(Received for publication, December 31, 1996)
From the We have previously reported that a 19-base pair
element of the 5 Tissue hypoxia is a condition of decreased oxygen (O2)
levels that elicits homeostatic responses aimed at counteracting the negative effects of O2 depletion (1). Hypoxia induces the
expression of several genes, such as erythropoietin
(Epo)1 (2), vascular endothelial growth
factor (3), and glycolytic enzymes (4, 5) that favor the adaptation of
the organism to the decreased availability of O2.
Significant progress has been made in the understanding of the
molecular mechanism underlying hypoxia-inducible gene expression. A
hypoxia-responsive enhancer (HRE) has been identified in the
3 Nitric oxide (NO), a free radical gas generated by the enzyme nitric
oxide synthase, is involved in the antimicrobial and antitumor
activities of murine macrophages (12-14). Macrophage nitric oxide
synthase is the product of a transcriptionally inducible gene (iNOS),
whose maximal expression requires stimulation by IFN- Little is known about the signals that can modulate the inducibility of
iNOS by these different pathways. It has been recently shown that iron
can down-regulate iNOS transcription induced by IFN- We found that DFX induced binding activity to the HIF-1 consensus
sequence of the iNOS promoter and expression of the iNOS-HRE in the
murine macrophage cell line ANA-1. DFX, although ineffective by itself,
was also a potent co-stimulus of iNOS transcription and enhanced iNOS
mRNA expression in IFN- The mouse macrophage cell line ANA-1 was
established by infecting fresh bone marrow-derived cells from C57BL/6
mice with the J2 (v-raf/v-myc) recombinant
retrovirus (26, 27). ANA-1 macrophages were cultured in Dulbecco's
modified Eagle's medium (Whittaker Bioproducts, Walkersville, MD)
supplemented with 10% heat-inactivated fetal calf serum (HyClone
Laboratories, Logan, UT), 2 mM L-glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin (Life Technologies, Inc.) (complete medium). Cells were maintained at 37 °C in a humidified incubator containing 5% CO2 in
air. Mouse rIFN- Nuclear extracts were
prepared by modification of a standard protocol (29) as described
previously (23). Briefly, cells were washed twice with cold Dulbecco's
phosphate-buffered saline and pelleted by centrifugation at 1,200 rpm
for 5 min at 4 °C. The cell pellet was washed with buffer A (10 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2,
10 mM KCl) and incubated on ice for 10 min in buffer A. The
cell suspension was homogenized with a glass Dounce homogenizer, and
the nuclei were pelleted by centrifugation at 2,000 rpm for 5 min at
4 °C and resuspended in buffer C (0.42 M KCl, 20 mM Tris-HCl, pH 7.5, 20% glycerol, 1.5 mM
MgCl2). The suspension was mixed on a rotor at 4 °C for
30 min, and nuclear debris was pelleted by centrifugation for 30 min at
13,000 rpm. The supernatant was dialyzed against two changes of buffer
D (25 mM Tris-HCl, pH 7.5, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl) and clarified by
centrifugation at 15,000 rpm for 10 min at 4 °C. Aliquots were
frozen and stored at Probes were
generated using Klenow fragment of DNA polymerase and
[ The 1,749-bp fragment of the 5 ANA-1
macrophages were transfected by a modification of the DEAE-dextran
method (30), as described (23). Briefly, 10 µg of plasmid DNA were
added to 107 cells in 1 ml of Dulbecco's modified Eagle's
medium without serum and containing 250 µg/ml DEAE-dextran and 50 mM Tris-HCl, pH 7.5. The cells were incubated at 37 °C
for 1 h followed by a 2-min shock with 10% Me2SO at
room temperature. The cells were washed, plated in 6-well plates at
1 × 106/ml in 2.5 ml of complete medium, and
incubated at 37 °C in 5% CO2. Twenty-four hours later,
the cells were incubated with the appropriate stimulus for an
additional 18 h. The cells were then washed, resuspended in 0.25 M Tris-HCl, pH 7.5, and lysed by three cycles of rapid
freezing and thawing. The lysates were centrifuged (11,000 × g for 10 min), and the supernatants were assayed for CAT
activity by TLC (31). Protein content was determined as described by
Bradford (32), using the Bio-Rad Protein Assay. To control for
differences in the uptake of transfected DNA, cells were cotransfected
with 5 µg of pGL2 plasmid (pGL2 control, Promega), which contains the
luciferase reporter gene under control of SV40 promoter and enhancer.
Cell lysates were then assayed for luciferase activity.
Total cellular RNA was harvested and
processed as described previously (28). Briefly, cells were
solubilized with guanidine isothiocyanate, and the total cellular RNA
was purified by centrifugation through a cushion of cesium chloride.
