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H I Swanson

Publications and source records attributed to H I Swanson.

17 recordsLinked to original sources
PubMed2000

The aryl hydrocarbon receptor interacts with estrogen receptor alpha and orphan receptors COUP-TFI and ERRalpha1.

The molecular mechanisms underlying the apparent "cross-talk" between estrogen receptor (ER)- and arylhydrocarbon receptor (AHR)-mediated activities are unknown. To determine how AHR ligand 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may inhibit ER action and, conversely, to examine how 17-beta-estradiol (E(2)) affects AHR activity, we examined discrete activities of each receptor, i.e., protein-protein interactions, DNA binding, and transcriptional activation. We report that AHR interacts directly with ERalpha, COUP-TF, and ERRalpha1, in a ligand-specific manner in vitro. Unoccupied or beta-napthoflavone (beta-NF)-occupied AHR showed stronger interaction with ERalpha, COUP-TF, and ERRalpha1 than when AHR was occupied by the partial antagonist alpha-naphthoflavone (alpha-NF), indicating a role for ligand in AHR interaction with these proteins. We also report that AHR interacts with COUP-TF in transfected CV-1 cells. In contrast, the AHR nuclear translocator protein (ARNT) did not interact with COUP-TF, ERRalpha1, or ERalpha. We next examined the interaction of either ERalpha or COUP-TF with a consensus xenobiotic response element (XRE). Purified ERalpha did not bind the consensus XRE, but COUP-TFI bound the consensus XRE, suggesting a role for COUP-TF as a AHR/ARNT competitor for XRE binding. In transiently transfected MCF-7 human breast cancer cells, overexpression of COUP-TFI inhibited TCDD-activated reporter gene activity from the CYP1A1 promoter. TCDD inhibited estradiol (E(2))-activated reporter gene activity from a consensus ERE and from the EREs in the pS2 and Fos genes, and COUP-TFI did not block the antiestrogenic activity of TCDD. The specific interaction of COUP-TF with XREs and AHR together with the inhibition of TCDD-induced gene expression by COUP-TF suggests that COUP-TF may regulate AHR action both by direct DNA binding competition and through protein-protein interactions.

Amino Acid Sequence↗
PubMed2000

Role of heat shock protein 90 dissociation in mediating agonist-induced activation of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a cytosolic basic helix-loop-helix protein that associates with a chaperone complex that includes two molecules of heat shock protein 90 (HSP90). It has been hypothesized that after ligand binding, the AhR dissociates from its chaperone complex and translocates into the nucleus, where it heterodimerizes with its DNA binding partner, the AhR nuclear translocator (ARNT), and activates specific genes. However, it remains unclear whether nuclear translocation of the AhR occurs before or after dissociation of the HSP90/chaperone complex. Because sodium molybdate stabilizes the AhR-HSP90 interaction and inhibits the gene activation of a number of steroid receptors, we reasoned that molybdate would be a useful tool in delineating the role of HSP90 dissociation in AhR nuclear translocation. In this study, we demonstrate that molybdate inhibits AhR gene activation in both HepG2 and Hepa-1 cells in a concentration-dependent manner and protects the AhR against agonist-induced proteolysis. In addition, we demonstrate that AhR/ARNT dimerization, but not nuclear translocation of the AhR, is inhibited by molybdate. This indicates that 1) HSP90 dissociation is not required for nuclear translocation of the AhR, 2) HSP90 dissociation is essential for formation of the AhR/ARNT heterodimer, and 3) an additional undefined regulatory step is required for AhR/ARNT dimerization in the nucleus.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1999

The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements.

