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Gene Transcription
*
Department of Paediatrics, Teikyo University School of Medicine, Tokyo,
Japan
Department of Physiology, University of Birmingham, Birmingham, United
Kingdom
Department of Immunology, National Children's Medical Centre, Tokyo,
Japan.
Correspondence to Dr. Akio Nakamura, Department of Paediatrics, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173, Japan. Phone: 03-3964-1211 (ext. 1480); Fax: 03-3579-8212; E-mail: akio{at}med.teikyo-u.ac.jp
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-). It has
been shown that Stx-2 induces TNF- production and that activation of
ß2-adrenoceptors downregulates TNF-. However, little is
known about the signaling pathway by which ß2-adrenoceptor
agonists suppress the Stx-2—induced TNF- gene transcription. The
possible signaling components involved in this pathway were investigated.
Human adenocarcinoma—derived renal tubular epithelial cells (ACHN) were
exposed to Stx-2 in the presence or absence of a
ß2-adrenoceptor agonist. Mitogen-activated protein kinase
(MAPK), activating protein—1 (AP-1), and nuclear factor—
B
(NF-B) were measured to evaluate the regulatory mechanisms involved in
TNF- gene transcription. Stx-2 (4 pg/ml) stimulated MAPK (p42/p44, p38)
and AP-1 and increased TNF- promoter activity by 2.4-fold. The increase
in TNF- was attenuated by both a p42/p44 inhibitor, PD098059
(10-6 M), and a p38 inhibitor, SB203580 (10-6 M), and
AP-1—binding activity was inhibited by PD098059. Terbutaline
(10-6 M to 10-8 M) suppressed MAPK (p42/p44, p38),
NF-B (p50, p65), and TNF- promoter activity in a dose-dependent
way that was prevented by the ß2-adrenoceptor antagonist,
ICI118,551. However, inhibition of MAPK (p42/p44) and TNF- promoter
activity was partially prevented by the cAMP-protein kinase (PKA) inhibitors,
H-89 (5 x 10-6 M) and KT5720 (10-5 M), whereas the
suppression of p38 MAPK or NF-B (p50) was not blocked by these
inhibitors. The suppression of NF-B (p65) was completely overcome by
H-89 or KT5720. In summary, the downregulation of TNF- transcription by
terbutaline was mediated by an inhibitory effect of
ß2-adrenoceptor activation on MAPK (p42/p44, p38) and
NF-B (p50/p65), which were exerted through a cAMP-PKA pathway and a
cAMP-independent mechanism. It is likely that cAMP-PKA and MAPK (p42/p44, p38)
may play a critical role in the regulation of the Stx-2—induced
TNF- transcription via ß2-adrenoceptor activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hughes et al. (9)
have indicated that proximal tubules are exquisitely sensitive to Stx-1
cytotoxicity and that inflammatory factors can increase their responsiveness
to the toxin through a variety of mechanisms. They furthermore indicated that
Stx-1 stimulates the production of inflammatory cytokines, such as
interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF) by the proximal
tubule (10). It has been found
that IL-1 and TNF- induce expression of Gb3 on the surface of several
cells and can upregulate cell sensitivity to Stx cytotoxicity
(3,11,12).
Importantly, TNF- has been shown to enhance Stx-mediated apoptosis in a
human renal tubular epithelium—derived cell line, ACHN
(7). In an additional report
that used a mouse model of HUS, it was found that Stx induced TNF synthesis
within the kidney and at the same time increased the renal sensitivity to the
toxic effects of TNF (13). One
explanation may be that Stx stimulation of TNF- production in the renal
tubule increases its sensitivity to the tubular cytotoxic effect of Stx.
However, the mechanism by which Stx is able to stimulate TNF production
remains uncertain at present.
ß2-adrenoceptor agonists have been known to inhibit the
production of TNF- in response to an endotoxin challenge
(14,15).
Recently, we reported that Stx-2 caused a marked increase in TNF-
protein release from ACHN cells, which could be blunted in the presence of a
ß2-adrenoceptor agonist, and that the cytotoxic effects of
Stx-2 could be prevented when ß2-adrenoceptors were activated
(16). These findings suggest
that the mechanism by which Stx-stimulated TNF- production might
involve intracellular signaling events triggered by
ß2-adrenoceptors. It would seem that
ß2-adrenoceptor activation prevents TNF- production as
a consequence of the inhibition of mitogen-activated protein kinase (MAPK,
p42/p44) and enhanced cAMP generation
(17). The MAPK and
cAMP/cAMP-dependent protein kinase (PKA) pathways have been reported to
regulate the nuclear binding activity of activating protein—1 (AP-1) and
nuclear factor—B (NF-B), which involves TNF- gene
transcription (18). However,
it remains unclear whether activation of nuclear factors through these
pathways is necessary for the induction of TNF- by Stx.
