Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells

Dae Goon Yoo; Cuk Seong Kim; Sang Ki Lee; Hyo Shin Kim; Eun Jung Cho; Myoung Soo Park; Sang Do Lee; Jin Bong Park; Byeong Hwa Jeon

doi:10.4196/kjpp.2010.14.3.139

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Yoo, Kim, Lee, Kim, Cho, Park, Lee, Park, and Jeon: Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells

Original Article

Korean J Physiol Pharmacol 2010;14(3):139-144.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.3.139

Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells

Dae Goon Yoo^1,^∗, Cuk Seong Kim^2,^∗, Sang Ki Lee³, Hyo Shin Kim¹, Eun Jung Cho¹, Myoung Soo Park¹, Sang Do Lee¹, Jin Bong Park¹, Byeong Hwa Jeon^1,

¹Department of Physiology, Infection Signaling Network Research Center, and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon 301-131, Korea

²Cardiovascular Institute, University of Pittsburgh Medical Center, Pittsburgh PA 15213, USA

³Department of Sports Science, Chungnam National University, Daejeon 305-765, Korea

Corresponding to: Byeong Hwa Jeon, Infection Signaling Network Research Center, Department of Physiology, School of Medicine, Chungnam National University, 6, Munhwa-dong, Jung-gu, Daejeon 301-131, Korea. (Tel) 82-42-580-8214, (Fax) 82-42-585-8440, (E-mail) bhjeon@cnu.ac.kr

^∗ First two authors contributed equally to this article.

Received 26 April 2010 Revised 14 May 2010 Accepted 14 May 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-α (TNF-α) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-α induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-α dose-dependent (5∼100 ng/ml). TNF-α-stimulated A549 cells evidenced increased Ref-1 expression in a dose-dependent manner. The adenoviral transfection of cells with AdRef-1 inhibited TNF-α-induced COX-2 expression relative to that seen in the control cells (Adβgal). Pretreatment with 10 μM of SB203580 suppressed TNF-α-induced COX-2 expression, thereby suggesting that p38 MAPK might be involved in COX-2 expression in A549 cells. The phosphorylation of p38 MAPK was increased significantly after 5 minutes of treatment with TNF-α, reaching a maximum level at 10 min which persisted for up to 60 min. However, p38MAPK phosphorylation was markedly suppressed in the Ref-1-overexpressed A549 cells. Taken together, our results appear to indicate that Ref-1 negatively regulates COX-2 expression in response to cytokine stimulation via the inhibition of p38 MAPK phosphorylation. In the lung cancer cell lines, Ref-1 may be involved as an important negative regulator of inflammatory gene expression.

Keywords: Redox factor-1, Cyclooxygenase-2, A549, Lung cancer, p38 MAPK

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Fig. 1.

Tumor necrosis factor-α (TNF-α) induced cyclooxygenase-2 (COX-2) expression in the A549 cells. (A) The effect of TNF-α on COX-2 expression in BEAS-2B and A549 cells. COX-2 expression was analyzed via Western blotting 24 hr after exposure to TNF-α (30∼100 ng/ml). (B) COX-2 expression in A549 cells evidenced dose-dependency to TNF-α (5∼100 ng/ml). (C) The time-dependent change of COX-2 expression in the response to TNF-α (30 ng/ml) in A549 cells.

Fig. 2.

Tumor necrosis factor-α (TNF-α) induced Ref-1 expression in the A549 cells. (A) Effect of TNF-α on the Ref-1 expression in the A549 cells. Ref-1 expression was analyzed via Western blotting 24 hr after exposure to TNF-α (30∼100 ng/ml). β-actin was used as a loading control. (B) Densitometry analysis for Ref-1 expression. Each bar shows the mean±S.E. (n=4), ^∗p<0.05, ^∗∗p<0.01.

Fig. 3.

Ref-1 inhibited COX-2 expression in the A549 cells. (A) Western blots for COX-2 and Ref-1. After the cells were treated with AdRef-1 (0∼500 MOI), the cells were stimulated with TNF-α (30 ng/ml) for 18 hr. Western blotting was conducted using anti-COX-2 and anti-Ref-1 antibodies. The total adenovirus titer was balanced with Adβgal (500 MOI). (B) Densitometry analysis for COX-2 expression. Each bar showed the mean±S.E. (n=3), ^∗p<0.05, ^∗∗p<0.01.

Fig. 4.

Ref-1 inhibited p38 MAPK activation in the A549 cells. (A) TNF-α induced COX-2 expression was inhibited by SB203580 (10 μM), an inhibitor of p38 MAPK. (B) Time-dependent change of p38 MAPK activation (p-p38 MAPK) and ERK activation (p-ERK) in the response of TNF-α in the Adβgal or AdRef-1-transfected A549 cells. (C) Densitometry analysis for phospho-p38 MAPK expression. Each bar showed the mean±S.E. (n=4), ^∗∗p<0.01.

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