From the Institut für Medizinische
Strahlenkunde und Zellforschung (MSZ) and ¶ Klinik und Poliklinik
für Haut und Geschlechtskrankheiten, Universität
Würzburg, D-97078 Würzburg, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Tumor necrosis factor a (TNF- Tumor necrosis factor The diverse stimuli that up-regulate TNF- The family of MAPKs consists of at least three subgroups:
(a) the extracellular signal-regulated kinase (ERK),
(b) the Jun N-terminal kinase, which is also known as
stress-activated protein kinase (JNK/SAPK), and (c) the p38
subgroup of MAPKs (for a review, see Ref. 13). The human homolog of p38
designated CSBP has been identified as the binding protein of the
pyridinyl-imidazole compound SB203580 that was shown to have an
inhibitory effect on LPS-stimulated TNF- Whereas MAPK-activating pathways have been implicated in LPS-induced
TNF- Two findings suggest a cell type-specific involvement of intracellular
signaling pathways inducing TNF- To investigate T-cell-specific regulation of the TNF- Cell Lines and Antibodies--
A3.01 human T lymphoma cells were
grown in RPMI 1640 medium supplemented with 10% fetal calf serum to a
density of 8 × 105 cells/ml. Cells were incubated at
37 °C in humidified air with 7% CO2. Antibodies raised
against ERK2 (sc-154), JNK1 (sc-474), and p38 (sc-535) were purchased
from Santa Cruz Biotechnology, Inc. The monoclonal antibodies against
the HA tag (12CA5) were produced and purified according to a standard
protocol. The TNF- DNA Constructs and Cloning--
The human TNF-
The eukaryotic expression vector for HA-SAPK
The pRSPA vector system was used for the expression of all cDNAs in
eukaryotic cells. pRSPA is an expression vector with the Rous sarcoma
virus promoter and the simian virus 40 polyadenylation signal region in
a pBluescript backbone (33). The cDNA of MLK3 and the corresponding
kinase inactive mutant were kindly provided by K. Gallo and P. Godowski
(34). Raf-BXB-CX (constitutively active Raf) lacks the N-terminal
negative regulatory domain and contains the C-terminal membrane
targeting 17 amino acids of Ki-Ras fused to the kinase domain of c-Raf
l (18, 35). MKK6(EE) is a constitutively active mutant of MKK6 with two
serines involved in the activation of the kinase replaced by glutamic
acid (26). The interfering mutants of ERK2, SAPK Transient Transfections and Reporter Gene Assays--
Cells
were split to a density of 4 × 105 cells/ml 1 day before
transfection. A DMRIETM-C based transfection protocol was
used according to the manufacturer's instructions (Life Technologies).
Cells were seeded in 6-well plates (7 × 105
cells/well) in 1.5 ml of Opti-MEM (Life Technologies) containing 3 µl
of DMRIETM and up to 3 µg of vector DNA. Transfections
for luciferase assays were performed with 0.5 µg of reporter
construct plus 2 µg of pRSPA containing diverse cDNAs. Unless
otherwise indicated, cells in each well were harvested in 100 µl of
lysis buffer (50 mM
Na-2-(N-morpholino)ethanesulfonic acid, pH 7.8, 50 mM Tris-HCl, pH 7.8, 10 mM dithiothreitol, and 2% Triton X-100) 24 h after transfection. The crude cell lysates were cleared by centrifugation, and 50 µl of precleared cell extracts were added to 50 µl of luciferase assay buffer (125 mM
Na-2-(N-morpholino)ethanesulfonic acid, pH 7.8, 125 mM Tris-HCl, pH 7.8, 25 mM magnesium acetate, and 2 mg/ml ATP). Immediately after the injection of 50 µl of 1 mM D-luciferin (AppliChem) into each sample,
the luminescence was measured for 5 s in a luminometer (Berthold).
The luciferase activities were normalized on the basis of protein
content as well as on the
A3.01 T cells were stimulated with 10 ng/ml TPA (Sigma) or 0.5 µM ionomycin (Sigma) for up to 24 h. The
MEK-specific inhibitor PD098059 (Calbiochem) was used in a 20 µM concentration of a 20 mM stock solution in
DMSO. The p38-specific inhibitor SB203580 (Calbiochem) was used at a
concentration of 2-20 µM of a 20 mM stock
solution in DMSO. Actinomycin D (Sigma) was used at a concentration of
2 µg/ml of a 0.4 mg/ml stock solution in 10% ethanol, and
cyclosporin A (CsA) (Sigma) was used at a concentration of 200 ng/ml of
a 10 mg/ml stock solution in DMSO. Cells were preincubated with these
inhibitors 30 min before stimulation.
TNF-specific Flow Cytometry Analysis--
To determine the
expression of TNF- Immunoprecipitation, Kinase Assay, and Immunoblotting--
Cells
were lysed in radioimmunoprecipitation buffer (25 mM
Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 0.1% SDS,
0.5% deoxycholate, 1% Nonidet P-40, 2 mM EDTA, 1 mM Pefabloc, 1 mM sodium orthovanadate, 5 mM benzamidine, 5 µg/ml aprotinin, and 5 µg/ml
leupeptin), and cell debris was removed by centrifugation. Supernatants
were incubated with different antisera for 2 h at 4 °C. The
immunocomplexes were precipitated with protein A-agarose (Boehringer)
and washed twice with high-salt radioimmunoprecipitation buffer
containing 500 mM NaCl. Immunocomplexes were used for
in vitro kinase assays as described previously (36) with
myelin basic protein (MBP), 3pK(K-M), and glutathione
S-transferase (GST)-c-Jun(1-135) as substrates for ERK,
p38, and JNK, respectively. Proteins were separated by
SDS-polyacrylamide gel electrophoresis, blotted onto polyvinylidene
difluoride membranes, and detected with a BAS 2000 Bio Imaging Analyzer
(Fuji) and by autoradiography. The appropriate primary antibodies and
peroxidase-coupled protein A were used for detection of the
immunoprecipitated proteins in immunoblots, followed by a standard
enhanced chemiluminescence reaction (Amersham).
