The signalling pathway that comprises JAK kinases and STAT proteins (for signal transducer and activator of transcription) is important for relaying signals from various cytokines outside the cell to the inside1,2,3. The feedback mechanism responsible for switching off the cytokine signal has not been elucidated. We now report the cloning and characterization of an inhibitor of STAT activation which we name SSI-1 (for STAT-induced STAT inhibitor-1). We found that SSI-1 messenger RNA was induced by the cytokines interleukins 4 and 6 (IL-4, IL-6), leukaemia-inhibitory factor (LIF), and granulocyte colony-stimulating factor (G-CSF). Stimulation by IL-6 or LIF of murine myeloid leukaemia cells (M1 cells) induced SSI-1 mRNA expression which was blocked by transfection of a dominant-negative mutant of Stat3, indicating that the SSI-1 gene is a target of Stat3 (refs 4, 5, 6, 7). Forced overexpression of SSI-1 complementary DNA interfered with IL-6- and LIF-mediated apoptosis and macrophage differentiation of M1 cells, as well as IL-6 induced tyrosine-phosphorylation of a receptor glycoprotein component, gp130, and of Stat3. When SSI-1 is overexpressed in COS7 cells, it can associate with the kinases Jak2 and Tyk2. These findings indicate that SSI-1 is responsible for negative-feedback regulation of the JAK–STAT pathway induced by cytokine stimulation.
Most cytokine receptors consist of two or three polypeptide chains, namely a ligand-specific receptor chain and a signal transducer that is commonly used by various cytokines8,9,10,11. The nature of this receptor system explains the functional redundancy of cytokines. Homo- or heterodimerization of receptor components with ligands or ligand–receptor complexes stimulates a unique cytokine signalling cascade, the JAK–STAT pathway1,2,3,12 which causes the activation of target genes in the cell nucleus; little is known about the direct targets of the STAT family. One feature of cytokines is the transient expression of their activity, which suggests that negative-feedback regulation operates in cytokine-signal transduction: possible candidates for exerting this control are the SH2-domain-containing phosphotyrosine phosphatase SHP-1, which associates with the tyrosine-phosphorylated IL-3 receptor β-chain and with the erythropoietin receptor (EPO-R)13,14,15, and a cytokine-inducible SH2-containing protein (CIS) which binds to the Stat5-binding sites of EPO-R (refs 16, 17, and A. Yoshimura et al., personal communication). We have now cloned a cDNA encoding an SH2-domain-containing protein that is inducible by Stat3 and inhibits Stat3 function.
To clone other members of the STAT family, we prepared a monoclonal antibody against a sequence motif (GTFLLRFS) found in the SH2 domain of Stat3 (refs 4, 5). Using this antibody, we screened a murine thymus cDNA library and isolated 20 unknown genes, two of which contained an SH2 domain. One was later identified as CIS16 and the other was a new gene (named SSI-1). SSI-1 cDNA has a single open reading frame that encodes a 212-amino-acid polypeptide and contains an SH2 domain in the middle of its sequence (at codons 79–167). No other consensus motifs like SH3 domains were found in SSI-1 (Fig. 1a). The SH2 domain of SSI-1 was homologous to that of CIS 36% but showed no significant homology with those of Stat3 or Stat6 except at the phosphotyrosine recognition site4,5,18,19 (Fig. 1b).
SSI-1 expression was examined in various murine tissues. SSI-1 mRNA was ubiquitously expressed, with expression being strong in lung, spleen and testis, and weak in all other tissues (Fig. 2a). We examined SSI-1 induction in several factor-dependent cell lines (Fig. 2b). Hybridoma MH60 and myeloid leukaemia M1 cells both expressed SSI-1 mRNA, with expression peaking 60–120 min after treatment with IL-6 plus soluble IL-6 receptor (sIL-6R). IL-4-dependent CT4S cells20 and G-CSF-dependent NFS60 cells expressed SSI-1 mRNA in response to IL-4 and G-CSF, respectively. These results show that SSI-1 is induced not only by IL-6 and G-CSF, both of which activate Stat3 (refs 4, 21), but also by IL-4, a cytokine that activates Stat6 (ref. 19). Consistent with these findings, the promoter region of the SSI-1 gene was found to contain Stat3 and Stat6 binding sequences4,19,22 (data not shown).
