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Department of Internal Medicine, University of Innsbruck, Innsbruck, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsReferences |
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and/or LPS, was reduced by iron, and
was enhanced by the iron chelator desferrioxamine. Introduction of a
double mutation into the NF-IL6 binding site (-153/-142) of an iNOS
promoter construct resulted in a reduction of IFN-/LPS inducibility
by >90% and also impaired iron mediated regulation of the iNOS
promoter. Our results provide evidence that this NF-IL6 binding site is
of central importance for maintaining a high transcriptional rate of
the iNOS gene after IFN-/LPS stimulation, and that NF-IL6 may
cooperate with hypoxia inducible factor-1 in the orchestration of
iron-mediated regulation of iNOS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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, TNF-
, IL-1ß, and/or LPS. NO
formation is a major effector mechanism of macrophages against invading
micro-organisms and tumor cells, and many of the cytotoxic actions of
NO can be referred to its high affinity to iron (7). As an example of
this NO exerts cytostatic effects toward target cells by direct
interference with the catalytic iron-combining centers of enzymes in
the citric acid cycle, mitochondrial respiration, or DNA synthesis such
as aconitase, succinate oxidoreductase, or ribonuclotide reductase (8, 9). Moreover, NO directly affects the regulation of cellular iron
homeostasis via its stimulatory effect on the binding activity of iron
regulatory proteins to cis-acting RNA stem loop structures,
the so-called iron-responsive elements, within the untranslated regions
of ferritin and transferrin receptor mRNA, thus controlling the
expression of those proteins at the translational/post-transcriptional
level (10, 11). On the contrary, iron by itself regulates the
expression of iNOS transcriptionally (12). These data provided evidence
for the existence of an autoregulatory feedback loop in macrophages
that links maintenance of iron homeostasis with optimal formation of NO
for host defense (13). However, the underlying mechanism by which iron
exerts transcriptional regulation of iNOS has remained elusive to date,
although recent data suggested that activation of hypoxia inducible
factor-1 (HIF-1) may be involved (14).
The murine iNOS promoter contains numerous consensus sequences for
known transcription factors. These are located in two clusters: one
called region I (ranging from +10 to -300 bp upstream of the TATA
box), and the other called region II (-1100 to -800 bp) (15, 16).
Region II was supposed to be primarily important for IFN--mediated
induction of iNOS because it contains binding sites for IFN regulatory
factor-1 (IRF-1), STAT1, and NF-
B (17, 18, 19). On the other hand,
region I has been shown to be the principal target region for
LPS-mediated iNOS induction since it contains three NF-IL6 binding
sites, one TNF response element, one NF-B binding site, and one
octamer binding site. A number of groups have demonstrated the
importance of this proximal promoter region for LPS-mediated induction
of murine iNOS transcription (15, 16, 20).
This study was initiated to identify the regulatory regions and transcription factors responsible for iron-mediated regulation of iNOS in cytokine-stimulated macrophage-like cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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The J774A.1 murine macrophage cell line and the murine
fibroblast cell line NIH3T3 were obtained from the American Type
Culture Collection (Manassas, VA). Cells were grown in DMEM
supplemented with 10% heat-inactivated FCS (very low endotoxin,
Biochrom-Seromed, Berlin, Germany), 2 mM L-glutamine, 100
U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were then
supplemented with 100 µM ferric iron (Fe(3+); applied as
FeCl3·6H2O) or 200 µM desferrioxamine (all
from Sigma, Munich, Germany) before stimulation with 100 U/ml murine
rIFN- (sp. act., 107 U/mg; Life Technologies, Vienna,
Austria) and/or with up to 10 µg/ml Escherichia coli LPS
(serotype 055:B5, Sigma) or were left untreated.
Preparation of nuclear extracts
J774A.1 cells were grown to 50% confluence, then seeded at a
density of 5 x 106/culture flask and left untreated
overnight. Previous to stimulation cells were washed with fresh medium
and pretreated with iron and desferrioxamine for 30 min. After
stimulation with IFN- and/or LPS for 0.5–24 h, cells were washed
twice with prewarmed PBS and harvested by scrabbing, and nuclear
extracts were prepared as previously described (21).
