From the Department of Geriatric Medicine, Kyoto University Graduate School of Medicine, Kyoto, 606-8507, Japan
Received for publication, October 4, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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Transmigration of monocytes to the
subendothelial space is the initial step of atherosclerotic plaque
formation and inflammation. Integrin activation and chemotaxis are two
important functions involved in monocyte transmigration. To delineate
the signaling cascades leading to integrin activation and chemotaxis by
monocyte chemoattractant protein-1 (MCP-1), we have investigated the
roles of MAPK and Rho GTPases in THP-1 cells, a monocytic cell
line. MCP-1 stimulated Several lines of evidence indicate that monocyte chemoattractant
protein-1 (MCP-1)1 is
involved in the pathogenesis of atherosclerosis by promoting directed
migration of inflammatory cells, such as monocytes and T lymphocytes
(1, 2). During the progression of atherosclerosis, there is an
accumulation of low-density lipoprotein within macrophages present in
the intimal layer. Deposition of lipids within these cells leads to the
formation and eventual enlargement of atherosclerotic lesions. Boring
et al. (3) noted an overall decrease in atherosclerotic lesion size in mice deficient for the MCP-1 receptor, CCR2, when they
are crossed with ApoE knockout mice. Gu et al. (4) also found decreased atherosclerotic lesions in MCP-1-deficient mice when
they are crossed with the low-density lipoprotein receptor knockout mice. These studies have demonstrated that MCP-1 and CCR2 play
a crucial role in the initiation of atherosclerosis by recruiting
monocytes to the vessel wall.
According to the multistep theory, monocytes roll on the endothelial
cells, interact with E-selectin, adhere to the endothelial cells by
firm adhesion to ICAM-1 and VCAM-1, and then migrate into the
subendothelium (5). Rolling of monocytes on endothelial cells is
dependent on the binding of E-selectin and sialyl Lewis X, and adhesion
to the endothelium is dependent on the interaction of integrins on
monocytes and adhesion molecules on the endothelial cells, such as
VCAM-1 and ICAM-1. Integrins consist of several subtypes, and each
subtype is specific for its ligand. For example, We recently demonstrated that the Reagents--
RPMI medium was obtained from Nissui
Pharmaceuticals Co. Ltd. (Tokyo, Japan). Fetal calf serum was purchased
from Grand Cayman (British West Indies). L-Glutamine and
penicillin/streptomycin were obtained from Bio Whittaker (Walkersville,
MD). Recombinant human MCP-1 was obtained from PeproTech EC Ltd.
(London, England). Recombinant human soluble VCAM-1, ICAM-1, and
E-selectin were from Genzyme/Techne (Minneapolis, MN). Fibronectin,
BSA, arginine-glycine-aspartate-serine (RGDS) peptides, and
arginine-glycine-glutamate-serine peptides were from Sigma.
Anti-human Cell Lines--
The monocytic cell line THP-1 was a generous
gift from Dr. K. Nishida (Daiichi Pharmaceuticals Co. Ltd., Tokyo,
Japan) and was cultured in RPMI supplemented with
L-glutamine and penicillin/streptomycin plus 10% fetal
calf serum in an atmosphere of 95% air and 5% CO2 at
37 °C.