Twenty µg of RNA were size-fractionated in a 1.2% agarose gel,
blotted onto a Nytran membrane (Schleicher & Schuell), and incubated
overnight at 42 °C in Hybrisol I hybridization solution (Oncor,
Gaithersburg, MD). The cDNA probes that were specific for mouse
macrophage nitric oxide synthase (33) or for human
glyceraldehyde-3-phosphate dehydrogenase (CLONTECH
Laboratories Inc.) were radiolabeled with [32P]dCTP
(Amersham Corp.) by using an RTS RadPrime DNA labeling system (Life
Technologies, Inc.) according to the manufacturer's procedure. The
blot was hybridized individually with the radiolabeled probes (1 to
2 × 106 cpm/ml) during an overnight incubation and
washed three times for 10 min at room temperature with 2 × SSC,
0.1% SDS and two times for 20 min at 60 °C with 0.2 × SSC,
0.1% SDS. The blot was autoradiographed at ANA-1 cells were treated as
indicated for 12 h, and nuclei were isolated as described (28).
Thawed nuclei were mixed with 150 µl of 2 × transcription mix
(1 × = 100 mM sucrose, 10% glycerol, 10 mM Tris-HCl, pH 8.0, 2.5 mM MgCl2,
2.5 mM dithiothreitol, 0.5 mM each of ATP, CTP,
and GTP) and incubated with 100 µCi of [32P]UTP (800 Ci/mmol; Amersham Corp.) at 30 °C for 30 min. Twenty µl of 100 mM CaCl2 and 20 units of RNase-free DNase I
were added and incubated 10 min at 30 °C with gentle mixing every 2 min. The nuclei were then lysed with 1 ml of Trizol (Life Technologies, Inc.), and the RNA was isolated according to the manufacturer's procedure. Approximately 2 × 106 cpm of RNA were used
in hybridization for 48 h with 2.5 µg of each slot-blotted
denatured plasmid (pGEM-3Z vector alone, vector containing the
3.9-kilobase cDNA insert of macrophage iNOS (33), and vector
containing a chicken To address
whether DFX induces DNA-binding activity to the iNOS-HRE, EMSA was
performed on nuclear extract prepared from ANA-1 macrophages treated
with medium or DFX, using as radiolabeled probe a 19-base pair (bp)
oligonucleotide (AB.2) containing the HIF-1 binding site of the iNOS
promoter. As shown in Fig. 1, ANA-1 macrophages
expressed a constitutive binding activity. Treatment with DFX caused
the appearance of an inducible complex that was specifically inhibited
by excess unlabeled probe (AB.2) but not by an unrelated probe (AB.1).
In addition, the DFX-inducible binding complex was not competed for by
a probe mutated in three bases within the putative HIF-1 binding site
(Mu.AB2). Both the constitutive and the DFX-inducible binding
activities were completely inhibited by a probe encompassing the
canonical HIF-1 binding site (Epo) of the Epo enhancer, which differs
from the AB.2 probe in five flanking bases. These results demonstrate
that DFX induces in macrophages a specific DNA-binding activity to the
HIF-1 binding site of the iNOS promoter.
To establish
whether DFX was a stimulus for the activation of the iNOS-HRE in
macrophages, ANA-1 cells were transiently transfected with two CAT
constructs containing three tandem copies of the iNOS-HRE, either wild
type (pBL-WT.iNOS) or mutated at the HIF-1 binding site (pBL-Mu.iNOS),
5
To determine whether DFX can be a stimulus for the
expression of iNOS mRNA, ANA-1 macrophages were incubated with
medium, IFN-
Nuclear run-on experiments were performed to
demonstrate that the increase in iNOS mRNA in response to IFN-
Transcriptional activation of the iNOS gene is blocked in the presence
of protein synthesis inhibitors (17, 28). To establish whether the
induction of iNOS mRNA expression by DFX was dependent on de
novo protein synthesis, ANA-1 macrophages were incubated in
medium, IFN- To identify the region(s) of the iNOS
promoter responsible for the DFX-dependent transcriptional
activation of the iNOS gene and, in particular, to investigate the
functional role of the iNOS-HRE, ANA-1 macrophages were transiently
transfected with plasmids containing the full-length or deletion
mutants of the 5
It
has been previously reported that induction of HIF-1 binding activity
by DFX in the Hep3B cell line was due to chelation of iron (25).
Therefore, we investigated the role of iron chelation in the
DFX-dependent induction of HIF-1 binding and iNOS-HRE
activity in murine macrophages. ANA-1 macrophages were treated with
medium or DFX in the presence or absence of iron sulfate
(FeSO4), and the induction of HIF-1 binding (Fig.
7A) or iNOS-HRE activity (Fig. 7B)
was measured. The addition of FeSO4 completely blocked the
appearance of the DFX inducible complex to the HIF-1 binding site of
the iNOS-HRE (Fig. 7A) and dramatically inhibited the inducibility of the pBLWT.iNOS construct (Fig. 7B). We
then tested whether the iron-dependent inhibition of
iNOS-HRE activation also impaired the ability of IFN-
The mechanism by
which PA activates the iNOS-HRE is unknown. However, PA shares with DFX
the property of being an iron chelator (34, 35), and this property
seems to account for its antiproliferative effects on tumor cell lines
(34). To address whether PA induces HIF-1 binding and iNOS-HRE
activation via chelation of iron, ANA-1 macrophages were treated with
medium or PA in the presence or absence of FeSO4, and HIF-1
binding (Fig. 8A) or iNOS-HRE activity (Fig.
