Functional role of a constitutively active dioxin/Ah receptor in a transgenic mouse model

Abstract: The dioxin/ Aryl hydrocarbon receptor is a ligand-activated transcription factor that mediates most (if not all) of the toxic effects of the group of highly potent environmental pollutants collectively called dioxins and PCBs, including the highly toxic compound 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). The toxic effects include immune suppression, endocrine disruption, impaired reproduction and carcinogenesis. In addition, the Ah receptor regulates expression of several genes, most of which are xenobiotic metabolizing enzymes such as CYP1A1 Although the high toxicity of these compounds has been known for more than three decades, the mechanism(s) of action behind the wide spectrum of effects is yet not known. Moreover, although the Ah receptor was cloned more than ten years ago, a physiological role or ligand has not unambiguously been identified. A transgenic mouse model that expresses a constitutively active Ah receptor (CA-AhR) was therefore developed. The aim of this study was to characterize the functional role of this receptor in these mice. Homozygous CA-AhR mice were fertile and the CA-AhR was expressed in several tissues such as thymus, spleen, liver, lungs and heart as well as in all parts of the gastrointestinal tract. In CA-AhR mice, unexposed to any exogenous Ah receptor ligand, expression of the Ah receptor target gene CYP1A1 was induced in all of these organs, indicating that the CA-AhR was transcriptionally active in a constitutive manner. In addition, effects observed in most experimental animals after TCDD exposure, such as decreased thymus weight and increased liver weight were also observed in the CA- AhR mice. Increased mortality was observed from six months of age in the CA-AhR mice. This correlated with the development of cystic stomach tumors that penetrated all layers of the glandular stomach. These tumors consisted of differentiated cells, such as foveolar, parietal and cardio-pyloral type of cells. Immunohistochemical analysis demonstrated expression of CYP1A1 and the proliferation marker PCNA. Intestinal and squamous metaplasia was also observed in the tumors. Some tumors were surrounded by a region of connective and fatty tissue together with lymphatic foci and vessels, reminiscent of hamartomatous lesions observed in humans. Wild-type animals orally treated with TCDD demonstrated an expansion of the zone of proliferating cells normally found in the narrow isthmus-region of the stomach to include the entire parietal-chief cell region. Moreover, the parietal/chief cell region was decreased in the stomach of CA-AhR mice and this was associated with foveolar hyperplasia. To identify dysregulated genes in the CA-AhR mouse stomachs, RNA from three months old animals was subjected suppressive subtraction analysis. Osteopontin gene expression was found to be down- regulated in the glandular stomach but also in other organs of CA-AhR mice. TCDD exposure of wild- type and mutant mouse hepatoma cells demonstrated a rapid but transient decrease in osteopontin expression that was dependent on functional Ah receptor and Amt. Moreover, FACS analysis of lymphoid cells revealed that the CD5-expressing peritoneal population of B cells (B1a) was substantially reduced in CA-AhR mice. Taken together, these results demonstrate that expression of the CA-AhR in mice resulted in a number of effects. In particular, the development of wen- differentiated stomach tumors indicates a role of the receptor in regulation of gastric epithelial cell homeostasis.