Iron activates NF-kappa B in Kupffer cells

Hongyun She1, Shigang Xiong1, Min Lin1, Ebrahim Zandi2, Cecilia Giulivi3, and Hidekazu Tsukamoto1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

10.1152/ajpgi.00108. 2002.--- Iron exacerbates various types of liver injury in which nuclear factor (NF)-kappa B-driven genes are implicated. This study tested a hypothesis that iron directly elicits the signaling required for activation of NF-kappa B and stimulation of tumor necrosis factor (TNF)-alpha gene expression in Kupffer cells. Addition of Fe2+ but not Fe3+ (~5-50 µM) to cultured rat Kupffer cells increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappa B-dependent manner. Cu+ but not Cu2+ stimulated TNF-alpha protein release and promoter activity but with less potency. Fe2+ caused a disappearance of the cytosolic inhibitor kappa Balpha , 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-kappa B but coinciding with activation of inhibitor kappa B kinase (IKK) but not c-Jun NH2-terminal kinase. In conclusion, Fe2+ serves as a direct agonist to activate IKK, NF-kappa B, and TNF-alpha promoter activity and to induce the release of TNF-alpha 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-alpha -dependent liver injury.

tumor necrosis factor-alpha ; free radical; promoter; inhibitor kappa B kinase; electron paramagnetic resonance; nuclear factor-kappa B


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)-kappa 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-kappa 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<UP><SUB>2</SUB><SUP>−</SUP></UP>· or oxidative destruction of ferritin proteins (6, 39). Alternatively, · NO may cause mobilization of intracellular iron (11, 13, 21) by targeting iron-sulfur groups contained in several key enzymes (12, 17). Thus it is conceivable that in liver diseases in which mild iron accumulation, oxidative stress, and TNF-alpha induction commonly coexist, the transient release of catalytically active iron may serve to facilitate oxidative signaling for proinflammatory NF-kappa B activation.

The present study tested whether direct addition of ionic iron to cultured Kupffer cells leads to activation of NF-kappa B and induction of TNF-alpha expression. Our results demonstrate that Fe2+ but not Fe3+ at concentrations as low as 5 µM stimulates TNF-alpha release. It also induces TNF-alpha promoter activity in an NF-kappa B-dependent manner, and this effect is associated with time-dependent activation of inhibitor kappa B (Ikappa B) kinase (IKK) and NF-kappa 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-kappa B activation in Kupffer cells in a redox status-dependent manner.


    MATERIALS AND METHODS
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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-alpha and TNF-alpha promoter activity. For activation of IKK and NF-kappa 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-kappa 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 kappa B consensus sequence (3), the kappa B site from TNF-alpha 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.

Ikappa Balpha and p65 immunoblot analysis. Cytoplasmic and nuclear extracts of iron-stimulated, cultured Kupffer cells were examined for Ikappa Balpha 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% beta -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 Ikappa Balpha (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 Ikappa Balpha 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 alpha -(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 beta -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 IKKalpha 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)-Ikappa Balpha , and [gamma -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-Ikappa Balpha 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-alpha promoter analysis. To assess the effects of ionic iron and copper on TNF-alpha promoter activity, cultured Kupffer cells were transiently transfected with a TNF-alpha promoter-luciferase construct using Targefect F-2 (Targeting System, San Diego, CA). The construct was created by ligating a 1.4-kb mouse TNF-alpha 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-kappa B, the cells were also cotransfected with the Ikappa Balpha super repressor plasmid, which expresses Ikappa Balpha 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-alpha RT-PCR. For RT-PCR analysis for TNF-alpha , 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 beta -actin and 43 for TNF-alpha ) cycles of denaturation (95°C, 30 s), annealing (58°C, 30 s), and extension (72°C, 60 s). Primers used for TNF-alpha were sense, 5'-ATGAGCACAGAAAGCATGATG and antisense, 5'-TACAGGCTTGTCACTCGAATT, and for beta -actin they were sense, 5'-CACGGCATTGTAACCAACTG and antisense, 5'-AGGGCAACATAGCACAGCTT.

