Functional role of an endogenous inhibitor of lipid peroxidation

• Main Authors: Ching-Jiunn Chen, 陳清俊
• Other Authors: Wen-Chang Chang
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/36u485

博士 === 國立成功大學 === 基礎醫學研究所 === 90 === 英 文 摘 要 The accumulation of oxygen radicals injures the functions of cells and leads to the pathogenesis of some organs including vascular, brain, and kidney and so on. Important sources of the oxygen radicals are reactions associated with the metabolism of unsaturated fatty acids, such as lipoxygenases, cyclooxygenases (COXs), and monooxygenases. If the regulation of these enzymes is abnormal, or the downstream degradation enzymes lose their catalysis, it will result in the accumulation of oxygen radicals and affect the normal functions of the cells. Therefore, to study how cells regulate the expression of these enzymes and maintain cells away from the damage of oxygen radicals was the main aim of this study. In our preliminary studies, we found that a factor in the cytosolic fraction of human epidermoid carcinoma A431 cells could inhibit the activity of arachidonate 12-lipoxygenase (12-LOX) in vitro. After serial studies on its purification, characterization, analysis of amino acid sequence, and specific activity, we identified that the inhibitor as phospholipid hydroperoxide glutathione peroxidase (PHGPx). Depletion of glutathione (GSH) in cells resulted in up-regulation of the enzyme activities of COX and 12-LOX, especially on COX. In terms of the inhibitory effect on 12-LOX activity, PHGPx was more sensitive to GSH concentrations than glutathione peroxidase 1, suggesting that treatment of cells with GSH depleting agents would essentially abolish the enzyme activity of PHGPx. Additionally, the high activity of 12-LOX in the cytosolic fraction of human platelet might due to the lack of PHGPx activity. It appears that PHGPx is a major regulator of lipid oxygenation in A431 cells. The accumulated evidence indicates that hydroperoxide plays an important role in the initial reaction of these oxygenases, and that the PHGPx may inhibit the start reaction of these enzymes by removing hydroperoxides. The lipid oxygenation stimulated by 13-hydroperoxyoctadecadienoic acid in GSH-depleted cells supported this notion. To get more direct evidence of this hypothesis, we constructed a serial of plasmids and transformed into A431 cells. A stable transfectant overexpressing tag-PHGPx which could be recognized by either Xpress or PHGPx antibodies and contains the specific enzyme activity of PHGPx was established. The metabolism of arachidonic acid by COX and 12-LOX significantly decreased in stable transfectants overexpressing PHGPx compared to that in a vector-control cell line, suggesting that overexpressing PHGPx in cells could inhibit the lipid oxygenation. In addition, inhibition of PHGPx activity by the treatment of cells with antisense oligonucleotide of PHGPx mRNA increased the enzymatic catalysis of both COX and 12-LOX. The metabolism of arachidonic acid by COX and 12-LOX significantly increased in stable transfectants overexpressing antisense of PHGPx mRNA compared to that in a vector-control cell line, suggesting that blocking the biosynthesis of PHGPx by antisense of PHGPx mRNA would up-regulate lipid oxygenation in cells. Consequently, we conclude that PHGPx can regulate the enzyme activity of oxygenases in A431 cells.