Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells

Rong He, Masakatsu Nanamori, Hairong Sang, Hong Yin, Mary C. Dinauer, Richard D. Ye

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38 Scopus citations

Abstract

A whole-cell-based reconstitution system was developed to study the signaling mechanisms underlying chemoattractant-induced activation of NADPH oxidase. This system takes advantage of the lack of formyl peptide receptor-mediated response in COS-phox cells expressing gp91phox, p22phox, p67phox, and p47phox, which respond to phorbol ester and arachidonic acid with O2.- production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCδ, but not PKCα, -βII, and -ζ, is necessary for the COS-phox cells to respond to fMLP. A role of PKCδ in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCδ translocation and the sensitivity of fMLP-induced O2.- production to rottlerin, a PKCδ-selective inhibitor. Optimal reconstitution also requires phospholipase C-β2 and PI3K-γ. We found that formyl peptide receptor could use the endogenous Rac1 as well as exogenous Rac1 and Rac2 for NADPH oxidase activation. Exogenous expression of p40phox potentiated fMLP-induced O2 .- production and raised the level of O2.- in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O2.- production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.

Original languageEnglish
Pages (from-to)7462-7470
Number of pages9
JournalJournal of Immunology
Volume173
Issue number12
DOIs
StatePublished - Dec 15 2004

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