The Ah receptor (AHR) mediates the metabolic adaptation to a number of planar aromatic chemicals. Essential steps in this adaptive mechanism include AHR binding of ligand in the cytosol, translocation of the receptor to the nucleus, dimerization with the Ah receptor nuclear translocator, and binding of this heterodimeric transcription factor to dioxin-responsive elements (DREs) upstream of promoters that regulate the expression of genes involved in xenobiotic metabolism. The AHR is also involved in other aspects of mammalian biology, such as the toxicity of molecules like 2,3,7,8-tetrachlorodibenzo-p-dioxin as well as regulation of normal liver development. In an effort to test whether these additional AHR-mediated processes require a nuclear event, such as DRE binding, we used homologous recombination to generate mice with a mutation in the AHR nuclear localization/DRE binding domain. These Ahr nls mice were found to be resistant to all 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxic responses that we examined, including hepatomegaly, thymic involution, and cleft palate formation. Moreover, aberrations in liver development observed in these mice were identical to that observed in mice harboring a null allele at the Ahr locus. Taken in sum, these data support a model where most, if not all, of AHR-regulated biology requires nuclear localization. The aryl hydrocarbon receptor (AHR)1 regulates an adaptive metabolic response to a variety of planar aromatic chemicals that are widely dispersed in the environment. Over the last 20 years, the mechanistic details of this adaptive signaling pathway have been well characterized (1-4). The AHR is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor. Upon binding agonists, the AHR translocates from the cytoplasm to the nucleus, where it forms a heterodimer with another bHLH-PAS protein known as the aryl hydrocarbon nuclear translocator (ARNT). This heterodimeric complex interacts with dioxin-responsive elements (DREs) within the genome and upregulates the transcription of a battery of xenobiotic metabolizing enzymes (XMEs). These regulated XMEs include the cytochrome P450s Cyp1a1, Cyp1b1, and Cyp1a2 and the phase II enzymes Gst-a1 and Ugt1-06 (reviewed in Refs. 2 and 3).In addition to regulating an adaptive metabolic response, the AHR also mediates toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and plays an important role in normal development. Early genetic and pharmacological experiments provided evidence that the AHR mediates toxic responses to TCDD and related pollutants (5). Highly reproducible toxic endpoints in rodent species include thymic involution, hepatomegaly, epithelial hyperplasia, and teratogenesis. More recently, generation of null alleles at the Ahr locus in mice revealed that the AHR also plays an important role in normal mammalian development (6 -9). Across laboratories, the most reproducible phenotype associated with the homozygous null allele is a smaller liver. We have proposed that smaller liver size is the result of the pers...

The aryl hydrocarbon receptor-associated protein 9, ARA9 (also known as XAP2 or AIP1), is a chaperone that is found in complexes with certain xenobiotic receptors, such as the aryl hydrocarbon receptor (AHR) and the peroxisome proliferatoractivated receptor ␣ (PPAR␣). In an effort to better understand the physiological role of ARA9 outside of its role in xenobiotic signal transduction, we generated a null allele at the Ara9 locus in mice. Mice with a homozygous deletion of this gene die at various time points throughout embryonic development. Embryonic lethality is accompanied by decreased blood flow to head and limbs, as well as a range of heart deformations, including double outlet right ventricle, ventricular-septal defects, and pericardial edema. The early cardiovascular defects observed in Ara9-null mice suggest an essential role for the ARA9 protein in cardiac development. The observation that the developmental aberrations in Ara9-null mice are distinct from those observed for disrupted alleles at Ahr or Ppar␣ indicates that the role of ARA9 in cardiac development is independent of its interactions with its known xenobiotic receptor partners.The aryl hydrocarbon receptor-associated protein 9 (ARA9, 2 also known as AIP1) is found in association with two mammalian client proteins, the aryl hydrocarbon receptor (AHR) and the peroxisome proliferator-activated receptor ␣ (PPAR␣) (1-4). The ARA9 protein has also been characterized in association with the hepatitis-B virus X-protein, HBVx. As the result of its interaction with this viral protein, it is also known by the name hepatitis B virus X-associated protein or XAP2 (5).The ARA9 protein is structurally related to the immunophilin family of proteins, harboring an N-terminal FK506 binding (FKBP) domain and C-terminal tetratricopeptide repeats (TPRs) (1,3,6). The FKBP domains of many immunophilin proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity that can be blocked by the binding of immunosuppressants such as FK506 (7,8). Despite the sequence similarity between the N terminus of ARA9 and the FKBP domains of other immunophilins, ARA9 does not appear to possess isomerase activity or affinity for FK506 (1). We postulate that the structural similarity of ARA9 to the immunophilin family is a reflection of a shared capacity to act as a cellular chaperone, and play a role in the folding and localization of client proteins.The ARA9 protein has been extensively studied in association with the AHR. As a chaperone, ARA9 maintains the AHR in a cytosolic localization, decreases AHR degradation, and increases its ligand binding capacity (9 -11). Although little is known regarding the functional role of ARA9 in PPAR␣ and HBVx function, initial data are consistent with ARA9 acting as a chaperone for these proteins as well (4, 5). We hypothesize that in addition to xenobiotic signaling, the ARA9 protein is also a common chaperone to many proteins involved in a variety of essential cellular functions. This idea is supported by results from whole mount in ...

