Biomedical subjects

Masahiko Negishi

Publications and source records attributed to Masahiko Negishi.

At least 19 recordsLinked to original sources

PubMed2006

Orphan nuclear receptor constitutive active/androstane receptor-mediated alterations in DNA methylation during phenobarbital promotion of liver tumorigenesis.

Altered DNA methylation is an epigenetic mechanism that plays a key role in the carcinogenesis process, and the nongenotoxic rodent hepatocarcinogen phenobarbital (PB) alters the methylation status of DNA in mouse liver. The constitutive active/androstane nuclear receptor (CAR) mediates half of the PB-induced hepatic gene expression changes and it is essential for liver tumor promotion in PB-treated mice. Here, a technique involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis was utilized to detect PB-induced regions of altered DNA methylation (RAMs) in CAR wildtype (WT) mice that are sensitive to promotion by PB and resistant CAR knockout (KO) mice. The CAR WT mice developed preneoplastic lesions after 23 weeks of PB treatment (precancerous) and liver tumors after 32 weeks, while the CAR KO mice did not develop tumors (Y. Yamamoto, et al., 2004, Cancer Res. 64, 7197-7200). Our goal was to discern those RAMs which are playing important roles in tumor formation by comparing the RAMs that form in sensitive and resistant groups of mice. Using this novel approach, 42 unique RAMs were identified in the precancerous as compared to the CAR KO, 23-week PB-treated tissue. Of these 42 RAMs, 14 carried forward to the tumor tissue, and additionally, 104 total unique RAMs were observed in the tumor tissue. These results indicate that there are unique RAMs occurring in the sensitive CAR WT mice and that a portion of these are seen in both the precancerous and tumor tissue. We hypothesize that these unique RAMs may be facilitating the tumorigenesis process, and these data support the view that DNA methylation plays a causative role in PB-induced tumorigenesis.

Analysis of Variance↗

PubMed2006

The nuclear receptor constitutively active/androstane receptor regulates type 1 deiodinase and thyroid hormone activity in the regenerating mouse liver.

We observed that the level of reverse triiodothyronine (rT3) was significantly increased after partial hepatectomy (PH) in both wild-type and constitutively active/androstane receptor (CAR) knockout (KO) mice, and treatment with phenobarbital (PB), a CAR activator, after PH decreased rT3 to restore its original level only in wild-type mice. On the other hand, no significant changes in the level of total T3 or free T3 in the serum were observed in either wild-type or CAR KO mice after PH or treatment with PB. Type 1 deiodinase (D1) activity and expression were significantly reduced by PH and up-regulated by PB in a CAR-dependent manner. In addition, known T3-regulated genes [tyrosine aminotransferase (TAT) and basic transcription element binding protein (BTEB)] were also significantly decreased by PH and induced by PB. Injection of rT3 into normal mice revealed that rT3 is capable of repressing the known thyroid hormone-regulated genes Tat, Bteb, and Cpt-1 in the liver. Our results suggest that PH decreases D1 activity leading to increased rT3 level, resulting in the repression of T3 target genes. Subsequent treatment with PB decreases rT3 in a CAR-dependent manner through the up-regulation of the D1 gene.

Animals↗

PubMed2006

Relative activation of human pregnane X receptor versus constitutive androstane receptor defines distinct classes of CYP2B6 and CYP3A4 inducers.