EMSA
For performing EMSAs different oligonucleotide probes,
representing standard consensus sequences of known transcription
factors or specific sequences within the murine iNOS promoter, were
used: NF-IL6-153/-142 sense, 5'-CCACAGAGTGATGTAATCA-3';
antisense, 5'-GTGCTT GATTCCATCACTCTG-3'; NF-IL6-153/-142m
sense, 5'-CCACAGAGTGAAATAATCA-3'; antisense,
5'-GTGCTTGATTATTTCACTCTG-3'; NF-IL6cons. sense,
5'-AAGCTGCAGATT GCGCAATCTGCA-3'; antisense,
5'-TGCTGCAGATTGCGCAATCTGCA-3'; and NF-B sense,
5'-AGCTTCAGAGGGGACTTTCCGAGAGG-3'; antisense, 5'-TCGACCTCTCGGA
AAGTCCCCTCTG-3'. For preparation of double-stranded probes oligomers
were annealed, and overhanging ends were filled with
[-32P]dCTP (Amersham, Aylesbury, U.K.) and the three
other corresponding nonradiolabeled dNTPs (from Pharmacia Biotech,
Piscataway, NJ) using the Klenow fragment of DNA polymerase I. For
preparation of unlabeled competitors dCTP instead of
[-32P]dCTP was used. Nuclear proteins (10 µg) were
preincubated with 2 µg of double-stranded poly(dI-dC)·poly(dI-dC)
(Pharmacia Biotech) on ice for 10 min before addition of 2 ng of the
radiolabeled oligonucleotide probe (sp. act., 50,000 cpm/ng). The DNA
binding reactions were conducted in the presence of 200 mM HEPES (pH
7.8), 10 mM EDTA, and 10 mM DTT for 30 min at room temperature (final
volume, 10 µl). After addition of 50 ng of heparin to the binding
reaction and subsequent incubation at room temperature for 10 min, 1.7
µl of 87% glycerol were added, and samples were analyzed on a 6%
nondenaturing polyacrylamide gel (22).
For competition studies a 30- to 50-fold molar excess of unlabeled double-stranded oligonucleotides was added to the nuclear extracts 10 min before addition of the radiolabeled oligonucleotide probe. For supershift assays, nuclear proteins (10 µg) were preincubated with 1 µg of a specific rabbit polyclonal IgG Ab (Santa Cruz Biotechnology, Santa Cruz, CA) on ice for 1 h before addition of the radiolabeled oligonucleotide probe.
Plasmid construction and transient transfection assays
A 1749-bp HincII fragment, corresponding to the
5'-flanking region of murine iNOS fused to a promoterless
chloramphenicol acetyltransferase (CAT) reporter gene (p1iNOS-CAT) (5)
and mutants thereof, p10.5iNOS-CAT, with a deletion of the 5' region up
to -975 bp, and p1(IRFm), with a site-specific mutation in
the IRF-1 binding site (at position -920) (17) were provided by Drs.
E. Martin, Q.-w. Xie, and C. Nathan (Cornell University, Ithaca, NY).
Further deletion mutants were constructed by stepwise digestion of the
5' region of the iNOS promoter using restriction endonucleases as
indicated below (Fig. 1
). Progressive
deletions of the 5' region were obtained using the restriction enzyme
NheI up to position -764 bp (p1del
NheI-764), BstXI up to position
-206 bp (p1del BstXI-206), and PstI
up to position -47 bp (p1del PstI-47),
respectively. Moreover, the 1.6-kb full-length promoter fragment
(-1588 to +15 bp) and further deletion mutants were generated by PCR
and subcloned into the pGL3 promoterless firefly luciferase reporter
gene vector (pGL3-basic, Promega, Madison, WI). Point mutations of the
transcription factor consensus sequences NF-IL6-153/-142
and the HRE-224/-216 were introduced by site-directed
mutagenesis via overlap extension using PCR (23) and then subcloned
into the pGL3-basic vector. All plasmids were checked for accuracy by
sequencing.
