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Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla, Avda. Sánchez Pizjuán 4, E-41009 Sevilla, Spain
1 Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
2 Unidad de Hipertensión, Hospital Universitario Virgen Macarena, Sevilla, Spain
(Correspondence should be addressed to F Sobrino; Email: fsobrino{at}us.es)
* R El Bekay and G Alba contributed equally to this work 
R El Bekay is now at Laboratorio de Investigación, Fundación IMABIS, Hospital Carlos Haya, Málaga, CIBER de obesidad y Nutrición (CB06/03/0018)
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production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca2+ elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca2+/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.
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Introduction |
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Given that chronic inflammation of vessel walls is a pathological indicator of hypertension (Touyz 2003) and that reactive oxygen species (ROS) such as superoxide anion 
and H2O2 constitute the main intermediary molecules responsible for inflammation (Finkel 1998), a link between the development of hypertension and ROS production has been postulated (El Bekay et al. 2003). NADPH oxidase in phagocytic cells is a multi-component enzyme composed of at least two membrane proteins, gp91phox and p22phox, which together form the flavocytochrome b558, and four cytosolic proteins, namely p47phox, p67phox, p40phox and Rac. In resting cells, this oxidase is inactive and its components are distributed separately between the cell membrane and the cytosol. When neutrophils are exposed to appropriate stimuli, NADPH oxidase becomes activated through the association of all its components at the plasma membrane, which is followed by 
production (Inanami et al. 1998a,b, 2001, Johnson et al. 1998, Babior 2000).
The Rac small GTPases, members of the Rho-GTPases family, including Rho, Cdc42 and Rac, constitute a growing subgroup of Ras proteins which act as molecular switches upon their cycling between active GTP- and inactive GDP-bound states (Van Aelst & D'Souza-Schorey 1997). Previous studies have shown that Rho-GTPases are involved in multiple cellular processes, such as actin polymerization and cytoskeleton rearrangement, regulation of gene transcription, cell cycle progression and cell survival (Etienne-Manneville & Hall 2002). The Rac subfamily of Rho-GTPases includes three highly homologous members, namely Rac1, Rac2 and Rac3. Unlike Rac1 and Rac3, which are widely expressed, Rac2 is found only in haematopoietic cells (Didsbury et al. 1989, Haataja et al. 1997) In addition to their reported roles in actin remodelling, Rac proteins have been implicated in the generation of 
via the phagocytic NADPH oxidase complex (Segal & Abo 1993). Rac1 was identified as an oxidase-related factor from peritoneal macrophages (Abo et al. 1991), whereas Rac2 was demonstrated to fulfil such a role in human neutrophils (Knaus et al. 1991, Bokoch 1994). Rac exists in the cytosol as a complex with a GDP dissociation inhibitor (Abo et al. 1991), and upon activation of NADPH oxidase Rac separates from this inhibitor and becomes stably associated with NADPH oxidase components at the plasma membrane (Northup et al. 1982, Curnutte et al. 1987). The GTP-bound form of Rac binds to p67phox, and also likely to flavocytochrome b558, in the assembled active oxidase complex (Diekmann et al. 1994, Heyworth et al. 1994, Koga et al. 1999).
In a cell-free system, Rac1 and Rac2 recombinant proteins can reconstitute a fully active NADPH oxidase complex able to produce 
(Abo et al. 1991, 1992, Knaus et al. 1991). Moreover, it has recently been reported that neutrophils from Rac2-deficient mice exhibit a diminished NADPH oxidase activity (Roberts et al. 1999, Ambruso et al. 2000, Williams et al. 2000, Kim & Dinauer 2001). This finding is consistent with other previous results suggesting that Rac2 is a primary GTPase modulator of NADPH oxidase activation (Heyworth et al. 1994). However, upstream signals and regulatory proteins controlling Rac activity in neutrophils remain unknown, and only an involvement of phosphatidylinositol-3-kinase (PI3K) in Rac2 activation elicited by the respiratory burst stimulator, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), has been demonstrated so far (Akasaki et al. 1999, Benard et al. 1999). Therefore, the role of Rac2 in Ang II-stimulated neutrophils remains to be analysed.
Recently, the mitogen-activated protein kinases (MAPKs) family has become a focus of interest in cardiovascular research. In human neutrophils, we have described that Ang II induces a robust phosphorylation of p38MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2), which is hindered by inhibitors of NADPH oxidase and tyrosine kinases, as well as by ROS scavengers (El Bekay et al. 2003). Also, we have reported that Ang II enhances the synthesis de novo and activity of calcineurin (CaN) and that the immunosuppressant cyclosporin A (CsA) inhibits Ang II-induced CaN activity, but not CaN synthesis (El Bekay et al. 2003). In this study, we present experimental evidence that Ang II promotes Rac2 translocation from the cytosol to the plasma membrane in human neutrophils. Conversely, prevention of Rac2 translocation by Clostridium difficile toxin A, an inhibitor of Rho-GTPases, including Rho, Rac and Cdc42 (Aktories et al. 2000, Voth & Ballard 2005), abolishes ROS production and activation of p38MAPK, ERK1/2 and JNK1/2 elicited by Ang II in human neutrophils. The present data thus indicate that Rac2 translocation to the plasma membrane plays a critical role in Ang II-dependent signalling pathways in human neutrophils.
