Leukotriene D4 Activates Mitogen-activated Protein Kinase through a Protein Kinase Calpha -Raf-1-dependent Pathway in Human Monocytic Leukemia THP-1 Cells*

Mitsunobu Hoshino, Takashi Izumi, and Takao ShimizuDagger

From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Leukotriene D4 (LTD4) is a major lipid mediator involved in inflammatory and allergic disorders including bronchial asthma. Despite its potent biological activity, little is known about the receptor and intracellular signaling pathways. Here we analyzed the signal transduction mechanisms through LTD4 receptors using human monocytic leukemia THP-1 cells. When these cells were stimulated with LTD4, intracellular calcium concentration was increased and mitogen-activated protein kinase (MAP kinase) was activated severalfold. This activation was inhibited by staurosporine or GF109203X treatment or abolished by protein kinase C depletion. Cytosolic protein kinase Calpha was translocated to the membrane, and Raf-1 was activated by LTD4 treatment in a similar time course. LTD4-induced Raf-1 activation was diminished by protein kinase C depletion in the cells. A chemotactic response of THP-1 cells toward LTD4 was observed which was inhibited by pertussis toxin (PTX) pretreatment. Thus, LTD4 has at least two distinct signaling pathways in THP-1 cells, a PTX-insensitive mitogen-activated protein kinase activation through protein kinase Calpha and Raf-1 and a PTX-sensitive chemotactic response. This cellular signaling can explain in part the versatile activities of LTD4 in macrophages under inflammatory and allergic conditions.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Leukotriene D4 (LTD4),1 a metabolite of arachidonate via the 5-lipoxygenase pathway, has biological activities such as bronchial constriction and increase in vascular permeability. It is related to the onset and progression of bronchial asthma and other allergic disorders (1-3). Although the cell-surface LTD4 receptor is presumed to be a seven-transmembrane receptor coupling to a heterotrimeric GTP-binding protein(s) (G protein(s)) (4, 5), it has not yet been purified or cDNA-cloned. Moreover, cellular events that evoke various biological effects have not been clarified. We report here that in human monocytic THP-1 cells, LTD4 increased the intracellular calcium concentration and activated MAP kinase through a PTX-insensitive G protein, PKCalpha and Raf-1 pathway. Furthermore, THP-1 cells showed a chemotaxis toward LTD4 through a PTX-sensitive G protein. Thus, LTD4 receptors have at least two signal transduction pathways in THP-1 cells, the PTX-insensitive MAP kinase cascade and the PTX-sensitive pathway leading to chemotaxis.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells and Materials-- LTD4 was obtained from Cascade Biochem (Reading, UK). A human monocytic leukemia cell line, THP-1 cells, obtained from American Type Culture Collection (Rockville, MD), was kept in RPMI 1640 medium (Nissui Pharmaceutical Co, Tokyo) supplemented with 10% fetal bovine serum (Moregate, Melbourne, Australia) at 37 °C, 5% CO2. Serum-starved cells were obtained by incubation for 24 h in the medium without fetal bovine serum. TPA was purchased from Sigma. Staurosporine, GF109203X, and bovine fibronectin were from Wako (Osaka, Japan). [3H]LTD4 (specific activity, 8,880 GBq/mmol) was from NEN Life Science Products (Tokyo). [gamma -32P]ATP (specific activity, 222 TBq/mmol), transfer membrane (Hybond-N+), p42/p44 MAP kinase enzyme assay (RPN 84), anti-PKCalpha antibody, and ECL detection reagents were from Amersham Corp. (Buckinghamshire, UK). Probond resin was from Invitrogen (NV Leek, The Netherlands). Protein A-agarose CL-4B and glutathione-Sepharose 4B were from Pharmacia Biotech (Uppsala, Sweden). BAPTA/AM and Fura-2/AM were from Dojin (Kumamoto, Japan). Pertussis toxin (PTX) was from Funakoshi (Tokyo), and wortmannin was from Kyowa Medex Co (Tokyo). Tyrphostin, methyl 2,5-dihydroxycinnamate, and genistein were from Life Technologies, Inc. Anti-Raf-1 antibody (C-12) and anti-PKCdelta antibody (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Erk2 antibody was from Upstate Biotechnology (Lake Placid, NY). Bovine serum albumin fraction V (fatty acid-free) was from Bayer (Kankakee, IL). Polycarbonate filters with 8-µm pores were from Neuroprobe (Cabin John, MD). A Diff-Quick staining kit was from International Reagents Corp. (Kobe, Japan). MK-571 was from Biomol (Plymouth Meeting, PA). Protein determination reagents (BCA kit) were from Pierce. Other chemicals and reagents were of analytical grade. ONO-1078 was a kind gift from Ono Pharmaceutical Co (Osaka, Japan). A His-tagged MAP kinase kinase and a GST fusion kinase-negative (GST-kn) MAP kinase were generous gifts from Drs. Y. Gotoh and E. Nishida (Kyoto University).

