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Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase.

ABSTRACT: In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.

SUBMITTER: Bissonnette SA

PROVIDER: S-EPMC2755574 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase.

Bissonnette Sarah A SA Glazier Christina M CM Stewart Mary Q MQ Brown Glenn E GE Ellson Chris D CD Yaffe Michael B MB

The Journal of biological chemistry 20071120 4

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The rol ...[more]

PMID: 18029359

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Project description:Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

Dataset Information

Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase.

Publications

Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase.

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