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<ul><li> 1. 2005 FASEB The FASEB Journal express article 10.1096/fj.04-2377fje. Published online January 10, 2005. The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2 Claus Kerkhoff,*, Wolfgang Nacken,*, Malgorzata Benedyk,* Marie Claire Dagher, Claudia Sopalla,*, and Jacques Doussiere *Institute of Experimental Dermatology, University of Mnster, Mnster, Germany; Laboratoire de Biochimie et Biophysique des Systmes Intgrs, UMR 5092 CEA-CNRS-UJF, Dpartement Rponse et Dynamique Cellulaires, Grenoble, France; and Interdisciplinary Center for Clinical Research (IZKF), Mnster, Germany Corresponding author: Claus Kerkhoff, Ph.D., Institute of Experimental Dermatology, Rntgenstr. 21, 48149 Mnster, Germany. E-mail: kerkhoc@uni-muenster.de ABSTRACT The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9/ mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation. Key words: neutrophils cytosolic phox proteins MRP8/14 superoxide O2.- gene silencing T he NADPH oxidase of phagocytes is a multisubunit enzyme complex that produces the superoxide anion (O2.-) (1). This system is activated by a variety of stimuli for the destruction of pathogens, but it also exerts toxic effects in most inflammatory processes (2). Individuals deficient in superoxide production due to a genetic lesion in any of four components of this system (p22phox, gp91phox, p47phox, or p67phox) experience severe recurrent infections, often from catalase-positive microbes. This condition is known as chronic granulomatous disease (3, 4). Page 1 of 28 (page number not for citation purposes) </li> <li> 2. The NADPH oxidase consists of six subunits that are partitioned between different subcellular locations in the resting state. Two of these subunits, p22phox and gp91phox, are integral membrane proteins and form a heterodimeric flavocytochrome, also known as cytochrome b558, which constitutes the catalytic core of the enzyme. The remaining oxidase components reside in the cytosol and include the small GTPase Rac as well as a complex of p40phox, p47phox, and p67phox. Activation of the NADPH oxidase is initiated by phosphorylation of phox proteins, which is believed to induce conformational changes that subsequently lead to rearrangements affecting both intra- and intermolecular interactions within the cytosolic p40-p47-p67phox complex. These events culminate in the translocation of this complex to the membrane and association with both Rac-GTP and cytochrome b558 to form the active enzyme (5). In previous studies, we showed that the Ca2+- and arachidonic acid (AA-) binding S100A8/A9 protein complex enhanced the NADPH oxidase activation in a cell-free system comprising neutrophil membranes, recombinant cytosolic factors of oxidase activation (p47phox, p67phox and GTPS preloaded Rac-2), and AA. The enhancing effect of S100A8/A9 on the NADPH oxidase activation has been ascribed to the binding of the S100A8/A9 heterodimer to cytosolic oxidase activating factors, in particular to p67phox and Rac-2 (6, 7). S100A8/A9 interacts with the NADPH oxidase complex and probably enhances the enzyme activity by transferring the cofactor AA, thus stabilizing the activated conformation of the enzyme. S100A8 and S100A9 belong to the S100 family of calcium binding proteins (for review, see refs 8, 9). Their expression is restricted to a specific stage of myeloid differentiation and is probably driven by a recently characterized regulatory element (10). S100A8 and S100A9 are predominantly localized in the cytoplasm. Upon elevation of the intracellular calcium level, they are translocated from the cytosol to the cytoskeleton and the plasma membrane (1113). Although the exact functions of these proteins remain unknown, they are normally associated as heteromeric complexes, which are able to bind polyunsaturated fatty acids in a calcium- dependent manner (14, 15), whereas the individual S100 proteins do not bind fatty acids. The unique C-tail of S100A9, containing the three consecutive histidine residues (His103-His105), is involved in the binding of the fatty acid carboxyl-group to the protein complex (16). The binding of calcium to the two EF