Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dioxin receptor is a ligand-dependent transcription factor that binds to target DNA sequences (xenobiotic responsive elements, XREs) following ligand-dependent dimerization with its partner factor, Arnt (aryl hydrocarbon receptor nuclear translocator). Both factors contain an N-terminal basic region helix-loop-helix motif mediating dimerization and subsequent DNA binding. In this study we investigate the possible role of Arnt in agonistic and antagonistic effects of the dioxin receptor ligand alpha-naphthoflavone (ANF). Using specific antisera for the ligand binding dioxin receptor and Arnt, respectively, we show that exposure of the dioxin receptor to ANF in vitro induced recruitment of Arnt, thus stimulating binding of the heteromeric complex to XRE. In transient transfection assays, ANF at high concentrations stimulated expression of an XRE-driven reporter gene. This agonistic effect of ANF is, therefore, most likely attributable to ANF stimulation of dioxin receptor-Arnt heterodimerization and subsequent binding of the complex to XRE. Using a minimal XRE-driven reporter gene construct, we could further confirm earlier studies showing that ANF antagonizes the effect of a dioxin receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Next we employed chimeric receptor constructs containing amino acids 1-500 of the human glucocorticoid receptor fused to dioxin receptor fragments lacking the very N-terminal basic region helix-loop-helix dimerization and DNA binding motif. These chimeric receptor constructs show dioxin responsiveness upon transient transfection into mutant Arnt-deficient hepatoma cells and are, thus, functionally uncoupled from Arnt. Importantly, dioxin-dependent activation of the chimeric receptors was inhibited in the presence of ANF, demonstrating that dimerization of dioxin receptor with Arnt was not necessary for manifestation of the antagonistic effect of ANF. Rather, dioxin receptor sequences, which confer dioxin regulation upon a heterologous DNA binding and transactivating domain, also mediated the antagonistic effects of ANF.
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PMID:Agonistic and antagonistic effects of alpha-naphthoflavone on dioxin receptor function. Role of the basic region helix-loop-helix dioxin receptor partner factor Arnt. 803 60

The intracellular basic helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). In analogy to nuclear receptors that are members of the steroid hormone receptor superfamily the dioxin receptor is a ligand-inducible transcriptional regulator that directly binds to response elements within regulated genes. The most commonly studied dioxin receptor ligands are dioxin itself and structurally related environmental contaminants. A physiological ligand has not yet been identified. Interestingly, however, indolo[3,2-b]carbazole, a compound formed from precursors in the diet, has been shown to bind the murine dioxin receptor with high affinity in vitro. In the present study we show that this compound and its methylated derivative 5,11-dimethylindolo[3,2-b]carbazole very potently activated transcription from a dioxin or xenobiotic response element (XRE)-driven reporter gene in both murine and human hepatoma cells. This effect was not observed in mutant, dioxin-resistant hepatoma cells which are either deficient in expression of dioxin receptor or the bHLH receptor partner factor Arnt. In vitro indolocarbazoles induced XRE binding activity by the human dioxin receptor-Arnt complex in a dose-dependent manner. Thus, both dioxin- and indolocarbazole-activated forms of dioxin receptor regulate target gene expression by the same mechanism involving recruitment of the bHLH factor Arnt and recognition of the XRE element. Finally, the indolo[3,2-b]carbazole-activated human dioxin receptor appeared to generate more stable complexes with the XRE target sequence relative to those produced by the dioxin-activated receptor form, indicating interesting mechanistic differences between different classes of dioxin receptor ligands in their abilities to modulate human dioxin receptor function.
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PMID:Regulation of human dioxin receptor function by indolocarbazoles, receptor ligands of dietary origin. 810 94

