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• W2094885091 abstract "This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. An ICSBP binding site in the iNOS promoter region (−923 to −913) was identified using an electrophoretic mobility shift assay and chromatin co-immunoprecipitation. Overexpression of ICSBP greatly enhanced IFN-γ-induced iNOS promoter activation in RAW264.7 cells, and IFN-γ-induced iNOS promoter activation was abolished in ICSBP−/− macrophages. Furthermore, transduction of retrovirus-ICSBP in ICSBP−/− macrophages rescued IFN-γ-induced iNOS gene expression. However, transduction of retrovirus-ICSBP in the absence of IFN-γ activation did not induce iNOS expression in either RAW264.7 cells or ICSBP−/− macrophages. Interestingly, ICSBP alone transduced into ICSBP−/− macrophages did not bind to IFN-stimulated response element site (−923 to −913) of the iNOS promoter region, although following activation with IFN-γ, a DNA·protein complex was formed that contains ICSBP and IRF-1. Co-transduction of ICSBP with IRF-1 clearly induces nitric oxide production. In addition, interleukin-4 inhibits IFN-γ-induced iNOS gene expression by attenuating the physical interaction of ICSBP with IRF-1. Complex formation of ICSBP with IRF-1 is essential for iNOS expression, and interleukin-4 attenuates the physical interaction of ICSBP with IRF-1 resulting in the inhibition of INOS gene expression. This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. An ICSBP binding site in the iNOS promoter region (−923 to −913) was identified using an electrophoretic mobility shift assay and chromatin co-immunoprecipitation. Overexpression of ICSBP greatly enhanced IFN-γ-induced iNOS promoter activation in RAW264.7 cells, and IFN-γ-induced iNOS promoter activation was abolished in ICSBP−/− macrophages. Furthermore, transduction of retrovirus-ICSBP in ICSBP−/− macrophages rescued IFN-γ-induced iNOS gene expression. However, transduction of retrovirus-ICSBP in the absence of IFN-γ activation did not induce iNOS expression in either RAW264.7 cells or ICSBP−/− macrophages. Interestingly, ICSBP alone transduced into ICSBP−/− macrophages did not bind to IFN-stimulated response element site (−923 to −913) of the iNOS promoter region, although following activation with IFN-γ, a DNA·protein complex was formed that contains ICSBP and IRF-1. Co-transduction of ICSBP with IRF-1 clearly induces nitric oxide production. In addition, interleukin-4 inhibits IFN-γ-induced iNOS gene expression by attenuating the physical interaction of ICSBP with IRF-1. Complex formation of ICSBP with IRF-1 is essential for iNOS expression, and interleukin-4 attenuates the physical interaction of ICSBP with IRF-1 resulting in the inhibition of INOS gene expression. nitric oxide lipopolysaccharide interferon nuclear factor κB IFN-stimulated response element IFN consensus sequence-binding protein interleukin electrophoretic mobility shift assay reverse transcriptase green fluorescent protein Nitric oxide (NO)1 has been identified as an important signaling molecule involved in regulating a wide range of biological activities in the immune, vascular, and neural system (1Lowenstein C.J. Dinerman J.L. Snyder S.H. Ann. Intern. Med. 1994; 120: 227-237Crossref PubMed Scopus (853) Google Scholar, 2Nathan C. Xie Q.W. Cell. 1994; 78: 915-918Abstract Full Text PDF PubMed Scopus (2758) Google Scholar, 3Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (817) Google Scholar). NO and its metabolites are central to the antimicrobial and tumoricidal activity of activated macrophages and are implicated in the pathogenesis of tissue damage associated with acute and chronic inflammation (4Nathan C.F. Hibbs J.B., Jr. Curr. Opin. Immunol. 1991; 3: 65-70Crossref PubMed Scopus (1328) Google Scholar, 5MacMicking J. Xie Q.W. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3498) Google Scholar, 6Chan J. Xing Y. Magliozzo R.S. Bloom B.R. J. Exp. Med. 1992; 175: 1111-1122Crossref PubMed Scopus (862) Google Scholar, 7Nathan C. Shiloh M.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8841-8848Crossref PubMed Scopus (1161) Google Scholar, 8Fang F.C. J. Clin. Invest. 