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DNase I footprinting
DNase I footprinting was conducted as described by Leblanc and Moss (24). Nuclear extracts were prepared as outlined above. Probes for DNase I footprinting representing position -220 to +14 bp (relative to the transcription start site) of the murine iNOS promoter were generated by PCR. The binding reaction was performed in a total volume of 50 µl containing 2–3 ng of end-labeled DNA fragment (15,000 cpm), 10 µg of nuclear extracts, and 2 µg of competitor double-stranded poly(dI-dC)·poly(dI-dC). DNase I (from bovine pancreas, Sigma) digestion was conducted with 0.005 and 0.02 Kunitz units for naked DNA and 0.08 and 0.16 Kunitz units for DNA and proteins at room temperature for 2 min. Probes were then analyzed on a sequencing gel (6% PAGE, 7 M urea, and 1x TBE). To localize the position of the footprint, G and G+A chemical sequencing ladders of the same end-labeled DNA probe were generated as previously described (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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In cells transiently transfected with a plasmid containing the
whole murine iNOS promoter (p1iNOS-CAT) CAT activity is inducible upon
treatment with IFN-/LPS. This 2.5-fold induction compared with that
in the untreated cells was reduced after the treatment of cells with
iron, but was significantly enhanced by addition of the iron chelator
desferrioxamine (DFO; p < 0.01). These data are in
agreement with our previous results, showing transcriptional regulation
of iNOS expression by iron perturbation (12). We then tried to identify
the region within the iNOS promoter that is responsible for
iron-mediated regulation of the iNOS promoter. Recent reports suggested
that IRF-1 may be the central player in cytokine-mediated iNOS
expression (17, 18, 26). Since knockout of IRF-1 resulted in dramatic
reduction of iNOS expression without any effect on MHC class II
expression (18), an observation consistent with the findings observed
after iron perturbations (12), we investigated an iNOS promoter
construct bearing a mutation within the IRF-1 binding region
(p1IRFm). Although, inducibility by IFN-/LPS was reduced
compared with that of the parent plasmid (p1iNOS-CAT), regulation of
promoter activity by iron perturbation was still present (Fig. 1
), thus
excluding IRF-1 as a potential candidate for iron-mediated regulation.
Therefore, we performed a series of further deletions of the 5' region
of the iNOS promoter. Interestingly, deletions of the region of the
iNOS gene down to position -206 bp (p1del
BstXI-206) still showed inducibility by
IFN-/LPS and regulation by iron and DFO, although to a lesser extent
than with the parent plasmid (p1iNOS-CAT, Fig. 1). Further deletion of
the iNOS promoter down to position -47 bp (p1del
PstI-47) resulted in loss of both IFN-/LPS
inducibility and iron-mediated regulation of CAT activity (Fig. 1).
Structural and functional analysis of the 5'-flanking region -220 to +15 bp of the murine iNOS gene by DNase I footprinting
Since our transient transfection experiments indicated that the
region between -206 and -47 bp upstream from the transcriptional
start site of the iNOS promoter may bear the regulatory motif that
mediates regulation of iNOS expression by cytokines and iron, we
conducted DNase I footprinting analysis to identify a transcription
factor responsible for this. DNase I footprinting analysis of this
region with nuclear extracts from J774A.1 cells after treatment with
IFN-/LPS revealed an inducible protection between position -160 to
-140 bp of the iNOS promoter (Fig. 2,
compare lane 4 with lane 3). This protection
peaked after 4 and 8 h of cytokine stimulation, but could not be
observed after treatment of cells for shorter periods (2 h; data not
shown). A protection in the DNase I footprint assay is indicative for
an interaction of a transcription factor with its DNA target sequence.
The interaction of this protein with the iNOS promoter was weakened
upon addition of iron and enhanced by DFO (Fig. 2, compare lanes
4, 5, and 6).
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To investigate whether the observed protection in the footprint
analysis can indeed be referred to inducible binding of a transcription
factor we next performed EMSA with labeled oligonucleotides
representing the protected region (-140 to -160 bp) within the iNOS
promoter. Using the same nuclear extracts as that for footprint
analysis we identified IFN-- and LPS-inducible binding of a protein
to this region (Fig. 3A).
Treatment with IFN- or LPS resulted in induction of protein binding
to the labeled DNA oligonucleotide compared with the untreated control,
and both stimuli when applied together were synergistic in this
respect. Nevertheless, perturbation of cells with iron decreased
binding of this protein to its DNA sequence, while DFO even enhanced
protein/DNA interaction. Notably, this effect was less pronounced when
cells were treated with IFN- and LPS at the same time, indicating
that a strong stimulus may weaken regulation by iron perturbation (Fig. 3A). Finally, this protein/DNA interaction could be
completely abolished by competition with the cold oligonucleotide
construct of the same region, pointing to the specificity of the
observed protein/DNA interaction (Fig. 3A).
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, and NF-B, could potentially bind to this region.
Upon EMSA supershift analysis only an anti-NF-IL6 (C/EBPß) Ab
produced a supershift (Fig. 3B), while no altered migration
of protein/DNA complexes was observed upon incubation of nuclear
extracts with anti-NF-B-p50, anti-NF-B-p65,
anti-RelC, or anti-STAT1 Abs (data not shown). The complex
supershifted with anti-NF-IL6 Ab was completely abolished by
competition with a 30-fold excess of the appropriate unlabeled duplex
oligonucleotide, now called NF-IL6-153/-142 (Fig. 3B, compare lanes 3 and 4). The
specificity of this interaction was further sustained by the
observation that an excess of cold oligonucleotide with a double
mutation within the NF-IL6 binding site, called
NF-IL6-153/-142m, was not able to compete the supershift
(Fig. 3B, lane 5).
