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• W2011537701 abstract "We have previously reported that a 19-base pair element of the 5′-flanking region of the inducible nitric oxide synthase (iNOS) gene containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE) mediates picolinic acid (PA)- or hypoxia-induced activation of the iNOS promoter in interferon-γ (IFN-γ)-treated murine macrophages. The iron chelator desferrioxamine (DFX) induces the activity of the human erythropoietin enhancer in Hep3B cells. We have investigated the influence of DFX on the activation of the iNOS promoter and iNOS gene expression in ANA-1 macrophages. We have found that DFX induced DNA-binding activity to the hypoxia-inducible factor 1 (HIF-1) consensus sequence of the iNOS promoter and activated the iNOS-HRE in murine macrophages. These activities of DFX were associated with a synergistic induction of iNOS mRNA expression and iNOS transcription in IFN-γ-treated ANA-1 macrophages. Functional analysis of the 5′-flanking region of the iNOS gene demonstrated that IFN-γ plus DFX activated the full-length iNOS promoter and that the iNOS-HRE was required for DFX-induced iNOS transcriptional activity. We also investigated the role of iron metabolism in the DFX- or PA-dependent induction of HIF-1 activity and iNOS expression. We demonstrate that addition of iron sulfate completely abolished DFX or PA induction of HIF-1 binding and iNOS-HRE activation and abrogated IFN-γ plus either DFX- or PA-induced iNOS expression. These data establish that DFX is a co-stimulus for the transcriptional activation of the iNOS gene in IFN-γ-treated macrophages, and they provide evidence that the iNOS-HRE is required for the DFX-dependent activation of the iNOS promoter. Furthermore, our results indicate that the iNOS-HRE is a regulatory element of the iNOS promoter responsive to iron chelation. We have previously reported that a 19-base pair element of the 5′-flanking region of the inducible nitric oxide synthase (iNOS) gene containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE) mediates picolinic acid (PA)- or hypoxia-induced activation of the iNOS promoter in interferon-γ (IFN-γ)-treated murine macrophages. The iron chelator desferrioxamine (DFX) induces the activity of the human erythropoietin enhancer in Hep3B cells. We have investigated the influence of DFX on the activation of the iNOS promoter and iNOS gene expression in ANA-1 macrophages. We have found that DFX induced DNA-binding activity to the hypoxia-inducible factor 1 (HIF-1) consensus sequence of the iNOS promoter and activated the iNOS-HRE in murine macrophages. These activities of DFX were associated with a synergistic induction of iNOS mRNA expression and iNOS transcription in IFN-γ-treated ANA-1 macrophages. Functional analysis of the 5′-flanking region of the iNOS gene demonstrated that IFN-γ plus DFX activated the full-length iNOS promoter and that the iNOS-HRE was required for DFX-induced iNOS transcriptional activity. We also investigated the role of iron metabolism in the DFX- or PA-dependent induction of HIF-1 activity and iNOS expression. We demonstrate that addition of iron sulfate completely abolished DFX or PA induction of HIF-1 binding and iNOS-HRE activation and abrogated IFN-γ plus either DFX- or PA-induced iNOS expression. These data establish that DFX is a co-stimulus for the transcriptional activation of the iNOS gene in IFN-γ-treated macrophages, and they provide evidence that the iNOS-HRE is required for the DFX-dependent activation of the iNOS promoter. Furthermore, our results indicate that the iNOS-HRE is a regulatory element of the iNOS promoter responsive to iron chelation. Tissue hypoxia is a condition of decreased oxygen (O2) levels that elicits homeostatic responses aimed at counteracting the negative effects of O2 depletion (1Helfman T. Falanga V. Am. J. Med. Sci. 1993; 306: 37-41Google Scholar). Hypoxia induces the expression of several genes, such as erythropoietin (Epo) 1The abbreviations used are: Epo, erythropoietin; EMSA, electrophoretic mobility shift assay; HRE, hypoxia-responsive enhancer; HIF-1, hypoxia-inducible factor 1; iNOS, inducible nitric oxide synthase; IFN-γ, interferon-γ; LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; DFX, desferrioxamine; PA, picolinic acid; bp, base pair(s); CHX, cycloheximide. 