MLK3 regulates fMLP-stimulated neutrophil motility

O Polesskaya, C Wong, L Lebron, JM Chamberlain… - Molecular …, 2014 - Elsevier
O Polesskaya, C Wong, L Lebron, JM Chamberlain, HA Gelbard, V Goodfellow, M Kim
Molecular immunology, 2014Elsevier
Introduction Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that
connects extracellular cytokine or mitogen signals received through G-protein coupled
receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial
cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell
types remains unknown. Since neutrophils play a crucial role in innate immunity and
contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 …
Introduction
Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP.
Methods
The expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3−/− mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3−/− mice.
Results
The expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3−/− mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3−/− mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3−/− mice—thereby confirming the role of MLK3 in neutrophil migration.
Conclusions
Mlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.
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