To further confirm the specific involvement of the transcription factor
NF-IL6 we performed EMSAs using nuclear extracts from J774.A1 cells and
a synthetic oligonucleotide bearing the NF-IL6 consensus sequence
(NF-IL6cons.; Fig. 3B, lanes 6–11).
As with the NF-IL6 sequence within the iNOS promoter
(NF-IL6-153/-142), stimulation of cells with IFN-
and/or LPS induced binding of the protein to the NF-IL6 consensus DNA
motif (Fig. 3B, lanes 6 and 7).
Addition of a polyclonal Ab against NF-IL6 was able to supershift this
protein/DNA complex (Fig. 3B, lane 8). DNA
binding of supershifted NF-IL6 was completely abolished upon
competition with a 30-fold excess of the appropriate unlabeled
consensus NF-IL6 oligonucleotide (NF-IL6cons.) and with a
30-fold excess of cold NF-IL6-153/-142, but not with the
mutated NF-IL6-153/-142m oligonucleotide (Fig. 3B, lanes 9–11).
Our data obtained to date indicated that the protein binding to the
iNOS promoter as shown by in vitro footprints and EMSAs, which is both
cytokine inducible and iron regulated, is NF-IL6. However, since the
NF-IL6 Ab was not sufficient to supershift all protein/DNA binding
complexes with the NF-IL6-153/-142 oligonucleotide (Fig. 3B, lane 3), it is possible that other
unrecognized transcription factors may also bind to this region,
although to a much lesser extent than NF-IL6.
Functional analysis of the NF-IL6-153/-142 promoter
element for IFN- and/or LPS inducibility and its regulation by iron
To determine whether iron-mediated regulation of NF-IL6 binding to
its consensus sequence NF-IL6-153/-142 accounts for
altered expression of iNOS we constructed a series of new plasmids and
performed transient transfections. When transfecting cells with a
plasmid containing the region from -230 to +15 bp (piNOS-230) of the
iNOS promoter, we found inducibility by IFN-/LPS, which was in
agreement with our previous data (Fig. 4). However, iNOS promoter activity in
response to IFN-/LPS was reduced by about 30% upon deletion of the
5' end between -230 and -1588 bp compared with the parent plasmid
(piNOS-LUC) and to a lesser extend with IFN- and LPS when applied
alone (Fig. 4). Beside the NF-IL6 motif at position -153 to -142 bp,
the plasmid piNOS-230 also contains a hypoxia-responsive element
(position -224 to -216 bp; HRE-224/-216) that binds
HIF-1. The latter has been shown previously to participate in iron- and
IFN--mediated regulation of iNOS in ANA-1 macrophages (14). To
investigate the impact of these two transcription factors on the
induction of iNOS in response to IFN- and/or LPS treatment we first
transfected cells with a plasmid containing a double mutation in the
HIF-1 binding site. As shown in Fig. 4 no difference in the stimulatory
potential of IFN- and/or LPS on iNOS promoter activity was observed
compared with that in cells transfected with the plasmid without the
mutation. When we introduced a mutation into the NF-IL6 site while HRE
remained unaffected, we observed an almost complete loss of IFN-/LPS
inducibility of the promoter (Fig. 4). The same was observed by further
deletion of the promoter down to position -140 bp, thus eliminating
the NF-IL6 site between -153 to -142 bp (Fig. 4). This indicated that
NF-IL6 is essential for IFN-/LPS-mediated induction of iNOS.
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/LPS. Using the piNOS-230
plasmid for transfection of NIH-3T3 cells, which were then stimulated
with IFN- and/or LPS, we found hyperinducibility of promoter
activity upon treatment with DFO by a factor between 2.1 and 3.3 (Fig. 5, A–C). However, in this
setting iron had no effect on IFN-/LPS-treated cells, which could
probably be due to the high iron content of the medium supplemented
with FCS (Fig. 5). When cells were transfected with a plasmid bearing a
double mutation within the HRE region (piNOS-230HREm), we
observed a reduction in the stimulatory potential of DFO on luciferase
activity by about 30–40% compared with that of the parent plasmid
(piNOS-230). However, the difference in luciferase activity between
IFN-/LPS and IFN-/LPS plus DFO in cells transfected with
piNOS-230HREm was still significant
(p < 0.05). A similar observation was made
when cells were transfected with the plasmid piNOS-170 (Fig. 5).
