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• W2040409641 abstract "SOCS-3 (suppressor of cytokine signaling 3) is an intracellular protein that is selectively and rapidly induced by appropriate agonists and that modulates responses of immune cells to cytokines by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observations that interferon γ (IFNγ) up-regulates SOCS-3 gene and protein expression in primary mouse macrophages, J774 macrophage cell line and embryonal fibroblasts, we investigated which sequences of the 5′ SOCS-3 gene are responsive to IFNγ. By promoter deletion analysis we identified a functional IFNγ-responsive element, located at nucleotides -72/-64 upstream from the transcription initiation, whose presence and integrity is necessary to ensure responsiveness to IFNγ. This element contains a STAT consensus binding sequence (SOCS-3/STAT-binding element (SBE)) whose specific mutation totally abolished the responsiveness to IFNγ. In contrast, discrete deletion of other 5′ regions of the SOCS-3 promoter did not substantially modify the inducibility by IFNγ. Electromobility shift assay analyses revealed that IFNγ promotes specific DNA binding activities to an oligonucleotide probe containing the SOCS-3/SBE sequence. Even though IFNγ triggered tyrosine phosphorylation of both STAT1 and STAT3 in macrophages and J774 cells, only STAT1 was appropriately activated and thus found to specifically bind to the SOCS-3/SBE oligonucleotide probe. Accordingly, IFNγ-induced SOCS-3 protein expression was not impaired in STAT3-deficient embryonal fibroblasts. Taken together, these results demonstrate that the induction of SOCS-3 by IFNγ depends upon the presence of a STAT-binding element in the SOCS-3 promoter that is specifically activated by STAT1. SOCS-3 (suppressor of cytokine signaling 3) is an intracellular protein that is selectively and rapidly induced by appropriate agonists and that modulates responses of immune cells to cytokines by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observations that interferon γ (IFNγ) up-regulates SOCS-3 gene and protein expression in primary mouse macrophages, J774 macrophage cell line and embryonal fibroblasts, we investigated which sequences of the 5′ SOCS-3 gene are responsive to IFNγ. By promoter deletion analysis we identified a functional IFNγ-responsive element, located at nucleotides -72/-64 upstream from the transcription initiation, whose presence and integrity is necessary to ensure responsiveness to IFNγ. This element contains a STAT consensus binding sequence (SOCS-3/STAT-binding element (SBE)) whose specific mutation totally abolished the responsiveness to IFNγ. In contrast, discrete deletion of other 5′ regions of the SOCS-3 promoter did not substantially modify the inducibility by IFNγ. Electromobility shift assay analyses revealed that IFNγ promotes specific DNA binding activities to an oligonucleotide probe containing the SOCS-3/SBE sequence. Even though IFNγ triggered tyrosine phosphorylation of both STAT1 and STAT3 in macrophages and J774 cells, only STAT1 was appropriately activated and thus found to specifically bind to the SOCS-3/SBE oligonucleotide probe. Accordingly, IFNγ-induced SOCS-3 protein expression was not impaired in STAT3-deficient embryonal fibroblasts. Taken together, these results demonstrate that the induction of SOCS-3 by IFNγ depends upon the presence of a STAT-binding element in the SOCS-3 promoter that is specifically activated by STAT1. Interferon γ (IFNγ) 1The abbreviations used are: IFN, interferon; STAT, signal transducers and activators of transcription; Jak, Janus kinase; SBE, STAT-binding element; EMSA, electromobility shift assay; IL, interleukin; PEM, peritoneal elicited macrophage(s); MEF, mouse embryonal fibroblast(s); Ab, antibody. 