The role of Tyk2, Stat1 and Stat4 in LPS-induced endotoxin signals
Kenjirou Kamezaki1,2,
Kazuya Shimoda1,2,
Akihiko Numata1,2,
Tadashi Matsuda3,
Kei-Ichi Nakayama4,5 and
Mine Harada1,2
1 First Department of Internal Medicine, Faculty of Medicine, Kyushu University and 2 Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
3 Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-Ku Kita 12 Nishi 6, Sapporo 060-0812, Japan
4 Department of Molecular and Cellular Biology, Laboratory of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Japan
5 CREST, Japan Science and Technology Corporation (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Correspondence to: K. Shimoda; E-mail: kshimoda{at}intmed1.med.kyushu-u.ac.jp
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Abstract
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Mice lacking Tyk2, Stat1 or Stat4, which are members of the JakStat signaling cascade, were resistant to LPS-induced endotoxin shock. Interestingly, Tyk2-deficient mice had higher resistance to LPS challenge than mice lacking either Stat1 or Stat4. The activation of MAPK and NF-
B by LPS, and the production of TNF-
and IL-12 after LPS injection, were not abrogated by the absence of Tyk2, Stat1 or Stat4. In Stat1-deficient mice, the induction of IFN-ß by LPS in macrophages was severely reduced, although the serum level of IFN-
was elevated after LPS injection. In contrast, in Stat-4 deficient mice, the induction of IFN-ß by LPS was normal, but the serum level of IFN-
remained low after LPS injection. Interestingly, the induction of both IFN-ß and IFN-
by LPS was severely reduced in Tyk2-deficient mice. Therefore, Stat1 and Stat4 independently play substantial roles in the susceptibility to LPS. Tyk2 is essential for LPS-induced endotoxin shock, and this signaling pathway is transduced by the activation of Stat1 and Stat4.
Keywords: IFN, IL-12, Jak
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Introduction
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The gram-negative bacterial wall component, endotoxin (LPS), is the major active agent in the pathogenesis of endotoxin shock. The binding of LPS to toll-like receptor 4 (TLR4) leads to the activation of monocytes and macrophages, which then release cytokines and nitric oxide (NO). LPS activates both MyD88-dependent and MyD88-independent pathways, each of which leads to the activation of MAPKs and nuclear factor
B (NF-
B). (1) In addition to the signaling cascade downstream of TLR4, IFN-ß and IFN-
are also involved in sensitivity to LPS. Deficiency of the IFN-ß (2) or IFN-
receptor (3) results in resistance to high dose LPS challenge.
Tyk2 is a member of the Jak kinase family, whose members are activated by cytokine binding to cell surface receptors. Activated Jaks phosphorylate tyrosine residues of the receptors, thereby recruiting signal transducers and activators of transcription (Stats) into the activated receptor complex. Stats are then phosphorylated and activated by Jaks, and subsequently translocate into the nucleus where they affect gene expression (4,5). Tyk2 has been identified as a tyrosine kinase which is able to compensate for a mutation making fibroblasts unresponsive to IFN-
(6). Using Tyk2-deficient mice, we and others have shown that Tyk2 has only minor functions in the IFN-
signaling pathway. These functions include roles in NO production in macrophages induced by LPS and the suppression of the growth of bone marrow progenitor cells. In contrast, almost all IL-12 function was abrogated by the lack of Tyk2 (7,8). In addition, IL-18-induced IFN-
production from T and NK cells, and IL-18-induced NK cell function were abrogated in Tyk2-deficient mice (9). The binding of IL-18 to its receptor activates the DNA binding activity of both NF-
B and AP-1. (10) These data indicate that Tyk2 is involved in the IL-18 signaling cascade. As the intracellular signaling cascades induced by LPS were shared with the cascades activated by IL-18, and because the production of IFN-ß was involved in causing sensitivity to LPS (2), we have examined the roles of Tyk2, Stat1 and Stat4 in LPS signaling. We report here that Tyk2-deficient mice were extremely resistant to LPS shock, and Stat1- and Stat4-deficient mice were moderately resistant to LPS shock compared to their wild-type littermates. Furthermore, LPS signaling was transduced via Tyk2 and the subsequent downstream activation of Stat1 and Stat4.