TNF-alpha immunoassay. The effects of iron and copper on the release of TNF-alpha by cultured Kupffer cells were examined by analyzing the TNF-alpha protein in the media with a commercially available mouse TNF-alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fe2+ but not Fe3+ stimulates release of TNF-alpha . We first tested whether iron stimulates the release of TNF-alpha by cultured Kupffer cells. As shown in Fig. 1, the addition of Fe2+ but not Fe3+ increased TNF-alpha 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-alpha 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-alpha 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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Fig. 1.   Fe2+ but not Fe3+ stimulates tumor necrosis factor (TNF)-alpha release. Cultured Kupffer cells in serum-free medium were treated with increasing concentrations of ferrous sulfate, ferric ammonium sulfate, cuprous chloride, and cupric sulfate for 4 h, followed by determination of TNF-alpha protein in the medium by ELISA. Note significantly increased release of TNF-alpha protein with Fe2+ but not Fe3+ at the concentrations as low as 5 µM, reaching the maximal 8-fold stimulation at 10 µM. Cu+ also stimulates the release, but the dose response is shifted to the right, indicating less potency. Data are obtained from ~3-6 different experiments and expressed as %control (no metal addition). TNF-alpha released under the control condition was 5.55 ± 2.88 pg/ml (mean ± SD, n = 6). *P < 0.05 and **P < 0.01 vs. control.

Iron stimulates TNF-alpha promoter activity. We then tested whether Fe2+ stimulates the TNF-alpha 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-alpha promoter activity, but Cu2+ and Fe3+ did not (Fig. 2A). Cotransfection of a super repressor Ikappa Balpha 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-alpha promoter in a NF-kappa B-dependent manner.


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Fig. 2.   Fe2+ increases TNF-alpha promoter activity and mRNA level. A: cultured Kupffer cells were transfected with a TNF-alpha promoter-luciferase construct followed by the treatment with Fe2+, Fe3+, Cu+, or Cu2+ for 14 h. The promoter activity was normalized by transfection efficiency as determined by Renilla luciferase activity. Note that Fe2+ induces the promoter activity by 2-fold at 10 and 50 µM. Cu+ slightly induces the promoter, but the oxidized metals (Fe3+ and Cu2+) do not. B: cells were cotransfected with the promoter-luciferase construct with a LacZ vector or dominant-negative inhibitor kappa Balpha (Ikappa Balpha ), followed by addition of Fe2+ (50 µM). Iron treatment stimulated the TNF-alpha promoter activity, and this effect was completely blocked by cotransfection with a super repressor Ikappa Balpha (DN-Ikappa B). Lipopolysaccharide (LPS)-stimulated promoter activity is shown as a positive control. *P < 0.01 vs. control. C: iron treatment increases TNF-alpha mRNA levels in Kupffer cells. The effects of Fe2+ on TNF-alpha mRNA levels in cultured Kupffer cells were examined by RT-PCR. Note increased mRNA levels with Fe2+. The last lane shows a robust induction by LPS as a positive control.

Fe2+ increases TNF-alpha mRNA levels. We then examined whether TNF-alpha 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-alpha message. Densitometric analysis and standardization with beta -actin data showed 2.3- and 2.0-fold increases in TNF-alpha message by 10 and 50 µM Fe2+, respectively.

Fe2+ activates NF-kappa B in cultured Kupffer cells. Next, we examined whether Fe2+ increases the binding of nuclear proteins to the kappa 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 kappa B site from the TNF-alpha 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-kappa 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-kappa B DNA binding was due to activation of the transcription factor, we performed Western blot analysis for cytosolic Ikappa Balpha and nuclear p65. As shown in the representative blots in Fig. 4, the cytosolic level of Ikappa Balpha 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 Ikappa Balpha degradation, NF-kappa B activation, and nuclear translocation of the RelA protein, resulting in increased DNA binding by NF-kappa B, all commencing at 30 min. In addition, the lack of the AP-1 response suggests that the effect of Fe2+ on NF-kappa B is rather selective.


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Fig. 3.   Treatment with Fe2+ increases the binding of nuclear factor (NF)-kappa B in cultured rat Kupffer cells. A: typical response of increased binding of both p65/p50 heterodimer and p50/p50 homodimer in cultured Kupffer cells exposed to Fe2+ (50 µM) for ~30 min-2 h. However, activator protein (AP)-1 binding is not affected by the treatment. B: supershift assays of the nuclear extracts from iron-treated Kupffer cells reveal the components of the NF-kappa B binding complexes to be a p65/p50 heterodimer and a p50/p50 homodimer.