8B) was measured. PA induced a DNA-binding activity to the iNOS-HRE that was completely abolished by addition of FeSO4
(Fig. 8A). PA also induced a 10-fold expression of the
pBL-WT.iNOS construct over untreated cells that was almost completely
abrogated in the presence of FeSO4 (Fig. 8B). As
previously reported, PA alone does not induce the expression of iNOS
promoter or iNOS mRNA (data not shown). However, the
PA-dependent induction of iNOS promoter activity (Fig.
8C) and iNOS mRNA expression (Fig. 8D) in
IFN-
We have investigated the role of DFX in the regulation of iNOS
promoter transcriptional activity in murine macrophages. We have found
that DFX induced HIF-1 binding and iNOS-HRE expression. The presence of
a functional iNOS-HRE was required for IFN- Wang and Semenza (25) have provided evidence that DFX induces HIF-1
activity in Hep3B cells. HIF-1 binds to a hypoxia-inducible enhancer
that was originally described in Hep3B cells as a 50-nucleotide functionally tripartite element (7). However, the homology between the
human 50-nucleotide element and the murine iNOS-HRE is limited to the
HIF-1 binding site plus six flanking bases. We have found that DFX
induced a specific DNA complex to an oligonucleotide probe (AB.2)
encompassing the HIF-1 binding site of the iNOS promoter in ANA-1
macrophages. The appearance of the binding complex was completely
inhibited by an oligonucleotide containing the canonical HIF-1
consensus sequence of the Epo enhancer (Epo) (7) but not by an
oligonucleotide mutated in 3 bp within the HIF-1 binding site (Mu.AB2),
indicating that the integrity of the HIF-1 binding site was critical
for the occurrence of the binding activity. Accordingly, DFX also
induced in macrophages the expression of a tk promoter CAT-reporter
gene construct containing three copies of the wild type (pBL-WT.iNOS)
but not mutated (pBL-Mu.iNOS) iNOS-HRE. The same 3-bp mutation within
the HIF-1 binding site inhibited hypoxia- or DFX-induced
transcriptional activation of the Epo enhancer in Hep3B (7, 25) and
abolished the hypoxia or picolinic acid inducibility of the iNOS-HRE in
murine macrophages (23). These results suggest that the HIF-1 binding
site, conserved between the Epo enhancer and the iNOS-HRE, is
sufficient to confer inducibility by DFX in murine macrophages.
Consistent with the finding that DFX was a potent activator of the
iNOS-HRE in macrophages, DFX synergistically induced the expression of
the full-length iNOS promoter in IFN- The DFX-dependent induction of HIF-1 binding and iNOS
promoter activity was associated with increased expression of the
endogenous iNOS gene in ANA-1 macrophages. DFX induced iNOS mRNA
expression and increased the rate of transcription of the iNOS gene in
combination with IFN- Iron chelation appeared to be the mechanism by which DFX induced HIF-1
activity in Hep3B cells (25). Cellular iron has been involved in the
control of iNOS transcriptional activity induced by IFN- The data presented here, together with previously published
observations (23), demonstrate that the involvement of HIF-1 binding
and iNOS-HRE expression is the common feature of the induction of iNOS
promoter activity by hypoxia, picolinic acid, and DFX, and they suggest
the existence of a common pathway of activation of iNOS transcription.
This pathway of iNOS expression appears to be clearly distinct from
that induced by LPS, which is largely dependent on NF- We thank Dr. John R. Ortaldo for the critical
review of the manuscript. We also thank Drs. Q. Xie and C. Nathan,
Cornell University Medical College, New York, NY, for kindly providing
the plasmid p1-iNOS-CAT containing the full-length iNOS promoter.
Laboratory of Experimental Immunology,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-flanking region of the inducible nitric oxide
synthase (iNOS) gene containing a sequence homology to a
hypoxia-responsive enhancer (iNOS-HRE) mediates picolinic acid (PA)- or
hypoxia-induced activation of the iNOS promoter in interferon-
(IFN-
)-treated murine macrophages. The iron chelator desferrioxamine
(DFX) induces the activity of the human erythropoietin enhancer in
Hep3B cells. We have investigated the influence of DFX on the
activation of the iNOS promoter and iNOS gene expression in ANA-1
macrophages. We have found that DFX induced DNA-binding activity to the
hypoxia-inducible factor 1 (HIF-1) consensus sequence of the iNOS
promoter and activated the iNOS-HRE in murine macrophages. These
activities of DFX were associated with a synergistic induction of iNOS
mRNA expression and iNOS transcription in IFN-
-treated ANA-1
macrophages. Functional analysis of the 5
-flanking region of the iNOS
gene demonstrated that IFN-
plus DFX activated the full-length iNOS
promoter and that the iNOS-HRE was required for DFX-induced iNOS
transcriptional activity. We also investigated the role of iron
metabolism in the DFX- or PA-dependent induction of HIF-1
activity and iNOS expression. We demonstrate that addition of iron
sulfate completely abolished DFX or PA induction of HIF-1 binding and
iNOS-HRE activation and abrogated IFN-
plus either DFX- or
PA-induced iNOS expression. These data establish that DFX is a
co-stimulus for the transcriptional activation of the iNOS gene in
IFN-
-treated macrophages, and they provide evidence that the
iNOS-HRE is required for the DFX-dependent activation of
the iNOS promoter. Furthermore, our results indicate that the iNOS-HRE
is a regulatory element of the iNOS promoter responsive to iron
chelation.