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Methods
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Mice
The generation of Tyk2-deficient mice has been previously described (7). Stat1-deficient mice were provided by Dr R. Schreiber (11). Stat4-deficient mice were provided by Dr Ihle (12). Tyk2-, Stat1- and Stat-4 deficient mice were maintained on a mixed background of 129/SV and C57BL/6. Mice were housed and bred in the Kyushu University Animal Center.
Antibodies and reagents
Lipopolysaccharide (Salmonella typhimurium) was purchased from Sigma (MO). Recombinant murine IFN-ß was purchased from Chemicon (CA). Anti-I
B-
, -phospho-I
B-
, -ERK1/2, -phospho-ERK1/2, -p38, -phospho-p38 and -phospho-Stat1 antibodies were purchased from Cell Signaling (MA). Anti-phospho-Stat4 and -IRF3 antibodies were purchased from Zymed Laboratories Inc. (CA). Anti-Stat1 and -Stat4 antibodies were purchased from Santa Cruz Biotechnology (CA).
LPS challenge
Mice were injected intraperitoneally with the indicated doses of LPS dissolved in sterile PBS. The mice were monitored for lethality for 120 h after LPS challenge. Five 6-week-old male mice were in each experimental group.
Preparation of peritoneal macrophages
Mice were injected intraperitoneally with 2 ml of 4% thioglycollate. After 72 h, peritoneal macrophages were obtained by peritoneal lavage with 10 ml of ice-cold PBS. Cells were incubated in plastic dishes for 12 h and washed with PBS to eliminate non-adherent cells. Adherent cells were used for further experiments.
Western blotting
Peritoneal macrophages were stimulated with 100 ng/ml of LPS for 30 min or 1 h, or stimulated with IFN-ß for 30 min in RPMI1640 containing 10% FCS. After LPS stimulation, cells were lysed as previously described (13). Cell lysates were centrifuged at 12 000 g for 15 min to remove debris. Total cell lysates were resolved by SDS10% PAGE and transferred to a nitrocellulose membrane. Membranes were probed using appropriate antibodies and visualized by ECL (Amersham, Uppsala, Sweden).
Measurement of cytokines
Six- to eight-week-old male mice were injected intraperitoneally with the indicated doses of LPS and blood was collected at the indicated times after LPS injection. The serum concentration of cytokines was determined using ELISA kits. The IL-12p40 ELISA kit was purchased from BioSource (CA). The IFN-
and TNF-
ELISA kits were purchased from Genzyme (MN).
RNA extraction and cDNA synthesis
Peritoneal macrophages were stimulated with 100 ng/ml of LPS in RPMI1640 containing 10% FCS. Total RNA was extracted from cells using ISOGEN reagent (Nippongene, Tokyo, Japan) according to the manufacturer's protocol. Target RNA (1 µg) was reverse transcribed using 0.25 U AMV Reverse Transcriptase XL (TaKaRa, Otsu, Japan) at 42°C for 30 min in the presence of 50 mM KCl, 10 mM TrisHCl (pH 8.3), 5 mM MgCl2, 1 mM dNTPs, 0.25 U RNase inhibitor, 0.125 µM Oligo dT-Adaptor primer.
