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The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67^phox and Rac-2

Claus Kerkhoff^*,,1, Wolfgang Nacken^*,, Malgorzata Benedyk^*, Marie Claire Dagher, Claudia Sopalla^*, and Jacques Doussiere

^* Institute of Experimental Dermatology, University of Münster, Germany;
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, UMR 5092 CEA-CNRS-UJF, Département Réponse et Dynamique Cellulaires, Grenoble, France; and
Interdisciplinary Center for Clinical Research (IZKF) Münster, Germany

¹Correspondence: Institute of Experimental Dermatology, Röntgenstr. 21, Münster 48149, Germany. E-mail: kerkhoc{at}uni-muenster.de

SPECIFIC AIM

The aim of the present study was to establish the functional role of S100A8/A9 in the activation of the phagocyte NADPH oxidase and to investigate the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation.

PRINICIPAL FINDINGS

1. Gene silencing of S100A9 decreases NADPH oxidase in NB4 cells
Neutrophil-like NB4 cells were used as a cellular model to investigate the effect of specific blockage of S100A9 expression, using morpholino antisense oligonucleotides, on NADPH oxidase activity. Western blot analysis confirmed that S100A9 was nearly undetectable in cells treated with antisense oligonucleotides. S100A9 gene expression was not affected in cells preincubated with either S100A9 sense oligonucleotides or the carrier. S100A9 gene silencing did not affect the level of phox gene transcription in neutrophil-like NB4 cells, indicating that gene silencing of S100A9 was specific. We could not detect S100A8, the heterodimerization partner of S100A9, in antisense-treated NB4 cells.

Estimation of superoxide generation in neutrophil-like NB4 cells after stimulation with phorbolester or LPS/fMLP proved that NADPH oxidase was significantly decreased in NB4 cells treated with antisense oligonucleotides compared with NB4 cells exposed to sense oligonucleotides.

2. Oxidative burst is altered in S100A9 KO-mice
Bone marrow polymorphonuclear neutrophils (PMNs) from S100A9^–/– or S100A9⁺/⁺ mice were analyzed upon the generation of superoxides. Incubation of PMNs with phorbolester resulted in a time-dependent increase in DCF fluorescence related to ROS production (Fig. 1 A). The maximal DCF fluorescence increase was 2-fold lower in PMNs from S100A9^–/– mice than for PMNs from S100A9^+/+ mice (n=7; P<0.003; Fig. 1B ). PMNs from S100A9^–/– mice also showed significantly reduced ROS generation after PAF stimulation compared with PMNs from wild-type mice (n=6; P=0.044; Fig. 1C ).

View larger version (27K): [in this window] [in a new window]   Figure 1. NADPH oxidase activity of PMNs from S100A9–/– or S100A9+/+ mice. Intracellular reactive oxygen species production was continuously monitored using 2',7'-dichlorofluorescein (DCF) fluorescence. A) Overlay of DCF fluorescence increase of wild-type or S100A9–/– cells. B, C) Statistical analysis of the net increase in DCF fluorescence per 100 s, after stimulation with either PMA (B) or PAF (C).

3. S100A8/A9 enhances NADPH oxidase by transferring arachidonic acid
S100A8/A9 protein complexes bind polyunsaturated fatty acids in a calcium-dependent manner, and arachidonic acid (AA) is indispensable for in vivo and in vitro oxidase activation. Therefore, NADPH oxidase of neutrophil membranes was activated in the semi-recombinant system with the optimal concentration of AA in either the absence of S100 proteins (control) or the presence of wild-type or two mutant S100A8/A9 complexes, both unable to bind AA.

Bovine as well as human S100A8/A9 enhanced the NADPH oxidase activity by 1.6-fold and 1.5-fold, respectively (Fig. 2 ). In contrast, the two mutant S100A8/A9 complexes only exhibited a slightly enhanced NADPH oxidase activity or a slightly decreased NADPH oxidase activity, respectively. These data clearly indicate that binding of AA to S100A8/A9 represents an important molecular mechanism by which S100A8/A9 facilitates NADPH oxidase activation, probably by transferring AA to the NADPH oxidase complex.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of various S100A8/A9 protein complexes on NADPH oxidase activation. The human S100A8/A9 complexes were reconstituted using wild-type S100A8 and the different S100A9 proteins (wild-type, S100A9(H103,104,105K106A), or S100A9(1-100)). The mole ratio of S100 proteins to p67phox was adjusted to 1:1. Oxidase activity is expressed as % deviation from control.

