From the Department of a Pathology and g Molecular Microbiology and Immunology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, the e Department of Internal Medicine and Center for Gene Therapy, University of Iowa College of Medicine, Iowa City, Iowa 52242, the f Division of Biochemistry and Molecular Biology, University of Southampton, Southampton S016 6YD, United Kingdom, the h Department of Chemistry, University of Minnesota, Duluth, Minnesota 55812, and the i Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, California 90073
Received for publication, October 24, 2002, and in revised form, February 25, 2003
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ABSTRACT |
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Iron chelators inhibit
endotoxin-induced NF- NF- Hepatic macrophages (HMs), i.e. Kupffer cells,
represent the largest population of macrophages, accounting for as much
as 70-80% to total body macrophages. In addition to their crucial role in the first line of defense against invading microorganisms or
bacterial products via splanchnic circulation, HMs represent the major
site for both regulation of inflammatory and immune responses and
metabolism of iron. For the former, HMs release a wide array of soluble
factors, including cytokines and chemokines, that are mostly encoded by
NF- The finding that iron chelators inhibit activation of NF- Materials--
Cell lines employed in this study included
RAW264.7 cells obtained from the American Tissue Culture Collection
(Rockville, MD), R37 cells, stably Nramp1G169 expressing RAW264.7, and
R21 cells stably transfected RAW264.7 with a vector encoding the gene in an antisense orientation, both provided by Dr. C. H. Barton (University of Southampton, UK). Fetal bovine serum was obtained from
Sigma Chemical Co. (St. Louis, MO). Adenoviral vectors, AdN17Rac1 (Ad.DN-Rac1), Ad.CuZn-SOD, and Ad.LacZ, expressing dominant negative mutant Rac1, Cu,Zn-SOD, or Cell Preparation--
HMs were isolated from male Wistar rats by
the Non-parenchymal Liver Cell Core of the Research Center for
Alcoholic Liver and Pancreatic Diseases as previously published (24,
25). Briefly, the liver was digested in situ by sequential
perfusion with Pronase and collagenase, and non-parenchymal liver cells were fractionated by discontinuous gradient ultracentrifugation using
arabinogalactan. An HM-enriched fraction was further purified by the
adherence method to achieve the final purity exceeding 95%. After 3 days of culture, the cells were treated with LPS (100 or 500 ng/ml) or
TNF Fe59 Labeling and [LMW·Fe]i
Measurement--
Freshly isolated HMs, hepatocytes, liver
myofibroblastic cells, or RAW264.7 cells were plated onto a 60-mm dish.
The cells were cultured with 5 µCi/ml FeCl3 for 14-16 h
in 5 ml of DMEM containing 5% fetal bovine serum and antibiotics.
Labeled cells were sequentially washed with 5 ml of warm PBS once, PBS
containing 100 µM bathophenanthroline sulfate once, and
PBS twice. The washed cells were treated with LPS (500 ng/ml) or TNF Measurement of Intracellular Chelatable Iron in HMs--
The
chelatable pool of iron in HMs was measured as previously described
(27). Briefly, primary cultures of rat HMs were washed with PBS twice,
scraped gently, and incubated in PBS (5 × 106/3 ml)
to which an iron chelator, desferrioxamine or 1,10-phenanthroline, was
added to form a stable complex with iron. The formed complexes were
spectrophotometrically detected by their absorption at 430 and 510 nm,
respectively. The kinetic of complex formation was assessed with a
Beckman spectrophotometer equipped with a flow cuvette connected with a
vessel with constant stirring. The blank absorption was obtained by
using the mixture of all reagents except chelators.
IKK Assay--
To assay the activity of IKK, HMs cultured in a
100-mm dish were treated with LPS (500 ng/ml) for 15-30 min in the
presence and absence of the iron chelators, L-NIL, or FeTTPS, washed
once with PBS, and lysed with a lysis buffer (20 mM
Tris-HCl, pH 7.5, 20 mM NaF, 20 mM
Immunoblot Analysis--
For immunoblot analysis of IKK and
nitrated I Nuclear Protein Extraction and Electrophoretic Mobility Shift
Assay--
To examine DNA binding by NF- Analysis of Nramp1G169-expressing RAW264.7--
A
recent study (33) demonstrates that RAW264.7 cells stably transfected
with a vector expressing a wild type Nramp1G169 (a
transformant designated as R37) have lower cellular iron load and a
reduced chelatable iron pool as compared with those stably transfected
with a vector encoding the gene in an antisense orientation (designated
as R21). We have obtained these two stable transformants from Dr.
C. H. Barton's laboratory (University of Southampton, UK) and
tested whether the altered chelatable pool of iron in R37 affects the
[LMW·Fe]i signaling, IKK activation, NF- LPS-induced [LMW·Fe]i--
We previously showed
that the treatment of cultured HMs with an iron chelator, L1, prevents
LPS-induced NF- [LMW·Fe]i Response Is Unique to
Macrophages--
We next tested whether TNF Inhibition of iNOS and NADPH Oxidase Abrogates Inhibit
[LMW·Fe]i Rise--
To further examine the association
between the [LMW·Fe]i response and NF- Peroxynitrite Is Responsible for [LMW·Fe]i
Signaling--
The results presented so far suggest that ·NO
produced by iNOS and NADPH oxidase-derived O LPS and Peroxynitrite Increase Tyrosine Nitration of
I Nramp1 Overexpression Modulates [LMW·Fe]i
Signaling--
Nramp1 is a 90- to 100-kD membrane-spanning protein
that is known to confer the natural resistance of macrophages to
infection by intracellular pathogens (39) through mechanisms that are believed to regulate iron transport into or out of late endosomes (Ref.
