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Infection and Immunity, February 2001, p. 931-936, Vol. 69, No. 2
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.2.931-936.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Inhibition of p38 Mitogen-Activated Protein Kinase
Augments Lipopolysaccharide-Induced Cell Proliferation in
CD14-Expressing Chinese Hamster Ovary Cells
Dipshikha
Chakravortty,
Yutaka
Kato,
Tsuyoshi
Sugiyama,
Naoki
Koide,
Mya Mya
Mu,
Tomoaki
Yoshida, and
Takashi
Yokochi*
Department of Microbiology and Immunology and
Division of Bacterial Toxin, Research Center for Infectious Disease,
Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
Received 7 August 2000/Returned for modification 28 September
2000/Accepted 8 November 2000
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ABSTRACT |
CD14-expressing Chinese hamster ovary (CD14-CHO) cells, established
by transfection of human CD14 DNA, acquired high responsiveness to
lipopolysaccharide (LPS) through membrane-bound CD14 expression. LPS
induced DNA synthesis and activated a series of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2 (Erk1/2), p38, and c-Jun N-terminal kinase/stress-activated protein kinase, in CD14-CHO cells but not in mock-transfected CHO cells. Anti-CD14 antibody completely abrogated both LPS-induced DNA synthesis and LPS-induced phosphorylation of those MAP kinases, suggesting a
critical role of membrane-bound CD14 in LPS signaling. A p38 MAP kinase
inhibitor, SB203580, markedly augmented LPS-induced DNA synthesis in
CD14-CHO cells, whereas an Erk1/2 inhibitor, PD98059, had no affect. On
the other hand, SB203580 exhibited no effect on epidermal growth
factor-induced DNA synthesis in CD14-CHO cells, although PD98059
inhibited it significantly. The activation and inactivation of p38 MAP
kinase with dominant negative and dominant positive mutants also
suggested the participation of p38 MAP kinase in LPS-induced DNA
synthesis. It was therefore suggested that the activation of p38 MAP
kinase can negatively regulate LPS-induced cell proliferation in
CD14-CHO cells.
| |
INTRODUCTION |
Lipopolysaccharide (LPS) is present
on the outer membranes of all gram-negative bacteria and known to cause
the systemic inflammatory response syndrome septic shock and
disseminated intravascular coagulation, leading finally to multiorgan
failure (1, 21, 24, 33). The activation of
monocytes/macrophages by LPS leads to inflammatory response via various
cellular signaling events. LPS binds to monocytes/macrophages via
membrane-bound CD14, which is the glycosylphosphatidylinositol-linked
glycoprotein (37). The integration of membrane-bound CD14
renders various cell types highly sensitive to LPS. In fact, Chinese
hamster ovary (CHO) cells which are transfected with the CD14 gene and
express membrane-bound CD14 acquire the high responsiveness to LPS
(7-9, 13-15, 18, 20, 22, 31, 38, 39). Membrane-bound
CD14-expressing CHO (CD14-CHO) cells can respond to a low concentration
of LPS and exhibit various responses, such as release of arachidonic acid metabolites (8), translocation of nuclear factor
kappa B (NF-
B) (5, 9, 23) and production of interleukin
6 (10, 15), like LPS-responsive monocytes/macrophages.
CD14-CHO cells may provide an experimental system useful for LPS
signaling. LPS signaling is transduced by intracellular signal pathways
using NF-B and a series of mitogen-activated protein (MAP) kinases. In particular, the phosphorylation of three major MAP kinases i.e., extracellular signal-regulated kinase 1/2 (Erk1/2), p38, and
c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), is
rapidly induced by LPS in target cells such as macrophages (34,
35). There is no report on the activation of MAP kinases in
LPS-stimulated CD14-CHO cells. In the present study, we examined whether MAP kinases were activated in LPS-stimulated CD14-CHO cells and
what function the activation of MAP kinases had in those cells. Here we
discuss the relationship between the activation of p38 MAP kinases and
cell proliferation in LPS-stimulated CD14-CHO cells.
| |
MATERIALS AND METHODS |
Materials.