1The abbreviations used are: Epo, erythropoietin; EMSA, electrophoretic mobility shift assay; HRE, hypoxia-responsive enhancer; HIF-1, hypoxia-inducible factor 1; iNOS, inducible nitric oxide synthase; IFN-γ, interferon-γ; LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; DFX, desferrioxamine; PA, picolinic acid; bp, base pair(s); CHX, cycloheximide. (2Jelkmann W. Physiol. Rev. 1992; 72: 449-489Google Scholar), vascular endothelial growth factor (3Goldberg M.A. Schneider T.J. J. Biol. Chem. 1994; 269: 4355-4359Google Scholar), and glycolytic enzymes (4Semenza G.L. Roth P.H. Fang H.-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Google Scholar, 5Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Google Scholar) that favor the adaptation of the organism to the decreased availability of O2. Significant progress has been made in the understanding of the molecular mechanism underlying hypoxia-inducible gene expression. A hypoxia-responsive enhancer (HRE) has been identified in the 3′-flanking region of the Epo gene in a region required for Epo transcriptional activation (6Semenza G.L. Nejfelt M.K. Chi S.M. Antonarakis S.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5680-5684Google Scholar, 7Semenza G.L. Wang G.L. Mol. Cell. Biol. 1994; 12: 5447-5454Google Scholar). This regulatory region, which is highly conserved between human and mouse, also plays a critical role in the hypoxia-induced transcriptional activation of genes encoding glycolytic enzymes (4Semenza G.L. Roth P.H. Fang H.-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Google Scholar, 5Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Google Scholar). The HRE contains the consensus sequence for the binding of a transactivating factor, which is inducible by hypoxia (hypoxia-inducible factor 1, HIF-1) (7Semenza G.L. Wang G.L. Mol. Cell. Biol. 1994; 12: 5447-5454Google Scholar, 8Wang G.L. Semenza G.L. J. Biol. Chem. 1993; 268: 21513-21518Google Scholar, 9Wang G.L. Jiang B. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Google Scholar). Induction of HIF-1 binding and functional activation of the HRE was demonstrated in a variety of mammalian cell lines, regardless of their ability to express Epo, suggesting a general involvement of HIF-1 in the regulation of gene expression by O2 tension (10Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 90: 4304-4308Google Scholar, 11Maxwell P.H. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2423-2427Google Scholar). Nitric oxide (NO), a free radical gas generated by the enzyme nitric oxide synthase, is involved in the antimicrobial and antitumor activities of murine macrophages (12Nathan C.F. Hibbs Jr., J.B. Curr. Opin. Immunol. 1991; 3: 65-70Google Scholar, 13Nathan C. FASEB J. 1992; 6: 3051-3064Google Scholar, 14Moncada S. Higgs A. N. Engl. J. Med. 1993; 329: 2002-2012Google Scholar). Macrophage nitric oxide synthase is the product of a transcriptionally inducible gene (iNOS), whose maximal expression requires stimulation by IFN-γ plus a second signal (15Nathan C. Xie Q. J. Biol. Chem. 1994; 269: 13725-13728Google Scholar, 16Xie Q. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Google Scholar, 17Lorsbach R.B. Murphy W.J. Lowenstein C.J. Snyder S.H. Russell S.W. J. Biol. Chem. 1993; 268: 1908-1913Google Scholar). Three regulatory elements of the 5′-flanking region of the iNOS gene play a functional role in the control of its transcriptional activation (18Xie Q. Whisnant R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Google Scholar, 19Lowenstein C.L. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russel S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 90: 9730-9734Google Scholar). The interferon regulatory factor 1 binding site, located at position −923 to −913, is required for the synergistic activity of IFN-γ, as shown in vivo in interferon regulatory factor 1 knock-out mice (20Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D. Le J. Koh S.I. Kimura T. Green S.J. Mak T.W. Taniguchi T. Vilcek J. Science. 