Regulation of TNF-induced oxygen radical production in human neutrophils: role of δ-PKC

Abstract

In human neutrophils, TNF-elicited O₂⁻ production requires adherence and integrin activation. How this cooperative signaling between TNFRs and integrins regulates O₂⁻ generation has yet to be fully elucidated. Previously, we identified δ-PKC as a critical early regulator of TNF signaling in adherent neutrophils. In this study, we demonstrate that inhibition of δ-PKC with a dominant-negative δ-PKC TAT peptide resulted in a significant delay in the onset time of TNF-elicited O₂⁻ generation but had no effect on Vmax, indicating an involvement of δ-PKC in the initiation of O₂⁻ production. In contrast, fMLP-elicited O₂⁻ production in adherent and nonadherent neutrophils was δ-PKC-independent, suggesting differential regulation of O₂⁻ production. An important step in activation of the NADPH oxidase is phosphorylation of the cytosolic p47phox component. In adherent neutrophils, TNF triggered a time-dependent association of δ-PKC with p47phox, which was associated with p47phox phosphorylation, indicating a role for δ-PKC in regulating O₂⁻ production at the level of p47phox. Activation of ERK and p38 MAPK is also required for TNF-elicited O₂⁻ generation. TNF-mediated ERK but not p38 MAPK recruitment to p47phox was δ-PKC-dependent. δ-PKC activity is controlled through serine/threonine phosphorylation, and phosphorylation of δ-PKC (Ser643) and δ-PKC (Thr505) was increased significantly by TNF in adherent cells via a PI3K-dependent process. Thus, signaling for TNF-elicited O₂⁻ generation is regulated by δ-PKC. Adherence-dependent cooperative signaling activates PI3K signaling, δ-PKC phosphorylation, and δ-PKC recruitment to p47phox. δ-PKC activates p47phox by serine phosphorylation or indirectly through control of ERK recruitment to p47phox.