1The abbreviations used are: IFN, interferon; STAT, signal transducers and activators of transcription; Jak, Janus kinase; SBE, STAT-binding element; EMSA, electromobility shift assay; IL, interleukin; PEM, peritoneal elicited macrophage(s); MEF, mouse embryonal fibroblast(s); Ab, antibody. is a pluripotent cytokine involved in the regulation of nearly all the different phases of both innate and adaptive immune responses. Produced by activated T and natural killer cells, IFNγ has a crucial role in several processes, including host defense against viruses and microorganisms, cell proliferation, phagocyte activation, control of apoptosis, promotion of antigen processing and presentation, and T helper type 1 (TH1) differentiation (1Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Google Scholar). IFNγ has a number of activating properties on cells of the immune system but can also exert important immunosuppressive actions by modulating cellular responses to different cytokines and inflammatory stimuli (2Muhl H. Pfeilschifter J. Int. Immunopharmacol. 2003; 3: 1247-1255Google Scholar). For example, under specific conditions, IFNγ may inhibit proinflammatory cytokine release by activated human peripheral blood mononuclear cells and polymorphonuclear neutrophils (3Ghezzi P. Dinarello C.A. J. Immunol. 1988; 140: 4238-4244Google Scholar, 4Meda L. Gasperini S. Ceska M. Cassatella M.A. Cell Immunol. 1994; 157: 448-461Google Scholar) and up-regulates the release of cytokine antagonists such as IL-1Ra, type II IL-1 receptors, and IL-18BP from mononuclear phagocytes (5Sihvola M. Hurme M. Scand. J. Immunol. 1989; 29: 689-698Google Scholar, 6McDonald P.P. Gasperini S. Calzetti F. Cassatella M.A. Cell Immunol. 1998; 184: 45-50Google Scholar, 7Dinarello C.A. Blood. 1996; 87: 2095-2147Google Scholar, 8Corbaz A. ten Hove T. Herren S. Graber P. Schwartsburd B. Belzer I. Harrison J. Plitz T. Kosco-Vilbois M.H. Kim S.H. Dinarello C.A. Novick D. van Deventer S. Chvatchko Y. J. Immunol. 2002; 168: 3608-3616Google Scholar, 9Veenstra K.G. Jonak Z.L. Trulli S. Gollob J.A. J. Immunol. 2002; 168: 2282-2287Google Scholar). The biologic activities of IFNγ are mainly mediated through the regulation of gene expression. This is achieved upon interaction of IFNγ with its specific cell surface receptor(s) and activation of different intracellular signaling cascades (10Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Google Scholar). It is well established that the immediate transcriptional responses induced by IFNγ are achieved primarily through the activation of the Jak/STAT signaling pathway (11Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Google Scholar). STAT1 plays a major role in mediating the immune and proinflammatory actions of IFNγ (12Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Google Scholar, 13Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Google Scholar), but also STAT3 and STAT5 can be activated by IFNγ in certain cell types (14Woldman I. Varinou L. Ramsauer K. Rapp B. Decker T. J. Biol. Chem. 2001; 276: 45722-45728Google Scholar, 15Meinke A. Barahmand-Pour F. Wohrl S. Stoiber D. Decker T. Mol. Cell Biol. 1996; 16: 6937-6944Google Scholar, 16Caldenhoven E. Buitenhuis M. van Dijk T.B. Raaijmakers J.A. Lammers J.W. Koenderman L. de Groot R.P. J. Leukocyte Biol. 1999; 65: 391-396Google Scholar). One of the genes rapidly induced by IFNγ is SOCS-3 (suppressor ofcytokine signaling 3) (17Sakamoto H. Yasukawa H. Masuhara M. Tanimura S. Sasaki A. Yuge K. Ohtsubo M. Ohtsuka A. Fujita T. Ohta T. Furukawa Y. Iwase S. Yamada H. Yoshimura A. Blood. 1998; 92: 1668-1676Google Scholar, 18Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Google Scholar, 19Cassatella M.A. Gasperini S. Bovolenta C. Calzetti F. Vollebregt M. Scapini P. Marchi M. Suzuki R. Suzuki A. Yoshimura A. Blood. 1999; 94: 2880-2889Google Scholar, 20Lang R. Pauleau A.L. Parganas E. Takahashi Y. Mages J. Ihle J.N. Rutschman R. Murray P.J. Nat. Immunol. 2003; 4: 546-550Google Scholar, 21Croker 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: 540-545Google Scholar, 22Hong F. Jaruga B. Kim W.H. Radaeva S. El-Assal O.N. Tian Z. Nguyen V.A. Gao B. J. Clin. Invest. 2002; 110: 1503-1513Google Scholar). SOCS-3 is a member of a family of intracellular proteins that negatively regulate responses of immune cells to cytokines by inhibiting the Jak/STAT pathway (23Yasukawa H. Sasaki A. Yoshimura A. Annu. Rev. Immunol. 