Selective Activation of ERK, JNK, or p38 Signaling Pathways
Stimulates TNF-
Expression of each of these kinases in A3.01 cells is sufficient to
induce strong TNF-
We next investigated the role of MAPK pathways in TNF- Expression of TNF-
Therefore, we analyzed the time dependence of TNF-
As shown previously (17, 18), TPA/ionomycin treatment leads to a strong
activation of the MAPK family members ERK, JNK, and p38 in T cells,
whereas TPA stimulation leads solely to a maximal activation of ERK. To
correlate the kinetics of MAPK activation with the level of TNF- Selective Inhibition of Distinct MAPKs in A3.01 T Cells--
To
test whether MAPK pathways are functionally involved in
TPA/ionomycin-induced TNF-
The PD98059 compound has been described as a specific inhibitor of MEK
activation (39). Indeed, titration experiments in A3.01 cells (data not
shown) revealed maximal inhibitory effects of this inhibitor at a
concentration of 20 µM, at which it acts specifically on
ERK without affecting JNK or p38 activity (Fig. 4). Preincubation of A3.01 cells with
this inhibitor resulted in a 90% inhibition of TPA/ionomycin-induced
ERK activation as determined by immunocomplex kinase assays (Fig.
4A, top panel). In contrast, JNK and p38 activity
remained unaffected by PD98059 (Fig. 4A, middle
and bottom panels).
Because TNF-
Specific inhibition of JNK/SAPK activity was achieved by overexpression
of the protein JIP-1 (Ref. 16; see "Experimental Procedures" for
details). To determine the inhibitory efficiency of JIP-1 in
transiently transfected A3.01 T cells, we coexpressed JIP-1 with
HA-SAPK
As an inhibitor of the p38 pathway, compound SB203580 has been
successfully used in a variety of cellular systems (for a review, see
Ref. 40). This compound binds to the ATP-binding site of p38, thereby
inhibiting its catalytical activity (41). The inhibitory efficiency of
SB203580 in A3.01 cells was evaluated by p38-dependent transcription of a reporter gene. For this purpose, a construct carrying four copies of a combined AP-1/Ets binding site of the polyoma
virus enhancer in front of a luciferase gene was used. This enhancer
element has been previously described to be responsive to
constitutively active Raf in NIH3T3 cells (42). In A3.01 T cells, the
activator protein- 1 (AP-1)/Ets reporter gene construct is induced
by the expression of either Raf-BXB-CX, MLK3, or MKK6(EE), providing a
useful tool for the identification of selective inhibitors of distinct
MAPK signaling pathways. The enhancer induction by MKK6(EE) was reduced
to more than 95% by treating A3.01 cells with increasing amounts of
SB203580 (Fig. 4C). However, concentrations of SB203580
higher than 4 µM also resulted in an unspecific
inhibition of Raf-BXB-CX- and MLK3-induced AP-1/Ets enhancer activity,
indicating the loss of specificity of this compound at higher
concentrations (Fig. 4C). This was confirmed by monitoring
TPA/ionomycin-stimulated ERK activity, which was repressed by 80% when
the cells were pretreated with 10-20 µM SB203580 (data
not shown). Specificity of the inhibitory effect of SB203580 for p38
but not ERK or JNK/SAPK was achieved at a concentration of 4 µM, at which it inhibits MKK6(EE)-induced AP-1/Ets
enhancer activity to 93% (Fig. 4C).
The inhibitory effect of dominant interfering mutants of ERK2
(ERK2(B3)), SAPK ERK-, JNK-, and p38-activating Pathways Critically Contribute to
TPA/Ionomycin-induced TNF-
Blockage of ERK and p38 activation by PD98059 and SB203580,
respectively, results in a significant decrease in TNF-
Inhibition of ERK signaling by ERK2(B3) overexpression or pretreatment
with PD98059 also impaired the TNF-
These data point to a cooperation of MAPK signaling pathways in the
regulation of the TNF-
These data suggest that all three MAPK signaling pathways cooperate in
the regulation of the TNF- Distinct Regions of the TNF-
The intact promoter up to nucleotide
These data indicate that there are overlapping but distinct regions of
the TNF- In this report, we show for the first time that selective
activation of ERK, JNK, or p38 signaling pathways is sufficient for
rapid transcriptional activation of the TNF- According to our data, the first regulatory step of induced TNF- Our findings demonstrate that TNF- The JNK pathway also appears to be of critical importance for TNF- Our study also defined a functional role of the p38 pathway in the
transcriptional induction of TNF- The role of the p38 pathway in transcription of the TNF- Although selective activation of either pathway is sufficient to induce
TNF- It was reported earlier that CsA affects JNK but not ERK activity in
activated T cells (17). Here we show that p38 activity is also
partially inhibited by CsA (Fig. 4A), suggesting a similar activation route for p38 and JNK (49) Because CsA only inhibits TNF- The different observations concerning the contribution of MAPK pathways
to the transcriptional regulation of TNF- Deletion of the promoter from nucleotide In conclusion, our data indicate that TNF-) is a potent
proinflammatory cytokine and plays a crucial role in early events of
inflammation. TNF-
is primarily produced by monocytes and T
lymphocytes. In particular, T-cell-derived TNF-
plays a critical
role in autoimmune inflammation and superantigen-induced septic shock.
