From the Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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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 cytokine-stimulated 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
.
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INTRODUCTION |
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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 (2-6). 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, 17-19), 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 3.8 kilobases (kb) in the 5'-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
85 to
76 base pairs (bp) (24) and an interferon regulatory factor-1
(IRF-1) site at positions
923 to
913 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
5'-flanking region is not sufficient for iNOS induction. Instead,
inducible NF-
B 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-
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.
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EXPERIMENTAL PROCEDURES |
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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) + IL-1
(100 units/ml) + IFN
(250 units/ml) in the presence or absence of the
NF-
B 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 (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 5'-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 +33 to 7242 kb (GenBankTM 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 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 (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 MgCl2, 10 mM KCl, 25 µg/ml chymostatin, 25 µg/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 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 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) and adding 50 µl 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 × 2 min, 65 °C × 15 min, 37 °C × 15 min, 23 °C × 15 min, and 4 °C × 15 min. Probes were end-labeled with [-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 mM
MgCl2, 2.5% glycerol, 2% Ficoll, and 21 mM
dithiothreitol (final volume of 30 µl). 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 0.4× TBE
running buffer (450 mM Tris borate and 1 µM
EDTA, pH 8.0). After electrophoresis, gels were dried and subjected to
autoradiography.
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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 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-
B 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-
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(mut7.2
B)Luc in order to further confirm the mutation, and
similar results were obtained regardless of the method used to create
piN0S(mut7.2
B)Luc. To reduce errors in the reactions, PCR was
performed using the Expand Long Template PCR system (Boehringer Mannheim). The piNOS(mut7.2
B)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-
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 µg 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 < 0.01.
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RESULTS |
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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.
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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.
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The NF-B Element at
115 to
106 bp Is Not Required for
Cytokine Induction of the Human iNOS Promoter--
An NF-
B element
at
85 bp upstream in the murine iNOS promoter has been shown to
confer LPS responsiveness in the murine iNOS gene (24). Analysis of the
5'-flanking region of the human iNOS gene revealed a putative NF-
B
element located at
115 to
106 bp that differs by only one
nucleotide from the functional murine NF-
B element at
85 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(7.2
B)Luc) and a mutated
NF-
B construct (piNOS(mut7.2
B)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
(
115 to
106 bp) (piNOS(mut7.2
B)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
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-
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.
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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 3.8
kb in the 5'-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 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 (16). 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.
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DNA Sequence Analysis from 3.8 to
7.2 kb Reveals Putative
Cis-regulatory NF-
B Elements, Which May Confer Cytokine
Inducibility--
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 (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
115 bp, we
sequenced the 5'-flanking region from
3761 to
7242 kb looking for
other specific NF-
B sites. Computer analysis revealed five potential
NF-
B sites in the functionally active upstream region. Fig.
5 depicts the sequence of
the human iNOS 5'-flanking region from
3.8 to
7.2 kb and localizes
the five putative NF-
B sites at
5219 (
5.2 kb),
5467 (
5.5
kb),
5808 (
5.8 kb),
6080 (
6.1 kb), and
6476 (
6.5 kb), which
are located upstream of
4.7 kb. Also shown are the relevant
restriction enzyme sites and putative NF-
B sites between
3.8 and
4.7 kb, which are non-functional by deletional analysis.
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Mutational Analysis of Upstream NF-B Sites Reveal That Four
NF-
B Sites Are Requisite for Cytokine Induction of the Human iNOS
Promoter--
To determine whether any of the putative NF-
B
elements in the upstream region between
4.7 and
7.2 kb were
functional, we used site-directed mutagenesis to generate five
additional 7.2-kb constructs, each with a 2-base point mutation in the
core sequence of each NF-
B element (as shown in Fig. 6). The NF-
B
mutant at site
6.5 kb retained the full CM inducibility typically
seen with the 7.2-kb construct. Mutation
of the cis-regulatory sites at
5.2,
5.5, and
6.1 kb resulted in
loss of inducible promoter activity of 60%, 45%, and 65%,
respectively. Interestingly, the mutation at
5.8 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
5.8 kb is absolutely required for iNOS promoter activity and that the
sites at
5.2,
5.5, and
6.1 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
5.8 kb in the 5'-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
5.8 kb, providing further evidence that this site is important for
cytokine-induced iNOS expression (Fig.