2000; 18: 143-164Google Scholar). Although in vitro overexpression studies have reported that the SOCS proteins are pleiotropic inhibitors of the Jak/STAT pathways activated by several cytokines, targeted gene disruption has shown that these molecules have more specific roles in vivo (20Lang R. Pauleau A.L. Parganas E. Takahashi Y. Mages J. Ihle J.N. Rutschman R. Murray P.J. Nat. Immunol. 2003; 4: 546-550Google Scholar, 21Croker 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: 540-545Google Scholar, 24Alexander 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: 597-608Google Scholar, 25Marine J.C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J.N. Cell. 1999; 98: 609-616Google Scholar, 26Metcalf D. Greenhalgh C.J. Viney E. Willson T.A. Starr R. Nicola N.A. Hilton D.J. Alexander W.S. Nature. 2000; 405: 1069-1073Google Scholar, 27Roberts 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: 9324-9329Google Scholar, 28Yasukawa 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: 551-556Google Scholar). In this regard, conditional gene targeting studies have recently suggested that SOCS-3 has a nonreduntant function in macrophages and hepatocytes, in which it selectively regulates IL-6 and gp130 signaling (20Lang R. Pauleau A.L. Parganas E. Takahashi Y. Mages J. Ihle J.N. Rutschman R. Murray P.J. Nat. Immunol. 2003; 4: 546-550Google Scholar, 21Croker 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: 540-545Google Scholar), in accordance with previous observations showing that SOCS-3 binds phosphorylated gp130 and regulates the subsequent activation of STAT3 induced by IL-6 (29De Souza D. Fabri L.J. Nash A. Hilton D.J. Nicola N.A. Baca M. Biochemistry. 2002; 41: 9229-9236Google Scholar, 30Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Google Scholar, 31Nicholson S.E. De Souza D. Fabri L.J. Corbin J. Willson T.A. Zhang J.G. Silva A. Asimakis M. Farley A. Nash A.D. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6493-6498Google Scholar). Furthermore, these recent reports revealed that SOCS-3 not only inhibits STAT3-dependent IL-6 signaling but also precludes IL-6 from eliciting STAT1-dependent responses, suggesting that this mediator has a critical role in the balance between these two pathways. In this study, we investigated the mechanisms regulating SOCS-3 expression by IFNγ in mouse peritoneal macrophages, J774 mouse macrophage cell line, and embryonal fibroblasts. Analysis of ≈7-kb of the genomic 5′-flanking region of the mouse SOCS-3 gene revealed that the most proximal STAT-binding site present in the SOCS-3 promoter is necessary for IFNγ-induced transcriptional activity. Deletion or point mutation of this STAT-binding element (SBE) completely abrogated IFNγ action. Accordingly, IFNγ promoted specific DNA binding activities to an oligonucleotide corresponding to the SOCS-3/SBE sequence that exclusively contained STAT1. Any contribution of STAT3 was excluded, as demonstrated by the inability of IFNγ-activated STAT3 to bind the SOCS-3/SBE sequence and by the evidence that IFNγ fully retains the capacity of inducing SOCS-3 expression in STAT3-/- embryonal fibroblasts. These data further reinforce the finding that induction of SOCS-3 expression by IFNγ is STAT1-dependent. On the basis of these observations, it is conceivable that defining the molecular mechanism and the signaling pathway responsible for the induction of SOCS-3 gene expression in response to IFNγ might help in clarifying how IFNγ can modulate cellular responses to different cytokines. Cell Culture—C57BL/6J mice, purchased from Harlan Italy (Correzzana, Milan, Italy), were injected with 1 ml of 4% thioglycollate 4 days before harvesting peritoneal elicited macrophages (PEM). Red cells were removed by hypotonic lysis, and macrophages were seeded in 6-well plates in RPMI supplemented with 10% heat-inactivated low endotoxin fetal bovine serum (0.01 endotoxin units/ml; Biochrom Seromed, Berlin, Germany), 2 mm ultraglutamine 1,100 units/ml penicillin, 100 mg/ml streptomycin (Biowhittaker, Verviers, Belgium) and