Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF- κB signaling

Background: Retinoic acid receptor alpha (RAR alpha) plays an essential role in the regulation of many biological processes, such as hematopoietic cell differentiation, while abnormal RAR alpha function contributes to the pathogenesis of certain diseases including cancers, especially acute promyelocytic leukemia (APL). Recently, oridonin, a natural diterpenoid isolated from Rabdosia rubescens, was demonstrated to regulate RAR alpha by increasing its protein level. However, the underlying molecular mechanism for this action has not been fully elucidated. Methods: In the APL cell line, NB4, the effect of oridonin on RAR alpha protein was analyzed by western blot and real-time quantitative RT-PCR analyses. Flow cytometry was performed to detect intracellular levels of reactive oxygen species (ROS). The association between nuclear factor-kappa B (NF-kappa B) signaling and the effect of oridonin was assessed using specific inhibitors, shRNA gene knockdown, and immunofluorescence assays. In addition, primary leukemia cells were treated with oridonin and analyzed by western blot in this study. Results: RAR alpha possesses transcriptional activity in the presence of its ligand, all-trans retinoic acid (ATRA). Oridonin remarkably stabilized the RAR alpha protein, which retained transcriptional activity. Oridonin also moderately increased intracellular ROS levels, while pretreatment with the ROS scavenger, N-acetyl-l-cysteine (NAC), dramatically abrogated RAR alpha stabilization by oridonin. More intriguingly, direct exposure to low concentrations of H2O2 also increased RAR alpha protein but not mRNA levels, suggesting a role for ROS in oridonin stabilization of RAR alpha protein. Further investigations showed that NAC antagonized oridonin-induced activation of NF-kappa B signaling, while the NF-kappa B signaling inhibitor, Bay 11-7082, effectively blocked the oridonin increase in RAR alpha protein levels. In line with this, over-expression of I kappa B alpha (A32/36), a super-repressor form of I kappa B alpha, or NF-kappa B-p65 knockdown inhibited oridonin or H2O2 induced RAR alpha stability. Finally, tumor necrosis factor alpha (TNF alpha), a classical activator of NF-kappa B signaling, modulated the stability of RAR alpha protein. Conclusions: Oridonin stabilizes RAR alpha protein by increasing cellular ROS levels, which causes activation of the NF-kappa B signaling pathway.