We next examined SSI-1 induction in M1 cells transfected with wild-type Stat3 (M1/Stat3) and a Stat3 mutant (M1/Y705F) (Fig. 2c). Stat3(Y705F) is a dominant-negative form of Stat3, in which a tyrosine at residue 705 that is phosphorylated by a JAK kinase has been substituted by phenylalanine6,7. In this experiment, transfectants containing only the neomycin-resistance gene (M1/Neo) were used as controls. SSI-1 mRNA was more strongly induced by IL-6 plus sIL-6R or LIF in M1/Stat3 cells than in M1/Neo control cells, but was not induced in M1/Y705F cells. These results indicate that the SSI-1 gene is one of the target genes of Stat3 and is induced by the JAK–STAT pathway.
To test the effect of SSI-1 on the gp130-mediated signalling pathway, we established M1 transfectants that constitutively express wild-type SSI-1 (M1/SSI-1) or a mutant SSI-1 (M1/TR) that is truncated at the C-terminal region which includes the SH2 domain. As shown in Fig. 3a,b, M1/Neo and M1/TR underwent growth arrest and cell death after treatment with IL-6 plus sIL-6R or LIF, as did the parental M1 cells (M1). The dead cells showed features of apoptosis such as chromatin condensation and apoptotic bodies (Fig. 3c). In contrast, growth of M1/SSI-1 cells did not arrest following stimulation (Fig. 3a–c). Expression of the receptor for the immunoglobulin fragment Fcγ (FcγR) on days 0, 1, 2 and 3 was examined with flow cytometry in M1/Neo, M1/SSI-1 and M1/TR cells treated with IL-6 plus sIL-6R. Despite a partial inhibition of Stat3 phosphorylation (Fig. 4a), SSI-1 almost completely blocked IL-6-induced FcγR expression, which is a direct target gene of Stat3 in M1cells (Fig. 3d)6. These results suggest that SSI-1 inhibits the Stat3-mediated signalling pathway.
Stimulation of cytokines of the IL-6 family induces tyrosine-phosphorylation of the signal-transducing receptor component gp130 to activate Stat3 by JAK kinases23,24,25. To determine how SSI-1 exerts its inhibitory effect on the gp130-mediated signalling pathway, we measured the tyrosine-phosphorylation of gp130 and Stat3 in M1 cells after stimulation with IL-6 plus sIL-6R. As shown in Fig. 4a, tyrosine-phosphorylation of these molecules was much reduced in M1/SSI-1 cells compared with that in control M1/Neo and M1/TR cells. Next we tested whether SSI-1 interacts directly with JAK kinases. The cDNAs encoding SSI-1 and Jak2 or Tyk2 were co-transfected into COS7 cells; as shown in Fig. 4b, SSI-1 co-precipitated with Jak2 or Tyk2. These results indicate that the activity of JAK kinases is suppressed by SSI-1. To see whether the inhibitory effect of SSI-1 is specific to JAK kinases, we immunoblotted M1/Neo, M1/SSI-1 and M1/TR cell lysates before and after IL-6 plus sIL-6R stimulation with anti-phosphotyrosine. In the case of M1/SSI-1, the intensity of the bands corresponding to gp130 and Stat3 was significantly reduced compared with the same bands from control M1/Neo and M1/TR cells. The intensity of other bands was comparable among M1/Neo, M1/SSI-1 and M1/TR cell lysates. In addition, there was no difference in the phosphorylation of Flt-3 and the insulin-receptor-β among these cells after stimulation with the corresponding ligands. These results indicate that SSI-1 specifically inhibits the JAK–STAT signalling pathway (Fig. 4c).
