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• W2100286113 abstract "The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of −3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-κB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-κB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-κB is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5′-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to −5.8 and −7.2 kb revealed 2–3-fold and 4–5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from −3.8 to −7.2 kb identified five putative NF-κB cis-regulatory transcription factor binding sites upstream of −4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-κB motif at −5.8 kb is required for cytokine-induced promoter activity, while the sites at −5.2, −5.5, and −6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-α or interleukin-1β, but not interferon-γ, exhibited inducible DNA binding activity for NF-κB. These data indicate that NF-κB activation is required for cytokine induction of the human iNOS gene and identifies four NF-κB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-α and interleukin-1β. The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of −3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-κB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-κB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-κB is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5′-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to −5.8 and −7.2 kb revealed 2–3-fold and 4–5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from −3.8 to −7.2 kb identified five putative NF-κB cis-regulatory transcription factor binding sites upstream of −4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-κB motif at −5.8 kb is required for cytokine-induced promoter activity, while the sites at −5.2, −5.5, and −6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-α or interleukin-1β, but not interferon-γ, exhibited inducible DNA binding activity for NF-κB. These data indicate that NF-κB activation is required for cytokine induction of the human iNOS gene and identifies four NF-κB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-α and interleukin-1β. The expression of the inducible nitric oxide synthase (iNOS) 1The abbreviations used are: iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; IFNγ, interferon-γ; IRF, interferon regulatory factor; ANOVA, analysis of variance; IL, interleukin; TNFα, tumor necrosis factor-α; CM, cytokine mixture; EMSA, electrophoretic mobility shift assay; PDTC, pyrrolidine dithiocarbamate; DDTC, diethyldithiocarbamate. gene is an important part of the immune response to infection (1Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4157) Google Scholar, 2Nussler A.K. Billiar T.R. J. Leukocyte Biol. 1993; 54: 171-178Crossref PubMed Scopus (830) Google Scholar). Overexpression of the iNOS gene is seen in many acute and chronic human diseases including septic shock, hemorrhagic shock, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, and its associated cancer diathesis (2Nussler A.K. Billiar T.R. J. Leukocyte Biol. 1993; 54: 171-178Crossref PubMed Scopus (830) Google Scholar, 3Heirholzer C. Harbrecht B. Menezes J. Kane J. MacMicking J. Nathan C. Peitzman A. Billiar T.R. Tweardy D.J. J. Exp. Med. 1998; 187: 917-928Crossref PubMed Scopus (425) Google Scholar, 4McCartney-Francis N. Allen J.B. Mizel D.E. Albina J.E. Xie Q. Nathan C.F. Wahl S.M. J. Exp. Med. 1993; 178: 749-754Crossref PubMed Scopus (598) Google Scholar, 5Koprowski H. Zheng Y.M. Heber-Katz E. Fraser N. Rorke L. Fu Z.F. Hanlon C. Dietzhold B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3024-3027Crossref PubMed Scopus (472) Google Scholar, 6Middleton S.J. Shorthouse M. Hunter J.O. Lancet. 1993; 341: 465-466Abstract PubMed Scopus (340) Google Scholar). Although it is constitutively expressed in some epithelial cell types (7Asano K Chee C.B. Gaston B. Lilly C.M. Gerard C. Drazen J.M. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10089-10093Crossref PubMed Scopus (533) Google Scholar, 8Mannick J.B. Asano K. Izumi K. Kieff E. Stamler J.S. Cell. 1994; 74: 1137-1146Abstract Full Text PDF Scopus (459) Google Scholar), iNOS expression in most cell types requires exposure to inflammatory stimuli such as cytokines and/or lipopolysaccharide (LPS) (9Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 10Nussler A. Di Silvio M. Billiar T.R. Hoffman R.A. Geller D.A. Selby R. Madriaga J. Simmons R.L. J. Exp. Med. 1992; 176: 261-264Crossref PubMed Scopus (386) Google Scholar, 11Geller D.A. Nussler A.K. Di Silvio M. Lowenstein C.L. Shapiro R.A. Wang S.C. Simmons R.L. Billiar T.