However, little is known about the intracellular signaling pathways
that regulate TNF expression in T cells. Here we show that
extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase
(JNK), and p38-mitogen-activated protein kinase (MAPK) pathways control
the transcription and synthesis of TNF-
in A3.01 T cells that
produce the cytokine upon T cell activation by costimulation with
12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin.
Selective activation of each of the distinct MAPK pathways by
expression of constitutively active kinases is sufficient for TNF-
promoter induction. Furthermore, blockage of all three pathways almost
abolishes TPA/ionomycin-induced transcriptional activation of the
TNF-
promoter. Selective inhibition of one or more MAPK pathways
impairs TNF-
induction by TPA/ionomycin, indicating a cooperation
between these signal transduction pathways. Our approach revealed that
the MAPK kinase 6 (MKK6)/p38 pathway is involved in both
transcriptional and posttranscriptional regulation of TNF expression.
Moreover, analysis of the progressive 5' deletion mutants of the
TNF-
promoter indicates that distinct promoter regions are targeted
by either ERK-, JNK-, or p38-activating pathways. Thus, unlike what has
been reported for other TNF-
-producing cells, all three MAPK
pathways are critical and cooperate to regulate transcription of the
TNF-
gene in T lymphocytes, suggesting a T-cell-specific
regulation of the cytokine.
INTRODUCTION
Top
Abstract
Introduction
References
(TNF-
)1 is primarily
produced by cells of hematopoietic origin, such as lymphocytes,
monocytes, and mast cells. T lymphocytes produce the cytokine when they
are activated via their antigen receptor, and cells of the
monocyte/macrophage lineage generate it upon lipopolysaccharide (LPS)
stimulation (1). Mast cells also secrete TNF-
after high-affinity
IgE receptor aggregation (2). TNF-
is among the earliest activated cytokines in inflammation, and its production is crucial for the development of an early defense against many pathogens (reviewed in
Ref. 1). However, these beneficial effects of TNF-
are dependent on
the strength and duration of its expression. High systemic levels of
TNF-
induced by stimulation of T cells with bacterial superantigen
(3) or by LPS stimulation of macrophages (1, 4) cause subsequent septic
shock. Furthermore, the critical role of TNF-
in the generation of
autoimmune inflammation has been defined by the targeted disruption of
the TNF gene (5). In addition to autoimmune diseases (6) and
superantigen-induced septic shock (7), the pivotal role of
T-cell-produced TNF-
in the modulation of inflammatory responses is
further reflected in the observation that T-cell membrane-bound TNF-
is of particular importance for the regulation of monocytic interleukin
10 and TNF-
production (8).
expression in different
cells are known to be activators of MAPK-activating signaling pathways.
Indeed, in monocytes, macrophages, and mast cells, it was shown that
MAPK activation plays a central role in the induced TNF-
expression
(2, 9-12), whereas little is known about the regulation of this
cytokine in T lymphocytes.
production by human
monocytes (10). JNK and p38 differ from ERK in that they are
predominantly regulated by cellular stress inducers and proinflammatory
cytokines (14, 15). Whereas in T cells, T cell receptor (TCR) ligation
or TPA treatment is sufficient to maximally induce ERK activity, JNK
and p38 activation requires a costimulatory signal such as CD28 ligand
binding or ionomycin cotreatment, respectively (16-18). MAPKs are
activated by other kinases functioning in a kinase cascade. The direct
upstream kinase of ERK is MEK, which is regulated via phosphorylation
by Raf (19). JNK is activated by SAPK/ERK kinase (SEK, also known as
MKK4) as well as by the recently identified kinase MKK7 (20-22). The
activation of JNK is further controlled by a putative scaffold protein,
JNK-interacting protein 1 (JIP-1), which binds to JNK and several other
components of the JNK pathway (23). Overexpression of JIP-1 or the
JNK-binding domain of JIP-1 leads to the cytoplasmic retention of JNK
and the inhibition of JNK-dependent gene expression (16).
One of the SAPK/ERK kinase activators (reviewed by Fanger et
al., Ref. 20) is the mixed lineage kinase 3 (MLK3) also known as
the SH3 domain-containing proline-rich kinase (SPRK) (24). MAPK kinase
6 (MKK6) functions as an activating kinase for all known p38 isoforms
(25-27), whereas MKK3 predominantly activates the isoform p38
(28).
Until this time, a specific physiological activator of MKK6 has not
been identified.
expression by monocytes and macrophages at diverse control
levels (10-12), and two reports show that the JNK and ERK pathways
play a role in TNF-
expression by mast cells (2, 9), the
contribution of MAPKs to the regulation of TNF-
expression in T
lymphocytes is still unclear.
expression: (a) in lymphocytes versus monocytes, different sets of
transcription factors are recruited to the promoter of the TNF-
gene
(29, 30), and (b) extracellular stimuli with a cell
type-specific function trigger TNF-
expression in different cells,
such as LPS in monocytes, Fc
RI receptor aggregation in mast cells,
or activation of the antigenic receptor in T lymphocytes. Antigenic activation of T lymphocytes, which can be mimicked by costimulation with a phorbol ester such as TPA and a calcium ionophore such as
ionomycin (for a review, see Ref. 31), leads to a rapid induction of
TNF-
transcription that does not require new protein biosynthesis (32).
gene, we analyzed the involvement of distinct MAPK pathways in TNF-
transcription and biosynthesis upon activation of the human T-cell line
A3.01. We demonstrate that ERK, JNK, and p38 pathways that are
activated upon stimulation with TPA and ionomycin (TPA/ionomycin) are
critical for and cooperatively contribute to the induction of TNF-
expression in these T cells.