-flanking region of the Epo gene in a region required for Epo
transcriptional activation (6, 7). This regulatory region, which is
highly conserved between human and mouse, also plays a critical role in
the hypoxia-induced transcriptional activation of genes encoding
glycolytic enzymes (4, 5). The HRE contains the consensus sequence for
the binding of a transactivating factor, which is inducible by hypoxia (hypoxia-inducible factor 1, HIF-1) (7-9). Induction of HIF-1 binding
and functional activation of the HRE was demonstrated in a variety of
mammalian cell lines, regardless of their ability to express Epo,
suggesting a general involvement of HIF-1 in the regulation of
gene expression by O2 tension (10, 11).
plus a second
signal (15-17). Three regulatory elements of the 5
-flanking region of
the iNOS gene play a functional role in the control of its
transcriptional activation (18, 19). The interferon regulatory factor 1 binding site, located at position
923 to
913, is required for the
synergistic activity of IFN-
, as shown in vivo in
interferon regulatory factor 1 knock-out mice (20) and in
vitro in the RAW 264.7 cell line (21). The consensus sequence for
the binding of NF-
B family members, located at position
85 to
76, mediates iNOS transcriptional activation in response to bacterial
lipopolysaccharide (LPS) (22). We recently reported that the promoter
region of the iNOS gene contains a sequence homology to the HRE
(referred to hereafter as iNOS-HRE) (23). Functional studies in murine
macrophages demonstrated that the iNOS-HRE was required for the
activation of the iNOS promoter by IFN-
plus picolinic acid, a
catabolite of L-tryptophan, and by IFN-
plus hypoxia
(1% O2). These results provided the first indication that
iNOS is a hypoxia-inducible gene. The iNOS-HRE contained a 100%
homology to the consensus sequence for the binding of HIF-1
(5
-TACGTGCT-3
) (7), and the HIF-1 binding site was essential for the
induction of iNOS promoter activity by picolinic acid or hypoxia in
IFN-
-treated ANA-1 macrophages (23). Furthermore, we demonstrated
that a CAT construct containing three copies of the iNOS-HRE was
inducible by hypoxia or picolinic acid and that mutation of the HIF-1
binding site abolished hypoxia- or picolinic acid-dependent
transcriptional activation (23). These data established that a
hypoxia-responsive element mediates a novel pathway of iNOS
transcriptional activation induced by picolinic acid or hypoxia that is
different from that mediated by NF-
B induced by LPS. Thus, at least
two distinct pathways of iNOS expression are elicited in response to
different signals and multiple regulatory elements control its
transcriptional activation.
plus LPS,
whereas desferrioxamine (DFX), an iron chelator, can augment such a
response (24). These results suggested the existence of a regulatory
loop between iron metabolism and iNOS induction by the IFN-
plus LPS
pathway, although analysis of the activation of the iNOS promoter was
not performed in these experiments. However, the discovery of the
hypoxia-dependent activation of iNOS expression supports
the hypothesis of a different mechanism of action of DFX on iNOS
expression, independent from the IFN-
plus LPS pathway. In fact,
recent evidence has been provided that DFX induces HIF-1 binding
activity in Hep3B cells (25). DFX also induces the expression of
reporter genes carrying the human Epo enhancer, and mutations in the
HIF-1 binding site eliminated DFX-inducible transcriptional activation
(25). These results suggest that DFX can directly activate the HRE, at
least in Hep3B cells. It follows, then, that DFX itself could be a
co-stimulus for the activation of iNOS expression in IFN-
-treated
macrophages along the hypoxia pathway. It was important to test this
possibility because it would change our perspective on the connections
between iron metabolism, iNOS induction, and macrophage activation.
Therefore, we studied the functional activation of the iNOS-HRE and
iNOS gene by DFX in macrophages.
-treated cells. Functional analysis of
the 5
-flanking region of the iNOS gene in macrophages stimulated with
IFN-
plus DFX demonstrated that the integrity of the HIF-1 binding
site was required for the induction of iNOS promoter activity. Finally,
addition of iron sulfate completely abrogated the expression of HIF-1
activity and iNOS mRNA in response to either DFX or PA. These
results demonstrate that DFX is in itself a co-stimulus for the
transcriptional activation of iNOS expression in IFN-
-treated murine
macrophages, and they identify the iNOS-HRE as a regulatory sequence of
the iNOS promoter that is activated by iron deprivation.
Cells and Reagents
(specific activity
107 units/mg) was
purchased from Life Technologies, Inc. Desferrioxamine, ferrous
sulfate, and cycloheximide were purchased from Sigma. Picolinic acid
was purchased from Sigma (purity ~ 99%) and was prepared as
described previously (28).
70 °C for subsequent analysis of DNA
binding proteins.
