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. 2005 Apr 4;145(4):460–468. doi: 10.1038/sj.bjp.0706205

Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways

Yu-Hsiang Kuan 1, Ruey-Hseng Lin 1,2, Lo-Ti Tsao 3, Chun-Nan Lin 4, Jih-Pyang Wang 3,5,*
PMCID: PMC1576157  PMID: 15806113

Abstract

  1. Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system.

  2. Pretreatment of neutrophils with pertussis toxin (1 μg ml−1), 50 μM 2′-amino-3′-methoxyflavone (PD 98059), or 1 μM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 μM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation.

  3. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3±0.3 μM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation.

  4. The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 μM 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 μM 2-aminoethyldiphenyl borate (2-APB). ART (30 μM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation.

  5. 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8±1.0 nM). ART increased the recruitment of PKC-α, -βI, and -βII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation.

  6. ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47phox, which was inhibited by GF 109203X and SB 203580.

  7. These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways in rat neutrophils.

Keywords: Artocarpol A, superoxide anion, intracellular free-Ca2+ concentration, mitogen-activated PK, PKC, neutrophils

Introduction

Neutrophils constitute the first line of host defense against the invasion of microorganisms through an array of microbicidal mechanisms including chemotaxis, phagocytosis, degranulation, and generation of reactive oxygen species, including superoxide anion and its toxic metabolites. The enzyme responsible for superoxide anion generation is NADPH oxidase. Inherited deficiencies of this enzyme result in chronic granulomatous disease, characterized by enhanced susceptibility to microbial infection (Smith & Curnutte, 1991). The mechanism of activation of superoxide anion generation in neutrophils is complex and incompletely understood. Upon neutrophils activation, the cytosol components of NADPH oxidase (p47phox, p67phox, p40phox, and Rac) are translocated to membrane and associated with the flavocytochrome b558, which contains FAD and heme redox centers, to form a functional oxidase complex, which catalyzes the reduction of oxygen to superoxide anion by using NADPH as the electron donor (Segal & Abo, 1993). Phosphorylation of p47phox, which induces a conformational change leading to the assembly of a functional oxidase, is a crucial step in oxidase activation (Babior et al., 2002). Previous reports demonstrated that the stimulation of neutrophils by receptor-binding ligands activates mitogen-activated PK (MAPK), including p38 MAPK and extracellular signal-regulated kinase (ERK, or p44/42 MAPK), during superoxide anion generation (El Benna et al., 1996). PLC is also activated by receptor-binding ligands, leading to the hydrolysis of PI(4,5)P2 to generate D-myo-inositol 1,4,5-trisphosphate (IP3), which increases in [Ca2+]i, and diacylglycerol, which activates PKC (Berridge, 1987). These two messengers act synergistically for superoxide anion generation.

The Moraceous plant Artocarpus rigida has been used as a folk veterinary remedy for the treatment of wounds in Asia. Artocarpol A (ART), a natural phenolic compound isolated from the root bark of this plant, was shown to inhibit phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion generation in rat neutrophils and TNF-α formation in macrophage-like cell lines in our previous report (Chung et al., 2000). In this study, we found to our surprise that ART alone stimulates superoxide anion generation in rat neutrophils, and we herein describe the involvement of PLC/Ca2+, PKC, and p38 MAPK signaling pathways in the ART-induced response by a combination of pharmacological and immunological approaches.

Methods

Isolation of neutrophils

Rat (Sprague–Dawley) blood was collected from the abdominal aorta and the neutrophils were purified by dextran sedimentation, centrifugation through Ficoll-Paque, and hypotonic lysis of erythrocytes (Wang et al., 1995). Purified neutrophils containing >95% viable cells were normally resuspended in Hanks' balanced salt solution (HBSS) containing 10 mM HEPES, pH 7.4, and 4 mM NaHCO3, and kept in an icebath before use. All experiments in the present study were performed under the guidelines of the Institutional Experimental Laboratory Animal Committee and were in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.

Measurement of superoxide anion generation

Superoxide anion generation was determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c (Wang et al., 1995). Briefly, the assay mixture contained neutrophils (2 × 106 cells) and 40 μM of ferricytochrome c in a final volume of 1.5 ml. The reference cuvette also received 17.5 U ml−1 of superoxide dismutase. Absorbance changes in the reduction of ferricytochrome c were monitored continuously at 550 nm in a double-beam spectrophotometer (Hitachi, U-3210).