In response to dioxin, the nuclear basic helix-loop-helix (bHLH) dioxin receptor forms a complex with the bHLH partner factor Arnt that regulates target gene transcription by binding to dioxin-responsive sequence motifs. Previously, we have demonstrated that the latent form of dioxin receptor present in extracts from untreated cells is stably associated with molecular chaperone protein hsp90, and Arnt is not a component of this complex. Here, we used a coimmunoprecipitation assay to demonstrate that the in vitro-translated dioxin receptor, but not Arnt, is stably associated with hsp90. Although it showed ligand-binding activity, the in vitro-translated dioxin receptor failed to dissociate from hsp90 upon exposure to ligand. Addition of a specific fraction from wild-type hepatoma cells, however, to the in vitro-expressed receptor promoted dioxin-dependent release of hsp90. This stimulatory effect was mediated via the bHLH dimerization and DNA-binding motif of the receptor. Moreover, ligand-dependent release of hsp90 from the receptor was not promoted by fractionated cytosolic extracts from mutant hepatoma cells which are deficient in the function of bHLH dioxin receptor partner factor Arnt. Thus, our results provide a novel model for regulation of bHLH factor activity and suggest that derepression of the dioxin receptor by ligand-induced release of hsp90 may require bHLH-mediated concomitant recruitment of an additional cellular factor, possibly the structurally related bHLH dimerization partner factor Arnt. In support of this model, addition of in vitro-expressed wild-type Arnt, but not a mutated form of Arnt lacking the bHLH motif, promoted release of hsp90 from the dioxin receptor in the presence of dioxin.
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PMID:A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor. 813 47

The dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and binds to DNA target sequences as a heterodimer of the approximately 100 kDa ligand binding receptor and the approximately 85 kDa auxiliary factor, Arnt. Both of these factors encompass an N-terminal basic helix-loop-helix (bHLH) motif required for DNA binding and dimerization. In this study we describe the construction of glucocorticoid/dioxin receptor fusion proteins which allow the regulation of glucocorticoid receptor activity by dioxin in transient transfections of CHO and hepatoma cells. Thus, in the absence of dioxin, chimeric receptor constructs which contain large 500-720 amino acid C-terminal dioxin receptor fragments, but lack the N-terminal bHLH motif, confer repression upon the transcriptional activity of a glucocorticoid receptor derivative, tau DBD, containing its N-terminal strong transactivating signal (tau) and its DNA binding domain (DBD). In the presence of dioxin, this repression is reversed. Importantly, these chimeric receptors did not require the bHLH Arnt co-factor for function. A considerably smaller region of the dioxin receptor, located between amino acids 230 and 421, showed specific dioxin binding activity in vitro. Moreover, dioxin binding in vitro correlated with the ability of receptor fragments to form stable complexes in vitro with the molecular chaperone hsp90. These findings support the notion that hsp90 may be important for folding of a dioxin binding configuration of the receptor. Finally, tau DBD activity was constitutively repressed in a dioxin non-responsive manner by dioxin receptor fragments which failed to bind ligand but also failed to bind hsp90 in vitro, indicating that alternative mechanisms in addition to hsp90 binding may contribute to the inactivation function. In summary, the dioxin receptor system provides a novel and complex model of regulation of bHLH factors that may also give important insights into the mechanism of action of ligand-activated nuclear receptors.
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PMID:Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor. 822 32

The basic region/helix-loop-helix dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). Upon ligand binding the dioxin receptor is converted from a latent, non-DNA binding form to a form that directly interacts with target genes by binding to dioxin-responsive transcriptional control elements. We have purified by conventional and DNA affinity chromatographic procedures the ligand-activated, DNA binding form of dioxin receptor to examine its architecture and functional properties. We observed that the DNA binding activity of the receptor was labile. Most notably, this activity was lost following DNA affinity purification. In complementation experiments we have identified an auxiliary factor(s) that exhibited very poor, if any, intrinsic affinity for the DNA target sequence in vitro but strongly increased the DNA binding activity of the purified receptor-containing material identified by immunoblot analysis. In a similar fashion the in vitro expressed basic region/helix-loop-helix factor Arnt (that has been postulated to modulate the nuclear translocation function of the receptor) reconstituted the DNA binding function of the purified receptor, and the purified auxiliary factor reconstituted receptor activity upon addition to an extract from mutant, Arnt-deficient hepatoma cells. Conversely, purified dioxin receptor reconstituted DNA binding activity in extracts from receptor-deficient hepatoma cells which express bona fide levels of Arnt. Interestingly, UV cross-linking studies using a BrdU-substituted DNA target sequence indicated that primarily the receptor protein was bound to DNA. Moreover, we demonstrate that purified receptor or Arnt exhibited virtually no detectable affinity for the target sequence individually but, in the presence of one another, showed a strong synergy in DNA binding activity in vitro. Importantly, simultaneous expression of the receptor and Arnt resulted in synergistic induction of gene expression in vivo. These data demonstrate that Arnt plays a central role in control of dioxin receptor function by cooperatively modulating the DNA binding activity of the receptor in vitro and dioxin-dependent transactivation in vivo.
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PMID:Purification of the DNA binding form of dioxin receptor. Role of the Arnt cofactor in regulation of dioxin receptor function. 830 14