hands, of high and low affinity, respectively, of each S100 protein, is the prerequisite for the binding of AA to the complex S100A8/A9 (15). Moreover, the estimated calcium concentration required to induce fatty acid binding is within the physiological range (14). The S100A8/A9 protein complex accounts for the entire AA binding capacity of the neutrophil cytosol (14), indicating that S100A8/A9 plays an essential role in the cellular AA metabolism. S100A8/A9 may serve either as a transport vehicle for AA to neighboring cells at inflammatory loci (17) or to transfer AA to both AA-dependent enzymes and AA-consuming pathways. Interestingly, AA is indispensable for in vivo and in vitro NADPH oxidase activation. This was demonstrated by invalidation of the cytosolic phospholipase A2 with antisense RNA in myeloid PLB-985 cells. The PMA-induced O2.- generation, abolished in invalidated cells, was restored by addition of AA (18). Furthermore, AA induces a significant structural change in cytochrome b558 (19, 20). Therefore, the aim of the present study was to establish the functional role of S100A8/A9 in the activation of the NADPH oxidase and to investigate the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. We demonstrated the relevance of S100A8/A9 in NADPH oxidase activation by (1) the specific blockage of S100A9 Page 2 of 28 (page number not for citation purposes) </li> <li> 3. expression in all-trans-retinoic acid (ATRA)-treated NB4 cells using morpholino antisense oligonucleotides and (2) the measurement of oxidative burst in bone marrow polymorphonuclear neutrophils (PMNs) from S100A9/ mice. Further, we provide strong evidence that S100A8/A9 acts by binding to cytosolic factors of oxidase activation and by delivering AA to the membrane- bound flavocytochrome b. MATERIALS AND METHODS Culture of NB4 cells and induction of differentiation The NB4 acute promyelocytic leukemia cells, capable of differentiating into nonmalignant neutrophils with ATRA, were obtained from the American Type Culture Collection. The cells were maintained in suspension in RPMI medium supplemented with 10% fetal calf bovine serum and kept at 37C in a 5% CO2 atmosphere. The cells were passaged by dilution in fresh medium to a density of ~0.2 106 cells/ml. Before induction of differentiation by ATRA, the cells were maintained at a logarithmic growth rate and seeded at a density of 0.2 106 cells/ml. ATRA was added at a final concentration of 1 M by dilution from a 10 mM stock solution prepared in Me2SO. Control cells were treated with a similar dilution of Me2SO, which had no effect on the differentiation or the rate of cell division. Inhibition of S100A9 expression by antisense oligonucleotides Morpholino antisense and sense oligonucleotides were synthesized by Gene Tools (Philomath, OR) to target the sequence of human S100A9 mRNA (GenBankTM accession number NM_002965) (21). We selected the sequence 5-AAGTCATCGTCTTGCACTCTGT-3, which targets the sequence -18 thru +7 relative to the first ATG. The sequence has minimal secondary structure and the lowest self-complementarity, based on the GC content. The invert of the antisense (sense) (5-TGTCTCACGTTCTGCTACTGAA-3) was used as control. Equimolar concentrations (1.4 M) of the oligonucleotides and ethoxylated polyethylenimine (EPEI) were first combined and incubated for 20 min at room temperature, and then serum-free RPMI medium (Invitrogen) was added. NB4 cells were suspended in this solution at 1.0 106/ml and incubated for 3 h at 37C in a 5% CO2 atmosphere. The cells were then centrifuged at 1,000 rpm for 5 min, resuspended in RPMI with 10% serum at 0.2 106/ml, and returned to the incubator. After treatment with the oligonucleotides for different times as indicated, the cells were incubated for 3 days in the presence of ATRA (1.0 M, Sigma) and finally harvested for both Western blot analysis and measurement of NADPH oxidase activity. Determination of mRNA expression by quantitative PCR To test whether S100A9 gene silencing affected the expression of phox proteins during differentiation, gene expression analysis was performed using real-time PCR (GeneAmp 5700 Sequence Detection System, PE Applied Biosystems). Isolation