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J. Biol. Chem., Vol. 279, Issue 3, 1621-1626, January 16, 2004

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Stress Induces Mitochondria-mediated Apoptosis Independent of SAPK/JNK Activation in Embryonic Stem Cells^*

Gen Nishitai, Nao Shimizu, Takahiro Negishi, Hiroyuki Kishimoto, Kentaro Nakagawa, Daiju Kitagawa, Tomomi Watanabe, Haruka Momose, Shinya Ohata, Shuhei Tanemura, Satoshi Asaka, Junko Kubota, Ryota Saito, Hiroki Yoshida, Tak W. Mak¶, Teiji Wada¶, Josef M. Penninger¶, Noriyuki Azuma||, Hiroshi Nishina**, and Toshiaki Katada

From the Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan, the Department of Biomolecular Sciences, Saga Medical School, Saga 849-8501, Japan, the ^¶University Health Network, Departments of Medical Biophysics and Immunology, the University of Toronto, Toronto, Ontario M5G 2C1, Canada, and the ^||Department of Ophthalmology, National Center for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan

Received for publication, September 17, 2003 , and in revised form, October 24, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1^–/– mkk7^–/– ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1^–/– ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1^–/– mkk7^–/– ES cells. Instead, interleukin-1 (IL-1)-induced IL-6 gene expression was greatly suppressed in sek1^–/– mkk7^–/– fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Apoptosis or programmed cell death is critical for many biological events such as embryonic development, immune responses, and tissue homeostasis in multicellular organisms. In mammalian cells, apoptotic signaling cascades can be divided into two broad categories: the intrinsic (mitochondria-dependent) and the extrinsic (death receptor-mediated) pathways. The initiation of mitochondria-dependent pathway requires a change in the organella membrane permeability that is prevented by anti-apoptotic molecules such as Bcl-2 and Bcl-X_L and promoted by pro-apoptotic molecules including Bax and Bak, and the permeability change results in the release of mitochondrial proteins. One of the released proteins, cytochrome c, associates with Apaf1 and caspase 9 to activate the effector caspase 3 (1, 2). Cellular stresses such as UV irradiation and heat shock mediate apoptosis through the mitochondria-dependent pathway (3). However, upstream signaling that regulates the pro-apoptotic molecules remains to be elucidated.

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK),¹ which belongs to the family of mitogen-activated protein kinase (MAPK), is activated not only by many types of cellular stresses including UV irradiation, heat shock, cisplatinum, etoposide, thapsigargin, and tunicamycin but also by inflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor (TNF-). The activated SAPK/JNK phosphorylates a number of substrates including the c-Jun component of the activator protein-1 transcription factor to regulate gene expression for the stress responses. Activation of SAPK/JNK requires the dual phosphorylation of Tyr and Thr residues located in a Thr-Pro-Tyr motif of the MAPK, and two kinases, SEK1 (also known as MKK4) and MKK7 (SEK2), are responsible for the phosphorylation (4–6). SEK1 and MKK7 preferentially phosphorylate the Tyr and Thr residues of SAPK/JNK, respectively (7). Interestingly, the Tyr phosphorylation by SEK1 is sequentially followed by the Thr phosphorylation by MKK7 in stress-stimulated mouse embryonic stem (ES) cells (8, 9).

Targeted gene-disruption experiments in mice demonstrate that both SEK1 and MKK7 are required for embryonic development. Sek1^–/– embryos die between embryonic day 10.5 (E10.5) and E12.5 with impaired liver formation and massive apoptosis (10, 11). We have recently shown that SEK1-mediated SAPK/JNK pathway downstream TNF- receptor 1 participates in embryonic hepatoblast proliferation and survival via a pathway different from NF-B-induced anti-apoptosis. On the other hand, mkk7^–/– embryos die between E11.5 and 12.5 with similar defects in liver formation. These results indicate that SAPK/JNK activation mediated through SEK1 plus MKK7 plays indispensable roles in hepatoblast proliferation and survival during mouse embryogenesis (12).

