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C A Bradfield

Publications and source records attributed to C A Bradfield.

At least 19 recordsLinked to original sources
PubMed2001

Identification of toxicologically predictive gene sets using cDNA microarrays.

We have developed an approach to classify toxicants based upon their influence on profiles of mRNA transcripts. Changes in liver gene expression were examined after exposure of mice to 24 model treatments that fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Analysis of 1200 transcripts using both a correlation-based approach and a probabilistic approach resulted in a classification accuracy of between 50 and 70%. However, with the use of a forward parameter selection scheme, a diagnostic set of 12 transcripts was identified that provided an estimated 100% predictive accuracy based on leave-one-out cross-validation. Expansion of this approach to additional chemicals of regulatory concern could serve as an important screening step in a new era of toxicological testing.

Animals↗
PubMed2001

Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis.

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists, are a group of structurally diverse nongenotoxic hepatocarcinogens including the fibrate class of hypolipidemic drugs that induce peroxisome proliferation in liver parenchymal cells. Sustained activation of PPARalpha by these agents leads to the development of liver tumors in rats and mice. To understand the molecular mechanisms responsible for the pleiotropic effects of these agents, we have utilized the cDNA microarray to generate a molecular portrait of gene expression in the liver of mice treated for 2 weeks with Wy-14,643, a potent peroxisome proliferator. PPARalpha activation resulted in the stimulation of expression (fourfold or greater) of 36 genes and decreased the expression (fourfold or more decrease) of 671 genes. Enhanced expression of several genes involved in lipid and glucose metabolism and many other genes associated with peroxisome biogenesis, cell surface function, transcription, cell cycle, and apoptosis has been observed. These include: CYP2B9, CYP2B10, monoglyceride lipase, pyruvate dehydrogenase-kinase-4, cell death-inducing DNA-fragmentation factor-alpha, peroxisomal biogenesis factor 11beta, as well as several cell recognition surface proteins including annexin A2, CD24, CD39, lymphocyte antigen 6, and retinoic acid early transcript-gamma, among others. Northern blotting of total RNA extracted from the livers of PPARalpha-/- mice and from mice lacking both PPARalpha and peroxisomal fatty acyl-CoA oxidase (AOX), that were fed control and Wy-14,643-containing diets for 2 weeks, as well as time course of induction following a single dose of Wy-14,643, revealed that upregulation of genes identified by microarray procedure is dependent upon peroxisome proliferation vis-à-vis PPARalpha. However, cell death-inducing DNA-fragmentation factor-alpha mRNA, which is increased in the livers of wild-type mice treated with peroxisome proliferators, was not enhanced in AOX-/- mice with spontaneous peroxisome proliferation. These observations indicate that the activation of PPARalpha leads to increased and decreased expression of many genes not associated with peroxisomes, and that delayed onset of enhanced expression of some genes may be the result of metabolic events occurring secondary to PPARalpha activation and alterations in lipid metabolism.

Animals↗
PubMed2000

Mop3 is an essential component of the master circadian pacemaker in mammals.

Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light-dark (LD) cycles is impaired and activity levels are reduced in Mop3-/- mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a nonredundant and essential component of the circadian pacemaker in mammals.

ARNTL Transcription Factors↗
PubMed2000

Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice.

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

Animals↗
PubMed2000

The basic helix-loop-helix-PAS protein MOP9 is a brain-specific heterodimeric partner of circadian and hypoxia factors.

PAS (PER, ARNT, SIM) proteins play important roles in adaptation to low atmospheric and cellular oxygen levels, exposure to certain environmental pollutants, and diurnal oscillations in light and temperature. In an attempt to better understand how organisms sense environmental changes, we have characterized a novel member of the PAS superfamily, MOP9 (member of PAS superfamily), that maps to human chromosome 12p11.22-11.23. This protein displays significant homology to the Drosophila circadian factor CYCLE and its putative mammalian ortholog MOP3/bMAL1. Like its homologs, MOP9 forms a transcriptionally active heterodimer with the circadian CLOCK protein, the structurally related MOP4, and hypoxia-inducible factors, such as HIF1alpha. In a manner consistent with its role as a biologically relevant partner of these proteins, MOP9 is coexpressed in regions of the brain such as the thalamus, hypothalamus, and amygdala. Importantly, MOP9 is coexpressed with CLOCK in the suprachiasmatic nucleus, the site of the master circadian oscillator in mammals.

ARNTL Transcription Factors↗
PubMed2000

ARA9 modifies agonist signaling through an increase in cytosolic aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3, 7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone; i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress.

Animals↗
PubMed2000

The PAS superfamily: sensors of environmental and developmental signals.

