©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Cytokine Responsive Vascular Smooth Muscle Cell Enhancer of Inducible Nitric Oxide Synthase
ACTIVATION BY NUCLEAR FACTOR-kappaB (*)

(Received for publication, August 31, 1995; and in revised form, October 5, 1995)

Jayne Spink Jonathan Cohen Tom J. Evans (§)

From the Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, London W12 0NN, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The production of inducible nitric oxide synthase (iNOS) within vascular smooth muscle (VSM) cells following exposure to proinflammatory cytokines is a major cause of the vasorelaxation and hypotension of septic shock. We have defined the cytokine-responsive element of the murine iNOS promoter, transfected into a VSM cell line, and the role of the NF-kappaB/Rel family of proteins in iNOS gene activation in these cells. The combination of interleukin-1, interferon-, and tumor necrosis factor-alpha stimulates promoter activity by a factor of 8.1-fold; single cytokines show little activity, while pairs of cytokines produce an intermediate effect. Using a series of promoter deletion mutants, we have defined the cytokine-responsive element from position -890 to -1002; this region contains an NF-kappaB-binding site as well as a number of interferon response elements. Nuclear proteins from cytokine-stimulated VSM cells which bind to an oligonucleotide containing this kappaB site are composed of p65 together with an unidentified protein of 50 kDa, which is not a known Rel family member. A promoter mutant with a 2-base pair change within this kappaB site, which abolishes NF-kappaB binding, has an activity of only approximately 34% (S.E. ± 1.5) of the wild-type promoter. In addition, protein binding to this site is abolished by a specific inhibitor of NF-kappaB activation, which also abrogates iNOS activity. Residual inducibility in such mutant promoters is attributable to the presence of an independently functioning downstream kappaB site (-85 to -75). The mechanism by which NF-kappaB activates the iNOS promoter in VSM cells in response to cytokines appears to be markedly different to that operative in macrophages in response to lipopolysaccharide.


INTRODUCTION

Nitric oxide (NO) (^1)is a potent vasorelaxant(1) , but also plays a role in physiological processes as diverse as neurotransmission (2) and host defense(3) . There are three types of enzymes which synthesize NO, the NO synthases (4) . Of these, two are constitutive, but the third is normally produced only following transcriptional activation of its gene(5, 6) . This inducible NO synthase (iNOS) is produced in a variety of cell types following stimulation with a number of different factors, the most important of which are proinflammatory cytokines and lipopolysaccharide (4, 7) . Normal VSM produces no NO in the resting state. However, following stimulation with cytokines, such as interleukin-1 (IL-1), interferon- (IFN-), and tumor necrosis factor-alpha (TNF-alpha), iNOS is synthesized(8, 9, 10) , leading to NO production, vasorelaxation, and the hypotension so characteristic of septic shock(11, 12) .

The promoter of the murine gene for iNOS contains numerous potential sites for the binding of a number of different transcription factors (13) . This 1.7-kilobase pair DNA element confers inducibility by IFN- and lipopolysaccharide (LPS) in macrophages. A key region of the promoter in mediating the response to LPS is a downstream kappaB site, which extends from position -85 to -76(14) . The synergic effect of IFN-, however, requires the presence of the 5` region of the promoter which has not been functionally mapped in any detail(13, 15) . All studies on the promoter of iNOS have so far been performed in macrophages and have described the effect of only LPS and IFN-.

A number of different cell types can produce iNOS after stimulation by LPS and IFN-, as well as proinflammatory cytokines such as IL-1, TNF-alpha, and IL-2(10) . Cytokines are rarely produced in isolation, and their ability to interact with one another increases the range of biological effects which they mediate. Given the complexity of the iNOS promoter and the necessity to integrate the stimulatory effects of a number of proinflammatory cytokines, different mechanisms might apply in cell types other than macrophages and following different stimuli.

Of particular interest is the control of iNOS gene activity within VSM cells where the production of NO plays an important part in the pathophysiology of septic shock. We chose to analyze the effects of three proinflammatory cytokines, TNF-alpha, IFN-, and IL-1 on iNOS promoter function, since these cytokines are all produced during septic shock from a variety of different causes(16) , and are known to synergize in the production of iNOS from VSM cells(10) . In contrast to the promoter function in macrophages, we find that an upstream kappaB site is of key importance in mediating the synergic effect of cytokines on iNOS gene activity within VSM cells.


