Cooperation of STAT-1 and IRF-1 in Interferon-γ-induced Transcription of the gp91 Gene
2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês
10.1074/jbc.m109803200
ISSN1083-351X
AutoresAtsushi Kumatori, Dan Yang, Shoichi Suzuki, Michio Nakamura,
Tópico(s)Immune Response and Inflammation
ResumoInterferon (IFN)-γ induces the expression of the gp91phox gene both during myeloid differentiation and also in mature phagocytes through several cis-elements and their binding proteins. To find new cis-elements for this induction, transient expression assays were performed using a reporter gene driven by serially truncated gp91phox promoters in U937 cells. The results suggest that a critical cis-element for induction exists in the region from bp −115 to −96 of the promoter. Site-directed mutagenesis showed that a γ-activated sequence (GAS) element at bp −100 (−100GAS) of the gp91phox promoter plays a pivotal role for the IFN-γ-dependent activity of the bp −115 to +12 region of the gp91phox promoter. Electrophoretic mobility shift assays using several GAS competitors and specific antibodies indicated that phosphorylated STAT-1α specifically binds to the −100GAS. Site-directed mutagenesis showed that an interferon-stimulated response element (ISRE) at bp −88 (−88ISRE) mediates the induction of the gene by IFN-γ in cooperation with −100GAS. Electrophoretic mobility shift assay showed that IRF-1 dominantly binds to −88ISRE in an IFN-γ-dependent fashion. These results demonstrate a new mechanism for IFN-γ-induced transcription of the gp91phox gene by the cooperation of STAT-1α and IRF-1 binding to −100GAS and −88ISRE, respectively. Interferon (IFN)-γ induces the expression of the gp91phox gene both during myeloid differentiation and also in mature phagocytes through several cis-elements and their binding proteins. To find new cis-elements for this induction, transient expression assays were performed using a reporter gene driven by serially truncated gp91phox promoters in U937 cells. The results suggest that a critical cis-element for induction exists in the region from bp −115 to −96 of the promoter. Site-directed mutagenesis showed that a γ-activated sequence (GAS) element at bp −100 (−100GAS) of the gp91phox promoter plays a pivotal role for the IFN-γ-dependent activity of the bp −115 to +12 region of the gp91phox promoter. Electrophoretic mobility shift assays using several GAS competitors and specific antibodies indicated that phosphorylated STAT-1α specifically binds to the −100GAS. Site-directed mutagenesis showed that an interferon-stimulated response element (ISRE) at bp −88 (−88ISRE) mediates the induction of the gene by IFN-γ in cooperation with −100GAS. Electrophoretic mobility shift assay showed that IRF-1 dominantly binds to −88ISRE in an IFN-γ-dependent fashion. These results demonstrate a new mechanism for IFN-γ-induced transcription of the gp91phox gene by the cooperation of STAT-1α and IRF-1 binding to −100GAS and −88ISRE, respectively. inteferon electrophoretic mobility gel shift assay CCAAT displacement protein binding increased during differentiation interferon-stimulated response element hematopoietic-associated factor IFN consensus-binding protein IFN consensus sequence-binding protein in adult T-cell leukemia interferon-stimulated gene factor-3γ signal transducers and activators of transcription the γ-activated sequence IFP53/tryptophanyl-tRNA synthetase promoter the guanylate-binding protein the c-fos cis-inducible element the Fcγ receptor I promoter interferon regulatory factor differentiation-induced factor CREB-binding protein Phagocytes, such as macrophages and granulocytes, generate superoxide anions by the phagocyte NADPH oxidase to kill ingested microorganisms (1Morel F. Doussiere J. Vignais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (526) Google Scholar). The gp91phox gene encodes an essential component of the oxidase, and mutations including a deletion (2Faizunnessa N.N. Tsuchiya T. Kumatori A. Kurozumi H. Imajoh-Ohmi S. Kanegasaki S. Nakamura M. Hum. Genet. 