Cell Adhesion Assay--
Polystyrene 96-well flat-bottomed
microtiter plates (Costar 3595, Corning Incorporated, Corning, NY) were
coated with 25 µl of soluble VCAM-1 (2.5 µg/ml), soluble ICAM-1
(2.5 µg/ml), soluble E-selectin (2.5 µg/ml), or fibronectin (10 µg/ml) for 1 h at room temperature. After incubation, wells were
blocked by incubation with 225 µl of 10 mg/ml heat-denatured BSA for
30 min at room temperature (10). Control wells were filled with 10 mg/ml heat-denatured BSA. 100 µl of THP-1 cells suspended at a
concentration of 106/ml in 0.1% BSA-RPMI were incubated
for the indicated times in a CO2 incubator at 37 °C in
the presence or absence of MCP-1. After incubation nonadherent cells
were removed by centrifugation (top side down) at 48 × g for 5 min (11). Attached cells were fixed with 5%
glutaraldehyde for 30 min at room temperature. Cells were washed three
times with water, and 100 µl of 0.1% crystal violet in 200 mM MES (pH 6.0) was added to each well and incubated at
room temperature for 20 min. Excess dye was removed by washing with
water three times, and the bound dye was solubilized with 100 µl of
10% acetic acid (12, 13). The absorbance of each well at 595 nm was
then measured using a multiscan enzyme-linked immunosolvent assay
reader (SPECTRA classic; TECAN). Each sample was assayed in
triplicate. The absorbance was linear to the cell number up to an
OD of 1.9 (data not shown). For example, 0.05 of OD represents
adhesion of about 2,000 cells, and 0.5 of OD represents adhesion of
about 25,000 cells.
Chemotaxis Assay--
The migration of THP-1 cells was
determined using a modification of the method of Campbell et
al. (14). Briefly, THP-1 cells were resuspended in 0.1% BSA-RPMI.
After adjusting the cell density to 1 × 106 cells/ml,
100,000 cells in 100 µl were added to the top chamber of a
24-transwell apparatus (6.5-mm diameter, 5-µm pore size, Costar
#3421; Corning Incorporated, Corning, NY) and incubated for 2 h at
37 °C in an atmosphere containing 5% CO2. Cells that passed through the membrane were collected from the lower well and
counted in a FACScan (Becton-Dickinson, San Jose, CA).
Intracellular [Ca2+]
Measurement--
Agonist-dependent increases in
cytoplasmic Ca2+ were determined in THP-1 cells as
described previously (15).
MAPK Assay--
After serum starvation for 24 h in 0.1%
BSA-RPMI with or without C3 exoenzyme, the cells were stimulated with
MCP-1 and lysed with equal volumes of ice-cold 500-µl lysis buffer
(20 mM HEPES (pH 7.4), 50 mM NaCl, 1% Triton
X-100, 20 µM leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 mM
sodium orthovanadate, 50 mM sodium fluoride). After
centrifugation at 13,000 × g for 15 min, equal amounts
of cell lysates were analyzed by Western blot with anti-ERK1,
anti-phospho-ERK, anti-p38-MAPK, and anti-phospho-p38-MAPK antibodies.
Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and the enhanced
chemiluminescence (ECL) system (Amersham Pharmacia Biotech).
MCP-1 Increased Adhesion of THP-1 Cells to VCAM-1 and
Fibronectin--
To determine the regulation of integrin avidity by
MCP-1, we studied adhesion of THP-1 cells to purified adhesion
molecules. Cell adhesion to soluble E-selectin, soluble ICAM-1, and
soluble VCAM-1 was determined in the presence or absence of 10 nM MCP-1 under static conditions. MCP-1 increased adhesion
of THP-1 cells to VCAM-1 by more than 2-fold but not to E-selectin or
ICAM-1 (Fig. 1), indicating increased
avidity of the Inhibition of ERK but Not p38-MAPK Abrogated MCP-1-induced
Adhesion--
To determine whether MAPK activation is involved in
MCP-1-mediated integrin activation, we next pretreated the cells with MEK- or p38-MAPK-specific inhibitors and examined the effect of ERK and
p38-MAPK on MCP-1-dependent integrin activation.
Pretreatment of THP-1 cells with the MEK inhibitor, PD98059, inhibited
MCP-1-induced adhesion to VCAM-1 and fibronectin in a
dose-dependent manner (Fig.
5). In contrast, pretreatment with
p38-MAPK inhibitor, SB203580, did not affect MCP-1-induced cell
adhesion, indicating the involvement of ERK but not p38-MAPK in
MCP-1-dependent integrin activation.
