Suppressor of Cytokine Signaling-1 (SOCS-1) Suppresses Tumor Necrosis Factor ␣-Induced Cell Death in Fibroblasts
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Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)͞suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor ␣-induced cell death in fibroblasts Yoshiaki Morita*†, Tetsuji Naka*, Yoshinori Kawazoe*, Minoru Fujimoto*, Masashi Narazaki*, Reiko Nakagawa‡, Hidehiro Fukuyama§, Shigekazu Nagata§, and Tadamitsu Kishimoto¶ʈ Departments of *Medicine III, ‡Microbiology, and §Genetics, Osaka University Medical School, 2-2, Yamada-oka, Suita-city, Osaka 565-0871, Japan; ¶Osaka University, 1-1 Yamada-oka, Suita-city, Osaka 565-0871, Japan; and †Department of Immunology and Inflammation Medicinal Biology Research Laboratory, Fujisawa Pharmaceutical Company, Ltd., 1-6 Kashima 2 Chome Yodogawa-ku, Osaka 532-8514, Japan Contributed by Tadamitsu Kishimoto, February 28, 2000 Signal transducers and activators of transcription (STAT)-induced Ϫ͞Ϫ mice. Moreover, murine embryonic fibroblasts (MEFs) STAT inhibitor-1 [SSI-1; also known as suppressor of cytokine lacking the SSI-1 gene become sensitive to TNF-␣-induced cell signaling-1 (SOCS-1)] was identified as a negative feedback regu- death. In contrast, L929 cells forced to express SSI-1 (L929͞ lator of Janus kinase-STAT signaling. We previously generated SSI-1) show resistance to TNF-␣-induced cell death. In addition, mice lacking the SSI-1 gene (SSI-1 ؊͞؊) and showed that thymo- SB203580, a highly specific inhibitor of p38 mitogen-activated cytes and splenocytes in SSI-1 ؊͞؊ mice underwent accelerated protein (MAP) kinase, eliminates resistance to TNF-␣-induced apoptosis. In this paper, we show that murine embryonic fibro- cell death in L929͞SSI-1 cells. Also, SSI-1 is revealed to relate blasts lacking the SSI-1 gene are more sensitive than their litter- to the activation of p38 MAP kinase. Other families of SSI, mate controls to tumor necrosis factor-␣ (TNF-␣)-induced cell however, such as SSI-3 and SOCS-5, do not show these effects. death. In addition, L929 cells forced to express SSI-1 (L929͞SSI-1), The primary nature of SSI-1 is a negative feedback regulator of but not SSI-3 or SOCS-5, are resistant to TNF-␣-induced cell death. JAK-STAT signaling. However, the experiment using STAT1 Furthermore L929͞SSI-1 cells treated with TNF-␣ sustain the acti- Ϫ͞Ϫ or STAT6 Ϫ͞Ϫ MEFs reveals that JAK-STAT signaling is vation of p38 mitogen-activated protein (MAP) kinase. In contrast, not related to TNF-␣-induced cell death signals. Thus, our SSI-1 ؊͞؊ murine embryonic fibroblasts treated with TNF-␣ show results show that SSI-1 suppresses TNF-␣-induced cell death hardly any activation of p38 MAP kinase. These findings suggest through regulation of p38 MAP kinase instead of through that SSI-1 suppresses TNF-␣-induced cell death, which is mediated regulation of JAK-STAT signaling, and they reveal an important by p38 MAP kinase signaling. role of SSI-1 on TNF-␣ signaling. ytokines play important roles in controlling homeostasis of Materials and Methods Corganisms through cell differentiation, proliferation, and Measurement of Serum TNF-␣, IFN-␥, and IL-1. Whole blood was apoptosis, and these effects are mainly brought about by Janus collected from SSI-1 Ϫ͞Ϫ mice or SSI-1 ϩ͞ϩ mice under kinase-signal transducers and activators of transcription (JAK- anesthesia at 10 days after birth, and then was centrifuged. STAT) signaling. Serum of SSI-1 ϩ͞ϩ mice injected intravenously with Con A at Recently, several groups independently have cloned negative a dose of 10 g͞g of body weight was used as positive control. feedback regulators of JAK-STAT signaling. STAT-induced The serum was stored at Ϫ80°C. TNF-␣ serum samples were STAT inhibitor-1 (SSI-1)͞suppressor of cytokine signaling-1 measured by using a mouse TNF-␣ ELISA kit (Genzyme) (SOCS-1) is known as one of the negative feedback regulators of according to the manufacturer’s instructions. IFN-␥ or IL-1 was JAK-STAT signaling (1–3). SSI-1 is induced by various cyto- measured with an ELISA kit (BioSource International, Cama- kines, such as IL-2, IL-3, IL-4, IL-6, IL-13, granulocyte– rillo, CA). macrophage colony-stimulating factor, erythropoietin, IFN-␥, and leukemia inhibitory factor, can bind to all four members of Plasmid Construction and DNA Transfection. Mouse SSI-1 cDNA the JAKs, and inhibits signals of various cytokines by suppressing was subcloned into the mammalian vector pEF-BOS and ex- the activation of the JAKs (4). However, the specificity of SSI-1 pressed under the control of an elongation factor gene promoter. for the JAKs in vitro is not well defined. SSI-1 has two charac- An Arg-105 point mutation of SSI-1 yielding Gln was introduced teristic domains, an Src homology 2 domain and a carboxyl- by using a quick-change site-directed mutagenesis kit (Strat- terminal conserved domain, which