1997; 99: 469-473PubMed Google Scholar, 3Kumatori A. Faizunnessa N.N. Suzuki S. Moriuchi T. Kurozumi H. Nakamura M. Genomics. 1998; 53: 123-128Crossref PubMed Scopus (27) Google Scholar) of the gene result in X-linked chronic granulomatous disease characterized by severe and recurrent infections due to lack of superoxide generation (4Tauber A.I. Borregaard N. Simons E. Wright J. Medicine (Baltimore). 1983; 62: 286-309Crossref PubMed Scopus (172) Google Scholar). The gp91phox gene is expressed almost uniquely in differentiated myeloid cells (5Royer-Pokora B. Kunkel L.M. Monaco A.P. Goff S.C. Newburger P.E. Baehner R.L. Cole F.S. Curnutte J.T. Orkin S.H. Nature. 1986; 322: 32-38Crossref PubMed Scopus (594) Google Scholar). The human gp91phox promoter from bp −450 to +12 directs the transcription of a reporter gene in a subset of mouse monocytes/macrophages in transgenic mice (6Skalnik D.G. Dorfman D.M. Perkins A.S. Jenkins N.A. Copeland N.G. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8505-8509Crossref PubMed Scopus (55) Google Scholar). Eosinophils, in particular, express gp91phox in a patient with chronic granulomatous disease (7Kuribayashi F. Kumatori A. Suzuki S. Nakamura M. Matsumoto T. Tsuji Y. Biochem. Biophys. Res. Commun. 1995; 209: 146-152Crossref PubMed Scopus (28) Google Scholar). These observations indicate that the expression of the gp91phox gene is both lineage- and differentiation stage-specific. Transcription of the gp91phoxgene is also modulated by inflammatory mediators such as interferon (IFN)-γ,1 tumor necrosis factor-α, and lipopolysaccharide in myeloid cells (8Newburger P.E. Ezekowitz R.A. Whitney C. Wright J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5215-5219Crossref PubMed Scopus (109) Google Scholar, 9Cassatella M.A. Hartman L. Perussia B. Trinchieri G. J. Clin. Invest. 1989; 83: 1570-1579Crossref PubMed Scopus (62) Google Scholar). Although enhancer elements for the expression of gp91phox gene in mature myeloid cells have been suggested to be in a 50-kbp region located upstream of a transcription start site of the gene (10Lien L.L. Lee Y. Orkin S.H. Mol. Cell. Biol. 1997; 17: 2279-2290Crossref PubMed Scopus (23) Google Scholar), principalcis-regulatory elements are clustered in a 450-bp proximal promoter region of the gene. Several transcription factors regulate gp91phox gene expression through these cis-elements. A transcriptional repressor, CCAAT displacement protein (CDP/cut), binds to multiple sites in the proximal promoter in immature myeloid cells (11Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 12Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Coincident with induction of gp91phox gene expression, the binding activity of CDP to these binding sites decreases during differentiation of the cells (11Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 12Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 13Lievens P.M. Donady J.J. Tufarelli C. Neufeld E.J. J. Biol. Chem. 1995; 270: 12745-12750Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14Marziali G. Perrotti E. Ilari R. Coccia E.M. Mantovani R. Testa U. Battistini A. Blood. 1999; 93: 519-526Crossref PubMed Google Scholar). Moreover, the induction of the gp91phox gene is prevented by the constitutive overexpression of CDP in a myeloid cell line (13Lievens P.M. Donady J.J. Tufarelli C. Neufeld E.J. J. Biol. Chem. 1995; 270: 12745-12750Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Finally, CDP itself disappears in the maturation course to peripheral monocytes and macrophages (14Marziali G. Perrotti E. Ilari R. Coccia E.M. Mantovani R. Testa U. Battistini A. Blood. 1999; 93: 519-526Crossref PubMed Google Scholar). Although general transcriptional activators such as those with increased binding during differentiation, like (BID)/YY1, CCAAT box-binding factors, CP1, and NF-Y, bind to the promoter, these factors can work only after CDP has been released because their binding sites overlap those for CDP (12Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 14Marziali G. Perrotti E. Ilari R. Coccia E.M. Mantovani R. Testa U. Battistini A. Blood. 