To determine the molecular mechanisms underlying the "cross talk" between the activity of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), which binds to arylhydrocarbon receptor (AHR) and estradiol (E2)-liganded estrogen receptor (ER), we first examined the initial step of estrogen action, ligand binding to ER. None of the AHR ligands tested, i.e. TCDD, benzo[a]pyrene, 3,3',4,4',5-pentachlorobiphenyl, beta-naphthoflavone, or alpha-naphthoflavone, bound to ER alpha. We report the first examination of TCDD interaction with ER beta: TCDD did not displace E2 from ER beta. We then examined a second possible mechanism, i.e. direct inhibition of ER alpha binding to estrogen response elements (EREs) by the AHR/AHR nuclear translocator (ARNT) complex. The AHR/ARNT heterodimer did not bind either a full or half-site ERE. However, AHR/ARNT bound specifically to oligomers containing naturally occurring EREs derived from the human c-fos, pS2, and progesterone receptor (PR) gene promoters that include xenobiotic response element (XRE)-like sequences. In contrast, neither purified E2-liganded-ER from calf uterus or recombinant human ER alpha bound a consensus XRE. TCDD inhibited E2-activated reporter gene activity from a consensus ERE and from EREs in the pS2, PR, and Fos genes in transiently transfected MCF-7 human breast cancer cells. However, this inhibition was not reciprocal since E2 did not inhibit TCDD-stimulated luciferase activity from the CYP1A1 promoter in transiently transfected MCF-7 or human endometrial carcinoma HEC-1A cells. We propose that at least part of the mechanism by which the AHR/ARNT complex inhibits estrogen action is by competitively inhibiting ER alpha binding to imperfect ERE sites, adjacent to or overlapping XREs.

Animals↗
PubMed1999

Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site.

Basic helix-loop-helix proteins that interact with the DNA recognition site CACGTG include the c-Myc/Max heterodimer and the ARNT (Ahreceptornucleartranslocator) homodimer. We have utilized a PCR-based protocol to identify high affinity binding sites of either the c-Myc/Max or ARNT/ARNT dimers and analyzed the ability of these dimers to interact with their derived consensus sequences and activate genes. chi(2)analysis of the selected DNA recognition sites revealed that DNA binding of the ARNT homodimer is symmetric, resulting in the consensus sequence RTCACGTGAY. Gel shift analysis demonstrated that the flanking nucleotides play an important role in dictating DNA binding affinity of the ARNT homodimer. These flanking sequences also regulate the ability of ARNT to competitively displace the c-Myc/Max heterodimer from a CACGTG-containing sequence. However, transient transfection analyses in CV-1 cells revealed that ARNT and c-Myc/Max exhibited similar abilities to activate transcription through each other's consensus sequences. Taken together, these results indicate that although binding affinity of these dimers for the CACGTG core sequences may be differentially influenced by flanking nucleotides, transcriptional activity may also be determined by other factors, such as cellular concentrations of these proteins and their co-activators.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1998

The aryl hydrocarbon receptor interacts with transcription factor IIB.

The aryl hydrocarbon receptor (AHR) and its DNA binding partner, the AHR nuclear translocator (ARNT), are basic helix-loop-helix transcription factors that mediate many of the toxic and carcinogenic effects of polyhalogenated aromatic hydrocarbons. The basic regions of the AHR and ARNT contact the GCGTG recognition site, whereas both their helix-loop-helix domains and periodicity-ARNT-single-minded domains participate in heterodimerization. To delineate the transcription factors that may facilitate DNA binding and transcriptional activation of the AHR/ARNT heterodimer, we questioned whether transcription factor IIB (TFIIB) may interact with either the AHR or ARNT and whether this interaction may affect the ability of the AHR/ARNT complex to bind DNA. Coaffinity precipitation assays demonstrated that both the AHR and ARNT were capable of interacting with TFIIB. Domain mapping experiments revealed that TFIIB interacts with the periodicity-ARNT-single-minded and carboxyl-terminal regions of the AHR. To determine whether the interaction between TFIIB and the AHR may affect DNA binding of the AHR and ARNT complex, we performed gel shift experiments in the absence and presence of TFIIB. The addition of TFIIB significantly increased the formation of the AHR/ARNT DNA binding complex, but only if TFIIB was first allowed to interact with the AHR before the addition of ARNT. These results indicate that TFIIB interacts with the AHR and may stabilize the DNA binding form of the AHR and thereby augment the ability of the AHR/ARNT complex to interact with its DNA recognition site.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1996

Mapping the protein/DNA contact sites of the Ah receptor and Ah receptor nuclear translocator.