This study was undertaken to investigate the mechanisms of
Stx-2—induced TNF- gene transcription and the inhibitory
mechanisms after ß2-adrenoceptor activation. We attempted to
clarify the possible roles of cAMP-PKA, p42/p44 MAPK, p38 MAPK, c-Jun
N-terminal protein kinase (JNK), AP-1, or NF-B (p50, p65) in
TNF- gene transcription in ACHN cells, which are an in vitro
model of renal tubular epithelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enzyme-linked immunosorbent assay kit was from Biosource
International Inc. (Camarillo, CA). The MAPK (p42/p44) assay kit and isotopes
([-33P]dCTP, [
-33P]ATP) were purchased
from Amersham Japan (Tokyo, Japan). p38 MAPK and JNK assay kits were purchased
from Stratagene (La Jolla, CA). AP-1 and NF—B (p50, p65) assay
kits (NUSHIFT) were purchased from Geneka (Montreal, Quebec, Canada). PAGEL
was obtained from ATTO Corp. (Tokyo, Japan). BAS III and BAS2000 were produced
by Fuji Photo Film Co. Ltd. (Tokyo, Japan). Unless otherwise stated, reagents
were from Sigma Chemical Co. (St. Louis, MO).
Culture Conditions
The ACHN line was obtained from the American Type Culture Collection (ATCC
CRL 1611). The cells express the renal tubular marker CD24 as well as CD77,
the receptor for Stx (7).
Furthermore, the cells were identified by means of immunostaining by using
labeling with ß2-adrenoceptor antibody-FITC conjugate. The
cells were maintained at 37°C in 5% CO2 in DMEM that contained
10% FCS, 0.225% NaHCO3, 0.1 mM nonessential amino acids, and 0.03%
glutamine and were cultured on 25-cm2 tissue culture flasks and
grown until approximately 80% confluent. After the dishes were washed with
phosphate-buffered saline to remove the nonadherent cells, the cells were
placed in the medium for 2 h before the Stx-2 challenge.
Cell Viability
Cells were removed from the culture flasks, and plated onto 96-well
microtiter plates at a density of 1.5 x 104 cells per well in
200 µl of medium. The cells were incubated for 4, 24, 48 h after the Stx-2
(0 to 512 pg/ml) challenge in either the absence or presence of terbutaline
(Ter; 10-6 M to 10-8 M), formoterol (For;
10-6 M), ICI118,551 (ICI; 10-6 M), forskolin
(10-5 M), prostaglandin E2 (PG; 10-6 M), PD
(10-6 M), SB (10-6 M), H-89 (5 x 10-6
M), and/or KT5720 (KT; 10-5 M). Cell viability was determined by
the uptake of neutral red as described previously
(20).
Assay for TNF- Release
To confirm the stimulatory effects of Stx-2 on TNF- release from
ACHN cell, cells were exposed to varying concentration of Stx-2 (2 to 512
pg/ml). Furthermore, TNF- release was assayed up to 24 h after the
Stx-2 (4 pg/ml) challenge in either the absence or presence of the
ß2-adrenoceptor agonists, Ter (10-6 M or
10-7 M) or For (10-6 M), the
ß2-adrenoceptor antagonist, ICI (10-6 M), the
cAMP-elevating agents, forskolin (10-5 M) or PG (10-6
M), and/or the cAMP-PKA inhibitors, H-89 (5 x 10-6 M) or KT
(10-5 M). The agonists (activators) or antagonists (inhibitors)
were added 10 min or 20 min before the Stx-2 (4 pg/ml) challenge,
respectively. To examine the influence of MAPK (p42/p44, p38) inhibition on
TNF- in the cells, the MAPK/extracellular signal—regulated kinase
(MEK 1) inhibitor, PD (10-6 M) or the specific p38 inhibitor, SB
(10-6 M) was added 20 min before the Stx-2 (4 pg/ml) challenge.
TNF- levels in the incubate were estimated with a commercially
available enzyme-linked immunosorbent assay kit.