7).
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NF-B Proteins Bind to the iNOS
5.8 kb Promoter
Element--
To determine whether nuclear proteins could bind to the
sequence at
5.8 kb, EMSA was performed on nuclear extracts of
CM-stimulated AKN-1 and A549 cells using an oligonucleotide containing
the NF-
B sequence at
5808 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
+ TNF
+ 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
5808 is composed of both p50 and p65 proteins in AKN-1 cells.
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DISCUSSION |
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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 5'-flanking region
upstream of
4.7 kb. The cytokine-responsive region from
4.7 to
7.2 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
4.7 to
7.2 kb, four
were shown to be functional by mutational analysis. The site at
5.8 kb is required for both basal and cytokine-induced promoter activity, whereas the sites at
5.2,
5.5, and
6.1 kb exert a cooperative effect on cytokine-stimulated iNOS expression. This work identifies a
unique far upstream cytokine-responsive enhancer region from
5.2 to
6.1 kb in the 5'-flanking region of the human iNOS gene and
underscores one of the major differences between the human and murine
iNOS promoters.
Contained within ~1.7 kb of the murine iNOS 5'-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 7.2 kb of the human iNOS promoter. Previous work by
Xie et al. (24) and Murphy et al. (23) determined that both the proximal NF-
B at
85 bp and the upstream NF-
B at
974 bp in the murine iNOS promoter are functionally important in
LPS-stimulated RAW 264.7 cells. The upstream NF-
B is also responsible for the responsiveness to triple cytokine induction in
murine vascular smooth muscle cells (28). In contradistinction to the
murine system, neither deletion or mutation of the NF-
B site at
115 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 5'-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 3.8 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 5'-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
4.7 kb, that they function in a position-independent
fashion, and that they consist of a novel enhancer region of ~800 bp,
which contains four functional NF-
B sites. Sequence analysis of the 5'-flanking region from ~
1.0 to
3.8 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
5.8 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
7.2 kb that are
currently being investigated. Linn et al. (44) recently
reported an enhancer region from
8.7 to
10.7 kb in the human iNOS
promoter, which confers IL-1
and IFN
responsiveness in DLD-1
cells.
NF-B elements are present in the 5'-flanking regions of several
inflammatory response genes, including cell adhesion molecules ELAM-1,
VCAM-1, and ICAM-1 (45-49) 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 I
B
promoter. Our
data localize multiple NF-
B elements in the human iNOS gene to a
segment of DNA that spans ~800 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
5.2,
5.5, and
5.8 kb, we observe a 2-3-fold increase in
luciferase activity. The addition of the functional element at
6.1 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
5.2 to
6.1 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.
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ACKNOWLEDGEMENTS |
---|
We thank Debra Williams for excellent
technical assistance. We thank Hideaki Tahara for the kind gift of the
plasmid containing the -galactosidase gene.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM-52021 (to D. A. G.), GM-37753 (to R. L. S.) and GM-44100 (to T. R. B.) and a scholarship from the Association of Academic Surgery (to B. S. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF049872.
To whom correspondence should be addressed: 497 Scaife Hall, Dept.
of Surgery, University of Pittsburgh, Pittsburgh, PA 15261. Tel.:
412-648-1935; Fax: 412-624-1172; E-mail: bsta{at}med.pitt.edu.
1
The abbreviations used are: iNOS,
inducible nitric oxide synthase; LPS, lipopolysaccharide; kb, kilobase
pair(s); bp, base pair(s); PCR, polymerase chain reaction; IFN,
interferon-
; IRF, interferon regulatory factor; ANOVA, analysis of
variance; IL, interleukin; TNF
, tumor necrosis factor-
; CM,
cytokine mixture; EMSA, electrophoretic mobility shift assay;
PDTC, pyrrolidine dithiocarbamate; DDTC, diethyldithiocarbamate.
2 H. Kleinert and U. Forstermann, personal communication.
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REFERENCES |
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