The Ah receptor (AHR) and its DNA binding partner, the Ah receptor nuclear translocator (ARNT), are basic helix-loop-helix proteins distinguished by their PER, AHR, ARNT, and SIM (PAS) homology regions. To identify the amino acids of the AHR.ARNT heterodimer that contact the TNGCGTG recognition sequence, we have performed deletion mapping and amino acid substitutions within the N termini of both the AHR and ARNT. The ability of the variant AHR and ARNT proteins to bind DNA and activate gene transcription was determined by the gel shift analysis and transient transfection assays. We have found that the amino acids of ARNT that contact DNA are similar to those of other basic/helix-loop-helix proteins and include glutamic acid residue 83 and arginine residues 86 and 87. Although our initial experiments indicated that DNA binding of the AHR may involve two regions that are bordered by amino acids 9-17 and amino acids 34-42, further analysis demonstrated that only amino acids 34-39 are critical for the AHR.TNGC interaction. These experiments indicate that while the structural features of the ARNT.GTG complex may closely resemble that deduced for proteins such as Max, E47, and USF, the AHR.TNGC complex may represent a unique DNA binding form of basic/helix-loop-helix proteins.

Amino Acid Sequence↗
PubMed1995

DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins.

The Ah receptor (AHR), the Ah receptor nuclear translocator protein (ARNT), and single-minded protein (SIM) are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatory proteins. In this study, we examine the DNA half-site recognition and pairing rules for these proteins using oligonucleotide selection-amplification and coprecipitation protocols. Oligonucleotide selection-amplification revealed that a variety of bHLH-PAS protein combinations could interact, with each generating a unique DNA binding specificity. To validate the selection-amplification protocol, we demonstrated the preference of the AHR.ARNT complex for the sequence commonly found in dioxin-responsive enhancers in vivo (TNGCGTG). We then demonstrated that the ARNT protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence, CACGTG. Further examination indicated that ARNT may have a relaxed partner specificity, since it was also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and ARNT were consistent with the idea that the ARNT protein has a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AHR, SIM, and ARNT are T(C/T)GC, GT(G/A)C (5'-half-sites), and GTG (3'-half-sites), respectively.

Amino Acid Sequence↗
PubMed1995

DNA binding of the transformed guinea pig hepatic Ah receptor complex: identification and partial characterization of two high-affinity DNA-binding forms.

We have examined and characterized the binding of transformed guinea pig hepatic Ah receptor to its specific DNA recognition site, the dioxin-responsive element (DRE), using gel retardation analysis. Saturation binding analysis of transformed TCDD:AhR complexes were indicative of a single high-affinity binding site (Kd = 2.5 +/- 0.8 nM); however, DNA-binding analysis revealed the presence of two distinct TCDD-inducible protein-DRE complexes. Sucrose gradient centrifugation and subsequent gel retardation analysis of the fractions demonstrated a similarity in the distribution of [3H]TCDD-specific binding and TCDD-inducible protein-DNA complex formation, supporting the presence of the AhR in both complexes. In addition, the formation of both DNA-binding complexes exhibited the same nucleotide specificity previously determined for the AhR complex. Since labeling studies using a radio-iodinated photoaffinity dioxin agonist demonstrated that guinea pig cytosol contains a single ligand binding subunit of 105 kDa, the difference in migration of the complexes is due to other proteins associated with each complex. Overall, our results demonstrate the presence of two distinct high affinity DNA-binding forms of transformed guinea pig AhR complex which exhibit similar DNA-binding affinity and nucleotide specificity.

Animals↗
PubMed1993

Binding of transformed Ah receptor complex to a dioxin responsive transcriptional enhancer: evidence for two distinct heteromeric DNA-binding forms.

