The heteromeric unliganded aryl hydrocarbon receptor complex (AHRC) contains the aryl hydrocarbon receptor monomer (AHR). Binding of polycyclic or halogenated aromatic hydrocarbon (PAH and HAH) ligand causes release of AHR, which then associates with the AHR nuclear translocator protein (ARNT) to generate the heterodimeric "transformed" AHRC. AHR and ARNT belong to a novel subclass of basic helix-loop-helix-containing transcription factors. The transformed AHRC binds xenobiotic responsive elements in responsive genes and turns on their transcription. Certain of these genes encode enzymes involved in the metabolic activation of PAHs to mutagenic derivatives. HAHs are not genotoxic: Their pathogenicity depends on the AHRC but not on their metabolism. Current research includes investigations directed towards delineating the pathways of HAH pathogenesis, ascertaining whether AHR can mediate signal transduction independently of DNA binding, understanding the mechanism of transcriptional activation, and investigating the potential roles of AHR and ARNT in development.
Recent studies of tissue culture cells have defined a widespread system of oxygen-regulated gene expression based on the activation of a heterodimeric transcription factor termed hypoxia-inducible factor-1 (HIF-1). To determine whether the HIF-1 transcriptional response is activated within solid tumors and to define the consequences, we have studied tumor xenografts of a set of hepatoma (Hepa-1) cells that are wild type (wt), deficient (c4), and revertant (Rc4) for an obligatory component of the HIF-1 heterodimer, HIF-1. Because HIF-1 is also essential for the xenobiotic response (in which it is termed the aryl hydrocarbon receptor nuclear translocator), we also studied c31 cells, which have a different defect in the xenobiotic response and form the HIF-1 complex normally. Two genes that show different degrees of HIF-1-dependent hypoxia-inducible expression in cell culture were selected for analysis-the glucose transporter, GLUT3, and vascular endothelial growth factor (VEGF). In situ hybridization showed intense focal induction of gene expression in tumors derived from wt, Rc4, and c31 cells, which was reduced (VEGF) or not seen (GLUT3) in those derived from c4 cells. In association with these changes, tumors of c4 cells had reduced vascularity and grew more slowly. These findings show that HIF-1 activation occurs in hypoxic regions of tumors and demonstrate a major inf luence on gene expression, tumor angiogenesis, and growth.
The aryl hydrocarbon (Ah) receptor binds various environmental pollutants, such as polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds (dioxins, dibenzofurans, and biphenyls), and mediates the carcinogenic effects of these agents. The complementary DNA and part of the gene for an 87-kilodalton human protein that is necessary for Ah receptor function have been cloned. The protein is not the ligand-binding subunit of the receptor but is a factor that is required for the ligand-binding subunit to translocate from the cytosol to the nucleus after binding ligand. The requirement for this factor distinguishes the Ah receptor from the glucocorticoid receptor, to which the Ah receptor has been presumed to be similar. Two portions of the 87-kilodalton protein share sequence similarities with two Drosophila proteins, Per and Sim. Another segment of the protein shows conformity to the consensus sequence for the basic helix-loop-helix motif found in proteins that bind DNA as homodimers or heterodimers.
The Ah (dioxin) receptor binds a number of widely disseminated environmental pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons, and mediates their carcinogenic effects. The ligand-bound receptor activates Cyp 1a1 gene transcription through interaction with specific DNA sequences, termed xenobiotic responsive elements (XREs). The Ah receptor nuclear translocator protein (Arnt) is required for Ah receptor function. Arnt is now shown to be a structural component of the XRE binding form of the Ah receptor. Furthermore, Arnt and the ligand-binding subunit of the receptor were extracted as a complex from the nuclei of cells treated with ligand. Arnt contains a basic helix-loop-helix motif, which may be responsible for interacting with both the XRE and the ligand-binding subunit.