LPS from Escherichia coli O55:B5 and
epidermal growth factor (EGF) were obtained from Sigma Chemical Co.,
St. Louis, Mo. SB203580, PD98059, and
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) were purchased from
Calbiochem, San Diego, Calif. They were dissolved in dimethyl sulfoxide
and diluted in the culture medium. Anti-human CD14 antibody CLB-MON/1
was purchased from Nichirei (Tokyo, Japan).
Establishment of CD14-CHO cells.
CHO-K1 fibroblasts,
obtained from the American Type Culture Collection (Manassas, Va.),
were maintained in Ham's F-12 (Sigma) containing 5% heat-inactivated
fetal calf serum and antibiotics. The plasmid carrying human CD14 DNA
was a kind gift from R. J. Ulevitch, The Scripps Research
Institute, La Jolla, Calif. CHO cells were transfected with the CD14
plasmid by the lipofection method (8). CD14-CHO cells were
selected positively by using anti-CD14 antibody-coated beads and
further cultured with the addition of Geneticin (750 µg/ml). CD14-CHO
cells were maintained in Ham's F-12 with 5% fetal calf serum. CHO
cells transfected by the control vector plasmid served as
mock-transfected control CHO cells.
Laser flow cytometric analysis of CD14 expression and LPS
binding.
CD14-CHO cells were incubated with a 1:200 dilution of
fluorescein isothiocyanate (FITC)-conjugated anti-human CD14 monoclonal antibody (Coulter, Miami, Fla.) or 1 µg of FITC-conjugated LPS (Sigma) per ml at 4°C for 1 h. The cells were washed and
suspended in phosphate-buffered saline. Fluorescence was analyzed by a
laser flow cytometer (FACScaliber; Becton Dickinson, San Jose, Calif.).
DNA synthesis.
DNA synthesis in CD14-CHO cells was assayed
by [3H]thymidine incorporation into the nucleus. Cells
(3 × 104/100 µl) were plated in 96-well plates and
incubated with LPS (1 µg/ml or various concentrations) in the
presence or absence of anti-CD14 antibody (10 µg/ml) for 24 h.
[3H]thymidine (1 µCi/well; Amersham, London, England)
was added to the cultures. Eighteen hours later, the cells were
harvested on glass fiber filters Radioactivity was determined as counts per minute in a Beckman 
counter. In some experiments, CD14-CHO or
mock-transfected control CHO cells were also pretreated with 20 µM
SB203580 or PD98059, or various concentrations of SB203580, for 1 h before LPS treatment.
Immunoblotting.
CD14-CHO cells were seeded in 35-mm-diameter
plastic dishes (4 × 105 cells/dish) and incubated
with LPS (0.1 µg/ml) for 30 min. Cells were lysed in the lysis buffer
and boiled for 5 min at 100°C as described previously
(3). Aliquots (20 µg/lane) containing equal amounts of
protein were electrophoresed under reducing conditions in a 4 to 20%
gradient polyacrylamide gel and transferred to a polyvinylidene
difluoride membrane filter. The membranes were treated with 5% bovine
serum albumin for 1 h to block nonspecific binding, rinsed, and
incubated with a panel of rabbit polyclonal antibodies against Erk1/2,
phospho-Erk1/2, p38, phospho-p38, JNK/SAPK, and phospho-JNK/SAPK (New
England Biolabs, Beverly, Mass.) for 1 h. The membranes were
further treated with a 1:3,000 dilution of horseradish
peroxidase-conjugated protein G for 1 h. Immune complexes were
detected with an enhanced chemiluminescence substrate (New England
Nuclear, Boston, Mass.) and exposed to Kodak XAR X-ray film. A
prestained molecular weight standard kit (Nippon Bio-Rad, Tokyo, Japan)
was used as a reference. In some experiments, CD14-CHO cells were
pretreated with 20 µM SB203580 or PD98059 for 1 h before LPS treatment.
Transfection of dominant negative mutants of Erk1/2, p38, and
JNK/SAPK and a dominant positive mutant of MKK6.