Guinea pig hepatic Ah receptor (AhR) complex was transformed in vitro to its DNA-binding form by incubation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Transformed TCDD-AhR was covalently cross-linked by UV-irradiation to a bromodeoxyuridine-substituted oligonucleotide containing its specific DNA recognition site, the dioxin responsive element (DRE). Denaturing gel electrophoresis and autoradiography identified four TCDD-inducible protein-DNA complexes, with molecular masses of approximately 97, 105, and 115 kDa and a somewhat broader complex at 247 kDa. The 247-kDa complex appears to contain two distinct protein-DNA complexes of approximately 232 and 256 kDa and represents two proteins covalently cross-linked to a single DRE oligonucleotide, while the 97, 105, and 115-kDa complexes represent single protein-DRE cross-links. UV cross-linking to DRE oligonucleotides containing variable numbers of BrdU residues revealed that the 105-kDa protein, identified as the AhR ligand-binding subunit by photoaffinity labeling with a radioiodinated AhR agonist, cross-links to the DRE core consensus (5'-GCGTG-3'); the 97- and 115-kDa non-ligand-binding proteins differentially cross-link immediately 5'-ward of the core. Overall, our results not only demonstrate that the critical protein-DNA contacts which occur between the AhR complex and the DRE are made primarily by the ligand-binding subunit but also indicate that the AhR complex exists as two distinct heteromeric DNA-binding forms, containing one 105-kDa ligand-binding subunit and either one 115- or one 97-kDa non-ligand-binding subunit.

Animals↗
PubMed1993

In vitro analysis of Ah receptor domains involved in ligand-activated DNA recognition.

The Ah receptor (AHR) is a basic helix-loop-helix protein that mediates the effects of 2,3,7,8-tetrachloro-dibenzo-p-dioxin. In this report, we describe a rabbit reticulocyte system that allows functional expression of both the AHR and its dimeric partner, the AHR nuclear translocator protein (ARNT). By using this in vitro system, we were able to reconstitute agonist binding to the AHR and agonist-induced AHR-ARNT recognition of a cognate DNA enhancer sequence. Expression of AHR deletion mutants revealed the location of N-terminal domains responsible for ligand and DNA recognition and C-terminal domains that play roles in agonist-induced DNA recognition.

Animals↗
PubMed1993

Half-life of aryl hydrocarbon receptor in Hepa 1 cells: evidence for ligand-dependent alterations in cytosolic receptor levels.

The rate of turnover of the Ah receptor (AhR) was determined using the density shift method in Hepa 1 and in a Hepa 1 mutant line, c4, which fails to accumulate AhR complexes in the nucleus. The half-life of the AhR was found to be 7.7 and 9.7 h in Hepa 1 and c4 cells, respectively. The effect of AhR occupation with either an agonist, beta-naphthoflavone (beta NF), or a partial antagonist, alpha-naphthoflavone (alpha NF), on AhR half-life and concentration in the cytosolic fraction was examined. In Hepa 1 cells, a 12-h exposure to beta NF resulted in a 62% decrease in AhR concentration. The same treatment, using alpha NF as the ligand, resulted in a 14% decrease. The half-life of the AhR increased from 7.7 to 9.3 h during beta NF treatment and was essentially the same during alpha NF treatment in Hepa 1 cells. In c4 cells, a 12-h exposure to beta NF resulted in a 44% decrease in AhR concentrations, whereas exposure to alpha NF resulted in an 8% decrease. The half-life of the AhR in c4 cells during beta NF exposure increased from 9.7 to 14.6 h, and alpha NF exposure increased half-life to 17.6 h. These results indicate: (a) cytosolic AhR concentrations are modulated by ligand occupation, (b) exposure to AhR ligands, after an initial decrease in AhR levels, resulted in an increase in AhR half-life, and (c) similar results were obtained in Hepa 1 and c4 cells, this would indicate that AhR occupation with ligand and subsequent AhR-ligand nuclear translocation does not appear to play a significant role in regulation of AhR half-life in Hepa 1 cells.

Animals↗
PubMed1993

Protein kinase C is not involved in Ah receptor transformation and DNA binding.

Induction of cytochrome P4501A1 by 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) is mediated by the Ah receptor (AhR) complex, a ligand-dependent DNA-binding transactivator. Recently a role for protein kinase C (PKC) in the induction response has been reported in which PKC or a related kinase positively modulates AhR activity. We have examined the role of PKC by determining the effect of two nonspecific PKC inhibitors, H7 and staurosporine, and one specific PKC inhibitor, calphostin c, on AhR functionality. Although no kinase activity was detectable in cytosol, under the conditions used for our assays, AhR transformation and DNA binding still occurred. Addition of relatively high concentrations of the kinase inhibitors also had no significant effect on TCDD:AhR:DRE complex formation. Thus, our results indicate that protein kinase activity does not appear to be necessary for TCDD-dependent AhR transformation and DNA binding and they imply that protein kinases must play a role in another step(s) in the AhR-dependent mechanism of P4501A1 induction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

The AH-receptor: genetics, structure and function.