40, for review). A recent study (33) using a stable transformant of
RAW264.7 cells expressing high level of Nramp1 revealed that these
cells (R37) have less total iron content and a reduced chelatable pool
of iron as compared with the mock transfected cells (R21). We
considered that the use of these cells would offer a unique opportunity
to assess the effects of the reduced chelatable iron pool on
LPS-stimulated [LMW·Fe]i response and IKK activation. The
total non-heme iron content in R37 cells was indeed reduced by 25%
(Fig. 7A, left
panel). Following Fe59 loading, the specific activity
achieved with the isotope was comparable between the two cell lines
(Fig. 7A, middle panel). The chelatable pool of
iron as assessed by an increase in the radioactivity in the low
molecular weight fraction after L1 treatment of the cell lysate was
significantly reduced in R37 cells (Fig. 7A, right
panel), confirming the finding by Atkinson and Barton (33). When
these cells were treated with LPS, R37 cells showed attenuated
[LMW·Fe]i response as compared with R21 cells (Fig.
7B), and this alteration was associated with inhibited IKK activity (Fig. 7C), NF- Iron Directly Activates IKK--
To test the cause and effect
relationship, we next examined the effect of direct addition of ionic
iron to cultured HMs on NF- The present study demonstrated a novel transient rise in the
intracellular labile level of iron ([LMW·Fe]i) that leads
to IKK and NF- The source of [LMW·Fe]i remains unknown. Three interesting
possibilities are suggested. First, ferritin, the main intracellular
site for sequestration and storage of excess iron, may be a target for
oxygen (42, 43) and nitrogen (44) radicals for release of iron. The
ONOO An equally important question is how the transient rise in
[LMW·Fe]i leads to IKK activation. One possibility is iron-mediated release of calcium. Oxidant stress is known to induce calcium mobilization in a variety of cell types (49-51). This mode of
calcium mobilization is prevented with cell-membrane permeable iron
chelators (52), suggesting the involvement of iron in this process. In
fact, excessive iron loading is known to perturb mitochondrial calcium
homeostasis and to induce the release of calcium from this storage site
(53). More recently, micromolar concentrations of ferrous iron were
shown to cause a transient release of calcium from liver mitochondria
without either damaging the organelle or decreasing its membrane
potential, suggesting the physiological implication of iron-mediated
calcium mobilization (54). Moreover, ONOO Peroxynitrite was recently shown to activate NF- It is interesting that the [LMW·Fe]i response was
evident only in HMs and the murine macrophage cell line but not in
hepatocytes and myofibroblastic cells, despite the finding that the
latter cells also activated NF-B activation in hepatic macrophages (HMs),
suggesting a role for the intracellular chelatable pool of iron in
NF-
B activation. The present study tested this hypothesis.
Analysis of Fe59-loaded HMs stimulated with
lipopolysaccharide (LPS), revealed a previously unreported, transient
rise in intracellular low molecular weight (LMW)·Fe59
complex ([LMW·Fe]i) at
2 min returning to the
basal level within 15 min. The [LMW·Fe]i response preceded I
B kinase (IKK) (
15 min) and NF-
B (
30 min) activation. Iron chelators (1,2-dimethyl-3-hydroxypyridin-4-one and
N,N'-bis-2-hydroxybenzylethylenediamine-N,N'-diacetic acid) abrogated the [LMW·Fe]i response and IKK and
NF-
B activation. The [LMW·Fe]i response was also
observed in tumor necrosis factor
(TNF
)-stimulated HMs and
RAW264.7 cells treated with LPS and interferon-
but not in primary
rat hepatocytes or myofibroblastic cells exposed to LPS or TNF
. Both [LMW·Fe]i response and IKK activation in LPS-stimulated HMs
were inhibited by diphenylene iodonium (nonspecific inhibitor for
flavin-containing oxidases),
L-N6-(1-iminoethyl)lysine
(selective iNOS inhibitor), and adenoviral-mediated expression of a
dominant negative mutant of Rac1 or Cu,Zn-superoxide dismutase,
suggesting the role of ·NO and O
,
5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride. Conversely, ONOO
alone induced both
[LMW·Fe]i response and IKK activation. Finally, direct
addition of ferrous iron to cultured HMs activated IKK and NF-
B.
These results support a novel signaling role for [LMW·Fe]i
in IKK activation, which appears to be induced by ONOO
and selectively operative in macrophages.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B is the prototypic transcription factor in eukaryotic cells
known to play a pivotal role in transactivation of promoters for genes
involved in inflammation, immune responses, and anti-apoptotic mechanisms (Ref. 1, for review). At least two levels of redox regulation of NF-
B appear to exist: one in the nucleus and another in the cytoplasm. The former involves direct redox modification of
specific cysteine residues in the DNA binding domain of NF-
B. In
particular, oxidation of cysteine at the position 62 in p50 inhibits
DNA binding activity (2). Conversely, reduction of NF-
B by
thioredoxin and Ref-1 appear to increase its activity (3, 4).
For the cytosolic regulation, upstream signaling that is yet to
be determined, leads to activation of
IKK,1 resulting in
phosphorylation of two serine residues (Ser-32 and Ser-36) on I
B
,
its polyubiquitination, and degradation by 26 S proteasome.