EXPERIMENTAL PROCEDURES
monoclonal antibody was purchased from
PharMingen, Inc.
promoter
(
1057/+131; a generous gift of Dr. S. A. Nedospasov, Laboratory
of Molecular Immunoregulation, PRI/DynCorp; National Cancer Institute,
Frederick Cancer Research and Development Center, MD) was cloned into
the HindIII site of pGL3 basic luciferase expression vector
(Promega). The diverse 5' deletion mutants of the TNF-
promoter were
produced by restriction digestion or PCR amplification of regions of
the human TNF-
promoter from nucleotide +131 relative to the
transcriptional start site to various deletion end points described in
Fig. 6. The promoter fragments were cloned into the pGL3 basic
luciferase expression vector, and successful cloning was confirmed by sequencing.
and the prokaryotic
expression vector pGEX-KG-c-Jun(1-135) were gifts from J. Kyriakis and
L. Zon.
, MKK6, Raf-BXB-CX,
and MLK3 are ATP-binding site mutants generated by the replacement of
lysine with arginine (ERK2(B3), SAPK
(K-R), and Raf-BXB-CX375) or
alanine (MKK6(A) and MLK3 K144A) (26, 34, 36, 37). JIP-1 is a
cytoplasmic protein that was identified as a putative scaffold protein
that binds to several components of the JNK pathway and regulates JNK
activity (23). Overexpression of JIP-1 or the JNK-binding domain of
JIP-1 inhibits JNK activity by causing cytoplasmic retention of JNK
that leads to the subsequent inhibition of JNK-regulated gene
expression (16). The JIP-1 cDNA used in this study consists of the
JNK-binding domain fused to a Flag-Tag and was kindly provided by R. Davies. All cDNAs were subcloned in the pRSPA vector.
-galactosidase activity of cotransfected
Rous sarcoma virus LTR
-gal vector. The
-galactosidase assay was performed with 20 µl of precleared cell lysate according to a standard protocol (38). Mean and standard deviations of at least three
independent experiments are shown in the figures.
, an intracellular immunostaining procedure and
subsequent flow cytometry analysis were applied. A3.01 T cells were
split 24 h before stimulation. The stimulation was carried out in
the presence of 2 mM monensin (Sigma), which prohibits the
secretion of proteins, thereby leading to intracellular retention of
the produced protein. Treatment of monensin did not affect the basal or
induced MAPK activity (data not shown). After a stimulation time of 2 or 10 h, cells were harvested, washed once in phosphate-buffered
saline, fixed with 4% (w/v) paraformaldehyde in phosphate-buffered
saline at 4 °C for 20 min, and subject to the incubation and washing
steps described below in permeabilization buffer containing 1% fetal
calf serum and 0.1% (w/v) saponin in phosphate-buffered saline.
According to the manufacturer's instructions (PharMingen), cells were
then incubated with the primary antibody in permeabilization buffer supplemented with 2% goat serum. A mouse IgG1 antiserum (Dako) was
used as an isotype-specific control for the monoclonal mouse anti-human
TNF-
antibody of isotype IgG1 (PharMingen). After two washing steps,
cells were exposed to biotin-SP-conjugated goat anti-mouse IgG
F(ab')2 (Dianova), washed again, and stained with
streptavidin-Cy-chrome (PharMingen). Fluorescence was measured on
10,000 cells/sample using a FACScan (Beckton Dickinson).
RESULTS
Promoter-dependent
Transcription--
To investigate the role of the ERK, JNK, or p38
signaling pathways as mediators of induced TNF-
transcription, we
performed transient cotransfection experiments and measured the human
TNF-
promoter activity as promoter-dependent luciferase
expression in the human T-cell line A3.01. Previously, we have
established an approach to selectively activate ERK, JNK, or p38 in
A3.01 T cells by expressing constitutively active versions of
corresponding upstream kinases (18). Briefly, a constitutively active
kinase mutant of Raf (Raf-BXB-CX) serves as a specific ERK activator. Overexpression of MLK3 results in a strong activation of JNK without affecting ERK and p38 activities. Finally, an active mutant of MKK6
(MKK6(EE)) is a specific activator of p38 (18).
promoter activity in a
concentration-dependent manner (Fig.
1A-C, see also Fig. 6),
although they exert no effect on a nonspecific thymidine kinase minimal
promoter (data not shown). The corresponding catalytically inactive
kinase versions showed no significant effect on the TNF-
promoter,
even at the highest input (Fig. 1, A-C). Moreover,
combining MKK6(EE) with either Raf-BXB-CX or MLK3 synergistically
enhanced the promoter activity (Fig. 1D), suggesting
cooperation between these signaling pathways in the regulation of
TNF-
-specific transcription.
View larger version (20K):
[in a new window]
Fig. 1.
Expression of Raf-BXB-CX, MLK3, and MKK6(EE)
induces TNF- -dependent reporter gene expression.