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 522-526Crossref PubMed Scopus (638) Google Scholar, 12Geller D.A. Lowenstein C.J. Shapiro R.A. Nussler A.K. Di Silvio M. Wang S.C. Nakayama D.K. Simmons R.L. Snyder S.H. Billiar T.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3491-3495Crossref PubMed Scopus (814) Google Scholar, 13Geller D.A. de Vera M.E. Russell D.A. Shapiro R.A. Nussler A.K. Simmons R.L. Billiar T.R. J. Immunol. 1995; 155: 4890-4898PubMed Google Scholar). We and others have shown that iNOS up-regulation in response to LPS and cytokines is transcriptionally regulated (14Xie Q. Whisnant R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Crossref PubMed Scopus (1030) Google Scholar, 15Lowenstein 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, 16de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (359) Google Scholar). The nitric oxide (NO) generated by iNOS from its substratel-arginine has beneficial effects (e.g. antimicrobial, anti-atherogenic, anti-apoptotic) (8Mannick J.B. Asano K. Izumi K. Kieff E. Stamler J.S. Cell. 1994; 74: 1137-1146Abstract Full Text PDF Scopus (459) Google Scholar, 17Granger D.L. Hibbs J.B. Perfect J.R. Durack D.T. J. Clin. Invest. 1988; 85: 264-273Crossref Scopus (288) Google Scholar, 18Hayashi T. Fukuto J.M. Ignarro L.J. Chaudhuri G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11259-11263Crossref PubMed Scopus (452) Google Scholar, 19Saavedra J.E. Billiar T.R. Williams D.L. Kim Y.L. Watkins S.C. Keefer L.K. J. Med. Chem. 1997; 40: 1947-1954Crossref PubMed Scopus (254) Google Scholar), whereas overproduction of induced NO can have detrimental consequences (e.g. direct cellular injury, pro-inflammatory) (20Stuehr D.J. Nathan C. J. Exp. Med. 1989; 169: 1543-1555Crossref PubMed Scopus (1599) Google Scholar, 21Harbrecht B.G. Wu B. Watkins S.C. Marshall Jr., H.P. Peitzman A.B. Billiar T.R. Shock. 1995; 4: 332-337Crossref PubMed Scopus (100) Google Scholar). Thus, elucidating the mechanisms that govern iNOS gene expression should provide insight into the molecular mechanisms of gene regulation in several pathophysiologic states and may even lead to novel therapeutic strategies to modulate iNOS expression. Previously, we reported that transcriptional activation of the human iNOS gene required the presence of cytokine-responsive elements upstream of −3.8 kilobases (kb) in the 5′-flanking region of the human iNOS gene (16de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (359) Google Scholar). These findings contrast markedly with the murine iNOS promoter, where two regions within 1 kb of the transcription start site have been identified as essential for the induction of iNOS in RAW 264.7 murine macrophages by LPS and IFNγ (14Xie Q. Whisnant R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Crossref PubMed Scopus (1030) Google Scholar, 15Lowenstein 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, 22Goldring C.E.P. Reveneau S. Algarte M. Leannin J.-F. Nucleic Acids Res. 1996; 24: 1682-1687Crossref PubMed Scopus (87) Google Scholar, 23Murphy W.J. Muroi M. Zhang C.X. Suzuki T. Russell S.W. J. Endotoxin Res. 1996; 3: 381-393Crossref Scopus (5) Google Scholar). Deletional analysis of the murine gene identified an NF-κB element at positions −85 to −76 base pairs (bp) (24Xie Q.-W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar) and an interferon regulatory factor-1 (IRF-1) site at positions −923 to −913 kb (25Martin E. Nathan C. Xie Q. J. Exp. Med. 1994; 180: 977-984Crossref PubMed Scopus (456) Google Scholar, 26Kamijo 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: 1612-1615Crossref PubMed Scopus (787) Google Scholar) that mediate iNOS induction by LPS and IFNγ, respectively. The involvement of NF-κB in the induction of the murine iNOS gene is consistent with the well described role of this transcription factor in regulating inflammation-associated genes. NF-κB has been shown to be required for iNOS induction in both rodent macrophages (24Xie Q.-W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar, 27Griscavage J.M. Wilk S. Ignarro L.