Both the human pregnane X receptor (hPXR) and constitutive androstane receptor (hCAR) are capable of regulating CYP3A4 and CYP2B6 gene expression. However, the majority of currently identified CYP3A4 and CYP2B6 inducers are confirmed activators of hPXR but not hCAR. To compare these receptors with respect to their chemical selectivities, 16 drugs known to induce CYP3A4 and/or CYP2B expression were evaluated for relative activation of hPXR versus hCAR. Because of the high basal but low chemical-induced activation of hCAR in immortalized cells, alternative methods were used to evaluate hCAR activation potential. Thirteen of the 16 compounds were classified as moderate to strong hPXR activators. In contrast, carbamazepine (CMZ), efavirenz (EFV), and nevirapine (NVP) were classified as negligible or weak hPXR activators at concentrations associated with efficacious CYP2B6 reporter or endogenous gene induction in primary human hepatocytes, suggesting potential activation of hCAR. Subsequent experiments demonstrated that these three drugs efficiently induced nuclear accumulation of in vivo-transfected enhanced yellow fluorescent protein-hCAR and significantly increased expression of a CYP2B6 reporter gene when hCAR was expressed in CAR-/- mice. In addition, using a recently identified, chemically responsive splice variant of hCAR (hCAR3), the hCAR activation profiles of the 16 compounds were evaluated. By combining results from the hPXR- and hCAR3-based reporter gene assays, these inducers were classified as hPXR, hCAR, or hPXR/hCAR dual activators. Our results demonstrate that CMZ, EFV, and NVP induce CYP2B6 and CYP3A4 preferentially through hCAR and that hCAR3 represents a sensitive tool for in vitro prediction of chemical-mediated human CAR activation.

Alkynes↗

PubMed2006

The role of the nuclear receptor constitutive androstane receptor in the pathogenesis of non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease is a common liver injury, but the pathophysiological mechanisms leading to the development of non-alcoholic steatohepatitis (NASH) remain unclear. The pathological roles of the nuclear receptor constitutive androstane receptor (CAR), a key regulator of drug-metabolising enzymes, in the development of NASH were investigated. METHODS AND RESULTS: CAR(+/+) and CAR(-/-) mice were given a methionine and choline-deficient (MCD) diet to establish a dietary model of NASH. Increases in serum alanine aminotransferase (ALT) and in infiltration of inflammatory cells were dominant in CAR(+/+) mice at 8 weeks. There was no significant difference in the lipid concentration of the liver - namely, the first hit between CAR(+/+) and CAR(-/-) mice. The index of lipid peroxidation increased in liver of the CAR(+/+) mice, as demonstrated by 8-iso-prostaglandin F2alpha (F2-isoprostanes). Western blotting analysis showed that nuclear translocation of CAR occurred in CAR(+/+) mice fed the MCD diet. As a result, the CAR activation caused the lipid peroxidation - namely, the second hit. The expressions of cytochrome P450 (CYP)2B10, 2C29, 3A11 all increased considerably in the CAR(+/+) mice. Furthermore, alpha smooth muscle actin immunohistochemistry and Sirius red staining showed an increase in the degree of fibrosis in CAR(+/+) mice fed the MCD diet at 16 weeks. The mRNA expressions of collagen alpha1(1) and the tissue inhibitor of metalloproteinase-1 were found to be elevated in CAR(+/+) mice. CONCLUSION: CAR caused the worsening of the hepatic injury and fibrosis in the dietary model of NASH. Our results suggest that the CAR nuclear receptor may thus play a critical role in the pathogenesis of NASH.

Alanine Transaminase↗

PubMed2006

Phenytoin induction of the cyp2c37 gene is mediated by the constitutive androstane receptor.

The CYP2C subfamily of cytochrome P450 monooxygenases is responsible for the metabolism of approximately 20% of therapeutic drugs and many endogenous compounds in humans. These enzymes can be induced by prior treatment with drugs, resulting in changes in drug efficacy. Induction of human CYP2C enzymes by xenobiotics occurs at the transcriptional level and is reported to involve the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). In the present study, we report that murine CYP2C37 mRNA is induced by phenobarbital and phenytoin. In contrast, the mouse PXR agonist 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile did not induce CYP2C37 mRNA, suggesting that PXR does not regulate this gene. The induction of CYP2C37 mRNA by phenobarbital and phenytoin is essentially abolished in CAR-null mice; thus, induction of Cyp2c37 by these xenobiotics is CAR-dependent. A functional CAR response element (CAR-RE) was identified at -2791 base pairs from the translation start site of the Cyp2c37 gene. Mutation of this CAR-RE abolished mouse CAR transactivation of a Cyp2c37 -2.9-kilobase pair luciferase reporter construct in HepG2 cells.