Transfection of cells with the NF-IL6 mutated construct
(piNOS-230NF-IL6 m) reduced inducibility by cytokines by
>90% as shown above (Fig. 4). However, at this very low level,
treatment of piNOS-230NF-IL6 m-transfected cells with
IFN-/LPS plus DFO increased the albeit little promoter activity by
2.5-fold compared with stimulation with IFN-/LPS alone (Fig. 5A). This phenomenon should be due to activation of HRE
binding affinity by DFO (14), since a double-knockout deletion of HRE
and NF-IL6 by using the plasmid piNOS170NF-IL6m failed
to show even this little of an effect of the drug (Fig. 5). Regulation
of promoter activity by DFO was similar in cells treated with IFN-
or LPS alone and in those treated with a combination of both (Fig. 5).
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and
along with other transcription factors, such as HIF-1 and NF-IL6,
mediates iron regulation of the iNOS gene (Figs. 4 and 5). Discussion
In the present study we investigated the underlying mechanism responsible for the previously observed transcriptional regulation of iNOS by iron perturbation (12). By means of transient transfections, deletion, and mutational analysis and in vitro DNase I footprint, gel shift, and supershift assays, we could identify a trans-activating factor that targets to a NF-IL6 consensus motif within the murine iNOS promoter and whose DNA binding affinity is modulated by iron.
Even more importantly, NF-IL6 appears to be essential for
cytokine/LPS-mediated induction of iNOS, since mutation of the
NF-IL6-153/-142 binding motif almost fully abolished
inducibility of iNOS promoter by IFN- and/or LPS.
NF-IL6, a member of the CCAAT/enhancer binding protein (C/EBP) family
of transcription factors, belongs to a class of DNA binding proteins
called basic leucine zipper protein family, which includes C/EBP,
C/EBPß (NF-IL6), C/EBP, and C/EBP
(NF-IL6ß) (29). These
proteins are all characterized by a leucine zipper domain and a
DNA-binding basic region located in the C-terminus of the proteins.
Members of the C/EBP family can associate through the leucine zipper
domain to form homo- and heterodimers with each other and bind with
similar affinity to various C/EBP binding sites (27, 28, 29).
The binding affinity of NF-IL6 to its DNA consensus sequence is induced
in various tissues after stimulation with LPS or proinflammatory
cytokines such as IL-1, TNF-, and IL-6 (30, 31). NF-IL6 expression
is also induced during macrophage differentiation. NF-IL6 consensus
binding motifs are found in the functional regulatory regions of genes
that are induced in activated macrophages, such as IL-6, IL-1, IL-8,
TNF-, G-CSF, lysozyme genes, and iNOS (15, 32, 33, 34).
Interestingly, the data presented here demonstrate that NF-IL6 binding
to the iNOS promotor is induced by LPS and IFN-; however, the latter
stimulus is not able to sufficiently stimulate the induction of iNOS at
least in transiently transfected NIH3T3 or J774A.1 cells, which is in
accordance with previously published data (35). This implies that
although NF-IL6 appears to be an essential component for iNOS
expression as shown by our mutational analysis, other transcription
factors must be activated in addition to gain full induction of iNOS
expression. This is not surprising, since numerous consensus binding
sequences for known transcription factors have been identified within
the iNOS promoter. While the essential transcription factor for
induction of iNOS by IFN- is IRF-1 (19), numerous
trans-activating factors have been identified to transduce
LPS-mediated stimulation of iNOS (15, 16, 20, 36). Beside binding of
transcription factor NF-B/Rel to promoter elements (37) at position
-85 to -76 bp (15, 16, 20) or position -971 to -962 bp (15, 16),
other LPS-responsive elements within the iNOS promoter, e.g., at
position -62 to -56, and a binding site for an octamer-like binding
protein in an as yet unidentified complex with other factors have been
identified (20, 36).
Although NF-B/Rel was suggested to be essential for the LPS-mediated
induction of iNOS, it is not sufficient (37, 38). It has become
increasingly evident that combinatory effects of transcription factors
are very important in gene regulation. NF-B frequently associates
with other transcription factors to induce gene expression (39). NF-IL6
could be a candidate for this. Evidence has been provided for a
functional and physical interaction between NF-IL6 and NF-B (40, 41, 42)
through cooperative binding of the two transcription factors (41),
direct associations between Rel and C/EBP proteins (41, 43), or
specific interactions between these proteins and the basal
transcription machinery (44, 45). It was shown that C/EBP and Rel
proteins activate the IL-12/p40 promoter in a synergistic manner, as
specific mutations in either recognition site reduced promoter activity
considerably (46). Accordingly, both the C/EBP factors and NF-B
cis sequences have been identified in the regulatory regions
of many genes involved in inflammation and immune regulation (47).