The AH-receptor is a ligand-activated transcription factor that regulates a number of biological responses to planar aromatic hydrocarbons. Interest in this receptor is related to its role in the toxic action of a variety of environmental chemicals, the simplicity and elegance of the murine genetics that led to its characterization and the distinctive mechanism by which this receptor activates gene expression. Recent cloning experiments have demonstrated that the AH-receptor is structurally related to the Per, ARNT and Sim proteins. Members of this newly described gene family are characterized by two N-terminal domains, the most characteristic of which is a motif referred to as a PAS domain. In the AH-receptor, this domain harbours sequences involved in the formation of a hydrophobic pocket that bind receptor agonists. Adjacent to the PAS domain in the AH-receptor, ARNT and Sim proteins is a basic/helix-loop-helix (bHLH) domain that appears to mediate heterodimerization and sequence specific DNA binding properties. The observation that the bHLH domain is present in the AH-receptor and the ARNT protein, a factor required for proper AH-receptor function, suggests that these proteins are heterodimeric partners that activate gene expression in a manner similar to Myc/Max and MyoD/E2A. The objectives of this review are to describe recent experimental results in this field and to use this information to develop a molecular model of AH-receptor mediated signal transduction.

Animals↗
PubMed1993

Cloning and expression of a human Ah receptor cDNA.

In this report, we describe the cloning and expression of a cDNA encoding a human Ah receptor, a basic/helix-loop-helix protein that mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison of this human cDNA with a murine homologue (Ahb1 allele) indicates that the molecular mass variation observed between the receptors found in these two species results from hypervariability of amino acid sequences in the carboxyl termini (< 60% conserved over 450 amino acids). Differential usage of stop codons generates proteins with molecular masses that differ by 6 kDa. In contrast, the amino-terminal halves of these proteins are highly conserved and show 90% amino acid sequence identity. Northern blot analysis indicates that the human Ah receptor mRNA is expressed at its highest levels in placenta and is also highly expressed in lung, heart, pancreas, and liver, with lower levels of expression found in brain, kidney, and skeletal muscle. Expression of the human cDNA in a rabbit reticulocyte lysate system allowed functional analysis of ligand binding, agonist-induced and Ah receptor nuclear translocator-dependent DNA binding, and receptor stabilization by sodium molybdate.

Amino Acid Sequence↗
PubMed1991

Detection of the Ah receptor in rainbow trout: use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin in cell culture.

The Ah receptor was detected in RTG-2 cells (rainbow trout embryonic gonad cells) following the addition of the photoaffinity ligand, [125I]2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, to cells in culture. Cytosolic and nuclear extracts were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed one radiolabeled band. Very little non-specific binding was observed under the conditions employed when compared to photoaffinity labeling RTG-2 cytosolic extracts in vitro. The photoaffinity-labeled Ah receptor in RTG-2 cytosol was analyzed by sucrose density centrifugation. The cytosolic form was observed to sediment at approximately 9.8S and the high salt nuclear extract form at approximately 7.5S. The relative molecular weight of the Ah receptor was determined to be 145 kDa under denaturing conditions and is considerably larger than the Ah receptor from mammalian sources. Inhibition of photoaffinity ligand binding to the RTG-2 cytosolic Ah receptor by competing ligands revealed the same rank order of ligand affinity as that previously demonstrated for the mouse Ah receptor.

Affinity Labels↗
PubMed1990

Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B1 (AFB1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500-2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM2 and LM4b, as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HCl) to act as scavengers for the direct alkylating agent AFB1-8,9-Cl2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB1-8,9-Cl2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB1. RXM increased the apparent Km for the AFB1-DNA binding reaction without changing the associated Vmax; the apparent Km values at 0, 3.5, 35, and 350 microM RXM were 35, 38, 66, and 86 microM for trout liver microsomes. RXM also inhibited the activation of AFB1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by beta-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

7-Alkoxycoumarin O-Dealkylase↗