Animals↗

PubMed2006

Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 gene by xenobiotics.

CAR (constitutive active/androstane receptor) regulates both the distal enhancer PBREM (phenobarbital-responsive enhancer module) and the proximal element OARE [OA (okadaic acid) response element] to synergistically up-regulate the endogenous CYP2B6 (where CYP is cytochrome P450) gene in HepG2 cells. In this up-regulation, CAR acts as both a transcription factor and a co-regulator, directly binding to and enhancing PBREM upon activation by xenobiotics such as TCPOBOP {1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene} and indirectly associating with the OARE in response to OA [Swales, Kakizaki, Yamamoto, Inoue, Kobayashi and Negishi (2005) J. Biol. Chem. 280, 3458-3466]. We have now identified the cohesin protein SMC1 (structural maintenance of chromosomes 1) as a CAR-binding protein and characterized it as a negative regulator of OARE activity, thus repressing synergy. Treatment with SMC1 small interfering RNA augmented the synergistic up-regulation of CYP2B6 expression 20-fold in HepG2 cells, while transient co-expression of spliced form of SMC1 abrogated the synergistic activation of a 1.8 kb CYP2B6 promoter. SMC1 indirectly binds to a 19 bp sequence (-236/-217) immediately downstream from the OARE in the CYP2B6 promoter. Both DNA affinity and chromatin immunoprecipitation assays showed that OA treatment dissociates SMC1 from the CYP2B6 promoter, reciprocating the indirect binding of CAR to OARE. These results are consistent with the conclusion that SMC1 binding represses OARE activity and its dissociation allows the recruitment of CAR to the OARE, synergizing PBREM activity and the expression of the CYP2B6 gene.

Animals↗

PubMed2006

Estrogen receptor alpha mediates 17alpha-ethynylestradiol causing hepatotoxicity.

Estrogens are known to cause hepatotoxicity such as intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal replacement therapy. Enterohepatic nuclear receptors including farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive active/androstane receptor (CAR) are important in maintaining bile acid homeostasis and protecting the liver from bile acid toxicity. However, no nuclear receptor has been implicated in the mechanism for estrogen-induced hepatotoxicity. Here Era(-/-), Erb(-/-), Fxr(-/-), Pxr(-/-), and Car(-/-) mice were employed to show that Era(-/-) mice were resistant to synthetic estrogen 17alpha-ethynylestradiol (EE2)-induced hepatotoxicity as indicated by the fact that the EE2-treated Era(-/-) mice developed none of the hepatotoxic phenotypes such as hepatomegaly, elevation in serum bile acids, increase of alkaline phosphatase activity, liver degeneration, and inflammation. Upon EE2 treatment, estrogen receptor alpha (ERalpha) repressed the expression of bile acid and cholesterol transporters (bile salt export pump (BSEP), Na(+)/taurocholate cotransporting polypeptide (NTCP), OATP1, OATP2, ABCG5, and ABCG8) in the liver. Consistently, biliary secretions of both bile acids and cholesterol were markedly decreased in EE2-treated wild-type mice but not in the EE2-treated Era(-/-) mice. In addition, ERalpha up-regulated the expression of CYP7B1 and down-regulated the CYP7A1 and CYP8B1, shifting bile acid synthesis toward the acidic pathway to increase the serum level of beta-muricholic acid. ERbeta, FXR, PXR, and CAR were not involved in regulating the expression of bile acid transporter and biosynthesis enzyme genes following EE2 exposure. Taken together, these results suggest that ERalpha-mediated repression of hepatic transporters and alterations of bile acid biosynthesis may contribute to development of the EE2-induced hepatotoxicity.

Animals↗

PubMed2006

Differential regulation of hepatic CYP2B6 and CYP3A4 genes by constitutive androstane receptor but not pregnane X receptor.

