Novel pathways of F-actin polymerization in the human neutrophil

Abstract

Recently we demonstrated the existence of a phosphatidylinositol 3-kinase (PI3K)-independent F-actin polymerization during neutrophil pseudopod extension. Here we examine the use of the PI3K-dependent and PI3K-independent pathways of activation by the N-formyl peptide receptor and the chemokine receptors, and the priming of the 2 pathways by granulocytemacrophage colony-stimulating factor (GM-CSF) and insulin. The inhibition of PI3K activity with wortmannin showed that rate of pseudopod extension stimulated with N-formyl-Met-Leu-Phe (fMLP was mostly dependent on PI3K, while the rate of interleukin-8 (IL-8)-stimulated pseudopod extension was less dependent on PI3K. The incubation of cells with either GM-CSF or insulin increased the rate of pseudopod extension by 50% when the cells were stimulated with IL-8 but not with fMLP. The stimulation with IL-8 phosphorylated the PI3K regulatory subunit. This phosphorylation was enhanced by GM-CSF, which increased PI3K activity and total phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) production. The effect of GM-CSF was blocked with wortmannin. In contrast, insulin did not increase p85 phosphorylation and did not enhance PI3K activity or PtdIns(3,4,5)P 3 production. The effect of insulin was insensitive to wortmannin; however, it was blocked by an Src homology 2 (SH2) - IntroductionDirectional F-actin polymerization in the lamella region of a migrating human neutrophil is signaled by G-protein-coupled chemotactic receptors (GPCRs). It is known that the ligation of these receptors triggers the release of G ␤␥ . 1 Downstream from the GPCRs, the regulation of F-actin polymerization depends on PI3K activation and PtdIns(3,4,5)P 3 production, and involves the activation of protein kinase B (Akt/PKB) and the guanosine triphosphatases (GTPases) Cdc42 and Rac2. 2,3 Cdc42 and Rac2 form complexes with the Wiskott-Aldrich syndrome protein (WASp) family proteins and actin-related protein 2/3 (Arp2/3) to promote the formation of free barbed ends, 4 which in turn initiate cytoskeletal F-actin polymerization in the lamella region. 3 This mechanism of F-actin polymerization provides a useful framework for the understanding of the signaling of actin dynamics in the living cell during motility; however, it is far from complete. The earlier findings that F-actin polymerization is independent of the release of intracellular calcium and the activation of phospholipase C-␤ suggest that the signaling of migration is distinct from signaling of secretion. [5][6][7] In contrast, there is strong supporting evidence for the dependence of neutrophil migration on the activation of phosphatidylinositol 3-kinase-␥ (PI3K␥) in mice. 6,8,9 Similar dependence on PI3K␥ is expected for the migration of the human neutrophil on the basis of the dramatic change of PI3K␥ activity after neutrophil stimulation with chemoattractant 10 and the ability of the PI3K␥ product, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), to induce crawling. 11 However, mouse neutrophils lacking PI3K␥ activity...

Experimental Evidence for the Limiting Role of Enzymatic Reactions in Chemoattractant-induced Pseudopod Extension in Human Neutrophils

“…Cell Preparation-Human neutrophils were freshly isolated as described elsewhere (9). Briefly, whole blood was obtained by venipuncture from consenting, healthy adult volunteers (mean age 29 Ϯ 9.5 years) into Vacutainers anticoagulated with K 2 EDTA.…”

“…Effect of Chemoattractant Concentration-We have shown that the rate of pseudopod extension is dependent upon chemoattractant concentration at room temperature and at 37°C (9,31). The increase of chemoattractant concentration increases the number of ligand-bound GPCRs and was expected to increase the apparent concentration of downstream substrates synthesized after receptor activation.…”

“…PI3Ks and their products play key roles in the signaling of chemotaxis (6,7); however, there are alternative pathways of chemotaxis that do not require PI3K activation (8 -11). Both PI3K-dependent and -independent signaling of chemotaxis are dependent upon tyrosine kinase activation, which modulates the GPCR-mediated activation by means of receptor tyrosine kinases (9), intracellular tyrosine kinases (12), or both (13,14). The complexity of the signaling of neutrophil chemotaxis is essential for the functioning of the cells during innate immune responses, and its further elucidation will allow for better control over the immune responses in pathology and disease.…”

“…The addition of new filaments to the F-actin network at the tip of the pseudopod results in the extension of pseudopod length without a significant change in pseudopod thickness. Thus, the measurement of the rate of pseudopod extension in different experimental conditions is expected to provide valuable information for the dynamics of chemoattractant-induced F-actin polymerization (8,9,27).…”

“…Here we use a phenomenological approach, in which the temperature dependencies of two recently identified pathways of pseudopod extension, the PI3K-dependent and PI3K-independent pathways (8,9), are assumed to obey the Arrhenius law, and the PI3K-independent pathway is assumed to have a negligible contribution to the overall process of F-actin polymerization. These conditions are met for pseudopod extension stimulated with fMLP, where 80% of the rate of pseudopod extension at 37°C is PI3K-dependent (8).…”