Activation of the aryl hydrocarbon receptor (AHR) alters mammary gland *development by impairing epithelial cell proliferation
The aryl hydrocarbon receptor (AHR) is a bHLH-PAS transcription factor that is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related ligands such as 2,3,7,8-tetrachlorodibenzofuran (TCDBF). Previous studies have implicated AHR in epithelial development and reduced proliferation of mammary epithelial cell lines. Treatment of mammary glands from C57BL/6 mice in organ culture with TCDBF (30 nM) suppressed terminal end bud (TEB) development. Concomitant decreases in lobule number and size ($>$2-fold), and in (H$\sp3$) thymidine incorporation (35-45%) were observed. Immunohistochemical staining patterns for AHR, ARNT (the heterodimerization partner of AHR) and two AHR-regulated genes, Cyp1A1 and Cyp1B1, were identical and not altered by ligand treatment. Highest expression levels were seen in developing lobules, with minimal staining in central ductal regions. In vivo, AHR mRNA and protein were expressed at moderate levels during estrous-stimulated growth and TEBs branching. During pregnancy-induced development, only proteolytic fragments of AHR protein were detectable, suggesting that increased AHR turnover is associated with and may be permissive for large increases in lobule proliferation and differentiation. The ligand-induced phenotype that occurred in organ culture in part reflected the mammary gland phenotype of AHR $-$/$-$ mice. AHR-mediated decreases in lobule proliferation were further analyzed using mammary epithelial cell lines. In MCF-7 cells, AHR-ligand treatment produced similar decreases in cell proliferation, with the accumulation of a large, slow cycling G$\sb1$ population after 24 h. Appearance of this population correlated with decreases in Rb phosphorylation and cyclin dependent kinase (cdk) 4 and Cdk 2 activity. Levels of cyclins, cdks, or cdk inhibitors were not altered by TCDD treatment; however, a decrease in association of cyclin D1/Cdk 4 into active kinase complexes was observed. AHR was associated with the Cdk 4 complex, and upon TCDD treatment, this association decreased and an increased interaction with Rb was noted. Taken together, these results support the involvement of AHR in normal mammary gland development and in mediating the suppressive developmental response to AHR ligands. The AHR-ligand-induced decreases in cell proliferation may be due to (1) decreased association of AHR with Cdk 4, destabilizing and decreasing Cdk 4 activity; (2) increased association with Rb, preventing its phosphorylation, or both. ^
Major Professor: William Greenlee, Purdue University.
Biology, Molecular|Biology, Cell|Health Sciences, Toxicology|Health Sciences, Pharmacology