Assay of MAPK (p42/p44, p38, JNK)
MAPK activities were assayed up to 100 min after the Stx-2 (4 pg/ml)
challenge in either the absence or presence of the
ß2-adrenoceptor agonists, Ter (10-6 M to
10-8 M) or For (10-6 M), the
ß2-adrenoceptor antagonist, ICI (10-6 M), the
cAMP-elevating agents, forskolin (10-5 M) or PG (10-6
M), and/or the cAMP-PKA inhibitors, H-89 (5 x 10-6 M) or KT
(10-5 M). The agonists (activators) or antagonists (inhibitors)
were added 10 min or 20 min before the Stx-2 (4 pg/ml) challenge,
respectively. The assay system is based on the MAPK-catalyzed transfer of the
-phosphate group of adenosine-5'-triphosphate to a peptide that
is selective for MAPK. Cells were lysed in 10 mM Tris, 150 mM NaCl, 2 mM
ethylenegly-cotetraacetic acid, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM
phenylmethyl sulfonyl fluoride, and 10 µg/ml aprotinin (pH 7.4) and then
precipitated at 25,000 x g for 20 min. The supernatant that
contained the cytoplasmic MAPK was retained. The reaction was initiated by the
addition of [-33P]ATP, and the incorporation of
33P into the peptide at 30°C for 30-min incubation was assayed
as a MAPK activity in a scintillation counter.
Electrophoretic Mobility Shift Assay
AP-1 and NF—B (p50, p65) were assayed 2 h after the Stx-2 (4,
16, 64 pg/ml) challenge in either the absence or presence of the
ß2-adrenoceptor agonists, Ter (10-6 M to
10-8 M) or For (10-6 M), the
ß2-adrenoceptor antagonist, ICI (10-6 M), and/or
the cAMP-PKA inhibitor, H-89 (5 x 10-6 M). The agonists or
antagonists were added 10 min or 20 min before the Stx-2 challenge,
respectively. To examine the influence of MAPK (p42/p44, p38), inhibition on
AP-1 and NF—B (p50, p65)—binding activities in the cells,
PD (10-6 M) or SB (10-6 M) was added 20 min before the
Stx-2 challenge. Nuclear extracts from ACHN cells were prepared by using
methods described previously
(21). The nuclear proteins
were stored in aliquots at -70°C. Double-stranded oligonucleotides that
contained AP-1 and NF—B (p50, p65) consensus—binding sites
were radiolabeled by using T4 polynucleotide kinase and
[33P]-ATP and were then purified by centrifugation over a
G-25 purification column. Specific oligonucleotide probes, NF-B (p50)
probe (5'-GCCATGGGGGGATCCCCGAAGTCC-3'), NF-B (p65) probe
(5'-AGCTTGGGGTATTTCCAGCCG-3'), AP-1 probe (5'-
CGCTTGATGAGTCAGCCGGAA-3') and its mutation probe, NF-B (p50)
(5'-GCCATGGGCCGATCCCCGAAGTCC-3'), NF-B (p65)
(5'-AGCTTGGCATAGGTCCAGCCG-3'), AP-1
(5'-CGCTTGATGACCCAGCCGGAA-3'), were radiolabeled and used to
examine the specificity of the nuclear factor binding. Five micrograms of
nuclear protein were incubated with the 33P-labeled probes for 20
min at 4 to 10°C according to the manufacturer's instructions. The samples
were loaded onto a 5% polyacrylamide (38:2) gel (PAGEL) with Tris-glycine
buffer, and electrophoresis was performed at 100 V for 1 h. The gel was vacuum
dried and exposed on an imaging plate (BAS III), and the incorporated
radioactivity was measured with a Bioimage analyzer (BAS 2000). The
specificity of the reaction was determined by competition reactions in which a
100-fold molar excess of unlabeled AP-1 or NF-B oligonucleotide probe
was added to the binding reaction. The composition of activated AP-1 or
NF-B was analyzed by using a supershift assay. In the study, 2 µl of
antibodies reactive to the human c-Fos, c-Jun, p50, and p65 proteins were
incubated with the reaction mixture for 20 min and then added to radiolabeled
AP-1 or NF-B probe.
Reporter Plasmid and Luciferase Assay
pGV-B2-TNFprom was generated in which a rat TNF- promoter
(22) drives the expression of
luciferase of pGV-B2 (pica gene basic vector-2) and prepared as described in
our previous manuscript (23).
pGV-B2-TNFprom (0.7 kb) contains potential recognition sites for several
transcription factors, including AP-1 and NF-B, and comparison of the
region with human TNF- promoter revealed a high degree of conversion
(24). Transfection of
pGV-B2-TNFprom into the cells was performed by using Transfectam according to
the manufacturer's recommendations. For the luciferase assay, the cells were
incubated for 3 h with 10% FCS-DMEM after the Stx-2 (4 pg/ml) challenge in
either the absence or presence of the ß2-adrenoceptor agonist,
Ter (10-6 M or 10-7 M), the cAMP-PKA stimulators,
forskolin (10-5 M) or PG (10-6 M), the MAPK (p42/p44,
p38) inhibitors, PD (10-6 M) or SB (10-6 M), the
NF-B inhibitors, pyrrolidine dithiocarbamate (PDTC; 1.5 x
10-4 M), and/or the cAMP-PKA inhibitors, H-89 (5 x
10-6 M) or KT (10-5 M). The agonists (stimulators) or
antagonists (inhibitors) were added 10 min or 20 min before the Stx-2
challenge, respectively. The luciferase activities were normalized on the
basis of ß-galactosidase activities, which were assayed as described
previously (23).
