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Originally published In Press as doi:10.1074/jbc.M308386200 on February 17, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21589-21597, May 14, 2004
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Rho Is Involved in Superoxide Formation during Phagocytosis of Opsonized Zymosans*

Jun-Sub Kim{ddagger}, Becky A. Diebold§, Jong-Il Kim{ddagger}, Jaebong Kim{ddagger}, Jae-Yong Lee{ddagger}, and Jae-Bong Park{ddagger}

From the {ddagger}Department of Biochemistry, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea and the §Department of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, July 31, 2003 , and in revised form, January 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. We wanted to determine whether Rho is also involved in the production of superoxide during phagocytosis. Inhibition of Rho by Tat-C3 exoenzyme (Tat-C3) blocked superoxide formation and curtailed the phagocytosis of serum- (SOZ), C3bi- (COZ), and IgG-opsonized zymosan (IOZ) particles. Tat-C3 did not affect superoxide formation in response to phorbol myristate acetate (PMA), formyl Met-Leu-Phe (fMLP), or macrophage colony-stimulating factor (M-CSF). Superoxide formation was also reduced in J774 cells transfected with a cDNA expressing dominant-negative form of RhoA (N19RhoA). However, purified prenylated recombinant RhoA did not activate NADPH oxidase in vitro, suggesting that Rho does not interact directly with NADPH oxidase. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo. It also inhibited the phosphorylation of p47PHOX, one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47PHOX.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytosis by macrophages is essential for the uptake and degradation of pathogens. It also triggers immune responses and participates in development and tissue remodeling. Phagocytosis can be triggered by the macrophage receptors Fc{gamma}Rs, which recognize the Fc domain of immunoglobulin G (IgG) (1). Another type of receptor through which phagocytosis occurs is the complement receptor (CR),1 which recognizes C3b/C3bi fragments. CR1 is thought to participate mainly in particle binding, whereas CR3 (also referred to as CD11b/CD18, Mac-1, and {alpha}M{beta}2) and CR4 (CD11c/CD18 and {alpha}X{beta}2), which are heterodimers of integrin, are responsible for internalizing particles (1, 2). A third receptor involved in phagocytosis is the mannose receptor that recognizes mannose and fucose saccharides in the capsule or on the lipopolysaccharide of invading bacteria (1, 3, 4).

Rho family GTPases are essential for the actin changes needed for phagocytosis and engulfment (4). Cdc42/Rac regulates the macrophage phagocytosis mediated by Fc{gamma}R, whereas Rho is thought to regulate that mediated by CR3 (5, 6, 7). Nevertheless it has been reported that C3 exoenzyme abrogates Fc{gamma}R-mediated phagocytosis (8).

After phagocytosis in macrophages, there is an abrupt increase in superoxide formation, known as the oxidative burst, which is catalyzed by the membrane-associated NADPH oxidase enzyme complex, which generates superoxide () by the one-electron reduction of oxygen, using NADPH as electron donor (9). The redox core of the phagocyte NADPH oxidase is a membrane-spanning heterodimeric flavocytochrome b558 composed of p22PHOX and gp91PHOX. It is present in the membranes of secretory granules that fuse with the plasma membrane upon phagocytosis (10). The other components, p40PHOX, p47PHOX, and p67PHOX form a cytoplasmic complex (11). p47PHOX is phosphorylated by protein kinases (12), primarily by protein kinase C (PKC) (13). When phagocytes are activated, the p40PHOX, p47PHOX, and p67PHOX complex translocates to the membrane, where it associates with cytochrome b558 to form the active NADPH oxidase (14, 15). In addition, Ras-related small GTP-binding proteins, Rac1 (16) or Rac2 (17), are essential for activating NADPH oxidase. Rac2 is a critical regulator of NADPH oxidase in response to formyl methionylleucyl-phenylalanine (fMLP) and antibody-coated sheep red blood cells (SRBC) (IgG-SRBC), whereas it is not responsible for superoxide formation induced by serum-opsonized zymosans (SOZ) (18). In the resting state, Rac is localized in the cytoplasm in a dimeric complex with Rho GDP dissociation inhibitor (RhoGDI), and when the phagocyte is activated, GTP-bound Rac translocates to the membrane independently of the translocation of the p40PHOX, p47PHOX, and p67PHOX cytosolic complex (17, 19). Rap1, another Ras-related GTPase, is rapidly and transiently activated upon stimulation by fMLP, platelet-activating factor, granulocyte-macrophage colony-stimulating factor, and IgG-coated particles. Rap1 activation is, however, independent of the respiratory burst (20).

Rac acts downstream of Fc{gamma}R activation in stimulating NADPH oxidase (18). Superoxide production is also induced by interaction of CR3 with anti-CR3 antibody-coated particles (21), by Staphylococcus particles coated with anti-CD18 antibodies (22), and by non-opsonized zymosan (23). However, it is not clear whether Rho is involved in the superoxide formation during these phagocytotic responses, and the aim of the present work was to investigate this question using the mouse macrophage cell line, J774 and primary phagocytic cells. To do this we exploited the observation that C3 exoenzyme modifies Rho by ADP-ribosylation and specifically inhibits its activity (24). However, since C3 exoenzyme is not easily transduced into cells, we conjugate it with Tat penetratin, a domain of the human immunodeficiency virus-1 (HIV-1). The resulting Tat-C3 is readily transduced into cells and efficiently and specifically inhibits Rho. Using Tat-C3 we were able to demonstrate that Rho was involved in the formation of superoxide during phagocytosis by both the Fc{gamma}R and CR3 pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Zymosan A particles, PD98059, SB203580, LY294002, phorbol myristate acetate (PMA), phenylmethylsulfonyl fluoride (PMSF), dimyristoylphosphatidyl choline (DMPC), 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), methylviologen (MV), Triton X-100, glutathione (GSH), fMLP, macrophage colony-stimulating factor-1 (MCSF-1), Y-27632, isopropyl {beta}-D-thiogalactoside (IPTG), and tetramethylrhodamine B isothiocyanate (TRITC)-conjugated phalloidin were purchased from Sigma Chemicals. BA85 membrane filter (25-mm diameter) was from Schleicher & Schuell (Keene, NH). Protein A-agarose beads were from Pierce. Catalase, horseradish peroxidase, and superoxide dismutase (SOD) were from Roche Applied Science, and complement C3bi was from Calbiochem. IgG against zymosan, 2',7'-dichlorofluorescein diacetic acid (DCFH-DA), and FluoSpheres carboxylate-modified microspheres containing red and yellow green fluorescence (1 and 2 µm) were from Molecular Probes (Eugene, OR), and GSH-Sepharose 4B beads from Amersham Biosciences. Anti-RhoA, -Rac1, and -His tag antibodies were purchased from Santa Cruz Bio-technology, and anti-phospho-ERK, -phospho-JNK, -phospho-p38 MAPK, and -p38 MAPK antibodies from Cell Signaling (Beverly, MA). Anti-p47PHOX antibody and pGEX-1{lambda}T plasmid containing the fusion between GSH S-transferase (GST) and 47PHOX were kind gifts from Dr. J. W. Park of Kyoungbuk National University, Korea. Anti-p47PHOX and -Rac2 antibodies were obtained from Dr. Gary M. Bokoch of The Scripps Research Institute. Tat-peptide (KKKRRQRRR) was synthesized from Peptron, Korea. Plasmids encoding Tat-yeast cytosine deaminase (Tat-YCD) and -green fluorescence protein (Tat-GFP) were obtained from Dr. J. Park of Hallym University, Korea.

