Benzo[a]pyrene inhibits osteoclastogenesis by affecting RANKL-induced activation of NF-κB

Exposure to polycyclic aryl hydrocarbons is linked to cancer, immunosuppression and other numerous health problems. We previously demonstrated that exposure to benzo[a]pyrene (BaP), an environmental pollutant present in high concentrations in urban smog and cigarette smoke, inhibits osteoclast differentiation and bone resorption. We hypothesized that this inhibition could be due to crosstalk between the receptor activator of NF-κB ligand (RANKL) and AhR signaling cascades competing for NF-κB, a common transcription factor for both pathways. RAW264.7 cells (a mouse macrophage cell line capable of differentiating into osteoclasts in the presence of RANKL) were exposed to different concentrations of RANKL and BaP and the effect on NF-κB activation, nuclear translocation, as well as the effect of NF-κB inhibitors on BaP-mediated CYP1B1 gene expression was measured. The results demonstrated that BaP inhibited both RANKL-induced NF-κB activation and nuclear translocation. At the same time, BaP-induced CYP1B1 gene expression was inhibited by two NF-κB inhibitors in a dose-dependent manner, demonstrating that NF-κB is involved in a BaP-mediated signaling pathway. A reporter gene assay showed that both BaP and RANKL-induced luciferase reporter gene transcription under the control of NF-κB response elements. Co-immunoprecipitation results demonstrated that AhR interacted with NF-κB p65 in RAW cells and BaP appeared to enhance this interaction. However, in the presence of RANKL, we did not observe any interaction between AhR and p65. These results support our hypothesis that BaP-mediated inhibition of osteoclastogenesis is a consequence of crosstalk between AhR and RANKL signaling pathways competing for the common transcription factor NF-κB.