Diversity as Opportunity: Insights from 600 Million Years of AHR Evolution.

The aryl hydrocarbon receptor (AHR) was for many years of interest only to pharmacologists and toxicologists. However, this protein has fundamental roles in biology that are being revealed through studies in diverse animal species. The AHR is an ancient protein. AHR homologs exist in most major groups of modern bilaterian animals, including deuterostomes (chordates, hemichordates, echinoderms) and the two major clades of protostome invertebrates [ecdysozoans (e.g. arthropods and nematodes) and lophotrochozoans (e.g. molluscs and annelids)]. AHR homologs also have been identified in cnidarians such as the sea anemone Nematostella and in the genome of Trichoplax, a placozoan. Bilaterians, cnidarians, and placozoans form the clade Eumetazoa, whose last common ancestor lived approximately 600 million years ago (MYA). The presence of AHR homologs in modern representatives of all these groups indicates that the original eumetazoan animal possessed an AHR homolog. Studies in invertebrates and vertebrates reveal parallel functions of AHR in the development and function of sensory neural systems, suggesting that these may be ancestral roles. Vertebrate animals are characterized by the expansion and diversification of AHRs, via gene and genome duplications, from the ancestral protoAHR into at least five classes of AHR-like proteins: AHR, AHR1, AHR2, AHR3, and AHRR. The evolution of multiple AHRs in vertebrates coincided with the acquisition of high-affinity binding of halogenated and polynuclear aromatic hydrocarbons and the emergence of adaptive functions involving regulation of xenobiotic-metabolizing enzymes and roles in adaptive immunity. The existence of multiple AHRs may have facilitated subfunction partitioning and specialization of specific AHR types in some taxa. Additional research in diverse model and non-model species will continue to enrich our understanding of AHR and its pleiotropic roles in biology and toxicology.

Interaction of fish aryl hydrocarbon receptor paralogs (AHR1 and AHR2) with the retinoblastoma protein.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. In some mammalian cell lines, TCDD induces G1 cell cycle arrest, which depends on an interaction between the AHR and the retinoblastoma tumor suppressor (RB). Mammals possess one AHR, whereas fishes possess two or more AHR paralogs that differ in the domains important for AHR-RB interactions in mammals. To test the hypothesis that fish AHR paralogs differ in their ability to interact with RB, we cloned RB cDNA from Atlantic killifish, Fundulus heteroclitus, and studied the interactions of killifish RB protein with killifish AHR1 and AHR2. In coimmunoprecipitation experiments, in vitro-expressed killifish RB coprecipitated with both AHR1 and AHR2. Consistent with these results, both killifish AHR1 and AHR2 interacted with RB in mammalian two-hybrid assays. These results suggest that both fish AHR1 and AHR2 paralogs may have the potential to influence cell proliferation through interactions with RB.

The developmentally-regulated Smoc2 gene is repressed by Aryl-hydrocarbon receptor (Ahr) signaling.

SPARC-Related Modular Calcium Binding Protein-2 (Smoc-2) is a broadly-expressed matricellular protein which contributes to mitogenesis via activation of Integrin-Linked Kinase (ILK). Here we show that expression of Smoc2 is repressed in cultured cells following treatment with Aryl-hydrocarbon receptor (Ahr) ligands including the ubiquitous environmental pollutants Benzo[a]pyrene (B[a]P) and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The Smoc2 promoter contains two consensus putative Ahr-binding sites and Smoc2 promoter-driven reporter genes are repressed in response to B[a]P in an Ahr-dependent manner in cultured cells. Using organ culture experiments we show that TCDD also represses Smoc2 mRNA expression in testes from Ahr(+/+) but not Ahr(-/-) mice. Therefore, exposure to Ahr ligands is likely to affect Smoc2 expression in vivo. Taken together our results indicate that Smoc2 is a novel transcriptional target of activated Ahr. Perturbation of Smoc2 expression may mediate the adverse developmental effects of environmental aryl-hydrocarbon exposure.

Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: insights from comparative genomics.

