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

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 leuke...

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Autores principales: Cao, Yang, Wei, Wei, Zhang, Nan, Yu, Qing, Xu, Wen-Bin, Yu, Wen-Jun, Chen, Guo-Qiang, Wu, Ying-Li, Yan, Hua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403721/
https://www.ncbi.nlm.nih.gov/pubmed/25886043
http://dx.doi.org/10.1186/s12885-015-1219-8
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author Cao, Yang
Wei, Wei
Zhang, Nan
Yu, Qing
Xu, Wen-Bin
Yu, Wen-Jun
Chen, Guo-Qiang
Wu, Ying-Li
Yan, Hua
author_facet Cao, Yang
Wei, Wei
Zhang, Nan
Yu, Qing
Xu, Wen-Bin
Yu, Wen-Jun
Chen, Guo-Qiang
Wu, Ying-Li
Yan, Hua
author_sort Cao, Yang
collection PubMed
description 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 H(2)O(2) 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 H(2)O(2)-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.
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spelling pubmed-44037212015-04-21 Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling Cao, Yang Wei, Wei Zhang, Nan Yu, Qing Xu, Wen-Bin Yu, Wen-Jun Chen, Guo-Qiang Wu, Ying-Li Yan, Hua BMC Cancer Research Article 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 H(2)O(2) 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 H(2)O(2)-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. BioMed Central 2015-04-10 /pmc/articles/PMC4403721/ /pubmed/25886043 http://dx.doi.org/10.1186/s12885-015-1219-8 Text en © Cao et al.; licensee BioMed Central. 2015 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Cao, Yang
Wei, Wei
Zhang, Nan
Yu, Qing
Xu, Wen-Bin
Yu, Wen-Jun
Chen, Guo-Qiang
Wu, Ying-Li
Yan, Hua
Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
title Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
title_full Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
title_fullStr Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
title_full_unstemmed Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
title_short Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling
title_sort oridonin stabilizes retinoic acid receptor alpha through ros-activated nf-κb signaling
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403721/
https://www.ncbi.nlm.nih.gov/pubmed/25886043
http://dx.doi.org/10.1186/s12885-015-1219-8
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