Therefore, NF-IL6 may cooperate with NFB to maintain full iNOS
induction. As suggested by other authors, NF-IL6 could maintain a high
transcription rate of iNOS rather than triggering the initial induction
(48). This is in agreement with the observation that after cytokine/LPS
stimulation of cells the activation of NF-IL6 occurs later than that of
NF-B. Therefore, NF-B could act as a first inducer of iNOS
transcription, and this stimulus is preserved by the action of NF-IL6.
This is in agreement with the induction kinetics of the acute phase
genes (1-acid glycoprotein and serum amyloid A and P) by
LPS, where STAT3 functions as an inducer of transcription, and NF-IL6
then maintains a high rate of transcription (49). After stimulation of
cells with LPS the binding affinity of NF-B p50/p65 and p50/c-Rel to
the iNOS promoter peaks at 30 min after treatment and is then decreased
(37); however, synthesis of iNOS mRNA continues for >24 h (2, 50). In
contrast, induction of NF-IL6 binding to the iNOS promoter, as shown by
our DNase I footprint analysis, occurs at later time points, with
maximum binding affinities 8 h after cytokine/LPS stimulation of
J774.A1 cells. This would fit into the hypothesis that NF-B acts as
an inducer of iNOS gene transcription, and NF-IL6 maintains the active
transcription state.
Recent work has demonstrated that macrophages from
NF-IL6-/- mice produce similar amounts of NO after
stimulation with IFN-/LPS as those from NF-IL6+/+ mice
(51). However, this does not automatically exclude the potential
importance of NF-IL6 for iNOS transcription in vivo, since, as
suggested by these authors, other members of the C/EBP transcription
factor family, such as NF-IL6ß (52), could overcome a lack of NF-IL6,
thus leading to similar transcriptional activities toward target genes
such as iNOS.
Moreover, NF-IL6 participates in iron-mediated regulation of iNOS as
shown herein and may cooperate with other transcription factors, such
as HIF-1, in this respect. The binding of HIF-1 to target sequences has
been shown to be inducible by hypoxia and DFO (14), and it has been
suggested to be a major player in iron-mediated regulation of iNOS, at
least in ANA-1 cells stimulated with IFN- alone (14). This results
were also confirmed by us; however, knockout of HRE still showed
significant regulation of iNOS by iron perturbation, which is mainly
due to modulation of NF-IL6 binding to its consensus sequence upon iron
depletion (Fig. 5). In contrast, the binding affinity of NF-B to the
iNOS promoter appears to be unaffected by iron perturbations (data not
shown).
However, DFO when combined with IFN- was able to induce iNOS
promoter activity even when IFN- alone was quite uneffective.
Nevertheless, DFO when applied alone did not have a direct effect on
transcriptional iNOS expression as shown by nuclear run-off analysis
(12, 14), iNOS mRNA stability (12), or iNOS enzymatic activity (12).
Moreover, all these experiments provided evidence that the modulating
effects of DFO on iNOS mRNA expression are exclusively due to the
transcriptional mechanism investigated in this paper and are not due to
modulation of iNOS mRNA half-life by the drug (12). Thus, DFO may not
be a direct inducer of gene transcription in the absence of
inflammatory stimuli. More likely, DFO acts as a costimulus able to
modulate or strengthen a signal induced by a cytokine or LPS and thus
contributes to gene expression via its stimulatory effect on binding of
transcription factors. The latter effect could be due in part to the
potential of DFO to inhibit iron-catalyzed formation of radicals (14, 53), since radicals are also involved in activation/deactivation of
transcription factors (54). This is in accordance with the data showing
that DFO induces HIF-1 binding activity and could thus be a costimulus
for the activation of iNOS expression in IFN--treated macrophages
along the hypoxia pathway.
In summary, our data demonstrate that the NF-IL6-153/-142 element in the iNOS promoter plays a central role for the induction of iNOS gene expression in response to cytokine stimulation, and that it is centrally involved in iron-mediated regulation of the iNOS promoter.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Guenter Weiss, Department of Internal Medicine, University Hospital, Anichstr. 35, A-6020 Innsbruck, Austria. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; HIF-1, hypoxia-induced factor-1; IRF, IFN regulatory factor; CAT, chloramphenicol acetyltransferase; HRE, hypoxia-responsive element; DFO, desferrioxamine; LUC, luciferase; C/EBP, CCAAT enhancer binding protein.