Measurement of NADPH oxidase activity in a cell-free system

Neutrophils (1 × 108 cells ml−1) were treated with 2.5 mM diisopropyl fluorophosphate and disrupted in Tris buffer (10 mM Tris–HCl, pH 7.0, 0.34 M sucrose, 10 mM benzamidine, and 2 mM phenylmethylsulfonyl fluoride) by sonication (Wang et al., 1997a). After centrifugation (48,000 × g for 45 min at 4°C), supernatants were pooled as the cytosolic fraction, and pellets were collected and resuspended in Tris buffer, as the membrane fraction. Plasma membrane and cytosolic fractions were mixed in 1.5 ml of assay buffer (0.17 M sucrose, 2 mM NaN3, 1 mM MgCl2, 1 mM EGTA, 65 mM KH2PO4–NaOH, pH 7.0) supplemented with 10 μM FAD, 3 μM GTPγS, 40 μM ferricytochrome c, 50 μM NADPH, and activated by 100 μM arachidonic acid (AA). Superoxide dismutase was present in the reference cuvette. Absorbance changes in the reduction of ferricytochrome c were monitored.

Immunoblot analysis of MAPK phosphorylation

After stimulation of cells (2 × 107 cells 0.5 ml−1) with test drugs, the reactions were terminated by the addition of Laemmli sample buffer (Chang & Wang, 1999), and the solution was boiled for 5 min. Proteins (60 μg per lane) were resolved by 10% SDS–PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% (w v−1) nonfat dried milk and probed with anti-phospho-p44/42 MAPK or anti-phospho-p38 MAPK. The blots were then stripped and reprobed with anti-pan ERK or anti-p38 MAPK antibody to standardize protein loading in each lane. Detection was performed with the enhanced chemiluminescence reagent. Quantification was by densitometry.

Measurement of p38 MAPK activity

Neutrophils (2 × 107 cells) were lysed on ice in 0.2 ml of lysis buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 50 mM NaF, 10 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 0.1% 2-mercaptoethanol, 0.5 mM Na3VO4, 0.1 mM phenylmethylsulfonyl fluoride, and 1 μg ml−1 each of aprotinin, pepstatin, and leupeptin), and then clarified by centrifugation (Hsu et al., 2004). p38 MAPK in lysate was immunoprecipitated by the addition of rabbit polyclonal anti-p38 MAPK and 50% slurry of protein A-Sepharose beads, and the samples were rotated at 4°C overnight. The beads were washed twice in lysis buffer and once in kinase assay buffer (40 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM Na3VO4, and 2 mM dithiothreitol). The kinase activity of p38 MAPK was assayed using 10 μg of myelin basic protein (MBP) as substrate and 200 μM ATP in an assay volume of 20 μl, which was incubated at 30°C for 20 min. The reaction was stopped by the addition of Laemmli sample buffer and the solution was boiled for 5 min. Proteins were resolved by 12.5% SDS–PAGE, transferred to membranes, and probed with anti-phospho-MBP and p38 MAPK antibody. Detection was performed with the enhanced chemiluminescence reagent. Quantification was by densitometry.

Measurement of [Ca2+]i

Neutrophils (5 × 107 cells ml−1) were incubated with 5 μM 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester (fluo-3/AM) for 45 min at 37°C. After being washed, the cells were resuspended in HBSS to 5 × 106 cells ml−1. Fluorescence changes were monitored with a fluorescence spectrophotometer at 535 nM with excitation at 488 nm. [Ca2+]i was calibrated from the fluorescence intensity as follows:

graphic file with name 145-0706205e1.gif

where F is the observed fluorescence intensity. The values Fmax and Fmin were obtained at the end of experiments by the sequential addition of 0.33% Triton X-100 and 50 mM EGTA. The Kd was taken as 400 nM.

Determination of inositol phosphates

Neutrophils (3 × 107 cells ml−1) were incubated with myo-[3H]inositol (3,071 GBq mmol−1) at 37°C for 2 h (Wang et al., 1997a). Cells were then washed twice with HBSS containing 10 mM LiCl. After stimulation of cells with test drugs, the reactions were stopped by addition of CHCl3 : CH3OH (1 : 1, v v−1) mixture and acidification with 2.4 M HCl. The aqueous phase was removed, neutralized with 0.4 M NaOH, and then applied to an AG 1-X8 resin (format) column. Inositol-1-phosphate, inositol bisphosphate, and IP3 were eluted sequentially using 0.2, 0.4, and 1.0 M ammonium formate in 0.1 M formic acid, respectively, as eluents. Each of the column fractions was collected and counted in liquid scintillation counter.

Measurement of PKC and p47phox membrane translocation

Neutrophils (5 × 107 cells) were disrupted in 0.3 ml Tris buffer supplemented with 10 μg ml−1 each of pepstatin and leupeptin by sonication. After removing the unbroken cells, the lysate was then further centrifuged (100,000 × g for 30 min at 4°C) to collect pellets as membrane fractions. Membrane proteins were resolved by 7.5 and 10% SDS–PAGE for PKC and p47phox, respectively, and were transferred to polyvinylidene difluoride membranes. The polyvinylidene difluoride membranes were then probed with anti-PKC-α, anti-PKC-βI, anti-PKC-βII, or anti-p47phox antibody, and also with anti-CD88 antibody to standardize protein loading in each lane. Detection was performed with the enhanced chemiluminescence reagent. Quantification was by densitometry.

Measurement of PKC activity

Neutrophils (5 × 107 cells) were suspended in 0.5 ml of buffer A (20 mM Tris–HCl, pH 7.5, 0.25 M sucrose, 50 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 10 mM EGTA, and 0.01% leupeptin) and disrupted by sonication (Wang et al., 1995). After centrifugation (100,000 × g for 1 h at 4°C), the cytosolic fraction was removed. The kinase activity of PKC was assayed using 8 μg of MBP as substrate in an assay volume of 80 μl (50 μM ATP, 80 μg ml−1 of phosphatidylserine, 3 mM MgCl2, 25 mM Tris–HCl, pH 7.5, cytosolic fraction as PKC source, and test drugs), which was incubated at 25°C for 30 min, in the presence of 5 mM CaCl2. The reaction was stopped by the addition of Laemmli sample buffer and the solution was boiled for 5 min. Proteins were resolved by 15% SDS–PAGE, transferred to membranes, and probed with anti-phospho-MBP and anti-PKC-βI antibody. Detection was performed with the enhanced chemiluminescence reagent. Quantification was by densitometry.

Measurement of p47phox phosphorylation

Neutrophils (2 × 107 cells) were suspended in 0.5 ml of lysis buffer (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.05% SDS, 0.5% sodium deoxycholate, 2 mM Na3VO4, 1 mM dithiothreitol, and 1 μg ml−1 each of aprotinin, pepstatin, and leupeptin) for 30 min at 4°C, and then clarified by centrifugation. The supernatants were removed and incubated with a specific anti-p47phox antibody for 2 h at 4°C with constant mixing. Protein A-Sepharose was then added and incubated overnight at 4°C with constant mixing. The beads were sedimented and were washed twice in lysis buffer. After addition of Laemmli sample buffer, the solution was boiled for 5 min. Proteins were resolved by 10% SDS–PAGE, and immunoblot analysis with anti-phosphoserine antibody. The blots were then stripped and reprobed with anti-p47phox antibody. Detection was performed with the enhanced chemiluminescence reagent. Quantification was by densitometry.

Materials

ART (purity >99%) was purified as previously described (Chung et al., 2000). Dextran T-500, myo-[3H]inositol, and enhanced chemiluminescence reagent were purchased from Amersham Pharmacia Biotech (Buckinghamshire, U.K.). HBSS was obtained from Invitrogen (Carlsbad, CA, U.S.A.). 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580), 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X), 2′-amino-3′-methoxyflavone (PD 98059), calcein/AM, fluo-3/AM, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122), and 2-aminoethyldiphenyl borate (2-APB) were purchased from Calbiochem-Novabiochem (San Diego, CA, U.S.A.). Rabbit polyclonal antibodies to phospho-p44/42 MAPK and phospho-p38 MAPK were purchased from New England Biolabs (Beverly, MA, U.S.A.). Rabbit polyclonal antibodies to CD88, mouse polyclonal antibodies to p38 MAPK, and mouse monoclonal p47phox antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Mouse monoclonal pan ERK antibody was purchased from BD Biosciences Pharmingen (San Diego, CA, U.S.A.). Mouse monoclonal phosphoserine antibody was purchased from Alexis (Carlsbad, CA, U.S.A.). Rabbit polyclonal antibodies to phospho-MBP was obtained from Upstate Biotechnology (Lake Placid, NY, U.S.A.). Polyvinylidene difluoride membrane was obtained from Millipore (Bedford, MA, U.S.A.). Other chemicals were purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). The final percentage of dimethylsulfoxide (DMSO) in the reaction mixture was ⩽0.5% (v v−1).

Statistical analysis

Statistical analyses were performed using Student's t-test for two group comparisons test or ANOVA followed by the Bonferroni t-test for multigroup comparisons test; P<0.05 was considered significant for all tests. The curve estimation regression analysis with logarithmic model (SPSS) was used to calculate IC50 values.

Results and discussion

ART stimulated superoxide anion generation in neutrophil suspension and in a cell-free system

It is well documented that formyl-Met-Leu-Phe (fMLP) stimulates superoxide anion generation by binding to the G protein-coupled receptor. Several signaling pathways, initiated by receptor–ligand interaction, have been reported to be involved in the regulation of NADPH oxidase. Conversely, phorbol ester bypasses the membrane receptor, activates PKC directly (Castagna et al., 1982) and thus stimulates the superoxide anion generation. Exposure of neutrophils to ART evoked superoxide anion generation in a concentration-dependent manner (Figure 1a). Unlike fMLP, which stimulated a transient and rapid superoxide anion generation, ART and PMA induced a slow and long-lasting response, which was preceded by a lag. The finding that the ART-induced response was greatly diminished in the presence of diphenylene iodonium (DPI) (Figure 1b), the NADPH oxidase inhibitor (Cross & Jones, 1986), suggests that the superoxide anion generation was through the NADPH oxidase activation. Cell viability was ⩾90% during the incubation of cells with 30 μM ART at 37°C for 10 min as assessed in lactate dehydrogenase (LDH) release assay and in calcein fluorescence assay. These data imply that ART is probably not responsible for the anti-inflammatory effect of A. rigida, or alternatively that ART may exert its anti-inflammatory action through the inhibition of other biological functions of the inflammatory cells.

Figure 1.

Figure 1

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Stimulation of superoxide anion generation by ART. (a) Neutrophils were stimulated with the indicated concentrations of ART, 1 μM fMLP plus 5 μg ml−1 of cytochalasin B, or 1 nM PMA. Superoxide anion generation was continuously monitored at 550 nm. Traces are representative of 3–4 independent experiments. (b) The concentration dependence of the superoxide anion generation by stimulaton with ART for 10 min is expressed as means±s.d. of 4 independent experiments. In some experiments, cells were incubated with 1 μM diphenylene iodonium (DPI) for 3 min before stimulation with ART. (c) In a cell-free system, DMSO (as vehicle control), 30 μM ART, or 100 μM AA was added into the reaction mixture of cytosolic and membrane fractions. Values of superoxide anion generation are expressed as means±s.d. of 4 independent experiments. *P<0.05, as compared with the vehicle control value.

To determine whether ART directly stimulates NADPH oxidase activity, an experiment with NADPH oxidase in a cell-free system was performed. Addition of 100 μM AA to the mixture of neutrophil membrane and cytosolic fractions mimics the effect of phosphorylation of p47phox upon cell activation, and leads to the assembly and activation of NADPH oxidase (Fuchs et al., 1995). ART (30 μM) alone, however, only slightly evoked superoxide anion generation in the cell-free system (Figure 1c). It is plausible that ART acts via signaling-mediated mechanisms, while the direct stimulation of oxidase by ART might play a minor role in the superoxide anion generation in intact cells. Pretreatment of neutrophils with 1 μg ml−1 of pertussis toxin, a Gi/o protein inhibitor, for 90 min nearly abrogated the fMLP-induced superoxide anion generation but had no significant inhibitory effect on ART-induced response (data not shown), suggesting that the pertussis toxin-sensitive G protein is not linked to the effect of ART.

Role of ERK activation in ART-stimulated superoxide anion generation

Three distinct mammalian MAPKs have been identified: ERK (p44/42 MAPK), p38 MAPK, and c-Jun N-terminal kinase (JNK), each with different physiological roles. Cell activation induces a signaling cascade that leads to the activation of MAPK via phosphorylation of both tyrosine and threonine residues (Derijard et al., 1995). It has been reported that both ERK and p38 MAPK, but not JNK, phosphorylate p47phox (El Benna et al., 1996). To address the relevance of the ERK signal pathway to ART activation, we first examined the effect of the ERK signal blocker on ART-induced superoxide anion generation. U0126 and PD 98059, both MEK1/2 inhibitors (Favata et al., 1998), had no significant inhibition of fMLP- and ART-induced superoxide anion generation (Figure 2a). The role of ERKs in formyl peptide-stimulated superoxide anion generation is controversial (Avdi et al., 1996; Kuroki & O'Flaherty, 1997; Tamura et al., 1999). This variation may arise from the differences in experimental conditions. Nevertheless, the present results suggest that the ERK signaling pathway is not linked to ART-induced superoxide anion generation in rat neutrophils. Moreover, unlike fMLP, ART did not stimulate ERKs phosphorylation during the period of superoxide anion generation based on the immunoblot analysis with anti-phospho-p44/42 MAPK antibodies (Figure 2b). The absence of ERK phosphorylation in ART stimulation also points to an ERK-independent mechanism.

Figure 2.

Figure 2

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Effect of U0126 and PD 98059 on ART-stimulated superoxide anion generation, and the effect of ART on ERK phosphorylation. (a) Neutrophils were pretreated with DMSO, the indicated concentrations of U0126 or PD 98059 for 3 min at 37°C before stimulation with 30 μM ART or 1 μM fMLP plus 5 μg ml−1 of cytochalasin B for 10 min. Values are expressed as means±s.d. of 6–8 independent experiments. (b) Neutrophils were incubated with DMSO, fMLP plus cytochalasin B for 1 min, or with ART for the indicated time. ERK phosphorylation was detected by immunoblot analysis using anti-phospho-p44/42 MAPK. The blots above were then stripped and reprobed with anti-pan ERK. The ratio of immunointensity between the ERK and the phosphorylation of ERK is shown. Values are expressed as means±s.d. of 3−4 independent experiments. *P<0.05, as compared with the vehicle control value (lane 2).

Role of p38 MAPK activation in ART-stimulated superoxide anion generation

Pretreatment of neutrophils with SB 203580, a p38 MAPK inhibitor (Cuenda et al., 1995), diminished the fMLP-stimulated superoxide anion generation, confirming a previous report (Zu et al., 1998), and also inhibited ART-induced response (IC50 value of 4.3±0.3 μM) (Figure 3a). These results appear to reflect the involvement of the p38 MAPK signaling pathway. Furthermore, artocarpol stimulated p38 MAPK phosphorylation, as assessed by immunostaining with anti-phospho-p38 MAPK antibody, in a concentration- and time-dependent manner (Figure 3b). A significant increase in band immunointensity was observed at 10 μM ART for 10 min reaction time and at 30 μM ART for 4 min reaction time, which is compatible with the results of superoxide anion generation as shown in Figure 1a. Therefore, the immunoprecipitation of cell lysates of ART-stimulated cells with p38 MAPK-specific antibody and the kinase activity was assessed. Both fMLP and ART stimulated p38 MAPK activity (both P<0.05), which was greatly attenuated by SB 203580 (30 μM) treatment, at a concentration that greatly blocked superoxide anion generation, either before or after the immunoprecipitation of p38 MAPK (Figure 3c). It is likely that the p38 MAPK signaling pathway is implicated in ART-induced superoxide anion generation. There are at least four members of the p38 MAPK family, including p38 MAPK-α, -β, -γ, and -δ isoforms, in which p38 MAPK-α and -δ are expressed in neutrophils (Hale et al., 1999). SB 203580 inhibited p38 MAPK-α and -β, but not -γ and -δ (Wang et al., 1997b), implying that ART stimulated superoxide anion generation via p38 MAPK-α activation.

Figure 3.

Figure 3

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Effect of SB 203580 (SB) on ART-stimulated superoxide anion generation, and the effect of ART on p38 MAPK activation. (a) Neutrophils were pretreated with DMSO (as control) or the indicated concentrations of SB for 3 min at 37°C before stimulation with 30 μM ART or 1 μM fMLP plus 5 μg ml−1 of cytochalasin B for 10 min. Values are expressed as means±s.d. of 3–5 independent experiments. *P<0.05, as compared with the corresponding control values. (b) Cells were incubated with DMSO, 30 μM ART for the indicated time or with the indicated concentrations of ART for 10 min. The ratio of immunointensity between the p38 MAPK and the phosphorylation of p38 MAPK is shown. Values are expressed as means±s.d. of 3–4 independent experiments. *P<0.05, as compared with the vehicle control value (lane 1). (c) Cells were preincubated with DMSO or 30 μM SB for 3 min before stimulation with fMLP plus cytochalasin B for 1 min (SB+fMLP) or with ART for 10 min (SB+ART). Cell lysates were immunoprecipitated with anti-p38 MAPK antibody, and assayed for MAPK activity using MBP as substrate. In some experiments, the cell lysates from fMLP- or ART-activated cells were immunoprecipitated with anti-p38 MAPK antibody, and assayed for MAPK activity in the presence of SB (fMLP+SB or ART+SB). The ratio of immunointensity between the p38 MAPK and the phosphorylation of MBP is shown. Values are expressed as means±s.d. of 3–4 independent experiments. *P<0.05, as compared to the vehicle control value (lane 1); #P<0.05, as compared to the corresponding activated control values (lanes 2 and 5).

Role of the PLC/Ca2+ signaling pathway in ART-stimulated superoxide anion generation

It is conceivable that the PLC/Ca2+ signal pathway is implicated in fMLP-induced superoxide anion generation. Like fMLP, the ART-induced response was nearly abrogated in a Ca2+-free medium (Figure 4a). Thus, we sought to determine whether the PLC/Ca2+ signal pathway could be involved. The finding that U-73122 (1 μM), an inhibitor of PLC-coupled processes, fully inhibited the fMLP- and ART-induced superoxide anion generation confirms this possibility. Moreover, a comparable inhibition was evident with 2-APB (100 μM), a cell-permeant IP3 receptor blocker (Maruyama et al., 1997), providing further support for the notion (Figure 4a). Recent evidence indicates that the principal antagonistic effect of 2-APB is on Ca2+ entry rather than Ca2+ release (Bootman et al., 2002). Nevertheless, stimulation of superoxide anion generation by ART has been shown to be Ca2+-dependent.

Figure 4.

Figure 4

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Effect of U-73122 and 2-APB on ART-stimulated superoxide anion generation, and the effect of ART on [Ca2+]i changes and cellular IP3 formation. (a) Neutrophils were pretreated with DMSO (as control), 1 μM U-73122, or 100 μM 2-APB in a Ca2+ (1 mM)-containing medium or with DMSO in a Ca2+-free medium (−Ca2+) for 3 min at 37°C before stimulation with 30 μM ART or 1 μM fMLP plus 5 μg ml−1 of cytochalasin B for 10 min. Values are expressed as means±s.d. of 3–5 independent experiments. *P<0.05, as compared with the corresponding control values. (b) Fluo-3-loaded cells were stimulated with ART (arrow) in a Ca2+ (1 mM)-containing medium (upper panel) or a Ca2+-free medium (lower panel). In some experiments, cells were preincubated with 10 μM U-73122 for 1 min before stimulation with ART in a Ca2+-free medium. The traces are representative of 3 independent experiments. (c) Myo-[3H]inositol-loaded neutrophils were exposed to DMSO (as vehicle control) or fMLP for 10 s, or to ART for 15 s at 37°C. In some experiments, myo-[3H]inositol-loaded cells were preincubated with 1 μM U-73122 for 1 min before stimulation with ART. The radioactivity of IP3 generation was counted. Values are expressed as means±s.d. of 5–6 independent experiments. *P<0.05, as compared to the vehicle control value. #P<0.05.

In fact, ART (30 μM), at effective concentration, stimulated a slow rate of [Ca2+]i rise in a Ca2+-containing medium, preceded by a 20–40 s lag, reached a maximal level at 1–1.5 min after stimulation, then gradually declined as assessed by the increase in fluo 3 fluorescence (Figure 4b, upper panel). Although the increase in [Ca2+]i is neither sufficient nor always required for superoxide production, this messenger acts synergistically with other signals for superoxide anion generation. Cell activation causes an increase in [Ca2+]i due to the release of Ca2+ from internal stores and/or an influx of Ca2+ across the plasma membrane. In the absence of external Ca2+, ART stimulated a small [Ca2+]i rise (Figure 4b, lower panel), suggesting that ART increased internal Ca2+ release and external Ca2+ entry.

The role of IP3 in mediating Ca2+ release from internal stores is firmly established, but the mechanisms responsible for Ca2+ entry are less clear. The finding that U-73122 (10 μM) fully blocked the ART-induced Ca2+ rise implies the involvement of the PLC/IP3 signal. This notion was further supported by the result that the exposure of myo-[3H]inositol-loaded neutrophils to ART for 15 s significantly increased the IP3 formation and this effect was inhibited by U-73122 (1 μM) (Figure 4c). The IP3 formation coincided with the onset of [Ca2+]i rise by ART. The mammalian PLC isoenzymes can be divided into four major families: PLC-β, -γ, -δ, and -ɛ (Rhee, 2001), which are regulated through different mechanisms. PLC-β2 has been reported to be the major PLC isoform in neutrophils (Li et al., 2000). However, a 10-fold higher concentration of U-73122 was required to diminish ART-induced Ca2+ release than the fMLP-induced Ca2+ response (Wang, 1996) and the ART-induced IP3 formation. At present, we cannot clearly explain the difference in the U-73122 effects. One possibility is that a putative messenger other than IP3 is probably also involved in ART-induced Ca2+ signals, which can be blocked by the higher concentration of U-73122, but this remains to be investigated.

