Polyunsaturated fatty acids modulate NOX 4 anion superoxide production in human fibroblasts.
The strong ROS (reactive oxygen species) production, part of an antioxidant response of human fibroblasts triggered by DHA (docosahexaenoic acid; C(22:6,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) (approximately 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.
Rossary
Adrien
A
UF 21455, Stress Oxydant et Vitamines, Fédération de Biochimie, Hôpital E. Herriot, 5, Place d'Arsonval, F-69437 Lyon, France.
Arab
Khelifa
K
Steghens
Jean-Paul
JP
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D004996:Ethidium / Q000032:analysis; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D013058:Mass Spectrometry; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors
D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013481:Superoxides; D004281:Docosahexaenoic Acids; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C086986:CYBA protein, human; D004996:Ethidium