-32P]dCTP. The following oligonucleotide probes were
used: AB.2 (5
-GTGACTACGTGCTGCCTAG-3
) encompassing the
consensus sequence for the binding of HIF-1, shown in bold; AB.1
(5
-TGAGTCCCAGTTTTGAAGTG-3
) from an unrelated sequence of the iNOS
promoter; Mu-AB.2 (5
-GTGACTAGCTGCCTAG-3
), with the three mutated bases underlined; Epo
(5
-GCCCTACGTGCTGCCTCA-3
) from the 3
-flanking region of
the human Epo gene (7). Binding reactions were performed in buffer
containing 25 mM Tris-HCl, pH 7.5, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, with 5 µg of nuclear extract, and 0.4 µg of denatured calf thymus DNA on ice. After incubation for 10 min, probe (1 × 104 cpm) was added,
and the incubation was continued for an additional 20 min. Samples were
loaded onto 5% nondenaturing polyacrylamide gel, and electrophoresis
was performed at 180 V in 0.3 × TBE at 4 °C.
-flanking region of
the murine iNOS gene inserted upstream of the promoterless
chloramphenicol acetyltransferase (CAT) gene in pCAT-Basic (Promega
Corp.) (i.e. p1-iNOS-CAT) (18) was kindly provided by Q. Xie
and C. Nathan (Cornell University Medical College, New York, NY).
Deletion mutants of the iNOS promoter were obtained as described
previously (23). For functional studies in the context of a
heterologous promoter, three tandem copies of a 21-base pair (bp)
double-strand oligonucleotide encompassing the iNOS-HRE (
231 to
211) were subcloned in the HindIII/BamHI sites
of pBLCAT2 plasmid, containing the CAT reporter gene under the control
of a herpes simplex virus thymidine kinase promoter fragment
spanning from
105 to +51,. The constructs were sequenced using
Sequenase version 2.0 (United States Biochemical Corp.).
70 °C on XAR-5 film
(Eastman Kodak) with the use of Lightning Plus intensifying screens
(DuPont NEN).
-actin cDNA insert). Filters were then
washed and autoradiographed as for the Northern blot analysis.
DFX Induces DNA-binding Activity to the iNOS-HRE
Fig. 1.
DFX induces a DNA-binding activity to the
HIF-1 binding site of the iNOS-HRE. Nuclear extracts from ANA-1
macrophages treated with medium or DFX (400 µM) for
12 h were incubated with AB.2 probe in the absence (0)
or presence of 100-fold molar excess of the indicated unlabeled
competitor oligonucleotide (described under "Materials and
Methods") and analyzed by EMSA. Competitor DNAs were incubated with
nuclear extracts for 10 min on ice prior to addition of labeled probe.
Binding activities are labeled as follows: C, constitutive;
I, induced; Free, free labeled probe.
[View Larger Version of this Image (48K GIF file)]
of the herpes simplex virus thymidine kinase minimal promoter. The
pBL-WT.iNOS construct was constitutively expressed in macrophages (Fig.
2). Treatment with DFX caused a 10-fold increase in the
expression of CAT activity, relative to untreated cells. In contrast,
plasmid pBL-Mu.iNOS was not inducible by DFX. These results, consistent
with the data obtained in EMSA, demonstrate that the iNOS-HRE is
sufficient to confer inducibility by DFX in murine macrophages and that
mutation of the HIF-1 binding site abolishes DFX inducible
transcriptional activation.
Fig. 2.
DFX induces iNOS-HRE activity in murine
macrophages. ANA-1 macrophages were transfected with the indicated
plasmids containing three copies of the iNOS-HRE either wild type
(5-AGTGACTACGTGCTGCCTAGG3
, pBL-WT.iNOS)
or mutated
(5
-AGTGACTAGCTGCCTAGG-3
,
pBLMu.iNOS) in the pBLCAT2 vector. Bold type indicates
the HIF-1 binding site, with the mutation underlined.
Twenty-four hours after the transfection, ANA-1 cells were treated with
medium or DFX (400 µM) for additional 18 h, and CAT
activity was assayed. Results are expressed as fold increase of CAT
activity (% acetylation) relative to that expressed by pBL-WT.iNOS in
untreated cells (arbitrarily considered to be equal to 1). Results were
normalized for the expression of the parental vector (pBLCAT2) and are
from one representative experiment.
[View Larger Version of this Image (11K GIF file)]
in the Induction of iNOS mRNA
Expression
(100 units/ml), and DFX (400 µM) alone or
in combination, and total RNA was harvested after 18 h. No
expression of iNOS mRNA was observed in cells treated with medium
or DFX alone, and very low levels of iNOS mRNA were detected in
macrophages stimulated with IFN-
alone (Fig.
3A). In contrast, addition of DFX caused a
major increase of IFN-
-induced iNOS mRNA expression that was detectable after 6 h of treatment (see Fig. 5) and increased up to
18 h (Fig. 3A). To determine the dose dependence of DFX
effects, ANA-1 macrophages were incubated with increasing
concentrations of DFX in the presence or absence of IFN-
for 18 h. DFX alone, at doses ranging from 50 µM (data not
shown) to 800 µM, failed to induce iNOS mRNA. The
combination of IFN-
and DFX increased the levels of iNOS mRNA at
a dose of 100 µM DFX (3-4-fold above IFN-
alone)
(Fig. 3B) and reached its maximum (15-20-fold above IFN-
alone) at a dose of 800 µM DFX. These data demonstrate
that DFX acts synergistically with IFN-
in a
dose-dependent fashion for the induction of iNOS mRNA
expression in murine macrophages.