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 3308-3312Crossref Scopus (245) Google Scholar) and vascular smooth muscle cells (28Spink J. Cohen J. Evans T.J. J. Biol. Chem. 1995; 270: 29541-29547Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). NF-κB has been implicated in the induction of the human iNOS gene as well, but its role has not been clearly defined (29Spitsin S.V. Farber J.L. Bertovich M. Moehren G. Koprowski H. Michaels F.H. Mol. Med. 1997; 3: 315-326Crossref PubMed Scopus (4) Google Scholar, 30Nunokawa Y. Oikawa S. Tanaka S. Biochem. Biophys. Res. Commun. 1996; 223: 347-352Crossref PubMed Scopus (82) Google Scholar, 31Kleinert H. Euchenhofer C. Ihrig-Biedert I. Forstermann U. Mol. Pharmacol. 1996; 49: 15-21PubMed Google Scholar). In the A549 and DLD-1 human epithelial cell lines, inhibitors of NF-κB activation minimally decreased iNOS expression (29Spitsin S.V. Farber J.L. Bertovich M. Moehren G. Koprowski H. Michaels F.H. Mol. Med. 1997; 3: 315-326Crossref PubMed Scopus (4) Google Scholar, 30Nunokawa Y. Oikawa S. Tanaka S. Biochem. Biophys. Res. Commun. 1996; 223: 347-352Crossref PubMed Scopus (82) Google Scholar). In contradistinction, others have shown that the same inhibitors do not inhibit cytokine-stimulated iNOS expression in DLD-1 cells. 2H. Kleinert and U. Forstermann, personal communication. Failure to identify a homologous functional NF-κB site in the human iNOS promoter raises the possibility that NF-κB may not be involved in the expression of the human gene by cytokines. Therefore, studies were performed to determine if NF-κB plays a role in the transcriptional activation of the human iNOS gene in human liver (AKN-1) and lung (A549) cell lines. In this study, we not only demonstrate that NF-κB plays a crucial role in human iNOS gene regulation, we also identify NF-κB response elements in the human iNOS promoter. Unlike the murine iNOS promoter, however, the first 1.0 kb of the human iNOS gene 5′-flanking region is not sufficient for iNOS induction. Instead, inducible NF-κB elements upstream of −4.7 kb are required for cytokine activation of the promoter. Specifically, we have identified a cytokine-responsive enhancer region from −5.2 to −6.1 kb in the human iNOS gene that contains four cis-acting NF-κB elements. Furthermore, gel shift assays and mutational analysis of these regulatory elements indicate that they play a functional role in the trans-activation of the human iNOS gene by NF-κB in response to cytokines. Human recombinant TNFα and IFNγ were obtained from R&D Systems, and IL-1β was provided by Craig Reynolds of the National Cancer Institute. LipofectAMINE was purchased from Life Technologies, Inc. Gel shift antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other reagents were obtained from Sigma. The AKN-1 human liver cell line was grown in modified HCD medium supplemented with 5% bovine calf serum as described previously (16de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (359) Google Scholar). The A549 cells (American Type Culture Collection, Rockville, MD) were cultured in F-12k medium supplemented with 10% fetal bovine serum. AKN-1 and A549 cells were plated onto 100-mm Petri dishes (Corning Co., Corning, NY) and stimulated with a cytokine mixture (CM) of TNFα (1,000 units/ml) + IL-1β (100 units/ml) + IFNγ (250 units/ml) in the presence or absence of the NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC, 20 or 100 μm) or diethyldithiocarbamate (DDTC, 10 mm) at the indicated time points. NO production was quantitated by measuring nitrite plus nitrate (NO2−and NO3−) in the culture supernatant by an automated procedure based on the Griess assay (32Green L. Wagner D.A. Glogowski J. Skipper P.I. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10824) Google Scholar). RNA extraction and Northern blot analysis were performed as described (12Geller D.A. Lowenstein C.J. Shapiro R.A. Nussler A.K. Di Silvio M. Wang S.C. Nakayama D.K. Simmons R.L. Snyder S.H. Billiar T.