Statistical Analyses
Statistical analyses were undertaken by using nonparametric analysis with
the Kruskal-Wallis test followed by the Mann-Whitney U test. Results
are expressed as mean ± SE of the mean.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis. In contrast, Figure
1B and 1C (line graphs) show that 24 to 48 h exposure to >2
pg/ml Stx-2 caused a reduction in cell viability, which was significantly
attenuated by Ter (10-6 M) over the dose range of 2 to 128 pg/ml
Stx-2. The Ter inhibition of Stx-2 (4 pg/ml)—induced cell cytotoxicity
at 24 h was completely abolished by the ß2-adrenoceptor
antagonist, ICI (10-6 M), and the inhibitors of the cAMP-PKA
pathway, H-89 (5 x 10-6 M) and KT (10-5 M), but
the Ter inhibition at 48 h was incompletely blocked by the cAMP-PKA inhibitors
(Figure 1B and 1C, bar graphs).
These findings suggest that Stx-2 (4 pg/ml)—induced cell cytotoxicity
and its modulation by ß2-adrenoceptor activation seem to
involve not only cAMP-PKA pathway but some other factors.
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Effect of Stx-2 Stimulation and ß2-Adrenoceptor
Activation on TNF- Release
Stx-2—induced TNF- release at 2 h was significantly increased
by the dose of >4 pg/ml (P < 0.05), whereas 2 pg/ml Stx-2 was
not able to increase TNF- release at 2 h from the ACHN cells (data not
shown). Therefore, the minimal dose (4 pg/ml) that significantly stimulated
TNF- release was used for this study.
Figure 2A shows the time course
of the effect of 4 pg/ml Stx-2 on TNF- protein supernatant levels in
the ACHN cell cultures when incubated alone and in the presence of
ß2-adrenoceptor agonist and MAPK (p42/p44, p38) inhibitors.
After 4 h of exposure to 4 pg/ml Stx-2, there was a sharp increase in
TNF- release of some six-fold, which was maintained after 24 h. Ter
(10-6 M), SB (10-6 M), and PD (10-6M)
significantly suppressed these responses (P < 0.05). In
Figure 2B, it can been seen
that the level of Stx-2 (4pg/ml)—induced TNF- release at 2 h was
suppressed in a dose-dependent way by 10-6 M and 10-7 M
Ter (P < 0.05). The inhibitors of the cAMP-PKA pathway, H-89 (5
x 10-6 M) and KT (10-5 M), and the
ß2-adrenoceptor antagonist, ICI (10-6 M),
significantly blocked the inhibitory action of Ter on Stx-2 (4
pg/ml)—mediated TNF- release (all P < 0.05).
Forskolin (10-5 M), PG (10-6 M), and For
(10-6 M) also suppressed the release of TNF- (P
< 0.05). These findings indicate that ß2-adrenoceptor
activation during an Stx-2 (4 pg/ml) challenge prevented TNF- release
through the cAMP-PKA pathway.
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Effect of Stx-2 Stimulation and ß2-Adrenoceptor
Activation on MAPK Activity
Figure 3A illustrates the
time course of the action of 4 pg/ml Stx-2 and/or Ter (10-6 M to
10-8 M) on MAPK (p42/p44) activity in the cells. MAPK (p42/p44)
activity was stimulated by the Stx-2 challenge, beginning after the initial
exposure, reaching a peak level of some six-fold at 20 min, and then returning
to the baseline at 60 min. In the presence of Ter, the peak level of
Stx-2—induced p42/p44 activity was suppressed in a dose-dependent
manner. The comparisons of p42/p44 activity at 20 min among these groups are
shown as bar graphs in Figure
3B. H-89 (5 x 10-6 M) and KT (10-5 M)
overcame the inhibitory action of Ter on Stx-2—mediated MAPK (p42/p44)
activity (P < 0.05). However, the magnitude of the block was
small, and the MAPK (p42/p44) activity was still significantly suppressed by
10-6 M Ter (H-89, 44% inhibition; KT, 42% inhibition; both
P < 0.05). On the other hand, the addition of ICI (10-6
M) significantly blocked the inhibitory action of Ter on Stx-2—mediated
p42/p44 MAPK activity (P < 0.05), indicating that the inhibitory
action of Ter on the Stx-2—mediated p42/p44 activity was dependent on
activation of ß2-adrenoceptors. Forskolin (10-5 M),
PG (10-6 M), and For (10-6 M) also significantly
suppressed Stx-2—mediated p42/p44 MAPK activity (P < 0.05).