Inhibition of p38-MAPK but Not ERK Abrogated MCP-1-induced
Chemotaxis--
To examine the role of MAPK in MCP-1-mediated
chemotaxis, we pretreated the cells with MAPK inhibitors before the
chemotaxis assay. We found that in the presence of MCP-1, THP-1 cells
showed a typical bell-shaped pattern of chemotactic responses, and the maximal response was achieved at 1 nM MCP-1 in the lower
chamber (data not shown). In contrast to the results in the cell
adhesion assays, pretreatment of THP-1 cells with SB203580 abrogated
MCP-1-induced chemotaxis in a dose-dependent manner (Fig.
6). In contrast, pretreatment with
PD98059 did not affect the chemotaxis, indicating the involvement of
p38-MAPK but not of ERK in MCP-1-mediated chemotaxis. We have also
tested the effect of anti-
Next, to examine the role of Rho GTPase and the Rho kinase, we
pretreated the cells with C3 exoenzyme and a Rho kinase inhibitor, Y-27632, and performed cell adhesion and chemotaxis assays.
Pretreatment of the cells with C3 exoenzyme and Y-27632 abrogated
MCP-1-mediated chemotaxis but not cell adhesion to fibronectin (Fig.
7) or VCAM-1 (not shown), indicating that
Rho is involved in chemotaxis but not in integrin activation. To
examine whether Rho and a Rho kinase are upstream of p38-MAPK, the
effect of C3 exoenzyme and Y-27632 on MCP-1-induced p38-MAPK activation
was determined. We found that MCP-1 could phosphorylate ERK and
p38-MAPK in THP-1 cells. However, pretreatment of the cells with C3
exoenzyme and Y-27632 abrogated MCP-1-induced phosphorylation of
p38-MAPK but not of ERK (Fig. 8).
SB203580 or PD98059 Did Not Affect Calcium Flux by MCP-1--
To
rule out the possibility of nonspecific inhibition of MCP-1-induced
signaling by these inhibitors, we checked MCP-1-dependent calcium flux after pretreatment with SB203580 and PD98059. As shown in
Fig. 9, MCP-1-induced calcium flux was
not affected by this pretreatment.
In this study we have elucidated the role of MAPK and Rho GTPase
in MCP-1-mediated cell adhesion and chemotaxis. We show that MCP-1
induced activation of the integrins 1 integrin-dependent, but not
2 integrin-dependent cell adhesion in a
time-dependent manner. MCP-1-mediated cell adhesion was
inhibited by a MEK inhibitor but not by a p38-MAPK inhibitor. In
contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK
inhibitor but not by the MEK inhibitor. The inhibitor of Rho GTPase, C3
exoenzyme, and a Rho kinase inhibitor abrogated MCP-1-dependent chemotaxis but not
integrin-dependent cell adhesion. Further, C3 exoenzyme and
the Rho kinase inhibitor blocked MCP-1-dependent p38-MAPK
activation. These data indicate that ERK is responsible for integrin
activation, that p38-MAPK and Rho are responsible for chemotaxis
mediated by MCP-1, and that Rho and the Rho kinase are upstream of
p38-MAPK in MCP-1-mediated signaling. This study demonstrates that two
distinct MAPKs regulate two dependent signaling cascades leading to
integrin activation and chemotaxis induced by MCP-1 in THP-1 cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
1 integrin,
very late antigen-4, binds to VCAM-1, and
2 integrins bind to
ICAM-1. Fibronectin, one of the extracellular matrix proteins, is also
known to bind to
1 integrins, mainly to
5
1 integrin. In these
serial events of the multistep theory, MCP-1 can play a key role in
monocyte recruitment by both integrin activation and by promoting
migration to the vessel wall. However, the signal transduction pathways
leading to integrin activation and chemotaxis have not been fully elucidated.