Real-time quantitative PCR
Relative quantification of IFN-ß and IRF-7 in cells was performed using real-time quantitative PCR with a TaqMan assay on an ABI 7000 system (Applied Biosystems, CA). Real-time quantitative PCR utilized a cDNA template with the appropriate primers as follows. Primers used to amplify murine IFN-ß were: 5'-ATGAGTGGTGGTTGCAGGC-3' and 5'-TGACCTTTCAAATGCAGTAGATTCA-3', with a murine IFN-ß probe, FAM-5'-AAGCATCAGAGGCGGACTCTGGGA-3'-TAMRA. Primers used to amplify murine IRF-7 were: 5'-CTGGAGCCATGGGTATGCA-3' and 5'-AAGCACAAGCCGAGACTGCT-3', with a murine IRF-7 probe, FAM-5'-CTGGAGGGCGTGCAGCGTGA-3'-TAMRA. Primers used to amplify murine GAPDH were 5'-ACGGCAAATTCAACGGCA-3' and 5'-AGATGGTGATGGGCTTCCC-3', with a murine GAPDH probe, FAM-5'-AGGCCGAGAATGGGAAGCTTGTCATC-3'-TAMRA. PCR amplifications were performed in a 50 µl volume, containing 1 µl cDNA template, 50 mM KCl, 10 mM TrisHCl (pH 8.3), 10 mM EDTA, 200 µM dNTPs, 3 mM MgCl2, 200 nM of each primer, 0.625 U AmpliTaqGold and 0.25 U AmpErase Uracil N-Glycosylase. Each amplification also contained 100 nM of the appropriate detection probe. Each PCR amplification was performed in duplicate, using conditions of 50°C for 2 min preceding 95°C for 10 min, followed by 40 cycles of amplification (95°C for 15 sec, 60°C for 1 min). In each reaction, GAPDH was amplified as a housekeeping gene to calculate a standard curve and allow for the correction for variations in target sample quantities. Relative copy numbers were calculated for each sample from the standard curve after normalization to GAPDH using the instrument software.
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Results
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Tyk2-deficient mice are resistant to LPS-induced endotoxin shock
Injection of LPS into mice causes the activation of macrophages and the resulting secretion of a variety of cytokines and mediators; the consequence is lethal endotoxin shock. To analyze the effects of Tyk2 deficiency on LPS sensitivity in vivo, we injected a high dose of LPS (50 mg/kg) into wild-type and Tyk2-deficient mice (Fig. 1A). All of the wild-type control mice died within 24 h after LPS injection, whereas all of the Tyk2-deficient mice survived. Because Stat4 activation by IL-12 is drastically reduced and Stat1 activation by IFN-
is partially abrogated in the absence of Tyk2 (7,8), we next examined the roles of Stat1 and Stat4 in endotoxin shock in vivo. We injected Stat1-deficient and Stat4-deficient mice intraperitoneally with a high dose of LPS (50 mg/kg). About half of Stat1- or Stat4-deficient mice were still alive within 24 h after LPS injection, although all of them died by 48 h after LPS injection (Fig. 1A). When mice were treated with a moderate dose of LPS (30 mg/kg), all of the Tyk2-deficient mice and about half of the Stat1- or Stat4-deficient mice were alive after LPS injection, although all of the wild-type mice died within 48 h after LPS injection (Fig. 1B). Furthermore, 100% of Tyk2-, Stat1- or Stat4-deficient mice survived challenge with a low dose of LPS (12.5 mg/kg) (Fig. 1C), whereas this low dose of LPS was lethal to wild-type mice. These data indicate that Tyk2-deficient mice were extremely resistant to LPS shock, and Stat1- and Stat4-deficient mice were moderately resistant to LPS shock when compared with their wild-type littermates.

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Fig. 1. Resistance of Tyk2-, Stat1- and Stat4-deficient mice to LPS challenge in vivo. Six-week-old, male wild-type, Tyk2-deficient, Stat1- or Stat4-deficient mice (n = 5) were injected intraperitoneally with (A) 50 mg/kg, (B) 30 mg/kg, or (C) 12.5 mg/kg of LPS. Survival of these mice was monitored until 120 h after LPS challenge. The data are representative of four independent experiments with similar results.
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Intracellular signaling pathways downstream of LPS
LPS activates I
B kinase and MAP kinases, which results in the activation of NF-
B and AP-1. We examined the phosphorylation of I
B, ERK1/2 and p38 MAP kinases. LPS treatment for 30 min induced the phosphorylation of I
B in macrophages from wild-type mice, and also Tyk2-, Stat1- and Stat4-deficient mice, and also resulted in a decrease of I
B protein in these mice (Fig. 2A). Furthermore, the phosphorylation of ERK1/2 and p38 occurred to the same extent in wild-type, Tyk2-, Stat1- and Stat4-deficient mice (Fig. 2B and C). These results demonstrate that neither the NF-
B pathway nor the AP-1 pathway was disrupted in Tyk2-, Stat1- and Stat4-deficient mice.