Importantly, suppression of Stx-2—induced p42/p44 MAPK activity by
ß2-adrenoceptor activation was partly mediated by the cAMP-PKA
pathway, but it was apparent that other mechanisms were also involved in the
suppression.
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Figure 4A presents the time course of the effect of 4 pg/ml Stx-2 and/or Ter (10-6 M to 10-8 M) on p38 MAPK activity in the cells. p38 MAPK activity was stimulated by the Stx-2 challenge, beginning some 40 min after the initial exposure, reaching a peak level of some 2.7-fold at 60 min, and then returning toward the baseline at 100 min. In the presence of Ter, the peak level of Stx-2—induced p42/p44 activity was suppressed in a dose-dependent manner. In Figure 4B, the comparisons of p38 MAPK activity at 60 min among these groups are shown as bar graphs, and it is evident that Ter (10-6 M) and For (10-6 M) significantly suppressed the Stx-2—mediated p38 MAPK activity (P < 0.05). The addition of ICI (10-6 M) significantly blocked the inhibitory action of Ter on Stx-2—mediated p38 MAPK activity (P < 0.05). This would be consistent with Ter suppressing the Stx-2—mediated p38 MAPK activity via ß2-adrenoceptor activation. Forskolin (10-5 M) or PG (10-6 M) was unable to suppress the Stx-2—induced p38 MAPK activity. Furthermore, H-89 (5 x 10-6 M) and KT (10-5 M) were unable to block the inhibitory action of Ter on Stx-2—mediated p38 MAPK activity. Thus, the inhibitory effect of the ß2-adrenoceptor agonist on p38 MAPK activity was independent of the cAMP-PKA pathway. The time course of action of 4 pg/ml Stx-2 with or without Ter (10-6 M to 10-8 M) on JNK activity in the cells was examined. Neither Stx-2 nor ß2-adrenoceptor activation were able to influence JNK activity in the cells (data not shown).
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Effect of Stx-2 Stimulation and ß2-Adrenoceptor
Activation on AP-1 and NF-B Activity
To address the question of whether Stx-2 stimulation and
ß2-adrenoceptor activation could lead to nuclear AP-1—
and NF-B translocation, an electrophoretic mobility shift assay was
performed on nuclear extracts from the cell. The AP-1—binding activity
of the nuclear extracts was markedly enhanced after 60-min treatment with
4pg/ml Stx-2 and reached a peak level at 2 h, after which it was sustained,
but NF-B (p50, p65) was not induced by this Stx-2 (4pg/ml) challenge
(data not shown). In Figure 5,
a competition analysis was performed to investigate the sequence specificity
of the protein-DNA interaction in the cell that was exposed by Stx-2 (4
pg/ml). Furthermore, to define the subunit composition of the
Stx-2—induced AP-1— and NF-B—binding complexes,
supershift assays were performed by using selective antibodies specifically
for c-Fos, c-Jun, NF-B/p50, and NF-B/p65. Incubation with
antibodies to c-Fos elicited a supershift complex, whereas the antibody to
c-Jun did not alter the gel-shift band, which suggests that Stx-2 induced an
AP-1 binding complex containing c-Fos. For the anti-p50 antibody, the
supershifted complex showed only a moderate extension. The situation with the
p65, was that the supershifted bands could be easily and clearly detected.