1999; 93: 519-526Crossref PubMed Google Scholar, 15Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar, 16Jacobsen B.M. Skalnik D.G. J. Biol. Chem. 1999; 274: 29984-29993Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Interferon regulatory factor (IRF)-2 also activates the gp91phox promoter through two interferon-stimulated response elements (ISRE) in the absence of CDP binding (15Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar, 17Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). PU.1 works as a pivotal transcription factor of the gp91phox gene by binding a PU.1/hematopoietic-associated factor (HAF)-1-binding element centered at bp −53 in human neutrophils, monocytes, and B-lymphocytes (18Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar). HAF-1 is a multiprotein complex, and its components are still not defined, but PU.1, IRF-1, ICSBP, and Elf-1 are picked out as candidates (19Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Voo K.S. Skalnik D.G. Blood. 1999; 93: 3512-3520Crossref PubMed Google Scholar). GATA-1, GATA-2, and GATA-3 regulate transcription of the gp91phox gene in eosinophilic cells (21Yang D. Suzuki S. Hao L.J. Fujii Y. Yamauchi A. Yamamoto M. Nakamura M. Kumatori A. J. Biol. Chem. 2000; 275: 9425-9432Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22Sadat M.A. Kumatori A. Suzuki S. Yamaguchi Y. Tsuji Y. Nakamura M. FEBS Lett. 1998; 436: 390-394Crossref PubMed Scopus (14) Google Scholar). IFN-γ is a cytokine that plays an important role in both innate and adaptive immunities (23Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2495) Google Scholar). IFN-γ binds to the specific cell surface receptor and activates the receptor-associated Janus family tyrosine kinase (JAK)1 and JAK2. The kinases phosphorylate and activate a latent cytoplasmic transcription factor, STAT-1α, which is then translocated to the nucleus as a transcriptionally active homodimer (γ-interferon activation factor) and binds to the γ-activated sequence (GAS) element of IFN-γ-responsive genes resulting in their activation (24Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1480) Google Scholar). IFN-γ also induces gp91phox gene expression, coinciding with monocytic differentiation of committed progenitors (9Cassatella M.A. Hartman L. Perussia B. Trinchieri G. J. Clin. Invest. 1989; 83: 1570-1579Crossref PubMed Scopus (62) Google Scholar) and increases gp91phox transcription in mature monocytes ex vivo(8Newburger P.E. Ezekowitz R.A. Whitney C. Wright J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5215-5219Crossref PubMed Scopus (109) Google Scholar). Two positive regulation mechanisms for the IFN-γ-induced transcription of the gp91phox gene have been proposed. One mechanism is the binding of BID/YY1 to the four binding sites that are scattered in a region from bp −90 to −355 of the promoter after dissociation of CDP from the gene (15Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar, 16Jacobsen B.M. Skalnik D.G. J. Biol. Chem. 1999; 274: 29984-29993Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The other is the binding of an undefined multiprotein complex of HAF-1 to the PU.1/HAF-1-binding element centered at −53 of the promoter (19Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 25Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 26Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). In this study, we show that STAT-1α bound to the GAS element at bp −100 of the gp91phox gene has an unequivocal role in IFN-γ-induced transcription of the gene. We further show that IRF-1 participates in the STAT-1-mediated transcription through binding to the ISRE at bp −88 of the gene. These results suggest a new mechanism for IFN-γ-induced transcription of the gp91phox gene in which STAT-1α and IRF-1 cooperate with each other. U937 cells (Japanese Collection of Research Bioresources, Tokyo) were maintained in RPMI 1640 