Multiple NF-κB Enhancer Elements Regulate Cytokine Induction of the Human Inducible Nitric Oxide Synthase Gene*

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-B in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-B inhibitors significantly suppressed cytokinestimulated iNOS mRNA expression and NO synthesis, indicating that NF-B 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-B cis-regulatory transcription factor binding sites upstream of ؊4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-B 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-␣ or interleukin-1␤, but not interferon-␥, exhibited inducible DNA binding activity for NF-B. These data indicate that NF-B activation is required for cytokine induction of the human iNOS gene and identifies four NF-B enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-␣ and interleukin-1␤.
The expression of the inducible nitric oxide synthase (iNOS) 1 gene is an important part of the immune response to infection (1,2). 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 (2)(3)(4)(5)(6). Although it is constitutively expressed in some epithelial cell types (7,8), iNOS expression in most cell types requires exposure to inflammatory stimuli such as cytokines and/or lipopolysaccharide (LPS) (9 -13). We and others have shown that iNOS up-regulation in response to LPS and cytokines is transcriptionally regulated (14 -16). The nitric oxide (NO) generated by iNOS from its substrate L-arginine has beneficial effects (e.g. antimicrobial, anti-atherogenic, anti-apoptotic) (8,(17)(18)(19), whereas overproduction of induced NO can have detrimental consequences (e.g. direct cellular injury, pro-inflammatory) (20,21). 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 Ϫ3.8 kilobases (kb) in the 5Ј-flanking region of the human iNOS gene (16). 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 IFN␥ (14,15,22,23). Deletional analysis of the murine gene identified an NF-B element at positions Ϫ85 to Ϫ76 base pairs (bp) (24) and an interferon regulatory factor-1 (IRF-1) site at positions Ϫ923 to Ϫ913 kb (25,26) that mediate iNOS induction by LPS and IFN␥, respectively.
The involvement of NF-B 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-B has been shown to be required for iNOS induction in both rodent macrophages (24,27) and vascular smooth muscle cells (28). NF-B has been implicated in the induction of the human iNOS gene as well, but its role has not been clearly defined (29 -31). In the A549 and DLD-1 human epithelial cell lines, inhibitors of NF-B activation minimally decreased iNOS expression (29,30). In contradistinction, others have shown that the same inhibitors do not inhibit cytokine-stimulated iNOS expression in DLD-1 cells. 2 Failure to identify a homologous functional NF-B site in the human iNOS promoter raises the possibility that NF-B may not be involved in the expression of the human gene by cytokines. Therefore, studies were performed to determine if NF-B 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-B plays a crucial role in human iNOS gene regulation, we also identify NF-B response elements in the human iNOS promoter. Unlike the murine iNOS promoter, however, the first 1.0 kb of the human iNOS gene 5Ј-flanking region is not sufficient for iNOS induction. Instead, inducible NF-B elements upstream of Ϫ4.7 kb are required for cytokine activation of the promoter. Specifically, we have identified a cytokine-responsive enhancer region from Ϫ5.2 to Ϫ6.1 kb in the human iNOS gene that contains four cis-acting NF-B 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-B in response to cytokines.

EXPERIMENTAL PROCEDURES
Materials-Human recombinant TNF␣ and IFN␥ were obtained from R&D Systems, and IL-1␤ 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.
Cell Culture-The AKN-1 human liver cell line was grown in modified HCD medium supplemented with 5% bovine calf serum as described previously (16). 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 TNF␣ (1,000 units/ml) ϩ IL-1␤ (100 units/ml) ϩ IFN␥ (250 units/ml) in the presence or absence of the NF-B inhibitors pyrrolidine dithiocarbamate (PDTC, 20 or 100 M) or diethyldithiocarbamate (DDTC, 10 mM) at the indicated time points. NO production was quantitated by measuring nitrite plus nitrate (NO 2 Ϫ and NO 3 Ϫ ) in the culture supernatant by an automated procedure based on the Griess assay (32).