Treatment of the cells with the antioxidant,
N-acetyl-L-cysteine or pyrrolidine
dithiocarbamate prevents activation of NF-
B (5-7), whereas addition
of H2O2 or the generation of O
B in certain cell types (5, 6, 8). TNF
-induced
activation of NF-
B is abrogated by inhibition of ROS production by
the electron transport chain in mitochondria suggesting oxidant stress
from this organelle as a signal for this mode of activation (9).
Inhibition of NADPH oxidase blocks or attenuates activation of NF-
B
in monocytic cells (10, 11), whereas inhibitors for 5-lipoxygenase
reduce both ROS generation and NF-
B activation in lymphoid cells
(10), indicating the possible cell type-dependent
differences in the source of ROS signals for activation of this
transcription factor. It is also important to note that intracellular
ROS generation may not be necessary for NF-
B activation in all cell
types. Indeed, in epithelial cell lines, no detectable increase in ROS
generation is observed in association with activation of NF-
B (12).
Moreover, anti-oxidants and metal chelators are often ineffective in
inhibiting NF-
B activation in these cells (13). Thus, it appears
that a tighter association exists between oxidant stress and NF-
B
activation in monocytic or lymphoid cells.
B-responsive genes as well as growth factors, lipid metabolites,
and gaseous mediators (O
and LPS (22). On the contrary,
the treatment with iron chelators inhibits LPS-mediated NF-
B
activation and expression of TNF
and interleukin-6 by cultured peritoneal macrophages (23) and HMs (24).
B in
macrophages suggests a role for a chelatable pool of iron in the signal
transduction for this molecular event. In testing this hypothesis, the
present study disclosed a novel and transient rise in the intracellular
level of low molecular weight·iron complex ([LMW·Fe]i) in
macrophages stimulated with LPS or TNF
. This response appears
specific to macrophages and precedes IKK activation and increased
NF-
B binding. The [LMW·Fe]i response is tightly
correlated with NF-
B activation and abrogated by inhibition of iNOS
and NADPH oxidase or overexpression of SOD. Our results support the
notion that ONOO
is the effector molecule for inducing
[LMW·Fe]i release. Last, addition of ionic iron to HMs
caused activation of IKK, establishing that iron can serve as an
independent agonist and as a potential signaling molecule for IKK activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase, respectively, were obtained from Dr. J. F. Engelhardt (University of Iowa).
Recombinant murine interferon-
, TNF
, and a TNF
enzyme-linked
immunosorbent assay kit were purchased from R&D Systems (Minneapolis,
MN). Anti-human I
B
and antibodies against p50 and p65 were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Iron 59 (FeCl3), [
-32P]ATP, and
[
-32P]dATP were obtained from PerkinElmer Life
Sciences (Boston, MA). Peroxynitrite (ONOO
) and its
scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III)
chloride (FeTTPS) were obtained from Calbiochem (San Diego, CA).
S-Nitroso-N-acetylpenicillamine (SNAP) was
purchased from Molecular Probes, Inc. (Eugene, OR). Pronase was
obtained from Roche Applied Science (Indianapolis, IN). Nitrocellulose filters was obtained from Bio-Rad (Hercules, CA). ECL kit was obtained
from Amersham Corp. (Arlington Heights, IL). Size-exclusion column was
purchased from Millipore Corp. (Bedford, MA).
L-N6-(1-Iminoethyl)lysine
(L-NIL) was obtained from Alexis Biochemicals (San Diego,
CA). Goat anti-rabbit IgG-horseradish peroxidase, collagenase type IV,
arabinogalactan, LPS (Escherichia coli 055:B5), sodium
bathophenanthroline disulfonate, N-acetylcysteine (NAC), diphenylene iodonium (DPI), and phosphatase inhibitor mixture were
purchase from Sigma Chemical Co. (St. Louis, MO). Iron chelators, 1,2-dimethyl-3-hydroxypyridin-4-one (L1) and
N,N'-bis-2-hydrobenzylethlenediamine-N,N'-diacetic acid (HBED) were generous gifts from Dr. Gary Brittenham (College of
Physicians and Surgeons, Columbia University, NY).
(10 ng/ml) in serum-free DMEM in the presence or absence of
various inhibitors as described below for subsequent collection of cell
lysate for IKK assay and nuclear protein extraction for NF-
B
electrophoretic mobility shift assay. Hepatocytes were isolated using
the standard collagenase digestion technique (26) by the Cell Culture
Core of the USC Research Center for Liver Diseases. The cells were
cultured in DMEM with 10% FCS and used immediately after overnight
culture for the experiments described below. RAW264.7 cells were
obtained from the American Tissue Culture Collection and cultured in
DMEM with 25 mM glucose and 10% FCS. Rat hepatic
myofibroblastic cells were isolated from a rat with biliary liver
fibrosis by the method described for HMs and by collecting a
fraction from the gradient interface between the medium and
1.035. They became spontaneously immortalized, and subsequently
cell cones were established by the limiting dilution method. They are
cultured in DMEM with 10% FCS.