1994; 263: 1612-1615Google Scholar) and in vitro in the RAW 264.7 cell line (21Martin E. Nathan C. Xie Q. J. Exp. Med. 1994; 180: 977-984Google Scholar). The consensus sequence for the binding of NF-κB family members, located at position −85 to −76, mediates iNOS transcriptional activation in response to bacterial lipopolysaccharide (LPS) (22Xie Q. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Google Scholar). We recently reported that the promoter region of the iNOS gene contains a sequence homology to the HRE (referred to hereafter as iNOS-HRE) (23Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Google Scholar). Functional studies in murine macrophages demonstrated that the iNOS-HRE was required for the activation of the iNOS promoter by IFN-γ plus picolinic acid, a catabolite of l-tryptophan, and by IFN-γ plus hypoxia (1% O2). These results provided the first indication that iNOS is a hypoxia-inducible gene. The iNOS-HRE contained a 100% homology to the consensus sequence for the binding of HIF-1 (5′-TACGTGCT-3′) (7Semenza G.L. Wang G.L. Mol. Cell. Biol. 1994; 12: 5447-5454Google Scholar), and the HIF-1 binding site was essential for the induction of iNOS promoter activity by picolinic acid or hypoxia in IFN-γ-treated ANA-1 macrophages (23Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Google Scholar). Furthermore, we demonstrated that a CAT construct containing three copies of the iNOS-HRE was inducible by hypoxia or picolinic acid and that mutation of the HIF-1 binding site abolished hypoxia- or picolinic acid-dependent transcriptional activation (23Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Google Scholar). These data established that a hypoxia-responsive element mediates a novel pathway of iNOS transcriptional activation induced by picolinic acid or hypoxia that is different from that mediated by NF-κB induced by LPS. Thus, at least two distinct pathways of iNOS expression are elicited in response to different signals and multiple regulatory elements control its transcriptional activation. Little is known about the signals that can modulate the inducibility of iNOS by these different pathways. It has been recently shown that iron can down-regulate iNOS transcription induced by IFN-γ plus LPS, whereas desferrioxamine (DFX), an iron chelator, can augment such a response (24Weiss G. Werner-Felmayer G. Werner E.R. Grunewald K. Wachter H. Hentze M.W. J. Exp. Med. 1994; 180: 969-976Google Scholar). These results suggested the existence of a regulatory loop between iron metabolism and iNOS induction by the IFN-γ plus LPS pathway, although analysis of the activation of the iNOS promoter was not performed in these experiments. However, the discovery of the hypoxia-dependent activation of iNOS expression supports the hypothesis of a different mechanism of action of DFX on iNOS expression, independent from the IFN-γ plus LPS pathway. In fact, recent evidence has been provided that DFX induces HIF-1 binding activity in Hep3B cells (25Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Google Scholar). DFX also induces the expression of reporter genes carrying the human Epo enhancer, and mutations in the HIF-1 binding site eliminated DFX-inducible transcriptional activation (25Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Google Scholar). These results suggest that DFX can directly activate the HRE, at least in Hep3B cells. It follows, then, that DFX itself could be a co-stimulus for the activation of iNOS expression in IFN-γ-treated macrophages along the hypoxia pathway. It was important to test this possibility because it would change our perspective on the connections between iron metabolism, iNOS induction, and macrophage activation. Therefore, we studied the functional activation of the iNOS-HRE and iNOS gene by DFX in macrophages. We found that DFX induced binding activity to the HIF-1 consensus sequence of the iNOS promoter and expression of the iNOS-HRE in the murine macrophage cell line ANA-1. DFX, although ineffective by itself, was also a potent co-stimulus of iNOS transcription and enhanced iNOS mRNA expression in IFN-γ-treated cells. Functional analysis of the 5′-flanking region of the iNOS gene in macrophages