let adhere overnight. Mouse macrophage cell line J774 (kindly provided by Dr. V. Kruys, Universitè Libre de Brussels, Brussels, Belgium) was cultured in Dulbecco's modified Eagle's medium (from Biowhittaker Europe, Verviers, Belgium) supplemented with 5% heat-inactivated low endotoxin fetal bovine serum and passaged twice weekly. Immortalized STAT3 wild type (floxed/floxed, fl/fl) and STAT3-/- (deleted/deleted, Δ/Δ) MEFs, generated as previously described (32Costa-Pereira A.P. Tininini S. Strobl B. Alonzi T. Schlaak J.F. Is'harc H. Gesualdo I. Newman S.J. Kerr I.M. Poli V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8043-8047Google Scholar), were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and passaged every 3 days. Construction of Reporter Plasmids—The 5′ genomic region of murine SOCS-3 was cloned from 129 genomic library (33Marine J.C. McKay C. Wang D. Topham D.J. Parganas E. Nakajima H. Pendeville H. Yasukawa H. Sasaki A. Yoshimura A. Ihle J.N. Cell. 1999; 98: 617-627Google Scholar) in pBlueScript (Stratagene, La Jolla, CA). Cloning of the XhoI/NotI fragment into pBS was followed by orientation verification, restriction enzyme digestion, and subcloning of a ≈7-kb construct into the KpnI/BamHI sites of the promoterless luciferase reporter vector pGL2basic (Promega, Madison, WI), generating construct 1. The SOCS-3 genomic fragment cloned in construct 1 spans from -6298 to +884 relative to the transcription initiation, which is defined as +1 (GenBank™ accession number AF314501) and contains the untranslated exon 1 and the fragment of exon 2 upstream the ATG. Truncated forms of SOCS-3 promoter were generated by restriction enzyme digestion of construct 1 as follows: construct 2, deleted the KpnI fragment; construct 3, deleted the KpnI/NheI fragment; construct 4, deleted the XhoI/SpeI fragment; construct 5, deleted the KpnI/SacII fragment. Construct 6 was generated by KOD Plus 2″ PCR (Toyobo, Tokyo, Japan) and spans -50 to +968 nucleotides. 3′-deleted construct 7 was generated by removing the proximal SacII/NotI fragment from construct 1. A mutated form of the -72/-64 STAT-binding element in construct 5 was produced by substituting the TTCCAGGAA sequence with TTCCAGGTT by site-directed mutagenesis. Transient Transfection and Dual Luciferase Assay—J774 (4 × 105 cells/well) were seeded in 12-well plates and transfected 24 h later with SuperFect transfection reagent (Qiagen). 3μg of each SOCS-3 promoter/luciferase construct were mixed at a 50:1 ratio with the Renilla-encoding pRL-null vector (Promega) and incubated with 7.5 μl of SuperFect transfection reagent according to the manufacturer's instruction. 12 h before stimulation, the cells were split into equal aliquots and replated. The cells were then treated with 100 units/ml IFNγ (PeproTech, London, UK) or left untreated as a control, for the time indicated. After stimulation, the cells were harvested, washed twice with phosphate-buffered saline, and lysed in 30 μl of Passive lysis buffer (Promega) followed by two freeze-thaw cycles. Luciferase assays of both firefly and Renilla reniformis luciferases were performed using a dual luciferase reporter assay system (Promega) according to the manufacturer's instructions, and the enzymatic activities of both luciferases were quantified using a Packar LumiCount Microplate Luminometer (Packard Instrument Co., Meriden, CT). The values of firefly luciferase activity were divided for the R. reniformis luciferase activity, to normalize for differences caused by unequal transfection efficiency. Western Blot—The cells were seeded the day before stimulation as follows: 1.5 × 106 J774, 6.5 × 106 PEM and 0.75 × 106 MEFs in 6-well plates. After stimulation for the times indicated with 100 units/ml IFNγ or 200 units/ml IL-6 (PeproTech), whole or nuclear cell lysates were prepared, and immunoblot analysis was performed as previously described (34Crepaldi L. Gasperini S. Lapinet J.A. Calzetti F. Pinardi C. Liu Y. Zurawski S. de Waal Malefyt R. Moore K.W. Cassatella M.A. J. Immunol. 