medium containing 10% (v/v) fetal calf serum. The cells were either untreated or treated with 100 units/ml human IFN-γ (kindly provided by Shionogi Corp., Osaka, Japan) for the indicated periods. Total RNA was prepared from cells with Trizol LS reagent (Invitrogen) according to the manufacturer's protocol. The RNA (5 μg/lane) was electrophoresed on formaldehyde-containing 0.9% agarose gels, transferred to HybondTM-N+ nylon membranes (Amersham Biosciences Inc.), and fixed by ultraviolet light. A probe of 2.9 kb of human gp91phox cDNA (2Faizunnessa N.N. Tsuchiya T. Kumatori A. Kurozumi H. Imajoh-Ohmi S. Kanegasaki S. Nakamura M. Hum. Genet. 1997; 99: 469-473PubMed Google Scholar) and an 800-bp cDNA of rat glyceraldehyde-3-phosphate dehydrogenase cDNA were labeled with [32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences). Messenger RNAs were hybridized with these labeled probes. The hybridization patterns were visualized, and the relative radioactivity of each band was estimated on a Molecular Imager FX (Bio-Rad). Constructs p−487/Luc and p−301/Luc were prepared by the exonuclease III deletion method (27Henikoff S. Methods Enzymol. 1987; 155: 156-165Crossref PubMed Scopus (676) Google Scholar) from pGV-5635/Luc, which had the fragment of the gp91phox gene extending from its initiation codon to upstream bp −5635. Constructs p−267/Luc, p−115/Luc, p−95/Luc, and p−84/Luc were prepared using the PCR method using pGV-267/Luc which had the fragment of the gene extending from its initiation codon to upstream bp −267 as the template as described previously in detail (21Yang D. Suzuki S. Hao L.J. Fujii Y. Yamauchi A. Yamamoto M. Nakamura M. Kumatori A. J. Biol. Chem. 2000; 275: 9425-9432Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). A mutant of GAS (p−115GASm/Luc) with a two-point mutation (TC to AG) from the bp −103 to −102 and a mutant of ISRE (p−115IRFm/Luc) with a five-point mutation (AGGG to TCCC) from bp −87 to −84 were prepared from p−115/Luc by PCR using a mutated primer. U937 cells (5 × 106) were electroporated at 950 microfarads and 280 V in a 4-mm gap cuvette containing 10 μg of gp91phoxpromoter/firefly luciferase plasmid and 0.5 μg of herpes simplex virus thymidine kinase promoter/Renilla luciferase plasmid in 0.25 ml of 20 mm Hepes/RPMI 1640 medium (pH 7.2) using a Bio-Rad Gene Pulser II (Bio-Rad). After a 15-min incubation on ice, the cells were incubated at 37 °C under 5% CO2, 95% air for 5 h in RPMI 1640 containing 10% fetal calf serum with or without 100 units/ml IFN-γ. Reporter activities were measured as described previously (18Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar). Nuclear extracts were prepared from U937 cells treated or untreated with IFN-γ as described previously (18Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar) with some modifications. Approximately 1 × 107 cells were swollen in 400 μl of ice-cold buffer A (10 mm Hepes (pH 7.9), 10 mm KCl, 0.1 mm EGTA, 0.1 mm EDTA, 1 mmdithiothreitol/CompleteTM Protease Inhibitor Mixture (Roche Molecular Biochemicals)/Phosphatase Inhibitor Mixture I and II (Sigma)). After a 15-min incubation at 4 °C, the cells were lysed in 0.6% Nonidet P-40 by vigorous vortexing for 10 s. After centrifugation at 5,000 rpm (1,700 × g) for 1 min, supernatants were discarded, and precipitated nuclei were resuspended in 100 μl of ice-cold buffer C (20 mm Hepes (pH 7.9), 0.4m NaCl, 1 mm EGTA, 1 mm EDTA, 1 mm dithiothreitol, CompleteTM Protease Inhibitor Mixture/Phosphatase Inhibitor Mixture I and II). Nuclear extracts were separated by centrifugation at 15,000 rpm (18,000 ×g) for 5 min at 4 °C and stored in aliquots at −80 °C. Protein concentration was determined with the Bio-Rad protein assay kit. Preparation of the double-stranded oligonucleotide probes and EMSAs were performed as described previously (18Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar). In competition assays, a 100-molar excess of unlabeled competitor oligonucleotides was added prior to the addition of the probe to the