IFN expression and cytokine production
In addition to the components downstream of LPS signaling, which include TLR4, MyD88 and IRAK-4, the lack of IFN-ß (2) and IFN-
receptors (3) results in resistance to high dose LPS challenge. IFN-
/ß and IFN-
signals also modulate sensitivity to LPS. Therefore, we next analyzed the serum concentrations of several inflammatory cytokines after LPS challenge. In wild-type mice, serum concentrations of TNF-
and IL-12 were dramatically increased 2 h after LPS challenge. In Tyk2-, Stat1- or Stat-4 deficient mice, the elevation of serum concentrations of TNF-
and IL-12 after LPS challenge was moderately decreased, measuring
5070% of the levels observed in wild-type mice (Fig. 3A and B).
The expression of IFNs (IFN
/ß and IFN-
) is induced by LPS, and IFNs play a major role in the response to LPS (3,14). The serum concentration of IFN-
was elevated 4 h after LPS challenge in wild-type mice (Fig. 3C). In Stat1-deficient mice, IFN-
was elevated by LPS challenge, but the degree of elevation was smaller than that observed in wild-type mice. In contrast, the level of serum IFN-
remained low after LPS injection in Tyk2- or Stat4-deficient mice. We next measured LPS-induced expression of IFN-ß mRNA in peritoneal macrophages by real-time PCR (Fig. 4A). The expression of IFN-ß mRNA is normalized to the levels of GAPDH mRNA expression. IFN-ß mRNA was induced by LPS stimulation in macrophages from wild-type mice. In contrast, very little IFN-ß mRNA induction was observed upon LPS stimulation of macrophages from Tyk2- or Stat1-deficient mice (Fig. 4A). In Stat4-deficient macrophages, IFN-ß mRNA was induced by LPS to a similar degree as was observed in wild-type macrophages.

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Fig. 4. The induction of IFN-ß by LPS in macrophages. Wild-type, Tyk2-, Stat1- or Stat4-deficient macrophages were stimulated with 100 ng/ml of LPS for the indicated times or left untreated. (A) RNA was extracted from cells, and the expression of IFN-ß mRNA was analyzed by real-time quantitative PCR. Results have been normalized to the levels of GAPDH mRNA expression. (B) Cells were lysed, and the phosphorylation of IRF-3 was analyzed by western blotting. (C) The induction of IRF-7 mRNA was measured by real-time quantitative PCR.
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During viral infection, cells defective for both IRF-3 and IRF-7 totally fail to induce the IFN-
/ß genes (15). It is possible that the activation or induction of both IRF-3 and IRF-7 may be essential for the maximum production of IFN-ß by LPS challenge or bacterial infection. Therefore, we examined the phosphorylation of IRF-3 and the induction of IRF-7 by LPS treatment in Tyk2-, Stat1- or Stat4-deficient cells (Fig. 4B and C). IRF-3 was phosphorylated after LPS treatment in wild-type macrophages; this phosphorylation of IRF-3 was not altered by the absence of Tyk2, Stat1 or Stat4 (Fig. 4B). The basal level of IRF-7 mRNA was very low in Stat1-deficient cells, and was not induced by stimulation with LPS. In contrast, wild-type and Stat4-deficient macrophages responded to LPS by inducing IRF-7 mRNA (Fig. 4C). In Tyk2-deficient macrophages, the basal level of IRF-7 was about half that in wild-type cells. In addition, IRF-7 was somewhat induced by LPS in the absence of Tyk2; however, the degree of induction was substantially lower than that observed in wild-type cells. These results indicate that the induction of IRF-7 by LPS is partially abrogated in the absence of Tyk2, and completely abrogated in the absence of Stat1.
Intracellular signaling pathways downstream of IFN
In addition to analyzing the production of IFN-
/ß or IFN-
after LPS challenge, we also examined the intracellular signaling pathways downstream of IFN-ß in Tyk2- or Stat1-deficient mice. IFN-ß induced the phosphorylation of Stat1 and Stat4 in bone marrow cells from wild-type mice (Fig. 5). In Tyk2-deficient cells, the phosphorylation of Stat1 was decreased, and the phosphorylation of Stat4 was abrogated. The phosphorylation of Stat4 by IFN-ß in Stat1-deficient cells was similar to that observed in wild-type cells. These data indicate that in Tyk2-deficient mice, there is not only a defect in the production of IFN-
/ß or IFN-
after LPS challenge, but there is also a disruption of signaling downstream of IFN-ß.