Measurement of Intracellular Ca2+ Concentrations-- THP-1 cells suspended in Hepes-Tyrode's-BSA buffer (Hepes-Tyrode's-BSA buffer contains the following: 140 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 0.49 mM MgCl2, 12 mM NaHCO3, 5.6 mM D-glucose, 0.37 mM NaH2PO4, 10 mM Hepes-NaOH (pH 7.4), containing 0.1% (w/v) of fatty acid-free BSA) were loaded with 3 µM Fura-2/AM at 37 °C for 1 h, washed twice, and resuspended in Hepes-Tyrode's-BSA buffer to a concentration of 1.0 × 107 cells/ml. After 5 min of stirring at 37 °C, ligands were added, and elevations in intracellular Ca2+ concentrations were measured using a spectrofluorometer (model CAF-100, JASCO, Tokyo), with emission wavelength set at 510 nm and excitation wavelengths at 340 and 380 nm.

Measurement of Cyclic AMP-- Cells were seeded on 24-well dishes 24 h prior to start of experiments. After aspirating off the medium, the cells were incubated for 20 min in Hepes-Tyrode's-BSA buffer containing 0.5 mM 3-isobutyl-1-methylxanthine at 37 °C. Next, the cells were exposed for 20 min to 50 µM forskolin in the presence of ligands. The supernatant was aspirated after centrifugation, and the reaction was terminated by adding 200 µl of 30% perchloric acid. After a 20-min incubation, the supernatant was collected by centrifugation. The concentrations of cyclic AMP (cAMP) in the supernatant were determined using a radioimmunoassay kit from Yamasa (Chiba, Japan).

MAP Kinase Assay-- Aliquots (3.0 × 106 cells in 1 ml of Hepes-Tyrode's-BSA buffer) of serum-starved cells were stimulated with ligands at 37 °C. The supernatant was aspirated after centrifugation, and the reaction was terminated by adding an ice-cold lysis buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM beta -glycerophosphate, 1 mM sodium orthovanadate, 2 mM EGTA, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml aprotinin (final concentration) in a total volume of 200 µl. Aliquots were then assayed for the MAP kinase as described previously (6). When examining the effects of PTX, cells were incubated overnight with 100 ng/ml PTX.

PKC Translocation Analysis-- Aliquots (3.0 × 106 cells in 1 ml of Hepes-Tyrode's-BSA buffer) of serum-starved cells were stimulated with ligands at 37 °C. The supernatant was aspirated after centrifugation, and the reaction was terminated by adding an ice-cold lysis buffer (20 mM Tris-HCl (pH 8.0), 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin). After sonication at 30 watts for 30 s and removal of the nuclei and unbroken cells by centrifugation (1,000 × g for 15 min), the supernatant was further centrifuged at 100,000 × g for 60 min to obtain the cytosol (supernatant) and the membrane (pellet) fractions. The pellet was extracted with the lysis buffer containing 1% Triton X-100 and sonicated. After leaving on ice for 30 min, the membrane extract was obtained by centrifugation at 10,000 × g for 30 min. Proteins were separated on a 10% SDS-PAGE gel and transferred onto Hybond-N+ membranes. After blocking the membrane with 10% BSA, immunoblot analysis was performed with an anti-PKC antibody. Blots were visualized with ECL detection reagents.

