The Comparative Roles of Suppressor of Cytokine Signaling-1 and -3 in the Inhibition and Desensitization of Cytokine Signaling
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m509595200
ISSN1083-351X
AutoresSamuel Wormald, Jian‐Guo Zhang, Danielle L. Krebs, Lisa A. Mielke, Jeremy D. Silver, Warren S. Alexander, Terence P. Speed, Nicos A. Nicola, Douglas J. Hilton,
Tópico(s)Medicinal Plant Pharmacodynamics Research
ResumoNegative feedback is a mechanism commonly employed in biological processes as a means of maintaining homeostasis. We have investigated the roles of suppressor of cytokine signaling (SOCS) proteins in regulating the kinetics of negative feedback in response to cytokine signaling. In mouse livers and bone marrow-derived macrophages, both interferon-γ (IFNγ) and interleukin-6 (IL-6) rapidly induced the tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT1) and STAT3. STAT3 tyrosine phosphorylation was bi-phasic in response to continuous IL-6 signaling. In macrophages lacking Socs3, however, continuous IL-6 signaling induced uniformly high levels of STAT3 tyrosine phosphorylation, and early IL-6-inducible genes were inappropriately expressed at intermediate time points. SOCS3 therefore imposes bi-phasic kinetics upon IL-6 signaling. Compared with Socs3 mRNA, Socs1 mRNA was induced relatively slowly, and SOCS1 simply attenuated the duration of IFNγ signaling. Surprisingly, heightened Socs1 mRNA expression but minimal STAT1 tyrosine phosphorylation was observed after prolonged stimulation with IFNγ, indicating that STAT1 may not play a large role in inducing Socs1 mRNA during steady-state IFNγ signaling. We also demonstrate that both SOCS1 and SOCS3 can desensitize primary bone marrow-derived macrophages to IFNγ and IL-6 signaling, respectively. Consistent with the kinetics with which Socs1 and Socs3 mRNAs were induced, SOCS3 desensitized cells to IL-6 rapidly, whereas SOCS1-mediated desensitization to IFNγ occurred at later time points. The kinetics with which SOCS proteins are induced by cytokine may therefore be a parameter that is "hard-wired" into specific cytokine signaling pathways as a means of tailoring the kinetics with which cells become desensitized. Negative feedback is a mechanism commonly employed in biological processes as a means of maintaining homeostasis. We have investigated the roles of suppressor of cytokine signaling (SOCS) proteins in regulating the kinetics of negative feedback in response to cytokine signaling. In mouse livers and bone marrow-derived macrophages, both interferon-γ (IFNγ) and interleukin-6 (IL-6) rapidly induced the tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT1) and STAT3. STAT3 tyrosine phosphorylation was bi-phasic in response to continuous IL-6 signaling. In macrophages lacking Socs3, however, continuous IL-6 signaling induced uniformly high levels of STAT3 tyrosine phosphorylation, and early IL-6-inducible genes were inappropriately expressed at intermediate time points. SOCS3 therefore imposes bi-phasic kinetics upon IL-6 signaling. Compared with Socs3 mRNA, Socs1 mRNA was induced relatively slowly, and SOCS1 simply attenuated the duration of IFNγ signaling. Surprisingly, heightened Socs1 mRNA expression but minimal STAT1 tyrosine phosphorylation was observed after prolonged stimulation with IFNγ, indicating that STAT1 may not play a large role in inducing Socs1 mRNA during steady-state IFNγ signaling. We also demonstrate that both SOCS1 and SOCS3 can desensitize primary bone marrow-derived macrophages to IFNγ and IL-6 signaling, respectively. Consistent with the kinetics with which Socs1 and Socs3 mRNAs were induced, SOCS3 desensitized cells to IL-6 rapidly, whereas SOCS1-mediated desensitization to IFNγ occurred at later time points. The kinetics with which SOCS proteins are induced by cytokine may therefore be a parameter that is "hard-wired" into specific cytokine signaling pathways as a means of tailoring