From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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ABSTRACT |
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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 C 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 C
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.
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INTRODUCTION |
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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, PKC 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.
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EXPERIMENTAL PROCEDURES |
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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). [-32P]ATP
(specific activity, 222 TBq/mmol), transfer membrane (Hybond-N+), p42/p44 MAP kinase enzyme assay (RPN 84), anti-PKC
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-PKC
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 -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 -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.
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).
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RESULTS |
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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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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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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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Translocation of PKC and PKC
following LTD4
Stimulation--
We then examined which PKC isozyme is involved in the
signaling pathway. PKC
and PKC
were detected in THP-1 cells by
immunoblot analysis. In the resting state, almost all PKC
and PKC
were present in the cytosol, but 100 nM LTD4
caused a translocation of PKC
and PKC
from the cytosol to the
membrane in 10 min (Fig. 4, A
and B). As a positive control, TPA also caused a
translocation of PKC
and PKC
from the cytosol to the membrane.
This translocation of PKC
was inhibited by pretreatment with
BAPTA/AM but that of PKC
was not affected by the same pretreatment
(data not shown).
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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 PKC, the Raf-1
immunoprecipitate failed to activate the exogenous substrates (Fig.
5B). These data strongly suggest that activation of Raf-1 by
PKC
plays a dominant role in LTD4-elicited MAP kinase
activation.
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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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DISCUSSION |
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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 PKC and Raf-1, a
well-known MAP kinase kinase kinase. The involvement of Gq,
PKC
, and Raf-1 in this sequence was demonstrated by the following
observations. 1) The activations of MAP kinase and PKC
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 PKC
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 PKC
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
C
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, PKC, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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