Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis.
Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.
Tampo
Yoshiko
Y
Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wis 53226, USA.
Kotamraju
Srigiridhar
S
Chitambar
Christopher R
CR
Kalivendi
Shasi V
SV
Keszler
Agnes
A
Joseph
Joy
J
Kalyanaraman
B
B
D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005452:Fluoresceins / Q000378:metabolism / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000378:metabolism / Q000493:pharmacokinetics; D005978:Glutathione / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007501:Iron / Q000378:metabolism* / Q000493:pharmacokinetics; D007502:Iron Chelating Agents / Q000494:pharmacology; D035941:Iron Regulatory Protein 1 / Q000378:metabolism; D008054:Lipid Peroxides / Q000494:pharmacology; D008665:Metalloporphyrins / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011990:Receptors, Transferrin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007502:Iron Chelating Agents; D008054:Lipid Peroxides; D008665:Metalloporphyrins; D016877:Oxidants; D011990:Receptors, Transferrin; C065013:2',7'-dichlorodihydrofluorescein; D006861:Hydrogen Peroxide; D007501:Iron; D053148:Caspase 3; D020169:Caspases; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1; D005978:Glutathione