2001; 167: 2312-2322Google Scholar) using the following primary Abs: anti-NH2 terminus SOCS-3 (Immuno-Biological Laboratories, Tokyo, Japan) diluted at 5 μg/ml; anti-phosphotyrosine STAT3, anti-STAT3, anti-phosphotyrosine STAT1, and anti-p38 mitogen-activated protein kinase (Cell Signaling Technology, Beverly, MA) diluted as recommended by the manufacturer; anti-STAT1 diluted at 2 μg/ml and anti-lamin A/C (1/100 diluted) (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-actin (diluted 1/2000) (Sigma). Antibody binding was detected by using horseradish peroxidase-conjugated anti-rabbit IgG (1/5000 diluted) (Amersham Biosciences) and was revealed using ECL (Amersham Biosciences). In selected experiments, quantitative analysis of STAT3 phosphorylation was carried out utilizing the Odyssey Infrared Imager (Li-Cor). In the latter case, antibody binding was detected by using Alexa Fluor 680-conjugated goat anti-rabbit IgG (1/5000 diluted) (Molecular Probes, Eugene, OR) or IRDye 800-conjugated goat anti-mouse IgG (1/2000 diluted) (Rockland, Gilbertsville, PA), according to the manufacturer's instructions. Northern Blot—After stimulation with IFNγ, J774 were harvested at different times, and total RNA was extracted by the guanidinium isothiocyanate method and processed for Northern blot analysis, as already described (35Cassatella M.A. Bazzoni F. Flynn R.M. Dusi S. Trinchieri G. Rossi F. J. Biol. Chem. 1990; 265: 20241-20246Google Scholar). Northern blot analysis was performed on 15 μg of RNA/lane. Specific SOCS-3 mRNA was detected by autoradiography after Northern blot hybridization with the cDNA probe encoding SOCS-3, labeled by the Ready-to-go kit (Amersham Biosciences). Electromobility Shift Assay—4 × 106 cells/well J774, 20 × 106 PEM, and 5 × 106 MEF were seeded in 60-mm culture dishes overnight. After IFNγ or IL-6 stimulation for the times indicated, the cells were harvested, and the nuclear extracts were prepared as described previously (36Dusi S. Donini M. Lissandrini D. Mazzi P. Bianca V.D. Rossi F. Eur. J. Immunol. 2001; 31: 929-938Google Scholar). Protein-DNA complexes were detected by EMSA analysis as previously described (19Cassatella M.A. Gasperini S. Bovolenta C. Calzetti F. Vollebregt M. Scapini P. Marchi M. Suzuki R. Suzuki A. Yoshimura A. Blood. 1999; 94: 2880-2889Google Scholar). 5 μg of nuclear extracts were incubated with a 32P-labeled double-stranded oligonucleotide probe containing the STAT-binding element (SOCS-3/SBE) located at -72/-64 nucleotides of the promoter of the SOCS-3 gene (5′-CAGTTCCAGGAATCGGGGGGC-3′) or with the mutated form of this sequence (5′-CAGTTCCAGGTTTCGGGGGGC-3′) for 15 min. Supershift experiments were performed by incubating extracts with 2 μg of anti-STAT1, anti-STAT3, or anti-CRE Abs (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at room temperature before adding the labeled probe. The reaction mixtures were then subjected to electrophoresis in a 5% nondenaturing polyacrylamide gel, dried, and analyzed in an Instant Imager (Packard Instruments, Meridien, CT). Induction of SOCS-3 Gene and Protein Expression by IFNγ—We initially investigated the ability of IFNγ to induce SOCS-3 protein expression in the mouse macrophage cell line J774 and PEM. SOCS-3 protein expression was not detectable in resting J774 and PEM cultures (Fig. 1, A and B, medium). The addition of 100 units/ml IFNγ to each cell culture induced high levels of SOCS-3 protein expression, which was maximal within 3 h, remained stable for up to 6 h and declined thereafter (Fig. 1, A and B). Northern blot analysis indicated that barely detectable levels of SOCS-3 mRNA were present under non stimulated conditions in J774 cells (Fig. 1C). Following IFNγ stimulation SOCS-3 mRNA was rapidly induced, reaching maximum levels within 1 h (Fig. 1C). These results extend at the protein level previous observations showing that IFNγ is able to transiently induce SOCS-3 mRNA in different cell types (17Sakamoto H. Yasukawa H. Masuhara M. Tanimura S. Sasaki A. Yuge K. Ohtsubo M. Ohtsuka A. Fujita T. Ohta T. Furukawa Y. Iwase S. Yamada H. Yoshimura A. Blood. 