the kinetics with which cells become desensitized. Interleukin-6 (IL-6) 2The abbreviations used are: IL-6, interluekin-6; IFNγ, interferon-γ; JAK, Janus kinase; STAT, signal transducers and activators of transcription; P-STAT, phosphorylated STAT; SOCS1, suppressor of cytokine signaling-1; LCM, L-cell-conditioned medium; PBS, phosphate-buffered saline; MTPBS, mouse-tonicity PBS; Q-PCR, quantitative PCR. and interferon-γ (IFNγ) are cytokines with key roles in regulating the immune response. Signaling by IL-6 and IFNγ begins at the surface of the cell where the cytokines associate with their respective receptor complexes (1Nicola N.A. Guidebook to Cytokines and Their Receptors. Oxford University Press, New York1994Google Scholar). The IFNγ-receptor complex consists of the ligand-binding IFNγ-receptor α subunit and the signal-transducing IFNγ-receptor β subunit, whereas the IL-6-receptor complex consists of the ligand-binding IL-6-receptor α subunit and the signal-transducing glycoprotein-130 subunit (1Nicola N.A. Guidebook to Cytokines and Their Receptors. Oxford University Press, New York1994Google Scholar). Associated with the receptor subunits are the Janus kinases (JAKs), which become activated upon receptor dimerization, and phosphorylate signal transducers and activators of transcription (STAT) transcription factors (2Ihle J.N. Semin. Immunol. 1995; 7: 247-254Crossref PubMed Scopus (57) Google Scholar, 3Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar). Both IFNγ and IL-6 can activate JAK1 (4Kaplan D.H. Greenlund A.C. Tanner J.W. Shaw A.S. Schreiber R.D. J. Biol. Chem. 1996; 271: 9-12Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (376) Google Scholar, 6Lutticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. 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Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar, 16Wormald S. Hilton D.J. J. Biol. Chem. 2003; 279: 821-824Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). STAT3 activates the transcription of Socs3 mRNA by associating with the 5′ promoter region of the Socs3 gene (17Auernhammer C.J. Bousquet C. Melmed S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6964-6969Crossref PubMed Scopus (250) Google Scholar). The regulation of Socs1 mRNA by STAT1 is poorly understood, and it remains to be clarified whether STAT1 directly activates the transcription of Socs1. Interferon regulatory factor-1, which is induced by STAT1, does, however, activate the transcription of Socs1 (18Saito H. Morita Y. Fujimoto M. Narazaki M. Naka T. Kishimoto T. J. Immunol. 2000; 164: 5833-5843Crossref PubMed Scopus (87) Google Scholar). SOCS1 and SOCS3 proteins can both inhibit JAK phosphorylation of STAT, thus creating a negative feedback loop that attenuates cytokine signal transduction, although the mechanisms by which they act appear to differ. Whereas SOCS1 functions by binding directly to JAK proteins, SOCS3 inhibits signaling by binding to phosphorylated tyrosine sites on the cytoplasmic domain of the receptor (19Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar, 20Nicholson S.E. Willson T.A. Farley A. Starr R. Zhang J.G. Baca M. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. EMBO J. 1999; 18: 375-385Crossref PubMed Scopus (370) Google Scholar). Although the signaling pathways activated by IFNγ and IL-6 share common features, their biological outcomes are quite different. IFNγ acts on cells to induce inflammatory and anti-viral responses, whereas IL-6 acts on cells to regulate the production of acute phase proteins (primarily in hepatocytes) as well as the growth and differentiation of a variety of cell types. Classically, this paradox has been explained by an understanding that IFNγ primarily signals via STAT1, whereas IL-6 primarily signals via STAT3 (21Ihle J.N. Curr. Opin. Cell Biol. 2001; 13: 211-217Crossref PubMed Scopus (596) Google Scholar). Physiologically, SOCS1 is the main inducible inhibitor of IFNγ signaling and in neonatal mice prevents the establishment of a lethal inflammatory disease characterized by heightened levels of circulating IFNγ, fatty degeneration of the liver, and necrosis of the liver and other organs (22Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). Mice lacking Socs3 die during embryogenesis (23Roberts A.W. Robb L. Rakar S. Hartley L. Cluse L. Nicola N.A. Metcalf D. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9324-9329Crossref PubMed Scopus (260) Google Scholar); however, the generation of mice conditionally deficient for Socs3 revealed SOCS3 to be a potent physiological suppressor of IL-6 but not IFNγ signaling (24Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. Nat. Immunol. 2003; 4: 540-545Crossref PubMed Scopus (667) Google Scholar, 25Lang R. Pauleau A.L. Parganas E. Takahashi Y. Mages J. Ihle J.N. Rutschman R. Murray P.J. Nat. Immunol. 2003; 4: 546-550Crossref PubMed Scopus (372) Google Scholar, 26Yasukawa H. Ohishi M. Mori H. Murakami M. Chinen T. Aki D. Hanada T. Takeda K. Akira S. Hoshijima M. Hirano T. Chien K.R. Yoshimura A. Nat. Immunol. 2003; 4: 551-556Crossref PubMed Scopus (628) Google Scholar). Surprisingly, IL-6 signaling in the absence of SOCS3 resulted in the prolonged phosphorylation of STAT1 and a qualitative shift in the transcriptional response of IL-6 toward that typical of IFNγ (16Wormald S. Hilton D.J. J. Biol. Chem. 2003; 279: 821-824Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 24Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. Nat. Immunol. 2003; 4: 540-545Crossref PubMed Scopus (667) Google Scholar, 25Lang R. Pauleau A.L. Parganas E. Takahashi Y. Mages J. Ihle J.N. Rutschman R. Murray P.J. Nat. Immunol. 2003; 4: 546-550Crossref PubMed Scopus (372) Google Scholar). This indicates that signaling by IFNγ and IL-6 is fundamentally similar, and that the activity of SOCS3 is required to sculpt the cellular response to IL-6 and thereby discriminate it from that of IFNγ. In addition to their role as inhibitors of cytokine signaling, a role for SOCS proteins in the desensitization of cytokine signaling was recently postulated (27Fischer P. Lehmann U. Sobota R.M. Schmitz J. Niemand C. Linnemann S. Haan S. Behrmann I. Yoshimura A. Johnston J.A. Muller-Newen G. Heinrich P.C. Schaper F. Biochem. J. 2004; 378: 449-460Crossref PubMed Scopus (64) Google Scholar). Desensitization, as opposed to inhibition, refers to the process by which an initial signaling event causes the cell to become refractory to subsequent signals. In the case of IFNγ signaling, the T-cell protein-tyrosine phosphatase has been shown to potently desensitize cells to repeated stimulation (28Sakamoto S. Qin J. Navarro A. Gamero A. Potla R. Yi T. Zhu W. Baker D.P. Feldman G. Larner A.C. J. Biol. Chem. 2004; 279: 3245-3253Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Although SOCS1 and SOCS3 are attractive candidates as mediators of desensitization to IFNγ and IL-6, respectively, detection of this activity has so far proved elusive (27Fischer P. Lehmann U. Sobota R.M. Schmitz J. Niemand C. Linnemann S. Haan S. Behrmann I. Yoshimura A. Johnston J.A. Muller-Newen G. Heinrich P.C. Schaper F. Biochem. J. 2004; 378: 449-460Crossref PubMed Scopus (64) Google Scholar). In this study, we have explored the relationship between the kinetics of STAT tyrosine phosphorylation and the kinetics of Socs mRNA expression for both IFNγ and IL-6 signaling. We demonstrate marked differences in the kinetics with which SOCS1 and SOCS3 inhibit IFNγ and IL-6 signaling, respectively, and, furthermore, we show that both SOCS1 and SOCS3 can desensitize cells to IFNγ and IL-6 signaling, respectively. Mouse Injections—C57BL/6 mice housed at The Walter and Eliza Hall Institute animal facility received an intraperitoneal injection at 6–8 weeks of age with 100 μl of either saline, recombinant murine IL-6 (500 μg/kg, a gift from Dr. Richard Simpson, Ludwig Institute for Cancer Research, Melbourne, Australia), or IFNγ (250 μg/kg weight, Peprotech, Rocky Hill, NJ). Mice were asphyxiated in CO2, and livers were dissected and frozen in liquid nitrogen at various time points following injection. Experiments were performed with the approval of the Melbourne Health Research Directorate Animal Ethics Committee. Culture and Stimulation of C57BL/6, Socs3-/Δ, Ifnγ-/-, and Socs1-/--Ifnγ-/- Macrophages—Socs3-deficient macrophages (Socs3-/Δ) were derived as previously described (24Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. Nat. Immunol. 