Therefore, the results suggest that the NF-B complex contained p65 and
small amounts of p50 proteins. Figures
6A and
7A show that AP-1—binding
activity in response to Stx-2 (4, 16, 64 pg/ml) was increased in a
dose-dependent manner and that Ter (10-8 M) was unable to change
the AP-1 activity. Furthermore, the high doses of Ter (10-6 M or
10-7 M) and For (10-6 M) did not alter the
AP-1—binding activity (Figure
8A). Figures 6B and
7B show that NF-B
(p50)—binding activity was not induced after the Stx-2 (4, 16, 64 pg/ml)
challenge. By contrast, Ter (10-8 M) was able to downregulate
NF-B (p50)—binding activity in both the presence and absence of
Stx-2 (Figure 6B). Moreover,
the high dose of Ter (10-6 M or 10-7 M) significantly
suppressed NF-B (p50)—binding activity in a concentration-related
way (Figure 8B). This response
was blocked by ICI (10-6 M) but not by H-89 (5 x
10-6 M). Thus the suppression of NF-B (p50)—binding
activity by Ter was mediated by ß2-adrenoceptors and was
independent of the cAMP-PKA pathway. Figure
6C and
7C show that NF-B
(p65)—binding activity could not be induced by the Stx-2 (4, 16, 64
pg/ml) challenge. The high doses of Ter (10-6 M or 10-7
M) were able to suppress NF-B (p65)—binding activity
significantly and in a concentration-related way
(Figure 8C), whereas the low
dose of Ter (10-8 M) could not modify NF-B
(p65)—binding activity in either the presence or absence of Stx-2
(Figure 6C). These responses
were blocked by ICI (10-6 M) and H-89 (5 x 10-6
M). Thus, the suppression of NF-B (p65)—binding activity by the
high dose of ß2-adrenoceptor agonist was mediated by
ß2-adrenoceptor activation and was dependent on the cAMP-PKA
pathway. To investigate which pathways of MAPK are involved in the regulation
of Stx-2—induced AP-1— and NF-B—binding activities,
SB (10-6 M) and PD (10-6 M) were added to the cells
exposed to 16 pg/ml Stx-2. The data in
Figure 7 indicate that
AP-1—binding activity induced by the Stx-2 challenge was significantly
suppressed by PD but not by SB, which suggests that the Stx-2—induced
AP-1 was influenced by the p42/p44 MAPK cascade. On the other hand,
NF-B (p50, p65)—binding activity was not significantly suppressed
by either PD or SB, which suggests that NF-B (p50, p65) was not
dependent on a MAPK pathway.
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Regulation of TNF- Promoter Activity
Figure 9A shows that
TNF- promoter activity was stimulated by the Stx-2 challenge, reaching
a peak level of some 2.4-fold at 2 h and then returning toward basal at 3 h.
In Figure 9B, the comparisons
of TNF- promoter activity at 2 h among these groups are given as bar
graphs. Ter (10-6 M or 10-7 M) significantly suppressed
Stx-2—mediated TNF- promoter activity in a dose-dependent manner
(P < 0.05). PD (10-6 M) and SB (10-6 M)
significantly suppressed the Stx-2—induced TNF- promoter activity
(both P < 0.05). Forskolin (10-5 M) and PG
(10-6 M) also depressed Stx-2—mediated TNF- promoter
activity (P < 0.05). H-89 (5 x 10-6 M) and KT
(10-5 M) blocked the inhibitory action of Ter on
Stx-2—mediated TNF- promoter activity (P < 0.05), but
the magnitude of the block was small and the promoter activity was still
suppressed significantly by 10-6 M Ter (H-89, 25% inhibition; KT,
24% inhibition; both P < 0.05). Thus
ß2-adrenoceptor activation and MAPK (p42/p44 and p38)
inhibition resulted in suppression of the Stx-2—induced TNF-
promoter activity. The cAMP-PKA pathway was partially involved in the
suppression. Under these conditions, in which the cells were not treated with
Stx-2, TNF- promoter activity was significantly inhibited (P
< 0.05) by Ter in a concentration-dependent manner by approximately 70% and
59% at 10-6 M and 10-7 M, respectively
(Figure 9C). The Ter
suppression of TNF- promoter activity was abolished by the addition of
H-89 (5 x 10-6 M) and KT (10-5 M), which was most
likely caused by cAMP-PKA pathway activation. The NF-B inhibitor, PDTC
(1.5 x 10-4 M), also significantly suppressed TNF-
promoter activity in the cells (P < 0.05). Therefore, the
constitutive production of TNF- in the cell was regulated through
cAMP-PKA and NF-B activation. However, the inhibition of MAPK (p42/p44,
p38) activities by PD (10-6 M) and SB (10-6 M) did not
attenuate the constitutive expression of TNF- gene in the cell.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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produced by macrophage
(25) and proximal tubule cells
(10) in response to Stx may
have a direct impact on the degree of Stx-mediated cytotoxicity
(9). However, the signal
transduction mechanism whereby TNF- is produced as a result of Stx
stimulation was unknown. In this study, we have demonstrated that Stx-2
induces both p42/p44 MAPK/AP-1 and p38 MAPK pathways and, subsequently,
TNF- production in the ACHN cells. p42/p44 MAPK phosphorylates Elk-1
and activates a serum response element, which leads to c-Fos induction, which
is a component of AP-1 (26).