Cytokine binding to the receptor induces homo- or heterodimerization of the receptor components and activates JAK kinases that are constitutively associated with the intracellular domains of the receptor components; once activated, these kinases tyrosine-phosphorylate receptor components. STATs then associate with phosphotyrosine residues on the receptor components through their SH2 domains and in turn are tyrosine-phosphorylated by JAK kinases1,2,3,18,25. Here we have cloned the cDNA encoding SSI-1, an SH2-domain-containing protein that is inducible by Stat3 and inhibits Stat3. It has been reported that CIS participates in negative-feedback regulation of the JAK–STAT signalling pathway16,17. CIS inhibits Stat5 function by directly associating with the Stat5-binding region of the EPO receptor, but the mechanism of SSI-mediated inhibition of the JAK–STAT pathway is quite different. SSI-1 reduces tyrosine-phosphorylation of Stat3 as well as of gp130; as tyrosine-phosphorylation of gp130 is a prerequisite for its association with SSI-1 at the Stat3-binding region, SSI-1 is unlikely to compete with Stat3 for binding to gp130. SSI-1 may block an earlier step in the JAK–STAT signalling pathway than CIS, probably by inhibiting JAK kinases. We have shown that SSI-1 associated with Jak2 and Tyk2, and this molecule has recently been cloned as a JAK-binding protein in a yeast two-hybrid system and also shown to bind Jak1 and Jak3 (A. Yoshimura et al., personal communication). Another point is that SSI-1 is not induced by all STATs, ruling out a role as a general inhibitor of the JAK–STAT pathway.
We have shown that SSI-1 is induced in response to cytokines that act through gp130, and to IL-4. In our experiments, NFS60 and TF-1 cells did not express SSI-1 in response to IL-3 or EPO stimulation, respectively (data not shown). The physiological significance of the cytokine-specific induction of SSI-1 needs to be further investigated.
Preparation of the monoclonal antibody. A hybridoma clone producing the monoclonal antibody (mAb) FL-238, which is reactive against the synthetic oligopeptide (Fmoc) GTFLLRFS (this sequence is highly conserved in the STAT family), was established by fusing spleen cells from BALB/c mice immunized with the synthetic oligopeptide TKPPGTFLLRFSESSKEG (amino acids 600 to 617 in the SH2 domain of Stat3) coupled to keyhole limpet haemocyanin with mouse myeloma cells P3-X63-Ag8-653. The mAb was purified by protein A affinity chromatography from the ascitic fluid of BALB/c mice.
Isolation of SSI-1 cDNA. Using the monoclonal antibody against the GTFLLRFS motif, SSI-1 cDNA was isolated from a murine thymus cDNA library (lambda ZAP, Stratagene) by using a PicoBlue Immunoscreening kit.
Cell culture. Myeloid leukaemia M1 cells were cultured in Eagle's minimal essential medium supplemented with double the normal concentrations of amino acids and vitamins and 10% (vol/vol) fetal calf serum (FCS). The IL-6-dependent myeloma MH60 cells were maintained in RPMI 1640 medium supplemented with IL-6 (5 ng ml−1) and 10% FCS. IL-2/IL-4-dependent CT4Scells20 were cultured in RPMI 1640 medium supplemented with IL-4 (10 U ml−1) and 10% FCS. IL-3-dependent myeloid NFS60 cells were maintained in RPMI 1640 medium supplemented with 10% FCS and 10% conditioned medium from the WEHI-3B cell line as a source of IL-3. We had previously constructed dominant-negative forms of Stat3 and established MI cell lines that constitutively express them6. Transfectant M1 cells were cultured with 250 μg ml−1Geneticin (Gibco).