MATERIALS AND METHODS

Cells

A7r5 rat VSM cells were obtained from the European Collection of Animal Cell Cultures at Porton Down, UK. Rat peritoneal macrophages were harvested 3 days after intraperitoneal inoculation of 10 ml of thioglycollate broth (Oxoid, UK). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine, 20% fetal calf serum (10% for macrophage), 100 units ml penicillin, and 100 µg ml streptomycin at 37 °C in an atmosphere of humidified 5% CO(2). All cell culture reagents were from ICN/Flow, Thame, UK. Cytokines were from Genzyme, Boston, MA. Concentrations used were: IL-1, 10 ng ml; IFN-, 200 units ml; TNF-alpha, 25 ng ml.

Cloning of iNOS Promoter Region

The murine iNOS promoter was amplifed by PCR (nucleotides -1577 to +165) using oligonucleotides based on the published sequence (13) from 1 µg of mouse genomic DNA. Forward primer was GACTGCAGGCTGAAATCCATAAGCTGTG (tagged with a PstI site), and the reverse primer was CTGTCGACAGTTGACTAGGCTACTCCG (tagged with a SalI site). The product was cloned into pUC18 and sequenced (Sequenase, Amersham, UK). The iNOS promoter was liberated as a HindIII/SalI fragment and ligated into pCAT-basic (Promega, Southampton, UK), to create pNOS-CAT.

Construction of Deletion Mutants of pNOS-CAT

Stepwise deletions of the promoter sequence of pNOS-CAT was constructed using controlled digestion with exonuclease III using the Erase-a-base system (Promega) according to the manufacturer's instructions. In all cases, deletion boundaries were determined by DNA sequencing. PCR-generated deletion mutants of the promoter were created using the reverse primer (+165) and the following forward primers: mutant 55, GGGAAGCTTCTCAGACAAGGGCAAAACACG(-1052); mutant 80, GCCAAGCTTAGATGAGTGGACCCTGGCAGG(-1002); mutant 79, GGCAAGCTTCTCTCTGTTTGTTCCTTTTCC(-959); and pNOS-kappaB, GCCAAGCTTGTGCTAGGCCGATTTTCCCTC, and its isogenic wild type, GCCAAGCTTGTGCTAGGGGGATTTTCCCTC. Each of these primers incorporates a HindIII site. Template DNA was pNOS-CAT. The PCR products were digested with SalI and HindIII and ligated into the multiple cloning site of pCAT-basic. The downstream kappaB mutation was generated by recombinant PCR(17) , using the primer set CCCAACTGCCGACTCTCC and GGAGAGTCGGCAGTTGGG, and cloned as a replacement cassette (SpeI/SalI) into pNOS-kappaB and its isogenic wild type. Mutant 43 was generated by digestion of pCAT-NOS with SphI and NheI (Delta -1542 to -767). Using the primers GACTGCAGGCTGAAATCCATAAGCTGTG (forward) and GCCGCTAGCCATCCTGCCAGGGTCCACTC (reverse, tagged with an NheI site), a PCR cassette (-979 to -1577) was generated. The product was cleaved at an internal restriction site at -1542 with SphI, digested with NheI, and ligated into the truncated pCAT-NOS to create an internal deletion (-767 to -979). Product fidelity was ascertained by sequencing.

Nuclear Extracts

Cells were grown to 90% confluence and washed with Hanks' balanced salt solution before incubation for 2 h in fresh medium containing cytokines. Cells were harvested into ice-cold phosphate-buffered saline, resuspended in 400 µl of ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol) and left to swell on ice for 15 min. Nonidet P-40 was added to 0.6%, and the cells were vortexed and centrifuged at 10,000 times g for 30 s. The pellet was resuspended in 50 µl of ice-cold buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA) and left on ice for 15) with intermittent mixing. Following centrifugation (10,000 times g at 4 °C for 5 min), the supernatant was transferred to a fresh tube. All buffers contained 1 µg ml pepstatin, antipain and aprotonin, 10 µg ml chymotrypsin inhibitor and 1 mM phenylmethylsulfonyl fluoride.

Preparation of Labeled DNA Fragments and Electrophoretic Mobility Shift Assay (EMSA)

The putative upstream kappa-B site from the iNOS promoter was constructed by annealing equimolar (10 pm) quantities of oligo1 (GAGAGGGAAAATCCCCCTAGCAC) and oligo 2 (GTGCTAGGGGGAT). The resultant overhang was in-filled with Klenow fragment using [P]dCTP as the label. The probe was resuspended in 60 µl of TE buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA).