A-C, A3.01 cells were cotransfected with 0.5 µg of the
TP(
1057) TNF-
promoter construct together with either increasing
amounts (1.0 or 2.0 µg DNA/transfection) of Raf-BXB-CX
(A), MLK3 (B), or MKK6 (EE) (C)
expression vector or the corresponding empty expression vector pRSPA
(2.0 µg; con.). Transfection of 1.0 or 2.0 µg of empty
expression vector pRSPA results in the same basal luciferase activity
(data not shown). As a control, the same experiments were performed
with corresponding kinase-inactive mutants (neg.) for Raf
(Raf-BXB-CX375W; A), MLK3 (MLK3 K144A; B), and
MKK6 (MKK6(Ala); C) (2.0 µg DNA). The figures show a mean
of three independent transfection experiments. D, the same
experiments as described above were performed with a combination of
different kinases in a 1:1 ratio as indicated. For control purposes,
pRSPA-GFP (green fluorescent protein) was used to equalize the amount
of expressed protein. The figure shows a mean of six independent
experiments. At 24 h posttransfection, cells were harvested, and
luciferase assays were performed (see above). The relative luciferase
activities of cells transfected with each cDNA expression vector
are based on the activities of cells transfected with the same amount
of control expression vector (pRSPA).
gene
expression of T cells activated by TPA/ionomycin in more detail.
by Activated A3.01 T Cells--
To
characterize the regulation of TNF-
expression, we stimulated A3.01
T cells with TPA, ionomycin, or a combination of both. The inducibility
of TNF-
expression was determined at both the translational and
transcriptional levels by TNF-
-specific flow cytometry analysis and
a TNF-
promoter reporter gene assay, respectively. Reporter gene
analysis allows for the assessment of TNF-
promoter activity
independent of TNF-
mRNA stability, another control level of
expression. Unstimulated A3.01 cells do not produce any detectable
amount of TNF-
(Fig. 5A). Stimulation of these cells with
TPA results in a weak induction of TNF-
transcription (Fig. 2B) and synthesis (Fig.
2A). A high induction of both transcription and protein
synthesis was observed by cotreatment with TPA and ionomycin (Fig. 2).
TPA/ionomycin-induced TNF-
production is sensitive to
cyclosporin A, whereas TPA-induced protein synthesis is unaffected
(Fig. 2A). TNF-
protein synthesis was detectable as early
as 2 h after TPA/ionomycin stimulation only in the absence of the
transcriptional inhibitor actinomycin D (Fig. 2A),
indicating that TPA/ionomycin-induced TNF-
synthesis requires
de novo transcription of the TNF-
gene.
View larger version (22K):
[in a new window]
Fig. 2.
Expression of TNF- by A3.01 T cells.
A, to measure TNF-
production upon stimulation, A3.01 T
cells were stained intracellularly with an anti-TNF-
monoclonal
antibody and analyzed by fluorescence-activated cell-sorting analysis
(see "Experimental Procedures"). The cells were stimulated with TPA
(T) or TPA/ionomycin (T+I) or left untreated
(w/o) for the times indicated. Some of the cells were
pretreated with CsA or the transcriptional inhibitor actinomycin D. The
mean of the TNF-
specific fluorescence intensity of stimulated cells
is based on that of unstimulated cells and is given in fold
stimulation. The experiment was repeated three times. B, to
analyze the induction of TNF-
promoter-dependent
transcription, the promoter construct TP(
1057) was cotransfected with
a Rous sarcoma virus LTR-driven
-galactosidase expression vector.
Cells were stimulated with TPA (T), ionomycin
(I), or both (T+I) for 16 h or left
untreated (w/o). The relative luciferase activity equalized
on
-galactosidase activity is given in fold stimulation based on
untreated cells (w/o). The figure shows a mean of three
independent transfections and is representative of four independent
experiments performed in triplicates.
transcription
compared with TNF-
production. TNF-
promoter activity as well as
protein synthesis is induced after 2 h of TPA/ionomycin stimulation and reaches maximal induction levels after 6 and 8 h,
respectively (Fig. 3, A and
B). These results indicate that the regulation of TNF-
expression in A3.01 cells is similar to that of primary T cells and
other T-cell lines (reviewed in Ref. 1).
View larger version (32K):
[in a new window]
Fig. 3.
The activation of ERK, JNK, and p38 precedes
the induction of TNF- transcription and production. A,
TNF-
production was measured as described in the Fig. 2 legend. Fold
stimulation of the relative fluorescence intensity is based on that of
unstimulated cells (time point 0). B, TNF-
promoter
activity was measured as described in the Fig. 2 legend. Cells were
harvested 24 h after transfection. Fold stimulation of the
relative luciferase activity is based on that of unstimulated cells
(time point 0). C-E, A3.01 cells were stimulated with
TPA/ionomycin for the indicated times. After cell lysis, kinase
activities were determined in immunocomplex kinase assays with MBP,
GST-c-Jun(1-135), and 3pK(K-M) as substrates for ERK, JNK, and p38,
respectively. Immunoblots show equal amounts of immunoprecipitated
kinases.
transcription, we determined the kinase activities of ERK, JNK, and p38
in time course experiments. ERK activity is maximally induced within 5 min of TPA/ionomycin treatment, whereas JNK and p38 activation reach
maximal induction after 15 min. After 60 min, MAPK activities drop to
levels that are still detectable after 4 h. This rapid stimulation
of MAPK activity preceding the induction of TNF-
transcription
suggests a functional connection of both processes.
expression, we established conditions to
selectively inhibit the activation of each of the three MAPKs. For this
purpose, specific kinase inhibitors and negative interfering kinase
mutants were tested for efficiency and specificity.
View larger version (44K):
[in a new window]
Fig. 4.
PD98059, SB203580, and JIP-1 are selective
inhibitors for ERK, p38, and JNK, respectively, in A3.01 T cells.
