The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. The toxicity of the potent AhR ligand 2,3,7,8-tetrachlorodibenzop-dioxin is almost exclusively mediated through this receptor. However, the key alterations in gene expression that mediate toxicity are poorly understood. It has been established through characterization of AhR-null mice that the AhR has a required physiological function, yet how endogenous mediators regulate this orphan receptor remains to be established. A picture as to how the AhR/ARNT heterodimer actually mediates gene transcription is starting to emerge. The AhR/ ARNT complex can alter transcription both by binding to its cognate response element and through tethering to other transcription factors. In addition, many of the coregulatory proteins necessary for AhR-mediated transcription have been identified. Cross talk between the estrogen receptor and the AhR at the promoter of target genes appears to be an important mode of regulation. Inflammatory signaling pathways and the AhR also appear to be another important site of cross talk at the level of transcription. A major focus of this review is to highlight experimental efforts to characterize nonclassical mechanisms of AhR-mediated modulation of gene transcription.

The effect of activation and over-expression of the nuclear receptor PPARβ/δ in human MDA-MB-231 (ER−) and MCF7 (ER+) breast cancer cell lines was examined. Target gene induction by ligand activation of PPARβ/δ was increased by over-expression of PPARβ/δ compared to controls. Over-expression of PPARβ/δ caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared to controls while ligand activation of PPARβ/δ further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Over-expression and/or ligand activation of PPARβ/δ in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 over-expressing PPARβ/δ in response to ligand activation of PPARβ/δ as compared to controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells over-expressing PPARβ/δ were significantly smaller and ligand activation of PPARβ/δ caused an even greater reduction in tumor volume as compared to controls. Interestingly, the decrease in MDA-MB-231 tumor size after over-expressing PPARβ/δ and ligand activation of PPARβ/δ correlated with increased necrosis. These data show that ligand activation and/or over-expression of PPARβ/δ in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPARβ/δ to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPARβ/δ in breast cancer and evaluating the effects of PPARβ/δ agonists.

The concept of selective receptor modulators has been established for the nuclear steroid hormone receptors. Such selective modulators have been used therapeutically with great success in the treatment of cancer. However, this concept has not been examined with regard to the aryl hydrocarbon receptor (AHR) because of the latent toxicity commonly associated with AHR activation. AHR-mediated toxicity is primarily derived from AHR binding to its dioxin response element (DRE) and driving expression of CYP1 family members, which have the capacity to metabolize procarcinogens to genotoxic carcinogens. Recent evidence using a non-DRE binding AHR mutant has established the DRE-independent suppression of inflammatory markers by the AHR. We wished to determine whether such DRE-independent repression with wild-type AHR could be dissociated from canonical DRE-dependent transactivation in a ligand-dependent manner and, in doing so, prove the concept of a selective AHR modulator (SAhRM). Here, we identify the selective estrogen receptor (ER) modulator Way-169916 as a dually selective modulator, binding both ER and AHR. Inflammatory gene expression associated with the cytokine-inducible acute-phase response (e.g., SAA1 and CRP) are diminished by Way-169916 in an AHR-dependent manner. Furthermore, activation of AHR by Way-169916 fails to stimulate canonical DREdriven AHR-mediated CYP1A1 expression, thus eliminating the potential for AHR-mediated genotoxic stress. Such anti-inflammatory activity in the absence of DRE-mediated expression fulfills the major criteria of an SAhRM, which suggests that selective modulation of AHR is possible and renders the AHR a therapeutically viable drug target for the amelioration of inflammatory disease.

The regulation of the aryl hydrocarbon receptor (AhR) protein levels has been an area of keen interest, given its important role in mediating the cellular adaptation and toxic response to several environmental pollutants. The carboxyl terminus of hsc70-interacting protein (CHIP) ubiquitin ligase was previously associated with the regulation of the aryl hydrocarbon receptor, although the mechanisms were not directly demonstrated. In this study we established that CHIP could associate with the AhR at cellular levels of these two proteins, suggesting a potential role for CHIP in the regulation of the AhR complex. The analysis of sucrose gradient fractionated in vitro translated AhR complexes revealed that CHIP can mediate hsp90 ubiquitination, while cooperating with unidentified factors to promote the ubiquitination of mature unliganded AhR complexes. In addition, the immunophilin-like protein XAP2 was able to partially protect the AhR from CHIP mediated ubiquitination in vitro. This protection required the direct interaction of the XAP2 with the AhR complex. Surprisingly, CHIP silencing in Hepa-1c1c7 cells by siRNA methods did not reveal the function of CHIP in the AhR complex, as it did not affect well characterized activities of the AhR nor affected its steady-state protein levels. However, the presence of potential compensatory mechanisms may beconfounding this particular observation. Our results suggests a model where the E3 ubiquitin ligase CHIP cooperates with other ubiquitination factors to remodel native AhR-hsp90 complexes and that co-chaperones such as the XAP2 may affect the ability of CHIP to arget AhR complexes for ubiquitination.