1 Departments of Pathology and 2 Molecular Microbiology and Immunology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033-9141; and 3 Department of Chemistry, University of Minnesota, Duluth, Minnesota 55812
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ABSTRACT |
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10.1152/ajpgi.00108. 2002. Iron exacerbates various types of liver injury in which nuclear
factor (NF)-
B-driven genes are implicated. This study tested a
hypothesis that iron directly elicits the signaling required for
activation of NF-
B and stimulation of tumor necrosis factor
(TNF)-
gene expression in Kupffer cells. Addition of
Fe2+ but not Fe3+ (~5-50 µM) to
cultured rat Kupffer cells increased TNF-
release and TNF-
promoter activity in a NF-
B-dependent manner. Cu+ but
not Cu2+ stimulated TNF-
protein release and promoter
activity but with less potency. Fe2+ caused a disappearance
of the cytosolic inhibitor
B
, a concomitant increase in nuclear
p65 protein, and increased DNA binding of p50/p50 and p65/p50 without
affecting activator protein-1 binding. Addition of Fe2+ to
the cells resulted in an increase in electron paramagnetic resonance-detectable ·OH peaking at 15 min, preceding activation of
NF-
B but coinciding with activation of inhibitor
B kinase (IKK)
but not c-Jun NH2-terminal kinase. In conclusion,
Fe2+ serves as a direct agonist to activate IKK, NF-
B,
and TNF-
promoter activity and to induce the release of TNF-
protein by cultured Kupffer cells in a redox status-dependent manner.
We propose that this finding offers a molecular basis for iron-mediated accentuation of TNF-
-dependent liver injury.
tumor necrosis factor-; free radical; promoter; inhibitor
B
kinase; electron paramagnetic resonance; nuclear factor-
B
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INTRODUCTION |
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IRON POTENTIATES VARIOUS
FORMS of liver injury (4, 19, 28, 41), and chelation
of iron or decreasing iron content conversely ameliorates the injury
(9, 22, 30, 32). The most accepted explanation for iron's
effects is an iron-catalyzed Fenton pathway resulting in the generation
of ·OH and consequent oxidative tissue injury. In particular, if the
generation of reactive oxygen species (ROS) is already enhanced by
underlying disease processes, a slight increase in hepatic iron content
may suffice for robust production of ·OH and accentuation of
oxidative damage, as exemplified in experimental alcoholic liver injury
(41). This accentuation of liver injury is accompanied by
enhanced nuclear factor (NF)-B activation and expression of
proinflammatory mediators (43). The latter events may
merely reflect a consequence of enhanced hepatocellular necrosis or may
also be considered as causal processes. In fact, at nontoxic
concentrations, iron is known to promote macrophage functions,
including antimicrobial effects (18) and tumor necrosis
factor (TNF)-mediated cytotoxicity (46). More
specifically, recent evidence suggests the role of iron in promoting
cytokine expression (7, 14) and NF-
B activation (42) by hepatic macrophages.
Even though a catalytically active pool of iron is estimated to be
extremely small in normal tissues, the pathological conditions may
cause a transient release of iron from the intracellular compartments into the microenvironment. For instance, oxidative stress is known to
release iron from ferritin through either reduction of Fe3+
by O induction
commonly coexist, the transient release of catalytically active iron
may serve to facilitate oxidative signaling for proinflammatory NF-
B activation.
The present study tested whether direct addition of ionic iron to
cultured Kupffer cells leads to activation of NF-B and induction of
TNF-
expression. Our results demonstrate that Fe2+ but
not Fe3+ at concentrations as low as 5 µM stimulates
TNF-
release. It also induces TNF-
promoter activity in an
NF-
B-dependent manner, and this effect is associated with
time-dependent activation of inhibitor
B (I
B) kinase (IKK) and
NF-
B without affecting activator protein (AP)-1 binding.
Collectively, these results support a notion that iron can serve as a
direct agonist to induce intracellular signaling for NF-
B activation
in Kupffer cells in a redox status-dependent manner.
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MATERIALS AND METHODS |
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Kupffer cell isolation and culture.
