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Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor.

Bunger MK, Glover E, Moran SM, Walisser JA, Lahvis GP, Hsu EL, Bradfield CA - Toxicol. Sci. (2008)

Bottom Line: The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes.Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs.These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Wisconsin 53706, USA.

ABSTRACT
The aryl hydrocarbon receptor (AHR) is known for its role in the adaptive and toxic responses to a large number of environmental contaminants, as well as its role in hepatovascular development. The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes. The AHR has also been implicated in signaling events independent of nuclear localization and DNA binding, and it has been suggested that such pathways may play important roles in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs. Using this model, we provide evidence that DNA binding is required AHR-mediated liver development, as Ahr(dbd/dbd) mice exhibit a patent ductus venosus, similar to what is seen in Ahr(-/-) mice. Furthermore, Ahr(dbd/dbd) mice are resistant to TCDD-induced toxicity for all endpoints tested. These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

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Cellular characterization of the AHRdbd protein. (A) Luciferase assay for DRE-driven transcription. Ahr−/− 3T3 fibroblasts were transfected with equal amounts of DRE-Luc (PL256) and either AHR or AHRdbd recombinant cDNAs. Cells were then treated with 1nM TCDD (black bars) or 0.1% DMSO alone (white bars) for 24 h. Values represent relative luciferase units normalized to total protein levels. (B) Subcellular localization of AHRdbd. Indirect immunofluorescence was used to identify the subcellular localization of AHRdbd in Ahr−/− 3T3 fibroblasts transiently transfected with either Ahr+/+ or Ahrdbd/dbd. Prior to staining, nuclear translocation was induced by exposure of cells to 1nM TCDD for 2 h prior to staining. (C) Mammalian 2-hybrid analysis of AHRdbd interactions. The schematic diagram depicts the reporter construct (pG5luc), the “bait” construct (Gal-ARNT), and the “fish” construct (AHR), showing the amino acid sequence of the basic region in wild-type (wt) and AHRdbd (dbd) recombinant proteins. The two-hybrid analysis was carried out using equal amounts (0.33 μg) of transiently transfected Gal-ARNT and either wild-type AHR or AHRdbd, followed by incubation with 0.1% DMSO or 1nM TCDD. Values are expressed as relative luciferase units (*p < 0.001).
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fig3: Cellular characterization of the AHRdbd protein. (A) Luciferase assay for DRE-driven transcription. Ahr−/− 3T3 fibroblasts were transfected with equal amounts of DRE-Luc (PL256) and either AHR or AHRdbd recombinant cDNAs. Cells were then treated with 1nM TCDD (black bars) or 0.1% DMSO alone (white bars) for 24 h. Values represent relative luciferase units normalized to total protein levels. (B) Subcellular localization of AHRdbd. Indirect immunofluorescence was used to identify the subcellular localization of AHRdbd in Ahr−/− 3T3 fibroblasts transiently transfected with either Ahr+/+ or Ahrdbd/dbd. Prior to staining, nuclear translocation was induced by exposure of cells to 1nM TCDD for 2 h prior to staining. (C) Mammalian 2-hybrid analysis of AHRdbd interactions. The schematic diagram depicts the reporter construct (pG5luc), the “bait” construct (Gal-ARNT), and the “fish” construct (AHR), showing the amino acid sequence of the basic region in wild-type (wt) and AHRdbd (dbd) recombinant proteins. The two-hybrid analysis was carried out using equal amounts (0.33 μg) of transiently transfected Gal-ARNT and either wild-type AHR or AHRdbd, followed by incubation with 0.1% DMSO or 1nM TCDD. Values are expressed as relative luciferase units (*p < 0.001).

Mentions: To determine whether the AHRdbd could signal effectively in cell culture, we performed transient transfections of AHR or AHRdbd with a DRE-driven luciferase reporter in immortalized Ahr−/− 3T3 fibroblasts. Upon transfection of wild-type AHR cDNA, luciferase activity increased relative to cells transfected with reporter alone. This response was enhanced 2.5-fold by exposure of the cells to 1nM TCDD. In comparison, luciferase activity in cells transfected with AHRdbd did not increase upon exposure of cells to TCDD (Fig. 3A).


Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor.