(10 ng/ml) in 5 ml of warm PBS for 2, 5, 10, or 20 min in the presence
or absence of iron chelators (L1, 100 µM; HBED, 100 µM), N-acetylcysteine (NAC, 500 µM), a nonspecific inhibitor for flavin-containing
oxidases, including NADPH oxidase and iNOS (DPI, 1 µM), a
selective iNOS inhibitor (L-NIL, 20 µM), and
a cell-permeable decomposition catalyst for ONOO
(FeTTPS,
200 µM). These inhibitors were added 0.5~4 h prior
to addition of LPS. For adenoviral-mediated expression of a dominant negative mutant Rac1 (Ad.N17Rac1), Cu,Zn-SOD (Ad.CuZn-SOD), or
-galactosidase (Ad.LacZ) as a control, the cultured HMs were infected with the vectors at the multiplicity of infection of 50 ~24
h before addition of LPS. The cells were also treated with a
spontaneous nitric oxide donor (SNAP) to examine their effects on
[LMW·Fe]i. The incubation was stopped at the respective time point by removing PBS completely and adding 200 µl of lysis buffer containing 1.4 M NaCl, 0.1 M HEPES (pH
7.4), 1.5% Triton X-100, and 1 mM phenylmethylsulfonyl
fluoride. A low molecular mass fraction (<5000 Da) was prepared
by a centrifugation of the lysate in a size-exclusion column
(Millipore, MA) at 8600 × g for 30 min at 4 °C.
Radioactivity of ultrafiltrate or total lysate was determined by a
liquid scintillation counter.
-glycerophosphate, 0.5 mM
Na3VO4, 2.5 mM metabisulfite, 5 mM benzamidine, 1 mM EDTA, 0.5 mM
EGTA, 10% glycerol and protease inhibitors, 300 mM NaCl,
and 1.5% Triton X-100). The lysates were immediately frozen in liquid
nitrogen and stored at
80 °C until assayed. IKK activity was
determined as previously described (28). HMs were also treated with
SNAP, ONOO
, and FeSO4 at the indicated
concentrations to test their direct effects on IKK. 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 dithiothreitol, 20 µM ATP, 2 µg of
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 analyzing
the membrane with a PhosphorImager (Amersham
Biosciences).
B
, cytosolic extracts were prepared as previously
reported (29). Proteins were resolved on a 10%
SDS-polyacrylamide gel (SDS-PAGE), transferred to a nitrocellulose
membrane, incubated with anti-IKK
antibodies, and detected by
chemiluminescence. For immunoblot analysis of nitrated I
B
,
I
B
was first immunoprecipitated with anti- I
B
antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA), and the immune complex was
resolved on a 10% SDS-PAGE and transferred to a membrane for
immunoblot analysis using anti-nitrotyrosine antibodies (Upstate
Biotechnology, Lake Placid, NY).
B, nuclear proteins were
extracted using the method of Schreiber et al. (30) from
cultured HMs, RAW264.7 cells, hepatic myofibroblastic cells, or
hepatocytes exposed to LPS or TNF
in the presence or absence of the
iron chelators. 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, 20% glycerol,
200 µg/ml poly(dI-dC)) on ice with the double-strand
B consensus
sequence (31) or the
B site from TNF
promoter (32) labeled with
32P. After a 20-min incubation, the reaction mixture was
resolved on a 6% non-denaturing polyacrylamide gel, and the gel was
dried for subsequent autoradiography. For the supershift assays,
antibodies against p50 and p65 (Santa Cruz Biotechnology) were added to
the reaction mixture for an additional 30 min.
B binding, and
TNF
release following LPS stimulation. TNF
immunoassay was
performed with a commercially available mouse TNF
enzyme-linked
immunosorbent assay kit (R&D Systems, Minneapolis, MN). For the
measurement of total non-heme iron content, R37 or R21 cells (3 × 107) were washed with ice-cold PBS twice. The cells were
treated with 1 ml of solution of 10% (w/v) trichloroacetic acid
dissolved in 3% (v/v) thioglycolic acid and 1.94 M HCl,
vortexed, and put on ice for 10 min. After centrifugation at
14,000 × g for 5 min at 4 °C, 100 µl of
supernatant was incubated for 30 min at 37 °C with the equal volume
of 0.42 mM bathophenanthroline sulfonate in 2 M
sodium acetate. The reaction mixture was then measured for the
absorbance at 550 nm on a microtiter plate scanning spectrophotometer (Power Wave 200TM, Bio-Tek Instruments, Winooski, VT). An
iron atomic absorption standard solution (Aldrich) was used as iron
standard. The labeling with Fe59 was performed as described
for HMs, the specific activity of Fe59 in the cells was
determined by dividing the radioactivity by iron content, and the
chelatable pool of iron was determined by measuring the mobilization of
Fe59 radioactivity into the low molecular weight fraction
following the treatment of the cell lysate with L1 (100 µM) for 5 min at room temperature.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation (24), suggesting the role of the
chelatable pool of iron in activation of this transcription factor. To
examine this possibility, HMs were loaded with Fe59, washed
stringently, and stimulated with LPS to determine
time-dependent changes in the Fe59
radioactivity in the low molecular weight cytosolic fraction ([LMW·Fe]i). Addition of LPS (500 ng/ml) resulted in a
transient rise in [LMW·Fe]i at
2 min, followed by a
decline to its basal level by 10-15 min (Fig.
1A). The radioactivity of
[LMW·Fe]i, separated by a size exclusion column with the
molecular mass cutoff of 5000 Da, was 1~2% of total cell-associated radioactivity and increased 2-fold by LPS stimulation. Because the
earliest time point we were able to process the cells for this
analysis was 2 min, this response might have taken place earlier than 2 min. Kinetic analysis of this event in relation to IKK and NF-
B
activation revealed that the [LMW·Fe]i response preceded
the beginning of increased IKK activity at 15 min and that of NF-
B
binding at 30 min (Fig. 1, B and C). Addition of
an iron chelators, L1 (100 µM, Fig.
