Ah Receptor Binding to its Cognate Response Element is Required for Dioxin-Mediated Toxicity

The aryl hydrocarbon receptor (AHR) is a ligand-activated member of the bHLH-PAS family of transcription factors. Members of this family include HIF1a, EPAS, and SIM, which are involved in hypoxia and nervous system development. Another member of this family, aryl hydrocarbon nuclear translocator (ARNT), is the dimerization partner for the AHR. The AHR is often classified as a sensor of a wide range of xenobiotics, leading to induction of xenobiotic metabolism through enhanced expression of phase I/II enzymes. The environmental contaminant 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) is a prototypic ligand for the AHR and is often used to study the effects of prolonged AHR activation. Rodent exposure to TCDD results in a plethora of toxic effects, including wasting syndrome, tumor promotion, developmental defects, and liver toxicity (reviewed in Vanden Heuvel and Lucier, 1993). The key target genes that lead to these toxic end points are largely unknown. AHR activation can occur through a growing list of chemicals that appear to be structurally diverse and include many polycyclic aromatic hydrocarbons, tryptophan metabolites, and flavones (Denison and Nagy, 2003). Soluble receptors such as the AHR could possibility alter transcription of target genes through several distinct mechanisms. The work of Bunger and coworkers (Bunger et al., 2008) has examined whether TCDD-mediated liver toxicity can be mediated by the AHR in the absence of DNA binding. The AHR exist in the cytoplasm as a core tetrameric complex, composed of the ligand-binding subunit, a dimer of hsp90, the X-associated protein 2 (also referred to as ARA9 or AIP), and p23 (reviewed in Petrulis and Perdew, 2002). The hsp90 and XAP2 are considered a chaperone complex that stabilizes the AHR in the cytoplasm, protecting it from proteolysis and helping the receptor maintain its ligand-binding conformation. Upon binding an agonist, a conformational change in the AHR occurs that allows access to its nuclear localization sequence, and the receptor rapidly translocates into the nucleus. In the nucleus, ARNT appears to cause displacement of hsp90, leading to the formation of the AHR/ ARNT complex, which can then bind to dioxin-responsive elements (DRE) and regulate many of the receptor’s target genes. Some of these targets include phase I drug metabolism genes such as CYP1A1, CYP1B1, and CYP1A2. The transcription of several important phase II enzymes, such as UGT1A1 and NADPH-quinone reductase, are also directly regulated by the AHR through DRE. The AHR also regulates genes with diverse functions such as p27, epiregulin, IGFBP-1, and Bax. Establishment of ahr-null mouse models has revealed that most, if not all, of the toxic effects of TCDD are mediated through the activation of the AHR (Fernandez-Salguero et al., 1996). The lack of AHR expression leads to lower reproductive success, reduced life span, immunological defects, and reduced liver size (Abbott et al., 1999; Rodriguez-Sosa et al., 2005; Schmidt et al., 1996). The mechanism mediating the reduced liver size appears to be smaller hepatocytes and a portosystemic shunting of blood due to a persistent fetal vascular structure (Lahvis et al., 2000). Through the use of microspheres, the portal blood flow that bypasses the ahr-null liver was calculated to be about 50% (Lahvis et al., 2000). Using ahr mice and Cre-lox technology, where the AHR was selectively disrupted in either hepatocytes or endothelial cells, revealed that the deletion of AHR expression in endothelial cells leads to a lack of developmental closure of the ductus venosus (Walisser et al., 2005). Furthermore, TCDD-mediated hepatotoxicity requires the expression of the AHR in hepatocytes. Several research groups have performed DNA microarray studies on liver after exposure to an AHR ligand. In these experiments a large number of genes exhibit increases in messenger RNA (mRNA) levels, while interestingly an equal number of genes demonstrated a decrease in mRNA levels. These results would suggest that the AHR could both increase and decrease transcriptional levels of various genes after ligand 1 To whom correspondence should be addressed at Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, 309 LSB, University Park, PA 16802. Fax: (814)-863-1696. E-mail: [email protected].