Expression and Purification of the Tat-C3, -YCD, and -GFP—In order to introduce C3 exoenzyme efficiently into cells, we fused HIV-1 Tat transduction domain (amino acid residues 49-57, KKKRRQRRR) to the N terminus of the C3 exoenzyme containing a His tag (25). A similar Tat-C3 (amino acid residue 47-57) was used to test the function of Rho in smooth muscle cell proliferation (26). Cultures of Escherichia coli BL21 (Amersham Biosciences) transformed with plasmid C3 or plasmid Tat-C3 were incubated with 0.1 mM IPTG for 5 h. To prepare denatured Tat-C3 fusion proteins, the induced bacteria were harvested and sonicated in binding buffer (20 mM Tris-HCl, pH 7.9, 5 mM imidazole, and 500 mM NaCl) containing 6 M urea and protease inhibitors (20 µg/ml soybean trypsin inhibitor, 2 µg/ml aprotinin, 5 µg/ml leupeptin, and 100 µg/ml PMSF). After removing cell debris by centrifugation, the clarified cell extract was loaded onto a Ni2+-IDA column (Novagen), and the column was washed first with binding buffer without 6 M urea, and then with washing buffer (20 mM Tris-HCl, pH 7.9, 80 mM imidazole, and 0.5 M NaCl). Proteins were eluted with elution buffer (20 mM Tris-HCl, pH 7.9, 1 M imidazole, and 0.5 M NaCl), followed by desalting with a PD10 column (Amersham Biosciences). Tat-YCD and -GFP were purified by the same procedure, and purified proteins were confirmed by Western blotting by using His tag antibody.

Cell Culture and Transduction of Tat-C3 Fusion Protein—Mouse macrophage J774 cells were cultured in DMEM containing 20 mM Hepes/NaOH (pH 7.4), 5 mM NaHCO3, 10% FBS, and antibiotics (100 units/ml streptomycin and 100 units/ml penicillin) at 37 °C in 5% CO2. For transduction of Tat-C3, the cells were grown to confluence in a 6-well plate, the culture medium was replaced with 1 ml of serum-free medium, and the cells were incubated with 10 µg/ml Tat-C3 for 30 min at 37 °C.

Peritoneal Macrophage Cell Culture—Male ICR mice (6 weeks) were purchased from the Experimental Animal Center, Central Laboratory Management, Hallym University. Resident macrophages were collected from the peritoneal cavity of mice with DMEM medium, washed, suspended in DMEM medium supplemented with 10% FBS, penicillin G (100 units/ml), and streptomycin (100 µg/ml), and plated in 100-mm culture plates at a density of 2 x 106 cells/plate. After a 2-h incubation at 37 °C in 5% CO2, the attached cells were washed three times with DMEM medium to remove non-adhering cells and grown for another 24 h. After three more washes, they were used in experiments.

Human Neutrophil Preparation—Human neutrophils were obtained from freshly drawn blood of healthy volunteers. After dextran sedimentation, centrifugation through Ficoll-Paque and hypotonic lysis of red blood cells, neutrophils were washed, and the adherent neutrophils were incubated in DMEM containing 10% FBS as previously described with a slight modification (27).

Preparation of FITC-Zymosan—Zymosan A particles were labeled with FITC without addition of gelatin, pelleted by centrifugation, washed more than seven times, and resuspended in PBS (5 x 108 zymosan particles/ml). Aliquots were stored at -70 °C and thawed immediately before use (28). Zymosan particles were opsonized with 1 µg/ml mouse serum (SOZ), C3bi (COZ), or IgG (IOZ) (29).

Phagocytosis Assay and Binding of Zymosans to the Surface of Macrophages—Cells were plated in 35-mm dishes at a density of 2 x 105 cells and grown overnight. After incubation in DMEM medium without FBS for 16 h at 37 °C in a CO2 incubator they were exposed for 30 min to 5 x 105 FITC-conjugated zymosan particles previously opsonized in fresh mouse serum, C3bi, or IgG, and washed and resuspended in 2 ml of PBS. Phagocytosis was assayed by measuring the fluorescence intensity of the cells at an excitation wavelength of 490 nm and an emission wavelength of 520 nm (Kontron SFM25 spectrometer) (30). The fluorescence of FITC-zymosan bound to the surface of the cells was quenched by adding crystal violet to 10 µM final concentration. The surface-bound FITC-zymosan was calculated by subtracting net trans-located fluorescence from total fluorescence of the cells. Where fluorescent beads were employed, the tagged engulfed beads were observed by fluorescence microscopy.

Determination of Superoxide—To measure intracellular superoxide, J774 cells (2 x 103) were harvested, washed three times with PBS, and resuspended in 1 ml of KRG buffer (120 mM NaCl, 5 mM KCl, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, 10 mM glucose, and 1 mM CaCl2) containing 50 units/ml SOD, 2000 units/ml catalase, and 50 µM luminol (31). The reaction was started by adding opsonized zymosan particles (2 x 104), and the chemiluminescence generated was measured with a luminometer (Lumat LB 9507, EG&G, Berthold, Germany). Prenylated recombinant RhoA was tested for ability to induce superoxide production in the cell-free NADPH oxidase system previously described (32).