2003; 4: 540-545Crossref PubMed Scopus (667) Google Scholar, 29Croker B.A. Metcalf D. Robb L. Wei W. Mifsud S. DiRago L. Cluse L.A. Sutherland K.D. Hartley L. Williams E. Zhang J.G. Hilton D.J. Nicola N.A. Alexander W.S. Roberts A.W. Immunity. 2004; 20: 153-165Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) from the bone marrow of mice in which one allele of Socs3 was deleted, and one allele of Socs3 was conditionally deleted from hematopoietic progenitors (29Croker B.A. Metcalf D. Robb L. Wei W. Mifsud S. DiRago L. Cluse L.A. Sutherland K.D. Hartley L. Williams E. Zhang J.G. Hilton D.J. Nicola N.A. Alexander W.S. Roberts A.W. Immunity. 2004; 20: 153-165Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) or macrophages (24Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. Nat. Immunol. 2003; 4: 540-545Crossref PubMed Scopus (667) Google Scholar). Ifnγ-/- and Socs1-/-Ifnγ-/- macrophages were derived from the bone marrow of Ifnγ-/- and Socs1-/-Ifnγ-/- mice (22Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). C57BL/6 (wild-type), Socs3-/Δ, Ifnγ-/-, and Socs1-/-Ifnγ-/- bone marrow cells were cultured in 8-cm tissue culture-treated plates (10 × 106/dish) overnight at 37 °C, 10% CO2 in 9 ml of Dulbecco's modified Eagle's medium with 10% fetal calf serum and 20% L-cell-conditioned medium (LCM, a source of macrophage colony-stimulating factor). Cells and media were then transferred to 10-cm Petri dishes and incubated for a further 6 days, with 1 ml of LCM added to the cells on day 3. Adherent cells were then washed in mouse-tonicity phosphate-buffered saline (MTPBS) and resuspended in 4 ml of cell dissociation buffer (Invitrogen). After incubation at 37 °C for 10 min, cells were resuspended in Dulbecco's modified Eagle's medium/10% fetal calf serum/20% LCM. Cells, of which more than 95% were macrophages (as determined by morphology), were then plated into 12-well tissue culture-treated plates (5 × 105 cells/well) and incubated at 37 °C, 10% CO2. The following day, cells were stimulated with maximal concentrations of either IL-6 (250 ng/ml) or IFNγ (125 ng/ml). Pulsed stimulations were performed by washing cells twice in MTPBS (37 °C), 15 min after stimulation, then adding Dulbecco's modified Eagle's medium/10% fetal calf serum/20% LCM (37 °C). For mRNA stability experiments, inhibition of transcription was achieved by adding 2 μg/ml actinomycin-D (Sigma-Aldrich) in 5 μl of dimethyl sulfate (Sigma-Aldrich) to cells plated in 250 μl of media. Immunoprecipitation and Western Blotting—Frozen livers were lysed in radioimmune precipitation assay buffer (30Brysha M. Zhang J.G. Bertolino P. Corbin J.E. Alexander W.S. Nicola N.A. Hilton D.J. Starr R. J. Biol. Chem. 2001; 276: 22086-22089Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and lysates were subjected to immunoprecipitation and then Western blotted for STAT proteins (30Brysha M. Zhang J.G. Bertolino P. Corbin J.E. Alexander W.S. Nicola N.A. Hilton D.J. Starr R. J. Biol. Chem. 2001; 276: 22086-22089Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and SOCS proteins (24Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. Nat. Immunol. 