Dominant negative
mutants of Erk1/2 (17), p38 (17), and
JNK/SAPK (16) and a dominant positive mutant of MAP kinase
kinase 6 (MKK6) (16) were kind gifts from J. D. Lee,
The Scripps Research Institute. CD14-CHO cells were transfected with 5 µg of dominant negative mutants by the Lipofectamine (GIBCO-BRL,
Gaithersburg, Md.) method (16, 17) for 8 h, and then
the culture medium was replaced by Ham's F-12 containing 5% fetal
calf serum. After 48 h of incubation, transfected cells were used
for experiments. CD14-CHO cells were also transfected with a dominant
positive mutant of MKK6 (5 µg/ml). To confirm overexpression of
transfected MKK6, phospho-p38 was analyzed by immunoblotting using
anti-phospho-p38 antibody. Anti-Flag (M2) antibody (Sigma) was also
used to test transfection efficiency.
Statistical analysis.
Results are expressed as means ± standard deviations of triplicate determinations. Statistical
significance was determined by Student's t test.
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RESULTS |
Establishment of CHO cells constitutively expressing membrane-bound
CD14.
The expression of membrane-bound CD14 on CD14-CHO cells,
which were transfected with human CD14 DNA, was analyzed by laser flow
cytometry (Fig. 1). CD14-CHO cells
expressed a high level of membrane-bound CD14. However, no significant
CD14 expression was detected on mock-transfected control CHO cells. In
an assay of the binding of FITC-conjugated LPS, the fluorescence
intensity on CD14-CHO cells was approximately 10 times as high as that
on mock-transfected CHO cells, suggesting enhanced binding of LPS to
CD14-CHO cells.
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FIG. 1.
Expression of membrane-bound CD14 and binding of LPS in
CD14-CHO cells. CD14-CHO cells and mock-transfected control CHO cells
were treated with FITC-conjugated anti-CD14 antibody (A) or LPS (B).
Fluorescence intensity in CD14 expression and LPS binding was analyzed
by laser flow cytometry.
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Augmented DNA synthesis in LPS-stimulated CD14-CHO cells and its
inhibition by anti-CD14 antibody.
LPS at a concentration of 0.01 to 10 µg/ml augmented [3H]thymidine uptake in CD14-CHO
cells; on the other hand, LPS at any concentration tested did not
enhance DNA synthesis in mock-transfected control CHO cells (Fig.
2A). LPS was suggested to augment DNA synthesis via membrane-bound CD14 expression. To confirm the
participation of membrane-bound CD14, the effect of anti-CD14 antibody
on LPS-induced DNA synthesis in CD14-CHO cells was studied by
[3H]thymidine incorporation (Fig. 2B). Anti-CD14 antibody
abolished the augmentation of DNA synthesis in LPS-stimulated CD14-CHO
cells. DNA synthesis in mock-transfected control CHO cells was not
altered in the presence or absence of anti-CD14 antibody, thus
confirming that the enhancement of DNA synthesis in CD14-CHO cells by
LPS was mediated with membrane-bound CD14. Anti-CD14 antibody at a concentration of 5 µg/ml completely abrogated LPS-induced
augmentation of DNA synthesis in CD14-CHO cells. The inhibition was
roughly dependent on the concentration of anti-CD14 antibody added
(data not shown).
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FIG. 2.
Augmented DNA synthesis in LPS-stimulated CD14-CHO cells
and its inhibition by anti-CD14 antibody. (A) Augmented DNA synthesis
in LPS-stimulated CD14-CHO cells. [3H]thymidine
incorporation was determined in CD14-CHO cells or mock-transfected
control CHO cells cultured with various concentrations of LPS. (B)
Inhibition of LPS-induced DNA synthesis by anti-CD14 antibody.
[3H]thymidine incorporation was determined in CD14-CHO
cells or mock-transfected control CHO cells cultured with LPS (1 µg/ml) in the presence of anti-CD14 antibody (10 µg/ml).
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Activation of a series of MAP kinases in LPS-stimulated CD14-CHO
cells and its inhibition by anti-CD14 antibody.
We have
demonstrated that LPS augments DNA synthesis in CD14-CHO cells. Based
on the fact that the MAP kinases Erk1/2, p38, and JNK/SAPK are reported
to be involved in signal transduction of cell growth (6, 26-28,
40), it was of interest to determine the activation of MAP
kinases in LPS-stimulated CD14-CHO cells. The phosphorylation of
Erk1/2, p38, and JNK/SAPK in LPS-stimulated CD14-CHO cells was studied
by immunoblotting using anti-phospho-MAP kinase antibodies (Fig.