Raf-1 Analysis-- A His-tagged MAP kinase kinase and a GST-kn MAP kinase were expressed in Escherichia coli and purified by column chromatography as described (7). Aliquots (3.0 × 106 cells in 1 ml of Hepes-Tyrode's-BSA buffer) of serum-starved cells were stimulated with ligands at 37 °C. The supernatant was aspirated after centrifugation, and the reaction was terminated by adding an ice-cold lysis buffer containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 20 mM beta -glycerophosphate, 1 mM sodium orthovanadate, 2 mM EGTA, 2 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10% glycerol, 1% Triton X-100, and 0.1% SDS. The homogenates were centrifuged at 10,000 × g for 15 min, and the supernatants were precleared with 70 µl of 1:1 slurry of protein A-Sepharose beads. They were then incubated for 2 h with 20 µl of an anti-Raf-1 antibody and 70 µl of 1:1 slurry of protein A-Sepharose beads at 4 °C. The immune complex on the beads was washed three times with the lysis buffer, twice with a solution containing 100 mM Tris-HCl (pH 8.0) and 0.5 M LiCl, and once with a solution containing 20 mM Tris-HCl (pH 8.0), 2 mM EGTA, and 10 mM MgCl2. This preparation was referred to as the immunoprecipitate.

To assay Raf-1 kinase toward an exogenous substrate, the His-tagged MAP kinase kinase (1.5 µg) and the GST-kn MAP kinase (2.5 µg) were incubated with the immunoprecipitate at 30 °C in a 24-µl solution containing 20 mM Tris-HCl (pH 8.0), 2 mM EGTA, 10 mM MgCl2, and 100 µM [gamma -32P]ATP (370 kBq). The reaction was stopped by adding Laemmli's sample buffer and boiling. After SDS-PAGE separation, the radioactivity incorporated in the His-tagged MAP kinase kinase or the GST-kn MAP kinase was detected and quantified using an image analyzer (Fujix BAS 2000).

Chemotaxis Assay-- Polycarbonate filters with 8-µm pores were coated with 10 µg/ml fibronectin in phosphate-buffered saline for 60 min. A dry coated filter was placed on a 96-blind well chamber containing the indicated amounts of LTD4, and the THP-1 cells (200 µl, 1.0 × 106 cells) were added to the top wells. The ligand solution and cell suspension were prepared in the same buffer (RPMI 1640 medium containing 0.1% BSA). After incubation at 37 °C in 5% CO2 for 4 h, the filter was disassembled. Cells on the filter were fixed with methanol and stained with a Diff-Quick staining kit. The upper side of the filter was then scraped free of cells. The number of cells that had migrated to the lower side was determined by measuring optical densities at 595 nm using a 96-well microplate reader (Bio-Rad model 3550) (8).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Occurrence of LTD4 Receptors in THP-1 Cells-- The membrane fractions of THP-1 cells were examined in a binding assay as described (4, 9). [3H]LTD4-specific binding to THP-1 cell membranes was estimated, and the value for the equilibrium dissociation constant (Kd) was 1.8 nM and for the maximum number of binding sites (Bmax) was 31 fmol/mg protein. LTD4 induced a rapid increase of intracellular calcium concentration in THP-1 cells in a dose-dependent manner (Fig. 1A). ONO-1078, a specific LTD4 receptor antagonist (10, 11), inhibited the response (Fig. 1A). 100 nM LTD4 induced an increase in intracellular calcium concentration about 250 nM above basal levels (Fig. 1B). Pretreatment of the cells with PTX reduced the LTD4-elicited calcium response by 30% and the ATP-elicited response by 20%, whereas it did not inhibit the thrombin-elicited calcium response (data not shown). Next, we determined the effects of LTD4 on cAMP accumulation. LTD4 dose-dependently inhibited the forskolin-induced cAMP accumulation with an IC50 value of 100 nM and did not increase cAMP level in the absence of forskolin (data not shown). This inhibitory effect of LTD4 was completely blocked by PTX or by ONO-1078 pretreatment (data not shown), indicating that the LTD4 receptor couples to PTX-sensitive G protein(s). These data show that LTD4 receptors are functionally active in THP-1 cells and couple to both PTX-sensitive and -insensitive G proteins.


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Fig. 1.   Increase of intracellular calcium ion induced by LTD4 in THP-1 cells. A, calcium responses induced by addition of LTD4 (1, 10, and 100 nM) or 100 µM ONO-1078, followed by 10 nM LTD4, as monitored by Fura-2/AM. Ligands were applied at the points indicated by the arrows. B, a dose-dependent curve of LTD4-induced increases of intracellular calcium measured as described under "Experimental Procedures." Data are representative of three independent experiments, which gave essentially identical results.