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Fig. 4.   Iron treatment causes cytosolic Ikappa Balpha degradation and nuclear translocation of p65. Cytosolic and nuclear proteins prepared from Kupffer cells treated with iron sulfate (50 µM) for ~0-4 h were analyzed by immunoblotting for Ikappa Balpha (A) and p65 (B) levels, respectively. These data, which are representative of 3 independent experiments, demonstrate that Fe2+ induces a transient disappearance of Ikappa Balpha in cytosol and an increase of p65 in nuclear extracts at 30 min after addition, the time point that correlates with the increased NF-kappa B binding shown in Fig. 3. Equal loading of proteins is supported by the staining of fractionated proteins on the filters, as shown below the Western blot data.

Direct addition of Fe2+ to nuclear proteins does not increase RelA binding. Even though our Western blot results strongly supported that activation of NF-kappa 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-kappa B to the kappa 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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Fig. 5.   Direct addition of Fe2+ to the nuclear proteins does not increase RelA binding. Addition of ferrous sulfate (~0.1-50 µM) to the nuclear extracts prepared from the resting Kupffer cells does not increase the binding of p65/p50 but enhances p50/p50 binding.

Iron activates IKK. To investigate the mechanisms of iron-mediated activation of NF-kappa 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-Ikappa Balpha , 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 Ikappa Balpha 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-kappa B.


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Fig. 6.   Fe2+ activates Ikappa B kinase (IKK) but not c-Jun NH2-terminal kinase (JNK) before NF-kappa B activation. IKK and JNK activity assays were performed on the Kupffer cell lysate samples collected at different time points after FeSO4 treatment. Note that IKK activity as assessed by phosphorylation of glutathione-S-transferase (GST)-Ikappa Balpha (P-Ikappa Balpha ) is increased at 15 min after addition of FeSO4. No activation of JNK is seen after the iron treatment, as assessed by phosphorylation of GST-c-Jun (P-c-Jun). LPS-induced activation of IKK (15 min) and JNK (30 min) is shown as positive controls in the last lanes. Relatively equal levels of IKKalpha , JNK p54, and JNK p46 are shown by immunoblots.

Iron increases EPR-detectable radicals before NF-kappa B activation. NF-kappa 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-kappa B. In fact, Fe2+ can react with oxygen in aqueous solution to produce Fe3+ and O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, and this ROS may be responsible for the observed effect. Fe2+ may also catalyze the formation of ·OH from H2O2, which is generated from basal NADPH oxidase activity of cultured Kupffer cells. To address these possibilities, the cells were treated with Fe2+ for ~0-30 min, ROS was trapped with POBN, and EPR spectra were analyzed. Kupffer cells without iron treatment exhibited an EPR spectrum constituted by an equal mixture of three spin adducts: methyl, hydroxyl, and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (Fig. 7B). Addition of 50 µM Fe2+ to these cells resulted in an enhancement of the hydroxyl and methyl-POBN adduct signals (Fig. 7A). The formation of these adducts must have relied on the production of hydroxyl radical from an iron-catalyzed Fenton reaction. The methyl adduct was likely produced at the attack of the ·OH on the methyl moiety of DMSO and the subsequent trap of this methyl radical by POBN. Both signals increased with incubation time (Fig. 7C) up to a maximum at ~15-20 min, regaining the initial values after 30 min. These increases in the steady-state concentration of these radicals indicate that the transient increases probably occurred as part of a response mechanism or signal transduction pathway on stimulus of exogenous iron. In particular, the fact that the peak of the radical generation at 15-20 min coincided with IKK activation and preceded activation of NF-kappa 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-kappa 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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Fig. 7.   Iron increases electron paramagnetic resonance (EPR)-detectable radicals before NF-kappa B activation. Cultured Kupffer cells were treated with iron sulfate (50 µM) for ~0-30 min, reactive oxygen species were trapped with alpha -(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN), and EPR spectra were analyzed. The cells without iron treatment exhibited an EPR spectrum constituted by an equal mixture of 3 spin adducts: methyl, hydroxyl, and superoxide anion (B). Addition of iron to the cells increased the hydroxyl and methyl-POBN adduct signals (A). The methyl adduct was likely produced on the attack of hydroxyl radical on the methyl moiety of DMSO and the subsequent trap of this methyl radical by POBN. Both signals increased with incubation time (C) up to a maximum at ~15 min, regaining the initial values after 30 min.


    DISCUSSION
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ABSTRACT
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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-kappa 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-alpha protein expression. These results suggest a possibility that iron may serve as an independent agonist for activation of NF-kappa B and induction of NF-kappa 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-gamma and LPS (7). We have previously demonstrated that the treatment of cultured Kupffer cells with an iron chelator effectively suppressed activation of NF-kappa B (22). Therefore, the evidence presented by the current study offers the pivotal molecular basis for the link between iron and NF-kappa 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-kappa B in Kupffer cells in vivo.