1997; 99: 2818-2825Crossref PubMed Google Scholar, 9Lorsbach R.B. Murphy W.J. Lowenstein C.J. Snyder S.H. Russell S.W. J. Biol. Chem. 1993; 268: 1908-1913Abstract Full Text PDF PubMed Google Scholar). Macrophages generate NO froml-arginine through the inducible isoform of nitric oxide synthase (iNOS), which is regulated transcriptionally. iNOS gene expression is induced by a variety of stimuli, including lipopolysaccharide (LPS), bacterial wall products, and cytokines. In particular, interferon-γ (IFN-γ) is a potent inducer of iNOS gene expression (10Xie Q.W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1741) Google Scholar, 11Ding A.H. Nathan C.F. Stuehr D.J. J. Immunol. 1988; 141: 2407-2412PubMed Google Scholar), although the molecular mechanism for the effect is still not understood fully. The murine iNOS promoter region contains several binding sites for transcription factors implicated in iNOS regulation, including two NF-κB binding sites. The NF-κB site situated closer to the transcriptional start site is required for iNOS gene induction by LPS, because deleting the downstream site essentially abolishes iNOS transcription (12Xie Q.W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar). Deletion of the upstream NF-κB site reduces but does not abolish iNOS transcription (12Xie Q.W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar, 13Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russell S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1008) Google Scholar, 14Xie Q.W. Whisnant R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Crossref PubMed Scopus (1030) Google Scholar). The upstream portion of the iNOS promoter contains an enhancer region, with several transcription factor binding sites that mediate transcriptional responses to IFN-γ. These sites include a γ-interferon-activated site element and two IFN-stimulated response elements (ISRE) (15Gao J. Morrison D.C. Parmely T.J. Russell S.W. Murphy W.J. J. Biol. Chem. 1997; 272: 1226-1230Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 16Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. et al.Science. 1994; 263: 1612-1615Crossref PubMed Scopus (787) Google Scholar). Deletion and mutational analysis have shown the importance of the promoter sequence from −1029 to −913 in the induction of iNOS by IFN-γ (13Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russell S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1008) Google Scholar). Site-directed mutagenesis and competition experiments have shown that the ISRE site between −923 and −913 is required for the full synergistic effects of IFN-γ and LPS in the transcription of the iNOS gene (16Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. et al.Science. 1994; 263: 1612-1615Crossref PubMed Scopus (787) Google Scholar). The cytokine IFN-γ plays an essential role in innate and adaptive immunity (17Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar). IFN signaling, which involves a variety oftrans- and cis-acting factors, is mediated through DNA motifs, designated ISRE and IFN-γ-activated sequence, which are found in promoters of IFN-inducible genes. The interferon consensus sequence-binding protein (ICSBP or IRF-8) is a transcription factor belonging to the IFN regulatory factor (IRF) family, which also includes the IRF-1, IRF-2, IRF-3, interferon-stimulated gene factor 3γ, and pip/IRF-4 proteins (18Taniguchi T. Ogasawara K. Takaoka A. Tanaka N. Annu. Rev. Immunol. 2001; 19: 623-655Crossref PubMed Scopus (1295) Google Scholar,19Tamura T. Ozato K. J. Interferon Cytokine Res. 2002; 22: 145-152Crossref PubMed Scopus (166) Google Scholar). Unlike the other members of the IRF family, ICSBP expression is limited to activated macrophages, B cells, and T cells (20Driggers P.H. Ennist D.L. Gleason S.L. Mak W.H. Marks M.S. Levi B.Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar, 21Nelson N. Marks M.S. Driggers P.H. Ozato K. Mol. Cell. Biol. 1993; 13: 588-599Crossref PubMed Scopus (174) Google Scholar, 22Nelson N. Kanno Y. Hong C. Contursi C. Fujita T. Fowlkes B.J. O'Connell E., Hu-Li, J. Paul W.E. Jankovic D. Sher A.F. Coligan J.E. Thornton A. Appella E. Yang Y. Ozato K. J. Immunol. 1996; 156: 3711-3720PubMed Google Scholar, 23Politis A.D. Ozato K. Coligan J.E. Vogel S.N. J. Immunol. 