The AP-1 site is present on the TNF- promoter, and AP-1 plays an
important role in the transcriptional regulation of TNF-
(27). It has been reported
that activation of p42/p44 MAPK and the subsequent TNF- production is
mediated via the Ras/Raf-1 pathway in RAW 264.7 macrophage cells
(28). Therefore, we speculated
that a tyrosine kinase might mediate the Stx-2—induced TNF-
production through the Ras/MAPK/AP-1 pathway. The view was supported by the
observation that the MEK1 inhibitor, PD, which selectively inhibited
Stx-2—induced p42/p44 MAPK phosphorylation and AP-1 activation in this
study, had an inhibitory effect on Stx-2—induced TNF- promoter
activity and protein release from the cells. These results indicate that
p42/p44 MAPK and AP-1 activation are both involved in the important pathway
that mediates the Stx-2—induced TNF- production in the cells. On
the other hand, Stx-2 did not influence the levels of JNK activation, c-Jun
induction, or NF-B (p50 and p65) translocation, which suggests that JNK
and NF-B activation are probably not involved in the major pathway
whereby Stx-2 leads to TNF- gene transcription. This study also
indicates that p38 MAPK in the cells was stimulated by Stx-2. It is also
evident that SB inhibited Stx-2—induced TNF- promoter activity
and protein release from the cells. However, neither DNA binding of
NF-B nor Stx-2—induced AP-1 was affected by SB. In addition, the
p38 MAPK signaling pathway was not influenced at a time when the cAMP-PKA
pathway was activated. Thus, Stx-2 induces p38 MAPK activation with the
subsequent initiation of TNF- gene transcription through routes other
than those that use AP-1, NF-B (p50 and p65), or cAMP-PKA.
An additional observation that arises from this study is that
Stx-2—induced TNF- gene transcription and protein release were
depressed by ß2-adrenoceptor activation. Importantly, however,
this was exerted through both the cAMP-PKA cascade and a cAMP-independent
pathway. Previous investigators
(15,29,30)
have reported that the inhibition of TNF- production by
ß2-adrenoceptor activation was dependent on an increase in
intracellular cAMP levels. This view is consistent with our study in that the
depressed TNF- protein release in response to Stx-2 after exposure to
ß2-adrenoceptor agonists could be abolished by the cAMP-PKA
inhibitor, H-89, or KT. Moreover, at the transcriptional level, one component
of the mechanisms that inhibit TNF- gene transcription could be
attributed to an action of intracellular cAMP. The constitutive activity of
TNF- promoter in the cells was suppressed by the NF-B inhibitor,
PDTC, but not by the MAPK inhibitors, PD and SB. Thus, the suppression of
TNF- gene transcription in the absence of Stx-2 appeared to be mediated
by NF-B (p65) translocation via a cAMP-PKA pathway. Conversely, in the
presence of Stx-2, the suppression of TNF- gene transcription is
partially mediated by MAPK as well as NF-B translocation via a cAMP-PKA
pathway. These observations would support the reports of Olivier et
al. (31) and Zhong et
al. (32), who found that
an increase in intracellular cAMP concentration decreased the
NF-B—mediated function of TNF- gene transcription.
Furthermore, Burgering et al.
(33) demonstrated that
elevated cAMP could block MAPK activation of oncogenic Ras proteins. Together,
these pieces of evidence suggest that the cAMP pathway leads to an inhibition
of Stx-2—induced MAPK (p42/p44) activation that indirectly suppresses
TNF- gene transcription, whereas an inhibition of NF-B (p65)
translocation by intracellular cAMP would lead to a decrease in TNF-
promoter activity. Interestingly, Zhong et al.
(32) reported that cAMP-PKA
not only regulates activation of NF-B, but also that of AP-1. However,
Grassl et al. (34)
found that AP-1 DNA-binding activity was enhanced after treatment of cAMP. We
were, however, unable to detect a significant change in AP-1—binding
activity after ß2-adrenoceptor activation. The reasons for
this discrepancy between their results and our data in this study are unclear.
It is evident from our findings that the inhibition of MAPK (p42/p44) by
ß2-adrenoceptor stimulation did not regulate AP-1 induction in
these cells.
Conversely, MAPK (p38) and NF-B (p50) were found to be involved in
the suppression of TNF- gene transcription by a cAMP-independent
pathway. Moreover, the Ter-induced downregulation of MAPK (p42/p44) activity
was only partly abolished by the addition of H-89 or KT, which suggests that
the inhibition of MAPK (p42/p44) activity by ß2-adrenoceptor
activation was partially independent of the cAMP-PKA pathway. Together, these
findings would suggest that ß2-adrenoceptor stimulation during
the Stx-2 challenge is able to prevent TNF- gene transcription not only
as a consequence of the enhanced cAMP generation but also as a consequence of
MAPK (p42/p44 and p38) and NF-B (p50) inhibition. This view would be
consistent with the report of Seldon et al.