stimulated with IFN-γ plus DFX demonstrated that the integrity of the HIF-1 binding site was required for the induction of iNOS promoter activity. Finally, addition of iron sulfate completely abrogated the expression of HIF-1 activity and iNOS mRNA in response to either DFX or PA. These results demonstrate that DFX is in itself a co-stimulus for the transcriptional activation of iNOS expression in IFN-γ-treated murine macrophages, and they identify the iNOS-HRE as a regulatory sequence of the iNOS promoter that is activated by iron deprivation. The mouse macrophage cell line ANA-1 was established by infecting fresh bone marrow-derived cells from C57BL/6 mice with the J2 (v-raf/v-myc) recombinant retrovirus (26Blasi E. Mathieson B.J. Varesio L. Cleveland J.L. Borchert P.A. Rapp U.R. Nature. 1985; 318: 667-670Google Scholar, 27Cox G.W. Mathieson B.J. Gandino L. Blasi E. Radzioch D. Varesio L. J. Natl. Cancer Inst. 1989; 81: 1492-1496Google Scholar). ANA-1 macrophages were cultured in Dulbecco's modified Eagle's medium (Whittaker Bioproducts, Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (HyClone Laboratories, Logan, UT), 2 mml-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin (Life Technologies, Inc.) (complete medium). Cells were maintained at 37 °C in a humidified incubator containing 5% CO2 in air. Mouse rIFN-γ (specific activity ≥107 units/mg) was purchased from Life Technologies, Inc. Desferrioxamine, ferrous sulfate, and cycloheximide were purchased from Sigma. Picolinic acid was purchased from Sigma (purity ∼ 99%) and was prepared as described previously (28Melillo G. Cox G.W. Biragyn A. Sheffler L.A. Varesio L. J. Biol. Chem. 1994; 269: 8128-8133Google Scholar). Nuclear extracts were prepared by modification of a standard protocol (29Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar) as described previously (23Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Google Scholar). Briefly, cells were washed twice with cold Dulbecco's phosphate-buffered saline and pelleted by centrifugation at 1,200 rpm for 5 min at 4 °C. The cell pellet was washed with buffer A (10 mm Tris-HCl, pH 7.5, 1.5 mm MgCl2, 10 mm KCl) and incubated on ice for 10 min in buffer A. The cell suspension was homogenized with a glass Dounce homogenizer, and the nuclei were pelleted by centrifugation at 2,000 rpm for 5 min at 4 °C and resuspended in buffer C (0.42 m KCl, 20 mm Tris-HCl, pH 7.5, 20% glycerol, 1.5 mmMgCl2). The suspension was mixed on a rotor at 4 °C for 30 min, and nuclear debris was pelleted by centrifugation for 30 min at 13,000 rpm. The supernatant was dialyzed against two changes of buffer D (25 mm Tris-HCl, pH 7.5, 20% glycerol, 0.2 mm EDTA, 0.1 m KCl) and clarified by centrifugation at 15,000 rpm for 10 min at 4 °C. Aliquots were frozen and stored at − 70 °C for subsequent analysis of DNA binding proteins. Probes were generated using Klenow fragment of DNA polymerase and [α-32P]dCTP. The following oligonucleotide probes were used: AB.2 (5′-GTGACTACGTGCTGCCTAG-3′) encompassing the consensus sequence for the binding of HIF-1, shown in bold; AB.1 (5′-TGAGTCCCAGTTTTGAAGTG-3′) from an unrelated sequence of the iNOS promoter; Mu-AB.2 (5′-GTGACTAAAAGCTGCCTAG-3′), with the three mutated bases underlined; Epo (5′-GCCCTACGTGCTGCCTCA-3′) from the 3′-flanking region of the human Epo gene (7Semenza G.L. Wang G.L. Mol. Cell. Biol. 1994; 12: 5447-5454Google Scholar). Binding reactions were performed in buffer containing 25 mm Tris-HCl, pH 7.5, 20% glycerol, 0.2 mm EDTA, 0.1 m KCl, with 5 μg of nuclear extract, and 0.4 μg of denatured calf thymus DNA on ice. After incubation for 10 min, probe (1 × 104 cpm) was added, and the incubation was continued for an additional 20 min. Samples were loaded onto 5% nondenaturing polyacrylamide gel, and electrophoresis was performed at 180 V in 0.3 × TBE at 4 °C. The 1,749-bp fragment of the 5′-flanking region of the murine iNOS gene inserted upstream of the promoterless chloramphenicol acetyltransferase (CAT) gene in pCAT-Basic (Promega Corp.) (i.e. p1-iNOS-CAT) (18Xie Q. Whisnant R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Google Scholar) was kindly provided by Q. Xie and C. Nathan (Cornell