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Materials and methods |
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Ang II, SP6000125 and chemicals of general use were purchased from Sigma–Aldrich or Calbiochem and eprosartan was from Solvay Pharma. The rabbit polyclonal antibodies to phosphorylated p38MAPK (Thr180/Tyr182) and total p38MAPK, and mouse monoclonal antibodies to phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 were obtained from New England Biolabs (Beverly, MA, USA). Mouse antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) to phosphorylated JNK1/2 (Thr183/Tyr185) and total JNK1/2 (sc-6254 and sc-571 respectively), and rabbit antibodies to Rac2 (sc-96) and ß-actin (sc-7210) were also used. Goat polyclonal antibodies against p22phox were kindly donated by T L Leto (NIH, Bethesda, MD, USA). The p21-activated kinase-1 p21-binding domain (PAK-1 PBD) conjugated to agarose, GTP
S and GDP were products from Upstate Cell Signaling. The inhibitors PD089059, SB203580 and NSC23766 were obtained from Calbiochem. The rabbit antiserum against CaN was kindly provided by Dr C B Klee (NIH).
Human neutrophil extraction and processing
Human peripheral neutrophils were obtained from healthy blood donors and processed as described in Carballo et al. (1999). Neutrophils were suspended in KR-HEPES buffer and subjected for 30–60 min at 37 °C to the treatments indicated in each figure legend, except for the experiments shown in Figs 5 and 7, in which the cells were preincubated at 37 °C for 7 h in Krebs Ringer (KR)-HEPES before additions. Under these conditions, cell viability ranged between 90 and 97%, as estimated from the lactic dehydrogenase release assay (Gualberto et al. 1998). The Universidad de Sevilla Ethics Committee approved this study and each subject gave informed consent prior to its undertaking.
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Human neutrophils (107 cells) were lysed on ice for 30 min in 60 µl buffer containing 100 mM HEPES (pH 7.3), 100 mM KCl, 3 mM NaCl, 3 mM MgCl2, 1.25 mM EGTA, and the protease inhibitors phenylmethylsulphonyl fluoride (1 mM), aprotinin (20 µg/ml), leupeptin (20 µg/ml) and benzamidine (156 µg/ml). Then, the cells were disrupted by sonication (20 W, three bursts of 5 sec each separated by 30-s interval), and unbroken cells and debris were removed by centrifugation at 10 000 g for 5 min at 4 °C. The supernatant obtained after further ultracentrifugation at 100 000 g for 30 min at 4 °C constituted the cytosolic fraction. The pellet was resuspended in a buffer containing 120 mM NaH2PO4 (pH 7.4), 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 20% (v/v) glycerol, 40 mM octylglucoside and the protease inhibitors as indicated above, and then recentrifuged at 20 000 g for 40 min at 4 °C. The new supernatant obtained, containing solubilized membranes, was used for immunoblotting analysis of Rac2 mobilization (El Bekay et al. 2003). With this purpose, this fraction was subjected to SDS-PAGE on 10% polyacrylamide gels (50 µg protein/lane) and electroblotted onto polyvinylidene difluoride membranes using a semi-dry device (Bio-Rad). Thereafter, antibody probing was carried out overnight without need of prior blocking (Mansfield 1995) with rabbit anti-Rac2 IgG, at a 1:2000 dilution in PBS supplemented with 0.02% Tween 20 and 1% BSA, and the 21.429 kDa Rac2 band (Didsbury et al. 1989) was detected by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG at a 1:20 000 dilution in PBS with 0.5% (w/v) BSA, followed by enhanced chemiluminescence as previously indicated (Carballo et al. 1999). Briefly, the membranes were incubated for 1 min in 10 ml fresh luminescent reagent solution, composed 10 mM Tris–HCl (pH 8.5), 2.25 mM luminol, 0.015% (v/v) H2O2 and 0.45 mM 4-iodophenol, the latter acting as an enhancer of the chemiluminescence reaction, and the signals were recorded on X-ray-sensitive films. The membrane levels of the p22phox subunit of NADPH oxidase were monitored as a loading control, as previously described (Mankelow & Henderson 2001).
Rac2 GTP-binding activity
Rac2 activity pull-down assays were carried out essentially as described previously (Price et al. 2003), on the basis of the capacity of PAK proteins to bind to GTP-activated Rac2, but not to Rac2 bound to GDP. After the treatments indicated in each case, neutrophils (107 cells) were washed and then lysed in a magnesium-containing lysis buffer composed 25 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 25 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 1% (v/v) Nonidet P-40, 2% (v/v) glycerol, 100 µM phenylarsine oxide and the protease inhibitors described above. After centrifugation at 15 000 g, the supernatant obtained was incubated with 5–10 µg PAK-1 PBD agarose beads, and the reaction mixture was gently rocked at 4 °C for 1 h. In parallel, lysates from untreated neutrophils were loaded with 100 µM GTPS (positive control) or 1 mM GDP (negative control), for 15 min at 30 °C before addition of PAK-1 PBD agarose. The beads were then pelleted and washed, and immunoblotting analysis was carried out with anti-Rac2 antibodies as described above.
p38MAPK, ERK1/2 and JNK1/2 phosphorylation
In order to reduce the MAPK basal phosphorylation levels usually found in our preparations of human neutrophils, these cells were preincubated in KR-HEPES at 37 °C for 7 h. After the treatments indicated in each case, the cells were lysed and immunoblotting analysis with antibodies to phosphorylated and total forms of the three MAPKs was carried out as described (El Bekay et al. 2003). Relative protein levels were determined by scanning densitometry analysis using the Scion Image software (Frederick, MD, USA).
and total ROS production
The production of ROS was analysed by two separate methods, each with a different specificity for the location and type(s) of ROS produced. (i) The lucigenin-based luminescence method was specific for 
, whereas (ii) luminol-based luminescence correlated well with total ROS produced by the cells (Li et al. 1998). Since both luminol and lucigenin can permeate freely through the cell membrane, their luminescence was an indication of the sum of intracellular plus extracellular ROS. The assays were carried out as described previously (Monteseirín et al. 1996), except that HRP (8 mU/ml) was included when luminol was used.