It is also known that iron overload inhibits functions of macrophages, including expression of proinflammatory cytokines (24, 26, 45). These effects are likely due to cytotoxicity of the cells exposed to either high or chronic iron loading. Indeed, acute iron overload via phagocytosis of erythrocytes is shown to cause cell toxicity in cultured Kupffer cells (20). In our study, Kupffer cells exposed to Fe2+ iron at different concentrations up to 50 µM did not show signs of cytotoxicity, and under such conditions, the direct agonistic effect on NF-kappa B was evident.

Our results also demonstrate that the peak of ·OH generation coincides with activation of IKK in iron-treated Kupffer cells, suggesting that either this most potent radical or downstream molecules may be the potential effectors for IKK activation. It is presumed that this radical is generated by Fe2+ via a Fenton pathway catalyzing one electron reduction of H2O2. The role of metal-catalyzed generation of ·OH in NF-kappa B activation has previously been proposed (22, 37, 42), and our present data further support the notion. However, it remains to be determined whether and how ·OH indeed activates IKK. It may exert direct effects on IKK, such as oxidation of cysteine residues within the activation loop of IKKalpha and -beta and a tighter conformation of the complex for phosphorylation of Ikappa Balpha via disulfide bond formation (33). It may also mediate IKK activation via its effects on upstream kinases. For instance, thioredoxin can be oxidized by ·OH, and this may cause a release of apoptosis signal-regulating kinase 1 (ASK1), which is usually bound to thioredoxin as an inactive form (31). Released ASK1 can then be oligomerized for activation of p38, which may in turn lead to activation of NF-kappa B (23). However, since ·OH is extremely reactive, it is difficult to conceive that such selective oxidation of target molecules can be achieved with this radical without additional regulatory mechanisms. ·OH may also target other unknown inhibitors of IKK. Alternatively, ·OH may induce intracellular lipid peroxidation, and lipid peroxides or their end products, such as aldehydes, may regulate signaling for IKK activation. Indeed, 4-hydroxynonenal, one such aldehydic product, has been shown to activate JNK (27, 44) and p38 MAPKs (44). However, in our study, Fe2+ activated IKK independently of JNK (Fig. 6) or p38 (unpublished data) MAPK activities. Obviously, future studies are needed to better delineate the molecular steps connecting iron and IKK activation.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, MT, Staal FJ, Gitler C, Herzenberg LA, and Herzenberg LA. Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway. Proc Natl Acad Sci USA 91: 11527-11531, 1994[Abstract/Free Full Text].

2.   Angel, P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, and Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49: 729-739, 1987[ISI][Medline].

3.   Baeuerle, PA, and Baltimore D. A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB. Genes Dev 3: 1689-1698, 1989[Abstract].

4.   Bassett, SE, Di Bisceglie AM, Bacon BR, Sharp RM, Govindarajan S, Hubbard GB, Brasky KM, and Lanford RE. Effects of iron loading on pathogenicity in hepatitis C virus-infected chimpanzees. Hepatology 29: 1884-1892, 1999[ISI][Medline].

5.   Bonkovsky, HL, Jawaid Q, Tortorelli K, LeClair P, Cobb J, Lambrecht RW, and Banner BF. Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis. J Hepatol 31: 421-429, 1999[ISI][Medline].

6.   Cairo, G, Tacchini L, Pogliaghi G, Anzon E, Tomasi A, and Bernelli-Zazzera A. Induction of ferritin synthesis by oxidative stress. Transcriptional and post-transcriptional regulation by expansion of the "free" iron pool. J Biol Chem 270: 700-703, 1995[Abstract/Free Full Text].

7.   Chaudhri, G, and Clark IA. Reactive oxygen species facilitate the in vitro and in vivo lipopolysaccharide-induced release of tumor necrosis factor. J Immunol 143: 1290-1294, 1989[Abstract/Free Full Text].

8.   Collart, MA, Baeuerle P, and Vassalli P. Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Mol Cell Biol 10: 1498-1506, 1990[ISI][Medline].

9.   Colletti, LM, Remick DG, and Campbell DA, Jr. Desferal attenuates TNF release following hepatic ischemia/reperfusion. J Surg Res 57: 447-453, 1994[ISI][Medline].

