A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites.
Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM-CSF-induced phosphorylation of Ser345, while p38 MAPK inhibitor abrogated TNF-alpha-induced phosphorylation of Ser345. Transfection of HL-60 cells with a mutated p47phox (S345A) inhibited GM-CSF- and TNF-alpha-induced priming of ROS production. This event was also inhibited in neutrophils by a cell-permeable peptide containing a TAT-p47phox-Ser345 sequence. Furthermore, ROS generation, p47phox-Ser345 phosphorylation, and ERK1/2 and p38 MAPK phosphorylation were increased in synovial neutrophils from rheumatoid arthritis (RA) patients, and TAT-Ser345 peptide inhibited ROS production by these primed neutrophils. This study therefore identifies convergent MAPK pathways on Ser345 that are involved in GM-CSF- and TNF-alpha-induced priming of neutrophils and are activated in RA. Inhibition of the point of convergence of these pathways might serve as a novel antiinflammatory strategy.
Dang
Pham My-Chan
PM
INSERM, U.773, CRB3, Paris, France.
Stensballe
Allan
A
Boussetta
Tarek
T
Raad
Houssam
H
Dewas
Cedric
C
Kroviarski
Yolande
Y
Hayem
Gilles
G
Jensen
Ole N
ON
Gougerot-Pocidalo
Marie-Anne
MA
El-Benna
Jamel
J
D000595:Amino Acid Sequence; D001172:Arthritis, Rheumatoid / Q000276:immunology; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D019833:Genistein / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013582:Synovial Fluid / Q000166:cytology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004791:Enzyme Inhibitors; D010455:Peptides; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019833:Genistein; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D020928:Mitogen-Activated Protein Kinases