3A). LPS induced the phosphorylation of Erk1/2, p38, and JNK/SAPK in CD14-CHO cells but not in mock-transfected control CHO cells. In addition, LPS did not affect constitutive levels
of Erk1/2, p38, and JNK/SAPK expression. Next, we examined whether
LPS-induced phosphorylation of MAP kinases in CD14-CHO cells was
dependent on membrane-bound CD14 (Fig. 3B). The addition of anti-CD14
antibody (5 µg/ml) completely blocked the phosphorylation of all the
three MAP kinases. Treatment with anti-CD14 antibody alone did not
affect the phosphorylation of MAP kinases. This suggested that LPS
activated Erk1/2, p38, and JNK/SAPK via membrane-bound CD14.
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FIG. 3.
Phosphorylation of Erk1/2, p38, and JNK/SAPK MAP kinases
in LPS-stimulated CD14-CHO cells and its inhibition by anti-CD14
antibody. (A) Phosphorylation of the MAP kinases in LPS-stimulated
CD14-CHO cells. CD14-CHO cells and mock-transfected control CHO cells
were cultured with LPS (0.1 µg/ml) for 30 min. (B) Inhibition
LPS-induced phosphorylation of the MAP kinases by anti-CD14 antibody.
CD14-CHO cells were pretreated with anti-CD14 antibody (5 µg/ml) for
4 h and then treated with LPS (0.1 µg/ml) for 30 min. The cells
were lysed, and the lysates were analyzed by immunoblotting using an
antibody to Erk1/2, phospho-Erk1/2, p38, phospho-p38, JNK/SAPK, or
phospho -JNK/SAPK.
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Enhancement of LPS-induced DNA synthesis in CD14-CHO cells by the
p38 inhibitor SB203580.
We tried to clarify the relationship
between LPS-induced DNA synthesis and LPS-induced MAP kinase activation
in CD14-CHO cells (Fig. 4). The effects
of the p38 inhibitor SB203580, the Erk1/2 inhibitor PD98059, and the
JNK/SAPK and Erk1/2 inhibitor (4) NPPB on DNA synthesis in
LPS-stimulated CD14-CHO cells were assessed by
[3H]thymidine incorporation. The addition of SB203580
resulted in striking enhancement of DNA synthesis in LPS-stimulated
CD14-CHO cells, whereas neither PD98059 nor NPPB affected DNA synthesis (Fig. 4A). The inhibitor solvent, dimethyl sulfoxide, exhibited no
effect on LPS-induced DNA synthesis. The concentration-dependent effect
of SB203580 on DNA synthesis in LPS-stimulated CD14-CHO cells was
studied (Fig. 4B). SB203580 at 10 µM exhibited a stronger enhancing
effect on LPS-induced DNA synthesis than SB203580 at 1 µM did. The
enhancing effect of SB203580 on DNA synthesis was also seen in the
absence of LPS (>5 µM, P < 0.01). In addition, we
confirmed that SB203580 and PD98059 completely abrogated the phosphorylation of p38 and Erk1/2, respectively (Fig. 4C). Results of
the experiment using the p38 inhibitor SB203580 suggested that the
inactivation of p38 MAP kinase might augment DNA synthesis in
LPS-stimulated CD14-CHO cells.
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FIG. 4.
Augmentation of LPS-induced DNA synthesis by a p38
inhibitor, SB203580. CD14-CHO cells or mock-transfected control CHO
cells were pretreated with SB203580 (20 µM), PD98059 (20 µM), or
NPPB (10 µM) (A) or with various concentrations of SB203580 (B) for
1 h and then treated with LPS (0.1 µg/ml). DNA synthesis was
determined by [3H]thymidine incorporation. (C) Inhibitory
action of SB203580 or PD98059 on the phosphorylation of p38 and Erk1/2
was confirmed by immunoblotting using an antibody to p38, phospho-p38,
Erk1/2, or phospho-Erk1/2.
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Enhancement of LPS-induced DNA synthesis by a dominant negative
mutant of p38 MAP kinase.
Since the inhibition of p38 MAP kinase
by SB203580 resulted in the enhancement of LPS-induced DNA synthesis,
we examined the effect of a dominant negative mutant of p38 MAP kinase
on LPS-induced DNA synthesis in CD14-CHO cells (Fig.