Northern hybridization. Cells (except M1 cells) were factor-depleted for 4 h in RPM1 medium containing 1% BSA, and then all cells were stimulated with cytokines at the following concentrations for various periods: IL-2, 10 ng ml−1; IL-3, 5 ng ml−1; IL-4, 10 U ml−1; IL-6, 100 ng ml−1; sIL-6R, 50 ng ml−1; G-CSF, 20 ng ml−1; LIF, 1,000 U ml−1. Cytoplasmic RNA was extracted using Iso-Gen (Nippon Gene). Total RNA (5 μg ml−1) was electrophoresed on agarose gels and transferred to a nylon membrane (Hybond N+, Amersham). The membrane was hybridized with radiolabelled cDNA probes.
Flow cytometry analysis. Transfectants (1 × 105) were cultured with IL-6 (300 ng ml−1) plus sIL-6R (500 ng ml−1) for 3 days. After collection, cells were incubated with 1 μg mouse IgG2a (Capel) for 20 min on ice. After washing with PBS, cells were incubated with fluroescein-isothiocyanate-conjugated anti-mouse IgG antibody (Zymed) for 20 min on ice. Cells were analysed in a fluorescence-activated cell sorter (Becton Dickinson FACS).
Plasmid construction and DNA transfection. Constructs were cloned into pEF-BOS, a mammalian expression vector. Briefly, SSI-1 cDNA was digested with the restriction enzymes XbaI and PvuII and then inserted into the XbaI site of the pEF-BOS vector (pEF-BOS/SSI-1 (SSI-1)). For the construction of pEF-BOS/SSI-1 (TR), 360 bp of a BssHII-digested fragment was deleted. cDNAs encoding Jak2 and Tyk2 were inserted into the XbaI site of the pEF-BOS vector.
M1 cells were transfected by electroporation with the SSI-1(SSI-1/TR) expression vector and pSV2 Neo at a 20 : 1 ratio, and neomycin-resistant clones were selected in growth medium containing Geneticin (Gibco) at 750 μg ml−1.
Western blot analysis. Cells were treated with IL-6 (300 ng ml−1) plus sIL-6R (500 ng ml−1), Flt-3 ligand (300 ng ml−1; Genzyme) or insulin (1 μM; Becton Dickinson) for 5 min or were untreated, and were solubilized with lysis buffer (0.5% Nonidet P-40, 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM Na2VO4) containing protease inhibitors. Immunoprecipitates obtained with anti-gp130 (Upstate Biotechnology), anti-Stat3 (ref. 4), anti-Flt-3 (Santa Cruz Biotechnology) or anti-insulin-receptor-β antibody (Santa Cruz Biotechnology) were resolved by SDS–PAGE under reducing conditions and transferred to nitrocellulose. Filters were probed with anti-phosphotyrosine monoclonal antibody (4G10; Upstate Biotechnology) and reprobed with anti-gp130, anti-Stat3, anti-Flt-3 or anti-insulin-receptor-β antibody, respectively, after stripping the first blots. Blots were visualized with the ECL detection system (Amersham). COS7 cells (1 × 106) were transfected with the designated plasmids encoding SSI-1 (2 μg), Jak2 (20 μg), or Tyk2 (20 μg) by using the DEAE–dextran method. After 48 h, cells were solubilized in lysis buffer and the lysates used for western blot analysis.
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We thank T. Tanaka for IL-4 and CT4S cells; S. Nagata for pEF-BOS vector and Jak2 cDNA; J. Krolewski for Tyk2 cDNA; K. Yasukawa for recombinant IL-6 and sIL-6R; Y. Shima, H. Danno, K. Kunisada and H. Tagoh for technical assistance; H. Saito for discussion; and A. Nobuhara for secretarial assistance. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
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Naka, T., Narazaki, M., Hirata, M. et al. Structure and function of a new STAT-induced STAT inhibitor. Nature 387, 924–929 (1997). https://doi.org/10.1038/43219
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