University Medical College, New York, NY). Deletion mutants of the iNOS promoter were obtained as described previously (23Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Google Scholar). For functional studies in the context of a heterologous promoter, three tandem copies of a 21-base pair (bp) double-strand oligonucleotide encompassing the iNOS-HRE (−231 to −211) were subcloned in the HindIII/BamHI sites of pBLCAT2 plasmid, containing the CAT reporter gene under the control of a herpes simplex virus thymidine kinase promoter fragment spanning from −105 to +51,. The constructs were sequenced using Sequenase version 2.0 (United States Biochemical Corp.). ANA-1 macrophages were transfected by a modification of the DEAE-dextran method (30Golub E.I. Kim H. Volsky D.J. Nucleic Acids Res. 1989; 17: 4902Google Scholar), as described (23Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Google Scholar). Briefly, 10 μg of plasmid DNA were added to 107 cells in 1 ml of Dulbecco's modified Eagle's medium without serum and containing 250 μg/ml DEAE-dextran and 50 mm Tris-HCl, pH 7.5. The cells were incubated at 37 °C for 1 h followed by a 2-min shock with 10% Me2SO at room temperature. The cells were washed, plated in 6-well plates at 1 × 106/ml in 2.5 ml of complete medium, and incubated at 37 °C in 5% CO2. Twenty-four hours later, the cells were incubated with the appropriate stimulus for an additional 18 h. The cells were then washed, resuspended in 0.25m Tris-HCl, pH 7.5, and lysed by three cycles of rapid freezing and thawing. The lysates were centrifuged (11,000 ×g for 10 min), and the supernatants were assayed for CAT activity by TLC (31Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Google Scholar). Protein content was determined as described by Bradford (32Bradford M.M. Annal. Biochem. 1976; 72: 248-254Google Scholar), using the Bio-Rad Protein Assay. To control for differences in the uptake of transfected DNA, cells were cotransfected with 5 μg of pGL2 plasmid (pGL2 control, Promega), which contains the luciferase reporter gene under control of SV40 promoter and enhancer. Cell lysates were then assayed for luciferase activity. Total cellular RNA was harvested and processed as described previously (28Melillo G. Cox G.W. Biragyn A. Sheffler L.A. Varesio L. J. Biol. Chem. 1994; 269: 8128-8133Google Scholar). Briefly, cells were solubilized with guanidine isothiocyanate, and the total cellular RNA was purified by centrifugation through a cushion of cesium chloride. Twenty μg of RNA were size-fractionated in a 1.2% agarose gel, blotted onto a Nytran membrane (Schleicher & Schuell), and incubated overnight at 42 °C in Hybrisol I hybridization solution (Oncor, Gaithersburg, MD). The cDNA probes that were specific for mouse macrophage nitric oxide synthase (33Lyons C.R. Orloff G.J. Cunningham J.M. J. Biol. Chem. 1992; 267: 6370-6374Google Scholar) or for human glyceraldehyde-3-phosphate dehydrogenase (CLONTECHLaboratories Inc.) were radiolabeled with [32P]dCTP (Amersham Corp.) by using an RTS RadPrime DNA labeling system (Life Technologies, Inc.) according to the manufacturer's procedure. The blot was hybridized individually with the radiolabeled probes (1 to 2 × 106 cpm/ml) during an overnight incubation and washed three times for 10 min at room temperature with 2 × SSC, 0.1% SDS and two times for 20 min at 60 °C with 0.2 × SSC, 0.1% SDS. The blot was autoradiographed at −70 °C on XAR-5 film (Eastman Kodak) with the use of Lightning Plus intensifying screens (DuPont NEN). ANA-1 cells were treated as indicated for 12 h, and nuclei were isolated as described (28Melillo G. Cox G.W. Biragyn A. Sheffler L.A. Varesio L. J. Biol. Chem. 1994; 269: 8128-8133Google Scholar). Thawed nuclei were mixed with 150 μl of 2 × transcription mix (1 × = 100 mm sucrose, 10% glycerol, 10 mm Tris-HCl, pH 8.0, 2.5 mm MgCl2, 2.5 mm dithiothreitol, 0.5 mm each of ATP, CTP, and GTP) and incubated with 100 μCi of [32P]UTP (800 Ci/mmol; Amersham Corp.) at 30 °C for 30 min. Twenty μl of 100 mm CaCl2 and 20 units of RNase-free DNase I were added and incubated 10 min at 30 °C with gentle mixing every 2 min. The nuclei were then lysed with 1 ml of Trizol (Life