Northern Blot Analysis-RNA extraction and Northern blot analysis were performed as described (12). Northern blot hybridizations were carried out using a 2.3-kb BamHI fragment of human iNOS cDNA (10).
DNA Sequencing and Analysis of the 5Ј-Flanking Region of the Human iNOS Gene-Using a series of both sense and antisense PCR primers, an EagI-BamHI fragment of the human iNOS promoter extending from ϩ33 to Ϫ7242 kb (GenBank™ accession number AF049872) was sequenced using the Sanger dideoxynucleotide sequencing method (33). 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.
Preparation of Nuclear Extracts-AKN-1 and A549 cells were treated with CM at the indicated times. In some experiments, PDTC (100 M) or DDTC (10 mM) was added 1 h prior to the addition of CM. Nuclear extracts were prepared as described by Ohlsson and Edlund (34) 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 MgCl 2 , 10 mM KCl, 25 g/ml chymostatin, 25 g/ml leupeptin, 0.2 mM phenylmethylsulfonyl 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 l of Buffer C (20 mM HEPES (pH 7.9), 10% glycerol, 1.5 mM MgCl 2 , 10 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) and adding 50 l of Buffer D (Buffer C but with 1.6 M KCl) 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).
Electromobility Shift Assays-The sequences of the oligonucleotides used in the gel shift assays are outlined in Table I. 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 M NaCl in one PCR cycle of 85°C ϫ 2 min, 65°C ϫ 15 min, 37°C ϫ 15 min, 23°C ϫ 15 min, and 4°C ϫ 15 min. Probes were end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase (Boehringer Mannheim) and purified by native polyacrylamide gel electrophoresis on a 15% polyacrylamide gel in 1x TBE. Five g of nuclear extracts were incubated with ϳ100,000 cpm of 32 P-labeled oligonucleotide (ϳ0.5 ng) for 45 min at room temperature in a buffer containing 2 g poly (dI-dC) (Boehringer Mannheim), 4.2 mM HEPES (pH 7.4), 4.2 mM KCl, 0.02 mM EDTA, 1 mM MgCl 2 , 2.5% glycerol, 2% Ficoll, and 21 mM dithiothreitol (final volume of 30 l). In some experiments, nuclear extracts were incubated with excess unlabeled oligonucleotides or antibodies against the different subunits of NF-B 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 0.4ϫ TBE running buffer (450 mM Tris borate and 1 M EDTA, pH 8.0). After electrophoresis, gels were dried and subjected to autoradiography.
Plasmids-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 (16). The piNOS(NA)Luc construct was generated by using the restriction enzymes NcoI and AflII to cut the DNA from Ϫ2.1 to Ϫ4.7 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-B construct with mutations at Ϫ0.11, Ϫ5.2, Ϫ5.5, Ϫ5.8, Ϫ6.1, and Ϫ6.5 kb, site-directed mutagenesis was performed on individual NF-B 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 (35) was also used to generate a piNOS(mut7.2B)Luc in order to further confirm the mutation, and similar results were obtained regardless of the method used to create piN0S(mut7.2B)Luc. To reduce errors in the reactions, PCR was performed using the Expand Long Template PCR system (Boehringer Mannheim). The piNOS(mut7.2B)Luc constructs were verified by restriction enzyme analysis and nucleotide sequencing of the initial ϳ800 bp of the 5Ј-flanking region of iNOS promoter, whereas restriction digests and sequencing were performed to confirm the orientation and validity of the upstream NF-B constructs. The promoter plasmid wild-type and mutated sequences in the NF-B elements of interest are listed in Figs. 3 and 6.
Transfections and Reporter Gene Assays-Transient transfections and activity assays were carried out as described previously (16). To control for transfection efficiency between groups, iNOS promoter constructs were co-transfected with 0.5 g of pIEP-lacZ, a plasmid encoding a CMV promoter-driven ␤-galactosidase gene (gift of Hideaki Tahara, University of Pittsburgh), and results were normalized to total protein content and to ␤-galactosidase activity.