Preparation of Serum-opsonized IOZ Particle-bound Protein A-Agarose Beads (SO-IOZ-beads)—Protein A-agarose beads (10 µl) were washed and resuspended in 40 µl of IOZ particles in PBS (8 µg of particles). Binding of agarose beads and IOZ (or FITC-IOZ) were performed for 4 h at 4 °C with continuous rotation. The IOZ-bound beads were washed and opsonized with 1 µg/ml mouse serum. Finally, serum-opsonized IOZ-bound beads (SO-IOZ-beads) were washed several times with a brief centrifugation to remove unbound serum and zymosans, and resuspended in 100 µl of PBS.

Observation of Superoxide Produced by SO-IOZ-bead on a Confocal Microscope—Cells were preincubated in serum-free medium for 16 h at 37 °C, incubated with 10 µM DCFH-DA for 30 min at 37 °C, washed, and then incubated with serum- and phenol red-free medium. Cells were stimulated with SO-IOZ- or SO-FITC-IOZ-beads for 15 min in the presence or absence of 10 µg/ml Tat-C3. Cells were washed with PBS followed by addition of serum- and phenol red-free medium. FITC or DCF fluorescence at an excitatory wavelength of 495 nm was observed on a confocal microscope system (Bio-Rad) (33).

Transient Transfection with RhoA cDNA Constructs—90% confluent J774 cells in 35-mm dishes were transiently transfected with 5 µg of pcDNA3 plasmids encoding RhoA constructs (wild-type RhoA and the dominant-negative form, N19RhoA), or co-transfected with 5 µg of pcDNA3 RhoA constructs and 5 µg of pEGFP-C2 (Clontech), using the GenePORTER2 transfection reagent (Gene Therapy Systems, San Diego, CA). After 48 h, the cells were resuspended in 1 ml of KRG buffer containing 50 units/ml SOD, 2000 units/ml catalase, and 50 µM luminol for measuring superoxide formation, or resuspended in 2 ml of PBS for phagocytosis assay. Phagocytosis and superoxide formation were started by adding opsonized zymosan particles.

Observation of Actin Filaments—When appropriate, 103-104 cells were preincubated with 10 µg/ml of Tat-C3 or 30 µM Y-27632 for 30 min, and subsequently stimulated with 100 ng/ml M-CSF or 100 nM fMLP for 10 min. The cells were fixed in 4% formaldehyde/PBS (v/v). To visualize actin filaments, they were permeabilized in 0.1% Triton X-100/PBS (v/v), blocked in 0.5% bovine serum albumin/PBS (w/v) for 45 min, and stained with 80 ng/ml TRITC-conjugated phalloidin in PBS for 1 h. Images were generated by confocal laser scanning microscopy as described previously (34, 35).

Quantitation of Ruffling—Ruffling was defined by the presence of F-actin-rich submembranous folds seen by confocal microscopy. The extent of ruffling of each cell was scored using a scale of 0-2, where 0 indicates no ruffles, 1 indicates ruffling confined to one area of the cell (<25% of the cell circumference), and 2 indicates two or more discrete areas containing ruffles. The ruffling index is recorded as the sum of the ruffling scores of 100 cells (7).

GST Pull-down Assay for Activated RhoA, Rac1, and Rac2—GST pull-downs assay was conducted as described previously (36, 37). Briefly, a total of 2x106 cells cultured in 100-mm plates were washed in ice-cold PBS and harvested. The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.2, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 500 mM NaCl, 10 mM MgCl2, 5 µg/ml each of leupeptin and aprotinin, and 1 mM PMSF). After centrifugation (15,000 rpm, 15 min, 4 °C), aliquots of the supernatant were added to the GST-Rho binding domain of Rhotekin (GST-RBD) or the GST-GTPase binding domain of p21-activated kinase-1 (PAK-1) (GST-PBD), which was previously incubated for 1 h with 50 µg of GST fusion proteins. The beads were incubated with cell lysates and washed, and the proteins on the beads were run on SDS-PAGE. RhoA, Rac1, and Rac2 were determined by Western blotting. For the preparation of GST-RBD and -PBD proteins, RBD and PBD cDNAs produced by polymerase chain reaction (PCR) were incorporated into pGEX4T-1, E. coli DH5{alpha} was transfected with plasmids, the expression of proteins was induced with IPTG, and the proteins were purified with GSH-Sepharose beads.

Expression and Purification of GST-RhoA and -Rac1 Fusion Proteins and Measurement of GTP Binding by Fusion Proteins—To prepare GST-RhoA and -Rac1, E. coli DH5{alpha} was transformed with pGEX4T1-RhoA and -Rac1 plasmids, and GST-RhoA and -Rac1 proteins were purified from the bacteria with GSH-Sepharose beads. To measure the binding of GTP to the fusion proteins, 1 µg of GST-RhoA or -Rac1 was incubated for 10 min at 30 °C with 0.1 µM {gamma}35S-GTP in 50 µl of GTP binding buffer (10 mM Hepes, pH 7.4, 0.5 µM MgCl2,1mM dithiothreitol, and 1 mM dimyristoylphosphatidyl choline) containing 30 µg of bovine serum albumin as a carrier protein. Assay of GTP binding to proteins was performed by the membrane filtering method with BA85 membrane (25 mm in diameter), according to Kikuchi et al. (38) with a slight modification (39).

Translocation of RhoA and Rac1 Proteins—J774 macrophages (2 x 105 cells) were treated with various agents for 30 min at 37 °C and stimulated with SOZ and IOZ for 10 min. They were then harvested and lysed by sonication in 50 µl of lysis buffer (20 mM Hepes, pH 7.4, 150 mM NaF, 2 mM NaVO4,20mM Na4P2O7,50 µg/ml PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, and 0.05% Triton-X 100) (40). Cell membranes were collected with a tabletop ultracentrifuge at 100,000 x g for 30 min and 4 °C and resuspended in 50 µl of lysis buffer. Western blotting with anti-RhoA and Rac-1 antibodies was employed to measure the translocation of RhoA and Rac1.

Expression and Purification of GST-p47PHOX, and Phosphorylation Assay of Recombinant GST-p47PHOX Fusion Protein—Recombinant GST-p47PHOX fusion protein was prepared for use as a kinase substrate by the procedure of Park et al. (41). Kinase assays of the recombinant GST-p47PHOX fusion protein were performed by the method of Yamamori et al. (42). Briefly, macrophages (2 x 106) stimulated with SOZ or IOZ for 15 min were washed and lysed in cold lysis buffer. The GST-p47PHOX protein was bound to GSH-Sepharose beads and incubated with aliquots of cell lysates. The beads were washed, resuspended, and incubated in kinase buffer (20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM L-glycerophosphate, 0.1 mM Na3VO4, and 2 mM dithiothreitol) containing 20 µM ATP and 10 µCi of [{gamma}-32P]ATP. Phosphorylated GST-p47PHOX was analyzed by SDS-PAGE, followed by autoradiography.