subunit of heterotrimeric G
protein, Gi, plays a key role in MCP-1-induced chemotaxis (6). In that
study, we reported that activation of ERK was not involved in
chemotaxis by MCP-1. MAPK family members, ERK, JNK, and p38-MAPK, have
been implicated in events necessary for proliferation, differentiation,
apoptosis, and certain kinds of stress responses (7). These MAPKs are
activated by specific cascades responsible for certain stimuli and
eventually induce a variety of cell responses. Recently, several groups
have reported on the involvement of MAPK and Rho in chemotaxis (8,
9). Most of the studies on signal transduction of chemotaxis and
cell adhesion have been conducted in adherent cells. However, in
adherent cells it would be difficult to separate chemotaxis and
integrin activation, because these two functions are closely connected. Therefore, the aim of our study was to examine whether integrin activation and chemotaxis can be separated by studying the signaling cascades leading to integrin activation and chemotaxis mediated by
MCP-1 in human monocytic THP-1 cells and to elucidate the role of MAPK
and Rho in these biological functions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 (very late antigen-4) antibody was from Upstate
Biotechnology (Lake Placid, NY). PD98059 and SB203580 were from
Calbiochem. C3 exoenzyme was a generous gift from Dr. S. Narumiya
(Kyoto University). A Rho kinase inhibitor, Y-27632, was a generous
gift from Welfide Corporation (Osaka, Japan).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 integrin by MCP-1. In the absence of MCP-1, more
THP-1 cells adhered to VCAM-1 than control. In time course experiments,
we also examined cell adhesion to fibronectin as a ligand for
5
1
integrin and found that MCP-1 increased cell adhesion to both VCAM-1
and fibronectin by more than 3-fold in a time-dependent
manner (Fig. 2).
MCP-1-dependent adhesion to VCAM-1 was also increased in a
dose-dependent manner, reaching a plateau at 1 nM MCP-1 (Fig. 3). Adhesion
to fibronectin was also increased by MCP-1 stimulation in a
dose-dependent manner (data not shown). To show that this
MCP-1-mediated adhesion is dependent on
4
1 and
5
1
integrins, we preincubated the cells with anti-
4 antibody and the
RGDS peptide. Preincubation of THP-1 cells with anti-
4 antibody
inhibited MCP-1-dependent and -independent cell adhesion to
VCAM-1 by about 80% but not with control IgG. Preincubation with the
RGDS peptide, but not with the RGES peptide, inhibited
MCP-1-dependent and -independent cell adhesion to
fibronectin (Fig. 4). These data indicate
that cell adhesion in our assay depends on the interaction between
integrins and their ligands and that MCP-1 increased the avidity of
both
4
1 and
5
1 integrins on THP-1 cells.
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Fig. 1.
MCP-1 stimulates cell adhesion to VCAM-1 but
not ICAM-1 or E-selectin in THP-1 cells. THP-1 cells were
subjected to adhesion assays on heat-denatured BSA (as control),
E-selectin, ICAM-1, or VCAM-1 for 10 min in the presence (open
columns) or absence (closed columns) of 10 nM MCP-1. The adhesion assays were done as described under
"Experimental Procedures." Data represent the mean ± S.D. of
triplicate measurements. Results are representative of four separate
experiments.
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Fig. 2.
Time dependence of MCP-1-mediated adhesion of
THP-1 cells to VCAM-1 and fibronectin. THP-1 cells were subjected
to adhesion assays on VCAM-1 (A) or fibronectin
(B) in the presence (squares) or absence
(circles) of 10 nM MCP-1 for the indicated
times. The adhesion assays were done as described under "Experimental
Procedures." Data represent the mean ± S.D. of triplicate
measurements. Results are representative of six separate
experiments.
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Fig. 3.
Dose dependence of MCP-1-mediated adhesion of
THP-1 cells to VCAM-1. THP-1 cells were subjected to adhesion
assays on VCAM-1 in the presence of MCP-1 at the indicated
concentrations for 20 min. The adhesion assays were done as described
under "Experimental Procedures." Data represent the mean ± S.D. of triplicate measurements. Results are representative of three
separate experiments.