EMSA

The binding reactions were performed at room temperature in a volume of 20 µl containing 10% glycerol, 25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.1 M NaCl, 2 µg ml poly(dI-dC), 1 µg ml pepstatin, antipain, and aprotonin, 10 µg ml chymotrypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, and 5-10 µg nuclear extract. A preincubation of 5 min was allowed prior to adding 0.5 µl of labeled DNA probe, and the incubation continued for a further 20 min. The DNA-protein complexes were separated on native 5% polyacrylamide gels in 0.5 times Tris borate-EDTA buffer. For supershift experiments, 1.0 µg of specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the incubations of nuclear extract and oligonucleotide probe, and the incubation continued for an additional 20-40 min on ice.

Ultraviolet-induced Cross-linking of Proteins to Labeled NF-kappaB Probe

EMSA was carried out as above, substituting bromo-dUTP for dTTP in synthesis of the radiolabeled probe. The specifically shifted band was localized by autoradiography and excised. Proteins were cross-linked to the DNA by exposing the slice to UV light on a transilluminator box for 10 min. Cross-linked proteins were eluted into SDS-gel sample buffer containing 6 M urea and then analyzed on an 8% SDS-polyacrylamide gel as described in Sambrook et al.(18) .

Transfection of VSM Cells

Cells were grown to 90% confluence in 90-mm tissue culture Petri dishes. They were washed twice with calcium- and magnesium-free Hanks' balanced salt solution prior to addition of 10 µg of plasmid DNA in a final volume of 568 µl of phosphate-buffered saline containing 0.5 mg ml DEAE-Dextran (Promega). Plates were incubated at 37 °C for 30 min, 6 ml of DMEM were then added together with chloroquine (Promega) (final concentration of 80 µM), and plates were incubated at 37 °C for another 2 h. Cells were treated with 10% dimethyl sulfoxide in phosphate-buffered saline for 1 min. This solution was immediately replaced with 10 ml of fresh DMEM. After overnight growth, cells were trypsinized and seeded into 60-mm Petri dishes, precoated with a solution of 14 µg ml of Pronectin F (Stratagene, Cambridge, UK), and allowed to attach for about 6-8 h prior to cytokine stimulation for 16 h.

Determination of CAT Activity in Transfected Cells

The CAT content of cell lysates was assayed by a liquid scintillation counting-based assay (Promega), as described in the manufacturer's protocol. Values from control reactions were subtracted from all results. Determinations were standardized for protein concentration using a modified Bradford dye reagent (Bio-Rad, Hemel-Hempstead, UK). Results are expressed as the -fold increase in CAT activity following the specified cytokine treatment.

NO Output from VSM Cells

2 times 10^5 A7r5 cells were seeded into each well of a 24-well plate and grown to confluence. The medium was replaced with 0.25 ml of fresh DMEM containing the specified cytokine mixtures. After 20 h of incubation, the nitrite present in the medium was assayed using the Griess reaction (19) .


RESULTS

Transfection of Smooth Muscle Cells with Full-length iNOS Promoter

We chose to analyze the effects of IL-1, IFN-, and TNF-alpha, since they are all produced during septic shock and are known to synergize in the production of iNOS from vascular smooth muscle cells(10) . The degree of NOS activity, as reflected by nitrite accumulation is shown in Fig. 2A. The addition of single cytokines resulted in only a modest stimulation of NOS activity in the vascular smooth muscle cell line, but combinations of 2 or 3 cytokines produce much greater stimulation. In separate experiments using fixed concentrations of 2 cytokines and varying the concentration of the third, we demonstrated that the concentration of each of the cytokines used in Fig. 2was producing a maximal effect on NOS activity (data not shown).


Figure 2: A, NOS activity in A7r5 cells following various cytokine treatments. Values are the means of three determinations; error bars are ± 1 S.E. of the mean. The nitrite levels for untreated cells have been subtracted in all cases and did not differ significantly from medium alone. B, iNOS promoter reporter gene activity within vascular smooth muscle cells following various cytokine treatments. Cytokines were used at the concentrations as in Panel A. Values are expressed as the -fold increase in CAT activity seen following cytokine treatment relative to the CAT activity seen with no added cytokines. Results are the means of three to five estimations; error bars are ± 1 S.E. of the mean.



