Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells.
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) production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCdelta, but not PKCalpha, -betaII, and -zeta, is necessary for the COS-phox cells to respond to fMLP. A role of PKCdelta in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCdelta translocation and the sensitivity of fMLP-induced O()(2) production to rottlerin, a PKCdelta-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) production and raised the level of O()(2) in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O()(2) production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.
He
Rong
R
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612, USA.
Nanamori
Masakatsu
M
Sang
Hairong
H
Yin
Hong
H
Dinauer
Mary C
MC
Ye
Richard D
RD
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D010084:Oxidation-Reduction; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000502:physiology; D054799:Phospholipase C beta; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology; D051745:Protein Kinase C-delta; D044042:Receptors, Formyl Peptide / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013481:Superoxides / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D010738:Type C Phospholipases / Q000235:genetics / Q000502:physiology; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000502:physiology; D000097766:RAC2 GTP-Binding Protein
D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D007527:Isoenzymes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D054799:Phospholipase C beta; D010750:Phosphoproteins; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D044042:Receptors, Formyl Peptide; D013481:Superoxides; D010738:Type C Phospholipases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C085072:neutrophil cytosol factor 40K; C497258:PRKCD protein, human; C518036:PLCB2 protein, human