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Background: The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand., Objectives: We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand., Methods: The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts., Results: We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes., Conclusions: The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of its xenobiotic ligands and acts as an environmental checkpoint during the cell cycle. We expressed stably integrated, Tet-Off-regulated AHR variants in fibroblasts from AHR-null mice to further investigate the AHR role in cell cycle regulation. Ahr+/+ fibroblasts proliferated significantly faster than Ahr-/- fibroblasts did, and exposure to a prototypical AHR ligand or deletion of the ligand-binding domain did not change their proliferation rates, indicating that the AHR function in cell cycle was ligand independent. Growth-promoting genes, such as cyclin and cyclin-dependent kinase genes, were significantly down-regulated in Ahr-/- cells, whereas growth-arresting genes, such as the transforming growth factor beta1 (TGF-beta1) gene, extracellular matrix (ECM)-related genes, and cyclin-dependent kinase inhibitor genes, were up-regulated. Ahr-/- fibroblasts secreted significantly more TGF-beta1 into the culture medium than Ahr+/+ fibroblasts did, and Ahr-/- showed increased levels of activated Smad4 and TGF-beta1 mRNA. Inhibition of TGF-beta1 signaling by overexpression of Smad7 reversed the proliferative and gene expression phenotype of Ahr-/- fibroblasts. Changes in TGF-beta1 mRNA accumulation were due to stabilization resulting from decreased activity of TTP, the tristetraprolin RNA-binding protein responsible for mRNA destabilization through AU-rich motifs. These results show that the Ah receptor possesses interconnected intrinsic cellular functions, such as ECM formation, cell cycle control, and TGF-beta1 regulation, that are independent of activation by either exogenous or endogenous ligands and that may play a crucial role during tumorigenesis.

The molecular basis for the adverse biological effects of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD), a pervasive environmental toxin, is largely unknown. TCDD is a ligand for the cytosolic aromatic hydrocarbon receptor (AHR) which mediates the transcriptional induction of the xenobiotic metabolizing genes in the CYP1 family of cytochromes P450. Previous studies have suggested that the AHR may carry out important functions in the cell in addition to metabolizing toxins. We present gene expression profiles of smooth muscle cells from wild type and Ahr(-/-) mice that show significant changes in the RNA levels of the transforming growth factor-beta3 (Tgfb3) gene and genes involved in the modulation and processing of TGF-beta. The RNA expression profiles support a hypothesis that in the wild type, the AHR represses Tgfb gene expression and affects the gene expression of several TGF-beta-modulating and processing genes. We also observed that RNA levels increased for TGF-beta2, CYP1b1, and TGF-beta-related genes in Ahr(-/-) smooth muscle cells exposed to TCDD. These data are consistent with a hypothesis that TCDD stimulates the TGF-beta2 signaling pathway in the absence of the AHR to activate the Cyp1b1 gene. The above results provide a possible explanation for some of the multiple biological effects of TCDD and the physiological role played by the AHR in the absence of environmental agents.

Benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands for the aryl hydrocarbon receptor (AHR). High-density oligonucleotide microarrays were used to generate global gene expression profiles of wild-type and Ahr(-/-) vascular smooth muscle cells (SMCs) from mouse aorta. To determine whether there are signaling pathways other than the AHR involved in B[a]P metabolism, wild-type and AHR knockout (Ahr(-/-) SMCs were exposed to B[a]P. Two signaling pathways, represented by TGF-beta2 and IGF-1, were identified as potential candidates of an AHR alternate pathway for cells to respond to B[a]P. The wild-type SMCs responded similarly to B[a]P and TCDD in the regulation of a small set of common genes known to respond to the activated AHR (e.g., glutamine S-transferase). However, wild-type SMCs responded in a way that involves many additional genes, suggesting that a very divergent cellular response may be involved when SMCs are exposed to the two classic inducers of the AHR. In contrast, many more genes in the Ahr(-/-) cells responded similarly to B[a]P and TCDD, including Cyp1b1, than responded differently, which indicates that eliminating the AHR is effective for investigating potential alternate cellular mechanisms that respond to B[a]P and TCDD.