Roles of aryl hydrocarbon receptor in aromatase-dependent cell proliferation in human osteoblasts

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and its expression is influenced by environmental compounds, such as 3-methylcholanthrene (3-MC) and β-naphthoflavone (β-NF). AhR and its downstream genes, such as CYP1A1, are considered to play a pivotal role in xenobiotic responses. AhR signaling has also been proposed to mediate osteogenesis in experimental animals, but its details have remained unclear. Therefore, in this study, we examined the possible roles of AhR in human bone. Immunohistochemical analysis revealed that AhR was detected in both osteoblasts and osteoclasts. We then screened AhR-target genes using a microarray analysis in human osteoblastic hFOB cells. Results of microarray and subsequent PCR analysis did reveal that estrogen metabolizing and synthesizing enzymes, such as CYP1B1 and aromatase, were increased by 3-MC in hFOB and osteosarcoma cell line, MG-63. The subsequent antibody cytokine analysis also demonstrated that interleukin-1β and -6 expression was increased by 3-MC and β-NF in hFOB cells and these interleukins were well known to induce aromatase. We then examined the cell proliferation rate of hFOB and MG-63 cells co-treated with 3-MC and testosterone as an aromatase substrate. The status of cell proliferation in both hFOB and MG-63 cells was stimulated by 3-MC and testosterone treatment, which was also inhibited by an estrogen blocker, aromatase inhibitor, or AhR antagonist. These findings indicated that AhR could regulate estrogen synthesis and metabolism in bone tissues through cytokine/aromatase signaling.