2A) or HBED (100 µM, data not shown), prior to LPS stimulation completely
abrogated the response as well as IKK (Fig. 2B) and NF-
B
(Fig. 2C) activation. Because the assessment of a cytosolic
labile pool of iron after detergent lysis of the cells may cause the
release of iron from solubilized vesicles, organelles, or proteins, we
have also employed another approach to determine changes in the
chelatable pool of iron in intact cells. This method involved chelation
of iron with desferrioxamine or 1,10-phenanthroline and
spectrophotometric determination of the iron·chelator complexes in
cell suspension. Using this method, we confirmed a transient rise in
the chelatable iron level with either chelator following LPS
stimulation (Fig. 3). However, the
response curve shifted to right with a peak occurring between 5 and 7 min, instead of 2 min or earlier when detected by the Fe59
method. This delay probably reflected the fact that the chelators were
not reaching liberated iron immediately and that this
spectrophotometric method has a higher detection threshold than the
radioactive method. Nevertheless, the early, transient iron response in
LPS-stimulated HMs was confirmed by this method in the intact
cells.
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Fig. 1.
LPS induces a transient rise in the
intracellular level of low molecular weight·iron complexes
([LMW·Fe]i), which precedes activation of IKK and
NF- B. A, cultured hepatic
macrophages (HMs) were loaded with Fe59, washed, and
stimulated with LPS (500 ng/ml) in PBS. Cell lysate collected at
different time points was subjected to the size exclusion column
(<5000 kDa), followed by scintillation counting of the filtrate. The
[LMW·Fe]i data are expressed as the percentage to the basal
level following standardization by total cellular Fe59
radioactivity. Note a distinct and transient rise in
[LMW·Fe]i at 2 min. B, HMs were similarly
stimulated with LPS, and cell lysate was collected for an IKK assay
with GST-I
B
as substrate. Note the induction of IKK activity
commences at 15 min. C, nuclear proteins were extracted from
LPS-treated HMs for electrophoretic mobility shift assay using a
B
probe. Note an increase in NF-
B binding occurs at 30 min. The data
presented in A represent means ± S.D. from six
experiments. IKK and NF-
B binding assays were performed on samples
from at least four separate experiments.
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Fig. 2.
Iron chelators block the
[LMW·Fe]i response, IKK activity, and NF-
B binding in LPS-stimulated HMs. A,
note the pretreatment of cultured HMs with L1 abrogates LPS-induced
increase in the [LMW·Fe]i. B, the treatment of
HMs with L1 or HBED completely prevents LPS-induced IKK activity.
C, similarly, L1 and HBED prevent increased binding of the
p50/p65 heterodimer to the probe. The data presented in A
represent means ± S.D. from four experiments. IKK and NF-
B
binding assays were performed on samples from at least four separate
experiments.
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Fig. 3.
The release of iron is confirmed in the
intact HMs. To confirm the intracellular release of iron in the
intact HMs without detergent lysis of the cells, the cells were
stimulated with LPS, and the formation of complexes between iron and
added chelators (desferrioxamine (top) and 1-10
phenanthroline (bottom)) was monitored
spectrophotometrically in cell suspension. Note the similar transient
rise in the formation of the iron·chelator complex using either
chelator. The peak is slightly shifted right between 5 and 7 min as
compared with the response detected with the Fe59 method.
The data represent means ± S.D. from three separate
experiments.
, another agonist for
NF-
B activation, causes the [LMW·Fe]i response in HMs.
After addition of TNF
, the identical, transient response to that
seen after LPS treatment was reproduced at 2 min (data not shown). We
then tested whether the [LMW·Fe]i response is observed in other cell types when NF-
B is activated. The [LMW·Fe]i was examined in cultured hepatocytes treated with LPS or TNF
. Despite the fact that these treatments caused robust NF-
B
activation, no [LMW·Fe]i response was observed (data not
shown). Next, we examined rat liver myofibroblastic cells stimulated
with TNF
and RAW264.7 cells, the murine macrophage cell line,
treated with interferon
(10 units/ml) and LPS (100 ng/ml). Again,
no significant changes occurred in the [LMW·Fe]i in
myofibroblastic cells, although NF-
B binding was increased (data not
shown). On the other hand, RAW264.7 cells showed a conspicuous
[LMW·Fe]i peak at 2 min after the treatment preceding
activation of NF-
B (data not shown). Therefore, these results
suggest that the [LMW·Fe]i response is unique to macrophages.
B activation and
to explore potential mechanisms underlying induction of the
[LMW·Fe]i response, we tested whether the known inhibitors
of NF-
B activation could regulate the [LMW·Fe]i
increase. Cultured HMs were stimulated with LPS in the presence of
N-acetylcysteine (NAC, 500 µM), diphenylene iodonium (DPI, 1 µM), a nonspecific inhibitor for
flavin-containing oxidases such as NADPH oxidase and iNOS, and
L-NIL (20 µM), a selective iNOS inhibitor.