MAP Kinase Activation after LTD4 Stimulation-- We then investigated LTD4-induced signal transduction mechanisms in THP-1 cells. After stimulation with 100 nM LTD4, the bands of MAP kinase (Erk2) were time-dependently gel-shifted in the immunoblot analysis (Fig. 2A). This gel shift became most evident 5 min after LTD4 stimulation. MAP kinase assay with an epidermal growth factor receptor peptide as a substrate showed a severalfold activation with 100 nM LTD4 (data not shown). As shown in Fig. 2B, the EC50 value of MAP kinase activation was about 0.1 nM, and the activation was saturable at a higher dose (higher than 1 nM). When cells were pretreated with MK-571 or ONO-1078, both specific antagonists for the LTD4 receptor (Cys-LTR1, see Ref. 12), LTD4 did not elicit the MAP kinase activation (Fig. 2B). These data suggest that the LTD4-elicited MAP kinase activation is Cys-LTR1-mediated.


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Fig. 2.   LTD4-elicited MAP kinase activation. A, a gel shift of MAP kinase (Erk2) in the immunoblot analysis. Serum-depleted cells were stimulated with 100 nM LTD4 for the indicated times or to TPA (100 ng/ml for 20 min as a positive control) at 37 °C. Cell lysates were separated by SDS-PAGE, transferred to a membrane, and probed with an anti-Erk2 antibody. The upper bands show phosphorylated Erk2 (P-Erk2) and the lower bands (Erk2) show basal Erk2. Data are representative of three independent experiments, which gave essentially identical results. B, a dose-dependent curve of LTD4-elicited MAP kinase activation. Serum-depleted cells were stimulated with various amounts of LTD4 for 5 min at 37 °C. The MAP kinase activities of all the cell lysates were assayed as described (6). A triangle and a hexagon represent 1 nM LTD4-elicited MAP kinase activation pretreated with 50 µM MK-571 or 50 µM ONO-1078-pretreated cells, respectively. Symbols and vertical bars denote the mean and S.E., respectively (n = 3).

Effects of Inhibitors on MAP Kinase Activation-- Next, effects of various inhibitors on LTD4-elicited MAP kinase activation were examined. When the cells were pretreated overnight with PTX, the LTD4-elicited MAP kinase activation was slightly inhibited (Fig. 3). Treatment of the cells with BAPTA/AM (intracellular calcium chelator) inhibited the enzyme activity by half (Fig. 3). Pretreatment with a nanomolar order of wortmannin (phosphatidylinositol 3-kinase inhibitor) (13-15) or with several nonreceptor type tyrosine kinase inhibitors (i.e. tyrphostin, methyl 2,5-dihydroxycinnamate, and genistein) did not inhibit the LTD4-elicited MAP kinase activation (data not shown). 10 µM or 30 µM GF109203X or 300 nM staurosporine, both inhibitors of protein kinase C (PKC), inhibited LTD4-elicited MAP kinase activation (Fig. 3). We thus considered that PKC might be involved in LTD4-elicited MAP kinase activation. To confirm this notion, cells were pretreated overnight with TPA to deplete endogenous PKCs. MAP kinase activation by LTD4 was completely blocked by this treatment (Fig. 3). Thus, in THP-1 cells, LTD4 receptors mediate MAP kinase activation via a signaling pathway that may depend on the activity of PKCs.


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Fig. 3.   Inhibition of LTD4-elicited MAP kinase activation by various treatments. Serum-depleted cells were preincubated as follows: overnight with 100 ng/ml PTX, for 30 min with 20 µM BAPTA/AM, for 30 min with 10 or 30 µM GF109203X, for 30 min with 300 nM staurosporine; or overnight with 1 µM TPA and stimulated with 100 nM LTD4 for 5 min at 37 °C. The MAP kinase activities of all the cell lysates were assayed as described (6). Vertical bars denote the mean ± S.E., respectively (n = 3). § indicates p < 0.01 as compared with the control values.

Translocation of PKCalpha and PKCdelta following LTD4 Stimulation-- We then examined which PKC isozyme is involved in the signaling pathway. PKCalpha and PKCdelta were detected in THP-1 cells by immunoblot analysis. In the resting state, almost all PKCalpha and PKCdelta were present in the cytosol, but 100 nM LTD4 caused a translocation of PKCalpha and PKCdelta from the cytosol to the membrane in 10 min (Fig. 4, A and B). As a positive control, TPA also caused a translocation of PKCalpha and PKCdelta from the cytosol to the membrane. This translocation of PKCalpha was inhibited by pretreatment with BAPTA/AM but that of PKCdelta was not affected by the same pretreatment (data not shown).