we have called the SC-motif (also referred to as the SOCS box). So far the cloned SSI families (5–6) comprise at least eight members on the basis of the two Abbreviations: STAT, signal transducers and activators of transcription; SSI-1, STAT-induced characteristic domains, even if overlapping is excluded. STAT inhibitor-1; SOCS-1, suppressor of cytokine signaling-1; TNF-␣, tumor necrosis fac- tor-␣; JAK, Janus kinase; MEFs, murine embryonic fibroblasts; RQ, an Arg-105 point muta- Our previous study revealed that mice lacking the SSI-1 gene tion of SSI-1 yielding Gln; TRAF, TNF receptor-associated factor; MAP kinase, mitogen- (SSI-1 Ϫ͞Ϫ) were healthy and normal at birth, but exhibited activated protein kinase; MKK, MAP kinase kinase; ERK, extracellular signal-regulated growth retardation with aging and died within 3 weeks after birth kinase; JNK, c-Jun amino-terminal kinase; ATF2, activating transcription factor 2; WST-8, (7). In addition, lymphocytes underwent accelerated apoptosis in 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; 1-methoxy-PMS, 1-methoxyphenazine methosulfate. lymphoid organs such as thymus and spleen of SSI-1 Ϫ͞Ϫ mice ʈTo whom reprint requests should be sent at ¶ address. E-mail: [email protected] at 10 days after birth. Because apoptosis generally is brought u.ac.jp. about by death signals, which are produced from tumor necrosis ␣ ␣ The publication costs of this article were defrayed in part by page charge payment. This factor- (TNF- ) or FasL signaling, it was thought that accel- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. erated apoptosis of thymocytes and splenocytes in SSI-1 Ϫ͞Ϫ §1734 solely to indicate this fact. ␣ IMMUNOLOGY mice might result from dysregulated TNF- or FasL signaling. In Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.090084797. this study, we observe an increase in the serum TNF-␣ of SSI-1 Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.090084797 PNAS ͉ May 9, 2000 ͉ vol. 97 ͉ no. 10 ͉ 5405–5410 Downloaded by guest on September 29, 2021 agene) according to the manufacturer’s instructions and desig- Table 1. Serum TNF-␣ protein levels, but not IFN-␥ or IL-1,of nated as RQ. Delta49 was generated by introducing two SalI sites SSI-1 ؊͞؊ mice increase in comparison with those of SSI-1 ؉͞؉ at two predetermined points of the sequence and excising the mice SalI-SalI fragment. All constructs were cloned into the pEF- Cytokine conc., pg͞ml BOS vector and confirmed by DNA sequencing. The mutants were transfected into L929 cells by electroporation in combina- Mice TNF-␣ IFN-␥ IL-1 tion with pSV2 neo, and neomycin-resistant clones were selected Ϫ͞Ϫ Ϯ Ͻ Ͻ ͞ SSI-1 42.8 10.2* 1.0 7.0 in DMEM supplemented with 500 g ml G418 (Nacalai Tesque, SSI-1 ϩ͞ϩ 12.7 Ϯ 4.3 Ͻ1.0 Ͻ7.0 Kyoto) and 10% FCS. Con A-injected (positive control) ND 220.1 59.7 Cytotoxicity Assays. MEFs were derived from embryonic day 16 Serum TNF-␣, IFN-␥, and IL-1 concentrations were determined in ten pairs embryos by the fibroblast-migration method. MEFs were grown of SSI-1 Ϫ͞Ϫ mice and their littermate controls, SSI-1 ϩ͞ϩ mice, by ELISA. Mice in DMEM supplemented with 10% FCS, 100 g͞ml streptomy- were sacrificed at the age of 10 days. Cytokine protein levels are shown as ͞ mean Ϯ SE. ND, not determined. cin sulfate, 100 units ml penicillin G potassium, and MEM ␣ Ϫ͞Ϫ Ͻ nonessential amino acid solution. Cells were seeded 24 h before *Serum TNF- levels of SSI-1 mice show significant difference at P 0.1 as compared with those of SSI-1 ϩ͞ϩ mice. the assay in 96-well plates at a density of 2.5 ϫ 104 cells per well in 5% FCS growth medium. Mouse recombinant TNF-␣ (Pep- ␥ rotech, London) or mouse recombinant IFN- (Peprotech) at g͞ml glutathione S-transferase͞activating transcription factor the indicated concentration was applied to the cells in the ͞ 2 (ATF2) fusion protein as a substrate at 30°C for 30 min. presence of cycloheximide at 10 g ml (Sigma). After treatment Reactions were stopped by adding a 5ϫ SDS͞PAGE sample for 24 h, cell viability was assessed with a Cell Counting Kit buffer, and the samples were run on an SDS͞PAGE gel, followed (Dojin Laboratories, Kumamoto, Japan) to count living cells by by Western blotting. Incorporation of the phosphorus residue combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- into the glutathione S-transferase͞ATF2 protein was detected (2,4-disulfophenyl )-2H-tetrazolium (WST-8) and 1-methoxy- with the phospho-specific ATF2 (Thr71) antibody (NEB). phenazine methosulfate (1-methoxy-PMS). L929 cells were seeded 24 h before the assay in 96-well plates Results ϫ 4 at a density of 2.5 10 cells per well in DMEM supplemented We previously reported that thymocytes and splenocytes lacking with 10% FCS, high glucose, pyridoxine hydrochloride, and 500 Ϫ͞Ϫ ͞ the SSI-1 gene (SSI-1 ) underwent accelerated apoptosis g ml G418, without phenol red, sodium pyruvate, or L- (7).