期刊
JOURNAL OF IMMUNOLOGY
卷 173, 期 12, 页码 7462-7470出版社
AMER ASSOC IMMUNOLOGISTS
DOI: 10.4049/jimmunol.173.12.7462
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资金
- NHLBI NIH HHS [HL45635, HL69974] Funding Source: Medline
- NIAID NIH HHS [AI33503] Funding Source: Medline
- NIDDK NIH HHS [T32 DK07739] Funding Source: Medline
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 gp91(phox), p22(phox), p67(phox), and p47(phox), which respond to phorbol ester and arachidonic acid with O-2((center dot) over bar) production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCS, but not PKCalpha, -betaII, and -zeta, is necessary for the COS-phox cells to respond to fMLP. A role of PKCS in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCdelta translocation and the sensitivity of fMLP-induced O-2((center dot) over bar) production to rottlerin, a PKCS-selective inhibitor. Optimal reconstitution also requires phospholipase C-beta2 and PI3K-gamma. 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 p40(phox) potentiated fMLP-induced O-2((center dot) over bar) production and raised the level of O-2((center dot) over bar) in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O-2((center dot) over bar) production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.
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