From the Department of Immunology, Keio University
School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan and
the § Department of Biophysics, Graduate School of Science,
Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-01, Japan
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ABSTRACT |
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p38/CSBP, a subgroup member of mitogen-activated protein kinase (MAPK) superfamily molecules, is known to be activated by proinflammatory cytokines and environmental stresses. We report here that p38 is specifically activated by signals that lead to interleukin-2 (IL-2) production in T lymphocytes. A p38 activator MKK6 was also markedly activated by the same stimulation. Pretreatment of cells with SB203580, a specific inhibitor of p38, as well as expression of a dominant-negative mutant of MKK6, suppressed the transcriptional activation of the IL-2 promoter. We also demonstrated that MKK7, a recently described MAPK kinase family member, plays a major role in the activation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in T lymphocytes. Moreover, a dominant-negative mutant of MKK7 abrogated the transcriptional activation of the distal nuclear factor of activated T cells response element in the IL-2 promoter. Cyclosporin A, a potent immunosuppressant, inhibited activation of both p38 and SAPK/JNK pathways but not the MAPK/extracellular signal-regulated kinase (ERK) pathway. Our results indicate that both MKK6 to p38 and MKK7 to SAPK/JNK signaling pathways are activated in a cyclosporin A-sensitive manner and contribute to IL-2 gene expression in T lymphocytes.
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INTRODUCTION |
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The mitogen-activated protein kinase
(MAPK)1 pathway is a
conserved eukaryotic signalling cascade that mediates the effects of
extracellular stimuli on a wide array of biological processes. This
pathway consists of three protein kinases, MAPK, MAPK kinase (MAPKK),
and MAPKK kinase; MAPKK kinase phosphorylates and activates MAPKK,
which in turn phosphorylates and activates MAPK (1-6). Three distinct
subgroups of the MAPK superfamily have been identified in mammalian
cells (7). These are classical MAPK (also termed extracellular
signal-regulated protein kinase, ERK), stress-activated protein kinase
(SAPK; also termed c-Jun NH2-terminal kinase (JNK)), and
p38 (also termed MPK2, RK, CSBP, or HOG1). Studies on the MAPK/ERK and
SAPK/JNK subgroups have led to insights into their physiological
functions (8). For example, previous studies have implicated a role of
MAPK/ERK and SAPK/JNK in activation of T lymphocytes leading to
interleukin-2 (IL-2) production (9-12). On the other hand, the
contribution of p38 to cellular responses to extracellular stimuli has
been poorly defined (13). It has been shown that p38 is activated by
proinflammatory cytokines (e.g. IL-1 and tumor necrosis
factor-) and cellular stresses (e.g. osmotic shock and
ultraviolet light) (14-18). p38 has been shown to phosphorylate
in vitro MAPKAP kinase-2 (16, 17) and transcription factors such as ATF-2, Elk-1, and CHOP (18-20).
However, its role in T lymphocyte activation has been
unclear.
IL-2 production, which is a critical step for T lymphocyte activation and proliferation, requires two coordinate signals; the primary stimulus is the interaction of antigen with the T cell receptor (TCR)·CD3 complex (21), and the secondary stimulus, called costimulation, is generated by the interaction of CD28 auxiliary receptors on T cells with its ligand on antigen-presenting cells (22). Various studies indicate that activation signals for IL-2 production can be bypassed by phorbol esters (such as TPA) and Ca2+ ionophore (such as A23187) (23-25).
Here we report that either treatment with TPA and Ca2+ ionophore or simultaneous activation of the TCR and CD28 results in synergistic activation of MKK6 (26-28) to the p38 pathway in T lymphocytes in a cyclosporin A (29)-sensitive manner. We also demonstrate that newly identified MKK7 (30-34) plays a major role as an upstream activator of SAPK/JNK in T cells. Several lines of evidence for involvement of p38 and SAPK/JNK in IL-2 gene expression are presented.
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EXPERIMENTAL PROCEDURES |
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Activation of Cells--
Jurkat cells were grown in RPMI 1640 with 10% fetal calf serum, 50 units/ml penicillin, 200 µg/ml
kanamycin, and 5 × 105 M
-mercaptoethanol. Monoclonal antibodies (mAbs) to human CD3 (NU-T3) and human CD28 (KOLT-2) were purchased from Nichirei. PD98059,
SB203580, and CsA were purchased from New England Biolabs, Calbiochem,and Wako, respectively. mAb NU-T3 (5 µg) was immobilized on a 35-mm dish by incubation for 12 h at 4 °C. mAb KOLT-2 (5 µg) was preincubated with 5 µg of rabbit anti-mouse antibodies for
12 h at 4 °C to induce cross-linking and then was added to Jurkat cells (2 × 106), or cells were stimulated with
various reagents such as TPA (Sigma) and Ca2+ ionophore
A23187 (Wako). After a 24-h treatment of Jurkat cells with various
stimuli, IL-2 production was measured using ELISA (Endogen) according
to the manufacturer's instructions.
Preparation of Cell Extracts--
Following stimulation, cells
were washed once with ice-cold Hepes-buffered saline and then lysed in
a buffer consisting of 20 mM Tris-HCl (pH 7.5), 2 mM
EGTA, 25 mM -glycerophosphate, 1% Triton X-100, 2 mM dithiothreitol, 1 mM vanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
pepstatin A, and 1% aprotinin and were centrifuged at 16,000 × g for 30 min at 2 °C. The supernatant (approximately 0.1 mg/ml cellular protein) was subjected to in-gel kinase assays or immune
complex kinase assays.
Immune Complex Kinase Assay-- Immunoprecipitation and immune complex kinase assays were performed as described previously (34, 35). Anti-p38 antibody was purchased from Santa Cruz. To assay for p38 activity, p38 was immunoprecipitated from cell lysates in the presence of 0.1% SDS and assayed for kinase activity using ATF-2 as a substrate. Anti-SEK1 (36), anti-MKK6 (28, 29), and anti-MKK7 (34) antibodies were previously produced and characterized.
Plasmids--
pME-MKK6, an expression plasmid for hemagglutinin
(HA)-tagged MKK6 under a control of the SR promoter, has previously
been characterized (28). A dominant-negative MKK6 (MKK6(AA)) was constructed by replacing Ser-207 and Thr-211 with alanine residues. The
mutation was made by site-directed mutagenesis (Quick
ChangeTM, Stratagene) and was confirmed by DNA sequencing.
An HA-tagged kinase-negative MKK7 (MKK7(KL)), constructed by replacing
ATP-binding lysine with leucine residue, has previously been reported
(34). A luciferase reporter plasmid carrying the human IL-2
enhancer/promoter element (encompassing nucleotides
326 to +47),
IL-2-Luc, was kindly provided by Dr. M. Iwashima (Mitsubishi Chemical
Corporation Yokohama Research Center, Japan). Another luciferase
reporter plasmid with three tandem repeats of the NFAT site, NFAT-Luc
(37), was kindly provided by Dr. G. Crabtree (Stanford University,
Stanford, CA).
Transfection and Luciferase Assay-- Transient transfection of Jurkat cells (5 × 106) was performed using SuperFectTM (Qiagen) with luciferase reporter plasmids (either 1 µg of IL-2-Luc or 0.5 µg of NFAT-Luc) along with various amounts of expression plasmids. The total amount of DNA transfected was maintained at 5 µg with pcDNA 3.1+ (Invitrogen). After 24 h of incubation at 37 °C, cells were stimulated for 8 h with indicated reagents and then lysed for luciferase assay. Luciferase in cell lysates was measured in duplicate by a luminometer (LB9507, Berthold), using a luciferase assay system (Promega). The protein concentration was determined with a protein assay kit (Bio-Rad) and used for normalization of the luciferase assays. Expression of HA-tagged proteins was analyzed by immunoblotting with the 12CA5 mouse mAb against the HA epitope.
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RESULTS AND DISCUSSION |
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Activation of p38 and MKK6 by Costimulatory Signals-- In a human T cell line, Jurkat, the activity of p38 markedly increased in response to signals generated by TPA and Ca2+ ionophore, whereas each stimulus alone resulted in little or no activation (Fig. 1A, shaded bar). Similar to its effect on IL-2 production (37), CsA inhibited the activation of p38 (Fig. 1A, shaded bar). SAPK/JNK was also activated in a similar manner as reported previously (Fig. 1A, black bar (9)). In contrast, incubation with TPA was sufficient for efficient activation of MAPK/ERK, which was neither enhanced by Ca2+ ionophore nor inhibited by CsA (Fig. 1A, white bar). Interestingly, treatment with TPA and Ca2+ ionophore did not induce the activation of p38 in non-lymphoid cells such as an epidermoid carcinoma cell line, KB (data not shown). We next examined whether MKK6, an upstream activator of p38 (27-29, 38), is activated in parallel with p38 activation during T cell activation. Whereas TPA alone had little effect on MKK6 activity, it induced marked activation of MKK6 in combination with Ca2+ ionophore (Fig. 1B). Furthermore, CsA inhibited the activation of MKK6 as observed in the p38 activation (Fig. 1B). Although treatment with Ca2+ ionophore alone induced slight activation of MKK6 in the Jurkat cells used here (Fig. 1B), this is not the case with another Jurkat line, TAg Jurkat (data not shown). The reason for this inconsistency remains to be determined. Whereas incubation of Jurkat cells with anti-CD3 or anti-CD28 mAb alone had little effect on either p38 activity or MKK6 activity, simultaneous incubation with both mAbs resulted in synergistic activation of both MKK6 and p38 and IL-2 production (Fig. 1C). Activation of MKK6 and p38 induced by simultaneous stimulation with anti-CD3 and anti-CD28 mAbs was also inhibited by CsA (data not shown).
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Activation of MKK7 during T Cell Activation--
One of the
MAPKKs, SEK1 (also termed as MKK4 or JNKK) has previously been reported
to function as a direct activator of SAPK/JNK (19, 39, 40). In addition
to SEK1, we have previously identified an activity to phosphorylate and
activate SAPK distinct from SEK1 (36). This novel kinase has been
identified by cDNA cloning and named MKK7 (34). We thus examined
whether SEK1 or MKK7 plays a major role in SAPK/JNK activation during T
cell activation. Whereas incubation of Jurkat cells with either
anti-CD3 or anti-CD28 mAb alone had a weak effect on MKK7 activity,
simultaneous incubation with both mAbs resulted in strong synergistic
activation of MKK7, which was inhibited in the presence of CsA (Fig.
2A). SEK1 activity was only
slightly activated in response to TPA and Ca2+ ionophore,
whereas activation of MKK7 activity was more prominent to the same
stimulation (Fig. 2B), which was also sensitive to CsA (data
not shown). Note that both SEK1 and MKK7 activities markedly increased
when Jurkat cells were exposed to hyperosmolar media as expected (Fig.
2B) (34, 36). Similar results were obtained in mouse
thymocytes (Fig. 2C) and splenic T cells (data not shown).
These results suggest that MKK7 acts as a major upstream activator of
SAPK/JNK during T cell activation, as is the case with tumor necrosis
factor- and Fas signaling pathway (34, 41). It has recently been
reported that SAPK/JNK activation is not induced in thymocytes of
SEK1-deficient mice in response to TPA and Ca2+ ionophore
(42, 43). The activation pathway of SAPK/JNK might be developmentally
regulated such that SEK1 but not MKK7 activates SAPK/JNK in the thymus.
Functions of SEK1 and MKK7 in SAPK/JNK pathway in thymocytes remain to
be investigated in further studies.
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Inhibition of MAPK Superfamily Pathway Blocks the Transcriptional Activation of IL-2 Gene-- Activation of MAPK/ERK alone was not sufficient to induce IL-2 production (Fig. 1A) (9). To evaluate the role of MAPK/ERK in IL-2 production directly, we employed PD98059, a specific inhibitor of classical MAPKK (also termed as MEK) (44, 45). As shown in Fig. 3A, preincubation with PD98059 prevented IL-2 production induced by simultaneous stimulation with TPA and Ca2+ ionophore in a dose-dependent manner. The concentration of PD98059, which inhibits IL-2 production by 50% in Jurkat cells (<5 µM), is similar to that inhibiting activation of MAPKK/MEK in vitro and in vivo (44). We also found that IL-2 production induced by stimulation via CD3 and CD28 was almost completely inhibited by pretreatment of the cells with 20 µM PD98059 (Fig. 3B). Furthermore, prevention of MAPK/ERK activation resulted in the suppression of IL-2 promoter-driven transcriptional activity (data not shown (46)). It is noteworthy that preteatment of the cells with 20 µM PD98059 did not prevent the activation of SAPK/JNK or p38 (data not shown). These results clearly indicated that the activation of MAPK/ERK pathway is required for IL-2 production.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Y. Gotoh for the initial involvement in this study. We are grateful to Dr. G. Crabtree (Stanford University) and Dr. M. Iwashima (Mitsubishi Chemical Corp.) for NFAT-Luc and IL-2-Luc, respectively. We thank Dr. S. Yonehara (Kyoto University) for critical reading of the manuscript, A. Minowa for technical support, and members of the Nishida laboratory for helpful discussion.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to E. N. and S. K.) and grants-in-aid from the Takeda Science Foundation (to S. K.) and the Toray Science Foundation (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed: Dept. of Immunology, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. Fax: 81-3-5361-7658.
To whom correspondence may be addressed: Dept. of Biophysics,
Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-01, Japan. Fax: 81-75-753-4235.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MAPKK, MAPK kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; CSBP, CSAIDTM binding protein; CsA, cyclosporin A; IL-2, interleukin-2; mAb, monoclonal antibody; TCR, T cell receptor; NFAT, nuclear factor of activated T cell; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; Luc, luciferase.
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REFERENCES |
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