5). The dominant negative mutant of p38,
but not that of Erk1/2 or JNK/SAPK, markedly enhanced LPS-induced DNA
synthesis in CD14-CHO cells. No dominant negative mutants affected DNA
synthesis in the absence of LPS.
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FIG. 5.
Effect of the dominant negative mutant of Erk1/2, p38,
or JNK/SAPK on DNA synthesis in LPS-stimulated CD14-CHO cells. CD14-CHO
cells were transfected with a dominant negative mutant of Erk1/2, p38,
or JNK/SAPK, and LPS-induced DNA synthesis was determined by
[3H]thymidine incorporation.
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Inhibition of LPS-induced DNA synthesis by p38 activation via a
dominant positive mutant of MKK6.
The effect of p38 activation on
LPS-induced DNA synthesis in CD14-CHO cells was examined by the
overexpression of MKK6, an upstream kinase of p38. The introduction of
dominant positive mutants of MKK6 into CD14-CHO cells led to reduced
DNA synthesis in LPS-stimulated CD14-CHO cells (Fig.
6A). The treatment also inhibited DNA
synthesis in the absence of LPS (P < 0.01). The phosphorylation of p38 by transfection of a dominant positive mutant of
MKK6 was confirmed by immunoblotting using anti-phospho-p38 antibody
(Fig. 6B).
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FIG. 6.
Effect of the dominant positive mutant of MKK6 on
LPS-induced DNA synthesis in CD14-CHO cells. (A) CD14-CHO cells were
transfected with a dominant positive mutant of MKK6, and DNA synthesis
was determined in the presence or absence of LPS. (B) Phosphorylation
of p38 MAP kinase in CD14-CHO cells transfected with a dominant
positive mutant of MKK6 was confirmed by immunoblotting.
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Effect of SB203580 on DNA synthesis in EGF-stimulated CD14-CHO
cells.
We determined whether p38 MAP kinase was involved in DNA
synthesis in CD14-CHO cells stimulated with agents other than LPS. The
effect of SB203580 on DNA synthesis in EGF-stimulated CD14-CHO cells
was assessed by [3H]thymidine incorporation (Fig.
7). EGF-induced DNA synthesis in CD14-CHO
cells was markedly inhibited by PD98059 but was not affected by
SB203580. Next, the effect of EGF on phosphorylation of MAP kinases was
studied (Fig. 8A). EGF induced the phosphorylation of Erk1/2 but not
p38 and JNK/SAPK in CD14-CHO cells, and PD98059 completely inhibited
EGF-induced Erk1/2 phosphorylation (Fig. 8B). In addition, there was no
significant difference between CD14-CHO cells and mock-transfected
control CHO cells in response to EGF (data not shown). This finding
suggested that Erk1/2 and p38 MAP kinases might be involved in the
signal transduction of EGF- and LPS-induced DNA synthesis in CD14-CHO
cells, respectively.
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FIG. 7.
Effect of SB203580 on DNA synthesis in EGF-stimulated
CD14-CHO cells. CD14-CHO cells were pretreated with SB203580 (20 µM)
or PD98059 (20 µM) for 1 h and then treated with EGF (50 ng/ml).
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FIG. 8.
Effect of EGF on the phosphorylation of Erk1/2, p38, and
JNK/SAPK MAP kinases in CD14-CHO cells and inhibition by PD98059. (A)
Phosphorylation of Erk1/2 MAP kinase by EGF. CD14-CHO cells were
cultured with EGF (50 ng/ml) for 30 min. The cells were lysed, and the
lysates were analyzed by immunoblotting using an antibody to Erk1/2,
phospho-Erk1/2, p38, phospho-p38, JNK/SAPK, or phospho-JNK/SAPK. (B)
Inhibition of EGF-induced Erk1/2 phosphorylation by PD98059. CD14-CHO
cells were pretreated with 20 µM PD98059 for 1 h and then
treated with EGF for 30 min. The cells were lysed, and the lysates were
analyzed by immunoblotting.
