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Peroxynitrite modulates MnSOD gene expression in lung epithelial cells
R M Jackson1, G Parish, E S Helton
1Birmingham VA Medical Center and University of Alabama at Birmingham, 35294-0006, USA. rjackson@uab.edu
Free Radical Biology & Medicine
|September 19, 1998
Summary
Peroxynitrite (ONOO-) stimulates manganese superoxide dismutase (MnSOD) gene expression in lung cells. This reactive nitrogen species increases MnSOD mRNA but not protein levels, suggesting a transcriptional regulation mechanism.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- Peroxynitrite (ONOO-) is a potent oxidant formed from nitric oxide (NO) and superoxide (O2.-).
- Oxidative stress, including from TNF-alpha and hyperoxia, is known to induce manganese superoxide dismutase (MnSOD) gene expression.
- MnSOD is a critical antioxidant enzyme protecting against reactive oxygen species (ROS).
Purpose of the Study:
- To investigate whether peroxynitrite (ONOO-) regulates manganese superoxide dismutase (MnSOD) gene expression in human lung epithelial cells (A549).
Main Methods:
- Human lung epithelial cells (A549) were treated with SIN-1 (a peroxynitrite generator) or authentic peroxynitrite.
- Messenger RNA (mRNA) levels for MnSOD and hypoxanthine guanine phosphoribosyl transferase (HPRT) were quantified.
- Luciferase gene expression driven by the MnSOD promoter was measured.
- Inhibitors of reactive nitrogen species (L-cysteine, N-acetyl cysteine, pyrrole dithiocarbamate) were used.
- Nitric oxide (NO) donors and breakdown products of peroxynitrite (H2O2, NO2-) were tested.
- Western blots and enzymatic activity assays were performed to assess MnSOD protein and activity.
Main Results:
- SIN-1 and authentic peroxynitrite significantly increased MnSOD mRNA levels but not HPRT mRNA.
- Peroxynitrite stimulated luciferase expression under the control of the MnSOD gene promoter.
- MnSOD gene induction by peroxynitrite was inhibited by L-cysteine and partially by N-acetyl cysteine and pyrrole dithiocarbamate.
- Nitric oxide donors, H2O2, and NO2- did not affect MnSOD mRNA levels.
- Neither peroxynitrite nor SIN-1 increased detectable MnSOD protein levels or enzymatic activity.
Conclusions:
- Peroxynitrite directly stimulates MnSOD transcript expression in lung epithelial cells.
- The effect of peroxynitrite on MnSOD appears to be at the transcriptional level.
- While peroxynitrite increases MnSOD mRNA, it does not lead to a detectable increase in MnSOD protein or activity under these experimental conditions.