Kupffer cells were isolated from normal Wistar rats by in situ
sequential digestion of the liver with pronase and collagenase and
arabinogalactan gradient ultracentrifugation as previously described
(22, 42). The adherence purification method was performed
to raise the purity of Kupffer cells cultured onto a 100-mm dish to
>96% as determined by phagocytosis of 1-µm latex beads. The
viability was tested by the trypan blue exclusion test and always
exceeded 97%. The cells were incubated with DMEM containing 5% fetal
calf serum for 2 days, following the adherence method for in vitro
experiments. For iron or copper treatment, the cells were washed twice
with PBS, incubated in serum-free DMEM, and exposed to ferrous sulfate,
ferric ammonium sulfate, cuprous chloride, or cupric sulfate
(~1-50 µM) for 4 h to assess their effects on the release
of TNF- and TNF-
promoter activity. For activation of IKK and
NF-
B, as well as electron paramagnetic resonance (EPR) detection of
radicals, the cells were incubated for shorter periods (from ~5 min
to 4 h) as specified below and in the figure legends. As a
positive control, the cells were treated with lipopolysaccharide (LPS;
Escherichia coli 055:B5, 500 ng/ml, Sigma, St. Louis, MO).
Nuclear protein extraction and EMSA.
To examine the effects of Fe2+ on DNA binding by NF-B
and AP-1, nuclear proteins were extracted from cultured Kupffer cells by using the method of Schreiber et al. (35). The extracts
(5 µg) were incubated in a reaction mixture [20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 2 mM dithiothreitol (DTT), 20% glycerol, and
200 µg/ml poly(dI-dC)] on ice with the double-strand
B consensus sequence (3), the
B site from TNF-
promoter
(8), or the AP-1 binding site (2) labeled
with 32P. After a 20-min incubation, the reaction mixture
was resolved on a 6% nondenaturing polyacrylamide gel and the gel was
dried for subsequent autoradiography. Densitometric analysis of the intensity of shifted bands was performed by using the Kodak
Electrophoresis Documentation and Analysis System and imaging analysis
software (Eastman Kodak, Rochester, NY). For the supershift assays,
antibodies against p50 and p65 (Santa Cruz Biotechnology, Santa Cruz,
CA) were added to the reaction mixture for an additional 30 min.
IB
and p65 immunoblot analysis.
Cytoplasmic and nuclear extracts of iron-stimulated, cultured Kupffer
cells were examined for I
B
and p65 levels by immunoblot analysis,
respectively. Cytoplasmic or nuclear proteins (10 µg) were mixed with
2× sample buffer (100 mM Tris · HCl, pH 6.8, 4% SDS, 20%
glycerol, and 10%
-mercaptoethanol) and separated by 10% PAGE
under reducing conditions. The proteins were transferred to
nitrocellulose filters (Bio-Rad, Hercules, CA) and treated overnight at
4°C with 5% BLOTTO [5% nonfat milk with (in mM) 50 Tris · HCl, pH 7.5, 50 NaCl, 1 EDTA, and 1 DTT]. The filters
were then incubated with rabbit polyclonal anti-human p65 (Biomol, Plymouth Meeting, PA) or anti-human I
B
(Santa Cruz Biotechnology) at 1:1,000 dilution in TBST (10 mM Tris · HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) with 1% BSA at room temperature for 2 h, followed by three washes with TBS and 0.2% Tween 20. The filters
were then incubated with horseradish peroxidase-conjugated goat
anti-rabbit IgG (Sigma) at 1:2,000 dilution at room temperature for
2 h. The immobilized p65 and I
B
antibody complexes were detected by chemiluminescence by using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL).
EPR spectra of iron-treated Kupffer cells.
To determine time-dependent changes in the generation of free radicals
by iron-treated Kupffer cells, the cells (107 cells/ml)
were suspended in PBS containing 5-10 mM glucose with or without
ferrous sulfate (50 µM). At different time points (0, 5, 10, 20, and
30 min), aliquots of the samples were withdrawn from the reaction
mixtures, mixed with 50 mM
-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN)
and 0.1% (vol/vol) DMSO, and immediately transferred to bottom-sealed
Pasteur pipettes. The EPR spectra were recorded at room temperature in
a Bruker ECS 106 spectrometer operating at 9.8 GHz. Instrument
conditions were as follows: modulation frequency, 100 kHz; time
constant, 1.3s; sweep scan, 18 G/min; modulation amplitude, 0.9 G; and
microwave power, 20 mW. The spectra were compared with simulated ones
obtained by using the published hyperfine splitting constants and the
simulation program from Oklahoma Research Center.
IKK and JNK assays.