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Fig. 4.   LTD4-elicited translocation of PKC. Serum-depleted cells were stimulated with 100 nM LTD4 for the indicated times or to TPA (100 ng/ml for 20 min as a positive control) at 37 °C. The cytosol and membrane fractions of the cell lysates were separated by SDS-PAGE, transferred to a membrane, and probed with an anti-PKCalpha (A) or an anti-PKCdelta (B) antibody. Data are representative of three independent experiments, which gave essentially identical results.

Involvement of Raf-1 in MAP Kinase Activation-- Finally, we examined the role of Raf-1 in LTD4-elicited MAP kinase activation. After stimulation with LTD4, THP-1 cells were treated with the ice-cold lysis buffer, and the lysates were immunoprecipitated with an anti-Raf-1 antibody. The Raf-1 immunoprecipitate activated the His-tagged MAP kinase kinase and the GST-kn MAP kinase by about 2-fold in several minutes (Fig. 5A). When cells were pretreated overnight with TPA to deplete endogenous PKCalpha , the Raf-1 immunoprecipitate failed to activate the exogenous substrates (Fig. 5B). These data strongly suggest that activation of Raf-1 by PKCalpha plays a dominant role in LTD4-elicited MAP kinase activation.


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Fig. 5.   Raf-1 is concerned with the LTD4-elicited MAP kinase activation. A, phosphorylation of a His-tagged MAP kinase kinase and a GST-kn MAP kinase by Raf-1 immunoprecipitates. Serum-depleted cells were stimulated with 100 nM LTD4 for indicated periods at 37 °C. The radioactivities incorporated into a His-tagged MAP kinase kinase (solid line) and a GST-kn MAP kinase (dashed line) were measured as described under "Experimental Procedures." Data are representative of three independent experiments, which gave essentially identical results. B, PKC depletion abolishes the phosphorylation of a His-tagged MAP kinase kinase and a GST-kn MAP kinase by Raf-1 immunoprecipitates. Serum-depleted cells were stimulated with 100 nM LTD4 for 5 min or to 100 ng/ml TPA for 20 min (as a positive control). For PKC depletion, serum-depleted cells were preincubated overnight with 1 µM TPA and stimulated with 100 nM LTD4. Filled columns and hatched columns show incorporated radioactivities in His-tagged MAP kinase kinases and GST-kn MAP kinases, respectively.

LTD4-induced Chemotaxis-- It has been reported that LTD4 induces a chemotaxis of eosinophils (16, 17). Thus, we examined whether LTD4 would induce a chemotaxis of THP-1 cells. As shown in Fig. 6, LTD4 did induce chemotaxis in THP-1 cells with the maximal response at 30 nM. After the cells were pretreated overnight with PTX, the LTD4-induced chemotaxis completely disappeared (Fig. 6). These data indicate that LTD4-stimulated chemotaxis occurs through a PTX-sensitive mechanism.


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Fig. 6.   LTD4-induced chemotaxis. Chemotaxis of THP-1 cells to LTD4 was determined by microchamber assay. Control cells (solid line) and PTX-pretreated cells (100 ng/ml, overnight; dashed line) were assayed using various concentrations of LTD4. Symbols and vertical bars denote the mean ± S.E., respectively (n = 4). § indicates p < 0.01 as compared with the control values.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