1998; 92: 1668-1676Google Scholar, 18Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Google Scholar, 20Lang R. Pauleau A.L. Parganas E. Takahashi Y. Mages J. Ihle J.N. Rutschman R. Murray P.J. Nat. Immunol. 2003; 4: 546-550Google Scholar, 37Gil M.P. Bohn E. O'Guin A.K. Ramana C.V. Levine B. Stark G.R. Virgin H.W. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6680-6685Google Scholar, 38Ding Y. Chen D. Tarcsafalvi A. Su R. Qin L. Bromberg J.S. J. Immunol. 2003; 170: 1383-1391Google Scholar, 39Ramana C.V. Gil M.P. Schreiber R.D. Stark G.R. Trends Immunol. 2002; 23: 96-101Google Scholar). Responses of SOCS-3 Reporter Gene to IFNγ—To identify the region(s) responsive to IFNγ in the SOCS-3 gene, we carried out a functional analysis of the SOCS-3 promoter. The murine SOCS-3 gene structure has been previously determined (40Auernhammer C.J. Bousquet C. Melmed S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6964-6969Google Scholar) and comprises an untranslated exon 1 (+1 to 299) separated from exon 2 (starting at +856) by an intron (+300 to +855). The main transcription site identified has been referred as +1, and the translation initiation site for murine SOCS-3 has been identified in exon 2 at +946 (Fig. 2) (18Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Google Scholar). A series of 5′-deletions of the mouse SOCS-3 promoter/reporter constructs (schematically represented in Fig. 2) were generated as described under “Experimental Procedures.” Construct 1 spans the -6298 to +884 5′ genomic region of mouse SOCS-3 linked to the luciferase reporter in pGL2Basic vector. The 5′ truncations of construct 1 are: construct 2, nucleotides -3980 to +884; construct 3, nucleotides -2661 to +884; construct 4, nucleotides -2056 to +884; construct 5, nucleotides -456 to +884; and construct 6, nucleotides -50 to +968. The 3′ truncation of construct 1 is: construct 7, nucleotides -6298 to -457. Basal and IFNγ-induced luciferase activity were assayed after transient transfection of J774 cells with the different constructs together with pRL-null, used to normalize transfection efficiency. Luciferase activity was expressed as fold induction by IFNγ-treated over unstimulated cells (Fig. 3). Stimulation with 100 units/ml IFNγ for 6 h did not increase the luciferase activity in J774 transfected with pGL2Basic alone (1.3 ± 0.1-fold relative to unstimulated pGL2Basic-transfected cells, n = 3). In contrast, basal luciferase activity was increased in IFNγ-treated cells by 8 ± 2-fold when the full SOCS-3 promoter (-6298/+884 nucleotides, construct 1) was used (Fig. 3). Progressive 5′ deletion of the SOCS-3 promoter from -6298 down to -454 nucleotides did not significantly alter responsiveness to IFNγ, which remained substantially similar with construct 2 (7 ± 1.5-fold, n = 4), construct 3 (6.96 ± 2-fold, n = 4), construct 4 (6.7 ± 1.7-fold, n = 4), and construct 5 (5.3 ± 2.8-fold, n = 4, p > 0.05) (Fig. 3). A further 5′ removal of 404 nucleotides resulted in the complete loss of responsiveness to IFNγ. Indeed, stimulation with IFNγ caused no further increase of luciferase activity in J774 cells transfected with construct 6 (-50/+968 nucleotides) as compared with J774 transfected with pGL2Basic alone (1.4 ± 0.08 and 1.3 ± 0.1-fold, respectively) (Fig. 3), indicating that the region from nucleotides -50 to +968 is likely not involved in promoter activity. Collectively, these data show that the promoter region mediating IFNγ induction is restricted within nucleotides -452 to -50. To further confirm these data, we assayed IFNγ-induced luciferase activity after transfecting J774 with construct 7 (-6298/-453 nucleotides) carrying a 3′deletion of the full-length promoter that removes the proximal 1381 nucleotides, where the minimal IFNγ-responsive elements are located. Removal of this region reduced luciferase activity of IFNγ-stimulated versus untreated cells to 1.9 ± 1-fold, indicating that the sequences located upstream nucleotide -454 do not significantly contribute to IFNγ responsiveness.Fig. 3Induction of luciferase activity in J774 cells in response to IFNγ: effects of SOCS-3 promoter deletion. J774 cells were transiently transfected with each of the SOCS-3 promoter reporter constructs illustrated in Fig. 2 and the Renilla-encoding pRL-null vector. 