Ah Receptor Pathway Intricacies; Signaling Through Diverse Protein Partners and DNA-Motifs.

The Ah receptor is a transcription factor that modulates gene expression via interactions with multiple protein partners; these are reviewed, including the novel NC-XRE pathway involving KLF6.

Molecular regulation of genes encoding xenobiotic-metabolizing enzymes: mechanisms involving endogenous factors.

It is widely recognized that xenobiotic-metabolizing enzymes play a fundamental role in the basic processes of carcinogenesis and toxicity on one hand, and chemoprevention and drug efficacy on the other. Realization that different factors can profoundly affect the expression of these enzymes at the genome level has resulted in an enhanced appreciation of the importance these genes play in our modern industrialized age. There continues to be rapid proliferation of studies addressing the molecular regulation of these genes. The discovery of common signal transduction pathways and transcription factors that dictate tissue and developmental-specific expression, as well as variation in expression within a given tissue, suggest that there may be significant interaction among these various regulatory systems. This report is a summary of a symposium that was part of the Structure, Function and Regulation of Cytochromes P450 and Xenobiotic Metabolizing Enzymes satellite meeting of the 2000 joint meeting of the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the French Pharmacological Society, and the Pharmacological Society of Canada held in Boston, Massachusetts. This symposium brought together several speakers who addressed specific receptor-mediated signal transduction pathways involved in the regulation of xenobiotic-metabolizing enzymes, as well as other molecular mechanisms whereby endogenous factors are involved in controlling tissue- and developmental-specific expression.

Maximal aryl hydrocarbon receptor activity depends on an interaction with the retinoblastoma protein.

The aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix/periodicity/AhR nuclear translocator/simple-minded (Per-Arnt-Sim) family of transcription factors that regulate critical functions during development and tissue homeostasis. Within this family, the AhR is the only member conditionally activated in response to ligand binding, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We recently demonstrated that the AhR interacts with the retinoblastoma protein (pRb). This report presents evidence that a LXCXE motif in the AhR protein confers pRb binding, which is necessary for maximal TCDD induced G(1) arrest in rat 5L hepatoma cells. The data support a mechanism whereby pRb seems to regulate G(1) cell cycle progression distinct from the direct repression of E2F-mediated transcription. Furthermore, the results indicate that the AhR-pRb interaction regulates TCDD induction of CYP1A1, suggesting that pRb may be a general AhR coactivator.

A direct interaction between the aryl hydrocarbon receptor and retinoblastoma protein. Linking dioxin signaling to the cell cycle.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In 5L hepatoma cells, TCDD induces a G1 cell cycle arrest through a mechanism that involves the AhR. The retinoblastoma tumor suppressor protein (pRb) controls cell cycle progression through G1 in addition to promoting differentiation. We examined whether the human AhR or its dimerization partner, the AhR nuclear translocator, interacts with pRb as a basis of the TCDD-induced cell cycle arrest. In vivo and in vitro assays reveal a direct interaction between pRb and the AhR but not the AhR nuclear translocator protein. Binding between the AhR and pRb occurs through two distinct regions in the AhR. A high affinity site lies within the N-terminal 364 amino acids of the AhR, whereas a lower affinity binding region colocalizes with the glutamine-rich transactivation domain of the receptor. AhR ligand binding is not required for the pRb interaction per se, although immunoprecipitation experiments in 5L cells reveal that pRb associates preferentially with the liganded AhR, consistent with a requirement for ligand-induced nuclear translocation. These observations provide a mechanistic insight into AhR-mediated cell cycle arrest and a new perspective on TCDD-induced toxicity.

Quantitative RT-PCR on CYP1A1 heterogeneous nuclear RNA: a surrogate for the in vitro transcription run-on assay.

A quantitative reverse transcription polymerase chain reaction (RT-PCR) assay was developed to amplify a region of the CYP1A1 heterogeneous nuclear RNA (hnRNA) transcript encompassing the first intron-exon boundary. The RT-PCR protocol uses a CYP1A1 recombinant RNA internal standard identical to the target hnRNA except for an engineered unique internal restriction site. Its inclusion enables normalization between reactions and a measurement of the absolute number of target hnRNA transcripts. Specificity for the hnRNA was achieved by using intron-directed primers in both the RT and the PCR. Nuclear run-on assays and the hnRNA RT-PCR assay detected an equivalent increase in transcription of Cyp1a-1 in cultured murine Hepa 1c1c7 cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The RT-PCR assay also revealed TCDD-dependent transcriptional activation of the Cyp1a-1 gene in murine skin, a tissue unsuited to the nuclear run-on assay because of inherent difficulties associated with the isolation of nuclei. These examples demonstrate that the hnRNA RT-PCR assay is a facile surrogate for the nuclear run-on assay. Moreover, the sensitivity and design characteristics of the RT-PCR assay suggest the potential for its broad application in general transcriptional research.