To assay the activity of IKK, Kupffer cells cultured in 100-mm dishes
were treated with ferrous sulfate for ~0-45 min or LPS (500 ng/ml) for 15 min, washed with PBS once, and lysed with a lysis buffer
(in mM: 20 Tris · HCl, pH 7.5, 20 NaF, 20 -glycerophosphate, 0.5 Na3VO4, 2.5 metabisulfite, 5 benzamidine, 1 EDTA, 0.5 EGTA, and 300 NaCl, with 10% glycerol and protease
inhibitors and 1.5% Triton X-100). The lysates were immediately frozen
in liquid nitrogen and stored at
80°C until assay. IKK activity was
determined as previously described (29). Briefly, IKK was
immunoprecipitated by IKK
antibodies and protein G-Sepharose. The
assay was performed at 30°C for 1 h in buffer containing 20 mM
Tris · HCl, pH 7.5, 20 mM MgCl2, 2 mM DTT, 20 µM
ATP, 2 µg/30 µl glutathione-S-transferase (GST)-I
B
, and [
-32P]ATP (0.5 µCi). The
reaction was stopped by addition of Laemmli buffer and was resolved by
10% SDS-PAGE followed by a transfer onto a nitrocellulose membrane.
Phosphate incorporated into GST-I
B
was visualized by exposing the
membrane to a PhosphorImager. The c-Jun NH2-terminal kinase
(JNK) assay was performed similarly, except that antibodies against
JNK-1 (Santa Cruz Biotechnology) and protein G-Sepharose were used to
immunoprecipitate JNK-1 and that GST-c-Jun (Santa Cruz Biotechnology)
was used as a substrate. For both IKK and JNK, total protein levels
were assessed by immunoblot analysis of the cell lysates.
Transfection and TNF- promoter analysis.
To assess the effects of ionic iron and copper on TNF-
promoter
activity, cultured Kupffer cells were transiently transfected with a
TNF-
promoter-luciferase construct using Targefect F-2 (Targeting
System, San Diego, CA). The construct was created by ligating a 1.4-kb
mouse TNF-
promoter (a KpnI and HindIII
fragment) (15) into the pGL3-Basic plasmid (Promega,
Madison, WI). For determination of transfection efficiency, Renilla
phRL-TK vector was used. For transfection, 3-day-cultured Kupffer cells
in six-well plates were treated with 2 µg of the reporter gene, 0.02 µg Renilla phRL-TK, and 2 µl of F-2 reagent in 1 ml serum-free RPMI
for 2 h. Then 1 ml of RPMI with 10% FCS was added to achieve the
final FCS concentration of 5% for overnight incubation. On the next day, the medium was changed to new DMEM with 10% FCS and the cells were incubated for 24 h. During the last 14 h of the
incubation, the medium was changed to serum-free RPMI with or without
ferrous sulfate, ferric ammonium sulfate, cuprous chloride, or cupric sulfate (10 or 50 µM), and the cell lysate was collected for
luciferase assay by using the Dual-Luciferase Reporter assay system
(Promega). Four experiments were performed independently, and all
results were normalized for transfection efficiency as determined by
Renilla luciferase activity. To determine the dependence of iron's
effects on NF-
B, the cells were also cotransfected with the I
B
super repressor plasmid, which expresses I
B
with S32A/S36A
mutations (16), or the empty vector. These plasmids were
kindly provided by Dr. Richard Rippe (University of North Carolina at
Chapel Hill).
TNF- RT-PCR.
For RT-PCR analysis for TNF-
, 3 µg of total RNA was reverse
transcribed into cDNA by a Moloney murine leukemia virus
reverse transcriptase and oligo(dT)15 at 37°C for 60 min.
Synthesized cDNA was amplified by denaturation at 94°C for 4 min,
followed by multiple (25 for
-actin and 43 for TNF-
) cycles of
denaturation (95°C, 30 s), annealing (58°C, 30 s), and
extension (72°C, 60 s). Primers used for TNF-
were sense,
5'-ATGAGCACAGAAAGCATGATG and antisense, 5'-TACAGGCTTGTCACTCGAATT, and
for
-actin they were sense, 5'-CACGGCATTGTAACCAACTG and antisense,
5'-AGGGCAACATAGCACAGCTT.
TNF- immunoassay.
The effects of iron and copper on the release of TNF-
by cultured
Kupffer cells were examined by analyzing the TNF-
protein in the
media with a commercially available mouse TNF-
immunoassay kit (R&D
Systems, Minneapolis, MN).
Statistical analysis. The numerical data were expressed as means ± SD, and comparison between treated and control groups was performed by Student's t-test.
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RESULTS |
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Fe2+ but not
Fe3+ stimulates release of TNF-.
We first tested whether iron stimulates the release of TNF-
by
cultured Kupffer cells. As shown in Fig.
1, the addition of Fe2+ but
not Fe3+ increased TNF-
release by twofold at 5 µM and
eightfold at 10 and 50 µM during the 4-h treatment period.