Received for publication December 1, 1998. Accepted for publication February 18, 1999.
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 F. B. Sow, W. C. Florence, A. R. Satoskar, L. S. Schlesinger, B. S. Zwilling, and W. P. Lafuse Expression and localization of hepcidin in macrophages: a role in host defense against tuberculosis J. Leukoc. Biol., October 1, 2007; 82(4): 934 - 945. [Abstract] [Full Text] [PDF]
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 G. Y. Suh, Y. Jin, A.-K. Yi, X. M. Wang, and A. M. K. Choi CCAAT/Enhancer-Binding Protein Mediates Carbon Monoxide-Induced Suppression of Cyclooxygenase-2 Am. J. Respir. Cell Mol. Biol., August 1, 2006; 35(2): 220 - 226. [Abstract] [Full Text] [PDF]
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 W.-G. Deng, S.-T. Tang, H.-P. Tseng, and K. K. Wu Melatonin suppresses macrophage cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52 acetylation and binding Blood, July 15, 2006; 108(2): 518 - 524. [Abstract] [Full Text] [PDF]
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 M. Torroella-Kouri, X. Ma, G. Perry, M. Ivanova, P. J. Cejas, J. L. Owen, V. Iragavarapu-Charyulu, and D. M. Lopez Diminished Expression of Transcription Factors Nuclear Factor {kappa}B and CCAAT/Enhancer Binding Protein Underlies a Novel Tumor Evasion Mechanism Affecting Macrophages of Mammary Tumor-Bearing Mice Cancer Res., November 15, 2005; 65(22): 10578 - 10584. [Abstract] [Full Text] [PDF]
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 S.-B. Lee and J. S. Schorey Activation and Mitogen-Activated Protein Kinase Regulation of Transcription Factors Ets and NF-{kappa}B in Mycobacterium-Infected Macrophages and Role of These Factors in Tumor Necrosis Factor Alpha and Nitric Oxide Synthase 2 Promoter Function Infect. Immun., October 1, 2005; 73(10): 6499 - 6507. [Abstract] [Full Text] [PDF]
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 K. K. Wu, J.-Y. Liou, and K. Cieslik Transcriptional Control of COX-2 via C/EBP{beta} Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 679 - 685. [Abstract] [Full Text] [PDF]
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E.-Y. Choi, E.-C. Kim, H.-M. Oh, S. Kim, H.-J. Lee, E.-Y. Cho, K.-H. Yoon, E.-A Kim, W.-C. Han, S.-C. Choi, et al. Iron Chelator Triggers Inflammatory Signals in Human Intestinal Epithelial Cells: Involvement of p38 and Extracellular Signal-Regulated Kinase Signaling Pathways J. Immunol., June 1, 2004; 172(11): 7069 - 7077. [Abstract] [Full Text] [PDF]
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 S. A. Godambe, K. M. Knapp, E. A. Meals, and B. K. English Role of vav1 in the Lipopolysaccharide-Mediated Upregulation of Inducible Nitric Oxide Synthase Production and Nuclear Factor for Interleukin-6 Expression Activity in Murine Macrophages Clin. Vaccine Immunol., May 1, 2004; 11(3): 525 - 531. [Abstract] [Full Text] [PDF]
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 S. Okada, S. Obata, M. Hatano, and T. Tokuhisa Dominant-negative effect of the c-fos family gene products on inducible NO synthase expression in macrophages Int. Immunol., November 1, 2003; 15(11): 1275 - 1282. [Abstract] [Full Text] [PDF]
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 H. Bowen, A. Lapham, E. Phillips, I. Yeung, M. Alter-Koltunoff, B.-Z. Levi, V. H. Perry, D. A. Mann, and C. H. Barton Characterization of the Murine Nramp1 Promoter: REQUIREMENTS FOR TRANSACTIVATION BY Miz-1 J. Biol. Chem., September 19, 2003; 278(38): 36017 - 36026. [Abstract] [Full Text] [PDF]
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G. Fritsche, M. Dlaska, H. Barton, I. Theurl, K. Garimorth, and G. Weiss Nramp1 Functionality Increases Inducible Nitric Oxide Synthase Transcription Via Stimulation of IFN Regulatory Factor 1 Expression J. Immunol., August 15, 2003; 171(4): 1994 - 1998. [Abstract] [Full Text] [PDF]