Dioxin-dependent, DNA sequence-specific binding of a multiprotein complex containing the Ah receptor.

Fractionation of rat liver cytosol, using as an affinity reagent the DNA recognition sequence for the liganded aromatic hydrocarbon (Ah) receptor, enriches for proteins that are about 110, 106, 98/96, 57, and 54 kDa in size. The proteins display 2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent, DNA sequence-specific binding that is characteristic of the liganded Ah receptor. Immunological studies imply: 1. That the 98/96-kDa protein is the Ah receptor nuclear translocator (Arnt); 2. That the 110- and 106-kDa proteins are not immunologically related either to each other or to Arnt; and 3. That the 110-, the 106-kDa, and the Arnt proteins are members of a multicomponent protein complex. cDNA cloning studies indicate that the 106-kDa protein is the rat Ah receptor. The N-terminal 384 amino acids of the rat receptor show substantial sequence homology to the mouse and human Ah receptor. The sequence conservation across species imputes functional importance to this region of the receptor. In contrast, the remainder of the protein is substantially less well conserved among rat, mouse, and human. The tissue distribution of Ah receptor mRNA is consistent with previous studies of the distribution of the receptor protein. Our findings demonstrate the use of DNA recognition site chromatography for purification of the Ah receptor and imply that the ligand receptor binds to DNA as part of a multiprotein complex.

Characterization of multiple forms of the Ah receptor: recognition of a dioxin-responsive enhancer involves heteromer formation.

We have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetrachlorodibenzo-p- dioxin-(TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chromatographic fractions of rat hepatic cytosol indicated that TCDD-dependent and sequence-specific DNA binding coeluted with a 200-kDa form of the Ah receptor (peak 2) previously characterized as being multimeric and having high affinity for calf thymus DNA. The TCDD-bound, 100-kDa form of the receptor (peak 1) bound weakly to the DNA recognition motif. These results indicated that the DNA-binding form of the Ah receptor is a multimer. SDS-polyacrylamide gel electrophoresis of peak 2 cross-linked to a bromodeoxyuridine-substituted DNA recognition motif indicated that this form of the receptor present in rat hepatic cytosol is composed of at least two DNA-binding proteins of approximately 100 and 110 kDa. Using the chemical cross-linking agent dimethyl pimelimidate, we further established that the 100-kDa form of the receptor (peak 1) associates with a different protein to generate the receptor form (peak 2) that binds to the dioxin-responsive enhancer. Photoaffinity-labeling studies indicated that only the 100-kDa protein (peak 1), and not the 110-kDa protein, binds ligand. Together, these observations imply that the DNA-binding form of the Ah receptor exists as a heteromer.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces cytochrome P450IA1 enzyme activity by activating transcription of the corresponding gene.

We have reviewed the transcriptional regulation of the CYP1A1 gene, which encodes the cytochrome P450IA1 enzyme. TCDD induces CYP1A1 transcription via a receptor- and enhancer-dependent mechanism that involves multiple protein-DNA interactions. The system provides an example of enzyme regulation at the level of gene transcription.

Protein-DNA interactions at a dioxin-responsive enhancer. Evidence that the transformed Ah receptor is heteromeric.