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Fig. 5. The phosphorylation of Stat1 and Stat4 in bone marrow cells by IFN-ß stimulation. Bone marrow cells from wild-type, Tyk2- or Stat1-deficient mice were stimulated with 1000 U/ml of IFN-ß for 30 min, and the phosphorylation of (A) Stat1 and (B) Stat4 was analyzed by western blotting.
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Discussion
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Tyk2 is a member of the Jak kinase family, and has been previously demonstrated to play a restricted role in IFN-
/ß signaling and an important role in IL-12 signaling (7,8). Some IFN-
/ß-induced biological activities, such as inhibition of the growth of bone marrow progenitor cells, and NO production from macrophages after LPS stimulation were abrogated in the absence of Tyk2, whereas almost all other IFN-
/ß-induced biological activities, such as resistance to viral infection and MHC class I expression were normal in the absence of Tyk2 (7,8). Almost all IL-12-induced functions, except for T cell proliferation, were defective in Tyk2-deficient cells (7,8). In addition, IL-18-induced IFN-
production from T and NK cells, and IL-18-induced NK cell function were abrogated in Tyk2-deficient mice (9), which indicates that Tyk2 is involved in IL-18 signaling cascades. LPS binds to cell surface TLR4, and generates intracellular signaling (16). The LPSTLR4 response is mediated by both MyD88-dependent and MyD88-independent pathways, each of which leads to the activation of MAPKs and NF-
B (1). The MyD88-dependent cascade involves the downstream molecules IRAK and TRAF6. Disruption of these LPS signaling components induces resistance to high-dose LPS challenge (1719). As the LPS signaling pathway seems to be shared with IL-18 (10,20), we examined the role of Tyk2 in LPS signaling. Resistance to high-dose LPS challenge was observed in Tyk2-deficient mice, which is consistent with a report by Karaghiosoff et al. (2). Surprisingly, however, the LPS-induced activation of NF
B and AP-1 was not affected by the absence of Tyk2 (Fig. 2). This raises the possibility that LPS-induced signals affect cytokine signaling via Tyk2. Stats are the substrates of Tyk2; Stat1 transduces the majority of IFN-
/ß-induced biological activities, and Stat4 transduces all reported IL-12-induced biological activities. Therefore, we challenged Stat1- and Stat4-deficient mice with LPS.
Strikingly, Stat1- and Stat4-deficient mice are resistant to lower dose LPS challenge, but they are sensitive to some extent to high-dose LPS challenge (Fig. 1). Interestingly, the TLR4 signaling cascade, which results in the downstream activation of I
-B and MAP kinase, was not affected by the absence of Stat1 or Stat4 (Fig. 2). In addition to the primary LPS signaling components such as TLR4, MyD88 and IRAK-4, the lack of IFN-ß (2) or IFN-
receptor (3) can confer resistance to high dose LPS challenge. IFN-
/ß and IFN-
signals also affect the sensitivity to LPS.
LPS stimulation induced the expression of IFN-ß mRNA in macrophages (Fig. 4A) and elevated serum IFN-
(Fig. 3C). In Stat1-deficient macrophages, the expression of IFN-ß mRNA by LPS is severly reduced; thus IFN-ß-induced LPS sensitivity might be abolished in Stat1-deficient mice. Serum IFN-
levels were elevated by LPS challenge in Stat1-deficient mice (Fig. 3). As the production of IFN-
is mainly induced by IL-12 and IL-18 in T and NK cells, the absence of Stat1 may not affect the production of IFN-
by IL-12 or IL-18. In Stat1-deficient cells, some genes were reported to be induced by IFN-
, although Stat1 is an essential transcriptional factor for the transduction of IFN-
signals (21,22). Then almost all IFN-ß-induced activities and many IFN-
-induced activities are almost abrogated upon LPS challenge in Stat1-deficient mice. However, some IFN-
-induced biological activities, which are independent of Stat1, may be preserved.
