Immune Netw.  2011 Dec;11(6):376-382. 10.4110/in.2011.11.6.376.

CO/HO-1 Induces NQO-1 Expression via Nrf2 Activation

• Kim HJ
• Zheng M
• Kim SK
• Cho JJ
• Shin CH
• Joe Y
• Chung HT

• KMID: 2150723
• DOI: http://doi.org/10.4110/in.2011.11.6.376

Abstract

BACKGROUND
Carbon monoxide (CO) is a cytoprotective and homeostatic molecule with important signaling capabilities in physiological and pathophysiological situations. CO protects cells/tissues from damage by free radicals or oxidative stress. NAD(P)H:quinone oxidoreductase (NQO1) is a highly inducible enzyme that is regulated by the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway, which is central to efficient detoxification of reactive metabolites and reactive oxygen species (ROS).
METHODS
We generated NQO1 promoter construct. HepG2 cells were treated with CO Releasing Molecules-2 (CORM-2) or CO gas and the gene expressions were measured by RT-PCR, immunoblot, and luciferase assays.
RESULTS
CO induced expression of NQO1 in human hepatocarcinoma cell lines by activation of Nrf2. Exposure of HepG2 cells to CO resulted in significant induction of NQO1 in dose- and time-dependent manners. Analysis of the NQO1 promoter indicated that an antioxidant responsible element (ARE)-containing region was critical for the CO-induced Nrf2-dependent increase of NQO1 gene expression in HepG2 cells.
CONCLUSION
Our results suggest that CO-induced Nrf2 increases the expression of NQO1 which is well known to detoxify reactive metabolites and ROS.

Keyword

Carbon monoxide; Heme oxygenase-1; Nrf2; NQO1

MeSH Terms

Carbon Monoxide
Cell Line
Free Radicals
Gene Expression
Heme Oxygenase-1
Hep G2 Cells
Humans
Luciferases
Oxidative Stress
Reactive Oxygen Species
Response Elements
Carbon Monoxide
Free Radicals
Heme Oxygenase-1
Luciferases
Reactive Oxygen Species

Figure

• 

  Figure 1 Dose-dependent NQO1 mRNA induction by exogenous CO. (A, B) HepG2 cells were treated with various concentrations of CORM-2 as exogenous CO donor or Rucl2 as control for 6 h and the cells were harvested for semi-quantitative RT-PCR. The mRNA expression of NQO1 was determined by semi-quantitative RT-PCR. GAPDH was used as internal controls.

• 

  Figure 2 Time dependent NQO1 mRNA induction by exogenous CO. (A, B) HepG2 cells were treated with 50 uM CORM-2 or 250 ppm CO gas for indicated time points and analyzed. The mRNA expression of NQO1 was determined by semi-quantitative RT-PCR as described under Material and methods. GAPDH was used as internal controls.

• 

  Figure 3 Effects of exogenous CO on the NQO1 and HO-1 protein induction. HepG2 cells were treated with various concentrations of CORM-2 as exogenous CO donor for 6 h and the cell lysates were used for immunoblot with antibody against NQO1, HO-1, and β-actin.

• 

  Figure 4 Effects of CORM-2 on the NQO1 promoter activity. HepG2 cells were transfected with hNQO1p-501 containing the NQO1 promoter (-501 to +117). After 24 h cells were treated with various concentrations of CORM-2. At 6 h post-treatment, the level of firefly luciferase activity was normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments (**p<0.01).

• 

  Figure 5 Effects of CORM-2 on Nrf2-mediated NQO1 promoter activity. HepG2 cells were co-transfected with pGL3/NQO1p-ARE and pcDNA3-Nrf2. After 24 h, cells were treated with 30 uM CORM-2 and at 6 h post-treatment, luciferase activity was determined. The levels of firefly luciferase activity were normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments (*p<0.05, ***p<0.001).

• 

  Figure 6 Effects of CoPP and curcumin on NQO1 expression. (A, B) HepG2 cells were treated with 10 µM CoPP and 10 µM curcumin for the indicated time period. Total RNA were extracted and expression of NQO1 and GAPDH mRNA was detected by semiquantitative RT-PCR.

• 

  Figure 7 Effects of silencing of Nrf2 on CO-induced NQO1 promoter activity HepG2 cells were transfected with pGL3/NQO1p-501 containing the NQO1 promoter (-501 to +117), Nrf2 specific (siNRf2), and PERK specific (siPERK) or scrambled siRNA (scRNA). After 24 h cells were treated with various concentrations of CORM-2. At 6 h post-treatment, the level of firefly luciferase activity was normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments (*p<0.05, **p<0.01).

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