A, cells were left untreated (c, w/o)
or were treated with the solvent DMSO, PD98059, or CsA before
stimulation with TPA/ionomycin. Kinase activities of ERK (A, top
panel), JNK (A, middle panel), and p38 (A, bottom
panel) were determined as described above. Numbers in
bold in the autoradiogramms indicate kinase activation in fold compared
with the unstimulated control (c). Corresponding immunoblots
verify equal amounts of immunoprecipitated kinases. B, cells
were cotransfected with HA-SAPK and MLK3 and/or JIP-1 and stimulated
with TPA/ionomycin as indicated. HA-SAPK
was immunoprecipitated from
each sample using a HA-specific monoclonal antibody, and its activity
was determined in immunocomplex kinase assays with GST-c-Jun(1-135) as
a substrate. Equal protein load was verified by immunoblotting.
C, the 4x AP-1/Ets promoter construct was used to determine
the specificity of PD98059 and SB203580. This reporter construct is
inducible by transfection of either Raf-BXB-CX, MLK3, or MKK6(EE) by
95-, 400-, or 250-fold, respectively (data not shown). A3.01 cells were
cotransfected with the 4x AP-1/Ets promoter construct and either
Raf-BXB-CX, MLK3, or MKK6(EE). Cells were left untreated or treated
immediately after transfection with either DMSO (solvent control),
PD98059 (PD), or increasing amounts of SB203580 as
indicated. Promoter activities are expressed as the percentage of
relative luciferase activity based on that of untreated controls
(w/o) of each kinase. The figure shows the mean of three
independent experiments.
production (Fig. 2A) as well as
TNF-
promoter-dependent transcription (data not
shown) is sensitive to cyclosporin A, we measured the effects of
this inhibitor on the MAPK activities. Interestingly, JNK as well as
p38 activities were significantly inhibited, whereas ERK was not
affected (Fig. 4A). JNK inhibition by CsA has been observed
previously in Jurkat T cells (17); however, p38 was not included in
those studies.
. SAPK activity was markedly reduced by the coexpression of
JIP-1 when the cells were stimulated with TPA/ionomycin or
cotransfected with MLK3, a strong JNK/SAPK activator (Fig. 4B).
(SAPK
(K-R)), and MKK6 (MKK6(A)) was confirmed in
our previous studies by kinase assays and reporter gene analysis in
different cell systems including A3.01 T cells (18, 43). These studies
established ERK2(B3), SAPK
(K-R), and MKK6(A) as efficient dominant
negative mutants selective for the ERK, SAPK, and p38 signaling
pathways, respectively.
Expression--
After establishing tools
for the selective disruption of signaling through specific MAPK
cascades, we tested the effects of these inhibitors on the
TPA/ionomycin-induced TNF-
promoter activity (Fig.
5B) and TNF-
biosynthesis
measured by TNF-
specific fluorescence (Fig. 5A).
View larger version (27K):
[in a new window]
Fig. 5.
TPA/ionomycin-induced TNF- synthesis and
transcription are inhibited by dominant negative mutants or specific
kinase inhibitors of different MAPK signaling pathways. A,
A3.01 T cells were either left untreated (w/o) or stimulated
for 10 h with TPA/ionomycin (T+I). 20 min before
stimulation, cells were pretreated with either 20 µM
PD98059 (PD), 4 µM SB203580 (SB),
or DMSO or left untreated (w/o). Cells were subsequently
analyzed by flow cytometry. The data shown represent the flow cytometry
profiles of one of four independent experiments. TNF-
-specific
fluorescence is indicated by the filled curve, and the mean
fluorescence intensity is given in bold numbers. The
open curve indicates the fluorescence of the isotype
control. B, the promoter construct TP(
1057) was
cotransfected in A3.01 T cells with either corresponding empty
expression vector or ERK2(B3), SAPK
(K-R), JIP-1, or MKK6(A) as
indicated. Cells were either left untreated (
) or treated (+) with
TPA/ionomycin (T+I) 10 h before harvesting. Kinase
inhibitors PD98059 (20 µM) or SB203580 (4 µM, if not otherwise indicated) were added 30 min before
stimulation. The solvent of the inhibitors (DMSO) was also included in
this study, without showing effects on the promoter activity (data not
shown). Promoter activities are given as the fold stimulation of
relative luciferase activity based on the unstimulated control treated
or transfected in the same way. The figure shows the mean of four
independent transfection experiments.
specific fluorescence (Fig. 5A), indicating the crucial role of ERK
and p38 pathways in TNF-
biosynthesis in A3.01 T cells.
promoter activity (Fig.
5B). Moreover, we observed a partial reduction of induced promoter activity when JNK/SAPK activation is blocked by the expression of dominant negative SAPK
(K-R) or the inhibitory protein JIP-1. In
contrast, blockage of signaling through p38 by the expression of
MKK6(A) or incubation with up to 4 µM SB203580 did not
impair TPA/ionomycin-induced TNF-
promoter activity. The induction
of TNF-
promoter is almost abolished (Fig. 5B) using
higher concentrations of SB203580 (up to 20 µM), at which
ERK and JNK activities are also inhibited.
promoter. To prove this assumption, we
blocked p38 with specific concentrations of SB203580 (4 µM) combined with expression of JIP-1 and/or PD98059
treatment to block two or all three MAPK pathways. Although treatment
with SB203580 alone did not exert any effect on the induced promoter activity, the compound enhances the inhibitory effects of JIP-1 and
PD98059 (Fig. 5B). Blockage of all three pathways by the
combined action of JIP-1, SB203580, and PD98059 almost abolished the
TNF-
promoter induction by TPA/ionomycin (Fig. 5B).
promoter during T-cell activation.