mixture, which was then preincubated on ice for 15 min. For the inhibition assay with antibodies, an aliquot of nuclear extracts was incubated on ice with 2–4 μg of the indicated antibody for 1 h before the addition of the probe. Bound and free DNA were separated by electrophoresis in a native 6% polyacrylamide gel in 0.4× TBE buffer (36 mmTris, 36 mm boric acid, and 8 mm EDTA (pH 8.3)) at 4 °C. Sequences of the upper strands of double-strand oligonucleotides are as follows. Mutated bases are underlined and names used in the text are indicated in bold: W-107A (−107 to −85), 5′-AATTTCTGATAAAAGAAAAGGAA-3′; W-107B (−107 to −65), 5′-AATTTCTGATAAAAGAAAAGGAAACCGATTGCCCCAGGGCTGC-3′;W-115 (−115 to −90), 5′-TATTAGCCAATTTCTGATAAAAGAAA-3′;GASmut (−115 to −90), 5′-TATTAGCCAATTGATGATAAAAGAAA-3′; W-102A (−102 to −65), 5′-CTGATAAAAGAAAAGGAAACCGATTGCCCCAGGGCTGC-3′;W-102B (−102 to −73), 5′-CTGATAAAAGAAAAGGAAACCGATTGCCCC-3′; IRFmut (−102 to −73), 5′-CTGATAAAAGAAAAGTCCCCCGATTGCCCC-3′;BIDmut (−102 to −73), 5′-CTGATAAAAGAAACTGAAACCGATTGCCCC-3′; W-95 (−95 to −65), 5′-AAGAAAAGGAAACCGATTGCCCCAGGGCTGC-3′. The following four oligonucleotides are GAS elements used as competitors (28Eilers A. Baccarini M. Horn F. Hipskind R.A. Schindler C. Decker T. Mol. Cell. Biol. 1994; 14: 1364-1373Crossref PubMed Scopus (62) Google Scholar):GAS/IFP (GAS site from the IFP53/tryptophanyl-tRNA synthetase promoter), 5′-TCACCCAGATTCTCAGAAACACTT-3′;GAS/GBP (GAS site from the guanylate-binding protein promoter), 5′-AGTTTCATATTACTCTAAATC-3′; GAS/SIE (GAS site in the c-fos cis-inducible element), 5′-ATTCAGCAGTTCCCGTCAATCCCT-3′; GAS/GRR (GAS site from the Fcγ receptor I promoter), 5′-GATGTATTTCCCAGAAAAGG-3′. Additional oligonucleotides as competitors used in this study are as follows: a heterogeneous competitor (Hetero), 5′-CACAACCACATTCAACCTCTGCCACC-3′; IRF-1 cons (IRF-1 consensus binding site), 5′-ACGGATCCGGCATATTCAAAACCGAAACCAAGTCCCTCGAGAC-3′ (29Tanaka N. Kawakami T. Taniguchi T. Mol. Cell. Biol. 1993; 13: 4531-4538Crossref PubMed Scopus (385) Google Scholar). Specific antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA) were used in immunoassays as follows: murine monoclonal antibodies against STAT-1p84/p91 (C-136), STAT1αp91 (C-111), and Tyr-701-phosphorylated STAT-1 (A-2), rabbit polyclonal antibodies against IRF-1 (C-20), IRF-2 (C-19), and ISGF-3γ/p48 (C-20), and goat polyclonal antibodies against ICSAT that is also designated PU.1 interaction partner, lymphoid-specific IRF (ISIRF), or IRF-4 (M-17), and ICSBP (C-19). Western blot analysis was carried out using standard methods (30Imajoh-Ohmi S. Tokita K. Ochiai H. Nakamura M. Kanegasaki S. J. Biol. Chem. 1992; 267: 180-184Abstract Full Text PDF PubMed Google Scholar). Nuclear extracts (24 μg of protein) were mixed with an equal volume of 250 mm Tris-HCl (pH 6.8) containing 4.6% (w/v) SDS, 20% (w/v) glycerol, 10% (v/v) 2-mercaptoethanol, and 20 μg/ml bromphenol blue, and heated at 100 °C for 5 min. Samples separated by SDS-PAGE in 10% gel were transferred onto an ImmobilonTM polyvinylidene difluoride membrane (Millipore, Bedford, MA) using a semi-dry type electroblotting apparatus (model BE-300, Bio-Craft, Tokyo, Japan). The protein-blotted polyvinylidene difluoride membrane was stained with Coomassie Brilliant Blue, photographed, and destained with methanol. The membranes were pretreated with Block Ace (Yukijirushi Co., Sapporo, Japan) containing 0.1% Tween 20 for 1 h at room temperature followed by washing with TBS (20 mm Tris (pH 7.4), 150 mm NaCl) containing 0.1% Tween 20 (TBS/Tween) and then incubated with 1 μg/ml anti-IRF-1 antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) or nonimmunized rabbit IgG in TBS/Tween containing 20 mg/ml bovine serum albumin for 1 h at room temperature. After incubation, the membrane was washed with TBS/Tween and incubated with 1/2500 diluted anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Biosciences) for 1 h at room temperature. The membrane was then washed extensively with TBS/Tween and then rinsed with TBS. The