A schematic diagram of the murine iNOS promoter region is shown in Fig. 1with the putative transcription factor-binding sequences highlighted. This complete sequence was cloned upstream of the bacterial CAT gene in the reporter plasmid pCAT-basic, to produce an iNOS reporter vector, pNOS-CAT. This vector was transfected into vascular smooth muscle cells. CAT activity from such transfected cells after a variety of different cytokine stimuli is shown in Fig. 2B. Cytokine stimulation of CAT activity from this construct was analogous to nitrite output under the same conditions. When added singly, only IL-1 showed a significant stimulation of CAT activity of 3-fold over basal levels (S.E. ± 0.47). However, the combination of two or three cytokines together increased the levels of CAT activity in the transfectants, to a maximum of 8.1 (S.E. ± 0.86)-fold over basal levels with the combination of all three cytokines. Pairs of cytokines showed levels of stimulation broadly intermediate between single cytokines and the combination of all three; the addition of TNF-alpha to IL-1, however, made no significant difference to the result obtained with IL-1 alone. The interaction of the cytokines in these experiments was borderline between an additive and a synergistic effect.


Figure 1: Schematic representation of the 5` upstream elements of the murine iNOS gene. Putative transcription factor binding elements are illustrated: activator protein-1 (AP-1), TNF response element (TNF-RE), ISRE, GAS, IFN- response element (IFN-RE), and basal transcription complex recognition site (TATA).



CAT Activity following Transfection of Deletion Mutants of pCAT-NOS

Three large scale deletion mutants of pNOS-CAT were made using suitable restriction sites within the cloned promoter region; these are shown in Fig. 1. CAT activity following transfection of these mutant plasmids was assayed after a variety of cytokine treatments. Deletion of all but the 3` most 46 bases of the iNOS promoter (using the internal PstI site) led to a complete loss of cytokine inducibility of CAT (data not shown). Cells transfected with mutant 2.1 (Delta -172 to -1588) showed a reduced maximum level cytokine induction of CAT activity of 2.5-fold (S.E. ± 0.42) compared to a wild-type value of 8.1-fold over basal level (Fig. 3B). However, mutant 1.1 (Delta -331 to -172) retained virtually the same pattern of CAT activity following cytokine treatment as the full-length promoter (Fig. 3A). Hence the cytokine-responsive element or enhancer lay in the region -331 to -1588.


Figure 3: A CAT activity following transfection of vascular smooth muscle cells with plasmids 1.1 (Panel A) and 2.1 (Panel B). Values are expressed as the -fold increase in CAT activity seen following cytokine treatment relative to the CAT activity seen with no added cytokines. Cytokine concentrations are as in Fig. 2. Values are the means of three determinations; error bars are ± 1 S.E. of the mean.



Deletion Mapping of the Cytokine-responsive Element in the iNOS Promoter

In order to delineate the cytokine-responsive element within the 5` portion of the promoter of the iNOS gene, two sets of mutants were generated: first, a set which progressively removed the most 5` sequences of the promoter and second, a set which progressively removed the most 3` sequences of the 5` half of the promoter region. This combined approach was adopted to find the 5` and 3` limits of the smallest stretch of promoter DNA which retained cytokine inducibility. An initial set of mutants was constructed using exonuclease III digestion. However, the region -1080 to -899 proved to be exonuclease hypersensitive. Therefore, in order to analyze this area more fully, a second set of directed mutants was constructed using the PCR. The results of CAT activity following transfection of these various constructs is shown in Fig. 4. Experiments were performed in triplicate with consistent results; representative results are given. Stimulation of the transfectants was performed using the combination of all three cytokines, and the results are expressed as the percentage of the maximal stimulation achieved with the full-length promoter (pNOS-CAT) following such cytokine treatment. With the set of mutants progressing into the 5` portion of the promoter, full stimulation with the combination of cytokines was retained until the kappaB site starting at position -971 was removed (compare mutant 79 with 80). This reduced the induction of promoter activity following cytokine stimulation to 43% of that obtained with the full-length promoter. Deletion of the various GAS, ISRE, and IRF-1 sites starting at position -923 produced a further fall in induction after cytokine treatment to about a fifth of the amount produced by the full-length promoter (Fig. 4, mutant 3). This is about the level of induction seen in the mutant lacking the whole 5` section of the promoter (Fig. 3B, mutant 2.1).