Statistical Analysis-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 Ͻ 0.01.

Dithiocarbamates Block Cytokine Induction of Human iNOS-To determine whether iNOS induction in human lung
and liver cells is NF-B dependent, A549 cells and AKN-1 cells were treated with the CM of TNF␣, IL-1␤, and IFN␥ in the presence or absence of the established dithiocarbamate NF-B inhibitors PDTC or DDTC (36). 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-B is necessary for cytokine activation of iNOS gene expression in these human cell types.
EMSA Reveals Inducible NF-B DNA Binding Activity in Cytokine-stimulated Human Liver and Lung Cells-To document that the transcription factor NF-B 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-B (37). In both AKN-1 and A549 cells, basal levels of NF-B DNA binding were seen in control cells ( Fig. 2A). The addition of single cytokines, TNF␣ or IL-1␤ alone, induced a strong gel shift complex for NF-B, whereas IFN␥ had no effect. The CM of all three agents also induced DNA binding activity for NF-B, and the appearance of this complex was markedly suppressed by the addition of PDTC, further implicating NF-B 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-B was demonstrated by competition with 100-fold excess of unlabeled oligonucleotide ( Fig. 2A). Competition studies with excess unlabeled mutant oligonucleotide did not abolish the NF-B DNA complex, further demonstrating specificity for NF-B. Supershift studies with specific antibodies demonstrated the presence of both p50 and p65 subunits of NF-B in the complex (Fig. 2B). Antibody against the transcription factor AP-1 failed to induce a supershift.
The NF-B Element at Ϫ115 to Ϫ106 bp Is Not Required for Cytokine Induction of the Human iNOS Promoter-An NF-B element at Ϫ85 bp upstream in the murine iNOS promoter has been shown to confer LPS responsiveness in the murine iNOS gene (24). Analysis of the 5Ј-flanking region of the human iNOS gene revealed a putative NF-B element located at Ϫ115 to Ϫ106 bp that differs by only one nucleotide from the functional murine NF-B element at Ϫ85 bp (38). Because the first 400 bp of the human and murine iNOS promoter have 66% homology (38), and because this proximal corresponding NF-B element in the human iNOS promoter is relatively conserved, we sought to evaluate the functional role of this putative NF-B element. Transient transfections in AKN-1 cells were performed with a wild-type iNOS 7.2-kb promoter construct (piNOS(7.2B)Luc) and a mutated NF-B construct (piNOS(mut7.2B)Luc) generated by site-directed mutagenesis bearing a 2-bp mutation of the corresponding proximal NF-B element (Fig. 3A). 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-B (Ϫ115 to Ϫ106 bp) (piNOS(mut7.2B)Luc) was transfected, there was no significant decrease in either basal (data not shown) or stimulated reporter gene activity (Fig. 3B). In addition, the inducible activity of both the 7.2-kb wild-type and mutant constructs was inhibited by PDTC (Fig. 3B) and DDTC (data not shown). Interestingly, the addition of NF-B inhibitors did not change basal, unstimulated luciferase activity, suggesting that NF-B 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 Ϫ36 to Ϫ133 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-B element failed to abrogate cytokine-induced iNOS promoter activity. These results indicate that the promoter-proximal NF-B site is not required for maximal cytokine induction, suggesting that the functional NF-B elements are further upstream in the human iNOS promoter.

Cytokine-responsive Elements Are Localized to Regions More than 4.7 kb Upstream of the iNOS Transcription Start
Site-We have previously reported that the activation of the human iNOS promoter required the presence of cytokine-responsive elements upstream of Ϫ3.8 kb in the 5Ј-flanking region of the human iNOS gene (16). To further localize the cytokine-responsive region an additional deletion construct containing the first 4.7 kb of the 5Ј-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 2-3fold 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 (16). To confirm the presence of these elements upstream of Ϫ4.7 kb and demonstrate position-independence of this cytokine responsive enhancer region, the segment from Ϫ2.1 to Ϫ4.7 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 Ϫ4.7 kb in the human iNOS promoter.