Calcineurin protein and phosphatase activity levels
CaN phosphatase activity was measured as previously described (El Bekay et al. 2003). For immunoblotting analysis of CaN subunits A and B, neutrophils were lysed and subjected to SDS-PAGE (50 µg protein/lane) as described earlier (El Bekay et al. 2003). Detection was carried out by enhanced chemiluminescence as indicated above. To verify even protein loading, the blots were subsequently stripped and reprobed with rabbit polyclonal antibodies against ß-actin at a 1:1000 dilution.
Immunofluorescence microscopy analysis of Rac2 membrane translocation
The membrane translocation of Rac2 in human neutrophils was also assessed by immunofluorescence cell staining, as described previously (Vega et al. 2004) with minor modifications. After stimulation, neutrophils (107 cells) were harvested, washed with PBS and smeared onto poly-L-lysine-coated glass slides. The cells were fixed at room temperature with 2% paraformaldehyde for 30 min. After washing with PBS, unspecific binding was blocked with PBS containing 0.2% gelatin. Further, the cells were permeabilized with 0.1% Triton X-100 for 4 min, and thereafter incubated with rabbit anti-Rac2 IgG at a 1:100 dilution for 30 min, washed extensively and stained with FITC-conjugated anti-rabbit IgG at a 1:500 dilution for 30 min. After final washing, coverslips were mounted on the slides using 50% glycerol in PBS. Immunostained cells were observed and photographed using a Nikon EFD-3 microscope.
Intracellular Ca2+ levels
Cytosolic [Ca2+] was measured in cell populations using the fluorescent probe Fura2 as described previously (Sage et al. 1990).
Statistical analysis
Data are expressed as the mean±S.E.M. from a minimum number of three independent experiments. Protein expression levels were determined by densitometry of the bands using Scion Image software. This software detects the bands obtained by western blot and gives individual values which are dependent on the light quantification of the corresponding band. Measurements are expressed as arbitrary units. The results were normalized for unstimulated control. The numerical data obtained from Ang II stimulated and the corresponding controls were statistically analysed using Statgraphics plus 5.0 software (Manugistic Inc., Rockville, MD, USA) from ANOVA and the paired t-test. Asterisks indicate P values <0.01.
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Results |
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Ang II exogenously added promoted the accumulation of Rac2 at the plasma membrane in human neutrophils, with maximal levels being reached at a 10 nM concentration of Ang II and with concomitant disappearance of the Rac2 signal from the cytosol, as shown in Fig. 1A. The effect of this hormone on 
production by human neutrophils was also examined, and the values found were 0.043±0.009 (in control cells without additions), 0.165±0.022 (with 1 nM Ang II), 0.897±0.103 (with 10 nM Ang II) and 1.123±0.236 (with 100 nM Ang II) relative units of chemiluminescence. It was thus observed that Ang II elicited 
synthesis in a dose-dependent manner, confirming our previous report (El Bekay et al. 2003). Short-time kinetic experiments (Fig. 1B) revealed that an effective Ang II-dependent translocation of Rac2 from the cytosol to the plasma membrane took place as soon as within 1 min of incubation with the hormone, with maximal membrane levels being reached after 5 min of treatment. Rac2 translocation in Ang II-stimulated neutrophils was prevented by a 10 ng/ml dose of eprosartan (Fig. 1C), an inhibitor of the AT1 Ang II receptor (Brooks et al. 1999), indicating a specific role for this receptor in Ang II-induced Rac2 translocation in human neutrophils. In control experiments, the same effects were obtained when the chemoattractant fMLP at 100 nM substituted for Ang II (data not shown). The membrane levels of p22phox at the membrane, in keeping with it being an integral membrane protein (Mankelow & Henderson 2001), were similar in both resting and Ang II-stimulated neutrophils (Fig. 1A–C). We further investigated the effect of Ang II on the subcellular localization of Rac2 using immunofluorescence microscopy. As shown in Fig. 1D, this protein was primarily cytoplasmic in unstimulated cells (Fig. 1a and b), but was found predominantly located at the plasma membrane upon incubation of neutrophils with 10 nM Ang II for 10 min (Fig. 1c and d). These results corroborated that Ang II promotes the translocation of Rac2 to the cell membrane in human neutrophils.
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We have recently shown that Ang II induces an increase of both de novo synthesis and activity of CaN in human neutrophils (El Bekay et al. 2003). In order to determine whether inhibition of CaN activity affected Rac2 translocation to the plasma membrane, we pretreated neutrophils with CsA for 1 h. Figure 2A and B shows that CsA at a 0.3 µg/ml concentration was sufficient to suppress Rac2 translocation induced by Ang II in these cells indirectly suggesting an involvement of CaN in Ang II-dependent Rac2 mobilization to the plasma membrane in human neutrophils.
