However, the AhR profoundly controls the nuclear levels of HuR in response to CSE. HuR translocation from the nucleus to the cytoplasm is critical to its ability to stabilize target mRNA. This may be why in AhR+/+ cells, where HuR remains within the nucleus, HuR knockdown had no effect on Cox-2 mRNA stability. Results in C5N cells, a mouse keratinocyte cell line with exclusive nuclear HuR support this notion, as reduction in HuR expression had no effect on ornithine decarboxylase mRNA stability. However in AhR2/2 cells whereby HuR translocates to the cytoplasm, HuR was a key factor involved in Cox-2 mRNA stability, as siRNA-knockdown resulted in enhanced Cox-2 mRNA degradation. Our results support that retention of nuclear HuR is an important feature in the destabilization of Cox-2 mRNA by the AhR. In addition to Cox-2, HuR has thousands of target genes and stabilizes mRNAs that encode proteins associated with a variety of cellular functions including cell cycle, proliferation, apoptosis and inflammation. The AhR regulation of these functions is established opening the possibility that AhR retention of nuclear HuR may have important implications for the regulation of genes beyond the control of Cox-2. Our results are also the first to show in vivo evidence of pulmonary HuR translocation in response to cigarette smoke. In the lungs of AhR2/2 mice, there was no Cox2 mRNA induction despite concordant COX-2 protein and profound cytoplasmic HuR. It was surprising to note considerable levels of HuR in the cytoplasm of pulmonary cells without smoke exposure. Cytoplasmic HuR has been reported in the lungs of adult A/J mice, consistent with our data, and HuR expression is required for proper lung development. It may be that in the lung, an organ continuously exposed to the environment and one that is highly susceptible to Tubacin oxidative damage, a constitutive level of cytoplasmic HuR is required to ensure optimum immunological responsiveness. Although our results reveal a novel pathway in which the AhR regulates COX-2 protein expression by controlling the cellular localization of HuR, it remains to be established precisely how the AhR retains HuR in the nucleus. Our finding that the AhR regulates HuR localization in response to CSE, but not BP indicates divergent mechanism of AhR activation in maintaining HuR localization despite the ability of both to cause Cyp1a1 mRNA induction. It also indicates that BP, which is present in cigarette smoke, is not the component causing HuR translocation to the cytoplasm in the absence of AhR expression. Cigarette smoke is a complex mixture, containing more than 4800 compounds including metals, oxidants and free radicals, the latter of which are a potent source of oxidative stress. Given that the AhR protects against oxidative damage due to smoke exposure, it reasonable to speculate that the high oxidant conditions exerted by cigarette smoke contributes to HuR translocation in the absence of AhR expression. AhR activity was required to retain HuR within the nucleus, but did not require DNA-binding. It has been speculated that the DREindependent anti-inflammatory abilities of the AhR may involve multiple protein-protein interactions. The AhR localizes to the nucleus in the absence of exogenous ligand, a cellular phenomenon that depends on cell-cell contact. Adherent cells grown to sub-confluence, methodologically similar to the experiments conducted here, exhibit both cytoplasmic and nuclear AhR, making interaction with AhR and HuR within the nucleus a plausible assumption. Thus, while there is no known physical association between AhR and HuR, it is interesting to speculate that the AhR may interact with HuR to prevent its nuclear export, a notion we are actively Everolimus pursuing. It is believed that the AhR plays an important role in physiology independent of its ability to respond to dioxin.