Figure 4: CAT activity of a series of deletion mutants of the iNOS promoter. Mutant plasmid designations are shown to the left with the boundaries of the mutation shown measured relative to the mRNA start site. A schematic diagram of this region of the promoter is shown above with the various putative transcription factor-binding sites, and their orientation (5` to 3`) is indicated by the arrow. CAT activity is measured as the -fold increase in activity seen following addition of all three cytokines in the concentrations shown in Fig. 2; it is expressed as a percentage of the value achieved with the full-length iNOS promoter reporter gene construct (pNOS-CAT) in the same experiment. Values shown are for one experiment; similar results were obtained on two further repeats.



Mutants extending into the 3` end of this region of the promoter were also analyzed. Virtually full induction following cytokine treatment was seen in mutant 7, which contains a deletion extending from position -480 to -890, to the 3` side of the interferon regulatory sites. However, when the area deleted extended to position -979 (mutant 43), the level of induction following cytokine treatment dropped to one-third of the level seen with the full-length promoter (Fig. 4). This removes the GAS/ISRE/IRF-1 sites and the adjacent kappaB site. Thus, these combined sets of mutants show that a 112-base pair region of the promoter extending from position -890 to -1002 is required for full iNOS transcriptional activity following treatment of vascular smooth muscle cells with IL-1, TNF-alpha, and IFN-.

Role of NF-kappaB in the Induction of NOS

The presence of a kappaB site within this cytokine-responsive element suggests that proteins of the NF-kappaB/Rel family are involved in the induction of NOS within vascular smooth muscle cells following cytokine stimulation. We constructed a mutant promoter construct, termed pNOS-kappaB, in which two of the G residues in the kappaB sequence were changed to C residues. This mutant kappaB site no longer bound the specific nuclear proteins which bind to the wild-type sequence (see Fig. 6and data not shown). The transcriptional activity of this mutant promoter was tested as before using the CAT reporter construct transfected into vascular smooth cells (Fig. 5). The wild-type isogenic promoter (termed pNOS-kappaB) in this particular experiment mediated a 7.02-fold (S.E. ± 0.51) stimulation following stimulation with cytokines, whereas pNOS-kappaB mediated a 2.4-fold (S.E. ± 0.11) stimulation, an activity of 34% of the wild type. This is in good agreement with the activity of the deletion mutant 79 which lacks the kappaB site.


Figure 6: EMSA using nuclear extract (2 µg) from A7r5 cells and an oligonucleotide containing the upstream kappaB site (-957 to -979). A, nuclear extract from untreated cells (lane a) or cytokine-treated cells (IL-1, TNF-alpha, IFN-; lanes b-d) was mixed with no added competitor DNA (lanes a and b); or with a 5-fold molar excess of unlabeled nonspecific competitor DNA (an oligonucleotide containing the upstream GAS/ISRE sites; lane c); or a 5-fold molar excess of unlabeled specific DNA (the same oligonucleotide as used for labeling; lane d). The specific shifted band is shown with an arrow; a nonspecific band is indicated by an arrowhead. B, the same radiolabeled NF-kappaB-containing oligonucleotide was mixed with a nuclear extract of A7r5 cells which had received no treatment (lane a), the cytokine mixture, as described in Panel A (lane c), or this cytokine mixture with 60 µM PDTC, following a 10-min preincubation with the same concentration of the drug (lane b).




Figure 5: The -fold stimulation of CAT activity in VSM cells of reporter gene constructs with either wild-type or mutant kappaB sequences. The -fold stimulation in CAT activity following IL-1, TNF-alpha, and IFN- stimulation is shown relative to that seen with no cytokine additions, expressed as a percentage of the value achieved with the wild-type sequence (pNOS-kappaB). Bars represent the mean of two separate experiments; the error bars are 1 S.E. of the mean.



Comparison of the Relative Importance of the the Proximal and Distal kappaB Sites

In addition to the upstream kappaB site described above, the iNOS promoter also contains a more proximal downstream kappaB site located at position -85 to -76. To address the question of whether this downstream site participates in the inducibility of the iNOS promoter in VSM cells, it was mutated in both pNOS-kappaB and its isogenic wild type to create pNOS-kappaB and pNOS-kappaB, respectively. When transfected into VSM cells, pNOS-kappaB had a promtoer activity of 65% (S.E. ± 6.2) of the wild-type promoter pNOS-kappaB. The promoter construct in which both kappaB sites had been mutated (pNOS-kappaB) had only 15.7% (S.E. ± 7.7) of the activity of the wild-type construct (Fig. 5).