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production as well as translocation of the p47phox and p67phox NADPH oxidase subunits to the plasma membrane (El Bekay et al. 2003). As shown in Fig. 3A, we now found that these two inhibitors were also able to efficiently prevent Rac2 membrane translocation in Ang II-stimulated neutrophils. In a similar manner, the Ang II-promoted Rac2 translocation was effectively hindered by SP600125, an inhibitor of JNK1/2, in human neutrophils (Fig. 3B).
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Ang II promoted Rac2 GTP-binding activity in human neutrophils
In order to determine whether Ang II-induced Rac2 membrane translocation correlated with an increase in Rac2 GTP-binding activity, a PAK-1 PBD pull-down assay was carried out which was based on the property exhibited by PAK-1/3 Ser/Thr kinases of becoming stimulated upon their binding of active Rac GTPases (Bokoch 2003). Figure 4A shows that the treatment of human neutrophils with Ang II at 10 nM resulted in an up-regulation of Rac2 GTP binding by up to 40-fold. This enhancement was inhibited by eprosartan, indicating that this phenomenon depended on Ang II binding to AT1 receptors. Rac2 GTP-binding activity was also increased upon cell stimulation with 100 nM fMLP (Fig. 4A), as previously described in human neutrophils (Akasaki et al. 1999). As a control, when human neutrophil lysates were incubated with GTPS, Rac2 was found to massively bind to PAK-1 PBD, whereas no such association was detected when lysates were incubated with GDP (Fig. 4A). We also examined the effect of p38MAPK and ERK1/2 inhibitors on Rac2 GTP binding in Ang II-stimulated neutrophils. Figure 4B shows that both SB203580 and PD098059 hindered GTP binding by Rac2 suggesting an implication of both MAPKs in the signalling pathways modulating the Ang II-elicited targeting of Rac2 to the plasma membrane. Besides, Rac2 GTP-binding activity was totally inhibited by the PI3K inhibitor, wortmannin (Fig. 4C). As expected, Rac2 GTP-binding activity was also abolished in the presence of toxin A from C. difficile, confirming previous data (Aktories et al. 2000).
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In order to determine whether Rac2 translocation to the plasma membrane elicited by Ang II was mediated by signalling molecules other than MAPKs, the effect of pretreatment with the CaN inhibitor, CsA, prior to Ang II addition was assessed. We found that CsA at 1 µg/ml suppressed Rac2 GTP-binding activity induced by Ang II in human neutrophils, as revealed by its hindering of Rac2 GTP-binding activity (Fig. 4D). In addition, given that CsA was also able to inhibit Rac2 translocation to the plasma membrane (Fig. 2), and that crosstalk between CaN and MAPKs has been reported in myocytes (Lim et al. 2001) and in neutrophils (El Bekay et al. 2003), the inhibitory effect of CsA could be indirectly exerted through MAPK inhibition. In order to test this possibility, we examined the effect of CsA on Ang II-induced p38MAPK, ERK1/2 and JNK1/2 activation in human neutrophils. As shown in Fig. 5A and B, CsA prevented the phosphorylation, and hence the activation, of these three MAPKs by Ang II. These results allowed us to infer that CaN has an implication in both Rac2 and MAPK activation elicited by Ang II in human neutrophils.
Ang II-dependent Rac2 GTPase activity regulated 
production, cytosolic Ca2+ release and calcineurin activity
The treatment of cells with C. difficile toxin A (5–10 ng/ml), an inhibitor of the GTPase activity of GTP-binding proteins such as Rac, Rho and Cdc42 (Aktories et al. 2000, Voth & Ballard 2005), was found to reduce 
and total ROS release by 
81 and 97% respectively, in Ang II-stimulated human neutrophils when compared with untreated cells (Table 1), this indicating that GTPase activity plays a pivotal role in NADPH oxidase activation by Ang II in human neutrophils. In parallel, C. difficile toxin A was found to abolish Ang II-dependent Rac2 translocation to the plasma membrane (Fig. 6A). Since it has previously been shown that Ang II increases cytosolic Ca2+ in human neutrophils (El Bekay et al. 2003), we set to test whether Ca2+ signalling was involved in Ang II-dependent Rac2 activation. With this aim, neutrophils were pretreated simultaneously with thapsigargin, in order to deplete intracellular Ca2+ stores, and with EGTA to chelate extracellular Ca2+, and then the cells were activated by the addition of Ang II. Figure 6B shows that under these conditions Ang II-elicited Rac2 mobilization became drastically inhibited, indicating that cytosolic Ca2+ elevation is crucial for Rac2 translocation to the plasma membrane. C. difficile toxin A treatment also inhibited CaN activity (Fig. 6C), but did not affect the de novo synthesis of CaN induced by Ang II (Fig. 6D). Conversely, we also assessed whether elevation of intracellular Ca2+ required activation of Rac2 GTPase. As shown in Fig. 6E, when increasing doses of C. difficile toxin A were added to cells prior to Ang II stimulation, the Ca2+ signal was hindered, suggesting that the Ca2+ mobilization elicited by Ang II in human neutrophils depends, at least in part, on the activation of Rac2. In this context, Rac2 has also been shown to participate in actin polymerization and cytoskeleton rearrangement. Present observations on the inhibition on Ca2+ mobilization could be thus linked to the disruption of the actin cytoskeleton organization induced by several toxins, as previously observed (Bozem et al. 2000).
