p38 Mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways are not essential regulators of formyl peptide-stimulated p47(phox) activation in neutrophils.
Three structurally unrelated p38 mitogen-activated protein kinase (MAPK) inhibitors, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580), 1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl] urea (BIRB 796) and 5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl]thio]-6H-pyrimido[1,6-b]pyridazin-6-one (VX 745) showed approximately 40% inhibition of formyl-Met-Leu-Phe (fMLP)-stimulated neutrophil superoxide anion (O2(•-)) generation at concentrations that greatly diminished p38 MAPK activity. However, a significant inhibition of p47(phox) activation occurred at concentrations much higher than the corresponding IC50 values of these inhibitors in blocking p38 MAPK activity. 4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole (SB202474), an inactive analogue of SB203580, at a concentration (30μM) which significantly attenuated p38 MAPK activity, had no effect on p47(phox) activation, whereas it inhibited O2(•-) generation with an IC50 value of approximately 16μM. Moreover, both SB203580 and BIRB 796 had no effect on protein kinase B (PKB)/Akt Ser473 phosphorylation and S100A9 protein membrane translocation at concentrations that effectively blocked p38 MAPK activity. Pretreatment of cells with two structurally unrelated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors, 2-(2-amino-3-methoxy-phenyl)-chromen-4-one (PD 98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), at concentrations that effectively blocked MEK activity, attenuated p47(phox) phosphorylation but did not affect the recruitment of p47(phox) to p22(phox) or O2(•-) generation. Both p47(phox) activation and O2(•-) generation were attenuated by a protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) in the concentration range that effectively blocked PKC activity. Taken together, these results suggest that the ERK-mediated Ser phosphorylation of p47(phox) is not implicated in the assembly of NADPH oxidase or O2(•-) generation, and that O2(•-) generation is partly attributable to p38 MAPK signaling through mechanisms other than p47(phox) activation, Akt activation and S100A9 membrane recruitment in fMLP-stimulated neutrophils.
Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China.
Ya-Ru
YR
Tsai
Yi-Jen
YJ
Wang
Miau-Rong
MR
Lee
Mei-Feng
MF
Hsu
Jih-Pyang
JP
Wang
D000818:Animals; D040502:Calgranulin B / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D013481:Superoxides / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D040502:Calgranulin B; D047428:Protein Kinase Inhibitors; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases