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(Journal of Leukocyte Biology. 2001;69:659-665.)
© 2001 by Society for Leukocyte Biology

Signal transduction pathways for activation of extracellular signal-regulated kinase by arachidonic acid in rat neutrophils

Ling-Chu Chang* and Jih-Pyang Wang*,{dagger}

* Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan 407, and
{dagger} Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan 404, Republic of China

Correspondence: Jih P. Wang, Department of Education and Research, Taichung Veterans General Hospital, 160 Chung Kang Road, Sec. 3, Taichung, Taiwan 407, Republic of China. E-mail: w1994{at}vghtc.vghtc.gov.tw


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ABSTRACT
 
Phosphorylation of extracellular signal-regulated kinase (ERK) in response to arachidonic acid (AA) was rapid and transient, peaking at 1 min and disappearing after 3 min, and it was accompanied by an increase in ERK activity in rat neutrophils. We examined the upstream regulation of AA-stimulated ERK activation using one of the following signaling pathway inhibitors to pretreat rat cells: the ERK kinase inhibitor U0126 or PD98059, the Gi/o inhibitor pertussis toxin (PTX), the tyrosine kinase inhibitor genistein, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or LY294002, the Ca2+ chelator 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, or the phospholipase C (PLC) inhibitor U73122. All of these inhibitors attenuated AA-induced ERK activation. Activation of ERK was also effectively attenuated by the cyclooxygenase and lipoxygenase inhibitor BW755C and by the leukotriene biosynthesis inhibitor MK886, but the cyclooxygenase inhibitor indomethacin did not attenuate ERK activation. After exposing cells to three distinct protein kinase C (PKC) inhibitors, we found that Gö6976 significantly attenuated ERK phosphorylation but potentiated ERK activity. Neither Gö6983 nor GF109203X affected AA-induced responses. These data suggest that the lipoxygenase metabolite(s) produced mediates AA-stimulated ERK activation and that this effect is upstream regulated by PT-sensitive G protein, nonreceptor tyrosine kinase, PI3K, and PLC/Ca2+ signaling pathways in rat neutrophils.

Key Words: phosphorylation • kinase activity • protein kinases • lipoxygenase • phosphatidylinositol 3-kinase • phospholipase C


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INTRODUCTION
 
Extracellular signal-regulated kinases (ERKs), a serine/threonine kinase family of the mitogen-activated protein kinase (MAPK) superfamily, may play a crucial role in cell growth and differentiation [1 ]. Several isoforms of ERK have been described, and at least two of them, ERK1 (p44 MAPK) and ERK2 (p42 MAPK), are expressed in neutrophils [2 ]. In mammalian cells, ERKs are activated by MAPK/ERK kinases (MEKs), a family with dual specificity for tyrosine and threonine phosphorylation within the TEY motif. In turn, serine phosphorylation by MEK kinase or Raf [3 ] activates MEKs. G protein, phospholipase C (PLC), tyrosine kinase, phosphatidylinositol 3-kinase (PI3K), or protein kinase C (PKC) may regulate Raf in some mammalian cell types [3 ]. The activated ERKs phosphorylate and regulate other protein kinases or stimulate a variety of transcription factors [4 ]. Stimulation of neutrophils by chemoattractants and granulocyte-macrophage colony-stimulating factor has been shown to activate ERKs through a Raf/MEK/MAPK pathway [5 , 6 ]. It has been proposed that ERKs mediate the important signaling pathways in neutrophils regulating aggregation, phagocytosis, degranulation, respiratory burst, and the release of cytokines [7 8 9 ].

The action of phospholipase A2 at the membrane phospholipids [10 ] releases arachidonic acid (AA), which plays a pivotal role in signal transduction pathways activated by a variety of extracellular stimuli [11 , 12 ]. AA and its metabolites mediate a number of biological processes of activated neutrophils including adhesion, chemotaxis, aggregation, degranulation, and respiratory burst [7 , 13 , 14 ]. However, the mechanisms of activation by AA remain unclear. AA and its metabolites have been shown to activate ERK in a variety of cell types, including rat liver [15 ], rabbit renal proximal tubule epithelium [12 ], rat vascular smooth muscle cells [16 , 17 ], and human neutrophils [6 , 7 , 18 ]. However, the mechanism of ERK activation by AA is not yet fully understood. Characterization in human neutrophils has demonstrated that AA or its lipoxygenase metabolites stimulate ERK activation through pertussis toxin (PTX)-sensitive [6 , 7 ], Ca2+-independent [6 ], and PKC-dependent [18 ] or -independent [7 ] signaling pathways.

In this study, we characterized the signaling pathways of ERK activation by AA in rat neutrophils. We examined the effects of selective inhibitors of signaling pathways on ERK activation, using immunoblot analysis of ERK phosphorylation with anti-phospho-p44/42 MAPK antibodies and an ERK activity assay.


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MATERIALS AND METHODS
 
Materials
Rabbit polyclonal phospho-p44/42 MAPK antibody was obtained from New England Biolabs (Beverly, MA). Mouse monoclonal antibodies against ERK1, ERK2, and pan-ERK were purchased from Transduction Laboratories (Lexington, KY). Anti-phospho-myelin basic protein (MBP), MAPK 1 and 2 antibodies, and MBP were purchased from Upstate Biotechnology (Lake Placid, NY). PTX was purchased from Research Biochemicals International Laboratories (Natick, MA). Hank’s balanced salt solution was obtained from Gibco Life Technologies (Grand Island, NY). AACOCF3, U73122, MK886, and LY294002 were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). GF109203X, Gö6976, Gö6983, PD98059, and wortmannin were purchased from Calbiochem-Novabiochem Co. (San Diego, CA). U0126 was obtained from Promega (Madison, WI). 1,2-Bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-tetraacetyoxmethyl ester (BAPTA-AM) was purchased from Molecular Probes Inc. (Eugene, OR). Polyvinylidene difluoride membrane was obtained from Millipore Corp. (Bedford, MA). Dextran T-500 and enhanced chemiluminescence reagents were obtained from Amersham-Pharmacia Biotech (Buckinghamshire, United Kingdom). BW755C was generously provided by Wellcome Research Laboratories (Kent, United Kingdom). All other reagents and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Preparation of rat neutrophils
Neutrophils were isolated from Sprague-Dawley rats as described previously [19 ]. Briefly, fresh whole blood was obtained from the abdominal aorta and immediately mixed with ethylenediaminetetraacetate. The neutrophils were purified by dextran sedimentation followed by centrifugation through Ficoll-Hypaque and hypotonic lysis of erythrocytes. Purified neutrophils of >95% viability were suspended in Hank’s balanced salt solution containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4) and 4 mM NaHCO3, and kept in an ice bath before use.

Detection of ERK phosphorylation
The ERK phosphorylation assay was carried out using a modification of a previously described method [20 ]. Neutrophils (2 x 107 cells/mL) were incubated with the vehicle or test drugs at 37°C for the indicated time before stimulation with AA. Reactions were terminated by adding a stop solution (20% trichloroacetic acid, 1 mM phenylmethylsulfonyl fluoride, 7 µg/mL of aprotinin and pepstatin, 2 mM N-ethylmaleimide, 10 mM NaF, 2 mM Na3VO4, and 2 mM p-nitrophenyl phosphate). Protein pellets were washed twice with ice-cold stop solution (without trichloroacetic acid), lysed in Laemmli sample buffer, and boiled for 5 min. Proteins (60 µg/lane) were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. After the membranes were blocked with 5% fat-free dried milk in TBST (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Tween 20) for 1 h, they were probed with anti-phospho-p44/42 MAPK antibodies in TBST with 0.1% fat-free dried milk at room temperature for 1 h and then incubated in sheep anti-rabbit immunoglobulin G conjugated with horseradish peroxidase for 1 h. The phospho-p44/42 MAPKs were visualized by chemiluminescence and quantified by densitometry. To standardize protein loading in each lane, blots were incubated with strip buffer (62.5 mM Tris-HCl [pH 6.8], 100 mM ß-mercaptoethanol, 2% sodium dodecyl sulfate) at 50°C for 30 min, followed by reprobing with antibodies against ERK1 and ERK2 or pan-ERK in TBST with 0.1% fat-free dried milk. The membranes were incubated in secondary antibody and treated with chemiluminescence as in the above procedure.

Immunoprecipitation of ERK and ERK activity
Neutrophils (2 x 107 cells) were lysed for 30 min on ice in 0.2 mL of lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM ethylenediaminetetraacetate, 1 mM EGTA, 0.1% Triton X-100, 50 mM NaF, 10 mM sodium ß-glycerophosphate, 5 mM sodium pyrophosphate, 0.1% ß-mercaptoethanol, 0.5 mM Na3VO4, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin, pepstatin and leupeptin). Samples were clarified by centrifugation at 4°C for 10 min at 12,000 g. ERKs were immunoprecipitated by the addition of 4 µL of rabbit polyclonal anti-MAPK 1/2 and 20 µL of a 50% slurry of protein A beads in 0.2 ml of lysis buffer, and the samples were rotated at 4°C for 2 h. The beads were washed twice in 0.5 mL of lysis buffer and twice in 0.2 mL of kinase assay buffer (20 mM morpholinopropane sulfonic acid [pH 7.2], 25 mM sodium ß-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM dithiothreitol). The kinase activity of ERK was assayed using 10 µg of 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 boiled for 5 min. Samples were separated using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with anti-phospho-MBP or ERK2 monoclonal antibodies.


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RESULTS
 
AA-stimulated ERK activation and the roles of G protein and tyrosine kinase
Based on immunoblot analysis with anti-phospho-p44/42 MAPK antibodies, we found that AA induced ERK phosphorylation in a concentration- and time-dependent manner (Fig. 1A B ). After exposure of cells to AA for 1 min, ERK phosphorylation was evident at all concentrations of AA >= 10 µM. When cells were treated with 30 µM AA for various times, ERK phosphorylation peaked at 1 min, declined thereafter, and was eliminated after 3 min. Thus, rat neutrophils were stimulated with 30 µM AA for 1 min in the subsequent ERK activation experiments. The AA trifluoromethyl ketone analog AACOCF3 (at concentrations up to 100 µM) failed to stimulate ERK phosphorylation (data not shown).



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  		Figure 1. Concentration- and time-dependent AA-stimulated ERK phosphorylation in rat neutrophils. Cells were (A) incubated with various concentrations of AA for 1 min or (B) stimulated with 30 µM AA for various time intervals. Phosphorylation of ERK was detected by probe with anti-phospho-p44/42 MAPK antibodies as described in Materials and Methods. To confirm each loading, the blots were reprobed with anti-ERK1 and anti-ERK2 antibodies. Similar results were obtained from five independent experiments.

Cells were pretreated with the selective MEK inhibitor [21 , 22 ], either U0126 (0.1 or 1 µM) for 10 min or PD98059 (1 or 10 µM) for 30 min. Both reduced AA-induced ERK phosphorylation (about 90% inhibition for 1 µM U0126 and 80% inhibition for 10 µM PD98059) (Fig. 2A B ). Parallel changes in kinase activity were also demonstrated in neutrophils. Pretreatment of cells for 30 min with 30 µM genistein, a general tyrosine kinase inhibitor [23 ], or for 2 h with 1 µg/mL PTX, an endotoxin that inhibits Gi/o protein activation, also attenuated the ERK activation by AA (Fig. 2C 2D) . None of the four inhibitors alone had any effect on the basal intensity of ERK phosphorylation (data not shown).



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  		Figure 2. Effects of U0126, PD98059, genistein, and PTX on ERK activation. Cells were preincubated with (A) 0.1 or 1 µM U0126 for 10 min, (B) 1 or 10 µM PD98059 for 30 min, (C) 30 µM genistein for 30 min, or (D) 1 µg/mL of PT for 2 h before stimulation with 30 µM AA for 1 min. Phosphorylation of ERK was detected by probe with anti-phospho-p44/42 MAPK antibodies as described in Materials and Methods. To confirm each loading, the blots were reprobed by anti-pan-ERK antibodies. Lysates were also immunoprecipitated with anti-MAPK 1/2 antibodies and assayed for ERK activity using MBP as substrate (see Materials and Methods). To confirm each loading, the blots were probed by anti-ERK2 antibodies. Similar results were obtained from four independent experiments.

Role of AA metabolites and PI3K in AA-stimulated ERK activation
To assess the role of AA metabolites in ERK activation, cells were treated for 10 min with 10 µM BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase [24 ], or 30 nM MK886, a potent inhibitor of leukotriene synthesis [25 ], before addition of AA. AA-stimulated ERK phosphorylation was attenuated under these conditions (about 85% and 70% inhibition, respectively) (Fig. 3A C ). However, pretreatment with 10 µM indomethacin, a cyclooxygenase inhibitor, had no effect on the AA-induced response (Fig. 3B) . None of the three inhibitors alone had any effect on the basal intensity of ERK phosphorylation (data not shown). Comparable effects were also observed in the kinase activity assay in neutrophils (Fig. 3D) .



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  		Figure 3. Effects of BW755C (BW), indomethacin (Indo), and MK886 (MK) on AA-stimulated ERK activation. Cells were preincubated with (A) 10 µM BW, (B) 10 µM Indo, or (C) 30 nM MK for 10 min before stimulation with 30 µM AA for 1 min. Phosphorylation of ERK was detected by probing with anti-phospho-p44/42 MAPK antibodies as described in Materials and Methods. To confirm each loading, the blots were reprobed with anti-pan-ERK antibodies. (D) Lysates were also immunoprecipitated with anti-MAPK 1/2 antibodies and assayed for ERK activity using MBP as substrate (see Materials and Methods). To confirm each loading, the blots were probed with anti-ERK2 antibodies. Similar results were obtained from three to four independent experiments.

Two PI3K inhibitors, wortmannin and LY294002 [26 , 27 ], were used to study the role of PI3K in AA activation of ERK. Cells pretreated for 10 min with wortmannin or LY294002 showed concentration-dependent inhibition of AA-stimulated ERK phosphorylation (Fig. 4A B ). The 50% inhibitory concentration (IC50) value for wortmannin was < 0.1 µM (61% inhibition at 0.1 µM) and for LY294002 was 4.7 µM. Neither PI3K inhibitor alone had a significant effect on the basal intensity of ERK phosphorylation. The kinase activity of ERK in neutrophils was also greatly reduced, by either 0.1 µM wortmannin or 30 µM LY294002 (Fig. 4C 4D) .



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  		Figure 4. Effects of wortmannin and LY294002 on ERK activation induced by AA. (A, B) Cells were preincubated with various concentrations of wortmannin or LY294002 for 10 min before stimulation with 30 µM AA for 1 min or without stimulation. Phosphorylation of ERK was detected by probe with anti-phospho-p44/42 MAPK antibodies as described in Materials and Methods. To confirm each loading, the blots were reprobed with anti-pan-ERK antibodies. (C, D) Cells were preincubated with 0.1 µM wortmannin or 30 µM LY294002 for 10 min before stimulation with 30 µM AA for 1 min or without stimulation. Lysates were immunoprecipitated with anti-MAPK 1/2 antibodies and assayed for ERK activity using MBP as a substrate (see Materials and Methods). To confirm each loading, the blots were probed with anti-ERK2 antibodies. Similar results were obtained from three to four independent experiments.

Role of PLC/Ca2+ and PKC in AA-stimulated ERK activation
Treatment of cells for 10 min with 1 µM U73122, a PLC inhibitor [28 ], greatly reduced AA-stimulated ERK phosphorylation (89% inhibition). Cells preincubated for 1 h with 10 µM BAPTA-AM, a cell permeable Ca2+-chelator [29 ], also decreased the response to AA stimulation (81% inhibition) (Fig. 5A B ). Parallel inhibition of kinase activity by U73122 and 1,2-bis(O-aminopheoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) was also observed. BAPTA-AM is hydrolyzed by cytosolic esterases and is trapped intracellularly as the active chelator BAPTA. Neither U73122 nor BAPTA alone had a significant effect on the basal level of ERK phosphorylation (data not shown).



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  		Figure 5. Effects of U73122 and BAPTA on ERK activation induced by AA. Cells were pretreated with (A) 1 µM U73122 for 10 min or (B) 10 µM BAPTA-AM (BAPTA) for 1 h before stimulation with 30 µM AA for 1 min or without stimulation. Phosphorylation of ERK was detected by probing with anti-phospho-p44/42 MAPK antibodies as described in Materials and Methods. To confirm each loading, the blots were reprobed with anti-pan-ERK antibodies. Lysates were also immunoprecipitated with anti-MAPK 1/2 antibodies and assayed for ERK activity using MBP as substrate (see Materials and Methods). To confirm each loading, the blots were probed with anti-ERK2 antibodies. Similar results were obtained from three to four independent experiments.

The role of the PKC signaling pathway in AA-stimulated ERK activation was explored using three selective PKC inhibitors, Gö6976, Gö6983, and GF109203X [30 , 31 ]. As shown in Figure 6A , preincubation for 10 min with Gö6976 reduced the AA-stimulated ERK phosphorylation in a concentration-dependent manner with an IC50 value of about 7.8 µM. Significant inhibition was observed at concentrations of Gö6976 >= 1 µM. Unexpectedly, Gö6976 did not inhibit but instead potentiated the AA-stimulated ERK activity. Pretreatment of cells for 10 min with up to 10 µM Gö6983 or GF109203X failed to inhibit ERK activation by AA (Fig. 6B 6C 6D) . None of the three PKC inhibitors alone affected the basal level of phosphorylation of ERK.



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  		Figure 6. Effects of Gö6976, Gö6983, and GF109203X on ERK activation induced by AA. Cells were preincubated with (A) various concentrations of Gö6976, (B) 10 µM Gö6983, or (C) 10 µM GF109203X for 10 min before stimulation with 30 µM AA for 1 min or without stimulation. Phosphorylation of ERK was detected by probe with anti-phospho-p44/42 MAPK antibodies as described in Materials and Methods. To confirm each loading, the blots were reprobed with anti-pan-ERK antibodies. (D) Cells were preincubated with Gö6976, Gö6983, or GF109203X (each at 10 µM) for 10 min before stimulation with 30 µM AA for 1 min or without stimulation. Lysates were immunoprecipitated with anti-MAPK 1/2 antibodies and assayed for ERK activity using MBP as a substrate (see Materials and Methods). To confirm each loading, the blots were probed with anti-ERK2 antibodies. Similar results were obtained from three to four independent experiments.


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DISCUSSION
 
Our data demonstrated that AA stimulates ERK activation in rat neutrophils. Rapid and transient ERK phosphorylation in response to AA is similar to the response induced by formylmethionyl-leucyl-phenylalanine (fMLP) (data not shown). A similar response has also been reported for AA-stimulated ERK activity and AA metabolite-stimulated ERK mobility shift in human neutrophils [6 , 7 ]. However, Hii et al. [18 ] demonstrated that AA caused a slow, long-lasting increase in ERK activity, which peaked at 15 min in human neutrophils. The reason for this discrepancy in results is not clear, but it might be attributable to differences in the experimental or assay conditions. The effect of AA on ERK might not be a nonspecific lipid effect, since many cis-6 and trans-polyunsaturated fatty acids have failed to stimulate ERK activity [7 ]. Our finding that AACOCF3, a trifluoromethyl ketone analog of AA, had no effect on ERK phosphorylation suggests that the free-carboxyl group of AA plays an essential role in the activation of ERK and confirms the specific effect of AA. Because the mechanism of activation of ERK by AA is incompletely understood, we examined the upstream regulation of ERK activation by AA using selective inhibitors of signal transduction pathways.

Chemoattractant-stimulated ERK activation in human neutrophils may involve the activation of Ras, Raf, and MEK [5 , 6 ]. Stimulation of ERK by AA, accompanied by Raf and MEK activation, has also been reported in human neutrophils [7 ]. PD98059 and U0126 are selective and noncompetitive inhibitors of MEK [21 , 22 ]. Inhibition of AA-induced response by both MEK inhibitors indicates that ERK phosphorylation is also induced by the MEK/ERK pathway in rat neutrophils.

Previous studies have shown that AA stimulates GTP{gamma}S loading of the heterotrimeric G proteins in human neutrophil membrane fractions [11 ]. Our data show that PTX treatment greatly reduced AA-stimulated ERK activation in rat neutrophils. This is consistent with the involvement of PTX-sensitive G protein in the ERK cascade in human neutrophils [6 , 7 ]. In the G protein-coupled, receptor-mediated signaling pathway leading to activation of ERK in cardiac myocytes, the adapter protein Shc is phosphorylated by Src-related tyrosine kinases [32 ]. In addition, it has been reported that the mechanism of AA-stimulated ERK activation in renal proximal tubule epithelial cells involves the tyrosine phosphorylation of Shc by some nonreceptor tyrosine kinases followed by activation of Ras [33 ]. Our results demonstrate that AA-stimulated ERK activation is inhibited by the general tyrosine kinase inhibitor genistein [23 ], which suggests that activated nonreceptor tyrosine kinase is also involved in the AA-induced response in rat neutrophils.

It appears that AA does not act directly via a G protein-linked cell surface receptor but acts indirectly by serving as a substrate for the generation of intracellular metabolites. Therefore, we investigated the role of AA metabolites in AA-stimulated activation of ERK. It is well known that AA is metabolized by different enzymes into a variety of derivatives. In rat neutrophils, AA is metabolized to prostaglandins D2 and E2, 6-keto prostaglandin F1{alpha}, thromboxane A2, leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (HETE) [34 ]. Previous reports have indicated that ERK activation in response to AA is cyclooxygenase independent and requires AA metabolism to 5(S)-HPETE, 5-oxoHETE, and 5-HETE but not to leukotrienes in human neutrophils [6 , 7 ]. In addition, the responses to lipoxygenase metabolites are coupled through PTX-sensitive G protein. Although LTB4 itself stimulates human neutrophils to activate ERK [6 ], HETE probably does not operate through the LTB4 receptor. In this study, the dual cyclooxygenase and lipoxygenase inhibitor BW755C [24 ] and the leukotriene biosynthesis inhibitor MK886 [25 ] both greatly attenuated AA-stimulated ERK activation in rat neutrophils. However, the cyclooxygenase inhibitor indomethacin did not affect the AA-stimulated response. These results support the hypothesis that ERK activation by AA is mediated by the metabolites of lipoxygenase but not the metabolites of cyclooxygenase.

It has been reported that AA activation of ERK in rabbit vascular smooth muscle cells and renal proximal epithelium requires cytochrome P450 [12 , 17 ] and that the exogenous addition of 20-HETE and 15-HETE stimulates ERK via Ras activation [16 , 17 ]. Thus, cytochrome P450-dependent metabolites may provide another Ras-dependent pathway upstream of ERK activation. A cytochrome P450-dependent mechanism of AA metabolism has also been reported in human neutrophils [35 ]. In our study, BW755C eliminated ERK activation. Since the formation of cytochrome P450 AA metabolites was not affected by BW755C [35 ], this excludes cytochrome P450 metabolites from a role in AA activation of ERK. Since activated ERK directly phosphorylates cytosolic phospholipase A2 in activated cells [36 ], the activation of ERK by AA may amplify phospholipase A2 activity, resulting in the release of additional AA by a positive-feedback mechanism.

PI3K participation in ERK cascade activation has been reported after the stimulation of G protein-coupled receptors in COS-7 and CHO-K1 cells [37 , 38 ]. Neutrophils contain two classes of PI3K, the classical tyrosine kinase-regulated PI3K{alpha} (p85/p110{alpha}) and a novel Gß{gamma}-regulated PI3K{gamma} (p101/p110{gamma}) [39 ]. Activation of the nonreceptor tyrosine kinase Lyn by fMLP occurs via a G protein-mediated pathway, which in turn forms a complex with PI3K in human neutrophils [40 ]. In this study, two PI3K inhibitors, wortmannin and LY294002 [26 , 27 ], were used to assess the role of PI3K in AA-stimulated ERK activation. Both PI3K inhibitors attenuated the AA-stimulated response, indicating the participation of a PI3K pathway in ERK activation. The less potent inhibitory effect of LY294002, as compared with wortmannin, has also been reported in the inhibition of fMLP-stimulated ERK activation in HL60 cells [41 ].