Accumulated evidence suggests that cross-talk between the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) results in shared transcriptional activation of CYP2B and CYP3A genes. Although most data imply symmetrical cross-regulation of these genes by rodent PXR and CAR, the actual selectivities of the corresponding human receptors are unknown. The objective of this study was to evaluate the symmetry of human (h) PXR and hCAR cross-talk by comparing the selectivities of these receptors for CYP2B6 and CYP3A4. Human hepatocyte studies revealed nonselective induction of both CYP2B6 and CYP3A4 by hPXR activation but marked preferential induction of CYP2B6 by selective hCAR activation. Gel shift assays demonstrated that hPXR exhibited strong and relatively equal binding to all functional response elements in both CYP2B6 and CYP3A4 genes, whereas hCAR displayed significantly weak binding to the CYP3A4 proximal ER6 motif. In cell-based transfection assays, hCAR displayed greater activation of CYP2B6 reporter gene expression compared with CYP3A4 with constructs containing both proximal and distal regulatory elements. Furthermore, in agreement with binding observations, transfection assays using promoter constructs containing repeats of CYP2B6 DR4 and CYP3A4 ER6 motifs revealed an even greater difference in reporter activation by hCAR. In contrast, hPXR activation resulted in less discernible differences between CYP2B6 and CYP3A4 reporter gene expression. These results suggest asymmetrical cross-regulation of CYP2B6 and CYP3A4 by hCAR but not hPXR in that hCAR exhibits preferential induction of CYP2B6 relative to CYP3A4 because of its weak binding and functional activation of the CYP3A4 ER6.

Aryl Hydrocarbon Hydroxylases↗

PubMed2006

Characterization of specific donor binding to alpha1,4-N-acetylhexosaminyltransferase EXTL2 using isothermal titration calorimetry.

Glycosyltransferases encompass one of the largest families of enzymes found in nature. Their principle function is to catalyze the transfer of activated donor-sugar molecules to various acceptor substrates. The molecular basis that governs this specific transfer reaction, such as how a given transferase determines donor-sugar specificity, remains to be elucidated. Human alpha1,4-N-acetylhexosaminyltransferase (EXTL2) transfers N-acetylglucosamine and N-acetylgalactosamine but does not transfer glucose or galactose. Isothermal titration calorimetry (ITC) is a powerful technique used to characterize various binding reactions, including both protein-ligand and protein-protein interactions. ITC provides the binding stoichiometry, affinity, and the thermodynamic parameters free energy (DeltaG), enthalpy (DeltaH), and entropy (DeltaS) of these binding interactions. This chapter describes our ITC study demonstrating the two-step mechanism that regulates the specific binding of N-acetylhexosamines to EXTL2.

Acetylglucosamine↗

PubMed2006

Phenobarbital confers its diverse effects by activating the orphan nuclear receptor car.

In the early 1960s, phenobarbital (PB) was shown to induce hepatic drug metabolism and the induction was implicated in the molecular mechanism of drug tolerance development. Since then, it has become evident that PB not only induces drug metabolism, but also triggers pleiotropic effects on liver function, such as cell growth and communication, proliferation of the endoplasmic reticulum, tumor promotion, glucose metabolism, steroid/thyroid hormone metabolism, and bile acid synthesis. Upon activation by PB and numerous PB-type inducers, the nuclear receptor CAR mediates those pleiotropic actions by regulating various hepatic genes, utilizing multiple regulatory mechanisms.

Animals↗

PubMed2005

Localization of the nuclear receptor CAR at the cell membrane of mouse liver.

The nuclear receptor constitutive active/androstane receptor CAR is a drug-sensing transcription factor. Upon activation by various drugs such as phenobarbital (PB), CAR translocates from the cytoplasm into the nucleus to regulate the genes that encode enzymes and proteins involved in hepatic metabolism. Here, we have shown the presence of CAR at the cell membrane of mouse livers, using Car+/+ and Car-/- mice. Levels of the cell membrane CAR increased after PB treatment. The CAR exists as a large approximately 160 kDa complex. Thus, CAR undergoes PB-induced translocation to the cell membrane, indicating that CAR may exert a non-genomic action.