antigen antibody complexes were detected using an Enhanced Chemiluminescence Kit (Amersham Biosciences Inc.). Data are expressed as mean ± S.E. Statistical analysis was done on data from at least three independent experiments by Student's t test. We analyzed the time course of gp91phoxmRNA levels in human promonocytic U937 cells after addition of 100 units/ml IFN-γ by Northern blot analysis. As shown in Fig. 1, the amount of gp91phoxmRNA increased ∼4.5-fold within 5 h and reached its maximal level (∼7.5-fold) at 48 h after the addition of IFN-γ as demonstrated in previous studies (8Newburger P.E. Ezekowitz R.A. Whitney C. Wright J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5215-5219Crossref PubMed Scopus (109) Google Scholar, 9Cassatella M.A. Hartman L. Perussia B. Trinchieri G. J. Clin. Invest. 1989; 83: 1570-1579Crossref PubMed Scopus (62) Google Scholar, 26Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar) with U937 cells and cultured human monocyte-derived macrophages. Similar studies previously done (15Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar, 25Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 26Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar) for IFN-γ induction of the gp91phox gene were focused on the later phase of induction (at 24–48 h after the addition of IFN-γ). In this study, we focused on the early phase of IFN-γ induction of the gp91phox gene because the changes in this phase are the most pronounced. The bp −450 to +12 fragment of the gp91phox promoter directs IFN-γ-inducible transcription in a stably transfected PLB-985 myeloid cell line (25Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Therefore, we initially prepared a p−487/Luc construct that contains the gp91phox gene fragment from bp −487 to +12 in front of a luciferase reporter gene and transiently transfected it with the reference plasmid pRL-TK into promonocytic U937 cells. The reporter activity of the cells was assessed following treatment with IFN-γ (100 units/ml) for 5 h and was compared with that of untreated transfectants (Fig. 2). Reporter activity of the p−487/Luc was induced 8.2 (±0.6)-fold by IFN-γ treatment and reached levels 12 times higher than that of the control, untreated wild-type p−115/Luc, indicating that the bp −487 to +12 region of the gp91phox promoter directs transcriptional induction of the gene by IFN-γ in U937 cells. To determine regions responsible for the IFN-γ induction in the bp −487 to +12 fragment of the gp91phox promoter, five serial deletion constructs were generated from the p−487/Luc construct. Progressive 5′ deletions from bp −487 to −116 still retained both a high inducibility and high relative promoter activity in response to IFN-γ treatment (compare p−301/Luc, p−267/Luc, and p−115/Luc with p−487/Luc). The removal of an additional 20-bp fragment from bp −115 to −96 remarkably reduced the IFN-γ-dependent inducibility and the activity of the promoter (compare p−95/Luc with p−115/Luc), suggesting that a pivotalcis-element(s) for IFN-γ induction lies within the region from bp −115 to −96 of the gp91phox promoter. Although bp −95/Luc and bp −84/Luc showed significantly higher activity than that of pXP2N, their inducibilities were not significantly different from that of pXP2N. Therefore, further deleted constructs were not analyzed.Figure 2Identification of a region for IFN -γ-induced activation of the gp91phox promoter in U937 cells. Luciferase reporter constructs containing serially truncated gp91phox promoter are illustrated on the left. These constructs were transfected into U937 cells incubated with (I) or without (N) IFN-γ (100 units/ml) for 5 h. Firefly luciferase activities were normalized by accompanied activities of the co-transfectedRenilla luciferase reporter gene (see "Materials and Methods"). Each column and bar in thecenter graph are the relative means of three or four independent data sets and S.E., respectively. The activity ofp−115/Luc N was set to 1. Fold induction was calculated as the ratio of