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Fig. 4.
1
integrin-dependent adhesion of THP-1 cells to VCAM-1 and
fibronectin. THP-1 cells were pre-incubated with 10 µg/ml
anti-
4 antibody and control IgG (cont. IgG)
(A) or 2 mM RGDS and RGES peptides
(B) for 1 h in an atmosphere of 95% air and 5%
CO2 at 37 °C. After the incubation, cells were subjected
to adhesion assays on VCAM-1 (A) or fibronectin
(B) in the presence (open columns) or absence
(closed columns) of 10 nM MCP-1 for 20 min
(A) or 10 min (B). The adhesion assays were done
as described under "Experimental Procedures." Data represent the
mean ± S.D. of triplicate measurements. Results are
representative of four (A) and three (B) separate
experiments. To make comparison easier, the level of adhesion obtained
without MCP-1 without antibody (A) or competitors
(B) was taken as 100%.
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Fig. 5.
MEK inhibitor (PD98059) blocks
MCP-1-dependent adhesion to VCAM-1 and fibronectin in a
dose-dependent manner. THP-1 cells were pre-incubated
with PD98059 or SB203580 at the indicated concentrations for 1 h
in an atmosphere of 95% air and 5% CO2 at 37 °C. Both
inhibitors were dissolved in Me2SO, and its final
concentration was 0.2% including control. After the incubation, cells
were subjected to adhesion assays on VCAM-1 (A) or
fibronectin (B) in the presence (open columns) or
absence (closed columns) of 10 nM MCP-1 for 20 min. The adhesion assays were done as described under "Experimental
Procedures." Data represent the mean ± S.D. of triplicate
measurements. Results are representative of five separate experiments.
To make comparison easier, the level of adhesion obtained without MCP-1
without inhibitors was taken as 100%.
4 antibody and the RGDS peptide in the
chemotaxis assay, but both of them had no effect on chemotaxis induced
by MCP-1 (data not shown), indicating that integrin activation does not
play a role in chemotaxis induced by MCP-1 in THP-1 cells.
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Fig. 6.
p38-MAPK inhibitor (SB203580) blocks
MCP-1-dependent chemotaxis of THP-1 cells in a
dose-dependent manner. THP-1 cells were pre-incubated
with PD98059 or SB203580 at the indicated concentrations for 1 h
in an atmosphere of 95% air and 5% CO2 at 37 °C. After
incubation, cells were subjected to chemotaxis assays as described
under "Experimental Procedures." The concentrations of MCP-1 in the
lower chamber were 0 (closed columns) and 1 nM
(open columns). Data represent the mean ± S.D. of
duplicate measurements. Results are representative of six separate
experiments.
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Fig. 7.
C3 exoenzyme and Y-27632 blocks
MCP-1-dependent chemotaxis of THP-1 cells, but
MCP-1-dependent adhesion is not affected by C3
exoenzyme. THP-1 cells were pre-incubated with 15 µg/ml C3
exoenzyme for 24 h or with the indicated concentrations of Y-27632
for 1 h (A) in an atmosphere of 95% air and 5%
CO2 at 37 °C. After the incubation, cells were subjected
to chemotaxis assays (A) or adhesion assays to fibronectin
for 20 min (B) as described under "Experimental
Procedures." In chemotaxis assays (A), the concentrations
of MCP-1 in the lower chamber were 0 (closed columns) and 1 nM (open columns). In adhesion assays
(B), cells were stimulated with 10 nM MCP-1
(open columns) or left unstimulated (closed
columns). Data represent the mean ± S.D. of duplicate
(A) or triplicate (B) measurements. Results are
representative of four separate experiments. In B, the level
of adhesion obtained without MCP-1 without C3 exoenzyme was taken as
100%.
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Fig. 8.