10.   Di Bisceglie, AM, Bonkovsky HL, Chopra S, Flamm S, Reddy RK, Grace N, Killenberg P, Hunt C, Tamburro C, Tavill AS, Ferguson R, Krawitt E, Banner B, and Bacon BR. Iron reduction as an adjuvant to interferon therapy in patients with chronic hepatitis C who have previously not responded to interferon: a multicenter, prospective, randomized, controlled trial. Hepatology 32: 135-138, 2000[ISI][Medline].

11.   Drapier, JC, and Hibbs JB, Jr. Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible. J Clin Invest 78: 790-797, 1986[ISI][Medline].

12.   Drapier, JC, and Hibbs JB, Jr. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells. J Immunol 140: 2829-2838, 1988[Abstract/Free Full Text].

13.   Drapier, JC, Hirling H, Wietzerbin J, Kaldy P, and Kuhn LC. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMBO J 12: 3643-3649, 1993[Abstract].

14.   Fuhrman, B, Oiknine J, and Aviram M. Iron induces lipid peroxidation in cultured macrophages, increases their ability to oxidatively modify LDL, and affects their secretory properties. Atherosclerosis 111: 65-78, 1994[ISI][Medline].

15.   Han, J, Huez G, and Beutler B. Interactive effects of the tumor necrosis factor promoter and 3'-untranslated regions. J Immunol 146: 1843-1848, 1991[Abstract/Free Full Text].

16.   Hellerbrand, C, Jobin C, Iimuro Y, Licato L, Sartor RB, and Brenner DA. Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. Hepatology 27: 1285-1295, 1998[ISI][Medline].

17.   Hibbs, JB, Jr, Taintor RR, and Vavrin Z. Iron depletion: possible cause of tumor cell cytotoxicity induced by activated macrophages. Biochem Biophys Res Commun 123: 716-723, 1984[ISI][Medline].

18.   Jiang, X, and Baldwin CL. Iron augments macrophage-mediated killing of Brucella abortus alone and in conjunction with interferon-gamma. Cell Immunol 148: 397-407, 1993[ISI][Medline].

19.   Junge, B, Carrion Y, Bosco C, Galleano M, Puntarulo S, Tapia G, and Videla LA. Effects of iron overload and lindane intoxication in relation to oxidative stress, Kupffer cell function, and liver injury in the rat. Toxicol Appl Pharmacol 170: 23-28, 2001[ISI][Medline].

20.   Kondo, H, Saito K, Grasso JP, and Aisen P. Iron metabolism in the erythrophagocytosing Kupffer cell. Hepatology 8: 32-38, 1988[ISI][Medline].

21.   Lancaster, JR, Jr, and Hibbs JB, Jr. EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proc Natl Acad Sci USA 87: 1223-1227, 1990[Abstract].

22.   Lin, M, Rippe RA, Niemela O, Brittenham G, and Tsukamoto H. Role of iron in NF-kappa B activation and cytokine gene expression by rat hepatic macrophages. Am J Physiol Gastrointest Liver Physiol 272: G1355-G1364, 1997[Abstract/Free Full Text].

23.   Liu, H, Nishitoh H, Ichijo H, and Kyriakis JM. Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin. Mol Cell Biol 20: 2198-2208, 2000[Abstract/Free Full Text].

24.   Loegering, DJ, Raley MJ, Reho TA, and Eaton JW. Macrophage dysfunction following the phagocytosis of IgG-coated erythrocytes: production of lipid peroxidation products. J Leukoc Biol 59: 357-362, 1996[Abstract].

25.   Mendler, MH, Turlin B, Moirand R, Jouanolle AM, Sapey T, Guyader D, Le Gall JY, Brissot P, David V, and Deugnier Y. Insulin resistance-associated hepatic iron overload. Gastroenterology 117: 1155-1163, 1999[ISI][Medline].

26.   Olynyk, JK, and Clarke SL. Iron overload impairs pro-inflammatory cytokine responses by Kupffer cells. J Gastroenterol Hepatol 16: 438-444, 2001[ISI][Medline].

27.   Parola, M, Robino G, Marra F, Pinzani M, Bellomo G, Leonarduzzi G, Chiarugi P, Camandola S, Poli G, Waeg G, Gentilini P, and Dianzani MU. HNE interacts directly with JNK isoforms in human hepatic stellate cells. J Clin Invest 102: 1942-1950, 1998[Abstract/Free Full Text].