Purification of Prenylated RhoA and Rac2 from Sf-9 Cells—To purify prenylated RhoA and Rac2, we used the method described by Diebold and Bokoch (32). Briefly, cDNA, encoding RhoA or Rac2, was ligated to the baculovirus transfer vector pAcGLT (BD PharMingen, San Diego, CA). Purified pAcGLT plasmid carrying the GST-RhoA or GST-Rac2 fusion constructs were used for homologous recombination with BaculoGold baculovirus, and Sf9 cells were infected with the baculovirus encoding RhoA or Rac2. GST-RhoA and -Rac2 were purified from Sf9 cell extracts with GSH-Sepharose, followed by cleavage with thrombin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transduction of Tat-C3 into Cells—In an attempt to establish whether Rho is involved in phagocytosis and superoxide formation in macrophages, J774 cells were treated with Tat-C3, which is readily transduced into cells and inhibits Rho. Tat-C3 and -YCD were efficiently transduced into J774 cells (Fig. 1A). Tat-GFP also entered the cells and displayed fluorescence (Fig. 1, B and C). The introduction of Tat-C3 led to an alteration of electrophoretic mobility of RhoA on SDS-PAGE indicating that Tat-C3 modified the RhoA, possibly generating ADP-ribosylated RhoA. No such effect was observed with C3 exoenzyme or Tat-YCD and -GFP. The Tat fusion proteins were also transduced into peritoneal macrophages of mouse (Fig. 1B), and Tat-C3 again induced a mobility shift of RhoA on SDS-PAGE. Tat-C3, -YCD, and -GFP were present at higher densities on cellular membranes than in the cytosol. Much of each protein was dissociated from the membranes in the presence of 1 M NaCl, which blocks the nonspecific ionic interaction between the Tat-proteins and membranes; thereafter, the Tat-proteins remained inside the cells (Fig. 1C).



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  		FIG. 1.
Tat-C3 is transduced into macrophage cells. Tat-C3 (1-30 µg/ml) and Tat-YCD (1-30 µg/ml) were added to J774 cells (2 x 106) for 30 min at 37 °C, and the cells were washed with PBS. Transduced Tat-proteins and RhoA were detected by Western blotting using anti-His tag and -RhoA antibodies, respectively (A). Tat-C3, -YCD, and -GFP (10 µg/ml) were added to peritoneal (2 x 106) and J774 macrophages (2 x 106) for 30 min at 37 °C. Tat proteins and RhoA were detected by Western blotting as in A and B. C3 exoenzyme, Tat-C3, -YCD, and -GFP (10 µg/ml, respectively) were added to J774 cells for 30 min at 37 °C, washed with or without 1 M NaCl, and the cells were fixed, permeabilized, and stained for the His tag epitope with the anti-His tag antibody followed by Texas red-tagged secondary antibody with Tat-C3- and -YCD-treated cells. C, Texas red and GFP fluorescence was observed by confocal microscopy.

 
Tat-C3 Inhibits Superoxide Formation Induced by SOZ—To elucidate whether Rho is involved in the induction of superoxide formation, we examined the effect of Tat-C3 on superoxide formation induced by SOZ. C3b in serum rapidly attaches to zymosans, and about 80% of it is converted to C3bi when the zymosans are opsonized with fresh FBS at 37 °C (43). In addition, yeast particles opsonized in normal rabbit serum could be stained with anti-C3c sera, but not with anti-IgG sera (44). Therefore, SOZ may be regarded as identical to C3bi-opsonized zymosan (COZ). SOZ particles induced superoxide production in macrophages after a 5-7-min lag, and maximal intracellular superoxide formation was achieved in about 30 min (Fig. 2A). The lag may be due to the time needed for SOZ to bind to the CRs of the macrophages. Indeed, treatment of cells with Tat-C3 at 0.1-10 µg/ml without preincubation significantly reduced superoxide formation. Furthermore, preincubation of the macrophages with Tat-C3 (even at a concentration as low as 0.01 µg/ml) for 30 min almost completely blocked superoxide formation induced by SOZ particles (Fig. 2A). On the other hand, Tat-YCD even at 10 µg/ml did not block SOZ particle-induced superoxide formation (Fig. 2A), although it was efficiently transduced into macrophages (Fig. 1). In addition, Tat-peptide did not affect superoxide formation at concentrations up to 800 nM (Fig. 2A, inset). C3 exoenzyme at 200 µg/ml and for 24 h, which did not significantly change the cell viability, did inhibit superoxide formation somewhat (Fig. 2B), suggesting that the Tat domain itself is not involved in the inhibition of superoxide production. Some C3 exoenzyme can be translocated when a high concentration of it is used over a long period of incubation (45). Tat-C3 also almost completely abrogated superoxide induction by SOZ in primary peritoneal mouse macrophages (Fig. 3A). In addition, Tat-C3 again blocked superoxide formation by SOZ and IOZ in human neutrophils (Fig. 3B).



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  		FIG. 2.
Tat-C3 inhibits superoxide formation in macrophages stimulated by SOZ particles. To measure intracellular superoxide, J774 cells (2 x 103) were resuspended in 1 ml of KRG buffer containing 50 units/ml SOD, 2000 units/ml catalase, and 50 µM luminol. Superoxide formation was started by addition of SOZ particles (2 x 104), and subsequent chemiluminescence was measured with a luminometer. Tat-C3, Tat-YCD, and synthesized Tat-peptide (800 nM) alone (inset in A) were first added to J774 cells for 30 min at 37 °C (A). J774 cells (2 x 103) were pretreated with C3 exozyme without the Tat-penetratin domain (50-200 µg/ml) for 24 h at 37 °C, and superoxide formation was measured as in A. The lower Western blot shows a small mobility shift of RhoA in response to exposure to a high concentration of C3 for an extended period (B).

 



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  		FIG. 3.
Tat-C3 inhibits superoxide formation by opsonized zymosan particles in primary cells. Mouse peritoneal macrophages (A) and human neutrophils (2 x 103)(B) were preincubated with or without Tat-C3 (10 µg/ml) for 30 min at 37 °C, and superoxide formation induced by SOZ (2 x 104) (A and B) and IOZ (2 x 104) (B) particles was determined by measuring chemiluminescence as described in the legend to Fig. 2.

 
Rho Is Not Involved in Superoxide Formation Unrelated to Phagocytosis—Macrophages form superoxide in response to PMA, fMLP, and M-CSF, which are not phagocytosed. The amount of superoxide formed in these cases was much lower (10-20%) than that formed by SOZ, but its formation was unaffected by Tat-C3 (Fig. 4). This suggests that Tat-C3 specifically affects superoxide formation associated with phagocytosis, not that resulting from direct stimulation of PKC or triggering of cell-surface receptor.



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  		FIG. 4.
Tat-C3 does not inhibit superoxide formation induced by PMA, fMLP, and M-CSF. J774 cells (2 x 103) were preincubated with or without Tat-C3 (10 µg/ml) for 30 min at 37 °C. Superoxide formation was induced by adding 1 µM PMA, 100 nM fMLP, or 100 ng/ml M-CSF to the cells and determined by measuring chemiluminescence.

 
Tat-C3 Inhibits Phagocytosis of SOZ by Macrophages—We measured phagocytosis of SOZ particles by J774 cells and the effect of Tat-C3 on these processes. Tat-C3 inhibited phagocytosis of SOZ particles, whereas Tat-YCD did not (Fig. 5A). Treatment of the cells with high concentration of C3 exoenzyme had a small effect on phagocytosis (Fig. 5A). Although we could not detect any C3 exoenzyme band on Western blots (Fig. 5B), small amounts of C3 exoenzyme appear to be transduced into cells at a high concentration of external dosage (Fig. 2B and Ref. 45). Indeed, Tat-C3 gave rise to an inhibitory effect even at a concentration as low as 0.01 µg/ml (Fig. 2A). Tat-C3 inhibited binding (data not shown) and phagocytosis of SOZ particles to a similar extent, suggesting that binding and phagocytosis of the particles are closely linked. It is important to note that whereas phagocytosis was reduced by only about 50% (Fig. 5A), superoxide formation was almost completely abolished by the same concentration of Tat-C3 (Fig. 2A).



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  		FIG. 5.
Involvement of Rho in phagocytosis of SOZ particles. J774 cells (2 x 105) were treated with 50 µg/ml of C3 exoenzyme (C3), 10 µg/ml Tat-C3, and 10 µg/ml Tat-YCD for 30 min at 37 °C. For phagocytosis, the cells were incubated for 30 min with 5 x 105 of FITC-conjugated zymosan particles previously opsonized in fresh serum, and then washed. Phagocytosis was assayed by measuring the intensity of fluorescence of FITC bound to zymosan with fluorescence spectrophotometry (A). Tat-C3 and Tat-YCD were translocated into macrophage cells and detected with anti-His tag antibody (B). Values are means ± S.E. of three experiments. *, p < 0.05; **, p < 0.01.

 
Superoxide Does Not Affect Phagocytosis of SOZ—Next, we examined the possibility that superoxide plays a role as a second messenger for phagocytosis. NAC (superoxide scavengers), DPI, and AEBSF (NADPH oxidase inhibitors) (46) had no significant effect on the phagocytosis and binding of SOZ particles, whereas they inhibited superoxide formation. In addition, more superoxide was detected during phagocytosis in the presence of MV (a superoxide generator) than control, and MV did not affect phagocytosis and binding of SOZ particles (Supplemental Data, Fig. 1).

Effect of RhoA Plasmids on Superoxide Formation in Response to SOZ—To test whether RhoA is involved in superoxide formation during macrophage phagocytosis, we transfected J774 cells with plasmids encoding RhoA. Cells transfected with wild-type RhoA produced 36% more superoxide than control cells during phagocytosis, whereas those transfected with N19RhoA produced about 40% less. Cells transfected with mock vector yielded similar levels of superoxide formation as non-transfected cells (Fig. 6B). Phagocytosis of SOZ by macrophages transfected with N19RhoA was reduced by about 20%, and that with wild-type RhoA increased by about 15% (Fig. 6A). Visualization of cells transfected with EGFP-/RhoA-cDNA constructs by fluorescence microscopy indicated that the efficiencies of transfection into macrophages were about 40% (Fig. 6C).



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  		FIG. 6.
RhoA is involved in superoxide formation in macrophages. J774 cells (2 x 103) were transfected with 5 µg of cDNA construct encoding wild-type RhoA or dominant negative, N19RhoA, or mock vector. After 48 h, phagocytosis of SOZ particles was determined (A), and the chemiluminescence produced in response to the addition of SOZ was measured with a luminometer (B). 5 µg of EGFP and 5 µg of RhoA cDNA constructs were added to cells, and cells and fluorescence of EGFP were observed with phase contrast and fluorescence microscopy (C). Values are means ± S.E. of three experiments. *, p < 0.05 (A). B and C, representative of three independent experiments.

 
Recombinant Prenylated RhoA Does Not Directly Activate NADPH Oxidase—We attempted to elucidate how Rho is involved in superoxide formation during phagocytosis. To test whether RhoA directly activates NADPH oxidase, we measured the superoxide formation in vitro after adding GTP-bound prenylated recombinant RhoA to a semi-recombinant, cell-free NADPH oxidase system (32). Table I shows that GTP-bound RhoA did not stimulate superoxide production by NADPH oxidase, whereas Rac2 did.


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  		TABLE I
Recombinant prenylated RhoA does not stimulate NADPH oxidase activity

The assay consisted of 3 nM cytochrome b558, 140 nM recombinant p47PHOX, 35 nM recombinant p67PHOX, 15 nM prenylated Rac2 or RhoA, 166 nM FAD, 100 µM cytochrome c, 60 µM SDS, and relax buffer (10 mM Pipes, pH 7.4, 3 mM NaCl, 130 mM KCl, 3.5 MgCl2) in a total volume of 150 µl. NADPH (0.33 mM) was added to initiate the reaction. The production of superoxide anion was monitored by the reduction of cytochrome c, which was recorded at 550 nm against time using a plate reader, and the Vmax was calculated using software. Superoxide dismutase (50 units) was added as an inhibitor in a duplicate well to show that the reduction of cytochrome c was due to the production of superoxide anion. Activity (Vmax) is expressed as milli OD/min and converted to nmol of /min using an extinction coefficient of 21 mM–1 cm–1 for cytochrome c.

 
Tat-C3 Inhibits Induction of Superoxide Formation by Both COZ and IOZ Particles—In order to distinguish the Fc{gamma}R- and CRs-pathways of phagocytosis and superoxide formation, we analyzed phagocytosis of COZ and IOZ particles and subsequent superoxide formation separately. As shown in Fig. 7A, COZ or IOZ particles were equally well engulfed by the macrophages, while NOZ particles were engulfed about half as efficiently. Tat-C3 significantly inhibited the phagocytosis of all three particles, though phagocytosis of COZ particles was more strongly inhibited than that of IOZ particles (Fig. 7A). This result is in good agreement with the report that uptake of COZ particles by macrophages is mediated by RhoA (5).



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  		FIG. 7.
Involvement of Rho in the phagocytosis and superoxide formation in J774 macrophages induced by both C3bi- and IgG-opsonized zymosan particles. For phagocytosis, the cells were incubated with FITC-conjugated NOZ, COZ, and IOZ. The zymosan particles were estimated to bind ~0.1 µg/ml C3bi and IgG proteins. A, phagocytosis was evaluated from the FITC fluorescence in macrophages pretreated with (gray bar) or without (black bar)10 µg/ml Tat-C3 for 30 min at 37 °C. B, superoxide formation was initiated by adding NOZ, COZ, or IOZ to J774 cells (2 x 103) pretreated with (gray bar) or without (black bar) 10 µg/ml Tat-C3 for 30 min at 37 °C. Superoxide formation was started by adding 0.1 µg/ml of C3bi and IgG protein ligands separately, and chemiluminescence was subsequently measured. C, J774 cells were pretreated with (gray bar) or without (black bar) 10 µg/ml Tat-C3 for 30 min at 37 °C. D, superoxide formed by COZ and IOZ particles (black bar), or by 0.1 µg/ml C3bi and IgG proteins (gray bar) were compared by replotting B and C. Values are means ± S.E. of three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Phagocytosis of IOZ particles led to the production of about 160% more superoxide than that of COZ particles, and Tat-C3 also completely inhibited superoxide formation in both cases. The total amount of superoxide formed when IOZ and COZ particles were added together did not surpass that formed with only IOZ particles (Fig. 7B). NOZ induced a low level of superoxide production, and it was inhibited by Tat-C3 (Fig. 7B). In addition, stimulation of Fc{gamma}R and CRs by adding IgG and C3bi ligands on their own to macrophages without zymosans, again produced a low level of superoxide that was also inhibited by Tat-C3 (Fig. 7C). However, superoxide formation during phagocytosis of COZ and IOZ particles was much greater (about 6-fold) than that stimulated by C3bi and IgG ligands (Fig. 7D).

Tat-C3 Inhibits Superoxide Formation Induced by Non-phagocytotic Ligands—In order to clarify that reduction of superoxide formation by Tat-C3 is not a simple reflection of reduced phagocytosis, cells were stimulated with non-phagocytotic ligands. Non-phagocytotic ligands were prepared by linking serum-opsonized IOZ to protein A-agarose beads through anti-zymosan antibody (SO-IOZ-beads) exploiting the fact that agarose beads are too large to be phagocytosed into macrophages. Here zymosan particles appeared to be coated with both C3bi and IgG as ligands. SO-FITC-IOZ particles were found to bind to the surface of beads (data not shown). Cells stimulated with the SO-IOZ-beads could not engulf the beads, but gave fluorescence of DCF, which can be observed in response to superoxide formation. However, cells pretreated with Tat-C3 did not show it despite of binding of the cells to the beads (Fig. 8). This result indicates that superoxide formation induced by SOZ and IOZ occurs irrespective of phagocytosis.



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  		FIG. 8.
Observation of superoxide produced by SO-IOZ-bead on a confocal microscope. J774 cells were preincubated in serum-free medium for 16 h at 37 °C, incubated with 10 µM DCFH-DA for 30 min at 37 °C, washed and then incubated with serum and phenol red-free medium. Cells were stimulated with SO-IOZ-bead for 15 min in the presence or absence of 10 µg/ml Tat-C3. Cells were washed with PBS and added serum and phenol red-free medium. DCF fluorescence at an excitatory wavelength of 495 nm was observed on a confocal microscopy system (Bio-Rad).

 
Tat-C3 Does Not Inhibit Rac Activity—To exclude the possibility that Tat-C3 at high concentration also inhibits Rac in vivo (47), we compared the GTP binding activities of RhoA and Rac1, and found that Tat-C3 completely abolished the GTP binding activity of RhoA, but not that of Rac1 (Fig. 9A). Treatment with fMLP or M-CSF to J774 cells led to membrane ruffling via Rac1 and Cdc42, which is in agreement with previous reports (7, 48). Membrane ruffling was stimulated rather than inhibited by Tat-C3, suggesting that Tat-C3 at 10 µg/ml does not affect Rac activity in vivo, and that inactivation of Rho lead to induction of membrane ruffling. The ruffling in response to fMLP and M-CSF, and the effect on it of Tat-C3 were measured quantitatively, and the results are given in Fig. 9B. Y-27632, an inhibitor of Rho kinase also did not affect membrane ruffling induced by M-CSF and fMLP (data not shown).



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  		FIG. 9.
Tat-C3 does not affect the activity of Rac. For ADP-ribosylation of GST fusion proteins, 10 µg/ml Tat-C3 (gray bar) or control (black bar), 3 µM NAD, 6 µM GDP, 1 µg/ml GST-RhoA or -Rac1 in 50 µl buffer were premixed on ice and incubated for 1 h at 37 °C. For binding of GTP, GST-RhoA and -Rac1 were incubated with 0.1 µM {gamma}35S-GTP in 50 µl of GTP binding buffer at 30 °C for 10 min. GTP binding was assayed by membrane filtration with BA 85 membrane (A). F-actin-rich ruffles in J774 cells were fixed, and actin was stained with rhodamine-phalloidin. Cells were incubated with (gray bar) or without (black bar) 10 µg/ml of Tat-C3 for 30 min, and then stimulated with 100 nM fMLP or 100 ng/ml M-CSF for 10 min. Ruffling of J774 cells was assessed quantitatively: ruffling index is arbitrarily set at 1 when ruffling was below 25%, and 2 when two or more discrete areas contained ruffles. Total ruffling index is the sum of indices of 100 cells (B). J774 macrophages (2 x 106 cells) were either pretreated with or without Tat-C3 (10 µg/ml) for 30 min and then treated with SOZ and IOZ to allow phagocytosis for the indicated time. J774 macrophages were harvested and lysed in a buffer containing 1% Triton X-100. GST-RBD and -PBD were mixed with cell extracts, precipitated with GSH-Sepharose beads, and washed, and proteins bound to the beads were fractionated on SDS-PAGE. Thereafter, Western blotting was performed with anti-RhoA, -Rac1, and -Rac2 antibodies (C). A and B, values are means ± S.E. of three experiments. *, p < 0.05; **, p < 0.01; p < 0.001 ***. C, results are from one of three independent experiments.

 
Both RhoA and Rac Are Activated during Phagocytosis of SOZ and IOZ—To assess the involvement of Rho GTPases in phagocytosis and superoxide formation, we assayed activation of RhoA, Rac1, and Rac2. As shown in Fig. 9C, RhoA, Rac1, and Rac2 were all activated during phagocytosis of SOZ and IOZ particles. Upon stimulation with SOZ and IOZ the levels of GTP-RhoA and -Rac1 increased gradually for 15-20 min, and then fell slightly, whereas Rac2 was activated for about 5-10 min after stimulation with SOZ and IOZ. RhoA was more activated than Rac1 and Rac2 in response to SOZ, while Rac1 and Rac2 were more activated than RhoA in response to IOZ. Tat-C3 strongly inhibited activation of RhoA but not that of Rac1 and Rac2 after stimulation with SOZ and IOZ particles (Fig. 9C).

Tat-C3 Inhibits Phosphorylation of p47PHOX—In order to reveal the linkage of Rho with NADPH oxidase, we tested whether inhibition of Rho by Tat-C3 affected the phosphorylation of p47PHOX, one of the cytosolic components of NADPH oxidase complex. As shown in Fig. 10, lysates of cells phagocytosing SOZ and IOZ stimulated the phosphorylation of p47PHOX, while lysates of the cells pretreated with Tat-C3, SB203580 (an inhibitor of p38MAPK), or PD98059 (an inhibitor of MAP kinase kinase (MEK)), strongly inhibited its phosphorylation. p38MAPK and ERK have already been reported to phosphorylate p47PHOX (49). Furthermore, Tat-C3 abrogated activation of ERK1/2 and p38MAPK of macrophages in response to SOZ (data not shown).



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  		FIG. 10.
Inhibition of GST-p47PHOX phosphorylation by Tat-C3 exoenzyme. The J774 cells were pretreated with none (-), 10 µg/ml Tat-C3, 30 µM SB203580, and 50 µM PD98059 for 30 min. J774 macrophages (2 x 106 cells) were stimulated with SOZ (S) or IOZ (I) (5 x 106 particles), and the cell extracts were mixed with GSH-Sepharose suspension to which GST-p47PHOX was bound. The mixture was rotated, and the beads were washed and suspended in 30 µl of kinase buffer (20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM L-glycerophosphate, 0.1 mM Na3VO4, and 2 mM dithiothreitol) containing 20 µM ATP and 10 µCi of [{gamma}-32P]ATP. Thereafter phosphorylated GST-p47PHOX was analyzed by autoradiography. These results represent one of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Rho Is Involved in Superoxide Formation by Macrophages—In the present study, we found that Tat-C3, virtually abolished superoxide generation induced by SOZ and IOZ particles (Figs. 2, 3, and 7) and that transient transfection with the dominant-negative N19RhoA cDNA construct reduced superoxide formation whereas wild-type RhoA cDNA construct led to enhancement of it (Fig. 6). Since Tat-C3 specifically inhibits Rho, this is clear evidence that RhoA is involved in SOZ particle-induced superoxide formation.

Tat-C3 only inhibited phagocytosis of SOZ by 50% (Fig. 5) and the N19RhoA construct had at best a marginal effect on phagocytosis (Fig. 6). In addition, Tat-C3 reduced phagocytosis of IOZ by only 25% (Fig. 7A). However, Tat-C3 almost completely abrogated superoxide formation induced by COZ and IOZ (Fig. 7B), and N19RhoA reduced more superoxide production than phagocytosis. These results suggest that superoxide formation by SOZ and IOZ is not strictly dependent on phagocytosis. On the other hand, cytocalasin D (CD) inhibited both phagocytosis and superoxide production in response to SOZ, and the inhibition levels of the two processes were similar in CD concentration-dependent manner (Supplemental Data, Fig. 2). In this case, superoxide formation appears to be very closely linked to phagocytosis. Indeed, phagocytosis needs remodeling of the actin cytoskeleton (4), and the cytoskeleton also plays a role in the signaling pathway that activates NADPH oxidase (21, 50). In contrast, superoxide is not required for phagocytosis of SOZ (Supplemental Data, Fig. 1). As shown in Fig. 8, cells stimulated with non-phagocytotic SO-IOZ-beads formed superoxide, while Tat-C3 completely blocked superoxide formation. This suggests that inhibition of superoxide formation by Tat-C3 is not a simple consequence of a reduction in phagocytosis. Although RhoA is not directly associated with the NADPH oxidase complex (Table I), it must participate indirectly in superoxide formation. Therefore, the activation of RhoA triggered by COZ and IOZ particles may participate in both phagocytosis and superoxide formation via activation of p47PHOX (Fig. 10). The concept that phagocytosis and superoxide formation are regulated through different signaling pathways was supported by the observation that the PKC inhibitor (GF109203X) inhibits superoxide formation, but slightly enhances phagocytosis of serum-opsonized particles (42). We also revealed that Tat-C3 did not inhibit superoxide formation in response to PMA, fMLP, and M-CSF (Fig. 4), suggesting that Tat-C3 is not cytotoxic and that Rho is not involved in superoxide formation in response to those agents. Regarding the effect of the Tat-peptide domain, it does not change superoxide formation in response to SOZ (Fig. 2A), although basic polyamino acids inhibit NADPH oxidase (51). It is also demonstrated by the fact that Tat-RacV12 induces production of reactive oxygen species (ROS) (52); Tat-domain of this fusion protein does not block ROS formation. In regard to the effect of PMA on superoxide formation, it activates PKC, and PKC in turn activates p47PHOX by phosphorylating its serine residues (53). In addition, PMA stimulates translocation of p47PHOX, p67PHOX, and Rac1 to membranes (19). fMLP, on the other hand, was reported to activate phospholipase D (PLD) through receptor to form phosphatidic acid (PA), which in turn stimulates PA-activated protein kinase, resulting in activation of p22PHOX by phosphorylation (54). PLD induced superoxide formation in J774 cells, and Tat-C3 did not inhibit it (data not shown), suggesting that Rho is not involved in the signaling pathway via PLD to activate NADPH oxidase.

With regard to the effect of Tat-C3 on Fc{gamma}R-versus CR3-mediated phagocytosis and superoxide formation, we found that it inhibited the phagocytosis of COZ particles much more strongly than that of IOZ (Fig. 7A). However, Rho was also required for the Fc{gamma}R-mediated pathway of phagocytosis and superoxide formation as well as for CR3-mediated processes (Fig. 7, A and B). It also proved to be involved in signaling leading to superoxide formation through CR3 and Fc{gamma}R stimulation not leading to phagocytosis (Fig. 7C). However, superoxide formation induced by PMA, fMLP, and M-CSF, which is unrelated to phagocytosis, was insensitive to Tat-C3 (Fig. 4). We believe, therefore, that Rho is required specifically for CR3- and Fc{gamma}R-mediated superoxide formation.

NOZ particles, which are recognized by mannose receptors, {alpha}-glucan receptors (55), and CR3 (56), also induced both phagocytosis and superoxide formation (Fig. 7). On the other hand, mannose receptor-mediated phagocytosis is not accompanied by superoxide production (57). These observations together suggest that phagocytosis and superoxide formation induced with NOZ particles may be mediated through CR3 or other receptors that regulate Rho. Phagocytosis of IO- and CO-microspheres also triggered superoxide in the macrophages (data not shown).

Tat-C3 neither inhibited GTP binding to Rac (Fig. 9A) nor membrane ruffling (Fig. 9B). Tat-C3 thus was not cytotoxic. Microinjection of C3 into Bac1 macrophages induces cell flattening and radial spreading, and these cells still possess lamellipodia, showing that C3 does not inhibit Rac-regulated processes (48). RhoA, Rac1, and Rac2 were all activated during phagocytosis of SOZ and IOZ, and only RhoA was inhibited by Tat-C3 (Fig. 9C). These results suggest that Tat-C3 does not affect Rac activity, but specifically inhibits Rho and that Rac may be upstream of Rho (48, 58), or the two may work in parallel.

Superoxide formation with fMLP and IgG-SRBC in neutrophils of Rac2 knockout (rac2-/-) mice is reduced, whereas that elicited by SOZ particles is preserved (18). This suggests that the requirement for Rac2 in superoxide formation is not absolute and that the specific signaling pathway leading to activation of neutrophil respiratory burst is selectively regulated by Rac2 (18). Rac2 appears to be involved in neutrophil migration and NADPH oxidase function, whereas Rac1 plays a role in controlling cell spreading (59). Likewise Rac1 and Rac2 activated by SOZ and IOZ may play different roles in phagocytosis and superoxide formation. However, which Rac is involved in the activation of NADPH oxidase in J774 cells is not yet clear. Recently it was reported that human monocytes use Rac1, not Rac2, in the NADPH oxidase (60).

It is well known that the onset of respiratory burst activity during phagocytosis is linked to phosphorylation of p47PHOX and its translocation to phagosome (61): ERK1/2 and p38 MAPK participates in the phosphorylation of p47PHOX (42, 62, 63), but JNK is unable to phosphorylate p47PHOX (48). In addition, other protein kinases such as phosphatidylinositol 3-kinase, Rho kinase, and myosin light chain kinase (MLCK) have been reported to regulate superoxide formation (64-67). Remarkably MLCK regulates superoxide release and the phosphorylation and translocation of p47PHOX (66). In the present study, Tat-C3, PD98059, and SB203580 all inhibited phosphorylation of p47PHOX (Fig. 10). This indicates that Rho, p38MAPK, and ERK1/2 are involved in the signaling pathway leading to the phosphorylation of p47PHOX. Moreover, Tat-C3 abrogated activation of ERK1/2 and p38MAPK (data not shown), suggesting that Rho is involved in the regulation of ERK1/2 and p38MAPK activity. Tat-C3 also blocked translocation of RhoA to membranes (data not shown), suggesting that translocation of RhoA may be essential for superoxide formation.

Taken together, Rho participates in the regulation of not only phagocytosis but also superoxide formation. Despite the multiple events associated with superoxide formation, details of how Rho regulates these processes remain to be clarified. We can be confident however that Rho is critically involved in regulation of superoxide formation via phosphorylation of p47PHOX.


   FOOTNOTES
 
* This study was supported by Grant 02-PJ10-PG6-AG01-0003 from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Data. Back

To whom correspondence should be addressed: Dept. of Biochemistry, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea. Tel.: 82-33-248-2542; Fax: 82-33-244-8425; E-mail: jbpark{at}hallym.ac.kr.

1 The abbreviations used are: CR, complement receptor; COZ, C3bi-opsonized zymosan; DCFH-DA, 2',7'-dichlorofluorescein diacetate; ERK1/2, extracellular signal-regulated kinase 1/2; FITC, fluorescein isothiocyanate; IPTG, isopropyl-1-thio-{beta}-D-galactopyranoside; IOZ, IgG-opsonized zymosan; luminol, 5-amino-2,3-dihydroxy-1,4-phtalazinedione; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MAPK, mitogen-activated protein kinase; MV, methylviologen; PA, phosphatidic acid; PD98059, 2'-amino-3'-methoxyflavone; PMA, phorbol 12-myristate 13-acetate; SB203580, 4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SOD, superoxide dismutase; SOZ, serum-opsonized zymosan; PMSF, phenylmethylsulfonyl fluoride; TRITC, tetramethylrhodamine B isothiocyanate; GST, glutathione S-transferase; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PKC, protein kinase C; {gamma}S-GTP, guanosine 5'-3-O-(thio)triphosphate; HIV-1, human immunodeficiency virus-1; fMLP, formyl Met-Leu-Phe; M-CSF, macrophage colony-stimulating factor; SRBC, sheep red blood cells. Back


   ACKNOWLEDGMENTS
 
We thank Dr. J. W. Park at Kyoungbuk National University, Korea, for the anti-p47PHOX antibody and pGEX-1{lambda}T plasmid containing an insert of p47PHOX cDNA. Dr. J. Park at Hallym University kindly provided Tat-YCD and -GFP plasmids. We thank Dr. Gary M. Bokoch at The Scripps Research Institute for supplying anti-p47PHOX and -Rac2 antibodies.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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