Effect of Pyrrolidine Dithiocarbamate (PDTC) on NO Production kappaB Site Binding Activity in Nuclear Extracts

The role of transcription factors which bind to these kappaB sites was examined further by the use of PDTC, an antioxidant which prevents the release of NF-kappaB proteins from their cytoplasmic inhibitor, IkappaB(20) . We also used the electrophoretic mobility shift assay to detect potential NF-kappaB binding proteins, using as a probe an oligonucleotide extending from -957 to -979, which contains just the kappaB site of the cytokine-responsive element. Fig. 6shows that, following treatment with IL-1, TNF-alpha, and IFN-, a nuclear factor which binds specifically to this probe is produced (Fig. 6A, arrow). However, when the cells are pretreated with 60 µm of PDTC, the binding of this factor is abolished (Fig. 6B, lanes b and c). NO output of A7r5 cells after treatment with IL-1, TNF-alpha, and IFN- (cytomix) was measured 20 h after stimulation in the presence or absence of 60 µm of PDTC. The inclusion of PDTC was found to reduce nitrite output from 57.3 µM (S.E. ± 3.3) to 5.0 µM (S.E. ± 0.02).

IL-1 and TNF-alpha but Not LPS Activate NF-kappaB Binding in VSM Cells

The effect of a variety of agents on nuclear factors binding to this kappaB site is shown in Fig. 7. IL-1 and TNF-alpha on their own can both induce binding of nuclear proteins to the kappaB site (Fig. 7, lanes a and b and lanes e and f). At the concentrations used, IL-1 induces a greater effect than TNF-alpha. IFN- has no effect (lanes c and d). The combination of cytokines gives a greater response (lanes i and j); the magnitude of the increased protein binding is borderline between an additive and synergistic effect. LPS treatment of the vascular smooth muscle cells does not produce any binding to this kappaB site (lanes g and h).


Figure 7: EMSA using 2 µg (lanes a, c, e, g, and i) or 4 µg (lanes b, d, f, h, and j) of nuclear extract from A7r5 cells and the same oligonucleotide as used in Fig. 6. Cells were treated for 2 h with the following reagents: IL-1 (lanes a and b); IFN- (lanes c and d); TNF-alpha (lanes e and f); LPS (2 µg ml; lanes g and h); or a mixture of IL-1, IFN-, and TNF-alpha (lanes i and j). The specific shifted band is shown with an arrow; a nonspecific band is indicated by an arrowhead.



Comparison of the Components of Factors Binding to the -957 to -979 kappaB Site in Macrophages and VSM Cells

LPS produces little additional effect on iNOS production in A7r5 cells over that seen with the combination of proinflammatory cytokines. However, in macrophages, LPS is a potent activator of iNOS transcription, synergizing strongly with IFN-, and activating a number of NF-kappaB/Rel family proteins which bind to a downstream kappaB site at position -85 to -76 in the iNOS promoter. The lack of effect of LPS on the binding of A7r5 vascular smooth muscle nuclear proteins to the upstream kappaB site may be because of a lack of NF-kappaB activation by LPS in these cells or because this site does not bind the NF-kappaB components which are activated by LPS. To investigate this further, we tested the ability of nuclear proteins from rat peritoneal macrophages to bind to the upstream NF-kappaB site following LPS treatment (Fig. 8). Both VSM cell and macrophage nuclear extracts contain different elements which bind to the probe in a nonspecific manner (Fig. 8, lower bands) the intensity of which do not diminish in the presence of excess unlabeled probe (data not shown). In addition, there are specific complexes formed (Fig. 8, upper bands). The composition of these NF-kappaB-protein complexes was analyzed using specific antibodies to various members of the NF-kappaB/Rel family to supershift the protein-DNA complexes. In the VSM cell extracts anti-p65 produced a clear supershifted band in addition to decreasing the intensity of the specific shifted band (lane a). Anti-p50 (lane b) under the same conditions was without effect, as were the antibodies to the other rel gene family members (data not shown). These antibodies are all known to bind the relevant rat proteins. (^2)In addition, extracts from LPS-treated rat peritoneal macrophages produced a number of specifically shifted bands which are not completely resolved on the gel system used (Fig. 8, lanes c-e). However, both anti-p65 and anti-p50 clearly produced a supershift of a component of these complexes (Fig. 8, lanes d and e).