1994; 152: 2270-2278PubMed Google Scholar). Proteins of the IRF family, including ICSBP, bind to the ISRE (18Taniguchi T. Ogasawara K. Takaoka A. Tanaka N. Annu. Rev. Immunol. 2001; 19: 623-655Crossref PubMed Scopus (1295) Google Scholar). The tyrosine-phosphorylated form of ICSBP does not bind to DNA independently but requires complex formation with other members of the IRF family (IRF-1 and IRF-2) or with other transcriptional elements (24Levi B.Z. Hashmueli S. Gleit-Kielmanowicz M. Azriel A. Meraro D. J. Interferon Cytokine Res. 2002; 22: 153-160Crossref PubMed Scopus (65) Google Scholar). Previous studies found that ICSBP represses transcriptional activity of ISRE promoters in various cell types. However, recent studies suggest that ICSBP can activate genes required for the development and function of macrophages (25Wang I.M. Contursi C. Masumi A., Ma, X. Trinchieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (160) Google Scholar, 26Tamura T. Nagamura-Inoue T. Shmeltzer Z. Kuwata T. Ozato K. Immunity. 2000; 13: 155-165Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). ICSBP−/− mice were shown to be highly susceptible to infection with several pathogens including Listeria monocytogenes (27Fehr T. Schoedon G. Odermatt B. Holtschke T. Schneemann M. Bachmann M.F. Mak T.W. Horak I. Zinkernagel R.M. J. Exp. Med. 1997; 185: 921-931Crossref PubMed Scopus (136) Google Scholar, 28Holtschke T. Lohler J. Kanno Y. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K.P. Gabriele L. Waring J.F. Bachmann M.F. Zinkernagel R.M. Morse III, H.C. Ozato K. Horak I. Cell. 1996; 87: 307-317Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar) and are impaired in the production by macrophages of IFN-γ-induced NO (29Contursi C. Wang I.M. Gabriele L. Gadina M. O'Shea J. Morse III, H.C. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 91-96Crossref PubMed Scopus (65) Google Scholar). However, the molecular mechanism for the regulation of NO by ICSBP is not clear. In the present study we have analyzed iNOS gene expression in macrophages induced by IFN-γ and have identified an ICSBP binding site in the iNOS promoter region. Transduction of ICSBP with a retroviral vector in ICSBP−/− macrophages rescues IFN-γ-induced iNOS expression. Surprisingly, iNOS expression is not induced by transduction of ICSBP alone but requires co-transduction of ICSBP with IRF-1. This study provides new insights into the molecular mechanism of iNOS gene induction by IFN-γ, which indicates the importance of an ICSBP·IRF-1 complex in the regulation of iNOS gene expression. iNOS promoter fragments were inserted into the luciferase reporter vector pGL2B (Promega) as described previously (13Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russell S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1008) Google Scholar). The IRF-1 expression plasmid was a gift from T. Taniguchi (University of Tokyo, Tokyo, Japan). All site-directed mutant plasmids were generated by two-step polymerase chain reaction using overlapping internal primers containing the mutant site (30Plevy S.E. Gemberling J.H. Hsu S. Dorner A.J. Smale S.T. Mol. Cell. Biol. 1997; 17: 4572-4588Crossref PubMed Scopus (273) Google Scholar). The RAW264.7 murine macrophage cell line (American Type Culture Collection) was maintained in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin. ICSBP−/− macrophages (CL-2 cells) were established from bone marrow cells obtained from ICSBP−/− mice following coincubation with the murine J2 virus, harboring v-raf and v-myc genes (25Wang I.M. Contursi C. Masumi A., Ma, X. Trinchieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (160) Google Scholar). The CL-2 cells were maintained in the same media as above, supplemented with recombinant mouse M-CSF (6 ng/ml; R & D Systems, Minneapolis, MN) and recombinant mouse GM-CSF (6 ng/ml; Peprotech, Rocky Hill, NJ) (25Wang I.M. Contursi C. Masumi A., Ma, X. Trinchieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (160) Google Scholar). IFN-γ and IL-4 were obtained from R & D Systems. LPS was purchased from Sigma. Antibodies against iNOS, ICSBP, IRF-1, and IRF-2 were from Santa Cruz Biotechnology. Nitrite concentration, a measure of NO synthesis, was assayed by a standard Griess reaction adapted to microplates, as described previously (31Xiong H. Kawamura I. Nishibori T. Mitsuyama M. Cell. Immunol. 