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Subsequent experiments were designed to examine whether the inhibition of Rac2 affected MAPK activation in Ang II-stimulated neutrophils. Figure 7A and B shows that C. difficile toxin A at a 5–10 ng/ml dose efficiently prevented Ang II-dependent p38MAPK, ERK1/2 and JNK1/2 activation. In order to gain insight on the specificity of this inhibitory effect, we also used a chemical compound, NSC23766, identified as a Rac-specific small-molecule inhibitor (Gao et al. 2004). NSC23766 exhibited an inhibitory effect on the phosphorylation of p38MAPK, JNK1/2 and ERK1/2 similar to that exerted by C. difficile toxin A (Fig. 7C and D). These results thus led support to the idea that the signalling pathways mediated by these three MAPKs are involved in Rac2 targeting to the cell membrane.
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Discussion |
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production, consistently with the translocation to the cell membrane of the cytosolic components of NADPH oxidase, p47phox and p67phox (El Bekay et al. 2003). Despite Rac2 having been reported as participating in a wide array of signalling pathways in neutrophils and other cells (Roberts et al. 1999, Yang et al. 2000), its role in the stimulation of these cells by Ang II has yet not been addressed. In the present work, we report for the first time the ability of Ang II to induce translocation of Rac2 from the cytosol to the plasma membrane, and to enhance its GTP-binding activity in a dose-dependent manner via AT1 receptors in human neutrophils. Previous and present data also indicate that translocation to the plasma membrane of the p47phox and p67phox subunits of NADPH oxidase and of Rac2 occur in parallel in Ang II-activated human neutrophils. In this context, it has been reported that Rac2 mobilization to the cell membrane is diminished in p47phox-deficient neutrophils from chronic granulomatous disease (CGD) patients (El Benna et al. 1994). However, Rac2 translocation is not affected in CGD neutrophils lacking p67phox (Dusi et al. 1995a). In contrast, other authors have shown that Rac2 is able to translocate independently of other cytosolic factors, and to migrate to the cell membrane in the absence of both p67phox and p47phox in human neutrophils (Heyworth et al. 1994, Dusi et al. 1995b). Also, we have observed that both Ang II-dependent Rac2 translocation and its GTPase activity increase were abrogated by CsA, a CaN inhibitor and immunosuppressant, and that this compound prevented p38MAPK, ERK1/2 and JNK1/2 activation by Ang II. These data thus underscore a pivotal role of CaN both in Rac2 translocation and the activation of these three MAPKs in Ang II-stimulated human neutrophils. Other authors have also shown that Rac is an upstream mediator of PAK-1 and JNK1/2 activation during Ang II signalling in vascular smooth muscle cells (Schmitz et al. 2001, Woolfolk et al. 2005). However, the crosstalk between Rac2 and MAPKs in Ang II-stimulated human neutrophils had not been hitherto investigated. Here, we describe that both Rac2 membrane translocation and enhanced GTP-binding activity elicited by Ang II were blocked by SB203580 and PD098059, two compounds acting as inhibitors of p38MAPK and ERK1/2 respectively. Rac2 mobilization was also shown to be prevented by the JNK1/2 inhibitor, SP600125. Taken together, the present results lead us to infer that these MAPKs are likely to play a relevant role in the targeting of Rac2, p47phox and p67phox to the plasma membrane. We have also observed that both Ang II-dependent Rac2 enhanced GTP-binding activity and translocation to plasma membrane in these cells were inhibited by wortmannin, a PI3K inhibitor. This indicates that PI3K is also likely to participate in Rac2 activation promoted by Ang II, in agreement with previous studies showing that in human neutrophils Rac2 becomes activated by fMLP in a PI3K-dependent manner (Akasaki et al. 1999).
C. difficile produces two toxins, which cause notable pathological disorders. Both, toxins A and B, translocate to the cytosol of target cells and inactivate small GTP-binding proteins, which include Rho, Rac and Cdc42. Inactivation of these substrates occurs through monoglycosylation of a single reactive threonine, which lies within their effector-binding loop and coordinates a divalent cation critical for GTP binding. By glycosylating small GTPases, toxins A and B cause actin condensation and cell rounding, which is followed by cell death (Just et al. 1995, Voth & Ballard 2005). Our data illustrate that toxin A dramatically inhibited both 
and ROS production and p38MAPK, ERK1/2 and JNK1/2 phosphorylation, in agreement with the pivotal role assigned to Rac2 in NADPH oxidase activation (Kim & Dinauer 2001, 2006) and in MAPK phosphorylation (Yu et al. 2001). These results indicate that Rac2 is necessary both for optimal activity of the assembled oxidase complex towards ROS production and MAPK-mediated signalling elicited by Ang II.
In order to obtain a more specific inhibitory effect on Rac2 activity, we also tested a chemical compound, NSC23766, identified as a Rac-specific small-molecule inhibitor (Gao et al. 2004). We have observed that this molecule evoked an inhibition of the Ang II-dependent phosphorylation of both p38MAPK, ERK1/2 and JNK1/2 and similar to that provoked by toxin A. These data are in agreement with a previous study showing that T cells from Rac2-deficient mice exhibit a decreased phosphorylation of p38MAPK and ERK1/2, in parallel to a reduced antigen-induced Ca2+ efflux (Yu et al. 2001).