Interestingly, Cu+ but not Cu2+ also stimulated
TNF-
release at 10 and 50 µM, but its effect seemed less potent
compared with Fe2+. Thus these results demonstrate direct
stimulation of Kupffer cell TNF-
release by iron and copper in a
redox status-dependent manner. It should also be noted that no toxicity
was observed in Kupffer cells exposed to ~1-50 µM of iron or
copper as assessed by lactate dehydrogenase release or Sytox
green nucleic acid staining (Molecular Probes, Eugene, OR).
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Iron stimulates TNF- promoter activity.
We then tested whether Fe2+ stimulates the TNF-
promoter
in cultured Kupffer cells. The promoter activity was indeed increased ~2-3 fold with 10 and 50 µM Fe2+ (Fig.
2A). Cu+ (50 µM)
also slightly increased TNF-
promoter activity, but Cu2+
and Fe3+ did not (Fig. 2A). Cotransfection of a
super repressor I
B
vector completely abrogated the stimulation
with 50 µM Fe2+, whereas cotransfection with a LacZ
vector did not (Fig. 2B). Stimulation of the promoter
activity by 50 µM Fe2+ was about half of the maximal
response achieved by LPS (500 ng/ml) in a serum-free condition (Fig.
2B). These results establish that Fe2+ activates
TNF-
promoter in a NF-
B-dependent manner.
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Fe2+ increases TNF- mRNA levels.
We then examined whether TNF-
promoter activity induced by treatment
with Fe2+ is associated with increased mRNA levels for this
cytokine. As shown in RT-PCR data in Fig. 2C, the iron
treatment increased TNF-
message. Densitometric analysis and
standardization with
-actin data showed 2.3- and 2.0-fold increases
in TNF-
message by 10 and 50 µM Fe2+, respectively.
Fe2+ activates NF-B in cultured
Kupffer cells.
Next, we examined whether Fe2+ increases the binding of
nuclear proteins to the
B site in cultured rat Kupffer cells. At 10 and 50 µM, there was increased DNA binding regardless of whether we
used the consensus sequence (Fig. 3) or
the
B site from the TNF-
promoter (data not shown). Figure
3A shows the representative EMSA results obtained with 50 µM Fe2+. Increased binding was noted from 30 min
following the iron addition and lasted for 2-4 h. Densitometric
analysis of three sets of EMSA results demonstrated 3.4 ± 1.0-fold and 2.1 ± 0.8-fold increases (n = 3, P < 0.05) in p65/p50 and p50/p50 binding at 30 min
after the treatment with Fe2+, respectively. At 2 h,
the intensities of both bands were only moderately increased by 67%
for p65/p50 and 86% for p50/p50. AP-1 binding was analyzed by using
the same nuclear extracts, but no changes were noted (Fig.
3A). Similar results were observed with 10 µM
Fe2+ (data not shown). The supershift assay was performed
to identify the proteins encompassing the two sizes of the DNA-protein
complexes detected by NF-
B EMSA. This assay revealed that they were
a p50/p50 homodimer and a p65/p50 heterodimer (Fig. 3B). To
confirm that iron-induced enhancement in NF-
B DNA binding was due to
activation of the transcription factor, we performed Western blot
analysis for cytosolic I
B
and nuclear p65. As shown in the
representative blots in Fig. 4, the
cytosolic level of I
B
was transiently reduced at 30 min-1 h
while the nuclear p65 level increased from 30 min to ~2-4 h
after the iron addition. Loading of cytosolic or nuclear proteins was
equal, as shown by the staining of the proteins on the filters (Fig.
4). These results were confirmed in three independent experiments.
These results support an interpretation that the iron treatment caused
I
B
degradation, NF-
B activation, and nuclear translocation of
the RelA protein, resulting in increased DNA binding by NF-
B, all
commencing at 30 min. In addition, the lack of the AP-1 response
suggests that the effect of Fe2+ on NF-
B is rather
selective.
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Direct addition of Fe2+ to nuclear
proteins does not increase RelA binding.
Even though our Western blot results strongly supported that activation
of NF-B was most likely responsible for iron-induced enhancement in
DNA binding of this transcription factor, it was still possible that
iron directly increased the association of the nuclear NF-
B to the
B site in the nucleus. To test this possibility, Fe2+
was added to the nuclear extracts prepared from the resting cultured Kupffer cells at 0.1, 1, 10, and 50 µM and the effects were analyzed by EMSA. The results demonstrated that the binding of p50/p50 but not
of p65/p50 was apparently increased by the treatment (Fig. 5), and densitometric analysis of three
sets of data showed 25 ± 7, 46 ± 11, 97 ± 18, and
121 ± 21% increases in p50/p50 binding at 0.1, 1, 10, and 50 µM, respectively, and confirmed no increase in p65/p50 binding. These
data suggested that this direct effect of iron on the nuclear extracts
could not explain the increased binding of p65/p50 observed in the
iron-treated cells.