Fig. 3.
DFX synergizes with IFN- for induction of
iNOS mRNA expression in murine macrophages. ANA-1 macrophages
were cultured at 1 × 106 cells/ml in 60-mm tissue
culture plates and treated with medium (Med), IFN-
(100 units/ml), DFX (400 µM) alone or in combination with
IFN-
(A) or with medium, IFN-
(100 units/ml) alone,
DFX (800 µM) alone, or IFN-
plus the indicated
concentrations of DFX (B) for 18 h. Total cellular RNA
was examined for iNOS mRNA expression as described under
"Materials and Methods." The same filters were hybridized with the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to
monitor RNA loading. Results shown are from one representative
experiment.
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Protein synthesis is required for IFN-
plus DFX induction of iNOS mRNA expression. ANA-1 macrophages
(1 × 106 cells/ml) were incubated with medium,
IFN-
(100 units/ml), DFX (400 µM) alone or in
combination with IFN-
, in the absence or presence of 7.5 µg/ml
CHX. Total cellular RNA was harvested 6 h later and examined for
iNOS mRNA expression as described. Data are from one of two similar
experiments.
[View Larger Version of this Image (61K GIF file)]
Synergistically Induce iNOS
Transcription
plus DFX was due to transcriptional activation of the gene. ANA-1
macrophages were treated with medium, IFN-
, DFX alone or in
combination with IFN-
. The iNOS gene was not constitutively
transcribed in ANA-1 macrophages, and treatment with IFN-
or DFX
alone failed to induce iNOS transcription (Fig. 4). In
contrast, ANA-1 cells treated with IFN-
plus DFX showed a
significant increase in the rate of transcription of the iNOS gene.
These data establish that DFX synergistically induces iNOS
transcriptional activation in IFN-
-treated macrophages.
Fig. 4.
DFX synergistically induces iNOS
transcription in IFN--treated ANA-1 macrophages. ANA-1
macrophages (2.5 × 107) were treated with medium,
IFN-
(100 units/ml), DFX (400 µM) alone or in
combination with IFN-
. Nuclei were isolated after 12 h, and the
rate of transcription of the iNOS gene was then determined by nuclear
run-on analysis as described. The following plasmid DNA were used:
piNOS, pGEM vector containing a 3.9-kilobase cDNA insert of mouse
macrophage nitric oxide synthase; p-actin, vector containing a chicken
-actin cDNA insert; pGEM, vector lacking any cDNA
insert.
[View Larger Version of this Image (49K GIF file)]
, DFX alone or in combination with IFN-
, in the presence or absence of CHX (7.5 µg/ml) for 6 h. As shown in Fig. 5, the addition of CHX almost completely abrogated the
synergistic induction of iNOS mRNA expression by IFN-
plus DFX,
indicating that de novo protein synthesis was required for
iNOS mRNA induction by IFN-
plus DFX.
-flanking region of the iNOS gene linked to the CAT
reporter gene. Cells were stimulated with medium, IFN-
(100 units/ml), or DFX (400 µM) alone or in combination. ANA-1
cells transfected with plasmid p1, containing the full-length iNOS
promoter, did not express significant levels of CAT activity either
constitutively or after treatment with IFN-
alone (Fig.
6). Low but consistent induction of CAT expression was
induced by treatment with DFX alone. However, a strong synergistic
induction of CAT activity was observed following stimulation with
IFN-
and DFX. Deletion of the upstream region of the iNOS promoter
(
1588 to
721, plasmid p3), containing the IFN-
-responsive
region, completely abrogated the synergistic interaction between DFX
and IFN-
in the induction of iNOS promoter activity (data not
shown). These data indicated that maximal activation of the iNOS
promoter required the presence of the IFN-
-responsive region and was
induced by the combination of IFN-
plus DFX. Transfection of plasmid
p162, deleted of the region from
721 to
410, did not affect the
induction of CAT activity by IFN-
plus DFX. In contrast, plasmids
p167 (deleted from
721 to
201) and p201 (deleted from
328 to
201) were no longer inducible following stimulation of ANA-1
macrophages with the combination of IFN-
and DFX (Fig. 6). To
establish whether DFX-dependent activation of the iNOS
promoter required the presence of a functional iNOS-HRE, ANA-1
macrophages were transfected with two iNOS promoter-CAT reporter gene
constructs (23), containing either a deletion of the iNOS-HRE (
227 to
209, plasmid p220) or a mutation of two bases required for HIF-1
binding to DNA (
223
222, plasmid p209) (7). A reduction greater
than 80% relative to the wild type iNOS promoter was observed in the
induction of CAT expression with either plasmid. These data demonstrate
that the synergistic interaction between IFN-
and DFX in the
transcriptional activation of the iNOS promoter is mediated, at least
in part, by the iNOS-HRE.
Fig. 6.
Functional requirement of the iNOS-HRE for
DFX-dependent induction of iNOS promoter activity.
