Biochem. J. (2007) 406
(77–83) (Printed in Great Britain)
Polyunsaturated fatty acids modulate NOX 4 anion superoxide production in human fibroblasts
Adrien ROSSARY*†, Khelifa ARAB‡ and Jean-Paul STEGHENS*†1
*UF 21455, Stress Oxydant et Vitamines, Fédération de Biochimie, Hôpital E. Herriot, 5, Place d'Arsonval, F-69437 Lyon, France, †EA 3090, Claude Bernard University Lyon 1, Lyon, France, and ‡Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Neuenheimer Feld 280, 69120 Heidelberg, Germany
The strong ROS (reactive oxygen species) production, part of an antioxidant response of human fibroblasts triggered by DHA (docosahexaenoic acid; C22:6,n-3), served as a model for deciphering the relative contribution of NOX (NADPH oxidase) to ROS production, as the role of this enzymatic system remains controversial. Using hydroxyethidium fluorescence for fibroblast ROS production, RT (reverse transcriptase)–PCR for NOX 4 mRNA quantification and mRNA silencing, we show that ROS production evolves in parallel with the catalytic activity of NOX and is suppressed by siNOX 4 (small interference oligonucleotide RNA directed against NOX 4) silencing. Apocynin and plumbagin, specific inhibitors of NOX, prevent ROS production in this cellular model and confirm the role of NOX 4 for this production. Furthermore, we show that, in cell lysates, NOX 4 activity can be modulated by PUFAs (polyunsaturated fatty acids) at the micromolar level in the presence of calcium: NOX 4 activity is increased by arachidonic acid (C20:4,n-6) (~175% of the control), and conjugated linoleic acid (C18:2 [9Z,11E]) is a potent inhibitor (50% of the control). Unexpectedly, intracellular superoxide dismutase does not participate in the modulation of this ROS production and the opposite effects of some PUFAs, described in our experiments, could suggest another way of regulating NOX activity.
Key words: docosahexaenoic acid, fibroblast, haem oxygenase, NADPH oxidase (NOX), polyunsaturated fatty acid, reactive oxygen species.
Abbreviations used: AA, arachidonic acid (C20:4,n-6); BCA, bicinchoninic acid; CLA, conjugated linoleic acid (C18:2 [9Z,11E]); DHA, docosahexaenoic acid (C22:6,n-3); DHA-met, docosahexaenoic methyl ester; DMEM, Dulbecco's modified Eagle's medium; DPI, diphenyl iodonium; DUOX, dual oxidase; E+, ethidium; E_OH+, hydroxyethidium; EPA, eicosapentaenoic acid (C20:5,n-3); gp91phox, one of the two integral membrane proteins making up flavocytochrome b558; HE, hydroethidine; HO-1, haem oxygenase 1; IU, international unit; LC/MS, liquid chromatography MS; NOX, NADPH oxidase; p22phox, the other subunit making up flavocytochrome b558; PUFA, polyunsaturated fatty acid; QPCR, quantitative PCR; ROS, reactive oxygen species; RT, retention time; RT–PCR, reverse transcriptase–PCR; siNOX 4, small interference oligonucleotide RNA directed against NOX 4; siRNA, small interfering RNA; SOD, superoxide dismutase; TIC, total ion current; XO, xanthine oxidase.
1To whom correspondence should be addressed (email jean-paul.steghens@chu-lyon.fr).
Received 3 July 2006/5 April 2007; accepted 2 May 2007
Published as BJ Immediate Publication 2 May 2007, doi:10.1042/BJ20061009
© The Authors Journal compilation © 2007 Biochemical Society
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