p38 Mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways are not essential regulators of formyl peptide-stimulated p47ᵖʰᵒˣ 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 p47ᵖʰᵒˣ 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 p47ᵖʰᵒˣ 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 p47ᵖʰᵒˣ phosphorylation but did not affect the recruitment of p47ᵖʰᵒˣ to p22ᵖʰᵒˣ or O₂ •⁻ generation. Both p47ᵖʰᵒˣ 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 p47ᵖʰᵒˣ 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 p47ᵖʰᵒˣ activation, Akt activation and S100A9 membrane recruitment in fMLP-stimulated neutrophils.