Inhibition of arachidonate metabolism in human epidermoid carcinoma A431 cells overexpressing phospholipid hydroperoxide glutathione peroxidase
Received: 22 March 2002 Accepted: 03 April 2002 DOI:
Cite this article as: Chen, CJ., Huang, HS. & Chang, WC. J Biomed Sci (2002) 9: 453. doi:10.1007/BF02256540 Abstract
Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including phospholipid hydroperoxides, fatty acid hydroperoxides and cholesterol hydroperoxides, and can reduce them to hydroxide compounds. It also seems to be a major regulator of lipid oxygenation in human epidermoid carcinoma A431 cells. In order to study the functional role of PHGPx in the regulation of 12-lipoxygenase and cyclooxygenase, cDNA of PHGPx was inserted into pcDNA3.1/His, and a plasmid designated as S4 with the His-tag sequence inserted between PHGPx and its 3′-untranslated region was constructed. A number of stable transfectants of A431 cells that could express the tag-PHGPx were generated using plasmid S4. Using an intact cell assay system, the metabolism of arachidonic acid to prostaglandin E
2 significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line. If the intact cell assay was carried out in the presence of 13-hydroperoxyoctadecadienoic acid as a stimulator of lipid peroxidation, formation of 12-hydroxyeicosatetraenoic acid from arachidonic acid also significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line, indicating that PHGPx could downregulate the 12-lipoxygenase activity in cells. These results support the hypothesis that PHGPx plays a pivotal role in the regulation of arachidonate metabolism in A431 cells. Key Words Phospholipid hydroperoxide glutathione peroxidase Cyclooxygenase 12-Lipoxygenase A431 cells References
Chang WC, Nakao J, Orimo H, Murota S. Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets. Biochem J 202:771–776;1982.
Chang WC, Ning CC, Lin MT, Huang JD. Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. J Biol Chem 267:3657–3666;1992.
Chen CJ, Huang HS, Lee YT, Yang CY, Chang WC. Characterization and purification of a lipoxygenase inhibitor in human epidermoid carcinoma A431 cells. Biochem J 327:193–198;1997.
Chen CJ, Huang HS, Lin SB, Chang WC. Regulation of cyclooxygenase and 12-lipoxygenase catalysis by phospholipid hydroperoxide glutathione peroxidase in A431 cells. Prostaglandins Leukot Essent Fatty Acids 62:261–268;2000.
Chu FF, Doroshow JH, Esworthy RS. Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI. J Biol Chem 268:2571–2576;1993.
Esworthy RS, Doan K, Doroshow JH, Chu FF. Cloning and sequencing of the cDNA encoding a human testis phospholipid hydroperoxide glutathione peroxidase. Gene 144:317–318;1994.
Ghyselinck NB, Dufaure JP. A mouse cDNA sequence for epididymal androgen-regulated proteins related to glutathione peroxidase. Nucleic Acids Res 18:7144;1990.
Huang HS, Chen CJ, Lu HS, Chang WC. Identification of a lipoxygenase inhibitor in A431 cells as a phospholipid hydroperoxide glutathione peroxidase. FEBS Lett 424:22–26;1998.
Imai H, Narashima K, Arai M, Sakamoto H, Chiba N, Nakagawa Y. Suppression of leukotriene formation in RBL-2H3 cells that overexpressed phospholipid hydroperoxide glutathione peroxidase. J Biol Chem 273:1990–1997;1998.
Imai H, Sumi D, Hanamoto A, Arai M, Sugiyama A. Molecular cloning and functional expression of a cDNA for rat phospholipid hydroperoxide glutathione peroxidase: 3′-Untranslated region of the gene is necessary for functional expression. J Biochem 118:1061–1067;1995.
Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292;1986.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275;1951.
Maddipati KR, Gasparski C, Marnett LJ. Characterization of the hydroperoxide-reducing activity of human plasma. Arch Biochem Biophys 254:9–17;1987.
Maiorino M, Gregolin C, Ursini F. Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol 186:448–457;1990.
Marshall PJ, Kulmacz RJ, Lands WE. Constraints on prostaglandin biosynthesis in tissues. J Biol Chem 262:3510–3517;1987.
Nelson MJ, Chase DB, Seitz SP. Photolysis of ‘purple’ lipoxygenase: Implications for the structure of the chromophore. Biochemistry 34:6159–6163;1995.
Pushpa-Rekha TR, Burdsall AL, Oleksa LM, Chisolm GM, Driscoll DM. Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites. J Biol Chem 270:26993–26999;1995.
Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 179:588–590;1973.
Sakamoto H, Imai H, Nakagawa Y. Involvement of phospholipid hydroperoxide glutathione peroxidase in the modulation of prostaglandin D2 synthesis. J Biol Chem 275:40028–40035;2000.
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1989.
Schnurr K, Belkner J, Ursini F, Schewe T, Kuhn H. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase controls the activity of the 15-lipoxygenase with complex substrates and preserves the specificity of the oxygenation products. J Biol Chem 271:4653–4658;1996.
Schnurr K, Borchert A, Gerth C, Anton M, Kuhn H. Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale. Protein Expr Purif 19:403–410;2000.
Thomas JP, Maiorino M, Ursini F, Girotti AW. Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides. J Biol Chem 265:454–461;1990.
Ursini F, Maiorino M, Gregolin C. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Biochim Biophys Acta 839:62–70;1985.
Yamamoto S. ‘Enzymatic’ lipid peroxidation: Reactions of mammalian lipoxygenases. Free Radic Biol Med 10:149–159;1991.
Google Scholar Copyright information
© National Science Council 2002