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

Cao Y, Wei W, Zhang N, Yu Q, Xu WB, Yu WJ, Chen GQ, Wu YL, Yan H - BMC Cancer (2015)

Bottom Line: However, the underlying molecular mechanism for this action has not been fully elucidated.Flow cytometry was performed to detect intracellular levels of reactive oxygen species (ROS).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. yang9yue8ri@gmail.com.

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.

Show MeSH

Related in: MedlinePlus

Oridonin-induced RARα stability requires the activation and nuclear translocation of p65. (A) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h. The intracellular localization of p65 was analyzed using anti-p65 antibodies (red) with DAPI staining (blue) for nuclei. Scale bars represent 20 μm. (B, C) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h (B) or treated with 5 μM H2O2 for the times indicated (C), and the cell lysates were western blotted for the indicated proteins. (D) NB4 cells were infected with pSIREN-RetroQ-derived retroviruses carrying shRNA for p65 or scrambled shRNA as a control, and the cell lysates were western blotted for the indicated proteins. (E, F) NB4-NC and NB4-sh-p65 cells were treated with 10 μM oridonin for 12 h (E) or with 5 μM H2O2 for the times indicated (F), and the cell lysates were western blotted for proteins as indicated. All experiments were repeated three times and gave consistent results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4403721&req=5

Fig5: Oridonin-induced RARα stability requires the activation and nuclear translocation of p65. (A) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h. The intracellular localization of p65 was analyzed using anti-p65 antibodies (red) with DAPI staining (blue) for nuclei. Scale bars represent 20 μm. (B, C) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h (B) or treated with 5 μM H2O2 for the times indicated (C), and the cell lysates were western blotted for the indicated proteins. (D) NB4 cells were infected with pSIREN-RetroQ-derived retroviruses carrying shRNA for p65 or scrambled shRNA as a control, and the cell lysates were western blotted for the indicated proteins. (E, F) NB4-NC and NB4-sh-p65 cells were treated with 10 μM oridonin for 12 h (E) or with 5 μM H2O2 for the times indicated (F), and the cell lysates were western blotted for proteins as indicated. All experiments were repeated three times and gave consistent results.

Mentions: To confirm that oridonin stabilizes RARα through the NF-κB pathway, we used NB4/GFP-MAD cells to perform further experiments. This engineered cell line stably expresses the GFP-tagged super-repressor form of IκΒα, namely IκΒα (A32/36), which confers cellular resistance to signal-induced phosphorylation and subsequent proteasome-mediated degradation of IκΒα, resulting in the constitutive suppression of NF-κB activation by sequestering it in the cytoplasm [36]. As shown in Figure 5A, the over-expression of IκΒα (A32/36) blocked oridonin-induced nuclear translocation of p65. As expected, both oridonin- and H2O2-induced RARα stability were inhibited in NB4/GFP-MAD cells compared with NB4/GFP cells (Figure 5B and C). Furthermore, we stably transfected NB4 cells with shRNA specifically against the p65 subunit of the NF-κB family, which effectively silenced the expression of p65 but not p50 (Figure 5D). Notably, p65 knockdown prevented oridonin and H2O2-induced RARα stability in NB4 cells (Figure 5E and F). Collectively, these results suggest that the activation and nuclear translocation of p65 is essential for oridonin to stabilize RARα.Figure 5


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

Cao Y, Wei W, Zhang N, Yu Q, Xu WB, Yu WJ, Chen GQ, Wu YL, Yan H - BMC Cancer (2015)

Oridonin-induced RARα stability requires the activation and nuclear translocation of p65. (A) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h. The intracellular localization of p65 was analyzed using anti-p65 antibodies (red) with DAPI staining (blue) for nuclei. Scale bars represent 20 μm. (B, C) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h (B) or treated with 5 μM H2O2 for the times indicated (C), and the cell lysates were western blotted for the indicated proteins. (D) NB4 cells were infected with pSIREN-RetroQ-derived retroviruses carrying shRNA for p65 or scrambled shRNA as a control, and the cell lysates were western blotted for the indicated proteins. (E, F) NB4-NC and NB4-sh-p65 cells were treated with 10 μM oridonin for 12 h (E) or with 5 μM H2O2 for the times indicated (F), and the cell lysates were western blotted for proteins as indicated. All experiments were repeated three times and gave consistent results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403721&req=5

Fig5: Oridonin-induced RARα stability requires the activation and nuclear translocation of p65. (A) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h. The intracellular localization of p65 was analyzed using anti-p65 antibodies (red) with DAPI staining (blue) for nuclei. Scale bars represent 20 μm. (B, C) NB4/GFP and NB4/GFP-MAD cells were treated with 10 μM oridonin for 12 h (B) or treated with 5 μM H2O2 for the times indicated (C), and the cell lysates were western blotted for the indicated proteins. (D) NB4 cells were infected with pSIREN-RetroQ-derived retroviruses carrying shRNA for p65 or scrambled shRNA as a control, and the cell lysates were western blotted for the indicated proteins. (E, F) NB4-NC and NB4-sh-p65 cells were treated with 10 μM oridonin for 12 h (E) or with 5 μM H2O2 for the times indicated (F), and the cell lysates were western blotted for proteins as indicated. All experiments were repeated three times and gave consistent results.
Mentions: To confirm that oridonin stabilizes RARα through the NF-κB pathway, we used NB4/GFP-MAD cells to perform further experiments. This engineered cell line stably expresses the GFP-tagged super-repressor form of IκΒα, namely IκΒα (A32/36), which confers cellular resistance to signal-induced phosphorylation and subsequent proteasome-mediated degradation of IκΒα, resulting in the constitutive suppression of NF-κB activation by sequestering it in the cytoplasm [36]. As shown in Figure 5A, the over-expression of IκΒα (A32/36) blocked oridonin-induced nuclear translocation of p65. As expected, both oridonin- and H2O2-induced RARα stability were inhibited in NB4/GFP-MAD cells compared with NB4/GFP cells (Figure 5B and C). Furthermore, we stably transfected NB4 cells with shRNA specifically against the p65 subunit of the NF-κB family, which effectively silenced the expression of p65 but not p50 (Figure 5D). Notably, p65 knockdown prevented oridonin and H2O2-induced RARα stability in NB4 cells (Figure 5E and F). Collectively, these results suggest that the activation and nuclear translocation of p65 is essential for oridonin to stabilize RARα.Figure 5

Bottom Line: However, the underlying molecular mechanism for this action has not been fully elucidated.Flow cytometry was performed to detect intracellular levels of reactive oxygen species (ROS).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. yang9yue8ri@gmail.com.

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.

Show MeSH
Related in: MedlinePlus