DNA Sequence Analysis from Ϫ3.8 to Ϫ7.2 kb Reveals Putative Cis-regulatory NF-B Elements, Which May Confer Cytokine Inducibility-The DNA sequence from the transcription start site to Ϫ3761 bp of the human iNOS gene has been previously published by our group and others (38,40,41). Because our data indicated a strong role for NF-B in mediating TNF␣-and IL-1␤-stimulated activation of the human iNOS gene, and because we were not able to demonstrate a functional role for the proximal NF-B element at Ϫ115 bp, we sequenced the 5Ј-flanking region from Ϫ3761 to Ϫ7242 kb looking for other specific NF-B sites. Computer analysis revealed five potential NF-B sites in the functionally active upstream region. construct. Mutation of the cis-regulatory sites at Ϫ5.2, Ϫ5.5, and Ϫ6.1 kb resulted in loss of inducible promoter activity of 60%, 45%, and 65%, respectively. Interestingly, the mutation at Ϫ5.8 kb resulted in loss of both basal and inducible promoter activity (Fig. 6). These data indicate that, within the context of the 7.2-kb construct, the site at Ϫ5.8 kb is absolutely required for iNOS promoter activity and that the sites at Ϫ5.2, Ϫ5.5, and Ϫ6.1 kb are also functionally important and regulate iNOS gene expression. Previously, we reported a 10-fold increase in cytokine-stimulated iNOS promoter activity with a full-length 16-kb iNOS promoter construct (16). To further evaluate the role of the site at Ϫ5.8 kb in the 5Ј-flanking region of the human iNOS gene, we created the same 2-nucleotide point mutations in the 16-kb construct. Upon transient transfection and CM-stimulation, we observed a 9-fold increase in promoter activity, similar to our previous findings (16). This was reduced 40% by mutating the site at Ϫ5.8 kb, providing further evidence that this site is important for cytokine-induced iNOS expression (Fig. 7).
NF-B Proteins Bind to the iNOS Ϫ5.8 kb Promoter Element-To determine whether nuclear proteins could bind to the sequence at Ϫ5.8 kb, EMSA was performed on nuclear extracts of CM-stimulated AKN-1 and A549 cells using an oligonucleotide containing the NF-B sequence at Ϫ5808 kb in the human iNOS gene. In A549 cells, NF-B DNA binding activity was detectable in control cells. The addition of single cytokines (IL-1␤ or TNF␣) and CM (IL-1␤ ϩ TNF␣ ϩ IFN␥) resulted in an increase in DNA binding activity, which was inhibited by PDTC (Fig. 8A). Competition assays confirmed specificity for NF-B. Similar results were obtained with nuclear extracts from cytokine-stimulated AKN-1 liver cells, although a smaller nonspecific protein-DNA complex was also observed. Supershift studies revealed the presence of p65 and p50 in the NF-B complex (Fig. 8B). Antibodies against AP1 did not result in a supershift. These data show that the CM-inducible NF-B complex at Ϫ5808 is composed of both p50 and p65 proteins in AKN-1 cells.

