Regulation of phagocyte NADPH oxidase by hydrogen peroxide through a Ca2+/c-Abl signaling pathway

The importance of H₂O₂ as a cellular signaling molecule has been demonstrated in a number of cell types and pathways. Here we explore a positive feedback mechanism of H₂O₂-mediated regulation of the phagocyte respiratory burst NADPH oxidase (NOX2). H₂O₂ induced a dose-dependent stimulation of superoxide production in human neutrophils, as well as in K562 leukemia cells overexpressing NOX2 system components. Stimulation was abrogated by the addition of catalase, the extracellular Ca²⁺ chelator BAPTA, the T-type Ca²⁺ channel inhibitor mibefradil, the PKCδ inhibitor rottlerin, or the c-Abl nonreceptor tyrosine kinase inhibitor imatinib mesylate or by overexpression of a dominant-negative form of c-Abl. H₂O₂ induced phosphorylation of tyrosine 311 on PKCδ and this activating phosphorylation was blocked by treatment with rottlerin, imatinib mesylate, or BAPTA. Rac GTPase activation in response to H₂O₂ was abrogated by BAPTA, imatinib mesylate, or rottlerin. In conclusion, H₂O₂ stimulates NOX2-mediated superoxide generation in neutrophils and K562/NOX2 cells via a signaling pathway involving Ca²⁺ influx and c-Abl tyrosine kinase acting upstream of PKCδ. This positive feedback regulatory pathway has important implications for amplifying the innate immune response and contributing to oxidative stress in inflammatory disorders.