In the search of other intracellular signalling events related to Rac2, increased intracellular Ca2+ seems sufficient to induce Rac2 activation in several epithelial cell lines (Price et al. 2003). However, fMLP-induced Rac2 activation is independent of intracellular Ca2+ in human neutrophils (Geijsen et al. 1999). From our data, a mutual interdependence between Rac2 and Ca2+ mobilization seems operative, since clostridial toxin A inhibited Ang II-stimulated cytosolic Ca2+ elevation and, conversely, Ang II-elicited Rac2 activation was blocked upon the chelation of intra- and extracellular Ca2+ using thapsigargin plus EGTA. These findings thus strongly suggest that cytosolic Ca2+ increase induced by Ang II is necessary for Rac2 activation. Our results are also consistent with recently published data showing that Ang II induces CaN activation in human neutrophils (El Bekay et al. 2003), and suggest the existence of a pathway linking Ca2+/CaN and MAPK signalling pathways, as it occurs in cardiac myocytes (Lim et al. 2001). This notion is further supported by our previous observations that Ang II enhances CaN expression and NF-
B DNA-binding activity in human neutrophils, which constitute two crucial intracellular signalling events (El Bekay et al. 2003). It has also been shown that Ang II modulates the cellular immune response through a CaN-dependent pathway (Nataraj et al. 1999). Interestingly, CaN has been shown to synergize with protein kinase C (PKC) to activate Rac in T cells (Werlen et al. 1998), and to engage in crosstalk with PKC and MAPK activation in transducing the Ang II stimulus in cardiomyocytes (Murat et al. 2000). Both proteins, Rac2 and PKC, are constituents of the machinery responsible for the activation of NADPH oxidase (Kwong et al. 1993, Bokoch & Knaus 1994) and are thus amenable to participate in Ang II-dependent signal transduction pathways. Also, it has been reported that the rapid CsA-induced depression of the respiratory burst in neutrophils stimulated by fMLP is due to a primary reduction of Ca2+ signalling (Nguyen et al. 1998). In this context, present observations illustrate for the first time the prevention of Ang II-dependent intracellular Ca2+ elevation through Rac2 inhibition (by C. difficile toxin A), with simultaneous decrease of CaN activity, events which are consistent with the view that Rac2 activation is coupled to the elicitation of intracellular oxidative stress by Ang II in human neutrophils.
Finally, our results also illustrate that MAPK activation and both Rac2 translocation to the plasma membrane and GTP-binding activity in Ang II-stimulated neutrophils are mutually regulated processes, given that specific pharmacological inhibitors acting on either the MAPK or the Rac2 pathway reciprocally abrogated activation of the other. Although the precise hierarchy is difficult to determine, the complex response elicited by Ang II in neutrophils suggests that the presumptive crosstalk between Rac2 and MAPK activation mechanisms is probably dependent on the participation of other putative signalling pathways, such as those mediated by PI3K, Ca2+ elevation or CaN activation.
Summarizing, present data provide a framework linking Rac2 activation to the signalling pathways switched on by Ang II in human neutrophils, and allow to extend our previous observations on the crosstalk between NADPH oxidase activation and other signalling pathways which become activated by phosphorylation. Elucidation of the specific mechanisms whereby Rac GTPases act to modulate the respiratory burst in neutrophils should provide insight on the precise role of the various signalling pathways involved in inflammatory processes, and in turn be useful to design novel therapies targeting the chronic inflammation of vessel walls typical of atherosclerosis and hypertension.
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Acknowledgements |
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References |
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Abo A, Boyhan A, West I, Thrasher AJ & Segal AW 1992 Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245. Journal of Biological Chemistry 267 16767–16770
Van Aelst L & D'Souza-Schorey C 1997 Rho GTPases and signaling networks. Genes and Development 11 2295–2322
Akasaki T, Koga H & Sumimoto H 1999 Phosphoinositide 3-kinase-dependent and -independent activation of the small GTPase Rac2 in human neutrophils. Journal of Biological Chemistry 274 18055–18059
Aktories K, Schmidt G & Just I 2000 Rho GTPases as targets of bacterial protein toxins. Biological Chemistry 381 421–426[CrossRef][Web of Science][Medline]
Ambruso DR, Knall C, Abell AN, Panepinto J, Kurkchubasche A, Thurman G, Gonzalez-Aller C, Hiester A, deBoer M, Harbeck RJ et al. 2000 Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. PNAS 97 4654–4659
Babior BM 2000 Phagocytes and oxidative stress. American Journal of Medicine 109 33–44[CrossRef][Web of Science][Medline]
El Bekay R, Álvarez M, Monteseirin J, Alba G, Chacón P, Vega A, Martín-Nieto J, Jiménez J, Pintado E, Bedoya FJ et al. 2003 Oxidative stress is a critical mediator of the angiotensin II signal in human neutrophils: involvement of mitogen-activated protein kinase, calcineurin, and the transcription factor NF-B. Blood 102 662–671