The aryl hydrocarbon receptor (Ahr) 1 binds a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons, and certain halogenated aromatic hydrocarbons, such as TCDD. Following ligand treatment, induction of CYP1A1 and several other enzymes involved in xenobiotic metabolism occurs in most tissues. Certain of these enzymes (including CYP1A1) are involved in the metabolism of polycyclic aromatic hydrocarbons to active genotoxic metabolites, and Ahr therefore plays an important role in carcinogenesis by these compounds. Ahr also mediates most, if not all, of the carcinogenic and toxic effects of the halogenated aromatic hydrocarbons, although metabolism of these compounds does not appear to be involved (reviewed in Ref. 1).The unliganded Ahr is located in the cytoplasm of the mouse hepatoma cell line, Hepa-1, as part of a complex that also contains two molecules of the 90-kDa heat shock protein (HSP90) and perhaps another protein of approximately 43 kDa (2). It is not known whether both, one, or neither of the HSP90 molecules bind Ahr directly. HSP90 appears to be required for ligand binding by Ahr (3). Binding of ligand leads to dissociation of Ahr from HSP90. In ligand-treated cells, Ahr is found in the nuclear fraction, from which it can be extracted in the form of a complex with the aryl hydrocarbon receptor nuclear translocator protein (Arnt) (4, 5). This complex is probably a heterodimer of the two proteins, although the presence of additional small protein(s) has not been rigorously excluded. Arnt appears to be a nuclear protein in Hepa-1 cells (5, 6). Some evidence indicates that dissociation of Ahr from HSP90 occurs in the nucleus and that HSP90 may play a direct role in translocating Ahr into this organelle (7,8). It has been proposed that Arnt promotes the dissociation of Ahr from HSP90 (9). Transcriptional activation of the cyp1a1 gene results from the binding of the Ahr/Arnt heterodimer to short DNA sequences, termed xenobiotic responsive elements (XREs), located in the 5Ј-flanking region of the gene (4, 10 -12). Both Ahr and Arnt bind directly to the XRE sequence (13).Mouse Ahr and Arnt are 20% identical in amino acid sequence. They also show a striking resemblance in overall structure (14 -16). Both proteins contain bHLH motifs toward their amino termini. However, Ahr and Arnt represent a novel subclass of bHLH-containing transcription factors because they differ from most or all other such proteins in that (i) activation of the Ahr complex requires ligand, (ii) the XRE sequence differs from the E-box sequence, which is the recognition sequence for nearly all other bHLH-containing transcription factors (reviewed in Ref. 17), and (iii) both proteins contain an approximately 300-amino acid segment of sequence similarity, called the PAS domain. The PAS domains of each protein contain two copies of an approximately 50-amino acid degenerate direct repeat, referred to as the PAS A and PAS B repeats. The Drosophila proteins single-minded (Sim) and period * This work was sup...
The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g.,2,3,7,), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second a helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT.The aryl hydrocarbon receptor (AHR) is a component of a soluble, intracellular protein complex that binds a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons (found in cigarette smoke and smog) and certain halogenated hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls. Ligand triggers transformation of the AHR complex to a form which can activate transcription of the CYPlAJ and CYP1A2 genes and induce several other enzymes of xenobiotic metabolism. The CYPlAl and CYP1A2 enzymes are directly involved in the conversion of polycyclic aromatic hydrocarbons and heterocyclic amines (found in cooked food), respectively, to their active genotoxic metabolites, and the AHR therefore plays an important role in carcinogenesis by both classes of compounds (reviewed in references 35, 36, and 48). TCDD is metabolized only very poorly, and the parent compound is probably the active agent. TCDD acts as a tumor promoter, or nongenotoxic carcinogen (reviewed in reference 47). Several polycyclic aromatic and halogenated hydrocarbons have also been shown to be teratogenic, and teratogenesis is mediated by the AHR (reviewed in reference 6). TCDD also affects the differentiation of keratinocytes (reviewed in reference 18). These last observations suggest that the AHR plays a role...
We used homologous recombination in embryonic stem cells to generate mice heterozygous for an aryl hydrocarbon nuclear translocator (ARNT) null mutation. These mice were intercrossed, but no live homozygous Arnt-/- knockout mice were produced among 64 newborns. Homozygotes die in utero between 9.5 and 10.5 days of gestation. Abnormalities included neural tube closure defects, forebrain hypoplasia, delayed rotation of the embryo, placental hemorrhaging, and visceral arch abnormalities. However, the primary cause of lethality appears to be failure of the embryonic component of the placenta to vascularize and form the labyrinthine spongiotrophoblast. This may be related to ARNT's known role in hypoxic induction of angiogenesis. We found no defects in yolk sac circulation.
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