1996; 172: 118-125Crossref PubMed Scopus (25) Google Scholar). Nitrite accumulation was determined by using NaNO2 as standard, and the data were expressed as concentration of nitrite. RAW267.4 cells were transiently transfected using Superfect (Qiagen). For each transfection, 2.5 μg of plasmid was mixed with 100 μl of RPMI 1640 (without serum and antibiotics) and 10 μl of Superfect reagent. The mixture was incubated at room temperature for 10 min, 600 μl of RPMI 1640 complete medium was added and immediately placed onto the cells in 6-well plates, and luciferase activity was measured 16–24 h later. For CL-2 cell transfection, reporter (10 μg) and expression vectors (30 μg) were transfected into 107 CL-2 cells by electroporation, and luciferase activity was measured 16 h later. When indicated, murine IFN-γ (10 ng/ml) was added to the culture for 6–12 h before harvest. The cells were harvested with reporter lysis buffer (Promega), and 20 μl of extract was assayed for luciferase as described (30Plevy S.E. Gemberling J.H. Hsu S. Dorner A.J. Smale S.T. Mol. Cell. Biol. 1997; 17: 4572-4588Crossref PubMed Scopus (273) Google Scholar). Cells were co-transfected with a constitutively active cytomegalovirus promoter-β-galactosidase reporter plasmid to normalize experiments for transfection efficiency. For retroviral transduction we used a derivative of the Moloney murine leukemia virus vector pMMP412 (32Pickl W.F. Pimentel-Muinos F.X. Seed B. J. Virol. 2001; 75: 7175-7183Crossref PubMed Scopus (141) Google Scholar), courtesy of Dr. Adrain Ting (Mount Sinai School of Medicine). To prepare pseudotyped virus human 293 EbnaT cells were seeded at a density of 4 × 106 cells in a 10-cm dish. The next day, cells were transfected using calcium phosphate with a mixture of 2.5 μg of plasmid pMD.G encoding vesicular stomatitis virus G protein, 7.5 μg of plasmid pMD.OGP encoding gag-pol, and 10 μg of the retroviral expression construct. 48 h post-transfection, the viral supernatant was collected, centrifuged at 800 ×g, and used to infect cells. RAW264.7 and CL-2 cells (5 × 106 cells) were resuspended in 12 ml of viral supernatant in the presence of polybrene (4 μg/ml), aliquoted into six-well plates, and spun at 800 × g in a microtiter rotor for 1 h at room temperature. RAW264.7 cell nuclear extracts were prepared as described previously (33Lo K. Landau N.R. Smale S.T. Mol. Cell. Biol. 1991; 11: 5229-5243Crossref PubMed Scopus (180) Google Scholar). EMSA probes were prepared by annealing complementary single-stranded oligonucleotides with 5′GATC overhangs (Genosys Biotechnologies, Inc.) and were labeled by filling in with [α-32P]dGTP and [α-32P]dCTP using Klenow enzyme. Labeled probes were purified with Nuctrap purification columns (Roche Molecular Biochemicals). The sequence of the EMSA probe (−935 to −905) is 5′gatcACACTGTCTCA-ATATTTCACTTTCATAATGGAAAAT. Electrophoretic mobility shift assays were performed as described previously (34Zhu C. Gagnidze K. Gemberling J.H. Plevy S.E. J. Biol. Chem. 2001; 276: 18519-18528Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), using 105-cpm probe and 5 μg of nuclear extracts per reaction. DNA binding complexes were separated by 5% polyacrylamide-Tris/glycine-EDTA gel run at 4 °C for 4 h at 150 volts. Gels were dried and exposed to Eastman Kodak Co. X-AR 5 film at −80 °C in the presence of an intensifying screen. Nuclear protein extracts and pre-stained molecular weight markers were denatured in Laemmli buffer (10% glycerol, 2% SDS, 0.1 m dithiothreitol, 65 mmTris, 0.01 mg/ml bromphenol blue) at 90 °C, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. After protein transfer, the membranes were blocked with 5% nonfat milk in TBST (Triton-containing Tris-buffered saline), incubated with anti-iNOS, anti-ICSBP, anti-IRF-1, or anti-IRF-2 antibodies (1:6000) for 1–2 h, washed with TBST, and stained with anti-rabbit or anti-mouse IgG conjugated to peroxidase (1:6000). Immunoreactivity was visualized by enhanced