ANA-1 macrophages were transfected with the indicated plasmids as
described under "Materials and Methods." Twenty-four hours later,
cells were treated with medium, IFN- (100 units/ml), DFX (400 µM) alone or in combination with IFN-
for an
additional 18 h, and CAT activity was assayed by TLC. Plasmid p220
contains a 19-bp deletion encompassing the iNOS-HRE
(5
-aCGCCTaGggg-3
; the sequence homology between iNOS promoter and erythropoietin (Epo) gene is shown in
uppercase, and the binding site of the HIF-1 is
underlined). In plasmid p209 a 2-bp mutation
(5
-TATGCT-3
, underlined) was created at
position
223
222 internal to the binding site for HIF-1. Results
are expressed as relative CAT activity, obtained by dividing CAT
activity (% acetylation) by relative light units for luciferase. Data
shown are from one representative experiment.
[View Larger Version of this Image (14K GIF file)]
plus DFX to
induce the activity of the full-length iNOS promoter and the expression
of iNOS mRNA. We measured the inducibility of plasmid p1 (Fig.
7C) or the levels of iNOS mRNA (Fig. 7D) in
ANA-1 macrophages treated with IFN-
plus DFX in the presence or
absence of FeSO4. Iron sulfate caused a greater than 90%
reduction in the IFN-
plus DFX-dependent induction of
iNOS promoter activity (Fig. 7C) and completely inhibited
the synergistic activation of iNOS mRNA expression (Fig.
7D). We conclude that iron was involved in the mechanism by
which DFX induced iNOS-HRE activity and iNOS mRNA expression in
murine macrophages.
Fig. 7.
Ferrous sulfate inhibits
DFX-dependent induction of HIF-1 binding and iNOS
expression. A, electrophoretic mobility shift assay. ANA-1
macrophages were treated with medium or DFX (200 µM) in
the absence or presence of FeSO4 (300 µM) for
12 h. Nuclei were isolated, and EMSA was performed with
radiolabeled AB.2 probe as described. B, iNOS-HRE activity.
ANA-1 macrophages were transfected with plasmid pBL-WT.iNOS.
Twenty-four hours after the transfection cells were treated with medium
or DFX (400 µM) in the absence or presence of
FeSO4 (300 µM) for an additional 18 h,
and CAT activity was assayed as described. C, iNOS promoter activity. ANA-1 cells were transfected with plasmid p1, containing the
full-length iNOS promoter. Cells were treated with medium, IFN- (100 units/ml) plus DFX (400 µM) in the presence or absence of
FeS04 (300 µM) for 18 h, and CAT
activity was assayed. D, Northern blot analysis. ANA-1
macrophages were treated with medium, IFN-
(100 units/ml) plus DFX
(200 µM) in the presence or absence of FeS04
(300 µM) for 18 h, and total cellular RNA was
examined for iNOS mRNA expression as described.
[View Larger Version of this Image (44K GIF file)]
-treated ANA-1 macrophages was dramatically inhibited in the
presence of FeSO4. As shown in Fig. 8D,
FeSO4 either alone or in combination with IFN-
did not
induce the expression of iNOS mRNA. These results indicate that the
mechanism by which PA activates iNOS expression in murine macrophages
is dependent, at least in part, on chelation of iron.
Fig. 8.
Ferrous sulfate inhibits
PA-dependent induction of iNOS-HRE activity and iNOS
expression. A, electrophoretic mobility shift assay. ANA-1
macrophages were treated with medium or PA (4 mM) in the
absence or presence of FeSO4 (300 µM) for
12 h. Nuclei were isolated, and EMSA was performed with
radiolabeled AB.2 probe as described. B, iNOS-HRE activity.
ANA-1 macrophages, transfected with plasmid pBL-WT.iNOS, were treated
with medium or PA (4 mM) in the absence or presence of
FeSO4 (300 µM) for 18 h, and CAT
activity was assayed as described. C, iNOS promoter activity. ANA-1 cells, transfected with plasmid p1, were treated with
medium, IFN- plus PA (4 mM) in the presence or absence
of FeS04 (300 µM) for 18 h, and CAT
activity was assayed. D, Northern blot analysis. ANA-1
macrophages were treated with medium, IFN-
, FeS04 (300 µM) alone or in combination with IFN-
, IFN-
plus PA
(4 mM) in the presence or absence of FeS04 (300 µM) for 18 h, and total cellular RNA was examined
for iNOS mRNA expression as described.
[View Larger Version of this Image (37K GIF file)]
plus DFX-dependent iNOS promoter activation in ANA-1
macrophages. These activities were paralleled by a synergistic
induction of iNOS transcription and iNOS mRNA expression in cells
treated with IFN-
plus DFX. These data demonstrated that DFX is in
itself an activator of the iNOS-HRE in murine macrophages and that the
hypoxia-responsive element is involved in the DFX-dependent
transcriptional activation of the iNOS gene. Thus, DFX is a co-stimulus
with IFN-
for iNOS induction along the hypoxia/picolinic acid
pathway.