Benard V, Bohl BP & Bokoch GM 1999 Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. Journal of Biological Chemistry 274 13198–13204
El Benna J, Ruedi JM & Babior BM 1994 Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation. Journal of Biological Chemistry 269 6729–6734
Bokoch GM 1994 Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins. Current Opinion in Cell Biology 6 212–218[CrossRef][Web of Science][Medline]
Bokoch GM 2003 Biology of the p21-activated kinases. Annual Review of Biochemistry 72 743–781[CrossRef][Web of Science][Medline]
Bokoch GM & Knaus UG 1994 Ras-related GTP-binding proteins and leukocyte signal transduction. Current Opinion in Hematology 1 53–60[Medline]
Bozem M, Kuhlmann S, Blum R, Feick P & Schulz I 2000 Hormone-stimulated calcium release is inhibited by cytoskeleton-disrupting toxins in AR4-2J cells. Cell Calcium 28 73–82[CrossRef][Web of Science][Medline]
Brooks DP, Ohlstein EH & Ruffolo RR Jr 1999 Pharmacology of eprosartan, an angiotensin II receptor antagonist: exploring hypotheses from clinical data. American Heart Journal 138 246–251[CrossRef][Medline]
Carballo M, Márquez G, Conde M, Martín-Nieto J, Monteseirín J, Conde J, Pintado E & Sobrino F 1999 Characterization of calcineurin in human neutrophils. Inhibitory effect of hydrogen peroxide on its enzyme activity and on NF-B DNA binding. Journal of Biological Chemistry 274 93–100
Curnutte JT, Kuver R & Babior BM 1987 Activation of the respiratory burst oxidase in a fully soluble system from human neutrophils. Journal of Biological Chemistry 262 6450–6452
Didsbury J, Weber RF, Bokoch GM, Evans T & Snyderman R 1989 Rac, a novel ras-related family of proteins that are botulinum toxin substrates. Journal of Biological Chemistry 264 16378–16382
Diekmann D, Abo A, Johnston C, Segal AW & Hall A 1994 Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science 265 531–533
Dusi S, Donini M & Rossi F 1995a Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes. Biochemical Journal 308 991–994[Web of Science][Medline]
Dusi S, Donini M & Rossi F 1995b Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox. Biochemical Journal 314 409–412[Web of Science]
Etienne-Manneville S & Hall A 2002 Rho GTPases in cell biology. Nature 420 629–635[CrossRef][Medline]
Finkel T 1998 Oxygen radicals and signaling. Current Opinion in Cell Biology 10 248–253[CrossRef][Web of Science][Medline]
Gao Y, Dickerson JB, Guo F, Zheng J & Zheng Y 2004 Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. PNAS 101 7618–7623
Geijsen N, van Delft S, Raaijmakers JA, Lammers JW, Collard JG, Koenderman L & Coffer PJ 1999 Regulation of p21rac activation in human neutrophils. Blood 94 1121–1130
Gualberto A, Marquez G, Carballo M, Youngblood GL, Hunt SW, Baldwin AS & Sobrino F 1998 p53 transactivation of the HIV-1 long terminal repeat is blocked by PD 144795, a calcineurin-inhibitor with anti-HIV properties. Journal of Biological Chemistry 273 7088–7093
Haataja L, Groffen J & Heisterkamp N 1997 Characterization of RAC3, a novel member of the Rho family. Journal of Biological Chemistry 272 20384–20388
Hahn AW, Johas U, Buhler FR & Resink TJ 1994 Activation of human peripheral monocytes by angiotensin II. FEBS Letters 347 178–180[CrossRef][Web of Science][Medline]
Heyworth PG, Bohl BP, Bokoch GM & Curnutte JT 1994 Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavocytochrome b558. Journal of Biological Chemistry 269 30749–30752
Inanami O, Johnson JL & Babior BM 1998a The leukocyte NADPH oxidase subunit p47PHOX: the role of the cysteine residues. Archives of Biochemistry and Biophysics 350 36–40[CrossRef][Web of Science][Medline]
Inanami O, Johnson JL, McAdara JK, Benna JE, Faust LR, Newburger PE & Babior BM 1998b Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47PHOX on serine 303 or 304. Journal of Biological Chemistry 273 9539–9543
Inanami O, Yamamori T, Takahashi TA, Nagahata H & Kuwabara M 2001 ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-L-pyrroline-N-oxide. Free Radical Research 34 81–92[CrossRef][Web of Science][Medline]
Ito H, Takemory K & Suzuki T 2001 Role of angiotensin II type I receptor in the leukocytes and endothelial cells of brain microvessels in the pathogenesis of hypertensive cerebral injury. Journal of Hypertension 19 591–597[CrossRef][Web of Science][Medline]
Johnson JL, Park JW, Benna JE, Faust LP, Inanami O & Babior BM 1998 Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity. Journal of Biological Chemistry 273 35147–35152
Just I, Selzer J, Eichel-Streiber C & Aktories K 1995 The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile. Journal of Clinical Investigation 95 1026–1031[Web of Science][Medline]
Kim C & Dinauer MC 2001 Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways. Immunology 166 1223–1232
Kim C & Dinauer MC 2006 Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid. Journal of Leukocyte Biology 79 223–234
Knaus UG, Heyworth PG, Evans T, Curnutte JT & Bokoch GM 1991 Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 254 1512–1515
Koga H, Terasawa H, Nunoi H, Takeshige K, Inagaki F & Sumimoto H 1999 Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase. Journal of Biological Chemistry 274 25051–25060