2003; 4: 540-545Crossref PubMed Scopus (667) Google Scholar). Immunoprecipitations were performed with antibodies to STAT1 (BD Transduction Laboratories, Lexington, KY), STAT3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and SOCS1 or SOCS3 (generated at the Walter and Eliza Hall Institute). Western blotting was carried out with antibodies specific for either tyrosine 701-phosphorylated STAT1 (P-STAT1, Cell Signaling, Beverly, MA), STAT1, tyrosine 705-phosphorylated STAT3 (P-STAT3, Cell Signaling), STAT3, SOCS1, or SOCS3. RNA Isolation and cDNA Synthesis—Total RNA for real-time PCR and microarray analysis was isolated from cells using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Single-stranded cDNA was synthesized from 20 μg of total RNA (for microarray analysis) or 1.7-fold between treated and control samples in two consecutive samples were considered to be differentially expressed. This cut-off was selected based on visual inspection of normalized quantile-quantile plots of the expression differences between pairs of microarrays. Genes displaying differential expression between control samples (either non-treated or PBS-treated) were removed. GeneCluster2 (31Reich M. Ohm K. Angelo M. Tamayo P. Mesirov J.P. Bioinformatics. 2004; 20: 1797-1798Crossref PubMed Scopus (81) Google Scholar) 3M. Reich, K. Ohm, M. Angelo, P. Tamayo, and J. P. Mesirov (2004) GeneCluster 2.0, available at www.broad.mit.edu/cancer/software/genecluster2/gc2.html. was then employed to identify sets of differentially expressed genes with common expression profiles. Differential Induction of STAT1 and STAT3 Tyrosine Phosphorylation by IFNγ and IL-6 in the Liver—Gene deletion studies in mice have revealed the importance of SOCS1 for regulating IFNγ and SOCS3 for regulating IL-6 signaling, however little is known of the comparative induction of SOCS proteins by IFNγ and IL-6. To investigate the in vivo activation of STAT1 and STAT3 and expression of Socs1 and Socs3 in response to these cytokines, we injected C57BL/6 mice with IFNγ or IL-6 and examined proteins in the livers of mice after various time periods. Immunoprecipitation (9Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Science. 1994; 263: 92-95Crossref PubMed Scopus (849) Google Scholar) and Western blotting demonstrated that, in response to either cytokine, tyrosine phosphorylation (a readout of STAT activation) of both STAT1 and STAT3 was maximal or near-maximal by 15 min after injection (Fig. 1, A and B). STAT3 tyrosine phosphorylation declined to near basal levels after 4 h in response to either cytokine, and a much higher level was detected following injection of IL-6 compared with injection of IFNγ (Fig. 1A). In contrast, while the maximal levels of STAT1 tyrosine phosphorylation were similar following injections of both cytokines, phosphorylation declined to basal levels after 4 h in response to IFNγ but after only 1 h in response to IL-6 (Fig. 1B). These differences in the phosphorylation of STAT1 and STAT3 by IFNγ and IL-6 are consistent with the role of STAT1 as an important mediator of the response to IFNγ and the role of STAT3 as an important mediator of the response to IL-6 (21Ihle J.N. Curr. Opin. Cell Biol. 2001; 13: 211-217Crossref PubMed Scopus (596) Google Scholar). IFNγ induced the robust expression of SOCS1 protein, with levels reaching a maximum at 2–4 h after injection of IFNγ, at a time when STAT1 and STAT3 phosphorylation was waning (Fig. 1C; compare with Fig. 1, A and B). SOCS1 protein expression was maintained at a high level for at least 8 h, and declined to near basal levels by 16 h. In contrast, whereas the kinetics of induction of SOCS1 by IL-6 were similar to those induced by IFNγ, the magnitude was greatly reduced. In the case of SOCS3, the opposite appeared true; IL-6 induced the expression of SOCS3 protein very rapidly, with maximal levels detected 30 min to 1 h following injection, at a time that correlates with a decline in STAT1/STAT3 tyrosine phosphorylation (Fig. 1D; compare with Fig. 1, A and B), whereas IFNγ induced a barely detectable amount of SOCS3 (Fig. 1D). We confirmed the pattern of SOCS1 and