Ligand-activated receptors are well-known targets of environmental chemicals that disrupt endocrine signaling. Genomic approaches are providing new opportunities to understand the comparative biology and molecular evolution of these receptors. One example of this is the aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) transcription factor through which planar aromatic hydrocarbons cause altered gene expression and toxicity. In contrast to humans and other mammals, which possess a single AHR, teleosts such as the Atlantic killifish (Fundulus heteroclitus) have at least two AHRs (AHR1 and AHR2). Analysis of sequenced genomes has revealed additional, unexpected AHR diversity in non-mammalian vertebrates, including the chicken Gallus gallus (three predicted AHR genes), bony fishes such as the pufferfish Takifugu (formerly Fugu) rubripes (five AHR genes) and zebrafish Danio rerio (three AHR genes), and cartilaginous fishes such as the spiny dogfish Squalus acanthias (three AHR genes). In contrast, invertebrates appear to possess single AHRs that do not bind typical ligands of vertebrate AHRs. We suggest that AHR diversity in vertebrates arose through both gene and whole-genome duplications combined with lineage-specific gene loss, and that sensitivity to the developmental toxicity of planar aromatic hydrocarbons may have had its origin in the evolution of the ligand-binding capacity of the AHR in the chordate lineage. Comparative molecular and genomic studies are providing new insights into AHR diversity and function in non-mammalian species, revealing additional complexity in mechanisms by which environmental chemicals interfere with receptor-dependent signaling.

Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus.

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.

The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells.

The aryl hydrocarbon receptor (AhR) is an environmental carcinogen-activated transcription factor associated with tumorigenesis. High levels of apparently active AhR characterize a variety of tumors, even in the absence of environmental ligands. Despite this association between transformation and AhR upregulation, little is known of the transcriptional consequences of constitutive AhR activation. Here, the effects of constitutively active and environmental ligand-induced AhR on c-myc, an oncogene whose promoter contains six AhR-binding sites (AhREs (aryl hydrocarbon response elements)), were investigated. A reporter containing the human c-myc promoter, with its six AhREs and two NF-kappaB-binding sites, was constructed. This vector, and variants with deletions in the NF-kappaB and/or AhR-binding sites, was transfected into a human breast cancer cell line, Hs578T, which expresses high levels of apparently active, nuclear AhR. Results indicate that: (1) the AhR constitutively binds the c-myc promoter; (2) there is a low but significant baseline level of c-myc promoter activity, which is not regulated by NF-kappaB and is not affected by an environmental AhR ligand; (3) deletion of any one of the AhREs has no effect on constitutive reporter activity, while deletion of all six increases reporter activity approximately fivefold; (4) a similar increase in reporter activity occurs when constitutively active AhR is suppressed by transfection with an AhR repressor plasmid (AhRR); (5) AhRR transfection significantly increases background levels of endogenous c-myc mRNA and c-Myc protein. These results suggest that the AhR influences the expression of c-Myc, a protein critical to malignant transformation.

Aryl hydrocarbon receptor polymorphisms and dioxin resistance in Atlantic killifish (Fundulus heteroclitus).

The aryl hydrocarbon receptor (AHR) gene encodes a ligand-activated transcription factor through which planar halogenated aromatic hydrocarbons (HAHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as polynuclear aromatic hydrocarbons (PAHs) cause altered gene expression and toxicity. To understand the role of AHR genetic variability in differential sensitivity to HAHs and PAHs, we are currently studying a population of the teleost Fundulus heteroclitus (Atlantic killifish) that has evolved genetic resistance to the toxic and biochemical effects of these compounds. Here, we report that the killifish AHR1 locus is highly polymorphic and that the frequencies of the major allele types differ between dioxin-sensitive and dioxin-resistant populations. Twenty-five single nucleotide polymorphisms (SNPs), nine of which are non-synonymous, were identified in the AHR1 coding sequence. Seven identified alleles were assigned to three groups, designated AHR1*1, AHR1*2 and AHR1*3. AHR1*1 alleles were under-represented in a population of dioxin- and polychlorinated biphenyl (PCB)-resistant fish from a PCB-contaminated Superfund site (New Bedford Harbor, Massachusetts, USA) compared to dioxin-sensitive fish from a less contaminated reference site (Scorton Creek, Massachusetts, USA). To determine the possible role of these AHR1 variants in differential HAH sensitivity, we expressed representative variant proteins from the two most divergent allelic groups (AHR1*1 and AHR1*3) by in-vitro transcription and translation and assessed their functional properties. AHR1*1A and AHR1*3A proteins displayed similar binding capacities and affinities for [H]TCDD. In transient transfection assays using mammalian cells, AHR1*1A and AHR1*3A exhibited similar abilities to support TCDD-dependent transactivation of a luciferase reporter gene under control of AHR-responsive enhancer elements. We discuss the possibility of other functional differences in AHR1 variants or their interaction with other killifish loci (AHR2, AHRR) that may contribute to differences in dioxin sensitivity.