28.   Powell, LW. The role of alcoholism in hepatic iron storage disease. Ann NY Acad Sci 252: 124-134, 1975[ISI][Medline].

29.   Rothwarf, DM, Zandi E, Natoli G, and Karin M. IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. Nature 395: 297-300, 1998[ISI][Medline].

30.   Sadrzadeh, SM, Nanji AA, and Price PL. The oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one reduces hepatic-free iron, lipid peroxidation and fat accumulation in chronically ethanol-fed rats. J Pharmacol Exp Ther 269: 632-636, 1994[Abstract].

31.   Saitoh, M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, and Ichijo H. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J 17: 2596-2606, 1998[Abstract/Free Full Text].

32.   Sakaida, I, Kayano K, Wasaki S, Nagatomi A, Matsumura Y, and Okita K. Protection against acetaminophen-induced liver injury in vivo by an iron chelator, deferoxamine. Scand J Gastroenterol 30: 61-67, 1995[ISI][Medline].

33.   Schoonbroodt, S, and Piette J. Oxidative stress interference with the nuclear factor-kappa B activation pathways. Biochem Pharmacol 60: 1075-1083, 2000[ISI][Medline].

34.   Schreck, R, and Baeuerle PA. Assessing oxygen radicals as mediators in activation of inducible eukaryotic transcription factor NF-kappa B. Methods Enzymol 234: 151-163, 1994[ISI][Medline].

35.   Schreiber, E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989[ISI][Medline].

36.   Schulze-Osthoff, K, Beyaert R, Vandevoorde V, Haegeman G, and Fiers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J 12: 3095-3104, 1993[Abstract].

37.   Shi, X, Dong Z, Huang C, Ma W, Liu K, Ye J, Chen F, Leonard SS, Ding M, Castranova V, and Vallyathan V. The role of hydroxyl radical as a messenger in the activation of nuclear transcription factor NF-kappaB. Mol Cell Biochem 194: 63-70, 1999[ISI][Medline].

38.   Shibanuma, M, Kuroki T, and Nose K. Inhibition by N-acetyl-L-cysteine of interleukin-6 mRNA induction and activation of NF kappa B by tumor necrosis factor alpha in a mouse fibroblastic cell line, Balb/3T3. FEBS Lett 353: 62-66, 1994[ISI][Medline].

39.   Tacchini, L, Recalcati S, Bernelli-Zazzera A, and Cairo G. Induction of ferritin synthesis in ischemic-reperfused rat liver: analysis of the molecular mechanisms. Gastroenterology 113: 946-953, 1997[ISI][Medline].

40.   Tapia, G, Troncoso P, Galleano M, Fernandez V, Puntarulo S, and Videla LA. Time course study of the influence of acute iron overload on Kupffer cell functioning and hepatotoxicity assessed in the isolated perfused rat liver. Hepatology 27: 1311-1316, 1998[ISI][Medline].

41.   Tsukamoto, H, Horne W, Kamimura S, Niemela O, Parkkila S, Yla-Herttuala S, and Brittenham GM. Experimental liver cirrhosis induced by alcohol and iron. J Clin Invest 96: 620-630, 1995[ISI][Medline].

42.   Tsukamoto, H, Lin M, Ohata M, Giulivi C, French SW, and Brittenham G. Iron primes hepatic macrophages for NF-kappaB activation in alcoholic liver injury. Am J Physiol Gastrointest Liver Physiol 277: G1240-G1250, 1999[Abstract/Free Full Text].

43.   Tsukamoto, H, Lin M, Pham TV, Nanji A, and Fong TL. Role of inflammation in liver fibrogenesis. In: Therapy in Liver Disease. The Pathophysiological Basis of Therapy, edited by Arroyo V, Bosch J, and Rodes J.. Barcelona: Masson, 1997, p. 173-177.

44.   Uchida, K, Shiraishi M, Naito Y, Torii Y, Nakamura Y, and Osawa T. Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. J Biol Chem 274: 2234-2242, 1999[Abstract/Free Full Text].

45.   Van Asbeck, BS, Marx JJ, Struyvenberg A, and Verhoef J. Functional defects in phagocytic cells from patients with iron overload. J Infect 8: 232-240, 1984[ISI][Medline].

46.   Warren, S, Torti SV, and Torti FM. The role of iron in the cytotoxicity of tumor necrosis factor. Lymphokine Cytokine Res 12: 75-80, 1993[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 283(3):G719-G726




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