There is conflicting evidence regarding the role of PLC/Ca2+ in G protein-mediated MEK/ERK activation. One report says that ERK activation by fMLP was greatly attenuated (85%) by U73122 in HL60 cells [41 ]. Others report that the depletion of [Ca2+]i by BAPTA resulted in partial inhibition of MEK activation by fMLP in human neutrophils [42 ] or ERK activation by platelet-activating factor in guinea pig neutrophils [43 ]. Yet another report describes a Ca2+-independent mechanism for ERK activation in human neutrophils stimulated with the lipoxygenase metabolites 5-oxoHETE and LTB4 [6 ]. The reasons for this discrepancy are not clear but might be attributable to differences in experimental conditions. The latter study [6 ] utilized Fura-2-loaded human neutrophils and detected ERK mobility changes as a measure of the extent of phosphorylation. In this study, both the PLC inhibitor U73122 [28 ] and the cell-permeable Ca2+ chelator BAPTA-AM [29 ] attenuated AA-stimulated ERK activation, suggesting the involvement of the PLC/Ca2+ signaling pathway in rat neutrophils.

A recent report indicated that PKC{alpha} may directly phosphorylate and activate Raf both in vitro and in NIH 3T3 fibroblasts and implicated PKC{alpha} as an upstream regulator of the ERK cascade [44 ]. AA is important for activating PKC [45 ], but we are uncertain whether AA stimulates ERK activation through a PKC-dependent or -independent signaling pathway. Previous reports have indicated that exposure of vascular smooth muscle cells [16 ], rat liver cells [15 ], or human neutrophils [18 ] to AA results in ERK activation in a PKC-dependent fashion. In contrast, ERK activation by AA is independent of PKC in rabbit renal proximal tubule epithelial cells [12 ] and human neutrophils [7 ]. The conflicting evidence on the role of PKC in AA-stimulated ERK activation in human neutrophils may arise from the utilization of different kinase assay conditions and PKC inhibitors. Our previous report demonstrated that rat neutrophils express PKC{alpha}, ß, {delta}, {varepsilon}, {theta}, µ, {iota}/{lambda}, and {zeta}, although {lambda} and {zeta} are barely detectable [46 ]. In this study, we used three PKC inhibitors, Gö6976, Gö6983, and GF109203X [30 , 31 ]. Gö6976 preferentially inhibits PKC{alpha}, ß, and µ; Gö6983 inhibits PKC{alpha}, ß, {gamma}, {delta}, and {zeta}; and GF109203X inhibits PKC{alpha}, ß, {delta}, {varepsilon}, µ, and {zeta} in in vitro kinase assays. Thus both Gö6983 and GF109203X inhibit a broader spectrum of PKC isoforms than Gö6976. Yet in the current study, only the latter significantly attenuated AA-induced ERK phosphorylation, and, contrary to expectations, it potentiated AA-stimulated ERK activity. Immunoblot analysis of AA-induced ERK phosphorylation in the lysate, prepared from cells pretreated with Gö6976 before immunoprecipitation, was also greatly enhanced (data not shown). The reasons for this discrepancy are not clear but might be attributable to differences in experimental processes or the composition of the stop solution in Western blot analysis and the lysis buffer in immunoprecipitation analysis, which may have changed the character of Gö6976. Therefore, studies of the role of PKC in ERK activation carried out with Gö6976 should be evaluated cautiously. Because the other two PKC inhibitors failed to significantly alter the AA-stimulated ERK activation, it is plausible that the PKC signaling pathway is not involved.

In conclusion, this study demonstrated that AA induces a rapid and transient stimulation of ERK phosphorylation in rat neutrophils. Based on the effects of selective inhibitors on signaling pathways, we concluded that AA-stimulated ERK activation occurs through a cyclooxygenase- and cytochrome P450-independent pathway, is mediated by its lipoxygenase metabolites, and is upstream-regulated not only by PTX-sensitive G protein as previously described in human neutrophils but also by nonreceptor tyrosine kinase, PI3K, and PLC/Ca2+ as demonstrated in this study. PKC probably does not contribute to AA-stimulated ERK activation in rat neutrophils.


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ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the National Science Council (NSC88-2314-B-075A-002) and Taichung Veterans General Hospital (TCVGH-887323C), Republic of China.

Received August 21, 2000; revised November 14, 2000; accepted November 16, 2000.


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