Technologies, Inc.), and the RNA was isolated according to the manufacturer's procedure. Approximately 2 × 106 cpm of RNA were used in hybridization for 48 h with 2.5 μg of each slot-blotted denatured plasmid (pGEM-3Z vector alone, vector containing the 3.9-kilobase cDNA insert of macrophage iNOS (33Lyons C.R. Orloff G.J. Cunningham J.M. J. Biol. Chem. 1992; 267: 6370-6374Google Scholar), and vector containing a chicken β-actin cDNA insert). Filters were then washed and autoradiographed as for the Northern blot analysis. To address whether DFX induces DNA-binding activity to the iNOS-HRE, EMSA was performed on nuclear extract prepared from ANA-1 macrophages treated with medium or DFX, using as radiolabeled probe a 19-base pair (bp) oligonucleotide (AB.2) containing the HIF-1 binding site of the iNOS promoter. As shown in Fig. 1, ANA-1 macrophages expressed a constitutive binding activity. Treatment with DFX caused the appearance of an inducible complex that was specifically inhibited by excess unlabeled probe (AB.2) but not by an unrelated probe (AB.1). In addition, the DFX-inducible binding complex was not competed for by a probe mutated in three bases within the putative HIF-1 binding site (Mu.AB2). Both the constitutive and the DFX-inducible binding activities were completely inhibited by a probe encompassing the canonical HIF-1 binding site (Epo) of the Epo enhancer, which differs from the AB.2 probe in five flanking bases. These results demonstrate that DFX induces in macrophages a specific DNA-binding activity to the HIF-1 binding site of the iNOS promoter. To establish whether DFX was a stimulus for the activation of the iNOS-HRE in macrophages, ANA-1 cells were transiently transfected with two CAT constructs containing three tandem copies of the iNOS-HRE, either wild type (pBL-WT.iNOS) or mutated at the HIF-1 binding site (pBL-Mu.iNOS), 5′ of the herpes simplex virus thymidine kinase minimal promoter. The pBL-WT.iNOS construct was constitutively expressed in macrophages (Fig.2). Treatment with DFX caused a 10-fold increase in the expression of CAT activity, relative to untreated cells. In contrast, plasmid pBL-Mu.iNOS was not inducible by DFX. These results, consistent with the data obtained in EMSA, demonstrate that the iNOS-HRE is sufficient to confer inducibility by DFX in murine macrophages and that mutation of the HIF-1 binding site abolishes DFX inducible transcriptional activation. To determine whether DFX can be a stimulus for the expression of iNOS mRNA, ANA-1 macrophages were incubated with medium, IFN-γ (100 units/ml), and DFX (400 μm) alone or in combination, and total RNA was harvested after 18 h. No expression of iNOS mRNA was observed in cells treated with medium or DFX alone, and very low levels of iNOS mRNA were detected in macrophages stimulated with IFN-γ alone (Fig.3 A). In contrast, addition of DFX caused a major increase of IFN-γ-induced iNOS mRNA expression that was detectable after 6 h of treatment (see Fig. 5) and increased up to 18 h (Fig. 3 A). To determine the dose dependence of DFX effects, ANA-1 macrophages were incubated with increasing concentrations of DFX in the presence or absence of IFN-γ for 18 h. DFX alone, at doses ranging from 50 μm (data not shown) to 800 μm, failed to induce iNOS mRNA. The combination of IFN-γ and DFX increased the levels of iNOS mRNA at a dose of 100 μm DFX (3–4-fold above IFN-γ alone) (Fig. 3 B) and reached its maximum (15–20-fold above IFN-γ alone) at a dose of 800 μm DFX. These data demonstrate that DFX acts synergistically with IFN-γ in a dose-dependent fashion for the induction of iNOS mRNA expression in murine macrophages.Figure 5Protein synthesis is required for IFN-γ plus DFX induction of iNOS mRNA expression. ANA-1 macrophages (1 × 106 cells/ml) were incubated with medium, IFN-γ (100 units/ml), DFX (400 μm) alone or in combination with IFN-γ, in the absence or presence of 7.5 μg/ml CHX. Total cellular RNA was harvested 6 h later and examined for iNOS mRNA expression as described. Data are from one of two similar experiments.View Large Image Figure ViewerDownload (PPT) Nuclear run-on experiments were performed to demonstrate that the increase in iNOS mRNA in