Role of PKC activation in ART-stimulated superoxide anion generation

Based on the observation that ART stimulated the PLC/Ca2+ signaling pathway, we assumed the detection of PKC signals through the concomitantly generated diacylglycerol in ART-stimulated cells. Our previous report demonstrated that rat neutrophils express conventional isoforms (PKC-α and -β), novel isoforms (PKC-δ, -ɛ, and -θ), atypical isoforms (PKC-ι), and PKC-μ (Chang & Wang, 1999). Phosphorylation of p47phox in vitro is mediated by all families of PKC isoforms (Regier et al., 1999), although it is not known which specific isoform of PKC is responsible in vivo. It has been reported that the depletion of PKC-β inhibited fMLP-induced phosphorylation of p47phox and superoxide anion generation in differentiated HL-60 cells (Korchak et al., 1998), and neutrophils from PKC-β knockout mice markedly reduced the level of superoxide production (Dekker et al., 2000). Antisense-PKC-α-transfected THP-1 cells showed a decreased release of superoxide anion (Dieter & Schwende, 2000). Moreover, the requirement of PKC-δ in the regulation of NADPH oxidase activity in neutrophils has been reported recently (Brown et al., 2003). The respiratory burst caused by fMLP was inhibited by GF 109203X (0.1 μM), a broad PKCs inhibitor, in neutrophils (Tamura et al., 1999). The critical role of PKC in ART-induced response is supported by data that GF 109203X was potent against ART (IC50 value of 7.8±1.0 nM) (Figure 5a).

Figure 5.

Figure 5

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Effect of GF 109203X (GF) on ART-stimulated superoxide anion generation, and the effect of ART on PKC activation. (a) Neutrophils were pretreated with DMSO (as control) or the indicated concentrations of GF for 3 min at 37°C before stimulation with 30 μM ART or 1 μM fMLP plus 5 μg ml−1 of cytochalasin B for 10 min. Values are expressed as means±s.d. of 3–4 independent experiments. *P<0.05, as compared with the corresponding control values. (b) Neutrophils were treated with 30 μM ART for the indicated time or with the indicated concentrations of ART for 7 min at 37°C. The membrane-associated PKC was immunoblotted with the specific monoclonal antibody to PKC-α, -βI, or -βII. The polyvinylidene difluoride membranes were also probed with anti-CD88 antibody as the loading control. The fold increase in the immunointensity as compared with the corresponding control values (lane 1) is shown. Values are expressed as means±s.d. of 6–7 independent experiments. *P<0.05, as compared with the corresponding control values. (c) Cytosolic PKC was incubated with DMSO or 0.1 μM GF for 3 min at 37°C before the addition of DMSO, 30 nM PMA or ART for 30 min at 25°C, using MBP as substrate. The ratio of immunointensity between the PKC-βI and the phosphorylation of MBP is shown. Values are expressed as means±s.d. of 4 independent experiments. *P<0.05, as compared with the vehicle control value (lane 1); #P<0.05, as compared with the corresponding activated control values (lanes 2 and 4).

In general, PKC activation is characterized by association of cytosolic PKC to the membrane fraction in intact cells. Since the ART stimulation of superoxide anion generation is via a Ca2+-dependent mechanism, it is likely that the Ca2+-independent PKC play a minor role. Therefore, immunoblot analysis was carried out to determine the subcellular distribution of Ca2+-dependent PKC (α, βI, and βII). Only a slight immunointensity for these PKC isoforms was detected in the membrane fractions of unstimulated cells. Addition of ART resulted in the membrane recruitment of PKCs in a time- and concentration-dependent manner (Figure 5b). A significant increase in band immunointensity of PKC-α was observed at 30 μM ART for 2 min reaction time, reached a maximal level at 7 min, and then declined, whereas the band intensity of PKC-βI increased at 5 min, reached a maximal level at 7 min and remained elevated to 10 min after stimulation. The band immunointensity of PKC-βII was reached a maximal level at 2 min after ART stimulation and remained elevated to 10 min at least. The kinetics of PKC activation is compatible with the results of superoxide anion generation as shown in Figure 1a, supporting the possibility that PKC regulates ART-induced response. Moreover, that the superoxide anion generation in response to ART stimulation in cells was accompanied by an increase in PKC activation is further supported by the observation that the addition of 30 μM ART significantly induced cytosolic PKC activation in the presence of Ca2+ (Figure 5c), which was fully inhibited by 0.1 μM GF 109203X. Pretreatment of cells with ART desensitized the PKC activation stimulated by the subsequent addition of PMA that may account for the inhibition of PMA-induced superoxide anion generation in our previous report (Chung et al., 2000).