DPI and L-NIL effectively blocked the [LMW·Fe]i
rise, whereas NAC or Me2SO as a vehicle had no
effect (Fig. 4A). DPI was
shown to inhibit NF-
B activation in HMs (11), whereas NAC did not
inhibit activation and nuclear translocation of NF-
B but suppressed
NF-
B-mediated transcription in HMs (34). Thus, our results for
[LMW·Fe]i correlate with these findings. L-NIL
was shown to attenuate LPS-induced NF-
B binding in RAW264.7 cells
(35), and our study demonstrates suppression by L-NIL of
LPS-induced IKK activity in HMs (Fig. 4B). Thus, these
results further tighten the relationship between the
[LMW·Fe]i response and activation of NF-
B, supporting the signaling role of [LMW·Fe]i. They also suggest the involvement of iNOS and NADPH oxidase in facilitating this signaling event. Because DPI is not a specific inhibitor, to further test the
role of NADPH oxidase, we next used an adenoviral vector expressing a
dominant negative mutant of Rac1 (Ad.DN-Rac1) and a vector expressing
-galactosidase (Ad.LacZ) as a control. Ad.DN-Rac1 has recently been
used to block activation of NADPH oxidase and subsequent activation of
NF-
B in LPS-stimulated macrophages via its dominant negative effect
on the recruitment of the regulatory units of the oxidase to the
membrane (36). HMs were infected with Ad.DN-Rac1 followed by
stimulation with LPS, and the [LMW·Fe]i rise was measured.
As shown in Fig. 4C, Ad.DN-Rac1 infection abrogated the
[LMW·Fe]i rise, whereas Ad.LacZ did not. This suggests the
importance of Rac1-mediated NADPH oxidase activation in generating the
[LMW·Fe]i rise. To test whether O
B activation in macrophages (36), and
indeed our experiment demonstrated complete inhibition of the
[LMW·Fe]i rise by Ad.SOD infection (Fig.
4D).
View larger version (27K):
[in a new window]
Fig. 4.
A, diphenylene iodonium (DPI,
1 µM), a nonspecific inhibitor for
flavin-containing oxidases such as NADPH oxidase and iNOS, and
L-N6-(1-iminoethyl)-lysine, a selective iNOS
inhibitor (L-NIL, 20 µM), abrogate the
LPS-induced [LMW·Fe]i response in HMs. B,
L-NIL also inhibits LPS-stimulated IKK activity in HMs
(upper panel) while not affecting the IKK level as shown by
immunoblotting (lower panel). The data in A
represent means ± S.D. from four experiments. *,
p < 0.05 as compared with the basal; +,
p < 0.05 as compared with LPS plus Me2SO.
Expression of a dominant negative mutant of Rac1 (C) or
Cu,Zu-SOD (D) blocks LPS-induced [LMW·Fe]i
response in HMs. Cultured HMs were infected with an adenoviral vector
expressing a dominant negative mutant Rac1 (Ad.DN-Rac1), Cu/Zu-SOD
(Ad.SOD), or LacZ (Ad.LacZ) as a control. Twenty-four hours later, the
cells were stimulated with LPS (500 ng/ml) for the
[LMW·Fe]i analysis as described under "Experimental
Procedures." The data were obtained from four and three sets of
experiment for C and D, respectively. *,
p < 0.05 as compared with the basal; +,
p < 0.05 as compared with Ad.LacZ.
B activation in HMs. Indeed, both radical species are required
for this iron-mediated signaling pathway, because ·NO alone
produced by addition of SNAP to the cells caused no transient rise in
[LMW·Fe]i but a gradual and sustained increase in this
parameter (Fig. 5A). In
addition, SNAP did not activate IKK (Fig. 5B). Thus, these
results indicate that ONOO
, a radical species
generated from ·NO, and O
. The
treatment abrogated LPS-induced [LMW·Fe]i rise (Fig.
6A) and IKK activation (Fig.
6B). Finally, HMs were directly treated with
ONOO
. Peroxynitrite at the concentrations of 5-50
µM activated IKK as early as 5 min (Fig. 6C).
This induction of IKK activity is earlier than the timing observed for
LPS stimulation, suggesting that ONOO
is indeed an
intermediate signaling molecule. More importantly, ONOO
induced [LMW·Fe]i rise (Fig. 6D).
View larger version (15K):
[in a new window]
Fig. 5.
SNAP induces a gradual and sustained increase
in LMW·Fe59 (A) and does not activate
IKK (B) in HMs. Fe59-loaded, cultured
HMs were treated with SNAP (500 µM) to determine their
effects on the [LMW·Fe]i and IKK activity as described
under "Experimental Procedures." Note a gradual and sustained
increase in [LMW·Fe]i during 2-20 min after addition of
the NO donor, SNAP (A). SNAP does not activate IKK at 30 and
45 min, while LPS shows strong induction of IKK activity at 30 min
(B).
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Fig. 6.
LPS-induced [LMW·Fe]i response
(A) and IKK activity (B) in HMs are
blocked with FeTTPS, a cell-permeable decomposition catalyst for
ONOO . FeTTPS (200 µM) was added 30 min
prior to addition of LPS to cultured HMs for both the
[LMW·Fe]i analysis and IKK assay. Direct addition of
peroxynitrite induces IKK activity in dose- and
time-dependent manners in HMs (C). Peroxynitrite
(10 µM) also induces an increase in [LMW·Fe]i
(D). LPS induces nitration of I
B
from 5 to 15 min, and
this effect is abrogated by FeTTPS or L1 (E). Peroxynitrite
also increases I
B
nitration at 15-60 min (F).