LTD4 is produced from arachidonic acid, triggered by the activation of 5-lipoxygenase. LTD4 together with other cysteinyl LTs constitute a slow reacting substance of anaphylaxis, a major mediator in bronchial asthma and various allergic disorders (1-3). We and others (4, 5) previously provided indirect evidence that the LTD4 receptor couples to heterotrimeric G protein(s) in the cells and mobilizes intracellular calcium (18). However, the biochemical properties of the LTD4 receptor are unknown, and intracellular signaling mechanisms through the LTD4 receptor are not well understood. This is mostly because only scanty numbers of LTD4 receptors are expressed in the cells, and suitable cell lines that respond strongly to LTD4 stimulation have not been readily available. We studied various macrophage and monocytic cell lines (RAW264.7, P388D1, J774A.1, PU5-1.8, THP-1 cells, etc.) by examining the LTD4-induced calcium response and MAP kinase activation. Among them, a human monocytic leukemia cell line, THP-1 cells, showed most obvious responses toward LTD4. THP-1 cells exhibited a specific binding to LTD4, an increase in intracellular calcium concentration, and activation of MAP kinase. By comparing the potency of LTD4 and (5S,6R,7E,9E,11Z,14Z)-5-hydroxy-6-(S-glutathionyl)-icosatetraen-1-oic acid (LTC4) (data not shown), the receptor expressed on the THP-1 cell membranes can be classified as Cys-LTR1 (12). To our knowledge, this is the first detailed description of MAP kinase activation by LTD4. Since MAP kinase is a key enzyme that transmits signals from the cell surface to the nucleus (19) and activates several molecules including a cytosolic phospholipase A2 (20), we further studied the mechanisms of how LTD4 caused an activation of MAP kinase. The MAP kinase is activated by a variety of extracellular stimuli, including those mediated by receptor tyrosine kinases and by G protein-coupled receptors (21-24). The mechanisms from the G protein-coupled receptors to MAP kinase involve one or more molecules as follows: PKC (22, 25), Pyk2 (26-28), or phosphatidylinositol 3-kinase (29), depending on the cell types or ligands. Some experiments were performed using receptor overexpression systems, and this may differ from the native cells possessing endogenous receptors. In this study we provided evidence that LTD4 receptors couple to the PTX-insensitive G protein (Gq or G11) and cause a transient intracellular calcium mobilization, which in turn activates PKCalpha and Raf-1, a well-known MAP kinase kinase kinase. The involvement of Gq, PKCalpha , and Raf-1 in this sequence was demonstrated by the following observations. 1) The activations of MAP kinase and PKCalpha were scarcely inhibited by PTX treatment (Fig. 3, data not shown), although inhibition of adenylate cyclase was completely restored by the same treatment (data not shown). 2) LTD4 induced a translocation of PKCalpha from the cytosol to the membrane and activation of Raf-1 (Figs. 4A and 5A). 3) The LTD4-elicited MAP kinase activation was inhibited by staurosporine or GF109203X but not by wortmannin or tyrosine kinase inhibitors (Fig. 3, data not shown). 4) The activation of Raf-1 and MAP kinase was inhibited by PKC depletion in the cells (Fig. 3 and Fig. 5B). Moreover, calcium-independent PKCdelta was also translocated by LTD4 in a similar time course (Fig. 4B), and this may be also the consequence of an elevation in diacylglycerol levels through a Gq-phospholipase Cbeta pathway.

To date, the role of MAP kinase in macrophages/monocytic cells remains elusive. The activation of cytosolic phospholipase A2 may yield a production of various eicosanoids (i.e. prostaglandins or leukotrienes) or platelet-activating factor, which either amplify or regulate inflammatory reactions (30, 31). Furthermore, MAP kinase activation may induce various genes that relate to tumoricidal or bactericidal activity of macrophages.

Another finding in this study relates to the chemotactic activity of LTD4 in THP-1 cells. LTD4 has been considered to function as a potent chemoattractant for human eosinophils (16, 17). Considering that chemotactic responses by other ligands, such as interleukin-8 (32, 33), C5a (34), and LTB4 (8), are also inhibited by PTX-pretreatment, the common molecule(s) probably locates downstream of Gi-like protein(s).

In conclusion, THP-1 cells have functional LTD4 receptors (Cys-LTR1) that couple to both Gq-like and Gi-like proteins. LTD4 activates MAP kinase mostly through a Gq, PKCalpha , and Raf-1 pathway, whereas the chemotactic response is mediated through Gi-like protein. THP-1 cells proved to be an appropriate cell line for elucidation of LTD4-induced cellular signaling as well as for purification of the Cys-LTR1 molecules.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Y. Gotoh and E. Nishida (Kyoto University) for providing a His-tagged MAP kinase kinase and a GST-kn MAP kinase. We thank Drs. K. Matsushima, M. Aihara, and I. Waga (University of Tokyo) for suggestions and M. Ohara for comments.

    FOOTNOTES

* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Yamanouchi Foundation for Metabolic Disorders, Human Science Foundation, and Senri Life Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail; tshimizu{at}m.u-tokyo.ac.jp.

1 The abbreviations used are: LTD4, leukotriene D4, (5S,6R,7E,9E,11Z,14Z)-5-hydroxy-6-(S-cysteinylglycinyl)-icosatetraen-1-oic acid; BAPTA/AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; BSA, bovine serum albumin; DTT, dithiothreitol; G protein, GTP-binding protein; LT, leukotriene; MAP kinase, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; PTX, pertussis toxin; TPA, 12-O-tetradecanoylphorbol-13-acetate.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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