chemiluminescence reaction (enhanced chemiluminescence kit; Santa Cruz Biotechnology). For RT-PCR assays, total RNA was extracted using TRIzol (Invitrogen). cDNA was prepared from 1–3 μg of RNA with Superscript II RT (Invitrogen) and random hexamer primers (Promega). The following primers were used for PCR amplification: IL-12 p40, 5′-TCGCAGCAAGATGTG-3′ and 5′-GAGCAGCAGATGT-GAGTGGC; iNOS, 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′ and 5′-GGCTGTC-AGAGCCTCGTGGCTTTGG-3′. PCR was carried out by a standard protocol for appropriate cycles. An equal aliquot of cDNA was amplified with β-actin primers. Aliquots of PCR reactions were resolved on a 1% agarose gel and visualized with UV light after staining with ethidium bromide. RAW264.7 cells were activated with IFN-γ for 4 h. Then cells were washed with cold phosphate-buffered saline and lysed for 15 min on ice in 0.5 ml of lysis buffer (50 mm Tris, pH 8.0, 280 mm NaCl, 0.5% Nonidet P-40, 0.2 mm EDTA, 2 mm EGTA, 10% glycerol, and 1 mm dithiothreitol) containing protease inhibitors. Cell lysates were clarified by centrifugation at 4 °C for 15 min at 14,000 rpm. Cells lysates were incubated with 2 μg of IRF-1 antibody (Santa Cruz Biotechnology) in the presence of 20 μl of 50% (v/v) protein G-agarose overnight at 4 °C with gentle rocking. After three washings with lysis buffer, precipitated complexes were solubilized by boiling in SDS buffer, fractionated by 10% SDS-PAGE, and transferred to nitrocellulose. Western blotting was performed using ICSBP antibody. The experiments were performed using a chromatin immunoprecipitation (Chip) assay kit from Upstate Biotechnology according to the manufacturer's protocol. RAW264.7 cells were cultured in complete Dulbecco's modified Eagle's medium, activated with IFN-γ for 4 h, fixed with 1% formaldehyde for 30 min at room temperature, and immunoprecipitated with anti-ICSBP antibody. The immunoprecipitated DNA was amplified by PCR with primers spanning from −1000 to −788 in the murine iNOS p40 promoter region. Macrophages from ICSBP−/− mice are impaired in IFN-γ-induced NO production (29Contursi C. Wang I.M. Gabriele L. Gadina M. O'Shea J. Morse III, H.C. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 91-96Crossref PubMed Scopus (65) Google Scholar), which suggests that ICSBP is involved directly in the regulation of iNOS gene expression. Because ICSBP is a transcription factor, we hypothesize that the murine iNOS promoter region may have an ICSBP site to which ICSBP can bind, resulting in the IFN-γ-induced activation of the iNOS promoter. The mouse iNOS promoter has an enhancer and basal promoter, within which a number of response elements have been localized. Those known to be active include NF-κB sites located both in the enhancer and basal promoter and two ISRE. The distal ISRE element (−923 to −913) is a strong activator, whereas the proximal one has no significant effect on iNOS promoter activation (16Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. et al.Science. 1994; 263: 1612-1615Crossref PubMed Scopus (787) Google Scholar, 35Martin E. Nathan C. Xie Q.W. J. Exp. Med. 1994; 180: 977-984Crossref PubMed Scopus (455) Google Scholar). In the present study, the ISRE site between −923 and −913 was selected for the analysis, because this site is important for full induction of iNOS gene expression by IFN-γ. This site was also reported previously (16Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. et al.Science. 1994; 263: 1612-1615Crossref PubMed Scopus (787) Google Scholar) as a binding site for IRF-1. RAW264.7 cells were activated with IFN-γ, LPS, or IFN-γ plus LPS for 4 h, and nuclear proteins were extracted for EMSA, which was performed by using probe spanning −935 to −905 of the iNOS promoter region containing −923 to −913 of the ISRE site. A protein·oligonucleotide complex was formed that is inducible by IFN-γ and unaffected by LPS (Fig. 1 A). When we used probe (−935 to −905) with mutations of ISRE site (TTTCACTTTC to GCCGTAGGCA), there was no DNA·protein complex formation (data not shown). Furthermore, ICSBP antibody blocked the formation of DNA·protein complex (Fig. 1 B), which suggests that ICSBP can bind to this ISRE site. To confirm