Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury.
c-Src has been shown to activate NF-kappaB (nuclear factor kappaB) following H/R (hypoxia/reoxygenation) by acting as a redox-dependent IkappaBalpha (inhibitory kappaB) tyrosine kinase. In the present study, we have investigated the redox-dependent mechanism of c-Src activation following H/R injury and found that ROS (reactive oxygen species) generated by endosomal Noxs (NADPH oxidases) are critical for this process. Endocytosis following H/R was required for the activation of endosomal Noxs, c-Src activation, and the ability of c-Src to tyrosine-phosphorylate IkappaBalpha. Quenching intra-endosomal ROS during reoxygenation inhibited c-Src activation without affecting c-Src recruitment from the plasma membrane to endosomes. However, siRNA (small interfering RNA)-mediated knockdown of Rac1 prevented c-Src recruitment into the endosomal compartment following H/R. Given that Rac1 is a known activator of Nox1 and Nox2, we investigated whether these two proteins were required for c-Src activation in Nox-deficient primary fibroblasts. Findings from these studies suggest that both Nox1 and Nox2 participate in the initial redox activation of c-Src following H/R. In summary, our results suggest that Rac1-dependent Noxs play a critical role in activating c-Src following H/R injury. This signalling pathway may be a useful therapeutic target for ischaemia/reperfusion-related diseases.
Li
Qiang
Q
Department of Anatomy and Cell Biology and the Center for Gene Therapy, The College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Zhang
Yulong
Y
Marden
Jennifer J
JJ
Banfi
Botond
B
Engelhardt
John F
JF
D015687:Cell Hypoxia; D002478:Cells, Cultured; D004705:Endocytosis; D011992:Endosomes / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D016328:NF-kappa B; D017382:Reactive Oxygen Species; D019000:Phosphotyrosine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D016392:Proto-Oncogene Proteins pp60(c-src); D051550:I-kappa B Kinase; D020830:rac1 GTP-Binding Protein; D010100:Oxygen