response to IFN-γ plus DFX was due to transcriptional activation of the gene. ANA-1 macrophages were treated with medium, IFN-γ, DFX alone or in combination with IFN-γ. The iNOS gene was not constitutively transcribed in ANA-1 macrophages, and treatment with IFN-γ or DFX alone failed to induce iNOS transcription (Fig. 4). In contrast, ANA-1 cells treated with IFN-γ plus DFX showed a significant increase in the rate of transcription of the iNOS gene. These data establish that DFX synergistically induces iNOS transcriptional activation in IFN-γ-treated macrophages. Transcriptional activation of the iNOS gene is blocked in the presence of protein synthesis inhibitors (17Lorsbach R.B. Murphy W.J. Lowenstein C.J. Snyder S.H. Russell S.W. J. Biol. Chem. 1993; 268: 1908-1913Google Scholar, 28Melillo G. Cox G.W. Biragyn A. Sheffler L.A. Varesio L. J. Biol. Chem. 1994; 269: 8128-8133Google Scholar). To establish whether the induction of iNOS mRNA expression by DFX was dependent on de novo protein synthesis, ANA-1 macrophages were incubated in medium, IFN-γ, DFX alone or in combination with IFN-γ, in the presence or absence of CHX (7.5 μg/ml) for 6 h. As shown in Fig.5, the addition of CHX almost completely abrogated the synergistic induction of iNOS mRNA expression by IFN-γ plus DFX, indicating that de novo protein synthesis was required for iNOS mRNA induction by IFN-γ plus DFX. To identify the region(s) of the iNOS promoter responsible for the DFX-dependent transcriptional activation of the iNOS gene and, in particular, to investigate the functional role of the iNOS-HRE, ANA-1 macrophages were transiently transfected with plasmids containing the full-length or deletion mutants of the 5′-flanking region of the iNOS gene linked to the CAT reporter gene. Cells were stimulated with medium, IFN-γ (100 units/ml), or DFX (400 μm) alone or in combination. ANA-1 cells transfected with plasmid p1, containing the full-length iNOS promoter, did not express significant levels of CAT activity either constitutively or after treatment with IFN-γ alone (Fig.6). Low but consistent induction of CAT expression was induced by treatment with DFX alone. However, a strong synergistic induction of CAT activity was observed following stimulation with IFN-γ and DFX. Deletion of the upstream region of the iNOS promoter (−1588 to −721, plasmid p3), containing the IFN-γ-responsive region, completely abrogated the synergistic interaction between DFX and IFN-γ in the induction of iNOS promoter activity (data not shown). These data indicated that maximal activation of the iNOS promoter required the presence of the IFN-γ-responsive region and was induced by the combination of IFN-γ plus DFX. Transfection of plasmid p162, deleted of the region from −721 to −410, did not affect the induction of CAT activity by IFN-γ plus DFX. In contrast, plasmids p167 (deleted from −721 to −201) and p201 (deleted from −328 to −201) were no longer inducible following stimulation of ANA-1 macrophages with the combination of IFN-γ and DFX (Fig. 6). To establish whether DFX-dependent activation of the iNOS promoter required the presence of a functional iNOS-HRE, ANA-1 macrophages were transfected with two iNOS promoter-CAT reporter gene constructs (23Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Google Scholar), containing either a deletion of the iNOS-HRE (−227 to −209, plasmid p220) or a mutation of two bases required for HIF-1 binding to DNA (−223 −222, plasmid p209) (7Semenza G.L. Wang G.L. Mol. Cell. Biol. 1994; 12: 5447-5454Google Scholar). A reduction greater than 80% relative to the wild type iNOS promoter was observed in the induction of CAT expression with either plasmid. These data demonstrate that the synergistic interaction between IFN-γ and DFX in the transcriptional activation of the iNOS promoter is mediated, at least in part, by the iNOS-HRE. It has been previously reported that induction of HIF-1 binding activity by DFX in the Hep3B cell line was due to chelation of iron (25Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Google Scholar). Therefore, we investigated the role of iron chelation in the DFX-dep" @default.
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