ART stimulates the phosphorylation of p47phox and the translocation of p47phox to the plasma membrane

Stimulation of neutrophils by fMLP and phorbol ester results in extensive phosphorylation of p47phox (DeLeo et al., 1999). p47phox is phosphorylated on multiple sites through the action of several serine/threonine kinases including PKC, MAPK, p21rac-activated PK, and a novel phosphatidic acid-activated PK (El Benna et al., 1996; Regier et al., 1999), resulting in a conformational rearrangement, exposing SH3 domain, proline-rich regions, and a PX domain those together mediate interactions both with cytochrome b558 and p67phox (Lambeth, 2000). Thus, p47phox appears to serve as an adaptor protein, providing a platform for the assembly of a functional oxidase. Like PMA, ART (30 μM) induced the serine phosphorylation of p47phox (Figure 6a), which was attenuated by 0.1 μM GF 109203X, implying the participation of PKC signaling. Moreover, ART evoked the membrane association of p47phox (Figure 6b), which was inhibited by GF 109203X and 30 μM SB 203580, whereas the 2 μM PMA-induced translocation of p47phox to the plasma membrane was suppressed by GF 109203X but not by SB 203580. These data imply the involvement of PKC and p38 MAPK signaling in ART-stimulated recruitment of p47phox to membrane. Previous reports demonstrated that SB 203580 did not prevent the fMLP-induced translocation of p47phox/p67phox to the plasma membrane of neutrophils (Lal et al., 1999) but inhibited that of the serum-opsonized zymosan (OZ)-induced response (Yamamori et al., 2002).

Figure 6.

Figure 6

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ART stimulated the phosphorylation of p47phox and the translocation of p47phox to the plasma membrane. (a) Neutrophils were treated with DMSO or 0.1 μM GF109203X (GF) for 3 min at 37°C before stimulation or no stimulation with 30 μM ART or 2 μM PMA for 10 min. The p47phox in cell lysates were immunoprecipitated with a specific antibody to p47phox. Phosphorylation of p47phox was detected by immunoblot analysis using anti-phosphoserine antibody. The blots above were then stripped and reprobed with anti-p47phox antibody. The ratio of immunointensity between the p47phox and the phosphorylation of p47phox is shown. Values are expressed as means±s.d. of 3–4 independent experiments. *P<0.05, as compared with the vehicle control value (lane 1); #P<0.05, as compared with the corresponding activated control values (lanes 2 and 3). (b) Neutrophils were treated with DMSO, 30 μM SB 203580 (SB) or GF for 3 min at 37°C before stimulation with ART or PMA for 10 min. The membrane-associated p47phox was immunoblotted with anti-p47phox antibody. The polyvinylidene difluoride membranes were also probed with anti-CD88 antibody as the loading control. The fold increase in the immunointensity as compared with the vehicle control value (lane 1) is shown. Values are expressed as means±s.d. of 6–7 independent experiments. *P<0.05, as compared with the vehicle control value; #P<0.05, as compared with the corresponding activated control values (lanes 2 and 5).

In conclusion, ART stimulates superoxide anion generation in rat neutrophils probably through the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways to promote the phosphorylation of p47phox and the assembly of the NADPH oxidase in the plasma membrane, which leads to release of the superoxide anion to the extracellular compartment. The direct activation of NADPH oxidase may play a minor role in the ART-induced response.

Acknowledgments

This study was supported in part by grants from the National Science Council (NSC-91-2320-B-075A-004) and Taichung Veterans General Hospital (TCVGH-927303C), Taiwan, Republic of China.

Abbreviations

AA

arachidonic acid

2-APB

2-aminoethyldiphenyl borate

ART

artocarpol A

ERK

extracellular signal-regulated kinase

fluo3/AM

1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester

fMLP

formyl-Met-Leu-Phe

GF 109203X

2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide

HBSS

Hanks' balanced salt solution

IP3

D-myo-inositol 1,4,5-trisphosphate

MAPK

mitogen-activated PK

MBP

myelin basic protein

PD 98059

2′-amino-3′-methoxyflavone

PMA

phorbol 12-myristate 13-acetate

SB 203580

4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole

U0126

1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene

U-73122

1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione

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