4. S100A8 interacts with both p67^phox and Rac-2
Two experimental approaches were used to investigate to which phox protein(s) the S100 proteins were bound. First, human recombinant S100A8 or S100A9 alone was added at various concentrations to the mixture of neutrophil membranes and recombinant cytosolic phox proteins. NADPH oxidase activity was only slightly affected with increasing concentrations of S100A9, whereas S100A8 decreased elicited oxidase activity in a dose-dependent manner.

Second, we used the in vitro pull-down assay to confirm the specific interactions of S100 proteins with cytosolic phox proteins. These studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67^phox and the Rac proteins, whereas S100A9 bound only to Rac. Neither S100A8 nor S100A9 interacted with p47^phox. This finding confirmed the ability of the S100A8/A9 complex to bind to the complex of cytosolic factors of NADPH oxidase activation (p67^phox, p47^phox and Rac).

CONCLUSIONS AND SIGNIFICANCE

The functional relevance of S100A8/A9 in the phagocyte NADPH oxidase activation was demonstrated in intact cells by the impaired NADPH oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow-derived PMNs from S100A9^–/– mice. This conclusion is in contrast to a recent report of Hobbs et al. They observed no difference in the ability of neutrophils (or monocytes) from S100A9^+/+ and S100A9^–/– mice to generate superoxides. The discrepancy with our results might be due to different experimental protocols or methods used. However, we confirmed the impaired NADPH oxidase activity in PMNs from S100A9^–/– mice (compared with wild-type mice) using isoluminol amplified chemiluminescence and the Amplex assay. Additional evidence was given by the finding that the impaired oxidase activation could be mimicked in a cell-free system by pretreatment of neutrophil cytosol with a S100A9-specific antibody. The significance of our finding was further increased by the demonstration that NADPH oxidase activity was also impaired after stimulation with physiological agonists.

Further analyses gave detailed insights into the molecular mechanisms by which S100A8/A9 promotes NADPH oxidase activation. S100A8/A9 transferred the cofactor AA to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex (unable to bind AA) to enhance NADPH oxidase. This is in accordance to earlier reports showing that AA is indispensable for in vivo and in vitro oxidase activation. Additional evidence is given by the report by Roulin et al. that S100A8/A9 shuttles AA between the cytosol and the plasma membrane upon neutrophil stimulation.

It has been assumed that S100A8/A9 enhances NADPH oxidase activation via direct interaction with cytochrome b₅₅₈ (Berthier et al.). In contrast, we found that S100A8/A9 interacts with the cytosolic factors of oxidase activation, p67^phox and Rac2, as shown by functional in vitro assays of NADPH oxidase activation and protein-protein interaction assays. This finding is indicated by the growing body of evidence that cytosolic phox proteins interact with cytoskeletal proteins, cytoskeletal reorganization is involved in NADPH oxidase activation, and S100A8/A9 are in close association with cytoskeletal structures. These analyses revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67^phox and Rac-2 but not to p47^phox, whereas S100A9 did not interact with either p67^phox or p47^phox.

A model of oxidase assembly has been put forward in which phox activation, resulting in superoxide production, is probably the consequence of a conformational change in gp91^phox, caused by the interaction of cytochrome b₅₅₉ with one or several of the cytosolic oxidase components. Herein, p67^phox functions as the "activator," whereas p47^phox (due to binding to p22^phox) and Rac (due to tether p67^phox to the membrane) serve as "organizers." Based on our findings we proposed a working model (Fig. 3 ) in which S100A8/A9 is involved in phagocyte NADPH oxidase activation by transferring AA to gp91^phox during interactions with two cytosolic factors of oxidase activation: p67^phox and Rac-2. Binding of AA to gp91^phox induces a structural change in cytochrome b₅₅₈, which results to the final formation of the active complex.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram.

We established the functional role of S100A8/A9 during NADPH oxidase activation in intact cells and analyzed the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. This study provides new insight into the NADPH oxidase activation and links an important feature of phagocytes with proteins abundant in phagocytes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2377fje;

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