Representative data from at least three separate experiments are shown
for IKK activity (B and C) and I
B
nitration
(E and F). The data from four and five separate
experiments are shown as means ± S.D. for A and
D, respectively. *, p < 0.05 as compared
with the basal.
B
--
Peroxynitrite is recently shown to activate NF-
B by
the mechanism that appears to involve nitration of I
B
(29). If
this nitration occurs at Tyr-42, this may abrogate negative regulation of I
B
degradation facilitated by phosphorylation of this tyrosine residue (37), resulting in accelerated I
B
degradation.
Alternatively, any nitrated I
B
may be subjected to its
preferential degradation (38). Thus, we then examined whether the
treatment of cultured HMs with LPS or ONOO
results in
nitration of I
B
. For this analysis, HM cytosolic extracts were
immunoprecipitated with an antibody against I
B
followed by
immunoblotting with an anti-nitrotyrosine antibody. As shown in Fig.
6E, LPS induced nitration of I
B
at 5 and 15 min, and
this effect was abrogated when the cells were pretreated with FeTTPS, a
decomposition catalyst for ONOO
or L1, an iron chelator.
The treatment of the cells with ONOO
(10 µM) also increased nitration of I
B
from 15 to 60 min (Fig. 6F). Thus these results support the previous
findings on the role of I
B
nitration as a signaling event for
I
B
degradation besides IKK-mediated phosphorylation of this
inhibitory protein (29) and suggest that ONOO
and iron
are involved in this signaling process in the LPS-stimulated cells.
B binding (Fig. 7D),
and TNF
release by R37 (Fig. 7E). Thus, these results
provide an example of overexpression of a protein that regulates a
chelatable pool of iron and in turn modifies iron-mediated signaling,
NF-
B activation, and expression of an NF-
B-responsive gene.
View larger version (25K):
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Fig. 7.
Overexpression of Nramp1 in RAW264.7 cells
reduces total cellular non-heme iron content, the chelatable pool of
iron, LPS-induced LMW·Fe59 response, IKK activity,
NF- B binding and TNF
release. RAW264.7 cells stably transfected with a vector
encoding wild type Nramp1 in a sense (R37) or antisense
(R21) orientation were compared. The non-heme iron content
is reduced in R37 cells (A, left panel, *,
p < 0.05 compared with R21, n = 3),
whereas the specific activity achieved with Fe59 is
comparable between the two cell lines (A, middle
panel). Note L1-induced mobilization of iron into the low
molecular weight fraction (LMW·Fe59) is significantly
lower in R37, demonstrating the chelatable pool of iron is reduced in
these cells (A, right panel, **,
p < 0.01 compared with R21, n = 3).
They were also examined for LPS-stimulated changes in the
[LMW·Fe]i following Fe59 loading. The data are
expressed as the percentage of the basal level of [LMW·Fe]i
following standardization by total cellular Fe59
radioactivity. Note the [LMW·Fe]i response is attenuated in
R37 cells (B, *, p < 0.05 as compared with
the basal; **, p < 0.05 as compared with R21,
n = 3). LPS-induced IKK activity (C),
NF-
B binding (D), and TNF
release (E, *,
p < 0.05; **, p < 0.01 compared with
R21, n = 3) are also reduced in R37 cells.
B activation. Ferrous sulfate was added
to HMs in the serum-free medium. Addition of ferrous sulfate at the
concentration above 10 µM induced IKK activity (Fig.
8A), and this effect was
apparent as early as 5 min (Fig. 8B). These results
demonstrate that iron can serve as an independent agonist for IKK
activation.
View larger version (16K):
[in a new window]
Fig. 8.
Direct addition of ferrous iron induces IKK
activity in HMs in dose- and time-dependent manners.
Ferrous sulfate was added to cultured HMs at the indicated
concentrations. Note that, at 10 and 50 µM, it induces
IKK activity (A). Addition of FeSO4 (50 µM) begins to activate IKK at 5-15 min
(B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation in response to LPS or TNF
in macrophages. This response occurs rapidly (
2 min) and is transient in
nature, much like the intracellular calcium response seen in agonist-induced cells that initiates calcium-mediated signal
transduction. Our results suggest that both iNOS-derived ·NO and
NADPH oxidase-derived O
is a likely effector molecule for the cytosolic
release of iron. Peroxynitrite has been shown to induce expression of
IL-8 by human blood cells, the effect that was inhibited with
pyrrolidine dithiocarbamate, a classic inhibitor of NF-
B (41). More
recently, the direct evidence for ONOO
-induced NF-
B
binding was presented using human monocytes (29). Our results
demonstrate for the first time that ONOO
activates IKK,
the upstream kinase responsible for phosphorylation of I
B
at
serine residues 32 and 36, facilitating NF-
B nuclear translocation
via polyubiquitination and subsequent degradation of I
B
by
26 S proteasome. Furthermore, we demonstrate that iron directly
activates IKK in cultured HMs. Together, these results support the
notion that ONOO
activates IKK through the release of
iron, placing this novel [LMW·Fe]i signaling event
downstream of ONOO
and upstream of IKK. We further
present an intriguing example of the reduced chelatable pool of iron in
Nramp1-overexpressing RAW264.7 cells that is associated with
attenuation of LPS-induced [LMW·Fe]i response, IKK
activity, NF-
B binding, and TNF
expression. This highlights the
physiological importance of the [LMW·Fe]i response that can
be modified by the changes in cellular iron homeostasis rendered by one
particular protein. We have previously reported an opposite example of
an enhanced chelatable pool of iron in HMs. In these HMs, which were isolated from a rat model of alcoholic liver disease, the expanded chelatable pool of iron was tightly associated with increased NF-
B
binding and TNF
expression, the changes of which could be entirely
normalized by the treatment with the iron chelator, L1 (25). In fact,
these cells exhibit a heightened rise in the labile pool of iron after
LPS stimulation as compared with the cells from control
animals.2 Thus, these
findings collectively demonstrate that the chelatable iron pool within
the cells can modulate the [LMW·Fe]i signaling for IKK and
NF-
B activation and that this mode of regulation has physiological
and pathological relevance.