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H. Oexle, A. Kaser, J. Most, R. Bellmann-Weiler, E. R. Werner, G. Werner-Felmayer, and G. Weiss Pathways for the regulation of interferon-{gamma}-inducible genes by iron in human monocytic cells J. Leukoc. Biol., August 1, 2003; 74(2): 287 - 294. [Abstract] [Full Text] [PDF]
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J. D. Schilling, S. M. Martin, D. A. Hunstad, K. P. Patel, M. A. Mulvey, S. S. Justice, R. G. Lorenz, and S. J. Hultgren CD14- and Toll-Like Receptor-Dependent Activation of Bladder Epithelial Cells by Lipopolysaccharide and Type 1 Piliated Escherichia coli Infect. Immun., March 1, 2003; 71(3): 1470 - 1480. [Abstract] [Full Text] [PDF]
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 K Nishiya, H Wang, K Tahara, and K Hashimoto Enhancement by iron of interleukin 1 induced granulocyte macrophage colony stimulating factor (GM-CSF) production by human synovial fibroblasts Ann Rheum Dis, January 1, 2003; 62(1): 89 - 90. [Full Text] [PDF]
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K. Cieslik, Y. Zhu, and K. K. Wu Salicylate Suppresses Macrophage Nitric-oxide Synthase-2 and Cyclo-oxygenase-2 Expression by Inhibiting CCAAT/Enhancer-binding Protein-beta Binding via a Common Signaling Pathway J. Biol. Chem., December 13, 2002; 277(51): 49304 - 49310. [Abstract] [Full Text] [PDF]
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 E. Martelin, R. Lapatto, and K. O. Raivio Regulation of xanthine oxidoreductase by intracellular iron Am J Physiol Cell Physiol, December 1, 2002; 283(6): C1722 - C1728. [Abstract] [Full Text] [PDF]
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H. Bowen, T. E. Biggs, E. Phillips, S. T. Baker, V. H. Perry, D. A. Mann, and C. H. Barton c-Myc Represses and Miz-1 Activates the Murine Natural Resistance-associated Protein 1 Promoter J. Biol. Chem., September 13, 2002; 277(38): 34997 - 35006. [Abstract] [Full Text] [PDF]
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 J. Zoll, W. J. G. Melchers, J. M. D. Galama, and F. J. M. van Kuppeveld The Mengovirus Leader Protein Suppresses Alpha/Beta Interferon Production by Inhibition of the Iron/Ferritin-Mediated Activation of NF-{kappa}B J. Virol., August 28, 2002; 76(19): 9664 - 9672. [Abstract] [Full Text] [PDF]
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 X. Teng, D. Li, J. D. Catravas, and R. A. Johns C/EBP-{beta} Mediates iNOS Induction by Hypoxia in Rat Pulmonary Microvascular Smooth Muscle Cells Circ. Res., February 8, 2002; 90(2): 125 - 127. [Abstract] [Full Text] [PDF]
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E. M. Coccia, E. Stellacci, G. Marziali, G. Weiss, and A. Battistini IFN-{gamma} and IL-4 differently regulate inducible NO synthase gene expression through IRF-1 modulation Int. Immunol., July 1, 2000; 12(7): 977 - 985. [Abstract] [Full Text] [PDF]
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 V.C. Allport, D.M. Slater, R. Newton, and P.R. Bennett NF-{kappa}B and AP-1 are required for cyclo-oxygenase 2 gene expression in amnion epithelial cell line (WISH) Mol. Hum. Reprod., June 1, 2000; 6(6): 561 - 565. [Abstract] [Full Text] [PDF]
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A. S. Kristof, J. Marks-Konczalik, and J. Moss Mitogen-activated Protein Kinases Mediate Activator Protein-1-dependent Human Inducible Nitric-oxide Synthase Promoter Activation J. Biol. Chem., March 9, 2001; 276(11): 8445 - 8452. [Abstract] [Full Text] [PDF]
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W. Xiao, L. Wang, X. Yang, T. Chen, D. Hodge, P. F. Johnson, and W. Farrar CCAAT/Enhancer-binding Protein beta Mediates Interferon-gamma -induced p48 (ISGF3-gamma ) Gene Transcription in Human Monocytic Cells J. Biol. Chem., June 22, 2001; 276(26): 23275 - 23281. [Abstract] [Full Text] [PDF]
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X. Teng, D. Li, J. D. Catravas, and R. A. Johns C/EBP-{beta} Mediates iNOS Induction by Hypoxia in Rat Pulmonary Microvascular Smooth Muscle Cells Circ. Res., February 8, 2002; 90(2): 125 - 127. [Abstract] [Full Text] [PDF]
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