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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Figure 2

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Coupled assay of MKK4 activation. Neutrophils were stimulated with LPS (...
Coupled assay of MKK4 activation. Neutrophils were stimulated with LPS (100 ng/ml) for 20 min at 37°C. (a) Phosphorylation of MKK4. Under identical conditions to the experiments shown in Fig. 1, MKK4 was immunoprecipitated from cell lysates and submitted to SDS-PAGE and Western blotting with an anti-phosphorylated MKK4 antibody. Blot is representative of three experiments. (b) Activation of MKK4. 32P phosphorylation of rhp38 MAPk from blots was quantified by phosphor screen autoradiography. Lysates in the absence of rhp38 MAPk (lanes 1 and 2) were compared with lysates in the presence of rhp38 MAPk (lanes 3 and 4) and a cell-free control to demonstrate the lack of specific activation of MKK4 in response to LPS as determined by 32P phosphorylation of rhp38 MAPk. (c) Coupled activation of rhp38 MAPk by MKK4. The ability of MKK4 to activate rhp38 MAPk was determined by the ability of phosphorylated rhp38 MAPk to phosphorylate the substrate ATF-21-110. Lysates in the absence of rhp38 MAPk (lanes 1 and 2) represent baseline phosphorylation of the ATF-21-110, whereas lysates in the presence of rhp38 MAPk (lanes 3 and 4) demonstrate little increase in phosphorylation of ATF-21-110 via coupled activation of rhp38 MAPk by MKK4 in response to LPS. The cell-free control quantifies intrinsic activity of the rhp38 MAPk and is equivalent to the unstimulated and LPS-stimulated lysates in lanes 3 and 4. Plots depict mean values and SEM from three consecutive experiments expressed in arbitrary units.