The Ah receptor in rat hepatic cytosol was transformed to a DNA-binding form by incubation in vitro with the ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. The transformed receptor was covalently cross-linked to a bromodeoxyuridine-substituted DNA recognition motif by exposure to ultraviolet irradiation. Analyses of the cross-linked protein-DNA complexes by gel electrophoresis and autoradiography imply that the DNA-binding form of the liganded Ah receptor is composed of two protein components, whose molecular masses are about 110 and 100 kDa. Protease digestion studies suggest that the two components have different primary structures. Photoaffinity labeling studies imply that the smaller protein is the ligand-binding component of the receptor. These findings constitute biochemical evidence that the DNA-binding form of the Ah receptor is a heterodimer.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-inducible, Ah receptor-mediated bending of enhancer DNA.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin produces its biological effects by binding to an intracellular protein, the Ah receptor. The liganded receptor activates transcription by binding to a specific recognition motif within a dioxin-responsive enhancer upstream of the target CYP1A1 gene. Here, we have used gel retardation to analyze the interaction between the liganded Ah receptor and five circularly permuted DNA fragments that contain a receptor recognition motif. Our findings indicate that the binding of the liganded receptor to its recognition motif bends the DNA at (or near) the site of the protein-DNA interaction. This observation implies that Ah receptor-induced DNA distortion may contribute to the activation of CYP1A1 transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Regulation of 5-aminolevulinate synthase in mouse erythroleukemic cells is different from that in liver.

We have measured the transcriptional gene activity of 5-aminolevulinate synthase, the first enzyme of the heme biosynthetic pathway, together with corresponding mRNA and protein levels in mouse erythroleukemic cells induced to differentiate with dimethyl sulfoxide. When the heme biosynthetic pathway was blocked by succinylacetone there was a large increase in both 5-aminolevulinate synthase activity and protein levels, and this was reversed by the addition of exogenous hemin. Transcriptional activity of the 5-aminolevulinate synthase gene and mRNA levels were both significantly increased during differentiation of cells by dimethyl sulfoxide but were not markedly altered by succinylacetone or hemin treatment. The results demonstrate that levels of 5-aminolevulinate synthase in mouse erythroleukemic cells are regulated by a significant post-transcriptional mechanism possibly at the translational level. Evidence is also presented for a less significant post-transcriptional control by heme of mRNA levels for 5-aminolevulinate synthase. These results indicate that the regulation of 5-aminolevulinate synthase in differentiating erythroid cells is complex but differs from that in liver cells where heme controls the level of 5-aminolevulinate synthase by acting primarily to inhibit gene transcription.

Regulation of 5-aminolevulinate synthase mRNA in different rat tissues.

cDNA clones for rat liver 5-aminolevulinate synthase have been isolated and used to examine mRNA levels in different rat tissues. Northern hybridization analysis of total RNA from various rat tissues showed the presence of a single 5-aminolevulinate synthase mRNA species of estimated length 2.3 kilobases. Primer extension and RNase mapping studies indicated that the mRNA is identical in all tissues. Highest basal levels were seen in liver and heart. Administration of hemin to rats reduced the basal level of this mRNA only in liver but the heme precursor, 5-aminolevulinate (or its methyl ester), repressed the basal levels in liver, kidney, heart, testis, and brain. The drug 2-allyl-2-isopropylacetamide increased the mRNA level in liver and kidney only while human chorionic gonadotropin hormone elevated the level in testis. Administration of the heme precursor 5-aminolevulinate prevented these inductions. Nuclear transcriptional run-off experiments in liver cell nuclei showed that 2-allyl-2-isopropylacetamide and 5-aminolevulinate exert their effect by altering the rate of transcription of the 5-aminolevulinate synthase gene. The results indicate that a single 5-aminolevulinate synthase mRNA is expressed in all tissues and that its transcription is negatively regulated by heme.

A unique gene for 5-aminolevulinate synthase in chickens. Evidence for expression of an identical messenger RNA in hepatic and erythroid tissues.

Reports to date have led to the conclusion that there are isozymes for 5-aminolevulinate synthase in the liver and erythroid tissue of chicken. Indeed, the existence of a multigene family for chicken 5-aminolevulinate synthase has been proposed. We find no evidence to support these proposals. In this work we show that 5-aminolevulinate synthase mRNA from chicken liver and reticulocytes is identical as determined by RNase mapping and primer extension studies and that the 5-aminolevulinate synthase protein from these tissues is the same size as judged by immunoblot analysis. We also show that a single mRNA species for 5-aminolevulinate synthase is present in chicken liver, reticulocytes, brain, and heart and an avian erythroblastosis virus-transformed chicken erythroblast cell line. Southern analysis shows the presence of only one gene copy for 5-aminolevulinate synthase in the chicken haploid genome. Overall, these results lead to the conclusion that in chickens 5-aminolevulinate synthase is encoded by a unique gene and is expressed as a single mRNA species in all tissues.