Figure 8: EMSA performed as in Fig. 7with nuclear extracts (2 µg) from cytokine-treated A7r5 cells (lanes a and b) or peritoneal macrophages treated with 2 µg ml LPS for 2 h (lanes c-e). The following additions were made to the incubations; none (lanes c and f), anti-p65 (lanes a and d), anti-p50 (lanes b and e). The specific shifted band in the A7r5 cells is shown by the long arrow. The short arrows indicate the supershifted bands



The molecular composition of the specific protein-DNA complex produced in VSM cells on the upstream kappaB site was further analyzed by cross-linking the proteins in this complex to the radiolabeled DNA probe and analyzing the components on an SDS-polyacrylamide gel (Fig. 9). As expected, the main protein component of the complex had a molecular mass in reasonable agreement with the molecular mass of p65 complexed to a short DNA fragment (upper band). However, we also reproducibly found an additional protein component of molecular mass of about 50 kDa as part of this complex (lower band). The identity of this protein is not known.


Figure 9: Autoradiograph of a SDS-polyacrylamide gel analysis of proteins cross-linked to the radiolabeled NF-kappaB probe. The specific band produced from vascular smooth muscle cell nuclear cell extracts as seen in Fig. 8, long arrow, was cut from the gel, and the proteins contained within it were cross-linked to the radiolabeled probe by ultraviolet irradiation. These proteins were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiographed. Molecular mass markers in kilodaltons are shown to the left.




DISCUSSION

A particular feature of iNOS activity within VSM cells is that full activity requires the presence of combinations of cytokines (Fig. 2B). This additive/synergic effect of IL-1, TNF-alpha, and IFN- is also seen on the transcriptional rate following cytokine treatment (Fig. 2A). Such differences that do exist between changes in levels of NO output and promoter activity may be attributable to post-transcriptional/translational modifications. We were careful to choose concentrations of cytokines which were saturating for their stimulatory effect on iNOS production and that fall within the range found in disease states. This suggests that each cytokine produces a unique effect in enhancing iNOS gene transcription which cannot be substituted by increasing the concentration of another cytokine. Since NO is highly potent its production needs to be tightly regulated, such that it is only produced under the appropriate conditions. Relying on more than one signal for gene activation reduces the likelihood of inappropriate transcription of the gene.

The 1.7-kilobase pair 5` upstream sequence of the iNOS gene has only been shown to function as a promoter within macrophages(13, 15) . We have shown here that the same sequence functions as a cytokine-inducible promoter within VSM cells. We have defined an enhancer type element responsible for the maximal effect of these three cytokines as a 112-base pair region extending from position -890 to -1002, which contains consensus sequences for a kappaB site, a GAS/ISRE element and binding sites for IRF1.

We have provided evidence that the iNOS promoter is regulated differently in VSM cells stimulated with IL-1, TNF-alpha, and IFN- compared to macrophages stimulated with LPS and IFN- (summarized in Table 1). The combination of IL-1, TNF-alpha, and IFN- increased the transcriptional rate of the promoter within VSM cells by a factor of 8.1. This compares to an enhancement of 44-fold reported for the same promoter following LPS and IFN- treatment within the macrophage cell line RAW 264(13) . These results correlate with the output of NO from each of these cells: RAW 264 cells produce approximately 7.5 nmol of nitrite h per 10^6 cells, while A7r5 cells produce about 2 nmol of nitrite h per 10^6 cells. (^3)The effects of LPS on the transcription of iNOS within macrophages are mediated through the binding of NF-kappaB/Rel proteins to the downstream kappaB site(14) . In contrast to those results most of the stimulatory effects of the three cytokines IL-1, TNF-alpha, and IFN- are mediated through an upstream 112-base pair element. The 112-base pair element described in this study contains an NF-kappaB binding sequence, which when deleted or mutated by two nucleotides, reduces the promoter's activity to 34% of the wild type ( Fig. 4and Fig. 5). This residual activity is virtually completely removed when the second downstream kappaB site is mutated as well (Fig. 5). However, mutation of the downstream kappaB site alone (mutant pNOS-kappaB) only reduces activity to 65% of the wild type. Thus although both kappaB sites contribute to full iNOS gene activity in VSM cells, the upstream site is more important. Moreover, since promoter constructs mutated in the downstream site alone retain considerable promoter activity (Fig. 5), this indicates that there is no absolute requirement for this site for effective promoter activity within VSM cells. Inhibition of NF-kappaB activation with PDTC prevents NOS production within vascular smooth cells and inhibits the binding of a nuclear factor to sequences containing the upstream kappaB site (Fig. 6).