Whereas the p38-activating pathway appears to be indispensable for
posttranscriptional events in TNF-
biosynthesis, as demonstrated by
intracellular TNF-
staining in stimulated cells pretreated with
SB203580 (Fig. 5A), it can be substituted by another
signaling pathway in transcriptional processes induced by
TPA/ionomycin. ERK and JNK pathways, however, are necessary for maximal
induction of the promoter activity.
Promoter Are Responsive to Each
MAPK Activating Cascade--
To assess whether the contribution of
each MAPK pathway is connected to distinct promoter elements, a series
of 5' deletion mutants of the TNF-
promoter (Fig.
6A) were cloned in front of the luciferase cDNA in the pGL3 vector. These reporter constructs were used in cotransfection experiments with the constitutively active
kinases described above. Fig. 6B shows the fold stimulation of promoter activity induced by Raf-BXB-CX, MLK3, and MKK6(EE) compared
with empty expression vector. The basic empty luciferase vector pGL3
served as a negative control and showed no significant induction by the
kinase activators.
View larger version (32K):
[in a new window]
Fig. 6.
Raf-BXB-CX, MLK3, and MKK6(EE) induced
transactivation of different 5' deletion mutants of the TNF-
promoter. A, a schematic representation of the different 5'
deletion mutants used in the study. B, the promoter
constructs were cotransfected with empty expression vector or either
Raf-BXB-CX (B), MLK3 (C), or MKK6(EE)
(D). Equal transfection efficiency of the promoter
constructs was monitored by
-galactosidase expression as described
under "Experimental Procedures." The data are shown as the fold
stimulation of relative luciferase activities based on
vector-transfected cells and represent the mean of four independent
experiments.
1057 (TP-1057) relative to the
transcriptional start site showed high inducibility by all three
activators (Fig. 6B). A deletion from nucleotide
1057 to
600 (TP-600) resulted in a slight decrease in induction by active Raf
and a strong decrease if cells were transfected with MKK6(EE) or MLK3.
Whereas there is a drop in Raf-induced promoter activity by the
deletion of the region up to nucleotide
120 (TP-120) (Fig.
6B), no such decline is observed for MKK6(EE)- and
MLK3-induced transcription. However, the MKK6(EE) and MLK3 inducibility
of the promoter is almost abolished when the deletion is extended to
nucleotide
105 (TP-105).
promoter targeted by the different pathway activators. The
region between
1057 and
600 is responsible for induction by all
three pathways; Raf also targets elements within nucleotides
200 to
120. MLK3 and MKK6(EE) overlap in their responsive regions, which
require nucleotides
120 to
100.
DISCUSSION
promoter and that these
pathways are critical and act in concert to mediate TPA/ionomycin-induced TNF-
transcription and production in A3.01 T-cells. This indicates that in contrast to other TNF-producing cell
lines, all three MAPK pathways play a pivotal role in the induction of
TNF-
transcription in activated T cells.
expression in activated T cells is transcriptional initiation, because
the onset of TNF-
synthesis is abolished by treatment with the
transcriptional inhibitor actinomycin D (Fig. 2A).
Therefore, the transcriptional regulation of TNF expression may be the
most critical step. This assumption is supported by earlier
observations in a monocytic cell line, where a large increase in
secreted TNF-
levels is primarily due to transcriptional activation
of the TNF-
gene by LPS (44).
promoter activity is regulated by
all three MAPK signaling pathways in T cells. A critical role for the
ERK pathway is illustrated by the observation that selective activation
(Figs. 1A and 6) or inhibition of this pathway (Fig. 5)
positively or negatively interferes with the induction of TNF-
expression, respectively. In macrophages the Raf/MEK/ERK pathway has
also been reported to be critical for LPS-induced TNF-
transcription
(11). However, in contrast to our data in this study, constitutively
active Raf alone was not sufficient to transactivate the promoter, as
was the case in a mast cell line (9). In mast cells, the role of the
ERK pathway in TNF production is still unclear, because one report
favors the involvement of ERK (2), whereas others suggest that the
Raf/MEK/ERK pathway is not required (9). This difference may be
accounted for by the use of two different mast cell lines in these studies.
transcription in A3.01 T cells because both selective activation (Figs.
1B and Fig. 6) or inhibition of JNK signaling (Fig.
5B) induces or blocks the induced promoter activity,
respectively. Similar observations have been made in a mast cell line
(9) where TNF-
promoter-dependent reporter gene activity
was induced by expression of the JNK activator MEK kinase (MEKK), and
this induced activity was partially blocked by a dominant negative mutant of JNK. In macrophages, the involvement of the JNK pathway in
the regulation of LPS-induced TNF-
biosynthesis was reported to be
on the translational rather than the transcriptional level (12),
because a dominant negative JNK mutant blocked the translation of
TNF-
mRNA but not the LPS-induced transactivation of the TNF-
promoter.
promoter in T cells for the first
time. Selective activation of this pathway readily induces
transcription (Figs. 1C and 6); however, blockage of p38 activity by SB203580 only exerts inhibiting effects on the induced promoter activity in the absence of either JNK or ERK signaling (Fig.
5B). A plausible mechanism might be that the signal is at least partially mediated by SB203580-insensitive p38 isoforms such as
p38
, which is expressed in T cells (45). Nevertheless, SB203580-sensitive p38 isoforms are also involved, because the inhibitor clearly shows synergistic effects if either ERK or JNK signaling is blocked. This may be due to a dosage effect with regard to
the number of MAPKs involved. The blockage of some p38 isoforms may be
overcome by other unaffected MAPKs. However, if the number of active
MAPKs is further reduced by other inhibitors, such as PD98059 or JIP-1,
the blockage of p38 activity could not be bypassed anymore.
gene has
not been elucidated in macrophages; however, similar to our observation
in T cells, p38 appears to regulate posttranscriptional processes (10).
In contrast, no critical role for the p38 pathway in TNF-
transcription and biosynthesis has been observed in mast cells (2,
9).
promoter activity (Fig. 1), the function of each MAPK pathway
appears not to be redundant. Several findings support a cooperation
between the MAPK pathways: (a) TPA stimulation that
maximally induces ERK but not JNK or p38 activity (18) is not
sufficient to achieve full promoter activity (Fig. 2B), (b) synergistic activation of the promoter is observed by
the coexpression of active MKK6 with either active Raf or MLK3 (Fig. 1D), and (c) promoter induction is reduced when
either JNK or ERK activation was inhibited and is almost abolished
after a blockage of all three pathways (Fig. 5B). While
preparing this article, a report has been published showing that the
serine/threonine kinase Cot up-regulates TNF-
promoter-driven
expression in Jurkat T cells (46). Cot activates both ERK and JNK via
MEK and SEK kinase, respectively (47, 48). Cot-induced TNF-
promoter activity was partially inhibited by the MEK inhibitor PD98059, which is
consistent with our finding that the ERK signaling pathway is involved
in the regulation of TNF-
gene transcription. According to our data,
the remaining promoter activity might be due to the JNK signal, which
is not blocked by PD98059.
production if cells are cooperatively treated with both TPA and
ionomycin (Fig. 2A), it is most likely that the inhibiting effect of CsA is mediated by an inhibition of p38 and JNK, which are
strongly activated in the presence of the costimulus ionomycin (Fig. 3,
D and E) (18).
in mast cells, monocytes,
and lymphocytes are probably due to cell lineage specificities. A cell
type-specific regulation of the cytokine has been suggested earlier
when comparing TNF-
synthesis in a dendritic cell line
versus a mast cell line (50). T-cell specificity is
postulated to be due to an involvement of the T-cell-specific transcription factor NFATp controlling the TNF-
promoter activity (30, 51). NFATp bound to the
factor binding site 3 (k3) acts
cooperatively with the CRE binding factors ATF-2/Jun. The combined
CRE/k3 site is located at nucleotide
106 to
88 relative to the
transcriptional start site and is required for T-cell stimulation by
the antigen receptor (30, 51). Because ATF-2 is a substrate for p38,
and both c-Jun and ATF-2 are JNK substrates (15, 52-54), it is most
likely that the JNK and p38 signaling pathways contribute to the
regulation of the CRE/k3 element. Supporting this assumption, a
promoter construct without an intact CRE site (TP-105) is much less
inducible by MLK3 or MKK6(EE) compared with a promoter construct with
an intact CRE/k3 site (TP-120) (Fig. 6), suggesting that the CRE/k3
site is targeted by JNK- and p38-activating pathways. In contrast, a
Raf-responsive region appears to be located between nucleotides
200
and
120 (Fig. 6). Thus far, binding sites for Krox-24/Egr-1 (55),
SP-1, and NFAT (30, 51) have been identified in this region, and a
promoter element containing the Egr-1 site was found to be essential
for TPA-induced promoter activity in T cells (56).
600 to
200 does not result
in remarkable changes of promoter activity, which is consistent with an
earlier report (32). Interestingly, there is a significant reduction in
the inducibility of the TNF-
promoter by active Raf, MLK3, or MKK6
when the region between
1057 and
600 is deleted (Fig. 6). These
data indicate that there are one or more responsive sites to these
kinases in T cells. Two
factor binding sites (k1 and k2) are
located within this region at nucleotides
650 and
610. These are
extremely conserved and are important for LPS responsiveness in
monocytes of several species (57). These sites may also contribute to
induced TNF expression in activated T cells. Furthermore, there might
be other regulatory regions upstream of
600 within the human TNF-
promoter that have yet to be identified.
expression in T cells is
regulated by several distinct MAPK pathways that functionally cooperate
and are critical for transcriptional as well as for posttranscriptional
processes. The involvement of ERK, JNK, and p38 pathways in
transcriptional regulation of the TNF-
gene suggests a
T-cell-specific regulation. These data might be helpful with regard to
cell type-specific therapeutic modulation of the TNF-
expression in
a beneficial way.
![]() |
ACKNOWLEDGEMENTS |
---|
We are very thankful to Dr. S. A. Nedospasov for providing the TNF- promoter construct. We also thank
Dr. Peter Krammer and Dr. Henning Walczak (Deutsches
Krebsforschungszentrum (DKFZ), Heidelberg, Germany) and Bruce Jordan
(Institut für Medizinische Strahlenkunde und Zellforschung,
Würzburg (MSZ), Germany) for critical reading of the manuscript
and helpful suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft (Grant Lu477/2-3) and by the Fritz Thyssen Stiftung (Grant 1998 20 60).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.
§ These authors contributed equally to this study.
To whom correspondence should be addressed: Institut für
Medizinische Strahlenkunde und Zellforschung (MSZ), Versbacherstrasse 5, D-97078 Würzburg, Germany. Tel.: 49-931-2013851; Fax:
49-931-2013887; E-mail: IMSD019{at}rzbox.uni-wuerzburg.de.
The abbreviations used are: TNF, tumor necrosis factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; MKK6, MAPK kinase 6; MLK3, mixed-lineage kinase 3; TPA, 12-O-tetradecanoylphorbol-13-acetate; CsA, cyclosporin A; JIP-1, JNK-interacting protein 1; CRE, cAMP responsive element; LPS, lipopolysaccharide; HA, hemagglutinin; GST, glutathione S-transferase; MBP, myelin basic protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|