DISCUSSION
The purpose of this study was to identify and characterize the NF-B elements responsible for cytokine-induced transcriptional activation of the human iNOS gene. Our data indicate that cytokine-induced iNOS expression in human liver and lung epithelial cell lines is dependent on the transcription factor NF-B and that the active response elements are localized in the 5Ј-flanking region upstream of Ϫ4.7 kb. The cytokine-responsive region from Ϫ4.7 to Ϫ7.2 kb functions in a position-independent fashion, thereby exhibiting the characteristics of an enhancer element. Of the five potential NF-B binding sites localized from Ϫ4.7 to Ϫ7.2 kb, four were shown to be functional by mutational analysis. The site at Ϫ5.8 kb is required for both basal and cytokine-induced promoter activity, whereas the sites at Ϫ5.2, Ϫ5.5, and Ϫ6.1 kb exert a cooperative effect on cytokine-stimulated iNOS expression. This work identifies a unique far upstream cytokine-responsive enhancer region from Ϫ5.2 to Ϫ6.1 kb in the 5Ј-flanking region of the human iNOS gene and underscores one of the major differences between the human and murine iNOS promoters.
Contained within ϳ1.7 kb of the murine iNOS 5Ј-flanking region are a number of functional cis-regulatory elements including two NF-B elements, an IRF-1 element, a ␥-interferon activated sequence (GAS) site (42), and a hypoxia-responsive element (43), which is lacking within the first 7. cular smooth muscle cells (28). In contradistinction to the murine system, neither deletion or mutation of the NF-B site at Ϫ115 bp of the human iNOS promoter had a significant effect on cytokine-induced promoter activity, suggesting that this promoter-proximal NF-B motif is neither necessary nor sufficient for transcriptional activation of the human iNOS gene by cytokines. In addition, the NF-B inhibitor PDTC completely blocks the cytokine-induced promoter activity even in the absence of the proximal NF-B promoter element, which is consistent with the location of functional NF-B elements further upstream. Our data indicate that, unlike the promoter-proximal NF-B site in the murine iNOS gene, the promoter-proximal NF-B site in the human 5Ј-flanking region does not confer cytokine inducibility.
We have previously shown that the functional promoter elements that regulate cytokine-inducible human iNOS expression are located upstream of Ϫ3.8 kb (16). Laubach et al. (39) has also demonstrated no inducible activity with a 3.7-kb promoter fragment in DLD-1 cells. However, two groups have reported approximately 2-fold induction of reporter gene expression in cytokine-stimulated A549 cells transfected with constructs containing 1.8 -3.7 kb of the 5Ј-flanking region of the human iNOS gene (30,40) The principle difference between our experiments and those of the other groups appears to be the use of different luciferase reporter plasmids, raising the possibility that the sequence elements within the plasmid vector itself may be affecting the transcriptional activity of the first 1.8 -3.7 kb of the iNOS promoter. Our results show that the functional promoter elements are upstream of Ϫ4.7 kb, that they function in a position-independent fashion, and that they consist of a novel enhancer region of ϳ800 bp, which contains four functional NF-B sites. Sequence analysis of the 5Ј-flanking region from ϳϪ1.0 to Ϫ3.8 kb shows a paucity of potential cis-regulatory elements, which may account for the lack of cytokine responsiveness in this region.
Transfection of a 16-kb human iNOS promoter construct produced a 9-fold increase in luciferase activity following cytokine stimulation. By mutating the NF-B element at Ϫ5.8 kb within the 16-kb construct, we observed a 40% decrease in promoter activity. This decrease is consistent with data we have published previously, demonstrating only a 2-fold increase in promoter activity when comparing the 7.2-kb construct with the 16-kb construct (16). Thus, there are additional functional elements even further upstream from Ϫ7.2 kb that are currently being investigated. Linn et al. (44) recently reported an enhancer region from Ϫ8.7 to Ϫ10.7 kb in the human iNOS promoter, which confers IL-1␤ and IFN␥ responsiveness in DLD-1 cells.