However, the physiological role of SAPK/JNK activation in cell survival and apoptosis is still controversial, being suggested to have an anti-apoptotic, pro-apoptotic, or no function in these processes (13). Mice lacking both JNK1 and JNK2 (Jnk1^–/– Jnk2^–/–) die around E11 with defective neural tube morphogenesis and altered apoptosis (14, 15). These results assign both pro- and anti-apoptotic functions to JNK1 and JNK2 in the development. It also appears that the SAPK/JNK pathway functions in a manner dependent on the types of cells and stimulus, and its different components can sometimes play opposing roles in apoptosis. The most convincing evidence to date that shows that the involvement of SAPK/JNK activation in pro-apoptotic function comes from Jnk1^–/– Jnk2^–/– and mkk4^–/– mkk7^–/– mouse embryonic fibroblasts (MEFs). Both Jnk1^–/– Jnk2^–/– and mkk4^–/– mkk7^–/– MEFs exhibited profound defects in stress-induced apoptosis (16, 17). Furthermore, it has been reported that active JNK causes the release of apoptogenic factors such as cytochrome c and Smac from isolated mitochondria in a cell-free system (18, 19). These results strongly indicate that the SAPK/JNK activation directly regulates mitochondria-dependent apoptosis in pro-apoptotic direction.

To evaluate the exact role of SAPK/JNK activation in mitochondria-dependent apoptosis, we here utilized mouse ES cells in terms of the following advantages. 1) ES cells are a prototype of all cell lineages and can be differentiated into MEF-like cells with retinoic acid. 2) ES cells do not express death receptors including Fas and TNF- receptor 1 but have stress-induced mitochondria-dependent apoptotic pathway. 3) The molecular mechanism of SAPK/JNK activation is well characterized in ES cells. The present results clearly show that SAPK/JNK activation is not required for the stress-induced mitochondria-dependent apoptosis in ES and MEF-like cells. Instead, we found that IL-1-induced IL-6 gene expression was greatly impaired in MEF derived from sek1^–/– mkk7^–/– ES cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture and Materials—The murine ES cell line E14K (wild type) was maintained in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum and leukemia inhibitory factor as described previously (20). The generation of apaf1^–/– ES cells was described previously (3). Sek1^–/– mkk7^–/– ES cells were newly generated as described in Fig. 2. MEF-like cells are prepared from ES cells by culture with 10 µM retinoic acid for 14 days without leukemia inhibitory factor. For thermotolerance, cells were incubated at 44 °C for 10 min and further cultured for 6 h.

View larger version (40K): [in this window] [in a new window]   FIG. 2. Targeted gene disruption of both sek1 and mkk7 in mouse ES cells. a, flow chart of the disruption of sek1 and mkk7 genes and a novel mkk7 (puromycin)-targeting vector. The genomic mkk7 3'-flanking probe used for Southern blotting is indicated as gray boxes, and the predicted structures of targeted alleles are shown as puro. Restriction enzymes used for the vector construction (HincII) and genomic Southern blotting (XbaI) are indicated by arrows. See "Experimental Procedures" for further explanation. b, Western blot analysis of SEK1, MKK7, JNK, ERK2, and p38 in wild-type and sek1–/– mkk7–/– ES cells. c, SAPK/JNK activity in wild-type and sek1–/– mkk7–/– ES cells. ES cells were stimulated with heat shock (44 °C for 20 min), UV (1 kJ/m2 for 20 min), etoposide (2 µg/ml for 8 h), cisplatinum (50 µM for 8 h), thapsigargin (200 nM for 30 min), tunicamycin (10 µg/ml for 30 min), or anisomycin (5 µg/ml for 30 min). The amounts of JNK, ERK2, and p38 in sek1–/– mkk7–/– ES cells were comparable with those in wild-type cells.

Generation of ES Cells Lacking Both SEK1 and MKK7—sek1 and mkk7 genes were disrupted as shown in Fig. 2a. Steps 1 and 2 were done by using a sek1 (neomycin)-targeting vector and gene-dosage effect, respectively (20). Steps 3 and 4 were performed by using a mkk7 (loxP-hygromycin)-targeting vector and an adenovirus-encoding Cre recombinase, respectively (9, 21). Step 5 was done by using a novel mkk7 (puromycin)-targeting vector as shown in Fig. 2a.

Assay of SAPK/JNK Activity—ES cells were plated at 1.5 x 10⁶ cells/35-mm dish and cultured for overnight. The cells were stimulated by anisomycin (Sigma, 5 µg/ml), cisplatinum (Sigma, 50 µM), etoposide (Sigma, 2 µg/ml), thapsigargin (Sigma, 200 nM), tunicamycin (Sigma, 10 µg/ml), UV (1 kJ/m², UV Stratalinker 1800, Stratagene), and heat shock (44 °C for 20 min). SAPK/JNK proteins were immunoprecipitated at 4 °C for 2 h using the anti-SAPK/JNK antibody (C-17, Santa Cruz Biotechnology, Inc.). The SAPK/JNK activity in the precipitated fractions was measured with glutathione S-transferase-c-Jun as an in vitro substrate in the presence of 60 µM [-³²P]ATP (8, 9). The amount of the precipitated SAPK/JNK that had been monitored by immunoblotting with the anti-SAPK/JNK (FL) antibody was almost constant in a series of the present experiments.

Immunoprecipitation and Immunoblotting—ES cells (2 x 10⁶ cells) were suspended at 4 °C in 0.2 ml of a lysis buffer consisting of 1% Nonidet P-40, 80 mM Tris-HCl (pH 7.5), 10 mM EDTA, and 4 µg/ml aprotinin. The cell lysates were incubated for 30 min and centrifuged at 15,000 rpm for 15 min. The supernatants were analyzed by SDS-PAGE and immunoblotting. Proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) and probed with antibodies against anti-SAPK/JNK1, anti-ERK2, anti-p38, and anti-phospho-SAPK/JNK. The bands were visualized by SuperSignal West Pico chemiluminescent substrate for the development of immunoblots utilizing a horseradish peroxidase-conjugated second antibody according to the manufacturer's instructions (Pierce). For detection of phospho-p38, cells were lysed directly in Laemmli sample buffer and sonicated by Ultrasonic Disruptor (TOMY) followed by SDS-PAGE and immunoblotting. Endogenous SEK1 and MKK7 were immunoprecipitated with anti-SEK1 (KN-001) and anti-MKK7 (KN-004) and detected with anti-SEK1 (C-20) and anti-MKK7 (T-19) antibodies, respectively (8, 9).

DNA Fragmentation Assay—ES cells (2 x 10⁶ cells), which had been attached to and detached from culture dishes after stimuli, were collected by means of incubation with trypsin/EDTA and centrifugation, respectively. The cells were mixed, washed twice with phosphate-buffered saline, and collected by centrifugation. After removing the supernatants, the cells were lysed in 0.33 ml of a buffer consisting of 5 mM Tris-HCl (pH 7.4), 20 mM EDTA, and 0.5% Triton X-100. After centrifugation at 27,000 x g for 20 min at 4 °C to remove nuclei and insoluble fraction, the cell lysates were subjected to phenol extraction and ethanol precipitation for DNA purification. The precipitated DNA was suspended in 20 µl of H₂O and treated with 50 µg/ml RNase for 30 min at 37 °C. The DNA samples (10 µl) were subjected to electrophoresis on 2% agarose gels and visualized by a UV illuminator (22).

Assay of Caspase 3 Activity—ES cells were harvested as described above and washed twice with phosphate-buffered saline. The cells, after being resuspended in 50 µl of a buffer consisting of 10 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, and 1 mM phenylmethanesulfonyl fluoride, were frozen in liquid nitrogen and thawed at 37 °C three times. The cell lysates (50 µg) were incubated at 37 °C for 1 h with 20 µM acetyl-Asp-Glu-Val-Asp -(4-methyl-coumaryl-7-amide) (DEVD-MCA; Peptide Institute, Inc.) in the final volume of 50 µl in 20 mM Hepes-NaOH (pH 7.4), 2 mM dithiothreitol, and 10% glycerol. The reaction was terminated by adding 450 µl of ice-cold H₂O, and substrate cleavage leading to the release of free MCA was monitored (excitation 355 nm and emission 460 nm) at room temperature (22).

Sub-G₁ Assay—For flow-cytometric analysis, cells were first fixed with 70% ethanol and further incubated with 10 µg/ml RNase A at 37 °C for 1 h. The cells were stained in a solution (50 µg of propidium iodide/ml, 0.1% sodium citrate, and 0.1% Nonidet P-40) for 30 min at 4 °C. The apoptotic sub-G₁ population was determined by FACScan flow cytometer (BD Biosciences) with Cell Quest software (23).

Subcellular Fractionation—ES cells (1 x 10⁷ cells) were harvested by cell scraper (SUMILON) and washed twice with phosphate-buffered saline. The cells, after being resuspended in 100 µl of buffer A consisting of 250 mM sucrose, 20 mM HEPES-KOH (pH 7.4), 10 mM KCl, 1.5 mM Na-EGTA, 1.5 mM Na-EDTA, 1 mM MgCl₂, 1 mM dithiothreitol, and 2 µg/ml aprotinin, were homogenized 10 strokes by a 27-gauge syringe. After homogenization, cells were centrifuged at 600 x g for 10 min at 4 °C to remove nuclei, unbroken cells, and large debris. Supernatants containing mitochondria were transferred to a new tube and further centrifuged at 10,000 x g for 10 min at 4 °C. Mitochondrial pellets were washed in 100 µl of buffer A followed by centrifugation at 10,000 x g for 10 min at 4 °C and lysed in 1.5x Laemmli sample buffer. The samples (10 µl) were analyzed by SDS-PAGE, and immunoblotting was probed with the anti-Bax and mitochondrial heat shock protein 70 antibodies.

Northern Blotting—Total RNA was prepared from differentiated ES cells by TRIzol reagent (Invitrogen), separated by formamide agarose gel, and transferred to a Hybond-XL (Amersham Biosciences). IL-6 and -actin were detected by Northern blotting using the specific DNA probes. The cDNA fragments corresponding to mouse IL-6 and -actin were amplified using the following primers: IL-6, 5'-ATG AAG TTC CTC TCT GCA AGA GAC T-3' and 5'-CAC TAG GTT TGC CGA GTA GAT CTC-3', and -actin, 5'-CAT CAC TAT TGG CAA CGA GC-3' and 5'-ACG CAG CTC AGT AAC AGT CC-3'.

All of the experiments were repeated at least three times with different batches of the cell samples, and the results were fully reproducible. Hence, most of the data shown are representative of several independent experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Various Stresses Induce Apoptosis through Mitochondria in Mouse ES Cells—We first investigated the thermotolerance effect on apoptosis in ES cells, which protects cells against successive stress by the pretreatment of mild heat shock. For this mechanism, a recent study revealed that HSP70 and HSP27 induced by thermotolerance suppress mitochondria-dependent apoptosis by directly associating with Apaf1 and cytochrome c and blocking the assembly of a functional apoptosome (24–26). As shown in Fig. 1, various stresses could induce DNA ladder formation (Fig. 1a) and caspase 3 activation, which was measured by DEVD cleavage (Fig. 1b) in wild-type ES cells. However, these apoptotic responses were markedly attenuated by the thermotolerance treatment. To confirm the involvement of Apaf1 in the stress-induced apoptosis, ES cells lacking Apaf1 (apaf1^–/–) (3) were subjected to the same experiments. As shown in Fig. 1c, apoptosis in response to various stresses including heat shock, UV, cisplatinum, etoposide, thapsigargin, and tunicamycin was almost completely blocked in apaf1^–/– ES cells. These results clearly show that the stress-induced apoptosis is mediated through mitochondria in ES cells.

View larger version (56K): [in this window] [in a new window]   FIG. 1. Characterization of stress-induced apoptosis in ES cells. Wild-type ES cells were treated with (+) or without (–) heat shock at 44 °C for 10 min (thermotolerance), incubated for 6 h, and stimulated with the indicated stresses for 12 h. a, agarose-gel electrophoresis of DNA fragmentation. b, caspase 3 activation measured by DEVD cleavage. c, wild-type and apaf1–/– ES cells were stimulated with heat shock (44 °C for 20 min), UV (20 J/m2), cisplatinum (50 µM), etoposide (2 µg/ml), thapsigargin (200 nM), or tunicamycin (10 µg/ml) and further incubated for 12 h. DNA fragmentation was measured by agarose-gel electrophoresis. The data shown are representative of three independent experiments.

Stress-induced Apoptosis Is Still Observed in sek1^–/– mkk7^–/– ES Cells—To examine the SAPK/JNK-inactivation effect, various apoptotic responses including caspase 3 activation, DNA fragmentation, and sub-G₁ population were further analyzed in sek1^–/– mkk7^–/– ES cells in comparison with wild-type and apaf1^–/– ES cells (Fig. 3). Surprisingly, the apoptotic responses were still observed clearly in sek1^–/– mkk7^–/– ES cells at the same levels as wild-type ES cells. In sharp contrast, such responses were almost completely abolished in apaf1^–/– cells. The dose dependence and time course of etoposide-induced apoptosis were also the same between sek1^–/– mkk7^–/– and wild-type ES cells (Fig. 3d and data not shown). Thus, the SAPK/JNK inactivation appears to exert no influence on the stress-induced apoptosis in ES cells.

View larger version (56K): [in this window] [in a new window]   FIG. 3. Stress-induced apoptosis in wild-type, sek1–/– mkk7–/–, and apaf1–/– ES cells. ES cells were stimulated with UV (20 J/m2), etoposide (2 µg/ml), tunicamycin (10 µg/ml), or anisomycin (5 µg/ml) and further incubated for 12 h, and caspase 3 activation (a), DNA fragmentation (b), and sub-G1 population (c) were measured. ES cells were stimulated with the indicated concentrations of etoposide, and caspase 3 activation (d) was measured.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Effect of p38 inhibition on stress-induced apoptosis in ES cells. a, ES cells were incubated with (+) or without (–) 10 µM SB203580 for 1 h, stimulated with UV (20 J/m2), and further incubated for 12 h. UV-induced apoptosis was estimated by caspase 3 activation. b, ES cells were stimulated with UV (1 kJ/m2 for 30 min) in the presence (+) or absence (–) of 10 µM SB203580, and active forms of p38 and JNK were detected by immunoblotting with anti-phospho-p38 and anti-phospho-JNK antibodies.

View larger version (53K): [in this window] [in a new window]   FIG. 5. Stress-induced Bax translocation in wild-type, sek1–/– mkk7–/–, and apaf1–/– ES cells. ES cells were stimulated with UV (200 J/m2) and further incubated for 4, 8, and 12 h. Mitochondrial membranes were prepared and immunoblotted (IB) with anti-Bax and anti-mitochondrial heat shock protein 70 (mt-HSP70) antibodies.

View larger version (53K): [in this window] [in a new window]   FIG. 6. Stress-induced apoptosis in wild-type, sek1–/– mkk7–/–, and apaf1–/– MEF-like cells. ES cells were treated with 10 µM retinoic acid for 14 days and differentiated into MEF-like cells. The cells were stimulated UV (200 J/m2), etoposide (20 µg/ml), tunicamycin (50 µg/ml), or anisomycin (50 µg/ml) and further incubated for 20 h. UV-induced apoptosis was measured by DNA fragmentation.

View larger version (37K): [in this window] [in a new window]   FIG. 7. IL-1-induced IL-6 gene expression is impaired in sek1–/– mkk7–/– MEF-like cells. a, wild-type and sek1–/– mkk7–/– MEF-like cells were stimulated with 100 ng/ml IL-1 for 1 and 6 h. b, wild-type MEF-like cells were incubated with (+) or without (–) 50 µM SP600125 for 1 h and further stimulated with IL-1 for 1 h. Total RNAs were prepared and applied on agarose gel. IL-6 and -actin mRNAs were detected by Northern blot analysis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

First, SAPK/JNK activation is completely dependent on the existence of both SEK1 and MKK7 in mouse ES cells in which stress-induced apoptosis is mediated through mitochondria (Fig. 1). The mutant sek1^–/– mkk7^–/– ES cells had a defect in the SAPK/JNK activation in response to a variety of stimuli (Fig. 2) but displayed normal stress-induced apoptosis as the same level as wild-type cells (Fig. 3). Another stress-responsive MAPK, p38, was not involved in the stress-induced apoptosis (Fig. 4). Second, Bax was capable of translocating into mitochondrial membranes in response to UV irradiation in sek1^–/– mkk7^–/– ES cells as observed in the wild-type cells (Fig. 5). Third, the apoptosis in response to various stresses such as UV, etoposide, tunicamycin, and anisomycin occurred even in sek1^–/– mkk7^–/– MEF-like cells. Therefore, there must be a SAPK/JNK-independent signaling that is required for the initiation of stress-induced apoptosis in ES and MEF-like cells.

To examine the involvement of MKK7 in cell cycle and senescence, we have recently prepared mkk7^–/– MEFs from mouse embryos. Interestingly, early passaged wild-type and mkk7^–/– MEFs showed comparable extent and kinetics of cell death in response to UV irradiation; however, at late time points of culture (after getting cellular immortality), mkk7^–/– MEFs displayed a resistance to the UV-induced apoptosis.² These results suggested that the resistance to UV response observed in Jnk1^–/– Jnk2^–/– and mkk4^–/– mkk7^–/– MEFs (16, 17) might have some relations to cellular immortality. Because SAPK/JNK activation is crucial for hepatoblast proliferation (12), the immortalized MEFs may lose the regulation of checkpoint in cell cycle and result in resistance to stress-induced apoptosis due to impaired G₁ growth arrest. Therefore, it will be interesting to find out whether re-expression of JNK1 plus JNK2 in Jnk1^–/– Jnk2^–/– or MKK4 plus MKK7 in mkk4^–/– mkk7^–/– MEFs can restore the apoptotic response to UV stress.

However, recent reports with Jnk1^–/– Jnk2^–/– mice clearly show that SAPK/JNK activation is required not only for anti-apoptosis but also for pro-apoptosis in the development of mouse fetal brain (14, 15). There was a reduction of cell death in the lateral edges of hind brain prior to neural tube closure. In contrast, there was increased apoptosis and caspase activation in the forebrain in Jnk1^–/– Jnk2^–/– mouse embryos. Thus, SAPK/JNK activation plays a pro-apoptotic role under some circumstances. SAPK/JNK activation may be involved in regulating apoptosis indirectly rather than directly.

SAPK/JNK is activated in T cells by co-stimulation with antigen and CD28 receptors and regulates the production of growth factor IL-2 and cell proliferation (29). Previously, we reported impaired CD28-mediated IL-2 production and proliferation in sek1^–/– T lymphocytes (23). It has recently been reported that activation of wild-type CD8⁺ T cells in the presence of different concentrations of SP600125 caused a dose-dependent inhibition of IL-2 production and cell proliferation (30). Thus, SAPK/JNK activation plays an important role in T cell functions. IL-1 is a pro-inflammatory cytokine in inflamed tissues, and the molecular mechanism of IL-1-induced gene expression will yield novel molecular targets for anti-inflammatory therapy. It has been reported that SAPK/JNK activation is required for the gene expression of a cytokine IL-6 and a chemokine IL-8 in a human epidermal carcinoma cell line by using overexpression of a catalytically inactive mutant or antisense RNA of SAPK (31, 32). These results indicate that IL-1-mediated activation of SAPK/JNK induces the phosphorylation of a Jun family of component(s) of activator protein-1, resulting in the gene expression of IL-6 and IL-8. In this study, we have also observed the impaired IL-1-mediated IL-6 gene expression both in sek1^–/– mkk7^–/– MEF-like cells and in SP600125-treated wild-type cells (Fig. 7). These results provide the first genetic link between SAPK/JNK activation and IL-6 gene expression using gene-disrupted cells and also indicate that SAPK/JNK activation provides a crucial and specific signal for immune responses. Thus, the molecular switch, SAPK/JNK activation, regulates cell proliferation and gene expression in embryonic development and immune responses rather than stress-induced apoptosis.

	FOOTNOTES

 
^* This work was supported in part by research grants from the Ministry of Education, Science, Sports, and Culture and the Ministry of Health, Labor and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4754; Fax: 81-3-5841-4751; E-mail: nishina{at}mol.f.u-tokyo.ac.jp.

¹ The abbreviations used are: SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; SEK, stress-activated protein kinase/extracellular signal-regulated kinase kinase; ES, embryonic stem; MEF, mouse embryonic fibroblast; IL, interleukin; TNF-, tumor necrosis factor ; E, embryonic day; ERK, extracellular signal-regulated kinase.

² T. Wada, N. Joza, H.-Y. M. Cheng, T. Sasaki, I. Kozieradzki, M. Nghiem, K. Bachmaier, T. Katada, H. Nishina, and J. M. Penninger, submitted for publication.

	ACKNOWLEDGMENTS

 
We thank Dr. Izumu Saito and RIKEN BRC for providing us with adenovirus-encoding Cre recombinase. We also thank Dr. James R. Woodgett for suggestions and criticism.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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