Activation of p38-MAPK, but not ERK by MCP-1,
is blocked by C3 exoenzyme and Y-27632. THP-1 cells were
pre-incubated with or without 15 µg/ml of C3 exoenzyme for 24 h
(A) or 10 µM Y-27632 for 1 h
(B) in an atmosphere of 95% air and 5% CO2 at
37 °C. After the incubation, cells were stimulated with 10 nM MCP-1 for 1 min and subjected to the MAPK assay as
described under "Experimental Procedures." Results are
representative of three (A) and two (B)
independent experiments.
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Fig. 9.
MCP-1-dependent Ca2+
flux is not affected by PD98059 or SB203580. THP-1 cells were
pre-incubated with 50 µM PD98059 (B) or 10 µM SB203580 (C) for 1 h in an atmosphere
of 95% air and 5% CO2 at 37 °C. In A, cells
were pre-incubated with 0.2% Me2SO. After the incubation,
cells were stimulated with 10 nM MCP-1, and
Ca2+ flux was measured as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
1 and
5
1 in THP-1 cells and that the integrin activation is dependent on ERK activation. In contrast, MCP-1-dependent chemotaxis was dependent on
activation of Rho and p38-MAPK. Thus, as depicted in Fig.
10 two important biological functions
mediated by MCP-1 utilize two distinct MAPK-dependent signaling pathways.
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Fig. 10.
A model proposed to explain MCP-1-mediated
chemotaxis and integrin activation. MCP-1-mediated chemotaxis is
mediated through Rho, Rho kinase, and p38-MAPK, and MCP-1-mediated
activation of 1 integrin is dependent on ERK. These two distinct
signaling cascades are supposed to be required for transmigration of
monocytes.
In this study we used sensitive cell adhesion assays to demonstrate important functions of MCP-1. Although Weber et al. (16) demonstrated that binding of monocytes to VCAM-1 was reduced at 15 min under stimulation with MCP-1 in a similar assay, we found greater than a 2-fold increase in cell adhesion to this molecule at 10 to 20 min. In preliminary experiments, we have found a basal increase in cell adhesion after labeling the cells with fluorescent dye and washing the cells. We speculate that this is because of some stress on the cells. Further, a basal increase in cell adhesion after spinning down and washing the cells might be because of increased MAPK activation during these procedures. In support of this hypothesis, MacKenna et al. (17) reported that in cardiac fibroblasts ERK and JNK are activated by mechanical stretch. Therefore, it would be important to avoid stress on the cells as much as possible in this experiment. Work is now in progress to determine the effect of mechanical stress on MAPK activation and cell adhesion.
We found that in the monocytic cell line 1 integrins but not
2
integrins are activated by MCP-1. However, Weber et al. (18) have reported that MCP-1 induces a prolonged increase in the binding of
monocytes to ICAM-1 in a static adhesion assay. This difference may be
because of a difference in the way to remove nonadherent cells. We
removed nonadherent cells by centrifugation, but they did it by plate
washer. So we speculate that binding of
2 integrin and ICAM-1 is not
strong enough to overcome the centrifugation force. Further, Chan
et al. (19) have reported that activation of
1 integrin
by chemokines might be much stronger than that of
2 integrin and
that
1 integrin/VCAM-1 interaction activates
2 integrin-mediated
cell adhesion in human T cells. Therefore, in in vivo
situations, activation of
1 integrins might be stronger and more
important in the early phase of cell migration.
In the cell adhesion assay, we could not abrogate MCP-1-dependent adhesion to fibronectin by the RGDS peptide, even though we have used sufficient concentrations of the peptide. We speculate that the inhibitory effect of this peptide on the interaction between fibronectin and integrins is not so strong (20). Because RGDS-independent adhesion of fibronectin has been reported (21), it is also possible that RGDS-independent cell adhesion is induced by MCP-1.
In this study we showed that in THP-1 cells ERK is responsible for integrin activation by MCP-1 but not p38-MAPK or Rho. Laudanna et al. (22), however, reported that Rho is also involved in integrin activation by interleukin-8 in neutrophils and lymphocytes. The reasons for these differences are not clear, but signaling through integrin activation in response to chemokines might be cell type-specific. It is also possible that different types of integrins in leukocytes might be activated in response to each chemokine.