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3491-3495Crossref PubMed Scopus (814) Google Scholar). Northern blot hybridizations were carried out using a 2.3-kb Bam HI fragment of human iNOS cDNA (10Nussler A. Di Silvio M. Billiar T.R. Hoffman R.A. Geller D.A. Selby R. Madriaga J. Simmons R.L. J. Exp. Med. 1992; 176: 261-264Crossref PubMed Scopus (386) Google Scholar). Using a series of both sense and antisense PCR primers, an Eag I-Bam HI fragment of the human iNOS promoter extending from +33 to −7242 kb (GenBank™ accession numberAF049872) was sequenced using the Sanger dideoxynucleotide sequencing method (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 13.42-13.74Google Scholar). Sequencing was conducted by Lark Technologies, Inc. (Houston, TX) and the University of Pittsburgh DNA Sequencing Facility. Putative cis-regulatory elements were detected by comparison with the TRANSFAC data base and the MatInspector Release 2.1 data base using a threshold factor of 85.0. AKN-1 and A549 cells were treated with CM at the indicated times. In some experiments, PDTC (100 μm) or DDTC (10 mm) was added 1 h prior to the addition of CM. Nuclear extracts were prepared as described by Ohlsson and Edlund (34Ohlsson H. Edlund T. Cell. 1986; 45: 35-44Abstract Full Text PDF PubMed Scopus (170) Google Scholar) with some modifications. Briefly, the cells were washed, scraped into phosphate-buffered solution, and centrifuged at 3,000 rpm for 10 min in a Sorvall SS-34 rotor. The pelleted cells were resuspended in Buffer A (10 mm HEPES (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 25 μg/ml chymostatin, 25 μg/ml leupeptin, 0.2 mmphenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, and 0.5% Nonidet P-40) at 5 times the packed cell volume and disrupted by 10 strokes in a Dounce homogenizer. Nuclei were recovered by microcentrifugation at 5,000 rpm for 15 min, resuspended in the same volume of Buffer B (Buffer A without Nonidet P-40) and re-centrifuged at 5,000 rpm. Nuclear proteins were extracted at 4 °C by gently mixing the nuclei in 150 μl of Buffer C (20 mm HEPES (pH 7.9), 10% glycerol, 1.5 mm MgCl2, 10 mm KCl, 0.2 mm EDTA, 0.2 mmphenylmethylsulfonyl fluoride, and 0.5 mm dithiothreitol) and adding 50 μl of Buffer D (Buffer C but with 1.6 mKCl) in a dropwise fashion. Supernatants were collected after 1 h by microcentrifugation at 13,000 rpm for 30 min. Protein concentration was measured using bicinchoninic acid protein assay reagent (Pierce). The sequences of the oligonucleotides used in the gel shift assays are outlined in TableI. Complementary strands were synthesized by the DNA Synthesis Facility of the University of Pittsburgh and annealed in 50 mm Tris-HCl (pH 7.6) and 0.1 mNaCl in one PCR cycle of 85 °C × 2 min, 65 °C × 15 min, 37 °C × 15 min, 23 °C × 15 min, and 4 °C × 15 min. Probes were end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (Boehringer Mannheim) and purified by native polyacrylamide gel electrophoresis on a 15% polyacrylamide gel in 1x TBE. Five μg of nuclear extracts were incubated with ∼100,000 cpm of 32P-labeled oligonucleotide (∼0.5 ng) for 45 min at room temperature in a buffer containing 2 μg poly (dI-dC) (Boehringer Mannheim), 4.2 mm HEPES (pH 7.4), 4.2 mm KCl, 0.02 mm EDTA, 1 mmMgCl2, 2.5% glycerol, 2% Ficoll, and 21 mmdithiothreitol (final volume of 30 μl). In some experiments, nuclear extracts were incubated with excess unlabeled oligonucleotides or antibodies against the different subunits of NF-κB and AP-1 for 15 min before the addition of the labeled probe. DNA-protein complexes were resolved on a 4% nondenaturing polyacrylamide gel in 0.4× TBE running buffer (450 mm Tris borate and 1 μmEDTA, pH 8.0). After electrophoresis, gels were dried and subjected to autoradiography.Table IOligonucleotides used in electromobility shift assaysNF-κB consensus5′-AGTTGAGGGGACTTTCCCAGGC-3′NF-κB mutant consensus5′-AGTTGAGGTAACTTTCCCAGGC-3′NF-κB-5.8 wild-type5′-AGAGGGCTTTCCCAGAACCA-3′NF-κB-5.8 mutant5′-AGAGGGCTCGCCCAGAACCA-3′ Open table in a new tab Construction of a 1.3-kb (piNOS(1.3)Luc) and a 7.2-kb (piNOS(7.2)Luc) iNOS promoter-luciferase construct has been described previously (16de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (359) Google Scholar). The piNOS(NA)Luc construct was generated by using the restriction enzymes Nco I and Afl II to cut the DNA from −2.1 to −4.7 from the 7.2-kb promoter construct. The plasmid DNA was ligated, and the promoter construct was then sequenced to confirm the deletion. To generate the mutated NF-κB construct with mutations at −0.11, −5.2, −5.5, −5.8, −6.1, and −6.5 kb, site-directed mutagenesis was performed on individual NF-κB elements in the context of the full-length reporter construct piNOS(7.2)Luc, using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). PCR mutagenesis (35Michael S.F. BioTechniques. 