relative luciferase activity of IFN-γ-treated samples to that of the untreated sample and is shown as the mean ± S.E. on the right. Asterisks indicate a statistically significant difference (p < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To analyze binding of protein complexes to the bp −115 to −96 region, we prepared nuclear extracts from U937 cells at various times after the addition of 100 units/ml IFN-γ, and we performed EMSAs with the fragment from bp −107 to −85 (W-107A in Fig.3) as a probe. As shown in Fig.4, binding of a protein complex (shown by an arrow) to the probe was induced within 1 h of stimulation with IFN-γ (Fig. 4 A) and could be continuously detected over the entire 48 h after the IFN-γ addition (Fig.4 B). The binding of this protein complex to the probe was specific because the binding was abolished by an excess of homologous competitor (Ho), with a sequence identical to that of the probe, but not by an excess of heterogeneous competitor (He), with a sequence completely different from that of the probe (Fig. 4 B). To identify the DNA-binding protein complex, we searched for an element possibly recruiting a certain transcription factor in the region from bp −107 to −85 of the gp91phox promoter. As shown in Fig. 3, the bp −104 to −96 region of the gene (5′-TTCTGATAA-3′, −100GAS) perfectly conforms to the GAS element at bp −137 of the human MHC-II transactivator CIIA (5′-TTCTGATAA-3′) (31Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (435) Google Scholar) and was present in the region from bp −115 to −96, which was shown to be important for the IFN-γ-induced gp91phox promoter activity in the functional assay (Fig. 2). Therefore, we focused on GAS-binding proteins and did further experiments. We examined the effect of mutations in the −100GAS element for the binding of this protein complex to the W-107A probe using a fragment from bp −115 to −90 (W-115 in Fig. 3) and a −100GAS mutant version of W-115 (GASmut in Fig. 3) as competitors. As shown in Fig. 5, the binding of this protein complex (shown by an arrow) was abolished by the W-115 competitor (lane 4) but not by the GASmut competitor (lane 5), indicating that the −100GAS element is essential for binding of the protein complex to the W-107A probe. Previous studies (32Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (340) Google Scholar, 33Lee Y.J. Benveniste E.N. J. Immunol. 1996; 157: 1559-1568PubMed Google Scholar, 34Dong Y. Rohn W.M. Benveniste E.N. J. Immunol. 1999; 162: 4731-4739PubMed Google Scholar, 35Nguyen V.T. Benveniste E.N. J. Biol. Chem. 2000; 275: 23674-23684Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) showed that the STAT-1α transcription factor frequently binds to GAS elements in the genes of IFN-γ-induced proteins such as IRF-1, class II transactivator, and CD40. Differentiation-induced factor (DIF) also binds to GAS elements of several IFN-γ-induced genes such as the Fcγ receptor I (FcγR) in U937 cells after the addition of IFN-γ (28Eilers A. Baccarini M. Horn F. Hipskind R.A. Schindler C. Decker T. Mol. Cell. Biol. 1994; 14: 1364-1373Crossref PubMed Scopus (62) Google Scholar). To examine whether either one or both factors bind to the −100GAS of the gp91phoxpromoter in the IFN-γ-treated U937 cells, we performed EMSAs using IFP GAS and GBP GAS as common competitors to both STAT-1α and DIF and FcγRI GAS and c-fos SIE GAS as specific competitors to STAT-1α (28Eilers A. Baccarini M. Horn F. Hipskind R.A. Schindler C. Decker T. Mol. Cell. Biol. 1994; 14: 1364-1373Crossref PubMed Scopus (62) Google Scholar). As shown in Fig. 5, binding of the protein complex to the W-107A probe was abolished by all four GAS competitors (lanes 6–9), suggesting that this protein complex includes STAT-1α but not DIF. To confirm the binding of STAT-1α to the −100GAS, we performed gel shift immunoassays using three different anti-STAT-1 antibodies (Fig. 5) as follows: an anti-STAT-1 antibody specific for both STAT-1α and STAT-1β (l
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