(35), who found that the
inhibitory effects of ß2-adrenoceptor agonists on TNF-
gene transcription and/or translation could be regulated by both a cAMP-PKA
cascade and a cAMP-independent mechanism. Intracellular signaling pathways
other than cAMP-PKA could also be involved in the modulation of MAPK and
NF-B by ß2-adrenoceptors. Crespo et al.
(36) reported that signaling
that arises from ß2-adrenoceptor stimulation leading to MAPK
activation involves an initiating signal that requires ß subunits
of G protein and a Gs-induced inhibitory signal mediated by the
cAMP-PKA pathway. However, we were unable to observe an elevation of MAPK as a
result of exposure of the cells to the ß2-adrenoceptor agonist
alone (17).
Stx consists of an A subunit of 32 kD associated with five B subunits, each
of 7.5 kD. The B subunits bind specifically to the cell surface Gb3 receptor,
also known as CD77, and facilitate the entry of the holotoxin into susceptible
cells (37). ACHN cells express
the receptor for Stx, Gb3/CD77, and binding to the CD77 antigen induces cell
cytotoxicity/apoptosis (11).
Taga et al. (38) have
reported that ligand binding to CD77 causes an increase of intracellular
Ca2+ concentration and a rapid increase in cAMP in Burkitt lymphoma
cell, which seems to be a critical step in the apoptotic process. However, in
ACHN cells, intracellular cAMP generation was not found to be elevated by the
addition of Stx-2 (data not shown), and the activation of the cAMP-PKA pathway
via ß2-adrenoceptor appears to be part of a protective
response against Stx-2—induced cell cytotoxicity. Katagiri et
al. (39) showed that Stx
binding to Gb3 causes activation of the Src family tyrosine kinase Yes.
Whether these intracellular signaling pathways are part of the process that
leads TNF- gene transcription and induces cell death is still unclear.
Hughes et al. (10)
examined the effect of protein synthesis inhibitor, cycloheximide, on
TNF- mRNA and protein release in human glomerular epithelial cells and
suggested that the mechanism of Stx-1—induced TNF- production
was, in part, independent of global inhibition of protein synthesis or
cytotoxicity. We were able to demonstrate that Stx binding to the Gb3/CD77
receptor stimulated TNF- production in ACHN cells through both the
p42/p44MAPK/AP-1 pathway and the p38MAPK pathway. However, it has to be
accepted that other intracellular signaling pathways induced by Stx may be
involved in the activation of TNF- gene transcription. The mechanisms
that transduce the signal mediated by Stx binding to Gb3/CD77 on ACNH require
further examination. In the renal tubule, it is quite clear that
ß2-adrenoceptors are present and able to influence tubular
function (40). We have also
confirmed in this study that functional ß2-adrenoceptors are
expressed on the ACHN cells. In an in vivo study, it has been found
that ß2-adrenoceptors are distributed predominantly in the
outer and inner stripe of the outer medulla in the rat kidney
(41). A significant inhibitory
effect of ß2-adrenoceptor agonists on
lipopolysaccharide-induced TNF- mRNA has been observed in the medullary
region of rat (42). However,
it is unclear whether ß2-adrenoceptor agonists are able to
inhibit TNF- production in the human kidney in response to Stx.
Therefore, the importance of ß2-adrenoceptor stimulation as a
means whereby TNF- can be inhibited must be investigated further by
using human tissue and then in humans. It may offer a therapeutic approach to
overcome the impairment of renal tubular function in HUS.
In summary, we found that Stx-2 induced p42/p44 MAPK/AP-1 and p38 MAPK
activation with subsequent TNF- gene transcription. The
ß2-adrenoceptor agonist inhibited NF-B (p50, p65)
activation and Stx-2—induced p42/p44 MAPK and p38 MAPK activation,
which, in turn, was correlated with reduction in TNF- gene
transcription. The inhibitory effect was regulated by both a cAMP-PKA pathway
and a cAMP-independent mechanism.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß and p38
mitogen-activated protein kinase on tumor necrosis factor- production.
Endocrinology 140:3562
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cells and endothelial cells. J Biol Chem271
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P < 0.05 versus cells exposed with
4pg/ml Stx-2 plus Ter (10-6 M).
, control; [UNK], Stx-2; 
, Stx-2 + Ter
(10-6 M); 
, Stx-2 + PD (10-6 M); 
, Stx-2 +
SB (10-6 M). (B) The inhibitory effect of Ter or formoterol (For)
on TNF-