-treated macrophages. Although
DFX alone did not induce detectable iNOS transcription, as assessed by
nuclear run-on analysis, we found that DFX alone induced low but
consistent levels of expression of the iNOS promoter in transient
transfection assays. This discrepancy has been reported previously for
PA- or LPS-induced activation of iNOS promoter and might be explained
by negative regulatory regions that control the expression of the
endogenous gene or by differences in the sensitivity of the assays (19,
23). Induction of iNOS promoter activity by DFX was the result, at
least in part, of the cooperative interaction between the iNOS-HRE and
IFN-
-responsive sequences. Deletion or mutation of the HIF-1 binding
site of the iNOS promoter demonstrated that the
DFX-dependent iNOS transcriptional activation required the
integrity of the HIF-1 binding site, providing further evidence of the
involvement of HIF-1 in the regulation of iNOS promoter activity.
. The synergistic interaction between IFN-
and DFX in the induction of iNOS mRNA was
dose-dependent and required de novo protein
synthesis, as demonstrated by inhibition of DFX-induced iNOS mRNA
expression in the presence of CHX. HIF-1 binding activity observed in
hypoxia- or DFX-treated Hep3B cells was also abolished by addition of
CHX (7, 25). This finding is consistent with the involvement of
interferon regulatory factor 1 and HIF-1, both of which require ongoing
protein synthesis, in the induction of iNOS transcriptional activity
induced by IFN-
plus DFX (7, 20). The augmented expression of iNOS
mRNA induced by IFN-
plus DFX in ANA-1 macrophages was also
associated with increased levels of NO production and with the
expression of tumoricidal activity against the tumor necrosis
factor-
-resistant tumor target cell line
P815.2 These data demonstrate that DFX
functions as a macrophage co-stimulator in combination with
IFN-
.
plus LPS in
the J774 macrophage-like cell line (24). Data presented here suggest
that the augmenting effect of DFX on IFN-
plus
LPS-dependent iNOS transcription can be accounted for by
induction of HIF-1 binding and activation of the iNOS-HRE and that DFX
and LPS activate iNOS transcription through distinct pathways. However,
a possible effect of DFX on LPS activation cannot be ruled out at the
present. Addition of iron sulfate completely abolished the DFX- or
PA-dependent induction of HIF-1 binding and iNOS-HRE
expression in ANA-1 macrophages. The abrogation of these activities was
paralleled by lack of iNOS promoter activation and iNOS mRNA
expression following stimulation with IFN-
plus either DFX or PA.
These data indicate that the iNOS-HRE may be activated by iron
chelation, and they provide evidence of a molecular mechanism by which
iron metabolism may affect iNOS promoter activation in murine
macrophages. Furthermore these data provide the first indication of the
mechanism by which PA acts on murine macrophages to induce the
expression of the iNOS gene.
B activation
(22). Hypoxia and DFX are nonclassical macrophage activating agents
because their signals are delivered through an intracellular sensor
rather than through cell surface receptors. Our results are consistent
with the possibility that hypoxia, picolinic acid, and DFX use a common
sensor in the signal transduction pathway. The putative hemoprotein,
which would function as intracellular O2 sensor, may be
responsive to changes in O2 tension as well as to chelation
of iron (36). Picolinic acid shares with DFX the property of acting as
a co-stimulus with IFN-
for the induction of tumoricidal activity
(37), NO production (38), and iNOS mRNA expression (28) in murine
macrophages. In contrast, IFN-
plus hypoxia induce significant
levels of iNOS mRNA and iNOS protein but not of NO production,
which can be achieved only by subsequent reoxygenation (39). Therefore,
the biological implications of the induction of iNOS expression by
hypoxia, picolinic acid, or DFX are quite different. In fact, hypoxia
may occur in inflammatory and neoplastic lesions (14, 40, 41), whereas picolinic acid might be induced in those conditions in which elevated levels of tryptophan metabolites, such as kynurenine and quinolinic acid, have been detected (42-44). In contrast, DFX is a
pharmacological agent used for the treatment of several pathological
conditions, including iron overload (45), cancer (46-48), and
Alzheimer's disease (49, 50). The notion that, in the presence of
IFN-
, DFX might be responsible for the induction of iNOS expression is important for the potential role of nitric oxide in mediating undesired side effects or beneficial therapeutic activities.
*
This work was supported in part by a grant from Associazione
Italiana per la Ricerca sul Cancro and from Telethon.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.
§
To whom correspondence should be addressed at present address: NCI,
National Institutes of Health, Medicine Branch, Bldg. 10, Rm. 12N226,
Bethesda, MD 20892. Tel.: 301-496-4916; Fax: 301-402-0172.
Present address: Laboratory of Molecular Biology, Institute G. Gaslini, Genova, Italy.
1
The abbreviations used are: Epo, erythropoietin;
EMSA, electrophoretic mobility shift assay; HRE, hypoxia-responsive
enhancer; HIF-1, hypoxia-inducible factor 1; iNOS, inducible nitric
oxide synthase; IFN-, interferon-
; LPS, lipopolysaccharide; CAT,
chloramphenicol acetyltransferase; DFX, desferrioxamine; PA, picolinic
acid; bp, base pair(s); CHX, cycloheximide.
2
G. Melillo, L. S. Taylor, A. Brooks, T. Musso,
G. W. Cox, and L. Varesio, unpublished observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.