggests that the P450cam's substrate pocket may be conserved in the mammalian P450 and can accommodate a steroid molecule, and that residue 209 appears to be located at the critical site that determines the steroid-substrate specificity of a P450 depending on the type of group at the 11-position of steroid molecule.

Pregnane X receptor (PXR) was originally characterized as a transcription factor that induces hepatic drug metabolism by activating cytochrome P450 genes. Here we have now demonstrated a novel function of PXR, that of eliciting p38 mitogen-activated protein kinase (MAPK) phosphorylation for cell migration. Upon xenobiotic activation of ectopic human PXR, human hepatocellular carcinoma HepG2 cells were found to exhibit increased phosphorylation of p38 MAPK and to subsequently change morphology and migrate. p38 MAPK was responsible for the regulation of these morphological changes and cell migration because the p38 MAPK inhibitor SB239063 repressed both. Prior to this phosphorylation, PXR directly activated the early response GADD45β gene by binding to a distal direct repeat 4 site of the GADD45β promoter. Ectopic expression of GADD45β increased p38 MAPK phosphorylation, whereas siRNA knockdown of GADD45β decreased the PXR-induced p38 MAPK phosphorylation, confirming that GADD45β can regulate PXR-induced p38 MAPK phosphorylation in HepG2 cells. These results indicate that PXR activates the GADD45β gene, increasing p38 MAPK phosphorylation, and leading HepG2 cells to change morphology and migrate. The GADD45β gene is a direct target for PXR, eliciting cell signals to regulate various cellular functions.

Mouse CYP2C55 has been characterized as an enzyme that catalyzes synthesis of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid metabolite known to have important physiological functions such as regulation of renal vascular tone and ion transport. We have now found that CYP2C55 is induced by phenobarbital (PB) and pregnenolone 16alpha-carbonitrile (PCN) in both mouse kidney and liver. The nuclear xenobiotic receptors constitutive active/androstane receptor (CAR) and pregnane X receptor (PXR) regulate these drug inductions: CYP2C55 mRNA was increased 25-fold in PB-treated Car(+/+) but not in Car(-/-) mice and was induced in Pxr(+/+) but not Pxr(-/-) mice after PCN treatment. Cell-based promoter analysis and gel shift assays identified the DNA sequence (-1679)TGAACCCAGTTGAACT(-1664) as a DR4 motif that regulates CAR- and PXR-mediated transcription of the Cyp2c55 gene. Chronic PB treatment increased hepatic microsomal CYP2C55 protein and serum 19-HETE levels. These findings indicate that CAR and PXR may play a role in regulation of drug-induced synthesis of 19-HETE in the mouse.

BackgroundPhenobarbital (PB) is the most well-known among numerous non-genotoxic carcinogens that cause the development of hepatocellular carcinoma (HCC). PB activates nuclear xenobiotic receptor Constitutive Active/Androstane Receptor (CAR; NR1I3) and this activation is shown to determine PB promotion of HCC in mice. The molecular mechanism of CAR-mediated tumor promotion, however, remains elusive at the present time. Here we have identified Growth Arrest and DNA Damage-inducible 45beta (GADD45B) as a novel CAR target, through which CAR represses cell death.Methodology/principal findingsPB activation of nuclear xenobiotic receptor CAR is found to induce the Gadd45b gene in mouse liver throughout the development of HCC as well as in liver tumors. Given the known function of GADD45B as a factor that represses Mitogen-activated protein Kinase Kinase 7 - c-Jun N-terminal Kinase (MKK7-JNK) pathway-mediated apoptosis, we have now demonstrated that CAR interacts with GADD45B to repress Tumor Necrosis Factor alpha ( TNFalpha)-induced JNK1 phosphorylation as well as cell death. Primary hepatocytes, prepared from Car(+/+), Car(-/-), Gadd45b(+/+) and Gadd45b(-/-) mice, were treated with TNFalpha and Actinomycin D to induce phosphorylation of JNK1 and cell death. Co-treatment with the CAR activating ligand TCPOBOP (1,4 bis[2-(3,5-dichloropyridyloxy)]benzene) has resulted in repression of both phosphorylation and cell death in the primary hepatocytes from Car(+/+) but not Car(-/-) mice. Repression by TCPOBOP was not observed in those prepared from Gadd45b(-/-) mice. In vitro protein-protein interaction and phosphorylation assays have revealed that CAR interacts with MKK7 and represses the MKK7-mediated phosphorylation of JNK1.Conclusions/significanceCAR can form a protein complex with GADD45B, through which CAR represses MKK7-mediated phosphorylation of JNK1. In addition to activating the Gadd45b gene, CAR may repress death of mouse primary hepatocytes by forming a GADD45B complex and repressing MKK7-mediated phosphorylation of JNK1. The present finding that CAR can repress cell death via its interaction with GADD45B provides an insight for further investigations into the CAR-regulated molecular mechanism by which PB promotes development of HCC.