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| |
DISCUSSION |
In this study we have demonstrated that LPS augments DNA synthesis
and activates a series of MAP kinases, Erk1/2, p38, and JNK/SAPK, in
CD14-CHO cells via membrane-bound CD14. Furthermore, a p38-specific
inhibitor, SB203580, strikingly enhanced LPS-induced DNA synthesis in
CD14-CHO cells. This suggests that the activation of p38 MAP kinase
inhibits LPS-induced DNA synthesis in CD14-CHO cells and that the
inhibition of p38 MAP kinase removes the inhibitory effect. This idea
is also supported by the results of experiments with the dominant
negative mutant of p38 and the dominant positive mutant of MKK6. Thus,
LPS-activated p38 MAP kinase may negatively regulate the proliferative
activity of CD14-CHO cells. This is to our knowledge the first report
on the inhibitory action of p38 MAP kinase on LPS-induced cell
proliferation. On the other hand, Erk1/2 did not seem to be involved in
LPS-induced DNA synthesis of CD14-CHO cells, although Erk1/2 plays a
pivotal role in EGF-induced DNA synthesis of CD14-CHO cells. Therefore,
p38 MAP kinase is a key molecule in regulation of LPS-induced cell
growth in CD14-CHO cells.
The p38 MAP kinase signal pathway plays an essential role in regulation
of many cellular processes, including proliferation, differentiation,
and cell death (6, 27-29, 40). Previously we reported
that LPS reduced DNA synthesis in LPS-sensitive bovine aortic
endothelial cells (3) and that SB203580 prevented
LPS-induced reduction of DNA synthesis in those cells (3).
The activation of p38 also regulates DNA synthesis negatively in bovine
aortic endothelial cells. Although different cell types respond
differentially to LPS in DNA synthesis, the inhibition of p38 MAP
kinase essentially seems to have an enhancing effect on DNA synthesis.
It has been reported that p38 MAP kinase provides a negative signal on
cell proliferation of various cell types by various stimuli other than LPS (25-27, 30). On the other hand, the constitutive
activation of p38 is reported to inhibit DNA synthesis in various cell
types (6, 32). The exact role of p38 in cell proliferation
requires further characterization.
Cell surface expression of CD14 provides the high responsiveness to LPS
on CD14-CHO cells (7-9, 13-15, 18, 20, 22, 31, 38, 39).
CD14-CHO fibroblasts become responsive to an extremely low
concentration of LPS (8). Therefore, CD14-CHO cells have been studied as a model of LPS-responsive phagocytic and nonphagocytic cells since they appear to contain many elements responsible for LPS-induced signaling. In particular, CD14-CHO cells have been used for
LPS signaling for NF-B activation (5, 9), interleukin 6 production (10, 15), arachidonic acid release
(8), and endotoxin tolerance (11). In the
present study, we found that LPS induced the activation of a series of
MAP kinases, Erk1/2, p38, and JNK/SAPK, in CD14-CHO cells. There is the
first report on the activation of MAP kinases in LPS-stimulated
CD14-CHO cells. Our experimental system using CD14-CHO cells might
provide a new model for characterization of LPS-induced MAP kinase activation.
LPS modulates the growth of gingival fibroblasts in the presence of
growth factors (12). LPS also acts as a mitogen in chicken embryo (36) and intestinal (2) fibroblasts.
In CD14-CHO cells, LPS induces the up-regulation of an apparently
extracellular protein, H411, which contains EGF-like repeats and
promotes growth (10). Microinjection of mRNA of its highly
homologous protein led to an autocrine/paracrine stimulation of DNA
synthesis (19). LPS may induce DNA synthesis in CD14-CHO
cells by up-regulation of H411 proteins. Although the biological
significance of LPS-induced DNA synthesis in CD14-CHO cells is unknown,
this system might be useful for characterization of the growth and
proliferation of macrophages by stimulation of LPS.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of
Japan and Daiko Foundation.
We thank M. Naruse for laser flow cytometric analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Aichi Medical University School of
Medicine, Aichi 480-1195, Japan. Fax: 81 (561) 63-9187. Phone: 81 (561) 62-3311. E-mail: yokochi{at}amugw.aichi-med-u.ac.jp.
Editor:
R. N. Moore
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Infection and Immunity, February 2001, p. 931-936, Vol. 69, No. 2
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.2.931-936.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
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