Over the past decade, PAS domains have been identified in dozens of signal transduction molecules and various forms have been found in animals, plants, and prokaryotes. In this review, we summarize this rapidly expanding research area by providing a detailed description of three signal transduction pathways that utilize PAS protein heterodimers to drive their transcriptional output. It is hoped that these model pathways can provide a framework for use in understanding the biology of the less well-understood members of this emerging superfamily, as well as of those to be characterized in the days to come. We use this review to develop the idea that most eukaryotic PAS proteins can be classified by functional similarities, as well as by predicted phylogenetic relationships. We focus on the alpha-class proteins, which often act as sensors of environmental signals, and the beta-class proteins, which typically act as broad-spectrum partners that target these heterodimers to their genomic targets.

Amino Acid Sequence↗
PubMed1999

Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation.

The aryl hydrocarbon receptor (AHR) and the alpha-class hypoxia inducible factors (HIF1alpha, HIF2alpha, and HIF3alpha) are basic helix-loop-helix PAS (bHLH-PAS) proteins that heterodimerize with ARNT. In response to 2,3,7,8-tetrachlorodibenzo-p-dioxin, the AHR. ARNT complex binds to "dioxin responsive enhancers" (DREs) and activates genes involved in the metabolism of xenobiotics, e.g. cytochrome P4501A1 (Cyp1a1). The HIF1alpha.ARNT complex binds to "hypoxia responsive enhancers" and activates the transcription of genes that regulate adaptation to low oxygen, e.g. erythropoietin (Epo). We postulated that activation of one pathway would inhibit the other due to competition for ARNT or other limiting cellular factors. Using pathway specific reporters in transient transfection assays, we observed that DRE driven transcription was markedly inhibited by hypoxia and that hypoxia responsive enhancer driven transcription was inhibited by AHR agonists. When we attempted to support this cross-talk model using endogenous loci, we observed that activation of the hypoxia pathway inhibited Cyp1a1 up-regulation, but that activation of the AHR actually enhanced the induction of Epo by hypoxia. To explain this unexpected additivity, we examined the Epo gene and found that its promoter harbors DREs immediately upstream of its transcriptional start site. These experiments outline conditions where inhibitory and additive cross-talk occur between the hypoxia and dioxin signal transduction pathways and identify Epo as an AHR-regulated gene.

Base Sequence↗
PubMed1998

Characterization of the Ah receptor-associated protein, ARA9.

The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9. We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex. Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9. Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR. In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GR-Hsp90 complexes. In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes. The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist beta-napthoflavone, decreasing the EC50 by greater than 5-fold and increasing the maximal response 2.5-fold. In contrast, co-expression of FKBP52 had no effect on AHR signaling. In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3H-FK506 could not be detected. Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.

Animals↗
PubMed1998

Ahr null alleles: distinctive or different?

Two independent laboratories have generated Ahr "null" or "knockout" mice that share some common characteristics but also have distinct phenotypes. In this Commentary, we will discuss our view of the candidate variables that might account for these differences. More importantly, we hope that this discussion can identify important parameters to be assessed by investigators in the process of characterizing their own modified loci. The variables that we have considered include the possibility that different targeting strategies can result in altered products with unsuspected function or that the targeting event itself can alter the function of neighboring genes. Further, genetic background can have an important influence on phenotype, and differences in genome can be introduced during derivation by the type of embryonic stem cells used and by the random segregation of parental genes in the F2 generation of line propagation. In addition, phenotype may be acutely sensitive to environmental variables, such as pathogen and chemical exposure and stress introduced by crowding and disease. Finally, we discuss approaches to resolving differences between null mice and propose a partial solution, the institution of a repository for detailed information on targeted alleles that may not typically be allowed in today's "fast paced" scientific publications.

Alleles↗
PubMed1998

The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors.

We report that MOP3 is a general dimerization partner for a subset of the basic-helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) superfamily of transcriptional regulators. We demonstrated that MOP3 interacts with MOP4, CLOCK, hypoxia-inducible factor 1alpha (HIF1alpha), and HIF2alpha. A DNA selection protocol revealed that the MOP3-MOP4 heterodimer bound a CACGTGA-containing DNA element. Transient transfection experiments demonstrated that the MOP3-MOP4 and MOP3-CLOCK complexes bound this element in COS-1 cells and drove transcription from a linked luciferase reporter gene. We also deduced the high-affinity DNA binding sites for MOP3-HIF1alpha complex (TACGTGA) and used transient transfection experiments to demonstrate that the MOP3-HIF1alpha and MOP3-HIF2alpha heterodimers bound this element, drove transcription, and responded to cellular hypoxia. Finally, we found that MOP3 mRNA expression overlaps in a number of tissues with each of its four potential partner molecules in vivo.

Animals↗
PubMed1998

Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization partner the aryl hydrocarbon receptor nuclear translocator.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants. Here, we describe orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematode Caenorhabditis elegans, encoded by the genes ahr-1 and aha-1, respectively. The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate that C. elegans AHR-1, like vertebrate AHR, requires some form of post-translational activation. Moreover, this requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor: C. elegans AHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by beta-naphthoflavone in the yeast system. The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators.