NF-B elements are present in the 5Ј-flanking regions of several inflammatory response genes, including cell adhesion molecules ELAM-1, VCAM-1, and ICAM-1 (45)(46)(47)(48)(49) and the cytokines IL-1, IL-6, and IL-8 (50 -54). However, each of these promoters has only one or two proximally-located functional NF-B binding sites. Unique from this, Cheng et al. (55) demonstrated six functional NF-B sites located within the first 360 bp of the porcine IB␣ promoter. Our data localize multiple NF-B elements in the human iNOS gene to a segment of DNA that spans ϳ800 base pairs and is located more than 5.2 kb upstream of the TATA box. Therefore, the presence of multiple functional NF-B binding sites so far upstream is unique to the human iNOS promoter. Four of the five sites in the iNOS promoter are functional, although to different degrees. Because iNOS is expressed in a number of different cell types and under different conditions, we speculate that this arrangement may serve as a means for cell type specificity and tight control in iNOS regulation. For example, the number of binding sites in a promoter may influence the intensity of the response to a given transcription factor depending on the concentration of that factor in that given cell type. In this scenario, low concentrations would be expected to evoke only a minimal response; however, with increasing levels of NF-B translocating into the nucleus, more NF-B sites would become occupied and a greater response elicited. Another potential mechanism may be that more NF-B binding sites over a segment of DNA serve to recruit the transcription factor to that portion of the genome and concentrate them toward the active elements. This idea is supported by our data, which show that, with the 5.8-kb promoter construct, which contains the NF-B sites at Ϫ5.2, Ϫ5.5, and Ϫ5.8 kb, we observe a 2-3-fold increase in luciferase activity. The addition of the functional element at Ϫ6.1 kb in the 7.2-kb construct increases this activity to 4 -5-fold.
It has been shown that the mechanisms involved in functional synergy between transcription factors in promoter activation involve protein-protein interactions (56,57). Through direct physical interactions between proteins, both DNA binding affinity and complex stability are enhanced, resulting in a highly stable multi-protein complex (58,59). In addition, it has also been shown that the arrangement in transcription factor binding site spacing, as well as intervening sequence between consensus elements, plays an important role in promoter activation (59). The NF-B elements from Ϫ5.2 to Ϫ6.1 kb in the human iNOS gene are spaced in approximate multiples of nucleosome units (200 bp) and this spacing may contribute to the three-dimensional structure necessary for efficient iNOS transcription.
Another noteworthy finding in this study is that a combination of three cytokines (TNF␣, IL-1␤, and IFN␥) was required to achieve a significant increase in iNOS promoter activity in both AKN-1 and A549 cells. Although, either TNF␣ or IL-1␤ alone induced NF-B DNA binding activity, this induction was not sufficient to activate iNOS transcription suggesting that induction or activation of additional transcription factors are required for iNOS expression. For example, members of the B and STAT family have been shown to exhibit both functional and physical interactions with other transcription factors, including NF-IL-6, C-EBP, Jun, and Sp1 (56, 60 -63). Recently, Ohmori et al. (64) demonstrated that IFN␥-activated STAT1␣ can cooperate with TNF␣-induced NF-B to promote transcription of a number of inflammatory response genes, including the interferon regulatory factor-1 (IRF-1), intercellular adhesion molecule-1 (ICAM-1), monokine induced by interferon-␥ (MIG), and regulated on activation normal T cell expressed and secreted (RANTES) genes. Gao et al. (42) have shown that STAT1␣ is also involved in mediating IFN␥ inducibility in the murine iNOS promoter. Whether STAT1␣ plays a similar role in the human iNOS promoter is unknown and is currently being investigated. Another recent study utilizing in vivo footprinting of the murine iNOS promoter suggested a functional role for Oct-1 in mediating transcription (22). We are currently performing in vivo footprinting of the human iNOS promoter to further our understanding of the complex transcription factor synergy that regulates iNOS gene expression.
Because iNOS expression has such profound physiologic effects, its regulation is strictly controlled. The expression of iNOS and subsequent production of NO serves a protective role by increasing perfusion to the viscera and sites of inflammation. However, sustained overproduction can have detrimental effects including refractory hypotension and death. Thus, the combination of cytokine-inducible transcription factors working in synergy increases the diversity and complexity of the regulation of iNOS gene expression and reduces the chance of inappropriate transcription.