Bunger MK, Glover E, Moran SM, Walisser JA, Lahvis GP, Hsu EL, Bradfield CA - Toxicol. Sci. (2008)

Cellular characterization of the AHRdbd protein. (A) Luciferase assay for DRE-driven transcription. Ahr−/− 3T3 fibroblasts were transfected with equal amounts of DRE-Luc (PL256) and either AHR or AHRdbd recombinant cDNAs. Cells were then treated with 1nM TCDD (black bars) or 0.1% DMSO alone (white bars) for 24 h. Values represent relative luciferase units normalized to total protein levels. (B) Subcellular localization of AHRdbd. Indirect immunofluorescence was used to identify the subcellular localization of AHRdbd in Ahr−/− 3T3 fibroblasts transiently transfected with either Ahr+/+ or Ahrdbd/dbd. Prior to staining, nuclear translocation was induced by exposure of cells to 1nM TCDD for 2 h prior to staining. (C) Mammalian 2-hybrid analysis of AHRdbd interactions. The schematic diagram depicts the reporter construct (pG5luc), the “bait” construct (Gal-ARNT), and the “fish” construct (AHR), showing the amino acid sequence of the basic region in wild-type (wt) and AHRdbd (dbd) recombinant proteins. The two-hybrid analysis was carried out using equal amounts (0.33 μg) of transiently transfected Gal-ARNT and either wild-type AHR or AHRdbd, followed by incubation with 0.1% DMSO or 1nM TCDD. Values are expressed as relative luciferase units (*p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563146&req=5

fig3: Cellular characterization of the AHRdbd protein. (A) Luciferase assay for DRE-driven transcription. Ahr−/− 3T3 fibroblasts were transfected with equal amounts of DRE-Luc (PL256) and either AHR or AHRdbd recombinant cDNAs. Cells were then treated with 1nM TCDD (black bars) or 0.1% DMSO alone (white bars) for 24 h. Values represent relative luciferase units normalized to total protein levels. (B) Subcellular localization of AHRdbd. Indirect immunofluorescence was used to identify the subcellular localization of AHRdbd in Ahr−/− 3T3 fibroblasts transiently transfected with either Ahr+/+ or Ahrdbd/dbd. Prior to staining, nuclear translocation was induced by exposure of cells to 1nM TCDD for 2 h prior to staining. (C) Mammalian 2-hybrid analysis of AHRdbd interactions. The schematic diagram depicts the reporter construct (pG5luc), the “bait” construct (Gal-ARNT), and the “fish” construct (AHR), showing the amino acid sequence of the basic region in wild-type (wt) and AHRdbd (dbd) recombinant proteins. The two-hybrid analysis was carried out using equal amounts (0.33 μg) of transiently transfected Gal-ARNT and either wild-type AHR or AHRdbd, followed by incubation with 0.1% DMSO or 1nM TCDD. Values are expressed as relative luciferase units (*p < 0.001).
Mentions: To determine whether the AHRdbd could signal effectively in cell culture, we performed transient transfections of AHR or AHRdbd with a DRE-driven luciferase reporter in immortalized Ahr−/− 3T3 fibroblasts. Upon transfection of wild-type AHR cDNA, luciferase activity increased relative to cells transfected with reporter alone. This response was enhanced 2.5-fold by exposure of the cells to 1nM TCDD. In comparison, luciferase activity in cells transfected with AHRdbd did not increase upon exposure of cells to TCDD (Fig. 3A).

Bottom Line: The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes.Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs.These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Wisconsin 53706, USA.

ABSTRACT
The aryl hydrocarbon receptor (AHR) is known for its role in the adaptive and toxic responses to a large number of environmental contaminants, as well as its role in hepatovascular development. The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes. The AHR has also been implicated in signaling events independent of nuclear localization and DNA binding, and it has been suggested that such pathways may play important roles in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs. Using this model, we provide evidence that DNA binding is required AHR-mediated liver development, as Ahr(dbd/dbd) mice exhibit a patent ductus venosus, similar to what is seen in Ahr(-/-) mice. Furthermore, Ahr(dbd/dbd) mice are resistant to TCDD-induced toxicity for all endpoints tested. These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

Show MeSH
Related in: MedlinePlus