An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells

被引:103
作者
Pak, JH [1 ]
Manevich, Y [1 ]
Kim, HS [1 ]
Feinstein, SI [1 ]
Fisher, AB [1 ]
机构
[1] Univ Penn, Sch Med, Inst Environm Med, Philadelphia, PA 19104 USA
关键词
D O I
10.1074/jbc.M204222200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H2O2. To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (-18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pMol/10(6) cells for antisense versus 70 pmol/10(6) cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium. iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.
引用
收藏
页码:49927 / 49934
页数:8
相关论文
共 38 条
[1]   Characterization of acidic Ca2+-independent phospholipase A2 of bovine lung [J].
Akiba, S ;
Dodia, C ;
Chen, X ;
Fisher, AB .
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 1998, 120 (02) :393-404
[2]  
BLIGH EG, 1959, CAN J BIOCHEM PHYS, V37, P911
[3]   1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities [J].
Chen, JW ;
Dodia, C ;
Feinstein, SI ;
Jain, MK ;
Fisher, AB .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (37) :28421-28427
[4]   Crystal structure of a novel human peroxidase enzyme at 2.0 Å resolution [J].
Choi, HJ ;
Kang, SW ;
Yang, CH ;
Rhee, SG ;
Ryu, SE .
NATURE STRUCTURAL BIOLOGY, 1998, 5 (05) :400-406
[5]  
DOUGLAS WHJ, 1974, IN VITRO CELL DEV B, V10, P230
[6]   Free radicals in the physiological control of cell function [J].
Dröge, W .
PHYSIOLOGICAL REVIEWS, 2002, 82 (01) :47-95
[7]   Phospholipid hydroperoxidase are substrates for non-selenium glutathione peroxidase [J].
Fisher, AB ;
Dodia, C ;
Manevich, Y ;
Chen, JW ;
Feinstein, SI .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (30) :21326-21334
[8]   Augmented expression of peroxiredoxin VI in rat lung and kidney after birth implies an antioxidative role [J].
Fujii, T ;
Fujii, J ;
Taniguchi, N .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 2001, 268 (02) :218-224
[9]   Reactive oxygen species and protein oxidation in aging: A look back, a look ahead [J].
Hensley, K ;
Floyd, RA .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 2002, 397 (02) :377-383
[10]   Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product [J].
Hirotsu, S ;
Abe, Y ;
Okada, K ;
Nagahara, N ;
Hori, H ;
Nishino, T ;
Hakoshima, T .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (22) :12333-12338