The intracellular basic region/helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to dioxin response elements. Here we show that the partially purified, ligand-bound receptor alone could not bind target DNA. In contrast, DNA binding by the receptor could be induced by addition of a cytosolic auxiliary activity which functionally and biochemically corresponded to the bHLH factor Arnt. While Arnt exhibited no detectable affinity for the dioxin response element in the absence of the dioxin receptor, it strongly promoted the DNA binding function of the ligand-activated but not the ligand-free receptor forms. Arnt also functionally reconstituted in vitro the DNA binding activity of a mutant, nuclear translocation-deficient dioxin receptor phenotype in cytosolic extracts from a dioxin-resistant hepatoma cell line. Importantly, coimmunoprecipitation experiments showed that Arnt physically interacted in solution with the ligand-activated dioxin receptor but failed to heterodimerize with the ligand-free, hsp90-associated receptor form. Mutational analysis suggested that the functional interaction between these two factors occurred via the bHLH motif of Arnt. These data suggest that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt. The dioxin receptor system also provides the first example of signal-controlled dimerization of bHLH factors.
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PMID:Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor. 838 9

Oxygen is an important regulator of gene expression in mammalian cells, though the extent of operation and the organization of the inducible mechanisms involved are still largely undetermined. To define better the response to hypoxia, we have used differential display PCR to identify genes whose expression is induced in HeLa cells exposed to 1% oxygen. Among six genes whose induction by hypoxia was newly defined in this way, three were of known function, encoding the glucose transporter isoform 3 (Glut-3), adenylate kinase isoenzyme 3 (AK-3), and tissue factor, two were expressed sequence tags (ESTs), and one corresponded to a new sequence. One regulator of the transcriptional response to hypoxia has recently been identified as a heterodimeric DNA-binding complex termed hypoxia-inducible factor-1 (HIF-1), which is also inducible by the iron chelator, desferrioxamine. Of the six hypoxically regulated genes, at least four were also induced by exposure of the cells to desferrioxamine. To analyse further the mechanisms underlying induction of the genes identified in the differential display, inducible expression was compared in wild-type mouse hepatoma cells (Hepa-1), and mutant derivatives (c4) which fail to generate HIF-1, due to a functional defect in one component, HIF-1 beta. Two types of response were defined. For Glut-3 and AK-3, mutant (c4) cells showed almost complete loss of the inducible response to both hypoxia and desferrioxamine. In contrast, tissue factor mRNA was more inducible by both stimuli in c4 than wild-type cells. These studies demonstrate the critical importance of HIF-1 beta in newly recognized responses to hypoxia, and provide further evidence of the importance of this system of gene regulation in mammalian cells; they also demonstrate responses to both hypoxia and desferrioxamine which are independent of HIF-1 beta and which appear exaggerated in HIF-1 beta-deficient cells.
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PMID:Identification of hypoxically inducible mRNAs in HeLa cells using differential-display PCR. Role of hypoxia-inducible factor-1. 891 36