of total RNA from isolated cells was carried out using the RNA isolation kit (Qiagen) according to the manufacturers instructions. Table 1 shows the human amplification primers used. The primers were obtained from MWG. Page 3 of 28 (page number not for citation purposes) </li> <li> 4. Measurement of NADPH oxidase by the nitroblue tetrazolium (NBT) method At the indicated times of continuous exposure to ATRA, the NB4 cells were pelleted by centrifugation at 700 g for 5 min. NADPH oxidase activity of NB4 cells was measured by adding 1 ml of cell suspension (0.5-2106 cells) to a solution containing 2 mg/ml of NBT and 20 ng/ml of phorbol myristate acetate (PMA) phosphate-buffered saline. The incubation was allowed to proceed for 1 h at 37C and was stopped by adding 0.4 ml cold 2 M HCl. The formazan product was obtained by centrifugation of the sample at 700 g for 10 min. The supernatant was discarded, and the formazan was dissolved in 1 ml of Me2SO. The absorbance of the solution was measured at 590 nm. The data are expressed as absorbance units/106 cells. Animals S100A9/ mice were generated as described earlier (22). Both the S100A9+/+ (wild-type) and S100A9-deficient animals were housed under specific pathogen-free conditions according to federal and state regulations and studied from 6 to 12 wk of age. Measurement of intracellular reactive oxygen species production The production of intracellular reactive oxygen species like superoxide and hydrogen peroxide was determined by measuring changes in the fluorescence of 2,7-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes), an oxidation-sensitive fluorescence probe (23). Briefly, the cell suspension was incubated in fresh serum-free RPMI 1640 with 5 M DCFH-DA at room temperature for 40 min. The loaded cells were then washed twice with Hanks balanced salt solution containing 140 mg/l CaCl2 and 200 mg/l MgSO4*7H2O (Biochrom, Germany); 5 106 cells were then placed in a cuvette in a thermostatically controlled cell holder at 37C and stirred continuously. Fluorescence was excited at 488 nm, and emission was recorded at 530 nm. The change in fluorescence intensity was monitored by a spectrofluorometer (Fluoromax, Jobin Yvon GmbH) over a time period of 10 min. After 400 s, PMA (1 M) was added to elicit the generation of reactive oxygen species. At the end of the experiment, 0.1% Triton and 100 M H2O2 were added to verify that the increase in fluorescence with time correlates with the production of reactive oxygen species and was not limitative. The rate of increase before stimulation was subtracted from the rate of increase after stimulation to give the rate of increase due to induction by the stimulating agent. This value was taken as a relative measure of the activity of the NADPH oxidase complex. Preparation of plasma membranes, cytosol, and native S100A8/A9 A particulate fraction enriched in plasma membrane, termed neutrophil membrane, and a soluble fraction, termed neutrophil cytosol, were prepared by density gradient fractionation of a sonicated homogenate of resting bovine neutrophils in a saline phosphate buffer (PBS), consisting of 2.7 mM KCl, 136.7 mM NaCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4 (24). The amount of S100A8/A9 in the membrane fraction of resting neutrophils was found to be negligible as analyzed by Western blot analysis. The bovine S100A8/A9 protein complex was purified from bovine neutrophil cytosol as described earlier (6). The cytosolic fraction was supplemented with 1 M KCl to favor the Page 4 of 28 (page number not for citation purposes) </li> <li> 5. detachment of cytosolic factors of oxidase activation from the S100 complex. The absence of Rac-2, p67phox, or p47phox in the purified fraction was confirmed by Western blot analysis. The human S100A8/A9 protein complex was purified as described by van den Bos et al. (25) with some modifications from human neutrophils prepared from leukocyte rich blood fractions ("buffy coat") according to Mller et al. (26). Before use, the proteins were re-chromatographed by anion-exchange chromatography using a UnoQ column (Bio-Rad, Mnchen, Germany). The protein concentration in the various b...</li></ul>