Kwong CH, Malech HL, Rotrosen D & Leto TL 1993 Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins. Biochemistry 32 5711–5717[CrossRef][Medline]
Li Y, Zhu H, Kuppusamy P, Roubaud V, Zweier JL & Trush MA 1998 Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. Journal of Biological Chemistry 273 2015–2023
Lim HW, New L, Han J & Molkentin JD 2001 Calcineurin enhances MAPK phosphatase-1 expression and p38 MAPK inactivation in cardiac myocytes. Journal of Biological Chemistry 276 15913–15919
Mankelow TJ & Henderson LM 2001 Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites. Biochemical Journal 358 315–324[CrossRef][Web of Science][Medline]
Mansfield MA 1995 Rapid immunodetection on polyvinylidene fluoride membrane blots without blocking. Analytical Biochemistry 229 140–143[CrossRef][Web of Science][Medline]
Mazzone A, De Servi S, Ricevuti G, Mazzucchelli I, Fossati G, Pasotti D, Bramucci E, Angoli L, Marsico F & Specchia G 1993 Increased expression of neutrophil and monocyte adhesion molecules in unstable coronary artery disease. Circulation 88 358–363
Monteseirín J, Camacho MJ, Montaño R, Llamas E, Conde M, Carballo M, Guardia P, Conde J & Sobrino F 1996 Enhancement of antigen-specific functional responses by neutrophils from allergic patients. Journal of Experimental Medicine 183 2571–2579
Murat A, Pellieux C, Brunner HR & Pedrazzini T 2000 Calcineurin blockade prevents cardiac mitogen-activated protein kinase activation and hypertrophy in renovascular hypertension. Journal of Biological Chemistry 275 40867–40873
Nataraj C, Oliverio MI & Mannon RB 1999 Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway. Journal of Clinical Investigation 104 1693–1701[Web of Science][Medline]
Nguyen NS, Pulido SM & Ruegg UT 1998 Biphasic effects of cyclosporin A on formyl-methionyl-leucyl-phenylalanine stimulated responses in HL-60 cells differentiated into neutrophils. British Journal of Pharmacology 124 1774–1780[CrossRef][Web of Science][Medline]
Northup JK, Smigel MD & Gilman AG 1982 The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding. Journal of Biological Chemistry 257 11416–11423
Price LS, Langeslag M, ten Klooster JP, Hordijk PL, Jalink K & Collard JG 2003 Calcium signaling regulates translocation and activation of Rac. Journal of Biological Chemistry 278 39413–39421
Roberts AW, Kim C, Zhen L, Lowe JB, Kapur R, Petryniak B, Spaetti A, Pollock JD, Borneo JB, Bradford GB et al. 1999 Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 10 183–196[CrossRef][Web of Science][Medline]
Ross R 1999 Atherosclerosis-an inflammatory disease. New England Journal of Medicine 340 115–126
Sage SO, Pintado E, Mahaut-Smith MP & Merritt JE 1990 Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils. Biochemical Journal 265 915–918[Web of Science][Medline]
Schmitz U, Thommes K, Beier I, Wagner W, Sachinidis A, Dusing R & Vetter H 2001 Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck. Journal of Biological Chemistry 276 22003–22010
Segal AW & Abo A 1993 The biochemical basis of the NADPH oxidase of phagocytes. Trends in Biochemical Sciences 18 43–47[CrossRef][Web of Science][Medline]
Shimada K & Yakazi Y 1978 Binding sites for angiotensin II in human mononuclear leucocytes. Journal of Biochemistry 84 1013–1015
Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JA & Smith RD 1993 Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacological Reviews 45 205–251[Web of Science][Medline]
Touyz RM 2003 Recent advances in intracellular signalling in hypertension. Current Opinion in Nephrology and Hypertension 12 165–174[CrossRef][Web of Science][Medline]
Vega A, Chacón P, Monteseirín J, El Bekay R, Alvarez M, Alba G, Conde J, Martín-Nieto J, Bedoya FJ, Pintado E et al. 2004 A new role for monoamine oxidases in the modulation of macrophage-inducible nitric oxide synthase gene expression. Journal of Leukocyte Biology 75 1093–1101
Voth DE & Ballard JD 2005 Clostridium difficile toxins: mechanism of action and role in disease. Clinical Microbiology Reviews 18 247–263
Werlen G, Jacinto E, Xia Y & Karin M 1998 Calcineurin preferentially synergizes with PKC-theta to activate JNK and IL-2 promoter in T lymphocytes. EMBO Journal 17 3101–3111[CrossRef][Web of Science][Medline]
Williams DA, Tao W, Yang F, Kim C, Gu Y, Mansfield P, Levine JE, Petryniak B, Derrow CW, Harris C et al. 2000 Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency. Blood 96 1646–1654
Woolfolk EA, Equchi S, Ohtsu H, Nakashima H, Ueno H, Gerthoffer WT & Motley ED 2005 Angiotensin II-induced activation of p21-activated kinase 1 requires Ca2+ and protein kinase C {delta} in vascular smooth muscle cells. American Journal of Physiology. Cell Physiology 289 C1286–C1294[CrossRef]
Yang FC, Kapur R, King AJ, Tao W, Kim C, Borneo J, Breese R, Marshall M, Dinauer MC & Williams DA 2000 Rac2 stimulates Akt activation affecting BAD/Bcl-XL expression while mediating survival and actin function in primary mast cells. Immunity 12 557–568[CrossRef][Web of Science][Medline]
Yu H, Leitenberg D, Li B & Flavell RA 2001 Deficiency of small GTPase Rac2 affects T cell activation. Journal of Experimental Medicine 194 915–926
Received in final form 7 September 2007
Accepted 24 September 2007
Made available online as an Accepted Preprint 26 September 2007
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