SOCS3 protein expression in response to IFNγ and IL-6 at the mRNA level using a Q-PCR assay (Fig. 1E). Again, IFNγ induced Socs1 to greater levels than did IL-6, whereas IL-6 induced Socs3 to greater levels than did IFNγ. IL-6 induced both Socs1 and Socs3 mRNA to a similar extent, but more SOCS3 protein than SOCS1 protein, indicating that synthesis of SOCS3 protein may be more efficient than synthesis of SOCS1 protein. The kinetics of Socs3 mRNA and SOCS3 protein expression were similar, suggesting that the SOCS3 protein is highly labile, a finding that is consistent with the reported half-life of this protein (33Siewert E. Muller-Esterl W. Starr R. Heinrich P.C. Schaper F. Eur. J. Biochem. 1999; 265: 251-257Crossref PubMed Scopus (94) Google Scholar). Expression of SOCS1 protein, however, appeared to persist at 4 h and 8 h, despite declining levels of Socs1 mRNA, suggesting that the half-life of SOCS1 during active cytokine signaling in vivo is substantially greater than that of SOCS3. Continuous IL-6 Signaling in Bone Marrow-derived Macrophages Produces a Bi-phasic Response—The in vivo analysis of signaling confirmed the reciprocal relationship of IFNγ with SOCS1, and IL-6 with SOCS3. However, the intrinsic variability when working with mice, and the difficulty in precisely regulating cytokine levels following injection led us to investigate the kinetics of signaling in more tractable systems: cultures of primary cells. We initially examined IL-6 signaling in Socs3-deficient primary hepatocytes, but like Socs3-deficient mouse embryonic fibroblasts (27Fischer P. Lehmann U. Sobota R.M. Schmitz J. Niemand C. Linnemann S. Haan S. Behrmann I. Yoshimura A. Johnston J.A. Muller-Newen G. Heinrich P.C. Schaper F. Biochem. J. 2004; 378: 449-460Crossref PubMed Scopus (64) Google Scholar), these cells exhibited signs of basal STAT3 activation (albeit to a lesser extent), including low level STAT3 tyrosine phosphorylation and induction of IL-6-inducible genes (data not shown). Fortunately, Socs3-deficient primary bone marrow-derived macrophages did not show signs of basal STAT3 activation (Fig. 2A, lane 10). In bone marrow-derived macrophages stimulated with IL-6, tyrosine phosphorylation of STAT3 was observed during two distinct phases in response to IL-6 (Fig. 2, A and B). The first phase occurred between 15 and 30 min, and the second from 120 min onward. The decline in STAT3 phosphorylation from the initial peak at 15 min was rapid, and near-basal P-STAT3 levels were achieved by 60 min, suggesting that the half-life of STAT3 phosphorylation must be very short during this period. Expression of Socs3 mRNA was maximal at 30 min, 15 min after maximal STAT3 activation was observed (Fig. 3A). This was followed by a rapid reduction of Socs3 mRNA expression, with minimal expression observed between 60 to 90 min, and subsequent re-establishment of expression by 180 min. SOCS3 Is Responsible for Bi-phasic Tyrosine Phosphorylation of STAT3 and Expression of Genes in Response to IL-6—The observation of two separate phases of signaling by IL-6 in bone marrow-derived macrophages suggested that negative feedback by SOCS3 might impose a period of oscillation upon signaling by IL-6. To test this hypothesis, levels of phosphorylated STAT3 were determined in response to IL-6 in Socs3-/Δ bone marrow-derived macrophages (Fig. 2, A and B). In the absence of Socs3, STAT3 phosphorylation was strongly induced after 15 min and was not substantially reduced for the remainder of the time course. SOCS3 is therefore responsible for imposing bi-phasic kinetics upon the signaling of IL-6. To determine how the expression levels of IL-6-responsive genes are affected by SOCS3-mediated regulation of IL-6 signaling, the responses of the IL-6-inducible genes growth arrest and DNA damage-inducible gene 45γ (Gadd45γ) (34Nakayama K. Hara T. Hibi M. Hirano T. Miyajima A. J. Biol. 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