The aryl hydrocarbon receptor (AHR) plays an essential role in the toxic response to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), in the adaptive up-regulation of xenobiotic metabolizing enzymes, and in hepatic vascular development. In our model of AHR signaling, the receptor is found in a cytosolic complex with a number of molecular chaperones, including Hsp90, p23, and the aryl hydrocarbon receptor-interacting protein (AIP), also known as ARA9 and XAP2. To understand the role of AIP in adaptive and toxic aspects of AHR signaling, we generated a conditional mouse model where the Aip locus can be deleted in hepatocytes. Using this model, we demonstrate two important roles for the AIP protein in AHR biology. (i) The expression of AIP in hepatocytes is essential to maintain high levels of functional cytosolic AHR protein in the mammalian liver. (ii) Expression of the AIP protein is essential for dioxin-induced hepatotoxicity. Interestingly, classical AHR-driven genes show differential dependence on AIP expression. The Cyp1b1 and Ahrr genes require AIP expression for normal up-regulation by dioxin, whereas Cyp1a1 and Cyp1a2 do not. This differential dependence on AIP provides evidence that the mammalian genome contains more than one class of AHR-responsive genes and suggests that a search for AIP-dependent, AHR-responsive genes may guide us to the targets of the dioxin-induced hepatotoxicity.

The aryl hydrocarbon receptor-associated protein-9 (ARA9) is a chaperone of the aryl hydrocarbon receptor (AHR). The AHR has been shown to play a late developmental role in the normal closure of a fetal hepatovascular shunt known as the ductus venosus (DV). Given that Ara9-null mice display early embryonic lethality, we generated a hypomorphic Ara9 allele (designated Ara9 fxneo ) that displays reduced ARA9 protein expression. In an effort to demonstrate the role of ARA9 protein in AHR-mediated DV closure, we used combinations of Ara9 wild-type [Ara9(ϩ/ϩ)], null [Ara9(Ϫ/Ϫ)], and hypomorphic [Ara9(fxneo/fxneo)] alleles to produce mice with a graded expression of the ARA9 protein. Liver perfusion studies demonstrated that although none of the Ara9(ϩ/ϩ) mice displayed a patent DV, the shunt was observed in 10% of the Ara9(ϩ/fxneo) mice, 55% of the Ara9(ϩ/Ϫ) mice, and 83% of the Ara9(fxneo/fxneo) mice. That expression level of ARA9 correlates with the frequency of a phenocopy of the Ahr-null allele supports the conclusion that the ARA9 protein is essential for AHR signaling during development.

Intercellular communication that controls the developmental fate of multipotent cells is commonly mediated by the Notch family of transmembrane receptors. Speci®c transmembrane ligands activate Notch receptors on neighboring cells inducing the proteolytic liberation and nuclear translocation of the intracellular domain of Notch (N IC ). Nuclear N IC associates with a transcriptional repressor known as C-promoter binding factor/RBP-Jk, suppressor of hairless, or LAG-1, converting it from a repressor into an activator. Through physical interactions with chromatin remodeling enzymes and potentially with components of the transcriptional machinery, N IC activates target genes that mediate cell fate decisions. As Notch1 is disrupted via a chromosomal translocation in a subset of human T-cell leukemia, leading to a truncated polypeptide resembling N IC , deregulated chromatin remodeling and transcription may fuel uncontrolled cell proliferation in this hematopoietic malignancy. This review summarizes the mechanics of Notch signaling and focuses on prospective molecular mechanisms for how constitutively active Notch might derail nuclear processes as an initiating step in T-cell leukemogenesis.