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Iron activates IKK.
To investigate the mechanisms of iron-mediated activation of NF-B,
we examined the effect of Fe2+ on IKK activity in cultured
Kupffer cells at different time points. As shown in Fig.
6, IKK activity, as assessed by
phosphorylation of GST-I
B
, was increased at 15 min, whereas the
total IKK level was unchanged. As a positive control, LPS-stimulated
IKK activity is shown. The timing of IKK activation preceded the
disappearance of cytosolic I
B
at 30 min after addition of iron
(Fig. 4). In contrast, iron did not induce JNK activity (Fig. 6), and
this result corroborated unchanged AP-1 binding by iron (Fig.
3A). Another stress-activated mitogen-activated protein
kinase (MAPK), p38, was also assessed. The level of phosphorylated p38
was also unaffected by the iron treatment, suggesting that
Fe2+ did not activate this MAPK (H. She, unpublished
observations). The results on IKK and JNK were confirmed in at least
three independent experiments. Thus these results demonstrate for the
first time that Fe2+ activates IKK and support a notion
that Fe2+ serves as an agonist to stimulate signal
transduction, which is rather selective for activation of NF-
B.
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Iron increases EPR-detectable radicals before NF-B activation.
NF-
B is a redox-sensitive transcription factor, and ROS are
implicated in its activation (1, 34, 36, 38). Thus we postulated that Fe2+ stimulates ROS production in Kupffer
cells preceding activation of NF-
B. In fact, Fe2+ can
react with oxygen in aqueous solution to produce Fe3+ and
O
B at 30 min suggests the signaling role of the
former in the latter events. In fact, this notion was developed in
previous studies (37) that demonstrated activation of
NF-
B by ·OH-gener- ating systems and a reversal of this effect by
·OH scavengers or metal chelators in Jurkat cells.
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DISCUSSION |
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Biological and mechanistic implications.
The results presented by the current study demonstrate a direct
stimulatory effect of Fe2+ on signal transduction for
NF-B activation in cultured Kupffer cells. The effect is seen at
least at the level of IKK activation and extended to the most
downstream level of TNF-
protein expression. These results suggest a
possibility that iron may serve as an independent agonist for
activation of NF-
B and induction of NF-
B-responsive genes in
Kupffer cells in vivo. In fact, iron supplementation aggravates liver
injury induced by alcohol (41) or hepatitis viral
infection (4) in experimental animals. In a clinical setting, the increased hepatic iron content frequently accompanies many
different types of liver disease, such as alcoholic liver disease
(28), viral hepatitis (10), and nonalcoholic
steatohepatitis (5, 25), and iron reduction modalities
often ameliorate such liver damage (10). Acute iron
loading to the isolated perfused rat liver results in early increases
in Kupffer cell-dependent respiratory activity (40), and
iron directly enhances interleukin-1 secretion by macrophages
stimulated by interferon-
and LPS (7). We have
previously demonstrated that the treatment of cultured Kupffer cells
with an iron chelator effectively suppressed activation of NF-
B
(22). Therefore, the evidence presented by the current study offers the pivotal molecular basis for the link between iron and
NF-
B activation suggested by the earlier studies. Indeed, in
pathological livers, iron that is compartmentalized into protein-bound forms may be released transiently into the microenvironment due to
oxidative (6, 39) or nitrosative (11, 13, 21)
stress. This catalytically active pool of iron may directly activate
NF-
B in Kupffer cells in vivo.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health grants R37-AA-06603, P50-AA-11999 (USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases), P30-DK-48522 (USC Research Center for Liver Diseases), R24-AA-12885 (Non-Parenchymal Liver Cell Core), and the Medical Research Service of the Department of Veterans Affairs. S. Xiong was supported by a Cooley's Anemia Foundation Postdoctoral Award.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Tsukamoto, Keck School of Medicine, Univ. of Southern California, 1333 San Pablo St., MMR-402, Los Angeles, CA 90033-9141 (E-mail: htsukamo{at}hsc.usc.edu).
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.
10.1152/ajpgi.00108.2002
Received 19 March 2002; accepted in final form 12 May 2002.
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