Elucidating the molecular mechanisms of benzo[a]pyrene-mediated inhibition of osteoclastogenesis

Exposure to polycyclic aryl hydrocarbons is linked to cancer, immunosuppression and other numerous health problems. Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant present in high concentrations in urban smog and cigarette smoke. The focus of this thesis was to examine the effects of BaP on osteoclasts, the bone resorbing cells, and elucidate the molecular mechanisms of these effects. Results obtained from the experiments using the authentic rabbit osteoclasts demonstrated that BaP was most likely affecting osteoclasts indirectly via the stromal cells present in the cultures. Depending on initial seeding cell density, BaP had either an inhibitory (high cell density) or a stimulatory (low cell density) effect. To examine the direct effects of BaP on osteoclastogenesis, RAW 264.7 cells, a mouse macrophage cell line capable of differentiating into osteoclasts in the presence of receptor activator of NF-κB ligand (RANKL), were used. The results demonstrated that BaP inhibited both osteoclast differentiation and bone resorption. This inhibition of osteoclastogenesis was reversed by the aryl hydrocarbon receptor (AhR) antagonist resveratrol as well as by high concentrations of RANKL. Moreover, high concentrations of RANKL also inhibited BaP-induced cytochrome P450 1B1 (CYP1B1) gene expression. These observations lead to a hypothesis that there is a crosstalk between RANKL and BaP signaling cascades competing for common transcription factors. To elucidate which transcription factors could be involved in the signaling cross-talk, the roles of NF-κB, NFATc1, c-Myc in both signaling cascades were investigated. Studies using transcription factor inhibitors demonstrated that NF-κB and not NFATc1 is involved in BaP-induced CYP1B1 expression. Moreover, BaP inhibited RANKL-induced NF-κB activation and nuclear translocation, confirming that NF-κB is the transcription factor responsible for the signaling cross-talk. Furthermore, co-immunoprecipitation studies demonstrated that AhR is present in a complex with NF-κB/p65 and NκBα; however, the presence of RANKL disrupted this interaction. Finally, both BaP and RANKL induced luciferase reporter gene transcription under the control of NF-κB. These results support the hypothesis that BaP-mediated inhibition of osteoclastogenesis is a consequence of crosstalk between BaP and RANKL signaling pathways competing for NF-κB.

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