Cytochrome b5 stimulates the coumarin 7-hydroxylation activity of P450coh. A mutation of Arg-129 in P450coh, however, abolishes the stimulation. Moreover, this mutant P450coh binds loosely to cytochrome b5-conjugated Sepharose 4B, whereas wild-type P450coh binds tightly. Consistent with this, the mutation increases the Ka value for b5 binding approximately 6-fold. The identity of residue 209 also alters the stimulation of the activity of P450coh depending on the type of the substrates used and products formed. Coumarin 7-hydroxylation activity is greatly stimulated by cytochrome b5 only when Phe is at position 209, while cytochrome b5 stimulates testosterone hydroxylation activity of P450coh in which Phe, Asn, Ser or Lys substitutes residue 209. P450coh changes its rate of hydrogen peroxide formation depending on the identity of residue 209 and substrate used. Cytochrome b5 decreases the hydrogen peroxide formation of some P450coh whose activities are stimulated by the cytochrome; however, the decrease does not always result in stimulating the activity. The results indicate, therefore, that residues 129 and 209 play different roles in stimulating P450coh activity by cytochrome b5; Arg-129 is a key residue in the cytochrome b5-binding domain and is essential for the stimulation. Residue 209, however, alters the efficiency of electron transport for substrate oxidation as a residue which resides near the sixth ligand of heme and in the substrate-binding site.

Objective We previously found that the lack of nuclear xenobiotic receptor, PXR, decreases femoral bone mineral density (BMD) in Pxr -/- mice. Our present study aims to elucidate the inherited phenotype that correlates with the decreased BMD and to identify the PXR-regulated gene that may link with this phenotype. Methods Pxr +/+ and Pxr -/- mice were used to measure the serum levels of inorganic phosphate (Pi), calcium and vitamin D 3 . Real time PCR and western blots were used to determine the intestinal and renal expressions of Pi and calcium transporters and various other genes involved in bone homeostasis. Cell-based reporter and gel shift assays were performed to characterize the promoter of the identified PXR-regulated gene. Results In both Pxr -/- male and female mice, lumbar, sternum, and skull were all also found to have decreased their BMD values. Serum Pi levels, but not calcium levels, are attenuated in Pxr -/- mice, exhibiting a phenotype of hypophosphatemia. Among the members of the Na/Pi contransporter family, only the SLC34A2 mRNA and protein are repressed in Pxr -/- mice. PXR can directly activate the transcription of the SLC34A2 gene through an ER6 motif on its promoter. Conclusion Pxr -/- mice show the inherited phenotype of hypophosphatemia. The lack of PXR results in a severe repression of the Na/Pi cotransporter NaPi-IIb/Npt2b (SLC34A2), thus leading Pxr -/- males and females to develop a type of hypophosphatemia.

Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing liver diseases such as hepatocellular carcinoma. Here we have characterized the conserved threonine 38 of human CAR as the primary residue that regulates nuclear translocation and activation of CAR. Protein kinase C phosphorylates threonine 38 located on the alpha-helix spanning from residues 29-42 that constitutes a part of the first zinc finger and continues into the region between the zinc fingers. Molecular dynamics study has revealed that this phosphorylation may destabilize this helix, thereby inactivating CAR binding to DNA as well as sequestering it in the cytoplasm. We have found, in fact, that helix-stabilizing mutations reversed the effects of phosphorylation. Immunohistochemical study using an anti-phospho-threonine 38 peptide antibody has, in fact, demonstrated that the classic CAR activator phenobarbital dephosphorylates the corresponding threonine 48 of mouse CAR in the cytoplasm of mouse liver and translocates CAR into the nucleus. These results define CAR as a cell signal-regulated constitutive active nuclear receptor. These results also provide phosphorylation/dephosphorylation of the threonine as the primary drug target for CAR activation.

Nuclear xenobiotic receptor CAR activates transcription of the CYP2B6 gene by directly binding to the distal enhancer PB responsive enhancer module (PBREM). This CAR-mediated activation is synergized by transcription factors early growth response 1 (EGR1) and hepatocyte-enriched nuclear factor 4alpha (HNF4alpha) that bind to the proximal element OA response element KI (OARE(KI)) [Inoue, K., & Negishi, M. (2008). Nuclear receptor CAR requires early growth response 1 to activate the human cytochrome P450 2B6 gene. J. Biol. Chem. 283, 10425-10432]. Two additional EGR1 binding sites have now been found just downstream from PBREM. Internal deletion of EGR1 sites within the context of the -1.8 kb CYP2B6 promoter, which contains both PBREM and OARE(KI), revealed that the distal and proximal EGR1 sites are essential for EGR1 to synergize CAR-mediated transcription. Chromatin conformation capture 3C assays demonstrated that ERG1 may loop the distal PBREM towards the proximal OARE(KI) so that together, CAR and HNF4alpha synergistically activate the CYP2B6 promoter.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

The induction mechanism by pyrazole or phenobarbital of coumarin 7-hydroxylase (cytochrome P450coh) was investigated in DBA/2J male mice. The P450coh mRNA in the pyrazole-induced mice was increased gradually to a 20-fold higher level within 48 h, yet transcription of the P450coh gene was not affected. The half-life of P450coh mRNA, on the other hand, was at least 4-fold longer in the pyrazole-induced DBA/2J (approximately 6.0 h) than in control DBA/2J (approximately 1.5 h) male mice. The stabilization of P450coh mRNA, therefore, is the primary mechanism for the induction by pyrazole of coumarin 7-hydroxylase. Phenobarbital, on the other hand, regulates the induction either translationally or posttranslationally. This drug affected neither the P450coh mRNA nor the P450coh gene's transcription levels in the DBA/2J male mice, although Western blots showed approximately a 3-fold increase of the P450coh protein in the liver microsomes of the drug-treated mice. The results indicate, therefore, that both phenobarbital and pyrazole regulate the P450coh induction posttranscriptionally; the former inducer enhances the translational efficiency of P450coh mRNA or alters the degradation rate of P450coh aproprotein, while the latter stabilizes P450coh mRNA.