Amino Acid Sequence↗
PubMed1998

Expression of ARNT, ARNT2, HIF1 alpha, HIF2 alpha and Ah receptor mRNAs in the developing mouse.

The basic helix-loop-helix-PAS (bHLH-PAS) protein ARNT is a dimeric partner of the Ah receptor (AHR) and hypoxia inducible factor 1 alpha(HIF1 alpha). These dimers mediate biological responses to xenobiotic exposure and low oxygen tension. The recent cloning of ARNT and HIF1(homologues (ARNT2 and HIF2 alpha) indicates that at least six distinct bHLH-PAS heterodimeric combinations can occur in response to a number of environmental stimuli. In an effort to understand the biological relevance of this combinatorial complexity, we characterized their relative expression at a number of developmental time points by parallel in situ hybridization of adjacent tissue sections. Our results reveal that in general there is limited redundancy in the expression of these six transcription factors and that each of these bHLH-PAS members displays a unique pattern of developmental expression emerging as early as embryonic day 9.5.

Animals↗
PubMed1998

Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel alpha-class hypoxia inducible factor, HIF3alpha. The HIF3alpha open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1alpha and HIF2alpha (57% and 53% identity, respectively). The C-terminus of HIF3alpha contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1alpha. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3alpha dimerizes with a prototype beta-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3alpha-ARNT interaction can occur in vivo, and that the activity of HIF3alpha is upregulated in response to cobalt chloride or low oxygen tension.

Amino Acid Sequence↗
PubMed1997

Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo.

In an effort to identify regulators of aryl hydrocarbon receptor (AHR) signaling, we have employed the yeast two-hybrid system to screen for human proteins that interact in a ligand-dependent manner with the AHR. After screening 1.4 x 10(6) clones from a human B cell library, two distinct clones were identified that associated specifically with the liganded receptor. No clones were identified that interacted preferentially with the unliganded AHR. One of the ligand-dependent clones, ARA9, encodes a novel 330-amino acid protein with regions of amino acid sequence similarity to the 52-kDa FK506-binding protein known to be associated with the glucocorticoid receptor. Yeast two-hybrid experiments with ARA9 demonstrated a strong interaction with the AHR that is enhanced 11-fold in the presence of the ligand beta-naphthoflavone. In vitro experiments using proteins generated in reticulocyte lysates confirmed this interaction and indicated that ARA9 can be co-immunoprecipitated with the AHR using antisera raised specifically for either the AHR or the 90-kDa heat shock protein. The observation that ARA9 has a high affinity for both the 90-kDa heat shock protein-associated and ligand-activated forms of the AHR suggests that ARA9 is a component of the AHR-signaling pathway in vivo.

Amino Acid Sequence↗
PubMed1997

Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway.

In an effort to better understand the mechanism of toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, we employed an iterative search of human expressed sequence tags to identify novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AHR) or the Ah receptor nuclear translocator (ARNT). We characterized five new "members of the PAS superfamily," or MOPs 1-5, that are similar in size and structural organization to the AHR and ARNT. MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5 contained the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains a bHLH domain, but the entire open reading frame has yet to be completed. Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments demonstrated that MOP1 and MOP2 dimerize with ARNT and that these complexes are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 was found to associate with the AHR in vitro but not in vivo. This observation, coupled with the fact that MOP3 formed tighter associations with the 90-kDa heat shock protein than the human AHR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AHR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share ARNT as a common dimeric partner, suggests that the cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway.

Amino Acid Sequence↗
PubMed1997

Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT.

The arylhydrocarbon-receptor nuclear translocator (ARNT) is a member of the basic-helix-loop-helix-PAS family of heterodimeric transcription factors which includes the arylhydrocarbon receptor (AHR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the Drosophila single-minded protein (Sim). ARNT forms heterodimeric complexes with the arylhydrocarbon receptor, HIF-1alpha, Sim and the PAS protein Per. In response to environmental pollutants, AHR-ARNT heterodimers regulate genes involved in the metabolism of xenobiotics, whereas ARNT-HIF-1alpha heterodimers probably regulate those involved in the response to oxygen deprivation. By generating a targeted disruption of the Arnt locus in the mouse, we show here that Arnt-/- embryonic stem cells fail to activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond to a decrease in glucose concentration, indicating that ARNT is crucial in the response to hypoxia and to hypoglycaemia. Arnt-/- embryos were not viable past embryonic day 10.5 and showed defective angiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. The defect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reported for mice deficient in vascular endothelial growth factor or tissue factor. On the basis of these findings, we propose a model in which increasing tissue mass during organogenesis leads to the formation of hypoxic/nutrient-deprived cells, the subsequent activation of ARNT, and a concomitant increase in the expression of genes (including that encoding vascular endothelial growth factor) that promote vascularization of the developing yolk sac and solid tissues.

Animals↗