this result, we performed chromatin co-immunoprecipitation experiments. PCR analysis showed that ICSBP antibody precipitated the iNOS promoter region (−2000 to −788) from RAW264.7 cells activated with IFN-γ for 4 h. (Fig. 1 C). Western blotting experiments revealed that IFN-γ induced ICSBP protein expression in RAW264.7 cells (Fig. 1 D). These results strongly suggest that the ISRE site (−923 to −913) of the iNOS promoter region is a binding site for ICSBP. The above data indicate that ICSBP can bind to the ISRE site (−923 to −913) in the iNOS promoter region. The effect of overexpressing ICSBP on iNOS promoter activity was analyzed next. RAW264.7 cells were co-transfected with an iNOS promoter luciferase reporter construct and an ICSBP expression plasmid for 12 h and activated with IFN-γ for 8 h prior to determination of luciferase activity. Co-transfection of ICSBP strongly enhanced IFN-induced iNOS promoter activity (Fig. 2 A). Showing the importance of the ISRE site, IFN-γ-induced activity from an iNOS promoter with a mutated ISRE site (CTTTC into TGCCT) was inhibited 90%, and co-transfection with the ICSBP expression plasmid had no effect on the IFN-γ-induced activity (Fig. 2, A and B). The synergistic induction of the mutated iNOS promoter activation by IFN-γ and LPS was reduced by 40%, but the mutation has no effect on LPS-induced iNOS promoter activity (Fig. 2 B). The results indicate that ICSBP is important for IFN-γ-induced iNOS promoter activation. To analyze further the importance of ICSBP in the activation of the iNOS promoter, we tested ICSBP−/− macrophages for IFN-γ induced NO production and iNOS mRNA and protein expression. Both RAW264.7 cells and CL-2 cells were activated with IFN-γ (10 ng/ml) for 12 h (protein) or 72 h (nitrite). CL-2 cells had a reduced iNOS protein expression and nitrite accumulation induced by IFN-γ compared with RAW264.7 cells (Fig. 3, A and B). To determine iNOS promoter activation in CL-2 cells, RAW264.7 cells and CL-2 cells were transfected with iNOS promoter luciferase reporter construct. iNOS promoter activation induced by IFN-γ was strongly impaired in CL-2 cells (Fig. 3 C). However, co-transfection of ICSBP moderately rescued the iNOS promoter activation induced by IFN-γ in CL-2 cells. All these results suggest that ICSBP is important for iNOS expression induced by IFN-γ. The data above showed that CL-2 cells were deficient in NO production induced by IFN-γ. To demonstrate the importance of ICSBP for iNOS gene expression, we transduced ICSBP into ICSBP−/− macrophages to rescue iNOS gene expression. The transduction efficiency judged by GFP fluorescence was around 70–80% by fluorescence-activated cell sorter (data not shown). Cells were activated with IFN-γ and analyzed for iNOS mRNA and protein and nitrite accumulation. After transduction of ICSBP, IFN-γ-induced iNOS expression and nitrite accumulation were rescued (Fig. 4, A–C). In addition, the synthesis of IL-12 expression, another IFN-γ-regulated gene, was rescued, as well (Fig. 4 D). Unexpectedly, transduction of ICSBP alone did not rescue iNOS expression, although ICSBP protein was clearly expressed (Fig. 4, A–C). These results suggest that ICSBP alone is not sufficient for iNOS gene expression. To explain the requirement for both IFN-γ and ICSBP to rescue iNOS transcription and NO production in CL-2 cells, we suggest that either another transcription factor may be needed or that IFN-γ induces post-translational modification of ICSBP. We first examined whether ICSBP can bind to the ISRE site of the iNOS promoter. After transduction with ICSBP or GFP as a control, CL-2 cells were incubated with or without IFN-γ for 4 h, nuclear protein was extracted, and EMSA was performed with a probe (−935 to −905) containing the iNOS promoter ISRE site (−923 to −913). Interestingly, EMSA experiments indicated that transduction with ICSBP alone did not result in DNA·protein complex formation, which was, however, seen after activation with IFN-γ (Fig. 5 A). Furthermore, cycloheximide prevented the formation of this complex (Fig. 5 B), suggesting that synthesis of another IFN-induced transcription factor is needed for ICSBP to bind to the ISRE site of iNOS promoter. ICSBP is known to form complexes with IRF-1 or IRF-2. When RAW264.7 cells were activated with IFN-γ for 4 h, Western blotting results indicated that IFN-γ strongly induces IRF-1 protein expression (Fig. 6 A). However, because IFN-γ has no effect on IRF-2 protein expression (Fig. 6 A), IRF-1 is therefore the more likely candidate as the ICSBP partner. The ISRE site in the iNOS promoter region (−923 to −913) was identified as an IRF-1 binding site in previous studies (16Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D., Le, J. Koh S.I. Kimura T. Green S.J. et al.Science. 