NF-kappaB activation is crucial to iNOS transcriptional activation in both macrophages, after IFN- and LPS stimulation, and in vascular smooth muscle cells after TNF-alpha, IL-1, and IFN- treatment. However, a different kappaB site is involved in each case. The kappaB site binding protein in VSM cells consists of p65 together with a protein of molecular mass of 50 kDa, which is not recognized by antibodies to p50, p52, relB, p65 or c-rel ( Fig. 8and data not shown). Western blots have indicated that p105 (the precursor of p50) is not processed in A7r5 cells following TNF-alpha and IL-1 stimulation (data not shown). The antibody to p52 is known to recognize rat p52 in Western blots and supershift experiments,^2 but in fact, immunoprecipitation and Western analysis have indicated that A7r5 cells are devoid of p52 (data not shown). It is an intriguing possibility, therefore, that this unidentified 50-kDa protein may represent another rel family member, but clearly purification and cloning of the cDNA of this protein will be required to establish its nature. It is unlikely that this protein is a breakdown product of p65; while the presence of this protein is consistently demonstrable by UV cross-linking, no heterogeneity in the electrophoretic mobility of the protein-DNA complex has ever been observed.

In the cells used in this study, LPS gives little additional increase over the induction of iNOS activity seen with the combination of three cytokines, and has little effect on its own. (^4)In addition, LPS did not produce NFkappaB binding activity in these cells (Fig. 7). In some primary VSM cell preparations, LPS is active(8) , while in others it is not(10) . This may reflect the presence or absence of the LPS receptor CD14 within the cell population(21) .

The region we have delineated as necessary for the cytokine induction of iNOS transcription by TNF-alpha, IL-1, and IFN- contains two consensus sequences for IRF-1. This is a transcription factor whose level is increased following stimulation of cells with IFNs(22, 23) . Mice with a targeted disruption of the IRF-1 gene fail to induce iNOS within macrophages, suggesting that IRF-1 is essential for iNOS activation in these cells(24) . Studies using the iNOS promoter transfected into the macrophage cell line RAW 264 have shown that sequences within the 5` portion of the promoter mediate the synergic effect of IFN- with LPS on iNOS gene transcription(15, 13) . This effect was lost when sequences from position -1029 to -913 were deleted(15) ; however, the 3` limit of this element was not defined. Recent studies have shown the importance of IRF-1 binding to a site at position -913 to -923 in the induction of iNOS within macrophages by IFN-(25) . It remains to be determined if IRF-1 is involved in the induction of iNOS transcription in vascular smooth muscle cells. However, the close proximity of the IRF-1 binding sites and the kappaB site in this enhancer suggests that interactions may occur between transcription factors binding to these sites, as has been shown for a similar region in the HLA class I heavy chain gene promoter(26) . Closely adjacent to the IRF-1 sites is an ISRE/GAS site (Fig. 1). IFN- has been shown to promote phosphorylation of the transcription factor Stat91(27, 28) , which then undergoes translocation to the nucleus where it binds to the GAS site and activates transcription(27, 29) . We are currently examining the role that Stat91 and IRF-1 might play in the induction of iNOS by IFN-.

The production of NO within vascular smooth muscle produces profound vasodilatation. This may be beneficial in a local area of inflammation, by facilitating the delivery of inflammatory cells and immune effectors such as antibody and complement. However, the need for a tight control of this process is demonstrated by the widespread inappropriate vasodilatation that occurs in septic shock, leading to hypotension, multiorgan failure, and death(16) . The need for the simultaneous presence of several proinflammatory cytokines to produce maximal iNOS production ensures that the iNOS protein within smooth muscle is only produced as part of a vigorous inflammatory response, hopefully near the site of the inflammatory stimulus. The current study has begun to unravel the molecular mechanisms underlying this cooperative effect.


FOOTNOTES

*
This work was supported in part by the award of a Medical Research Council Clinician/Scientist fellowship (to T. J. E.), and through a grant from the Biotech Program of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Du Cane Rd., London W12 0NN, UK. Tel.: 44-181-740-3243; Fax: 44-181-40-3394; tevans@rpms.ac.uk.

(^1)
The abbreviations used are: NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; VSM, vascular smooth muscle; IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; EMSA, electrophoretic mobility shift assay; PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; ISRE, IFN stimulus response element; GAS, -IFN-activated site; IRF, IFN regulatory factor; NF, nuclear factor.

(^2)
L. Heerdt, personal communication.

(^3)
T. J. Evans, unpublished data.

(^4)
J. Spink, J. Cohen, and T. J. Evans, unpublished observations.


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