Animals↗

PubMed2005

Role of nuclear receptor CAR in carbon tetrachloride-induced hepatotoxicity.

AIM: To investigate the precise roles of CAR in CCl(4)-induced acute hepatotoxicity. METHODS: To prepare an acute liver injury model, CCl(4) was intraperitoneally injected in CAR+/+ and CAR-/- mice. RESULTS: Elevation of serum alanine aminotransferase and extension of centrilobular necrosis were slightly inhibited in CAR-/- mice compared to CAR+/+ mice without PB. Administration of a CAR inducer, PB, revealed that CCl(4)-induced liver toxicity was partially inhibited in CAR-/- mice compared with CAR+/+ mice. On the other hand, androstanol, an inverse agonist ligand, inhibited hepatotoxicity in CAR+/+ but not in CAR-/- mice. Thus, CAR activation caused CCl(4) hepatotoxicity while CAR inhibition resulted in partial protection against CCl(4)-induced hepatotoxicity. There were no differences in the expression of CYP2E1, the main metabolizing enzyme for CCl(4), between CAR+/+ and CAR-/- mice. However, the expression of other CCl(4)-metabolizing enzymes, such as CYP2B10 and 3A11, was induced by PB in CAR+/+ but not in CAR-/- mice. Although the main pathway of CCl(4)-induced acute liver injury is mediated by CYP2E1, CAR modulates its pathway via induction of CYP2B10 and 3A11 in the presence of activator or inhibitor. CONCLUSION: The nuclear receptor CAR modulates CCl(4)-induced liver injury via induction of CCl(4)-metabolizing enzymes in the presence of an activator. Our results suggest that drugs interacting with nuclear receptors such as PB might play critical roles in drug-induced liver injury or drug-drug interaction even though such drugs themselves are not hepatotoxic.

Androstanols↗

PubMed2005

Differential UGT1A1 induction by chrysin in primary human hepatocytes and HepG2 Cells.

Chrysin, a dietary flavonoid, has been shown to markedly induce UGT1A1 expression and activity in HepG2 and Caco-2 cell lines; thus, it has been suggested to have clinical utility in the treatment of UGT1A1-mediated deficiencies, such as unconjugated hyperbilirubinemia or the prevention of 7-ethyl-10-hydroxycamptothecin (SN-38) toxicity. However, little is known about its induction potential in a more physiologically relevant model system, such as primary hepatocyte culture. In this study, induction of UGT1A1 expression (mRNA, protein, and activity) was investigated in primary human hepatocyte cultures after treatment with chrysin and other prototypical inducers. Endogenous nuclear receptor-mediated UGT1A1 induction was studied using transient transfection reporter assays in primary human hepatocytes and HepG2 cells. Results indicated that induction of UGT1A1 expression was minimal in human hepatocytes treated with chrysin compared with that in HepG2 cells (1.2-versus 11-fold, respectively). Subsequent experiments to determine whether the differential response was due to its metabolic stability revealed strikingly different elimination rate constants between the two cell systems (half-life of 13 min in human hepatocytes versus 122 min in HepG2 cell suspensions). Further study demonstrated that UGT1A1 mRNA expression could be induced in human hepatocyte cultures by either increasing the chrysin dosing frequency or by modulating chrysin metabolism, suggesting that the differential induction observed in hepatocytes and HepG2 cells was due to differences in the metabolic clearance of chrysin. In conclusion, this study suggests that the metabolic stability of chrysin likely would limit its ability to induce UGT1A1 in vivo.

Adolescent↗

PubMed2005

Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4alpha.

