TY - JOUR
T1 - Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells
AU - He, Rong
AU - Nanamori, Masakatsu
AU - Sang, Hairong
AU - Yin, Hong
AU - Dinauer, Mary C.
AU - Ye, Richard D.
PY - 2004/12/15
Y1 - 2004/12/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=10344246590&partnerID=8YFLogxK
U2 - 10.4049/jimmunol.173.12.7462
DO - 10.4049/jimmunol.173.12.7462
M3 - Article
C2 - 15585872
AN - SCOPUS:10344246590
SN - 0022-1767
VL - 173
SP - 7462
EP - 7470
JO - Journal of Immunology
JF - Journal of Immunology
IS - 12
ER -