1994; 16: 410PubMed Google Scholar) was also used to generate a piNOS(mut7.2κB)Luc in order to further confirm the mutation, and similar results were obtained regardless of the method used to create piN0S(mut7.2κB)Luc. To reduce errors in the reactions, PCR was performed using the Expand Long Template PCR system (Boehringer Mannheim). The piNOS(mut7.2κB)Luc constructs were verified by restriction enzyme analysis and nucleotide sequencing of the initial ∼800 bp of the 5′-flanking region of iNOS promoter, whereas restriction digests and sequencing were performed to confirm the orientation and validity of the upstream NF-κB constructs. The promoter plasmid wild-type and mutated sequences in the NF-κB elements of interest are listed in Figs. 3 and 6.Figure 6Site-directed mutational analysis of the upstream NF-κB cis-regulatory elements. The site-directed mutational constructs utilized in the study are shown along with the wild-type sequence. A549 cells and AKN-1 cells were stimulated with CM following transient transfections of the constructs. Luciferase activity is expressed as light units/μg of protein in cell lysates. Values are expressed as mean ± S.E. (n = number of experiments).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Transient transfections and activity assays were carried out as described previously (16de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (359) Google Scholar). To control for transfection efficiency between groups, iNOS promoter constructs were co-transfected with 0.5 μg of pIEP-lacZ, a plasmid encoding a CMV promoter-driven β-galactosidase gene (gift of Hideaki Tahara, University of Pittsburgh), and results were normalized to total protein content and to β-galactosidase activity. Significance of differences was determined by ANOVA using the Statview statistics program (Abacus Concepts, Inc., Berkeley, CA). Statistical significance was established at a p value < 0.01. To determine whether iNOS induction in human lung and liver cells is NF-κB dependent, A549 cells and AKN-1 cells were treated with the CM of TNFα, IL-1β, and IFNγ in the presence or absence of the established dithiocarbamate NF-κB inhibitors PDTC or DDTC (36Schreck R. Meier B. Mannel D.N. Droge W. Bauerle P. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar). High levels of iNOS mRNA were elicited from the CM-stimulated cells, whereas the addition of PDTC and DDTC inhibited iNOS mRNA expression in a concentration-dependent fashion (Fig.1). As expected, nitrite and nitrate release was inhibited in a similar manner. These data demonstrate that NF-κB is necessary for cytokine activation of iNOS gene expression in these human cell types. To document that the transcription factor NF-κB is translocating into the nucleus of A549 and AKN-1 cells in the presence of CM, gel shifts were performed on nuclear extracts of CM-stimulated AKN-1 and A549 cells using a consensus oligonucleotide for NF-κB (37Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1258) Google Scholar). In both AKN-1 and A549 cells, basal levels of NF-κB DNA binding were seen in control cells (Fig. 2 A ). The addition of single cytokines, TNFα or IL-1β alone, induced a strong gel shift complex for NF-κB, whereas IFNγ had no effect. The CM of all three agents also induced DNA binding activity for NF-κB, and the appearance of this complex was markedly suppressed by the addition of PDTC, further implicating NF-κB in iNOS expression. This inducible band was seen as early as 30 min after cytokine stimulation, peaked at the 1-h time point, and began to diminish at 2 h (data not shown). Specificity of the DNA-protein interaction for NF-κB was demonstrated by competition with 100-fold excess of unlabeled oligonucleotide (Fig.2 A ). Competition studies with excess unlabeled mutant oligonucleotide did not abolish the NF-κB DNA complex, further demonstrating specificity for NF-κB. Supershift studies with specific antibodies demonstrated the presence of both p50 and p65 subunits of NF-κB in the complex (Fig. 2 B ). Antibody against the transcription factor AP-1 failed to induce a supershift. An NF-κB element at −85 bp upstream in the murine iNOS promoter has been shown to confer LPS responsiveness in the murine iNOS gene (24Xie Q.