Cytochrome P450 (P450) enzymes play important roles in the metabolism of endogenous and xenobiotic substrates in humans. CYP2C8 is an important member of the CYP2C subfamily, which metabolizes both endogenous compounds (i.e., arachidonic acids and retinoic acid) and xenobiotics (e.g., paclitaxel). Induction of P450 enzymes by drugs can result in tolerance as well as drug-drug interactions. CYP2C8 is the most strongly inducible member of the CYP2C subfamily in human hepatocytes, but the mechanism of induction by xenobiotics has not been delineated. To determine the mechanisms controlling the regulation of this important P450, we cloned the 5'-flanking region of CYP2C8 and investigated its transcriptional regulation by nuclear factors such as the pregnane X receptor (PXR), constitutive androstane receptor (CAR), glucocorticoid receptor (GR), and hepatic nuclear factor 4 (HNF4alpha) that are known to be involved in the induction of other P450 enzymes using both cell lines and primary hepatocyte models. We initially identified a distal PXR/CAR-binding site in the CYP2C8 promoter that confers inducibility of CYP2C8 via the PXR agonist/ligand rifampicin and the CAR agonist/ligand CITCO [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime]. A glucocorticoid-responsive element was identified that mediates dexamethasone induction via the GR. We finally identified an HNF4alpha-binding site within the CYP2C8 basal promoter region that is cis-activated by cotransfected HNF4alpha. In summary, the present studies show that CAR, PXR, GR, and HNF4alpha can regulate CYP2C8 expression and identify specific cis-elements within the promoter that control these regulatory pathways.

Androstanes↗

PubMed2005

Thr176 regulates the activity of the mouse nuclear receptor CAR and is conserved in the NR1I subfamily members PXR and VDR.

The mouse nuclear receptor CAR (constitutively active receptor) is a transcription factor that is activated by phenobarbital-type inducers such as TCPOBOP {1,4 bis[2-(3,5-dichloropyridyloxy)]benzene} in liver in vivo. However, CAR is constitutively active in cell-based transfection assays, the molecular mechanism for which has not been elucidated yet. In the model structure of CAR, Thr176 constitutes a part of the ligand-binding surface, but its side chain is not directed toward the surface, instead it forms a hydrogen bond with Thr350 in the AF2 (activation function 2) domain of CAR. Thr350 is known to regulate CAR activity [Ueda, Kakizaki, Negishi, and Sueyoshi (2002) Mol. Pharmacol. 61, 1284-1288]. Thr176 was mutated to various amino acids to examine whether this interaction played a role in conferring the constitutive activity. Hydrophobic and positively charged amino acids at position 176 abrogated the constitutive activity, whereas polar and negatively charged amino acids retained it. When one of the small hydrophobic amino acids, such as alanine or valine, was substituted for threonine, the mutants were fully activated by TCPOBOP. The co-activator SRC-1 (steroid receptor co-activator-1) regulated the activity changes associated with the mutations. Thr248 and Ser230 are the Thr176-corresponding residues in human pregnane X receptor and mouse vitamin D3 receptor respectively, interacting directly with the conserved threonine in the AF2 domains. Thr248 and Ser230 also regulated the ligand-dependent activity of these receptors by augmenting binding of the receptors to SRC-1. Thr176, Thr248 and Ser230 are conserved residues in the NR1I (nuclear receptor 1I) subfamily members and determine their activity.

Amino Acid Sequence↗

PubMed2005

The nuclear receptors constitutive androstane receptor and pregnane X receptor cross-talk with hepatic nuclear factor 4alpha to synergistically activate the human CYP2C9 promoter.

