Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils
Jerry A. Nick, … , Gary L. Johnson, G. Scott Worthen
Jerry A. Nick, … , Gary L. Johnson, G. Scott Worthen
Published March 15, 1999
Citation Information: J Clin Invest. 1999;103(6):851-858. https://doi.org/10.1172/JCI5257.
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Article

Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Abstract

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38α and p38δ were detected in neutrophils. LPS stimulation selectively activated p38α. Specific inhibitors of p38α MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-κB) activation, and synthesis of tumor necrosis factor-α (TNF-α). Inhibition of p38α MAPk resulted in a transient decrease in TNF-α mRNA accumulation but persistent loss of TNF-α synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38α MAPk, ultimately regulating adhesion, NF-κB activation, enhanced gene expression of TNF-α, and regulation of TNF-α synthesis.

Authors

Jerry A. Nick, Natalie J. Avdi, Scott K. Young, Lisa A. Lehman, Patrick P. McDonald, S. Courtney Frasch, Marcella A. Billstrom, Peter M Henson, Gary L. Johnson, G. Scott Worthen

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Inhibition of NF-κB activation by SB203580-induced inhibition of p38α MA...
Inhibition of NF-κB activation by SB203580-induced inhibition of p38α MAPk. (a) Effect of in vivo inhibition of p38α MAPk on NF-κB activation. Neutrophils were suspended in the presence or absence of SB203580 (10 μM) for 60 min at 37°C and subsequently stimulated with LPS (100 ng/ml) for 20 min at 37°C or left unstimulated. Nuclear extracts were then prepared and analyzed in EMSA using an NF-κB oligonucleotide probe. This blot is representative of three consecutive experiments. (b) Lack of a direct effect of SB203580 toward the binding of NF-κB to its cognate sequence. Nuclear extracts from LPS-stimulated neutrophils were coincubated with SB203580 (100 μM) for 30 min at room temperature in the binding mixture, before EMSA analysis. This blot is representative of three consecutive experiments. (c) Release of TNF-α at the time of NF-κB activation. The quantity of TNF-α released per 106 neutrophils from the samples studied in a was plotted for each condition. Bars depict the mean value and SEM of TNF-α release (per 106 neutrophils) from three independent experiments.EMSA, electrophoretic mobility shift assay; NF-κB, nuclear factor-kappa B.