-W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar). Analysis of the 5′-flanking region of the human iNOS gene revealed a putative NF-κB element located at −115 to −106 bp that differs by only one nucleotide from the functional murine NF-κB element at −85 bp (38Chartrain N.A. Geller D.A. Koty P.P. Sitrin N.F. Nussler A.K. Hoffman E.P. Billiar T.R. Hutchinson N.I. Mudgett J.S. J. Biol. Chem. 1994; 269: 6765-6772Abstract Full Text PDF PubMed Google Scholar). Because the first 400 bp of the human and murine iNOS promoter have 66% homology (38Chartrain N.A. Geller D.A. Koty P.P. Sitrin N.F. Nussler A.K. Hoffman E.P. Billiar T.R. Hutchinson N.I. Mudgett J.S. J. Biol. Chem. 1994; 269: 6765-6772Abstract Full Text PDF PubMed Google Scholar), and because this proximal corresponding NF-κB element in the human iNOS promoter is relatively conserved, we sought to evaluate the functional role of this putative NF-κB element. Transient transfections in AKN-1 cells were performed with a wild-type iNOS 7.2-kb promoter construct (piNOS(7.2κB)Luc) and a mutated NF-κB construct (piNOS(mut7.2κB)Luc) generated by site-directed mutagenesis bearing a 2-bp mutation of the corresponding proximal NF-κB element (Fig. 3 A ). CM treatment of cells transfected with the wild-type construct piNOS(7.2)Luc resulted in a 6-fold increase in luciferase activity. When the 7.2-kb construct containing the mutated proximal NF-κB (−115 to −106 bp) (piNOS(mut7.2κB)Luc) was transfected, there was no significant decrease in either basal (data not shown) or stimulated reporter gene activity (Fig. 3 B ). In addition, the inducible activity of both the 7.2-kb wild-type and mutant constructs was inhibited by PDTC (Fig. 3 B ) and DDTC (data not shown). Interestingly, the addition of NF-κB inhibitors did not change basal, unstimulated luciferase activity, suggesting that NF-κB does not play a dominant role in mediating basal transcription of the human iNOS gene in this cell type. Furthermore, deletion of the region from −36 to −133 bp maintained a 3-fold induction in iNOS promoter activity, which also exhibited PDTC inhibition (data not shown). Thus, either mutation or deletion of the proximal NF-κB element failed to abrogate cytokine-induced iNOS promoter activity. These results indicate that the promoter-proximal NF-κB site is not required for maximal cytokine induction, suggesting that the functional NF-κB elements are further upstream in the human iNOS promoter. We have previously reported that the activation of the human iNOS promoter required the presence of cytokine-responsive elements upstream of −3.8 kb in the 5′-flanking region of the human iNOS gene (16de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (359) Google Scholar). To further localize the cytokine-responsive region an additional deletion construct containing the first 4.7 kb of the 5′-flanking region was generated. Transient transfections of the piNOS(4.7)Luc construct into AKN-1 and A549 cells revealed no increase in promoter activity in the presence of CM. However, transfection of piNOS(5.8)Luc followed by CM stimulation resulted in a 2–3-fold induction in luciferase activity in both cell types (Fig. 4). Transfection of the 7.2-kb construct (piNOS(7.2)Luc) produced a 4–5-fold increase in promoter activity, consistent with our previous findings (16de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (359) Google Scholar). To confirm the presence of these elements upstream of −4.7 kb and demonstrate position-independence of this cytokine responsive enhancer region, the segment from −2.1 to −4.7 kb was deleted to create piNOS(NA)Luc. Transfection studies with this construct in CM-stimulated cells revealed the same 4-fold induction of activity, thereby demonstrating position-independence of this enhancer element and confirming the presence of cytokine-responsive cis-regulatory motifs upstream of −4.7 kb in the human iNOS promoter. The DNA sequence from the transcription start site to −3761 bp of the human iNOS gene has been previously published by our group and others (38Chartrain N.A. Geller D.A. Koty P.P. Sitrin N.F. Nussler A.K. Hoffman E.P. Billiar T.R. Hutchinson N.I. Mudgett J.S. J. Biol. Chem. 1994; 269: 6765-6772Abstract Full Text PDF PubMed Google Scholar, 40Spitsin S.V. Koprowski H. Michaels F.H. Mol. Med. 1996; 2: 226-235Crossref PubMed Google Scholar, 41Nunokawa Y. Ishida N. Tanaka S. Biochem. Biophys. Res. Commun. 1994; 200: 802-807Crossref PubMed Scopus (114) Google Scholar). Because our data indicated a strong role for NF-κB in mediating TNFα- and IL-1β-stimulated activation of the human iNOS gene, and because we were not able to demonstrate a functional role for the proxi" @default.
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• W2100286113 title "Multiple NF-κB Enhancer Elements Regulate Cytokine Induction of the Human Inducible Nitric Oxide Synthase Gene" @default.
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