12 h before stimulation, the cells were split into two equal aliquots and replated. The cells were then either stimulated for 6 h with 100 units/ml IFNγ or left untreated as a control. Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity. Responsiveness to IFNγ was calculated as the ratio of normalized luciferase activity of stimulated over unstimulated cells. The mean values with S.D. as indicated are from four independent experiments.View Large Image Figure ViewerDownload (PPT) Previous analysis of the region encompassing -454 to -50 nucleotides revealed the presence of two putative STAT-binding elements (TTX5AA, where X is any nucleotide), found at -95 to -87 and at -72 to -64 (40Auernhammer C.J. Bousquet C. Melmed S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6964-6969Google Scholar). The -72/-64 proximal STAT consensus element (TTCCAGGAA) had been shown to be essential for leukemia inhibitory factor-induced SOCS-3 promoter transactivation in ACTH-secreting corticotroph AtT-20 cells (40Auernhammer C.J. Bousquet C. Melmed S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6964-6969Google Scholar) and to be involved in insulin-induced SOCS-3 expression (41Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Google Scholar). Therefore, in subsequent experiments, we focused on this SOCS-3/SBE. To analyze whether SOCS-3/SBE was necessary for SOCS-3 induction by IFNγ, we generated a mutated form of this sequence by site-directed mutagenesis, introducing AA → TT substitution of -65/-64 nucleotides into the -454/+884 nucleotides promoter of construct 5 (Fig. 4, Construct 5mut), thus destroying the specific TTCCAGGAA. Luciferase activity induced by IFNγ was then assayed in J774 cells transiently transfected with the luciferase expression plasmid carrying the mutated form of the -454/+884 SOCS-3 promoter (construct 5mut) and was compared with that obtained with the -454/+884 wild type promoter (construct 5) (Fig. 4). Luciferase activity in response to IFNγ was completely abrogated in the presence of mutant STAT-binding sequence at any time assayed (Fig. 4). Taken together, our SOCS-3 promoter analysis indicated that (i) the region from nucleotides -50 to +968 (construct 6) is likely not involved in promoter activity; (ii) the 5′ truncated constructs 1–5 display similar inducibility by IFNγ, indicating that the region upstream nucleotide -454 is not responsible for IFNγ-induced SOCS-3 promoter activity; (iii) the region responsive to IFNγ is localized at nucleotides -454 to -50, as indicated by the complete loss of responsiveness to IFNγ upon its removal (in construct 6 and construct 7); and (iv) within the region from -454 to -50, a STAT-binding element from nucleotides -72 to -64 is fully responsible for the induction of the SOCS-3 reporter gene in response to IFNγ. STAT1-dependent Induction of SOCS-3 by IFNγ—SOCS-3 induction has been shown to be strictly dependent on both STAT1 and STAT3 activation in leukemia inhibitory factor-stimulated AtT-20 cells (40Auernhammer C.J. Bousquet C. Melmed S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6964-6969Google Scholar) and on STAT3 activation in granulocyte colony-stimulating factor-activated granulocytes (42Lee C.K. Raz R. Gimeno R. Gertner R. Wistinghausen B. Takeshita K. DePinho R.A. Levy D.E. Immunity. 2002; 17: 63-72Google Scholar). Because our data indicated that the induction of the SOCS-3-reporter gene by IFNγ was dependent on a STAT-binding sequence, we determined which was/were the STAT(s) involved in th" @default.
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• W2040409641 date "2004-04-01" @default.
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• W2040409641 title "Analysis of SOCS-3 Promoter Responses to Interferon γ" @default.
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• W2040409641 doi "https://doi.org/10.1074/jbc.m308999200" @default.
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