Recently, several reports have shown that p38-MAPK is involved in chemotaxis induced by serum, lysophosphatidylcholine, and chemokines in leukocytes and smooth muscle cells (8, 9). Our study has also shown that p38-MAPK is involved in chemotaxis induced by MCP-1 in THP-1 cells. In contrast, Yen et al. (23) showed that ERK is responsible for MCP-1-mediated chemotaxis. Knall et al. (24), on the other hand, have shown that ERK or p38-MAPK is not involved in interleukin-8-mediated chemotaxis. The reason for these differences is not clear, but in the system of Yen et al. (23) the activation of integrin might have been required for monocyte chemotaxis. Rho family GTPases have also been shown to be involved in cell migration (25). Thus our data are consistent with the others that p38-MAPK and Rho are involved in chemotaxis. However, the relationship between Rho and MAPK is quite complicated. For example, Zhang et al. (26) have shown that p38-MAPK is downstream of Rho in interleukin-1-mediated signaling. In contrast, Hippenstiel et al. (27) have reported that LPS-induced activation of p38-MAPK is not affected by Clostridium difficile toxin B-10463, a specific inhibitor of Rho. In terms of ERK and Rho, some reports have claimed that Rho is upstream of ERK (28-30), whereas others have demonstrated that Rho and ERK are activated independently (31). However, few studies have been conducted to examine whether ERK and p38-MAPK are differentially affected by Rho. In this paper, we clearly show that MCP-1 phosphorylates ERK and p38-MAPK in THP-1 cells and that Rho is upstream of p38-MAPK but not of ERK in MCP-1-mediated signal transduction. We have also shown that the Rho kinase (32) is between Rho and p38-MAPK and is a key molecule for chemotaxis. However, downstream targets of p38-MAPK leading to chemotaxis still remain to be determined. Although we found that MCP-1 also activated JNK (data not shown), the role of JNK activation in MCP-1-mediated signaling was not determined in this study.
In summary, we have provided clear evidence that two distinct signaling
cascades are present to mediate MCP-1-induced activation of 1
integrins and chemotaxis in THP-1 cells. These two distinct signaling
cascades would be important for transmigration of monocytes through
endothelial cells. The most intriguing aspect of this study is that we
could separate two important biological functions of leukocytes,
integrin activation and chemotaxis, by different assays and found that
two distinct signaling cascades mediate these two functions. In
adherent cells, however, segregation of integrin activation and
chemotaxis would be very difficult to assess. As depicted in Fig. 10,
identification of signaling molecules located at the bifurcation to ERK
and p38-MAPK would be important to delineate the signaling cascades
through CCR2. Further, it would be intriguing to determine the
signaling cascades in a condition closer to in vivo situations.
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ACKNOWLEDGEMENTS |
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We thank Dr. Israel F. Charo for critical reading of the manuscript.
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FOOTNOTES |
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* This study was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan; International Scientific Research Program grants from the Japanese Ministry of Education, Science, Sports, and Culture; Center of Excellence grants from the Japanese Ministry of Education, Science, Sports, and Culture; and a research grant for health sciences from the Japanese Ministry of Health and Welfare.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 the correspondence should be addressed: Dept. of Geriatric
Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. Tel.: 81-75-751-3463; Fax: 81-75-751-3463; E-mail:
harai@kuhp.kyoto-u.ac.jp.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M009068200
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ABBREVIATIONS |
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The abbreviations used are: MCP-1, monocyte chemoattractant protein-1; MAPK, mitogen-activated protein kinase; CCR, cysteine-cysteine chemokine receptor; BSA, bovine serum albumin; ERK, extracellular regulated kinase; MEK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; MES, 4-morpholineethanesulfonic acid; RGDS, arginine-glycine-aspartate-serine.
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REFERENCES |
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