title: Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling.
abstract: BACKGROUND
  Retinoic acid receptor alpha (RARα) plays an essential role in the regulation of many biological processes, such as hematopoietic cell differentiation, while abnormal RARα 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 RARα 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 RARα 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-κB) 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
  RARα possesses transcriptional activity in the presence of its ligand, all-trans retinoic acid (ATRA). Oridonin remarkably stabilized the RARα 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 RARα stabilization by oridonin. More intriguingly, direct exposure to low concentrations of H2O2 also increased RARα protein but not mRNA levels, suggesting a role for ROS in oridonin stabilization of RARα protein. Further investigations showed that NAC antagonized oridonin-induced activation of NF-κB signaling, while the NF-κB signaling inhibitor, Bay 11-7082, effectively blocked the oridonin increase in RARα protein levels. In line with this, over-expression of IκΒα (A32/36), a super-repressor form of IκΒα, or NF-κB-p65 knockdown inhibited oridonin or H2O2-induced RARα stability. Finally, tumor necrosis factor alpha (TNFα), a classical activator of NF-κB signaling, modulated the stability of RARα protein.
  
  
  CONCLUSIONS
  Oridonin stabilizes RARα protein by increasing cellular ROS levels, which causes activation of the NF-κB signaling pathway.
authors.affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. yang9yue8ri@gmail.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Department of Hematology, Xinhua Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. chubbywinnie@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. 755641678@qq.com.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. yq11267@rjh.com.cn.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yu
authors.affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. francoisxu@hotmail.com.
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Xu
authors.affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. ywj_office@163.com.
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Yu
authors.affiliation: Department of Pathophysiology, Chemical Biology Division of Shanghai Universities E-Institutes, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. chengq@shsmu.edu.cn.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
authors.affiliation: Department of Pathophysiology, Chemical Biology Division of Shanghai Universities E-Institutes, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. wuyingli@shsmu.edu.cn.
authors.forename: Ying-Li
authors.initials: YL
authors.lastname: Wu
authors.affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. yanhua_candy@163.com.
authors.forename: Hua
authors.initials: H
authors.lastname: Yan
mesh_terms: D000818:Animals; D002460:Cell Line; D045786:Diterpenes, Kaurane / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D045786:Diterpenes, Kaurane; D016328:NF-kappa B; C000607071:RARA protein, human; D017382:Reactive Oxygen Species; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; C011959:oridonin