Different Mitogen-activated Protein Kinase Signaling Pathways Cooperate to Regulate Tumor Necrosis Factor alpha  Gene Expression in T Lymphocytes*

Angelika HoffmeyerDagger §, Anne Grosse-WildeDagger §, Egbert FloryDagger , Bernd NeufeldDagger , Manfred Kunz, Ulf R. RappDagger , and Stephan LudwigDagger parallel

From the Dagger  Institut für Medizinische Strahlenkunde und Zellforschung (MSZ) and  Klinik und Poliklinik für Haut und Geschlechtskrankheiten, Universität Würzburg, D-97078 Würzburg, Germany

    ABSTRACT
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Abstract
Introduction
References

Tumor necrosis factor a (TNF-alpha ) is a potent proinflammatory cytokine and plays a crucial role in early events of inflammation. TNF-alpha is primarily produced by monocytes and T lymphocytes. In particular, T-cell-derived TNF-alpha 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-alpha 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-alpha promoter induction. Furthermore, blockage of all three pathways almost abolishes TPA/ionomycin-induced transcriptional activation of the TNF-alpha promoter. Selective inhibition of one or more MAPK pathways impairs TNF-alpha 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-alpha 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-alpha -producing cells, all three MAPK pathways are critical and cooperate to regulate transcription of the TNF-alpha gene in T lymphocytes, suggesting a T-cell-specific regulation of the cytokine.

    INTRODUCTION
Top
Abstract
Introduction
References

Tumor necrosis factor alpha  (TNF-alpha )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-alpha after high-affinity IgE receptor aggregation (2). TNF-alpha 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-alpha are dependent on the strength and duration of its expression. High systemic levels of TNF-alpha 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-alpha 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-alpha in the modulation of inflammatory responses is further reflected in the observation that T-cell membrane-bound TNF-alpha is of particular importance for the regulation of monocytic interleukin 10 and TNF-alpha production (8).

The diverse stimuli that up-regulate TNF-alpha 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-alpha expression (2, 9-12), whereas little is known about the regulation of this cytokine in T lymphocytes.

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-alpha 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 p38delta (28). Until this time, a specific physiological activator of MKK6 has not been identified.

Whereas MAPK-activating pathways have been implicated in LPS-induced TNF-alpha 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-alpha expression by mast cells (2, 9), the contribution of MAPKs to the regulation of TNF-alpha expression in T lymphocytes is still unclear.

Two findings suggest a cell type-specific involvement of intracellular signaling pathways inducing TNF-alpha expression: (a) in lymphocytes versus monocytes, different sets of transcription factors are recruited to the promoter of the TNF-alpha gene (29, 30), and (b) extracellular stimuli with a cell type-specific function trigger TNF-alpha expression in different cells, such as LPS in monocytes, Fcepsilon 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-alpha transcription that does not require new protein biosynthesis (32).

To investigate T-cell-specific regulation of the TNF-alpha gene, we analyzed the involvement of distinct MAPK pathways in TNF-alpha 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-alpha expression in these T cells.

    EXPERIMENTAL PROCEDURES

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-alpha monoclonal antibody was purchased from PharMingen, Inc.

DNA Constructs and Cloning-- The human TNF-alpha 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-alpha promoter were produced by restriction digestion or PCR amplification of regions of the human TNF-alpha 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.

The eukaryotic expression vector for HA-SAPKbeta and the prokaryotic expression vector pGEX-KG-c-Jun(1-135) were gifts from J. Kyriakis and L. Zon.

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, SAPKbeta , MKK6, Raf-BXB-CX, and MLK3 are ATP-binding site mutants generated by the replacement of lysine with arginine (ERK2(B3), SAPKbeta (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.

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 beta -galactosidase activity of cotransfected Rous sarcoma virus LTR beta -gal vector. The beta -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.

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-alpha , 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-alpha 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).

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).

    RESULTS

Selective Activation of ERK, JNK, or p38 Signaling Pathways Stimulates TNF-alpha Promoter-dependent Transcription-- To investigate the role of the ERK, JNK, or p38 signaling pathways as mediators of induced TNF-alpha transcription, we performed transient cotransfection experiments and measured the human TNF-alpha 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).

Expression of each of these kinases in A3.01 cells is sufficient to induce strong TNF-alpha 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-alpha 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-alpha -specific transcription.


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Fig. 1.   Expression of Raf-BXB-CX, MLK3, and MKK6(EE) induces TNF-alpha -dependent reporter gene expression. A-C, A3.01 cells were cotransfected with 0.5 µg of the TP(-1057) TNF-alpha 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).

We next investigated the role of MAPK pathways in TNF-alpha gene expression of T cells activated by TPA/ionomycin in more detail.

Expression of TNF-alpha by Activated A3.01 T Cells-- To characterize the regulation of TNF-alpha expression, we stimulated A3.01 T cells with TPA, ionomycin, or a combination of both. The inducibility of TNF-alpha expression was determined at both the translational and transcriptional levels by TNF-alpha -specific flow cytometry analysis and a TNF-alpha promoter reporter gene assay, respectively. Reporter gene analysis allows for the assessment of TNF-alpha promoter activity independent of TNF-alpha mRNA stability, another control level of expression. Unstimulated A3.01 cells do not produce any detectable amount of TNF-alpha (Fig. 5A). Stimulation of these cells with TPA results in a weak induction of TNF-alpha 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-alpha production is sensitive to cyclosporin A, whereas TPA-induced protein synthesis is unaffected (Fig. 2A). TNF-alpha 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-alpha synthesis requires de novo transcription of the TNF-alpha gene.


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Fig. 2.   Expression of TNF-alpha by A3.01 T cells. A, to measure TNF-alpha production upon stimulation, A3.01 T cells were stained intracellularly with an anti-TNF-alpha 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-alpha 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-alpha promoter-dependent transcription, the promoter construct TP(-1057) was cotransfected with a Rous sarcoma virus LTR-driven beta -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 beta -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.

Therefore, we analyzed the time dependence of TNF-alpha transcription compared with TNF-alpha production. TNF-alpha 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-alpha expression in A3.01 cells is similar to that of primary T cells and other T-cell lines (reviewed in Ref. 1).


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Fig. 3.   The activation of ERK, JNK, and p38 precedes the induction of TNF-alpha transcription and production. A, TNF-alpha 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-alpha 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.

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-alpha 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-alpha transcription suggests a functional connection of both processes.

Selective Inhibition of Distinct MAPKs in A3.01 T Cells-- To test whether MAPK pathways are functionally involved in TPA/ionomycin-induced TNF-alpha 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.

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).


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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-SAPKbeta and MLK3 and/or JIP-1 and stimulated with TPA/ionomycin as indicated. HA-SAPKbeta 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.

Because TNF-alpha production (Fig. 2A) as well as TNF-alpha 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.

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-SAPKbeta . 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).

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)), SAPKbeta (SAPKbeta (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), SAPKbeta (K-R), and MKK6(A) as efficient dominant negative mutants selective for the ERK, SAPK, and p38 signaling pathways, respectively.

ERK-, JNK-, and p38-activating Pathways Critically Contribute to TPA/Ionomycin-induced TNF-alpha 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-alpha promoter activity (Fig. 5B) and TNF-alpha biosynthesis measured by TNF-alpha specific fluorescence (Fig. 5A).


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Fig. 5.   TPA/ionomycin-induced TNF-alpha 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-alpha -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), SAPKbeta (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.

Blockage of ERK and p38 activation by PD98059 and SB203580, respectively, results in a significant decrease in TNF-alpha specific fluorescence (Fig. 5A), indicating the crucial role of ERK and p38 pathways in TNF-alpha biosynthesis in A3.01 T cells.

Inhibition of ERK signaling by ERK2(B3) overexpression or pretreatment with PD98059 also impaired the TNF-alpha 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 SAPKbeta (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-alpha promoter activity. The induction of TNF-alpha promoter is almost abolished (Fig. 5B) using higher concentrations of SB203580 (up to 20 µM), at which ERK and JNK activities are also inhibited.

These data point to a cooperation of MAPK signaling pathways in the regulation of the TNF-alpha 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-alpha promoter induction by TPA/ionomycin (Fig. 5B).

These data suggest that all three MAPK signaling pathways cooperate in the regulation of the TNF-alpha promoter during T-cell activation. Whereas the p38-activating pathway appears to be indispensable for posttranscriptional events in TNF-alpha biosynthesis, as demonstrated by intracellular TNF-alpha 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.

Distinct Regions of the TNF-alpha 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-alpha 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.


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Fig. 6.   Raf-BXB-CX, MLK3, and MKK6(EE) induced transactivation of different 5' deletion mutants of the TNF-alpha 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 beta -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.

The intact promoter up to nucleotide -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).

These data indicate that there are overlapping but distinct regions of the TNF-alpha 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

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-alpha promoter and that these pathways are critical and act in concert to mediate TPA/ionomycin-induced TNF-alpha 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-alpha transcription in activated T cells.

According to our data, the first regulatory step of induced TNF-alpha expression in activated T cells is transcriptional initiation, because the onset of TNF-alpha 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-alpha levels is primarily due to transcriptional activation of the TNF-alpha gene by LPS (44).

Our findings demonstrate that TNF-alpha 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-alpha expression, respectively. In macrophages the Raf/MEK/ERK pathway has also been reported to be critical for LPS-induced TNF-alpha 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.

The JNK pathway also appears to be of critical importance for TNF-alpha 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-alpha 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-alpha 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-alpha mRNA but not the LPS-induced transactivation of the TNF-alpha promoter.

Our study also defined a functional role of the p38 pathway in the transcriptional induction of TNF-alpha 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 p38delta , 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.

The role of the p38 pathway in transcription of the TNF-alpha 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-alpha transcription and biosynthesis has been observed in mast cells (2, 9).

Although selective activation of either pathway is sufficient to induce TNF-alpha 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-alpha 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-alpha 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-alpha gene transcription. According to our data, the remaining promoter activity might be due to the JNK signal, which is not blocked by PD98059.

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-alpha 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).

The different observations concerning the contribution of MAPK pathways to the transcriptional regulation of TNF-alpha 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-alpha 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-alpha promoter activity (30, 51). NFATp bound to the kappa  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).

Deletion of the promoter from nucleotide -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-alpha 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 kappa  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-alpha promoter that have yet to be identified.

In conclusion, our data indicate that TNF-alpha 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-alpha gene suggests a T-cell-specific regulation. These data might be helpful with regard to cell type-specific therapeutic modulation of the TNF-alpha expression in a beneficial way.

    ACKNOWLEDGEMENTS

We are very thankful to Dr. S. A. Nedospasov for providing the TNF-alpha 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.

parallel 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
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Abstract
Introduction
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