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

Kim HJ, Zheng M, Kim SK, Cho JJ, Shin CH, Joe Y, Chung HT - Immune Netw (2011)

Bottom Line: 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.Our results suggest that CO-induced Nrf2 increases the expression of NQO1 which is well known to detoxify reactive metabolites and ROS.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Ulsan, Ulsan 680-749, Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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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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).

Mentions: As shown in Fig. 4, CORM-2 treatment of HepG2 cells was able to significantly stimulate the NQO1 promoter at 6 h after the incubation. To confirm the involvement of Nrf2 for CO-induced NQO1 promoter activity, we inhibited Nrf2 expression by siRNA against Nrf2. Down-regulation of Nrf2 by siRNA abrogated the CO-induced NQO1 promoter activity (Fig. 7). These data indicate that the NQO1 promoter activity is under regulation of CO and Nrf2 in HepG2 cells. In addition, our previous result showed that CO up-regulates Nrf2-dependent HO-1 expression via PERK activation (27). Similar to the reduction of NQO1 promoter activity by siRNA against Nrf2, CO-induced NQO1 promoter activities were abrogated by PERK siRNA suggesting that induction of NQO1 promoter activity by CORM-2 is mediated via a PERK pathway. Thus, our data suggest that NQO1 expression is induced by CO through PERK-Nrf2 pathway.


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

Kim HJ, Zheng M, Kim SK, Cho JJ, Shin CH, Joe Y, Chung HT - Immune Netw (2011)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275707&req=5

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).
Mentions: As shown in Fig. 4, CORM-2 treatment of HepG2 cells was able to significantly stimulate the NQO1 promoter at 6 h after the incubation. To confirm the involvement of Nrf2 for CO-induced NQO1 promoter activity, we inhibited Nrf2 expression by siRNA against Nrf2. Down-regulation of Nrf2 by siRNA abrogated the CO-induced NQO1 promoter activity (Fig. 7). These data indicate that the NQO1 promoter activity is under regulation of CO and Nrf2 in HepG2 cells. In addition, our previous result showed that CO up-regulates Nrf2-dependent HO-1 expression via PERK activation (27). Similar to the reduction of NQO1 promoter activity by siRNA against Nrf2, CO-induced NQO1 promoter activities were abrogated by PERK siRNA suggesting that induction of NQO1 promoter activity by CORM-2 is mediated via a PERK pathway. Thus, our data suggest that NQO1 expression is induced by CO through PERK-Nrf2 pathway.

Bottom Line: 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.Our results suggest that CO-induced Nrf2 increases the expression of NQO1 which is well known to detoxify reactive metabolites and ROS.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Ulsan, Ulsan 680-749, Korea.

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.

No MeSH data available.


Related in: MedlinePlus