Jacqueline McGuire

A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.

A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor.

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.

Distinct Roles of the Molecular Chaperone hsp90 in Modulating Dioxin Receptor Function via the Basic Helix-Loop-Helix and PAS Domains

The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.

A constitutively active aryl hydrocarbon receptor causes loss of peritoneal B1 cells

The dioxin/aryl hydrocarbon (Ah) receptor functions as a ligand-activated transcription factor that mediates toxicity of dioxins and related environmental pollutants. We have developed a transgenic mouse model that expresses a constitutively active Ah receptor. The immune system is one of the most sensitive target organs for dioxin toxicity and we have therefore investigated alterations of different lymphocyte populations in these mice. The population of mature bone-marrow derived B cells was enlarged, consistent with previous findings in dioxin exposed mice. In contrast, the peritoneal population of CD5-expressing B cells (B1 cells) was significantly diminished. This is the first study that demonstrates the effect of an activated Ah receptor on B1 cells. Since these cells are important mediators of innate immunity against pathogens such as Influenza virus, these results may explain the decreased resistance against infections that has been documented after dioxin exposure.

Conditional expression of a constitutively active aryl hydrocarbon receptor in MCF-7 human breast cancer cells.

In addition to inducing transcription of a battery of target genes encoding drug-metabolizing enzymes, the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce antiestrogenic responses. However, the mechanisms underlying such complex biologic responses affecting growth and differentiation remain unclear. In the present study we have investigated biological effects of a constitutively active mutant of the aryl hydrocarbon (Ah) receptor (CA-AhR), in particular whether it modulates estrogen receptor function in human MCF-7 breast cancer cells. To this end, the CA-AhR protein was conditionally expressed using the tet repressor. Expression of CA-AhR resulted in constitutive formation of a DNA-binding AhR-aryl hydrocarbon receptor nuclear translocator heterodimeric complex and enhanced expression of the Ah receptor target gene CYP1A1 in the absence of TCDD. Moreover, expression of CA-AhR inhibited estrogen-dependent cathepsin D expression and growth of these cells. Thus, the present model system conditionally expressing the CA-AhR protein provides a novel tool for the investigation of AhR-mediated signaling pathways.

Cloning and regulation by glucocorticoid receptor ligands of a rat hsp90

We have isolated a full length cDNA that encodes a heat shock protein, hsp90, from a rat brain library and present the nucleotide sequence and deduced amino acid sequence. Comparison of the entire nucleotide sequence with mouse hsp84 and human hsp90 beta cDNAs reveal sequence similarities of 92 and 87%, respectively. The coding region of 2172 nucleotides corresponds to a polypeptide chain of 724 amino acids. Comparison with mouse hsp84 and human hsp90 beta amino acid sequences indicates a similarity of 97%, respectively. Characterization of the constitutive expression of this cDNA both by RNA blot hybridization and immunoblotting, reveals that it is expressed in all rat tissues examined. Hsp90 has been shown to form a transient complex with steroid hormone receptors. In order to further elucidate the role of hsp90 in the endocrine response of cells, we have examined the effects of dexamethasone and RU38486 on the level of hsp90 mRNA in a system in which glucocorticoids down-regulate glucocorticoid receptor mRNA levels. In this system, a subtle but reproducible approx. 2-fold decrease in hsp90 mRNA levels is observed after 48 h treatment with dexamethasone.