The discovery that the oxygen-regulated transcription factor HIF-1 alpha and the dioxin receptor AhR share the common heterodimerization partner ARNT (HIF-1 beta) raised the question whether a cross-talk between oxygen and dioxin signal transduction pathways exists. To answer this question we investigated an ARNT-deficient mutant cell line (Hepa1C4), which has lost its capability of responding to dioxin. The results demonstrate that the presence of ARNT is indispensable for hypoxia-inducible HIF-1 DNA binding as well as for oxygen-regulated reporter gene activity mediated by the EPO 3' hypoxia response element (HRE). Hypoxic induction of the vascular endothelial growth factor (VEGF) gene, however, was only partially abrogated in Hepa1C4 cells, suggesting that HIF-1-independent oxygen signaling pathways might exist. We further studied HIF-1 and AhR/ARNT DNA binding activity as well as the regulation of oxygen- and xenobiotic-responsive genes by treating mouse Hepa1 hepatoma cells with hypoxia and/or the dioxin analogue ICZ. Hypoxia-inducible VEGF expression was found to be independent of ICZ-treatment, whereas ICZ-inducible cytochrome P-450IA1 expression was slightly reduced by hypoxic treatment of the cells. Interestingly, the enhancer function of a xenobiotic response element (XRE) linked to a reporter gene was induced by hypoxia, but expression of a HRE-containing reporter gene was not affected by ICZ treatment.
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PMID:Oxygen- and dioxin-regulated gene expression in mouse hepatoma cells. 902 41

A 3,4-benzopyrene-resistant mutant clone (c4) of the mouse hepatoma Hepa-1c1c7 cell line was examined for the mutation that causes the defective function of aryl-hydrocarbon receptor (AHR) nuclear translocator (Arnt). Arnt dimerizes with AHR and mediates the induction signal of aryl-hydrocarbon hydroxylase activity. The Arnt cDNAs of c4 cells were cloned by reverse-transcription/PCR to compare the sequences with that of wild-type Arnt cDNA. The Arnt cDNA of c4 cells was found to have a single point mutation, leading to replacement of Gly326 with Asp between two internal repeats in the highly conserved Per-Arnt-Sim (PAS) domain, PAS A and PAS B. The inability of [Asp326]Arnt/AHR heterodimers to enhance reporter gene transcription under the control of the CYP1A1 gene promoter and enhancer confirmed that the G326-->D substitution was a causative mutation. While fluorescence microscopy and coimmunoprecipitation experiments showed that this mutant form of Arnt was not changed from wild-type Arnt in terms of nuclear localization or heterodimer formation with AHR, the binding activity of the [Asp326]Arnt x AHR heterodimer to the xenobiotic-responsive element was reduced markedly. Determination of the turnover rate in COS-7 cells transfected with expression plasmids for mutant Arnt or normal Arnt showed that the mutant protein turned over with an accelerated rate compared with that of the normal. Moreover, the mutant protein displayed increased proteolytic digestibility in vitro with various proteases.
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PMID:A point mutation responsible for defective function of the aryl-hydrocarbon-receptor nuclear translocator in mutant Hepa-1c1c7 cells. 920 42

Western blot analysis was used to determine the concentration of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and aryl hydrocarbon receptor (AHR) in 11 mammalian cell culture lines derived from hepatic and nonhepatic tissues. The strategy was to first use Western blot analysis to determine the expression of ARNT or AHR in each cell line relative to its concentration in murine wild-type Hepa-1c1c7 (Hepa-1) cells. Actual ARNT and AHR concentrations in known amounts of total cell lysates were then determined by generating a standard curve with defined amounts of a highly purified ARNT or AHR protein and performing regression analysis. The results show that the level of ARNT expression in each of the cell lines is similar and represents approximately 0.001-0.002% of total cellular protein. The range of expression was only approximately 3-fold with wild-type Hepa-1 cells expressing the highest level of ARNT (33,000/cell) and canine kidney cells (MDCK line) expressing 14,000 ARNT molecules/cell. In contrast, the concentration of AHR varied by 65-fold over the different cell lines with the wild-type Hepa-1 expressing 323,000 AHR/cell and rat hepatoma cells (H4IIE) expressing 4700. The ratio of AHR to ARNT ranged from 0.3 in H4IIE cells to 10 in the Hepa-1 line with the majority of cells expressing 1-5 times more AHR than ARNT protein. Immunocytochemical staining of each cell line showed that ARNT was exclusively localized to the nuclear compartment and that a conserved nuclear localization signal mapped to the NH-terminal portion of the protein.
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PMID:Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates. 927 42


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