likely achieves the same effect. Second,
iron-containing proteins can be an important source. Even though
·NO is known to mobilize iron from proteins containing the
[Fe-S] cluster such as IRP-1 (45), a recent study demonstrates that ONOO
is more efficient than ·NO in this effect
(46). Lastly, iron may be mobilized by ·NO or ONOO
from the transitional pool prior to incorporation into ferritin (47).
Regardless of the source, the [LMW·Fe]i response has to be
early and transient to serve as the signaling event, because a gradual
and sustained increase in [LMW·Fe]i seen after the
treatment with a ·NO donor, SNAP, did not activate IKK (Fig. 5,
A and B). This is very analogous to the
differential effects rendered by a transient versus
sustained increase in the intracellular calcium concentration (48).
at a low,
physiologically relevant concentration (20 µM), induced rapid intracellular mobilization of calcium in thymocytes (55). Because
the current study demonstrated the release of iron by ONOO
(Fig. 6D), an increase in the
intracellular calcium concentration may be an immediate consequence
initiating the known calcium-mediated signal transduction leading to
activation of IKK. Conversely, calcium mobilization may occur upstream
of iron. Currently, these possibilities are being investigated. The
analogous kinetic patterns of both iron and calcium mobilization may
also suggest that iron may signal via the mechanism analogous to that
shown for calcium. The transiently released calcium binds to acceptor
proteins such as calmodulin, and they in turn recruit and activate
signaling proteins. This mode of iron-mediated signaling has never been demonstrated but cannot be ruled out.
B in monocytes in a
manner that appears to involve tyrosine nitration of I
B
(29),
possibly preventing negative regulation mediated via phosphorylation of
the tyrosine residues such as Tyr-42 (37). Furthermore, nitrated I
B
becomes a target for degradation by intracellular enzymes (38).
Thus, this nitration-based mode of NF-
B activation does not require
activation of IKK and phosphorylation of Ser-32 and Ser-36 of I
B
.
In fact, our own analysis demonstrates increased nitration of I
B
by the treatment with LPS or ONOO
(Fig. 6, E
and F), supporting this notion. Furthermore, the nitration induced by LPS is prevented by FeTTPS or an iron chelator, L1, suggesting the involvement of ONOO
and iron in this
signaling event. It has yet to be determined how nitration and
phosphorylation I
B
interact to facilitate NF-
B activation in
LPS-stimulated HMs and which sites of I
B
are critical regulatory
targets of nitration.
B in response to agonists. These
results suggest that the iron-mediated signaling may be unique to the
macrophage or similar cell type. This notion may not be so out of line.
After all, the macrophages are the major site of both iron metabolism
and the initiation of inflammatory and immune responses. They
specifically utilize iron in their anti-microbial defense by releasing
iron extracellularly and promoting Fenton pathway-mediated killing of
microbes (20). They also express Nramp1, which regulates efflux of iron
from or influx of this metal into late endosomes to help eliminate
intracellular pathogens. We propose that macrophages are also evolved
to uniquely develop the mechanism by which iron can be utilized as a
signaling molecule to support transcriptional regulation of
inflammatory and immune-related genes.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the invaluable advice and guidance of Dr. Gary Brittenham and earlier technical support of Dr. Min Lin.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants R37AA06603, P50AA11999 (USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases), P30DK48522 (USC Research Center for Liver Diseases), and R24AA12885 (Non-parenchymal Liver Cell Core) and by the Medical Research Service of the Department of Veterans Affairs.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.
b Both authors contributed equally to this work.
c Supported by Cooley's Anemia Foundation Postdoctoral award.
d Supported by National Institute on Alcohol Abuse and Alcoholism Institutional Training Grant T32-AA07578.
j To whom correspondence should be addressed: Keck School of Medicine, University of Southern California, 1333 San Pablo St., MMR-402, Los Angeles, CA 90033-9141. Tel.: 323-442-5107; Fax: 323-442-3126; E-mail: htsukamo@usc.edu.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M210905200
2 S. Xiong, H. She, and H. Tsukamoto, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IKK, IB kinase;
HMs, hepatic macrophages;
[LMW·Fe]i, intracellular low
molecular weight·iron complex;
TNF
, tumor necrosis factor
;
LPS, lipopolysaccharide;
NF-
B, nuclear factor-
B;
AP-1, activator
protein 1;
ROS, reactive oxygen species;
O
, peroxynitrite;
FeTTPS, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) chloride;
Cu, Zn-SOD, copper-zinc superoxide dismutase;
SNAP, S-nitroso-N-acetylpenicillamine;
iNOS, inducible
nitric-oxide synthase;
NAC, N-acetylcysteine;
DPI, diphenylene iodonium;
DMEM, Dulbecco's modified Eagle's medium;
FCS, fetal calf serum;
PBS, phosphate-buffered saline;
GST, glutathione
S-transferase.
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