Rac1, and not Rac2, is involved in the regulation of the intracellular hydrogen peroxide level in HepG2 cells.
In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.
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Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Rheinlanddam 201, D-44139 Dortmund, Postfach 102664, D-44026 Dortmund, Germany. robbert.cool@mpi-dortmund.mpg.de
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D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D008845:Microinjections; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000502:physiology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020744:rac GTP-Binding Proteins
D009363:Neoplasm Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D009243:NAD; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins