SOCS-1 suppresses TNF-
-induced apoptosis through the regulation of Jak activation
Akihiro Kimura1,
Tetsuji Naka1,
Shigekazu Nagata2,
Ichiro Kawase1 and
Tadamitsu Kishimoto3
1 Department of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka, 565-0871, Japan 2 Department of Genetics, Osaka University Medical School, 2-2, Yamada-oka, Suita, Osaka, 565-0871, Japan 3 Laboratory of Immune Regulation, Graduate School of Frontier Biosciences, Osaka University, 1-3, Yamada-oka, Suita, Osaka, 565-0871, Japan
Correspondence to: T. Kishimoto; E-mail: a.kimura{at}imed3.med.osaka-u.ac.jp
Transmitting editor: T. Watanabe
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Abstract
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Suppressor of cytokine signaling-1 (SOCS-1) was identified as one of the negative feedback regulators of Janus kinase (Jak)-signal-transducer-and-activator-of-transcription (STAT) signaling. So far, it has been reported that SOCS-1 inhibits the action of multiple cytokines at least in vitro. We previously showed that SOCS-1 suppresses tumor necrosis factor-
(TNF-
)-induced apoptosis in murine embryonic fibroblast, but the mechanism of suppression was not fully clarified. In this study, we show that Jaks bind to TNF receptor-1 (TNFR-1) and are activated by TNF-
. We also show that the activations of Jaks and caspases by TNF-
are suppressed by SOCS-1. Furthermore, in Jak-deficient cell lines, DNA fragmentation and caspase-8 activation by TNF-
are suppressed, indicating that Jaks participate in TNF-
-induced apoptosis signaling. Taken together, these results suggest that SOCS-1 inhibits TNF-
-induced apoptosis through regulation of Jaks.
Keywords: caspase, cell death, negative feedback regulator, TNFR-1, tyrosine kinase
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Introduction
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The proinflammatory cytokine tumor necrosis factor (TNF) has diverse biological effects, such as induction of other cytokines, cell proliferation, differentiation and apoptosis (1,2). The intracellular signal is initiated by the binding of TNF-
to one of two specific and ubiquitously expressed receptors, TNFR-1 and TNFR-2. Both TNFR-1 and TNFR-2 belong to the TNF/nerve-growth factor (NGF) receptor superfamily and are characterized by multiple cysteine-rich domains in the extracellular region (3,4). Along with several other members of this superfamily such as Fas, DR3, DR4 and DR5, TNFR-1 is also known as a death receptor (5,6).
The engagement of TNFR-1 triggers the recruitment of the death domain (DD)-containing adaptor molecule known as TNF receptor-associated death domain protein (TRADD) followed by that of the DD-containing Fas-associated death domain protein (FADD). This signaling complex, the so-called death-inducing signaling complex (DISC), allows for caspase-8 recruitment and activation that lead to apoptosis (7). TNFR-1 occupation not only triggers the apoptosis pathway, but can also lead to the NF-
B and JNK signaling pathways by instead binding the DD-containing Ser/Thr kinase receptor-interacting protein (RIP) (via TRADD), followed by binding to the TNFR-associated factor (TRAF2/5) and c-inhibitor of apoptosis proteins (c-IAP) (1,8,9). On the other hand, ligation of TNFR-2 can affect the expression of genes involved in immediate early prosurvival responses, and this also depends on the activation of NF-
B (10).
Suppressor of cytokine signaling-1 (SOCS-1), also called STAT-induced STAT inhibitor-1 (SSI-1) and Jak-binding protein (JAB), was initially identified as an intracellular negative feedback molecule that inhibits Jak-STAT signaling initiated by various stimuli, including IFN-
, IL-4, IL-6 and leukemia-inhibitory factor (LIF) (1113). SOCS-1 can be induced by various cytokines and binds to Jaks to inhibit the subsequent signal transduction (1417). SOCS-1-deficient (SOCS-1 KO) mice are born healthy but develop various abnormalities as they grow, including growth retardation, thymic atrophy and fulminant hepatitis accompanied by serious fatty degeneration and lung damage through infiltration of mononuclear cells, and all die within three weeks after birth (18,19). These pathological alterations are reduced in IFN-
-deficient SOCS-1 KO mice (20,21). SOCS-1 is therefore a key molecule for negative feedback regulator of IFN-
signaling in vivo. Recently, however, it has been reported that SOCS-1 is essential for in vivo crosstalk inhibition in cytokine between IFN-
and IL-4, that SOCS-1 has the capacity to associate in vitro with other types of intracellular molecule such as Vav, Tec, Syk, ITAM motif of CD8, and Kit receptor, and that SOCS-1 plays important roles in the innate immune response by regulating lipopolysaccharide (LPS) signaling (2227). In addition, we previously reported that SOCS-1 is induced by TNF-
and inhibits TNF-
-induced apoptosis (28,29), although the molecular mechanisms involved in the related signaling have not yet been clearly identified.
Here, we demonstrate that SOCS-1 inhibits only the apoptosis pathway, but not the NF-
B pathway, in TNF-
signaling. To explore the related mechanism, we first investigated whether Jaks are involved in TNF-
-induced apoptosis signaling, as Jaks are target proteins of SOCS-1 and it has been reported that TNF-
activates Jak-STAT signaling (30,31). As expected, we found that Jaks were activated by TNF-
and bound with the domain-like box-1 in the TNFR-1 cytoplasmic region. We also found that, in Jak-deficient cell lines, TNF-
-induced apoptotic processes such as caspase-8 activation and DNA fragmentation were inhibited. Viewing the findings as a whole, it suggests that SOCS-1 suppresses TNF-
-induced apoptosis by inhibiting Jak activation.
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Methods
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Cells and cell culture
The human leukemic T-cell line Jurkat was cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum, 100 µg/ml streptomycin, 100 units/ml penicillin G potassium at 37°C and 5% CO2. Stable transfected Jurkat mutant lines (Jurkat/Neo, Jurkat/SOCS-1) were maintained in the presence of 500 µg/ml G418 (Nacalai Tesque, Kyoto, Japan). The human fibrosarcoma 2fTGH cells, and mutant cell lines derived from this clone deficient in Jak1 (U4A), Tyk2 (U1A), Stat1 (U3A) (a gift from G. R. Stark, The Cleveland Clinic Foundation, OH) and COS7 cells were cultured in high glucose Dulbeccos modified Eagles medium containing 10% fetal calf serum and antibiotics at 37°C and 5% CO2. Reconstituted 2fTGH mutant lines were also maintained in the presence of 500 µg/ml G418.
Measurement of apoptosis by flow cytometry
Cells were left untreated or stimulated with 100 ng/ml hTNF-
and 100 µM cycloheximide (CHX), 100 ng/ml hTNF-
, 100 µM CHX and 100 µM Genistein or 100 ng/ml anti-Fas, CH-11 (MBL, Nagoya, Japan). According to MEBCYTO Apoptosis Kit (MBL), cells were washed in PBS, and resuspended in 100 µl of binding buffer. Cells were then incubated with 10 µl of annexin V-FITC for 15 min at room temperature in the dark, followed by addition of 400 µl of binding buffer, and analyzed by Cytomics FC500 (Beckman Coulter, Inc.).
Caspase-3 and caspase-8 activity
Cells were plated in 60-mm dishes and stimulated with 100 ng/ml hTNF-
and 5 µM CHX. Cells were lysed with lysis buffer (1% NP-40, 20 mM TrisHCl pH 7.5, 150 mM NaCl, 10 mM Na2VO4, 0.5mM DTT, 1/100 protease inhibitor cocktail), and then samples were resolved by SDSPAGE. Caspase-3 and caspase-8 were detected by mouse monoclonal anti-caspase-3 antibody (Santa Cruz, CA), rabbit polyclonal anti-caspase-8 antibody and HRP-conjugated anti-mouse (or rabbit) Ig antibody. Signals were visualized by enhanced chemiluminescence reagent (Perkin Elmer, Boston, MA).
Coimmunoprecipitation and western blotting
Jurkat/Neo, Jurkat/SOCS-1 and Jurkat were treated with 100 ng/ml hTNF-
and 5 µM CHX for the indicated times. Cells were lysed as described above and then lysates were immunoprecipitated for 2h at 4°C by 2 µg of anti-hTNFR-1 antibody (R&D System, Inc., MN) or anti-caspase-8 antibody (MBL). Following the incubation for 1h at 4°C with protein GSepharose, samples were analyzed by western blotting using anti-TRADD antibody (MBL) or anti-Jak1 (BD Transduction Laboratories).
COS7 cells were transfected with 5 µg of pEF-BOS-Jak1 and 5 µg of pcDNA3.1-TNFR-1 or 5 µg of pcDNA3.1-mutant TNFR-1 [TNFR-1
344426, TNFR-1
315426, TNFR-1
243426 and TNFR-1
box(229323)] by DEAEdextran method. After incubation for 2 days, cells were lysed as described above and immunoprecipitated by anti-TNFR-1 antibody, followed by incubation with protein GSepharose. Immunocomplex was resolved by SDSPAGE and Jak1 was detected by anti-Jak1 antibody.
NF-
B activation
Cells were stimulated with 100 ng/ml hTNF-
for the indicated times. Nuclear extracts were prepared by a modified method of Haglund and Rothblum (32). Briefly, cells were washed in PBS and pelleted before resuspension in 200 µl of cold buffer A (10 mM HEPES pH 7.9, 50 mM NaCl, 1mM DTT, 0.1mM EDTA, 0.1mM PMSF) and incubated for 20 min on ice prior to the addition of 200 µl of cold buffer B (buffer A with 0.1% NP-40). Cells were gently pipetted and returned to ice for another 20 min. Nuclei were pelleted, washed in buffer A, and pelleted again, and nuclear proteins were extracted in 25 µl of buffer D (400 mM NaCl, 20 mM HEPES pH 7.9, 1mM EDTA, 1mM EGTA, 1mM DTT, 1mM PMSF). The tubes were put on ice for at least 30 min, followed by centrifugation at 4°C for 10 min. Nuclear extracts were resolved by SDSPAGE, samples were detected by anti-NF-
B p65 antibody (Santa Cruz).
Cytotoxicity assay
Cells were seeded 24 h before the assay in 96-well plates at a density of 2 x 104 cells per well. Cells were treated with hTNF-
at the indicated concentration for 18 h in the presence of 5 µM CHX. After treatment for 18 h, cell viability was assessed with a Cell Counting Kit (Dojin Laboratories, Kumamoto, Japan).
In vitro kinase assay
Jak1 was immunoprecipitated as described above from equal numbers of untreated and TNF-treated cells (2 x 107). Immunoprecipitates were washed once with lysis buffer, once with 0.5 M LiCl and 20 mM Tris (pH 7.5), and once with distilled water. Samples were resuspended in 30 µl of 20 mM Tris (pH 7.5), 10 mM MnCl and 10 µCi of [
-32P]ATP (Amersham) per kinase assay, and incubated at room temperature for 15 min. Samples were then washed with distilled water, and proteins were separated by SDSPAGE, transferred onto nitrocellulose, and analyzed by autoradiography. Equal loading of samples was assessed by immunoblotting the same membranes with anti-Jak1 antibodies.
DNA fragmentation
Cells were treated with hTNF-
or anti-Fas, CH-11 in the presence of CHX. After washed by PBS, cells were centrifugated and supernatants were removed. Pellets were lysed with lysis buffer (10 mM TrisHCl pH 7.4, 10 mM EDTA pH 8, 0.5% Triton X-100). After incubation at 4°C for 10 min, samples were centrifuged and then supernatants were transferred to new tubes. Following treatment with RNase A for 1 h, samples were treated with proteinase K for 30 min, analyzed on 2% agarose gels, and visualized by ethidium bromide staining.
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Results
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SOCS-1 inhibits only TNF-
-induced apoptosis, but not TNF-
-induced NF-
B activation or FasL-induced apoptosis
We previously reported that TNF-
-induced apoptosis was more potent in SOCS-1 KO murine embryonic fibroblasts (MEF) than in wild-type (WT) MEF (29). To clarify how SOCS-1 inhibits TNF-
-induced apoptosis, we established human acute T-cell leukemia cell lines (Jurkat cells) that constitutively expressed SOCS-1 (Jurkat/SOCS-1). The expression of SOCS-1 was confirmed by western blotting (Fig. 1D). With Jurkat/Neo as control, Jurkat/SOCS-1 was treated with TNF-
in the presence of CHX and, after 3 h, apoptosis was quantitated by flow-cytometric detection of annexin-V positive cells. The annexin-V positive cell count was significantly reduced in Jurkat/SOCS-1 compared to that in the control (Fig. 1A). It is also known that FasL induces apoptosis by forming DISC in a similar manner to TNF-
-induced apoptosis. However, there was no difference in FasL-induced apoptosis between Jurkat/SOCS-1 and Jurkat/Neo (Fig. 1B). We also investigated whether SOCS-1 affects the NF-
B pathway also known to be involved in TNF-
signaling. Consistent with a previous report (27), SOCS-1 could not inhibit the NF-
B pathway (Fig. 1C).
Recruitment of TRADD and FADD following ligation of TNFR-1 with TNF-
leads to the recruitment of procaspase-8 and results in its autocatalytic activation to mature active caspase-8, which cleaves downstream caspases including procaspase-3 to initiate apoptosis (1,3335). To investigate whether SOCS-1 affects this caspase-8 and caspase-3 activation, Jurkat/Neo and Jurkat/SOCS-1 were treated with TNF-
plus CHX. It is known that both caspase-3 and caspase-8 are cleaved, leading to their activation. Cell extracts from Jurkat/Neo and Jurkat/SOCS-1 after treatment with TNF-
plus CHX were subjected to western blot analysis with anti-caspase-3 and anti-caspase-8 antibodies. In Jurkat/SOCS-1, TNF-
-induced cleavage of procaspase-3 was delayed compared to Jurkat/Neo (Fig. 1D). In addition, SOCS-1 inhibited TNF-
-induced caspase-8 cleavage (Fig. 1E). Taken together, these results suggest that SOCS-1 inhibits TNF-
-induced apoptosis involving caspase-3 and caspase-8 activation by regulating an event concerned only with TNF-
signaling, and not with FasL signaling, and moreover that SOCS-1 acts on caspase-8 in TNF-
-induced apoptosis.
SOCS-1 affects interaction between TRADD and caspase-8, but not between TNFR-1 and TRADD
To investigate whether SOCS-1 affects DISC formation in TNF-
-induced apoptosis, we first examined the interaction between TNFR-1 and TRADD using Jurkat/SOCS-1 and Jurkat/Neo. After TNF-
stimulation for 15 min, cell lysates were immunoprecipitated by anti-TNFR-1 antibody and samples were analyzed by western blotting using anti-TRADD antibody. There was no difference in the interaction between TNFR-1 and TRADD observed in the two cell lines (Fig. 2A). As a new model of TNF-
signaling and TNF-
-induced apoptosis, it has recently been demonstrated that, after TNF-
stimulation, TRADD, TRAF2 and RIP1 dissociate from TNFR-1, and that the dissociated complex finally binds to FADD and caspase-8, resulting in apoptosis (36,37). Consistent with this model, we could not detect TNF-
-induced interaction between TNFR-1 and FADD (or caspase-8) (data not shown). We therefore next examined the interaction between caspase-8 and TRADD. After TNF-
stimulation in Jurkat/Neo and Jurkat/SOCS-1, cell lysates were immunoprecipitated by anti-caspase-8 and analyzed by western blotting using anti-TRADD antibody. While TNF-
-induced interaction between caspase-8 and TRADD was detected in Jurkat/Neo, the interaction between caspase-8 and TRADD was inhibited in Jurkat/SOCS-1 (Fig. 2B). These data suggest that SOCS-1 may inhibit TNF-
-induced DISC formation in TRADD/caspase-8, but not in TNFR-1/TRADD.

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Fig. 2. SOCS-1 inhibits interaction between TRADD and caspase-8. (A) After stimulation with 100 ng/ml hTNF- and 100 µM CHX for 15 min in Jurkat/Neo cells or Jurkat/SOCS-1 cells, cell lysates were immunoprecipitated by anti-TNFR-1 antibody and then immunoblotted by anti-TRADD antibody. (B) As shown in (A), after stimulation for 1 h, 3 h and 5 h, interaction between TRADD and caspase-8 was determined.
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Jaks act downstream of TNFR-1 in TNF-
signaling
It has been reported that TNF-
activates Jaks through TNFR-1 (30,31). First we tested the interaction between TNFR-1 and Jak1. After treatment of Jurkat cells with TNF-
, cell lysates were immunoprecipitated by anti-TNFR-1 antibody and the immunoprecipitated samples were analyzed by western blotting using anti-Jak1 antibody. Whether stimulated with TNF-
or not, Jak1 interacted with TNFR-1 (Fig. 3A). It is believed that Jaks associate with the membrane-proximal domain of specific cytokine receptor subunits (3840). Two short motifs with limited homology, designated box 1 and box 2, have been described in the membrane-proximal regions of several cytokine receptors. Box 1 is defined as a proline-rich motif (41), whereas box 2 is characterized as a cluster of hydrophobic amino acids (39). The cytoplasmic region of TNFR-1 contains a box 1-like motif (Fig. 3B). We therefore prepared deletion mutants of TNFR-1 [TNFR-1
344426, TNFR-1
315426, TNFR-1
243426 and TNFR-1
box (229323)] and used them to investigate whether Jak1 associates with the box 1-like domain of TNFR-1 (Fig. 3C). Co-transfection of COS7 cells with Jak1 and each of the TNFR-1 variants revealed that Jak1 bound with TNFR-1 WT, TNFR1
344426, TNFR-1
315426 and TNFR-1
243426, but that its interaction with TNFR-1
box was relatively impaired (Fig. 3D). These results suggest that Jaks may associate with TNFR-1 through the box-like domain in TNFR-1, like other cytokine receptors such as IL-4R and IFN-
R
.
To assess whether Jak1 is activated by TNF-
, we next examined Jak1 kinase activity by in vitro kinase assay. As previously shown (31), we found that Jak1 kinase was activated by TNF-
(Fig. 3E). Moreover, TNF-
-induced Jak1 kinase activity was inhibited by SOCS-1 (Fig. 3E). Here, we speculated that Jaks may participate in TNF-
-induced apoptosis signaling. To verify this hypothesis, we co- incubated Genistein, a tyrosine kinase inhibitor, with TNF-
plus CHX in Jurkat cells, and examined changes in sensitivity to TNF-
-induced apoptosis. Interestingly, Jurkat cells with Genistein displayed greater resistance to TNF-
-induced apoptosis than those without Genistein (Fig. 3F). It is therefore in principle conceivable that Jaks participate in TNF-
-induced apoptosis signaling through TNFR-1. We then proceeded to investigate the relationship between Jaks and TNF-
-induced apoptosis.
Jaks participate in TNF-
-induced apoptosis signaling
In order to investigate whether Jaks participate in TNF-
-induced apoptosis, we first analyzed DNA fragmentation as a hallmark of apoptosis by using the mutant cell lines U4A (Jak1/), U1A (Tyk2/), U3A (STAT1/) and 2fTGH (parental cell line). After treatment of these cell lines with TNF-
plus CHX, qualitative analysis of DNA damage was undertaken from the electrophoretic pattern of DNA fragmentation on agarose gels. DNA fragmentation was detected in 2fTGH and U3A, but not in U4A or U1A (Fig. 4A). Identical data were obtained in MTT assay (Fig. 4B). We tested for caspase-8 activation in these mutant cell lines. As in Jurkat cells, after treatment with TNF-
plus CHX, cell lysates were analyzed by western blotting using anti-caspase-8 antibody. As expected, caspase-8 activation was inhibited in U4A and U1A when compared to 2fTGH (Fig. 4C). To determine if apoptotic function could be restored in these cell variants by restoring the wild-type form of these proteins, Jak1 and tyk2 were expressed stably in U4A and U1A cell lines, respectively (U4A/Jak1 and U1A/Tyk2). Expression of the missing protein (Jak) restored TNF-
-induced caspase-8 activation (Fig. 4D). These results suggest that Jaks are necessary for TNF-
-induced apoptosis while STAT is dispensable for TNF-
-induced apoptosis.
As described above, TNF-
-induced NF-
B activation and FasL-induced apoptosis were unaffected by SOCS-1. We therefore asked whether Jaks would affect TNF-
-induced NF-
B activation and FasL-induced apoptosis. After treatment of 2fTGH, U4A and U1A cells with anti-Fas, DNA fragmentation was detected in all three cell lines (Fig. 4E). NF-
B activation consists of following events, the activation of I
B kinase, the degradation of I
Bs and the nuclear translocation of NF-
B. There was no difference in the nuclear translocation of NF-
B induced by TNF-
stimulation, whether Jaks were present or not (Fig. 4F). Overall, these findings suggest that Jaks participate only in TNF-
-induced apoptosis, and Jaks seem to be dispensable for TNF-
-induced NF-
B activation or FasL-induced apoptosis. Moreover, they indicate that Jaks may be involved in TNF-
-induced apoptosis independent of the JakSTAT pathway, as STATs were found to be unrelated to TNF-
-induced apoptosis. This points up the possibility that Jaks precipitate TNF-
-induced apoptosis by activating a target protein other than STAT in TNF-
signaling.
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Discussion
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Although it has been shown previously that SOCS-1 inhibits TNF-
-induced apoptosis, the suppressant mechanism has not been fully clarified (29,42). In the present study, we investigated how SOCS-1 inhibits TNF-
-induced apoptosis. Interestingly, SOCS-1 inhibited only TNF-
-induced apoptosis, but not TNF-
-induced NF-
B activation or FasL-induced apoptosis. We therefore began by exploring whereabouts SOCS-1 operates in TNF-
-induced apoptosis signaling. TNF-
triggers the activation of caspase-8 and caspase-3, known as apoptosis execute factors, and thereby leads cells to apoptosis. SOCS-1 inhibited the activation of both caspase-8 and caspase-3 (Fig. 1D and E). These findings brought us to the conclusion that SOCS-1 must operate upstream of caspase-8. When we examined whether SOCS-1 affects TNF-
-induced DISC formation, we found that SOCS-1 disturbed the interaction between TRADD and caspase-8 (Fig. 2B), but did not prevent TRADD from interacting with TNFR-1 (Fig. 2A), which was consistent with the finding that TNF-
-induced NF-
B activation was unaffected by SOCS-1 (Fig. 1C). From these results, we concluded that SOCS-1 was involved in regulating the upstream of TNF-
-induced apoptosis signaling.
What then was the target of SOCS-1 in TNF-
-induced apoptosis signaling? It is well known that Jaks are the target proteins of SOCS-1, and it has recently been reported that TNF-
activates Jaks through TNFR-1 (30,31). We therefore expected that Jaks might participate in TNF-
-induced apoptosis. Our analysis revealed the relationship between Jaks and TNF-
-induced apoptosis as follows. TNFR-1 had a box-1-like motif (Fig. 3B) through which Jak1 associated with it (Fig. 3A and D). Jak1 was activated by TNF-
, and SOCS-1 inhibited TNF-
-induced Jak1 activation (Fig. 3E). Moreover, cells treated with Genistein, a tyrosine kinase inhibitor, displayed resistance to TNF-
-induced apoptosis (Fig. 3F). This suggested that tyrosine kinase, that is to say Jak, might participate in TNF-
-induced apoptosis. To investigate this possibility, we used Jak (or STAT)-deficient cell lines. TNF-
-induced DNA fragmentation and caspase-8 activation were reduced in U4A (Jak1/) and U1A (Tyk2/) cell lines compared to 2fTGH (parental) and U3A (STAT1/) (Fig. 4C). STATs are known to be downstream molecules of Jaks. It was once thought that STAT1 was essential for TNF-
-induced apoptosis (43,44), but this has been demonstrated not to be so (29,45). The above result is consistent with the later report. Moreover, the restoration of Jak was sufficient to restore the sensitivity to apoptosis by TNF-
(Fig. 4D). Like SOCS-1, Jaks were not required for NF-
B activation by TNF-
. These results suggest that Jaks play an important role in TNF-
-induced apoptosis signaling independent of STAT.
Our overall findings indicate that SOCS-1 suppresses TNF-
-induced apoptosis by regulating Jaks. Although the molecular mechanisms of TNF-
-induced activation of the prosurvival pathway (NF-
B, JNK pathway) have been reasonably well elucidated (8,46), the principle which decides whether TNF signals cell survival or cell death remains largely unknown. Our data suggest that Jaks may be one of the factors regulating between the survival and the death signal. But how are Jaks involved in TNF-
signaling? It has recently been demonstrated as a new model of TNF-
-induced apoptosis that TRADD interacts with caspase-8 following the dissociation of TRADD from TNFR-1 (36,37). And we expect the following about the involvement of Jaks in TNF-
signaling: as SOCS-1 was found to inhibit the interaction between TRADD and caspase-8, we believe that Jaks may promote to dissociate TRADD from TNFR-1. For instance, through the phosphorylation of TRADD or TNFR-1 by Jaks in TNF-
signaling, TRADD may dissociate from TNFR-1 and interact with caspase-8 followed by the new DISC formation (apoptosis pathway). On the other hand, when TRADD or TNFR-1 was not phosphorylated by Jaks, TRADD and TRAF2 may associate with TNFR-1 followed by the NF-
B pathway. Jaks and SOCS-1 may thereby act on the switch of survival or death signaling by TNF-
. Thereby Jaks and SOCS-1 may regulate survival or death signaling by TNF-
. Further analysis may lead to the discovery of new functions of Jaks other than JakSTAT signaling. As SOCS-1 is one of the regulatory proteins of Jaks, it is likely that it operates against such Jak signaling in various signal transductions including cytokines.
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Acknowledgements
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We gratefully acknowledge the provision of 2fTGH, U4A, U1A and U3A cell lines by Dr G. R. Stark at The Cleveland Clinic Foundation, Ohio, and TNFR-1 cDNA by Dr Y. Niitsu (Sapporo Medical University). We thank Dr T. Taga (Kumamoto University) for helpful discussion and Ms Ito for her secretarial assistance. This work was supported in part by grants, a grant-in-aid, and a Hitec Research Center grant from the Ministry of Education, Science, and Culture, Japan.
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Abbreviations
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CHXcycloheximide
DISCdeath-inducing signaling complex
FADDFas-associated death domain protein
JakJanus kinase
STATsignal-transducer-and-activator-of-transcription
SOCS-1suppressor of cytokine signaling-1
TRADDTNF receptor-associated death domain protein
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