1994; 263: 1612-1615Crossref PubMed Scopus (787) Google Scholar), and IRF-1 −/− mice are defective in NO production. In addition, IRF-1 binds to the same ISRE site that we identified as an ICSBP site. To confirm the binding of IRF-1 to the ISRE site, we transduced ICSBP−/− macrophages with ICSBP and activated the cells with IFN-γ for 4 h. Nuclear protein was extracted, and EMSA was performed using a probe (−935 to −905) bracketing the ISRE site (−923 to −913). Both ICSBP and IRF-1 antibodies prevented the DNA·protein complex formation (Fig. 6 B), showing the presence of both proteins in the DNA·protein complex. In addition, co-immunoprecipitation experiments demonstrated that a complex formed between ICSBP and IRF-1 in nuclear proteins from IFN-γ-activated RAW264.7 cells (Fig. 6 C). When RAW264.7 cells were co-transfected with an iNOS luciferase reporter and both ICSBP and IRF-1, strong luciferase activity was observed, whereas transfection with ICSBP or IRF-1 alone only weakly induced iNOS promoter activation (Fig. 6 D). Co-transduction with ICSBP and IRF-1 induced iNOS expression and nitrite accumulation, whereas transduction of with either ICSBP or IRF-1 alone was not sufficient to induce iNOS expression (Fig. 6 F). These results suggest that the complex formation of ICSBP with IRF-1 is essential for iNOS gene expression. IL-4 is a Th2 cytokine that can counteract the effects of IFN-γ and thus serves as an anti-inflammatory molecule. IL-4 can suppress iNOS expression in both primary macrophages and RAW264.7 cells treated with IFN-γ. In the present study, we demonstrated that complex formation of ICSBP with IRF-1 is essential for iNOS induction by IFN-γ. To test whether the ICSBP·IRF-1 complex is a target of IL-4, we first analyzed the effect of IL-4 on iNOS gene expression. RAW264.7 cells pretreated with IL-4 (10 ng/ml) for 1 h were activated with IFN-γ, after which iNOS mRNA and protein expression and nitrite accumulation were determined. IL-4 clearly inhibited iNOS expression and nitrite accumulation induced by IFN-γ (Fig. 7, A–C). We next analyzed the effect of IL-4 on IFN-γ-induced DNA·protein complex formation by EMSA with probe covering iNOS promoter ISRE site (−923 to −913). The results showed that IL-4 prevented the DNA·protein complex formation between ICSBP and IRF-1 (Fig. 7 D). Co-immunoprecipitation experiments indicated that the physical binding of ICSBP with IRF-1 was attenuated by the pretreatment with IL-4 (Fig. 7 E). These results suggest that the interaction of ICSBP with IRF-1 may be the target for the IL-4-mediated inhibition of IFN-γ-induced iNOS expression. This study describes the role of ICSBP in the regulation of murine macrophage iNOS gene expression. An ICSBP binding site in the iNOS promoter region (−923 to −913) was identified, and we show that the complex formed by ICSBP and IRF-1 is essential for IFN-γ-induced iNOS gene expression. IL-4, a Th2 cytokine that inhibits IFN-γ-induced iNOS gene expression, attenuates the physical interaction of ICSBP with IRF-1. Thus, complex formation of ICSBP with IRF-1 is a central element in i" @default.
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• W2094885091 date "2003-01-01" @default.
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• W2094885091 title "Complex Formation of the Interferon (IFN) Consensus Sequence-binding Protein with IRF-1 Is Essential for Murine Macrophage IFN-γ-induced iNOS Gene Expression" @default.
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• W2094885091 doi "https://doi.org/10.1074/jbc.m209583200" @default.
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