In Stat4-deficient macrophages, the transcription of IFN-ß is induced by LPS stimulation to a similar extent as observed in wild-type mice (Fig. 4A). In this case, almost all IFN-ß-induced biological activities are unaffected because these activities are mainly dependent on Stat1 and Stat2. However, some signals are abrogated because they require Stat4 activation (23). One example of this is the requirement for activated Stat4 for IFN-
production from T cells by IFN-ß. And also, the production of IFN-
by IL-12 or IL-18 needs Stat4 activation. Based on them, it is reasonable that the serum IFN-
level after LPS injection in Stat4-deficient mice was much lower than that observed in wild-type mice (Fig. 3C). Thus, some IFN-ß-induced biological activities and almost all IFN-
-induced biological activities are absent in Stat4-deficient mice.
Neither the induction of IFN-ß transcript in macrophages nor the elevation of serum IFN-
levels was observed after LPS challenge in the absence of Tyk2 (Figs 3C and 4A). Furthermore, the phosphorylation of Stat1 by IFN-
/ß was weak, and IFN-ß did not phosphorylate Stat4 in the absence of Tyk2 (Fig. 5). Taken together, these data indicate that both type I and type II IFN signals activated by LPS challenge are severely affected in Tyk2-deficient mice. This results in higher resistance to LPS challenge in the Tyk2-deficient mice than in mice deficient for only one of Stat1 or Stat4.
The induction of IFN-ß by LPS was severly reduced in Tyk2- and Stat1-deficient macrophages (Fig. 4A). As cells defective for both IRF-3 and IRF-7 totally fail to induce the IFN-
/ß genes in response to viral infections (15), we examined the activation of IRF-3 and the induction of IRF-7 after stimulation with LPS in Tyk2-, Stat1- and Stat4-deficient mice. The phosphorylation of IRF-3 is observed in all mice (Fig. 4B). In Stat1-deficient mice, the basal level of IRF-7 mRNA is very low, and it is not induced by stimulation with LPS (Fig. 4C). This may be the reason why induction of IFN-ß by LPS is severly reduced in Stat1-deficient mice. In Tyk2-deficient mice, the basal level of IRF-7 is about half that seen in wild-type mice, and the degree of IRF-7 induction by LPS is about one-third of that observed in wild-type mice. Therefore, Stat1 is essential for, and Tyk2 plays a partial role in the regulation of transcription of IRF-7 modulated by LPS. As some induction of IRF-7 did occur in Tyk2-deficient cells, the mechanisms resulting in the severe reduction of IFN-ß mRNA induction by LPS stimulation may differ in the Tyk2- and Stat1-deficient mice.
The signaling pathways downstream of IFN-
/ß are complicated. Many IFN-
/ß-induced biological activities are preserved in the absence of Tyk2, but are affected in Stat1-deficient mice (7,8,11). Some IFN-
/ß-induced biological activities, such as the inhibition of B cell progenitor growth by IL-7, are absent in Tyk2-deficient mice, but are not affected in Stat1-deficient mice (24,25). Therefore, it seems that two signaling pathways exist, one which is dependent on Tyk2, but independent of Stat1, and the second, which is independent of Tyk2, but dependent on Stat1. Stat1 is phosphorylated by IFN-
/ß in Tyk2-deficient cells, although the phosphorylation is weaker than in wild-type cells. The induction of IRF-1 and IRF-7 by IFN-
occurred in spite of the absence of Tyk2 (7) (Fig. 4B), whereas it was severly reduced in the absence of Stat1. Taken together, these results and the fact that Tyk2-deficient mice have high resistance to LPS challenge compared to Stat1-deficient mice, suggest that the signaling pathway via Tyk2 plays a substantial role in sensitivity to LPS, and this signal is transduced by the activation of both Stat1 and Stat4.
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Acknowledgements
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This work was supported in part by a Grant from the Japan Leukemia Foundation, a Grant for Clinical Research, and Grants-in-Aid for Scientific Research (number 13218096, 15390302) from the Ministry of Education, Science, Sports, and Culture in Japan.
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Notes
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Transmitting editor: T. Hirano
Received 3 March 2004,
accepted 27 May 2004.
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