CYP2C9 is an important human drug-metabolizing enzyme that is expressed primarily in liver. Recent studies in our laboratory have shown that the nuclear receptor pregnane X receptor (PXR) is important in the transcriptional activation of the CYP2C9 promoter by drugs such as rifampicin and that the essential element is a constitutive androstane receptor (CAR)/PXR site -1839 bp upstream of the translation start site. Both CAR and PXR transcriptionally up-regulate the CYP2C9 promoter via these elements. In the present study, we ask whether additional sites in the proximal promoter also play a role in this induction. We identify two proximal hepatic nuclear factor (HNF) 4alpha binding sites at -152 and -185 bp of the CYP2C9 promoter, both of which bind HNF4alpha in gel shift assays and transcriptionally up-regulate this promoter in response to HNF4alpha in HepG2 cells. HNF4alpha synergizes with CAR and with PXR in HepG2 cells treated with rifampicin. The synergy only occurs when the CAR/PXR binding site at -1839 bp is present. Mutation of the two HNF4alpha binding sites differentially prevented up-regulation of CYP2C9 promoter by both CAR as well as HNF4alpha, synergy between the two receptors, and essentially abolished induction by rifampicin in HepG2 cells transfected with PXR. These studies strongly support the hypothesis that there is cross talk between distal CAR/PXR sites and HNF4alpha binding sites in the CYP2C9 promoter and that the HNF4alpha sites are required for maximal induction of the CYP2C9 promoter.

Aryl Hydrocarbon Hydroxylases↗

PubMed2005

Two-step mechanism that determines the donor binding specificity of human UDP-N-acetylhexosaminyltransferase.

In its x-ray crystal structures, alpha-1,4-N-acteylhexosaminyltransferase (exostosin-like protein 2 (EXTL2)) forms no direct interaction with the N-acetyl group of the UDP-N-acetylhexosamine. Mutation of the residues that interact with the hydroxyl groups of the donor not only failed to abrogate donor binding but in fact increased binding affinity. Isothermal titration calorimetry is now used to examine the binding nature of various UDP-sugars in H2O and D2O solutions. UDP-N-acetylhexosamines bind to EXTL2 with a high affinity in both solutions, resulting in a relatively large increase of entropy, whereas the weak binding of UDP-galactose and -glucose, which occurred only in D2O solution, only slightly increased entropy. Thus, specific donor binding appears to undergo two distinct steps, beginning with the N-acetyl group expelling water from the donor. enzyme complex into the bulk solvent followed by positioning of the donor into the binding site for the subsequent interactions with the enzyme.

Base Sequence↗

PubMed2005

Identification of HMG-CoA reductase inhibitors as activators for human, mouse and rat constitutive androstane receptor.

Constitutive active (or androstane) receptor (CAR, NR1I3), a member of the nuclear receptor family, is a major regulator for induction of cytochrome P450 2B (CYP2B) genes by phenobarbital. Phenobarbital-like inducer, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is a potent mouse CAR ligand that has been used to study CAR target genes in mice but does not activate human CAR (hCAR) or rat CAR (rCAR). Although 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) was reported to be an hCAR agonistic ligand, activation of hCAR by CITCO in cell-based reporter assay was weak. Therefore, we performed a screening of 50 drugs and chemicals using cell-based reporter assays to identify activators of hCAR. Among them, HMG-CoA reductase inhibitors (cerivastatin, simvastatin, fluvastatin, and atorvastatin) enhanced the hCAR-mediated transcriptional activation of phenobarbital-responsive enhancer module reporter gene by up to 3-fold. Similar activation by HMG-CoA reductase inhibitors was also observed with mouse and rat CARs. On the other hand, pravastatin did not activate hCAR at the concentrations tested (up to 30 microM). The extent of activation by the HMG-CoA reductase inhibitors was stronger than that by CITCO. Cerivastatin, simvastatin, fluvastatin, and atorvastatin induced CYP2B6 mRNA in stable hCAR-expressed FLC7 cells but not in original FLC7 cells. Therefore, we concluded that CAR mediates the effects of HMG-CoA reductase inhibitors on the induction of CYP2B genes, although HMG-CoA reductase inhibitors also activate pregnane X receptor. HMG-CoA reductase inhibitors such as cerivastatin would be useful to study for elucidating molecular and cellular mechanisms of hCAR.

Androgens↗